[CANCER RESEARCH 36, 2790-2794, August 1976]

SUMMARY

We have used the spleen colony assay system and survival duration studies in male DBA/2 mice with P388 leukemia to study the effects of allopurinol pretreatment on theantileukemic activity of cyclophosphamide and its bonemarrow toxicity. Allopurinol drinking water (0.5 mg/mI) wasgiven for 7 days prior to cyclophosphamide (10 to 200 mg/kg i.p.). Average daily albopurinol intake per mouse was 1.25mg (equivalent to 4 mg/kg/day human dosage). Dose-response curves with and without allopurinol pretreatmentshowed an almost constant 0.9-log increase in the toxicityof cyclophosphamide to leukemic colony-forming units,whereas allopurinol had no effect on the toxicity of cyclophosphamide to normal bone marrow colony-forming units.Parallel survival studies revealed no difference in the antileukemic activity of cyclophosphamide as a result of albopurinol pretreatment. The allopurinol-induced change in theantitumor activity of cyclophosphamide as seen in thespleen colony assay was not explainable on a pharmacokinetic basis. Flow microfluorometric analysis of P388 leukemia tumor cell cycle parameters revealed no change in theblockading effects of cycbophoshamide as a result of allopurinol preexposure. Although we have failed to explain theunderlying mechanism of this drug interaction, our datasuggest that allopurinol may increase the antitumor activityof cyclophosphamide without increasing its bone marrowtoxicity.

INTRODUCTION

Cycbophosphamide is a major anticancer drug with activity against a wide variety of human and animal neoplasms(19). This drug is inactive in vitro, requiring hepatic microsomal enzymes to convert it into its active forms (3, 7, 15). Anumber of routine drugs (e.g. , sedatives, narcotics, antibiotics, etc.) may increase or decrease hepatic microsomal

,ThisinvestigationwassupportedinpartbyClinicalCancerResearchCenter Grant CA-i 1067 (to the University of California) and Medical OncologyProgram Project Grant CA-i7094 (to the University of Arizona) from theNational Cancer Institute, NIH, Bethesda, Md; the Faculty Training Grant inClinical Pharmacology from the Pharmaceutical Manufacturing Association,Washington, D. C.; and the Cancer Coordinating Committee Grant from theUniversity of California, Berkeley, Calif. Presented in part at the 64th AnnualMeeting of the American Association of Cancer Research, San Diego, Calif.May1975(1).

2To whom requests for reprints should be addressed.3 Present address: Department of Microbiology, University of California,

School of Medicine, San Francisco, Calif. 94143.Received November 6, 1975; accepted April 20, 1976.

enzyme activity and thereby alter cycbophosphamide metabolism (6, 25). Allopurinob [4-hydroxypyrazobo(3,4d)- pyrimidine], a xanthine oxidase inhibitor which is commonlyused for the prophylaxis of hyperuricemia in patients withhematobogical neoplasms (17, 20), has been said to increase the suppression of granulopoiesis caused by cylophosphamide and 6-mercaptopurine (2, 5, 18, 24). The Boston Collaborative Drug Surveillance Program has used retrospective data to show that cancer patients, especiallythose with hematological cancers, treated with a combination of allopurinol and cycbophosphamide experiencedgreater leukopenia and more thrombocytopenia than thosetreated with the anticancer drug alone (2, 24). Ragab et al.(22), using in vivo mouse and associated in vitro bonemarrow culture techniques, demonstrated that allopurinolat greater than 100 mg/kg caused important depressions ofgranulocytic colony-forming cells. We have studied the invivo effects of allopurinol pretreatment on cyclophosphamide antileukemic and bone marrow stem cell toxicity usinga mouse spleen colony assay system and tumor-bearingmouse survival studies. In addition we have attempted torelate changes observed in cyclophosphamide antileukemicactivity to changes in its pharmacokinetics or its effects ontumor cell cycle kinetics caused by prior treatment of themice with allopurinol (1).

MATERIALS AND METHODS

Mice. Six- to 8-week old male DBA/2 mice (The JacksonLaboratory, Bar Harbor, Maine) weighing approximately 25g were used in these experiments.

Mouse Tumor Line. P388 lymphocytic leukemia was supplied by Dr. John Harris (Department of Radiobiology, University of California, San Francisco, San Francisco, Calif.and serially transplanted as an ascites tumor at weeklyintervals (i0@ cells every 7 days in Medium 199) (GrandIsland Biological Co., Grand Island, N. Y.). It was selectedfor these studies because it more accurately predicts clinical efficacy of anticancer drugs than do other mouse tumorlines(27).

Chemotherapeutic Agents. Albopurinol(Burroughs Wellcome Company, Research Triangle Park, N. C.) in powderform was dissolved in 1.0 N sodium hydroxide, and the pHwas adjusted to 10.5 by the addition of 2 N hydrochloricacid. A concentration of 0.5 mg allopurinol per ml solutionwas obtained by the addition of distilled water. Cycbophosphamide (Mead Johnson and Company, Evansville, Ind.) inpowder form was dissolved in sterile water to the desiredconcentration.

CANCERRESEARCHVOL. 362790

The Effect of Auopurinol on Cyclophosphamide AntitumorActivity1

David S. Alberts2 and Theodoor van Daalen Wetters3

Section of Hematology-Oncology, Department of Internal Medicine, College of Medicine, University of Arizona, Tucson, Arizona 85724 fD. S. A.), and TheCancer Research Institute, University of California School of Medicine, San Francisco, California 94143 fT. v. 0. W.)

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Allopurinol-Cyclophosphamide Interaction

The Administration Schedule of ChemotherapeuticAgents. The mice were divided into 4 groups of 5 animals.On Day 0, Groups 2 and 4 were started on albopurinoldrinking water; Groups 1 and 3 stayed on a regular acidwater diet (21). On Day 7, Groups 3 and 4 received cycbophosphamide i.p. (10 to 200 mg/kg) in a constant volume of0.01 mg/g body weight. All groups were sacrificed on Day10, and their femurs were assayed for leukemic or normalbone marrow spleen colony-forming units. Mice averaged2.5 mI/day of either acid or albopurinol drinking water;therefore, daily total albopurinol dosage averaged 1.25 mgImouse or approximately 50 mg/kg.

LCFU4Assay. Our assay for LCFU was adapted from thatof Bruce and van der Gaag (4) and was carried out in thefollowing way: On Day 5, 106 P388 cells were injected intothe tail veins of each of the control and abbopurinol-pretreated mice. In the appropriate groups, cycbophosphamidewas injected i.p 2 days (Day 7) after tumor cell injection(when p. P388 tumor had a 93+% growth fraction) (14). OnDay 10, the mice were sacrificed, femurs were isolated, andbone marrow cells were washed out with Medium 199.Appropriate dilutions of the femoral bone marrow cells wereinjected in 0.2-mI volumes into the tail veins of groups of 15to 20 recipient mice. Nine days later these mice were sacrificed, spleens were removed and fixed in Bouin's solution,the macroscopic LCFU were counted using a dissectingmicroscope, and the fractions of surviving LCFU per femur(compared to the controls) were determined.

NCFU Assay. NCFU were assayed in a way similar to thatfor LCFU except that 3 days after therapy with i.p. cycbophosphamide the mice were sacrificed and appropriate dilutions of normal femoral bone marrow cells were injectedi.v. into groups of 15 to 20 whole-body-irradiated (750 radsfrom a 250-ky source)mice.

Mean Survival Time Studies. Groups of 7 DBA/2 micewere given i06 P388 ascitescellsiv.ina volume of0.2 ml,and the mean survival times of each of the control andtreatment groups were calculated by observing for deaths at12-hr intervals. The administration schedule of chemotherapeutic agents was similar to that for the LCFU and NCFUassays.

Statistical Analysis. A simple t test was usedto determinestatistical sign ificance between d ifferent experimentalgroups (8). Dose-response curves were constructed on thebasis of linear regression analysis using the Wang 2200computer.

Determination of Cyclophosphamide Alkylating Metabolites. Allopurinol-pretreated and untreated mice were givencyclophosphamide i.p. in a dosage of 200 mg/kg. At eachtime point after cyclophosphamide administration, groupsof mice were sacrificed by decapitation and their blood wascollected in iced, heparinized centrifuge tubes. The bloodsamples were centrifuged at 2000 rpm for 10 mm at 0°,andthe resulting 2.5 to 3.5 ml plasma (for each group of 7 mice)were immediately separated and frozen at —20°.

Cyclophosphamide metabolites in the mouse plasmasamples were assayed using a modification of the methodof Friedman and Boger (11). Two ml of each plasma sample

4The abbreviations used are: LCFU, leukemic bone marrow colony-forming units; NCFU, normal bone marrow spleen colony-forming units; FMF,flow microfluorometer.

were placed into a centrifuge tube along with 2 ml of 4%perchloric acid. The samples were centrifuged at 200 rpmfor 10 mm at 0°.One-mI aliquots of the resulting supernatants were transferred to clean test tubes to which wereadded, in succession, 1 ml of 2 M sodium acetate, pH 5.5,and 0.5 ml of 5% 4-(p-nitrobenzyl)pyridine (Aldrich Chemical Company, Milwaukee, Wis.) in acetone. These mixtureswere heated at 100°for 25 mm and then cooled in ice for 20mm. To release the colored chromophore for absorbancedeterminations, each sample was treated as foIIow@:(a) 2 mlof acetone, 5 ml of ethyl acetate, and 2 ml of 1.0 N sodiumhydroxide were added; (b) samples were immediately mixedfor 20 sec in a Vortex mixer; (C) samples were spun in atabletop centrifuge for 20 sec; and (d) an aliquot of theupper phase of each sample was placed in a 1-cm quartzcell, and absorbance was read on a Beckman Kintrac spectrophotometer at 540 nm 80 sec after addition of the NaOH.

Because cycbophosphamide is inactive in vitro, it wasnecessary to construct a standard curve using nitrogenmustard in human plasma. Standard curves were determined in quadruplicate. The absorbances obtained from themouse plasma samples were compared to the absorbancesof the standards, and the @tg/mlplasma of nitrogen mustardequivalents were determined.

Tumor Cell Cycle Parameter Analysis. Cell cycle parameters for the P388 leukemia cells in ascites form were determined using the FMF at the Biomedical Division of theLawrence Livermore Laboratories, Livermore, Calif. (underthe direction of Dr. J. W. Gray) (13, 26).

The flow facilities used in this study have been describedin detail by Van Dilla et al. (26). The monodisperse cellsuspension stained with acriflavin, a fluorescent DNA dye,is hydrodynamically focused so that the cells pass in singlefile through the exciting light beam from an argon ion laser.Each cell upon excitation by the laser beam yields a shortfluorescent light pulse the amplitude of which is a measureof the stain content (and therefore DNA content) of the cell.The light pulses are collected by an optical system, converted into electrical pulses, and analyzed by a multichannel pulse-height analyzer. The histogram resulting from theanalysis of a large number (10@to 10@)of cells gives thedistribution of cellular fluorescent stain content over thecell population. Using this technique it is possible rapidly todetermine DNA histograms of tumor cell populations following therapy in vivo with anticancer drugs. These DNAhistograms were analyzed using computer systems to determine cell cycle parameters for the tumor cells and the exactpoints of blockade by drugs (i.e, noncancer and anticancer)(13).

The FMF studies for in vivo effects of drugs on cell cycleparameters were performed in the following way: (a) i0@P388 ascites cells were injected i.p. into groups of DBA/2mice, some of which had been treated with albopurinol intheir drinking water at a concentration of one-half the totaldaily dose per ml (the average DBA/2 mouse drinks 2.5 mlwater/day); (b) the anticancer drug was injected i.p. atvarying dosages (drug dose required to kill iO to 50% of atumor) 2 days after cell administration; (c) sequential cellsamples were taken by sacrificing the tumor-bearing miceat frequent time intervals up to 24 hr following drug administration; (d) the ascites cells were harvested and washed

AUGUST 1976 2791

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Survival time (days)at cycbophosphamidedoseof0

mg/kg20 mg/kg40 mg/kg50 mg/kg70mg/kgCyclophospha

5.5 ±0.00―8.5 ±0.221 1.6 ±1.714.1 ±0.5514.6 ±0.25mideonlyAlbopurinol―

plus5.5 ±0.008.1 ±0.1911.8 ±0.2814.1 ±0.3314.5 ±0.21cyclophospha

mide

D. S. Alberts and T. van Daalen Wetters

with media, the RBC were lysed with ammonium chloridebuffer, and the counted cells were placed in 10% formalin ata concentration of 5 x 106 cells/mI and (e) the cells werestained with acriflavin and FMF analysis was performed.

RESULTS

Allopurinol drinking water pretreatment changed neitherthe total LCFU or NCFU per femur from those of the untreated control mice; however, albopurinol prior to cyclophosphamide (10 to 27.5 mg/kg) caused a constant increase (approximately 0.7 to 0.9 log) in cyclophosphamideantileukemic stem cell effect. Chart 1 shows the dose-response curve with respect to the fraction of surviving LCFUper mouse femur for cycbophosphamide alone and allopurinol plus cyclophosphamide at dosages of 10 to 27.5 mg/kg.

At cyclophosphamide dosages of 10 to 200 mg/kg, allo

purinol pretreatment did not significantly change the doseresponse curve of cyclophosphamide for NCFU (Chart 2).The dose-response curves in each case are relatively flatwith only little more than a i-log decrease in NCFU over adosage range of 10 to 200 mg/kg.

Table 1 shows the mean survival time study results fortumor-bearing mice for albopurinol alone, cycbophosphamide alone, and the combination of these drugs. Thosegroups of animals treated with allopurinol or acid water andnot cyclophosphamide had mean survival times of 5.5 days.At cyclophosphamide dosages of 20, 40, 50, and 70 mg/kg,allopurinol pretreatment had no effect on a change in survival duration over that achieved with cycbophosphamidealone.

As shown in Chart 3, albopurinol prior to cycbophosphamide injection caused no change in the plasma alkylatingmetabolite curve as compared to that for cyclophosphamide alone. Plasma half-lives for cycbophosphamide aloneand allopurinol prior to cycbophosphamide as well as theareas under their respective plasma alkylating metabolitecurves were similar (59 @tg-hr/mlfor cyclophosphamide and53 @g-hr/mlfor the combination).

FMF analysis of P388 leukemia cells following in vivo

100 .@

102 I 10 100

DOSE CYCLOPHOSPHAMIDE (mg/Kg)

Chart 2. Surviving fraction of NCFU per femur as a function of the cyclophosphamide dosage with or without prior allopurinol drinking water treatment. Bars, SE. Dose-response curves were constructed on the basis oflinear regression analysis using the Wang 2200 computer. CFU, colonyformingunits.

Table 1

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Chart 1. Surviving fraction of LCFU per femur as a function of the cyclophosphamide dosage.with or without prior allopurinol drinking water treatment. Bars, SE. Dose-response curves were constructed on the basis oflinear regression analysis using the Wang 2200 computer. CFU, colonyforming units.

Survival data for Ieukemic mice after drug therapy

a Mean ± SE.

B Allopurinol drinking water (0.5 mg/mI) for 7 days prior to cyclophosphamide.

CANCER RESEARCH VOL. 362792

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Allopurinol-Cyclophosphamide Interaction

therapy with allopurinol or allopurinol plus cyclophosphamide revealed no measurable cell cycle blockade by thenoncancer drug (Chart 4). There was no evidence of potentiation of the blockading effect of cycbophosphamide. Cyclophosphamide alone caused a late G2 blockade and anearly, partial S-phase block, the peak effect occurring at 16hr after anticancer drug injection (Chart 4).

DISCUSSION

Allopurinol, a xanthine oxidase inhibitor, is the standarddrug used for the prophylaxis of hyperuricemia associatedwith rapid tumor cell lysis secondary to anticancer drugtherapy (17, 20). Because 6-mercaptopurine is metabolizedto its inactive form (thiouric acid) by xanthine oxidase, it is

60 -

50 -

40

accepted clinical practice to decrease the dose of 6-mercaptopurine in the presence of albopurinol usage (5, 18).Ragab et a!. (22) have used an in vivo-in vitro mouse bonemarrow stem cell assay to demonstrate that allopurinolpotentiates the suppression of de novo granulopoiesiscaused by 6-mercaptopurine. In addition, they showed thatallopurinol alone in considerably higher dosage (100 to 400mg/kg) than would be used in clinical medicine causedsevere myelosuppression and inhibition of bone marrowcolony formation (22). We have used the in vivo mousespleen colony assay system to measure the changes in thecytotoxic effects of cyclophosphamide on leukemic andnormal bone marrow colony formation as a result of priorexposure of the mice to allopurinol. In this setting albopurinol pretreatment caused an almost 1-log increase in theinhibition of cyclophosphamide of leukemic spleen colonyformation at a range of 10- to 27.5-mg/kg dosages. In contrast, allopurinol at dosages equivalent to those commonlyused in man for the prophylaxis of hyperuricemia caused noincreased toxicity to normal bone marrow stem cell production at a wide range of cyclophosphamide dosage. Weconclude that this commonly used mouse model (P388 leukemia in DBA/2 mice) for the screening of potential anticancer activity against human neoplasms (27) does not reflect adecrease in the therapeutic ratio of cyclophosphamide as aresult of allopurinol pretreatment.

The mechanisms for the potentiation of the antileukemiceffect of cyclophosphamide by allopurinol are at this timestill unexplained. Using the standard alkylating agent assaytechniques, we were unable to demonstrate a change in thepharmacokinetics of cyclophosphamide in mice as a resultof prior allopurinol exposure. Coffey et al. (5), studying the

4,0

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DNA Conten? DNA Content

AUGUST1976 2793

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Chart 3. Metabolism of cyclophosphamide in vivo. Points, alkylating activity per ml of plasma in terms of HN2 equivalents and the mean of at least 7male DBA/2 mice. Cyclophosphamide dosage was 200 mg/kg.

Chart 4. DNA histograms of P388 leukemia cells exposed in vivo to water, allopurinol, cyclophosphamide,or allopurinol plus cyclophosphamide. The 16-hr timepoint after cyclophosphamide administration was included because it represented the period of the peak“blockadingeffect―of the anticancer drug on the tumor cell cycle. d, day.

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D. S. Alberts and T. van Daalen Wetters

effect of allopurinol on the pharmacokinetics of 6-mercaptopurine in cancer patients, were also unable to show achange in the anticancer drug half-life or area under theplasma concentration-time curve as a result of the druginteraction effect. Our FMF analysis of the in vivo effects ofallopurinol and allopurinol plus cyclophopshamide on P388leukemia tumor cell cycle parameters demonstrated no allopurinol-associated changes (Chart 4); however, our analytical techniques may not be sensitive enough to detect smallchanges in DNA repair synthesis caused by the drug interaction. Although allopurinol appears to inhibit purine synthesis (23) and may be converted to an allopurinol ribonucleotide that can inhibit enzymes normally regulated bypurine or pyrimidine ribonucleotides (10, 16), there is noevidence that these metabolic effects are related to its apparent interactions with cyclophosphamide. In addition,whereas allopurinol inhibits de novo purine synthesis, italso increases utilization of preformed purines (e.g. , hypothanine and xanthine) and does not affect purine or pyrimidine nucleotide pool sizes (9).

One possible explanation of the allopurinol-cyclophosphamide interaction effect is that the former drug interfereswith the ability of the P388 leukemic cells to repair thesublethal damage caused by cyclophosphamide. The parallel nature of the the cyclophosphamide and allopurinolcyclophosphamide dose-response curves is a phenomenonoften seen in radiobiology as a result of the prevention ofDNA repair synthesis. The slopes of such curves reflect thesensitivity of the cells to the drug, and the extrapolatedintercept on the abscissa represents the ability with which acell repairs sublethal damage (12).

lt is also possible that the plasma levels of an alkylatingagent may not correlate with the antitumor activity of thesedrugs. It is conceivable that allopurinol alters the fraction ofbound RNA, DNA, and protein receptors, thereby potentiating the antitumor effects of cyclophosphamide.

ACKNOWLEDGMENTS

We wish to thank Le Roy Kondo for his technical assistance, Dr. MalcolmRowland for his advice concerning experimental design and pharmacokinetic analysis, and Margo Walter for editing and typing.

REFERENCES

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2. Boston Collaborative Drug Surveillance Program. Allopurinol and Cytotoxic Drugs. Interaction and Relation to Bone Marrow Suppression. J.Am. Med. Assoc., 227: 1036-1040, 1974.

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7. Dixon, R. L. Effect of Chloramphenicol on the Metabolism and Lethalityof Cyclophosphamide in Rats. Proc. Soc. Exptl. Biol. Med.. 127: 1151-1155, 1968.

8. Dixon, W. J. BMD Biomedical Computer Programs. In: W. J. Dixon (ed),BMD Biomedical Computer Programs. pp. 67-181 . Berkeley: Universityof California Press, 1973.

9. Elion, G. B., and Nelson, D. J. In: 0. Sperling, A. DeVries, and J. B.Wyngaaden (eds.), Purine Metabolism in Man, Vol. 41B, p. 639. NewYork: Plenum Publishing Comp. , 1974.

10. Fox, R. M., Royse-Smith, 0. , and O'Sullivan, W. J. Orotidinuria Inducedby Allopurinol. Science, 168: 861-862, 1970.

11. Friedman, 0., and Boger, E. Colorimetric Estimation of Nitrogen Mustards in Aqueous Media. Anal. Chem., 33: 906-910, 1961.

12. Frindel, E., and Tubiana, M. Radiobiology and the Cell Cycle. In: R.Baserga (ed), The Cell Cycle and Cancer, pp. 389-436. New York:Marcel Dekker, 1971.

13. Gray, J. Cell Cycle Analysis from Computer Synthesis of Deoxyribonucleic Acid Histograms. J. Histochem. Cytochem.. 22: 642-650, 1974.

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16. KeIley, W. N., and Beardmore, T. D. Allopurinol: Alteration in PyrmidineMetabolism in Man. Science, 169: 388-390, 1970.

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18. Levine, A. S., Sharp, H. L., Mitchell, J., Krivit, W., and Nesbit, M. E.Combination Therapy with 6-Mercaptopurine (NSC-755) and Allopurinol(NSC-139O) during induction and Maintenance of Remission of AcuteLeukemia in Children. Cancer Chemotherapy Rept. , 53: 53-57, 1969.

19. Livingston, R. B. , and Carter, S. K. Single Agents in Cancer Chemotherapy, pp. 25-81. New York: Plenum Publishing Corp., 1970.

20. Lowrey, M. E. Metabolic and Therapeutic Effects of Allopurinol in Patients with Leukemia and Gout. Am. J. Med., 40: 548—559,1966.

21. McPherson, C. W. Reduction of Pseudomonas Aeruginosa and ColiformBacteria in Mouse Drinking Water following treatment with HydrochloricAcid or Chlorine. Lab. Animal Care, 13: 737-744, 1963.

22. Ragab, A. H. , Gilkerson, E.. and Myers, M. The Effects of 6-Mercaptopurine and Allopurinol on Granulopoiesis. Cancer Res. , 34: 2246-2249.1974.

23. Rundles, R. W., Wyngaarden. J. B., Hitchings, G. H., Elion, G. B., andSilberman, H. R. Effects of a Xanthine Oxidase Inhibitor on ThiopurineMetabolism, Hyperuricemia. and Gout. Trans. Assoc. Am. Physicians.76: 126-140, 1963.

24. Shaprio, S. Allopurinol and Bone Marrow Suppression. Lancet, 2: 412,1974.

25. Sladek, N. E. Therapeutic Efficacy of Cyclophosphamide as a Functionof Its Metabolism. Cancer Res.. 32: 535-542, 1972.

26. Van Dilla, M. A., Steinmetz, L. L. , Davis, D. T., Calvert, R. N., and Gray, J.w. HighSpeedCellAnalysisandSortingwithFlowSystems:BiologicalApplication and New Approaches. IEEE Trans. NucI. Sci. NS, 21: 714-728,1974.

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2794 CANCER RESEARCH VOL. 36

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1976;36:2790-2794. Cancer Res   David S. Alberts and Theodoor van Daalen Wetters  ActivityThe Effect of Allopurinol on Cyclophosphamide Antitumor

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