Effects of Allopurinol on the Therapeutic Efficacy of Methotrexate1

(CANCER RESEARCH 35, 1702 1705, July 1975] Effects of Allopurinol on the Therapeutic Efficacy of Methotrexate1

Gerald B. Grindey and Richard G. Moran

Department of Experimental Therapeutics, and Grace Cancer Drug Center, Roswell Park Memorial Institute. Buffalo, New York 14263

SUMMARY In the present study, the relevancy of such effects to the therapeutic selectivity of MTX in vivo was approached. The antitumor effects of methotrexate against early Hughes et al. (13) and Christine et al. (4) reported that the leukemia L1210 were partially reversed by the coadminis- level of TdR in normal mouse serum is 1 to 2 /IM. Such tration of allopurinol in vivo, even though allopurinol did levels of TdR suggest that, in the mouse, an increased not alter the growth-inhibitory effects of methotrexate availability of preformed purines might be all that is against L1210 cells in culture. These data suggest that this required to alter the toxicity and/or the therapeutic efficacy alteration in antitumor activity results from a decreased of MTX. As has been shown by Pomales et al. (18, 19), catabolism of preformed systemic purines by allopurinol, a HPP, a potent inhibitor of xanthine oxidase, markedly potent inhibitor of xanthine oxidase. On the other hand, the increased the utilization of exogenous Hx for synthesis of therapeutic effect of methotrexate against the P288 leu cellular nucleic acids in vivo. Hence, the effects of the kemia was not significantly altered by allopurinol treat concurrent administration of HPP on the toxicity and ment. In addition, coadministration of allopurinol did not antitumor efficacy of MTX were examined. significantly alter the toxicity of methotrexate in normal mice. The data are consistent with the hypothesis that, in the mouse, the antileukemic effects of methotrexate are more MATERIALS AND METHODS related to a purineless rather than a thymineless death. Female DBA/2 Ha-DD mice (18 to 21 g) were obtained INTRODUCTION from the breeding colony of the Roswell Park Memorial Institute, while the female DBA/2J mice were purchased The studies by Hakala and Taylor (8, 9) demonstrated from The Jackson Laboratory, Bar Harbor, Maine. The leukemias L1210 and P-288 used were as described previ that inhibition of the growth of mouse Sarcoma 180 cells in culture by MTX2 could be completely prevented by the ously (15, 20). In all experiments, 1 x 10" tumor cells were addition of the end products of folate metabolism to the inoculated i.p. into recipient mice and treatments were culture medium, i.e., by a source of preformed purine such begun, except where indicated, 24 hr after tumor cell as Hx, as well as exogenous TdR and the otherwise inoculation. In all experiments, HPP was administered I hr nonessential amino acid glycine. When Eagle's medium was prior to MTX. The mean survival was counted from the day supplemented with Hx, the concentration of MTX required of inoculation, which was considered Day 0. Drugs were to bring about half-maximal inhibition of the growth of obtained from Sigma Chemical Co., St. Louis, Mo., or Sarcoma 180 cells was twice that observed in the absence of from the pharmacy of the Roswell Park Memorial Institute. Hx; the addition of TdR alone had no such sparing effect. In S.D. and / tests were as described by Snedecor (21). a subline of Sarcoma 180 selected in the presence of TdR for Leukemia L1210 cells (7) were grown in culture in resistance to MTX, the addition of TdR to the test medium Roswell Park Memorial Institute Medium 1640 supple decreased the growth-inhibitory effects of MTX, while mented with 10% dialyzed fetal calf serum (Grand Island purine alone was without effect (10). This same specificity Biological Co., Grand Island, N. Y.), which was further dialyzed for 3 days after purchase, and with /V-2-hydroxy- has been observed in a subline of mouse fibroblast cells ethylpiperazine-Ari-2-ethanesulfonicacid and 2-(Ar-mor- (LOOT) by Borsa and Whitmore (2). The lethality of MTX to L5178Y mouse lymphoma cells has been reported (11, pholino)ethanesulfonic acid (Sigma Chemical Co.) at a final 12) to be significantly decreased by exogenous purine, while concentration of 16 and 8 mM, respectively. Cell number addition of preformed purine to the culture medium appears was determined with a Coulter Model B particle counter to augment the MTX-induced decrease in the cloning (Coulter Electronics, Hialeah, Fla.) with a 70-^m window. efficiency of L60T cells (3). Thus, the exogenous supply of Under these conditions, L1210 grew logarithmically for about 48 hr with a doubling time of 10 to 12 hr. Leukemia one or all of the end products of folate metabolism can P-288 cells grown in culture originated from a 6-day ascites significantly alter the cytotoxicity of MTX. tumor in female DBA/2J mice. The cells were washed and suspended in Roswell Park Memorial Institute Medium 'This work was supported in part by USPHS Grants CA-13038 and 1640, supplemented as above at an initial density of 2 to 2.5 CA-05298. A preliminary report of this investigation has appeared (5). x IO4 cells/ml. Such primary cultures, when incubated at 2The abbreviations used are: MTX, methotrexate: Hx, hypoxanthine; 37Â°,grew logarithmically for about 72 hr with a mean TdR, thymidine; HPP, allopurinol. Received December 2. 1974; accepted March 31, 1975. doubling time of about 18 hr after a slight lag.

1702 CANCER RESEARCH VOL. 35

RESULTS 14 L I2IO

The ability of various concentrations of TdR and Hx to - 10 support the growth of cultures of L1210 cells in the presence of a high concentration of MTX is shown in Chart I. Under I 8 CONTROLS culture conditions that allowed the continuous logarithmic I 6 growth of control cultures, the optimal concentration of O MTX TdR required for the reversal of MTX toxicity was 3 to 6 Â¿IM O < â€¢¿MTX+ ALLOPURINOL(20mg/kgi6) in the presence of adequate Hx (32 jtM). The optimal a: concentration of Hx in the presence of adequate TdR (5.6 H\i) was 18 to 50 /iM (Chart 1). The same range of 0.75 1.5' 3.0 4.5 60 9.0 concentrations of both TdR and Hx were also found to be adequate for the reversal of the toxicity of 10~*M MTX for MTX (mg/kg x3) Chart 2. Partial reversal of the antitumor effects of MTX by HPP mouse leukemia P-288 cells in culture; such levels of TdR against leukemia LI210. in DBA/2 Ha-DD female mice. MTX was and Hx have previously been shown to protect Sarcoma 180 administered i.p. I hr after HPP every other day for 3 days starting on Day cells in culture from the effects of MTX (9). 1. HPP was administered Â¡.p.,daily for 6 days starting on Day 0. Each As shown in Chart 2, HPP, given daily for 6 days at 20 group contained 20 mice. HPP reversal of the therapeutic effects of MTX mg/kg, partially reversed the antitumor effects of various at 0.75 mg/kg was significant at/Â»< 0.05; at 1.5 mg/kg,p < 0.001; at 3.0 doses of MTX against early leukemia L1210 in DBA/2 mg/kg,p < 0.00l;at4.5mg/kg,p < 0.001; and at 6.0 mg/kg,p < 0.05. H-DD mice. HPP alone on this schedule had no effect on the survival of tumor-bearing animals, nor was it toxic to normal mice, as indicated by both weight loss and mortality. Table 1 MTX alone at the optimal dosage (3 mg/kg) on this Effect of HPP on the activity of MTX in the treatment of leukemia LI 210 schedule increased the life-span of leukemic mice by 73%, in DBA Â¡2Ha-DD mice while only a 33% increase in life-span was observed in mice MTX" survival treated with the combination of MTX and HPP. The (mg/kg)33333HPP'(mg/kg)305102030Av. (days)9.0 difference in survival time due to HPP coadministration was significant at p < 0.001. The effects of varying doses of Â±1.29e8.5 Â±1.2614.8 HPP on the therapeutic activity of MTX given every other Â±1.5712.5 day for 3 days (3 mg/kg) are shown in Table 1. While HPP Â±0.92"11.8 at 5 mg/kg afforded a slight reversal of MTX activity, Â±1.98"10.3 maximum effects were observed at 20 mg/kg (p < 0.01). Â±2.29-11.7 Â±0.75' HPP alone at 30 mg/kg had no effect on tumor lethality. The effects of administration of HPP on the lethal po * MTX was administered i.p. every other day for 3 days starting on Day tency of MTX were examined in normal DBA/2Ha-DD 1. Each group consisted of 6 mice. mice. The results of such a study, in which MTX was 'HPP was administered i.p. I hr prior to MTX daily for 6 days starting on Day 0. administered daily for 5 days and HPP was administered c Mean Â±S.D. daily for 6 days, are shown in Chart 3. When these data d Significantly different from the survival of MTX-treated animals (3 were analyzed by the method of Litchfield and Wilcoxon mg/kg) at p < 0.05. (14), the lethal doses for 50% of the mice for MTX alone 'Significantly different from the survival of MTX-treated animals (3 (4.0 mg/kg) and that for MTX plus HPP (3.2 mg/kg) were mg/kg) at/? < 0.01. found not to differ significantly (p = 0.05). In addition, no definite effects of HPP on the lethality of MTX were ob served when both drugs were administered daily for 15 100 r NORMAL MICE days.

I z 15 1.75 2.5 3.5 5.0 7.0 100 140 200 280 400 IO MTX (mg/kg K 5) Chart 3. Effect of the administration of HPP on the toxicity of MTX in normal DBA/2 Ha-DD female mice. HPP (20 mg/kg) was administered i.p. daily for 6 days. MTX was administered i.p. I hr after HPP daily for 5 days starting 24 hr after the 1st injection of HPP. Each group contained at IO'' ICT least 10 mice, while the groups treated with 2.5, 3.5, or 5.0 mg/kg of MTX TdR or Hx or MTX plus HPP contained 20 mice. The lethal dose for 50% of the mice Chart I. Reversal of the growth-inhibitory effects of MTX by TdR and for MTX alone was not significantly different from that of MTX plus HPP Hx. (p = 0.05).

JULY 1975 1703

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1975 American Association for Cancer Research. G. B. Grindey and R. G. Moran The above data demonstrate that the coadministration of DISCUSSION HPP alters the therapeutic efficacy of MTX against mouse leukemia L1210 in vivo. When the effect of HPP on the Murray (16) has proposed that, in the normal mouse, the antiproliferative effects of MTX against L1210 cells grow presence of significant levels of Hx would be unlikely, due to ing in culture was examined, the presence of noninhibitory the high xanthine oxidase activity in serum (1). This is or even toxic concentrations of HPP did not alter the consistent with the observations by Pomales et al. (18, 19) potency of MTX (Chart 4). These data constitute evidence that administration of HPP, a potent inhibitor of xanthine against a direct reversal of the effects of MTX by HPP at oxidase, dramatically increases the incorporation of both the level of the target cell and suggest that an indirect effect Hx and xanthine into cellular nucleic acids in the mouse. of HPP is responsible for the decreased antitumor efficacy Thus the decreased therapeutic activity of MTX observed of MTX seen in vivo (Chart 2). following concurrent administration of HPP (Chart 2), In an attempt to completely reverse the therapeutic although apparently not attributable to a direct antagonism activity of MTX against leukemia L1210in DBA/2 Ha-DD at the level of the target cell (Chart 4), might well be the mice, HPP was administered daily for 11 days starting 3 result of increased levels of circulating Hx. days prior to tumor inoculation. This was followed by the As described by Hakala and Taylor (8, 9), the growth- administration of MTX every other day for 3 days starting inhibitory effects of MTX against mammalian cells in 24 hr after tumor inoculation. However, this intensive culture can be completely reversed by the addition of TdR schedule of HPP still afforded only a partial reversal of the and Hx to the culture medium. The concentration of TdR in therapeutic activity of MTX against leukemia L1210. On normal mouse serum (1 to 2 ^M), as reported by Hughes et the other hand, administration of HPP only very slightly al. (13) and Christine et al. (4), is sufficient for substantial reversed the therapeutic effects of MTX against leukemia reversal of the growth-inhibitory effects of MTX in the P-288 (Chart 5). At no dose of MTX was this reversal presence of adequate preformed purine (Chart 1). Thus, in significant (p > 0.05). Thus, depending on the type of mice, an increased availability of preformed purine might be tumor, treatment with HPP can result in either no effect or all that is required to reduce the therapeutic effectiveness of a partial reversal of the antitumor activity of MTX. MTX. As shown in Chart 2, concurrent administration of HPP reduced the therapeutic effectiveness of MTX against leukemia L1210. On the other hand, treatment with HPP neither reduces the antitumor effect of MTX against HPP(pM) leukemia P-288 (Chart 5) nor significantly alters the toxicity of MTX in normal mice (Chart 3). It is difficult to attribute the partial reversal of the cytocidal effects of MTX by HPP strictly on the basis of the exogenous supply of the end products of folate metabolism, since the same concentra tions of TdR and Hx will reverse MTX toxicity against both L1210 and P-288 in culture. In addition, it is difficult to evaluate the effects of HPP on host immunity to these transplanted tumors in decreasing the therapeutic activity of (0 30 MTX (15). MTX (Mx IO'9) It would seem impossible to predict a priori the effect of Chart 4. Effect of HPP on the growth-inhibitory activity of MTX MTX therapy on the circulating levels of TdR or Hx against leukemia LI210 cells in culture. Numbers above each curve, following HPP treatment. Such levels might increase due to concentrations of HPP (^M); â€¢¿.dose-responsecurve for MTX alone. gross cellular cytotoxicity, with resultant release of salvage metabolites, or they might decrease due to a decreased P-288 overall rate of purine and thymidylate synthesis in tissues that normally would release such compounds into the serum. The partial reversal of MTX effects by HPP might also be due not to end product reversal by TdR and Hx but rather to the presence of circulating Hx by itself. In mouse fibroblasts in culture, it has been reported that the O MTX l MTX t ALLOPURINOL addition of purine to the culture medium increased the (20mg/kg x II) (E lethality of MTX (3). On the other hand, Hryniuk and * Berlino (11, 12) have reported that, in L5178Y murine 1.5 3.0 4.5 6.0 9.0 18.0 lymphoblasts in culture, the addition of Hx to the medium MTX (mg/kg x3) partially protected the cells against the lethal effects of Chart 5. Slight reversal of the antitumor effects of MTX by HPP MTX. against leukemia P-288 in DBA/2J female mice. MTX was administered In preliminary studies (6) we found that injection of levels i.p. 1 hr after HPP every other day for 3 days starting on Day I. HPP (20 mg/kg) was administered i.p. daily for 11 days starling 3 days prior to of TdR phosphorylase, partially purified from Escherichia tumor inoculation. Each group contained 10 mice. The average survival of coli B, which supressed the incorporation of TdR into tumor the groups treated with MTX plus HPP was not significantly different >90%, had little effect on the therapeutic efficacy of MTX from that of MTX alone (p > 0.05). towards mouse leukemia L1210. However, the toxicity of

1704 CANCER RESEARCH VOL.35

MTX towards the host was substantially increased by this 5. Grindey, G. B., and Moran, R. G. Antitumor Effects of Methotrexate enzyme therapy. This suggests that the normal levels of (MTX): Partial Protection by Allopurinol (HPP) in vivo. Proc. circulating TdR in the mouse partially spare those tissues Am. Assoc. Cancer Res., 14: 27, 1973. critical for survival from the toxicity of MTX. In addition, a 6. Grindey, G. B., Moran, R. G., and Werkheiser, W. C. Approaches to the Rational Combination of Antimetabolites for Cancer Chemother report has recently appeared (22) that administration of apy. In: E. J. Ariens (Ã©d.).DrugDesign. Vol. 5, pp. 160 249. New additional TdR further decreases the host toxicity of MTX York: Academic Press, Inc. 1975. in the mouse, while not affecting the response of an early 7. Grindey, G. B., and Nichol, C. A. Interaction of Drugs Inhibiting L1210 tumor to MTX therapy. Different Steps in the Synthesis of DNA. Cancer Res., 32: 527-531, While these results support the concept that, in the 1972. mouse, the therapeutic index of MTX might well be 8. Hakala, M. T. Prevention of Toxicity of Amethopterin for Sarcoma improved by coadministration of TdR, it is clear that this 180 Cells in Tissue Culture. Science, 126: 255, 1957. concept cannot be directly applied in the clinic without 9. Hakala, M. T., and Taylor. E. The Ability of Purine and Thymine further studies. Whereas the high levels of xanthine oxidase Derivatives and of Glycine to Support the Growth of Mammalian in mouse serum make significant levels of circulating Hx Cells in Culture. J. Biol. Chem., 234: 126 128, 1959. unlikely in the mouse (1, 16), substantial concentrations of 10. Hakala, M. T., Zakrzewski, S. F., and Nichol, C. A. Relation of Folie Hx ( > 40 p M) have been found in human serum (17), which Acid ReducÃaseto Amethopterin Resistance in Cultured Mammalian Cells. J. Biol. Chem., 236: 952 958, 1961. lacks xanthine oxidase activity (1). In the presence of TdR, 11. Hryniuk, W. M. Purineless Death as a Link between Growth Rate and these concentrations of Hx are adequate to reverse the Cytotoxicity by Methotrexate. Cancer Res., 32: 1506 1511, 1972. growth-inhibitory effects of MTX against 2 human malig 12. Hryniuk. W. M., and Berlino, J. R. Growth Rate and Cell Kill. Ann. nant cell lines (HeLa and J-lll) in culture (9). Recently, N. Y. Acad. Sci., 186: 330-342, 1971. Then and Angehrn (23), utilizing a biological assay, re 13. Hughes. W. L., Christine, M., and Stollar, B. D. A Radioimmunoas ported that the concentration of TdR in human blood was say for Measurement of Serum Thymidine. Anal. Biochem., 55: extremely low (<2 x 10^' M). They concluded that the 468 478, 1973. concentration of TdR in human blood is too low to prevent 14. Litchfield, J. T., Jr., and Wilcoxon, F. A Simplified Method of Evaluating Dose-Effect Experiments. J. Pharmacol. Exptl. Therap., the bactericidal action of the antifolate, Trimethoprim. Thus, coadministration of MTX and TdR to human 96. 99 113, 1949. 15. Mihich, E. Combined Effects of Chemotherapy and Immunity against subjects (who may already have sufficient levels of circulat Leukemia L1210 in DBA/2 Mice. Cancer Res., 29: 848 854, 1969. ing purines) might well result in a total product reversal of 16. Murray, A. W. The Biological Significance of Purine Salvage. Ann. MTX and may compromise or negate the therapeutic Rev. Biochem., 40: 811 826, 1971. effects of this agent. 17. Orsulak, P. J., Haab, W., and Appleton, M. D. Quantitative In summary, while the therapeutic effects of MTX in the Estimation of Uric Acid, Xanthine and Hypoxanthine in Plasma mouse may be more related to a purineless death, efficacy Using Thin-Layer Chromatography. Anal. Biochem., 23: 156 162, may be dependent on a thymineless death in the clinic. 1968. Further studies are needed to document the levels of TdR 18. Pomales, R., Bieber, S., Friedman, R., and Hitchings, G. H. and Hx in plasma in cancer patients and to evaluate the role Augmentation of the Incorporation of Hypoxanthine into Nucleic of various salvage metabolites in clinical MTX therapy. Acids by the Administration of an Inhibitor of Xanthine Oxidase. Biochim. Biophys. Acta, 72: 119 120, 1963. 19. Pomales, R., Elion, G. B., and Hitchings, G. H. Xanthine as a Precursor of Nucleic Acid Purines in the Mouse. Biochim. Biophys. Acta, 95: 505-506, 1965. REFERENCES 20. Schwartz, H. S., and Grindey, G. B. Adriamycin and Daunorubicin: A Comparison of Antitumor Activities and Tissue Uptake in 1. Al-Khalidi, U. A. S., and Chaglassian, T. H. The Species Distribution Mice following Immunosuppression. Cancer Res., 33: 1837 1844, of Xanthine Oxidase. Biochem. J., 97: 318-320, 1965. 1973. 2. Borsa. J.. and Whitmore. G. F. Studies Relating to the Mode of 21. Snedecor, G. W. In: Statistical Methods, Ed. 5, pp. 42 52. Ames, Action of Methotrexate. II. Studies on Sites of Action in L-cells in Iowa: Iowa State College Press, 1956. vitro. Mol. Pharmacol., 5: 303-317, 1969. 22. Tattersall, M. H. N.. Brown, B., and Frei, E., III. The Reversal of 3. Borsa, J., and Whitmore, G. F. Cell Killing Studies on the Mode of Methotrexate Toxicity by Thymidine with Maintenance of Antitumor Action of Methotrexate on L-cells in vitro. Cancer Res., 29: Effects. Nature, 253: 198 200, 1975. 737.744, 1969. 23. Then, R., and Angehrn, P. The Biochemical Basis of the Antimicrobial 4. Christine, M., Stollar, D., and Hughes. W. L. A Radioimmunoassay Action of Sulfonamides and Trimethoprim In Vivo. I. Action of for the Measurement of Serum Thymidine. Federation Proc., 31: 807, Sulfonamides and Trimethoprim in Blood and Urine. Biochem. 1972. Pharmacol., 23: 2977-2982, 1974.

JULY 1975 1705

Gerald B. Grindey and Richard G. Moran

Cancer Res 1975;35:1702-1705.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/35/7/1702

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/35/7/1702. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.