Low-Dose Methotrexate Results in the Selective Accumulation of Aminoimidazole Carboxamide Ribotide in an Erythroblastoid Cell Line

Abstract

Therapeutic and toxic response to low-dose methotrexate (MTX) in the treatment of autoimmune disease continues to be highly variable, resulting in a critical need to identify predictive biomarkers of response. Biomarker development has been hampered by an incomplete understanding of the molecular pharmacology of low-dose MTX. To address this issue, accumulation of the substrates for aminoimidazole carboxamide ribonucleotide transformylase (AICART) and thymidylate synthase (TS) was measured as markers of pharmacological activity of MTX in an erythroblastoid cell line. A 115-fold increase in the AICART substrate and anti-inflammatory mediator, 5-aminoimidazole-4-carboxamide-1-b-D-ribofuranosyl 59-monophosphate (ZMP), was observed following exposure to 10 nM MTX but subsequently decreased with increasing MTX concentrations, declining to baseline levels with 1000 nM MTX. In contrast, the TS substrate, 29-deoxyuridine 59-monophosphate disodium salt (dUMP), displayed concentration-dependent accumulation, increasing 29-, 342-, and 471-fold over baseline with 10, 100, and 1000 nM MTX, respectively. Cellular levels of dUMP correlated with levels of the parent drug (MTX-PG 1 ; r 5 0.66, P , 0.001) and its polyglutamates (MTX-PG 2-6 ) (r 5 0.81, P , 0.001), whereas cellular levels of ZMP were only moderately correlated with MTX-PG 1 (r 5 0.34, P , 0.01). In contrast, accumulation of ZMP at 10 nM MTX was associated with a 2.9-fold increase in the AICART inhibitor dihydrofolate (DHF), represented primarily by long-chain DHF polyglutamates. Selectivity, defined as the ratio of ZMP to dUMP, was maximal following exposure to 6 nM MTX. Characterizing the range of MTX concentrations that selectively promote ZMP accumulation while preserving pyrimidine biosynthesis may lead to optimization of low-dose MTX therapy.

LD-Aminopterin in the Canine Homologue of Human Atopic Dermatitis: A Randomized, Controlled Trial Reveals Dosing Factors Affecting Optimal Therapy

“…These observations led these investigators to predict clinical anti-inflammatory responses due to AICAR might be paradoxically related to antifolate dose, whereas a dose-proportional response would be seen if due to inhibition of thymidylate synthase. Toxicity is observed in all subjects administered a sufficiently high dose of LD-aminopterin or methotrexate [12], [14], consistent with the proposal that antifolate toxicity is mediated by thymidylate synthase inhibition [40]. In contrast, the dose-response data for efficacy in this study mirrors the in vitro concentration-response findings for AICAR described by Funk et al .…”

“…recently demonstrated AICAR increased 115-fold following exposure of an erythroblastoid cell line to 10 nM methotrexate, but decreased with increasing methotrexate concentrations, declining to baseline with 1000 nM methotrexate [40]. In contrast, the substrate for thymidylate synthase, 2′-deoxyuridine 5′-monophosphate (dUMP), displayed concentration-dependent accumulation over the same range of methotrexate concentration.…”

Folate Depletion and Increased Glutamation in Juvenile Idiopathic Arthritis Patients Treated With Methotrexate

“…K562 cells actively transport and metabolize MTX to its polyglutamated metabolites (33-35). Cells were maintained in RPMI-1640 medium (Life Technologies, 61870-127) supplemented with 10% fetal bovine serum (Atlanta Biologicals, S11150) in a 37°C and 5% CO 2 controlled incubator at a density between 2×10 5 and 1×10 6 cells/mL.…”

“…Cells were treated with MTX over 24-hours as previously published (35). Folic acid (Sigma, F7876) was added to folic acid-free RPMI-1640 (Life Technologies, 27016-021) to achieve concentrations between 0 and 20 μM, and supplemented with 10% fetal bovine serum.…”

Molecular Basis for Pharmacokinetics and Pharmacodynamics of Methotrexate in Rheumatoid Arthritis Therapy

“…2) Biochemical studies have shown that MTX-PGs can inhibit the activity of AICART more strongly than MTX, indicating that MTX-PG is the biologically active form of MTX. 19) The inhibition of AICART by MTX-PGs promotes the accumulation of its substrate, AICART ribonucleotide (ZMP), 20) which is a potent inhibitor of adenosine deaminase, and thereby causes an increase in adenosine triphosphate (ATP) and adenosine levels. These intracellular compounds are released into the extracellular space, where activation of signaling pathways by stimulation of adenosine receptors leads to anti-inflammatory action of MTX.…”