QUINAZOLINE THYMIDYLATE SYNTHASE INHIBITORS - METHODS FOR ASSESSING THE CONTRIBUTION OF POLYGLUTAMATION TO THEIR IN-VITRO ACTIVITY

Many quinazoline thymidylate synthase (TS) inhibitors undergo intracel lular metabolism to polyglutamate forms which can significantly alter their activity and pharmacodynamics through improved TS inhibition and drug retention. When a series of quinazolines was tested for inhibito ry activity towards TS (IC50 0.001-2 mu M) and the growth of L1210 cel ls (IC50 0.005-10 mu M), no direct correlation was observed. However, a very good correlation was apparent if a L1210 variant cell line (L12 10: R(D1694)) was used. This line is deficient in its ability to form antifolate polyglutamates. A number of other intact cell methods have also been developed which estimate the contribution that intracellular polyglutamation makes to a compound's activity. These assays were val idated using a series of quinazoline-based TS inhibitors with well-def ined activity for TS, folylpolyglutamate synthetase (FPGS) and the red uced-folate cell membrane carrier (RFC). Short-exposure growth-inhibit ion assays or the measurement of TS activity in situ after various inc ubation times, followed by different lengths of time in drug-free medi um, can indicate both the speed and extent of appearance of retentive forms (usually polyglutamates). Continuous-exposure growth-inhibition assays, in the presence of leucovorin (LV), are also useful, since onl y the growth-inhibitory potency of polyglutamated analogues is signifi cantly decreased by LV. Highly polyglutamated compounds, e.g. ZD1694, are virtually inactive in the presence of a high concentration of LV. It is proposed that these methods, when considered together, provide a greater degree of information concerning the rate and extent of polyg lutamation of a particular compound than isolated FPGS assays alone.