The induction and inhibition of UDP-glycosyltransferases in Haemonchus contortus and their role in the metabolism of albendazole.

Albendazole (ABZ) is an anthelmintic frequently used to treat haemonchosis, a common parasitosis of ruminants caused by the gastrointestinal nematode Haemonchus contortus. This parasite is able to protect itself against ABZ via the formation of inactive ABZ-glycosides. The present study was designed to deepen the knowledge about the role of UDP-glycosyltransferases (UGTs) in ABZ glycosylation in H. contortus. The induction effect of phenobarbital, a classical inducer of UGTs, as well as ABZ and ABZ-sulphoxide (ABZSO, the main active metabolite of ABZ) on UGTs expression and UGT activity toward ABZ was studied ex vivo in isolated adult nematodes. The effect of three potential UGT inhibitors (5-nitrouracil, 4,6-dihydroxy-5-nitropyrimidine and sulfinpyrazone) on ABZ glycosylation was tested. Pre-incubation of nematodes with ABZ and ABZSO led to increased expression of several UGTs as well as ABZ-glycosides formation in subsequent treatment. Phenobarbital also induced UGTs expression, but did not affect ABZ biotransformation. In the nematode's subcellular fraction, sulfinpyrazone inhibited UGT activity toward ABZ, although no effect of other inhibitors was observed. The inhibitory potential of sulfinpyrazone on the formation of ABZ-glycosides was also proved ex vivo in living nematodes. The obtained results confirmed the role of UGTs in ABZ biotransformation in H. contortus adults and revealed sulfinpyrazone as a potent inhibitor of ABZ glycosylation in this parasite. The possible use of sulfinpyrazone with ABZ in combination therapy merits further research.

Usefulness of Optimized Human Fecal Material in Simulating the Bacterial Degradation of Sulindac and Sulfinpyrazone in the Lower Intestine.

The first aim of this study was to evaluate the usefulness of optimized human fecal material in simulating sulforeductase activity in the lower intestine by assessing bacterial degradation of sulindac and sulfinpyrazone, two sulforeductase substrates. The second aim was to evaluate the usefulness of drug degradation half-life generated in simulated colonic bacteria (SCoB) in informing PBPK models. Degradation experiments of sulfinpyrazone and of sulindac in SCoB were performed under anaerobic conditions using recently described methods. For sulfinpyrazone, the abundance of clinical data allowed for construction of a physiologically based pharmacokinetic (PBPK) model and evaluation of luminal degradation clearance determined from SCoB data. For sulindac, the availability of sulindac sulfide and sulindac sulfone standards allowed for evaluating the formation of the main metabolite, sulindac sulfide, during the experiments in SCoB. Both model compounds degraded substantially in SCoB. The PBPK model was able to adequately capture exposure of sulfinpyrazone and its sulfide metabolite in healthy subjects, in ileostomy and/or colectomy subjects, and in healthy subjects pretreated with metoclopramide by implementing degradation half-lives in SCoB to calculate intrinsic colon clearance. Degradation rates of sulindac and formation rates of sulindac sulfide in SCoB were almost identical, in line with in vivo data suggesting the sulindac sulfide is the primary metabolite in the lower intestine. Experiments in SCoB were useful in simulating sulforeductase related bacterial degradation activity in the lower intestine. Degradation half-life calculated from experiments in SCoB is proven useful for informing a predictive PBPK model for sulfinpyrazone.

Characterization of the comparative drug binding to intra- (liver fatty acid binding protein) and extra- (human serum albumin) cellular proteins.

1. This study compared the extent, affinity, and kinetics of drug binding to human serum albumin (HSA) and liver fatty acid binding protein (LFABP) using ultrafiltration and surface plasmon resonance (SPR). 2. Binding of basic and neutral drugs to both HSA and LFABP was typically negligible. Binding of acidic drugs ranged from minor (fu > 0.8) to extensive (fu < 0.1). Of the compounds screened, the highest binding to both HSA and LFABP was observed for the acidic drugs torsemide and sulfinpyrazone, and for ß-estradiol (a polar, neutral compound). 3. The extent of binding of acidic drugs to HSA was up to 40% greater than binding to LFABP. SPR experiments demonstrated comparable kinetics and affinity for the binding of representative acidic drugs (naproxen, sulfinpyrazone, and torsemide) to HSA and LFABP. 4. Simulations based on in vitro kinetic constants derived from SPR experiments and a rapid equilibrium model were undertaken to examine the impact of binding characteristics on compartmental drug distribution. Simulations provided mechanistic confirmation that equilibration of intracellular unbound drug with the extracellular unbound drug is attained rapidly in the absence of active transport mechanisms for drugs bound moderately or extensively to HSA and LFABP.

Use of cryopreserved transiently transfected cells in high-throughput pregnane X receptor transactivation assay.

Cryopreserved, transiently transfected HepG2 cells were compared to freshly transfected HepG2 cells for use in a pregnane X receptor (PXR) transactivation assay. Assay performance was similar for both cell preparations; however, cryopreserved cells demonstrated less interassay variation. Validation with drugs of different PXR activation potencies and efficacies demonstrated an excellent correlation (r(2) > 0.95) between cryopreserved and fresh cells. Cryopreservation did not change the effect of known CYP3A4 inducers that have poor cell permeability, indicating that cryopreservation had little effect on membrane permeability. In addition, cryopreserved HepG2 cells did not exhibit enhanced susceptibility to cytotoxic compounds compared to transiently transfected control cells. The use of cryopreserved cells enables this assay to run with enhanced efficiency.

Sulfinpyrazone C-glucuronidation is catalyzed selectively by human UDP-glucuronosyltransferase 1A9.

The uricosuric agent sulfinpyrazone (SFZ) is metabolized via C-glucuronidation, an uncommon metabolic pathway, in humans. The present study aimed to characterize SFZ glucuronidation by human liver microsomes (HLMs) and identify the hepatic forms of UDP-glucuronosyltransferase responsible for this pathway. Incubations of SFZ with HLMs formed a single glucuronide that was resistant to beta-glucuronidase and acid hydrolysis, consistent with formation of a C-glucuronide. Mass spectral analysis confirmed the identity of the metabolite as SFZ glucuronide (sulfinpyrazone beta-D-glucuronide; SFZG). SFZ C-glucuronidation by HLMs exhibited Michaelis-Menten kinetics, with mean (+/- S.D.) Km and Vmax values of 51 +/- 21 microM and 2.6 +/- 0.6 pmol/min . mg, respectively. Fifteen recombinant human UDP-glucuronosyltransferases (UGTs), expressed in HEK293 cells, were screened for their capacity to catalyze SFZ C-glucuronidation. Of the hepatically expressed enzymes, only UGT1A9 formed SFZG. UGTs 1A7 and 1A10, which are expressed in the gastrointestinal tract, also metabolized SFZ, but rates of metabolism were low compared with UGT1A9. SFZ glucuronidation by UGT1A9 exhibited "weak" negative cooperative kinetics, which was modeled by the Hill equation (S50 16 microM). The data indicate that UGT1A9 is the enzyme responsible for hepatic SFZ C-glucuronidation and that SFZ may be used as a substrate "probe" for UGT1A9 activity in HLMs.

Metabolism of sulfinpyrazone sulfide and sulfinpyrazone by human liver microsomes and cDNA-expressed cytochrome P450s.

Human liver microsomes catalyze the oxidation of sulfinpyrazone sulfide (SPZS) to a variable mixture of sulfinpyrazone (SPZ) enantiomers and two minor phenolic metabolites. In one, the thiophenyl ring is hydroxylated, whereas in the second an N-phenyl ring is hydroxylated. SPZ is further oxidized to sulfinpyrazone sulfone (SPZO) and a minor polar metabolite that also has an N-phenyl ring hydroxylated. Determination of the metabolism of SPZ and SPZS under modified incubation conditions of prior heat treatment, higher pH, and the presence of detergent indicated that the formation of SPZ was cytochrome P450 (P450)- but not flavin monooxygenase-dependent. Specific P450 inhibitors (sulfaphenazole, quinidine sulfate, coumarin, diethyldithiocarbamic acid, troleandomycin, and furafylline) and specific cDNA-expressed P450s were used to identify the major isoforms responsible for the oxidation of SPZS to SPZ and SPZ to SPZO. Both P450 2C9 and P450 3A4 were responsible for the oxidation of SPZS to SPZ, whereas P450 3A4 alone catalyzed the further oxidation of SPZ to SPZO. SPZS was found to be metabolized by P450 2C9 to SPZ with a high degree of enantiomeric selectivity (9:1) and a K(m) comparable with its previously determined K(i) for inhibition of the P450 2C9-dependent 7-hydroxylation of (S)-warfarin (WARF). In contrast, the P450 3A4-catalyzed oxidation of SPZS to SPZ proceeded with the same enantioselectivity but to a much lesser degree (58:42). These results provide evidence that the metabolism of both (S)-WARF and SPZS is mediated by a common enzyme, P450 2C9, which is central to understanding the WARF-SPZ interaction and SPZS-mediated drug interactions in general.

Vinblastine and sulfinpyrazone export by the multidrug resistance protein MRP2 is associated with glutathione export.

The multidrug resistance proteins MRP1 and MRP2 are members of the same subfamily of ATP-binding cassette transporters. Besides organic molecules conjugated to negatively charged ligands, these proteins also transport cytotoxic drugs for which no negatively charged conjugates are known to exist. In polarized MDCKII cells, MRP1 routes to the lateral plasma membrane, and MRP2 to the apical plasma membrane. In these cells MRP1 transports daunorubicin, and MRP2 vinblastine; both transporters export reduced glutathione (GSH) into the medium. We demonstrate that glutathione transport in MDCKII-MRP1 cells is inhibited by the inhibitors of organic anion transporters sulfinpyrazone, indomethacin, probenecid and benzbromarone. In MDCKII-MRP2 cells, GSH export is stimulated by low concentrations of sulfinpyrazone or indomethacin, whereas export is inhibited down to control levels at high concentrations. We find that unmodified sulfinpyrazone is a substrate for MRP2, also at concentrations where GSH export is inhibited. We also show that GSH export in MDCKII-MRP2 cells increases in the presence of vinblastine, and that the stoichiometry between drug and GSH exported is between two and three. Our data indicate that transport of sulfinpyrazone and vinblastine is associated with GSH export. However, at high sulfinpyrazone concentrations this compound is transported without GSH. Models of MRP action are discussed that could explain these results.

Inhibition of (S)-warfarin metabolism by sulfinpyrazone and its metabolites.

Sulfinpyrazone markedly potentiates the anticoagulant effect of warfarin. The increased clotting time is accompanied by a marked decrease in the clearance of (S)-warfarin by virtue of a decrease in the P4502C9-catalyzed formation clearance to its major and inactive metabolite (S)-7-hydroxywarfarin. These data suggested that the mechanism of the drug interaction might be mediated through the inhibition of the catalytic activity of P4502C9 by sulfinpyrazone. However, initial human liver microsomal studies indicated that the in vitro Ki, for inhibition of (S)-7-hydroxywarfarin formation by sulfinpyrazone is at least 25-fold higher than the therapeutic concentration of sulfinpyrazone in vivo. This result implied that other inhibitors probably contribute to the interaction. Kinetic studies conducted on sulfinpyrazone and two major metabolites, sulfinpyrazone sulfide and sulfinpyrazone sulfone, in microsomes prepared from three human livers give mean Ki's of 230 microM, 17 microM, and 73 microM respectively. Because sulfinpyrazone and its sulfide metabolite attain comparable plasma concentrations during the course of therapy, our inhibition results suggest that the sulfide metabolite is likely to be the primary species responsible for the inhibition of P4502C9-catalyzed formation of (S)-7-hydroxywarfarin and the decrease in (S)-warfarin clearance in vivo.

Sulphoxide reduction by rat and rabbit tissues in vitro.

The reduction of sulindac, sulphinpyrazone and diphenyl sulphoxide to their thioether analogues has been studied in vitro using rat and rabbit tissues. Sulindac reduction was about 10-fold higher in homogenates of rat kidney and liver than in other tissues although the tissue differences decreased when dithiothreitol was used as a co-factor. The greatest sulindac reducing activity in rat liver was in the cytosolic fraction whereas reoxidation of the thioether back to the sulphoxide was largely in the microsomal fraction. Studies using NADPH/NADH, acetaldehyde and dithiothreitol as cofactors showed that aldehyde oxidase was the main sulindac reducing system in rat and rabbit liver cytosols but not in renal cytosols where reduction was probably linked to the thioredoxin system, as reported previously. Menadione and hydralazine caused essentially complete inhibition of sulindac reduction by hepatic but not renal cytosol and the inhibition was dependent on preincubation of the enzyme with the inhibitor, which is indicative of aldehyde oxidase activity. Little reduction of sulphinpyrazone or diphenyl sulphoxide was detected with rat or rabbit kidney or renal cytosols, although increased reduction was detected when acetaldehyde was added as a cofactor to rabbit and rat liver cytosols. The data indicate that different enzyme systems are responsible for sulphoxide reduction in the liver and kidney.

Sulphoxide reduction by rat intestinal flora and by Escherichia coli in vitro.

The caecal microflora from female rats show a greater ability to reduce the sulphoxide group of sulindac than either the liver or kidneys. Studies on sulphoxide reduction by Escherichia coli showed that NADH, NADPH and dithiothreitol (DTT), but not acetaldehyde could act as cofactors. The cytosolic fraction was responsible for about 90%, 80% and 60% of the total reducing activity with sulindac, diphenyl sulphoxide and sulphinpyrazone, respectively. The main NADPH linked activity in the E. coli cytosol was dependent on thioredoxin, since the activity was essentially abolished by passing through a G50 column or by the addition of anti-thioredoxin anti-serum. Partial purification and separation of sulphoxide reducing activity by DEAE-cellulose chromatography separated two main protein bands, each of which possessed sulindac reducing activity. The importance of thioredoxin for much of the NADPH dependent activity was confirmed but the eluate fractions also showed the presence of other activities with NADH, NADPH and DTT that were independent of thioredoxin. Incubation of the DEAE-cellulose eluate fractions with flosequinan and sulphinpyrazone showed that the reducing activity in the two main protein peaks showed different substrate specificities and that there were multiple sulphoxide reductase systems present in E. coli cytosol.

The reduction of sulphinpyrazone and sulindac by intestinal bacteria.

1. Incubation of human or rabbit faeces with sulphinpyrazone gave greater reduction under anaerobic than under aerobic conditions. Reduction of sulindac by human faeces was more extensive than that of sulphinpyrazone. 2. Growth of mixed cultures of intestinal bacteria in nutrient media containing antibiotics produced a marked inhibition in their ability to reduce sulphinpyrazone. Sulphide formation was inhibited by metronidazole and lincomycin for human faeces and by tetracycline for rabbit faeces/caecal contents. 3. The formation of the sulphides of sulindac and sulphinpyrazone ex vivo was decreased in faeces from patients treated with metronidazole. Metronidazole, but not tetracycline, decreased the extent of reduction of sulphinpyrazone by rabbits in vivo. No reduction of either substrate occurred on incubation with ileostomy effluent. These data indicate that anaerobic intestinal bacteria are important in the reduction of these sulphoxide-containing drugs. 4. However, when incubated anaerobically with over 200 strains of bacteria isolated from human faeces, sulphinpyrazone was reduced by most of the aerobic but not the anaerobic organisms. Sulindac was reduced more extensively by the same aerobes and by some anaerobes. 5. The discrepancy between the apparent importance of anaerobes in vivo and in vitro may be due to their very large number present in the hind gut and to the production of an anaerobic environment suitable for the enzymic activity of other organisms, such as aerobes or facultative anaerobes.

Effects of ischaemic heart disease, Crohn's disease and antimicrobial therapy on the pharmacokinetics of sulphinpyrazone.

The renewed interest in sulphinpyrazone in recent years has arisen from its potential to inhibit platelet aggregation. In vivo much of the activity is probably due to the thioether or sulphide metabolite which has a greater potency and a longer half-life than the parent compound. The sulphide metabolite is formed exclusively by the gut microflora in man. The pharmacokinetics of sulphinpyrazone (200 mg orally) have been studied, with particular attention to the formation of the sulphide metabolite, in groups of patients who might be expected to show abnormal formation of this active metabolite due to altered delivery of the drug to the lower gut or altered gut flora. Five patients studied 1 month after a myocardial infarction did not differ markedly from young, normal volunteers with respect to either sulphinpyrazone or its metabolite. Crohn's disease in the quiescent phase did not significantly alter the pharmacokinetics or metabolism of the drug, but 1 patient who had undergone a hemicolectomy formed negligible concentrations of the active metabolite. Antimicrobial therapy produced highly variable results with almost complete suppression of sulphide formation in some subjects but no apparent effect in others.

Prostaglandinas, ácido acetilsalicílico, dipiridamol e sulfinpirazona na doença coronária / Prostaglandins acetylsalicylic acid, dipyridamol and sulfinpyrazone in coronary disease

As prostaglandinas säo substâncias existentes em todos os tecidos do organismo. Säo atuantes à pequena distância do sítio de produçäo. Têm efeitos biológicos marcantes, sobretudo na circulaçäo coronária, com grandes implicaçöes na doença arterial coronária. Drogas como ácido acetilsalicílico, dipiridamol e sulfinpirazona exercem seus benefícios porque têm efeitos farmacológicos nas prostaglandinas

Renal clearance of sulphinpyrazone in man.

Six healthy young volunteers received a single dose of sulphinpyrazone 200 mg p.o. Plasma concentration and urinary excretion rate curves showed large intersubject variation for sulphinpyrazone and its metabolites. The sulphide metabolite could only be detected in plasma and not before 3-7 h after ingestion. The total recovery in urine of all compounds varied from 30-56% of the dose. In two subjects the mean residence time of sulphinpyrazone was twice as long as in the other subjects (10.4 h compared with 4.6 h), but the area under the plasma concentration-time curve was comparable to that in the others (mean: 3.0 mg X ml-1 X min), indicating that drug absorption was quantitatively similar but delayed. The renal clearance of sulphinpyrazone varied from 14-40 ml X min-1 (mean: 28 ml X min-1). In view of the very high plasma protein binding of sulphinpyrazone, active tubular secretion is the predominant mechanism in its renal clearance. The same holds for the sulphone metabolite, which has a mean renal clearance of 24 ml X min-1, and even more for the p-hydroxysulphinpyrazone metabolite, which has a renal clearance of 118 ml X min-1. No unambiguous evidence was found in favour of concentration-dependent renal clearance of sulphinpyrazone or its metabolites over the concentration range studied. The renal clearance, especially of sulphinpyrazone, appeared to be dependent on urine pH and not on urine flow rate.

Dose-related cardiac electrophysiologic effects of sulfinpyrazone.

To test the hypothesis that sulfinpyrazone exerts cardiac electrophysiologic effects, the drug was intravenously injected into 20 subjects during invasive electrophysiologic testing. Sulfinpyrazone was given intravenously as a bolus and by infusion to achieve two different and stable serum levels. The 20 subjects who were treated with drug were assigned to either a low- (N = 10) or high- (N = 10) dose regimen. The resultant four serum levels of sulfinpyrazone were 102 +/- 45, 199 +/- 75, 278 +/- 57, and 352 +/- 77 X 10(-3) mumol (means +/- SD). Electrophysiologic measurements were made during a baseline electrophysiologic study and at each of the sulfinpyrazone levels and at equivalent times in an untreated control group (N = 11). Two electrophysiologic measurements differed when measured at the highest level of sulfinpyrazone and in control subjects: increased HV interval in sinus rhythm and shortened atrial functional refractory period in sinus rhythm (only for those values below the median). Serum levels of sulfinpyrazone correlated with increased sinoatrial conduction time (only for those values above the median; r = 0.64) and with shortened atrial functional refractory periods (r = 0.37). The latter was stronger (r = 0.67) when only values below the median were included in analysis. Shortening of atrial functional refractory period correlated with serum sulfinpyrazone levels during atrial pacing at fixed cycle lengths of 600 and 500 msec. Serum levels of sulfinpyrazone did not correlate with changes in HV interval. HV intervals did not increase in subjects receiving sulfinpyrazone during atrial pacing and, therefore, the effect on HV interval in sinus rhythm is felt to be spurious.(ABSTRACT TRUNCATED AT 250 WORDS)

Aspirin and other platelet-aggregation inhibiting drugs.

The biochemistry of platelets is surprisingly complex, and offers the opportunity for numerous platelet-aggregation inhibiting ("antiplatelet") drugs to interfere with different aspects of their metabolism and function. Thus, aspirin inhibits platelet aggregation by irreversibly inactivating cyclo-oxygenase, a key enzyme in platelet prostaglandin metabolism, while the other nonsteroidal anti-inflammatory drugs and sulphinpyrazone cause reversible and dose-dependent inhibition of the same enzyme. Dipyridamole can inhibit both platelet adhesion and aggregation by raising the platelet cyclic AMP level through phosphodiesterase inhibition. The use of aspirin, sulphinpyrazone, and dipyridamole as antithrombotic agents has now been extensively evaluated. In general, treatment with these drugs has been more likely to prevent arterial than venous thromboembolism, and aspirin or the combination of aspirin and dipyridamole has been more effective in this respect than has sulphinpyrazone. Recent evidence strongly suggests that aspirin reduces the risk of non-fatal myocardial infarction in patients with unstable angina, and that the administration of aspirin in combination with dipyridamole significantly improves graft patency after aortocoronary bypass. Aspirin also appears to reduce the likelihood of stroke or death in men with transient cerebral ischaemic attacks.

Pharmacokinetics of sulphinpyrazone and its major metabolites after a single dose and during chronic treatment.

The pharmacokinetics of sulphinpyrazone and its major metabolites (sulfide, sulfone, p-hydroxysulfone and p-hydroxy-sulphinpyrazone) were investigated in 9 volunteers after a single oral dose as well as after chronic treatment for 23 days. Chronic administration of sulphinpyrazone, in comparison with a single oral dose, led to significant changes in plasma AUC (115.86 to 42.90 mg/l . h), in renal clearance (1.06 to 1.80 l/h), in hepatic intrinsic clearance (319.0 to 598.0 l/h), and in the unbound fraction in plasma 1.15 to 1.69%) and in tissue (2.73 to 1.31%). The volume of distribution changed from 20.24 to 52.04 l. The steady state concentrations predicted from the single dose were significantly higher than the values found after chronic treatment. The results suggest that sulphinpyrazone induces its own metabolism. The metabolism of the sulfone, p-hydroxysulfone and the p-hydroxy-sulphinpyrazone to further degradation products was also induced. Chronic treatment with sulphinpyrazone reduced the plasma AUC of the sulfide and caused a decrease in its elimination half-life (20.9 to 14.3 h). Since considerable amounts of the sulfide are formed in the G.I. tract, it is suggested that besides the induction of metabolism, bacteria which reduce sulphinpyrazone to the sulfide may also be responsible for the observed pharmacokinetic changes.

The site of reduction of sulphinpyrazone in the rabbit.

Comparison of oral and i.v. administration of sulphinpyrazone (10 mg/kg) to rabbits showed that the oral route was associated with an incomplete bioavailability and a six-fold greater formation of the active sulphide metabolite. The bile was an important route of elimination of unchanged sulphinpyrazone in rabbits (18% of an i.v. dose in four hours). Only small amounts of the sulphide appeared in the bile after i.v. administration. Pretreatment with oral antibiotics decreased the area under the plasma concentration-time curve (AUC) for the sulphide but increased that of the parent drug. Excretion of the p-hydroxysulphide metabolite in urine was decreased 30-fold by antibiotic treatment. The contents of the caecum showed the greatest capacity for sulphinpyrazone reduction in vitro. The liver possessed a slight ability to reduce sulphinpyrazone in vitro under anaerobic, but not aerobic, conditions. The gut bacteria are the main site of reduction of sulphinpyrazone to the active sulphide metabolite in the rabbit. These findings contrast with those obtained for sulindac which was reduced extensively under both aerobic and anaerobic conditions by rabbit-liver soluble fraction in vitro. The sulphide metabolites of both sulphinpyrazone and sulindac were oxidized to the parent drug by rabbit-liver microsomes.