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EP0706375A4 - Excipients proteinoidiques - Google Patents

Excipients proteinoidiques

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Withdrawn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)
English (en)
Other versions
EP0706375A1 (fr
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/fr
Publication of EP0706375A4 publication Critical patent/EP0706375A4/fr
Withdrawn legal-status Critical Current

Links

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

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Abstract

L'invention concerne des excipients proténoïdiques perfectionnés, ainsi que leurs procédés de fabrication et leur utilisation; ces excipients permettent de délivrer des agents pharmaceutiques par voie orale. Les excipients proténoïdiques sont solubles dans des conditions de pH sélectionnées, correspondant à celles existant dans le tube digestif, et sont plus résistants, dans le temps, à la photolyse ou à la décomposition, voire aux deux. Les excipients de proténoïdiques sont élaborés à partir de protéinoïdes contenant de 2 à 20 acides aminés et ayant une masse moléculaire allant de 250 à 2400 daltons environ.
EP94920205A 1993-06-14 1994-06-14 Excipients proteinoidiques Withdrawn EP0706375A4 (fr)

Applications Claiming Priority (3)

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

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EP0706375A1 EP0706375A1 (fr) 1996-04-17
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US6344213B1 (en) 1996-03-29 2002-02-05 Emisphere Technologies, Inc. Compounds and compositions for delivering active agents
US5667806A (en) * 1995-06-07 1997-09-16 Emisphere Technologies, Inc. Spray drying method and apparatus
US6358504B1 (en) 1997-02-07 2002-03-19 Emisphere Technologies, Inc. Compounds and compositions for delivering active agents
EP2353588B1 (fr) * 2010-01-21 2015-04-15 Agricultural Technology Research Institute Préparation à libération prolongée de facteur IX
US10300024B2 (en) 2015-08-10 2019-05-28 Bar-Ilan University Proteinoid compounds, process of preparing same and uses thereof
CN115501346B (zh) * 2022-09-28 2024-04-23 安徽农业大学 一种茶渣蛋白-ε-聚赖氨酸纳米材料和花色苷纳米复合物及制备方法

Citations (5)

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Publication number Priority date Publication date Assignee Title
GB2095994A (en) * 1981-03-06 1982-10-13 Toyo Jozo Kk Preparation having excellent absorption property
EP0068314A1 (fr) * 1981-06-19 1983-01-05 Ciba-Geigy Ag Traitement des troubles arthritiques
WO1988001213A1 (fr) * 1986-08-18 1988-02-25 Clinical Technologies Associates, Inc. Systemes de distribution pour agents pharmacologiques
US4873087A (en) * 1982-01-14 1989-10-10 Toyo Jozo Company, Ltd. Suppository preparation having excellent absorption property
WO1993025583A2 (fr) * 1992-06-15 1993-12-23 Emisphere Technologies, Inc. Proteinoides vecteurs ainsi que leurs procedes de preparation et d'utilisation

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CA1077842A (fr) * 1975-10-09 1980-05-20 Minnesota Mining And Manufacturing Company Excipient albumineux pour medicament
US4684524A (en) * 1984-03-19 1987-08-04 Alza Corporation Rate controlled dispenser for administering beneficial agent

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2095994A (en) * 1981-03-06 1982-10-13 Toyo Jozo Kk Preparation having excellent absorption property
EP0068314A1 (fr) * 1981-06-19 1983-01-05 Ciba-Geigy Ag Traitement des troubles arthritiques
US4873087A (en) * 1982-01-14 1989-10-10 Toyo Jozo Company, Ltd. Suppository preparation having excellent absorption property
WO1988001213A1 (fr) * 1986-08-18 1988-02-25 Clinical Technologies Associates, Inc. Systemes de distribution pour agents pharmacologiques
WO1993025583A2 (fr) * 1992-06-15 1993-12-23 Emisphere Technologies, Inc. Proteinoides vecteurs ainsi que leurs procedes de preparation et d'utilisation

Non-Patent Citations (1)

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Title
See also references of WO9428878A1 *

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JPH08511545A (ja) 1996-12-03
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WO1994028878A1 (fr) 1994-12-22
CA2164957A1 (fr) 1994-12-22
AU697044B2 (en) 1998-09-24

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