JP2005225836A - Hepatocyte growth promoter and therapeutic medicine for liver disease - Google Patents
Hepatocyte growth promoter and therapeutic medicine for liver disease Download PDFInfo
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本発明は、高い肝実質細胞増殖促進作用を有する肝実質細胞増殖促進剤とそれを必須成分として含む肝疾患治療薬に関する。 The present invention relates to a hepatocyte proliferation promoter having a high hepatocyte proliferation promoting effect and a therapeutic agent for liver diseases comprising the same as an essential component.
グリチルリチンは、マメ科の植物（Glycyrrhiza glabra）の根から単離・精製され、生薬の甘草の有効成分の一つとして知られている。その構造は１８位の水素がβ配位をとるステロイドに類似した基本骨格をもち、Ｃ−３位に２つの糖が結合した配糖体である。
グリチルリチンとそのいくつかの誘導体は、多くの薬理作用、例えば鎮痙、鎮痛、抗炎症、抗消化性潰瘍作用などを有することが知られている。また、そのアグリコンであるグリチルリチン酸にも、抗炎症、抗アレルギー、高脂血症改善作用などの種々の薬理作用があることが知られている（非特許文献１参照。）。
本邦において、グリチルリチン製剤は、肝炎（hepatitis）や肝硬変（hepatic cirrhosis）などの慢性肝疾患に対する治療薬（肝庇護薬）として、２０数年来、臨床的に使用されている。また、ヨーロッパにおいても、慢性Ｃ型肝炎患者に対するグリチルリチン製剤の有効性と安全性が臨床的に評価されており、静脈内投与で慢性Ｃ型肝炎患者の血清アラニンアミノトランスフェラーゼ活性の亢進が減少することが報告されている（非特許文献２参照。）。
Glycyrrhizin is isolated and purified from the roots of leguminous plants (Glycyrrhiza glabra) and is known as one of the active ingredients of herbal licorice. The structure is a glycoside having a basic skeleton similar to a steroid in which hydrogen at the 18-position takes a β-coordinate, and two sugars bonded to the C-3 position.
Glycyrrhizin and some of its derivatives are known to have many pharmacological actions such as antispasmodic, analgesic, anti-inflammatory, anti-peptic ulcer effects and the like. In addition, glycyrrhizic acid, which is an aglycon, is known to have various pharmacological actions such as anti-inflammatory, anti-allergic, and hyperlipidemia improving actions (see Non-Patent Document 1).
In Japan, glycyrrhizin preparations have been used clinically for more than 20 years as therapeutic agents (hepatic protection drugs) for chronic liver diseases such as hepatitis and hepatic cirrhosis. In Europe, the efficacy and safety of glycyrrhizin preparations for chronic hepatitis C patients are clinically evaluated, and the increase in serum alanine aminotransferase activity in chronic hepatitis C patients is reduced by intravenous administration. Has been reported (see Non-Patent Document 2).
グリチルリチン製剤の主な薬理効果は、その抗炎症作用にあるものと考えられているが、肝炎などの主な炎症部位である肝実質細胞に対する増殖促進作用の有無については、有効なモデル実験系がなかったこともあり、詳しく検討した報告は極めて少なかった。
最近、グリチルリチンとそのいくつかの誘導体がラット肝実質細胞の増殖にどのような影響を与えるかについて、ラットの初代培養肝実質細胞系（ｉｎ ｖｉｔｒｏ実験系）を用いた検討がなされ、その結果、グリチルリチンと一部の誘導体は、肝実質細胞の上皮増殖因子（ＥＧＦ）受容体を直接的に刺激し、その増殖を促進させるＥＧＦ様の増殖促進作用を有することが見出された（非特許文献３参照）。
Recently, the effect of glycyrrhizin and some of its derivatives on the proliferation of rat liver parenchymal cells has been studied using the rat primary hepatocyte culture system (in vitro experimental system). It has been found that glycyrrhizin and some derivatives have an EGF-like growth-promoting action that directly stimulates and promotes the growth of epithelial growth factor (EGF) receptors in hepatocytes. 3).
本発明は、肝実質細胞の増殖促進作用が従来公知のグリチルリチンやその誘導体よりも高く、肝疾患の治療薬として有効であり、肝疾患によりダメージを受けた肝臓の再生促進や生体肝移植後の肝臓再生などに有効な肝実質細胞増殖促進剤とそれを必須成分として含む肝疾患治療薬の提供を目的とする。 The present invention has a liver parenchymal cell growth promoting action higher than that of conventionally known glycyrrhizin and its derivatives, and is effective as a therapeutic agent for liver disease. An object of the present invention is to provide a liver parenchymal cell growth promoter effective for liver regeneration and the like, and a therapeutic agent for liver disease containing it as an essential component.
本発明者らは、グリチルリチンをリード化合物とした幾つかの新規化合物について、前述した非特許文献３におけるグリチルリチンと同様な肝実質細胞の増殖に促進的な影響を与えることができるか否かを、ラット初代培養肝実質細胞系で検討した。その結果、従来公知のグリチルリチンやその誘導体よりも肝実質細胞増殖促進効果が高く、肝疾患治療薬などとして有効に使用することができる化合物を見出し、本発明を完成させた。
The present inventors, for some novel compounds using glycyrrhizin as a lead compound, whether or not it can have a proliferative effect on the proliferation of hepatocytes similar to glycyrrhizin in Non-Patent
前記目的を達成するため、本発明は、式（１） To achieve the above object, the present invention provides the following formula (1).
（式中、Ｒ１は水素原子を表し、Ｒ２はメチル基を表す。）
で表される肝実質細胞増殖促進剤を提供する。
また本発明は、前述した本発明に係る肝実質細胞増殖促進剤を有効成分として含むことを特徴とする肝疾患治療薬を提供する。
(In the formula, R 1 represents a hydrogen atom and R 2 represents a methyl group.)
The hepatocyte growth promoter represented by these is provided.
The present invention also provides a therapeutic agent for liver disease comprising the aforementioned liver parenchymal cell growth promoter according to the present invention as an active ingredient.
本発明によれば、肝実質細胞の増殖促進作用が従来公知のグリチルリチンやその誘導体よりも高く、肝疾患の治療薬として有効であり、肝疾患によりダメージを受けた肝臓の再生促進や生体肝移植後の肝臓再生などに有効な肝実質細胞増殖促進剤及びそれを有効成分として含む肝疾患治療薬を提供することができる。 According to the present invention, the liver parenchymal cell proliferation promoting action is higher than that of conventionally known glycyrrhizin and its derivatives, and is effective as a therapeutic agent for liver disease, promoting regeneration of liver damaged by liver disease and living liver transplantation. It is possible to provide a liver parenchymal cell growth promoter effective for later liver regeneration and a therapeutic agent for liver disease containing the same as an active ingredient.
本発明に係る肝実質細胞増殖促進剤は、前記式（１）で表され、従来公知のグリチルリチンのモノメチルエステルに相当する。ただし、本発明の肝実質細胞増殖促進剤は、式（１）においてＲ２で表される部分、すなわち、アグリコンのＣ−３位から近傍にある２番目の糖のカルボキシル基にメチル基がエステル結合したものである。後述するが、本発明者らが行った初代培養肝実質細胞の増殖に関する実験の結果、式（１）においてＲ１、Ｒ２の一方及び両方がメチルエステル化された各化合物の中で、本発明に係る化合物（式（１）においてＲ１が水素原子、Ｒ２がメチル基であるグリチルリチンモノメチルエステル）のみが、グリチルリチン（ＧＬ）よりも高い肝細胞の増殖促進作用を示すことが見出されている。 The hepatocyte growth promoter according to the present invention is represented by the formula (1) and corresponds to a conventionally known monomethyl ester of glycyrrhizin. However, the liver parenchymal cell growth promoter of the present invention is a compound in which a methyl group is esterified to the moiety represented by R 2 in formula (1), that is, the carboxyl group of the second sugar in the vicinity from the C-3 position of aglycone. It is a combination. As will be described later, as a result of experiments on the proliferation of primary cultured hepatocytes performed by the present inventors, among the compounds in which one or both of R 1 and R 2 in formula (1) are methyl esterified, It was found that only the compound according to the invention (glycyrrhizin monomethyl ester in which R 1 is a hydrogen atom and R 2 is a methyl group in formula (1)) exhibits a higher hepatocyte proliferation promoting effect than glycyrrhizin (GL). ing.
本発明に係る肝実質細胞増殖促進剤は、市販のグリチルリチンのメチルエステル化により容易に合成することができる。例えば、適当量のグリチルリチンとメタノールを混合し、塩酸や硫酸などの酸触媒を加え、室温又は加温下で撹拌してエステル化反応を行い、反応後、分離、精製して製造することができる。エステル化反応により、式（１）においてＲ１、Ｒ２の一方及び両方がメチルエステル化された各化合物の混合物が生じる。本発明に係る肝実質細胞増殖促進剤は、これらの化合物が混合された状態で提供することもできるが、各化合物の混合物から、液体クロマトグラフィーなどの従来公知の分離精製手段を用い、本発明に係る化合物を濃縮又は純化して用いることが望ましい。 The hepatocyte growth promoter according to the present invention can be easily synthesized by methyl esterification of commercially available glycyrrhizin. For example, it can be produced by mixing an appropriate amount of glycyrrhizin and methanol, adding an acid catalyst such as hydrochloric acid or sulfuric acid, stirring at room temperature or warming, carrying out an esterification reaction, separating and purifying after the reaction. . The esterification reaction produces a mixture of compounds in which one or both of R 1 and R 2 are methyl esterified in formula (1). The hepatocyte growth promoter according to the present invention can be provided in a state where these compounds are mixed, but the present invention can be provided by using a conventionally known separation and purification means such as liquid chromatography from the mixture of each compound. It is desirable to concentrate or purify the compound according to the above.
本発明に係る肝実質細胞増殖促進剤は、カリウム、ナトリウムなどの金属イオン等と薬理的に許容される塩を形成し得る。本発明の肝実質細胞増殖促進剤は、このような薬理学的に許容される塩の形態となった化合物をも包含する。 The hepatocyte growth promoter according to the present invention can form pharmacologically acceptable salts with metal ions such as potassium and sodium. The hepatocyte growth promoter of the present invention also includes a compound in the form of such a pharmacologically acceptable salt.
本発明に係る肝実質細胞増殖促進剤は、肝実質細胞の増殖促進作用が従来公知のグリチルリチンやその誘導体よりも高く、肝疾患の治療薬として有効であり、肝疾患によりダメージを受けた肝臓の再生促進や生体肝移植後の肝臓再生などに有効利用することができる。 The liver parenchymal cell proliferation promoter according to the present invention has a liver parenchymal cell proliferation promoting action higher than that of conventionally known glycyrrhizin and its derivatives, and is effective as a therapeutic agent for liver disease. It can be used effectively for promoting regeneration and liver regeneration after living-donor liver transplantation.
また本発明に係る肝疾患治療薬は、前記本発明に係る肝実質細胞増殖促進剤を必須の成分として含むことを特徴としている。本発明の肝疾患治療薬は、常法により錠剤、散剤、カプセル剤、顆粒剤、細粒剤、注射剤、吸入剤等の製剤形態、または軟膏、塗布液等の皮膚外用剤等の製剤とすることができ、経口または非経口投与により肝疾患治療として臨床に供し得る。投与量は治療するべき症状及び投与方法により左右されるが、通常は、成人１日あたり１μｇから１０ｇを単一投与または１日数回に分けて投与することができる。 The therapeutic agent for liver disease according to the present invention is characterized by containing the above-mentioned hepatocyte growth promoter according to the present invention as an essential component. The therapeutic agent for liver disease of the present invention can be prepared by conventional methods such as tablets, powders, capsules, granules, fine granules, injections, inhalants and the like, or preparations such as ointments and skin external preparations such as coating solutions. It can be used clinically as a treatment for liver disease by oral or parenteral administration. The dose depends on the symptoms to be treated and the method of administration, but usually 1 μg to 10 g per day for an adult can be administered as a single dose or divided into several times a day.
前記のように経口投与の製剤形態としては、錠剤、散剤、カプセル剤、顆粒剤、細粒剤などがあり、これらの製剤には製薬上許容される賦形剤として、澱粉、乳糖、マンニット、エチルセルロース、ナトリウムカルボキシメチルセルロース、ヒドロキシプロピルセルロース等が配合され、滑沢剤としてステアリン酸マグネシウムまたはステアリン酸カルシウムが添加される。またゼラチン、アラビアゴム、セルロースエステル、ポリビニルピロリドン等を結合剤として用い得る。注射剤、吸入剤、外用剤等の非経口のための製剤としては無菌の水性または非水性の溶剤、または乳濁剤が挙げられる。非水性の溶液剤または懸濁剤の基剤としてはプロピレングリコール、ポリエチレングリコール、グリセリン、オリーブ油、コーン油、オレイン酸エチル等が用いられる。一方、坐剤の基剤としてはカカオ脂、マクロゴール等を用いることができる。 As mentioned above, the dosage forms for oral administration include tablets, powders, capsules, granules, fine granules and the like, and pharmaceutically acceptable excipients for these preparations include starch, lactose, mannitol. Ethyl cellulose, sodium carboxymethyl cellulose, hydroxypropyl cellulose and the like are blended, and magnesium stearate or calcium stearate is added as a lubricant. Gelatin, gum arabic, cellulose ester, polyvinyl pyrrolidone and the like can be used as a binder. Examples of parenteral preparations such as injections, inhalants, and external preparations include sterile aqueous or non-aqueous solvents or emulsions. As the base of the non-aqueous solution or suspension, propylene glycol, polyethylene glycol, glycerin, olive oil, corn oil, ethyl oleate and the like are used. On the other hand, cocoa butter, macrogol and the like can be used as a base for suppositories.
本発明の肝疾患治療薬は、肝実質細胞の増殖促進作用が高い本発明に係る肝実質細胞増殖促進剤を必須の成分として含むものなので、例えば、Ｃ型肝炎などの肝炎、肝硬変などの各種の肝疾患治療薬として有効であり、肝疾患によりダメージを受けた肝臓の再生促進を図ることができる。またこの肝疾患治療薬を生体肝移植後の患者に用いることにより、移植肝臓の成長促進を図ることができる。 The therapeutic agent for hepatic diseases of the present invention contains the hepatocyte proliferation promoter according to the present invention having a high hepatocyte proliferation promoting action as an essential component. It is effective as a therapeutic agent for liver diseases, and can promote the regeneration of liver damaged by liver diseases. Moreover, the growth of transplanted liver can be promoted by using this therapeutic agent for liver disease in a patient after living-related liver transplantation.
＜グリチルリチンメチルエステルの調製＞
グリチルリチン１．７ｇ（２．１ｍｍｏｌ）とメタノール４０ｍＬの混合物に、撹拌しながら１０％塩酸−メタノール溶液を０．８５ｍＬ加えた。反応液は室温で８時間撹拌させた後、炭酸銀を加えて中和させた。精製した沈殿物を濾過し、濾液を濃縮乾固させて１．７５ｇの反応混合物を得た。
反応混合物はＨＰＬＣ（カラム：Ｉｎｅｒｔｓｉｌ ＰＲＥＰ−ＯＤＳ，２０φ×２５０；溶媒：４５％アセトニトリル−水；検出：ＵＶ ２５４ｎｍ）を用いて分離精製し、次の（Ａ）〜（Ｃ）の化合物；
（Ａ） 式（１）においてＲ１がメチル基、Ｒ２が水素原子である化合物（グリチルリチンモノメチルエステル、以下、ＧＬ−ｉｎｎｅｒと記す。）２６０ｍｇ、
（Ｂ） 式（１）においてＲ１が水素原子、Ｒ２がメチル基である化合物（本発明に係るグリチルリチンモノメチルエステル、以下、ＧＬ−ｏｕｔｅｒと記す。）２４０ｍｇ、
（Ｃ） 式（１）においてＲ１とＲ２がともにメチル基である化合物（グリチルリチンジメチルエステル、以下、ＧＬ−ｄｉｍｅｔｈｙｌと記す。）２００ｍｇ、
をそれぞれ得た。
ＧＬ−ｉｎｎｅｒ ＭＳ；（ＥＳ+）：ｍ／ｚ ８３７［Ｍ+Ｈ］＋，６６１［（Ｍ+Ｈ）−Ｃ６Ｈ９Ｏ６］＋，４７１［（Ｍ+Ｈ）−Ｃ１３Ｈ１９Ｏ１２］＋
ＧＬ−ｏｕｔｅｒ ＭＳ；（ＥＳ+）：ｍ／ｚ ８３７［Ｍ+Ｈ］＋，６４６［（Ｍ+Ｈ）−Ｃ６Ｈ９Ｏ６］＋，４７１［（Ｍ+Ｈ）−Ｃ１３Ｈ１９Ｏ１２］＋
ＧＬ−ｄｉｍｅｔｈｙｌ ＭＳ；（ＥＳ+）：ｍ／ｚ ８５１［Ｍ+Ｈ］＋
なお、比較のために、グリチルリチン（以下、ＧＬと記す。）についても同様に試験を行い、結果を比較した。
<Preparation of glycyrrhizin methyl ester>
0.85 mL of 10% hydrochloric acid-methanol solution was added to a mixture of 1.7 g (2.1 mmol) of glycyrrhizin and 40 mL of methanol while stirring. The reaction solution was stirred at room temperature for 8 hours and then neutralized by adding silver carbonate. The purified precipitate was filtered, and the filtrate was concentrated to dryness to obtain 1.75 g of reaction mixture.
The reaction mixture was separated and purified using HPLC (column: Inertsil PREP-ODS, 20φ × 250; solvent: 45% acetonitrile-water; detection: UV 254 nm), and the following compounds (A) to (C):
(A) A compound in which R 1 is a methyl group and R 2 is a hydrogen atom in formula (1) (glycyrrhizin monomethyl ester, hereinafter referred to as GL-inner) 260 mg,
(B) Compound (1) in which R 1 is a hydrogen atom and R 2 is a methyl group (glycyrrhizin monomethyl ester according to the present invention, hereinafter referred to as GL-outer) 240 mg,
(C) A compound in which R 1 and R 2 in the formula (1) are both methyl groups (glycyrrhizin dimethyl ester, hereinafter referred to as GL-dimethyl) 200 mg,
Respectively.
GL-inner MS; (ES +): m / z 837 [M + H] + , 661 [(M + H) -C 6 H 9 O 6 ] + , 471 [(M + H) -C 13 H 19 O 12 ] +
GL-outer MS; (ES +): m / z 837 [M + H] + , 646 [(M + H) -C 6 H 9 O 6 ] + , 471 [(M + H) -C 13 H 19 O 12 ] +
GL-dimethyl MS; (ES +): m / z 851 [M + H] +
For comparison, glycyrrhizin (hereinafter referred to as GL) was similarly tested and the results were compared.
＜肝細胞の単離及び培養＞
Ｗｉｓｔａｒ系雄性ラット（体重２００−２５０ｇ）を用い、Ｓｅｇｌｅｎらの方法（Ｍｅｔｈｏｄｓ Ｃｅｌｌ Ｂｉｏｌ．，１３，２９：１９７５）に従い行った。Ｉｎ ｓｉｔｕコラゲナーゼ還流法により単離した肝実質細胞を５％ウシ新生児血清含有Ｗｉｌｌｉａｍｓ’Ｅ培地（０．１μｇ／ｍＬアプロチニン、１００Ｕ／ｍＬペニシリン、０．１μｇ／ｍＬストレプトマイシン、０．１ｎＭデキサメタゾン含有）に懸濁し、コラーゲンコート培養ディッシュ（３５−ｍｍφ）に播き、３７℃、５％ＣＯ２存在下で３時間培養した。培養開始３時間後、ウシ新生児血清不含Ｗｉｌｌｉａｍｓ’Ｅ培地（無血清培地）に換え、グリチルリチンとその誘導体や種々の薬物を加え、さらに０−２１時間培養した。
<Isolation and culture of hepatocytes>
Wistar male rats (body weight 200-250 g) were used according to the method of Seglen et al. (Methods Cell Biol., 13, 29: 1975). Liver parenchymal cells isolated by the in situ collagenase reflux method were added to Williams'E medium containing 0.1% bovine serum (containing 0.1 μg / mL aprotinin, 100 U / mL penicillin, 0.1 μg / mL streptomycin, 0.1 nM dexamethasone). The suspension was suspended, seeded in a collagen-coated culture dish (35-mmφ), and cultured at 37 ° C. in the presence of 5% CO 2 for 3 hours. Three hours after the start of the culture, the medium was replaced with a newborn bovine serum-free Williams'E medium (serum-free medium), glycyrrhizin, its derivatives and various drugs were added, and further cultured for 0-21 hours.
＜核数の測定＞
Ｎａｋａｍｕｒａらの方法（Ｊ．Ｂｉｏｃｈｅｍ．，９４，１０２９；１９８３）を一部改変し行った。一定時間培養した肝実質細胞をリン酸緩衝液（ＰＢＳ：ｐＨ７．４）で洗浄し、０．１％ＴｒｉｔｏｎＸ−１００含有０．１Ｍクエン酸溶液処理を行い、裸核を得た。そこへ、同容量の０．３％トリパンブルー−ＰＢＳ溶液を加え、血球計算盤にて計測をした。
<Measurement of the number of nuclei>
The method of Nakamura et al. (J. Biochem., 94, 1029; 1983) was partially modified. Liver parenchymal cells cultured for a certain period of time were washed with a phosphate buffer (PBS: pH 7.4) and treated with a 0.1 M citric acid solution containing 0.1% Triton X-100 to obtain naked nuclei. To this, the same volume of 0.3% trypan blue-PBS solution was added, and the measurement was performed with a hemocytometer.
＜ＤＮＡ合成能の測定＞
ＭｏｒｌｅｙとＫｉｎｇｄｏｎの方法（Ａｎａｌ．Ｂｉｏｃｈｅｍ．，４５，２９８；１９７２）に従い、ＤＮＡ画分に取り込まれた３Ｈ−チミジンの取り込み量を測定し、ＤＮＡ合成能を求めた。特異的３Ｈ−チミジン量は１０μＭ ａｐｈｉｄｉｃｏｌｉｎ共存下の計数量を差し引いて求めた。ＤＮＡ合成能としては単位時間、単位蛋白質当たりの３Ｈ−チミジン量（ｄｐｍ／ｍｇ ｐｒｏｔｅｉｎ／ｈ）で表した。
<Measurement of DNA synthesis ability>
According to the method of Morley and Kingdon (Anal. Biochem., 45, 298; 1972), the amount of 3 H-thymidine incorporated into the DNA fraction was measured to determine the DNA synthesis ability. The amount of specific 3 H-thymidine was determined by subtracting the amount counted in the presence of 10 μM aphidicolin. The ability to synthesize DNA was expressed in terms of unit time and 3 H-thymidine amount per unit protein (dpm / mg protein / h).
＜蛋白質の定量＞
Ｌｏｗｒｙらの変法（Ａｎａｌ．Ｂｉｏｃｈｅｍ．，４７，１８４；１９７２）に従い、ウシ血清アルブミンを標準物質にして測定した。
<Quantification of protein>
According to a modified method of Lowry et al. (Anal. Biochem., 47, 184; 1972), bovine serum albumin was measured as a standard substance.
＜実験結果及び考察＞
実験結果を図１及び図２に示す。
図１は、グリチルリチン（ＧＬ）とその誘導体（ＧＬ−ｉｎｎｅｒ、ＧＬ−ｏｕｔｅｒ、ＧＬ−ｄｉｍｅｔｈｙｌ）により誘導される肝細胞ＤＮＡ合成能と増殖の刺激に関する経時変化を示すグラフであり、ＡはＤＮＡ合成能、Ｂは核数の測定結果をそれぞれ示している。
この実験において、薬物処理条件は次の通りとした。
・薬物添加濃度：ＧＬ １０−６Ｍ；ＧＬ−ｉｎｎｅｒ １０−６Ｍ；ＧＬ−ｏｕｔｅｒ １０−７Ｍ；ＧＬ−ｄｉｍｅｔｈｙｌ １０−６Ｍ。
・Ｓｅｅｄｉｎｇ密度：３．３×１０４ｃｅｌｌｓ／ｃｍ２（薬物添加３時間前に播種）。
図１Ａ，Ｂ中の矢印は、無血清培地への培地交換と薬物を添加した時間（０ ｔｉｍｅ）を示す。
また図１Ａ，Ｂ中の記号★，★★は、Ｃｏｎｔｒｏｌ（薬剤添加直後；Ｏ ｔｉｍｅの測定値）に対する有意差を示す。★Ｐ＜０．０５、★★Ｐ＜０．０１（ｍｅａｎ±Ｓ．Ｅ．Ｍ．，ｎ＝３）。
<Experimental results and discussion>
The experimental results are shown in FIGS.
FIG. 1 is a graph showing temporal changes in hepatocyte DNA synthesis ability and proliferation stimulation induced by glycyrrhizin (GL) and its derivatives (GL-inner, GL-outer, GL-dimethyl), and A is DNA synthesis. No. and B indicate the measurement results of the number of nuclei, respectively.
In this experiment, the drug treatment conditions were as follows.
Drug addition concentration: GL 10 −6 M; GL-inner 10 −6 M; GL-outer 10 −7 M; GL-dimethyl 10 −6 M.
Seeding density: 3.3 × 10 4 cells / cm 2 (seeding 3 hours before drug addition).
The arrows in FIGS. 1A and 1B indicate the time (0 time) when the medium was replaced with a serum-free medium and the drug was added.
In addition, symbols ★ and ★★ in FIGS. 1A and 1B indicate a significant difference with respect to Control (immediately after addition of a drug; measured value of O time). ★ P <0.05, ★★ P <0.01 (mean ± SEM, n = 3).
図２は、肝細胞のＤＮＡ合成及び増殖におけるＧＬｓ（ＧＬとその誘導体（ＧＬ−ｉｎｎｅｒ、ＧＬ−ｏｕｔｅｒ、ＧＬ−ｄｉｍｅｔｈｙｌ））の用量依存効果を示すグラフであり、ＡはＤＮＡ合成能、Ｂは核数についての用量依存効果を示す。
この実験条件は次の通りとした。
・Ｓｅｅｄｉｎｇ密度：３．３×１０４ｃｅｌｌｓ／ｃｍ２（薬物添加３時間前に播種）。
・培養時間：無血清培地への交換後４時間。
図２Ａ，Ｂ中の記号★，★★は、Ｃｏｎｔｒｏｌ（薬物無処理群）に対する有意差を示す。★Ｐ＜０．０５、★★Ｐ＜０．０１（ｍｅａｎ±Ｓ．Ｅ．Ｍ．，ｎ＝３）。
この結果、ＧＬｓのそれぞれのＥＣ５０値は表１に示す通りであった。
FIG. 2 is a graph showing the dose-dependent effect of GLs (GL and its derivatives (GL-inner, GL-outer, GL-dimethyl)) on hepatocyte DNA synthesis and proliferation. The dose-dependent effect on the number of nuclei is shown.
The experimental conditions were as follows.
Seeding density: 3.3 × 10 4 cells / cm 2 (seeding 3 hours before drug addition).
-Culture time: 4 hours after replacement with serum-free medium.
Symbols ★ and ★★ in FIGS. 2A and 2B indicate a significant difference with respect to Control (drug-untreated group). ★ P <0.05, ★★ P <0.01 (mean ± SEM, n = 3).
As a result, each EC 50 value of GLs was as shown in Table 1.
初代培養肝実質細胞の増殖に対して、ＧＬ−ｉｎｎｅｒ、ＧＬ−ｏｕｔｅｒ及びＧＬは、それぞれ単独で、時間依存的な肝細胞の増殖促進作用を示した。すなわち、低密度培養条件下で早期に増殖が始まり（薬物添加後、ＤＮＡ合成が３時間、核数が３．５時間で有意）、４時間から２１時間で一定となる変化を示した。さらに、それらの変化パターンは、ＤＮＡ合成能の増加が核数の増加に先立って発現した（図１参照。）。
また、これらの化合物は、用量依存的に増殖促進作用を示した。すなわち、各々の化合物とグリチルリチンの効力は、ＥＣ５０値より、ＧＬ−ｏｕｔｅｒ、ＧＬ、ＧＬ−ｉｎｎｅｒの順に強力（ＤＮＡ合成と核数のＥＣ５０値は、ほぼ一致していた。）で、ＧＬ−ｄｉｍｅｔｈｙｌは有意な効果を示さなかった（図２及び表１参照。）。
以上のことから、実験したグリチルリチンメチルエステルのうち、アグリコンのＣ−３位から近傍にある１番目の糖のカルボキシル基にメチル基がエステル結合したもの（ＧＬ−ｉｎｎｅｒ）及び１番目と２番目の両糖のカルボキシル基に各々メチル基がエステル結合したもの（ＧＬ−ｄｉｍｅｔｈｙｌ）は増殖促進効果が弱く、一方、２番目の糖のカルボキシル基にメチル基がエステル結合した本発明に係る化合物（ＧＬ−ｏｕｔｅｒ）は、グリチルリチン（ＧＬ）よりも約１０倍、初代培養肝実質細胞の増殖促進効果を強く発現することが見出された。
GL-inner, GL-outer, and GL each independently showed a time-dependent hepatocyte proliferation promoting effect on the proliferation of primary cultured hepatocytes. That is, proliferation started early under low-density culture conditions (after drug addition, DNA synthesis was significant for 3 hours and the number of nuclei was significant for 3.5 hours), and showed a constant change from 4 hours to 21 hours. Furthermore, these change patterns were expressed prior to the increase in the number of nuclei when the DNA synthesis ability increased (see FIG. 1).
In addition, these compounds showed a growth promoting effect in a dose-dependent manner. That is, the potency of each compound and glycyrrhizin, from The EC 50 values, with GL-outer, GL, strong in the order of GL-inner, (is The EC 50 values of DNA synthesis and number of nuclei were substantially consistent.), GL -Dimethyl has no significant effect (see Figure 2 and Table 1).
From the above, among the glycyrrhizin methyl esters tested, those in which a methyl group is ester-bonded to the carboxyl group of the first sugar in the vicinity from the C-3 position of the aglycone (GL-inner) and the first and second A compound in which a methyl group is ester-bonded to the carboxyl groups of both sugars (GL-dimethyl) has a weak growth-promoting effect, whereas the compound according to the present invention in which a methyl group is ester-bonded to the carboxyl group of the second sugar (GL- outer) was found to be about 10 times stronger than glycyrrhizin (GL), and strongly expresses the effect of promoting the growth of primary cultured hepatocytes.
Claims (2)
で表される肝実質細胞増殖促進剤。 Formula (1)
A hepatocyte growth promoter represented by:
A therapeutic agent for liver disease, comprising the liver parenchymal cell growth promoter according to claim 1 as an active ingredient.
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| JP2004038414A JP2005225836A (en) | 2004-02-16 | 2004-02-16 | Hepatocyte growth promoter and therapeutic medicine for liver disease |
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| Application Number | Priority Date | Filing Date | Title |
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| JP2004038414A JP2005225836A (en) | 2004-02-16 | 2004-02-16 | Hepatocyte growth promoter and therapeutic medicine for liver disease |
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| JP2005225836A true JP2005225836A (en) | 2005-08-25 |
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2004
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