JP4454051B2 - Novel glycolipid, production method and use thereof - Google Patents

Description

により表される糖脂質。
【００２１】
（２）紅藻スギノリ属ギガルティナ・テネラから上記式（Ｉ）により表される糖脂質を製造する方法において、前記紅藻スギノリ属ギガルティナ・テネラをアセトン、酢酸エチル、クロロホルム、メタノール又はクロロホルム−メタノール混液からなる群から選ばれる有機溶媒又は有機溶媒混液の１つにより抽出する工程と、前記有機溶媒抽出物を減圧濃縮する工程と、前記減圧濃縮した抽出物を酸性条件下に酢酸エチル−水による層分配に供し、水層を分離する工程と、前記分離した水層をアルカリ性条件下に酢酸エチルとの層分配に供し、酢酸エチル層を分離する工程と、前記分離した酢酸エチル層を減圧濃縮し、得られた緑色の粗粉末を回収する工程と、前記粗粉末を精製する工程と、前記精製工程で得られた溶液を減圧濃縮し、得られた白色粉末を回収する工程とを具備することを特徴とする方法。
【００２２】
（３）上記式（Ｉ）で表される糖脂質を有効成分とする医薬。
（４）上記式（Ｉ）で表される糖脂質を有効成分とするＤＮＡ合成酵素β阻害剤。
【００２３】
（５）上記式（Ｉ）で表される糖脂質を有効成分とするＨＩＶ由来逆転写酵素阻害剤。
（６）上記式（Ｉ）で表される糖脂質を有効成分とする免疫抑制剤。
【００２４】
【発明の実施の形態】
以下、本発明の式（Ｉ）で表される糖脂質（以下ＫＭ０４３ともいう）について詳細に説明する。
本発明のＫＭ０４３の製造方法の一例を挙げる。
【００２５】
すなわち、海藻類から有機溶媒により抽出される物質を、酸性条件下に有機溶媒と水との液層分配に供する。得られた水層をアルカリ性条件下に有機溶媒との液層分配に供する。得られた有機層をさらに精製することにより本発明のＫＭ０４３を製造することができる。
【００２６】
海藻としては、例えば紅藻スギノリ属ギガルティナ・テネラ(Gigartina tenella) を用いることができる。
抽出溶媒としては、例えばアセトン、酢酸エチル、クロロホルム、メタノール又はクロロホルム−メタノール混液（２：１（体積比））を用いることができる。抽出溶媒としては、アセトンがＫＭ０４３の収量の観点から好ましい。
【００２７】
抽出溶媒は、ＫＭ０４３の抽出効率の観点からできるだけ多く用いることが好ましい。抽出溶媒は、通常、海藻乾燥重量の５倍以上を用い、海藻乾燥重量の３０倍の抽出溶媒を用いるとより抽出効率が上がる。しかしながら、抽出溶媒の量は、抽出効率とコスト等の観点から適宜設定することができる。
【００２８】
本発明のＫＭ０４３の製造方法をより詳細に説明する。ギガルティナ・テネラ藻体の乾燥物を粉砕処理し、アセトンで抽出する。得られたアセトン層を減圧濃縮後、好ましくはpH２〜５、さらに好ましくはpH２の条件下に酢酸エチルと酢酸エチルに対して１〜３倍体積の水とにより液層分配を行う。液層分配に用いる水の量は、酢酸エチルに対して３倍体積が好ましい。得られた水層に対して１〜３倍体積、好ましくは３倍体積の酢酸エチルを添加、混合し、好ましくはpH９〜１１、さらに好ましくはpH９の条件下に液層分配を行い、酢酸エチル層を得る。これを減圧濃縮すると緑色の粗粉末が得られる。
【００２９】
この粗粉末は、例えば次のような工程にさらに供することにより精製することができ、白色粉末状の本発明の糖脂質の精製物が得られる。
すなわち、得られた粗粉末を、次の精製工程であるシリカゲルカラムクロマトグラフィーの展開溶媒として用いる有機溶媒に溶解し、シリカゲルカラムクロマトグラフィーに付す。展開溶媒としては、例えば酢酸エチル、メタノールを用いることができる。酢酸エチル−メタノール−水（１００：３０〜１５：５、好ましくは１００：２０：５（いずれも体積比））の混合溶媒で溶出する分画を集める。これを減圧濃縮後、高速液体クロマトグラフィーに付し、アセトニトリル８０〜７０％水溶液、好ましくは７０％水溶液により溶出する分画を得る。これを減圧濃縮後、さらに高速液体クロマトグラフィーに付し、アセトニトリル７０〜６０％、好ましくは６５％水溶液により溶出する分画を得る。この分画を減圧濃縮することにより、白色粉末状の本発明の糖脂質の精製物が得られる。
【００３０】
ＫＭ０４３は、上記の方法以外に、セファデックスＬＨ−２０（ファルマシア社製）等による分配クロマトグラフィー、他の活性アルミナ樹脂、イオン交換樹脂のような適当な既知の吸着剤に吸着させて溶離させる方法、あるいは高速液体クロマトグラフィー法などを組み合わせることにより精製することもできる。
【００３１】
本発明のＫＭ０４３及びその薬学的に許容し得る塩は、医薬として用いることができる。具体的には、本発明のＫＭ０４３及びその薬学的に許容し得る塩（以下、医薬の説明において、単に「本発明のＫＭ０４３」ともいう）は、免疫抑制剤、ＨＩＶに対する抗ウイルス剤、抗癌剤又は既存の抗癌剤と共に治療効果を促進するための助剤として用いることができる。
【００３２】
ＫＭ０４３の薬学的に許容し得る塩の例としては、ナトリウム及びカリウムのような一価の陽イオンの塩を挙げることができる。
本発明のＫＭ０４３を医薬として用いる場合、ＫＭ０４３を単独で用いることもできるが、薬学的製剤として用いることが好ましい。薬学的製剤において、本発明のＫＭ０４３は、薬学的に許容され得る適切な担体または希釈剤等と組み合わせて製剤することができる。また、本発明のＫＭ０４３は、薬学的製剤において、薬学的に許容され得る他の活性化合物と共に用いることもできる。
【００３３】
本発明のＫＭ０４３を含有する薬学的製剤は、経口投与、非経口投与又は直腸内投与することができる。薬学的製剤は、これらの各の投与経路に対して適切な、それ自体は既知の通常用いられる方法により固体、半固体、液体又は気体の剤形にすることができる。具体的には、錠剤、散剤、顆粒剤、カプセル剤、シロップ剤、エリキシル剤、懸濁剤・乳剤、坐剤、注射剤、吸入剤及びエアロゾルなどの剤形とすることができる。しかしながら、本発明のＫＭ０４３を含有する薬学的製剤の剤形はこれらに限定されるものではない。
【００３４】
本発明のＫＭ０４３を経口投与する場合、錠剤、散剤、顆粒剤、カプセル剤、シロップ剤、エリキシル剤、懸濁剤・乳剤等に製剤することができる。
錠剤、散剤、顆粒剤、カプセル剤には、本発明のＫＭ０４３を適切な担体と共に処方することにより製剤することができる。好適な担体として用い得るものには、例えばラクトース、マンニトール、コーンスターチ又は馬鈴薯デンプンのような通常用いられる担体；結晶セルロース、セルロース誘導体、コーンスターチ又はゼラチンのようなバインダー；コーンスターチ、馬鈴薯デンプン又はカルボキシメチルセルロースナトリウムのような錠剤崩壊剤；タルク又はステアリン酸マグネシウムのような潤滑剤等が含まれ、さらに、必要に応じて、希釈剤、緩衝剤、浸潤剤、保存剤及びフレーバー剤等を添加することができる。
【００３５】
シロップ剤、エリキシル剤及び懸濁剤・乳剤には、所定量のＫＭ０４３と注射用滅菌水や規定生理食塩水のような薬学的に許容され得る溶媒を用い単位剤型に調製することができる。本明細書において、「単位剤型」との用語は、投与対象のための単位となる容量として適する物理的に個別の単位を指し、各単位は薬学的に許容され得る希釈剤、担体又は溶媒等と共に、必要な薬効を与えるのに十分な量に計算されたＫＭ０４３を所定量含有するものをいう。
【００３６】
また、本発明のＫＭ０４３は、液体又は微細粉末の形態で吸入用製剤として処方することができる。吸入用製剤には、気体又は液体の噴霧剤と共に、必要に応じて浸潤性付与剤のような従来用いられる助剤を添加することができる。吸入用製剤としては、本発明のＫＭ０４３をエアロゾル容器に充填することもできるし、ネブライザーのような非加圧容器に充填することもできる。エアロゾル容器に充填する場合、本発明のＫＭ０４３は、ジクロロフルオロメタン、プロパン、窒素等の薬学的に許容し得る加圧ガス中に添加したものとすることができる。
【００３７】
非経口投与する場合、本発明のＫＭ０４３は、植物性油、合成樹脂酸グリセリド、高級脂肪酸のエステル又はプロピレングリコールのような水性又は非水性の溶媒中に溶解、懸濁又は乳化し、さらに、必要に応じて可溶化剤、浸透圧調節剤、乳化剤、安定剤及び保存料のような従来の添加剤を添加することにより注射用製剤として処方することができる。
【００３８】
直腸内投与する場合、本発明のＫＭ０４３を乳化基剤又は水溶性基剤のような種々の基剤と混合することにより坐薬製剤として処方することができる。基剤の一例を挙げると、体温で融解するが室温では固化しているカカオバター、カーボンワックス及びポリエチレングリコールなどがある。
【００３９】
本発明のＫＭ０４３の投与量は、その用途すなわち、免疫抑制剤、ＨＩＶに対する抗ウイルス剤、抗癌剤及び既存の抗癌剤と共に治療効果を促進するための助剤、並びに投与経路、対象とする疾病の程度や段階等に応じて適宜設定することができる。一例を挙げると、ＫＭ０４３を経口剤として投与する場合には、好ましくは１〜８０ｍｇ／ｋｇ体重／日に設定することができる。ＫＭ０４３を注射剤として投与する場合には、１〜３０ｍｇ／ｋｇ体重／日に設定することができる。これらの投与量は、医者等のような治療を行う者により、治療対象者の病状等に対応させて適宜調節することができる。
【００４０】
【実施例】
以下に本発明の実施例を説明するが、本発明は、これらに限定されるものではない。
（実施例１）
本発明の一般式（Ｉ）で表される糖脂質の抽出及び精製
海浜より採取したギガルティナ・テネラ藻体の乾燥物約２００グラムを粉砕処理し、２リットルのアセトンで１週間抽出した。
【００４１】
抽出物を減圧濃縮後、酢酸エチル−水（１：１（体積比））を用いてpH２条件下に液層分配を行い、水層を得た。残りの酢酸エチル層にこれと同体積の水を添加し、pH２条件下に液層分配を行い、水層を得た。再度残りの酢酸エチル層にこれと同体積の水を添加し、pH２条件下に液層分配を行い、水層を得た。得られた水層を集め、水層に対して同体積の酢酸エチルを添加し、pH９の条件下に液層分配を行い、酢酸エチル層を得た。残りの水層にこれと同体積の酢酸エチルを添加し、pH９の条件下に液層分配を行い、酢酸エチル層を得た。再度残りの水層にこれと同体積の酢酸エチルを添加し、pH９の条件下液層分配を行い、酢酸エチル層を得た。得られた酢酸エチル層を集め、これを減圧濃縮し、緑色の粗粉末を得た。この緑色の粉末を酢酸エチルに再溶解し、シリカゲルカラムクロマトグラフィー(4cm×40cm、ワコーゲルC ２００ 和光純薬社製）に付し、酢酸エチル−メタノール−水（１００：２０：５（体積比））の混合溶媒で溶出する分画を集めた。これを減圧濃縮することにより得られる黄色の粗粉末を、高速液体クロマトグラフィー(C18カラム YMC D-ODS-5-ST 120A （株）ワイエムシイ社製) に付し、アセトニトリル７０％水溶液により溶出する分画を得た。これを減圧濃縮後、さらに高速液体クロマトグラフィー(C8 カラム InertsilC8 ジーエルサイエンス（株）製）に付し、アセトニトリル６５％水溶液により溶出する分画を得た。この分画を減圧濃縮し、本発明の式（Ｉ）により表される糖脂質（ＫＭ０４３）を白色粉末として約２ミリグラム得た。
【００４２】
上記実施例１で得られたＫＭ０４３は、結晶化させることができず、融点を測定することができなかった。
＜測定例１＞
上記実施例１で得た白色粉末を高速原子衝撃質量分析(FAB-MS)に供した。得られたスペクトルを図１に示す。
【００４３】
図１のスペクトルより、FAB-MS m/z 839.2(M-H) ^- が得られ、ＫＭ０４３の分子量（測定値）として８４０を得た。（分子式：Ｃ₄₅Ｈ₇₆Ｏ₁₂Ｓからの計算値：８４０）。
【００４４】
＜測定例２＞
実施例１で得たＫＭ０４３について、溶媒としてＣＤ₃ ＯＤ（重メタノール）、内部標準にＴＭＳ（テトラメチルシラン）を用い、¹ Ｈ−ＮＭＲスペクトルを測定した。
【００４５】
得られたスペクトルを図２に示す。
図２において、（ａ）〜（ｆ）を付したシグナルは、それぞれ次のものと思われる。
【００４６】
（ａ）メチル基、 （ｂ）メチレン鎖、
（ｃ）＝ＣＨ−ＣＨ ₂ −ＣＨ₂ −、（ｄ）−ＣＨ ₂ −ＣＯ−、
（ｅ）＝ＣＨ−ＣＨ ₂ −ＣＨ＝、 （ｆ）−ＣＨ₂ −ＨＣ＝ＣＨ−ＣＨ₂ −
上記シグナル（ａ）〜（ｆ）は、ＫＭ０４３の構造において不飽和高級脂肪酸鎖の存在を示すものであり、式（Ｉ）の構造と合致した。シグナル（ｄ）は、ＫＭ０４３において脂肪酸と糖がグリセロール骨格を介して結合する「グリセロ糖脂質」であることを示唆し、式（Ｉ）の構造と合致する。
【００４７】
図２において、シグナル（ｇ）及び（ｇ' ）は、それぞれが糖のプロトンを示すものである。このことから、実施例１で得られたＫＭ０４３の糖部分が単糖であり、その１位と２位以下のプロトンの組み合わせを示すものであり、式（Ｉ）の構造と合致する。
【００４８】
＜測定例３＞
実施例１で得たＫＭ０４３を中性メタノールに溶解し、ＵＶスペクトルを測定した。
【００４９】
得られたスペクトルを図３に示す。
＜測定例４＞
実施例１で得たＫＭ０４３を以下に示す薄層クロマトグラフィーに付し、Ｒf 値を求めた。
【００５０】
吸着剤：シリカゲルプレート(Kieselgel60F254 メルク社製）
展開溶媒が酢酸エチル−メタノール−水（１００：２０：５）の場合のＲf 値は０．２０であった。
【００５１】
展開溶媒がイソプロパノール−メタノール（５：１）の場合のＲf 値は０．1 ０であった。
＜測定例５＞
実施例１で得たＫＭ０４３について、化学分解後、ガスクロマトグラフィーを行い、結合する脂肪酸を調べた。
【００５２】
得られたガスクロマトグラフを図４に示す。
図４のガスクロマトグラフから、Ｃ_16:0とＣ_20:5の脂肪酸に対応するピークの面積が同じであることが分かり、従って１：１の割合でそれぞれの脂肪酸がグリセロール骨格に存在することを示すものである。
【００５３】
＜測定例６＞
実施例１で得たＫＭ０４３の旋光度を測定し、［α］_D＝＋５７．０２°（ｃ＝０．１１ ＭｅＯＨ）を得た。
【００５４】
＜定性試験１＞
上記実施例１で得たＫＭ０４３は、メタノール、ジメチルスルフォキシドに易溶であり、水、クロロホルムに難溶であった。
【００５５】
＜定性試験２＞
上記実施例１で得たＫＭ０４３は、硫酸オルシノール、ヨードに対して陽性であった。この結果は、ＫＭ０４３に炭素化合物が含有されることを示すものであり、式（Ｉ）の構造と合致する。
【００５６】
＜定性試験３＞
上記実施例１で得たＫＭ０４３は、ニンヒドリンに対して陰性であった。この結果は、ＫＭ０４３には蛋白、ペプチド、アミノ酸が含有されていないことを示すものであり、式（Ｉ）の構造と合致する。
【００５７】
＜測定例７＞
上記実施例１で得られたＫＭ０４３のＤＮＡ合成酵素群に対する活性を次の方法で調べた。
【００５８】
ＤＮＡ合成酵素群としてＤＮＡ合成酵素α、ＤＮＡ合成酵素β及びＨＩＶ由来のＤＮＡ合成酵素（逆転写酵素）を試験した。ＤＮＡ合成酵素αは、牛胸腺から常法により抽出精製した標品を、ＤＮＡ合成酵素βは、ラット由来の該当遺伝子を通常の遺伝子組み換え法により大腸菌に組み込み、生産させた標品を、ＨＩＶ由来のＤＮＡ合成酵素（逆転写酵素）は、市販（Worthington Biochemical 社製）の標品を用いた。
【００５９】
これらのＤＮＡ合成酵素に対するＫＭ０４３の阻害作用の測定には、一般的なＤＮＡ合成酵素反応系（日本生化学会編、新生化学実験講座２、核酸ＩＶ、東京化学同人、第６３頁〜６６頁）を用いた。すなわち、放射性同位元素で標識した［³ Ｈ］−ＴＴＰを含む系においてＤＮＡ合成酵素反応を行い、放射比活性を生成物（合成ＤＮＡ鎖）量の指標とするものである。
【００６０】
阻害率は、（ａ）コントロールでの合成ＤＮＡ量、（ｂ）実施例１に示した方法で生成したＫＭ０４３の存在下での合成ＤＮＡ量について、
（ａ−ｂ）／ａ×１００＝阻害率（％）
として評価した。
得られた結果を次の表１に示す。
【００６１】
【表１】

【００６２】
表１の結果から、ＫＭ０４３は、ＤＮＡ合成酵素βに対して高い阻害作用を有することが分かる。
上記従来の技術の欄において説明したように、ＤＮＡ合成酵素βは、免疫反応のＤＮＡ再構成において抗原特異的に反応する抗体あるいはレセプター分子を作り出す根源に関与するとともに、変異による抗体の多様性に一定の役割を果たしているともいわれている。これらのことから、ＤＮＡ合成酵素βは、免疫反応に密接に関連し、ＤＮＡ合成酵素βを抑制することは、免疫反応の抑制につながると推測される。よって、ＤＮＡ合成酵素β抑制作用を有するＫＭ０４３は、免疫抑制剤としての作用を有することが期待される。
【００６３】
表１の結果から、ＫＭ０４３は、ＨＩＶ由来ＤＮＡ合成酵素（逆転写酵素）に対しても高い阻害作用を有することが分かる。
＜測定例８＞
上記実施例１で得られたＫＭ０４３のＤＮＡ合成酵素阻害の作用機構をLineweaver-Burk プロットにより調べた。
【００６４】
ＤＮＡ合成酵素βに関してＫＭ０４３は、活性化ＤＮＡ及びｄＴＴＰに対し共に非拮抗的阻害を示した。
＜測定例９＞
上記実施例１で得られたＫＭ０４３の癌細胞増殖抑制効果を次の方法を用いて評価した。
【００６５】
i)細胞培養方法
本実験に用いた細胞は、ヒト子宮癌由来Ｈｅｌａ−Ｓ３細胞である。
培地としては、イーグル最少（ＭＥＭ）培地（日水製薬社製）に、０．２％（Ｗ／Ｖ）炭酸水素ナトリウム、０．０００３％（Ｗ／Ｖ）グルタミン、及び子牛血清１０％（V ／V ）を添加したものを用いた。培養は、５％ＣＯ₂ インキュベータにて３７℃で行った。
【００６６】
ii) ＫＭ０４３の添加および細胞生存率の測定
上記に示した培地に、予め各試験濃度になるようにＫＭ０４３を溶解した。ただし、ＫＭ０４３は水に難溶であるため、一度ＤＭＳＯ（ジメチルスルフォキシド）に溶解し、そのものを上記の培地に溶かした。なお、培養中の培地内に存在するＤＭＳＯの終濃度は、すべての試験区で１％以下になっており、本測定例で用いたＨｅｌａ−Ｓ３細胞の増殖の抑制にＤＭＳＯが関わる可能性は否定できる状態である。
【００６７】
本試験のための培養は、９６穴マイクロプレートで行った。各ウエルに２．０×１０³ 個の細胞を植込み、１つの試験濃度に対し３ウエルずつ与えた。またポジティブコントロールとして培地に１％のＤＭＳＯを含むものを用いた。
【００６８】
ＫＭ０４３添加後は、５％ＣＯ₂ インキュベータ内、３７℃で４８時間培養し、各試験区の細胞生存率の判定を行った。
生存率の判定は、文献（「Rapid Colorimetric Assay for Cellular Growth and Survival: Application to Proliferation and Cytotoxicity Assays 」、Tim Mosmann, Journal of Immunological Methods, 65 (1983) 55 〜63）に記載されているＭＴＴアッセイ法を用いた。即ち、上記４８時間培養後テトラゾリウム塩ＭＴＴを添加し、更に４時間培養した。生細胞による還元を経て生産するホルマザン量を生細胞数に比例するとみなし、５７０ｎｍの光学密度（Ｏ．Ｄ．）で定量した。
【００６９】
細胞生存率は次の式により算出した。
細胞生存率（％）
＝試験区のＯ. Ｄ. ［５７０ｎｍ］／対照区のＯ. Ｄ. ［５７０ｎｍ］
得られた結果を図５に示す。なお図５に示すデータは３ウエルの平均値である。
【００７０】
図５の結果から、ＫＭ０４３は、濃度依存的に癌細胞の増殖を抑えることが分かる。しかしながら、その細胞増殖抑制作用は、ブレオマイシンのような既知抗癌剤に比べると低い。
【００７１】
＜測定例１０＞
上記実施例１で得られたＫＭ０４３が他の抗癌剤と併用された場合に示す抗癌作用を次の方法を用いて測定した。
【００７２】
i)細胞培養
細胞培養条件は、上記測定例９と同じに行った。
ii) ＫＭ０４３及びブレオマイシン(Bleomycin) の添加
予め５０μＭのＫＭ０４３（上記測定例９と同じ方法で溶解した）と所定濃度のブレオマイシンを溶解した培地（１）：「ＫＭ０４３ ５０μＭ＋ブレオマイシンの試験区」、及び所定濃度のブレオマイシンのみを溶解した培地（２）：「ブレオマイシンのみの試験区」）を用意した。
【００７３】
各ブレオマイシン濃度に対しては、３ウエルずつを与えた。各ウエルには２．０×１０³ 個の細胞を植込み、続いて薬剤を含む培地を加えた。
「ＫＭ０４３ ５０μＭ＋ブレオマイシンの試験区」、「ブレオマイシンのみの試験区」について、それぞれ５％ＣＯ₂ インキュベータ内、３７℃で４８時間培養後、細胞生存率を測定した。（測定方法は、上記測定例９と同じ）。
【００７４】
得られた結果を図６に示す。図６において、破線により結ばれる黒い丸は、培地（１）：「ＫＭ０４３ ５０μＭ＋ブレオマイシンの試験区」の結果を、実線により結ばれる黒い四角は培地（２）：「ブレオマイシンのみの試験区」の結果を表す。
【００７５】
図６の結果から、実施例１で得たＫＭ０４３は、測定例９に示したようにその濃度が癌細胞の増殖にほとんど影響がないほどの低濃度（５０μＭ）であるにもかかわらず、低濃度のブレオマイシンと併用することによりブレオマイシンのみの場合より癌細胞の増殖を強く抑えることが分かる。
【００７６】
現在、抗癌剤治療においてその副作用が問題となっているが、実施例１で得たＫＭ０４３を併用することにより、抗癌剤の量をへらしつつ同等の治療効果を維持することができると考えられる。
【００７７】
【発明の効果】
本発明によれば、新規な糖脂質、その製造方法及びその用途が提供される。
本発明の式（Ｉ）で表される糖脂質は特にβ型のＤＮＡ合成酵素に対する高い阻害作用を有し、かつ非拮抗型の作用機序を有する。
【００７８】
このようなＤＮＡ合成酵素βは、免疫反応の根元に関与する酵素であるので、ＤＮＡ合成酵素βの阻害活性を有する式（Ｉ）で表される糖脂質は、免疫抑制剤として作用すると考えられる。さらに、その作用機序が非拮抗型であるので、式（Ｉ）で表される糖脂質は、生体に対する副作用が少ないことが予想される。
【００７９】
また、式（Ｉ）で表される糖脂質は、ＨＩＶ由来逆転写酵素阻害作用を有する。
さらに、式（Ｉ）で表される糖脂質は、特に他の抗癌剤との併用により強い癌細胞増殖抑制作用を有する。
【図面の簡単な説明】
【図１】本発明の式（Ｉ）で表される糖脂質の高速原子衝撃質量分析スペクトル。
【図２】本発明の式（Ｉ）で表される糖脂質の¹ Ｈ−ＮＭＲスペクトル。
【図３】本発明の式（Ｉ）で表される糖脂質のＵＶスペクトル。
【図４】本発明の式（Ｉ）で表される糖脂質のガスクロマトグラフ。
【図５】本発明の式（Ｉ）で表される糖脂質（単独）の癌細胞増殖抑制作用を表すグラフ。
【図６】本発明の式（Ｉ）で表される糖脂質（併用）の癌細胞増殖抑制作用を表すグラフ。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a novel glycolipid, a production method thereof, and its use as an inhibitor, immunosuppressant and anticancer agent for higher animal DNA synthase and HIV (human immunodeficiency virus) -derived reverse transcriptase.
[0002]
[Prior art]
The DNA synthase group of higher organisms is an essential element for maintaining the homeostasis of organisms because it is fundamentally involved in cell division and differentiation in vivo. For the five types of DNA synthases α, β, γ, δ and ε in the group of DNA synthases, the characteristics of each enzyme reaction have been clarified mainly from in vitro proofs in the last 10 years. .
[0003]
For example, DNA synthase β is said to be an enzyme that is deeply involved in recombination of DNA strands. Here, “recombination” refers to recombination of DNA in one cell, and is different from “recombination” in genetic engineering in which DNA encoding a specific protein is incorporated into E. coli or the like.
[0004]
DNA recombination involving the DNA synthase β is considered to be necessary mainly in the following two situations. One is for repairing wounds scattered in DNA, and the other is for expressing new genetic information in cells. The former is called DNA recombination repair, and the latter is called somatic DNA recombination.
[0005]
Of the two types of recombination involving DNA synthase β, somatic DNA recombination is still not fully understood in what kind of tissue or in any scene of biological reaction, but at least in immunocompetent cells. It has become clear that this is happening.
[0006]
For example, Hirose et al., “Difference in the expression level of DNA synthase β in mouse tissue” (Experimental Cell Research, 181 (1989), 169-180) describes that DNA synthase β In the spleen and cerebrum, it has been reported that both the amount of enzyme and the amount of mRNA expressed are prominently present (FIGS. 2, 3, 4, Table 1). Hirose et al. Describes that DNA synthase β in thymus and spleen tissues can control somatic DNA recombination (page 178, lines 31-38).
[0007]
Among the four tissues reported by Hirose et al. That a large amount of DNA synthase β is present, the thymus and spleen are tissues in which many immunocompetent cells are present. The thymus is the T lymphocyte and the spleen is the B lymphocyte. B lymphocytes and T lymphocytes differentiate from progenitor cells to produce antibodies that are most specific for invading antigens (in T lymphocytes, cell surface antigen receptors). DNA synthase β is involved in the mechanism of producing antibodies (or T lymphocyte antigen receptors) that specifically react to various (different countless) antigens from limited genetic information in immune reactions. It is believed that
[0008]
More specifically, in B lymphocytes, somatic cell type DNA recombination involving the DNA synthase β described above occurs between the V gene and J gene of DNA encoding the antibody protein in the course of differentiation. This recombination is called VJ recombination. By such recombination, various antibody proteins can be produced by changing combinations from limited genetic information. This is called DNA reconstruction.
[0009]
Similar to antibodies, T-lymphocyte antibody receptor proteins can produce various specific receptors that can respond to differences in individual antigens by recombination and reconstitution of the relevant DNA.
[0010]
Thus, it can be said that the DNA synthase β is involved in the source of producing an antibody or receptor molecule that reacts specifically with an antigen in the DNA reconstitution of an immune reaction.
Therefore, it is speculated that inhibition of DNA synthase β suppresses immune reactions centered on DNA reconstitution.
[0011]
As described above, while VJ recombination exists as a mechanism for producing antibodies that specifically react to various antigens from limited genetic information in immune reactions, in addition to acquiring diversity, There is another molecular mechanism. That is, when mutation occurs in the antibody gene, diversity can be further combined with recombination. The “mutation” here is not a large-scale one in which, for example, a part of the antibody protein is completely deleted, but a part of the base sequence is replaced or a new sequence is inserted, and the amino acid composition gradually changes. It ’s like making different antibodies. Therefore, mutation is not harmful, but rather it is more convenient for an immune response if it occurs to some extent.
[0012]
It is considered that DNA synthase β plays a certain role in this mutation.
For example, Thomas et al. “Proceedings National Academy of Science of the United States of America, vol. 83, pp.1867-1871, March 1986” describes TdT ( The result of measuring the frequency of mutation in an experimental system in which terminal deoxyribonucleotidyl transferase) and DNA synthase β coexist has been reported. TdT is a special DNA synthase that is different from the above five DNA synthases α to ε, and according to Thomas et al., Is an enzyme that is closely related to the appearance of mutation or the frequency thereof. . In Table 2 of Thomas et al., The mutation frequency increases depending on the concentration of TdT, and the maximum mutation frequency of 44% is higher than that of DNA synthase β alone than that of TdT alone. Has been reported. Further, Thomas et al. Describes that both TdT and DNA synthase β react with each other in a competitive manner to cause mutation (page 1868, left column).
[0013]
From the report of Thomas et al., It can be interpreted that the DNA synthase β plays a role of at least auxiliary amplification of the mutation induced by TdT. That is, DNA synthase β plays a certain role in the diversity of antibodies due to mutation. This also suggests that inhibition of DNA synthase β leads to suppression of immune reaction.
[0014]
Currently, dideoxy TTP is known as a compound that selectively inhibits DNA synthase β (Tersa et al., Annual Review of Biochemistry 1991 60 513-552). However, dideoxy TTP is highly toxic in vivo and no marked physiological activity has been observed.
[0015]
On the other hand, HIV-derived reverse transcriptase among the enzymes of the DNA synthase group is an enzyme that plays a central role during HIV infection, as is well known. The fact that inhibiting HIV-derived reverse transcriptase can bring about a therapeutic effect on HIV has also been demonstrated by already marketed clinical drugs such as azidothymidine (edited by the Japan Pharmaceutical Information Center, Yakuho Kaihosha Co., Ltd.). Issued, “1996 edition of Japan Pharmaceutical Collection”, page 599). However, it is difficult to cope with easily mutated HIV with only existing drugs such as azidothymidine, and development of a new inhibitor for HIV-derived reverse transcriptase is desired.
[0016]
Many DNA synthetase inhibitors including the above-mentioned dideoxy TTP and azidothymidine are analogs of DNA in structure, and show antagonistic inhibition in the DNA synthesis reaction. While antagonistic inhibitors have good inhibition rates, they have the disadvantage that they can be incorporated into other various DNA metabolizing enzymes and cause side effects on living bodies.
[0017]
[Problems to be solved by the invention]
Therefore, an object of the present invention is to provide a novel physiologically active substance having a high inhibitory action on a β-type DNA synthase and a non-antagonistic action mechanism, and a method for producing the same.
[0018]
[Means for Solving the Problems]
The present inventors have found that glycolipids extracted from specific algae have a high inhibitory action on DNA synthase β and have a non-antagonistic action mechanism, and further, this glycolipid is derived from HIV. The present inventors have found that it has an inhibitory effect on reverse transcriptase and, in particular, a cancer cell growth inhibitory effect when used in combination with a specific anticancer agent.
[0019]
As described above, the DNA synthase β is an enzyme involved in the root of the immune reaction. Therefore, it is considered that the glycolipid of the present invention having an inhibitory activity on DNA synthase β acts as an immunosuppressant.
That is, the present invention provides the following (1) to (6).
(1) The following formula (I)
[0020]
[Chemical 7]

A glycolipid represented by
[0021]
(2) In the method for producing the glycolipid represented by the above formula (I) from the red alga Sugiori genus Gigartina tenella, the red alga Sugiori genus Gigartina tenella is mixed with acetone, ethyl acetate, chloroform, methanol, or a chloroform-methanol mixture. A step of extracting with one of an organic solvent or an organic solvent mixture selected from the group consisting of: a step of concentrating the organic solvent extract under reduced pressure; and a layer of ethyl acetate-water under acidic conditions for the extract under reduced pressure The separated aqueous layer is subjected to partitioning, the separated aqueous layer is subjected to layer partitioning with ethyl acetate under alkaline conditions, the ethyl acetate layer is separated, and the separated ethyl acetate layer is concentrated under reduced pressure. Recovering the obtained green coarse powder, purifying the coarse powder, and concentrating the solution obtained in the purification step under reduced pressure, Method characterized by comprising the step of collecting the white powder.
[0022]
(3) A pharmaceutical comprising the glycolipid represented by the above formula (I) as an active ingredient.
(4) A DNA synthase β inhibitor comprising the glycolipid represented by the above formula (I) as an active ingredient.
[0023]
(5) An HIV-derived reverse transcriptase inhibitor comprising the glycolipid represented by the above formula (I) as an active ingredient.
(6) An immunosuppressant comprising the glycolipid represented by the above formula (I) as an active ingredient.
[0024]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the glycolipid represented by the formula (I) of the present invention (hereinafter also referred to as KM043) will be described in detail.
An example of the manufacturing method of KM043 of this invention is given.
[0025]
That is, a substance extracted from seaweed with an organic solvent is subjected to a liquid layer partition between the organic solvent and water under acidic conditions. The obtained aqueous layer is subjected to liquid layer distribution with an organic solvent under alkaline conditions. By further purifying the obtained organic layer, KM043 of the present invention can be produced.
[0026]
As the seaweed, for example, the red alga Gigartina tenella can be used.
As the extraction solvent, for example, acetone, ethyl acetate, chloroform, methanol, or a chloroform-methanol mixture (2: 1 (volume ratio)) can be used. As the extraction solvent, acetone is preferable from the viewpoint of the yield of KM043.
[0027]
It is preferable to use as many extraction solvents as possible from the viewpoint of the extraction efficiency of KM043. The extraction solvent is usually 5 times or more the dry weight of seaweed, and the extraction efficiency is further increased by using 30 times the dry weight of seaweed. However, the amount of the extraction solvent can be appropriately set from the viewpoints of extraction efficiency and cost.
[0028]
The manufacturing method of KM043 of this invention is demonstrated in detail. The dried material of Gigartina tenella is pulverized and extracted with acetone. After concentration of the obtained acetone layer under reduced pressure, liquid layer partitioning is performed with ethyl acetate and water having a volume of 1 to 3 times that of ethyl acetate under the conditions of preferably pH 2 to 5, more preferably pH 2. The amount of water used for liquid layer distribution is preferably 3 times the volume of ethyl acetate. 1 to 3 times volume, preferably 3 times volume of ethyl acetate is added to and mixed with the aqueous layer thus obtained, and liquid layer partitioning is preferably carried out under conditions of pH 9 to 11, more preferably pH 9, to obtain ethyl acetate. Get a layer. When this is concentrated under reduced pressure, a green crude powder is obtained.
[0029]
This crude powder can be purified, for example, by further subjecting it to the following steps, and a purified product of the glycolipid of the present invention in the form of a white powder is obtained.
That is, the obtained crude powder is dissolved in an organic solvent used as a developing solvent for silica gel column chromatography, which is the next purification step, and subjected to silica gel column chromatography. As the developing solvent, for example, ethyl acetate or methanol can be used. Fractions eluted with a mixed solvent of ethyl acetate-methanol-water (100: 30 to 15: 5, preferably 100: 20: 5 (both volume ratio)) are collected. This is concentrated under reduced pressure, and then subjected to high performance liquid chromatography to obtain a fraction that is eluted with an acetonitrile 80-70% aqueous solution, preferably a 70% aqueous solution. This is concentrated under reduced pressure and further subjected to high performance liquid chromatography to obtain a fraction eluted with 70 to 60% acetonitrile, preferably 65% aqueous solution. By concentrating this fraction under reduced pressure, a purified product of the glycolipid of the present invention in the form of a white powder can be obtained.
[0030]
In addition to the above method, KM043 is a method of elution by adsorbing on a suitable known adsorbent such as partition chromatography using Sephadex LH-20 (Pharmacia), other activated alumina resin, or ion exchange resin. Alternatively, it can be purified by a combination of high performance liquid chromatography.
[0031]
The KM043 of the present invention and a pharmaceutically acceptable salt thereof can be used as a medicine. Specifically, the KM043 of the present invention and a pharmaceutically acceptable salt thereof (hereinafter, also simply referred to as “KM043 of the present invention” in the description of the medicine) are an immunosuppressant, an antiviral agent against HIV, an anticancer agent or It can be used as an auxiliary agent for promoting the therapeutic effect together with existing anticancer agents.
[0032]
Examples of pharmaceutically acceptable salts of KM043 include salts of monovalent cations such as sodium and potassium.
When KM043 of the present invention is used as a medicine, KM043 can be used alone, but is preferably used as a pharmaceutical preparation. In the pharmaceutical preparation, the KM043 of the present invention can be formulated in combination with a suitable pharmaceutically acceptable carrier or diluent. The KM043 of the present invention can also be used in pharmaceutical formulations with other pharmaceutically acceptable active compounds.
[0033]
The pharmaceutical preparation containing KM043 of the present invention can be administered orally, parenterally or rectally. The pharmaceutical preparations can be made into solid, semi-solid, liquid or gaseous dosage forms by any commonly used method known per se, suitable for each of these administration routes. Specifically, dosage forms such as tablets, powders, granules, capsules, syrups, elixirs, suspensions / emulsions, suppositories, injections, inhalants and aerosols can be used. However, the dosage form of the pharmaceutical preparation containing KM043 of the present invention is not limited thereto.
[0034]
When KM043 of the present invention is orally administered, it can be formulated into tablets, powders, granules, capsules, syrups, elixirs, suspensions / emulsions and the like.
Tablets, powders, granules, and capsules can be formulated by formulating KM043 of the present invention together with an appropriate carrier. Those which can be used as suitable carriers include commonly used carriers such as lactose, mannitol, corn starch or potato starch; binders such as crystalline cellulose, cellulose derivatives, corn starch or gelatin; corn starch, potato starch or sodium carboxymethylcellulose Such tablet disintegrating agents; lubricants such as talc or magnesium stearate and the like, and further, diluents, buffering agents, wetting agents, preservatives and flavoring agents can be added as necessary.
[0035]
The syrup, elixir and suspension / emulsion can be prepared into unit dosage forms using a predetermined amount of KM043 and a pharmaceutically acceptable solvent such as sterile water for injection and normal saline. As used herein, the term “unit dosage form” refers to a physically discrete unit suitable as a unit volume for a subject to be administered, each unit being a pharmaceutically acceptable diluent, carrier or solvent. And the like containing a predetermined amount of KM043 calculated to an amount sufficient to give the necessary medicinal effect.
[0036]
Further, KM043 of the present invention can be formulated as a preparation for inhalation in the form of liquid or fine powder. A conventional auxiliary agent such as an infiltrating agent can be added to the inhalation preparation together with a gas or liquid propellant, if necessary. As a preparation for inhalation, the KM043 of the present invention can be filled in an aerosol container or a non-pressurized container such as a nebulizer. When filling an aerosol container, the KM043 of the present invention can be added to a pharmaceutically acceptable pressurized gas such as dichlorofluoromethane, propane, or nitrogen.
[0037]
For parenteral administration, the KM043 of the present invention is dissolved, suspended or emulsified in an aqueous or non-aqueous solvent such as vegetable oil, synthetic resin acid glyceride, higher fatty acid ester or propylene glycol, and further required. Depending on the formulation, conventional additives such as solubilizers, osmotic pressure regulators, emulsifiers, stabilizers and preservatives can be added to formulate as an injectable preparation.
[0038]
When administered rectally, KM043 of the present invention can be formulated as a suppository formulation by mixing with various bases such as emulsifying bases or water-soluble bases. Examples of bases include cocoa butter, carbon wax and polyethylene glycol that melt at body temperature but solidify at room temperature.
[0039]
The dose of KM043 of the present invention depends on its use, that is, an immunosuppressant, an antiviral agent against HIV, an anticancer agent and an auxiliary agent for promoting a therapeutic effect together with an existing anticancer agent, an administration route, the degree of the target disease, It can be set as appropriate according to the stage. For example, when KM043 is administered as an oral preparation, it can be preferably set to 1 to 80 mg / kg body weight / day. When KM043 is administered as an injection, it can be set to 1 to 30 mg / kg body weight / day. These doses can be appropriately adjusted by a person such as a doctor or the like according to the medical condition of the person to be treated.
[0040]
【Example】
Examples of the present invention will be described below, but the present invention is not limited thereto.
Example 1
Extraction and purification of glycolipids represented by general formula (I) of the present invention
About 200 grams of dried Gigartina Tenella algae collected from the beach were ground and extracted with 2 liters of acetone for 1 week.
[0041]
After the extract was concentrated under reduced pressure, liquid layer partitioning was performed under pH 2 conditions using ethyl acetate-water (1: 1 (volume ratio)) to obtain an aqueous layer. The same volume of water was added to the remaining ethyl acetate layer, and liquid layer partitioning was performed under pH 2 conditions to obtain an aqueous layer. The same volume of water was again added to the remaining ethyl acetate layer, and liquid layer partitioning was performed under pH 2 conditions to obtain an aqueous layer. The obtained aqueous layer was collected, the same volume of ethyl acetate was added to the aqueous layer, and the liquid layer was partitioned under pH 9 conditions to obtain an ethyl acetate layer. The same volume of ethyl acetate was added to the remaining aqueous layer, and liquid layer partitioning was performed under the condition of pH 9 to obtain an ethyl acetate layer. Again, the same volume of ethyl acetate was added to the remaining aqueous layer, and the liquid layer was partitioned under pH 9 conditions to obtain an ethyl acetate layer. The obtained ethyl acetate layers were collected and concentrated under reduced pressure to obtain a green crude powder. This green powder was redissolved in ethyl acetate and subjected to silica gel column chromatography (4 cm × 40 cm, Wako Gel C 200, manufactured by Wako Pure Chemical Industries, Ltd.), and ethyl acetate-methanol-water (100: 20: 5 (volume ratio)). Fractions eluting with the mixed solvent were collected. The yellow crude powder obtained by concentrating this under reduced pressure was subjected to high performance liquid chromatography (C18 column YMC D-ODS-5-ST 120A manufactured by YMC Co., Ltd.) and eluted with a 70% acetonitrile aqueous solution. A picture was obtained. This was concentrated under reduced pressure, and further subjected to high performance liquid chromatography (C8 column, Inertsil C8, manufactured by GL Science Co., Ltd.) to obtain a fraction eluted with an aqueous 65% acetonitrile solution. This fraction was concentrated under reduced pressure to obtain about 2 milligrams of a glycolipid (KM043) represented by the formula (I) of the present invention as a white powder.
[0042]
KM043 obtained in Example 1 above could not be crystallized and the melting point could not be measured.
<Measurement Example 1>
The white powder obtained in Example 1 was subjected to fast atom bombardment mass spectrometry (FAB-MS). The obtained spectrum is shown in FIG.
[0043]
From the spectrum in Fig. 1, FAB-MS m / z 839.2 (M-H)^- And 840 was obtained as the molecular weight (measured value) of KM043. (Molecular formula: C₄₅H₇₆O₁₂Calculated from S: 840).
[0044]
<Measurement Example 2>
For KM043 obtained in Example 1, CD was used as a solvent._Three OD (heavy methanol), TMS (tetramethylsilane) as internal standard,¹ 1 H-NMR spectrum was measured.
[0045]
The obtained spectrum is shown in FIG.
In FIG. 2, the signals marked with (a) to (f) are considered as follows.
[0046]
(A) a methyl group, (b) a methylene chain,
(C) = CH—CH ₂ -CH₂ -, (D) -CH ₂ -CO-,
(E) = CH—CH ₂ -CH =, (f) -CH₂ −HC = CH-CH₂ −
The above signals (a) to (f) indicate the presence of an unsaturated higher fatty acid chain in the structure of KM043, which is consistent with the structure of formula (I). The signal (d) suggests that in KM043 a fatty acid and a sugar are “glyceroglycolipids” that are linked via a glycerol skeleton, consistent with the structure of formula (I).
[0047]
In FIG. 2, signals (g) and (g ′) each represent a sugar proton. From this, the saccharide part of KM043 obtained in Example 1 is a monosaccharide, which indicates a combination of protons at the 1-position and the 2-position or less, and matches the structure of the formula (I).
[0048]
<Measurement Example 3>
KM043 obtained in Example 1 was dissolved in neutral methanol, and the UV spectrum was measured.
[0049]
The obtained spectrum is shown in FIG.
<Measurement Example 4>
The KM043 obtained in Example 1 was subjected to the following thin layer chromatography to determine the Rf value.
[0050]
Adsorbent: Silica gel plate (Kieselgel60F254 manufactured by Merck)
When the developing solvent was ethyl acetate-methanol-water (100: 20: 5), the Rf value was 0.20.
[0051]
The Rf value when the developing solvent was isopropanol-methanol (5: 1) was 0.110.
<Measurement Example 5>
KM043 obtained in Example 1 was subjected to gas chromatography after chemical decomposition, and the bound fatty acid was examined.
[0052]
The obtained gas chromatograph is shown in FIG.
From the gas chromatograph in FIG._{16: 0}And C_{20: 5}It can be seen that the areas of the peaks corresponding to the fatty acids are the same, thus indicating that each fatty acid is present in the glycerol skeleton in a ratio of 1: 1.
[0053]
<Measurement Example 6>
The optical rotation of KM043 obtained in Example 1 was measured, and [α]_D= + 57.02 ° (c = 0.11 MeOH).
[0054]
<Qualitative test 1>
KM043 obtained in Example 1 was easily soluble in methanol and dimethyl sulfoxide, and hardly soluble in water and chloroform.
[0055]
<Qualitative test 2>
KM043 obtained in Example 1 was positive for orcinol sulfate and iodine. This result indicates that KM043 contains a carbon compound, which is consistent with the structure of formula (I).
[0056]
<Qualitative test 3>
KM043 obtained in Example 1 was negative for ninhydrin. This result indicates that KM043 contains no protein, peptide, or amino acid, which is consistent with the structure of formula (I).
[0057]
<Measurement Example 7>
The activity of KM043 obtained in Example 1 above against the DNA synthase group was examined by the following method.
[0058]
As a group of DNA synthases, DNA synthase α, DNA synthase β and HIV-derived DNA synthase (reverse transcriptase) were tested. DNA synthase α is a sample extracted and purified from bovine thymus by a conventional method, and DNA synthase β is a sample obtained by incorporating a rat-derived gene into Escherichia coli by a conventional genetic recombination method and producing it. As a DNA synthase (reverse transcriptase), a commercially available sample (manufactured by Worthington Biochemical) was used.
[0059]
In order to measure the inhibitory action of KM043 on these DNA synthases, a general DNA synthase reaction system (edited by the Japanese Biochemical Society, Shinsei Chemistry Laboratory 2, Nucleic Acid IV, Tokyo Kagaku Dojin, pages 63-66) Using. That is, labeled with a radioisotope [^Three The DNA synthase reaction is performed in a system containing H] -TTP, and the radioactivity is used as an indicator of the amount of product (synthetic DNA chain).
[0060]
The inhibition rate is (a) the amount of synthetic DNA in the control, (b) the amount of synthetic DNA in the presence of KM043 produced by the method shown in Example 1.
(Ab) / a × 100 = inhibition rate (%)
As evaluated.
The obtained results are shown in Table 1 below.
[0061]
[Table 1]

[0062]
From the results in Table 1, it can be seen that KM043 has a high inhibitory action on DNA synthase β.
As explained in the above-mentioned section of the prior art, DNA synthase β is involved in the source of producing an antibody or receptor molecule that reacts antigen-specifically in the DNA reconstitution of immune reaction, and also contributes to the diversity of antibodies due to mutation. It is said to play a certain role. From these facts, it is speculated that DNA synthase β is closely related to immune reaction, and that suppression of DNA synthase β leads to suppression of immune reaction. Therefore, KM043 having a DNA synthase β inhibitory action is expected to have an action as an immunosuppressive agent.
[0063]
From the results in Table 1, it can be seen that KM043 also has a high inhibitory action on HIV-derived DNA synthase (reverse transcriptase).
<Measurement Example 8>
The action mechanism of DNA synthase inhibition of KM043 obtained in Example 1 was examined by Lineweaver-Burk plot.
[0064]
With respect to DNA synthase β, KM043 showed non-antagonistic inhibition of both activated DNA and dTTP.
<Measurement Example 9>
The cancer cell proliferation inhibitory effect of KM043 obtained in Example 1 was evaluated using the following method.
[0065]
i) Cell culture method
The cells used in this experiment are human uterine cancer-derived Hela-S3 cells.
As the medium, Eagle's minimum (MEM) medium (manufactured by Nissui Pharmaceutical), 0.2% (W / V) sodium bicarbonate, 0.0003% (W / V) glutamine, and calf serum 10% ( What added V / V) was used. Culture is 5% CO₂ It performed at 37 degreeC with the incubator.
[0066]
ii) Addition of KM043 and measurement of cell viability
In the medium shown above, KM043 was dissolved in advance at each test concentration. However, since KM043 is hardly soluble in water, it was once dissolved in DMSO (dimethyl sulfoxide) and dissolved in the above medium. In addition, the final concentration of DMSO present in the culture medium during culture is 1% or less in all the test sections, and there is a possibility that DMSO is involved in the suppression of the growth of Hela-S3 cells used in this measurement example. It can be denied.
[0067]
The culture for this test was performed in a 96-well microplate. 2.0 x 10 per well^Three Cells were implanted and given 3 wells for each test concentration. As a positive control, a medium containing 1% DMSO was used.
[0068]
5% CO after adding KM043₂ The cells were cultured for 48 hours at 37 ° C. in an incubator, and the cell viability of each test group was determined.
The survival rate is determined by the MTT assay method described in the literature (“Rapid Colorimetric Assay for Cellular Growth and Survival: Application to Proliferation and Cytotoxicity Assays”, Tim Mosmann, Journal of Immunological Methods, 65 (1983) 55-63). Was used. That is, after the 48-hour culture, tetrazolium salt MTT was added, and further cultured for 4 hours. The amount of formazan produced through reduction by viable cells was considered to be proportional to the number of viable cells, and was quantified with an optical density (OD) of 570 nm.
[0069]
Cell viability was calculated by the following formula.
Cell viability (%)
= OD of test group [570 nm] / OD of control group [570 nm]
The obtained results are shown in FIG. The data shown in FIG. 5 is an average value of 3 wells.
[0070]
From the results of FIG. 5, it can be seen that KM043 suppresses the growth of cancer cells in a concentration-dependent manner. However, its cytostatic activity is low compared to known anticancer agents such as bleomycin.
[0071]
<Measurement Example 10>
The anticancer activity shown when KM043 obtained in Example 1 was used in combination with another anticancer agent was measured using the following method.
[0072]
i) Cell culture
Cell culture conditions were the same as those in Measurement Example 9 above.
ii) Addition of KM043 and Bleomycin
Medium (1) in which 50 μM KM043 (dissolved in the same manner as in Measurement Example 9 above) and bleomycin at a predetermined concentration were dissolved (1): “KM043 50 μM + Bleomycin test zone” and medium in which only a predetermined concentration of bleomycin was dissolved (2 ): “Bleomycin-only test section”).
[0073]
Three wells were given for each bleomycin concentration. 2.0 x 10 per well^Three Cells were implanted, followed by the addition of medium containing the drug.
For “KM043 50 μM + Bleomycin test group” and “Bleomycin-only test group”, 5% CO₂ Cell viability was measured after culturing at 37 ° C. for 48 hours in an incubator. (The measurement method is the same as the measurement example 9).
[0074]
The obtained result is shown in FIG. In FIG. 6, the black circle connected by a broken line indicates the result of the medium (1): “KM043 50 μM + bleomycin test group”, and the black square connected by the solid line indicates the result of the medium (2): “bleomycin-only test group”. Represents.
[0075]
From the results shown in FIG. 6, KM043 obtained in Example 1 has a low concentration (50 μM) as shown in Measurement Example 9, although the concentration is low enough to hardly affect the growth of cancer cells. It can be seen that when used in combination with bleomycin at a concentration, the growth of cancer cells is suppressed more strongly than with bleomycin alone.
[0076]
Currently, side effects are a problem in anticancer drug treatment, but it is considered that the combined use of KM043 obtained in Example 1 can maintain the same therapeutic effect while reducing the amount of anticancer drug.
[0077]
【The invention's effect】
According to the present invention, a novel glycolipid, its production method and its use are provided.
The glycolipid represented by the formula (I) of the present invention has a particularly high inhibitory action on β-type DNA synthase and has a non-antagonistic action mechanism.
[0078]
Since such a DNA synthase β is an enzyme involved in the root of the immune reaction, the glycolipid represented by the formula (I) having an inhibitory activity on the DNA synthase β is considered to act as an immunosuppressant. . Furthermore, since the mechanism of action is non-antagonistic, the glycolipid represented by formula (I) is expected to have fewer side effects on the living body.
[0079]
In addition, the glycolipid represented by the formula (I) has an HIV-derived reverse transcriptase inhibitory action.
Furthermore, the glycolipid represented by the formula (I) has a strong cancer cell growth inhibitory action particularly in combination with other anticancer agents.
[Brief description of the drawings]
FIG. 1 is a fast atom bombardment mass spectrometry spectrum of a glycolipid represented by the formula (I) of the present invention.
FIG. 2 shows the glycolipid represented by the formula (I) of the present invention.¹ H-NMR spectrum.
FIG. 3 is a UV spectrum of a glycolipid represented by the formula (I) of the present invention.
FIG. 4 is a gas chromatograph of a glycolipid represented by the formula (I) of the present invention.
FIG. 5 is a graph showing the cancer cell proliferation inhibitory action of the glycolipid represented by formula (I) of the present invention (alone).
FIG. 6 is a graph showing the cancer cell growth inhibitory action of the glycolipid represented by the formula (I) of the present invention (in combination).

Claims (2)

The following formula (I) from the red alga Suginori genus Gigartina Tenella

In a method for producing a glycolipid represented by
Extracting the red algae Sugiori Gengartina Tenella with one of an organic solvent or an organic solvent mixture selected from the group consisting of acetone, methanol and chloroform-methanol mixture;
Concentrating the organic solvent extract under reduced pressure;
Subjecting the extract concentrated under reduced pressure to a layer partition with ethyl acetate-water under acidic conditions, and separating the aqueous layer;
Subjecting the separated aqueous layer to layer partitioning with ethyl acetate under alkaline conditions and separating the ethyl acetate layer;
Concentrating the separated ethyl acetate layer under reduced pressure and recovering the resulting green crude powder;
Purifying the coarse powder;
And a step of concentrating the solution obtained in the purification step under reduced pressure and recovering the white powder obtained. The following formula (I)

The immunosuppressive agent which uses the glycolipid represented by this as an active ingredient.

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