Xanthine Oxidase Inhibitors in the Management of Gout By JAMESB. WYNGAARDEN, M.D. Duke University Medical School The forms of therapy for gout available in the past decade have been very successful in about two-thirds of gouty patients. Those who have not responded satisfactorily to uricosuric therapy are by and large patients who have some degree of renal insufficiency, or who continue to use salicylates concurrently, or who are intolerant of the drugs. It has been a goal in the therapy of gout to be able to regulate the production of uric acid, as well as its excretion. Experiments directed toward this goal have been conducted with azaserine by Zuckerman, Drell and Levin,l and with diazo-oxonorleucine by Grayzel, Seegmiller and Love.2 Both agents are glutamine antagonists and block an early reaction of purine synthesis. When these agents are administered to man the rate of production of uric acid is reduced and the levels of urate in serum and urine fall. The compounds are too toxic to be therapeutically useful in gout but the experiments are of interest. The pyrazolopyrimidines resemble the purines in structure except that nitrogen 7 and carbon 8 of the purine ring are reversed (Fig. 118). There are several pyrazolopyrimidine derivatives. The adenine analogue was explored as a cancer chemotherapeutic agent some time ago, with some preliminary success. When the studies were extended to man this compound proved to be hepatotoxic and its use was abandoned. 4-Hydroxypyrazolo ( 3,4-d)pyrimidine is an analogue of hypoxanthine which appeared to be biologically ineffective in tumor trials. It was shelved until recently when interest in its use as a xanthine oxidase inhibitor developed. Many purine analogues bind at the active site of xanthine oxidase and by this competitive mechanism inhibit the enzyme. The pyrazolopyrimidines were shown to be inhibitors of xanthine oxidase by Feigelson, Davidson and Robins." This demonstration rested in the literature for some time until the group at Burroughs Wellcome Laboratories became interested in the possible advantage of inhibiting xanthine oxidase in patients who were receiving 6-mercaptopurine as cancer chemotherapy. This latter compound is normally oxidized to 6-thiouric acid by xanthine oxidase. The question raised was whether one could provide a smoother chemotherapeutic course by giving a xanthine inhibitor together with 6-mercaptopurine ( 6-MP) . Xanthine oxidase is the enzyme responsible for oxidation of hypoxanthine to xanthine, and of xanthine to uric acid (Fig. 119). These reactions are on the degradative side of purine nucleotide metabolism, so that inhibition of xanthine oxidase should not have any direct effect on nucleotide production, and nucleic acid metabolism. Figure 120 shows one of the early studies done by Dr. Wayne Rundles4 at Duke University in which he gave 6-mercaptopurine to a patient with chronic granulocytic leukemia, and determined the 883 ARTHRITISAND RHEUMATISM,VOL. 8, N O . 5-PART 1 (OCTOBER), 1965 884 GOUT AND PURINE METABOLISM Adenine 4-Aminopyrazolo(3,4-d)pyr imid ine NH2 NH2 I I Hypoxanthine OH 4-Hydroxypyrazol0(3,4 -d)pyrimidine OH Fig. 118.--Structural formulae of pyrazolo (3,4d)pyrimidines. XANTHINE OXIDASE Hypoxonthine Fig. 119.-Action Xonthine Uric Acid of xanthine oxidase on oxypurines. amount which is excreted unchanged and the amount excreted as thiouric acid. Later the experiment was repeated after 300 mg. of hydroxypyrazolopyrimidine (HPP) had been given. In the second instance a larger percentage of 6-MP was excreted unchanged and the fraction converted to 6-thiouric acid was drastically reduced. Because of this effect on xanthine oxidase the question of what HPP would do to uric acid levels was raised. Appropriate study disclosed that the serum and urinary uric acid levels both fell and that there was a concomitant increase in the excretion of xanthine and hypoxanthine. The changes in this particular subject were not great but they indicated the desirability of extending these studies to other hyperuricemic patients, particularly those with goutS5z6 Figure 121 shows one of the gouty patients who was treated with HPP shortly thereafter. In this particular patient, who was asymptomatic at the time, the serum urate level of 6 to 7 mg. per cent was reduced by HPP to about 3 to 4 mg. per cent, and the urinary uric acid which was in the range of 600 mg./24 hours fell to between 200 and 300 mg./24 hours in about 5 885 XANTHINE OXIDASE INHIBITORS e n x CHRONIC GRANULOCYTIC LEUKEMIA Hgb VPRC WBC yo x K.L.C. F-72928 gm,/ m o A URINE SERUk mercaptopurine. = A Serum CHRONIC NON-TOPHACEOUS Unrestricted Diet o URINE W.R.B., F45318 GOUT Uric Acid mg.% 10 8 6 4 2 I 01 0=10/12/62 0: , 0 , , , ;-y ~, 5 12 . ! 15 , . , , 20 , . .,? 25 I 55 i 85 115 -/ 175 GOUT AND PURINE METABOLIShf 886 Uric Acid % Uric Acid Oxy purin G.B.C., 874431. ACUTE GOUTY ARTHRITIS 14, W. Nightwatchman Subsiding Second Attack Treated With HPP Alone. 24hrs. 24hi 12 1200 I 0 - 1000- 8- 800- 6 - 600- O =3/28/63 ig/da y Them I 900 Perform.Status %,I 70 75 75 85 90 400 600 95 95 ' too 200 r--95 Fig. 122.-Effect of large dose of HPP on urate levels in gouty subjects. days. There was an associated rise in oxypurine excretion. When the drug was discontinued the uric acid values returned to their previous levels. Figure 122 shows another gouty patient who was admitted for an attack of acute gouty arthritis. HPP was given in an initial dose of 900 mg./day. At that level his urinary uric acid fell from 1,OOO mg./24 hours to about 400 mg./24 hours and his serum uric acid from 8 or 10 mg. per cent to about 3 or 4 mg. per cent. With reduction of dosage an increase in urate levels occurred. Table 38 presents a summary of our first eight patient^.^ The Duke series now comprises 40 patients with gout, but the experience shown here is representative of the total group. In this group of patients the serum urate level was brought to or toward normal in from 5 to 20 days. With the drop of uricemia the urinary uric acid generally fell to an average of about half of previous levels. The dosage used ranged from 200 to 900 mg. per day. Oxypurine excretion increased in all patients. If xanthine oxidase inhibition were the only effect of HPP and renal function were good, the increase in hypoxanthine and xanthine excretions would be expected to balance the decrease in urate output. We have rarely found this. In our initial studies we may have encountered some loss of xanthine by precipitation from certain urine samples, and incomplete recoveries. We have since found that 887 XANTHINE OXIDASE INHIBITORS Table 38.-Eflect of 4-Hydroxypyrazolopyrimidine (HPP) on Purine Metabolism in Gout Patient, No. Age, Race, Sex Diagnosis HPP Days mg./day Rx Serum Uric Acid Urine Uric Acid mg./day Urine Oxypurine mg./day H. C. S., A65086 55, W, M Acute and Chr. Arth. 0 4U0 4 8.7 4.4 560 212 21 102 W. R. B.,F45318 41, W, M Chronic arthritis 0 400 5 6.5 3.2 590 364 24 108 M. N. B.,84618 58, W, M Renal stones acute and Chr. Arth. 0 203 400 12 10 8.7 6.25 3.7 610 440 290 11 52 100 J. E. E..A?Z670 40. W, M Severe Arth. tophi ++ + 0 x00 10 x.7 5.6 455 260 7= 75 J . C. P.,F88837 59, w, M Acute and Chr. Arth. nephropathy 0 300 400 6 20 11.9 6.4 3.9 280 220 180 10 35 J . L. C., E22211 60, C, M Chr. Arth. tophi 0 800 7 9.3 7.2 430 575 8’ 32 K. E. S.,F92133 w, M Chr. Arth. tophi 0 300 5 8.4 5.8 480 295 16* 51 G. B. C., I374431 44, w, M 2nd acute attack 0 900 6 11.1 4.6 890 335 14 75 75, ++++ + 2* *Constant Protein -Constant Purine (“Nutrament” based) Diet. the balance may occasionally be nearly perfect in patients with good renal function immediately after HPP is given, but generally a major deficit of oxypurines does exist. In patients with renal damage or with marked tophaceous involvement the oxypurine increment/urate decrement ratio is generally well below 1, perhaps below 0.5. We have also observed a number of different kinds of response which suggest that total purine excretion may decline as a function of time. This may sometimes be a matter of depletion of stored urates but, curiously, the change may be in the oxypurines rather than in the urate, so that after some months the balance may not be the same as it was initially. Paper and column chromatographic analyses of urine have indicated that there are only trivial changes of other purine bases in the urine. One would not anticipate any major change of these on the basis of the action of the drug. We have observed a few patients who have not responded particularly well. In one patient, for example (Table 38, J. L. C . ) ,with chronic tophaceous gout of severe degree and some limitation of renal function, who received 800 mg. of HPP per day, the serum urate level dropped from 9.3 mg. per cent to 7.2 mg. per cent, but the urinary uric acid actually increased, and there was only a modest increase in the urinary oxypurines. The atypical responses have, by and large, been in patients with tophaceous gout with some degree of renal involvement. We observed a few attacks of gout early in the period after HPP was started. In our first series of 14 subjects three had attacks in the first 888 GOUT AND PURINE METABOLISM 40 days. At that time we were giving HPP alone. It did not seem that the rate of attacks was any greater than it had been before therapy, but in some patients attacks of gout continued with about the established frequency. Because of this, we have subsequently added maintenance colchicine to the therapy of many subjects and the frequency of acute attacks has been far less. The acute attacks have occurred at serum levels well within the normal range; in fact we have observed attacks with serum levels as low as 2 mg. per cent. One patient was of particular interest. H e had had gout for some years, with hypertension and renal involvement, and was not satisfactorily controlled by uricosuric therapy. H e was given HPP and almost immediately began having staccato attacks of gout. These continued for about six weeks in spite of regulation of the urate level at 5 mg. per cent. It was necessary to terminate HPP therapy. On return to uricosuric therapy he has now had only one attack of any severity in about a year and a half. At the time we were unwilling to increase the dose of HPP to a level which might have brought his serum urate value lower, because we were uncertain whether we might have produced hyperxanthinemia of significant degree and whether he was now having a different type of gouty attack based on xanthine crystals rather than sodium urate crystals. We have subsequently measured the plasma oxypurine levels in patients receiving HPP. The normal values range from about 0.1 to 0.3 mg. per cent. During HPP therapy values have generally been around 0.4 and 0.5 mg. per cent. Others7g8 have seen values as high as 0.9 mg. per cent or rarely even 2 mg. per cent but xanthine levels certainly do not rise to match the uric acid levels that existed before therapy. The absence of a more dramatic rise of oxypurines apparently has its basis in a very high renal clearance of hypoxanthine and xanthine.9 One of the fears has been that the increase in urinary oxypurines might give rise to complications based on the insolubility of xanthine. The increment in oxypurines favors xanthine slightly; in our experience perhaps 55 to 60 per cent of the increase has been in xanthine. Hypoxanthine is a very soluble compound; one would not be much concerned about even very high levels of hypoxanthine in the urine, but xanthine is not very soluble in acid or neutral urine. Furthermore, the pK of xanthine is 7.7 compared with a pK of uric acid which is about 5.4. Alkalinization of the urine, therefore, would have to be carried to impractically high levels to have any appreciable effect on xanthine solubility. The urinary levels of xanthine that may be observed with HPP therapy approach those seen in xanthinuric subjects who form xanthine stones. In spite of this we have not seen any xanthine sludge or stones. We have watched for signs of toxicity very carefully, but have seen no increase in urinary sedimentary abnormalities in any of the patients treated. Three of some 60 patients who have received HPP for various reasons have shown minor signs of toxicity. In one case there was a skin rash, which did not recur when the drug was later restarted; another patient had a drug fever which did not recur when HPP was reinstituted; 889 XANTHINE OXIDASE INHIBITORS and one patient had a transient drop in white count which did not recur when the drug was readministered. In all three of these instances the patients were receiving other compounds as well as HPP, and it was not certain in our minds that HPP was involved. Some patients had minimal gastric irritation at one time or another. This has not been a serious problem in our minds and DY.Rundles, who has been in the field of cancer chemotherapy for a long time and is very alert to the problems of intolerance and toxicity, is extremely sanguine about this compound up to this point. Our experience now ranges up to almost two and a half years. No really serious side effects have been observed during this time. Of course one must temper one’s enthusiasm in recognition of the possibility of late toxic effects; for example, it is possible that this compound is incorporated into nucleic acids, although it is converted to a ribonucleotide so sparingly, at least in vitro, that it does not seem likely that much HPP is going to enter the nucleic acid pool. One might view this poor conversion to the ribonucleotide stage as unfortunate in one sense, because the ribonucleotide derivative is a very potent pseudo-feedback inhibitor of the first step of purine biosynthesis in 0itr0.l~ This inhibition may be an explanation for what appears to be a total reduction in rate of purine synthesis in some patients, but at the present time that is not certain. Perhaps reutilization of hypoxanthine and a resetting of the feedback by natural purine ribonucleotides also occur. ACKNOWLEDGMENTS Figures 118, 120, 121, 122, and Table 38 reprinted from Rundles et a14 with permission of the editor and publisher of the Transactions of the Association of American Physicians. REFERENCES 1. Zuckerman, R., Drell, W., and Levin, M. H.: Urinary Purines in Gout; Effect of Azaserine. Arth. iL7 Rheumat. 2:46, 1959. 2. Grayzel, A., Seegmiller, J. E., and Love, E.: Suppression of Uric Acid Synthesis in the Gouty Human by the Use of 6-diazo-5-0x0-L-norleucine. J. Clin. Invest. 39:447, 1960. 3. Feigelson, P., Davidson, J. D., and Robins, R. K.: Pyrazolopyrimidines as Inhibitors and Substrates of Xanthine Oxidase. J. Biol. C h m . 226: 993, 1957. 4. Rundles, R. W., Wyngaarden, J. B., Hitchings, G. H., Elion, G. B., and Silberman, H. R.: Effects of a Xanthine Oxidase Inhibitor on Thiopurine Metabolism, Hyperuricemia and Gout. Trans. Assn. of Am. Phys. LXXVI, 126: 1963. 5. Wyngaarden, J. B., Rundles, R. W., Silberman, H. R., and Hunter, S.: Control of Hyperuricemia with Hydroxypyrazolopyrimidine, a Purine Analogue which Inhibits Uric Acid Synthesis. Arth. 67 Rheumat. @:306, 1963. 6. Rundles, R. W., Silberman, H. R., Hitchings, G. W., and Elion, G. B.: Eftects of Xanthine Oxidase Inhibitor on Clinical Manifestations and Purine Metabolism in Gout. Ann. Int. Med. 60:717, 1964. 7. Klinenberg, J. R., Goldfinger, S., Miller, J., and Seegmiller, J. E.: The Eff-ectiveness of a Xanthine Oxidase Inhibitor in the Treatment of Gout, 890 Arth. G Rheumat. 6:779, 1963. 8. Yii, T. F., and Gutman, A. B.: Effect of Allopurinol (4-hydroxyppazolo (3,4d)-pyrimidine) on Serum and Urinary Uric Acid in Primary and Secondary Gout. Am. J . Med. 37:885, 1964. 9. Goldfinger, S., Klinenberg, J., and Seegmiller, J. E.: The Renal Excretion of GOUT AND PURINE METABOLISM Oxypurines. Clin. Res. 12:252, 1964. 10. McCollister, R. J., Gilbert, W. R., Jr., Ashton, D. M., and Wyngaarden, J. B.: Pseudofeedback Inhibition of Purine Synthesis of 6-Mercaptopurine Ribonucleotide and other Purine Analogues. J . Biol. Chem. 239:1560, 1964. DR. SMYTH: We are all grateful to Dr. Wyngaarden for introducing for consideration this exciting new area for research in purine metabolism. I think it opens tremendous vistas for further exploration. One of the teams that has been in the forefront of this exciting field of exploration has been the group at the NIH, Dr. Klinenberg, Dr. Seegmiller and Dr. Goldfinger, who presented some of their preliminary studies in Boston in December, 1963. We would like to ask Dr. Klinenberg to bring us up to date on their experiences with this subject.