Effect of zamicastat on blood pressure and heart rate response to cold pressor test: A double-blind, randomized, placebo-controlled study in healthy subjects.

AIMS: Dopamine beta-hydroxylase (DßH) inhibitors, like zamicastat, hold promise for treating pulmonary arterial hypertension. This study aimed to validate the mechanism of action of zamicastat by studying its effect on the overdrive of the sympathetic nervous system (SNS). METHODS: A single-centre, prospective, double-blind, randomized, placebo-controlled, crossover study evaluated the effect of 400 mg zamicastat in 22 healthy male subjects. Cold pressor test (CPT) was performed at screening and each treatment period on Days -1 and 10. Plasma and 24 h-urine levels of dopamine (DA), epinephrine (EPI) and norepinephrine (NE), and plasma DßH activity, were measured. RESULTS: Compared to placebo, zamicastat showed a - 4.62 mmHg decrease in systolic blood pressure during the cold stimulus vs. rest phases on Day 10 of CPT (P = .020). Zamicastat decreased mean arterial pressure response to cold stimulus during CPT (-2.62 mmHg; P = .025). At Day 10, zamicastat significantly increased plasma DA, before CPT (12.63 ng/L; P = .040) and after CPT (19.22 ng/L; P = .001) as well as the estimated plasma EPI change from baseline after CPT (P = .040). Inhibition of plasma DßH activity ranged from 19.8% to 25.0%. At Day 10, significant reductions in 24-h urinary excretion of EPI (P = .002) and NE (P = .001) were observed. Zamicastat Cτ geometric mean ± GSD ranged from 45.86 ± 1.46 ng/mL on Day 3 to 58.64 ± 1.52 ng/mL on Day 10, with moderate inter-individual variability (CV: 32.6%-36.6%). Steady state was already achieved on Day 6. CONCLUSIONS: Our results demonstrated the effect of zamicastat on the overdrive sympathetic response to cold stimulus, confirming its potential as SNS modulator.

Long-term effect of Toll-like receptor-2, -4, -5, -7 and NOD2 stimulation on Na+,K+-ATPase activity and expression in intestinal epithelial cells.

Inappropriate activation of Toll-like receptors (TLRs) and nucleotide-binding oligomerization domain receptors (NOD) is involved in many chronic disorders, including inflammatory bowel disease (IBD). Altered function and/or expression of Na+,K+-ATPase (NKA) and epithelial ion channels are the main cause of electrolyte absorption imbalance in patients with IBD, leading to diarrhea. We aimed to evaluate the effect of TLRs and NOD2 stimulation upon NKA activity and expression in human intestinal epithelial cells (IECs) using RT-qPCR, Western blot, and electrophysiology techniques. TLR2, TLR4, and TLR7 activation inhibited NKA activity [(means ± SE) -20.0 ± 1.2%, -34.0 ± 1.5%, and -24.5 ± 2.0% in T84 cells; and -21.6 ± 7.4%, -37.7 ± 3.5%, and -11.0 ± 2.3% in Caco-2 cells]. On the other hand, activation of TLR5 increased NKA activity (16.2 ± 2.9% in T84 and 36.8 ± 5.2% in Caco-2 cells) and ß1-NKA mRNA levels (21.8 ± 7.8% in T84 cells). The TLR4 agonist synthetic monophosphoryl lipid A (MPLAs) reduced α1-NKA mRNA levels in both T84 and Caco-2 cells (-28.5 ± 3.6% and -18.7 ± 2.8%), and this was accompanied by a decrease in α1-NKA protein expression (-33.4 ± 11.8% and -39.4 ± 11.2%). NOD2 activation upregulated NKA activity (12.2 ± 5.1%) and α1-NKA mRNA levels (6.8 ± 1.6%) in Caco-2 cells. In summary, TLR2, TLR4, and TLR7 activation induce downregulation of NKA in IECs, whereas TLR5 and NOD2 activation has the opposite effect. A comprehensive understanding of the cross talk between TLRs, NOD2, and NKA is of utmost relevance for developing better IBD treatments.

Effect of Toll-like receptor-2, -4, -5, -7, and NOD2 stimulation on potassium channel conductance in intestinal epithelial cells.

Disproportionate activation of pattern recognition receptors plays a role in inflammatory bowel disease (IBD) pathophysiology. Diarrhea is a hallmark symptom of IBD, resulting at least in part from an electrolyte imbalance that may be caused by changes in potassium channel activity. We evaluated the impact of Toll-like receptors (TLRs) and nucleotide-binding oligomerization domain 2 (NOD2) stimulation on potassium conductance of the basolateral membrane in human intestinal epithelial cells (IECs) and the role of potassium channels through electrophysiological assays under short-circuit current in Ussing chambers. TLRs and NOD2 were stimulated using specific agonists, and potassium channels were selectively blocked using triarylmethane-34 (TRAM-34), adenylyl-imidodiphosphate (AMP-PNP), and BaCl2. Potassium conductance of the basolateral membrane decreased upon activation of TLR2, TLR4, and TLR7 in T84 cells (means ± SE, -11.2 ± 4.5, -40.4 ± 7.2, and -19.4 ± 5.9, respectively) and in Caco-2 cells (-13.1 ± 5.7, -55.7 ± 7.4, and -29.1 ± 7.2, respectively). In contrast, activation of TLR5 and NOD2 increased basolateral potassium conductance, both in T84 cells (18.0 ± 4.1 and 18.4 ± 2.8, respectively) and in Caco-2 cells (21.2 ± 8.4 and 16.0 ± 3.6, respectively). TRAM-34 and AMP-PNP induced a decrease in basolateral potassium conductance upon TLR4 stimulation in both cell lines. Both KCa3.1- and Kir6-channels appear to be important mediators of this effect in IECs and could be potential targets for therapeutic agent development.NEW & NOTEWORTHY This study highlights that PRRs stimulation directly influences K+-channel conductance in IECs. TLR-2, -4, -7 stimulation decreased K+ conductance, whereas TLR5 and NOD2 stimulation had the opposite effect, leading to an increase of it instead. This study reports for the first time that KCa3.1- and Kir6-channels play a role in K+ transport pathways triggered by TLR4 stimulation. These findings suggest that KCa3.1- and Kir6-channels modulation may be a potential target for new therapeutic agents in IBD.

Inactivation Mechanism of the Fatty Acid Amide Hydrolase Inhibitor BIA 10-2474.

BIA 10-2474 is a time-dependent inhibitor of fatty acid amide hydrolase (FAAH) that was under clinical development for the treatment of neurological conditions when the program was terminated after one subject died and four were hospitalized with neurological symptoms during a first-in-human clinical study. The present work describes the mechanism of FAAH inhibition by BIA 10-2474 as a target-specific covalent inhibition, supported by quantum mechanics and molecular modelling studies. The inhibitor incorporates a weakly reactive electrophile which, upon specific binding to the enzyme's active site, is positioned to react readily with the catalytic residues. The reactivity is enhanced on-site by the increased molarity at the reaction site and by specific inductive interactions with FAAH. In the second stage, the inhibitor reacts with the enzyme's catalytic nucleophile to form a covalent enzyme-inhibitor adduct. The hydrolysis of this adduct is shown to be unlikely under physiological conditions, therefore leading to irreversible inactivation of FAAH. The results also reveal the important role played by FAAH Thr236 in the reaction with BIA 10-2474, which is specific to FAAH and is not present in other serine hydrolases. It forms a hydrogen bond with the imidazole nitrogen of the inhibitor and helps lowering the activation free energy of the first step of the reaction, by pre-orienting and stabilizing the inhibitor in a near-reactive configuration. In the second step, Thr236 can also serve as a mechanistic alternative to protonate the leaving group.

Effect of Opicapone on Levodopa Pharmacokinetics in Patients with Fluctuating Parkinson's Disease.

BACKGROUND: Inhibiting catechol-O-methyltransferase extends the plasma half-life of levodopa, potentially allowing physicians to optimize the levodopa regimen in patients with Parkinson's disease (PD) experiencing motor fluctuations. OBJECTIVES: To evaluate the effects of once-daily opicapone on levodopa plasma pharmacokinetics and motor response when added to two different levodopa dosing regimens. METHODS: A total of 24 patients with PD and motor fluctuations were enrolled in an exploratory, open-label, modified cross-over trial. Participants first received levodopa/carbidopa 500/125 mg (five intakes) for 2 weeks and were then randomly assigned (1:1) to levodopa/carbidopa 400/100 mg given over either four or five daily intakes plus opicapone 50 mg for an additional 2 weeks. Levodopa 12-hour pharmacokinetics was the primary outcome (ie, excluding the effect of last/evening levodopa/carbidopa intake), with motor complications evaluated as secondary outcomes. RESULTS: Over 12-hour pharmacokinetics and compared with five-intake levodopa/carbidopa 500/125 mg without opicapone, maximal levodopa concentrations were similar or nonsignificantly higher on both levodopa/carbidopa 400/100 mg regimens plus opicapone. Despite a 100 mg lower total levodopa/carbidopa daily dose, adding opicapone 50 mg at least doubled the levodopa plasma half-life and minimal concentrations, with a significant ≈30% increase in total exposure. The levodopa fluctuation index was only significantly lower for the five intakes plus opicapone regimen (difference of -71.8%; P < 0.0001). Modifications to levodopa pharmacokinetics were associated with decreased off time and increased on time. CONCLUSIONS: Combining opicapone 50 mg with a 100 mg lower daily dose of levodopa provides higher levodopa bioavailability with avoidance of trough levels. Despite the lower levodopa dose, modifying the levodopa pharmacokinetic profile with opicapone was associated with decreased off time and increased on time. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Absorption, metabolism and excretion of opicapone in human healthy volunteers.

AIMS: The absorption, metabolism and excretion of opicapone (2,5-dichloro-3-(5-[3,4-dihydroxy-5-nitrophenyl]-1,2,4-oxadiazol-3-yl)-4,6-dimethylpyridine 1-oxide), a selective catechol-O-methyltransferase inhibitor, were investigated. METHODS: Plasma, urine and faeces were collected from healthy male subjects following a single oral dose of 100 mg [14 C]-opicapone. The mass balance of [14 C]-opicapone and metabolic profile were evaluated. RESULTS: The recovery of total administered radioactivity averaged >90% after 144 hours. Faeces were the major route of elimination, representing 70% of the administered dose; 5% and 20% were excreted in urine and expired air, respectively. The Cmax of total radioactivity matched that of unchanged opicapone, whereas the total radioactivity remained quantifiable for a longer period, attributed to the contribution of opicapone metabolites, involving primarily 3-O-sulfate conjugation (58.6% of total circulating radioactivity) at the nitrocatechol ring. Other circulating metabolites, accounting for <10% of the radioactivity exposure, were formed by glucuronidation, methylation, N-oxide reduction and gluthatione conjugation. Additionally, various other metabolites resulting from combinations with the opicapone N-oxide reduced form at the 2,5-dichloro-4,6-dimethylpyridine 1-oxide moiety, including nitro reduction and N-acetylation, reductive opening and cleavage of the 1,2,4-oxadiazole ring and the subsequent hydrolysis products were identified, but only in faeces, suggesting the involvement of gut bacteria. CONCLUSION: [14 C]-opicapone was fully excreted through multiple metabolic pathways. The main route of excretion was in faeces, where opicapone may be further metabolized via reductive metabolism involving the 1,2,4-oxadiazole ring-opening and subsequent hydrolysis.

Opicapone versus placebo in the treatment of Parkinson's disease patients with end-of-dose motor fluctuation-associated pain: rationale and design of the randomised, double-blind OCEAN (OpiCapone Effect on motor fluctuations and pAiN) trial.

BACKGROUND: Optimisation of dopaminergic therapy may alleviate fluctuation-related pain in Parkinson's disease (PD). Opicapone (OPC) is a third-generation, once-daily catechol-O-methyltransferase inhibitor shown to be generally well tolerated and efficacious in reducing OFF-time in two pivotal trials in patients with PD and end-of-dose motor fluctuations. The OpiCapone Effect on motor fluctuations and pAiN (OCEAN) trial aims to investigate the efficacy of OPC 50 mg in PD patients with end-of-dose motor fluctuations and associated pain, when administered as adjunctive therapy to existing treatment with levodopa/dopa decarboxylase inhibitor (DDCi). METHODS: OCEAN is a Phase IV, international, multicentre, randomised, double-blind, placebo-controlled, parallel-group, interventional trial in PD patients with end-of-dose motor fluctuations and associated pain. It consists of a 1-week screening period, 24-week double-blind treatment period and 2-week follow-up period. Eligible patients will be randomised 1:1 to OPC 50 mg or placebo once daily while continuing current treatment with levodopa/DDCi and other chronic, stable anti-PD and/or analgesic treatments. The primary efficacy endpoint is change from baseline in Domain 3 (fluctuation-related pain) of the King's Parkinson's disease Pain Scale (KPPS). The key secondary efficacy endpoint is change from baseline in Domain B (anxiety) of the Movement Disorder Society-sponsored Non-Motor rating Scale (MDS-NMS). Additional secondary efficacy assessments include other domains and total scores of the KPPS and MDS-NMS, the Parkinson's Disease Questionnaire (PDQ-8), the MDS-sponsored Unified Parkinson's Disease Rating Scale (MDS-UPDRS) Parts III and IV, Clinical and Patient's Global Impressions of Change, and change in functional status via Hauser's diary. Safety assessments include the incidence of treatment-emergent adverse events. The study will be conducted in approximately 140 patients from 50 clinical sites in Germany, Italy, Portugal, Spain and the United Kingdom. Recruitment started in February 2021 and the last patient is expected to complete the study by late 2022. DISCUSSION: The OCEAN trial will help determine whether the use of adjunctive OPC 50 mg treatment can improve fluctuation-associated pain in PD patients with end-of-dose motor fluctuations. The robust design of OCEAN will address the current lack of reliable evidence for dopaminergic-based therapy in the treatment of PD-associated pain. TRIAL REGISTRATION: EudraCT number 2020-001175-32 ; registered on 2020-08-07.

Opicapone Use in Clinical Practice across Germany: A Sub-Analysis of the OPTIPARK Study in Parkinson's Disease Patients with Motor Fluctuations.

INTRODUCTION: The OPTIPARK study confirmed the effectiveness and safety of opicapone as adjunct therapy to levodopa in patients with Parkinson's disease (PD) and motor fluctuations under real-world conditions. The aim of this sub-analysis was to evaluate opicapone in the German patient cohort of OPTIPARK in order to provide country-specific data. METHODS: OPTIPARK was an open-label, single-arm study conducted in routine clinical practice across Germany and the UK. Patients with PD and motor fluctuations received once-daily opicapone 50 mg for 3 months in addition to levodopa. The primary endpoint was Clinicians' Global Impression of Change (CGI-C). Secondary assessments included Patients' Global Impressions of Change (PGI-C), Unified Parkinson's Disease Rating Scale (UPDRS) I-IV, Parkinson's Disease Questionnaire (PDQ-8), and Non-Motor Symptoms Scale (NMSS). This sub-analysis reports outcomes from the German patients only. RESULTS: Overall, 363 (97.6%) of the 372 patients included in the German cohort received ≥1 dose of opicapone and 291 (80.2%) completed the study. Improvements on CGI-C and PGI-C were reported by 70.8% and 76.3% of patients, respectively. UPDRS scores improved for activities of daily living during OFF time by -3.3 ± 4.5 points and motor scores during ON time by -5.3 ± 7.9 points. PDQ-8 and NMSS scores also demonstrated improvements. Treatment emergent adverse events considered at least possibly related to opicapone occurred in 37.7% of patients, with most being of mild or moderate intensity. CONCLUSION: Opicapone added to levodopa in patients with PD and motor fluctuations was effective and generally well tolerated in routine clinical practice across Germany.

Non-clinical toxicology evaluation of BIA 10-2474.

In 2016, one subject died and four were hospitalized with neurological symptoms during a clinical trial with the fatty acid amide hydrolase (FAAH) inhibitor BIA 10-2474. The present paper reviews the regulatory toxicology studies that were carried out to support the clinical trial application for BIA 10-2474. Animal studies complied with national and international standards including European regulatory guidelines (e.g. EEC Council Directive 75/318/EEC and subsequent amendments). The CNS effects seen in the rat and mouse appear to be common in rodents in such studies and do not in principle seem to be of the type to generate a signal. In the same way in non-human primates, insignificant alterations in the mesencephalon, and especially of the autonomic nervous system (Meissner's plexus in the bowel) in rodents and monkeys were observed in some animals treated with a high dose. Overall, these data, as well as the extensive additional data generated since the accident, support the conclusion that the tragic fatality that occurred during the clinical trial with BIA 10-2474 was unpredictable and that the mechanism responsible remains unknown, from a non-clinical toxicological perspective.

Complex effects of eslicarbazepine on inhibitory micro networks in chronic experimental epilepsy.

OBJECTIVE: Many antiseizure drugs (ASDs) act on voltage-dependent sodium channels, and the molecular basis of these effects is well established. In contrast, how ASDs act on the level of neuronal networks is much less understood. METHODS: In the present study, we determined the effects of eslicarbazepine (S-Lic) on different types of inhibitory neurons, as well as inhibitory motifs. Experiments were performed in hippocampal slices from both sham-control and chronically epileptic pilocarpine-treated rats. RESULTS: We found that S-Lic causes an unexpected reduction of feed-forward inhibition in the CA1 region at high concentrations (300 µM), but not at lower concentrations (100 µM). Concurrently, 300 but not 100 µM S-Lic significantly reduced maximal firing rates in putative feed-forward interneurons located in the CA1 stratum radiatum of sham-control and epileptic animals. In contrast, feedback inhibition was not inhibited by S-Lic. Instead, application of S-Lic, in contrast to previous data for other drugs like carbamazepine (CBZ), resulted in a lasting potentiation of feedback inhibitory post-synaptic currents (IPSCs) only in epileptic and not in sham-control animals, which persisted after washout of S-Lic. We hypothesized that this plasticity of inhibition might rely on anti-Hebbian potentiation of excitatory feedback inputs onto oriens-lacunosum moleculare (OLM) interneurons, which is dependent on Ca2+ -permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. Indeed, we show that blocking Ca2+ -permeable AMPA receptors completely prevents upmodulation of feedback inhibition. SIGNIFICANCE: These results suggest that S-Lic affects inhibitory circuits in the CA1 hippocampal region in unexpected ways. In addition, ASD actions may not be sufficiently explained by acute effects on their target channels, rather, it may be necessary to take plasticity of inhibitory circuits into account.

Antagonistic modulation of SIK1 and SIK2 isoforms in high blood pressure and cardiac hypertrophy triggered by high-salt intake.

Salt-inducible kinases (SIKs) represent a subfamily of AMPK family kinases. SIK1 has been shown to act as a mediator during the cellular adaptation to variations in intracellular sodium in a variety of cell types. SIK2, as an isoform of the SIK family, modulates various biological functions and acts as a signal transmitter in various pathways. To evaluate the role of both SIK1 and SIK2 isoforms in blood pressure (BP), body fluid regulation and cardiac hypertrophy development, we made use of constitutive sik1-/- (SIK1-KO), sik2-/- (SIK2-KO), double sik1-/-sik2-/- (double SIK1*2-KO) knockout and wild-type (WT) mice challenged to a standard (0.3% NaCl) or chronic high-salt (HS, 8% NaCl) diet intake for 12 weeks.Mice, under a standard diet intake, had similar and normal BP. On a chronic HS intake, SIK1-KO and double SIK1*2-KO mice showed increased BP, but not WT and SIK2-KO mice. A chronic HS intake led to the development of cardiac left ventricle hypertrophy (LVH) in normotensive WT and hypertensive SIK1-KO mice, but not in SIK2-KO mice. Double SIK1*2-KO mice under standard diet intake show normal BP but an increased LV mass. Remarkably, in response to a dietary stress condition, there is an increase in BP but LVH remained unchanged in double SIK1*2-KO mice.In summary, SIK1 isoform is required for maintaining normal BP in response to HS intake. LVH triggered by HS intake requires SIK2 isoform and is independent of high BP.

Long-term efficacy and safety of eslicarbazepine acetate monotherapy for adults with newly diagnosed focal epilepsy: An open-label extension study.

OBJECTIVE: To assess the efficacy, safety, and tolerability of eslicarbazepine acetate (ESL) monotherapy during long-term treatment. METHODS: An open-label extension (OLE) study was conducted in adults completing a phase 3, randomized, double-blind, noninferiority trial, during which they had received monotherapy with either once-daily ESL or twice-daily controlled-release carbamazepine (CBZ-CR) for newly diagnosed focal epilepsy. In the OLE study, all patients received ESL (800-1600 mg/d) for 2 years. Primary efficacy outcome was retention time (from baseline of the OLE study). Secondary efficacy assessments included seizure freedom rate (no seizures during the OLE study) and responder rate (≥50% seizure frequency reduction from baseline of double-blind trial). Safety assessments included evaluation of treatment-emergent adverse events (TEAEs). RESULTS: Of 206 randomized patients, 96 who received ESL in the double-blind trial (ESL/ESL) and 88 who received CBZ-CR in the double-blind trial (CBZ-CR/ESL) were treated with ESL monotherapy (89.3% overall). Treatment retention time was similar between groups, with low probability of ESL withdrawal overall (<0.07 at any time). After 24 months, the probability of ESL withdrawal was 0.0638 (95% confidence interval [CI] = 0.0292-0.1366) in the ESL/ESL group and 0.0472 (95% CI = 0.0180-0.1210) in the CBZ-CR/ESL group. Seizure freedom rates were 90.6% (ESL/ESL) and 80.7% (CBZ-CR/ESL; P = .0531). Responder rates remained >80% in both groups throughout the study. Incidence of serious TEAEs was similar between groups (7.3% vs 5.7%; 0% vs 1.1% possibly related), as were the incidences of TEAEs considered at least possibly related to treatment (17.7% vs 18.2%) and TEAEs leading to discontinuation (3.1% vs 4.5%). The types of TEAEs were generally consistent with the known safety profile of ESL. SIGNIFICANCE: ESL monotherapy was efficacious and generally well tolerated over the long term, including in patients who transitioned from CBZ-CR monotherapy. No new safety concerns emerged.

Analysis of cutaneous allergic reactions in clinical trials of eslicarbazepine acetate.

OBJECTIVES: To evaluate cutaneous allergic reactions in clinical trials of adjunctive eslicarbazepine acetate (ESL) for focal seizures. MATERIALS AND METHODS: Data were analyzed from three phase III randomized, double-blind, placebo-controlled studies of adjunctive ESL in adults (placebo, n = 426; ESL, n = 1021) and two randomized, double-blind, placebo-controlled studies (and open-label extensions [OLEs]) of adjunctive ESL in children aged 4-17 years (placebo, n = 160; ESL, n = 202; OLE, n = 337). RESULTS: Adult studies: Rash (ESL 1.9%, placebo 0.9%) and pruritus (ESL 1.2%, placebo 0.9%) were the most frequent rash-related treatment-emergent adverse events (TEAEs). Most rash-related TEAEs were mild or moderate in severity. Incidence of rash increased with increasing ESL dose, but was not higher for patients who initiated treatment with higher ESL doses. Pediatric studies: Allergic dermatitis (ESL 3.0%, placebo 0) and rash (controlled studies: ESL 1.0%, placebo 1.3%; OLE periods: ESL ≤1.2%) were the most frequent rash-related TEAEs. There was one case of DRESS in the ESL group. Most rash-related TEAEs were mild or moderate in severity and judged as not related to treatment with ESL. CONCLUSIONS: Serious skin rashes were rare during adult and pediatric clinical trials of ESL. Although the incidence of rash with ESL was low, it is important for patients/caregivers to be made aware of the potential signs and symptoms associated with serious skin rashes.

Efficacy and safety of eslicarbazepine acetate as adjunctive therapy for refractory focal-onset seizures in children: A double-blind, randomized, placebo-controlled, parallel-group, multicenter, phase-III clinical trial.

PURPOSE: This was a phase-III, randomized, double-blind, placebo-controlled study aimed to evaluate efficacy and tolerability of eslicarbazepine acetate (ESL) as adjunctive therapy in pediatric patients with refractory focal-onset seizures (FOS). METHODS: Children (2-18 years old) with FOS, receiving 1-2 antiepileptic drugs, were randomized to ESL or placebo. Treatment was started at 10 mg/kg/day, up-titrated up to 20-30 mg/kg/day, and maintained for 12 weeks, followed by one-year open-label follow-up. Primary efficacy endpoints were relative reduction in standardized seizure frequency (SSF) and proportion of responders (≥50% SSF reduction) from baseline. Safety was evaluated by the incidence of treatment-emergent adverse events (TEAEs). RESULTS: The intention-to-treat (ITT) set included 134 patients randomized to ESL and 129 to placebo; 89.6% and 91.5%, respectively, completed the trial. An unbalanced number of seizures at baseline were observed between groups. Least square (LS) mean relative change in SSF from baseline was higher in the ESL group (-18.1%) than in placebo (-8.6%). Proportion of responders between ESL and placebo groups was not statistically different. A post hoc analysis showed greater relative reduction in SSF in patients above 6 years old treated with ESL 20 or 30 mg/kg/day compared with placebo; this was significant in patients above 6 years old treated with ESL 30 mg/kg/day (LS mean difference: 31.9%; p = 0.0478). The observed safety profile in children was consistent with that established in adult studies. CONCLUSIONS: Adjunctive ESL treatment was well-tolerated, but this trial failed to demonstrate that ESL was more effective than placebo in the predefined efficacy endpoints; factors that may have contributed to this outcome, affecting particularly the young age group, include etiological heterogeneity, difficulty in recognizing simple partial seizures, high seizure frequency with risk of imbalance, and underestimation of the efficacious dose range.

Effects of nepicastat upon dopamine-ß-hydroxylase activity and dopamine and norepinephrine levels in the rat left ventricle, kidney, and adrenal gland.

Background and Objective: Evaluate the activity of dopamine-ß-hydroxylase (DßH) as well as the effect of the DßH inhibitor nepicastat upon enzyme activity and levels of dopamine (DA) and norepinephrine (NE) in the rat left ventricle, kidney, and adrenal glands.Methods: DßH assay consisted of the enzymatic hydroxylation of tyramine into octopamine, and DA and NE tissues levels were quantified by HPLC-ED.Results: Nepicastat (30 mg/kg, p.o.) reduced DßH activity by 93% and 80% in the adrenals at 4 h and 8 h postdrug administration, accompanied by significant reductions in NE and epinephrine tissue levels and an increase in DA levels and of DA/NE tissue ratios, with similar findings for NE, DA and of DA/NE tissue ratios in left ventricle and kidney. DßH activity in the left ventricle and kidney showed a high degree of variability, which does not allow corroboration of the effects of nepicastat upon catecholamine tissue levels.Conclusion: The assay of DßH activity in heart and kidney lacks the necessary robustness, but DßH activity in the adrenals appears to be an appropriate marker. However, the effect size upon DA/NE tissue ratios (an indirect measure of DßH activity) as induced by nepicastat was very similar in sympathetically innervated tissues, left ventricle and kidney, and the adrenal medulla.

Polyamine Modulation of Anticonvulsant Drug Response: A Potential Mechanism Contributing to Pharmacoresistance in Chronic Epilepsy.

Despite the development of numerous novel anticonvulsant drugs, ∼30% of epilepsy patients remain refractory to antiepileptic drugs (AEDs). Many established and novel AEDs reduce hyperexcitability via voltage- and use-dependent inhibition of voltage-gated Na+ channels. For the widely used anticonvulsant carbamazepine (CBZ), use-dependent block of Na+ channels is significantly reduced both in experimental and human epilepsy. However, the molecular underpinnings of this potential cellular mechanism for pharmacoresistance have remained enigmatic.Here, we describe the mechanism that leads to the emergence of CBZ-resistant Na+ channels. We focused on the endogenous polyamine system, which powerfully modulates Na+ channels in a use-dependent manner. We had shown previously that the intracellular polyamine spermine is reduced in chronic epilepsy, resulting in increased persistent Na+ currents. Because spermine and CBZ both bind use-dependently in spatial proximity within the Na+ channel pore, we hypothesized that spermine loss might also be related to diminished CBZ response. Using the pilocarpine model of refractory epilepsy in male rats and whole-cell patch-clamp recordings, we first replicated the reduction of use-dependent block by CBZ in chronically epileptic animals. We then substituted intracellular spermine via the patch pipette in different concentrations. Under these conditions, we found that exogenous spermine significantly rescues use-dependent block of Na+ channels by CBZ. These findings indicate that an unexpected modulatory mechanism, depletion of intracellular polyamines, leads both to increased persistent Na+ currents and to diminished CBZ sensitivity of Na+ channels. These findings could lead to novel strategies for overcoming pharmacoresistant epilepsy that target the polyamine system.SIGNIFICANCE STATEMENT Pharmacoresistant epilepsy affects ∼18 million people worldwide, and intense efforts have therefore been undertaken to uncover the underlying molecular and cellular mechanisms. One of the key known candidate mechanisms of pharmacoresistance has been a loss of use-dependent Na+ channel block by the anticonvulsant carbamazepine (CBZ), both in human and experimental epilepsies. Despite intense scrutiny, the molecular mechanisms underlying this phenomenon have not been elucidated. We now show that a loss of intracellular spermine in chronic epilepsy is a major causative factor leading to the development of CBZ-resistant Na+ currents. This finding can be exploited both for the screening of anticonvulsants in expression systems, and for novel strategies to overcome pharmacoresistance that target the polyamine system.

Bioelectrical impedance analysis of body composition for the anesthetic induction dose of propofol in older patients.

BACKGROUND: Older people are currently the fastest growing segment of the worldwide population. The present study aimed to estimate propofol dose in older patients based on size descriptors measured by bioelectrical impedance analysis (BIA). METHODS: A cross sectional study in adult and older patients with body mass index equal to or lower than 35 kg/m2 was carried out. BIA and Clinical Frail Scale scoring were performed during pre-operative evaluation. Propofol infusion was started at 2000 mg/h until loss of consciousness (LOC) which was defined by "loss of eye-lash reflex" and "loss of response to name calling". Total dose of propofol at LOC was recorded. Propofol plasma concentration was measured using gas chromatography/ion trap-mass spectrometry. RESULTS: Forty patients were enrolled in the study. Total propofol dose required to LOC was lower in Age ≥ 65 group and a higher plasma propofol concentration was measured in this group. 60% of old patients were classified as "apparently vulnerable" or "frail" and narrow phase angle values were associated with increasing vulnerability scores. In the Age ≥ 65 group, the correlation analysis showed that the relationship between propofol dose and total body weight (TBW) scaled by the corresponding phase angle value is stronger than the correlation between propofol dose and TBW or fat free mass (FFM). CONCLUSIONS: This study demonstrates that weight-based reduction of propofol is suitable in older patients; however FFM was not seen to be more effective than TBW to predict the propofol induction dose in these patients. Guiding propofol induction dose according to baseline frailty score should also be considered to estimate individualized dosage profiles. Determination of phase angle value appears to be an easy and reliable tool to assess frailty in older patients. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02713698 . Registered on 23 February 2016.

Effects of eslicarbazepine on slow inactivation processes of sodium channels in dentate gyrus granule cells.

OBJECTIVE: Pharmacoresistance is a problem affecting ∼30% of chronic epilepsy patients. An understanding of the mechanisms of pharmacoresistance requires a precise understanding of how antiepileptic drugs interact with their targets in control and epileptic tissue. Although the effects of (S)-licarbazepine (S-Lic) on sodium channel fast inactivation are well understood and have revealed maintained activity in epileptic tissue, it is not known how slow inactivation processes are affected by S-Lic in epilepsy. METHODS: We have used voltage clamp recordings in isolated dentate granule cells (DGCs) and cortical pyramidal neurons of control versus chronically epileptic rats (pilocarpine model of epilepsy) and in DGCs isolated from hippocampal specimens from temporal lobe epilepsy patients to examine S-Lic effects on sodium channel slow inactivation. RESULTS: S-Lic effects on entry into and recovery from slow inactivation were negligible, even at high concentrations of S-Lic (300 µmol/L). Much more pronounced S-Lic effects were observed on the voltage dependence of slow inactivation, with significant effects at 100 µmol/L S-Lic in DGCs from control and epileptic rats or temporal lobe epilepsy patients. For none of these effects of S-Lic could we observe significant differences either between sham-control and epileptic rats, or between human DGCs and the two animal groups. S-Lic was similarly effective in cortical pyramidal neurons from sham-control and epileptic rats. Finally, we show in expression systems that S-Lic effects on slow inactivation voltage dependence are only observed in Nav 1.2 and Nav 1.6 subunits, but not in Nav 1.1 and Nav 1.3 subunits. SIGNIFICANCE: From these data, we conclude that a major mechanism of action of S-Lic is an effect on slow inactivation, primarily through effects on slow inactivation voltage dependence of Nav 1.2 and Nav 1.6 channels. Second, we demonstrate that this main effect of S-Lic is maintained in both experimental and human epilepsy and applies to principal neurons of different brain areas.

Efficacy and safety of eslicarbazepine acetate versus controlled-release carbamazepine monotherapy in newly diagnosed epilepsy: A phase III double-blind, randomized, parallel-group, multicenter study.

OBJECTIVE: We assessed the efficacy and safety of once-daily eslicarbazepine acetate in comparison with twice-daily (BID) controlled-release carbamazepine (carbamazepine-CR) monotherapy in newly diagnosed focal epilepsy patients. METHODS: This randomized, double-blind, noninferiority trial (NCT01162460) utilized a stepwise design with 3 dose levels. Patients who remained seizure-free for the 26-week evaluation period (level A: eslicarbazepine acetate 800 mg/carbamazepine-CR 200 mg BID) entered a 6-month maintenance period. If a seizure occurred during the evaluation period, patients were titrated to the next target level (level B: eslicarbazepine acetate 1200 mg/carbamazepine-CR 400 mg BID, level C: eslicarbazepine acetate 1600 mg/carbamazepine-CR 600 mg BID) and the evaluation period began again. The primary endpoint was the proportion of seizure-free patients for 6 months after stabilization in the per protocol set. The predefined noninferiority criteria were -12% absolute and -20% relative difference between treatment groups. RESULTS: Eight hundred fifteen patients were randomly assigned; 785 (388 in the eslicarbazepine acetate group and 397 in the carbamazepine-CR group) were included in the per protocol set, and 813 (401 in the eslicarbazepine acetate group and 412 in the carbamazepine-CR group) were included in the full analysis set for the primary analysis. Overall, 71.1% of eslicarbazepine acetate-treated patients and 75.6% of carbamazepine-CR-treated patients were seizure-free for ≥6 months at the last evaluated dose (average risk difference = -4.28%, 95% confidence interval [CI] = -10.30 to 1.74; relative risk difference = -5.87%, 95% CI = -13.50 to 2.44) in the per protocol set. Rates of treatment-emergent adverse events were similar between groups for patients in the safety set. Noninferiority was also demonstrated in the full analysis set, as 70.8% of patients with eslicarbazepine acetate and 74.0% with carbamazepine-CR were seizure-free at the last evaluated dose (average risk difference = -3.07, 95% CI = -9.04 to 2.89). SIGNIFICANCE: Treatment with eslicarbazepine acetate was noninferior to BID carbamazepine-CR. With its once-daily formulation, eslicarbazepine acetate provides a useful option for first-line monotherapy for adults with newly diagnosed epilepsy and focal onset seizures.

Elucidation of the Impact of P-glycoprotein and Breast Cancer Resistance Protein on the Brain Distribution of Catechol-O-Methyltransferase Inhibitors.

P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) are clinically important efflux transporters that act cooperatively at the blood-brain barrier, limiting the entry of several drugs into the central nervous system (CNS) and affecting their pharmacokinetics, therapeutic efficacy, and safety. In the present study, the interactions of catechol-O-methyltransferase (COMT) inhibitors (BIA 9-1059, BIA 9-1079, entacapone, nebicapone, opicapone, and tolcapone) with P-gp and BCRP were investigated to determine the contribution of these transporters in their access to the brain. In vitro cellular accumulation and bidirectional transport assays were conducted in Madin-Darby canine kidney (MDCK) II, MDCK-MDR1, and MDCK-BCRP cells. In vivo pharmacokinetic studies were carried out for tolcapone and BIA 9-1079 in rats, with and without elacridar, a well-known P-gp and BCRP modulator. The results suggest that BIA 9-1079, nebicapone, and tolcapone inhibit BCRP in a concentration-dependent manner. Moreover, with net flux ratios higher than 2 and decreased over 50% in the presence of verapamil or Ko143, BIA 9-1079 was identified as a P-gp substrate while BIA 9-1059, entacapone, opicapone, and nebicapone were revealed to be BCRP substrates. In vivo, brain exposure was limited for tolcapone and BIA 9-1079, although tolcapone crossed the blood-brain barrier at a greater rate and to a greater extent than BIA 9-1079. The extent of brain distribution of both compounds was significantly increased in the presence of elacridar, attesting to the involvement of efflux transporters. These findings provide relevant information and improve the understanding of the mechanisms that govern the access of these COMT inhibitors to the CNS.