Sequential combination of methotrexate and vindesine in previously treated children with acute leukemia. A phase I-II study.

A therapeutic synergistic effect is seen in animal models when vinca alkaloids are administered after methotrexate. To examine further this interaction in clinical studies, a phase I-II trial was conducted in children with hematologic malignancies in the Department of Pediatrics at Memorial Sloan-Kettering Cancer Center. A schedule of sequential of methotrexate and vindesine was developed which showed effect in acute lymphoblastic leukemia in children in relapse and which was relatively nontoxic. The regimen has also been useful for reinduction for patients who are candidates for bone marrow transplant.

Synthesis of an N-aminopyrazinonium analogue of cytidine.

An N-aminated pyrazine analogue of cytidine, in which the pyrimidine N(3) ring nitrogen and C(4) amino group were replaced by a C-amino and an N-amino function, respectively, was prepared as a potential deaminase-resistant cytidine antimetabolite. The nucleoside 1,2-diamino-4-beta-D-ribofuranosylpyrazin-2-onium chloride (6) was a mild cytostatic agent but was neither a substrate for nor an inhibitor of mouse kidney cytidine deaminase. It ionized with a lower pKa than expected. The anion did not undergo the dimerization usually observed with N-imino heterocyclic ylides but unerwent hydrolysis of the 2-amino group to yield a 1-aminopyrazine-2,3-dione nucleoside.

Initial rate kinetics and evidence for duality of mediated transport of adenosine, related purine nucleosides, and nucleoside analogues in L1210 cells.

In studies using a rapid kinetic technique, evidence was derived for multiplicity of systems mediating [3H]adenosine transport in L1210 cells. A variety of approaches were used in discriminating between transport and kinase-mediated phosphorylation. Under these conditions, two systems mediating influx were delineated which exhibited high-affinity [Km = 13.9 +/- 2 (S.E.) microM] or low-affinity [Km = 199 +/- 27 microM] for [3H]-adenosine. Both systems exhibited high capacities, but that associated with the low-affinity system (V 37 degrees max = 263 +/- 43 nmol = 99.6 +/- 12 nmol sec/g, dry weight). The relative difference in affinity of these two systems during influx was also reflected in the values for influx Ki obtained with other nucleosides and nucleoside analogues. Influx of [3H]-adenosine by each mediated system was inhibited by 6-(2-hydroxy-5-nitrobenzyl)thioguanosine, a specific transport inhibitor, and by 9-beta-D-arabinofuranosylpurine-6(1H)thione which is not phosphorylated in L1210 cells. Influx kinetics were the same in L1210 cells, in adenosine triphosphate-depleted L1210 cells (L1210/ara-C/MMPR) which have substantially reduced ability for [3H]adenosine phosphorylation, and in the presence of 2'-deoxycoformycin, a potent inhibitor of adenosine deaminase. The same multiplicity in mediated influx of [3H]adenosine was shown at 0 degrees when transport became rate limiting to total uptake. The high-affinity system mediating [3H]adenosine influx was also elucidated in L1210 cell plasma membrane vesicles in the presence or absence of 2'-deoxycoformycin. Almost all of the natural nucleosides examined competed less effectively with [3H]adenosine for influx by the high-affinity system than by the low-affinity system. These results are discussed with respect to possible pharmacological implications.

Further studies stereospecificity at carbon 6 for membrane transport of tetrahydrofolates. Diastereoisomers of 5-methyltetrahydrofolates as competitive inhibitors of transport of methotrexate in L1210 cells.

The unnatural d diastereoisomer at carbon 6 of 5-methyltetrahydrofolate was only slightly less effective than the natural 1 diastereoisomer as a competitive inhibitor of the carrier-mediated membrane transport of [3H]methotrexate into L1210 murine leukemia cells. The apparent Ki for a mixture containing equal amounts of both natural and unnatural diastereoisomers was not significantly different from that found for the unnatural form. These results show that the reduced folate carrier system in these cells has a strong affinity for the unnatural stereoisomer, a finding in contrast to that obtained with the corresponding diastereoisomer of 5-formyltetrahydrofolate.

Syntheses of alpha- and gamma-substituted amides, peptides, and esters of methotrexate and their evaluation as inhibitors of folate metabolism.

N-[4-[[(Benzyloxy)carbonyl]methylamino]benzoyl]-L-glutamic acid alpha-benzyl ester (2) and gamma-benzyl ester (6) served as key intermediates in syntheses of precursors to amides and peptides of methotrexate (MTX) involving both the alpha- and gamma-carboxyl groupings of the glutamate moiety. Coupling of 2 and 6 at the open carboxyl grouping with amino compounds was affected by the mixed anhydride method (using isobutyl chloroformate); carboxyl groupings of amino acids coupled with 2 and 6 were protected as benzyl esters. N-[4-[[(Benzyloxy)carbonyl]methylamino]benzoyl]-L-glutamic acid gamma-methyl ester (5), a precursor to MTX gamma-methyl ester, was prepared from L-glutamic acid gamma-methyl ester and 4-[[(benzyloxy)carbonyl]methylamino]benzoyl chloride (1) in a manner similar to that used to prepare 2 and 6. The precursor to MTX alpha-methyl ester was prepared from gamma-benzyl ester 6 by treatment with MeI in DMF containing (i-Pr)2NEt. Benzyl and (benzyloxy)carbonyl protective groupings were removed by hydrogenolysis, and the deprotected side-chain precursors were converted to alpha- and gamma-substituted amides, peptides, and esters of MTX by alkylation with 6-(bromomethyl)-2,4-pteridinediamine hydrobromide (12). Biochemical-pharmacological studies on the prepared compounds aided in establishing that the alpha-carboxyl grouping of the glutamate moiety contributes to the binding of MTX to dihydrofolate reductase while the gamma-carboxyl does not. Other studies on the peptide MTX-gamma-Glu (13h) are concerned with the contribution toward antifolate activity of this metabolite of MTX. The compounds prepared were also evaluated and compared with MTX with respect to cytotoxicity toward H.Ep.-2 cells and effect on L1210 murine leukemia.

Increased schedule-dependent synergism of vindesine versus vincristine in combination with methotrexate against L1210 leukemia.

BD2F1 mice were inoculated ip with 10(6) L1210 leukemia cells and treated with vincristine or vindesine alone or in combination with methotrexate. Drug administration was begun on Day 1 and was continued every 4 days until a total of five doses were given or death occurred. Methotrexate (48 or 72 mg/kg ip) produced a 199% and a 222% increase in lifespan, respectively, as compared with untreated animals (6.9 +/- 0.5 days). When given as single agents, vincristine (0.5-1.0 mg/kg ip) or vindesine (0.5-1.5 mg/kg ip) produced between a 27% and an 88% increase in lifespan. The therapeutic benefit observed when either vinca alkaloid was used with methotrexate was schedule-dependent. With the exception of vindesine plus 72 mg/kg of methotrexate, the increase in lifespan produced by the simultaneous administration of methotrexate and either vinca alkaloid was additive. When vindesine was administered with 72 mg/kg of methotrexate, the increase in lifespan was greater than expected from an additive effect of the two agents. However, none of the trials employing single-agent therapy or simultaneous combination therapy produced long-term survivors (greater than or equal to 90 days after therapy). When either vinca alkaloid was given 24 hours after the folate analog, the increase in lifespan was almost 100% greater than that observed when the agents were given simultaneously; moreover, long-term survivors were produced. Vindesine in combination with 48 mg/kg of methotrexate produced 10%-25% long-term survivors, as compared to 5%-7% long-term survivors obtained with vincristine. In combination with 72 mg/kg of methotrexate, vindesine produced 27%-60% long-term survivors, as compared to 10%-20% long-term survivors obtained with vincristine. When either vinca alkaloid was administered 72 hours after methotrexate, the regimen was still synergistic, but the overall effect was less than with a 24-hour delay. When two doses of either vinca alkaloid were injected at 24 and 72 hours after the folate analog, the result was either highly therapeutic or very toxic. Two doses of 0.5 mg/kg of vindesine or vincristine with 48 mg/kg of methotrexate produced 35% and 20% long-term survivors, respectively. All other regimens were toxic.

Differential synthesis of methotrexate polyglutamates in normal proliferative and neoplastic mouse tissues in vivo.

Synthesis of poly-gamma-glutamyl metabolites of methotrexate was demonstrated in mouse small intestine, liver, and bone marrow and in L1210 leukemia, Sarcoma 180, and Ehrlich tumor cells after s.c. injections of [3H]methotrexate to tumor-bearing mice. Ion-exchange chromatography of tissue extracts resolved six peaks of radioactivity believed to represent methotrexate and metabolites with up to five additional glutamyl residues. Polyglutamate formation in L1210 cells and small intestine was shown to be independent of dose at least to 400 mg/kg as long as intracellular levels of drug in excess of the dihydrofolate reductase-binding capacity (exchangeable) were maintained. Both the total amount of polyglutamates and the average length of the polyglutamyl chain increased with time as long as exchangeable level of drug was present intracellularly. The results also showed differences in the extent of metabolism of methotrexate polyglutamates among the tissues examined. Although these differences were at times very large, there was no consistent correlation between these differences and other pharmacological parameters or cytotoxicity. Tumor cells appeared to synthesize more polyglutamates than did the normal tissues examined. However, differences in total drug persistence and sensitivity to drug among tumor cells and among normal tissues did not reflect the relative extent of polyglutamate synthesis in each group. It is concluded that the extent of polyglutamate synthesis per se may not be a determinant of drug sensitivity in murine tissues. However, the accumulation of these metabolites may contribute in some way to overall therapeutic response or relative cytotoxicity.

Synthesis and properties of the sulfonyl analogues of 4(5)-aminoimidazole-5(4)-carboxamide, 4(5)-(formylamino)imidazole-5(4)-carboxamide, guanine, and xanthine.

Reduction of 4(5)-nitroimidazole-5(4)-sulfonamide afforded the sulfonamide analogue of 4(5)-aminoimidazole-5(4)-carboxamide (AICA). This was formylated to afford the sulfonamide analogue of formyl-AICA and was ring closed to the unsubstituted 6-sulfonyl analogue of guanine, 3-aminoimidazo[4,5-e]-1,2,4-thiadizine 1,1-dioxide. Diazotiz ation of the latter afforded the corresponding 6-sulfonyl analogue of xanthine. None of the imidazole-sulfonamides or the purine 6-sulfonyl analogues inhibited the growth of L1210 cells in culture nor were they substrates for or significant inhibitors of human hypoxanthine--guanine phosphoribosyltransferase or milk xanthine oxidase.

Schedule-dependent synergism of methotrexate and vincristine against murine L1210 leukemia.

BD2F1 mice were inoculated with 10(6) L1210 murine lymphocytic leukemia cells and treated simultaneously with methotrexate and vincristine or with methotrexate followed by vincristine greater than or equal to 24 hours later. Four to six doses of methotrexate, 48 mg/kg ip, administered every 4 days beginning on Day 1 resulted in a 168%-228% increase in lifespan, while vincristine, at 0.5 mg/kg ip, given on the same schedule until death (two or three doses) gave only a 37% increase in lifespan. Simultaneous administration of both agents resulted in a therapeutic effect which was approximately additive. When vincristine was given 24 hours or 24 and 72 hours after the methotrexate, a further increase (70%-100%) in lifespan over that expected from an additive effect and long-term survivors (greater than 90 days) were obtained. Synergism between the two agents and long-term survivors were also seen with higher methotrexate concentrations (72 or 96 mg/kg) given in four or five courses. If therapy was initiated on Day 2 when the peritoneal tumor burden was approximately 2 x 10(7) cells, the combination of methotrexate with delayed vincristine still resulted in an increased therapeutic effect over that obtained with either drug alone, or that expected on an additive basis.