JPH10194976A - Immunosuppressant - Google Patents
ImmunosuppressantInfo
- Publication number
- JPH10194976A JPH10194976A JP652497A JP652497A JPH10194976A JP H10194976 A JPH10194976 A JP H10194976A JP 652497 A JP652497 A JP 652497A JP 652497 A JP652497 A JP 652497A JP H10194976 A JPH10194976 A JP H10194976A
- Authority
- JP
- Japan
- Prior art keywords
- heat
- treated
- curdlan
- immunosuppressant
- acid
- 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.)
- Granted
Links
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Saccharide Compounds (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
Abstract
Description
【0001】[0001]
【発明の属する技術分野】本発明は、重篤な副作用のな
い免疫抑制剤に関する。[0001] The present invention relates to an immunosuppressant having no serious side effects.
【0002】[0002]
【従来の技術】生体内の免疫系は、細菌、酵母、カビ、
ウイルスなどの微生物による感染や、腫瘍に対する防御
に重要な役割を果たしており、その主要な機構は、Tリ
ンパ球およびBリンパ球が、これらの微生物や腫瘍を、
抗原受容体を介して認識することにより刺激を受け、抗
原特異的に活性化し、これの異物を排除する能力を高め
ることである。しかし、自己免疫疾患や、臓器移植の拒
否反応などの治療、抑制においては、免疫応答を抑制す
ることが必要であり、そのために免疫抑制剤が使用され
る。現在、免疫抑制剤としては、抗原非特異性のステロ
イド剤や核酸合成系に作用する薬剤が多く使用されてい
るが、これらは、重篤な副作用を生ずることがある。ま
た、シクロスポリン等、臓器移植の拒否反応の抑制に使
用される免疫抑制剤にも、様々な副作用を伴うものがあ
る。2. Description of the Related Art The in vivo immune system is composed of bacteria, yeast, mold,
It plays an important role in infection by viruses and other microorganisms and in defense against tumors. The main mechanism is that T lymphocytes and B lymphocytes
To stimulate by recognizing via an antigen receptor, activate it in an antigen-specific manner, and enhance its ability to eliminate foreign substances. However, in the treatment and suppression of autoimmune diseases and rejection of organ transplantation, it is necessary to suppress the immune response, and immunosuppressants are used for that purpose. At present, many non-antigen steroids and drugs acting on nucleic acid synthesis systems are used as immunosuppressants, but these may cause serious side effects. In addition, some immunosuppressants such as cyclosporin used for suppressing rejection of organ transplantation have various side effects.
【0003】[0003]
【発明が解決しようとする課題】本発明は、重篤な副作
用なしに優れた免疫抑制作用を示す免疫抑制剤を提供す
ることを目的とする。SUMMARY OF THE INVENTION An object of the present invention is to provide an immunosuppressant which exhibits excellent immunosuppressive action without serious side effects.
【0004】[0004]
【課題を解決するための手段】本発明者らは、糖類およ
び免疫に関する研究を進める上で、加熱処理した直鎖の
β−1,3グルカンが、リンパ球抑制作用を示すことを
見いだした。かかる加熱処理β−1,3グルカンは、免
疫担当細胞が抗原特異的および抗原非特異的な刺激を受
けたときのBおよびTリンパ球の活性化を抑制する作用
を有する。すなわち、該加熱処理β−1,3グルカンを
脾臓細胞の培養系に添加すると、マイトジェン刺激を加
えずに培養したときの生細胞数および細胞代謝活性はそ
れほど損なわず、Bリンパ球マイトジェン刺激下で培養
したときのリンパ球の生細胞数、特に、Bリンパ球の生
細胞数の増加および細胞代謝活性の上昇を強度に抑制
し、Tリンパ球マイトジェン刺激下で培養したときの細
胞代謝活性の上昇を強度に抑制した。この抑制作用は、
リンパ球が活性化されるときにより選択的に働くことか
ら、従来の免疫抑制剤が示した非特異性な作用とは異な
ることを示しており、また、Bリンパ球の活性化を強度
に抑制することから、近年提案された免疫抑制剤のTリ
ンパ球に対する選択的な作用とも異なることを示してい
る。そのため、公知のステロイド剤に認められる様々な
副作用、核酸合成系に作用する薬剤に認められる造血器
などの重篤な副作用、また、シクロスポリン、FK50
6に認められる腎障害、肝障害等の副作用はないと考え
られる。また、該加熱処理β−1,3グルカンはBリン
パ球の活性化を強度に抑制するため、Bリンパ球の異常
によって引き起こされる悪性リンパ腫、全身性エリテマ
トーデス、慢性関節リウマチ等の自己免疫疾患またはア
レルギー疾患の治療にきわめて有用である。さらにTリ
ンパ球の活性化も抑制するため、臓器移植の拒絶反応の
予防にも有用であると考えられる。Means for Solving the Problems The present inventors have found that a heat-treated linear β-1,3 glucan exhibits a lymphocyte inhibitory effect in advancing research on saccharides and immunity. Such heat-treated β-1,3 glucan has an action of suppressing the activation of B and T lymphocytes when the immunocompetent cells are stimulated with antigen-specific and non-antigen-specific stimuli. That is, when the heat-treated β-1,3 glucan is added to the culture system of spleen cells, the number of viable cells and the cell metabolic activity when cultured without adding mitogen stimulation are not significantly impaired, and the cells are stimulated under B lymphocyte mitogen stimulation. Live cell count of lymphocytes when cultured, especially increase in live cell count of B lymphocytes and increase of cell metabolic activity are strongly suppressed, and cell metabolic activity is increased when cultured under T lymphocyte mitogen stimulation Was strongly suppressed. This suppression effect
It shows that it is different from the non-specific action exhibited by conventional immunosuppressants because it acts more selectively when lymphocytes are activated, and also strongly suppresses the activation of B lymphocytes. Thus, the results show that the immunosuppressive agents proposed in recent years differ from the selective action on T lymphocytes. Therefore, various side effects observed in known steroid drugs, serious side effects such as hematopoietic organs observed in drugs acting on the nucleic acid synthesis system, cyclosporine, FK50
It is considered that there are no side effects such as renal damage and liver damage observed in No. 6. In addition, since the heat-treated β-1,3 glucan strongly suppresses the activation of B lymphocytes, autoimmune diseases such as malignant lymphoma, systemic lupus erythematosus, rheumatoid arthritis, etc. or allergy caused by B lymphocyte abnormalities. It is extremely useful for treating diseases. Furthermore, since it suppresses the activation of T lymphocytes, it is considered to be useful for preventing rejection of organ transplantation.
【0005】本発明は、かかる知見に基づいて完成され
たものであって、加熱処理されたβ−1,3−グルコシ
ド結合からなる直鎖の糖類を有効成分とする免疫抑制剤
を提供するものである。用いるβ−1,3−グルコシド
結合からなる直鎖の糖類としては、カードラン加水分解
物が挙げられる。特に、酸加水分解物、とりわけ蟻酸加
水分解物や、β−1,3−グルカナーゼのような酵素に
よる加水分解物で、数平均分子量が340から4000
の範囲にあるものが好ましい。加熱処理としては、水溶
液中で80℃以上での加熱が好ましい。本発明の免疫賦
活剤は、副作用がなく、常用に適しており、免疫抑制用
の医薬として好適である。The present invention has been completed on the basis of the above findings, and provides an immunosuppressant comprising, as an active ingredient, a heat-treated linear saccharide comprising a β-1,3-glucoside bond. It is. Examples of the linear saccharide comprising a β-1,3-glucoside bond include a curdlan hydrolyzate. In particular, acid hydrolysates, especially formic acid hydrolysates, and hydrolysates of enzymes such as β-1,3-glucanase, having a number average molecular weight of 340 to 4000
Are preferred. As the heat treatment, heating at 80 ° C. or higher in an aqueous solution is preferable. The immunostimulant of the present invention has no side effects, is suitable for ordinary use, and is suitable as a drug for immunosuppression.
【0006】[0006]
【発明の実施の形態】本発明において、加熱処理に供さ
れるβ−1,3−グルコシド結合からなる直鎖の糖類と
しては、グルコース分子が分岐せずに、β−1,3−グ
ルコシド結合により結合したオリゴ糖ないしは多糖であ
り、代表的な例としては、アルカリゲネス(Alkaligene
s)属又はアグロバクテリウム(Agrobacterium)属の細
菌が産生する、β−1,3−グルコシド結合を有する多
糖類であるカードランの加水分解物が挙げられる。以
下、カードランの加水分解物を例として本発明を説明す
る。カードランの加水分解物は、自体公知の多糖類の加
水分解法により調製できるが、得られる加水分解物の免
疫抑制活性から、蟻酸、酢酸のような有機酸、塩酸、硫
酸のような無機酸、特に、蟻酸あるいはβ−1,3−グ
ルカナーゼのような酵素で行うことが好ましく、数平均
分子量が、340〜4000の範囲のものが好ましい。BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, as a linear saccharide comprising a β-1,3-glucoside bond subjected to a heat treatment, a glucose molecule is not branched, and a β-1,3-glucoside bond is formed. Oligosaccharides or polysaccharides linked by the following are typical examples: Alkaligenes (Alkaligene)
s) A hydrolyzate of curdlan, a polysaccharide having a β-1,3-glucoside bond, produced by a bacterium of the genus or Agrobacterium genus. Hereinafter, the present invention will be described using a hydrolyzate of curdlan as an example. The hydrolyzate of curdlan can be prepared by a known polysaccharide hydrolysis method.However, from the immunosuppressive activity of the obtained hydrolyzate, organic acids such as formic acid and acetic acid, and inorganic acids such as hydrochloric acid and sulfuric acid are used. In particular, it is preferable to use an enzyme such as formic acid or β-1,3-glucanase, and the number average molecular weight is preferably in the range of 340 to 4000.
【0007】本明細書における数平均分子量は、以下の
条件で高速液体クロマトグラフィー(HPLC)により
測定したものである。測定装置としては、東ソー(株)
製の高速液体クロマトグラフィー装置を使用した。 検出器:RI−8022 ポンプ:CCPM−II カラムオーブン:CO−8020 脱気装置:SD−8022 オートサンプラー:AS−8020 測定カラムは、TSKゲルのカラム(G−OLIGO−
PWまたはG−3000PWXL、7.8φmm×30c
m)を用い、流速0.6〜0.7ml/分、温度40℃で測
定した。試料の0.02%水溶液を調製し、その200
μlを用いた。溶出は純水で行った。一方、分子量既知
のプルラン標準品を同様に測定して較正曲線を作成し、
この較正曲線を基に、東ソー(株)のGPC−LALL
Sプログラムとマイクロソフト(株)の計算プログラム
「エクセル」によって分子量分布関数を求め、数平均分
子量として換算した。[0007] The number average molecular weight in the present specification is measured by high performance liquid chromatography (HPLC) under the following conditions. As a measuring device, Tosoh Corporation
A high performance liquid chromatography device manufactured by KK was used. Detector: RI-8022 Pump: CCPM-II Column oven: CO-8020 Deaerator: SD-8022 Autosampler: AS-8020 The measurement column is a TSK gel column (G-OLIGO-
PW or G-3000PWXL, 7.8φmm × 30c
m) at a flow rate of 0.6 to 0.7 ml / min and a temperature of 40 ° C. A 0.02% aqueous solution of the sample was prepared and its 200
μl was used. Elution was performed with pure water. On the other hand, a calibration curve was created by similarly measuring a pullulan standard product with a known molecular weight,
Based on this calibration curve, GPC-LALL of Tosoh Corporation
The molecular weight distribution function was determined by the S program and the calculation program “Excel” of Microsoft Corporation, and converted as a number average molecular weight.
【0008】酸加水分解は、例えば、1〜85重量%程
度の酸を含有するカードランの0.5〜5.0重量%水溶
液を、70〜100℃にて10分〜3時間加熱すること
により行うことができる。加水分解反応液を水酸化ナト
リウム等のアルカリ剤で中和し、遠心分離して上澄を
得、必要に応じて、活性炭処理、透析、溶媒分画、加熱
によるホルミル基の除去等の自体公知の方法で精製する
ことにより、所望の加水分解物が得られる。酵素による
加水分解は、例えば、用いる酵素に適したpH、温度で
所定時間、カードランの0.5〜3重量%水溶液を酵素
処理することにより行うことができる。ついで、酵素を
失活させた後、遠心分離して上澄を得、必要に応じて活
性炭処理、透析、溶媒分画等の自体公知の方法で精製す
ることにより、所望の加水分解物が得られる。In the acid hydrolysis, for example, a 0.5 to 5.0% by weight aqueous solution of curdlan containing about 1 to 85% by weight of an acid is heated at 70 to 100 ° C. for 10 minutes to 3 hours. Can be performed. The hydrolysis reaction solution is neutralized with an alkali agent such as sodium hydroxide and centrifuged to obtain a supernatant, and if necessary, activated carbon treatment, dialysis, solvent fractionation, removal of formyl group by heating, etc. are known per se. The desired hydrolyzate can be obtained by the purification according to the above method. Enzymatic hydrolysis can be performed, for example, by subjecting a 0.5-3% by weight aqueous solution of curdlan to an enzyme treatment at a pH and temperature suitable for the enzyme to be used for a predetermined time. Then, after inactivating the enzyme, a supernatant is obtained by centrifugation, and if necessary, purified by a method known per se such as activated carbon treatment, dialysis, or solvent fractionation, to obtain a desired hydrolyzate. Can be
【0009】得られた加水分解物は、水溶液の状態で加
熱処理に付される。加熱処理は、一般に、80℃以上、
好ましくは、90〜120℃にて、通常、10〜30分
間加熱することにより行う。加熱手段は、特に限定する
ものではなく、加熱後、直ちに室温まで冷却する。[0009] The obtained hydrolyzate is subjected to a heat treatment in the state of an aqueous solution. The heat treatment is generally performed at 80 ° C. or higher,
Preferably, it is carried out by heating at 90 to 120 ° C., usually for 10 to 30 minutes. The heating means is not particularly limited, and is immediately cooled to room temperature after heating.
【0010】加熱処理されたカードラン加水分解物は、
そのまま本発明の免疫抑制剤として使用できる。また、
自体公知の医薬担体または賦形剤と自体公知の方法で合
して、免疫抑制用の医薬とすることができる。用いる、
医薬担体または賦形剤は特に限定するものではなく、当
該免疫抑制剤の具体的用途に応じて当業者が適宜選択で
きる。また、免疫抑制剤の形態も特に限定する物ではな
く、具体的用途に応じて、種々の固体や液体の形態とす
ることができる。本発明の免疫抑制剤は、経口投与、非
経口投与いずれでもよく、その投与量は、カードラン加
水分解物の固形分量として1日当たり4mg〜40gであ
る。本発明の免疫抑制剤は、ステロイド剤に認められる
様々な副作用、核酸合成系に作用する薬剤に認められる
造血器などの重篤な副作用、また、シクロスポリン、F
K506に認められる腎障害、肝障害等の副作用はな
く、また、本発明はBリンパ球の活性化を強度に抑制す
るため、Bリンパ球の異常によって引き起こされる悪性
リンパ腫、全身性エリテマトーデス、慢性関節リウマチ
等の自己免疫疾患またはアレルギー疾患の治療にきわめ
て有用である。さらにTリンパ球の活性化も抑制するた
め、臓器移植の拒絶反応の予防にも有用である。[0010] The heat-treated curdlan hydrolyzate is
It can be used as it is as the immunosuppressant of the present invention. Also,
It can be combined with a known pharmaceutical carrier or excipient by a known method to obtain a drug for immunosuppression. Use,
The pharmaceutical carrier or excipient is not particularly limited, and can be appropriately selected by those skilled in the art according to the specific use of the immunosuppressant. Also, the form of the immunosuppressant is not particularly limited, and can be in various solid or liquid forms depending on the specific application. The immunosuppressant of the present invention may be administered orally or parenterally, and its dosage is 4 mg to 40 g per day as a solid content of the curdlan hydrolyzate. The immunosuppressive agent of the present invention can be used for various side effects observed in steroids, serious side effects such as hematopoietic organs observed in drugs acting on the nucleic acid synthesis system, cyclosporine, F
K506 has no side effects such as renal damage and liver damage, and the present invention strongly suppresses the activation of B lymphocytes, so that malignant lymphomas caused by B lymphocyte abnormalities, systemic lupus erythematosus, chronic joint It is extremely useful for treating autoimmune diseases such as rheumatism or allergic diseases. Furthermore, it suppresses the activation of T lymphocytes, and is therefore useful for preventing rejection of organ transplantation.
【0011】[0011]
【実施例】つぎに、実施例および試験例を挙げて、本発
明をさらに具体的に説明するが、本発明は、これらに限
定されるものではない。 実施例1 酵素によるカードラン加水分解物の調製 カードラン4gを0.05M酢酸緩衝液200mlに分散
し、ヒイロタケ酵素18ユニット/mlを添加した。これ
を40℃に昇温し、4時間インキュベートした後沸騰水
浴中に15分間保持して酵素を失活させた。ついで、冷
却、遠心分離して沈澱部分を除去し、上澄を直径5cmの
高さ30cmのカラムに詰めた活性炭に吸着させ、100
0mlの水で洗浄後、さらに4%エタノール2000mlで
洗浄した。ついで、20%エタノール2000mlで吸着
成分を溶出し、これをエバポレーターで濃縮した後、凍
結乾燥した。このものはHPLCで測定したときの数平
均分子量が340(プルラン換算値)であった。このも
のを、水溶液中で、100℃にて、10分間加熱し、つ
いで冷却して免疫抑制剤を得た。Next, the present invention will be described more specifically with reference to examples and test examples, but the present invention is not limited to these examples. Example 1 Preparation of Curdlan Hydrolyzate with Enzyme 4 g of curdlan was dispersed in 200 ml of 0.05 M acetate buffer, and 18 units / ml of Hilotake enzyme was added. This was heated to 40 ° C., incubated for 4 hours, and then kept in a boiling water bath for 15 minutes to inactivate the enzyme. Then, the precipitate was removed by cooling and centrifugation, and the supernatant was adsorbed on activated carbon packed in a column having a diameter of 5 cm and a height of 30 cm.
After washing with 0 ml of water, it was further washed with 2000 ml of 4% ethanol. Subsequently, the adsorbed component was eluted with 2000 ml of 20% ethanol, and this was concentrated by an evaporator and freeze-dried. This had a number average molecular weight of 340 (pullulan equivalent) as measured by HPLC. This was heated in an aqueous solution at 100 ° C. for 10 minutes, and then cooled to obtain an immunosuppressant.
【0012】実施例2 蟻酸によるカードラン加水分解物の調製 カードラン30グラムを85%蟻酸3000ml中に分散
し、90℃まで加温して20分間保持した。ついで、容
器ごと冷水にさらして室温まで冷却し、エバポレーター
で濃縮した後、5N NaOHで中和してpH7とし、遠
心分離した。上澄はホルミル基を除去するため沸騰水浴
中で120分間加熱したが、このとき、pHが低下した
ので、2N NaOHを添加して7に戻した。このものを
ビスキングチューブ中にいれ純水10リットルに対して
一夜透析し、透析内液を凍結乾燥した。このものをHP
LCで測定したときの数平均分子量はプルラン換算で約
2800であった。このものを、水溶液中で100℃に
て10分間加熱し、ついで冷却して免疫抑制剤を得た。Example 2 Preparation of Curdlan Hydrolyzate with Formic Acid 30 grams of curdlan was dispersed in 3000 ml of 85% formic acid, heated to 90 ° C. and kept for 20 minutes. Then, the whole container was exposed to cold water to cool to room temperature, concentrated by an evaporator, neutralized with 5N NaOH to pH 7, and centrifuged. The supernatant was heated in a boiling water bath for 120 minutes to remove formyl groups. At this time, the pH dropped, and 2N NaOH was added to return to 7. This was placed in a bisking tube, dialyzed overnight against 10 liters of pure water, and the dialysate was freeze-dried. This is HP
The number average molecular weight measured by LC was about 2,800 in terms of pullulan. This was heated in an aqueous solution at 100 ° C. for 10 minutes, and then cooled to obtain an immunosuppressant.
【0013】試験例1 実施例1で得た酵素分解カードランおよび実施例2で得
た蟻酸分解カードランを用いて、マウス脾臓リンパ球増
殖反応に対する酵素分解カードラン熱処理品および蟻酸
分解カードラン熱処理品の作用を調べることにより、酵
素分解カードラン熱処理品および蟻酸分解カードラン熱
処理品のリンパ球代謝活性上昇およびリンパ球増殖の抑
制効果を検証した。マウス(C57BL/6、雌、14
週齢)から無菌的に脾臓を摘出し、RPMI1640培
地中で脾臓を押しつぶし、200メッシュの篩に通し脾
臓細胞浮遊液を得た。脾臓細胞浮遊液の細胞数を自動血
球計測装置により測定した後、細胞数を5×106/ml
の濃度にRPMI1640培地で調製し、96穴組織培
養プレートに1穴あたり100マイクロリットルを播種
した。Bリンパ球増殖刺激物質のリポポリサッカライド
を200マイクログラム/mlの濃度でRPMI1640
培地に溶解した液、Tリンパ球増殖刺激物質のコンカナ
バリンAを8マイクログラム/mlの濃度でRPMI16
40培地に溶解した液、あるいはRPMI1640培地
を、それぞれ1穴当たり50マイクロリットル播種した
脾臓細胞浮遊液に加えて、Bリンパ球刺激群、Tリンパ
球刺激群、無刺激群とした。これらの3群にリン酸緩衝
生理食塩水を100℃、10分間加熱して冷却した液
(対照)あるいは酵素分解カードランを8mg/mlの濃度
でリン酸緩衝生理食塩水に溶解し100℃、10分間加
熱して冷却した液、蟻酸分解カードランを8mg/mlの濃
度でリン酸緩衝生理食塩水に溶解し100℃、10分間
加熱して冷却した液をそれぞれ1穴当たり50マイクロ
リットル加え、37℃の5%炭酸ガス培養器内で2日間
培養し、培養後の生細胞数と細胞代謝活性を調べた。Test Example 1 Using the enzyme-degraded curdlan obtained in Example 1 and the formate-degraded curdlan obtained in Example 2, heat-treated products of enzymatically degraded curdlan and proliferation of formate-degraded curdlan for mouse spleen lymphocyte proliferation reaction By examining the action of the products, the heat-treated products decomposed with enzymatically decomposed curdlan and heat-treated products with decomposed formic acid were examined to increase the lymphocyte metabolic activity and suppress lymphocyte proliferation. Mouse (C57BL / 6, female, 14
Aged), the spleen was aseptically removed, crushed in an RPMI1640 medium, and passed through a 200-mesh sieve to obtain a spleen cell suspension. After measuring the number of cells in the spleen cell suspension using an automatic blood cell counter, the number of cells was determined to be 5 × 10 6 / ml.
Was prepared in RPMI1640 medium to a concentration of 100 μl, and 100 μl per well was seeded on a 96-well tissue culture plate. The B lymphocyte proliferation stimulator lipopolysaccharide was added at a concentration of 200 micrograms / ml to RPMI 1640.
A solution dissolved in a medium, T-lymphocyte proliferation stimulating substance concanavalin A was added at a concentration of 8 microgram / ml to RPMI16.
A solution dissolved in 40 media or an RPMI 1640 medium was added to the spleen cell suspension seeded at 50 microliters per well, to prepare a B lymphocyte stimulation group, a T lymphocyte stimulation group, and a non-stimulation group. A phosphate buffered saline solution heated at 100 ° C. for 10 minutes and cooled (control) or an enzyme-degraded curdlan at a concentration of 8 mg / ml was dissolved in phosphate buffered saline solution at 100 ° C. A liquid cooled by heating for 10 minutes, formic acid decomposed curdlan was dissolved in a phosphate buffered saline at a concentration of 8 mg / ml, and heated and cooled for 10 minutes at 100 ° C., and 50 microliters were added to each well. The cells were cultured in a 5% carbon dioxide incubator at 37 ° C. for 2 days, and the number of living cells and the cell metabolic activity after the culture were examined.
【0014】生細胞数の測定は、培養細胞液の細胞数を
自動血球計測装置で測定した後に、培養細胞液200マ
イクロリットルにR−フィコエリトリンで標識したマウ
スBリンパ球に対する特異抗体の抗マウスCD45R抗
体を1マイクログラム、フルオロセインイソチオシアネ
ートで標識したマウスTリンパ球に対する特異抗体の抗
マウスT細胞レセプター(アルファ/ベータ)抗体を1
マイクログラム、および死細胞を特異的に染色する7−
アミノアクチノマイシンDを1マイクログラム加え、5
℃で30分間放置した後、RPMI1640培地で洗浄
し、フローサイトメーターで総細胞に占めるBリンパ球
およびTリンパ球の割合ならびにBリンパ球およびTリ
ンパ球に占める死細胞の割合を測定し、Bリンパ球およ
びTリンパ球の生細胞数を算出した。細胞代謝活性は、
培養の終わる3時間前に臭化3−(4,5−ジメチル−
2−チアゾリル)−2,5−ジフェニル−2Hテトラゾ
リウムを5mg/mlの濃度でRPMI1640培地に溶解
した液を1穴当たり10マイクロリットル加え、培養終
了時に20%ドデシル硫酸ナトリウム溶液を1穴当たり
50マイクロリットル加え、37℃で1日放置後、マイ
クロプレートリーダーで培養液の吸光度550nmを測定
することにより細胞代謝活性を求めた。表1にその結果
を示す。The number of viable cells was determined by measuring the number of cells in the cultured cell solution using an automatic hemacytometer, and then adding 200 μl of the cultured cell solution to the anti-mouse CD45R specific antibody against mouse B lymphocytes labeled with R-phycoerythrin. One microgram of antibody was used as an anti-mouse T cell receptor (alpha / beta) antibody specific for mouse T lymphocytes labeled with fluorescein isothiocyanate.
7- Specific staining of micrograms and dead cells
Add 1 microgram of aminoactinomycin D and add
After being left at 30 ° C. for 30 minutes, the plate was washed with RPMI1640 medium, and the proportion of B lymphocytes and T lymphocytes in the total cells and the proportion of dead cells in B lymphocytes and T lymphocytes were measured with a flow cytometer. Viable cell counts of lymphocytes and T lymphocytes were calculated. Cell metabolism activity
Three hours before the end of the culture, 3- (4,5-dimethyl-bromide)
A solution of 2-thiazolyl) -2,5-diphenyl-2H tetrazolium in RPMI1640 medium at a concentration of 5 mg / ml was added at 10 μl per well, and at the end of the culture, a 20% sodium dodecyl sulfate solution was added at 50 μl per well. After adding 1 liter and left at 37 ° C. for 1 day, the cell metabolic activity was determined by measuring the absorbance of the culture at 550 nm using a microplate reader. Table 1 shows the results.
【0015】[0015]
【表1】 [Table 1]
【0016】表1に示すごとく、無刺激群においては、
酵素分解カードラン熱処理品および蟻酸分解カードラン
熱処理品はいずれもBリンパ球の生細胞数を軽度にしか
減少させず細胞代謝活性にはほとんど影響を及ぼさなか
ったが、Bリンパ球刺激群においては、酵素分解カード
ラン熱処理品および蟻酸分解カードラン熱処理品はいず
れもBリンパ球の生細胞数を大幅に減少させ細胞代謝活
性の上昇を完全に抑制した。Tリンパ球刺激群において
は、酵素分解カードラン熱処理品および蟻酸分解カード
ラン熱処理品はいずれも細胞代謝活性の上昇を強度に抑
制した。この様に、酵素分解カードラン熱処理品および
蟻酸分解カードラン熱処理品のいずれにも、リンパ球が
活性化されるときにより選択的に働き、強度な抑制作用
が認められた。As shown in Table 1, in the non-stimulated group,
Both the heat-treated products decomposed with enzymatically decomposed curdlan and the heat-treated products with degraded formic acid curd only slightly reduced the viable cell count of B lymphocytes and had little effect on the cell metabolic activity. Both the heat-treated products decomposed with enzymatically decomposed curdlan and the heat-treated products decomposed with formic acid decomposed significantly reduced the number of viable B lymphocytes and completely suppressed the increase in cell metabolic activity. In the T lymphocyte stimulation group, both the heat-treated products decomposed with enzymatically decomposed curdlan and the heat-treated products degraded with formic acid decomposed significantly suppressed the increase in cell metabolic activity. As described above, both the heat-treated products decomposed with enzymatically decomposed curdlan and the heat-treated products decomposed with formic acid degraded more selectively when lymphocytes were activated, and exhibited a strong inhibitory action.
【0017】試験例2 ラミナリビオース、ラミナリトリオース、ラミナリテト
ラオース、ラミナリペンタオースおよびラミナリヘキサ
オース(焼津水産化学)の各試薬を用いて、マウス脾臓
リンパ球増殖反応に対する加熱処理ラミナリビオース、
加熱処理ラミナリトリオース、加熱処理ラミナリテトラ
オース、加熱処理ラミナリペンタオース、加熱処理ラミ
ナリヘキサオースの作用を調べることにより、加熱処理
ラミナリビオース、加熱処理ラミナリトリオース、加熱
処理ラミナリテトラオース、加熱処理ラミナリペンタオ
ース、加熱処理ラミナリヘキサオースのリンパ球代謝活
性上昇抑制効果を検証した。マウス(C57BL/6、
雌、18週齢)から無菌的に脾臓を摘出し、RPMI1
640培地中で脾臓を押しつぶし、200メッシュの篩
に通し脾臓細胞浮遊液を得た。脾臓細胞浮遊液の細胞数
を自動血球計測装置により測定した後、細胞数を5×1
06/mlの濃度にRPMI1640培地で調製し、96
穴組織培養プレートに1穴あたり100マイクロリット
ルを播種した。Bリンパ球増殖刺激物質のリポポリサッ
カライドを200マイクログラム/mlの濃度でRPMI
1640培地に溶解した液、Tリンパ球増殖刺激物質の
コンカナバリンAを8マイクログラム/mlの濃度でRP
MI1640培地に溶解した液、あるいはRPMI16
40培地を、それぞれ1穴当たり50マイクロリットル
播種した脾臓細胞浮遊液に加えて、Bリンパ球刺激群、
Tリンパ球刺激群、無刺激群とした。これらの3群にリ
ン酸緩衝生理食塩水を121℃、20分間加熱して冷却
した液(対照)あるいはラミナリビオース、ラミナリト
リオース、ラミナリテトラオース、ラミナリペンタオー
ス、ラミナリヘキサオースを8mg/mlの濃度あるいは1
mg/mlの濃度でリン酸緩衝生理食塩水に溶解し121
℃、20分間加熱して冷却した液、をそれぞれ1穴当た
り50マイクロリットル加え、37℃の5%炭酸ガス培
養器内で1日間培養し、培養後の細胞代謝活性を調べ
た。Test Example 2 Laminaribiose, laminaritriose, laminaritetraose, laminaripentaose and laminarihexaose (Yaitsu Fisheries Chemical) were used to heat-treat the spleen lymphocyte proliferation reaction in mice. Liviose,
By examining the effects of heat-treated laminaritriose, heat-treated laminaritetraose, heat-treated laminaripentaose, and heat-treated laminarihexaose, heat-treated laminaribiose, heat-treated laminaritriose, and heat-treated laminaritetraose The effect of aose, heat-treated laminaripentaose and heat-treated laminarihexaose on the increase in lymphocyte metabolic activity was verified. Mouse (C57BL / 6,
Female, 18 weeks old).
The spleen was crushed in 640 medium and passed through a 200-mesh sieve to obtain a spleen cell suspension. After measuring the number of cells in the spleen cell suspension using an automatic blood cell counter, the number of cells was determined to be 5 × 1.
In RPMI1640 medium was prepared to a concentration of 0 6 / ml, 96
100 microliters per well were seeded in a well tissue culture plate. The B lymphocyte proliferation stimulator lipopolysaccharide was added at a concentration of 200 micrograms / ml in RPMI.
A solution dissolved in 1640 medium, a T lymphocyte proliferation stimulating substance, concanavalin A, was added at a concentration of 8 microgram / ml to RP.
Solution dissolved in MI1640 medium or RPMI16
Forty B medium was added to the spleen cell suspension sown at 50 microliters per well, and the B lymphocyte stimulation group was added.
A T lymphocyte stimulation group and a non-stimulation group were set. To these three groups, a solution prepared by heating and cooling phosphate buffered saline at 121 ° C. for 20 minutes (control) or laminaribiose, laminaritriose, laminaritetraose, laminaripentaose and laminarihexaose were added. 8mg / ml concentration or 1
dissolved in phosphate buffered saline at a concentration of mg / ml
The solution cooled by heating at 20 ° C. for 20 minutes was added in an amount of 50 μl / hole, and cultured in a 5% CO 2 incubator at 37 ° C. for 1 day, and the cell metabolic activity after the culture was examined.
【0018】細胞代謝活性は、培養の終わる3時間前に
臭化3−(4,5−ジメチル−2−チアゾリル)−2,5
−ジフェニル−2Hテトラゾリウムを5mg/mlの濃度で
RPM11640培地に溶解した液を1穴当たり10マ
イクロリットル加え、培養終了時に20%ドデシル硫酸
ナトリウム溶液を1穴当たり50マイクロリットル加
え、37℃で2日放置後、マイクロプレートリーダーで
培養液の吸光度550nmを測定することにより細胞代謝
活性を求めた。表2にその結果を示す。The cell metabolic activity was determined by measuring 3- (4,5-dimethyl-2-thiazolyl) -2,5 bromide 3 hours before the end of the culture.
10 microliters per well of a solution of diphenyl-2H tetrazolium dissolved in RPM11640 medium at a concentration of 5 mg / ml was added at the end of the culture, and 50 microliters per well of a 20% sodium dodecyl sulfate solution was added at 37 ° C for 2 days. After standing, the cell metabolic activity was determined by measuring the absorbance of the culture at 550 nm using a microplate reader. Table 2 shows the results.
【0019】[0019]
【表2】 [Table 2]
【0020】表2に示すごとく、無刺激群においては、
加熱処理ラミナリビオース、加熱処理ラミナリトリオー
ス、加熱処理ラミナリテトラオース、加熱処理ラミナリ
ペンタオース、加熱処理ラミナリヘキサオースはいずれ
も細胞代謝活性にはほとんど影響を及ぼさなかったが、
Bリンパ球刺激群においては、加熱処理ラミナリビオー
ス、加熱処理ラミナリトリオース、加熱処理ラミナリテ
トラオース、加熱処理ラミナリペンタオース、加熱処理
ラミナリヘキサオースはいずれも2mg/mlおよび0.4
mg/mlの濃度において細胞代謝活性の上昇を完全に抑制
した。Tリンパ球刺激群においては、加熱処理ラミナリ
ビオース、加熱処理ラミナリトリオース、加熱処理ラミ
ナリテトラオース、加熱処理ラミナリペンタオース、加
熱処理ラミナリヘキサオースはいずれも2mg/mlの濃度
において細胞代謝活性の上昇を完全に抑制したが、0.
4mg/mlの濃度においては、加熱処理ラミナリビオー
ス、加熱処理ラミナリトリオースのみが細胞代謝活性の
上昇を部分抑制した。この様に、加熱処理ラミナリビオ
ース、加熱処理ラミナリトリオース、加熱処理ラミナリ
テトラオース、加熱処理ラミナリペンタオース、加熱処
理ラミナリヘキサオースはいずれも、リンパ球が活性化
されるときにより選択的に働き、強度な抑制作用を示
し、特に、Bリンパ球に対する作用が強く、また、リン
パ球抑制活性は加熱処理ラミナリビオースおよび加熱処
理ラミナリトリオースが強かった。As shown in Table 2, in the unstimulated group,
Heat-treated laminaribiose, heat-treated laminaritriose, heat-treated laminaritetraose, heat-treated laminalipentaose, and heat-treated laminalihexaose hardly affected cell metabolic activity,
In the B lymphocyte stimulation group, heat-treated laminaribiose, heat-treated laminaritriose, heat-treated laminaritetraose, heat-treated laminaripentaose, and heat-treated laminarihexaose were all 2 mg / ml and 0.4 mg / ml.
At a concentration of mg / ml, the increase in cell metabolic activity was completely suppressed. In the T lymphocyte stimulation group, heat-treated laminaribiose, heat-treated laminaritriose, heat-treated laminaritetraose, heat-treated laminaripentaose, and heat-treated laminarihexaose were all cells at a concentration of 2 mg / ml. Although the increase in metabolic activity was completely suppressed, it was confirmed that the metabolic activity was increased by 0.1%.
At a concentration of 4 mg / ml, only heat-treated laminaribiose and heat-treated laminaritriose partially suppressed the increase in cell metabolic activity. Thus, the heat-treated laminaribiose, the heat-treated laminaritriose, the heat-treated laminaritetraose, the heat-treated laminaripentaose, and the heat-treated laminarihexaose are all more selective when lymphocytes are activated. In particular, it exhibited a strong inhibitory action, and particularly had a strong action on B lymphocytes. The lymphocyte inhibitory activity was strong in heat-treated laminaribiose and heat-treated laminaritriose.
【0021】[0021]
【発明の効果】本発明によれば、重篤な副作用のない、
優れた免疫抑制剤が提供される。According to the present invention, there are no serious side effects,
An excellent immunosuppressant is provided.
───────────────────────────────────────────────────── フロントページの続き (72)発明者 日下 博昭 兵庫県宝塚市安倉南3丁目3番1−202号 ────────────────────────────────────────────────── ─── Continuation of front page (72) Inventor Hiroaki Kusaka 3-3-1202 Asakuraminami, Takarazuka City, Hyogo Prefecture
Claims (8)
結合からなる直鎖の糖類を有効成分とする免疫抑制剤。An immunosuppressant comprising a heat-treated linear saccharide comprising a β-1,3-glucoside bond as an active ingredient.
鎖の糖類がカードラン加水分解物である請求項1記載の
免疫抑制剤。2. The immunosuppressant according to claim 1, wherein the linear saccharide comprising a β-1,3-glucoside bond is a curdlan hydrolyzate.
2記載の免疫抑制剤。3. The immunosuppressant according to claim 2, wherein the hydrolyzate is an acid hydrolyzate.
求項3記載の免疫抑制剤。4. The immunosuppressant according to claim 3, wherein the acid used for the acid hydrolysis is formic acid.
による加水分解物である請求項2記載の免疫抑制剤。5. The immunosuppressant according to claim 2, wherein the hydrolyzate is a hydrolyzate by β-1,3-glucanase.
鎖の糖類の数平均分子量が340から4000の範囲に
ある請求項1〜5いずれか1項記載の免疫抑制剤。6. The immunosuppressant according to claim 1, wherein the linear saccharide comprising a β-1,3-glucoside bond has a number average molecular weight in the range of 340 to 4000.
である請求項1〜6いずれか1項記載の免疫抑制剤。7. The immunosuppressant according to claim 1, wherein the heat treatment is performed at a temperature of 80 ° C. or higher in an aqueous solution.
請求項1〜7いずれか1項記載の免疫抑制。8. The immunosuppression according to claim 1, wherein the immunosuppression action is a lymphocyte suppression action.
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JP00652497A JP4091137B2 (en) | 1997-01-17 | 1997-01-17 | Immunosuppressant |
Applications Claiming Priority (1)
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JP00652497A JP4091137B2 (en) | 1997-01-17 | 1997-01-17 | Immunosuppressant |
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JPH10194976A true JPH10194976A (en) | 1998-07-28 |
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ID=11640767
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Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2002098433A1 (en) * | 2001-06-01 | 2002-12-12 | Ajinomoto Co., Inc. | Drugs for intestinal diseases |
JP2009148206A (en) * | 2007-12-20 | 2009-07-09 | Kirin Food-Tech Co Ltd | Composition for activating dendritic cell |
WO2009068996A3 (en) * | 2007-11-26 | 2009-12-23 | Novartis Ag | Conjugated beta-1,3-linked glucans |
JP2022537533A (en) * | 2019-06-14 | 2022-08-26 | 正大制▲薬▼(青▲島▼)有限公司 | β-Glucan composition and use thereof |
-
1997
- 1997-01-17 JP JP00652497A patent/JP4091137B2/en not_active Expired - Lifetime
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2002098433A1 (en) * | 2001-06-01 | 2002-12-12 | Ajinomoto Co., Inc. | Drugs for intestinal diseases |
WO2009068996A3 (en) * | 2007-11-26 | 2009-12-23 | Novartis Ag | Conjugated beta-1,3-linked glucans |
US9439954B2 (en) | 2007-11-26 | 2016-09-13 | Glaxosmithkline Biologicals Sa | Conjugated beta-1,3-linked glucans |
US9439955B2 (en) | 2007-11-26 | 2016-09-13 | Glaxosmithkline Biologicals Sa | Conjugated β-1,3-linked glucans |
JP2009148206A (en) * | 2007-12-20 | 2009-07-09 | Kirin Food-Tech Co Ltd | Composition for activating dendritic cell |
JP2022537533A (en) * | 2019-06-14 | 2022-08-26 | 正大制▲薬▼(青▲島▼)有限公司 | β-Glucan composition and use thereof |
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