Sequential methotrexate/5-FU: FdUMP formation and TS inhibition in a transplantable rodent colon adenocarcinoma

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Abstract
Parameters for inhibition of thymidylate synthetase were studied after sequential methotrexate/5-fluorouracil (5-FU) administration in a dimethylhydrazine (DMH)-induced transplantable rat colon carcinoma. Tumor-bearing rats were treated with methotrexate (MTX) 40 mg/kg IP Bolus 5-FU, 100 mg/kg IP, was injected after 24 h. Micromethods for assay of 5-fluoro-2′-deoxyuridylate (FdUMP) and thymidylate synthetase (TS) were used to study the in vivo intracellular pharmacokinetics of 5-FU. Formation of FdUMP was equally rapid in tumors regardless of MTX pretreatment, with peak values found at 30 min. Although MTX pretreatment did not increase peak FdUMP levels, it appeared to result in increased persistence of FdUMP, well in excess of available TS-binding sites, at 24 and 48 h. The combination therapy was less effective in terms of TS inhibition over the first 8 h after 5-FU administration, but may have been associated with improved TS inhibition at later time points. Total levels of TS (TStot) steadily increased from a pre-5-FU treatment level of 18.8 pmol to more than 40 pmol/g at 24 h. MTX per se had no apparent effect on baseline TStot levels or on the 5-FU-mediated increases in TStot. We conclude that MTX and 5-FU were antagonistic in terms of TS inhibition over the first 8 h after 5-FU in this DMH-induced rat colon carcinoma, but were possibly synergistic in increasing persistent levels of FdUMP and TS inhibition at later time points. The observation that 5-FU treatment can result in progressive increases in TS levels in some tumors suggests that this may be an important mechanism of 5-FU resistance.