Nuno Marques Pires

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/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 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.

The hyperactivity of the sympathetic nervous system (SNS) plays a major role in the development and progression of several cardiovascular diseases. One strategy to mitigate the SNS overdrive is by restricting the biosynthesis of norepinephrine via the inhibition of dopamine β-hydroxylase (DBH). Zamicastat is a new DBH inhibitor that decreases norepinephrine and increases dopamine levels in peripherally sympathetic-innervated tissues. The cardiometabolic and inflammatory effects of sympathetic down-regulation were evaluated in 50 week old male spontaneously hypertensive rats (SHRs) receiving zamicastat (30 mg/kg/day) for 9 weeks. After 8 weeks of treatment, the blood pressure (BP) and heart rate (HR) were assessed by tail cuff plethysmography. At the end of the study, 24 h urine, plasma, heart, and kidney were collected for biochemical and morphometric analyses. Zamicastat-induced sympathetic down-regulation decreased the high BP in SHRs, with no observed effect on HR. The heart-to-body weight ratio was lower in SHRs treated with zamicastat, whereas the body weight and kidney-to-body weight ratio were similar between both SHR cohorts. Zamicastat-treated SHRs showed reduced 24 h urine output, but the urinary amount of protein excreted and creatinine clearance rate remained unchanged. Zamicastat treatment significantly decreased plasma triglycerides, free fatty acids, and aspartate aminotransferase levels. Aged SHRs showed higher plasma levels of inflammatory markers as compared with age-matched normotensive Wistar-Kyoto rats. The inflammatory benefits attained with DBH inhibition were expressed by a decrease in CRP, MCP-1, IL-5, IL-17α, GRO/KC, MIP-1α, and RANTES plasma levels as compared with untreated SHRs. In conclusion, DBH inhibition decreased norepinephrine levels, reduced end-organ damage, and improved cardiometabolic and inflammatory biomarkers in aged male SHRs.

Fatty acid amide hydrolase (FAAH) can be targeted for the treatment of pain associated with various medical conditions. Herein we report the design and synthesis of a novel series of heterocyclic‐N‐carboxamide FAAH inhibitors that have a good alignment of potency, metabolic stability and selectivity for FAAH over monoacylglycerol lipase (MAGL) and carboxylesterases (CEs). Lead optimization efforts carried out with benzotriazolyl‐ and imidazolyl‐N‐carboxamide series led to the discovery of clinical candidate 8 l (3‐(1‐(cyclohexyl(methyl)carbamoyl)‐1H‐imidazol‐4‐yl)pyridine 1‐oxide; BIA 10‐2474) as a potent and long‐acting inhibitor of FAAH. However, during a Phase I clinical trial with compound 8 l, unexpected and unpredictable serious neurological adverse events occurred, affecting five healthy volunteers, including the death of one subject.

Novel 5-(2,4-difluorophenoxy)-3-aryl-1,3,4-oxadiazol-2(3H)-ones were prepared and in vivo SAR are discussed. Ionisable substituents on the N-phenyl ring provided compounds with significantly improved aqueous solubility. In addition, these analogues retained equivalent or improved potency against FAAH enzyme compared to the parent phenols 2–3. FAAH inhibition by the 2-(piperazin-1-yl)ethyl and 3-(piperazin-1-yl)propyl derivatives 24 and 30 was confined to the periphery in mice (30 mg/kg), whereas hepatic FAAH activity was inhibited by over 90%.

The aim of this study was to evaluate the effect of carbamazepine (CBZ) upon chemically induced absence seizures and in a genetic absence seizures model in the mouse. The γ-butyrolactone (GBL)-induced acute absence seizures and the stargazer spontaneous absence seizures mice models were used to characterize the aggravation of absence seizures induced by oral CBZ treatment. The effect of CBZ upon GABA inward-currents in Ltk cells expressing human recombinant α1β2γ2, α2β2γ2, α3β2γ2 and α5β2γ2 GABAA receptors was evaluated by means of patch clamp. GBL administration induced motor impairment in NMRI mice. High dose CBZ (25mg/kg body weight) had no effect on motor performance but exacerbated the behavioral incoordination observed for GBL. Also, coadministration of a high dose CBZ and GBL impaired spontaneous locomotion. Moreover, CBZ was investigated after oral administration to evaluate the potential to aggravate GBL-induced acute spike-and-wave discharges (SWD) in the electroencephalogram. High dose CBZ significantly aggravated SWD induced by GBL. Likewise, in the stargazer mouse model of genetic spontaneous absence seizures, CBZ significantly aggravated SWD frequency and duration. Pre-treatment with the T-type Ca(2+) channel blocker ethosuximide (200mg/kg body weight) prevented the CBZ aggravation of SWD induced by GBL and in the stargazer mouse. CBZ increased in a concentration dependent manner sub-maximal α1β2γ2 and α3β2γ2 GABA currents. CBZ aggravates absence seizures as assessed in two dedicated mouse models of absence seizures. Facilitation of sub-maximal α1β2γ2, and α3β2γ2 GABA currents by CBZ may play a role in CBZ-induced GABA-mediated aggravation of absence seizures.

1. This study explores the impact of permeability and P-glycoprotein (P-gp) efflux, upon brain exposure to etamicastat, a new dopamine-β-hydroxylase (DBH) inhibitor and consequently brain levels of catecholamines. 2. Brain exposure to etamicastat (10 mg/kg), following intravenous administration to mice, was residual and upon oral administration of the same dose no compound was detected, concurring with the absence of effects upon brain catecholamines. The intravenous co-administration of elacridar (1.0 mg/kg), a known P-gp/BCRP dual modulator, significantly increased brain etamicastat exposure, but the levels attained were very low when compared to those of nepicastat, a centrally active DBH inhibitor. 3. In vitro permeability studies from apical-to-basal direction conducted in Caco-2 cells and MDCK-II cells showed that etamicastat apparent permeability was 1.2 × 10⁻⁵ and 1.1 × 10⁻⁶cm/s, respectively, 5- and 50-fold lower as compared to nepicastat. The secretory efflux ratio in MDCK-II cells overexpressing human P-gp showed an efflux ratio greater than 2, for both compounds, which was significantly decreased by elacridar. Despite its lower bioavailability and higher clearance, as compared to nepicastat, etamicastat showed preferential distribution to peripheral tissues and high plasma free fraction (15.5%), which may explain its effects upon peripheral DBH and catecholamine levels. 4. Though P-gp-mediated efflux may contribute to the limited brain penetration of etamicastat, the low permeability along with the pharmacokinetic properties of etamicastat may be perceived as the main contributors for its peripheral selectivity, which is advantageous for a cardiovascular drug candidate.

Overactivity of the sympathetic nervous system has an important role in the development and progression of arterial hypertension. Catheter-based renal nerve ablation for the treatment of drug-resistant hypertension has recently been developed. An alternative strategy for the modulation of sympathetic nerve function is to reduce the biosynthesis of noradrenaline (NA) by inhibiting dopamine β-hydroxylase (DβH), the enzyme that catalyzes the conversion of dopamine (DA) to NA in the sympathetic nerves. Renal denervation (RDN) surgery was performed in spontaneously hypertensive rats (SHR) to evaluate the effect of RDN on the DA and NA levels and on blood pressure over a 28-day period. The selective peripheral DβH inhibitor etamicastat (30 mg kg ⁻¹day⁻¹) was administered to another cohort of SHR. RDN and etamicastat treatment had no effect on the renal function, as assessed by measuring the water balance response, renal function and urinary electrolyte levels. RDN significantly decreased the systolic blood pressure (SBP) and the diastolic blood pressure (DBP). A gradual return of the SBP and the DBP to the high baseline levels was observed over time. Conversely, treatment with etamicastat resulted in a significant decrease in the SBP and the DBP at all time points. On the last day of the assessment, NA levels in renal tissue were significantly decreased in both RDN and etamicastat-treated groups. In contrast, the NA levels in the left ventricle were decreased only in the etamicastat-treated group. Thus, RDN produces transitory decreases in blood pressure, whereas prolonged downregulation of sympathetic drive with the DβH inhibitor etamicastat results in a sustained decrease in the SBP and the DBP.

Eslicarbazepine acetate (ESL) is a once daily antiepileptic drug (AED) approved by the European Medicines Agency (EMA), the Food and Drug Administration (FDA) and Health Canada as an adjunctive therapy in adults with partial‐onset seizures (POS). In humans and in relevant animal laboratory species,ESLundergoes extensive first pass hydrolysis to its major active metabolite eslicarbazepine that represents ~95% of circulating active moieties.ESLand eslicarbazepine showed anticonvulsant activity in animal models.ESLmay not only suppress seizure activity but may also inhibit the generation of a hyperexcitable network. Data reviewed here suggest thatESLand eslicarbazepine demonstrated the following in animal models: (1) the selectivity of interaction with the inactive state of the voltage‐gated sodium channel (VGSC), (2) reduction inVGSCavailability through enhancement of slow inactivation, instead of alteration of fast inactivation ofVGSC, (3) the failure to cause a paradoxical upregulation of persistent Na⁺current (I_NaP), and (4) the reduction in firing frequencies of excitatory neurons in dissociated hippocampal cells from patients with epilepsy who were pharmacoresistant to carbamazepine (CBZ). In addition, eslicarbazepine effectively inhibited high‐ and low‐affinity hCa_V3.2 inward currents with greater affinity thanCBZ. These preclinical findings may suggest the potential for antiepileptogenic effects; furthermore, the lack of effect uponK_V7.2 outward currents may translate into a reduced potential for eslicarbazepine to facilitate repetitive firing.

Rationale:: In human genetic studies a single nucleotide polymorphism within the salt-inducible kinase 1 ( SIK1 ) gene was associated with hypertension. Lower SIK1 activity in vascular smooth muscle cells (VSMCs) leads to decreased sodium-potassium ATPase activity, which associates with increased vascular tone. Also, SIK1 participates in a negative feedback mechanism on the transforming growth factor-β1 signaling and downregulation of SIK1 induces the expression of extracellular matrix remodeling genes. Objective:: To evaluate whether reduced expression/activity of SIK1 alone or in combination with elevated salt intake could modify the structure and function of the vasculature, leading to higher blood pressure. Methods and Results:: SIK1 knockout ( sik1 ^−/− ) and wild-type ( sik1 ^+/+ ) mice were challenged to a normal- or chronic high-salt intake (1% NaCl). Under normal-salt conditions, the sik1 ^−/− mice showed increased collagen deposition in the aorta but similar blood pressure compared with the sik1 ^+/+ mice. During high-salt intake, the sik1 ^+/+ mice exhibited an increase in SIK1 expression in the VSMCs layer of the aorta, whereas the sik1 ^−/− mice exhibited upregulated transforming growth factor-β1 signaling and increased expression of endothelin-1 and genes involved in VSMC contraction, higher systolic blood pressure, and signs of cardiac hypertrophy. In vitro knockdown of SIK1 induced upregulation of collagen in aortic adventitial fibroblasts and enhanced the expression of contractile markers and of endothelin-1 in VSMCs. Conclusions:: Vascular SIK1 activation might represent a novel mechanism involved in the prevention of high blood pressure development triggered by high-salt intake through the modulation of the contractile phenotype of VSMCs via transforming growth factor-β1-signaling inhibition.