AU687423B2

AU687423B2 - Physiologically active substance-prolonged releasing-type pharmaceutical preparation

Info

Publication number: AU687423B2
Application number: AU73055/94A
Authority: AU
Inventors: Takaaki Baba; Ritsuko Ehama; Shoji Fukushima; Toshihiko Kaminuma; Masahiro Tajima; Kazuo Watabe; Takashi Yoshimoto
Original assignee: Shiseido Co Ltd
Current assignee: Shiseido Co Ltd
Priority date: 1993-09-20
Filing date: 1994-09-19
Publication date: 1998-02-26
Anticipated expiration: 2014-09-19
Also published as: AU7305594A; AU5538098A; CA2132396A1; US5637309A; KR950007873A; EP0645136A2; EP0645136A3

Description

AUSTRALIA

Patents Act COMPLETE SPECIFICATION

(ORIGINAL)

Class Int. Class Application Number: Lodged: Complete Specification Lodged: Accepted: Published: Priority Related Art: 0 00 o Name of Applicant: 0**0 Shiseido Company, Ltd.

Actual Inventor(s): SMasahiro Tajima Takashi Yoshimoto Shoji Fukushima Toshihiko Kaminuma Ritsuko Ehama Takaaki Baba Kazuo Watabe Address for Service: PHILLIPS ORMONDE FITZPATRICK Patent and Trade Mark Attorneys 367 Collins Street Melbourne 3000 AUSTRALIA Invention Title: PHYSIOLOGICALLY ACTIVE SUBSTANCE-PROLONGED RELEASING-TYPE PHARMACEUTICAL PREPARATION Our Ref 384275 POF Code: 1349/237681 The following statement is a full description of this invention, including the best method of performing it known to applicant(s): -1- 1A- PHYSIOLOGICALLY ACTIVE SUBSTANCE-PROLONGED RELEASING-TYPE PHARMACEUTICAL PREPARATION BACKGROUND OF THE INVENTION 1. Field of the Invention This invention relates to a pharmaceutical preparation capable of prolonged releasing a physiologically active substance in a controlled state. The pharmaceutical preparation is, particularly, directed to physiologically active peptides such as calcitonin generelated peptide (CGRP) and maxadilans (MAXs). The pharmaceutical preparation of the invention is useful as an 10 intracorporeal implantation-type, particularly intrathec al implantation-type physiologically active substance- 0 prolonged releasing preparation.

2. Background of the Invention Prolonged releasing (hereinafter, the same as gradually or delayed releasing) preparations, wherein when the drugs or physiologically active substances are administered into living bodies, elution of the drugs in the living bodies is controlled and their absorption is adjusted, have been investigated from long ago. For example, a method which comprises coating drugs with various coats, a method which comprises incorporating drugs into matrices of waxes or macromolecules, etc. have been known.

However, when intrathecal diseases are treated, in the case of intravenous administration of physiologically active substances, migration of these physiologically active substances into the brains is prevented by the blood brain barriers. As a method for direct administration of a physiologically active substance into brains, there is a method which comprises seating a catheter at the time of the operation and gradually supplying the drug into the brain, but since the apparatus is expensive and in addition there is a large 2 danger of infection, it is hard to say that the method is a reliable method. For example, in delayed cerebral vasospasm occurring after subarachnoid hemorrhage, the pathosis is retardingly manifested and moreover is lasting, and thus a method which comprises inserting a catheter for administration of drugs and a method which comprises continuously administering a drug into the vein are used. However, it is the present state of things that a method for obtaining sure therapeutic effects has not yet been developed.

When one's eyes are turned to physiologically active peptides, particularly calcitonin gene-related peptide (CGRP) and maxadilans (MAXs) about which the present inventors have contemplated development of phar- 15 maceutical preparations effective for various diseases, prolonged releasing pharmaceutical preparations for active substances specifically effective in relation to targeted diseases have not been proposed. When CGRP and maxadilans are specifically taken up, these are extremely interesting as proteins capable of inducing vasodilative and temporary immune suppression in mammals, as disclosed in E. A. Lerner et al., International Publication No, WO 91/00293, but after the report of Lerner et al., prolonged releasing pharmaceutical preparations therefor S 25 have not been proposed.

In this connection, the "maxadilans" were described in the above publication as proteins derived from the salivary gland lysate of a sand fly Lutzomyia longipalpis, and, thereafter, named "maxadilans" by them (for example, J. Biol. Chem., vol. 267, 1062-1066, 1992).

Lerner et al. exhibit, in the just above literature, through expression of recombinant maxadilans, that the analogues of maxadilan disclosed in WO 91/00293 also have a vasodilative activity. Other maxadilan analogues having a vasodilative activity are disclosed in M.

Ohnuma, E. A. Lerner et al., Peptide Chemistry 1993: Y.

II I I I~ 3- Okada 145-148.

As an interesting report from the aspect of the pharmacological actions of CGRP, H. Shimizu et al., Nou Shinkei Geka (Neurosurgery), 22(2): 131-139, 1994 discloses that when subarachnoid hemorrhage models of rabbits are used, and portions of an aseptic solution of CGRP (human alpha CGRP: Bachem Feinchemikalien, AG, Budendorf, Switzerland) are injected into the cisterna magnas of the animals, respectively, dilation effects on the contracted blood vessels are obtained.

However, this method is not always satisfactory in the effects of prophylaxis or treatment of cerebral vasospasm where the pathosis is retardingly manifested as stated above, and moreover, it is necessary to continue Sto strictly monitor the injection of the aqueous solution of the physiologically active substance into the cisterna magna, since said injection is very dangerous, during the injection operation.

Thus, the object of this invention lies in providing a pharmaceutical preparation effective for prophylaxis or treatment of, especially cerebral vasospasm, and a method therefor.

SUMMARY OF THE INVENTION The present inventors, in order to accomplish Sthe above object, have investigated above combinations of the contemplated physiologically active substances with various carriers, and have found, unexpectedly, that when a cellulosic polymer-based carrier is used, the physiologically active substance, particularly the physiologically active peptide exhibits controlled releasability.

As a further result of this research, they also found that MAXs themselves are effective for prophylaxis and treatment of cerebral vasospasm, irrespective of their dosage forms.

Another important finding found by the present 4 inventors is that a method to implant a prolcnged releasing pharmaceutical preparation containing a compound having a vasodilative action into the brain, which method has hitherto not been tried at all as a method to present or treat cerebral vasospasm, and has not been disclosed nor suggested in scientific literatures, etc., is extremely effective for prophylaxis or treatment of the disease.

Thus, this invention is directed to a pharmaceutical preparation carrying an effective amount of a physiologically active substance and capable of prolonged releasing the physiologically active substance, wherein the carrier of the physiologically active substance comprises a combination of a cellulosic polymer and at least one auxiliary component selected from fats and oils, waxes, fatty acids, saccharides and polyacrylate ester derivatives.

As a more specific embodiment, this invention is directed to a pharmaceutical preparation carrying an effective amount of a physiologically active substance and capable of prolonged releasing the physiologically active substance, wherein the physiologically active substance is one or more selected from the group consisting of CGRP and MAXs, 25 and the carrier of the physiologically active substance comprises 10 to 90 by weight of a cellulose ether derivative, 1 to 30 by weight of a fat or oil or a wax, and 1 to 30 by weight of a fatty acid, based on the total weight of the pharmaceutical preparation; or the carrier of the physiologically active substance comprises 10 to 90 by weight of a cellulose ether derivative and 1 to 40 by weight of a saccharide, based on the total weight of the pharmaceutical preparation; or the carrier of the physiologically active 5 substance is a combination of 10 to 90 by weight of crystalline cellulose and 0.01 to 10 by weight of a polyacrylate ester derivative with at least one or more selected from the group consisting of 1 to 30 by weight of a fatty acid, 1 to 30 by weight of a fat or an oil and 1 to 30 by weight of a wax, based on the total weight of the pharmaceutical preparation; or the carrier of the physiologically active substance comprises about 50 by weight of hyaluronic acid and about 50 by weight of a cationic polyacrylic acid derivative, based on the total weight of the pharmaceutical preparation.

As still another embodiment, this invention is directed to a method for prophylaxis or treatment of 15 cerebral vasospasm which comprises administering an effective amount of at least one of MAXs into the body.

As still another embodiment, this invention is directed to a method for prophylaxis or treatment of cerebral vasospasm by use of a compound having a vasodilative action as a physiologically active substance, which comprises a step to implant a prolonged releasing (gradually releasing) pharmaceutical preparation carrying the compound into the brain.

As still another embodiment, this invention is 25 directed to the use of a compound having a vasodilative action for preparing an intrathecal implantation-type prolonged releasing pharmaceutical preparation for prophylaxis or treatment of cerebral vasospasm.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a graph showing the behaviors of release of CGRP from pharmaceutical preparation of the invention, P-1, P-II and P-III (prepared in Example 1, Example 2 and Example 3, respectively) in the in vitro test; Fig. 2 is a graph showing the behaviors of 6 release of maxadilan (SEQ ID NO 3) from pharmaceutical preparations of the invention, P-VII, P-VIII and P-IX (prepared in Example 7, Example 8 and Example 9, respectively) in the in vitro test; Fig. 3 is a graph showing the behavior of release of CGRP from a pharmaceutical preparation of the invention, P-XI (prepared in Example 11); Fig. 4 is a graph showing the behaviors of release of CGRP from pharmaceutical preparations of the invention, P-XIII and P-XIV (prepared in Example 13 and Example 14, respectively) in the in vitro test; Figs. 5, and 6 to 8 are a graph showing the Sbehaviors of release of CGRP from pharmaceutical preparatione of the invention, P-XV-1 and P-XV-2 (both prepared *15 in Example 15), and P-XVX to P-XX (prepared in Examples S' 16 to 20), in the in vitro test; Fig. 9 is a graph showing the behavior of release of CGRP from a pharmaceutical preparation of the invention, P-I, implanted into the brain of a rabbit; Figs. 10 and 11 show results obtained by implanting preparations of the invention, P-V and P-VI, into the brain, particularly at the subarachnoid region of model animals of subarachnoid hemorrhage, respectively, and observing the behaviors of vasodilative; 25 Fig. 12 is a graph showing change of the CGRP concentration in the CSF when an aqueous CGRP solution was administered to a rabbit by a oisternal puncture method, and showing stability of CGRP in the CSF; Fig. 13 shows the results of the vasodilative test by administration of the aqueous CGRP solution by the cisternal puncture method; Fig. 14 shows the results of the vasodilative test similar to those in Figs. 10 and 11, on a preparation of the invention, P-VIII (containing an N-terminusmodified maxadilan, SEQ ID NO: 3); Fig. 15 is a graph showing the results of an in 7 vitro release test on a preparation of the invention, P-II by change of the CGRP concentration in the CSF; and Fig. 16 is a graph showing the effect of inhibition of vasospasm when an aqueous solution of a modified-type maxadilan, SEQ ID NO: 3 was administered to a model animal of cerebral vasospasm by a cisternal puncture method.

DETAILED DESCRIPTION OF THE INVENTION Cellulosic polymers used in the invention include, for example, cellulose ether derivatives such as hydroxypropylcellulose, methylcellulose, hydroxypropylmethylcellulose, hydroxyethylcellulose and carboxymethylcellulose; and crystalline cellulose. Preferred among 15 the cellulose ether derivatives is hydroxypropylcellulose. The compounding amount of this polymer in the pharmaceutical preparation cannot be limited since it varies depending on the kinds of auxiliaries to be combined and physiologically active substances to be carried 20 thereon, and is determined taking release initiation time and release sustainment time into account, but is generally 10 to 90 by weight, preferably 40 to 60 by weight, based on the total weight of the pharmaceutical preparation.

25 Fat and oils or waxes include hardened oils, cacao butter, beef tallow, lard, beeswax, carnauba wax, white wax, etc. Preferred among them are hardened oils.

The compounding amount thereof is not limitative, either, and can be determined taking release initiation time and release sustainment time into account, but is generally 1 to 30 by weight, preferably 10 to 20 by weight, based on the total "eight of the pharmaceutical preparation.

Hereafter, expressions of by weight are based on the total weights of the pharmaceutical preparations, respectively, unless otherwise defined.

Fatty acids include saturated or unsaturated 8 carboxylic acids having 12 to 22 carbon atoms such as stearic acid, lauric acid, myristic acid, isostearic acid, palmitic acid and behenic acid. Stearic acid is r'eferred among them. The compounding amount thereof is limitative, and can also be determined taking release initiation time and release sustainment time into account, but is generally 1 to 30 by weight, preferably to 20 by weight.

Saccharides include sucrose, lactose, glucose, fructose, maltose, dextrin, trehalose, pullulan, etc.

Preferred among them are lactose and glucose. The disintegrability of the gradually releasing preparation can be adjusted by addition of a saccharide. Namely, the disintegration velocity can be accelerated by increasing 1c its compounding amount. The compounding amount is not limitative, and can also be determined taking release initiation time and release sustainment time into account, but is generally 1 to 40 by weight, preferably 10 to 30 by weight. When a saccharide is used as an 20 auxiliary, especially in the case where it is combined with the above cellulose ether derivative, desired effects can be obtained without incorporating a fat or oil or a wax, or a fatty acid, which is an auxiliary component.

25 When crystalline cellulose is used as a cellulosic polymer, a combination thereof with a polyacrylate ester derivative such as poly (methacrylic acid-co-ethyl acrylate), poly (methaorylic acid-co-methyl methacrylate) and poly (methyl methacrylate-co-ethyl acrylate), preferably Eudragid L30D-5.5, and L100 (trade name; available from Lame Co., Germany) is recommended. The compounding amount of the polyacrylate ester derivative is generally 0.01 to 10 by weight, preferably 0.1 to 5 by weight.

As a carrier suitably usable in combination with CGRP or MAXs among later-described physiologically active substances, an ion complex comprising hyaluronic 9 acid and a cationic polyacrylic acid derivative can also be mentioned. As to hyaluronic acid, any of those (straight-chain high molecular polysaccharides formed through alternate bonds derived from B-N-acetyl-D-glucosamine and B-D-glucuronio acid) derived from wide natural origins, for example, the connective tissues of mammals, cockscombs of chickens, capsules of streptococci, etc.

can also be used. Suitable cationic polyacrylio acid derivatives such as poly (methyl methaorylate-co-butyl methacrylate-co-dimethylamino-ethyl methacrylate) and poly (ethyl acrylate-co-methyl methacrylate-co-trimethylaminonium ethyl methacrylate hydrochloride) include, for example, Eudragid E and Eudragid ES (trade names; available from Lame Co., Germany). As to the rate of mixing of them, they are suitably used almost in equal amounts.

Various physiologically active substances can be used, without particular limitation about kinds and action patterns contemplated thereon, so long as they can r be prepared as a pharmaceutical preparation of the inven- 20 tion and can attain significant effects through continuous release. Such physiologically active substances include, for example, adrenaline, abscisic acid, arginine vasotocin, angiotensinogen, angiotensin, angiotensin I converting enzyme, succus gastricus-inhibiLing polypep- 25 tides, insulin, insulin-like growth factors, S factor, erythropoietin, luteinizing hormone, luteinizing hormonereleasing hormone, progestogen, oxytocin, 2-octyl-Tbromoacetoacetate, autacoids, gastrin, gastrin secretionaccelerating peptide, gastron, activated vitamin D3, kallidin, calcitonin, calcitonin gene-related peptide (CGRP), kininogen, thymus hormone, gluoagon, gluoocorticoids, vasoactive small intestinal peptide, plasma kallikrein, serum factor, blood glucose-elevating hormone, thyroid-stimulating hormone, thyrotropin-releasing hormone, thyroid hormone, melanosite-stimulating hormone, melanosite-stimulating hormone-releasing hormone, melano- 10 site-stimulating hormone release-inhibiting hormone, corticotropin-like middl~e lobe peptide, urokinase, cholecystokinin octapeptide, oholecystokinin tetrapeptide, cholecystokinin variant, cholecystokinin-12, cholecystopancreothymine, cholecystokinin, growth factor, substance P, female sex hormones, adipokinin, chorionic gonadotropin, nerve growth factor, pancreatic polypeptides, reproduction nest-stimulating substanoe, gonadotropic hormones, growth hormone, growth hormone-releasing factor, seoretin, caeruleln, serotonin, fibroblast growth factor, kalllkrein glandularis, somatostatin, somatomedins A and B, placental lactogen, thymosin, thymopoletin, thyrogl.obulii, traumatic acid, endothelial cell growth factor, mollusc heart stimulant nervous peptide, 15 neurotensin, equine serum gonadotropic hormone, brain hormones, noradrenaline, vasopressin, estrogenic hormone, histamine, epidermic cell growth factor, parathyroid hormone, parathyroid-stimulating hormone, corticotropinreleasing factor, adrenal cortical hormone, PACAP, bradykinin, bradykinin-like peptide, proinsulin, proopiomelanocortin, prostaglanadins, pro PTH, prolactin, prolactinrel~easing hormone, prolactin release-inhibiting hormone, *fee 6 Sbo. florigene, human menopausal gonadotropin, bombesine, 464:06maxadilans (MAXs), mineral corticoid, light-adapted of :25 horomone, rethionyllysyibradykinin, 1-methyladrenine, melatonin, motilin, androgen, diuretic hormone, lipotropinj renin, relaxin and follicle maturation hormone.

Among physiologically active substances including those enumerated above, those usable for prophylaxis or treatment of cerebral vasospasm, whnich is a preferred embodiment of the invention, specifically include CGRP, MAXs, deferoxamine, methylprednisolone, nioorandil, nioaraben, magnesium sulfate, actinomycin D, 21-aminosteroid, isopr'oterenol, tPA, nimodipine, hydrocortisone, nicardipine, nifedipine, diltiazem, dllazp, teprothid, 11 AA861, papaverine, OKY 1581, amyl nitrite, erythrityl tetranitrate, isosorbide dinitrate, nitroglycerin, pentaerythritol tetronitrate, VIP, vasopressin, bradykinin, PACAP, SOD, catalase, bepridil, nadololol, felodipine, isradipine, varapamil, atenolol, metoprolol and propanolol.

Among them, compounds having a vasodilative action, particularly CGRP and MAXs can be mentioned as those which exert significant effects, particularly in prophylaxis or treatment of cerebral vasospasm, in combination with carriers in accordance with the invention of the present application, or in combination with other carriers capable of continuously releasing physiologically active substances, specifically by intracorporeal 15 implantation, particularly by intrathecal implantation.

The abbreviation of MAXs is used herein in a conception of including a natural-occurring peptide (or protein) (SEQ ID NO: 1) derived from the sand fly Lutzomyia longipalpis disclosed in above-mentioned E. A.

20 Lerner et al,, WO 91/00293 and its recombinant peptides; and the GIL-modified maxadilan (see, SEQ ID NO: 2; E. A.

Lerner et al., J. Bio. Chem., Vol. 267 pp. 1062- 1066, 1992) wherein its N-terminus is modified with a sequence consisting of three amino acid residues, GIL-; 25 and their analogues. A prepresentative analogue includes a peptide fragment obtained, according to the method disclosed in E. A. Lerner et al., ibid., namely by obtaining a particular modified-type maxadilan-fused protein, and then disgesting it with a protease such as factor Xa or thrombin. Representative among them are a peptide (SEQ ID NO: 3) wherein an amino acid sequence residue GSIL- is bonded to the N-terminus of SEQ ID NO: 1 maxadilan, and a peptide (SEQ ID NO: 4) wherein an amino acid sequence residue LVPRGSIL- is bonded thereto. MAXs also include those wherein one or more amino acid residues in the amino acid sequence are deleted or replaced, 12 and those wherein one or more amino acid residues are added to the N-terminus or C-terminus, and may be converted amino acid residues Lys Ala Gly Lys at the C-terminus thereof to Lys Ala-NH 2 A person skilled in the art will be able to obtain MAXs usable in the invention, referring to the amino acid sequences specifically disclosed in SEQ ID NOs.: 1 to 4 of the above Sequence Listing, by a liquid phase or solid phase peptide synthesis method known per se, or by a recombinant method wherein a nucleotide sequence is used which encodes a sequence formed by deleting one or more amino acid residues in its amino *i acid sequence or their amino acid sequences, or replacing them by other amino acid residue(s), or adding other amino acid residue(s) into the sequence(s).

The level of a physiologically active substance which can be contained in the pharmaceutical preparations of the invention composed of the above-mentioned carriers and optionally used auxiliaries is not limited since the optimal amount varies depending on kinds of carriers and active substances to be used, and methods for application of the preparations. However, generally, it is possible to incorporate 1 x 1012 to 30 by weight, preferably 1 x 10 4 to 5 by weight of a physiologically active substance, based on the total weight of the pharmaceutical preparation, into the preparation.

The pharmaceutical preparations of the invention can be prepared by compounding the above-mentioned carrier-constituting components and physiologically active substances at levels described above, respectively, and using formulation techniques known per se. In such formulation, it is possible to incorporate one or more of optional additives conventionally used in the art, for example, disintegration-adjusting agents, stabilizers, antioxidants, wetting agents, binders, lubricants, etc., in accordance with the forms of use of 13 the pharmaceutical preparations. The dosage forms of preparations thus prepared can usually be tablets, pills or capsules. However, the dosage form of the preparations may also be a liquid obtained by pulverizing a solid agent prepared above so that it can be used as an injection, and suspending the powder in a suitable fluid sterilized distilled water, physiological saline, etc.).

On the other hand, it is known that part of MAXs among the above physiologically active substances have a vasodilative action, as is the case with CORP, as disclosed by the above E. A. Lerner et al. For example, the maxadilan of SE ID NO: 2 exhibits an extremely interesting vasodilative action 80 to 100 times higher than that of CORP. An N-terminus-modified-type maxadilan denoted by SEQ ID NO: 3 is known to exhibit a vasodilative action (particularly, erytheka activities) further about 10 times higher than that of SEQ ID NO: 2 (the above M. Ohnuma et al., Peptide Chemistry 1993 Y. Okada 20 pp. 1145-118), and is a particularly interesting peptide.

~However, it has not so far been disclosed in technical literatures that these MAXs can be used for prophylaxis or treatment of cerebral vasospasm.

Thus, according to this invention, although cerebral vasospasm can be prevented or treated by implanting a pharmaceutical preparation comprising the above carriers having contained therein at least one of MAXs, preferably into a living body, particularly into the brain, a method to use MAXs for treating the disease without using these carriers is also disclosed.

Namely, this invention also provides a method for prophylaxis or treatment of cerebral vasospasm which comprises a step of administering an effective amount of at least one of MAXs into the body of a patient to whom cerebral vasospasm may occur or a patient to whom cere- 14 bral vasospasm occurred. This administration step can, for example, be performed by administering an injection obtained by merely dissolving or suspending at least one of MAXs in sterilized distilled water, physiological saline or a buffered solution, into the vein or artery, or can also be performed by administering preparations obtained by adding various inorganic salts as an ionic strength-adjusting agent, and other excipients, for example, dextrin, lactose, starch, etc. thereto, and formulating the mixtures.

Administration time varies depending on dosage forms adopted, administration routes, and the purpose of use (prophylaxis or treatment), but can usually be immediately to 10 days after the operation of subarach- 15 noid hemorrhage.

Thus, this invention also provides as another embodiment the use of at least one of MAXs for preparing a pharmaceutical preparation for prophylaxis or treatment of cerebral vasospasm. The use of prolonged releasing pharmaceutical preparations of CGRP or MAXs for intrathecal implantation has not so far been disclosed in technical literatures, either, and, in addition, has a significantly excellent advantage, for example, over set: administration of a sterile aqueous solution of CGRP into 25 the cisterna magna, as disclosed in the above H. Shimizu et al., No Shinkei Geka, 22(2) 131-139, 1994.

A further important thing is that effective prophylaxis or treatment of cerebral vasospasm can be performed not only by combinations of the above-mentioned carriers for pharmaceutical preparations with CGRP or MAXs, but also by implanting into the brains pharmaceutical preparations prepared by combinations of physiologically active substances exhibiting a vasodilative action, as mentioned above with other carriers.

Thus, from this viewpoint, as stated above, this invention also provides a method for prophylaxis or 15 treatment of cerebral vasospasm which comprises a step of implanting a prolonged releasing pharmaceutical preparation comprising certain carriers having contained therein a physiologically active substance having a vasodilative action, preferably at least one selected from the group consisting of CGRP and MAXs, into the brain of a patient to whom cerebral vasospasm may occur or a patient to whom cerebral vasospasm occurred. As an alternative embodiment of this method, this invention also provides the use f a physiologically active substance (or compound) having a vasodilative action, preferably at least one selected from the group consisting of CGRP and MAXs, for preparing an intrathecal implantation-type pharmaceutical preparation for prophylaxis or treatment of cerebral vasospasm.

The pharmaceutical preparation of the invention of the present application can be administered in a predetermined effective amount in a prolonged releasable form, particularly parenterally, but preferably, the 20 characteristic of the preparation lies in a point that it exhibits a remarkable effect when implanted into a living o e* 0 body, particularly into the brain. The pharmaceutical 00..

*,?Joe preparation of the invention, when thus implanted into the brain, prolonged releases the active substance with 25 retention of the dosage form for 7 days or more, mostly "10 days or more, and thus the active substance distributes only on the administration site and does not disperse throughout the brain. Therefore, there is not waste of the active substance, and moreover, there is less possibility that it has unnecessary actions on other sites. Moreover, the preparation has desired properties that it does not hurt the cells at the administration site, and releases that active substance stably over a long term as long as 2 days to 2 weeks after the administration, ard thereafter the base (carriers and auxiliaries) is absorbed in the living body. When the pre-

I

16 paration is intrathecally implanted as a tablet, it is desirable to place it in the subarachnoid space and/or in the furrow of the brain surface so that it is not let to flow by the reflux of the cerebrospinal fluids. It is also desirable that the thickness of the preparation is mm or less so that it can be intrathecally implanted without any trouble, and since when the expansion coefficient of the tablet is too large, it may do damages such as cell detachment to the cells and/or tissues around the administration site, and therefore, it is desirable to adjust the carrier components and the auxiliary components to make the expansion coefficient 200 or less.

A person skilled in the art will be able to readily determine the optimal amounts of physiologically 15 active substances which can be incorporated in these prolonged releasing pharmaceutical preparations, according to in vitro tests or in vivo tests as described later.

This invention is further illustrated below 20 according to specific examples, but it should not be construed that they are provided for the purpose of limiting the scope of the invention.

Example 1 Preparation of a prolonged releasing pharmaceu- 25 tical preparation (P-I) 10 g of stearic acid and 19 g of hardened oil were mixed, 2.0 g of 0.4 CGRP solution (corresponding to 8 mg of CGRP) and 20 g of lactose were added thereto and the "Yixture was mixed, and 60 g of hydroxypropylcellulose was added thereto. The mixture was mixed adequately, and then subjected to a KBr compressor (150 kg, 1 minute) to prepare tabular tablets having a diameter of 13 mm.

Example 2 Preparation of a prolonged releasing pharmaceutical preparation (P-II) 17 g of stearic acid and 20 g of hardened oil were mixed, 2.5 g of 0.4 CGRP solution (corresponding to 10 mg of CGRP) and 20 g of lactose were added thereto and the mixture was mixed, and 40 g of hydroxypropylcellulose was added thereto. The mixture was mixed adequately, and then pressure molded using a Correct 19 K compressor (KIKUSUI CLEAN PRESS) (6 mmO x 2 mm).

Example 3 Preparation of a prolonged releasing pharmaceutical preparation (P-III) g of palmitic acid and 15 g of beeswax were mixed, 2.0 g of 0.4 CGRP solution (corresponding to 8 mg of CGRP) was added thereto and the mixture was mixed, and 70 g of hydroxypropylcellulose was added thereto.

15 The mixture was mixed adequately, and then pressure S"molded using a Correct 19 K compressor (KIKUSUI CLEAN PRESS) (6 mm6 x 2 mm).

Example 4 Preparation of a prolonged releasing pharmaceutical preparation (P-IV) 10 g of steario acid and 10 g of hardened oil were mixed, 2.0 g of 0.4 CGRP solution (corresponding to 8 mg of CGRP) and 20 g of glucose were added thereto and the mixture was mixed, and 60 g of hydroxypropylcellulose was added thereto. The mixture was mixed adequately, and then pressure molded using a Correct 19 K compressor (KIKUSUI CLEAN PRESS) (6 mmd x 2 mm).

Example Preparation of a prolonged releasing pharmaceutical preparation

(P-V)

g of stearic acid and 10 g of hardened oil were mixed, 2.5 g of 1.6 CGRP solution (corresponding to 40 mg of CGRP) and 20 g of lactose were added thereto and the mixture was mixed, and 60 g of hydroxypropylcellulose was added thereto. The mixture was mixed adequately, and then pressure molded using a Correct 19 K

I

18 compressor (KIKUSUI CLEAN PRESS) (6 mm x 2 mm).

Example 6 Preparation of a prolonged releasing pharmaceutical preparation (P-VI) 10 g of stearic acid and 10 g of hardened oil were mixed, 2.5 g of 10 CGRP solution (corresponding to 250 mg of CGRP) and 20 g of lactose were added thereto and the mixture was mixed, and 60 g of hydroxypropylcellulose was added thereto. The mixture was mixed adequately, and then pressure molded using a Correct 19 K compressor (KIKUSUI CLEAN PRESS) (6 mm x 2 mm).

Example 7 Preparation of a prolonged releasing pharmaceutical preparation (P-VII) 15 10 g of stearic acid and 10 g of hardened oil were mixed, 2.5 g of 0.6 solution of a GSIL-modifiedtype maxadilan denoted by SEQ ID NO: 3 (corresponding to mg of SEQ ID NO: 3) and 20 g of lactose were added thereto and the mixture was mixed, and 60 g of hydroxypropylcellulose was added thereto. The mixture was mixed adequately, and then pressure molded using a Correct 19 K compressor (KIKUSUI CLEAN PRESS), and the moldings were pulverized and mixed and pressure molded again using the same compressor (6 mmO x 2 mm).

Example 8 SPreparation of a prolonged releasing pharmaceutical preparation (P-VIII) g of stearic acid and 10 g of hardened oil were mixed, 2.5 g of 1.0 solution of a GSIL-modifiedtype maxadilan denoted by SEQ ID NO: 3 (corresponding to mg of SEQ ID NO: 3) and 20 g of lactose were added thereto and the mixture was mixed, and 60 g of hydroxypropylcellulose was added there:.o. The mixture was mixed adequately, and then pressure molded using a Correct 19 K compressor (KIKUSUI CLEAN PRESS), and the moldings were pulverized and mixed and pressure molded again using the 19 sa8me compressor (6 mmO x 2 mm).

Example 9 Preparation of a prolonged releasing pharmaceutical preparation (P-IX) 10 g of stearic acid and 10 g of hardened oil were mixed, 2.5 g of 10 solution of a GSIL-modifiedtype maxadilan denoted by SEQ ID NO: 3 (corresponding to 250 mg of SEQ ID NO: 3) and 20 g of lactose were added thereto and the mixture was mixed, and 60 g of hydroxypropylcellulose was added thereto. The mixture was mixed adequately, and then pressure molded using a Correct 19 K compressor (KIKUSUI CLEAN PRESS), and the moldings were !1 pulverized and mixed and pressure molded again using the 99 same compressor (6 mmO x 2 mm).

Example Preparation of a prolonged releasing pharmaceutical preparation (P-X) 15 g of palmitic acid and 15 g of beeswax were 9.9.

mixed, 3.0 g of 0.4 solution of a GSIL-modified-type 20 maxadilan denoted by SEQ ID NO: 3 (corresponding to 10 mg of SEQ ID NO: 3) was added thereto and the mixture was mixed, and 70 g of hydroxypropyloellulose was added 9 thereto. The mixture was mixed adequately, and then pressure molded using a Correct 19 K compressor (KIKUSUI 25 CLEAN PRESS) (6 mmO x 2 mm).

Example 11 Preparation of a prolonged releasing pharmaceutical preparation (P-XI) g of 0.4 CGRP solution (corresponding to 8 mg of CGRP) and 20 g of lactose were mixed adequately, and 80 g of hydroxypropylcellulose was added thereto.

The mixture was mixed adequately, and then subjected to a KBr compressor (150 kg, 1 minute) to prepare tabular tablets (P-XI) having a diameter of 13 mm.

20 Example 12 Preparation of a prolonged releasing pharmaceutical preparation (P-XII) g of 0.4 CGRP solution (corresponding to 10 mg of CGRP) and 20 g of glucose were mixed adequately, and 80 g of methyloellulose was added thereto. The mixture was mixed adequately, and then subjected to a KBr compressor (150 kg, 1 minute) to prepare tabular tablets (P-XII) having a diameter of 13 mm.

Example 13 Preparation of a prolonged releasing pharmaceutical preparation (P-XIII) 2.5 g of 0.4 CGRP solution (corresponding to 0.6 10 mg of CGRP) and 13 g of poly (methacrylic acid-co- W'0* 15 methyl methacrylate), Eudragid L-100 (trade name; available from Lame Co., Germany), were mixed, and 87 g of crystalline cellulose was added thereto. The mixture was mixed adequately, and then subjected to a KBr compressor (150 kg, 1 minute) to prepare tablets (P-XIII) having a 20 diameter of 13 mm.

.ic. Example 14 Preparation of a prolonged releasing pharmaceu- Sical preparation (P-XIV) 17 g of stearic acid and 17 g of hydrogenated 25 oil (hydrogenated castor oil) were mixed, 2.0 g of 0.4 CORP solution (corresponding to 3 mg of CGRP) and 0.3 g of poly (methacrylic acid-co-ethyl acrylate), of Eudragid L30D-5.5 (trade name; available from Lame Co., Germany), were added and the mixture was mixed, and 66 g of crystalline cellulose was added thereto. The mixture was mixed adequately, and then subjected to a KBr compressor (150 fkg/em 2 1 minute) to prepare tablets (P-XIV) having a diameter of 13 mm.

,Example Preparation of a prolonged releasing pharmaceutical preparation (P-XV)

I

21 100 g of aqueous 2 hyaluronic acid solution and 100 g of aqueous 2 Eudragid E solution were subjected to reaction at room temperature for 2 hours under stirring. The reaction mixture was centrifuged at 3,000 rpm for 10 minutes, and the p!'oduct was recovered and vacuum dried to give a polyion complex of hyaluronic acid Eudragid E.

The obtained solid was pulverized and classified to give powder having a particle size of 150 microns or less. 100 g of the powder and 0.75 of 0.4 CGRP solution (corresponding to 3 mg of CGRP) were mixed, and then pressure molded using a Correct 19 K compressor (KIKUSUI CLEAN PRESS), and the moldings were pulverized and mixed and pressure molded again using the same com- 15 pressor (6 mmd x 2 mm).

Examples 16 to Procedures described in the aforementioned Examples, particularly Examples 14 and 15, are repeated, except that compositions of the preparation are those 0000 20 used in the following table 1.

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C C *C* C *C C C C C Table 1 Example Nos. (preparation Nos.) Compositions 16 (P-XVI) 17 (P-XVII) 18 (P-XVIII) 19 (P-XIX) 20 (P-XX) Crystalline cellulose (mg) 1600 1450 1300 2000 0 Hydrogenated oil (hydrogenated caster oil) (mg) 200 270 330 0 0 Stearic acid (mg) 200 270 330 0 0 EudragitW L30D-5.5 tl) 120 110 100 300 300 EudragiW LI00 0 0 o.14 0.2 C, -Na 0 0 0 0 Lacto.-- 0 0 0 0 CGRP (mg) 0.2 0.2 0.2 0.2 0.2 H 2 0 (ml) 0.2 0.2 0.2 0.2 0.2 Ethanol (ml) 0 0 0 5 0 23 Examples 21 to 34 In vitro release test on physiologically active substances ml portions of Hartmann's Solution (available from The Green Cross Corp. Japan) were aseptically put in 15-mi tubes, respectively.

The following pharmaceutical preparations were aseptically put in these tubes, respectively: P-I, P-I, P-IV (Fig. 1) P-VXI, P-VIII, P-IX (Fig. 2) P-XI (Fig. 3) P.-XIIII P-XXV (Fig. 4) P-XV (Fig. P-XVII P-XVII, P-XIX (Fig. 6) P-XVIII (Fig. 7) P-XX (Fig. 8) The mixtures were shaken at 37 0 C and 120 rpm, sampling was performed 1, 2, 3, 7, 11 and 14 days thereafter, and each sample was subjected to determination of the release amount of the physiologically active substance by high performance liquid chromatography. The results are shown in Figs. 1 to 8, respectively (The relations between the figures and the pharmaceutical preparations are shown in the above parentheses).

Numbers in the figures express the disintegration states of the tablets, and the meanings are as follows: Disintegration state 1 No change 2 20 swelling is observed 3 Small cracks are formed 4 Large cracks are formed Disintegrated into pieces Example In vivo release test on a physiologically active substance An in vivo test on drug release effect with 3 rabbits according to the method shown below was performed, using the tablets (P-II) obtained in Example 2.

The results are shown in Fig. 9.

Test. method Method of intrathecally implantation of the tablets into the brains The rabbits 3.0 k(g) after sodium pentobarbital anesthesia were fixed on their faces, the occipital bone membranes (dura maters) were exposed by incision, and the occipital bones were shaved by a drill to :expose the dura maters further wider. Thereafter, the :dura maters and the arachnoidmembrane were incised to a 99e9 length of about 8 mm, the tablets of the prolonged releasing pharmaceutical preparation of Example 2 were implanted there on two of the three animals, a placebo tablet was implanted there on the other one, the dura maters, the muscles and~ the skins were sutured, and an appropriate amount of an antibiotic was administered to the incision sites, respectively.

The cerebrospinal fluids were sampled every day from the rabbits by the following sampling method, respectively, and the samples were assayed for the CORP :concentrations (nM) in the cerebrospinal fluids according to the following assay method.

Method of sampling of the cerebrospinal fluids The rabbits after sodium pentobarbital anesthesia were fixed on their faces, the occipital bone membranes (dura maters) were exposed by incision, the occipital bone membranes were Incised, and the celebrospinal fluids were sampled therefrom.

Method OP assay of the concentration of the physiologically active substance In the oelebrospirial fluids The concentrations were assayed by the following radioimmunoansay.

25 4,000 cpm portions of a labeling compound 12-( 125 1-iodohistidyl 10 )CGRP] were put in measuring tubes, and separately from these, 100 i portions of7 1, 2, 5, 10, 50, 100, 500 and 100 fmol standard solutions were prepared using synthesized CGRP (available from Bachem 100 pl portions of an antibody (obtained by dissolving RPN 1841 available from Amarsham Co. in 2 ml and diluting the solution to 12.5 ml) and 600 g1 portions of an analyzing buffer [50 mM sodium phosphate (pH 7.4), 0.3 bovine serum albumin, 10 mM EDTA] were added, respectively, to the tubes containing 100 pl portions of the test samples, the standard solutions or water, respectively, the covers of the tubes were shut, and the .mixtures were allowed to stand at 40C for 5 days, 250 p1 15 of a dextran/active carbon solution [50 mM sodium phosphate (pH 0.25 gelatin, 10 mM EDTAJ was added to each mixture, and the resultant mixture was immediately centrifuged at 2,000 x g for 20 minutes. Both of the S :precipitate and the supernatant were measured for 200 seconds by a T-counter, and the concentration of the :'physiologically active substance (CGRP) in the .erebrospinal fluid was assayed based on the standard curve obtained from the standard substance solutions.

'..':Separately, an in vivo test related to the disintegration state of a tablet was performed using the tablet of P-IT as the tablet and a rabbit other than the above rabbits. In this connection, implantation of the tablet into the brain was performed in the same manner as above, and the disintegration state of the tablet was observed through araniotomy. The results are shown below. The following numerical values exhibit the disintegration state of the tablet, and the meanings are the same as defined above.

After implantation 1st day 5th day 10th day Disintegration state 2 2 11 26 It is understood from the results that the prolonged releasing cerebral vasospasm inhibitor of the invention keeps the dosage from in vivo even 10 days after the intrathecal implantation, Examples 36 and 37 Vasodilative test (I) A vasodilative test was performed according the following method, using the tablets obtained J,n Example and Example 6, i.e, P-V and P-VI, respectively, and tablets not containing a physiologically active peptide CGRP (placebo tablets). The results are shown in Fig. about the tablet of P-V and in Fig. 11 about the tablet ~:of P-VI.

Process of preparation of the placebo tablet Prolonged releasing pharmaceutical preparations as placebos were prepared in the same manners as in the preparation of the prolonged releasing pharmaceutical preparation of Example 5 and in the preparation of the prolonged releasing pharmaceutical preparation of Example 6 respectively, except for replacement of *o o CGRP by hydroxypropylcellulose.

0o*o veto Vasodilative test An experiment was performed according to the •following procedure using 8 rabbits about the tablet of 25 P-V and 7 rabbits about the tablet of P-VI, these rabbits weighing 2.5 to 3 kg.

After an X-ray photograph of the basilar artery of each rabbit was taken, subarachnoid hemorrhage models were prepared according to a known method G.

Vollmoer et al., Neurosurgery 28 27-32 (1991)]. (Day 0) 24 hours later (Day each animal was anesthetized with sodium pentobarbital, and the occipital region was incised from the occipital bone to the primary cervical vertebrae along the median line.

The muscles attached to the occipital bone, the primary cervical vertebrae and the occipital 27 bone membrane were carefully detached by a knife so as not to injure the vein.

(14) "'he exposed occipital bone was shaved by a surgical drill to a thiokness of the order of' 2 to 5 mm the lower part.

Thereafter, the occipital bone membrane was incised by a knife to a length of the order of 8 to mm.

The tablet was plaesd in the subarachnoid region through this site using a pair of tweezers.

After the insertion of the tablet, the ~occipital bone membrane was sutured with silk thread.

The out portion was further shut with Aron Alpha (trade mark) along the suture line.

Thereafter, the muscle and the skin were sutured with silk thread, and an appropriate amount of antibiotic was administered.

The blood vessel calibers were measured a;every day from immediately after the administration to days thereafter (Day 6) by angiography.

Comparative example Comparative tests against Example 35 and Examples 36 and 37 As a comparative example, an aqueous CGRP solution was administered to one rabbit by a cisternal puncture method in accordance with the known method in the above and the CGEP concentration (nM) in the cerebrospinal. fluid was assayed over the lapse of time in the same manner as in Example 35. The results are shown In F'ig. 12.

As another comparative example, portions of an aqueous CORP solution or portions of distilled water were administered) respectively to rabbit models (2 animals about the aqueous COP solution and 14 animals about the distilled water) In which spasm of the basilar artery had been clearly observed after subarachnoid hemorrhage, also 28 by a cisternal puncture method in accordance with the known method in the above and the blood vessel calibers were measured in the same manner as in the Examples 36 and 37. The results are shown in Fig. 13.

Example 38 Vasodilative test (II) A vasodilative test was performed in the same manner as in Examples 36 and 37 using the tablet obtained in Example 8 (P-VIII) and a tablet (placebo tablet) not containing a GSIL-modified-type maxadilan denoted by SEQ ID NO: 3 as a physiologically active peptide. The results are shown in Fig. 14. The placebo tablet was prepared in the same manner as in Example 8 except for replacement of the GSIL-modified-type maxadilan by ".15 hydroxypropylcellulose.

0 15 Example 39 In vivo release test on a physiologically active substance 1 In addition to Example 35, this experiment was performed aiming to obtain statistically more meaningful data. The operations described in Examnple 35 were repeated except that 30 rabbits (5 animals each for Normal, 0000 day 1, day 2, day 3, day 4 and day 5) were used, and the cerebrospinal fluids (CSF) were collected 1 day (day 1), 025 2 days (day 3 days (day 4 days (day 4) and 5 days (day 5) after the implantation of the tablet (P-II) from animals ecch for the respective days, and thereby the CGRP concentrations in the CSFs were assayed. The results are shown in Fig. In the above Normal means a case where the tablet (P-II) was not implanted.

Example In vivo disappearance test on a pharmaceutical preparation An in vivo test related to the disappearance effect of a prolonged releasing pharmaceutical prepara- 30 Test results Table 2 Days from the day when Assessment of the tablets was disappearance of implanted the tablet 1st day 2nd day 3rd day Nv+ 4th day day o 10th day c* 1st month 3rd month 6th month There was not any rabbit at all which died during the term when the disappearance effect test on 5 this prolonged releasing pharmaceutical preparation (tablet) was performed.

Example 41 Cerebral vasospasm inhibition test on maxadilan (SEQ ID NO: 3) Preparation of a cerebral vasospasm model animal A Japanese white rabbit (male, weighing 2-3 kg) was subjected to general anesthesia by injection of sodium pentabarbital through the auricular vein. After the rabbit was retained so as not to move, the surroundings of the punctured vein were disinfected with sterilized ethanol, a self-retaining needle was punctured into the vein, and immediately thereafter, a siliconemade extension tube equipped, at one end, with a three way cock connected at the two ways with 10-mi syringes each containing physiological saline and connected at the 29 tion (tablet) in the brains was performed by the following method, using the tablet (P-II) obtained in Example 2. In this test, 18 rabbits (2 animals each for day 1, day 2, day 3, day 4, day 5, day 10, month 1, month 3 and month 6) were used. The operations and the results are shown below.

Method of implantation of the tablet into the brain Each of 18 rabbits (2.5 3.0 kg) was anesthetized with sodium pentobarbital and fixed on its face, the occipital bone membrane (dura mater) was exposed by incision, and the occipital bone was shaved by a drill to expose the dura mater further wider. Thereafter, the dura mater and the arachnoidea were incised to a length of about 8 mm, the tablet of Example 2 as a prolonged releasing pharmaceutical preparation was implanted, the dura mater, the muscle and the skin were sutured, and an appropriate amount of an antibiotic was administered to the incision site.

20 Assay method and assessment criterion S• On 1st day, 2nd day, 3rd day, 4th day, 5th day, day, 1st month, 3rd month, 6th month after the implantation of the tablet into the rabbits, the two rabbits each for the respective days were subjected to a 25 craniotomy operation to expose the portion where the tablet was implanted, and observation was performed visually. The assessment criterion was as follows.

The tablet scarcely disappears Nearly half of the tablet disappeared Almost all the tablet disappeared The tablet completely disappeared 31 residual way with a 10-ml syringe containing sodium pentobarbital was connected to the self-retaining needle (when the blood vessel is thin, the blood vessel is stimulated by fingers to dilate it). After the connection, the physiological saline was flashed and it was confirmed that the needle was in the vein, and then, the sodium pentobarbital was injected into the rabbit so that the amount became 75 mg per kg of the weight of the rabbit. At this time, since the animal went under anesthesia within one minute, the respiratory tract was previously secured so as to make respiration possible.

Insertion of catheter (Seldinger's method) The femoral region artery was exposed by incision of the femoral region, and a catheter was inserted 15 into the vertebral artery from the exposed portion under ftransillumination, using a guide wire. Separately, 0.2 ml of heparin was injected so as to prevent formation of thrombus at the time of insertion of the catheter.

Angiography A spot for angiography was determined by transillumination, the head was fast fixed, back flow of the blood was confirmed, and 0.8 ml of a contrast medium was injected under a certain pressure (2.3 kg/cm 2 At the time when 0.6 ml thereof was injected, a photograph of 25 the blood vessel was taken.

Cisterna puncture This procedure was performed in common when blood of a test preparation was injected into the rabbit.

The animal was placed on its face, and the head was leaned downward so as to make an angle of 30° against a horizontal plane. After the occipital bone was confirmed, the primary cervical vertebrae was confirmed, and a 26G butterfly needle was stuck at an angle of about 600 between them. When the needle hit the occipital bone, the needle was made vertical to 900, and punctured furtner deeply. At this point of time, it was confirmed 32 that the cerebrospinal fluid flew out by reducing pressure. The fresh blood of the artery was injected through this needle at a rate of 1 ml/min in an amount of 1 ml per kg of the weight of the rabbit. After the injection, back flow of the cerebrospinal fluid was again confirmed, and thb rabbit was allowed to stand, leaving it intact, for 15 minutes or more. Photographs of the cerebral blood vessel of the animal were taken 3 days after the blood injection.

Administration of a test preparation Groups of rabbits, one group consisting of animals, were treated as mentioned above, and among them, rabbits were selected wherein when the basilar artery was divided, starting from the junction from the vertebral 15 artery, into three equal parts, and the diameter at the middle point was measured, spasm occurred at a level of 18 or more. About the selected rabbits, portions of an aqueous solution of a modified-type maxadilan denoted by SE ID NO: 3 or portions of distilled water as a comparative example were administered 3 days after the injection S. of the blood, respectively. After The administration, angiography of the basilar artery was performed, on each rabbit, over the lapse of time, and the ratio of the diameter of the blood vessel to that before subarachnoid 25 hemorrhage was investigated. As to the model animals, rabbits weighing 2.5 3.0 kg were used as follows: 8 animals in the modified-type maxadilan administration group and 4 animals in the sterile water administration group. The dose of the modified-type maxadilan was 7 [g per kg of the weight of the rabbit.

The results are shown in Fig. 16.

As apparent from Fig. 16, in the aqueous modified-type maxadilan solution administration group, spasm was significantly inhibited during from immediately after the administration to about 4 hours thereafter, compared to the sterile water administration group.

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SS 55

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33 Sequence Listing SEQ ID NO: 1 SEQUENCE LENGTH 63 TYPE amino acid TOPOLOGY linear MOLECULE TYPE peptide

SEQUENCE

Cys Asp Ala Thr Cys Gin Phe Ar'g Lys Ala Ile Asp Asp Cys Gin Lys 1 5 10 Gin Ala His His Ser Asn Val. Leu Gin Thr Ser Val Gin Thr Thr Ala 20 25 Thr Phe Thr Ser Met Asp Thr Ser Gin Leu Pro Gly Asn Ser Val Phe 35 40 15 Lys Glu Cys Met Lys Gin Lys Lys Lys Giu Phe Lys Ala Gly Lys 55 SEQ ID NO: 2 SEQUENCE LENGTH 66 20 SEQUENCE TYPE amino acid TOPOLOGY linear MOLECULE TYPE ,peptide

SEQUENCE

Gly Ile Leu Cys Asp Ala Thr Cys Gin Phe Arg Lys Ala le Asp Asp 1 5 10 Cys Gin Lys Gin Ala His His Ser Asn Val Leu Gin Thr Ser Val Gin 25 Thr Thr Ala Thr Phe Thr Ser Met Asp Thr Ser Gin Leu Pro Gly Asn 140 145 Ser Val Phe Lys Giu Cys Met Lys Gin Lys Lys Lys Giu Phe Lys Ala 55 Giy Lys 34 SEQ ID NO: 3 SEQUENCE LENGTH 67 SEQUENCE TYPE amino acid TOPOLOGY linear MOLECULE TYPE peptide

SEQUENCE

Gly Ser Ile Leu Cys Asp Ala Thr Cys Gin Phe Arg Lys Ala lie Asp 16 1 5 10 Asp Cys Gin Lys Gin Ala His His Ser Asn Val Leu Gln Thr Ser Val 32 20 25 Gln Thr Thr Ala Thr Phe Thr Ser Met Asp Thr Ser Gln Leu Pro Gly 48 35 40 Asn Ser Val Phe Lys Glu Cys Met Lys Gin Lys Lys Lys Glu Phe Lys 64 50 55 Ala Gly Lys 67 SEQ ID NO: 4 SEQUENCE LENGTH 71 SEQUENCE TYPE amino acid TOPOLOGY linear MOLECULE TYPE peptide

SEQUENCE

Leu Val Pro Arg Gly Ser Ile Leu Cys Asp Ala Thr Cys Gln Phe Arg 16 25 5 10 Lys Ala Ile Asp Asp Cys Gln Lys Gin Ala His His Ser Asn Val Leu 32 25 Gin Thr Ser Val Gin Thr Thr Ala Thr Phe Thr Ser Met Asp Thr Ser 48 40 Gin Leu Pro Gly Asn Ser Val Phe Lys Glu Cys Met Lys Gin Lys Lys 64 25 Lys Glu Phe Lys Ala Gly Lys 71

Claims

1. A pharmaceutical preparation carrying an effective amount of a physiologically active substance and capable of prolonged releasing the physiologically active substance, wherein the carrier of the physiologically active substance comprises a combination of 10 to 90% by weight, based on the total weight of the pharmaceutical preparation, a cellulosic polymer selected from the group consisting of cellulose ether derivatives and crystalline celluloses, and at least one auxiliary component selected from the group consisting of 1 to 30% by weight of the fat or oil or the wax and 1 to 30% by weight of a fatty acid; with the proviso that when the cellulosic polymer is a crystalline cellulose, the auxiliary component is a polyacrylate ester,

2. A pharmaceutical preparation according to claim 1, directed to intracorporeal implantation.

3. A pharmaceutical preparation according to claim 2 wherein said i intracorporeal is intrathecal. e* 4

4. A pharmaceutical preparation according to any one of claims 1 to 3 i wherein the physiologically active substance is a physiologically active peptide. 4.

5. A pharmaceutical preparation according to claim 4 wherein the physiologically active peptide is calcitonin gene-related peptide (CGRP). 44

6. A pharmaceutical preparation according to claim 4 wherein the physiologically active peptide is any one of maxadilans (MAXs). 4 S7. A pharmaceutical preparation carrying an effective amount of a physiologically active substance and capable of prolonged releasing the physiologically active substance, wherein the physiologically active substance is S"1\ one or more selected from the group consisting of CGRP and MAXs, and l i 36 7 wherein the physiologically active peptide is any one of' maxadilans (MM~s). A pharmaceu~tical preparation carrying an ef'fec- tive amount of' a physiologically active substance and capable of' prolonged releasing the physiologically active substance, wherein the physiologically active substance is one or, more selected from the group consisting of' CORP and MAXs, and the carrier of' the physiologically active substance comprises 10 to 90 by weight of' a cellulose ether derivative, 1 to 30 by weight of' a fat or oil or a wax, and 1 to 30 by weight of a f'atty acid, based on the total weight of' the pharmaceutical preparation. *fe11. A pharmaceutical preparation carrying an ef'fec- tive amouti of' a physiologically active substance and capable of prolonged releasing the physiologically active substance, wherein the physiologically active substance is one or more selected f'rom the group consisting of' CORP and MAXs, and the carrier of' the physiologically active substance comprises 10 to 90 by weight of' a cellulose ether derivative, and 1 to 40 by weight of a saacha- ride, based on the total weight of the pharmaceutical preparation.

12. A pharmaceutical preparation carrying an ef'feo- tive amount of' a physiologioa.ly active substance and capable of' prolonged releasing the physiologically active substance, wherein the physiologically active substance is one or more selected P'rom the group consisting of' CORP and MAXs) and the carrier of' the physiologically active substance is a combination of' 10 to 90 by weight of' orystalline cellulose and 0.1 to 10 by weight of a polyacrylate ester derivative with one or more selected 37 from the group consisting of 1 to 30 by weight of a fatty acid, 1 to 30 by weight of a fat or an oil and 1 to 30 by weight of a wax, based on the total weight of the pharmaceutical preparation.

13. A pharmaceutical preparation carrying an effec- tive amount of a physiologically active substance and capable of prolonged releasing the physiologically active substance, wherein the physiologically active substance is one or more selected from the group consisting of CGRP and MAXs, and the carrier of the physiologically active substance comprises about 50 by weight of hyaluronic acid and about 50 by weight of a cationic polyacrylic V**oa acid derivative, based on the total amount of the pharma- oeutical preparation.

14. A method for prophylaxis or treatment of cere- bral vasospasm which comprises administering an effective amount of at least one of MAXs into the body. A method for prophylaxis or treatment of cere- B bral vasospasm by use of a compound having a vasodilative l action as a physiologically active substance, which comprises a step of implanting a prolonged releasing pharmaceutical preparation carrying the compound into the brain.

16. A method for prophylaxis or treatment according to claim 15 wherein the compound is selected from the group consisting of CGRP, MAXs, deferoxamine, methyl- prednisolone, nicorandil, nicaraben, magnesium sulfate, actinomyoin D, 21-aminosteroid, isoproterenol, tPA, nimodipine, hydrocortisone, nicardipine, nifedipine, diltiazem, dilazp, teprothid, AA861, papaverine, OKY 1581, amyl nitrite, erythrityl tetranitrate, isosorbide dinitrate, nitroglycerin, pentaerythritol tetronitrate, VIP, vasopressin, bradykinin, PACAP, SOD, oatalase, bepridil, nadololol, felodipine, isradipine, varapamil, 38 atenolol, metoprolol and propanolol.

17. A method for prophylaxis or treatment according to claim 15 wherein the compound is selected from the group oonsisttng of CGRP and MAXs.

18. A method for prophylaxis or treatment according to claim 15 wherein the compound is CGRP.

19. A method for prophylaxis or treatment according to claim 15 wherein the compound is at least one of MAXs. A method for prophylaxis or treatment according to claim 15 wherein the compound is a maxadilan denoted by SEQ ID NO: 3.

21. A meth,.' for prophylaxis or treatment according *5 to claim 15 wherein the carrier of the prolonged releas- ing pharmaceutical preparation comprises a combination of a cellulosic polymer and at least one auxiliary component selected from fats and oils, waxes, fatty acids, saccha- rides and polyacrylate ester derivatives.

22. A method for prophylaxis or treatment according to claim 15 wherein the carrier of the pharmaceutical preparation comprises 10 to 90 by weight of a cellulose ether derivative, 1 to 30 by weight of a fat or oil or a wax, and 1 to 30 by weight of a fatty acid, based on the total weight of the pharmaceutical preparation.

23. A method for prophylaxis or treatment according #@see: to claim 15 wherein the carrier, of the pharmaceutical preparation comprises 10 to 90 by weight of a cellulose ether derivative and 1 to 40 by weight of a saceharide, based on the total weight of the pharmaceutical prepara- tion.

24. A method for prophylaxis or treatment according to claim 15 wherein the carrier of the pharmaceutical preparation comprises 10 to 90 by weight of crystalline cellulose and 0.01 to 10 by weight of a polyacrylate ester, based on the total weight of the pharmaceutical preparation. A method for prophylaxis or treatment according 39 to claim 15 wherein the carrier of the pharmaceutical preparation comprises about 50 by weight of hyaluronic acid and about 50 by weight of a cationic polyacrylic acid derivative, based on the total weight of the pharma- ceutical preparation.

26. A method for prophylaxis or treatment according to claim 15 wherein the carrier of the pharmaceutical preparation comprises 10 to 90 by weight of a cellulose ether derivative, 1 to 30 by weight of a fat or oil or a wax, and I to 30 by weight of a fatty acid, based on the total weight of the pharmaceutical preparation; and :the compound is selected from the group consisting of an effective amount of CORP and MAXs.

27. A method for prophylaxis or treatment according to claim 15 wherein the carrier of the pharmaceutical preparation comprises 10 to 90 by weight of a cellulose ether derivative and 1 to 4I0 by weight of a saceharlde, based on the total weight of the pharmaceutical prepara- 00. tion; and the compound is selected from the group con- sisting of an effective amount of CGRP and MAXs. S.28. A method for prophylaxis or treatment according to claim 15 wherein the carrier of the pharmaceutical preparation comprises 10 to 90 by weight of crystelline cellulose and 0.01 to 10 by weight of a polyacrylate ester, based on the total weight of the pharmaceutical preparation; and the compound is selected from the group consisting of an effective amount of CORP and MAXs.

29. A method for prophylaxis or treatment according to claim 15 wherein the carrier of the pharmaceutical preparation comprises about 50 by weight of hyaluronic acid and about 50 by weight o. a cationic polyacrylic acid derivative, based on the total weight of the pharma- ceutical preparation; and the compound is selected from the group consisting of an effective amount of CORP and MAXsa DATED.- 19th Soptember, 1994 PHILLIPS ORMOND PTZATRI0 "'CK AttOOyAN t i i ii l iiI Abstract of the Disclosure A prolonged releasing pharmaceutical prepara- tion is provided carrying a physiologically active sub- stance, particularly, caloitonin gene-re3ated peptide (CGRP) or a maxadilan (MAX), This pharmaceutical prepa- ration can attain the expected effects by incorpolating the physiologically active substance into a combination, as carriers for the physiologically active substance, of a cellulosic polymer and at least one auxiliary component selected from the group consisting of fats and oils, waxes, fatty acids, saccharides and polyacrylate ester derivatives. The pharmaceutical preparation can con- veniently be used, in living bodies, particularly as an intrathecal implantation-type preparation. *'Se 0, 0*. *0 *S COOO** 9 e