JP3682475B2 - Water-soluble anticancer agent - Google Patents
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- JP3682475B2 JP3682475B2 JP21605993A JP21605993A JP3682475B2 JP 3682475 B2 JP3682475 B2 JP 3682475B2 JP 21605993 A JP21605993 A JP 21605993A JP 21605993 A JP21605993 A JP 21605993A JP 3682475 B2 JP3682475 B2 JP 3682475B2
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【０００１】
【産業上の利用分野】
本発明は、ブロック共重合体と、アドリアマイシンとの複合体からなる水溶性抗癌剤に関する。
【０００２】
【従来の技術】
親水性高分子構造部分とアドリアマイシンを結合せしめた高分子構造部分とを有するブロック共重合体からなる水溶性高分子化抗癌剤については、特開平２−３００１３３号公報に記載されている。
【０００３】
【発明が解決しようとする課題】
特開平２−３００１３３号公報に記載された方法によって得られる水溶性高分子化抗癌剤は優れた抗癌活性を示すが、至適投与量がアドリアマイシンに比べかなり高くなっていた。
【０００４】
【課題を解決するための手段】
本発明者らは、前記水溶性高分子化抗癌剤の抗癌活性を改良するために鋭意検討した結果本発明を完成した。
【０００５】
即ち、本発明は、
（１）式（１）、（２）またはこれらの塩の構造を有するブロック共重合体と、アドリアマイシンとの複合体からなる水溶性抗癌剤、
【０００６】
【化３】
（式中、Ｒ1 は水素または低級アルキル基を表し、Ｒ2 は結合基を表し、Ｒ3 はメチレン基またはエチレン基を表し、Ｙは水素または保護基を表し、またＲはそれぞれ独立して水酸基または式（３）の構造を有するアドリアマイシンの残基を表すものとし、ｎは５〜１，０００、ｍは２〜３００、ｘは０〜３００の整数を示すが、ｘはｍより大きくないものとする。）
【０００８】
【化４】
（２）ブロック共重合体が、ポリエチレングリコール構造部分を外側に、ポリアミノ酸またはその塩構造部分を内側とするミセルを形成し、ミセル内にアドリアマイシンを含有するものである上記（１）記載の水溶性抗癌剤、
（３）Ｒ1 がメチル基である上記（１）または（２）記載の水溶性抗癌剤、
（４）Ｒ2 が炭素数２〜４のアルキレン基である上記（１）、（２）または（３）記載の水溶性抗癌剤、
（５）Ｒ3 がメチレン基である上記（１）、（２）、（３）または（４）記載の水溶性抗癌剤、
に関する。
【００１０】
本発明の水溶性抗癌剤は高い薬理効果を有する。
【００１１】
以下、本発明について詳細に説明する。
【００１２】
前記式（１）または（２）において、Ｒ1 は水素または低級アルキル基を表すが、好ましいものはメチル基である。Ｒ2 は本発明の水溶性抗癌剤の水溶性を損なわない限り（好ましくは、さらに本発明の水溶性抗癌剤のミセル形成能を損なわない限り）、特に限定されず、ポリエチレングリコール構造部分の末端にポリアミノ酸構造部分を形成させる際、ポリエチレングリコール構造部分を構成することになる化合物の末端を該形成に適した構造に変換させるために使用した方法及び化合物に対応した構造をとる。例えばメチレン基（−ＣＨ2 −）、エチレン基（−ＣＨ2 ＣＨ2 −）、プロピレン基（−ＣＨ（ＣＨ3 ）ＣＨ2 −）、トリメチレン基（−ＣＨ2 ＣＨ2 ＣＨ2 −）、イソブチレン基（−ＣＨ2 ＣＨ（ＣＨ3 ）ＣＨ2 −）等の炭素数１〜８、好ましくは炭素数１〜４のアルキレン基等が挙げられる。ポリアミノ酸の塩としては、ナトリウム塩、カリウム塩等が挙げられるが、特に限定されるものではない。前記式（１）において、Ｙは水素または保護基を表し、保護基としてはアセチル基等が挙げられるが、特に限定されない。
【００１３】
水溶性抗癌剤に用いられるブロック共重合体は、水溶性である限りその分子量は特に限定されないが、好ましくは１，０００〜１００，０００、特に好ましくは５，０００〜５０，０００である。ブロック共重合体中の、ポリエチレングリコール構造部分とアドリアマイシンを結合せしめたポリアミノ酸構造部分の割合は、ブロック共重合体の水溶性が保たれる限り特に限定されないが、好ましくは１：０．１〜１０（重量比）、特に好ましくは１：０．２〜５（重量比）である。また、ｎは５〜１，０００であるが好ましくは１５〜４００であり、ｍは２〜３００であるが好ましくは１０〜１００であり、ｘは０〜３００であるが好ましくは０〜１００である。ブロック共重合体に結合させるアドリアマイシンの量は特に限定されないが、通常ブロック共重合体中の量が３〜８０重量％となる量であり、好ましくは５〜６０重量％となる量である。ブロック共重合体に結合しているアドリアマイシンの量と結合していないアドリアマイシンの量の比は特に限定されないが、通常１：０．０１〜１０（重量比）であり、好ましくは１：０．１〜２である。
【００１４】
ブロック共重合体におけるアドリアマイシンを結合せしめる担持用担体は種々の方法により製造することができる。例えばポリエチレングリコールまたはその末端を化学修飾したものにポリアミノ酸を反応させ、その後保護基を含むものは保護基を除去することにより、またはポリエチレングリコールまたはその末端を化学修飾したものと重合性アミノ酸またはアミノ酸誘導体モノマーを反応させ、保護基を含むものは保護基を除去することにより担持用担体が得られる。
【００１５】
ポリエチレングリコールの末端の化学修飾は、公知の方法によって行うことができ、例えば水酸基をアミノ基に変換する方法として、エチレンイミンを反応させる方法、アクリロニトリルやメタクリロニトリルをマイケル付加後、ニトリル基を還元しアミノ基に変換する方法、水酸基をハロゲン基に置換した後、エタノールアミン等のアルコールアミンを反応する方法、または水酸基を直接ニトリルに変換後、還元しアミノ基に変換する方法等で行うことができる。また、水酸基をカルボキシル基に変換する方法として、通常の酸化反応、縮合反応、付加反応、加水分解反応、またはこれらを組み合わせた反応等を採用できる。例えば、水酸基を金属ナトリウムでアルコラートとした後、ブロモ酢酸エチル等のハロゲン化脂肪酸エステルを付加し、その後加水分解する方法で水酸基をカルボキシル基に変換することができる。
【００１６】
また、保護基を除去する方法は、アルカリによる方法、酸による方法及び還元法で可能である。アルカリ法で用いるアルカリ性物質としては、カセイソーダ、カセイカリ、ヒドラジン、アンモニア等通常のアルカリ性物質を用いることができる。酸法で用いる酸性物質としては、トリフルオロメタンスルホン酸、メタンスルホン酸、トリフルオロ酢酸、酢酸、ギ酸、フッ化水素酸、臭化水素酸、塩化水素酸等の通常の酸性物質を用いることができる。また副反応を防止するため、アニソール、チオアニソール、ｍ−クレゾール、ｏ−クレゾール等を加えることもできる。還元法としては、接触還元法、接触水素移動還元法等一般的な方法を用いることができる。
【００１７】
また、ポリアミノ酸構造部分が、末端にアミノ基を有する場合、末端アミノ基を修飾したものを担持用担体として用いることもできる。修飾法としては酸無水物または酸ハロゲン化物等を用いる方法が挙げられる。修飾は保護基を除去する前でも後でもどちらでも可能である。
【００１８】
このようにして得られる担持用担体に必要によりアドリアマイシンを反応させることにより本発明で用いられるブロック共重合体が得られる。例えば式（１）のブロック共重合体でアドリアマイシンが結合したものを得るには、式（１）においてすべてのＲが水酸基である担持用担体にアドリアマイシンを反応させればよい。担持用担体にアミド結合でアドリアマイシンを結合させる際、反応はペプチド結合生成法として知られる常法に準じて行うことができる。例えば、酸ハロゲン化物法、酸無水物法、カップリング法等が使用できるが、縮合剤を使用するカップリング法が望ましい。縮合剤としては、１−エチル−３−（３−ジメチルアミノプロピル）カルボジイミド（ＥＤＣ）、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（ＥＤＣ・ＨＣｌ）、ジシクロヘキシルカルボジイミド（ＤＣＣ）、カルボニルジイミダゾール（ＣＤＩ）、１−エトキシカルボニル−２−エトキシ−１，２−ジヒドロキシキノリン（ＥＥＤＱ）、ジフェニルホスホリルアジド（ＤＰＰＡ）等が使用できる。縮合剤はアドリアマイシンに対して０．５〜２０倍モル用いるのが好ましく、特に１〜１０倍モル用いるのが好ましい。またこの際、Ｎ−ヒドロキシサクシンイミド（ＨＯＮＳｕ）、１−ヒドロキシベンゾトリアゾール（ＨＯＢｔ）、Ｎ−ヒドロキシ−５−ノルボルネン−２，３−ジカルボン酸イミド（ＨＯＮＢ）等を共存させてもよい。
【００１９】
アドリアマイシンの使用量は特に限定されないが、通常担持用担体のカルボキシル基１当量に対し、０．１〜２モル用いる。
【００２０】
縮合反応は溶媒中で行うのが好ましく、溶媒としては、例えば、Ｎ，Ｎ−ジメチルホルムアミド（ＤＭＦ）、ジメチルスルホキシド（ＤＭＳＯ）、ジオキサン、テトラヒドロフラン（ＴＨＦ）、水及びそれらの混合溶媒等種々のものが使用でき、特に限定されない。溶媒の使用量は特に限定されないが、通常担持用担体に対して１０〜５００重量倍用いる。
【００２１】
縮合反応は、−１０〜４０℃で行うのが好ましく、特に、−５〜３０℃で行うのが好ましい。反応は２〜４８時間行えば充分である。
【００２２】
本発明で用いられるブロック共重合体に、アドリアマイシンを添加した後、適当な処理をすることにより、本発明の水溶性抗癌剤が得られる。あるいはアドリアマイシンを添加する代わりに、縮合反応の条件により、すなわちアドリアマイシンの量を多めにする、縮合剤の量を少なめにする、反応時間を短くするといった方法により、未反応のアドリアマイシンを残した後、適当な処理をすることにより得ることもできる。
【００２３】
処理の方法としては、縮合反応液にアドリアマイシンまたはアドリアマイシンの溶液を添加したもの、あるいは未反応のアドリアマイシンを残した縮合反応液を透析、限外濾過することにより、水溶液とする方法が挙げられる。あるいは縮合反応液をイソプロピルエーテル（ＩＰＥ）等の貧溶媒で沈析した後適当な溶媒に溶解したもの、もしくはブロック共重合体を適当な溶媒に溶解したものにアドリアマイシンまたはアドリアマイシンの溶液を添加し、透析、限外濾過してもよい。あるいは縮合反応液を透析、限外濾過した後、アドリアマイシンまたはアドリアマイシンの溶液を添加し、再度透析、限外濾過してもよい。ブロック共重合体とアドリアマイシンを混合する際用いる溶媒としては、ブロック共重合体とアドリアマイシンを共によく溶解するものが好ましい。例えばＤＭＦ、ＤＭＦと水の混合溶媒等が挙げられる。また、ブロック共重合体とアドリアマイシンを混合する際、超音波照射等の処理を行ってもよい。
【００２４】
限外濾過を行った後でも、ブロック共重合体に結合していないアドリアマイシンが存在することは、種々の分析手段により確認できる。例えば、液体クロマトグラフ、質量分析等の手段が挙げられる。定量することも可能である。
【００２５】
以下に、ポリエチレングリコール構造部分とポリアスパラギン酸構造部分とからなる担持用担体で、アドリアマイシンをポリアスパラギン酸の側鎖に結合させたブロック共重合体にアドリアマイシンを添加した水溶性抗癌剤を例にとり、その合成法を詳しく述べる。
【００２６】
この水溶性抗癌剤に用いるブロック共重合体の合成は、以下の反応式に示すごとくβ−ベンジル−Ｌ−アスパルテート−Ｎ−カルボン酸無水物（ＢＬＡ−ＮＣＡ）を、片末端にメトキシ基を有し、他の片末端に３−アミノプロピル基を有するポリエチレングリコール（ＰＥＧ−ＮＨ2 ）を開始剤として、ＤＭＦ、ＤＭＳＯ、ジオキサン、クロロホルム、ＴＨＦ、アセトニトリル等の溶媒中で開環重合させ、ポリエチレングリコール−ポリ（β−ベンジル−Ｌ−アスパルテート）ブロック共重合体（ＰＥＧ−ＰＢＬＡ）を得、次いでこのＰＥＧ−ＰＢＬＡのベンジルエステルを加水分解してポリエチレングリコール−ポリアスパラギン酸ブロック共重合体（ＰＥＧ−Ｐ（Ａｓｐ．））を得る。このＰＥＧ−Ｐ（Ａｓｐ．）に抗癌剤のアドリアマイシンとＥＤＣ、ＤＣＣ等の縮合剤を加え、溶液中で反応させることにより、アドリアマイシンの一級アミノ基とポリアスパラギン酸のカルボキシル基とをアミド結合で結合させたブロック共重合体（ＰＥＧ−Ｐ（Ａｓｐ．）ＡＤＲを得る。
【００２７】
【化５】
（式中、Ｒは水酸基あるいは式（３）を表し、ｎは５〜１，０００、ｍは２〜３００、ｘは０〜３００の整数を示すが、ｘはｍより大きくないものとする。）
【００２９】
【化６】
縮合反応液を透析、限外濾過し、アドリアマイシン換算で２０ｍｇ／ｍｌ程度の水溶液とし、アドリアマイシンのＤＭＦ溶液を添加し、１０分間超音波照射を行い、その後再度透析、限外濾過を行うことにより、本発明の水溶性抗癌剤を得ることができる。あるいは縮合反応液をＩＰＥで沈析した後ＤＭＦに溶解し、アドリアマイシンのＤＭＦ−水混合溶液を添加し、１０分間超音波照射を行い、透析、限外濾過を行っても得ることができる。
【００３１】
本発明の水溶性抗癌剤は高いアドリアマイシン含量にもかかわらず良好な水溶性を有しており、凍結乾燥したり濃縮してもその水溶性は保たれている。
【００３２】
この水溶性抗癌剤の抗癌活性は、表１に示すように元のアドリアマイシン自体よりも高いものである。しかもその高い抗癌活性はアドリアマイシンよりも少ない副作用の範囲でも達成される。また特開平２−３００１３３号公報に記載されている水溶性高分子化抗癌剤に比べ、少ない投与量で同等の抗癌活性を示している。
【００３３】
本発明の水溶性抗癌剤は、一般的に使用される種々の剤型、例えば固形剤、軟膏、液剤などの形で使用しうるが、通常注射剤として使用され、その投与量は、１週間当り１〜３回投与で、総量５０〜１，０００ｍｇ／ｍ2 週程度である。
【００３４】
【実施例】
次に実施例により本発明を具体的に説明する。
【００３５】
実施例１
β−ベンジル−Ｌ−アスパルテート−Ｎ−カルボン酸無水物（ＢＬＡ−ＮＣＡ）７．１ｇをＮ，Ｎ−ジメチルホルムアミド（ＤＭＦ）７０ｍｌに溶解した。片末端メトキシ基片末端３−アミノプロピル基のポリエチレングリコール（ＰＥＧ−ＮＨ2 ）（分子量５，１００）５．０ｇをＤＭＦ５０ｍｌに溶解し、その溶液をＢＬＡ−ＮＣＡ溶液に加えた。４０時間後に反応混合物をイソプロピルエーテル（ＩＰＥ）１，０００ｍｌに滴下して沈澱したポリマーを濾過で回収し、ＩＰＥで洗浄した後に真空乾燥してポリエチレングリコール−ポリ（β−ベンジル−Ｌ−アスパルテート）ブロック共重合体（ＰＥＧ−ＰＢＬＡ）９．３ｇを得た。ＰＥＧ−ＰＢＬＡ７．０ｇを０．５Ｎ水酸化ナトリウムに懸濁しながら室温でベンジルエステルを加水分解した。ポリマーが溶解した後、酢酸でｐＨを酸性とし、ＡＤＶＡＮＴＥＣ ＵＫ−１０（分画分子量＝１０，０００）の限外濾過膜で限外濾過した。濃縮液を凍結乾燥してポリエチレングリコール−ポリアスパラギン酸ブロック共重合体（ＰＥＧ−Ｐ（Ａｓｐ．））５．１ｇを得た。このＰＥＧ−Ｐ（Ａｓｐ．）１，２３０ｍｇを水１５ｍｌに溶解した。アドリアマイシン塩酸塩１，５００ｍｇをＤＭＦ１５０ｍｌに懸濁し、氷冷下トリエチルアミン３５３μｌを加えた後ＰＥＧ−Ｐ（Ａｓｐ．）水溶液を加えた。この混合溶液にＮ−ヒドロキシサクシンイミド３７５ｍｇ、および１−エチル−３−（３−ジメチルアミノプロピル）カルボジイミド（ＥＤＣ）５２５μｌを加え、氷冷下４時間反応させた。その後ＥＤＣ５２５μｌを追加し室温で１８時間反応させた。反応後、反応混合液中には全アドリアマイシンに対して２．５％の未反応のアドリアマイシンが残っていた。反応混合液をシリカゲルカラム（ワコーゲルＣ−１００）およびイオン交換樹脂（強酸性イオン交換樹脂ＰＫ−２１６）を用いて精製し、未反応のアドリアマイシン等の低分子物質を除去した。精製後の液を透析膜（分画分子量＝１２，０００）を用いて０．１Ｍ酢酸中で３時間透析した。透析後、ＡＤＶＡＮＴＥＣ ＵＫ−５０（分画分子量＝５０，０００）の限外濾過膜で限外濾過した。水洗と濃縮を繰り返し、アドリアマイシン換算で２０ｍｇ／ｍｌ（紫外分光光度計で４８５ｎｍの吸収より算出）の水溶液３５ｍｌを得た。得られたＰＥＧ−Ｐ（Ａｓｐ．）ＡＤＲは前記式（１）の構造を有し、Ｒ1 はメチル基、Ｒ2 はトリメチレン基、Ｒ3 はメチレン基、Ｙは水素を表す。ｎ＝１１５、ｍ＝２０、ｘ＝４でＲの一部は水酸基で残りは前記残基式（３）である。アドリアマイシン含有率は４７重量％であるが良好な水溶性を示した。このＰＥＧ−Ｐ（Ａｓｐ．）ＡＤＲの２０ｍｇ／ｍｌ（アドリアマイシン換算）の水溶液１０ｍｌにアドリアマイシン塩酸塩２０ｍｇ、トリエチルアミン５μｌをＤＭＦ２０ｍｌに溶解した溶液を添加し、１０分間の超音波照射を行った後、透析膜（分画分子量＝１２，０００）を用いて０．０１Ｍ酢酸中で３時間透析した。透析後、ＡＤＶＡＮＴＥＣ ＵＫ−５０（分画分子量＝５０，０００）の限外濾過膜で限外濾過した。水洗と濃縮を繰り返し、アドリアマイシン換算で２０ｍｇ／ｍｌ（紫外分光光度計で４８５ｎｍの吸収より算出）の水溶液１０．２ｍｌを得た。液体クロマトグラフによる分析の結果、ブロック共重合体に結合していないアドリアマイシンの量は全アドリアマイシン中の８．５％であった。このものも元のＰＥＧ−Ｐ（Ａｓｐ．）ＡＤＲと同様良好な水溶性を示した。
【００３６】
実施例２
実施例１で得たＰＥＧ−Ｐ（Ａｓｐ．）ＡＤＲの２０ｍｇ／ｍｌ（アドリアマイシン換算）の水溶液１０ｍｌにアドリアマイシン塩酸塩５０ｍｇ、トリエチルアミン１２μｌをＤＭＦ２０ｍｌに溶解した溶液を添加し、１０分間の超音波照射を行った後、透析膜（分画分子量＝１２，０００）を用いて０．０１Ｍ酢酸中で３時間透析した。透析後、ＡＤＶＡＮＴＥＣ ＵＫ−５０（分画分子量＝５０，０００）の限外濾過膜で限外濾過した。水洗と濃縮を繰り返し、アドリアマイシン換算で２０ｍｇ／ｍｌ（紫外分光光度計で４８５ｎｍの吸収より算出）の水溶液１１．５ｍｌを得た。液体クロマトグラフによる分析の結果、ブロック共重合体に結合していないアドリアマイシンの量は全アドリアマイシン中の１８．４％であった。このものも元のＰＥＧ−Ｐ（Ａｓｐ．）ＡＤＲと同様良好な水溶性を示した。
【００３７】
実施例３
β−ベンジル−Ｌ−アスパルテート−Ｎ−カルボン酸無水物（ＢＬＡ−ＮＣＡ）６．４ｇをＮ，Ｎ−ジメチルホルムアミド（ＤＭＦ）６０ｍｌに溶解した。片末端メトキシ基片末端３−アミノプロピル基のポリエチレングリコール（ＰＥＧ−ＮＨ2 ）（分子量５，１００）３．０ｇをＤＭＦ３０ｍｇに溶解し、その溶液をＢＬＡ−ＮＣＡ溶液に加えた。４０時間後に反応混合物をイソプロピルエーテル（ＩＰＥ）７００ｍｌに滴下して沈澱したポリマーを濾過で回収し、ＩＰＥで洗浄した後に真空乾燥してポリエチレングリコール−ポリ（β−ベンジル−Ｌ−アスパルテート）ブロック共重合体（ＰＥＧ−ＰＢＬＡ）７．２ｇを得た。ＰＥＧ−ＰＢＬＡ５．０ｇを０．５Ｎ水酸化ナトリウムに懸濁しながら室温でベンジルエステルを加水分解した。ポリマーが溶解した後、酢酸でｐＨを酸性とし、ＡＤＶＡＮＴＥＣ ＵＫ−１０（分画分子量＝１０，０００）の限外濾過膜で限外濾過した。濃縮液を凍結乾燥してポリエチレングリコール−ポリアスパラギン酸ブロック共重合体（ＰＥＧ−Ｐ（Ａｓｐ．））３．１ｇを得た。このＰＥＧ−Ｐ（Ａｓｐ．）１，０１０ｍｇを水１５ｍｌに溶解した。アドリアマイシン塩酸塩１，５００ｍｇをＤＭＦ１５０ｍｌに懸濁し、氷冷下トリエチルアミン３５３μｌを加えた後ＰＥＧ−Ｐ（Ａｓｐ．）水溶液を加えた。この混合溶液にＮ−ヒドロキシサクシンイミド３７５ｍｇ、および１−エチル−３−（３−ジメチルアミノプロピル）カルボジイミド（ＥＤＣ）５２５μｌを加え、氷冷下４時間反応させた。その後ＥＤＣ５２５μｌを追加し、室温で１８時間反応させた。反応後、反応混合液中には全アドリアマイシンに対して２．３％の未反応のアドリアマイシンが残っていた。反応混合液をシリカゲルカラム（ワコーゲルＣ−１００）およびイオン交換樹脂（強酸性イオン交換樹脂ＰＫ−２１６）を用いて精製し、未反応のアドリアマイシン等の低分子物質を除去した。精製後の液を透析膜（分画分子量＝１２，０００）を用いて０．１Ｍ酢酸中で３時間透析した。透析後、ＡＤＶＡＮＴＥＣ ＵＫ−５０（分画分子量＝５０，０００）の限外濾過膜で限外濾過した。水洗と濃縮を繰り返し、アドリアマイシン換算で２０ｍｇ／ｍｌ（紫外分光光度計で４８５ｎｍの吸収より算出）の水溶液３７ｍｌを得た。得られたＰＥＧ−Ｐ（Ａｓｐ．）ＡＤＲは前記式（１）の構造を有し、Ｒ1 はメチル基、Ｒ2 はトリメチレン基、Ｒ3 はメチレン基、Ｙは水素を表す。ｎ＝１１５、ｍ＝３０、ｘ＝６でＲの一部は水酸基で残りは前記式（３）である。アドリアマイシン含有率は５４重量％であるが良好な水溶性を示した。このＰＥＧ−Ｐ（Ａｓｐ．）ＡＤＲの２０ｍｇ／ｍｌ（アドリアマイシン換算）の水溶液１０ｍｌにアドリアマイシン塩酸塩１５ｍｇ、トリエチルアミン４μｌをＤＭＦ２０ｍｌに溶解した溶液を添加し、１０分間の超音波照射を行った後、透析膜（分画分子量＝１２，０００）を用いて０．０１Ｍ酢酸中で３時間透析した。透析後、ＡＤＶＡＮＴＥＣ ＵＫ−５０（分画分子量＝５０，０００）の限外濾過膜で限外濾過した。水洗と濃縮を繰り返し、アドリアマイシン換算で２０ｍｇ／ｍｌ（紫外分光光度計で４８５ｎｍの吸収より算出）の水溶液９．８ｍｌを得た。液体クロマトグラフによる分析の結果、ブロック共重合体に結合していないアドリアマイシンの量は全アドリアマイシン中の５．５％であった。このものも元のＰＥＧ−Ｐ（Ａｓｐ．）ＡＤＲと同様良好な水溶性を示した。
【００３８】
実施例４
実施例３で得たＰＥＧ−Ｐ（Ａｓｐ．）ＡＤＲの２０ｍｇ／ｍｌ（アドリアマイシン換算）の水溶液１０ｍｌにアドリアマイシン塩酸塩５０ｍｇ、トリエチルアミン１２μｌをＤＭＦ２０ｍｌに溶解した溶液を添加し、１０分間の超音波照射を行った後、透析膜（分画分子量＝１２，０００）を用いて０．０１Ｍ酢酸中で３時間透析した。透析後、ＡＤＶＡＮＴＥＣ ＵＫ−５０（分画分子量＝５０，０００）の限外濾過膜で限外濾過した。水洗と濃縮を繰り返し、アドリアマイシン換算で２０ｍｇ／ｍｌ（紫外分光光度計で４８５ｎｍの吸収より算出）の水溶液１０．５ｍｌを得た。液体クロマトグラフによる分析の結果、ブロック共重合体に結合していないアドリアマイシンの量は全アドリアマイシン中の１４．４％であった。このものも元のＰＥＧ−Ｐ（Ａｓｐ．）ＡＤＲと同様良好な水溶性を示した。
【００３９】
実施例５
実施例３で得たＰＥＧ−ＰＢＬＡ２．０ｇを０．５Ｎ水酸化ナトリウムに懸濁しながら室温でベンジルエステルを加水分解した。ポリマーが溶解した後、酢酸でｐＨをほぼ中性とし、アミコンＹＭ−１（分画分子量＝１，０００）の限外濾過膜で限外濾過した。限外濾過を続けながら、０．０５Ｎ塩酸で洗浄し、さらに水洗を充分に行った。濃縮液を凍結乾燥してポリエチレングリコール−ポリアスパラギン酸ブロック共重合体（ＰＥＧ−Ｐ（Ａｓｐ．））１．３ｇを得た。このＰＥＧ−Ｐ（Ａｓｐ．）１００ｍｇをＤＭＦ１０ｍｌに溶解し、氷冷下アドリアマイシン塩酸塩７１．２ｍｇ、トリエチルアミン２７．３μｌ、および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（ＥＤＣ・ＨＣｌ）６７．２ｍｇを加え４時間反応させた。さらに室温で２０時間反応させた。反応後、反応混合液中には未反応のアドリアマイシンはほとんど残っていなかった。反応混合液をイソプロピルエーテル（ＩＰＥ）１００ｍｌに滴下して沈澱したポリマーを濾過で回収し、ＩＰＥで洗浄した後真空乾燥した。水に溶解し、アミコンＹＭ−３（分画分子量＝１，０００）の限外濾過膜で限外濾過した。限外濾過を続けながら、水、メタノール、酢酸の混合液で洗浄し、さらに水洗を充分に行った後濃縮して、固形分重量で約５０ｍｇ／ｍｌ（一部を凍結乾燥してその重量より算出）の水溶液３．１ｍｌを得た。得られたＰＥＧ−Ｐ（Ａｓｐ．）ＡＤＲは前記式（１）の構造を有し、Ｒ1 はメチル基、Ｒ2 はトリメチレン基、Ｒ3 はメチレン基、Ｙは水素を表す。ｎ＝１１５、ｍ＝３０、ｘ＝６でＲの一部は水酸基で残りは前記式（３）である。アドリアマイシン含有率は４０％であるが良好な水溶性を示した。このＰＥＧ−Ｐ（Ａｓｐ．）ＡＤＲの５０ｍｇ／ｍｌ（固形分）の水溶液１ｍｌにアドリアマイシン塩酸塩１０ｍｇ、トリエチルアミン２．５μｌをＤＭＦ１０ｍｌに溶解した溶液を添加し、１０分間室温で撹拌した後、透析膜（分画分子量＝１２，０００）を用いて水中で３時間透析した。透析後、ＡＤＶＡＮＴＥＣ ＵＫ−５０（分画分子量＝５０，０００）の限外濾過膜で限外濾過した。水洗と濃縮を繰り返し、２ｍｌの水溶液を得た。液体クロマトグラフによる分析の結果、ブロック共重合体に結合していないアドリアマイシンの量は全アドリアマイシン中の１８％であった。このものも元のＰＥＧ−Ｐ（Ａｓｐ．）ＡＤＲと同様良好な水溶性を示した。
【００４０】
応用例１
ＣＤＦ１メスのマウスの背側部皮下にマウス大腸癌Ｃｏｌｏｎ ２６細胞を移植し、腫瘍の体積が１００ｍｍ3 前後に達した時点から実施例１、２、３または４で得られた水溶性抗癌剤、または実施例１および２または３および４に対応する抗癌剤（フリーのアドリアマイシン塩酸塩）を添加しないＰＥＧ−Ｐ（Ａｓｐ．）ＡＤＲ、またはアドリアマイシン塩酸塩を４日間隔１回、計３回静脈内に投与し、進行癌に対する効果を検討した。各薬剤は生理食塩水に用時溶解して用いた。また投与量はすべて全アドリアマイシンの量に換算して用いた。薬剤の抗腫瘍効果は、腫瘍消失マウス数と腫瘍増殖曲線から判定した。結果を表１と図１〜７に示す。図から明らかなように、アドリアマイシン塩酸塩を投与した場合、移植した腫瘍の増殖抑制効果は認められるが腫瘍の縮小はほとんど認められないのに対し、本発明の水溶性抗癌剤を投与した場合、移植した腫瘍の増殖抑制効果に優れ、移植した腫瘍が消失したケースも認められた。また、投与量については、ブロック共重合体に結合していないアドリアマイシンの割合が増えるに従って、低い投与量で抗癌活性が見られた。また、毒性の一つの指標である体重減少については、本発明の水溶性抗癌剤はアドリアマイシンに比べ低い体重変化の範囲でも、腫瘍の消失が見られるマウスが存在した。
【００４１】
【表１】
【発明の効果】
本発明の水溶性抗癌剤は、良好な水溶性を有し、しかも遊離のアドリアマイシンに比較して低い毒性の範囲でも高い抗腫瘍効果を示す。また、従来の水溶性高分子化抗癌剤に比べ、低い投与量で同等の抗癌活性を示すことより、本発明により極めて有用な医薬を提供できるものである。
【図面の簡単な説明】
【図１】アドリアマイシン塩酸塩を投与した場合の、マウス大腸癌Ｃｏｌｏｎ ２６の腫瘍増殖曲線。
【図２】実施例１の水溶性抗癌剤を投与した場合の、マウス大腸癌Ｃｏｌｏｎ ２６の腫瘍増殖曲線。
【図３】実施例２の水溶性抗癌剤を投与した場合の、マウス大腸癌Ｃｏｌｏｎ ２６の腫瘍増殖曲線。
【図４】実施例１および２に対応する抗癌剤（フリーのアドリアマイシン塩酸塩）を添加しないＰＥＧ−Ｐ（Ａｓｐ．）ＡＤＲを投与した場合の、マウス大腸癌Ｃｏｌｏｎ ２６の腫瘍増殖曲線。
【図５】実施例３の水溶性抗癌剤を投与した場合の、マウス大腸癌Ｃｏｌｏｎ ２６の腫瘍増殖曲線。
【図６】実施例４の水溶性抗癌剤を投与した場合の、マウス大腸癌Ｃｏｌｏｎ ２６の腫瘍増殖曲線。
【図７】実施例３および４に対応する抗癌剤（フリーのアドリアマイシン塩酸塩）を添加しないＰＥＧ−Ｐ（Ａｓｐ．）ＡＤＲを投与した場合の、マウス大腸癌Ｃｏｌｏｎ ２６の腫瘍増殖曲線。[0001]
[Industrial application fields]
The present invention relates to a water-soluble anticancer agent comprising a complex of a block copolymer and adriamycin.
[0002]
[Prior art]
A water-soluble polymerized anticancer agent comprising a block copolymer having a hydrophilic polymer structure portion and a polymer structure portion to which adriamycin is bonded is described in JP-A No. 2-300133.
[0003]
[Problems to be solved by the invention]
Although the water-soluble polymerized anticancer agent obtained by the method described in JP-A No. 2-300133 shows excellent anticancer activity, the optimum dose is considerably higher than that of adriamycin.
[0004]
[Means for Solving the Problems]
The inventors of the present invention have completed the present invention as a result of intensive studies to improve the anticancer activity of the water-soluble polymerized anticancer agent.
[0005]
That is, the present invention
(1) A water-soluble anticancer agent comprising a complex of a block copolymer having the structure of formula (1), (2) or a salt thereof and adriamycin,
[0006]
[Chemical 3]
(Wherein R 1 Represents hydrogen or a lower alkyl group, R 2 Represents a linking group and R Three Represents a methylene group or an ethylene group, Y represents a hydrogen atom or a protecting group, and each R independently represents a hydroxyl group or a residue of adriamycin having the structure of the formula (3), and n is from 5 to 1, 000, m represents an integer of 2 to 300, and x represents an integer of 0 to 300, but x is not larger than m. )
[0008]
[Formula 4]
(2) The water-soluble solution according to the above (1), wherein the block copolymer forms a micelle having a polyethylene glycol structure portion on the outside and a polyamino acid or a salt structure portion on the inside, and contains adriamycin in the micelle. Sex cancer drugs,
(3) R 1 The water-soluble anticancer agent according to the above (1) or (2), wherein is a methyl group,
(4) R 2 The water-soluble anticancer agent according to (1), (2) or (3), wherein is an alkylene group having 2 to 4 carbon atoms,
(5) R Three The water-soluble anticancer agent according to (1), (2), (3) or (4), wherein is a methylene group,
About.
[0010]
The water-soluble anticancer agent of the present invention has a high pharmacological effect.
[0011]
Hereinafter, the present invention will be described in detail.
[0012]
In the formula (1) or (2), R 1 Represents hydrogen or a lower alkyl group, and preferred is a methyl group. R 2 Is not particularly limited as long as the water solubility of the water-soluble anticancer agent of the present invention is not impaired (preferably, unless the ability of micelle formation of the water-soluble anticancer agent of the present invention is further impaired). When the portion is formed, the structure corresponding to the method and the compound used to convert the terminal of the compound constituting the polyethylene glycol structural portion into a structure suitable for the formation is adopted. For example, a methylene group (—CH 2 -), Ethylene group (-CH 2 CH 2 -), Propylene group (-CH (CH Three ) CH 2 -), Trimethylene group (-CH 2 CH 2 CH 2 -), Isobutylene group (-CH 2 CH (CH Three ) CH 2 Examples thereof include alkylene groups having 1 to 8 carbon atoms, preferably 1 to 4 carbon atoms such as-). Examples of the polyamino acid salt include, but are not particularly limited to, a sodium salt and a potassium salt. In the formula (1), Y represents hydrogen or a protecting group, and examples of the protecting group include an acetyl group, but are not particularly limited.
[0013]
The molecular weight of the block copolymer used for the water-soluble anticancer agent is not particularly limited as long as it is water-soluble, but it is preferably 1,000 to 100,000, particularly preferably 5,000 to 50,000. The ratio of the polyamino acid structural part in which the polyethylene glycol structural part and adriamycin are combined in the block copolymer is not particularly limited as long as the water solubility of the block copolymer is maintained, but preferably 1: 0.1 to 0.1 10 (weight ratio), particularly preferably 1: 0.2 to 5 (weight ratio). N is 5 to 1,000, preferably 15 to 400, m is 2 to 300, preferably 10 to 100, and x is 0 to 300, preferably 0 to 100. is there. The amount of adriamycin to be bound to the block copolymer is not particularly limited, but is usually an amount such that the amount in the block copolymer is 3 to 80% by weight, and preferably 5 to 60% by weight. The ratio of the amount of adriamycin bound to the block copolymer to the amount of unbound adriamycin is not particularly limited, but is usually 1: 0.01 to 10 (weight ratio), preferably 1: 0.1. ~ 2.
[0014]
The carrier for supporting adriamycin in the block copolymer can be produced by various methods. For example, a polyamino acid is reacted with polyethylene glycol or a chemically modified end thereof, and then a protecting group is removed by removing a protecting group, or polyethylene glycol or a chemically modified end thereof and a polymerizable amino acid or amino acid A carrier for support can be obtained by reacting a derivative monomer and removing the protecting group from those containing a protecting group.
[0015]
Chemical modification of the end of polyethylene glycol can be carried out by a known method. For example, as a method of converting a hydroxyl group to an amino group, a method of reacting ethyleneimine, Michael addition of acrylonitrile or methacrylonitrile, and reduction of the nitrile group This can be carried out by a method for converting to an amino group, a method for reacting an alcohol amine such as ethanolamine after substitution of a hydroxyl group with a halogen group, or a method for converting a hydroxyl group directly to a nitrile and then reducing to a amino group. it can. Moreover, as a method for converting a hydroxyl group into a carboxyl group, a normal oxidation reaction, condensation reaction, addition reaction, hydrolysis reaction, or a combination of these can be employed. For example, the hydroxyl group can be converted to a carboxyl group by a method in which the hydroxyl group is converted to an alcoholate with metallic sodium, a halogenated fatty acid ester such as ethyl bromoacetate is added, and then hydrolyzed.
[0016]
Moreover, the method of removing a protecting group is possible by the method by an alkali, the method by an acid, and the reduction method. Usable alkaline substances such as caustic soda, caustic potash, hydrazine, and ammonia can be used as the alkaline substance used in the alkali method. As the acidic substance used in the acid method, ordinary acidic substances such as trifluoromethanesulfonic acid, methanesulfonic acid, trifluoroacetic acid, acetic acid, formic acid, hydrofluoric acid, hydrobromic acid, and hydrochloric acid can be used. . In order to prevent side reactions, anisole, thioanisole, m-cresol, o-cresol and the like can be added. As the reduction method, a general method such as a catalytic reduction method or a catalytic hydrogen transfer reduction method can be used.
[0017]
Moreover, when the polyamino acid structure part has an amino group at the terminal, a modified terminal amino group can be used as a carrier for support. Examples of the modification method include a method using an acid anhydride or an acid halide. Modification can be done either before or after removal of the protecting group.
[0018]
The block copolymer used in the present invention can be obtained by reacting the thus obtained supporting carrier with adriamycin as necessary. For example, in order to obtain a block copolymer of formula (1) to which adriamycin is bonded, adriamycin may be reacted with a carrier for support in which all Rs are hydroxyl groups in formula (1). When adriamycin is bound to the support by an amide bond, the reaction can be performed according to a conventional method known as a peptide bond generation method. For example, an acid halide method, an acid anhydride method, a coupling method and the like can be used, but a coupling method using a condensing agent is desirable. Examples of the condensing agent include 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC / HCl), and dicyclohexylcarbodiimide (DCC). Carbonyldiimidazole (CDI), 1-ethoxycarbonyl-2-ethoxy-1,2-dihydroxyquinoline (EEDQ), diphenylphosphoryl azide (DPPA) and the like can be used. The condensing agent is preferably used in an amount of 0.5 to 20 times, particularly 1 to 10 times the mol of adriamycin. At this time, N-hydroxysuccinimide (HONSu), 1-hydroxybenzotriazole (HOBt), N-hydroxy-5-norbornene-2,3-dicarboxylic acid imide (HONB), etc. may coexist.
[0019]
Although the amount of adriamycin used is not particularly limited, it is usually used in an amount of 0.1 to 2 mol per 1 equivalent of the carboxyl group of the carrier for support.
[0020]
The condensation reaction is preferably carried out in a solvent. Examples of the solvent include N, N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), dioxane, tetrahydrofuran (THF), water, and mixed solvents thereof. Can be used and is not particularly limited. The amount of the solvent used is not particularly limited, but is usually 10 to 500 times by weight with respect to the carrier for support.
[0021]
The condensation reaction is preferably performed at −10 to 40 ° C., particularly preferably −5 to 30 ° C. It is sufficient to carry out the reaction for 2 to 48 hours.
[0022]
The water-soluble anticancer agent of the present invention can be obtained by adding adriamycin to the block copolymer used in the present invention and then performing an appropriate treatment. Alternatively, instead of adding adriamycin, after leaving unreacted adriamycin according to the conditions of the condensation reaction, that is, by increasing the amount of adriamycin, decreasing the amount of condensing agent, and shortening the reaction time, It can also be obtained by appropriate processing.
[0023]
Examples of the treatment method include a method of adding an adriamycin or a solution of adriamycin to the condensation reaction solution, or a method of preparing an aqueous solution by dialysis and ultrafiltration of the condensation reaction solution leaving unreacted adriamycin. Alternatively, a solution of adriamycin or adriamycin is added to a solution obtained by precipitating the condensation reaction solution with a poor solvent such as isopropyl ether (IPE) and then dissolving in a suitable solvent, or a solution obtained by dissolving the block copolymer in a suitable solvent, Dialysis and ultrafiltration may be performed. Alternatively, after dialysis and ultrafiltration of the condensation reaction solution, adriamycin or a solution of adriamycin may be added, followed by dialysis and ultrafiltration again. The solvent used when mixing the block copolymer and adriamycin is preferably a solvent that well dissolves both the block copolymer and adriamycin. Examples thereof include DMF, a mixed solvent of DMF and water, and the like. Further, when the block copolymer and adriamycin are mixed, a treatment such as ultrasonic irradiation may be performed.
[0024]
Even after ultrafiltration, the presence of adriamycin not bound to the block copolymer can be confirmed by various analytical means. For example, means such as liquid chromatograph and mass spectrometry can be used. It is also possible to quantify.
[0025]
The following is an example of a water-soluble anticancer agent in which adriamycin is added to a block copolymer in which adriamycin is bonded to a side chain of polyaspartic acid, which is a carrier for support composed of a polyethylene glycol structural part and a polyaspartic acid structural part. The synthesis method will be described in detail.
[0026]
The block copolymer used for this water-soluble anticancer agent was synthesized with β-benzyl-L-aspartate-N-carboxylic acid anhydride (BLA-NCA) and a methoxy group at one end as shown in the following reaction formula. And polyethylene glycol having a 3-aminopropyl group at the other end (PEG-NH 2 ) As an initiator and ring-opening polymerization in a solvent such as DMF, DMSO, dioxane, chloroform, THF, acetonitrile, and the like, and a polyethylene glycol-poly (β-benzyl-L-aspartate) block copolymer (PEG-PBLA) The benzyl ester of PEG-PBLA is then hydrolyzed to obtain a polyethylene glycol-polyaspartic acid block copolymer (PEG-P (Asp.)). An anticancer agent, adriamycin, and a condensing agent such as EDC and DCC are added to this PEG-P (Asp.) And reacted in solution to bond the primary amino group of adriamycin and the carboxyl group of polyaspartic acid with an amide bond. Block copolymer (PEG-P (Asp.) ADR) is obtained.
[0027]
[Chemical formula 5]
(In the formula, R represents a hydroxyl group or formula (3), n represents 5 to 1,000, m represents an integer of 2 to 300, and x represents an integer of 0 to 300, but x is not larger than m. )
[0029]
[Chemical 6]
By dialysis and ultrafiltration of the condensation reaction solution to make an aqueous solution of about 20 mg / ml in terms of adriamycin, add a DMF solution of adriamycin, perform ultrasonic irradiation for 10 minutes, and then perform dialysis and ultrafiltration again. The water-soluble anticancer agent of the present invention can be obtained. Alternatively, the condensation reaction solution is precipitated with IPE, dissolved in DMF, adriamycin in DMF-water mixed solution is added, ultrasonic irradiation is performed for 10 minutes, and dialysis and ultrafiltration can be performed.
[0031]
The water-soluble anticancer agent of the present invention has a good water solubility despite a high adriamycin content, and the water solubility is maintained even when lyophilized or concentrated.
[0032]
As shown in Table 1, the anticancer activity of this water-soluble anticancer agent is higher than that of the original adriamycin itself. Moreover, its high anticancer activity is achieved even in the range of fewer side effects than adriamycin. Moreover, compared with the water-soluble polymerized anticancer agent described in JP-A No. 2-300133, the same anticancer activity is shown with a small dose.
[0033]
The water-soluble anticancer agent of the present invention can be used in various commonly used dosage forms, for example, solids, ointments, liquids, etc., but is usually used as an injection, and its dosage is per week. 1 to 3 doses, total 50 to 1,000 mg / m 2 About a week.
[0034]
【Example】
Next, the present invention will be described specifically by way of examples.
[0035]
Example 1
7.1 g of β-benzyl-L-aspartate-N-carboxylic anhydride (BLA-NCA) was dissolved in 70 ml of N, N-dimethylformamide (DMF). One end methoxy group One end 3-aminopropyl group polyethylene glycol (PEG-NH 2 ) (Molecular weight 5,100) 5.0 g was dissolved in 50 ml of DMF, and the solution was added to the BLA-NCA solution. After 40 hours, the reaction mixture was dropped into 1,000 ml of isopropyl ether (IPE), and the precipitated polymer was collected by filtration, washed with IPE, and then vacuum-dried to polyethylene glycol-poly (β-benzyl-L-aspartate). 9.3 g of a block copolymer (PEG-PBLA) was obtained. The benzyl ester was hydrolyzed at room temperature while suspending 7.0 g of PEG-PBLA in 0.5N sodium hydroxide. After the polymer was dissolved, the pH was acidified with acetic acid, and ultrafiltration was performed with an ultrafiltration membrane of ADVANTEC UK-10 (fraction molecular weight = 10,000). The concentrated solution was lyophilized to obtain 5.1 g of a polyethylene glycol-polyaspartic acid block copolymer (PEG-P (Asp.)). 1,230 mg of this PEG-P (Asp.) Was dissolved in 15 ml of water. 1,500 mg of adriamycin hydrochloride was suspended in 150 ml of DMF, 353 μl of triethylamine was added under ice cooling, and then an aqueous PEG-P (Asp.) Solution was added. To this mixed solution, 375 mg of N-hydroxysuccinimide and 525 μl of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) were added and reacted for 4 hours under ice cooling. Thereafter, 525 μl of EDC was added and reacted at room temperature for 18 hours. After the reaction, 2.5% of unreacted adriamycin remained in the reaction mixture with respect to the total adriamycin. The reaction mixture was purified using a silica gel column (Wakogel C-100) and an ion exchange resin (strongly acidic ion exchange resin PK-216) to remove low-molecular substances such as unreacted adriamycin. The purified solution was dialyzed for 3 hours in 0.1 M acetic acid using a dialysis membrane (fraction molecular weight = 12,000). After dialysis, ultrafiltration was performed with an ultrafiltration membrane of ADVANTEC UK-50 (fraction molecular weight = 50,000). Washing with water and concentration were repeated to obtain 35 ml of an aqueous solution of 20 mg / ml in terms of adriamycin (calculated from absorption at 485 nm with an ultraviolet spectrophotometer). The obtained PEG-P (Asp.) ADR has the structure of the above formula (1), and R 1 Is a methyl group, R 2 Is trimethylene group, R Three Represents a methylene group and Y represents hydrogen. n = 115, m = 20, x = 4, a part of R is a hydroxyl group and the rest is the residue formula (3). Although the content of adriamycin was 47% by weight, it showed good water solubility. A solution prepared by dissolving 20 mg of adriamycin hydrochloride and 5 μl of triethylamine in 20 ml of DMF was added to 10 ml of an aqueous solution of 20 mg / ml (in terms of adriamycin) of PEG-P (Asp.) ADR, followed by dialysis for 10 minutes. The membrane (fraction molecular weight = 12,000) was dialyzed in 0.01 M acetic acid for 3 hours. After dialysis, ultrafiltration was performed with an ultrafiltration membrane of ADVANTEC UK-50 (fraction molecular weight = 50,000). Washing with water and concentration were repeated to obtain 10.2 ml of an aqueous solution of 20 mg / ml in terms of adriamycin (calculated from absorption at 485 nm with an ultraviolet spectrophotometer). As a result of analysis by liquid chromatography, the amount of adriamycin not bound to the block copolymer was 8.5% of the total adriamycin. This also showed good water solubility, similar to the original PEG-P (Asp.) ADR.
[0036]
Example 2
A solution of 50 mg of adriamycin hydrochloride and 12 μl of triethylamine in 20 ml of DMF was added to 10 ml of an aqueous solution of 20 mg / ml (converted to adriamycin) of PEG-P (Asp.) ADR obtained in Example 1, and subjected to ultrasonic irradiation for 10 minutes. Then, the mixture was dialyzed for 3 hours in 0.01 M acetic acid using a dialysis membrane (fraction molecular weight = 12,000). After dialysis, ultrafiltration was performed with an ultrafiltration membrane of ADVANTEC UK-50 (fraction molecular weight = 50,000). Washing with water and concentration were repeated to obtain 11.5 ml of an aqueous solution of 20 mg / ml in terms of adriamycin (calculated from absorption at 485 nm with an ultraviolet spectrophotometer). As a result of analysis by liquid chromatography, the amount of adriamycin not bound to the block copolymer was 18.4% of the total adriamycin. This also showed good water solubility, similar to the original PEG-P (Asp.) ADR.
[0037]
Example 3
6.4 g of β-benzyl-L-aspartate-N-carboxylic acid anhydride (BLA-NCA) was dissolved in 60 ml of N, N-dimethylformamide (DMF). One end methoxy group One end 3-aminopropyl group polyethylene glycol (PEG-NH 2 ) (Molecular weight 5,100) 3.0 g was dissolved in 30 mg of DMF, and the solution was added to the BLA-NCA solution. After 40 hours, the reaction mixture was added dropwise to 700 ml of isopropyl ether (IPE), and the precipitated polymer was collected by filtration, washed with IPE, and then vacuum-dried to dry the polyethylene glycol-poly (β-benzyl-L-aspartate) block. The polymer (PEG-PBLA) 7.2g was obtained. The benzyl ester was hydrolyzed at room temperature while suspending 5.0 g of PEG-PBLA in 0.5N sodium hydroxide. After the polymer was dissolved, the pH was acidified with acetic acid, and ultrafiltration was performed with an ultrafiltration membrane of ADVANTEC UK-10 (fraction molecular weight = 10,000). The concentrated solution was freeze-dried to obtain 3.1 g of a polyethylene glycol-polyaspartic acid block copolymer (PEG-P (Asp.)). This PEG-P (Asp.) 1,010 mg was dissolved in 15 ml of water. 1,500 mg of adriamycin hydrochloride was suspended in 150 ml of DMF, 353 μl of triethylamine was added under ice cooling, and then an aqueous PEG-P (Asp.) Solution was added. To this mixed solution, 375 mg of N-hydroxysuccinimide and 525 μl of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) were added and reacted for 4 hours under ice cooling. Thereafter, 525 μl of EDC was added and reacted at room temperature for 18 hours. After the reaction, 2.3% of unreacted adriamycin remained in the reaction mixture with respect to the total adriamycin. The reaction mixture was purified using a silica gel column (Wakogel C-100) and an ion exchange resin (strongly acidic ion exchange resin PK-216) to remove low-molecular substances such as unreacted adriamycin. The purified solution was dialyzed for 3 hours in 0.1 M acetic acid using a dialysis membrane (fraction molecular weight = 12,000). After dialysis, ultrafiltration was performed with an ultrafiltration membrane of ADVANTEC UK-50 (fraction molecular weight = 50,000). Washing with water and concentration were repeated to obtain 37 ml of an aqueous solution of 20 mg / ml in terms of adriamycin (calculated from absorption at 485 nm with an ultraviolet spectrophotometer). The obtained PEG-P (Asp.) ADR has the structure of the above formula (1), and R 1 Is a methyl group, R 2 Is trimethylene group, R Three Represents a methylene group and Y represents hydrogen. n = 115, m = 30, x = 6, a part of R is a hydroxyl group and the rest is the formula (3). The adriamycin content was 54% by weight but showed good water solubility. A solution prepared by dissolving 15 mg of adriamycin hydrochloride and 4 μl of triethylamine in 20 ml of DMF was added to 10 ml of an aqueous solution of 20 mg / ml (converted to adriamycin) of PEG-P (Asp.) ADR. The membrane (fraction molecular weight = 12,000) was dialyzed in 0.01 M acetic acid for 3 hours. After dialysis, ultrafiltration was performed with an ultrafiltration membrane of ADVANTEC UK-50 (fraction molecular weight = 50,000). Washing with water and concentration were repeated to obtain 9.8 ml of an aqueous solution of 20 mg / ml in terms of adriamycin (calculated from absorption at 485 nm with an ultraviolet spectrophotometer). As a result of analysis by liquid chromatography, the amount of adriamycin not bound to the block copolymer was 5.5% of the total adriamycin. This also showed good water solubility, similar to the original PEG-P (Asp.) ADR.
[0038]
Example 4
A solution of 50 mg of adriamycin hydrochloride and 12 μl of triethylamine in 20 ml of DMF was added to 10 ml of an aqueous solution of 20 mg / ml (converted to adriamycin) of PEG-P (Asp.) ADR obtained in Example 3, and subjected to ultrasonic irradiation for 10 minutes. Then, the mixture was dialyzed for 3 hours in 0.01 M acetic acid using a dialysis membrane (fraction molecular weight = 12,000). After dialysis, ultrafiltration was performed with an ultrafiltration membrane of ADVANTEC UK-50 (fraction molecular weight = 50,000). Washing with water and concentration were repeated to obtain 10.5 ml of an aqueous solution of 20 mg / ml in terms of adriamycin (calculated from absorption at 485 nm with an ultraviolet spectrophotometer). As a result of analysis by liquid chromatography, the amount of adriamycin not bound to the block copolymer was 14.4% of the total adriamycin. This also showed good water solubility, similar to the original PEG-P (Asp.) ADR.
[0039]
Example 5
The benzyl ester was hydrolyzed at room temperature while suspending 2.0 g of PEG-PBLA obtained in Example 3 in 0.5N sodium hydroxide. After the polymer was dissolved, the pH was made almost neutral with acetic acid, and ultrafiltration was performed with an ultrafiltration membrane of Amicon YM-1 (fraction molecular weight = 1,000). While continuing ultrafiltration, it was washed with 0.05N hydrochloric acid and further washed thoroughly with water. The concentrated solution was lyophilized to obtain 1.3 g of a polyethylene glycol-polyaspartic acid block copolymer (PEG-P (Asp.)). 100 mg of this PEG-P (Asp.) Was dissolved in 10 ml of DMF, and 71.2 mg of adriamycin hydrochloride, 27.3 μl of triethylamine, and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC. HCl) 67.2 mg was added and reacted for 4 hours. Furthermore, it was made to react at room temperature for 20 hours. After the reaction, almost no unreacted adriamycin remained in the reaction mixture. The reaction mixture was dropped into 100 ml of isopropyl ether (IPE), and the precipitated polymer was collected by filtration, washed with IPE, and then vacuum dried. It melt | dissolved in water and ultrafiltered with the ultrafiltration membrane of Amicon YM-3 (fraction molecular weight = 1,000). While continuing ultrafiltration, wash with a mixture of water, methanol, and acetic acid, and after washing thoroughly with water, concentrate and concentrate to a solid content weight of about 50 mg / ml (partly freeze-dried, 3.1 ml of an aqueous solution was obtained. The obtained PEG-P (Asp.) ADR has the structure of the above formula (1), and R 1 Is a methyl group, R 2 Is trimethylene group, R Three Represents a methylene group and Y represents hydrogen. n = 115, m = 30, x = 6, a part of R is a hydroxyl group and the rest is the formula (3). Although the content of adriamycin was 40%, it showed good water solubility. A solution of 10 mg of adriamycin hydrochloride and 2.5 μl of triethylamine in 10 ml of DMF was added to 1 ml of an aqueous solution of 50 mg / ml (solid content) of PEG-P (Asp.) ADR, and the mixture was stirred for 10 minutes at room temperature. Dialysis was carried out in water for 3 hours using (fraction molecular weight = 12,000). After dialysis, ultrafiltration was performed with an ultrafiltration membrane of ADVANTEC UK-50 (fraction molecular weight = 50,000). Washing with water and concentration were repeated to obtain 2 ml of an aqueous solution. As a result of analysis by liquid chromatography, the amount of adriamycin not bound to the block copolymer was 18% of the total adriamycin. This also showed good water solubility, similar to the original PEG-P (Asp.) ADR.
[0040]
Application example 1
Mouse colon cancer Colon 26 cells were implanted subcutaneously in the dorsal part of a CDF1 female mouse, and the tumor volume was 100 mm. Three PEG-P without the addition of the water-soluble anticancer agent obtained in Example 1, 2, 3 or 4 or the anticancer agent corresponding to Example 1 and 2 or 3 and 4 (free adriamycin hydrochloride) from the time point reached before or after (Asp.) ADR or adriamycin hydrochloride was administered intravenously once every 4 days for a total of 3 times to examine the effect on advanced cancer. Each drug was dissolved in physiological saline before use. All doses were converted to total adriamycin. The antitumor effect of the drug was determined from the number of tumor-eliminated mice and the tumor growth curve. The results are shown in Table 1 and FIGS. As is apparent from the figure, when adriamycin hydrochloride was administered, the growth suppression effect of the transplanted tumor was observed but the tumor was hardly reduced, whereas when the water-soluble anticancer agent of the present invention was administered, In some cases, the transplanted tumor disappeared because of its excellent growth-suppressing effect. As for the dose, anticancer activity was observed at a lower dose as the proportion of adriamycin not bound to the block copolymer increased. In addition, regarding weight loss, which is one index of toxicity, there was a mouse in which the disappearance of the tumor was observed even in the range of weight change in which the water-soluble anticancer drug of the present invention was lower than that of adriamycin.
[0041]
[Table 1]
【The invention's effect】
The water-soluble anticancer agent of the present invention has good water-solubility and exhibits a high antitumor effect even in a low toxicity range compared to free adriamycin. In addition, the present invention can provide an extremely useful pharmaceutical because it exhibits an equivalent anticancer activity at a lower dose compared to conventional water-soluble polymerized anticancer agents.
[Brief description of the drawings]
FIG. 1 shows the tumor growth curve of mouse colon cancer Colon 26 when adriamycin hydrochloride is administered.
FIG. 2 is a tumor growth curve of mouse colon cancer Colon 26 when the water-soluble anticancer agent of Example 1 is administered.
FIG. 3 shows the tumor growth curve of mouse colon cancer Colon 26 when the water-soluble anticancer agent of Example 2 was administered.
FIG. 4 is a tumor growth curve of mouse colon cancer Colon 26 when PEG-P (Asp.) ADR without the addition of an anticancer agent (free adriamycin hydrochloride) corresponding to Examples 1 and 2 was administered.
FIG. 5 is a tumor growth curve of mouse colon cancer Colon 26 when the water-soluble anticancer agent of Example 3 was administered.
6 is a tumor growth curve of mouse colon cancer Colon 26 when the water-soluble anticancer agent of Example 4 is administered. FIG.
FIG. 7 is a tumor growth curve of mouse colon cancer Colon 26 when PEG-P (Asp.) ADR without addition of an anticancer agent (free adriamycin hydrochloride) corresponding to Examples 3 and 4 was administered.
Claims (5)
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| JP21605993A JP3682475B2 (en) | 1993-08-31 | 1993-08-31 | Water-soluble anticancer agent |
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| JP21605993A JP3682475B2 (en) | 1993-08-31 | 1993-08-31 | Water-soluble anticancer agent |
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| US6093391A (en) * | 1992-10-08 | 2000-07-25 | Supratek Pharma, Inc. | Peptide copolymer compositions |
| US6153193A (en) * | 1993-04-28 | 2000-11-28 | Supratek Pharma Inc. | Compositions for targeting biological agents |
| CN1069099C (en) * | 1995-09-29 | 2001-08-01 | 科学技术振兴事业团 | Novel anthraeycline compound derivatives and its medical preparation |
| WO2003000771A1 (en) | 2001-06-20 | 2003-01-03 | Nippon Kayaku Kabushiki Kaisha | Block copolymer reduced in impurity content, polymeric carrier, pharmaceutical preparations in polymeric form and process for the preparation of the same |
| JP2004010479A (en) * | 2002-06-03 | 2004-01-15 | Japan Science & Technology Corp | New solid pharmaceutical preparation comprising block copolymer and anthracycline anticancer agent, and method for producing the same |
| JP2004018494A (en) * | 2002-06-19 | 2004-01-22 | Japan Science & Technology Corp | Method for producing block copolymer-medicine composite material |
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| JP5183478B2 (en) * | 2006-08-31 | 2013-04-17 | ナノキャリア株式会社 | Transdermal composition containing active ingredient-containing polymer micelle, transdermal pharmaceutical composition, and transdermal cosmetic composition |
| KR101444274B1 (en) | 2006-10-19 | 2014-09-26 | 나노캬리아 가부시키가이샤 | Block copolymer for drug complex and pharmaceutical composition |
| CN104434877A (en) * | 2013-09-16 | 2015-03-25 | 成都市绿科华通科技有限公司 | Poly methyl olefinic acid-containing macromolecule drug microcapsule |
| CN104434878A (en) * | 2013-09-16 | 2015-03-25 | 成都市绿科华通科技有限公司 | Polymethacrylic acid-containing self-assembled drug carrier microcapsule system |
| CA2974936A1 (en) * | 2015-02-23 | 2016-09-01 | Nippon Kayaku Kabushiki Kaisha | Block copolymer conjugate of physiologically active substance |
| JP6817956B2 (en) * | 2015-11-18 | 2021-01-20 | 日本化薬株式会社 | Compositions containing novel glutamic acid derivatives and block copolymers and their uses |
| CA3009130A1 (en) | 2015-12-22 | 2017-06-29 | Nippon Kayaku Kabushiki Kaisha | Polymer conjugate of sulfoxide derivative-coordinated platinum(ii) complex |
-
1993
- 1993-08-31 JP JP21605993A patent/JP3682475B2/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| JPH0769900A (en) | 1995-03-14 |
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