AP665A

AP665A - Novel "burst free" sustained release poly-(lactide/glycolide microspheres).

Info

Publication number: AP665A
Application number: APAP/P/1997/001088A
Authority: AP
Inventors: Jeyanthi Ramasubbu
Original assignee: United States Governmnet Represented By The Secretary Of The Army
Priority date: 1996-01-24
Filing date: 1996-11-18
Publication date: 1998-08-19
Also published as: EP0817619A1; NZ325561A; WO1997026869A1; BR9607752A; AU722884B2; AP9701088A0; JPH11509862A; CN1188408A; AU1410497A; EP0817619A4; BR9607752B1; CN100391445C

Abstract

Novel burst-free, sustained release biocompatible and biodegradable microcapsules which can be programmed to release their active core for variable durations ranging from 1-100 days in an aqueous physiological environment. The microcapsules are comprised of a core of polypeptide or other biologically active agent encapsulated in a matrix of poly(lactide/glycolide)copolymer as a blend of uncapped (free carboxyl end group)and end-capped forms ranging in rations from 100/0 to 1/99.

Description

NOVEL, BURST-FREE SUSTAINED RELEASE POLY(LACTIDE/GLYCOLIDE) MICROSPHERES

Z. GOVERNMENT INTERESTS

The invention described herein nay be manufactured, licensed and used by or for governmental purposes without the payment of any royalties to us there n.

ZZ. CROSS REFERENCE

This application is a continuation-in-part of U.S. Patent application Serial No. 08/446,149, filed May 22, 1995.

ZZI. FIELD OF THE INVENTION

This invention relates to providing novel biocompatible and biodegradable microspheres for burst-free programmable sustained release of biologically active agents, inclusive of polypeptides, over a period of up to 100 days in an aqueous physiological environment.

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IV. BACKGROUND OP TBS INVENTION

Several publications and patents are available for sustained release of active agents iron biodegradable polymers, particularly, poly(lactide/glycolides) (PLGA). Prior usages of PLGA for controlled release of polypeptides have involved the use of molar ratios of lactide/glycolide (L/C) of 75/25 to 100/0 for molecular weights >20,000. Further prior art preparations of PLGA utilized fillers or additives in the inner aqueous layer to improve the stability and encapsulation efficency and/or to increase the viscosity of the aqueous layer, thereby modulating polymer hydrolysis and the biologically active agent or polypeptide release.

In addition, the prior art use of PLGA copolymers were endcapped, in that the terminal carboxyl end groups were blocked. In these end-capped co-polymers, the microcapsule preparations exhibited a low to moderate burst release of - 10-40% of the entrapped polypeptide in the first 24 hours after placement in an aqueous physiological environment. In part, these characteristics are due to the use of fillers in the inner aqueous phase.

Further, a 1-month release of polypeptide is known with the use of a 75/25 co-polymer of PLGA of Mw <20,000.

Investigations in controlled release research has been proceeding especially to obtain a 1 to 2 month delivery system

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Printed from Mimosa 09/24/1997 15:10:05 page -4ΑΡ. ύ 0 6 6 5 for biologically active agents or polypeptides using poly(lactide/glycolide) polymers. However, most of these systems have one or more of the following problems: Poor encapsulation efficency and large 'burst release' followed by an intermediate 'no release' or 'lag phase' until the polymer degrades. In general, release from these polymers occur over a period from about 4 weeks to about several months. In addition, in order to achieve this release a 50/50 copolymer of HH > 30,000 or s 75/25 copolymer of Mw > 10,000 are employed which often results in residual polymer remaining at the site of administration long after the release of active core.

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A

V. SUMMARY or THE INVENTION

This invention provides biocompatible and biodegradable nicrospheres that have been designed for novel, burst free, programmable sustained release of biologically active agents, including polypeptides over a period of up to 100 days in an aqueous physiological environment.

Unlike currently available release systems, which rely on 10 the use of fillers/additives such as gelatin, albumin, dextran, pectin, polyvinyl pyrrolidone, polyethylene glycol, sugars, etc., and .are still prone to low encapsulation efficiencies and burst effects, this invention achieves high encapsulation and burstfree release without the use of any additive. In this invention, burst-free, programmable sustained release is achieved through the use of a unique blend of the 'uncapped' and end-capped forms of poly(lactide/glycolide) polymer in the molecular weight range of 2,000 to 60,000 daltons.

In general, microspheres described in this invention are produced by a unique emulsification technique wherein an inner water-in-oil (w/o) emuleion is stabilized by dispersing in a solvent-saturated aqueous phase containing an emulsion stabilizer. A ternary w/o/w emulsion ie then formed by emulsifying the above w/o emulsions in an external pre-cooled aqueous phase containing an o/w emulsifier. Essentially, the

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Printed from Mimosa 09/24/1997 15:10:05 page-6AP.00665 *»« Γ inner w/o emulsion is comprised of an aqueous layer containing from - 2 to about 20% (w/w) of the active agent to be entrapped and an oil layer containing poly(lactide/glycolide) copolymer in concentrations ranging from - 5 to about— 50% (w/w oil phase).

The copolymer includes molecular weight ranging from 2,000 to about 60,000 daltons, with molar composition of lactide/glycolide from 90/10 to 40/60 and a blend of its uncapped and end-capped forms in a ratio of 100/0 to 1/99. Very high encapsulation efficiencies of about 80 to 100% are achieved depending on polymer molecular weight and structural form.

Programmable release of active core over variable durations between 1-100 days is achieved by a judicious selection of process parameters such as polymer concentration, peptide concentration and the agueous/oil phase ratio.

This invention is particularly suitable for high encapsulation efficiencies and burst-free, continuous programmable release of polypeptides of molecular weights ranging from 1,000 to about 250,000 daltons, and also other biologically active agents over a period of 1-100 days. A uniqueness of the invention is that when using a 100/0 blend of the uncapped and capped polymer, the final phase of active core release is concurrent with the complete solubilization of the polymer to innocuous components, such as lactic and glycolic acids. This is ΐ

a significant advantage over the currently available 30 day 25 release systems wherein a major regulatory concern is about toxicity of residual polymer at the site of administration, long

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Printed from Mimosa 09/24/1997 15:10:05 page -7AP.00665 s+i after release of the active core.

The microcapsules described in this invention are suitable for administration via several routes such as parenteral (intramuscular, subcutaneous), oral, topical, nasal, rectal and vaginal routes.

▼X. BRIEF DESCRIPTION 07 DRAWINGS

FIG. 1 shows a comparison of drug release from a conventional system versus a controlled release system. Peak and valley levels from conventional administrations are shown, in contrast to the steady therapeutic levels from the controlled release administration.

FIG. 2 shows a scanning electron micrograph of PLGA microspheres prepared by the process described in the invention using 50/50 uncapped polymer of Mw 8-12k dalton and shows superior sphere morphology, sphere integrity, and narrow size distribution.

FIG. 2a shows a scanning electron micrograph of PLGA microspheres prepared by conventional solvent evaporation method using a 50/50 12k uncapped polymer of Mw 8-12k dalton.

FIG. 3 shows cumulative Histatin release from PLGA microspheres, wherein release profiles from several batches are prepared using 50/50, uncapped polymer (of Mw 8-12k dalton) and wherein the process parameters are varied to modulate release between 1 and 35 days.

FIG. 4 shows a scanning electron micrograph of solid, smooth

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Printed from Mimosa 09/24/1997 15:10:05 page -8AP . Ο Ο 6 6 5 spherical surfaces of PLGA microspheres prepared by the method of in the invention using 50/50, end-capped polymer (of Mw 30-40k daIton).

FIG. 5 shows cumulative Histatin release from PLGA 5 microspheres, wherein the release profiles are from several batches prepared using 50/50, uncapped and end-capped polymer of Mw 30-40k daltons, and wherein the process parameters are varied to modulate release between 2β to €0 days.

FIG. 6 shows cumulative Histatin release from PLGA 10 microspheres, wherein combined release profiles from several batches have been prepare' using 50/50, uncapped and end-capped polymer of Mw 8-40k daltons, while varying the process parameters to modulate release between 1 and 60 days.

FIG. 7 shows a cumulative percent release of LHRH from PLGA 15 microspheres prepared using uncapped polymer of Mw 8-12 daltons.

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to the design of biocompatible and biodegradable microspheres for novel, programmable sustained release of biologically active agents, including polypeptides over a period of up to 100 days in an aqueous physiological environment with little or no burst release.

Unlike currently available release systems which rely on the use of fillers/additives such as gelatin, albumin, dextran, pectin, polyvinyl pyrrolidone, polyethylene glycol, sugars, etc., and are still prone to low encapsulation efficiencies and burst effects, this invention achieves high encapsulation efficiency

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Printed from Mimosa 09/24/1997 15:10:05 page -9AP.00665 and 'burst-free' release without the use of any additive. In this invention, burst-free, programmable sustained release is achieved through the use of a unique blend of the 'uncapped' and endcapped forms of poly (lactide/glycolide) polymer.

The 'uncapped' form refers to poly(lactide/glycolide) with free carboxyl end groups which renders the polymer more hydrophilic compared to the routinely used end-capped form. Currently used 'end-capped' polymer hydrates between 4-12 weeks depending on the molecular weight, resulting in an intermediate 'no release' or a 'lag phase'. The uncapped polymer hydrates typically between 5 to 60 days depending on the molecular weight, thue releasing its core continuously without a lag phase, λ careful blend of the two forms and appropriate molecular weights and L/G ratios, results in a continuous release between 1 to 100 days. In addition, release within this time is programmable by a judicious selection of process parameters such as polymer concentration, peptide concentration and the agueous/oil phase ratio.

The coploymer in this invention includes molecular weight ranging from 2,000 to 60,000 daltons, a lactide/glycolide ratio of 90/10 to 40/60 and a blend of the uncapped/capped forms in the ratio of 100/0 to 1/99. The molecular weight of the polypeptide may be in the range of 1000 to 250,000 daltons while that of other biologically active agents may range from 100 to 100,000 daltons.

Microcapsules described in this invention are prepared by a

0 I 0/Z6 ,'d/dV

Printed from Mimosa 09/24/1997 15:10:05 page-10AP . Ο Ο 6 6 5 unique aqueous emulsification techinique which has been developed for use with the uncapped polymer to provide superior sphere morphology, sphere integrity and narrow size distribution. This is accomplished by first preparing an inner water-in-oil (w/o) by mixing the solutions of polymer in an organic solvent such as methylene chloride and the biologically active agent in water. This is followed by stabilization of the w/o emulsion in a solvent-saturated aqueous solution containing an o/w emulsifier such as polyvinyl alcohol. A ternary emulsion is then formed by emulsifying the w/o emulsion in an external aqueous phase containing the same emulsifier as above at concentrations ranging from 0.25 - 1% w/v. Microcapsules are hardened upon solvent removal by evaporation, rinsed to remove residual emulsifier and lyophilized. Low temperature is used both at the time of primary emulsification (w/o emulsion formation) and during the formation of the final w/o/w emulsion to achieve stable emulsion and superior sphere characteristics.

In the context of the invention, a biologically active agent is any water-soluble hormone drugs, antibiotics, antitumor agents, antiinflammatory agents, antipyretics, analgesics, antitussives, expectorants, sedatives, muscle relaxants, antiepileptics, antiulcer agents, antidepressants, antiallergic drugs, cardiotonics, antiarrhythmic drugs, vasodilators, antihypertensives, diuretics, anticoagulants, antinarcotics, etc.

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More precisely, applicants have discovered a pharmaceutical composition and process with the following itemized features:

1. A controlled release aicrocapsule pharmaceutical formulation for burst-free, sustained, programmable release of a biologically active agent over a duration from 1-100 days, comprising an active agent and a blend of uncapped and end-capped biodegradable poly(lactide/glycolide) .

2. The pharmaceutical formulation of item 1, wherein the biodegradable poly(lactide/glycolide) is a blend of uncapped and capped forms, in ratios ranging from 100/0 to 1/99.

3. The microcapsules of items 1 or 2 wherein the copolymer (lactide to glycolide L/G) ratio for uncapped and endcapped polymer is 52/48 to 48/52.

4. The microcapsules of items l or 2 wherein the copolymer

L/G ratio for uncapped and end-capped polymer is 90/10 to 40/60.

5. The microcapsules of items 1 or 2 or 3 or 4 wherein the molecular weight of the copolymer is between 2,000-60,000 daltons.

6. The microcapsules of items 1 or 2 or 3 or 4 or 5 wherein the biologically active agent is a peptide or polypeptide.

7. The microcapsules of item 6, wherein said polypeptide is histatin consisting of 12 amino acids and having a molecular weight of 1563.

8. The microcapsules of items 1 or 2 or 3 or 4 or 5 or 6 characterized by the capacity to completely release histatin in

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Printed from Mimosa 09/24/1997 15:10:05 page -12AP.00665 an aqueous physiological environment from 1-35 days with a 100/0 blend of uncapped and end-capped poly(lactide/glycolide) having a L/G ratio of 48/52 to 52/48, and a molecular weight <15,000.

9. The microcapsules of items 1 or 2 or 3 or 4 or 5 or 6 5 characterized by the capacity to completely release histatin in an aqueous physiological environment from 18-40 days with a 100/0 blend of uncapped and end-capped poly(lactide/glycolide) having a L/G ratio of 48/52 to 52/48 and a molecular weight range of 28,000-40,000.

10. The microcapsules of items l or 2 or 3 or 4 or 5 or 6 characterized by the capacity to release up to 90* of the histatin in an aqueous physiological environment from 28-70 days with a 0/100 blend of uncapped and end-capped poly(lactide/glycolide) having a L/G ratio of 48/52 to 52/48 and a molecular weight range of 10,000-40,000 daltons.

11. The microcapsules of items 1 or 2 or 3 or 4 or 5 or 6 characterized by the capacity to release up to 80% of histatin in an aqueous physiological environment from 56-100 days with a 0/100 blend of uncapped and end-capped poly{lactide/glycolide) having a L/G ratio of 75/25 and a molecular weight of < 15,000 daltons.

12. The microcapsules of items 7 or 8 or 9 or 10 or 11 having analogs of histatin with chain lengths of from 11-24 amino acids of molecular weights from 1,500-3,000 daltons and characterized by the following structures:

1. dshaxrhhgyxrxfhexhhshrgy

8 0 t 0 / L 6 /d/dV

Printed from Mimosa 09/24/1997 15:10:05 page-13AP.00665

XRHHGYXRKFHEKHHSHRGYR

XRHHGYKRKFHEKHHSHR

RKFHEXHHSHRGYR

AKRHHGYKRKFH «AKRHHGYKRKFH

KRHHGYKRKF

2.

3.

4.

5.

6.

7.

* D-amino acid

13. The microcapsules of items 1 or 2 or 3 or 4 or 5 wherein the biologically active agent is a polypeptide Leutinizing hormone releasing hormone (LHRH) that is a decapeptide of molecular weight 1182 in its acetate form, and having the structure:

p-EHWSYGLRPG

14. The microcapsule of items 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 having a molecular weight of from 1,000 to 250,000 daltons.

15. The microcapsules of items 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 wherein release profiles of variable rates and durations are achieved by blending uncapped and capped nicrospheres as a cocktail in variable amounts.

16. The microcapsules of items 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 wherein release of profiles of variable rates and duration are achieved by blending uncapped and capped polymer in different ratios within the same microshreres.

17. The microcapsules of items 6 or 7 or 8 or 9 or 10 or 11

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Printed from Mimosa 09/24/1997 15:10:05 page-14AP.00665 ¹³ or 12 or 13 or 14 or 15 or 16 wherein the entrapped polypeptide is any of the vaccine agents against enterotoxigenic E. coli (ETEC) such as CFA/I,CFA/II,CS1,CS3,CS6 and CS17 and other ETECrelated enterotoxins.

IB. The nicrocapsules of items 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 wherein the entrapped polypeptide consists of peptide antigens of molecular weight range of about 800-5000 daltons for immunization against enterotoxigenic E. coli (ETEC).

19. The microcapsules of items 1 or 2 or 3 or 4 or 5 wherein said biologically active agents are selected from the group consisting of water-soluLle hormone drugs, antibiotics, antitumor agents, anti inflammatory agents, antipyretics, analgesics, antitussives, expectorants, sedatives, muscle relaxants, antiepileptics, antiulcer agents, antidepressants, antiallergic drugs, cardiotonics, antiarrhythmic drugs, vasodilators, antihypertensives, diuretics, anticoagulants, and antinarcotics, in the molecular weight range of 100-100,000 daltons.

20. The microcapsules of items 1 or 2 or 3 or 4 or 5 or 6 or

7 or 8 wherein said biodegradable poly (lactide/glycolide, is in an oil phase, and is present in about 1-50% (v/v).

21. The microcapsules of items 1 or 2 or 3 or 4 or 5 or 6 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 wherein concentration of the active agent is in the range of o.l to about

60% (v/v).

22. The microcapsules of items 1 or 2 or 3 or 4 or 5 or 6 or

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Printed from Mimosa 09/24/1997 15:10:05 page -15AP.00665 or 9 or io or ll or 12 or 13 or 14 or 15 or 16 or 17 wherein a ratio of the inner aqueous to oil phases is about 1/4 to l/40(v/v).

23. λ process for preparing controlled release microcapsule 5 formulations characterized by burst-free, sustained, programmable release of biologically active agents comprising: Dissolving biodegradable poly (lactide/glycolide), in uncapped form in methylene chloride, and dissolving a biologically active agent or active core in water; adding the aqueous layer to the polymer solution and emulsifying to provide an inner water-in-oil (w/o, emulsion; stabilizing the w/o emulsion in a solvent-saturated aqueous phase containing a oil-in-water (o/w) emulsifier; adding said w/o emulsion to an external aqueous layer containing oil-inwater emulsifier to form a ternary emulsion; and stirring the resulting water-in-oil-in-water (w/o/w) emulsion for sufficient time to remove said solvent, and rinsing hardened roicrocapsules with water and lyophilizing said hardened microcapsules.

24. A process for preparing controlled release microcapsule formulations characterized by burst-free, sustained, programmable release of biologically active agents comprising:

dissolving biodegradable poly(lactide/glycolide) in endcapped form in methylene chloride, and dissolving a biologically active agent or active core in water; adding the aqueous layer to the polymer solution and emulsifying to provide an inner water25 in-oil emulsion; stabilizing the w/o emulsion in a solventsaturated aqueous phase containing a oil-in-water (o/w)

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Printed from Mimosa 09/24/1997 15:10:05 page -16AP.00665 emulsifier; adding said v/o emulsion to an external aqueous layer containing oil-in-water emulsifier to form a ternary emulsion; end stirring a resulting water-in-oil-water (w/o/w) emulsion for sufficient time to remove said solvent; and rinsing hardened microcapsules with water; and lyophilizing said hardened microcapsules.

25. The process of items 23 or 24 wherein a solventsaturated external aqueous phase is added to emulsify the inner v/o emulsion prior to addition of the external aqueous layer, to provide microcapsules of narrow size distribution range between 0.05-500μΠ.

'26. The process of items 23 or 24, wherein a low temperature of about 0-4*C is provided during preparation of the inner w/o emulsion, and a low temperature of about 4-20*c is provided during preparation of the w/o/w emulsion to provide a stable emulsion and high encapsulation efficiency.

27. The process of items wherein a 100/0 blend of uncapped and end-capped polymer is used to provide release of the active core in a continous and sustained manner without a lag phase.

28. The microcapsules of items 6, wherein, when the entrapped polypeptide is active at a low pH, such as LHRH, « adrenocorticotropic hormone, epidermal growth factor, calcitonin released polypeptide is bioactive.

29. The microcapsules of items 6 or 7 or 8 or 9 or 10 or ll, 25 wherein, when entrapped peptide such as histatin is inactive at a low pH, a pH-stabilizing agent of inorganic salts are added to

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Printed from Mimosa 09/24/1997 15:10:05 page -17AP.00665 the inner aqueous phase to maintain biological activity of the released peptide.

30. The microcapsules of items 6 or 7 or 8 or 9 or 10 or 11 wherein, when entrapped polypeptide such as histatin is inactive at a low pH, a non-ionic surfactant such as polyoxyethylene sorbitan fatty acid eeters (Tween 80, Tween 60 and Tween 20) and polyoxyethylene - polyoxypropylene block copolymers (Pluronice) is added to the inner aqueous phase to maintain biological activity of the released polypeptide.

31. The microcapsules of items 29, wherein placebo spheres loaded with the pH-stabilizing agents are coadministered with polypeptide-loaded spheres to maintain the solution pH around the microcapsules and preserve the biological activity of the released peptide in instances where the addition of pH15 stabilizing agents in the inner aqueous phase is undesirable for the successful encapsulation of the acid pH sensitive polypeptide.

32. The microcapsules of item 30 wherein placebo spheres loaded with non-ionic surfactant are coadministered with polypeptide-loaded spheres to maintain biological activity of the released peptide where the addition of non-ionic surfactants in t the inner aqueous phase is undesirable for successful encapsulation of the acid pH sensitive polypeptide.

33. The microcapsules of items 1 or 2 or 3 or 4 or 5 or 6 or

8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 comprising a blend of uncapped and capped polymer, wherein complete

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Printed from Mimosa 09/24/1997 15:10:05 page-18AP.00665 solubilization of the copolymer leaves no residual polymer at the site of administration and occurs concurrently with the complete release of the entrapped agent.

34. A process of using microcapsules of items 1 or 2 or 3 or

4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or or 17 or 18 or 19 or 20 for human administration via parenteral routes, such as intramuscular and subcutaneous.

35. A process of using microcapsules of items l or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or

16 or 17 or 18 or 19 or 20 for human administration via topical route.

-36. A process of using microcapsules of items l or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or or 17 or IB or 19 or 20 for human administration via oral routes.

37. A process of using microcapsules of items 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 for human admininstration via nasal, transdermal, rectal, and vaginal routes.

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Conservation of bloaetlvltv of polypeptides As the polymer degrades rapidly, there ie a preciptitous drop in pH accompanied by the release of soluble oligomers in the 5 microenvironment which may affect the biological activity of acid pH-sensitive peptides/proteins. Zn such instances, biological activity can be maintained by the use of inorganic salts yor buffering agents in the inner aqueous phase codissolved with the peptide.

The following unique advantages are characteristics of this invention:

1. Burst-free, prolonged, sustained release of polypeptides and other biocompatible and biodegradable microcapsulee up to 100 days in an aqueous physiological environment without the use of additives in the core.

2. Release of active core programmable for variable durations over 1-100 days, by using a blend of uncapped and capped polymer of different molecular weights and copolymer ratio, and by manipulating the process parameters.

3. Complete release of the active core is concurrent with complete solubilization of the carrier polymer to innocuous components, such as lactic and glycolic acids, especially when using a 100/0 blend of uncapped/capped polymer. This is of tremendous significance, as most biodegradable polymers currently used for 1-30 day delivery, do not degrade completely at the end of the intended release duration, thereby causing serious concern

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Printed from Mimosa 09/24/1997 15:10:05 page -20AP.00665 of regulatory authorities on the effects of residual polymer at the site of administration.

4. Ease of administration of the microcapsules in various dosage forms via several routes, such as parenteral (intramuscular and sucutaneous), oral, topical, nasal, vaginal, etc.

The hydrophilic horao-and co-polymers based on D,L-lactide and glycolide contains hydrophilic adjusted homo-and co-polymers with free carboxylic end groups, and is characterized by the formula:

Poly (D,T.-lactide-eo*gly col Ide) 50:50

Γ fll		Γ fi 1
ί		fl

J L ^CH> J	n

(C₃H₄O₂)_n(.C₂H₂O₂)_n ^n!B ’ ^1:1

Wherein Z= Molecular Weight/130; for example Z-92 for Mw 12,000 and 262 for Mw 34,000.

While the molar ratio of the lactide to glycolide may vary, it is most preferred that the lactide to glycolide copolymer ratio be 50:50.

Reference is now made to FIG. 1 which depicts a blood-drug concentration versus time graph that shows conventional drug administration using a series of dosages compared to an ideal controlled release system. Unfortunately, many drugs have a

AP/P/ 97/01088

Printed from Mimosa 09/24/1997 15:10:05 page-21AP.00665 therapeutic range, above which they are toxic and below which they are ineffective. Oscillating drug levels that are commonly observed following systemic administration causes alternating periods of ineffectiveness and toxicity. A sustained-release encapsulated biologically active agent or polypeptide preparation, ideally, will maintain the drug in the desired therapeutic range by means of a single dose, as depicted in the THERAPEUTIC RANGE in FIG.l , where the ideal case for controlled release is shown.

In FIG. 2, there is shown a scanning electron micrograph of

PLGA microspheres prepared using 50/50 uncapped polymer of Hw 812k dalton. The uncapped polymer has solid, smooth spherical surfaces, and is suited to provide a burst free” release system.

Table I is a summarization of the microsphere process description for preparing a peptide system (Histatin peptide) having a controlled release over the course of from 1 to 100 days.

Release profiles can be modified by a judicious blend of uncapped and capped polymers either in separate microspheres or in the same microspheres. Release from microcapsule formulations 1 through 21 listed in Table 1, occur independently of each other and hence the cumulative release from blends of these formulations are additive. By blending several formulations of uncapped and end-capped microspheres, release curves of any desired duration can be tailored. In addition, based on the release characteristics of uncapped and end-capped polymers,

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Printed from Mimosa 09/24/1997 15:10:05 page-22AP.00665 blending of the two forme in a single formulation comprising different ratios of uncapped to capped polymer, would significantly influence the polymer hydration and hence release of the active core thereby providing release curves of any desirable pattern. Manipulation of polymer hydration and degradation resulting in modulation of release of active core is achieved by the addition of uncapped polymer to end-capped polymer in amounts as low as It up to loot.

Λ

I . : -Js

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Printed from Mimosa 09/24/1997 15:10:05 page-23AP.00665 ?2

Table 1- Hlcrocapeule compositions containing Histatin polypeptide

Entulaif icatl on Technique		<	<	<	<
c u « 0 ~ W · «Η o Λ9 « 4J C «C * 2 w d a. κ *		1:20	o ΓΜ	O CM	«d
Theoretic al peptide Core Load (»1		in	CM	in	m
	υ 2 0 C S'? u .-io	β n	tn eo	01	oo m
e
0 4-> c- kl ϋ β w o	X . Κ.Γ* 0 j o X 9 2 *	CM W	CM e*	**1	CM w
u 1 o 0b	L/C Ratio t Type	n' os/os	n* os/os	5O/SO	O in %. O 9 in »
o ^4 w4 •
υ c			CM

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AP.00665

a	a	«	a	a	a	a	a	a
o ‘	o	o	©	o	o	o	o	o
w			**				r-l
	•4
rS			w	*4			W
								n

in	*n	in	in	in	in	in	m	PM
				m
	o	O	O	w	o		o
T*·			w	PM		r»		f*
V	9		CM	hi	PM	PM	CM	PM
r>	r>	m	e-t		rH	w		rM
O	©	o	in	in	o	O	©	o
in	in	m	PW	ΓΜ	in	in	in	in
	*4.	*>►	**»					***.
O	o	o	in	in	o	o	O ©	O ©
in	in	m	r*	f*	in	in	m »	in *

•	•	•	•	•	o		PM	m
in	©	r*	a	σ»	K			w

AP/P/ 9 7 / 0 1 0 88

Printed from Mimosa 09/24/1997 15:10:05 page-25AP.00665

B	β	B	B	B	B	a	B
o	©	O	O	O	o	©	©
Ft	Ft	•t	Ft	Ft	w	Ft

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AP/P/ 9 7 / 0 1 0 88

Printed from Mimosa 09/24/1997 15:10:05 page -27·

AP.00665

While referring to Table 1 in conjunction with FIG. 3, it can be seen that the cumulative Histatin release from PLGA microspheres from several batches prepared using 50/50 and 75/25 uncapped and end-capped, polymer modulates release between 1 to

100 days by varying the process parameters. 1-35 days by uncapped

50/50, 18-56 days by capped 50/50 and 56-100 days by capped 75/25.

In referring to FIG. 4, a view is provided through a scanning electron micrograph of PLGA microspheres designed for a one to two month release system prepared using end-capped polymer of Mw 30-40k daltons.

FIG. 5 depicts the cumulative Histatin release from PLGA microspheres, in which the release profiles are from several batches prepared using 50/50, uncapped and capped polymer, and varying the process parameters to modulate release between 28 to 60 days.

Figure 6 represents cumulative Histatin release from PLGA microspheres --- these combined release profiles are from several batches prepared using 50/50 uncapped and capped polymer, and varying the process parameters to modulate release between 1-60 days.

In the context of the invention, a biologically active agent is any water-soluble antibiotics, antitumor agents, antipyretics analgesics, anti-inflammatory agents, antitussives, expectorants, sedatives, muscle relaxants, anti epileptics, antiulcer agents,

AP/P/ 8 7/01088

Printed from Mimosa 09/24/1997 15:10:05 page-28AP.00665 anti-depressants, anti-allergic drugs, cardiotonics, antiarrhythmics drugs, vasodilators, antihypertensives, diuretics, anticoagulants, hormone drugs, anti-narcotics, etc.

In general, burst free sustained release delivery of biologically active agents from PLGA microshperes is accomplished in the context of this invention using of 90/10 to 40/60 molar ratios, and ratios of uncapped polymer to end-capped polymer of 100/0 to 1/99.

In general, the approaches for designing the biologically active agents encapsulated in the uncapped and combination uncapped/end-capped PLGA microspheres and characteristics of these encapsulants are briefly set forth below as follows:

1. Providing PLGA microspheres of surface morphologies using 50/50 uncapped and capped polymers of Mw - 8-40K daltons as shown in Figs. 2 and 4.

2. Providing in vitro release of a polypeptide, Histatin from PLGA microspheres, as shown in Figs. 3 and 5, using uncapped and capped polymer of Mw - 8-40K daltons and molar ratios such as 50/50 and 75/25.

For example, design of a 1-12 weeek bioactive compound release system is achieved using PLGA with the following specifications:

1. Polymer molecular weight:

-about 2-60K daltons

2. Copolymer molar ratio (L/G) :

- 90/10 to 40/60

3. Polymer end groups:

- uncapped and /or end-capped and combining judiciously within the following parameters:

4. Polymer concentration

- from 5 to 50%

5. Inner aqueous to oil phase ratio:

- 1:5 to 1:20 (v/v)

6. Peptide loads:

- from 2 to about 40% (w/w polymer)

AP/P/ 9 7 / 0 1 0 88

Printed from Mimosa 09/24/1997 15:10:05 page-29AP.00665 and by using the unique aqueous emulsification method described in the invention.

The uniqueness and novelty of invention may generally be 5 summarized in a brief way as follows:

1. Use of uncapped poly(lactide/glycolide) to achieve burstfree, continuous, sustained, programmable release of biologically active agents over 1-100 days.

2. Use of a unique aqueous emulsification system to achieve superior microsphere characteristics such as uniform sphere morphology end narrow size distribution.

3. Burst-free, prolonged, sustained release of polypeptides and other biologically actice agents from biocompatible and biodegradable microcapsules up to 100 days in an aqueous physiological environment without the use of additives in the inner core.

4. Release of active core programmable for variable durations over 1-100 days by using a blend of uncapped and capped polymer for different molecular weights and copolymer rations and manipulating the process parameters.

5. Complete release of the active core concurrent with complete solubilization of carrier polymer to innocuous components such as lactic and glycolic acids, especially when using a 100/0 blend of uncapped/capped polymer. This is of tremendous significance as most biodegradable polymers currently in use for 1-30 day delivery, do not degrade completely et the end of the intended release duration causing serious concern for regulatory authorities on the effects of residual polymer at the site of administration.

6. Ease of administration of the microcapsules in various dosages forms via several routes such as parenteral (intramusclar and subcutaneous), oral, topical, nasal, vaginal, etc.

The following examples are illustrative of, but not limitations upon the microcapsule compositions pertaining to this invention.

AP/P/ 9 7 / 0 1 0 88

Example l

Polylactide/glycolide (PLGA) microcapsules are prepared by a unique aqueous emulsification technique which has been developed for use with the uncapped polymer to provide superior sphere morphology, sphere integrity and narrow size distribution (See

Figures la and lb). This is accomplished by dissolving the polymer in a chlorinated hydrocarbon solvent such as methylene chloride and dissolving the biologically active agent in water. A

Printed from Mimosa 09/24/1997 15:10:05 page -30AP.00665 w/o emulsion is then formed by mixing the solutions of polymer and the active agent by sonication, followed by emulsion stabilization in a solvent - saturated aqueous solution containing polyvinyl alcohol. A ternary emulsion is then formed by emulsifying the w/o emulsion in an external, pre-cooled aqueous phase containing polyvinyl alcohol (0.25 - 1% w/v). Microcapsules are hardened upon removal of solvent by evaporation, rinsed to remove any residual emulsifier, and then lyophilized.

Table 1 lists the microcapsule compositions, Nos. 1-21 thus prepared, consisting of a biologically active polypeptide,

Kistatin (composed of 12 amino acids and a molecular weight of 1563) and blends of uncapped and capped polymer of ratios 100/0 to 1/99, and having a lactide/glycolide ratio of 90/10 to 40/60, and a molecular weight range between 2000 to 60,000 daltons.

0 Example.,!

Microcapsule compositions are prepared as described in Example 1 wherein the copolymer L/G ratio is 48/52 to 52/48, and the ratio of uncapped/capped polymer is 100/0. The active core is

Histatin (Mw 1563), the polymer molecular weight is < 15,000 and the polymer concentrations vary from 7% to - 401 w/w.

Compositions 1,2,4 12-14 and 16-18 are listed in Table 1.

Release profiles of the active core from the compositions in an aqueous physiological environment, such as phosphate-buffered saline, pH 7.0 maintained at 37 ± 1*C are plotted as cumulative percentage release versus time, and presented in Figure 2.

Burst-free, variable release from 1-35 days is achieved by varying the polymer concentration from 7 to - 40% w/w in the oil phase.

Example 3

Microcapsule compositions are prepared as described in Example 2, wherein the aqueous /oil ratio is varied from 1/4 to 1/20 (v/v). Compositions 1,2,4 and 12 are listed in Table 1.

Release profiles of the active core from the compositions in an aqueous physiological environment described in Example 2 are plotted as cumulative percentage release versus time, and presented in Figure 2.

Burst-free, continuous release from 1-35 days, with different onset and completion times are achieved by selecting

AP/P/ 9 7 / 0 1 0 88

Printed from Mimosa 09/24/1997 15:10:05 page-31AP . 0 0 6 6 5 different v/o ratios in the inner core.

Example 4

Microcapsule compositions are prepared as described in

Example 2, wherein the polymer molecular weight is 28,000-40,000 and polymer concentrations vary from 5% to - 15% v/w.

Compositions 19-21 are listed in Table 1.

Release profiles of the active core from the compositions in an aqueous physiological environment are described in Example 2 are plotted as cumulative percentage release versus time and presented in Figure 3.

Burst-free, variable release from 18-40 days is achieved by varying the polymer concentration.

Example 5

Microcapsule compositions are prepared as described in

Example 2, wherein the ratio of uncapped/capped polymer is 1/99 and polymer concentrations vary between 5% to - 12% w/w. Compositions 10 and 11 are listed in Table 1.

Release profiles of the active core from the compositions in an aqueous physiological environment are described in Example 2, and plotted as cumulative percentage release versus time and presented in Figure 2.

Burst-free, variable release from 28-70 days is achieved by varying the polymer concentration in the oil phase.

Example 6

Microcapsule compositions are prepared as described in

Example 5, wherein polymer molecular weight is 28,000-40,000 and polymer concentrations vary between 5% to - 12% w/w. Compositions 5 and 6 are listed in Table 1.

Release profiles of the active core from the compositions in an aqueous physiological environment are described in Example 2 and are plotted as cumulative percentage release versus time, and presented in Figure 3.

Burst-free, variable release from 28-70 days is achieved by varying the polymer concentration.

AP/P/ 9 7/01088

Example 7

Microcapsule compositions are prepared as described in Example 6, wherein the aqueous/oil ratio varies between 1/5 to

Printed from Mimosa 09/24/1997 15:10:05 page-32AP.00665

1/25 (v/v). Compositions 3 and 7 are listed in Table l.

Release profiles of the active core from the compositions in an aqueous physiological environment are described in Example 2, and plotted as cumulative percentage release versus time, and presented in Figure 3.

Burst-free, variable release from 28-70 days is achieved by varying the aqueous/oil ratios.

Example 8

Microcapsule compositions are prepared as described in 15 Example 5, wherein the copolymer ratio is 75/25 and polymer concentrations vary between 5¾ to - 25* w/w. Compositions 8 and 9 are listed in Table 1.

Release profiles of the active core from the compositions in 2 0 an aqueous physiological environment' are described in Example 2, and are plotted as cumulative percentage release versus time, and presented in Figure 2.

Burst-free, variable release from 56->90 days is achieved by 25 varying the polymer concentration in the oil phase.

Example 9

Microcapsule compositions are described in Example 2, wherein the active core is leutinizing hormone releasing hormone (LHRH, a decapeptide of molecular weight 1182) and the polymer concentration is -40% w/w. Release profiles of the active core from the composition in an aqueous physiological environment is described in Example 2, and is plotted as cumulative percentage release versus time, and presented in Figure 4.

Burst-free, continuous and complete release is achieved within 35 days, similar to Histatin acetate.

Example 10

Microcapsule compositions are prepared as described in Example 2, wherein an additive such as sodium salt (carbonate or bicarbonate) is added to the inner aqueous phase at concentrations of 1-10* w/w to maintain the biological activity of the released polypeptide.

Burst-free, variable release from 1-28 days is achieved similar to Examples 283, and the released polypeptide is biologically active until 30 days, due to the presence of the sodium salt.

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Printed from Mimosa 09/24/1997 15:10:05 page -33AP.00665

Example li

Microcapsule compositions are prepared as described in Example 2, wherein an additive such as a nonionic surfactant, polyoxyethylene/polyoxypropylene block copolymer (Pluronics F6B and F127) is added to either the inner oil or the aqueous phase at concentrations from 10-1001 w/w, to maintain the biological activity of the released polypeptide.

Burst-free, continuous release from 1-35 days is achieved similar to Examples 2 & 3, and the released polypeptide is bioactive due to the presence of the surfactant.

Example 12

Cumulative histatin release from the microcapsule compositions described in Examples 1 through ll and release profiles plotted in Figures 2 and 3 show the burst-free, programmable peptide release for variable duration from 1-100 days. Virtually any pattern of cumulative release is achievable over'a 100 day duration by a judicious blending of several compositions, as shown in these figures.

Claims

WE CLAIM

1. DSHAKRHHGYKRKFHEKHHSHRGY

1. A controlled release microcapsule pharmaceutical formulation for burst-free, sustained, programmable release of a

2. KRHHGYKRKFHEKHHSHRGYR

25 3. KRHHGYKRKFHEKHHSHR

2. The pharmaceutical formulation of claim 1, wherein the biodegradable poly(lactide/glycolide) is a blend of uncapped and

10 capped forms, in ratios ranging from 100/0 to 1/99.

3. The nicrocapsules of claims 1 or 2 wherein the copolymer (lactide to glycolide L/C) ratio for uncapped and endcapped polymer is 52/48 to 48/52.

4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 13 or 14 for human administration via oral routes.

37. A process of using microcapsules of items l or 2 or 3 or

4. RKFHEKHHSHRGYR

0 8 0 1 0/76 /d/dV

Printed from Mimosa 09/24/1997 15:10:05 page-36AP

4. The microcapsules of claims 1 or 2 wherein the copolymer 15 L/G ratio for uncapped and end-capped polymer is 90/10 to 40/60.

5 administration via topical route.

36. A process of using microcapsules of items 1 or 2 or 3 or

5 31. The microcapsules of claim 29, wherein placebo spheres loaded with the pH-stabilizing agents are coadministered with polypeptide-loaded spheres to maintain the solution pH around the microcapsules and preserve the biological activity of the released peptide in instances where the addition of pH10 stabilizing agents in the inner aqueous phase is undesirable for the successful encapsulation of the acid pH sensitive polypeptide.

32. The microcapsules of claim 30 wherein placebo spheres loaded with non-ionic surfactant are coadministered with

15 polypeptide-loaded spheres to maintain biological activity of the released peptide where the addition of non-ionic surfactants in the inner aqueous phase is undesirable for successful encapsulation of the acid pH sensitive polypeptide.

33. The microcapsules of claims 1 or 2 or 3 or 4 or 5 or 6

20 or 8 or 9 or 10 or ii or 13 or 14 comprising a blend of uncapped and capped polymer, wherein complete solubilization of the copolymer leaves no residual polymer at the site of administration and occurs concurrently with the complete release of the entrapped agent.

25 34. A process of using microcapsules of claims 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 13 or 14 for human

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ΛΟ administration via parenteral routes, such as intramuscular and subcutaneous.

35. A procese of using microcapsules of claims 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 13 or 14 for human

5 0.05-500gm.

26. The process of claims 23 or 24, wherein a low temperature of about 0-4’C is provided during preparation of the inner w/o emulsion, and a low temperature of about 4-2O*C is provided during preparation of the w/o/w emulsion to provide a

10 stable emulsion and high encapsulation efficiency.

27. The process of claim 23 wherein a 100/0 blend of uncapped and end-capped polymer is used to provide release of the active core in a continous and sustained manner without a lag phase.

15 28. The microcapsules of claim 6, wherein, when the entrapped polypeptide is active at a low pH, such as LHRH, adrenocorticotropic hormone, epidermal growth factor, calcitonin released polypeptide is bioactive.

29. The microcapsules of claims 6 or 7 or 8 or 9 or 10 or

20 11, wherein, when entrapped peptide such as histatin is inactive at a low pH, a pH-stabilizing agent of inorganic salts are added to the inner aqueous phase to maintain biological activity of the released peptide.

30. The microcapsules of claims 6 or 7 or 8 or 9 or 10 or 11

25 wherein, when entrapped polypeptide such as histatin is inactive at a low pH, a non-ionic surfactant such as polyoxyethylene

AP/P/ 9 7 / 0 1 0 88

Printed from Mimosa 09/24/1997 15:10:05 page-40AP.00665 . 39 sorbitan fatty acid esters (Tween 80, Tween 60 and Tween 20) and polyoxyethylene - polyoxypropylene block copolymers (Pluronics) is added to the inner aqueous phase to maintain biological activity of the released polypeptide.

5 emulsion; stabilizing the w/o emulsion in a solvent-saturated aqueous phase containing a oil-in-water (o/w) emulsifier; adding said w/o emulsion to an external aqueous layer containing oil-inwater emulsifier to form a ternary emulsion; and stirring the resulting water-in-oil-in-water (w/o/w) emulsion for sufficient io time to remove said solvent, and rinsing hardened microcapsules with water and lyophilizi-g said hardened microcapsules.

15 dissolving biodegradable poly (lactide/glycolide) in endcapped form in methylene chloride, and dissolving a biologically active agent or active core in water; adding the aqueous layer to the polymer solution and emulsifying to provide an inner waterin-oil emulsion; stabilizing the w/o emulsion in a solvent20 saturated aqueous phase containing a oil-in-water (o/w) emulsifier; adding said w/o emulsion to an external aqueous layer containing oil-in-water emulsifier to form a ternary emulsion; and stirring a resulting water-in-oil-water (w/o/w) emulsion for sufficient time to remove said solvent; and rinsing hardened

25 microcapsules with water; and lyophilizing said hardened microcapsules.

AP/P/

5 19. The microcapsules of claims 1 or 2 or 3 or 4 or 5 wherein said biologically active agents are selected from the group consisting of water-soluble hormone drugs, antibiotics, antitumor agents, anti inflammatory agents, antipyretics, analgesics, antitussives, expectorants, sedatives, muscle

10 relaxants, antiepileptics, antiulcer agents, antidepressants, antiallergic drugs, cardiotonics, antiarrhythmic drugs, vasodilators, antihypertensives, diuretics, anticoagulants, and antinarcotics, in the molecular weight range of 100-100,000 daltons.

15 20. The microcapsules of claims 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 wherein said biodegradable poly(lactide/glycolide) is in an oil phase, and is present in about 1-50% (w/w).

21. The microcapsules of claims 1 or 2 or 3 or 4 or 5 or 6 or 8 or 9 or 10 or 11 or 13 or 14 wherein concentration of the

20 active agent is in the range of 0.1 to about 60% (w/w).

22. The microcapsules of claims 1 or 2 or 3 or 4 or 5 or 6 or 8 or 9 or 10 or ll or 13 or 14 wherein a ratio of the inner aqueous to oil phases is about 1/4 to 1/40(v/v).

23. A process for preparing controlled release microcapsule 25 formulations characterized by burst-free, sustained, programmable release of biologically active agents comprising: Dissolving

5 13. The microcapsules of claim 6 wherein the biologically active agent ie a polypeptide Leutinizing hormone releasing hormone (LHRH) that ie a decapeptide of molecular weight lie2 in its acetate form, and having the structure:

p-EHWSYGLRPG

14. The microcapsule of claim 6 having a molecular weight of from-1,000 to 250,000 daltons.

15. The microcapsules of claims 6 or 7 or 8 or 9 or 10 or 11 or 13 or 14 wherein release profiles of variable rates and

15 durations are achieved by blending uncapped and capped microspheres as a cocktail in variable amounts.

16. The microcapsules of claims 6 or 7 or 8 or 9 or 10 or 11 or 13 or 14 wherein release of profiles of variable rates and duration are achieved by blending uncapped and capped polymer in

20 different ratios within the same microshreres.

17. The microcapsules of claims 6 or 7 or 8 or 9 or 10 or 11 or 13 or 14 wherein the entrapped polypeptide is any of the vaccine agents against enterotoxigenic E. coli (ETEC) such as CFA/I,CFA/II,CS1,CS3,CS6 and CS17 and other ETEC-related

25 enterotoxins.

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18. The microcapsules of claims 6 or 7 or 8 or 9 or 10 or ll 13 or 14 wherein the entrapped polypeptide consists of peptide antigens of molecular weight range of about 800-5000 daltons for immunization against enterotoxigenic E. coli (ETEC).

5. AKRHHGYKRKFH

5 uncapped and end-capped poly(lactide/glycolide) having a L/C ratio of 48/52 to 52/48 and a molecular weight range of 28,00040,000.

10. The microcapsules of claim 7 characterized by the capacity to release up to 90% of the histatin in an aqueous

10 physiological environment from 28-70 days with a 0/100 blend of uncapped and end-capped poly(lactide/glycolide) having a L/G ratio of 48/52 to 52/48 and a molecular weight range of 10,00040,000 daltons.

11. The microcapsules of claim 7 characterized by the

15 capacity to release up to 80% of histatin in an aqueous physiological environment from 56-100 days with a 0/100 blend of uncapped and end-capped poly(lactide/glycolide) having a L/G ratio of 75/25 and a molecular weight of < 15,000 daltons.

12. The microcapsules of claims 7 or 8 or 9 or 10 or 11

20 having analogs of histatin with chain lengths of from 11-24 amino acids of molecular weights from 1,500-3,000 daltons and characterized by the following structures:

5. The microcapsules of claim 3 wherein the molecular weight of the copolymer is between 2,000-60,000 daltons.

5 biologically active agent over a duration from 1-100 days, comprising an active agent and a blend of uncapped and end-capped biodegradable poly(lactide/glycolide) .

6. «AKRHHGYKRKFH

6. The microcapsules of claim 1 wherein the biologically active agent is a peptide or polypeptide.

20 7. The microcapsules of claim 6, wherein said polypeptide is histatin consisting of 12 amino acids and having a molecular weight of 1563.

7. KRHHCYKRKF * D-amino acid

8 8 0 I 0 / L 6 /d/dV

Printed from Mimosa 09/24/1997 15:10:05 page -38AP.00665 . 37 biodegradable poly (lactide/glycolide), in uncapped form in methylene chloride, and dissolving a biologically active agent or active core in water; edding the aqueous layer to the polymer solution and emulsifying to provide an inner water-in-oil (w/o)

8. The microcapsules of claim 7 characterized by the capacity to completely release histatin in an aqueous

25 physiological environment from 1-35 days with a ΙΟΟ/Ο blend of uncapped and end-capped poly(lactide/glycolide) having a L/G

AP/P/ 9 7 / 0 1 0 88

Printed from Mimosa 09/24/1997 15:10:05 page -35AP.00665 ratio of 48/52 to 52/48, and a molecular weight -<15,000.

9 7 / 0 1 0 88

Printed from Mimosa 09/24/1997 15:10:05 page-39AP.00665

25. The process of claims 23 or 24 wherein a solventsaturated external aqueous phase is added to emulsify the inner w/o emulsion prior to addition of the external aqueous layer, to provide microcapsules of narrow size distribution range between

9. The microcapeules of claim 7 characterized by the capacity to completely release histatin in an aqueous physiological environment from 18-40 days with a 100/0 blend of

10 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 13 or 14 for human admininstration via nasal, transderma1, rectal, and vaginal routes.

AP/P/ 9 7/01088