JP2020117540A - Urethral stricture therapeutic agent and urethral stricture treatment method - Google Patents
Urethral stricture therapeutic agent and urethral stricture treatment method Download PDFInfo
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- JP2020117540A JP2020117540A JP2020081974A JP2020081974A JP2020117540A JP 2020117540 A JP2020117540 A JP 2020117540A JP 2020081974 A JP2020081974 A JP 2020081974A JP 2020081974 A JP2020081974 A JP 2020081974A JP 2020117540 A JP2020117540 A JP 2020117540A
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- Prior art keywords
- urethral stricture
- hydrogel
- therapeutic agent
- forming polymer
- urethral
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- 238000006467 substitution reaction Methods 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- 230000008733 trauma Effects 0.000 description 1
- 201000005112 urinary bladder cancer Diseases 0.000 description 1
- 229920002554 vinyl polymer Polymers 0.000 description 1
- NLVXSWCKKBEXTG-UHFFFAOYSA-N vinylsulfonic acid Chemical compound OS(=O)(=O)C=C NLVXSWCKKBEXTG-UHFFFAOYSA-N 0.000 description 1
- 239000013585 weight reducing agent Substances 0.000 description 1
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Abstract
Description
本発明は、尿道狭窄症の治療に有用な治療剤に関する。本発明はまた尿道狭窄症の治療方法に関する。 The present invention relates to a therapeutic agent useful for treating urethral stricture. The invention also relates to a method of treating urethral stricture.
尿道狭窄症は、前立腺肥大症や膀胱がんに対する尿道内視鏡による手術の後遺症、交通事故や労働作業中の事故の外傷、先天的な尿道の疾患である尿道下裂など、さまざまな要因で生じる。怪我や炎症により尿道粘膜に傷がついて、その傷が修復される過程で尿道粘膜や尿道粘膜を取り囲む尿道海綿体に瘢痕化が起こり、尿道が狭くなる疾患である。 Urethral stenosis can be caused by a variety of factors, including aftereffects of urethroscopic surgery for benign prostatic hyperplasia and bladder cancer, trauma from traffic accidents and labor accidents, and hypospadias, a congenital urethral disease. Occurs. It is a disease in which the urethral mucosa is damaged due to injury or inflammation, and in the process of repairing the wound, the urethral mucosa and the cavernosa of the urethra surrounding the urethral mucosa are scarred and the urethra is narrowed.
尿道狭窄症の治療方法として、外科的に尿道を再建する治療法が存在するが、侵襲性が高く長期の入院が必要とされることになるため、近年は低侵襲で簡便なブジー（尿道拡張具）やバルーンカテーテル、コールドナイフ、レーザー等を利用した経尿道内視鏡的拡張手技が実施されている（林田重昭、桐山啻夫、広中弘、吉川静、男子尿道狭窄症に対する内尿道切開術の経験、泌尿器科紀要（1972）、18（8）：588-593）。 There is a surgical method to reconstruct the urethra as a treatment method for urethral stricture, but it is highly invasive and requires long-term hospitalization. Instrument), balloon catheter, cold knife, laser, etc. are used to perform transurethral endoscopic dilation procedures (Shigeaki Hayashida, Tetsuo Kiriyama, Hiroshi Hironaka, Shizuka Yoshikawa, endourethral resection for male urethral stricture) Experience, Bulletin of Urology (1972), 18(8):588-593).
しかしながら、上記の従来の治療法では、瘢痕組織化した尿道内面に上皮細胞を再建する能力が極めて低いために、尿道狭窄を再発してしまうことが問題となっている。本発明は、尿道狭窄治療において低侵襲性の経尿道内視鏡的手技で再狭窄を回避できる尿道狭窄治療剤および尿道狭窄治療方法を提供することを目的としている。 However, the above-mentioned conventional treatment methods have a problem of recurrent urethral stricture because the ability to reconstruct epithelial cells on the inner surface of the urethra in which scars are organized is extremely low. An object of the present invention is to provide a therapeutic agent for urethral stricture and a method for treating urethral stricture which can avoid restenosis by a minimally invasive transurethral endoscopic procedure in the treatment of urethral stricture.
本発明者らは、尿道狭窄症の治療法として、経尿道内視鏡的手技で切開処置した尿道内面に特定の物性を持ったハイドロゲルを留置することが、切開処置部位の上皮化を促進し、該部位が瘢痕組織化によって尿道狭窄を再発することを有効に防止することを見出し、本発明を完成した。 As a treatment method for urethral stenosis, the present inventors promoted epithelialization of the incision-treated site by placing a hydrogel having specific physical properties on the inner surface of the urethra incised by a transurethral endoscopic procedure. However, they have found that the site effectively prevents recurrence of urethral stricture due to scar organization, and completed the present invention.
さらに本発明者らは、動物細胞を上記ハイドロゲル中に含有させることが、上記課題の解決に効果的であることを見出した。特に動物細胞が患者自身の口腔粘膜細胞であることが効果的であることを見出した。 Furthermore, the present inventors have found that the inclusion of animal cells in the hydrogel is effective in solving the above problems. In particular, it has been found that it is effective that the animal cells are the oral mucosa cells of the patients themselves.
すなわち、本発明の課題は、ハイドロゲル形成性高分子を少なくとも含み、１０℃における貯蔵弾性率が５０Ｐａ以下かつ３７℃における貯蔵弾性率が１００Ｐａ以上であることを特徴とする尿道狭窄治療剤によって解決される。 That is, the problem of the present invention is solved by a therapeutic agent for urethral stricture, which comprises at least a hydrogel-forming polymer and has a storage elastic modulus at 10° C. of 50 Pa or less and a storage elastic modulus at 37° C. of 100 Pa or more. To be done.
さらに本発明の課題は、上記尿道狭窄治療剤が動物細胞を含むことを特徴とする尿道狭窄治療剤によって解決される。 Further, the object of the present invention is solved by a therapeutic agent for urethral stricture, wherein the therapeutic agent for urethral stricture contains animal cells.
また、本発明の課題は、前記動物細胞が患者自身の口腔粘膜細胞であることを特徴とする尿道狭窄治療剤によって解決される。 Further, the object of the present invention is solved by a therapeutic agent for urethral stricture, wherein the animal cells are oral mucosal cells of the patient himself.
さらに本発明の課題は、経尿道内視鏡的手技で切開処置した尿道内面に、ハイドロゲル形成性高分子を少なくとも含み、１０℃における貯蔵弾性率が５０Ｐａ以下かつ３７℃における貯蔵弾性率が１００Ｐａ以上であることを特徴とする尿道狭窄治療剤を１０℃以下の温度に冷却して注入し、室温以上の温度で該尿道内面に留置する操作を少なくとも含むことを特徴とする尿道狭窄治療方法によって解決される。 A further object of the present invention is to include at least a hydrogel-forming polymer on the inner surface of the urethra incised by a transurethral endoscopic procedure and have a storage elastic modulus at 10°C of 50 Pa or less and a storage elastic modulus at 37°C of 100 Pa. According to the method for treating urethral stricture, which comprises at least the operation of injecting the therapeutic agent for urethral stricture characterized by the above to be cooled to a temperature of 10° C. or lower and indwelling on the inner surface of the urethra at a temperature of room temperature or higher. Will be resolved.
以上説明したように、本発明によれば、経尿道内視鏡的手技で切開処置した尿道内面に特定の物性を持ったハイドロゲルを留置することによって、切開処置部位の上皮化を促進し、該部位が瘢痕組織化によって尿道狭窄を再発することを有効に防止することができる。 As described above, according to the present invention, by indwelling the hydrogel having specific physical properties on the inner surface of the urethra treated by transurethral endoscopic procedure, promoting epithelialization of the incision treatment site, It is possible to effectively prevent recurrence of urethral stricture due to scar organization at the site.
さらに、該ハイドロゲルが動物細胞（特に患者自身の口腔粘膜細胞）を含有することによって、尿道内面の切開処置部位の上皮化がより促進される。 Furthermore, the hydrogel containing animal cells (particularly oral mucosa cells of the patient itself) further promotes epithelialization of the incision treatment site on the inner surface of the urethra.
以下、本発明の内容について詳細に説明する。
（ハイドロゲル形成性の高分子）
本発明の「ハイドロゲル形成性高分子」とは、架橋（ｃｒｏｓｓｌｉｎｋｉｎｇ）構造ないし網目構造を熱可逆的に生成し、該構造に基づき、その内部に水等の分散液体を保持するハイドロゲルを熱可逆的に形成可能な性質を有する高分子をいう。又、「ハイドロゲル」とは高分子からなる架橋ないし網目構造と該構造中に支持ないし保持された水を含むゲルをいう。
Hereinafter, the content of the present invention will be described in detail.
(Hydrogel-forming polymer)
The "hydrogel-forming polymer" of the present invention means that a hydrogel that reversibly forms a crosslinking structure or a network structure and that holds a dispersed liquid such as water therein is heated based on the structure. A polymer having the property of being reversibly formed. The term "hydrogel" refers to a gel containing a crosslinked or network structure composed of a polymer and water supported or retained in the structure.
（貯蔵弾性率）
本発明においてハイドロゲルの貯蔵弾性率の測定は、文献（H.Yoshioka ら、Journal of Macromolecular Science, A31(1), 113 (1994））に記載された方法により行うことができる。即ち、観測周波数１Ｈｚにおける試料の動的弾性率を所定の温度（１０℃、２５℃または３７℃）で測定し、該試料の貯蔵弾性率（Ｇ′、弾性項）を求める。この測定に際しては、下記の測定条件が好適に使用可能である。
(Storage elastic modulus)
In the present invention, the storage elastic modulus of the hydrogel can be measured by the method described in the literature (H. Yoshioka et al., Journal of Macromolecular Science, A31(1), 113 (1994)). That is, the dynamic elastic modulus of the sample at an observation frequency of 1 Hz is measured at a predetermined temperature (10° C., 25° C. or 37° C.) to obtain the storage elastic modulus (G′, elastic term) of the sample. In this measurement, the following measurement conditions can be preferably used.
く動的・損失弾性率の測定条件＞
測定機器（商品名）：ストレス制御式レオメーター AR500、TAインスツルメント社製
試料溶液の量：約０．８ｇ
測定用セルの形状・寸法：アクリル製平行円盤（直径４．０ｃｍ）、ギャップ６００μｍ
測定周波数：１Ｈｚ
適用ストレス：線形領域内。
Dynamic/loss elastic modulus measurement conditions>
Measuring instrument (brand name): Stress control rheometer AR500, TA Instruments sample solution amount: approx. 0.8 g
Shape and dimensions of measuring cell: acrylic parallel disk (diameter 4.0 cm), gap 600 μm
Measurement frequency: 1 Hz
Applied stress: Within the linear region.
本発明の尿道狭窄治療剤は、その貯蔵弾性率が１０℃において５０Ｐａ以下、好ましくは３０Ｐａ以下（特には１０Ｐａ以下）であり、かつその貯蔵弾性率が３７℃において１００Ｐａ以上、好ましくは２００Ｐａ以上（特には３００Ｐａ以上）であることが好ましい。 The therapeutic agent for urethral stricture of the present invention has a storage elastic modulus of 50 Pa or less at 10° C., preferably 30 Pa or less (particularly 10 Pa or less), and a storage elastic modulus of 100 Pa or more at 37° C., preferably 200 Pa or more ( In particular, it is preferably 300 Pa or more).
本発明の尿道狭窄治療剤は１０℃以下の低温で尿道狭窄治療部位に注入し、体温で尿道狭窄治療剤を尿道狭窄治療部位に留置する。尿道狭窄治療剤の１０℃における貯蔵弾性率が５０Ｐａを超えるとその固さが大き過ぎ、カテーテルを介しての注入が困難となる。 The therapeutic agent for urethral stricture of the present invention is injected into the urethral stricture treatment site at a low temperature of 10° C. or lower, and the urethral stricture treatment agent is placed at the urethral stricture treatment site at body temperature. If the storage elastic modulus at 10° C. of the therapeutic agent for urethral stricture exceeds 50 Pa, its hardness is too high, and it becomes difficult to inject it through a catheter.
一方、本発明の尿道狭窄治療剤の３７℃における貯蔵弾性率がｌ００Ｐａを下回る場合、その強度が不足し、尿道狭窄治療部位に長期間留置することが困難となる。 On the other hand, when the storage elastic modulus at 37° C. of the therapeutic agent for urethral stricture of the present invention is less than 100 Pa, its strength is insufficient and it becomes difficult to indwell it at the treatment site for urethral stricture for a long time.
さらに男性患者の場合は尿道狭窄治療部位が陰茎部にあることが多く、体外に露出するため外気温の影響を受け易い。本発明の「ハイドロゲル形成性高分子」水溶液の室温（２５℃）における貯蔵弾性率が１００Ｐａを下回る場合、外気温の低下により容易に流動化するため、動物細胞の尿道狭窄治療部位への留置が不完全となる。その結果、瘢痕組織化した尿道内面に上皮細胞を再建する能力が低いために、尿道狭窄を再発してしまうことが問題となる。従って、本発明の尿道狭窄治療剤の貯蔵弾性率は２５℃において１００Ｐａ以上、好ましくは２００Ｐａ以上（特には３００Ｐａ以上）であることが好ましい。 Furthermore, in the case of male patients, the treatment site for urethral stricture is often located in the penis and is exposed to the outside of the body, so that it is easily affected by the outside temperature. When the storage elastic modulus of the aqueous solution of the “hydrogel-forming polymer” of the present invention at room temperature (25° C.) is less than 100 Pa, it easily fluidizes due to a decrease in outside air temperature, and therefore the animal cells are placed in the urethral stricture treatment site. Is incomplete. As a result, recurrence of urethral stricture becomes a problem because the ability to reconstruct epithelial cells on the inner surface of the urethra in which scars are organized is low. Therefore, the storage elastic modulus of the therapeutic agent for urethral stricture of the present invention is preferably 100 Pa or more, preferably 200 Pa or more (particularly 300 Pa or more) at 25°C.
本発明の尿道狭窄治療剤に上記のような好適な貯蔵弾性率を与える「ハイドロゲル形成性の高分子」は、後述するような具体的な化合物の中から、上記したスクリーニング方法（貯蔵弾性率測定法）に従って容易に選択することができる。 The “hydrogel-forming polymer” which gives the above-mentioned preferred storage elastic modulus to the therapeutic agent for urethral stricture of the present invention is selected from the above-mentioned screening methods (storage elastic modulus) from the specific compounds described below. It can be easily selected according to the measurement method).
そのハイドロゲルがより低い温度で可逆的に流動性を示す高分子の具体例としては、例えば、ポリプロピレンオキサイドとポリエチレンオキサイドとのブロック共重合体等に代表されるポリアルキレンオキサイドブロック共重合体；メチルセルロース、ヒドロキシプロピルセルロース等のエーテル化セルロース；キトサン誘導体（K,R,Holme,et al. Macromolecules，２４，３８２８（１９９１））等が知られている。 Specific examples of the polymer whose hydrogel exhibits reversible fluidity at a lower temperature include, for example, polyalkylene oxide block copolymers represented by block copolymers of polypropylene oxide and polyethylene oxide; methyl cellulose. , Etherified cellulose such as hydroxypropyl cellulose; chitosan derivative (K, R, Holme, et al. Macromolecules, 24, 3828 (1991)) and the like are known.
（好適なハイドロゲル形成性高分子）
本発明の「ハイドロゲル形成性の高分子」として好適に使用可能な、架橋形成に疎水結合を利用したハイドロゲル形成性高分子は、曇点を有する複数のブロックと親水性のブロックが結合してなることが好ましい。
(Preferable hydrogel-forming polymer)
The hydrogel-forming polymer that utilizes a hydrophobic bond for cross-linking, which can be preferably used as the “hydrogel-forming polymer” of the present invention, has a structure in which a plurality of blocks each having a cloud point are combined with a hydrophilic block. It is preferable that
該親水性のブロックは、より低い温度で該ハイドロゲルが水溶性になるために存在することが好ましく、また曇点を有する複数のブロックは、ハイドロゲルがより高い温度でゲル状態に変化するために存在することが好ましい。
換言すれば、曇点を有するブロックは該曇点より低い温度では水に溶解し、該曇点より高い温度では水に不溶性に変化するために、曇点より高い温度で、該ブロックはゲルを形成するための疎水結合からなる架橋点としての役割を果たす。
The hydrophilic block is preferably present for the hydrogel to become water soluble at lower temperatures, and the plurality of blocks having a cloud point causes the hydrogel to change to a gel state at higher temperatures. Is preferably present.
In other words, a block having a cloud point dissolves in water at a temperature below the cloud point and becomes insoluble in water at a temperature above the cloud point, so that at a temperature above the cloud point, the block does not form a gel. It acts as a cross-linking point consisting of hydrophobic bonds to form.
本発明に用いるハイドロゲルは、疎水性結合が温度の上昇と共に強くなるのみならず、その変化が温度に対して可逆的であるという性質を利用したものである。１分子内に複数個の架橋点が形成され、安定性に優れたゲルが形成される点からは、「ハイドロゲル形成性の高分子」が「曇点を有するブロック」を複数個有することが好ましい。 The hydrogel used in the present invention utilizes the property that not only the hydrophobic bond becomes stronger as the temperature rises but also its change is reversible with temperature. From the viewpoint that a gel having excellent stability is formed by forming a plurality of cross-linking points in one molecule, the "hydrogel-forming polymer" may have a plurality of "blocks having cloud points". preferable.
一方、上記「ハイドロゲル形成性の高分子」中の親水性ブロックは、前述したように、該「ハイドロゲル形成性の高分子」がより低い温度で水溶性に変化させる機能を有し、上記転移温度より高い温度で疎水性結合力が増大しすぎて上記ハイドロゲルが凝集沈澱してしまうことを防止しつつ、含水ゲルの状態を形成させる機能を有する。 On the other hand, the hydrophilic block in the “hydrogel-forming polymer” has a function of changing the “hydrogel-forming polymer” to be water-soluble at a lower temperature, as described above, It has the function of forming a hydrogel state while preventing the hydrogel from coagulating and precipitating due to an excessive increase in hydrophobic binding force at a temperature higher than the transition temperature.
さらに本発明に用いる「ハイドロゲル形成性の高分子」は、生体内で分解、吸収されるものであることが望ましい。すなわち、本発明の「ハイドロゲル形成性の高分子」が生体内で加水分解反応や酵素反応により分解されて、生体に無害な低分子量体となって吸収、***されることが好ましい。 Further, the “hydrogel-forming polymer” used in the present invention is preferably one that is decomposed and absorbed in the living body. That is, it is preferable that the “hydrogel-forming polymer” of the present invention is decomposed in a living body by a hydrolysis reaction or an enzymatic reaction to be absorbed and excreted as a low molecular weight body which is harmless to the living body.
本発明の「ハイドロゲル形成性の高分子」が曇点を有する複数のブロックと親水性のブロックが結合してなるものである場合には、曇点を有するブロックと親水性のブロックの少なくともいずれか、好ましくは両方が生体内で分解、吸収されるものであることが好ましい。 When the “hydrogel-forming polymer” of the present invention is one in which a plurality of blocks having a cloud point and a hydrophilic block are bonded, at least one of the block having a cloud point and the hydrophilic block However, it is preferable that both are decomposed and absorbed in vivo.
（曇点を有する複数のブロック）
曇点を有するブロックとしては、水に対する溶解度−温度係数が負を示す高分子のブロックであることが好ましく、より具体的には、ポリプロピレンオキサイド、プロピレンオキサイドと他のアルキレンオキサイドとの共重合体、ポリＮ−置換アクリルアミド誘導体、ポリＮ−置換メタアクリルアミド誘導体、Ｎ−置換アクリルアミド誘導体とＮ−置換メタアクリルアミド誘導体との共重合体、ポリビニルメチルエーテル、ポリビニルアルコール部分酢化物からなる群より選ばれる高分子が好ましく使用可能である。
(Multiple blocks with cloud point)
As the block having a cloud point, solubility in water-preferably a polymer block having a negative temperature coefficient, more specifically, polypropylene oxide, a copolymer of propylene oxide and another alkylene oxide, Polymers selected from the group consisting of poly N-substituted acrylamide derivatives, poly N-substituted methacrylamide derivatives, copolymers of N-substituted acrylamide derivatives and N-substituted methacrylamide derivatives, polyvinyl methyl ether, polyvinyl alcohol partial acetyl chloride Can be preferably used.
曇点を有するブロックを生体内で分解、吸収されるものとするには、曇点を有するブロックを疎水性アミノ酸と親水性アミノ酸から成るポリペプチドとすることが有効である。あるいはポリ乳酸やポリグリコール酸などのポリエステル型生分解性ポリマーを生体内で分解、吸収される曇点を有するブロックとして利用することもできる。 In order to decompose and absorb the block having the cloud point in vivo, it is effective to use the block having the cloud point as a polypeptide composed of a hydrophobic amino acid and a hydrophilic amino acid. Alternatively, a polyester type biodegradable polymer such as polylactic acid or polyglycolic acid can be used as a block having a cloud point that is decomposed and absorbed in vivo.
上記の高分子（曇点を有するブロック）の曇点が４℃より高く４０℃以下であることが、本発明に用いる高分子（曇点を有する複数のブロックと親水性のブロックが結合した化合物）の貯蔵弾性率を所定温度で所望の値とする点から好ましい。 The cloud point of the above polymer (block having a cloud point) is higher than 4° C. and 40° C. or less, and the polymer used in the present invention (a compound in which a plurality of blocks having a cloud point and a hydrophilic block are bound to each other It is preferable in that the storage elastic modulus of 1) is a desired value at a predetermined temperature.
ここで曇点の測定は、例えば、上記の高分子（曇点を有するブロック）の約１質量％の水溶液を冷却して透明な均一溶液とした後、除々に昇温（昇温速度約１℃／ｍｉｎ）して、該溶液がはじめて白濁する点を曇点とすることによって行うことが可能である。 Here, the cloud point is measured, for example, by cooling an aqueous solution of about 1% by mass of the above-mentioned polymer (block having a cloud point) to obtain a transparent uniform solution, and then gradually raising the temperature (heating rate about 1 C./min), and the cloud point is the point at which the solution becomes cloudy for the first time.
本発明に使用可能なポリＮ−置換アクリルアミド誘導体、ポリＮ−置換メタアクリルアミド誘導体の具体的な例を以下に列挙する。
ポリ−Ｎ−アクロイルピペリジン；ポリ−Ｎ−ｎ−プロピルメタアクリルアミド；ポリ−Ｎ−イソプロピルアクリルアミド；ポリ−Ｎ，Ｎ−ジエチルアクリルアミド；ポリ−Ｎ−イソプロピルメタアクリルアミド；ポリ−Ｎ−シクロプロピルアクリルアミド；ポリ−Ｎ−アクリロイルピロリジン；ボリ−Ｎ，Ｎ−エチルメチルアクリルアミド；ポリ−Ｎ−シクロプロピルメタアクリルアミド；ポリ−Ｎ−エチルアクリルアミド。
上記の高分子は単独重合体（ホモポリマー）であっても、上記重合体を構成する単量体と他の単量体との共重合体であってもよい。このような共重合体を構成する他の単量体としては、親水性単量体、疎水性単量体のいずれも用いることができる。一般的には、親水性単量体と共重合すると生成物の曇点は上昇し、疎水性単量体と共重合すると生成物の曇点は下降する。従って、これらの共重合すべき単量体を選択することによっても、所望の曇点（例えば４℃より高く４０℃以下の曇点）を有する高分子を得ることができる。
Specific examples of poly N-substituted acrylamide derivatives and poly N-substituted methacrylamide derivatives that can be used in the present invention are listed below.
Poly-N-acroylpiperidine; poly-N-n-propylmethacrylamide; poly-N-isopropylacrylamide; poly-N,N-diethylacrylamide; poly-N-isopropylmethacrylamide; poly-N-cyclopropylacrylamide; Poly-N-acryloylpyrrolidine; poly-N,N-ethylmethylacrylamide; poly-N-cyclopropylmethacrylamide; poly-N-ethylacrylamide.
The polymer may be a homopolymer or a copolymer of a monomer constituting the polymer and another monomer. As the other monomer constituting such a copolymer, either a hydrophilic monomer or a hydrophobic monomer can be used. Generally, copolymerization with a hydrophilic monomer raises the cloud point of the product, and copolymerization with a hydrophobic monomer lowers the cloud point of the product. Therefore, by selecting these monomers to be copolymerized, a polymer having a desired cloud point (for example, a cloud point higher than 4° C. and lower than 40° C.) can be obtained.
（親水性単量体）
上記親水性単量体としては、Ｎ−ビニルピロリドン、ビニルピリジン、アクリルアミド、メタアクリルアミド、Ｎ−メチルアクリルアミド、ヒドロキシエチルメタアクリレート、ヒドロキシエチルアクリレート、ヒドロキシメチルメタアクリレート、ヒドロキシメチルアクリレート、酸性基を有するアクリル酸、メタアクリル酸およびそれらの塩、ビニルスルホン酸、スチレンスルホン酸等、並びに塩基性基を有するＮ，Ｎ−ジメチルアミノエチルメタクリレート、Ｎ，Ｎ−ジエチルアミノエチルメタクリート、Ｎ，Ｎ−ジメチルアミノプロピルアクリルアミドおよびそれらの塩等が挙げられるが、これらに限定されるものではない。
(Hydrophilic monomer)
Examples of the hydrophilic monomer include N-vinylpyrrolidone, vinylpyridine, acrylamide, methacrylamide, N-methylacrylamide, hydroxyethyl methacrylate, hydroxyethyl acrylate, hydroxymethyl methacrylate, hydroxymethyl acrylate, and acrylic having an acidic group. Acid, methacrylic acid and salts thereof, vinyl sulfonic acid, styrene sulfonic acid, etc., and N,N-dimethylaminoethyl methacrylate, N,N-diethylaminoethyl methacrylate, N,N-dimethylaminopropyl having a basic group Examples include acrylamide and salts thereof, but are not limited thereto.
（疎水性単量体）
一方、上記疎水性単量体としては、エチルアクリレート、メチルメタクリレート、グリシジルメタクリレト等のアクリレート誘導体およびメタクリレート誘導体、Ｎ−ｎ−ブチルメタアクリルアミド等のＮ−置換アルキルメタアクリルアミド誘導体、塩化ビニル、アクリロニトリル、スチレン、酢酸ビニル等が挙げられるが、これらに限定されるものではない。
(Hydrophobic monomer)
On the other hand, examples of the hydrophobic monomer include acrylate and methacrylate derivatives such as ethyl acrylate, methyl methacrylate, and glycidyl methacrylate, N-substituted alkyl methacrylamide derivatives such as Nn-butyl methacrylamide, vinyl chloride, and acrylonitrile. Examples thereof include, but are not limited to, styrene and vinyl acetate.
（親水性のブロック）
一方、上記した曇点を有するブロックと結合すべき親水性のブロックとしては、具体的には、メチルセルロース、デキストラン、ポリエチレンオキサイド、ポリビニルアルコール、ポリＮ−ビニルピロリドン、ポリビニルピリジン、ポリアクリルアミド、ポリメタアクリルアミド、ポリＮ−メチルアクリルアミド、ポリヒドロキシメチルアクリレート、ポリアクリル酸、ポリメタクリル酸、ポリビニルスルホン酸、ポリスチレンスルホン酸およびそれらの塩；ポリＮ，Ｎ−ジメチルアミノエチルメタクリレート、ポリＮ，Ｎ−ジエチルアミノエチルメタクリレート、ポリＮ，Ｎ−ジメチルアミノプロピルアクリルアミドおよびそれらの塩等が挙げられる。
(Hydrophilic block)
On the other hand, specific examples of the hydrophilic block to be combined with the block having the cloud point include methyl cellulose, dextran, polyethylene oxide, polyvinyl alcohol, poly N-vinylpyrrolidone, polyvinyl pyridine, polyacrylamide and polymethacrylamide. , Poly N-methyl acrylamide, poly hydroxymethyl acrylate, poly acrylic acid, poly methacrylic acid, polyvinyl sulfonic acid, polystyrene sulfonic acid and salts thereof; poly N,N-dimethylaminoethyl methacrylate, poly N,N-diethylaminoethyl methacrylate , Poly N,N-dimethylaminopropyl acrylamide and salts thereof.
また親水性のブロックは生体内で分解、代謝、***されることが望ましく、アルブミン、ゼラチンなどのたんぱく質、ヒアルロン酸、ヘパリン、キチン、キトサンなどの多糖類などの親水性生体高分子が好ましく用いられる。 Further, it is desirable that the hydrophilic block be decomposed, metabolized and excreted in vivo, and proteins such as albumin and gelatin, hydrophilic biopolymers such as polysaccharides such as hyaluronic acid, heparin, chitin and chitosan are preferably used. ..
曇点を有するブロックと上記の親水性のブロックとを結合する方法は特に制限されないが、例えば、上記いずれかのブロック中に重合性官能基（例えばアクリロイル基）を導入し、他方のブロックを与える単量体を共重合させることによって行うことができる。また、最点を有するブロックと上記の親水性のブロックとの結合物は、曇点を有するブロックを与える単量体と、親水性のブロックを与える単量体とのブロック共重合によって得ることも可能である。また、曇点を有するブロックと親水性のブロックとの結合は、予め両者に反応活性な官能基（例えば水酸基、アミノ基、力ルポキシル基、イソシアネート基等）を導入し、両者を化学反応により結合させることによって行うこともできる。この際、親水性のブロック中には通常、反応活性な官能基を複数導入する。また、曇点を有するポリプロピレンオキサイドと親水性のブロックとの結合は、例えば、アニオン重合またはカチオン重合で、プロピレンオキサイドと「他の親水性ブロック」を構成するモノマー（例えばエチレンオキサイド）とを繰り返し逐次重合させることで、ポリプロピレンオキサイドと「親水性ブロック」（例えばポリエチレンオキサイド）が結合したブロック共重合体を得ることができる。このようなブロック共重合体は、ポリプロピレンオキサイドの末端に重合性基（例えばアクリロイル基）を導入後、親水性のブロックを構成するモノマーを共重合させることによっても得ることができる。更には、親水性のブロック中に、ポリプロピレンオキサイド末端の官能基（例えば水酸基）と結合反応し得る官能基を導入し、両者を反応させることによっても、本発明に用いる高分子を得ることができる。また、ポリプロピレングリコールの両端にポリエチレングリコールが結合した、プルロニック Ｆ−１２７（商品名、旭電化工業株式会社製）等の材料を連結させることによっても、本発明に用いる「ハイドロゲル形成性の高分子」を得ることができる。 The method of combining the block having a cloud point and the hydrophilic block is not particularly limited, but for example, a polymerizable functional group (for example, an acryloyl group) is introduced into any of the above blocks to give the other block. It can be performed by copolymerizing monomers. Further, the combined product of the block having the highest point and the hydrophilic block may also be obtained by block copolymerization of a monomer giving a block having a cloud point and a monomer giving a hydrophilic block. It is possible. In addition, the block having a cloud point and the hydrophilic block are bonded to each other in advance by introducing a reactive functional group (for example, a hydroxyl group, an amino group, a forcepoxyl group, an isocyanate group, etc.) into them and chemically bonding them. It can also be done by At this time, usually, a plurality of reactive functional groups are introduced into the hydrophilic block. In addition, the bond between the polypropylene oxide having a cloud point and the hydrophilic block is, for example, anionic polymerization or cationic polymerization, and propylene oxide and a monomer (for example, ethylene oxide) that constitutes "another hydrophilic block" are repeatedly and sequentially. By polymerizing, a block copolymer in which polypropylene oxide and a “hydrophilic block” (for example, polyethylene oxide) are bound can be obtained. Such a block copolymer can also be obtained by introducing a polymerizable group (for example, an acryloyl group) into the terminal of polypropylene oxide and then copolymerizing a monomer that constitutes a hydrophilic block. Furthermore, the polymer used in the present invention can be obtained by introducing into the hydrophilic block a functional group capable of undergoing a binding reaction with a functional group at the terminal of polypropylene oxide (for example, a hydroxyl group) and reacting both. .. In addition, by connecting materials such as Pluronic F-127 (trade name, manufactured by Asahi Denka Kogyo Co., Ltd.) in which polyethylene glycol is bonded to both ends of polypropylene glycol, the “hydrogel-forming polymer used in the present invention is also used. Can be obtained.
この曇点を有するブロックを含む態様における本発明の高分子は、曇点より低い温度においては、分子内に存在する上記「曇点を有するブロック」が親水性のブロックとともに水溶性であるので、完全に水に溶解し、ゾル状態を示す。しかし、この高分子の水溶液の温度を上記曇点より高い温度に加温すると、分子内に存在する「曇点を有するブロック」が疎水性となり、疎水的相互作用によって、別個の分子間で会合する。 The polymer of the present invention in the embodiment including the block having the cloud point has a structure in which the “block having the cloud point” present in the molecule is water-soluble together with the hydrophilic block at a temperature lower than the cloud point, It completely dissolves in water and shows a sol state. However, when the temperature of the aqueous solution of this polymer is heated to a temperature higher than the cloud point, the “block having a cloud point” existing in the molecule becomes hydrophobic, and the hydrophobic interaction causes association between different molecules. To do.
一方、親水性のブロックは、この時（曇点より高い温度に加温された際）でも水溶性であるので、本発明の高分子は水中において、曇点を有するブロック間の疎水性会合部を架橋点とした三次元網目構造を持つハイドロゲルを生成する。このハイドロゲルの温度を再び、分子内に存在する「曇点を有するブロック」の曇点より低い温度に冷却すると、該曇点を有するブロックが水溶性となり、疎水性会合による架橋点が解放され、ハイドロゲル構造が消失して、本発明の「ハイドロゲル形成性の高分子」は、再び完全な水溶液となる。このように、好適な態様における本発明の高分子の物性変化は、分子内に存在する曇点を有するブロックの該曇点における可逆的な親水性、疎水性の変化に基づくものであるので、温度変化に対応して、完全な可逆性を有する。 On the other hand, since the hydrophilic block is water-soluble even at this time (when heated to a temperature higher than the cloud point), the polymer of the present invention has a hydrophobic association site between blocks having the cloud point in water. A hydrogel having a three-dimensional network structure with a cross-linking point is generated. When the temperature of this hydrogel is cooled again to a temperature lower than the cloud point of the “block having a cloud point” existing in the molecule, the block having the cloud point becomes water-soluble and the cross-linking point by the hydrophobic association is released. The hydrogel structure disappears, and the “hydrogel-forming polymer” of the present invention becomes a complete aqueous solution again. As described above, the physical property change of the polymer of the present invention in the preferred embodiment is based on the reversible hydrophilicity/hydrophobicity change at the cloud point of the block having the cloud point existing in the molecule. It has complete reversibility in response to temperature changes.
本発明者らの検討によれば、上記した「ハイドロゲル形成性の高分子」の水中における微妙な親水性−疎水性のバランスは、細胞を培養する際の細胞の安定性に寄与しているものと考えられる。 According to the study by the present inventors, the delicate hydrophilic-hydrophobic balance in water of the above-mentioned "hydrogel-forming polymer" contributes to the stability of cells when culturing the cells. Thought to be a thing.
（ゲルの溶解性）
上述したように本発明のハイドロゲル形成性の高分子は、体温（３７℃）で実質的に水不溶性を示し、氷冷下で可逆的に水可溶性を示す。上記「実質的に水不溶性」とは、３７℃において、水ｌ００ｍＬに溶解する上記高分子の量が、５．０ｇ以下（更には０．５ｇ以下、特に０．１ｇ以下）であることが好ましい。
(Gel solubility)
As described above, the hydrogel-forming polymer of the present invention is substantially water-insoluble at body temperature (37° C.) and reversibly water-soluble under ice cooling. The term “substantially water-insoluble” means that the amount of the polymer dissolved in 100 mL of water at 37° C. is 5.0 g or less (further 0.5 g or less, particularly 0.1 g or less). ..
一方、上記氷冷下「水可溶性」とは、１０℃において、水ｌ００ｍＬに溶解する上記高分子の量が、０．５ｇ以上（更には１．０ｇ以上）であることが好ましい。
更に「可逆的に水可溶性を示す」とは、上記「ハイドロゲル形成性の高分子」の水溶液が一旦３７℃で「実質的に水不溶性」のゲル状態となった後においても、１０℃においては、上記した水可溶性を示すことをいう。
On the other hand, the term “water-soluble” under ice cooling preferably means that the amount of the polymer dissolved in 100 mL of water at 10° C. is 0.5 g or more (further 1.0 g or more).
Furthermore, "reversibly exhibits water solubility" means that even after the aqueous solution of the "hydrogel-forming polymer" once becomes a "substantially water-insoluble" gel state at 37°C, at 10°C. Means to exhibit the above-mentioned water solubility.
上記高分子は、その１０％水溶液が５℃で、１０〜３，０００センチポイズ（更には５０〜１．０００センチポイズ）の粘度を示すことが好ましい。このような粘度は、例えば以下のような測定条件下で測定することが好ましい。
粘度計：ストレス制御式レオメータ（機種名：AR500、TAインスツルメンツ社製）
ローター直径：６０ｍｍ
ローター形状：平行平板
The polymer preferably has a viscosity of 10 to 3,000 centipoise (further, 50 to 1.000 centipoise) when its 10% aqueous solution is 5°C. Such viscosity is preferably measured, for example, under the following measurement conditions.
Viscometer: Stress control rheometer (Model name: AR500, TA Instruments)
Rotor diameter: 60mm
Rotor shape: parallel plate
本発明の「ハイドロゲル形成性の高分子」の水溶液は、３７℃で多量の水中に浸潰しても、該ゲルは実質的に溶解しない。上記「ハイドロゲル形成性の高分子」が形成するハイドロゲルの上記特性は、例えば、以下のようにして確認することが可能である。すなわち、「ハイドロゲル形成性の高分子」０．１５ｇを、氷冷下で、蒸留水１．３５ｇに溶解してｌ０ｗｔ％の水溶液を作製し、該水溶液を径が３５ｍｍのプラスチックシャーレ中に注入し、３７℃に加湿することによって、厚さ約１．５ｍｍのゲルを該シャーレ中に形成させた後、該ゲルを含むシャーレ全体の重量（ｆグラム）を測定する。次いで、該ゲルを含むシャーレ全体を２５０ｍｌ中の水中に３７℃で１０時間静置した後、該ゲルを含むシャーレ全体の重量（ｇグラム）を測定して、ゲル表面からの該ゲルの溶解の有無を評価する。この際、本発明のハイドロゲル形成性の高分子においては、上記ゲルの重量減少率、すなわち（ｆ−ｇ）／ｆが、５．０％以下であることが好ましく、更には１．０％以下（特に０．１％以下）であることが好ましい。 The aqueous solution of the "hydrogel-forming polymer" of the present invention does not substantially dissolve the gel even when it is immersed in a large amount of water at 37°C. The above properties of the hydrogel formed by the above-mentioned “hydrogel-forming polymer” can be confirmed, for example, as follows. That is, 0.15 g of "hydrogel-forming polymer" was dissolved in 1.35 g of distilled water under ice cooling to prepare a 10 wt% aqueous solution, and the aqueous solution was poured into a plastic petri dish having a diameter of 35 mm. Then, a gel having a thickness of about 1.5 mm is formed in the petri dish by humidifying at 37° C., and then the weight (f gram) of the entire petri dish containing the gel is measured. Then, the whole petri dish containing the gel was allowed to stand in water in 250 ml at 37° C. for 10 hours, and then the weight (g grams) of the whole petri dish containing the gel was measured to measure the dissolution of the gel from the gel surface. Evaluate the presence or absence. At this time, in the hydrogel-forming polymer of the present invention, the weight reduction rate of the gel, that is, (f-g)/f is preferably 5.0% or less, and further 1.0%. It is preferably below (particularly below 0.1%).
本発明の「ハイドロゲル形成性の高分子」の水溶液は、３７℃でゲル化させた後、多量（体積比で、ゲルの０．１〜１００倍程度）の水中に浸潰しても、長期間に亘って該ゲルは溶解することがない。このような本発明に用いる高分子の性質は、例えば、該高分子内に曇点を有するブロックが２個以上（複数個）存在することによって達成される。 The aqueous solution of the “hydrogel-forming polymer” of the present invention, even after being gelled at 37° C., is long-lived even if it is immersed in a large amount (about 0.1 to 100 times the volume of the gel). Over time the gel does not dissolve. Such properties of the polymer used in the present invention are achieved, for example, by having two or more (plural) blocks having a cloud point in the polymer.
これに対して、ポリプロピレンオキサイドの両端にポリエチレン才キサイドが結合してなる前述のプルロニック Ｆ−１２７を用いて同様のゲルを作成した場合には、数時間の静置で該ゲルは完全に水に溶解することを、本発明者らは見出している。 On the other hand, when a similar gel was prepared by using the above-mentioned Pluronic F-127 in which polyethylene oxide was bound to both ends of polypropylene oxide, the gel was completely dissolved in water after standing for several hours. The present inventors have found that they dissolve.
非ゲル化時の細胞毒性をできる限り低いレベルに抑える点からは、水に対する濃度、すなわち｛（高分子）／（高分子＋水）｝×１００（％）で、２０％以下（更には１５％以下、特に１０％以下）の濃度でゲル化が可能な「ハイドロゲル形成性の高分子」を用いることが好ましい。 From the viewpoint of suppressing the cytotoxicity at the time of non-gelling to a level as low as possible, the concentration in water, that is, {(polymer)/(polymer+water)}×100(%), is 20% or less (further 15 % Or less, especially 10% or less), it is preferable to use a "hydrogel-forming polymer" capable of gelation.
本発明に用いられる「ハイドロゲル形成性の高分子」の分子量は３万以上３，０００万以下が好ましく、より好ましくは１０万以上１，０００万以下、さらに好ましくは５０万以上５００万以下である。 The "hydrogel-forming polymer" used in the present invention has a molecular weight of preferably 30,000 or more and 30 million or less, more preferably 100,000 or more and 10 million or less, and further preferably 500,000 or more and 5 million or less. is there.
本発明の尿道狭窄治療剤の貯蔵弾性率を好ましい範囲に調整するには、上記したように「ハイドロゲル形成性の高分子」の種類を選択することと合わせ、尿道狭窄治療剤中の「ハイドロゲル形成性の高分子」の濃度を調整することによっても行うことができる。通常、本発明の尿道狭窄治療剤の貯蔵弾性率は、「ハイドロゲル形成性の高分子」の濃度を上げると増大し、該濃度を下げると減少する。 In order to adjust the storage elastic modulus of the therapeutic agent for urethral stricture of the present invention to a preferable range, in combination with selecting the type of “hydrogel-forming polymer” as described above, “hydro” in the therapeutic agent for urethral stricture is combined. It can also be performed by adjusting the concentration of the "gel-forming polymer". Generally, the storage elastic modulus of the therapeutic agent for urethral stricture of the present invention increases when the concentration of the “hydrogel-forming polymer” is increased, and decreases when the concentration is decreased.
（添加塩）
本発明の尿道狭窄治療剤は、上記した「ハイドロゲル形成性の高分子」を少なくとも含むものであるが、生体の体液に近いｐＨや浸透圧を持たせるために、ｐＨ緩衝液や生理食塩水などの塩類を添加することが望ましい。
(Added salt)
The therapeutic agent for urethral stricture of the present invention contains at least the above-mentioned "hydrogel-forming polymer", but in order to have a pH and an osmotic pressure close to the body fluid of a living body, a pH buffer solution or a physiological saline solution is used. It is desirable to add salts.
（動物細胞）
本発明の尿道狭窄治療剤は動物細胞を分散させて使用することができる。動物細胞としては、尿道粘膜上皮細胞が最も好ましい。採取の容易性と免疫拒絶反応を避けるためには、自己口腔粘膜細胞が好ましく利用される。
(Animal cell)
The therapeutic agent for urethral stricture of the present invention can be used by dispersing animal cells. Most preferred animal cells are urethral mucosal epithelial cells. Autologous oral mucosal cells are preferably used in order to facilitate collection and avoid immune rejection.
動物細胞として免疫寛容性が高いという観点から、分化細胞以外に未分化細胞を利用することもできる。未分化細胞の例として、ＥＳ細胞、ｉＰＳ細胞などの多分化能幹細胞や間葉系幹細胞を挙げることができる。 From the viewpoint of high immunological tolerance as animal cells, undifferentiated cells can be used in addition to differentiated cells. Examples of undifferentiated cells include pluripotent stem cells such as ES cells and iPS cells and mesenchymal stem cells.
本発明の尿道狭窄治療剤では、動物細胞を尿道狭窄治療剤として使用する直前に混合することも出来るが、予備的に細胞を尿道狭窄治療剤に分散させ、細胞を増殖させてから尿道狭窄部位に適用することもできる。 In the therapeutic agent for urethral stricture of the present invention, animal cells can be mixed immediately before use as a therapeutic agent for urethral stricture, but cells are preliminarily dispersed in the therapeutic agent for urethral stricture, and cells are proliferated before the urethral stricture site. Can also be applied to.
上記細胞が未分化細胞の場合には、未分化のまま増殖させた後に粘膜上皮細胞への系譜に分化誘導しておくこともできる。 When the above-mentioned cells are undifferentiated cells, they can be allowed to proliferate in an undifferentiated state and then differentiated into a lineage of mucosal epithelial cells.
（サイトカイン）
本発明の尿道狭窄治療剤には、尿道狭窄部位の粘膜細胞上皮化を促進させる目的で、各種サイトカインを含有させても良い。サイトカインは水溶性であるので、そのまま尿道狭窄部位に注入しても容易に拡散してしまうが、本発明の尿道狭窄治療剤は「ハイドロゲル形成性の高分子」により繊密な高分子網目を形成しているので、サイトカインの拡散を抑制する。そのため、尿道狭窄部位周辺にサイトカインを高濃度で保持することが出来るため、サイトカインの効果を長期間維持することができる。
(Cytokine)
The therapeutic agent for urethral stricture of the present invention may contain various cytokines for the purpose of promoting mucosal cell epithelialization of the urethral stricture site. Since the cytokine is water-soluble, it can easily diffuse even if it is directly injected into the urethral stricture site, but the therapeutic agent for urethral stricture of the present invention has a delicate polymer network due to the "hydrogel-forming polymer". As it is formed, it suppresses the diffusion of cytokines. Therefore, since the cytokine can be retained at a high concentration around the urethral stricture site, the effect of the cytokine can be maintained for a long period of time.
本発明で好適に用いられるサイトカインは、尿道狭窄部位の粘膜細胞上皮化を促進するものであれば特に制限なく用いることが出来るが、その作用として細胞の未分化維持、増殖促進、尿道粘膜上皮細胞系譜への分化誘導促進などの作用を持つものが好ましく用いられる。 The cytokine preferably used in the present invention can be used without particular limitation as long as it promotes the mucosal cell epithelialization of the urethral stricture site, but its effect is to maintain undifferentiation of cells, promote proliferation, urethral mucosal epithelial cells. Those having an action of promoting differentiation induction into a lineage are preferably used.
（尿道狭窄治療方法）
本発明の尿道狭窄治療剤は、経尿道内視鏡的手技で切開処置した尿道内面に留置することで切開処置部位の上皮化を促進し、該部位の瘢痕組織化によって尿道狭窄が再発することを有効に防止する。
(Urethral stricture treatment method)
The therapeutic agent for urethral stricture of the present invention promotes the epithelialization of the incision-treated site by placing it on the inner surface of the urethra that has been incised by a transurethral endoscopic procedure, and the urethral stricture recures due to the scar organization of the site. Effectively prevent.
患者に対し、通常の経尿道内視鏡的切開術により、尿道狭窄部位の切開を行い、尿道カテーテルを挿入後、尿道カテーテル周囲と尿道内面切開部位の間に氷冷した本発明の尿道狭窄治療剤を尿道カテーテルとは別のカテーテルを介して注入する。本発明の尿道狭窄治療剤は体温により暖められて即座にゲル化、尿道内面切開部位全体を覆うように留置される。術後約３週間で尿道カテーテルを抜去すると、狭窄切開部位は瘢痕化することなく粘膜上皮細胞で覆われ、良好な尿の流通が確保される。 The urethral stricture treatment of the present invention, in which the urethral stricture site is incised by a normal transurethral endoscopic incision for the patient, and after inserting the urethral catheter, ice-cooled between the urethral catheter periphery and the urethral inner surface incision site The drug is infused via a catheter separate from the urethral catheter. The therapeutic agent for urethral stricture of the present invention is warmed by body temperature and immediately gelled, and is placed so as to cover the entire incised site on the inner surface of the urethra. When the urethral catheter is removed about 3 weeks after the operation, the stenotic incision site is covered with mucosal epithelial cells without scarring, and good urine circulation is secured.
本発明の尿道狭窄治療剤は、氷冷水に溶解する性質を有するので、術後何らかの理由で本発明の尿道狭窄治療剤を除去したい場合には氷冷水で容易に洗い流すことができる。 Since the therapeutic agent for urethral stricture of the present invention has a property of dissolving in ice-cold water, if it is desired to remove the therapeutic agent for urethral stricture of the present invention for some reason after surgery, it can be easily washed off with ice-cold water.
以下に「ハイドロゲル形成性の高分子」の製造例および本発明の実施例を示し、本発明を更に具体的に説明するが、本発明の範囲は特許請求の範囲により限定されるものであり、以下の実施例によって限定されるものではない。 Hereinafter, the present invention will be described in more detail with reference to production examples of "hydrogel-forming polymer" and examples of the present invention, but the scope of the present invention is limited by the claims. However, the present invention is not limited to the following examples.
製造例１
ポリプロピレンオキサイド−ポリエチレンオキサイド共重合体（プロピレンオキサイド／エチレンオキサイド平均重合度約６０／１８０、旭電化工業株式会社製：プルロニック Ｆ−１２７）ｌ０ｇを乾燥クロロホルム３０ｍｌに溶解し、五酸化リン共存下、ヘキサメチレンジイソシアネート０．１３ｇを加え、沸点還流下に６時間反応させた。溶媒を減圧留去後、残さを蒸留水に溶解し、分画分子量５０万の限外濾過膜を用いて限外濾過を行い、高分子量重合体と低分子量重合体を分画した。得られた水溶液を凍結して、Ｆ−１２７高重合体およびＦ−１２７低重合体を得た。
Production example 1
Polypropylene oxide-polyethylene oxide copolymer (propylene oxide/ethylene oxide average degree of polymerization about 60/180, Asahi Denka Kogyo Co., Ltd.: Pluronic F-127) 10 g was dissolved in 30 ml of dry chloroform, and then, in the presence of phosphorus pentoxide, hexa 0.13 g of methylene diisocyanate was added, and the mixture was reacted under reflux of boiling point for 6 hours. After distilling off the solvent under reduced pressure, the residue was dissolved in distilled water and subjected to ultrafiltration using an ultrafiltration membrane having a molecular weight cutoff of 500,000 to fractionate a high molecular weight polymer and a low molecular weight polymer. The obtained aqueous solution was frozen to obtain an F-127 high polymer and an F-127 low polymer.
上記により得たＦ−１２７高重合体（本発明のハイドロゲル形成性高分子、「ハイドロゲル形成性の高分子」−１）１ｇを、９ｇの蒸留水に氷冷下で溶解し、ｌ０ｗｔ％の水溶液を得た。この水溶液の貯蔵弾性率をストレス制御式レオメーター（AR500、TAインスツルメント社製）を用い、適用周波数１Ｈｚで測定したところ、１０℃で４Ｐａ、２５℃で１８９０Ｐａ、３７℃で６６６０Ｐａであった。この温度依存性貯蔵弾性率変化は、可逆的に繰り返し観測された。一方、上記Ｆ−１２７低重合体を、氷点下ｌ０ｗｔ％の濃度で蒸留水に溶解したものは、６０℃以上に加熱しても全くゲル化しなかった。 1 g of the F-127 high polymer (hydrogel-forming polymer of the present invention, “hydrogel-forming polymer”-1) obtained as described above was dissolved in 9 g of distilled water under ice cooling to give 10 wt%. An aqueous solution of The storage elastic modulus of this aqueous solution was measured using a stress control rheometer (AR500, manufactured by TA Instruments) at an applied frequency of 1 Hz, and was 4 Pa at 10°C, 1890 Pa at 25°C, and 6660 Pa at 37°C. .. This temperature-dependent storage elastic modulus change was repeatedly observed reversibly. On the other hand, the above F-127 low polymer dissolved in distilled water at a concentration of 10 wt% below freezing did not gel at all even when heated to 60°C or higher.
製造例２
トリメチロールプロパン１モルに対し、エチレンオキサイド１６０モルをカチオン重合により付加して、平均分子量約７０００のポリエチレンオキサイドトリオールを得た。
上記により得たポリエチレンオキサイドトリオール１００ｇを蒸留水１０００ｍｌに溶解した後、室温で濾過マンガン酸カリウム１２ｇを徐々に加えて、そのまま約１時間、酸化反応させた。固形物を濾過により除いた後、生成物をクロロホルムで抽出し、溶媒（クロロホルム）を減圧留去してポリエチレンオキサイドトリカルボキシル体９０ｇを得た。
上記により得たポリエチレンオキサイドトリカルボキシル体ｌ０ｇと、ポリプロピレンオキサイドジアミノ体（プロピレンオキサイド平均重合度約６５、米国ジェファーソンケミカル社製、商品名：ジェファーミンＤ−４０００、曇点：約９℃）ｌ０ｇとを四塩化炭素１０００ｍｌに溶解し、ジシクロヘキシルカルボジイミド１．２ｇを加えた後、沸点還流下に６時間反応させた。反応液を冷却し、固形物を濾過により除いた後、溶媒（四塩化炭素）を減圧留去し、残さを真空乾燥して、複数のポリプロピレンオキサイドとポリエチレンオキサイドとが結合した本発明のハイドロゲル形成性高分子（「ハイドロゲル形成性の高分子」−２）を得た。この高分子１ｇを、１９ｇの蒸留水に氷冷下で溶解し、５ｗｔ％の水溶液を得た。この水溶液の貯蔵弾性率をストレス制御式レオメーター（AR500、TAインスツルメント社製）を用い、適用周波数１Ｈｚで測定したところ、１０℃でｌＰａ、２５℃で５５０Ｐａ、３７℃で３３６０Ｐａであった。この温度依存性貯蔵弾性率変化は、可逆的に繰り返し観測された。
Production example 2
160 mol of ethylene oxide was added by cationic polymerization to 1 mol of trimethylolpropane to obtain polyethylene oxide triol having an average molecular weight of about 7,000.
After dissolving 100 g of the polyethylene oxide triol obtained above in 1000 ml of distilled water, 12 g of filtered potassium manganate was gradually added at room temperature, and the oxidation reaction was continued for about 1 hour. After removing the solid matter by filtration, the product was extracted with chloroform, and the solvent (chloroform) was distilled off under reduced pressure to obtain 90 g of a polyethylene oxide tricarboxylate.
10 g of the polyethylene oxide tricarboxylate obtained as described above and 10 g of polypropylene oxide diamino derivative (propylene oxide average degree of polymerization of about 65, manufactured by Jefferson Chemical Co., USA, trade name: Jeffamine D-4000, cloud point: about 9° C.) It was dissolved in 1000 ml of carbon tetrachloride, 1.2 g of dicyclohexylcarbodiimide was added, and the mixture was reacted under reflux of boiling point for 6 hours. After cooling the reaction solution and removing the solid matter by filtration, the solvent (carbon tetrachloride) is distilled off under reduced pressure, the residue is vacuum dried, and the hydrogel of the present invention in which a plurality of polypropylene oxides and polyethylene oxides are bound A forming polymer (“hydrogel-forming polymer”-2) was obtained. 1 g of this polymer was dissolved in 19 g of distilled water under ice cooling to obtain a 5 wt% aqueous solution. The storage elastic modulus of this aqueous solution was measured with a stress control rheometer (AR500, manufactured by TA Instruments Co., Ltd.) at an applied frequency of 1 Hz and found to be 1 Pa at 10° C., 550 Pa at 25° C., and 3360 Pa at 37° C. .. This temperature-dependent storage elastic modulus change was repeatedly observed reversibly.
製造例３
Ｎ−イソプロピルアクリルアミド（イーストマンコダック社製）９６ｇ、Ｎ−アクリロキシスクシンイミド（国産化学株式会社製）１７ｇ、およびｎ−ブチルメタクリレート（関東化学株式会社製）７ｇをクロロホルム４０００ｍｌに溶解し、窒素置換後、Ｎ，Ｎ´−アゾビスイソブチロニトリル１．５ｇを加え、６０℃で６時間重合させた。反応液を濃縮した後、ジエチルエーテルに再沈（再沈殿）した。濾過により固形物を回収した後、真空乾燥して、７８ｇのポリ（Ｎ−イソプロピルアクリルアミド−コ−Ｎ−アクリロキシスクシンイミド−コ−ｎ−ブチルメタクリレート）を得た。
上記により得たポリ（Ｎ−イソプロピルアクリルアミド−コ−Ｎ−アクリロキシスクシンイミド−コ−ｎ−ブチルメタクリレート）に、過剰のイソプロピルアミンを加えてポリ（Ｎ−イソプロピルアクリルアミド−コ−ｎ−ブチルメタクリレート）を得た。このポリ（Ｎ−イソプロピルアクリルアミド−コ−ｎ−ブチルメタクリレート）の水溶液の曇点は１９℃であった。
Production Example 3
96 g of N-isopropylacrylamide (manufactured by Eastman Kodak), 17 g of N-acryloxysuccinimide (manufactured by Kokusan Kagaku Co., Ltd.), and 7 g of n-butyl methacrylate (manufactured by Kanto Kagaku Co., Ltd.) were dissolved in 4000 ml of chloroform, and after nitrogen substitution. , N,N′-azobisisobutyronitrile (1.5 g) was added, and the mixture was polymerized at 60° C. for 6 hours. The reaction solution was concentrated and then reprecipitated (reprecipitated) in diethyl ether. The solid matter was collected by filtration and then dried in vacuum to obtain 78 g of poly(N-isopropylacrylamide-co-N-acryloxysuccinimide-co-n-butyl methacrylate).
To the poly(N-isopropylacrylamide-co-N-acryloxysuccinimide-co-n-butylmethacrylate) obtained above, excess isopropylamine was added to give poly(N-isopropylacrylamide-co-n-butylmethacrylate). Obtained. The cloud point of this aqueous solution of poly(N-isopropylacrylamide-co-n-butyl methacrylate) was 19°C.
前記のポリ（Ｎ−イソプロピルアクリルアミド−コ−Ｎ−アクリロキシスクシンイミド−コ−ｎ−ブチルメタクリレート）ｌ０ｇ、および両末端アミノ化ポリエチレンオキサイド（分子量６，０００、川研ファインケミカル株式会社製）５ｇをクロロホルム１０００ｍｌに溶解し、５０℃で３時間反応させた。室温まで冷却した後、イソプロピルアミン１ｇを加え、１時間放置した後、反応液を濃縮し、残渣をジエチルエーテル中に沈澱させた。濾過により固形物を回収した後、真空乾燥して、複数のポリ（Ｎ−イソプロピルアクリルアミド−コ−ｎ−ブチルメタクリレート）とポリエチレンオキサイドとが結合した本発明のハイドロゲル形成性高分子（「ハイドロゲル形成性の高分子」−３）を得た。
この高分子１ｇを、９ｇの蒸留水に氷冷下で溶解し、ｌ０ｗｔ％）水溶液を得た。この水溶液の貯蔵弾性率をストレス制御式レオメーター（AR500、TAインスツルメント社製）を用い、適用周波数ｌＨｚで測定したところ、１０℃でｌＰａ以下、２５℃で３０Ｐａ、３７℃で２５０Ｐａであった。この温度依存性貯蔵弾性率変化は、可逆的に繰り返し観測された。
10 g of the above poly(N-isopropylacrylamide-co-N-acryloxysuccinimide-co-n-butylmethacrylate) and 5 g of aminated polyethylene oxide at both ends (molecular weight 6,000, manufactured by Kawaken Fine Chemicals Co., Ltd.) in 1000 ml of chloroform. And was reacted at 50° C. for 3 hours. After cooling to room temperature, 1 g of isopropylamine was added, and the mixture was allowed to stand for 1 hour, then the reaction solution was concentrated, and the residue was precipitated in diethyl ether. The solid matter is collected by filtration and then dried in a vacuum to form a hydrogel-forming polymer (“hydrogel” of the present invention in which a plurality of poly(N-isopropylacrylamide-co-n-butylmethacrylate) and polyethylene oxide are bonded. Forming polymer”-3) was obtained.
1 g of this polymer was dissolved in 9 g of distilled water under ice cooling to obtain a 10 wt% aqueous solution. The storage elastic modulus of this aqueous solution was measured with a stress control rheometer (AR500, manufactured by TA Instruments Co., Ltd.) at an applied frequency of 1 Hz to find that it was 1 Pa or less at 10° C., 30 Pa at 25° C. and 250 Pa at 37° C. It was This temperature-dependent storage elastic modulus change was repeatedly observed reversibly.
製造例４
（滅菌方法）
上記した本発明のハイドロゲル形成性高分子（「ハイドロゲル形成性の高分子」−３）の２．０ｇを、ＥＯＧ（エチレンオキサイドガス）滅菌バッグ（ホギメディカル社製、商品名：ハイブリッド滅菌バッグ）に入れ、ＥＯＧ滅菌装置（イージーパック、井内盛栄堂製）でＥＯＧをバッグに充填し、室温にて一昼夜放置した。さらに４０℃で半日放置した後、ＥＯＧをバッグから抜き、エアレーションを行った。バッグを真空乾燥器（４０℃）に入れ、時々エアレーションしながら半日放置することにより滅菌した。
この滅菌操作により高分子水溶液の貯蔵弾性率が変化しないことを、別途確認した。
Production Example 4
(Sterilization method)
2.0 g of the above-mentioned hydrogel-forming polymer of the present invention (“hydrogel-forming polymer”-3) was added to an EOG (ethylene oxide gas) sterilization bag (manufactured by Hogy Medical Co., Ltd., trade name: Hybrid Sterilization Bag). ), the bag was filled with EOG using an EOG sterilizer (Easy Pack, manufactured by Inei Seieidou), and left at room temperature for 24 hours. Further, after leaving it at 40° C. for half a day, EOG was taken out from the bag and aerated. The bag was placed in a vacuum dryer (40° C.) and left for half a day with occasional aeration to sterilize.
It was separately confirmed that the storage elastic modulus of the aqueous polymer solution was not changed by this sterilization operation.
製造例５
Ｎ−イソプロピルアクリルアミド７１．０ｇおよびｎ−ブチルメタクリレート４，４ｇをエタノール１１１７ｇに溶解した。これにポリエチレングリコールジメタクリレート（ＰＤＥ６０００、日本油脂株式会社製）２２．６ｇを水７７３ｇに溶解した水溶液を加え、窒素気流下７０℃に加湿した。窒素気流下７０℃を保ちながら、Ｎ，Ｎ，Ｎ´，Ｎ´−テトラメチルエチレンジアミン（ＴＥＭＥＤ）０．８ｍｌと１０％濾過硫酸アンモニウム（ＡＰＳ）水溶液８ｍｌを加え３０分間機梓反応させた。さらにＴＥＭＥＤ Ｏ．８ｍｌと１０％ＡＰＳ水溶液８ｍｌを３０分間隔で４回加えて重合反応を完結させた。反応液をｌ０℃以下に冷却後、１０℃の冷却蒸留水５Ｌを加えて希釈し、分画分子量１０万の限外濾過膜を用いて１０℃で２Ｌまで濃縮した。
該濃縮液に冷却蒸留水４Ｌを加えて希釈し、上記限外濾過濃縮操作を再度行った。上記の希釈、限外濾過濃縮操作を更に５回繰り返し、分子量Ｉ０万以下のものを除去した。この限外濾過により濾過されなかったもの（限外濾過膜内に残留したもの）を回収して凍結乾燥し、分子量１０万以上の本発明のハイドロゲル形成性高分子（「ハイドロゲル形成性の高分子」−５）７２ｇを得た。
Production Example 5
71.0 g of N-isopropylacrylamide and 4,4 g of n-butyl methacrylate were dissolved in 1117 g of ethanol. An aqueous solution prepared by dissolving 22.6 g of polyethylene glycol dimethacrylate (PDE6000, manufactured by NOF CORPORATION) in 773 g of water was added thereto, and the mixture was humidified at 70°C under a nitrogen stream. While maintaining the temperature at 70° C. under a nitrogen stream, 0.8 ml of N,N,N′,N′-tetramethylethylenediamine (TEMED) and 8 ml of a 10% filtered ammonium sulfate (APS) aqueous solution were added and agitated for 30 minutes. In addition, TEMED O. The polymerization reaction was completed by adding 8 ml and 10 ml of 10% APS aqueous solution 4 times at intervals of 30 minutes. The reaction solution was cooled to 10° C. or lower, diluted with 5 L of 10° C. cold distilled water, and concentrated to 2 L at 10° C. using an ultrafiltration membrane having a molecular weight cutoff of 100,000.
4 L of cold distilled water was added to the concentrated solution to dilute it, and the ultrafiltration concentration operation was repeated. The above dilution and ultrafiltration concentration operations were repeated 5 times to remove those having a molecular weight of 100,000 or less. Those which have not been filtered by this ultrafiltration (those remaining in the ultrafiltration membrane) are collected and freeze-dried to obtain the hydrogel-forming polymer of the present invention having a molecular weight of 100,000 or more (“hydrogel-forming polymer”). Polymer "-5) 72 g was obtained.
上記により得た本発明のハイドロゲル形成性高分子（「ハイドロゲル形成性の高分子」−５）１ｇを、９ｇの蒸留水に氷冷下で溶解し、ｌ０ｗｔ％もの水溶液を得た。この水溶液の貯蔵弾性率をストレス制御式レオメーター（AR500、TAインスツルメント社製）を用い、適用周波数１Ｈｚで測定したところ、１０℃でｌＰａ以下、２５℃で８０Ｐａ、３７℃で４６０Ｐａであった。この温度依存性貯蔵弾性率変化は、可逆的に繰り返し観測された。 1 g of the hydrogel-forming polymer of the present invention (“hydrogel-forming polymer”-5) obtained above was dissolved in 9 g of distilled water under ice cooling to obtain an aqueous solution of 10 wt%. The storage elastic modulus of this aqueous solution was measured with a stress-controlled rheometer (AR500, manufactured by TA Instruments Co., Ltd.) at an applied frequency of 1 Hz. It was This temperature-dependent storage elastic modulus change was repeatedly observed reversibly.
製造例６
Ｎ−イソプロピルアクリルアミド４２，０ｇおよびｎ−ブチルメタクリレート４，０ｇをエタノール５９２ｇに溶解した。これにポリエチレングリコールジメタクリレート（ＰＤＥ６０００、日本油脂株式会社製）１１．５ｇを水６５．１ｇに溶解した水溶液を加え、窒素気流下７０℃に加温した。窒素気流下７０℃を保ちながら、Ｎ，Ｎ，Ｎ´，Ｎ´−テトラメチルエチレンジアミン（ＴＥＮＥＤ）０．４ｍｌと１０％過硫酸アンモニウム（ＡＰＳ）水溶液４ｍｌを加え３０分間隔で反応させた。さらにＴＥＮＥＤ ０．４ｍｌと１０％ＡＰＳ水溶液４ｍｌを３０分間隔で４回加えて重合反応を完結させた。反応液を５９Ｃ以下に冷却後、５℃の冷却蒸留水５Ｌを加えて希釈し、分画分子量１０万の眼外濾過膜を用いて５℃で２Ｌまで濃縮した。
該濃縮液に冷却蒸留水４Ｌを加えて希釈し、上記眼外濾過濃縮操作を再度行った。上記の希釈、限外濾過濃縮操作を更に５回繰り返し、分子量１０万以下のものを除去した。この限外濾過により濾過されなかったもの（限外濾過膜内に残留したもの）を回収して凍結乾燥し、分子量１０万以上の本発明のハイドロゲル形成性高分子（「ハイドロゲル形成性の高分子」−６）４０ｇを得た。
上記により得た本発明のハイドロゲル形成性高分子（「ハイドロゲル形成性の高分子」−６）１ｇを、９ｇの蒸留水に氷冷下で溶解しｌ０ｗｔ％の水溶液を得た。この水溶液の貯蔵弾性率をストレス制御式レオメーター（AR500、TAインスツルメント社製）を用い、適用周波数１Ｈｚで測定したところ、１０℃で４３Ｐａ、２５℃で６８０Ｐａ、３７℃で１３１０Ｐａであった。この温度依存性貯蔵弾性率変化は、可逆的に繰り返し観測された。
Production Example 6
42.0 g of N-isopropylacrylamide and 40 g of n-butyl methacrylate were dissolved in 592 g of ethanol. An aqueous solution prepared by dissolving 11.5 g of polyethylene glycol dimethacrylate (PDE6000, manufactured by NOF CORPORATION) in 65.1 g of water was added thereto, and the mixture was heated to 70°C under a nitrogen stream. While maintaining the temperature at 70° C. under a nitrogen stream, 0.4 ml of N,N,N′,N′-tetramethylethylenediamine (TENED) and 4 ml of a 10% ammonium persulfate (APS) aqueous solution were added and reacted at intervals of 30 minutes. Furthermore, TENED 0.4 ml and 10% APS aqueous solution 4 ml were added 4 times at intervals of 30 minutes to complete the polymerization reaction. The reaction solution was cooled to 59 C or lower, diluted by adding 5 L of cold distilled water at 5° C., and concentrated to 2 L at 5° C. using an extraocular filtration membrane having a cutoff molecular weight of 100,000.
4 L of cold distilled water was added to the concentrated solution to dilute it, and the above-mentioned extracorporeal filtration concentration operation was repeated. The above-mentioned dilution and ultrafiltration concentration operation were repeated 5 times to remove those having a molecular weight of 100,000 or less. Those which have not been filtered by this ultrafiltration (those remaining in the ultrafiltration membrane) are collected and freeze-dried to obtain the hydrogel-forming polymer of the present invention having a molecular weight of 100,000 or more (“hydrogel-forming polymer”). Polymer"-6) 40 g was obtained.
1 g of the hydrogel-forming polymer of the present invention (“hydrogel-forming polymer”-6) obtained above was dissolved in 9 g of distilled water under ice cooling to obtain a 10 wt% aqueous solution. The storage elastic modulus of this aqueous solution was measured with a stress-controlled rheometer (AR500, manufactured by TA Instruments) at an applied frequency of 1 Hz, and was 43 Pa at 10°C, 680 Pa at 25°C, and 1310 Pa at 37°C. .. This temperature-dependent storage elastic modulus change was repeatedly observed reversibly.
製造例７
Ｎ−イソプロピルアクリルアミド４５，５ｇおよびｎ−ブチルメタクリレート０．５６ｇをエタノール５９２ｇに溶解した。これにポリエチレングリコールジメタクリレート（ＰＤＥ６０００、日本油脂株式会社製）Ｉ１．５ｇを水６５，１ｇに溶解した水溶液を加え、窒素気流下７０℃に加温した。窒素気流下７０℃を保ちながら、Ｎ，Ｎ，Ｎ´，Ｎ´−テトラメチルエチレンジアミン（ＴＥＮＥＤ）０．４ｍｌと１０％濾過硫酸アンモニウム（ＡＰＳ）水溶液４ｍｌを加え３０分間撹拌反応させた。さらにＴＥＮＥＤ ４．０ｍｌと１０％ＡＰＳ水溶液４ｍｌを３０分間隔で４回加えて重合反応を完結させた。反応液を１０℃以下に冷却後、１０℃の冷却蒸留水５Ｌを加えて希釈し、分画分子量１０万の限外濾過膜を用いて１０℃で２Ｌまで濃縮した。
該濃縮液に冷却蒸留水４Ｌを加えて希釈し、上記限外濾過濃縮操作を再度行った。上記の希釈、限外濾過濃縮操作を更に５回繰り返し、分子量１０万以下のものを除去した。この限外濾過により濾過されなかったもの（限外濾過膜内に残留したもの）を回収して凍結乾燥し、分子量１０万以上の本発明のハイドロゲル形成性高分子（「ハイドロゲル形成性の高分子」−７）２２ｇを得た。
上記により得たハイドロゲル形成性高分子（「ハイドロゲル形成性の高分子」−７）１ｇを、９ｇの蒸留水に氷冷下で溶解しｌ０ｗｔ％）水溶液を得た。この水溶液の貯蔵弾性率をストレス制御式レオメーター（AR500、TAインスツルメント社製）を用い、適用周波数１Ｈｚで測定したところ、１０℃でｌＰａ以下、２５℃でｌＰａ以下、３７℃で９０Ｐａであった。この温度依存性貯蔵弾性率変化は、可逆的に繰り返し観測された。
Production Example 7
45.5 g of N-isopropylacrylamide and 0.56 g of n-butyl methacrylate were dissolved in 592 g of ethanol. An aqueous solution prepared by dissolving 1.5 g of polyethylene glycol dimethacrylate (PDE6000, manufactured by NOF CORPORATION) in 65,1 g of water was added thereto, and the mixture was heated to 70° C. under a nitrogen stream. While maintaining the temperature at 70° C. under a nitrogen stream, 0.4 ml of N,N,N′,N′-tetramethylethylenediamine (TENED) and 4 ml of a 10% filtered ammonium sulfate (APS) aqueous solution were added, and the mixture was stirred and reacted for 30 minutes. Further, 4.0 ml of TENED and 4 ml of 10% APS aqueous solution were added 4 times at intervals of 30 minutes to complete the polymerization reaction. The reaction solution was cooled to 10° C. or less, diluted with 5 L of 10° C. cold distilled water, and concentrated to 2 L at 10° C. using an ultrafiltration membrane having a molecular weight cutoff of 100,000.
4 L of cold distilled water was added to the concentrated solution to dilute it, and the ultrafiltration concentration operation was repeated. The above-mentioned dilution and ultrafiltration concentration operation were repeated 5 times to remove those having a molecular weight of 100,000 or less. Those which have not been filtered by this ultrafiltration (those remaining in the ultrafiltration membrane) are collected and freeze-dried to obtain the hydrogel-forming polymer of the present invention having a molecular weight of 100,000 or more (“hydrogel-forming polymer”). Polymer "-7) 22 g was obtained.
1 g of the hydrogel-forming polymer (“hydrogel-forming polymer”-7) obtained above was dissolved in 9 g of distilled water under ice cooling to obtain an aqueous solution of 10 wt %. The storage elastic modulus of this aqueous solution was measured with a stress control rheometer (AR500, manufactured by TA Instruments Co., Ltd.) at an applied frequency of 1 Hz. It was 1 Pa or less at 10° C., 1 Pa or less at 25° C. and 90 Pa at 37° C. there were. This temperature-dependent storage elastic modulus change was repeatedly observed reversibly.
実施例１
製造例６で得た凍結乾燥ハイドロゲル形成性高分子−６を製造例４と同様にしてＥＯＧ滅菌した。ＥＯＧ滅菌後のハイドロゲル形成性高分子−６をｌ０ｗｔ％の濃度でリン酸緩衝液に氷冷下で溶解した。男性尿管狭窄患者の口腔粘膜組織（２ｃｍ×ｌｃｍ）を採取し、コラゲナーゼ処理した細胞に患者本人の血清（細胞と等量）を添加し、上記ハイドロゲル形成性高分子−６のリン酸緩衝液に氷冷下で分散させた。この細胞分散液を３７℃に昇温してゲル化させ、そのまま１０日間培養し細胞数を約１０倍に増殖させた。患者に対し、経尿道内視鏡的切開術により、尿道狭窄部位の切開を行い、尿道カテーテルを挿入後、尿道カテーテル周囲と尿道内面切開部位の間に氷冷した前記細胞培養後の本発明の尿道狭窄治療剤を注入した。本発明の尿道狭窄治療剤は体温により暖められて即座にゲル化、尿道内面切開部位全体を覆うように留置された。術後３週間で尿道カテーテルを抜去した。狭窄切開部位は癒痕化することなく粘膜上皮細胞で覆われ、良好な尿の流通が確保された。１０名の患者に同様の治療を行い、全ての患者で再狭窄は認められなかった。
Example 1
The freeze-dried hydrogel-forming polymer-6 obtained in Production Example 6 was EOG-sterilized in the same manner as in Production Example 4. Hydrogel-forming polymer-6 after EOG sterilization was dissolved in a phosphate buffer at a concentration of 10 wt% under ice cooling. Oral mucosal tissue (2 cm x 1 cm) of a male ureteral stenosis patient was collected, and the serum of the patient himself (the same amount as the cells) was added to the collagenase-treated cells, and the phosphate buffer of the above hydrogel-forming polymer-6 was added. The solution was dispersed under ice cooling. This cell dispersion liquid was heated to 37° C. to cause gelation, and the mixture was cultured as it was for 10 days to proliferate the cell number by about 10 times. To the patient, by transurethral endoscopic incision, to make an incision in the urethral stricture site, after inserting the urethral catheter, after the cell culture of the present invention after ice-cooled between the urethral catheter periphery and the urethral incision site A therapeutic agent for urethral stricture was injected. The therapeutic agent for urethral stricture of the present invention was warmed by body temperature and immediately gelled, and was placed so as to cover the entire urethral incision site. The urethral catheter was removed 3 weeks after the operation. The stricture incision site was covered with mucosal epithelial cells without scarring, and good circulation of urine was secured. Ten patients received similar treatment and no restenosis was noted in any of the patients.
実施例２
男性尿管狭窄患者の口腔粘膜細胞と血清を添加しないこと以外は実施例１と同じ治療を１０人の患者に行った結果、全ての患者で再狭窄は認められなかったが、２例で狭窄切開部位の癒痕化を認めた。
Example 2
The same treatment as in Example 1 was performed on 10 patients except that oral mucosal cells and serum of male ureteral stenosis patients were not added, and as a result, restenosis was not observed in all patients, but stenosis was observed in 2 patients. A scar at the incision site was observed.
実施例３
製造例６で得た凍結乾燥ハイドロゲル形成性高分子−６の代わりに製造例５で得た凍結乾燥ハイドロゲル形成性高分子−５を使用すること以外は実施例２と同じ治療を１０人の患者に行った結果、２例で再狭窄を認めた。
Example 3
The same treatment as in Example 2 was carried out by 10 persons except that the freeze-dried hydrogel-forming polymer-5 obtained in Production Example 6 was used instead of the freeze-dried hydrogel-forming polymer-6 obtained in Production Example 6. As a result, the restenosis was observed in 2 cases.
比較例１
製造例６で得た凍結乾燥ハイドロゲル形成性高分子−６の代わりに製造例７で得た凍結乾燥ハイドロゲル形成性高分子−７を使用すること以外は実施例１と同じ治療を１０人の患者に行った結果、全ての患者で狭窄切開部位の瘢痕化を認めた。ハイドロゲル形成性高分子−７の３７℃での貯蔵弾性率が１００Ｐａ未満と低いため、本発明の尿道狭窄治療剤として機能しなかったものと考えられる。
Comparative Example 1
The same treatment as in Example 1 was performed on 10 persons except that the freeze-dried hydrogel-forming polymer-7 obtained in Production Example 6 was used instead of the freeze-dried hydrogel-forming polymer-6 obtained in Production Example 6. As a result, the scarring of the stricture incision site was observed in all the patients. Since the storage elastic modulus of the hydrogel-forming polymer-7 at 37° C. was as low as less than 100 Pa, it is considered that it did not function as the therapeutic agent for urethral stricture of the present invention.
比較例２
実施例１において、ＥＯＧ滅菌後の凍結乾燥ハイドロゲル形成性高分子−６を１１ｗｔ％の濃度でリン酸緩衝液に氷冷下で溶解した。この水溶液の貯蔵弾性率をストレス制御式レオメーター（AR500、TAインスツルメント社製）を用い、適用周波数１Ｈｚで測定したところ、１０℃で７５Ｐａであった。この溶液は氷冷下でも流動性が低く、カテーテルを介して尿道狭窄部位へ注入することが出来ず、本発明の尿道狭窄治療剤として機能しなかった。
Comparative example 2
In Example 1, freeze-dried hydrogel-forming polymer-6 after EOG sterilization was dissolved in a phosphate buffer at a concentration of 11 wt% under ice cooling. The storage elastic modulus of this aqueous solution was measured with a stress control rheometer (AR500, manufactured by TA Instruments) at an applied frequency of 1 Hz and found to be 75 Pa at 10°C. This solution had low fluidity even under ice cooling, could not be injected into the urethral stricture site through a catheter, and did not function as a therapeutic agent for urethral stricture of the present invention.
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| WO2003006081A1 (en) * | 2001-07-13 | 2003-01-23 | Mebiol Inc. | Material for tissue/organ regeneration and method of tissue/organ regeneration |
| JP2006141436A (en) * | 2004-11-16 | 2006-06-08 | Mebiol Kk | Percutaneous vascular puncture sealing material and percutaneous vascular puncture sealing apparatus |
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Free format text: JAPANESE INTERMEDIATE CODE: C21 Effective date: 20230123 |
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| A912 | Re-examination (zenchi) completed and case transferred to appeal board |
Free format text: JAPANESE INTERMEDIATE CODE: A912 Effective date: 20230324 |
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| C211 | Notice of termination of reconsideration by examiners before appeal proceedings |
Free format text: JAPANESE INTERMEDIATE CODE: C211 Effective date: 20230328 |