||||||||||||||| USOO5330767A United States Patent (19) 11 Patent Number: 5,330,767 Yamamoto et al. 45) Date of Patent: "Jul. 19, 1994

54 SUSTAINED RELEASE MICROCAPSULE 4,675, 189 6/1987 Kent et al...... 424/462X 4,710,384 12/1987 Rotman ...... 424/465 75 Inventors: Masaki Yamamoto; Shigeyuki 4,767,628 8/1988 Hutchinson ...... 424/426 Takada; Yasuaki Ogawa, all of Osaka, 4,917,893 4/1990 Okada et al...... 428/402.2 X Japan 4,954,298 9/1990 Yamamoto et al...... 264/4.6 73) Assignee: Takeda Chemical Industries, Ltd., FOREIGN PATENT DOCUMENTS Osaka, Japan 1169090 6/1984 Canada ...... 514/4 * Notice: The portion of the term of this patent 0145240 6/1985 European Pat. Off. . subsequent to Mar. 27, 2004 has been 0.145240 6/1985 European Pat. Off. . disclaimed. 24935 4/982 France . 929402 6/1963 United Kingdom. (21) Appl. No.: 936,726 1405108 9/1975 United Kingdom ...... 264/4.6 22 Filed: Aug. 31, 1992 2088314 6/1982 United Kingdom . OTHER PUBLICATIONS Related U.S. Application Data Ogawa et al.: "Controlled Release of LHRH Agonist, 60 Continuation of Ser. No. 541,067, Jun. 20, 1990, aban Leuprolide Acetate from Microcapsules...", J. Pharm. doned, which is a division of Ser. No. 249, 198, Sep. 23, Pharmacol., 1989, 41:439-440. 1988, Pat. No. 4,954,298, which is a continuation of Sanders et al.: "Controlled Release of a Luteinizing Ser. No. 826,968, Feb. 7, 1986, abandoned. Hormone-Releasing Hormone Analog from Poly(d- 30 Foreign Application Priority Data l-Lactide-Co-Glycolide) Microspheres", Journal of Pharmaceutical Sciences, vol. 73, No. 9, Sep. 1984. Feb. 7, 1985 (JP Japan ...... 60-22978 The Theory and Practice of Industrial Pharmacy, edited Nov. 27, 1985 JP Japan ...... 60-267977 by L. Lachman et al., Lea & Febiger, Philadelphia, 51 Int. Cl...... A61K9/50; A61K9/52; 1970, pp. 384-391. BOJ 3/04 European Search Report, European Patent Applin. No. 52 U.S. Cl...... 424/497; 424/461; 863OO3O8. 428/402.2; 428/402.21; 428/402.22; 514/963 58) Field of Search ...... 264/4.1, 4.6; Primary Examiner-Richard D. Lovering 428/402.2, 402.21, 402.22; 424/461, 462, 486, Attorney, Agent, or Firm-Wegner, Cantor, Mueller & 493, 497; 514/963, 965 Player (56) References Cited 57 ABSTRACT U.S. PATENT DOCUMENTS Microcapsules are advantageously produced with high 3,043,782 7/1962 Jensen ...... 427A23.33 X take-up of a water-soluble drug by preparing a W/O 3,092,553 6/1963 Fisher, Jr. et al...... 424/489 emulsion composed of a water-soluble drug-containing 3,523,906 8/1970 Vrancken et al...... 264/4.6 solution as the inner aqueous phase and a polymer-con 3,539,465 A1970 Hiestand et al...... 264/4.3 X taining solution as the oil phase, dispersing said emul 3,691,090 9/1972 Kitajima et al...... 252/316 sion in an aqueous phase and subjecting the resulting 3,922,338 11/1975 Estevenel et al...... 424/469 W/O/W emulsion to an in-water drying, wherein the 4,234,571 1 1/1980 Nestor et al...... 514/15 viscosity of the W/O emulsion used in preparing the 4,273,920 6/1981 Nevin ...... 528/361 4,384,975 5/1983 Fong ...... 424/496 X W/O/W emulsion is adjusted to about 150 to about 4,389,330 6/1983 Tice et al...... 427A23.36 10,000 centipoises. 4,555,399 1/1985 Hsiao ...... 424/465 4,652,441 3/1987 Okada et al...... 264/4.6 X 13 Claims, No Drawings 5,330,767 2 thereof having LH-RH like activity, i.e. the polypep SUSTAINED RELEASE MICROCAPSULE tides of the formula: This application is a continuation of U.S. application Ser. No. 07/541,067 filed Jun. 20, 1990, now abandoned, Pro-Rs (I) which is a divisional of Ser. No. 07/249, 198, filed Sep. 23, 1988, U.S. Pat. No. 4,954,298, which is a continua wherein R is His, Tyr, Trp or p-NH2-Phe, R2 is Tyr or tion of Ser. No. 06/826,968, filed Feb. 7, 1986, aban Phe, R3 is Gly or a D-amino acid residue, R4 is Leu, Ile doned. or Nile and R5 is Gly-NH-R6 (R5 is H or a lower alkyl This invention relates to a method for producing O group which may optionally be substituted by hydroxy) sustained-release microcapsules containing a water-sol or NH-R6 (R6is as defined above), and salts thereof (see uble drug. U.S. Pat. Nos. 3,853,837, 4,008,209 and 3,972,859, Brit For drugs required to be administered for a pro ish Patent No. 1,423,083, and Proceedings of the Na longed period, various dosage forms have been pro tional Academy of Sciences of the United States of posed. Among them, there is disclosed in European 15 America, volume 78, pages 6509-6512 (1981). Patent Application Publication No. 52,510A a method Referring to the above formula (I), the D-amino acid of microencapsulation by phase separation using a coac residue represented by R3 is, for example, an a-D-amino ervation agent such as a mineral oil or a vegetable oil. acid residue containing up to 9 carbon atoms (e.g. D Microcapsules obtained by the above-mentioned Leu, Ile, Nile, Val, NVal, Abu, Phe, Phg, Ser, Tyr, Met, method have a drawback in that the particles are apt to 20 Ala, Trp, al-Aibu). It may have an appropriate protec adhere to one another in their production process. tive group (e.g. t-butyl, t-butoxy, t-butoxycarbonyl, Under these circumstances, the intensive studies were naphtyl). An acid addition salt or metal complex of the curried out in order to develop sustained release drug peptide (I) can of course be used in the same manner as preparations. As a result, it was found that microcap the peptide (I). sules having favorable properties can be obtained effi 25 In abbreviating the amino acids, peptides, protective ciently with a high rate of drug take-up into the micro groups and so on as used in specifying the polypeptides capsules when, in the process of forming a three-phase of formula (I), there are used the abbreviations accord emulsion for microencapsulation by an in-water drying, ing to the IUPAC-IUB Commission on Biological No the viscosity of the W/O emulsion for preparing the menclature or the abbreviations commonly used in the three-phase W/O/W emulsion is adjusted to about 150 30 relevant field of art. For those amino acids which in to about 10,000 cp. Further research work based on this volve optical isomerism, each abbreviation, unless oth finding has now led to completion of the present inven erwise indicated, refers to the L-form. to. In this specification, the acetate of the polypeptide of Thus this invention is directed to: a method of prepar the above formula (I) wherein Rise His, R2 =Tyr, ing sustained-release microcapsules containing a water 35 R3 =D-Leu, R4=Leu and R5 = NHCH2-CH3 is called soluble drug, which comprises preparing a W/O emul “TAP-144'. Said polypeptide in acetate form has the sion composed of a water-soluble drug-containing solu generic name "leuprolide'. tion as the inner aqueous phase and a polymer-contain Said polypeptides further include LH-RH antagonists ing solution as the oil phase adjusting the viscosity of (see U.S. Pat. Nos. 4,086,219, 4,124,577, 4,253,997, the W/O emulsion used in preparing the W/O/W emul 40 4,317,815, 329,526 and 368,702). sion to from about 150 to about 10,000 cp, dispersing Said polypeptides also include, for example, insulin, said emulsion in an aqueous phase and subjecting the somatostatin, somatostatin derivatives (see U.S. Pat. resulting W/O/W emulsion to an in-water drying. Nos. 4,087,390, 4,093,574, 4,100,117 and 4,253,998), The viscosity value mentioned herein is measured growth hormones, , adrenocorticotropic hor with an Ubbelohde viscometer in accordance with the 45 mone (ACTH), melanocyte-stimulating hormone Japanese Pharmacopeia. This is dynamic viscosity (MSH), thyrotropin-releasing hormone (TRH), salts value, and "cp' stands for centipoise. and derivatives thereof (see U.S. Pat. Nos. 3,957,247 The water-soluble drug to be used in the practice of and 4,100,152), thyroid-stimulating hormone (TSH), this invention is highly hydrophilic and has a small luteinizing hormone (LH), follicle-stimulating hormone oil/water distribution coefficient which, when given in 50 (FSH), vasopressin, vasopressin derivatives de terms of octanol/water distribution coefficient, for in smopressin see Folia Endocrinologica Japonica, vol stance, is not greater than about 0.1. ume 54, No. 5, pages 676-691 (1978)), oxytocin, calci Said water-soluble drug includes, but is not particu tonin, parathyroid hormone, glucagon, gastrin, secretin, larly limited to, physiologically active polypeptides, pancreozymin, cholecystokinin, angiotensin, human other antibiotics, antitumor agents, antipyretics, analge 55 placental lactogen, human chorionic sics, antiinflammatory agents, antitussives and expector (HCG), enkephalin, enkephalin derivatives (see U.S. rants, sedatives, muscle relaxants, antiepileptics, anti Pat. No. 4,277,394 and European Patent Application ulcer agents, antidepressants, antiallergic agents, car Publication No. 31567A), endorphin, kyotorphin, inter diotonics, antiarrhythmic agents, vasodilators, an ferons (a, 3 and y), interleukins (I, II and III), taftsin, tihypotensive diuretics, antidiabetic agents, anticoagu thymopoietin, thymosin, thynostimulin, thymic hu lants, henostatic agents, antitubercular agents, hor moral factor (THF), serum thymic factor (STF or FTS) mones and narcotic antagonists. and derivatives thereof (see U.S. Pat. No. 4,229,438), The physiologically active polypeptides usable in the and other thymic factors Igaku no Ayumi (Medicine in practice of this invention contain two or more amino Progress), volume 125, No. 10, pages 835-843 (1983)), acids and preferably have a molecular weight of about 65 tumor necrosis factor (TNF), colony stimulating factor 200 to about 80,000. (CSF), motilin, dinorphin, bombesin, neurotensin, ceru Examples of said polypeptides include luteinizing lein, bradykinin, urokinase, asparaginase, kallikrein, hormone releasing hormone (LH-RH), derivatives substance P, nerve growth factor, blood coagulation 5,330,767 3 4. factors VIII and IX, lysozyme chloride, polymixin B, arin and sodium citrate, among others. The hemostatic colistin, gramicidin, bacitracin, protein synthesis agents include thromboplastin, thrombin, menadione stimulating peptides (British Patent No. 8,232,082), gas sodium bisulfite, acetomenaphthone, e-aminocaproic tric inhibitory polypeptide (GIP), vasoactive intestinal acid, tranexamic acid, carbozochrome sodium sulfate, polypeptide (VIP), platelet-derived growth factor adrenochrome monoaminoguanidine methanesulfonate, (PDGF), growth hormone-releasing factor (GRF, and so forth. The antituberculous agents include, among somatocrinin), bone morphagenetic protein (BMP) and others, isoniazide, ethanbutol and sodium p-aminosali epidermal growth factor (EGF). cylate. The hormones include, among others, predniso The antitumor agents mentioned above include, lone succinate, prednisolone sodium phosphate, dexa among others, bleomycin hydrochloride, methotrexate, 10 methasone sodium sulfate, betanethasone sodium phos actinomycin D, mitomycin C, vinblastine sulfate, vin phate, diphosphate, and cristine sulfate, daunorubicin hydrochloride, adriamy methimazole. The narcotic antagonists include levallor cin, neocarcinostatin, cytosine arabinoside, fluorouracil, phan tartrate, nalorphine hydrochloride and neloxone tetrahydrofuryl-5-fluorouracil, krestin, picibanil lenti hydrochloride, among others. nan, levanisole, bestatin, azimexon, glycyrrhizin, poly 15 The above-mentioned water-soluble drugs are used in I:C, poly A:U and poly ICLC. amounts selected depending on the kind of drug, de The antibiotics mentioned above include, among sired pharmacological effects and duration of the ef. others, gentamicin, dibekacin, kanendomycin, livido fects, among others, and the concentration in the inner mycin, tobramycin, amikacin, fradiomycin, sisomicin, aqueous phase is selected generally within the range tetracycline hydrochloride, oxytetracycline hydrochlo 20 about 0.001% to about 70% (weight/weight), prefera ride, rollitetracycline, doxycycine hydrochloride, ampi bly within the range of 0.01%. to 50% (weight/weight). cillin, piperacillin, ticarcillin, cephalotin, cephaloridine, In carrying out the method according to this inven cefotiarn, cefsulodine, cefnenoxime, cefnetazole, tion, the viscosity of the inner aqueous phase may be cefazolin, cefotaxime, cefoperazone, ceftizoxime, mox increased by further adding a drug retaining substance alactam, thienamycin, sulfazecin and azthreonan. 25 to the inner aqueous phase. The antipyretic, analgesic and antiinflammatory The drug retaining substance mentioned above is a agents mentioned above include, among others, sodium substance which is soluble in water but is hardly soluble salicylate, sulpyrine, sodium flufenamate, sodium di in the organic solvent in the oil phase and, when dis clofenac, sodium indomethacin, morphine hydrochlo solved in water, gives a highly viscous semisolid or, ride, pethidine hydrochloride, levorphanol tartrate and 30 when placed in a water-dissolved state under the action oxymorphone. The antitussives and expectorants in of some external factor, for instance temperature, pH, clude ephedrine hydrochloride, methylephedrine hy metal ion (e.g. Cut, Al, Zn), organic acid drochloride, noscapine hydrochloride, codeine phos (e.g. tartaric acid, citric acid, tannic acid) or salt thereof phate, dihydrocodeine phosphate, alloclamide hydro (e.g. calcium citrate) or chemical condensing agent (e.g. chloride, chlophedianol hydrochloride, picoperidamine 35 glutaraldehyde, acetaldehyde), gives a senisolid or hydrochloride, cloperastine, protokylol hydrochloride, solid matrix as a result of marked increase of viscosity isoproterenol hydrochloride, salbutamol sulfate and caused by said external factor. terbutaline sulfate, among others. The sedatives include Examples of said drug retaining substance are natural hydrochloride, prochlorperazine, tri or synthetic gums or high-molecular compounds. fluoperazine, atropine sulfate, scopolamine methyl bro The natural gums include gum acacia, Irish moss, mide, and so forth. The muscle relaxants include, for karayagum, gum tragacanth, gum guaiac, Xanthan gum example, pridinol methanesulfonate, tubocurarine chlo and locust bean gum. The natural high molecular com ride and pancuronium bromide. The antiepileptics in pounds include proteins, such as casein, gelatin, colla clude sodium , ethosuximide, sodium acetaz gen, albumin (e.g. human serum albumin), globulin and olamide chlordiazepoxide hydrochloride, etc. The anti 45 fibrin, and carbohydrates, such as cellulose, dextrin, ulcer agents include, among others, pectin, starch, agar and mannan. They may be used and histidine hydrochloride. The antidepressants in either as such or in the form of synthetic guns resulting clude imipramine, clomipramine, noxiptiline and phen from partial chemical modification, for example esters elzine sulfate, amongst others. The antiallergic agents or ethers derived from the above-mentioned natural include, for instance, diphenhydramine hydrochloride, 50 gums (e.g. methylcellulose, ethylcellulose, carboxy chlorpheniramine maleate, tripelenamine hydrochlo methylcellulose, gelatin succinate), hydrolyzates ride, methdilazine hydrochloride, clemizole hydrochlo thereof (e.g. sodium alginate, sodium pectinate), or salts ride, diphenylpyraline hydrochloride and methoxy of these. phenamine hydrochloride. The cardiotonics include, The synthetic high-molecular compounds include, among others, trans-at-oxocamphor, theophillol, ami 55 among others, polyvinyl compounds (e.g. polyvinyl hophylline and etillefrine hydrochloride. The antiar pyrrolidone, polyvinyl alcohol, polyvinyl methyl ether, rhythmic agents include propranolol hydrochloride, polyvinyl ether), polycarboxylic acids (e.g. polyacrylic alprenolol hydrochloride, bufetolol hydrochloride, ox acid, polymethacrylic acid, Carbopol (Goodrich)), yprenolol hydrochloride, etc. The vasodilators include polyethylene compounds (e.g. polyethylene glycol), oxyfedrine hydrochloride, diltiazem hydrochloride, polysaccharides (e.g. polysucrose, polyglucose, poly tolazoline hydrochloride, hexobendine and bamethan lactose), and salts of these. sulfate, among others. The hypotensive diuretics in Also included within the scope of drug retaining clude hexamethonium bromide, pentolinium, mecamyl substances are substances capable of giving high amine hydrochloride, ecarazine hydrochloride, cloni molecular compounds as a result of condensation or dine hydrochloride, etc. The antidiuretic agents in 65 cross-linking which proceeds under the action of the clude, among others, sodium glymidine, glypizide, external factor mentioned above. phenformin hydrochloride, buformin hydrochloride Among these drug retaining substances, there are and metformin. The anticoagulants include sodium hep particularly preferable gelatin, albumin, pectin and 5,330,767 5 6 agar. The drug retaining substances may be used either the above-mentioned drug retaining substance may alone or in combination. further be added. To said inner aqueous phase, there The polymer to be contained in the oil phase in carry may be added a pH-adjusting agent for maintaining the ing out the method according to this invention is a stability or solubility of the water-soluble drug, such as polymer which is scarcely soluble or insoluble in water carbonic acid, acetic acid, oxalic acid, citric acid, tar and is biocompatible. Examples are such biodegradable taric acid, succinic acid, phosphoric acid, the sodium or polymers as aliphatic polymers (e.g. polylactic acid, potassium salt of the above compound, hydrochloric polyglycolic acid, polycitric acid, polymalic acid), acid or sodium hydroxide. There may further be added poly-a-cyanoacrylic acid esters, poly-g-hydrox a stabilizer for the water-soluble drug such as albumin, ybutyric acid, polyalkylene oxalate (e.g. polytrimethy 10 gelatin, citric acid, sodium ethylenediaminetetraacetate, lene oxalate, polytetramethylene oxalate), polyorthoest dextrin or sodium hydrogen sulfite, or a preservative ers, polyorthocarbonates and other polycarbonates (e.g. such as a para-hydroxybenzoic acid ester (e.g. methyl polyethylene carbonate, polyethylene-propylene car , propylparaben), benzyl alcohol, chlorobutanol bonate), and polyamino acids (e.g. poly-y-benzyl-L- or thimerosal. glutamic acid, poly-L-alanine, poly-y-methyl-L- 15 The thus-obtained aqueous solution for use as the glutamic acid). Other biocompatible high polymers are inner aqueous phase is added to a polymer-containing polystyrene, polyacrylic acid, polymethacrylic acid, solution (oil phase), followed by an emulsification pro acrylic acid-methacrylic acid copolymers, polyamides cedure to give a W/O emulsion. (nylon), polyethylene terephthalate (tetron), polyamino For said emulsification procedure, a known method acids, silicone polymers, dextran stearate, ethylcellu 20 of effecting dispersion is used. Said method is, for exam lose, acetylcellulose, nitrocellulose, polyurethanes, ma ple, the intermittent shaking method, the mixer method leic anhydride-based copolymers, ethylenesvinyl ace using a propeller-shaped stirrer, a turbine-shaped stirrer tate copolymers, polyvinyl acetate, polyvinyl alcohol, or the like, the colloid mill method, the homogenizer polyacrylamide, etc. These polymers may be homopol method or the ultrasonication method. ymers or copolymers of two or more monomers, or 25 The thus-prepared W/O emulsion is them emulsified mixtures of the polymers. They may also be in the salt into a W/O/W triplicate-phase emulsion and subjected form. to an in-water drying. Thus, said W/O emulsion is fur Among these polymers, particularly preferred for use ther added to a third aqueous phase to give a W/O/W in injections are biodegradable polymers, most prefera emulsion and thereafter the solvent in the oil phase is bly polylactic acid, lactic acid-glycolic acid copolymer 30 removed to give microcapsules. and mixtures thereof. To the external aqueous phase, there may be added an The ratio of lactic acid to glycolic acid in the copoly emulsifying agent. As the emulsifying agent, there may mer is preferably about 100/0 to 50/50 (weight %) be used any one capable of forming generally a stable preferably about 50 to 95 weight % of lactic acid and O/W emulsion, for example-an anionic surfactant (e.g. about 50 to 5 weight % of glycolic acid, more prefera 35 sodium oleate, sodium stearate, sodium laurylsulfate), a bly about 60 to 95 weight % of lactic acid and about 40 nonionic surfactant (e.g. polyoxyethylenesorbitan fatty to 5 weight % of glycolic acid, still more preferably acid ester (Tween 80, Tween 60, products of Atlas about 60 to 85 weight % of lactic acid and about 40 to Powder Co., U.S.A.), a polyoxyethylene castor oil de 15 weight % of glycolic acid. The ratio is especially rivative (HCO-60, HCO-50, products of Nikko Chemi preferably about 75-2 mole % of lactic acid and about cals, Japan), polyvinyl pyrrolidone, polyvinyl alcohol, 25-t2 mole % of glycolic acid. carboxymethylcellulose, lecithin or gelatin. Such emul The polymers for use in this invention preferably sifiers may be used either alone or in combination of have an average molecular weight of about 1,000 to some of them. The emulsifying agent concentration about 800,000, more preferably about 2,000 to about may suitably be selected within the range of about 100,000. 45 0.01% to 20%, preferably within the range of about The lactic acid-glycolic acid copolymers still more 0.05% to 10%. preferably have an average molecular weight of about The viscosity of the W/O emulsion for preparing the 5000 to about 30000. W/O/W emulsion is adjusted to about 150 cp to 10,000 These polymers are used in amounts to be selected cp, preferably about 150 cp to 5,000 cp. In adjusting the depending on the intensity of pharmacological activity 50 viscosity, there may be used the following means or a of the water-soluble drug, drug release rate, the dura combination thereof for instance: tion and other factors. For instance, these are used as To increase the polymer concentration in the oil the microcapsule bases in an amount of about 3 to phase; 10,000 parts by weight, preferably about 5 to about 100 To adjust the ratio in amount between the aqueous parts by weight, per part by-weight of the water-soluble 55 phase and the oil phase; drug. To adjust the temperature of said W/O emulsion; The solution (oil phase) containing the above poly To adjust the temperature of the-external aqueous mer is that of the polymer in an organic solvent. phase; or Said organic solvent may be any organic sovlent To adjust the temperature of the W/O emulsion with which has a boiling point not higher than about 120° C. a line heater or cooler or the like in infusing the and hardly miscible with water. Examples are haloge W/O emulsion into the external aqueous phase. nated alkanes (e.g. dichloronethane, chloroform, chlo What is important in taking such measures as men roethane, trichloroethane, carbon tetrachloride), ethyl tioned above is only that the W/O emulsion has a vis acetate, ethyl ether, cyclohexane, benzene, and toluene. cosity of about 150 cp to 10,000 cp when it is made up These may be used in admixture of two or more. 65 into a W/O/W emulsion. In carrying out the microencapsulation method ac In adjusting the viscosity of the W/O emulsion by cording to this invention, water is added to the water taking one or more of the above procedures, the poly soluble drug to prepare the inner aqueous phase. Here, mer concentration in the oil phase, when adjusted, is 5,330,767 7 8 preferably adjusted to about 10 to 80% (weight by In this manner, the rate of take-up of the water-solu weight), although the preferable range of such concen ble drug, which is the active ingredient, into microcap tration is not specified generally but may vary depend sules can be increased by using the method according to ing on the kind of polymer, kind of solvent and other this invention. Furthermore, the use of a smaller amount factors. of organic solvent in the production process is sufficient The adjusting the viscosity of the W/O emulsion in as compared with the process involving drying in the the above manner is preferably carried out so that the oil phase. From the above and other viewpoints, the W/O ratio falls within the range of about 1% to 50% method according to this invention is advantageous in (volume by volume), although the preferable range of commercial microcapsule production. such ratio is not specified generally but may depend on 10 The microcapsules produced by the method accord the kind and amount of water-soluble drug and proper ing to this invention have many advantages. For in ties of the oil phase. stance, they scarcely undergo aggregation or cohesion In adjusting the viscosity of the W/O emulsion in the to one another during the production step. There can be above manner, the temperature of the W/O emulsion is obtained microcapsules which are satisfactorily spheri 15 cal in shape. The step of removing the solvent from the generally regulated to from about -20° C. to the boil oil phase is easy to control, whereby the surface struc ing point of the organic solvent used, preferably about ture of microcapsules, which is decisive for the rate of O C. to 30 C. drug release (inclusive, e.g. of the number and size of In cases where the polymer concentration in the oil pores which are to serve as main routes of drug release), phase has been adjusted or in cases where the ratio 20 can be controlled. between the aqueous phase and the oil phase has been The microcapsules produced by the method accord adjusted, the viscosity of the W/O emulsion can be also ing to this invention can be administered to the living adjusted on the occasion of preparing the W/O emul body by implantation thereof as such. They may also be SO administered in various dosage forms and thus can be In cases where the viscosity of the W/O emulsion is 25 used as raw material in producing such dosage forms. adjusted by regulating the temperature of the W/O The injection form is preferably as the dosage form emulsion, the temperature of said W/O emulsion is mentioned above. adjusted, for example on the Occasion of adding the For instance, in making up the microcapsules accord W/O emulsion to the external aqueous phase. The vis ing to this invention for an injection, the microcapsules cosity adjustment may also be effected by adjusting in 30 according to the invention are dispersed in an aqueous advance the temperature of the external aqueous phase medium together with a dispersing agent (e.g. Tween on the occasion of adding the W/O emulsion to the 80, HCO-60, carboxymethylcellulose, sodium alginate), external aqueous phase so that the temperature of the a preservative (e.g. methylparaben, propylparaben), an W/O emulsion can be adjusted when the W/O/W isotonizing agent (e.g. sodium chloride, mannitol, Sorbi emulsion is prepared. 35 tol, glucose). Such suspension can serve as a sustained For removing the solvent from the oil phase in sub release injection. jecting the W/O/W emulsion to an in-water drying, Furthermore, the above microencapsulated sus any of the common methods in general use is employed. tained-release injection can be converted to a more Thus, the solvent is removed, for example by simply stable, sustained-release injection by adding an addi allowing the W/O/W emulsion to stand under stirring, tional excipient (e.g. mannitol, sorbitol, lactose, glu by heating slowly said emulsion, by blowing nitrogen cose), redispersing the resulting mixture and effecting gas or the like onto said emulsion, by gradually reduc solidification by freeze drying or spray drying with ing the pressure while stirring with a propeller-shaped simultaneous addition of distilled water for injection or stirrer or a magnetic stirrer, or by using a rotary evapo some appropriate dispersing agent. rator while adjusting the degree of vacuum. In the step 45 The dose of the sustained-release preparation accord of solvent removal, the required time can be reduced by ing to this invention may vary depending on the kind gradually warming the W/O/W emulsion after the and amount of the water-soluble drug, which is the progress of solidification of the polymer to a certain active-ingredient, dosage form, duration of drug re extent, to thereby rendering the solvent removal more lease, recipient animal (e.g. warm-blooded animals such complete. 50 as mouse, rat, horse, cattle, human) and purpose of The thus-produced microcapsules are collected by administration but should be within the range of effec centrifugation or filtration, rinsed several times with tive dose of said active ingredient. For example, the distilled water to thereby remove the free water-soluble single dose per said animal of the microcapsules can drug portion adhering to the microcapsule surface and adequately be selected within the range of about 0.01 to other substances, and, if necessary, warmed under re 55 200 mg/kg body weight, preferably about 0.2 to 40 duced pressure to thereby remove the moisture in mi mg/kg, still more preferably about 0.2 to 20 mg/kg or crocapsules and the solvent in the microcapsule wall 0.2 to 6 mg/kg. The volume of the suspension for ad more completely. ministering as the above-mentioned injection can ade The microcapsules obtained in the above manner are quately be selected within the range of about 0.1 to 10 sieved as necessary to eliminate excessively large micro ml, preferably about 0.1 to 5 ml, more preferably about capsules. For use in the form of suspensions depending 0.5 to3 n1. on the extent of the sustained-release property, the mi In this manner, there is obtained a pharmaceutical crocapsules may have a grain size within the range in composition prepared in the form of microcapsules which the dispersibility and penetration requirements which comprises an effective but greater amount of the are met. Thus, for example, they may have an average 65 water-soluble drug as compared with the ordinary sin grain size within the range of about 0.5 to 400 um, gle dose and a biocompatible high polymer and is capa desirably and preferably within the range of about 2 to ble of releasing the drug continuously over a prolonged 200 um, more preferably about 2 to 100 um. period of time. 5,330,767 10 The sustained-release preparation according to the emulsion was then dispersed in 500 ml of a 5% aqueous present invention has the following advantages, among solution of polyvinyl alcohol (PVA) using a homoge others: nizer to give a (W/O)/W emulsion. On that occasion, (1) Sustained-release of the water-soluble drug can be the homogenizer was operated at 4,000 rpm for 1 min attained in various dosage forms. In particular, where a ute. Thereafter, the (W/O)/W emulsion was stirred long-term treatment with an injection is required, the gently with an ordinary stirrer for 2 hours to thereby desired pharmacological effects can be achieved in a allow the evaporation of methylene chloride, hence the stable manner by injection of the preparation once a solidification of microcapsules, to proceed. The micro week, once a month, or even once a year, instead of capsules were then collected by centrifugation and daily administration. Thus, said preparation can achieve O rinsed with purified water on the same occasion. The a sustained drug release over a longer period as com microcapsules collected were lyophilized to obtain a pared with the prior art sustained-release preparations. powder. (2) When the preparation in which a biodegradable The content of interferon a taken up in the microcap polymer is used is administered in the form of an injec sules was 11.5%, the recovery rate (take-up rate) being tion, such surgical operation as implantation is no more 15 92.0%. required but the preparation can be administered subcu taneously or intramuscularly with ease in quite the same EXAMPLE 2 manner as the ordinary suspension injections. There is Leuprolide (450 mg) and 50 mg of sodium carboxy no need for taking it out again from the body, because methylcellulose (Na-CMC) were dissolved in 500 mg of the biodegradable polymer is used. water at 60' C. The solution was added to a solution of The preparation can also be administered directly to 4,000 mg of lactic acid-glycolic acid copolymer (lactic turnors, the site of inflammation or the site where there acid/glycolic acid=75 mole %/25 mole %, weight is a receptor, for instance, whereby systemic side effects average molecular weight: 12,000) in 4.5 ml of methy can be reduced and the drug can be allowed to act on a lene chloride and the mixture was stirred in a Polytron target organ efficiently for a long period of time. Poten 25 homogenizer for 20 seconds. The thus-obtained W/O tiation of the drug activity is thus expected. Further emulsion had a viscosity of 3,300 cp at 15 C. The subse more, the preparation can be administered intraarteri quent steps were operated in the manner of Example 1 ally in the vasoembolic therapy for kidney cancer, lung to give microcapsules. The content of leuprolide in the cancer and so forth as proposed by Kato et al. Lancet, microcapsules was 9.8%, the recovery (take-up) being volume II, pages 479-480 (1979). 98%. (3) The release of the active ingredient is continuous, so that, in case of, for instance, hormone antagonists or EXAMPLE 3 receptor antagonists stronger pharmacological effects Cefotian dihydrochloride (50mg) and 20 mg of gela are obtained as compared with daily or frequent admin tin were dissolved in 250 mg of water at 40 C. The istration. 35 solution was mixed with a solution of 4 g of polylactic (4) As compared with the conventional method of acid (weight-average molecular weight: 30,000) in 6.3 production of microcapsules which comprises prepar ml of chloroform and the mixture was stirred to give a ing a W/O/W triple-phase emulsion and subjecting the W/O emulsion. This W/O emulsion was placed in a emulsion to an in-water drying method, the method glass syringe and adjusted to 16 C. Then, the emulsion according to this invention makes it possible to allow was injected into 1,000 ml of a water phase containing the water-soluble drug, which is the active ingredient, 0.1% (weight/weight) of Tween 80 and having a tem to be taken up into microcapsules efficiently. In addi perature of 16 C. while stirring at 7,000 rpm for 1 tion, there can be obtained fine microcapsules having a minute for emulsification. The chloroform was then good degree of sphericity. allowed to evaporate while stirring at 2,000 rpm for 3 In accordance with the method of this invention, the 45 hours, followed by filtration, which gave microcapsules rate of water-soluble drug take-up into microcapsules of 5 to 80 um in size. In this example, the W/O emulsion can be increased markedly by adjusting the viscosity of had a viscosity of about 180 cp. The take-up of cefotiam the W/O emulsion to a value higher than that employed into the microcapsules amounted to 85%. in the conventional processes. Accordingly, sustained release microcapsules containing a water-soluble drug 50 EXAMPLE 4 can be produced with advantage. Leuprolide (450 mg) and 90 mg of gelatin were dis The following examples illustrate the invention in solved in 1 ml of distilled water to give an aqueous further detail. In the following Examples, the weight phase. A solution of 4 g of lactic acid-glycolic acid average molecular weight is based on standard of poly copolymer (lactic acid/glycolic acid = 75 mole %/25 styrene. 55 mole %, weight-average molecular weight: 14,000) in a mixture of 6 ml of methylene chloride and 1.5 ml of EXAMPLE 1 n-pentane was used as the oil phase. The aqueous phase Interferon a (500 mg) was dissolved in 300 mg of was gradually added to the oil phase while stirring at water at 50 C. The solution was added to a solution of room temperature with a turbine-shaped mixer. The 3,500 mg of polylactic acid (weight-average molecular 60 thus-produced W/O emulsion showed a viscosity of 70 weight: 21,000) in 4 ml of methylene chloride and the cp at 24 C. mixture was stirred in a small-size homogenizer(Poly A 0.5% aqueous solution of polyvinyl alcohol (500 tron, product of Kinematica, Switzerland) for 20 sec ml) was cooled to 15 C. Into this solution, there was onds. The thus-obtained W/O emulsion was cooled to injected gradually the above W/O emulsion while stir 15 C. in a hermetically closed vessel and defoaming 65 ring with a homogenizer. The thus-produced (W/O)/W and liquid temperature adjustment were conducted. emulsion was stirred gently with a propeller-shaped The emulsion cooled to 15 C. had a viscosity of 4,500 stirrer at room temperature for about 4 hours to thereby cp as measured with an Ubbelohde viscometer. This cause evaporation of methylene chloride and n-pentane 5,330,767 11 12 and solidification of the oil phase. The oil phase in solid like in infusing the W/O emulsion into the external form was collected by centrifugation. The leuprolide aqueous phase; or carrying out the above procedures in containing microcapsules thus obtained were rinsed combination, dispersing said emulsion in an aqueous with water and lyophilized into a powder. The take up phase and subjecting the resulting W/O/W emulsion to of leuprolide into the microcapsules amounted to 89%. an in-water drying to form microcapsules. 2. A microcapsule as claimed in claim 1, wherein the EXAMPLE 5 viscosity of the W/O emulsion in preparing the Leuprolide (495 mg) and 80 mg of gelatin were dis W/O/W emulsion is adjusted to about 150 to 5,000 solved in 0.5 ml of distilled water to give an aqueous centipoises. phase. A solution of 3,970 mg of lactic acid-glycolic 10 3. A microcapsule as claimed in claim 1, wherein the acid copolymer (lactic acid/glycolic acid = 75 mol viscosity of the W/O emulsion in preparing the %/25 mole %, weight-average molecular weight: 14,000) in 5.5 ml of methylene chloride was used as the W/O/W emulsion is adjusted to about 150 to about oil phase. The aqueous phase was gradually added to 10,000 centipoises by the manner of regulating the ten the oil phase while stirring at room temperature with a 5 peratures of (i) the W/O emulsion or (ii) both the W/O turbine-shaped mixer and the emulsion was cooled to emulsion and the external aqueous phase to from about 18 C. The thus-produced W/O emulsion showed a -20 C. to the boiling point of the organic solvent used. viscosity of 310 cp. 4. A microcapsule as claimed in claim 3, wherein the A 0.1% aqueous solution of polyvinyl alcohol (1,000 temperature is about 0° C. to 30' C. ml) was cooled to 18 C. Into this solution, there was 20 5. A microcapsule as claimed in claim 1, wherein the injected gradually the above W/O emulsion while stir polymer concentration in the oil phase is increased to ring with a homogenizer. The thus-produced (W/O)/W about 10 to 80% emulsion was stirred gently with a propeller-shaped 6. A microcapsule as claimed in claim 1, wherein the stirrer at room temperature for about 3 hours to thereby ratio of the aqueous phase to the oil phase is adjusted to cause evaporation of methylene chloride and solidifica 25 the range of about 1% to 50%. tion of the oil phase. The oil phase in solid form was 7. A microcapsule as claimed in claim 1, wherein the collected by centrifugation. The leuprolide-containing water-soluble drug is a biologically active polypeptide. microcapsules thus obtained were rinsed with water 8. A microcapsule as claimed in claim 7, wherein the and lyophilized into a powder. The take up of leupro biologically active polypeptide is (Pyr)Glu-His-Trp lide into the microcapsules amounted to 94%. 30 Ser-Tyr-D-Leu-Leu-Arg-Pro-NH-C2H5.acetate. What we claim is: 9. A microcapsule as claimed in claim 1, wherein the 1. A sustained-release microcapsule for injection con polymer is polylactic acid. taining a water-soluble drug, which is produced by a 10. A microcapsule as claimed in claim 1, wherein the process consisting essentially of preparing a W/O emul polymer is a copolymer of lactic acid and glycolic acid. sion composed of a water-soluble drug-containing solu 35 11. A microcapsule as claimed in claim 10, wherein tion as the inner aqueous phase and a biodegradable the ratio of lactic acid: glycolic acid of the copolymer is polymer-containing solution as the oil phase, adjusting about 75-2 mole %: about 252 mole %. the viscosity of the W/O emulsion used in preparing the 12. A microcapsule as claimed in claim 1, wherein the W/O/W emulsion to from about 150 to about 10,000 in-water drying is conducted by allowing the W/O/W centipoises by the procedures of increasing the polymer emulsion to stand under stirring. concentration in the oil phase; adjusting the ratio of the 13. A microcapsule as claimed in claim 1, wherein the aqueous phase to the oil phase; adjusting the tempera in-water drying is conducted by gradually reducing the ture of said W/O emulsion; adjusting the temperature of pressure while stirring with a propeller-shaped stirrer, the external aqueous phase; adjusting the temperature of magnetic stirrer or rotary evaporator. the W/O emulsion with a line heater or cooler or the 45

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