PHARMACOLOGY OF ANTIRHEUMATIC AGENTS

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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.