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Analysis of "early" thymidine/inosine protection as an adjunct to methotrexate therapy.

The feasibility of "early" thymidine and inosine protection of methotrexate (MTX) toxicity is evaluated in this paper. This approach is based on the proposition that the most vulnerable period for susceptible host cells to MTX toxicity is an interval following a pulse of MTX when the MTX monoglutamate level is high. In contrast, tumor cells that accumulate active MTX polyglutamyl derivatives are exposed to the cytotoxic effects of MTX, not only when the monoglutamate levels are high, but also over the much longer interval during which polyglutamyl derivatives are retained within these cells. The protecting agents employed, thymidine and inosine, circumvent the antipurine and antipyrimidine effects of MTX, but neither agent inhibits the transport or the polyglutamylation of MTX. Nucleoside protection given over 6 hours after MTX markedly decreased toxicity to normal BDF1 mice at MTX doses up to 150 mg/kg/day for 3 consecutive days. The LD10 for unprotected mice was 14 mg/kg/day, while the LD10 for the protected mice was 114 mg/kg/day, a greater than eightfold increase in the drug dose delivered. MTX with early nucleoside protection produced a 50% increase in median life span in tumor-bearing mice at doses of drug that were toxic to unprotected mice. Flow cytometric analyses indicated that consecutive daily pulses of MTX with early nucleoside protection alter the cell cycle distribution. By 24 hours after the last of three daily pulses of MTX, the G1 component was maximal (80% of the cell population), with less than 2% of the cells in G2M and 17% of the cells in S phase. This distribution persisted for 24 hours, after which a normal pattern emerged, a process that was accelerated by neither thymidine/inosine nor leucovorin. These studies support the concept that toxicity to the host is initiated largely in the interval shortly after MTX administration. The data indicate that early nucleoside protection permits the repetitive administration of much higher doses of MTX than can be given with MTX alone. This approach may be useful not only in the treatment of human neoplasms but in the treatment of other disorders, such as rheumatoid arthritis, psoriatic arthritis, or psoriasis. Finally, this paper considers the potential advantages of early thymidine/inosine protection in comparison to leucovorin rescue with the administration of low or moderate doses of MTX.

Animals↗
PubMed1985

Phenotypic heterogeneity in cultured human head and neck squamous cell carcinoma lines with low-level methotrexate resistance.

Low-level methotrexate (MTX) resistance (less than 20-fold) was induced by gradual selection pressure in four human head and neck squamous cell carcinoma lines established in culture from biopsies of patients not previously treated with MTX. Each parental and resistant line was characterized with respect to MTX uptake and polyglutamylation, dihydrofolate reductase (DHFR) content, and growth rate. Relative DHFR gene copy numbers and amounts of DHFR-related cytoplasmic messenger RNA were analyzed by plasmid complementary DNA hybridization in a dot blot assay and were correlated with the amount of gene product. The resistant lines were not cloned in order to simulate in vitro the conditions which might exist in an in vivo setting, where multiple resistant subpopulations of cells may be present in a tumor. The study was restricted to cells with low-level resistance since these are likely to be the clinically most relevant type. Of the four resistant lines characterized, one showed a severe defect in MTX uptake and polyglutamylation, another was a DHFR overproducer with only small changes in uptake and polyglutamylation, a third was likewise a DHFR overproducer but also showed lower MTX uptake, and the fourth was minimally altered except for growth rate. The diversity in resistance phenotype among these cells in vitro suggests that in vivo resistance in patients with head and neck carcinoma who are treated with MTX may similarly involve multiple mechanisms and that further therapeutic intervention using MTX or other antifolates should take this into account.

Biological Transport↗
PubMed1985

Role of methotrexate polyglutamates in methotrexate- and sequential methotrexate-5-fluorouracil-mediated cell kill.

The synthesis of methotrexate (MTX) polyglutamates has been investigated in a human leukemia cell line, CCRF-CEM. An increase in either the extracellular MTX concentration (0.1-10 microM) or exposure time (4-24 h) allowed a greater accumulation of polyglutamate derivatives. The species which predominated (di-, tri-, or tetraglutamate) was also time and concentration dependent. Derivatives up to the pentaglutamate were readily detectable, with hexaglutamate present under some conditions. Polyglutamate derivatives were preferentially retained when the cells were transferred to drug-free medium. MTX itself rapidly exited the cells under these conditions. In the absence of extracellular MTX, the performed intracellular MTX polyglutamates continued to be elongated. Accumulation of MTX polyglutamates, particularly triglutamate and longer species, positively correlated with cytotoxicity to CCRF-CEM cells as measured by a clonogenic assay. Investigation of the methotrexate-fluorouracil interaction revealed no effect of 5-fluorouracil on MTX polyglutamate accumulation under various conditions where widely different degrees of cytotoxicity were found. The synergy observed when MTX precedes fluorouracil treatment is therefore not an effect on MTX polyglutamylation. MTX polyglutamates may, however, play an indirect role in this synergy.

Cell Survival↗
PubMed1985

Role of polyglutamates in methotrexate action.

Numerous cell systems and tissues contain methotrexate (MTX) polyglutamates after MTX and HDMTX (high dose) treatment. The formation of MTX polyglutamates is concentration- and time-dependent. Great differences in the length of the poly-y-glutamyl side chains are observed. Efflux kinetics of MTX and MTX polyglutamates vary considerably depending on the kind of cells and the length of the side chain. In erythrocytes accumulation of MTX and MTX polyglutamates results in intracellular concentrations much higher than the serum MTX levels. Efflux differences with long-lasting storage of the polyglutamate forms of MTX up to the total life-span of the single erythrocyte. Even 6-14 days after HDMTX therapy, high MTX levels could be determined in sarcoma tissue. In each case besides unchanged MTX there was a formation of MTX polyglutamates. The portion of polyglutamates varied considerably, amounting from 3% to 68% of total tumor MTX. A relation between the formation of MTX polyglutamates and the clinical effectiveness of HDMTX therapy seems to be possible. MTX polyglutamates play a major role in the principles and effectiveness of HDMTX therapy.

Erythrocytes↗
PubMed1986

Hydrolytic cleavage of methotrexate gamma-polyglutamates by folylpolyglutamyl hydrolase derived from various tumors and normal tissues of the mouse.

Folylpolyglutamate hydrolase (folyl hydrolase) activity derived from murine tumors and various normal tissues was measured by means of high performance liquid chromatography using methotrexate polyglutamates as substrate. Enzyme-mediated hydrolysis was considerably greater (10-20-fold) on a specific activity basis in extracts from all normal mouse tissues (kidney greater than bone marrow greater than small intestine approximately equal to liver) than from tumor cells (Sarcoma 180 greater than Ehrlich approximately equal to L1210 cells). Enzyme preparations from purified absorptive and crypt cell epithelium from mouse small intestine exhibited comparable levels of specific activity and were greater than that derived from the total organ. Folyl hydrolase from mouse kidney showed mixed endo- and exopeptidase activity while that derived from all other normal tissues and tumor cells was consistent with endopeptidase activity. Levels of cell-free folyl hydrolase activity derived from tumor cells varied substantially with the phase of growth in vivo. Also, levels were appreciably lower from the same cells grown in vitro. Hydrolysis by crude or partially purified enzyme preparations from mouse small intestine or tumor cells conformed to Michaelis-Menten kinetics (single saturable component). Rates of hydrolysis and Km values were proportional to gamma-glutamyl chain length in the case of L1210 cell-derived enzyme but not for enzyme derived from small intestine. Km values derived for 4-amino-10-methylpteroyldiglutamate were the same [Km = 80.4 +/- 9 (SE) microM] for small intestine and L1210 cells. However, with 4-amino-10-methylpteroyltetraglutamate Km values were 3-fold lower for tumor cell preparations and 8-fold lower for preparations derived from small intestine. Fourfold lower Km values for 4-amino-10-methylpteroyldiglutamate were obtained with enzyme derived from Sarcoma 180 cells as compared to the enzyme from L1210 or intestinal cells. Varying levels of folyl hydrolase activity for methotrexate polyglutamates in cell-free preparations from different tumor cells appeared to reflect differences in in situ hydrolytic activity shown for the same substrate when internalized. The relevance of these results to antifolate pharmacology and, specifically, to a role for polyglutamates of 4-aminofolate compounds in determining cytotoxicity and selective antitumor activity of these agents is discussed.

Animals↗
PubMed1986

Conversion of methotrexate to 7-hydroxymethotrexate and 7-hydroxymethotrexate polyglutamates in cultured rat hepatic cells.

The formation of 7-hydroxymethotrexate and its diglutamate has been established in rat hepatic parenchymal cells in culture. The formation of 7-hydroxymethotrexate increases with the extracellular methotrexate concentration (1-50 microM) and with time over a 24-h period. The majority of the 7-hydroxy derivative is found in the medium after 6- and 24-h incubations at all concentrations examined. At high methotrexate concentration (50 microM) 7% of the total extracellular methotrexate was the 7-hydroxy derivative. 7-Hydroxymethotrexate diglutamate is accumulated within the cell, although longer chain length derivatives are not observed. The inability to form longer chain length polyglutamate derivatives is consistent with a limited capacity of hepatocytes to convert radiolabeled 7-hydroxymethotrexate to the tri- and tetraglutamates although the diglutamate is readily formed. A hepatoma cell line has an extremely limited capacity to form 7-hydroxy derivatives from methotrexate but form measurable amounts of mono-, di-, and triglutamates of 7-hydroxymethotrexate. 7-Hydroxymethotrexate was a good substrate for glutamylation in the hepatoma cells with large amounts of tri- through pentaglutamate derivatives. These studies confirm that 7-hydroxymethotrexate and the diglutamate must be considered when evaluating methotrexate pharmacology and demonstrate that this will be necessary with regard to methotrexate hepatotoxicity.

Animals↗
PubMed1986

Inhibitory action of 10-deazaaminopterins and their polyglutamates on human thymidylate synthase.

The action of 10-deazaaminopterin, its 10-alkyl derivatives, and their polyglutamates against thymidylate synthase (TMPS) from human acute myeloblastic leukemia was examined. Comparison of aminopterin with methotrexate showed that the methylation of the N10-position (methotrexate) increased the inhibitory effect of aminopterin on TMPS. In contrast, alkylation of the 10-position of 10-deazaaminopterin decreased inhibition of TMPS, and the 50% inhibitory concentration values were progressively higher, in the order 10,10-dimethyl-, 10-methyl-, and 10-ethyl-derivatives. The addition of gamma-glutamyl moieties to both 10-deazaaminopterin, and one of its alkylated analogs, 10-ethyl-10-deazaaminopterin, enhanced inhibition. The maximum inhibition was achieved with the addition of three glutamyl moieties to 10-deazaaminopterin and two glutamyl moieties to 10-ethyl-10-deazaaminopterin, respectively. Thus, 10-deazaaminopterin-tetraglutamate was 138-fold and 10-ethyl-10-deazaaminopterin-triglutamate was greater than 51-fold more active than their respective parental compound. The compounds 10-deazaaminopterin and its polyglutamates, 10-methyl- and 10,10-dimethyl-analogs, inhibited TMPS in a noncompetitive fashion with respect to 5,10-methylene-tetrahydropteroylglutamate. Ki values for the monoglutamates were 220 microM, 310 microM, and 225 microM, respectively. In contrast, 10-ethyl-10-deazaaminopterin and its polyglutamates inhibited TMPS in a competitive fashion with a Ki value of 410 microM for the monoglutamate. With 5,10-methylene-tetrahydropteroylpentaglutamate as a substrate, 10-deazaaminopterin and its polyglutamates behaved as mixed type inhibitors, and 10-ethyl-10-deazaaminopterin, monoglutamate and diglutamate, behaved as noncompetitive inhibitors, whereas its pentaglutamate behaved as a mixed-type inhibitor. These results suggest that the addition of gamma-glutamyl moieties to the substrate also caused the change in the mode of inhibitory action of these compounds. These findings also show that both replacement of the N10-position of the 4-aminopteroyl structure with a methylene group and its alkylation caused interesting and unexpected changes in the structure-activity relationships and the mode of action for these 4-aminopteroyl antifolates as inhibitors of TMPS, which may be therapeutically relevant.

Alkylation↗
PubMed1987

L-asparaginase-induced modulation of methotrexate polyglutamylation in murine leukemia L5178Y.

The modulation of methotrexate polyglutamylation by L-asparaginase has been examined in mice bearing sublines of leukemia L5178Y that have different sensitivities to asparaginase. A single i.p. injection of 200 IU/kg of asparaginase completely inhibited ascites tumor cell growth in the parental L5178Y/S+ tumor for 120 h compared to 72 and 30 h in the L5178Y/S and L5178Y/S+/- sublines, respectively. Similarly, DNA and protein synthesis were completely inhibited by asparaginase for 96 h in L5178Y/S+ cells, but only for 72 and 24 h in L5178Y/S and L5178Y/S+/- cells. In each tumor the temporal patterns of depletion and recovery of S-phase cells were similar to the patterns of suppression and recovery of DNA and protein synthesis observed in that tumor. When methotrexate was administered at either 96 or 24 h after asparaginase during the asparaginase-induced S-phase nadirs of L5178Y/S+ and L5178Y/S+/- cells, respectively, subsequent methotrexate polyglutamylation was inhibited 83 and 92% compared to tumor cells exposed to methotrexate only. Recovery of methotrexate polyglutamylation in both tumors following L-asparaginase pretreatment coincided in time with the return in the fraction of S-phase cells towards the pretreatment values. The inhibition of methotrexate polyglutamate accumulation by asparaginase was associated with decreased retention of methotrexate in tumor cells. In contrast, asparaginase had no significant effect on methotrexate polyglutamate accumulation and methotrexate retention when administered after methotrexate. These data indicated that the asparaginase-induced modulation of methotrexate polyglutamylation in mice was directly related to the time course of inhibition and recovery of tumor cell proliferation by asparaginase, and thus varied with the intrinsic sensitivity of the individual tumor to the enzyme.

Animals↗
PubMed1987

Toxicity, elimination, and metabolism of 10-ethyl-10-deazaaminopterin in rats and dogs.

10-Ethyl-10-deazaaminopterin (10-EdAM) is an antifolate compound with greater therapeutic activity than methotrexate against transplanted tumors in mice. When given weekly for 3 weeks, the 10% lethal dose in rats was 125 mg/kg (i.p.) and in dogs it was 2.5 mg/kg (i.v.). The major histopathological findings in intoxicated animals were damage to the mucosa of the gastrointestinal tract in rats and dogs and hypocellularity of the marrow in rats. The elimination of 50 mg/kg of 10-EdAM from the plasma of rats was triexponential with a terminal phase t1/2 of 18.5 h but a mean residence time of 0.7 h. The primary route of elimination in rats was biliary secretion of parent compound and eventual excretion of the parent compound and the deglutamate metabolite in the feces; the 7-hydroxy metabolite was also present in plasma, bile, and feces. Biliary elimination was independent of dose over a 5-fold range. The elimination of 10-EdAM from the plasma of dogs was also triexponential with a mean terminal phase t1/2 of 9.1 h and a mean residence time of 2.5 h; nonrenal clearance was the primary route of elimination. The pharmacokinetic parameters were independent of dose over the range of 0.25 to 5.0 mg/kg. High tissue concentrations of 10-EdAM were observed initially in liver, kidney, and small intestine of rats, while concentrations in bone marrow were low. Some polyglutamate formation was observed in these tissues as early as 0.5 h after drug administration but declined over 72 h.

Aminopterin↗
PubMed1988

Proliferation-dependent cytotoxicity of methotrexate in murine L5178Y leukemia.

The basis for the proliferation-dependent cytotoxicity of methotrexate has been investigated in mice bearing the L5178Y ascites leukemia. Methotrexate at 60 mg/kg i.p. reduced the viability of logarithmically growing ascites cells (55% active S phase cells) to 28% of control, whereas the viability of the slowly growing cells (18% active S phase) was decreased to only 59% of control. Log phase tumor cells accumulated 8-fold higher levels of methotrexate polyglutamates compared to cells that had approached the stationary phase. However, no differences between log phase and slowly growing tumor cells were observed in the cellular levels of unmetabolized methotrexate. Intestinal mucosa and bone marrow from non-tumor-bearing mice resembled slowly growing tumor cells and had markedly lower levels of methotrexate polyglutamates than logarithmically growing cells. The greater accumulation of methotrexate polyglutamates in the logarithmically growing tumor cells was consistent with an increased synthesis of methotrexate polyglutamates in these cells. The enhanced methotrexate polyglutamylation in log phase versus slowly growing cells was not related to changes in the rates of either cellular methotrexate transport, transmembrane efflux of methotrexate, or hydrolysis of methotrexate polyglutamates. Thymidylate synthase activity measured in situ and in extracts from log phase cells was 4- and 2-fold higher, respectively, than in the more slowly growing cells. Methotrexate produced a 2.4-fold greater depletion of poly-gamma-glutamyl derivatives of 5,10-methylenetetrahydropteroylglutamate in log phase cells compared to slowly growing cells, and this was a function of both the increased methotrexate polyglutamate accumulation and thymidylate synthase activity in the rapidly proliferating cells. These results provide further evidence that the selectivity of methotrexate for tumors with a high growth fraction is a consequence of the rapid rates of both cellular methotrexate polyglutamate synthesis and oxidation of 5,10-methylenetetrahydropteroyl polyglutamates by thymidylate synthase.

Animals↗
PubMed1988

Methotrexate suppresses passive adjuvant arthritis: studies on the metabolism of methotrexate in mononuclear cells derived from normal and adjuvant arthritic rats.

Passive transfer of adjuvant arthritis by spleen cells is suppressed by methotrexate. Mononuclear cells derived from spleens of normal and adjuvant arthritic Lewis rats were incubated with [3H]-methotrexate and harvested at various periods of time. The amount of methotrexate and its various polyglutamates were quantitated. The results of these studies indicate that the kinetics of uptake of methotrexate by mononuclear cells from normal and adjuvant arthritic rats are similar. However, the amount of methotrexate polyglutamates accumulating in the mononuclear cells of adjuvant arthritic rats was significantly lower than that observed in mononuclear cells derived from normal rats.

Animals↗
PubMed1989

Biochemical and growth inhibitory effects of the erythro and threo isomers of gamma-fluoromethotrexate, a methotrexate analogue defective in polyglutamylation.

We previously reported (J. Galivan et al., Proc. Natl. Acad. Sci. USA, 82: 2598-2602, 1985) the synthesis and characterization of DL-erythro,threo-gamma-fluoromethotrexate (FMTX). The individual diastereomers, DL-erythro-FMTX (eFMTX) and DL-threo-FMTX (tFMTX), and their radiolabeled counterparts have now been prepared and characterized. Transport of eFMTX (Km = 9.3 microM; Vmax = 7.5 pmol/min/10(7) cells) was similar to that of methotrexate (MTX: Km = 6.6-9.9 microM; Vmax = 11.4-14.2 pmol/min/10(7) cells), while tFMTX (Km = 65.1 microM; Vmax = 8.4 pmol/min/10(7) cells) was transported less efficiently. Both isomers were able to saturate intracellular dihydrofolate reductase and accumulate further as unbound intracellular drug. Based on competition experiments and studies with MTX transport-defective cell lines, both isomers utilized the reduced folate/MTX transport system. Efflux half-times for the isomers were similar to those of MTX. Each isomer was equivalent to MTX in its ability to inhibit dihydrofolate reductase activity and bind to intracellular dihydrofolate reductase when the intracellular drug concentration was limiting. Both isomers had drastically diminished capacity to be metabolized to poly(gamma-glutamyl) metabolites by isolated folylpolyglutamate synthetase and in whole cells; tFMTX was metabolized to a slightly lesser extent than eFMTX. Using the CCRF-CEM human leukemia and H35 rat hepatoma cell lines, the growth-inhibitory effects of eFMTX were almost the same as those of MTX during continuous exposure, while tFMTX was slightly less potent. This difference in growth-inhibitory potency of the two isomers correlated with their ability to inhibit de novo thymidylate synthesis in the H35 cell line. These results indicate that both diastereomers of FMTX are similar in their properties to MTX, except that both are incapable of being readily converted to polyglutamate derivatives. As a result of these properties, both isomers could be used under appropriate conditions in comparative studies with MTX to define the roles of MTX polyglutamates.

Animals↗
PubMed1989

Inherent resistance of human squamous carcinoma cell lines to methotrexate as a result of decreased polyglutamylation of this drug.

Three human squamous carcinoma cell lines (FaDu, A253, and SQCC/Y1) were tested for sensitivity to methotrexate (MTX) and trimetrexate, a second generation folate antagonist in clinical trials. Two of the three cell lines (A253 and SQCC/Y1) showed inherent resistance to methotrexate, when cytotoxicity was evaluated after short term exposure (4 and 24 h). In contrast, all three cell lines were markedly sensitive to trimetrexate, an antifolate which is taken up by cells by a different transport system than is methotrexate and which is not polyglutamylated. The basis for the natural resistance to methotrexate shown by two of the three cell lines was examined. Levels of dihydrofolate reductase activity, inhibition of this enzyme by methotrexate, and influx of MTX did not differ significantly between the three cell lines; however, resistance was correlated to the amounts of polyglutamates of methotrexate synthesized by the three cell lines. After a 24-h incubation with 10 microM MTX, the A253 cell line was able to form only 35.0 pmol/10(7) cells of polyglutamates, compared to 250 pmol/10(7) cells synthesized by the FaDu cell line, while the SQCC/Y1 cell line, intermediate in sensitivity to methotrexate, was able to form 145 pmol/10(7) cells of MTX polyglutamates. The A253 cell line contained less folylpolyglutamate synthetase activity compared to the FaDu and SQCC/Y1 cell lines. However, it is not clear if this difference is sufficient to explain the marked differences in polyglutamates of methotrexate found between the cell lines. We conclude that decreased polyglutamylation of methotrexate in some human squamous cell carcinomas may be the major contributing factor in inherent resistance to high dose pulse administration of this drug.

Antineoplastic Agents↗
PubMed1989

Wild-type and drug-resistant Leishmania major hydrolyze methotrexate to N-10-methyl-4-deoxy-4-aminopteroate without accumulation of methotrexate polyglutamates.

We have examined the metabolism of the folate analog methotrexate (MTX) in the human parasite Leishmania major. These cells readily hydrolyzed MTX to N-10-methyl-4-deoxy-4-aminopteroate (MAPA), such that following a 24-h incubation in 1 microM [3H]MTX approximately 30% of the cell-associated radioactivity was MAPA. MAPA also accumulated in the culture medium, exceeding the concentration of MTX after 24 h. Neither 7-hydroxy-methotrexate nor MTX polyglutamates were observed in cells or medium, even after a 72-h incubation with MTX. In contrast to MTX, folate is extensively polyglutamylated in L. major (Santi, D. V., Nolan, P., and Shane, B. (1987) Biochem. Biophys. Res. Commun. 146, 1089-1092). MAPA was found to be 190-fold less potent than MTX as an inhibitor of leishmanial growth and to bind less tightly than MTX to leishmanial dihydrofolate reductase-thymidylate synthase. We therefore examined the possibility that MTX resistance is mediated by increased MTX hydrolysis to MAPA in drug-resistant Leishmania. However, enzymatic assays show that the rate of MTX hydrolysis was unaltered in the MTX-resistant R1000-3 line and the primaquine-resistant PQ-R30 line (which is 24-fold cross-resistant to MTX). In addition to MAPA, several minor unidentified metabolites were observed in the LT252, R1000-5B, and PQR30 cells but no consistent differences in the amounts of these metabolites were evident among these lines. These data indicate that alterations in the rate of MTX hydrolysis in vitro, or in the characteristics of MTX metabolites formed in vivo, do not underly the MTX resistance displayed by the H region-amplified R1000-5B and PQ-R30 lines.

Animals↗