Effect of antiperoxidative drugs on gastric damage induced by ethanol in rats

Lesion formation due to oral administration of absolute ethanol could be prevented by parenteral pretreatment with antiperoxidative drugs such as butylated hydroxytoluene (BHT), quercetin and quinacrine. Also effective were allopurinol and oxypurinol, inhibitors of xanthine oxidase, but not superoxide dismutase (SOD) and hydroxyl radical scavengers, such as sodium benzoate and dimethyl sulfoxide (DMSO). BHT, quercetin, quinacrine and sulfhydryl compounds such as reduced glutathione and cysteamine which offer gastroprotection in vivo against ethanol inhibited lipid peroxidation induced in vitro by ferrous ion in porcine gastric mucosal homogenate, but SOD, sodium benzoate, DMSO, allopurinol and oxypurinol did not. These results suggest the possibility that an active species, probably derived from free iron mobilized by the xanthine oxidase system, other than oxygen radicals such as hydroxyl radicals, contributes to lipid peroxidation and lesion formation in the gastric mucosa after absolute ethanol administration.     

Effect of Wen-Pi-Tang extract on lung damage by influenza virus infection

The effect of Wen-Pi-Tang extract on influenza virus infection in mice was investigated. The administration of Wen-Pi-Tang extract at a dose of 100 mg/kg body wt. for 8 consecutive days to influenza virus-infected mice reversed the lack of body wt. gain and prevented the increase in lung weight caused by the infection in comparison with uninfected mice, while allopurinol, a xanthine oxidase (XOD) inhibitor, did not show these effects. The serum levels of uric acid and allantoin in influenza virus-infected mice were reduced by Wen-Pi-Tang extract administration. Moreover, Wen-Pi-Tang extract reduced the uric acid level more as the dose increased, although it exerted lower activity than allopurinol. The XOD activity of the lungs was elevated by influenza virus infection, but Wen-Pi-Tang extract administration inhibited this activity, indicating prevention of lung damage by oxygen free radicals generated by XOD. After the administration of Wen-Pi-Tang extract to influenza virus-infected mice, the lung superoxide dismutase activity was not significantly different from that of uninfected mice, whereas lung catalase activity was lower in the former than the latter, but slightly higher than that of influenza virus-infected mice, suggesting that Wen-Pi-Tang extract may prevent the generation of highly toxic hydroxyl radicals in the lung. In addition, the administration of both Wen-Pi-Tang extract and allopurinol reduced the degree of lung consolidation caused by influenza virus infection. In particular, Wen-Pi-Tang extract reduced the consolidation score in a dose-dependent manner and more markedly than allopurinol did. This study suggests that Wen-Pi-Tang extract could improve pathological conditions of the lungs induced by influenza virus infection.     

Stimulation of uric acid release from the perfused rat liver by platelet activating factor or potassium.

The stimulation of hepatic glycogenolysis by platelet activating factor (AGEPC) or increased perfusate potassium concentration ([K+]o), but not phenylephrine, causes a transient increase in uric acid release into the effluent perfusate of perfused rat livers. Uric acid was identified in chromatograms of perfusate samples using reversed-phase h.p.l.c., which show a peak which co-elutes with authentic uric acid, and by the fact that the A293 of perfusate samples decreases in the presence of uricase. Uric acid release is dose-dependent with respect to both AGEPC and [K+]o, and is blocked completely by prior exposure of the perfused liver to 5 mM-allopurinol, a specific inhibitor of xanthine oxidase (XOD). Allopurinol inhibits the increase in portal vein pressure induced by AGEPC, increased [K+]o or phenylephrine; the inhibitory effect increases with increasing concentrations of the agents. Also, allopurinol inhibits the second phase of O2 uptake and glucose release characteristic of concentrations of AGEPC or increased [K+]o equal to or greater than their reported half-maximal concentration for glucose release. The ratio of xanthine dehydrogenase (XDH) to XOD activity in extracts of freeze-clamped perfused livers is not affected by treatment of the livers with AGEPC or increased [K+]o. The results suggest that uric acid production may be an indicator of ischaemia within localized hepatic sinusoids, and that allopurinol partially protects the hepatocyte from the effects of AGEPC or increased [K+]o by inhibiting XOD-dependent superoxide production. We propose that the second phase of the glycogenolytic response to these agents results from ischaemia and subsequent reperfusion. Activation of XOD in vivo and hence O2-derived free radical production may be involved in the response of the liver to vasoactive agonists under a variety of pathophysiological conditions.     

Purpurogallin is a more powerful protector of kidney cells than Trolox and allopurinol.

Phase contrast and electron microscopic experiments demonstrated that oxyradicals generated with xanthine oxidase and hypoxanthine markedly damage rat kidney mesangial and porcine tubular epithelial cells. Purpurogallin, a phenol found in oak nutgalls, prolongs survival of the xanthine oxidase exposed renal cells three- to nine-fold longer than those without purpurogallin present. At levels equimolar to purpurogallin, either Trolox or allopurinol is less effective in delaying cell necrosis. Purpurogallin scavenges not only xanthine oxidase generated oxyradicals, but also non-enzymatically produced peroxyl radicals, more actively than equimolar levels of Trolox or allopurinol. Purpurogallin inhibits xanthine oxidase with severalfold higher potency than allopurinol and its more active metabolite oxypurinol. Therefore, purpurogallin is a stronger antioxidant than Trolox and a more potent inhibitor of xanthine oxidase than allopurinol as well as oxypurinol.     

Allopurinol, an inhibitor of xanthine oxidase, improves the development of IVM/IVF bovine embryos (>4 cell) in vitro under certain culture conditions

To determine the origin of free oxygen radicals in the culture medium of bovine embryos, the effect of allopurinol, an inhibitor of xanthine oxidase, on the development of embryos (>4 cell) in modified synthetic oviduct fluid (m-SOF) medium was examined. When embryos were cultured in the presence of 0.2 mM allopurinol under high oxygen tension (5% CO2 in air), the blastocyst rate significantly (P<0.05) increased compared with the absence of allopurinol (allopurinol (+) 42 vs. (-) 25%; Day 6, 63 vs. 51%; Day 7, 69 vs. 58%; Day 8). However, allopurinol had no effect on embryo development under low oxygen tension (5% CO2, 5% O2, 90% N2). Moreover, it was found that the developmental rate and the total cell number of blastocysts decreased (development rate: 60 vs. 28%, cell number: 132 vs. 74) when the embryos were cultured in medium containing 0.01 U/mL xanthine oxidase (XOD) and 0.1 mM hypoxanthine (HXT), and the damaging effect of XOD and HXT was removed by the addition of 0.2 mM allopurinol. The beneficial effect of allopurinol was also observed when the glucose concentration was increased to 4.5 mM from 1.5 mM (control: 22% vs. allopurinol: 34%; Day 8), but no beneficial effects were observed in the media without glucose (control: 55% vs. allopurinol: 59%). Taken together, these results suggested that a portion of the free oxygen radicals are generated from the XOD and HXT reactions under culture conditions, and this generation is enhanced by high oxygen tension in the gas atmosphere or by high glucose concentrations in the medium.     

Reactive oxygen metabolite-induced toxicity to cultured bovine endothelial cells: Status of cellular iron in mediating injury

We aimed to determine the status of iron in mediating oxidant-induced damage to cultured bovine aortic endothelial cells. Chromium-51-labeled cells were exposed to reaction mixtures of xanthine oxidase/hypoxanthine and glucose oxidase/glucose; these produce superoxide and hydrogen peroxide, or hydrogen peroxide, respectively. Xanthine oxidase caused a dose dependent increase of ⁵¹Cr release. Damage was prevented by allopurinol, oxypurinol, and extracellular catalase, but not by superoxide dismutase. Prevention of xanthine oxidase-in-duced damage by catalase was blocked by an inhibitor of catalase, aminotriazole. Glucose oxidase also caused a dose-dependent increase of ⁵¹Ci release. Glucose oxidase-induced injury, which was catalase-inhibitable, was not prevented by extracellular superoxide dismutase. Both addition of and pretreatment with deferoxamine (a chelator of Fe³⁺) prevented glucose oxidase-induced injury. The presence of phenanthroline (a chelator of divalent Fe²⁺) prevented glucose oxidase-induced ⁵¹Cr release, whereas pretreatment with the agent did not. Apotransferrin (a membrane impermeable iron binding protein) failed to influence damage. Neither deferoxamine nor phenanthroline influenced cellular antioxidant defenses, or inhibited lysis by non-oxidant toxic agents. Treatment with allopurinol and oxypurinol, which inhibited cellular xanthine oxidase, failed to prevent glucose oxidase injury. We conclude that (1) among the oxygen species extracellularly generated by xanthine oxidase/hypoxanthine, hydrogen peroxide induces damage via a reaction on cellular iron; (2) deferoxamine and phenanthroline protect cells by chelating Fe³⁺ and Fe²⁺, respectively; and (3) reduction of cellular stored iron (Fe³⁺) to Fe²⁺ may be a prerequisite for mediation of oxidantinduced injury, but this occurs independently of extracellular superoxide or cellular xanthine oxidase-derived superoxide. © 1994 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Further evidence for the role of free radicals in the limb teratogenicity of L-NAME

BACKGROUND: L-NAME (N(G)-nitro-(L)-arginine methyl ester), a nitric oxide synthase inhibitor, causes severe limb reduction malformations when gravid rats are treated intraperitoneally on gd-17. Hemorrhages, appearing within hours of L-NAME administration, and defects at term can be significantly reduced by co-treatment with PBN (alpha-phenyl-N-t-butylnitrone), a spin trap antioxidant. We have proposed that limb defects result from ischemia-reperfusion injury. We examine the role of xanthine oxidase and ROS formation in the limb effects of L-NAME. METHODS: Gravidas were treated with L-NAME (50 mg/kg) in the presence or absence of allopurinol, a xanthine oxidase inhibitor. Spatial patterns of limb hemorrhage were determined promptly and at term as was digit length at the latter interval. Xanthine oxidase activities were assayed in control and treated limbs with and without allopurinol co-treatment. RESULTS: Allopurinol significantly reduced hemorrhage severity in a dose-responsive fashion when fetuses were examined at term. Higher doses of allopurinol significantly preserved digit length. Xanthine oxidase activities in fetal limb were significantly increased by L-NAME treatment whereas co-treatment with allopurinol restored activities to near-control levels. CONCLUSIONS: These findings support the role of excess reactive oxygen species (ROS) formation in L-NAME-induced limb reduction. We propose that nitric oxide (NO) depletion by L-NAME interferes with vascular integrity, and causes vasoconstriction. Resultant hypoxia stimulates superoxide formation and nitric oxide formation catalyzed by the inducible isoform of nitric oxide synthase. The reduction products of superoxide or the products of its reaction with nitric oxide oxidize or nitrate endothelial components resulting in limb reduction defects.     

Xanthine oxidase-dependent ROS production mediates vitamin A pro-oxidant effects in cultured Sertoli cells

Several studies have suggested that vitamin A (retinol, ROH) presents pro-oxidant properties in biological systems. Recent studies point out that xantine oxidase, a ROS-generating enzyme, catalyses ROH oxidation to RA in vitro. These works stimulated the authors to investigate whether xanthine oxidase could be involved on the ROH pro-oxidative effects reported in cultured Sertoli cells. In vitro, it was demonstrate that xanthine oxidase generates superoxide in the presence of ROH as assessed by superoxide mediated-NBT reduction. Superoxide production is potentiated in the presence of NADH and inhibited by allopurinol. In Sertoli cells, ROH treatment increased xanthine oxidase activity and inhibition of the enzyme with allopurinol attenuated ROH-induced ROS production, protein damage and cytotoxicity. Moreover, inhibition of ROH oxidation to RA by retinaldehyde dehydrogenase inhibitor potentiated both xanthine oxidase-dependent ROS production and cell damage in ROH-treated cells. The data show that xanthine oxidase may play a role on vitamin A pro-oxidant effects.     

Xanthine oxidase-dependent ROS production mediates vitamin A pro-oxidant effects in cultured Sertoli cells

Several studies have suggested that vitamin A (retinol, ROH) presents pro-oxidant properties in biological systems. Recent studies point out that xantine oxidase, a ROS-generating enzyme, catalyses ROH oxidation to RA in vitro. These works stimulated the authors to investigate whether xanthine oxidase could be involved on the ROH pro-oxidative effects reported in cultured Sertoli cells. In vitro, it was demonstrate that xanthine oxidase generates superoxide in the presence of ROH as assessed by superoxide mediated-NBT reduction. Superoxide production is potentiated in the presence of NADH and inhibited by allopurinol. In Sertoli cells, ROH treatment increased xanthine oxidase activity and inhibition of the enzyme with allopurinol attenuated ROH-induced ROS production, protein damage and cytotoxicity. Moreover, inhibition of ROH oxidation to RA by retinaldehyde dehydrogenase inhibitor potentiated both xanthine oxidase-dependent ROS production and cell damage in ROH-treated cells. The data show that xanthine oxidase may play a role on vitamin A pro-oxidant effects.     

Modulation of radiation-induced changes in the xanthine oxidoreductase system in the livers of mice by its inhibitors

The xanthine oxidoreductase (XOD) system, which consists of xanthine dehydrogenase (XDH) and xanthine oxidase (XO), is one of the major sources of free radicals in biological systems. The XOD system is present predominantly in the normal tissues as XDH. In damaged tissues, XDH is converted into XO, the form that generates free radicals. Therefore, the XO form of the XOD system is expected to be found mainly in radiolytically damaged tissue. In this case, XO may catalyze the generation of free radicals and potentiate the effect of radiation. Inhibition of the XOD system is likely to attenuate the detrimental effects of ionizing radiation. We have examined this possibility using allopurinol and folic acid, which are known inhibitors of the XOD system. Swiss albino mice (7-8 weeks old) were given single doses of allopurinol and folic acid (12.5-50 mg/kg) intraperitoneally and irradiated with different doses of gamma radiation at a dose rate of 0.023 Gy/s. The XO and XDH activities as well as peroxidative damage and lactate dehydrogenase (LDH) were determined in the liver. An enhancement of the activity of XO and a simultaneous decrease in the activity of XDH were observed at doses above 3 Gy. The decrease in the ratio XDH/XO and the unchanged total activity (XDH + XO) suggested the conversion of XDH into XO. The enhanced activity of XO may potentiate radiation damage. The increased levels of peroxidative damage and the specific activity of LDH in the livers of irradiated mice supported this possibility. Allopurinol and folic acid inhibited the activities of XDH and XO, decreased their ratio (XDH/XO), and lowered the levels of peroxidative damage and the specific activity of LDH. These results suggested that allopurinol and folic acid have the ability to inhibit the radiation-induced changes in the activities of XDH and XO and to attenuate the detrimental effect of this conversion, as is evident from the diminished levels of peroxidative damage and the decreased activity of LDH.     

Re-evaluation of superoxide scavenging capacity of xanthohumol

The chemopreventive chalcone xanthohumol (Xh) has been reported to decrease xanthine oxidase (XOD) catalysed formation of formazan from nitroblue tetrazolium (NBT) and is discussed as a potent scavenger of superoxide. Re-evaluation of the scavenging capacity indicated that Xh disturbed detection of superoxide with NBT, in case of an insufficient NBT/Xh ratio. Xh lacked superoxide scavenging activity in contrast to the Xh-derivative 3'-hydroxy-Xh with catechol substructure, used as positive control. This was shown by the use of sufficient concentration of NBT and other detectors such as hydroxylamine, XTT, cytochrome c and hydroethidine. HPLC analysis of reaction products in a xanthine/XOD/peroxidase system demonstrated beside enhanced inhibition of NBT-formazan by Xh that NBT even prevented oxidation of Xh. p-coumaric acid or ferulic acid could replace Xh in that system, indicating that superoxide detection using NBT is likely jeopardized by interference of phenoxyl-radicals. Furthermore, this study provides evidence that Xh can moderately generate superoxide via auto-oxidation.     

Nitric Oxide Reversibly Suppresses Xanthine Oxidase Activity

The effects of nitric oxide (NO) on xanthine oxidase (XOD) activity and the site(s) of the redox center(s) affected were investigated. XOD activity was determined by superoxide (O₂^-) generation and uric acid formation. NO reversibly and dose-dependently suppressed XOD activity in both determination methods. The suppression interval also disclosed a dose-dependent prolongation. The suppression occurred irrespective of the presence or absence of xanthine; indicating that the reaction product of NO and O₂^-, peroxynitrite, is not responsible for the suppression. Application of synthesized peroxynitrite did not affect XOD activity up to 2 μM. Methylene blue, which is an electron acceptor from Fe/S center, prevented the NO-induced inactivation. The results indicate that NO suppresses XOD activity through reversible alteration of the flavin prosthetic site.     

Hypouricemic Actions of Exopolysaccharide Produced by Cordyceps militaris in Potassium Oxonate-Induced Hyperuricemic Mice

The hypouricemic actions of exopolysaccharide produced by Cordyceps militaris (EPCM) in potassium oxonate-induced hyperuricemia in mice were examined. Hyperuricemic mice were administered intragastrically with EPCM (200, 400 and 800 mg/kg body weight) or allopurinol (5 mg/kg body weight) once daily. Serum uric acid, blood urea nitrogen and liver xanthine oxidase (XOD) activities of each treatment were measured after administration for 7 days. EPCM showed dose-dependent uric acid-lowering actions. EPCM at a dose of 400 mg/kg body weight and allopurinol showed the same effect in serum uric acid, blood urea nitrogen and liver XOD activities in hyperuricemic mice. An increase in liver XOD activities was observed in hyperuricemic mice due to administration of EPCM at a dose of 200 mg/kg body weight. EPCM at a dose of 800 mg/kg body weight did not show significant effects on serum uric acid and XOD activities. We conclude that EPCM has a hypouricemic effect caused by decreases in urate production and the inhibition of XOD activities in hyperuricemic mice, and this natural product exhibited more potential efficacy than allopurinol in renal protection.     

Xanthine oxidase and endothelium dependent relaxation

Superoxide anion (O2-) generated from xanthine oxidase/xanthine has been used to decrease the half life of endothelium derived relaxing factor (EDRF). However, by itself, xanthine oxidase causes endothelium dependent relaxation. This relaxation is unrelated to the oxidative property of the enzyme since it is not inhibited by allopurinol. In addition, the relaxation is not inhibited by the cyclooxygenase inhibitor, indomethacin, or the phospholipase A2 inhibitor, p-bromophenacyl bromide. On the other hand the relaxation is inhibited by the trypsin inhibitor (TI) from chicken egg white. A similar endothelium dependent relaxation elicited by pancreatin and trypsin is also inhibited by TI. Pancreatin used in the preparation of xanthine oxidase contains trypsin, chymotrypsin and carboxypeptidase. When compared to trypsin both chymotrypsin and carboxypeptidase elicit little relaxation. Thus the endothelium dependent relaxation elicited by xanthine oxidase is likely due to contamination with trypsin. Our results emphasize that when the superoxide generating system, xanthine oxidase/xanthine is used to study the effect of oxygen radicals on EDRF, it is advantageous to ensure that only purified preparations of xanthine oxidase are used.     

Re-evaluation of superoxide scavenging capacity of xanthohumol

Abstract

The chemopreventive chalcone xanthohumol (Xh) has been reported to decrease xanthine oxidase (XOD) catalysed formation of formazan from nitroblue tetrazolium (NBT) and is discussed as a potent scavenger of superoxide. Re-evaluation of the scavenging capacity indicated that Xh disturbed detection of superoxide with NBT, in case of an insufficient NBT/Xh ratio. Xh lacked superoxide scavenging activity in contrast to the Xh-derivative 3′-hydroxy-Xh with catechol substructure, used as positive control. This was shown by the use of sufficient concentration of NBT and other detectors such as hydroxylamine, XTT, cytochrome c and hydroethidine. HPLC analysis of reaction products in a xanthine/XOD/peroxidase system demonstrated beside enhanced inhibition of NBT-formazan by Xh that NBT even prevented oxidation of Xh. p-coumaric acid or ferulic acid could replace Xh in that system, indicating that superoxide detection using NBT is likely jeopardized by interference of phenoxyl-radicals. Furthermore, this study provides evidence that Xh can moderately generate superoxide via auto-oxidation.     

Intracellular utilization of superoxide anion by indoleamine 2,3-dioxygenase of rabbit enterocytes.

The participation of superoxide anion (O2-) in the intracellular indoleamine 2,3-dioxygenase activity was studied using the dispersed cell suspension of the rabbit small intestine. The dioxygenase activity was assayed by measuring [14C]formate released from DL-[ring-2-14C]tryptophan. The addition of diethyldiethiocarbamate, a superoxide dismutase inhibitor, markedly accelerated the intracellular dioxygenase activity while the superoxide dismutase activity decreased concomitantly. Furthermore, substrates of xanthine oxidase such as inosine, adenosine, and hypoxanthine also increased the dioxygenase activity in the cells, particularly in the presence of methylene blue. This increase was completely abolished by the addition of allopurinol, a specific inhibitor of xanthine oxidase. These results, taken together, indicate that the intracellular accumulation of O2- results in acceleration of the in situ dioxygenase activity, and that indoleamine 2,3-dioxygenase utilizes O2- in the isolated intestinal cells.     

Antioxidant activity in vitro of two aromatic compounds from Caesalpinia sappan L

Two antioxidant compounds were isolated from C. sappan L by multiple steps of column chromatography and thin layer chromatography in succession with superoxide scavenging assay as activity monitor. Structures of the two compounds were determined by spectroscopic methods as 1',4'-dihydro-spiro[benzofuran-3(2H),3'-[3H-2]benzopyran]-1',6',6',7'-tetrol (compound 1) and 3-[[4,5-dihydroxy-2(hydroxymethyl) phenyl]-methyl]-2,3-dihydro-3,6-benzofurandiol (compound 2). Characterization of antioxidant properties of these two compounds was done by determining the inhibitory effect on xanthine oxidase activity as well as scavenging effect on superoxide anion and hydroxyl radicals. Our results indicated that compounds 1 and 2 inhibited xanthine oxidase activity and scavenged superoxide anion and hydroxyl radicals. Compounds 1 and 2 possessed similar radical scavenging activities as ascorbic acid, and they were more effective than other well-known antioxidants such as alpha-tocopherol, beta-carotene, and BHT. As inhibitors of free radical formation, compounds 1 and 2 were more effective than all the other antioxidants tested. In conclusion, compounds 1 and 2 can be regarded as primary antioxidants with radical-scavenging and chain-breaking activities as well as secondary antioxidants with inhibitory effect on radical generation.     

Effects of superoxide radicals on transport (Na+K) adenosine triphosphatase and protection by superoxide dismutase

Membrane (Na+K)ATPase isolated from rat brain was preincubated in a medium in which superoxide radicals were generated enzymatically. Exposure to superoxide radicals caused an irreversible inactivation, which could be prevented by further addition of superoxide dismutase. (Na+K)ATPase was also protected by addition of allopurinol, a xanthine oxidase inhibitor, during preincubation. The K-activated nitrophenylphosphatase associated with (Na+K)ATPase was also found to be inactivated by preincubation with superoxide radicals, which could be prevented by superoxide dismutase.     

Inhibition of mammalian xanthine oxidase by folate compounds and amethopterin

We have examined the effects of folate compounds and the folate analog amethopterin (methotrexate) as inhibitors of mammalian xanthine oxidase and have found that they offer potent inhibition of the enzyme. We have compared the inhibitory potency of folic acid and its coenzyme derivative tetrahydrofolic acid to that of allopurinol, a known inhibitor of xanthine oxidase, and have demonstrated that folic acid and tetrahydrofolic acid are severalfold more potent than allopurinol as inhibitors of xanthine oxidase. Comparative inhibition constants calculated were 5.0 X 10(-7) M for folic acid. 1.25 X 10(-6) M for tetrahydrofolic acid, and 4.88 X 10(-6) M for allopurinol. Incubation of xanthine oxidase with folic acid at a concentration of 10(-6) M abolished 94% of the enzymic activity within 1 min of incubation with the enzyme. At the same concentration, allopurinol was almost ineffective as an inhibitor of xanthine oxidase. The substrate xanthine protected the enzyme against total inhibition by folic acid. Reversibility of the enzymic inhibition by folic acid was demonstrated. Folic acid-inactivated enzyme was totally regenerated either by filtration through Sephadex G-200 or by precipitation with ammonium sulfate. 2-Amino-4-hydroxypteridine was a poor substrate for the enzyme but a potent inhibitor for the oxidation of xanthine by the enzyme. The inhibition constant calculated was 1.50 X 10(-6) M. In the presence of an excess of xanthine oxidase, neither folic acid nor tetrahydrofolic acid and allopurinol exhibited any change in intensity of their absorbance or in the wavelength of their maximal absorbance that might have been suggestive of substrate utility. The folate analog amethopterin was also determined a potent inhibitor of mammalian xanthine oxidase. The inhibition constant calculated was 3.0 X 10(-5) M.     

Biochemical characterization of some pyrazolopyrimidine-based inhibitors of xanthine oxidase

Inhibition of xanthine oxidase-catalyzed conversion of xanthine to uric acid by various pyrazolopyrimidine-based inhibitors (allopurinol derivatives) was evaluated and compared with the standard inhibitor allopurinol. Three compounds out of the seven compounds used in the study were found to be reasonably good inhibitors of xanthine oxidase (XO). 4-Amino-6-mercaptopyrazolo-3,4-d-pyrimidine was found to be the most potent inhibitor of XO (IC₅₀=0.600±0.009 µM). 4-Mercapto-1H-pyrazolo-3,4-d-pyrimidine (IC₅₀=1.326±0.013 µM) and 4-amino-6-hydroxypyrazolo-3,4-d-pyrimidine (IC₅₀=1.564±0.065 µM) also showed inhibitory activity comparable to that of allopurinol (IC₅₀ = 0.776 ± 0.012 µM). All three compounds showed competitive type of inhibition with comparable K_i values. Induction of the electron transfer reaction catalyzed by XO in the presence of these compounds monitored as reduction of 2,6-dichlorophe nolindophenol (DCPIP) revealed that electron transfer by 4-amino-6-mercaptopyrazolo-3,4-d-pyrimidine is comparable to that obtained by allopurinol or xanthine. However, 4-mercapto-1H-pyrazolo-3,4-d-pyrimidine and 4-amino-6-hydroxypyrazolo-3,4-d-pyrimidine did not show DCPIP reduction. On the other hand, enzymatic reduction of cytochrome c in the presence of the three compounds was found to be insignificant and much less in comparison to allopurinol and xanthine. Therefore, both 4-amino-6-hydroxypyrazolo-3,4-d-pyrimidine and 4-mercapto-1H-pyrazolo-3,4-d-pyrimidine displayed the inhibitory property and also did not produce XO-mediated reactive oxygen species (ROS). Since 4-mercapto-1H-pyrazolo-3,4-d-pyrimidine was found to have some toxicity, the effect of 4-amino-6-hydroxypyrazolo-3,4-d-pyrimidine on the enzymatic formation of uric acid and ROS was investigated and it was found that this compound inhibited enzymatic generation of both uric acid and ROS. It can be noted that the standard inhibitor, allopurinol, inhibits uric acid formation but produces ROS.