Faessel Hélène M, Slocum Harry K, Rustum Youcef M, Greco William R
Groton Laboratories, Pfizer Inc., Clinical PK-PD, Groton, CT 06340, USA.
Int J Oncol. 2003 Aug;23(2):401-9.
In order to examine the intracellular locus of the folic acid (PteGlu)-enhanced synergies of trimetrexate (TMQ) plus the thymidylate synthase (TS) inhibitor, raltitrexed (RTX), and TMQ plus the glycinamide ribonucleotide formyltransferase (GARFT) inhibitor, AG2034, comprehensive protection studies with thymidine (dThd) and hypoxanthine (HX) were conducted in a 96-well plate cell growth inhibition (sulforhodamine B) assay. Current modeling techniques were extended to characterize these protection patterns involving multiple-agent interaction. Wild-type human ileocecal HCT-8 cells and DW2, a subline deficient in folylpoly-gamma-glutamate synthetase (FPGS) were individually treated for 96 h with TMQ, AG2034 and a 1:1 mixture of TMQ:AG2034 or with TMQ, RTX, and a 1:1 mixture of TMQ:RTX in the presence of PteGlu (2.3 or 40 micro M) and the protection agents (10 micro M dThd and/or 100 micro M HX). Drug treatments were randomly assigned to wells. Both isobols and 3-dimensional concentration-effect surfaces were used to assess the nature and the intensity of drug interactions. The structural Hill model was fitted to data with weighted non-linear regression for most cases. A so-called 'double Hill' model was sometimes more appropriate when a plateau in the middle of the concentration-effect curve was found. In HCT-8 and DW2 cells at 2.3 and 40 micro M PteGlu, inhibition of DHFR by TMQ induced antithymidylate and antipurine effects; AG2034 and RTX selectively inhibited de novo purine or thymidine synthesis, respectively. dThd protection increased the PteGlu-enhancement of the TMQ + AG2034 synergy, whereas HX protection increased the PteGlu-enhancement of the TMQ + RTX synergy. The PteGlu-enhanced synergies of TMQ + AG2034 and TMQ + RTX occur primarily through inhibition of purine synthesis and inhibition of thymidylate synthesis, respectively. These results further substantiate the hypothesis that the nonpolyglutamylatable DHFR inhibitor, TMQ, acts as a modulator by decreasing the protection by PteGlu of cells against the polyglutamylatable AG2034 and RTX.
为了研究叶酸（PteGlu）增强的三甲曲沙（TMQ）与胸苷酸合成酶（TS）抑制剂雷替曲塞（RTX）以及TMQ与甘氨酰胺核糖核苷酸甲酰基转移酶（GARFT）抑制剂AG2034协同作用的细胞内位点，在96孔板细胞生长抑制（磺酰罗丹明B）试验中进行了用胸苷（dThd）和次黄嘌呤（HX）的全面保护研究。当前的建模技术得到扩展，以表征这些涉及多药相互作用的保护模式。野生型人回盲部HCT-8细胞和缺乏叶酰聚γ-谷氨酸合成酶（FPGS）的亚系DW2分别在PteGlu（2.3或40 μM）和保护剂（10 μM dThd和/或100 μM HX）存在的情况下，用TMQ、AG2034以及TMQ:AG2034的1:1混合物或用TMQ、RTX以及TMQ:RTX的1:1混合物处理96小时。药物处理随机分配到各孔。等效应线和三维浓度-效应表面均用于评估药物相互作用的性质和强度。在大多数情况下，用加权非线性回归将结构希尔模型拟合到数据。当在浓度-效应曲线中间发现一个平台时，有时所谓的“双希尔”模型更合适。在2.3和40 μM PteGlu条件下的HCT-8和DW2细胞中，TMQ对二氢叶酸还原酶（DHFR）的抑制诱导了抗胸苷酸和抗嘌呤效应；AG2034和RTX分别选择性抑制嘌呤或胸苷的从头合成。dThd保护增强了PteGlu对TMQ + AG2034协同作用的增强作用，而HX保护增强了PteGlu对TMQ + RTX协同作用的增强作用。TMQ + AG2034和TMQ + RTX的PteGlu增强协同作用分别主要通过抑制嘌呤合成和抑制胸苷酸合成而发生。这些结果进一步证实了以下假设：不可聚谷氨酸化的DHFR抑制剂TMQ通过降低PteGlu对细胞针对可聚谷氨酸化的AG2034和RTX的保护作用而起到调节剂的作用。
