PHARMACOLOGY OF ANTIRHEUMATIC AGENTS

我们最近有了一个惊人的发现，腺苷介导了 甲氨蝶呤（MTX），最有效的 用于治疗类风湿性关节炎的药物。 我们的观察 提示MTX通过增加炎症部位的腺苷浓度， 抑制性5-氨基咪唑-4-甲酰胺核苷酸 转化酶，从而增加AICAR的细胞内浓度。 AICAR的细胞内积累增加细胞外腺苷 通过一种未知的机制。 为了确定氨甲喋呤是如何减少 炎症我们建议：1。研究生物化学机制， 甲氨蝶呤增加甲氨蝶呤（及其受体）的细胞外腺苷浓度 聚谷氨酸盐）在人成纤维细胞和内皮细胞中积累 细胞中AICAR、肌苷和腺嘌呤核苷酸的浓度 MTX处理的细胞和MTX处理对活性的直接影响 最直接参与调节腺苷释放的酶： 腺苷脱氨酶、腺苷激酶和AMP脱氨酶。 我们将 测定腺苷量中核苷酸的浓度 外周血中腺苷调节酶的释放和活性 RA患者发病前及发病后6周外周血单个核细胞 MTX的治疗。 2.检查信号转导通路， 白细胞腺苷A/2/A受体。 尽管cAMP被认为 介导腺苷受体连接的影响，我们已经发现证据， 表明腺苷通过cAMP抑制白细胞功能， 蛋白磷酸酶（最可能是蛋白磷酸酶）的独立活化 磷酸酶1，pp 1）。 为了更好地理解信号转导， 腺苷受体我们将研究腺苷受体刺激蛋白 磷酸酶活性直接，腺苷介导的蛋白质易位 细胞内的磷酸酶，腺苷受体占有率对 pp 1与调控蛋白的结合及其分子鉴定 pp 1调节蛋白和底物在中性粒细胞。 3.测试 补充假设MTX的抗肿瘤作用导致， 部分原因是抑制了后 蛋白质和脂质的翻译修饰， 直接进行甲基化反应。 4.研究MTX的作用机制 抑制动物模型中的慢性炎症（胶原性关节炎）， 类风湿性关节炎患者。 我们将研究 单克隆抗腺苷受体抗体对MTX抑制 在适当的动物模型中的炎症。 为…提供证据 腺苷介导的炎症抑制 MTX，我们将比较白细胞上腺苷受体的表达， 外周血与从滑液中获得的那些，以建立 是否存在配体介导的腺苷下调 受体。 我们设计这些研究是为了提高我们对 甲氨蝶呤的毒性我们相信 从这些研究中获得的信息将允许开发更具体和 用于治疗类风湿性关节炎的毒性较小的治疗方案 关节炎和其他慢性炎症性疾病。

We have recently made the surprising discovery that adenosine mediates the antiinflammatory effects of methotrexate (MTX), one of the most effective agents used in the treatment of rheumatoid arthritis. Our observations suggest that MTX increases adenosine concentrations at inflammed sites by inhibiting 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) transformylase thereby increasing intracellular concentrations of AICAR. Intracellular accumulation of AICAR increases extracellular adenosine concentration by an unknown mechanism. To determine how MTX diminishes inflammation we propose to: 1. Examine the biochemical mechanism by which MTX increases extracellular adenosine concentrations of MTX (and its polyglutamates) that accumulate in human fibroblasts and endothelial cells, the concentrations of AICAR, inosine and adenine nucleotides in MTX-treated cells and the direct effect of MTX treatment on the activity of the enzymes most directly involved in regulation of adenosine release: adenosine deaminase, adenosine kinase and AMP deaminase. We will determine the concentration of nucleotides in the amount of adenosine release from and activity of adenosine-regulating enzymes in peripheral blood mononuclear cells of patients with RA before and 6 weeks after onset of treatment with MTX. 2. Examine signal transduction pathway at leukocyte adenosine A/2/A receptors. Although cAMP has been thought to mediate the effects of adenosine receptor ligation we have found evidence to indicate that adenosine inhibits leukocyte function via the cAMP- independent activation of a protein phosphatase (most likely protein phosphatase 1, pp1). To better understand signal transduction at adenosine receptors we will study adenosine receptor-stimulated protein phosphatase activity directly, adenosine-mediated translocation of protein phosphatases within the cell, effects of adenosine receptor occupancy on association of pp1 with regulatory proteins and molecular identification of pp1 regulatory proteins and substrates in the neutrophil. 3. Test the complementary hypothesis that the antiinflammatory effects of MTX result, in part, from inhibition of transmethylation reactions required for post- translational modification of proteins and lipids by examining transmethylation reactions directly. 4. Study the mechanism by which MTX inhibits chronic inflammation in animal models (collagen arthritis) and patients with rheumatoid arthritis. We will study the effect of monoclonal antiadenosine receptor antibodies on MTX inhibition of inflammation in appropriate animal models. To show evidence for adenosine-mediated suppression of inflammation in patients treated with MTX we will compare adenosine receptor expression on leukocytes from peripheral blood to those obtained from synovial fluid to establish whether there has been ligand-mediated down-regulation of adenosine receptors. We have designed these studies to improve our understanding of the antiinflammatory properties of MTX and we believe that the information gained from these studies will permit the development of more specific and less toxic therapeutic regimens for use in the therapy of rheumatoid arthritis and other chronic inflammatory diseases.