WO2002098451A1 - Mixtures of calcitonin drug-oligomer conjugates comprising polyalkylene glycol, uses thereof, and methods of making same - Google Patents

Mixtures of calcitonin drug-oligomer conjugates comprising polyalkylene glycol, uses thereof, and methods of making same Download PDF

Info

Publication number
WO2002098451A1
WO2002098451A1 PCT/US2002/017575 US0217575W WO02098451A1 WO 2002098451 A1 WO2002098451 A1 WO 2002098451A1 US 0217575 W US0217575 W US 0217575W WO 02098451 A1 WO02098451 A1 WO 02098451A1
Authority
WO
WIPO (PCT)
Prior art keywords
mixture
oligomer
calcitonin
moiety
polyethylene glycol
Prior art date
Application number
PCT/US2002/017575
Other languages
French (fr)
Inventor
Nnochiri N. Ekwuribe
Christopher H. Price
Aslam M. Ansari
Amy L. Odenbaugh
Original Assignee
Nobex Corporation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Nobex Corporation filed Critical Nobex Corporation
Priority to DK02732030.8T priority Critical patent/DK1404360T3/en
Priority to DE60235010T priority patent/DE60235010D1/en
Priority to JP2003501489A priority patent/JP4272510B2/en
Priority to EP02732030A priority patent/EP1404360B1/en
Priority to KR1020037015912A priority patent/KR100930606B1/en
Priority to MXPA03011283A priority patent/MXPA03011283A/en
Priority to AT02732030T priority patent/ATE454160T1/en
Priority to CA002449686A priority patent/CA2449686A1/en
Publication of WO2002098451A1 publication Critical patent/WO2002098451A1/en

Links

Classifications

    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • A61K38/16Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22Hormones
    • A61K38/23Calcitonins
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575Hormones
    • C07K14/585Calcitonins
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00Drugs for skeletal disorders
    • A61P19/08Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00Drugs for skeletal disorders
    • A61P19/08Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis

Definitions

  • the present invention relates to drug-oligomer conjugates, and, more particularly, to calcitonin drug-oligomer conjugates.
  • Calcitonin is a naturally occurring hormone with a short half-life that is believed to act directly on osteoclasts (via receptors on the cell surface for calcitonin). This action may directly inhibit osteoclastic bone resorption, which may lead to hypocalcemic and/or hypophosphatemic serum effects. Calcitonin may be useful in treating various bone disorders including, but not limited to, osteoporosis and Paget's disease. Osteoporosis is a bone disease in which bone tissue is normally mineralized, but the amount of bone is decreased and the structural integrity of trabecular bone is impaired. Cortical bone becomes more porous and thinner. This makes the bone weaker and more likely to fracture.
  • Calcitonin given as a subcutaneous injection has shown significant improvements in bone density; however, a high incidence of side effects, including pain at the injection site, flushing and nausea, have been reported which may limit the use of the drug.
  • Paget's disease of bone is a metabolic bone disorder of unknown origin which normally affects older people.
  • the disease causes an increased and irregular formation of bone as the bone cells, which are responsible for dissolving the body's old bone and replacing it with new, become out of control.
  • the deformed new bone becomes larger, weaker and has more blood vessels than normal bone.
  • the structure is irregular and consequently weaker, which makes it prone to fracture even after a minor injury.
  • Calcitonin may be effective in treating disorders of increased skeletal remodeling, such as Paget's disease. In treating Paget's disease, chronic use of calcitonin may produce long-term reduction in symptoms; however, side effects of calcitonin administration may include nausea, hand swelling, urticaria, and intestinal cramping.
  • conjugating polypeptides such as calcitonin with polydispersed mixtures of polyethylene glycol or polyethylene glycol-containing polymers.
  • U.S. Patent No. 5,359,030 to Ekwuribe proposes conjugating polypeptides such as calcitonin with polydispersed mixtures of polyethylene glycol modified glycolipid polymers and polydispersed mixtures of polyethylene glycol modified fatty acid polymers.
  • the number average molecular weight of polymer resulting from each combination is preferred to be in the range of from about 500 to about 10,000 Daltons.
  • PEG polydispersed polyethylene glycol
  • conjugates described in Ekwuribe is likely a result of the use of polydispersed polyethylene glycol in the polymer synthesis.
  • PEG is typically produced by base-catalyzed ring-opening polymerization of ethylene oxide. The reaction is initiated by adding ethylene oxide to ethylene glycol, with potassium hydroxide as catalyst. This process results in a polydispersed mixture of polyethylene glycol polymers having a number average molecular weight within a given range of molecular weights.
  • PEG products offered by Sigma- Aldrich of Milwaukee, Wisconsin are provided in polydispersed mixtures such as PEG 400 (M span 380-420); PEG 1,000 (M ⁇ 950-1,050); PEG 1,500 (M n 1,400-1,600); and PEG 2,000 (M n 1,900-2,200).
  • a mixture of calcitonin-oligomer conjugates comprising polyethylene glycol according to embodiments of the present invention may lower serum calcium levels by 10, 15 or even 20 percent or more. Moreover, a mixture of calcitonin-oligomer conjugates comprising polyethylene glycol according to embodiments of the present invention may be more effective at surviving an in vitro model of intestinal digestion than non-conjugated calcitonin. Furthermore, mixtures of calcitonin-oligomer conjugates comprising polyethylene glycol according to embodiments of the present invention may exhibit a higher bioavailability than non-conjugated calcitonin.
  • a substantially monodispersed mixture of conjugates each comprising a calcitonin drug coupled to an oligomer that comprises a polyethylene glycol moiety is provided.
  • the polyethylene glycol moiety preferably has at least 2, 3, or 4 polyethylene glycol subunits and, most preferably, has at least 7 polyethylene glycol subunits.
  • the oligomer preferably further comprises a lipophilic moiety.
  • the calcitonin . drug is preferably salmon calcitonin.
  • Oligomers are preferably coupled at Lys 11 and Lys 18 of the salmon calcitonin.
  • the conjugate is preferably amphiphilically balanced such that the conjugate is aqueously soluble and able to penetrate biological membranes.
  • each conjugate includes salmon calcitonin covalently coupled at Lys 11 of the salmon calcitonin to a carboxylic acid moiety of a first oligomer that comprises octanoic acid covalently coupled at the end distal to the carboxylic acid moiety to a methyl terminated polyethylene glycol moiety having at least 7 polyethylene glycol subunits, and covalently coupled at Lys ls of the salmon calcitonin to a carboxylic acid moiety of a second oligomer that comprises, octanoic acid covalently coupled at the end distal to the carboxylic acid moiety to a methyl terminated polyethylene glycol moiety having at least 7 polyethylene glycol subunits.
  • a substantially monodispersed mixture of conjugates where each conjugate comprises a calcitomn drug coupled to an oligomer comprising a polyethylene glycol moiety, and the mixture is capable of lowering serum calcium levels in a subject by at least 5 percent.
  • a substantially monodispersed mixture of conjugates where each conjugate comprises a calcitonin drug coupled to an oligomer comprising a polyethylene glycol moiety, and the mixture has an increased resistance to degradation by chymotrypsin and/or trypsin when compared to the resistance to degradation by chymotrypsin and/or trypsin of the calcitonin drug which is not coupled to the oligomer.
  • a substantially monodispersed mixture of conjugates where each conjugate comprises a calcitonin drug coupled to an oligomer comprising a polyethylene glycol moiety, and the mixture has a higher bioefficacy than the bioeff ⁇ cacy of the calcitonin ' drug which is not coupled to the oligomer.
  • each conjugate includes a calcitonin drug coupled to an oligomer that comprises a polyethylene glycol moiety, and the mixture has a molecular weight distribution with a standard deviation of less than about 22 Daltons.
  • each conjugate includes a calcitonin drug coupled to an oligomer that comprises a polyethylene glycol moiety, and the mixture has a dispersity coefficient (DC) greater than 10,000 where
  • n is the number of different molecules in the sample
  • ⁇ i is the number of i ⁇ molecules in the sample
  • Mj is the mass of the i ⁇ molecule.
  • each conjugate includes a calcitonin drug coupled to an oligomer and has the same number of polyethylene glycol subunits.
  • each conjugate has the same molecular weight and has the formula: Calcitonin I ug ⁇ B-L j —G k -R-G' m -—R'—G'* n —T " 1 (A)
  • B is a bonding moiety
  • L is a linking group
  • G, G and G" are individually selected spacer groups
  • R is a lipophilic group and R' is a polyalkylene glycol group, or R' is the lipophilic group and R is the polyalkylene oxide group; T is a terminating group; j, k, m and n are individually 0 or 1 ; and p is an integer from 1 to the number of nucleophilic residues on the calcitonin drug.
  • compositions comprising conjugate mixtures of the present invention as well as methods of treating osteoporosis in a subject in need of such treatment by administering an effective amount of such pharmaceutical compositions are also provided. Additionally, methods of synthesizing such conjugate mixtures are provided.
  • Calcitonin-oligomer conjugate mixtures according to embodiments of the present . invention may lower serum calcium levels by 20 percent or more. Moreover, such conjugates may provide decreased degradation by intestinal enzymes and/or provide increased bioavailability when compared to non-conjugated calcitonin.
  • Figure 1 illustrates a generic scheme for synthesizing a mixture of activated polymers comprising a polyethylene glycol moiety and a fatty acid moiety according to embodiments of the present invention
  • Figure 2 illustrates a scheme for synthesizing a mixture of mPEG according to embodiments of the present invention
  • Figure 3 illustrates a scheme for synthesizing a mixture of activated mPEG7-hexyl oligomers according to embodiments of the present invention
  • Figure 4 illustrates a scheme for synthesizing a mixture of activated mPEG7-octyl oligomers according to embodiments of the present invention
  • Figure 5 illustrates a scheme for synthesizing a mixture of activated mPEG-decyl oligomers according to embodiments of the present invention
  • Figure 6 illustrates a scheme for synthesizing a mixture of activated stearate-PEG6 oligomers according to embodiments of the present invention
  • Figure 7 illustrates a scheme for synthesizing a mixture of activated stearate-PEG8 oligomers according to embodiments of the present invention
  • Figure 8 illustrates a scheme for synthesizing a mixture of activated PEG3 oligomers according to embodiments of the present invention
  • Figure 9 illustrates a scheme for synthesizing a mixture of activated palmitate-PEG3 oligomers according to embodiments of the present invention.
  • Figure 10 illustrates a scheme for synthesizing a mixture of activated PEG6 oligomers according to embodiments of the present invention
  • Figure 11 illustrates a scheme for synthesizing various propylene glycol monomers according to embodiments of the present invention
  • Figure 12 illustrates a scheme for synthesizing various propylene glycol polymers according to embodiments of the present invention
  • Figure 13 illustrates a scheme for synthesizing various propylene glycol polymers according to embodiments of the present invention
  • Figure 14 illustrates a comparison of the average AUCs for various mixtures of calcitonin-oligomer conjugates according to embodiments of the present invention with non- conjugated calcitonin, which is provided for comparison purposes only and does not form part of the invention;
  • Figure 15 illustrates a dose-response curve for a mixture of mPEG7-octyl-calcitonin diconjugates according to embodiments of the present invention compared with a dose- response curve for calcitonin, which is provided for comparison purposes and is not a part of the present invention
  • Figure 16 illustrates a dose-response curve after oral administration of a mixture of mPEG7-octyl-calcitonin diconjugates according to embodiments of the present invention
  • Figure 17 illustrates a dose-response curve after subcutaneous administration of a mixture of mPEG7-octyl-calcitonin diconjugates according to embodiments of the present ⁇ invention
  • Figure 18 illustrates a dose-response curve after subcutaneous administration of salmon calcitomn, which is provided for comparison purposes and is not part of the present invention.
  • non-polydispersed is used to describe a mixture of compounds having a dispersity that is in contrast to the polydispersed mixtures described in U.S. Patent No. 5,359,030 to Ekwuribe.
  • substantially monodispersed is used to describe a mixture of compounds wherein at least about 95 percent of the compounds in the mixture have the same molecular weight.
  • the term "monodispersed” is used to describe a mixture of compounds wherein about 100 percent of the compounds in the mixture have the same molecular weight.
  • the term “substantially purely monodispersed” is used to describe a mixture of compounds wherein at least about 95 percent of the compounds in the mixture have the same molecular weight and have the same molecular structure.
  • a substantially purely monodispersed mixture is a substantially monodispersed mixture, but a substantially monodispersed mixture is not necessarily a substantially purely monodispersed mixture.
  • the term "purely monodispersed” is used to describe a mixture of compounds wherein about 100 percent of the compounds in the mixture have the same molecular weight and have the same molecular structure. Thus, a purely monodispersed mixture is a monodispersed mixture, but a monodispersed mixture is not necessarily a purely monodispersed mixture.
  • the term "weight average molecular weight” is defined as the sum of the products of the weight fraction for a given molecule in the mixture times the mass of the molecule for each molecule in the mixture. The "weight average molecular weight" is represented by the symbol M w -
  • number average molecular weight is defined as the total weight of a mixture divided by the number of molecules in the mixture and is represented by the symbol M n .
  • DC dispersity coefficient
  • intra-subject variability means the variability in activity occurring within the same subject when the subject is administered the same dose of a drug or pharmaceutical composition at different times.
  • inter-subject variability means the variability in activity between two or more subjects when each subject is admimstered the same dose of a given drug or pharmaceutical formulation.
  • bioefficacy means the ability of a drug or drug conjugate to interact with one or more desired receptors in vivo.
  • calcitonin drug means a drug possessing all or some of the biological activity of calcitonin.
  • calcitonin means chicken calcitomn, eel calcitonin, human calcitonin, porcine calcitonin, rat calcitonin or salmon calcitonin provided by natural, synthetic, or genetically engineered sources.
  • calcitonin analog means calcitonin wherein one or more of the amino acids have been replaced while retaining some or all of the activity of the calcitonin.
  • the analog is described by noting the replacement amino acids with the position of the replacement as a superscript followed by a description of the calcitonin.
  • Pro 2 calcitonin, human means that the glycine typically found at the 2 position of a human calcitonin molecule has been replaced with proline.
  • Calcitonin analogs may be obtained by various means, as will be understood by those skilled in the art.
  • amino acids may be substituted for other amino acids in the calcitonin structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules.
  • structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules.
  • the hydropathic index of amino acids may be considered.
  • the importance of the hydropathic amino acid index in conferring interactive biologic function on a polypeptide is generally understood in the art. It is accepted that the . relative hydropathic character of the amino acid contributes to the secondary structure of the resultant polypeptide, which in turn defines the interaction of the polypeptide with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.
  • Each amino acid has been assigned a hydropathic index on the basis of its .
  • hydrophobicity and charge characteristics as follows: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).
  • amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a polypeptide with similar biological activity, i.e., still obtain a biological functionally equivalent polypeptide.
  • substitution of amino acids whose hydropathic indices are within ⁇ 2 of each other is preferred, those which are within ⁇ 1 of each other are particularly preferred, and those within ⁇ 0.5 of each other are even more ' particularly preferred.
  • hydrophilicity values have been assigned to arnino acid residues: arginine (+3.0); lysine ( ⁇ 3.0); aspartate (+3.0 ⁇ 1); glutamate (+3.0 ⁇ 1); seine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ⁇ 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4).
  • an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent polypeptide.
  • substitution of amino acids whose hydrophilicity values are within ⁇ 2 of each other is preferred, those which are within ⁇ 1 of each other are particularly preferred, and those within ⁇ 0.5 of each other are even more particularly preferred.
  • amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.
  • Exemplary substitutions i.e., amino acids that may be interchanged without significantly altering the biological activity of the polypeptide
  • amino acids that may be interchanged without significantly altering the biological activity of the polypeptide include, for example: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
  • calcitonin fragment means a segment of the amino acid sequence found in the calcitonin that retains some or all of the activity of the calcitonin.
  • calcitonin fragment analog means a segment of the amino acid sequence found in the calcitonin molecule wherein one or more of the amino acids in the segment have been replace while retaining some or all of the activity of the calcitonin.
  • PEG refers to straight or branched polyethylene glycol polymers, and includes the monomethylether of polyethylene glycol (mPEG).
  • PEG subunit and polyethylene glycol subunit refer to a single polyethylene glycol unit, i.e., — (CH 2 CH 2 O)— .
  • lipophilic means the ability to dissolve in lipids and/or the ability to penetrate, interact with and/or traverse biological membranes
  • lipophilic moiety or “lipophile” means a moiety which is lipophilic and/or which, when attached to another chemical entity, increases the lipophilicity of such chemical entity. Examples of lipophilic moieties include, but are not limited to, alkyls, fatty acids, esters of fatty acids, chplesteryl, adamantyl and the like.
  • lower alkyl refers to substituted or unsubstituted alkyl moieties having frqm 1 to 5 carbon atoms.
  • the term "higher alkyl” refers to substituted or unsubstituted alkyl moieties having 6 or more carbon atoms.
  • a substantially monodispersed mixture of calcitonin drug-oligomer conjugates is provided.
  • Each calcitonin drug-oligomer conjugate in the monodispersed mixture includes a calcitonin drug coupled to an oligomer that comprises a polyethylene glycol moiety.
  • at least about 96, 97, 98 or 99 percent of the conjugates in the mixture have the same molecular weight.
  • the mixture is a monodispersed mixture.
  • the mixture is a substantially purely monodispersed mixture.
  • at least about 96, 97, 98 or 99 percent of the conjugates in the mixture have the same molecular weight and have the same molecular structure.
  • the mixture is a purely monodispersed mixture.
  • the calcitonin drug is preferably calcitonin. More preferably, the calcitonin drug is salmon calcitonin. However, it is to be understood that the calcitonin drug may be selected from various calcitonin drugs known to those skilled in the art including, for example, calcitomn precursor peptides, calcitonin, calcitonin analogs, calcitonin fragments, and calcitonin fragment analogs.
  • Calcitonin precursor peptides include, but are not limited to, katacalcin (PDN-21) (C-procalcitonin), and N-proCT (amino-terminal procalcitonin cleavage peptide), human.
  • Calcitonin analogs may be provided by substitution of one or more amino acids in calcitonin as described above.
  • Calcitonin fragments include, but are not limited to, calcitomn 1-7, human; and calcitonin 8-32, salmon.
  • Calcitonin fragment analogs may be provided by substitution of one or more of the amino acids in a calcitonin fragment as described above.
  • the oligomer may be various oligomers comprising a polyethylene glycol moiety as will be understood by those skilled in the art.
  • the polyethylene glycol moiety of the oligomer has at least 2, 3 or 4 polyethylene glycol subunits. More preferably, the polyethylene glycol moiety has at least 5 or 6 polyethylene glycol subunits and, most preferably, the polyethylene glycol moiety has at least 7 polyethylene glycol subunits.
  • the oligomer may comprise one or more other moieties as will be understood by those skilled in the art including, but not limited to, additional hydrophilic moieties, lipophilic moieties, spacer moieties, linker moieties, and terminating moieties.
  • the various moieties in the oligomer are covalently coupled to one another by either hydrolyzable or non- hydrolyzable bonds.
  • the oligomer may further comprise one or more additional hydrophilic moieties (i.e., moieties in addition to the polyethylene glycol moiety) including, but not limited to, sugars, polyalkylene oxides, and polyamine/PEG copolymers.
  • additional hydrophilic moieties i.e., moieties in addition to the polyethylene glycol moiety
  • polyethylene glycol is a polyalkylene oxide
  • the additional hydrophilic moiety may be a polyethylene glycol moiety. Adjacent polyethylene glycol moieties will be considered to be the same moiety if they are coupled by an ether bond.
  • the moiety i.e., moieties in addition to the polyethylene glycol moiety
  • oligomers according to embodiments of the present invention comprise a polyethylene glycol moiety and no additional hydrophilic moieties.
  • the oligomer may further comprise one or more lipophilic moieties as will be understood by those skilled in the art.
  • the hpophilic moiety is preferably a saturated or unsaturated, linear or branched alkyl moiety or a saturated or unsaturated, linear or branched fatty acid moiety.
  • the lipophilic moiety is an alkyl moiety, it is preferably a linear, saturated or unsaturated alkyl moiety having 1 to 28 carbon atoms. More preferably, the alkyl moiety has 2 to 12 carbon atoms.
  • the lipophilic moiety is a fatty acid moiety, it is preferably a natural fatty acid moiety that is linear, saturated or unsaturated, having 2 to 18 carbon atoms.
  • the fatty acid moiety has 3 to 14 carbon atoms. Most preferably, the fatty acid moiety has at least 4, 5 or 6 carbon atoms.
  • rhe oligomer may further comprise one or more spacer moieties as will be understood ' by those skilled in the art. Spacer moieties may, for example, be used to separate a hydrophilic moiety from a Hpophilic moiety, to separate a lipophilic moiety or hydrophihc moiety from the calcitomn drug, to separate a first hydrophilic or lipophilic moiety from a second hydrophilic or lipophilic moiety, or to separate a hydrophilic moiety or lipophilic moiety from a linker moiety.
  • Spacer moieties are preferably selected from the group consisting of sugar, cholesterol and glycerine moieties.
  • the oligomer may further comprise one or more linker moieties that are used to couple the oligomer with the calcitonin drug as will be understood by those skilled in the art.
  • Linker moieties are preferably selected from the group consisting of alkyl and fatty acid moieties.
  • the oligomer may further comprise one or more terminating moieties at the one or more ends of the oligomer which are not coupled to the calcitonin drug.
  • the terminating moiety is preferably an alkyl or alkoxy moiety, and is more preferably a lower alkyl or lower alkoxy moiety. Most preferably, the terminating moiety is methyl or methoxy. While the terminating moiety is preferably an alkyl or alkoxy moiety, it is to be understood that the terminating moiety may be various moieties as will be understood by those skilled in the art including, but not limited to, sugars, cholesterol, alcohols, and fatty acids.
  • the oligomer is preferably covalently coupled to the calcitonin drug. In some
  • the. calcitonin drug is coupled to the oligomer utilizing a hydrol zable bond (e.g., an ester or carbonate bond).
  • a hydrolyzable coupling may provide a calcitomn drug- oligomer conjugate that acts as a prodrug.
  • a hydrolyzable coupling may provide for a time-release or controlled-release effect, administering the calcitonin drug over a given time period as one or more oligomers are cleaved from their respective calcitomn drug-oligomer conjugates to provide the active drug.
  • the calcitonin drug is coupled to the oligomer utilizing a non-hydrolyzable bond (e.g., a carbamate, amide, or ether bond).
  • the oligomer further comprises one or more bonding moieties that are used to covalently couple the oligomer with the calcitonin drug as will be understood by those skilled in the art.
  • Bonding moieties are preferably selected from the group consisting of covalent bond(s), ester moieties, carbonate moieties, carbamate moieties, amide moieties and secondary amine moieties. More than one moiety on the oligomer may be covalently coupled to the calcitonin drug.
  • oligomer is preferably covalently coupled to the calcitonin drug
  • the oligomer may be non-covalently coupled to the calcitonin drug to form a non-covalently conjugated calcitonin drug-oligomer complex.
  • non-covalent couplings include, but are not limited to, hydrogen bonding, ionic bonding, Van der Waals bonding, and micellular or liposomal encapsulation.
  • oligomers may be suitably constructed, modified and/or appropriately functionalized to impart the ability for non-covalent conjugation in a selected manner (e.g., to impart hydrogen bonding capability), as will be understood by those skilled in the art.
  • oligomers maybe derivatized with various compounds including, but not limited to, amino acids, oligopeptides, peptides, bile acids, bile acid derivatives, fatty acids, fatty acid derivatives, salicylic acids, salicylic acid derivatives, aminosalicylic acids, and aminosalicylic acid derivatives.
  • the resulting oligomers can non-covalently couple (complex) with drug molecules, pharmaceutical products, and/or pharmaceutical excipients.
  • the resulting complexes preferably have balanced lipophilic and hydrophilic properties.
  • oligomers may be derivatized with amine and/or alkyl amines. Under suitable acidic conditions, the resulting oligomers can form non- covalently conjugated complexes with drug molecules, pharmaceutical products and/or pharmaceutical excipients.
  • the products resulting from such complexation preferably have balanced lipophilic and hydrophilic properties.
  • More than one oligomer may be coupled to the calcitonin drug.
  • the oligomers in the plurality are preferably the same. However, it is to be understood that the oligomers in the plurality may be different from one another, or, alternatively, some of the oligomers in the plurality may be the same and some may be different.
  • all of the bonds coupling the plurality of oligomers to the calcitonin drug may be hydrolyzable, but have varying degrees of hydrolyzability such that, for example, one or more of the oligomers is rapidly removed from the calcitonin drug by hydrolysis in the body and one or more of the oligomers is slowly removed from the - calcitonin drug by hydrolysis in the body.
  • the oligomer may be coupled to the calcitonin drug at various nucleophilic residues of the calcitonin drug including, but not limited to, nucleophilic hydroxyl functions and/or amino functions.
  • nucleophilic hydroxyl function may be found, for example, at serine and/or tyrosine residues
  • a nucleophilic amino function may be found, for example, at histidine and/or lysine residues, and/or at the one or more N-termini of the polypeptide.
  • the coupling preferably forms a secondary amine.
  • the oligomer may be coupled to an amino functionality of the salmon calcitonin, including the amino functionality of Lys l , Lys and/or the N-terminus. While one or more oligomers may be coupled to the salmon calcitonin, a higher bioefficacy, such as improved serum calcium lowering ability, is observed for the di-conjugated salmon calcitonin where an oligomer is coupled to the amino functionalities of Lys 11 and the Lys 18 . Substantially monodispersed mixtures of calcitonin drug-oligomer conjugates of the present invention may be synthesized by various methods.
  • a substantially monodispersed mixture of oligomers consisting of carboxylic acid and polyethylene glycol is synthesized by contacting a substantially monodispersed mixture of carboxylic acid with a substantially monodispersed mixture of polyethylene glycol under conditions sufficient to provide a substantially monodispersed mixture of oligomers.
  • the oligomers of the substantially monodispersed mixture are then activated so that they are capable of reacting, with a calcitonin drug to provide a calcitonin drug-oligomer conjugate.
  • a synthesis route for providing a substantially monodispersed. mixture of oligomers is illustrated in Figure 3 and described in Examples 11-18 hereinbelow.
  • FIG. 4 Another embodiment of a synthesis route for providing a substantially monodispersed mixture of oligomers is illustrated in Figure 4 and described in Examples 19-24 hereinbelow. Still another embodiment of a synthesis route for providing a substantially monodispersed mixture of oligomers is illustrated in Figure 5 and described in Examples 25-29 hereinbelow. Yet another embodiment of a synthesis route for providing a substantially monodispersed mixture of oligomers is illustrated in Figure 6 and described in Examples 30-31 hereinbelow. Another embodiment of a synthesis route for providing a substantially monodispersed mixture of oligomers is illustrated in Figure 7 and described in Examples 32-37 hereinbelow.
  • Still another embodiment of a synthesis route for providing a substantially monodispersed mixture of oligomers is illustrated in Figure 8 and described in Example 38 hereinbelow.
  • Yet another embodiment of a synthesis route forproviding a substantially monodispersed mixture of oligomers is illustrated in Figure 9 and described in Example 39 hereinbelow.
  • Another embodiment of a synthesis route for providing a substantially monodispersed mixture of oligomers is illustrated in Figure 10 and described in Example 40 hereinbelow.
  • the substantially monodispersed mixture of activated oligomers maybe reacted with a substantially monodispersed mixture of calcitonin drugs under conditions sufficient to provide a mixture of calcitonin drug-oligomer conjugates.
  • a preferred synthesis is described in Example 41 hereinbelow.
  • the reaction conditions e.g., selected molar ratios, solvent mixtures and/or pH
  • the reaction conditions may be controlled such that the mixture of calcitonin drug-oligomer conjugates resulting from the reaction of the substantially monodispersed mixture of activated oligomers and the substantially monodispersed mixture of calcitonin drugs is a substantially monodispersed mixture.
  • conjugation at the amino functionality of lysine may be suppressed by maintaining the pH of the reaction solution below the pK a of lysine.
  • the mixture of calcitonin drug-oligomer conjugates may be separated and isolated utilizing, for example, HPLC to provide a substantially monodispersed mixture of calcitonin drug-oligomer conjugates, for example mono-, di-, or tri-conjugates.
  • the degree of conjugation (e.g., whether the isolated molecule is a mono-, di-, or tri-conjugate) of a particular isolated conjugate may be determined and/or verified utilizing various techniques as will be understood by those skilled in the art including, but not limited to, mass spectroscopy.
  • the particular conjugate structure (e.g., whether the oligomer is at Lys 11 , Lys 18 or the N-teiminus of a salmon calcitonin monoconjugate) may be determined and/or verified utilizing various techniques as will be understood by those skilled in the art including, but not limited to, sequence analysis, peptide mapping, selective enzymatic cleavage, and/or endopeptidase cleavage.
  • one or more of the reaction sites on the calcitonin drug may be blocked by, for example, reacting the calcitonin drug with a suitable blocking reagent such as N-tert-butoxycarbonyl (t-BOC), or N-(9- fluorenylmethoxycarbonyl) (N-FMOC).
  • a suitable blocking reagent such as N-tert-butoxycarbonyl (t-BOC), or N-(9- fluorenylmethoxycarbonyl) (N-FMOC).
  • t-BOC N-tert-butoxycarbonyl
  • N-FMOC N-(9- fluorenylmethoxycarbonyl)
  • the substantially monodispersed mixture of blocked calcitonin drugs may be reacted with the substantially monodispersed mixture of activated oligomers to provide a mixture of calcitomn drug-oligomer conjugates having oligomer(s) coupled to one or more nucleophilic residues and having blocking moieties coupled to other nucleophilic residues.
  • the calcitonin drug-oligomer conjugates may be de-blocked as will be understood by those skilled in the art. If necessary, the mixture of calcitonin drug-oligomer conjugates may then be separated as described above to provide a substantially monodispersed mixture of calcitonin drug- oligomer conjugates. Alternatively, the mixture of calcitonin drug-oligomer conjugates may be separated prior to de-blocking.
  • Substantially monodispersed mixtures of calcitonin drug-oligomer conjugates according to embodiments of the present invention preferably have improved properties when compared with those of conventional mixtures.
  • a substantially monodispersed mixture of calcitonin-oligomer conjugates preferably is capable of lowering serum calcium levels by at least 5 percent.
  • the mixture of conjugates is capable of lowering serum calcium levels by at least 10, 11, 12, 13 or 14 percent. More preferably, the mixture of conjugates is capable of lowering serum calcium levels by at least 15, 16, 17, 18 or 19 percent, and, most preferably, the mixture of conjugates is capable of lowering serum calcium levels by at least 20 percent.
  • a substantially monodispersed mixture of calcitonin-oligomer conjugates preferably has an increased resistance to degradation by chymotrypsin and/or trypsin when compared to the resistance to degradation by chymotrypsin and/or trypsin, respectively, of the calcitonin drug which is not coupled to the oligomer.
  • Resistance to chymotrypsin or trypsin corresponds to the percent remaining when the molecule to be tested is digested in the applicable enzyme using the procedure outlined in Example 51 below.
  • the resistance to degradation by chymotrypsin of the mixture of calcitonin drug- oligomer conjugates is about 10 percent greater than the resistance to degradation by chymotrypsin of the mixture of calcitonin drugs that is not conjugated with the oligomer.
  • the resistance to degradation by chymotrypsin of the mixture of calcitonin drug-oligomer conjugates is about 15 percent greater than the resistance to degradation by chymotrypsin of the mixture of calcitonin drug that is not conjugated with the oligomer, and, most preferably, the resistance to degradation by chymotrypsin of the mixture of calcitonin drug-oligomer conjugates is about 20 percent greater than the resistance to degradation by chymotrypsin of the mixture of calcitonin drug that is not conjugated with the oligomer.
  • the resistance to degradation by trypsin of the mixture of calcitonin drug-oligomer conjugates is about 10 percent greater than the resistance to degradation by trypsin of the mixture of calcitonin drug that is not conjugated with the oligomer.
  • the resistance to degradation by trypsin of the mixture of calcitonin drug-oligomer conjugates is about 20 percent greater than the resistance to degradation by trypsin of the mixture of calcitomn drug that is not conjugated with the oligomer, and, most preferably, the resistance to degradation by trypsin of the mixture of calcitonin drug-oligomer conjugates is about 30 percent greater than the resistance to degradation by trypsin of the mixture of calcitomn drug that is not conjugated with the oligomer.
  • a substantially monodispersed mixture of calcitonin- oligomer conjugates preferably has a higher bioefficacy than the bioefficacy of the calcitonin drug which is not coupled to the oligomer.
  • the bioefficacy of a particular compound corresponds to its area-under-the-curve (AUC) value.
  • AUC area-under-the-curve
  • the bioefficacy of the mixture is about 5 percent greater than the bioefficacy of the calcitonin drug which is not coupled to the oligomer. More preferably, the bioefficacy of the mixture is about 10 percent greater than the bioefficacy of the calcitonin drug which is not coupled to the oligomer.
  • a substantially monodispersed mixture of calcitonin-oligomer conjugates preferably has an in vivo activity that is greater than the in vivo activity of a polydispersed mixture of calcitonin drug-oligomer conjugates having the same number average molecular weight as the substantially monodispersed mixture.
  • the number average molecular weight of a mixture may be measured by various methods including, but not limited to, size exclusion chromatography such as gel permeation chromatography as described, for example, in H.R. Allcock & F.W. Lampe, CONTEMPORARY POLYMER CHEMISTRY 394-402 (2d. ed., 1991).
  • a substantially monodispersed mixture of calcitonin-oligomer conjugates preferably has an in vitro activity that is greater than the in vitro activity of a polydispersed mixture of calcitonin drug-oligomer conjugates having the same number average molecular weight as the substantially monodispersed mixture.
  • the number average molecular weight of a mixture may be measured by various methods including, but not limited to, size exclusion chromatography.
  • a substantially monodispersed mixture of calcitonin- oligomer conjugates preferably has an increased resistance to degradation by chymotrypsin and/or trypsin when compared to the resistance to degradation by chymotrypsin and/or trypsin of a polydispersed mixture of calcitonin drug-oligomer conjugates having the same number average molecular weight as the substantially monodispersed mixture.
  • the number average molecular weight of a mixture may be measured by various methods including, but not limited to, size exclusion chromatography.
  • a substantially monodispersed mixture of calcitonin-oligomer conjugates preferably has an inter-subject variability that is less than the inter-subject variability of a polydispersed mixture of calcitonin drug-oligomer conjugates having the same number average molecular weight as the substantially monodispersed mixture.
  • the number average molecular weight of a mixture may be measured by various methods including, but not limited to, size exclusion chromatography.
  • the inter-subject variability may be measured by various methods, as will be understood by those skilled in the art.
  • the inter-subject variability is preferably calculated as follows.
  • the area under a dose response curve (i.e., the area between the dose- response curve and a baseline value) is determined for each subject.
  • the average AUC for all subjects is determined by summing the AUCs of each subject and dividing the sum by the number of subjects.
  • the absolute value of the difference between the subject's AUC and the average AUC is then determined for each subject.
  • the absolute values of the differences obtained are then summed to give a value that represents the inter-subject variability. Lower values represent lower inter-subject variabilities and higher values represent higher inter- subject variabilities.
  • Substantially monodispersed mixtures of calcitonin drug-oligomer conjugates according to embodiments of the present invention preferably have two or more of the above- described improved properties. More preferably, substantially monodispersed mixtures of calcitonin drug-oligomer conjugates according to embodiments of the present invention have three or more of the above-described improved properties. Most preferably, substantially monodispersed mixtures of calcitonin drug-oligomer conjugates according to embodiments of the present invention have four or more of the above-described improved properties.
  • Each conjugate in the mixture includes a calcitonin drug coupled to an oligomer that comprises a polyethylene glycol moiety.
  • the standard deviation is preferably less than about 14 Daltons and is more preferably less than about 11 Daltons.
  • the molecular weight distribution may be determined by methods known to those skilled in the art including, but not limited to, size exclusion chromatography such as gel permeation chromatography as described, for example, in H.R. Allcock & F.W. Lampe, CONTEMPORARY POLYMER CHEMISTRY 394-402 (2d. ed., 1991). The standard deviation of the molecular weight distribution may then be determined by statistical methods as will be understood by those skilled in the art.
  • the calcitonin drug is preferably calcitonin. More preferably, the calcitonin drug is salmon calcitonin. However, it is to be understood that the calcitonin drug may be selected from various calcitonin drugs known to those skilled in the art including, for example, calcitonin precursor peptides, calcitonin, calcitonin analogs, calcitonin fragments, and calcitonin fragment analogs. Calcitonin precursor peptides include, but are not limited to, katacalcin (PDN-21) (C-procalcitonin), and N-proCT (ammo-terminal procalcitonin cleavage peptide), human.
  • Calcitonin analogs may be provided by substitution of one or more amino acids in calcitonin as described above.
  • Calcitonin fragments include, but are not limited to, calcitomn 1-7, human; and calcitonin 8-32, salmon.
  • Calcitonin fragment analogs may be provided by substitution of one or more of the amino acids in a calcitonin fragment as described above.
  • the oligomer may be various oligomers comprising a polyethylene glycol moiety as will be understood by those skilled in the art.
  • the polyethylene glycol moiety of the oligomer has at least 2, 3 or 4 polyethylene glycol subunits. More preferably, the polyethylene glycol moiety has at least 5 or 6 polyethylene glycol subunits and, most preferably, the polyethylene glycol moiety has at least 7 polyethylene glycol subunits.
  • the oligomer may comprise one or more other moieties as will be understood by those skilled in the art including, but not limited to, additional hydrophilic. moieties, lipophilic moieties, spacer moieties, linker moieties, and terminating moieties.
  • the various moieties in the oligomer are covalently coupled to one another by either hydrolyzable or non- hydrolyzable bonds.
  • the oligomer may further comprise one or more additional hydrophilic moieties (i.e., moieties in addition to the polyethylene glycol moiety) including, but not limited to, sugars, polyalkylene oxides, and polyamine/PEG copolymers.
  • additional hydrophilic moieties i.e., moieties in addition to the polyethylene glycol moiety
  • polyethylene glycol is a ⁇ polyalkylene oxide
  • the additional hydrophilic moiety may be a polyethylene glycol moiety. Adjacent polyethylene glycol moieties will be considered to be the same moiety if they are coupled by an ether bond.
  • the moiety i.e., moieties in addition to the polyethylene glycol moiety
  • oligomers according to embodiments of the present invention comprise a polyethylene glycol moiety and no additional hydrophilic moieties.
  • the oligomer may further comprise one or more lipophilic moieties as will be understood by those skilled in the art.
  • the lipophilic moiety is preferably a saturated or unsaturated, linear or branched alkyl moiety or a saturated or unsaturated, linear or branched fatty acid moiety.
  • the lipophilic moiety is an alkyl moiety, it is preferably a linear, saturated or unsaturated alkyl moiety having 1 to 28 carbon atoms. More preferably, the alkyl moiety has 2 to 12 carbon atoms.
  • the lipophihc moiety is a fatty acid moiety, it is preferably a natural fatty acid moiety that is linear, saturated or unsaturated, having 2 to 18 carbon atoms. More preferably, the fatty acid moiety has 3 to 14 carbon atoms. Most preferably, the fatty acid moiety has at least 4, 5 or 6 carbon atoms.
  • the oligomer may further comprise one or more spacer moieties as will be understood by those skilled in the art.
  • Spacer moieties may, for example, be used to separate a hydrophilic moiety from a lipophilic moiety, to separate a lipophihc moiety or hydrophilic moiety from the calcitomn drug, to separate a first hydrophilic or hpophilic moiety from a second hydrophilic or lipophilic moiety, or to separate a hydrophihc moiety or lipophilic moiety from a linker moiety.
  • Spacer moieties are preferably selected from the group consisting of sugar, cholesterol and glycerine moieties.
  • the oligomer may further comprise one or more linker moieties that are used to couple the oligomer with the calcitonin drug as will be understood by those skilled in the art.
  • Linker moieties are preferably selected from the group consisting of alkyl and fatty acid moieties.
  • the oligomer may further comprise one or more terminating moieties at the one or more ends of the oligomer which are not coupled to the calcitonin drug.
  • the terminating moiety is preferably an alkyl or alkoxy moiety, and is more preferably a lower alkyl or lower alkoxy moiety. Most preferably, the terminating moiety is methyl or methoxy. While the terminating moiety is preferably an alkyl or alkoxy moiety, it is to be understood that the terminating moiety may be various moieties as will be understood by those skilled in the art including, but not limited to, sugars, cholesterol, alcohols, and fatty acids.
  • the oligomer is preferably covalently coupled to the calcitonin drug.
  • the calcitonin drug is coupled to the oligomer utilizing a hydrolyzable bond (e.g., an ester or carbonate bond).
  • a hydrolyzable coupling may provide a calcitonin drug- oligomer conjugate that acts as a prodrug.
  • a hydrolyzable • coupling may provide for a time-release or controlled-release effect, administering the .
  • the calcitonin drug is coupled to the oligomer utilizing a non-hydrolyzable bond (e.g., a carbamate, amide, or ether bond).
  • a non-hydrolyzable bond e.g., a carbamate, amide, or ether bond.
  • Use of a non-hydrolyzable bond may be preferable when it is desirable to allow the calcitonin drug-oligomer conjugate to circulate in the bloodstream for an extended period of time, preferably at least 2 hours.
  • the oligomer When the oligomer is covalently coupled to the calcitonin drug, the oligomer further comprises .one or more bonding moieties that are used to covalently couple the oligomer with the calcitonin drug as will be understood by those skilled in the art.
  • Bonding moieties are preferably selected from the group consisting of covalent bond(s), ester moieties, carbonate moieties, carbamate moieties, amide moieties and secondary amine moieties. More than one moiety on the oligomer may be covalently coupled to the calcitonin drug.
  • oligomer is preferably covalently coupled to the calcitonin drug
  • the oligomer may be non-covalently coupled to the calcitonin drug to form a non-covalently conjugated calcitonin drug-oligomer complex.
  • non-covalent couplings include, but are not limited to, hydrogen bonding, ionic bonding, Van der Waals bonding, and micellular or liposomal encapsulation.
  • oligomers may be suitably constructed, modified and/or appropriately functionalized to impart the ability for non-covalent conjugation in a selected manner (e.g., to impart hydrogen bonding capability), as will be understood by those skilled in the art.
  • oligomers may be derivatized with various compounds including, but not limited to, amino acids, oligopeptides, peptides, bile acids, bile acid derivatives, fatty acids, fatty acid derivatives, salicylic acids, salicylic acid derivatives, aminosalicylic acids, and aminosalicylic acid derivatives.
  • the resulting oligomers can non-covalently couple (complex) with drug molecules, pharmaceutical products, and/or pharmaceutical excipients.
  • the resulting complexes preferably have balanced lipophilic and hydrophilic properties.
  • oligomers may be derivatized with amine and/or alkyl amines. Under suitable acidic conditions, the resulting oligomers can form non- covalently conjugated complexes with drug molecules, pharmaceutical products and/or pharmaceutical excipients.
  • the products resulting from such complexation preferably have balanced lipophilic and hydrophilic properties.
  • More than one oligomer may be coupled to the calcitonin drug.
  • the oligomers in the plurality are preferably the same. However, it is to be understood that the oligomers in the plurality may be different from one another, or, alternatively, some of the oligomers in the plurality may be the same and some may be different.
  • all of the bonds coupling the plurality of oligomers to the calcitonin drug may be hydrolyzable, but have varying degrees of hydrolyzability such that, for example, one or more of the oligomers is rapidly removed from the calcitonin drug by hydrolysis in the body and one or more of the oligomers is slowly removed from the calcitonin drug by hydrolysis in the body.
  • the oligomer may be coupled to the calcitonin drug at various nucleophilic residues of the calcitonin drug including, but not limited to, nucleophilic hydroxyl functions and/or amino functions.
  • nucleophilic hydroxyl function may be found, for example, at serine and/or tyrosine residues
  • a nucleophilic amino function may be found, for example, at histidine and/or lysine residues, and/or at the one or more N-termini of the polypeptide.
  • the coupling preferably forms a secondary amine.
  • the oligomer may be coupled to an amino functionality of the salmon calcitonin, including the amino functionality of Lys 1 ! , Lys and/or the N-terminus. While one or more oligomers may be coupled to the salmon calcitonin, a higher bioefficacy, such as improved serum calcium lowering ability, is observed for the di-conjugated salmon calcitonin where an oligomer is coupled to the amino functionalities of Lys 11 and the Lys 18 . Mixtures of calcitonin drug-oligomer conjugates having a molecular weight distribution with a standard deviation of less than about 22 Daltons may be synthesized by various methods.
  • a mixture of oligomers having a molecular weight distribution with a standard deviation of less than about 22 Daltons consisting of carboxylic acid and polyethylene glycol is synthesized by contacting a mixture of carboxylic acid having a molecular weight distribution with a standard deviation of less than about 22 Daltons with a mixture of polyethylene glycol having a molecular weight distribution with a standard deviation of less than about 22 Daltons under conditions sufficient to provide a mixture of oligomers having a molecular weight distribution with a standard deviation of less than about 22 Daltons.
  • the oligomers of the mixture having a molecular weight distribution with a standard deviation of less than about 22 Daltons are then activated so that they are capable of - reacting with a calcitonin drug to provide a calcitonin drug-oligomer conjugate.
  • a synthesis route for providing a mixture of activated oligomers having a molecular weight distribution with a standard deviation of less than about 22 Daltons is illustrated in Figure 3 and described in Examples 11-18 hereinbelow.
  • Another embodiment . of a synthesis route for providing a mixture of activated. oligomers having a molecular weight distribution with a standard deviation of less than about 22 Daltons is illustrated in Figure 4 and described in Examples 19-24 hereinbelow.
  • Still another embodiment of a synthesis route for providing a mixture of activated oligomers having a molecular weight distribution with a standard deviation of less than about 22 Daltons is illustrated in Figure 5 and described in Examples 25-29 hereinbelow.
  • Yet another embodiment of a synthesis route for providing a mixture of activated oligomers having a molecular weight distribution with a standard deviation of less than about 22 Daltons is illustrated in Figure 6 and described in Examples 30-31 hereinbelow.
  • Another embodiment of a synthesis route for providing a mixture of activated oligomers having a molecular weight distribution with a standard deviation of less than about 22 Daltons is illustrated in Figure 7 and described in Examples 32-37 hereinbelow.
  • Still another embodiment of a synthesis route for providing a mixture of activated oligomers having a molecular weight distribution with a standard deviation of less than about 22 Daltons is illustrated in Figure 8 and described in Example 38 hereinbelow.
  • Yet another embodiment of a synthesis route for providing a mixture of activated oligomers having a molecular weight distribution with a standard deviation of less than about 22 Daltons is illustrated in Figure 9 and described in Example 39 hereinbelow.
  • Another embodiment of a synthesis route for providing a mixture of activated oligomers having a molecular weight distribution with a standard deviation of less than about 22 Daltons is illustrated in Figure 10 and described in Example 40 hereinbelow.
  • the mixture of activated oligomers having a molecular weight distribution with a standard deviation of less than about 22 Daltons is reacted with a mixture of calcitonin drugs having a molecular weight distribution with a standard deviation of less than about 22 Daltons under conditions sufficient to provide a mixture of calcitonin drug-oligomer conjugates.
  • a preferred synthesis is described in Example 41 hereinbelow.
  • the reaction conditions e.g., selected molar ratios, solvent mixtures and/or pH
  • the mixture of calcitonin drug- oligomer .conjugates resulting from the reaction of the mixture of activated oligomers having a molecular weight distribution with a standard deviation of less than about 22 Daltons and the mixture of calcitonin drugs having a molecular weight distribution with a standard deviation of less than about 22 Daltons is a mixture having a molecular weight distribution with a standard deviation of less than about 22 Daltons.
  • conjugation at the amino functionality of lysine may be suppressed by maintaining the pH of the reaction solution below the pK a of lysine.
  • the mixture of calcitonin drug-oligomer conjugates may be separated and isolated utilizing, for example, HPLC to provide a mixture of calcitonin drug-oligomer conjugates, for example mono-, di-, or tri-conjugates, having a molecular weight distribution with a standard deviation of less than about 22 Daltons.
  • the degree of conjugation e.g., whether the isolated molecule is a mono-, di-, or tri-conjugate
  • a particular isolated conjugate may be determined and/or verified utilizing various techniques as will be understood by those skilled in the art including, but not limited to, mass spectroscopy.
  • the particular conjugate structure (e.g., whether the oligomer is at Lys 11 , Lys 18 or the N-terminus of a salmon calcitonin monoconjugate) may be determined and/or verified utilizing various techniques as will be understood by those skilled in the art including, but not limited to, sequence analysis, peptide mapping, selective enzymatic cleavage, and/or endopeptidase cleavage.
  • one or more of the reaction sites on the calcitonin drug may be blocked by, for example, reacting the calcitonin drug with a suitable blocking reagent such as N-tert-butoxycarbonyl (t-BOC), or N-(9- fluorenylmethoxycarbonyl) (N-FMOC).
  • a suitable blocking reagent such as N-tert-butoxycarbonyl (t-BOC), or N-(9- fluorenylmethoxycarbonyl) (N-FMOC).
  • t-BOC N-tert-butoxycarbonyl
  • N-FMOC N-(9- fluorenylmethoxycarbonyl)
  • the mixture of blocked calcitonin drugs having a molecular weight distribution with a standard deviation of less than about 22 Daltons may be reacted with the mixture of activated oligomers having a molecular weight distribution with a standard deviation of less than about 22 Daltons to provide a mixture of calcitonin drug-oligomer conjugates having oligomer(s) coupled to one or more nucleophilic residues and having blocking moieties coupled to other nucleophilic residues.
  • the calcitonin drug-oligomer conjugates may be de-blocked as will be understood by those skilled in the art.
  • the mixture of calcitonin drug-oligomer conjugates may then be separated as described above to provide a mixture of calcitonin drug- oligomer conjugates having a molecular weight distribution with a standard deviation of less than about 22 Daltons.
  • the mixture of calcitonin drug-oligomer conjugates may be separated prior to de-blocking.
  • Mixtures of calcitonin drug-oligomer conjugates having a molecular weight distribution with a standard deviation of less than about 22 Daltons preferably have improved properties when compared with those of conventional mixtures.
  • a mixture of calcitonin drug-oligomer conjugates having a molecular weight distribution with a standard deviation of less than about 22 Daltons preferably is capable of lowering serum calcium levels by at least 5 percent.
  • the mixture of conjugates is capable of lowering serum calcium levels by at least 10, 11, 12, 13 or 14 percent. More preferably, the mixture of conjugates is capable of lowering serum calcium levels by at least 15, 16, 17, 18 or 19 percent, and, most preferably, the mixture of conjugates is capable of lowering serum calcium levels by at least 20 percent.
  • a mixture of calcitomn drug-oligomer conjugates having a molecular weight distribution with a standard deviation of less than about 22 Daltons preferably has an increased resistance to degradation by chymotrypsin and/or trypsin when compared to the resistance to degradation by chymotrypsin and/or trypsin, respectively, of the calcitonin drug which is not coupled to the oligomer.
  • Resistance to chymotrypsin or trypsin corresponds to the percent remaining when the molecule to be tested is digested in the applicable enzyme using a procedure similar to the one outlined in Example 51 below.
  • the resistance to degradation by chymotrypsin of the mixture of calcitonin drug- oligomer conjugates is about 10 percent greater than the resistance to degradation by chymotrypsin of the mixture of calcitonin drugs that is not conjugated with the oligomer.
  • the resistance to degradation by chymotrypsin of the mixture of calcitonin drug-oligomer conjugates is about 15 percent greater than the resistance to degradation by chymotrypsin of the mixture of calcitonin drug that is not conjugated with the oligomer, and, most preferably, the resistance to degradation by chymotrypsin of the mixture of calcitonin drug-oligomer conjugates is about 20 percent greater than the resistance to degradation by chymotrypsin of the mixture of calcitonin drug that is not conjugated with the oligomer.
  • the resistance to degradation by trypsin of the mixture of calcitonin drug-oligomer conjugates is about 10 percent greater than the resistance to degradation by trypsin of the mixture of calcitonin drug that is not conjugated with the oligomer. More preferably, the resistance to degradation by trypsin of the mixture of calcitonin drug-oligomer conjugates is about 20 percent greater than the resistance to degradation by trypsin of the mixture of calcitonin drug that is not conjugated with the oligomer, and, most preferably, the resistance to degradation by trypsin of the mixture of calcitonin drug-oligomer conjugates is about 30 percent greater than the resistance to degradation by trypsin of the mixture of. calcitonin drug that is not conjugated with the oligomer.
  • a mixture of calcitonin drug-oligomer conjugates having a molecular weight distribution with a standard deviation of less than about 22 Daltons preferably has a higher bioefficacy than the bioefficacy of the calcitonin drug which is not coupled to the ohgomer.
  • the bioefficacy of a particular compound corresponds to its area- under-the-curve (AUC) value.
  • AUC area- under-the-curve
  • the bioefficacy of the mixture is about 5 percent greater than the bioefficacy of the calcitonin drug which is not coupled to the oligomer. More preferably, the bioefficacy of the mixture is about 10 percent greater than the bioefficacy of the calcitonin drug which is not coupled to the oligomer.
  • a mixture of calcitonin drug-oligomer conjugates having a molecular weight distribution with a standard deviation of less than about 22 Daltons preferably has an in vivo activity that is greater than the in vivo activity of a polydispersed mixture of calcitonin drug-oligomer conjugates having the same number average molecular weight as the mixture of calcitonin drug-oligomer conjugates having a molecular weight distribution with a standard deviation of less than about 22 Daltons.
  • the number average molecular weight of a mixture may be measured by various methods including, but not limited to, size exclusion chromatography such as gel permeation chromatography as described, for example, in H.R. Allcock & F.W. Lampe, CONTEMPORARY POLYMER CHEMISTRY 394-402 (2d. ed., 1991).
  • a mixture of calcitomn drug-oligomer conjugates having a molecular weight distribution with a standard deviation of less than about 22 Daltons preferably has an in vitro activity that is greater than the in vitro activity of a polydispersed mixture of calcitonin drug-oligomer conjugates having the same number average molecular weight as the mixture of calcitonin drug-oligomer conjugates having a molecular weight distribution with a standard deviation of less than about 22 Daltons.
  • the number average molecular weight of a mixture may be measured by various methods including, but not limited to, size exclusion chromatography.
  • a mixture of calcitonin drug-oligomer conjugates having a molecular weight distribution with a standard deviation of less than about 22 Daltons preferably has an increased resistance to degradation by chymotrypsin and/or trypsin when compared to the resistance to degradation by chymotrypsin and/or trypsin of a polydispersed mixture of calcitonin drug-oligomer conjugates having the same number average molecular weight as the mixture of calcitonin drug-oligomer conjugates having a molecular weight distribution with a standard deviation of less than about 22 Daltons.
  • the number average molecular weight of a mixture may be measured by various methods including, but hot limited to, size exclusion chromatography.
  • a mixture of calcitonin drug-oligomer conjugates having a molecular weight distribution with a standard deviation of less than about 22 Daltons preferably has an inter-subject variability that is less than the inter-subject variability of a polydispersed mixture of calcitonin drug-oligomer conjugates having the same number average molecular weight as the mixture of calcitonin drug-oligomer conjugates having a molecular weight distribution with a standard deviation of less than about 22 Daltons.
  • the number average ' molecular weight of a mixture may be measured by various methods including, but not limited to, size exclusion chromatography.
  • the inter-subject variability may be measured by various methods, as will be understood by those skilled in the art.
  • the inter-subject variability is preferably calculated as follows.
  • the area under a dose response curve (AUC) i.e., the area between the dose- response curve and a baseline value
  • the average AUC for all subjects is determined by summing the AUCs of each subject and dividing the sum by the number of subjects.
  • the absolute value of the difference between the subject's AUC and the average AUC is then determined for each subject.
  • the absolute values of the differences obtained are then summed to give a value that represents the inter-subject variability.
  • Lower values represent lower inter-subject variabilities and higher values represent higher inter- subject variabilities.
  • Mixtures of calcitonin drug-oligomer conjugates having a molecular weight distribution with a standard deviation of less than about 22 Daltons according to embodiments of the present invention preferably have two or more of the above-described improved properties. More preferably, mixtures of calcitonin drug-oligomer conjugates .having a molecular weight distribution with a standard deviation of less than about 22 Daltons according to embodiments of the present invention have three or more of the above- described improved properties. Most preferably, mixtures of calcitonin drug-oligomer conjugates having a molecular weight distribution with a standard deviation of less than about 22 Daltons according to embodiments of the present invention have four or more of the above-described improved properties.
  • a mixture of conjugates ⁇ where each conjugate includes a calcitonin drug coupled to an oligomer comprising a polyethylene glycol moiety, and the mixture has a dispersity coefficient (DC) greater than 10,000 wherein: n is the number of different molecules in the sample; Nj is the number of i- molecules in the sample; and Mj is the mass of the i ⁇ molecule.
  • DC dispersity coefficient
  • the mixture of conjugates preferably has a dispersity coefficient greater than 100,000. More preferably, the dispersity coefficient of the conjugate mixture is greater than 500,000 and, most preferably, the dispersity coefficient is greater than 10,000,000.
  • the variables n, Ni, and Mi may be determined by various methods as will be understood by those skilled in the art, including, but not limited to, methods described below in Example 49.
  • the calcitonin drug is preferably calcitonin. More preferably, the calcitonin drug is salmon calcitonin. However, it is to be understood that the calcitonin drug may be selected from various calcitonin drugs known to those skilled in the art including, for example, calcitonin precursor peptides, calcitonin, calcitonin analogs, calcitonin fragments, and calcitonin fragment analogs. Calcitonin precursor peptides include, but are not limited to, katacalcin (PDN-21) (C-procalcitonin), andN-proCT (ammo-terminal procalcitonin cleavage peptide), human.
  • Calcitonin analogs may be provided by substitution of one or more amino acids in calcitonin as described above.
  • Calcitonin fragments include, but are not limited to, calcitonin 1-7, human; and calcitonin 8-32, salmon.
  • Calcitonin fragment analogs maybe provided by substitution of one or more of the amino acids in a calcitonin fragment as described above.
  • the oligomer may be various oligomers comprising a polyethylene glycol moiety as will be understood by those skilled in the art.
  • the polyethylene glycol moiety of the oligomer has at least 2, 3 or 4 polyethylene glycol subunits. More preferably, the polyethylene glycol moiety has at least 5 or 6 polyethylene glycol subunits and, most preferably, the polyethylene glycol moiety has at least 7 polyethylene glycol subunits.
  • the oligomer may comprise one or more other moieties as will be understood by those skilled in the art including, but not limited to, additional hydrophihc moieties, lipophilic moieties, spacer moieties, linker moieties, and terminating moieties.
  • additional hydrophihc moieties include hydrophihc moieties, lipophilic moieties, spacer moieties, linker moieties, and terminating moieties.
  • the various moieties in the ohgomer are covalently coupled to one another by either hydrolyzable or non- hydrolyzable bonds.
  • the oligomer may further comprise one or more additional hydrophihc moieties (i.e., moieties in addition to the polyethylene glycol moiety) including, but not limited to, sugars, polyalkylene oxides, and polyamine/PEG copolymers.
  • additional hydrophihc moieties i.e., moieties in addition to the polyethylene glycol moiety
  • sugars i.e., polyalkylene oxides, and polyamine/PEG copolymers.
  • polyethylene glycol is a polyalkylene oxide
  • the additional hydrophilic rnoiety may be a polyethylene glycol moiety. Adjacent polyethylene glycol moieties will be considered to be the same moiety if they are coupled by an ether bond.
  • the moiety i.e., moieties in addition to the polyethylene glycol moiety
  • oligomers according to embodiments of the "present invention comprise a polyethylene glycol moiety and no additional hydrophilic moieties.
  • the oligomer may further' comprise one or more lipophilic moieties as will be understood by those skilled in the art.
  • the lipophihc moiety is preferably a saturated or unsaturated, linear or branched alkyl moiety or a saturated or unsaturated, linear or branched fatty acid moiety.
  • the lipophilic moiety is an alkyl moiety, it is preferably a linear, saturated or unsaturated alkyl moiety having 1 to 28 carbon atoms. More preferably, the alkyl moiety has 2 to 12 carbon atoms.
  • the lipophilic moiety is a fatty acid moiety, it is preferably a natural fatty acid moiety that is linear, saturated or unsaturated, having 2 to 18 carbon atoms. More preferably, the fatty acid moiety has 3 to 14 carbon atoms. Most preferably, the fatty acid moiety has at least 4, 5 or 6 carbon atoms.
  • the ohgomer may further comprise one or more spacer moieties as will be understood by those skilled in the art.
  • Spacer moieties may, for example, be used to separate a hydrophilic moiety from a lipophilic moiety, to separate a lipophilic moiety or hydrophilic moiety from the calcitonin drug, to separate a first hydrophilic or lipophilic moiety from a second hydrophilic or lipophihc moiety, or to separate a hydrophilic moiety or lipophilic moiety from a linker moiety.
  • Spacer moieties are preferably selected from the group consisting of sugar, cholesterol and glycerine moieties.
  • the oligomer may further comprise one or more linker moieties that are used to couple the oligomer with the calcitonin drug as will be understood by those skilled in the art.
  • Linker moieties are preferably selected from the group consisting of alkyl and fatty acid moieties.
  • the oligomer may further comprise one or more terminating moieties at the one or more ends of the oligomer which are not coupled to the calcitonin drug.
  • the terminating moiety is preferably an alkyl or alkoxy moiety, and is more preferably a lower alkyl or lower alkoxy moiety. Most preferably, the terminating moiety is methyl or methoxy. While the terminating moiety is preferably an alkyl or alkoxy moiety, it is to be understood that the terminating moiety may be various moieties as will be understood by those skilled in the art • including, but not limited to, sugars, cholesterol, alcohols, and fatty acids.
  • the oligomer is preferably covalently coupled to the calcitonin drug.
  • the calcitonin drug is coupled to the oligomer utilizing a hydrolyzable bond (e.g., an ester or carbonate bond).
  • a hydrolyzable coupling may provide a calcitonin drug- oligomer conjugate that acts as a prodrug.
  • a hydrolyzable coupling may provide for a time-release, or controlled-release effect, administering the calcitonin drug over a given time period as one or more oligomers are cleaved from their respective calcitonin drug-oligomer conjugates to provide the active drug.
  • the calcitonin drug is coupled to the oligomer utilizing a non-hydrolyzable bond (e.g., a carbamate, amide, or ether bond).
  • the oligomer When the oligomer is covalently coupled to the calcitonin drug, the oligomer further comprises one or more bonding moieties that are used to covalently couple the oligomer with the calcitonin drug as will be understood by those skilled in the art. Bonding moieties are preferably selected from the group consisting of covalent bond(s), ester moieties, carbonate moieties, carbamate moieties, amide moieties and secondary amine moieties. More than one moiety on the oligomer may be covalently coupled to the calcitonin drug.
  • oligomer is preferably covalently coupled to the calcitonin drug
  • the ohgomer may be non-covalently coupled to the calcitonin drug to form a non-covalently conjugated calcitonin drug-oligomer complex.
  • non-covalent couplings include, but are not limited to, hydrogen bonding, ionic bonding, Van der Waals bonding, and micellular or liposomal encapsulation.
  • oligomers may be suitably constructed, modified and/or appropriately functionalized to impart the ability for non-covalent conjugation in a selected manner (e.g., to impart hydrogen bonding capability), as will be understood by those skilled in the art.
  • oligomers may be derivatized with various compounds including, but not limited to, amino acids, oligopeptides, peptides, bile acids, bile acid derivatives, fatty acids, fatty acid derivatives, salicylic acids, salicylic acid derivatives, aminosalicylic acids, and aminosalicylic acid derivatives.
  • the resulting oligomers can non-covalently couple (complex) with drug molecules, pharmaceutical products, and or pharmaceutical excipients.
  • the resulting complexes preferably have balanced lipophilic and hydrophilic properties.
  • oligomers may be derivatized with amine and/or alkyl amines. Under, suitable acidic conditions, the resulting oligomers can form non- covalently conjugated complexes with drug molecules, pharmaceutical products and/or pharmaceutical excipients.
  • the products resulting from such complexation preferably have balanced lipophilic and hydrophilic properties.
  • More than one oligomer i.e., a plurality of oligomers
  • the oligomers in the plurality are preferably the same. However, it is to be understood that the oligomers in the plurality may be different from one another, or, alternatively, some of the oligomers in the plurality may be the same and some may be different.
  • all of the bonds coupling the plurahty of oligomers to the calcitonin drug may be hydrolyzable, but have varying degrees of hydrolyzability such that, for example, one or more of the oligomers is rapidly removed from the calcitonin drug by hydrolysis in the body and one or more of the oligomers is slowly removed from the calcitonin drug by hydrolysis in the body.
  • the oligomer may be coupled to the calcitonin drug at various nucleophilic residues of the calcitonin drug including, but not limited to, nucleophilic hydroxyl functions and/or amino functions.
  • a nucleophihc hydroxyl function may be found, for example, at serine and/or tyrosine residues
  • a nucleophilic amino function may be found, for example, at histidine and/or lysine residues, and/or at the one or more N-termini of the polypeptide.
  • the coupling preferably forms a secondary amine.
  • the oligomer may be coupled to an amino functionality of the salmon calcitonin, including the amino functionality of Lys 11 , Lys 18 and/or the N-terminus. While one or more oligomers may be coupled to the salmon calcitonin, a higher bioefficacy, such as improved serum calcium lowering ability, is observed for the di-conjugated salmon calcitonin where an oligomer is coupled to the amino functionalities of Lys 11 and the Lys 18 .
  • Mixtures of calcitonin drug-oligomer conjugates having a dispersity coefficient greater than 10,000 may be synthesized by various methods. For example, a mixture of oligomers having a dispersity coefficient greater than 10,000 consisting of carboxylic acid and polyethylene glycol is synthesized by contacting a mixture of carboxylic acid having a dispersity coefficient greater than 10,000 with a mixture of polyethylene glycol having a dispersity coefficient greater than 10,000 under conditions sufficient to provide a mixture of oligomers having a dispersity coefficient greater than 10,000. The oligomers of the mixture having a dispersity coefficient greater than 10,000 are then activated so that they are capable of reacting with a calcitonin drug to provide a calcitonin drug-oligomer conjugate.
  • a synthesis route for providing a mixture of activated oligomers having a dispersity coefficient greater than 10,000 is illustrated in Figure 3 and described in Examples 11-18 hereinbelow.
  • Another embodiment of a synthesis route for providing a mixture of activated oligomers having a dispersity coefficient greater than 10,000 is illustrated in Figure 4 and described in Examples 19-24 hereinbelow.
  • Still another embodiment of a synthesis route for providing a mixture of activated oligomers having a dispersity coefficient greater than 10,000 is illustrated in Figure 5 and described in Examples 25-29 hereinbelow.
  • Yet another embodiment of a synthesis route for providing a mixture of activated oHgomers having a dispersity coefficient greater than 10,000 is illustrated in Figure 6 and described in Examples 30-31 hereinbelow.
  • FIG. 7 Another embodiment of a synthesis route for providing a mixture of activated oligomers having a dispersity coefficient greater than 10,000 is illustrated in Figure 7 and described in Examples 32-37 hereinbelow. Still another embodiment of a synthesis route for providing a mixture of activated oligomers having a dispersity coefficient greater than 10,000 is illustrated in Figure 8 and described in Example 38 hereinbelow. Yet another embodiment of a synthesis route for providing a mixture of activated oligomers having a dispersity coefficient greater than 10,000 is illustrated in Figure 9 and described in Example 39 hereinbelow. Another embodiment of a synthesis route for providing a mixture of activated oligomers having a dispersity coefficient greater than 10,000 is illustrated in Figure 10 and described in Example 40 hereinbelow.
  • the mixture of activated oligomers having a dispersity coefficient greater than 10,000 is reacted with a mixture of calcitonin drugs having a dispersity coefficient greater than 10,000 under conditions sufficient to provide a mixture of calcitonin drug-oligomer conjugates.
  • a preferred synthesis is described in Example 41 hereinbelow.
  • reaction conditions e.g., selected molar ratios, solvent mixtures and/or pH
  • the mixture of calcitonin drug- oligomer conjugates resulting from the reaction of the mixture of activated oligomers having a dispersity coefficient greater than 10,000 and the mixture of calcitonin drugs having a dispersity coefficient greater than 10,000 is a mixture having a dispersity coefficient greater than 10,000.
  • conjugation at the amino functionality of lysine maybe suppressed by maintaining the pH of the reaction solution below the pK a of lysine. .
  • the mixture of calcitonin drug-oligomer conjugates maybe separated and isolated utilizing, for example, HPLC to provide a mixture of calcitonin drug-oligomer conjugates, for example mono-, di-, or tri-conjugates, having a dispersity coefficient greater than 10,000.
  • the degree of conjugation e.g., whether the isolated molecule is a mono-, di-, or tri-conjugate
  • a particular isolated conjugate may be determined and/or verified utilizing various techniques as will be understood by those skilled in the art including, but not limited to, mass spectroscopy.
  • the particular conjugate structure (e.g., whether the oligomer is at Lys 11 , Lys 18 or the N-terminus of a salmon calcitonin monoconjugate) may be determined and/or verified utilizing various techniques as will be understood by. those skilled in the art including, but not limited to, sequence analysis, peptide mapping, selective enzymatic cleavage, and/or endopeptidase cleavage.
  • one or more of the reaction sites on the calcitonin drug may be blocked by, for example, reacting the calcitonin drug with a suitable blocking reagent such as N-tert-butoxycarbonyl (t-BOC), or N-(9- fluorenylmethoxycarbonyl) (N-FMOC).
  • a suitable blocking reagent such as N-tert-butoxycarbonyl (t-BOC), or N-(9- fluorenylmethoxycarbonyl) (N-FMOC).
  • t-BOC N-tert-butoxycarbonyl
  • N-FMOC N-(9- fluorenylmethoxycarbonyl)
  • the mixture of blocked calcitomn drugs having a dispersity coefficient greater than 10,000 may be reacted with the mixture of activated oligomers having a dispersity coefficient greater than 10,000 to provide a mixture of calcitonin drug-oligomer conjugates having oligomer(s) coupled to one or more nucleophilic residues and having blocking moieties coupled to other nucleophilic residues.
  • the calcitonin drug-oligomer conjugates may be de-blocked as will be understood by those skilled in the art.
  • the mixture of calcitonin drug- oligomer conjugates may then be separated as described above to provide a mixture of calcitonin drug-oligomer conjugates having a dispersity coefficient greater than 10,000.
  • the mixture of calcitonin drug-oligomer conjugates may be separated prior to de-blocking.
  • Mixtures of calcitonin drug-oligomer conjugates having a dispersity coefficient greater than 10,000 according to embodiments of the present invention preferably have improved properties when compared with those of conventional mixtures.
  • a mixture of calcitonin drug-oligomer conjugates having a dispersity coefficient greater than 10,000 preferably is capable of lowering serum calcium levels by at least 5 percent.
  • the mixture of conjugates is capable of lowering serum calcium levels by at least 10, 11, 12, 13 or 14 percent. More preferably, the mixture of conjugates is capable of lowering serum calcium levels by at least 15, 16, 17, 18 or 19 percent, and, most preferably, the mixture of conjugates is capable of lowering serum calcium levels by at least 20 percent.
  • a mixture of calcitonin drug-oligomer conjugates having a dispersity coefficient greater than 10,000 preferably has an increased resistance to degradation by chymotrypsin and/or trypsin when compared to the resistance to degradation by chymotrypsin and/or trypsin, respectively, of the calcitonin drug which is not coupled to the oligomer.
  • Resistance to chymotrypsin or trypsin corresponds to the percent remaining when the molecule to be tested is digested in the applicable enzyme using a procedure similar to the one outlined in Example 51 below.
  • the resistance to degradation by chymotrypsin of the mixture of calcitonin drug-oligomer conjugates is about 10 percent greater than the resistance to degradation by chymotrypsin of the mixture of calcitonin drugs that is not conjugated with the oligomer.
  • the resistance to degradation by chymotrypsin of the mixture of calcitonin drug-oligomer conjugates is about 15 percent greater than the resistance to degradation by chymotrypsin of the mixture of calcitonin drug that is not conjugated with the oligomer, and, most preferably, the resistance to degradation by chymotrypsin of the mixture of calcitomn drug-oligomer conjugates is about 20 percent greater than the resistance to degradation by chymotrypsin of the mixture of calcitonin drug that is not conjugated with the oligomer.
  • the resistance to degradation by trypsin of the mixture of calcitonin drug-oligomer conjugates is about 10 percent greater than the resistance to degradation by trypsin of the mixture of calcitonin drug that is not conjugated with the oligomer.
  • the resistance to degradation by trypsin of the mixture of calcitonin drug-oligomer conjugates is about 20 percent greater than the resistance to degradation by trypsin of the mixture of calcitonin drug that is not conjugated with the oligomer, and, most preferably, the resistance to degradation by trypsin of the mixture of calcitonin drug-oligomer conjugates is about 30 percent greater than the resistance to degradation by trypsin of the mixture of calcitonin drug that is not conjugated with the oligomer.
  • a mixture of calcito in drug-oligomer conjugates having a dispersity coefficient greater than 10,000 preferably has a higher bioefficacy than the bioefficacy of the calcitonin drug which is not coupled to the oligomer.
  • the bioefficacy of a particular compound corresponds to its area-under-the-curve (AUC) value.
  • AUC area-under-the-curve
  • the bioefficacy of the mixture is about 5 percent greater than the bioefficacy of the calcitonin drug which is not coupled to the oligomer. More preferably, the bioefficacy of the mixture is about 10 percent greater than the bioefficacy of the calcitonin drug which is not coupled to the oligomer.
  • a mixture of calcitomn drug-oligomer conjugates having a dispersity coefficient greater than 10,000 preferably has an in vivo activity that is greater than the in vivo activity of a polydispersed mixture of calcitomn drug-oligomer conjugates having the same number average molecular weight as the mixture of calcitonin drug-oligomer conjugates having a dispersity coefficient greater than 10,000.
  • the number average molecular weight of a mixture may be measured by various methods including, but not limited to, size exclusion chromatography such as gel permeation chromatography as described, for example, in H.R. Allcock & F.W. Lampe, CONTEMPORARY POLYMER CHEMISTRY 394-402 (2d. ed., 1991).
  • a mixture of calcitonin drug-oligomer conjugates having a dispersity coefficient greater than 10,000 preferably has an in vitro activity that is greater than the in vitro activity of a polydispersed mixture of calcitonin drug-oligomer conjugates having the same number average molecular weight as the mixture of calcitonin drug-oligomer conjugates having a dispersity coefficient greater than 10,000.
  • the number average molecular weight of a mixture may be measured by various methods including, but not limited to, size exclusion chromatography.
  • a mixture of calcitonin drug-oligomer conjugates having a dispersity coefficient greater than 10,000 preferably has an increased resistance to degradation by chymotrypsin and or trypsin when compared to the resistance to degradation by chymotrypsin and/or trypsin of a polydispersed mixture of calcitonin drug-oligomer conjugates having the same number average molecular weight as the mixture of calcitonin drug-oligomer conjugates having a dispersity coefficient greater than 10,000.
  • the number average molecular weight of a mixture may be measured by various methods including, but not limited to, size exclusion chromatography.
  • a mixture of calcitonin drug-oligomer conjugates having a dispersity coefficient greater than 10,000 preferably has an inter-subject variability that is less than the inter-subject variability of a polydispersed mixture of calcitonin drug-oligomer conjugates having the same number average molecular weight as the mixture of calcitonin drug-oligomer conjugates having a dispersity coefficient greater than 10,000.
  • the number average molecular weight of a mixture may be measured by various methods including, but not limited to, size exclusion chromatography.
  • the inter-subject variability may be measured by various methods, as will be understood by those skilled in the art.
  • the inter-subject variability is preferably calculated as follows.
  • the area under a dose response curve (i.e., the area between the dose- response curve and a baseline value) is deteimined for each subject.
  • the average AUC for all subjects is determined by summing the AUCs of each subject and dividing the sum by the number of subjects.
  • the absolute value of the difference between the subject's AUC and the average AUC is then determined for each subject.
  • the absolute values of the differences obtained are then summed to give a value that represents the inter-subject variability. Lower values represent lower inter-subject variabilities and higher values represent higher inter- subject variabilities.
  • Mixtures of calcitonin drug-oligomer conjugates having a dispersity coefficient greater than 10,000 according to embodiments of the present invention preferably have two or more of the above-described improved properties. More preferably, mixtures of calcitonin drug-oligomer conjugates having a dispersity coefficient greater than 10,000 according to embodiments of the present invention have three or more of the above-described improved properties. Most preferably, mixtures of calcitonin drug-oligomer conjugates having a dispersity coefficient greater than 10,000 according to embodiments of the present invention have four or more of the above-described improved properties.
  • each conjugate includes a calcitomn drug coupled to ah oligomer and has the same number of polyethylene glycol subunits.
  • the calcitonin drug is preferably calcitonin. More preferably, the calcitonin drug is salmon calcitonin. However, it is to be understood that the calcitonin drug may be selected from various calcitomn drugs known to those skilled in the art including, for example, calcitonin precursor peptides, calcitonin, calcitonin analogs, calcitonin fragments, and calcitonin fragment analogs. Calcitonin precursor peptides include, but are not limited to, katacalcin (PDN-21) (C-procalcitonin), and N-proCT (ammo-terminal procalcitonin cleavage peptide), human.
  • Calcitonin analogs may be provided by substitution of one or more amino acids in calcitonin as described above.
  • Calcitonin fragments include, but are not limited to, calcitonin 1-7, human; and calcitonin 8-32, salmon.
  • Calcitonin fragment analogs may be provided by substitution of one or more of the amino acids in a calcitonin fragment as described above.
  • the oligomer may be various oligomers comprising a polyethylene glycol moiety as will be understood by those skilled in the art.
  • the polyethylene glycol moiety of the oligomer has at least 2, 3 or 4 polyethylene glycol subunits. More preferably, the polyethylene glycol moiety has at least 5 or 6 polyethylene glycol subunits and, most preferably, the polyethylene glycol moiety has at least 7 polyethylene glycol subunits.
  • the oligomer may comprise one or more other moieties as will be understood by those skilled in the art including, but not limited to, additional hydrophilic moieties, lipophilic moieties, spacer moieties, linker moieties, and terminating moieties.
  • the various moieties in the oligomer are covalently coupled to one another by either hydrolyzable or non- hydrolyzable bonds.
  • the oligomer may further comprise one or more additional hydrophilic moieties (i.e., moieties in addition to the polyethylene glycol moiety) including, but not limited to, sugars, polyalkylene oxides, and polyamine/PEG copolymers.
  • additional hydrophilic moieties i.e., moieties in addition to the polyethylene glycol moiety
  • polyethylene glycol is a polyalkylene oxide
  • the additional hydrophilic moiety may be a polyethylene glycol moiety. Adjacent polyethylene glycol moieties will be considered to be the same moiety if they are coupled by an ether bond.
  • the moiety i.e., moieties in addition to the polyethylene glycol moiety
  • O-C 2 H 4 -O-C 2 H 4 -O-C 2 H 4 -O-C 2 H 4 -O-C 2 H 4 -O-C 2 H 4 — is a single polyethylene glycol moiety having six polyethylene glycol subunits. If this moiety were the only hydrophilic moiety in the oligomer, the ohgomer would not contain an additional hydrophilic moiety. Adjacent polyethylene glycol moieties will be considered to be different moieties if they are coupled by a bond other than an ether bond. For example, the moiety
  • oligomers according to embodiments of the present invention comprise a polyethylene glycol moiety and no additional hydrophilic moieties.
  • the oligomer may further comprise one or more lipophilic moieties as will be understood by those skilled in the art.
  • the lipophilic moiety is preferably a saturated or unsaturated, linear or branched alkyl moiety or a saturated or unsaturated, linear or branched fatty acid moiety.
  • Hpophilic moiety is an alkyl moiety, it is preferably a linear, saturated or unsaturated alkyl moiety having 1 to 28 carbon atoms. More preferably, the alkyl moiety has 2 to 12 carbon atoms.
  • the lipophilic moiety is a fatty acid moiety, it is preferably a natural fatty acid moiety that is linear, saturated or unsaturated, having 2 to 18 carbon atoms. More preferably, the fatty acid moiety has 3 to 14 carbon atoms. Most preferably, the fatty acid moiety has at least 4, 5 or 6 carbon atoms.
  • the oligomer may further comprise one or more spacer moieties as will be understood by those skilled in the art.
  • Spacer moieties may, for example, be used to separate a hydrophilic moiety from a lipophilic moiety, to separate a lipophilic moiety or hydrophilic moiety from the calcitonin drug, to separate a first hydrophilic or lipophilic moiety from a second hydrophilic or lipophilic moiety, or to separate a hydrophilic moiety or lipophilic moiety from a linker moiety.
  • Spacer moieties are preferably selected from the group consisting of sugar,, cholesterol and glycerine moieties.
  • the oligomer may further comprise one or more linker moieties that are used to couple the oligomer with the calcitonin drug as will be understood by those skilled in the art.
  • Linker moieties are preferably selected from the group consisting of alkyl and fatty acid moieties.
  • the oligomer may further comprise one or more terminating moieties at the one or more ends of the oligomer which are not coupled to the calcitonin drug.
  • the terminating moiety is preferably an alkyl or alkoxy moiety, and is more preferably a lower alkyl or lower alkoxy moiety. Most preferably, the terminating moiety is methyl or methoxy. While the terminating moiety is preferably an alkyl or alkoxy moiety, it is to be understood that the terminating moiety may be various moieties as will be understood by those skilled in the art including, but not limited to, sugars, cholesterol, alcohols, and fatty acids.
  • the oligomer is preferably covalently coupled to the calcitonin drug.
  • the calcitonin drug is coupled to the oligomer utilizing a hydrolyzable bond (e.g., an ester or carbonate bond).
  • a hydrolyzable coupling may provide a calcitonin drug- oligomer conjugate that acts as a prodrug.
  • a hydrolyzable coupling may provide for a time-release or controlled-release effect, administering the calcitonin drug over a given time period as one or more oligomers are cleaved from their respective calcitonin drug-oligomer conjugates to provide the active drug.
  • the calcitonin drug is coupled to the oligomer utilizing a non-hydrolyzable bond (e.g., a carbamate, amide, or ether bond).
  • the oligomer When the oligomer is covalently coupled to the calcitonin drug, the oligomer further comprises one or more bonding moieties that are. used to covalently couple the oligomer with the calcitonin drug as will be understood by those skilled in the art. Bonding moieties are preferably selected from the group consisting of covalent bond(s), ester moieties, carbonate moieties, carbamate moieties, amide moieties and secondary amine moieties. More than one moiety on the oligomer may be covalently coupled to the calcitonin drug.
  • oligomer is preferably covalently coupled to the calcitonin drug
  • the oligomer may be non-covalently coupled to the calcitonin drug to form a non-covalently conjugated calcitonin drug-oligomer complex.
  • non-covalent couplings include, but are not limited to, hydrogen bonding, ionic bonding, Van der Waals bonding, and micellular or liposomal encapsulation.
  • oligomers may be suitably constructed, modified and/or appropriately functionalized to impart the ability for non-covalent conjugation in a selected manner (e.g., to impart hydrogen bonding capability), as will be understood by those skilled in the art.
  • oligomers may be derivatized with various compounds including, but not limited to, amino acids, oligopeptides, peptides, bile acids, bile acid derivatives, fatty acids, fatty acid derivatives, salicylic acids, salicylic acid derivatives, aminosalicylic acids, and aminosalicylic acid derivatives.
  • the resulting oHgomers can non-covalently couple (complex) with drug molecules, pharmaceutical products, and/or pharmaceutical excipients.
  • the resulting complexes preferably have balanced lipophilic and hydrophilic properties.
  • oligomers may be derivatized with amine and/or alkyl amines. Under suitable acidic conditions, the resulting oHgomers can form non- covalently conjugated complexes with drug molecules, pharmaceutical products -and/or pharmaceutical excipients.
  • the products resulting from such complexation preferably have balanced lipophilic and hydrophilic properties.
  • More than one oligomer may be coupled to the calcitonin drug.
  • the oligomers in the plurality are preferably the same. However, it is to be understood that the oligomers in the plurality may be different from one another, or, alternatively, some of the oligomers in the plurality may be the same and some may be different.
  • all of the bonds coupling the plurality of oHgomers to the calcitonin drug may be hydrolyzable, but have varying degrees of hydrolyzability such that, for example, one or more of the oligomers is rapidly removed from the calcitomn drug by hydrolysis in the body and one or more of the oligomers is slowly removed from the calcitonin drug by hydrolysis in the body.
  • the oligomer may be coupled to the calcitonin drug at various nucleophilic residues . of the calcitonin drug including, but not Hmited to, nucleophilic hydroxyl functions and/or amino functions.
  • a nucleophilic hydroxyl function may be found, for example, at serine and/or tyrosine residues
  • a nucleophilic amino function may be found, for example, at histidine and/or lysine residues, and/or at the one or more N-termini of the polypeptide.
  • the coupling preferably forms a secondary amine.
  • the oligomer may be coupled to an amino functionality of the salmon calcitomn, including the amino functionality of Lys 11 , Lys 18 and/or the N-terminus. While one or more oligomers may be coupled to the salmon calcitonin, a higher bioefficacy, such as improved serum calcium lowering ability, is observed for the di-conjugated salmon calcitonin where an oligomer is coupled to the amino functionalities of Lys 11 and the Lys 18 .
  • Mixtures of calcitonin drug-oligomer conjugates where each conjugate in the mixture has the same number of polyethylene glycol .subunits may be synthesized by various methods. For example, a mixture of oligomers consisting of carboxylic acid and polyethylene glycol where each oligomer in the mixture has the same number of polyethylene glycol subunits is synthesized by contacting a mixture of carboxylic acid with a mixture of polyethylene glycol where each polyethylene glycol molecule in the mixture has the same number of polyethylene glycol subunits under conditions sufficient to provide a mixture of oligomers where each oligomer in the mixture has the same number of polyethylene glycol subunits.
  • the oligomers of the mixture where each oligomer in the mixture has the same number of polyethylene glycol subunits are then activated so that they are capable of reacting with a calcitonin drug to provide a calcitonin drug-oligomer conjugate.
  • a synthesis route for providing a mixture of activated oligomers where each oligomer in the mixture has the same number of polyethylene glycol subunits is illustrated in Figure 3 and described in Examples 11-18 hereinbelow.
  • Another embodiment of a synthesis route for providing a mixture of activated oligomers where each oligomer in the mixture has the same number of polyethylene glycol subunits is illustrated in Figure 4 and described in Examples 19-24 hereinbelow.
  • Still another embodiment of a synthesis route for providing a mixture of activated oligomers where each oligomer in the mixture has the same number of polyethylene glycol subunits is illustrated in Figure 5 and described in Examples 25-29 hereinbelow.
  • Yet another embodiment of a synthesis route for providing a mixture of activated oligomers where each oligomer in the mixture has the same number of polyethylene glycol subunits is illustrated in Figure 6 and described in Examples 30-31 hereinbelow.
  • Another embodiment of a synthesis route for providing a mixture of activated oligomers where each oligomer in the mixture has the same number of polyethylene glycol subunits is illustrated in Figure 7 and described in Examples 32-37 hereinbelow.
  • Still another embodiment of a synthesis route for providing a mixture of activated oligomers where each oligomer in the mixture has the same number of polyethylene glycol subunits is illustrated in Figure 8 and described in Example 38 hereinbelow.
  • Yet another embodiment of a synthesis route for providing a mixture of activated oligomers where each oligomer in the mixture has the same number of polyethylene glycol subunits is illustrated in Figure 9 and described in Example 39 hereinbelow.
  • Another embodiment of a synthesis route for providing a mixture of activated oligomers having a mixture of activated oligomers where each oligomer in the mixture has the same number of polyethylene glycol subunits is illustrated in Figure 10 and described in Example 40 hereinbelow.
  • reaction conditions e.g., selected molar ratios, solvent mixtures and/or pH
  • the mixture of calcitonin drug-oligomer conjugates resulting from the reaction of the mixture of activated oHgomers where each oligomer in the mixture has the same number of polyethylene glycol subunits and the mixture of calcitonin drugs is a mixture of conjugates where each conjugate in the mixture has the same number of polyethylene glycol subunits.
  • conjugation at the amino functionality of lysine may be suppressed by mamtaining the pH of the reaction solution below the pK a of lysine.
  • the mixture of calcitomn drug-oligomer conjugates may be separated and isolated utilizing, for example, HPLC to provide a mixture of calcitonin drug-oligomer conjugates, for example mono-, di-, or tri-conjugates, where each conjugate in the mixture has the same number of polyethylene glycol subunits.
  • the degree of conjugation e.g., whether the isolated molecule is a mono-, di-, or tri-conjugate
  • a particular isolated conjugate may be determined and/or verified utilizing various techniques as will be understood by those skilled in the art including, but not limited to, mass spectroscopy. The .
  • conjugate structure e.g., whether the oligomer is at Lys 11 , Lys 18 or the N-terminus of a salmon calcitonin monoconjugate
  • conjugate structure may be determined and/or verified utilizing various techniques as will be understood by those skilled in the art including, but not limited to, sequence analysis, peptide mapping, selective enzymatic cleavage, and/or endopeptidase cleavage.
  • one or more of the reaction sites on the calcitonin drug may be blocked by, for example, reacting the calcitonin drug with a suitable blocking reagent such as N-tert-butoxycarbonyl (t-BOC), or N-(9- fluorenylmethoxycarbonyl) (N-FMOC).
  • a suitable blocking reagent such as N-tert-butoxycarbonyl (t-BOC), or N-(9- fluorenylmethoxycarbonyl) (N-FMOC).
  • t-BOC N-tert-butoxycarbonyl
  • N-FMOC N-(9- fluorenylmethoxycarbonyl)
  • the mixture of blocked calcitonin drugs may be reacted with the mixture of activated oligomers where each oligomer in the mixture has the same number of polyethylene glycol subunits to provide a mixture of calcitomn drug-ohgomer conjugates having oligomer(s) coupled to one or more nucleophilic residues and having blocking moieties coupled to other nucleophilic residues.
  • the calcitonin drug-oligomer conjugates may be de-blocked as will be understood by those skilled in the art.
  • the mixture of calcitomn drug-oligomer conjugates may then be separated as described above to provide a mixture of calcitonin drug- oligomer conjugates where each conjugate in the mixture has the same number of polyethylene glycol subunits.
  • the mixture of calcitonin drug-oligomer conjugates may be separated prior to de-blocking.
  • Mixtures of calcitonin drug-oligomer conjugates where each conjugate in the mixture has the same number of polyethylene glycol subunits according to embodiments of the present invention preferably have improved properties when compared with those of conventional mixtures.
  • a mixture of calcitonin drug-oligomer conjugates where each conjugate in the mixture has the same number of polyethylene glycol subunits preferably is capable of lowering serum calcium levels by at least 5 percent.
  • the mixture of conjugates is capable of lowering serum calcium levels by at least 10, 11, 12, 13 or 14 percent. More preferably, the mixture of conjugates is capable of lowering serum calcium levels by at least 15, 16, 17, 18 or 19 percent, and, most preferably, the mixture of conjugates is capable of lowering serum calcium levels by at least 20 percent.
  • a mixture of calcitonin drug-oligomer conjugates where each conjugate in the mixture has the same number of polyethylene glycol subunits preferably has an increased resistance to degradation by chymotrypsin and/or trypsin when compared to the resistance to degradation by chymotrypsin and/or trypsin, respectively, of the calcitonin drug which is not coupled to the oligomer.
  • Resistance to chymotrypsin or trypsin corresponds to the percent remaining when the molecule to be tested is digested in the applicable enzyme using a procedure similar to the one outlined in Example 51 below.
  • the resistance to degradation by chymotrypsin of the mixture of calcitonin drug-oligomer conjugates is about 10 percent greater than the resistance to degradation by chymotrypsin of the mixture of calcitonin drugs that is not conjugated with the oligomer.
  • the resistance to degradation by chymotrypsin of the mixture of calcitonin drug-oligomer conjugates is about 15 percent greater than the resistance to degradation by chymotrypsin of the mixture of calcitonin drug that is not conjugated with the oligomer, and, most preferably, the resistance to degradation by chymotrypsin of the mixture of calcitonin drug-oligomer conjugates is about 20 percent greater than the resistance to degradation by chymotrypsin of the mixture of calcitonin drug that is not conjugated with the oligomer.
  • the resistance to degradation by trypsin of the mixture of calcitonin drug-oligomer conjugates is about 10 percent greater than the resistance to degradation by trypsin of the mixture of calcitonin drug that is not conjugated with the oligomer.
  • the resistance to degradation by trypsin of the mixture of calcitonin drug-oligomer conjugates is about 20 percent greater than the resistance to degradation by trypsin of the mixture of calcitonin drug that is not conjugated with the oligomer, and, most preferably, the resistance to degradation by trypsin of the mixture of calcitomn drug-oligomer conjugates is about 30 percent greater than the resistance to degradation by trypsin of the mixture of calcitonin drug that is not conjugated with the oligomer.
  • a mixture of calcitonin drug-oligomer conjugates where each conjugate in the mixture has the same number of polyethylene glycol subunits preferably has a higher bioefficacy than the bioefficacy of the calcitonin drug which is not coupled to the ohgomer.
  • the bioefficacy of a particular compound corresponds to its area- . under-the-curve (AUC) value.
  • AUC under-the-curve
  • the bioefficacy of the mixture is about 5 percent greater than the bioefficacy of the calcitonin drug which is not coupled to the oligomer. More preferably, the bioefficacy of the mixture is about 10 percent greater than the bioefficacy of the calcitonin drug which is not coupled to the oligomer.
  • a mixture of calcitonin drug-oligomer conjugates where. each conjugate in the mixture has the same number of polyethylene glycol subunits preferably has an in vivo activity that is greater than the in vivo activity of a polydispersed mixture of calcitonin drug-oligomer conjugates having the same number average molecular weight as the mixture of calcitonin drug-oligomer conjugates where each conjugate in the mixture has the same number of polyethylene glycol subunits.
  • the number average molecular weight of a mixture may be measured by various methods including, but not limited to, size exclusion chromatography such as gel permeation chromatography as described, for example, in H.R. Allcock & F.W. Lampe, CONTEMPORARY POLYMER CHEMISTRY 394-402 (2d. ed., 1991).
  • a mixture of calcitonin drug-oligomer conjugates where each conjugate in the mixture has the same number of polyethylene glycol subunits preferably has an in vitro activity that is greater than the in vitro activity of a polydispersed mixture of calcitonin drug-oligomer conjugates having the same number average molecular weight as the mixture of calcitonin drug-oligomer conjugates where each conjugate in the mixture has the same number of polyethylene glycol subunits.
  • the number average molecular weight of a mixture may be measured by various methods including, but not limited to, size exclusion chromatography.
  • a mixture of calcitonin drug-oligomer conjugates where each conjugate in the mixture has the same number of polyethylene glycol subunits preferably has an increased resistance to degradation by chymotrypsin and/or trypsin when . compared to the resistance to degradation by chymotrypsin and/or trypsin of a polydispersed mixture of calcitonin drug-oligomer conjugates having the same number average molecular weight as the mixture of calcitonin drug-oligomer conjugates where each conjugate in the mixture has the same number of polyethylene glycol subunits.
  • the number average molecular weight of a mixture may be measured . by various methods including, but not limited to, size exclusion chromatography.
  • a mixture of calcitonin drug-oligomer conjugates where each conjugate in the mixture has the same number of polyethylene glycol subunits preferably has an inter-subject variability that is less than the inter-subject variability of a polydispersed mixture of calcitonin drug-oligomer conjugates having the same number average molecular weight as the mixture of calcitonin drug-oligomer conjugates where each conjugate in the mixture has the same number of polyethylene glycol subunits.
  • the number average molecular weight of a mixture may be measured ' by various methods including, but not limited to, size exclusion chromatography.
  • the inter- subject variability may be measured by various methods, as will be understood by those skilled in the art.
  • the inter-subject variability is preferably calculated as follows.
  • the area under a dose response curve (AUC) i.e., the area between the dose-response curve and a baseline value
  • AUC dose response curve
  • the average AUC for all subjects is determined by summing the AUCs of each subject and dividing the sum by the number of subjects.
  • the absolute value of the difference between the subject's AUC and the average AUC is then determined for each subject.
  • the absolute values of the differences obtained are then summed to give a value that represents the inter-subject variability.
  • Lower values represent lower inter-subject variabilities and higher values represent higher inter-subject variabilities.
  • Mixtures of calcitonin drug-oligomer conjugates where each conjugate in the mixture has the same number of polyethylene glycol subunits according to embodiments of the present invention preferably have two or more of the above-described improved properties. More preferably, mixtures of calcitonin drug-oligomer conjugates where each conjugate in - the mixture has the same number of polyethylene glycol subunits according to embodiments of the present invention have three or more of the above-described improved properties. Most preferably, mixtures of calcitonin- drug-oligomer conjugates where each conjugate in the mixture has the same number of polyethylene glycol subunits according to embodiments of the present invention have four or more of the above-described improved properties. . According to still other embodiments of the present invention, a mixture of conjugates is provided in which each conjugate has the same molecular weight and has the structure of Formula A:
  • B is a bonding moiety
  • L is a linker moiety
  • G, G' and G" are individually selected spacer moieties
  • R is a lipophilic moiety and R' is a polyalkylene glycol moiety, or R' is the lipophilic moiety and R is the polyalkylene glycol moiety; T is a terminating moiety; j, k, m and n are individually 0 or 1; and p is an integer from 1 to the number of nucleophilic residues on the calcitonin drug.
  • the calcitonin drug is preferably calcitonin. More preferably, the calcitonin drug is salmon calcitonin.
  • the calcitonin drag may be selected from various calcitonin drugs known to those skilled in the art including, for example, calcito in precursor peptides, calcitonin, calcitonin analogs, calcitonin fragments, and calcitonin fragment analogs.
  • Calcitonin precursor peptides include, but are not limited to, katacalcin (PDN-21) (C-procalcitonin), andN-proCT (ammo-terminal procalcitonin cleavage peptide), human.
  • Calcitonin analogs may be provided by substitution of one or more amino acids in calcitonin as described above.
  • Calcitonin fragments include, but are not limited to, calcitonin 1-7, human; and calcitonin 8-32, salmon. Calcitonin fragment analogs may be provided by substitution of one or more of the amino acids in a calcitonin fragment as described above.
  • the polyalkylene glycol moiety of the oligomer preferably has at least 2, 3 or 4 polyalkylene glycol subunits. More preferably, the polyalkylene glycol moiety has at least 5 or 6 polyalkylene glycol subunits and, most preferably, the polyethylene glycol moiety has at least 7 polyalkylene glycol subunits.
  • the polyalkylene glycol moiety is preferably a lower polyalkylene glycol moiety such as a polyethylene glycol moiety, a polypropylene glycol moiety, or a polybutylene glycol moiety. More preferably, the polyalkylene glycol moiety is a polyethylene glycol moiety or a polypropylene glycol moiety. Most preferably, the polyalkylene glycol moiety is a polyethylene glycol moiety. When the polyalkylene glycol moiety is a polypropylene glycol moiety, the moiety preferably has a uniform (i.e., not random) structure.
  • An exemplary polypropylene glycol moiety having a uniform structure is as follows:
  • This uniform polypropylene glycol structure may be described as having only one methyl substituted carbon atom adjacent each oxygen atom in the polypropylene glycol chain.
  • Such uniform polypropylene glycol moieties may exhibit both lipophilic and hydrophilic characteristics and thus be useful in providing amphiphilic calcitonin drug-oligomer conjugates without the use of lipophilic polymer moieties.
  • coupling the secondary alcohol moiety of the polypropylene glycol moiety with a calcitonin drug may provide the calcitonin drug (e.g., salmon calcitonin) with improved resistance to degradation caused by enzymes such as trypsin and chymotrypsin found, for example, in the gut.
  • Uniform polypropylene glycol according to embodiments of the present invention is preferably synthesized as illustrated in Figures 11 through 13, which will now be described.
  • 1,2-propanediol 53 is reacted with a primary alcohol blocking reagent to provide a secondary alcohol extension monomer 54.
  • the primary alcohol blocking reagent may be various primary alcohol blocking reagents as will be understood by those skilled in the art including, but not limited to, silylchloride compounds such as t- butyldiphenylsilylchloride and t-butyldimethylsilylchloride, and esterification reagents such as Ac 2 O.
  • the primary alcohol blocking reagent is a primary alcohol blocking reagent that is substantially non-reactive with secondary alcohols, such as t- butyldiphenylsilylchloride or t-butyldimethylsilylchloride.
  • the secondary alcohol extension monomer (54) maybe reacted with methanesulfonyl chloride (MeSO 2 Cl) to provide a primary extension alcohol monomer mesylate 55.
  • the secondary alcohol extension monomer 54 may be reacted with a secondary alcohol blocking reagent to provide compound 56.
  • the secondary alcohol blocking reagent may be various secondary alcohol blocking reagents as will be understood by those skilled in the art including, but not limited to, benzyl chloride.
  • the compound 56 may be reacted with a Bi de-blocking reagent to remove the blocking moiety Bi and provide a primary alcohol extension monomer 57.
  • the Bi de-blocking reagent may be selected from various de-blocking reagents as will be understood by one skilled in the art.
  • the B ⁇ de-blocking reagent is a de- esterif ⁇ cation reagent, such as a base (e.g., potassium carbonate).
  • a base e.g., potassium carbonate
  • the de-blocking reagent is preferably tetrabutylammonium fluoride (TBAF).
  • TBAF tetrabutylammonium fluoride
  • the primary alcohol extension monomer 54 and the secondary alcohol extension monomer 57 may be capped as follows.
  • the secondary alcohol extension monomer 54 may be reacted with a capping reagent to provide a compound 59.
  • the capping reagent may be various capping reagents as will be understood by those skilled in the art including, but not limited to, alkyl halides such as methyl chloride.
  • the compound 59 may be reacted with a B ⁇ de-blocking agent as described above to provide a primary alcohol capping monomer 60.
  • the primary alcohol capping monomer 60 may be reacted with methane sulfonyl chloride to provide the secondary alcohol capping monomer mesylate 61.
  • the primary alcohol extension monomer 57 may be reacted with a capping reagent to provide a compound 62.
  • the capping reagent maybe various capping reagents as described above.
  • the compound 62 may be reacted with a B 2 de-blocking reagent to- remove the blocking moiety B 2 and provide a . secondary alcohol capping monomer 63.
  • the B de-blocking reagent may be various de- blocking agents as will be understood by those skilled in the art including, but not limited to, H 2 in the presence of a palladium/activated carbon catalyst.
  • the secondary alcohol capping monomer may be reacted with methanesulfonyl chloride to provide a.primary alcohol capping monomer mesylate 64. While the embodiments illustrated in Figure 11 show the . synthesis of capping monomers, it is to be understood that similar reactions may be performed to provide capping polymers.
  • chain extensions may be effected by reacting a primary alcohol extension mono- or poly-mer such as the primary alcohol extension monomer 57 with a primary alcohol extension mono- or poly-mer mesylate such as the primary alcohol extension monomer mesylate 55 to provide various uniform polypropylene chains or by reacting a secondary alcohol extension mono- or poly-mer such as the secondary alcohol extension monomer 54 with a secondary alcohol extension mono-or poly-mer mesylate such as the secondary alcohol extension monomermesylate 58.
  • the primary alcohol extension monomer mesylate 55 is reacted with the primary alcohol extension monomer 57 to provide a dimer compound 65.
  • the secondary alcohol extension monomer mesylate 58 may be reacted with the secondary alcohol extension monomer 54 to provide the dimer compound 65.
  • the Bi blocking moiety on the dimer compound 65 may be removed using a B ! de-blocking reagent as described above to provide a primary alcohol extension dimer 66.
  • the primary alcohol extension dimer 66 may be reacted with methane sulfonyl chloride to provide a secondary alcohol extension dimer mesylate 67.
  • the B 2 blocking moiety on the dimer compound 65 may be removed using the B de-blocking reagent as described above to provide a secondary alcohol extension dimer 69.
  • the secondary alcohol extension dimer 69 may be reacted with methane sulfonyl chloride to provide a primary alcohol extension dimer mesylate 70.
  • the chain extension process may be repeated to achieve various other chain lengths.
  • the primary alcohol extension dimer 66 may be reacted with the primary alcohol extension dimer mesylate 70 to provide a tetramer compound 72.
  • a generic chain extension reaction scheme involves reacting the primary alcohol extension mono- or poly-mer 73 with the primary alcohol extension mono- or poly-mer mesylate 74 to provide the uniform polypropylene polymer 75.
  • the values of m and n may each range from 0 to 1000 or more. Preferably, m and n are each from 0 to 50.
  • the primary alcohol extension dimer mesylate 70 is reacted with the primary alcohol capping monomer 60 to provide the capped/blocked primary alcohol extension trimer 71.
  • the Bi blocking moiety may be removed and the resulting capped primary alcohol extension trimer may be reacted with a primary alcohol extension mono- or poly-mer mesylate to extend the chain of the capped trimer 71.
  • An end of a secondary alcohol extension mono-or poly-mer or an end of a secondary alcohol extension mono-or poly-mer mesylate may be reacted with a secondary alcohol capping mono-or poly-mer mesylate or a secondary alcohol capping mono- or poly-mer, respectively, to provide a capped uniform polypropylene chain.
  • the secondary alcohol extension dimer mesylate 67 is reacted with the secondary alcohol capping monomer 63 to provide the capped/blocked primary alcohol extension trimer 68.
  • the B 2 blocking moiety may be removed as described above and the resulting capped secondary alcohol extension trimer may be reacted with a secondary alcohol extension mer mesylate to extend the chain of the capped trimer 68.
  • Uniform polypropylene glycol moieties maybe coupled to a calcitonin drug, a lipophilic moiety such as a carboxylic acid, and/or various other moieties by various methods as will be understood by those skilled in the art including, but not limited to, those described herein with respect to polyethylene glycol moieties.
  • the lipophilic moiety is a lipophilic moiety as will be understood by those skilled in the art.
  • the lipophilic moiety is preferably a saturated or unsaturated, linear or branched alkyl moiety or a saturated or unsaturated, linear or branched fatty acid moiety.
  • the lipophilic moiety is an alkyl moiety, it is preferably a linear, saturated or unsaturated alkyl moiety having 1 to 28 carbon atoms. More preferably, the alkyl moiety has 2 to 12 carbon atoms.
  • the lipophilic moiety is a fatty acid moiety, it is preferably a natural fatty acid moiety that is linear, saturated or unsaturated, having 2 to 18 carbon atoms. More preferably, the fatty acid moiety has 3 to 14 carbon atoms. Most preferably, the fatty acid moiety has at least 4, 5 or 6 carbon atoms.
  • the spacer moieties, G, G' and G are spacer moieties as will be understood by those skilled in the art.
  • Spacer moieties are preferably selected from the group consisting of sugar, cholesterol and glycerine moieties.
  • oligomers of these embodiments do not include spacer moieties (i.e., k, m and n are preferably 0).
  • linker moiety may be used to couple the oligomer with the drug as will be understood by those skilled in the art.
  • Linker moieties are preferably selected from the group consisting of alkyl and fatty acid moieties.
  • the terminating moiety is preferably an alkyl or alkoxy moiety, and is more preferably a lower alkyl or lower alkoxy moiety. Most preferably, the terminating moiety is methyl or methoxy. While the terminating moiety is preferably an alkyl or alkoxy moiety, it is to be understood that the terminating moiety may be various moieties as will be understood by those skilled in the art including, but not limited to, sugars, cholesterol, alcohols, and fatty acids.
  • the oligomer which is represented by the bracketed portion of the structure of Formula A, is covalently coupled to the calcitonin drag.
  • the calcitonin drag is coupled to the oligomer utilizing a hydrolyzable bond (e.g., an ester or carbonate bond).
  • a hydrolyzable coupling may provide a calcitomn drug-oligomer conjugate that acts as a prodrag.
  • a hydrolyzable coupling may provide for a time-release or controlled-release effect, administering the calcitonin drug over a given time period as one or more oligomers are cleaved from their respective calcitonin drug-oligomer conjugates to provide the active drug, hi other embodiments, the calcitonin drug is coupled to the oligomer utilizing a non- hydrolyzable bond (e.g., a carbamate, amide, or ether bond).
  • a non- hydrolyzable bond e.g., a carbamate, amide, or ether bond
  • the bonding moiety, B may be various bonding moieties that may be used to covalently couple the oligomer with the calcitonin drug as will be understood by those skilled in the art. Bonding moieties are preferably selected from the group consisting of covalent bond(s), ester moieties, carbonate moieties, carbamate moieties, amide moieties and secondary amine moieties.
  • the variable p is an integer from 1 to the number of nucleophilic residues on the calcitonin drag.
  • oligomers When p is greater than 1, more than one oligomer (i.e., a plurality of oligomers) is coupled to the drag. According the these embodiments of the present invention,- the oligomers in the plurality are the same.
  • a plurality of oligomers When a plurality of oligomers are coupled to the drug, it may be preferable to couple one or more of the oligomers to the drug with hydrolyzable bonds and couple one or more of the oligomers to the drug with non- hydrolyzable bonds.
  • all of the bonds coupling the plurality of oligomers to the drag may be hydrolyzable, but have varying degrees of hydrolyzability such that, for example, one or more of the oligomers is rapidly removed from the drug by hydrolysis in the body and one or more of the oHgomers is slowly removed from the drug by hydrolysis in the body.
  • p is preferably 1 or 2, and is more preferably 2.
  • the oligomer may be coupled to the calcitonin drug at various nucleophilic residues of the calcitonin drug including, but not limited to, nucleophilic hydroxyl functions and/or amino functions.
  • a nucleophilic hydroxyl function may be found, for example, at serine and/or tyrosine residues, and a nucleophilic amino function maybe found, for example, at histidine and/or lysine residues, and/or at the one or more N-termini of the polypeptide.
  • the coupling preferably forms a secondary amine.
  • the oligomer may be coupled to an amino functionality of the salmon calcitonin, including the amino functionality of Lys 11 , Lys 18 and/or the N-terminus. While one or more oligomers may be coupled to the salmon calcitonin, a higher bioefficacy, such as improved serum calcium lowering ability, is observed for the di-conjugated salmon calcitonin where an oligomer is coupled to the amino functionalities of Lys 11 and the Lys 18 .
  • Mixtures of calcitonin drug-oligomer conjugates where each conjugate in the mixture has the same molecular weight and has the structure of Formula A may be synthesized by various methods.
  • a mixture of oligomers consisting of carboxylic acid and polyethylene glycol is synthesized by contacting a mixture of carboxylic acid with a mixture of polyethylene glycol under conditions sufficient to provide a mixture of oligomers.
  • the oligomers of the mixture are then activated so that they are capable of reacting with a calcitonin drug to provide a calcitonin drug-oligomer conjugate.
  • a synthesis route for providing a mixture of activated oligomers where each oligomer has the same molecular weight and has a structure of the oligomer of Formula A is illustrated in Figure 3 and described in Examples 11-18 hereinbelow.
  • FIG. 4 Another embodiment of a synthesis route for providing a mixture of activated oligomers where each oligomer has the same molecular weight and has a structure of the oligomer of Formula A is illustrated in Figure 4 and described in Examples 19-24 hereinbelow. Still another embodiment of a synthesis route for providing a mixture of activated oligomers where each oligomer has the same molecular weight and has a structure of the oligomer of Formula A is illustrated in Figure 5 and described in Examples 25-29 hereinbelow. Yet another embodiment of a synthesis route for providing a mixture of activated oligomers where each oligomer has the same molecular weight and has a structure of the oligomer of Formula A is illustrated in Figure 6 and described in Examples 30-31 hereinbelow.
  • FIG. 7 Another embodiment of a synthesis route for providing a mixture of activated oligomers where each oligomer has the same molecular weight and has a structure of the oligomer of Formula A is illustrated in Figure 7 and described in Examples 32-37 hereinbelow. Still another embodiment of a synthesis route for providing a mixture of activated oligomers where each oligomer has the same molecular weight and has a structure of the oligomer of Formula A is illustrated in Figure 8 and described in Example 38 hereinbelow. Yet another embodiment of a synthesis route for providing a mixture of activated oligomers where each oligomer has the same molecular weight and has a structure of the oligomer of Formula A is illustrated in Figure 9 and described in Example 39 hereinbelow. Another embodiment of a synthesis route for providing a mixture of activated oligomers where each ohgomer has the same molecular weight and has a structure of the oligomer of Formula A is illustrated in Figure 10 and described in Example 40 hereinbe
  • reaction conditions e.g., selected molar ratios, solvent mixtures and/or pH
  • the mixture of calcitonin drug-oligomer conjugates resulting from the reaction of the mixture of activated oligomers where each oligomer has the same molecular weight and has a structure of the oligomer of Formula A and the mixture of calcitonin drags is a mixture of conjugates where each conjugate has the same molecular weight and has the structure Formula A.
  • conjugation at the amino functionality of lysine may be suppressed by maintaining the pH of the reaction solution below the pK a of lysine.
  • the mixture of calcitonin drug-oligomer conjugates may be separated and isolated utilizing, for example, HPLC to provide a mixture of calcitonin drug-oligomer conjugates, for example mono-, di-, or tri-conjugates, where each conjugate in the mixture has the same number molecular weight and has the structure of Formula A.
  • the degree of conjugation e.g., whether the isolated molecule is a mono-, di-, or tri-conjugate
  • a particular isolated conjugate may be determined and/or verified utilizing various techniques as will be understood by those skilled in the art including, but not limited to, mass spectroscopy.
  • the particular conjugate structure (e.g., whether the oligomer is at Lys 11 , Lys 18 or the N-terminus of a salmon calcitonin monoconjugate) may be determined and/or verified utilizing various techniques as will be understood by those skilled in the art including, but not limited to, sequence analysis, peptide mapping, selective enzymatic cleavage, and/or endopeptidase cleavage.
  • one or more of the reaction sites ori the calcitonin drug maybe blocked by, for example, reacting the calcitonin drag with a suitable blocking reagent such as N-tert-butoxycarbonyl (t-BOC), or N-(9- fluorenylmethoxycarbonyl) (N-FMOC).
  • a suitable blocking reagent such as N-tert-butoxycarbonyl (t-BOC), or N-(9- fluorenylmethoxycarbonyl) (N-FMOC).
  • t-BOC N-tert-butoxycarbonyl
  • N-FMOC N-(9- fluorenylmethoxycarbonyl)
  • the mixture of blocked calcitonin drugs may be reacted with the mixture of activated oligomers where each oligomer in the mixture has the same molecular weight and has a structure of the oligomer of Formula A to provide a mixture of calcitonin drug-oligomer conjugates having oligomer(s) coupled to one or more nucleophilic residues and having blocking moieties coupled to other nucleophilic residues.
  • the calcitonin drug-oligomer conjugates may be de- blocked as will be understood by those skilled in the art.
  • the mixture of calcitonin drug-oligomer conjugates may then be separated as described above to provide a mixture of calcitonin drug-oligomer conjugates where each conjugate in the mixture has the same number molecular weight and has the structure of Formula A.
  • the mixture of calcitonin drug-oligomer conjugates may be separated prior to de-blocking.
  • Mixtures of calcitonin drag-oligomer conjugates where each conjugate in the mixture has the same molecular weight and has the structure of Formula A according to embodiments of the present invention preferably have improved properties when compared with those of conventional mixtures.
  • a mixture of calcitomn drug-oligomer conjugates where each conjugate in the mixture has the same molecular weight and has the structure of Formula A preferably is capable of lowering serum calcium levels by at least 5 percent.
  • the mixture, of conjugates is capable of lowering serum calcium levels by at least 10, 11, 12, 13 or 14 percent.
  • the mixture of conjugates is capable of lowering serum calcium levels by at least 15, 16, 17, 18 or 19 percent, and, most preferably, the mixture of conjugates is capable of lowering serum calcium levels by at least 20 percent.
  • a mixture of calcitonin drug-oligomer conjugates where each conjugate in the mixture has the same molecular weight and has the structure of Formula A preferably has an increased resistance to degradation by chymotrypsin and/or trypsin when compared to the resistance to degradation by chymotrypsin and/or trypsin, respectively, of the calcitonin drag which is not coupled to the oligomer.
  • Resistance to chymotrypsin or trypsin corresponds to the percent remaining when the molecule to be tested is digested in the applicable enzyme using a procedure similar to the one outlined in Example 51 below.
  • the resistance to degradation by chymotrypsin of the mixture of calcitonin drug- oligomer conjugates is about 10 percent greater than the resistance to degradation by - chymotrypsin of the mixture of calcitonin drugs that is not conjugated with the oligomer.
  • the resistance to degradation by chymotrypsin of the mixture of calcitonin drug-oligomer conjugates is about 15 percent greater than the resistance to degradation by chymotrypsin of the mixture of calcitonin drug that is not conjugated with the oligomer, and, most preferably, the resistance to degradation by chymotrypsin of the mixture of calcitonin drag-oligomer conjugates is about 20 percent greater than the resistance to degradation by chymotrypsin of the mixture of calcitonin drug that is not conjugated with the oligomer.
  • the resistance to degradation by trypsin of the mixture of calcitonin drug-oligomer conjugates is about 10 percent greater than the resistance to degradation by trypsin of the mixture of calcitonin drug that is not conjugated with the oligomer.
  • the resistance to degradation by trypsin of the mixture of calcitonin drag-oligomer conjugates is about 20 percent greater than the resistance to degradation by trypsin of the mixture of calcitonin drug that is not conjugated with the oligomer, and, most preferably, the resistance to degradation by trypsin of the mixture of calcitomn drag-oligomer conjugates is about 30 percent greater than the resistance to degradation by trypsin of the mixture of calcitonin drug that is not conjugated with the oligomer.
  • a mixture of calcitonin drug-oligomer conjugates where each conjugate in the mixture has the same molecular weight and has the structure of Formula A preferably has a higher bioefficacy than the bioefficacy of the calcitonin drag which is not coupled to the oligomer.
  • the bioefficacy of a particular compound corresponds to its area-under-the-curve (AUC) value.
  • AUC area-under-the-curve
  • the bioefficacy of the mixture is about 5 percent greater than the bioefficacy of the calcitonin drug which is not coupled to the oligomer. More preferably, the bioefficacy of the mixture is about 10 percent greater than the bioefficacy of the calcitonin drug which is not coupled to the oligomer.
  • a mixture of calcitonin drug-oligomer conjugates where each conjugate in the mixture has the same molecular weight and has the structure of Formula A preferably has an in vivo activity that is greater than the in vivo activity of a polydispersed mixture of calcitonin drug-oligomer conjugates having the same number average molecular weight as the mixture of calcitonin drug-oligomer conjugates where each conjugate in the mixture has the same molecular weight and has the structure of Formula A.
  • the number average molecular weight of a mixture may be measured by various methods including, but not limited to, size exclusion chromatography such as gel permeation chromatography as described, for example, in H.R. Allcock & F.W. Lampe, CONTEMPORARY POLYMER CHEMISTRY 394-402 (2d. ed., 1991).
  • a mixture of calcitonin drug-oligomer conjugates where each conjugate in the mixture has the same molecular weight and has the structure of Formula A preferably has an in vitro activity that is greater than the in vitro activity of a polydispersed mixture of calcitomn drug-oligomer conjugates having the same number average molecular weight as the mixture of calcitonin drug-oligomer conjugates where each conjugate in the mixture has the same molecular weight and has the structure of Formula A.
  • the number average molecular weight of a mixture may be measured by various methods including, but not Hmited to, size exclusion chromatography.
  • a mixture of calcitonin drug-oligomer conjugates where each conjugate in the mixture has the same molecular weight and has the stracture of Formula A preferably has an increased resistance to degradation by chymotrypsin and/or trypsin when compared to the resistance to degradation by chymotrypsin and/or trypsin of a polydispersed mixture of calcitonin drug-oligomer conjugates having the same number average molecular weight as the mixture of calcitonin drag-oligomer conjugates where each conjugate in the mixture has the same molecular weight and has the structure of Formula A.
  • the number average molecular weight of a mixture may be measured by various methods including, but not limited to, size exclusion chromatography.
  • a mixture of calcitonin drag-oligomer conjugates where each conjugate in the mixture has the same molecular weight and has the structure of Formula A preferably has an inter-subject variability that is less than the inter-subject variability of a polydispersed mixture of calcitonin drug-oligomer conjugates having the same number average molecular weight as the mixture of calcitonin drag-oligomer conjugates where each conjugate in the mixture has the same molecular weight and has the structure of Formula A.
  • the number average molecular weight of a mixture maybe measured by various methods including, but not limited to, size exclusion chromatography.
  • the inter-subject variability may be measured by various methods, as will be understood by those skilled in the art.
  • the inter-subject variability is preferably calculated as follows.
  • the area under a dose response curve (AUC) i.e., the area between the dose- response curve and a baseline value
  • the average AUC for all subjects is determined by summing the AUCs of each subject and dividing the sum by the number of subjects.
  • the absolute value of the difference between the subject's AUC and the average AUC is then determined for each subject.
  • the absolute values of the differences obtained are then summed to give a value that represents the inter-subject variability.
  • Lower values represent lower inter-subject variabihties and higher values represent higher inter- subject variabilities.
  • Mixtures of calcitonin drug-oligomer conjugates where each conjugate in the mixture has the same molecular weight and has the stracture of Formula A according to embodiments of the present invention preferably have two or more of the above-described improved properties. More preferably, mixtures of calcitonin drug-oligomer conjugates where each conjugate in the mixture has the same molecular weight and has the stracture of Formula A according to embodiments of the present invention have three or more of the above-described improved properties. Most preferably, mixtures of calcitonin drag-oligomer conjugates where each conjugate in the mixture has the same molecular weight and has the structure of Formula A according to embodiments of the present invention have four or more of the above-described improved properties.
  • compositions comprising a conjugate mixture according to embodiments of the present invention are also provided.
  • the mixtures of calcitonin drug- oligomer conjugates described above may be formulated for administration in a pharmaceutical carrier in accordance with known techniques. See, e.g., Remington, The Science And Practice of Pharmacy (9 th Ed. 1995).
  • the mixture of calcitonin drug-oligomer conjugates is typically admixed with, z'nter alia, a pharmaceutically acceptable carrier.
  • the carrier must, of course, be acceptable in the sense of being compatible with any other ingredients in the pharmaceutical composition and should not be deleterious to the patient.
  • the carrier may be a solid or a Hquid, or both, and is preferably formulated with the mixture of calcitonin drug-oligomer conjugates as a unit-dose formulation, for example, a tablet, which may contain from about 0.01 or 0.5% to about 95% or 99% by weight of the mixture of calcitonin drug-oligomer conjugates.
  • the pharmaceutical compositions maybe prepared by any of the well known techniques of pharmacy including, but not Hmited to, admixing the components, optionally including one or more accessory ingredients.
  • compositions according to embodiments of the present invention include those suitable for oral, rectal, topical, inhalation (e.g., via an aerosol) buccal (e.g., sub-lingual), vaginal, parenteral (e.g., subcutaneous, intramuscular, intradermal, intraarticular, intrapleural, intraperitoneal, inracerebral, intraarterial, or intravenous), topical (i.e., both skin and mucosal surfaces, including airway surfaces) and transdermal administration, although the most suitable route in any given case will depend on the nature and severity of the condition being treated and on the nature of the particular mixture of calcitonin drag-oligomer conjugates which is being used.
  • buccal e.g., sub-lingual
  • vaginal e.g., parenteral (e.g., subcutaneous, intramuscular, intradermal, intraarticular, intrapleural, intraperitoneal, inracerebral, intraarterial, or intravenous)
  • parenteral
  • compositions suitable for oral administration maybe presented in discrete units, such as capsules, cachets, lozenges, or tables, each containing a predetermined amount of the mixture of calcitonin drug-oligomer conjugates; as a powder or granules; as a solution or a suspension in an aqueous or non-aqueous liquid; or as an oil-in-water or water- in-oil emulsion.
  • Such formulations may be prepared by any suitable method of pharmacy which includes the step of bringing into association the mixture of calcitonin drag-oligomer conjugates and a suitable carrier (which may contain one or more accessory ingredients as noted above).
  • the pharmaceutical composition according to embodiments of the present invention are prepared by uniformly and intimately admixing- the mixture of calcitonin drag-oligomer conjugates with a liquid or finely divided solid carrier, or both, and then, if necessary, shaping the resulting mixture.
  • a tablet may be prepared by compressing or molding a powder or granules containing the mixture of calcitonin drug- oligomer conjugates, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared by compressing, in a suitable machine, the mixture in a free-flowing form, such as a powder or granules optionally mixed with a binder, lubricant, inert diluent, and/or surface active/dispersing agent(s). Molded tablets may be made by molding, in a suitable machine, the powdered compound moistened with an inert liquid binder.
  • Pharmaceutical compositions suitable for buccal (sub-lingual) administration include lozenges comprising the mixture of calcitonin drug-oligomer conjugates in a flavoured base, usually sucrose and acacia or tragacanth; and pastilles comprising the mixture of calcitonin.
  • compositions according to embodiments of the present invention suitable for parenteral administration comprise sterile aqueous and non-aqueous injection solutions of the mixture of calcitonin drug-oligomer conjugates, which preparations are preferably isotonic with the blood of the intended recipient. These preparations may contain anti-oxidants, buffers, bacteriostats and solutes which render the composition isotonic with the blood of the intended recipient.
  • Aqueous and non-aqueous sterile suspensions may include suspending agents and thickening agents.
  • compositions may be presented in unit ⁇ dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, saline or water-for-injection immediately prior to use.
  • sterile liquid carrier for example, saline or water-for-injection immediately prior to use.
  • Extemporaneous injection solutions and suspensions maybe prepared from sterile powders, granules and tablets of the kind previously described.
  • an injectable, stable, sterile composition comprising a mixture of calcitonin drug-oligomer conjugates in a unit dosage form in a sealed container may be provided.
  • the mixture of calcitonin drug-oligomer conjugates is provided in the form of a lyophilizate which is capable of being reconstituted with a suitable pharmaceutically acceptable carrier to form a liquid composition suitable for injection thereof into a subject.
  • the unit dosage form typically comprises from about 10 mg to about 10 grams of the mixture of calcitonin drug-oligomer conjugates.
  • a sufficient amount of • emulsifying agent which is physiologically acceptable may be employed in sufficient quantity to emulsify the mixture of calcitonin drug-oligomer conjugates in an aqueous carrier.
  • a sufficient amount of • emulsifying agent which is physiologically acceptable may be employed in sufficient quantity to emulsify the mixture of calcitonin drug-oligomer conjugates in an aqueous carrier.
  • a useful emulsifying agent is phosphatidyl choline.
  • compositions suitable for rectal administration are preferably presented as unit dose suppositories. These may be prepared by admixing the mixture of calcitonin drug-oligomer conjugates with one or more conventional solid carriers, for example, cocoa butter, and then shaping the resulting mixture.
  • compositions suitable for topical application to the skin preferably take the form of an ointment, cream, lotion, paste, gel, spray, aerosol, or oil.
  • Carriers which may be used include petroleum jelly, lanohne, polyethylene glycols, alcohols, transdermal enhancers, and combinations of two or more thereof.
  • compositions suitable for transdermal administration may be presented as discrete patches adapted to remain in intimate contact with the epidermis of the recipient for a prolonged period of time.
  • Compositions suitable for transdermal administration may also be delivered by iontophoresis (see, for example, Pharmaceutical Research 3 (6):318 (1986)) and typically take the form of an optionally buffered aqueous - solution ofthe mixture of calcitonin drug-oligomer conjugates.
  • Suitable formulations comprise citrate or bis ⁇ tris buffer (pH 6) or ethanol/water and contain from 0.1 to 0.2M active ingredient.
  • the bone disorder is preferably characterized by excessive osteoclastic bone resorption and/or hypercalcemic serum effects.
  • Bone disorders that may be treated and/or prevented by methods of the present invention include, but are not limited to, osteoporosis, Paget's disease, and hypercalcernia.
  • any mixture of calcitonin drug-oligomer conjugates will vary somewhat from mixture to mixture, and patient to patient, and will depend upon factors such as the age and condition of the patient and the route of delivery. Such dosages can be determined in accordance with routine pharmacological procedures known to those skilled in the art. As a general proposition, a dosage from about 0.1 to about 50 mg/kg will have therapeutic efficacy, with all weights being calculated based upon the weight of the mixture of calcitonin drug-oligomer conjugates. Toxicity concerns at the higher level may restrict intravenous dosages to a lower level such as up to about 10 mg/kg, with all weights being calculated based upon the weight of the active base.
  • a dosage from about 10 mgkg to about 50 mg/kg may be employed for oral administration.
  • a dosage from about 0.5 mgkg to 5 mg/kg may be employed for intramuscular injection.
  • the frequency of administration is usually one, two, or three times per day or as necessary to control the condition.
  • the drug-oligomer • conjugates may be administered by continuous infusion. The duration of treatment depends on the type of bone disorder being treated and may be for as long as the life of the patient.
  • reaction 1 A substantially monodispersed mixture, of polymers comprising polyethylene glycol moieties is provided as illustrated in reaction 1 :
  • R 1 is H or a HpophiHc moiety.
  • R 1 is preferably H, alkyl, aryl alkyl, an aromatic moiety, a fatty acid moiety, an ester of a fatty acid moiety, cholesteryl, or adamantyl.
  • R is more preferably H, lower alkyl, or an aromatic moiety.
  • R 1 is most preferably H, methyl, or benzyl.
  • n is from 1 to 25.
  • n is from 1 to 6.
  • X + is a positive ion.
  • X + is any positive ion in a compound, such as a strong base, that is capable of ionizing a hydroxyl moiety on PEG.
  • positive ions include, but are not limited to, sodium ions, potassium ions, lithium ions, cesium ions, and thallium ions.
  • R 2 is.H or a lipophilic moiety.
  • R 2 is preferably linear or branched alkyl, aryl alkyl, an aromatic moiety, a fatty acid moiety, or an ester of a fatty acid moiety.
  • R 2 is more preferably lower alkyl, benzyl, a fatty acid moiety having 1 to 24 carbon atoms, or an ester of a fatty acid moiety having 1 to 24 carbon atoms.
  • R is most preferably methyl, a fatty acid moiety having 1 to 18 carbon atoms or an ethyl ester of a fatty acid moiety having 1 to 18 carbon atoms.
  • m is from 1 to 25. Preferably m is from 1 to 6.
  • Ms is a mesylate moiety (i.e., CH 3 S(O 2 )-).
  • a mixture of compounds having the stracture of Formula I is reacted with a mixture of compounds having the structure of Formula II to provide a mixture of polymers comprising polyethylene glycol moieties and having the structure of Formula HI.
  • the mixture of compounds having the structure of Formula I is a substantially monodispersed mixture.
  • at least about 96, 97, 98 or 99 percent of the compounds in the mixture of compounds of Formula I have the same molecular weight, and, more preferably, the mixture of compounds of Formula I is a monodispersed mixture.
  • the mixture of compounds of Formula II is a substantially monodispersed mixture.
  • the mixture of compounds of Formula II has the same molecular weight, and, .more preferably, the mixture of compounds of Formula II is a monodispersed mixture.
  • the mixture of compounds of Formula HI is a substantially monodispersed mixture.
  • at least about 96, 97, 98 or 99 percent of the compounds in the mixture of compound of Formula HI have the same molecular weight. More preferably, the mixture of compounds of Formula HI is a monodispersed mixture.
  • Reaction 1 is preferably performed between about 0°C and about 40°C, is more preferably performed between about 15°C and about 35°C, and is most preferably performed at room temperature (approximately 25°C). Reaction 1 may be performed for various periods of time as will be understood by those skilled in the art. Reaction 1 is preferably performed for a period of time between about 0.25, 0.5 or 0.75 hours and about 2, 4 or 8 hours.
  • Reaction 1 is preferably carried out in an aprotic solvent such as, but not limited to, N,N-dimethylacetamide (DMA), N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), hexamethylphosphoric triamide, tefrahydrofuran (THF), didxane, diethyl ether, methyl t-butyl ether (MTBE), toluene, benzene, hexane, pentane, N-methylpyrollidinone, tetrahydronaphthalene, decahydronaphthalene, 1,2-dichlorobenzene, l,3-dimethyl-2- imidazolidinone, or a mixture thereof. More preferably, the solvent is DMF, DMA or toluene.
  • DMA N,N-dimethylacetamide
  • DMF N,N-dimethylformamide
  • DMSO dimethyl sulfoxide
  • the molar ratio of the compound of Formula I to the compound of Formula H is preferably greater than about 1:1. More preferably, the molar ratio is at least about 2:1.
  • R 1 and X + are as described above and the mixture of compounds of Formula IV is substantially monodispersed; preferably, at least about 96, 97, 98 or 99 percent of the compounds in the mixture of compounds of Formula IV have the same molecular weight; and, more preferably, the mixture of compounds of Formula TV is a monodispersed mixture.
  • Various compounds capable of ionizing a hydroxyl moiety on the PEG moiety of the compound of Formula IV will be understood by those skilled in the art.
  • the compound capable of ionizing a hydroxyl moiety is preferably a strong base. More preferably, the compound capable of ionizing a hydroxyl moiety is selected from the group consisting of sodium hydride, potassium hydride, sodium t-butoxide, potassium t-butoxide, butyl lithium (BuLi), and lithium diisopropylamine.
  • the compound capable of ionizing a hydroxyl moiety is more preferably sodium hydride.
  • the molar ratio of the compound capable of ionizing a hydroxyl moiety on the PEG moiety of the compound of Formula JV to the compound of Formula IV is preferably at least about 1:1, and is more preferably at least about 2:1.
  • Reaction 2 is preferably performed between about 0°C and about 40°C, is more preferably performed between about 0°C and about 35°C, and is most preferably performed between about 0°C and room temperature (approximately 25°C).
  • Reaction 2 may be performed for various periods of time as will be understood by those skilled in the art. Reaction 2 is preferably performed for a period of time between about 0.25, 0.5 or 0.75 hours and about 2, 4 or 8 hours.
  • Reaction 2 is preferably carried out in an aprotic solvent such as, but not limited to, N,N-dimethylacetamide (DMA), N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), hexamethylphosphoric triamide, tetrahydrofuran (THF), dioxane, diethyl ether, methyl t-butyl ether (MTBE), toluene, benzene, hexane, pentane, N-methylpyrollidinone, dichloromethane, chloroform, tetrahydronaphthalene, decahydronaphthalene, 1,2- dichlorobenzene, l,3-dimethyl-2-imidazolidinone, or a mixture thereof. More preferably, the solvent is DMF, dichloromethane or toluene.
  • DMA N,N-dimethylacetamide
  • DMF N,N-dimethylformamide
  • R 2 and Ms are as described above and the compound of Formula V is present as a substantially monodispersed mixture of compounds of Formula V; preferably at least about 96, 97, 98 or 99 percent of the compounds in the mixture of compounds of Formula V have the same molecular weight; and, more preferably, the mixture of compounds of Formula V is a monodispersed mixture.
  • Q is a halide, preferably chloride or fluoride.
  • CH 3 S(O 2 )Q is methanesulfonyl halide.
  • the methanesulfonyl halide is preferably methanesulfonyl chloride or methanesulfonyl fluoride. More preferably, the methanesulfonyl halide is methanesulfonyl chloride.
  • the molar ratio of the methane sulfonyl halide to the compound of Formula V is preferably greater than about 1:1, and is more preferably at least about 2:1.
  • Reaction 3 is preferably performed between about -10°C ' and about 40°C, is more preferably performed between about 0°C and about 35°C, and is most preferably performed between about 0°C and room temperature (approximately 25°C).
  • Reaction 3 may be performed for various periods of time as will be understood by those skilled in the art. Reaction 3 is preferably performed for a period of time between about 0.25, 0.5 or 0.75 hours and about 2, 4 or 8 hours.
  • Reaction 3 is preferably carried, out in the presence of an aliphatic amine including, but not limited to, monomethylamine, dimethylamine, trimethylamine, monoethylamine, diethylamine, triethylamine, monoisopropylamine, diisopropylamine, mono-n-butylamine, di- n-butylamine, tri-n-butylamine, monocyclohexylamine, dicyclohexylamine, or mixtures thereof. More preferably, the aliphatic amine is a tertiary amine such as triethylamine.
  • the compounds of Formula V are PEG or mPEG compounds, respectively, which are commercially available from Aldrich of Milwaukee, Wisconsin; Fluka of Switzerland, and/or TCI America of Portland, Oregon.
  • R 2 is a lipophilic moiety such as, for example, higher alkyl, fatty acid, an ester of a fatty acid, cholesteryl, or adamantyl
  • the compounds of Formula V may be provided by various methods as will be understood by those skilled in the art.
  • the compounds of Formula V are preferably provided as follows:
  • R is a lipophilic moiety, preferably higher alkyl, fatty acid ester, cholesteryl, or adamantyl, more preferably a lower alkyl ester of a fatty acid, and most preferably an ethyl ester of a fatty acid having from 1 to 18 carbon atoms.
  • R 3 is H, benzyl, trityl, tetrahydropyran, or other alcohol protecting groups as will be understood by those skilled in the art.
  • X 2 + is a positive ion as described above with respect to X + .
  • a mixture of compounds of Formula VI is reacted with a mixture of compounds of Formula VH under reaction conditions similar to those described above with reference to reaction 1.
  • the mixture of compounds of Formula VI is a substantially monodispersed mixture.
  • at least about 96, 97, 98 or 99 percent of the compounds in the mixture of compounds of Formula VI have the same molecular weight.
  • the mixture of compounds of Formula VI is a monodispersed mixture.
  • the - mixture of compounds of Formula VH is a substantially monodispersed mixture.
  • Formula VH have the same molecular weight. More preferably, the mixture of compounds of Formula VH is a monodispersed mixture.
  • the compound of Formula VIH may be hydrolyzed to convert the R 3 moiety into an alcohol by various methods as will be understood by those skilled in the art.
  • R 3 is benzyl or trityl
  • the hydrolysis is preferably performed utilizing H 2 in the presence of a palladium-charcoal catalyst as is known by those skilled in the art.
  • reaction 5 is unnecessary.
  • the compound of Formula VI may be commercially available or be provided as described above with reference to reaction 3.
  • the compound of Formula VH may be provided as described above with reference to reaction 2.
  • Substantially monodispersed mixtures of polymers comprising PEG moieties and having the stracture of Formula HI above can further be reacted with other substantially monodispersed polymers comprising PEG moieties in order to extend the PEG chain.
  • the following scheme may be employed: O t i 'I 2
  • Ms, m and n are as described above with reference to reaction 1; p is similar to n and m, and X 2 + is similar to X + as described above with reference to reaction 1.
  • Q is as described above with reference to reaction 3.
  • R is as described above with reference to reaction 1 and is preferably lower alkyl.
  • R 1 is H.
  • Reaction 6 is preferably performed in a manner similar to that described above with reference to reaction 3.
  • Reaction 7 is preferably performed in a manner similar to that described above with reference to reaction 1.
  • at least about 96, 91, 98 or 99 percent of the compounds in the mixture of compounds of Formula HI have the same molecular weight, and, more preferably, the mixture of compounds of Formula HI is a monodispersed mixture.
  • the mixture of compounds of Formula X is a substantially monodispersed mixture.
  • at least about 96, 97, 98 or 99 percent of the compounds in the mixture of compounds of Formula X have the same molecular weight, and, more preferably, the mixture of compounds of Formula X is a monodispersed mixture.
  • a process according to embodiments of the present invention is illustrated by the scheme shown in Figure 1, which will now be described.
  • the synthesis of substantially monodispersed polyethylene glycol-containing oligomers begins by the preparation of the monobenzyl ether (1) of a substantially monodispersed polyethylene glycol.
  • the mesylate (5) of this alcohol may be prepared by addition of methanesulfonyl chloride and used as the electrophile in the reaction with the sodium salt of the monomethyl ether of a substantially monodispersed polyethylene glycol derivative, thereby extending the polyethylene glycol portion of the oligomer to the desired length, obtaining the elongated ester (6).
  • the ester may be hydrolyzed to the acid (7) in aqueous base and transformed into the activated ester (8) by reaction with a carbodiimide and N-hydroxysuccinimide.
  • oligomer illustrated in Figure 1 is activated using N-hydroxysuccinimide
  • various other reagents may be used to activate oligomers of the present invention including, but not limited to, active phenyl chloroformates such as ⁇ r ⁇ -nitrophenyl chloroformate, phenyl chloroformate, 3, 4-phenyldichloro formate, and 3, 4-phenyldichloro formate; tresylation; and acetal formation.
  • q is from 1 to 24.
  • q is from 1 to 18, and q is more preferably from 4 to 16.
  • R 4 is a moiety capable of undergoing hydrolysis to provide the carboxylic acid.
  • R 4 is preferably lower alkyl and is more preferably ethyl.
  • the variables n and m are as described above with reference to reaction 1.
  • reaction mixture was diluted with another 75 mL of dichloromethane and washed successively with saturated NaHCO 3 , water and brine. The organics were dried over Na 2 SO 4 , filtered and concentrated in vacuo to give a non-polydispersed mixture of compounds 9 as a clear oil (5.31g, 86%).
  • non-polydispersed compound 11 (35.7 mmol) in dry DMF (25.7 mL), under N 2 was added in portion a 60% dispersion of NaH in mineral oil, and the mixture was stirred at room temperature for 1 hour.
  • this salt 12 was added a solution of . non-polydispersed mesylate 9 (23.36) in dry DMF (4 ml) in a single portion, and the mixture was stirred at room temperature for 3.5 hours. Progress of the reaction was monitored by TLC (12% CH 3 OH-CHCl 3 ). The reaction mixture was diluted with an equal amount of IN HCl, and extracted with ethyl acetate (2 x 20 ml) and discarded. Extraction of aqueous solution and work-up gave non-polydispersed polymer 10 (82 -84% yield).
  • the non-polydispersed compounds 15 were prepared from a diol by using the procedure described above for compound 10.
  • the crude product mixture was purified via flash chromatography (silica gel, gradient elution: ethyl acetate to 9/1 ethyl acetate/methanol) to yield 8.099 g (70 %) of non-polydispersed 16 as a yellow oil.
  • the non-polydispersed mesylate 17 (19.21 g, 80.6 mmol) in 80 ml dry toluene was added to the NaH alcohol mixture, and the combined solutions were stirred at room temperature for three days.
  • the reaction mixture was quenched with 50 ml methanol and filtered through basic alumina.
  • the filtrate was concentrated in vacuo and purified by flash chromatography (silica gel, gradient elution: 3/1 ethyl acetate/hexanes to ethyl acetate) to yield the non-polydispersed product as a pale yellow oil (16.52 g, 44 %).
  • Non-polydispersed benzyl ether 18 (1.03 g, 2.0 mmol) was dissolved in 25 ml ethanol. To this solution was added 270 mg 10 % Pd/C, and the mixture was placed under a hydrogen atmosphere and stirred for four hours, at which time TLC showed the complete disappearance of the starting material. The reaction mixture was filtered through Celite 545 to remove the catalyst, and the filtrate was concentrated in vacuo to yield the non- polydispersed title compound as a clear oil (0.67 g, 79 %). FAB MS: m/e 425 (M+H), 447 (M+Na). Example 15
  • the non-polydispersed alcohol 19 (0.835 g, 1.97 mmol) was dissolved in 3.5 ml dry ' dichloromethane and placed under a nitrogen atmosphere. Triethylamine (0.301 ml, 0.219 g, 2.16 mmol) was added and the mixture was chilled in an ice bath. After two minutes, the methanesulfonyl chloride (0.16 ml, 0.248 g, 2.16 mmol) was added. The mixture was stirred for 15 minutes at 0 C, then at room temperature for two hours.
  • Non-polydispersed ester 21 (0.25 g, 0.46 mmol) was stirred for 18 hours in 0.71 ml of I N NaOH. After 18 hours, the mixture was concentrated in vacuo to remove the alcohol and the residue dissolved in a further 10 ml of water. The aqueous solution was acidified to pH 2 with 2 N HCl and the product was extracted into dichloromethane (30 ml x 2). The combined organics were then washed with brine (25 ml x 2), dried over Na 2 SO 4 , filtered and concentrated in vacuo to yield the non-polydispersed title compound as a yellow oil (0.147 g, 62 %). FAB MS: m/e 499 (M+H), 521 (M+Na).
  • Non-polydispersed acid 22 (0.209 g, 0.42 mmol) were dissolved in 4 ml of dry dichloromethane and added to a dry flask already containing NHS (N-hydroxysuccinimide) (57.8 mg, 0.502 mmol) and EDC (l-(3-dime ylammopropyl)-3-e1hylcarbodiimide hydrochloride) (98.0 mg, 0.502 mmol) under a N 2 atmosphere.
  • NHS N-hydroxysuccinimide
  • EDC l-(3-dime ylammopropyl)-3-e1hylcarbodiimide hydrochloride
  • the crude reaction mixture was filtered through Celite (washed CH 2 C1 2 -200 mL), then washed with H 2 O (300 mL), 5% NaHCO 3 (300 mL), H 2 O (300 mL), sat. -NaCl (300 mL), dried MgSO , and evaporated to dryness.
  • the oil was then placed on a vacuum line for ⁇ 2h to ensure dryness and afforded the non-polydispersed title compound as a yellow oil (29.15 g, 80% yield).
  • the oil was then dissolved in HCl (250 mL, 1 N) and washed with ethyl acetate (250 mL) to remove excess 24. Additional washings of ethyl acetate (125 mL) may be required to remove remaining 24.
  • the aqueous phase was washed repetitively with CH 2 C1 2 (125 mL volumes) until most of the 25 has been removed from the aqueous phase.
  • the first extraction will contain 24, 25, and dicoupled side product and should be back extracted with HCl (125 L, IN).
  • the organic layers were combined and evaporated to dryness.
  • the resultant oil was then dissolved in CH 2 ⁇ 2 (100 mL) and washed repetitively with H 2 O (50 mL volumes) until 25 was removed.
  • Example 23 MPEG7-C8 acid (28) . .
  • the crude reaction mixture was concentrated, acidified (pH ⁇ 2), saturated with NaCl, and washed CH 2 C1 2 (2 x 50 mL).
  • the organic layers were combined, washed sat. NaCl, dried MgSO 4 , and evaporated to dryness to afford the non-polydispersed title compound as a clear oil (0.35 g, 53% yield).
  • Non-polydispersed mPEG7-C8-acid 28 (0.3 lg, 0.64 mmol) was dissolved in 3 ml of anhydrous methylene chloride and then solution of N-hydroxysuccinimide (0.079g, 0.69 mmol) and EDCI-HC1 (135.6 mg, 0.71 mmol) in anhydrous methylene chloride added. Reaction was stirred for several hours, then washed with IN HCl, water, dried over MgSO 4 , filtered and concentrated. Crude material was purified by column chromatography, concentrated to afford the non-polydispersed title compound as a clear oil and dried via vacuum. Examples 25 through 29 refer to the scheme illustrated in Figure 5.
  • the crude reaction mixture was filtered through Celite (washed CH 2 C1 2 , 80 mL) and the filtrate was washed H 2 O (100 mL), 5% NaHCO 3 (2 x 100 mL), H 2 O (100 mL), sat. NaCl (100 mL), dried MgSO , and evaporated to dryness to afford the non-polydispersed title compound as a yellowish oil (7.42 g, 97% yield).
  • Example 28 To the oil of non-polydispersed mPEGrdo ester 32 (0.570 g, 1.1 mmol) was added IN NaOH (1.6 mL) and the reaction mixture was stirred overnight. The crade reaction mixture was concentrated, acidified (pH ⁇ 2), saturated with NaCl, and washed CH 2 CI2 (2 50 mL). The organic layers were combined, washed sat. NaCl (2 x 50 mL), dried MgSO 4 , and ⁇ evaporated to dryness to afford the non-polydispersed title compound as a clear oil (0.340 g, 62% yield).
  • the non-polydispersed acid 33 was activated using procedures similar to those described above in Example 24.
  • Non-polydispersed stearoyl chloride 35 (0.7g, 2.31 mmol) was added slowly to a mixture of PEG6 (5 g, 17.7 mmol) and pyridine (0.97g, 12.4 mmol) in benzene. The reaction mixture was stirred for several hours ( ⁇ 5). The reaction was followed by TLC using ethylacetate/methanol as a developing solvent. Then the reaction mixture was washed with water, dried over MgSO 4 , concentrated and dried via vacuum. Purified non-polydispersed compound 36 was analyzed by FABMS: m/e 549/ M1H. Example 31 Activation of C18(PEG6) Oligomer .
  • Non-polydispersed stearoyl-PEG6 36 ( 0.8 g, 1.46 mmol ) was dissolved in toluene and added to a phosgene solution (10 ml, 20 % in toluene) which was cooled with an ice bath. The reaction mixture was stirred for 1 h at 0 C and then for 3 h at room temperature. Then ' phosgene and toluene were distilled off and the remaining non-polydispersed stearoyl PEG6 chloroformate 37 was dried over P 2 O 5 overnight.
  • Example 32 Tetraethylene glycol monobenzylether (39) To the oil of non-polydispersed tetraethylene glycol (19.4 g, 0.10 mol) was added a solution of NaOH (4.0 g in 4.0 mL) and the reaction was stirred for 15 mm. Then benzyl chloride (3.54 mL, 30.8 mmol) was added and the reaction mixture was heated to 100°C and stirred overnight. The reaction mixture was cooled to room temperature, diluted with sat. NaCl (250 L), and washed CH 2 C1 2 (2 x 200 mL). The organic layers were combined, washed sat. NaCl, dried MgSO , and chromatographed (silica, ethyl acetate) to afford the non-polydispersed title compound as a yellow oil (6.21 g, 71% yield).
  • Example 33 Mesylate of tetraethylene glycol monobenzylether (40) To a solution of CH 2 CI 2 (20 mL) was added non-polydispersed tetraethylene glycol monobenzylether 39 (6.21 g, 22 mmol) and cooled to 0°C in an ice bath. Then Iriemylamine (3.2 mL, 24 mmol) was added and the reaction mixture was stirred for 15 min at 0°C.
  • the crude reaction mixture was filtered through Celite (washed, CH 2 C1 , 250 mL) and the filtrate was washed H 2 O, dried MgSO 4 , and evaporated to dryness.
  • the resultant oil was chromatographed (silica, ethyl acetate/methanol, 10:1) and chromatographed (silica, chloroform methanol, 25:1) to afford the non-polydispersed title compound as a clear oil (2.62 g, 34% yield).
  • non-polydispersed octaethylene glycol monobenzylether 41 (0.998 g, 2.07 mmol) and pyridine (163.9 mg, 2.07 mmol) was added non-polydispersed stearoyl chloride 42 (627.7 mg, 2.07 mmol) in benzene.
  • the reaction mixture was stirred overnight (18 hours). The next day the reaction mixture was washed with water, dried over MgSO 4 , concentrated and dried via vacuum. Then the crude product was chromatographed on flash silica gel column, using 10% methanol/90% chloroform. The fractions containing the product were combined, concentrated and dried via vacuum to afford - the non-polydispersed title compound.
  • Example 36 Hydrogenolysis of Stearate-PEG8-Benzyl
  • Example 38 Synthesis of Activated Triethylene Glycol Monomethyl Oligomers The following description refers to the scheme illustrated in Figure 8.
  • a solution of toluene containing 20% phosgene (100 ml, approximately 18.7 g, 189 mmol phosgene) was chilled to 0°C under a N 2 atmosphere.
  • Non-polydispersed mTEG triethylene glycol, monomethyl ether, 7.8 g, 47.5 mmol
  • the mixture was stirred for one hour at 0°C, then allowed to warm to room temperature and stirred for another two and one half hours.
  • the remaining phosgene, ethyl acetate and toluene were removed via vacuum distillation to leave the non-polydispersed mTEG chloroformate 46 as a clear oily residue.
  • the non-polydispersed residue 46 was dissolved in 50 mL of dry dichloromethane to which was added TEA (triethyleamine, 6.62 mL, 47.5 mmol) and NHS (N-hydroxysuccinimide, 5.8 g, 50,4 mmol). The mixture was stirred at room temperature under a dry atmosphere for twenty hours during which time a large amount of white precipitate appeared. The mixture was filtered to remove this precipitate and concentrated in vacuo. The resultant oil 47 was taken up in dichloromethane and washed twice with cold deionized water, twice with IN HCl and once with brine.
  • TEA triethyleamine, 6.62 mL, 47.5 mmol
  • NHS N-hydroxysuccinimide
  • the organics were dried over MgSO , filtered and concentrated to provide the non-polydispersed title compound as a clear, light yellow oil. If necessary, the NHS ester could be further purified by flash chromatography on silica gel using EtOAc as the elutant.
  • Non- polydispersed palmitic anhydride (5 g; 10 mmol) was dissolved in dry THE (20 mL) and stirred at room temperature. To the stirring solution, 3 mol excess of pyridine was added followed by non-polydispersed triethylene glycol (1.4 mL). The reaction mixture was stirred for 1 hour (progress of the reaction was monitored by TLC; ethyl acetate-chloroform; 3:7). At the end of the reaction, THF was removed and the product was mixed with 10% H 2 SO 4 acid and extracted ethyl acetate (3 x 30 L).
  • non-polydispersed product 48 was washed sequentially with water, brine, dried over MgSO 4 , and evaporated to give non-polydispersed product 48.
  • a solution of N,N'-disuccinimidyl carbonate (3 mmol) in DMF (-10 L) is added to a solution of the non-polydispersed product 48 (1 mmol) in 10 mL of anydrous DMF while stirring.
  • Sodium hydride (3 mmol) is added slowly to the reaction mixture. The reaction mixture is stirred for several hours (e.g., 5 hours). Diethyl ether is added to precipitate the activated oligomer. This process is repeated 3 times and the product is finally dried.
  • Example 40 Synthesis of Activated Hexaethylene Glycol Monomethyl Oligomers The following description refers to the scheme illustrated in Figure 10.
  • Non- polydispersed activated hexaethylene glycol monomethyl ether was prepared analogously to that of non-polydispersed triethylene glycol in Example 39 above.
  • a 20% phosgene in toluene solution 35 mL, 6.66 g, 67.4 mmol phosgene was chilled under a N 2 atmosphere in an ice/salt water bath.
  • Non-polydispersed hexaethylene glycol 50 (1.85 mL, 2.0 g, 6.74 mmol) was dissolved in 5 mL anhydrous EtOAc and added to the phosgene solution via syringe. The reaction mixture was kept stirring in the ice bath for one hour, removed and stirred a further 2.5 hours at room temperature. The phosgene, EtOAc, and toluene were removed by vacuum distillation, leaving non-polydispersed compound 51 as a clear, oily residue. The non-polydispersed residue 51 was dissolved in 20 mL dry dichloromethane and placed under a dry, inert atmosphere.
  • Example 41 The procedure of Example 41 was used to conjugate salmon calcitonin with the activated ohgomer of Example 29. MS for PEG7-decyl-sCT, mono-conjugate: 3926. MS for PEG7-decyl-sCT, di-conjugate: 4420.
  • Example 41 The procedure of Example 41 was used to conjugate salmon calcitonin with the activated oligomer of Example 31. MS for stearate-PEG6-sCT, mono-conjugate: 4006. MS for stearate-PEG6-sCT, di-conjugate: 4582.
  • Example 44
  • Example 41 The procedure, of Example 41 was used to conjugate salmon calcitonin with the activated ohgomer of Example 37. MS for stearate-PEG8-sCT, mono-conjugate: 4095.
  • Example 41 The procedure of Example 41 is used to conjugate salmon calcitonin with the activated oligomer of Example 18.
  • Example 46 The procedure of Example 41 is used to conjugate salmon calcitonin with the activated oligomer of Example 38.
  • Example 41 The procedure of Example 41 is used to conjugate salmon calcitonin with the activated oligomer of Example 39.
  • Example 41 The procedure of Example 41 is used to conjugate salmon calcitonin with the activated oligomer of Example 40.
  • the dispersity coefficient of a mixture of salmon calcitonin-oligomer conjugates is determined as follows. A mixture of salmon calcitonin-oligomer conjugates is provided, for example as described above in Example 41. A first sample of the mixture is purified via
  • the mixture may include one or more of the following conjugates, which are described by stating the conjugation position followed by the degree of conjugation: Lys 11 monoconjugate; Lys 18 monoconjugate; N- terminus monoconjugate; Lys 11 ' 18 diconjugate; Lys 11 , N-terminus diconjugate; Lys 1 , N- terminus diconjugate; and/or Lys 11,18 , N-terminus triconjugate.
  • Each isolated fraction of the mixture is analyzed via mass spectroscopy to determine the mass of the fraction, which allows each isolated fraction to be categorized as a mono-, di-, or tri-conjugate and provides a value for the variable "M,-" for each conjugate in the sample.
  • a second sample of. the mixture is analyzed via HPLC to provide an HPLC trace. Assuming that the molar absorptivity does not change as a result of the conjugation, the , weight percent of a particular conjugate in the mixture is provided by the area under the peak of the HPLC trace corresponding to the particular conjugate as a percentage of the total area under all peaks of the HPLC trace.
  • the sample is collected and lyophilized to dryness to determine the anhydrous gram weight of the sample.
  • the gram weight of the sample is multiplied by the weight percent of each component in the sample to determine the gram weight of each conjugate in the sample.
  • the variable "Ni" is determined for a particular conjugate (the i th conjugate) by dividing the gram weight of the particular conjugate in the sample by the mass of the particular conjugate and multiplying the quotient by Avagadro's number (6.02205 x 10 23 mole “1 ), Mi, determined above, to give the number of molecules of the particular conjugate, Ni, in the sample..
  • the dispersity coefficient is then calculated using n, Mj as determined for each conjugate, and Ni as determined for each conjugate.
  • Riinning buffer was then pumped across the cells at a rate of 100 ⁇ L/rnin except during 30-second intervals when the flow was stopped, and acidification of the running buffer in the sensor chamber was measured. Acidification rates were determined every 2 minutes. The temperature of the sensor chambers was 37°C. Cells were allowed to equilibrate in the sensor chambers for 2-3 hours prior to the start of the experiment during which time basal acidification rates were monitored. Cells were then exposed to test compounds. (Salmon Calcitonin or Octyl-Di-Calcitonin) diluted in ranning buffer at various nM concentration. Exposure of cells to test compounds occurred for the first 40 seconds of each 2 minute pump cycle in a repeating pattern for a total of 20 minutes.
  • a control compounds are treated with 2 ⁇ L of 1 mM HCl.
  • a sampling procedure is repeated at various time intervals depending on the gut enzyme used. Chymotrypsin has 15, 30 and 60 ⁇ minute samples. Trypsin has 30, 60, 120 and 180 minute samples. Once all points have been acquired, a final sample is removed from the control tube to make sure that observed degradation is not temperature or buffer related.
  • the chymotrypsin and trypsin samples may be collected directly into HPLC vials.
  • RP-HPLC acetonitrile gradient
  • AUC % degradation
  • mice Male CF-1 mice (Charles River, Raleigh, NC) weighing 20-25 g were housed in the Nobex vivarium in a light- (L:D cycle of 12:12, lights on at 0600 h), temperature- (21-23°C), and humidity- (40-60 % relative humidity) controlled room. Animals were permitted free access to laboratory chow (PMI Nutrition) and tap water. Mice were allowed to acclimate to housing conditions for 48-72 hours prior to the day of experiment.
  • mice Prior to dosing, mice were fasted overnight and water was provided ad libitum. Mice were randomly distributed into groups of five animals per time point and were administered a single oral dose of a PEG7-octyl-sCT, diconjugate (Octyl Di) according to the present invention or salmon calcitonin (sCT or Calcitonin) for comparison purposes. Oral doses were administered using a gavaging needle (Popper #18, 5 cm from hub to bevel) at 10 mL/kg in the following 0.2 ⁇ g mL phosphate-buffered PEG7-octyl-sCT, diconjugate, formulation:
  • the buffered formulation was prepared by adding 80 mL of phosphate buffer in a clean tared glass beaker. The sodium cholate was slowly added to the phosphate buffer with stirring until dissolved. The deoxy cholate was then added and stirring was continued until dissolved.
  • mice were ether-anesthetized, the vena cavae exteriorized, and blood samples were obtained via a syringe fitted with a 25-gauge needle. Blood aliquots were allowed to clot at 22°C for 1 hour, and the sera removed and pipetted into a clean receptacle. Total serum calcium was determined for each animal using a calibrated Vitros
  • Serum calcium data were plotted and pharmacokinetic parameters determined via curve-fitting techniques using SigmaPlot software (Version 4.1). Means and standard deviations (or standard errors) were calculated and plotted to determine effect differences among dosing groups. Average serum calcium data for various conjugates are provided in Table 5 below.
  • Example 50 Despite an in vitro activity as determined in Example 50 above that may not be comparable with the in vitro activity of PEG7-octyl-sCT and PEG7-decyl-sCT mono- and diconjugates, the stearate-PEG6-sCT, diconjugate, and stearate-PEG8-sCT, diconjugate, appear to have in vivo activity (as evidenced by the drops in % baseline calcium from Table 5 above) that are comparable with the in vivo activity observed for the PEG7-oetyl-sCT and PEG7-decyl-sCT, mono- and di-conjugates.
  • the improved in vivo activity of the stearate containing conjugates may indicate that these conjugates are undergoing hydrolysis in vivo to provide an active salmon calcitonin or active salmon calcitonin-PEG conjugate.

Abstract

A mixture of conjugates in which each conjugate in the mixture comprises a calcitonin drug coupled to an oligomer that includes a polyalkylene glycol moiety is disclosed. The mixture may lower serum calcium levels in a subject by 10, 15 or even 20 percent or more. Moreover, the mixture may be more effective at surviving an in vitro model of intestinal digestion than non-conjugated calcitonin. Furthermore, the mixture may exhibit a higher bioavailability than non-conjugated calcitonin.

Description

MIXTURES OF CALCITONIN DRUG-OLIGOMER
CONJUGATES COMPRISING POLYALKYLENE GLYCOL,
USES THEREOF, AND METHODS OF MAKING SAME
Field Of The Invention
The present invention relates to drug-oligomer conjugates, and, more particularly, to calcitonin drug-oligomer conjugates.
Background Of The Invention Calcitonin is a naturally occurring hormone with a short half-life that is believed to act directly on osteoclasts (via receptors on the cell surface for calcitonin). This action may directly inhibit osteoclastic bone resorption, which may lead to hypocalcemic and/or hypophosphatemic serum effects. Calcitonin may be useful in treating various bone disorders including, but not limited to, osteoporosis and Paget's disease. Osteoporosis is a bone disease in which bone tissue is normally mineralized, but the amount of bone is decreased and the structural integrity of trabecular bone is impaired. Cortical bone becomes more porous and thinner. This makes the bone weaker and more likely to fracture. In the United States, about 21% of postmenopausal women have osteoporosis (low bone density), and about 16% have had a fracture, in women older than 80, about 40% have experienced a fracture of the hip, vertebra, arm, or pelvis. The population of older men and women has been increasing, and therefore the number of people with osteoporosis is increasing.
Calcitonin given as a subcutaneous injection has shown significant improvements in bone density; however, a high incidence of side effects, including pain at the injection site, flushing and nausea, have been reported which may limit the use of the drug.
Paget's disease of bone is a metabolic bone disorder of unknown origin which normally affects older people. The disease causes an increased and irregular formation of bone as the bone cells, which are responsible for dissolving the body's old bone and replacing it with new, become out of control. Over a period of time the deformed new bone becomes larger, weaker and has more blood vessels than normal bone. Unlike normal bone, the structure is irregular and consequently weaker, which makes it prone to fracture even after a minor injury.
In its mildest form the disease has no symptoms. In more severe cases the pain can be intense. The relentless progression of the disease may cause bones to bow, the skull may increase in size and the spinal column may curve. As the bones enlarge they may cause pressure on nearby nerves which can result in muscle weakness. In the case of severe skull - enlargement this pressure can result in deafness, disturbed vision, dizziness and tinnitus. Calcitonin may be effective in treating disorders of increased skeletal remodeling, such as Paget's disease. In treating Paget's disease, chronic use of calcitonin may produce long-term reduction in symptoms; however, side effects of calcitonin administration may include nausea, hand swelling, urticaria, and intestinal cramping. Various references have proposed conjugating polypeptides such as calcitonin with polydispersed mixtures of polyethylene glycol or polyethylene glycol-containing polymers. For example, U.S. Patent No. 5,359,030 to Ekwuribe proposes conjugating polypeptides such as calcitonin with polydispersed mixtures of polyethylene glycol modified glycolipid polymers and polydispersed mixtures of polyethylene glycol modified fatty acid polymers. The number average molecular weight of polymer resulting from each combination is preferred to be in the range of from about 500 to about 10,000 Daltons.
The polydispersity of the polymer mixtures and conjugates described in Ekwuribe is likely a result of the use of polydispersed polyethylene glycol in the polymer synthesis. PEG is typically produced by base-catalyzed ring-opening polymerization of ethylene oxide. The reaction is initiated by adding ethylene oxide to ethylene glycol, with potassium hydroxide as catalyst. This process results in a polydispersed mixture of polyethylene glycol polymers having a number average molecular weight within a given range of molecular weights. For example, PEG products offered by Sigma- Aldrich of Milwaukee, Wisconsin are provided in polydispersed mixtures such as PEG 400 (M„ 380-420); PEG 1,000 (Mπ 950-1,050); PEG 1,500 (Mn 1,400-1,600); and PEG 2,000 (Mn 1,900-2,200).
It is desirable to provide non-polydispersed mixtures of calcitonin drug-oligomer conjugates where the oligomer comprises polyethylene glycol. Summary Of The Invention
It has unexpectedly been discovered that a mixture of calcitonin-oligomer conjugates comprising polyethylene glycol according to embodiments of the present invention may lower serum calcium levels by 10, 15 or even 20 percent or more. Moreover, a mixture of calcitonin-oligomer conjugates comprising polyethylene glycol according to embodiments of the present invention may be more effective at surviving an in vitro model of intestinal digestion than non-conjugated calcitonin. Furthermore, mixtures of calcitonin-oligomer conjugates comprising polyethylene glycol according to embodiments of the present invention may exhibit a higher bioavailability than non-conjugated calcitonin.
According to embodiments of the present invention, a substantially monodispersed mixture of conjugates each comprising a calcitonin drug coupled to an oligomer that comprises a polyethylene glycol moiety is provided. The polyethylene glycol moiety preferably has at least 2, 3, or 4 polyethylene glycol subunits and, most preferably, has at least 7 polyethylene glycol subunits. The oligomer preferably further comprises a lipophilic moiety. The calcitonin. drug is preferably salmon calcitonin. Oligomers are preferably coupled at Lys11 and Lys18 of the salmon calcitonin. The conjugate is preferably amphiphilically balanced such that the conjugate is aqueously soluble and able to penetrate biological membranes. According to other embodiments of the present invention, a substantially monodispersed mixture of conjugates is provided where each conjugate includes salmon calcitonin covalently coupled at Lys11 of the salmon calcitonin to a carboxylic acid moiety of a first oligomer that comprises octanoic acid covalently coupled at the end distal to the carboxylic acid moiety to a methyl terminated polyethylene glycol moiety having at least 7 polyethylene glycol subunits, and covalently coupled at Lysls of the salmon calcitonin to a carboxylic acid moiety of a second oligomer that comprises, octanoic acid covalently coupled at the end distal to the carboxylic acid moiety to a methyl terminated polyethylene glycol moiety having at least 7 polyethylene glycol subunits.
According to still other embodiments of the present invention, a substantially monodispersed mixture of conjugates is provided where each conjugate comprises a calcitomn drug coupled to an oligomer comprising a polyethylene glycol moiety, and the mixture is capable of lowering serum calcium levels in a subject by at least 5 percent. According to yet other embodiments of the present invention, a substantially monodispersed mixture of conjugates is provided where each conjugate comprises a calcitonin drug coupled to an oligomer comprising a polyethylene glycol moiety, and the mixture has an increased resistance to degradation by chymotrypsin and/or trypsin when compared to the resistance to degradation by chymotrypsin and/or trypsin of the calcitonin drug which is not coupled to the oligomer.
According to other embodiments of the present invention, a substantially monodispersed mixture of conjugates is provided where each conjugate comprises a calcitonin drug coupled to an oligomer comprising a polyethylene glycol moiety, and the mixture has a higher bioefficacy than the bioeffϊcacy of the calcitonin 'drug which is not coupled to the oligomer.
According to still other embodiments of the present invention, a mixture of conjugates is provided where each conjugate includes a calcitonin drug coupled to an oligomer that comprises a polyethylene glycol moiety, and the mixture has a molecular weight distribution with a standard deviation of less than about 22 Daltons.
According to yet other embodiments of the present invention, a mixture of conjugates is provided where each conjugate includes a calcitonin drug coupled to an oligomer that comprises a polyethylene glycol moiety, and the mixture has a dispersity coefficient (DC) greater than 10,000 where
Figure imgf000005_0001
wherein: n is the number of different molecules in the sample; Νi is the number of i~ molecules in the sample; and Mj is the mass of the i~ molecule. According to other embodiments of the present invention, a mixture of conjugates is provided in which each conjugate includes a calcitonin drug coupled to an oligomer and has the same number of polyethylene glycol subunits.
According to still other embodiments of the present invention, a mixture of conjugates is provided in which each conjugate has the same molecular weight and has the formula: Calcitonin I ug^B-Lj—Gk-R-G'm-—R'—G'*n—T "1 (A)
L J p wherein:
B is a bonding moiety; L is a linking group; G, G and G" are individually selected spacer groups;
R is a lipophilic group and R' is a polyalkylene glycol group, or R' is the lipophilic group and R is the polyalkylene oxide group; T is a terminating group; j, k, m and n are individually 0 or 1 ; and p is an integer from 1 to the number of nucleophilic residues on the calcitonin drug.
Pharmaceutical compositions comprising conjugate mixtures of the present invention as well as methods of treating osteoporosis in a subject in need of such treatment by administering an effective amount of such pharmaceutical compositions are also provided. Additionally, methods of synthesizing such conjugate mixtures are provided. Calcitonin-oligomer conjugate mixtures according to embodiments of the present . invention may lower serum calcium levels by 20 percent or more. Moreover, such conjugates may provide decreased degradation by intestinal enzymes and/or provide increased bioavailability when compared to non-conjugated calcitonin.
Brief Description of the Drawings
Figure 1 illustrates a generic scheme for synthesizing a mixture of activated polymers comprising a polyethylene glycol moiety and a fatty acid moiety according to embodiments of the present invention;
Figure 2 illustrates a scheme for synthesizing a mixture of mPEG according to embodiments of the present invention;
Figure 3 illustrates a scheme for synthesizing a mixture of activated mPEG7-hexyl oligomers according to embodiments of the present invention;
Figure 4 illustrates a scheme for synthesizing a mixture of activated mPEG7-octyl oligomers according to embodiments of the present invention; Figure 5 illustrates a scheme for synthesizing a mixture of activated mPEG-decyl oligomers according to embodiments of the present invention; Figure 6 illustrates a scheme for synthesizing a mixture of activated stearate-PEG6 oligomers according to embodiments of the present invention;
Figure 7 illustrates a scheme for synthesizing a mixture of activated stearate-PEG8 oligomers according to embodiments of the present invention; Figure 8 illustrates a scheme for synthesizing a mixture of activated PEG3 oligomers according to embodiments of the present invention;
Figure 9 illustrates a scheme for synthesizing a mixture of activated palmitate-PEG3 oligomers according to embodiments of the present invention;
Figure 10 illustrates a scheme for synthesizing a mixture of activated PEG6 oligomers according to embodiments of the present invention;
Figure 11 illustrates a scheme for synthesizing various propylene glycol monomers according to embodiments of the present invention;
Figure 12 illustrates a scheme for synthesizing various propylene glycol polymers according to embodiments of the present invention; Figure 13 illustrates a scheme for synthesizing various propylene glycol polymers according to embodiments of the present invention;
Figure 14 illustrates a comparison of the average AUCs for various mixtures of calcitonin-oligomer conjugates according to embodiments of the present invention with non- conjugated calcitonin, which is provided for comparison purposes only and does not form part of the invention;
Figure 15 illustrates a dose-response curve for a mixture of mPEG7-octyl-calcitonin diconjugates according to embodiments of the present invention compared with a dose- response curve for calcitonin, which is provided for comparison purposes and is not a part of the present invention; Figure 16 illustrates a dose-response curve after oral administration of a mixture of mPEG7-octyl-calcitonin diconjugates according to embodiments of the present invention;
Figure 17 illustrates a dose-response curve after subcutaneous administration of a mixture of mPEG7-octyl-calcitonin diconjugates according to embodiments of the present invention; and Figure 18 illustrates a dose-response curve after subcutaneous administration of salmon calcitomn, which is provided for comparison purposes and is not part of the present invention. Detailed Description Of Preferred Embodiments
The invention will now be described with respect to preferred embodiments described herein. It should be appreciated however that these embodiments are for the purpose of illustrating the invention, and are not to be construed as limiting the scope of the invention as defined by the claims.
As used herein, the term "non-polydispersed" is used to describe a mixture of compounds having a dispersity that is in contrast to the polydispersed mixtures described in U.S. Patent No. 5,359,030 to Ekwuribe. As used herein, the term "substantially monodispersed" is used to describe a mixture of compounds wherein at least about 95 percent of the compounds in the mixture have the same molecular weight.
As used herein, the term "monodispersed" is used to describe a mixture of compounds wherein about 100 percent of the compounds in the mixture have the same molecular weight. As used herein, the term "substantially purely monodispersed" is used to describe a mixture of compounds wherein at least about 95 percent of the compounds in the mixture have the same molecular weight and have the same molecular structure. Thus, a substantially purely monodispersed mixture is a substantially monodispersed mixture, but a substantially monodispersed mixture is not necessarily a substantially purely monodispersed mixture. As used herein, the term "purely monodispersed" is used to describe a mixture of compounds wherein about 100 percent of the compounds in the mixture have the same molecular weight and have the same molecular structure. Thus, a purely monodispersed mixture is a monodispersed mixture, but a monodispersed mixture is not necessarily a purely monodispersed mixture. As used herein, the term "weight average molecular weight" is defined as the sum of the products of the weight fraction for a given molecule in the mixture times the mass of the molecule for each molecule in the mixture. The "weight average molecular weight" is represented by the symbol Mw-
As used herein, the term "number average molecular weight" is defined as the total weight of a mixture divided by the number of molecules in the mixture and is represented by the symbol Mn.
As used herein, the term "dispersity coefficient" (DC) is defined by the formula:
Figure imgf000009_0001
wherein: n is the number of different molecules in the sample; Nj is the number of i~ molecules in the sample; and Mj is the mass of the i- molecule.
As used herein, the term "intra-subject variability" means the variability in activity occurring within the same subject when the subject is administered the same dose of a drug or pharmaceutical composition at different times.
As used herein, the term "inter-subject variability" means the variability in activity between two or more subjects when each subject is admimstered the same dose of a given drug or pharmaceutical formulation.
As used herein, the term "bioefficacy" means the ability of a drug or drug conjugate to interact with one or more desired receptors in vivo.
As used herein, the term "calcitonin drug" means a drug possessing all or some of the biological activity of calcitonin.
As used herein, the term "calcitonin" means chicken calcitomn, eel calcitonin, human calcitonin, porcine calcitonin, rat calcitonin or salmon calcitonin provided by natural, synthetic, or genetically engineered sources.
As used herein, the term "calcitonin analog" means calcitonin wherein one or more of the amino acids have been replaced while retaining some or all of the activity of the calcitonin. The analog is described by noting the replacement amino acids with the position of the replacement as a superscript followed by a description of the calcitonin. For example, "Pro2 calcitonin, human" means that the glycine typically found at the 2 position of a human calcitonin molecule has been replaced with proline. Calcitonin analogs may be obtained by various means, as will be understood by those skilled in the art. For example, certain amino acids may be substituted for other amino acids in the calcitonin structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules. As the interactive capacity and nature of calcitonin defines its biological functional activity, certain amino acid sequence substitutions can be made in the amino acid sequence and nevertheless remain a polypeptide with like properties.
In making such substitutions, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a polypeptide is generally understood in the art. It is accepted that the . relative hydropathic character of the amino acid contributes to the secondary structure of the resultant polypeptide, which in turn defines the interaction of the polypeptide with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like. Each amino acid has been assigned a hydropathic index on the basis of its . hydrophobicity and charge characteristics as follows: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). As will be understood by those skilled in the art, certain amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a polypeptide with similar biological activity, i.e., still obtain a biological functionally equivalent polypeptide. In making such changes, the substitution of amino acids whose hydropathic indices are within ±2 of each other is preferred, those which are within ±1 of each other are particularly preferred, and those within ±0.5 of each other are even more ' particularly preferred.
It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. U.S. Patent 4,554,101 provides that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein. As detailed in U.S. Patent . 4,554, 101 , the following hydrophilicity values have been assigned to arnino acid residues: arginine (+3.0); lysine (±3.0); aspartate (+3.0 ± 1); glutamate (+3.0 ± 1); seine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ± 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). As is understood by those skilled in the art, an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent polypeptide. In such changes, the substitution of amino acids whose hydrophilicity values are within ±2 of each other is preferred, those which are within ±1 of each other are particularly preferred, and those within ±0.5 of each other are even more particularly preferred.
As outlined above, amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions (i.e., amino acids that may be interchanged without significantly altering the biological activity of the polypeptide) that take various of the foregoing characteristics into consideration are well known to those of skill in the art and include, for example: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
As used herein, the term "calcitonin fragment" means a segment of the amino acid sequence found in the calcitonin that retains some or all of the activity of the calcitonin.
As used herein, the term "calcitonin fragment analog" means a segment of the amino acid sequence found in the calcitonin molecule wherein one or more of the amino acids in the segment have been replace while retaining some or all of the activity of the calcitonin.
As used herein, the term "PEG" refers to straight or branched polyethylene glycol polymers, and includes the monomethylether of polyethylene glycol (mPEG). The terms "PEG subunit" and polyethylene glycol subunit refer to a single polyethylene glycol unit, i.e., — (CH2CH2O)— . As used herein, the term "lipophilic" means the ability to dissolve in lipids and/or the ability to penetrate, interact with and/or traverse biological membranes, and the term, "lipophilic moiety" or "lipophile" means a moiety which is lipophilic and/or which, when attached to another chemical entity, increases the lipophilicity of such chemical entity. Examples of lipophilic moieties include, but are not limited to, alkyls, fatty acids, esters of fatty acids, chplesteryl, adamantyl and the like.
As used herein, the term "lower alkyl" refers to substituted or unsubstituted alkyl moieties having frqm 1 to 5 carbon atoms.
As used herein, the term "higher alkyl" refers to substituted or unsubstituted alkyl moieties having 6 or more carbon atoms. In embodiments of the present invention, a substantially monodispersed mixture of calcitonin drug-oligomer conjugates is provided. Each calcitonin drug-oligomer conjugate in the monodispersed mixture includes a calcitonin drug coupled to an oligomer that comprises a polyethylene glycol moiety. Preferably, at least about 96, 97, 98 or 99 percent of the conjugates in the mixture have the same molecular weight. More preferably, the mixture is a monodispersed mixture. Even more preferably, the mixture is a substantially purely monodispersed mixture. Still more preferably, at least about 96, 97, 98 or 99 percent of the conjugates in the mixture have the same molecular weight and have the same molecular structure. Most preferably, the mixture is a purely monodispersed mixture.
The calcitonin drug is preferably calcitonin. More preferably, the calcitonin drug is salmon calcitonin. However, it is to be understood that the calcitonin drug may be selected from various calcitonin drugs known to those skilled in the art including, for example, calcitomn precursor peptides, calcitonin, calcitonin analogs, calcitonin fragments, and calcitonin fragment analogs. Calcitonin precursor peptides include, but are not limited to, katacalcin (PDN-21) (C-procalcitonin), and N-proCT (amino-terminal procalcitonin cleavage peptide), human. Calcitonin analogs may be provided by substitution of one or more amino acids in calcitonin as described above. Calcitonin fragments include, but are not limited to, calcitomn 1-7, human; and calcitonin 8-32, salmon. Calcitonin fragment analogs may be provided by substitution of one or more of the amino acids in a calcitonin fragment as described above.
The oligomer may be various oligomers comprising a polyethylene glycol moiety as will be understood by those skilled in the art. Preferably, the polyethylene glycol moiety of the oligomer has at least 2, 3 or 4 polyethylene glycol subunits. More preferably, the polyethylene glycol moiety has at least 5 or 6 polyethylene glycol subunits and, most preferably, the polyethylene glycol moiety has at least 7 polyethylene glycol subunits.
The oligomer may comprise one or more other moieties as will be understood by those skilled in the art including, but not limited to, additional hydrophilic moieties, lipophilic moieties, spacer moieties, linker moieties, and terminating moieties. The various moieties in the oligomer are covalently coupled to one another by either hydrolyzable or non- hydrolyzable bonds.
The oligomer may further comprise one or more additional hydrophilic moieties (i.e., moieties in addition to the polyethylene glycol moiety) including, but not limited to, sugars, polyalkylene oxides, and polyamine/PEG copolymers. As polyethylene glycol is a polyalkylene oxide, the additional hydrophilic moiety may be a polyethylene glycol moiety. Adjacent polyethylene glycol moieties will be considered to be the same moiety if they are coupled by an ether bond. For example, the moiety
— O-C2H4— O-C2H4— O-C2H4— O-C2H4— O-C2H4— O-C2H4 — is a single polyethylene glycol moiety having six polyethylene glycol subunits. If this moiety were the only hydrophilic moiety in the oligomer, the oligomer would not contain an additional hydrophilic moiety. Adjacent polyethylene glycol moieties will be considered to be different moieties if they are coupled by a bond other than an ether bond. For example, the moiety
O
II —O—C2H4—O—C2H4—O—C2H4—O—C2H4—C-O—C2H4—O—C2H4— is a polyethylene glycol moiety having four polyethylene glycol subunits and an additional hydrophilic moiety having two polyethylene glycol subunits. Preferably, oligomers according to embodiments of the present invention comprise a polyethylene glycol moiety and no additional hydrophilic moieties.
The oligomer may further comprise one or more lipophilic moieties as will be understood by those skilled in the art. The hpophilic moiety is preferably a saturated or unsaturated, linear or branched alkyl moiety or a saturated or unsaturated, linear or branched fatty acid moiety. When the lipophilic moiety is an alkyl moiety, it is preferably a linear, saturated or unsaturated alkyl moiety having 1 to 28 carbon atoms. More preferably, the alkyl moiety has 2 to 12 carbon atoms. When the lipophilic moiety is a fatty acid moiety, it is preferably a natural fatty acid moiety that is linear, saturated or unsaturated, having 2 to 18 carbon atoms. More preferably, the fatty acid moiety has 3 to 14 carbon atoms. Most preferably, the fatty acid moiety has at least 4, 5 or 6 carbon atoms. rhe oligomer may further comprise one or more spacer moieties as will be understood' by those skilled in the art. Spacer moieties may, for example, be used to separate a hydrophilic moiety from a Hpophilic moiety, to separate a lipophilic moiety or hydrophihc moiety from the calcitomn drug, to separate a first hydrophilic or lipophilic moiety from a second hydrophilic or lipophilic moiety, or to separate a hydrophilic moiety or lipophilic moiety from a linker moiety. Spacer moieties are preferably selected from the group consisting of sugar, cholesterol and glycerine moieties. The oligomer may further comprise one or more linker moieties that are used to couple the oligomer with the calcitonin drug as will be understood by those skilled in the art. Linker moieties are preferably selected from the group consisting of alkyl and fatty acid moieties.
The oligomer may further comprise one or more terminating moieties at the one or more ends of the oligomer which are not coupled to the calcitonin drug. The terminating moiety is preferably an alkyl or alkoxy moiety, and is more preferably a lower alkyl or lower alkoxy moiety. Most preferably, the terminating moiety is methyl or methoxy. While the terminating moiety is preferably an alkyl or alkoxy moiety, it is to be understood that the terminating moiety may be various moieties as will be understood by those skilled in the art including, but not limited to, sugars, cholesterol, alcohols, and fatty acids. The oligomer is preferably covalently coupled to the calcitonin drug. In some
. embodiments, the. calcitonin drug is coupled to the oligomer utilizing a hydrol zable bond (e.g., an ester or carbonate bond). A hydrolyzable coupling may provide a calcitomn drug- oligomer conjugate that acts as a prodrug. In certain instances, for example where the calcitonin drug-oligomer conjugate is inactive (i.e., the conjugate lacks the ability to affect the body through the calcitonin drug's primary mechanism of action), a hydrolyzable coupling may provide for a time-release or controlled-release effect, administering the calcitonin drug over a given time period as one or more oligomers are cleaved from their respective calcitomn drug-oligomer conjugates to provide the active drug. In other embodiments, the calcitonin drug is coupled to the oligomer utilizing a non-hydrolyzable bond (e.g., a carbamate, amide, or ether bond). Use of a non-hydrolyzable bond may be . preferable when it is desirable to allow the calcitonin drug-oligomer conjugate to circulate in the bloodstream for an extended period of time, preferably at least 2 hours. When the oligomer is covalently coupled to the calcitonin drug, the oligomer further comprises one or more bonding moieties that are used to covalently couple the oligomer with the calcitonin drug as will be understood by those skilled in the art. Bonding moieties are preferably selected from the group consisting of covalent bond(s), ester moieties, carbonate moieties, carbamate moieties, amide moieties and secondary amine moieties. More than one moiety on the oligomer may be covalently coupled to the calcitonin drug.
While the oligomer is preferably covalently coupled to the calcitonin drug, it is to be understood that the oligomer may be non-covalently coupled to the calcitonin drug to form a non-covalently conjugated calcitonin drug-oligomer complex. As will be understood by those skilled in the art, non-covalent couplings include, but are not limited to, hydrogen bonding, ionic bonding, Van der Waals bonding, and micellular or liposomal encapsulation. According to embodiments of the present invention, oligomers may be suitably constructed, modified and/or appropriately functionalized to impart the ability for non-covalent conjugation in a selected manner (e.g., to impart hydrogen bonding capability), as will be understood by those skilled in the art. According to other embodiments of present invention, oligomers maybe derivatized with various compounds including, but not limited to, amino acids, oligopeptides, peptides, bile acids, bile acid derivatives, fatty acids, fatty acid derivatives, salicylic acids, salicylic acid derivatives, aminosalicylic acids, and aminosalicylic acid derivatives. The resulting oligomers can non-covalently couple (complex) with drug molecules, pharmaceutical products, and/or pharmaceutical excipients. The resulting complexes preferably have balanced lipophilic and hydrophilic properties. According to still other embodiments of the present invention, oligomers may be derivatized with amine and/or alkyl amines. Under suitable acidic conditions, the resulting oligomers can form non- covalently conjugated complexes with drug molecules, pharmaceutical products and/or pharmaceutical excipients. The products resulting from such complexation preferably have balanced lipophilic and hydrophilic properties.
More than one oligomer (i.e., a plurality of oligomers) may be coupled to the calcitonin drug. The oligomers in the plurality are preferably the same. However, it is to be understood that the oligomers in the plurality may be different from one another, or, alternatively, some of the oligomers in the plurality may be the same and some may be different. When a plurality of oligomers are coupled to the calcitonin drug, it may be preferable to couple one or more of the oligomers to the calcitonin drug with hydrolyzable bonds and couple one or more of the oligomers to the calcitonin drug with non-hydrolyzable bonds. Alternatively, all of the bonds coupling the plurality of oligomers to the calcitonin drug may be hydrolyzable, but have varying degrees of hydrolyzability such that, for example, one or more of the oligomers is rapidly removed from the calcitonin drug by hydrolysis in the body and one or more of the oligomers is slowly removed from the - calcitonin drug by hydrolysis in the body.
The oligomer may be coupled to the calcitonin drug at various nucleophilic residues of the calcitonin drug including, but not limited to, nucleophilic hydroxyl functions and/or amino functions. When the calcitonin drug is a polypeptide, a nucleophilic hydroxyl function may be found, for example, at serine and/or tyrosine residues, and a nucleophilic amino function may be found, for example, at histidine and/or lysine residues, and/or at the one or more N-termini of the polypeptide. When an oligomer is coupled to the one or more N- terminus of the calcitonin polypeptide, the coupling preferably forms a secondary amine. When the calcitonin drug is salmon calcitonin, for example, the oligomer may be coupled to an amino functionality of the salmon calcitonin, including the amino functionality of Lys l, Lys and/or the N-terminus. While one or more oligomers may be coupled to the salmon calcitonin, a higher bioefficacy, such as improved serum calcium lowering ability, is observed for the di-conjugated salmon calcitonin where an oligomer is coupled to the amino functionalities of Lys11 and the Lys18. Substantially monodispersed mixtures of calcitonin drug-oligomer conjugates of the present invention may be synthesized by various methods. For example, a substantially monodispersed mixture of oligomers consisting of carboxylic acid and polyethylene glycol is synthesized by contacting a substantially monodispersed mixture of carboxylic acid with a substantially monodispersed mixture of polyethylene glycol under conditions sufficient to provide a substantially monodispersed mixture of oligomers. The oligomers of the substantially monodispersed mixture are then activated so that they are capable of reacting, with a calcitonin drug to provide a calcitonin drug-oligomer conjugate. One embodiment of a synthesis route for providing a substantially monodispersed. mixture of oligomers is illustrated in Figure 3 and described in Examples 11-18 hereinbelow. Another embodiment of a synthesis route for providing a substantially monodispersed mixture of oligomers is illustrated in Figure 4 and described in Examples 19-24 hereinbelow. Still another embodiment of a synthesis route for providing a substantially monodispersed mixture of oligomers is illustrated in Figure 5 and described in Examples 25-29 hereinbelow. Yet another embodiment of a synthesis route for providing a substantially monodispersed mixture of oligomers is illustrated in Figure 6 and described in Examples 30-31 hereinbelow. Another embodiment of a synthesis route for providing a substantially monodispersed mixture of oligomers is illustrated in Figure 7 and described in Examples 32-37 hereinbelow. Still another embodiment of a synthesis route for providing a substantially monodispersed mixture of oligomers is illustrated in Figure 8 and described in Example 38 hereinbelow. Yet another embodiment of a synthesis route forproviding a substantially monodispersed mixture of oligomers is illustrated in Figure 9 and described in Example 39 hereinbelow. Another embodiment of a synthesis route for providing a substantially monodispersed mixture of oligomers is illustrated in Figure 10 and described in Example 40 hereinbelow.
The substantially monodispersed mixture of activated oligomers maybe reacted with a substantially monodispersed mixture of calcitonin drugs under conditions sufficient to provide a mixture of calcitonin drug-oligomer conjugates. A preferred synthesis is described in Example 41 hereinbelow. As will be understood by those skilled in the art, the reaction conditions (e.g., selected molar ratios, solvent mixtures and/or pH) may be controlled such that the mixture of calcitonin drug-oligomer conjugates resulting from the reaction of the substantially monodispersed mixture of activated oligomers and the substantially monodispersed mixture of calcitonin drugs is a substantially monodispersed mixture. For example, conjugation at the amino functionality of lysine may be suppressed by maintaining the pH of the reaction solution below the pKa of lysine. Alternatively, the mixture of calcitonin drug-oligomer conjugates may be separated and isolated utilizing, for example, HPLC to provide a substantially monodispersed mixture of calcitonin drug-oligomer conjugates, for example mono-, di-, or tri-conjugates. The degree of conjugation (e.g., whether the isolated molecule is a mono-, di-, or tri-conjugate) of a particular isolated conjugate may be determined and/or verified utilizing various techniques as will be understood by those skilled in the art including, but not limited to, mass spectroscopy. The particular conjugate structure (e.g., whether the oligomer is at Lys11, Lys18 or the N-teiminus of a salmon calcitonin monoconjugate) may be determined and/or verified utilizing various techniques as will be understood by those skilled in the art including, but not limited to, sequence analysis, peptide mapping, selective enzymatic cleavage, and/or endopeptidase cleavage.
As will be understood by those skilled in the art, one or more of the reaction sites on the calcitonin drug may be blocked by, for example, reacting the calcitonin drug with a suitable blocking reagent such as N-tert-butoxycarbonyl (t-BOC), or N-(9- fluorenylmethoxycarbonyl) (N-FMOC). This process may be preferred, for example, when the calcitonin drug is a polypeptide and it is desired to form an unsaturated conjugate (i.e., a conjugate wherein not all nucleophilic residues are conjugated) having an oligomer at the N- terminus of the polypeptide. Following such blocking, the substantially monodispersed mixture of blocked calcitonin drugs may be reacted with the substantially monodispersed mixture of activated oligomers to provide a mixture of calcitomn drug-oligomer conjugates having oligomer(s) coupled to one or more nucleophilic residues and having blocking moieties coupled to other nucleophilic residues. After the conjugation reaction, the calcitonin drug-oligomer conjugates may be de-blocked as will be understood by those skilled in the art. If necessary, the mixture of calcitonin drug-oligomer conjugates may then be separated as described above to provide a substantially monodispersed mixture of calcitonin drug- oligomer conjugates. Alternatively, the mixture of calcitonin drug-oligomer conjugates may be separated prior to de-blocking.
Substantially monodispersed mixtures of calcitonin drug-oligomer conjugates according to embodiments of the present invention preferably have improved properties when compared with those of conventional mixtures. For example, a substantially monodispersed mixture of calcitonin-oligomer conjugates preferably is capable of lowering serum calcium levels by at least 5 percent. Preferably, the mixture of conjugates is capable of lowering serum calcium levels by at least 10, 11, 12, 13 or 14 percent. More preferably, the mixture of conjugates is capable of lowering serum calcium levels by at least 15, 16, 17, 18 or 19 percent, and, most preferably, the mixture of conjugates is capable of lowering serum calcium levels by at least 20 percent.
As another example, a substantially monodispersed mixture of calcitonin-oligomer conjugates preferably has an increased resistance to degradation by chymotrypsin and/or trypsin when compared to the resistance to degradation by chymotrypsin and/or trypsin, respectively, of the calcitonin drug which is not coupled to the oligomer. Resistance to chymotrypsin or trypsin corresponds to the percent remaining when the molecule to be tested is digested in the applicable enzyme using the procedure outlined in Example 51 below. Preferably, the resistance to degradation by chymotrypsin of the mixture of calcitonin drug- oligomer conjugates is about 10 percent greater than the resistance to degradation by chymotrypsin of the mixture of calcitonin drugs that is not conjugated with the oligomer. More preferably, the resistance to degradation by chymotrypsin of the mixture of calcitonin drug-oligomer conjugates is about 15 percent greater than the resistance to degradation by chymotrypsin of the mixture of calcitonin drug that is not conjugated with the oligomer, and, most preferably, the resistance to degradation by chymotrypsin of the mixture of calcitonin drug-oligomer conjugates is about 20 percent greater than the resistance to degradation by chymotrypsin of the mixture of calcitonin drug that is not conjugated with the oligomer.„ Preferably, the resistance to degradation by trypsin of the mixture of calcitonin drug-oligomer conjugates is about 10 percent greater than the resistance to degradation by trypsin of the mixture of calcitonin drug that is not conjugated with the oligomer. More preferably, the resistance to degradation by trypsin of the mixture of calcitonin drug-oligomer conjugates is about 20 percent greater than the resistance to degradation by trypsin of the mixture of calcitomn drug that is not conjugated with the oligomer, and, most preferably, the resistance to degradation by trypsin of the mixture of calcitonin drug-oligomer conjugates is about 30 percent greater than the resistance to degradation by trypsin of the mixture of calcitomn drug that is not conjugated with the oligomer.
As still another example, a substantially monodispersed mixture of calcitonin- oligomer conjugates preferably has a higher bioefficacy than the bioefficacy of the calcitonin drug which is not coupled to the oligomer. The bioefficacy of a particular compound corresponds to its area-under-the-curve (AUC) value. Preferably, the bioefficacy of the mixture is about 5 percent greater than the bioefficacy of the calcitonin drug which is not coupled to the oligomer. More preferably, the bioefficacy of the mixture is about 10 percent greater than the bioefficacy of the calcitonin drug which is not coupled to the oligomer.
As yet another example, a substantially monodispersed mixture of calcitonin-oligomer conjugates preferably has an in vivo activity that is greater than the in vivo activity of a polydispersed mixture of calcitonin drug-oligomer conjugates having the same number average molecular weight as the substantially monodispersed mixture. As will be understood by those skilled in the art, the number average molecular weight of a mixture may be measured by various methods including, but not limited to, size exclusion chromatography such as gel permeation chromatography as described, for example, in H.R. Allcock & F.W. Lampe, CONTEMPORARY POLYMER CHEMISTRY 394-402 (2d. ed., 1991).
As another example, a substantially monodispersed mixture of calcitonin-oligomer conjugates preferably has an in vitro activity that is greater than the in vitro activity of a polydispersed mixture of calcitonin drug-oligomer conjugates having the same number average molecular weight as the substantially monodispersed mixture. As will be understood by those skilled in the art, the number average molecular weight of a mixture may be measured by various methods including, but not limited to, size exclusion chromatography. As still another example, a substantially monodispersed mixture of calcitonin- oligomer conjugates preferably has an increased resistance to degradation by chymotrypsin and/or trypsin when compared to the resistance to degradation by chymotrypsin and/or trypsin of a polydispersed mixture of calcitonin drug-oligomer conjugates having the same number average molecular weight as the substantially monodispersed mixture. As will be understood by those skilled in the art, the number average molecular weight of a mixture may be measured by various methods including, but not limited to, size exclusion chromatography.
As yet another example, a substantially monodispersed mixture of calcitonin-oligomer conjugates preferably has an inter-subject variability that is less than the inter-subject variability of a polydispersed mixture of calcitonin drug-oligomer conjugates having the same number average molecular weight as the substantially monodispersed mixture. As will be understood by those skilled in the art, the number average molecular weight of a mixture may be measured by various methods including, but not limited to, size exclusion chromatography. The inter-subject variability may be measured by various methods, as will be understood by those skilled in the art. The inter-subject variability is preferably calculated as follows. The area under a dose response curve (AUC) (i.e., the area between the dose- response curve and a baseline value) is determined for each subject. The average AUC for all subjects is determined by summing the AUCs of each subject and dividing the sum by the number of subjects. The absolute value of the difference between the subject's AUC and the average AUC is then determined for each subject. The absolute values of the differences obtained are then summed to give a value that represents the inter-subject variability. Lower values represent lower inter-subject variabilities and higher values represent higher inter- subject variabilities.
Substantially monodispersed mixtures of calcitonin drug-oligomer conjugates according to embodiments of the present invention preferably have two or more of the above- described improved properties. More preferably, substantially monodispersed mixtures of calcitonin drug-oligomer conjugates according to embodiments of the present invention have three or more of the above-described improved properties. Most preferably, substantially monodispersed mixtures of calcitonin drug-oligomer conjugates according to embodiments of the present invention have four or more of the above-described improved properties.
In still other embodiments according to the present invention, a mixture of conjugates having a molecular weight distribution with a standard deviation of less than about 22
Daltons is provided. Each conjugate in the mixture includes a calcitonin drug coupled to an oligomer that comprises a polyethylene glycol moiety. The standard deviation is preferably less than about 14 Daltons and is more preferably less than about 11 Daltons. The molecular weight distribution may be determined by methods known to those skilled in the art including, but not limited to, size exclusion chromatography such as gel permeation chromatography as described, for example, in H.R. Allcock & F.W. Lampe, CONTEMPORARY POLYMER CHEMISTRY 394-402 (2d. ed., 1991). The standard deviation of the molecular weight distribution may then be determined by statistical methods as will be understood by those skilled in the art.
The calcitonin drug is preferably calcitonin. More preferably, the calcitonin drug is salmon calcitonin. However, it is to be understood that the calcitonin drug may be selected from various calcitonin drugs known to those skilled in the art including, for example, calcitonin precursor peptides, calcitonin, calcitonin analogs, calcitonin fragments, and calcitonin fragment analogs. Calcitonin precursor peptides include, but are not limited to, katacalcin (PDN-21) (C-procalcitonin), and N-proCT (ammo-terminal procalcitonin cleavage peptide), human. Calcitonin analogs may be provided by substitution of one or more amino acids in calcitonin as described above. Calcitonin fragments include, but are not limited to, calcitomn 1-7, human; and calcitonin 8-32, salmon. Calcitonin fragment analogs may be provided by substitution of one or more of the amino acids in a calcitonin fragment as described above.
The oligomer may be various oligomers comprising a polyethylene glycol moiety as will be understood by those skilled in the art. Preferably, the polyethylene glycol moiety of the oligomer has at least 2, 3 or 4 polyethylene glycol subunits. More preferably, the polyethylene glycol moiety has at least 5 or 6 polyethylene glycol subunits and, most preferably, the polyethylene glycol moiety has at least 7 polyethylene glycol subunits.
The oligomer may comprise one or more other moieties as will be understood by those skilled in the art including, but not limited to, additional hydrophilic. moieties, lipophilic moieties, spacer moieties, linker moieties, and terminating moieties. The various moieties in the oligomer are covalently coupled to one another by either hydrolyzable or non- hydrolyzable bonds.
The oligomer may further comprise one or more additional hydrophilic moieties (i.e., moieties in addition to the polyethylene glycol moiety) including, but not limited to, sugars, polyalkylene oxides, and polyamine/PEG copolymers. As polyethylene glycol is a ■ polyalkylene oxide, the additional hydrophilic moiety may be a polyethylene glycol moiety. Adjacent polyethylene glycol moieties will be considered to be the same moiety if they are coupled by an ether bond. For example, the moiety
— O-C2H4— O-C2H4— O-C2H4— O-C2H4— O-C2H4— O-C2H4 — is a single polyethylene glycol moiety having six polyethylene glycol subunits. If this moiety were the only hydrophilic moiety in the oligomer, the oligomer would not contain an additional hydrophilic moiety. Adjacent polyethylene glycol moieties will be considered to be different moieties if they are coupled by a bond other than an ether bond. For example, the moiety
O
II —O-C2H4—O—C2H4—O—C2H4—O—C2H4—C-O—C2H4—O—C2H4— is a polyethylene glycol moiety having four polyethylene glycol subunits and an additional hydrophilic moiety having two polyethylene glycol subunits. Preferably, oligomers according to embodiments of the present invention comprise a polyethylene glycol moiety and no additional hydrophilic moieties.
The oligomer may further comprise one or more lipophilic moieties as will be understood by those skilled in the art. The lipophilic moiety is preferably a saturated or unsaturated, linear or branched alkyl moiety or a saturated or unsaturated, linear or branched fatty acid moiety. When the lipophilic moiety is an alkyl moiety, it is preferably a linear, saturated or unsaturated alkyl moiety having 1 to 28 carbon atoms. More preferably, the alkyl moiety has 2 to 12 carbon atoms. When the lipophihc moiety is a fatty acid moiety, it is preferably a natural fatty acid moiety that is linear, saturated or unsaturated, having 2 to 18 carbon atoms. More preferably, the fatty acid moiety has 3 to 14 carbon atoms. Most preferably, the fatty acid moiety has at least 4, 5 or 6 carbon atoms.
The oligomer may further comprise one or more spacer moieties as will be understood by those skilled in the art. Spacer moieties may, for example, be used to separate a hydrophilic moiety from a lipophilic moiety, to separate a lipophihc moiety or hydrophilic moiety from the calcitomn drug, to separate a first hydrophilic or hpophilic moiety from a second hydrophilic or lipophilic moiety, or to separate a hydrophihc moiety or lipophilic moiety from a linker moiety. Spacer moieties are preferably selected from the group consisting of sugar, cholesterol and glycerine moieties. The oligomer may further comprise one or more linker moieties that are used to couple the oligomer with the calcitonin drug as will be understood by those skilled in the art. Linker moieties are preferably selected from the group consisting of alkyl and fatty acid moieties.
The oligomer may further comprise one or more terminating moieties at the one or more ends of the oligomer which are not coupled to the calcitonin drug. The terminating moiety is preferably an alkyl or alkoxy moiety, and is more preferably a lower alkyl or lower alkoxy moiety. Most preferably, the terminating moiety is methyl or methoxy. While the terminating moiety is preferably an alkyl or alkoxy moiety, it is to be understood that the terminating moiety may be various moieties as will be understood by those skilled in the art including, but not limited to, sugars, cholesterol, alcohols, and fatty acids. The oligomer is preferably covalently coupled to the calcitonin drug. In some embodiments, the calcitonin drug is coupled to the oligomer utilizing a hydrolyzable bond (e.g., an ester or carbonate bond). A hydrolyzable coupling may provide a calcitonin drug- oligomer conjugate that acts as a prodrug. In certain instances, for example where the calcitonin drug-oligomer conjugate is inactive (i.e., the conjugate lacks the ability to affect the. body through the calcitonin drug's primary mechanism of action), a hydrolyzable • coupling may provide for a time-release or controlled-release effect, administering the . calcitonin drug over a given time period as one or more oligomers are cleaved from their respective calcitonin drug-oligomer conjugates to provide the active drug. In other embodiments, the calcitonin drug is coupled to the oligomer utilizing a non-hydrolyzable bond (e.g., a carbamate, amide, or ether bond). Use of a non-hydrolyzable bond may be preferable when it is desirable to allow the calcitonin drug-oligomer conjugate to circulate in the bloodstream for an extended period of time, preferably at least 2 hours. When the oligomer is covalently coupled to the calcitonin drug, the oligomer further comprises .one or more bonding moieties that are used to covalently couple the oligomer with the calcitonin drug as will be understood by those skilled in the art. Bonding moieties are preferably selected from the group consisting of covalent bond(s), ester moieties, carbonate moieties, carbamate moieties, amide moieties and secondary amine moieties. More than one moiety on the oligomer may be covalently coupled to the calcitonin drug.
While the oligomer is preferably covalently coupled to the calcitonin drug, it is to be understood that the oligomer may be non-covalently coupled to the calcitonin drug to form a non-covalently conjugated calcitonin drug-oligomer complex. As will be. understood by those skilled in the art, non-covalent couplings include, but are not limited to, hydrogen bonding, ionic bonding, Van der Waals bonding, and micellular or liposomal encapsulation. According to embodiments of the present invention, oligomers may be suitably constructed, modified and/or appropriately functionalized to impart the ability for non-covalent conjugation in a selected manner (e.g., to impart hydrogen bonding capability), as will be understood by those skilled in the art. According to other embodiments of present invention, oligomers may be derivatized with various compounds including, but not limited to, amino acids, oligopeptides, peptides, bile acids, bile acid derivatives, fatty acids, fatty acid derivatives, salicylic acids, salicylic acid derivatives, aminosalicylic acids, and aminosalicylic acid derivatives. The resulting oligomers can non-covalently couple (complex) with drug molecules, pharmaceutical products, and/or pharmaceutical excipients. The resulting complexes preferably have balanced lipophilic and hydrophilic properties. According to still other embodiments of the present invention, oligomers may be derivatized with amine and/or alkyl amines. Under suitable acidic conditions, the resulting oligomers can form non- covalently conjugated complexes with drug molecules, pharmaceutical products and/or pharmaceutical excipients. The products resulting from such complexation preferably have balanced lipophilic and hydrophilic properties.
More than one oligomer (i.e., a plurality of oligomers) may be coupled to the calcitonin drug. The oligomers in the plurality are preferably the same. However, it is to be understood that the oligomers in the plurality may be different from one another, or, alternatively, some of the oligomers in the plurality may be the same and some may be different. When a plurality of oligomers are coupled to the calcitonin drug, it may be preferable to couple one or more of the oligomers to the calcitonin drug with hydrolyzable bonds and couple one or more of the oligomers to the calcitonin drug with non-hydrolyzable bonds. Alternatively, all of the bonds coupling the plurality of oligomers to the calcitonin drug may be hydrolyzable, but have varying degrees of hydrolyzability such that, for example, one or more of the oligomers is rapidly removed from the calcitonin drug by hydrolysis in the body and one or more of the oligomers is slowly removed from the calcitonin drug by hydrolysis in the body.
The oligomer may be coupled to the calcitonin drug at various nucleophilic residues of the calcitonin drug including, but not limited to, nucleophilic hydroxyl functions and/or amino functions. When the calcitonin drug is a polypeptide, a nucleophilic hydroxyl function may be found, for example, at serine and/or tyrosine residues, and a nucleophilic amino function may be found, for example, at histidine and/or lysine residues, and/or at the one or more N-termini of the polypeptide. When an oligomer is coupled to the one or more N- terminus of the calcitonin polypeptide, the coupling preferably forms a secondary amine. When the calcitonin drug is salmon calcitonin, for example, the oligomer may be coupled to an amino functionality of the salmon calcitonin, including the amino functionality of Lys1 !, Lys and/or the N-terminus. While one or more oligomers may be coupled to the salmon calcitonin, a higher bioefficacy, such as improved serum calcium lowering ability, is observed for the di-conjugated salmon calcitonin where an oligomer is coupled to the amino functionalities of Lys11 and the Lys18. Mixtures of calcitonin drug-oligomer conjugates having a molecular weight distribution with a standard deviation of less than about 22 Daltons may be synthesized by various methods. For example, a mixture of oligomers having a molecular weight distribution with a standard deviation of less than about 22 Daltons consisting of carboxylic acid and polyethylene glycol is synthesized by contacting a mixture of carboxylic acid having a molecular weight distribution with a standard deviation of less than about 22 Daltons with a mixture of polyethylene glycol having a molecular weight distribution with a standard deviation of less than about 22 Daltons under conditions sufficient to provide a mixture of oligomers having a molecular weight distribution with a standard deviation of less than about 22 Daltons. The oligomers of the mixture having a molecular weight distribution with a standard deviation of less than about 22 Daltons are then activated so that they are capable of - reacting with a calcitonin drug to provide a calcitonin drug-oligomer conjugate. One embodiment of a synthesis route for providing a mixture of activated oligomers having a molecular weight distribution with a standard deviation of less than about 22 Daltons is illustrated in Figure 3 and described in Examples 11-18 hereinbelow. Another embodiment . of a synthesis route for providing a mixture of activated. oligomers having a molecular weight distribution with a standard deviation of less than about 22 Daltons is illustrated in Figure 4 and described in Examples 19-24 hereinbelow. Still another embodiment of a synthesis route for providing a mixture of activated oligomers having a molecular weight distribution with a standard deviation of less than about 22 Daltons is illustrated in Figure 5 and described in Examples 25-29 hereinbelow. Yet another embodiment of a synthesis route for providing a mixture of activated oligomers having a molecular weight distribution with a standard deviation of less than about 22 Daltons is illustrated in Figure 6 and described in Examples 30-31 hereinbelow. Another embodiment of a synthesis route for providing a mixture of activated oligomers having a molecular weight distribution with a standard deviation of less than about 22 Daltons is illustrated in Figure 7 and described in Examples 32-37 hereinbelow. Still another embodiment of a synthesis route for providing a mixture of activated oligomers having a molecular weight distribution with a standard deviation of less than about 22 Daltons is illustrated in Figure 8 and described in Example 38 hereinbelow. Yet another embodiment of a synthesis route for providing a mixture of activated oligomers having a molecular weight distribution with a standard deviation of less than about 22 Daltons is illustrated in Figure 9 and described in Example 39 hereinbelow. Another embodiment of a synthesis route for providing a mixture of activated oligomers having a molecular weight distribution with a standard deviation of less than about 22 Daltons is illustrated in Figure 10 and described in Example 40 hereinbelow.
The mixture of activated oligomers having a molecular weight distribution with a standard deviation of less than about 22 Daltons is reacted with a mixture of calcitonin drugs having a molecular weight distribution with a standard deviation of less than about 22 Daltons under conditions sufficient to provide a mixture of calcitonin drug-oligomer conjugates. A preferred synthesis is described in Example 41 hereinbelow. As will be understood by those skilled in the art, the reaction conditions (e.g., selected molar ratios, solvent mixtures and/or pH) may be controlled such that the mixture of calcitonin drug- oligomer .conjugates resulting from the reaction of the mixture of activated oligomers having a molecular weight distribution with a standard deviation of less than about 22 Daltons and the mixture of calcitonin drugs having a molecular weight distribution with a standard deviation of less than about 22 Daltons is a mixture having a molecular weight distribution with a standard deviation of less than about 22 Daltons. For example, conjugation at the amino functionality of lysine may be suppressed by maintaining the pH of the reaction solution below the pKa of lysine. Alternatively, the mixture of calcitonin drug-oligomer conjugates may be separated and isolated utilizing, for example, HPLC to provide a mixture of calcitonin drug-oligomer conjugates, for example mono-, di-, or tri-conjugates, having a molecular weight distribution with a standard deviation of less than about 22 Daltons. The degree of conjugation (e.g., whether the isolated molecule is a mono-, di-, or tri-conjugate) of a particular isolated conjugate may be determined and/or verified utilizing various techniques as will be understood by those skilled in the art including, but not limited to, mass spectroscopy. The particular conjugate structure (e.g., whether the oligomer is at Lys11, Lys18 or the N-terminus of a salmon calcitonin monoconjugate) may be determined and/or verified utilizing various techniques as will be understood by those skilled in the art including, but not limited to, sequence analysis, peptide mapping, selective enzymatic cleavage, and/or endopeptidase cleavage.
As will be understood by those skilled in the art, one or more of the reaction sites on the calcitonin drug may be blocked by, for example, reacting the calcitonin drug with a suitable blocking reagent such as N-tert-butoxycarbonyl (t-BOC), or N-(9- fluorenylmethoxycarbonyl) (N-FMOC). This process may be preferred, for example, when the calcitonin drug is a polypeptide and it is desired to form an unsaturated conjugate (i.e., a conjugate wherein not all nucleophilic residues are conjugated) having an oligomer at the N- terminus of the polypeptide. Following such blocking, the mixture of blocked calcitonin drugs having a molecular weight distribution with a standard deviation of less than about 22 Daltons may be reacted with the mixture of activated oligomers having a molecular weight distribution with a standard deviation of less than about 22 Daltons to provide a mixture of calcitonin drug-oligomer conjugates having oligomer(s) coupled to one or more nucleophilic residues and having blocking moieties coupled to other nucleophilic residues. After the conjugation reaction, the calcitonin drug-oligomer conjugates may be de-blocked as will be understood by those skilled in the art. If necessary, the mixture of calcitonin drug-oligomer conjugates may then be separated as described above to provide a mixture of calcitonin drug- oligomer conjugates having a molecular weight distribution with a standard deviation of less than about 22 Daltons. Alternatively, the mixture of calcitonin drug-oligomer conjugates may be separated prior to de-blocking.
Mixtures of calcitonin drug-oligomer conjugates having a molecular weight distribution with a standard deviation of less than about 22 Daltons according to embodiments of the present invention preferably have improved properties when compared with those of conventional mixtures. For example, a mixture of calcitonin drug-oligomer conjugates having a molecular weight distribution with a standard deviation of less than about 22 Daltons preferably is capable of lowering serum calcium levels by at least 5 percent. Preferably, the mixture of conjugates is capable of lowering serum calcium levels by at least 10, 11, 12, 13 or 14 percent. More preferably, the mixture of conjugates is capable of lowering serum calcium levels by at least 15, 16, 17, 18 or 19 percent, and, most preferably, the mixture of conjugates is capable of lowering serum calcium levels by at least 20 percent.
As another example, a mixture of calcitomn drug-oligomer conjugates having a molecular weight distribution with a standard deviation of less than about 22 Daltons preferably has an increased resistance to degradation by chymotrypsin and/or trypsin when compared to the resistance to degradation by chymotrypsin and/or trypsin, respectively, of the calcitonin drug which is not coupled to the oligomer. Resistance to chymotrypsin or trypsin corresponds to the percent remaining when the molecule to be tested is digested in the applicable enzyme using a procedure similar to the one outlined in Example 51 below. Preferably, the resistance to degradation by chymotrypsin of the mixture of calcitonin drug- oligomer conjugates is about 10 percent greater than the resistance to degradation by chymotrypsin of the mixture of calcitonin drugs that is not conjugated with the oligomer. More preferably, the resistance to degradation by chymotrypsin of the mixture of calcitonin drug-oligomer conjugates is about 15 percent greater than the resistance to degradation by chymotrypsin of the mixture of calcitonin drug that is not conjugated with the oligomer, and, most preferably, the resistance to degradation by chymotrypsin of the mixture of calcitonin drug-oligomer conjugates is about 20 percent greater than the resistance to degradation by chymotrypsin of the mixture of calcitonin drug that is not conjugated with the oligomer. Preferably, the resistance to degradation by trypsin of the mixture of calcitonin drug-oligomer conjugates is about 10 percent greater than the resistance to degradation by trypsin of the mixture of calcitonin drug that is not conjugated with the oligomer. More preferably, the resistance to degradation by trypsin of the mixture of calcitonin drug-oligomer conjugates is about 20 percent greater than the resistance to degradation by trypsin of the mixture of calcitonin drug that is not conjugated with the oligomer, and, most preferably, the resistance to degradation by trypsin of the mixture of calcitonin drug-oligomer conjugates is about 30 percent greater than the resistance to degradation by trypsin of the mixture of. calcitonin drug that is not conjugated with the oligomer.
As still another example, a mixture of calcitonin drug-oligomer conjugates having a molecular weight distribution with a standard deviation of less than about 22 Daltons preferably has a higher bioefficacy than the bioefficacy of the calcitonin drug which is not coupled to the ohgomer. The bioefficacy of a particular compound corresponds to its area- under-the-curve (AUC) value. Preferably, the bioefficacy of the mixture is about 5 percent greater than the bioefficacy of the calcitonin drug which is not coupled to the oligomer. More preferably, the bioefficacy of the mixture is about 10 percent greater than the bioefficacy of the calcitonin drug which is not coupled to the oligomer.
As yet another example, a mixture of calcitonin drug-oligomer conjugates having a molecular weight distribution with a standard deviation of less than about 22 Daltons preferably has an in vivo activity that is greater than the in vivo activity of a polydispersed mixture of calcitonin drug-oligomer conjugates having the same number average molecular weight as the mixture of calcitonin drug-oligomer conjugates having a molecular weight distribution with a standard deviation of less than about 22 Daltons. As will be understood by those skilled in the art, the number average molecular weight of a mixture may be measured by various methods including, but not limited to, size exclusion chromatography such as gel permeation chromatography as described, for example, in H.R. Allcock & F.W. Lampe, CONTEMPORARY POLYMER CHEMISTRY 394-402 (2d. ed., 1991).
As another example, a mixture of calcitomn drug-oligomer conjugates having a molecular weight distribution with a standard deviation of less than about 22 Daltons preferably has an in vitro activity that is greater than the in vitro activity of a polydispersed mixture of calcitonin drug-oligomer conjugates having the same number average molecular weight as the mixture of calcitonin drug-oligomer conjugates having a molecular weight distribution with a standard deviation of less than about 22 Daltons. As will be understood by those skilled in the art, the number average molecular weight of a mixture may be measured by various methods including, but not limited to, size exclusion chromatography. As still another example, a mixture of calcitonin drug-oligomer conjugates having a molecular weight distribution with a standard deviation of less than about 22 Daltons preferably has an increased resistance to degradation by chymotrypsin and/or trypsin when compared to the resistance to degradation by chymotrypsin and/or trypsin of a polydispersed mixture of calcitonin drug-oligomer conjugates having the same number average molecular weight as the mixture of calcitonin drug-oligomer conjugates having a molecular weight distribution with a standard deviation of less than about 22 Daltons. As will be understood by those skilled in the art, the number average molecular weight of a mixture may be measured by various methods including, but hot limited to, size exclusion chromatography. As yet another example, a mixture of calcitonin drug-oligomer conjugates having a molecular weight distribution with a standard deviation of less than about 22 Daltons preferably has an inter-subject variability that is less than the inter-subject variability of a polydispersed mixture of calcitonin drug-oligomer conjugates having the same number average molecular weight as the mixture of calcitonin drug-oligomer conjugates having a molecular weight distribution with a standard deviation of less than about 22 Daltons. As will be understood by those skilled in the art, the number average' molecular weight of a mixture may be measured by various methods including, but not limited to, size exclusion chromatography. The inter-subject variability may be measured by various methods, as will be understood by those skilled in the art. The inter-subject variability is preferably calculated as follows. The area under a dose response curve (AUC) (i.e., the area between the dose- response curve and a baseline value) is determined for each subject. The average AUC for all subjects is determined by summing the AUCs of each subject and dividing the sum by the number of subjects. The absolute value of the difference between the subject's AUC and the average AUC is then determined for each subject. The absolute values of the differences obtained are then summed to give a value that represents the inter-subject variability. Lower values represent lower inter-subject variabilities and higher values represent higher inter- subject variabilities.
Mixtures of calcitonin drug-oligomer conjugates having a molecular weight distribution with a standard deviation of less than about 22 Daltons according to embodiments of the present invention preferably have two or more of the above-described improved properties. More preferably, mixtures of calcitonin drug-oligomer conjugates .having a molecular weight distribution with a standard deviation of less than about 22 Daltons according to embodiments of the present invention have three or more of the above- described improved properties. Most preferably, mixtures of calcitonin drug-oligomer conjugates having a molecular weight distribution with a standard deviation of less than about 22 Daltons according to embodiments of the present invention have four or more of the above-described improved properties.
According to yet other embodiments of the present invention, a mixture of conjugates is provided where each conjugate includes a calcitonin drug coupled to an oligomer comprising a polyethylene glycol moiety, and the mixture has a dispersity coefficient (DC) greater than 10,000 where
Figure imgf000031_0001
wherein: n is the number of different molecules in the sample; Nj is the number of i- molecules in the sample; and Mj is the mass of the i~ molecule.
The mixture of conjugates preferably has a dispersity coefficient greater than 100,000. More preferably, the dispersity coefficient of the conjugate mixture is greater than 500,000 and, most preferably, the dispersity coefficient is greater than 10,000,000. The variables n, Ni, and Mi may be determined by various methods as will be understood by those skilled in the art, including, but not limited to, methods described below in Example 49.
The calcitonin drug is preferably calcitonin. More preferably, the calcitonin drug is salmon calcitonin. However, it is to be understood that the calcitonin drug may be selected from various calcitonin drugs known to those skilled in the art including, for example, calcitonin precursor peptides, calcitonin, calcitonin analogs, calcitonin fragments, and calcitonin fragment analogs. Calcitonin precursor peptides include, but are not limited to, katacalcin (PDN-21) (C-procalcitonin), andN-proCT (ammo-terminal procalcitonin cleavage peptide), human. Calcitonin analogs may be provided by substitution of one or more amino acids in calcitonin as described above. Calcitonin fragments include, but are not limited to, calcitonin 1-7, human; and calcitonin 8-32, salmon. Calcitonin fragment analogs maybe provided by substitution of one or more of the amino acids in a calcitonin fragment as described above.
The oligomer may be various oligomers comprising a polyethylene glycol moiety as will be understood by those skilled in the art. Preferably, the polyethylene glycol moiety of the oligomer has at least 2, 3 or 4 polyethylene glycol subunits. More preferably, the polyethylene glycol moiety has at least 5 or 6 polyethylene glycol subunits and, most preferably, the polyethylene glycol moiety has at least 7 polyethylene glycol subunits.
The oligomer may comprise one or more other moieties as will be understood by those skilled in the art including, but not limited to, additional hydrophihc moieties, lipophilic moieties, spacer moieties, linker moieties, and terminating moieties. The various moieties in the ohgomer are covalently coupled to one another by either hydrolyzable or non- hydrolyzable bonds.
The oligomer may further comprise one or more additional hydrophihc moieties (i.e., moieties in addition to the polyethylene glycol moiety) including, but not limited to, sugars, polyalkylene oxides, and polyamine/PEG copolymers. As polyethylene glycol is a polyalkylene oxide, the additional hydrophilic rnoiety may be a polyethylene glycol moiety. Adjacent polyethylene glycol moieties will be considered to be the same moiety if they are coupled by an ether bond. For example, the moiety
— O-C2H4— O-C2H4— O-C2H4— O-C2H4— O-C2H4— O-C2H4 — is a single polyethylene glycol moiety having six polyethylene glycol subunits. If this moiety were the only hydrophilic moiety in the oligomer, the oligomer would not contain an additional hydrophilic moiety. Adjacent polyethylene glycol moieties will be considered to be different moieties if they are coupled by a bond other than an ether bond. For example, the moiety
O
II
-O-C2H4—O—C2H4—O—C2H4—O—C2H4—C-O—C2H4—O—C2H4 is a polyethylene glycol moiety having four polyethylene glycol subunits and an additional hydrophilic moiety having two polyethylene glycol subunits. Preferably, oligomers according to embodiments of the "present invention comprise a polyethylene glycol moiety and no additional hydrophilic moieties. The oligomer may further' comprise one or more lipophilic moieties as will be understood by those skilled in the art. The lipophihc moiety is preferably a saturated or unsaturated, linear or branched alkyl moiety or a saturated or unsaturated, linear or branched fatty acid moiety. When the lipophilic moiety is an alkyl moiety, it is preferably a linear, saturated or unsaturated alkyl moiety having 1 to 28 carbon atoms. More preferably, the alkyl moiety has 2 to 12 carbon atoms. When the lipophilic moiety is a fatty acid moiety, it is preferably a natural fatty acid moiety that is linear, saturated or unsaturated, having 2 to 18 carbon atoms. More preferably, the fatty acid moiety has 3 to 14 carbon atoms. Most preferably, the fatty acid moiety has at least 4, 5 or 6 carbon atoms.
The ohgomer may further comprise one or more spacer moieties as will be understood by those skilled in the art. Spacer moieties may, for example, be used to separate a hydrophilic moiety from a lipophilic moiety, to separate a lipophilic moiety or hydrophilic moiety from the calcitonin drug, to separate a first hydrophilic or lipophilic moiety from a second hydrophilic or lipophihc moiety, or to separate a hydrophilic moiety or lipophilic moiety from a linker moiety. Spacer moieties are preferably selected from the group consisting of sugar, cholesterol and glycerine moieties. The oligomer may further comprise one or more linker moieties that are used to couple the oligomer with the calcitonin drug as will be understood by those skilled in the art. Linker moieties are preferably selected from the group consisting of alkyl and fatty acid moieties.
The oligomer may further comprise one or more terminating moieties at the one or more ends of the oligomer which are not coupled to the calcitonin drug. The terminating moiety is preferably an alkyl or alkoxy moiety, and is more preferably a lower alkyl or lower alkoxy moiety. Most preferably, the terminating moiety is methyl or methoxy. While the terminating moiety is preferably an alkyl or alkoxy moiety, it is to be understood that the terminating moiety may be various moieties as will be understood by those skilled in the art • including, but not limited to, sugars, cholesterol, alcohols, and fatty acids.
The oligomer is preferably covalently coupled to the calcitonin drug. In some embodiments, the calcitonin drug is coupled to the oligomer utilizing a hydrolyzable bond (e.g., an ester or carbonate bond). A hydrolyzable coupling may provide a calcitonin drug- oligomer conjugate that acts as a prodrug. In certain instances, for example where the calcitonin drug-oligomer conjugate is inactive (i.e., the conjugate lacks the ability to affect the body through the calcitonin drug's primary mechanism of action), a hydrolyzable coupling may provide for a time-release, or controlled-release effect, administering the calcitonin drug over a given time period as one or more oligomers are cleaved from their respective calcitonin drug-oligomer conjugates to provide the active drug. In other embodiments, the calcitonin drug is coupled to the oligomer utilizing a non-hydrolyzable bond (e.g., a carbamate, amide, or ether bond). Use of a non-hydrolyzable bond may be preferable when it is desirable to allow the calcitonin drug-oligomer conjugate to circulate in the bloodstream for an extended period of time, preferably at least 2 hours. When the oligomer is covalently coupled to the calcitonin drug, the oligomer further comprises one or more bonding moieties that are used to covalently couple the oligomer with the calcitonin drug as will be understood by those skilled in the art. Bonding moieties are preferably selected from the group consisting of covalent bond(s), ester moieties, carbonate moieties, carbamate moieties, amide moieties and secondary amine moieties. More than one moiety on the oligomer may be covalently coupled to the calcitonin drug.
While the oligomer is preferably covalently coupled to the calcitonin drug, it is to be understood that the ohgomer may be non-covalently coupled to the calcitonin drug to form a non-covalently conjugated calcitonin drug-oligomer complex. As will be understood by ' those skilled in the art, non-covalent couplings include, but are not limited to, hydrogen bonding, ionic bonding, Van der Waals bonding, and micellular or liposomal encapsulation. According to embodiments of the present invention, oligomers may be suitably constructed, modified and/or appropriately functionalized to impart the ability for non-covalent conjugation in a selected manner (e.g., to impart hydrogen bonding capability), as will be understood by those skilled in the art. According to other embodiments of present invention, oligomers may be derivatized with various compounds including, but not limited to, amino acids, oligopeptides, peptides, bile acids, bile acid derivatives, fatty acids, fatty acid derivatives, salicylic acids, salicylic acid derivatives, aminosalicylic acids, and aminosalicylic acid derivatives. The resulting oligomers can non-covalently couple (complex) with drug molecules, pharmaceutical products, and or pharmaceutical excipients. The resulting complexes preferably have balanced lipophilic and hydrophilic properties. According to still other embodiments of the present invention, oligomers may be derivatized with amine and/or alkyl amines. Under, suitable acidic conditions, the resulting oligomers can form non- covalently conjugated complexes with drug molecules, pharmaceutical products and/or pharmaceutical excipients. The products resulting from such complexation preferably have balanced lipophilic and hydrophilic properties.
More than one oligomer (i.e., a plurality of oligomers) maybe coupled to the calcitonin drug. The oligomers in the plurality are preferably the same. However, it is to be understood that the oligomers in the plurality may be different from one another, or, alternatively, some of the oligomers in the plurality may be the same and some may be different. When a plurality of oligomers are coupled to the calcitonin drug, it may be preferable to couple one or more of the oligomers to the calcitonin drug with hydrolyzable bonds and couple one or more of the oligomers to the calcitonin drug with non-hydrolyzable bonds. Alternatively, all of the bonds coupling the plurahty of oligomers to the calcitonin drug may be hydrolyzable, but have varying degrees of hydrolyzability such that, for example, one or more of the oligomers is rapidly removed from the calcitonin drug by hydrolysis in the body and one or more of the oligomers is slowly removed from the calcitonin drug by hydrolysis in the body.
The oligomer may be coupled to the calcitonin drug at various nucleophilic residues of the calcitonin drug including, but not limited to, nucleophilic hydroxyl functions and/or amino functions. When the calcitonin drug is a polypeptide, a nucleophihc hydroxyl function may be found, for example, at serine and/or tyrosine residues, and a nucleophilic amino function may be found, for example, at histidine and/or lysine residues, and/or at the one or more N-termini of the polypeptide. When an'oligomer is coupled to the one or more N- terminus of the calcitonin polypeptide, the coupling preferably forms a secondary amine. When the calcitonin drug is salmon calcitonin, for example, the oligomer may be coupled to an amino functionality of the salmon calcitonin, including the amino functionality of Lys11, Lys18 and/or the N-terminus. While one or more oligomers may be coupled to the salmon calcitonin, a higher bioefficacy, such as improved serum calcium lowering ability, is observed for the di-conjugated salmon calcitonin where an oligomer is coupled to the amino functionalities of Lys11 and the Lys18.
Mixtures of calcitonin drug-oligomer conjugates having a dispersity coefficient greater than 10,000 may be synthesized by various methods. For example, a mixture of oligomers having a dispersity coefficient greater than 10,000 consisting of carboxylic acid and polyethylene glycol is synthesized by contacting a mixture of carboxylic acid having a dispersity coefficient greater than 10,000 with a mixture of polyethylene glycol having a dispersity coefficient greater than 10,000 under conditions sufficient to provide a mixture of oligomers having a dispersity coefficient greater than 10,000. The oligomers of the mixture having a dispersity coefficient greater than 10,000 are then activated so that they are capable of reacting with a calcitonin drug to provide a calcitonin drug-oligomer conjugate. One embodiment of a synthesis route for providing a mixture of activated oligomers having a dispersity coefficient greater than 10,000 is illustrated in Figure 3 and described in Examples 11-18 hereinbelow. Another embodiment of a synthesis route for providing a mixture of activated oligomers having a dispersity coefficient greater than 10,000 is illustrated in Figure 4 and described in Examples 19-24 hereinbelow. Still another embodiment of a synthesis route for providing a mixture of activated oligomers having a dispersity coefficient greater than 10,000 is illustrated in Figure 5 and described in Examples 25-29 hereinbelow. Yet another embodiment of a synthesis route for providing a mixture of activated oHgomers having a dispersity coefficient greater than 10,000 is illustrated in Figure 6 and described in Examples 30-31 hereinbelow. Another embodiment of a synthesis route for providing a mixture of activated oligomers having a dispersity coefficient greater than 10,000 is illustrated in Figure 7 and described in Examples 32-37 hereinbelow. Still another embodiment of a synthesis route for providing a mixture of activated oligomers having a dispersity coefficient greater than 10,000 is illustrated in Figure 8 and described in Example 38 hereinbelow. Yet another embodiment of a synthesis route for providing a mixture of activated oligomers having a dispersity coefficient greater than 10,000 is illustrated in Figure 9 and described in Example 39 hereinbelow. Another embodiment of a synthesis route for providing a mixture of activated oligomers having a dispersity coefficient greater than 10,000 is illustrated in Figure 10 and described in Example 40 hereinbelow.
The mixture of activated oligomers having a dispersity coefficient greater than 10,000 is reacted with a mixture of calcitonin drugs having a dispersity coefficient greater than 10,000 under conditions sufficient to provide a mixture of calcitonin drug-oligomer conjugates. A preferred synthesis is described in Example 41 hereinbelow. As will be understood by those skilled in the art, the reaction conditions (e.g., selected molar ratios, solvent mixtures and/or pH) may be controlled such that the mixture of calcitonin drug- oligomer conjugates resulting from the reaction of the mixture of activated oligomers having a dispersity coefficient greater than 10,000 and the mixture of calcitonin drugs having a dispersity coefficient greater than 10,000 is a mixture having a dispersity coefficient greater than 10,000. For example, conjugation at the amino functionality of lysine maybe suppressed by maintaining the pH of the reaction solution below the pKa of lysine. . Alternatively, the mixture of calcitonin drug-oligomer conjugates maybe separated and isolated utilizing, for example, HPLC to provide a mixture of calcitonin drug-oligomer conjugates, for example mono-, di-, or tri-conjugates, having a dispersity coefficient greater than 10,000. The degree of conjugation (e.g., whether the isolated molecule is a mono-, di-, or tri-conjugate) of a particular isolated conjugate may be determined and/or verified utilizing various techniques as will be understood by those skilled in the art including, but not limited to, mass spectroscopy. The particular conjugate structure (e.g., whether the oligomer is at Lys11, Lys18 or the N-terminus of a salmon calcitonin monoconjugate) may be determined and/or verified utilizing various techniques as will be understood by. those skilled in the art including, but not limited to, sequence analysis, peptide mapping, selective enzymatic cleavage, and/or endopeptidase cleavage.
As will be understood by those skilled in the art, one or more of the reaction sites on the calcitonin drug may be blocked by, for example, reacting the calcitonin drug with a suitable blocking reagent such as N-tert-butoxycarbonyl (t-BOC), or N-(9- fluorenylmethoxycarbonyl) (N-FMOC). This process may be preferred, for example, when the calcitonin drug is a polypeptide and it is desired to form an unsaturated conjugate (i.e., a conjugate wherein not all nucleophilic residues are conjugated) having an oligomer at the N- terminus of the polypeptide. Following such blocking, the mixture of blocked calcitomn drugs having a dispersity coefficient greater than 10,000 may be reacted with the mixture of activated oligomers having a dispersity coefficient greater than 10,000 to provide a mixture of calcitonin drug-oligomer conjugates having oligomer(s) coupled to one or more nucleophilic residues and having blocking moieties coupled to other nucleophilic residues. After the conjugation reaction, the calcitonin drug-oligomer conjugates may be de-blocked as will be understood by those skilled in the art. If necessary, the mixture of calcitonin drug- oligomer conjugates may then be separated as described above to provide a mixture of calcitonin drug-oligomer conjugates having a dispersity coefficient greater than 10,000. Alternatively, the mixture of calcitonin drug-oligomer conjugates may be separated prior to de-blocking. Mixtures of calcitonin drug-oligomer conjugates having a dispersity coefficient greater than 10,000 according to embodiments of the present invention preferably have improved properties when compared with those of conventional mixtures. For example, a mixture of calcitonin drug-oligomer conjugates having a dispersity coefficient greater than 10,000 preferably is capable of lowering serum calcium levels by at least 5 percent. Preferably, the mixture of conjugates is capable of lowering serum calcium levels by at least 10, 11, 12, 13 or 14 percent. More preferably, the mixture of conjugates is capable of lowering serum calcium levels by at least 15, 16, 17, 18 or 19 percent, and, most preferably, the mixture of conjugates is capable of lowering serum calcium levels by at least 20 percent. As another example, a mixture of calcitonin drug-oligomer conjugates having a dispersity coefficient greater than 10,000 preferably has an increased resistance to degradation by chymotrypsin and/or trypsin when compared to the resistance to degradation by chymotrypsin and/or trypsin, respectively, of the calcitonin drug which is not coupled to the oligomer. Resistance to chymotrypsin or trypsin corresponds to the percent remaining when the molecule to be tested is digested in the applicable enzyme using a procedure similar to the one outlined in Example 51 below. Preferably, the resistance to degradation by chymotrypsin of the mixture of calcitonin drug-oligomer conjugates is about 10 percent greater than the resistance to degradation by chymotrypsin of the mixture of calcitonin drugs that is not conjugated with the oligomer. More preferably, the resistance to degradation by chymotrypsin of the mixture of calcitonin drug-oligomer conjugates is about 15 percent greater than the resistance to degradation by chymotrypsin of the mixture of calcitonin drug that is not conjugated with the oligomer, and, most preferably, the resistance to degradation by chymotrypsin of the mixture of calcitomn drug-oligomer conjugates is about 20 percent greater than the resistance to degradation by chymotrypsin of the mixture of calcitonin drug that is not conjugated with the oligomer. Preferably, the resistance to degradation by trypsin of the mixture of calcitonin drug-oligomer conjugates is about 10 percent greater than the resistance to degradation by trypsin of the mixture of calcitonin drug that is not conjugated with the oligomer. More preferably, the resistance to degradation by trypsin of the mixture of calcitonin drug-oligomer conjugates is about 20 percent greater than the resistance to degradation by trypsin of the mixture of calcitonin drug that is not conjugated with the oligomer, and, most preferably, the resistance to degradation by trypsin of the mixture of calcitonin drug-oligomer conjugates is about 30 percent greater than the resistance to degradation by trypsin of the mixture of calcitonin drug that is not conjugated with the oligomer.
As still another example, a mixture of calcito in drug-oligomer conjugates having a dispersity coefficient greater than 10,000 preferably has a higher bioefficacy than the bioefficacy of the calcitonin drug which is not coupled to the oligomer. The bioefficacy of a particular compound corresponds to its area-under-the-curve (AUC) value. Preferably, the bioefficacy of the mixture is about 5 percent greater than the bioefficacy of the calcitonin drug which is not coupled to the oligomer. More preferably, the bioefficacy of the mixture is about 10 percent greater than the bioefficacy of the calcitonin drug which is not coupled to the oligomer. A yet another example, a mixture of calcitomn drug-oligomer conjugates having a dispersity coefficient greater than 10,000 preferably has an in vivo activity that is greater than the in vivo activity of a polydispersed mixture of calcitomn drug-oligomer conjugates having the same number average molecular weight as the mixture of calcitonin drug-oligomer conjugates having a dispersity coefficient greater than 10,000. As will be understood by those skilled in the art, the number average molecular weight of a mixture may be measured by various methods including, but not limited to, size exclusion chromatography such as gel permeation chromatography as described, for example, in H.R. Allcock & F.W. Lampe, CONTEMPORARY POLYMER CHEMISTRY 394-402 (2d. ed., 1991).
As another example, a mixture of calcitonin drug-oligomer conjugates having a dispersity coefficient greater than 10,000 preferably has an in vitro activity that is greater than the in vitro activity of a polydispersed mixture of calcitonin drug-oligomer conjugates having the same number average molecular weight as the mixture of calcitonin drug-oligomer conjugates having a dispersity coefficient greater than 10,000. As will be understood by those skilled in the art, the number average molecular weight of a mixture may be measured by various methods including, but not limited to, size exclusion chromatography.
As still another example, a mixture of calcitonin drug-oligomer conjugates having a dispersity coefficient greater than 10,000 preferably has an increased resistance to degradation by chymotrypsin and or trypsin when compared to the resistance to degradation by chymotrypsin and/or trypsin of a polydispersed mixture of calcitonin drug-oligomer conjugates having the same number average molecular weight as the mixture of calcitonin drug-oligomer conjugates having a dispersity coefficient greater than 10,000. As will be understood by those skilled in the art, the number average molecular weight of a mixture may be measured by various methods including, but not limited to, size exclusion chromatography.
As yet another example, a mixture of calcitonin drug-oligomer conjugates having a dispersity coefficient greater than 10,000 preferably has an inter-subject variability that is less than the inter-subject variability of a polydispersed mixture of calcitonin drug-oligomer conjugates having the same number average molecular weight as the mixture of calcitonin drug-oligomer conjugates having a dispersity coefficient greater than 10,000. As will be understood by those skilled in the art, the number average molecular weight of a mixture may be measured by various methods including, but not limited to, size exclusion chromatography. The inter-subject variability may be measured by various methods, as will be understood by those skilled in the art. The inter-subject variability is preferably calculated as follows. The area under a dose response curve (AUC) (i.e., the area between the dose- response curve and a baseline value) is deteimined for each subject. The average AUC for all subjects is determined by summing the AUCs of each subject and dividing the sum by the number of subjects. The absolute value of the difference between the subject's AUC and the average AUC is then determined for each subject. The absolute values of the differences obtained are then summed to give a value that represents the inter-subject variability. Lower values represent lower inter-subject variabilities and higher values represent higher inter- subject variabilities.
Mixtures of calcitonin drug-oligomer conjugates having a dispersity coefficient greater than 10,000 according to embodiments of the present invention preferably have two or more of the above-described improved properties. More preferably, mixtures of calcitonin drug-oligomer conjugates having a dispersity coefficient greater than 10,000 according to embodiments of the present invention have three or more of the above-described improved properties. Most preferably, mixtures of calcitonin drug-oligomer conjugates having a dispersity coefficient greater than 10,000 according to embodiments of the present invention have four or more of the above-described improved properties.
According to other embodiments of the present invention, a mixture of conjugates in which each conjugate includes a calcitomn drug coupled to ah oligomer and has the same number of polyethylene glycol subunits is provided.
The calcitonin drug is preferably calcitonin. More preferably, the calcitonin drug is salmon calcitonin. However, it is to be understood that the calcitonin drug may be selected from various calcitomn drugs known to those skilled in the art including, for example, calcitonin precursor peptides, calcitonin, calcitonin analogs, calcitonin fragments, and calcitonin fragment analogs. Calcitonin precursor peptides include, but are not limited to, katacalcin (PDN-21) (C-procalcitonin), and N-proCT (ammo-terminal procalcitonin cleavage peptide), human. Calcitonin analogs may be provided by substitution of one or more amino acids in calcitonin as described above. Calcitonin fragments include, but are not limited to, calcitonin 1-7, human; and calcitonin 8-32, salmon. Calcitonin fragment analogs may be provided by substitution of one or more of the amino acids in a calcitonin fragment as described above. The oligomer may be various oligomers comprising a polyethylene glycol moiety as will be understood by those skilled in the art. Preferably, the polyethylene glycol moiety of the oligomer has at least 2, 3 or 4 polyethylene glycol subunits. More preferably, the polyethylene glycol moiety has at least 5 or 6 polyethylene glycol subunits and, most preferably, the polyethylene glycol moiety has at least 7 polyethylene glycol subunits.
The oligomer may comprise one or more other moieties as will be understood by those skilled in the art including, but not limited to, additional hydrophilic moieties, lipophilic moieties, spacer moieties, linker moieties, and terminating moieties. The various moieties in the oligomer are covalently coupled to one another by either hydrolyzable or non- hydrolyzable bonds.
The oligomer may further comprise one or more additional hydrophilic moieties (i.e., moieties in addition to the polyethylene glycol moiety) including, but not limited to, sugars, polyalkylene oxides, and polyamine/PEG copolymers. As polyethylene glycol is a polyalkylene oxide, the additional hydrophilic moiety may be a polyethylene glycol moiety. Adjacent polyethylene glycol moieties will be considered to be the same moiety if they are coupled by an ether bond. For example, the moiety
— O-C2H4-O-C2H4-O-C2H4-O-C2H4-O-C2H4-O-C2H4 — is a single polyethylene glycol moiety having six polyethylene glycol subunits. If this moiety were the only hydrophilic moiety in the oligomer, the ohgomer would not contain an additional hydrophilic moiety. Adjacent polyethylene glycol moieties will be considered to be different moieties if they are coupled by a bond other than an ether bond. For example, the moiety
O
-O-C2H4— O— C2H4— O-C2H4— O— C2H4— C- O - C2H4— O-C2H4- is a polyethylene glycol moiety having four polyethylene glycol subunits and an additional hydrophilic moiety having two polyethylene glycol subunits. Preferably, oligomers according to embodiments of the present invention comprise a polyethylene glycol moiety and no additional hydrophilic moieties. The oligomer may further comprise one or more lipophilic moieties as will be understood by those skilled in the art. The lipophilic moiety is preferably a saturated or unsaturated, linear or branched alkyl moiety or a saturated or unsaturated, linear or branched fatty acid moiety. When the Hpophilic moiety is an alkyl moiety, it is preferably a linear, saturated or unsaturated alkyl moiety having 1 to 28 carbon atoms. More preferably, the alkyl moiety has 2 to 12 carbon atoms. When the lipophilic moiety is a fatty acid moiety, it is preferably a natural fatty acid moiety that is linear, saturated or unsaturated, having 2 to 18 carbon atoms. More preferably, the fatty acid moiety has 3 to 14 carbon atoms. Most preferably, the fatty acid moiety has at least 4, 5 or 6 carbon atoms.
The oligomer may further comprise one or more spacer moieties as will be understood by those skilled in the art. Spacer moieties may, for example, be used to separate a hydrophilic moiety from a lipophilic moiety, to separate a lipophilic moiety or hydrophilic moiety from the calcitonin drug, to separate a first hydrophilic or lipophilic moiety from a second hydrophilic or lipophilic moiety, or to separate a hydrophilic moiety or lipophilic moiety from a linker moiety. Spacer moieties are preferably selected from the group consisting of sugar,, cholesterol and glycerine moieties. The oligomer may further comprise one or more linker moieties that are used to couple the oligomer with the calcitonin drug as will be understood by those skilled in the art. Linker moieties are preferably selected from the group consisting of alkyl and fatty acid moieties.
The oligomer may further comprise one or more terminating moieties at the one or more ends of the oligomer which are not coupled to the calcitonin drug. The terminating moiety is preferably an alkyl or alkoxy moiety, and is more preferably a lower alkyl or lower alkoxy moiety. Most preferably, the terminating moiety is methyl or methoxy. While the terminating moiety is preferably an alkyl or alkoxy moiety, it is to be understood that the terminating moiety may be various moieties as will be understood by those skilled in the art including, but not limited to, sugars, cholesterol, alcohols, and fatty acids.
The oligomer is preferably covalently coupled to the calcitonin drug. In some embodiments, the calcitonin drug is coupled to the oligomer utilizing a hydrolyzable bond (e.g., an ester or carbonate bond). A hydrolyzable coupling may provide a calcitonin drug- oligomer conjugate that acts as a prodrug. In certain instances, for example where the calcitonin drug-oligomer conjugate is inactive (i.e., the conjugate lacks the abiHty to affect the body through the calcitonin drug's primary mechanism of action), a hydrolyzable coupling may provide for a time-release or controlled-release effect, administering the calcitonin drug over a given time period as one or more oligomers are cleaved from their respective calcitonin drug-oligomer conjugates to provide the active drug. In other embodiments, the calcitonin drug is coupled to the oligomer utilizing a non-hydrolyzable bond (e.g., a carbamate, amide, or ether bond). Use of a non-hydrolyzable bond may be preferable when it is desirable to allow the calcitonin drug-oligomer conjugate to circulate in the bloodstream for an extended period of time, preferably at least 2 hours. When the oligomer is covalently coupled to the calcitonin drug, the oligomer further comprises one or more bonding moieties that are. used to covalently couple the oligomer with the calcitonin drug as will be understood by those skilled in the art. Bonding moieties are preferably selected from the group consisting of covalent bond(s), ester moieties, carbonate moieties, carbamate moieties, amide moieties and secondary amine moieties. More than one moiety on the oligomer may be covalently coupled to the calcitonin drug.
While the oligomer is preferably covalently coupled to the calcitonin drug, it is to be understood that the oligomer may be non-covalently coupled to the calcitonin drug to form a non-covalently conjugated calcitonin drug-oligomer complex. As will be understood by those skilled in the art, non-covalent couplings include, but are not limited to, hydrogen bonding, ionic bonding, Van der Waals bonding, and micellular or liposomal encapsulation. According to embodiments of the present invention, oligomers may be suitably constructed, modified and/or appropriately functionalized to impart the ability for non-covalent conjugation in a selected manner (e.g., to impart hydrogen bonding capability), as will be understood by those skilled in the art. According to other embodiments of present invention, oligomers may be derivatized with various compounds including, but not limited to, amino acids, oligopeptides, peptides, bile acids, bile acid derivatives, fatty acids, fatty acid derivatives, salicylic acids, salicylic acid derivatives, aminosalicylic acids, and aminosalicylic acid derivatives. The resulting oHgomers can non-covalently couple (complex) with drug molecules, pharmaceutical products, and/or pharmaceutical excipients. The resulting complexes preferably have balanced lipophilic and hydrophilic properties. According to still other embodiments of the present invention, oligomers may be derivatized with amine and/or alkyl amines. Under suitable acidic conditions, the resulting oHgomers can form non- covalently conjugated complexes with drug molecules, pharmaceutical products -and/or pharmaceutical excipients. The products resulting from such complexation preferably have balanced lipophilic and hydrophilic properties.
More than one oligomer (i.e., a plurality of oligomers) may be coupled to the calcitonin drug. The oligomers in the plurality are preferably the same. However, it is to be understood that the oligomers in the plurality may be different from one another, or, alternatively, some of the oligomers in the plurality may be the same and some may be different. When a plurality of oligomers are coupled to the calcitonin drug, it may be preferable to couple one or more of the oligomers to the calcitonin drug with hydrolyzable bonds and couple one or more of the oligomers to the. calcitonin drug with non-hydrolyzable bonds. Alternatively, all of the bonds coupling the plurality of oHgomers to the calcitonin drug may be hydrolyzable, but have varying degrees of hydrolyzability such that, for example, one or more of the oligomers is rapidly removed from the calcitomn drug by hydrolysis in the body and one or more of the oligomers is slowly removed from the calcitonin drug by hydrolysis in the body.
The oligomer may be coupled to the calcitonin drug at various nucleophilic residues . of the calcitonin drug including, but not Hmited to, nucleophilic hydroxyl functions and/or amino functions. When the calcitonin drug is a polypeptide, a nucleophilic hydroxyl function may be found, for example, at serine and/or tyrosine residues, and a nucleophilic amino function may be found, for example, at histidine and/or lysine residues, and/or at the one or more N-termini of the polypeptide. When an oligomer is coupled to the one or more N- terminus of the calcitonin polypeptide, the coupling preferably forms a secondary amine. When the calcitonin drug is salmon calcitonin, for example, the oligomer may be coupled to an amino functionality of the salmon calcitomn, including the amino functionality of Lys11, Lys18 and/or the N-terminus. While one or more oligomers may be coupled to the salmon calcitonin, a higher bioefficacy, such as improved serum calcium lowering ability, is observed for the di-conjugated salmon calcitonin where an oligomer is coupled to the amino functionalities of Lys11 and the Lys18.
Mixtures of calcitonin drug-oligomer conjugates where each conjugate in the mixture has the same number of polyethylene glycol .subunits may be synthesized by various methods. For example, a mixture of oligomers consisting of carboxylic acid and polyethylene glycol where each oligomer in the mixture has the same number of polyethylene glycol subunits is synthesized by contacting a mixture of carboxylic acid with a mixture of polyethylene glycol where each polyethylene glycol molecule in the mixture has the same number of polyethylene glycol subunits under conditions sufficient to provide a mixture of oligomers where each oligomer in the mixture has the same number of polyethylene glycol subunits. The oligomers of the mixture where each oligomer in the mixture has the same number of polyethylene glycol subunits are then activated so that they are capable of reacting with a calcitonin drug to provide a calcitonin drug-oligomer conjugate. One embodiment of a synthesis route for providing a mixture of activated oligomers where each oligomer in the mixture has the same number of polyethylene glycol subunits is illustrated in Figure 3 and described in Examples 11-18 hereinbelow. Another embodiment of a synthesis route for providing a mixture of activated oligomers where each oligomer in the mixture has the same number of polyethylene glycol subunits is illustrated in Figure 4 and described in Examples 19-24 hereinbelow. Still another embodiment of a synthesis route for providing a mixture of activated oligomers where each oligomer in the mixture has the same number of polyethylene glycol subunits is illustrated in Figure 5 and described in Examples 25-29 hereinbelow. Yet another embodiment of a synthesis route for providing a mixture of activated oligomers where each oligomer in the mixture has the same number of polyethylene glycol subunits is illustrated in Figure 6 and described in Examples 30-31 hereinbelow. Another embodiment of a synthesis route for providing a mixture of activated oligomers where each oligomer in the mixture has the same number of polyethylene glycol subunits is illustrated in Figure 7 and described in Examples 32-37 hereinbelow. Still another embodiment of a synthesis route for providing a mixture of activated oligomers where each oligomer in the mixture has the same number of polyethylene glycol subunits is illustrated in Figure 8 and described in Example 38 hereinbelow. Yet another embodiment of a synthesis route for providing a mixture of activated oligomers where each oligomer in the mixture has the same number of polyethylene glycol subunits is illustrated in Figure 9 and described in Example 39 hereinbelow. Another embodiment of a synthesis route for providing a mixture of activated oligomers having a mixture of activated oligomers where each oligomer in the mixture has the same number of polyethylene glycol subunits is illustrated in Figure 10 and described in Example 40 hereinbelow.
The mixture of activated oligomers where each oligomer in the mixture has the same number of polyethylene glycol subunits is reacted with a mixture of calcitonin drugs under conditions sufficient to provide a mixture of calcitomn drug-oligomer conjugates. A preferred synthesis is described in Example 41 hereinbelow. As will be understood by those skilled in the art, the reaction conditions (e.g., selected molar ratios, solvent mixtures and/or pH) may be controlled such that the mixture of calcitonin drug-oligomer conjugates resulting from the reaction of the mixture of activated oHgomers where each oligomer in the mixture has the same number of polyethylene glycol subunits and the mixture of calcitonin drugs is a mixture of conjugates where each conjugate in the mixture has the same number of polyethylene glycol subunits. For example, conjugation at the amino functionality of lysine may be suppressed by mamtaining the pH of the reaction solution below the pKa of lysine. Alternatively, the mixture of calcitomn drug-oligomer conjugates may be separated and isolated utilizing, for example, HPLC to provide a mixture of calcitonin drug-oligomer conjugates, for example mono-, di-, or tri-conjugates, where each conjugate in the mixture has the same number of polyethylene glycol subunits. The degree of conjugation (e.g., whether the isolated molecule is a mono-, di-, or tri-conjugate) of a particular isolated conjugate may be determined and/or verified utilizing various techniques as will be understood by those skilled in the art including, but not limited to, mass spectroscopy. The . particular conjugate structure (e.g., whether the oligomer is at Lys11, Lys18 or the N-terminus of a salmon calcitonin monoconjugate) may be determined and/or verified utilizing various techniques as will be understood by those skilled in the art including, but not limited to, sequence analysis, peptide mapping, selective enzymatic cleavage, and/or endopeptidase cleavage.
As will be understood by those skilled in the art, one or more of the reaction sites on the calcitonin drug may be blocked by, for example, reacting the calcitonin drug with a suitable blocking reagent such as N-tert-butoxycarbonyl (t-BOC), or N-(9- fluorenylmethoxycarbonyl) (N-FMOC). This process may be preferred, for example, when the calcitonin drug is a polypeptide and it is desired to form an unsaturated conjugate (i.e., a conjugate wherein not all nucleophilic residues are conjugated) having an oligomer at the N- terminus of the polypeptide. Following such blocking, the mixture of blocked calcitonin drugs may be reacted with the mixture of activated oligomers where each oligomer in the mixture has the same number of polyethylene glycol subunits to provide a mixture of calcitomn drug-ohgomer conjugates having oligomer(s) coupled to one or more nucleophilic residues and having blocking moieties coupled to other nucleophilic residues. After the conjugation reaction, the calcitonin drug-oligomer conjugates may be de-blocked as will be understood by those skilled in the art. If necessary, the mixture of calcitomn drug-oligomer conjugates may then be separated as described above to provide a mixture of calcitonin drug- oligomer conjugates where each conjugate in the mixture has the same number of polyethylene glycol subunits. Alternatively, the mixture of calcitonin drug-oligomer conjugates may be separated prior to de-blocking.
Mixtures of calcitonin drug-oligomer conjugates where each conjugate in the mixture has the same number of polyethylene glycol subunits according to embodiments of the present invention preferably have improved properties when compared with those of conventional mixtures. For example, a mixture of calcitonin drug-oligomer conjugates where each conjugate in the mixture has the same number of polyethylene glycol subunits preferably is capable of lowering serum calcium levels by at least 5 percent. Preferably, the mixture of conjugates is capable of lowering serum calcium levels by at least 10, 11, 12, 13 or 14 percent. More preferably, the mixture of conjugates is capable of lowering serum calcium levels by at least 15, 16, 17, 18 or 19 percent, and, most preferably, the mixture of conjugates is capable of lowering serum calcium levels by at least 20 percent.
As anther example, a mixture of calcitonin drug-oligomer conjugates where each conjugate in the mixture has the same number of polyethylene glycol subunits preferably has an increased resistance to degradation by chymotrypsin and/or trypsin when compared to the resistance to degradation by chymotrypsin and/or trypsin, respectively, of the calcitonin drug which is not coupled to the oligomer. Resistance to chymotrypsin or trypsin corresponds to the percent remaining when the molecule to be tested is digested in the applicable enzyme using a procedure similar to the one outlined in Example 51 below. Preferably, the resistance to degradation by chymotrypsin of the mixture of calcitonin drug-oligomer conjugates is about 10 percent greater than the resistance to degradation by chymotrypsin of the mixture of calcitonin drugs that is not conjugated with the oligomer. More preferably, the resistance to degradation by chymotrypsin of the mixture of calcitonin drug-oligomer conjugates is about 15 percent greater than the resistance to degradation by chymotrypsin of the mixture of calcitonin drug that is not conjugated with the oligomer, and, most preferably, the resistance to degradation by chymotrypsin of the mixture of calcitonin drug-oligomer conjugates is about 20 percent greater than the resistance to degradation by chymotrypsin of the mixture of calcitonin drug that is not conjugated with the oligomer. Preferably, the resistance to degradation by trypsin of the mixture of calcitonin drug-oligomer conjugates is about 10 percent greater than the resistance to degradation by trypsin of the mixture of calcitonin drug that is not conjugated with the oligomer. More preferably, the resistance to degradation by trypsin of the mixture of calcitonin drug-oligomer conjugates is about 20 percent greater than the resistance to degradation by trypsin of the mixture of calcitonin drug that is not conjugated with the oligomer, and, most preferably, the resistance to degradation by trypsin of the mixture of calcitomn drug-oligomer conjugates is about 30 percent greater than the resistance to degradation by trypsin of the mixture of calcitonin drug that is not conjugated with the oligomer.
As still another example, a mixture of calcitonin drug-oligomer conjugates where each conjugate in the mixture has the same number of polyethylene glycol subunits preferably has a higher bioefficacy than the bioefficacy of the calcitonin drug which is not coupled to the ohgomer. The bioefficacy of a particular compound corresponds to its area- . under-the-curve (AUC) value. Preferably, the bioefficacy of the mixture is about 5 percent greater than the bioefficacy of the calcitonin drug which is not coupled to the oligomer. More preferably, the bioefficacy of the mixture is about 10 percent greater than the bioefficacy of the calcitonin drug which is not coupled to the oligomer.
As yet another example, a mixture of calcitonin drug-oligomer conjugates where. each conjugate in the mixture has the same number of polyethylene glycol subunits preferably has an in vivo activity that is greater than the in vivo activity of a polydispersed mixture of calcitonin drug-oligomer conjugates having the same number average molecular weight as the mixture of calcitonin drug-oligomer conjugates where each conjugate in the mixture has the same number of polyethylene glycol subunits. As will be understood by those skilled in the art, the number average molecular weight of a mixture may be measured by various methods including, but not limited to, size exclusion chromatography such as gel permeation chromatography as described, for example, in H.R. Allcock & F.W. Lampe, CONTEMPORARY POLYMER CHEMISTRY 394-402 (2d. ed., 1991).
As another example, a mixture of calcitonin drug-oligomer conjugates where each conjugate in the mixture has the same number of polyethylene glycol subunits preferably has an in vitro activity that is greater than the in vitro activity of a polydispersed mixture of calcitonin drug-oligomer conjugates having the same number average molecular weight as the mixture of calcitonin drug-oligomer conjugates where each conjugate in the mixture has the same number of polyethylene glycol subunits. As will be understood by those skilled in the art, the number average molecular weight of a mixture may be measured by various methods including, but not limited to, size exclusion chromatography.
As still another example, a mixture of calcitonin drug-oligomer conjugates where each conjugate in the mixture has the same number of polyethylene glycol subunits preferably has an increased resistance to degradation by chymotrypsin and/or trypsin when . compared to the resistance to degradation by chymotrypsin and/or trypsin of a polydispersed mixture of calcitonin drug-oligomer conjugates having the same number average molecular weight as the mixture of calcitonin drug-oligomer conjugates where each conjugate in the mixture has the same number of polyethylene glycol subunits. As will be understood by those skilled in the art, the number average molecular weight of a mixture may be measured . by various methods including, but not limited to, size exclusion chromatography.
As yet another example, a mixture of calcitonin drug-oligomer conjugates where each conjugate in the mixture has the same number of polyethylene glycol subunits preferably has an inter-subject variability that is less than the inter-subject variability of a polydispersed mixture of calcitonin drug-oligomer conjugates having the same number average molecular weight as the mixture of calcitonin drug-oligomer conjugates where each conjugate in the mixture has the same number of polyethylene glycol subunits. As will be understood by those skilled in the art, the number average molecular weight of a mixture may be measured ' by various methods including, but not limited to, size exclusion chromatography. The inter- subject variability may be measured by various methods, as will be understood by those skilled in the art. The inter-subject variability is preferably calculated as follows. The area under a dose response curve (AUC) (i.e., the area between the dose-response curve and a baseline value) is determined for each subject. The average AUC for all subjects is determined by summing the AUCs of each subject and dividing the sum by the number of subjects. The absolute value of the difference between the subject's AUC and the average AUC is then determined for each subject. The absolute values of the differences obtained are then summed to give a value that represents the inter-subject variability. Lower values represent lower inter-subject variabilities and higher values represent higher inter-subject variabilities.
Mixtures of calcitonin drug-oligomer conjugates where each conjugate in the mixture has the same number of polyethylene glycol subunits according to embodiments of the present invention preferably have two or more of the above-described improved properties. More preferably, mixtures of calcitonin drug-oligomer conjugates where each conjugate in - the mixture has the same number of polyethylene glycol subunits according to embodiments of the present invention have three or more of the above-described improved properties. Most preferably, mixtures of calcitonin- drug-oligomer conjugates where each conjugate in the mixture has the same number of polyethylene glycol subunits according to embodiments of the present invention have four or more of the above-described improved properties. . According to still other embodiments of the present invention, a mixture of conjugates is provided in which each conjugate has the same molecular weight and has the structure of Formula A:
Calcitomn r ug--B-Lj—Gk-R-G'm—R'—G"n-—T "1 (A)
L J p wherein:
B is a bonding moiety; L is a linker moiety;
G, G' and G" are individually selected spacer moieties;
R is a lipophilic moiety and R' is a polyalkylene glycol moiety, or R' is the lipophilic moiety and R is the polyalkylene glycol moiety; T is a terminating moiety; j, k, m and n are individually 0 or 1; and p is an integer from 1 to the number of nucleophilic residues on the calcitonin drug. The calcitonin drug is preferably calcitonin. More preferably, the calcitonin drug is salmon calcitonin. However, it is to be understood that the calcitonin drag may be selected from various calcitonin drugs known to those skilled in the art including, for example, calcito in precursor peptides, calcitonin, calcitonin analogs, calcitonin fragments, and calcitonin fragment analogs. Calcitonin precursor peptides include, but are not limited to, katacalcin (PDN-21) (C-procalcitonin), andN-proCT (ammo-terminal procalcitonin cleavage peptide), human. Calcitonin analogs may be provided by substitution of one or more amino acids in calcitonin as described above. Calcitonin fragments include, but are not limited to, calcitonin 1-7, human; and calcitonin 8-32, salmon. Calcitonin fragment analogs may be provided by substitution of one or more of the amino acids in a calcitonin fragment as described above. According to these embodiments of the present invention, the polyalkylene glycol moiety of the oligomer preferably has at least 2, 3 or 4 polyalkylene glycol subunits. More preferably, the polyalkylene glycol moiety has at least 5 or 6 polyalkylene glycol subunits and, most preferably, the polyethylene glycol moiety has at least 7 polyalkylene glycol subunits. The polyalkylene glycol moiety is preferably a lower polyalkylene glycol moiety such as a polyethylene glycol moiety, a polypropylene glycol moiety, or a polybutylene glycol moiety. More preferably, the polyalkylene glycol moiety is a polyethylene glycol moiety or a polypropylene glycol moiety. Most preferably, the polyalkylene glycol moiety is a polyethylene glycol moiety. When the polyalkylene glycol moiety is a polypropylene glycol moiety, the moiety preferably has a uniform (i.e., not random) structure. An exemplary polypropylene glycol moiety having a uniform structure is as follows:
— O-CH-CH2— O-CH-CH2— O-CH-CH2-O-CH-CH2— O — I I 1 I
CH3 CH3 CH3 CH3
This uniform polypropylene glycol structure may be described as having only one methyl substituted carbon atom adjacent each oxygen atom in the polypropylene glycol chain. Such uniform polypropylene glycol moieties may exhibit both lipophilic and hydrophilic characteristics and thus be useful in providing amphiphilic calcitonin drug-oligomer conjugates without the use of lipophilic polymer moieties. Furthermore, coupling the secondary alcohol moiety of the polypropylene glycol moiety with a calcitonin drug may provide the calcitonin drug (e.g., salmon calcitonin) with improved resistance to degradation caused by enzymes such as trypsin and chymotrypsin found, for example, in the gut.
Uniform polypropylene glycol according to embodiments of the present invention is preferably synthesized as illustrated in Figures 11 through 13, which will now be described. As illustrated in Figure 11, 1,2-propanediol 53 is reacted with a primary alcohol blocking reagent to provide a secondary alcohol extension monomer 54. The primary alcohol blocking reagent may be various primary alcohol blocking reagents as will be understood by those skilled in the art including, but not limited to, silylchloride compounds such as t- butyldiphenylsilylchloride and t-butyldimethylsilylchloride, and esterification reagents such as Ac2O. Preferably, the primary alcohol blocking reagent is a primary alcohol blocking reagent that is substantially non-reactive with secondary alcohols, such as t- butyldiphenylsilylchloride or t-butyldimethylsilylchloride. The secondary alcohol extension monomer (54) maybe reacted with methanesulfonyl chloride (MeSO2Cl) to provide a primary extension alcohol monomer mesylate 55.
Alternatively, the secondary alcohol extension monomer 54 may be reacted with a secondary alcohol blocking reagent to provide compound 56. The secondary alcohol blocking reagent may be various secondary alcohol blocking reagents as will be understood by those skilled in the art including, but not limited to, benzyl chloride. The compound 56 may be reacted with a Bi de-blocking reagent to remove the blocking moiety Bi and provide a primary alcohol extension monomer 57. The Bi de-blocking reagent may be selected from various de-blocking reagents as will be understood by one skilled in the art. When the primary alcohol has been blocked by forming an ester, the BΪ de-blocking reagent is a de- esterifϊcation reagent, such as a base (e.g., potassium carbonate). When the primary alcohol has been blocked using a silylchloride, the
Figure imgf000052_0001
de-blocking reagent is preferably tetrabutylammonium fluoride (TBAF). The primary alcohol extension monomer 57 may be reacted with methane sulfonyl chloride to provide a secondary alcohol extension monomer mesylate 58.
The primary alcohol extension monomer 54 and the secondary alcohol extension monomer 57 may be capped as follows. The secondary alcohol extension monomer 54 may be reacted with a capping reagent to provide a compound 59. The capping reagent may be various capping reagents as will be understood by those skilled in the art including, but not limited to, alkyl halides such as methyl chloride. The compound 59 may be reacted with a B\ de-blocking agent as described above to provide a primary alcohol capping monomer 60. The primary alcohol capping monomer 60 may be reacted with methane sulfonyl chloride to provide the secondary alcohol capping monomer mesylate 61. The primary alcohol extension monomer 57 may be reacted with a capping reagent to provide a compound 62. The capping reagent maybe various capping reagents as described above. The compound 62 may be reacted with a B2 de-blocking reagent to- remove the blocking moiety B2 and provide a . secondary alcohol capping monomer 63. The B de-blocking reagent may be various de- blocking agents as will be understood by those skilled in the art including, but not limited to, H2 in the presence of a palladium/activated carbon catalyst. The secondary alcohol capping monomer may be reacted with methanesulfonyl chloride to provide a.primary alcohol capping monomer mesylate 64. While the embodiments illustrated in Figure 11 show the . synthesis of capping monomers, it is to be understood that similar reactions may be performed to provide capping polymers.
In general, chain extensions may be effected by reacting a primary alcohol extension mono- or poly-mer such as the primary alcohol extension monomer 57 with a primary alcohol extension mono- or poly-mer mesylate such as the primary alcohol extension monomer mesylate 55 to provide various uniform polypropylene chains or by reacting a secondary alcohol extension mono- or poly-mer such as the secondary alcohol extension monomer 54 with a secondary alcohol extension mono-or poly-mer mesylate such as the secondary alcohol extension monomermesylate 58. For example, in Figure 13, the primary alcohol extension monomer mesylate 55 is reacted with the primary alcohol extension monomer 57 to provide a dimer compound 65. Alternatively, the secondary alcohol extension monomer mesylate 58 may be reacted with the secondary alcohol extension monomer 54 to provide the dimer compound 65. The Bi blocking moiety on the dimer compound 65 may be removed using a B! de-blocking reagent as described above to provide a primary alcohol extension dimer 66. The primary alcohol extension dimer 66 may be reacted with methane sulfonyl chloride to provide a secondary alcohol extension dimer mesylate 67. Alternatively, the B2 blocking moiety on the dimer compound 65 may be removed using the B de-blocking reagent as described above to provide a secondary alcohol extension dimer 69. The secondary alcohol extension dimer 69 may be reacted with methane sulfonyl chloride to provide a primary alcohol extension dimer mesylate 70.
As will be understood by those skilled in the art, the chain extension process may be repeated to achieve various other chain lengths. For example, as illustrated in Figure 13, the primary alcohol extension dimer 66 may be reacted with the primary alcohol extension dimer mesylate 70 to provide a tetramer compound 72. As further illustrated in Figure 13, a generic chain extension reaction scheme involves reacting the primary alcohol extension mono- or poly-mer 73 with the primary alcohol extension mono- or poly-mer mesylate 74 to provide the uniform polypropylene polymer 75. The values of m and n may each range from 0 to 1000 or more. Preferably, m and n are each from 0 to 50. While the embodiments illustrated in Figure 13 show primary alcohol extension mono- and/or poly-mers being reacted with primary alcohol extension mono- and/or poly-mer mesylates, it is to be understood that similar reactions may be carried out using secondary alcohol extension mono- and/or poly-mers and secondary alcohol extension mono- and/or poly-mer mesylates. An end of a primary alcohol extension mono- or poly-mer or an end of a primary alcohol extension mono- or poly-mer mesylate may be reacted with a primary alcohol capping mono- or poly-mer mesylate or a primary alcohol capping mono- or poly-mer, respectively, to provide a capped uniform polypropylene chain. For example, as illustrated in Figure 12, the primary alcohol extension dimer mesylate 70 is reacted with the primary alcohol capping monomer 60 to provide the capped/blocked primary alcohol extension trimer 71. As will be understood by those skilled in the art, the Bi blocking moiety may be removed and the resulting capped primary alcohol extension trimer may be reacted with a primary alcohol extension mono- or poly-mer mesylate to extend the chain of the capped trimer 71.
An end of a secondary alcohol extension mono-or poly-mer or an end of a secondary alcohol extension mono-or poly-mer mesylate may be reacted with a secondary alcohol capping mono-or poly-mer mesylate or a secondary alcohol capping mono- or poly-mer, respectively, to provide a capped uniform polypropylene chain. For example, as illustrated in Figure 12, the secondary alcohol extension dimer mesylate 67 is reacted with the secondary alcohol capping monomer 63 to provide the capped/blocked primary alcohol extension trimer 68. The B2 blocking moiety may be removed as described above and the resulting capped secondary alcohol extension trimer may be reacted with a secondary alcohol extension mer mesylate to extend the chain of the capped trimer 68. While the syntheses illustrated in Figure 12 show the reaction of a dimer with a capping monomer to provide a trimer, it is to be understood that the capping process may be performed at any point in the synthesis of a uniform polypropylene glycol moiety, or, alternatively, uniform polypropylene glycol moieties may be provided that are not capped. While the embodiments illustrated in Figure 12 show the capping of a polybutylene oligomer by synthesis with a capping monomer, it is to be understood that polybutylene oligomers of the present invention may be capped directly (i.e., without the addition of a capping monomer) using a capping reagent as described above in Figure 11. Uniform polypropylene glycol moieties according to embodiments of the present invention maybe coupled to a calcitonin drug, a lipophilic moiety such as a carboxylic acid, and/or various other moieties by various methods as will be understood by those skilled in the art including, but not limited to, those described herein with respect to polyethylene glycol moieties. According to these embodiments of the present invention, the lipophilic moiety is a lipophilic moiety as will be understood by those skilled in the art. The lipophilic moiety is preferably a saturated or unsaturated, linear or branched alkyl moiety or a saturated or unsaturated, linear or branched fatty acid moiety. When the lipophilic moiety is an alkyl moiety, it is preferably a linear, saturated or unsaturated alkyl moiety having 1 to 28 carbon atoms. More preferably, the alkyl moiety has 2 to 12 carbon atoms. When the lipophilic moiety is a fatty acid moiety, it is preferably a natural fatty acid moiety that is linear, saturated or unsaturated, having 2 to 18 carbon atoms. More preferably, the fatty acid moiety has 3 to 14 carbon atoms. Most preferably, the fatty acid moiety has at least 4, 5 or 6 carbon atoms.
According to these embodiments of the present invention, the spacer moieties, G, G' and G", are spacer moieties as will be understood by those skilled in the art. Spacer moieties are preferably selected from the group consisting of sugar, cholesterol and glycerine moieties. Preferably, oligomers of these embodiments do not include spacer moieties (i.e., k, m and n are preferably 0).
According to these embodiments of the present invention, the linker moiety, L, may be used to couple the oligomer with the drug as will be understood by those skilled in the art. Linker moieties are preferably selected from the group consisting of alkyl and fatty acid moieties.
According to these embodiments of the present invention, the terminating moiety is preferably an alkyl or alkoxy moiety, and is more preferably a lower alkyl or lower alkoxy moiety. Most preferably, the terminating moiety is methyl or methoxy. While the terminating moiety is preferably an alkyl or alkoxy moiety, it is to be understood that the terminating moiety may be various moieties as will be understood by those skilled in the art including, but not limited to, sugars, cholesterol, alcohols, and fatty acids.
According to these embodiments of the present invention, the oligomer, which is represented by the bracketed portion of the structure of Formula A, is covalently coupled to the calcitonin drag. In some embodiments, the calcitonin drag is coupled to the oligomer utilizing a hydrolyzable bond (e.g., an ester or carbonate bond). A hydrolyzable coupling may provide a calcitomn drug-oligomer conjugate that acts as a prodrag. In certain instances, for example where the calcitonin drug-oligomer conjugate is inactive (i.e., the conjugate lacks the ability to affect the body through the calcitonin drug's primary mechanism of action), a hydrolyzable coupling may provide for a time-release or controlled-release effect, administering the calcitonin drug over a given time period as one or more oligomers are cleaved from their respective calcitonin drug-oligomer conjugates to provide the active drug, hi other embodiments, the calcitonin drug is coupled to the oligomer utilizing a non- hydrolyzable bond (e.g., a carbamate, amide, or ether bond). Use of a non-hydrolyzable bond may be preferable when it is desirable to allow the calcitonin drug-oligomer conjugate to circulate in the bloodstream for an extended period of time, preferably at least 2 hours. The bonding moiety, B, may be various bonding moieties that may be used to covalently couple the oligomer with the calcitonin drug as will be understood by those skilled in the art. Bonding moieties are preferably selected from the group consisting of covalent bond(s), ester moieties, carbonate moieties, carbamate moieties, amide moieties and secondary amine moieties. The variable p is an integer from 1 to the number of nucleophilic residues on the calcitonin drag. When p is greater than 1, more than one oligomer (i.e., a plurality of oligomers) is coupled to the drag. According the these embodiments of the present invention,- the oligomers in the plurality are the same. When a plurality of oligomers are coupled to the drug, it may be preferable to couple one or more of the oligomers to the drug with hydrolyzable bonds and couple one or more of the oligomers to the drug with non- hydrolyzable bonds. Alternatively, all of the bonds coupling the plurality of oligomers to the drag may be hydrolyzable, but have varying degrees of hydrolyzability such that, for example, one or more of the oligomers is rapidly removed from the drug by hydrolysis in the body and one or more of the oHgomers is slowly removed from the drug by hydrolysis in the body. When the calcitonin drag is salmon calcitonin, p is preferably 1 or 2, and is more preferably 2.
The oligomer may be coupled to the calcitonin drug at various nucleophilic residues of the calcitonin drug including, but not limited to, nucleophilic hydroxyl functions and/or amino functions. When the calcitonin drug is a polypeptide, a nucleophilic hydroxyl function may be found, for example, at serine and/or tyrosine residues, and a nucleophilic amino function maybe found, for example, at histidine and/or lysine residues, and/or at the one or more N-termini of the polypeptide. When an oligomer is coupled to the one or more N- terminus of the calcitonin polypeptide, the coupling preferably forms a secondary amine. When the calcitonin drug is salmon calcitonin, for example, the oligomer may be coupled to an amino functionality of the salmon calcitonin, including the amino functionality of Lys11, Lys18 and/or the N-terminus. While one or more oligomers may be coupled to the salmon calcitonin, a higher bioefficacy, such as improved serum calcium lowering ability, is observed for the di-conjugated salmon calcitonin where an oligomer is coupled to the amino functionalities of Lys11 and the Lys18. Mixtures of calcitonin drug-oligomer conjugates where each conjugate in the mixture has the same molecular weight and has the structure of Formula A may be synthesized by various methods. For example, a mixture of oligomers consisting of carboxylic acid and polyethylene glycol is synthesized by contacting a mixture of carboxylic acid with a mixture of polyethylene glycol under conditions sufficient to provide a mixture of oligomers. The oligomers of the mixture are then activated so that they are capable of reacting with a calcitonin drug to provide a calcitonin drug-oligomer conjugate. One embodiment of a synthesis route for providing a mixture of activated oligomers where each oligomer has the same molecular weight and has a structure of the oligomer of Formula A is illustrated in Figure 3 and described in Examples 11-18 hereinbelow. Another embodiment of a synthesis route for providing a mixture of activated oligomers where each oligomer has the same molecular weight and has a structure of the oligomer of Formula A is illustrated in Figure 4 and described in Examples 19-24 hereinbelow. Still another embodiment of a synthesis route for providing a mixture of activated oligomers where each oligomer has the same molecular weight and has a structure of the oligomer of Formula A is illustrated in Figure 5 and described in Examples 25-29 hereinbelow. Yet another embodiment of a synthesis route for providing a mixture of activated oligomers where each oligomer has the same molecular weight and has a structure of the oligomer of Formula A is illustrated in Figure 6 and described in Examples 30-31 hereinbelow. Another embodiment of a synthesis route for providing a mixture of activated oligomers where each oligomer has the same molecular weight and has a structure of the oligomer of Formula A is illustrated in Figure 7 and described in Examples 32-37 hereinbelow. Still another embodiment of a synthesis route for providing a mixture of activated oligomers where each oligomer has the same molecular weight and has a structure of the oligomer of Formula A is illustrated in Figure 8 and described in Example 38 hereinbelow. Yet another embodiment of a synthesis route for providing a mixture of activated oligomers where each oligomer has the same molecular weight and has a structure of the oligomer of Formula A is illustrated in Figure 9 and described in Example 39 hereinbelow. Another embodiment of a synthesis route for providing a mixture of activated oligomers where each ohgomer has the same molecular weight and has a structure of the oligomer of Formula A is illustrated in Figure 10 and described in Example 40 hereinbelow.
The mixture of activated oligomers where each oligomer has the same molecular weight and has a structure of the oligomer of Formula A is reacted with a mixture of calcitonin drugs where each drug in the mixture has the same molecular weight under ' conditions sufficient to provide a mixture of calcitonin drug-oligomer conjugates. A preferred synthesis is described in Example 41 hereinbelow. As will be understood by those skilled in the art, the reaction conditions (e.g., selected molar ratios, solvent mixtures and/or pH) may be controlled such that the mixture of calcitonin drug-oligomer conjugates resulting from the reaction of the mixture of activated oligomers where each oligomer has the same molecular weight and has a structure of the oligomer of Formula A and the mixture of calcitonin drags is a mixture of conjugates where each conjugate has the same molecular weight and has the structure Formula A. For example, conjugation at the amino functionality of lysine may be suppressed by maintaining the pH of the reaction solution below the pKa of lysine. Alternatively, the mixture of calcitonin drug-oligomer conjugates may be separated and isolated utilizing, for example, HPLC to provide a mixture of calcitonin drug-oligomer conjugates, for example mono-, di-, or tri-conjugates, where each conjugate in the mixture has the same number molecular weight and has the structure of Formula A. The degree of conjugation (e.g., whether the isolated molecule is a mono-, di-, or tri-conjugate) of a particular isolated conjugate may be determined and/or verified utilizing various techniques as will be understood by those skilled in the art including, but not limited to, mass spectroscopy. The particular conjugate structure (e.g., whether the oligomer is at Lys11, Lys18 or the N-terminus of a salmon calcitonin monoconjugate) may be determined and/or verified utilizing various techniques as will be understood by those skilled in the art including, but not limited to, sequence analysis, peptide mapping, selective enzymatic cleavage, and/or endopeptidase cleavage.
As will be understood by those skilled in the art, one or more of the reaction sites ori the calcitonin drug maybe blocked by, for example, reacting the calcitonin drag with a suitable blocking reagent such as N-tert-butoxycarbonyl (t-BOC), or N-(9- fluorenylmethoxycarbonyl) (N-FMOC). This process may be preferred, for example, when the calcitonin drag is a polypeptide and it is desired to form an unsaturated conjugate (i.e., a conjugate wherein not all nucleophilic residues are conjugated) having an oligomer at the N- terminus of the polypeptide. Following such blocking, the mixture of blocked calcitonin drugs may be reacted with the mixture of activated oligomers where each oligomer in the mixture has the same molecular weight and has a structure of the oligomer of Formula A to provide a mixture of calcitonin drug-oligomer conjugates having oligomer(s) coupled to one or more nucleophilic residues and having blocking moieties coupled to other nucleophilic residues. After the conjugation reaction, the calcitonin drug-oligomer conjugates may be de- blocked as will be understood by those skilled in the art. If necessary, the mixture of calcitonin drug-oligomer conjugates may then be separated as described above to provide a mixture of calcitonin drug-oligomer conjugates where each conjugate in the mixture has the same number molecular weight and has the structure of Formula A. Alternatively, the mixture of calcitonin drug-oligomer conjugates may be separated prior to de-blocking.
Mixtures of calcitonin drag-oligomer conjugates where each conjugate in the mixture has the same molecular weight and has the structure of Formula A according to embodiments of the present invention preferably have improved properties when compared with those of conventional mixtures. For example, a mixture of calcitomn drug-oligomer conjugates where each conjugate in the mixture has the same molecular weight and has the structure of Formula A preferably is capable of lowering serum calcium levels by at least 5 percent. Preferably, the mixture, of conjugates is capable of lowering serum calcium levels by at least 10, 11, 12, 13 or 14 percent. More preferably, the mixture of conjugates is capable of lowering serum calcium levels by at least 15, 16, 17, 18 or 19 percent, and, most preferably, the mixture of conjugates is capable of lowering serum calcium levels by at least 20 percent. As another example, a mixture of calcitonin drug-oligomer conjugates where each conjugate in the mixture has the same molecular weight and has the structure of Formula A preferably has an increased resistance to degradation by chymotrypsin and/or trypsin when compared to the resistance to degradation by chymotrypsin and/or trypsin, respectively, of the calcitonin drag which is not coupled to the oligomer. Resistance to chymotrypsin or trypsin corresponds to the percent remaining when the molecule to be tested is digested in the applicable enzyme using a procedure similar to the one outlined in Example 51 below. Preferably, the resistance to degradation by chymotrypsin of the mixture of calcitonin drug- oligomer conjugates is about 10 percent greater than the resistance to degradation by - chymotrypsin of the mixture of calcitonin drugs that is not conjugated with the oligomer. More preferably, the resistance to degradation by chymotrypsin of the mixture of calcitonin drug-oligomer conjugates is about 15 percent greater than the resistance to degradation by chymotrypsin of the mixture of calcitonin drug that is not conjugated with the oligomer, and, most preferably, the resistance to degradation by chymotrypsin of the mixture of calcitonin drag-oligomer conjugates is about 20 percent greater than the resistance to degradation by chymotrypsin of the mixture of calcitonin drug that is not conjugated with the oligomer. Preferably, the resistance to degradation by trypsin of the mixture of calcitonin drug-oligomer conjugates is about 10 percent greater than the resistance to degradation by trypsin of the mixture of calcitonin drug that is not conjugated with the oligomer. More preferably, the resistance to degradation by trypsin of the mixture of calcitonin drag-oligomer conjugates is about 20 percent greater than the resistance to degradation by trypsin of the mixture of calcitonin drug that is not conjugated with the oligomer, and, most preferably, the resistance to degradation by trypsin of the mixture of calcitomn drag-oligomer conjugates is about 30 percent greater than the resistance to degradation by trypsin of the mixture of calcitonin drug that is not conjugated with the oligomer.
As still another example, a mixture of calcitonin drug-oligomer conjugates where each conjugate in the mixture has the same molecular weight and has the structure of Formula A preferably has a higher bioefficacy than the bioefficacy of the calcitonin drag which is not coupled to the oligomer. The bioefficacy of a particular compound corresponds to its area-under-the-curve (AUC) value. Preferably, the bioefficacy of the mixture is about 5 percent greater than the bioefficacy of the calcitonin drug which is not coupled to the oligomer. More preferably, the bioefficacy of the mixture is about 10 percent greater than the bioefficacy of the calcitonin drug which is not coupled to the oligomer.
As yet another example, a mixture of calcitonin drug-oligomer conjugates where each conjugate in the mixture has the same molecular weight and has the structure of Formula A preferably has an in vivo activity that is greater than the in vivo activity of a polydispersed mixture of calcitonin drug-oligomer conjugates having the same number average molecular weight as the mixture of calcitonin drug-oligomer conjugates where each conjugate in the mixture has the same molecular weight and has the structure of Formula A. As will be understood by those skilled in the art, the number average molecular weight of a mixture may be measured by various methods including, but not limited to, size exclusion chromatography such as gel permeation chromatography as described, for example, in H.R. Allcock & F.W. Lampe, CONTEMPORARY POLYMER CHEMISTRY 394-402 (2d. ed., 1991).
As another example, a mixture of calcitonin drug-oligomer conjugates where each conjugate in the mixture has the same molecular weight and has the structure of Formula A preferably has an in vitro activity that is greater than the in vitro activity of a polydispersed mixture of calcitomn drug-oligomer conjugates having the same number average molecular weight as the mixture of calcitonin drug-oligomer conjugates where each conjugate in the mixture has the same molecular weight and has the structure of Formula A. As will be understood by those skilled in the art, the number average molecular weight of a mixture may be measured by various methods including, but not Hmited to, size exclusion chromatography.
As still another example, a mixture of calcitonin drug-oligomer conjugates where each conjugate in the mixture has the same molecular weight and has the stracture of Formula A preferably has an increased resistance to degradation by chymotrypsin and/or trypsin when compared to the resistance to degradation by chymotrypsin and/or trypsin of a polydispersed mixture of calcitonin drug-oligomer conjugates having the same number average molecular weight as the mixture of calcitonin drag-oligomer conjugates where each conjugate in the mixture has the same molecular weight and has the structure of Formula A. As will be understood by those skilled in the art, the number average molecular weight of a mixture may be measured by various methods including, but not limited to, size exclusion chromatography.
As yet another example, a mixture of calcitonin drag-oligomer conjugates where each conjugate in the mixture has the same molecular weight and has the structure of Formula A preferably has an inter-subject variability that is less than the inter-subject variability of a polydispersed mixture of calcitonin drug-oligomer conjugates having the same number average molecular weight as the mixture of calcitonin drag-oligomer conjugates where each conjugate in the mixture has the same molecular weight and has the structure of Formula A. As will be understood by those skilled in the art, the number average molecular weight of a mixture maybe measured by various methods including, but not limited to, size exclusion chromatography. The inter-subject variability may be measured by various methods, as will be understood by those skilled in the art. The inter-subject variability is preferably calculated as follows. The area under a dose response curve (AUC) (i.e., the area between the dose- response curve and a baseline value) is determined for each subject. The average AUC for all subjects is determined by summing the AUCs of each subject and dividing the sum by the number of subjects. The absolute value of the difference between the subject's AUC and the average AUC is then determined for each subject. The absolute values of the differences obtained are then summed to give a value that represents the inter-subject variability. Lower values represent lower inter-subject variabihties and higher values represent higher inter- subject variabilities. Mixtures of calcitonin drug-oligomer conjugates where each conjugate in the mixture has the same molecular weight and has the stracture of Formula A according to embodiments of the present invention preferably have two or more of the above-described improved properties. More preferably, mixtures of calcitonin drug-oligomer conjugates where each conjugate in the mixture has the same molecular weight and has the stracture of Formula A according to embodiments of the present invention have three or more of the above-described improved properties. Most preferably, mixtures of calcitonin drag-oligomer conjugates where each conjugate in the mixture has the same molecular weight and has the structure of Formula A according to embodiments of the present invention have four or more of the above-described improved properties.
Pharmaceutical compositions comprising a conjugate mixture according to embodiments of the present invention are also provided. The mixtures of calcitonin drug- oligomer conjugates described above may be formulated for administration in a pharmaceutical carrier in accordance with known techniques. See, e.g., Remington, The Science And Practice of Pharmacy (9th Ed. 1995). In the manufacture of a pharmaceutical composition according to embodiments of the present invention, the mixture of calcitonin drug-oligomer conjugates is typically admixed with, z'nter alia, a pharmaceutically acceptable carrier. The carrier must, of course, be acceptable in the sense of being compatible with any other ingredients in the pharmaceutical composition and should not be deleterious to the patient. The carrier may be a solid or a Hquid, or both, and is preferably formulated with the mixture of calcitonin drug-oligomer conjugates as a unit-dose formulation, for example, a tablet, which may contain from about 0.01 or 0.5% to about 95% or 99% by weight of the mixture of calcitonin drug-oligomer conjugates. The pharmaceutical compositions maybe prepared by any of the well known techniques of pharmacy including, but not Hmited to, admixing the components, optionally including one or more accessory ingredients.
The pharmaceutical compositions according to embodiments of the present invention include those suitable for oral, rectal, topical, inhalation (e.g., via an aerosol) buccal (e.g., sub-lingual), vaginal, parenteral (e.g., subcutaneous, intramuscular, intradermal, intraarticular, intrapleural, intraperitoneal, inracerebral, intraarterial, or intravenous), topical (i.e., both skin and mucosal surfaces, including airway surfaces) and transdermal administration, although the most suitable route in any given case will depend on the nature and severity of the condition being treated and on the nature of the particular mixture of calcitonin drag-oligomer conjugates which is being used.
Pharmaceutical compositions suitable for oral administration maybe presented in discrete units, such as capsules, cachets, lozenges, or tables, each containing a predetermined amount of the mixture of calcitonin drug-oligomer conjugates; as a powder or granules; as a solution or a suspension in an aqueous or non-aqueous liquid; or as an oil-in-water or water- in-oil emulsion. Such formulations may be prepared by any suitable method of pharmacy which includes the step of bringing into association the mixture of calcitonin drag-oligomer conjugates and a suitable carrier (which may contain one or more accessory ingredients as noted above). In general, the pharmaceutical composition according to embodiments of the present invention are prepared by uniformly and intimately admixing- the mixture of calcitonin drag-oligomer conjugates with a liquid or finely divided solid carrier, or both, and then, if necessary, shaping the resulting mixture. For example, a tablet may be prepared by compressing or molding a powder or granules containing the mixture of calcitonin drug- oligomer conjugates, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing, in a suitable machine, the mixture in a free-flowing form, such as a powder or granules optionally mixed with a binder, lubricant, inert diluent, and/or surface active/dispersing agent(s). Molded tablets may be made by molding, in a suitable machine, the powdered compound moistened with an inert liquid binder. Pharmaceutical compositions suitable for buccal (sub-lingual) administration include lozenges comprising the mixture of calcitonin drug-oligomer conjugates in a flavoured base, usually sucrose and acacia or tragacanth; and pastilles comprising the mixture of calcitonin. drug-oligomer conjugates in an inert base such as gelatin and glycerin or sucrose and acacia. Pharmaceutical compositions according to embodiments of the present invention suitable for parenteral administration comprise sterile aqueous and non-aqueous injection solutions of the mixture of calcitonin drug-oligomer conjugates, which preparations are preferably isotonic with the blood of the intended recipient. These preparations may contain anti-oxidants, buffers, bacteriostats and solutes which render the composition isotonic with the blood of the intended recipient. Aqueous and non-aqueous sterile suspensions may include suspending agents and thickening agents. The compositions may be presented in unit\dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, saline or water-for-injection immediately prior to use. Extemporaneous injection solutions and suspensions maybe prepared from sterile powders, granules and tablets of the kind previously described. For example, an injectable, stable, sterile composition comprising a mixture of calcitonin drug-oligomer conjugates in a unit dosage form in a sealed container may be provided. The mixture of calcitonin drug-oligomer conjugates is provided in the form of a lyophilizate which is capable of being reconstituted with a suitable pharmaceutically acceptable carrier to form a liquid composition suitable for injection thereof into a subject. The unit dosage form typically comprises from about 10 mg to about 10 grams of the mixture of calcitonin drug-oligomer conjugates. When the mixture of calcitonin drug-oligomer conjugates is substantially water-insoluble, a sufficient amount of emulsifying agent which is physiologically acceptable may be employed in sufficient quantity to emulsify the mixture of calcitonin drug-oligomer conjugates in an aqueous carrier. One such useful emulsifying agent is phosphatidyl choline.
Pharmaceutical compositions suitable for rectal administration are preferably presented as unit dose suppositories. These may be prepared by admixing the mixture of calcitonin drug-oligomer conjugates with one or more conventional solid carriers, for example, cocoa butter, and then shaping the resulting mixture.
Pharmaceutical compositions suitable for topical application to the skin preferably take the form of an ointment, cream, lotion, paste, gel, spray, aerosol, or oil. Carriers which may be used include petroleum jelly, lanohne, polyethylene glycols, alcohols, transdermal enhancers, and combinations of two or more thereof.
Pharmaceutical compositions suitable for transdermal administration may be presented as discrete patches adapted to remain in intimate contact with the epidermis of the recipient for a prolonged period of time. Compositions suitable for transdermal administration may also be delivered by iontophoresis (see, for example, Pharmaceutical Research 3 (6):318 (1986)) and typically take the form of an optionally buffered aqueous - solution ofthe mixture of calcitonin drug-oligomer conjugates. Suitable formulations comprise citrate or bis\tris buffer (pH 6) or ethanol/water and contain from 0.1 to 0.2M active ingredient. ' Methods of treating a bone disorder in a subject in need of such treatment by
' administering an effective amount of such pharmaceutical compositions are also provided. The bone disorder is preferably characterized by excessive osteoclastic bone resorption and/or hypercalcemic serum effects. Bone disorders that may be treated and/or prevented by methods of the present invention include, but are not limited to, osteoporosis, Paget's disease, and hypercalcernia.
The effective amount of any mixture of calcitonin drug-oligomer conjugates, the use of which is in the scope of present invention, will vary somewhat from mixture to mixture, and patient to patient, and will depend upon factors such as the age and condition of the patient and the route of delivery. Such dosages can be determined in accordance with routine pharmacological procedures known to those skilled in the art. As a general proposition, a dosage from about 0.1 to about 50 mg/kg will have therapeutic efficacy, with all weights being calculated based upon the weight of the mixture of calcitonin drug-oligomer conjugates. Toxicity concerns at the higher level may restrict intravenous dosages to a lower level such as up to about 10 mg/kg, with all weights being calculated based upon the weight of the active base. A dosage from about 10 mgkg to about 50 mg/kg may be employed for oral administration. Typically, a dosage from about 0.5 mgkg to 5 mg/kg may be employed for intramuscular injection. The frequency of administration is usually one, two, or three times per day or as necessary to control the condition. Alternatively, the drug-oligomer conjugates may be administered by continuous infusion. The duration of treatment depends on the type of bone disorder being treated and may be for as long as the life of the patient.
Methods of synthesizing conjugate mixtures according to embodiments of the present invention are also provided. While the following embodiments of a synthesis route are directed to synthesis of a monodispersed mixture, similar synthesis routes may be utilized for synthesizing other calcitonin drug-oligomer conjugate mixtures according to embodiments of the present invention.
A substantially monodispersed mixture, of polymers comprising polyethylene glycol moieties is provided as illustrated in reaction 1 :
R1(OC2H4)nO-X+ + R2(OC2H4)mO s *-
Figure imgf000065_0001
i
CD OD cm)
' R1 is H or a HpophiHc moiety. R1 is preferably H, alkyl, aryl alkyl, an aromatic moiety, a fatty acid moiety, an ester of a fatty acid moiety, cholesteryl, or adamantyl. R is more preferably H, lower alkyl, or an aromatic moiety. R1 is most preferably H, methyl, or benzyl.
In Formula I, n is from 1 to 25. Preferably n is from 1 to 6. X+ is a positive ion. Preferably X+ is any positive ion in a compound, such as a strong base, that is capable of ionizing a hydroxyl moiety on PEG. Examples of positive ions include, but are not limited to, sodium ions, potassium ions, lithium ions, cesium ions, and thallium ions. R2 is.H or a lipophilic moiety. R2 is preferably linear or branched alkyl, aryl alkyl, an aromatic moiety, a fatty acid moiety, or an ester of a fatty acid moiety. R2 is more preferably lower alkyl, benzyl, a fatty acid moiety having 1 to 24 carbon atoms, or an ester of a fatty acid moiety having 1 to 24 carbon atoms. R is most preferably methyl, a fatty acid moiety having 1 to 18 carbon atoms or an ethyl ester of a fatty acid moiety having 1 to 18 carbon atoms.
In Formula H, m is from 1 to 25. Preferably m is from 1 to 6. Ms is a mesylate moiety (i.e., CH3S(O2)-).
As illustrated in reaction 1, a mixture of compounds having the stracture of Formula I is reacted with a mixture of compounds having the structure of Formula II to provide a mixture of polymers comprising polyethylene glycol moieties and having the structure of Formula HI. The mixture of compounds having the structure of Formula I is a substantially monodispersed mixture. Preferably, at least about 96, 97, 98 or 99 percent of the compounds in the mixture of compounds of Formula I have the same molecular weight, and, more preferably, the mixture of compounds of Formula I is a monodispersed mixture. The mixture of compounds of Formula II is a substantially monodispersed mixture. Preferably, at least about 96, 97, 98 or 99 percent of the compounds in the mixture of compounds of Formula II have the same molecular weight, and, .more preferably, the mixture of compounds of Formula II is a monodispersed mixture. The mixture of compounds of Formula HI is a substantially monodispersed mixture. Preferably, at least about 96, 97, 98 or 99 percent of the compounds in the mixture of compound of Formula HI have the same molecular weight. More preferably, the mixture of compounds of Formula HI is a monodispersed mixture.-
Reaction 1 is preferably performed between about 0°C and about 40°C, is more preferably performed between about 15°C and about 35°C, and is most preferably performed at room temperature (approximately 25°C). Reaction 1 may be performed for various periods of time as will be understood by those skilled in the art. Reaction 1 is preferably performed for a period of time between about 0.25, 0.5 or 0.75 hours and about 2, 4 or 8 hours. Reaction 1 is preferably carried out in an aprotic solvent such as, but not limited to, N,N-dimethylacetamide (DMA), N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), hexamethylphosphoric triamide, tefrahydrofuran (THF), didxane, diethyl ether, methyl t-butyl ether (MTBE), toluene, benzene, hexane, pentane, N-methylpyrollidinone, tetrahydronaphthalene, decahydronaphthalene, 1,2-dichlorobenzene, l,3-dimethyl-2- imidazolidinone, or a mixture thereof. More preferably, the solvent is DMF, DMA or toluene.
The molar ratio of the compound of Formula I to the compound of Formula H is preferably greater than about 1:1. More preferably, the molar ratio is at least about 2:1. By providing an excess of the compounds of Formula I, one can ensure that substantially all of the compounds of Formula II are reacted, which may aid in the recovery of the compounds of Formula IH as discussed below.
Compounds of Formula I are preferably prepared as illustrated in reaction 2: i compound capable of 1 . +
R1(OC2H4)nOH + ionizing a hydroxyl moiety ** R (OC2H4)nO X 2 on the PEG moiety of V Formula IV (I) R1 and X+ are as described above and the mixture of compounds of Formula IV is substantially monodispersed; preferably, at least about 96, 97, 98 or 99 percent of the compounds in the mixture of compounds of Formula IV have the same molecular weight; and, more preferably, the mixture of compounds of Formula TV is a monodispersed mixture. Various compounds capable of ionizing a hydroxyl moiety on the PEG moiety of the compound of Formula IV will be understood by those skilled in the art. The compound capable of ionizing a hydroxyl moiety is preferably a strong base. More preferably, the compound capable of ionizing a hydroxyl moiety is selected from the group consisting of sodium hydride, potassium hydride, sodium t-butoxide, potassium t-butoxide, butyl lithium (BuLi), and lithium diisopropylamine. The compound capable of ionizing a hydroxyl moiety is more preferably sodium hydride.
The molar ratio of the compound capable of ionizing a hydroxyl moiety on the PEG moiety of the compound of Formula JV to the compound of Formula IV is preferably at least about 1:1, and is more preferably at least about 2:1. By providing an excess of the compound capable of ionizing the hydroxyl moiety, it is assured that substantially all of the compounds of Formula IV are reacted to provide the compounds of Formula I. Thus, separation difficulties, which may occur if both compounds of Formula IV and compounds of Formula I were present in the reaction product mixture, may be avoided.
Reaction 2 is preferably performed between about 0°C and about 40°C, is more preferably performed between about 0°C and about 35°C, and is most preferably performed between about 0°C and room temperature (approximately 25°C).
Reaction 2 may be performed for various periods of time as will be understood by those skilled in the art. Reaction 2 is preferably performed for a period of time between about 0.25, 0.5 or 0.75 hours and about 2, 4 or 8 hours.
Reaction 2 is preferably carried out in an aprotic solvent such as, but not limited to, N,N-dimethylacetamide (DMA), N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), hexamethylphosphoric triamide, tetrahydrofuran (THF), dioxane, diethyl ether, methyl t-butyl ether (MTBE), toluene, benzene, hexane, pentane, N-methylpyrollidinone, dichloromethane, chloroform, tetrahydronaphthalene, decahydronaphthalene, 1,2- dichlorobenzene, l,3-dimethyl-2-imidazolidinone, or a mixture thereof. More preferably, the solvent is DMF, dichloromethane or toluene.
Compounds of Formula H are preferably prepared as illustrated in reaction 3:
O
R2(OC2H4)mOH + CH3SQ > R2(OC2H4)mOMs 3
(V) ° (II)
R2 and Ms are as described above and the compound of Formula V is present as a substantially monodispersed mixture of compounds of Formula V; preferably at least about 96, 97, 98 or 99 percent of the compounds in the mixture of compounds of Formula V have the same molecular weight; and, more preferably, the mixture of compounds of Formula V is a monodispersed mixture.
Q is a halide, preferably chloride or fluoride.
CH3S(O2)Q is methanesulfonyl halide. The methanesulfonyl halide is preferably methanesulfonyl chloride or methanesulfonyl fluoride. More preferably, the methanesulfonyl halide is methanesulfonyl chloride.
The molar ratio of the methane sulfonyl halide to the compound of Formula V is preferably greater than about 1:1, and is more preferably at least about 2:1. By providing an excess of the methane sulfonyl halide, it is assured that substantially all of the compounds of Formula V are reacted to provide the compounds of Formula π. Thus, separation difficulties, which may occur if both compounds of Formula V and compounds of Formula H were present in the reaction product mixture, may be avoided.
Reaction 3 is preferably performed between about -10°C'and about 40°C, is more preferably performed between about 0°C and about 35°C, and is most preferably performed between about 0°C and room temperature (approximately 25°C).
Reaction 3 may be performed for various periods of time as will be understood by those skilled in the art. Reaction 3 is preferably performed for a period of time between about 0.25, 0.5 or 0.75 hours and about 2, 4 or 8 hours.
Reaction 3 is preferably carried, out in the presence of an aliphatic amine including, but not limited to, monomethylamine, dimethylamine, trimethylamine, monoethylamine, diethylamine, triethylamine, monoisopropylamine, diisopropylamine, mono-n-butylamine, di- n-butylamine, tri-n-butylamine, monocyclohexylamine, dicyclohexylamine, or mixtures thereof. More preferably, the aliphatic amine is a tertiary amine such as triethylamine.
As will be understood by those skilled in the art, various substantially monodispersed mixtures of compounds of Formula V are commercially available. For example, when R2 is H or methyl, the compounds of Formula V are PEG or mPEG compounds, respectively, which are commercially available from Aldrich of Milwaukee, Wisconsin; Fluka of Switzerland, and/or TCI America of Portland, Oregon.
When R2 is a lipophilic moiety such as, for example, higher alkyl, fatty acid, an ester of a fatty acid, cholesteryl, or adamantyl, the compounds of Formula V may be provided by various methods as will be understood by those skilled in the art. The compounds of Formula V are preferably provided as follows:
R'-OMs + R3(OC2H4)m-O-X2 + ► R3(OC2H4)m-OR2 4
(VI) (VII) (VIH)
R3(OC2H4)m-OR2 ► H(OC2H4)m-OR2
Figure imgf000069_0001
R is a lipophilic moiety, preferably higher alkyl, fatty acid ester, cholesteryl, or adamantyl, more preferably a lower alkyl ester of a fatty acid, and most preferably an ethyl ester of a fatty acid having from 1 to 18 carbon atoms.
R3 is H, benzyl, trityl, tetrahydropyran, or other alcohol protecting groups as will be understood by those skilled in the art.
X2 +is a positive ion as described above with respect to X+.
The value of m is as described above.
Regarding reaction 4, a mixture of compounds of Formula VI is reacted with a mixture of compounds of Formula VH under reaction conditions similar to those described above with reference to reaction 1. The mixture of compounds of Formula VI is a substantially monodispersed mixture. Preferably, at least about 96, 97, 98 or 99 percent of the compounds in the mixture of compounds of Formula VI have the same molecular weight. More preferably, the mixture of compounds of Formula VI is a monodispersed mixture. .The - mixture of compounds of Formula VH is a substantially monodispersed mixture. Preferably, at least about 96, 97, 98 or 99 percent of the compounds in the mixture of compounds of
Formula VH have the same molecular weight. More preferably, the mixture of compounds of Formula VH is a monodispersed mixture.
Regarding reaction 5, the compound of Formula VIH may be hydrolyzed to convert the R3 moiety into an alcohol by various methods as will be understood by those skilled in the art. When R3 is benzyl or trityl, the hydrolysis is preferably performed utilizing H2 in the presence of a palladium-charcoal catalyst as is known by those skilled in the art. Of course, when R is H, reaction 5 is unnecessary.
The compound of Formula VI may be commercially available or be provided as described above with reference to reaction 3. The compound of Formula VH may be provided as described above with reference to reaction 2.
Substantially monodispersed mixtures of polymers comprising PEG moieties and having the stracture of Formula HI above can further be reacted with other substantially monodispersed polymers comprising PEG moieties in order to extend the PEG chain. For example, the following scheme may be employed: O t i 'I 2
R2(OC2H4r n-OR1 + CH3SQ R2(OC2H4Wπ-OMs
(in) ° σx)
R2(OC2H4)m n-OMs + R (OC2H4)p-O-χ2 + ~- R2(OC2H4)ιn+n+p-OR4
(IX) (X) (XI)
Ms, m and n are as described above with reference to reaction 1; p is similar to n and m, and X2 + is similar to X+ as described above with reference to reaction 1. Q is as described above with reference to reaction 3. R is as described above with reference to reaction 1 and is preferably lower alkyl. R1 is H. Reaction 6 is preferably performed in a manner similar to that described above with reference to reaction 3. Reaction 7 is preferably performed in a manner similar to that described above with reference to reaction 1. Preferably, at least about 96, 91, 98 or 99 percent of the compounds in the mixture of compounds of Formula HI have the same molecular weight, and, more preferably, the mixture of compounds of Formula HI is a monodispersed mixture. The mixture of compounds of Formula X is a substantially monodispersed mixture. Preferably, at least about 96, 97, 98 or 99 percent of the compounds in the mixture of compounds of Formula X have the same molecular weight, and, more preferably, the mixture of compounds of Formula X is a monodispersed mixture. A process according to embodiments of the present invention is illustrated by the scheme shown in Figure 1, which will now be described. The synthesis of substantially monodispersed polyethylene glycol-containing oligomers begins by the preparation of the monobenzyl ether (1) of a substantially monodispersed polyethylene glycol. An excess of a commercially available substantially monodispersed polyethylene glycol is reacted with benzyl chloride in the presence of aqueous sodium hydroxide as described by Coudert et al (Synthetic Communications, 16(1): 19-26 (1986)). The sodium salt of 1 is then prepared by the addition of NaH, and this sodium salt is allowed to react with the mesylate synthesized from the ester of a hydroxyalkanoic acid (2). The product (3) of the displacement of the mesylate is debenzylated via catalytic hydrogenation to obtain the alcohol (4). The mesylate (5) of this alcohol may be prepared by addition of methanesulfonyl chloride and used as the electrophile in the reaction with the sodium salt of the monomethyl ether of a substantially monodispersed polyethylene glycol derivative, thereby extending the polyethylene glycol portion of the oligomer to the desired length, obtaining the elongated ester (6). The ester may be hydrolyzed to the acid (7) in aqueous base and transformed into the activated ester (8) by reaction with a carbodiimide and N-hydroxysuccinimide. While the oligomer illustrated in Figure 1 is activated using N-hydroxysuccinimide, it is to be understood that various other reagents may be used to activate oligomers of the present invention including, but not limited to, active phenyl chloroformates such as αrα-nitrophenyl chloroformate, phenyl chloroformate, 3, 4-phenyldichloro formate, and 3, 4-phenyldichloro formate; tresylation; and acetal formation.
Still referring to Figure 1, q is from 1 to 24. Preferably, q is from 1 to 18, and q is more preferably from 4 to 16. R4 is a moiety capable of undergoing hydrolysis to provide the carboxylic acid. R4 is preferably lower alkyl and is more preferably ethyl. The variables n and m are as described above with reference to reaction 1.
All starting materials used in the procedures described herein are either commercially available or can be prepared by methods known in the art using commercially available starting materials.
The present invention will now be described with reference to the following examples. It should be appreciated that these examples are for the purposes of illustrating aspects of the present invention, and do not limit the scope of the invention as defined by the claims. EXAMPLES
Examples 1 through 10
Reactions in Examples 1 through 10 were carried out under nitrogen with magnetic stirring, unless otherwise specified. "Work-up" denotes extraction with an organic solvent, washing of the organic phase with saturated NaCl solution, drying (MgSO4), and evaporation (rotary evaporator). Thin layer chromatography was conducted with Merck glass plates precoated with silica gel 60°F - 254 and spots were visualized by iodine vapor. All mass spectra were determined by Macromolecular Resources Colorado State University, CO and are reported in the order m/z, (relative intensity). Elemental analyses and melting points were performed by Galbraith Laboratories, Inc., Knoxville, TN. Examples 1-10 refer to the scheme illustrated in Figure 2. Example 1 8-Methoxy-l-(methylsuIfonyl)oxy-3,6-dioxaoctane (9)
A solution of non-polydispersed triethylene glycol monomethyl ether molecules (4.00 mL, 4.19 g, 25,5 mmol) and triethylamine (4.26 mL, 3.09 g, 30.6 mmol) in dry dichloromethane (50 mL) was chilled in an ice bath and place under a nitrogen atmosphere. A solution of methanesulfonyl chloride (2.37 mL, 3.51 g, 30.6 mmol) in dry dichloromethane (20 mL) was added dropwise from an addition funnel. Ten minutes after the completion of the chloride addition, the reaction mixture was removed from the ice bath and allowed to come to room temperature. The mixture was stirred for an additional hour, at which time TLC (CHCI3 with 15% MeOH as the elutant) showed no remaining triethylene glycol monomethyl ether.
The reaction mixture was diluted with another 75 mL of dichloromethane and washed successively with saturated NaHCO3, water and brine. The organics were dried over Na2SO4, filtered and concentrated in vacuo to give a non-polydispersed mixture of compounds 9 as a clear oil (5.31g, 86%).
Example 2 Ethylene glycol mono methyl ether (10) (m=4,5,6)
To a stirred solution of non-polydispersed compound 11 (35.7 mmol) in dry DMF (25.7 mL), under N2 was added in portion a 60% dispersion of NaH in mineral oil, and the mixture was stirred at room temperature for 1 hour. To this salt 12 was added a solution of . non-polydispersed mesylate 9 (23.36) in dry DMF (4 ml) in a single portion, and the mixture was stirred at room temperature for 3.5 hours. Progress of the reaction was monitored by TLC (12% CH3OH-CHCl3). The reaction mixture was diluted with an equal amount of IN HCl, and extracted with ethyl acetate (2 x 20 ml) and discarded. Extraction of aqueous solution and work-up gave non-polydispersed polymer 10 (82 -84% yield).
Example 3 3,6,9,12,15,18,21-Heptaoxadocosanol (10) (m=4) Oil; Rf 0.46 (methanol : chloroform = 3 :22); MS m/z calc'd for C15H32O8.340.21
( ^l), found 341.2. Example 4 3,6,9,12,15,18,21,24-OctaoxapentacosanoI (10) (m=5)
Oil; Rf 0.43 (methanol : chloroform = 6:10); MS m/z calc'd for C17H36O9384.24 (M++l), found 385.3.
Example 5 3,6,9,12,15,18,21,24,27-Nonaoxaoctacosanol (10) (m=6)
Oil; Rf 0.42 (methanol : chloroform
Figure imgf000074_0001
428.26 (M÷+l), found 429.3.
Example 6 20-methoxy-l-(methylsulfonyl)oxy-3,6,9,12,15,18-hexaoxaeicosane (14)
Non-polydispersed compound 14 was obtained in quantitative yield from the alcohol 13 (m=4) and methanesulfonyl chloride as described for 9, as an oil; Rf 0.4 (ethyl acetate : acetonitrile= 1:5); MS m/z calc'd for C17H370433.21 (M*+l), found 433.469.
Example 7 Ethylene glycol mono methyl ether (15) (m=3,4,5)
The non-polydispersed compounds 15 were prepared from a diol by using the procedure described above for compound 10.
Example 8 3,6,9,12,15,18,21,24,27,30-Decaoxaheneicosanol (15) (m=3)
Oil; Rf 0.41 (methanol : chloroform = 6:10); MS m z calc'd for C21H44O11 472.29 (M++l), found 472.29. .
Example 9 3,6,9,12,15,18,21,24,27,30,33-Unecaoxatetratricosanol (15) (m=4)
Oil; Rf 0.41 (methanol : chloroform = 6:10); MS m/z calc'd for C23H48O12 516.31 (M++l), found 516.31. Example 10 3,6,9,12,15,18,21,24,27,30,33,36-Dodecaoxaheptatricosanol (15) (m=5)
Oil; Rf 0.41 (methanol : chloroform = 6:10); MS m z calc'd for C25H52O13 560.67 (M÷+1), found 560.67.
Examples 11 through 18 refer to the scheme illustrated in Figure 3.
Example 11 Hexaethylene glycol monobenzyl ether (16) An aqueous sodium hydroxide solution prepared by dissolving 3.99 g (100 mmol)
NaOH in 4 ml water was added slowly to non-polydispersed hexaethylene glycol (28.175 g, 25 ml, 100 mmol). Benzyl chloride (3.9 g, 30.8 mmol, 3.54 ml) was added and the reaction mixture was heated with stirring to 100°C for 18 hours. The reaction mixture was then cooled, diluted with brine (250 ml) and extracted with methylene chloride (200 ml x 2). The combined organic layers were washed with brine once, dried over Na2SO , filtered and concentrated in vacuo to a dark brown oil. The crude product mixture was purified via flash chromatography (silica gel, gradient elution: ethyl acetate to 9/1 ethyl acetate/methanol) to yield 8.099 g (70 %) of non-polydispersed 16 as a yellow oil.
Example 12
Ethyl 6-methylsulfonyloxyhexanoate (17)
A solution of non-polydispersed ethyl 6-hydroxyhexanoate (50.76 ml, 50.41 g, 227 mmol) in dry dichloromethane (75 ml) was chilled in a ice bath and placed under a nitrogen atmosphere. Triethylamine (34.43 ml, 24.99 g, 247 mmol) was added. A solution of methanesulfonyl chloride (19.15 ml, 28.3 g, 247 mmol) in dry dichloromethane (75 ml) was added dropwise from an addition funnel. The mixture was stirred for three and one half hours, slowly being allowed to come to room temperature as the ice bath melted. The mixture was filtered through silica gel, and the filtrate was washed successively with water, saturated NaHCO , water and brine. The organics were dried over Na2SO4, filtered and concentrated in vacuo to a pale yellow oil. Final purification of the crude product was achieved by flash chromatography (silica gel, 1/1 hexanes/ethyl acetate) to give the non- polydispersed product (46.13 g, 85 %) as a clear, colorless oil. FAB MS: m/e 239 (M+H),
Figure imgf000076_0001
Example 13 6-{2-[2-(2-{2-[2-(2-Benzyloxyethoxy)ethoxy] ethoxy}-ethoxy)- ethoxy]-ethoxy}-hexanoic acid ethyl ester (18)
Sodium hydride (3.225 g or a 60 % oil dispersion, 80.6 mmol) was suspended in 80 ml of anhydrous toluene, placed under a nitrogen atmosphere and cooled in an ice bath. A solution of the non-polydispersed alcohol 16 (27.3 g, 73.3 mmol) in 80 ml dry toluene was added to the NaH suspension. The mixture was stirred at 0°C for thirty minutes, allowed to come to room temperature and stirred for another five hours, during which time the mixture became a clear brown solution. The non-polydispersed mesylate 17 (19.21 g, 80.6 mmol) in 80 ml dry toluene was added to the NaH alcohol mixture, and the combined solutions were stirred at room temperature for three days. The reaction mixture was quenched with 50 ml methanol and filtered through basic alumina. The filtrate was concentrated in vacuo and purified by flash chromatography (silica gel, gradient elution: 3/1 ethyl acetate/hexanes to ethyl acetate) to yield the non-polydispersed product as a pale yellow oil (16.52 g, 44 %). FAB MS: m/e 515 (M+H).
Example 14
6-{2-[2-(2-{2-[2-(2-hydroxyethoxy)ethoxy]ethoxy}-ethoxy)- ethoxy]-ethoxy}-hexanoic acid ethyl ester (19)
Non-polydispersed benzyl ether 18 (1.03 g, 2.0 mmol) was dissolved in 25 ml ethanol. To this solution was added 270 mg 10 % Pd/C, and the mixture was placed under a hydrogen atmosphere and stirred for four hours, at which time TLC showed the complete disappearance of the starting material. The reaction mixture was filtered through Celite 545 to remove the catalyst, and the filtrate was concentrated in vacuo to yield the non- polydispersed title compound as a clear oil (0.67 g, 79 %). FAB MS: m/e 425 (M+H), 447 (M+Na). Example 15
6-{2-[2-(2-{2-[2-(2-methylsulfonylethoxy)ethoxy]ethoxy}- ethoxy)-ethoxy]-ethoxy}-hexanoic acid ethyl ester (20)
. The non-polydispersed alcohol 19 (0.835 g, 1.97 mmol) was dissolved in 3.5 ml dry ' dichloromethane and placed under a nitrogen atmosphere. Triethylamine (0.301 ml, 0.219 g, 2.16 mmol) was added and the mixture was chilled in an ice bath. After two minutes, the methanesulfonyl chloride (0.16 ml, 0.248 g, 2.16 mmol) was added. The mixture was stirred for 15 minutes at 0 C, then at room temperature for two hours. The reaction mixture was filtered through silica gel to remove the triethylammonium chloride, and the filtrate was washed successively with water, saturated NaHCO3, water and brine. The organics were dried over Na2SO , filtered and concentrated in vacuo. The residue was purified by column chromatography (silica gel, 9/1 ethyl acetate/methanoi) to give non-polydispersed compound 20 as a clear oil (0.819 g, 83 %). FAB MS: m/e 503 (M+H).
Example 16
6-(2-{2-[2-(2-{2-[2-(2-methoxyethoxy)ethoxy]-ethoxy}-ethoxy)- ethoxy]-ethoxy}-ethoxy)-hexanoic acid ethyl ester (21)
NaH (88 mg of a 60 % dispersion in oil, 2.2 mmol) was suspended in anhydrous toluene (3 ml) under N2 and chilled to 0 C. Non-polydispersed diethylene glycol monomethyl ether (0.26 ml, 0.26 g, 2.2 mmol) that had been dried via azeotropic distillation with toluene was added. The reaction mixture was allowed to warm to room temperature and ' stirred for four hours, during which time the cloudy grey suspension became clear and yellow and then turned brown. Mesylate 20 (0.50 g, 1.0 mmol) in 2.5 ml dry toluene was added.
After stirring at room temperature over night, the reaction was quenched by the addition of 2 ml of methanol and the resultant solution was filtered through silica gel. The filtrate was concentrated in vacuo and the FAB MS: m/e 499 (M+H), 521 (M+Na). Additional purification by preparatory chromatography (silica gel, 19/3 chloroform/methanol) provided the non-polydispersed product as a clear yellow oil (0.302 g 57 %). FAB MS: m/e 527
(M+H), 549 (M+Na). Example 17
6-(2-{2-[2-(2-{2-[2-(2-methoxyethoxy)ethoxy]-ethoxy}- ethoxy)-ethoxy]-ethoxy}-ethoxy)-hexanoic acid (22)
Non-polydispersed ester 21 (0.25 g, 0.46 mmol) was stirred for 18 hours in 0.71 ml of I N NaOH. After 18 hours, the mixture was concentrated in vacuo to remove the alcohol and the residue dissolved in a further 10 ml of water. The aqueous solution was acidified to pH 2 with 2 N HCl and the product was extracted into dichloromethane (30 ml x 2). The combined organics were then washed with brine (25 ml x 2), dried over Na2SO4, filtered and concentrated in vacuo to yield the non-polydispersed title compound as a yellow oil (0.147 g, 62 %). FAB MS: m/e 499 (M+H), 521 (M+Na).
Example 18
6-(2-{2-[2-(2-{2-[2-(2-methoxyethoxy)ethoxy]-ethoxy}-ethoxy)-ethoxy]-ethoxy}-ethoxy)- hexanoic acid 2,5-dioxo-pyrrolidin-l-yl ester (23) Non-polydispersed acid 22 (0.209 g, 0.42 mmol) were dissolved in 4 ml of dry dichloromethane and added to a dry flask already containing NHS (N-hydroxysuccinimide) (57.8 mg, 0.502 mmol) and EDC (l-(3-dime ylammopropyl)-3-e1hylcarbodiimide hydrochloride) (98.0 mg, 0.502 mmol) under a N2 atmosphere. The solution was stirred at room temperature overnight and filtered through silica gel to remove excess reagents and the urea formed from the EDC. The filtrate was concentrated in vacuo to provide the non- polydispersed product as a dark yellow oil (0.235 g, 94 %). FAB MS: m/e 596 (M+H), 618 (M+Na).
Examples 19 through 24 refer to the scheme illustrated in Figure 4.
Example 19 Mesylate of triethylene glycol monomethyl ether (24)
To a solution of CH2C12 (100 mL) cooled to 0°C in an ice bath was added non- polydispersed triethylene glycol monomethyl ether (25 g, 0.15 mol). Then triethylamine (29.5 mL, 0.22 mol) was added and the solution was stirred for 15 min at 0°C, which was followed by dropwise addition of methanesulfonyl chloride (13.8 mL, 0.18 mol, dissolved in 20 mL CH2C12). The reaction mixture was stirred for 30 min at 0°C, allowed to warm to room temperature, and then stirred for 2 h. The crude reaction mixture was filtered through Celite (washed CH2C12 -200 mL), then washed with H2O (300 mL), 5% NaHCO3 (300 mL), H2O (300 mL), sat. -NaCl (300 mL), dried MgSO , and evaporated to dryness. The oil was then placed on a vacuum line for ~2h to ensure dryness and afforded the non-polydispersed title compound as a yellow oil (29.15 g, 80% yield).
Example 20 Heptaethylene glycol monomethyl ether (25)
To a solution of non-polydispersed tetraethylene glycol (51.5 g, 0.27 mol) in THF (IL) was added potassium t-butoxide (14.8 g, 0.13 mol, small portions over -30 min). The reaction mixture was then stirred for lh and then 24 (29.15 g, 0.12 mol) dissolved in THF (90 mL) was added dropwise and the reaction mixture was stirred overnight. The cπ J^ reaction mixture was filtered through Celite (washed CH2C12, -200 mL) and evaporated to "dryness. The oil was then dissolved in HCl (250 mL, 1 N) and washed with ethyl acetate (250 mL) to remove excess 24. Additional washings of ethyl acetate (125 mL) may be required to remove remaining 24. The aqueous phase was washed repetitively with CH2C12 (125 mL volumes) until most of the 25 has been removed from the aqueous phase. The first extraction will contain 24, 25, and dicoupled side product and should be back extracted with HCl (125 L, IN). The organic layers were combined and evaporated to dryness. The resultant oil was then dissolved in CH2θ2 (100 mL) and washed repetitively with H2O (50 mL volumes) until 25 was removed. The aqueous fractions were combined, total volume 500 mL, and NaCl was added until the solution became cloudy and then was washed with CH2C1 (2 x 500 mL). The organic layers were combined, dried MgSO4, and evaporated to dryness to afford a the non- polydispersed title compound as an oil (16.9 g, 41% yield). It may be desirable to repeat one or more steps of the purification procedure to ensure high purity.
Example 21 8-Bromooctoanate (26)
To a solution of 8-bromooctanoic acid (5.0 g, 22 mmol) in ethanol (100 mL) was added H2SO (0.36 mL, 7.5 mmol) and the reaction was heated to reflux with stirring for 3 h. The crude reaction mixture was cooled to room temperature and washed H2O (100 mL), sat. NaHCO3 (2 x 100 mL), H2O (100 mL), dried MgSO4, and evaporated to dryness to afford a clear oil (5.5 g, 98% yield).
Example 22 Synthesis of MPEG7-C8 ester (27)
To a solution of the non-polydispersed compound 25 (3.0 g, 8.8 mmol) in ether (90 mL) was added potassium t-butoxide (1.2 g, 9.6 mmol) and the reaction mixture was stirred for 1 h. Then dropwise addition of the non-polydispersed compound 26 (2.4 g, 9.6 mmol), dissolved in ether (10 mL), was added and the reaction mixture was stirred overnight. The crude reaction mixture was filtered through Celite (washed CH2C12, -200 mL) and evaporated to dryness. The resultant oil was dissolved in ethyl acetate and washed H2O (.2 x 200 L), dried MgSO4, and evaporated to dryness. Column chromatography (Silica, ethyl acetate to ethyl acetate/methanol, 10:1) was performed and afforded the non-polydispersed title compound as a clear oil (0.843 g, 19% yield).
Example 23 MPEG7-C8 acid (28) . . To the oil of the non-polydispersed compound 27 (0.70 g, 1.4 mmol) was added IN NaOH (2.0 mL) and the reaction mixture was stirred for 4h. The crude reaction mixture was concentrated, acidified (pH~2), saturated with NaCl, and washed CH2C12 (2 x 50 mL). The organic layers were combined, washed sat. NaCl, dried MgSO4, and evaporated to dryness to afford the non-polydispersed title compound as a clear oil (0.35 g, 53% yield).
Example 24 Activation of MPEG7-C8 acid (29)
Non-polydispersed mPEG7-C8-acid 28 (0.3 lg, 0.64 mmol) was dissolved in 3 ml of anhydrous methylene chloride and then solution of N-hydroxysuccinimide (0.079g, 0.69 mmol) and EDCI-HC1 (135.6 mg, 0.71 mmol) in anhydrous methylene chloride added. Reaction was stirred for several hours, then washed with IN HCl, water, dried over MgSO4, filtered and concentrated. Crude material was purified by column chromatography, concentrated to afford the non-polydispersed title compound as a clear oil and dried via vacuum. Examples 25 through 29 refer to the scheme illustrated in Figure 5.
Example 25 10-hydroxydecanoate (30)
To a solution of non-polydispersed 10-hydroxydecanoic acid (5.0 g, 26.5 mmol) in ethanol (100 mL) was added H2S04 (0.43 mL, 8.8 mmol) and the reaction was heated to reflux with stirring for 3 h. The crude reaction mixture was cooled to room temperature and washed H2O (100 mL), sat. NaHCO3 (2 x 100 mL), H20 (100 mL), dried MgSO4, and. evaporated to dryness to afford the non-polydispersed title compound as a clear oil (6.9 g, 98% yield).
Example 26 Mesylate of 10-hydroxydecanoate (31) To a solution of CH2C12 (27 mL) was added non-polydispersed 10-hydroxydecanoate
30 (5.6 g, 26 mmol) and cooled to 0°C in an ice bath. Then triethylamine (5 L, 37 mmol) was added and the reaction mixture was stirred for 15 min at 0°C. Then methanesulfonyl chloride (2.7 mL, 24 mmol) dissolved in CH2C12 (3 mL) was added and the reaction mixture was stirred at 0°C for 30 min, the ice bath was removed and the reaction was stirred for an additional 2 h at room temperature. The crude reaction mixture was filtered through Celite (washed CH2C12, 80 mL) and the filtrate was washed H2O (100 mL), 5% NaHCO3 (2 x 100 mL), H2O (100 mL), sat. NaCl (100 mL), dried MgSO , and evaporated to dryness to afford the non-polydispersed title compound as a yellowish oil (7.42 g, 97% yield).
Example 27
MPEG7-Cιo Ester (32)
To a solution of non-polydispersed heptaethylene glycol monomethyl ether 25 (2.5 g, 7.3 mmol) in tetrahychofuran (100 mL) was added sodium hydride .(0.194 g, 8.1 mmol) and the reaction mixture was stirred for 1 h. Then drop wise addition of mesylate of non- polydispersed 10-hydroxydecanoate 31 (2.4 g, 8.1 mmol), dissolved in tetrahyάrofuran (10 mL), was added and the reaction mixture was stirred overnight. The crade reaction mixture was filtered through Celite (washed CH2C1 , -200 mL) and evaporated to dryness. The resultant oil was dissolved in ethyl acetate and washed H2O (2 x 200 mL), dried MgSO4, evaporated to dryness, chromatographed (sihca, ethyl acetate/methanol, 10:1), and chromatographed (silica, ethyl acetate) to afford the non-polydispersed title compound as a clear oil (0.570 g, 15% yield).
Example 28
Figure imgf000082_0001
To the oil of non-polydispersed mPEGrdo ester 32 (0.570 g, 1.1 mmol) was added IN NaOH (1.6 mL) and the reaction mixture was stirred overnight. The crade reaction mixture was concentrated, acidified (pH~2), saturated with NaCl, and washed CH2CI2 (2 50 mL). The organic layers were combined, washed sat. NaCl (2 x 50 mL), dried MgSO4, and evaporated to dryness to afford the non-polydispersed title compound as a clear oil (0.340 g, 62% yield).
Example 29
Activation of MPEG7-Cι0 Acid (34)
The non-polydispersed acid 33 was activated using procedures similar to those described above in Example 24.
Examples 30 and 31 refer to the scheme illustrated in Figure 6.
Example 30 Synthesis of C18(PEG6) Oligomer (36)
Non-polydispersed stearoyl chloride 35 (0.7g, 2.31 mmol) was added slowly to a mixture of PEG6 (5 g, 17.7 mmol) and pyridine (0.97g, 12.4 mmol) in benzene. The reaction mixture was stirred for several hours (~5). The reaction was followed by TLC using ethylacetate/methanol as a developing solvent. Then the reaction mixture was washed with water, dried over MgSO4, concentrated and dried via vacuum. Purified non-polydispersed compound 36 was analyzed by FABMS: m/e 549/ M1H. Example 31 Activation of C18(PEG6) Oligomer .
Activation of non-polydispersed C18(PEG6) oligomer was accomplished in two steps: 1) Non-polydispersed stearoyl-PEG6 36 ( 0.8 g, 1.46 mmol ) was dissolved in toluene and added to a phosgene solution (10 ml, 20 % in toluene) which was cooled with an ice bath. The reaction mixture was stirred for 1 h at 0 C and then for 3 h at room temperature. Then ' phosgene and toluene were distilled off and the remaining non-polydispersed stearoyl PEG6 chloroformate 37 was dried over P2O5 overnight. 2) To a solution of non-polydispersed stearoyl PEG6 chloroformate 36 ( 0.78g, 1.27 . mmol) and TEA (128 mg, 1.27 mmol) in anhydrous methylene chloride, N-hydroxy succinimide (NHS) solution in methylene chloride was added. The reaction mixture was stirred for 16 hours, then washed with water, dried over MgSO , filtered, concentrated and dried via vacuum to provide the non-polydispersed activated Cl 8(PEG6) oligomer 38.
Examples 32 through 37 refer to the scheme illustrated in Figure 7.
Example 32 Tetraethylene glycol monobenzylether (39) To the oil of non-polydispersed tetraethylene glycol (19.4 g, 0.10 mol) was added a solution of NaOH (4.0 g in 4.0 mL) and the reaction was stirred for 15 mm. Then benzyl chloride (3.54 mL, 30.8 mmol) was added and the reaction mixture was heated to 100°C and stirred overnight. The reaction mixture was cooled to room temperature, diluted with sat. NaCl (250 L), and washed CH2C12 (2 x 200 mL). The organic layers were combined, washed sat. NaCl, dried MgSO , and chromatographed (silica, ethyl acetate) to afford the non-polydispersed title compound as a yellow oil (6.21 g, 71% yield).
Example 33 Mesylate of tetraethylene glycol monobenzylether (40) To a solution of CH2CI2 (20 mL) was added non-polydispersed tetraethylene glycol monobenzylether 39 (6.21 g, 22 mmol) and cooled to 0°C in an ice bath. Then Iriemylamine (3.2 mL, 24 mmol) was added and the reaction mixture was stirred for 15 min at 0°C. Then methanesulfonyl chloride (1.7 mL, 24 mmol) dissolved in CH2CI2 (2 mL) was added and the reaction mixture was stirred at 0°C for 30 min, the ice bath was removed and the reaction was stirred for an additional 2 h at room temperature. The crude reaction mixture was filtered through Celite (washed CH2CI2, 80 mL) and the filtrate was washed H2O (100 mL), 5% NaHCO3 (2 x 100 mL), H20 (100 mL), sat. NaCl (100 mL), and dried MgSO4. The resulting yellow oil was chromatographed on a pad of silica containing activated carbon (10 g) to afford the non-polydispersed title compound as a clear oil (7.10 g, 89% yield).
Example 34 Octaethylene glycol monobenzylether (41)
To a solution, of tetrahydrofuran (140 L) containing sodium hydride (0.43 g, 18 mmol) was. added dropwise a solution of non-polydispersed tetraethylene glycol (3.5 g, 18 mmol) in tefrahyάxofuran (10 mL) and the reaction mixture was stirred for 1 h. Then mesylate of non-polydispersed tetraethylene glycol monobenzylether 40 (6.0 g, 16.5 mmol) dissolved in tetrahydrofuran (10 L) was added dropwise and the reaction mixture was stirred overnight. The crude reaction mixture was filtered through Celite (washed, CH2C1 , 250 mL) and the filtrate was washed H2O, dried MgSO4, and evaporated to dryness. The resultant oil was chromatographed (silica, ethyl acetate/methanol, 10:1) and chromatographed (silica, chloroform methanol, 25:1) to afford the non-polydispersed title compound as a clear oil (2.62 g, 34% yield).
Example 35 Synthesis of Stearate PEG8-Benzyl (43)
To a stirred cooled solution of non-polydispersed octaethylene glycol monobenzylether 41 (0.998 g, 2.07 mmol) and pyridine (163.9 mg, 2.07 mmol) was added non-polydispersed stearoyl chloride 42 (627.7 mg, 2.07 mmol) in benzene. The reaction mixture was stirred overnight (18 hours). The next day the reaction mixture was washed with water, dried over MgSO4, concentrated and dried via vacuum. Then the crude product was chromatographed on flash silica gel column, using 10% methanol/90% chloroform. The fractions containing the product were combined, concentrated and dried via vacuum to afford - the non-polydispersed title compound. Example 36 Hydrogenolysis of Stearate-PEG8-Benzyl
To a methanol solution of non-polydispersed stearate-PEG8-Bzl 43 (0.854g 1.138 mmol ) Pd/C(10%) (palladium, 10% wt. on activated carbon) was added. The reaction mixture was stirred overnight (18 hours) under hydrogen. Then the solution was filtered, concentrated and purified by flash column chromatography using 10% methanol/90% chloroform, fractions with Rt=0.6 collected, concentrated and dried to provide the non- polydispersed acid 44.
Example 37
Activation of C18(PEG8) Oligomer
Two step activation of non-polydispersed stearate-PEG8 oligomer was performed as described for stearate-PEG6 in Example 31 above to provide the non-polydispersed activated C18(PEG8) oligomer 45.
Example 38 Synthesis of Activated Triethylene Glycol Monomethyl Oligomers The following description refers to the scheme illustrated in Figure 8. A solution of toluene containing 20% phosgene (100 ml, approximately 18.7 g, 189 mmol phosgene) was chilled to 0°C under a N2 atmosphere. Non-polydispersed mTEG (triethylene glycol, monomethyl ether, 7.8 g, 47.5 mmol) was dissolved in 25 mL anhydrous ethyl acetate and added to the chilled phosgene solution. The mixture was stirred for one hour at 0°C, then allowed to warm to room temperature and stirred for another two and one half hours. The remaining phosgene, ethyl acetate and toluene were removed via vacuum distillation to leave the non-polydispersed mTEG chloroformate 46 as a clear oily residue.
The non-polydispersed residue 46 was dissolved in 50 mL of dry dichloromethane to which was added TEA (triethyleamine, 6.62 mL, 47.5 mmol) and NHS (N-hydroxysuccinimide, 5.8 g, 50,4 mmol). The mixture was stirred at room temperature under a dry atmosphere for twenty hours during which time a large amount of white precipitate appeared. The mixture was filtered to remove this precipitate and concentrated in vacuo. The resultant oil 47 was taken up in dichloromethane and washed twice with cold deionized water, twice with IN HCl and once with brine. The organics were dried over MgSO , filtered and concentrated to provide the non-polydispersed title compound as a clear, light yellow oil. If necessary, the NHS ester could be further purified by flash chromatography on silica gel using EtOAc as the elutant.
Example 39
Synthesis of Activated Palmitate-TEG Oligomers
The following description refers to the scheme illustrated in Figure 9. Non- polydispersed palmitic anhydride (5 g; 10 mmol) was dissolved in dry THE (20 mL) and stirred at room temperature. To the stirring solution, 3 mol excess of pyridine was added followed by non-polydispersed triethylene glycol (1.4 mL). The reaction mixture was stirred for 1 hour (progress of the reaction was monitored by TLC; ethyl acetate-chloroform; 3:7). At the end of the reaction, THF was removed and the product was mixed with 10% H2SO4 acid and extracted ethyl acetate (3 x 30 L). The combined extract was washed sequentially with water, brine, dried over MgSO4, and evaporated to give non-polydispersed product 48. A solution of N,N'-disuccinimidyl carbonate (3 mmol) in DMF (-10 L) is added to a solution of the non-polydispersed product 48 (1 mmol) in 10 mL of anydrous DMF while stirring. Sodium hydride (3 mmol) is added slowly to the reaction mixture. The reaction mixture is stirred for several hours (e.g., 5 hours). Diethyl ether is added to precipitate the activated oligomer. This process is repeated 3 times and the product is finally dried.
Example 40 Synthesis of Activated Hexaethylene Glycol Monomethyl Oligomers The following description refers to the scheme illustrated in Figure 10. Non- polydispersed activated hexaethylene glycol monomethyl ether was prepared analogously to that of non-polydispersed triethylene glycol in Example 39 above. A 20% phosgene in toluene solution (35 mL, 6.66 g, 67.4 mmol phosgene) was chilled under a N2 atmosphere in an ice/salt water bath. Non-polydispersed hexaethylene glycol 50 (1.85 mL, 2.0 g, 6.74 mmol) was dissolved in 5 mL anhydrous EtOAc and added to the phosgene solution via syringe. The reaction mixture was kept stirring in the ice bath for one hour, removed and stirred a further 2.5 hours at room temperature. The phosgene, EtOAc, and toluene were removed by vacuum distillation, leaving non-polydispersed compound 51 as a clear, oily residue. The non-polydispersed residue 51 was dissolved in 20 mL dry dichloromethane and placed under a dry, inert atmosphere. Triethylamine (0.94 mL, 0.68 g, 6.7 mmol) and then NHS (N-hydroxy succinimide, 0.82 g, 7.1 mmol) were added, and the reaction mixture was stirred at room temperature for 18 hours. The mixture was filtered through silica gel to remove the white precipitate and concentrated in vacuo. The residue was taken up in dichloromethane and washed twice with cold water, twice with 1 N HCl and once with brine. The organics were dried over Na2SO , filtered and concentrated. Final purification was done via flash chromatography (silica gel, EtOAc) to obtain the UV active non-polydispersed NHS ester 52.
Example 41
150 mg of salmon calcitonin (MW 3432, 0.043 mmol) was dissolved in 30 ml of anhydrous DMF. Then TEA (35 μL) and the activated oligomer of Example 24 (42 mg, 0.067 mmol) in anhydrous THF (2 mL) was added. The reaction was stirred for 1 hour, then quenched with 2 mL of 0.1 % TF A in water. The reaction was followed by HPLC . Then the reaction mixture was concentrated and purified by prep. HPLC (RC Vydac C18 Protein and peptide, 1x25 column, water/acetonitrile with 0.1% TFA, detection at 280 nm). Two peaks, corresponding to mono- and di-conjugate were isolated. Samples were analyzed by MALDI- MS. MS forPEG7-octyl-sCT, mono-conjugate: 3897. MS for PEG7-octyl-sCT, di- conjugate: 4361.
Example 42
The procedure of Example 41 was used to conjugate salmon calcitonin with the activated ohgomer of Example 29. MS for PEG7-decyl-sCT, mono-conjugate: 3926. MS for PEG7-decyl-sCT, di-conjugate: 4420.
Example 43
The procedure of Example 41 was used to conjugate salmon calcitonin with the activated oligomer of Example 31. MS for stearate-PEG6-sCT, mono-conjugate: 4006. MS for stearate-PEG6-sCT, di-conjugate: 4582. Example 44
The procedure, of Example 41 was used to conjugate salmon calcitonin with the activated ohgomer of Example 37. MS for stearate-PEG8-sCT, mono-conjugate: 4095.
Example 45
The procedure of Example 41 is used to conjugate salmon calcitonin with the activated oligomer of Example 18.
Example 46 The procedure of Example 41 is used to conjugate salmon calcitonin with the activated oligomer of Example 38.
Example 47
The procedure of Example 41 is used to conjugate salmon calcitonin with the activated oligomer of Example 39.
Example 48
The procedure of Example 41 is used to conjugate salmon calcitonin with the activated oligomer of Example 40.
Example 49
Determination of the Dispersity Coefficient for a Mixture of Salmon Calcitonin-Oligomer Conjugates
The dispersity coefficient of a mixture of salmon calcitonin-oligomer conjugates is determined as follows. A mixture of salmon calcitonin-oligomer conjugates is provided, for example as described above in Example 41. A first sample of the mixture is purified via
HPLC to separate and isolate the various salmon calcitonin-oligomer conjugates in the sample. Assuming that each isolated fraction contains a purely monodispersed mixture of conjugates, "n" is equal to the number of fractions collected. The mixture may include one or more of the following conjugates, which are described by stating the conjugation position followed by the degree of conjugation: Lys11 monoconjugate; Lys18 monoconjugate; N- terminus monoconjugate; Lys11' 18 diconjugate; Lys11, N-terminus diconjugate; Lys1 , N- terminus diconjugate; and/or Lys11,18, N-terminus triconjugate. Each isolated fraction of the mixture is analyzed via mass spectroscopy to determine the mass of the fraction, which allows each isolated fraction to be categorized as a mono-, di-, or tri-conjugate and provides a value for the variable "M,-" for each conjugate in the sample.
A second sample of. the mixture is analyzed via HPLC to provide an HPLC trace. Assuming that the molar absorptivity does not change as a result of the conjugation, the , weight percent of a particular conjugate in the mixture is provided by the area under the peak of the HPLC trace corresponding to the particular conjugate as a percentage of the total area under all peaks of the HPLC trace. The sample is collected and lyophilized to dryness to determine the anhydrous gram weight of the sample. The gram weight of the sample is multiplied by the weight percent of each component in the sample to determine the gram weight of each conjugate in the sample. The variable "Ni" is determined for a particular conjugate (the ith conjugate) by dividing the gram weight of the particular conjugate in the sample by the mass of the particular conjugate and multiplying the quotient by Avagadro's number (6.02205 x 1023 mole"1), Mi, determined above, to give the number of molecules of the particular conjugate, Ni, in the sample.. The dispersity coefficient is then calculated using n, Mj as determined for each conjugate, and Ni as determined for each conjugate.
Example 50 Cytosensor® Studies T-47D cells (mammary ductal carcinoma cell line, obtained from American Type
.Culture Collection were suspended at a density of 1 x 107 cells/mL in running buffer (low- buffered, serum-free, bicarbonate-free RPMI 1640 medium from Molecular Devices of Sunnyvale, Cahfornia. Approximately 100,000 cells were then immobilized in an agarose cell entrapment medium in a 10 μL droplet and sandwiched between two 3-μm polycarbonate membranes in a cytosensor capsule cup. Cytosensor capsule cups placed in sensor chambers on the Cytosensor® Microphysiometer were then held in very close proximity to pH-sensitive detectors. Riinning buffer was then pumped across the cells at a rate of 100 μL/rnin except during 30-second intervals when the flow was stopped, and acidification of the running buffer in the sensor chamber was measured. Acidification rates were determined every 2 minutes. The temperature of the sensor chambers was 37°C. Cells were allowed to equilibrate in the sensor chambers for 2-3 hours prior to the start of the experiment during which time basal acidification rates were monitored. Cells were then exposed to test compounds. (Salmon Calcitonin or Octyl-Di-Calcitonin) diluted in ranning buffer at various nM concentration. Exposure of cells to test compounds occurred for the first 40 seconds of each 2 minute pump cycle in a repeating pattern for a total of 20 minutes. This allowed sufficient exposure of the cells, to the test compounds to elicit a receptor-mediated response in cellular metabolism followed by approximately 50 seconds of flow of the running buffer containing no compounds. This procedure rinsed away test solutions (which had a slightly lower pH than ranning buffer alone) from the sensor chamber before measuring the acidification rate. Thus, the acidification rates were solely a measure of cellular activity. A similar procedure was used to obtain data for PEG7-octyl-sCT, monoconjugate (Octyl-Mono); PEG7-decyl-sCT, monoconjugate (Decyl-Mono); PEG7-decyl-sCT, diconjugate (Decyl-Di); stearate-PEG6- sCT, monoconjugate (PEG6 St. Mono); and stearate-PEG8-sCT, monoconjugate (PEG8 St. Mono). Data was analyzed for relative activity of compounds by calculating the Area Under the Curve (AUC) for each cytosensor chamber acidification rate graph and plotted as a bar chart illustrated in Figure 14 showing average AUC measurements taken from multiple experiments performed under the same experimental conditions.
Example 51 Enzymatic Stability
Compounds, supplied as lyophilized powders, are resuspended in 10 mM phosphate buffer pH 7.4 and then submitted for concentration determination by HPLC. The phosphate buffer is used to create a solution with a pH that is optimum for activity of each particular gut enzyme. Aliquots of the compound thus prepared are transferred to 1.7 mL microcentrifuge tubes and shaken in a 37°C water bath for 15 minutes to allow compounds to equilibrate to temperature. After 15 minutes, 2 μL of the appropriate concentrated gut enzyme is added to each tube to achieve the final concentration desired. Chymotrypsin and trypsin are resuspended in 1 mM HCl. Also, as a control, compounds are treated with 2 μL of 1 mM HCl. Immediately following additions, 100 μL of sample is removed from the control tube and quenched with either 25 μL of chymotrypsin/trypsin quenching solution ( 1 : 1 1 % TFAIsopropanol). This sample will serve as T=0 min. A sampling procedure is repeated at various time intervals depending on the gut enzyme used. Chymotrypsin has 15, 30 and 60 ■minute samples. Trypsin has 30, 60, 120 and 180 minute samples. Once all points have been acquired, a final sample is removed from the control tube to make sure that observed degradation is not temperature or buffer related. The chymotrypsin and trypsin samples may be collected directly into HPLC vials. RP-HPLC (acetonitrile gradient) is used to determine AUC for each sample and % degradation is calculated based from the T=0 min control. The results are provided below in Tables 1 to 4.
Table 1
% Remaining Following 0.5 U/mL Chymotrypsin Digest of PEG7-Octyl-Salmon Calcitonin, Diconjugate
Figure imgf000091_0001
Table 2
% Remaining Following 0.5 U/mL Chymotrypsin Digest of Salmon Calcitomn (for comparison purposes; not part of the invention)
Figure imgf000091_0002
Table 3
% Remaining following 1 U/mL Trypsin Digest of PEG7-Octyl-Sahnon Calcitonin, Diconjugate
Figure imgf000092_0001
Table 4
% Remaining following 1 U/mL Trypsin Digest of Salmon Calcitonin (for comparison purposes; not part of the invention)
Figure imgf000092_0002
Example 52 Activity and Inter-Subject Variability
Male CF-1 mice (Charles River, Raleigh, NC) weighing 20-25 g were housed in the Nobex vivarium in a light- (L:D cycle of 12:12, lights on at 0600 h), temperature- (21-23°C), and humidity- (40-60 % relative humidity) controlled room. Animals were permitted free access to laboratory chow (PMI Nutrition) and tap water. Mice were allowed to acclimate to housing conditions for 48-72 hours prior to the day of experiment.
Prior to dosing, mice were fasted overnight and water was provided ad libitum. Mice were randomly distributed into groups of five animals per time point and were administered a single oral dose of a PEG7-octyl-sCT, diconjugate (Octyl Di) according to the present invention or salmon calcitonin (sCT or Calcitonin) for comparison purposes. Oral doses were administered using a gavaging needle (Popper #18, 5 cm from hub to bevel) at 10 mL/kg in the following 0.2 μg mL phosphate-buffered PEG7-octyl-sCT, diconjugate, formulation:
Figure imgf000093_0001
The buffered formulation was prepared by adding 80 mL of phosphate buffer in a clean tared glass beaker. The sodium cholate was slowly added to the phosphate buffer with stirring until dissolved. The deoxy cholate was then added and stirring was continued until dissolved.
The PEG7-octyl-sCT, diconjugate, solution equivalent to 20 μg was added. Finally, the remaining phosphate buffer was added to achieve a final weight of 100 g. Vehicle-control mice were used in all experiments. Dose-response curves were constructed using a single time point 60 minutes after drug administration. These curves are illustrated in Figures 15-
18.
At appropriate time points, mice were ether-anesthetized, the vena cavae exteriorized, and blood samples were obtained via a syringe fitted with a 25-gauge needle. Blood aliquots were allowed to clot at 22°C for 1 hour, and the sera removed and pipetted into a clean receptacle. Total serum calcium was determined for each animal using a calibrated Vitros
DT60 H analyzer.
Serum calcium data were plotted and pharmacokinetic parameters determined via curve-fitting techniques using SigmaPlot software (Version 4.1). Means and standard deviations (or standard errors) were calculated and plotted to determine effect differences among dosing groups. Average serum calcium data for various conjugates are provided in Table 5 below.
Table 5
Figure imgf000094_0001
Despite an in vitro activity as determined in Example 50 above that may not be comparable with the in vitro activity of PEG7-octyl-sCT and PEG7-decyl-sCT mono- and diconjugates, the stearate-PEG6-sCT, diconjugate, and stearate-PEG8-sCT, diconjugate, appear to have in vivo activity (as evidenced by the drops in % baseline calcium from Table 5 above) that are comparable with the in vivo activity observed for the PEG7-oetyl-sCT and PEG7-decyl-sCT, mono- and di-conjugates. While not wanting to be bound by a particular theory, the improved in vivo activity of the stearate containing conjugates may indicate that these conjugates are undergoing hydrolysis in vivo to provide an active salmon calcitonin or active salmon calcitonin-PEG conjugate.
In the specification, there has been disclosed typical preferred embodiments of the invention and, although specific terms are employed, they are used in a generic and descriptive sense only and not for purposes of limitation, the scope of the invention being set forth in the following claims.

Claims

What is Claimed is:
1. A substantially monodispersed mixture ot conjugates, eacή conjugate comprising a calcitonin drag coupled to an oligomer that comprises a polyethylene glycol moiety.
2. The mixture according to Claim 1 , wherein the polyethylene glycol moiety has at least 2, 3 or 4 polyethylene glycol subunits.
3. The mixture according to Claim 1, wherein the polyethylene glycol moiety has at least 5 or 6 polyethylene glycol subunits.
4. The mixture according to Claim 1 , wherein the polyethylene glycol moiety has at least 7 polyethylene glycol subunits.
5. The mixture according to Claim 1, wherein the mixture of conjugates is a monodispersed mixture.
6. The mixture according to Claim 1, wherein the mixture of conjugates is a substantially purely monodispersed mixture.
7. The mixture according to Claim 1, wherein the mixture of conjugates is a purely monodispersed mixture.
8. The mixture according to Claim 1 , wherein the mixture has the capability of lowering serum calcium levels by at least 5 percent.
9. The mixture according to Claim 1, wherein the mixture has an increased resistance to degradation by chymotrypsin when compared to the resistance to degradation by chymotrypsin of the calcitonin drag which is not coupled to the oligomer.
10. The mixture according to Claim 1 , wherein the mixture has a bioefficacy that is greater than the bioefficacy of the calcitonin drag which is not coupled to the oligomer.
11. The mixture according to Claim 1, wherein the calcitonin drug is salmon calcitonin.
12. The mixture according to Claim 11, wherein the oligomer is covalently coupled to an amine function of the salmon calcitonin.
13. The mixture according to Claim 12, wherein the amine function is at Lys1 ' or Lys18 of the salmon calcitonin.
14. The mixture according to Claim 11, wherein the conjugate comprises a first oligomer and a second oligomer.
15. The mixture according to Claim 14, wherein the first ohgomer is covalently coupled to Lys11 of the salmon calcitonin and the second ohgomer is covalently coupled to Lys of the salmon calcitonin.
16. The mixture according to Claim 1 , wherein the calcitonin drug is covalently coupled to the oligomer.
17. The mixture according to Claim 1 , wherein the calcitonin drug is covalently coupled to the oligomer by a hydrolyzable bond.
18. The mixture according to Claim 1, wherein the calcitonin drug is covalently coupled to the polyethylene glycol moiety of the oligomer.
19. The mixture according to Claim 1 , wherein the oligomer further comprises a lipophilic moiety.
20. The mixture according to Claim 19, wherein the calcitonin drug is covalently coupled to the lipophilic moiety.
21. The mixture according to Claim 20, wherein the polyethylene glycol moiety is covalently coupled to the lipophilic moiety.
22. The mixture according to Claim 1, wherein the conjugate comprises a plurality of oligomers.
23. The mixture according to Claim 22, wherein each oligomer in the plurality of oligomers has the same molecular structure.
24. The mixture according to Claim 1, wherein the oligomer comprises a first polyethylene glycol moiety covalently coupled to the calcitonin drug by a non-hydrolyzable bond and a second polyethylene glycol moiety covalently coupled to the first polyethylene glycol moiety by a hydrolyzable bond.
25. The monodispersed mixture according to Claim 24, wherein the ohgomer further comprises a lipophilic moiety covalently coupled to the second polyethylene glycol moiety.
26. The monodispersed mixture according to Claim 1, wherein the conjugates are each amphiphilically balanced such that each conjugate is aqueously soluble and able to penetrate biological membranes.
27. A pharmaceutical composition comprising: the mixture according to Claim 1; and a pharmaceutically acceptable carrier.
28. A substantially monodispersed mixture of conjugates each comprising salmon calcitonin covalently coupled at Lys11 of the salmon calcitomn to the carboxyHc acid moiety of a carboxylic acid, which is covalently coupled at the end distal to the carboxyHc acid moiety to a methyl terminated polyethylene glycol moiety having at least 7 polyethylene glycol subunits, and covalently coupled at Lys of the salmon calcitonin to the carboxylic acid moiety of a carboxylic acid, which is covalently coupled at the end distal to the carboxylic acid moiety to a methyl terminated polyethylene glycol moiety having at least 7 polyethylene glycol subunits.
29. The mixture according to Claim 28, wherein the conjugates each consist of salmon calcitonin covalently coupled at Lys11 of the salmon calcitonin to the carboxyHc acid moiety of octanoic acid, which is covalently coupled at the end distal to the carboxylic acid moiety to a methyl terminated polyethylene glycol moiety having 7 polyethylene glycol
18 subunits, and covalently coupled at Lys of the salmon calcitonin to the carboxylic acid moiety of octanoic acid, which is covalently coupled at the end distal to the carboxylic acid moiety to a methyl terminated polyethylene glycol moiety having 7 polyethylene glycol subunits.
30. The mixture according to Claim 28, wherein the mixture is a monodispersed mixture.
31. The mixture according to Claim 28, wherein the mixture is a substantially purely monodispersed mixture.
32. The mixture according to Claim 28, wherein the mixture is a purely monodispersed mixture.
33. A method of treating a bone disorder in a subj ect in need of such treatment, said method comprising: administering an effective amount of a substantially monodispersed mixture of conjugates each comprising a calcitonin drug coupled to an oligomer that comprises a polyethylene glycol moiety to the subject to treat the bone disorder.
34. The method according to Claim 33, wherein the bone disorder is characterized by excessive osteoclastic bone resorption or hypercalcemic serum effects.
35. The method according to Claim 33, wherein the bone disorder is osteoporosis Paget's disease, or hypercalcemia.
36. A substantially monodispersed mixture of conjugates each conjugate comprising a calcitonin drag coupled to an oligomer comprising a polyethylene glycol moiety, said mixture capable of lowering serum calcium levels in a subject by at least 5 percent.
37. The mixture according to Claim 36, wherein the mixture is capable of lowering serum calcium levels in a subject by at least 10 percent.
38. The mixture according to Claim 36, wherein the mixture is capable of lowering serum calcium levels in a subject by at least 20 percent.
39. The mixture according to Claim 36, further having an increased resistance to degradation by chymotrypsin or trypsin when compared to the resistance to degradation by chymotrypsin or trypsin of the calcitonin drug which is not coupled to the oligomer.
40. The mixture according to Claim 36, further having a bioefficacy higher than the bioefficacy of the calcitonin drug which is not coupled to the oligomer.
41. A mixture of conjugates each conjugate comprising a calcitonin drag coupled o an oligomer that comprises a polyethylene glycol moiety having at least 4 polyethylene glycol subunits, said mixture having a molecular weight distribution with a standard deviation of less than about 22 Daltons.
42. The mixture of conjugates according to Claim 41 , wherein the standard deviation of the molecular weight distribution is less than about 14 Daltons.
43. The mixture of conjugates according to Claim 41 , wherein the standard deviation of the molecular weight distribution is less than about 11 Daltons.
44. The mixture of conjugates according to Claim 41, wherein each conjugate in the mixture comprises salmon calcitonin coupled at Lys11 to a first oligomer and coupled at
1 R
Lys . to a second oligomer, and wherein the first ohgomer and the second oligomer each have the formula:
O — C— (CH2)7— (OC2H4)7-OCH3 .
45. The mixture of conjugates according to Claim 41 , wherein each conjugate in the mixture comprises salmon calcitonin coupled at Lys11 to a first oligomer and coupled at Lys18 to a second oligomer, and wherein the first oligomer and the second oligomer each have the formula:
O O
II II
— C-(OCH2CH2)6-OC(CH2)16CH3
46. The mixture of conjugates according to Claim 41 , wherein each conjugate in 1 1 the mixture comprises salmon calcitonin coupled at Lys or Lys to an oligomer having the formula:
O — C— (CH2)9 — (OC2H4)7 - OCH3
47. A mixture of conjugates each comprising a calcitonin drag coupled to an oligomer that comprises a polyethylene glycol moiety, wherein the mixture has a dispersity coefficient (DC) greater than 10,000 where
Figure imgf000100_0001
wherein: n is the number of different molecules in the sample;
Nj is the number of i~ molecules in the sample; and Mj is the mass of the i- molecule.
48. The mixture of conjugates according to Claim 47, wherein the dispersity coefficient is greater than 100,000.
49. The mixture of conjugates according to Claim 47, wherein the dispersity coefficient is greater than 500,000.
50. The mixture of conjugates according to Claim 47, wherein each conjugate in the mixture comprises salmon calcitonin coupled at Lys11 to a first ohgomer and coupled at
Lys to a second oligomer, and wherem the first oligomer and the second ohgomer each . have the formula:
O
II — C— (CH2)7— (OC2H4)7— OCH3 .
51. The mixture of conjugates according to Claim 47, wherein each conjugate in the mixture comprises salmon calcitonin coupled at Lys11 to a first oligomer and coupled at Lys18 to a second oligomer, and wherein the first oligomer and the second oligomer each have the formula:
O O
II II
-C— (OCH2CH2)6-OC(CH2)16CH3
52. The mixture of conjugates according to Claim 41, wherein each conjugate in the mixture comprises salmon calcitonin coupled at Lys11 or Lys18 to an oligomer having the formula:
O
II
— C— (CH2)9 — (OC2H4)7-OCH3 .
53. A mixture of conjugates in which each conjugate: comprises a calcitonin drag coupled to an oligomer; and has the same number of polyethylene glycol subunits.
54. The mixture of conjugates according to Claim 53, wherein each conjugate in the mixture comprises salmon calcitonin coupled at Lys11 to a first oligomer and coupled at Lys18 to a second oligomer, and wherein the first oligomer and the second oligomer each have the formula:
O — C— (CH2)7— (OC2H4)7-OCH3 .
-55. The mixture of conjugates according to Claim 53, wherein each conjugate in the mixture comprises salmon calcitonin coupled at Lys11 to a first oligomer and coupled at Lys18 to a second oligomer, and wherein the first oligomer and the second ohgomer each have the formula:
O O
II II
— C-(OCH2CH2)6-OC(CH2)16CH3
56. The mixture of conjugates according to Claim 53, wherein each conjugate in the mixture comprises salmon calcitonin coupled at Lys11 or Lys18 to an oligomer having the formula:
O — C— (CH2)9— (OC2H4)7- OCH3 .
57. A mixture of conjugates in which each conjugate has the same molecular weight and has the structure of the formula:
Figure imgf000102_0001
wherein: B is a bonding moiety;
L is a linker moiety; G, G' and G" are individually selected spacer moieties; R is a lipophilic moiety and R' is a polyalkylene glycol moiety, or R' is the lipophilic moiety and R is the polyalkylene glycol moiety; T is a terminating moiety; j, k, m and n are individually 0 or 1; and p is an integer from 1 to the number of nucleophilic residues on the. calcitonin drug.
58. The mixture of conjugates according to Claim 57, wherein the polyalkylene glycol moiety is a polyethylene glycol moiety.
59. The mixture of conjugates according to Claim 58, wherein the polyethylene glycol moiety has at least 2, 3 or 4 polyethylene glycol subunits.
60. The mixture of conjugates according to Claim 58, wherein the polyethylene glycol moiety has at least 5 or 6 polyethylene glycol subunits.
61. The mixture of conjugates according to Claim 58, wherein the polyethylene glycol moiety has at least 7 polyethylene glycol subunits.
62. The mixture of conjugates according to Claim 57, wherein: R is alkyl or alkylene;
R' is a polyethylene glycol moiety having at least 6 polyethylene glycol subunits; T is alkyl or alkoxy; j is 1; and k, m and n are 0.
63. The mixture of conjugates according to Claim 57, wherein: B is carbonyl;
R is C7-C16 alkylene;
R' is a polyethylene glycol moiety having between 6 and 8 polyethylene glycol subunits;
T is methyl or methoxy; and k, m and n are 0.
64. A process for synthesizing a substantially monodispersed mixture of conjugates each conjugate comprising a calcitonin drug coupled to an oligomer that comprises a polyethylene glycol moiety, said process comprising: reacting a substantially monodispersed mixture comprising compounds having the structure of Formula I:
Figure imgf000104_0001
wherein R1 is H or a lipophilic moiety; m is from 1 to 25; and * is a positive ion, with a substantially monodispersed mixture comprising compounds having the structure of Formula II:
R2(OC2H4)„-OMs (H) wherein R2 is H or a lipophilic moiety; and n is from 1 to 25, under conditions sufficient to provide a substantially monodispersed mixture comprising polymers having the stracture of Formula HI: R2(OC2H4)m+n-OR1 (HI); activating the substantially monodispersed mixture, comprising polymers of Formula HI to provide a substantially monodispersed mixture of activated polymers capable of reacting with a calcitonin drug; and reacting the substantially monodispersed mixture of activated polymers with a substantially monodispersed mixture of calcitonin drugs under conditions sufficient to provide a substantially monodispersed mixture of conjugates each comprising a calcitonin drug coupled to an oligomer that comprises a polyethylene glycol moiety with m+n subunits.
65. The process according to Claim 64, wherein R2 is a fatty acid moiety or an ester of a fatty acid moiety.
66. The process according to Claim 65, wherein the fatty acid moiety or the ester of a fatty acid moiety comprises an alkyl moiety at least 5 carbon atoms in length.
67. The process according to Claim 64, wherein R1 is a methyl group.
68. The process according to Claim 64, further comprising: reacting a substantially monodispersed mixture comprising compounds having the stracture of Formula V:
R2(OC2H4)n-OH (V) with a methanesulfonyl halide under conditions sufficient to provide a substantially monodispersed mixture comprising compounds having the structure of Formula H:
R2(OC2H4)n-OMs (H).
69. The process according to Claim 68, further comprising: reacting a substantially monodispersed mixture comprising compounds having the stracture of Formula VI:
R2-OMs (VI) wherein R2 is a lipophilic moiety; with a substantially monodispersed mixture comprising compounds having the stracture of Formula VH:
R3(OC2H4)m-O-X2 + (VH) wherein R3 is benzyl, trityl, or THP; and X2 + is a positive ion; under conditions sufficient to provide a substantially monodispersed mixture comprising compounds having the structure of Formula VHI:
R3(OC2H4)m-OR2 (VHI); and reacting the substantially monodispersed mixture comprising compounds having the stracture of Formula VIH under conditions sufficient to provide a substantially monodispersed mixture comprising compounds having the stracture of Formula V:
R2(OC2H4)m-OH (V).
70. The process according to Claim 64, further comprising: reacting a substantially monodispersed mixture comprising compounds having the structure of Formula IV:
R^OCzH n-OH (TV) under conditions sufficient to provide a substantially monodispersed mixture comprising compounds having the structure of Formula I:
R1(OC2H4)n-O'X+ (I).
71. The process according to Claim 64, wherein the activating of the substantially monodispersed mixture comprises reacting the substantially monodispersed mixture of polymers of Formula IH with N-hydroxy succinimide to provide an activated polymer capable of reacting with a calcitonin drug.
72. The process according to Claim 64, wherein the calcitonin drug is salmon calcitonin, and wherein the reacting of the substantially monodispersed mixture of activated polymers with a substantially monodispersed mixture of salmon calcitonin comprises: reacting the substantially monodispersed mixture of activated polymers with Lys11 and Lys18 of the salmon calcitonin to provide a substantially monodispersed mixture of diconjugates each comprising a salmon calcitonin coupled to two oligomers that each comprise a polyethylene glycol moiety with m+n subunits.
PCT/US2002/017575 2001-06-04 2002-06-04 Mixtures of calcitonin drug-oligomer conjugates comprising polyalkylene glycol, uses thereof, and methods of making same WO2002098451A1 (en)

Priority Applications (8)

Application Number Priority Date Filing Date Title
DK02732030.8T DK1404360T3 (en) 2001-06-04 2002-06-04 Monodispersed mixtures of calcitonin drug-oligomer conjugates comprising polyethylene glycol, uses thereof, and methods for preparing these
DE60235010T DE60235010D1 (en) 2001-06-04 2002-06-04 MONODISPERSIC MIXTURES OF CALCITONIN MEDICAMENT OLIGOMER CONJUGATES WITH POLYETHYLENE GLYCOL, THEIR USE AND METHOD OF PREPARING THEM
JP2003501489A JP4272510B2 (en) 2001-06-04 2002-06-04 Mixtures of calcitonin drug-oligomer conjugates containing polyalkylene glycols, their use, and methods for their production
EP02732030A EP1404360B1 (en) 2001-06-04 2002-06-04 Monodisperse mixtures of calcitonin drug-oligomer conjugates comprising polyethylene glycol, uses thereof, and methods of making same
KR1020037015912A KR100930606B1 (en) 2001-06-04 2002-06-04 Mixtures of calcitonin drug-oligomeric conjugates comprising polyalkylene glycols, uses thereof and methods for their preparation
MXPA03011283A MXPA03011283A (en) 2001-06-04 2002-06-04 Mixtures of calcitonin drug-oligomer conjugates comprising polyalkylene glycol, uses thereof, and methods of making same.
AT02732030T ATE454160T1 (en) 2001-06-04 2002-06-04 MONODISPERSE MIXTURES OF CALCITONIN-DRUG OLIGOMER CONJUGATES WITH POLYETHYLENE GLYCOL, THEIR USE AND METHOD FOR THEIR PRODUCTION
CA002449686A CA2449686A1 (en) 2001-06-04 2002-06-04 Mixtures of calcitonin drug-oligomer conjugates comprising polyalkylene glycol, uses thereof, and methods of making same

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US09/873,777 US6713452B2 (en) 2001-06-04 2001-06-04 Mixtures of calcitonin drug-oligomer conjugates comprising polyalkylene glycol, uses thereof, and methods of making same
US09/873,777 2001-06-04

Publications (1)

Publication Number Publication Date
WO2002098451A1 true WO2002098451A1 (en) 2002-12-12

Family

ID=25362290

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2002/017575 WO2002098451A1 (en) 2001-06-04 2002-06-04 Mixtures of calcitonin drug-oligomer conjugates comprising polyalkylene glycol, uses thereof, and methods of making same

Country Status (13)

Country Link
US (2) US6713452B2 (en)
EP (1) EP1404360B1 (en)
JP (1) JP4272510B2 (en)
KR (1) KR100930606B1 (en)
CN (1) CN100515492C (en)
AT (1) ATE454160T1 (en)
CA (1) CA2449686A1 (en)
CY (1) CY1109932T1 (en)
DE (1) DE60235010D1 (en)
DK (1) DK1404360T3 (en)
ES (1) ES2339224T3 (en)
MX (1) MXPA03011283A (en)
WO (1) WO2002098451A1 (en)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005004792A2 (en) 2003-06-24 2005-01-20 Nobex Corporation Mixtures of calcitonin drug-oligomer conjugates and methods of use in pain treatment
JP2006515622A (en) * 2003-01-13 2006-06-01 ニュー リバー ファーマシューティカルズ インコーポレイテッド Carbohydrate conjugates to prevent the abuse of controlled substances
US8133881B2 (en) 2003-01-13 2012-03-13 Shire Llc Carbohydrate conjugates to prevent abuse of controlled substances
US9012469B2 (en) 2010-09-30 2015-04-21 Astrazeneca Ab Crystalline naloxol-peg conjugate
EP2905033A1 (en) * 2003-12-16 2015-08-12 Nektar Therapeutics Monodisperse PEGylated naloxol compositions
US9308263B2 (en) 2011-10-21 2016-04-12 Seachaid Pharmaceuticals, Inc. Pharmaceutical compositions and uses thereof
US11129794B2 (en) 2003-12-16 2021-09-28 Nektar Therapeutics Chemically modified small molecules

Families Citing this family (28)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6191105B1 (en) * 1993-05-10 2001-02-20 Protein Delivery, Inc. Hydrophilic and lipophilic balanced microemulsion formulations of free-form and/or conjugation-stabilized therapeutic agents such as insulin
US6703381B1 (en) * 1998-08-14 2004-03-09 Nobex Corporation Methods for delivery therapeutic compounds across the blood-brain barrier
US6638906B1 (en) * 1999-12-13 2003-10-28 Nobex Corporation Amphiphilic polymers and polypeptide conjugates comprising same
US8394813B2 (en) 2000-11-14 2013-03-12 Shire Llc Active agent delivery systems and methods for protecting and administering active agents
US6835802B2 (en) 2001-06-04 2004-12-28 Nobex Corporation Methods of synthesizing substantially monodispersed mixtures of polymers having polyethylene glycol moieties
US20090281023A9 (en) * 2001-06-04 2009-11-12 Nobex Corporation Mixtures Of Calcitonin Drug-Oligomer Conjugates And Methods Of Use In Pain Treatment
US6828305B2 (en) 2001-06-04 2004-12-07 Nobex Corporation Mixtures of growth hormone drug-oligomer conjugates comprising polyalkylene glycol, uses thereof, and methods of making same
US6828297B2 (en) * 2001-06-04 2004-12-07 Nobex Corporation Mixtures of insulin drug-oligomer conjugates comprising polyalkylene glycol, uses thereof, and methods of making same
US6713452B2 (en) * 2001-06-04 2004-03-30 Nobex Corporation Mixtures of calcitonin drug-oligomer conjugates comprising polyalkylene glycol, uses thereof, and methods of making same
US6858580B2 (en) * 2001-06-04 2005-02-22 Nobex Corporation Mixtures of drug-oligomer conjugates comprising polyalkylene glycol, uses thereof, and methods of making same
US7713932B2 (en) * 2001-06-04 2010-05-11 Biocon Limited Calcitonin drug-oligomer conjugates, and uses thereof
US7169752B2 (en) * 2003-09-30 2007-01-30 New River Pharmaceuticals Inc. Compounds and compositions for prevention of overdose of oxycodone
US20060014697A1 (en) * 2001-08-22 2006-01-19 Travis Mickle Pharmaceutical compositions for prevention of overdose or abuse
US7030082B2 (en) * 2001-09-07 2006-04-18 Nobex Corporation Pharmaceutical compositions of drug-oligomer conjugates and methods of treating disease therewith
US7196059B2 (en) * 2001-09-07 2007-03-27 Biocon Limited Pharmaceutical compositions of insulin drug-oligomer conjugates and methods of treating diseases therewith
US6770625B2 (en) * 2001-09-07 2004-08-03 Nobex Corporation Pharmaceutical compositions of calcitonin drug-oligomer conjugates and methods of treating diseases therewith
AU2003285200A1 (en) * 2002-11-09 2004-06-03 Nobex Corporation Modified carbamate-containing prodrugs and methods of synthesizing same
US7648962B2 (en) * 2002-11-26 2010-01-19 Biocon Limited Natriuretic compounds, conjugates, and uses thereof
CN100558398C (en) * 2002-11-26 2009-11-11 拜奥康有限公司 Natriuresis chemical compound, conjugate and the application thereof of modifying
WO2004073620A2 (en) * 2003-02-14 2004-09-02 Quanta Biodesign, Ltd The selective and specific preparation of discrete peg compounds
GB0305977D0 (en) * 2003-03-15 2003-04-23 Koninkl Philips Electronics Nv Control of a conditional access mechanism
US8329958B2 (en) 2004-07-02 2012-12-11 Biocon Limited Combinatorial synthesis of PEG oligomer libraries
DK2626368T3 (en) * 2004-07-19 2017-02-27 Biocon Ltd INSULIN OLIGOMS CONJUGATES, FORMULATIONS AND APPLICATIONS THEREOF
US20080207505A1 (en) * 2005-01-12 2008-08-28 James Kenneth D Bna Conjugates and Methods of Use
JP5868594B2 (en) * 2007-10-16 2016-02-24 バイオコン・リミテッドBiocon Limited Orally administrable solid pharmaceutical composition and process thereof
JP5892940B2 (en) 2009-11-25 2016-03-23 アリスジェン ソシエテ アノニム Mucosal delivery composition comprising a peptide complexed with a crown compound and / or counterion
CN102875427B (en) * 2012-09-18 2014-02-19 安徽世华化工有限公司 Synthetic method of 2-methoxy methylmesylate
CN113087623A (en) * 2021-04-13 2021-07-09 苏州昊帆生物股份有限公司 Synthesis method of 8-bromoethyl octanoate

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0511903A2 (en) * 1991-04-23 1992-11-04 Teva Pharmaceutical Industries Ltd. Conjugate of calcitonin and polyethylene glycol
US5349052A (en) * 1988-10-20 1994-09-20 Royal Free Hospital School Of Medicine Process for fractionating polyethylene glycol (PEG)-protein adducts and an adduct for PEG and granulocyte-macrophage colony stimulating factor
US5359030A (en) 1993-05-10 1994-10-25 Protein Delivery, Inc. Conjugation-stabilized polypeptide compositions, therapeutic delivery and diagnostic formulations comprising same, and method of making and using the same
WO1997014740A1 (en) 1995-10-19 1997-04-24 Receptagen Corporation Discrete-length polyethylene glycols
WO2000009073A2 (en) 1998-08-14 2000-02-24 Nobex Corporation Blood-brain barrier therapeutics
WO2000078302A1 (en) 1999-06-19 2000-12-28 Nobex Corporation Amphiphilic drug-oligomer conjugates with hydrolyzable lipophile components and methods for making and using the same
US6191105B1 (en) 1993-05-10 2001-02-20 Protein Delivery, Inc. Hydrophilic and lipophilic balanced microemulsion formulations of free-form and/or conjugation-stabilized therapeutic agents such as insulin

Family Cites Families (156)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3256153A (en) * 1963-02-08 1966-06-14 Smith Kline French Lab Method of stabilizing wax-fat coating materials and product thereof
US4003792A (en) * 1967-07-01 1977-01-18 Miles Laboratories, Inc. Conjugates of acid polysaccharides and complex organic substances
US3950517A (en) * 1970-05-08 1976-04-13 National Research Development Corporation Insulin derivatives
GB1381274A (en) * 1971-01-28 1975-01-22 Nat Res Dev Insulin derivatives
US3919411A (en) * 1972-01-31 1975-11-11 Bayvet Corp Injectable adjuvant and compositions including such adjuvant
US4044196A (en) * 1972-03-30 1977-08-23 Bayer Aktiengesellschaft Crosslinked copolymers of α,β-olefinically unsaturated dicarboxylic anhydrides
US4179337A (en) 1973-07-20 1979-12-18 Davis Frank F Non-immunogenic polypeptides
FR2408387A2 (en) * 1975-06-30 1979-06-08 Oreal COMPOSITIONS BASED ON AQUEOUS DISPERSIONS OF LIPID SPHERULES
US4087390A (en) * 1977-02-02 1978-05-02 Eli Lilly And Company Somatostatin analogs and intermediates thereto
US4093574A (en) * 1977-02-02 1978-06-06 Eli Lilly And Company Somatostatin analogs and intermediates thereto
GB1492997A (en) 1976-07-21 1977-11-23 Nat Res Dev Insulin derivatives
US4223163A (en) 1976-12-10 1980-09-16 The Procter & Gamble Company Process for making ethoxylated fatty alcohols with narrow polyethoxy chain distribution
JPS53116315A (en) * 1977-03-17 1978-10-11 Ueno Seiyaku Oyo Kenkyujo Kk Powder or granular containing improved sorbinic acid
US4100117A (en) * 1977-04-21 1978-07-11 Eli Lilly And Company Somatostatin analogs and intermediates thereto
US4253998A (en) * 1979-03-09 1981-03-03 American Home Products Corporation Peptides related to somatostatin
JPS54148722A (en) * 1978-05-12 1979-11-21 Takeda Chem Ind Ltd Nonapeptide and its preparation
US4277394A (en) * 1979-04-23 1981-07-07 Takeda Chemical Industries, Ltd Tetrapeptidehydrazide derivatives
GB2051574B (en) * 1979-05-10 1984-01-18 Kyoto Pharma Ind Adjuvant for promoting absorption of pharmacologically active substances through the rectum
US4469681A (en) * 1979-07-31 1984-09-04 The Rockefeller University Method and system for the controlled release of biologically active substances to a body fluid
US4348387A (en) * 1979-07-31 1982-09-07 The Rockefeller University Method and system for the controlled release of biologically active substances to a body fluid
FR2465486A1 (en) * 1979-09-21 1981-03-27 Roussel Uclaf NEW APPLICATION USING LH-RH OR AGONISTS
JPS5692846A (en) 1979-12-27 1981-07-27 Takeda Chem Ind Ltd Tetrapeptide derivative and its preparation
US4554101A (en) * 1981-01-09 1985-11-19 New York Blood Center, Inc. Identification and preparation of epitopes on antigens and allergens on the basis of hydrophilicity
US4698264A (en) * 1982-08-02 1987-10-06 Durkee Industrial Foods, Corp. Particulate composition and process for making same
IL68769A (en) * 1983-05-23 1986-02-28 Hadassah Med Org Pharmaceutical compositions containing insulin for oral administration
US4662392A (en) * 1983-07-29 1987-05-05 Intevep, S.A. Check valve
US4585754A (en) * 1984-01-09 1986-04-29 Valcor Scientific, Ltd. Stabilization of proteins and peptides by chemical binding with chondroitin
US4717566A (en) * 1984-03-19 1988-01-05 Alza Corporation Dosage system and method of using same
US4684524A (en) * 1984-03-19 1987-08-04 Alza Corporation Rate controlled dispenser for administering beneficial agent
US4849405A (en) * 1984-05-09 1989-07-18 Synthetic Blood Corporation Oral insulin and a method of making the same
US4963367A (en) * 1984-04-27 1990-10-16 Medaphore, Inc. Drug delivery compositions and methods
US4963526A (en) * 1984-05-09 1990-10-16 Synthetic Blood Corporation Oral insulin and a method of making the same
US4839341A (en) * 1984-05-29 1989-06-13 Eli Lilly And Company Stabilized insulin formulations
US4797288A (en) * 1984-10-05 1989-01-10 Warner-Lambert Company Novel drug delivery system
US4946828A (en) * 1985-03-12 1990-08-07 Novo Nordisk A/S Novel insulin peptides
US5157021A (en) * 1985-03-15 1992-10-20 Novo Nordisk A/S Insulin derivatives and pharmaceutical preparations containing these derivatives
US4917888A (en) * 1985-06-26 1990-04-17 Cetus Corporation Solubilization of immunotoxins for pharmaceutical compositions using polymer conjugation
SE457326B (en) * 1986-02-14 1988-12-19 Lejus Medical Ab PROCEDURES FOR PREPARING A QUICK SUBSTANTIAL CANDLES CONTAINING BLA MICROCRISTALLIN CELLULOSA
US4801575A (en) 1986-07-30 1989-01-31 The Regents Of The University Of California Chimeric peptides for neuropeptide delivery through the blood-brain barrier
CA1339955C (en) * 1986-10-14 1998-07-14 Richard Eugene Heiney Process for transforming a human insulin precursor to human insulin
GB8706313D0 (en) * 1987-03-17 1987-04-23 Health Lab Service Board Treatment & prevention of viral infections
US5093198A (en) * 1987-06-19 1992-03-03 Temple University Adjuvant-enhanced sustained release composition and method for making
DE3721721C1 (en) * 1987-07-01 1988-06-09 Hoechst Ag Process for coating granules
US5080891A (en) 1987-08-03 1992-01-14 Ddi Pharmaceuticals, Inc. Conjugates of superoxide dismutase coupled to high molecular weight polyalkylene glycols
US4774976A (en) * 1987-09-23 1988-10-04 Applied Power Inc. Modulating hydraulic pressure control valve and assembly method therefor
JPH01308231A (en) * 1988-06-03 1989-12-12 Takeda Chem Ind Ltd Stabilized pharmaceutical composition and production thereof
US5055300A (en) * 1988-06-17 1991-10-08 Basic Bio Systems, Inc. Time release protein
DK336188D0 (en) * 1988-06-20 1988-06-20 Nordisk Gentofte propeptides
US5306500A (en) * 1988-11-21 1994-04-26 Collagen Corporation Method of augmenting tissue with collagen-polymer conjugates
US5162430A (en) * 1988-11-21 1992-11-10 Collagen Corporation Collagen-polymer conjugates
WO1990007522A1 (en) * 1988-12-23 1990-07-12 Novo Nordisk A/S Human insulin analogues
US4994439A (en) * 1989-01-19 1991-02-19 California Biotechnology Inc. Transmembrane formulations for drug administration
US5089261A (en) * 1989-01-23 1992-02-18 Cetus Corporation Preparation of a polymer/interleukin-2 conjugate
US5182258A (en) * 1989-03-20 1993-01-26 Orbon Corporation Systemic delivery of polypeptides through the eye
US5324844A (en) 1989-04-19 1994-06-28 Enzon, Inc. Active carbonates of polyalkylene oxides for modification of polypeptides
US5122614A (en) 1989-04-19 1992-06-16 Enzon, Inc. Active carbonates of polyalkylene oxides for modification of polypeptides
US5286637A (en) * 1989-08-07 1994-02-15 Debiopharm, S.A. Biologically active drug polymer derivatives and method for preparing same
US5013556A (en) * 1989-10-20 1991-05-07 Liposome Technology, Inc. Liposomes with enhanced circulation time
DE3937797A1 (en) 1989-11-14 1991-05-16 Basf Ag METHOD FOR PRODUCING POLYETHERGLYCOLES
US5650388A (en) * 1989-11-22 1997-07-22 Enzon, Inc. Fractionated polyalkylene oxide-conjugated hemoglobin solutions
US5312808A (en) * 1989-11-22 1994-05-17 Enzon, Inc. Fractionation of polyalkylene oxide-conjugated hemoglobin solutions
CA2030174C (en) * 1990-01-10 1996-12-24 Anthony H. Cincotta Process for the long term reduction of body fat stores, insulin resistance, hyperinsulinemia and hypoglycemia in vertebrates
US5545618A (en) * 1990-01-24 1996-08-13 Buckley; Douglas I. GLP-1 analogs useful for diabetes treatment
US5126324A (en) * 1990-06-07 1992-06-30 Genentech, Inc. Method of enhancing growth in patients using combination therapy
IE912365A1 (en) 1990-07-23 1992-01-29 Zeneca Ltd Continuous release pharmaceutical compositions
DD297249A5 (en) 1990-08-07 1992-01-02 Veb Mineralwollewerk Flechtingen Bereich F/E Mineralwolle,De METHOD FOR AUTOMATIC MONITORING OF THE BONED GRADE ON MATERIAL RAILS
IL99699A (en) * 1990-10-10 2002-04-21 Autoimmune Inc Pharmaceutical oral, enteral or by-inhalation dosage form for suppressing an autoimmune response associated with type i diabetes
US5595732A (en) * 1991-03-25 1997-01-21 Hoffmann-La Roche Inc. Polyethylene-protein conjugates
CA2108266C (en) * 1991-04-19 2003-06-03 Albert J. Owen Convertible microemulsion formulations
US5304473A (en) * 1991-06-11 1994-04-19 Eli Lilly And Company A-C-B proinsulin, method of manufacturing and using same, and intermediates in insulin production
AU667316B2 (en) 1991-07-26 1996-03-21 Smithkline Beecham Corporation W/O microemulsions
US5206219A (en) * 1991-11-25 1993-04-27 Applied Analytical Industries, Inc. Oral compositions of proteinaceous medicaments
US5693769A (en) * 1991-12-13 1997-12-02 Transcell Technologies, Inc. Glycosylated steroid derivatives for transport across biological membranes and process for making and using same
ES2092808T3 (en) 1992-01-17 1996-12-01 Alfatec Pharma Gmbh SOLID BODIES WITH ACTIVE SUBSTANCE CONTENT WITH A STRUCTURE BASED ON HYDROPHILIC MACROMOLECULES AND PROCEDURE FOR ITS PRODUCTION.
GB9212511D0 (en) 1992-06-12 1992-07-22 Cortecs Ltd Pharmaceutical compositions
US5262172A (en) * 1992-06-19 1993-11-16 Digestive Care Inc. Compositions of gastric acid-resistant microspheres containing buffered bile acids
US5415872A (en) * 1992-06-22 1995-05-16 Digestive Care Inc. Compositions of gastric acid-resistant microspheres containing salts of bile acids
US6093391A (en) 1992-10-08 2000-07-25 Supratek Pharma, Inc. Peptide copolymer compositions
GB9316895D0 (en) 1993-08-13 1993-09-29 Guy S And St Thomas Hospitals Hepatoselective insulin analogues
US5298643A (en) 1992-12-22 1994-03-29 Enzon, Inc. Aryl imidate activated polyalkylene oxides
US5349001A (en) 1993-01-19 1994-09-20 Enzon, Inc. Cyclic imide thione activated polyalkylene oxides
US5321095A (en) 1993-02-02 1994-06-14 Enzon, Inc. Azlactone activated polyalkylene oxides
US5298410A (en) 1993-02-25 1994-03-29 Sterling Winthrop Inc. Lyophilized formulation of polyethylene oxide modified proteins with increased shelf-life
US5681811A (en) 1993-05-10 1997-10-28 Protein Delivery, Inc. Conjugation-stabilized therapeutic agent compositions, delivery and diagnostic formulations comprising same, and method of making and using the same
US5621039A (en) 1993-06-08 1997-04-15 Hallahan; Terrence W. Factor IX- polymeric conjugates
AU7113594A (en) 1993-06-21 1995-01-17 Enzon, Inc. Site specific synthesis of conjugated peptides
TW402506B (en) * 1993-06-24 2000-08-21 Astra Ab Therapeutic preparation for inhalation
IS1796B (en) 1993-06-24 2001-12-31 Ab Astra Inhaled polypeptide formulation composition which also contains an enhancer compound
US5747445A (en) * 1993-06-24 1998-05-05 Astra Aktiebolag Therapeutic preparation for inhalation
US5506203C1 (en) * 1993-06-24 2001-02-06 Astra Ab Systemic administration of a therapeutic preparation
US5830853A (en) * 1994-06-23 1998-11-03 Astra Aktiebolag Systemic administration of a therapeutic preparation
US6342225B1 (en) 1993-08-13 2002-01-29 Deutshces Wollforschungsinstitut Pharmaceutical active conjugates
HU217684B (en) * 1993-09-17 2000-03-28 Novo Nordisk A/S Acylated insulin-derivatives and pharmaceutical compositions comprising the same and their preparation
US5919455A (en) 1993-10-27 1999-07-06 Enzon, Inc. Non-antigenic branched polymer conjugates
US5643575A (en) 1993-10-27 1997-07-01 Enzon, Inc. Non-antigenic branched polymer conjugates
US5605976A (en) * 1995-05-15 1997-02-25 Enzon, Inc. Method of preparing polyalkylene oxide carboxylic acids
US5951974A (en) 1993-11-10 1999-09-14 Enzon, Inc. Interferon polymer conjugates
WO1995014037A1 (en) * 1993-11-17 1995-05-26 Ibah, Inc. Transparent liquid for encapsulated drug delivery
GB9406094D0 (en) 1994-03-28 1994-05-18 Univ Nottingham And University Polymer microspheres and a method of production thereof
US5461031A (en) * 1994-06-16 1995-10-24 Eli Lilly And Company Monomeric insulin analog formulations
US5504188A (en) * 1994-06-16 1996-04-02 Eli Lilly And Company Preparation of stable zinc insulin analog crystals
US6165976A (en) 1994-06-23 2000-12-26 Astra Aktiebolag Therapeutic preparation for inhalation
US5730990A (en) * 1994-06-24 1998-03-24 Enzon, Inc. Non-antigenic amine derived polymers and polymer conjugates
GB9417524D0 (en) * 1994-08-31 1994-10-19 Cortecs Ltd Pharmaceutical compositions
US5738846A (en) * 1994-11-10 1998-04-14 Enzon, Inc. Interferon polymer conjugates and process for preparing the same
US5646242A (en) * 1994-11-17 1997-07-08 Eli Lilly And Company Selective acylation of epsilon-amino groups
US5693609A (en) 1994-11-17 1997-12-02 Eli Lilly And Company Acylated insulin analogs
CA2206852A1 (en) * 1994-12-07 1996-06-13 Novo Nordisk A/S Polypeptide with reduced allergenicity
GB9424902D0 (en) 1994-12-09 1995-02-08 Cortecs Ltd Solubilisation Aids
SE9404468D0 (en) 1994-12-22 1994-12-22 Astra Ab Powder formulations
US5843866A (en) * 1994-12-30 1998-12-01 Hampshire Chemical Corp. Pesticidal compositions comprising solutions of polyurea and/or polyurethane
US5932462A (en) 1995-01-10 1999-08-03 Shearwater Polymers, Inc. Multiarmed, monofunctional, polymer for coupling to molecules and surfaces
US5907030A (en) 1995-01-25 1999-05-25 University Of Southern California Method and compositions for lipidization of hydrophilic molecules
KR0150565B1 (en) 1995-02-15 1998-08-17 김정재 A process for preparing human proinsulin by recombinant dna technology
US6251856B1 (en) 1995-03-17 2001-06-26 Novo Nordisk A/S Insulin derivatives
YU18596A (en) 1995-03-31 1998-07-10 Eli Lilly And Company Analogous formulations of monomer insulin
US5606038A (en) * 1995-04-10 1997-02-25 Competitive Technologies, Inc. Amphiphilic polyene macrolide antibiotic compounds
EP0741187A2 (en) 1995-05-05 1996-11-06 F. Hoffmann-La Roche Ag Recombinant obese (Ob) proteins
US5824638A (en) * 1995-05-22 1998-10-20 Shire Laboratories, Inc. Oral insulin delivery
US5700904A (en) * 1995-06-07 1997-12-23 Eli Lilly And Company Preparation of an acylated protein powder
US5631347A (en) * 1995-06-07 1997-05-20 Eli Lilly And Company Reducing gelation of a fatty acid-acylated protein
GB9516268D0 (en) 1995-08-08 1995-10-11 Danbiosyst Uk Compositiion for enhanced uptake of polar drugs from the colon
ES2190388T3 (en) 1995-09-21 2006-04-01 Genentech, Inc. VARIANTS OF HUMAN GROWTH HORMONE.
US5766620A (en) * 1995-10-23 1998-06-16 Theratech, Inc. Buccal delivery of glucagon-like insulinotropic peptides
US5639705A (en) 1996-01-19 1997-06-17 Arco Chemical Technology, L.P. Double metal cyanide catalysts and methods for making them
US5948751A (en) 1996-06-20 1999-09-07 Novo Nordisk A/S X14-mannitol
US5866538A (en) * 1996-06-20 1999-02-02 Novo Nordisk A/S Insulin preparations containing NaCl
GB9613858D0 (en) 1996-07-02 1996-09-04 Cortecs Ltd Hydrophobic preparations
US5905140A (en) 1996-07-11 1999-05-18 Novo Nordisk A/S, Novo Alle Selective acylation method
US5856369A (en) 1996-07-30 1999-01-05 Osi Specialties, Inc. Polyethers and polysiloxane copolymers manufactured with double metal cyanide catalysts
DE19632440A1 (en) 1996-08-12 1998-02-19 Basf Ag Easily prepared and separated catalyst giving pure alkoxylation product with narrow molecular weight distribution
US5874111A (en) * 1997-01-07 1999-02-23 Maitra; Amarnath Process for the preparation of highly monodispersed polymeric hydrophilic nanoparticles
US6011008A (en) 1997-01-08 2000-01-04 Yissum Research Developement Company Of The Hebrew University Of Jerusalem Conjugates of biologically active substances
US5830918A (en) * 1997-01-15 1998-11-03 Terrapin Technologies, Inc. Nonpeptide insulin receptor agonists
US6310038B1 (en) 1997-03-20 2001-10-30 Novo Nordisk A/S Pulmonary insulin crystals
US5898028A (en) * 1997-03-20 1999-04-27 Novo Nordisk A/S Method for producing powder formulation comprising an insulin
US6043214A (en) 1997-03-20 2000-03-28 Novo Nordisk A/S Method for producing powder formulation comprising an insulin
ZA984697B (en) 1997-06-13 1999-12-01 Lilly Co Eli Stable insulin formulations.
JP2001521006A (en) 1997-10-24 2001-11-06 イーライ・リリー・アンド・カンパニー Insoluble insulin composition
ZA989744B (en) 1997-10-31 2000-04-26 Lilly Co Eli Method for administering acylated insulin.
US5985263A (en) 1997-12-19 1999-11-16 Enzon, Inc. Substantially pure histidine-linked protein polymer conjugates
US5981709A (en) 1997-12-19 1999-11-09 Enzon, Inc. α-interferon-polymer-conjugates having enhanced biological activity and methods of preparing the same
US6211144B1 (en) 1998-10-16 2001-04-03 Novo Nordisk A/S Stable concentrated insulin preparations for pulmonary delivery
DE19908041A1 (en) 1999-02-24 2000-08-31 Hoecker Hartwig Covalently bridged insulin dimers
US6248363B1 (en) 1999-11-23 2001-06-19 Lipocine, Inc. Solid carriers for improved delivery of active ingredients in pharmaceutical compositions
KR100345214B1 (en) * 1999-08-17 2002-07-25 이강춘 The nasal transmucosal delivery of peptides conjugated with biocompatible polymers
US6323311B1 (en) 1999-09-22 2001-11-27 University Of Utah Research Foundation Synthesis of insulin derivatives
US6867183B2 (en) 2001-02-15 2005-03-15 Nobex Corporation Pharmaceutical compositions of insulin drug-oligomer conjugates and methods of treating diseases therewith
US7060675B2 (en) 2001-02-15 2006-06-13 Nobex Corporation Methods of treating diabetes mellitus
US6828305B2 (en) 2001-06-04 2004-12-07 Nobex Corporation Mixtures of growth hormone drug-oligomer conjugates comprising polyalkylene glycol, uses thereof, and methods of making same
US6713452B2 (en) * 2001-06-04 2004-03-30 Nobex Corporation Mixtures of calcitonin drug-oligomer conjugates comprising polyalkylene glycol, uses thereof, and methods of making same
US6835802B2 (en) 2001-06-04 2004-12-28 Nobex Corporation Methods of synthesizing substantially monodispersed mixtures of polymers having polyethylene glycol moieties
US6828297B2 (en) * 2001-06-04 2004-12-07 Nobex Corporation Mixtures of insulin drug-oligomer conjugates comprising polyalkylene glycol, uses thereof, and methods of making same
US6858580B2 (en) * 2001-06-04 2005-02-22 Nobex Corporation Mixtures of drug-oligomer conjugates comprising polyalkylene glycol, uses thereof, and methods of making same
US6913903B2 (en) 2001-09-07 2005-07-05 Nobex Corporation Methods of synthesizing insulin polypeptide-oligomer conjugates, and proinsulin polypeptide-oligomer conjugates and methods of synthesizing same
US6770625B2 (en) 2001-09-07 2004-08-03 Nobex Corporation Pharmaceutical compositions of calcitonin drug-oligomer conjugates and methods of treating diseases therewith

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5349052A (en) * 1988-10-20 1994-09-20 Royal Free Hospital School Of Medicine Process for fractionating polyethylene glycol (PEG)-protein adducts and an adduct for PEG and granulocyte-macrophage colony stimulating factor
EP0511903A2 (en) * 1991-04-23 1992-11-04 Teva Pharmaceutical Industries Ltd. Conjugate of calcitonin and polyethylene glycol
US5359030A (en) 1993-05-10 1994-10-25 Protein Delivery, Inc. Conjugation-stabilized polypeptide compositions, therapeutic delivery and diagnostic formulations comprising same, and method of making and using the same
US6191105B1 (en) 1993-05-10 2001-02-20 Protein Delivery, Inc. Hydrophilic and lipophilic balanced microemulsion formulations of free-form and/or conjugation-stabilized therapeutic agents such as insulin
WO1997014740A1 (en) 1995-10-19 1997-04-24 Receptagen Corporation Discrete-length polyethylene glycols
WO2000009073A2 (en) 1998-08-14 2000-02-24 Nobex Corporation Blood-brain barrier therapeutics
WO2000078302A1 (en) 1999-06-19 2000-12-28 Nobex Corporation Amphiphilic drug-oligomer conjugates with hydrolyzable lipophile components and methods for making and using the same

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
H.R. ALLCOCK; F.W. LAMPE: "CONTEMPORARY POLYMER CHEMISTRY", 1991, pages: 394 - 402
HIGGINS ET AL., BRITISH JOURNAL OF PHARMACOLOGY, vol. 134, November 2001 (2001-11-01), pages 53
RADHA KRISHNAN B. ET AL., PROCEEDINGS OF THE CONTROLLED RELEASE SOCIETY, vol. 26, 1999, pages 149 - 150
See also references of EP1404360A4

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006515622A (en) * 2003-01-13 2006-06-01 ニュー リバー ファーマシューティカルズ インコーポレイテッド Carbohydrate conjugates to prevent the abuse of controlled substances
US8133881B2 (en) 2003-01-13 2012-03-13 Shire Llc Carbohydrate conjugates to prevent abuse of controlled substances
WO2005004792A2 (en) 2003-06-24 2005-01-20 Nobex Corporation Mixtures of calcitonin drug-oligomer conjugates and methods of use in pain treatment
EP1643959A2 (en) * 2003-06-24 2006-04-12 Nobex Corporation Mixtures of calcitonin drug-oligomer conjugates and methods of use in pain treatment
JP2007521268A (en) * 2003-06-24 2007-08-02 バイオコン・リミテッド Mixtures of calcitonin drug-oligomer conjugates and methods of use in treating pain
EP1643959A4 (en) * 2003-06-24 2010-06-09 Biocon Ltd Mixtures of calcitonin drug-oligomer conjugates and methods of use in pain treatment
JP4829783B2 (en) * 2003-06-24 2011-12-07 バイオコン・リミテッド Mixtures of calcitonin drug-oligomer conjugates and methods of use in treating pain
EP2905033A1 (en) * 2003-12-16 2015-08-12 Nektar Therapeutics Monodisperse PEGylated naloxol compositions
US11129794B2 (en) 2003-12-16 2021-09-28 Nektar Therapeutics Chemically modified small molecules
US9012469B2 (en) 2010-09-30 2015-04-21 Astrazeneca Ab Crystalline naloxol-peg conjugate
US9149539B1 (en) 2010-09-30 2015-10-06 Astrazeneca Ab Crystalline naloxol-PEG conjugate
US9308263B2 (en) 2011-10-21 2016-04-12 Seachaid Pharmaceuticals, Inc. Pharmaceutical compositions and uses thereof

Also Published As

Publication number Publication date
US20040180831A1 (en) 2004-09-16
ES2339224T3 (en) 2010-05-18
US7084121B2 (en) 2006-08-01
EP1404360A1 (en) 2004-04-07
DE60235010D1 (en) 2010-02-25
EP1404360A4 (en) 2006-04-12
KR20040004694A (en) 2004-01-13
KR100930606B1 (en) 2009-12-09
MXPA03011283A (en) 2004-03-26
CA2449686A1 (en) 2002-12-12
US6713452B2 (en) 2004-03-30
EP1404360B1 (en) 2010-01-06
CY1109932T1 (en) 2014-09-10
DK1404360T3 (en) 2010-05-03
US20030060606A1 (en) 2003-03-27
CN1538851A (en) 2004-10-20
ATE454160T1 (en) 2010-01-15
CN100515492C (en) 2009-07-22
JP4272510B2 (en) 2009-06-03
JP2004534782A (en) 2004-11-18

Similar Documents

Publication Publication Date Title
US7084121B2 (en) Mixtures of calcitonin drug-oligomer conjugates comprising polyalkylene glycol, uses thereof, and methods of making same
US6828297B2 (en) Mixtures of insulin drug-oligomer conjugates comprising polyalkylene glycol, uses thereof, and methods of making same
US6828305B2 (en) Mixtures of growth hormone drug-oligomer conjugates comprising polyalkylene glycol, uses thereof, and methods of making same
US8030269B2 (en) Calcitonin drug-oligomer conjugates, and uses thereof
AU2002303961A1 (en) Mixtures of calcitonin drug-oligomer conjugates comprising polyalkylene glycol, uses thereof, and methods of making same
WO2005004792A2 (en) Mixtures of calcitonin drug-oligomer conjugates and methods of use in pain treatment
US20090281023A9 (en) Mixtures Of Calcitonin Drug-Oligomer Conjugates And Methods Of Use In Pain Treatment
AU2002310278A1 (en) Mixtures of growth hormone drug-oligomer conjugates compromising polyalkylene glycol, uses thereof, and methods of making same

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ OM PH PL PT RO RU SD SE SG SI SK SL TJ TM TN TR TT TZ UA UG US UZ VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
WWE Wipo information: entry into national phase

Ref document number: PA/a/2003/011283

Country of ref document: MX

Ref document number: 2003501489

Country of ref document: JP

Ref document number: 2449686

Country of ref document: CA

Ref document number: 1020037015912

Country of ref document: KR

WWE Wipo information: entry into national phase

Ref document number: 2002303961

Country of ref document: AU

WWE Wipo information: entry into national phase

Ref document number: 2002732030

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 20028153022

Country of ref document: CN

WWP Wipo information: published in national office

Ref document number: 2002732030

Country of ref document: EP

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642