JPH07287008A - Simplified test method for therapeutic agents for inflammatory diseases - Google Patents
Simplified test method for therapeutic agents for inflammatory diseasesInfo
- Publication number
- JPH07287008A JPH07287008A JP6078607A JP7860794A JPH07287008A JP H07287008 A JPH07287008 A JP H07287008A JP 6078607 A JP6078607 A JP 6078607A JP 7860794 A JP7860794 A JP 7860794A JP H07287008 A JPH07287008 A JP H07287008A
- Authority
- JP
- Japan
- Prior art keywords
- substance
- test method
- production inhibitory
- concentration
- cyclooxygenase activity
- 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.)
- Pending
Links
- 238000010998 test method Methods 0.000 title claims abstract description 11
- 239000003814 drug Substances 0.000 title description 8
- 208000027866 inflammatory disease Diseases 0.000 title 1
- 229940124597 therapeutic agent Drugs 0.000 title 1
- 239000000126 substance Substances 0.000 claims abstract description 25
- 102000004127 Cytokines Human genes 0.000 claims abstract description 17
- 108090000695 Cytokines Proteins 0.000 claims abstract description 17
- 238000012360 testing method Methods 0.000 claims abstract description 16
- 230000000694 effects Effects 0.000 claims abstract description 13
- 230000002401 inhibitory effect Effects 0.000 claims abstract description 13
- 102000004005 Prostaglandin-endoperoxide synthases Human genes 0.000 claims abstract description 12
- 108090000459 Prostaglandin-endoperoxide synthases Proteins 0.000 claims abstract description 12
- 210000002966 serum Anatomy 0.000 claims abstract description 10
- 230000016396 cytokine production Effects 0.000 claims abstract description 9
- 238000010171 animal model Methods 0.000 claims abstract description 8
- 210000004969 inflammatory cell Anatomy 0.000 claims abstract description 8
- 108090001005 Interleukin-6 Proteins 0.000 claims abstract description 6
- 230000004936 stimulating effect Effects 0.000 claims abstract description 6
- 101000862089 Clarkia lewisii Glucose-6-phosphate isomerase, cytosolic 1A Proteins 0.000 claims abstract description 4
- 108090001007 Interleukin-8 Proteins 0.000 claims abstract description 4
- KAQKFAOMNZTLHT-OZUDYXHBSA-N prostaglandin I2 Chemical compound O1\C(=C/CCCC(O)=O)C[C@@H]2[C@@H](/C=C/[C@@H](O)CCCCC)[C@H](O)C[C@@H]21 KAQKFAOMNZTLHT-OZUDYXHBSA-N 0.000 claims abstract description 4
- XEYBRNLFEZDVAW-ARSRFYASSA-N dinoprostone Chemical compound CCCCC[C@H](O)\C=C\[C@H]1[C@H](O)CC(=O)[C@@H]1C\C=C/CCCC(O)=O XEYBRNLFEZDVAW-ARSRFYASSA-N 0.000 claims abstract description 3
- 229960002986 dinoprostone Drugs 0.000 claims abstract description 3
- XEYBRNLFEZDVAW-UHFFFAOYSA-N prostaglandin E2 Natural products CCCCCC(O)C=CC1C(O)CC(=O)C1CC=CCCCC(O)=O XEYBRNLFEZDVAW-UHFFFAOYSA-N 0.000 claims abstract description 3
- 150000003180 prostaglandins Chemical class 0.000 claims abstract description 3
- 241001465754 Metazoa Species 0.000 claims description 8
- 239000002158 endotoxin Substances 0.000 claims description 7
- 229920006008 lipopolysaccharide Polymers 0.000 claims description 7
- 238000004519 manufacturing process Methods 0.000 claims description 5
- 108010033737 Pokeweed Mitogens Proteins 0.000 claims description 4
- 108060008682 Tumor Necrosis Factor Proteins 0.000 claims description 3
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 claims description 3
- 239000002904 solvent Substances 0.000 claims description 3
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 claims description 2
- 241000894006 Bacteria Species 0.000 claims description 2
- 210000003850 cellular structure Anatomy 0.000 claims description 2
- 239000000543 intermediate Substances 0.000 claims 1
- 239000000021 stimulant Substances 0.000 claims 1
- 230000028327 secretion Effects 0.000 abstract description 5
- 238000001990 intravenous administration Methods 0.000 abstract description 3
- 238000000338 in vitro Methods 0.000 abstract description 2
- 229940094443 oxytocics prostaglandins Drugs 0.000 abstract description 2
- 238000004113 cell culture Methods 0.000 abstract 1
- 229940079593 drug Drugs 0.000 description 7
- 210000004369 blood Anatomy 0.000 description 5
- 239000008280 blood Substances 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 102000000589 Interleukin-1 Human genes 0.000 description 4
- 108010002352 Interleukin-1 Proteins 0.000 description 4
- 102000004889 Interleukin-6 Human genes 0.000 description 4
- CGIGDMFJXJATDK-UHFFFAOYSA-N indomethacin Chemical compound CC1=C(CC(O)=O)C2=CC(OC)=CC=C2N1C(=O)C1=CC=C(Cl)C=C1 CGIGDMFJXJATDK-UHFFFAOYSA-N 0.000 description 4
- 238000000034 method Methods 0.000 description 4
- 210000004027 cell Anatomy 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- BSYNRYMUTXBXSQ-UHFFFAOYSA-N Aspirin Chemical compound CC(=O)OC1=CC=CC=C1C(O)=O BSYNRYMUTXBXSQ-UHFFFAOYSA-N 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- 229930105110 Cyclosporin A Natural products 0.000 description 2
- PMATZTZNYRCHOR-CGLBZJNRSA-N Cyclosporin A Chemical compound CC[C@@H]1NC(=O)[C@H]([C@H](O)[C@H](C)C\C=C\C)N(C)C(=O)[C@H](C(C)C)N(C)C(=O)[C@H](CC(C)C)N(C)C(=O)[C@H](CC(C)C)N(C)C(=O)[C@@H](C)NC(=O)[C@H](C)NC(=O)[C@H](CC(C)C)N(C)C(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)N(C)C(=O)CN(C)C1=O PMATZTZNYRCHOR-CGLBZJNRSA-N 0.000 description 2
- 108010036949 Cyclosporine Proteins 0.000 description 2
- VVNCNSJFMMFHPL-VKHMYHEASA-N D-penicillamine Chemical compound CC(C)(S)[C@@H](N)C(O)=O VVNCNSJFMMFHPL-VKHMYHEASA-N 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- HEFNNWSXXWATRW-UHFFFAOYSA-N Ibuprofen Chemical compound CC(C)CC1=CC=C(C(C)C(O)=O)C=C1 HEFNNWSXXWATRW-UHFFFAOYSA-N 0.000 description 2
- 102000004890 Interleukin-8 Human genes 0.000 description 2
- 241000699670 Mus sp. Species 0.000 description 2
- 239000012980 RPMI-1640 medium Substances 0.000 description 2
- 241000700159 Rattus Species 0.000 description 2
- 229960001138 acetylsalicylic acid Drugs 0.000 description 2
- 239000003435 antirheumatic agent Substances 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 229960001265 ciclosporin Drugs 0.000 description 2
- UREBDLICKHMUKA-CXSFZGCWSA-N dexamethasone Chemical compound C1CC2=CC(=O)C=C[C@]2(C)[C@]2(F)[C@@H]1[C@@H]1C[C@@H](C)[C@@](C(=O)CO)(O)[C@@]1(C)C[C@@H]2O UREBDLICKHMUKA-CXSFZGCWSA-N 0.000 description 2
- 229960003957 dexamethasone Drugs 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 229960001680 ibuprofen Drugs 0.000 description 2
- 238000003018 immunoassay Methods 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 229960000905 indomethacin Drugs 0.000 description 2
- 239000003112 inhibitor Substances 0.000 description 2
- 230000018276 interleukin-1 production Effects 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 210000005087 mononuclear cell Anatomy 0.000 description 2
- 229960001639 penicillamine Drugs 0.000 description 2
- AGHLUVOCTHWMJV-UHFFFAOYSA-J sodium;gold(3+);2-sulfanylbutanedioate Chemical compound [Na+].[Au+3].[O-]C(=O)CC(S)C([O-])=O.[O-]C(=O)CC(S)C([O-])=O AGHLUVOCTHWMJV-UHFFFAOYSA-J 0.000 description 2
- 206010002091 Anaesthesia Diseases 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 101800004538 Bradykinin Proteins 0.000 description 1
- QXZGBUJJYSLZLT-UHFFFAOYSA-N H-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-OH Natural products NC(N)=NCCCC(N)C(=O)N1CCCC1C(=O)N1C(C(=O)NCC(=O)NC(CC=2C=CC=CC=2)C(=O)NC(CO)C(=O)N2C(CCC2)C(=O)NC(CC=2C=CC=CC=2)C(=O)NC(CCCN=C(N)N)C(O)=O)CCC1 QXZGBUJJYSLZLT-UHFFFAOYSA-N 0.000 description 1
- 102000003777 Interleukin-1 beta Human genes 0.000 description 1
- 108090000193 Interleukin-1 beta Proteins 0.000 description 1
- 102000004125 Interleukin-1alpha Human genes 0.000 description 1
- 108010082786 Interleukin-1alpha Proteins 0.000 description 1
- 102100035792 Kininogen-1 Human genes 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 206010040070 Septic Shock Diseases 0.000 description 1
- UKNGDQSYPNBJAO-UHFFFAOYSA-N Tiaramide hydrochloride Chemical compound Cl.C1CN(CCO)CCN1C(=O)CN1C(=O)SC2=CC=C(Cl)C=C21 UKNGDQSYPNBJAO-UHFFFAOYSA-N 0.000 description 1
- 235000010489 acacia gum Nutrition 0.000 description 1
- 239000001785 acacia senegal l. willd gum Substances 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 230000037005 anaesthesia Effects 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 230000003356 anti-rheumatic effect Effects 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- QXZGBUJJYSLZLT-FDISYFBBSA-N bradykinin Chemical compound NC(=N)NCCC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N1[C@H](C(=O)NCC(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CO)C(=O)N2[C@@H](CCC2)C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)CCC1 QXZGBUJJYSLZLT-FDISYFBBSA-N 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 239000003246 corticosteroid Substances 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- YPHMISFOHDHNIV-FSZOTQKASA-N cycloheximide Chemical compound C1[C@@H](C)C[C@H](C)C(=O)[C@@H]1[C@H](O)CC1CC(=O)NC(=O)C1 YPHMISFOHDHNIV-FSZOTQKASA-N 0.000 description 1
- 231100000673 dose–response relationship Toxicity 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- 230000005484 gravity Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 239000002955 immunomodulating agent Substances 0.000 description 1
- 229940121354 immunomodulator Drugs 0.000 description 1
- 230000002584 immunomodulator Effects 0.000 description 1
- 229960003444 immunosuppressant agent Drugs 0.000 description 1
- 230000001861 immunosuppressant effect Effects 0.000 description 1
- 239000003018 immunosuppressive agent Substances 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000017306 interleukin-6 production Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000000041 non-steroidal anti-inflammatory agent Substances 0.000 description 1
- 229940021182 non-steroidal anti-inflammatory drug Drugs 0.000 description 1
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 1
- 210000003240 portal vein Anatomy 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 239000004576 sand Substances 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- HTJXMOGUGMSZOG-UHFFFAOYSA-N tiaramide Chemical compound C1CN(CCO)CCN1C(=O)CN1C(=O)SC2=CC=C(Cl)C=C21 HTJXMOGUGMSZOG-UHFFFAOYSA-N 0.000 description 1
- 229950010302 tiaramide Drugs 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Landscapes
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
(57)【要約】
【目的】従来のin vitro 試験における、数種サイトカ
インの分泌が均等に抑制されてしまう欠点を補い、細胞
の培養の労力と長時間を軽減する。
【構成】実験動物に被検物質を投与後、炎症性細胞刺激
物質を静脈内投与することにより惹起される、血清プロ
スタグランジン類（ＰＧＥ2もしくはＰＧＩ2）濃度、お
よび数種のサイトカイン（インターロイキン−１α［Ｉ
Ｌ−１α］、ＩＬ−１β、ＩＬ−６、ＩＬ−８およびＴ
ＮＦ−α）濃度を定量し、サイクロオキシゲナーゼ活性
および各種サイトカイン産生阻害作用を有する物質の簡
易試験法を提供する。(57) [Summary] [Purpose] To compensate for the drawback that the secretion of several cytokines is uniformly suppressed in conventional in vitro tests, and to reduce the labor and long time of cell culture. [Structure] Serum prostaglandins (PGE2 or PGI2) concentration and several cytokines (interleukin-) induced by intravenous administration of inflammatory cell stimulating substance after administration of test substance to experimental animals 1α [I
L-1α], IL-1β, IL-6, IL-8 and T
(EN) A simple test method for a substance having a cyclooxygenase activity and various cytokine production inhibitory effects by quantifying NF-α) concentration.
Description
【０００１】[0001]
【産業上の利用分野】この発明は実験動物、特にラット
またはマウスに炎症性細胞刺激物質を静脈内投与するこ
とにより起こる、血清中へのＰＧｓ分泌量および各種サ
イトカイン分泌量を定量することにより、サイクロオキ
シゲナーゼ活性阻害物質または各種サイトカイン産生阻
害物質の簡易試験法に関する。This invention relates to the quantification of the amount of PGs secreted into serum and the amount of various cytokines caused by the intravenous administration of an inflammatory cell stimulant to experimental animals, especially rats or mice. The present invention relates to a simple test method for a cyclooxygenase activity inhibitor or various cytokine production inhibitors.
【０００２】[0002]
【従来の技術】サイトカイン産生阻害作用を試験する方
法としては、これまでにヒトもしくは動物の単核球細胞
からのサイトカイン分泌を定量する方法がある。2. Description of the Related Art As a method for testing the cytokine production inhibitory effect, there has been a method for quantifying cytokine secretion from human or animal mononuclear cells.
【０００３】すなわち、ヒト末梢血単核球を用いる場合
を例にとると、健常人よりクエン酸存在下で静脈血を採
取した。この血液から比重遠心法により単核球細胞を得
た。That is, taking human peripheral blood mononuclear cells as an example, venous blood was collected from a healthy person in the presence of citric acid. Mononuclear cells were obtained from this blood by the gravity method.
【０００４】細胞をＲＰＭＩ１６４０培養液で３回洗浄
後、非動化牛胎児血清を１０％含んだＲＰＭＩ１６４０
培養液に２Ｘ１０6個／mlになるような細胞浮遊液を調
製した。 被検物質はエタノールに溶解し、エタノール
の最終濃度が０．５％になるように培養液で調製した。
被検薬調製液０．２５ｍｌを加え３７℃、５％二酸化炭
素／９５％空気で４０時間培養した。The cells were washed three times with RPMI1640 culture medium, and then RPMI1640 containing 10% immobilized fetal bovine serum.
A cell suspension was prepared so that the culture solution contained 2 × 10 6 cells / ml. The test substance was dissolved in ethanol and prepared in a culture medium so that the final concentration of ethanol was 0.5%.
0.25 ml of the test drug preparation solution was added, and the mixture was cultured at 37 ° C. in 5% carbon dioxide / 95% air for 40 hours.
【０００５】遠心分離（１５００ ｒｐｍ, ５分）によ
りその培養上清を採取し、上清中のサイトカイン（ＩＬ
−１，ＩＬ−６，ＩＬ−８）を免疫酵素法で定量する。The culture supernatant was collected by centrifugation (1500 rpm, 5 minutes), and the cytokine (IL
-1, IL-6, IL-8) is quantified by an immunoenzymatic method.
【０００６】一方被検物質を処置しない対照群について
も同じ時期に上記の測定をおこない比較して被検物質の
効果が判定される。On the other hand, the effect of the test substance is judged by comparing the above-mentioned measurements at the same time for the control group not treated with the test substance.
【０００７】[0007]
【発明が解決しようとする課題】上記の従来試験法にお
いては次の様な問題点がある。すなわちin vitro 試験
であるため、生体内におけるサイトカイン産生系と分泌
機序が異なると考えられ、数種サイトカインの分泌が均
等に抑制されてしまう。また細胞の培養に多くの労力と
長時間を要する。The above-mentioned conventional test methods have the following problems. That is, since it is an in vitro test, it is considered that the secretory mechanism is different from the cytokine production system in vivo, and the secretion of several cytokines is equally suppressed. Further, culturing cells requires a lot of labor and a long time.
【０００８】[0008]
【課題を解決するための手段】本発明者らは、この様な
問題点をするため、以前よりエンドトキシンショック時
にＰＧs（サーキュレーション、４５巻、１２４頁、１
９７２年）、ブラジキニン（サーキュレイション・リサ
ーチ、２２巻、１５５頁、１９６８年）、腫瘍壊死因子
（アニュアル・レビユー・バイオケミストリー、５７
巻、５０５頁、１９８８年）およびその他多くの生理活
性物質が動物の血中に分泌されていることに注目した。In order to solve such a problem, the present inventors have long been involved in PGs (circulation, 45, 124, 1) during endotoxin shock.
972), Bradykinin (circulation research, 22: 155, 1968), tumor necrosis factor (Annual Review Biochemistry, 57).
Vol. 505, p. 1988) and many other bioactive substances are noted in the blood of animals.
【０００９】そこで、炎症性細胞刺激物質を実験動物に
静脈内投与することにより、動物の血清中に数種の生理
活性物質が分泌されるのではないかと検討した結果、Ｐ
Ｇsの他、数種のサイトカインが分泌することを見い出
しこの発明をなすに至った。[0009] Therefore, it was investigated that intravenous administration of an inflammatory cell stimulating substance to an experimental animal might secrete several physiologically active substances into the serum of the animal.
In addition to Gs, it was found that several kinds of cytokines were secreted, and the present invention was completed.
【００１０】すなわち、本発明は、実験動物に被検物質
を投与後、炎症性細胞刺激物質を静脈内投与することに
より惹起される、血清プロスタグランジン類（ＰＧＥ2
もしくはＰＧＩ2）濃度、および数種のサイトカイン
（インターロイキン−１α［ＩＬ−１α］、ＩＬ−１
β、ＩＬ−６、ＩＬ−８およびＴＮＦ−α）濃度を定量
することを特徴とするサイクロオキシゲナーゼ活性およ
び各種サイトカイン産生阻害作用を有する物質の簡易試
験法である。That is, the present invention relates to serum prostaglandins (PGE2) which are induced by intravenously administering an inflammatory cell stimulating substance after administering a test substance to an experimental animal.
Or PGI2) concentration, and several cytokines (interleukin-1α [IL-1α], IL-1
β, IL-6, IL-8 and TNF-α) concentration is quantified, which is a simple test method for a substance having a cyclooxygenase activity and various cytokine production inhibitory effects.
【００１１】本発明において、炎症性細胞刺激物質がリ
ポポリサッカライド（ＬＰＳ）、ポークウイードマイト
ージェン（ＰＷＭ）またはグラム陰性菌の菌体成分であ
ることが好ましい。In the present invention, the inflammatory cell stimulating substance is preferably lipopolysaccharide (LPS), pokeweed mitogen (PWM) or a bacterial cell component of Gram-negative bacterium.
【００１２】また、実験動物がラットまたはマウスであ
ることが好ましい。Further, it is preferable that the experimental animal is a rat or a mouse.
【００１３】被検物質の投与時に使用した溶媒を投与し
た動物の血清中ＰＧｓ濃度および各種サイトカイン濃度
と比較することが好ましい。It is preferable to compare the serum PGs concentration and various cytokine concentrations of the animal to which the solvent used for administration of the test substance was administered.
【００１４】本発明の簡易試験法を詳述すれば、実験動
物に被検物質を投与し、体内に分布させるため一定時間
の放置させた後、炎症性細胞刺激物質（例えばＬＰＳ）
の一定用量を静脈内投与する。The simple test method of the present invention will be described in detail. After the test substance is administered to an experimental animal and allowed to stand for a certain period of time to be distributed in the body, an inflammatory cell stimulating substance (eg, LPS).
Is administered intravenously.
【００１５】その後一定時間放置後、エーテル麻酔下に
動物から採血し、遠心分離により血清を採取した。この
血清について適度の希釈を行い酵素免疫法により各種サ
イトカインおよびＰＧs量を定量し、溶媒投与の対照群
の値との比較により、被検物質のサイトカイン産生に及
ぼす作用を in vivoで評価するものである。Then, after standing for a certain period of time, blood was collected from the animal under ether anesthesia, and serum was collected by centrifugation. This serum is diluted appropriately and the amount of various cytokines and PGs is quantified by enzyme immunoassay, and the effect of the test substance on cytokine production is evaluated in vivo by comparison with the values of the control group of solvent administration. is there.
【００１６】[0016]
【発明の効果】本発明により、各種薬剤（抗リウマチ、
向免疫等）の前投与により、分泌抑制もしくは増強され
る各種サイトカインの種類が異なりこれらサイトカイン
分泌パターンを解析することにより薬剤の性質を明確に
することが可能となった。According to the present invention, various drugs (antirheumatic,
It has become possible to clarify the properties of the drug by analyzing the cytokine secretion pattern of various cytokines whose secretion is suppressed or enhanced by the pre-administration (such as immunity).
【００１７】[0017]
【実施例】以下に、実施例を挙げて本発明を具体的に説
明する。EXAMPLES The present invention will be specifically described below with reference to examples.
【００１８】実施例 （試験動物）体重約３０ｇのＩＣＲ系雄性マウスを１８
時間絶食後、１群２ー５匹で実験に使用した。アラビヤ
ゴム溶液に懸濁調製した被検物質の一定用量を投与（経
口、もしくは皮下投与）し、１時間の放置後ＬＰＳ（0.
01 - 30 mg/kg）を静脈内投与した。ＬＰＳ投与後さら
に３時間の放置し、クエン酸ナトリウム入りのシリンジ
で門脈より血液を採取した。遠心分離（3000 rpm, 10 m
in）により得た血清中のＰＧｓ濃度および各種サイトカ
イン（ＩＬ−１，ＩＬ−６）濃度を酵素免疫法により定
量した。Example (Test animal) ICR male mice weighing about 30 g
After fasting for 2 hours, 2 to 5 animals per group were used for the experiment. A fixed dose of the test substance suspended in arabic gum solution was administered (orally or subcutaneously), and left for 1 hour, then LPS (0.
01-30 mg / kg) was administered intravenously. After the LPS administration, the mixture was allowed to stand for another 3 hours and blood was collected from the portal vein with a syringe containing sodium citrate. Centrifuge (3000 rpm, 10 m
The concentration of PGs and various cytokines (IL-1, IL-6) in the serum obtained by (in) were quantified by enzyme immunoassay.
【００１９】（使用薬物）チアラミド（藤沢薬品）、ア
スピリン（シグマ）、インドメタシン（シグマ）、イブ
プロフェン（科研）、サイクロスポリンＡ（サンド薬
品）、Ｄ−ペニシラミン（大正製薬）、金チオリンゴ酸
ナトリウム（塩野義製薬）およびデキサメタゾン（シグ
マ）を使用した。(Drugs used) Thiaramide (Fujisawa Pharmaceutical), Aspirin (Sigma), Indomethacin (Sigma), Ibuprofen (Kaken), Cyclosporin A (Sand Chemicals), D-penicillamine (Taisho Pharmaceutical Co.), sodium gold thiomalate ( Shionogi Pharmaceutical) and dexamethasone (Sigma) were used.
【００２０】（実験結果） 非ステロイド系抗炎症剤のアスピリン、インドメタシ
ン、イブプロフェンおよびチアラミドについてサイクロ
オキシゲナーゼ阻害作用（ＰＧＩ2産生阻害作用） ＩＬ−１産生阻害作用、ＩＬ−６産生阻害作用を検討し
た結果、これら薬物はサイクロオキシゲナーゼを用量依
存的に抑制した。またこれら薬剤はＩＬ−１の産生につ
いてもある程度抑制作用を示すことがわかったが、ＩＬ
−６についたは全く抑制作用を示さなかった（表１）。(Experimental Results) Cyclooxygenase inhibitory action (PGI2 production inhibitory action) of the nonsteroidal anti-inflammatory agents aspirin, indomethacin, ibuprofen and tiaramide. These drugs inhibited cyclooxygenase in a dose-dependent manner. It was also found that these drugs have some inhibitory effect on the production of IL-1.
-6 had no inhibitory effect (Table 1).
【００２１】[0021]
【表１】 [Table 1]
【００２２】免疫抑制剤のサイクロスポリンＡは顕著な
ＩＬ−１産生抑制を示したが、サイクロオキシゲナーゼ
活性およびＩＬ−６の産生には影響を与えなかった。免
疫調節剤のＤ−ペニシラミンはサイクロオキシゲナーゼ
活性およびＩＬ−１産生に対し抑制作用がみられた。抗
リウマチ剤の金チオリンゴ酸ナトリウムはＩＬ−１の産
生に対し顕著な抑制作用を示した。副腎皮質ステロイド
のデキサメタゾンは、ＩＬ−１およびＩＬ−６の産生を
抑制したがサイクロオキシゲナーゼ活性には影響を与え
なかった（表２）。The immunosuppressant cyclosporin A showed a marked inhibition of IL-1 production, but had no effect on cyclooxygenase activity and IL-6 production. The immunomodulator D-penicillamine had an inhibitory effect on cyclooxygenase activity and IL-1 production. The antirheumatic drug sodium gold thiomalate showed a marked inhibitory effect on the production of IL-1. The corticosteroid dexamethasone suppressed the production of IL-1 and IL-6 but did not affect cyclooxygenase activity (Table 2).
【００２３】[0023]
【表２】 [Table 2]
【００２４】以上の様に、本試験法は各種薬剤により抑
制する炎症メデイエイター（数種サイトカイン）の種類
が異なることを明確にできることから、被検体の抗炎症
作用の性質を検討することが出来るものである。As described above, since it is possible to clarify that the types of inflammatory mediators (several cytokines) that are suppressed by various drugs are different in this test method, the properties of the anti-inflammatory action of the subject can be examined. It is a thing.
───────────────────────────────────────────────────── フロントページの続き (72)発明者 二木 伸子 東京都豊島区高田３丁目24番１号 大正製 薬株式会社内 (72)発明者 高橋 修哉 東京都豊島区高田３丁目24番１号 大正製 薬株式会社内 (72)発明者 横山 磨里 東京都豊島区高田３丁目24番１号 大正製 薬株式会社内 (72)発明者 山川 由紀 東京都豊島区高田３丁目24番１号 大正製 薬株式会社内 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Nobuko Niki 3-24-1, Takada, Toshima-ku, Tokyo Taisho Pharmaceutical Co., Ltd. (72) Inventor Shuya Takahashi 3-24-1, Takada, Toshima-ku, Tokyo Taisho Pharmaceutical Co., Ltd. (72) Inventor, Mari Mari Yokoyama, 3-24-1, Takada, Toshima-ku, Tokyo Taisho Pharmaceutical Co., Ltd. (72) Inventor, Yuki Yamakawa, 3-24-1, Takada, Toshima-ku, Tokyo Taisho Inside Pharmaceutical Co., Ltd.
Claims (4)
刺激物質を静脈内投与することにより惹起される、血清
プロスタグランジン類（ＰＧＥ2もしくはＰＧＩ2）濃
度、および数種のサイトカイン（インターロイキン−１
α［ＩＬ−１α］、ＩＬ−１β、ＩＬ−６、ＩＬ−８お
よびＴＮＦ−α）濃度を定量することを特徴とするサイ
クロオキシゲナーゼ活性および各種サイトカイン産生阻
害作用を有する物質の簡易試験法。1. A serum prostaglandin (PGE2 or PGI2) concentration, which is induced by intravenously administering an inflammatory cell stimulating substance after administration of a test substance to an experimental animal, and several types of cytokines (intermediates). Leukin-1
A simple test method for a substance having a cyclooxygenase activity and various cytokine production inhibitory effects, which comprises quantifying α [IL-1α], IL-1β, IL-6, IL-8 and TNF-α.
ド（ＬＰＳ）、ポークウイードマイトージェン（ＰＷ
Ｍ）またはグラム陰性菌の菌体成分である請求項１記載
のサイクロオキシゲナーゼ活性および各種サイトカイン
産生阻害作用を有する物質の簡易試験法。2. The inflammatory cell stimulants are lipopolysaccharide (LPS) and pokeweed mitogen (PW).
A simple test method for a substance having a cyclooxygenase activity and various cytokine production inhibitory activities according to claim 1, which is a cell component of M) or Gram-negative bacterium.
項１または請求項２記載のサイクロオキシゲナーゼ活性
および各種サイトカイン産生阻害作用を有する物質の簡
易試験法。3. A simple test method for a substance having a cyclooxygenase activity and various cytokine production inhibitory activities according to claim 1 or 2, wherein the experimental animal is a rat or a mouse.
た動物の血清中ＰＧｓ濃度および各種サイトカイン濃度
と比較することを特徴とする請求項１〜３項に記載のサ
イクロオキシゲナーゼ活性および各種サイトカイン産生
阻害作用を有する物質の簡易試験方法。4. The cyclooxygenase activity and various cytokines according to any one of claims 1 to 3, which are compared with the serum PGs concentration and various cytokine concentrations of the animal to which the solvent used when administering the test substance is administered. A simple test method for a substance having a production inhibitory action.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP6078607A JPH07287008A (en) | 1994-04-18 | 1994-04-18 | Simplified test method for therapeutic agents for inflammatory diseases |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP6078607A JPH07287008A (en) | 1994-04-18 | 1994-04-18 | Simplified test method for therapeutic agents for inflammatory diseases |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH07287008A true JPH07287008A (en) | 1995-10-31 |
Family
ID=13666578
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP6078607A Pending JPH07287008A (en) | 1994-04-18 | 1994-04-18 | Simplified test method for therapeutic agents for inflammatory diseases |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH07287008A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2001194369A (en) * | 1999-10-26 | 2001-07-19 | Toyama Chem Co Ltd | Screening method for drugs acting on the central nervous system and evaluation method for stress state |
| JP2003527566A (en) * | 1999-07-14 | 2003-09-16 | スペクトラル ダイアグノスティックス インコーポレイテッド | Preparation of diagnostic test spheres |
| JP2008539177A (en) * | 2005-04-26 | 2008-11-13 | トリオン ファーマ ゲーエムベーハー | Combination of antibodies and glucocorticoids for cancer treatment |
| JP2012254994A (en) * | 2012-07-23 | 2012-12-27 | Trion Pharma Gmbh | Antibody for cancer treatment, and combination with glucocorticoid |
-
1994
- 1994-04-18 JP JP6078607A patent/JPH07287008A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2003527566A (en) * | 1999-07-14 | 2003-09-16 | スペクトラル ダイアグノスティックス インコーポレイテッド | Preparation of diagnostic test spheres |
| JP2001194369A (en) * | 1999-10-26 | 2001-07-19 | Toyama Chem Co Ltd | Screening method for drugs acting on the central nervous system and evaluation method for stress state |
| JP4490576B2 (en) * | 1999-10-26 | 2010-06-30 | 富山化学工業株式会社 | Central nervous system drug screening method and stress state evaluation method |
| JP2008539177A (en) * | 2005-04-26 | 2008-11-13 | トリオン ファーマ ゲーエムベーハー | Combination of antibodies and glucocorticoids for cancer treatment |
| JP2012254994A (en) * | 2012-07-23 | 2012-12-27 | Trion Pharma Gmbh | Antibody for cancer treatment, and combination with glucocorticoid |
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