EP0706375A4 - Proteinoid carriers - Google Patents

Proteinoid carriers

Info

Publication number
EP0706375A4
EP0706375A4 EP94920205A EP94920205A EP0706375A4 EP 0706375 A4 EP0706375 A4 EP 0706375A4 EP 94920205 A EP94920205 A EP 94920205A EP 94920205 A EP94920205 A EP 94920205A EP 0706375 A4 EP0706375 A4 EP 0706375A4
Authority
EP
European Patent Office
Prior art keywords
proteinoid
composition
group
acid
active agent
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP94920205A
Other languages
German (de)
French (fr)
Other versions
EP0706375A1 (en
Inventor
Sam J Milstein
Martin L Kantor
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Emisphere Technologies Inc
Original Assignee
Emisphere Technologies Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US08/076,803 external-priority patent/US5578323A/en
Application filed by Emisphere Technologies Inc filed Critical Emisphere Technologies Inc
Publication of EP0706375A1 publication Critical patent/EP0706375A1/en
Publication of EP0706375A4 publication Critical patent/EP0706375A4/en
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/167—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction with an outer layer or coating comprising drug; with chemically bound drugs or non-active substances on their surface
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/02—Cosmetics or similar toiletry preparations characterised by special physical form
    • A61K8/0241—Containing particulates characterized by their shape and/or structure
    • A61K8/025—Explicitly spheroidal or spherical shape
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/02—Cosmetics or similar toiletry preparations characterised by special physical form
    • A61K8/11—Encapsulated compositions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1629—Organic macromolecular compounds
    • A61K9/1641—Organic macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, poloxamers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q13/00—Formulations or additives for perfume preparations
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C229/40—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino groups bound to carbon atoms of at least one six-membered aromatic ring and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C229/42—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino groups bound to carbon atoms of at least one six-membered aromatic ring and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton with carboxyl groups linked to the six-membered aromatic ring, or to the condensed ring system containing that ring, by saturated carbon chains
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C233/00—Carboxylic acid amides
    • C07C233/01—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms
    • C07C233/45—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by carboxyl groups
    • C07C233/53—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by carboxyl groups with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by a carbon atom of a six-membered aromatic ring
    • C07C233/55—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by carboxyl groups with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by a carbon atom of a six-membered aromatic ring having the carbon atom of the carboxamide group bound to a carbon atom of an unsaturated carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C233/00—Carboxylic acid amides
    • C07C233/57—Carboxylic acid amides having carbon atoms of carboxamide groups bound to carbon atoms of rings other than six-membered aromatic rings
    • C07C233/63—Carboxylic acid amides having carbon atoms of carboxamide groups bound to carbon atoms of rings other than six-membered aromatic rings having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by carboxyl groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C233/00—Carboxylic acid amides
    • C07C233/64—Carboxylic acid amides having carbon atoms of carboxamide groups bound to carbon atoms of six-membered aromatic rings
    • C07C233/81—Carboxylic acid amides having carbon atoms of carboxamide groups bound to carbon atoms of six-membered aromatic rings having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by carboxyl groups
    • C07C233/82—Carboxylic acid amides having carbon atoms of carboxamide groups bound to carbon atoms of six-membered aromatic rings having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by carboxyl groups with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom
    • C07C233/87—Carboxylic acid amides having carbon atoms of carboxamide groups bound to carbon atoms of six-membered aromatic rings having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by carboxyl groups with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom of a carbon skeleton containing six-membered aromatic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C235/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms
    • C07C235/02—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to acyclic carbon atoms and singly-bound oxygen atoms bound to the same carbon skeleton
    • C07C235/32—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to acyclic carbon atoms and singly-bound oxygen atoms bound to the same carbon skeleton the carbon skeleton containing six-membered aromatic rings
    • C07C235/38—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to acyclic carbon atoms and singly-bound oxygen atoms bound to the same carbon skeleton the carbon skeleton containing six-membered aromatic rings having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a six-membered aromatic ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C235/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms
    • C07C235/42—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to carbon atoms of six-membered aromatic rings and singly-bound oxygen atoms bound to the same carbon skeleton
    • C07C235/44—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to carbon atoms of six-membered aromatic rings and singly-bound oxygen atoms bound to the same carbon skeleton with carbon atoms of carboxamide groups and singly-bound oxygen atoms bound to carbon atoms of the same non-condensed six-membered aromatic ring
    • C07C235/58—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to carbon atoms of six-membered aromatic rings and singly-bound oxygen atoms bound to the same carbon skeleton with carbon atoms of carboxamide groups and singly-bound oxygen atoms bound to carbon atoms of the same non-condensed six-membered aromatic ring with carbon atoms of carboxamide groups and singly-bound oxygen atoms, bound in ortho-position to carbon atoms of the same non-condensed six-membered aromatic ring
    • C07C235/64—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to carbon atoms of six-membered aromatic rings and singly-bound oxygen atoms bound to the same carbon skeleton with carbon atoms of carboxamide groups and singly-bound oxygen atoms bound to carbon atoms of the same non-condensed six-membered aromatic ring with carbon atoms of carboxamide groups and singly-bound oxygen atoms, bound in ortho-position to carbon atoms of the same non-condensed six-membered aromatic ring having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a six-membered aromatic ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C235/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms
    • C07C235/70—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups and doubly-bound oxygen atoms bound to the same carbon skeleton
    • C07C235/84—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups and doubly-bound oxygen atoms bound to the same carbon skeleton with the carbon atom of at least one of the carboxamide groups bound to a carbon atom of a six-membered aromatic ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C237/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups
    • C07C237/02—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton
    • C07C237/22—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton having nitrogen atoms of amino groups bound to the carbon skeleton of the acid part, further acylated
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
    • A61K2800/60—Particulates further characterized by their structure or composition
    • A61K2800/65—Characterized by the composition of the particulate/core
    • A61K2800/652—The particulate/core comprising organic material
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2601/00—Systems containing only non-condensed rings
    • C07C2601/02—Systems containing only non-condensed rings with a three-membered ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2601/00—Systems containing only non-condensed rings
    • C07C2601/12—Systems containing only non-condensed rings with a six-membered ring
    • C07C2601/14—The ring being saturated
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2601/00—Systems containing only non-condensed rings
    • C07C2601/18—Systems containing only non-condensed rings with a ring being at least seven-membered

Definitions

  • This invention relates to proteinoids and protein ⁇ oid carriers made from them.
  • the proteinoid carriers releasably encapsulate active agents and have extended longer shelf life and/or photostability. Methods for the preparation of such proteinoid carriers are also disclosed.
  • chemi- cal or physical barriers or both which are imposed by the body.
  • oral delivery of many such agents would be the route of choice if not for the presence of chemical and physicochemical barriers such as extreme pH in the gut, exposure to powerful digestive enzymes, and impermeability of gastrointestinal membranes to the active ingredient.
  • adjuvants such as resorcinols and non-ionic surfactants polyoxyethylene oleyl ether and n-hexadecyl polyethylene ether to increase the permeability of the intestinal walls; and
  • enzymatic inhibitors such as pancreatic trypsin inhibitor, diisopropylfluorophosphate (DFF) and trasylol to avoid enzymatic degradation.
  • enzymatic inhibitors such as pancreatic trypsin inhibitor, diisopropylfluorophosphate (DFF) and trasylol.
  • Liposomes as drug delivery systems have also been described. They provide a layer of lipid around the encap ⁇ sulated pharmacological agent.
  • the use of liposomes con- taining heparin is disclosed in U.S. Patent No. 4,239,754 and several studies have been directed to the use of liposomes containing insulin; e.g., Patel et al . (1976) FEBS Letters Vol. 62, page 60 and Hashimoto et al . (1979) Endocrinol. Japan, Vol. 26, page 337.
  • the use of liposomes is still in the development stage and there are continuing problems, including:
  • proteinoid microspheres described in the '673 patent are useful for their intended purposes, the physicochemical properties of the proteinoid microspheres, such as light sensitivity, shelf life and the selectivity of their solubility in various portions of the gastrointestinal tract, could be improved. Additionally, there is a need in the art for microspheres that can encapsulate a broader range of active agents such as polar drugs.
  • the method employed in the '673 patent to prepare proteinoids produces a complex mixture of high molecular weight (MW) (> 1000 daltons) and low MW ( ⁇ .1000 daltons) peptide-like polymers which are difficult to separate. Moreover, the method produces a small amount of the low MW proteinoids which is the microsphere-forming fraction. Hence, an improved method of preparing of the proteinoids is also desired.
  • MW high molecular weight
  • ⁇ .1000 daltons low MW
  • the present invention relates to improved protein ⁇ oid carriers and methods of making and use thereof.
  • Proteinoids of a MW ranging between about 250 and about 2400 daltons and of defined amino acids are useful in preparing proteinoid carriers with improved stability against photodegradation and/or decomposition.
  • the proteinoids comprise a peptide polymer selected from the group consisting of:
  • peptide polymers made from at least one first monomer selected from the group consisting of tyrosine and phenylalanme; and from at least one second monomer selected from the group consisting of glutamic acid, pyroglutamic acid, glutamine, and aspartic acid;
  • peptide polymers made from at least one ⁇ first monomer selected from the group consisting of tyrosine and phenylalanme; and from at least one second monomer selected from the group consisting of glutamic acid, pyroglutamic acid, glutamine, and aspartic acid; and from at least one third monomer selected from the group consisting of lysine, arginine and ornithine, the proteinoid being a microsphere- and/or microcapsule-forming proteinoid and being soluble within a selected pH range.
  • the proteinoid molecules of the invention contain between about 2 and about 20 amino acid residues, preferably between about 2 and about 8 amino acid residues, and has a molecular weight which ranges between about 250 and about 2400 daltons, preferably between about 250 and about 600, and most preferably between about 250 and 400 daltons.
  • the proteinoid carriers are useful as delivery systems to releasably encapsulate and carry a broad range of cargoes including pharmaceutical agents, dye reagents and cosmetic ingredients.
  • the proteinoid carri ⁇ ers are useful as oral delivery systems of sensitive pharma- ceutical agents, which normally would not be administrable via the oral route, for selective release at targeted re ⁇ gions of the gastrointestinal tract.
  • dosage unit forms that include these compositions.
  • Figure 1 illustrates the molecular weight distri ⁇ bution as a function of monomer concentration of poly (Asp.Bz-co-Phe) polymer prepared by the NCA method as de- scribed in Example 3.
  • Figure 2 illustrates the molecular weight distri ⁇ bution of a function of monomer concentration of poly (Asp.Bz) polymer prepared by the DPPA method as described in Example 5.
  • Figure 3 illustrates the effect of reaction time duration on yields of poly (Asp.Bz) polymer prepared by the DPPA method as described in Example 5.
  • Figure 4 illustrates the effect of temperature of the molecular weight of poly (Asp.Bz) polymer prepared by the DPPA method as described in Example 5.
  • Figure 5 illustrates the effect of changing the molar ratios of [DPPA] / [M] on the molecular weight of poly (Asp.Bz) polymer by the DPPA method as described in Example 5.
  • Figure 6 is a photograph of an x-ray film of the western immunoblot analysis, as described in Example 9, of purified murine mAb 9BG5 (2 ⁇ g, lane 1; lmg, lane 2; and 0.25 ⁇ g, lane 3) ; empty proteinoid carrier supernatant after encapsulating process (no mAb) (lane 4) ; empty proteinoid carrier pellet (lane 5) ; proteinoid carrier encapsulated mAb supernatant after encapsulating process (lane 6) ; and pro ⁇ teinoid carrier encapsulated mAb pellet.
  • Lane MW contained standard molecular weight markers.
  • Figure 7 is a photograph of an x-ray film of a western immunoblot analysis of samples described in Example 10.
  • Figures 8 (a-c) illustrate the levels of serum proteins which bound to immobilized reovirus type 3 and V L SH under ELISA conditions as described in Example 11.
  • "Empty spheres” refers to animals orally administered empty pro ⁇ teinoid carriers (no mAb 9BG5) ;
  • mAb spheres refers to animals orally administered mAb 9BG5 encapsulated proteinoid carriers;
  • IV refers to animals intravenously administered unencapsulated mAb 9BG5; and
  • oral refers to animals orally administered unencapsulated mAb 9BG5.
  • Figure 9 show mAb binding under conventional ELISA procedures using immobilized reovirus type 3 and V L SH pro- teins with serial dilutions of purified mAb in 0.85 N ci ⁇ trate-0.5% gum ( Figure 9(a)) or phosphate buffered saline ( Figure 9 (b) ) as described in Example 11.
  • Figure 10 illustrates levels of erythropoietin (EPO) detected in rat serum taken from rats administered proteinoid carrier encapsulated EPO (15 ⁇ g EPO/kg body weight) and encapsulated EPO (15 ⁇ g EPO/kg body weight) as described in Example 15.
  • EPO erythropoietin
  • Figure 11 illustrates EPO serum levels in rats that were administered either erythropoietin (50 ⁇ g/kg) or encapsulated erythropoietin (50 ⁇ g/kg) directly into the proximal duodenum as described in Example 15. Serum eryth ⁇ ropoietin levels were determined over time with a erythro ⁇ poietin enzyme immunoassay kit.
  • Figure 12 illustrates EPO serum levels in rats who were orally gavaged with either encapsulated or unencapsu ⁇ lated erythropoietin (lOO ⁇ g/kg) or received a subcutaneous injection of either 2 ⁇ g/kg or lO ⁇ g/kg as described in Exam ⁇ ple 15. Serum erythropoietin levels were determined over time with an erythropoietin enzyme immunoassay kit.
  • Figure 13 illustrates serum calcium changes after oral administration of salmon calcitonin proteinoid carriers (0.25 mg calcitonin/kg body weight) in cynomolgus monkeys as described in Example 17. The results are expressed as abso ⁇ lute change in serum calcium from baseline values. The data represents means +/- SEM. ** Serum calcium levels significantly different from baseline values.
  • Figure 14 illustrates serum calcium changes fol ⁇ lowing oral administration of salmon calcitonin proteinoid carriers (0.60 mg/kg body weight) in rats as described in Example 18. The results are expressed as absolute change in serum calcium from baseline values. The data represents means +/- SEM. **Serum calcium levels significantly differ- ent compared to the control group at the corresponding time point.
  • Figure 15 illustrates serum calcium changes after intraduodenal administration of salmon calcitonin or calci ⁇ tonin proteinoid carriers (3 ug/kg body weight) in rats as described in Example 18. The results are expressed as abso ⁇ lute change in serum calcium from baseline values. The data represents means +/- SEM. ** Significantly different from the unencapsulated control group at the indicated time points.
  • Figure 16 illustrates clotting times after oral administration of proteinoid carrier encapsulated Factor IX (FIX sph PO) and IV administration of FIX solution (FIX IV) as described in Example 20.
  • FIX sph PO proteinoid carrier encapsulated Factor IX
  • FIX IV FIX solution
  • Figure 17 illustrates clotting times after oral administration of proteinoid carrier encapsulated Factor IX (FIX sph PO) and FIX solution (FIX unencap PO) or IV admin ⁇ istration of FIX solution (FIX IV) as described in Example 21.
  • FIX sph PO proteinoid carrier encapsulated Factor IX
  • FIX unencap PO FIX solution
  • IV admin ⁇ istration of FIX solution FIX IV
  • Figure 18 illustrates the percentage of intact alpha-interferon (IFN) remaining after incubating IFN and IFN proteinoid carriers in simulated gastric fluid (SGF) .
  • IFN alpha-interferon
  • Figure 19 illustrates the percentage of intact IFN remaining after incubating IFN and IFN proteinoid carriers in 0 . 08N HCl .
  • Figure 20 illustrates the percentage of intact IFN remaining after incubating IFN and IFN proteinoid carriers in simulated intestinal fluid (SIF) .
  • Figure 21 illustrates the clotting times in rats dosed with heparin or proteinoid/heparin, both in water. The data represents an average of 6 rats. The data repre ⁇ sents means +/- SEM.
  • Figure 22 illustrates clotting times in rats dosed ID with USP heparin or heparin proteinoid carriers, both in citric acid. Each time point is an average of 12 rats. The data represents means +/- SEM.
  • Figure 23 illustrates clotting times in rats dosed orally with heparin-spiked empty proteinoid carriers or heparin proteinoid carriers. Each time point is an average of 12 rats. The data represents means +/- SEM.
  • Figure 24 illustrates the average titers of rats immunized orally with Ml proteinoid carriers versus unencap ⁇ sulated Ml. Only responders in each group were averaged.
  • Figure 25 illustrates HA-NA titers of rats immu ⁇ nized orally with HA-NA microspheres versus unencapsulated HA-NA.
  • proteinoids of a MW of between about 250 and about 2400 daltons and of defined amino acid composition can be ob ⁇ tained by modifying known reactions and selecting starting materials. These proteinoids form proteinoid carriers with surprisingly enhanced stability against at least one of photodegradation and decomposition over time.
  • proteinoid carriers prepared from such proteinoids carry a broader range of pharmaceutical agents, including labile polypeptides such as insulin, alpha-interferon, calcitonin, antigens, e.g. influenza virus Ml-protein, and Factor IX and display a selective releasability within various portions of the gastrointestinal tract, relative to prior art proteinoid microspheres.
  • compositions of the subject invention are useful for administering biologically-active agents to any animals such as birds; mammals, such as primates and particularly humans; and insects.
  • the proteinoids of the invention comprise a pep ⁇ tide polymer selected from the group consisting of:
  • peptide polymers made from at least one first monomer selected from the group consisting of tyrosine and phenylalanme; and from at least one second monomer selected from the group consisting of glutamic acid, pyroglutamic acid, glutamine, and aspartic acid;
  • peptide polymers made from at least one first monomer selected from the group consisting of tyrosine and phenylalanme; at least one second monomer selected from the group consisting of glutamic acid, pyroglutamic acid, gluta ⁇ mine, and aspartic acid; and from at least one third monomer selected from the group consisting of lysine, arginine and ornithine, the proteinoid being a microsphere- or microcap- sule-forming proteinoid and being soluble within a selected pH range.
  • the proteinoid molecules of the invention contain between about 2 and about 20 amino acid residues, preferably between about 2 and about 8 amino acid residues, and have a molecular weight which ranges between 250 and about 2400 daltons, preferably between about 250 and about 600, and most preferably between about 250 and 400 daltons.
  • amino acid as used herein includes any carboxylic acid having at least one free amine group includ ⁇ ing naturally occurring and synthetic amino acids.
  • the preferred amino acids are oc-amino acids, and preferably are naturally occurring oc-amino acids although non-o;-amino acids are useful as well.
  • amino acids or components of a peptide are alanine, arginine, asparagine, aspartic acid, citrulline, cysteine, cystine, glutamine, glycine, histi- dine, isoleucine, leucine, lysine, methionine, ornithine, phenylalanme, proline, serine, threonine, tryptophan, tyrosine, valine, hydroxy proline, ⁇ -carboxyglutamate, or O-phosphoserine.
  • the most preferred amino acids are argi ⁇ nine, leucine, lysine, phenylalanme, tyrosine and valine.
  • amino acids or compo ⁇ nents of a peptide are ⁇ -alanine, phenylglycine, ⁇ -aminobutyric acid, ⁇ -amino butyric acid, 4- (4- aminophenyl)butyric acid, ⁇ .-amino isobutyric acid, e- aminocaproic acid, 7-aminoheptanoic acid, /3-aspartic acid, aminobenzoic acid, (aminomethyl)benzoic acid, aminophenylacetic acid, aminohippuric acid, ⁇ -glutamic acid, cysteine(ACM) , e-lysine, e-lysine (A-Fmoc) , methionine sulfone, norleucine, norvaline, ornithine, d-ornithine, p- nitrophenylalanine, hydroxy proline, and thioproline.
  • R 2 has the formula — 3 —C—- wherein R 3 is C ! to C M alkyl, Cj to C u alkenyl, phenyl, naphthyl, (Cj to C 10 alkyl) - phenyl, (Ci to C 10 alkenyl)phenyl, (Cj to C 10 alkyl) aphthyl,
  • R 3 is substituted with Cj to C 4 alkyl, Cj to C 4 alkenyl, Cj to C 4 alkoxy, -OH, -SH and -C0 2 R 5 or any combination thereof;
  • R 5 is hydrogen, Cj to C 4 alkyl or to C 4 alkenyl;
  • R 3 is optionally interrupted by oxygen, nitrogen, sulfur or any combination thereof.
  • R 4 is hydrogen, ⁇ to C 4 alkyl or Cj to C 4 alkenyl.
  • the phenyl or naphthyl groups can be optionally substituted. Suitable but non-limiting examples of substitutents are Cj to C 6 alkyl, C j to C 6 alkenyl, alkoxy having from 1 to 6 carbon atoms, hydroxy, thio, or C0 2 R 6 wherein R 6 is hydrogen, C ⁇ to C 6 alkyl, C x to C 6 alkenyl.
  • Proteinoid carriers prepared from the proteinoid molecules, in accordance with the present invention display a selective solubility at specific acidic or basic pH rang ⁇ es, depending on the choice and amount of the second and third monomers in the proteinoid.
  • Proteinoid carriers which are selectively soluble under alkaline pH environments, such as those found in the distal portion of the intestine, are prepared from base- soluble proteinoids. These proteinoids contain, as starting monomers in the reaction mixture, at least one second mono- mer selected from the group consisting of glutamic acid, glutamine, pyroglutamic acid, and aspartic acid. At a pH ranging between about 7.2 and about 11.0, the base-soluble proteinoid exists largely as the anion and is soluble. At a pH below about 7.0, the proteinoid is largely protonated and insoluble in water.
  • proteinoid carriers which are selec ⁇ tively soluble under acidic pH environments, such as the stomach, are prepared from acid-soluble proteinoids.
  • the proteinoid contain, as starting monomers in the proteinoid reaction mixture, at least one second monomer selected from the group consisting of glutamic acid, pyroglutamic acid, glutamine, and aspartic acid and at least one third monomer selected from the group consisting of lysine, arginine, and ornithine.
  • the base-soluble proteinoid exists largely as the cation and is soluble.
  • the proteinoid is largely unprotonated and insoluble in water.
  • the pH and the solubility characteristics of the acid-soluble proteinoid depends largely, but not exclusive ⁇ ly, upon the pH and solubility of the last amino acid added during the synthesis of the proteinoid.
  • a basic amino acid e.g., a third monomer, selected from the group consisting of lysine, arginine and ornithine in the acid-soluble proteinoid will result in the elevation of the pi (pH at the isoelectric point) of the proteinoid.
  • the proteinoids of the present invention are preparable by a thermal condensation reaction by heating mixtures of the appropriate amino acids under conditions described in the '673 patent.
  • mixtures of two to five specific amino acids with at least one selected from each of the aforementioned groups yield proteinoids which form proteinoid carriers with selective solubility at particular pH ranges and at high yields.
  • individual amino acids are added to a reaction flask containing tetramethylene sulfone (sulfolane) which has been heated to a temperature ranging between about 130°C and about 200°C, preferably about 175°C to 195°C, under an inert atmosphere of argon or nitrogen gas. After each addition, the solution is stirred for a period of time ranging between about 10 minutes and about 5 hours, depend- ing on the amino acid type and the order of addition.
  • tetramethylene sulfone sulfolane
  • the NCA method involves the preparation of N- carboxyanhydrides of alpha-amino acid esters and their subsequent polymerization, using low MW amines as initia ⁇ tors. It has been discovered that non-NCA derived amino esters, e.g., ce-methyl tyrosine ester, are effective initia ⁇ tors which are stable and soluble in many organic solvents such as tetrahydrofuran (THF) . The use of amino acids as initiators, presumably due to their poor solubility in organic solvents and their low stability, are not known. The NCA reaction produces a high yield of proteinoids with high purity.
  • the DPPA method involves the direct condensation of benzyl esters of alpha-amino acids in the presence of DPPA and a low MW amine, followed by removal of the protec ⁇ tive benzyl groups, contained in the proteinoid product, by alkaline hydrolysis. If catalytic hydrogenation is used in place of alkaline hydrolysis, low MW proteinoids of unex ⁇ pected high purities and yields are obtained.
  • Proteinoids prepared by any of the above methods can be used immediately to microencapsulate an active phar ⁇ macological agent or the proteinoid can be concentrated or dried by conventional means and stored for future use.
  • the proteinoids of the invention are purified as follows: crude proteinoids are slurried with water at room temperature, e.g. 25°C. While at this temperature, the pH of the slurry is adjusted to about pH 8 using an aqueous alkaline solution, e.g. 40% sodium hydroxide and 10% sodium bicarbonate solutions for an acid-soluble proteinoid. For a base-soluble proteinoid, the slurry is adjusted to an acidic pH with an aqueous acidic solution, e.g. 10% acetic acid solution. The mixture is then filtered and the filter cake washed with a volume of water. The washes and filtrate are then combined and evaporated to dryness in vacuo to afford proteinoids. If necessary, this process can be repeated until proteinoids of a desired purity level are obtained.
  • an aqueous alkaline solution e.g. 40% sodium hydroxide and 10% sodium bicarbonate solutions for an acid-soluble proteinoid.
  • the slurry is adjusted to an acidic pH with an
  • the proteinoid may be further purified by fractionating on a column containing solid supports which include silica gel or alumina, using methanol or propanol as mobile phase; ion exchange resin using water as the mobile phase; reverse phase column supports using trifluoroacetic acid/acetonitrile mixtures as mobile phase.
  • the proteinoids may also be purified by extraction with a lower alcohol such as propanol or butanol to remove low molecular weight con- taminants.
  • Proteinoid carriers are made from purified proteinoids as follows: proteinoids are dissolved in deion ⁇ ized water at a concentration ranging between about 75 and about 200 mg/ml, preferably about 100 mg/ml, at a tempera- ture between about 25°C and about 60°C, preferably about
  • Particulates remaining in the solution may be filtered out by conventional means such as gravity filtration over filter paper.
  • the proteinoid solution maintained at a temperature of about 40°C, is mixed with an aqueous acid solution (also at about 40°C) having an acid concentration ranging between about 1 N and about 2 N, preferably about 1.7 N.
  • the resulting mixture is further incubated at 40°C for a period of time effective for microsphere and microcap- sule formation as observed by light microscopy.
  • the preferred order of addition is adding the proteinoid solution to the aqueous acid solution.
  • Suitable acids include any acid which does not (a) adversely effect the proteinoid, e.g., chemical decomposi ⁇ tion; (b) interfere with microsphere or microcapsule forma ⁇ tion; (c) interfere with microsphere or microcapsule encap ⁇ sulation of cargo; and (d) adversely interact with the cargo.
  • Preferred acids for use in this invention include acetic acid, citric acid, hydrochloric acid, phosphoric acid, malic acid and maleic acid.
  • a proteinoid carrier stabilizing additives are preferably incorporated into the aqueous acid solution or into the proteinoid solution, prior to the microsphere or microcapsule formation process.
  • the presence of such additives promotes the stability and dispersibility of the proteinoid carriers in solution.
  • the additives may be employed at a concentration ranging between about 0.1 and 5 % (W/V) , preferably about
  • stabi ⁇ lizing additives include gum acacia, gelatin, polyethylene glycol, and polylysine.
  • the proteinoid carriers may be used immediately or may be stored at 4°C or lyophilized and stored under desiccant at room temperature or below.
  • the carrier forms hollow or solid matrix type microspheres wherein the cargo is distributed in a carrier matrix or capsule type microspheres encapsulating liquid or solid cargo.
  • a carrier matrix or capsule type microspheres encapsulating liquid or solid cargo.
  • the carrier microspheres are formed in the presence of a soluble material, e . g. , a pharmaceutical agent in the aforementioned aqueous acid solution, this material will be incorporated in the microspheres.
  • pharma- cologically active materials such as peptides, proteins, and polysaccharides as well as charged organic molecules, e . g. , antimicrobial agents, which normally have poor bioavailability by the oral route.
  • the amount of pharmaceu ⁇ tical agent which may be incorporated in the microsphere is dependent on a number of factors which include the concen ⁇ tration of agent in the microsphere forming solution, as well as the affinity of the cargo for the carrier.
  • the protein- oid molecules form spherical proteinoid carriers comprising proteinoid microcapsules and proteinoid microspheres of less than 10 micron diameter.
  • a "microsphere” is spherical homogeneous mesh work structure having no discrete inner chamber.
  • a “microcapsule” refers to a spher ⁇ ical structure having a proteinoid wall which forms a hollow or chamber.
  • the proteinoid carriers are formed in the presence of a soluble material, e.g., a pharmaceutical agent in the aforementioned aqueous acid solution, this material is believed to be encapsulated within the hollows of the microcapsules and confined within the proteinoid wall de ⁇ fined by the spherical structure or entrapped within the matrix of proteinoid molecules in the microsphere structure.
  • a soluble material e.g., a pharmaceutical agent in the aforementioned aqueous acid solution
  • this material is believed to be encapsulated within the hollows of the microcapsules and confined within the proteinoid wall de ⁇ fined by the spherical structure or entrapped within the matrix of proteinoid molecules in the microsphere structure.
  • pharmacologically active materials such as peptides, proteins, and polysaccharides as well as charged organic molecules, e.g., quinolones or antimicrobial agents, having poor bioavailability by the oral route.
  • the proteinoid carriers of the invention are pharmacologically harmless and do not alter the physiologi- cal and biological properties of the active agent. Further ⁇ more, the encapsulation process does not alter the pharmaco ⁇ logical properties of the active agent. While any suitable pharmacological agent can be encapsulated within proteinoid carriers, it is particularly valuable for delivering agents which otherwise would be destroyed or rendered less effec ⁇ tive by conditions encountered in the animal body before it reaches its target zone and which are poorly absorbed in the gastrointestinal tract.
  • the proteinoid carriers of the invention are particularly useful for the oral administration of certain pharmacological agents, e.g., small peptide hormones, which, by themselves, pass slowly or not at all through the gastro ⁇ intestinal mucosa and/or are susceptible to chemical cleav- age by acids and enzymes in the gastrointestinal tract.
  • pharmacological agents e.g., small peptide hormones
  • Non-limiting examples of such agents include human or bovine growth hormone, interferon and interleukin-II, calcitonin, atrial naturetic factor, antigens, monoclonal antibodies, and Factor IX, a vitamin K-dependent blood coagulation proenzyme.
  • Biologically-active agents suitable for use with carriers disclosed herein include, but are not limited to, peptides, and particularly small peptide hormones, which by themselves do not pass or only pass slowly through the gastro-intestinal mucosa and/or. are susceptible to chemical cleavage by acids and enzymes in the gastro-intestinal tract; polysaccharides and particularly mixtures of muco- polysaccharides; carbohydrates; lipids; or any combination thereof.
  • Examples include, but are not limited to, human growth hormone; bovine growth hormone; growth hormone re ⁇ leasing hormone; interferons; interleukin-I; insulin; hepa ⁇ rin, and particularly low molecular weight heparin; calcito ⁇ nin; erythropoietin; atrial naturetic factor; antigens; monoclonal antibodies; somatostatm; adrenocorticotropm; gonadotropin releasing hormone; oxytocin; vasopressin; cromolyn sodium (sodium or disodium cromoglycate) ; vancomy- cin; desferrioxamine (DFO) ; or any combination thereof.
  • human growth hormone bovine growth hormone
  • growth hormone re ⁇ leasing hormone interferons
  • interleukin-I insulin
  • hepa ⁇ rin and particularly low molecular weight heparin
  • calcito ⁇ nin erythropoietin
  • the carriers of the present invention can be used to deliver other active agents such as pesti ⁇ cides and the like.
  • the amount of active agent in the composition typically is a pharmacologically or biologically effective amount. However, the amount can be less than a pharmacolog- ically or biologically effective amount when the composition is used in a dosage unit form, such as a capsule, a tablet or a liquid, because the dosage unit form may contain a multiplicity of carrier/biologically-active agent composi ⁇ tions or may contain a divided pharmacologically or biologi- cally effective amount.
  • the total effective amounts will be administered by cumulative units containing in total pharma ⁇ cologically or biologically active amounts of biologically- active agent.
  • Dosage unit forms can also include any of excipients; diluents; disintegrants; lubricants; plasticizers; colorants; and dosing vehicles, including, but not limited to water, 1,2-propane diol, ethanol, olive oil, or any combination thereof.
  • proteinoids made from glutamic acid, aspartic acid, tyrosine, and phenylalanme are especially suitable for encapsulating polysaccharides like heparin.
  • the parti- cle size of the proteinoid carrier plays an important role in determining release of the active agent in the targeted area of the gastrointestinal tract.
  • Proteinoid carriers having diameters between about ⁇ . 0.1 microns and about 10 microns, preferably between about 5.0 microns and about 0.1 microns, and containing encapsulated or entrapped active agents are sufficiently small to effectively release the active agent at the targeted area within the gastrointesti ⁇ nal tract. Large proteinoid carriers (>10 microns) tend to be less effective as oral delivery systems.
  • the size of the proteinoid carriers formed by con ⁇ tacting proteinoids with water or aqueous solution contain ⁇ ing active agents can be controlled by manipulating a vari ⁇ ety of physical or chemical parameters, such as the pH, osmolarity or salt content of the encapsulating solution, and the choice of acid used in the encapsulating process.
  • active agent bearing proteinoid carriers can be produced from base-soluble proteinoids which are stable in the highly acidic stomach (normal pH of from about 2 to about 6) , but which dissolve in the distal portion of the intestines.
  • Such systems are suitable for oral administra- tion of peptide hormones, e.g., insulin, and polysac- charides, e.g., heparin, which otherwise would be quickly destroyed in the GI tract. They also are suitable for protecting the stomach from gastric irritants, such as aspirin.
  • aspirin-containing proteinoid carriers When such aspirin-containing proteinoid carriers are orally administered, they pass through the gastrointes ⁇ tinal mucosa and release the aspirin far more rapidly than conventional enterically coated aspirin, which first must traverse the stomach and then must enter the bloodstream from the intestine after the enteric coating has dissolved.
  • the proteinoid carriers of the invention may be orally administered alone as solids in the form of tablets, pellets, capsules, and granulates suitable for suspension in liquids such as edible oils.
  • the proteinoid carriers can be formulated into an orally administrable composition containing one or more physiologically compati ⁇ ble carriers or excipients. These compositions may contain conventional ingredients such as gelatin, polyvinylpyrrol ⁇ idone and fillers such as starch and methyl cellulose.
  • the proteinoid carriers of the invention may also be administered by injection.
  • Example 1 Preparation of a Base-soluble Proteinoid by a Thermal condensation Reaction
  • the cake was reslurried in 5 liters of water, filtered and the cake was again reslurried in 5 liters of water.
  • the pH of the slurry (at 25°C) was adjusted to 8 using 40% sodium hydroxide solution.
  • the mixture was filtered and the cake washed with a small amount of water.
  • the washes and fil ⁇ trate are combined and evaporated to dryness in vacuo to give Glu/Asp/Tyr/Phe proteinoid.
  • Appendices A, B, and C describe examples of other proteinoids prepared by the thermocondensation method.
  • Example 2 Preparation of an Acid-soluble Proteinoid by a Thermal Condensation Reaction 750 ml of tetramethylene sulfone is heated to
  • the cake is reslurried in 5 liters of water, filtered and the cake is again reslurried in 5 liters of water.
  • the pH of the slurry (at 25°C) was adjust ⁇ ed to 5 using 10% acetic acid solution.
  • the mixture is filtered and the cake is washed with a small amount of water.
  • the washes and filtrate are combined and evaporated to dryness m vacuo to give proteinoid.
  • Appendices A, B, and C describe examples of other proteinoids prepared by the thermocondensation method.
  • This example illustrates the NCA method for pre- paring copolypeptides consisting of Asp.Bz, Glu.Bz, Phe, and Tyr components.
  • the NCA monomers of these amino acids were prepared according to the reported method.
  • Polydispersity is defined herein as the molecular weight distribution of a sample. The distribution is as- signed a numerical value derived from the molecular weight (MW) divided by the molecular number (Mn) .
  • the polydispersity value for a homopolymer is 1 because the molecular weight is equal to the molecular number. Any polymer with a polydispersity value of 1 is considered to have a very narrow distribution. A polymer with polydispersity value of 1.6 to 1.7 is considered to have medium distribution. A polymer with a polydispersity value of 2.0-2.1 is considered to have a broad distribution.
  • Tyr.Me is a novel and effective initiator for the polymerization of amino acid NCA's.
  • Sample No.2-13 represents a polymerization initiated with -alanine and terminated with succinic anhydride.
  • ⁇ - alanine is insoluble in most organic solvents, the reaction was carried out in refluxing THF.
  • the polydispersity of the polymer obtained was broader than that of the polymers initiated by Tyr.Me.
  • Example 5 Preparation of Proteinoids bv the DPPA Method (#1)
  • DPPA dimethyl formamide
  • TEA distilled before use.
  • Solvents for polymer ⁇ ization were purified by conventional methods.
  • the direct polycondensation of Asp.Bz was carried out by stirring a dimethyl formamide (DMF) solution of the monomer in the pres ⁇ ence of DPPA and TEA. The mixture was stirred for 1 h at 0- 10°C followed by stirring at room temperature for two days. The resulting polymer was precipitated in a large amount of water, collected by filtration, and then dried in vacuo.
  • DMF dimethyl formamide
  • Table 3 Listed in Table 3 are the results for the polymeriza ⁇ tion of Asp.Bz in DMF at room temperature for two days. Poly(Asp.Bz)s were obtained from these direct polycondensations in high yield.
  • the molecular weight of the polymers was found to be dependent on the concentration of the monomer [M] .
  • Low molecu ⁇ lar weight polymers with broad distribution were obtained from a low [M] ( Figure 2, curve A) .
  • [M] was greater than 0.2 g/mL
  • a polymer with a bimodal molecular weight distribution was obtained ( Figure 2, curve B) .
  • the lower molecular weight oligomers (-1000) may be due to an intramolecular termination between the terminal amino and the ⁇ -carboxylic groups.
  • the example illustrates a preferred method for the removal of benzyl protective groups in poly(Asp.Bz) and poly(Glu.Bz) by catalytic hydrogenation.
  • the hydrogenation of the polymers was carried out according to the following procedure: To a solution of the polymer in THF/methanol (1:1, v/v), Pd on active carbon (10%) was added in the amount of 1/10 of the polymer weight. After the replacement of air by nitrogen, hydrogen gas was introduced into the system and maintained with a balloon. The reaction mixture was stirred at room temperature overnight. After removing the catalyst by filtration and concentrating the solution, the mixture was poured into a large amount of petro ⁇ leum ether to precipitate the polymer. The polymer obtained was then dried m vacuo.
  • This Example illustrates a method for the preparation and cleaning of empty proteinoid carriers.
  • This experiment describes encapsulation of anti- reovirus monoclonal antibody (mAb) 9BG5, an mAb directed against the sigma-1 gene product (Hemaglutinin, HA3) of the
  • HA3 binds to the cell surface receptor for
  • Reovirus type 3 and mAb 9GB5 interferes with viral binding to the receptor.
  • Mouse IgG monoclonal antibody 9BG5 was prepared and purified as described W.V. Williams et al. (1991) J. Biol. Chem.. Vol. 266(8), pages 5182-5190, as well as references cited therein, using a purified Reovirus type 3 preparation (W.V. Williams et al. (1988) Proc. Natl. Acad. Sci. U.S.A. Vol. 85, pages 6488-6492) .
  • the purified 9BG5 used in this Example had a protein concentration of 1.5 mg/ml in phosphate buffered saline (pH 7.2) .
  • Proteinoid carriers encapsulating mAb 9BG5 were prepared having final concentrations of Glu/Asp/Tyr/Phe proteinoid (1:1:1:1 mole ratio of Glu, Asp,Tyr, and Phe in the reaction mixture) 50 mg/ml, mAb 0.7 mg/ml and gum arabic 0.5% in 0.85 N citric acid. Empty proteinoid carriers were prepared to contain the same final concentrations, except mAb was omitted. Aliquots (0.5 ml), in duplicate, of both mAb and empty proteinoid carriers preparations were centrifuged at 5000 RPM. Pellets and supernatants were frozen prior to analysis by Western blotting to determine antibody encapsulation efficien ⁇ cy.
  • Figure 6 is an x-ray film of a western blot analysis of purified mAb 9BG5, empty proteinoid carriers (no mAb added) , and proteinoid carriers containing 9BG5.
  • the analysis was done by immunoblotting with anti-mouse IgG which specifically reacted with mAb 9BG5.
  • the lanes correspond to the following:
  • the relative mobility (molecular weight) of the pure mAb is slightly different than the mAb in the proteinoid carriers. This is most likely due to different salt concentra ⁇ tions in the samples, because the encapsulation process employed 0.8 M salt solution.
  • the mAb 9BG5 preparations used to prepare the encap ⁇ sulated proteinoid carriers had a protein concentration of approximately 2 mg/ml in phosphate buffered saline. Final proteinoid concentration was 50 mg/ml and 5%
  • Table 7 lists samples that were prepared. Numbers in parenthesis indicate amount of mAb added. TABLE 7
  • pellets of samples 9 and 10, and 11 and 12 contain between 5 and 10 ⁇ g of mAb.
  • the washed samples did not lose any significant amount of mAb, suggesting that the prote ⁇ inoid carriers remained intact after freeze-thawing.
  • Sample 17 had some mAb encapsulated which was lost after washing (see number 18) . This sphere preparation was not resistant to freeze-thawing. Additionally, a band at a MW of 150 kDa for sample 17 supernatants indicates that a significant amount of mAb is left behind after proteinoid carrier forma ⁇ tion. Based on these results, it appears that the mAb remains intact and therefore the encapsulating procedure does not degrade it. The empty proteinoid carrier controls did not produce any bands, as expected because they have no mAb.
  • the mAb 9BG5 (1 mg/ml), prepared as described in Example 9, was encapsulated in Glu/Asp/Tyr/Phe proteinoid (1:1:1:1 mole ratio of Glu, Asp,Tyr, and Phe in the reaction mixture) protein carrier formulation with gum arabic.
  • the mAb proteinoid carriers suspension contained 0.25 mg/ml mAb and 50 mg/ml proteinoid in 0.85 N citric acid-0.5% gum. Empty proteinoid carriers were prepared similarly, but did not contain mAb. Since 30% of the mAb was found to be encapsulated, the mAb proteinoid carriers were estimated to contain 0.075 mg/ml mAb and this value was used to determine dosages.
  • the mAb protein ⁇ oid carriers were examined microscopically and appear to be a fairly homogeneous preparation.
  • mAb 9BG5 proteinoid carriers 3.7 mg mAb/ kg body weight of rat by oral gavage (rat # 2287, 2288, 2290, and 2291) .
  • unencapsulated mAb 9BG5 3.7 mg/ kg body weight of rat by oral gavage (rats #2314 and 2315) .
  • Baseline blood samples (1 ml aliquots) were withdrawn from each rat just prior to dosing ("0" time). After dosing, blood samples were drawn at 1 h, 6 h and 24 h. The blood samples were processed immediately and sera were stored frozen at -20°C.
  • VLSH peptide (W.V. Williams et al (1991) J. Biol. Chem.. Vol. 266(8), pages 5182-5190). Control plates included wells having no immobilized reovirus and V L SH peptides to which mAb (lmg/ml) was added.
  • VLSH peptide (W.V. Williams et al. ibid, Table 1) is a synthetic variant of VL peptide, the latter which corresponds to a portion of the light chain variable CDR II region of 87.92.6 antibody.
  • the 87.92.6 antibody displays idiotypic and anti-idiotypic behavior towards reovirus type 3 receptor and mAb 9BG5, respectively (W.V. Williams et al. ibid) .
  • the bound protein content of each well were measured by standard protein methods, e.g., Lowry method, and the results for each multi-well plate are shown in Figures 8(a-c), respectively.
  • Figures 8 (a-c) illustrate the levels of serum proteins which bound to immobilized reovirus type 3 and V L SH as detected by measurement of protein concentration. These Figures show that the serum levels of bound proteins, after 24 hours post-dosing, were highest for animals orally administered mAb proteinoid carriers and animals administered unencapsulated mAb by the IV route. Lower levels of bound serum proteins were found in animals orally administered uncapsulated mAb. Serum taken from the animals receiving empty proteinoid carriers (no mAb) showed non-specific serum IgG protein binding, as expected, under the assay conditions.
  • Figure 9 show mAb binding under conventional ELISA procedures using immobilized reovirus type 3 and V L SH proteins.
  • Serial dilutions of mAb treated with 0.85 N citrate-0.5% gum ( Figure 9(a) or phosphate buffered saline ( Figure 9 (b) were employed.
  • the Figures show that the bound protein levels were higher for mAb in citrate buffer than for mAb in phosphate. Without being bound by any theory of operation for this inven- tion, it is believed that the binding enhancement may be due to changes in the three dimensional conformation resulting from citrate-protein binding.
  • serum levels of mAb were greater in animals receiving encapsulated mAb by the oral route or unencapsulated mAb by the IV route, than an animal receiving orally administered unencap ⁇ sulated mAb.
  • This Example describes a method for the preparation and cleaning of heparin proteinoid carriers.
  • PROCEDURE 1 Reagents: a. Proteinoid powder prepared as described in Example 1 b. Heparin c. Anhydrous citric acid (USP) d. Gum acacia NF e. Deionized water f. Desiccant g. Liquid nitrogen
  • Solution B (1.7 N citric acid with 1% gum): Dissolve 10 g of gum acacia and 109 g of citric acid in 1 liter of deionized water.
  • proteinoid carrier encapsulates prepared with citric acid solutions are preferably dialyzed against 5% acetic acid solution for at least two hours with at least four changes of the dialysis solution to remove citric acid by an exchange process.
  • Lyophilization a. Add one part of 50% trehalose (Sigma Chemical Co., St. Louis, MO, USA) into nine parts of dialyzed proteinoid carrier solution. Flash freeze protein ⁇ oid carriers in a freeze-drying flask using the shell freezer adjusted to rotate at ca. 190 rpm and immersed in a liquid nitrogen bath.
  • 50% trehalose Sigma Chemical Co., St. Louis, MO, USA
  • Resuspension a. Weigh the lyophilized powder and calculate the amount of proteinoid in the powder.
  • Reagents a. Proteinoid powder b. Anhydrous citric acid (USP) c. Gelatin (USP) d. Porcine insulin (Novo Nordisk) e. Deionized water (USP)
  • Proteinoid solution Dissolve 100 mg proteinoid per 1 ml deionized water at room temperature and desired volume. Using sy ⁇ ringe and 0.2 micron Acrodisk, filter the solution to ensure a clear liquid and incubate in a water bath at 40°C. See Section 5b.
  • Proteinoid carriers a. Proteinoid solution and insulin solution are com ⁇ bined at equal volumes sufficient to produce the final desired volume of proteinoid carriers.
  • EPO erythropoietin
  • Proteinoid and Insulin solutions should each be prepared at one-half the total volume of the final microsphere solution desired. citric acid with 1% gum was used in preparing the EPO-contain ⁇ ing proteinoid carrier.
  • an EPO-containing protein carrier prepared as described in Example 14, was evaluated in rats.
  • Rats weighing 150-200 grams are anesthetized with ketamine (8.5mg/kg) and thorazine 3.75mg/kg) with intramuscular injection.
  • the rat is then administered either unencapsulated erythropoietin or encapsulated erythropoietin by oral gavage.
  • an 8 french nelaton catheter is inserted down the esophagus of the rat until the 10cm mark on the catheter is even with the incisors.
  • the test or control solution is drawn up into a syringe and attached to the catheter. Holding the animal upright, the solution is expressed into the stomach of the rat.
  • the experimental results are summarized in Figures 10-12.
  • ⁇ Rats were foaming at nostrils.
  • Serum erythropoietin levels were determined over time with an erythropoietin enzyme immunoassay kit (Amgen, Thousand Oaks, CA, USA) .
  • the results show that EPO serum levels in rats administered erythropoietin proteinoid carriers were relatively higher at all time points compared to rats (control) which received unencapsulated material.
  • the EPO levels remained at approxi ⁇ mately 300 pg/mL serum in rats administered erythropoietin proteinoid carriers while the control rats had undetectable EPO levels.
  • Figure 11 illustrates EPO serum levels in rats that were administered either erythropoietin (50 ⁇ g/kg) or Gln/Asp/Tyr/Phe proteinoid (1:1:1:1 mole ratio of Gin, Asp,Tyr, and Phe in the reaction mixture) proteinoid carrier encapsulat- ed erythropoietin (50 ⁇ g/kg) directly into the proximal duodenum. Serum erythropoietin levels were determined over time with the aforementioned erythropoietin enzyme immunoassay kit.
  • Calcitonin a peptide hormone which acts predominantly on bone to lower serum calcium concentration
  • Calcitonin proteinoid carriers were prepared by mixing a 1:1 volume ratio of a lOOmg/ml aqueous solution of Gln/Asp/Tyr/Phe proteinoid (1:1:1:1 mole ratio of Gin, Asp, Tyr, and Phe used in the proteinoid reaction mixture) and a 150 ug/mL calcitonin solution in 1.7 N citric acid solution with 1% gum acacia, as described in Example 13. The efficiency of calcitonin encapsulation was approximately 40%. Calcitonin concentration was determined directly by HPLC after dissolving the calcitonin proteinoid carriers in 60% aqueous acetonitrile.
  • the calcitonin proteinoid carriers prepared as described in Example 16, were evaluated in cynomolgus monkeys.
  • a single oral dose of calcitonin proteinoid carriers (0.25 mg/kg body weight) was administered to each of four monkeys by nasogastric gavage. The dosage was based on the body weight taken on the morning of dosing.
  • the hypocalcemic response following oral calcitonin administration was used as an index of pharmacological response.
  • Serum calcium concentrations were quantitated by a conventional O-cresolphthalein complexone method.
  • Figure 13 demonstrates the response obtained in cynomolgus monkeys following naso-gastric gavage of microencap- sulated calcitonin. Significant changes from baseline serum calcium concentration were observed. Six hours following dosing, serum calcium concentrations decreased by 13 ⁇ g/ml. A significant pharmacological response was still apparent seven hours after the administration of calcitonin proteinoid carriers.
  • the calcitonin proteinoid carriers prepared in accordance with Example 16 are evaluated in fasted male Spraque Dawley rats weighing 100-150g. Calcitonin proteinoid carriers and calcitonin were administered by either oral gavage or intraduodenal injection. The rats are divided into the following groups:
  • calcitonin proteinoid carriers 60 ug calcitonin/kg body weight by oral gavage (3 rats) ; 2. calcitonin proteinoid carriers: 3 ug calcitonin/kg body weight by intraduodenal gavage (3 rats) ;
  • Calcitonin proteinoid carriers are prepared immediately prior to dosing and Groups 1 and 2 each receive an appropriate dosage of the proteinoid carrier suspension. Groups 3 and 4 receive the unencapsulated calcitonin (no proteinoid carriers) . Approxi ⁇ mately 0.5 ml of blood is serially withdrawn from the tail artery of each rat just prior to dosing ("0" time) and 1 h, 2 h and 3 h post-dosing. Serum from the blood samples are stored at -20°C for serum calcium concentration determination.
  • Figure 14 is the serum concentration-time curve for orally administeredmicroencapsulated calcitonin andunencapsu ⁇ lated calcitonin in rats.
  • serum calcium concentra ⁇ tions decreased 23 ⁇ g/ml in the rats receiving encapsulated calcitonin compared to a decrease of only 6.5 ⁇ g/ml in the control group.
  • the responses were dose-dependent (data not shown) .
  • Example 17 The results obtained in this Example and in Example 17 provide evidence that proteinoid encapsulation markedly improves the oral bioavailability of calcitonin. The data also indicate that the oral drug delivery system is not species- dependent.
  • Factor IX is a vitamin K-dependent blood coagulation proenzyme, MW 56 kD.
  • Factor IX deficiency known as hemophilia B, occurs in approximately 1 out of every 25,000 males. To date, treatment of this disorder is accomplished by intravenous administration of Factor IX, although a recent report details efforts to supplement by subcutaneous injection (Thompson (1986) Blood, Vol. 67(3), pages 565-572).
  • FIX Factor IX
  • FIX proteinoid carrier suspension A contained 50 mg/ml of proteinoid and 500 U/ml FIX (FIX is available from the American Red Cross, Rockville, Maryland, USA) solution containing 4% acetic acid, 2% gum acacia, 0.2% PEG 14 (avail ⁇ able from Union Carbide, Danbury, CT, USA) , 14 mM CaCl 2 , final pH 3.81.
  • the second suspension, FIX proteinoid carrier suspen- sion B contained 50 mg/ml proteinoid and 116 U/ml FIX solution containing 3.8% acetic acid, 1.5% gum acacia, 0.15% PEG 14, 11 mM CaCl 2 , final pH 4.58.
  • FIX proteinoid carrier preparations The stability of FIX proteinoid carrier preparations was assessed over a short time course in vitro.
  • the protein carriers encapsulating FIX were examined by optical microscopy and laser light scattering. Aliquots of proteinoid carrier suspension were withdrawn every 30 minutes for 1.5 hours, FIX proteinoid carriers were isolated by centrifugation at 4500Xg and dissolved in activated partial thromboplastin time (APTT) assay buffer (0.05M histidine-0.OlM NaCl-0.1% bovine serum albumin-0.01% TWEEN-40, pH 7.47) to release soluble FIX and proteinoid.
  • APTT activated partial thromboplastin time
  • Quantitation of FIX activity by APTT employed both FIX standards (0.025, 0.05, and 0.1 U/ml) and "empty" protein ⁇ oid carrier suspension as control.
  • APTT assay kits are commercially available, e.g. Sigma Diagnostics (St. Louis, MO, USA) .
  • FIX proteinoid carriers of greater stability are obtained by encapsulating FIX at a higher pH, e.g., pH 4.9. Furthermore, the efficiency of encapsulation is approximately 20% of available FIX units and activity levels remain constant for at least 1.5 hours when FIX proteinoid carrier pellets are stored at about 4°C.
  • FIX proteinoid carriers FIX sph PO
  • FIX sph PO Oral FIX proteinoid carriers
  • FIX IV Intravenous FIX (no proteinoid carriers)
  • FIX proteinoid carrier suspension and solution are prepared immediately prior to dosing.
  • One ml of blood was withdrawn from each rat just prior to dosing ("0" time) and 1 h, 2 h and 4 h (post-dosing) , a citrate anticoagulant was added to the blood, and plasma from the blood samples were stored at -70°C.
  • Plasma samples were assayed by a modified APTT assay using FIX coagulated deficient plasma (assay kit is available from Ortho Diagnosis (Raritan, New Jersey, USA) . Changes in clotting times were calculated by subtracting individual baseline (0 hr) values from subsequent clotting time values. The data shown in Figure 16 are the mean values for a given group. Values below baseline indicate the presence of exoge- nous FIX.
  • FIX proteinoid carriers As shown in Figure 16, significant amounts of FIX was delivered to blood via oral administration of FIX proteinoid carriers.
  • the relative plasma level is lower in the FIX proteinoid carriers group, however the dimunition in clotting time at 0.5, 1.0 and 2.0 hours is notable. This is achieved by oral dosing with approximately 14 times the IV dose.
  • Factor IX is an acid labile protein whose half-life is approximately less than one hour at 37°C at pH 5.0.
  • the FIX proteinoid carriers in this experiment were at pH 3.81 and encapsulated 14.8% of the available FIX units during prepara ⁇ tion. The results support that FIX proteinoid carriers remain viable in the GI tract to facilitate delivery.
  • FIX proteinoid carriers were prepared as described in Example 20. The rats are divided into two groups as follows:
  • FIX proteinoid carriers 1006U FIX/kg body weight by intragastric gavage (5 rats) .
  • FIX IV Intravenous FIX (no proteinoid carriers)
  • FIX IV Intravenous FIX
  • 3 rats received 0.3 ml FIX in 0.11 NaCl-0.02M sodium citrate, pH 6.85 by tail vein injection.
  • Oral FIX (no proteinoid carriers) (FIX unencap PO) 2760U FIX/kg body weight by intragastric gavage. 4 rats re ⁇ ceived 1.0 ml of FIX in saline solution containing 3.8% acetic acid, pH 6.85.
  • FIX proteinoid carrier suspension and solutions were prepared immediately prior to dosing. Plasma samples were obtained and assayed as described in Example 20. Changes in clotting times were calculated by subtracting individual baseline (0 hr) values from subsequent clotting time values. The data shown in Figure 17 are the mean values for a given group. Values below baseline indicate the presence of exoge ⁇ founded FIX.
  • Example 20 support that oral delivery of FIX can be accom- plished via the use of FIX proteinoid carriers. These proteinoid carriers appear to adequately protect FIX during transit through the GI tract and deliver FIX to the blood stream.
  • IFN-containing proteinoid carriers Encapsulation of IFN in proteinoid carriers was performed in the same manner described in Example 13.
  • Alpha- IFN is available from a number of commercial sources.
  • One commercial IFN product includes Roferon-A (Hoffman LaRoche) .
  • IFN proteinoid carriers were prepared with an aqueous solution of Glu/Asp/Tyr/-Phe proteinoid (1:1:1:1 mole ratio of Glu, Asp, Tyr and Phe used in the proteinoid reaction mixture) , and an IFN solution containing 1.7 N citric acid solution with 5% gelatin.
  • the IFN proteinoid carrier suspension contained 80 mg/ml proteinoid, 600 ug/ml IFN, 0.6N citric acid, and 2.5% gelatin, pH 3.0.
  • IFN proteinoid carriers were much more stable than IFN alone (in the absence of proteinoid) in SIF.
  • IFN alone at pH 7.4 was completely degraded within 10 minutes when incubated with SIF.
  • IFN alone was slightly more stable in SIF at pH 3 than at pH 7.4. After 6 hr incubation in SIF at pH 3, there was approximately 10% of the IFN remaining. The stability of IFN in SIF at pH 3 is attributed to the low pH, which appears to suppress enzymatic activity of the intestinal proteases.
  • proteinoid carriers are required for protective capability or whether (1) proteinoids (soluble proteinoids--not in carrier form) may be used and whether (2) alternative methods of carrier loading, such as incubating the therapeutic compound with preformed proteinoid carriers, are useful.
  • Heparin proteinoid carriers were prepared, following the procedure of Example 12, using a 1:1 volume ratio of 150 mg/ml of Glu/Asp/Tyr/Phe/Orn ⁇ 5 (1:1:1:1:0.5 mole ratio of Glu, Asp, Tyr, Phe, and Orn used in the proteinoid reaction mixture) proteinoid in deionized water, and an 20mg/mL aqueous heparin solution containing 1.7 N citric acid solution and 0.5% gum acacia.
  • the heparin proteinoid carrier suspension was dialyzed in acetic acid solution as described in Example 12.
  • Heparin proteinoid carriers were then centrifuged at 4800Xg (15 minutes) and total heparin was measured by assaying the pellet and the supernatant with a modification of the Azure A method (Gundry et al. Amer. J. of Surgery (1984) Vol. 148, pages 191- 194) . Proteinoid was assayed by dissolving the proteinoid carriers with 0.1 N NaOH and measuring absorbance at 294 nm.
  • Empty proteinoid carriers were prepared following the same procedure described above for the heparin proteinoid carriers, with the modification being that no heparin was present.
  • the lyophilized empty proteinoid carriers were resuspended in 0.85N citric acid and 0.5% gum containing heparin at a concentration of 20 mg/ml.
  • the amount of heparin co-isolated with the proteinoid carriers was measured as described above.
  • Rats Male Spaque Dawley rats weighing approximately 350g were dosed by oral gavage or intraduodenal (ID) injection (just anterior to the pyloric sphincter and into the duodenum) . Rats were dosed orally or ID with one of the following: lyophilized heparin proteinoid carriers, heparin-spiked empty proteinoid carriers, proteinoid/heparin in water, heparin in 0.85N citric acid and 0.5% gum and heparin alone in water. In both oral and ID injection experiments, weight ratios of heparin:proteinoid were constant. The total heparin dose in the oral studies was 100 mg/kg body weight; in ID injections studies, it was 50 mg/kg.
  • ID intraduodenal
  • the proteinoid dose was 40 mg/kg for oral gavages and 20 mg/kg for ID injections.
  • the dosing volume was approximate ⁇ ly 0.3 to 0.5 ml.
  • Approximately 0.5 ml of blood is serially withdrawn from the tail artery of each rat just prior to dosing ("0" time) and 1 h, 2 h and 4 h post-dosing. Serum from the blood samples are stored at -20°C for heparin activity determination.
  • Heparin proteinoid carriers gave the highest APTT values, indicated increased absorption of heparin when dosed orally, as well as when directly injected into the duodenum
  • influenza virus antigen-containing proteinoid carriers were prepared and evaluated in rats.
  • Ml protein a major internal component of influenza virus, was obtained by purification of a swine influenza vaccine donated by Drug Directorate, Health Protection Branch, Bureau of Biologies, Ottawa, Ontario Canada.
  • the vaccine was prepared with the high-yielding recombinant strain X-53Aa, which derives its HA and NA from the parent strain A/NJ/11/76 (H1N1) and its internal proteins, including Ml, from the parent strain A/PR/8/34 (R.B. Couc et al. (1983) Ann. Rev. Microbiol.. Vol. 37, pages 529-549 and B.R. Murphy (1982) Infec. Immun. , Vol.
  • Ml was purified as described by Khan et al ( (1982) J.Clin.Microbiol.. Vol. 16, pages 813-820) .
  • Ml proteinoid carriers were prepared, by mixing (at 40°C) , equivolumes of an aqueous solution of lOOmg/ml of Glu/Asp/Tyr/Phe proteinoid in deionized water and a lOmg/mL solution of Ml protein in 1.7N citric acid and 5% gum arabic (pH 2.0). The final Ml concentration in the suspension was 1.Omg/ml.
  • HA-NA antigen was isolated according to the procedure of Gallagher et al. ((1984) J.Clin.Microbiol. , Vol. 20, pages 80-93). Influenza virus (A/PR8/34) was centrifuged at 90,000 G for 60 min. The viral pellet was solubilized with 0.05M acetate buffer (pH 7.0) containing 7.5% octylglucoside and re- centrifuged under the same conditions. The resulting superna- tant contained approximately 90% HA and 10% NA as determined by SDS-PAGE.
  • HA-NAproteinoid carriers were prepared following the same protocol as for the Ml proteinoid carriers but substituted Ml for HA-NA.
  • the final concentration of HA-NA in the suspension was also 1.0 mg/ml.
  • mice Male Spraque Dawley rats (about 350g weight) were used in this experiment. Oral dosage was by gavage.
  • SC subcutaneously
  • Plasma samples from rats dosed orally with "empty" proteinoid carriers showed no significant antibody titer against either Ml or HA-NA antigens when assayed by ELISA (Table 8) .
  • rats dosed with 25 ug of either Ml or HA-NA antigen (with FCA) subcutaneously developed a vigorous antibody response with titers that ranged from 54,000-330,000 in the case of Ml and 176,750-909,000 in the case of HA-NA (Table 8) .
  • Plasma samples from three of the five rats dosed with Ml proteinoid carriers showed a significant primary response to Ml antigen. All three rats had titers ranging from 760 to 2150 as early as 14 days post-dosing, compared to ⁇ 30 in all rats that received the amount of unencapsulated Ml (Table 8) . Titers in the group that received proteinoid carriers increased to 1150-5200 by 42 days ( Figure 24) .
  • GLU2 ASP2 EQU GLU ASP EQU GLU ASP EQU 0
  • PA phosphoric acid
  • GLYC glycerol
  • PPA polyphosphoric acid

Abstract

Improved proteinoid carriers and methods for their preparation and use as oral delivery systems for pharmaceutical agents are described. The proteinoid carriers are soluble within selected pH ranges within the gastrointestinal tract and display enhanced stability towards at least one of photolysis or decomposition over time. The proteinoid carriers are prepared from proteinoids having between 2 and 20 amino acids and having a molecular weight of between about 250 and 2400 daltons.

Description

"PROTEINOID CARRIERS"
This application is a continuation-in-part of U.S. application serial no. 08/076,803, filed June 14, 1993 which in turn is a continuation-in-part of U.S. application serial no. 07/920,346, filed July 27, 1992, which in turn is a continuation-in-part of U.S. application serial no. 07/898,909, filed June 15, 1992, now abandoned.
Field of the Invention
This invention relates to proteinoids and protein¬ oid carriers made from them. The proteinoid carriers releasably encapsulate active agents and have extended longer shelf life and/or photostability. Methods for the preparation of such proteinoid carriers are also disclosed.
Background of the Invention
The available modes of delivery of pharmaceutical and therapeutic agents often are severely limited by chemi- cal or physical barriers or both, which are imposed by the body. For example, oral delivery of many such agents would be the route of choice if not for the presence of chemical and physicochemical barriers such as extreme pH in the gut, exposure to powerful digestive enzymes, and impermeability of gastrointestinal membranes to the active ingredient.
Among the numerous pharmacological agents which are known to be unsuitable for oral administration are biologically active peptides and proteins, such as insulin. These agents are rapidly destroyed in the gut by acid hydrolysis and/or by proteolytic enzymes.
A great deal of research has been devoted to developing effective oral drug delivery methods and systems for these vulnerable pharmacological agents. The proposed solutions have included :
(a) co-administration of adjuvants (such as resorcinols and non-ionic surfactants polyoxyethylene oleyl ether and n-hexadecyl polyethylene ether to increase the permeability of the intestinal walls; and
(b) co-administration of enzymatic inhibitors, such as pancreatic trypsin inhibitor, diisopropylfluorophosphate (DFF) and trasylol to avoid enzymatic degradation. The use of such substances, in drug delivery systems, is limited however either because of:
(a) their inherent toxicity when employed at ef¬ fective amounts;
(b) their failure to protect the active ingredi- ent or promote its absorption;
(c) their adverse interaction with the drug. Liposomes as drug delivery systems have also been described. They provide a layer of lipid around the encap¬ sulated pharmacological agent. The use of liposomes con- taining heparin is disclosed in U.S. Patent No. 4,239,754 and several studies have been directed to the use of liposomes containing insulin; e.g., Patel et al . (1976) FEBS Letters Vol. 62, page 60 and Hashimoto et al . (1979) Endocrinol. Japan, Vol. 26, page 337. The use of liposomes, however, is still in the development stage and there are continuing problems, including:
(a) poor stability;
(b) inadequate shelf life;
(c) limited to low MW (< 30,000) cargoes; (d) difficulty in manufacturing;
(e) adverse interactions with cargoes. More recently, synthetic amino acid polymers or proteinoids, forming icrospheres, have been described for encapsulating pharmaceuticals. For example, U.S. Patent No. 4,925,673 (the '673 patent), the disclosure which is hereby incorporated by reference in its entirety, describes such microsphere constructs as well as methods for their prepara- tion and use. The '673 patent also describes microspheres which encapsulate pharmaceutical agents for delivery into the gastrointestinal tract or into the blood.
While the proteinoid microspheres described in the '673 patent are useful for their intended purposes, the physicochemical properties of the proteinoid microspheres, such as light sensitivity, shelf life and the selectivity of their solubility in various portions of the gastrointestinal tract, could be improved. Additionally, there is a need in the art for microspheres that can encapsulate a broader range of active agents such as polar drugs.
The method employed in the '673 patent to prepare proteinoids produces a complex mixture of high molecular weight (MW) (> 1000 daltons) and low MW (<.1000 daltons) peptide-like polymers which are difficult to separate. Moreover, the method produces a small amount of the low MW proteinoids which is the microsphere-forming fraction. Hence, an improved method of preparing of the proteinoids is also desired.
Accordingly, there is a need in the art for im- proved proteinoid carriers as well as improved methods for their preparation.
Objects of the Invention
It is an object of this invention to provide proteinoids which forms proteinoid carriers as a delivery system with enhanced stability towards at least one of photodegradation and decomposition over time.
It is another object of the invention to provide a proteinoid that forms proteinoid carriers with more selec- tive solubility under various conditions such as pH.
It is yet another object of the invention to provide proteinoid carriers encapsulating biologically active agents which are selectively releasable within par- ticular portions of the gastrointestinal tract.
It is a further object of the invention to provide proteinoid carriers which promotes the bioavailability of pharmaceutical agents which otherwise display poor absorp- tion in the gastrointestinal tract.
It is yet a further object of the invention to provide an improved method for producing proteinoid carriers having particular characteristics and for improving yield of the desired proteinoid carriers. It has been found that these objects and other advantages, which will be apparent from this specification, are achieved by the invention described below.
Summary of the Invention The present invention relates to improved protein¬ oid carriers and methods of making and use thereof.
Proteinoids of a MW ranging between about 250 and about 2400 daltons and of defined amino acids are useful in preparing proteinoid carriers with improved stability against photodegradation and/or decomposition. The proteinoids comprise a peptide polymer selected from the group consisting of:
(i) peptide polymers made from at least one first monomer selected from the group consisting of tyrosine and phenylalanme; and from at least one second monomer selected from the group consisting of glutamic acid, pyroglutamic acid, glutamine, and aspartic acid;
(ii) peptide polymers made from at least one ^first monomer selected from the group consisting of tyrosine and phenylalanme; and from at least one second monomer selected from the group consisting of glutamic acid, pyroglutamic acid, glutamine, and aspartic acid; and from at least one third monomer selected from the group consisting of lysine, arginine and ornithine, the proteinoid being a microsphere- and/or microcapsule-forming proteinoid and being soluble within a selected pH range.
The proteinoid molecules of the invention contain between about 2 and about 20 amino acid residues, preferably between about 2 and about 8 amino acid residues, and has a molecular weight which ranges between about 250 and about 2400 daltons, preferably between about 250 and about 600, and most preferably between about 250 and 400 daltons. The proteinoid carriers are useful as delivery systems to releasably encapsulate and carry a broad range of cargoes including pharmaceutical agents, dye reagents and cosmetic ingredients. In particular, the proteinoid carri¬ ers are useful as oral delivery systems of sensitive pharma- ceutical agents, which normally would not be administrable via the oral route, for selective release at targeted re¬ gions of the gastrointestinal tract. Also contemplated by the present invention are dosage unit forms that include these compositions.
Description of the Drawings
Figure 1 illustrates the molecular weight distri¬ bution as a function of monomer concentration of poly (Asp.Bz-co-Phe) polymer prepared by the NCA method as de- scribed in Example 3.
Figure 2 illustrates the molecular weight distri¬ bution of a function of monomer concentration of poly (Asp.Bz) polymer prepared by the DPPA method as described in Example 5. Figure 3 illustrates the effect of reaction time duration on yields of poly (Asp.Bz) polymer prepared by the DPPA method as described in Example 5.
Figure 4 illustrates the effect of temperature of the molecular weight of poly (Asp.Bz) polymer prepared by the DPPA method as described in Example 5.
Figure 5 illustrates the effect of changing the molar ratios of [DPPA] / [M] on the molecular weight of poly (Asp.Bz) polymer by the DPPA method as described in Example 5. Figure 6 is a photograph of an x-ray film of the western immunoblot analysis, as described in Example 9, of purified murine mAb 9BG5 (2μg, lane 1; lmg, lane 2; and 0.25 μg, lane 3) ; empty proteinoid carrier supernatant after encapsulating process (no mAb) (lane 4) ; empty proteinoid carrier pellet (lane 5) ; proteinoid carrier encapsulated mAb supernatant after encapsulating process (lane 6) ; and pro¬ teinoid carrier encapsulated mAb pellet. Lane MW contained standard molecular weight markers.
Figure 7 is a photograph of an x-ray film of a western immunoblot analysis of samples described in Example 10.
Figures 8 (a-c) illustrate the levels of serum proteins which bound to immobilized reovirus type 3 and VLSH under ELISA conditions as described in Example 11. "Empty spheres" refers to animals orally administered empty pro¬ teinoid carriers (no mAb 9BG5) ; "mAb spheres" refers to animals orally administered mAb 9BG5 encapsulated proteinoid carriers; "IV" refers to animals intravenously administered unencapsulated mAb 9BG5; and "oral" refers to animals orally administered unencapsulated mAb 9BG5.
Figure 9 show mAb binding under conventional ELISA procedures using immobilized reovirus type 3 and VLSH pro- teins with serial dilutions of purified mAb in 0.85 N ci¬ trate-0.5% gum (Figure 9(a)) or phosphate buffered saline (Figure 9 (b) ) as described in Example 11.
Figure 10 illustrates levels of erythropoietin (EPO) detected in rat serum taken from rats administered proteinoid carrier encapsulated EPO (15μg EPO/kg body weight) and encapsulated EPO (15μg EPO/kg body weight) as described in Example 15.
Figure 11 illustrates EPO serum levels in rats that were administered either erythropoietin (50μg/kg) or encapsulated erythropoietin (50μg/kg) directly into the proximal duodenum as described in Example 15. Serum eryth¬ ropoietin levels were determined over time with a erythro¬ poietin enzyme immunoassay kit.
Figure 12 illustrates EPO serum levels in rats who were orally gavaged with either encapsulated or unencapsu¬ lated erythropoietin (lOOμg/kg) or received a subcutaneous injection of either 2μg/kg or lOμg/kg as described in Exam¬ ple 15. Serum erythropoietin levels were determined over time with an erythropoietin enzyme immunoassay kit.
Figure 13 illustrates serum calcium changes after oral administration of salmon calcitonin proteinoid carriers (0.25 mg calcitonin/kg body weight) in cynomolgus monkeys as described in Example 17. The results are expressed as abso¬ lute change in serum calcium from baseline values. The data represents means +/- SEM. ** Serum calcium levels significantly different from baseline values.
Figure 14 illustrates serum calcium changes fol¬ lowing oral administration of salmon calcitonin proteinoid carriers (0.60 mg/kg body weight) in rats as described in Example 18. The results are expressed as absolute change in serum calcium from baseline values. The data represents means +/- SEM. **Serum calcium levels significantly differ- ent compared to the control group at the corresponding time point.
Figure 15 illustrates serum calcium changes after intraduodenal administration of salmon calcitonin or calci¬ tonin proteinoid carriers (3 ug/kg body weight) in rats as described in Example 18. The results are expressed as abso¬ lute change in serum calcium from baseline values. The data represents means +/- SEM. ** Significantly different from the unencapsulated control group at the indicated time points. Figure 16 illustrates clotting times after oral administration of proteinoid carrier encapsulated Factor IX (FIX sph PO) and IV administration of FIX solution (FIX IV) as described in Example 20.
Figure 17 illustrates clotting times after oral administration of proteinoid carrier encapsulated Factor IX (FIX sph PO) and FIX solution (FIX unencap PO) or IV admin¬ istration of FIX solution (FIX IV) as described in Example 21.
Figure 18 illustrates the percentage of intact alpha-interferon (IFN) remaining after incubating IFN and IFN proteinoid carriers in simulated gastric fluid (SGF) .
Figure 19 illustrates the percentage of intact IFN remaining after incubating IFN and IFN proteinoid carriers in 0 . 08N HCl .
Figure 20 illustrates the percentage of intact IFN remaining after incubating IFN and IFN proteinoid carriers in simulated intestinal fluid (SIF) . Figure 21 illustrates the clotting times in rats dosed with heparin or proteinoid/heparin, both in water. The data represents an average of 6 rats. The data repre¬ sents means +/- SEM.
Figure 22 illustrates clotting times in rats dosed ID with USP heparin or heparin proteinoid carriers, both in citric acid. Each time point is an average of 12 rats. The data represents means +/- SEM.
Figure 23 illustrates clotting times in rats dosed orally with heparin-spiked empty proteinoid carriers or heparin proteinoid carriers. Each time point is an average of 12 rats. The data represents means +/- SEM.
Figure 24 illustrates the average titers of rats immunized orally with Ml proteinoid carriers versus unencap¬ sulated Ml. Only responders in each group were averaged. Figure 25 illustrates HA-NA titers of rats immu¬ nized orally with HA-NA microspheres versus unencapsulated HA-NA.
Detailed Description of the Invention All patents and literature references cited in this specification are hereby incorporated by reference in their entirety. In case of inconsistencies, the present description, including the definitions and interpretations, will prevail. The instant invention arose from the discovery that proteinoids of a MW of between about 250 and about 2400 daltons and of defined amino acid composition can be ob¬ tained by modifying known reactions and selecting starting materials. These proteinoids form proteinoid carriers with surprisingly enhanced stability against at least one of photodegradation and decomposition over time. In addition, proteinoid carriers prepared from such proteinoids carry a broader range of pharmaceutical agents, including labile polypeptides such as insulin, alpha-interferon, calcitonin, antigens, e.g. influenza virus Ml-protein, and Factor IX and display a selective releasability within various portions of the gastrointestinal tract, relative to prior art proteinoid microspheres.
The compositions of the subject invention are useful for administering biologically-active agents to any animals such as birds; mammals, such as primates and particularly humans; and insects. The proteinoids of the invention comprise a pep¬ tide polymer selected from the group consisting of:
(i) peptide polymers made from at least one first monomer selected from the group consisting of tyrosine and phenylalanme; and from at least one second monomer selected from the group consisting of glutamic acid, pyroglutamic acid, glutamine, and aspartic acid;
(ii) peptide polymers made from at least one first monomer selected from the group consisting of tyrosine and phenylalanme; at least one second monomer selected from the group consisting of glutamic acid, pyroglutamic acid, gluta¬ mine, and aspartic acid; and from at least one third monomer selected from the group consisting of lysine, arginine and ornithine, the proteinoid being a microsphere- or microcap- sule-forming proteinoid and being soluble within a selected pH range.
The proteinoid molecules of the invention contain between about 2 and about 20 amino acid residues, preferably between about 2 and about 8 amino acid residues, and have a molecular weight which ranges between 250 and about 2400 daltons, preferably between about 250 and about 600, and most preferably between about 250 and 400 daltons.
The term amino acid as used herein includes any carboxylic acid having at least one free amine group includ¬ ing naturally occurring and synthetic amino acids. The preferred amino acids are oc-amino acids, and preferably are naturally occurring oc-amino acids although non-o;-amino acids are useful as well.
The preferred naturally occurring amino acids for use in the present invention as amino acids or components of a peptide are alanine, arginine, asparagine, aspartic acid, citrulline, cysteine, cystine, glutamine, glycine, histi- dine, isoleucine, leucine, lysine, methionine, ornithine, phenylalanme, proline, serine, threonine, tryptophan, tyrosine, valine, hydroxy proline, γ-carboxyglutamate, or O-phosphoserine. The most preferred amino acids are argi¬ nine, leucine, lysine, phenylalanme, tyrosine and valine. The preferred non-naturally occurring amino acids for use in the present invention as amino acids or compo¬ nents of a peptide are β -alanine, phenylglycine, α-aminobutyric acid, γ-amino butyric acid, 4- (4- aminophenyl)butyric acid, α.-amino isobutyric acid, e- aminocaproic acid, 7-aminoheptanoic acid, /3-aspartic acid, aminobenzoic acid, (aminomethyl)benzoic acid, aminophenylacetic acid, aminohippuric acid, γ-glutamic acid, cysteine(ACM) , e-lysine, e-lysine (A-Fmoc) , methionine sulfone, norleucine, norvaline, ornithine, d-ornithine, p- nitrophenylalanine, hydroxy proline, and thioproline. The amino acids useful in the practice of the
subject invention have the formula:
HN (R4) - (R2)n-OH
0 1
R2 has the formula — 3 —C—- wherein R3 is C! to CM alkyl, Cj to Cu alkenyl, phenyl, naphthyl, (Cj to C10 alkyl) - phenyl, (Ci to C10 alkenyl)phenyl, (Cj to C10 alkyl) aphthyl,
(Cj to C10 alkenyl)naphthyl, phenyl (Cj to C10 alkyl), phenyl (Cj to Cjo alkenyl), naphthyl (Cj to C10 alkyl) and naphthyl (Cj to
C10 alkenyl) ; optionally R3 is substituted with Cj to C4 alkyl, Cj to C4 alkenyl, Cj to C4 alkoxy, -OH, -SH and -C02R5 or any combination thereof; R5 is hydrogen, Cj to C4 alkyl or to C4 alkenyl;
R3 is optionally interrupted by oxygen, nitrogen, sulfur or any combination thereof; and
R4 is hydrogen, ^ to C4 alkyl or Cj to C4 alkenyl.
The phenyl or naphthyl groups can be optionally substituted. Suitable but non-limiting examples of substitutents are Cj to C6 alkyl, Cj to C6 alkenyl, alkoxy having from 1 to 6 carbon atoms, hydroxy, thio, or C02R6 wherein R6 is hydrogen, Cλ to C6 alkyl, Cx to C6 alkenyl. Proteinoid carriers prepared from the proteinoid molecules, in accordance with the present invention, display a selective solubility at specific acidic or basic pH rang¬ es, depending on the choice and amount of the second and third monomers in the proteinoid. Proteinoid carriers which are selectively soluble under alkaline pH environments, such as those found in the distal portion of the intestine, are prepared from base- soluble proteinoids. These proteinoids contain, as starting monomers in the reaction mixture, at least one second mono- mer selected from the group consisting of glutamic acid, glutamine, pyroglutamic acid, and aspartic acid. At a pH ranging between about 7.2 and about 11.0, the base-soluble proteinoid exists largely as the anion and is soluble. At a pH below about 7.0, the proteinoid is largely protonated and insoluble in water.
Similarly, proteinoid carriers which are selec¬ tively soluble under acidic pH environments, such as the stomach, are prepared from acid-soluble proteinoids. In this case, the proteinoid contain, as starting monomers in the proteinoid reaction mixture, at least one second monomer selected from the group consisting of glutamic acid, pyroglutamic acid, glutamine, and aspartic acid and at least one third monomer selected from the group consisting of lysine, arginine, and ornithine. At a pH ranging between about 1 and about 7, the base-soluble proteinoid exists largely as the cation and is soluble. At a pH above about 7.2, the proteinoid is largely unprotonated and insoluble in water.
The pH and the solubility characteristics of the acid-soluble proteinoid depends largely, but not exclusive¬ ly, upon the pH and solubility of the last amino acid added during the synthesis of the proteinoid. For instance, the incorporation of a basic amino acid, e.g., a third monomer, selected from the group consisting of lysine, arginine and ornithine in the acid-soluble proteinoid will result in the elevation of the pi (pH at the isoelectric point) of the proteinoid. The proteinoids of the present invention are preparable by a thermal condensation reaction by heating mixtures of the appropriate amino acids under conditions described in the '673 patent. In contrast with the '673 patent procedures which use as many as eighteen amino acids, mixtures of two to five specific amino acids with at least one selected from each of the aforementioned groups yield proteinoids which form proteinoid carriers with selective solubility at particular pH ranges and at high yields.
In carrying out the thermal condensation reaction, it has now been discovered that inclusion of tetramethylene sulfone, an inert, high boiling, polar solvent, maximizes the yield (> 80%) of low MW proteinoids. Omission of sol¬ vent does not produce high yields of low MW proteinoids. Presumably this is due to the poor solubility of the amino acid monomers in these solvents and/or unavoidable side reactions between the monomers and the solvent under the reaction conditions.
In general, individual amino acids are added to a reaction flask containing tetramethylene sulfone (sulfolane) which has been heated to a temperature ranging between about 130°C and about 200°C, preferably about 175°C to 195°C, under an inert atmosphere of argon or nitrogen gas. After each addition, the solution is stirred for a period of time ranging between about 10 minutes and about 5 hours, depend- ing on the amino acid type and the order of addition.
Upon heating mixtures of amino acids to tempera¬ tures of about 190°C as described above, a reaction takes place and water, ammonia and carbon dioxide are produced as side-products. Water is removed from the reaction as formed and the reaction is terminated when water formation ceases. Thereafter, the proteinoid are precipitated out of the reaction solution by quenching with excess water, under vigorous stirring. After stirring for a period of about 1 hour, the proteinoids are collected by filtration, washed with water and dried under vacuum.
Chemical condensation methods which utilize derivatized amino acids are also useful for making the proteinoids of the present invention as they permit greater control of molecular weight. Such reactions are generally conducted at lower reaction temperature and with initiators. In particular, low MW proteinoids produced by the alpha- amino acid N-carboxyanhydride (NCA) method and the diphenylphosphoryl azide (DPPA) method (N. Nishi et al.
(1991) Makromol. Chem., Vol.192, pages 1789-1798) were found to form proteinoid carriers having selected solubility within a particular pH range.
The NCA method involves the preparation of N- carboxyanhydrides of alpha-amino acid esters and their subsequent polymerization, using low MW amines as initia¬ tors. It has been discovered that non-NCA derived amino esters, e.g., ce-methyl tyrosine ester, are effective initia¬ tors which are stable and soluble in many organic solvents such as tetrahydrofuran (THF) . The use of amino acids as initiators, presumably due to their poor solubility in organic solvents and their low stability, are not known. The NCA reaction produces a high yield of proteinoids with high purity. The DPPA method involves the direct condensation of benzyl esters of alpha-amino acids in the presence of DPPA and a low MW amine, followed by removal of the protec¬ tive benzyl groups, contained in the proteinoid product, by alkaline hydrolysis. If catalytic hydrogenation is used in place of alkaline hydrolysis, low MW proteinoids of unex¬ pected high purities and yields are obtained.
Proteinoids prepared by any of the above methods can be used immediately to microencapsulate an active phar¬ macological agent or the proteinoid can be concentrated or dried by conventional means and stored for future use.
The proteinoids of the invention are purified as follows: crude proteinoids are slurried with water at room temperature, e.g. 25°C. While at this temperature, the pH of the slurry is adjusted to about pH 8 using an aqueous alkaline solution, e.g. 40% sodium hydroxide and 10% sodium bicarbonate solutions for an acid-soluble proteinoid. For a base-soluble proteinoid, the slurry is adjusted to an acidic pH with an aqueous acidic solution, e.g. 10% acetic acid solution. The mixture is then filtered and the filter cake washed with a volume of water. The washes and filtrate are then combined and evaporated to dryness in vacuo to afford proteinoids. If necessary, this process can be repeated until proteinoids of a desired purity level are obtained.
If desired, the proteinoid may be further purified by fractionating on a column containing solid supports which include silica gel or alumina, using methanol or propanol as mobile phase; ion exchange resin using water as the mobile phase; reverse phase column supports using trifluoroacetic acid/acetonitrile mixtures as mobile phase. The proteinoids may also be purified by extraction with a lower alcohol such as propanol or butanol to remove low molecular weight con- taminants.
Proteinoid carriers are made from purified proteinoids as follows: proteinoids are dissolved in deion¬ ized water at a concentration ranging between about 75 and about 200 mg/ml, preferably about 100 mg/ml, at a tempera- ture between about 25°C and about 60°C, preferably about
40°C. Particulates remaining in the solution may be filtered out by conventional means such as gravity filtration over filter paper.
Thereafter, the proteinoid solution, maintained at a temperature of about 40°C, is mixed with an aqueous acid solution (also at about 40°C) having an acid concentration ranging between about 1 N and about 2 N, preferably about 1.7 N. The resulting mixture is further incubated at 40°C for a period of time effective for microsphere and microcap- sule formation as observed by light microscopy. In practic¬ ing this invention, the preferred order of addition is adding the proteinoid solution to the aqueous acid solution. Suitable acids include any acid which does not (a) adversely effect the proteinoid, e.g., chemical decomposi¬ tion; (b) interfere with microsphere or microcapsule forma¬ tion; (c) interfere with microsphere or microcapsule encap¬ sulation of cargo; and (d) adversely interact with the cargo. Preferred acids for use in this invention include acetic acid, citric acid, hydrochloric acid, phosphoric acid, malic acid and maleic acid.
In practicing the invention, a proteinoid carrier stabilizing additives are preferably incorporated into the aqueous acid solution or into the proteinoid solution, prior to the microsphere or microcapsule formation process. The presence of such additives promotes the stability and dispersibility of the proteinoid carriers in solution.
The additives may be employed at a concentration ranging between about 0.1 and 5 % (W/V) , preferably about
0.5 % (W/V). Suitable, but non-limiting, examples of stabi¬ lizing additives include gum acacia, gelatin, polyethylene glycol, and polylysine.
Thereafter, the proteinoid carriers may be used immediately or may be stored at 4°C or lyophilized and stored under desiccant at room temperature or below.
Under the above conditions, the carrier forms hollow or solid matrix type microspheres wherein the cargo is distributed in a carrier matrix or capsule type microspheres encapsulating liquid or solid cargo. If the carrier microspheres are formed in the presence of a soluble material, e . g. , a pharmaceutical agent in the aforementioned aqueous acid solution, this material will be incorporated in the microspheres. In this way, one can incorporate pharma- cologically active materials such as peptides, proteins, and polysaccharides as well as charged organic molecules, e . g. , antimicrobial agents, which normally have poor bioavailability by the oral route. The amount of pharmaceu¬ tical agent which may be incorporated in the microsphere is dependent on a number of factors which include the concen¬ tration of agent in the microsphere forming solution, as well as the affinity of the cargo for the carrier.
Under the aforementioned conditions, the protein- oid molecules form spherical proteinoid carriers comprising proteinoid microcapsules and proteinoid microspheres of less than 10 micron diameter. As defined herein, a "microsphere" is spherical homogeneous mesh work structure having no discrete inner chamber. A "microcapsule" refers to a spher¬ ical structure having a proteinoid wall which forms a hollow or chamber. If the proteinoid carriers are formed in the presence of a soluble material, e.g., a pharmaceutical agent in the aforementioned aqueous acid solution, this material is believed to be encapsulated within the hollows of the microcapsules and confined within the proteinoid wall de¬ fined by the spherical structure or entrapped within the matrix of proteinoid molecules in the microsphere structure. In this way, one can encapsulate or entrap pharmacologically active materials such as peptides, proteins, and polysaccharides as well as charged organic molecules, e.g., quinolones or antimicrobial agents, having poor bioavailability by the oral route. The amount of pharmaceu¬ tical agent which may be encapsulated or entrapped by the proteinoid carrier is dependent on a number of factors which include the concentration of agent in the encapsulating solution.
The proteinoid carriers of the invention are pharmacologically harmless and do not alter the physiologi- cal and biological properties of the active agent. Further¬ more, the encapsulation process does not alter the pharmaco¬ logical properties of the active agent. While any suitable pharmacological agent can be encapsulated within proteinoid carriers, it is particularly valuable for delivering agents which otherwise would be destroyed or rendered less effec¬ tive by conditions encountered in the animal body before it reaches its target zone and which are poorly absorbed in the gastrointestinal tract.
The proteinoid carriers of the invention are particularly useful for the oral administration of certain pharmacological agents, e.g., small peptide hormones, which, by themselves, pass slowly or not at all through the gastro¬ intestinal mucosa and/or are susceptible to chemical cleav- age by acids and enzymes in the gastrointestinal tract. Non-limiting examples of such agents include human or bovine growth hormone, interferon and interleukin-II, calcitonin, atrial naturetic factor, antigens, monoclonal antibodies, and Factor IX, a vitamin K-dependent blood coagulation proenzyme.
Biologically-active agents suitable for use with carriers disclosed herein include, but are not limited to, peptides, and particularly small peptide hormones, which by themselves do not pass or only pass slowly through the gastro-intestinal mucosa and/or. are susceptible to chemical cleavage by acids and enzymes in the gastro-intestinal tract; polysaccharides and particularly mixtures of muco- polysaccharides; carbohydrates; lipids; or any combination thereof. Examples include, but are not limited to, human growth hormone; bovine growth hormone; growth hormone re¬ leasing hormone; interferons; interleukin-I; insulin; hepa¬ rin, and particularly low molecular weight heparin; calcito¬ nin; erythropoietin; atrial naturetic factor; antigens; monoclonal antibodies; somatostatm; adrenocorticotropm; gonadotropin releasing hormone; oxytocin; vasopressin; cromolyn sodium (sodium or disodium cromoglycate) ; vancomy- cin; desferrioxamine (DFO) ; or any combination thereof.
Additionally the carriers of the present invention can be used to deliver other active agents such as pesti¬ cides and the like.
The amount of active agent in the composition typically is a pharmacologically or biologically effective amount. However, the amount can be less than a pharmacolog- ically or biologically effective amount when the composition is used in a dosage unit form, such as a capsule, a tablet or a liquid, because the dosage unit form may contain a multiplicity of carrier/biologically-active agent composi¬ tions or may contain a divided pharmacologically or biologi- cally effective amount. The total effective amounts will be administered by cumulative units containing in total pharma¬ cologically or biologically active amounts of biologically- active agent. Dosage unit forms can also include any of excipients; diluents; disintegrants; lubricants; plasticizers; colorants; and dosing vehicles, including, but not limited to water, 1,2-propane diol, ethanol, olive oil, or any combination thereof.
The choice of a particular proteinoid for use in encapsulating or entrapping a pharmacological agent depends on a number of factors which include:
(1) the acidity or basicity of the agent; (2) the targeted area for release in the gastro¬ intestinal tract;
(3) the solubility of the drug at certain pH ranges;
(4) efficiency of encapsulation; (5) interaction of drug with proteinoid.
For example, proteinoids made from glutamic acid, aspartic acid, tyrosine, and phenylalanme are especially suitable for encapsulating polysaccharides like heparin.
In addition to selective pH solubility, the parti- cle size of the proteinoid carrier plays an important role in determining release of the active agent in the targeted area of the gastrointestinal tract. Proteinoid carriers having diameters between about <. 0.1 microns and about 10 microns, preferably between about 5.0 microns and about 0.1 microns, and containing encapsulated or entrapped active agents are sufficiently small to effectively release the active agent at the targeted area within the gastrointesti¬ nal tract. Large proteinoid carriers (>10 microns) tend to be less effective as oral delivery systems. The size of the proteinoid carriers formed by con¬ tacting proteinoids with water or aqueous solution contain¬ ing active agents can be controlled by manipulating a vari¬ ety of physical or chemical parameters, such as the pH, osmolarity or salt content of the encapsulating solution, and the choice of acid used in the encapsulating process. By tailoring both the solubility characteristics of a proteinoid and the particle size of the proteinoid carriers, active agent bearing proteinoid carriers can be produced from base-soluble proteinoids which are stable in the highly acidic stomach (normal pH of from about 2 to about 6) , but which dissolve in the distal portion of the intestines. Such systems are suitable for oral administra- tion of peptide hormones, e.g., insulin, and polysac- charides, e.g., heparin, which otherwise would be quickly destroyed in the GI tract. They also are suitable for protecting the stomach from gastric irritants, such as aspirin. When such aspirin-containing proteinoid carriers are orally administered, they pass through the gastrointes¬ tinal mucosa and release the aspirin far more rapidly than conventional enterically coated aspirin, which first must traverse the stomach and then must enter the bloodstream from the intestine after the enteric coating has dissolved.
It also is possible to produce systems from acid- soluble proteinoids which are stable under weakly basic conditions (pH of about 8) , but which release active agent under acidic conditions (pH of about 2 to 5) . Such systems are suitable for the intravenous administration of pharmaco¬ logical agents such as calcium regulators and redox carrier systems for dopamine or gamma-aminobutyric acid.
The proteinoid carriers of the invention may be orally administered alone as solids in the form of tablets, pellets, capsules, and granulates suitable for suspension in liquids such as edible oils. Similarly, the proteinoid carriers can be formulated into an orally administrable composition containing one or more physiologically compati¬ ble carriers or excipients. These compositions may contain conventional ingredients such as gelatin, polyvinylpyrrol¬ idone and fillers such as starch and methyl cellulose.
The proteinoid carriers of the invention may also be administered by injection.
The following examples are illustrative of the invention but are not intended to limit the scope of the invention.
Example 1: Preparation of a Base-soluble Proteinoid by a Thermal condensation Reaction
750 ml of tetramethylene sulfone was heated to
190°C in an inert nitrogen atmosphere in a 4 liter flask with stirring. 294 g of glutamic acid was added and the mixture was heated for one-half hour. 266 g of aspartic acid was added and the mixture heated as rapidly as possible to 190°C and held there for 15 minutes. 362 g of tyrosine was added and the mixture heated at 190°C for 3 hours. 330 g of phenylalanme was added and the mixture heated at 190°C for 1.5 hours. The hot melt was then poured into 5 liters of water with vigorous stirring. After stirring for about 1 hour, the mixture was filtered and the filtrate discarded. The cake was reslurried in 5 liters of water, filtered and the cake was again reslurried in 5 liters of water. The pH of the slurry (at 25°C) was adjusted to 8 using 40% sodium hydroxide solution. The mixture was filtered and the cake washed with a small amount of water. The washes and fil¬ trate are combined and evaporated to dryness in vacuo to give Glu/Asp/Tyr/Phe proteinoid. Appendices A, B, and C describe examples of other proteinoids prepared by the thermocondensation method.
Example 2: Preparation of an Acid-soluble Proteinoid by a Thermal Condensation Reaction 750 ml of tetramethylene sulfone is heated to
190°C in an inert nitrogen atmosphere in a 4 liter flask with stirring. 294 g of glutamic acid is added and the mixture is heated for one-half hour. 362 g of tyrosine is added and the mixture is heated at 190°C for 3 hours. 330 g of phenylalanme is added and the mixture is heated at 190°C for 1.5 hours. 266 g of arginine is added and the mixture is heated for an additional 1.5 hours. The hot melt is then poured into 5 liters of water with vigorous stirring. After stirring for about 1 hour, the mixture is filtered and the filtrate is discarded. The cake is reslurried in 5 liters of water, filtered and the cake is again reslurried in 5 liters of water. The pH of the slurry (at 25°C) was adjust¬ ed to 5 using 10% acetic acid solution. The mixture is filtered and the cake is washed with a small amount of water. The washes and filtrate are combined and evaporated to dryness m vacuo to give proteinoid.
Appendices A, B, and C describe examples of other proteinoids prepared by the thermocondensation method.
Example 3: Preparation of Proteinoids by the NCA Method Using Amine Initiator
This example illustrates the NCA method for pre- paring copolypeptides consisting of Asp.Bz, Glu.Bz, Phe, and Tyr components. The NCA monomers of these amino acids were prepared according to the reported method.
The reactions were carried out in tetrahydrofuran (THF) or in dichloromethane using benzylamine (BzNH2) or 4- methylbenzyl amine (MeBzNH2) as initiator at room temperature ( [M] = 10%) . The characterization of the resulting copoly¬ mers was performed by ~E NMR and GPC. The results obtained are listed in Table 1.
As shown in Table 1, proteinoids having Asp and/or Glu as the second monomers and Phe and/or Tyr as the first monomers were obtained in high yield from the polymerization initiated with BzNH2 at the ratio of [M] / [I] = 5 (No. 2-1 to 2-7) .
The GPC curve (Figure 1) for poly(Asp.Bz-co-Phe) , from which a polydispersity of 1.91 was determined. Similar molecular weight distributions were observed for other copolymers.
Polydispersity is defined herein as the molecular weight distribution of a sample. The distribution is as- signed a numerical value derived from the molecular weight (MW) divided by the molecular number (Mn) . The polydispersity value for a homopolymer is 1 because the molecular weight is equal to the molecular number. Any polymer with a polydispersity value of 1 is considered to have a very narrow distribution. A polymer with polydispersity value of 1.6 to 1.7 is considered to have medium distribution. A polymer with a polydispersity value of 2.0-2.1 is considered to have a broad distribution. The homopolymerization of NCA of Asp.Bz and the copolymerizations of NCAs of Asp.Bz, Glu.Bz, Phe, and Tyr were also carried out using MeBzNH2 as initiator (No. 2-11, 2-15, and 2-16) . Similar results were obtained for reac¬ tions initiated by BzNH2.
TABLE 1
COPOLYMERIZATION OF NCAs INITIATED WITH AMINES STORED AT ROOM TEMPERATURE FOR 4 DAYS
Example 4: Preparation of Proteinoids by the NCA Method Using α-Methγl Tyrosine Ester as Initiator
This example illustrates the method of conducting NCA polymerizations, using α-methyl tyrosine ester (Tyr. e) as the initiator. The reaction conditions are essentially the same as described in Example 4 except tetrahydrofuran (THF) solvent was used. The results are listed in Table 2. TABLE 2
PROTEINOID SYNTHESIS BY NCA INITIATED WITH AMINO ACIDS STORED AT ROOM TEMPERATURE FOR 4 DAYS
It was found that the initiation by Tyr.Me is very fast (No. 2-17 to 2-20) and all the NCA has been converted after 2 hours. From GPC data, it was observed that the molecu¬ lar weight of the polymer increased with increasing ratio of [M] / [Tyr.Me] and the polydispersity is quite narrow. The existence of a Tyr.Me residue in the polymers was confirmed by 'H NMR spectra. In conclusion, Tyr.Me is a novel and effective initiator for the polymerization of amino acid NCA's.
Sample No.2-13 represents a polymerization initiated with -alanine and terminated with succinic anhydride. As β- alanine is insoluble in most organic solvents, the reaction was carried out in refluxing THF. As a result, the polydispersity of the polymer obtained was broader than that of the polymers initiated by Tyr.Me. Example 5: Preparation of Proteinoids bv the DPPA Method (#1)
This is an example of a direct polycondensation of Asp.Bz in the presence of DPPA and triethylamine (TEA) as a base under various polymerization conditions ( (a) , (b) , (c) , and (d) ) . The molecular weight of the polymers, as well as polydispersity, was evaluated in each case by GPC. The poly¬ mers were characterized by IR and NMR spectroscopy.
Asp.Bz was prepared by the esterification of L- aspartic acid as follows: L-aspartic acid (26.6 g, 0.2 mole) was suspended in 300 ml of freshly distilled benzyl alcohol in a 500 ml round bottom flask, followed by addition of 45 ml of concentrated hydrochloric acid (12N) . The mixture was heated up to 60°C under vigorous stirring for 30 minutes. Thereafter, the reaction solution cooled to room temperature. Triethyl amine (about 56 ml) was added to neutralize (to a pH of about 7) the solution. The crude product was collected by filtration, washed with ethanol and acetone, dried in vacuo, and crystal¬ lized twice from hot water. 18 g of product was obtained (% yield = 44%). M.pt = 217-C.
Commercial DPPA was used without further purifica¬ tion. TEA was distilled before use. Solvents for polymer¬ ization were purified by conventional methods. The direct polycondensation of Asp.Bz was carried out by stirring a dimethyl formamide (DMF) solution of the monomer in the pres¬ ence of DPPA and TEA. The mixture was stirred for 1 h at 0- 10°C followed by stirring at room temperature for two days. The resulting polymer was precipitated in a large amount of water, collected by filtration, and then dried in vacuo.
a. Effect of Monomer Concentration
Listed in Table 3 are the results for the polymeriza¬ tion of Asp.Bz in DMF at room temperature for two days. Poly(Asp.Bz)s were obtained from these direct polycondensations in high yield.
The molecular weight of the polymers was found to be dependent on the concentration of the monomer [M] . Low molecu¬ lar weight polymers with broad distribution were obtained from a low [M] (Figure 2, curve A) . On the other hand, when [M] was greater than 0.2 g/mL, a polymer with a bimodal molecular weight distribution was obtained (Figure 2, curve B) . The lower molecular weight oligomers (-1000) may be due to an intramolecular termination between the terminal amino and the β -carboxylic groups. After several reprecipitations from THF/methanol, a polymer with a higher molecular weight (M,. = 22,000) and narrow polydispersity (M„/^ = 1.68) was success¬ fully isolated from the polymer mixture prepared at [M] = lg/mL. The separation was also performed using GPC column with Bio-Beads.
TABLE 3
EFFECT OF THE MONOMER CONCENTRATION ON POLYMERIZATION
OF Asp.Bz BY DPPA IN DMF AT ROOM TEMPERATURE:
[DPPA]/[M] = 1.3; [TEA] [M] = 2.3
a) The polymer was collected by centrifugation after polymerization for 2 days; b) The polymer was collected by filtration after polymerization for 2.5 days. c) The values in parentheses are molar percentages.
b. Effect of Reaction Time and Temperature
The yield of the resulting polymer increased with the reaction time: 75% conversion in 5 h and 95% in 4 days (Figure 3, curve A) . The molecular weight of the resulting polymer also increased with time in the initial phase (up to 4 h) and then became almost constant (Figure 4) . The polymerization decreased with increasing temperature (Figure 3, curve B) . Polymers obtained at 60 and 80°C were of yellow color and insoluble in THF but soluble in DMF and DMSO. This may be due to the formation of an imide ring which has been reported to occur during thermal polycondensations of aspartic acid.
c. Effect of Molar Ratios [DPPA1 /CM] and [TEA] /[Ml
The dependence of the yield and the molecular weight of the polymer on the molar ratios of [DPPA] / [M] , as well as [TEA] / [M] , was investigated (Table 4). The highest yield was obtained at a [DPPA] / [M] of 1.3 and a [TEA] / [M] of 2.3 (Figure 5) . These observations are in agreement with the results reported by Nishi et al. Higher molecular weight products were obtained in the range of [DPPA] / [M] = 1.3-2.0 and [TEA] / [M] = 2.0-3.0, respectively.
TABLE 4
EFFECT OF THE MOLAR RATIOS OF DPPA AND TEA ON POLYMERIZATION OF Asp.Bz IN DMF AT ROOM TEMPERATURE: [M] = 0.50 g/ml
a) The value in parentheses are molar percentage.
d. Effect of Solvent A comparison of the polymerizations in different solvents is shown in Table 5. It can be seen from this table that the yield and the molecular weight of the polymer are influenced by the solvents used. Higher yields were obtained in DMF while higher molecular weights were obtained in THF and in bulk. On the other hand, the polymerization in dioxane gave a lower molecular weight product, and therefore is preferred.
TABLE 5
EFFECT OF THE SOLVENTS ON POLYMERIZATION OF
Asp . Bz AT ROOM TEMPERATURE FOR 2 DAYS
[M] / [DPPA] = 1.3 , [M] / [TEA] = 2 .3 [M] = 0 . 50 g/ml
a) Bulk polymerization.
b) The value in parentheses are molar percentage.
Example 6: Preparation of Proteinoids bv the DPPA Method (#2)
Copolymerizations of Asp.Bz with other amino acid monomers such as γ-benzyl glutamate (Glu.Bz) , jδ-alanine (Ala) , Phenylalanme (Phe) , and 0-benzyl tyrosine (Tyr.OBz) in the presence of DPPA were carried out using the same procedure as that for the homopolymerization of Asp.Bz (Example 5) . Random copoly(amino acids) were obtained in high yield (> 77%) as shown in Table 6. This indicates that the copolymerization of amino acids using DPPA is a useful approach to copolypeptide synthesis. Bimodal molecular weight distributions were also observed in these cases similarly to the homopolymerization of Asp.Bz. TABLE 6
COPOLYMERIZATION OF α-AMINO ACIDS IN THE PRESENCE OF DPPA AS CONDENSING AGENT IN DMF AT ROOM TEMPERATURE FOR 2
DAYS
Example 7: Reductive Debenzylation of Proteinoids Produced bv the DPPA Method
The example illustrates a preferred method for the removal of benzyl protective groups in poly(Asp.Bz) and poly(Glu.Bz) by catalytic hydrogenation.
The hydrogenation of the polymers was carried out according to the following procedure: To a solution of the polymer in THF/methanol (1:1, v/v), Pd on active carbon (10%) was added in the amount of 1/10 of the polymer weight. After the replacement of air by nitrogen, hydrogen gas was introduced into the system and maintained with a balloon. The reaction mixture was stirred at room temperature overnight. After removing the catalyst by filtration and concentrating the solution, the mixture was poured into a large amount of petro¬ leum ether to precipitate the polymer. The polymer obtained was then dried m vacuo.
The completion of the hydrogenation was confirmed by *H NMR of the polymer. In most cases, useful water-soluble polymers were produced. The hydrogenation is an effective and clean procedure for benzyl group removal. Example 8: Preparation of Empty Proteinoid carriers with Glu, Asp. Tyr, Phe Proteinoid
This Example illustrates a method for the preparation and cleaning of empty proteinoid carriers.
PROCEDURE
1. Reagents: a. Proteinoid powder prepared as described in Example
1 b. Anhydrous citric acid (USP) c. Gum acacia NF d. Deionized water e. Glacial acetic acid
2. Equipment: a. Ph meter b. Water bath, 40°C
3. Preparation of Solutions: a. Proteinoid solution - Dissolve lOOmg proteinoid in lml deionized water (or multiples thereof) . Filter through a Whatman #1 filter paper (if necessary) and keep at 40°C in a water bath. This is solution A.
b. 1.7 N citric acid with 0.5% acacia - Dissolve 5g of acacia and 109g of citric acid in 1 liter deionized water. Incubate at 40°C. This is solution B.
4. Preparation of Proteinoid carriers: a. Add all of solution A to solution B rapidly in one step while swirling solution B by hand, in a 40°C water bath. Example 9: Preparation of Murine IgG Monoclonal Antibody- containinα Proteinoid Carrier
This experiment describes encapsulation of anti- reovirus monoclonal antibody (mAb) 9BG5, an mAb directed against the sigma-1 gene product (Hemaglutinin, HA3) of the
Reovirus Type 3. HA3 binds to the cell surface receptor for
Reovirus type 3, and mAb 9GB5 interferes with viral binding to the receptor.
Mouse IgG monoclonal antibody 9BG5 was prepared and purified as described W.V. Williams et al. (1991) J. Biol. Chem.. Vol. 266(8), pages 5182-5190, as well as references cited therein, using a purified Reovirus type 3 preparation (W.V. Williams et al. (1988) Proc. Natl. Acad. Sci. U.S.A. Vol. 85, pages 6488-6492) . The purified 9BG5 used in this Example had a protein concentration of 1.5 mg/ml in phosphate buffered saline (pH 7.2) .
Proteinoid carriers encapsulating mAb 9BG5 were prepared having final concentrations of Glu/Asp/Tyr/Phe proteinoid (1:1:1:1 mole ratio of Glu, Asp,Tyr, and Phe in the reaction mixture) 50 mg/ml, mAb 0.7 mg/ml and gum arabic 0.5% in 0.85 N citric acid. Empty proteinoid carriers were prepared to contain the same final concentrations, except mAb was omitted. Aliquots (0.5 ml), in duplicate, of both mAb and empty proteinoid carriers preparations were centrifuged at 5000 RPM. Pellets and supernatants were frozen prior to analysis by Western blotting to determine antibody encapsulation efficien¬ cy.
Figure 6 is an x-ray film of a western blot analysis of purified mAb 9BG5, empty proteinoid carriers (no mAb added) , and proteinoid carriers containing 9BG5. The analysis was done by immunoblotting with anti-mouse IgG which specifically reacted with mAb 9BG5. The lanes correspond to the following:
Lane Sample
1 2 μg 9BG5 mAb 2 1 g 9BG5
3 0.25 g 9BG5
MW molecular weight markers
4 Emptyproteinoid carrier supernatant after encapsula¬ tion 5 Empty proteinoid carrier pellet
6 mAb containing supernatant after encapsulation 7 mAb containing protein carrier pellet
The data indicates that the 9BG5 proteinoid carriers contained about 40% of the mAb in the pellet and the remaining 60% did not incorporate in the proteinoid carriers and was left in the supernatant. The empty proteinoid carriers did not contain antibody in the supernatant or the pellet as was expected.
The relative mobility (molecular weight) of the pure mAb is slightly different than the mAb in the proteinoid carriers. This is most likely due to different salt concentra¬ tions in the samples, because the encapsulation process employed 0.8 M salt solution.
Example 10: Effect of Additives on Stability of Proteinoid
Carriers with Encapsulated Murine mAb 9BG5
Various proteinoid carrier formulations were screened, with or without additives, to determine optimal carrier-forming conditions and concentrations of mAb required for carrier formation.
The mAb 9BG5 preparations used to prepare the encap¬ sulated proteinoid carriers had a protein concentration of approximately 2 mg/ml in phosphate buffered saline. Final proteinoid concentration was 50 mg/ml and 5%
(w/w) gum acacia ("gum") or gelatin ("gel"). All proteinoid carriers were prepared in 0.85 N citric acid. Empty carriers were included for use as controls, and they were prepared in the same manner with the omission of mAb. Duplicate (0.5 ml) aliquots of proteinoid carrier suspension were centrifuged at 5000 RPM. Pellets and supernatants were frozen in dry ice prior to analysis.
Table 7 lists samples that were prepared. Numbers in parenthesis indicate amount of mAb added. TABLE 7
In order to test resistance to freeze and thawing on the integrity of the proteinoid carriers containing mAb, one of each pair of duplicate pellets were washed by gentle resuspension in 0.25 ml of 0.85 N citric acid. The pellets were then analyzed next to the unwashed pellets to test whether any mAb was lost in the washing. The samples were analyzed by conventional Western blotting as described in Example 9. Pellets were dissolved in sodium dodecyl sulfate with 0.05 N NaOH and analyzed under reducing conditions (breaks up the mAb into 50 kDa and 25 kDa bands) . Aliquots (50μl) of supernatants were analyzed under non-reducing conditions (expected intact 150 kDa mAb) . This was done to determine differentially whether the mAb left behind is denatured or intact.
As can be seen from the X-ray film from the Western Blots (Figure 7) , pellets of samples 9 and 10, and 11 and 12 contain between 5 and 10 μg of mAb. The washed samples did not lose any significant amount of mAb, suggesting that the prote¬ inoid carriers remained intact after freeze-thawing.
The supernatants of samples 9 and 11 had no signifi¬ cant amount of mAb, indicating that unincorporated material was lost during preparation.
Sample 17 had some mAb encapsulated which was lost after washing (see number 18) . This sphere preparation was not resistant to freeze-thawing. Additionally, a band at a MW of 150 kDa for sample 17 supernatants indicates that a significant amount of mAb is left behind after proteinoid carrier forma¬ tion. Based on these results, it appears that the mAb remains intact and therefore the encapsulating procedure does not degrade it. The empty proteinoid carrier controls did not produce any bands, as expected because they have no mAb.
Example 11: Efficacy of Encapsulated Murine
IgG Monoclonal Antibody
In this Experiment, a mAb 9BG5 proteinoid carrier preparation and unencapsulated mAb 9BG5 were evaluated in rats.
The mAb 9BG5 (1 mg/ml), prepared as described in Example 9, was encapsulated in Glu/Asp/Tyr/Phe proteinoid (1:1:1:1 mole ratio of Glu, Asp,Tyr, and Phe in the reaction mixture) protein carrier formulation with gum arabic. The mAb proteinoid carriers suspension contained 0.25 mg/ml mAb and 50 mg/ml proteinoid in 0.85 N citric acid-0.5% gum. Empty proteinoid carriers were prepared similarly, but did not contain mAb. Since 30% of the mAb was found to be encapsulated, the mAb proteinoid carriers were estimated to contain 0.075 mg/ml mAb and this value was used to determine dosages. The mAb protein¬ oid carriers were examined microscopically and appear to be a fairly homogeneous preparation.
For animal dosing, appropriate aliquots of proteinoid carriers were centrifuged at 5000 RPM for 15 minutes, and pellets were resuspended in 1.0 ml of 0.85 N citric acid-0.5% gum. A purified mAb solution (0.95 mg/ml mAb in 0.85 N citric acid-0.5% gum) was used for oral gavage. This solution was prewarmed to 40°C prior to administration. For IV adminis¬ tration, a purified mAb solution (1 mg/ml mAb in phosphate buffer saline) was used. The amounts and administration routes employed in the experiment are as follows: l. Empty proteinoid carriers (no mAb) : 1 ml aliquot contain¬ ing 50 mg empty proteinoid carriers by oral gavage (rats # 2312 and 2313) .
2. mAb 9BG5 proteinoid carriers: 3.7 mg mAb/ kg body weight of rat by oral gavage (rat # 2287, 2288, 2290, and 2291) .
3. unencapsulated mAb 9GB5: 0.73 mg/ kg body weight of rat by intravenous administration (rats #2292, 2293, and
2311) .
4. unencapsulated mAb 9BG5: 3.7 mg/ kg body weight of rat by oral gavage (rats #2314 and 2315) .
Baseline blood samples (1 ml aliquots) were withdrawn from each rat just prior to dosing ("0" time). After dosing, blood samples were drawn at 1 h, 6 h and 24 h. The blood samples were processed immediately and sera were stored frozen at -20°C.
Thawed serum taken from the experimental animals were analyzed by conventional ELISA techniques, in triplicate, using purified reovirus type 3 and VLSH dimeric peptides immobilized in multi-well plates (W.V. Williams et al (1991) J. Biol. Chem.. Vol. 266(8), pages 5182-5190). Control plates included wells having no immobilized reovirus and VLSH peptides to which mAb (lmg/ml) was added. VLSH peptide (W.V. Williams et al. ibid, Table 1) is a synthetic variant of VL peptide, the latter which corresponds to a portion of the light chain variable CDR II region of 87.92.6 antibody. The 87.92.6 antibody displays idiotypic and anti-idiotypic behavior towards reovirus type 3 receptor and mAb 9BG5, respectively (W.V. Williams et al. ibid) . The bound protein content of each well were measured by standard protein methods, e.g., Lowry method, and the results for each multi-well plate are shown in Figures 8(a-c), respectively. Figures 8 (a-c) illustrate the levels of serum proteins which bound to immobilized reovirus type 3 and VLSH as detected by measurement of protein concentration. These Figures show that the serum levels of bound proteins, after 24 hours post-dosing, were highest for animals orally administered mAb proteinoid carriers and animals administered unencapsulated mAb by the IV route. Lower levels of bound serum proteins were found in animals orally administered uncapsulated mAb. Serum taken from the animals receiving empty proteinoid carriers (no mAb) showed non-specific serum IgG protein binding, as expected, under the assay conditions.
Figure 9 show mAb binding under conventional ELISA procedures using immobilized reovirus type 3 and VLSH proteins. Serial dilutions of mAb treated with 0.85 N citrate-0.5% gum (Figure 9(a) or phosphate buffered saline (Figure 9 (b) were employed. The Figures show that the bound protein levels were higher for mAb in citrate buffer than for mAb in phosphate. Without being bound by any theory of operation for this inven- tion, it is believed that the binding enhancement may be due to changes in the three dimensional conformation resulting from citrate-protein binding.
In summary, serum levels of mAb, as reflected by the absorbance of bound proteins, were greater in animals receiving encapsulated mAb by the oral route or unencapsulated mAb by the IV route, than an animal receiving orally administered unencap¬ sulated mAb.
Example 12: Preparation of Proteinoid carrier containing Heparin
This Example describes a method for the preparation and cleaning of heparin proteinoid carriers.
PROCEDURE 1. Reagents: a. Proteinoid powder prepared as described in Example 1 b. Heparin c. Anhydrous citric acid (USP) d. Gum acacia NF e. Deionized water f. Desiccant g. Liquid nitrogen
2. Equipment: a. Magnetic stirrer b. Buret c. Microscope d. Clinical centrifuge e. Dialysis membrane tubing (Spectrum 6, 10 mm, 50,000 M.W. Cutoff) f. pH meter g. Lyophilizer (Labconco #75035) h. Lyophilizing flasks (150-300 mL) i. Rotating shell freezer j . Isopropanol/dry ice bath or liquid N2 k. Mortar and pestle 1. Storage containers (500 mL) m. Eppendorf pipet (0-100 uL) n. Plastic closures for dialysis tubing (Spectrum) o. 2 mL syringe with 0.45 urn Acrodisk
3. Preparation of Solutions: a. Proteinoid Solution A* (80 mg/ml) :
Dissolve 160 mg proteinoid in 1 ml of deionized water. Using a 2 ml syringe fitted with a 0.45 urn Acrodisk, the proteinoid solution was filtered into a 10 ml test tube and kept at 40° C.
b. Solution B (1.7 N citric acid with 1% gum) : Dissolve 10 g of gum acacia and 109 g of citric acid in 1 liter of deionized water.
c. Solution C (Heparin solution) :
Dissolve heparin in Solution B at 150 mg/mL and keep at 40° C.
4. Preparation of Proteinoid carriers: a. Add all of solution A to solution C quickly while swirling solution C slowly, by hand, in a 40°C water bath.
5. Dialysis of Heparin Proteinoid carriers:
or multiples thereof, It has been found the presence of citric acid in the encapsulated proteinoid carriers interferes with a subsequent lyophilization process. Hence, proteinoid carrier encapsulates prepared with citric acid solutions are preferably dialyzed against 5% acetic acid solution for at least two hours with at least four changes of the dialysis solution to remove citric acid by an exchange process. Thus, a. Transfer the suspension with a syringe (no needle) to dialysis tubing and seal with plastic closures. Tubing should be no more than 70% full.
b. Discard any amorphous material sedimented and/or aggregated on the surface.
c. Dialyze the proteinoid carrier suspension against acetic acid solution (using 20 mL of acetic acid solution per ml of proteinoid carrier suspension) while stirring the acetic acid solution with a magnetic stirrer.
d. Replace the acetic acid solution every hour. Continue dialyzing for a total of 3 hours.
6. Lyophilization: a. Add one part of 50% trehalose (Sigma Chemical Co., St. Louis, MO, USA) into nine parts of dialyzed proteinoid carrier solution. Flash freeze protein¬ oid carriers in a freeze-drying flask using the shell freezer adjusted to rotate at ca. 190 rpm and immersed in a liquid nitrogen bath.
b. Freeze dry for 24 hours or until dry as evidenced by lack of self-cooling.
c. Record weight of dry proteinoid carriers.
d. Grind to a fine powder with mortar and pestle. e. Transfer proteinoid into an amber container, seal with desiccant, and store at room temperature.
7. Resuspension: a. Weigh the lyophilized powder and calculate the amount of proteinoid in the powder.
b. Add aqueous 0.85 N citric acid into the lyophilized powder at 40°C. The final concentration of protein- oid in solution is 80 mg/ml.
Example 13: Preparation of Insuli -containing Proteinoid
Carrier This Example illustrates a method for the preparation of insulin proteinoid carriers.
PROCEDURE
1. Reagents: a. Proteinoid powder b. Anhydrous citric acid (USP) c. Gelatin (USP) d. Porcine insulin (Novo Nordisk) e. Deionized water (USP)
2. Equipment: a. Water bath b. 0.2 micron Acrodisk filter c. Sterile syringe (lOcc) d. Glass or plastic vessel of appropriate volume for desired amount of proteinoid carrier solution.
3. Preparation of Solutions: a. 1.7 N citric acid with 5.0% gelatin: Dissolve 109 mg anhydrous citric acid and 50 mg gelatin per 1 ml of deionized water at desired vol- ume" and incubate in water bath at 40°C until gela¬ tin is completely dissolved. This may be prepared and stored at 40°C for later use.
b. Insulin solution:
Dissolve 12 mg insulin per 1 ml of 1.7 N citric acid with 5% gelatin at 40°C at desired volume.
c. Proteinoid solution: Dissolve 100 mg proteinoid per 1 ml deionized water at room temperature and desired volume. Using sy¬ ringe and 0.2 micron Acrodisk, filter the solution to ensure a clear liquid and incubate in a water bath at 40°C. See Section 5b.
4. Preparation of Proteinoid carriers: a. Proteinoid solution and insulin solution are com¬ bined at equal volumes sufficient to produce the final desired volume of proteinoid carriers.
b. Rapidly add the filtered proteinoid solution to the insulin solution at 40°C while simultaneously and constantly swirling the insulin solution to ensure a thorough mixing.
Example 14: Procedure for Preparation of Erythropoietin
Containing Proteinoid carriers
Encapsulation of human erythropoietin (EPO) in proteinoid carriers was performed in the same manner described in Example 13. EPO was obtained from Genetic Institute
(Cambridge, MA, USA, now available from Amgen Corp., Thousand
Oaks, CA, USA) . A solution of Gln/Asp/Tyr/Phe (1:1:1:1 mole ratio of Gin, Asp, Tyr, and Phe in the proteinoid reaction mixture) proteinoid and a 150 ug/mL EPO solution in 1.7 N
** Proteinoid and Insulin solutions should each be prepared at one-half the total volume of the final microsphere solution desired. citric acid with 1% gum was used in preparing the EPO-contain¬ ing proteinoid carrier.
Example 15: Evaluation of Erythropoietin-containing Proteinoid Carrier
In this Example, an EPO-containing protein carrier, prepared as described in Example 14, was evaluated in rats.
An EPO experimental synopsis is given below.
Rats weighing 150-200 grams are anesthetized with ketamine (8.5mg/kg) and thorazine 3.75mg/kg) with intramuscular injection. The rat is then administered either unencapsulated erythropoietin or encapsulated erythropoietin by oral gavage. In brief, an 8 french nelaton catheter is inserted down the esophagus of the rat until the 10cm mark on the catheter is even with the incisors. The test or control solution is drawn up into a syringe and attached to the catheter. Holding the animal upright, the solution is expressed into the stomach of the rat. The experimental results are summarized in Figures 10-12.
ERYTHROPOIETIN EXPERIMENTAL SYNOPSIS
Batch Dose Rats Responding Comments
0/4 Fasted 15 hours. 0/4 Access to bedding. 2/4 Gavaged
0/2 0/2 Fasted 36 hours. 1/4 5% sucrose. 1/3 No bedding. 3/3 Gavaged.
1/5 Fasted 24 hours. 1/4 Access to bedding. 1/6 Gavaged.
0/5 Fasted 24 hours. 3*/6 No bedding.
0/3 Fasted 24 hours 1/4 No bedding. 1/3 Direct injection 1/4 into the stomach.
0/3
0/4 Direct injection 2/4 into the intestine.
0/4
1/5 Multiple Dosing 1/5 (5 dosing intervals 2/2 at t 1/2) 2/2 Gavage by stomach tube.
♦Rats were foaming at nostrils.
Figure 10 illustrates levels of erythropoietin (EPO) detected in rat serum taken from rats administered Gln/Asp/Tyr/Phe proteinoid carrier encapsulated EPO (15μg EPO/kg body weight) and encapsulated EPO (15μg EPO/kg body weight) at t = 0.5, 1, and 2 hours. Serum erythropoietin levels were determined over time with an erythropoietin enzyme immunoassay kit (Amgen, Thousand Oaks, CA, USA) . The results show that EPO serum levels in rats administered erythropoietin proteinoid carriers were relatively higher at all time points compared to rats (control) which received unencapsulated material. At t = 2 hours, the EPO levels remained at approxi¬ mately 300 pg/mL serum in rats administered erythropoietin proteinoid carriers while the control rats had undetectable EPO levels.
Figure 11 illustrates EPO serum levels in rats that were administered either erythropoietin (50μg/kg) or Gln/Asp/Tyr/Phe proteinoid (1:1:1:1 mole ratio of Gin, Asp,Tyr, and Phe in the reaction mixture) proteinoid carrier encapsulat- ed erythropoietin (50μg/kg) directly into the proximal duodenum. Serum erythropoietin levels were determined over time with the aforementioned erythropoietin enzyme immunoassay kit. The results show that EPO serum levels in rats adminis¬ tered erythropoietin proteinoid carriers steadily increased at a rate of approximately 50 pg/mL per hour over a range of two hours. In contrast, rats (control) which received unencapsu¬ lated EPO had EPO levels peaked at 100 pg/mL at 1 hour following administration and steadily decreased to about 50 pg/mL at the end of 2 hours. Figure 12 illustrates EPO serum levels in rats who were orally gavaged with either Gln/Asp/Tyr/Phe proteinoid
(1:1:1:1 mole ratio of Gin, Asp,Tyr, and Phe in the reaction mixture) proteinoid carrier encapsulated or unencapsulated erythropoietin (lOOμg/kg) ; or received a subcutaneous injection of either 2μg/kg or lOμg/kg. Serum erythropoietin levels were determined over time with the aforementioned erythropoietin enzyme immunoassay kit. The results show that EPO serum levels in rats (#640-645) orally administered erythropoietin protein¬ oid carriers were relatively higher up to t = 2 hours, compared to rats (EPO) which received unencapsulated material.
The results obtained in this Example provide evidence that proteinoid encapsulation markedly improved the oral bioavailability of EPO.
Example 16: Preparation of Calcitonin-containing
Proteinoid carrier
Encapsulation of salmon calcitonin in proteinoid carriers was performed in the same manner described in Example 13. Calcitonin, a peptide hormone which acts predominantly on bone to lower serum calcium concentration, was obtained from Sandoz (Basil, Switzerland) . Calcitonin proteinoid carriers were prepared by mixing a 1:1 volume ratio of a lOOmg/ml aqueous solution of Gln/Asp/Tyr/Phe proteinoid (1:1:1:1 mole ratio of Gin, Asp, Tyr, and Phe used in the proteinoid reaction mixture) and a 150 ug/mL calcitonin solution in 1.7 N citric acid solution with 1% gum acacia, as described in Example 13. The efficiency of calcitonin encapsulation was approximately 40%. Calcitonin concentration was determined directly by HPLC after dissolving the calcitonin proteinoid carriers in 60% aqueous acetonitrile.
Example 17: Evaluation of Calcitonin-containing Proteinoid carriers in Monkeys
In this Example, the calcitonin proteinoid carriers, prepared as described in Example 16, were evaluated in cynomolgus monkeys. Male cynomolgus monkeys weighing 4-5 kg were fasted overnight, anesthetized (approximately lOmg/kg ketamine HCl) and placed into a primate restraint chair for dosing and blood sampling. A single oral dose of calcitonin proteinoid carriers (0.25 mg/kg body weight) was administered to each of four monkeys by nasogastric gavage. The dosage was based on the body weight taken on the morning of dosing. Blood samples were collected from saphenous vein catheters at hourly intervals, starting at t = 0 prior to administration of the proteinoid carriers, and hourly, from 1 to 7 hours post-dose for serum calcium determination. The hypocalcemic response following oral calcitonin administration was used as an index of pharmacological response. Serum calcium concentrations were quantitated by a conventional O-cresolphthalein complexone method.
Figure 13 demonstrates the response obtained in cynomolgus monkeys following naso-gastric gavage of microencap- sulated calcitonin. Significant changes from baseline serum calcium concentration were observed. Six hours following dosing, serum calcium concentrations decreased by 13 μg/ml. A significant pharmacological response was still apparent seven hours after the administration of calcitonin proteinoid carriers.
Example 18: Evaluation of Calcitonin-containing Proteinoid Carriers in Rats
In this Example, the calcitonin proteinoid carriers prepared in accordance with Example 16 are evaluated in fasted male Spraque Dawley rats weighing 100-150g. Calcitonin proteinoid carriers and calcitonin were administered by either oral gavage or intraduodenal injection. The rats are divided into the following groups:
1. calcitonin proteinoid carriers: 60 ug calcitonin/kg body weight by oral gavage (3 rats) ; 2. calcitonin proteinoid carriers: 3 ug calcitonin/kg body weight by intraduodenal gavage (3 rats) ;
3. calcitonin: 60 ug calcitonin/kg body weight by oral gavage (3 rats) (Control) .
4. calcitonin: 3 ug calcitonin/kg body weight by intraduo- denal gavage (3 rats) (Control) .
Oral gavage dosing of rats is performed. Calcitonin proteinoid carriers are prepared immediately prior to dosing and Groups 1 and 2 each receive an appropriate dosage of the proteinoid carrier suspension. Groups 3 and 4 receive the unencapsulated calcitonin (no proteinoid carriers) . Approxi¬ mately 0.5 ml of blood is serially withdrawn from the tail artery of each rat just prior to dosing ("0" time) and 1 h, 2 h and 3 h post-dosing. Serum from the blood samples are stored at -20°C for serum calcium concentration determination. Figure 14 is the serum concentration-time curve for orally administeredmicroencapsulated calcitonin andunencapsu¬ lated calcitonin in rats. Experimental results in rats demon¬ strate a significant increase in pharmacological response (i.e., decreasing serum calcium levels) when proteinoid encap- sulated calcitonin is compared to the unencapsulated vehicle control group. One hour after dosing, serum calcium concentra¬ tions decreased 23 μg/ml in the rats receiving encapsulated calcitonin compared to a decrease of only 6.5 μg/ml in the control group. Furthermore, the responses were dose-dependent (data not shown) .
The results of intraduodenal injection of encapsulat¬ ed or unencapsulated calcitonin in rats is shown in Figure 15. The results demonstrate a time-dependent decrease in serum calcium levels for the encapsulated preparation. The control group showed no response. One hour after intraduodenal admin¬ istration, serum calcium levels in the calcitonin proteinoid carrier group decreased by 18μg/ml, whereas unencapsulated calcitonin was unchanged. These results indicate that transmembrane transport of calcitonin is enhanced by proteinoid encapsulation.
The results obtained in this Example and in Example 17 provide evidence that proteinoid encapsulation markedly improves the oral bioavailability of calcitonin. The data also indicate that the oral drug delivery system is not species- dependent.
Example 19: Preparation and Evaluation of Factor I -con- taining Proteinoid Carrier
Factor IX is a vitamin K-dependent blood coagulation proenzyme, MW 56 kD. Factor IX deficiency, known as hemophilia B, occurs in approximately 1 out of every 25,000 males. To date, treatment of this disorder is accomplished by intravenous administration of Factor IX, although a recent report details efforts to supplement by subcutaneous injection (Thompson (1986) Blood, Vol. 67(3), pages 565-572).
Encapsulation of Factor IX (FIX) in proteinoid carriers was performed, following the procedure described in Example 13, by mixing (1:1 v/v) 100 mg/mL of Glu/Asp/Tyr/Phe proteinoid (1:1:1:1 mole ratio of Glu, Asp, Tyr, and Phe used in the proteinoid reaction mixture) in deionized water and an aqueous solution of FIX. Two proteinoid carrier suspensions were prepared and evaluated in vivo separately as described in Examples 20 and 21.
FIX proteinoid carrier suspension A contained 50 mg/ml of proteinoid and 500 U/ml FIX (FIX is available from the American Red Cross, Rockville, Maryland, USA) solution containing 4% acetic acid, 2% gum acacia, 0.2% PEG 14 (avail¬ able from Union Carbide, Danbury, CT, USA) , 14 mM CaCl2, final pH 3.81.
The second suspension, FIX proteinoid carrier suspen- sion B, contained 50 mg/ml proteinoid and 116 U/ml FIX solution containing 3.8% acetic acid, 1.5% gum acacia, 0.15% PEG 14, 11 mM CaCl2, final pH 4.58.
The stability of FIX proteinoid carrier preparations was assessed over a short time course in vitro. The protein carriers encapsulating FIX were examined by optical microscopy and laser light scattering. Aliquots of proteinoid carrier suspension were withdrawn every 30 minutes for 1.5 hours, FIX proteinoid carriers were isolated by centrifugation at 4500Xg and dissolved in activated partial thromboplastin time (APTT) assay buffer (0.05M histidine-0.OlM NaCl-0.1% bovine serum albumin-0.01% TWEEN-40, pH 7.47) to release soluble FIX and proteinoid. Quantitation of FIX activity by APTT employed both FIX standards (0.025, 0.05, and 0.1 U/ml) and "empty" protein¬ oid carrier suspension as control. APTT assay kits are commercially available, e.g. Sigma Diagnostics (St. Louis, MO, USA) .
Based on the above analysis, it was determined that FIX proteinoid carriers of greater stability are obtained by encapsulating FIX at a higher pH, e.g., pH 4.9. Furthermore, the efficiency of encapsulation is approximately 20% of available FIX units and activity levels remain constant for at least 1.5 hours when FIX proteinoid carrier pellets are stored at about 4°C.
Example 20: Evaluation of FIX-containing Proteinoid carri¬ ers (A) in Rats
In this Example, FIX proteinoid carrier suspension
A, prepared as described in Example 19, were evaluated in male
Sprague Dawley rats (ave. weight 300g) . Appropriate aliquots of suspension were centrifuged at 4500Xg to pellet the FIX protein carriers, which were subsequently resuspended in the same buffer for animal dosing. The rats are divided into two groups as follows: 1. Oral FIX proteinoid carriers (FIX sph PO) : 2709 U FIX/kg body weight by intragastric gavage (4 rats) ;
2. Intravenous FIX (no proteinoid carriers) (FIX IV): 200 U/kg body weight by intravenous injection. 32 rats received 0.7 ml FIX in 0.11 NaCl-0.02M sodium citrate, pH 6.85 by tail vein injection.
The FIX proteinoid carrier suspension and solution are prepared immediately prior to dosing. One ml of blood was withdrawn from each rat just prior to dosing ("0" time) and 1 h, 2 h and 4 h (post-dosing) , a citrate anticoagulant was added to the blood, and plasma from the blood samples were stored at -70°C.
Plasma samples were assayed by a modified APTT assay using FIX coagulated deficient plasma (assay kit is available from Ortho Diagnosis (Raritan, New Jersey, USA) . Changes in clotting times were calculated by subtracting individual baseline (0 hr) values from subsequent clotting time values. The data shown in Figure 16 are the mean values for a given group. Values below baseline indicate the presence of exoge- nous FIX.
As shown in Figure 16, significant amounts of FIX was delivered to blood via oral administration of FIX proteinoid carriers. The relative plasma level is lower in the FIX proteinoid carriers group, however the dimunition in clotting time at 0.5, 1.0 and 2.0 hours is notable. This is achieved by oral dosing with approximately 14 times the IV dose. Moreover, these results are particularly interesting since Factor IX is an acid labile protein whose half-life is approximately less than one hour at 37°C at pH 5.0. The FIX proteinoid carriers in this experiment were at pH 3.81 and encapsulated 14.8% of the available FIX units during prepara¬ tion. The results support that FIX proteinoid carriers remain viable in the GI tract to facilitate delivery.
Example 21: Evaluation of FIX-containing Proteinoid Carri¬ ers (B) in Rats
In this Example, FIX proteinoid carrier suspension
B, prepared as described in Example 19, were evaluated in male Sprague Dawley rats (ave. weight 300g) . Resuspended FIX proteinoid carriers were prepared as described in Example 20. The rats are divided into two groups as follows:
1. Oral FIX proteinoid carriers (FIX sph PO) : 1006U FIX/kg body weight by intragastric gavage (5 rats) .
2. Intravenous FIX (no proteinoid carriers) (FIX IV): 185 U/kg body weight by intravenous injection. 3 rats received 0.3 ml FIX in 0.11 NaCl-0.02M sodium citrate, pH 6.85 by tail vein injection. 3. Oral FIX (no proteinoid carriers) (FIX unencap PO) : 2760U FIX/kg body weight by intragastric gavage. 4 rats re¬ ceived 1.0 ml of FIX in saline solution containing 3.8% acetic acid, pH 6.85.
The FIX proteinoid carrier suspension and solutions were prepared immediately prior to dosing. Plasma samples were obtained and assayed as described in Example 20. Changes in clotting times were calculated by subtracting individual baseline (0 hr) values from subsequent clotting time values. The data shown in Figure 17 are the mean values for a given group. Values below baseline indicate the presence of exoge¬ nous FIX. The FIX proteinoid carriers, prepared at pH 4.58, encapsulated 23.1% of the FIX units.
As shown in Figure 17, at oral dose levels of only
5 times that of the IV dose, significant oral delivery was observed. In addition, native FIX (pH 6.85) dosed at 15 times the IV dose level resulted in no detectable levels of exogenous
FIX in the plasma.
Thus, the results shown in this Example and in Example 20 support that oral delivery of FIX can be accom- plished via the use of FIX proteinoid carriers. These proteinoid carriers appear to adequately protect FIX during transit through the GI tract and deliver FIX to the blood stream.
Example 22: Preparation of alpha-Interferon (IFN) -contain¬ ing Proteinoid carrier
In this Example, a study was undertaken to evaluate the protective capability of proteinoid carriers on enzymatic degradation under simulated gastrointestinal condi¬ tions. The in vi tro stability of IFN in proteinoid carriers was examined in simulated gastric fluid (SGF) containing pepsin in 0.08 N HCl and simulated intestinal fluid (SIF) containing pancreatin in phosphate buffer. The reagents and stability assay procedure are described in the "United States Pharmacopoeia" (Vol. XXII, 1990, pages 1788 and 1789) .
Preparation of IFN-containing proteinoid carriers Encapsulation of IFN in proteinoid carriers was performed in the same manner described in Example 13. Alpha- IFN is available from a number of commercial sources. One commercial IFN product includes Roferon-A (Hoffman LaRoche) . IFN proteinoid carriers were prepared with an aqueous solution of Glu/Asp/Tyr/-Phe proteinoid (1:1:1:1 mole ratio of Glu, Asp, Tyr and Phe used in the proteinoid reaction mixture) , and an IFN solution containing 1.7 N citric acid solution with 5% gelatin. The IFN proteinoid carrier suspension contained 80 mg/ml proteinoid, 600 ug/ml IFN, 0.6N citric acid, and 2.5% gelatin, pH 3.0.
Stability of IFN proteinoid carriers in SGF
SGF (2 ml) was added into 1 ml of IFN proteinoid carrier suspension. The solution was incubated at 40°C with shaking, and aliquots were taken serially after SGF addition as described in the "U.S. Pharmacocopia" (ibid) . An equal volume of stopper solution (pepstatin A in phosphate buffer, was added to each aliquot immediately after sampling to stop the enzymatic degradation and to open the proteinoid carriers. The IFN concentration in all samples was then determined by HPLC. As a comparison, the stability of IFN alone in SGF was evaluated. The experiment were performed as described above, without the proteinoid carriers. As another control, the stability of IFN proteinoid carriers was evaluated in 0.08 N HCl.
Stability of IFN-containing proteinoid carriers in SIF
SIF (2 ml) was added into 1 ml IFN proteinoid ca- rriers. The solution was incubated at 40°C with shaking and samples were taken serially as described in the "United States Pharmacocopia" (ibid) . An equal volume of stopper solution (aprotinin and trypsin/chymotrypsin inhibitor in phosphate buffer) was added to each aliquot immediately after sampling to stop the enzymatic degradation. The IFN concentration was analyzed by HPLC.
To study the study the stability of IFN alone in SIF, 600 ug of IFN was dissolved in 0.85 N citric acid or 0.01 M phosphate buffer. SIF (2 ml) was added to 1 ml IFN solution. The solution was sampled and analyzed as described above.
Results and Discussion
(a) Protective Effects of Proteinoid carriers in SGF As shown in Figure 18, after 1 hour of SGF incuba¬ tion, approximately 50% of IFN remained intact. After incuba¬ tion in SGF for 6 hours, approximately 20% of IFN was not degraded. As expected, IFN alone (in the absence of proteinoid carriers) , was found to be completely destroyed by pepsin in SGF within 20 minutes.
Another control was performed using IFN alone in 0.08 N HCl. IFN alone was stable in SGF without pepsin (0.08 HCl) . There was only a slight decrease after 2 hour incubation. This suggests that IFN was rather stable in HCl at pH 1.2 up to six hours (Figure 19) .
The results suggest that proteinoid carriers can retard IFN from pepsin digestion, while IFN alone cannot survive in the stomach for more than 20 minutes. These obser¬ vations demonstrate the protective ability of proteinoid carriers on enzymatic digestion of protein drugs in the stomach. (b) Protective Effects of Proteinoid carriers in SIF
As shown in Figure 20, IFN proteinoid carriers were much more stable than IFN alone (in the absence of proteinoid) in SIF. IFN alone at pH 7.4 was completely degraded within 10 minutes when incubated with SIF. However, approximately 70% of the IFN/proteinoid carriers survived after 6 hours in SIF, indicating thatt considerable stability is provided by the proteinoid carrier.
IFN alone was slightly more stable in SIF at pH 3 than at pH 7.4. After 6 hr incubation in SIF at pH 3, there was approximately 10% of the IFN remaining. The stability of IFN in SIF at pH 3 is attributed to the low pH, which appears to suppress enzymatic activity of the intestinal proteases.
Example 23: Evaluation of Heparin-containing Proteinoid carriers in Rats
In this Example, a study was undertaken to ascertain whether proteinoid carriers are required for protective capability or whether (1) proteinoids (soluble proteinoids--not in carrier form) may be used and whether (2) alternative methods of carrier loading, such as incubating the therapeutic compound with preformed proteinoid carriers, are useful.
Preparation of Heparin-containing proteinoid carriers Encapsulation of heparin in proteinoid carriers was performed in the same manner described in Example 12. Heparin (USP grade) was used and this material is available from a variety of commercial sources including Eli Lilly (Indianapo¬ lis, USA) . Heparin proteinoid carriers were prepared, following the procedure of Example 12, using a 1:1 volume ratio of 150 mg/ml of Glu/Asp/Tyr/Phe/Ornα5 (1:1:1:1:0.5 mole ratio of Glu, Asp, Tyr, Phe, and Orn used in the proteinoid reaction mixture) proteinoid in deionized water, and an 20mg/mL aqueous heparin solution containing 1.7 N citric acid solution and 0.5% gum acacia. The heparin proteinoid carrier suspension was dialyzed in acetic acid solution as described in Example 12. Heparin proteinoid carriers were then centrifuged at 4800Xg (15 minutes) and total heparin was measured by assaying the pellet and the supernatant with a modification of the Azure A method (Gundry et al. Amer. J. of Surgery (1984) Vol. 148, pages 191- 194) . Proteinoid was assayed by dissolving the proteinoid carriers with 0.1 N NaOH and measuring absorbance at 294 nm.
Preparation of heparin-spiked empty proteinoid carriers
Empty proteinoid carriers were prepared following the same procedure described above for the heparin proteinoid carriers, with the modification being that no heparin was present. The lyophilized empty proteinoid carriers were resuspended in 0.85N citric acid and 0.5% gum containing heparin at a concentration of 20 mg/ml. The amount of heparin co-isolated with the proteinoid carriers was measured as described above.
Experimental Procedure
Male Spaque Dawley rats weighing approximately 350g were dosed by oral gavage or intraduodenal (ID) injection (just anterior to the pyloric sphincter and into the duodenum) . Rats were dosed orally or ID with one of the following: lyophilized heparin proteinoid carriers, heparin-spiked empty proteinoid carriers, proteinoid/heparin in water, heparin in 0.85N citric acid and 0.5% gum and heparin alone in water. In both oral and ID injection experiments, weight ratios of heparin:proteinoid were constant. The total heparin dose in the oral studies was 100 mg/kg body weight; in ID injections studies, it was 50 mg/kg. The proteinoid dose was 40 mg/kg for oral gavages and 20 mg/kg for ID injections. The dosing volume was approximate¬ ly 0.3 to 0.5 ml. Approximately 0.5 ml of blood is serially withdrawn from the tail artery of each rat just prior to dosing ("0" time) and 1 h, 2 h and 4 h post-dosing. Serum from the blood samples are stored at -20°C for heparin activity determination.
Results and Discussion
The results obtained suggest that heparin alone as well as soluble proteinoid and heparin (both in water, dosed orally or by ID injection) did not appear to be absorbed from the GI tract in amounts sufficient to increase APTT values
(Figure 21) . Heparin in citric acid elicited some increase in
APTT values, but only when dosed directly into the duodenum.
Heparin proteinoid carriers gave the highest APTT values, indicated increased absorption of heparin when dosed orally, as well as when directly injected into the duodenum
(Figures 22 and 23) . While the observed activity was lower than observed with heparin proteinoid carriers (Figure 23) , heparin-spiked empty proteinoid carriers showed increased APTTs over baselines. Both types of proteinoid carriers showed a much greater increase in APTT values than that observed with citric acid/heparin.
The results obtained in this Example suggest that, in the proteinoid system, proteinoid carriers are necessary for the observed increase in heparin absorption, as soluble proteinoid did not show detectable activity within the experimental limits.
Example 24: Preparation and Evaluation of Ml-containing Proteinoid carrier
In this Example, influenza virus antigen-containing proteinoid carriers were prepared and evaluated in rats.
Preparation of Ml Proteinoid carriers
Encapsulation of Ml in proteinoid carriers was performed in the same manner described in Example 13. Ml protein, a major internal component of influenza virus, was obtained by purification of a swine influenza vaccine donated by Drug Directorate, Health Protection Branch, Bureau of Biologies, Ottawa, Ontario Canada. The vaccine was prepared with the high-yielding recombinant strain X-53Aa, which derives its HA and NA from the parent strain A/NJ/11/76 (H1N1) and its internal proteins, including Ml, from the parent strain A/PR/8/34 (R.B. Couc et al. (1983) Ann. Rev. Microbiol.. Vol. 37, pages 529-549 and B.R. Murphy (1982) Infec. Immun. , Vol. 36, pages 1102-1108) . Ml was purified as described by Khan et al ( (1982) J.Clin.Microbiol.. Vol. 16, pages 813-820) . Ml proteinoid carriers were prepared, by mixing (at 40°C) , equivolumes of an aqueous solution of lOOmg/ml of Glu/Asp/Tyr/Phe proteinoid in deionized water and a lOmg/mL solution of Ml protein in 1.7N citric acid and 5% gum arabic (pH 2.0). The final Ml concentration in the suspension was 1.Omg/ml.
Preparation of HA-NA-containing Proteinoid carriers and unencapsulated antigens
HA-NA antigen was isolated according to the procedure of Gallagher et al. ((1984) J.Clin.Microbiol. , Vol. 20, pages 80-93). Influenza virus (A/PR8/34) was centrifuged at 90,000 G for 60 min. The viral pellet was solubilized with 0.05M acetate buffer (pH 7.0) containing 7.5% octylglucoside and re- centrifuged under the same conditions. The resulting superna- tant contained approximately 90% HA and 10% NA as determined by SDS-PAGE.
HA-NAproteinoid carriers were prepared following the same protocol as for the Ml proteinoid carriers but substituted Ml for HA-NA. The final concentration of HA-NA in the suspension was also 1.0 mg/ml.
"Empty" proteinoid carriers were prepared following the sample procedure described for the Ml proteinoid carriers, with the only modification being that a 1.7 N citric acid/gum solution was used in place of the Ml/citric acid/gum solution.
Unencapsulated antigens, Ml and HA-NA, was diluted in 1.7 N citric acid, 10 mg/ml gum arabic to the same final lmg/ml concentration.
Experimental procedure
Male Spraque Dawley rats (about 350g weight) were used in this experiment. Oral dosage was by gavage. Four groups of five rats each (the subcutaneous control group had 4) were dosed as follows: Group 1 was dosed orally with lmg of Ml proteinoid carriers per rat (1 ml) , Group 2 was dosed orally with 1 mg per rat of "empty" proteinoid carrier, Group 3 was dosed with 1 mg of unencapsulated Ml per rat of "empty" carrier, Group 3 was dosed with 1 mg of unencapsulated Ml per rat in 1 ml and Group 4 was dosed subcutaneously (SC) with 25 ug per rat of Ml in 0.3 ml. Blood samples (300 ul) were taken from each rat by tail bleeding before dosing and at 1, 2, 3 and 4 hours post-dose (to assay for antigen) and at 14, 28, and 42 days post-dose (for antibody assay) . Solutions for subcutane¬ ous control-Ml in TRIS (no SDS) was diluted to a concentration of 167 ug/mL. An equal amount of Freunds Complete Adjuvant (FCA, Sigma) was added and the mixture was thoroughly homoge¬ nized. The final concentration of Ml in the mixture was 83.3 ug/ml. HA-NA solutions for subcutaneous administration were prepared in the same manner except that phosphate buffered saline replaced TRIS-SDS buffer.
The same immunization and bleeding schedule was followed when dosing with HA-NA proteinoid carrier, with the following modifications: all rats received an oral booster with HA-NA proteinoid carrier (250 ug/rat) 42 days after the first oral dose and blood samples were again taken 14 days after the booster dose. Serum derived from the samples were stored at -20°C until assayed. Serum anti-Mi and anti-HA-NA specific IgGs were assayed by an ELISA method as described Khan et al. ((1982) J. Clin. Microbiol., vol. 16, pages 813-820).
Results and Discussion Attempts to measure antigen in plasma samples were unsuccessful. Ml antigen could not be detected in rat plasma samples taken 1-4 hours post-dosing in all groups, including the subcutaneous control.
Plasma samples from rats dosed orally with "empty" proteinoid carriers showed no significant antibody titer against either Ml or HA-NA antigens when assayed by ELISA (Table 8) . As expected, rats dosed with 25 ug of either Ml or HA-NA antigen (with FCA) subcutaneously developed a vigorous antibody response with titers that ranged from 54,000-330,000 in the case of Ml and 176,750-909,000 in the case of HA-NA (Table 8) .
Plasma samples from three of the five rats dosed with Ml proteinoid carriers showed a significant primary response to Ml antigen. All three rats had titers ranging from 760 to 2150 as early as 14 days post-dosing, compared to <30 in all rats that received the amount of unencapsulated Ml (Table 8) . Titers in the group that received proteinoid carriers increased to 1150-5200 by 42 days (Figure 24) .
Four out of six rats immunized with unencapsulated HA-NA did show a moderate anti-HA-NA IgG response, with titers of 3400-17,675, while two of six rats dosed with HA-NA proteinoid carrier showed a significant response (Figure 25) . The rats that did respond, however, reached titers at least eight times higher than those obtained in the controls. Although several rats showed higher titers after the oral booster with HA-NA proteinoid carriers given 42 days post-dose, most did not show a significant increase in titers. The results support that a single dose of Ml prote¬ inoid carriers was capable of inducing a significant IgG response to Ml as early as two weeks post-dosing, while rats dosed with same Ml (no proteinoid carriers) total dose showed no detectable antibody response. Similarly, a single dose of HA-NA proteinoid carriers induced a response in 33% of the rats used in the study. This response was up to eight times greater than rats dosed with unencapsulated HA-NA.
TABLE 8
ANTI M PROTEIN ANTIBODY TITERS IN SERUM FROM RATS DOSED WITH M PROTEINOID CARRIERS VS CONTROLS
14 day 28 day 42 day
Dosing rat # titer titer titer
oral M protein unencapsulated 1 mg/rat
empty carrier
M proteinoid carriers
1 mg/rat total
subcut. control 0.025 mg/rat in FCA
Appendix A Page 1 of 9
60
Proteinoid Batches
Glossary: a = amoφhous oil varying temperature cook time change < σ>
I ° S. g, ci t- < α. vø
*<* q o o o q q q qq q q q o q ά ό 9999 q q q o q q o q o o o n q o o o q ό d ό b ob b d o b b d 6 ά ά ό ό ά ό ά ά ά b bb b d d d b
H ~ Ξ U
P* o o q q b b b b
o o t o _ o o o o o o
H ι- X x S x x x x x x x X
o u ϋ o
<f > _ > _l _ > _1 f
OI
Z *D C) O r* 1 n - l-l fl -) G) 0 ω r» ω σ> o «- cM c *t co ι». ciθ cn o *-- cM c<_ <-ι* « C C*) ' ** ** <t 'I <* « 't ** ** l<. ... — ιw w ιv w ψ (0 <O I_ (D CD [*. ■ r. i ri***<« l*****» lf*.. ||*^« *r*.. ^i** 'c0o -_*al βalj i_*el ή_o oo o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o _ I O- O~ O- O_ O~ O~ O- O- O~ O~ O~ O~ O
Appendix A Page 3 of 9
62
Proteinoid Batches
Composition
-GLU2 LYSH2 PHE2 ASP
-GLU2 LYSH2 PHE2 ASP
-GLU2 LYSH2 PHE2 ASP
-ASP TYR PHE PGLU
GLU ASP-VAL LYSHB
GLU ASP-TYR PHE
GLN-ASPTYR PHE
GLU2ASP2 TYR2 PHE2 ORN
-TYR PHE ASP PGLU
-TYR PHE PGLU ASP
GLU2ASP2 TYR2 PHE2 ORN
GLU2 LYSH2 PHE2 ASP-
GLU ASPTYR PHE-
-GLU ASP TYR PHE
GLU2ASP2 TYR2 PHE2 ORN
-GLU ASP TYR PHE
-GLU ASP TYR PHE
-GLU ASP TYR PHE
-GLU ASP TYR PHE
-GLU ASP TYR PHE
ASP PHE
ASP2PHE
ASP3 PHE
-GLU ASP TYR PHE
-GLU ASP TYR PHE
-ASP PHE2
-GLU2 ASP2 TYR2 PHE2 ORN
-ASP2TYR
-ASP TYR
-ASP3 TYR
-GLU ASPTYR PHE
-GLU ASP TYR PHE
-ASP TYR2
-ASP TYR2
-GLU ASP TYR PHE
-GLU ASP TYR PHE
-ASP2 PHE
-GLN ASP TYR PHE
-GLU2 ASP2 TYR5 PHE5
-ASP2PHE
-PHE ASP2
-ASP2 PHE
-GLU2 TYR PHE
-GLU2ASP2 TYR2 PHE2 ORN -GLU2ASP2 TYR2 PHE2 ORN
Glossary: a = amorphous o = oil * = varying temperature + = cook time change ApperiBϋMo* Page 4 of 9
63
Proteinoid Batches
Composition
ASP
GLU ASP-TYR PHE
GLU LYS PHE ASP
GLU ORN ASP LYS PHE
GLU ASP-TYR PHE
GLU ASP-TYR PHE ORN0.5
-PGLU ASP.5TYR PHE
-GLU ASP.5TYR PHE
GLN ASP TYR PHE
GLU2 ASP2 EQU GLU ASP EQU GLU ASP EQU 0
GLU ASP EQU GLU ASP EQU GLU ASP EQU GLU ASP EQU GLU2 ASP2 EQU GLU2 ASP2 EQU GLU2 ASP2 EQU GLU2 ASP2 EQU GLU2 ASP2 EQU GLU2 ASP2 EQU GLU2 ASP2 EQU GLU2 ASP2 EQU GLU2 ASP2 EQU GLU ASP EQU GLU2 ASP2 EQU GLU2 ASP2 EQU GLU2 ASP2 EQU GLU2 ASP2 EQU GLU2 ASP2 EQU GLU2 ASP2 EQU GLU2 ASP2 ASPG GLU2 ASP2 SER GLU2 ASP2 EQU GLU2 ASP2 EQU GLU2 ASP2 EQU GLU2 ASP2 EQU GLU2 ASP2 EQU GLU2 ASP2 EQU GLU2 ASP2 EQU GLU2 ASP2 DQU GLU2 ASP2 EQU
Glossary: a = amoφhous o = oil * = varying temperature + = cook time change Appendix A Page 5 of 9
Proteinoid Batches
Bt No #AA Date
249 <3K 4
250 <3K 5
251 <3K 4 252-CP 4 253-CP 4 253 4 254-CP 5 255-CP 5 256-CP 4 257-CP 4
258 <3K 3
259 3K 4
2603K 4 261-CP 4 262-CP 4 263-CP 4 264-CP 4 266-CP 4 267-CP 3 268 4 269-CP 4 270-CP 4 271 3
272-CP 4 273-CP 4 274-CP 4 275-CP 4 276-CP 4 277-CP 3 278-CP 3 279-CP 3 280-CP 4 281-CP 4 282-CP 3 283-CP 4 284 5 285-CP 2 286-CP 2 287-CP 2 288-CP 3 289 2 290-CP 3 291-CP 3 292-CP 5 293-CP 4 294-CP 4
Glossary: a = amoφhous o = oil * = varying temperature + = cook time change Appendix A Page 6 of 9
65
Bt No #AA
Date
192-CP 3
193 > 6K 4
194-CP 3
195-CP 3
196-CP 4
197-CP 4
198-CP 3
200-CP 3
201 -CP 3
203-CP 4
204-CP 3
205-CP 4
208 3
209-CP 4
210-CP 4
213-CP 3
215-CP 3
216-CP 4
217 3
218-CP 4
219-CP 3
220-CP 4
221 -CP 3
222-CP 3
224-CP 4
225-CP 3
226 <3K 4
229 <3K 4
230-CP 2
231-CP 3
232-CP 3
233-CP 3
234-CP 4
235-CP 4
236-CP 3
237 < 3K 4
238 <3K 4 239-CP 3 240-CP 3 241 <3K 3 242-CP 3 243-CP 5 244-CP 3 245-CP 3 246 <3K 5 247-CP 3
Glossary: a = amorphous oil varying temperature + = cook time change Appendix A Page 7 of 9
Glossary: a = amorphous o = oil * = varying temperature + = cook time change Appendix A Page 8 of 9
67
Proteinoid Batches
Glossary: a = amoφhous o = oil * = varying temperature + = cook time change Appendix A Page 9 of 9
68
Proteinoid Batches
Temp Time Sphere Batch Size
Bt No #AA Composition Additive C (hr) Rating Molar Oper
Date
350 5 -(GLU ASP TYR PHE12 ORN SULFOLANE .0 .0
Sphere Rating: 0 = Worst 5 = Best
INT = insulin
MT = empty microsphere
HEP = heparin
Sul-M = sulfolane. medical grade
Sulfa = Sulf = Sul = Sulfolane
PA = phosphoric acid
Equ = equilents
GLYC = glycerol
TRIGL = triglyme
PPA = polyphosphoric acid
M.Oil = mineral o. = mineral oil
Glossary: a = amoφhous o = oil * = varying temperature + = cook time change i£F TABLES Appendix B
Proteinoid sorting, pKa and composition Page 2 of 2
Cherrocal basis for microsphere ODS
Sphere Max UV Max UV
Run} Material ID No. Composition Sphere frac Sphere pH Sphere IEF number range rating Matrix rating Frac. No. pH No. j — > (*no amp)
INSO HTO HEPO 19-20 5-3
47 257-CP Glu Asp ArgH OrnH no spher' INSO HTO HEPO 15-17 8-9-8.5
48 257-CP Glu Asp ArgH OrnH* no spher
Glu Asp ArgH no spher INSO HTO HEPO 1,17-20 9.8,4-2.6
49 258 >3
50 262-CP Glu Om Aβp LysFB 11-18 6.8-3.6 2 INS0 HT1 HEP4 15 4.8
51 262-FILT Glu Orn Asp LysFB 4-11 7.7-6.4 1-2 inβθ ntl hep4 1-2,12-20 9.4.6-1.8
Glu LyβFB Aβp LysBP no spher INSa Htc HEPc 14-20 6.3-3.8
52 267-cp
53 268-cp c Glu Aβp sul Tyr Phe 15-20 4.5-2.34 2-3 INS4oHT4oHEP4a 1-10, 18-20 12-2.5 nH Aβp LysFB 17-20 2.91-1.4 1 INSc HTc HEPc 4,7,9 9-7.5
54 269-cp Glu Or
55 273-cp Glu Leu LyβH Phe 17-20 3-1.2 1-2 INS2a MT2 HEP2a 19 2
— 3-9,13-15 9.8.6,8-8 66 272/273 Glu Leu LysH no spher
57 276 Glu Leu Arg Phe 12-18 3.67-1.4 1-2,2 INS2HT2HEP3 1-7,17-20 9-6,1.6-1 er INSc HTc HEPc 16-20 4.14- .4
58 274 Glu Leu Arg Tyr no sph
Glu Leu LysH Tyr ne spher INSc HTc HEPc 1-2 9.4-9.3
59 272 rg no spher — all free. —
60 274A Glu Leu A
61 278 Glu Lye Phe «ui 16-20 4.8-3.6 1-2 INSc HTc HEP4 all frac. —
62 284E GIuAβpTyrPheβulOrn 14-20 3.8-2.1 1-2 INS4oHT4oHEP3 16-20 3.3-2.1
63 287-cp Glu Phe 10-20 3.66-2.3 2 INS3 HT2HEP3 18-20.1-7 2.4-2.3,8 -8 2.3-7.76
64 284-E Glu2Aβp2Tyr2Phe20rn 14-20 3.95-1.6 2 wtoil INS4a HT4aHEP2a 3 Phe no spher INSc HTc HEPc 17-20 2.7-1.02
65 288-cp Glu Orn
66 293-cp Glu Aβp βul Tyr Phe 1-8 1.9-3.9 1-2 INS1 HT2 HEP1a
67 290-cp Glu Arg Phe 1-7 1.05-3.8 1-2 INS1 HT1 HEP1 18-20 12.1-12.6
68 g no βpher INS HT HEP 16-20 3.19-1.5 292-cp Glu Aβp Ar 69 300-cp Glu Orn Aβp Lys Phe 15-19 4.05-1.5 1-1 INS3 HT3 HEP37 all frac. 70 297-cp GLU ASP SUL TYR PHE 1-7 2.38-4.15 2-3 INS2 HT4 HEP 1-2 2.38-2 71 INS4 HT2 HEP3 1-20 — 301 <3J GLU ASP SUL TYR PHE GLU ASP SUL TYR PHE1-8 2_S3-3.76 2-3 INS4HT2HEP3a 1-3,18-20 2.88/1 GLU2 LYS2 PHE2 ASP 1-7 1.13-3.82 1 INS4a HT4 HEP2a 3-7 1.68-3 PGLU ASP.6 TYR PHE 1-12 2.12-4.20 2-3 INS3 HT2 HEP3 11-20 5.54-1 GL ASP TYR PHE 1-9 2.43-4.48 2-3 INS4o HT4HEP4β 1-13 2.43-7.0 PGLU ASP.έ TYR PHE 1-6 2.05-6.66 2-3 INS3 HT2 HEP3 4-7 3.3-7.0 GLU TYR VALΛ5LU2 LYS2 PHE 14 H3 H3Λ0H0H0 1 10.58 LYS-ARG LEU PGLU INSO HTO HEP2 SUL-U TYR PHE ASP PGLU 1-10 2.28-5.3 2-3o INS4aHT4hEP4a 2-8,19-20 2.3-4,12 SUL-TYR PHE ASP PGLU 1-11 1.93-6.30 2 INS2aHT4aHEP4a 18 8.95 SUL TYR PHE PGLU ASP 1-7 2,12-4.4 2a INS2βHT4aHEP4A 16-20 9.3-10.25 ASP2 TY PHE 1-6 1.86-6 Oa INSOHTOHEPO 18-20 12-12.6 GLU ASP 14-18 2.38-2.02 PARTICLES INSCHTCHEPC 14-18 2.38
ASP2 TYR PHEtGLU ORN PHE 14-17 4.9-3.1 1 -2 INSCHTCHEPC 17-19 3.1-1.65 GLU2 GLY - HTO 16-19 2.85-2.55
GLU ASP TYR PHE MO 3.17-13.48 1,0-1 INS5aHT3aHEP3« 14,2 ' 9.20,3.32
91 EHJP001 F2QB1 SA0° GLU ASP TYR PHE Rating 1-2 1-2 2-3 2-3 0 2-3 Desc. a a a a a pH
91 EHIPOOI F21 B1 SAQ7 GLU ASP TYR PHE Rating 4 4-5 Desc. a.ag a.P PH
91EHIPOO1F21B1SA07 GLU ASP TYR PHE Rating 3-4 4-5 Desc. »g.p a.P pH
91EHIP0Q1F22B1SA7 GLU ASP TYR PHE Rating 3-4 4-5 Desc. o PH
91EHIP011F23B1SA8 GLU ASP TYR PHE ORN Rating 4 3-4
Desc. a,o a.o
PH
91 EHIP011 F24SA7 GLU ASP TYR PHE ORN Rating 0-1
Desc. H
91EHIP001F26B1SA2A GLU ASP TYR PHE -- Rating 0-1 0 3-4 3-4 4-5 4-5 Desc. a.p a a.o a a PH
91EHIP001F25B1SA6 GLUASPTYR PHE - Rating 0-1 2-3 2-3 0-1 3-4 2-3 Desc. a a a a.o a pH
91EHIP001F26B1SA2A GLU ASP TYR PHE - Rating 2-3 2-3 3-4 2-3 3-4 Desc. a o PH
CA = citric acid a = amorphous HEP = heparin ag = aggregate p = paniculate GM = gum acacia CD = cyclodextrin INS = Insulin GL = gelatin o •= oil Rating: 0 = Worst 5 = Best
Appendix Page 2 of
Sphere Testing of Externally Prepared Proteinoids
SOI NO. COMPOSITION H 0.85 CA 6% AA 0.85 CA 5% AA IHS/CA IHS/AA HEP/CA +GH +GH GH/GL/CD GH/GL/CD +GH
91CTAP001F014B02 GLUASPTYRPHE
91CTAP001F014B02 GLUASPTYRPHE
91CTAP001F014B03 GLUASPTYRPHE
91CTAP001F014B03 GLUASPTYRPHE
91CTAP001F014B03 GLU ASPTYR PHE
91CTAPO01F014B03 GLUASPTYRPHE
91CTAP001F014B04 GLUASPTYR PHE
91CTAPOOIFθ14B05 GLUASPTYRPHE
91CTAP001F014B04 GLUASPTYRPHE
Appendix Page 3 of
Sphere Testing of Externally Prepared Proteinoids
SOI NO. COMPOSITION pH 0.85 CA 5% AA 0.85 CA 5% AA IHS/CA IHS/AA HEP/CA
+ GH + GH GH/GL/CD GH/GL/CD + GH
91EHIP011F16B1 GLU2ASP2TYR2PHE20RN 3-4
91 EHIP00 F20B1 SA07 GLU ASP TYR PHE 3 a.o
91EH1P00IF26B1SA3a
91CTAP001P012B01 GLU ASP TYR PHE
P006B01 GLU2 LYSH2 PHE2 ASP 3 3 a.o a.o
91CTAPR001F01OB01 GLU2LYSH2PHE2ASP 4 a
P003-B01 GLU2 LYSH2PHE2ASP 4-5 3 a a.o
P004-B01 GLU2LYSH2PHE2ASP 4 2-3 a a
91CTAP00QF011B01 GLUASPTYR PHEORN
Appendix Page 4 of
Sphere Testing of Externally Prepared Proteinoids
SOI NO. COMPOSITION pH 0.85 CA 5% AA 0.85 CA 5% AA IHS/CA IHS/AA HEP/CA
+ GH + GH GH/GL/CD GH/GL/CD +GH
91CTAP001F013B01 GLUASPTYR PHE
91CTAP001Fθ14B01 GLUASPTYR PHE
91CTAP001F014B01 GLUASPTYR PHE
91CTAP001P014B01 GLU ASP TYR PHE

Claims

WHAT IS CLAIMED IS;
1. A proteinoid comprising a peptide polymer selected from the group consisting of: i) peptide polymers made from at least one first monomer selected from the group consisting of tyrosine and phenylalanine and from at least one second monomer selected from the group consisting of glutamic acid, pyroglutamic acid, glutamine, and aspartic acid; and ii) peptide polymers made from at least one first monomer selected from the group consisting of tyrosine and phenylalanine; and from at least one second monomer select- ed from the group consisting of glutamic acid, pyroglutamic acid, glutamine, and aspartic acid; and from at least one third monomer selected from the group consisting of lysine, arginine, and ornithine, said proteinoid being a microsphere or microcap- sule-forming proteinoid and being soluble within a selected pH range.
2. The proteinoid of claim 1, said proteinoid having a molecular weight ranging between about 250 and about 2400.
3. The proteinoid of claim 2, said proteinoid having a molecular weight ranging between about 250 and about 400.
4. The proteinoid of claim 1, said proteinoid having between 2 to 20 amino acids.
5. The proteinoid of claim 4, said proteinoid having between 2 to 8 amino acids.
6. The proteinoid of claim 1, wherein said protein- oid is an acid-soluble proteinoid and said second monomer selected from the group consisting of glutamic acid, pyrogluta- mic acid, glutamine, and aspartic acid, and said third monomer is selected from the group consisting of lysine, arginine and ornithine.
7. The proteinoid of claim 1, wherein said protein- oid is a base-soluble proteinoid and said second monomer is selected from the group of glutamic acid, pyroglutamic acid, glutamine, and aspartic acid.
8. A proteinoid carrier comprising a proteinoid comprising i) peptide polymers made from at least one first monomer selected from the group consisting of tyrosine and phenylalanine and from at least one second monomer selected from the group consisting of glutamic acid, pyroglutamic acid, glutamine, and aspartic acid; and ii) peptide polymers made from at least one first monomer selected from the group consisting of tyrosine and phenylalanine; and from at least one second monomer select- ed from the group consisting of glutamic acid, pyroglutamic acid, glutamine, and aspartic acid; and at least one third monomer selected from the group consisting of lysine, arginine, and ornithine, said proteinoid being a microsphere- or microcapsule forming proteinoid and being soluble within a selected pH range.
9. The proteinoid carrier of claim 8, wherein said proteinoid carrier comprises a proteinoid microsphere.
10. The proteinoid carrier of claim 8, wherein said proteinoid carrier comprises a proteinoid microcapsule.
11. The proteinoid carrier of claim 8, wherein said proteinoid is an acid-soluble proteinoid and said second monomer is selected from the group consisting of glutamic acid, pyroglutamic acid, glutamine, and aspartic acid and said second monomer is selected from the group of lysine, arginine and ornithine. 12. The proteinoid carrier of claim 8, wherein said proteinoid is a base-soluble proteinoid and said second monomer is selected from the group of glutamic acid, pyroglutamic acid, glutamine and aspartic acid.
13. The proteinoid carrier of claim 8, wherein said proteinoid carrier having a diameter equal to or less than 10 microns.
14. The proteinoid carrier of claim 8, further encapsulating a cargo.
15. The proteinoid carrier of claim 14, wherein said cargo comprises a fragrance, cosmetic agent, dye, and water soluble vitamin.
16. The proteinoid carrier of claim 14, wherein said cargo is a biologically active agent.
17. The proteinoid carrier of claim 16, wherein said biologically active agent comprises an antigen, monoclonal antibody, calcitonin, erythropoietin, alpha interferon, heparin, insulin, growth hormone, atrial naturetic factor, factor IX, or interleukin-II.
18. A composition comprising a biologically active agent encapsulated within a proteinoid microsphere or microcap- sule, said microsphere or microcapsule comprising a proteinoid comprising i) peptide polymers made from at least one first monomer selected from the group consisting of tyrosine and phenylalanine and from at least one second monomer selected from the group consisting of glutamic acid, pyroglutamic acid, glutamine, and aspartic acid; and ii) peptide polymers made from at least one first monomer selected from the group consisting of tyrosine and phenylalanine; and from at least one second monomer select- ed from the group consisting of glutamic acid, pyroglutamic acid, glutamine, and aspartic acid; and at least one third monomer selected from the group consisting of lysine, arginine, and ornithine, said proteinoid being a microsphere- or microcapsule-forming proteinoid and being soluble within a selected pH range.
19. The composition of claim 18, wherein said pro- teinoid is an acid-soluble proteinoid and said second monomer is selected from the group of lysine, arginine and ornithine.
20. The composition of claim 18, wherein said pro- teinoid is a base-soluble proteinoid and said second monomer is selected from the group of glutamic acid, pyroglutamic acid, glutamine, and aspartic acid.
21. The composition of claim 18, wherein said biologically active agent comprises an antigen, monoclonal antibody, calcitonin, erythropoietin, alpha interferon, heparin, insulin, growth hormone, atrial naturetic factor, factor IX, or interleukin-II.
22. The composition of claim 16, wherein said biologically active agent comprises at least one protein.
23. The composition of claim 22, wherein said protein comprises erythropoietin.
24. The composition of claim 22, wherein said protein comprises alpha interferon.
25. The composition of claim 22, wherein said protein comprises calcitonin.
26. The composition of claim 22, wherein said protein comprises insulin.
27. The composition of claim 22, wherein said protein comprises atrial naturetic factor.
28. The composition of claim 22, wherein said protein comprises interleukin II.
29. The composition of claim 22, wherein said protein comprises -protein.
30. The composition of claim 22, wherein said protein comprises human growth hormone.
31. The composition of claim 22, wherein said protein comprises bovine growth hormone.
32. The composition of claim 16, wherein said biologically active agent comprises at least one polysaccha- ride.
33. The composition of claim 32, wherein said polysaccharide comprises heparin.
34. The composition of claim 16, wherein said biologically active agent comprises an antigen.
35. The composition of claim 16, wherein said biologically active agent comprises aspirin.
36. The composition of claim 16, wherein said biologically active agent comprises a quinolone.
37. The composition of claim 16, wherein said biologically active agent comprises an antimicrobial agent.
38. The composition of claim 17, wherein the monoclonal antibody is murine IgG.
39. The composition of claim 16, wherein said biologically active agent is Factor IX. 40. The composition of claim 16, wherein said biologically active agent is an antibody.
41. A dosage unit form comprising (A) a pharmacological composition according to claim 16; and (B) (a) an excipient, (b) a diluent, (c) a disintegrant, (d) a lubricant, (e) a plasticizer, (f) a colorant, (g) a dosing vehicle, or (h) any combination thereof.
42. A dosage unit form according to claim 41 comprising a tablet, a capsule, or a liquid.
43. A dosage unit form according to claim 41, wherein said dosing vehicle is selected from the group consisting of water, 1,2-propane diol, ethanol or any combina- tion thereof.
44. The composition of claim 16, wherein said biologically active agent comprises interleukin-1.
45. The composition of claim 16, wherein said biologically active agent comprises low molecular weight heparin.
46. The composition of claim 16, wherein said biologically active agent comprises somatostatin.
47. The composition of claim 16, wherein said biologically active agent comprises adrenocorticotropin.
48. The composition of claim 16, wherein said biologically active agent comprises gonadotropin releasing hormone .
49. The composition of claim 16, wherein said biologically active agent comprises oxytocin.
50. The composition of claim 16, wherein said biologically active agent comprises vasopressin.
51. The composition of claim 16, wherein said biologically active agent comprises cromolyn sodium.
52. The composition of claim 16, wherein said biologically active agent comprises vancomycin.
53. The composition of claim 16, wherein said biologically active agent comprises desferrioxamine.
54. The composition of claim 16, wherein said biologically active agent comprises an insecticide.
EP94920205A 1993-06-14 1994-06-14 Proteinoid carriers Withdrawn EP0706375A4 (en)

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US08/076,803 US5578323A (en) 1992-06-15 1993-06-14 Proteinoid carriers and methods for preparation and use thereof
US76803 1993-06-14
PCT/US1994/006735 WO1994028878A1 (en) 1993-06-14 1994-06-14 Proteinoid carriers

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EP0706375A4 true EP0706375A4 (en) 1996-11-06

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US5667806A (en) * 1995-06-07 1997-09-16 Emisphere Technologies, Inc. Spray drying method and apparatus
EP2353588B1 (en) * 2010-01-21 2015-04-15 Agricultural Technology Research Institute A sustained release preparation of factor IX
US10300024B2 (en) 2015-08-10 2019-05-28 Bar-Ilan University Proteinoid compounds, process of preparing same and uses thereof

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AU697044B2 (en) 1998-09-24
AU7108294A (en) 1995-01-03
EP0706375A1 (en) 1996-04-17
JPH08511545A (en) 1996-12-03
WO1994028878A1 (en) 1994-12-22

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