PHARMACOLOGY OF ANTIRHEUMATIC AGENTS

描述：（改编自申请人摘要）-研究者 他首先提出了这样一个假说，即腺苷作用于特定的受体， 在炎症细胞上，介导 甲氨蝶呤，最常用和有效的二线抗风湿药 代理可用。 在这个项目的前两年，他们实现了 四个原始目标中的三个。 他证明了腺苷的形成 细胞外的核苷酸去磷酸化，证实， 腺苷介导甲氨蝶呤的抗肿瘤作用 关节炎的体内模型，并描述了一种新的信号转导， 中性粒细胞腺苷A2 A受体。 他还展示了一个以前 腺苷作用于其受体在发病机制中的作用 甲氨蝶呤毒性（结节病），令人惊讶的是， 细胞因子（IL-1和TNF α）下调腺苷酸（A2 A） 受体mRNA。 他现在建议审查：一。生物化学机制， 甲氨蝶呤促进腺苷释放。 他将决定是否 甲氨蝶呤促进腺嘌呤核苷酸的细胞内积累 （HPLC），增加腺嘌呤核苷酸转运出细胞（HPLC， 腺嘌呤核苷酸库的放射性标记），或减少 细胞的腺苷（放射性标记的腺苷摄取）。 二. 相互作用 腺苷受体与炎症之间：调节和 反调节 他将研究甲氨蝶呤减少 A2 A、A3或两者缺陷小鼠的急性和慢性炎症 受体（基因敲除小鼠），以确定这些受体中哪一个更好， 负责腺苷的代谢作用。 他还将 确定IL-1和TNF α是否调节mRNA稳定性（核流出 测定）或转录（转染启动子构建体 A2 A受体mRNA。 三. 腺苷介导的甲氨蝶呤机制 毒性 甲氨蝶呤治疗最可怕的并发症是肝脏 纤维化 他的数据表明，腺苷，作用于受体， 成纤维细胞，促进基质生成并抑制基质分解。 他 将确定甲氨蝶呤是否促进腺苷释放， 肝细胞以及这些细胞释放的腺苷是否影响 通过培养的星状细胞（肝）产生胶原或胶原酶 成纤维细胞）。 拟议研究的结果将表明新的 治疗炎症性疾病药物的开发方法 并可能导致新的治疗方法的发展 和预防甲氨蝶呤毒性。

DESCRIPTION: (Adapted from the applicant's abstract) - The investigator first proposed the hypothesis that adenosine, acting at specific receptors on inflammatory cells, mediates the antiinflammatory effects of methotrexate, the most commonly used and effective second line antirheumatic agent available. During the first 2 years of this project they achieved three of the four original aims. He demonstrated that adenosine is formed extracellularly from the dephosphorylation of nucleotides, confirmed that adenosine mediates the antiinflammatory effects of methotrexate in an in vivo model of arthritis, and described a novel signal transduction at neutrophil adenosine A2A receptors. He also demonstrated a previously unsuspected role for adenosine, acting at its receptors, in the pathogenesis of methotrexate toxicity (nodulosis), and that, surprisingly, inflammatory cytokines (IL-1 and TNFalpha) downregulate antiinflammatory (A2A) adenosine receptor mRNA. He now proposes to examine: I. The biochemical mechanism by which methotrexate promotes adenosine release. He will determine whether methotrexate promotes intracellular accumulation of adenine nucleotides (HPLC), increases transport of adenine nucleotides out of cells (HPLC, radiolabelling of adenine nucleotide pools), or diminishes uptake of adenosine by cells (radiolabelled adenosine uptake). II. Interaction between adenosine receptors and inflammation: Regulation and counterregulation. He will study the capacity of methotrexate to diminish acute and chronic inflammation in mice deficient in A2A, A3, or both receptors (knockout mice) to determine better which of these receptors is responsible for the antiinflammatory actions of adenosine. He will also determine whether IL-1 and TNFalpha regulate mRNA stability (nuclear run-off assays) or transcription (transfection of promoter constructs) of adenosine A2A receptor mRNA. III. Adenosine-mediated mechanisms of methotrexate toxicity. The most feared complication of methotrexate therapy is hepatic fibrosis. His data indicate that adenosine, acting at its receptors on fibroblasts, promotes matrix generation and inhibits matrix breakdown. He will determine whether methotrexate promotes adenosine release from hepatocytes and whether the adenosine released from these cells affects collagen or collagenase production by cultured stellate cells (hepatic fibroblasts). The results of the proposed studies will suggest novel approaches to the development of drugs for the treatment of inflammatory arthritis and may lead to the development of new approaches to the treatment and prevention of methotrexate toxicity.