Valproic acid (VPA; 2-n-propylpentanoic acid) is widely used as a major drug in the treatment of epilepsy and in the control of several types of seizures. Being a simple fatty acid, VPA is a substrate for the fatty acid beta-oxidation (FAO) pathway, which takes place primarily in mitochondria. The toxicity of valproate has long been considered to be due primarily to its interference with mitochondrial beta-oxidation. The metabolism of the drug, its effects on enzymes of FAO and their cofactors such as CoA and/or carnitine will be reviewed. The cumulative consequences of VPA therapy in inborn errors of metabolism (IEMs) and the importance of recognizing an underlying IEM in cases of VPA-induced steatosis and acute liver toxicity are two different concepts that will be emphasized.

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Background and Purpose: In 2016, one person died and four others had mild-tosevere 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 ED 50 values of 13.5 and 6.2 μgÁkg −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/β-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 mgÁkg −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.Abbreviations: 2-AG, 2-arachidonoylglycerol; AA, arachidonic acid; ABHD6, α/β-hydrolase domain containing 6; ABPP, activity-based protein profiling; AEA, anandamide; AIG1, androgeninduced protein 1; BCRP, breast cancer resistance protein; CES, carboxyl esterase; CYP, cytochrome P450; DAG, diacylglycerol; FAAH, fatty acid amide hydrolase; LIPE, lipase E; MATE, multidrug and toxin extrusion; MDR, multidrug resistance; OAT, organic anion transporter; OCT, organic cation transporter; OEA, oleoylethanolamide; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PEA, palmitoylethanolamide; P-gp, P-glycoprotein; PLA2G15, PLA 2 Group 15; PLA2G6, PLA 2 Group 6; PNPLA6, patatin-like phospholipase domain containing 6; SHRSP, Spontaneously hypertensive rats stroke prone; TAMRA-FP, tetramethylrhodamine-fluorophosphonate.

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.

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), D 4 -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 D 4 -valproyl-CoA, at 1 mM). 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.

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In January 2016 one person died and others suffered adverse neurological effects during a Phase I clinical trial of the fatty acid amide hydrolase (FAAH) inhibitor BIA 10‐2474. We have compared the potency and selectivity of BIA 10‐2474 with two other mechanism‐based FAAH inhibitors PF‐04457845 and JNJ‐42165279 which appeared safe in human testing.In whole cells, BIA 10‐2474 inhibited FAAH with an IC50 of 74.4 and 18 nM after 1h and 24h incubation respectively. This compares with 26.2 and 41.5 nM for JNJ‐42165279 at 1 and 24h and 4.3 nm for PF‐04457845 at 1h. All three compounds were characterised as irreversible FAAH inhibitors. Interestingly, BIA 10‐2474 and JNJ‐42165279 were more potent FAAH inhibitors in whole cells as compared to a membrane preparation (4–6 fold more active) when tested after 1h incubation whereas PF‐04457845 was equipotent in the two models (4.3 and 2.0 nM respectively). Neither BIA 10‐2474 nor JNJ‐42165279 accumulated inside the cells.Activity based protein profiling in vitro identified only ABHD6 as an off‐target for BIA 10‐2474 with no activity against other rat brain serine hydrolase enzymes up to 100 mM. In contrast PF‐04457845 and JNJ‐42165279 both interacted with ABHD11. BIA 10‐2474 had no significant activity against 220 other human off‐targets in binding and enzyme studies up to 50 μM. In contrast, PF‐04457845 showed significant activity (>50% inhibition) at 37 sites at 50 μM, including cannabinoid receptors, serotonin receptors, and several ion channels.In vivo, oral administration of BIA 10‐2474 inhibited rat brain and liver FAAH with ED50s of 13.5 and 6.2 mg/kg PO respectively and increased brain levels of fatty acid amides over the same dose range. This compares with 4.3 and 2.0 mg/kg PO for PF‐04457845 and 12.5 and 3.7 mg/kg PO for JNJ‐42165279. Exposure levels of BIA 10‐2474 in plasma and brain were consistent with the potency identified in whole cells and there was no evidence for accumulation in the brain of either BIA 10‐2474 nor its main metabolites following repeated administration.These data suggest that the in situ and in vivo potency and the selectivity of BIA 10‐2474 is comparable to other clinically tested FAAH inhibitors. These data also demonstrate that there is no unusual accumulation of BIA 10‐2474 or its metabolites in cells nor in the CNS. In conclusion, these results fail to identify an off‐target activity or a mechanism that could explain the clinical trial accident.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.