WO1994004193A1 - Novel attachment of polyalkylene oxides to bio-effecting substances - Google Patents

Novel attachment of polyalkylene oxides to bio-effecting substances Download PDF

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Publication number
WO1994004193A1
WO1994004193A1 PCT/US1993/007579 US9307579W WO9404193A1 WO 1994004193 A1 WO1994004193 A1 WO 1994004193A1 US 9307579 W US9307579 W US 9307579W WO 9404193 A1 WO9404193 A1 WO 9404193A1
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Prior art keywords
bio
effecting
composition
polyalkylene oxide
agents
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PCT/US1993/007579
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French (fr)
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Richard B. Greenwald
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Enzon, Inc.
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Priority to AU50069/93A priority Critical patent/AU5006993A/en
Publication of WO1994004193A1 publication Critical patent/WO1994004193A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C331/00—Derivatives of thiocyanic acid or of isothiocyanic acid
    • C07C331/16—Isothiocyanates
    • C07C331/18—Isothiocyanates having isothiocyanate groups bound to acyclic carbon atoms
    • C07C331/20—Isothiocyanates having isothiocyanate groups bound to acyclic carbon atoms of a saturated carbon skeleton
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal 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
    • A61K47/51—Medicinal 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
    • A61K47/56—Medicinal 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
    • A61K47/59—Medicinal 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
    • A61K47/60—Medicinal 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C217/00—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton
    • C07C217/02—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C217/04—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C217/06—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one etherified hydroxy group and one amino group bound to the carbon skeleton, which is not further substituted
    • C07C217/08—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one etherified hydroxy group and one amino group bound to the carbon skeleton, which is not further substituted the oxygen atom of the etherified hydroxy group being further bound to an acyclic carbon atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C265/00—Derivatives of isocyanic acid
    • C07C265/02—Derivatives of isocyanic acid having isocyanate groups bound to acyclic carbon atoms
    • C07C265/04—Derivatives of isocyanic acid having isocyanate groups bound to acyclic carbon atoms of a saturated carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C265/00—Derivatives of isocyanic acid
    • C07C265/14—Derivatives of isocyanic acid containing at least two isocyanate groups bound to the same carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C271/00—Derivatives of carbamic acids, i.e. compounds containing any of the groups, the nitrogen atom not being part of nitro or nitroso groups
    • C07C271/06—Esters of carbamic acids
    • C07C271/32—Esters of carbamic acids having oxygen atoms of carbamate groups bound to carbon atoms of rings other than six-membered aromatic rings
    • C07C271/34—Esters of carbamic acids having oxygen atoms of carbamate groups bound to carbon atoms of rings other than six-membered aromatic rings with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C275/00—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups
    • C07C275/04—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of urea groups bound to acyclic carbon atoms
    • C07C275/06—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of urea groups bound to acyclic carbon atoms of an acyclic and saturated carbon skeleton
    • C07C275/10—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of urea groups bound to acyclic carbon atoms of an acyclic and saturated carbon skeleton being further substituted by singly-bound oxygen atoms

Definitions

  • the present invention relates to modifications of bio-effecting substances with polyalkylene oxides.
  • the invention relates to modifications which extend the circulating life and reduce the immunogenicity of bio-effecting substances.
  • PAO's polyalkylene oxides
  • PEG polyethylene glycol
  • Polyethylene glycol has several advantageous properties.
  • the toxicity of PEG'S of molecular weights greater than 1,000 daltons is very low. Consequently, the polymer can be safely administered intravenously in a wide variety of mammals.
  • the polymer also has a wide range of solubilities.
  • Polyalkylene oxides are soluble in most organic solvents as well as aqueous solutions.
  • Many polypeptides and other materials conjugated with PEG and PAO's not only retain their solubility properties, but also demonstrate enhanced water solubility as a result of the conjugation.
  • the biological activities of PEG conjugates are typically dominated by the non-PEG part of the conjugated molecule.
  • the hydroxyl end-groups of the polymers must first be converted or activated into reactive functional groups.
  • PEG-succinimidyl succinate SS-PEG
  • the activated polymer reacts with proteins under mild conditions while preserving biological activities.
  • the resultant ester linkage has limited stability in aqueous media.
  • urethane (carbamate) linkages between the amino groups of a protein and PEG has provided one solution to the problem of hydrolytic release of the polymer chains.
  • Attachment of PEG to a protein via a carbamate derivative was disclosed in Analytical Biochemistry 131, 25-33 (1983) using carbonyl diimidazole-activated PEG.
  • the activated polymer is not very reactive with proteins, often requiring two to three days to achieve sufficient modifications.
  • Another drawback associated with carbonyl diimidazole is its high relative cost.
  • Each of the activated forms of the PAO-based polymers described above has properties which, under certain circumstances, may be considered advantageous. Nevertheless, extending the PAO-modifying reagents and techniques to include additional proteins, peptides or chemotherapeutic agents which heretofore have not benefitted from such modifications because of unreactivity, harmful by-products and the like is desirable.
  • PAO - based compounds having the formula: (I) Z - (O - R 1 ) a - (O - R 2 ) b - (O - R 3 ) c - Y wherein:
  • R 1 , R 2 and R 3 are alkyl groups
  • a is an integer between 1 and 1000 and each of b and c is an integer between 0 and 1000, and the sum of a, b and c is between 10 and 1000;
  • Y is NCX, wherein X is one of O or S;
  • Z is one of an alkyl, cycloalkyl, branched alkyl, arylalkyl group or Y.
  • R 3 can be straight or branched alkyl groups and/or each of R 1 , R 2 , R 3 can be independently the same or different from each other.
  • the PAO-based compositions are polyethylene glycol-based, having a molecular weight of from about 2,000 to about 20,000.
  • compositions of (I) include reacting compounds having the formula:
  • a is an integer between 1 and 1000 and each of b and c is an integer between 0 and 1000, and the sum of a, b and c is between 10 and 1000;
  • A is one of an alkyl, cycloalkyl, branched alkyl, arylalkyl or amino group
  • phosgene or a phosgene-containing substance such as triphosgene in the presence of a suitable base.
  • a further aspect of the invention provides compositions containing bio-effecting substances covalently bound to at least one PAO-based derivative.
  • the covalent linkage achieved through the isocyanate and/or isothiocyanate portion of the activated PAO-based polymer derivatives (I) and an alcohol present in the bio-effecting substance produce a urethane and/or thiourethane linkage.
  • the covalent linkage between the reactants can also be achieved through the same isocyanate or isothiocyanate portions of the activated PAO-based polymers and amines present in the bio- effecting substance to produce urea and/or thiourea linkages.
  • a still further aspect of the invention is directed to methods for preparing such bio-effecting-PAO compositions.
  • the methods include reacting the PAO-based polymer with the bio-effecting substance under conditions which effect covalent attachment of the ingredients at the isocyato- and/or isothiocyato-portion of the polymer and amine and/or alcohol portion of the bio-effector.
  • the conditions are further defined as those which permit the covalent attachment of the reactants while substantially maintaining the desired effect of the bio-effecting substance.
  • the applications of the present invention are vast.
  • the activated PAO's of the present invention equip the artisan with a polymer-based material that is suitable both for amine-based and alcohol-based linkages. Indeed, it is now possible to attach the polymers to bio-effecting substances at both locations at will in order to maintain optimal activity of the native portion of the molecule.
  • the ability to effect such mixed attachments of the reactants allows the artisan to precisely attach the beneficial polymer where desired and more precisely modulate the activity of the therapeutic conjugates.
  • PAO-based polymers are provided which improve a wide range of therapeutic substances.
  • the conjugation prolongs the circulating life of the substances when compared to unmodified forms.
  • it is possible to substantially reduce the amount and frequency of administrations without diminishing the therapeutic effect.
  • Such reductions also reduce the concomitant toxicities often seen when high doses of agents are administered, especially over time.
  • the novel covalent conjugation of PAO's to bio-effecting substances also improves the aqueous solubility of the bio-effector and allows the artisan to provide therapeutic compositions in heretofore impractical dosage forms.
  • compositions of the present invention allow not only continued treatment but also substantial reductions in untoward reactions.
  • the present invention includes activated polyalkylene oxides having the general structure:
  • R 1 , R 2 and R 3 are alkyl groups
  • a is an integer between 1 and 1000 and each of b and c is an integer between 0 and 1000, and the sum of a, b and c is between 10 and 1000;
  • Y is NCX, wherein X is one of O or S;
  • Z is one of an alkyl, cycloalkyl, branched alkyl, arylalkyl group or Y.
  • the activated PAO's described herein is the presence of Y in a terminal position.
  • the NCO or isocyanate portion of the activated polymer reacts rapidly with nucleophiles such as the epsilon-amino group of lysine moieties or the hydroxyl or alcohol groups of various bio-effecting substances to produce stable, conjugated molecules.
  • the stability of the conjugate is achieved by covalent linkage between the isocyanate and/or isothiocyanate portion of the activated PAO-based polymer derivatives (I) and an alcohol present in the bio- effecting substance to produce a urethane and/or thiourethane linkage.
  • the covalent linkage between the reactants can also be achieved through the same isocyanate or isothiocyanate portions of the activated PAO-based polymers and amines present in the bio-effecting substance to produce urea and/or thiourea linkages. Conjugation with the isocyanate-activated PAO's is preferred.
  • conjugation reactions combining the novel activated PAO's described herein can be carried out with minimal or no pH adjustment to maintain an optimal reactivity range.
  • suitable alkyl groups include, for example, ethyl, propyl, isopropyl or butyl.
  • Cycloalkyl groups include cyclohexyls, for example;
  • suitable branched alkyIs include materials such as 3-methylhexyl, for example and
  • suitable arylalkyls groups include, for example, benzyl.
  • the present invention relates to PAO-based polymers which are water soluble and do not generate an immunogenic response when administered to mammals.
  • the polymeric substances included herein are also preferably water-soluble at room temperature.
  • a wide variety of materials are thus suitable for use herein.
  • a non-limiting list of such polymers include:
  • each of R 1 , R 2 and R 3 may be straight or branched alkyl groups.
  • each of R 1-3 may be independently the same as or different from the others of R 1-3 .
  • each of R 1-3 can be CH 2 CH 2 , CH 2 CHCH 3 or CH 2 CH 2 CH 2 CH 2 .
  • Polyethylene glycols are the preferred polyalkylene oxides (PAO's) described herein. Although polyethylene glycols come in a variety of molecular weights, averaging from about 600 to about 100,000 daltons, the preferred molecular weight range for modifying therapeutic agents is from about 2,000 to about 20,000 daltons. Molecular weights of from about 3,000 to about 7,000 are especially preferred.
  • the molecular weight of the polymer will vary depending upon the needs of the artisan and the particular bio-effecting substance to be modified. Those of ordinary skill in the art can determine the molecular weight ranges suitable for particular end use applications. The foregoing is merely illustrative not to be considered restrictive of the type of materials suitable for use herein. Those skilled in the art will realize that equivalent or substantially equivalent polymeric materials not specifically mentioned but having the qualities described herein are also contemplated.
  • new, activated polyethylene glycols are provided.
  • PEG- NCO poly(ethylene glycol)-isocyanates
  • PEG-NCS poly(ethylene glycol)-isothiocyanates
  • Heterobifunctional derivatives of PEG and PAO's are also possible.
  • Alternative functional groups for Z such as hydrogen, an alkyl group, a chloroformate or an N-dicarboximide are also contemplated.
  • the activated PAO's of (I) are generally prepared by reacting compounds having the formula:
  • A is one of an alkyl, cycloalkyl, branched alkyl, arylalkyl or amino group
  • phosgene or a phosgene-generating substance in the presence of a suitable base.
  • A an amino
  • a bifunctional diisocyanate is formed.
  • the reaction can be carried out over a broad temperature range of from about 10 to about 130° C.
  • methoxy PEG, mPEG, readily available from Aldrich Chemical of Milwaukee, WI is converted to mPEG-NH 2 by treatment with thionyl chloride to form mPEG-Cl followed by treatment with aqueous ammonia.
  • PEG isocyanate or PEG isothiocyanate formation is accomplished by reacting the PEG-amine with phosgene or thiophosgene or similar generating substances such as bis- (trichloromethyl) carbonate (triphosgene) in the presence of a base such as triethylamine.
  • Suitable alternative bases include pyridine, N,N-dimethylaniline, 1,4-diazabicyclo[2.2.2]octane or DABCOTM, available from Aldrich Chemical Co. of Milwaukee, WI. or any other suitable inert tertiary amine bases.
  • the amount of activated PAO required to modify and conjugate with a subject bio-effecting molecule will depend on several factors. Principally, however, the amount of polymer is dependent upon the needs of the artisan and the degree of modification desired.
  • the ratio of activated polymer to bio-effecting substance will accordingly vary widely. Molar ratios on the order of from about 0.1 : 1 to about 50 : 1 are contemplated, with ratios of from about 1 : 1 to about 25 : 1 being preferred and about 5 : 1 to about 20 : 1 being most preferred.
  • Protein modification for example, often requires a molar excess of the activated polymer. A 5 to 40-fold molar excess of PEG-NCO is sufficient to modify hemoglobin.
  • a is an integer between 1 and 1000 and each of b and c is an integer between 0 and 1000, and the sum of a, b and c is between 10 and 1000;
  • R" is a bio-effecting substance
  • Z' is an alkyl, cycloalkyl, branched alkyl, arylalkyl or -NH-CO-W-R"
  • bio-effecting substance is to be broadly construed and means any substance displaying a physiologic effect preferably in mammals after administration, whether oral, parenteral or otherwise.
  • bio-effecting there are included peptides, polypeptides and enzymes, both naturally and synthetically derived.
  • the present invention also contemplates attachment of the activated
  • PAO's to chemical moieties such as chemotherapeutic substances.
  • a non-limiting list of such bio-effecting substances includes:
  • a) enzymes such as asparaginase, arginase, adenosine deaminase, superoxide dismutase, catalase, chymotrypsin, lipase, uricase, bilirubin oxidase, glucose oxidase, glucuronidase, galactosidase, glucocerebrosidase and glucuronidase;
  • polypeptides such as Factor VIII, insulin, ACTH, glucagon, somatostatin, somatotropins, thymosin, parathyroid hormone, pigmentary hormones, somatomedins, erythropoietin, luteinizing hormone, hypothalamic releasing factors, antidiuretic hormones, prolactin, interleukins, interferons and colony stimulating factors, hemoglobin, cytokines and antibodies;
  • polypeptides such as Factor VIII, insulin, ACTH, glucagon, somatostatin, somatotropins, thymosin, parathyroid hormone, pigmentary hormones, somatomedins, erythropoietin, luteinizing hormone, hypothalamic releasing factors, antidiuretic hormones, prolactin, interleukins, interferons and colony stimulating factors, hemoglobin, cytokines and antibodies;
  • glycopolypeptides such as immunoglobulins, ovalbumin, lipase, glucocerebrosidase, lectins, tissue plasminogen activator and glycosilated interleukins, interferons and colony stimulating factors.
  • immunoglobulins such as IgG, IgE, IgM, IgA, IgD and fragments thereof.
  • chemotherapeutic agents of all therapeutic categories such as anti-inflammatory agents, antiinfective compositions such as antibiotics, including hydroxy- or amino-penicillins or cephalosporins, anti-tumor agents such as taxol or taxol derivatives including 2'-acetyltaxol or methotrexate and the like, analgesics such as opiates, cardiac agents, steroids such as corticosteriods, central nervous system agents, etc.
  • bio-effecting substances suitable for use herein contain at least one amine or alcohol substituent for covalent attachment of the activated PAO without substantially reducing or eliminating the therapeutic effect of the unmodified material.
  • the substance contain at least one amine or alcohol substituent for covalent attachment of the activated PAO without substantially reducing or eliminating the therapeutic effect of the unmodified material.
  • Scheme 3 when attachment is effected between the isocyanate portion of the activated PEG and alcohols on the bio-effecting substance, stable urethane linkages are formed. Tin-based catalysts such as dibutyltin dilaurate are advantageously included in such reactions.
  • isothiocyanate portions of the activated polymers are reacted with hydroxyls on the bio-effectors, thiourethane linkages are formed.
  • the above-described activated PAO's afford substantial modification of a wide variety of bio- effecting substances.
  • the modification reactions described above can be carried out in both aqueous and non-aqueous solvents.
  • protein modification is achievable under mild conditions using aqueous systems having a pH ranging from about 5.0 to about 11.0 and preferably from about 7.0 to about 8.0, moderate reaction temperatures of from about 0 to about 10° C, and reaction times of from about 0.25 to about 1 hour.
  • aqueous systems having a pH ranging from about 5.0 to about 11.0 and preferably from about 7.0 to about 8.0, moderate reaction temperatures of from about 0 to about 10° C, and reaction times of from about 0.25 to about 1 hour.
  • hydroxyl moieties are linked to the polymers
  • more rigorous temperature and time conditions are sustainable and will generally include tin-based catalysts to speed up the reaction.
  • mPEG-NH 2 was prepared using procedures similar to those described in Biotech. App. Biochem. 9, 258-268 (1987). In a 1 liter round bottom flask, 100 grams of mPEG-5000 was melted and then dried under high vacuum. Twenty grams of thionyl chloride was then added to the dried mPEG and the mixture was heated to 65-70 degrees C. for 5 hours using a reflux condenser. The unreacted thionyl chloride was removed with a rotary evaporator and high vacuum. A 2 gram sample of this melt was recrystallized from 2-propanol. The 13 C NMR spectrum (CDCl 3 ) showed a peak at 42 ppm corresponding to CH 2 -Cl. A peak at 61.5 ppm (corresponding to CH 2 -OH) could not be detected indicating that the conversion was greater than 95%.
  • the product was recrystallized from 1L of isopropanol.
  • the product was characterized by non-aqueous titration according to the method described in Eur. J. Biochem. 42, 151 (1974) and by 1 NMR and 13 C NMR.
  • the product showed 100% activity by non-aqueous titration.
  • mPEG-NCO The 7-urethane derivative in this Example, mPEG-NCO was prepared in situ by placing 515 mg (0.010 mmol) of mPEG-NH 2 in a 100 ml round bottom flask and undergoing drying by azeotropic toluene distillation and converted to mPEG-NCO as described above.
  • the FTIR showed the isocyanate peak at 2263 cm -1 of intensity similar to that obtained above (relative to polyethylene glycol peaks).
  • the reaction mixture was cooled to room temperature and to it were added 60 mg of 2'-acetyltaxol prepared as described in Biochem. Biophys. R.S. Commun.
  • the ether was decanted, and the precipitate was recrystallized from 20 milliliters of 2-propanol.
  • the 7-PEG urethane derivative was isolated by centrifuging. washing with two 20 milliliter portions of 2-propanol, and finally drying under high vacuum to obtain 508 mg of purer product containing less than 1% 2'-acetyltaxol and some non-functionalized PEG.
  • the 7-PEG urethane derivative could be further purified by HPLC using a semipreparative C 8 -column.
  • the FTIR spectrum of the purified compound had all the characteristic peaks of PEG in addition to peaks at 1748.6, 1741.2, 1726.5, 1663 cm -1 which are characteristic of the 2'-acetyltaxol molecule.
  • conjugation of the activated PEG-NCO to the protein bovine hemoglobin (Hb), obtained from California Biological and Protein Corp. of Huntington Beach, CA were carried out.
  • the reaction conditions included maintaining a constant pH of 7.8 and temperature of 8°C.
  • the hemoglobin was reacted in a solution having a concentration of about 10.6%.
  • PEG-NCO was added as a solid.
  • Hb (10.6%, 2ml) was diluted with 2 ml of pH 7.8 buffer in a jacketed reaction vessel kept at 8°C.
  • PEG-NCO (217.6 mg, 1.08 ⁇ 10 -5 M) was added to the reaction vessel containing the Hb.
  • the pH was adjusted to approximately 7.8 with 1.0N NaOH.
  • the reaction mixture was stirred uniformly and slowly for two hours at 8°C maintaining pH 7.8. Thereafter, 21 mg cysteine HCI (0.03M) and glycine solution (3.24 mg, 19 ⁇ l of stock 336/2ml of pH 7.0 phosphate buffer) which is equimolar with PEG-NCO were added, and the pH was re-adjusted to 7.8 with 1N NaOH.
  • the yield of the desired PEG-Hb was determined by HPLC retention time to be in excess of 73%
  • Hb (10.6%, 3ml) was diluted with 3 ml of pH 7.8 buffer in a jacketed reaction vessel kept at 8°C. The pH was adjusted to approximately 7.8 with 1.0N NaOH.
  • PEG-NCO 491.7 mg, 1.62 ⁇ 10 -2 M was added to the reaction vessel kept at 8°C with Hb. The reaction mixture was stirred uniformly and slowly for two hours at 8°C maintaining pH 7.8. Thereafter, 31.6 mg cysteine HCI (0.03M) and glycine solution (12.18 mg, 43.5 ⁇ l of stock 336/2ml of pH 7.0 phosphate buffer) which is equimolar with PEG-NCO were added. The pH was adjusted to 7. with 1N NaOH and allowed to stir for five minutes. The sample was analyzed by HPLC to determine the yield of desired PEG-Hb to be in excess of 83 %.

Abstract

Polyalkylene oxide (PAO)-based compositions containing isocyanate and/or isothiocyanate groups for covalent attachment to bio-effecting substances such as peptides or chemotherapeutics are disclosed. The compositions react readily with the bio-effecting substances to provide compositions having increased circulating lives in mammals, substantially reduced immunogenicity and enhanced aqueous solubility. Methods of preparing such PAO-based compositions are also disclosed.

Description

NOVEL ATTACHMENT OF POLYALKYLENE OXIDES
TO BIO-EFFECTING SUBSTANCES BACKGROUND OF THE INVENTION
The present invention relates to modifications of bio-effecting substances with polyalkylene oxides. In particular, the invention relates to modifications which extend the circulating life and reduce the immunogenicity of bio-effecting substances.
Some of the unique properties of polyalkylene oxides (PAO's) such as polyethylene glycol (PEG) to reduce the immunogenicity and antigenicity of therapeutic agents such as peptides have been set forth in U.S. Patent Number 4,179,337. The attachment of such non-immunogenic polymers has also been found to prolong the circulating life of several therapeutic products in the bloodstream when compared to unmodified or native material.
Polyethylene glycol has several advantageous properties. The toxicity of PEG'S of molecular weights greater than 1,000 daltons is very low. Consequently, the polymer can be safely administered intravenously in a wide variety of mammals. The polymer also has a wide range of solubilities. Polyalkylene oxides are soluble in most organic solvents as well as aqueous solutions. Many polypeptides and other materials conjugated with PEG and PAO's not only retain their solubility properties, but also demonstrate enhanced water solubility as a result of the conjugation. In addition, the biological activities of PEG conjugates are typically dominated by the non-PEG part of the conjugated molecule.
In order to covalently attach PAO's to bio-effecting materials such as proteins, the hydroxyl end-groups of the polymers must first be converted or activated into reactive functional groups. For example, the use of PEG-succinimidyl succinate (SS-PEG) as a conjugating agent has been suggested. The activated polymer reacts with proteins under mild conditions while preserving biological activities. The resultant ester linkage, however, has limited stability in aqueous media.
The formation of urethane (carbamate) linkages between the amino groups of a protein and PEG has provided one solution to the problem of hydrolytic release of the polymer chains. Attachment of PEG to a protein via a carbamate derivative was disclosed in Analytical Biochemistry 131, 25-33 (1983) using carbonyl diimidazole-activated PEG. The activated polymer, however, is not very reactive with proteins, often requiring two to three days to achieve sufficient modifications. Another drawback associated with carbonyl diimidazole is its high relative cost.
Commonly assigned U.S. Patent No.5,122,614 provides further improvements in PAO's which form urethane linkages with proteins. For example, the patent discloses protein modification with succinimidyl carbonates of PEG (SC-PEG) and bifunctional derivatives thereof (BSC-PEG) carried out within short periods of time over a broad pH range. The use of succinimidyl carbonates of PEG, however, cannot be universally applied to all bio-effecting materials. For example, under certain circumstances, some rearrangement of the SC-PEG has been observed, especially at higher temperatures. Beta alanine, often an unwanted by-product, is produced as a result of this rearrangement. In addition, substantial pH adjustment is required to maintain optimum reaction speed and minimize rearrangement.
Indirect attachment of isocyanate-containing substituents to PEG for the purpose of modifying bio- effecting agents has also been suggested. See, for example Eur.Polym.J. Vol.19. No.12, 1177-1183 (1983) and Eur.Polym.J. Vol.19. No.4, 341-346 (1983). The formation of PEG-O2CNH-(CH2)6-NCO or PEG-substituted- alkyl or aryl-isocyanate has been reported as a possible amine and/or alcohol group modifier. Such indirect polymer coupling technology, however, suffers from several significant shortcomings and drawbacks. For example, such PEG-diisocyanates are inefficiently prepared. Reacting PEG with the hexamethylene diisocyante is incomplete and yields mono- and diisocyanates while leaving significant amounts of unreacted products. In order to overcome this drawback, excess amounts of diisocyanates must be employed and then removed. Moreover, unlike technology using direct linkage of PEG to a target amine, the indirect attaching group which separates the PEG and amine is susceptible to breakdown into several undesirable byproducts, including potentially toxic aliphatics during in vivo metabolic processes.
Each of the activated forms of the PAO-based polymers described above has properties which, under certain circumstances, may be considered advantageous. Nevertheless, extending the PAO-modifying reagents and techniques to include additional proteins, peptides or chemotherapeutic agents which heretofore have not benefitted from such modifications because of unreactivity, harmful by-products and the like is desirable.
It would be advantageous to provide stable PEG-modified bio-effecting agents which maintain the activity of the functional moieties in the body for extended periods. It would also be beneficial to provide such PEG-conjugates in a form having substantially reduced immunogenicity, improved solubility and pH stability. It is therefore, an object of the present invention to provide improved therapeutic agents using PAO modification techniques. Other and further objects of the invention will be apparent from the disclosure set forth herein.
SUMMARY OF THE INVENTION
In accordance with the present invention, improvements in polyalkylene oxide(PAO)-based therapeutics are disclosed. In one aspect of the invention, PAO - based compounds are disclosed having the formula: (I) Z - (O - R1)a - (O - R2)b - (O - R3)c - Y wherein:
R1, R2 and R3 are alkyl groups;
a is an integer between 1 and 1000 and each of b and c is an integer between 0 and 1000, and the sum of a, b and c is between 10 and 1000;
Y is NCX, wherein X is one of O or S; and
Z is one of an alkyl, cycloalkyl, branched alkyl, arylalkyl group or Y.
In this aspect of the invention, each of R1, R2 and
R3 can be straight or branched alkyl groups and/or each of R1, R2, R3 can be independently the same or different from each other. In a particularly preferred aspect of the invention, the PAO-based compositions are polyethylene glycol-based, having a molecular weight of from about 2,000 to about 20,000.
In another aspect of the present invention, methods are provided for preparing the compositions of (I). The methods include reacting compounds having the formula:
(II) A-(O-R1)a-(O-R2)b-(O-R3)c-NH2 wherein R1, R2 and R3 are alkyl groups;
a is an integer between 1 and 1000 and each of b and c is an integer between 0 and 1000, and the sum of a, b and c is between 10 and 1000; and
A is one of an alkyl, cycloalkyl, branched alkyl, arylalkyl or amino group;
with phosgene or a phosgene-containing substance such as triphosgene in the presence of a suitable base.
A further aspect of the invention provides compositions containing bio-effecting substances covalently bound to at least one PAO-based derivative. The covalent linkage achieved through the isocyanate and/or isothiocyanate portion of the activated PAO-based polymer derivatives (I) and an alcohol present in the bio-effecting substance produce a urethane and/or thiourethane linkage. The covalent linkage between the reactants can also be achieved through the same isocyanate or isothiocyanate portions of the activated PAO-based polymers and amines present in the bio- effecting substance to produce urea and/or thiourea linkages.
A still further aspect of the invention is directed to methods for preparing such bio-effecting-PAO compositions. The methods include reacting the PAO-based polymer with the bio-effecting substance under conditions which effect covalent attachment of the ingredients at the isocyato- and/or isothiocyato-portion of the polymer and amine and/or alcohol portion of the bio-effector. The conditions are further defined as those which permit the covalent attachment of the reactants while substantially maintaining the desired effect of the bio-effecting substance.
The applications of the present invention are vast. The activated PAO's of the present invention equip the artisan with a polymer-based material that is suitable both for amine-based and alcohol-based linkages. Indeed, it is now possible to attach the polymers to bio-effecting substances at both locations at will in order to maintain optimal activity of the native portion of the molecule. The ability to effect such mixed attachments of the reactants allows the artisan to precisely attach the beneficial polymer where desired and more precisely modulate the activity of the therapeutic conjugates.
As a result of the present invention, PAO-based polymers are provided which improve a wide range of therapeutic substances. The conjugation prolongs the circulating life of the substances when compared to unmodified forms. Thus, it is possible to substantially reduce the amount and frequency of administrations without diminishing the therapeutic effect. Such reductions also reduce the concomitant toxicities often seen when high doses of agents are administered, especially over time. In addition, the novel covalent conjugation of PAO's to bio-effecting substances also improves the aqueous solubility of the bio-effector and allows the artisan to provide therapeutic compositions in heretofore impractical dosage forms.
The most dramatic of all improvements achieved as a result of the present invention is often the substantial reduction of the immunogenic and/or allergic responses of the compositions of the present invention when compared to the unmodified or native bio-effecting counterparts. Whereas in the past, such therapy-limiting consequences often would mean discontinuing treatment, the compositions of the present invention allow not only continued treatment but also substantial reductions in untoward reactions. DETAILED DESCRIPTION OF THE INVENTION
The present invention includes activated polyalkylene oxides having the general structure:
(I) Z - (O - R1)a - (O - R2)b - (O - R3)c - Y wherein:
R1, R2 and R3 are alkyl groups;
a is an integer between 1 and 1000 and each of b and c is an integer between 0 and 1000, and the sum of a, b and c is between 10 and 1000;
Y is NCX, wherein X is one of O or S; and
Z is one of an alkyl, cycloalkyl, branched alkyl, arylalkyl group or Y.
An important feature of the activated PAO's described herein is the presence of Y in a terminal position. The NCO or isocyanate portion of the activated polymer reacts rapidly with nucleophiles such as the epsilon-amino group of lysine moieties or the hydroxyl or alcohol groups of various bio-effecting substances to produce stable, conjugated molecules.
The stability of the conjugate is achieved by covalent linkage between the isocyanate and/or isothiocyanate portion of the activated PAO-based polymer derivatives (I) and an alcohol present in the bio- effecting substance to produce a urethane and/or thiourethane linkage. The covalent linkage between the reactants can also be achieved through the same isocyanate or isothiocyanate portions of the activated PAO-based polymers and amines present in the bio-effecting substance to produce urea and/or thiourea linkages. Conjugation with the isocyanate-activated PAO's is preferred.
The stability of the conjugates is superior to that of many other PEG conjugations which depend upon ester linkages, especially in the area of hydrolytic stability. In addition, conjugation reactions combining the novel activated PAO's described herein can be carried out with minimal or no pH adjustment to maintain an optimal reactivity range.
Within the compositions of (I), suitable alkyl groups include, for example, ethyl, propyl, isopropyl or butyl. Cycloalkyl groups include cyclohexyls, for example; suitable branched alkyIs include materials such as 3-methylhexyl, for example and suitable arylalkyls groups include, for example, benzyl.
In particular, the present invention relates to PAO-based polymers which are water soluble and do not generate an immunogenic response when administered to mammals. The polymeric substances included herein are also preferably water-soluble at room temperature. A wide variety of materials are thus suitable for use herein. A non-limiting list of such polymers include:
polyalkylene oxide homopolymers such as polyethylene glycol or polypropylene gylcols, polyoxyethylenated polyols, copolymers thereof and block copolymers thereof, provided that water solubility of the block copolymer is maintained. In addition, each of R1, R2 and R3 may be straight or branched alkyl groups. Alternatively, each of R1-3 may be independently the same as or different from the others of R1-3. For example, each of R1-3 can be CH2CH2, CH2CHCH3 or CH2CH2CH2CH2.
Polyethylene glycols are the preferred polyalkylene oxides (PAO's) described herein. Although polyethylene glycols come in a variety of molecular weights, averaging from about 600 to about 100,000 daltons, the preferred molecular weight range for modifying therapeutic agents is from about 2,000 to about 20,000 daltons. Molecular weights of from about 3,000 to about 7,000 are especially preferred.
The molecular weight of the polymer will vary depending upon the needs of the artisan and the particular bio-effecting substance to be modified. Those of ordinary skill in the art can determine the molecular weight ranges suitable for particular end use applications. The foregoing is merely illustrative not to be considered restrictive of the type of materials suitable for use herein. Those skilled in the art will realize that equivalent or substantially equivalent polymeric materials not specifically mentioned but having the qualities described herein are also contemplated.
In a particularly preferred aspect of the invention, new, activated polyethylene glycols are provided. Within this aspect are poly(ethylene glycol)-isocyanates (PEG- NCO) and their bifunctional derivatives, PEG bis-isocyanate, as well as poly(ethylene glycol)-isothiocyanates (PEG-NCS) and their bifunctional derivatives. Thus, for example, in (I), Z can be the same as Y, that is, Z = isocyanate or isothiocyanate. Heterobifunctional derivatives of PEG and PAO's are also possible. In this scenario, Z may be an alternative functional group to that of Y, that is, Z = isothiocyanate when Y = isocyanate. Alternative functional groups for Z such as hydrogen, an alkyl group, a chloroformate or an N-dicarboximide are also contemplated.
The activated PAO's of (I) are generally prepared by reacting compounds having the formula:
(II) A-(O-R1)a-(O-R2)b-(O-R3)c-NH2 wherein R1, R2 and R3 are alkyl groups; a is an integer between 1 and 1000 and each of b and c is an integer between 0 and 1000, and the sum of a, b and c is between 10 and 1000; and
A is one of an alkyl, cycloalkyl, branched alkyl, arylalkyl or amino group;
with phosgene or a phosgene-generating substance in the presence of a suitable base. In the case of A = an amino, a bifunctional diisocyanate is formed. The reaction can be carried out over a broad temperature range of from about 10 to about 130° C.
In the case of the preferred PEG-based polymers, initially, methoxy PEG, mPEG, readily available from Aldrich Chemical of Milwaukee, WI is converted to mPEG-NH2 by treatment with thionyl chloride to form mPEG-Cl followed by treatment with aqueous ammonia. See, for example, Biotech. App. Biochem. 9, 258-68 (1987). Thereafter, PEG isocyanate or PEG isothiocyanate formation is accomplished by reacting the PEG-amine with phosgene or thiophosgene or similar generating substances such as bis- (trichloromethyl) carbonate (triphosgene) in the presence of a base such as triethylamine. Suitable alternative bases include pyridine, N,N-dimethylaniline, 1,4-diazabicyclo[2.2.2]octane or DABCO™, available from Aldrich Chemical Co. of Milwaukee, WI. or any other suitable inert tertiary amine bases.
The amount of activated PAO required to modify and conjugate with a subject bio-effecting molecule will depend on several factors. Principally, however, the amount of polymer is dependent upon the needs of the artisan and the degree of modification desired. The ratio of activated polymer to bio-effecting substance will accordingly vary widely. Molar ratios on the order of from about 0.1 : 1 to about 50 : 1 are contemplated, with ratios of from about 1 : 1 to about 25 : 1 being preferred and about 5 : 1 to about 20 : 1 being most preferred. Protein modification, for example, often requires a molar excess of the activated polymer. A 5 to 40-fold molar excess of PEG-NCO is sufficient to modify hemoglobin.
Activation of the PAO-based polymer with isocyanate is depicted below in Scheme 1, using PEG as the representative polymer:
Figure imgf000015_0001
Activation of the polymer with isocyanate into the bifunctional derivative using PEG as the illustrative polymer is set forth below in Scheme 2:
Figure imgf000016_0001
X = O or S
The above-described activated PAO's can be covalently attached to a wide variety of bio-effecting substances to provide compositions having the general formula:
(III) Z'-(O - R1)a - (O - R2)b - (O - R3)c -NH-CO-W-R" wherein R1, R2 and R3 are alkyl groups;
a is an integer between 1 and 1000 and each of b and c is an integer between 0 and 1000, and the sum of a, b and c is between 10 and 1000;
W = O or NR, wherein R = H or an alkyl;
R" is a bio-effecting substance; and Z' is an alkyl, cycloalkyl, branched alkyl, arylalkyl or -NH-CO-W-R"
As used herein, the term "bio-effecting" substance is to be broadly construed and means any substance displaying a physiologic effect preferably in mammals after administration, whether oral, parenteral or otherwise. Within this broad gamut of substances, there are included peptides, polypeptides and enzymes, both naturally and synthetically derived. The present invention also contemplates attachment of the activated
PAO's to chemical moieties such as chemotherapeutic substances. A non-limiting list of such bio-effecting substances includes:
a) enzymes such as asparaginase, arginase, adenosine deaminase, superoxide dismutase, catalase, chymotrypsin, lipase, uricase, bilirubin oxidase, glucose oxidase, glucuronidase, galactosidase, glucocerebrosidase and glucuronidase;
b) polypeptides such as Factor VIII, insulin, ACTH, glucagon, somatostatin, somatotropins, thymosin, parathyroid hormone, pigmentary hormones, somatomedins, erythropoietin, luteinizing hormone, hypothalamic releasing factors, antidiuretic hormones, prolactin, interleukins, interferons and colony stimulating factors, hemoglobin, cytokines and antibodies;
c) glycopolypeptides such as immunoglobulins, ovalbumin, lipase, glucocerebrosidase, lectins, tissue plasminogen activator and glycosilated interleukins, interferons and colony stimulating factors.
d) immunoglobulins such as IgG, IgE, IgM, IgA, IgD and fragments thereof.
e) chemotherapeutic agents of all therapeutic categories such as anti-inflammatory agents, antiinfective compositions such as antibiotics, including hydroxy- or amino-penicillins or cephalosporins, anti-tumor agents such as taxol or taxol derivatives including 2'-acetyltaxol or methotrexate and the like, analgesics such as opiates, cardiac agents, steroids such as corticosteriods, central nervous system agents, etc.
The only limitation on the type of bio-effecting substances suitable for use herein is that the substance contain at least one amine or alcohol substituent for covalent attachment of the activated PAO without substantially reducing or eliminating the therapeutic effect of the unmodified material. As depicted in Scheme 3 below, when attachment is effected between the isocyanate portion of the activated PEG and alcohols on the bio-effecting substance, stable urethane linkages are formed. Tin-based catalysts such as dibutyltin dilaurate are advantageously included in such reactions. Similarly, when isothiocyanate portions of the activated polymers are reacted with hydroxyls on the bio-effectors, thiourethane linkages are formed.
Figure imgf000020_0001
As shown in Scheme 4 below, when the isocyante portions of the activated PEG'S are reacted with amines on the bio-effecting substance, stable urea linkages are formed. SCHEME 4
PEG-NCO MODIFICATION OF AMINE-CONTAINING
BIO-EFFECTING SUBSTANCE HEMOGLOBIN
Figure imgf000021_0001
The above-described activated PAO's afford substantial modification of a wide variety of bio- effecting substances. The modification reactions described above can be carried out in both aqueous and non-aqueous solvents. For example, protein modification is achievable under mild conditions using aqueous systems having a pH ranging from about 5.0 to about 11.0 and preferably from about 7.0 to about 8.0, moderate reaction temperatures of from about 0 to about 10° C, and reaction times of from about 0.25 to about 1 hour. In non-aqueous systems where hydroxyl moieties are linked to the polymers, more rigorous temperature and time conditions are sustainable and will generally include tin-based catalysts to speed up the reaction. EXAMPLES
The following examples serve to provide further appreciation of the invention but are not meant in any way to restrict the effective scope of the invention.
EXAMPLE 1
Preparation of mPEG-NH2 mPEG-NH2 was prepared using procedures similar to those described in Biotech. App. Biochem. 9, 258-268 (1987). In a 1 liter round bottom flask, 100 grams of mPEG-5000 was melted and then dried under high vacuum. Twenty grams of thionyl chloride was then added to the dried mPEG and the mixture was heated to 65-70 degrees C. for 5 hours using a reflux condenser. The unreacted thionyl chloride was removed with a rotary evaporator and high vacuum. A 2 gram sample of this melt was recrystallized from 2-propanol. The 13C NMR spectrum (CDCl3) showed a peak at 42 ppm corresponding to CH2-Cl. A peak at 61.5 ppm (corresponding to CH2-OH) could not be detected indicating that the conversion was greater than 95%.
Thereafter, 150 mL of deionized water and 250 mL concentrated ammonia were added to a flask containing the mPEG and the flask was heated at 60°C for 4 days using a reflux condenser. The reaction mixture was then stirred under aspirator vacuum at 60° C. for 4 hours to remove ammonia. The reaction mixture was then cooled to room temperature and to the flask were added 20 grams of K2CO3 and 120 grams of NaCl. After complete dissolution of the salts, mPEG-NH2 was extracted into two 500 ml portions of methylene chloride. The methylene chloride layer was dried over sodium sulfate and then evaporated to dryness. The product was recrystallized from 1L of isopropanol. The product was characterized by non-aqueous titration according to the method described in Eur. J. Biochem. 42, 151 (1974) and by 1 NMR and 13C NMR.
EXAMPLE 2
Preparation of mPEG-NCO
In this Example, 50 grams (10 mmol) of mPEG-NH2 was placed in a 500 ml flask and dried by azeotropic distillation with toluene. The flask was cooled to 65-70°C and 1.5 grams, (5 mmol), of bis-(trichloromethyl) carbonate (triphosgene) and 2 grams, (20 mmol), of triethylamine were added and stirred at this temperature for 2 hours before dry nitrogen was bubbled through the reaction mixture for 30 minutes. The mixture was then filtered at 40°C to remove triethylamine hydrochloride. The filtrate was evaporated to near dryness and precipitated with 250 milliliters of ether. The precipitate was dried under high vacuum. The product showed 100% activity by non-aqueous titration. The FTIR showed a strong peak at 2263 cm-1 and 13C NMR gave a peak at 43.08 ppm for CH2NCO and a weak peak at 121 ppm for N=C=O.
EXAMPLE 3
m-PEG-Isothiocyanate
To a solution of 50 g (0.01mol) of m-PEG-amine hydrochloride, in 500 milliliters of chloroform at room temperature was added 2.6 g (0.023mol) of thiophosgene and 6.1 g (0.06mol) of triethylamine. The resulting mixture was then refluxed for 2 hours, followed by removal of the solvent by distillation in vacuo to yield a semi-solid residue. This residue was recrystallized from 2-propanol to yield 45.6 g of the product (90% yield). FT-NMR 13C assignments: OCH3, 58.51 ppm; CH2NCS, 44.81. The purity of the sample was determined by non-aqueous titrations, as in the case of PEG-isocyanate, and was found to be greater than 90% pure. The FTIR showed a strong absorption at 2111.7 cm-1. EXAMPLE 4
PEGylation of 2'-acetyltaxol using mPEG-NCO:
The 7-urethane derivative In this Example, mPEG-NCO was prepared in situ by placing 515 mg (0.010 mmol) of mPEG-NH2 in a 100 ml round bottom flask and undergoing drying by azeotropic toluene distillation and converted to mPEG-NCO as described above. The FTIR showed the isocyanate peak at 2263 cm-1 of intensity similar to that obtained above (relative to polyethylene glycol peaks). The reaction mixture was cooled to room temperature and to it were added 60 mg of 2'-acetyltaxol prepared as described in Biochem. Biophys. R.S. Commun. 124, 329-336 (1984), which is incorporated by reference herein, and 10 mg Sn(II) octoate and the mixture was stirred at room temperature. The reaction was followed by HPLC on a C8 column with 3:1 methanol-H2O as eluent. The reaction appeared to be complete when about 75% of 2'-acetyltaxol was converted to the corresponding PEG-derivative. The reaction product was evaporated to near dryness and precipitated with ether. Most of the unreacted 2'-acetyltaxol and any 2', 7-diacetyltaxol present remained in the ether phase. The ether was decanted, and the precipitate was recrystallized from 20 milliliters of 2-propanol. The 7-PEG urethane derivative was isolated by centrifuging. washing with two 20 milliliter portions of 2-propanol, and finally drying under high vacuum to obtain 508 mg of purer product containing less than 1% 2'-acetyltaxol and some non-functionalized PEG.
The 7-PEG urethane derivative could be further purified by HPLC using a semipreparative C8-column. The FTIR spectrum of the purified compound had all the characteristic peaks of PEG in addition to peaks at 1748.6, 1741.2, 1726.5, 1663 cm-1 which are characteristic of the 2'-acetyltaxol molecule.
EXAMPLES 5 - 6
In these Examples, conjugation of the activated PEG-NCO to the protein bovine hemoglobin (Hb), obtained from California Biological and Protein Corp. of Huntington Beach, CA were carried out. The reaction conditions included maintaining a constant pH of 7.8 and temperature of 8°C. The hemoglobin was reacted in a solution having a concentration of about 10.6%. PEG-NCO was added as a solid.
EXAMPLE 5
Hb-PEG Conjugation using 12x molar excess PEG-NCO
Hb (10.6%, 2ml) was diluted with 2 ml of pH 7.8 buffer in a jacketed reaction vessel kept at 8°C. PEG-NCO (217.6 mg, 1.08 × 10-5M) was added to the reaction vessel containing the Hb. The pH was adjusted to approximately 7.8 with 1.0N NaOH. The reaction mixture was stirred uniformly and slowly for two hours at 8°C maintaining pH 7.8. Thereafter, 21 mg cysteine HCI (0.03M) and glycine solution (3.24 mg, 19μl of stock 336/2ml of pH 7.0 phosphate buffer) which is equimolar with PEG-NCO were added, and the pH was re-adjusted to 7.8 with 1N NaOH. The yield of the desired PEG-Hb was determined by HPLC retention time to be in excess of 73%
EXAMPLE 6
Hb conjugation using 18x molar excess PEG-NCO
Hb (10.6%, 3ml) was diluted with 3 ml of pH 7.8 buffer in a jacketed reaction vessel kept at 8°C. The pH was adjusted to approximately 7.8 with 1.0N NaOH. PEG-NCO (491.7 mg, 1.62×10-2M) was added to the reaction vessel kept at 8°C with Hb. The reaction mixture was stirred uniformly and slowly for two hours at 8°C maintaining pH 7.8. Thereafter, 31.6 mg cysteine HCI (0.03M) and glycine solution (12.18 mg, 43.5μl of stock 336/2ml of pH 7.0 phosphate buffer) which is equimolar with PEG-NCO were added. The pH was adjusted to 7. with 1N NaOH and allowed to stir for five minutes. The sample was analyzed by HPLC to determine the yield of desired PEG-Hb to be in excess of 83 %.
While there have been described what are presently believed to be the preferred embodiments of the invention, those skilled in the art will realize that changes and modifications may be made thereto without departing from the spirit of the invention and it is intended to claim all such changes and modifications as fall within the true scope of the invention.

Claims

WHAT IS CLAIMED IS:
1. A composition comprising the formula:
(III) Z'-(O - R1)a - (O - R2)b - (O - R3)c -NH-CO-W-R" wherein R1, R2 and R3 are alkyl groups;
a is an integer between 1 and 1000 and each of b and c is an integer between 0 and 1000, and the sum of a, b and c is between 10 and 1000;
W = O or NR, wherein R = H or an alkyl;
R" is a bio-effecting substance; and
Z' is an alkyl, cycloalkyl, branched alkyl, arylalkyl or
-NH-CO-W-R"
2. The composition of Claim 1, wherein said bio-effecting substance is a polypeptide.
3. The composition of Claim 1, wherein said bio-effecting substance is a chemotherapeutic agent.
4. The composition of Claim 2, wherein said polypeptide is an enzyme.
5. The composition of Claim 4, wherein said enzyme is selected from the group consisting of asparaginase, arginase, adenosine deaminase, superoxide dismutase, catalase, chymotrypsin, lipase, uricase, bilirubin oxidase, glucose oxidase, glucuronidase, galactosidase, glucocerebrosidase and glucuronidase.
6. The composition of Claim 2, wherein said polypeptide is selected from the group consisting of Factor VIII, insulin, ACTH, glucagon, somatostatin, somatotropins, thymosin, parathyroid hormone, pigmentary hormones, somatomedins, erythropoietin, luteinizing hormone, hypothalamic releasing factors, antidiuretic hormones, prolactin, interleukins, interferons and colony stimulating factors, hemoglobin, cytokines and antibodies.
7. The composition of Claim 2, wherein said polypeptide is a glycopolypeptide.
8. The composition of Claim 7, wherein said glycopolypeptide is selected from the group consisting of immunoglobulins, ovalbumin, lipase, glucocerebrosidase, lectins, tissue plasminogen activator and glycosilated interleukins, interferons and colony stimulating factors.
9. The composition of Claim 8, wherein said immunoglobulin is selected from the group consisting of IgG, IgE, IgM, IgA, IgD and fragments thereof.
10. The composition of Claim 3, wherein said chemotherapeutic agent is taxol or a taxol derivative.
11. The composition of Claim 3, wherein said chemotherapeutic agent is selected from the group consisting of anti-infectives, anti-inflammatories, antitumor agents, analgesics, cardiac agents, steroids and central nervous system agents.
12. A method of preparing a composition of Claim 1, comprising reacting a first composition corresponding to the formula:
(I) Z - (O - R1)a - (O - R2)b - (O - R3)c - Y wherein:
R1, R2 and R3 are alkyl groups;
a is an integer between 1 and 1000 and each of b and c is an integer between 0 and 1000, and the sum of a, b and c is between 10 and 1000;
Y is NCX, wherein X is one of O or S; and
Z is one of an alkyl, cycloalkyl, branched alkyl, arylalkyl group or Y. with a bio-effecting substance under conditions sufficient to effect conjugation of said composition and said bio-effecting molecule.
13. The method of Claim 12, wherein said conditions include reacting said first composition with said bio-effecting substance in a molar ratio of from about 0.1 : 1 to about 50 : 1.
14. The method of Claim 13, wherein said conditions include reacting said first composition with said bio-effecting substance in a molar ratio of from about 1 : 1 to about 25 : 1.
15. The method of Claim 14, wherein said conditions include reacting said composition of Claim 1 with said bio-effecting substance in a molar ratio of from about 5 : 1 to about 20 : 1.
16. The method of Claim 12, wherein said bio-effecting substance is a polypeptide.
17. The method of Claim 16, wherein said polypeptide is an enzyme.
18. The method of Claim 12, wherein said bio-effecting molecule is a chemotherapeutic agent.
19. The method of Claim 18, wherein saidchemotherapeutic agent is taxol or a taxol derivative.
20. The method of Claim 18, wherein said chemotherapeutic agent is selected from the group consisting of anti-infectives, anti-inflammatories, anti-tumor agents, analgesics, cardiac agents, steroids and central nervous system agents. AMENDED CLAIMS
[received by the International Bureau on 24 January 1994 (24.01.94); original claims 1-20 cancelled; original claims 21-36 amended;
(3 pages)]
21. A bio-effecting conjugate, comprising a chemotherapeutic agent linked to a polyalkylene oxide via a urethane linkage.
22. The bio-effecting conjugate of claim 21, wherein said chemotherapeutic agent is taxol or a taxol derivative.
23. The bio-effecting conjugate of claim 21, wherein said chemotherapeutic is selected from the group consisting of anti-infectives, anti-inflammatories, anti-tumor agents, analgesics, cardiac agents, steroids and central nervous system agents.
24. The bio-effecting conjugate of claim 21, wherein said polyalkylene oxide comprises polyethylene glycol.
25. The bio-effecting conjugate of claim 21, wherein said polyalkylene oxide has a molecular weight of from about 600 to about 100,000 daltons.
26. The bio-effecting conjugate of claim 25, wherein said polyalkylene oxide has a molecular weight of from about 2,000 to about 20,000 daltons.
27. The bio-effecting conjugate of claim 26, wherein said polyalkylene oxide has a molecular weight of from about 3,000 to about 7,000 daltons.
28. A method of preparing a bio-effecting conjugate containing a chemotherapeutic agent linked to a polyalkylene oxide urethane linkage, comprising: reacting an isocyanate-activated polyalkylene oxide with a chemotherapeutic agent in the presence of a tin-based catalyst.
29. The method of 28, wherein said chemotherapeutic agent is taxol or a taxol derivative.
30. The method of claim 28, wherein said chemotherapeutic is selected from the group consisting of anti-infectives, anti-inflammatories, anti-tumor agents, analgesics, cardiac agents, steroids and central nervous system agents.
31. The method of claim 28, wherein said polyalkylene oxide comprises polyethylene glycol.
32. The method of claim 28, wherein said polyalkylene oxide has a molecular weight of from about 600 to about 100,000 daltons.
33. The method of claim 28, wherein said polyalkylene oxide has a molecular weight of from about 2,000 to about 20,000 daltons.
34. The method of claim 28, wherein said polyalkylene oxide has a molecular weight of from about 3,000 to about 7,000 daltons.
35. The method of claim 28, wherein said tin-based catalyst is dibutyltin dilaurate or Sn(II) Octoate.
36. The method of claim 28, wherein further comprising conducting said reacting in a non-aqueous environment.
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Cited By (126)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0727992A1 (en) * 1993-10-20 1996-08-28 Enzon, Inc. 2'- and/or 7- substituted taxoids
EP0745390A2 (en) * 1995-05-31 1996-12-04 Bristol-Myers Squibb Company Polymeric prodrugs for beta-lactamase and uses thereof
US5585484A (en) * 1995-04-19 1996-12-17 Albert Einstein College Of Medicine Of Yeshiva University, A Division Of Yeshiva University Hemoglobin crosslinkers
WO1996040791A1 (en) * 1995-06-07 1996-12-19 Novo Nordisk A/S Modification of polypeptides
WO1996040792A1 (en) * 1995-06-07 1996-12-19 Novo Nordisk A/S Modification of polypeptides
EP0750633A1 (en) * 1994-03-16 1997-01-02 Enzon, Inc. Enhancement of antitumor therapy with hemoglobin-based conjugates
EP0807115A1 (en) * 1995-01-30 1997-11-19 Enzon, Inc. High molecular weight polymer-based prodrugs
WO1998006421A1 (en) * 1996-08-16 1998-02-19 Cancer Treatments International Therapeutic compositions for treatment of cancer
WO1999055376A1 (en) * 1998-04-28 1999-11-04 Applied Research Systems Ars Holding N.V. Peg-lhrh analog conjugates
JP2001508783A (en) * 1997-01-29 2001-07-03 ポリマスク・ファーマシューティカルズ・パブリック・リミテッド・カンパニー PEGylation method
US6495136B1 (en) 1998-03-26 2002-12-17 The Procter & Gamble Company Proteases having modified amino acid sequences conjugated to addition moieties
US6566115B1 (en) 1999-07-22 2003-05-20 The Procter & Gamble Company Protease conjugates having sterically protected clip sites
US6569663B1 (en) 1998-03-26 2003-05-27 The Procter & Gamble Company Serine protease variants having amino acid substitutions
US6586223B1 (en) 1999-07-22 2003-07-01 The Procter & Gamble Company Subtilisin protease variants having amino acid substitutions in defined epitope regions
US6586224B1 (en) 1999-07-22 2003-07-01 The Procter & Gamble Company Subtilisin protease variants having amino acid deletions and substitutions in defined epitope regions
WO2004000366A1 (en) 2002-06-21 2003-12-31 Novo Nordisk Health Care Ag Pegylated factor vii glycoforms
WO2004061094A1 (en) 2002-12-30 2004-07-22 Gryphon Therapeutics, Inc. Water-soluble thioester and selenoester compounds and methods for making and using the same
EP1455766A2 (en) * 2001-11-01 2004-09-15 SciClone Pharmaceuticals, Inc. Thymosin alpha 1 peptide/polymer conjugates
WO2004075923A3 (en) * 2003-02-26 2005-02-24 Nektar Therapeutics Al Corp Polymer-factor viii moiety conjugates
US6908757B1 (en) 1998-03-26 2005-06-21 The Procter & Gamble Company Serine protease variants having amino acid deletions and substitutions
US6914121B2 (en) 1998-04-28 2005-07-05 Applied Research Systems Ars Holding N.V. PEG-LHRH analog conjugates
US6946128B1 (en) 1999-07-22 2005-09-20 The Procter & Gamble Company Protease conjugates having sterically protected epitope regions
EP1588717A1 (en) * 1998-04-28 2005-10-26 Applied Research Systems ARS Holding N.V. PEG-LHRH analog conjugates
US6962954B2 (en) * 2002-03-25 2005-11-08 Albert Einstein College Of Medicine Of Yeshiva University Activated polyethylene glycol compounds
WO2006009901A2 (en) 2004-06-18 2006-01-26 Ambrx, Inc. Novel antigen-binding polypeptides and their uses
US7019117B2 (en) 2001-07-19 2006-03-28 Albert Einstein College Of Medicine Of Yeshiva University Size enhanced hemoglobins: surface decoration and crosslinking of the protein with polyoxy alkylene glycols
WO2006069220A2 (en) 2004-12-22 2006-06-29 Ambrx, Inc. Modified human growth hormone
WO2006069246A2 (en) 2004-12-22 2006-06-29 Ambrx, Inc. Compositions containing, methods involving, and uses of non-natural amino acids and polypeptides
WO2006134173A2 (en) 2005-06-17 2006-12-21 Novo Nordisk Health Care Ag Selective reduction and derivatization of engineered proteins comprising at least one non-native cysteine
US7338933B2 (en) 2004-01-08 2008-03-04 Neose Technologies, Inc. O-linked glycosylation of peptides
WO2008030558A2 (en) 2006-09-08 2008-03-13 Ambrx, Inc. Modified human plasma polypeptide or fc scaffolds and their uses
WO2008060780A2 (en) 2006-10-04 2008-05-22 Novo Nordisk A/S Glycerol linked pegylated sugars and glycopeptides
EP1982732A2 (en) 2000-02-11 2008-10-22 Maxygen Holdings Ltd. Factor VII or VIIA-like molecules
EP2042196A2 (en) 2001-10-10 2009-04-01 Neose Technologies, Inc. Remodelling and glycoconjugation of Granulocyte Colony Stimulating Factor (G-CSF)
EP2055189A1 (en) 2003-04-09 2009-05-06 Neose Technologies, Inc. Glycopegylation methods and proteins/peptides produced by the methods
WO2009067636A2 (en) 2007-11-20 2009-05-28 Ambrx, Inc. Modified insulin polypeptides and their uses
EP2080771A2 (en) 2001-02-27 2009-07-22 Maxygen Aps New interferon beta-like molecules
US7579444B2 (en) 2004-06-30 2009-08-25 Nektar Therapeutics Al, Corporation Polymer-factor IX moiety conjugates
EP2133099A2 (en) 2003-07-11 2009-12-16 Polytherics Limited Conjugated biological molecules and their preparation
EP2133098A1 (en) 2000-01-10 2009-12-16 Maxygen Holdings Ltd G-CSF conjugates
WO2010011735A2 (en) 2008-07-23 2010-01-28 Ambrx, Inc. Modified bovine g-csf polypeptides and their uses
US7714114B2 (en) 2005-02-16 2010-05-11 Nektar Therapeutics Conjugates of an EPO moiety and a polymer
US7736872B2 (en) 2004-12-22 2010-06-15 Ambrx, Inc. Compositions of aminoacyl-TRNA synthetase and uses thereof
EP2213733A2 (en) 2006-05-24 2010-08-04 Novo Nordisk Health Care AG Factor IX analogues having prolonged in vivo half life
US7795210B2 (en) 2001-10-10 2010-09-14 Novo Nordisk A/S Protein remodeling methods and proteins/peptides produced by the methods
US7803777B2 (en) 2003-03-14 2010-09-28 Biogenerix Ag Branched water-soluble polymers and their conjugates
US7816320B2 (en) 2004-12-22 2010-10-19 Ambrx, Inc. Formulations of human growth hormone comprising a non-naturally encoded amino acid at position 35
US7842661B2 (en) 2003-11-24 2010-11-30 Novo Nordisk A/S Glycopegylated erythropoietin formulations
WO2010144629A1 (en) 2009-06-09 2010-12-16 Prolong Pharmaceuticals, LLC Hemoglobin compositions
EP2263684A1 (en) 2003-10-10 2010-12-22 Novo Nordisk A/S IL-21 derivatives
EP2279756A2 (en) 2005-04-05 2011-02-02 Instituto di Ricerche di Biologia Molecolare p Angeletti S.P.A. Method for shielding functional sites or epitopes on proteins
EP2298354A2 (en) 2001-10-10 2011-03-23 BioGeneriX AG Remodelling and glycoconjugation of interferon-beta
US7932364B2 (en) 2003-05-09 2011-04-26 Novo Nordisk A/S Compositions and methods for the preparation of human growth hormone glycosylation mutants
US7947473B2 (en) 2004-12-22 2011-05-24 Ambrx, Inc. Methods for expression and purification of pegylated recombinant human growth hormone containing a non-naturally encoded keto amino acid
EP2327724A2 (en) 2004-02-02 2011-06-01 Ambrx, Inc. Modified human growth hormone polypeptides and their uses
US7956032B2 (en) 2003-12-03 2011-06-07 Novo Nordisk A/S Glycopegylated granulocyte colony stimulating factor
US8012931B2 (en) 2007-03-30 2011-09-06 Ambrx, Inc. Modified FGF-21 polypeptides and their uses
WO2011107591A1 (en) 2010-03-05 2011-09-09 Rigshospitalet Chimeric inhibitor molecules of complement activation
EP2386571A2 (en) 2005-04-08 2011-11-16 BioGeneriX AG Compositions and methods for the preparation of protease resistant human growth hormone glycosylation mutants
WO2011143274A1 (en) 2010-05-10 2011-11-17 Perseid Therapeutics Polypeptide inhibitors of vla4
US8076292B2 (en) 2001-10-10 2011-12-13 Novo Nordisk A/S Factor VIII: remodeling and glycoconjugation of factor VIII
US8093356B2 (en) 2005-06-03 2012-01-10 Ambrx, Inc. Pegylated human interferon polypeptides
US8114630B2 (en) 2007-05-02 2012-02-14 Ambrx, Inc. Modified interferon beta polypeptides and their uses
WO2012024452A2 (en) 2010-08-17 2012-02-23 Ambrx, Inc. Modified relaxin polypeptides and their uses
US8207112B2 (en) 2007-08-29 2012-06-26 Biogenerix Ag Liquid formulation of G-CSF conjugate
US8268967B2 (en) 2004-09-10 2012-09-18 Novo Nordisk A/S Glycopegylated interferon α
US8278418B2 (en) 2008-09-26 2012-10-02 Ambrx, Inc. Modified animal erythropoietin polypeptides and their uses
EP2514757A2 (en) 2005-01-10 2012-10-24 BioGeneriX AG Glycopegylated granulocyte colony stimulating factor
WO2013004607A1 (en) 2011-07-01 2013-01-10 Bayer Intellectual Property Gmbh Relaxin fusion polypeptides and uses thereof
WO2013006706A1 (en) 2011-07-05 2013-01-10 Bioasis Technologies Inc. P97-antibody conjugates and methods of use
EP2548967A2 (en) 2006-09-21 2013-01-23 The Regents of The University of California Aldehyde tags, uses thereof in site-specific protein modification
US8404809B2 (en) 2005-05-25 2013-03-26 Novo Nordisk A/S Glycopegylated factor IX
US8420792B2 (en) 2006-09-08 2013-04-16 Ambrx, Inc. Suppressor tRNA transcription in vertebrate cells
EP2586456A1 (en) 2004-10-29 2013-05-01 BioGeneriX AG Remodeling and glycopegylation of fibroblast growth factor (FGF)
EP2633866A2 (en) 2003-10-17 2013-09-04 Novo Nordisk A/S Combination therapy
WO2013185115A1 (en) 2012-06-08 2013-12-12 Sutro Biopharma, Inc. Antibodies comprising site-specific non-natural amino acid residues, methods of their preparation and methods of their use
US8632770B2 (en) 2003-12-03 2014-01-21 Novo Nordisk A/S Glycopegylated factor IX
US8633157B2 (en) 2003-11-24 2014-01-21 Novo Nordisk A/S Glycopegylated erythropoietin
WO2014022515A1 (en) 2012-07-31 2014-02-06 Bioasis Technologies, Inc. Dephosphorylated lysosomal storage disease proteins and methods of use thereof
WO2014036492A1 (en) 2012-08-31 2014-03-06 Sutro Biopharma, Inc. Modified amino acids comprising an azido group
US8716240B2 (en) 2001-10-10 2014-05-06 Novo Nordisk A/S Erythropoietin: remodeling and glycoconjugation of erythropoietin
US8716239B2 (en) 2001-10-10 2014-05-06 Novo Nordisk A/S Granulocyte colony stimulating factor: remodeling and glycoconjugation G-CSF
US8791070B2 (en) 2003-04-09 2014-07-29 Novo Nordisk A/S Glycopegylated factor IX
US8815232B2 (en) 2008-08-26 2014-08-26 Kyon Biotech Ag Compositions and methods for treating cancer
US8841439B2 (en) 2005-11-03 2014-09-23 Novo Nordisk A/S Nucleotide sugar purification using membranes
WO2014160438A1 (en) 2013-03-13 2014-10-02 Bioasis Technologies Inc. Fragments of p97 and uses thereof
EP2805964A1 (en) 2009-12-21 2014-11-26 Ambrx, Inc. Modified bovine somatotropin polypeptides and their uses
EP2805965A1 (en) 2009-12-21 2014-11-26 Ambrx, Inc. Modified porcine somatotropin polypeptides and their uses
US8911967B2 (en) 2005-08-19 2014-12-16 Novo Nordisk A/S One pot desialylation and glycopegylation of therapeutic peptides
US8916360B2 (en) 2003-11-24 2014-12-23 Novo Nordisk A/S Glycopegylated erythropoietin
WO2015006555A2 (en) 2013-07-10 2015-01-15 Sutro Biopharma, Inc. Antibodies comprising multiple site-specific non-natural amino acid residues, methods of their preparation and methods of their use
US8969532B2 (en) 2006-10-03 2015-03-03 Novo Nordisk A/S Methods for the purification of polypeptide conjugates comprising polyalkylene oxide using hydrophobic interaction chromatography
WO2015031673A2 (en) 2013-08-28 2015-03-05 Bioasis Technologies Inc. Cns-targeted conjugates having modified fc regions and methods of use thereof
US9005625B2 (en) 2003-07-25 2015-04-14 Novo Nordisk A/S Antibody toxin conjugates
WO2015054658A1 (en) 2013-10-11 2015-04-16 Sutro Biopharma, Inc. Modified amino acids comprising tetrazine functional groups, methods of preparation, and methods of their use
WO2015081282A1 (en) 2013-11-27 2015-06-04 Redwood Bioscience, Inc. Hydrazinyl-pyrrolo compounds and methods for producing a conjugate
US9050304B2 (en) 2007-04-03 2015-06-09 Ratiopharm Gmbh Methods of treatment using glycopegylated G-CSF
US9121025B2 (en) 2008-09-26 2015-09-01 Ambrx, Inc. Non-natural amino acid replication-dependent microorganisms and vaccines
US9133495B2 (en) 2006-09-08 2015-09-15 Ambrx, Inc. Hybrid suppressor tRNA for vertebrate cells
US9150848B2 (en) 2008-02-27 2015-10-06 Novo Nordisk A/S Conjugated factor VIII molecules
US9187532B2 (en) 2006-07-21 2015-11-17 Novo Nordisk A/S Glycosylation of peptides via O-linked glycosylation sequences
US9310374B2 (en) 2012-11-16 2016-04-12 Redwood Bioscience, Inc. Hydrazinyl-indole compounds and methods for producing a conjugate
US9434778B2 (en) 2014-10-24 2016-09-06 Bristol-Myers Squibb Company Modified FGF-21 polypeptides comprising an internal deletion and uses thereof
US9488660B2 (en) 2005-11-16 2016-11-08 Ambrx, Inc. Methods and compositions comprising non-natural amino acids
US9493499B2 (en) 2007-06-12 2016-11-15 Novo Nordisk A/S Process for the production of purified cytidinemonophosphate-sialic acid-polyalkylene oxide (CMP-SA-PEG) as modified nucleotide sugars via anion exchange chromatography
EP3103880A1 (en) 2008-02-08 2016-12-14 Ambrx, Inc. Modified leptin polypeptides and their uses
US9567386B2 (en) 2010-08-17 2017-02-14 Ambrx, Inc. Therapeutic uses of modified relaxin polypeptides
US9579390B2 (en) 2012-11-12 2017-02-28 Redwood Bioscience, Inc. Compounds and methods for producing a conjugate
EP3135690A1 (en) 2012-06-26 2017-03-01 Sutro Biopharma, Inc. Modified fc proteins comprising site-specific non-natural amino acid residues, conjugates of the same, methods of their preparation and methods of their use
US9605078B2 (en) 2012-11-16 2017-03-28 The Regents Of The University Of California Pictet-Spengler ligation for protein chemical modification
US10266578B2 (en) 2017-02-08 2019-04-23 Bristol-Myers Squibb Company Modified relaxin polypeptides comprising a pharmacokinetic enhancer and uses thereof
WO2019133399A1 (en) 2017-12-26 2019-07-04 Becton, Dickinson And Company Deep ultraviolet-excitable water-solvated polymeric dyes
WO2019191482A1 (en) 2018-03-30 2019-10-03 Becton, Dickinson And Company Water-soluble polymeric dyes having pendant chromophores
WO2020023300A1 (en) 2018-07-22 2020-01-30 Bioasis Technologies, Inc. Treatment of lymmphatic metastases
WO2020056066A1 (en) 2018-09-11 2020-03-19 Ambrx, Inc. Interleukin-2 polypeptide conjugates and their uses
WO2020082057A1 (en) 2018-10-19 2020-04-23 Ambrx, Inc. Interleukin-10 polypeptide conjugates, dimers thereof, and their uses
WO2020168017A1 (en) 2019-02-12 2020-08-20 Ambrx, Inc. Compositions containing, methods and uses of antibody-tlr agonist conjugates
WO2021183832A1 (en) 2020-03-11 2021-09-16 Ambrx, Inc. Interleukin-2 polypeptide conjugates and methods of use thereof
WO2021236526A1 (en) 2020-05-18 2021-11-25 Bioasis Technologies, Inc. Compositions and methods for treating lewy body dementia
WO2021255524A1 (en) 2020-06-17 2021-12-23 Bioasis Technologies, Inc. Compositions and methods for treating frontotemporal dementia
WO2022040596A1 (en) 2020-08-20 2022-02-24 Ambrx, Inc. Antibody-tlr agonist conjugates, methods and uses thereof
US11273202B2 (en) 2010-09-23 2022-03-15 Elanco Us Inc. Formulations for bovine granulocyte colony stimulating factor and variants thereof
WO2022212899A1 (en) 2021-04-03 2022-10-06 Ambrx, Inc. Anti-her2 antibody-drug conjugates and uses thereof
EP4155349A1 (en) 2021-09-24 2023-03-29 Becton, Dickinson and Company Water-soluble yellow green absorbing dyes
WO2024007016A2 (en) 2022-07-01 2024-01-04 Beckman Coulter, Inc. Novel fluorescent dyes and polymers from dihydrophenanthrene derivatives
WO2024044327A1 (en) 2022-08-26 2024-02-29 Beckman Coulter, Inc. Dhnt monomers and polymer dyes with modified photophysical properties

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0510356A1 (en) * 1991-03-25 1992-10-28 F. Hoffmann-La Roche Ag Polyethylene glycol protein conjugates

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0510356A1 (en) * 1991-03-25 1992-10-28 F. Hoffmann-La Roche Ag Polyethylene glycol protein conjugates

Cited By (244)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0727992A4 (en) * 1993-10-20 2001-01-31 Enzon Inc 2'- and/or 7- substituted taxoids
EP0727992A1 (en) * 1993-10-20 1996-08-28 Enzon, Inc. 2'- and/or 7- substituted taxoids
EP0750633A1 (en) * 1994-03-16 1997-01-02 Enzon, Inc. Enhancement of antitumor therapy with hemoglobin-based conjugates
EP0750633A4 (en) * 1994-03-16 1999-09-01 Enzon Inc Enhancement of antitumor therapy with hemoglobin-based conjugates
EP0807115A4 (en) * 1995-01-30 1998-10-07 Enzon Inc High molecular weight polymer-based prodrugs
EP0807115A1 (en) * 1995-01-30 1997-11-19 Enzon, Inc. High molecular weight polymer-based prodrugs
US5585484A (en) * 1995-04-19 1996-12-17 Albert Einstein College Of Medicine Of Yeshiva University, A Division Of Yeshiva University Hemoglobin crosslinkers
EP0745390A3 (en) * 1995-05-31 1999-03-10 Bristol-Myers Squibb Company Polymeric prodrugs for beta-lactamase and uses thereof
EP0745390A2 (en) * 1995-05-31 1996-12-04 Bristol-Myers Squibb Company Polymeric prodrugs for beta-lactamase and uses thereof
WO1996040792A1 (en) * 1995-06-07 1996-12-19 Novo Nordisk A/S Modification of polypeptides
WO1996040791A1 (en) * 1995-06-07 1996-12-19 Novo Nordisk A/S Modification of polypeptides
WO1998006421A1 (en) * 1996-08-16 1998-02-19 Cancer Treatments International Therapeutic compositions for treatment of cancer
JP2001508783A (en) * 1997-01-29 2001-07-03 ポリマスク・ファーマシューティカルズ・パブリック・リミテッド・カンパニー PEGylation method
US6569663B1 (en) 1998-03-26 2003-05-27 The Procter & Gamble Company Serine protease variants having amino acid substitutions
US6908757B1 (en) 1998-03-26 2005-06-21 The Procter & Gamble Company Serine protease variants having amino acid deletions and substitutions
US6495136B1 (en) 1998-03-26 2002-12-17 The Procter & Gamble Company Proteases having modified amino acid sequences conjugated to addition moieties
EP1588717A1 (en) * 1998-04-28 2005-10-26 Applied Research Systems ARS Holding N.V. PEG-LHRH analog conjugates
US7268210B2 (en) 1998-04-28 2007-09-11 Applied Research Systems Ars Holding N.V. PEG-LHRH analog conjugates
WO1999055376A1 (en) * 1998-04-28 1999-11-04 Applied Research Systems Ars Holding N.V. Peg-lhrh analog conjugates
US6914121B2 (en) 1998-04-28 2005-07-05 Applied Research Systems Ars Holding N.V. PEG-LHRH analog conjugates
US6433135B1 (en) 1998-04-28 2002-08-13 Applied Research Systems Ars Holding N.V. PEG-LHRH analog conjugates
US6566115B1 (en) 1999-07-22 2003-05-20 The Procter & Gamble Company Protease conjugates having sterically protected clip sites
US6586224B1 (en) 1999-07-22 2003-07-01 The Procter & Gamble Company Subtilisin protease variants having amino acid deletions and substitutions in defined epitope regions
US6946128B1 (en) 1999-07-22 2005-09-20 The Procter & Gamble Company Protease conjugates having sterically protected epitope regions
US6586223B1 (en) 1999-07-22 2003-07-01 The Procter & Gamble Company Subtilisin protease variants having amino acid substitutions in defined epitope regions
EP2133098A1 (en) 2000-01-10 2009-12-16 Maxygen Holdings Ltd G-CSF conjugates
EP1982732A2 (en) 2000-02-11 2008-10-22 Maxygen Holdings Ltd. Factor VII or VIIA-like molecules
EP2319541A1 (en) 2000-02-11 2011-05-11 Bayer HealthCare LLC Factor VII or VIIA-like conjugates
EP2080771A2 (en) 2001-02-27 2009-07-22 Maxygen Aps New interferon beta-like molecules
US7019117B2 (en) 2001-07-19 2006-03-28 Albert Einstein College Of Medicine Of Yeshiva University Size enhanced hemoglobins: surface decoration and crosslinking of the protein with polyoxy alkylene glycols
EP2322229A2 (en) 2001-10-10 2011-05-18 Novo Nordisk A/S Remodelling and glycoconjugation of Granulocyte Colony Stimulating Factor (G-CSF)
EP2305313A2 (en) 2001-10-10 2011-04-06 BioGeneriX AG Remodelling and glycoconjugation of interferon-alpha (IFNa)
EP2080525A1 (en) 2001-10-10 2009-07-22 BioGeneriX AG Remodeling and Glycoconjugation of Peptides
EP2279754A2 (en) 2001-10-10 2011-02-02 BioGeneriX AG Remodelling and glycoconjugation of human growth hormone (hGH)
US8716239B2 (en) 2001-10-10 2014-05-06 Novo Nordisk A/S Granulocyte colony stimulating factor: remodeling and glycoconjugation G-CSF
EP2305312A2 (en) 2001-10-10 2011-04-06 BioGeneriX AG Remodelling and glycoconjugation of follicle-stimulating hormone (FSH)
US8076292B2 (en) 2001-10-10 2011-12-13 Novo Nordisk A/S Factor VIII: remodeling and glycoconjugation of factor VIII
EP2279755A2 (en) 2001-10-10 2011-02-02 BioGeneriX AG Remodelling and glycoconjugation of Fibroblast Growth Factor (FGF)
EP2305314A2 (en) 2001-10-10 2011-04-06 BioGeneriX AG Remodelling and glycoconjugation of antibodies
EP2305311A2 (en) 2001-10-10 2011-04-06 BioGeneriX AG Glycoconjugation of peptides
US7795210B2 (en) 2001-10-10 2010-09-14 Novo Nordisk A/S Protein remodeling methods and proteins/peptides produced by the methods
US8716240B2 (en) 2001-10-10 2014-05-06 Novo Nordisk A/S Erythropoietin: remodeling and glycoconjugation of erythropoietin
EP2298354A2 (en) 2001-10-10 2011-03-23 BioGeneriX AG Remodelling and glycoconjugation of interferon-beta
EP2042196A2 (en) 2001-10-10 2009-04-01 Neose Technologies, Inc. Remodelling and glycoconjugation of Granulocyte Colony Stimulating Factor (G-CSF)
EP2279753A2 (en) 2001-10-10 2011-02-02 Novo Nordisk A/S Remodeling and glycoconjugation of peptides
EP1455766A4 (en) * 2001-11-01 2008-11-05 Sciclone Pharmaceuticals Inc Thymosin alpha 1 peptide/polymer conjugates
EP1455766A2 (en) * 2001-11-01 2004-09-15 SciClone Pharmaceuticals, Inc. Thymosin alpha 1 peptide/polymer conjugates
US7084112B2 (en) 2002-03-25 2006-08-01 Albert Einstein College Of Medicine Of Yeshiva University Activated polyethylene glycol compounds
US6962954B2 (en) * 2002-03-25 2005-11-08 Albert Einstein College Of Medicine Of Yeshiva University Activated polyethylene glycol compounds
WO2004000366A1 (en) 2002-06-21 2003-12-31 Novo Nordisk Health Care Ag Pegylated factor vii glycoforms
US8034900B2 (en) 2002-12-30 2011-10-11 Amylin Pharmaceuticals, Inc. Water-soluble thioester and selenoester compounds and methods for making and using the same
WO2004061094A1 (en) 2002-12-30 2004-07-22 Gryphon Therapeutics, Inc. Water-soluble thioester and selenoester compounds and methods for making and using the same
US8519102B2 (en) 2003-02-26 2013-08-27 Nektar Therapeutics Polymer Factor VIII moiety conjugates
US8143378B2 (en) 2003-02-26 2012-03-27 Nektar Therapeutics Polymer factor VIII moiety conjugates
US7199223B2 (en) 2003-02-26 2007-04-03 Nektar Therapeutics Al, Corporation Polymer-factor VIII moiety conjugates
EP2572733A1 (en) * 2003-02-26 2013-03-27 Nektar Therapeutics Polymer-factor VIII moiety conjugates
US7863421B2 (en) 2003-02-26 2011-01-04 Nektar Therapeutics Polymer-factor VIII moiety conjugates
US8133977B2 (en) 2003-02-26 2012-03-13 Nektar Therapeutics Polymer-factor VIII moiety conjugates
WO2004075923A3 (en) * 2003-02-26 2005-02-24 Nektar Therapeutics Al Corp Polymer-factor viii moiety conjugates
US8618259B2 (en) 2003-02-26 2013-12-31 Nektar Therapeutics Polymer-factor VIII conjugate compositions
US8889831B2 (en) 2003-02-26 2014-11-18 Nektar Therapeutics Unit dosage forms of pharmaceutical compositions comprising a polymer-factor VIII polypeptide conjugate
AU2004215912B2 (en) * 2003-02-26 2009-03-26 Nektar Therapeutics Polymer-factor VIII moiety conjugates
US8247536B2 (en) 2003-02-26 2012-08-21 Nektar Therapeutics Factor VIII compositions
US9999657B2 (en) 2003-02-26 2018-06-19 Nektar Therapeutics Polymer-factor VIII moiety conjugates
US7858749B2 (en) 2003-02-26 2010-12-28 Nektar Therapeutics Polymer-factor VIII moiety conjugates
US11141465B2 (en) 2003-02-26 2021-10-12 Nektar Therapeutics Method of making a water-soluble polymer-factor VIII moiety conjugate
US8247381B2 (en) 2003-03-14 2012-08-21 Biogenerix Ag Branched water-soluble polymers and their conjugates
US7803777B2 (en) 2003-03-14 2010-09-28 Biogenerix Ag Branched water-soluble polymers and their conjugates
EP2338333A2 (en) 2003-04-09 2011-06-29 BioGeneriX AG Glycopegylation methods and proteins/peptides produced by the methods
EP2055189A1 (en) 2003-04-09 2009-05-06 Neose Technologies, Inc. Glycopegylation methods and proteins/peptides produced by the methods
US8063015B2 (en) 2003-04-09 2011-11-22 Novo Nordisk A/S Glycopegylation methods and proteins/peptides produced by the methods
US8853161B2 (en) 2003-04-09 2014-10-07 Novo Nordisk A/S Glycopegylation methods and proteins/peptides produced by the methods
US8791070B2 (en) 2003-04-09 2014-07-29 Novo Nordisk A/S Glycopegylated factor IX
US7932364B2 (en) 2003-05-09 2011-04-26 Novo Nordisk A/S Compositions and methods for the preparation of human growth hormone glycosylation mutants
EP2277550A1 (en) 2003-07-11 2011-01-26 Polytherics Limited Conjugated biological molecules and their preparation
EP2253330A2 (en) 2003-07-11 2010-11-24 Polytherics Limited Conjugated biological molecules and their preparation
EP2133099A2 (en) 2003-07-11 2009-12-16 Polytherics Limited Conjugated biological molecules and their preparation
US9005625B2 (en) 2003-07-25 2015-04-14 Novo Nordisk A/S Antibody toxin conjugates
EP2263684A1 (en) 2003-10-10 2010-12-22 Novo Nordisk A/S IL-21 derivatives
EP2641611A2 (en) 2003-10-17 2013-09-25 Novo Nordisk A/S Combination therapy
EP2633866A2 (en) 2003-10-17 2013-09-04 Novo Nordisk A/S Combination therapy
US8916360B2 (en) 2003-11-24 2014-12-23 Novo Nordisk A/S Glycopegylated erythropoietin
US8633157B2 (en) 2003-11-24 2014-01-21 Novo Nordisk A/S Glycopegylated erythropoietin
US7842661B2 (en) 2003-11-24 2010-11-30 Novo Nordisk A/S Glycopegylated erythropoietin formulations
US7956032B2 (en) 2003-12-03 2011-06-07 Novo Nordisk A/S Glycopegylated granulocyte colony stimulating factor
US8632770B2 (en) 2003-12-03 2014-01-21 Novo Nordisk A/S Glycopegylated factor IX
US7338933B2 (en) 2004-01-08 2008-03-04 Neose Technologies, Inc. O-linked glycosylation of peptides
US9260472B2 (en) 2004-02-02 2016-02-16 Ambrx, Inc. Modified human four helical bundle polypeptides and their uses
EP2327724A2 (en) 2004-02-02 2011-06-01 Ambrx, Inc. Modified human growth hormone polypeptides and their uses
US8097702B2 (en) 2004-02-02 2012-01-17 Ambrx, Inc. Modified human interferon polypeptides with at least one non-naturally encoded amino acid and their uses
US8907064B2 (en) 2004-02-02 2014-12-09 Ambrx, Inc. Modified human four helical bundle polypeptides and their uses
US8906676B2 (en) 2004-02-02 2014-12-09 Ambrx, Inc. Modified human four helical bundle polypeptides and their uses
US8232371B2 (en) 2004-02-02 2012-07-31 Ambrx, Inc. Modified human interferon polypeptides and their uses
US9175083B2 (en) 2004-06-18 2015-11-03 Ambrx, Inc. Antigen-binding polypeptides and their uses
WO2006009901A2 (en) 2004-06-18 2006-01-26 Ambrx, Inc. Novel antigen-binding polypeptides and their uses
US7579444B2 (en) 2004-06-30 2009-08-25 Nektar Therapeutics Al, Corporation Polymer-factor IX moiety conjugates
US8586711B2 (en) 2004-06-30 2013-11-19 Nektar Therapeutics Polymer-factor IX moiety conjugates
US9347054B2 (en) 2004-06-30 2016-05-24 Nektar Therapeutics Polymer factor IX moiety conjugates
US8268967B2 (en) 2004-09-10 2012-09-18 Novo Nordisk A/S Glycopegylated interferon α
US9200049B2 (en) 2004-10-29 2015-12-01 Novo Nordisk A/S Remodeling and glycopegylation of fibroblast growth factor (FGF)
EP3061461A1 (en) 2004-10-29 2016-08-31 ratiopharm GmbH Remodeling and glycopegylation of fibroblast growth factor (fgf)
EP2586456A1 (en) 2004-10-29 2013-05-01 BioGeneriX AG Remodeling and glycopegylation of fibroblast growth factor (FGF)
US10874714B2 (en) 2004-10-29 2020-12-29 89Bio Ltd. Method of treating fibroblast growth factor 21 (FGF-21) deficiency
US7883866B2 (en) 2004-12-22 2011-02-08 Ambrx, Inc. Compositions of aminoacyl-tRNA synthetase and uses thereof
EP2284191A2 (en) 2004-12-22 2011-02-16 Ambrx, Inc. Process for the preparation of hGH
EP2399893A2 (en) 2004-12-22 2011-12-28 Ambrx, Inc. Compositions containing, methods involving, and uses of non-natural amino acids and polypeptides
US7816320B2 (en) 2004-12-22 2010-10-19 Ambrx, Inc. Formulations of human growth hormone comprising a non-naturally encoded amino acid at position 35
US8080391B2 (en) 2004-12-22 2011-12-20 Ambrx, Inc. Process of producing non-naturally encoded amino acid containing high conjugated to a water soluble polymer
US8143216B2 (en) 2004-12-22 2012-03-27 Ambrx, Inc. Modified human growth hormone
US7838265B2 (en) 2004-12-22 2010-11-23 Ambrx, Inc. Compositions of aminoacyl-tRNA synthetase and uses thereof
US8163695B2 (en) 2004-12-22 2012-04-24 Ambrx Formulations of human growth hormone comprising a non-naturally encoded amino acid
US8178108B2 (en) 2004-12-22 2012-05-15 Ambrx, Inc. Methods for expression and purification of recombinant human growth hormone
US8178494B2 (en) 2004-12-22 2012-05-15 Ambrx, Inc. Modified human growth hormone formulations with an increased serum half-life
WO2006069220A2 (en) 2004-12-22 2006-06-29 Ambrx, Inc. Modified human growth hormone
WO2006069246A2 (en) 2004-12-22 2006-06-29 Ambrx, Inc. Compositions containing, methods involving, and uses of non-natural amino acids and polypeptides
US7846689B2 (en) 2004-12-22 2010-12-07 Ambrx, Inc. Compositions of aminoacyl-tRNA synthetase and uses thereof
US7736872B2 (en) 2004-12-22 2010-06-15 Ambrx, Inc. Compositions of aminoacyl-TRNA synthetase and uses thereof
US7959926B2 (en) 2004-12-22 2011-06-14 Ambrx, Inc. Methods for expression and purification of recombinant human growth hormone mutants
US7858344B2 (en) 2004-12-22 2010-12-28 Ambrx, Inc. Compositions of aminoacyl-tRNA synthetase and uses thereof
US7829310B2 (en) 2004-12-22 2010-11-09 Ambrx, Inc. Compositions of aminoacyl-tRNA synthetase and uses thereof
US7939496B2 (en) 2004-12-22 2011-05-10 Ambrx, Inc. Modified human growth horomone polypeptides and their uses
US7947473B2 (en) 2004-12-22 2011-05-24 Ambrx, Inc. Methods for expression and purification of pegylated recombinant human growth hormone containing a non-naturally encoded keto amino acid
US9029331B2 (en) 2005-01-10 2015-05-12 Novo Nordisk A/S Glycopegylated granulocyte colony stimulating factor
EP2514757A2 (en) 2005-01-10 2012-10-24 BioGeneriX AG Glycopegylated granulocyte colony stimulating factor
US7714114B2 (en) 2005-02-16 2010-05-11 Nektar Therapeutics Conjugates of an EPO moiety and a polymer
EP2314320A2 (en) 2005-04-05 2011-04-27 Istituto di Richerche di Biologia Molecolare P. Angeletti S.p.A. Method for shielding functional sites or epitopes on proteins
EP2279756A2 (en) 2005-04-05 2011-02-02 Instituto di Ricerche di Biologia Molecolare p Angeletti S.P.A. Method for shielding functional sites or epitopes on proteins
US9187546B2 (en) 2005-04-08 2015-11-17 Novo Nordisk A/S Compositions and methods for the preparation of protease resistant human growth hormone glycosylation mutants
EP2386571A2 (en) 2005-04-08 2011-11-16 BioGeneriX AG Compositions and methods for the preparation of protease resistant human growth hormone glycosylation mutants
US8404809B2 (en) 2005-05-25 2013-03-26 Novo Nordisk A/S Glycopegylated factor IX
EP2975135A1 (en) 2005-05-25 2016-01-20 Novo Nordisk A/S Glycopegylated factor IX
US8093356B2 (en) 2005-06-03 2012-01-10 Ambrx, Inc. Pegylated human interferon polypeptides
EP2360170A2 (en) 2005-06-17 2011-08-24 Novo Nordisk Health Care AG Selective reduction and derivatization of engineered proteins comprinsing at least one non-native cysteine
WO2006134173A2 (en) 2005-06-17 2006-12-21 Novo Nordisk Health Care Ag Selective reduction and derivatization of engineered proteins comprising at least one non-native cysteine
US8911967B2 (en) 2005-08-19 2014-12-16 Novo Nordisk A/S One pot desialylation and glycopegylation of therapeutic peptides
US8841439B2 (en) 2005-11-03 2014-09-23 Novo Nordisk A/S Nucleotide sugar purification using membranes
US9488660B2 (en) 2005-11-16 2016-11-08 Ambrx, Inc. Methods and compositions comprising non-natural amino acids
EP2213733A2 (en) 2006-05-24 2010-08-04 Novo Nordisk Health Care AG Factor IX analogues having prolonged in vivo half life
US9187532B2 (en) 2006-07-21 2015-11-17 Novo Nordisk A/S Glycosylation of peptides via O-linked glycosylation sequences
US8618257B2 (en) 2006-09-08 2013-12-31 Ambrx, Inc. Modified human plasma polypeptide or Fc scaffolds and their uses
US9133495B2 (en) 2006-09-08 2015-09-15 Ambrx, Inc. Hybrid suppressor tRNA for vertebrate cells
US7919591B2 (en) 2006-09-08 2011-04-05 Ambrx, Inc. Modified human plasma polypeptide or Fc scaffolds and their uses
WO2008030558A2 (en) 2006-09-08 2008-03-13 Ambrx, Inc. Modified human plasma polypeptide or fc scaffolds and their uses
US8420792B2 (en) 2006-09-08 2013-04-16 Ambrx, Inc. Suppressor tRNA transcription in vertebrate cells
US8022186B2 (en) 2006-09-08 2011-09-20 Ambrx, Inc. Modified human plasma polypeptide or Fc scaffolds and their uses
US8053560B2 (en) 2006-09-08 2011-11-08 Ambrx, Inc. Modified human plasma polypeptide or Fc scaffolds and their uses
EP2548967A2 (en) 2006-09-21 2013-01-23 The Regents of The University of California Aldehyde tags, uses thereof in site-specific protein modification
US8969532B2 (en) 2006-10-03 2015-03-03 Novo Nordisk A/S Methods for the purification of polypeptide conjugates comprising polyalkylene oxide using hydrophobic interaction chromatography
WO2008060780A2 (en) 2006-10-04 2008-05-22 Novo Nordisk A/S Glycerol linked pegylated sugars and glycopeptides
US8383365B2 (en) 2007-03-30 2013-02-26 Ambrx, Inc. Methods of making FGF-21 mutants comprising non-naturally encoded phenylalanine derivatives
US9079971B2 (en) 2007-03-30 2015-07-14 Ambrx, Inc. Modified FGF-21 polypeptides comprising non-naturally occurring amino acids
US9517273B2 (en) 2007-03-30 2016-12-13 Ambrx, Inc. Methods of treatment using modified FGF-21 polypeptides comprising non-naturally occurring amino acids
US10961291B2 (en) 2007-03-30 2021-03-30 Ambrx, Inc. Modified FGF-21 polypeptides and their uses
US8012931B2 (en) 2007-03-30 2011-09-06 Ambrx, Inc. Modified FGF-21 polypeptides and their uses
US9975936B2 (en) 2007-03-30 2018-05-22 Ambrx, Inc. Nucleic acids encoding modified FGF-21 polypeptides comprising non-naturally occurring amino acids
US10377805B2 (en) 2007-03-30 2019-08-13 Ambrx, Inc. Modified FGF-21 polypeptides comprising non-naturally encoding amino acids and their uses
US9050304B2 (en) 2007-04-03 2015-06-09 Ratiopharm Gmbh Methods of treatment using glycopegylated G-CSF
US8114630B2 (en) 2007-05-02 2012-02-14 Ambrx, Inc. Modified interferon beta polypeptides and their uses
US9493499B2 (en) 2007-06-12 2016-11-15 Novo Nordisk A/S Process for the production of purified cytidinemonophosphate-sialic acid-polyalkylene oxide (CMP-SA-PEG) as modified nucleotide sugars via anion exchange chromatography
US8207112B2 (en) 2007-08-29 2012-06-26 Biogenerix Ag Liquid formulation of G-CSF conjugate
WO2009067636A2 (en) 2007-11-20 2009-05-28 Ambrx, Inc. Modified insulin polypeptides and their uses
EP2930182A1 (en) 2007-11-20 2015-10-14 Ambrx, Inc. Modified insulin polypeptides and their uses
US8946148B2 (en) 2007-11-20 2015-02-03 Ambrx, Inc. Modified insulin polypeptides and their uses
US9938333B2 (en) 2008-02-08 2018-04-10 Ambrx, Inc. Modified leptin polypeptides and their uses
EP3103880A1 (en) 2008-02-08 2016-12-14 Ambrx, Inc. Modified leptin polypeptides and their uses
US9150848B2 (en) 2008-02-27 2015-10-06 Novo Nordisk A/S Conjugated factor VIII molecules
EP3225248A1 (en) 2008-07-23 2017-10-04 Ambrx, Inc. Modified bovine g-csf polypeptides and their uses
WO2010011735A2 (en) 2008-07-23 2010-01-28 Ambrx, Inc. Modified bovine g-csf polypeptides and their uses
US10138283B2 (en) 2008-07-23 2018-11-27 Ambrx, Inc. Modified bovine G-CSF polypeptides and their uses
US8815232B2 (en) 2008-08-26 2014-08-26 Kyon Biotech Ag Compositions and methods for treating cancer
US9121024B2 (en) 2008-09-26 2015-09-01 Ambrx, Inc. Non-natural amino acid replication-dependent microorganisms and vaccines
US9644014B2 (en) 2008-09-26 2017-05-09 Ambrx, Inc. Modified animal erythropoietin polypeptides and their uses
US9156899B2 (en) 2008-09-26 2015-10-13 Eli Lilly And Company Modified animal erythropoietin polypeptides and their uses
US8278418B2 (en) 2008-09-26 2012-10-02 Ambrx, Inc. Modified animal erythropoietin polypeptides and their uses
US8569233B2 (en) 2008-09-26 2013-10-29 Eli Lilly And Company Modified animal erythropoietin polypeptides and their uses
US9121025B2 (en) 2008-09-26 2015-09-01 Ambrx, Inc. Non-natural amino acid replication-dependent microorganisms and vaccines
EP3216800A1 (en) 2008-09-26 2017-09-13 Ambrx, Inc. Modified animal erythropoietin polypeptides and their uses
US10428333B2 (en) 2008-09-26 2019-10-01 Ambrx Inc. Non-natural amino acid replication-dependent microorganisms and vaccines
EP3266463A1 (en) 2009-06-09 2018-01-10 Prolong Pharmaceuticals, LLC Hemoglobin compositions
WO2010144629A1 (en) 2009-06-09 2010-12-16 Prolong Pharmaceuticals, LLC Hemoglobin compositions
EP2805964A1 (en) 2009-12-21 2014-11-26 Ambrx, Inc. Modified bovine somatotropin polypeptides and their uses
EP2805965A1 (en) 2009-12-21 2014-11-26 Ambrx, Inc. Modified porcine somatotropin polypeptides and their uses
EP3815708A1 (en) 2010-03-05 2021-05-05 Omeros Corporation Chimeric inhibitor molecules of complement activation
WO2011107591A1 (en) 2010-03-05 2011-09-09 Rigshospitalet Chimeric inhibitor molecules of complement activation
WO2011143274A1 (en) 2010-05-10 2011-11-17 Perseid Therapeutics Polypeptide inhibitors of vla4
WO2012024452A2 (en) 2010-08-17 2012-02-23 Ambrx, Inc. Modified relaxin polypeptides and their uses
US8735539B2 (en) 2010-08-17 2014-05-27 Ambrx, Inc. Relaxin polypeptides comprising non-naturally encoded amino acids
US9567386B2 (en) 2010-08-17 2017-02-14 Ambrx, Inc. Therapeutic uses of modified relaxin polypeptides
US9962450B2 (en) 2010-08-17 2018-05-08 Ambrx, Inc. Method of treating heart failure with modified relaxin polypeptides
US11311605B2 (en) 2010-08-17 2022-04-26 Ambrx, Inc. Methods of treating heart failure and fibrotic disorders using modified relaxin polypeptides
US10751391B2 (en) 2010-08-17 2020-08-25 Ambrx, Inc. Methods of treatment using modified relaxin polypeptides comprising a non-naturally encoded amino acid
US11786578B2 (en) 2010-08-17 2023-10-17 Ambrx, Inc. Modified relaxin polypeptides and their uses
US9452222B2 (en) 2010-08-17 2016-09-27 Ambrx, Inc. Nucleic acids encoding modified relaxin polypeptides
EP4302783A2 (en) 2010-08-17 2024-01-10 Ambrx, Inc. Modified relaxin polypeptides and their uses
US10702588B2 (en) 2010-08-17 2020-07-07 Ambrx, Inc. Modified relaxin polypeptides comprising a non-naturally encoded amino acid in the A chain
US10253083B2 (en) 2010-08-17 2019-04-09 Ambrx, Inc. Therapeutic uses of modified relaxin polypeptides
US11439710B2 (en) 2010-08-17 2022-09-13 Ambrx, Inc. Nucleic acids encoding modified relaxin polypeptides
US11273202B2 (en) 2010-09-23 2022-03-15 Elanco Us Inc. Formulations for bovine granulocyte colony stimulating factor and variants thereof
US9382305B2 (en) 2011-07-01 2016-07-05 Bayer Intellectual Property Gmbh Relaxin fusion polypeptides and uses thereof
WO2013004607A1 (en) 2011-07-01 2013-01-10 Bayer Intellectual Property Gmbh Relaxin fusion polypeptides and uses thereof
EP3088005A1 (en) 2011-07-05 2016-11-02 biOasis Technologies Inc P97-antibody conjugates
WO2013006706A1 (en) 2011-07-05 2013-01-10 Bioasis Technologies Inc. P97-antibody conjugates and methods of use
EP3505534A1 (en) 2012-06-08 2019-07-03 Sutro Biopharma, Inc. Antibodies comprising sitespecific nonnatural amino acid residues, methods of their preparation and methods of their use
WO2013185115A1 (en) 2012-06-08 2013-12-12 Sutro Biopharma, Inc. Antibodies comprising site-specific non-natural amino acid residues, methods of their preparation and methods of their use
EP3135690A1 (en) 2012-06-26 2017-03-01 Sutro Biopharma, Inc. Modified fc proteins comprising site-specific non-natural amino acid residues, conjugates of the same, methods of their preparation and methods of their use
WO2014022515A1 (en) 2012-07-31 2014-02-06 Bioasis Technologies, Inc. Dephosphorylated lysosomal storage disease proteins and methods of use thereof
EP4074728A1 (en) 2012-08-31 2022-10-19 Sutro Biopharma, Inc. Modified peptides comprising an azido group
WO2014036492A1 (en) 2012-08-31 2014-03-06 Sutro Biopharma, Inc. Modified amino acids comprising an azido group
EP3584255A1 (en) 2012-08-31 2019-12-25 Sutro Biopharma, Inc. Modified amino acids comprising an azido group
US9579390B2 (en) 2012-11-12 2017-02-28 Redwood Bioscience, Inc. Compounds and methods for producing a conjugate
US9833515B2 (en) 2012-11-16 2017-12-05 Redwood Bioscience, Inc. Hydrazinyl-indole compounds and methods for producing a conjugate
US10314919B2 (en) 2012-11-16 2019-06-11 Redwood Bioscience, Inc. Hydrazinyl-indole compounds and methods for producing a conjugate
US9605078B2 (en) 2012-11-16 2017-03-28 The Regents Of The University Of California Pictet-Spengler ligation for protein chemical modification
US11426465B2 (en) 2012-11-16 2022-08-30 Redwiid Bioscience, Inc. Hydrazinyl-indole compounds and methods for producing a conjugate
US10888623B2 (en) 2012-11-16 2021-01-12 Redwood Bioscience, Inc. Hydrazinyl-indole compounds and methods for producing a conjugate
US9310374B2 (en) 2012-11-16 2016-04-12 Redwood Bioscience, Inc. Hydrazinyl-indole compounds and methods for producing a conjugate
WO2014160438A1 (en) 2013-03-13 2014-10-02 Bioasis Technologies Inc. Fragments of p97 and uses thereof
WO2015006555A2 (en) 2013-07-10 2015-01-15 Sutro Biopharma, Inc. Antibodies comprising multiple site-specific non-natural amino acid residues, methods of their preparation and methods of their use
EP3336103A1 (en) 2013-07-10 2018-06-20 Sutro Biopharma, Inc. Antibodies comprising multiple site-specific non-natural amino acid residues, methods of their preparation and methods of their use
WO2015031673A2 (en) 2013-08-28 2015-03-05 Bioasis Technologies Inc. Cns-targeted conjugates having modified fc regions and methods of use thereof
WO2015054658A1 (en) 2013-10-11 2015-04-16 Sutro Biopharma, Inc. Modified amino acids comprising tetrazine functional groups, methods of preparation, and methods of their use
WO2015081282A1 (en) 2013-11-27 2015-06-04 Redwood Bioscience, Inc. Hydrazinyl-pyrrolo compounds and methods for producing a conjugate
US11248031B2 (en) 2014-10-24 2022-02-15 Bristol-Myers Squibb Company Methods of treating diseases associated with fibrosis using modified FGF-21 polypeptides
US10189883B2 (en) 2014-10-24 2019-01-29 Bristol-Myers Squibb Company Therapeutic uses of modified FGF-21 polypeptides
US9434778B2 (en) 2014-10-24 2016-09-06 Bristol-Myers Squibb Company Modified FGF-21 polypeptides comprising an internal deletion and uses thereof
US9631004B2 (en) 2014-10-24 2017-04-25 Bristol-Myers Squibb Company Modified FGF-21 polypeptides comprising an internal deletion and uses thereof
US10377806B2 (en) 2014-10-24 2019-08-13 Bristol-Myers Squibb Company Methods of treating diseases associated with fibrosis using modified FGF-21 polypeptides and uses thereof
US10266578B2 (en) 2017-02-08 2019-04-23 Bristol-Myers Squibb Company Modified relaxin polypeptides comprising a pharmacokinetic enhancer and uses thereof
US11185570B2 (en) 2017-02-08 2021-11-30 Bristol-Myers Squibb Company Method of treating cardiovascular disease and heart failure with modified relaxin polypeptides
US11364281B2 (en) 2017-02-08 2022-06-21 Bristol-Myers Squibb Company Modified relaxin polypeptides comprising a pharmacokinetic enhancer and pharmaceutical compositions thereof
WO2019133399A1 (en) 2017-12-26 2019-07-04 Becton, Dickinson And Company Deep ultraviolet-excitable water-solvated polymeric dyes
WO2019191482A1 (en) 2018-03-30 2019-10-03 Becton, Dickinson And Company Water-soluble polymeric dyes having pendant chromophores
WO2020023300A1 (en) 2018-07-22 2020-01-30 Bioasis Technologies, Inc. Treatment of lymmphatic metastases
WO2020056066A1 (en) 2018-09-11 2020-03-19 Ambrx, Inc. Interleukin-2 polypeptide conjugates and their uses
WO2020082057A1 (en) 2018-10-19 2020-04-23 Ambrx, Inc. Interleukin-10 polypeptide conjugates, dimers thereof, and their uses
WO2020168017A1 (en) 2019-02-12 2020-08-20 Ambrx, Inc. Compositions containing, methods and uses of antibody-tlr agonist conjugates
WO2021183832A1 (en) 2020-03-11 2021-09-16 Ambrx, Inc. Interleukin-2 polypeptide conjugates and methods of use thereof
WO2021236526A1 (en) 2020-05-18 2021-11-25 Bioasis Technologies, Inc. Compositions and methods for treating lewy body dementia
WO2021255524A1 (en) 2020-06-17 2021-12-23 Bioasis Technologies, Inc. Compositions and methods for treating frontotemporal dementia
WO2022040596A1 (en) 2020-08-20 2022-02-24 Ambrx, Inc. Antibody-tlr agonist conjugates, methods and uses thereof
WO2022212899A1 (en) 2021-04-03 2022-10-06 Ambrx, Inc. Anti-her2 antibody-drug conjugates and uses thereof
EP4155349A1 (en) 2021-09-24 2023-03-29 Becton, Dickinson and Company Water-soluble yellow green absorbing dyes
WO2024007016A2 (en) 2022-07-01 2024-01-04 Beckman Coulter, Inc. Novel fluorescent dyes and polymers from dihydrophenanthrene derivatives
WO2024044327A1 (en) 2022-08-26 2024-02-29 Beckman Coulter, Inc. Dhnt monomers and polymer dyes with modified photophysical properties

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