863

PHARMACOLOGY OF ALLOPURINOL GEORGE H. HITCHINGS I t is now 8 years since allopurinol was first introduced for commercial distribution (in England) and about 12 years since the first Phase I human studies were carried out following several years of laboratory studies. I n the intervening years information about its origins, mode of action, general pharmacologic properties, and therapeutic applications has become rather widely known (1-3). A brief review of these topics is in order to provide a basis for more recent developments, and possibly also to give a somewhat more accurate perspective on some aspects of the drug’s development and properties. Allopurinol was chosen for trial as an inhibitor of xanthine oxidase in vivo for several reasons: a) like other inhibitors it was both an inhibitor and a substrate for the enzyme; but b) unlike other inhibitors the product also was a strong inhibitor; moreover c) as evaluated by the means then available it appeared not to become involved in purine anabolic reactions (4). At the time its laboratory development was begun, there were two possible explanations for the failure of previous attempts usefully to inhibit the xanthine oxidase in vivo. Westerfeld (5) had achieved only 50% inhibition of the enzyme with toxic levels of carbonyl reagents, and Byers’ strongest in vitro inGeorge H. Hitchings, Ph.D.: Vice-president, Wellcome Research Laboratories, Research Triangle Park, North Carolina 27709. Address reprint requests to Dr. Hitchings.

hibitor was inactive in vivo (6). It was possible that unsuitable agents had been chosen, but it was also possible that the prevailing conception of purine catabolism was nai’ve. This research was conducted shortly after the discovery that purine ring oxidation could take place at the ribonucleotide level (eg inosinate’ xanthylate, catalyzed by inosinate dehydrogenase), and one could not be certain of a predominate role for xanthine oxidase in the catabolism of the purine ring. T h e availability of mercaptopurine provided a simple model system that greatly simplified the detection of inhibition of xanthine oxidase in vivo, and the explanation of the effects of such an inhibition. Mercaptopurine has binding constants close to those of hypoxanthine for both xanthine oxidase and hypoxanthine-guanine phosphoribosyltransferase (HGPRT); it and its products give spectra well separated from those of interfering natural metabolites, eg the end product of the action of xanthine oxidase on the substrate was thiouric acid with strong absorption at 345 mp rather than allantoin, which is difficult to determine. Mercaptopurine has an effect on Adenocarcinoma 755 that is easily titrated, and it was felt that the inhibition of its degradation might be directly useful in connection with its antitumor effects. Studies in mice showed that the oxidation of mercaptopurine to thiouric acid could be inhibited by moderate doses of allopurinol and that this effect resulted in the potentiation of the antitumor effects of

Arthritis and Rheumatism, Vol. 18, No. 6 (November-December 1975), Supplement

HITCHINGS

864

several 6-substituted purines (7). T h e first experiment in man (8), in a patient with clironic granulocytic leukemia, confirmed the effect of allopurinol administration on the sparing of mercaptopurine. I t also showed a probably significant effect on serum and urinary urate that was substantiated b y tlie appearance of detectable levels of urinary Iiypoxanthine and xanthine. T h e observations were extended in logical sequence to patients with hyperuricemia, gout, tophaceous gout, and renal urate stones (1,4). Biologic half-life determinations using 14C-allopurinol in man and animals indicated that allopurinol itself was rapidly cleared but that radioactivity persisted in plasma for a prolonged period (9). T h e persistent material was primarily oxipurinol and it had a biologic half-life of anywhere from 17 to 44 hours in different patients. A later study confirmed the interpretation (10) that oxipurinol is subject to tubular reabsorption, as is uric acid, and that in a given individual there is a positive correlation between clearance of oxipurinol and of urate. T h e rapid clearance of allopurinol was misread by some as rapid clearance of xanthine oxidase-inhibitory activity, and multiple daily dosing was recommended. I n fact dosing with allopurinol on any schedule reaches the same equilibrium effect within a few days, ancl giving a single 300-mg tablet daily gives results identical in all parameters to 100 mg tid (11,lZ). T h e principal metabolite of allopurinol is oxipurinol, and it must be assumed that a considerable part of tlie effectiveness of allopurinol is attributable to the activity and pharmacokinetic properties of this metabolite. Nevertheless more consistent effects and lower closes are required with allopurinol than with oxipurinol (8,13,14), probably as the result of less complete and more erratic absorption of oxipurinol. T h e first question to be asked when an inhibitor is applied to one reaction in a metabolic sequence concerns the properties of the precursors of the inhibited reaction that might be expected to accumulate. Some bits of prior information were available. A few but by no means all xantliinuric patients formed xanthine urinary calculi (3), and the virtual absence of xanthine oxidase in such patients was soon documented (15). T h e renal clearances that had been reported for xanthine and hypoxanthine were not very high but were suspect for technical reasons’ ancl were soon shown, using allopurinol as a tool to over*Sce Hitchings (4), footnote 4.

Table 1. Serum and Urinnry Purines’ Eflecls of .4lloptirinol 1. 2. 3. 4. 5.

Decrease i n serum urate (dose dependent) Serum oxypurine remains below 10% of total Decrease in urinary uratc (dose dependent) Increased renal clearance (as percent oxypurine increases) Decreased total urinary purine (cnzymc depcndent)

come the difficulties, to be in the range of glomerular filtration rates or even higher (16). For this reason plasma oxypurines rise only minimally when allopurinol is given, and the net effect is a decrease in total serum purine. For the same reason tlie renal clearance of total purine end product increases as the percentage of oxypurines increases (Table 1). T h e solubility of hypoxanthine in both serum ancl urine is so much greater than that of urate that solid deposits of this precursor were not to be expected. T h e possibility of solid deposits of xanthine in tissues can be disposed of without very much comment. Xanthine is more soluble in serum than is urate; moreover the highest serum concentrations observed are only small fractions of the saturation level. T h e crystals reported in tissues are almost certainly artifacts of the metliod of preparation (17). T h e possibility of xantliine crystalluria was not so easily dismissed. Both Hitchings and Levin (4,18) felt that the solubility of xanthine in urine left only a moderate margin of safety, which might be exceeded under exceptional circumstances. I n fact the margin of safety seems to have been adequate. T h e 3 cases in which xantliine crystalluria have been reported are clearly exceptional-one Lesch-Nyhan syndrome (3) and 2 patients with lymphosarcoma, treated vigorously (19,20)-and the patients probably were subjected to less hazard with allopurinol than they would have been without it. A major factor limiting the output of xanthine -the decrease of total purine turnover that follows allopurinol treatment of most urate overproducerswas not fully anticipated. I n a general way the extent of the turndown bears a direct relationship to the degree of overproduction before treatment (4). T h e basis for what is believed to be the correct interpretation of this effect had been laid earlier; it rests on the demonstration that both hypoxanthine and xanthine are extensively reutilized only when they are protected by allopurinol from catabolic destruction (21-23). I n this view the reutilization of these two metabolites would enhance the normal feedback control of purine biosynthesis (Figure I), and any factor

PHARMACOLOGY OF ALLOPURINOL

565

-IMP

AR

XMP

JT H - X + It

1

HR 4

UA

Fig 1. Diagrammatic representation of control of purine biosynthesis. T h e synthesis of purines d e novo is controlled by adenylic acid (AMP) and guanylic acid (GMP) derivatives acting in concert. Reutilization of hypoxanthine (H) and xanthine (X) tends t o reduce synthesis d e novo because their nucleotides, inasinic acid (IMP) and xanthylic acid (XMP), are converted to A M P and G M P respectively.

that tended to increase the nucleotide pool levels would result in a compensatory decrease in synthesis de novo. I t is now clear that there is normally a rapid turnover of hypoxanthine, probably by way of the loop via the ribonucleotides (24). T h e normal turnover of xanthine is much lower (24). This lower turnover probably reflects mainly the fact that hypoxanthine is a much better substrate for H G P R T than is xanthine (25), whereas the two are comparable in substrate activity for xanthine oxidase (26). With hypoxanthine, conversion to the nucleotide would be somewhat preferred to oxidation, whereas with xanthine the main route would be oxidation. Most xanthinurics excrete mainly xanthine. T h e chief metabolite of allopurinol is oxipurinol. Because this is the product formed in vitro by xanthine oxidase, its formation in vivo was to be expected. T h e two inhibitors differ in the kinetics of their inhibitors of the enzyme. Allopurinol is competi-

OH

tive with the substrates. Oxipurinol in the presence of substrate brings about a noncompetitive inhibition and a reversible inactivation in vitro (27). This inactivation may or, more likely, may not have any bearing on its effectiveness in vivo. Among the minor metabolites of allopurinol the first to be discovered was 1-ribosylallopurinol (28) (Figure 2). Somewhat later an additional ribonucleoside, 7-ribosyloxipurinol, was detected in the urine of patients. But the third, 1-ribosyloxipurinol, has not been detected. All three ribonucleosides were synthesized enzymatically and allopurinol-1-ribonucleoside was synthesized chemically as well (29). Ribonucleosides can be formed by two routes, by the action of phosphatases on ribonucleotides or by the action of nucleoside phosphorylases in which the free base is condensed with ribose-I-phosphate (Figure 3). Until 1970 no nucleotides of allopurinol or oxipurinol had been found (28,30). Since then three ribonucleotides have been detected. Kinetic and

OH

RP ALLOPURINOL- I - RP

OH

RP OXIPURINOL-I-RP

I

RP 0x1PURl NOL-7-RP

Fig 2. Formulas for nucleotides derived from allopurinol and oxipurinol. R P = ribose-5'phosphate.

HITCHIN GS

866

Equation 1

H t Pyrophosphorylribose - 5-PO3H2

I R ibose

Equation 2

I Ribose - 5 - PO3 H2

I Ribose

Fig 5. Eqiiatioiis for biosynrhesis of O t l ~ l O gnticleosidrs. 111 Equation 1 the iiticleosirle is synthesized directly by a phosphorolysis. In Equation 2 i t is derived by the action of a phosphotase on the corresponding nucleotide.

other considerations make i t probable that the bulk of the ribonucleosides arise through the action of purine and pyrimidine nucleoside phosphorylases. T h e nucleotides were first detected through the coincidental observation that patients taking allopurinol had elevated excretions of orotic acid and orotidine (31,32). Both Fox and Beardmore (33,34) then showed that inhibitors of orotidylate decarboxylase were formed by incubation of allopurinol and oxipurinol with red cells. With the aid of high doses of highly active "C;-allopurinol and high-pressure liquid chromatography, three nucleotides (corresponding to the nucleosides of Figure 2) were isolated from rat tissue extracts (35). T h e availability of enzymatically synthesized reference nucleotides (36) facilitated the identifications. They were indeed inhibitors of orotidyla te decarboxylase, with oxipurinol-l-ribosepliospliate the niost potent and allopurinol-l-ribosephosphate the least potent (34). T h e concentrations reached appear to correlate closely with the levels of unsubstituted analogs in the plasma (37). I n acute experiments the inhibitory effects of these analogs apparently diminished the rate of de-

carboxylation of orotidylate, as measured by the liberation of ' C O , from 14C-carboxyl-orotate (38). T h e incorporation of infused 6-l4C-orotate into rat liver uridine phosphate pools showed a similar effect (35). Nevertheless, despite the rapid turnover of uricline pools, at the most critical time ( 1 hour after injection of 20 to 25 mg/kg of labeled allopurinol) decreases in UMP, UDP, and UDP-glucose pools barely attained significance ( P < 0.05); the effect had disappeared or rebound was present by 3 hours. Furthermore no effect on uridine phosphate levels was found in chronically treated animals (39). T h e chain of events finally unraveled is as follows: T h e initial inhibition of orotidylate clecarboxylase causes a rapid accumulation of the precursors of the inhibited reaction. I n rat liver for example, during the first hour after iv administration of 'C-allopurinol, the orotate concentration increases 19-fold and the orotidylate concentration increases 73-fold (40). In tracer experiments the dilution of the labeled substance must be taken into account, or it is implicitly assumed that the dilution is the same in treated and control subjects. I n this instance the rapid buildup of

PHARMACOLOG'I OF ALLOPURINOL

0 0.05 0.10

867

0.25

Allopurinol in Diet Per Cent Fig 4. The eflect of alloptirinol i n the diet on t;L< excrelio7z of orotate and orotidine.

precursor pools and dilution of the tracer account for much of the apparent inhibition even in the acute experiments. I n chronically treated animals pyrimicline turnover is normal as evidenced not only by the levels of uritline phosphate pools, but also in recent experiments in rate of synthesis of uridine jhospliates from bicarbonate (41). T h u s the rate of biosynthesis of uridylate derivates from intravenously administered NaH"C0, was calculated to be 4.87 ,Lmoles/g liver/ day in untreated animals, antl 5.33 in animals fed allopurinol in the diet for 4 weeks at a level of 0.2570 A dose-dependent increase in excretion of orotate orotidine reflects the expansion in the pools of these metabolites (Figure 4). T h u s the effects of allopurinol on pyrimidine metabolism are significant but seem primarily to be of biochemical interest. T h e possible effects of allopurinol on purine metabolism have been the subject of considerable speculation (42). Initial readings on a possible participation of allopurinol in anabolic reactions were persuasively negative (4). Both allopurinol antl oxipurinol are poor competitors of tlie natural substrates of H G P R T (25), but the formation in vivo of purinetype ribonucleotides from both has now been demon-

+

strated. I t seems unlikely that either nucleotide has measurable effects on purine or nucleic acid metabolism. T h e limits of detection have now been pushed to extremely lo\v levels. An analysis of many of the pertinent findings has been published by Elion and Nelson (37). Some transient changes in purine nucleotide pools were observed 1 hour 'tfter iv administration ot allopurinol at 20 mg/kg to rats, but these were in opposite directions in liver and kidney and absent in chronic experiments. T h e immediate fluctuations probably represent the events leading to the establishment of a new equilibrium in purine turnover that reflects the conservation of oxypurines. I n a chronic experiment allantoin excretion dropped 13.3 pmoles, whereas oxypurines replaced only 1.6 pmoles (per milligram of creatinine) of tl:is drop. T h e possibility ot direct feedback control of purine turnover by allopurinol ribonucleotide has been raised (43), but the levels of this nucleoticle by direct measurement fell short by 2 to 3 orders of magnitude of those necessary for inhibition of the amidotrmsferase that is the first conti olling step in purine biosynthesis. Kelley has pointed out that inhibition of purine synthesis de novo can occur in cultured cells by mechanisms not clepentlent on either xanthine oxidase or H G P R T (44). However a functional H G P R T seems to be a prerequisite for a reduction in purine biosynthesis in patients (3) although mechanisms other than H G P R T defects may also prevent the decrease (45). I n sum the primary controlling medianism is probably reutilization of oxypurines, but some nuances in tlie effects of the inhibitors on purine metabolism remain for further investigation. T h e availability of ribonucleotides of allopurino1 and oxipurinol has made possible the investigation of their participation in other purine anabolic reactions (Figure 5). T h e current status of unpublished work in this author's laboratories (by Elion, Krenitsky, Miller, Nelson, Spector, and others) is as follows: Allopurinol ribonucleotide binds to inosinate clehydrogenase (from Sarcoma 180) only 1/3,00Oth as well as inosinate. No product could be detected when twelve times as much allopurinol nucleotide as inosinate was present by a method that would have permitted the detection of 1/5,000th as much product. Neither analog nucleotide forms di- or triphosphates, i n consonance with the absence of inosinate and xanthylate kinases i n mammalian tissues. T h u s the possibility of incorporation of either analog per se into

HITCHINGS

868

/

AMP -ADP

/s-AMp -IMP+DP

\!

d-

-ATP

IADP

Reaction

\.

d-GDP

XMP

- -1GMP

GDP

Table 2. Competition of Pyrazolopyrimidines w i t h Purines

GTP

Fig 5. Diagrammatic representation of routes from inosinale (IMP) ultimately to deoxyadenosine diphosphale (d-ADP) and deoxyguanosine diphosphate (d-GMP). T h e double bar o n the arrows to diphosphates (DP) indicates that kinases that would convert to oxipurine nucleotides t o diphosphates have not been found.

nucleic acids is essentially nil. Oxipurinol ribonucleotide is converted to the guanine-analog nucleotide by xanthylate aminases from Escheiichia coli and Ehrlich ascites cells, but this reaction requires unrealistic concentrations of analog to produce any product; that is to say, the reaction runs about 1/50,00Oth as well with the analog as with xanthylate. T h e product has not been found in tissue extracts under conditions in which a concentration of 10-lo M would have been detected. T h e concentration of the adenine analog nucleotides also is below the limits of detection, ie < 10-lo M . Furthermore the guanine analog nucleotide seems not to be a substrate for human erythrocyte GMP kinase, the comparative activity with guanylate being less than 1OW5. T h e comparative activities of analog derivatives as substrates for purine anabolic reactions are summarized in Table 2. T h e direct search for incorporation of any allopurinol derivative into nucleic acids has been carried to progressively lower limits. T h e latest most sensitive test using 6-14C-allopurinol of very high specific activity (thirty times that originally used) gave purified RNA with a small but significant radioactivity (513 dpm/mg), although the DNA had less than 10 dpm/mg above background. T h e RNA was hydrolyzed and the radioactive components were characterized. Adenylic and guanylic acids accounted for all the radioactivity. T h e implications of this are that allopurinol ribonucleotide, like inosinate, is subject to opening of the pyrimidine ring and excision of formate, which found its way into purine biosynthesized de novo. This observation is consistent with the earlier finding of 'CO, in the expired air of mice given radioactive allopurinol. T h e analogs of aminoimidazole carboxamide and formylaminoimidazole

=+IMP X+XMP IMP-+XMP XMP+GMP G MP+G DP

M/allo* 1.6

x 103

>lo3 >lo5 5 x 104

>lo5

Source of Enzyme Human rbc Human rbc S180

Ehrlich ascites Human rbc

*M/allo: thc relative reaction rates of metabolites and pyrazolopyrimidine analogs as calculated from velocity (Vn) and dissociation (K,,)data.

carboxamide and/or their ribose derivatives may eventually be identified as minor (and inconsequential) metabolites of allopurinol. I n fact tlie unknown peak (37) in the chromatogram of rat liver extracts after giving IGallopurinol is probably one of these. T h e implications of these studies for clinical use of allopurinol are minimal, but tlie analogs have providecl the stimulus and tlie tools for the ,investigation of many details of purine and pyrimidine biochemistry. For clinical use the principal hazards remain those foreseen at the onset. I n more than a decade of use no new findings of clinical significance have appeared.

REFERENCES 1. Rundles R W , Elion GB, Hitchings G H : Allopurinol in the treatment of gout a n d secondary hyperuricemia. Bull Rheum Dis 16:400-403, 1966 2. Woodbury DM: Analgesic-antipyretics, anti-inflammatory agents, and inhibitors of uric acid synthesis, T h e Pharmacological Basis of Therapeutics. Fourth edition. Edited by LS Goodman, A Gilman. London and Toronto, hlacmillan, 1970, p p 341-344 3. Rundles RW, Wyngaarden JB, Hitchings G H , et al: Drugs and uric acid. Ann Rev Pharmacol 9:345-362, 1969 4. Hitchings G H : Effects 01 allopurinol i n relation to purine biosynthesis. Ann Rheum Dis 25:601-607, 1966 5. Westerfeld WW, Richert DA, Bloom KJ: T h e inhibition of xanthine and succinic oxidases by carbonyl reagents. J Biol Chem 34:1889-1896, 1959 6. Byers SO: Xanthine oxidase studies. J Am Pharm ASSOC 41:611-613, 1952 7. Elion GB, Callahan S, Nathan H , et al: Potentiation by inhibition of drug degradation: 6-substituted purines and xanthine oxidase. Biochem Pharmacol 1285-93, 1963 8. Rundles R W : Metabolic effects of allopurinol and alloxanthine. Ann Rheum Dis 25:615-620, 1966 9. Elion GB: Enzymatic and metabolic studies with allopurinol. A n n Rheum Dis 25:608-614, 1966

PHARMACOLOGY OF ALLOPURINOL

10. Elion GB, Yii T-F, Gutman AB, et al: Renal clearance of oxipurinol, the chief metabolite of allopurinol. Am J Med 45:69-77, 1968 1 1 . Rodnari GP, Robin JA, Tolchin S: EAicacy of single daily dose of allopurinol in gouty hyperuricemia, Purine Metabolism in Man. Edited by 0 Sperling, A DeVries, JB Wyngaaardcn. New York, Plenum, 1974, pp 571-575 12. Elion GB: Unpublished data 13. Chalmers RA, Kriimer H, Scott JT, et al: A comparative study of the xanthine oxidase inhibitors allopurinol and oxipurinol in man. Clin Sci 35:353-362, 1968 14. Elion GB, Hitchings G H : Comparative effects of allopurinol and oxipurinol in rats and dogs. Unpublished data 15. Engelman K, Watts RWE, Klinenberg JR, et al: Clinical, physiological, and biochemical studies of a patient with xanthinuria and pheochromocytoma. Am J Med 37:839-861, 1964 16. Goldfinger S, Klinenberg JR, Seegmiller JE: Renal excretion of oxypurines. J Clin Invest 44:623-628, 1965 17. Hitchings GH: Crystals in skeletal muscle. Br Med J 4:555-556, 1971 18. Levin NW, Abrahanis OL: Allopurinol in patients with impaired renal function. Ann Rheum Dis 25:681-687, 1966 19. Greene ML, Fujimoto W Y , Seegmiller JE: Urinary xanthine stones-a rare complication of allopurinol therapy. N Engl J Med 280:426427, 1969 20. Band PR, Silverberg DS, Henderson JF: Xanthine nephropathy in a patient with lymphosarcoma treated with allopurinol. N Engl J Med 283:354-357, 1970 21. Pomales R, Bieber S, Friedman R, et al: Augmentation of the incorporation of hypoxanthine into nucleic acids by the administration of an inhibitor of xanthine oxidase. Biochim Biophys Acta 72: 119-120, 1963 22. Pomales R, Elion GB, Hitchings GH: Xanthine as a precursor of nucleic acid purines in the mouse. Biochim Biophys Acta 95-96:505-506, 1965 23. Hitchings GH: T h e biochemical background to allopurinol. Niere une Stoffwechselkrankheiten. 10. Int Symp der Deutschen Gesellschaft fiir Fortschritte auf dem Gebiet der Inneren Medizin, Freiburg, October 1971, p p 107-118 24. Balis ME: Aspects of purine metabolism. Fed Proc 27: 1067-1074, 1968 25. Krenitsky TA, Papaioannou R, Elion GB: Human hypoxanthine phosphoribosyltransferase. I. Purification, properties, and specificity. J Biol Chem 244: 1263-1270, 1969 26. Krenitsky TA, Shannon MN, Elion GB, et al: A comparison of the specificities of xanthine oxidase and

869

27.

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

39.

40.

aldehyde oxidase. Arch Biochim Biophys 150:585-599, 1972 hlassey V, Komai H, Palmer G, et al: On the mechanism of inactivation of xanthine oxidase by allopurinol and other pyrazolo[3,4-d]pyrimidines. J Biol Chem 245: 2837-2844, 1970 Elion GB, Kovensky A, Hitchings GH, et al: hletabolic studies of allopurinol, an inhibitor of xanthine oxidase. Biochem Pharmacol 15:863-880, 1966 Krenitsky T A , Elion GB, Strelitz RA, et al: Ribonucleosides of allopurinol and oxoallopurinol. J Biol Chem 242:2675-2682, 1967 Kelley WN, Wyngaarden JB: Effects of allopurinol and oxipurinol on purine synthesis in cultured human cells. J Clin Invest 49:602-609, 1970 Fox RM, Royse-Smith D, O’Sullivan WJ, et al: Orotidinuria induced by allopurinol. Science 168:861-862, 1970 Kelley WN, Beardmore T D : Allopurinol: alteration in pyrimidine metabolism in man. Science 169:388-390, 1970 Fox RM, Wood MH, O’Sullivan WJ: Studies on the coordinate activity and lability of orotidylate phosphoribosyltransferase and decarboxylase in human erythrocytes, and the effects of allopurinol administration. J Clin Invest 50:1050-1060, 1971 Beardmore T D , Kelley WN: Mechanism of allopurinolmediated inhibition of pyrimidine biosynthesis. J Lab Clin Med 78:696-704, 1971 Nelson DJ, BuggP CJL, Krasny HC, et al: Formation of nucleotides of [6-1*C]allopurinol and oxipurinol in rat tissues and effects on uridine nucleotide pools. Biochem Pharmacol 22:2003-2022, I973 Fyfe JA, Miller RL, Krenitsky TA: Kinetic properties and inhibition of orotidine 5’-phosphate decarboxylase. Effects of some allopurinol metabolites on the enzyme. J Biol Chem 248:3801-3809, 1973 Elion GB, Nelson DJ: Ribonucleotides of allopurinol and oxipurinol in rat tissues and their significance in purine metabolism, Purine Metabolism in Man. Edited by 0 Sperling, A DeVries, JB Wyngaarden. New York, Plenum, 1974, pp 639-652 Kelley WN, Beardmore TD, Fox IH, et al: Effect of allopurinol and oxipurinol on pyrimidine synthesis in cultured human fibroblasts. Biochem Pharmacol 20: 1471-1478, 1971 Fyfe JA, Nelson DJ, Hitchings GH: T h e molecular basis for the effects of allopurinol on pyrimidine metabolism, Purine Metabolism in Man. Edited by 0 Sperling, .4 DeVries, JB Wyngaarden. New York, Plenum, 1974, p p 621-628 Hitchings GH: Indications for control mechanisms in purine and pyrimidine biosynthesis as revealed by

870

studies with inhibitors. Adv Enzyme Regul (in press) 41. Nelson DJ: Unpublished data 42. Fox IH, Wyngaarden JB, Kelley WN: Depletion of erythrocyte phosphoribosylpyrophosphate in man. N Engl J Med 283:1177-1182, 1970 43. McCollester RJ, Gilbert WR, Ashton ~ h f et , Pseudofeedback inhibition of purine synthesis by 6mercaptopurine ribonucleotide and other purine analogs. J Biol Chem 239:1560-1563, 1964

HITCHINGS

44. Kelley WN, Fox IH, Beardmore TD, et al: Allopurinol arid oxipurinol: alteration of purine and pyrimidine metabolism in cell culture. Ann NY Acad Sci 179:588595, 1971 45. Kelley WN, Rosenbloom FM, Miller

J, et a]: An en-

zymatic basis for variation in response to allopurinol: hypoxanthine-guanine phosphoribosyltransferase deficiency. N Engl J Med 278:287-293, 1968