Modulation of anti-metabolite effects. Effects of thymidine on the efficacy of the quinazoline-based thymidylate synthetase inhibitor, CB3717.

CB3717 (N-(4-(N-((2-amino-4- hydroxy-6-quinazolinyl)methyl)prop-2-ynylamino ) benzoyl)-L-glutamic acid) is an antitumour agent that inhibits thymidylate synthetase (TS). A dose-dependent fall in plasma thymidine (dThd) (1.43 microM to 0.47 microM) occurred in non-tumour-bearing mice following the administration of CB3717. Further, in mice carrying the L1210/CBRI tumour, the drug's antitumour properties were ablated by co-administration of dThd, an effect consistent with TS being the cytotoxic locus. In vitro studies of protection by dThd against CB3717 cytotoxicity were carried out in an attempt to quantify this reversal. The metabolism of [14C]-dThd was measured in cultures of L1210 cells (10(4)/ml) exposed to a completely cytotoxic dose of CB3717 (50 microM). The cytotoxicity of the drug was only expressed when the dThd concentration (0.5-2 microM) had fallen to less than 0.1 microM in the media. This reduction was due to: (1) dThd incorporation into DNA, (2) catabolism of dThd to thymine. By reducing the initial cell concentration to 10(3)/ml the depletion of dThd was substantially reduced and consequently cells continued to grow for a longer period. The critical concentration of dThd, below which growth in the presence of CB3717 could not be supported was estimated to be between 0.026 and 0.1 microM. Thus even the minimum level of dThd achieved in vivo was still in excess of that required for protection from CB3717 toxicity in vitro. There was a small accumulation of deoxyuridine (dUrd) (approximately 2-fold) in mouse plasma 24 hr after completion of a 5-day course of CB3717 (200 mg/kg) but in vitro studies demonstrated that this was unlikely to modulate CB3717 toxicity in the presence of dThd. We caution against the use of rodent tumour models (or human tumour xenografts) for antitumour or toxicity testing of compounds designed to inhibit the de novo synthesis of thymidylate; they may be misleading because the high dThd levels found in these animals compared with man may mask the cytotoxic effects of these drugs.