5-aminoimidazole-4-carboxamide ribotide (AICAR) and its metabolites: metabolic and cytotoxic effects and accumulation during methotrexate treatment

Abstract

Methotrexate is an antifolate useful in the treatment of neoplastic and non-neoplastic diseases. The mechanism of action of low dose methotrexate in the treatment of autoimmune diseases is unclear. It is known that methotrexate treatment blocks de novo purine biosynthesis, resulting in the accumulation of the intermediate 5-aminoimidazole-4-carboxamide ribotide and its metabolites. It is hypothesized that these substances are involved in the mechanism of action of low dose methotrexate therapy by inhibiting enzymes crucial to immune function. 5-aminoimidazole-4-carboxamide ribotide and its riboside were found to be irreversible inhibitors of S-adenosyl homocysteine hydrolase (EC 3.3.1.1) using enzyme from peripheral blood mononuclear cells of rheumatoid arthritis patients and from rabbit erythrocytes. 5-aminoimidazole-4-carboxamide was also found to be a competitive inhibitor (with respect to adenosine) of peripheral blood mononuclear cells adenosine deaminase (EC 3.5.4.4) and S-adenosyl homocysteine hydrolase. Methotrexote, at concentrations similar to blood levels of low-dose-treated patients (i.e., 10 and 20 nm), was cytotoxic to a culture of a human lymphoblast T cells but not to a culture of a human lymphoblast B cells. 5-aminoimidazole-4-carboxamide ribotide accumulated in T cells but not B cells treated with methotrexate, and 5-aminoimidazole-4-carboxamide-riboside potentiated methotrexate toxicity only in T cells. A reduction in the activity 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 cultured T cells was more sensitive to inhibition by 5-aminoimidazole-4-carboxamide riboside in the medium when compared with B cells. No substantial reduction of adenosine deaminase or S-adenosyl homocysteine hydrolase 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 interfering with both adenosine deaminase and S-adenosyl homocysteine hydrolase, enzymes crucial to normal immune function. Methotrexate cytotoxicity was associated with 5-aminoimidazole-4-carboxamide ribotide accumulation 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 sensitive to inhibition by 5-aminoimidazole-4-carboxamide-riboside. Dilution of inhibitors during enzyme assays may account for the fact that little or no decrease in the S-adenosyl homocysteine hydrolase or adenosine deaminase activities were observed in the methotrexate-treated cells. The data are consistent with a methotrexate-induced cytotoxicity targeted toward T cells, a cell type implicated in the pathology of autoimmune disease.