William T. Colwell

A new analogue of 10-deazaaminopterin with markedly enhanced curative effects against human tumor xenografts in mice

Purpose: These studies sought to evaluate the biochemical and cellular pharmacokinetic properties, cytotoxicity and antitumor efficacy of a new analogue of 10-deaza-aminopterin (PDX) against human tumors. Methods: Studies were conducted with a group of human tumor cell lines in culture examining PDX and other folate analogues as permeants for mediated membrane transport, as inhibitors of dihdrofolate reductase and as substrates for folylpolyglutamate synthetase. These same analogues were examined for their cytotoxicity following a 3-h pulse exposure, in experiments providing a value for IC50. Other studies with these analogues were conducted in nude mice bearing subcutaneously implanted human tumors. Treatment of the mice was initiated 4 days after implantation of the tumor using a schedule of administration of one dose per day for 5 days. The tumors were measured 6 days after cessation of therapy and compared to controls for assessment of response. Results: In the CCRF-CEM cell system, PDX was 2- to 3-fold less effective as an inhibitor of dihydrofolate reductase than aminopterin (AMT), methotrexate (MTX) or edatrexate (EDX) but much more effective as a permeant for one-carbon, reduced folate transport inward (PDX >AMT ≃ EDX >MTX) and substrate for folylpolyglutamate synthetase (PDX >AMT >EDX >MTX). As predicted by these results, PDX was 15- to 40-fold more cytotoxic than MTX and 3- to 4-fold more cytotoxic than the highly potent EDX following a 3-h pulse exposure in culture of CCRF-CEM cells and cells from a panel of three human breast and two human nonsmall-cell (NSC) lung cancers. The same relative differences were shown for the therapeutic efficacy of these three analogues at equitoxic doses in studies with the human MX-1 and LX-1 tumors and the human A549 NSC lung tumor xenografted in nude mice. On a schedule of qd × 5 given 3–4 days posttransplant, MTX was minimally active (modest tumor growth delay) against all three tumors. EDX was highly active (25–35% complete regressions and 5–10% cures) against the MX-1 and LX-1 tumors but very modestly active (no regressions) against the A549 tumor. In contrast, PDX was even more active (75–85% complete regressions and 25–30% cures) than EDX against the MX-1 and LX-1 tumors and highly active (30% complete regressions and 20% cures) against the A549 tumor. Conclusions: These studies showed significantly enhanced antitumor properties of PDX compared with MTX and EDX. Based upon these results, clinical trials of PDX in patients with metastatic breast and NSC lung cancer appear to be warranted.

Prostaglandins and pacemaker activity in isolated guinea pig SA node.

Prostaglandins PGE2, PGE1, PGF2alpha, and PGE1 substantially increase automaticity in SA-nodal, right atrial preparations excised from guinea pigs. This natural pacemaker tissue is sensitive to nanomolar doses of PG with, for example, 10(-8) M PGE2, increasing SA rate by about 20%. If these preparations are pretreated with 2 micrometer indomethacin, a blocker of endogenous prostaglandin synthesis, then spontaneous rate drops and subsequent rate increases due to PGE2 administration can be more easily demonstrated. Guinea pig pacemaker tissue differs from similar rabbit tissue not only in that it is directly responsive to PGE2, but also in that PGE2 does not depress the absolute response to transmural stimulation (adrenergically mediated rate increase). The positive chronotropic responses to PGE2 also occur when the guinea pig tissue is pretreated in 0.6 micrometer propranolol, which causes blockade of beta-adrenergic receptors. The pacemaker myocardium in the guinea pigs thus appears to be directly stimulated by exogenous PGE2 at very low doses. The observation that 2 micrometer indomethacin reduces SA-nodal rate suggests the presence of a very sensitive, functionally important, PGE-like system which modulates heart rate in this mammalian species.

Synthesis and evaluation of 8,10-dideazatetrahydrofolic acid and derivatives

Syntheses of 8,10-dideazatetrahydrofolic acid and its 5-N-methyl and 5-N-formyl derivatives are reported. Hydrolysis of 2,4-diamino-4-deoxy-8,10-dideazapteroic acid in hot alkali afforded 8, 10-dideazapteroic acid. Coupling with diethyl L-glutamate followed by saponification gave 8,10-dideazafolic acid. Hydrogenation in acidic media gave the tetrahydro compound, while hydrogenation in the presence of formaldehyde yielded the 5-N-CH 3 analog. The 5-N-CH 3 compound was more potent than 5,10-DDTHF as an inhibitor of growth for L1210 cells in culture. In contrast to 5,10-DDTHF, the locus of action was apparently unrelated to inhibition of GAR formyltransferase. Unfortunatly, 5-CH 3 -8,10-DDTHF was not active in vivo against an L1210 challenge in mice