CA1233414A - Method for producing microcapsule - Google Patents

Abstract

METHOD FOR PRODUCING MICROCAPSULE

Abstract of the Disclosure A microcapsule produced by preparing a water-in-oil emulsion comprising an inner aqueous layer containing said water-soluble drug and a drug retaining substance therefor and an oil layer containing a polymer substance, then thickening or solidifying said inner aqueous layer to a viscosity of not lower than about 5000 centiposes and finally subjecting the resulting emulsion to in water drying gives prolonged release of water-soluble drug.

Description

~233~ 24205-585 METHOD FOR PRODUCING ~lICROCAPSULE

This invention relates to a prolonged release micro-capsu1.e of water-soluble drug, and a method Eor producing the same.
A variety o:E dosaye forms have been proposed for drugs which requlre long-term oE repeated adm.inistration.
As one o such dosage worms, European Pat~llt ~p~ cation Publ.ication No. 52,510 discloses a microcapsule prepared by the phase separation technique using a coacervating agent such as mineral oils and vegetable oils. However, microcapsules prepared by this and other analogous processes have the disadvantage that there tends to occur an inter-adhesion of particles in the course of production.
Under the circumstances, the present inventors conducted studies to develop a prolonged release prepa-ration of water-soluble drug and found that a microcapsule having excellent properties can be produced with good efficiency by interposing a step of thickening or solidi-fying the inner aqueous layer of water in oil emulsion in the course of microencapsulation by in water drying process. The above finding was followed by further studies, which have resulted in the present invention.
The prolonged release microcapsule of the present invention is made by preparing a water-in-oil emulsion comprising an inner aqueous layer containing said water-soluble drug and a drug retaining substance therefor and an oil ,~j' ~L233~

layer containing a polymer suhstance (microcapsule wallsubstance) then thickening or solidi,ying said inner aqueous layer to a viscosity of not lower than about 5~00 centipoises and finally subjecting the resulting emulsion to in water drying process.

The water-soluble drug employed in the practice of this invention is a drug which is highly hydrophilic and has a low oil/water distribution ratio. The term "low oil/water distribution" ratio means that the octanol/water distribution ratio, for instance, is not greater than about 0.1.
There is no particular limitation on the kind and type ox said water-solubl~ dr~g~ Thus, for example, 15 biolocJically ackive polypep~.icles end othcr ~lltibiotic~
antitumor agen~.s, antipyretics, analgesics, anti:inflam-matory agents, antitussives and expectorants, sedatives, muscle relaxants, antiepileptics, antiulcer agents, anti-depressants, antiallergic drugs, cardiotonics, anti-arrhythmi.c agents, vasodilators, antihypertensive di-uretics, antidiabetics, anticoagulants, haemostatics, antituberculotics, hormone drugs, antinarcotics, etc. may be mentioned as the water-soluble drug.
The biologically active polypeptides which are employed in accordance with this invention are preferably those consisting oE two or more amino acid units and having a molecular weight between about 200 and about 80000.
Examples of such polypeptides include luteinizing hormone releasing hormone (LH-RH) and its derivatives having LH-RH live activity, i.e. the polypeptides of the formula (I):
)GlU-Rl-Trp-ser-R2-R3-R4 Arg-Pro-R5 (I) wherein Rl is His, Tyr, Trp or p-NH2-Phe; R2 is Tyr or Phe; R3 is Gly or a D-amino acid residue; R4 is Leu, Ile or Nle; R5 is Gly-NH-R6 (R6 is H or a lower alkyl group ~L23;3~

which may op-tionally be substituted by OH) or NH-R6 (R6 is as defined above) or salts thereof] [see United States Patents No. 3,853,837, 4,008,209 and No. 3,972,859, British Patent No. 1,423,083, Proceedings of the National Academy of Sciences of the United States of America 78, 6509-6512 (1981)].
Referring to the above formula (I), the D-amino acid residue designated by R3 may be an ~-D-amino acid residue of up to 9 carbon atoms (e.g. D-Leu, Ile, Nle, Val, Nval, Abu, Phe, Phg, Ser, Thr, Met, Ala, Trp, ~-Aibu), and these may have suitable protective groups (e.g. -t-butyl, t-butoxy, t-butoxycarbonyl, naphthyl). Of course, acid salts ancl metal complex compounds o pep~ldes (~) can also be veal ln the tame manner a k a~orem~nt:ion~d p~ptides In this sp~ci~lc~tion, when amino acids, peptides, protective groups, etc. are mentioned, in connection with polypeptides (I), by abbreviations, such abbreviations are those established by IUPAC-IUB Commission on Biochemical Nomenclature or those used commonly in the particular field of art and when there may exist optical isomers of such amino acids, L-forms are intended unless otherwise indicated.
It should be understood that the polypeptide of formula (I) wherein Rl = His, R2 = Tyr, R3 = D-Leu, R4 = Leu, and R5 - NHCH2-CH3 is hereinafter referred to as TAP-144.
As examples of such polypeptides, LEI-RH antagonists (United States Patents No. 4086219, No. 4124577, No. 4253977, No. 4317815, No. 329526, and No. 368702) may be mentioned.
As further examples of said polypeptides may be mentioned insulin, somatostatin, somatostatin derivatives (Uni-ted States Patent No. 4087390, No. 4093574, No. 4100117 and No. 4253998), growth hormones, prolactin, adrenocorticotropic hormone (ACTH), melanocyte stimulating hormone (MSH), thyroid hormone releasing hormone (TRH), ~LZ33~

its salts, and derivatiyes thereof (US. Patent No. 3,957,247 and U.S. Patent No. 4,10a,152), thyroid stimulating hormone ~TSH), luteinizing hormone (LH), follicle stimulating hormone (ASH), vasopressin, vaso~
pressin derivatives ~desmopressin [Folia Endocrinologica Japonica 54, No. 5, p. 676-691 (1978)]], oxytocin, calcitonin, parathyroid hormone, glucayon, gastrin, secretin, pancreozymin, cholecystokinin, angiotensin, human placental lactogen, human cholionic gonadotropin (HCG), enkephalin, enkephalin derivatives [United States Patent No. 4277394, European Patent Application Publication No. 31567], endorphin, kyo-torphin, interEerons (a, I, y), interleukins (I, II, and III), taftsin, thymopoie-tin, -thymosin, thymos~imulin, thymic humoral :Eactor ('rHF), serum thymic factor (F'~S), and its derivat:ives (United States Patent No. 4229438) and o-ther thymic fac-tors [Medicine in Progress 125, No. 10, p. 835-843 (1983)], tumor necrosis factor (TNF), colony stimulating factor (CSF), motilin, dinorphin, bombesin, neurotensin, cerulein,

2~ bradykinin, uxokinase, asparaginase, kallikrein, substance P analogue and antagonist, nerve growth factor, blood coagula-tion factors yIII~ ancl IX, lysozyme chloride, polymixin B, colistin, gramicidin, bacitracin, protein I synthesis stimulating peptides (British patent No. 82320~2), 25 I gastric inhibitory polypeptide (GIP), vasoac-tive intestinal polypeptide (VIP~, platelet-derived growth factor (PDGF), growth hormone releasing factor (GRF, somatocrinin), bone morphogenetic protein (BMP), epidemale growth factor (EGF), etc.

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As examples of said antitumor agents, may be mentioned bleomycin hydrochloride, methotrexate, actino-mycin D, mitomycin C, vinblastine sulfate, vincristine sulfate, daunorubicin hydrochloride, adriamycin, neocarcinostatin, cytosine arabinoside, fluorouracil, tetrahydrofuryl-5-fluorouracil, krestin, picibanil, lentinan, levamisole, bestatin, azimexon, glycyrrhizin, poly I:C, poly A:U and poly ICLC.
As examples of said antibiotics, may be mentioned gentamicin, dibekacin, kanendomycin, lividomycin, tobra-: mycin, amikacin, fradiomycin, sisomicin, -tetracycline hydrochloridç, oxytetracycline hydrochloride, rolit:etra-cycline, doxycycline hydrochloride, ampicillin, piperac.illin, ticarcillin, cephalothin, cephaloridine, cefotiam, cefsulodin, cefmenoxime, cefmetazole, cefazolin, cefotaxime, cefoper~zone, ceftizoxime, moxolactam, latamoxef, thienamycin, sulfazecin, and azthreonam.

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The aforementioned antipyretic, analgesic and anti-inflammatory drugs include, for instance, sodium salicylate, sulpyrine, sodium flufenamate, sodium diclofenac, sodium I indomethacin, morphine hydrochloride, pethidine hydro-chlorlde, levorphanol tartrate and oxymorphoneO Asexamples of said antitussives and expectorants may be mentioned ephedrine hydrochloride, methylephedrine hydro-chloride, noscapine hydrochloride, codeine phosphate, dihydrocodeine phosphate, alloclamide hydrochloride, 10 l chlophediano:L hydrochloride, picoperidamine hydrochloricle, cloper~stine, pro-to}~ylol hyd~ochloricle, isoproterenol hydrochloride, salbut~mol ~ul~a-te and terbu-tallnc suleate.
Examples of said seda-tives include chlorpromazine hydro-chloride, prochlorperazine, trifluoperazine, atropine sulfate and scopolamine methylbromide. The muscle relaxants include, among others, pridinol methanesulfonate, tubocurarine chloride and pancuronium bromide. The anti-epileptics include, for instance, sodium phenytoin, I ethosuximide, sodium acetazolamide and chlordiazepoxide 20 I hydrochloride. Examples of said antiulcer drugs include metoclopramide and L-histidine monohydrochloride.
Examples oE said antidepressants include imipramine, clomipramine, noxiptiline and phenelzine sulfate. The antiallergic drugs include, among others, diphenhydramine hydrochloride, chlorpheniramine maleate, tripelenamine hydrochloride, methdilazine hydrochloride, clemizole hydrochloride, diphenylpyraline hydrochloride and methoxy-phenamine hydrochloride. The cardiotonics include, among others, trans-~-oxocamphor, theophyllol, aminophylline and 30 j etilefrine hydrochloride. The antiarrythmic agents in-clude, for instance, propranolol hydrochloride, alprenolol hydrochloride, bufetolol hydrochloride and oxyprenolol ~%33~

hydrochloride. The vasodilators include, among others, oxyfedrine hydrochloride, diltiazem hydxochloride, tolazoline hydrochloride, hexobendine alld bamethan sulfate.
The antihypertensive diuretics include, among others, hexamethonium bromide, pentolinium, mecamylamine hydro-chloride, ecarazine hydrochloride and clonidine hydro-chloride. Examples of said antidiabetics include sodium glymidine, glypizide, phenformin hydrochloride, buformin hydrochloride and metformin. The anticoagulants include, among others, sodium heparin and sodium citrate. The haemostatic agents include, among otllers, thro~boplastin, thrombin, menadione sodium bisulfite, ace-tomenaphthone, ~-a~ino~caproic acid, tr~ne~amic acid, carbazoGhrom~
soclium sulfonate and ~dr~nochrome monoamino~uanidinc methanesulfonate. ~rnong said an-tituberculotics are iso-niazid, ethambutol and sodium p-aminosalicylate. The hormone druys are exemplified by prednisolone succinate, prednisolone sodium phosphate, dexamethasone sodium sulfate, betamethasone sodium phosphate, hexestrol phosphate, hexestrol acetate and methimazole. The antinarcotic agents include, among others, levallorphan tartrate, nalorphine hydrochloride and naloxone hydrochloride.
The proportion of said water-soluble drug depends on tlle Jcind of drug, expected pharmacological effect and its duration etc. but its concentration in the inner aqueous layer is selected from the range of about 0.001% to about 90% (w/w) and preferably 0.01% to 80% (w/w).
The drug retaining substance employed in accordance with this invention is either a substance which i5 water-soluble and hardly soluble in the organic solvent contained insaid oil layer and when dissolved in water assumes a viscous semi-solid consistency or a substance which gains considerably in viscosity to provide a semi-solid or solid matrix under the influence of an external factor such as temperature, pH, metal ions (e.g. Cu , Al , Zn , etc.), organic acids (e.g. tartaric acid, citric acid, tannic acid, ~;~3~

etc.), a salt thereof (e.g. calcium citrate, etc.), chemical condensing agents (e.g. glutaraldehyde, acetoaldehyde), e~c.
As examples of such drug retaining substance may be mentioned natural or synthetic mucilages and high molecular weight compounds.
Among such natural mucilages are gum acacia, Irish moss, gum karaya, gum tragacanth, gum guaiac, gum xanthane, locust bean gum, etc., while natural high molecular weight compounds include, among others, various proteins such as casein, gelatin, collagen, albumin (e.g. human serum albumin), globulin, fibrin, etc. and various carbohydrates such as cellulose, dextrin, pectin, starch, agar, mannan, etc. These substances may be usecl as they are or in chemically modified ~or~s, e.g. esteriEied or etherified Eorms (e.g. ~etllyleellulose, ~thyleellulose, carboxyln~hyl-cellulose, g~l~tin succinate, lo hydrolyzed eorms c sodium alginate, sodium pectinate, etc.) or sa:lts thereof.
As examples of said synthetic high molecular weight compounds may be mentioned polyvinyl compounds (e.g. poly-vinyl pyrrolidone, polyvinyl alcohol, polyvinyl methylether, polyvinyl ether, etc.), polyearboxylic ae ds (e.g.
polyaerylie aeid, polymethaerylie aeid, Carbopol [Goodrieh & Co., U. S. A.], ete.), polyethylene compounds (e.g.
polyethylene glycol, ete.) and polysaeeharides (e.g. poly-suerose, polyglucose, polylaetose, ete.) and salts thereof.
Also ineluded are those eompounds which undergoeondensation or cross-linking under the influence of said external faetors to give moleeular weight eompounds.
Among the aforementioned compounds, gelatin, albumin, peetin and agar are partieularly desirable.
These compounds may be used alone or in eombination and while the proportion of sueh eompounds depends on the kind of compound, it is seleeted from the range of about 0.05% to 80% (w/w) in terms of eoneentration in the inner aqueous layer, preferably from the range of about 0.1% to r k ~Z~3~

50% (w/w) on the same basis. It should, however, be understood that such compounds must be used in sufficient amounts to ensure that the initial viscosity of the inner aqueous layer in the water-in-oil emulsion described here-inafter will be not lower than about 50Q0 centipoises (cps),preferably not lower than about 10000 cps, or the inner aqueous layer may be increased in viscosity to not lower than about 5000 cps, preferably not lower than about 10000 cps, or be solidified by external factors.
The aforementioned polymer substance employed in the oil layer are polymers hardly soluble or insoluble in water and biocompatible. us examples o such polymer subs-tance may be mentionecl biodegradable aliphatic polymers (e.g. polylactic acid, poly-ylycolic acid, polycitric acid, polymalic acid, etc.), poly-~-cyanoacrylic acid esters, poly-~-hydroxybutyric acid, polyalkylene oxalate (e.g. polytrimethylene oxalate, polytetramethylene oxalate, etc.), poly(ortho esters), poly(ortho-carbonate), other polycarbonate (e.g. polyethylene carbonate, polyethylene propylene carbonate, etc.), poly-amino acids (e.g. poly-~-benzyl-L-glutamic acid, poly-l~alanine, poly-r-methyl-L-glutamic acid, etc.) and so on. As further examples of biocompa-tible polymer substance may be mentioned, polystyrene, polyacrylic acid, polymethacrylic acid, copolymer of acrylic acid and methacrylic acid, nylon, tetron, polyamino acid, silicone polymer, dextran stearate, ethylcellulose, acetylcellulose, nitrocellulose, poly-urethane, maleic anhydride copolymers, ethylene-vinyl acetate copolyrner,polyvinyl acetate, polyvinyl alcohol, polyacrylamide, etc. These polymer substances may be used alone or in the form of copolymers or mixtures ox two or more species or of salts.
When used in injectable preparations, biodegradable polymers, among said polymers substances, are especially desirable, and as preferable examples of such polymer substancernay be mentioned polylactic acid and a copolymer of lactic acid ~Z3~34~4 - 10_ and glycolic acid and these mixtures.
The average molecular weight of such a polymer substance as used in accordance with this invention preferably ranges from about 2000 to about 800000 and is more desirably selected from the range of about 5000 to about 200000.
When a lactic acid-glycolic acid copolymer is used as said polymer, its comonomer ratio is preferably in the range of about lO0/0 through about 50/50.
The proportion of such a polymer substance depends on the strength of pharmacological activity of the water soluble drug used and the ra-te and duration oE release of the drug. sy Jay of illus~ra-tion, toe proportion of this polymer substance may range :Erom l/5 to lO000 times in preEerably l to lO00 timcs if w~icJht of the wate:r-solubl~
drug.
The concentration of said polymer substance in the oil layer is about 0.5 to 90% (w/w) and preferably about 2 to 60~ (w/w).
The solution containing said polymer substance (oil layer) is a solution of the polymer substance in a solvent.
The solvent for this purpose should be one which boils at a temperature up to about 120C and is immiscible with water and capable of dissolving the polymer substance and as such there may be mentioned halogenated alkanes (e.g. di-chloromethane, chloroform, chloroe-thane, dichloroethane, trichloroethane, carbon tetrachloride, etc.), ethyl acetate, ethyl ether, cyclohexane, benzene, n-hexane and toluene. These solvents may be used alone or in combi-nation.
With regard to the microencapsulation procedure, the drug retaining substance in an amount sufficient to give the aforementioned concentration is first dissolved in water and, then, the water-soluble drug is added in an amount sufficient to give the aforementioned concentration, whereby an inner aqueous layer is provided.
As a pH-adjusting agent for maintaining the stability ~;~33~

and solubility of the water-soluble drug, there may be incorporated in this inner aqueous layer such an additive as carbonic acid, acetic acid, oxalic acid, citric acid, tartaric acid, succinic acid or phosphoric acid, sodium or potassium salts thereof, hydrochloric acid or sodium hydroxide. Moreover, as a stabilizer for the water-soluble drug, there may also be added such an agent as albumin, gelatin, citric acid, ethylenediamine sodium tetraacetate, dextrin, sodium hydrosulfite, etc. The inner aqueous layer may also contain a preservative such as p-oxybenzoic acid esters (e.g. me-thylparaben, propylparaben, etc.), benzyl alcohol, chlorobutanol, thi.merosal, e-tc.
l'he inner aqucous layer -thus prepared is poured into a solution of slid polymer subst~lnc~ (oil layer) end the lS mixture is emulsiEie~ to give a water-in-oil emlusion.
The emulsiEication can be effected by the conventional dispersion techniques. For example, intermittent shaking, mixing by means of a propeller mixer, turbine mixer or the like, colloid mill operation, mechanical homogeniz-ation, ultrasonication, etc. may be utilized.
When the viscosity of the inner aqueous layer insuch a water-in-oil emulsion is more than about 5000 centipoises or preferably over about 10000 centipoises from the beginning, the emulsion is immediately subjected to a desorption procedure but, otherwise, resort is had -to an external factor to thicken the inner aqueous layer to a viscosity over about 500Q centipoises or preferably over about 10000 centipoises or solidify the same.
Exemplary procedures for increasing the viscosity include a heat treatment, cooling to a low temperature, freezing, rendering the pH acidic or alkaline, or adding such an agent as metal ions (e.g. iron ion for gum acacia, copper ion for carboxymethylcellulose, or calcium or magnesium ion for sodium pectinate) or organic acids or salts thereof (e.g. calcium citrate for sodium alginate, or adipic acid or tartaric acid for polyvinyl alcohol).

~33~

There may also be mentioned the technique of cross-linking and condensing the polymer substance in t:he inner aqueous layer using a chemical condensing agent (e.g. glutaraldehyde~
acetaldehyde, etc.).
With regard to the heat treatment, the procedure must be carried out in a closed vessel so as to avoid evaporation of the solvent contained in the oil layer.
The temperature is virtually optional only if it is higher than the gelation temperature. Taking proteins as an example, the temperature is generally about 40 to 120C
and the time is about 5 minutes to about 8 hours. This treatment thickens or solidifies the inner aqueous layer the technique of cooling -the emulsion to a low temperature comprise cooling it to bout -SC to abouk 35C and maintaininy the low temper~-ture wi-th stirring Eor about l minute to 6 hours. In the case oE ajar whose gelation point is about 40C, the emulsification is conducted under heating at about 50 to 80C and, then, caused to gel at the above-mentioned temperature. For all types of inner aqueous layer, it may be frozen by cooling at about -60C to 0C hut the temperature should not be below the solidification point of the oil layer.
As regards the procedure of adding a metal ion, an organic acid or a salt thereof, the amount thereof depends on the amoun-t of the drug retaining substance in the inner aqueous layer and may range from about 1/4 to 20 molar equivalents and preferably from about 1 to 10 molar equivalents. The time required for said thickening or solidification is preferably not more than about 6 hours.
With regard to the technique of cross-linking and condensing the high molecular compound in the inner aqueous layer with chemical condensing agent, such condensing agent may for example be an aqueous solution of glutaralde-hyde or acetaldehyde or a solution of the same in an organic solvent such as halogenated alkanes (e.g. chloroform, dichloromethane, etc.), toluene, etc. Particularly,

3~4 -13^

a 501ution in the latter solvent which is miscible with the solvent used in the oil layer is desirable, because the particle size of the inner aqueous layer is not in-creased. The chemical condensing agent is added in a proportion ox about 2 to 5 molar equivalents based on the drug retaining substance in the inner aqueous layer and the mixture is reacted under stirring for about l to lO
hours.
More specifically, taking gelatin as an example of said drug retaining substance, a water-in-oil emulsion of predetermined particle size is first prepared and then cooled to about 0 to 10C for about 5 to 30 minu-tes with constant siring whqrcby the inner aq~leous layer is caused to cJel into ~cmi-solicl consistency. When agar is lS usecl as the drug retaininy substance, the desired semi-solid consistency can be obtained by using a somewhat lower concentration than in the case of gelatin and the same procedure as that for gelatin. When albumin is employedr solidification is effected with a condensing ~0 agent such as glutaraldehyde. In this case, the water-soluble drug is dissolved in a ca. 5 to 50% aqueous solution of human serum albumin and the resul-ting solution is added to -the organic solvent solution of high polymer to prepare a water-in-oil emulsion. Thereto is added a ca. l to 50% solution of glutaraldehyde in an organic solvent miscible with the oil layer and the mixture is reacted under stirring for about l to lO hours so as to solidify the inner aqueous layer. In this procedure, albumin may be replaced with other substances that can be thickened or solidified by cross-linking and condensation, such as globulin, gelatin, casein, collagen and other polyamino acids. After the reaction, a compound which is ready to react with the condensing agent, e.g. an amino compound such as ethanolamine, aminoacetic acid, etc., may be added so as to inactivate the residual condensing - agent.

1 233~
-14_ When a substance capable of increasing in viscosity on alteration of pH, such as carboxyvinyl polymer ~Carbopol, B. F. Goodrich, U.S.A.) is added to the inner aqueous layer, a ca. l to 20-o solution of sodium hydroxide in ethanol or methanol is separately prepared and a small quantity of the solution is added to the water-in-oil emulsion to increase the viscosity of the inner aqueous layer.
The water-in-oil emulsion thus prepared is subjected to in water drying- Thus, this water-in-oil emulsion is added to a third aqueous layer to give a W/O/W ternary layex emulsion and, finally, the solvent in the oil :layer is desorbed to give microcapsl1les.
An emulsifying aycnt may be added to -the third or outer ac1ueous layer. It may be virtually any emulsifier that forms a stable oil-in-water emulsion, and is thus exemplified by anionic surfactants ~e.g. scdium oleate, sodium stearate, sodium laurylsulfate, etc.), nonionic surfactants(e.g.
polyoxyethylene sorbitan fatty acid esters [Tween 80 and Tween 60, Atlas Powder, U.S.A.], polyoxyethylene castor oil derivatives [HCO-60 and HCO-50, Nikko Chemicals, Japan], etc.), polyvinyl pyrrolidone, polyvinyl alcohol, carboxyme-thyl-cellulose, lecithin, gela-tin, ekc. Such emulsifiers may be used ei-ther alone or in combination. The concentration of the emulsifier may be selected from the range of about O.Oln6 to 20% and is prefearbly in the range of about 0.05%O
to 10%.
The aforesaid desorption of the solvent from the oil layer can be accomplished by the conventional technique.
mus, such desorption is effected by gradual decrease of pressure under agitation with a propeller mlxer or magnetic stirrer or by adjusting the degree of vacc~m in a rotary evaporator. When the higher the stir-ring speed is, the smaller the diameter of the product microcapsule is.
The time required for such procedures can be shortened by wrung the Wow emulsion by degrees so as to make the solvent desorption thorough, after the solidification of the polymer has progressed to some extent and * Trade Mark ~X33~

the loss of the drug from the inner aqueous layer has decreased. When the thickening or solidification is effected by techniques other than temperature control, the desorption may be effected by allowing the W/O/W
emulsion to stand under stirring, warming the emulsion or blasting it with nitrogen gas. The process of desorption of the solvent is an important process having great bearings on the surface structure of microcapsules which governs the release of the drug. For example, when the desorption speed is increased, pits in the surface layer increase in number and size so that the release rate of the drug is increased.
The mi~rocapsules obtained in -the above manner are recovered by c~nkri~u~atiQn or ;eiltration, net thy E~eq water-501uble ~ruy, emulsi~yincJ a~Jents, etc. on the ~ur~ace are removed by repeated washing with water, then, if necessary, the microcapsules are warmed under reduced pressure to achieve a complete removal of moisture and of the solvent from the microcapsule wall.
The above microcapsules are gently crushed and sieved, if necessary, to remove coarse microcapsules.
The particle size of microcapsules depends on the desired degree oE prolonged release. When they are to be used as a suspension i-ts size may be within the range satisfying the recluired dispersibility and needle pass requirements.
For example, the average diame-ter may range from about O.S to ~00 em and preferably from about 2 to 200 em.
The method of this invention enables one to apply a high shear stress in the preparation of the W/O/W
emulsion with a less breakdown of the inner aqueous layer and facilitates particle size control to thereby provide fine microcapsules with good efficiency. The additional commercial advantage of this invention is that the re-quired quantity of organic solvent is as small as compared with the drying-in-oil technique.
Moreover, the microcapsules produced by the method ~Z3~

of this invention feature a reduced coalescence of individual microcapsules during production so that they are more truly spherical in configuration. In addition, desorption of the solvent from the oil layer can be easily controlled to adjust the surface structure tfor example, the number and size of fine holes which serve as main routes of the drug release) of microcapsules.
The microcapsules according to this invention can be administered in clinical practice directly as fine granu-lets or as formulated preparation into a variety ofpreparations. Thus, they can be used as raw materials for the production of final pharmaceutical preparations.
Such prepara-tions include, among o-thers, injections, oral preparations (e.g. powders, cJranules, capsules, talk e~c.), naval preparations, suppositories rectal, vaginal), and so on.
When the microcapsules according to this invention are to be processed into an injectable preparation, they are dispersed in an aqueous vehicle together with a dispersing agent te.g. Tween 80, HCO 60 (Nikko Chemicals), carboxymethylcellulose, sodium alginate, etc.), preserva-tive (e.g. methyl-paraben, propyl-paraben, benzyl alcohol, chlorobutanol, etc.), isotonizing agent (e.g. sodium chloride, glycerin, sorbitol, glucose, e-tc.), etc. The vehicle may also be a vegetable oil (e.g. olive oil, sesame oil, peanut oil, cottonseed oil, corn oil, etc.), propylene glycol or the like. In this rnanner, a prolonged release injection can be produced.
The prolonged release injection made from said microcapsules may be further supplemented with an ex-cipient (e.g. mannitol, sorbitol, lactose, glucose, etc.), redispersed, and then be solidified by freeze-drying or spray-drying, and on extemporaneous addition ox a dis-tilled water for injection or suitable vehicle or the reconstitution, such preparation gives a prolonged - release injection with greater stability.
* Trade Clark , ~33~

When the microcapsules according to this invention are to be processed into tablets, they are mixed with an excipient (e.g. lactose, sucrose, starch, etc.), dis-integrating agent (e.g. starch, calcium carbonate, etc.), binder (e.g. starch, gum arabic, carboxymethylcellulose, polyvinylpyrrolidone, hydroxypropylcellulose, etc.) or/and lubricant (e.g. talc, magnesium stearate, polyethylene glycol 6000, etc.), and the mixtures are compressed in molds.
To manufacture a nasal preparation from the micro-capsules according to this invention, they are provided solid, semi-solid or liquid sta-te in the conventional manner. Jo manuEacture the solid nasal prepara-tion for inskance, the microcapsules either ~5 they ore or togethcr with an excipient (e.cJ~ glucose, manni-tol, staxcll, micro-crystalline cellulose, etc.) and/or thickener (e.c~.
natural mucilages, cellulose derivatives, polyacrylates, etc.) are processed into a powdery composition. To make a liquid composition, the microcapsules are processed into an oily or aqueous suspension in substantially the same manner as in the case of injections. The semi-solid preparation may be an aqueous or oily gel or ointment.
In any case, there may be added a pH adjusting agent (e.g. carbonic acid, phosphoric acid, citric acid, hydro-chloric acid, sodium hydroxide, etc.), a preservative(e.g. p-hydroxybenzoic acid esters, chlorobutanol, benzalkonium chloride, etc.)l etc.
A suppository of the microcapsules according to this invention, whether in oily or aqueous solid or semi-solid state or in liquid state, can be produced in the per se conventional manner. The kind of oleagenous base for such composition is optional only if it will not dissolve the microcapsules. Thus, for example, higher fatty acid glycerides [e.g. cacao butter, Witepsol (Dynamit-Novel, West any), etc.], intermediate fatty acids [e.g. Miglyol~(Dynamit-Novel), etc.] and vegetable oils (e.g. sesame oil, soybean ~Tr~.l c~ ok ~L%334~

oil, cottonseed oil, etc.) may be mentioned. The aqueous base is exemplified by polyethylene glycol and propylene glycol, while the aqueous gel base may be selected from among natural mucilages, cellulose derivatives, vinyl polymers, polyacrylates, etc.
The dosage of the prolonged release preparation according to this invention depends on the kind and amount of the active ingredient (i.e. water-soluble drug), dosage form, duration of drug release, recipient animal (e.g.
warm-blooded animals such as mouse, rat, horse, cattle, man), and object of treatment. It is however, sufficient to ensure -that the effective dose of the active ingredient will be administered. For example, the amount per dose to humans may be selected from the range of about 1 mg to 15 10 g, preferably about 10 mg to 2 g, in terms of the weight of microcapsules.
When an injectable dosage form is employed, the volume of the suspension may be selected from the range of about 0.1 to 5 ml, preferably about 0.5 to 3 ml.
In this manner, there are provided pharmaceutical compositions made up of a water-soluble drug, a drug carrier substance and a biocompatible polymer which are capable of continuous and sustained release of the drug.
The prolonged release preparation according to this invention has the following and other advantages.
(1) The prolonged release of water-soluble drugs in various dosage forms can be ensured. Particularly when a long-term treatment with injections is required for the desired effect, the preparation helps achieve the desired pharmacological effects with an administration schedule of once a month or a week or even once a year, instead of giving injections every day. Moreover, compared with the conventional sustained release drugs, the prolonged release preparation according to this invention ensures longer sustained effects.
(2) When injections are prepared using biodegradable ~3~

polymers, surgical procedures such as implantation are not required but the preparations can be administered sub-cutaneously or intramuscularly as easily as ordinary injectable suspensions, not entailing the need for removal 5 f rom the body.
The preparation according to this invention can be directly administered to the tumor itself, the site of inflammation or the receptor region, so that systemic side effects can be controlled and the drug be allowed to act on the target organ for a longer time period with ef-ficiency, thus making for increased drug efficacy.
Moreover, it can be used in intra-arterial administration in the vascular embolic therapy proposed by Ka-to en al for cancer of -the kidney and of the lung (I,ancet II, ~79-~80, l 1979).
(3) The release of the active incJredient is continu-ous, and in the case of antihormones, receptor antagonists, etc., greater pharmacological effects are obtained than by daily pulsatiLe administration.

(4) Since a drug retaining substance is employed, the water-soluble drug can be incorporated in the micro-capsule more easily and efficiently than by the con-ventional drying-in-solvent technique. Moreover, the microcapsules are fine and more truly spherical in configuration.

(5) By varying the rate of desorption of the solvent from the polymer constituting the microcapsule wall, the number and size of fine holes in the surface layer of each microcapsule which are determinant of the rate of drug release can be freely controlled.

~233~

The following experimental and working examples are further illustrative of this invention.
Experimental Example 1 The repeated administration of TAP-144 to mature female rats in massive doses causes desensitization of the pituitary-gonad system to thereby halt the sexual cycle in the diestrous stage. It is known that this halt of the sexual cycle is promptly recovered as the adm~nis-tration of TAP-144 is discontinued. Therefore, using this halt of the sexual cycle of female rats as an indicator, the present inventors investiga-tes the durations of action oP 7 different microcapsules preparecl using different polymers, which are among the kinds oE microcapsules ~cco;rcli.n~ to ~mp~e l and two other microcapsules lS (No. 039 and No. 0310) which are produced by usiny TAP
for the inner aqueous layer in the amounts of l/2 and 2.5 times, respectively, but in otherwise the same manner as Example 1. Thus, using SD female rats (aged 14 to 16 weeks) which are confirmed to show a normal 4-day cycle by the examination of vaginal smears during the preceding period of at least 1 week in groups of 5 individuals, each of the above-mentioned microcapsules is injected subcutaneously at the back of the neck in a dose of 3 mg/kg as TAP-144.
Thereafter, the examination of vaginal smears is carried out every day to monitor changes in sexual cycle. The micro-capsules are administered either in the form ooily suspensions in purified sesame oil or in the form of aqueous suspensions prepared using a dispersed vehicle constituted with 0.2% Tween 80, 0.5%
sodium carboxymethylcellulose, 0.14~ methyl-paraben, 0.014%
propyl-paraben and 8% D-sorbitol in distilled water for injection.
The results are presented in Table 1. It will be apparent from Table 1 that all of the microcapsules according to this invention have every satisfactory durations of action.

33~

Table 1 .
Duration of actiona) Microcapsule No. Formulation (jays) 031 Aqueous _105 033 Aqueous19.0 + 0.6 033 Oily 19.2 + 0.5 034 Aqueous _123 035 Aqueous59.0 + 7.~
036 Aqueous117.2 + 1.8 037 Aqueous39.8 1.8 038 Aqueous35.0 1.3 , 039 ~queous60.0 6.2 0310 Aqueous19.6 0.~l a) Mean slander error Eor S rats.
Experimental Example 2 The repeated administration of TAP-144 to male rats in massive doses causes an atrophy (decrease of organ weight) of the inner genital organs due to desensitiza-tion of the pituitary-gonad system. Using this action, the duratior. of action of the TAP-144 microcapsules produced in Example 1 are investiyated. Thus, No. 032 and No. 035 microcapsules according to Example 1 are injected sub-cutaneously at the back of the neck of SD male rats (aged6 weeks) in the dose of 900 ~g as TAP-144 after 1, 2 and 4 weeks the inner genital organs are removed and weighed.
As control untrea-ted rats of the same age are used and the percentages of organ weights relative to the control organ weigh-ts are calculated. The results are shown in Table 2. In the group of rats given No. 032 microcapsules, no marked difference ls found between oily and aqueous formulations and a marked weight decrease persisting for 4 weeks is no-ted in the weight of testis. A marked weight decrease is also observed in the seminal vesicle, with a significant difference being noted even at 2 weeks. In ~Z33~

the No. 035 microcapsule group, too, marked weight decreases of both the testis and seminal visicle are found after 1 week. The above results indicate that the TAP-144 prolonged release injections according to this invention haze satisfactory durations of action.
Table 2 Microcapsule No. 032 No 035 Time Organ Oily Aqueous Aqueous 1 Week Testis 58.1 +8.3** 57.9 + 7.4** 62.4 +5.3**a) Prostate 94.6 + 4.7 92.9 ~9.9 ~6.7~ 7.9 Seminal 67.6 ~10.6 62.7 ~10.7* 66.4 +7.7*
vesicle 2 Weeks q'cstis 53.~ ~6.5** 65.1 ~10.6*
Prostate 67.2 16.3** 35.1 ' 7.3 Seminal 39.5 ~6.3** 56.2 t 7.8*
vesicLe _ 3 Weeks Testis 77.1 +5.7** 58.0-~5.7**
Prostate 97.4 +4.6 87.2 ~5.9 Seminal 89.3+2.7 80.5 +6.4*
vesicle a) Percentages relative to the organ weights of control rats (ur.treated, the same age ** A hi~h]y significant difference (P<0.01) from control by t-test.
* A significant difference (Pc0.05) from control by t test.
Example 1 In 2.5 ml of a 20% aqueous gelatin solution prepared by warming (at 60 to 70C) is dissolved 200 mg of TAP-144 and the whole solution is added to 10 ml of a 20~ dichloro-methane solution of one of the 7 different lactic acid or lactic acid-glycolic acid polymers (2 runs for polylactic acid with a molecular weight of 50000). The mixture is ultrasonicated (20 KHz, 100 W, a few minutes, the ultra-sonicator manufactured by Ohtake Seisakusho Inc., Japan) to give a 233A~LA

microfine W/O emulsion. This emulsion is immediately cooled with ice to eause gelation of the gelatin layer.
This is then added to 100 ml of 0.5% polyvinyl alcohol (Gosenol EG-40, the Nippon Synthetic Chemical Industry Co., Ltd.,Japan)-1/30 M
phosphate buffer (pH 6.0) and dispersed using a homogenizer (T.K. Homomixer, Tokushu Kika Kogyo Inc., Japan, 30 V)(3,000 r.p.m.) for 15 seconds to give a W/O/~ emulsion. This emulsion is quickly transferred to a rotary evaporator in which the dichloromethane is desorbed under ice-cooling. After foaming has subsided, the ernulsion is warmed to 30 to 40C in a constant-temperature water bath for ccmplete desorption of -the organie solvent. the hardened microcapsules are-~en ~;iltered through a gl~lss filter and washed 5 ~.im~s with 10 ml portion ox d.is-~illed waker. It i9 when ~pxeacl on a ~l~ss di~3h and allowed to dry under x~due~d pressure for 1 to 3 days.
The product is sieved through a 100-mesh screen (Sieve opening:
147 em) to give TAP-144 microcapsules.
In 10 ml of dichloromethane is dissolved 10 mg of the above microcapsules and TAP-144 in the solution is extracted wi-th 10 ml of distilled water under shaking for 10 minutes. The TAP-144 content of the aqueous layer is assayed by high per-formance liquid chromatography lHPLC) and the percentage of TAP-144 taken up in-to the microeapsules relative to the initial amount of TAP-144 added is ealeulated. The results are shown in Table 3.
Table 3 Microcapsule No. High polymer Takeup Lactic acid Mol. weight (%) _ _ glycolic acid Control 021 100/0 50000 6.7 023 100/0 50000 1.9 *trade Mark 33~
- 24 _ Microcapsule No. High polymer wakeup Lactic acid/ Mol. weight (%) glycolic acid _-This 031 100/073000 70.4 invention 032 100/0 50000 70.7 033 100/050000 71.5 034 100/015000 54.8 035 100/0 6800 55.
036 88.7/11.3 19000 44.0 037 78.1/21.9 10000 58.3 038 54.5/45~4 20000 53.1 _ It will be app~r~nt prom Table 3 tha-t when toe drying-in~water technique under the same conditionc; is carried out without gelation of the first aqueous layer (control), the takeup ratio are as low as 1.9 to 6.7%
whereas the takeup ratio for the microcapsules according to this invention are as high as 44.0 to 71.5%. In the repeated experiments by the same production procedure using polylactic acid with a molecular weight of 50000, to take up ratio are almost comparable.
Example 2 :Ln a 20~ aqueous gelatin solution prepared by warming (60 to 70C) is dissolved 200 mg of TAP-14~1. This solution is added ~7hen hot to a 20% dichloromethane solution of polylactic acid (average mol. wt. 50000) and the mixture is ultrasonicated in the same manner as Example 1 to give a fine W/O emulsion. Separately, 5 ml of a 25% aqueous solution of glu-taraldehyde is extracted with 5 ml of dichloromethane (using the aforementioned ultrasonicator, 50 W, 2 min.) and the organic layer is added to the emulsion prepared above. Using a four-blade rotary mixer the mixture is reacted at room temperature under agitation for 6 hours. Thereafter, 4 ml of ethanolamine is added and the reaction is further conducted for 1 hour. After ice-1233a~

cooling, the reaction mixture is poured in 100 ml of ice-cooled 0.5% polyvinyl alcohol-1/30 M phosphate buffer (pH 6.0).
Then, as in Example 1, a W/O/W emul.sion is prepared and the organic solvent is desorbed to give TAP-144 micro-capsules (Microcapsule No. 0311). In addition, a 30%
aqueous solution of human serum albumin in lieu of said 20%
gelatin is treated with glutaraldehyde in the same manner as above to prepare TAP-144 microcapsules ~Microcapsule No. 0312).
These microcapsules are dispersed in purified sesame oil and the d.lspersions are subcu-taneously injec-tecl lnto mature Çemale rats at a dose of 12 mg/kg as ~AP~ l in the same m2lnner a Experiment~1 Exam l -to asses8 -the durations ofaction of the respective p.rolongecl re].~ase preparations.
The results are presented in Table 4. It will be seen that their actions last for about 4 months, indicating that -these microcapsules are satisfactory prolonged release preparations.
Table 4 -MicrocapSule Drug retaining substance (days) 0311 20% gelatin ~147 0312 30% human serum albumin 114.8 + 5.9 a) Mean + standard error for 5 rats Example 3 ._, _ , ,_ In 10 ml of dichloromethane is dissolved 3 g of lactic acid-glycolic acid copolymer (monomer ratio: 88.7/11.3, average mol. wt. 19000) followed by addition of 3 ml of a 30% aqueous solution of gelatin (dissolved at 60C). Then, a solution of 200 mg of LH-RH antagonist (N-Ac-[D-P-Cl-Phel'2,D-Trp3 D-Arg6,D-Ala 0]-LH-RH) (European Patent Application Publication No. 81,877) is added and the mixture lZ33~4 is ultrasonicated in the same manner as Example 1 to give a W/O emulsion. This emulsion is immediately cooled with ice and dispersed in a water-cooled 0.5% aqueous solution of polyvinyl alcohol. Then, the dichloromethane is desorbed and LH-RH antagonist microcapsules recovered in the same manner as Example 1.
Example 4 In 2.5 ml of a 20~6 aqueous solution of gelatin (prepared at 60C) is dissolved 500 mg oil an enkephalin derivative (H-Tyr-D-Met(O)-Gly-EtPhe-NH-NHCOCH3-AcOH) (United States Patent No. 4277394,-TAI-1399) and the solution is added to 10 ml o a 209~ solu-tion oE polylac-tic acid (averaye mol. wt. S0000) in dichlocomethane. The mix-ture is Eurthex worked up in the tame manner as E'x~mpl~ 1 -to give W/O emulsion. Under ice-cooling, this emulsion is further worked up in 0.5% polyvinyl alcohol-1/30 M
phosphate buffer (pH 6.0) to give a W/O/W emulsion. The organic solvent is then desorbed under reduced pressure in a rotary evaporator and the mixture is warmed from ice-cooling to 35C and when foaming has subsided, it is sieved through a 100-mesh screen and filtered through a glass filter to give TAI-1399 microcapsules.
The microcapsules thus produced are washed 4 times with 10 ml portions of distilled water and redispersed in a mixed aqueous solution of 0.2~ Tween 80, 0.596 sodium carboxymethylcellulose and 10% mannitol, followed by freeze-drying to give a prolonged release preparation of TAI-1399 which is of a reconstituted suspension type and the action of which lasts ln vivo for more than about 2 weeks.
Example 5 In a 20% aqueous solution of gelatin (prepared at 60C) is dissolved 2.2 billion units of y-interferone and the solution is added to 10 ml of a 20% dichloromethane solution of polylactic acid (average mol. wt. 73000~. Then, the same procedure as Example 1 is followed to give a W/O/W
emulslon under ice-cooling, remove the solvent in a rotary *Trade Mark ;~33~

evaporator and recover solid microcapsules by filtration to give y-interferone microcapsules.
The above microcapsules are washed 4 times with 10 ml portions of distilled water and dispersed in 50 ml of a mixed aqueous solution of 0.2% Tween 80*, 0.5% sodium carboxymethylcellulose and 8~ D-sorbitol, and 1 ml portions of the dispersion are distributed into glass vials and freeze-dried. The contents of each vial are extemporaneously redispersed in 1 ml of distilled water for injection containing 0.4% methyl-paraben and 0.04% propyl-paraben to give a prolonged release injection containing about 25,000 thousand units of r-interferone per dose.
Example 6 In 2.5 ml of distilled water are dissolved 300 mg o synthetic serum thymic fac-tor (FTS: H-Glu-~la-L~s-Ser-Gln-Al~-Gl~Ser-Asn-OEI) and 750 mg of human serum albumin, and the whole solution is added -to 10 ml of a dichloro-methane solution containing 3 g of lactic acid-glycolic acid copolymer (monomer ratio: 78.1/21.9, average mol. wt.
10000). Thereafter, the same procedure as Example 1 is followed to prepare a W/O emulsion. To this emulsion is added 3 ml of a dichloromethane extract of 25~ aqueous ~lutaraldehyde (3 ml) and the reaction is carried out under stirring for 5 hours. Then, 3 ml of ethanolamine is added and the mixture is further stirred for 1 hour. This W/O
emulsion is poured in 100 ml of a 0.5% aqueous solution of polyvinyl alcohol and the solution is worked up in the same manner as Example 1 to give a W/O/W emulsion. Finally, the solvent is removed in rotary evaporator and the product microcapsules are recovered.
The microcapsules thus obtained are dispered in 20 ml of the same dispersing medium as the one used in Example 5 and 2 ml portions of -the dispersion are distrib-uted into glass vials and freeze-dried to give a prolonged release injection containing about 15 mg of FTS per dose.
*Trade Mark ~33~

- 2g -Example 7 In 2.5 ml of a 2% aqueous solution of agar (liquefied by warming at 60C) is dissolved citrate of a thyroid hormone derivative (DN-1417) of the followinq formula:

o CO - NH - fH -CO - No ON

H

This solution is added -to 10 m]. o:E a 20~ solution of poly-lac~icclcid(average mol. wt. 50000) in dichloromethan~.
The mix-kuxe is worked up in the same manner as Example 1 to give a W/O emulsion which is furthex processed into a W/O/W
emulsion under ice cooling. Finally, the organic solvent is removed to give DN-1417 microcapsules.
These microcapsules are recovered by filtration, dried in vacuo at 40C for 24 hours and sieved through a 100-mesh screen. The product (500 mg) is filled into a vial to give a prolonged release injection containing about 75 mg of DN-1417 which is used by a reconstituted suspension.
E
In 10 ml of dichloromethane is dissolved 2 g of lactic acid-glycolic acid copolymer monomer ratio: 54.5/
45.5, average mol. wt. 20000). To this solution is added 3 ml of a 20~ aqueous gelatin solution containing 400 mg of mitomycin C (liquified by warming at about 60C) and the mixture is worked up in the same manner as Example 1 to give mitomycin C microcapsules.
The microcapsules are dried in vacuo and sieved through a 100-mesh screen. A 200 mg of the product is taken as a prolonged release injection containing about 20 mg of - mitomycin C which is a reconstituted suspension for ~33~

administration.
Example 9 To a 20% dichloromethane solution of lactic acid/
glycolic acid copolymer (monomer ratio: ~8.1/21.9, average mol. wt. 10000) is added 3 ml of a 20% aqueous gelatin solution containing 1.5 g of gentamycin sulfate (liquefied by warming). The mixture is further worked up in the same manner as Example 1 to give microcapsules.
These microcapsules are dried in vacuo and sieved, and 350 mg of the product is taken as a prolonged release preparation containing about 100 mg of gentamycin for use by a reconstitu-ted suspension.
Example 10 In 10 ml ox dichloromethane is dissolvecl 3 o 15 polylactic acid (average mol. w-t. 15000) followed by addition of 3 ml of a 20% aqueous gelatin solution containing 43000 units of blood coagulation factor VIII and 15 mg of sodium citra-te. The mixture is worked up in the same manner as Example 4 to give microcapsules.
This product is dispersed in 20 ml of dispersing medium and 2 ml portions are filled into vials to freeze dry to give a prolonged release injection for a reconstituted suspension containing about 3000 units of blood coagulation factor VIII in each vial.
Example 11 In 3 ml of a 20~o aqueous gela-tin solution (liquified by warming) is dissolved 2 g of sulpyrine and the solution is added to 10 ml of a 25% dichloromethane solution of lactic acid-glycolic acid copolymer (54.5/45.5, 30 averaye mol. wt. 20000). Finally, the mixture is worked up in the same manner as Example 1 to give microcapsules.
Example 12 In 2.5 ml of 20O6 aqueous gelatin solution (liquified by ~7arming) is dissolved 500 mg of morphine hydrochloride and the solution 35 is added to 10 ml of a 206 dichloromethane solution of polylactic acid average mol. wt. 15000). Thereafter, the ~33~

same procedure as Example 1 is followed to give prolonged release microcapsules for injection.
Example 13 In 2.5 ml of 20% aqueous gelatin solution (li~lified by warming) is dissolved 150 mg of sodium diclofenac and the solution is added to 10 ml of a 20% dichloromethane solution of polylactic acid (average mol. wt. lS000). The mixture is worked up in the same manner as Example 1 to give microcapsules for injection.
Example 14 In 2.5 ml of a 20% aqueous gelatin solu-tion (liquified by warming) is dissolvcd 1 g of methylephedrine hydrochloride anal the soLu-tion is aclded to 10 ml of a 20~ clichlorometh~ne solution ox polo ic acid Vera mol. wt. S0000). k mix-ture is worked up in thy same manner as Example 1 -to give methylephedrine microcapsules for injection.
Example 15 In 3.0 ml of 20% aqueous gelatin solution (liquified by ~ming) is dissolved 1 g of chlorpromazine hydrochloride, and the solution is added to 10 ml of a 20% dichloromethane solution of lactic acid glycolic acid copolymer (88.7/11.3r average mol. we. 19000). The mixture is worked up in the same manner as Example 1 to give chlorpromazine hydro-chloride microcapsules for injection.
Example 16 In 3.0 ml of 30% gelatin (dissolved by warming) is dissolved 50 mg of pridinol methanesulfonate and the solution is added to 10 ml of a 30% dichloromethane solution of lactic acid-glycolic acid copolymer (78.1/21.9, average mol.
wt. 10000). The mixture is then worked up in the same manner as Example 1 to give pridinol methanesulfonate microcapsules for injection.
Example 17 Using 600 mg of chlordiazepoxide hydrochloride, the procedure of Example 11 is followed to give microcapsules.

~233~

Example l Using 800 mg of metoclopramide, the procedure of Example 12 is followed to give metoclopramide microcapsules for injection.
Example 19 Using 1 g of imipramine, the procedure of Example 15 is followed to give imipramine microcapsules for injection.
Example 20 Using 750 mg of diphenhydramine hydrochloride, the procedure of Example 14 is followed to give diphenhydramine hydrochloride microcapsules for injection.
Example 21 Using 750 mg of etilefrin hydrochloride/ the proceclure of Example 15 is followed to give etile~rin hydrochloride microc~psules or injection.
Example 22 Using 300 mg of propranolol hydrochloride, the procedure of Example 14 is followed to give propranolol hydrochloride microcapsules for injection.
Example 23 Using 250 mg of oxyfedrine hydrochloride, the procedure of Example 12 is followed to give oxyfedrine hydrochloride microcapsules for injection.
Example 2~
Using 300 mg of pentolinium, the procedure of Example 11 is followed to give pentolonium microcapsules.
Example 25 Using 1 g of phenformin hydrochloride, the procedure of Example 13 is followed to give phenformin hydrochloride microcapsules.
Example 26 Using 2 x 106 units of sodium heparin, the procedure of Example 15 is followed to give sodium heparin micro-capsules.
Example 27 Using 400 mg of adrenochrome monoaminoguanidine 33~

methanesullona~e, the procedure of Example 12 is ollowed to give adrenochrome monoaminoguanidine methanesulfonate microcapsules for injection.
Example 28 Using 800 mg of isoniazid, the procedure of Example 16 is followed to give isoniazid microcapsules for injec-tion.
Example 29 Using 750 mg of prednisolone sodium phosphate, the procedure of Example 15 is followed to give prednisolone sodium phosphate microcapsules for injection.
Example 30 Using 100 mg of levallorphan tartrate, the procedure of ~xampl~ 16 is ~ollow~d to ~iv~ levallorphan t~r~:ra-~e microcapsules or injection.

Claims (43)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A method for producing a prolonged release microcapsule of a water-soluble drug, which method comprises: preparing a water-in-oil emulsion comprising an inner aqueous layer containing said water-soluble drug and a drug-retaining substance and an outer oil layer containing a polymer substance, then thickening or solidifying said inner aqueous layer to a viscosity of not lower than about 5,000 centipoises and finally subjecting the resulting emulsion to in-water-drying.

2. A method as claimed in Claim 1, wherein the water-soluble drug is a biologically active polypeptide.

3. A method as claimed in Claim 2, the biologically active polypeptide is (Pyr)Glu-His-Trp-Ser-Tyr-D-Leu-Leu-Arg-Pro-NH-C2H5.

4. A method as claimed in Claim 1, wherein the substance for retaining the drug is a natural high molecular weight compound.

5. A method as claimed in Claim 4, wherein the natural high molecular weight compound is a protein.

6. A method as claimed in Claim 5, wherein the protein is gelatin.

7. A method as claimed in Claim 1, wherein the polymer substance in the oil layer is a copolymer of lactic acid and glycolic acid.

8. A method as claimed in Claim 7, wherein the ratio of lactic acid:glycolic acid is 75:25.

9. A method as claimed in Claim 1, wherein the polymer substance in the oil layer is polylactic acid.

10. A method as claimed in Claim 1, wherein the inner aqueous layer is thickened to a viscosity not less than about 10,000 centipoises.

11. A method as claimed in Claim 1, wherein the inner aqueous layer is solidified.

12. A method as claimed in Claim 1, wherein the average diameter of the microcapsule is 2 to 200 µm.

13. A method as claimed in Claim 1, wherein the inner aqueous layer contains as the drug-retaining substance a substance which gels when heated above a certain temperature and the inner layer is thickened or solidified by heating the emulsion in a closed vessel at a temperature higher than the gelation temperature of the inner aqueous layer.

14. A method as claimed in Claim 13, wherein the drug-retaining substance is a protein and the emulsion is heated in a closed vessel at a temperature of about 40 to 120°C for about 5 minutes to about 8 hours.

15. A method as claimed in Claim 1, wherein the inner aqueous layer cont-ains as the drug-retaining substance a substance which gels when cooled below a certain temperature and the inner layer is thickened or solidified by cooling the emulsion at a temperature lower than the gelation temperature of the inner aqueous layer but above the solidification point of the oil layer.

16. A method as claimed in Claim 15, wherein the drug-retaining substance is agar, the emulsion preparation is conducted under heating at about 50 to 80°C and the emulsion is cooled to below 40°C to gel the inner aqueous layer.

17. A method as claimed in Claim 15, wherein the emulsion is cooled at about 60 to 0°C to freeze the inner aqueous layer.

18. A method as claimed in Claim 1, wherein the inner aqueous layer contains as the drug-retaining substance a substance which increases the viscosity of the aqueous layer when a metal ion or an organic acid or a salt thereof is added thereto and the inner layer is thickened or solidified by adding to the emulsion a metal ion or an organic acid or a salt thereof.

19. A method as claimed in Claim 18, wherein the drug-retaining substance is gum acacia and iron ion is added: the drug-retaining substance is carboxymethylcellulose and copper iron is added: the drug-retaining substance is sodium pectinate and calcium or magnesium ion is added: the drug-retaining substance is sodium alginate and calcium citrate is added: or the drug-retaining substance is polyvinyl alcohol and adipic acid or tartaric acid is added.

20. A method as claimed in Claim 1, wherein the inner aqueous layer contains as the drug-retaining substance a substance which is cross-linkable with a chemical condensing agent, the substance is cross-linked and condensed by adding the chemical condensing agent.

21. A method as claimed in Claim 20, wherein the chemical condensing agent is glutaraldehyde or acetaldehyde.

22. A method as claimed in Claim 21, wherein the drug-retaining substance is a protein.

23. A method as claimed in Claim 1, wherein the inner aqueous layer contains as the drug-retaining substance a substance which increases the viscosity of the aqueous layer on alternation of the pH value and an acidic or alkaline substance is added to the emulsion.

24. A method as claimed in Claim 23, wherein the drug-retaining substance is a carboxyvinyl polymer which increases the viscosity of the aqueous layer as the pH value increases and a solution of sodium hydroxide in methanol or ethanol is added to the emulsion.

25. A method as claimed in Claim 1, wherein the water-in-oil emulsion in which the inner aqueous layer has been thickened or solidified is subjected to in-water-drying by adding the water-in-oil emulsion into a third aqueous layer to give a water/oil/water ternary layer emulsion and desorbing the solvent in the oil layer.

26. A method as claimed in Claim 25, wherein the third aqueous layer contains an emulsifier which is capable of forming a stable oil-in-water emulsion.

27. A method as claimed in Claim 25 or 26, wherein the desorption of the solvent is accomplished by gradual decrease of pressure with or without warming the water/oil/water emulsion.

28. A process as claimed in Claim 25 or 26, wherein the thickening or solidification is effected by techniques other than temperature control and the desorption is effected by stirring, warming or nitrogen gas blasting the water/oil/water emulsion.

29. A method for producing microcapsules of a water-soluble drug, which method comprises:
preparing a water-in-oil emulsion comprising an inner aqueous layer and an outer oil layer of a water-immiscible organic solvent wherein the inner aqueous layer has a viscosity of lower than about 5,000 centipoises and contains the water-soluble drug and a drug-retaining substance which is either a substance which is soluble in water and hardly soluble in the organic solvent in the outer oil and assumes a viscous semi-solid consistency when dissolved in water or a substance which gains considerably in viscosity to provide a semi-solid or solid matrix under the influence of an external factor selected from the group consisting of temperature, pH, metal ions, organic acid or salts thereof and chemical condensing agents;
and the outer oil layer contains in addition to the water-immiscible organic solvent a biocompatible polymer substance which is hardly soluble or insoluble in water but is soluble in the water-immiscible organic solvent and is capable of forming microcapsule shell when the solvent is desorbed;
thickening or solidifying the inner aqueous layer to a viscosity of not lower than about 5,000 centipoises by the influence of the external factor mentioned above, adding the water-in-oil emulsion in which the inner aqueous layer has been thickened or solidified into a third aqueous layer containing an emulsifier so as to form a water-in-oil-in-water ternary layer emulsion, and desorbing the organic solvent in the oil layer to give microcapsules.

30. A method as claimed in Claim 29, wherein the water-soluble drug is a biologically active polypeptide of molecular weight of about 200 to 80,000.

31. A method as claimed in Claim 29, wherein the water-soluble drug is a polypeptide of the formula:

(Pyr)Glu-R1-Trp-Ser-R2-R3-R4-Arg-Pro-R5 (I) wherein R1 is His, Tyr, Trp or p-NH2-Phe;
R2 is Tyr or Phe;
R3 is Gly or a D-amino acid residue;
R4 is Leu, Lle or Nle;
R5 is Gly-NH-R6 (R6 is H or a lower alkyl group which may be substituted by OH) or NH-R6 (R6 is as defined above), or a pharmaceutically acceptable salt thereof.

32. A method as claimed in Claim 31, wherein in formula (I) the D-amino acid residue is D-Leu, D-Ile, D-Nle, D-Val, D-Nval, D-Abu, D-Phe, D-Phg, D-Ser, D-Thr, D-Met, D-Ala, D-Trp or .alpha.-Aibu and the residue may have a protective group.

33. A method as claimed in Claim 29, 30 or 31, wherein the drug-retaining substance is a protein.

34. A method as claimed in Claim 29, 30 or 31, wherein the drug-retaining substance is gelatin.

35. A method as claimed in Claim 29, 30 or 31, wherein the drug-retaining substance is a protein and the polymer substance in the oil layer is polylactic acid or a copolymer of lactic acid and glycolic acid.

36. A method as claimed in Claim 29, 30 or 31, wherein the inner aqueous layer contains as the drug-retaining substance a substance which gels when heated above a certain temperature and the inner layer is thickened or solidified by heating the emulsion in a closed vessel at a temperature higher than the gelation temperature of the inner aqueous layer.

37. A method as claimed in Claim 29, 30 or 31, wherein the inner aqueous layer contains as the drug-retaining substance a substance which gels when cooled below a certain temperature and the inner layer is thickened or solidified by cooling the emulsion at a temperature lower than the gelation temperature of the inner aqueous layer but above the solidification point of the oil layer.

38. A method as claimed in Claim 29, 30 or 31, wherein the drug-retaining substance is gum acacia and iron ion is added the drug-retaining substance is carboxymethylcellulose and copper iron is added: the drug-retaining substance is sodium pectinate and calcium or magnesium ion is added: the drug-retaining substance is sodium alginate and calcium citrate is added: or the drug-retaining substance is polyvinyl alcohol and adipic acid or tartaric acid is added.

39. A method as claimed in Claim 28, 30 or 31, wherein the aqueous layer contains as the drug-retaining substance a substance which is cross-linkable with glutaraldehyde or acetaldehyde and the drug-retaining substance is cross-linked and condensed by adding glutaraldehyde or acetaldehyde to the emulsion.

40. A method as claimed in claim 29, 30 or 31, wherein the drug-retaining substance is a carboxyvinyl polymer which increases the viscosity of the aqueous layer as the pH value increases and a solution of sodium hydroxide in methanol or ethanol is added to the emulsion.

41. A method as claimed in claim 29, wherein the drug-retaining substance is gelatin, the polymer substance in the oil layer is a copolymer of lactic acid and glycolic acid; the amount of gelatin in the inner aqueous layer is about 0.1 to 50% (w/w); the amount of the lactic acid-glycolic acid copolymer in the oil layer is 2 to 60% (w/w); the organic solvent is a halogenated alkane; and the external factor for thickening or solidifying the inner aqueous layer is cooling the water-in-oil emulsion to about 0 to 10°C thereby causing gelation of the gelatin layer.

42. A method as claimed in claim 41, wherein the emulsifier is polyvinyl alcohol; and the third aqueous layer contains a pH-adjusting agent.

43. A method as claimed in claim 42, wherein the water-soluble drug is a polypeptide of the formula:

(Pyr)Glu-R1-Trp-Ser-R2-R3-R4-Arg-Pro-R5 (I) wherein R1 is His, Tyr, Trp or p-NH2-Phel;
R2 is Tyr or Phe;
R3 is Gly or a D-amino acid residue;
R4 is Leu, Ile or Nle;
R5 is Gly-NH-R6 (R6 is H or a lower alkyl group which may be substituted by OH) or NH-R6 (R6 is as defined above), or a pharmaceutically acceptable salt thereof.