JP2004168680A - Physiologically active substance nk33882p1, its production method and use - Google Patents
Physiologically active substance nk33882p1, its production method and use Download PDFInfo
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- JP2004168680A JP2004168680A JP2002333804A JP2002333804A JP2004168680A JP 2004168680 A JP2004168680 A JP 2004168680A JP 2002333804 A JP2002333804 A JP 2002333804A JP 2002333804 A JP2002333804 A JP 2002333804A JP 2004168680 A JP2004168680 A JP 2004168680A
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- JP
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- Prior art keywords
- nk33882p1
- active substance
- physiologically active
- acceptable salt
- pharmacologically acceptable
- Prior art date
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- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 12
- HEDRZPFGACZZDS-MICDWDOJSA-N Trichloro(2H)methane Chemical compound [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 claims description 12
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Abstract
Description
【０００１】
【発明の属する技術分野】
本発明は、生理活性物質ＮＫ３３８８２Ｐ１、その製造法及びその用途に関する。本発明のＮＫ３３８８２Ｐ１は、細胞増殖抑制作用を示し、悪性腫瘍に対する治療薬として使用される抗癌物質として期待される。
【０００２】
【従来の技術】
従来、抗癌抗生物質としては、アドリアマイシン等のアントラサイクリン類、ブレオマイシン等のブレオマイシン類、マイトマイシンＣ、アクチノマイシンＤ、ネオカルチノスタチン等が知られていた。（非特許文献１）
【０００３】
【非特許文献１】
龍原 徹著、ポケット医薬品集 ２００１年版、白文社、平成１３年１月発行、１２４頁〜１３３頁、
【０００４】
【発明が解決しようとする課題】
しかし、これらは副作用の面や抗癌効果の面から満足すべきものではなく、抗癌作用を示す新規化合物が望まれていた。
【０００５】
【課題を解決するための手段】
本発明者等は、これらの課題を解決するために微生物の代謝産物について種々検索した結果、放線菌に属する一菌株が、哺乳動物の癌細胞に対する細胞増殖抑制作用を有する生理活性物質ＮＫ３３８８２Ｐ１を産生し、ＮＫ３３８８２Ｐ１及びその薬理学上許容される塩が癌細胞に対する細胞増殖抑制作用を有することを見出し、本発明を完成した。
【０００６】
即ち、本発明は、以下の（１）〜（７）に関する。
（１）下記の理化学的性質をもつ生理活性物質ＮＫ３３８８２Ｐ１、又はその薬理学上許容される塩。
１）分子式：Ｃ１７Ｈ１５ＮＯ５
２）分子量：３１３（ＥＳＩ法により測定した［Ｍ＋Ｎａ］＋＝３３６及び［Ｍ＋Ｈ］＋＝３１４）
３）水素核磁気共鳴スペクトル：重クロロホルム中、４００ＭＨｚで測定したスペクトルを図１に示す。
４）炭素核磁気共鳴スペクトル：重クロロホルム中、１００ＭＨｚで測定したスペクトルを図２に示す。
５）溶解性：メタノール、アセトン、酢酸エチル、クロロホルム、ジメチルスルホキシドに易溶。水に難溶。
（２）式（Ｉ）で表される生理活性物質ＮＫ３３８８２Ｐ１、又はその薬理学上許容される塩。
【０００７】
【化２】
（３）上記（１）又は（２）に記載の生理活性物質ＮＫ３３８８２Ｐ１あるいはその薬理学上許容される塩、及び医薬用添加剤とを含む医薬。
（４）上記（１）又は（２）に記載の生理活性物質ＮＫ３３８８２Ｐ１あるいはその薬理学上許容される塩、及び医薬用添加剤とを含む抗癌剤。
（５）上記（１）又は（２）に記載の生理活性物質ＮＫ３３８８２Ｐ１あるいはその薬理学上許容される塩、及び医薬用添加剤とを含む乳癌治療剤。
（６）ストレプトマイセス属に属し、上記（１）又は（２）に記載された生理活性物質ＮＫ３３８８２Ｐ１を生産する能力を有する微生物を、栄養培地にて培養し、培養物中に生理活性物質ＮＫ３３８８２Ｐ１を生成蓄積せしめ、これを採取することを特徴とする生理活性物質ＮＫ３３８８２Ｐ１の製造法。
（７）上記（１）又は（２）に記載の生理活性物質ＮＫ３３８８２Ｐ１を生産する能力を有する微生物。
【０００９】
【発明の実施の形態】
本発明において、生理活性物質ＮＫ３３８８２Ｐ１は、ストレプトマイセス属に属するその生産菌を培養し、該化合物を生成せしめ、この培養物より採取する事により得られる。
【００１０】
ＮＫ３３８８２Ｐ１は、上記の理化学的性質を有する。
又、ＮＫ３３８８２Ｐ１は下記の平面構造式（１）を有する。
【００１１】
【化３】
ＮＫ３３８８２Ｐ１の生産菌の代表的なものとして、例えば土壌より分離したストレプトマイセス・エスピー（Ｓｔｒｅｐｔｏｍｙｃｅｓ ｓｐ．）ＮＡ ３３８８２株が挙げられる。以下に、本菌株の菌学的性状を示す。
【００１３】
１．形態的性質
２７℃で２週間後に観察した結果、気菌糸は単純分岐し、その先端はらせん状で、輪生枝の形成は認められない。又、胞子嚢及び遊走子も認められない。胞子表面はとげ状で、胞子は楕円形で、大きさは０．５〜０．８×０．６〜１．２μｍである。又、１０個以上の連鎖をなして胞子が形成される。
【００１４】
２．各種培地における生育
各種培地上、２７℃、２週間後の生育状態を下記表１に示す。
ＬＬ−ジアミノピメリン酸である。
【００１７】
以上を要約すると、本菌株は細胞壁がＬＬ−ジアミノピメリン酸であり、又、メナキノンの種類は、主にＭＫ−９（Ｈ８）である。インタ−ナショナル・ストレプトマイセス属・プロジェクト（略称ＩＳＰ）の方法によれば、胞子形成菌糸の形態は、Ｓｅｃｔｉｏｎ Ｓｐｉｒａｌｓに属し胞子表面はとげ状で、成熟した菌糸の色は灰色系統（Ｇｒａｙ ｃｏｌｏｒ−ｓｅｒｉｅｓ）である。メラニン様色素を生産し、培地中に茶色味の可溶性色素を呈する。又、基生菌糸の色はうす黄からうす黄茶を呈する。炭素源としてはＬ−アラビノ−ス、Ｄ−キシロース、Ｄ−グルコ−ス、Ｄ−フラクトース、シュクロース、イノシトール、Ｌ−ラムノース、Ｄ−マンニトール、ラフィノースを利用する。
【００１８】
以上の性質をもとにア−ル・イ−・ブッファナン・アンド・エヌ・イ−・ギボンズ編、バ−ジ−ズ・マニュアル・オブ・デタミネ−ティブ・バクテリオロジ−（Ｂｅｒｇｅｙ‘ｓ Ｍａｎｕａｌ ｏｆ Ｄｅｔｅｒｍｉｎａｔｉｖｅ Ｂａｃｔｅｒｉｏｌｏｇｙ）第８版、１９７４年に従って検索を行った結果、上記ＮＡ ３３８８２株はストレプトマイセス属に属することが判明した。よって、本菌をストレプトマイセス・エスピー（Ｓｔｒｅｐｔｏｍｙｃｅｓ ｓｐ．）ＮＡ３３８８２と命名した。
該菌株は、独立行政法人 産業技術総合研究所特許生物寄託センターに、寄託番号ＦＥＲＭ Ｐ−１８８２５として寄託されている。
【００１９】
本発明に用いるストレプトマイセス属に属する菌株は他のストレプトマイセス属の菌株と同様、変異させることが可能である。例えば、紫外線、エックス線、薬品等を用いる人工的な変異手段でも容易に変異しうる。このように得られたどの様な変異株であっても本発明である生理活性物質ＮＫ３３８８２Ｐ１の生産能を有するものは、全て本発明に使用する事ができる。
【００２０】
本発明によりＮＫ３３８８２Ｐ１を製造するには、まず前記菌株を放線菌が利用し得る栄養物を含有する培地で好気的に培養する。栄養源としては、従来から放線菌の培養に利用されている公知のものが使用でき、例えば炭素源としては、グルコース、グリセリン、ガラクトース、水飴、デキストリン、シュクロース、でんぷん、糖蜜、動・植物油等を使用できる。又、窒素源としては、大豆粉、小麦、小麦胚芽、コーンスティープ・リカー、肉エキス、ペプトン、酵母エキス、硫酸アンモニウム、硝酸ソーダ、尿素等を単独か又は組み合わせて用いることができる。その他、必要に応じ、ナトリウム、コバルト、塩素、硫酸、燐酸、及びその他のイオンを生成することのできる無機塩類を添加することは有効である。又、菌の生育を助け、生理活性物質ＮＫ３３８８２Ｐ１の生産を促進するような有機物及び無機物を適当に添加することができる。
【００２１】
培養法としては、液体培養法、特に深部攪拌培養法が適している。培養に適当な温度は１５℃〜３７℃であるが、多くの場合、２６℃〜３０℃付近で培養する。生理活性物質ＮＫ３３８８２Ｐ１の生産は、培地や培養条件により異なるが、振盪培養、タンク培養とも通常１〜１０日の間でその蓄積が最高に達する。培養物中の生理活性物質ＮＫ３３８８２Ｐ１の蓄積量が最高になった時に、培養を停止し、培養液から目的物質を単離する。
【００２２】
生理活性物質ＮＫ３３８８２Ｐ１の培養液からの採取にあたっては、その性状を利用した通常の分離手段を適宜組み合わせて精製することができる。すなわち、培養液を遠心分離あるいはろ過によりろ液と菌体部に分離し、ろ液からはそれを、ダイアイオンＨＰ−２０（三菱化成製）等に吸着させメタノ−ル等の低級アルコールやアセトン等により溶出するか、あるいは酢酸エチル又は１−ブタノ−ル等の有機溶媒で抽出することにより、一方の菌体部からはそれをメタノ−ル等の低級アルコールやアセトン等で抽出することによりＮＫ３３８８２Ｐ１の画分を得ることができる。
上述の方法に加え、物質の採取に用いられる公知の方法、たとえば吸着クロマトグラフィー、ゲル濾過クロマトグラフィー、薄層クロマトグラフィーよりのかき取り、高速液体クロマトグラフィー等を適宜組み合わせ、あるいは繰り返すことによってＮＫ３３８８２Ｐ１を純粋に単離することができる。
【００２３】
本発明において薬理学上許容される塩とは、通常とり得る塩であれば特に限定されないが、アルカリ金属塩が好ましく、具体的にはナトリウム塩、カリウム塩等が挙げられる。
【００２４】
医薬品として使用する場合の製剤化及び投与方法は従来公知の種々の方法が適用できる。すなわち、投与方法としては例えば注射、経口又は直腸投与等が可能である。製剤形態としては、例えば注射剤、粉末剤、顆粒剤、錠剤、座剤又はカプセル剤等をとり得る。
【００２５】
製剤化の際にＮＫ３３８８２Ｐ１又はそれらの薬理学上許容される塩に悪影響を与えない限り、医薬用に用いられる種々の医薬用添加剤、すなわち、担体やその他の助剤、例えば安定剤、防腐剤、無痛化剤、乳化剤等が必要に応じて使用され得る。製剤において、ＮＫ３３８８２Ｐ１又はその薬理学上許容される塩の含量は製剤形態等により広範囲に変えることが可能であり、一般には０．０１〜１００％（重量）、好ましくは０．１〜７０％（重量）含有し、残りは通常医薬用に使用される添加剤からなる。
【００２６】
ＮＫ３３８８２Ｐ１あるいはその薬理学上許容される塩、及び医薬用添加剤とを含む医薬は、好ましくは抗癌剤として固型癌、例えば食道癌、胃癌、肝臓癌、肺癌、乳癌、膵臓癌、大腸癌、子宮癌等に使用される。特に好ましくは、乳癌治療剤として使用される。
ＮＫ３３８８２Ｐ１又はその薬理学上許容される塩の投与量は症状、投与方法等により異なるが、成人１人１日あたり０．０１〜８００ｍｇ程度である。
【００２７】
以下に試験例を挙げて、ＮＫ３３８８２Ｐ１の、哺乳動物の癌細胞に対する細胞増殖抑制作用について述べる。
【００２８】
試験例 細胞増殖抑制作用
１０％ ＧＦ２１（和光純薬社製）を添加したＤａｉｇｏ’ｓ Ｔ培地を用いて、ヒト乳癌細胞Ｔ−４７Ｄを、３７℃、５％ＣＯ２下で培養した。この細胞を、９６穴プレートへ播種し、１日間培養した後、ＮＫ３３８８２Ｐ１を添加した。同時にβ−エストラジオールを添加し、６日間処理した。増殖度は、メチレンブルー法により評価した。そのＩＣ５０値は２．９μｇ／ｍｌだった。
【００２９】
試験例から明らかな様に、本発明のＮＫ３３８８２Ｐ１は、哺乳動物の癌細胞に細胞増殖抑制作用を示す。
【００３０】
【実施例】
以下に本発明の実施例を示すが、これは単なる一例示であって何等、本発明を限定するものではなく、種々の変法が可能である。
【００３１】
実施例１ ＮＫ３３８８２Ｐ１の醗酵法による生産
（１）発酵
種母作成用の培地として、グリセロール２．０％、グルコース１．０％、ペプトン０．５％、酵母エキス（日本製薬社製）０．５％、アジプロン（味の素社製）０．５％、燐酸水素二カリウム０．０５％、硫酸マグネシウム０．０５％、炭酸カルシウム０．２％、シリコン０．０１％から成る培地を用いた。なお、滅菌前の培地はｐＨ７．０に調整して使用した。
【００３２】
５００ｍｌ容三角フラスコに上記培地を１００ｍｌ分注し、１２０℃で２０分間滅菌し、これにストレプトマイセス・エスピー（Ｓｔｒｅｐｔｏｍｙｃｅｓ ｓｐ．）ＮＡ ３３８８２の斜面培養の１〜２白金耳を接種し、２５℃、２００回転／分の回転式振盪機にて２日間培養し、これを種母とした。
【００３３】
生産培養の培地として、グルコース１．０％、ガラクトース１．０％、デキストリン２．０％、コーン・スティープ・リカー１．０％、ファーマメディア１．０％、硫酸マグネシウム０．０５％、炭酸カルシウム０．２％、硫酸鉄０．０００１１％、硫酸銅０．０００６４％、塩化マンガン０．０００１５％、硫酸亜鉛０．０００７９％、塩化コバルト０．０００１％から成る培地を用いた。なお、滅菌前の培地はｐＨ７．０に調整して使用した。
【００３４】
上記培地１００ｍｌを含む３００本の５００ｍｌ容三角フラスコに、接種量が１％となるように接種し、５日間培養を行った。培養は同一条件下で３回行った。培養終了後、培養液に塩酸を加えｐＨ２に調整した後、遠心分離し上清液を各々３０リットル（計９０リットル）を得た。
【００３５】
（２）精製
この各々の遠心上清液を等量の酢酸エチルで抽出した。得られた酢酸エチル層を濃縮乾固しＮＫ３３８８２Ｐ１を含む画分をそれぞれ２３．２ｇ、１８．５ｇ、１７．５ｇ得た。
【００３６】
まず初めの培養で得られたＮＫ３３８８２Ｐ１を含む濃縮乾固物２３．２ｇをヘキサン−アセトン（１０：１）の混合溶媒に溶解後、あらかじめ同混合溶媒で充填したシリカゲルカラム（シリカゲル６０、メルク社製）１２００ｍｌ（φ４．７×５０ｃｍ）のカラムにかけ、同混合溶媒を３．３リットル、２：１の混合溶媒を３リットル流した後、１：１の混合溶媒３リットルで溶出、分画した。溶出した分画のうち、ＮＫ３３８８２Ｐ１を多く含む画分を濃縮乾固し、ＮＫ３３８８２Ｐ１を多く含む画分２６２０ｍｇを得た。
【００３７】
同様に２回めと３回めの培養で得られたＮＫ３３８８２Ｐ１を含む濃縮乾固物を合わせた３６ｇをヘキサン−アセトン（１０：１）の混合溶媒に溶解後、あらかじめ同混合溶媒で充填したシリカゲルカラム（シリカゲル６０、メルク社製）１６００ｍｌ（φ６．４×５０ｃｍ）のカラムにかけ、同混合溶媒を４．４リットル、２：１の混合溶媒を４リットル流した後、１：１の混合溶媒４リットルで溶出、分画した。溶出した分画のうち、ＮＫ３３８８２Ｐ１を多く含む画分を濃縮乾固し、ＮＫ３３８８２Ｐ１を多く含む画分３７７０ｍｇを得た。
【００３８】
次いでＮＫ３３８８２Ｐ１を多く含む画分２６２０ｍｇ、３７７０ｍｇをそれぞれ同一条件の中圧カラムクロマトマトグラフィーで精製した。即ち逆相カラムのＰＥＧＡＳＩＬ ＰＲＥＰ ＯＤＳ−５０１５−１２Ａ（φ３４×１５０ｍｍ、センシュー科学社製）を用い、２０％アセトニトリル水溶液１．５リットルで溶出した後、２０％アセトニトリル水溶液から８０％アセトニトリル水溶液でリニアグラジエント勾配をかけ、２．０リットル溶出した。溶出した分画のうちＮＫ３３８８２Ｐ１を多く含む画分を濃縮乾固し、それぞれ２３ｍｇ、４４ｍｇを得た。
【００３９】
更にＮＫ３３８８２Ｐ１を多く含む画分２３ｍｇと４４ｍｇをあわせた６７ｍｇを逆相高速液体クロマトグラフィーにて精製した。即ち、逆相カラムのＰＥＧＡＳＩＬ ＯＤＳ（φ１０×２５０ｍｍ、センシュー科学社製）を用い、流速７ｍｌ／分、３０％アセトニトリル水溶液で６０分間溶出した。ＮＫ３３８８２Ｐ１は、保持時間１４分から１５分に溶出された。溶出した分画のうち、ＮＫ３３８８２Ｐ１を多く含む画分をアセトニトリル留去後、同量の酢酸エチルで２回抽出した。酢酸エチル層を濃縮乾固し、純粋なＮＫ３３８８２Ｐ１（３．３ｍｇ）を得た。
【発明の効果】
本発明により、細胞増殖抑制作用を示す新規な生理活性物質ＮＫ３３８８２Ｐ１又はその薬理学上許容される塩が提供された。これらは、悪性腫瘍に対する治療薬等として利用できる。
【００４０】
【図面の簡単な説明】
【図１】ＮＫ３３８８２Ｐ１の重クロロホルム中で測定した４００ＭＨｚ水素核磁気共鳴スペクトルを示す。
【図２】ＮＫ３３８８２Ｐ１の重クロロホルム中で測定した１００ＭＨｚ炭素核磁気共鳴スペクトルを示す。[0001]
TECHNICAL FIELD OF THE INVENTION
TECHNICAL FIELD The present invention relates to a physiologically active substance NK33882P1, a method for producing the same, and its use. The NK33882P1 of the present invention exhibits a cell growth inhibitory effect and is expected as an anticancer substance used as a therapeutic agent for malignant tumors.
[0002]
[Prior art]
Conventionally, as anticancer antibiotics, anthracyclines such as adriamycin, bleomycins such as bleomycin, mitomycin C, actinomycin D, neocarzinostatin and the like have been known. (Non-Patent Document 1)
[0003]
[Non-patent document 1]
Toru Tatsuhara, Pocket Medicines 2001 Edition, Hakubunsha, published January 2001, pp. 124-133,
[0004]
[Problems to be solved by the invention]
However, these are not satisfactory in terms of side effects and anticancer effects, and new compounds exhibiting anticancer effects have been desired.
[0005]
[Means for Solving the Problems]
The present inventors have conducted various searches for metabolites of microorganisms in order to solve these problems, and as a result, one strain belonging to actinomycetes produced a physiologically active substance NK33882P1 having a cell growth inhibitory effect on cancer cells of mammals. However, they have found that NK33882P1 and a pharmacologically acceptable salt thereof have a cell growth inhibitory effect on cancer cells, and thus completed the present invention.
[0006]
That is, the present invention relates to the following (1) to (7).
(1) A physiologically active substance NK33882P1 having the following physicochemical properties, or a pharmacologically acceptable salt thereof.
1) Molecular formula: C 17 H 15 NO 5
2) Molecular weight: 313 ([M + Na] + = 336 and [M + H] + = 314 measured by ESI method)
3) Hydrogen nuclear magnetic resonance spectrum: FIG. 1 shows a spectrum measured at 400 MHz in deuterated chloroform.
4) Carbon nuclear magnetic resonance spectrum: FIG. 2 shows a spectrum measured at 100 MHz in deuterated chloroform.
5) Solubility: easily soluble in methanol, acetone, ethyl acetate, chloroform and dimethyl sulfoxide. Poorly soluble in water.
(2) NK33882P1, a physiologically active substance represented by the formula (I), or a pharmacologically acceptable salt thereof.
[0007]
Embedded image
(3) A medicine comprising the physiologically active substance NK33882P1 or the pharmaceutically acceptable salt thereof according to the above (1) or (2), and a pharmaceutical additive.
(4) An anticancer agent comprising the physiologically active substance NK33882P1 or the pharmaceutically acceptable salt thereof according to the above (1) or (2), and a pharmaceutical additive.
(5) A therapeutic agent for breast cancer comprising the physiologically active substance NK33882P1 or the pharmaceutically acceptable salt thereof according to the above (1) or (2), and a pharmaceutical additive.
(6) A microorganism belonging to the genus Streptomyces and capable of producing the physiologically active substance NK33882P1 described in (1) or (2) above is cultured in a nutrient medium, and the physiologically active substance NK33882P1 is contained in the culture. A method for producing a physiologically active substance NK33882P1 characterized by producing and accumulating NK33882P1.
(7) A microorganism having the ability to produce the physiologically active substance NK33882P1 according to (1) or (2).
[0009]
BEST MODE FOR CARRYING OUT THE INVENTION
In the present invention, the physiologically active substance NK33882P1 is obtained by culturing a producing bacterium belonging to the genus Streptomyces, producing the compound, and collecting the compound from the culture.
[0010]
NK33882P1 has the above physicochemical properties.
NK33882P1 has the following planar structural formula (1).
[0011]
Embedded image
A typical example of a NK33882P1 producing bacterium is Streptomyces sp. NA 33882 strain isolated from soil. The bacteriological properties of this strain are shown below.
[0013]
1. Morphological properties Observation after 2 weeks at 27 ° C. shows that the aerial mycelium is simply branched, its tip is spiral, and no ring-shaped branches are formed. Neither sporangia nor zoospores are observed. The spore surface is spiny, the spores are elliptical, and the size is 0.5-0.8 × 0.6-1.2 μm. Also, spores are formed in a chain of 10 or more.
[0014]
2. Growth in Various Media The growth status of each medium at 27 ° C. for 2 weeks is shown in Table 1 below.
[0017]
In summary, this strain has a cell wall of LL-diaminopimelic acid, and the type of menaquinone is mainly MK-9 (H 8 ). According to the method of the International Streptomyces genus project (abbreviated as ISP), the spore-forming hypha belongs to Section Spirals, the spore surface is spiny, and the mature hypha has a gray color (Gray color-). series). Produces a melanin-like pigment and presents a brown soluble pigment in the medium. In addition, the color of the underlying mycelium exhibits light yellow to light yellow brown. L-arabinose, D-xylose, D-glucose, D-fructose, sucrose, inositol, L-rhamnose, D-mannitol and raffinose are used as carbon sources.
[0018]
Bergey's Manual of Determinative, based on the above properties, edited by Ally Buffanand and N. Gibbons, Versions of Manual of Deterministic Bacteriology. As a result of conducting a search according to Bacteriology, 8th edition, 1974, it was found that the NA 33882 strain belongs to the genus Streptomyces. Therefore, this bacterium was named Streptomyces sp. NA33882.
The strain has been deposited at the National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary under the deposit number FERM P-18825.
[0019]
The strain belonging to the genus Streptomyces used in the present invention can be mutated similarly to other strains belonging to the genus Streptomyces. For example, mutation can be easily performed by artificial mutation using ultraviolet rays, X-rays, chemicals, or the like. Regardless of the thus obtained mutants, any of the mutants capable of producing the physiologically active substance NK33882P1 of the present invention can be used in the present invention.
[0020]
In order to produce NK33882P1 according to the present invention, the above strain is first aerobically cultured in a medium containing nutrients that can be used by actinomycetes. As the nutrient source, known ones conventionally used for cultivation of actinomycetes can be used. For example, as the carbon source, glucose, glycerin, galactose, starch syrup, dextrin, sucrose, starch, molasses, animal and vegetable oil, etc. Can be used. As the nitrogen source, soybean flour, wheat, wheat germ, corn steep liquor, meat extract, peptone, yeast extract, ammonium sulfate, sodium nitrate, urea and the like can be used alone or in combination. In addition, if necessary, it is effective to add sodium, cobalt, chlorine, sulfuric acid, phosphoric acid, and other inorganic salts capable of generating ions. In addition, organic substances and inorganic substances that assist the growth of bacteria and promote the production of the physiologically active substance NK33882P1 can be appropriately added.
[0021]
As a culture method, a liquid culture method, particularly a submerged stirring culture method, is suitable. A suitable temperature for the culture is 15 ° C to 37 ° C, but in many cases, the culture is performed at around 26 ° C to 30 ° C. The production of the physiologically active substance NK33882P1 varies depending on the culture medium and culture conditions, but usually the highest accumulation occurs in shaking culture and tank culture in 1 to 10 days. When the accumulated amount of the physiologically active substance NK33882P1 in the culture reaches the maximum, the culture is stopped and the target substance is isolated from the culture solution.
[0022]
When the physiologically active substance NK33882P1 is collected from the culture solution, it can be purified by appropriately combining ordinary separation means utilizing its properties. That is, the culture solution is separated into a filtrate and cells by centrifugation or filtration, and the filtrate is adsorbed on Diaion HP-20 (manufactured by Mitsubishi Chemical) or the like, and a lower alcohol such as methanol or acetone is absorbed. Or by extraction with an organic solvent such as ethyl acetate or 1-butanol, and then extracting it from one of the bacterial cells with a lower alcohol such as methanol or acetone. Can be obtained.
In addition to the above-mentioned methods, NK33882P1 can be obtained by appropriately combining or repeating known methods used for collecting a substance, for example, adsorption chromatography, gel filtration chromatography, scraping from thin-layer chromatography, high-performance liquid chromatography, and the like. It can be isolated purely.
[0023]
In the present invention, the pharmacologically acceptable salt is not particularly limited as long as it is a commonly available salt, but an alkali metal salt is preferable, and specific examples include a sodium salt and a potassium salt.
[0024]
Various methods known in the art can be applied to the preparation and administration methods for use as pharmaceuticals. That is, as the administration method, for example, injection, oral or rectal administration, etc. are possible. The formulation may be, for example, injections, powders, granules, tablets, suppositories or capsules.
[0025]
Various pharmaceutical additives used for medicine, ie, carriers and other auxiliaries, such as stabilizers and preservatives, as long as they do not adversely affect NK33882P1 or a pharmacologically acceptable salt thereof during formulation. A soothing agent, an emulsifier and the like can be used as needed. In the preparation, the content of NK33882P1 or a pharmacologically acceptable salt thereof can vary widely depending on the preparation form and the like, and is generally 0.01 to 100% (weight), preferably 0.1 to 70% ( Weight), with the balance consisting of additives normally used for pharmaceuticals.
[0026]
A medicine containing NK33882P1 or a pharmacologically acceptable salt thereof and a pharmaceutical additive is preferably a solid cancer as an anticancer agent, for example, esophageal cancer, stomach cancer, liver cancer, lung cancer, breast cancer, pancreatic cancer, colon cancer, uterus Used for cancer and the like. Particularly preferably, it is used as a therapeutic agent for breast cancer.
The dose of NK33882P1 or a pharmacologically acceptable salt thereof varies depending on symptoms, administration method, and the like, but is about 0.01 to 800 mg per adult per day.
[0027]
The cell growth inhibitory effect of NK33882P1 on mammalian cancer cells will be described below with reference to test examples.
[0028]
Test Example Human breast cancer cells T-47D were cultured at 37 ° C. in 5% CO 2 using Daigo's T medium supplemented with 10% GF21 (manufactured by Wako Pure Chemical Industries, Ltd.). The cells were seeded on a 96-well plate, cultured for one day, and then NK33882P1 was added. At the same time, β-estradiol was added and treated for 6 days. The degree of proliferation was evaluated by the methylene blue method. Its IC 50 value was 2.9 μg / ml.
[0029]
As is clear from the test examples, NK33882P1 of the present invention has a cell growth inhibitory effect on mammalian cancer cells.
[0030]
【Example】
Examples of the present invention will be described below, but these are merely examples, and do not limit the present invention in any way, and various modifications are possible.
[0031]
Example 1 Production of NK33882P1 by Fermentation Method (1) Glycerol 2.0%, glucose 1.0%, peptone 0.5%, yeast extract (manufactured by Nippon Pharmaceutical Co.) 0.5 %, Adipron (manufactured by Ajinomoto Co.) 0.5%, dipotassium hydrogen phosphate 0.05%, magnesium sulfate 0.05%, calcium carbonate 0.2%, and silicon 0.01%. The medium before sterilization was adjusted to pH 7.0 before use.
[0032]
100 ml of the above medium was dispensed into a 500 ml Erlenmeyer flask, sterilized at 120 ° C. for 20 minutes, and inoculated with 1 to 2 loops of a slope culture of Streptomyces sp. NA 33882. The culture was carried out for 2 days on a rotary shaker at 200 rpm, and this was used as a seed.
[0033]
As a culture medium for production culture, glucose 1.0%, galactose 1.0%, dextrin 2.0%, corn steep liquor 1.0%, pharma media 1.0%, magnesium sulfate 0.05%, calcium carbonate A medium consisting of 0.2%, iron sulfate 0.00011%, copper sulfate 0.00064%, manganese chloride 0.00015%, zinc sulfate 0.00079%, and cobalt chloride 0.0001% was used. The medium before sterilization was adjusted to pH 7.0 before use.
[0034]
300 500 ml Erlenmeyer flasks containing 100 ml of the above medium were inoculated so that the inoculum amount became 1%, and cultivation was performed for 5 days. Culture was performed three times under the same conditions. After completion of the culture, hydrochloric acid was added to the culture to adjust the pH to 2, and then centrifuged to obtain 30 liters of each supernatant liquid (a total of 90 liters).
[0035]
(2) Purification Each centrifugal supernatant was extracted with an equal volume of ethyl acetate. The obtained ethyl acetate layer was concentrated and dried to obtain 23.2 g, 18.5 g, and 17.5 g of fractions containing NK33882P1.
[0036]
First, 23.2 g of a concentrated and dried product containing NK33882P1 obtained in the first culture was dissolved in a mixed solvent of hexane-acetone (10: 1), and then filled in advance with a silica gel column (
[0037]
Similarly, 36 g of the concentrated and dried product containing NK33882P1 obtained in the second and third cultures was dissolved in a mixed solvent of hexane-acetone (10: 1), and silica gel previously filled with the same mixed solvent was used. A column (
[0038]
Next, 2620 mg and 3770 mg of the fractions rich in NK33882P1 were each purified by medium pressure column chromatography under the same conditions. That is, using a reverse phase column PEGASIL PREP ODS-5015-12A (φ34 × 150 mm, manufactured by Senshu Kagaku), elution was performed with 1.5 liter of a 20% acetonitrile aqueous solution, and then a linear gradient was performed with a 20% acetonitrile aqueous solution to an 80% acetonitrile aqueous solution. A gradient was applied and 2.0 liters eluted. Among the eluted fractions, the fraction containing a large amount of NK33882P1 was concentrated and dried to obtain 23 mg and 44 mg, respectively.
[0039]
67 mg of 23 mg and 44 mg of the fraction containing a large amount of NK33882P1 was purified by reversed-phase high performance liquid chromatography. That is, elution was carried out with a 30% aqueous solution of acetonitrile for 60 minutes at a flow rate of 7 ml / min using a reverse-phase column PEGASIL ODS (φ10 × 250 mm, manufactured by Senshu Kagaku). NK33882P1 was eluted with a retention time of 14 to 15 minutes. Among the eluted fractions, the fraction containing a large amount of NK33882P1 was distilled off with acetonitrile and then extracted twice with the same amount of ethyl acetate. The ethyl acetate layer was concentrated to dryness to obtain pure NK33882P1 (3.3 mg).
【The invention's effect】
According to the present invention, there is provided a novel physiologically active substance NK33882P1 or a pharmacologically acceptable salt thereof, which exhibits a cell growth inhibitory action. These can be used as therapeutic agents for malignant tumors and the like.
[0040]
[Brief description of the drawings]
FIG. 1 shows a 400 MHz hydrogen nuclear magnetic resonance spectrum of NK33882P1 measured in deuterated chloroform.
FIG. 2 shows a 100 MHz carbon nuclear magnetic resonance spectrum of NK33882P1 measured in deuterated chloroform.
Claims (7)
１）分子式：Ｃ１７Ｈ１５ＮＯ５
２）分子量：３１３（ＥＳＩ法により測定した［Ｍ＋Ｎａ］＋＝３３６及び［Ｍ＋Ｈ］＋＝３１４）
３）水素核磁気共鳴スペクトル：重クロロホルム中、４００ＭＨｚで測定したスペクトルを図１に示す。
４）炭素核磁気共鳴スペクトル：重クロロホルム中、１００ＭＨｚで測定したスペクトルを図２に示す。
５）溶解性：メタノール、アセトン、酢酸エチル、クロロホルム、ジメチルスルホキシドに易溶。水に難溶。A physiologically active substance NK33882P1 having the following physicochemical properties, or a pharmacologically acceptable salt thereof.
1) Molecular formula: C 17 H 15 NO 5
2) Molecular weight: 313 ([M + Na] + = 336 and [M + H] + = 314 measured by ESI method)
3) Hydrogen nuclear magnetic resonance spectrum: FIG. 1 shows a spectrum measured at 400 MHz in deuterated chloroform.
4) Carbon nuclear magnetic resonance spectrum: FIG. 2 shows a spectrum measured at 100 MHz in deuterated chloroform.
5) Solubility: easily soluble in methanol, acetone, ethyl acetate, chloroform and dimethyl sulfoxide. Poorly soluble in water.
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