Induction of profound hypouricemia by a non-sedating thiobarbiturate

Induction of Profound Hypouricemia Raymond

P. Warrell,

Jr, Josephia

Muindi, Yee-Wan

by a Non-Sedating Stevens,

Tbiobarbiturate

Marian Isaacs, and Charles W. Young

5-[N-phenylcarboxamido]-2-thiobarbituric acid (merbarone) is a non-sedating derivative of thiobarbituric acid originally developed for anticancer use. In the initial clinical study, a profound reduction in serum uric acid was observed. In 20 patients who received five daily doses of merbarone ranging from 100 to 750 mg/m’, serum uric acid concentration was reduced from a mean pretreatment value of 5.7 + 1.6 mg/dL to a mean lowest value of 1.3 ? 0.5 mg/dL. In most patients, the onset of the effect occurred with 24 hours and was maximal by 46 to 72 hours. Metabolic studies in two patients showed an increase in urinary uric acid excretion within 24 hours after initiation of drug treatment. A marked increase in fractional excretion of uric acid was sustained throughout the period of drug treatment. Urinary excretion of total oxypurines (xanthine and hypoxanthine) was increased twofold to threefold relative to baseline levels. Ultrafiltration studies showed that merbarone did not significantly displace binding of urate from albumin. When merbarone was incubated with xanthine oxidase in vitro. several reaction products were observed. including 2-oxo-2-desthio-merbarone and a compound with retention time similar to 4’-OH-merbarone. Both of these compounds have been described previously as metabolites of merbarone in human subjects. The parent drug and both metabolites were found to inhibit xanthine oxidase (Ki = 41, 36, and 240 pmol/L, respectively). However, this inhibitory effect was substantially less potent than allopurinol IK, = 0.025 jrmol/L). This study indicates that merbarone induces profound hypouricemia primarily by increasing uric acid excretion. Since urate binding to plasma protein is not affected, uricosuria is probably caused by a direct drug action on the renal tubule. Oxidation of merbarone by xanthine oxidase may be an important mechanism for drug metabolism in humans. Merbarone and both of its major metabolites inhibit xanthine oxidase in vitro. However, the magnitude of the increase in urinary oxypurine excretion is considerably less than that reported for allopurinol. Since merbarone does not cause adverse reactions at doses which induce marked hypouricemia, current studies are evaluating merbarone as treatment for hyperuricemia in patients who are intolerant of allopurinol. o 1989 by Grune & Stratton, Inc.

V

ARIOUS derivatives of barbituric acid have been in clinical use for more than one hundred years.’ Barbiturates which possess characteristic hypnotic and anticonvulsant properties are di-substituted at the C5 position (Fig 1). Compounds which have single substitutions at C5 do not have hypnotic activity,* and thus they have engendered little clinical interest. Recently, a group of thiobarbiturates was found to have antitumor activity in several animal models.‘*4 One of these compounds, 5-[N-phenylcarboxamidol-2-thiobarbituric acid (merbarone), has entered clinical trials as an antineoplastic agent (Fig 1). In the course of a pharmacologic study of merbarone, we unexpectedly observed profound hypouricemia in patients who received this drug.5 In this study, we show that merbarone induces hypouricemia by distinct mechanisms that include uricosuria and inhibition of xanthine oxidase.

From the Development Chemotherapy, HematologylLymphoma, and RenalfPhysiology Services, Department of Medicine. Memo rial Sloan-Kettering Cancer Center, the Ciinicai Pharmacology Laboratory, Sloan-Kettering Institute for Cancer Research, and the Cornell University Medical College, New York, NY. Supported in part by Grants CA-42445. CA-08748, CA-05826 and by Contract NOI-CM-52768 from the National Cancer Institute, DHHS. Published in part as Clin. Res. 35:805A, 1987. Address reprint requests to Raymond P. Warrell, Jr. MD, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021. o I989 by Grune & Stratton, Inc. 00260495/89/3806-0009$03.00/O

550

METHODS Clinical Study

Preliminary details of the initial clinical study have appeared elsewhere.6 In the first study, patients were hospitalized and received merbarone as a continuous intravenous (IV) infusion over 24 hours for five consecutive days. The dose ranged from 100 to 750 mg/m’of body surface area. After the hypouricemic effect was recognized, two patients were treated as part of a metabolic study. One patient received high doses (1.25 g/m’) by continuous IV infusion for five days. The second patient was treated with lower doses (150 mg/m’) administered over two hours daily for five days. Serum samples and complete 24-hour urine specimens were collected at baseline for two days and daily during the treatment period. Clinical

Biochemical

Measurements

Serum and urinary uric acid was measured by an autoanalyzer (Technicon, Tarrytown, NY) which used an enzymatic method (uricase). Lack of interference of merbarone with the uricase assay was confirmed by adding merabarone at different concentrations to samples of whole blood, and then blindly assaying the serum samples for uric acid. The decrease in uric acid was also confirmed by high-pressure liquid chromatography (HPLC) measurements, described below. Creatinine, electrolytes, total calcium and phosphorus in serum and urine were measured using an autoanalyzer. Reagents

Merbarone and its metabolites (2-oxo-2-desthio-merbarone and 4’-OH merbarone) was supplied by the National Cancer Institute, Bethesda, MD. Allopurinol, xanthine oxidase (EC 1.2.3.2; grade III from buttermilk), and hypoxanthine were obtained from Sigma Chemical (St Louis). All other reagents were of analytical grade and were obtained from Fisher Scientific (Fair Lawn, NJ) or J.T. Baker Chemical (Phillipsburg, NJ).

Metabolism, Vol 38, No 6 (June), 1989:

pp 550-554

551

HYPOURICEMIA INDUCED BY MERBARONE

to 8.2 with 0.6 mol/L

Allopurinol

Merbarone Fig 1. Chemical hypoxanthine.

structures

of merbarone,

Hypoxanthine allopurinol,

and

HPLC Quantitation of Merbarone and Metabolites from in vitro studies were also separated by a HPLC method’ which used a reversed phase Cl8 NovaPak column (3.9 mm x 15 cm; Waters Associates, Milford, MA) with an ODS-IO precolumn (4.6 mm x 3 cm; Biorad, Richmond, CA). The mobile phase consisted of 15% methanol in 60 mmol/L ammonium acetate, 32 mmol/L acetic acid, 42 mmol/L magnesium sulfate, 1 mmol/L SDS, and 0.1% triethylamine, at pH 7.1. The flow rate was 1 mL/min. Merbarone and its metabolic products were detected by ultraviolet (UV) spectroscopy at 279 and 306 nm. Merbarone

glycylglycine

buffer and digested

with 0.15

U/mL xanthine oxidase for 60 minutes. Uric acid generated from hypoxanthine and xanthine was measured spectrophotometrically by following absorbance decrease at 293 nm after digestion with uricase. Preliminary studies using an HPLC method (column: ubondapak C18; reverse phase: 3.9 mm x 30 cm [Waters Associates. Milford, MA]; mobile phase: 50 mmol/L ammonium hydrogen phosphate pH 6; flow rate 1.5 ml/min; isocratic mode) showed that, under these assay conditions, preformed uric acid in urine was completely digested, and the conversion of oxypurines to uric acid was 100%.

Ultrafiltration of Human Plasma

and metabolites

Pretreatment plasma (0.5 mL) was incubated with 0 to 190 pmol/L merbarone for two hours and then ultrafiltered by centrifugation at 1750 g for 20 minutes at 24” using a micropartition system (MPS-1) and an anisotropic hydrophilic YMT ultrafiltrate membrane (Amicon, Danver, MA). Ultrafiltrable uric acid was determined spectrophotometrically by uricase digestion in 0.1 mol/L sodium borate buffer, pH 8.5. Ultrafiltrable uric acid was expressed as the percentage of total uric acid in the plasma specimen.

RESULTS

Inhibition of Xanthine Oxidase Xanthine oxidase inhibition experiments were performed at 24°C in 50 mmol/L potassium phosphate buffer, pH 6.8, containing 0.3 mmol/L ethylenediaminetetra-acetate (EDTA) as previously described.* The assay mixture consisted of 0 to 20 pmol/L hypoxanthine and 0 to 152 rmol/L merbarone (or its metabolites) dissolved in dimethyl sulfoxide (DMSO). The concentration of DMSO in the reaction mixture was 5% vol/vol. The reaction was initiated by the addition of 0.006 U/mL of enzyme. No enzyme was added in the blank cuvette. The activity of the enzyme was measured spectrophotometrically by monitoring the rate of uric acid formation at 293 nm using a double beam Perkin Elmer recording spectrophotometer. For comparison of relative activities, the effects of allopurinol were compared in this assay using allopurinol0 to 1 rmol/L dissolved in H,O. The type of inhibition was determined by Lineweaver-Burk plot, and the inhibition constant was determined by either Dixon or Cornish-Bowden plots9

Hydroxylation of Merbarone by Xanthine Oxidase The metabolism of merbarone by xanthine oxidase was investigated in 0.1 mol/L sodium borate buffer, pH 8.5. The reaction mixture consisted of 95 pmol/L merbarone and 0.03 U/mL of the enzyme in the presence or absence of 0.4 mmol/L hypoxanthine. The reaction was stopped by the addition of 400 U/mL catalase after 0 to 60 minutes incubation at 37OC; 10 ILL aliquots were directly analyzed for merbarone and its metabolites by HPLC. Merbarone and 2-oxo-2-desthio-merbarone were identified by retention time and UV spectral characteristics. A compound similar to 4’-OHmerbarone was identified by characteristic retention time.

HPLC Quantitation of Uric Acid and Oxypurines Oxypurines (hypoxanthine and xanthine) were quantitated in urine by the method of Jorgensen and Paulsen.“’ Briefly, merbarone and its metabolites were removed from the urine using CF 25 ultrafiltration membrane cones. 100 PL of the ultrafiltered urine was then incubated with 0.02 U/mL uricase in 67 mmol/L glycine buffer, pH 9.3, for one hour at 24OC to digest preformed uric acid. Uricase was inactivated by alkalinization to pH 11.5 with NaOH for 15 minutes. Following neutralization with HCl, the pH was adjusted

Clinical Hypouricemic EJect The effect of various doses of merbarone on serum uric acid in 22 treatment courses in 20 patients is shown in Table 1. As noted, merbarone caused a striking fall in serum uric acid concentrations. Mean serum uric acid decreased from 5.7 + 1.6 mg/dL to 1.3 + 0.5 mg/dL. The onset of the effect occurred within 48 hours and became maximal within three to seven days. Since the effect was unanticipated and serum uric acid was not determined on a daily basis during the preliminary study, the day on which the lowest value of serum uric acid appeared probably occurred earlier than indicated by this table. Over the range of doses examined in this study (100 to 750 mg/m2), the hypouricemic effect was independent of the administered dose.

Urinary Uric Acid and Oxypurine Excretion

Detailed 24-hour urinary excretion of uric acid and oxypurines are shown for two patients at baseline and during drug treatment with high and low doses of merbarone (Fig 2A,B). The hypouricemic effect was associated with a sharp increase in urinary urate excretion during the first 24 hours after drug administration. Uricosuria was sustained, albeit to a lesser degree, throughout the duration of the drug infusion despite the very low serum uric acid levels (Fig 2A,B). The calculated fractional clearance of uric acid relative to creatinine was strikingly elevated throughout this period, increasing from a mean baseline level of 3.5% to a peak of 46.1% in the patient treated at the low-dose level, and from 10.3% to 97.8% in the patient treated at the high-dose level. At both the low-dose and high-dose levels, administration of merbarone was also associated with approximately a twofold or threefold increase in urinary oxypurine excretion (Fig 2A,B). In the patient who received the high dose infusion, oxypurine excretion appeared to increase progressively over the five-day treatment period.

552

WARRELL

ET AL

Table 1. Decrease in Serum Uric Acid Concentration During Treatment With Merbarone Administered Daily for Five Days SerumUricAcidConcentration (mg/dl) Subject No.

Dose(mglm’)

Initial Value

1

100

4.2

0.8 (7)

la

100

4.8

1.2 (4)

5.8 (10)

2

100

6.2

1.1 (5)

5.3 (17)

3

100

6.9

1.8 (6)

5.8 (8)

3a

100

6.6

1.0 (6)

5.1 (20)

4

150

6.2

2.0 (6)

4.4 (7)

Recovery Value(dl

Lowest Value(d)

4.8 (10)

5

150

6.2

1.9 (3)

4.6 (12)

6

150

3.4

1.0 (3)

3.5 (11)

7

200

5.7

0.8 17)

4.2 (12)

8

200

3.7

0.7 (3)

2.6 (10)

9

250

5.8

1.2 (61

6.2 (10)

10

250

4.4

0.8 (5)

4.0(11)

11

250

7.8

1.3 (5)

5.9 (8)

12

300

5.0

2.4 (3)

5.5 (10)

13

300

5.0

0.8 (3)

4.0(11)

14

300

9.9

1.7 (5)

7.4110)

15

400

4.4

1.0 (5)

3.0(6)

16

400

5.1

1.9 (8)

4.0(101

17

500

5.2

1.2 (5)

3.3 (8)

18

500

4.9

1.3 (5)

3.8 (11)

19

750

9.0

1.4 (5)

4.7 (15)

20

750

4.5

1.3 (4)

3.3 (32)

5.7 f 1.6

1.3 * 0.5

4.6 f 1.2

Mean f SD

Merbarone (150 mglmzld)

6.0

7.0-

6.0

Merbarone (1,250 mglm*/d)

6.0 -

n .30 * g % 5 z! 8 .I6 g

.24

0.9

a a

t .g 0.6-

0.3-

I '

A

4

'

‘DAY:

5

-.12

3

-.06

ii D

0 ii

3 0

z.

@

.E el

.12 5 A e .06 ij 5 =

0.6

0.3

5

2

B

’

’

‘DAY:

Fig 2. Change in serum uric acid (solid line) and urinary excretion of uric acid (open bars) and oxypurines (solid bars) after treatment with a low dose (A) and high dose (El) of merbarone.

553

HYPOURICEMIA INDUCED BY MERBARONE

Table 2. Inhibition of Xanthine Metabolites,

Oxidase by Merbarone,

Major

and Allopurinol

K,

Type of Inhibition

Merbarone

41 I.rmol/L

mixed

4’-OH-Merbarone

36 pmol/L

uncompetitive

2-Oxo-2-Desthiomerbarone

240 pmol/L

noncompetitive

Allopurinol

0.025

mixed

umol/L

Ultrafiltration of Plasma To evaluate whether the uricosuric effect was due to displacement of mate from serum protein, ultrafiltrable uric acid was measured before and after the addition of merbarone to human plasma. Over concentrations of merbarone which ranged from 0 to 190 hmol/L, the percent of ultrafiltered uric acid was not significantly altered, ranging from 67.0% to 69.0%. Interaction of Merbarone and Xanthine Oxidase Following the incubation of merbarone with xanthine oxidase and hypoxanthine, several metabolites were detected in the reaction mixture. One of these compounds, 2-0x02-desthio-merbarone, has been described previously as a principal metabolite of merbarone in human subjects.” As shown in Table 2, merbarone and both of these major metabolites inhibited xanthine oxidase. However, the degree of inhibition was substantially less than that observed for allopurinol. DISCUSSION

We have not found prior reports that barbiturates affect the formation or excretion of uric acid. A previous study has shown that administration of phenobarbital to rats did not cause a significant change in urinary excretion of uric acid.‘* Chlorprothixene, a structurally unrelated phenothiazine, was reported to cause hypouricemia and uricosuria.‘3,‘4 However, excessive sedation induced by that drug precluded further evaluation of its hypouricemic activity. The primary ring structure of the barbiturates resembles the pyrimidine ring of both hypoxanthine and allopurinol (Fig 1). The lack of hypouricemic effect of other barbiturates may be related to the nature of chemical substitution at the C5 position. While a variety of barbiturates have been synthesized, only compounds with hypnotic or anticonvulsant properties have been of a major clinical interest, and these drugs are disubstituted at the C5 position.’ Allopurinol was originally developed for anticancer use in conjunction with thiopurines. Rundles et al” and Rundles16 observed that the drug was associated with hypouricemia in cancer patients, and the compound is now the most widely used drug for the treatment of gout.” Allopurinol is metabolized by xanthine oxidase at the C2 position to form oxipurino], and both allopurinol and oxipurinol are competitive inhibitors of the enzyme.16 Similarly, xanthine oxidase catalyzes the oxidation of merbarone at C2 to form 2-0x02-desthio-merbarone. Together with the other major reaction product (4’-OH-merbarone), these compounds comprise the principal metabolites of merbarone which have been described previously. The parent drug and both metabolites

were found to inhibit xanthine oxidase. Although the Ki of merbarone for xanthine oxidase (41 pmol/L) is substantially higher than that of allopurinol (0.025 &mol/L), this concentration is readily achieved in vivo. The lowest dose of merbarone in this study (100 mg/m*) achieved steady-state plasma concentrations ranging from 11 to 33 pmol/L.6 Moreover, the half-life of merbarone in plasma exceeds that of allopurinol (12 hour v 2 hour)* and is thus more similar to that of oxipurinol. Hypouricemia induced by allopurinol is accompanied by decreased urinary excretion of uric acid and increased urinary excretion of oxypurines.” Conversely, administration of merbarone causes an increase in uricosuria. The fractional excretion of uric acid remained greatly elevated throughout the merbarone treatment period. Since merbarone did not significantly displace binding of urate from plasma protein, the uricosuric effect is probably due to direct effects on the renal tubule. Administration of merbarone did cause a minor (twofold to threefold) increase in urinary oxypurine excretion, suggesting some degree of inhibition of xanthine oxidase in vivo. However, the magnitude of this effect was substantially lower than seen with allopurinol. Although daily oxypurine excretion appeared to increase progressively during drug treatment, the relatively small change suggests that inhibition of xanthine oxidase is a less important effect in vivo. Alternatively, the change in oxypurine excretion may reflect an increased clearance of plasma oxypurines.” The activity of merbarone is somewhat analogous to benzbromarone, a benzofuran derivative that acts as a uricosuric in vivo and that inhibits xanthine oxidase in vitro.20-22 However, administration of benzbromarone to human subjects does not significantly affect urinary oxypurine excretion.” In the absence of direct comparisons, the potency of merbarone relative to other hypouricemic drugs can only be estimated. In studies of patients with gout and cancer, Yu and Gutman’* and Muggia et al*-’reported that allopurinol in doses ranging from 200 to 900 mg per day achieved mean lowest serum uric acid concentrations of 4.2 and 6. I mg/dL respectively. Thompson et al24 reported that serum uric acid in gouty subjects was reduced up to 45% of baseline levels in 41 of 53 patients treated with probenecid, and in ten of 15 patients treated with sulfinpyrazone. By contrast, even the lowest dose of merbarone in this study ( 100 mg/m2) caused a mean reduction in serum uric acid of 79% relative to baseline levels. Although the drug was administered IV, studies in dogs suggest that at least 70% of an orally administered dose is absorbed (C.W. Young, unpublished data). Further studies are planned to characterize dose-response (particularly at low doses), to quantify oxypurine clearance and excretion after prolonged administration, and to compare oral v IV bioavailability. These data indicate that merbarone is a unique new agent that acts on uric acid metabolism primarily by increasing urinary uric acid excretion and, to a lesser degree. by decreasing formation. Since merbarone has not been associated with adverse reactions at doses that induce this striking hypouricemic effect, the drug may be useful for treatment of hyperuricemia in patients who arc resistant to or intolerant of allopurinol.

554

WARRELL ET AL

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constituents determined during toxicological investigations and phenobarbital effect. Bull Inst Marit Trop Med Gydnia 27:33-37, 1975 13. Healey LA, Harrison M, Decker JL: Uricosuric effect of chlorprothixene. N Engl J Med 272:526-527, 1965 14. Weinshilboum RM, Goldstein JL, Kelley WN: Prolonged hypouricemia associated with acute chlorprothixene ingestion. Arthritis Rheum 18:739-741, 1975 15. Rundles RW, Wyngarden JB, Hitchings GH, et al: Effects of a xanthine oxidase inhibitor on thiopurine metabolism, hyperuricemia, and gout. Trans Assoc Am Physicians 76: 126- 140, 1963 16. Rundles RW: The development of allopurinol. Arch Intern Med 145:1492-1503, 1985 17. Wyngarden JB, Kelley WN: Gout, in Stanbury JB, Wyngarden JB, Frederickson DS (eds): The Metabolic Basis of Inherited Disease (ed 4). New York, McGraw-Hill, 1978, pp 916-1010 18. Yu TF, Gutman AB: Effect of allopurinol (4-hydropyrazolo(3,4d) pyrimidine) on serum and urinary uric acid in primary and secondary gout. Am J Med 37:885-898, 1964 19. Puig J, Mateos F, Jimenez M, et al: Impaired renal excretion of hypoxanthine and xanthine in primary gout. Pediatr Res 24: 131, 1988 (abstr) 20. Sinclair DS, Fox IH: The pharmacology of hypouricemic effect of benzbromarone. J Rheumatol2:437-445, 1975 21. Yu T-F: Pharmacokinetic and clinical studies of a new uricosuric agent-benzbromarone. J Rheumatol3:305-312, 1976 22. Jain AK, Ryan JR, McMahon FG, et al: Effect of single oral doses of benzbromarone on serum and urinary uric acid. Arthritis Rheum 17:149-157, 1974 23. Muggia FM, Ball TJ Jr, Ultmann JE: Allopurinol in the treatment of neoplastic disease complicated by hyperuricemia. Arch Intern Med 120:12-18, 1967 24. Thompson GR, Duff IF, Robinson WD, et al: Long term uricosuric therapy in gout. Arthritis Rheum 5:384-396, 1962