JPS5883619A - Anti-inflammatory agent - Google Patents

Abstract

PURPOSE:An anti-inflammatory agent, containing 3,4-dihydroxybenzaldehyde as an active constituent, and having the inhibitory action on granuloma, adjuvant arthritis, leukocytoplania and blood platelet agglutination with low toxicity. CONSTITUTION:An anti-inflammatory agent, containing 3,4-dihydroxybenzaldehyde as an active constituent, and applicable in various dosage forms or alone or a mixed form with a parmaceutically acceptable diluent and another drug. For improving the irritant action, the following methods may be used: The active constituent may be changed into a clathrate compound with a compound, e.g. cyclodextrin, having the entrapping ability or a mixture or condensate with various amines, amino acids or saccharide for use. The content of the active constituent in the pharmaceutical can be adjusted to a wide range of 0.01- 100wt%, preferably 0.1-70wt%.

Description

【発明の詳細な説明】 本発明は３．４−ジヒドロキシベンツアルデヒドを有効
成分とする抗炎症作用剤に係る。DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an anti-inflammatory agent containing 3,4-dihydroxybenzaldehyde as an active ingredient.

本発明５４郷は樵々の物質の抗炎症性について検討して
いる過程において、６．４−ジヒドロキシベンツアルデ
ヒド（以下、本物質と称する）が優れた抗炎症作用を有
することを見い出し、本＠明に至つ九。In the course of studying the anti-inflammatory properties of woodcutter substances, the present invention 54 Go discovered that 6,4-dihydroxybenzaldehyde (hereinafter referred to as the substance) has excellent anti-inflammatory properties, and 9 to light.

本物質自体は各種の食用植物中に存在する公知物質であ
り、制癌剤としての提＊（特開１１８５５−５１０１８
）がなされているｏしかしながら、その作用機序につい
ては殆んど明らかで・なく、また、本物質の抗炎症作用
についての報告はこれまでに見い出されない。This substance itself is a known substance that exists in various edible plants, and has been suggested as an anticancer agent* (Japanese Patent Application Laid-Open No. 11855-51018).
) However, its mechanism of action is largely unclear, and no reports have been found so far regarding the anti-inflammatory effect of this substance.

本発明者等は本物質の抗炎症作用を梅々の方法により検
鹸した結果、肉芽−増殖抑制作用、アジュバント関節炎
抑制作用、白血球遊走抑制作用及び皿小坂凝集抑制作用
を有し、又、毒性も−いことより、抗炎症、抗リウマチ
剤としての適性を有することの知見を得て本発明に至っ
た。As a result of testing the anti-inflammatory effect of this substance using the Umeme method, the present inventors found that it has an inhibitory effect on granulation proliferation, an inhibitory effect on adjuvant arthritis, an inhibitory effect on leukocyte migration, an inhibitory effect on Sarakosaka aggregation, and toxicity. More than that, we have found that it has suitability as an anti-inflammatory and anti-rheumatic agent, leading to the present invention.

以下、本物質の毒物学的特性及び薬理学的特性について
説明する０ （１）急性毒性 雌性ＪＯＬ−ＩＯＲ系マウスを用いそ経口及び腹腔内投
与経路における急性毒性を調べた。経口投与はα２　＠
　ｏｍｏ　Ｋ溶解分散したものを、腹腔内投与は生理食
塩水に溶解しえものをそれぞれ両ゾンデ又は注射筒を用
いて所定の量にｌＩ［シて与えた０ 投与後中毒症状の観察を続け７日間までの経時的死亡率
を求め、Ｌｉｔａｈｆｉ＊１ｄ−Ｗｉｌｏｏｘｏｎ図針
算法によりＬＤ５ｏ値を求め次。The toxicological and pharmacological properties of this substance will be explained below. (1) Acute Toxicity Female JOL-IOR mice were used to examine acute toxicity by oral and intraperitoneal administration routes. Oral administration is α2 @
For intraperitoneal administration, the dissolved and dispersed Omo K was dissolved in physiological saline and given to the prescribed amount using both probes or syringes.Continue to observe symptoms of toxicity after administration. The mortality rate over time was determined for up to 1 day, and the LD5o value was determined using the Litahfi*1d-Wilooxon chart calculation method.

（２）変異原性 次のような手法により調べた０ ［ＨＩＨｍ欠損株（Ｂａｃｉｌｌｕｓ　５ｕｂｔｉｌｉ
ｓ　Ｍ　４５　）と組換修復保持株（Ｂａｃｉｌｌｕｓ
　５ｕｂｔｉｌｉｓ　Ｈ１７）を用い、小型ピペットに
Ｂ−１寒天培地（＾エキス１０ｇ１ボリペゾトン１０１
１％Ｎａ０Ａ　５１１、寒天１５ｇ、蒸留水１０００ｍ
、−７Ｏ）上に出発点が接触しないように画線した０ 本物質をｌ）Ｍ２Ｏに醇解し、その０．０５−を直径８
ｍの円型Ｆ紙にしみこませ１１画縁の開始点をおおうよ
うＫｆｌｌき、６１℃で一晩培養後、生前阻止域の長さ
を測定した。陽性対照としてカナマイシン、陽性対照と
してマイトマイシンを用いた０結果を下表に示したが、
この結果から本物質は変異原性を示さながっ之。(2) Mutagenicity [HIHm-deficient strain (Bacillus 5ubtili
s M 45 ) and a recombinant repair carrier strain (Bacillus
5ubtilis H17) and B-1 agar medium (^Extract 10g1 Boripezoton 101) in a small pipette.
1% Na0A 511, agar 15g, distilled water 1000m
, -7O) with a line drawn so that the starting points do not touch. Dissolve this substance in M2O,
The mixture was soaked into a circular F paper of size 11, covered with the starting point of the 11th edge, and after culturing overnight at 61°C, the length of the antenatal inhibition zone was measured. The table below shows the results using kanamycin as a positive control and mitomycin as a positive control.
These results indicate that this substance is mutagenic.

□ （ｊ）　　血小板凝集抑制作用 血小板はウサギ（ぼうさぎ在来ｍｓ）の耳静脈よ４１８
％チト２−ト（ミドリ十字）存在下で採血し、遠心分離
して多血小板血ｖＲ（ＰＲＰ　）及び貧血小板血漿（Ｐ
ＰＰ　）を得、＃集反応に除し、ＰＲＰをＰＩ’Ｐで希
釈して血小板数３０万／μｔに調整し友ものを用い友。□ (j) Platelet aggregation inhibitory effect Platelets are found in the ear veins of rabbits (Bosagi ms)418
Blood was collected in the presence of %cyto2-to (green cross) and centrifuged to produce platelet-rich blood vR (PRP) and platelet-poor plasma (PRP).
PP) was obtained, divided into #collection reaction, diluted PRP with PI'P to adjust the platelet count to 300,000/μt, and used a platelet.

凝集剤とし−て用いたアラキドン酸はＭｕ塩（ｓｉｇｍ
ａ　ＩＩ　）を用い、生理食塩水に浴解し、血小板に添
加した。本物質は生理食塩水に溶解し、血小板に所定量
象加した０又、対照粂としてナノロキセンを用いたが、
水に不溶のため、Ｈａ塩として生理食塩水に溶解し血小
板に所定＊ｇｓ加した。Arachidonic acid used as a flocculant was Mu salt (sigm
a II) was dissolved in physiological saline and added to platelets. This substance was dissolved in physiological saline and added to platelets in a predetermined amount, and nanoloxene was used as a control.
Since it is insoluble in water, it was dissolved in physiological saline as a Ha salt and added to platelets in a predetermined amount *gs.

凝集反応は二元バイオサイエンス社製プレートレット・
アゲリゾ−ジョントレーサーＦＡＴ−４ムによ砂＃ｊ定
し九〇 本物質のｌ０１００は７５μＭまた対照桑ナゾロキセン
の工Ｃ１゜０は１４０μＭであつ九。この結果から本物
質が血小板凝集を抑制する効果を有することか明らかで
ある。The agglutination reaction was performed using Binary Bioscience platelets.
The 10100 of the 90 substances determined by agelisolution tracer FAT-4 was 75 μM, and the control C10 of the mulberry nazoloxene was 140 μM. From this result, it is clear that this substance has the effect of suppressing platelet aggregation.

（４）多形核白血球遊走抑制作用（ｉｎ　ｖｉｔｒｏ）
ラットの多核白血球を用いＢｏｙｌ＠ｎ法〔「日本臨床
Ｊ２７！、９号、１７２負（１９６９））に準拠して行
つ九〇即ち、酵引物質として大腸菌（ＩＩｌ、　０Ｏ１
ｉ　）の培ｇＩＰ液と血清と０１０：１の混合物を３７
℃で１時間インキエベートし、それを生理食塩水で５倍
に希釈したものを用い、供試薬は多核白血球浮遊液と１
０分間ルインキエペートしたのち、多核白血球の遊走を
測定した。(4) Polymorphonuclear leukocyte migration inhibitory effect (in vitro)
Using rat polynucleated leukocytes, the method was carried out according to the Boyl@n method [Japan Clinical J27!, No. 9, 172 Negative (1969)].
i) A mixture of culture medium gIP solution and serum at a ratio of 010:1 to 37%
℃ for 1 hour and then diluted 5 times with physiological saline.
After incubation for 0 minutes, the migration of polynuclear leukocytes was measured.

本物質は生理食塩水に溶解し、白血球層に１００μＭに
なるよう添加した。又、対照桑ナゾロキセン及びインド
メタシンは水に不溶のためＭ＆塩として系内の濃［１０
０μＭになるよう添加した。薬量１００声麓における本
物質、ナデロキ竜ン及びインドメタシンによる多形核白
血球の遊走の抑制率はそれぞれ４９，０．２６ｍ１で本
物質の遊走阻止活性はかなり強いことが判つ九〇 （５）多形核白血球遊走抑制作用（ｉｎ　ｖｉｖｏ）実
験は５退会の呑竜０１を用い、０Ｍ０ｐｏｕａｈ法（石
川ら、県誌８８１４７２，１９６８　＞に準拠し、炎症
部位への多形核白血球の浸出試験を行った。本物質及び
対照系インドメタシンは０．２−の〇ＭＯに分散させ所
定量を経口により与え九。This substance was dissolved in physiological saline and added to the white blood cell layer at a concentration of 100 μM. In addition, the control mulberry nazoloxene and indomethacin are insoluble in water, so they are used as M&salt to reduce the concentration [10
It was added to 0 μM. The inhibition rate of polymorphonuclear leukocyte migration by this substance, nadelochirin, and indomethacin at a dose of 100 doses was 49 and 0.26 m1, respectively, indicating that the migration-inhibiting activity of this substance is quite strong.90(5) Polymorphonuclear leukocyte migration inhibitory effect (in vivo) experiments were carried out using Donryu 01 with 5 withdrawals, according to the 0M0pouah method (Ishikawa et al., Prefectural Journal 881472, 1968), and an exudation test of polymorphonuclear leukocytes into the inflamed site was performed. The present substance and the reference indomethacin were dispersed in 0.2-〇MO and given orally in the prescribed amounts9.

ＯＭＯ注入後、３．＜Ｓ＋２４ｈｒｌに浸出を中の多形
核白血球数を測定した結果は次の通シであり、本物質は
多形核白血球の炎症部位への浸出を抑制す石ことが判っ
た。After OMO injection, 3. The results of measuring the number of polymorphonuclear leukocytes in the exudate at <S+24 hrl were as follows, indicating that this substance inhibits the exudation of polymorphonuclear leukocytes into the inflamed site.

（６）　　肉芽橿抑制作用 実験は５退会の呑竜（６）を用い、鋤村勢の方法〔応用
鉄塩１９（３）、３２９（１９８０））に準じて行なり
九。ペーパーディスクは８■φ×α２６■（１）のＰ紙
を２１１０ＭＯ溶液（ジヒドロキシストレ！トｉイシン
、ペニシリン１００万単位のもの（Ｌ　１　ｇｖ／−含
む）に浸漬処理し友ものを用い友。このディスク２枚を
ラットの背部皮下へエーテル麻酔下に埋め込んだ。被検
桑は０４％　　　　（ＯＭＯ溶液に分散させ１０日間投
与、１１日倣に肉芽摘出し、肉芽腫の重量を測定した。(6) Granulation suppression effect experiments were conducted using Donryu (6) with 5 withdrawals according to the method of Sei Sukumura [Applied Iron Salts 19 (3), 329 (1980)]. The paper disk was made by immersing P paper of 8 mm φ x α 26 mm (1) in a 2110 MO solution (dihydroxystretolysin, 1 million units of penicillin (including L 1 gv/-)). Two of these discs were implanted subcutaneously in the back of a rat under ether anesthesia. The mulberry to be tested was dispersed in 04% (OMO) solution and administered for 10 days. On the 11th day, the granulomas were removed and the weight of the granulomas was measured.

。 ラット菌数ニ一群５匹 上記結果から理解されるように本物質は体重抑制もなく
増殖性肉芽腫を抑制することが判る〇又、試験後解剖し
て胃粘膜の観察及び胸腺の重量一定を行つ九結果、イン
ドメタシン及びプレドニゾロン投与群に両粘膜の出血及
び債、環形成が見られ、又、プレドニゾロン投与群では
有意に胸腺の萎縮が見られ九〇本物質投与群は００ｎｔ
ｒＯｊ群に比較して何ら異常を誌めなかった。. As can be understood from the above results, this substance suppresses proliferative granulomas without suppressing body weight.After the test, dissection was performed to observe the gastric mucosa and to keep the weight of the thymus constant. As a result, bleeding of both mucous membranes, bonds, and ring formation were observed in the indomethacin and prednisolone administration group, and significant thymus atrophy was observed in the prednisolone administration group, and 00 nt was observed in the prednisolone administration group.
No abnormalities were observed compared to the rOj group.

７）　アジュバント関節炎抑制作用 アジュバント関節炎の発症予防効果を８退会のＪＯＬ−
１ｉｉＤ系ラツト（す★一群６匹として用い、藤平らの
方法（応用系［１５（２）、１６９．１９７１）に従っ
て−べた。即ち、エーテル麻酔し九ラットの尾にνｒ＠
ｕｎｄのｏｏｍｐｘｅｔｓアゾユパント（０，６岬／α
１−）を接種した０接種２遍間後、被検桑を一゛日−回
、２０日間連続して経口投与し九〇その結果を第１図に
示した。7) Adjuvant arthritis inhibitory effect Adjuvant arthritis prevention effect 8 withdrawal JOL-
1iiD rats (6 rats per group) were used according to Fujihira's method (Applied System [15(2), 169.1971). Namely, 9 rats were anesthetized with ether and νr@
und's oompxets Azoyu Panto (0,6 Misaki/α
After 2 inoculations with 1-), the test mulberry was orally administered once a day for 20 consecutive days.The results are shown in Figure 1.

結果から明らかなように本物質はアジュバント関節炎の
竺療効釆を示した。又、対照率ルドｘ＝　ｆ　１２ン投
与評は体重を有意に抑制し、解剖して摘出し九胸腺重量
の有意な萎縮が紹められたが本物質投与群では、体重抑
制及び胸ｓｅａｍは認められず、副作用も少ないことが
判った。As is clear from the results, this substance showed significant therapeutic efficacy for adjuvant arthritis. In addition, in the control rate Rudo x = f12, the body weight was significantly suppressed and a significant atrophy of the nine thymus weight was observed after dissection, but in the group administered with this substance, body weight was suppressed and the breast seam was significantly reduced. It was found that there were no side effects, and there were few side effects.

従って、本物質はリュウマチ勢の慢性炎症の治療剤とし
て有効である。Therefore, this substance is effective as a therapeutic agent for chronic rheumatic inflammation.

以上の結果よｐ１本物質はすぐれた肉芽−増殖抑制作用
、アジュバント関節炎抑制作用、白血球遊走抑制作用及
び血小板凝集抑制作用を有し、しかも低毒性であること
が理糎できる。従って、本物質は抗炎症剤及び慢性関節
リウマチ、全身性エリテマトーデス（ＳＬＲ）等の抗リ
ウマチ剤として極めて有用な用途を有する。From the above results, it can be concluded that this substance has excellent granulation-proliferation inhibitory activity, adjuvant arthritis inhibitory activity, leukocyte migration inhibitory effect, and platelet aggregation inhibitory effect, and is low in toxicity. Therefore, this substance has extremely useful uses as an anti-inflammatory agent and an anti-rheumatic agent for chronic rheumatoid arthritis, systemic lupus erythematosus (SLR), and the like.

次に１本物質を上記治療剤として適用するための製剤化
について６明する。Next, the formulation of this substance for use as the above-mentioned therapeutic agent will be explained.

本物質は種々の形態で適用できる。また、本物質は単独
又は１１桑上許軒しうる希釈剤および他の薬剤との混合
物形論でも使用できる。This substance can be applied in various forms. The substances can also be used alone or in mixtures with acceptable diluents and other agents.

この際、本物質の刺激性を教書するために、本物質をシ
クμデキストリン等の包ＩＩ耗を有する化合物を用い包
接化合物とすることや、各権のアイン、ア々ノ績もしく
はｍ類との混合物又は縮合物としてから使用することも
有効である。At this time, in order to explain the irritating properties of this substance, it is recommended to make this substance into a clathrate compound using a compound that has a claus II abolition such as cycloμ dextrin, or to make it a clathrate compound using a compound that has a class II content such as cycloμ dextrin, or to make it a clathrate compound. It is also effective to use it as a mixture or condensate with.

本物質は経口的又は非経口的にも適用できるので、それ
らの投与に適した、任意の形態をと９得る０さらに、本
物質燻投県単位形で提供する仁とができ、有効条量が含
有されていれば散剤、頼粒、錠剤、糖衣錠、カブ★ル、
座薬、＃Ｉ！濁剤、液剤、乳剤、アンプル、注射液など
の種々の形態をとり得る。Since the substance can be applied orally or parenterally, any form suitable for such administration can be obtained.In addition, the substance can be provided in smoked dosage form and administered in effective doses. If it contains powders, grains, tablets, sugar-coated tablets, capsules,
Suppositories, #I! It can take various forms such as suspension, liquid, emulsion, ampoule, and injection solution.

Ｌｔがって、本発明の薬剤は従来公知のいがなる製剤化
手段の適用によっても調製可能であると理解すべきであ
る。なお、本発明の薬剤における本物質（有効成分）の
含量はａ０１〜１００５Ｇ、好ましくは０．１〜７ｏ−
（重量）の広範囲に調整できる。It should therefore be understood that the medicaments of the invention can also be prepared by applying conventionally known pharmaceutical formulation means. The content of this substance (active ingredient) in the drug of the present invention is a01-1005G, preferably 0.1-7o-
(weight) can be adjusted over a wide range.

本発明の薬剤は前述したようにヒトおよび動物に対して
経口的もしくは非軽口的に投与されるが、特に経口投与
が好ま、しい。この場合、経口投与は舌下投与も包含す
るものであり、非経口投与は、皮下、筋肉、静脈などの
注射ならびに点滴を包含′する。As mentioned above, the drug of the present invention is administered orally or non-administered to humans and animals, and oral administration is particularly preferred. In this case, oral administration also includes sublingual administration, and parenteral administration includes subcutaneous, intramuscular, intravenous, etc. injections and infusions.

本実＠薬剤の投与ｔは対象が動物かヒトにより、又年令
、個人差、病状などに影響されるので、場合によっては
下記範囲外量を投与する場合も生ずるが、一般にヒトを
対象ナーる場合、本物質の紗目的投与量は体重１〜．１
日ａＧ０．１〜５００ｑ。The actual administration of a drug depends on whether the subject is an animal or a human, and is influenced by age, individual differences, medical conditions, etc. In some cases, doses outside the range shown below may be administered, but in general, humans are the subject. When using this substance, the intended dose of this substance is 1-. 1
Day aG0.1-500q.

好ましくはα５〜２ｏｏｑであり、非鹸口的投与量は体
重ＩＫｆ、１日当シ０．０１〜２００ｖａｐ、好ましく
は、０．１〜１００ｗを１回〜４回に分けて投与する。Preferably it is α5 to 2 ooq, and the non-oral dose is 0.01 to 200 vap per day based on body weight IKf, preferably 0.1 to 100 w, administered in 1 to 4 divided doses.

以下に実施例として本発明の薬剤の製剤化の具体例を示
す◇実施例中の部は特記しない眠り重量を示す。Specific examples of formulation of the drug of the present invention are shown below as Examples.◇The parts in Examples indicate sleeping weights not otherwise specified.

実施１１１ 本物質　　　　　　　　　　　　　１０部重質酸化マグ
ネシウム　　　　　　　　　１５ｇ乳１Ｉ１１７５Ｉ を均一に混合して粉末、又は″−粉粒状して散剤とする
、又この散剤をカプセル容器に入れてカプセルとする。Example 111 10 parts of this substance, 15 g of heavy magnesium oxide, 1I1175I of milk were uniformly mixed to form a powder or a powder, and the powder was placed in a capsule container to form a capsule.

実施例２ 本物質　　　　　　　　　　　　４５部デンプン　　　
　　　　　　　　１５Ｍ乳糖　　　　　　１６Ｉ 結晶セル四−ス　　　　　　　　２１Ｍ−リピニルアル
コール　　　　　　　　　６Ｉ水　　　　　　　　　　
　　　　　　　　　　　３０　Ｉ−を均一に混合して捏
和後、破砕造粒し、乾燥し、■別して顆粒剤とする。′ 実施例６ 実施例２で得られた顆粒剤９６部にステアリン酸カルシ
ウム４部を加え圧縮成形して直径１０ｍの錠剤とする。Example 2 This substance 45 parts starch
15M Lactose 16I Crystal Cellulose 21M-Lipinyl Alcohol 6I Water
After uniformly mixing and kneading 30 I-, the mixture is crushed and granulated, dried, and separated into granules. ' Example 6 4 parts of calcium stearate was added to 96 parts of the granules obtained in Example 2, and the mixture was compressed to form tablets with a diameter of 10 m.

実施例４ 本物質　　　　　　　　　　　　９４部ポリビニルアル
コール　　　　　　　　　６ｇ水　　　　　　　　　　
　　　　　　　　　　３０　＃を用いて実施例２と同様
にして顆粒剤とする。得られ九顆粒の９０部に結晶セル
ロース１０部を加えて圧縮成形して直径８■の錠剤とし
、これにシロッジゼラチン沈降性炭酸カルシウムを加え
翔衣錠とする■ 実施例５ 本物質　　　　　　　　　　　　１０部ペンシルアルコ
ール　　　　　　　６＃生理食塩゛水　　　　　　　　
　　８７Ｉを加え加温混合後滅劇して注射剤とする。Example 4 This substance 94 parts Polyvinyl alcohol 6g Water
Granules were prepared in the same manner as in Example 2 using #30. 10 parts of crystalline cellulose is added to 90 parts of the obtained nine granules and compressed to form tablets with a diameter of 8 cm. To this, silodge gelatin precipitated calcium carbonate is added to form tablets. Example 5 10 parts of this substance Pencil alcohol 6# physiological saline water
Add 87I, heat and mix, and sterilize the mixture to obtain an injection.

【図面の簡単な説明】[Brief explanation of the drawing]

第１図社アジエパント関節炎に対する抗発症作用の実験
結果を示す図である。 代理人　　　川　　　目　　　義　　　雄第１図 ↑　　　　→　Ｔジュバ〉トｊ１ゴ侵日数ｉＩＬ削根与
開始FIG. 1 is a diagram showing the experimental results of the anti-onset effect against Agiepantoarthritis. Agent Yoshio Kawame Figure 1 ↑ → T Juba〉Start reducing the number of days of intrusion into Japan

Claims (1)

【特許請求の範囲】 ＜１）５．４−ジヒドロキシベンツアルデヒドを主成分
とする抗炎症作用剤０ （２）抗リウマチ剤、であることを特徴とする特許請求
の範ｖ！Ａ第（１）項に記載の抗炎症作用剤０（３）抗
炎症剤であることを特徴とする特許請求の範囲第（１）
ＩＩに記載の抗炎症作用剤０[Scope of Claims] <1) An anti-inflammatory agent containing 5,4-dihydroxybenzaldehyde as a main component (2) An anti-rheumatic agent v! Anti-inflammatory agent 0 (3) Claim No. (1) characterized in that it is an anti-inflammatory agent as described in A. (1).
Anti-inflammatory agent 0 according to II