Uptake and Biotransformation of the Tire Rubber-derived Contaminants 6-PPD and 6-PPD Quinone in the Zebrafish Embryo (Danio rerio).

N-(1,3-Dimethylbutyl)-N'-phenyl-p-phenylenediamine (6-PPD) is a widely used antioxidant in tire rubber known to enter the aquatic environment via road runoff. The associated transformation product (TP) 6-PPD quinone (6-PPDQ) causes extreme acute toxicity in some fish species (e.g., coho salmon). To interpret the species-specific toxicity, information about biotransformation products of 6-PPDQ would be relevant. This study investigated toxicokinetics of 6-PPD and 6-PPDQ in the zebrafish embryo (ZFE) model. Over 96 h of exposure, 6-PPD and 6-PPDQ accumulated in the ZFE with concentration factors ranging from 140 to 2500 for 6-PPD and 70 to 220 for 6-PPDQ. A total of 22 TPs of 6-PPD and 12 TPs of 6-PPDQ were tentatively identified using liquid chromatography coupled to high-resolution mass spectrometry. After 96 h of exposure to 6-PPD, the TPs of 6-PPD comprised 47% of the total peak area (TPA), with 4-hydroxydiphenylamine being the most prominent in the ZFE. Upon 6-PPDQ exposure, >95% of 6-PPDQ taken up in the ZFE was biotransformed, with 6-PPDQ + O + glucuronide dominating (>80% of the TPA). Among other TPs of 6-PPD, a reactive N-phenyl-p-benzoquinone imine was found. The knowledge of TPs of 6-PPD and 6-PPDQ from this study may support biotransformation studies in other organisms.

Amino antioxidants: A review of their environmental behavior, human exposure, and aquatic toxicity.

Amino antioxidants (AAOs), a suite of emerging organic contaminants, have been widely used in numerous industrial and commercial products to inhibit oxidation and corrosion. Recently, their environmental ubiquity, health risks, bioaccumulative and toxic potential have led to mounting public concern. This review summarizes the current state of knowledge on the production and usage, environmental occurrence, bioavailability, human exposure, and aquatic toxicity of representative AAOs, and provides suggestions for future research directions. Previous studies have revealed widespread distribution of many AAOs in various environmental matrixes, including air, water, sediment, dust, and biota. In addition to parent compounds, their degradation products, such as 2-anilino-5-(1,3-dimethylbutylamino)-1,4-benzoquinone (6PPD-Q) and 4-nitrodiphenylamine (4-NO2-DPA), have also been detected at high levels in multiple compartments. Dust ingestion and air inhalation are the two most well-investigated routes for human exposure to AAOs and their transformation products, while studies on other pathways (e.g., skin contact and dietary intake) still remain extremely limited. Moreover, AAO burdens in human tissue have been poorly documented. Toxicological data have shown that a few AAOs may cause teratogenic, developmental, reproductive, endocrinic, neuronic, and genetic toxicity to aquatic organisms, and the toxic capacities of degradation products differ from their precursors. Future studies should focus on elucidating AAO exposure for humans and associated health risks. Additionally, more attention should be given to AAO transformation products (particularly those quinoid derivatives possessing substantial affinity with DNA) and to the effects of complex mixtures of these chemicals.

Pooled Population Pharmacokinetic Analysis of Tribendimidine for the Treatment of Opisthorchis viverrini Infections.

Opisthorchiasis, caused by the foodborne trematode Opisthorchis viverrini, affects more than 8 million people in Southeast Asia. In the framework of a phase 2b clinical trial conducted in Lao People's Democratic Republic, pharmacokinetic samples were obtained from 125 adult and adolescent O. viverrini-infected patients treated with 400 mg tribendimidine following the design of a sparse sampling scheme at 20 min and 2, 7.75, 8, and 30 h after treatment using dried blood spot sampling. Pharmacokinetic data for the metabolites deacetylated amidantel (dADT) and acetylated dADT (adADT) were pooled with data from two previous ascending-dose trials and evaluated using nonlinear mixed-effects modeling. The observed pharmacokinetic data were described using a flexible transit absorption model for the active metabolite dADT, followed by one-compartment disposition models for both metabolites. Significant covariates were age, body weight, formulation, and breaking of the enteric coating on the tablets. There were significant associations between O. viverrini cure and both the dADT maximum concentration and the area under the concentration-time curve (P < 0.001), with younger age being associated with a higher probability of cure. Modeling and simulation of exposures in patients with different weight and age combinations showed that an oral single dose of 400 mg tribendimidine attained therapeutic success in over 90% of adult patients. Our data confirmed that tribendimidine could be a valuable novel alternative to the standard treatment, praziquantel, for the treatment of O. viverrini infections.

In Vitro and In Vivo Drug-Drug Interaction Study of the Effects of Ivermectin and Oxantel Pamoate on Tribendimidine.

Soil-transmitted helminth (STH) infections still remain a major health problem in poor rural settings. The lack of efficacious drugs against all STH species raises interest in drug combinations. Drug-drug interactions (DDIs) are, however, of major concern, so careful in vitro and in vivo characterization is needed. The combination of tribendimidine with either ivermectin or oxantel pamoate targets a broad range of STHs and thus represents a promising treatment alternative. Drug-drug interactions, however, have not yet been investigated. Therefore, the effects of combinations of ivermectin, oxantel pamoate, and tribendimidine's active metabolite deacylated amidantel (dADT) on cytochrome P450 (CYP450) metabolism were evaluated, followed by a pharmacokinetic analysis of tribendimidine and ivermectin alone and in combination in healthy rats. Oxantel pamoate is only poorly absorbed and was therefore excluded from pharmacokinetic analysis. No evident effect was observed for tribendimidine-oxantel pamoate at the CYP450 metabolism level, whereas a combination of tribendimidine and ivermectin led to moderately increased CYP2D6 inhibition compared to ivermectin or tribendimidine alone. Coadministration of tribendimidine with ivermectin altered neither the time to maximum concentration of drug in plasma (Tmax) nor the elimination half-lives of dADT, the acetylated derivative of amidantel (adADT), and ivermectin. While the area under the concentration-versus-time curve (AUC) and maximum concentration of drug in plasma (Cmax) values of dADT, adADT, and ivermectin are reduced by coadministration, the change is insufficient to declare that a DDI has been detected. Further studies are necessary to understand the observed interaction of tribendimidine and ivermectin, which is not related to P450 metabolism, and its significance for the situation in humans.

Pharmacokinetics of a Pediatric Tribendimidine Dose-Finding Study To Treat Hookworm Infection in African Children.

Tribendimidine is a broad-spectrum anthelminthic available in China, which is currently being pursued for U.S. Food and Drug Administration approval for soil-transmitted helminth infections. Pharmacokinetic (PK) studies with tribendimidine in children, the main target group for treatment programs, have not been conducted to date. In the framework of a dose-ranging study in hookworm-infected school-aged children in Côte d'Ivoire, children were treated with either 100, 200, or 400 mg tribendimidine. Dried blood spot samples were collected up to 22 h after treatment. The active metabolite, deacetylated amidantel (dADT) and its metabolite acylated dADT (adADT) were quantified using liquid chromatography tandem mass spectrometry. PK parameters were calculated using a noncompartmental model, and univariate logistic regression was applied using maximal blood concentrations (Cmax) and area under the blood concentration-time curve for 0 to 22 h (AUC0-22) as predictors of drug efficacy. Dried blood spot samples of 101 children were analyzed. We observed a less than proportional and proportional exposure in dADT's median Cmax and AUC0-22, respectively, following administration of 100 mg (Cmax = 853 ng/ml; AUC0-22 = 3,019 h · ng/ml) and 400 mg (Cmax = 2,275 ng/ml; AUC0-22 = 12,530 h · ng/ml) tribendimidine. There were large, dose-independent variations in the time to Cmax (Tmax) and ratios of dADT to adADT. We did not detect an influence of Cmax or AUC0-22 of dADT or adADT on drug efficacy or adverse events. Since our study population was bearing hookworm infection of mainly low intensity, additional studies with heavy intensity infections might be required to confirm this observation.

Acute Effects of Riluzole and Retigabine on Axonal Excitability in Patients With Amyotrophic Lateral Sclerosis: A Randomized, Double-Blind, Placebo-Controlled, Crossover Trial.

Increased excitability of motor neurons in patients with amyotrophic lateral sclerosis (ALS) may be a relevant factor leading to motor neuron damage. This randomized, double-blind, three-way crossover, placebo-controlled study evaluated peripheral motor nerve excitability testing as a biomarker of hyperexcitability and assessed the effects of riluzole and retigabine in 18 patients with ALS. We performed excitability testing at baseline, and twice after participants had received a single dose of either 100 mg riluzole, 300 mg retigabine, or placebo. Between- and within-day repeatability was at least acceptable for 14 out of 18 recorded excitability variables. No effects of riluzole on excitability testing were observed, but retigabine significantly decreased strength-duration time-constant (9.2%) and refractoriness at 2 ms (10.2) compared to placebo. Excitability testing was shown to be a reliable biomarker in patients with ALS, and the acute reversal of previously abnormal variables by retigabine justifies long-term studies evaluating the impact on disease progression and survival.

Beneficial Combination of Lacosamide with Retigabine in Experimental Animals: An Isobolographic Analysis.

BACKGROUND/AIM: To isobolographically determine the types of interactions that occur between retigabine and lacosamide (LCM; two third-generation antiepileptic drugs) with respect to their anticonvulsant activity and acute adverse effects (sedation) in the maximal electroshock-induced seizures (MES) and chimney test (motor performance) in adult male Swiss mice. METHODS: Type I isobolographic analysis for nonparallel dose-response effects for the combination of retigabine with LCM (at the fixed-ratio of 1:1) in both the MES and chimney test in mice was performed. Brain concentrations of retigabine and LCM were measured by high-pressure liquid chromatography (HPLC) to characterize any pharmacokinetic interactions occurring when combining these drugs. RESULTS: Linear regression analysis revealed that retigabine had its dose-response effect line nonparallel to that of LCM in both the MES and chimney tests. The type I isobolographic analysis illustrated that retigabine combined with LCM (fixed-ratio of 1:1) exerted an additive interaction in the mouse MES model and sub-additivity (antagonism) in the chimney test. With HPLC, retigabine and LCM did not mutually change their total brain concentrations, thereby confirming the pharmacodynamic nature of the interaction. CONCLUSION: LCM combined with retigabine possesses a beneficial preclinical profile (benefit index ranged from 2.07 to 2.50) and this 2-drug combination is worth recommending as treatment plan to patients with pharmacoresistant epilepsy.

Validated LC-MS-MS Method for Determination of SF0034 in Mice Plasma: Application to a Pharmacokinetic Study in Mice.

A rapid and sensitive assay method has been developed and validated using liquid chromatography coupled to tandem mass spectrometry with electrospray ionization in the positive-ion mode for the estimation of SF0034 in mice plasma. The assay procedure involves a simple protein precipitation of SF0034 and tolbutamide (internal standard, IS) from mice plasma. Chromatographic separation was performed on an Atlantis dC18 column using an isocratic mobile phase comprising 0.2% formic acid:acetonitrile (10:90, v/v) at a flow rate of 0.60 mL/min. The total run time was 2.5 min. For mass spectrometric detection, the multiple reaction monitoring was used and ion transitions monitored were m/z 322 → 248 for SF0034 and 271 → 155 for IS. Method validation was performed as per regulatory guidelines and the results met the acceptance criteria. A calibration curve was constructed in the range of 2.08-2,078 ng/mL. The intra- and inter-day precision was in the range of 1.06-14.4% and 7.16-11.7%, respectively. This novel method has been applied to a pharmacokinetic study in mice.

Biotransformation of 2,4-toluenediamine in human skin and reconstructed tissues.

Reconstructed human epidermis (RHE) is used for risk assessment of chemicals and cosmetics and RHE as well as reconstructed human full-thickness skin (RHS) become important for e.g., the pre-clinical development of drugs. Yet, the knowledge regarding their biotransformation capacity is still limited, although the metabolic activity is highly relevant for skin sensitization, genotoxicity, and the efficacy of topical dermatics. The biotransformation of the aromatic amine 2,4-toluenediamine (2,4-TDA) has been compared in two commercially available RHS to normal human skin ex vivo, and in primary epidermal keratinocytes and dermal fibroblasts as well as in vitro generated epidermal Langerhans cells and dermal dendritic cells. The mono N-acetylated derivative N-(3-amino-4-methyl-phenyl)acetamide (M1) was the only metabolite detectable in substantial amounts indicating the predominance of N-acetylation. RHS exceeded human skin ex vivo in N-acetyltransferase activity and in cell cultures metabolite formation ranked as follows: keratinocytes > fibroblasts ~ Langerhans cells ~ dendritic cells. In conclusion, our results underline the principal suitability of RHS as an adequate test matrix for the investigation of N-acetylation of xenobiotics which is most relevant for risk assessment associated with cutaneous exposure to aromatic amines.

Population Pharmacokinetic Modeling of Tribendimidine Metabolites in Opisthorchis viverrini-Infected Adults.

There is a pressing need for alternative treatments against the liver fluke Opisthorchis viverrini Oral tribendimidine is a promising candidate, but its population pharmacokinetic properties are unknown. Two phase IIa trials were conducted in Laos in O. viverrini-infected adults receiving single oral doses of 25 to 600 mg tribendimidine administered as different formulations in each study (study 1 used 200-mg tablets, and study 2 used 50-mg tablets). Venous whole blood, plasma, and capillary dried blood spots were sampled frequently from 68 adults, and concentrations of the tribendimidine metabolites dADT (deacetylated amidantel) and adADT (acetylated dADT) were measured. Population pharmacokinetics were assessed by using nonlinear mixed-effects modeling. The relationship between drug exposure and cure (assessed at 21 days posttreatment) was evaluated by using univariable logistic regression. A six-transit compartment absorption model with a one-disposition compartment for each metabolite described the data well. Compared to the 50-mg formulation (study 2), the 200-mg formulation (study 1) had a 40.1% higher mean transit absorption time, a 113% higher dADT volume of distribution, and a 364% higher adADT volume of distribution. Each 10-year increase in age was associated with a 12.7% lower dADT clearance and a 21.2% lower adADT clearance. The highest cure rates (≥55%) were observed with doses of ≥100 mg. Higher dADT, but not adADT, peak concentrations and exposures were associated with cure (P = 0.004 and 0.003, respectively). For the first time, population pharmacokinetics of tribendimidine have been described. Known differences in the 200-mg versus 50-mg formulations were captured by covariate modeling. Further studies are needed to validate the structural model and confirm covariate relationships. (This study has been registered with the ISRCTN Registry under no. ISRCTN96948551.).

Single-Ascending-Dose Pharmacokinetic Study of Tribendimidine in Opisthorchis viverrini-Infected Patients.

Praziquantel is the only drug available for the treatment of Opisthorchis viverrini infections. Tribendimidine has emerged as a potential treatment alternative; however, its pharmacokinetic (PK) properties have not been sufficiently studied to date. Via two phase IIa dose-finding studies, 68 O. viverrini patients were treated with 25- to 600-mg doses of tribendimidine using 50- and 200-mg tablet formulations. Plasma, blood, and dried blood spots (DBS) were sampled at selected time points. The two main metabolites of tribendimidine, active deacetylated amidantel (dADT) and acetylated dADT (adADT), were analyzed in plasma, blood, and DBS. PK parameters were estimated by noncompartmental analysis. An acceptable agreement among plasma and DBS concentrations was observed, with a mean bias of ≤10%, and 60% dADT and 74% adADT concentrations being within ±20% margins. We found that 200-mg tribendimidine tablets possess immediate floating characteristics, which led to variable time to maximal concentration of drug (Tmax) values (2 to 24 h) between individuals. Dose proportionality was observed for dADT from 25 to 200 mg using 50-mg tablets, but at higher dosages (200 to 600 mg), saturation occurred. The median ratio of the area under the plasma concentration-time curve from 0 to 24 h (AUC0-24) of dADT to the AUC0- 24 of adADT ranged from 0.8 to 26.4, suggesting substantial differences in acetylation rates. Cure rates ranged from 11% (25-mg dose) to 100% (400-mg dose). Cured patients showed significantly higher dADT maximal serum concentrations (Cmax) and AUC0-24 values than uncured patients. Tribendimidine is a promising drug for the treatment of opisthorchiasis. However, the tablet formulation should be optimized to achieve consistent absorption among patients. Further studies are warranted to assess the large differences between individuals in the rate of metabolic turnover of dADT to adADT. (This study has been registered with the ISRCTN Registry under no. ISRCTN96948551.).

New antiepileptic drugs: focus on ezogabine, clobazam, and perampanel.

Ezogabine, clobazam, and perampanel are among the newest antiseizure drugs approved by the Food and Drug Administration between 2011 and 2012. Ezogabine and perampanel are approved for adjunctive treatment of partial epilepsy. Perampanel is also approved for adjunctive treatment of primary generalized tonic-clonic seizures. Ezogabine and perampanel have novel mechanisms of action. Ezogabine binds to voltage-gated potassium channels and increases the M-current thereby causing membrane hyperpolarization. Perampanel is a selective, non-competitive 2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid receptor antagonist, which reduces neuronal excitation. Clobazam has been used worldwide since the 1970s and is approved for adjunctive treatment of seizures associated with Lennox-Gastaut syndrome. Clobazam is the only 1,5-benzodiazepine currently in clinical use, which is less sedating than the commonly used 1,4-benzodiazepines. Phase III multicenter, randomized, double-blind, placebo-controlled trials demonstrated efficacy and good tolerability of these 3 new antiepileptic drugs. These drugs represent a welcome addition to the armamentarium of practitioners, but it remains to be seen how they will affect the landscape of pharmacoresistant epilepsy.

Real-time detection of p-phenylenediamine penetration into human skin by in vivo Raman spectroscopy.

BACKGROUND: Penetration, autoxidation and N-acetylation of p-phenylenediamine (PPD) have been studied in vitro and ex vivo. However, a clear understanding of in vivo PPD penetration and the formation of PPD derivatives is lacking. OBJECTIVES: To obtain insights into the in vivo penetration, clearance and formation of PPD derivatives in human skin. METHODS: Patch test chambers containing PPD 1% pet. were applied to the forearms of two human volunteers, with increasing application times. Non-invasive Raman microspectroscopy was used for detection of PPD (derivatives) in skin at several follow-up times. RESULTS: Application of a PPD 1% pet. patch for 30 min resulted in substantial amounts of PPD in the stratum corneum of 90 mg PPD/g keratin. PPD contents were highest after three applications for 1 h each (330 mg PPD/g keratin), followed by single applications for 2 h 40 min, 2 h, and 23 h. The PPD half-time in the skin was 3 h. No spectral contributions of Bandrowski's base, monoacetyl-PPD and diacetyl-PPD were detected. CONCLUSIONS: We have gained insights into the in vivo penetration of PPD in human skin by using non-invasive Raman spectroscopy. Penetration into the skin was fast, and the PPD concentrations detected in the stratum corneum were high. PPD was detected in both the stratum corneum and the viable epidermis. Oxidized or acetylated PPD derivatives could not be detected.

Effect of scavenger receptor class B type I antagonist ITX5061 in patients with hepatitis C virus infection undergoing liver transplantation.

Hepatitis C virus (HCV) entry inhibitors have been hypothesized to prevent infection of the liver after transplantation. ITX5061 is a scavenger receptor class B type I antagonist that blocks HCV entry and infection in vitro. We assessed the safety and efficacy of ITX5061 to limit HCV infection of the graft. The study included 23 HCV-infected patients undergoing liver transplantation. The first 13 "control" patients did not receive drug. The subsequent 10 patients received 150 mg of ITX5061 immediately before and after transplant and daily for 1 week thereafter. ITX5061 pharmacokinetics and plasma HCV RNA were quantified. Viral genetic diversity was measured by ultradeep pyrosequencing (UDPS). ITX5061 was well tolerated with measurable plasma concentrations during therapy. Although the median HCV RNA reduction was greater in ITX-treated patients at all time points in the first week after transplantation, there was no difference in the overall change in the area over the HCV RNA curve in the 7-day treatment period. However, in genotype (GT) 1-infected patients, treatment was associated with a sustained reduction in HCV RNA levels compared to the control group (area over the HCV RNA curve analysis, P = 0.004). UDPS revealed a complex and evolving pattern of HCV variants infecting the graft during the first week. ITX5061 significantly limited viral evolution where the median divergence between day 0 and day 7 was 3.5% in the control group compared to 0.1% in the treated group. In conclusion, ITX5061 reduces plasma HCV RNA after transplant notably in GT 1-infected patients and slows viral evolution. Following liver transplantation, the likely contribution of extrahepatic reservoirs of HCV necessitates combining entry inhibitors such as ITX5061 with inhibitors of replication in future studies.

Development and validation of an LC-MS/MS method for determination of p-phenylenediamine and its metabolites in blood samples.

In some developing countries, p-phenylenediamine (PPD) is used in combination with Henna as hair dye or skin decoration. A sensitive LC-MS/MS method was developed and validated for the simultaneous determination of p-phenylenediamine (PPD) and its metabolites N-acetyl-p-phenylenediamine (MAPPD) and N,N-diacetyl-p-phenylenediamine (DAPPD) in human blood. Acetanilide was used as an internal standard (IS). The LC-MS/MS was operated under multiple reaction-monitoring mode using the electrospray positive ionization technique. The transition ions m/z 109→92, m/z 151→92, m/z 193→92, and m/z 136→77 were selected for the quantification of PPD, MAPPD, DAPPD, and IS, respectively. The linear range was 10-2000ng/mL for all the compounds. The absolute recoveries were 51.94, 56.20 and 54.88% for PPD, MAPPD and DAPPD, respectively. Intra- and inter-assay imprecision were lower than 14% (RSD), and the bias of the assay was lower than 15% for all the compounds. The stability studies demonstrated that critical degradation for PPD in blood samples and autosampler occurred after 6h, while MAPPD and DAPPD were stable in blood samples and the autosampler up to 48h and 24h, respectively. This newly developed method allows for the detection of PPD and its metabolites in blood samples in the clinical and forensic setting.

Synergistic Interaction of Retigabine with Levetiracetam in the Mouse Maximal Electroshock-Induced Seizure Model: A Type II Isobolographic Analysis.

BACKGROUND/AIMS: To assess interactions between retigabine and levetiracetam in suppressing maximal electroshock-induced tonic seizures in Albino Swiss mice, type II isobolographic analysis was used. Total brain antiepileptic drug concentrations were measured with high pressure liquid chromatography. RESULTS: The combinations of retigabine with levetiracetam at the fixed-ratios of 1:5 and 1:10 were supra-additive (synergistic; p < 0.05) in terms of seizure suppression, while the combinations at the fixed-ratios of 1:1 and 1:2 were additive. No pharmacokinetic changes in total brain concentrations of levetiracetam and retigabine were documented, indicating the pharmacodynamic nature of interaction between these antiepileptic drugs in the mouse maximal electroshock-induced tonic seizure model. CONCLUSION: The combination of retigabine with levetiracetam at the fixed-ratios of 1:5 and 1:10 appears to be particularly beneficial combination exerting supra-additive interaction in suppressing maximal electroshock-induced tonic seizures.

Human systemic exposure to [¹4C]-paraphenylenediamine-containing oxidative hair dyes: Absorption, kinetics, metabolism, excretion and safety assessment.

Systemic exposure was measured in humans after hair dyeing with oxidative hair dyes containing 2.0% (A) or 1.0% (B) [(14)C]-p-phenylenediamine (PPD). Hair was dyed, rinsed, dried, clipped and shaved; blood and urine samples were collected for 48 hours after application. [(14)C] was measured in all materials, rinsing water, hair, plasma, urine and skin strips. Plasma and urine were also analysed by HLPC/MS/MS for PPD and its metabolites (B). Total mean recovery of radioactivity was 94.30% (A) or 96.21% (B). Mean plasma Cmax values were 132.6 or 97.4 ng [(14)C]-PPDeq/mL, mean AUC(0-∞) values 1415 or 966 ng [(14)C]-PPDeq/mL*hr in studies A or B, respectively. Urinary excretion of [(14)C] mainly occurred within 24 hrs after hair colouring with a total excretion of 0.72 or 0.88% of applied radioactivity in studies A or B, respectively. Only N,N'-diacetylated-PPD was detected in plasma and the urine. A TK-based human safety assessment estimated margins of safety of 23.3- or 65-fold relative to respective plasma AUC or Cmax values in rats at the NOAEL of a toxicity study. Overall, hair dyes containing PPD are unlikely to pose a health risk since they are used intermittently and systemic exposure is limited to the detoxified metabolite N,N'-diacetyl-PPD.

P-retigabine: an N-propargyled retigabine with improved brain distribution and enhanced antiepileptic activity.

Retigabine (RTG, [ethyl N-[2-amino-4-[(4-fluorophenyl)methyl]amino] phenyl] carbamate]) is a first-in-class antiepileptic drug that acts by potentiating neuronal KCNQ potassium channels; however, it has less than optimal brain distribution. In this study, we report that P-RTG (ethyl N-[2-amino-4-((4-fluorobenzyl)(prop-2-ynyl)amino)phenyl]carbamate), an RTG derivative that incorporates a propargyl group at the N position of the RTG linker, exhibits an inverted brain distribution compared with RTG. The brain-to-plasma concentration ratio of P-RTG increased to 2.30 compared with 0.16 for RTG. However, the structural modification did not change the drug's potentiation potency, subtype selectivity, or RTG molecular determinants on KCNQ channels. In addition, in cultured hippocampal neurons, P-RTG exhibited a similar capability as RTG for suppressing both induced and spontaneous action potential firing. Notably, P-RTG antiepileptic activity in the maximal electroshock (MES)-induced mouse seizure model was significantly enhanced to a value 2.5 times greater than that of RTG. Additionally, the neurotoxicity of P-RTG in the rotarod test was comparable with that of RTG. Collectively, our results indicate that the incorporation of a propargyl group significantly improves the RTG brain distribution, supporting P-RTG as a promising antiepileptic drug candidate. The strategy for improving brain-to-plasma distribution of RTG might be applicable for the drug development of other central nervous system diseases.

Effect of hemodialysis on pharmacokinetics of ezogabine/retigabine and its N-acetyl metabolite in patients with end stage renal disease.

Ezogabine (EZG)/retigabine (RTG) and its metabolites are mainly eliminated renally. This Phase I study assessed the effect of hemodialysis on the pharmacokinetics of EZG/RTG and its N-acetyl metabolite (NAMR) in patients with end-stage renal disease; tolerability of EZG/RTG was a secondary endpoint. Patients (N=8) received EZG/RTG 100 mg orally 4 hours before (Period 1) or following (Period 2) dialysis. Blood (both periods) and dialysate (Period 1) samples were taken up to 68 hours post dose. Tolerability was assessed throughout both periods. The area under the concentration- time curve (0-68 hours) for EZG/RTG was 33% lower (geometric mean ratio [90% confidence interval]: 0.67 [0.61, 0.73]) on dialysis versus off dialysis and 43% lower for NAMR (0.57 [0.53, 0.62]). Median (range) reductions in plasma concentrations from dialysis start to end were 52% (17-59%) for EZG/RTG and 51% (27-72%) for NAMR. EZG/RTG 100 mg was generally tolerated.

A novel design for a dose finding, safety, and drug interaction study of an antiepileptic drug (retigabine) in early clinical development.

OBJECTIVE: To obtain information on the acceptable doses of the antiepileptic drug (AED) retigabine (RTG), the maximum tolerated dose (MTD), drug interactions, safety and tolerability, and preliminary evidence of efficacy when administered as adjunctive therapy and as monotherapy. MATERIALS: Study 202 was an open-label, add-on study in patients with partial or generalized epilepsy treated with valproic acid (VPA), carbamazepine (CBZ), phenytoin (PHT), or topiramate (TPM) as monotherapy. Following baseline assessments, patients entered a dose titration phase of 28 – 56 days. The initial daily RTG dose was 100 or 200 mg (2 or 3 × daily). The RTG dose was increased every 1 - 2 weeks by 50 - 200 mg to a maximum of 1,600 mg/day. Once the RTG MTD had been attained, patients entered a 14-day maintenance period. Following this, the patient's background AED dose could be reduced, with the possibility of achieving RTG monotherapy. The final dosing regimen attained was maintained for an additional 14 days. Patients who completed study 202 could choose to continue treatment with RTG (with or without other AEDs) in study 208, the long-term extension of study 202. Safety assessments included adverse event (AE) monitoring, clinical laboratory evaluations, electrocardiograms, and physical and neurologic examinations. Patients' seizure diaries to assess the frequency and type of seizures, the percentage change in seizure rate, and the responder rate (>= 50% reduction in seizure rate from baseline) were evaluated. RESULTS: 60 patients (mean age 37.2, range 16 - 64 years) were enrolled in study 202, and 47 (78%) continued treatment with RTG in the extension study (208). In study 202, the most commonly reported AEs were: dizziness (53%), asthenia (42%), somnolence (33%), nausea (27%), speech disorder (27%), and tremor (27%). In the extension study, AEs were similar and included dizziness, somnolence, diplopia, feeling "drunk", confusion, fatigue, and dysarthria. The median percent reductions in 28-day seizure rate, relative to baseline in Studies 202 and 208, were ~ 20% and 47%, respectively. RTG did not alter the pharmacokinetics of the four monotherapy AEDs investigated. CBZ and PHT increased RTG clearance by 27% and 36%, respectively, whereas TPM and VPA had no effect on RTG clearance. CONCLUSIONS: Studies 202 and 208 provided critical information on RTG safety and tolerability, and reductions in seizure rates towards the design and conduct of subsequent pivotal clinical trials. Likewise, information regarding the appropriate dosage of RTG with VPA, CBZ, PHT, or TPM was obtained, which permitted the subsequent pivotal trials to be performed appropriately. *Currently at Shire Pharmaceuticals, Behavioral Health Business Unit, Wayne, PA, USA **Currently at University of Pennsylvania, Department of Neurology, Philadelphia, PA, USA.