Studies Relating to the Reversal of the Cytotoxicity of the Thymidylate Synthetase Inhibitor CB 3717

Abstract

The quinazoline based folate analogue CB 3717 is an antitumour agent, at present under clinical study, which has thymidylate synthetase (TS) as its sole cytotoxic locus. Unlike other antimetabolites affecting TMP synthesis the effects of the drug can be totally reversed by thymidine (TdR) alone both in vivo and in vitro. Our in vitro studies to define the requirements for the reversal of the cytotoxicity of CB 3717 have demonstrated that the methods generally used for this type of study are inaccurate and misleading. Cell counts (48hr) of L1210 suspension cultures (initial cell density 10⁴/ml) indicate that high levels (>2µ TdR) are required whereas colony assays suggest that low levels (>0.2µ M TdR) are sufficient. By monitoring the metabolism of TdR in suspension cultures this apparent anomaly has been resolved. Cytotoxicity was apparent when the concentration of TdR in the medium decreased to <0.05 M. Depletion of TdR occurred by (i) incorporation into DNA, which is dependent upon the cell concentration (for example 10⁴cells/ml supplemented with 0.5 moles/litre TdR incorporated 50% of this within 24 hours), (ii) catabolism to thymine by TdR phosphorylase. The nucleoside transport inhibitor dipyridamole (10µM) effectively blocked both the incorporation of TdR into DNA and the circumvention of CB 3717 inhibition of TS but did not affect the TdR phosphorylase activity present in the serum. We have shown that the level of TdR required to overcome CB 3717 toxicity to L1210 cells in vitro is within the range 0.05 - 0.17µM. Deoxyuridine (100µM) which is metabolised by TdR kinase and TdR phosphorylase did not affect the metabolism of TdR (1µM) nor its ability to overcome CB 3717 cytotoxicity.