RESUMO
BACKGROUND AND PURPOSE: In 2016, one person died and four others had mild-to-severe neurological symptoms during a phase I trial of the fatty acid amide hydrolase (FAAH) inhibitor BIA 10-2474. EXPERIMENTAL APPROACH: Pharmacodynamic and pharmacokinetic studies were performed with BIA 10-2474, PF-04457845 and JNJ-42165279 using mice, rats and human FAAH expressed in COS cells. Selectivity was evaluated by activity-based protein profiling (APBB) in rats. BIA 10-2474 effect in stroke-prone spontaneously hypertensive rats (SHRSP) was investigated. KEY RESULTS: BIA 10-2474 was 10-fold less potent than PF-04457845 in inhibiting human FAAH in situ but inhibited mouse brain and liver FAAH with ED50 values of 13.5 and 6.2 µg·kg-1 , respectively. Plasma and brain BIA 10-2474 levels were consistent with in situ potency and neither BIA 10-2474 nor its metabolites accumulated following repeat administration. FAAH and α/ß-hydrolase domain containing 6 were the primary targets of BIA 10-2474 and, at higher exposure levels, ABHD11, PNPLA6, PLA2G15, PLA2G6 and androgen-induced protein 1. At 100 mg·kg-1 for 28 days, the level of several lipid species containing arachidonic acid increased. Daily treatment of SHRSP with BIA 10-2474 did not affect mortality rate or increased the incidence of haemorrhage or oedema in surviving animals. CONCLUSIONS AND IMPLICATIONS: BIA 10-2474 potently inhibits FAAH in vivo, similarly to PF-04457845 and interacts with a number of lipid processing enzymes, some previously identified in human cells as off-targets particularly at high levels of exposure. These interactions occurred at doses used in toxicology studies, but the implication of these off-targets in the clinical trial accident remains unclear.
Assuntos
Amidoidrolases , Piridinas , Animais , Óxidos N-Cíclicos , Endocanabinoides , Inibidores Enzimáticos/farmacologia , Fosfolipases A2 do Grupo VI , Camundongos , Piridinas/farmacologia , RatosRESUMO
BACKGROUND & AIMS: Hyperammonemia is a frequent side-effect of valproic acid (VPA) therapy, which points to an imbalance between ammoniagenesis and ammonia disposal via the urea cycle. The impairment of this liver-specific metabolic pathway induced either by primary genetic defects or by secondary causes, namely associated with drugs administration, may result in accumulation of ammonia. To elucidate the mechanisms which underlie VPA-induced hyperammonemia, the aim of this study was to evaluate the effect of both VPA and its reactive intermediate, valproyl-CoA (VP-CoA), on the synthesis of N-acetylglutamate (NAG), a prime metabolite activator of the urea cycle. METHODS: The amount of NAG in livers of rats treated with VPA was quantified by HPLC-MS/MS. The NAG synthase (NAGS) activity was evaluated in vitro in rat liver mitochondria, and the effect of both VPA and VP-CoA was characterized. RESULTS: The present results clearly show that VP-CoA is a stronger inhibitor of NAGS activity in vitro than the parent drug VPA. The hepatic levels of NAG were significantly reduced in VPA-treated rats as compared with control tissues. CONCLUSIONS: These data strongly suggest that the hyperammonemia observed in patients under VPA treatment may result from a direct inhibition of the NAGS activity by VP-CoA. The subsequent reduced availability of NAG will impair the flux through the urea cycle and compromise the major role of this pathway in ammonia detoxification.
Assuntos
Acil Coenzima A/metabolismo , Aminoácido N-Acetiltransferase/antagonistas & inibidores , Hiperamonemia/induzido quimicamente , Hiperamonemia/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Ácido Valproico/toxicidade , Acil Coenzima A/toxicidade , Amônia/sangue , Animais , Anticonvulsivantes/toxicidade , Citrulina/biossíntese , Modelos Animais de Doenças , Glutamatos/metabolismo , Glutamina/sangue , Humanos , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Ratos , Ratos Wistar , Ureia/sangue , Ureia/metabolismoRESUMO
BACKGROUND/AIMS: Carnitine palmitoyl-transferase I (CPT I) catalyses the synthesis of long-chain (LC)-acylcarnitines from LC-acyl-CoA esters. It is the rate-limiting enzyme of mitochondrial fatty acid beta-oxidation (FAO) pathway and its activity is regulated by malonyl-CoA. The antiepileptic drug valproic acid (VPA) is a branched chain fatty acid that is activated to the respective CoA ester in the intra- and extra-mitochondrial compartments. This drug has been associated with a clear inhibition of mitochondrial FAO, which motivated our study on its potential effect on hepatic CPT I. METHODS: To investigate the effect of valproyl-CoA (VP-CoA) on CPT I, we performed in vitro studies using control human fibroblasts and rat CPT IA expressed in Saccharomyces cerevisiae. In addition to the wild-type enzyme, two mutant rCPT IAs were studied, one of which showing increased sensitivity towards malonyl-CoA (S24A/Q30A), whereas the other one is insensitive to malonyl-CoA (E3A). RESULTS: We demonstrate that VP-CoA inhibits the CPT I activity in control fibroblasts. Similar results were obtained using rCPT IA WT and S24A/Q30A. Importantly, VP-CoA also inhibited the activity of the rCPT IA E3A. We show that VP-CoA inhibits CPT IA competitively with respect to palmitoyl-CoA, and non-competitively to carnitine. Evidence is provided that VP-CoA interferes at the catalytic domain of CPT IA affecting the sensitivity for malonyl-CoA. CONCLUSIONS: The interference of VP-CoA with CPT IA, a pivotal enzyme in mitochondrial fatty acid beta-oxidation, may be a crucial mechanism in the drug-induced hepatotoxicity and the weight gain frequently observed in patients under VPA therapy.
Assuntos
Acil Coenzima A/farmacologia , Anticonvulsivantes/toxicidade , Carnitina O-Palmitoiltransferase/antagonistas & inibidores , Fígado Gorduroso/induzido quimicamente , Ácido Valproico/toxicidade , Animais , Carnitina O-Palmitoiltransferase/genética , Carnitina O-Palmitoiltransferase/metabolismo , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Ácidos Graxos/metabolismo , Fígado Gorduroso/enzimologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Humanos , Oxirredução , Plasmídeos , Ratos , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , TransfecçãoRESUMO
The pyruvate uptake rate in inverted submitochondrial vesicles prepared from rat liver was optimized and further characterized; the potential inhibitory effects of the anticonvulsive drug valproic acid or 2-n-propyl-pentanoic acid (VPA), Delta4-valproic acid or 2-n-propyl-4-pentenoic acid and the respective coenzyme A (CoA) conjugates were studied in the presence of a proton gradient. All tested VPA metabolites inhibited the pyruvate uptake, but the CoA esters were stronger inhibitors (40% and 60% inhibition, respectively, for valproyl-CoA and Delta4-valproyl-CoA, at 1mM). At the same concentration, the specific inhibitor 2-cyano-4-hydroxycinnamate decreased the pyruvate uptake rate by 70%. The reported inhibition of the mitochondrial pyruvate uptake may explain the significant impairment of the pyruvate-driven oxidative phosphorylation induced by VPA.
Assuntos
Anticonvulsivantes/farmacologia , Ácidos Graxos Monoinsaturados/farmacologia , Ácidos Graxos Insaturados/farmacologia , Membranas Mitocondriais/efeitos dos fármacos , Ácido Pirúvico/metabolismo , Ácido Valproico/farmacologia , Animais , Anticonvulsivantes/metabolismo , Transporte Biológico/efeitos dos fármacos , Coenzima A/farmacologia , Ácidos Graxos Monoinsaturados/metabolismo , Ácidos Graxos Insaturados/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/metabolismo , Membranas Mitocondriais/metabolismo , Fosforilação Oxidativa , Ratos , Ratos Wistar , Ácido Valproico/metabolismoRESUMO
The effect of the antiepileptic drug valproic acid (VPA) on mitochondrial oxidative phosphorylation (OXPHOS) was investigated in vitro. Two experimental approaches were used, in the presence of selected respiratory-chain substrates: (1) formation of ATP in digitonin permeabilized rat hepatocytes and (2) measurement of the rate of oxygen consumption by polarography in rat liver mitochondria. VPA (0.1-1.0 mM) was found to inhibit oxygen consumption and ATP synthesis under state 3 conditions with glutamate and 2-oxoglutarate as respiratory substrates. No inhibitory effect on OXPHOS was observed when succinate (plus rotenone) was used as substrate. We tested the hypothesis that dihydrolipoyl dehydrogenase (DLDH) might be a direct target of VPA, especially its acyl-CoA intermediates. Valproyl-CoA (0.5-1.0 mM) and valproyl-dephosphoCoA (0.5-1.0 mM) both inhibited the DLDH activity, acting apparently by different mechanisms. The decreased activity of DLDH induced by VPA metabolites may, at least in part, account for the impaired rate of oxygen consumption and ATP synthesis in mitochondria if 2-oxoglutarate or glutamate were used as respiratory substrates, thus limiting the flux of these substrates through the citric acid cycle.
Assuntos
Di-Hidrolipoamida Desidrogenase/antagonistas & inibidores , Di-Hidrolipoamida Desidrogenase/metabolismo , Ácidos Cetoglutáricos/farmacologia , Ácido Valproico/farmacologia , Trifosfato de Adenosina/metabolismo , Animais , Anticonvulsivantes/farmacologia , Hepatócitos/metabolismo , Ácidos Cetoglutáricos/metabolismo , Masculino , Mitocôndrias/metabolismo , Mitocôndrias Hepáticas/metabolismo , Fosforilação Oxidativa , Oxigênio/metabolismo , Consumo de Oxigênio , Ratos , Ratos WistarRESUMO
The hypothesis whether valproic acid (VPA) and its main microsomal metabolite, Delta(4)-valproic acid, can be activated to the respective CoA esters in the cell cytosol was investigated. The valproyl-CoA formation was measured in different subcellular fractions obtained by differential centrifugation of liver homogenates of rats treated with VPA (studies ex vivo) and digitonin fractionation of rat hepatocytes incubated with VPA and cofactors (studies in vitro). The results show that VPA activation may occur in the cytosol and is not restricted to the mitochondrial matrix as believed until now. Furthermore, the activation of Delta(4)-VPA is demonstrated in vitro. Valproyl-CoA and Delta(4)-valproyl-CoA were detected after in vitro incubations and the former also in the mitochondrial and cytosolic fractions obtained from liver cells of treated rats. The activation to valproyl-CoA was characterized in cytosolic fractions, optimized with respect to time and protein and the kinetic constants (K(m)(app)) were estimated for the reaction substrates. Other medium-chain fatty acids decreased the formation of valproyl-CoA suggesting a competition for both mitochondrial and extra-mitochondrial VPA activating enzymes. The present findings suggest additional mechanisms of mitochondrial dysfunction associated with VPA, and they may contribute to the further understanding of the toxic effects associated with this drug.