PHARMACOLOGY OF ANTIRHEUMATIC AGENTS

描述：(改编自申请人的摘要)-调查员 首次提出了腺苷作用于特定受体的假说 对炎性细胞的影响，介导其抗炎作用 甲氨蝶呤--最常用、最有效的二线抗风湿药 代理可用。在这个项目的头两年里，他们实现了 四个最初目标中的三个。他证明了腺苷是由 从细胞外的核苷酸去磷酸化，证实 腺苷介导甲氨蝶呤的抗炎作用 活体关节炎模型，并描述了一种新的信号转导途径 中性粒细胞腺苷A2a受体。他还展示了之前的 作用于其受体的腺苷在发病机制中的意外作用 甲氨蝶呤毒性(结节病)，令人惊讶的是，炎症性 细胞因子(IL-1和TNFpha)下调抗炎(A2A)腺苷 受体基因。他现在建议研究：一、生化机制 哪种甲氨蝶呤能促进腺苷的释放。他将决定是否 甲氨蝶呤促进腺嘌呤核苷酸在细胞内蓄积 (高效液相)，增加腺嘌呤核苷酸向细胞外的转运(高效液相色谱法， 腺嘌呤核酸池的放射性标记)，或减少摄取 细胞的腺苷(放射性标记的腺苷摄取)。二、互动 腺苷受体与炎症之间的关系：调节和 反监管。他将研究甲氨蝶呤降低 A2A和/或A3缺陷小鼠的急性和慢性炎症 受体(基因敲除小鼠)，以更好地确定这些受体中的哪一个 负责腺苷的抗炎作用。他还将 确定IL-1和TNFpha是否调节mRNA稳定性(核径流 检测)或转录(转导启动子构建体) A2A型受体基因。三、腺苷介导的甲氨蝶呤作用机制 毒性。甲氨蝶呤治疗最可怕的并发症是肝脏 纤维化症。他的数据表明，腺苷作用于它的受体 成纤维细胞，促进基质生成，抑制基质分解。他 将确定甲氨蝶呤是否促进腺苷从 肝细胞以及从这些细胞释放的腺苷是否影响 培养的星状细胞(肝脏)产生胶原或胶原酶 成纤维细胞)。拟议的研究结果将提出新的 治疗炎症性疾病的药物开发途径 关节炎，并可能导致开发新的治疗方法 和预防甲氨蝶呤中毒。

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.