PHARMACOLOGY OF ANTIRHEUMATIC AGENTS

我们最近有了一个令人惊讶的发现，腺苷介导了 甲氨蝶呤(MTX)的抗炎作用 用于治疗类风湿性关节炎的药物。我们的观察 提示MTX通过以下方式增加炎症部位的腺苷浓度 抑制5-氨基咪唑-4-甲酰胺核糖核苷酸(AICAR) 转化酶从而增加细胞内AICAR的浓度。 AICAR在细胞内的积累增加了细胞外的腺苷 以一种未知的机制进行集中。确定MTX如何减少 我们建议：1.检查炎症的生物化学机制 甲氨蝶呤增加甲氨蝶呤胞外腺苷浓度(及其 聚谷氨酸)在人成纤维细胞和内皮细胞中积聚 细胞内AICAR、肌苷和腺嘌呤核苷酸浓度 甲氨蝶呤处理的细胞及其对活性的直接影响 与腺苷释放的调节最直接相关的酶： 腺苷脱氨酶、腺苷激酶和AMP脱氨酶。我们会 腺苷量中核苷酸浓度的测定 外周血腺苷调节酶的释放和活性 类风湿关节炎患者发病前和发病后6周外周血单个核细胞的变化 使用甲氨蝶呤治疗。2.研究信号转导途径 白细胞腺苷A/2/A受体。尽管坎普一直被认为 介导腺苷受体结扎的作用我们发现了证据 为了表明腺苷通过cAMP抑制白细胞功能- 蛋白质磷酸酶(最有可能是蛋白质)的独立激活 磷酸酶1(PP1)。为了更好地了解信号转导 腺苷受体我们将研究腺苷受体刺激蛋白 直接磷酸酶活性，腺苷介导的蛋白质转位 细胞内的磷酸酶，腺苷受体占位的影响 PP1与调节蛋白的关系及分子鉴定 中性粒细胞中的PP1调节蛋白和底物。3.测试 补充假设认为MTX的抗炎作用是结果， 在一定程度上，由于抑制后-甲基化所需的转甲基化反应 蛋白质和脂类的翻译修饰 直接进行转甲基化反应。4.研究甲氨蝶呤的作用机制 抑制动物模型的慢性炎症(胶原性关节炎)和 类风湿性关节炎患者。我们将研究这些措施的效果 抗腺苷受体单抗对甲氨蝶呤抑制作用的研究 在适当的动物模型中出现炎症。拿出证据来证明 腺苷介导的抗炎作用 MTX我们将比较健康人白细胞上腺苷受体表达 外周血从滑液中获取的那些建立 是否存在配体介导的腺苷下调 感受器。我们设计这些研究是为了提高我们对 甲氨蝶呤的抗炎特性，我们认为信息 从这些研究中获得的成果将使我们能够制定更具体和 用于治疗类风湿的毒性较小的治疗方案 关节炎等慢性炎症性疾病。

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.