D. M. Moccio

New folate analogs of the 10-deaza-aminopterin series Basis for structural design and biochemical and pharmacologic properties

SummaryStructural modification of the N10 position of 4-amino folates affects mediated membrane transport in mammalian cells but has little or no effect on target enzyme (dihydrofolate reductase) inhibition. Some of these modifications have been associated with differential effects on transport in various cell types in a manner which favored greater accumulation and persistence of drug in reponsive tumor cells than in normal proliferative tissue. With the aim of identifying new structures with greater potential for differential mediated accumulation, we have studied three new 10-alkyl analogs of 10-deaza-aminopterin. Two of these analogs showed therapeutic efficacy substantially greater than 10-deaza-aminopterin, an analog with antitumor properties superior to methotrexate. These analogs, the 10-methyl, 10-ethyl, and 10,10-dimethyl derivatives, were equivalent to the parent compound, 10-deaza-aminopterin, and aminopterin, and slightly more potent than methotrexate, as inhibitors of L1210 cell dihydrofolate reductase. The three new analogs, 10-deaza-aminopterin, and aminopterin exhibited similar transport properties in L1210, Ehrlich, and S180 cells. Efflux and influx Vmax were similar to those of methotrexate, but influx Km was 4- to 14-fold lower than for methotrexate. That is, substitution at N10, but not at C10, reduced influx potential in these tumor cells. These differences in transport properties among this group of analogs which determine net accumulation were reflected in the individual values for growth-inhibitory potency. In contrast to that seen in tumor cells, alkylation at both N10 and C10 reduced influx potential (increased Km) in isolated intestinal epithelial cells from mouse small intestine. Influx was in the order aminopterin >10-deaza-aminopterin with further reduction in each series showing a magnitude in proportion to the size of the 10 substituent. Otherwise, influx Vmax and efflux were similar for the group. Accumulation of polyglutamates in small intestine was greater following aminopterin administration than following administration of other analogs (10-ethyl, 10-deaza-aminopterin < methotrexate < 10-deaza-aminopterin). Polyglutamate accumulation for all the analogs was greater in tumor cells, but accumulation of each varied between the two tumors (L1210 and S180) examined. Differences among the analogs were not as great in L1210 as in S180 cells, and their metabolism was not in the same relative order. Plasma pharmacokinetics for 10-methyl and 10-ethyl derivatives were similar to those for 10-deaza-aminopterin and methotrexate. The 10,10-dimethyl analog was cleared more rapidly.As in our prior reports, a greater selective action of some of the new analogs was associated with increased persistence of the analog in tumor versus small intestine. The greatest differential in persistence was found with analogs which had a lower value for influx Km in tumor but a higher value in intestinal epithelium. Two analogs (10-deaza-aminopterin and 10-ethyl, 10-deaza-aminopterin) which exhibited identical transport properties but different extents of polyglutamylation had pharmacokinetics in tumor that were indistinguishable. However, the analog that was more rapidly polyglutamylated (10-ethyl, 10-deaza-aminopterin) was therapeutically more effective.

Stereospecificity at carbon 6 of formyltetrahydrofolate as a competitive inhibitor of transport and cytotoxicity of methotrexate in vitro

Abstract The unnatural diastereoisomer of l -5-formyltetrahydrofolate was 20-fold less effective as a competitive inhibitor of [ 3 H] methotrexate influx than the natural diastereoisomer during carrier-mediated membrane transport in L1210, S180 and Ehrlich cells. Values derived for K i , were 1.84 to 2.29 μM for the natural derivative and 35.2 to 53.8 μM for the unnatural derivative. Values for K i derived with a chemically synthesized mixture containing equal amounts of both natural and unnatural diastereoisomers were 2-fold greater than values obtained for the natural diastereoisomer. The unnatural diastereoisomer was 100-fold less effective and the chemically synthesized mixture was 2-fold less effective than the natural diastereoisomer in preventing inhibition by methotrexate of L1210 cell growth in culture. These results indicate that the unnatural diastereoisomer competes relatively ineffectively with the natural diastereoisomer or methotrexate for transport in these murine tumor cells.

New folate analogs of the 10-deaza-aminopterin series

SummaryA group of folate analogs of the 10-deaza-aminopterin series, which were designed on the basis of the results of an intensive biochemical and pharmacokinetic program, have been examined in therapy experiments utilizing a group of murine tumor models. These new analogs were found to be markedly superior to methotrexate against four of five ascites tumors (L1210 leukemia, Sarcoma 180, Ehrlich carcinoma and Tapper carcinosarcoma) and against four of five solid tumors (S180, Tapper carcinosarcoma, E0771 mammary adenocarcinoma, Lewis lung carcinoma, and T241 sarcoma). Analogs alkylated (methyl or ethyl) at the 10 position of 10-deaza-aminopterin were found to be the most effective of the group. These analogs achieved log10 reduction in tumor burden several-fold greater in magnitude than methotrexate against L1210 and S180 ascites tumors and there were also long-term survivors. 10-Deaza-aminopterin itself gave a result intermediate between those obtained with the 10-alkyl derivatives and methotrexate. Against the solid forms of the Tapper tumor some partial regressions were obtained with methotrexate and 10-deaza-aminopterin, but a far greater number, extending over a longer period were obtained with the 10-ethyl derivative of 10-deaza-aminopterin. Against the E0771 tumor, 10-deaza-aminopterin was 2-fold and the ethyl derivative of 10-deaza-aminopterin was >5-fold more effective than methotrexate in retarding tumor growth. Evidence for partial regressions and marked effects against metastatic disease were also obtained in the case of the 10-alkyl derivative. Similar results were also obtained with the T241 sarcoma. For Lewis lung carcinoma the relative potency was about the same but overall antitumor effects were more modest.

Detection by high-performance liquid chromatography of methotrexate and its metabolites in tumor tissue from osteosarcoma patients treated with high-dose methotrexate/leucovorin rescue

Methotrexate (MTX) polyglutamates were detected in osteogenic sarcoma tumor samples obtained from patients 24 or 48 h after receiving high-dose MTX/leucovorin rescue therapy. Tumor samples were assayed by high-performance liquid chromatography, and polyglutamyl metabolites, along with MTX, were quantitated using both direct u.v. absorption at 313 nm and an enzyme titration assay. Good agreement between these two methods was found although the more sensitive enzyme assay detected peaks in some samples not detected by u.v. absorbance. A wide variation in MTX:MTX polyglutamate levels (1:1 to 25:1) was found among the six clinical samples studied. Also, no correlation between the extent of polyglutamate formation and plasma levels (determined at the time of tumor sampling) was observed. High intracellular levels of a derivative which appears to be the 7-hydroxy metabolite of MTX were also detected in four of six samples. This material coeluted with authentic standard, showed spectral properties like standard 7-OH-MTX, and did not inhibit dihydrofolate reductase.

Analog specific aberrancies in antifolate inhibition of L1210 cell dihydrofolate reductase

Abstract During studies with L1210 cells and a variety of folate analogs, large discrepancies were revealed between data on membrane transport, on inhibition of dihydrofolate reductase in cell-free extracts, and on inhibition of growth in culture for 10-oxa-, 10-benzyl- and 10-phenethyl-aminopterin, and for 3-deaza, 10-methyl-aminopterin. While aminopterin, 10-methyl (methotrexate)-, 10-ethyl- and 10-propyl-aminopterin were tight binding inhibitors ( K i : 2–3 × 10 −12 M) of dihydrofolate reductase in cell-free extracts from L1210 cells, the other four analogs were only weak competitive inhibitors ( K i = 3–300 × 10 −8 M). Similar differences among analogs were observed for inhibition of dihydrofolate reductase in cell-free extracts from Sarcoma 180 and Ehrlich cells, but not for this enzyme in microbial cell-free extracts. There were only small differences in the transport of all of the analogs by L1210 cells. Inhibition of L1210 cell growth in culture by 10-oxa-, 10-benzyl- and 10-phenethyl-aminopterin and by 3-deaza, 10-methyl-aminopterin, in contrast to the other analogs, was several orders of magnitude greater than that predicted from the data on dihydrofolate reductase inhibition. The extent of binding of 10-oxa-, 10-benzyl- and 10-phenethyl-aminopterin, and of 3-deaza and 10-methyl-aminopterin to dihydrofolate reductase in intact L1210 cells, in contradistinction to that seen for the cell-free enzyme preparations, approached that observed for methotrexate; these estimates of drug-enzyme interaction in situ were more predictive of the extent of inhibition by these analogs of L1210 cell growth in culture.