CA2318513A1 - Process for making absorbable microparticles - Google Patents

Process for making absorbable microparticles Download PDF

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CA2318513A1
CA2318513A1 CA002318513A CA2318513A CA2318513A1 CA 2318513 A1 CA2318513 A1 CA 2318513A1 CA 002318513 A CA002318513 A CA 002318513A CA 2318513 A CA2318513 A CA 2318513A CA 2318513 A1 CA2318513 A1 CA 2318513A1
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peptide
process according
polymer
absorbable
bound
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French (fr)
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Thomas Ciaran Loughman
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Kinerton Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/58—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. poly[meth]acrylate, polyacrylamide, polystyrene, polyvinylpyrrolidone, polyvinylalcohol or polystyrene sulfonic acid resin
    • A61K47/585—Ion exchange resins, e.g. polystyrene sulfonic acid resin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/593—Polyesters, e.g. PLGA or polylactide-co-glycolide

Abstract

This invention pertains to a process for making an encased bound microparticle sustained release complex comprising one or more peptides, one or more proteins or a combination thereof immobilized on an absorbable polymer microparticle having an absorbable polymer coating, where said process comprises nebulizing a dispersion of the bound microparticles.

Description

WO 99!38535 PCT/IE99/00007 _1_ Description PROCESS FOR MAKING ABSORBABLE MICROPARTICLES
Technical Field This invention pertains to a process for making an encased bound micropartide which is a sustained release complex of one or more peptide, one or more protein or a combination thereof immobilized on an absorbable polymer micropartide having an absorbable encasing polymer. The microparticle complex made by a process of this invention comprises a peptides) andlor proteins) which have at least one amino group and/or at least one carboxyl group per molecule, a solid absorbable polyester micropartide having surface and subsurtace carboxylic groups or amino groups in suffident amounts to bind the peptides) and/or proteins) so that the immobilized peptides) or proteins) represent 0.1 % to 30% of the total mass of the micropartide complex which is encased individually or in groups with an absorbable encasing polymer to control the release of the immobilized peptides) and/or protein(s).
Background Art Many drug delivery systems have been developed, tested and utilized for tt~e controlled in vivo release of pharmaceutical compositions. For example, polyesters such as poly(DL-lactic add), poly(glycolic add), poly(E-caprolactone) and various other copolymers have been used to release biologically active molecules such as progesterone; these have been in the form of miaocapsules, films or rods (M.
Chasin and R. Langer, editors, Biodegradable Polymers as Drug Delivery Systems, Dekker, NY
1990). Upon implantation of the potymerltherapeutic agent oomposidon, for example, subcutaneously or intramuscularly, the therapeutic agent is released over a speafic period of time. Such bio-compatible biodegradable polymeric systems are designed to permit the entrapped therapeutic agent to diffuse from the polymer matrix.
Upon release of the therapeutic agent, the poUer is degraded in vivo, obviating surgical removal of the implant. Although the factors that contribute to poller degradation are not well understood, it is believed that such degradation for polyesters may be regulated by the accessibility of ester linkages to non-enzymatic autocatalytic hydrolysis of the polymeric components.
For example, Deluca (EPO Publication 0 467 389 A2) describes a physical interaction between a hydrophobic biodegradable polymer and a protein or polypeptide.
The composifion formed was a mixture of a therapeutic agent and a hydrophobic polymer that sustained its diffusional release from the matrix after introduction into a subject.
Hutchinson (U.S. Pat. No. 4,767,628) controlled the release of a therapeutic agent by un'rformdispersion in a polymeric device. It is disclosed that this formulation provides for controlled continuous release by the overlap of two phases: first, a diffusion- , dependent leading of the drug from the surtace of the formulation; and second, releasing by aqueous channels induced by degradation of the potymer.
Other in-situ forming biodegradable implants and methods of forming them are described in U.S. Pat. Nos. 5,278,201 ('201 Patent) and U.S. Pat. No.
5,077,049 ('049 Patent), to Dunn et al. The Dunn et al. patents disclose methods for assisting the restoration of periodontal tissue in a periodontal pocket and for retarding a migration of epithelial cells along the root surface of a tooth. The '049 Patent discloses methods which involve placement of an in-situ forming biodegradable barrier adjacent to the surtace of the tooth. The barrier is miaoporous and includes pores of defined size and can include 7 5 biologically active agents. The barrier formation is achieved by plating a liquid solution of a biodegradable polymer, such as poly(dl-lactide-co-glyootide) water-ooagulatable, thermoplastic in a water misdble, non-toxic organic solvent such as N-methyl pyrrolidone (i.e., to achieve a typical polymer concentration of about 50%) into the periodontal pocket The organicsolvent dissipates into the periodontal fluids and the biodegradable, water ooagulatable polymer forms an in-situ solid biodegradable implant. The dissipation of solvent aeates pores within the solid biodegradable implant to promote cell ingrowth.
The '859 Patent likewise discloses methods for the same indications involving the formation of the biodegradable barrier from a liquid mixture of a biodegradable, curable thermosetting prepdymer, curing agent and water-soluble material such as salt, sugar, and water-soluble polymer. The curable thermosetting prepolymer is described as an acrylic-ester terminated absorbable polymer.
In addition, a number of systems for the controlled delivery of biologically active compounds to a variety of s'ttes are disclosed in the literature. For example, U.S. Patent No. S,Oi 1,692, to Fujioka et al., discloses a sustained pulsewise release pharmaceutical preparation which comprises drug-containing polymeric material layers. The polymeric - material layers contain the drug only in a slight amount, or free of the drug. The entire surface extends in a direction perpendicular to the layer plane and is coated with a polymeric material which is insoluble in water. These types of pulsewise-release pharmaceutical dosages are suitable for embedding beneath the skin.
U.S. Pat. No. 5,366,756, to Chesterfield ei al., describes a method of preparing porous bioabsorbable surgical implant materials. The method comprises providing a WO 99!38535 PCT/IE99/00007 quantity of particles of bioabsorbable implant material, and coating particles of bioabsorbable implant material with at least one growth factor. The implant can also contain antimicrobial agents.
U.S. Patent No. 5,385,738, to Yamhira et al., discloses a sustained-release injection system, comprising a suspension of a powder comprised of an active ingredient and a pharmaceutically acceptable biodegradable carrier(e.g., proteins, polysaccharides, and synthetic high molecular weight compounds, preferably collagen, atelo collagen, gelatin, and a mixture thereof) in a viscous solvent (e.g., vegetable oils, polyethylene glycol, propylene glycol, silicone oil, and medium-chain fatty acid figlycerides) for injection. The active ingredient in the pharmaceutical fom~ulation is incorporated into the biodegradable carrierin the following state: (i) the active ingredient is chemically bound to the cartiermatrix; (ii) the active ingredient is bound to the carrier matrix by intenndeallar action; or (iii) the active ingredient is physically embraced within the carriermatrix.
Moreover, such systems as those previously described in the literature, for example, such as by Dunn, et al. (U.S. Pat. No. 4,938,763), teach in-situ formations of biodegradable, miaoporous, solid implants in a living body through coagulation of a solution of a polymer in an organic solvent such as N-methyl-2-pyrrolidine.
However, the use of solvents, including those of low molecular organic ones, faalitates migration of the solution from the application site thereby causing damage to living tissue including oeB
dehydration and necxosis. Loss of the solvent mass can lead to shrinkage of the coagulum and separation from surrounding tissue.
US Patent No. 5,612,052 describes cation-exchanging miaopartides made typically of carboxyl-bearing polyester chains onto which basic bioadtve agents are immobilized to provide a control release system within an absorbable gel-forming liquid polyester. The contents of US Patent 5,612,052 is incorporated herein by reference.
Conjugating carboxylic entities, ionicalty, with basic polypeptide has been noted in the prior art as described in US Patent No. 5,672,659 and US Patent No. 5,665,702.
However, these complexes are soluble chemical entities formed by molecularly reading the individual basic and carboxylic components in their respective solutions to form a well-defined ion-conjugate as a new chemical entity with physi~emical properties.
Disclosure of Inventson The present invention is directed to a process (process A) for making an encased bound micropartide or micropartides wherein the encased bound micropartide or micropartides comprise bound miaopartide or miaopartides and an absorbable encasing polymer where the bound micropartide or miaopartides comprise an absorbable heterochain polymer core and one or more peptide, one or more protein or a combination thereof immobilized on said absorbable heterochain polymer core where each peptide is independently selected from growth hormone releasing peptide -(GHRP), luteinizing homnone-releasing hormone (LHRH), somatostatin, -bombesin, gastrin releasing peptide (GRP), calatonin, bradykinin, galanin, melanocyte stimulating hormone (MSH), growth hormone releasing factor (GRF), amylin, tachykinins, seaetin, parathyroid hormone (PTH), enkaphelin, endothelin, calatonin gene releasing peptide (CGRP), neuromedins, parathyroid hormone related protein (PTHrP), glucagon, neurotensin, adrenocaticothrophic hom~one (ACTH), peptide YY (PYY), glucagon releasing peptide (GLP), vasoactive intestinal peptide (VIP), pituitary adenylate cydase activating peptide (PACAP), motilin, substance P, neuropeptide Y (NPY), TSH and analogs and fragments thereof or a pharmaceutically acceptable salt thereof; and where each protein is independently selected from growth hormone, erythropoietin, granulocyte-colony stimulating factor, granulocyte-macrophage-colony stimulating factor and interferons;
said process comprising the steps of:
obtaining a dispersion, where the dispersion comprises solid bound miaopartides in a solution of the absorbable encasing polymer, by homogenizing and concurrently dispersing said solid bound micropartides into the solution of an absorbable encasing polymer; and nebulizing said dispersion through a nebulization probe, where said probe has an operating ultrasonic frequency range of 12 kHz to 36 kHz, into a medium, where the medium is a non-solvent of said absorbable casing polymer, at a flow rate of about 1 mUminto 15 >nlhnin.
A preferred process of process A, denoted as process B, is where the medium comprises isopropanol or ethanol.
A preferred process of process B, denoted as process C, is where the solution of the absorbable polymer consists of about 5% to 30% of the absorbable polymer and _ the temperature of the mediumis roomtemperatureto about -80°C.
A preferred process of process A, denoted process D, is where the peptide, peptides, protein or proteins or combination thereof of the bound miaopartide comprises 0.1 % to 30% of the total mass of the bound micropartide.
A preferred process of D, denoted process E, is where the absorbable polymer core comprises glycotate units and atrate residues wherein the ratio of glycolate units to atrate residues is about 7-1 to about 20-1 or glycolate units and tartrate residues wherein the ratio of glycolate units to tartrate residues is about 7-i to about 20-t or gfycolate units and malate residues wherein the ratio of glycolate units to malate residues is about 7-7 to about 20-1.
A preferred process of process E, denoted process F, is where the absorbable encasing polymer comprises (a) I-lactide based units and glyootide based units where the ratio of I-lactide based units to glycolide based units is about 60-40 to about 90-10, (b) d,l-lactide based units and glycolide based units where the ratio of d,l-lactide based units to glyoolide based units is about 60-40 to about 90-10, (c) d,l-lactide based units, a (d) I-lactide based units and d,l-lacctide based units where the ratio of I-lactide based units to d,l-lactide based units is about 80-20.
A preferred process of process F, denoted process G, is where the absorbable encasing polymer constitutes 5 to 70% of the total mass of the encased bound miaopartide or miaopartides.
A preferred process of process G, denoted process H, is where the absorbable encasing polymer constitutes 20-60% of the total mass of the encased bound miaopartide or miaopartides.
A preferred process of process H, denoted process I, is where the absorbable encasing polymer constitutes 30-50% of the total mass of the encased bound miaopartide or miaopartides.
A preferred process of process I, denoted process J, is where the peptide is an LHRH analog.
A preferred process of process J, denoted process K, is where the LHRH analog is p-Glu-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NHS.
A preferred process of process 1, denoted process L, is where the peptide is a somatostatin analog.
A preferred process of process L, denoted process M, is where the somatostatin analog is H-~-D-Nal-Cys-Tyr-D-Trp-Lys-Val-Cys-Thr-NHS, where the two Cys are . bonded by a disulfide bond.
Another preferred process of process L is where the somatostatin analog is N-hydroxyethylpipera2inyl-acetyl-D-Phe-Cys-Tyr-D-Trp-Lys-Abu-Cys-Thr-NHZ where the two Cys residues are bonded by a disutf'~de bond.

Yet another preferred process of process L is where the somatostatin analog is N-hydroxyethylpiperazinyl-ethylsulfonyl-Phe-Cys-Tyr-D-Trp-Lys-Abu-Cys-Thr-NHz -where the two Cys residues are bonded by a disulfide bond.
The term "absorbable" as used herein, means a water insoluble material such as a polymer which undergoes chain disassociation in the biological environment to water soluble by-products.
The term "microparlide" as used herein, refers to the parlides of absorbable polyester, which are preferably in essentially spherical form.
The term "bound miaopartide" as used herein, refers to a miaopartide having one or more peptide and/or one or more protein sonically immobilized on the micropartide.
The term "encased miaopartide" as used herein, refers to a bound miaopartide having a polymer coating, where the polymer coating is not necessarily completely occlusive.
The temp "polymer core" as used herein, is another way of referring to miaopartides.
The term"encasing polymerw as used herein, refers to the polymer that is used to encase a bound miaopartide.
The term "gel-forming liquid polyester" as used herein, refers to materials which absorb solvents such as water, undergo phase transformation and maintain three dimensional networks capable of reversible deformation.
The instant application denotes amino adds using the standard three letter abbreviation known to those skilled in the art, for example Ala = alanine.
A miaopartidethat is used in a process of the present invention is crystalline and is made of an absorbable polyester, such as polyglycolide having one or more carboxylic groups on the individual cthains which results in a suffident concentration of carboxylic groups on the surtace of the miaopartide and immediate subsurtace of the miaopartideto complex and sonically immobilize a peptides) and/or a proteins) having one or more basic groups. Or the carboxylic groups of the polyglycolide can be amidated, for example by a diamine, preferably a primary or secondary amine or a mixture thereof, wherein the amineforms a complex that sonically immobilizes a peptides) and/or a proteins) having one or more addic groups. Since the surtace of the micropartides is not necessarily homogeneous, the term "subsurtace" refers to the crevices and the like found on the surtace of the micropartides. The bound miaopartides provide a means for the controlled release of a peptides) and/or proteins) in a patient.
To further control the release of the immobilized peptides) and/or protein(s), a process of _7_ this invention encases the bound micropartides individually or in groups with an absorbable encasing polymer. The bound miaopartides release the peptides) andlor proteins) over a period of about two days to about three months in a patient, preferably about one week to about three months. The encased miaopartides release the - 5 peptides) andlor proteins) over a period of about three days to six months in a patient, preferably about two weeks to five months.
Typical examples of a peptide that can be immobilized or bound on a miaopartide used in this invention include but are not limited to growth hormone releasing peptide (GHRP), luteinizing hom~one-releasing hormone (LHRH), somatostatin, t0 bombesin, gastrin releasing peptide (GRP), calatonin, bradykinin, galanin, melanocyte stimulating hormone (MSH), growth hormone releasing factor (GRF), arnylin, tachykinins, seaetin, parathyroid homnone (PTH), enkaphelin, endothelin, calatonin gene releasing peptide (CGRP), neuromedins, parathyroid hormone related protein (PTHrP), glucagon, neurotensin, adrenocorticorthrophic hormone (ACTH), peptide YY (PYY), glucagon 15 releasing peptide (GLP), vasoacttve intestinal peptide (VIP), pituitary adenylate cydase activating peptide (PACAP), motilin, substance P, neuropeptide Y (NPY), TSH, and analogs and fragments thereof. Examples of proteins that can be immobilized or bound on a miaopartideused in this invention are growth hormone, erythropoietin, granulocyte-. _ colony stimulating factor, granulocyte-macrophage-colony stimulating factor and 20 interferons.
A miaopartidecan be made of a ladide based polymer or a slid semi-crystalline potylactone such as polyglycolide which can be formed by ring opening polymerization of acid-bearing hydroxylic initiators such as gtycolic, lacfic, malic, tartaric, and atric acid. A
micropartide used irt the present invention can be synthesized according to the following 25 procedure. In a reaction vessel are mixed a lactide based monomerand/or a ladone such as gtyoolide and an add initiator such as tartaric add, matic add a afic acid.
The reaction vessel is warmed to about 35-45°C, preferably 40°C and put under vacuum for about 20-60 minutes, preferably 30 minutes. The temperature of the reaction vessel is raised to about 105-115°C, preferably 110°C. Once this temperature is reached the vessel is 30 placed under an atmosphere of oxygen-free nitrogen, and the m6cture is stirred. Once the . mixture melts, a catalytic amount of an organometallic catalyst suitable for ring opening pdymerization, such as stannous 2-ethyl-hexanoate solution in a non-erotic solvent, such as toluene is added. A vacuum Is reapplied for about 30-90 seconds to remove toluene without signficant removal of monomer. The temperature of the mixture is raised 35 to about 115-125°C, preferably 120°C for about 5-10 minutes before further raising it to $_ about 145-150°C. It was kept at this temperature for about 3-5 hours, preferably 4 hours, under constant mechanicalstirring. -The resulting polymer is miaonized by initially grinding it using a Knife-grinder.
The polymer is then micxonited in an Afet Miaonizer using a pressurized dry nitrogen stream. The mean partide diameter size is analyzed in a Malvern MastersizerlE
using a volume distribution mode! and 2~I5 cS silicone oil as dispersant.
The polymer is purfied and the sodium salt thereof is formed by dispersing the miaonized polymer in acetone and pladng it in a sonicator, preferably for about 30 minutes. During this time the dispersion was also homogenized at about 8,000-24,000 rpm, preferably 9,500 rpm, using a homogenizes. After this sonicationlhomogenization step the dispersion is centrifuged at about 3,000-7,000 rpm, preferably 5,000 rpm preferably for about 30 minutes in a centrifuge. The supernatant is discarded, the centrifuge cakes re-suspended in fresh acetone, and the sonicationMomogenization step repeated. Once the second centrifugation is complete, the supernatant is discarded and the cakes were re-suspended in deionized water. One final sonication/homogenization step is then carried out to remove any remaining acetone and the dispersion is once again centr'rtuged at about 5,000 rpm for about 30 minutes.
The centrifuge cakes are re-suspended in fresh deionized water and the pH of the dispersion is monitored. Suffident volumes of a weak base such as 0.2M sodium carbonate solution are added with stirring to raise the pH to between about pH
8 and about pH 9. The dispersions are allowed to stir for about 30 minutes before being vacuum-filtered over filter paper. The titter cakes are rinsed with further deionized water, frozen, and lyophilized.
Purification is rrmnitored by differential scanning calorimetry (DSC) with a heating rate of about 5°Clmin to 15°Clmin, preferably 10°Clmin.
An anion-exchanger miaopartide is obtained by taking the ration-exchanger miaopartides and incubating it in a hot dilute solution (.-04°C) of a diamine, it is preferred that the amines can be both a primary amine or both a secondary amine or a mixture of a primary and a secondary amine, of known concentration in dioxane or THF under an inert gas such as argon. The concentration of the diarnine in dioxane or THF is determined b y acidimetry. When the reaction practically ceases to take place, the amidated miaopartides are separated by fif6ration, rinsed with dioxane or THF, and dried under reduced pressure.
A peptides) andlor proteins) can be immoblized on. a micropartide according to the following method. The sodium salt of a micropartide is dispersed in solutions containing the free-base of a peptides) and/or proteins) dissolved in water.
The WO 99138535 PCTItE99100007 _g_ dispersions are incubated at room temperature with stirring for about 2 hours before filtering out the bound miaopartides. The filter cakes are rinsed with further deionized water, frozen, and lyophilized. Samples are then analyzed for nitrogen by elemental analysis to determine the amount of the peptides) andlor proteins) immobilized.
The size of a micropartide plays a role in the amount of a peptide andlor protein that a miaopartide of the instant invention can immobilize. The smaller the size of a miaoparfide,the more surface area a mass of miaopartidespossess and, thus, the more peptide and/or protein can be immobilized per mass of micropartides. Size reduction of the micropartides to micron or sub-micron dimensions can be achieved as described above. The diameterof the miaopartidescan range in size from about 0.5 E.~m to 100 Nrrt preferably 1 L~m to 15 ~m and more preferably 3 t.~m to 10 pm The absorbable encasing polymer can be a crystalline or non-crystalline ladide/gtycolide copolymer, amorphous I-lactideld,l-lactide co-polymer, caprolactonel glycolide copolymer or trimethylene carbonatelglycolide copolymer, that is soluble in conventional organic solvents, such as chtoroform, methylene chloride, acetone, acetonitrile, ethyl acetate and ethyl formate or a combination thereof. Non-solvents of such an absorbable encasing polymer include water, low boiling temperature alcohols and hydrocarbons or a combination thereof. The absorbable encasing polymers can be synthesized by catalyzing ring-opening polymerization of lactones, or by polymerization of cyclic monomers such as ~-caprolactone, p-dioxanone, trimethylene carbonate, 1,5-dioxepan-2-one or 1,4-dioxepan-2-one in the presence of a chain initiator, such as a hydroxy polycarboxylic aad. Still another method involves reacting an organic polycarboxylic add with a pre-formed polyester, which is disclosed in U.S.
Patent No.
5,612,052, the contents of which are inoorpo<ated herein by reference.
The encasing of the bound micropartides can be achieved by a process of this invention whidi involves the use of an ultrasonic atomizer where a dispersion of the bound miaopartides in an absorbable encasing polymer solution is introduced as micro-droplets into a cooled non-solvent medium (the cooled non-solvent medium is a non-solvent of the absorbable encasing polymer). Bound miaopartides are encased with an absorbable encasing copolymer of ladide and glyoolide using coagulation of solid miaopartidesencased in a polymer solution and delivered through an ultrasonic atomizer (nebulizer) into a liquid mediumthat is a non-solvent forthe encasing polymer, but where the liquid medium non-solvent is capable of extracting the solvent of the encasing polymer solution about the encased solid miaopartides. The nebulizer probe nebulizes at a frequency of 12 to 36 kHz. It is preferred that a probe be used that can achieve a frequency of about 34 kHz to 36 kHz. The relation between the frequency a probe can generate and its affect on a process of this invention is that a higher frequency allows the process to be able to handle more viscous solutions and also higher flow rates of the dispersion of the bound micropartides in an absorbable encasing polymer solution.
Depending on the concentration of the polymer solution for encasing the miaopartides, the number of the original bound micropartides in the encased micropartides can vary from 1 to several hundred with an average diameter of an encased miaopartide ranging from 0.5 um to 100 ~
The following method relates to the preparation of encased peptide-loaded andbr protein-loaded (hereinafter peptide-loaded) ration exchangers (CE) by nebulization. The encasing copolymer of interest is dissolved in a solvent, such as either acetoniMle, ethyl acetate or ethyl formate. A suffident weight of this solution is used for dispersion of the peptide-loaded CE so that the weight ratio of peptide-loaded CE to encasing copolymer ranges from about 30:70 to about 80:20. Dispersion is achieved by high speed homogenization. Peptide-loaded ration exchanger is dispersed in acetonitrile or ethyl acetate solution of encasing copolymers. The concentration of encasing copolymer in solution varies from 10% to 25% (WIW) for ethyl acetate and from 12.5 to 30%
(W/W) for aoetonitrile depending on the par4de characteristics desired (morphology, size, specfic surface area). The solution is homogenized using a homogenizer, such as an Ultra-turrax T25 (IKA, Staufen, Germany) with dispersing tools attached. A 10-gauge dispersing tool is used for batch sizes of 1 ml to 50 ml while a 25-gauge toot is used for batch sizes of 50 ml to 2.5 L. The rotary speed of these dispersing tools can be varied from 8,000 rpm to 24,000 rpm. The homogenizationldispersion step ensures a uniform dispersal of the polypeptide-loaded ration exchanger in the encasing polymer solution without the need for sonication or vortexing. The dispersion is fed at a flow rate of between 1 mlhnin and 10 mUmin to an ultrasonic atomization nozzle with variable frequency - this frequency can be altered from 12kHz to 36kHz - higher frequency allows higher flow rates while maintaining particle characteristics. The dispersion is thus nebulized into a colleding sink made up of at feast 1 to 10 times excess of a medium such as isopropanol or ethanol (compared to the volume of encasing copolymer solvent used) at roomtemperature or cooled with suffiaent dry-ice pellets (usually 0.5 - 1 Kg by weight per literof IPA) so that the temperature of the slurry remains between -50°C and -80°C
throughoutthe nebuf~zation. This slurry is stirred at between 300 and 700 rpm dependirx~
on its volume. In the case of acetonitrile or ethyl acetate as solvent, the nebulization droplets will freeze immediately on contact with the scurry. Once nebulization is complete the entire dispersion is allowed to thaw of 'tts own accord to between 10°C and room temperature before vacuum filtering. The filter cakes are rinsed with de-ionized water to remove excess non-solvent. The particles obtained have the appearance of smooth microspheres in the case of a predominantly d,l-lactide encasing copolymer;
they appear slightly wrinkled when the encasing copolymer is mainly I-lactide based.
The binding capacity of a micropartide ion-exchanger can be determined as follows. For example, for a ration-exchanger micropartide, available carboxylic groups, in a predetermined mass of the micropartides, are neutralized using cold dilute aqueous sodium carbonate solution of known normality. The neutralized micropartides are isolated by filtration and rinsed thoroughly with cold deionized water and then air dried. The solid miaopartides are then incubated in dilute solution of Pilocarpine hydrochloride of known concentration so as to provide a slight excess of the basic drug over that predicted from the binding capacity data. The concentration of the remaining Pilocarpine HCI
in the aqueous medium is monitored for a period of time until no significant change in the base pick-up by the miaopartides can be recorded. The percent of immobilized base on the miaopartides is determined from the exhaustion data and then verfied by elemental analysis for nitrogen.
The binding capaaty of the anion-exchanger (amtdated particles) is determined by (1 ) elemental analysis for nitrogen and (2) extent of binding to Naproxen b y measuring the extent of Naproxen removed from a dilute solution using HPLC.
The latter is confirmed by release of the immobif~zed Naproxen with a dilute sodium hydroxide solution of known concentration.
The encased miaopartides made by a process of this invention can be administered to a patient v is administration routes well known to those of ordinary skill ~
the art, such as parenteral administration or aal administration. Preferably it is administered as a powder or a suspension via intranasal route or as an inhalant through the pulmonary system. When it is administered parenterally it is preferable that it is administered as a dispersion in an isotonic aqueous medium or in a non-aqueous, absorbable gel-forming liquid polyester.
The effective dosages of encased micropartides, made by a process of this invention, to be administered to a patient can be determined by the attending physician or veterinarian and wilt be dependent upon the proper dosages contemplated for the peptides) and/or proteins) and the quantity of the peptides) andlor proteins) immobilized on the miaopartides. Such dosages will either be known or can be determined by one of ordinary skill in the art.

_12_ Modes for Carrying Out the Invention Example 1 Preparation, Micronization, and Purification of Poly(glycolic acid) polymers initiated with Citric Acid (PGCA) for use as Cation Exchangers (CE) .
Example 1(a): 7/1 PGCA- A 500 ml glass reactor was loaded with 242.63 g of glycolide (Purac Biochem, Arkelsedijk, The Netherlands) and 57.37 g of atric acid (Akirich, Gillingham, Dorset, U.K.). The afic acid had been further dried over silica gel (Fisher Scientific, Loughborough, Leics., U.K.) in an Abderhalden apparatus (Aldrich, St. Louis, Missouri, USA). The reacts was immersed in an oil bath at about 40°C
and put under vacuum (0.04 mbar) for about 30 minutes. The bath was then lowered and it's temperature raised to about 110°C. Once this temperature was reached the reactor was placed under an atmosphere of oxygen-free nitrogen and re-immersed. The contents were stirred at about 100 rpm using a Heidolph stirrer (Heidolph Elektro GmbH, Kelheirn, Germany). Once the reactor contents melted 1.09 ml of a 0.1 M stannous 2-ethyl-hexanoate solution (Sigma, St. Louis, Missouri, USA) in toluene (Riedel de-Haen, Seelze, Germany) was added (stoichiometricratio of 50 ppm). A vacuum was reapplied via a liquid nitrogen trap for about 30 seconds to remove toluene without signficant removal of monomer. The oil bath temperature was then raised to about 120°~ for about 5 minutes before further raising it to about 150°C. It was kept at this temperature for about 4 hours under constant mechanicalstirting of about 100 rpm. The title polymer was obtained.
Example 1(b): 10/1 PGCA- The title polymer was obtained by fdlowing the procedure of Example la, but using 257.40 g of glycolide, 42.60 g of atricacid and 1.10 ml of a 0.1 M
stannous 2-ethyl-hexanoate solution in toluene (stoichiometricratio of 50 ppm).
Example 1(c): 15/1 PGCA- 15/1 PGCA- A flame-dried resin kettle equipped with a mechanical stirrer and an argon inlet was charged with glyoolide (2.586 mole, 300 g), anhydrous citricacid (0.172 mole, 33 g), and stannous octoate (0.2 M in toluene, 862m1, 0.172 mmole). The polymerization reactor and its contents were purged with dry argon several times. After melting the polymerization charge, the reactants were heated and _ stirred at about 160°C until the polymer started to precipitate from the mett_ Shortly after partial preapitation, the stirring was terminated and the reaction was continued at about 160°C for about 2 hours. At the condusion of the polymerization, the temperature was lowered below 120°C and excess monomerwas removed under reduced pressure. The composition of the isolated polymer was verified using infrared and NMR
spectroscopy. .

Micronization- Each of the polymers of Examples t(a), I(b) and I(c) were ground initially using a Knife-grinder (IKA, Staufen, Germany). They were then micronized in an Aljet Miaonizer (Fluid Energy Aljet, Plumsteadsville, Pennsylvania, USA) using a pressurized dry nitrogen stream. Example I(a) had a mean particle diameter size of 24.84 ~m by analysis in a Malvern MastersizerlE (Malvern, Worcs., U.K.) using a volume distribution model and 200/5 cS silicone oil (Dow Corning, Seneffe, Belgium) as dispersant. Examples I(b) and I(c) had mean particle diametersizes of 4.69 l.~m and 6.31 L~m, respectively, after miaonization.
PurificatioNSodium Salt Formation- Fifty gram batches of Examples I(a), I(b), and 1(c) were dispersed in 2L of acetone (Riedel de-Haen, Seelze, Germany) and placed in a sonicator (Branson Uttrasonics BV, Soest, The Netherlands) for about 30 minutes. During this time the dispersion was also homogenized at about 9,500 rpm using an Ultra-turrax T25 homogenizer (IKA, Staufen, Germany). After this sonication/ homogenization step the dispersion was centrifuged at about 5,000 rpm for about 30 minutes in a Sorvall centrifuge (Sorvall, Wilmington, Delaware, USA). The supernatant was discarded, the centrifuge cakes re-suspended in fresh acetone, and the sonicationlhomogenization step repeated. Once the second centrifugation was complete, the supernatant was discarded and ' the cakes were re-suspended in deionized water. One 6na1 sonicationlhomogenization step was then carried out to remove any remaining acetone and the dispersion was once again centrifuged at about 5,000 rpm for about 30 minutes.
The cenirfilge cakes were re-suspended in fresh deionized water and the pH of the dispersion was monitored. Sufficient volumes of 0.2M sodium carbonate solution were added in each case (with stirring) to raise the pH to between about pH B
and about pH 9. The dispersions were allowed to stir for about 30 minutes before being vacuum-filtered over a Whatman no.1 (24 an diameter) fitter paper (Whatman Intt.
Ltd., Maidstone, Kent, U.K.). The fitter cakes were rinsed with further deionized water, frozen, and lyophilized in an Edwards SuperModulyo Lyophitizer (Edwards, Crawley, West Sussex, U.K.).
Purfication was monitored by differential scanning calorimetry (DSC) using a TA
DSC912S (TA Instruments, New Castle, Delaware, USA) with a heating rate of 10°Chnin. The DSC thertnograms obtained in each case did not show any endothermic peak for monomericglycolide but showed endotherms at 176°C, 176°C, and 180°C for Examples I(a), I(b), and I(c), respectively.

_ WO 99/38535 PCTIIE99/00007 Example il Preparation of Microparticulate Cation-Exchanger of Glycolide/Malic Acid Copotymer PGMA , The title micropartide was synthesized according to the method described in Example t(c) but using glycolide (2.586 mole, 300 g), anhydrous malicacid {0.172 mole, _ 23 g), and stannous odoate (0.2 M in toluene, 862m1, 0.172 m mole).
Differential Scanning Calorimetry was used to determine the polymer melting temperature (Tm 206°C).
The solid polymer was ground to achieve average particle diameter of about 125 Eun using a Wiley mill. Further reduction of the particle size to about 5-10 Ntn diameter was achieved using a jet-mill receiving pressurized dry nitrogen. The resulting miaopartides were rinsed with acetone to remove trace monomer and low mdecular weight digomers.
The product was then dried under reduced pressure at 40°C until used.
The average diameterof the dry micropartidewas determined using a particle size analyzer.
Example 11t Preparation, Micronization, and Purification of a Poly(glycolic acid) polymer initiated with Tartaric Acid (PGTA) for use as a Cation Exchanger (CE) Example Itl(a): 10/1 PGTA- A 500 ml glass reactor was loaded with 264.fi5 g of glyaolide (Purac Biochem, Arkelsedijk, The Netherlands) and 34.22 g of L-Tartaric aad (Riedel de-Haen, Seelze, Germany). The tartaric add had been further dried over silica gel (Fisher Scientific, Loughborough, Leics., U.K.) in an Abdefialden apparatus (Aldrich, St. Louis, MO). The reactor was immersed in an oil bath at about 40°C
and put under vacuum (0.04 mbar) for about 30 minutes. The bath was then lowered and it's temperature raised to about 110°C. Once this temperature was reached the reactor was plaa~d under an atmosphere of oxygen-free nitrogen and re-immersed. The contents were stirred at about 100 rpm using a Heidolph stirrer (Heidolph Elektro GmbH, Kelheim, Germany). Once the reactor contents melted 1.14 ml of a 0.1 M stannous 2-ethyl-hexanoate solution (Sigma, St. Louis, Missouri, USA) in toluene (Riedel de-Haen, Seelze, Germany) was added (stoichiometric ratio of 50 ppm). A vacuum was reapplied via a liquid nitrogen trap for about 30 seconds to remove toluene without signficant removal of monomer. The oil bath temperahire was then raised to about 120°C for about 5 minutes before further raising it to about 150°C. It was kept at this temperature for about 4 hours under constant mechanicalstirring of about 100 rpm. The title polymer was obtained.
Micronaation-Example III(a) was ground initially using a Kn'rfe-grinder (IKA, Staufen, Germany). It was then miaonized in an Aljet . Miaonizer (Fluid Energy Aljet, Plumsteadsvilte, Pennsylvania, USA) using a pressurized dry nitrogen stream.
This gave a mean particle diameter of 12.42 L~m by analysis in a Malvern MastersizerlE
(Malvern, Worcs., U.K.) using a volume distribution model and 20015 cS
silicone oil (Dow Coming, Seneffe, Belgium) as dispersant.
Purificationl5odium Salt i=ormation-A 50 g batch of Example lll(a) was dispersed in 2L of acetone (Riedel de-Haen) and placed in a sonicator (Branson Ultrasonics BV, Soest, The Netherlands) for about 30 minutes. During this time the dispersion was also homogenized at about 9,500 rpm using an Ultra-turrax T25 homogenizer (IKA, Staufen, Germany). After this sonicationlhomogenization step the dispersion was centrifuged at about 5,000 rpm for about 30 minutes in a Sorvall centrifuge (Sorvall, wlmington, Delaware, USA). The supernatant was discarded, the centrifuge cakes re-suspended in fresh acetone, and the sonicationlhomogenization step repeated. Once the second centrifugation was complete, the supernatant was discarded and the cakes were re-suspended in deionized water. One final sonicationlhomogenization step was then carried out to remove any remaining acetone and the dispersion was once again centrifuged at about 5,000 rpm for about 30 minutes.
The centrifuge cakes were resuspended in fresh de-ionized water and the pH of the dispersion was monitored. A suffiaent volume of 0.2M sodium carbonate solution was added to raise the pH to between about pH 8 and about pH 9. The dispersion was allowed to stir for about 30 minutes before being vacuum-filtered over a Whatman no.1 (24 an diameter) fitter paper (Whatman Intl. Ltd., Maidstone, Kent, U.K.). The filter cake was rinsed with further deionized water, frozen, and lyophilized in an Edwards SuperModulyo Lyophilizer (Edwards, Crawtey, West Sussex, U.K.).
Purficationwas monitored by DSC using a TA DSC912S (TA Instruments New Castle, Delaware, USA) with a heating rate of about 10°Chnin. The DSC
them~ogram obtained did not show any endothermic peak for monomeric glycolide but showed an endotherm at 181 °C .
Example III(b): 15J1 PGTA- The tifJe polymer was synthesized according to the procedure described for Example I(c) but using glycolide (2.586 mole, 300 g), anhydrous tartaric acid (0.172 mole, 26.8 g) and stannous octoate (0.2 M in toluene, 862 mI, .0172 mmole). Differential Scanning Calorimetry was used to determine the polymer melting temperature (Tm = 204°C) .
The solid polymer was ground to ad~ieve average partide diameter of about 125 L~m using a Wiley mill. Further reduction of the particle size to about 5-10 l~m diameter was achieved using a jet-mill receiving pressurized dry nitrogen. The resulting miaopartides were rinsed with acetone to remove trace amounts of monomer and low molecular weight WO 99!38535 PCTIIE99l00007 oligomers. The product was then dried under reduced pressure at about 40°C until used.
The average diameter of the dry miaopartide was determined using a particle size analyzer.
Example IV
Preparation of Polyglycolide-based Microparticulate Anion-Exchanger (AE-1 ) The preparation of an anion-exchanger is achieved in two steps. First, low moleaalarweight polyglycolide is prepared using a similar procedure in Example I(c), but using the following polymerization charge: glyoolide (1 mote, 116 g), 1,3 propanediot as an initiator (30 mmole, 2.22 g) and stannous octoate (0.03 mmole). The size reduction and purification of the polymer are then conducted as also described in Example t(c). In the second step, the pracfically non-ionic miaopartides are incubated in hot dilute solution (~80°C) of a diamine, for example hexanediamine of known concentration in dioxane under argon. The concentration of the diamine in dioxane is determined by aadimetry.
When the reaction practically ceases to take place, the amidated micropartides are separated by filtration, rinsed with dioxane, and dried under reduced pressure. The binding capacity of the anion-exchanger (amidated particles) is determined by (1 ) elemental analysis for nitrogen and (2) extent of binding to Naproxen by measuring the extent of drug removed from a dilute solution using HPLC. The latter is confirmed b y release of the immobilized Naproxen with a dilute sodium hydroxide solution of known concentration.
Example V
Preparation of Poly(iactide co-gtycolide) copolymers initiated with propanediol (PLGPD) for use as encasing materials Example V(a): 75J25 P(I)LGPD- A 500 mlglass reactorwas loaded with 235.01 g of I-lactide(Purac Biochem, Arkelsedijk, The Netherlands), 63.09 g of gtycolide (Purac Biochem, Arkelsedijk, The Netherlands) and 1.90 g of propanediol (Riedel de-haen, Seelze, Germany) and then 3.96 mt of a 0.1 M stannous 2-ethyl-hexanoate solution (Sigma, St. Louis, Missouri, USA) in toluene (Riedel de-haen, Seelze, Germany) was _ added (stoichiometricratio of 200 ppm). After drying under vacuum for about one hour to remove the toluene, the reactorwas placed under an atmosphere of oxygen-free nitrogen and immersed in an oil bath preheated at about 160°C. The reactor contents were stirred at about 100 rpm with a Heidolph stirrer (Heidolph Elektro GmbH, Kelheim, Germany).
Once the contents had melted the temperature was increased to about 180°C and maintained at this level for about 3 hours. An amorphous copolymer was obtained. The _17.
copolymer was found to have a molecularweight (MW) of about 12,500 g/mol by gel permeation chromatography (GPC) on a Waters 510 Pump, Waters 410 D'rfferential Refradometer(Waters, Milford, Massachusetts, USA) with light-scattering detection on a Wyatt Minidawn Light Scattering Detector (Wyatt Technology Corporation, Santa Barbara, California, USA).
Example V(b): 90V'10 P(I)LGPD- The title product was synthesized according to the procedure of Example V(a) but using 274.31 g of I-ladide, 24.55 g of glycolide, 1.14 g of propanediol and 3.89 ml of a 0.1 M stannous 2-ethyl-hexanoate solution in toluene (stoichiometric ratio of 200 ppm). A crystalline copolymer was obtained. The copolymer was found to have a molecularweight of about 20,780 ghnol by GPC.
Example V(c): 90/10 P(d,l)LGPD- The title product was obtained by following the procedure of Example V(a) but using 274.31 g of d,l-lactide, 24.55 g of glycolide, i .14 g of propanediol and 3.86 ml of a 0.1 M stannous 2-ethyl-hexanoate solution in toluene (stoichiometricratio of 200 ppm). An amorphous copolymer was obtained. The copolymer was found to have a molecularweight of about 20,650 glmd by GPC.
Example V(d): Poly(I-lactide co-d,l-tactide) copolymer Initiated with propanediol (PLGPD) for use as coating material, 80/20 P(I)L(d,l)LPD
The title product was obtained by following the procedure of Example V(a) but using 239.098 of 1-ladide, 59.778 of d,hlactide (Purac Biochem, Arkelsedijk, The Netherlands) and 1.148 of propanediol and 3.96 ml of a 0.1 M stannous 2-ethyl hexanoate solution in toluene was added (stoichiometricratio of 200ppm). An amorphous copolymer was obtained. The copolymer was found to have a molecularweight (Mw) of 22,320 gllnol by GPC_ It showed a glassy transition at 48°C by DSC.
Purification- Examples V(a), V(b), and V(c) were each washed by nebulization of a 30°~ (WlW) solution in aoetonitrile (Labscan, Dublin, Ireland) at 8 mlltnin into deionized water cooled to about 2°C in a 6L jacketed reactor lin ked to a arculation bath and stirred at about 350 rpm with a Heidolph stirrer (Heidolph Elektro GmbH, Kelheim, Germany). The solutions were fed to a Vibra-Cell VC 50 Atomization nozzle (Biobtock, Illkirch, France) using a Masterflex pump (Cole Parmer Instrument Co., Niles, Illinois, USA) and nebulization was achieved using a sonication frequency of 12 kHz. The dispersions obtained were filtered over Whatman No.1 (24 an diameter) fitter papers (Whatman Intl.
Ltd., Maidstone, Kent, U.K.) and the fitter cakes were rinsed with deionized water, frozen, and lyophilized in an Edwards SuperModutyo Lyophitizer (Edwards, Crawley, West Sussex, U.K.).

_18_ Purity was confirmed by DSC using a TA DSC912s (TA Instruments, New Castle, Delaware, USA) with a heating rate of 10°Chnin which showed glass transitions (Tg) at 44°C, 49°C, 45°C and 48°C for Examples V(a), V(b), V(c) and V(d), respectively.
Example VI
Preparation of Peptide-Loaded Cation Exchangers Example VKa): Loading with Peptide A (p-Glu-His-Trp-Ser Tyr-D-Trp-Leu-Arg-Pro-Gly-NH~, an LHRH analog)- Fourgramsof each of the sodium salts of Examples I(a), I(b), I(c) and II(a) were dispersed in solutions containing 1.33 g of the free-base of Peptide A (Kinerton Ltd., Dublin, Ireland) dissolved in 70 ml of deionized water. The dispersions were incubated at room temperature with stirring for about 2 hours before filtering over a 9 cm diameter Whatman No.1 filter paper (Whatman Intl. Ltd., Maidstone, Kent, U_K.). The filter cakes were rinsed with further deionized water, frozen, and lyophilized in an Edwards SuperModulyo (Edwards, Crawley, West Sussex, U.K.).
Samples were then analyzed for nitrogen by elemental analysis to determine the amount of Peptide A bound. The following results were obtained ExampTe TC x. o ymer w . o ep~i'de~'~
BQUnd a y a . o Vl~a . o a vya~~m~ yc) ~ 5n rVLA 19.29 0 vya~yv~ m(a) ~ uli r~ 17:60 0 t A

Example VI(b): Loading with Peptide B (H-~-D-Nal-Cys-Tyr-D-Trp-Lys-Vai-Cys-Thr NHS the two Cys are bonded by a disulfide bond, a somatostatin analogue)- Following the procedure of Example VI(b) and using 4 g of each of the sodium salts of Examples I(a), I(b), I(c) and II(a) and 1.33 g of the free-base of Peptide B (Kinerton Ltd., Dublin, Ireland) bound miaopartidesof Examples I(a), I(b) and I(c) with peptide B immobilized thereon were obtained. Samples were analyzed for nitrogen content by elemental analysis to determine the amount of Peptide B bound. The results obtained are shown below xamp a x. E Polymer wt. % Pep~~e B
Bound vl(b)(I) I(a) in r~,c~H ~~.zu io V I(D)(II)I(D) l ui I ru~H13. I a io VI(b)(III)1(C) 7 5/l rLiCiH1 'J' .f74"/o VI(D)(IV)I11(a) 1011 rla 14.L3"/o I H

Example VII
Preparation of Encased Polypeptide-Loaded Cation Exchangers by Nebulization Polypeptide-loaded ration exchangers were dispersed in acetonitrile (t_abscan, Dublin, Ireland) solutions of encasing copolymers, indicated below. This dispersal was achieved by homogenizing with an Ultra-turrax T25 (tKA, Staufen, Germany) at about 9,500 rpm for about 5 minutes. The concentration of the encasing copolymerl aoetonitrile solutions ranged from 12.5% to 25% (W/W) and the ratio of encasing copolymer to polypeptide-loaded ration exchanger ranged from 1:1 to 1.3:1 by weight After dispersal, the dispersion was fed to a Vibra-Cell VC50 atomization nozzle (Bioblock, Illkirch, France) with a sonication frequency of 16 kHz using a ceramic piston pump (FMI, Oyster Bay, N.Y., USA) set at 2mtlmin flow rate. Upon reaching the nozzle the dispersion was nebulized into isopropyl alcohol (IPA) (Labscan, Dublin, Ireland) cooled to about -80°C by the addition of dry-ice pellets (A.LG., Dublin, Ireland). The IPA
acted as a collecting non-solvent and was stirred at about 300 rpm using a Heidolph stirrer (Heidolph Elektro GmbH, Kelheirn, Germany). Once nebulization was complete the entire dispersion was allowed to thaw to a temperature between about 10°C and about room temperature. The encased miaopartides were then recovered by vaamm filtration over a Whatman No.1 fitter paper (Whatman tntl. Ltd., Maidstone, Kent, U.K.).
The fitter cake was rinsed with deionized water, frozen and lyophilized in an Edwards SuperModulyo lyophilizer (Edwards, Crawley, West Sussex, U.K.). The resulting encased miaopartideswere analyzed for size using the Malvern MastersizeNE
(Malvern, Worcs., U.K.) and 1% Tween 20 in water as a dispersant. The encased micropartides were also analyzed for nitrogen content by elemental analysis to determine peptide content. The table below represents the various encasing experiments carried out yep e- ~casirig--~onc: ncasng can w .

Loaded Copolymerof EncasingCopolyme Partide Peptide CE: Ex-# Copolymer r : Peptide-Diameter Loading in Ex-# Aoetonitrileloaded CE

vu(a) m(a){II)v(a) z4.31io -1~ . Lrm . o P eptide A

vll(b) Vila){ii)V(b) 22.41% . , iarrr . o Peptide - A

vu{c) m(a)(nl)v(D) 1 Z.5~ 1:1 . urn . o Peptide A

vll(a) vl(a)(In)v(c) ~ z.5% 1:1 . ~

Peptide A

vn(e) vl(a)(IV)v(c) i4.y5~ 1:1 . ~rrl . o Peptide A

vlltr) vl(a)(I)v(c) ~4.yz~ 1.27:1- . L~m .

Peptide A

vu(g) v1(b)(u)v(a) 25.37% . . L~m . o Peptide B

vlt(n) W(b)(n) V(b) 20% . . . dun . o Peptide B

vll(I) vl(D)(ul)v(b) 12.5~ . . ~m .

Peptide B

VIIU) VI(D)(111)V(C) lj.'J% 1: . 1,1m . o Peptide B

VII(K) VI(D)(IV)V(C) 14.~Jb% 1:1 . NJTI . o Peptide B

vll(I) vl(D)(I)v(c) 14.~J2% 1.2s~ . Nm o Peptide B

All samples were sieved over a 180 l.lm sieve (Bioblodc, lllkirch, France) prior to in vivo and/or in vitro testing.
A bound miaopartideor encased miaopartidecan be tested in vitro to assess the release rate of a bound peptide or bound protein by the following method. An aliquot of a bound miaopartideor encased micropartide having a mass of about 50 mg is placed in a continuous flow-cell system where a buffered phosphate solution at about pH
7.2 and at about 37°C flow aaoss the entire mass of the bound miccopartides or encased micxopartides at a rate of about 45 mllhr. Samples of the buffer containing the released drug are collected at about 4°C and analyzed for the peptide or protein concentrations at 1- or 2-day intervals. The retease profile of each miaopartideis determined over a period of 2 weeks.
A bound miaopartide or encased micropartide can be tested to assess the release rate of a bound peptide or bound protein in an in vivo system by the following method. Samples are administered to male Wistar rats (Bioresources, Trinity College, Dublin, Ireland) by intramuscular injection to the thigh. The suspension medium consists of 3% carboxymethylcellulose and 1 % Tween 20 in saline solution. For Peptide A-loaded WO 99138535 PC'TIIE99/00007 samples the effective equivalent dose is 40 pg/Kg/day. The dose for Peptide B-loaded samples is 1 mglKg/day. Samples are~taken by cardiac puncture and the plasma peptide levels are monitored by radioimmunoassays (RIA) specific far Peptide A and Peptide B.
In the case of Peptide A-loaded samples (Peptide A is an LHRH analog), a testosterone RIA is also used to monitor testosterone suppression. As an alternative to the suspension medium,gel-formerscan be used in certain cases. The results are shown in Tables A and B, below.
Table A
ep i a ep ~ a > p m es os erone n Examples Days Days vii(a) zu zi c i~

unto) c vii~e~ c i3 a in ge - ormer Table B
t'ep Ide ~ I'epuae ~ (>>voo Examples pg/ml) Days V I I (g ) NOi teSteO

V11(h) NOI IeSteC1 VIII) NOt IeSteC1 Example VIII
Vlll(a): Nebulization using acetonitrile as solvent and room temperature iPA
as non-solvent About 1.06 g of the ration exchanger of Example i(c) (not bound to polypeptide) was dispersed in a 25.24% (W/W) solution of encasing copolymer of Example V(a) in acetonitrile (Labscan, Dublin, Ireland) such that the ratio of ration exchanger to encasing copolymer was about 1.03a by weight. This dispersal was achieved by homogenizing with an Ultra-turrax T25 (IKA, Staufen, Germany) at about 9,500rpm for about 5 minutes.
After dispersal, the dispersion was fed to a Vibra-Cell VC50 atomization nozzle (Bioblock, Iltkirch, France) with a sonication frequency of l6kHz using a ceramic piston pump (FMI, Oyster Bay, N.Y., U.S.A.) set at 2mllminflowrate. Upon reaching the nozzle the dispersion was nebulized into IPA (Labscan, Dublin, Ireland) at room temperature (17 to 22°C). This IPA acted as a collecting non-solvent and was stirred at about 300rpm using, a Heidolph stirrer(Heidolph ElektroGmbH, Kelheim,Germany). Once nebulization was complete the dispersion was left to stir for about another 60 minutes at room temperature before the encased particles were recovered by vacuum filtration over a Whatman No. 1 filter paper (Whatman Intl. Ltd., Maidstone, Kent, U.K.). The filter cake was rinsed with deionized water, frozen and lyophilized in an Edwards SuperModulyo lyophilizer (Edwards, Crawley, West Sussex, U.K.). The resulting particles were analyzed for particle size using the Malvern MastersizerlE (Malvern, Worcs., U.K.) and 1 % Tween 20 in water as a dispersant. The resulting particles had a mean particle size (d(0.5}) of 84.75tun Vllt(b): Nebulization using Ethyl Acetate as solvent and room-temperature IPA
as non-solvent The nebulization was carried out substantially according to the procedure of Example VI11(a) but using about 0.99g of canon exchanger of Example I(c) (not bound to polypeptide) dispersed in a 24.88°~ (W/IJI~ solution of encasing copolymer of Example V(a) in ethyl acetate (Riedel-de Haen, Seelze, Germany) such that the ratio of ration exchanger to encasing copolymer was about 0.96:1 by weight. The resulting particles had a mean particle size (d(0.5)) of 100.56t.~m VIII(c): Nebulization using Ethyl Acetate as solvent and a higher frequency probe About i .02g of ration exchanger of Example 1(c) (not bound to polypeptide) was dispersed in a 15.14% (WIW) solution of encasing copolymer of Example V(a) in ethyl acetate (Riedel-de Haen) such that the ratio of ration exchanger to encasing copolymer was about 1.05:1 by weight. This dispersal was achieved by homogenizing with an Ultra-iurrax T25 (IKA, Staufen, Germany) at about 9,500 rpm for about 5 minutes. .
After dispersal, the dispersion was fed to a Martin Walter 400 GSIP nebulizer (Sodeva, Le Bouget du Lac, France) with an ultrasonic frequency setting of about 34.6kHz using a ceramic piston pump (FMI, Oyster Bay, N.Y., U.S.A.) set at SmUlmin flow rate. Upon reaching the nozzle the dispersion was nebulized into IPA
(Labscan, Dublin, Ireland) cooled to about -77° by the addition of dry-ice pellets (A.I.G., Dublin, Ireland). This IPA acted as a collecting non-solvent and was stirred at 300rpm using a Heidolph stirrer (Heidotph Elektro GmbH, Kelheim, Germany). Once nebulization was complete the coated particles were recovered by vacuum filtration over a Whatman No. 1 filter paper (Whatman Intl. Ltd., Maidstone, Kent, U.K.). The filter cake was rinsed with deionized water, frozen and lyophilized in an Edwards SuperModulyo lyophilizer (Edwards, : Crawley, West Sussex, U.K.). The resulting particles were analyzed for particle size using the Malvern MasterizerlE (Malvern, Worcs., U.K.) and t %
Tween 20 in water as a dispersant. The resulting particles had a mean particle size (d(0.5)) of 95.691.~m Example IX
Binding to CE's and subsequent Encasing of Somatostatin Analog Peptides C
and D
IX(a): Loading with peptide C
About 1.01 g of the sodium salt of Example I(c) dispersed in a solution containing - 0.25g of the free base of peptide C, which has the structure N
hydroxyethylpiperazinyl-acetyl-D-Phe-Cys-Tyr-D-Trp-Lys-Abu-Cys-Thr-NH2 where the two Cys residues are bonded by a disulfide bond (Kinerton Ltd., Dublin, Ireland) dissolved in 40 m1 deionized water. The dispersion was incubated with stirring for about 2 hours before filtering over a 9 an diameter Whatman No. 1 filter paper (Whatman Intl.
Ltd., Maidstone, Kent, U.K.). The fitter cake was rinsed with further deionized water, frozen, and lyophilized in an Edwards SuperModulyo (Edwards, Crawley, West Sussex. U.K.). The sample was then sent for nitrogen analysis to determine the amountof peptide bound, 20.21 %. .
IX(b~: Loading w'tth peptide D
Using the procedure of Example IX(a) but using about 2.04 g of the sodium salt of , Example I(c) dispersed in a solution containing 0.51 g of the free base of peptide D, which has the structure N-hydroxyethylpiperazinyl-ethylsulfonyl-Phe-Cys-Tyr-D-Trp-Lys-Abu-Cys-Thr-NHZ where the two Cys residues are bonded by a disulfide bond (Kinerton Ltd., Dublin, Ireland) dissolved in 80rn1. The sample was then sent for nitrogen analysis to determinethe amount of peptide bound,19.53%.
Example X
The bound miaopartides of Examples IX(a) and IX(b) were encased as described in Example VII yielding the following results:
~X. Pepti e- coanng c:onc. (wnrv) coanng nnean wt.
No. of r loaded copolymercoating copolymercopolymerParticlePeptide CE

in aoetonitr~e:Peptide Size Loading loaded (Nrn) CE

x(a) ix(a) vtc) 7 L.51 "ro ~ :~ ts3.~~ ~.4ts~r -V(C) 12.48% -~~8a ~.~5 8.87%
-o j - 12.35% - . . .~3 _ 8 74%

Claims (16)

Claims:
1. A process for making an encased bound microparticle or microparticles wherein the encased bound microparticle or microparticles comprise bound microparticle or microparticles and an absorbable encasing polymer where the bound microparticle or microparticles comprise an absorbable heterochain polymer core and one or more peptide, one or more protein or a combination thereof immobilized on said absorbable heterochain polymer core where each peptide is independently selected from growth hormone releasing peptide (GHRP), luteinizing hormone-releasing hormone (LHRH), somatostatin, bombesin, gastrin releasing peptide (GRP), calcitonin, bradykinin, galanin, melanocyte stimulating hormone (MSH), growth hormone releasing factor (GRF), amylin, tachykinins, secretin, parathyroid hormone (PTH), enkaphelin, endothelin, calcitonin gene releasing peptide (CGRP), neuromedins, parathyroid hormone related protein (PTHrP), glucagon, neurotensin, adrenocorticothrophic hormone (ACTH), peptide YY (PYY), glucagon releasing peptide (GLP), vasoactive intestinal peptide (VIP), pituitary adenylate cyclase activating peptide (PACAP), motilin, substance P, neuropeptide Y (NPY), TSH and analogs and fragments thereof or a pharmaceutically acceptable salt thereof; and where each protein is independently selected from growth hormone, erythropoietin, granulocyte-colony stimulating factor, granulocyte-macrophage-colony stimulating factor and interferons;
said process comprising the steps of:
obtaining a dispersion, where the dispersion comprises solid bound microparticles in a solution of the absorbable encasing polymer, by homogenizing and concurrently dispersing said solid bound microparticles into the solution of an absorbable encasing polymer; and nebulizing said dispersion through a nebulization probe, where said probe has an operating ultrasonic frequency range of 12 kHz to 36 kHz, into a medium, where the medium is a non-solvent of said absorbable encasing polymer, at a flow rate of about 1 ml/min to 15 ml/min.
2. A process according to claim 1 wherein said medium comprises isopropanol or ethanol.
3. A process according to claim 2 wherein the solution of the absorbable polymer consists of about 5% to 30% of the absorbable polymer and the temperature of the medium is room temperature to about -80°C.
4. A process according to claim 1 wherein the peptide, peptides, protein a proteins or combination thereof of the bound microparticle comprises 0.1 % to 30% of the total mass of the bound microparticle.
5. A process according to claim 4 wherein the absorbable polymer core comprises glycolate units and citrate residues wherein the ratio of glycolate units to citrate residues is about 7-1 to about 20-1 a glycolate units and tartrate residues wherein the ratio of glycolate units to tartrate residues is about 7-1 to about 20-1 or glycolate units and malate residues wherein the ratio of glycolate units to malate residues is about 7-1 to about 20-1.
6. A process according to claim 5 wherein absorbable encasing polymer comprises (a) I-lactide based units and glycolide based units where the ratio of I-lactide based units to glycolide based units is about 60-40 to about 90-10, (b) d,I-lactide based units and glycolide based units where the ratio of d,I-lactide based units to glycolide based units is about 60-40 to about 90-10, (c) d,I-lactide based units, or (d) I-lactide based units and d,I-lactide based units where the ratio of I-lactide based units to d,I-lactide based units is about 80-20.
7. A process according to claim 6 wherein the absorbable encasing polymer constitutes 5 to 70% of the total mass of the encased bound microparticle or microparticles.
8. A process according to claim 7 wherein the absorbable encasing polymer constitutes 20-60% of the total mass of the encased bound microparticle or microparticles.
9. A process according to claim 8 wherein the absorbable encasing polymer constitutes 30-50% of the total mass of the encased bound microparticle or microparticles.
10. A process according to claim 9 where the peptide is an LHRH analog.
11. A process according to claim 10 where the LHRH analog is p-Glu-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NH2.
12. A process according to claim 9 where the peptide is a somatostatin analog.
13. A process according to claim 12 where the somatostatin analog is H-.beta.-D-Nal-Cys-Tyr-D-Trp-Lys-Val-Cys-Thr-NH2, where the two Cys are bonded by a disulfide bond.
14. A process according to claim 12 where the somatostatin analog is N-hydroxyethylpiperazinyl-acetyl-D-Phe-Cys-Tyr-D-Trp-Lys-Abu-Cys-Thr-NH2 where the two Cys residues are bonded by a disulfide band.
15. A process according to claim 12 where the somatostatin analog is N-hydroxyethylpiperazinyl-ethylsulfonyl-Phe-Cys-Tyr-D-Trp-Lys-Abu-Cys-Thr-NH2 where the two Cys residues are bonded by a disulfide bond.
16. A process according to claim 1 for making an encased bound micoparticle or microparticles, substantially as hereinbefore described and exemplified.
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