JPH07206670A - Immunosuppressive agent - Google Patents
Immunosuppressive agentInfo
- Publication number
- JPH07206670A JPH07206670A JP266894A JP266894A JPH07206670A JP H07206670 A JPH07206670 A JP H07206670A JP 266894 A JP266894 A JP 266894A JP 266894 A JP266894 A JP 266894A JP H07206670 A JPH07206670 A JP H07206670A
- Authority
- JP
- Japan
- Prior art keywords
- trichostatin
- immunosuppressive agent
- active component
- agent containing
- cells
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
Abstract
Description
【０００１】[0001]
【産業上の利用分野】本発明は新規な免疫抑制剤に関す
る。本発明の免疫抑制剤は自己免疫疾患、アレルギー性
疾患、臓器移植などの治療に有用である。FIELD OF THE INVENTION The present invention relates to a novel immunosuppressant. The immunosuppressive agent of the present invention is useful for treating autoimmune diseases, allergic diseases, organ transplants and the like.
【０００２】[0002]
【従来の技術】微生物の代謝産物であるトリコスタチン
Ａは、従来、抗真菌作用〔ジャーナル・オブ・アンチバ
イオチックス（J.Antibiotics)，29，1-6(1976) 〕、抗
原虫作用〔ジャーナル・オブ・アンチバイオチックス
（J.Antibiotics)，41,461-468(1988)〕および抗癌作用
（特開昭60−149520号公報）を有することが知られてい
るほか、分化誘導活性〔キャンサー・リサーチ ( Cance
r Research) ，47，3688-3691(1987)〕 、ヒストン脱ア
セチル化酵素阻害活性〔ジャーナル・オブ・バイオロジ
カル・ケミストリー (J. Biol. Chem.），265 ，17174-
17179(1990) 〕を有することが報告されている。BACKGROUND OF THE INVENTION Trichostatin A, which is a microbial metabolite, has been used for its antifungal action [J. Antibiotics, 29 , 1-6 (1976)], antiprotozoal action [Journal. Of antibiotics (J. Antibiotics, 41 , 461-468 (1988)] and anti-cancer action (JP-A-60-149520), as well as differentiation-inducing activity (cancer)・ Research (Cance
r Research), 47 , 3688-3691 (1987)], histone deacetylase inhibitory activity [Journal of Biological Chemistry (J. Biol. Chem.), 265 , 17174-].
17179 (1990)].
【０００３】[0003]
【発明が解決しようとする課題】免疫抑制剤としては、
副腎皮質ホルモン、代謝拮抗剤、アルキル化剤、アルカ
ロイド、抗生物質、抗リンパ球グロブリン、抗CD3モノ
クローナル抗体などが知られており、自己免疫疾患、ア
レルギー性疾患、臓器移植などの治療薬として用いられ
ている。しかしながら、有効性、持続性、副作用などの
点で必ずしも満足されるものではなく、さらに優れた免
疫抑制剤の開発が求められている。The immunosuppressive agents include
Corticosteroids, antimetabolites, alkylating agents, alkaloids, antibiotics, anti-lymphocyte globulin, anti-CD3 monoclonal antibodies, etc. are known and used as therapeutic agents for autoimmune diseases, allergic diseases, organ transplants, etc. ing. However, they are not always satisfactory in terms of efficacy, sustainability, side effects, etc., and there is a demand for the development of even more excellent immunosuppressive agents.
【０００４】[0004]
【課題を解決するための手段】本発明により式According to the present invention, the formula
【０００５】[0005]
【化２】 [Chemical 2]
【０００６】で表されるトリコスタチンＡを有効成分と
する免疫抑制剤が提供される。トリコスタチンＡはスト
レプトミセス属である放線菌を培地に培養し、培養物中
に生成蓄積させ、該培養物中から精製単離することによ
り得られる〔ジャーナル・オブ・アンチバイオチックス
（J.Antibiotics)，29,1-6(1976)〕。次にトリコスタチ
ンＡの免疫抑制作用について試験例で説明する。An immunosuppressive agent containing trichostatin A as an active ingredient is provided. Trichostatin A is obtained by culturing actinomycetes belonging to the genus Streptomyces in a medium, producing and accumulating in the culture, and purifying and isolating from the culture [J. Antibiotics (J. Antibiotics). ), 29 , 1-6 (1976)]. Next, the immunosuppressive action of trichostatin A will be described in Test Examples.
【０００７】試験例１．マウスリンパ球混合反応におけ
るＴ細胞増殖抑制試験 無菌的にAKRマウス [日本エス. エル. シー（株)]より
脾臓を摘出し、単細胞浮遊液とした。この浮遊液にマイ
トマイシンC (MMC)[協和発酵工業 (株)]を添加し（終濃
度50μg/ml）、37℃で30分間培養した。培養後、ハンク
スの平衡塩溶液(HBSS 、ギブコ社) 中に2.5%の牛胎児血
清(FCS、ギブコ社) を加えた溶液で3回洗浄を行ない、1
x 107 cells/mlに調製した。Test Example 1. T Cell Proliferation Inhibition Test in Mouse Lymphocyte Mixed Reaction Spleen was aseptically removed from AKR mouse [Nippon SLC, Inc.] to obtain a single cell suspension. Mitomycin C (MMC) [Kyowa Hakko Kogyo Co., Ltd.] was added to this suspension (final concentration 50 μg / ml), and the mixture was incubated at 37 ° C. for 30 minutes. After culturing, wash 3 times with a solution of 2.5% fetal calf serum (FCS, Gibco) in Hanks balanced salt solution (HBSS, Gibco).
It was adjusted to x 10 7 cells / ml.
【０００８】96穴マイクロタイタープレートの各ウエル
にB10.BRマウス[日本エス.エル.シー（株）]のリンパ節
細胞浮遊液 50 μl （1.5 x 105 cells含有）、AKRマウ
スの脾臓細胞浮遊液 50μl（5 x 105 cells含有）およ
び各試験濃度のトリコスタチンＡを含む培養液 100μl
を添加し、37℃のCO2インキュベーター内で72時間培養
した。尚、培養終了18時間前に[3H]-チミジン1.0μCiを
添加した。培養終了後、セルハーベスターで濾紙上に細
胞を補集し、乾燥後トルエン系シンチレーターを加え、
液体シンチレーションカウンターで細胞に取り込まれた
[3H]-チミジンの放射能量を測定した（試験群）。[0008] 96 B10.BR mouse to each well of a well microtiter plate [Japan SLC. El. Sea Corporation] lymph node cell suspension 50 μl (1.5 x 10 5 cells containing) of the spleen cell suspensions of AKR mice 50 μl (containing 5 x 10 5 cells) and 100 μl of culture containing Trichostatin A at each test concentration
Was added and the cells were cultured in a CO 2 incubator at 37 ° C for 72 hours. [ 3 H] -thymidine 1.0 μCi was added 18 hours before the end of the culture. After culturing, collect cells on filter paper with a cell harvester, add toluene scintillator after drying,
Incorporated into cells by liquid scintillation counter
The radioactivity of [ 3 H] -thymidine was measured (test group).
【０００９】対照群としてトリコスタチンＡを含まない
培養液を添加し、以下上記と同様に培養を行い細胞に取
り込まれた[3H]-チミジンの放射能量を測定した。Ｔ細
胞増殖抑制率は、次式に従って算出した。As a control group, a culture solution containing no trichostatin A was added, and the following culture was carried out in the same manner as above to measure the radioactivity of [ 3 H] -thymidine incorporated into cells. The T cell proliferation inhibition rate was calculated according to the following formula.
【００１０】[0010]
【数１】 [Equation 1]
【００１１】（式中、 MMC処理 AKRマウス放射能量は、
MMC処理した AKRマウスの脾臓細胞に取り込まれた[3H]
-チミジンの放射能量を、またB10.BRマウス放射能量
は、B10.BRマウスのリンパ節細胞に取り込まれた[3H]-
チミジンの放射能量を表す)試験結果を第１表に示す。(Wherein the MMC-treated AKR mouse radioactivity is
Incorporated into spleen cells of MKR-treated AKR mice [ 3 H]
-Thymidine radioactivity and B10.BR mouse radioactivity were incorporated into lymph node cells of B10.BR mice [ 3 H]-
The test results are shown in Table 1 (representing the amount of radioactivity of thymidine).
【００１２】[0012]
【表１】 [Table 1]
【００１３】第１表によれば、トリコスタチンＡはマウ
スリンパ球混合反応におけるT細胞増殖を抑制し、明ら
かな免疫抑制作用を示した。According to Table 1, Trichostatin A suppressed T cell proliferation in mouse lymphocyte mixed reaction and showed a clear immunosuppressive action.
【００１４】試験例２．マウスリンパ遅延型過敏症反応
抑制試験 トリニトロフェニル（ＴＮＰ）ハプテンに対する免疫応
答は広く研究されており、遅延型過敏症反応は、細胞媒
介免疫応答に関わる調節機構をマウスにおいて決定する
ものとして特徴づけられている。また抗ＴＮＰ抗体産生
も認められ、これらは、抗原特異的Ｔ細胞媒介炎症反応
であり、ヒトおよび他の哺乳類にも広く認められる。Test Example 2. Mouse Lymphoid Delayed Hypersensitivity Inhibition Assay The immune response to the trinitrophenyl (TNP) hapten has been extensively studied, and the delayed hypersensitivity response was characterized as determining the regulatory mechanism involved in cell-mediated immune responses in mice. Has been. Anti-TNP antibody production was also observed, these are antigen-specific T cell-mediated inflammatory responses, which are also widespread in humans and other mammals.
【００１５】マウスにおけるＴＮＰハプテンに対する免
疫応答 Balb/c系雄マウス（７週齢、チャールズリバー社）を１
群あたりマウス５匹を用いた。１０mMのトリニトロベン
ズスルホン酸（ＴＮＢＳ、和光純薬）0.1ml を用いて剃
毛背部皮内に感作した日を０日目とした。次に試験化合
物を10％ジメチルスルホキシド (DMSO) 中で調製し、０
日目〜４日目の期間を通じて１日１回、腹腔内投与 (投
与量:50mg/kg)した。また対照群として、10％DMSOを上
記と同様に投与した。5 日目に10mMのＴＮＢＳ 0.05ml
を用いてマウスの左足蹠内に遅延型過敏症反応を惹起せ
しめ、24時間後に足の腫張を厚み計 (ピーコックゲー
ジ)を用いて測定し、非惹起足である右足蹠との差を求
めた。 Immunity to TNP haptens in mice
Epidemiological response 1 male Balb / c mouse (7 weeks old, Charles River)
Five mice were used per group. Day 0 was the day of sensitization into the dorsal skin of the shaved skin with 0.1 ml of 10 mM trinitrobenzsulfonic acid (TNBS, Wako Pure Chemical Industries, Ltd.). Test compounds were then prepared in 10% dimethylsulfoxide (DMSO) and
Intraperitoneal administration (dose: 50 mg / kg) was carried out once a day throughout the period from day 4 to day 4. As a control group, 10% DMSO was administered in the same manner as above. 0.05 ml of 10 mM TNBS on the 5th day
Was used to elicit a delayed hypersensitivity reaction in the left footpad of the mouse, and after 24 hours, swelling of the foot was measured using a thickness gauge (Peacock gauge) to determine the difference from the right footpad, which is the non-induced foot. It was
【００１６】また、抗ＴＮＰ抗体産生を、足の腫張を測
定した後に採血した血清中の抗ＴＮＰ抗体量を酵素免疫
測定法で測定した。上記の遅延型過敏症反応および抗Ｔ
ＮＰ抗体産生による免疫抑制作用の指標として、対照群
と試験化合物群とを比較し、次式により抑制率を求め
た。The anti-TNP antibody production was measured by the enzyme immunoassay for the amount of anti-TNP antibody in the serum collected after measuring the swelling of the foot. Delayed type hypersensitivity reaction and anti-T described above
As an index of the immunosuppressive action by NP antibody production, the control group and the test compound group were compared, and the suppression rate was calculated by the following formula.
【００１７】[0017]
【数２】 [Equation 2]
【００１８】試験結果によれば、トリコスタチンＡの遅
延型過敏症反応における抑制率は47.1％、抗ＴＮＰ抗体
産生における抑制率は26.7％であった。また、トリコス
タチンＡの急性毒性（LD50) は、マウス経口投与におい
て 100mg/kg 以上である〔ジャーナル・オブ・アンチバ
イオチックス（J.Antibiotics)，41,461-468(1988)〕。According to the test results, the inhibition rate of delayed type hypersensitivity reaction of trichostatin A was 47.1%, and the inhibition rate of anti-TNP antibody production was 26.7%. Moreover, the acute toxicity (LD 50 ) of trichostatin A is 100 mg / kg or more in oral administration to mice [Journal of Antibiotics, 41 , 461-468 (1988)].
【００１９】以上の試験結果より、トリコスタチンＡは
優れた免疫抑制作用を有し、自己免疫疾患、アレルギー
性疾患、臓器移植などの治療剤として有用であることが
確認された。From the above test results, it was confirmed that trichostatin A has an excellent immunosuppressive action and is useful as a therapeutic agent for autoimmune diseases, allergic diseases, organ transplants and the like.
【００２０】トリコスタチンＡは、そのままあるいは各
種の医薬組成物として経口的または非経口的に投与され
る。このような医薬組成物の剤形としては、たとえば錠
剤、丸薬、散剤、顆粒剤、カプセル剤、坐剤、注射剤、
点滴剤などがあげられる。上記剤形の製剤化には、通常
知られている方法が適用され、たとえば各種の賦形剤、
潤滑剤、結合剤、崩壊剤、懸濁化剤、等張化剤、乳化
剤、吸収促進剤などを含有していてもよい。Trichostatin A is orally or parenterally administered as it is or as various pharmaceutical compositions. Examples of the dosage form of such a pharmaceutical composition include tablets, pills, powders, granules, capsules, suppositories, injections,
Examples include drops. For the formulation of the above dosage form, a generally known method is applied, for example, various excipients,
It may contain a lubricant, a binder, a disintegrating agent, a suspending agent, an isotonicity agent, an emulsifying agent, an absorption promoter and the like.
【００２１】医薬組成物に使用される担体としては、た
とえば水、注射用蒸留水、生理食塩水、グルコース、フ
ラクトース、白糖、マンニット、ラクトース、澱粉、コ
ーン・スターチ、ポテト・スターチ、セルロース、メチ
ルセルロース、カルボキシメチルセルロースカルシウ
ム、ヒドロキシプロピルセルロース、アルギン酸、タル
ク、クエン酸ナトリウム、炭酸カルシウム、リン酸水素
カルシウム、ステアリン酸マグネシウム、尿素、シリコ
ーン樹脂、ソルビタン脂肪酸エステル、グリセリン脂肪
酸エステルなどがあげられ、これらは製剤の種類に応じ
て適宜選択される。Examples of the carrier used in the pharmaceutical composition include water, distilled water for injection, physiological saline, glucose, fructose, sucrose, mannitol, lactose, starch, corn starch, potato starch, cellulose and methyl cellulose. , Carboxymethyl cellulose calcium, hydroxypropyl cellulose, alginic acid, talc, sodium citrate, calcium carbonate, calcium hydrogen phosphate, magnesium stearate, urea, silicone resin, sorbitan fatty acid ester, glycerin fatty acid ester, etc. It is appropriately selected according to the type.
【００２２】トリコスタチンＡの投与量は、目的とする
治療効果、投与方法、治療期間、患者の年齢、体重など
により決められるが、経口もしくは非経口（たとえば、
注射、点滴、座剤による直腸投与あるいは皮膚貼付な
ど）的投与方法により、通常成人１日当り0.01〜2 mg/k
gである。次に、実施例をあげて本発明の態様を説明す
る。The dose of trichostatin A is determined depending on the desired therapeutic effect, administration method, treatment period, patient's age, body weight, etc., but it may be oral or parenteral (eg,
It is usually 0.01 to 2 mg / k per day for adults, depending on the administration method such as injection, drip, rectal administration by suppository or skin application.
It is g. Next, embodiments of the present invention will be described with reference to examples.
【００２３】[0023]
実施例１．錠剤 トリコスタチンＡ100 g、ラクトース40 g 、コーンスタ
ーチ18 g およびカルボキシメチルセルロースカルシウ
ム10 g を混合し、10 %ヒドロキシプロピルセルロース
溶液を加えて練合した。この練合液を1.0 mmのバスケッ
トを取り付けた押しだし造粒機で造粒し、ステアリン酸
マグネシウムを加えて整粒して打錠用顆粒とし、常法に
より打錠を行って、１製剤（170 mg) 中にトリコスタチ
ンＡを100 mg含む8 mm径の錠剤を得た。Example 1. Tablets 100 g of trichostatin A, 40 g of lactose, 18 g of corn starch and 10 g of calcium carboxymethyl cellulose were mixed, and a 10% hydroxypropyl cellulose solution was added and kneaded. This kneading liquid is granulated by an extrusion granulator equipped with a 1.0 mm basket, and magnesium stearate is added thereto to form granules for tableting, which are then tableted by a conventional method to give 1 preparation (170 8 mg tablet containing 100 mg of trichostatin A was obtained.
【００２４】実施例２．カプセル剤 トリコスタチンＡ50 g、ラクトース 80 g およびポテト
スターチ 38 g からなる混合物に、10 %ヒドロキシプロ
ピルセルロース溶液を加えて練合し、以下実施例１と同
様に造粒し、ステアリン酸マグネシウムを加えてカプセ
ル充填機によりハードカプセルに充填し、常法により１
カプセル（170 mg) 中トリコスタチンＡを50 mg含むカ
プセル剤を得た。Example 2. Capsules To a mixture of 50 g of trichostatin A, 80 g of lactose and 38 g of potato starch, 10% hydroxypropylcellulose solution was added and kneaded, and granulated in the same manner as in Example 1 below, and magnesium stearate was added. Fill the hard capsules with a capsule filling machine and use the standard method to
A capsule containing 50 mg of trichostatin A in a capsule (170 mg) was obtained.
【００２５】実施例３．ソフトカプセル剤 10 gのトリコスタチンＡを100 gの大豆油に溶かし、得
られた溶液を常法によりカプセルに注入することによ
り、１カプセル当り10 mgのトリコスタチンＡを含むソ
フトカプセル剤を得た。Example 3. Soft capsule 10 g of trichostatin A was dissolved in 100 g of soybean oil, and the resulting solution was injected into capsules by a conventional method to obtain a soft capsule containing 10 mg of trichostatin A per capsule.
【００２６】[0026]
【発明の効果】本発明により優れた免疫抑制剤が提供さ
れる。The present invention provides an excellent immunosuppressant.
Claims (1)
剤。1. The formula: An immunosuppressive agent containing trichostatin A as an active ingredient represented by:
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP266894A JPH07206670A (en) | 1994-01-14 | 1994-01-14 | Immunosuppressive agent |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP266894A JPH07206670A (en) | 1994-01-14 | 1994-01-14 | Immunosuppressive agent |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH07206670A true JPH07206670A (en) | 1995-08-08 |
Family
ID=11535699
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP266894A Withdrawn JPH07206670A (en) | 1994-01-14 | 1994-01-14 | Immunosuppressive agent |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH07206670A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7235688B1 (en) | 2004-11-04 | 2007-06-26 | University Of Notre Dame Du Lac | Process for preparing histone deacetylase inhibitors and intermediates thereof |
| US7271198B2 (en) * | 2000-11-21 | 2007-09-18 | Wake Forest University | Method of treating autoimmune diseases |
| WO2016210292A1 (en) | 2015-06-25 | 2016-12-29 | Children's Medical Center Corporation | Methods and compositions relating to hematopoietic stem cell expansion, enrichment, and maintenance |
| WO2017161001A1 (en) | 2016-03-15 | 2017-09-21 | Children's Medical Center Corporation | Methods and compositions relating to hematopoietic stem cell expansion |
-
1994
- 1994-01-14 JP JP266894A patent/JPH07206670A/en not_active Withdrawn
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7271198B2 (en) * | 2000-11-21 | 2007-09-18 | Wake Forest University | Method of treating autoimmune diseases |
| US7557141B2 (en) | 2000-11-21 | 2009-07-07 | Wake Forest University Health Sciences | Method of treating autoimmune diseases |
| US7235688B1 (en) | 2004-11-04 | 2007-06-26 | University Of Notre Dame Du Lac | Process for preparing histone deacetylase inhibitors and intermediates thereof |
| WO2016210292A1 (en) | 2015-06-25 | 2016-12-29 | Children's Medical Center Corporation | Methods and compositions relating to hematopoietic stem cell expansion, enrichment, and maintenance |
| WO2017161001A1 (en) | 2016-03-15 | 2017-09-21 | Children's Medical Center Corporation | Methods and compositions relating to hematopoietic stem cell expansion |
| EP4049665A1 (en) | 2016-03-15 | 2022-08-31 | Children's Medical Center Corporation | Methods and compositions relating to hematopoietic stem cell expansion |
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