S9511: a Southwest Oncology Group phase II study of trimetrexate, 5-fluorouracil, and leucovorin in unresectable or metastatic adenocarcinoma of the stomach.

OBJECTIVE: The primary objective of this trial was to evaluate the response rate for trimetrexate in conjunction with 5-FU and leucovorin (LV) (= TFL) in the treatment of advanced gastric cancer in a phase II, cooperative group setting. METHODS: Patients with locally advanced, unresectable, or metastatic adenocarcinoma of the stomach received trimetrexate 110 mg/m IV over 60 minutes day 1, followed by 5-FU 500 mg/m IV bolus and LV 200 mg/m IV over 60 minutes day 2, followed by oral LV 15 mg every 6 hours x 7 doses, all weekly for 6 weeks followed by 2 weeks of rest, continued until progression. RESULTS: Characteristics for 37 eligible patients: median age 63 (range: 23-83); male/female: 69% of 31%; performance status 0/1/2 15/20/1. The confirmed response rate was 19%, and median overall survival was 6 months. Two patients died as a result of therapy, 1 because of infection without significant neutropenia, and 1 due to perforation of a responding gastric lesion. Seventy-two percent experienced grades 3 and 4 toxicity, most commonly diarrhea, fatigue, and lymphopenia. CONCLUSIONS: This regimen achieves response rates comparable to other 5-FU-based regimens, when used in treatment of incurable gastric cancer. Toxicity appears manageable.

Inhibition of apoptosis by overexpressing Bcl-2 enhances gene amplification by a mechanism independent of aphidicolin pretreatment.

To study the effect of apoptosis on gene amplification, we have constructed HeLa S3 cell lines in which the expression of bcl-2 (BCL2) can be controlled by tetracycline in the growth medium. Induction of Bcl-2 expression caused a temporary delay of apoptosis and resulted in roughly a 3-fold increase in the frequency of resistant colonies when cells were selected with trimetrexate. This resistance was due to amplification of the dihydrofolate reductase gene. Cells grown out of the pooled resistant colonies retained the same level of resistance to trimetrexate whether Bcl-2 was induced or repressed, consistent with the theory that Bcl-2 functions by facilitating gene amplification, rather than being the resistance mechanism per se. Pretreating cells with aphidicolin is another method to increase gene amplification frequency. When Bcl-2-expressing cells were pretreated with aphidicolin, the resulting increase in gene amplification frequency was approximately the product of the increases caused by aphidicolin pretreatment or Bcl-2 expression alone, indicating that Bcl-2 increases gene amplification through a mechanism independent of that of aphidicolin pretreatment. These results are consistent with the concept that gene amplification occurs at a higher frequency during drug-induced cell cycle perturbation. Bcl-2 evidently increases the number of selected amplified colonies by prolonging cell survival during the perturbation.

The design of novel antifolates against pneumocystis carinii

The pneumonia caused by Pneumocystis carinii is ultimately responsible for the death of many acquired immunodeficiency syndrome (AIDS) patients. Large doses of trimethoprim and pyrimethamine in combination with a sulphonamide and/or pentamidine suppress the infection but produce serious side-effects and seldom prevent recurrence after treatment withdrawal. However, the partial success of the aforementioned antifolates, and also trimetrexate used alone, does suggest dihydrofolate reductase (DHFR) as a target for the development of antipneumocystis agents. From the DHFR inhibitory activities of 3'-substituted pyrimethamine analogues it was suggested that the 3'-(3'',3''-dimethyltriazen-1''-yl) substituent may be responsible for the greater activity for the P.carinii over the mammalian enzyme. Crystallographic and molecular modeling studies revealed considerable geometrical and electronic differences between the triazene and the chemically related formamidine functions that may account for the differences in DHFR inhibitory profiles. Structural and electronic parameters calculated for a series of 3'-(3'',3''-disubstitutedtriazen-1''-yl) pyrimethamine analogues did not correlate with the DHFR inhibitory activities. However, the in vitro screening against P.carinii DHFR revealed that the 3''-hydroxyethyl-3''-benzyl analogue was the most active and selective. Models of the active sites of human and P.carinii DHFRs were constructed using DHFR sequence and structural homology data which had identified key residues involved in substrate and cofactor binding. Low energy conformations of the 3'',3''-dimethyl and 3''-hydroxyethyl-3''-benzyle analogues, determined from nuclear magnetic resonance studies and theoretical calculations, were docked by superimposing the diaminopyrimidine fragment onto a previously docked pyrimethamine analogue. Enzyme kinetic data supported the 3''-hydroxyethyl-3''-benzyl moiety being located in the NADPH binding groove. The 3''-benzyl substituent was able to locate to within 3 AA of a valine residue in the active site of P.carinii DHFR thereby producing a hydrophobic contact. The equivalent residue in human DHFR is threonine, more hydrophilic and less likely to be involved in such a contact. This difference may account for the greater inhibitory activity this analogue has for P.carinii DHFR and provide a basis for future drug design. From an in vivo model of PCP in immunosuppressed rats it was established that the 3"-hydroxyethyl-3"-benzyl analogue was able to reduce the.P.carinii burden more effectively with increasing doses, without causmg any visible signs of toxicity. However, equivalent doses were not as effective as pentamidine, a current treatment of choice for Pneumocystis carinii pneumonia.