Synthesis of novel benzbromarone derivatives designed to avoid metabolic activation.

We synthesized six novel BBR derivatives that were designed to avoid metabolic activation via ipso-substitution and evaluated for their degree of toxicity and hURAT1 inhibition. It was found that all of the derivatives demonstrate lower cytotoxicity in mouse hepatocytes and lower levels of metabolic activation than BBR, while maintaining their inhibitory activity toward the uric acid transporter. We propose that these derivatives could serve as effective uricosuric agents that have much better safety profiles than BBR.

Metabolic Epoxidation Is a Critical Step for the Development of Benzbromarone-Induced Hepatotoxicity.

Benzbromarone (BBR) is effective in the treatment of gout; however, clinical findings have shown it can also cause fatal hepatic failure. Our early studies demonstrated that CYP3A catalyzed the biotransformation of BBR to epoxide intermediate(s) that reacted with sulfur nucleophiles of protein to form protein covalent binding both in vitro and in vivo. The present study attempted to define the correlation between metabolic epoxidation and hepatotoxicity of BBR by manipulating the structure of BBR. We rationally designed and synthesized three halogenated BBR derivatives, fluorinated BBR (6-F-BBR), chlorinated BBR (6-Cl-BBR), and brominated BBR (6-Br-BBR), to decrease the potential for cytochrome P450-mediated metabolic activation. Both in vitro and in vivo uricosuric activity assays showed that 6-F-BBR achieved favorable uricosuric effect, while 6-Cl-BBR and 6-Br-BBR showed weak uricosuric efficacy. Additionally, 6-F-BBR elicited much lower hepatotoxicity in mice. Fluorination of BBR offered advantage to metabolic stability in liver microsomes, almost completely blocked the formation of epoxide metabolite(s) and protein covalent binding, and attenuated hepatic and plasma glutathione depletion. Moreover, the structural manipulation did not alter the efficacy of BBR. This work provided solid evidence that the formation of the epoxide(s) is a key step in the development of BBR-induced hepatotoxicity.

Inactivation of CYP3A4 by Benzbromarone in Human Liver Microsomes.

BACKGROUND: Benzbromarone is a uricosuric drug in current clinical use that can cause serious hepatotoxicity. Chemically reactive and/or cytotoxic metabolites of benzbromarone have been identified; however there is a lack of available information on their role in benzbromarone hepatotoxicity. The reactive metabolites of some hepatotoxic drugs are known to covalently bind, or alternatively are targeted, to specific cytochrome P450 (P450) enzymes, a process that is often described as mechanism-based inhibition. OBJECTIVE: We examined whether benzbromarone causes a mechanism-based inhibition of human P450 enzymes. METHOD: Microsomes from human livers were preincubated with benzbromarone and NADPH, followed by evaluation of CYP2C9 and CYP3A4 activities. RESULTS: Benzbromarone metabolism resulted in inhibition of CYP3A4 but not CYP2C9 in a time-dependent manner. Confirmation of pseudo-first order kinetics of inhibition, a requirement for NADPH, and a lack of protection by scavengers suggested that benzbromarone is a mechanism-based CYP3A4 inhibitor. CONCLUSION: Modification of the P450 enzyme by the reactive metabolite is a common trait of drugs that induce idiosyncratic hepatotoxicity, and might provide a speculative, mechanistic model for the rare occurrences of this type of drug toxicity.

Probenecid potentiates MPTP/MPP+ toxicity by interference with cellular energy metabolism.

The uricosuric agent probenecid is co-administered with the dopaminergic neurotoxin MPTP to produce a chronic mouse model of Parkinson's disease. It has been proposed that probenecid serves to elevate concentrations of MPTP in the brain by reducing renal elimination of the toxin. However, this mechanism has never been formally demonstrated to date and is questioned by our previous data showing that intracerebral concentrations of MPP(+), the active metabolite of MPTP, are not modified by co-injection of probenecid. In this study, we investigated the potentiating effects of probenecid in vivo and in vitro arguing against the possibility of altered metabolism or impaired renal elimination of MPTP. We find that probenecid (i) is toxic in itself to several neuronal populations apart from dopaminergic neurons, and (ii) that it also potentiates the effects of other mitochondrial complex I inhibitors such as rotenone. On a mechanistic level, we show that probenecid is able to lower intracellular ATP concentrations and that its toxic action on neuronal cells can be reversed by extracellular ATP. Probenecid can potentiate the effect of mitochondrial toxins due to its impact on ATP metabolism and could therefore be useful to model atypical parkinsonian syndromes.

[Drug-drug interactions and nephrotoxicity]. / Arzneimittelinteraktionen und Nephrotoxizität.

Nephrotoxicity is a common and often clinically relevant adverse drug reaction. Mechanisms include vascular, tubulo-toxic, tubulo-obstructive, and immunological effects. Drug-drug interactions may occur at a pharmacokinetic or pharmacodynamic level. Such interactions can both increase (cisplatin and aminoglycoside) but also protect from nephrotoxicity (cidofovir and probenecid).Important measures for preventing nephrotoxicity are (1) consideration of potential pharmacokinetic and pharmacodynamic interactions when prescribing a drug, (2) prescription of nephrotoxic drugs for the shortest possible period, (3) detection of high-risk patients, and (4) consideration of hydration and prophylactic comedication.

Sequential metabolism and bioactivation of the hepatotoxin benzbromarone: formation of glutathione adducts from a catechol intermediate.

Benzbromarone (BBR) is a uricosuric agent that has been used as a treatment for chronic gout. Although never approved in the United States, BBR was recently withdrawn from European markets due to several clinical cases linking the drug to an idiosyncratic hepatotoxicity that is sometimes fatal. We report here a possible mechanism of toxicity that involves the bioactivation of BBR through sequential hydroxylation of the benzofuran ring to a catechol, which can then be further oxidized to a reactive quinone intermediate capable of adducting protein. NADPH-supplemented human liver microsomes generated a single metabolite that was identified as 6-OH BBR by comparison with the synthesized chemical standard. CYP2C9 was the major recombinant enzyme capable of catalyzing the formation of 6-OH BBR, although CYP2C19 also showed a lower degree of activity. Further oxidation of either 6-OH BBR or 5-OH BBR by human liver microsomes resulted in the formation of a dihydroxy metabolite with identical chromatographic and mass spectral properties. This product of sequential metabolism of BBR was identified as the catechol, 5,6-dihydroxybenzbromarone. Incubation of the catechol with liver microsomes, in the presence of glutathione, resulted in the formation of two glutathione adducts that could derive from a single ortho-quinone intermediate. Isoform profiling with recombinant human P450s suggested that CYP2C9 is primarily responsible for the formation of this reactive quinone intermediate.

[Life-threatening adverse effects of pharmacologic antihyperuricemic therapy]. / Lebensbedrohliche Nebenwirkungen der Gichtbehandlung.

Minor hypersensitivity reactions to allopurinol presenting as skin rash occur in approximately 2% of patients. A more severe, albeit rare, hypersensitivity reaction with fever, eosinophilia, dermatitis, renal failure, vasculitis and hepatic dysfunction carries a mortality of up to 20%. The incidence of this severe reaction can probably be reduced by adjusting the dose of allopurinol in patients with impaired renal function. Azathioprine and mercaptopurine are metabolised by xanthine oxidase, the enzyme that is inhibited by allopurinol. Concomitant administration can result in life-threatening neutropenia unless the dose of allopurinol is reduced by approximately 75%. The uricosuric agent benzbromarone has recently been withdrawn from the market because of several cases of fulminant hepatic failure with subsequent death of the patient or liver transplantation.

Effects of endotoxin-induced fever and probenecid on disposition of enrofloxacin and its metabolite ciprofloxacin after intravascular administration of enrofloxacin in goats.

Pharmacokinetics of enrofloxacin and its active metabolite ciprofloxacin were investigated in normal, febrile and probenecid-treated adult goats after single intravenous (i.v.) administration of enrofloxacin (5 mg/kg). Pharmacokinetic evaluation of the plasma concentration-time data of enrofloxacin and ciprofloxacin was performed using two- and one-compartment open models, respectively. Plasma enrofloxacin concentrations were significantly higher in febrile (0.75-7 h) and probenecid-treated (5-7 h) goats than in normal goats. The sum of enrofloxacin and ciprofloxacin concentrations in plasma > or =0.1 microg /mL was maintained up to 7 and 8 h in normal and febrile or probenecid-treated goats, respectively. The t1/2beta, AUC, MRT and ClB of enrofloxacin in normal animals were determined to be 1.14 h, 6.71 microg .h/mL, 1.5 h and 807 mL/h/kg, respectively. The fraction of enrofloxacin metabolized to ciprofloxacin was 28.8%. The Cmax., t1/2beta, AUC and MRT of ciprofloxacin in normal goats were 0.45 microg /mL, 1.79 h, 1.84 microg .h/mL and 3.34 h, respectively. As compared with normal goats, the values of t1/2beta (1.83 h), AUC (11.68 microg ? h/mL) and MRT (2.13 h) of enrofloxacin were significantly higher, whereas its ClB (430 mL/h/kg) and metabolite conversion to ciprofloxacin (8.5%) were lower in febrile goats. The Cmax. (0.18 microg /mL) and AUC (0.99 microg .h/mL) of ciprofloxacin were significantly decreased, whereas its t1/2beta (2.75 h) and MRT (4.58 h) were prolonged in febrile than in normal goats. Concomitant administration of probenecid (40 mg/kg, i.v.) with enrofloxacin did not significantly alter any of the pharmacokinetic variables of either enrofloxacin or ciprofloxacin in goats.

Chemical structure and toxicity of diuretics in isolated hepatocytes.

The effects of several diuretics, including tienilic acid and indacrinone, on isolated rat heaptocytes were examined. Addition of tienilic acid and indacrinone at 1 mM to a suspension of freshly isolated cells caused dose-dependent loss of cell viability as judged by the LDH-latency test. Survey of 19 structurally related compounds revealed that the extent of cell injury and chemical structure were correlated, and an intense adverse effect was attributed to the 2-thienylcarbonyl moiety. Several other factors influencing cell viability are also disclosed. Further study revealed that tienilic acid and indacrinone were toxic to the primary culture of hepatocytes at a lower dose than that a freshly isolated hepatocytes. Thus, an isolated hepatocyte system can be used to select compounds displaying low hepatotoxicity, as for example is needed when screening diuretics.

Toxicity of uricosuric diuretics in rat hepatocyte culture.

Several aryloxyacetic acid diuretics have shown hepatotoxicity in humans, yet there continues to be interest in developing these compounds because of the uricosuric properties of some of them. This study was designed to test the utility of the hepatocyte monolayer culture as a model for studying these compounds. In addition, an attempt was made to define the structural components that are common to hepatotoxicity. Ticrynafen, indacrinone, ethacrynic acid and A-49816, an investigational compound, were found to be toxic in hepatocyte cultures; thus, with the exception of indacrinone, paralleling the experience in humans. The toxic compounds share a ketodichlorophenoxyacetic acid chemical structure. A-56234, an investigational uricosuric, was also found to be toxic in cultures but has not been demonstrated to be hepatotoxic in humans in limited clinical experience. It does not possess the ketodichlorophenoxyacetic acid structure proper but may be metabolized to a closely related structure. Furosemide, which does not have the ketodichlorophenoxyacetic acid structure, was not toxic in hepatocyte cultures and has not been hepatotoxic in humans. Thus, the structure common to the toxic compounds is ketodichlorophenoxyacetic acid or a closely related compound. The hepatocyte monolayer system appears to be a good model for demonstrating toxicity and, perhaps, for predicting toxicity of new compounds under development.