Tries S, Neupert W, Laufer S
Preclinical Development, Merckle GmbH, Blaubeuren, Germany.
Inflamm Res. 2002 Mar;51(3):135-43. doi: 10.1007/pl00000285.
We examined the effects of ML3000 and several non-steroidal antiinflammatory drugs (NSAIDs) on the synthesis of products of 5-LOX (LTB4, LTC4) and COX-1/2 (TXB2, PGE2) in vitro and ex vivo in order to further elucidate the mechanism of action of ML3000.
Using a human whole blood assay the effect of ML3000 on the shunt of arachidonic acid to the lipoxygenase pathway when COX is blocked was studied. ML3000 (0.3, 1, 3, 10, 30 microg/ml) and indomethacin (0.3, 1, 3, 10, 30 microg/ml) concentration-dependently inhibited the synthesis of PGE2 (IC50 = 3.9 and 4.5 microM). In contrast to ML3000, indomethacin produced an increase of LTC4 of up to 155.5% of control. 5-lipoxygenase inhibition was further tested in a basophilic leukemia cell assay using RBL-1 cells. ML3000 (1-10 microM) inhibited the synthesis of LTB4 in a concentration related manner (IC50: 3.6 microM). In carrageenan induced rat paw edema, ML3000 and indomethacin completely blocked the formation of PGE2 in the inflamed tissue. The LTB4 production in the inflamed paw was reduced to basal levels by ML3000 (10 +/- 1.4 pg/paw saline control and 7.5 +/- 1.3-5.9 +/- 3.2 pg/paw ML3000), whereas LTB4 levels remained markedly elevated as compared to saline control by indomethacin (30.7 pg/paw). 5-LOX inhibition in the inflamed rat colon was investigated by measuring LTB4 synthesis. MK-886 and ML3000 at 10 mg/kg p.o. reduced LTB4 production to 29.8 +/- 4.9 and 30.1 +/- 2.8 pg/mg tissue as compared to control (54.2 +/- 7.4 mg/kg tissue). LTB4 levels in the rat stomach were comparable to control (2.5 +/- 0.4 pg/mg protein) after oral administration of ML3000 (10, 30, 100 mg/kg), whereas oral treatment with indomethacin (0.3, 1, 3 mg/kg) or diclofenac (1, 3 mg/kg) increased LTB4 up to 9.2 +/- 2.3 or 8.9 +/- 1.6 pg/mg protein. This effect was significant at 1 mg/kg diclofenac and 0.3 mg/kg indomethacin.
These results provide further evidence, that ML3000 inhibits 5-LOX as well as COX-1 and COX-2 in vitro and in animal experiments. The favourable gastrointestinal (GI) tolerability of the compound is believed to be linked to the mechanism of combined 5-LOX and COX-1/2 inhibition of ML3000.
我们研究了ML3000和几种非甾体抗炎药（NSAIDs）在体外和体内对5-脂氧合酶（5-LOX）产物（白三烯B4、白三烯C4）和环氧化酶-1/2（COX-1/2）产物（血栓素B2、前列腺素E2）合成的影响，以进一步阐明ML3000的作用机制。
采用人全血试验研究了COX被阻断时ML3000对花生四烯酸分流至脂氧合酶途径的影响。ML3000（0.3、1、3、10、30微克/毫升）和吲哚美辛（0.3、1、3、10、30微克/毫升）浓度依赖性地抑制前列腺素E2的合成（半数抑制浓度[IC50]分别为3.9和4.5微摩尔）。与ML3000不同，吲哚美辛使白三烯C4增加高达对照的155.5%。使用RBL-1嗜碱性白血病细胞试验进一步检测了5-脂氧合酶抑制作用。ML3000（1-10微摩尔）以浓度相关方式抑制白三烯B4的合成（IC50：3.6微摩尔）。在角叉菜胶诱导的大鼠足肿胀模型中，ML3000和吲哚美辛完全阻断了炎症组织中前列腺素E2的形成。ML3000使炎症足爪中的白三烯B4产生量降至基础水平（生理盐水对照为10±1.4皮克/足爪，ML3000为7.5±1.3-5.9±3.2皮克/足爪），而与生理盐水对照相比，吲哚美辛使白三烯B4水平显著升高（30.7皮克/足爪）。通过测量白三烯B4合成研究了炎症大鼠结肠中的5-脂氧合酶抑制作用。口服10毫克/千克的MK-886和ML3000使白三烯B4产生量分别降至29.8±4.9和30.1±2.8皮克/毫克组织，而对照为（54.2±7.4皮克/毫克组织）。口服ML3000（10、30、100毫克/千克）后，大鼠胃中的白三烯B4水平与对照相当（2.5±0.4皮克/毫克蛋白质），而口服吲哚美辛（0.3、1、3毫克/千克）或双氯芬酸（1、3毫克/千克）使白三烯B4增加至9.2±2.3或8.9±1.6皮克/毫克蛋白质。双氯芬酸1毫克/千克和吲哚美辛0.3毫克/千克时此效应显著。
这些结果进一步证明，在体外和动物实验中，ML3000可抑制5-脂氧合酶以及COX-1和COX-2。该化合物良好的胃肠道耐受性被认为与ML3000联合抑制5-脂氧合酶和COX-1/2的机制有关。
