JP4530626B2 - Method for producing sustained-release composition - Google Patents

Description

（米国特許Ｎｏ．４，０２４，２４８、ドイツ特許第２４３８３５２号，特開昭和５１−４１３５９号）、
（２）トリプトレリン(Triptorelin)
【化２】

（米国特開第４０１０１２５号，特開昭５２−３１０７３号）、
（３）ナファレリン(Nafarelin)
【化３】

（米国特開第４２３４５７１号，特開昭５５−１６４６６３号，同昭６３−２６４４９８号，同昭６４−２５７９４号）、
（４）ヒストレリン(Histrelin)
【化４】

（５）デスロレリン(Deslorelin)
【化５】

（米国特開第４５６９９６７号，同４２１８４３９号）、
（６）メテレリン(Meterelin)
【化６】

（ＰＣＴ ＷＯ ９１/１８０１６）、
（７）ゴナドレリン(Gonadrelin)
【化７】

（ドイツ特許第２２１３７３７号）、
（８）ゴセレリン(Goserelin)
【化８】

（米国特開第４１００２７４号，特開昭５２−１３６１７２号）、
（９）レシレリン（Lecirelin）
【化９】

〔ベルギー特許第897455号、特開昭59-59654号〕など、またはそれらの塩などが用いられる。
【００１５】
本明細書中で使用される略号の意味は次のとおりである。
略号 名称
N(4H₂-furoyl)Gly： N-テトラヒドロフロイルグリシン残基
NAc ： N-アセチル基
D2Nal ： D-3-(2-ナフチル)アラニン残基
D4ClPhe ： D-3-(4-クロロ)フェニルアラニン残基
D3Pal ： D-3-(3-ピリジル)アラニン残基
NMeTyr ： N-メチルチロシン残基
Aph(Atz) ： N-[5'-(3'-アミノ-1'H-1',2',4'-トリアゾリル)]フェニルアラニン残基
NMeAph(Atz) ： N-メチル-[5'-(3'-アミノ-1'H-1',2',4'-トリアゾリル)]フェニルアラニン残基
DLys(Nic) ： D-(e-N-ニコチノイル)リシン残基
Dcit ： D-シトルリン残基
DLys(AzaglyNic) ： D-(アザグリシルニコチノイル)リシン残基
DLys(AzaglyFur) ： D-(アザグリシルフラニル)リシン残基
DhArg(Et₂) ： D-(N,N'-ジエチル)ホモアルギニン残基
DAph(Atz) ： D-N-[5'-(3'-アミノ-1'H-1',2',4'-トリアゾリル)]フェニルアラニン残基
DhCi ： D-ホモシトルリン残基
Lys(Nisp) ： (e-N-イソプロピル)リシン残基
hArg(Et₂) ： (N,N'-ジエチル)ホモアルギニン残基
その他アミノ酸に関し、略号で表示する場合、IUPAC-IUBコミッション・オブ・バイオケミカル・ノーメンクレーチュアー(Commission on Biochemical Nomenclature) （ヨーロピアン・ジャーナル・オブ・バイオケミストリー(European Journal of Biochemistry)第138巻、９〜３７頁（1984年）による略号または該当分野における慣用略号に基づくものとし、また、アミノ酸に関して光学異性体がありうる場合は、特に明示しなければＬ体を示すものとする。
【００１６】
本発明において、「生理活性物質および当該生理活性物質の約1.5倍モル以上の酸または塩基を含む水溶液」および「水溶液に対して約0.1〜約20重量%の酸または塩基」の「酸または塩基」としては一般に製造上用いられているものであれば特に限定されないが、無機酸、有機酸、無機塩基、有機塩基、酸性または塩基性アミノ酸等を用いることができる。好ましくは酸であり、より好ましくは有機酸である。
無機酸の好適な例としては、例えば塩酸、臭化水素酸、硝酸、硫酸、リン酸等が用いられる。有機酸の好適な例としては、例えばメタンスルホン酸、ベンゼンスルホン酸、ｐ−トルエンスルホン酸等のスルホン酸類；ギ酸、酢酸、プロピオン酸等の脂肪酸；シュウ酸、マロン酸、コハク酸などの脂肪族ジカルボン酸；アクリル酸、フマル酸、マレイン酸などの不飽和脂肪酸；フタル酸、イソフタル酸、テレフタル酸などの炭素環カルボン酸；その他、トリフルオロ酢酸、酒石酸、クエン酸、リンゴ酸、乳酸、グリコール酸等の置換カルボン酸が用いられ、より好適には脂肪酸、乳酸、グリコール酸が用いられ、特に好適には酢酸が用いられる。酸性アミノ酸の好適な例としては、例えばアスパラギン酸、グルタミン酸等が用いられ、塩基性アミノ酸の好適な例としては、例えばアルギニン、リジン、オルニチン等が用いられる。無機塩基の好適な例としては、例えばナトリウム、カリウム等のアルカリ金属；カルシウム、マグネシウム等のアルカリ土類金属；水素化リチウム、水素化カリウム、水素化ナトリウム等の水素化アルカリ金属類；酸化リチウム、水酸化カリウム、水酸化ナトリウム、水酸化カルシウム等の無機水酸化物；炭酸ナトリウム、炭酸カリウム、炭酸水素ナトリウム等の炭酸の塩等、並びにアルミニウム、アンモニウム等が用いられる。有機塩基の好適な例としては、例えばリチウムエトキシド、リチウム−tert−ブトキシド、ナトリウムメトキシド、ナトリウムエトキシド、カリウム−tert−ブトキシド等の炭素数１ないし６の金属アルコキシド類；カリウムフェノキシド、ナトリウムフェノキシド等の金属フェノキシド類；酢酸ナトリウム、酢酸カリウム等の酢酸の塩；n-ブチルリチウム、t-ブチルリチウム、ジエチルアミノリチウム等の有機リチウム塩；フェニルヒドラジン、p-トリルヒドラジン等のヒドラジン類；アミジン類；水酸化第四アンモニウム類；スルホニウム塩基類；トリメチルアミン、トリエチルアミン、ピリジン、ピコリン、２，６−ルチジン、エタノールアミン、ジエタノールアミン、トリエタノールアミン、シクロヘキシルアミン、ジシクロヘキシルアミン、Ｎ，Ｎ’−ジベンジルエチレンジアミン等のアミン類等を用いることができる。
酸が「有機酸」である場合、そのpKaは特に限定されないが、例えば、好ましくは約0.1〜約6.0であり、より好ましくは約1.0〜約6.0であり、さらに好ましくは約3.5〜約6.0である。
【００１７】
本発明において、当該「酸と塩基」は、生理活性物質と独立して酸または塩基を水溶液に含有せしめてもよいし、または生理活性物質の酸または塩基との塩の形で水溶液に含有せしめてもよく、さらに生理活性物質の酸または塩基との塩および、それとは独立した酸または塩基を水溶液に含有せしめてもよい。
【００１８】
このように本発明で用いられる生理活性物質はそれ自身であっても、薬理学的に許容される塩であってもよい。
このような塩としては、該生理活性物質がアミノ基等の塩基性基を有する場合、無機酸（無機の遊離酸とも称する）（例、炭酸、重炭酸、塩酸、硫酸、硝酸、ホウ酸等）、有機酸（有機の遊離酸とも称する）（例、コハク酸、酢酸、プロピオン酸、トリフルオロ酢酸等）など酸との塩が用いられる。該生理活性物質がＬＨＲＨ誘導体の場合、特に酢酸を添加することが好ましく、たとえば酢酸リュープロレリンなどが好ましい。
生理活性物質がカルボキシル基等の酸性基を有する場合、無機塩基（無機の遊離塩基とも称する）（例、ナトリウム、カリウム等のアルカリ金属、カルシウム、マグネシウム等のアルカリ土類金属など）や有機塩基（有機の遊離塩基とも称する）（例、トリエチルアミン等の有機アミン類、アルギニン等の塩基性アミノ酸類等）など塩基との塩が用いられる。また、生理活性ペプチドは金属錯体化合物（例、銅錯体、亜鉛錯体等）を形成していてもよい。
これらの化合物またはその塩は、前記文献あるいは公報記載の方法あるいはこれに準じる方法で製造することができる。
【００１９】
ここで「生理活性物質」に対する「酸または塩基」の使用量としては、生理活性物質１モルに対して、約1.5モル倍以上使用することができ、好ましくは約1.5倍モル以上約5倍モル以下であり、より好ましくは約1.65倍モル以上約3倍モル以下である。
また、本発明において「水溶液」に対する「酸または塩基」の使用量（重量％）としては、本願発明の効果を発揮すればよく特に限定されないが、水溶液に対して約0.1〜約20％が好ましく、より好ましくは約１〜約15％、さらに好ましくは約３〜約10％である。
前記で述べたように、この「酸または塩基」は、水溶液に外部から独立して含有せしめてもよいし、生理活性物質の塩の形で水溶液に含有せしめてもよく、さらに外部から独立して含有せしめる方法と生理活性物質の塩の形で水溶液に含有せしめる方法を組み合わせてもよい。
【００２０】
本発明における生体内分解性ポリマーとしては、例えば、生体内分解性の高分子重合物が好ましく、例えば脂肪族ポリエステル〔例えばα−ヒドロキシ酸類（例、グリコール酸、乳酸、２−ヒドロキシ酪酸、２−ヒドロキシ吉草酸、２−ヒドロキシ−３−メチル酪酸、２−ヒドロキシカプロン酸、２−ヒドロキシイソカプロン酸、２−ヒドロキシカプリル酸等）、α−ヒドロキシ酸の環状二量体類（例、グリコリド、ラクチド等）、ヒドロキシジカルボン酸類（例、リンゴ酸）、ヒドロキシトリカルボン酸（例、クエン酸）等の単独重合体（例、乳酸重合体等）または２種以上の共重合体（例えば、乳酸／グリコール酸共重合体，２−ヒドロキシ酪酸／グリコール酸共重合体等）、あるいはこれら単独重合体および／または共重合体の混合物（例、乳酸重合体と２−ヒドロキシ酪酸／グリコール酸共重合体との混合物等）〕、ポリ−α−シアノアクリル酸エステル、ポリアミノ酸（例、ポリ−γ−ベンジル−Ｌ−グルタミン酸，ポリ−Ｌ−アラニン，ポリ−γ−メチル−Ｌ−グルタミン酸等）、無水マレイン酸系共重合体（例、スチレン／マレイン酸共重合体等）等が用いられる。これらの中で脂肪族ポリエステル、ポリ−α−シアノアクリル酸エステルが好ましい。さらに、脂肪族ポリエステルが特に好ましい。脂肪族ポリエステルの中でα−ヒドロキシモノカルボン酸類（例、グリコール酸、乳酸等）、α−ヒドロキシジカルボン酸類（例、リンゴ酸）、α−ヒドロキシトリカルボン酸（例、クエン酸）等のα−ヒドロキシカルボン酸類の1種以上から合成され、遊離の末端カルボキシル基を有する重合体、共重合体、またはこれらの混合物；ポリ（α−シアノアクリル酸エステル）；ポリアミノ酸（例、ポリ（γ−ベンジル−L−グルタミン酸）等）；無水マレイン酸系共重合体（例、スチレン−マレイン酸共重合体等）などが用いられる。
モノマーの結合様式としては、ランダム、ブロック、グラフトのいずれでもよい。また、前記α−ヒドロキシモノカルボン酸類、α−ヒドロキシジカルボン酸類、α−ヒドロキシトリカルボン酸類が分子内に光学活性中心を有する場合、Ｄ−、Ｌ−、ＤＬ−体のいずれを用いてもよい。これらの中でも、乳酸−グリコール酸重合体（以下、ポリ（ラクチド−ｃｏ−グリコリド）、ポリ（乳酸−ｃｏ−グリコール酸）あるいは乳酸−グリコール酸共重合体と称することもあり、特に明示しない限り、乳酸、グリコール酸のホモポリマー（重合体）及びコポリマー（共重合体）を総称する。また乳酸ホモポリマーは乳酸重合体、ポリ乳酸、ポリラクチドなどと、またグリコール酸ホモポリマーはグリコール酸重合体、ポリグリコール酸、ポリグリコリドなどと称される場合がある）、ポリ（α−シアノアクリル酸エステル）などが好ましい。さらに好ましくは、乳酸−グリコール酸重合体であり、より好ましくは、遊離の末端カルボキシル基を有する乳酸−グリコール酸重合体である。
生体内分解性ポリマーは塩であってもよい。塩としては、例えば、無機塩基（例、ナトリウム、カリウム等のアルカリ金属、カルシウム、マグネシウム等のアルカリ土類金属など）や有機塩基（例、トリエチルアミン等の有機アミン類、アルギニン等の塩基性アミノ酸類等）などとの塩、または遷移金属（例，亜鉛，鉄，銅など）との塩および錯塩などが用いられる。
生体内分解性ポリマーとして乳酸−グリコール酸重合体を用いる場合、その組成比（モル％）は１００／０〜約４０／６０が好ましく、１００／０〜約５０／５０がより好ましい。また、２カ月以上にわたって生理活性物質を放出する長期徐放型マイクロカプセルの場合、組成比が１００／０である乳酸重合体も好ましく用いられる。
該「乳酸−グリコール酸重合体」の最小繰り返し単位の一つである乳酸の光学異性体比は、Ｄ−体／Ｌ−体（モル／モル％）が約７５／２５〜約２５／７５の範囲のものが好ましい。このＤ−体／Ｌ−体（モル／モル％）は、特に約６０／４０〜約３０／７０の範囲のものが汎用される。
該「乳酸−グリコール酸重合体」の重量平均分子量は、通常、約３０００〜約１０００００、好ましくは約５０００〜約５００００、より好ましくは約８０００〜約３００００、さらに好ましくは約１７０００〜約３００００のものが用いられる。
また、分散度（重量平均分子量／数平均分子量）は、通常約１．１〜約４．０が好ましく、さらには約１．２〜約３．５が好ましい。なお、本明細書で用いられる重量平均分子量および分散度は、ゲル浸透クロマトグラフィー（ＧＰＣ）で測定した値を意味する。重量平均分子量及び各重合体含有量は、単分散ポリスチレンを基準物質としてゲルパーミエーションクロマトグラフィー（ＧＰＣ）で測定したポリスチレン換算の重量平均分子量及びそれらから算出した各重合体含有量である。また、測定は全て高速ＧＰＣ装置（東ソー(株)製；ＨＬＣ−８１２０ＧＰＣ）で行い、カラムはSuperH4000×２及びSuperH2000(何れも東ソー(株)製)を使用し、移動相としてテトラヒドロフランを流速0.6mL/minで使用した。尚、検出方法は示差屈折率によるものである。
該「乳酸−グリコール酸重合体」の遊離の末端カルボキシル基量は、重合体の単位質量（グラム）あたり通常約２０〜約１０００μmol（マイクロモル）が好ましく、さらには約４０〜約３００μmol（マイクロモル）が特に好ましい。
本発明において、「生理活性物質」に対する生体内分解性ポリマーの遊離の末端カルボキシル基量は約0.1〜約５倍モルが好ましく、より好ましくは約0.2〜約４倍モル、さらに好ましくは約0.3〜約3.5倍モルである。
【００２１】
前記の遊離の末端カルボキシル基量とはラベル化法により求めたもの（以下、「ラベル化法によるカルボキシル基量」と称する）をいう。具体的にポリ乳酸の場合について述べると、ポリ乳酸 Ｗｍｇを５Ｎ塩酸／アセトニトリル（ｖ／ｖ＝４／９６）混液２ｍｌに溶解し、０．０１Ｍ ｏ−ニトロフェニルヒドラジン塩酸塩（ＯＮＰＨ）溶液（５Ｎ塩酸／アセトニトリル／エタノール＝１．０２／３５／１５）２ｍｌと０．１５Ｍ １−エチル−３−（３−ジメチルアミノプロピル）−カルボジイミド塩酸塩溶液（ピリジン／エタノール＝４ｖ／９６ｖ）２ｍｌを加えて４０℃で３０分反応させた後溶媒を留去する。残滓を水洗（４回）した後、アセトニトリル２ｍｌで溶解し、０．５ｍｏｌ／ｌのエタノール性水酸化カリウム溶液１ｍｌを加えて６０℃で３０分反応させる。反応液を１．５Ｎ水酸化ナトリウム水溶液で希釈してＹｍｌとし、１．５Ｎ水酸化ナトリウム水溶液を対象として５４４ｎｍ吸光度Ａ（／ｃｍ）を測定する。一方、ＤＬ−乳酸水溶液を基準物質として、その遊離カルボキシル基量 Ｃｍｏｌ／Ｌをアルカリ滴定で求め、またＯＮＰＨラベル化法でＤＬ−乳酸ヒドラジドとしたときの５４４ｎｍ吸光度を Ｂ（／ｃｍ）とするとき、重合体の単位質量（グラム）あたりの遊離の末端カルボキシル基のモル量は以下の数式で求められる。
［ＣＯＯＨ］（ｍｏｌ／ｇ）＝（ＡＹＣ）／（ＷＢ）
また、該「カルボキシル基量」は生体内分解性ポリマーをトルエン−アセトン−メタノール混合溶媒に溶解し、フェノールフタレインを指示薬としてこの溶液をアルコール性水酸化カリウム溶液でカルボキシル基を滴定して求めることもできる（以下、この方法によって求めた値を「アルカリ滴定法によるカルボキシル基量」と称する）が、滴定中にポリエステル主鎖の加水分解反応を競合する結果、滴定終点が不明確になる可能性があり前記ラベル化法で定量するのが望ましい。
該「乳酸−グリコール酸重合体」は、例えば、乳酸とグリコール酸からの無触媒脱水重縮合（特開昭６１−２８５２１号）あるいはラクチドとグリコリド等の環状ジエステル化合物からの触媒を用いた開環重合（Encyclopedic Handbook of Biomaterials and Bioengineering Part A: Materials, Volume 2, Marcel Dekker, Inc. 1995年）で製造できる。前記の公知の開環重合方法によって得られる重合体は、末端に遊離のカルボキシル基を有しているとは限らないが、例えば、ＥＰ−Ａ−０８３９５２５号に記載の加水分解反応に付すことにより、単位質量当たりにある程度のカルボキシル基量を有する重合体に改変することができ、これを用いることもできる。
前記の「遊離の末端カルボキシル基を有する乳酸−グリコール酸重合体」は公知の製造法（例えば無触媒脱水重縮合法、特開昭６１−２８５２１号公報参照）と同様の方法またはそれに準じた方法により製造できる。
なかでも、本発明に用いられる高分子重合物としては乳酸重合体（以下、本発明の乳酸重合体と略記する場合がある）が好ましく、例えば、乳酸のみから成る重合体、或いは乳酸とその他のモノマー（例えばグリコール酸等）との共重合体を含み、特に限定されないが、通常重量平均分子量５０００以下の重合体含有量が約１０重量％以下、好ましくは重量平均分子量５０００以下の重合体含有量が約５重量％以下のものが用いられる。
また、本発明の乳酸重合体の重量平均分子量は通常１５０００〜５００００、好ましくは１５０００〜４００００、より好ましくは１７０００〜３００００である。
【００２２】
本発明の乳酸重合体の原料となる高分子量の乳酸重合体は、市販品でも公知の方法で重合したものでもよく、その重量平均分子量は通常１５０００〜５０００００、好ましくは２００００〜１０００００である。公知の重合方法としては、例えば、乳酸及び要すればグリコール酸とを縮合重合させる方法、例えばラクチドを、要すればグリコリドと共に、例えばジエチル亜鉛、トリエチルアルミニウム、オクチル酸スズ等のルイス酸又は金属塩等の触媒を用いて開環重合させる方法、前記方法に更にカルボキシル基が保護されたヒドロキシカルボン酸誘導体を存在させてラクチドを開環重合させる方法（例えば特許国際公開WO00/35990等）、その他ラクチドに加熱下で触媒を添加して開環重合させる方法（例えばJ. Med. Chem, 16, 897(1973)等）、例えばラクチドとグリコリドとを共重合させる方法等が用いられる。
重合形態としては、ラクチド等を溶融させて重合反応に付すバルク重合、ラクチド等を適当な溶媒に溶解して重合反応に付す溶液重合が用いられるが、中でも溶液重合によって得られる重合体を本発明の乳酸重合体の原料として使用することが工業生産上好ましい。
溶液重合においてラクチドを溶解する溶媒としては、例えばベンゼン，トルエン，キシレン等の芳香族炭化水素類、デカリン、ジメチルホルムアミド等が用いられる。
【００２３】
前記の如くして得られた高分子量の乳酸重合体を加水分解するには、自体公知の加水分解方法が用いられ、例えば該高分子量の乳酸重合体を適当な溶媒に溶解した後、水及び要すれば酸を加えて反応させればよい。
高分子量の乳酸重合体を溶解する溶媒としては、具体的には、例えばクロロホルム，ジクロロメタン等のハロゲン化炭化水素、例えばトルエン，ｏ−キシレン，ｍ−キシレン，ｐ−キシレン等の芳香族炭化水素、例えばテトラヒドロフラン等の環状エーテル、アセトン、Ｎ，Ｎ−ジメチルホルムアミド等が用いられる。尚、高分子量の乳酸重合体の重合時に、高分子量の乳酸重合体の加水分解で使用できる溶媒を用いた場合には、重合した高分子量の乳酸重合体を単離せず、重合及び加水分解の操作を連続して行うことができる。
高分子量乳酸重合体を溶解する溶媒の使用量は、溶質である乳酸重合体に対して通常約０．１〜約１００倍、好ましくは約１〜約１０倍である。
添加する水の量は、高分子量乳酸重合体に対して通常約０．００１〜約１倍重量、好ましくは約０．０１〜約０．１倍重量である。
必要に応じて添加する酸としては、例えば塩酸，硫酸，硝酸等の無機酸、例えば乳酸，酢酸，トリフルオロ酢酸等の有機酸等が挙げられ、好ましくはトリフルオロ酢酸が用いられる。
添加する酸の量は、高分子量乳酸重合体に対して通常０〜約１０倍重量、好ましくは約０．１〜約１倍重量である
加水分解反応温度は、通常約０〜約１５０℃、好ましくは約２０〜約８０℃である。
加水分解反応時間は、高分子量の乳酸重合体の重量平均分子量及び反応温度によっても異なり、通常約１０分〜約１００時間、好ましくは約１〜約２０時間である。
加水分解処理の終了時期は、加水分解生成物の重量平均分子量に基づいて判断する。即ち、加水分解処理中に適宜サンプリングを行い、サンプル中の加水分解生成物の重量平均分子量をゲルパーミエーションクロマトグラフィー（ＧＰＣ）により測定し、当該分子量が約１５０００〜約５００００、好ましくは約１５０００〜約４００００、より好ましくは約１７０００〜約３００００となっていることが確認できたら加水分解処理を停止させる。
【００２４】
前記の如く高分子量の乳酸重合体を加水分解する操作に付すことにより得られる、加水分解生成物を含有する溶液から、そこに含有される目的の乳酸重合体を析出させる方法としては、該加水分解生成物含有溶液を、そこに含有される目的の乳酸重合体を析出させ得る溶媒と接触させる方法等が用いられる。
加水分解生成物含有溶液の好ましい態様としては、例えばクロロホルム，ジクロロメタン等のハロゲン化炭化水素、例えばトルエン，ｏ−キシレン，ｍ−キシレン，ｐ−キシレン等の芳香族炭化水素、例えばテトラヒドロフラン等の環状エーテル、アセトン、Ｎ，Ｎ−ジメチルホルムアミド等ジクロロメタンやキシレン等の高分子量乳酸重合体を溶解する溶媒に、重量平均分子量１５０００〜５００００、好ましくは１５０００〜４００００、より好ましくは１７０００〜３００００の乳酸重合体が約１０〜約５０ｗｔ％溶解しているもの等が用いられる。
加水分解生成物含有溶液中に含有される目的の乳酸重合体を析出させ得る溶媒としては、例えばメタノール，エタノール等のアルコール類、例えばイソプロピルエーテル等の鎖状エーテル類、例えばヘキサン等の脂肪族炭化水素、水等が用いられる。
【００２５】
目的とする乳酸重合体を析出させ得る溶媒の使用量は、加水分解生成物含有溶液の溶媒に対して通常約０．１〜約１００倍重量、好ましくは約１〜約１０倍重量である。
この様な各溶媒の種類と使用量の組み合わせの好ましい具体例としては、例えば溶質の約１〜約５倍重量のジクロロメタンを溶媒として用いられている加水分解生成物含有溶液に、溶解度を低下させる溶媒としてイソプロピルエーテルを、該ジクロロメタンに対して約２〜約１０倍重量使用する態様等が用いられる。
目的の乳酸重合体溶質を析出させ得る溶媒を加水分解生成物含有溶液に接触させる際の、溶媒の温度は、通常約−２０〜約６０℃、好ましくは約０〜約４０℃であり、加水分解生成物含有溶液の温度は通常約０〜約４０℃、好ましくは約１０〜約３０℃である。
溶媒と加水分解生成物含有溶液とを接触させる方法としては、加水分解生成物含有溶液を溶媒中に一度に加える方法、加水分解生成物含有溶液を溶媒中に滴下する方法、溶媒を加水分解生成物含有溶液中に一度に加える方法、或いは溶媒を加水分解生成物含有溶液中に滴下する方法等が用いられる。
前記のようにして得られた本発明の乳酸重合体は、末端カルボキシル基量が徐放性製剤用基材として好ましい範囲にあるため、徐放性製剤用基材として好ましいものである。さらに、生体適合性の高分子重合物としては、例えば、ポリスチレン、ポリメタアクリル酸、アクリル酸とメタアクリル酸との共重合体、ポリアミノ酸、デキストランステアレート、エチルセルロース、アセチルセルロース、ニトロセルロース、無水マレイン酸系共重合物、エチレンビニルアセテート系共重合物、ポリビニルアセテート、ポリアクリルアミドなどが用いられる。
これらの高分子重合物は１種でもよく、また２種以上の共重合物あるいは単なる混合物でもよく、またその塩でもよい。
【００２６】
以下に、本発明の生理活性物質またはその塩および本発明の乳酸重合体またはその塩を含有する徐放性組成物（例えば、マイクロカプセル）の製造法を例示する。
以下の製造工程中、必要に応じて、以下のものを自体公知の方法により添加してもよい。
（１）薬物保持剤
アルブミン、ゼラチン、クエン酸、サリチル酸、エチレンジアミン四酢酸ナトリウム、デキストリン、亜硫酸水素ナトリウム、ポリエチレングリコールなどのポリオール化合物、寒天、アルギン酸、ポリビニルアルコール、塩基性アミノ酸など。
（２）生理活性物質またはその塩の安定性、溶解性を保つためのｐＨ調整剤
炭酸、酢酸、シュウ酸、クエン酸、リン酸、塩酸、水酸化ナトリウム、アルギニン、リジンおよびそれらの塩など。
（３）生理活性物質またはその塩の安定化剤
アルブミン、ゼラチン、クエン酸、エチレンジアミン四酢酸ナトリウム、デキストリン、亜硫酸水素ナトリウム、ポリエチレングリコールなどのポリオール化合物など。
（４）保存剤
パラオキシ安息香酸エステル類(メチルパラベン、プロピルパラベンなど)、ベンジルアルコール、クロロブタノール、チメロサールなど
【００２７】
（Ｉ）Ｗ／Ｏ／Ｗ法
本方法においては、まず高分子重合物である生体内分解性ポリマーの溶解液（好ましくは有機溶媒溶液であり、より好ましくは水難溶性の有機溶媒である）を作製する。本発明の徐放性組成物（微粒子（マイクロパーティクル）の形態であることが好ましく、より好ましくはマイクロスフェアあるいはマイクロカプセルである）の製造の際に使用する溶解液は水難溶性の有機溶媒であり、沸点が約１２０℃以下であることが好ましい。
該有機溶媒としては、例えば、ハロゲン化炭化水素（例、ジクロロメタン、クロロホルム、ジクロロエタン、トリクロロエタン、四塩化炭素等）、エーテル類（例、エチルエーテル、イソプロピルエーテル等）、脂肪酸エステル(例、酢酸エチル、酢酸ブチル等）、芳香族炭化水素（例、ベンゼン、トルエン、キシレン等）、アルコール類（例えば、エタノール、メタノール等）、アセトニトリルなどが用いられる。なかでもハロゲン化炭化水素が好ましく、特にジクロロメタンが好適である。また、これらは適宜の割合で混合して用いてもよい。その場合は、ハロゲン化炭化水素とアルコール類との混液が好ましく、特にジクロロメタンとエタノールとの混液が好適である。
生体内分解性ポリマーの溶解液中の濃度は、生体内分解性ポリマーの分子量、溶解液の種類によって異なるが、例えば、ジクロロメタンを溶解液として用いた場合、一般的には約０.５〜約７０重量％、より好ましくは約１〜約６０重量％、特に好ましくは約２〜約５０重量％から選ばれる。
また、ジクロロメタンとの混有機溶媒としてエタノールを用いた場合の両者の比率は，一般的には約０.０１〜約５０％(v/v)、より好ましくは約０.０５〜約４０％(v/v)、特に好ましくは約０.１〜約３０％(v/v)から選ばれる。
次に生理活性物質は、既に記載したように当該生理活性物質の約1.5倍モル以上の酸または塩基を含む水溶液あるいは約0.1〜約20重量％の濃度の酸または塩基の水溶液に溶解する。生理活性物質は適当な酸あるいは塩基との塩として溶解する場合もあり、さらにはその水溶液が水、水とアルコール類（例、メタノール、エタノール等）などとの混液を溶媒として用いることもある。
生理活性物質またはその塩の添加濃度は一般的には約０．００１ｍｇ／ｍｌ〜約１０ｇ／ｍｌ、より好ましくは約０．１ｍｇ／ｍｌ〜約５ｇ／ｍｌで更に好ましくは約１０ｍｇ／ｍｌ〜約３ｇ／ｍｌである。
溶解補助剤、安定化剤として公知のものを用いてもよい。生理活性物質や添加剤の溶解あるいは分散には活性が失われない程度に加熱、振とう、撹拌などを行ってもよく、そうして得られた水溶液を内水相と称する。
前記により得られた内水相と油相とをホモジナイザーまたは超音波等の公知の方法で乳化し、W/Oエマルションを形成させる。
ここで混合する生体内分解性ポリマーと生理活性物質の混合比率として、生体内分解性ポリマーの単位質量（グラム）あたりの末端カルボキシル基量が生理活性物質に対して通常約0.01〜約１０倍モル、好ましくは約0.1〜約５倍モルである。
混合する油相の重量は内水相の重量に対し、約１〜約１０００倍、好ましくは約２〜約１００倍、より好ましくは約３〜約３０倍である。
得られたW/Oエマルションの粘度範囲は一般的には約１２〜約２５℃で、約１０〜約１０，０００ｃｐで、好ましくは約１００〜約５，０００ｃｐであり、より好ましくは約２００〜約３，０００ｃｐであり、さらに好ましくは約３００〜約２,０００ｃｐである。
本発明方法を工業的に実施する場合、W/Oエマルションの粘度範囲は一般的には約１２〜約２５℃で、約３，０００ｃｐ以下が好ましく、より好ましくは約２,０００ｃｐ以下であり、さらに好ましくは、約３００〜約２,０００ｃｐである。
次いで、得られたW/Oエマルションを水相中に加え、W（内水相）/O（油相）/ W（外水相）エマルションを形成させた後、油相中の溶媒を揮散ないしは外水相中に拡散させ、マイクロカプセルを調製する。この際の外水相重量は一般的には油相重量の約１倍〜約１０，０００倍、より好ましくは約５倍〜約５，０００倍、さらに好ましくは約１０倍〜約２，０００倍、特に好ましくは約２０倍〜約５００倍から選ばれる。
【００２８】
前記の外水相中には乳化剤を加えてもよい。該乳化剤は、一般に安定なW/O/Wエマルションを形成できるものであればいずれでもよい。具体的には、例えば、アニオン性界面活性剤（オレイン酸ナトリウム、ステアリン酸ナトリウム、ラウリル硫酸ナトリウムなど）、非イオン性界面活性剤（ポリオキシエチレンソルビタン脂肪酸エステル〔ツイーン(Tween)80、ツイーン(Tween)60、アトラスパウダー社〕、ポリオキシエチレンヒマシ油誘導体〔HCO−60、HCO−50、日光ケミカルズ〕など）、ポリビニルピロリドン、ポリビニルアルコール、カルボキシメチルセルロース、レシチン、ゼラチン、ヒアルロン酸などが用いられる。これらの中の１種類か、いくつかを組み合わせて使用してもよい。使用の際の濃度は、好ましくは約０．０１〜約１０重量％の範囲で、さらに好ましくは約０．０５〜約５重量％の範囲で用いられる。
前記の外水相中には浸透圧調節剤を加えてもよい。該浸透圧調節剤としては、水溶液とした場合に浸透圧を示すものであればよい。
該浸透圧調節剤としては、例えば、多価アルコール類、一価アルコール類、単糖類、二糖類、オリゴ糖およびアミノ酸類またはそれらの誘導体などが用いられる。
前記の多価アルコール類としては、例えば、グリセリン等の三価アルコール類、アラビトール，キシリトール，アドニトール等の五価アルコール類、マンニトール，ソルビトール，ズルシトール等の六価アルコール類などが用いられる。なかでも、六価アルコール類が好ましく、特にマンニトールが好適である。
前記の一価アルコール類としては、例えば、メタノール、エタノール、イソプロピルアルコールなどが挙げられ、このうちエタノールが好ましい。
前記の単糖類としては、例えば、アラビノース，キシロース，リボース，２−デオキシリボース等の五炭糖類、ブドウ糖，果糖，ガラクトース，マンオース，ソルボース，ラムノース，フコース等の六炭糖類が用いられ、このうち六炭糖類が好ましい。
前記のオリゴ糖としては、例えば、マルトトリオース，ラフィノース糖等の三糖類、スタキオース等の四糖類などが用いられ、このうち三糖類が好ましい。
前記の単糖類、二糖類およびオリゴ糖の誘導体としては、例えば、グルコサミン、ガラクトサミン、グルクロン酸、ガラクツロン酸などが用いられる。
前記のアミノ酸類としては、Ｌ−体のものであればいずれも用いることができ、例えば、グリシン、ロイシン、アルギニンなどが用いられる。このうちＬ−アルギニンが好ましい。
これらの浸透圧調節剤は単独で使用しても、混合して使用してもよい。これらの浸透圧調節剤は、外水相の浸透圧が生理食塩水の浸透圧の約１／５０〜約５倍、好ましくは約１／２５〜約３倍となる濃度で用いられる。浸透圧調節剤としてマンニトールを用いた場合、約０．５％〜約１．５％の濃度が好ましい。
有機溶媒を除去する方法としては、自体公知の方法あるいはそれに準じる方法が用いられる。例えば、プロペラ型撹拌機またはマグネチックスターラーなどで撹拌しながら常圧もしくは徐々に減圧にして有機溶媒を蒸発させる方法、ロータリーエヴァポレーターなどを用いて真空度を調節しながら有機溶媒を蒸発させる方法などが用いられる。
【００２９】
このようにして得られたマイクロカプセルは遠心分離または濾過して分取した後、マイクロカプセルの表面に付着している遊離の生理活性物質、乳化剤、浸透圧調節剤などを蒸留水で数回繰り返し洗浄し、再び蒸留水などに分散して凍結乾燥する。
製造工程中、粒子同士の凝集を防ぐために凝集防止剤を加えてもよい。該凝集防止剤としては、例えば、マンニトール，ラクトース，ブドウ糖，デンプン類（例、コーンスターチ等）などの水溶性多糖、グリシンなどのアミノ酸、フィブリン，コラーゲンなどのタンパク質などが用いられる。なかでも、マンニトールが好適である。
マンニトール等の凝集防止剤の添加量は、マイクロカプセル全体に対して、通常０〜約２４重量％である。
また、凍結乾燥後、必要であれば、減圧下マイクロカプセル同士が融着しない条件下で加温してマイクロカプセル中の水分および有機溶媒の除去を行ってもよい。好ましくは、毎分約１０〜約２０℃の昇温速度の条件下、示差走査熱量計で求めたマイクロカプセルの中間点ガラス転移温度付近あるいは若干高い温度で加温する。より好ましくはマイクロカプセルの中間点ガラス転移温度付近あるいはこれより約３０℃高い温度範囲内で加温する。とりわけ，生体内分解性ポリマーとして乳酸-グリコール酸重合体を用いる場合には好ましくはマイクロカプセルの中間点ガラス転移温度付近から中間点ガラス転移温度より約１０℃高い温度範囲，さらに好ましくは、中間点ガラス転移温度付近から中間点ガラス転移温度より約５℃高い温度範囲で加温する。
加温時間はマイクロカプセルの量などによって異なるものの、一般的にはマイクロカプセル自体が所定の温度に達した後、約１２時間〜約１６８時間、好ましくは約２４時間〜約１２０時間、特に好ましくは約４８時間〜約９６時間である。
加温方法は、マイクロカプセルの集合が均一に加温できる方法であれば特に限定されない。
該加温乾燥方法としては、例えば、恒温槽、流動槽、移動槽またはキルン中で加温乾燥する方法、マイクロ波で加温乾燥する方法などが用いられる。なかでも恒温槽中で加温乾燥する方法が好ましい。
（II）相分離法
本法によってマイクロカプセルを製造する場合には，前記（Ｉ）の水中乾燥法に記載した生理活性物質及び本発明の生体内分解性ポリマーから成る組成物を含むW/O乳化液にコアセルベーション剤を撹拌下徐々に加えてマイクロカプセルを析出，固化させる。該コアセルベーション剤は油相体積の約０．０１〜約１，０００倍、好ましくは約０．０５〜約５００倍、特に好ましくは約０．１〜約２００倍から選ばれる。
コアセルベーション剤としては、有機溶媒と混和する高分子系，鉱物油系または植物油系の化合物等で本発明の生体内分解性ポリマーを溶解しないものであれば特に限定はされない。具体的には、例えば、シリコン油，ゴマ油，大豆油，コーン油，綿実油，ココナッツ油，アマニ油，鉱物油，n-ヘキサン，n-ヘプタンなどが用いられる。これらは２種類以上混合して使用してもよい。
このようにして得られたマイクロカプセルを分取した後、ヘプタン等で繰り返し洗浄して生理活性物質及び本発明の生体内分解性ポリマーからなる組成物以外のコアセルベーション剤等を除去し、減圧乾燥する。もしくは、前記（Ｉ）（ｉ）の水中乾燥法で記載と同様の方法で洗浄を行った後に凍結乾燥、さらには加温乾燥する。
【００３０】
（III）噴霧乾燥法
本法によってマイクロカプセルを製造する場合には，前記（Ｉ）の水中乾燥法に記載した生理活性物質及び本発明の生体内分解性ポリマーから成る組成物を含有するW/O乳化液をノズルを用いてスプレードライヤー（噴霧乾燥器）の乾燥室内に噴霧し、極めて短時間内に微粒化液滴内の有機溶媒を揮発させ、マイクロカプセルを調製する。該ノズルとしては、例えば、二流体ノズル型，圧力ノズル型，回転ディスク型等がある。この後、必要であれば、前記（Ｉ）の水中乾燥法で記載と同様の方法で洗浄を行った後に凍結乾燥、さらには加温乾燥してもよい。
上述のマイクロカプセル以外の剤形としてマイクロカプセルの製造法（Ｉ）の水中乾燥法に記載した生理活性物質及び本発明の生体内分解性ポリマーから成る組成物を含む有機溶媒溶液または分散液を、例えば、ロータリーエヴァポレーターなどを用いて真空度を調節しながら有機溶媒および水を蒸発させて乾固した後、ジェットミルなどで粉砕して微粒子（マイクロパーティクル）としてもよい。
さらには、粉砕した微粒子をマイクロカプセルの製造法（Ｉ）の水中乾燥法で記載と同様の方法で洗浄を行った後に凍結乾燥、さらには加温乾燥してもよい。
【００３１】
本発明において、生理活性物質に対して約1.5〜約５倍モルの酸または塩基を使用することが好ましい。
生理活性物質が一般式
5-oxo-Pro-His-Trp-Ser-Tyr-Y-Leu-Arg-Pro-Z
〔式中、各記号は前記と同意義を示す。〕で表される化合物である場合、当該物質に対して約1.5〜約５倍モルの酸を使用することが好ましい。当該酸としては有機酸が好ましく、特に酢酸などが用いられる。
生体内分解性ポリマーとしては乳酸−グリコール酸重合体が好ましく、その組成モル比は１００対０〜約５０対５０のものが好ましく、特に１００対０の重合体も好適に用いられ、重量平均分子量５０００以下の重合体含有量が約５重量％以下である、重量平均分子量１５０００〜５００００の乳酸重合体が好ましい。
生体内分解性ポリマーの溶解液としては、水難溶性の有機溶媒が好ましく、特にジクロロメタンが好ましい。
本発明において、１）生理活性物質と酸または塩基を含む水溶液と、２）生体内分解性ポリマーの溶解液とを混合した混合液は均一に混合されていることが好ましく、この混合液はエマルションであることが好ましい。
さらに好ましくはエマルションがW/O型であり、そのエマルションサイズが微細なものである。
混合液の乾燥は水中乾燥法で行うことが好ましく、特に水中乾燥の外水相に浸透圧調節剤を用いることが好ましい。
浸透圧調節剤としてはマンニトールが好ましい。
【００３２】
本発明の徐放性組成物は、そのまままたはこれらを原料物質として種々の剤形に製剤化し、筋肉内、皮下、臓器などへの注射剤または埋め込み剤、鼻腔、直腸、子宮などへの経粘膜剤、経口剤（例、カプセル剤（例、硬カプセル剤、軟カプセル剤等）、顆粒剤、散剤等の固形製剤、シロップ剤、乳剤、懸濁剤等の液剤等）などとして投与することができる。
例えば、本発明の徐放性組成物を注射剤とするには、これらを分散剤（例、ツイーン（Tween）80，HCO−60等の界面活性剤、ヒアルロン酸ナトリウム，カルボキシメチルセルロース，アルギン酸ナトリウム等の多糖類など）、保存剤（例、メチルパラベン、プロピルパラベンなど）、等張化剤（例、塩化ナトリウム，マンニトール，ソルビトール，ブドウ糖，プロリンなど）等の分散媒と共に水性懸濁剤とするか、ゴマ油、コーン油などの植物油等の分散媒と共に分散して油性懸濁剤として実際に使用できる徐放性注射剤とすることができる。
本発明の徐放性組成物の粒子径は、懸濁注射剤として使用する場合には、その分散度、通針性を満足する範囲であればよく、例えば、平均粒子径として約０．１〜約３００μｍ、好ましくは約０．５〜約１５０μｍの範囲、さらに好ましくは約１から約１００μｍの範囲である。
本発明の徐放性組成物を無菌製剤にするには、製造全工程を無菌にする方法、ガンマ線で滅菌する方法、防腐剤を添加する方法等が用いられるが、特に限定されない。
さらに、前記の徐放性組成物の徐放性注射剤は、懸濁剤として、前記の組成以外に、賦形剤（たとえば、マンニトール、ソルビトール、ラクトース、ブドウ糖など）を加えて、再分散した後、凍結乾燥もしくは噴霧乾燥して固型化し、用時に、注射用蒸留水あるいは適当な分散媒を加えると、より安定した徐放性注射剤が得られる。
徐放性組成物の徐放性注射剤にマンニトール等の賦形剤を添加する場合、賦形剤の含有量は注射剤全体に対して、０〜約５０重量％、好ましくは約１〜約２０重量％である。
徐放性組成物の徐放性注射剤を用時、注射用蒸留水あるいは適当な分散媒に分散させる場合、徐放性組成物の含有量は分散媒と徐放性組成物の総量に対して、約１〜約８０重量％、好ましくは約１０〜約６０重量％である。
【００３３】
本発明の徐放性組成物を経口投与製剤にするには、自体公知の方法に従い、本発明の徐放性組成物を例えば賦形剤（例、乳糖、白糖、デンプンなど）、崩壊剤（例、デンプン、炭酸カルシウムなど）、結合剤（例、デンプン、アラビアゴム、カルボキシメチルセルロース、ポリビニルピロリドン、ヒドロキシプロピルセルロースなど）または滑沢剤（例、タルク、ステアリン酸マグネシウム、ポリエチレングリコール６０００など）などを添加して圧縮成形し、次いで必要により、味のマスキング，腸溶性あるいは持続性の目的のため自体公知の方法でコーティングすることにより経口投与製剤とすることができる。そのコーティング剤としては、例えばヒドロキシプロピルメチルセルロース，エチルセルロース，ヒドロキシメチルセルロース，ヒドロキシプロピルセルロース，ポリオキシエチレングリコール，ツイーン８０，ブルロニックＦ６８，セルロースアセテートフタレート，ヒドロキシプロピルメチルセルロースフタレート，ヒドロキシメチルセルロースアセテートサクシネート，オイドラギット（ローム社製，西ドイツ，メタアクリル酸・アクリル酸共重合）および酸化チタン，ベンガラ等の色素が用いられる。
【００３４】
本発明の徐放性組成物を経鼻投与製剤とするには、自体公知の方法に従い、本発明方法により製造された徐放性組成物を固状、半固状または液状の経鼻投与剤とすることができる。たとえば、前記固状のものとしては、該徐放性組成物をそのまま、あるいは賦形剤（例、グルコース、マンニトール、デンプン、微結晶セルロースなど）、増粘剤（例、天然ガム類、セルロース誘導体、アクリル酸重合体など）などを添加、混合して粉状の組成物とする。前記液状のものとしては、注射剤の場合とほとんど同様で、油性あるいは水性懸濁剤とする。半固状の場合は、水性または油性のゲル剤、あるいは軟膏状のものがよい。また、これらはいずれも、ｐＨ調整剤（例、炭酸、リン酸、クエン酸、塩酸、水酸化ナトリウムなど）、防腐剤（例、パラオキシ安息香酸エステル類、クロロブタノール、塩化ベンザルコニウムなど）などを加えてもよい。
【００３５】
本発明の徐放性組成物を坐剤とするには、自体公知の方法に従い、本発明方法により製造された徐放性組成物を油性または水性の固状、半固状あるいは液状の座剤とすることができる。前記組成物に用いる油性基剤としては、マイクロカプセルを溶解しないものであればよく、たとえば高級脂肪酸のグリセリド〔例、カカオ脂、ウイテプゾル類（ダイナマイトノーベル社）など〕、中級脂肪酸〔例、ミグリオール類（ダイナマイトノーベル社）など〕、あるいは植物油（例、ゴマ油、大豆油、綿実油など）などが用いられる。また、水性基剤としては、たとえばポリエチレングリコール類、プロピレングリコール、水性ゲル基剤としては、たとえば天然ガム類、セルロース誘導体、ビニール重合体、アクリル酸重合体などが用いられる。
本発明の徐放性組成物は、注射剤として用いることが好ましい。
【００３６】
本発明の徐放性組成物は、低毒性であるので、哺乳動物（例、ヒト、牛、豚、犬、ネコ、マウス、ラット、ウサギ等）に対して安全な医薬などとして用いることができる。
本発明の徐放性組成物またはその徐放性組成物の投与量は、主薬である生理活性物質またはその塩の種類と含量、剤形、生理活性物質またはその塩放出の持続時間、対象疾病、対象動物などによって種々異なるが、生理活性物質またはその塩の有効量であればよい。主薬である生理活性物質またはその塩の1回当たりの投与量としては、例えば、徐放性組成物が６カ月製剤である場合、好ましくは、成人１人当たり約０．０１ｍｇ〜約１０ｍｇ／ｋｇ体重の範囲，さらに好ましくは約０．０５ｍｇ〜約５ｍｇ／ｋｇ体重の範囲から適宜選ぶことができる。
1回当たりの徐放性組成物の投与量は、成人１人当たり好ましくは、約０．０５ｍｇ〜約５０ｍｇ／ｋｇ体重の範囲、さらに好ましくは約０．１ｍｇ〜約３０ｍｇ／ｋｇ体重の範囲から適宜選ぶことができる。
投与回数は、数週間に1回、1か月に1回、または数か月（例、３ヵ月、４ヵ月、６ヵ月など）に1回等、主薬である生理活性物質またはその塩の種類と含量、剤形、生理活性物質またはその塩放出の持続時間、対象疾病、対象動物などによって適宜選ぶことができる。
【００３７】
本発明の徐放性組成物またはその徐放性組成物は、含有する生理活性物質またはその塩の種類に応じて、種々の疾患などの予防・治療剤として用いることができるが、例えば、生理活性物質またはその塩がＬＨ−ＲＨ誘導体である場合には、ホルモン依存性疾患、特に性ホルモン依存性癌（例、前立腺癌、子宮癌、乳癌、下垂体腫瘍など）、前立腺肥大症、子宮内膜症、子宮筋腫、思春期早発症、月経困難症、無月経症、月経前症候群、多房性卵巣症候群等の性ホルモン依存性の疾患の予防・治療剤および避妊（もしくは、その休薬後のリバウンド効果を利用した場合には、不妊症の予防・治療）剤、アルツハイマー病や免疫不全等の疾患の予防・治療剤などとして用いることができる。さらに、性ホルモン非依存性であるがＬＨ−ＲＨ感受性である良性または悪性腫瘍などの予防・治療剤としても用いることができる。
乳癌細胞にはホルモン感受性があり、エストロゲンにより増殖する乳癌細胞があるため、酢酸リュープロレリンをはじめとするホルモン療法薬剤を閉経前の術後再発予防剤として用いることは困難であると考えられていたが、前記の公知の方法またはそれに準じた方法により製造されたLH-RHアゴニストまたはアンタゴニスト（好ましくは、リュープロレリンまたはその塩、より好ましくは酢酸リュープロレリン）を含有してなる剤（好ましくは、リュープロレリンまたはその塩（好ましくは酢酸リュープロレリン）を含有してなる徐放型マイクロカプセルを含有してなる剤）は、予想外にも、閉経前乳癌の術後再発予防剤または再発抑制剤として用いることができる。
前記のLH-RHアゴニストまたはアンタゴニスト（好ましくは、リュープロレリンまたはその塩、より好ましくは酢酸リュープロレリン）を含有してなる剤（好ましくは、リュープロレリンまたはその塩（好ましくは酢酸リュープロレリン）を含有してなる徐放型マイクロカプセルを含有してなる剤）を含有する徐放性組成物は、そのまま皮下、筋肉内、血管など（好ましくは皮下など）に容易に注射剤および埋め込み剤など（好ましくは注射剤など）として投与することができる。また、その他前記の種々の製剤に成形して投与することもでき、そのような製剤を製造する際の原料物質としても使用され得る。
また、前記LH-RHアゴニストまたはアンタゴニストを含有する徐放性組成物の投与量は、対象疾患、LH-RHアゴニストまたはアンタゴニスト（好ましくは、リュープロレリンまたはその塩、より好ましくは酢酸リュープロレリン）の含量、剤形、LH-RHアゴニストまたはアンタゴニスト（好ましくは、リュープロレリンまたはその塩、より好ましくは酢酸リュープロレリン）の持続時間、投与対象動物[例、温血哺乳動物(例、ヒト、マウス、ラット、ウサギ、ヒツジ、ブタ、ウシ、ウマなど)]により種々異なるが、該LH-RHアゴニストまたはアンタゴニスト（好ましくは、リュープロレリンまたはその塩、より好ましくは酢酸リュープロレリン）の有効量であればよい。たとえば、前記温血哺乳動物に１回あたり投与量として、約０.０１mgないし約１００mg／kg体重、好ましくは約０.０２mgないし約５０mg／kg体重、さらに好ましくは約０．０５ｍｇないし約２０ｍｇ／kg体重の範囲から適宜選ぶことができる。
【００３８】
また、前記LH-RHアゴニストまたはアンタゴニストを含有する徐放性組成物を注射剤として投与する場合、成人患者（体重６０ｋｇに対し）においては、一月あたりLH-RHアゴニストまたはアンタゴニスト（好ましくは、リュープロレリンまたはその塩、より好ましくは酢酸リュープロレリン）を通常約０.０１から約５０ｍｇ程度、好ましくは約０.１から約２０ｍｇ程度、より好ましくは約０.１から約１５ｍｇ程度を皮下あるいは筋肉内に投与すればよい。
LH-RHアゴニストまたはアンタゴニスト（好ましくは、リュープロレリンまたはその塩、より好ましくは酢酸リュープロレリン）を含有してなる剤（好ましくは、リュープロレリンまたはその塩（好ましくは酢酸リュープロレリン）を含有してなる徐放型マイクロカプセルを含有してなる剤）の投与期間は、特に限定されないが、通常約１〜約５年、好ましくは約２年である。
他の動物の場合も、体重６０ｋｇ当たりに換算した量を投与することができる。また、前記のLH-RHアゴニストまたはアンタゴニスト（好ましくは、式 5-oxo-Pro-His-Trp-Ser-Tyr-DLeu-Leu-Arg-Pro-NH-C₂H₅で表されるぺプチドまたはその塩（以下、単に「リュープロレリンまたはその塩」と称する場合がある））、より好ましくは酢酸リュープロレリンは、必要に応じて糖衣を施した錠剤、カプセル剤、エリキシル剤、徐放性製剤などとして経口的に、あるいは水もしくはそれ以外の薬学的に許容し得る液との無菌性溶液、または懸濁液剤、徐放性製剤（特に徐放型マイクロカプセル）などの注射剤、埋め込み剤（生体内分解性ポリマーを基材として成型されたもの、チタンなどの生体内適合性金属の筒に封入され、一定速度で活性成分を放出するもの）、生体に投与可能な有機溶媒に生体内分解性ポリマーおよび薬物を溶解あるいは分散した注射剤、または溶液、懸濁液剤などの経鼻投与製剤の形で非経口的に投与できるが、好ましくは徐放性製剤として、特に好ましくは徐放性注射剤として投与される。また、徐放性製剤が徐放型マイクロカプセルである場合、約２カ月以上にわたってLH-RHアゴニストあるいはアンタゴニストを放出する長期徐放型マイクロカプセルであることが好ましい。
リュープロレリンまたはその塩、より好ましくは酢酸リュープロレリンを生理学的に認められる公知の担体、香味剤、賦形剤、ベヒクル、防腐剤、安定剤、結合剤などとともに一般に認められた製剤実施に要求される単位用量形態で混和することによって前記製剤を製造することができる。
【００３９】
錠剤、カプセル剤などに混和することができる添加剤としては、例えばゼラチン、コーンスターチ、トラガント、アラビアゴムのような結合剤、結晶性セルロースのような賦形剤、コーンスターチ、ゼラチン、アルギン酸などのような膨化剤、ステアリン酸マグネシウムのような潤滑剤、ショ糖、乳糖またはサッカリンのような甘味剤、ペパーミント、アカモノ油またはチェリーのような香味剤などが用いられる。調剤単位形態がカプセルである場合には、前記タイプの材料にさらに油脂のような液状担体を含有することができる。注射のための無菌組成物は注射用水のようなベヒクル中の活性物質、胡麻油、椰子油などのような天然産出植物油などを溶解または懸濁させるなどの通常の製剤実施に従って処方することができる。注射用の水性液としては、例えば、生理食塩水、ブドウ糖やその他の補助薬を含む等張液（例えば、Ｄ−ソルビトール、Ｄ−マンニトール、塩化ナトリウムなど）などが用いられ、適当な溶解補助剤、例えば、アルコール（例、エタノール）、ポリアルコール（例、プロピレングリコール、ポリエチレングリコール）、非イオン性界面活性剤（例、ポリソルベート８０（ＴＭ）、ＨＣＯ−５０）などと併用してもよい。油性液としては、例えば、ゴマ油、大豆油などが用いられ、溶解補助剤である安息香酸ベンジル、ベンジルアルコールなどと併用してもよい。
また、前記製剤は、例えば、緩衝剤（例えば、リン酸塩緩衝液、酢酸ナトリウム緩衝液など）、無痛化剤（例えば、塩化ベンザルコニウム、塩酸プロカインなど）、安定剤（例えば、ヒト血清アルブミン、ポリエチレングリコールなど）、保存剤（例えば、ベンジルアルコール、フェノールなど）、酸化防止剤などと配合してもよい。調製された注射液は通常、適当なアンプル、バイアルなどの密封容器に充填される。
【００４０】
【実施例】
以下に実施例を挙げて、本発明をさらに具体的に説明する。
実施例１
ＤＬ−乳酸重合体（重量平均分子量２１，９００）２０６．６ｇをジクロロメタン３５４．８ｇで溶解した溶液を約３０℃に温調した。この溶液３８１．５ｇを量り取り、酢酸リュープロレリン１５．８ｇを１６．６ｇの酢酸水溶液（氷酢酸０．６ｇを蒸留水３１．７５ｇに溶解）に溶解して約５５℃に加温した水溶液と混合し、ミニミキサー（特殊機化製）を用いて乳化しW/O乳化物を形成した（回転数約１０，０００ｒｐｍ）。次いでこのW/O乳化物を約１８℃に冷却後、予め約１８℃に温調しておいた０．１％（ｗ／ｗ）ポリビニルアルコール（ＥＧ−４０、日本合成化学製）＋１％マンニトール水溶液２５リットル中に注入し、 ＨＯＭＯＭＩＣ ＬＩＮＥ ＦＬＯＷ（特殊機化製）を用いて二次乳化しW/O/W乳化物とした（タービン回転数約７，０００ｒｐｍ、循環ポンプ回転数約２０００ｒｐｍ）。このW/O/W乳化物を約３時間水中乾燥し、７５μｍの標準篩を用いて篩過し、次いで遠心機（Ｈ−６００Ｓ， 国産遠心器製）を用いて連続的にマイクロスフェアを沈降させて捕集した（回転数約２，０００ｒｐｍ、流量約６００ｍｌ／ｍｉｎ）。捕集されたマイクロスフェアは少量の蒸留水に再分散し、９０μｍの標準篩を用いて篩過した後、マンニトール１８．９ｇを添加し、凍結乾燥機（ＴＲＩＯＭＡＳＴＥＲ，共和真空製）で凍結乾燥して粉末（マイクロマイクロスフェア末）を得た。得られたマイクロスフェアの酢酸リュープロレリン含量は8.2％であり、回収率も約７５％であった。
酢酸の添加により良好にW/Oエマルションを得ることができ、外水相にマンニトールを添加することにより、得られたマイクロスフェアの分散性を改善することができる。
【００４１】
実験例１
ＤＬ−乳酸重合体（重量平均分子量２１，９００）１５１．３ｇをジクロロメタン２５９．９ｇで溶解した溶液を約３０℃に温調した。この溶液３７３．７ｇを量り取り、酢酸リュープロレリン１５．５ｇを１６．２ｇの酢酸水溶液（氷酢酸０．６ｇを蒸留水３１．７ｇに溶解）に溶解して約５５℃に加温した水溶液と混合し、ミニミキサー（特殊機化製）を用いて乳化しW/O乳化物を形成した（回転数約１０，０００ｒｐｍ）。乳化開始２，５，８分後にW/Oエマルションを一部回収し、粘度測定（振動粘度測定計）し、その結果を表１に示した。
【表１】

安定なW/Oエマルションが得られ、乳化時間8分ではやや粘度が高いものの、5分乳化でも製造上問題となるような粘度の上昇はなかった。
【００４２】
比較例１
ＤＬ−乳酸重合体（重量平均分子量２１，９００）１５１．１ｇをジクロロメタン２５９．８ｇで溶解した溶液を約３０℃に温調した。この溶液３７4．６ｇを量り取り、酢酸リュープロレリン１５．５ｇを１５．９ｇの蒸留水に溶解して約５５℃に加温した水溶液と混合し、ミニミキサー（特殊機化製）を用いて乳化しW/O乳化物を形成した（回転数約１０，０００ｒｐｍ）。乳化開始２，４分後にW/Oエマルションを一部回収し、粘度測定（振動粘度測定計）し、その結果を表２に示した。
【表２】

４分でW/Oエマルションの粘度が上昇した。酢酸添加系で行った実験例１と比較して、W/Oエマルションの粘度上昇が顕著であった。
【００４３】
実験例２
酢酸リュープロレリン（薬物含量９７．４％、酢酸含量６．０％）０．２０６１ｇを酢酸濃度の異なる酢酸水溶液０．２１１６ｇに溶解し、ＤＬ−乳酸重合体（重量平均分子量２１，９００）１．８２ｇをジクロロメタン３．１５ｇで溶解した溶液を添加後、ボルテックスミキサーで約３０秒間攪拌することによりW/Oエマルションを作製した。W/Oエマルションの様子を比較した。その結果を図１に示す。酢酸量が薬物に対して約１．８倍モルの場合小さなエマルション粒子が形成されているようであった。1.4倍モルでは薬物がゲル化、1.6倍モルではややゲル化傾向が認められた。1.8、2.3、2.8倍モルでは均一なエマルションが得られたが、1.8倍モルのエマルションは青みがかったほぼ透明色であるのに対し、2.3倍モル以上はやや白っぽいエマルション色であった。また約1.7倍モルにおいても青みのある透明色を呈したことを確認した。このことから青みのある透明色を呈した1.7〜1.8倍モルが最も微細なエマルション粒子を形成したと考えられた。
【００４４】
実験例３
酢酸リュープロレリン（薬物含量９７．４％、酢酸含量６．０％）０．２ｇを酢酸濃度の異なる酢酸水溶液０．２１１６ｇに溶解し、乳酸グリコール酸共重合体（重量平均分子量１０５００）１．８２ｇをジクロロメタン３．１５ｇで溶解した溶液を添加後、ボルテックスミキサーで約３０秒間攪拌することによりW/Oエマルションを作製した。W/Oエマルションの様子を比較したところ、酢酸量が薬物に対して約1.8倍モルの場合均一なエマルション粒子が形成されているようであった。1.3、1.4倍モルでは油相と内水相の分離が認められた。
【００４５】
実験例４
酢酸リュープロレリン（薬物含量９７．４％、酢酸含量６．０％）０．２ｇを酢酸濃度の異なる酢酸水溶液０．２１１６ｇに溶解し、ＤＬ−乳酸重合体（重量平均分子量１４５００）１．８２ｇをジクロロメタン３．１５ｇで溶解した溶液を添加後、ボルテックスミキサーで約３０秒間攪拌することによりW/Oエマルションを作製した。W/Oエマルションの様子を比較したところ、酢酸量が薬物に対して約1.8倍モルの場合均一なエマルション粒子が形成されているようであった。1.3、1.4倍モルでは油相と内水相の分離が認められた。
【００４６】
実験例５
実施例１で得られたマイクロスフェア約１１０ｍｇを０．３ｍｌの分散媒（０．１５ｍｇのカルボキシメチルセルロース，０．３ｍｇのポリソルベート８０，１５ｍｇのマンニトールを溶解した蒸留水）に分散して７週齢雄性ＳＤラットの背部皮下に２２Ｇ注射針で投与した。投与から所定時間後にラットを屠殺して投与部位に残存するマイクロスフェアを取り出し、この中のペプチドＡを定量してその初期含量で除して求めた残存率を表３に示す。

表３から明らかなように、ペプチドＡのみを処方して製造した実施例１のマイクロスフェアは、生理活性物質を高いトラップ効率で含むことができ、分散性も良好であり、生理活性物質の初期の過剰放出も抑止した。また、このマイクロスフェアは非常に長期にわたって生理活性物質を一定速度で放出している。
【００４７】
実験例６
ペプチドＡ酢酸塩0.6gを2wt%酢酸水溶液0.65gに溶解した(ペプチドＡに対して1.5倍モル以上)。これにポリ乳酸(重量平均分子量２１，０００)5.4gをジクロロメタン9.45gで溶解した溶液を添加し、手で軽く分散させた後ポリトロン(キネマティカ社製)で所定時間乳化しW/Oエマルションを作製した。乳化時間を変え、W/Oエマルションの粘度を測定した。その結果を図２に示す。
同様にして、ペプチドＡ0.6gを2wt%酢酸水溶液0.635gに溶解した(ペプチドＡに対して1.5倍モル未満)。これにポリ乳酸(重量平均分子量２１，０００)5.4gをジクロロメタン9.45gで溶解した溶液を添加し、手で軽く分散させた後ポリトロン(キネマティカ社製)で所定時間乳化しW/Oエマルションを作製した。
ペプチドＡに対して1.5倍モル未満の酢酸を使用したものは比較的短い乳化時間でW/Oエマルションの粘度が上昇することがあったが、ペプチドＡに対して1.5倍モル以上の酢酸を使用したものはW/Oエマルションが安定で、図２に示されるように短時間で粘度が上昇したりすることもなく容易にW/Oエマルションを製造することができた。
【００４８】
以上の実験結果から、薬物に対して約1.5倍モル以上の酢酸共存によりW/Oエマルションが安定化し、約1.65倍モル以上では比較的微細なエマルションが得られることがわかった。O相のポリマーとしては乳酸重合体や乳酸グリコール酸重合体で確認できており、最終製剤の製造性も改善できた。
【００４９】
【発明の効果】
本発明の徐放性組成物の製造方法により、W/Oエマルションを安定に形成させ、製造中の薬物リークを抑制することができ、徐放性組成物の製造性が向上し、また薬物を高含量で取り込め、安定した薬物放出をする徐放性組成物を得ることが可能となる。さらに本発明方法を用いて製造された徐放性組成物は薬物を高含量で保持し、安定した薬物の放出特性を示し、医薬品として有用である。
本発明方法により、生理活性物質を含む水溶液と生体内分解性ポリマーの溶解液の混合液を安定化することができ、通常条件においてその混合液の粘度を３０００ｃｐ以下にすることができる。
【図面の簡単な説明】
【図１】実験例２で作成したW/Oエマルションの様子を表す写真である。下記の数字は酢酸と薬物のモル比を表す数値である。
【図２】実験例６における、ペプチドＡに対して１．５倍モル以上の酢酸を使用した場合の、乳化時間とW/Oエマルション粘度の関係を示す図である。左から順に、酢酸／薬物モル比が1.4、1.6、1.8、2.3および2.8の例を示す。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for producing a sustained release composition comprising a physiologically active substance and a biodegradable polymer, a sustained release composition produced by the method, and the like.
[0002]
[Prior art]
Methods for producing sustained-release microcapsules from W / O emulsions using biodegradable polymers are described in, for example, Patent Document 1, Patent Document 2, and Patent Document 3.
The biodegradable polymer having sustained release is useful as a base material such as a microcapsule for encapsulating a physiologically active substance. As such a biodegradable polymer, it is known that polylactic acid, those containing a copolymer of lactic acid and glycolic acid, etc. (Patent Document 4 etc.) are useful.
Patent Document 5 discloses a sustained-release preparation comprising a bioactive peptide or a salt thereof and a biodegradable polymer having a terminal carboxyl group, and a production method thereof.
[0003]
[Patent Document 1]
Japanese Patent Laid-Open No. 57-118512
[Patent Document 2]
JP-A-57-150609
[Patent Document 3]
JP-A-6-145046
[Patent Document 4]
JP-A-11-269094
[Patent Document 5]
JP-A-7-97334
[0004]
[Problems to be solved by the invention]
An object of the present invention is to provide a method for producing a sustained-release composition for stably forming a W / O emulsion, a sustained-release composition produced by this method, and the like.
[0005]
[Means for Solving the Problems]
In view of such circumstances, the present inventors have conducted extensive research with the aim of developing a method for producing a sustained-release composition that stably forms a W / O emulsion. It was found that the W / O emulsion can be stabilized by using an aqueous solution containing an acid or base. As a result of further research based on this, the present invention was completed.
That is, the present invention
(1) A sustained release composition in which an aqueous solution containing a physiologically active substance and an acid or base at least about 1.5 times the mole of the physiologically active substance and a biodegradable polymer solution are mixed and then the mixture is dried. Manufacturing method of
(2) The method according to (1) above, wherein the aqueous solution is an aqueous solution obtained using a salt of a physiologically active substance and an acid or base;
(3) The method according to (1) above, wherein the weight ratio of the physiologically active substance in the sustained release composition is about 0.001 to about 50% by weight;
(4) A method for stabilizing a mixed solution of an aqueous solution containing a physiologically active substance and a biodegradable polymer solution, which contains about 1.5 times or more mole of acid or base of the physiologically active substance;
(5) The viscosity of a mixed solution of an aqueous solution containing a physiologically active substance and a biodegradable polymer solution is about 3000 cp or less, characterized by containing an acid or base that is about 1.5 times mol or more of the physiologically active substance How to make;
(6) The method according to (1), (4) or (5) above, wherein the physiologically active substance is a physiologically active peptide;
(7) The method according to (6) above, wherein the physiologically active peptide is an LH-RH derivative;
(8) LH-RH derivative is a general formula
5-oxo-Pro-His-Trp-Ser-Tyr-Y-Leu-Arg-Pro-Z
(In the formula, Y represents DLeu, DAla, DTrp, DSer (tBu), D2Nal or DHis (ImBzl), Z represents NH-C₂H_FiveOr Gly-NH₂Indicates. The method according to (7) above, which is a compound represented by the formula:
(9) The method according to (1), (4) or (5) above, wherein the amount of the acid or base relative to the physiologically active substance is about 1.5 to about 5 times mol;
(10) The method according to (1), (4) or (5) above, wherein the amount of the acid or base relative to the physiologically active substance is about 1.65 to about 3 times moles;
(11) The method according to (1), (4) or (5) above, wherein the acid is an organic acid;
(12) The method according to (11) above, wherein the organic acid is a fatty acid;
(13) The method according to (12), wherein the fatty acid is acetic acid;
(14) The method according to (1), (4) or (5) above, wherein the biodegradable polymer is an α-hydroxycarboxylic acid polymer;
(15) The method according to (14) above, wherein the α-hydroxycarboxylic acid polymer is a lactic acid-glycolic acid polymer;
(16) The method according to the above (15), wherein the composition molar ratio of lactic acid to glycolic acid in the lactic acid-glycolic acid polymer is from 100 to 0 to 50 to 50;
(17) The method according to (16) above, wherein the lactic acid-glycolic acid polymer has a molar ratio of lactic acid to glycolic acid of 100 to 0;
(18) The method according to the above (15), wherein the lactic acid-glycolic acid polymer has a weight average molecular weight of 5,000 to 50,000;
(19) The method according to (15) above, wherein the lactic acid-glycolic acid polymer has a weight average molecular weight of 17,000 to 30,000;
(20) The method according to (1) above, wherein the biodegradable polymer is a lactic acid polymer having a weight average molecular weight of 15,000 to 50,000, wherein the polymer content having a weight average molecular weight of 5000 or less is about 5% by weight or less;
(21) The method according to (1) above, wherein the biodegradable polymer is a lactic acid-glycolic acid polymer having a terminal carboxyl group amount of about 20 to about 1000 μmol (micromol) per unit mass (gram) of the polymer;
(22) The method according to (1) above, wherein the amount of terminal carboxyl groups of the biodegradable polymer is about 0.1 to about 5 times mol of the physiologically active substance;
(23) The method according to (1), (4) or (5) above, wherein the biodegradable polymer solution is a solution using a poorly water-soluble organic solvent;
(24) The method according to (23) above, wherein the poorly water-soluble organic solvent is dichloromethane;
(25) The method according to (1), (4) or (5), wherein the mixed solution is uniformly mixed;
(26) The method according to the above (25), wherein the uniformly mixed liquid is an emulsion;
(27) The method according to the above (26), wherein the emulsion is W / O type;
(28) The method according to the above (27), wherein the size of the W / O emulsion is fine;
(29) The method according to (1) above, wherein the drying of the mixed solution is drying in water;
(30) The method according to (29) above, wherein an aqueous solution of an osmotic pressure regulator is used for the outer aqueous phase dried in water;
(31) The method according to (30) above, wherein the osmotic pressure regulator is mannitol;
(32) The method according to (1) above, wherein the sustained-release composition is in the form of fine particles;
(33) The method according to (32), wherein the fine particles are microspheres or microcapsules;
(34) 1) A bioactive substance and 2) an aqueous solution containing about 0.1 to about 20% by weight acid or base with respect to the aqueous solution and a biodegradable polymer solution are mixed, and then the mixture is dried. A method for producing a sustained release composition;
(35) The method according to (34) above, wherein the aqueous solution is an aqueous solution obtained using a salt of a physiologically active substance and an acid or base;
(36) A sustained release composition produced using the method according to (1) above; and
(37) Use of an aqueous solution containing the physiologically active substance and an acid or base of about 1.5 times mol or more of the physiologically active substance for the production of a sustained-release preparation containing the physiologically active substance. .
[0006]
DETAILED DESCRIPTION OF THE INVENTION
The physiologically active substance used in the present invention is not particularly limited as long as it is pharmacologically useful, and may be a non-peptide compound or a peptide compound. As the non-peptide compound, agonists, antagonists, compounds having enzyme inhibitory action and the like are used. The peptide compound may be an agonist or an antagonist. For example, a physiologically active peptide is preferable, and a molecular weight of about 300 to about 40,000, preferably about 400 to about 30,000, more preferably about 500 to about 20,000. The physiologically active peptides of
The weight ratio of the physiologically active substance in the composition of the present invention varies depending on the type of physiologically active substance, the desired pharmacological effect and the duration of the effect, and is not particularly limited. 50 wt%, preferably about 0.02 to about 40 wt%, more preferably about 0.1 to about 30 wt%, more preferably about 0.1 to about 24 wt%, most preferably about 5 to about 24 % By weight.
[0007]
The physiologically active substance is not particularly limited, and is a physiologically active peptide, antitumor agent, antibiotic, antipyretic, analgesic, anti-inflammatory agent, antitussive expectorant, sedative, muscle relaxant, antiepileptic agent, antiulcer agent, anti Depressant, antiallergic agent, cardiotonic agent, antiarrhythmic agent, vasodilator, antihypertensive diuretic, antidiabetic agent, anticoagulant, hemostatic agent, antituberculosis agent, hormone agent, narcotic antagonist, bone resorption inhibitor, blood vessel Neonatal inhibitors and the like are used.
Examples of the physiologically active peptide include luteinizing hormone releasing hormone (LH-RH), insulin, somatostatin, growth hormone, growth hormone releasing hormone (GH-RH), prolactin, erythropoietin, corticosteroid, melanocyte stimulating hormone. , Thyroid hormone releasing hormone (TRH), thyroid stimulating hormone, luteinizing hormone, follicle stimulating hormone, vasopressin, oxytocin, calcitonin, gastrin, secretin, pancreosimimine, cholecystokinin, angiotensin, human placental lactogen, human chorionic gonadotropin , Enkephalin, Endorphin, Kyotorphin, Tuftsin, Thymopoietin, Thymosin, Thymothymline, Thymic factor, Blood thymic factor, Tumor necrosis factor, Colony inducer , Motilin, dynorphin, bombesin, neurotensin, cerulein, bradykinin, atrial natriuretic factor, nerve growth factor, cell growth factor, neurotrophic factor, peptides with endothelin antagonism, and their derivatives, and fragments thereof Alternatively, a fragment derivative or the like is used.
[0008]
Examples of peptides having physiological activity include LH-RH antagonists (US Pat. Nos. 4,086,219, 4,124,577, 4,253,997, 4,317, 815) is used.
Examples of peptides having further physiological activity include insulin, somatostatin, and somatostatin derivatives (US Pat. Nos. 4,087,390, 4,093,574, 4,100,117, and 4). , 253, 998), growth hormone, prolactin, corticotropin (ACTH), melanocyte stimulating hormone (MSH), thyroid hormone releasing hormone [(Pyr) Glu-His-ProNH2 And its derivatives (see JP 50-121273, JP 52-116465), thyroid stimulating hormone (TSH), luteinizing hormone (LH), follicle stimulating hormone. (FSH), vasopressin, vasopressin derivatives {desmopressin [The Journal of the Japan Endocrine Society, Vol. 54, No. 5, 67 6-691 (1978)]}, oxytocin, calcitonin, parathyroid hormone, glucagon, gastrin, secretin, pancreosimine, cholecystokinin, angiotensin, human placental lactogen, human chorionic gonadotropin (HCG), enkephalin, Enkephalin derivatives (see US Pat. No. 4,277,394, European Patent Application Publication No. 31567), endorphins, kyotorphins, interferons (eg, α-type, β-type, γ-type, etc.), interleukins (eg , I, II, III, etc.), tuftsin, thymopoietin, thymosin, thymostimulin, thymic humoral factor (THF), blood thymic factor (FTS) and its derivatives (see US Pat. No. 4,229,438), and Other thymic factors [Ayumi of Medicine, 125 No. 10, 835-843 (1983)], tumor necrosis factor (TNF), colony-inducing factor (CSF), motilin, dynorphin, bombesin, neurotensin, cerulein, bradykinin, urokinase, asparaginase, kallikrein, substance P , Nerve growth factor, cell growth factor, neurotrophic factor, blood coagulation factor factor VIII, factor IX, lysozyme chloride, polymyxin B, colistin, gramicidin, bacitracin and erythropoietin (EPO), peptides with antagonistic activity of endothelin ( European Patent Publication Nos. 436189, 457195, 496452, JP-A-3-94692 and JP-A-3-130299) are used.
[0009]
Examples of the antitumor agents include bleomycin, methotrexate, actinomycin D, mitomycin C, vinblastine sulfate, vincristine sulfate, daunorubicin, adriamycin, neocartinostatin, cytosine arabinoside, fluorouracil, tetrahydrofuryl-5-fluorouracil, krestin, picibanil , Lentinan, levamisole, bestatin, azimexone, glycyrrhizin, poly I: C, poly A: U, poly ICLC and the like.
Examples of the antibiotic include gentamicin, dibekacin, canendomycin, ribidomycin, tobramycin, amikacin, fradiomycin, sisomycin, tetracycline hydrochloride, oxytetracycline hydrochloride, loritetracycline, doxycycline hydrochloride, ampicillin, piperacillin, ticarcillin, cephalothin, cephalolidine , Cefotiam, cefsulosin, cefmenoxime, cefmethazole, cefazoline, cefotaxime, cefoperazone, ceftizoxime, moxalactam, thienamycin, sulfazecin, azuleonam, etc. are used.
Examples of antipyretic, analgesic and anti-inflammatory agents include salicylic acid, sulpyrine, flufenamic acid, diclofenac, indomethacin, morphine, pethidine hydrochloride, levorphanol tartrate, oxymorphone, and the like.
Antitussive expectorants include ephedrine hydrochloride, methylephedrine hydrochloride, noscapine hydrochloride, codeine phosphate, dihydrocodeine phosphate, aloclamide hydrochloride, clofedanol hydrochloride, picoperidamine hydrochloride, cloperastine, protochelol hydrochloride, isoproterenol hydrochloride, salbutamol hydrochloride, Terbutaline sulfate is used.
As the sedative, chlorpromazine, prochlorperazine, trifluoroperazine, atropine sulfate, methyl scopolamine bromide and the like are used.
As a muscle relaxant, pridinol methanesulfonate, tubocurarine chloride, pancuronium bromide and the like are used.
As the antiepileptic agent, phenytoin, ethosuximide, acetazolamide sodium, chlordiazepoxide and the like are used.
As the anti-canker, metoclopromide, histidine hydrochloride and the like are used.
As an antidepressant, imipramine, clomipramine, noxiptillin, phenelzine sulfate, etc. are used.
Examples of antiallergic agents include diphenhydramine hydrochloride, chlorpheniramine maleate, tripelenamine hydrochloride, methodirazine hydrochloride, clemizole hydrochloride, diphenylpyraline hydrochloride, methoxyphenamine hydrochloride, and the like.
[0010]
As a cardiotonic agent, transpyoxocamphor, theophylol, aminophylline, ethylephrine hydrochloride, etc. are used.
As the arrhythmia therapeutic agent, propranol, alprenolol, bufetrol, oxyprenolol and the like are used.
Examples of the vasodilator include oxyfedrine hydrochloride, diltiazem, tolazoline hydrochloride, hexobenzine, and bamethane sulfate.
Examples of antihypertensive diuretics include hexamethonium bromide, pentolinium, mecamylamine hydrochloride, ecarazine hydrochloride, clonidine, and the like.
Examples of the therapeutic agent for diabetes include sodium grimidine, glipizide, phenformin hydrochloride, buformin hydrochloride, metformin, and the like.
As the anticoagulant, heparin sodium, sodium citrate and the like are used.
As the hemostatic agent, thromboplastin, thrombin, menadione sodium bisulfite, acetomenaphton, ε-aminocaproic acid, tranexamic acid, sodium carbazochrome sulfonate, adrenochrome monoaminoguanidine methanesulfonate and the like are used.
As the antituberculosis agent, isoniazid, ethambutol, paraaminosalicylic acid and the like are used.
As the hormone agent, prednisolone, sodium prednisolone phosphate, dexamethasone sodium sulfate, betamethasone sodium phosphate, hexestrol phosphate, hexestrol acetate, methimazole and the like are used.
As narcotic antagonists, levalorphan tartrate, narolphine hydrochloride, naloxone hydrochloride, etc. are used.
As the bone resorption inhibitor, (sulfur-containing alkyl) aminomethylenebisphosphonic acid or the like is used.
Examples of the angiogenesis inhibitor include angiogenesis-inhibiting steroids (see Science, Vol. 221, page 719 (1983)), fumagillin (see European Patent Publication No. 325199), fumagillol derivatives (European Patent Publication No. 357061, No. 359036, No. 386667, No. 415294) are used.
[0011]
Preferred examples of the physiologically active peptide include LH-RH derivatives, which are hormone-dependent diseases, particularly sex hormone-dependent cancers (eg, prostate cancer, uterine cancer, breast cancer, pituitary tumor, etc.), prostatic hypertrophy, Endometriosis, uterine fibroids, precocious puberty, dysmenorrhea, amenorrhea, premenstrual syndrome, sex hormone-dependent diseases such as multilocular ovary syndrome and contraception (or its rebound effect after withdrawal) LH-RH derivatives or salts thereof effective for infertility), prevention of recurrence after premenopausal breast cancer surgery, or diseases such as Alzheimer's disease and immunodeficiency are used. Furthermore, LH-RH derivatives effective for benign or malignant tumors which are independent of sex hormone but are sensitive to LH-RH are also used.
Specific examples of LH-RH derivatives include, for example, Treatment with GnRH analogs: Controversies and perspectives (published by The Parthenon Publishing Group Ltd. 1996) Year], peptides described in JP-T-3-503165, JP-A-3-101695, JP-A-7-97334, and JP-A-8-259460 are used.
[0012]
As the LH-RH derivative, an LH-RH agonist or an LH-RH antagonist is used, and examples of the LH-RH antagonist include a compound represented by the general formula [I].
X-D2Nal-D4ClPhe-D3Pal-Ser-A-B-Leu-C-Pro-DAlaNH₂
[Where X is N (4H₂-furoyl) Gly or NAc, A is a residue selected from NMeTyr, Tyr, Aph (Atz), NMeAph (Atz), B is DLys (Nic), DCit, DLys (AzaglyNic), DLys (AzaglyFur), DhArg (Et₂), DAph (Atz) and DhCi, C represents Lys (Nisp), Arg or hArg (Et₂), Peptides, abarelix, degarelix, antarelix, itrelix, orntide, cetrorelix or ganirelix, or a salt thereof.
Non-peptidic LH-RH antagonists include WO 95/28405 (JP-A 8-295693), WO 97/14697 (JP-A 9-169767), WO 97/14682 ( JP-A-9-169735), WO 96/24597 (JP-A-9-169768), thienopyridine compounds [eg, 3- (N-Benzyl-N-methylaminomethyl) 4,7-dihydro-5-isobutyryl- 7- (2,6-difluorobenzyl) -2- [4-[(1-hydroxycyclopropyl) carbonylamino] phenyl] -4-oxothieno [2,3-b] pyridine etc. ", WO 00/00493 publication, thieno Pyrimidine compounds such as 5- (N-benzyl-N-methylaminomethyl) -1- (2,6-difluorobenzyl) -6- [4- (3-methoxyureido) phenyl] -3-phenylthieno [2,3-d WO 00/56739 describing 5-pyranidine-2,4 (1H, 3H) -dione etc., 5-[(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro- 2-naphthalenyl) methyl] -N- (2,4,6-trimethoxyphenyl) -2-furamide (American Cancer Society (AACR), 2002.4.6-10) is used.
[0013]
As the LH-RH agonist, for example, the general formula [II]
5-oxo-Pro-His-Trp-Ser-Tyr-Y-Leu-Arg-Pro-Z
[Wherein Y is a residue selected from DLeu, DAla, DTrp, DSer (tBu), D2Nal and DHis (ImBzl), Z is NH—C₂H_FiveOr Gly-NH₂Or the like] is used. In particular, Y is DLeu and Z is NH-C₂H_FiveOr a salt thereof (ie, 5-oxo-Pro-His-Trp-Ser-Tyr-DLeu-Leu-Arg-Pro-NH-C₂H_Five(Hereinafter also referred to as “peptide A”) or a salt thereof, particularly an acetate salt thereof (leuprorelin acetate)) and the like are suitable.
[0014]
As a LH-RH agonist, in addition to the above-mentioned leuprorelin, preferred specific examples include, for example,
(1) Buserelin
[Chemical 1]

(U.S. Patent No. 4,024,248, German Patent No. 2,438,352, JP-A-51-41359),
(2) Triptorelin
[Chemical 2]

(U.S. Pat. No. 4,010,125, JP-A 52-31073),
(3) Nafarelin
[Chemical Formula 3]

(U.S. Pat. No. 4,234,571, JP-A 55-164663, JP-A 63-264498, and JP-A 64-25794),
(4) Histrelin
[Formula 4]

(5) Deslorelin
[Chemical formula 5]

(U.S. Pat. Nos. 4,569,967 and 4,218,439),
(6) Meterelin
[Chemical 6]

(PCT WO 91/18016),
(7) Gonadrelin
[Chemical 7]

(German Patent No. 2213737),
(8) Goserelin
[Chemical 8]

(U.S. Pat. No. 4,100,204, JP-A-52-136172),
(9) Lecirelin
[Chemical 9]

[Belgium Patent No. 897455, Japanese Patent Laid-Open No. 59-59654] or a salt thereof is used.
[0015]
The meanings of the abbreviations used in this specification are as follows.
Abbreviation Name
N (4H₂-furoyl) Gly: N-tetrahydrofuroylglycine residue
NAc: N-acetyl group
D2Nal: D-3- (2-naphthyl) alanine residue
D4ClPhe: D-3- (4-chloro) phenylalanine residue
D3Pal: D-3- (3-pyridyl) alanine residue
NMeTyr: N-methyltyrosine residue
Aph (Atz): N- [5 '-(3'-amino-1'H-1', 2 ', 4'-triazolyl)] phenylalanine residue
NMeAph (Atz): N-methyl- [5 '-(3'-amino-1'H-1', 2 ', 4'-triazolyl)] phenylalanine residue
DLys (Nic): D- (e-N-nicotinoyl) lysine residue
Dcit: D-citrulline residue
DLys (AzaglyNic) ： D- (Azaglycylnicotinoyl) lysine residue
DLys (AzaglyFur): D- (Azaglycylfuranyl) lysine residue
DhArg (Et₂): D- (N, N'-diethyl) homoarginine residue
DAph (Atz): D-N- [5 '-(3'-amino-1'H-1', 2 ', 4'-triazolyl)] phenylalanine residue
DhCi: D-homocitrulline residue
Lys (Nisp): (e-N-isopropyl) lysine residue
hArg (Et₂): (N, N'-diethyl) homoarginine residue
Other abbreviations for amino acids, IUPAC-IUB Commission on Biochemical Nomenclature (European Journal of Biochemistry, Vol. 138, It shall be based on the abbreviations according to pages 9 to 37 (1984) or conventional abbreviations in the relevant field, and when there may be optical isomers with respect to amino acids, L forms will be indicated unless otherwise specified.
[0016]
In the present invention, the “acid or base” in “an aqueous solution containing a physiologically active substance and an acid or base of about 1.5-fold mol or more of the physiologically active substance” and “about 0.1 to about 20% by weight acid or base relative to the aqueous solution” "Is not particularly limited as long as it is generally used in production, and inorganic acids, organic acids, inorganic bases, organic bases, acidic or basic amino acids, and the like can be used. Preferably it is an acid, More preferably, it is an organic acid.
Preferable examples of the inorganic acid include hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid and the like. Preferable examples of the organic acid include sulfonic acids such as methanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid; fatty acids such as formic acid, acetic acid, and propionic acid; aliphatics such as oxalic acid, malonic acid, and succinic acid. Dicarboxylic acids; unsaturated fatty acids such as acrylic acid, fumaric acid and maleic acid; carbocyclic carboxylic acids such as phthalic acid, isophthalic acid and terephthalic acid; other trifluoroacetic acid, tartaric acid, citric acid, malic acid, lactic acid and glycolic acid Substituted carboxylic acids such as fatty acids, lactic acid and glycolic acid are used, and acetic acid is particularly preferably used. Preferable examples of acidic amino acids include aspartic acid and glutamic acid, and examples of basic amino acids include arginine, lysine and ornithine. Suitable examples of inorganic bases include, for example, alkali metals such as sodium and potassium; alkaline earth metals such as calcium and magnesium; alkali metal hydrides such as lithium hydride, potassium hydride and sodium hydride; lithium oxide; Inorganic hydroxides such as potassium hydroxide, sodium hydroxide, and calcium hydroxide; carbonates such as sodium carbonate, potassium carbonate, and sodium bicarbonate; and aluminum and ammonium are used. Preferable examples of the organic base include metal alkoxides having 1 to 6 carbon atoms such as lithium ethoxide, lithium tert-butoxide, sodium methoxide, sodium ethoxide, potassium tert-butoxide, etc .; potassium phenoxide, sodium phenoxide Metal phenoxides such as: salts of acetic acid such as sodium acetate and potassium acetate; organic lithium salts such as n-butyllithium, t-butyllithium and diethylaminolithium; hydrazines such as phenylhydrazine and p-tolylhydrazine; amidines; Quaternary ammonium hydroxides; sulfonium bases; trimethylamine, triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexyla Emissions, N, N'amines such as di-dibenzylethylenediamine and the like can be used.
When the acid is an “organic acid”, its pKa is not particularly limited. For example, it is preferably about 0.1 to about 6.0, more preferably about 1.0 to about 6.0, and further preferably about 3.5 to about 6.0. is there.
[0017]
In the present invention, the “acid and base” may contain an acid or a base in an aqueous solution independently of the physiologically active substance, or may be contained in an aqueous solution in the form of a salt of the physiologically active substance with an acid or base. Further, a salt of a physiologically active substance with an acid or base and an acid or base independent of the salt may be contained in the aqueous solution.
[0018]
Thus, the physiologically active substance used in the present invention may be itself or a pharmacologically acceptable salt.
As such salts, when the physiologically active substance has a basic group such as an amino group, an inorganic acid (also referred to as an inorganic free acid) (eg, carbonic acid, bicarbonate, hydrochloric acid, sulfuric acid, nitric acid, boric acid, etc.) ), Organic acids (also referred to as organic free acids) (eg, succinic acid, acetic acid, propionic acid, trifluoroacetic acid, etc.) and salts with acids. When the physiologically active substance is an LHRH derivative, it is particularly preferable to add acetic acid, such as leuprorelin acetate.
When the physiologically active substance has an acidic group such as a carboxyl group, an inorganic base (also referred to as an inorganic free base) (eg, an alkali metal such as sodium or potassium, an alkaline earth metal such as calcium or magnesium) or an organic base ( A salt with a base such as an organic free base (eg, organic amines such as triethylamine, basic amino acids such as arginine, etc.) is used. In addition, the physiologically active peptide may form a metal complex compound (eg, copper complex, zinc complex, etc.).
These compounds or salts thereof can be produced by the methods described in the above-mentioned documents or gazettes or a method analogous thereto.
[0019]
Here, the amount of the “acid or base” used relative to the “bioactive substance” can be about 1.5 times mole or more, preferably about 1.5 times mole or more and about 5 times mole per mole of the physiologically active substance. More preferably, it is about 1.65 times mol or more and about 3 times mol or less.
In the present invention, the amount (% by weight) of the “acid or base” used in the “aqueous solution” is not particularly limited as long as the effect of the present invention is exhibited, but is preferably about 0.1 to about 20% with respect to the aqueous solution. More preferably about 1 to about 15%, still more preferably about 3 to about 10%.
As described above, this “acid or base” may be contained in an aqueous solution independently from the outside, or may be contained in the aqueous solution in the form of a salt of a physiologically active substance, and further independent from the outside. And a method of adding it to an aqueous solution in the form of a salt of a physiologically active substance may be combined.
[0020]
The biodegradable polymer in the present invention is preferably a biodegradable polymer, for example, an aliphatic polyester [for example, α-hydroxy acids (eg, glycolic acid, lactic acid, 2-hydroxybutyric acid, 2- Hydroxyvaleric acid, 2-hydroxy-3-methylbutyric acid, 2-hydroxycaproic acid, 2-hydroxyisocaproic acid, 2-hydroxycaprylic acid, etc.), cyclic dimers of α-hydroxy acid (eg, glycolide, lactide) Etc.), hydroxydicarboxylic acids (eg, malic acid), homopolymers (eg, lactic acid polymer) such as hydroxytricarboxylic acid (eg, citric acid), or two or more copolymers (eg, lactic acid / glycolic acid) Copolymer, 2-hydroxybutyric acid / glycolic acid copolymer, etc.) or a mixture of these homopolymers and / or copolymers ( Examples, mixtures of lactic acid polymers and 2-hydroxybutyric acid / glycolic acid copolymers, etc.)], poly-α-cyanoacrylic acid esters, polyamino acids (eg, poly-γ-benzyl-L-glutamic acid, poly-L -Alanine, poly-γ-methyl-L-glutamic acid, etc.), maleic anhydride copolymers (eg, styrene / maleic acid copolymers, etc.) and the like are used. Of these, aliphatic polyesters and poly-α-cyanoacrylic acid esters are preferred. Furthermore, aliphatic polyesters are particularly preferred. Among aliphatic polyesters, α-hydroxy such as α-hydroxymonocarboxylic acids (eg, glycolic acid, lactic acid, etc.), α-hydroxydicarboxylic acids (eg, malic acid), α-hydroxytricarboxylic acid (eg, citric acid), etc. A polymer, copolymer, or mixture thereof synthesized from one or more of carboxylic acids and having a free terminal carboxyl group; poly (α-cyanoacrylate); polyamino acid (eg, poly (γ-benzyl-) L-glutamic acid), etc.); maleic anhydride copolymers (eg, styrene-maleic acid copolymers, etc.) are used.
The bonding mode of the monomers may be random, block, or graft. When the α-hydroxymonocarboxylic acids, α-hydroxydicarboxylic acids, and α-hydroxytricarboxylic acids have an optically active center in the molecule, any of D-, L-, and DL-forms may be used. Among these, lactic acid-glycolic acid polymer (hereinafter referred to as poly (lactide-co-glycolide), poly (lactic acid-co-glycolic acid) or lactic acid-glycolic acid copolymer, unless otherwise specified, Homopolymers (polymers) and copolymers (copolymers) of lactic acid and glycolic acid are collectively referred to as lactic acid homopolymers as lactic acid polymers, polylactic acid, polylactide, etc., and glycolic acid homopolymers as glycolic acid polymers, Glycolic acid, polyglycolide and the like) and poly (α-cyanoacrylic acid ester) are preferred. A lactic acid-glycolic acid polymer is more preferable, and a lactic acid-glycolic acid polymer having a free terminal carboxyl group is more preferable.
The biodegradable polymer may be a salt. Examples of the salt include inorganic bases (eg, alkali metals such as sodium and potassium, alkaline earth metals such as calcium and magnesium) and organic bases (eg, organic amines such as triethylamine, and basic amino acids such as arginine). Etc.), or salts and complex salts with transition metals (eg, zinc, iron, copper, etc.).
When a lactic acid-glycolic acid polymer is used as the biodegradable polymer, the composition ratio (mol%) is preferably 100/0 to about 40/60, and more preferably 100/0 to about 50/50. In the case of a long-term sustained release microcapsule that releases a physiologically active substance over 2 months or more, a lactic acid polymer having a composition ratio of 100/0 is also preferably used.
The optical isomer ratio of lactic acid, which is one of the minimum repeating units of the “lactic acid-glycolic acid polymer”, is about 75/25 to about 25/75 in D-form / L-form (mol / mol%). A range is preferred. As the D-form / L-form (mol / mol%), those having a range of about 60/40 to about 30/70 are generally used.
The weight average molecular weight of the “lactic acid-glycolic acid polymer” is generally about 3000 to about 100,000, preferably about 5000 to about 50000, more preferably about 8000 to about 30000, and further preferably about 17000 to about 30000. Is used.
Further, the dispersity (weight average molecular weight / number average molecular weight) is usually preferably about 1.1 to about 4.0, more preferably about 1.2 to about 3.5. In addition, the weight average molecular weight and dispersion degree used by this specification mean the value measured by gel permeation chromatography (GPC). The weight average molecular weight and the content of each polymer are the weight average molecular weight in terms of polystyrene measured by gel permeation chromatography (GPC) using monodisperse polystyrene as a reference substance and the contents of each polymer calculated from them. All measurements were performed with a high-speed GPC device (manufactured by Tosoh Corporation; HLC-8120GPC), the columns used were SuperH4000 × 2 and SuperH2000 (both manufactured by Tosoh Corporation), and the flow rate was 0.6 mL of tetrahydrofuran as the mobile phase. Used at / min. The detection method is based on the differential refractive index.
The amount of the free terminal carboxyl group of the “lactic acid-glycolic acid polymer” is usually preferably about 20 to about 1000 μmol (micromol), more preferably about 40 to about 300 μmol (micromol) per unit mass (gram) of the polymer. Is particularly preferred.
In the present invention, the amount of the free terminal carboxyl group of the biodegradable polymer relative to the “bioactive substance” is preferably about 0.1 to about 5 times mol, more preferably about 0.2 to about 4 times mol, more preferably about 0.3 to about About 3.5 times mole.
[0021]
The above-mentioned free terminal carboxyl group amount refers to a value obtained by a labeling method (hereinafter referred to as “carboxyl group amount by labeling method”). Specifically, in the case of polylactic acid, Wmg of polylactic acid is dissolved in 2 ml of 5N hydrochloric acid / acetonitrile (v / v = 4/96) mixed solution, and 0.01M o-nitrophenylhydrazine hydrochloride (ONPH) solution (5N 2 ml of hydrochloric acid / acetonitrile / ethanol = 1.02 / 35/15) and 2 ml of 0.15M 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride solution (pyridine / ethanol = 4v / 96v) were added. After reacting at 40 ° C. for 30 minutes, the solvent is distilled off. The residue is washed with water (4 times), dissolved in 2 ml of acetonitrile, 1 ml of 0.5 mol / l ethanolic potassium hydroxide solution is added, and the mixture is reacted at 60 ° C. for 30 minutes. The reaction solution is diluted with a 1.5N aqueous sodium hydroxide solution to give Yml, and the 544 nm absorbance A (/ cm) is measured for the 1.5N aqueous sodium hydroxide solution. On the other hand, when the DL-lactic acid aqueous solution is used as a reference substance, the free carboxyl group amount Cmol / L is obtained by alkali titration, and the absorbance at 544 nm when DL-lactic acid hydrazide is obtained by the ONPH labeling method is B (/ cm). The molar amount of the free terminal carboxyl group per unit mass (gram) of the polymer can be obtained by the following formula.
[COOH] (mol / g) = (AYC) / (WB)
The "carboxyl group amount" is obtained by dissolving a biodegradable polymer in a toluene-acetone-methanol mixed solvent and titrating this solution with an alcoholic potassium hydroxide solution using phenolphthalein as an indicator. (Hereafter, the value obtained by this method is referred to as “carboxyl group amount by alkali titration method”), but the end point of titration may become unclear as a result of competing with the hydrolysis reaction of the polyester main chain during titration. It is desirable to quantify by the labeling method.
The “lactic acid-glycolic acid polymer” is, for example, ring-opening using a catalyst from a cyclic diester compound such as non-catalytic dehydration polycondensation from lactic acid and glycolic acid (Japanese Patent Laid-Open No. 61-28521) or lactide and glycolide. It can be produced by polymerization (Encyclopedic Handbook of Biomaterials and Bioengineering Part A: Materials, Volume 2, Marcel Dekker, Inc. 1995). The polymer obtained by the above known ring-opening polymerization method does not necessarily have a free carboxyl group at the end, but for example, by subjecting it to a hydrolysis reaction described in EP-A-0839525. The polymer can be modified into a polymer having a certain amount of carboxyl groups per unit mass, and this can also be used.
The above-mentioned “lactic acid-glycolic acid polymer having a free terminal carboxyl group” is a method similar to a known production method (for example, non-catalytic dehydration polycondensation method, see JP-A-61-28521) or a method analogous thereto. Can be manufactured.
Among them, the polymer polymer used in the present invention is preferably a lactic acid polymer (hereinafter sometimes abbreviated as the lactic acid polymer of the present invention), for example, a polymer composed only of lactic acid, or lactic acid and other polymers. Including a copolymer with a monomer (for example, glycolic acid) and not particularly limited, but a polymer content with a weight average molecular weight of 5000 or less is usually about 10% by weight or less, preferably a weight average molecular weight of 5000 or less. Of about 5% by weight or less is used.
Moreover, the weight average molecular weight of the lactic acid polymer of this invention is 15000-50000 normally, Preferably it is 15000-40000, More preferably, it is 17000-30000.
[0022]
The high molecular weight lactic acid polymer used as a raw material for the lactic acid polymer of the present invention may be a commercial product or a polymerized by a known method, and its weight average molecular weight is usually 15,000 to 500,000, preferably 20,000 to 100,000. Known polymerization methods include, for example, a method of condensation polymerization of lactic acid and, if necessary, glycolic acid, for example, lactide, and glycolide, if necessary, together with Lewis acid or metal salt such as diethyl zinc, triethylaluminum, tin octylate, etc. A method of ring-opening polymerization using a catalyst such as the above, a method of further ring-opening polymerization of lactide in the presence of a hydroxycarboxylic acid derivative with a carboxyl group protected (for example, Patent International Publication WO00 / 35990), and other lactides A method of ring-opening polymerization by adding a catalyst under heating (for example, J. Med. Chem, 16,897(1973) etc.), for example, a method of copolymerizing lactide and glycolide, etc. is used.
As the polymerization form, bulk polymerization in which lactide or the like is melted and subjected to a polymerization reaction, or solution polymerization in which lactide or the like is dissolved in an appropriate solvent and subjected to the polymerization reaction are used. It is preferable for industrial production to be used as a raw material for the lactic acid polymer.
As a solvent for dissolving lactide in solution polymerization, for example, aromatic hydrocarbons such as benzene, toluene and xylene, decalin, dimethylformamide and the like are used.
[0023]
In order to hydrolyze the high molecular weight lactic acid polymer obtained as described above, a hydrolysis method known per se is used. For example, after dissolving the high molecular weight lactic acid polymer in a suitable solvent, water and If necessary, an acid may be added and reacted.
Specific examples of the solvent for dissolving the high molecular weight lactic acid polymer include halogenated hydrocarbons such as chloroform and dichloromethane, aromatic hydrocarbons such as toluene, o-xylene, m-xylene, and p-xylene. For example, cyclic ethers such as tetrahydrofuran, acetone, N, N-dimethylformamide and the like are used. In the polymerization of the high molecular weight lactic acid polymer, when a solvent that can be used for hydrolysis of the high molecular weight lactic acid polymer is used, the polymerized high molecular weight lactic acid polymer is not isolated, but is polymerized and hydrolyzed. The operation can be performed continuously.
The amount of the solvent for dissolving the high molecular weight lactic acid polymer is usually about 0.1 to about 100 times, preferably about 1 to about 10 times that of the lactic acid polymer as the solute.
The amount of water to be added is usually about 0.001 to about 1 times the weight, preferably about 0.01 to about 0.1 times the weight of the high molecular weight lactic acid polymer.
Examples of the acid to be added as necessary include inorganic acids such as hydrochloric acid, sulfuric acid, and nitric acid, and organic acids such as lactic acid, acetic acid, and trifluoroacetic acid. Preferably, trifluoroacetic acid is used.
The amount of acid to be added is usually 0 to about 10 times by weight, preferably about 0.1 to about 1 times by weight with respect to the high molecular weight lactic acid polymer.
The hydrolysis reaction temperature is usually about 0 to about 150 ° C, preferably about 20 to about 80 ° C.
The hydrolysis reaction time varies depending on the weight average molecular weight of the high molecular weight lactic acid polymer and the reaction temperature, and is usually about 10 minutes to about 100 hours, preferably about 1 to about 20 hours.
The end time of the hydrolysis treatment is determined based on the weight average molecular weight of the hydrolysis product. That is, sampling is performed appropriately during the hydrolysis treatment, the weight average molecular weight of the hydrolysis product in the sample is measured by gel permeation chromatography (GPC), and the molecular weight is about 15000 to about 50000, preferably about 15000. When it is confirmed that the amount is about 40000, more preferably about 17000 to about 30000, the hydrolysis treatment is stopped.
[0024]
As a method for precipitating the target lactic acid polymer contained therein from the solution containing the hydrolysis product obtained by subjecting the high molecular weight lactic acid polymer to hydrolysis, as described above, A method of bringing the decomposition product-containing solution into contact with a solvent capable of precipitating the target lactic acid polymer contained therein is used.
Preferred embodiments of the hydrolyzate-containing solution include halogenated hydrocarbons such as chloroform and dichloromethane, aromatic hydrocarbons such as toluene, o-xylene, m-xylene, and p-xylene, and cyclic ethers such as tetrahydrofuran. A lactic acid polymer having a weight average molecular weight of 15000 to 50000, preferably 15000 to 40000, more preferably 17000 to 30000 is dissolved in a solvent for dissolving a high molecular weight lactic acid polymer such as dichloromethane or xylene such as acetone or N, N-dimethylformamide. What is dissolved in about 10 to about 50 wt% is used.
Examples of the solvent capable of precipitating the target lactic acid polymer contained in the hydrolysis product-containing solution include alcohols such as methanol and ethanol, chain ethers such as isopropyl ether, and aliphatic carbonization such as hexane. Hydrogen, water or the like is used.
[0025]
The amount of the solvent capable of precipitating the target lactic acid polymer is usually about 0.1 to about 100 times by weight, preferably about 1 to about 10 times by weight with respect to the solvent of the hydrolysis product-containing solution.
As a preferred specific example of the combination of the kind and amount of each solvent, the solubility is lowered in a hydrolysis product-containing solution using, for example, about 1 to about 5 times the weight of dichloromethane as a solvent. An embodiment in which isopropyl ether is used as a solvent in an amount of about 2 to about 10 times the weight of the dichloromethane is used.
When the solvent capable of precipitating the target lactic acid polymer solute is brought into contact with the hydrolysis product-containing solution, the temperature of the solvent is usually about -20 to about 60 ° C, preferably about 0 to about 40 ° C. The temperature of the decomposition product-containing solution is usually about 0 to about 40 ° C, preferably about 10 to about 30 ° C.
As a method of bringing the solvent into contact with the hydrolysis product-containing solution, a method in which the hydrolysis product-containing solution is added to the solvent at once, a method in which the hydrolysis product-containing solution is dropped into the solvent, and a method in which the solvent is hydrolyzed. For example, a method of adding to a product-containing solution at a time or a method of dropping a solvent into a hydrolysis product-containing solution is used.
The lactic acid polymer of the present invention obtained as described above is preferable as a substrate for sustained-release preparations because the amount of terminal carboxyl groups is in the preferred range as a substrate for sustained-release preparations. Furthermore, examples of the biocompatible polymer include polystyrene, polymethacrylic acid, a copolymer of acrylic acid and methacrylic acid, polyamino acid, dextrantearate, ethyl cellulose, acetyl cellulose, nitrocellulose, anhydrous A maleic acid copolymer, an ethylene vinyl acetate copolymer, polyvinyl acetate, polyacrylamide, or the like is used.
These high molecular weight polymers may be one kind, two or more kinds of copolymers or a simple mixture, or a salt thereof.
[0026]
Below, the manufacturing method of the sustained release composition (for example, microcapsule) containing the bioactive substance or its salt of this invention and the lactic acid polymer of this invention or its salt is illustrated.
During the following production steps, the following may be added by a method known per se as necessary.
(1) Drug retention agent
Polyol compounds such as albumin, gelatin, citric acid, salicylic acid, ethylenediaminetetraacetic acid sodium, dextrin, sodium bisulfite, polyethylene glycol, agar, alginic acid, polyvinyl alcohol, basic amino acids and the like.
(2) pH adjuster for maintaining the stability and solubility of physiologically active substances or salts thereof
Carbonic acid, acetic acid, oxalic acid, citric acid, phosphoric acid, hydrochloric acid, sodium hydroxide, arginine, lysine and their salts.
(3) Stabilizer for physiologically active substance or salt thereof
Polyol compounds such as albumin, gelatin, citric acid, sodium ethylenediaminetetraacetate, dextrin, sodium bisulfite and polyethylene glycol.
(4) Preservative
Paraoxybenzoates (methylparaben, propylparaben, etc.), benzyl alcohol, chlorobutanol, thimerosal, etc.
[0027]
(I) W / O / W method
In this method, first, a biodegradable polymer solution that is a high molecular weight polymer (preferably an organic solvent solution, more preferably a poorly water-soluble organic solvent) is prepared. The solution used in the production of the sustained-release composition of the present invention (preferably in the form of fine particles (microparticles), more preferably microspheres or microcapsules) is a poorly water-soluble organic solvent. The boiling point is preferably about 120 ° C. or lower.
Examples of the organic solvent include halogenated hydrocarbons (eg, dichloromethane, chloroform, dichloroethane, trichloroethane, carbon tetrachloride, etc.), ethers (eg, ethyl ether, isopropyl ether, etc.), fatty acid esters (eg, ethyl acetate, Butyl acetate, etc.), aromatic hydrocarbons (eg, benzene, toluene, xylene, etc.), alcohols (eg, ethanol, methanol, etc.), acetonitrile and the like are used. Of these, halogenated hydrocarbons are preferable, and dichloromethane is particularly preferable. These may be mixed and used at an appropriate ratio. In that case, a mixed liquid of a halogenated hydrocarbon and an alcohol is preferable, and a mixed liquid of dichloromethane and ethanol is particularly preferable.
The concentration of the biodegradable polymer in the solution varies depending on the molecular weight of the biodegradable polymer and the type of the solution. For example, when dichloromethane is used as the solution, it is generally about 0.5 to about It is selected from 70% by weight, more preferably from about 1 to about 60% by weight, particularly preferably from about 2 to about 50% by weight.
When ethanol is used as a mixed organic solvent with dichloromethane, the ratio of the two is generally about 0.01 to about 50% (v / v), more preferably about 0.05 to about 40% ( v / v), particularly preferably selected from about 0.1 to about 30% (v / v).
Next, as already described, the physiologically active substance is dissolved in an aqueous solution containing about 1.5 times or more moles of the acid or base of the physiologically active substance or an aqueous solution of acid or base at a concentration of about 0.1 to about 20% by weight. The physiologically active substance may be dissolved as a salt with an appropriate acid or base, and the aqueous solution may be water or a mixture of water and alcohols (eg, methanol, ethanol, etc.) as a solvent.
The concentration of the physiologically active substance or salt thereof is generally from about 0.001 mg / ml to about 10 g / ml, more preferably from about 0.1 mg / ml to about 5 g / ml, and even more preferably from about 10 mg / ml to about 10 g / ml. 3 g / ml.
Known solubilizers and stabilizers may be used. Heating, shaking, stirring and the like may be performed to such an extent that the activity is not lost in the dissolution or dispersion of the physiologically active substance or additive, and the aqueous solution thus obtained is referred to as an inner aqueous phase.
The inner aqueous phase and the oil phase obtained as described above are emulsified by a known method such as a homogenizer or ultrasonic wave to form a W / O emulsion.
As a mixing ratio of the biodegradable polymer and the physiologically active substance to be mixed here, the amount of terminal carboxyl groups per unit mass (gram) of the biodegradable polymer is usually about 0.01 to about 10 times mol with respect to the physiologically active substance. , Preferably about 0.1 to about 5 times mol.
The weight of the oil phase to be mixed is about 1 to about 1000 times, preferably about 2 to about 100 times, more preferably about 3 to about 30 times the weight of the inner aqueous phase.
The viscosity range of the resulting W / O emulsion is generally from about 12 to about 25 ° C., from about 10 to about 10,000 cp, preferably from about 100 to about 5,000 cp, more preferably from about 200 to About 3,000 cp, more preferably about 300 to about 2,000 cp.
When the process of the present invention is carried out industrially, the viscosity range of the W / O emulsion is generally about 12 to about 25 ° C., preferably about 3,000 cp or less, more preferably about 2,000 cp or less, More preferably, it is about 300 to about 2,000 cp.
Next, the obtained W / O emulsion is added to the aqueous phase to form a W (inner aqueous phase) / O (oil phase) / W (outer aqueous phase) emulsion, and then the solvent in the oil phase is volatilized or removed. Diffusion into the external aqueous phase to prepare microcapsules. In this case, the weight of the outer aqueous phase is generally about 1 to about 10,000 times, more preferably about 5 to about 5,000 times, and further preferably about 10 to about 2,000 times the weight of the oil phase. Times, particularly preferably from about 20 times to about 500 times.
[0028]
An emulsifier may be added to the outer aqueous phase. The emulsifier may be any as long as it can generally form a stable W / O / W emulsion. Specifically, for example, anionic surfactants (sodium oleate, sodium stearate, sodium lauryl sulfate, etc.), nonionic surfactants (polyoxyethylene sorbitan fatty acid esters [Tween 80, Tween , 60, Atlas Powder Co., Ltd.], polyoxyethylene castor oil derivatives [HCO-60, HCO-50, Nikko Chemicals], etc.), polyvinylpyrrolidone, polyvinyl alcohol, carboxymethylcellulose, lecithin, gelatin, hyaluronic acid and the like. One of these or some of them may be used in combination. The concentration in use is preferably in the range of about 0.01 to about 10% by weight, more preferably in the range of about 0.05 to about 5% by weight.
An osmotic pressure regulator may be added to the outer aqueous phase. Any osmotic pressure adjusting agent may be used as long as it shows osmotic pressure when used as an aqueous solution.
Examples of the osmotic pressure regulator include polyhydric alcohols, monohydric alcohols, monosaccharides, disaccharides, oligosaccharides and amino acids, or derivatives thereof.
Examples of the polyhydric alcohol include trihydric alcohols such as glycerin, pentahydric alcohols such as arabitol, xylitol, and adonitol, and hexahydric alcohols such as mannitol, sorbitol, and dulcitol. Of these, hexahydric alcohols are preferable, and mannitol is particularly preferable.
As said monohydric alcohol, methanol, ethanol, isopropyl alcohol etc. are mentioned, for example, Of these, ethanol is preferable.
Examples of the monosaccharide include pentose saccharides such as arabinose, xylose, ribose and 2-deoxyribose, and hexose saccharides such as glucose, fructose, galactose, manose, sorbose, rhamnose and fucose. Carbosaccharides are preferred.
Examples of the oligosaccharide include trisaccharides such as maltotriose and raffinose sugar, and tetrasaccharides such as stachyose, among which trisaccharide is preferable.
Examples of the monosaccharide, disaccharide, and oligosaccharide derivatives include glucosamine, galactosamine, glucuronic acid, galacturonic acid, and the like.
Any amino acid can be used as the amino acid as long as it is L-form. For example, glycine, leucine, arginine and the like are used. Of these, L-arginine is preferred.
These osmotic pressure regulators may be used alone or in combination. These osmotic pressure regulators are used at a concentration such that the osmotic pressure of the external water phase is about 1/50 to about 5 times, preferably about 1/25 to about 3 times the osmotic pressure of physiological saline. When mannitol is used as the osmotic pressure regulator, a concentration of about 0.5% to about 1.5% is preferred.
As a method for removing the organic solvent, a method known per se or a method analogous thereto is used. For example, a method of evaporating an organic solvent under normal pressure or gradually reducing pressure while stirring with a propeller type stirrer or a magnetic stirrer, a method of evaporating an organic solvent while adjusting the degree of vacuum using a rotary evaporator or the like Etc. are used.
[0029]
The microcapsules thus obtained are separated by centrifugation or filtration, and free physiologically active substances, emulsifiers, osmotic pressure regulators, etc. adhering to the surface of the microcapsules are repeated several times with distilled water. Wash, re-disperse in distilled water and freeze-dry.
An anti-aggregation agent may be added during the production process in order to prevent the particles from aggregating. Examples of the aggregation inhibitor include water-soluble polysaccharides such as mannitol, lactose, glucose, and starches (eg, corn starch), amino acids such as glycine, proteins such as fibrin and collagen, and the like. Of these, mannitol is preferred.
The addition amount of an anti-aggregating agent such as mannitol is usually 0 to about 24% by weight based on the whole microcapsule.
In addition, after freeze-drying, if necessary, the microcapsules may be heated under the condition that the microcapsules are not fused together to remove moisture and the organic solvent in the microcapsules. Preferably, heating is performed at a temperature near or slightly higher than the midpoint glass transition temperature of the microcapsule obtained by a differential scanning calorimeter under a temperature rising rate of about 10 to about 20 ° C. per minute. More preferably, the heating is performed in the vicinity of the midpoint glass transition temperature of the microcapsule or in a temperature range higher by about 30 ° C. In particular, when a lactic acid-glycolic acid polymer is used as the biodegradable polymer, it is preferably a temperature range from about the midpoint glass transition temperature of the microcapsule to about 10 ° C. higher than the midpoint glass transition temperature, more preferably the midpoint. Heating is performed in the temperature range from about the glass transition temperature to about 5 ° C. higher than the intermediate glass transition temperature.
Although the heating time varies depending on the amount of microcapsules and the like, generally, after the microcapsules themselves reach a predetermined temperature, it is about 12 hours to about 168 hours, preferably about 24 hours to about 120 hours, particularly preferably. About 48 hours to about 96 hours.
The heating method is not particularly limited as long as the assembly of microcapsules can be uniformly heated.
As the heating and drying method, for example, a method of heating and drying in a thermostatic bath, a fluidized bath, a moving tank or a kiln, a method of heating and drying with a microwave, and the like are used. Of these, a method of heating and drying in a thermostatic bath is preferred.
(II) Phase separation method
When microcapsules are produced by this method, coacervation is applied to a W / O emulsion containing a composition comprising the bioactive substance described in (I) underwater drying method and the biodegradable polymer of the present invention. The agent is gradually added with stirring to precipitate and solidify the microcapsules. The coacervation agent is selected from about 0.01 to about 1,000 times, preferably about 0.05 to about 500 times, particularly preferably about 0.1 to about 200 times the oil phase volume.
The coacervation agent is not particularly limited as long as it is a polymer, mineral oil, or vegetable oil compound that is miscible with an organic solvent and does not dissolve the biodegradable polymer of the present invention. Specifically, for example, silicon oil, sesame oil, soybean oil, corn oil, cottonseed oil, coconut oil, linseed oil, mineral oil, n-hexane, n-heptane and the like are used. You may use these in mixture of 2 or more types.
After separating the microcapsules thus obtained, the coacervation agent other than the composition comprising the bioactive substance and the biodegradable polymer of the present invention is removed by repeated washing with heptane or the like, and the pressure is reduced. dry. Alternatively, washing is performed in the same manner as described in the above-mentioned (I) (i) underwater drying method, followed by freeze-drying and further heating and drying.
[0030]
(III) Spray drying method
When microcapsules are produced by this method, a W / O emulsion containing a composition comprising the bioactive substance described in (I) underwater drying method and the biodegradable polymer of the present invention is passed through a nozzle. It is sprayed into the drying chamber of a spray dryer (spray dryer) and the organic solvent in the atomized droplets is volatilized within a very short time to prepare microcapsules. Examples of the nozzle include a two-fluid nozzle type, a pressure nozzle type, and a rotating disk type. Thereafter, if necessary, washing may be performed by the same method as described in (I) underwater drying method, followed by freeze-drying and further heat drying.
An organic solvent solution or dispersion containing a composition comprising the bioactive substance described in the microcapsule production method (I) underwater drying method as a dosage form other than the above microcapsules and the biodegradable polymer of the present invention, For example, the organic solvent and water may be evaporated to dryness while adjusting the degree of vacuum using a rotary evaporator or the like, and then pulverized with a jet mill or the like to form fine particles (microparticles).
Further, the pulverized fine particles may be washed by the same method as described in the microcapsule production method (I) underwater drying method, and then freeze-dried and further heated and dried.
[0031]
In the present invention, it is preferable to use about 1.5 to about 5 times moles of acid or base with respect to the physiologically active substance.
Physiologically active substance is general formula
5-oxo-Pro-His-Trp-Ser-Tyr-Y-Leu-Arg-Pro-Z
[Wherein each symbol is as defined above. It is preferable to use about 1.5 to about 5 times moles of acid relative to the substance. The acid is preferably an organic acid, and particularly acetic acid or the like is used.
As the biodegradable polymer, a lactic acid-glycolic acid polymer is preferable, and a compositional molar ratio is preferably from 100 to 0 to about 50 to 50, and a polymer having a ratio of 100 to 0 is also preferably used. A lactic acid polymer having a weight average molecular weight of 15,000 to 50,000 and a polymer content of 5,000 or less is preferably about 5% by weight or less.
As the solution of the biodegradable polymer, a poorly water-soluble organic solvent is preferable, and dichloromethane is particularly preferable.
In the present invention, a mixed solution obtained by mixing 1) an aqueous solution containing a physiologically active substance and an acid or base and 2) a solution of a biodegradable polymer is preferably uniformly mixed, and this mixed solution is an emulsion. It is preferable that
More preferably, the emulsion is of the W / O type and the emulsion size is fine.
The mixed solution is preferably dried by an underwater drying method, and it is particularly preferable to use an osmotic pressure regulator in the outer water phase of underwater drying.
As the osmotic pressure regulator, mannitol is preferable.
[0032]
The sustained-release composition of the present invention is formulated into various dosage forms as they are or as raw materials, and is injected or implanted into muscles, subcutaneouss, organs, etc., transmucosal into nasal cavity, rectum, uterus, etc. And oral agents (eg, capsules (eg, hard capsules, soft capsules, etc.), granules, solid preparations such as powders, syrups, emulsions, suspensions, etc.) it can.
For example, in order to use the sustained-release composition of the present invention as an injection, these are used as a dispersant (eg, surfactant such as Tween 80, HCO-60, sodium hyaluronate, carboxymethyl cellulose, sodium alginate, etc. Polysaccharides, etc.), preservatives (eg, methyl paraben, propyl paraben, etc.), isotonic agents (eg, sodium chloride, mannitol, sorbitol, glucose, proline, etc.) It can be dispersed with a dispersion medium such as sesame oil and corn oil to produce a sustained-release injection that can be actually used as an oily suspension.
When used as a suspension injection, the particle size of the sustained-release composition of the present invention may be in a range satisfying the degree of dispersion and needle penetration. For example, the average particle size is about 0.1 To about 300 μm, preferably about 0.5 to about 150 μm, more preferably about 1 to about 100 μm.
In order to make the sustained-release composition of the present invention into a sterile preparation, a method of sterilizing all manufacturing steps, a method of sterilizing with gamma rays, a method of adding a preservative, and the like are used, but there is no particular limitation.
Further, the sustained-release injection of the sustained-release composition was redispersed as a suspension by adding an excipient (for example, mannitol, sorbitol, lactose, glucose, etc.) in addition to the composition described above. Thereafter, freeze-dried or spray-dried to solidify, and upon use, distilled water for injection or an appropriate dispersion medium is added to obtain a more stable sustained-release injection.
When an excipient such as mannitol is added to the sustained-release injection of the sustained-release composition, the content of the excipient is 0 to about 50% by weight relative to the whole injection, preferably about 1 to about 20% by weight.
When using the sustained-release injection of the sustained-release composition, when dispersed in distilled water for injection or an appropriate dispersion medium, the content of the sustained-release composition is relative to the total amount of the dispersion medium and the sustained-release composition. About 1 to about 80% by weight, preferably about 10 to about 60% by weight.
[0033]
In order to make the sustained-release composition of the present invention into a preparation for oral administration, according to a method known per se, the sustained-release composition of the present invention is treated with, for example, an excipient (eg, lactose, sucrose, starch, etc.), a disintegrant ( Eg, starch, calcium carbonate, etc.), binder (eg, starch, gum arabic, carboxymethylcellulose, polyvinylpyrrolidone, hydroxypropylcellulose, etc.) or lubricant (eg, talc, magnesium stearate, polyethylene glycol 6000, etc.), etc. It can be added and compression-molded, and if necessary, it can be made into an orally-administered preparation by coating by a method known per se for the purpose of masking of taste, enteric property or durability. Examples of the coating agent include hydroxypropylmethylcellulose, ethylcellulose, hydroxymethylcellulose, hydroxypropylcellulose, polyoxyethylene glycol, Tween 80, brulonic F68, cellulose acetate phthalate, hydroxypropylmethylcellulose phthalate, hydroxymethylcellulose acetate succinate, Eudragit (Rohm) Manufactured by West Germany, methacrylic acid / acrylic acid copolymer), and dyes such as titanium oxide and bengara.
[0034]
In order to make the sustained-release composition of the present invention into a nasal preparation, the sustained-release composition produced by the method of the present invention is solid, semi-solid or liquid according to a method known per se. It can be. For example, as the solid, the sustained-release composition is used as it is, or an excipient (eg, glucose, mannitol, starch, microcrystalline cellulose, etc.), a thickener (eg, natural gums, cellulose derivatives). , Acrylic acid polymer, etc.) are added and mixed to form a powdery composition. The liquid is almost the same as in the case of injections, and is oily or aqueous suspension. In the case of a semi-solid form, an aqueous or oily gel or an ointment is preferred. These are all pH adjusters (eg, carbonic acid, phosphoric acid, citric acid, hydrochloric acid, sodium hydroxide, etc.), preservatives (eg, paraoxybenzoic acid esters, chlorobutanol, benzalkonium chloride, etc.), etc. May be added.
[0035]
In order to use the sustained-release composition of the present invention as a suppository, according to a method known per se, the sustained-release composition produced by the method of the present invention is converted into an oily or aqueous solid, semi-solid or liquid suppository. It can be. The oily base used in the composition is not particularly limited as long as it does not dissolve the microcapsules. For example, higher fatty acid glycerides (eg, cacao butter, witepsols (Dynamite Nobel), etc.), intermediate fatty acids (eg, miglycols). (Dynamite Nobel, etc.)] or vegetable oils (eg, sesame oil, soybean oil, cottonseed oil, etc.) are used. Examples of the aqueous base include polyethylene glycols and propylene glycol, and examples of the aqueous gel base include natural gums, cellulose derivatives, vinyl polymers, and acrylic acid polymers.
The sustained-release composition of the present invention is preferably used as an injection.
[0036]
Since the sustained-release composition of the present invention has low toxicity, it can be used as a safe medicine for mammals (eg, humans, cows, pigs, dogs, cats, mice, rats, rabbits, etc.). .
The dosage of the sustained-release composition of the present invention or the sustained-release composition thereof depends on the type and content of the physiologically active substance or salt thereof as the main drug, the dosage form, the duration of release of the physiologically active substance or salt thereof, the target disease Depending on the target animal and the like, it may be an effective amount of a physiologically active substance or a salt thereof. For example, when the sustained-release composition is a 6-month preparation, the dose of the physiologically active substance or salt thereof as the active ingredient is preferably about 0.01 mg to about 10 mg / kg body weight per adult. And more preferably within the range of about 0.05 mg to about 5 mg / kg body weight.
The dose of the sustained-release composition per dose is preferably within the range of about 0.05 mg to about 50 mg / kg body weight, more preferably about 0.1 mg to about 30 mg / kg body weight per adult. You can choose.
The frequency of administration is once a few weeks, once a month, or once every few months (eg, 3 months, 4 months, 6 months, etc.) And content, dosage form, physiologically active substance or salt release duration, target disease, target animal, and the like.
[0037]
The sustained-release composition of the present invention or the sustained-release composition thereof can be used as a prophylactic / therapeutic agent for various diseases depending on the type of physiologically active substance or salt thereof contained. When the active substance or a salt thereof is an LH-RH derivative, hormone-dependent diseases, particularly sex hormone-dependent cancers (eg, prostate cancer, uterine cancer, breast cancer, pituitary tumor, etc.), prostatic hypertrophy, intrauterine Prophylactic / therapeutic and contraceptive agents for sex hormone dependent diseases such as membranous disease, uterine fibroids, precocious puberty, dysmenorrhea, amenorrhea, premenstrual syndrome, multilocular ovary syndrome When the rebound effect is used, it can be used as a prophylactic / therapeutic agent for infertility, a prophylactic / therapeutic agent for diseases such as Alzheimer's disease and immune deficiency. Furthermore, it can also be used as a prophylactic / therapeutic agent for benign or malignant tumors that are independent of sex hormones but are LH-RH sensitive.
Breast cancer cells are hormone sensitive and there are breast cancer cells that proliferate by estrogen, so it is considered difficult to use hormone therapy drugs such as leuprorelin acetate as postoperative menopausal postoperative recurrence prevention agents. However, an agent comprising an LH-RH agonist or antagonist (preferably leuprorelin or a salt thereof, more preferably leuprorelin acetate) produced by the aforementioned known method or a method analogous thereto (preferably Unexpectedly, an agent comprising a sustained-release microcapsule containing leuprorelin or a salt thereof (preferably leuprorelin acetate) is a prophylactic agent for postoperative recurrence of premenopausal breast cancer or It can be used as a recurrence inhibitor.
An agent (preferably leuprorelin or a salt thereof (preferably leuprorelin acetate) containing the LH-RH agonist or antagonist (preferably leuprorelin or a salt thereof, more preferably leuprorelin acetate). The sustained release composition containing the sustained release type microcapsule containing) is easily injected directly into the subcutaneous, intramuscular, blood vessel, etc. (preferably subcutaneously). Etc. (preferably injections etc.). Moreover, it can also be molded into the above-mentioned various preparations and administered, and can also be used as a raw material for producing such preparations.
The dose of the sustained release composition containing the LH-RH agonist or antagonist is the target disease, LH-RH agonist or antagonist (preferably leuprorelin or a salt thereof, more preferably leuprorelin acetate). Content, dosage form, duration of LH-RH agonist or antagonist (preferably leuprorelin or a salt thereof, more preferably leuprorelin acetate), subject animal [eg, warm-blooded mammal (eg, human, Mouse, rat, rabbit, sheep, pig, cow, horse, etc.)], but an effective amount of the LH-RH agonist or antagonist (preferably leuprorelin or a salt thereof, more preferably leuprorelin acetate) If it is. For example, the dose per dose to the warm-blooded mammal is about 0.01 mg to about 100 mg / kg body weight, preferably about 0.02 mg to about 50 mg / kg body weight, more preferably about 0.05 mg to about 20 mg / kg body weight. It can be selected appropriately from the range of kg body weight.
[0038]
In addition, when the sustained-release composition containing the LH-RH agonist or antagonist is administered as an injection, in an adult patient (for body weight of 60 kg), an LH-RH agonist or antagonist (preferably a rejuvenating agent) per month. Or about 0.1 mg to about 50 mg, preferably about 0.1 to about 20 mg, more preferably about 0.1 to about 15 mg subcutaneously or prorelin or a salt thereof, more preferably leuprorelin acetate. It can be administered intramuscularly.
An agent (preferably leuprorelin or a salt thereof (preferably leuprorelin acetate) comprising LH-RH agonist or antagonist (preferably leuprorelin or a salt thereof, more preferably leuprorelin acetate); The administration period of the agent containing the sustained-release microcapsule) is not particularly limited, but is usually about 1 to about 5 years, preferably about 2 years.
In the case of other animals, an amount converted per 60 kg body weight can be administered. Also, the LH-RH agonist or antagonist (preferably the formula 5-oxo-Pro-His-Trp-Ser-Tyr-DLeu-Leu-Arg-Pro-NH-C₂H_FiveOr a salt thereof (hereinafter sometimes simply referred to as “leuprorelin or a salt thereof”), more preferably leuprorelin acetate is a sugar-coated tablet or capsule if necessary. , Elixirs, sustained release formulations orally, or aseptic solutions or suspensions with water or other pharmaceutically acceptable liquids, sustained release formulations (especially sustained release microcapsules) Injections, implants (molded using biodegradable polymers as base materials, sealed in biocompatible metal cylinders such as titanium and releasing active ingredients at a constant rate), administered to living bodies It can be administered parenterally in the form of an injection prepared by dissolving or dispersing a biodegradable polymer and a drug in a possible organic solvent, or a nasal preparation such as a solution or suspension, but preferably sustained release As agents are administered particularly preferably as a sustained release injection. Further, when the sustained-release preparation is a sustained-release microcapsule, it is preferably a long-term sustained-release microcapsule that releases an LH-RH agonist or antagonist for about 2 months or more.
Leuprorelin or a salt thereof, more preferably leuprorelin acetate, together with known physiologically recognized carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders, etc. The formulation can be made by blending in the required unit dose form.
[0039]
Additives that can be mixed into tablets, capsules and the like include binders such as gelatin, corn starch, tragacanth and gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid and the like A swelling agent, a lubricant such as magnesium stearate, a sweetening agent such as sucrose, lactose or saccharin, a flavoring agent such as peppermint, red oil, or cherry are used. When the dispensing unit form is a capsule, a liquid carrier such as fats and oils can be further contained in the above-mentioned type of material. Sterile compositions for injection can be formulated according to conventional pharmaceutical practice such as dissolving or suspending active substances in vehicles such as water for injection, naturally occurring vegetable oils such as sesame oil, coconut oil and the like. As an aqueous solution for injection, for example, isotonic solutions (eg, D-sorbitol, D-mannitol, sodium chloride, etc.) containing physiological saline, glucose and other adjuvants are used. For example, alcohol (eg, ethanol), polyalcohol (eg, propylene glycol, polyethylene glycol), nonionic surfactant (eg, polysorbate 80 (TM), HCO-50) and the like may be used in combination. As the oily liquid, for example, sesame oil, soybean oil and the like are used, and they may be used in combination with solubilizing agents such as benzyl benzoate and benzyl alcohol.
In addition, the preparation includes, for example, a buffer (eg, phosphate buffer, sodium acetate buffer, etc.), a soothing agent (eg, benzalkonium chloride, procaine hydrochloride, etc.), a stabilizer (eg, human serum albumin). , Polyethylene glycol, etc.), preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants and the like. The prepared injection solution is usually filled in a sealed container such as an appropriate ampoule or vial.
[0040]
【Example】
The present invention will be described more specifically with reference to the following examples.
Example 1
A solution prepared by dissolving 206.6 g of a DL-lactic acid polymer (weight average molecular weight 21,900) in 354.8 g of dichloromethane was adjusted to about 30 ° C. 381.5 g of this solution was weighed, 15.8 g of leuprorelin acetate was dissolved in 16.6 g of an acetic acid aqueous solution (0.6 g of glacial acetic acid was dissolved in 31.75 g of distilled water), and the solution was heated to about 55 ° C. And was emulsified using a minimixer (manufactured by Tokushu Kika) to form a W / O emulsion (rotation speed: about 10,000 rpm). Next, after cooling this W / O emulsion to about 18 ° C., 0.1% (w / w) polyvinyl alcohol (EG-40, manufactured by Nippon Synthetic Chemical Co., Ltd.) + 1% mannitol, which was previously adjusted to about 18 ° C. It was poured into 25 liters of an aqueous solution and subjected to secondary emulsification using HOMOMIC LINE FLOW (made by Tokushu Kika) to obtain a W / O / W emulsion (turbine rotation speed about 7,000 rpm, circulation pump rotation speed about 2000 rpm). This W / O / W emulsion is dried in water for about 3 hours, sieved using a 75 μm standard sieve, and then microspheres are continuously settled using a centrifuge (H-600S, manufactured by a domestic centrifuge). And collected (rotation speed: about 2,000 rpm, flow rate: about 600 ml / min). The collected microspheres are re-dispersed in a small amount of distilled water, passed through a 90 μm standard sieve, added with 18.9 g of mannitol, and freeze-dried with a freeze dryer (TRIOMASTER, manufactured by Kyowa Vacuum). Thus, powder (micromicrosphere powder) was obtained. The resulting microsphere had a leuprorelin acetate content of 8.2% and a recovery rate of about 75%.
A W / O emulsion can be obtained satisfactorily by adding acetic acid, and the dispersibility of the obtained microspheres can be improved by adding mannitol to the outer aqueous phase.
[0041]
Experimental example 1
A solution in which 151.3 g of DL-lactic acid polymer (weight average molecular weight 21,900) was dissolved in 259.9 g of dichloromethane was adjusted to a temperature of about 30 ° C. 373.7 g of this solution was weighed, 15.5 g of leuprorelin acetate was dissolved in 16.2 g of acetic acid aqueous solution (0.6 g of glacial acetic acid was dissolved in 31.7 g of distilled water), and the solution was heated to about 55 ° C. And was emulsified using a minimixer (manufactured by Tokushu Kika) to form a W / O emulsion (rotation speed: about 10,000 rpm). A portion of the W / O emulsion was recovered 2, 5 and 8 minutes after the start of emulsification, and the viscosity was measured (vibration viscometer). The results are shown in Table 1.
[Table 1]

A stable W / O emulsion was obtained, and although the viscosity was slightly high at an emulsification time of 8 minutes, there was no increase in viscosity that would cause a manufacturing problem even when emulsified for 5 minutes.
[0042]
Comparative Example 1
A solution in which 151.1 g of DL-lactic acid polymer (weight average molecular weight 21,900) was dissolved in 259.8 g of dichloromethane was adjusted to a temperature of about 30 ° C. 374.6 g of this solution is weighed, 15.5 g of leuprorelin acetate is dissolved in 15.9 g of distilled water and mixed with an aqueous solution heated to about 55 ° C., and then used with a minimixer (made by Tokushu Kika). The mixture was emulsified to form a W / O emulsion (rotation speed: about 10,000 rpm). A portion of the W / O emulsion was recovered 2 and 4 minutes after the start of emulsification, and the viscosity was measured (vibration viscometer). The results are shown in Table 2.
[Table 2]

The viscosity of the W / O emulsion increased in 4 minutes. Compared with Experimental Example 1 performed in the acetic acid addition system, the viscosity increase of the W / O emulsion was remarkable.
[0043]
Experimental example 2
DL-lactic acid polymer (weight average molecular weight 21,900) 1 was prepared by dissolving 0.2061 g of leuprorelin acetate (drug content 97.4%, acetic acid content 6.0%) in 0.2116 g of acetic acid aqueous solution having different acetic acid concentrations. After adding a solution prepared by dissolving 0.82 g in 3.15 g of dichloromethane, the mixture was stirred with a vortex mixer for about 30 seconds to prepare a W / O emulsion. The state of the W / O emulsion was compared. The result is shown in FIG. When the amount of acetic acid was about 1.8 times mol of the drug, small emulsion particles appeared to be formed. The drug gelled at 1.4-fold mol, and a slight gelation tendency was observed at 1.6-fold mol. Uniform emulsions were obtained at 1.8, 2.3, and 2.8 moles, but the 1.8 moles emulsion was a bluish, almost transparent color, while 2.3 moles and more had a slightly whitish emulsion color. It was also confirmed that a transparent color with bluish color was exhibited even at about 1.7 times mole. From this, it was considered that the finest emulsion particles were formed in a molar amount of 1.7 to 1.8 times showing a bluish transparent color.
[0044]
Experimental example 3
0.2 g of leuprorelin acetate (drug content 97.4%, acetic acid content 6.0%) was dissolved in 0.2116 g of aqueous acetic acid solution having different acetic acid concentrations, and a lactic acid glycolic acid copolymer (weight average molecular weight 10500) was obtained. After adding a solution prepared by dissolving 82 g in 3.15 g of dichloromethane, a W / O emulsion was prepared by stirring with a vortex mixer for about 30 seconds. When the states of the W / O emulsion were compared, uniform emulsion particles appeared to be formed when the amount of acetic acid was about 1.8 times mol of the drug. Separation of the oil phase and the inner aqueous phase was observed at 1.3 and 1.4-fold moles.
[0045]
Experimental Example 4
0.22 g of leuprorelin acetate (drug content 97.4%, acetic acid content 6.0%) was dissolved in 0.2116 g of aqueous acetic acid solution having different acetic acid concentrations, and 1.82 g of DL-lactic acid polymer (weight average molecular weight 14500). After adding a solution prepared by dissolving 3.15 g of dichloromethane, the mixture was stirred with a vortex mixer for about 30 seconds to prepare a W / O emulsion. When the states of the W / O emulsion were compared, uniform emulsion particles appeared to be formed when the amount of acetic acid was about 1.8 times mol of the drug. Separation of the oil phase and the inner aqueous phase was observed at 1.3 and 1.4-fold moles.
[0046]
Experimental Example 5
About 110 mg of the microsphere obtained in Example 1 was dispersed in 0.3 ml of a dispersion medium (0.15 mg of carboxymethyl cellulose, 0.3 mg of polysorbate 80, distilled water in which 15 mg of mannitol was dissolved), and a 7-week-old male. The SD rat was subcutaneously administered to the back of the rat with a 22G needle. Table 3 shows the residual ratio obtained by killing the rats after a predetermined time from the administration, taking out the microspheres remaining at the administration site, quantifying the peptide A therein and dividing by the initial content.

As is apparent from Table 3, the microsphere of Example 1 prepared by prescribing only peptide A can contain a physiologically active substance with high trapping efficiency, has good dispersibility, and is an initial stage of the physiologically active substance. Was also suppressed. In addition, the microspheres release physiologically active substances at a constant rate for a very long time.
[0047]
Experimental Example 6
Peptide A acetate 0.6 g was dissolved in 2 wt% acetic acid aqueous solution 0.65 g (1.5 times mol or more with respect to peptide A). To this was added a solution obtained by dissolving 5.4 g of polylactic acid (weight average molecular weight 21,000) in 9.45 g of dichloromethane, and lightly dispersed by hand, and then emulsified with Polytron (manufactured by Kinematica) for a predetermined time to prepare a W / O emulsion. did. The viscosity of the W / O emulsion was measured by changing the emulsification time. The result is shown in FIG.
Similarly, 0.6 g of peptide A was dissolved in 0.635 g of a 2 wt% acetic acid aqueous solution (less than 1.5-fold mol with respect to peptide A). To this was added a solution obtained by dissolving 5.4 g of polylactic acid (weight average molecular weight 21,000) in 9.45 g of dichloromethane, and lightly dispersed by hand, and then emulsified with Polytron (manufactured by Kinematica) for a predetermined time to prepare a W / O emulsion. did.
Those using less than 1.5-fold mol of acetic acid relative to peptide A sometimes increased the viscosity of the W / O emulsion in a relatively short emulsification time, but more than 1.5-fold mol of acetic acid relative to peptide A was used. As a result, the W / O emulsion was stable, and the W / O emulsion could be easily produced without increasing the viscosity in a short time as shown in FIG.
[0048]
From the above experimental results, it was found that the W / O emulsion was stabilized by the coexistence of about 1.5 times mol or more of acetic acid with respect to the drug, and a relatively fine emulsion was obtained at about 1.65 times mol or more. The O-phase polymer was confirmed to be a lactic acid polymer or a lactic acid glycolic acid polymer, and the productivity of the final preparation could be improved.
[0049]
【The invention's effect】
According to the method for producing a sustained-release composition of the present invention, a W / O emulsion can be stably formed, drug leakage during production can be suppressed, the manufacturability of the sustained-release composition can be improved, and the drug can be added. It becomes possible to obtain a sustained-release composition that can be incorporated at a high content and stably release the drug. Furthermore, the sustained-release composition produced using the method of the present invention retains a high content of the drug, exhibits stable drug release characteristics, and is useful as a pharmaceutical product.
By the method of the present invention, a mixed solution of an aqueous solution containing a physiologically active substance and a biodegradable polymer solution can be stabilized, and the viscosity of the mixed solution can be reduced to 3000 cp or less under normal conditions.
[Brief description of the drawings]
1 is a photograph showing the state of a W / O emulsion prepared in Experimental Example 2. FIG. The following numbers are numerical values representing the molar ratio of acetic acid to drug.
FIG. 2 is a graph showing the relationship between emulsification time and W / O emulsion viscosity when 1.5 times or more of acetic acid is used with respect to peptide A in Experimental Example 6. In order from the left, examples of acetic acid / drug molar ratios of 1.4, 1.6, 1.8, 2.3 and 2.8 are shown.

Claims (28)

Formula: 5-oxo-Pro-His -Trp-Ser-Tyr-DLeu-Leu-Arg-Pro-NH-C 2 1.5~5 molar of a physiologically active substance and the physiologically active substance represented by H ₅ A method for producing a sustained-release composition comprising mixing an aqueous solution containing acetic acid and a biodegradable polymer solution, and then drying the mixture.   The method according to claim 1, wherein the aqueous solution is an aqueous solution containing a salt of a physiologically active substance and acetic acid.   The method according to claim 1, wherein the weight ratio of the physiologically active substance in the sustained-release composition is 0.001 to 50% by weight. Formula: allowed to contain 5-oxo-Pro-His- Trp-Ser-Tyr-DLeu-Leu-Arg-Pro-NH-C 2 1.5~5 moles of acetic acid of the biologically active substance represented by H ₅ A method for stabilizing a mixed solution of an aqueous solution containing the physiologically active substance and a biodegradable polymer solution. Formula: allowed to contain 5-oxo-Pro-His- Trp-Ser-Tyr-DLeu-Leu-Arg-Pro-NH-C 2 1.5~5 moles of acetic acid of the biologically active substance represented by H ₅ A method for reducing the viscosity of a mixed solution of an aqueous solution containing the physiologically active substance and a biodegradable polymer solution to 3000 cp or less.   6. The method according to claim 1, 4 or 5, wherein the amount of acetic acid relative to the physiologically active substance is 1.65 to 3 times mol.   The method according to claim 1, 4 or 5, wherein the amount of acetic acid with respect to the physiologically active substance is 1.7 to 1.8 times mol.   The method according to claim 1, 4 or 5, wherein the biodegradable polymer is an α-hydroxycarboxylic acid polymer.   The method according to claim 8, wherein the α-hydroxycarboxylic acid polymer is a lactic acid-glycolic acid polymer.   The method according to claim 9, wherein the lactic acid-glycolic acid polymer has a molar ratio of lactic acid to glycolic acid of 100: 0 to 50:50.   The method according to claim 10, wherein the lactic acid-glycolic acid polymer has a molar ratio of lactic acid to glycolic acid of 100 to 0.   The method according to claim 9, wherein the lactic acid-glycolic acid polymer has a weight average molecular weight of 5,000 to 50,000.   The method according to claim 9, wherein the lactic acid-glycolic acid polymer has a weight average molecular weight of 17,000 to 30,000.   The method according to claim 1, wherein the biodegradable polymer is a lactic acid polymer having a weight average molecular weight of 15,000 to 50,000, wherein the polymer content having a weight average molecular weight of 5,000 or less is 5% by weight or less.   The method according to claim 1, wherein the biodegradable polymer is a lactic acid-glycolic acid polymer having a terminal carboxyl group amount of 20 to 100 µmol (micromol) per unit mass (gram) of the polymer.   The method according to claim 1, wherein the amount of terminal carboxyl groups of the biodegradable polymer is 0.1 to 5 times mol of the physiologically active substance.   The method according to claim 1, 4 or 5, wherein the biodegradable polymer solution is a solution using a poorly water-soluble organic solvent.   The method according to claim 17, wherein the poorly water-soluble organic solvent is dichloromethane.   The method according to claim 1, 4 or 5, wherein the mixed solution is uniformly mixed.   The method according to claim 19, wherein the uniformly mixed liquid is an emulsion.   21. The method of claim 20, wherein the emulsion is W / O type.   The method according to claim 21, wherein the size of the emulsion is fine.   The method according to claim 1, wherein the drying of the mixed solution is drying in water.   24. The method of claim 23, wherein an aqueous solution of an osmotic pressure regulator is used in the outer water phase dried in water.   The method according to claim 24, wherein the osmotic pressure regulator is mannitol.   The method according to claim 1, wherein the sustained-release composition is in the form of fine particles.   27. A method according to claim 26, wherein the microparticles are microspheres or microcapsules.   A sustained release composition produced using the method according to claim 1.

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