JPH0769900A - Water-soluble anticancer agent - Google Patents
Water-soluble anticancer agentInfo
- Publication number
- JPH0769900A JPH0769900A JP5216059A JP21605993A JPH0769900A JP H0769900 A JPH0769900 A JP H0769900A JP 5216059 A JP5216059 A JP 5216059A JP 21605993 A JP21605993 A JP 21605993A JP H0769900 A JPH0769900 A JP H0769900A
- Authority
- JP
- Japan
- Prior art keywords
- adriamycin
- water
- group
- anticancer agent
- block copolymer
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
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- Medicinal Preparation (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
Description
【０００１】[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。The present invention relates to a block copolymer,
The present invention relates to a water-soluble anticancer agent composed of a complex with adriamycin.
【０００２】[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。2. Description of the Related Art A water-soluble polymerized anticancer agent comprising a block copolymer having a hydrophilic polymer structure part and a polymer structure part to which adriamycin is bound is described in JP-A No. 2-300133. There is.
【０００３】[0003]
【発明が解決しようとする課題】特開平２−３００１３
３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。DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention
Although the water-soluble polymerized anticancer drug obtained by the method described in JP-A No. 3 shows excellent anticancer activity, the optimum dose was considerably higher than that of adriamycin.
【０００４】[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。[Means for Solving the Problems] The present inventors have completed the present invention as a result of intensive investigations for improving the anticancer activity of the water-soluble polymerized anticancer agent.
【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、That is, according to the present invention, the expressions (1), (1) and (2) are used.
Or a block copolymer having a structure of these salts,
A water-soluble anticancer drug consisting of a complex with adriamycin,
【０００６】[0006]
【化３】 [Chemical 3]
【０００７】（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。）(Wherein R 1 represents hydrogen or a lower alkyl group, R 2 represents a bonding group, R 3 represents a methylene group or an ethylene group, Y represents hydrogen or a protecting group, and R is respectively Each independently represent a hydroxyl group or a residue of adriamycin having the structure of formula (3), n is 5 to
Although 1,000 and m are integers of 2 to 300 and x is 0 to 300, x is not larger than m. )
【０００８】[0008]
【化４】 [Chemical 4]
【０００９】（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。(2) The block copolymer forms micelles 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 (1). (3) The water-soluble anticancer agent according to the above (1), wherein R 1 is a methyl group.
Alternatively, the water-soluble anti-cancer agent according to (2), (4) the water-soluble anti-cancer agent according to (1), (2) or (3) above, wherein R 2 is an alkylene group having 2 to 4 carbon atoms, and (5) R 3 is The present invention relates to the water-soluble anticancer agent according to (1), (2), (3) or (4), which is a methylene group.
【００１０】本発明の水溶性抗癌剤は高い薬理効果を有
する。The water-soluble anticancer agent of the present invention has a high pharmacological effect.
【００１１】以下、本発明について詳細に説明する。The present invention will be described in detail below.
【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（−Ｃ
Ｈ2 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。In the above formula (1) or (2), R 1
Represents hydrogen or a lower alkyl group, with a methyl group being preferred. R 2 is not particularly limited as long as it does not impair the water solubility of the water-soluble anti-cancer agent of the present invention (preferably, unless it impairs the micelle forming ability of the water-soluble anti-cancer agent of the present invention). When forming the amino acid structure portion, the structure corresponding to the method and compound used for converting the terminal of the compound that constitutes the polyethylene glycol structure portion into a structure suitable for the formation is adopted. A methylene group (-CH 2
-), ethylene group (-CH 2 CH 2 -), propylene group (-CH (CH 3) CH 2 -), trimethylene group (-C
H 2 CH 2 CH 2 —), isobutylene group (—CH 2 CH
Examples include alkylene groups having 1 to 8 carbon atoms, such as (CH 3 ) CH 2 —, preferably 1 to 4 carbon atoms. Examples of the salt of polyamino acid include sodium salt and potassium salt, but are not particularly limited. In the above formula (1), Y represents hydrogen or a protecting group, and examples of the protecting group include an acetyl group and the like, but are not particularly limited.
【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１５〜４０
０であり、ｍは２〜３００であるが好ましくは１０〜１
００であり、ｘは０〜３００であるが好ましくは０〜１
００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。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 polyethylene glycol structure part and the polyamino acid structure part to which adriamycin is bound in the block copolymer is not particularly limited as long as the water solubility of the block copolymer is maintained, but is preferably 1: 0.1 to 0.1. 10 (weight ratio),
Particularly preferably, it is 1: 0.2 to 5 (weight ratio). Further, n is 5 to 1,000, but preferably 15 to 40.
0 and m is 2 to 300, preferably 10 to 1
00 and x is 0 to 300, preferably 0 to 1
00. The amount of adriamycin to be bound to the block copolymer is not particularly limited, but it is usually 3 to 80% by weight, preferably 5 to 60% by weight in the block copolymer. The ratio of the amount of adriamycin bound to the block copolymer to the amount of adriamycin not bound is not particularly limited, but is usually 1:
0.01 to 10 (weight ratio), preferably 1: 0.
It is 1-2.
【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。The carrier for supporting adriamycin in the block copolymer can be produced by various methods. For example, by reacting a polyamino acid with polyethylene glycol or a chemically modified terminal thereof, and then removing the protective group for those containing a protective group, or by removing polyethylene glycol or a chemically modified terminal thereof and a polymerizable amino acid or amino acid. A carrier for carrying can be obtained by reacting a derivative monomer and removing the protective group containing a protective group.
【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。The 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 into an amino group, a method of reacting ethyleneimine, Michael addition of acrylonitrile or methacrylonitrile, and then nitrile A method of reducing a group to convert it to an amino group, a method of replacing a hydroxyl group with a halogen group and then reacting an alcohol amine such as ethanolamine, or a method of directly converting a hydroxyl group to a nitrile and then reducing it to an amino group. It can be carried out. Further, as a method for converting a hydroxyl group into a carboxyl group, a usual oxidation reaction, condensation reaction, addition reaction, hydrolysis reaction, a reaction combining these, or the like can be adopted. For example, a hydroxyl group can be converted into a carboxyl group by a method in which a hydroxyl group is converted to an alcoholate with sodium metal, a halogenated fatty acid ester such as ethyl bromoacetate is added, and then hydrolysis is performed.
【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。The protecting group can be removed by an alkali method, an acid method or a reduction method. As the alkaline substance used in the alkaline method, usual alkaline substances such as caustic soda, caustic potash, hydrazine, and ammonia can be used. As the acidic substance used in the acid method, a usual acidic substance such as trifluoromethanesulfonic acid, methanesulfonic acid, trifluoroacetic acid, acetic acid, formic acid, hydrofluoric acid, hydrobromic acid, and hydrochloric acid can be used. . Further, 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】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。When the polyamino acid structural portion has an amino group at the terminal, a modified terminal amino group can be used as a carrier for supporting. Examples of the modification method include a method using an acid anhydride or an acid halide. Modification can be either before or after removal of the protecting groups.
【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（Ｅ
ＤＣ・ＨＣｌ）、ジシクロヘキシルカルボジイミド（Ｄ
ＣＣ）、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。The block carrier used in the present invention can be obtained by reacting the supporting carrier thus obtained with adriamycin if necessary. For example, in order to obtain a block copolymer of the formula (1) to which adriamycin is bound, adriamycin may be reacted with a supporting carrier in which all Rs in the formula (1) are hydroxyl groups. When adriamycin is bound to the supporting carrier by an amide bond, the reaction can be carried out according to a conventional method known as a peptide bond forming method. For example, an acid halide method, an acid anhydride method, a coupling method, etc. can be used,
A coupling method using a condensing agent is desirable. As the condensing agent, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC), 1-ethyl-3- (3
-Dimethylaminopropyl) carbodiimide hydrochloride (E
DC / HCl), dicyclohexylcarbodiimide (D
CC), 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, more preferably 1 to 10 times, the molar amount of adriamycin. At this time, N
-Hydroxysuccinimide (HONSu), 1-hydroxybenzotriazole (HOBt), N-hydroxy-5-norbornene-2,3-dicarboxylic acid imide (HONB), and the like may coexist.
【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。The amount of adriamycin used is not particularly limited, but is usually 0.1 to 2 mol per 1 equivalent of the carboxyl group of the carrier for carrying.
【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。The condensation reaction is preferably carried out in a solvent, and examples of the solvent include N, N-dimethylformamide (DMF), dimethylsulfoxide (DMSO), dioxane, tetrahydrofuran (THF), water and a mixed solvent thereof. Various materials can be used and are not particularly limited. Although the amount of the solvent used is not particularly limited, it is usually used in an amount of 10 to 500 times the weight of the carrier for carrying.
【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。The condensation reaction is preferably carried out 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】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。In the block copolymer used in the present invention,
The water-soluble anticancer agent of the present invention can be obtained by adding adriamycin and then performing an appropriate treatment. Alternatively, instead of adding adriamycin, depending on the conditions of the condensation reaction, that is, by increasing the amount of adriamycin, decreasing the amount of condensing agent, shortening the reaction time, etc., after leaving unreacted adriamycin, It can also be obtained by appropriate treatment.
【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。Examples of the treatment method include a method in which an adriamycin or a solution of adriamycin is added to the condensation reaction solution, or a condensation reaction solution in which unreacted adriamycin remains is dialyzed and ultrafiltered to form an aqueous solution. To be Alternatively, the condensation reaction solution is precipitated with a poor solvent such as isopropyl ether (IPE) and then dissolved in a suitable solvent, or the block copolymer is dissolved in a suitable solvent, and a solution of adriamycin or adriamycin is added, Dialysis and ultrafiltration may be performed. Alternatively, the condensation reaction solution may be dialyzed and ultrafiltered, then adriamycin or a solution of adriamycin may be added, and the mixture may be dialyzed and ultrafiltered again. The solvent used when mixing the block copolymer and adriamycin is preferably one that dissolves both the block copolymer and adriamycin well. Examples thereof include DMF and a mixed solvent of DMF and water. In addition, when mixing the block copolymer and adriamycin, a treatment such as ultrasonic irradiation may be performed.
【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。The presence of adriamycin not bound to the block copolymer can be confirmed by various analytical means even after ultrafiltration. For example, means such as liquid chromatography and mass spectrometry can be used. It is also possible to quantify.
【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。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 supporting composed of a polyethylene glycol structural part and a polyaspartic acid structural part. Now, the synthesis method will be described in detail.
【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、ＤＭＳ
Ｏ、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、Ｄ
ＣＣ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。The block copolymer used for the water-soluble anticancer agent is synthesized by β-benzyl-
L-aspartate-N-carboxylic acid anhydride (BLA-
NCA) having a methoxy group at one end and a polyethylene glycol (PEG-NH 2 ) having a 3-aminopropyl group at the other end as an initiator, DMF, DMS
Ring-opening polymerization is performed in a solvent such as O, dioxane, chloroform, THF or acetonitrile to obtain a polyethylene glycol-poly (β-benzyl-L-aspartate) block copolymer (PEG-PBLA).
-The benzyl ester of -PBLA is hydrolyzed to obtain a polyethylene glycol-polyaspartic acid block copolymer (PEG-P (Asp.)). This PEG-P
(Asp.) With anticancer agents adriamycin and EDC, D
A block copolymer (PEG-P (Asp.) ADR) in which a primary amino group of adriamycin and a carboxyl group of polyaspartic acid are bound by an amide bond is added by adding a condensing agent such as CC and reacting in a solution. obtain.
【００２７】[0027]
【化５】 [Chemical 5]
【００２８】（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０〜３
００の整数を示すが、ｘはｍより大きくないものとす
る。）(Wherein R represents a hydroxyl group or formula (3), n is 5 to 1,000, m is 2 to 300, and x is 0 to 3
Although an integer of 00 is shown, x is not larger than m. )
【００２９】[0029]
【化６】 [Chemical 6]
【００３０】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。The condensation reaction solution is dialyzed and ultrafiltered to obtain an aqueous solution of about 20 mg / ml in terms of adriamycin, a DMF solution of adriamycin is added, ultrasonic irradiation is performed for 10 minutes, and then dialysis and ultrafiltration are performed again. As a result, the water-soluble anticancer agent of the present invention can be obtained. Alternatively, the condensation reaction solution can be precipitated by IPE, dissolved in DMF, added with a DMF-water mixed solution of adriamycin, subjected to ultrasonic irradiation for 10 minutes, dialyzed, and subjected to ultrafiltration.
【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。The water-soluble anticancer agent of the present invention has good water solubility despite its high content of adriamycin, and its water solubility is maintained even when freeze-dried or concentrated.
【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２−３
００１３３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。The anticancer activity of this water-soluble anticancer agent is higher than that of the original adriamycin itself as shown in Table 1. Moreover, its high anticancer activity is achieved even in the range of fewer side effects than adriamycin. In addition, Japanese Patent Laid-Open No. 2-3
Compared with the water-soluble polymerized anticancer agent described in Japanese Patent No. 00133, it shows the same anticancer activity at a smaller dose.
【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５０〜１，０００
ｍｇ／ｍ2 週程度である。The water-soluble anticancer agent of the present invention may be used in various commonly used dosage forms, for example, solid dosage forms, ointments, liquid dosage forms, etc., but it is usually used as an injection, and its dose is 1 to 3 administrations per week for a total dose of 50 to 1,000
It is about mg / m 2 weeks.
【００３４】[0034]
【実施例】次に実施例により本発明を具体的に説明す
る。EXAMPLES The present invention will be described in detail with reference to examples.
【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）５．０
ｇをＤＭＦ５０ｍｌに溶解し、その溶液をＢＬＡ−ＮＣ
Ａ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−Ｐ
ＢＬＡ）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（Ａ
ｓｐ．））５．１ｇを得た。このＰＥＧ−Ｐ（Ａｓ
ｐ．）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４８５ｎ
ｍ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．）ＡＤ
Ｒ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（Ａｓ
ｐ．）ＡＤＲと同様良好な水溶性を示した。Example 1 7.1 g of β-benzyl-L-aspartate-N-carboxylic acid anhydride (BLA-NCA) was dissolved in 70 ml of N, N-dimethylformamide (DMF). One terminal methoxy group One terminal 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 BLA-NC.
A solution. After 40 hours, the reaction mixture was added dropwise to 1,000 ml of isopropyl ether (IPE), and the precipitated polymer was collected by filtration, washed with IPE, and vacuum dried to obtain polyethylene glycol-poly (β-benzyl-L-aspartate). Block copolymer (PEG-P
BLA) 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 ADVANTEC UK-10
Ultrafiltration was performed with an ultrafiltration membrane (molecular weight cut off = 10,000). Lyophilize the concentrated solution to give polyethylene glycol-
Polyaspartic acid block copolymer (PEG-P (A
sp. )) 5.1 g was obtained. This PEG-P (As
p. ) 1,230 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 solution of PEG-P (Asp.) Was added. To this mixed solution, 375 mg of N-hydroxysuccinimide, and 1
-Ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) (525 µl) was added, and the mixture was reacted under ice cooling for 4 hours. Then add 525 μl of EDC and add 18 at room temperature.
Reacted for hours. After the reaction, 2.5% of unreacted adriamycin remained with respect to the total amount of adriamycin in the reaction mixture. 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 unreacted low molecular substances such as adriamycin. The purified solution is 0.1M using a dialysis membrane (molecular weight cut-off = 12,000).
It was dialyzed in acetic acid for 3 hours. After dialysis, ADVANTEC
Ultrafiltration was carried out with an ultrafiltration membrane of UK-50 (molecular weight cut off = 50,000). Repeated washing with water and concentration, 20 mg / ml in terms of adriamycin (485 n in UV spectrophotometer)
35 ml of an aqueous solution of (calculated from absorption of m) was obtained. The obtained PEG-P (Asp.) ADR has the structure of the above formula (1), R 1 is a methyl group, R 2 is a trimethylene group, R 3 is a methylene group, and Y is hydrogen. n = 115, m = 20,
When x = 4, part of R is a hydroxyl group and the rest is the above-mentioned residue formula (3). The adriamycin content was 47% by weight, but it showed good water solubility. This PEG-P (Asp.) AD
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 (converted to adriamycin) of R, and after ultrasonic irradiation for 10 minutes, a dialysis membrane (molecular weight cutoff = 12, 000) in 0.01 M acetic acid for 3 hours. After dialysis, ADVANTEC UK-50
Ultrafiltration was performed using an ultrafiltration membrane (molecular weight cut off = 50,000). Washing with water and concentration were repeated to obtain 10.2 ml of an aqueous solution containing 20 mg / ml in terms of adriamycin (calculated from absorption at 485 nm by 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 is also the original PEG-P (As
p. ) Similar to ADR, it showed good water solubility.
【００３６】実施例２ 実施例１で得たＰＥＧ−Ｐ（Ａｓｐ．）ＡＤＲの２０ｍ
ｇ／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１
２μｌ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＝１
２，０００）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。Example 2 20 m of PEG-P (Asp.) ADR obtained in Example 1
50 mg of adriamycin hydrochloride and 1 of triethylamine in 10 ml of an aqueous solution of g / ml (converted to adriamycin)
A solution prepared by dissolving 2 μl in 20 ml of DMF was added, and 10
After performing ultrasonic irradiation for 1 minute, the dialysis membrane (molecular weight cut off = 1
2,000) for 3 hours in 0.01 M acetic acid. After dialysis, ultrafiltration was performed using an ADVANTEC UK-50 (molecular weight cut-off = 50,000) ultrafiltration membrane. Repeated washing with water and concentration, 20 mg / adriamycin equivalent
11.5 ml of an aqueous solution of ml (calculated by an ultraviolet spectrophotometer from the absorption at 485 nm) was obtained. 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 is also the original PEG-P (Asp.) ADR
It also showed good water solubility.
【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）３．０
ｇをＤＭＦ３０ｍｇに溶解し、その溶液をＢＬＡ−ＮＣ
Ａ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−ＰＢＬ
Ａ）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（Ａｓ
ｐ．））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ＰＥ
Ｇ−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 Ｕ
Ｋ−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Ｐ
ＥＧ−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２
０ｍｇ／ｍｌ（アドリアマイシン換算）の水溶液１０ｍ
ｌ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、１
０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。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 BLA-NC.
A 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 vacuum dried to obtain polyethylene glycol-poly (β-benzyl-L).
-Aspartate) block copolymer (PEG-PBL
A) 7.2 g was obtained. 5.0 g of PEG-PBLA was added to 0.
The benzyl ester was hydrolyzed at room temperature while suspended in 5N sodium hydroxide. After the polymer was dissolved, the pH was made acidic 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 a polyethylene glycol-polyaspartic acid block copolymer (PEG-P (As
p. )) 3.1 g were obtained. 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 PE was added.
An aqueous GP (Asp.) Solution was added. N in this mixed solution
-Hydroxysuccinimide (375 mg) and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) (525 µl) were added, and the mixture was reacted under ice cooling for 4 hours. After that, 525 μl of EDC was added, and the mixture was reacted at room temperature for 18 hours. After the reaction, 2.3% of unreacted adriamycin with respect to the total adriamycin remained in the reaction mixture. 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 unreacted low molecular substances such as adriamycin. The purified solution was dialyzed for 3 hours in 0.1 M acetic acid using a dialysis membrane (molecular weight cut-off = 12,000). After dialysis, ADVANTEC U
Ultrafiltration was carried out with an ultrafiltration membrane of K-50 (molecular weight cut off = 50,000). Repeated washing with water and concentration, 20 mg / ml in terms of adriamycin (485 nm with UV spectrophotometer)
37 ml of an aqueous solution of (calculated from absorption of 1) was obtained. Obtained P
EG-P (Asp.) ADR has the structure of the above formula (1), R 1 is a methyl group, R 2 is a trimethylene group, R 3 is a methylene group, and Y is hydrogen. n = 115, m = 30, x
= 6, part of R is a hydroxyl group and the rest is of the above formula (3).
The adriamycin content was 54% by weight, but it showed good water solubility. This PEG-P (Asp.) ADR 2
10 mg of 0 mg / ml (converted to adriamycin)
A solution of adriamycin hydrochloride (15 mg) and triethylamine (4 μl) dissolved in DMF (20 ml) was added to 1 l.
After performing ultrasonic irradiation for 0 minutes, a dialysis membrane (molecular weight cut off =
12,000) for 3 hours in 0.01 M acetic acid. After dialysis, ultrafiltration was performed using an ADVANTEC UK-50 (molecular weight cut-off = 50,000) ultrafiltration membrane. Repeated washing with water and concentration, 20 mg in terms of adriamycin
/ Ml (calculated from the absorption at 485 nm with an ultraviolet spectrophotometer)
9.8 ml of an aqueous solution of 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 product also showed good water solubility like the original PEG-P (Asp.) ADR.
【００３８】実施例４ 実施例３で得たＰＥＧ−Ｐ（Ａｓｐ．）ＡＤＲの２０ｍ
ｇ／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１
２μｌ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＝１
２，０００）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。Example 4 20 m of PEG-P (Asp.) ADR obtained in Example 3
50 mg of adriamycin hydrochloride and 1 of triethylamine in 10 ml of an aqueous solution of g / ml (converted to adriamycin)
A solution prepared by dissolving 2 μl in 20 ml of DMF was added, and 10
After performing ultrasonic irradiation for 1 minute, the dialysis membrane (molecular weight cut off = 1
2,000) for 3 hours in 0.01 M acetic acid. After dialysis, ultrafiltration was performed using an ADVANTEC UK-50 (molecular weight cut-off = 50,000) ultrafiltration membrane. Repeated washing with water and concentration, 20 mg / adriamycin equivalent
10.5 ml of an aqueous solution (ml, calculated from the absorption at 485 nm by an ultraviolet spectrophotometer) was obtained. 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 is also the original PEG-P (Asp.) ADR
It also showed good water solubility.
【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＝１，０
００）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（Ａ
ｓｐ．））１．３ｇを得た。このＰＥＧ−Ｐ（Ａｓ
ｐ．）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・ＨＣ
ｌ）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、Ｒ
3 はメチレン基、Ｙは水素を表す。ｎ＝１１５、ｍ＝３
０、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、ＡＤＶＡＮＴＥ
Ｃ 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。Example 5 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 Amicon YM-1 (fraction molecular weight = 1,0
The ultrafiltration membrane of (00) was ultrafiltered. While continuing the ultrafiltration, the plate was washed with 0.05N hydrochloric acid and further thoroughly washed with water. Lyophilize the concentrated solution to give polyethylene glycol-
Polyaspartic acid block copolymer (PEG-P (A
sp. )) 1.3 g were obtained. This PEG-P (As
p. ) 100 mg 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.HC) under ice cooling.
l) 67.2 mg was added and reacted for 4 hours. Further, the reaction was carried out at room temperature for 20 hours. After the reaction, almost no unreacted adriamycin remained in the reaction mixture. 100 ml of the reaction mixture is isopropyl ether (IPE)
The polymer precipitated by dripping was collected by filtration, washed with IPE and dried in vacuum. Dissolved in water, Amicon YM-
Ultrafiltration was carried out with an ultrafiltration membrane of 3 (molecular weight cut off = 1,000). Washing with a mixed solution of water, methanol, and acetic acid while continuing ultrafiltration, further thoroughly washing with water, and then concentrating, and the solid content is about 50 mg / ml (a part of which is lyophilized to 3.1 ml of an aqueous solution of (calculation) was obtained. The obtained PEG-P (Asp.) ADR has the structure of the above formula (1), R 1 is a methyl group, R 2 is a trimethylene group, R
3 represents a methylene group and Y represents hydrogen. n = 115, m = 3
0, x = 6, part of R is a hydroxyl group and the rest is of the above formula (3). The adriamycin content was 40%, but it showed good water solubility. A solution of 10 mg of adriamycin hydrochloride and 2.5 μl of triethylamine dissolved 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 at room temperature for 10 minutes, and then dialyzed. It dialyzed in water for 3 hours using (molecular weight cut off = 12,000). After dialysis, ADVANTE
Ultrafiltration was carried out with an ultrafiltration membrane of CUK-50 (molecular weight cut off = 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 product also showed good water solubility like the original PEG-P (Asp.) ADR.
【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Ｃｏ
ｌｏｎ 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。Application Example 1 Mouse colon cancer Co on the dorsal skin of a CDF1 female mouse
lon 26 cells were transplanted, and the tumor volume was 100 mm 3.
PEG-P without addition of the water-soluble anticancer agent obtained in Example 1, 2, 3 or 4 or the anticancer agent corresponding to Examples 1 and 2 or 3 and 4 (free adriamycin hydrochloride) (Asp.) ADR or adriamycin hydrochloride was intravenously administered once every 4 days for a total of 3 times, and the effect on advanced cancer was examined. Each drug was dissolved in physiological saline before use. All doses were converted to total adriamycin. The antitumor effect of the drug was judged from the number of tumor-free mice and the tumor growth curve. The results are shown in Table 1 and FIGS. As is clear from the figure, when adriamycin hydrochloride is administered, the growth-suppressing effect of the transplanted tumor is observed, but tumor shrinkage is hardly observed, whereas when the water-soluble anticancer agent of the present invention is administered, In some cases, the transplanted tumor disappeared and the growth inhibitory effect on the tumor was excellent. Regarding the dose, as the proportion of adriamycin not bound to the block copolymer increased, the anticancer activity was observed at the lower dose. Regarding weight loss, which is one of the indicators of toxicity, there was a mouse in which the water-soluble anticancer agent of the present invention showed disappearance of tumor even in the range of weight change lower than that of adriamycin.
【００４１】[0041]
【表１】 [Table 1]
【００４２】[0042]
【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。INDUSTRIAL APPLICABILITY The water-soluble anticancer agent of the present invention has good water solubility and exhibits a high antitumor effect even in the range of low toxicity as compared with free adriamycin. Further, since the same anticancer activity is exhibited at a lower dose as compared with the conventional water-soluble polymerized anticancer agent, the present invention can provide an extremely useful drug.
【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。FIG. 1 is a tumor growth curve of mouse colon cancer Colon 26 when adriamycin hydrochloride is administered.
【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。FIG. 2 shows a tumor growth curve of mouse colon cancer Colon 26 when the water-soluble anticancer agent of Example 1 is administered.
【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。FIG. 3 is a tumor growth curve of mouse colon cancer Colon 26 when the water-soluble anticancer agent of Example 2 is administered.
【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（Ａ
ｓｐ．）ＡＤＲを投与した場合の、マウス大腸癌Ｃｏｌ
ｏｎ ２６の腫瘍増殖曲線。FIG. 4 PEG-P (A) without addition of anti-cancer agent (free adriamycin hydrochloride) corresponding to Examples 1 and 2.
sp. ) Mouse colon cancer Col when ADR is administered
on 26 tumor growth curve.
【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。FIG. 5 is a tumor growth curve of mouse colon cancer Colon 26 when the water-soluble anticancer agent of Example 3 is administered.
【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。FIG. 6 is a tumor growth curve of mouse colon cancer Colon 26 when the water-soluble anticancer agent of Example 4 is administered.
【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（Ａ
ｓｐ．）ＡＤＲを投与した場合の、マウス大腸癌Ｃｏｌ
ｏｎ ２６の腫瘍増殖曲線。FIG. 7: PEG-P (A) without addition of anti-cancer agent (free adriamycin hydrochloride) corresponding to Examples 3 and 4.
sp. ) Mouse colon cancer Col when ADR is administered
on 26 tumor growth curve.
フロントページの続き (72)発明者 片岡 一則 千葉県柏市大室1083−４、柏ビレジ141− ９ (72)発明者 岡野 光夫 千葉県市川市国府台６−12−12 (72)発明者 桜井 靖久 東京都杉並区永福３−17−６ (72)発明者 ▲勢▼藤 隆 群馬県前橋市下川町45−３ (72)発明者 福島 重人 群馬県高崎市岩鼻町239 (72)発明者 山田 好美 群馬県多野郡新町1393−２ (72)発明者 浴本 久雄 東京都北区志茂２−11−１−803 (72)発明者 岡本 一也 東京都荒川区東尾久５−７−10−305 (72)発明者 真柴 洋子 東京都北区志茂３−29−11Front Page Continuation (72) Inventor Kazunori Kataoka 1083-4 Omuro, Kashiwa City, Chiba Prefecture 141-9 Kashiwa Village, (72) Inventor, Mitsuo Okano 6-12-12, Kokufudai, Ichikawa City, Chiba (72) Inventor, Yasuhisa Sakurai 3-17-6 Eifuku, Suginami-ku, Tokyo (72) Inventor ▲ Takashi Fuji, 45-3 Shimokawa-cho, Maebashi-shi, Gunma Prefecture (72) Inventor Shigeto Fukushima 239 Iwahana-cho, Takasaki-shi, Gunma Prefecture (72) Inventor Yamada Yoshimi 1393-2 Shinmachi, Tano-gun, Gunma Prefecture (72) Inventor Hisao Yumoto 2-11-1-803 Shimo, Kita-ku, Tokyo (72) Inventor Kazuya Okamoto 5-7-10-305 Higashiohisa, Arakawa-ku, Tokyo ( 72) Inventor Yoko Mashiba 3-29-11 Shimo, Kita-ku, Tokyo
Claims (5)
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。 【化１】 （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。） 【化２】 1. A water-soluble anticancer agent comprising a complex of a block copolymer having the structure of formula (1) or (2) or a salt thereof and adriamycin. [Chemical 1] (In the formula, R 1 represents hydrogen or a lower alkyl group, and R 2
Represents a bonding group, R 3 represents a methylene group or an ethylene group, Y represents hydrogen or a protecting group, and R independently represents a hydroxyl group or a residue of adriamycin having the structure of formula (3). And n is 5 to 1,000,
Although m represents an integer of 2 to 300 and x represents an integer of 0 to 300, x is not larger than m. ) [Chemical 2]
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請求項１記載の水溶性
抗癌剤。2. The block copolymer according to claim 1, which forms a micelle having a polyethylene glycol structure portion on the outside and a polyamino acid or a salt structure portion thereof on the inside, and contains adriamycin in the micelle. Water-soluble anticancer drug.
記載の水溶性抗癌剤。3. The method according to claim 1, wherein R 1 is a methyl group.
The water-soluble anticancer agent described.
る請求項１、２または３記載の水溶性抗癌剤。4. The water-soluble anticancer agent according to claim 1, 2 or 3, wherein R 2 is an alkylene group having 2 to 4 carbon atoms.
３または４記載の水溶性抗癌剤。5. A compound according to claim 1, wherein R 3 is a methylene group.
The water-soluble anticancer agent according to 3 or 4.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP21605993A JP3682475B2 (en) | 1993-08-31 | 1993-08-31 | Water-soluble anticancer agent |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP21605993A JP3682475B2 (en) | 1993-08-31 | 1993-08-31 | Water-soluble anticancer agent |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH0769900A true JPH0769900A (en) | 1995-03-14 |
| JP3682475B2 JP3682475B2 (en) | 2005-08-10 |
Family
ID=16682636
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP21605993A Expired - Lifetime JP3682475B2 (en) | 1993-08-31 | 1993-08-31 | Water-soluble anticancer agent |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP3682475B2 (en) |
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| WO1996040056A1 (en) * | 1995-06-07 | 1996-12-19 | Supratek Pharma Inc. | Polyether block copolymer micellar compositions for targeting biological agents |
| US6080396A (en) * | 1995-09-29 | 2000-06-27 | Japan Science And Technology Corporation | Anthracycline compound derivative and pharmaceutical preparation containing the same |
| 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 |
| EP1056469A4 (en) * | 1998-02-27 | 2003-05-07 | Supratek Pharma Inc | Novel peptide copolymer compositions |
| WO2003101465A1 (en) * | 2002-06-03 | 2003-12-11 | Nippon Kayaku Kabushiki Kaisha | Novel solid preparation containing block copolymer and anthracycline anticancer agent and process for producing the same |
| WO2004000362A1 (en) * | 2002-06-19 | 2003-12-31 | Nippon Kayaku Kabushiki Kaisha | Process for producing block copolymer/drug composite |
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| US8431143B2 (en) | 2005-08-05 | 2013-04-30 | Toto Ltd. | Therapeutic method of administering pharmaceutical titanium dioxide composite and light irradiation |
| 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 |
| WO2016136641A1 (en) * | 2015-02-23 | 2016-09-01 | 日本化薬株式会社 | Block copolymer conjugate of physiologically active substance |
| WO2017086392A1 (en) | 2015-11-18 | 2017-05-26 | 日本化薬株式会社 | Composition comprising novel glutamic acid derivative and block copolymer, and use thereof |
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-
1993
- 1993-08-31 JP JP21605993A patent/JP3682475B2/en not_active Expired - Lifetime
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1996040056A1 (en) * | 1995-06-07 | 1996-12-19 | Supratek Pharma Inc. | Polyether block copolymer micellar compositions for targeting biological agents |
| US6080396A (en) * | 1995-09-29 | 2000-06-27 | Japan Science And Technology Corporation | Anthracycline compound derivative and pharmaceutical preparation containing the same |
| EP1056469A4 (en) * | 1998-02-27 | 2003-05-07 | Supratek Pharma Inc | Novel peptide copolymer compositions |
| 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 |
| WO2003101465A1 (en) * | 2002-06-03 | 2003-12-11 | Nippon Kayaku Kabushiki Kaisha | Novel solid preparation containing block copolymer and anthracycline anticancer agent and process for producing the same |
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| US8431143B2 (en) | 2005-08-05 | 2013-04-30 | Toto Ltd. | Therapeutic method of administering pharmaceutical titanium dioxide composite and light irradiation |
| 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 |
| WO2008026776A1 (en) * | 2006-08-31 | 2008-03-06 | Nanocarrier Co., Ltd. | Transdermal composition, transdermal pharmaceutical composition and transdermal cosmetic composition comprising polymer micelle encapsulating active ingredient |
| WO2008047948A1 (en) | 2006-10-19 | 2008-04-24 | Nanocarrier Co., Ltd. | Block copolymer for drug complex and pharmaceutical composition |
| US8519051B2 (en) | 2006-10-19 | 2013-08-27 | Nanocarrier Co., Ltd. | Block copolymer for drug conjugates 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 |
| WO2016136641A1 (en) * | 2015-02-23 | 2016-09-01 | 日本化薬株式会社 | Block copolymer conjugate of physiologically active substance |
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| WO2017086392A1 (en) | 2015-11-18 | 2017-05-26 | 日本化薬株式会社 | Composition comprising novel glutamic acid derivative and block copolymer, and use thereof |
| JPWO2017086392A1 (en) * | 2015-11-18 | 2018-09-06 | 日本化薬株式会社 | Composition containing novel glutamic acid derivative and block copolymer and use thereof |
| WO2017110669A1 (en) * | 2015-12-22 | 2017-06-29 | 日本化薬株式会社 | Polymer conjugate of sulfoxide derivative-coordinated platinum(ii) complex |
| US10946028B2 (en) | 2015-12-22 | 2021-03-16 | Nippon Kayaku Kabushiki Kaisha | Polymer conjugate of sulfoxide derivative-coordinated platinum(II) complex |
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