JP2004123785A - N-alkanoylated hyaluronic acid or its salt, and its production method - Google Patents
N-alkanoylated hyaluronic acid or its salt, and its production method Download PDFInfo
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【0001】
【発明の属する技術分野】
本発明は、n−アルカノイル化ヒアルロン酸もしくはその塩およびその製造法に関する。さらに詳しくは、疎水部位と親水部位を併せ持ち、医療品、食品、化粧品分野などの新しい素材として有用なn−アルカノイル化ヒアルロン酸もしくはその塩およびその製造法に関する
【0002】
【従来の技術】
ヒアルロン酸はN−アセチルーD−グルコサミンとD−グルクロン酸が交互に結合してできた直鎖状の高分子多糖であり、多数の遊離カルボキシル基と多数の遊離ヒドロキシル基を有するために水に対する親和性に富み、任意の量の水に溶けて高粘度の水溶液を形成する。この遊離カルボキシル基と遊離ヒドロキシル基を封鎖する事により物性の異なるヒアルロン酸を入手することが期待される。そのため、医療品、食品、化粧品分野などに使用することを目的として、ヒアルロン酸またはその塩(以下、「ヒアルロン酸(塩)」と記述することがある)の化学修飾が行われている(例えば、特許文献1,2参照。)。しかしながら、従来の何れの反応も水分子の存在する系での反応であり、ヒアルロン酸(塩)の化学修飾に適用しうる有機反応が制限されていた。このため、医療品、食品、化粧品分野などの新しい素材となる疎水部位と親水部位を併せ持つ新しいタイプのヒアルロン酸(塩)の製造は非常に困難であった。
【0003】
【特許文献1】
特開平7−309902号公報
【特許文献2】
特開平8−53501号公報
【0004】
【発明が解決しようとする課題】
本発明者らは、医療品、食品、化粧品分野などの新しい素材として有用な、疎水部位と親水部位を併せ持つ新しいタイプのヒアルロン酸(塩)について鋭意研究した。その結果、ヒアルロン酸(塩)とカチオン性化合物との複合体を非水系溶媒に溶解し、次いで該複合体をn−アルカノイル基を有する酸ハロゲン化物と反応させることにより、疎水域と親水域を併せ持つ新しいタイプのヒアルロン酸(塩)が得られることを見出し、この知見に基づいて本発明を完成した。
以上の記述から明らかなように、本発明の目的は、医療品、食品、化粧品分野などの新しい素材として有用な、疎水部位と親水部位を併せ持つ新しいタイプのヒアルロン酸(塩)、特に、非水系での製造であるため発熱物質、抗原性物質などの混入が少なく、さらに体内で代謝されるため特に医薬分野で、例えば手術時等の保湿剤、潤滑剤、創傷被覆剤、さらにはDDS(ドラッグデリバリーシステム)材料として利用できるヒアルロン酸(塩)およびその製造法を提供することである。
【0005】
【課題を解決するための手段】
本発明は、以下の(1)〜(10)で示される。
(1)下記一般式(1)で表されるn−アルカノイル化ヒアルロン酸もしくはその塩。
(式中、R1、R2、R3、およびR4はそれぞれ水素または直鎖状のC5〜C15のn−アルカノイル基を表し、R5は水素、金属陽イオン、またはカチオン性化合物を表し、nは2〜7,500の整数を表す。)。
【0006】
(2)n−アルカノイル基が、下記一般式(2)で表されるn−アルカノイル基である前記(1)記載のn−アルカノイル化ヒアルロン酸もしくはその塩。
CH3−(CH2)m−CO− (2)
(式中、mは4〜10の整数を表す。)。
【0007】
(3)カチオン性化合物が第4級アンモニウム塩である前記(1)記載のn−アルカノイル化ヒアルロン酸もしくはその塩。
【0008】
(4)第4級アンモニウム塩がジステアリルジメチルアンモニウム塩である前記(3)記載のn−アルカノイル化ヒアルロン酸もしくはその塩。
【0009】
(5)ヒアルロン酸もしくはその塩とカチオン性化合物との複合体を非水系溶媒中でハロゲン化n−アルカノイルと反応させることを特徴とする下記一般式(3)で表されるn−アルカノイル化ヒアルロン酸もしくはその塩の製造法。
(式中、R1、R2、R3、およびR4はそれぞれ水素またはC4〜C18のn−アルカノイル基を表し、R5は水素、金属陽イオン、またはカチオン性化合物を表し、nは2〜7,500の整数を表す。)。
【0010】
(6)ハロゲン化n−アルカノイルが、下記一般式(4)で表されるハロゲン化n−アルカノイルである前記(5)記載のn−アルカノイル化ヒアルロン酸もしくはその塩の製造法。
CH3−(CH2)m−CO−A (4)
(式中、mは3〜15の整数を表し、Aはハロゲンを表す。)。
【0011】
(7)ハロゲン化n−アルカノイルが、下記一般式(5)で表されるハロゲン化n−アルカノイルである前記(5)記載のn−アルカノイル化ヒアルロン酸もしくはその塩の製造法。
CH3−(CH2)m−CO−A (5)
(式中、mは4〜10の整数であり、Aはハロゲンを表す。)。
【0012】
(8)非水系溶媒がDMF,エタノール、メタノール、アセトン、クロロホルム、トルエン、塩化メチレンまたはヘプタンから選ばれた1種以上の溶媒である前記(5)記載のn−アルカノイル化ヒアルロン酸もしくはその塩の製造方法。
【0013】
(9)カチオン性化合物が第4級アンモニウム塩である前記(5)記載のn−アルカノイル化ヒアルロン酸もしくはその塩の製造法。
【0014】
(10)第4級アンモニウム塩がジステアリルジメチルアンモニウム塩である前記(9)記載のn−アルカノイル化ヒアルロン酸もしくはその塩の製造法。
【0015】
【発明の実施の形態】
本発明に使用するヒアルロン酸(塩)は特に限定されるものではなく、鶏の鶏冠など各種動物組織由来であっても、ヒアルロン酸(塩)の生産能を有する微生物由来であってもよいが、本発明に使用するヒアルロン酸(塩)は微生物由来であることが好ましい。ヒアルロン酸生産能を有する微生物としては、例えばストレプトコッカス・ピオゲネス(Streptococcus pyogenes)、ストレプトコッカス・エクイシミリス(Streptococcus equisimilis)、ストレプトコッカス・エクイ(Streptococcus equi)、ストレプトコッカス・デイスガラクテイエ(Streptococcus dysgalactiae)、ストレプトコッカス・ズーエピデミカス(Streptococcus zooepidemicus)などを挙げることができる。
【0016】
微生物による生産は、例えば、1リットル当たりブドウ糖20〜30g、酵母エキス5g、リン酸1カリウム3g、リン酸2カリウム2g、チオ硫酸ナトリウム0.1g、硫酸マグネシュウム7水塩0.1g、亜硫酸ナトリウム0.02g、塩化コバルト0.01g、塩化マンガン0,01g、泡消剤5gを含む培地に予め準備しておいた種菌を添加し、pH6.0〜8.5、好ましくは7.0、培養温度25℃〜40℃、好ましくは30℃〜35℃で1〜3日間振とう培養もしくは通気培養し、ヒアルロン酸(塩)を該培養液中に生成蓄積させ、該培養液を遠心分離もしくはろ過して菌体を除去した後、ろ過液を限外ろ過もしくは透析することによって低分子量物質を除去し、低分子量物質を除去したろ過液に塩化ナトリウムを溶解後、水溶性有機溶剤を添加してヒアルロン酸ナトリウムを沈殿させ、該沈殿を遠心分離回収後、真空乾燥する方法を挙げることができる。また、和光純薬製、ナカライテスク製、東京化成製、生化学工業製、シグマ製など市販の試薬グレードのヒアルロン酸(塩)を使用しても特に差し支えない。
【0017】
また、本発明において原料として使用するヒアルロン酸(塩)はHPLC法により測定される平均分子量が、少なくとも5000から150万のものが好ましい。該HPLCによる平均分子量の測定には、多糖類の分子量測定に適する任意のカラムを用いることができるが、例えばShodex Ionpak KS806およびIonpak KS−G等のカラムを用いることが好ましい。この場合、溶出液としては0.2mol/L の塩化ナトリウム水溶液を用い、流速1.0ml/分で流し、ヒアルロン酸(塩)の検出は206nmで行うことができる。該平均分子量は極限粘度で求めた分子量既知のヒアルロン酸ナトリウムで作製した検量線を用いて計算により求めることができる。
【0018】
陽イオンとしては、アルカリ金属イオン、カリウムイオン,ナトリウムイオンなどを挙げることができ、本発明においてはナトリウムイオンが好ましい。
【0019】
本発明において用いるカチオン性化合物は、具体的には第4級アンモニウム塩、アミノ基を2個以上有するアミノ酸、ペプチド、ポリアミノ酸の塩、およびアミノ基を有する糖質の塩などを挙げることができ、好ましくは第4級アンモニウム塩である。
【0020】
第4級アンモニウム塩としては、アルキル基のうちの少なくとも1個が炭素数8個以上からなるものが好ましい。具体的には、ジステアリルジメチルアンモニウムクロライド(Distearyldimethylammonium Chloride)、ジオレイルジメチルアンモニウムクロライド(Dioleyldimethylammonium Chloride)、セチルピリジニウムクロライド(Cetylpyridinium Chloride)、セチルトリメチルアンモニウムクロライド(Cetyltrimethylammonium Chloride)、セチルトリメチルアンモニウムブロミド(Cetyltrimethylammonium Bromide)、ジテトラデシルジメチルアンモニウムブロマイド(Ditetradecyldimethylammonium Bromide)、ジドデシルジメチルアンモニウムブロミド(Didodecyldimethylammonium Bromide)、ジデシルジメチルアンモニウム ブロミド(Didecyldimetylammonium Bromide)、オクタデシルトリメチルアンモニウムクロライド(Octadecyltrimethylammonium Chloride)、ノルマルオクタデシルトリメチルアンモニウムブロミド(n−Octadecyltrimethylammonium Bromide)、トリドデシルメチルアンモニウムクロライド(Tridodecylmethylammonium Chloride)、トリオクチルメチルアンモニウムブロミド(Trioctylmethylammonium Bromide)、ジオクタノイルL−アルファーフォスファチジルコリン(Dioctanoyl L−α−Phosphatidylcholine)、ジラウロイル、L−アルファーフォスファチジルコリン(Dilauroyl L−α−Phosphatidylcholine)、ジパルミトイル D,L−アルファーフォスファチジルコリン(Dipalmitoyl D,L−α−Phosphatidylcholine)、1,2−ジミリストイル−3−トリメチルアンモニウムプロパン(1,2−Dimyristoyl−3−Trimethylammonium Propane)、1,2―ジオレオイル−3−トリメチルアンモニウムプロパン(1,2−Dioleoyl−3−Trimethylammonium Propane)、1,2−ジパルミトイル−3−トリメチルアンモニウムプロパン(1,2−Dipalmitoyl−3−Trimethylammonium Propane)、1,2−ジステアロイル−3−トリメチルアンモニウムプロパン(1,2−Distearoyl−3−Trimethylammonium Propane)、ベンザルコニウムクロライド(Benzalkonium Chloride)、ベンゼトニウムクロライド(Benzethonium Chloride)などを挙げることができる。
【0021】
ヒアルロン酸(塩)とカチオン性化合物との複合体とはヒアルロン酸のアニオン性部位(カルボキシル基)とカチオン性化合物とがイオン的に結合したものを言い、その調製法の一例を以下に示す。
【0022】
<ヒアルロン酸(塩)とカチオン性化合物との複合体の調製法>
(A) ヒアルロン酸(塩)を蒸留水またはこれに相当する純水に0.01〜10重量%の濃度、好ましくは0.05〜1重量%の濃度になるように溶解する。なお、本発明において、蒸留水に相当する純水とは、例えば、連続イオン交換(Electric Deionization)および逆浸透(Reverse Osmosis)等により精製した水を意味する。
(B) 一方、ヒアルロン酸(塩)と複合体を形成させるカチオン性化合物、好ましくは第4級アンモニウム塩を蒸留水または純水に添加し、均一に分散させる。
【0023】
(B)で調製したカチオン性化合物の水溶液中のカチオン基と、(A)で調製したヒアルロン酸(塩)の水溶液中のカルボキシル基とのモル比が、0.5〜5:1、好ましくは0.7〜1.5:1、例えば1:1になるように2つの水溶液を混合する。
【0024】
なお、混合する際の温度は室温でも良いが、好ましくは使用するカチオン性化合物のゲル−液晶転移温度以上に両液を加温して行う。
【0025】
混合により発生した水不溶物は通常化学実験で使用する分離法、例えば遠心分離、吸引ろ過、加圧ろ過等の方法で混合液より回収する。回収した水不溶物は、当該カチオン性化合物のゲル−液晶転移温度以上に加温した蒸留水または純水にて洗浄した後、乾燥に供する。乾燥は、通常化学実験で使用する乾燥手段、例えば常圧乾燥、真空乾燥、凍結乾燥等により行うことができる。
【0026】
また、本発明において使用しうる非水系溶媒は、例えば、DMF,エタノール、メタノール、アセトン、クロロホルム、塩化メチレン、トルエン、ヘプタン、プロピレングリコール、ポリエチレングリコール、DMSO、THF等、またはこれらの2種以上の混合溶媒が包含される。DMF,エタノール、メタノール、アセトン、クロロホルム、塩化メチレン、トルエンもしくはヘプタン、またはこれらの混合溶媒が好ましい。
【0027】
非水系溶媒に溶解させるヒアルロン酸(塩)とカチオン性化合物との複合体の濃度は、特に限定するものではないが、1〜1000mmol/Lの範囲であることが好ましい。
【0028】
該複合体の非水系溶媒への溶解後に行うO−アシル化反応(n−アルカノイル化反応)としては、ピリジンなど塩基性溶剤の存在下、ハロゲン化n−アルカノイルを用いるO−アシル化法(Schotten−Baumann法)などを挙げることができる。ここで、ハロゲン化n−アルカノイルとしては具体的には、n−ブチリルクロライド、n−ヘキサノイルクロライド、n−オクタノイルクロライド、n−デカノイルクロライド、n−ドデカノイルクロライド、n−ステアロイルクロライド等を挙げる事ができ、C4〜C18、好ましくはC5〜C17、より好ましくはC6〜C12のハロゲン化n−アルカノイルである。
【0029】
例えば、O−アシル化反応がn−ドデカノイル化、即ちn−ドデカノイル化ヒアルロン酸(塩)の製造の場合は、上述のように得られたヒアルロン酸(塩)のカチオン性化合物複合体を窒素雰囲気下、DMFに溶解し、ピリジン、n−ドデカノイルクロライドを加え攪拌し、反応液に酢酸ナトリウム飽和エタノールを加え、析出した沈殿をアセトン/水(9/1)で洗浄後、真空乾燥し、n−ドデカノイル化ヒアルロン酸(塩)を得ることができる。
【0030】
本発明のn−アルカノイル化ヒアルロン酸(塩)の用途は、特に限定されるものではないが、医薬品、食品、化粧品分野などに用いられる各種材料として使用可能である。特に本発明のn−アルカノイル化ヒアルロン酸(塩)は、非水系での製造であるため発熱物質、抗原性物質などの混入が少なく、さらに体内で代謝されるため特に医薬分野で、例えば手術時等の保湿剤、潤滑剤、創傷被覆剤、さらにはDDS(ドラッグデリバリーシステム)材料として用いることが可能である。中でもその子宮内における分解速度が、人体(子宮)内のバイオリズムと高い相関性を示すことが期待されていることから、例えば子宮内膜症治療薬を担持する子宮内もしくは膣内埋植用製剤のデバイスとして極めて有効である。
【0031】
【実施例】
以下、実施例をもって本発明を詳細に説明する。
各種のn−アルカノイル化ヒアルロン酸(塩)の製造例を以下に示す。
実施例1
1.n−ブチリル化ヒアルロン酸(塩)の製造
1−1. ヒアルロン酸とジステアリルジメチルアンモニウムクロライド錯体の作製
ヒアルロン酸ナトリウム(チッソ CHA、極限粘度より求めた平均分子量5万、以下、「CHA」と記載する)1.8gを純水300mlに溶解するとともに、ジステアリルジメチルアンモニウムクロライド(以下、DSCと略する)2.47gを560mlの純水にケン濁させた。両液を45℃に加温後攪拌しながら混合し、5分間攪拌を続けた。発生した錯体を遠心分離(5000rpm、室温)にて回収し、45℃の温水にて洗浄した。洗浄終了後、一夜凍結乾燥し、その後、50℃で一夜真空乾燥し、ヒアルロン酸とジステアリルジメチルアンモニウムクロライド錯体(以下CHA−DSCと略する)を得た。収量3.3g、収率85%。
【0032】
1−2.n−ブチリル化ヒアルロン酸の合成
得られたCHA−DSC464mgを窒素雰囲気下、DMF30mlに攪拌溶解させ、目的のDSに必要な量のn−ブチリルクロライド、ピリジンを加え60℃、2時間攪拌した。反応終了後、酢酸ナトリウム飽和エタノール溶液(60 ml)を加え、ゲルを析出させると共にDSCを除去した。これをアセトン/水=9:1の溶液で5回洗浄後、60℃で真空乾燥し、白色固体のn−ブチリル化ヒアルロン酸を得た。
【0033】
実施例2
n−ヘキサノイル化ヒアルロン酸の合成
実施例1の1−1.に準拠して得たCHA−DSC464mgを窒素雰囲気下、DMF30mlに攪拌溶解させ、目的のDSに必要な量のn−ヘキサノイルクロライド、ピリジンを加え60℃、2時間攪拌した。反応終了後、酢酸ナトリウム飽和エタノール溶液(60 ml)を加え、ゲルを析出させると共にDSCを除去した。これをアセトン/水=9:1の溶液で5回洗浄後、60℃真空乾燥し白色固体のn−ヘキサノイル化ヒアルロン酸を得た。
【0034】
実施例3
n−オクタノイル化ヒアルロン酸の合成
実施例1の1−1.に準拠して得たCHA−DSC464mgを窒素雰囲気下、DMF30mlに攪拌溶解させ、n−オクタノイルクロライド16.3mg、ピリジン30mlを加え60℃、2時間攪拌した。反応終了後、酢酸ナトリウム飽和エタノール溶液(60 ml)を加え、ゲルを析出させると共にDSCを除去した。これをアセトン/水=9:1の溶液で5回洗浄後、60℃真空乾燥し白色固体のn−オクタノイル化ヒアルロン酸を得た。
【0035】
実施例4
n−デカノイル化ヒアルロン酸の合成
実施例1の1−1.に準拠して得たCHA−DSC464mgを窒素雰囲気下、DMF30mlに攪拌溶解させ、n−デカノイルクロライド19.1mg、ピリジン30mlを加え60℃、2時間攪拌した。反応終了後、酢酸ナトリウム飽和エタノール溶液(60 ml)を加え、ゲルを析出させると共にDSCを除去した。これをアセトン/水=9:1の溶液で5回洗浄後、60℃真空乾燥し白色固体のn−デカノイル化ヒアルロン酸を得た。
【0036】
実施例5
n−ドデカノイル化ヒアルロン酸の合成
実施例1の1−1.に準拠して得たCHA−DSC464mgを窒素雰囲気下、DMF30mlに攪拌溶解させ、n−ドデカノイルクロライド21.9mg、ピリジン30mlを加え60℃、2時間攪拌した。反応終了後、酢酸ナトリウム飽和エタノール溶液(60 ml)を加え、ゲルを析出させると共にDSCを除去した。これをアセトン/水=9:1の溶液で5回洗浄後、60℃真空乾燥し白色固体のn−ドデカノイル化ヒアルロン酸を得た。
【0037】
実施例6
n−ステアロイル化ヒアルロン酸の合成
実施例1の1−1.に準拠して得たCHA−DSC464mgを窒素雰囲気下、60 ℃でDMF 30 mlに溶解し、ピリジン,目的のDSに必要な量のステアロイルクロライドを加え、2 hr攪拌した。反応溶液に酢酸ナトリウム飽和エタノール60 ml加え、析出した沈殿をアセトン / 水 (9 / 1 ) で5回洗浄した。これを真空乾燥して白色固体のn−ステアロイル化ヒアルロン酸を得た。
なお、ここでいうDSとは各反応条件のHA−DSCと酸ハロゲン化物のモル比で実施した際の理論上のヒアルロン酸二糖単位当たり存在する4個の水酸基へのアルカノイル基の導入割合を表わす。
【0038】
実施例7
実施例1〜6で得られた各n−アルカノル化ヒアルロン酸の溶解性及びゲル化の評価
各n−アルカノル化ヒアルロン酸5mgを各溶剤1mlに添加、試験管ミキサーで均一にした後、室温で一日放置し、その状態を観察し、その結果を表1〜3に示した。
各表中で溶液特性の表現は以下の状態を意味する
可溶:溶媒に完全に溶解し、ゲル等の形成しない状態
不溶:白色固体のまま溶媒に溶解しない状態
S−G(ゾル−ゲル):溶媒中に細かいゲルを形成した状態
不均一G(不均一ゲル):ゲルではあるが均一性を欠くゲル
G(ゲル):均一のゲル
【0039】
表 1 各種n−アルカノイル化ヒアルロン酸の溶解特性
【0040】
表 2 DSが異なるn−ブチリル化およびヘキサノイル化ヒアルロン酸の溶解特性
【0041】
表 3 DSの異なるn−ステアロイル化ヒアルロン酸の溶解特性
【0042】
【発明の効果】
本発明のn−アルカノイル化ヒアルロン酸もしくはその塩は、、疎水域と親水域を併せ持つ新しいタイプのヒアルロン酸(塩)であり、医療品、食品、化粧品分野などの新しい素材として極めて有用である。特に本発明のn−アルカノイル化ヒアルロン酸(塩)は、非水系での製造であるため発熱物質、抗原性物質などの混入が少なく、さらに体内で代謝されるため特に医薬分野で、例えば手術時等の保湿剤、潤滑剤、創傷被覆剤、さらにはDDS(ドラッグデリバリーシステム)材料として利用できる。また、本発明のn−アルカノイル化ヒアルロン酸もしくはその塩の製造法は、ヒアルロン酸(塩)とカチオン性化合物との複合体を非水系溶媒に溶解し、次いで該複合体をn−アルカノイル基を有する酸ハロゲン化物と反応させることにより、疎水部位と親水部位を併せ持ち、医療品、食品、化粧品分野などの新しい素材として有用なヒアルロン酸(塩)を容易に製造することができる。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to n-alkanoylated hyaluronic acid or a salt thereof and a method for producing the same. More specifically, the present invention relates to an n-alkanoylated hyaluronic acid or a salt thereof having both a hydrophobic portion and a hydrophilic portion and useful as a new material in the fields of medical products, foods and cosmetics, and a method for producing the same.
[Prior art]
Hyaluronic acid is a linear high molecular polysaccharide formed by alternately bonding N-acetyl-D-glucosamine and D-glucuronic acid, and has many free carboxyl groups and many free hydroxyl groups, and thus has an affinity for water. It is highly soluble and dissolves in any amount of water to form a highly viscous aqueous solution. It is expected that hyaluronic acid having different physical properties will be obtained by blocking the free carboxyl group and free hydroxyl group. Therefore, chemical modification of hyaluronic acid or a salt thereof (hereinafter, sometimes referred to as "hyaluronic acid (salt)") has been performed for the purpose of use in medical products, foods, cosmetics, and the like (for example, , Patent Documents 1 and 2). However, any of the conventional reactions is a reaction in a system in which water molecules are present, and an organic reaction applicable to chemical modification of hyaluronic acid (salt) is limited. For this reason, it has been very difficult to produce a new type of hyaluronic acid (salt) having both a hydrophobic part and a hydrophilic part, which is a new material for medical products, foods, cosmetics and the like.
[0003]
[Patent Document 1]
JP-A-7-309902 [Patent Document 2]
JP-A-8-53501
[Problems to be solved by the invention]
The present inventors have intensively studied a new type of hyaluronic acid (salt) having both a hydrophobic part and a hydrophilic part, which is useful as a new material in the fields of medical products, foods and cosmetics. As a result, the complex of the hyaluronic acid (salt) and the cationic compound is dissolved in a non-aqueous solvent, and then the complex is reacted with an acid halide having an n-alkanoyl group, whereby the hydrophobic region and the hydrophilic region are reduced. The inventors have found that a new type of hyaluronic acid (salt) can be obtained, and based on this finding, have completed the present invention.
As is apparent from the above description, an object of the present invention is to provide a new type of hyaluronic acid (salt) having both a hydrophobic portion and a hydrophilic portion, particularly a non-aqueous system, which is useful as a new material in the fields of medical products, foods and cosmetics. Since it is manufactured in a small amount, there is little contamination with pyrogenic substances, antigenic substances, etc., and it is metabolized in the body. In particular, in the field of medicine, for example, humectants, lubricants, wound dressings, and DDS (drugs) for surgery etc (Delivery system) It is to provide a hyaluronic acid (salt) usable as a material and a method for producing the same.
[0005]
[Means for Solving the Problems]
The present invention is shown by the following (1) to (10).
(1) An n-alkanoylated hyaluronic acid represented by the following general formula (1) or a salt thereof.
(Wherein, R 1 , R 2 , R 3 , and R 4 each represent hydrogen or a linear C 5 -C 15 n-alkanoyl group, and R 5 represents hydrogen, a metal cation, or a cationic compound. And n represents an integer of 2 to 7,500.)
[0006]
(2) The n-alkanoylated hyaluronic acid or a salt thereof according to the above (1), wherein the n-alkanoyl group is an n-alkanoyl group represented by the following general formula (2).
CH 3 - (CH 2) m -CO- (2)
(In the formula, m represents an integer of 4 to 10.)
[0007]
(3) The n-alkanoylated hyaluronic acid or a salt thereof according to the above (1), wherein the cationic compound is a quaternary ammonium salt.
[0008]
(4) The n-alkanoylated hyaluronic acid or a salt thereof according to the above (3), wherein the quaternary ammonium salt is distearyl dimethyl ammonium salt.
[0009]
(5) An n-alkanoylated hyaluronic acid represented by the following general formula (3), wherein a complex of hyaluronic acid or a salt thereof and a cationic compound is reacted with an n-alkanoyl halide in a non-aqueous solvent. A method for producing an acid or a salt thereof.
(Wherein R 1 , R 2 , R 3 , and R 4 each represent hydrogen or a C 4 -C 18 n-alkanoyl group, R 5 represents hydrogen, a metal cation, or a cationic compound; Represents an integer of 2 to 7,500.)
[0010]
(6) The method for producing an n-alkanoylated hyaluronic acid or a salt thereof according to the above (5), wherein the n-alkanoyl halide is an n-alkanoyl halide represented by the following general formula (4).
CH 3 - (CH 2) m -CO-A (4)
(In the formula, m represents an integer of 3 to 15, and A represents halogen.)
[0011]
(7) The method for producing an n-alkanoylated hyaluronic acid or a salt thereof according to the above (5), wherein the n-alkanoyl halide is an n-alkanoyl halide represented by the following general formula (5).
CH 3 - (CH 2) m -CO-A (5)
(In the formula, m is an integer of 4 to 10, and A represents halogen.)
[0012]
(8) The n-alkanoylated hyaluronic acid or a salt thereof according to the above (5), wherein the non-aqueous solvent is one or more solvents selected from DMF, ethanol, methanol, acetone, chloroform, toluene, methylene chloride and heptane. Production method.
[0013]
(9) The method for producing n-alkanoylated hyaluronic acid or a salt thereof according to the above (5), wherein the cationic compound is a quaternary ammonium salt.
[0014]
(10) The method for producing n-alkanoylated hyaluronic acid or a salt thereof according to (9), wherein the quaternary ammonium salt is distearyl dimethyl ammonium salt.
[0015]
BEST MODE FOR CARRYING OUT THE INVENTION
The hyaluronic acid (salt) used in the present invention is not particularly limited, and may be derived from various animal tissues such as chicken cockscomb or derived from a microorganism capable of producing hyaluronic acid (salt). The hyaluronic acid (salt) used in the present invention is preferably derived from a microorganism. The microorganism having a hyaluronic acid-producing ability, for example, Streptococcus pyogenes (Streptococcus pyogenes), Streptococcus equisimilis (Streptococcus equisimilis), Streptococcus equi (Streptococcus equi), Streptococcus Day scan galactosamine Teie (Streptococcus dysgalactiae), Streptococcus zooepidemicus (Streptococcus zooepidemicus).
[0016]
The production by microorganisms is, for example, 20-30 g of glucose per liter, 5 g of yeast extract, 3 g of 1 potassium phosphate, 2 g of 2 potassium phosphate, 0.1 g of sodium thiosulfate, 0.1 g of magnesium sulfate heptahydrate, 0 g of sodium sulfite. To a medium containing 0.02 g, 0.01 g of cobalt chloride, 0.01 g of manganese chloride, and 5 g of an antifoaming agent, a previously prepared inoculum was added, and pH 6.0 to 8.5, preferably 7.0, and culture temperature. Shaking culture or aeration culture at 25 ° C. to 40 ° C., preferably 30 ° C. to 35 ° C. for 1 to 3 days to form and accumulate hyaluronic acid (salt) in the culture solution, and centrifuge or filter the culture solution. After removing the cells by ultrafiltration or dialysis of the filtrate, low molecular weight substances are removed.Sodium chloride is dissolved in the filtrate from which the low molecular weight substances have been removed. Precipitating the sodium hyaluronate with the addition of sexual organic solvent, mention may be made after centrifugation recovered precipitate, a method of vacuum drying. Also, it is possible to use a commercially available reagent grade hyaluronic acid (salt) such as Wako Pure Chemical, Nacalai Tesque, Tokyo Kasei, Seikagaku, or Sigma.
[0017]
The hyaluronic acid (salt) used as a raw material in the present invention preferably has an average molecular weight of at least 5,000 to 1.5 million as measured by HPLC. For the measurement of the average molecular weight by the HPLC, any column suitable for measuring the molecular weight of the polysaccharide can be used. For example, a column such as Shodex Ionpak KS806 and Ionpak KS-G is preferably used. In this case, a 0.2 mol / L aqueous solution of sodium chloride is used as the eluate, the solution is flowed at a flow rate of 1.0 ml / min, and the detection of hyaluronic acid (salt) can be performed at 206 nm. The average molecular weight can be determined by calculation using a calibration curve prepared with sodium hyaluronate having a known molecular weight determined by the intrinsic viscosity.
[0018]
Examples of the cation include an alkali metal ion, a potassium ion, and a sodium ion. In the present invention, a sodium ion is preferable.
[0019]
Specific examples of the cationic compound used in the present invention include a quaternary ammonium salt, an amino acid having two or more amino groups, a peptide, a salt of a polyamino acid, and a saccharide salt having an amino group. And preferably a quaternary ammonium salt.
[0020]
As the quaternary ammonium salt, those in which at least one of the alkyl groups has 8 or more carbon atoms are preferable. Specifically, distearyl dimethyl ammonium chloride (Distearyldimethylammonium Chloride), dioleyl dimethyl ammonium chloride (Dioleyldimethylammonium Chloride), cetylpyridinium chloride (cetylpyridinium Chloride Useful), cetyltrimethylammonium chloride (cetyltrimethylammonium Chloride Useful), cetyltrimethylammonium bromide (cetyltrimethylammonium Bromide) , Ditetradecyldimethylammonium bromide, didodecyldimethylammonium bromide Umuburomido (Didodecyldimethylammonium Bromide), didecyldimethylammonium bromide (Didecyldimetylammonium Bromide), octadecyl trimethyl ammonium chloride (Octadecyltrimethylammonium Chloride), n-octadecyl bromide (n-Octadecyltrimethylammonium Bromide), tridodecylmethylammonium chloride (Tridodecylmethylammonium Chloride), trioctylmethylammonium Ammonium bromide (Trioctylmethylammonium Bromide), dioctanoyl L-al Diphosphanodyl choline (Dioctanoyl L-α-phosphatidylcholine), dilauroyl, L-alpha-phosphatidylcholine (Dilauroyl L-α-Phosphatidylcholine), dipalmitoyl D, L-alpha-dylcholine D, L-alpha-dicholine-D-L-alpha-dicholine-D-L-alpha-dicholine. α-phosphatidylcholine), 1,2-dimyristoyl-3-trimethylammonium propane, 1,2-dioleoyl-3-trimethylammonium propane (1,2-Dioleoyl-3-) Trimethylammonium Propane), 1,2-dipalmitoyl- -Trimethylammonium propane (1,2-Dipalmitoyl-3-Trimethylammonium Propane), 1,2-distearoyl-3-trimethylammonium propane (1,2-Distearoyl-3-Trimethylammonium Propane), Benzalkonium Chloride (Benzalkonium Chloride) , Benzethonium chloride and the like.
[0021]
The complex of hyaluronic acid (salt) and a cationic compound refers to a complex in which an anionic site (carboxyl group) of hyaluronic acid and a cationic compound are ionically bonded, and an example of a preparation method thereof is shown below.
[0022]
<Method for Preparing Complex of Hyaluronic Acid (Salt) and Cationic Compound>
(A) Hyaluronic acid (salt) is dissolved in distilled water or its equivalent pure water to a concentration of 0.01 to 10% by weight, preferably 0.05 to 1% by weight. In addition, in this invention, the pure water equivalent to distilled water means the water refine | purified by continuous ion exchange (Electric Deionization), reverse osmosis (Reverse Osmosis), etc., for example.
(B) On the other hand, a cationic compound capable of forming a complex with hyaluronic acid (salt), preferably a quaternary ammonium salt, is added to distilled water or pure water and uniformly dispersed.
[0023]
The molar ratio of the cationic group in the aqueous solution of the cationic compound prepared in (B) to the carboxyl group in the aqueous solution of hyaluronic acid (salt) prepared in (A) is 0.5 to 5: 1, preferably The two aqueous solutions are mixed so as to be 0.7-1.5: 1, for example 1: 1.
[0024]
In addition, the temperature at the time of mixing may be room temperature, but it is preferable to heat both solutions to a temperature higher than the gel-liquid crystal transition temperature of the cationic compound to be used.
[0025]
The water-insoluble matter generated by the mixing is recovered from the mixture by a separation method usually used in a chemical experiment, for example, a method such as centrifugation, suction filtration, or pressure filtration. The recovered water-insoluble matter is washed with distilled water or pure water heated to a temperature higher than the gel-liquid crystal transition temperature of the cationic compound, and then dried. Drying can be performed by a drying means usually used in a chemical experiment, for example, normal pressure drying, vacuum drying, freeze drying and the like.
[0026]
Non-aqueous solvents that can be used in the present invention include, for example, DMF, ethanol, methanol, acetone, chloroform, methylene chloride, toluene, heptane, propylene glycol, polyethylene glycol, DMSO, THF, and the like, or two or more of these. Mixed solvents are included. DMF, ethanol, methanol, acetone, chloroform, methylene chloride, toluene or heptane, or a mixed solvent thereof is preferred.
[0027]
The concentration of the complex of the hyaluronic acid (salt) and the cationic compound dissolved in the non-aqueous solvent is not particularly limited, but is preferably in the range of 1 to 1000 mmol / L.
[0028]
The O-acylation reaction (n-alkanoylation reaction) performed after dissolving the complex in a non-aqueous solvent includes an O-acylation method (Schotten) using an n-alkanoyl halide in the presence of a basic solvent such as pyridine. -Baumann method) and the like. Here, specific examples of the n-alkanoyl halide include n-butyryl chloride, n-hexanoyl chloride, n-octanoyl chloride, n-decanoyl chloride, n-dodecanoyl chloride, n-stearoyl chloride, and the like. And C 4 -C 18 , preferably C 5 -C 17 , more preferably C 6 -C 12 halogenated n-alkanoyl.
[0029]
For example, when the O-acylation reaction is n-dodecanoylation, that is, in the case of producing n-dodecanoylated hyaluronic acid (salt), the cationic compound complex of hyaluronic acid (salt) obtained as described above is mixed with a nitrogen atmosphere. Under a DMF solution, pyridine and n-dodecanoyl chloride were added and the mixture was stirred. Sodium acetate-saturated ethanol was added to the reaction mixture, and the precipitate was washed with acetone / water (9/1) and dried in vacuo. -Dodecanoylated hyaluronic acid (salt) can be obtained.
[0030]
The use of the n-alkanoylated hyaluronic acid (salt) of the present invention is not particularly limited, but it can be used as various materials used in the fields of pharmaceuticals, foods, cosmetics, and the like. In particular, the n-alkanoylated hyaluronic acid (salt) of the present invention is produced in a non-aqueous system, so that it is less contaminated with pyrogens and antigenic substances, and is further metabolized in the body. It can be used as a humectant, a lubricant, a wound dressing, and a DDS (drug delivery system) material. Above all, since the rate of degradation in the uterus is expected to show a high correlation with the biorhythm in the human body (uterus), for example, the preparation of an intrauterine or vaginal implant preparation carrying a therapeutic agent for endometriosis is recommended. It is extremely effective as a device.
[0031]
【Example】
Hereinafter, the present invention will be described in detail with reference to examples.
Production examples of various n-alkanoylated hyaluronic acids (salts) are shown below.
Example 1
1. Production of n-butyrylated hyaluronic acid (salt) 1-1. Preparation of Hyaluronic Acid and Distearyldimethylammonium Chloride Complex 1.8 g of sodium hyaluronate (nitrogen CHA, average molecular weight determined from intrinsic viscosity: 50,000; hereinafter, referred to as “CHA”) was dissolved in 300 ml of pure water. 2.47 g of stearyl dimethyl ammonium chloride (hereinafter abbreviated as DSC) was suspended in 560 ml of pure water. The two liquids were heated to 45 ° C. and mixed while stirring, and the stirring was continued for 5 minutes. The generated complex was recovered by centrifugation (5000 rpm, room temperature) and washed with hot water at 45 ° C. After the completion of the washing, the mixture was freeze-dried overnight, and then vacuum-dried at 50 ° C. overnight to obtain hyaluronic acid and distearyldimethylammonium chloride complex (hereinafter abbreviated as CHA-DSC). 3.3 g, 85% yield.
[0032]
1-2. Synthesis of n-butyryl hyaluronic acid 464 mg of the obtained CHA-DSC was dissolved in 30 ml of DMF with stirring under a nitrogen atmosphere, and necessary amounts of n-butyryl chloride and pyridine required for DS were added thereto, followed by stirring at 60 ° C for 2 hours. After the completion of the reaction, a saturated ethanol solution of sodium acetate (60 ml) was added to precipitate a gel and to remove DSC. This was washed five times with a solution of acetone / water = 9: 1, and then dried in vacuo at 60 ° C. to obtain a white solid n-butyrylated hyaluronic acid.
[0033]
Example 2
Synthesis of n-hexanoylated hyaluronic acid 1-1 in Example 1 Under a nitrogen atmosphere, 464 mg of CHA-DSC was dissolved in 30 ml of DMF with stirring, and necessary amounts of n-hexanoyl chloride and pyridine for the target DS were added, followed by stirring at 60 ° C. for 2 hours. After completion of the reaction, a saturated ethanol solution of sodium acetate (60 ml) was added to precipitate a gel and to remove DSC. This was washed five times with a solution of acetone / water = 9: 1, and dried in vacuo at 60 ° C. to obtain n-hexanoylated hyaluronic acid as a white solid.
[0034]
Example 3
Synthesis of n-octanoylated hyaluronic acid 1-1 of Example 1 Under a nitrogen atmosphere, 464 mg of CHA-DSC obtained in accordance with the above was dissolved under stirring in 30 ml of DMF, 16.3 mg of n-octanoyl chloride and 30 ml of pyridine were added, and the mixture was stirred at 60 ° C. for 2 hours. After completion of the reaction, a saturated ethanol solution of sodium acetate (60 ml) was added to precipitate a gel and to remove DSC. This was washed five times with a solution of acetone / water = 9: 1, and dried in vacuo at 60 ° C. to obtain n-octanoylated hyaluronic acid as a white solid.
[0035]
Example 4
Synthesis of n-decanoylated hyaluronic acid 1-1 in Example 1 Under a nitrogen atmosphere, 464 mg of CHA-DSC obtained in accordance with the above was dissolved in 30 ml of DMF with stirring, 19.1 mg of n-decanoyl chloride and 30 ml of pyridine were added, and the mixture was stirred at 60 ° C. for 2 hours. After completion of the reaction, a saturated ethanol solution of sodium acetate (60 ml) was added to precipitate a gel and to remove DSC. This was washed 5 times with a solution of acetone / water = 9: 1, and dried in vacuo at 60 ° C. to obtain n-decanoylated hyaluronic acid as a white solid.
[0036]
Example 5
Synthesis of n-dodecanoylated hyaluronic acid 1-1 in Example 1 Under a nitrogen atmosphere, 464 mg of CHA-DSC was dissolved in 30 ml of DMF with stirring, 21.9 mg of n-dodecanoyl chloride and 30 ml of pyridine were added, and the mixture was stirred at 60 ° C. for 2 hours. After the completion of the reaction, a saturated ethanol solution of sodium acetate (60 ml) was added to precipitate a gel and to remove DSC. This was washed five times with a solution of acetone / water = 9: 1, and dried in vacuo at 60 ° C. to obtain n-dodecanoylated hyaluronic acid as a white solid.
[0037]
Example 6
Synthesis of n-stearoylated hyaluronic acid 1-1 of Example 1 464 mg of CHA-DSC obtained in accordance with the above was dissolved in 30 ml of DMF at 60 ° C. under a nitrogen atmosphere, and pyridine and an amount of stearoyl chloride required for a target DS were added thereto, followed by stirring for 2 hours. To the reaction solution, 60 ml of ethanol saturated with sodium acetate was added, and the deposited precipitate was washed five times with acetone / water (9/1). This was vacuum-dried to obtain a white solid n-stearoylated hyaluronic acid.
Here, DS is the ratio of introduction of alkanoyl groups to four hydroxyl groups present per theoretical hyaluronic acid disaccharide unit when the reaction is carried out at a molar ratio of HA-DSC and acid halide under each reaction condition. Express.
[0038]
Example 7
Evaluation of solubility and gelation of each n-alkanolated hyaluronic acid obtained in Examples 1 to 6 5 mg of each n-alkanolated hyaluronic acid was added to 1 ml of each solvent, and after homogenizing with a test tube mixer, at room temperature After one day, the state was observed, and the results are shown in Tables 1 to 3.
In each table, the expression of the solution property means the following states: Soluble: completely dissolved in a solvent and no gel or the like formed Insoluble: white solid not dissolved in a solvent SG (sol-gel) : Fine gel formed in solvent Non-uniform G (heterogeneous gel): Gel that is a gel but lacks uniformity G (gel): Uniform gel
Table 1 Dissolution characteristics of various n-alkanoylated hyaluronic acids
[0040]
Table 2 Dissolution properties of n-butyrylated and hexanoylated hyaluronic acid with different DS
[0041]
Table 3 Dissolution characteristics of n-stearoylated hyaluronic acid with different DS
[0042]
【The invention's effect】
The n-alkanoylated hyaluronic acid of the present invention or a salt thereof is a new type of hyaluronic acid (salt) having both a hydrophobic region and a hydrophilic region, and is extremely useful as a new material in the fields of medical products, foods, cosmetics and the like. In particular, the n-alkanoylated hyaluronic acid (salt) of the present invention is produced in a non-aqueous system, so that it is less contaminated with pyrogens and antigenic substances, and is further metabolized in the body. And the like, and can be used as a humectant, a lubricant, a wound dressing, and a DDS (drug delivery system) material. Further, the method for producing an n-alkanoylated hyaluronic acid or a salt thereof of the present invention comprises dissolving a complex of hyaluronic acid (salt) and a cationic compound in a non-aqueous solvent, and then converting the complex to an n-alkanoyl group. By reacting with an acid halide having the compound, a hyaluronic acid (salt) having both a hydrophobic portion and a hydrophilic portion and useful as a new material in the fields of medical products, foods, and cosmetics can be easily produced.
Claims (10)
(式中、R1、R2、R3、およびR4はそれぞれ水素または直鎖状のC5〜C15のアルカノイル基を表し、R5は水素、金属陽イオン、またはカチオン性化合物を表し、nは2〜7,500の整数を表す。)。An n-alkanoylated hyaluronic acid represented by the following general formula (1) or a salt thereof.
(Wherein, R 1 , R 2 , R 3 , and R 4 each represent hydrogen or a linear C 5 -C 15 alkanoyl group, and R 5 represents hydrogen, a metal cation, or a cationic compound. , N represents an integer of 2 to 7,500.)
CH3−(CH2)m−CO− (2)
(式中、mは4〜10の整数を表す。)。The n-alkanoylated hyaluronic acid or a salt thereof according to claim 1, wherein the n-alkanoyl group is an n-alkanoyl group represented by the following general formula (2).
CH 3 - (CH 2) m -CO- (2)
(In the formula, m represents an integer of 4 to 10.)
(式中、R1、R2、R3、およびR4はそれぞれ水素またはC4〜C18のn−アルカノイル基を表し、R5は水素、金属陽イオン、またはカチオン性化合物を表し、nは2〜7,500の整数を表す。)。Reacting a complex of a hyaluronic acid or a salt thereof and a cationic compound with an n-alkanoyl halide in a non-aqueous solvent, wherein the n-alkanoylated hyaluronic acid represented by the following general formula (3) or Method for producing salt.
(Wherein R 1 , R 2 , R 3 , and R 4 each represent hydrogen or a C 4 -C 18 n-alkanoyl group, R 5 represents hydrogen, a metal cation, or a cationic compound; Represents an integer of 2 to 7,500.)
CH3−(CH2)m−CO−A (4)
(式中、mは3〜15の整数であり、Aはハロゲンを表す。)。The method for producing n-alkanoylated hyaluronic acid or a salt thereof according to claim 5, wherein the n-alkanoyl halide is an n-alkanoyl halide represented by the following general formula (4).
CH 3 - (CH 2) m -CO-A (4)
(In the formula, m is an integer of 3 to 15, and A represents halogen.)
CH3−(CH2)m−CO−A (5)
(式中、mは4〜10の整数であり、Aはハロゲンを表す。)。The method for producing an n-alkanoylated hyaluronic acid or a salt thereof according to claim 5, wherein the n-alkanoyl halide is an n-alkanoyl halide represented by the following general formula (5).
CH 3 - (CH 2) m -CO-A (5)
(In the formula, m is an integer of 4 to 10, and A represents halogen.)
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