5-AMINOIMIDAZOLE-4-CARBOXAMIDE RIBOTIDE (AICAR) AND ITS METABOLITES -METABOLIC AND CYTOTOXIC EFFECTS AND ACCUMULATION DURING METHOTREXATE TREATMENT

Methotrexate is an antifolate useful in the treatment of neoplastic an d non-neoplastic diseases. The mechanism of action of low dose methotr exate in the treatment of autoimmune diseases is unclear. It is known that methotrexate treatment blocks de novo purine biosynthesis, result ing in the accumulation of the intermediate 5-aminoimidazole-4-carboxa mide ribotide and its metabolites. It is hypothesized that these subst ances are involved in the mechanism of action of low dose methotrexate therapy by inhibiting enzymes crucial to immune function. 5-aminoimid azole-4-carboxamide ribotide and its riboside were found to be irrever sible inhibitors of S-adenosyl homocysteine hydrolase (EC 3.3.1.1) usi ng enzyme from peripheral blood mononuclear cells of rheumatoid arthri tis patients and from rabbit erythrocytes. 5-aminoimidazole-4-carboxam ide was also found to be a competitive inhibitor (with respect to aden osine) of peripheral blood mononuclear cells adenosine deaminase (EC 3 .5.4.4) and S-adenosyl homocysteine hydrolase. Methotrexote, at concen trations similar to blood levels of low-dose-treated patients (i.e., 1 0 and 20 nm), was cytotoxic to a culture of a human lymphoblast T cell s but not to a culture of a human lymphoblast B cells. 5-aminoimidazol e-4-carboxamide ribotide accumulated in T cells but not B cells treate d with methotrexate, and 5-aminoimidazole-4-carboxamide-riboside poten tiated methotrexate toxicity only in T cells. A reduction in the activ ity of phosphoribosyl-aminoimidazole carboxamide formyltransferase (EC 2.1.2.3) was observed in methotrexate-treated cultures of T cells but not B cells. The S-adenosyl homocysteine hydrolase activity of cultur ed T cells was more sensitive to inhibition by 5-aminoimidazole-4-carb oxamide riboside in the medium when compared with B cells. No substant ial reduction of adenosine deaminase or S-adenosyl homocysteine hydrol ase activities was observed in methotrexate treated cultures of T or B cells. The data suggest that 5-aminoimidazole-4-carboxamide ribotide and 5-aminoimidazole-4-carboxamide-riboside have the potential for int erfering with both adenosine deaminase and S-adenosyl homocysteine hyd rolase, enzymes crucial to normal immune function. Methotrexate cytoto xicity was associated with 5-aminoimidazole-4-carboxamide ribotide acc umulation and an inhibition of 5-aminoimidazole-4-carboxamide ribotide transformylase. The fact that 5-aminoimidazole-4-carboxamide-riboside potentiates methotrexate cytotoxicity in T cells may also be related to the fact that its S-adenosyl homocysteine hydrolase is relatively s ensitive to inhibition by 5-aminoimidazole-4-carboxamide-riboside. Dil ution of inhibitors during enzyme assays may account for the fact that little or no decrease in the S-adenosyl homocysteine hydrolase or ade nosine deaminase activities were observed in the methotrexate-treated cells. The data are consistent with a methotrexate-induced cytotoxicit y targeted toward T cells, a cell type implicated in the pathology of autoimmune disease.