Population pharmacokinetic-pharmacodynamic modeling of serum biomarkers as predictors of tumor dynamics following lenvatinib treatment in patients with radioiodine-refractory differentiated thyroid cancer (RR-DTC).

Lenvatinib is a receptor tyrosine kinase (RTK) inhibitor targeting vascular endothelial growth factor (VEGF) receptors 1-3, fibroblast growth factor (FGF) receptors 1-4, platelet-derived growth factor receptor-α (PDGFRα), KIT, and RET that have been implicated in pathogenic angiogenesis, tumor growth, and cancer. The primary objective of this work was to evaluate, by establishing quantitative relationships, whether lenvatinib exposure and longitudinal serum biomarker data (VEGF, Ang-2, Tie-2, and FGF-23) are predictors for change in longitudinal tumor size which was assessed based on data from 558 patients with radioiodine-refractory differentiated thyroid cancer (RR-DTC) receiving either lenvatinib or placebo treatment. Lenvatinib PK was best described by a 3-compartment model with simultaneous first- and zero-order absorption and linear elimination from the central compartment with significant covariates (body weight, albumin <30 g/dL, ALP>ULN, RR-DTC, RCC, HCC subjects, and concomitant CYP3A inhibitors). Except for body weight, none of the covariates have any clinically meaningful effect on exposure to lenvatinib. Longitudinal biomarker measurements over time were reasonably well defined by a PK/PD model with common EC50, Emax, and a slope for disease progression for all biomarkers. Longitudinal tumor measurements over time were reasonably well defined by a tumor growth inhibition Emax model, which in addition to lenvatinib exposure, included model-predicted relative changes from baseline over time for Tie-2 and Ang-2 as having significant association with tumor response. The developed PK/PD models pave the way for dose optimization and potential prediction of clinical response.

Population pharmacokinetic-pharmacodynamic analyses of amyloid positron emission tomography and plasma biomarkers for lecanemab in subjects with early Alzheimer's disease.

Lecanemab is a humanized immunoglobulin G1 monoclonal antibody that selectively binds to soluble Aß aggregate species, while demonstrating low affinity for Aß monomer. This article describes the population pharmacokinetic (PK) and PK/pharmacodynamic (PD) analyses for amyloid plaques, as measured using positron emission tomography (PET), and biomarkers of amyloid pathology as evidenced by Aß42/40 ratio and plasma p-tau181 following i.v. administration of lecanemab in subjects with early Alzheimer's disease. Lecanemab PKs were well-characterized with a two-compartment model with first-order elimination. Final PK model contained covariate effects of anti-drug antibody positive status, sex, body weight, and albumin on clearance. The time course of amyloid PET standard uptake ratio (SUVr), plasma Aß42/40 ratio, and p-tau181 were described using indirect response models with lecanemab exposure as a maximum effect function stimulating the reduction of SUVr, and as a linear function increasing Aß42/40 ratio and decreasing p-tau181 formation rates. PK/PD simulations show that 10 mg/kg biweekly dosing results in larger and faster decrease in SUVr and p-tau181 and increase in Aß42/40 ratio as compared to 10 mg/kg monthly dose. Furthermore, the PK/PD simulations showed that after treatment discontinuation the brain amyloid re-accumulation to baseline levels is slow with a recovery half-life of ~4 years, whereas plasma Aß42/40 ratio and p-tau181 return to baseline levels faster than amyloid. Given the relationship between changes in amyloid PET SUVr and soluble biomarkers, the developed PK/PD models can be used to inform lecanemab dose regimens in future clinical studies.

Phase 1 study on the safety and efficacy of E6011, antifractalkine antibody, in patients with Crohn's disease.

BACKGROUND AND AIM: E6011 is a humanized monoclonal antibody targeting fractalkine (FKN), a CX3C chemokine, which regulates leukocyte trafficking during inflammation. We evaluated the safety and pharmacokinetic profile of E6011 in patients with Crohn's disease (CD) and also performed preliminary pharmacodynamic (PD) and efficacy assessments. METHODS: This study included a 12-week multiple ascending dose (MAD) phase (2, 5, 10, and 15 mg/kg intravenously every 2 weeks, n = 6, 8, 7, and 7, respectively) and a 40-week Extension phase (n = 12) at the same dose as the MAD phase. Serum E6011, serum total FKN (free soluble FKN and E6011-FKN complex) as a PD marker and CD activity index were evaluated. The primary outcome was safety assessment in the MAD phase. RESULTS: Twenty-seven (96%) of 28 patients had previously been treated with anti-tumor necrosis factor α agents. During the MAD phase, adverse events (AEs) occurred in 18 (64%). The most common AE was nasopharyngitis (five patients, 18%). No severe AEs occurred. Serious AEs occurred in three patients, progression of CD in two, and anemia in one. Serum E6011 concentrations increased dose-dependently after infusion and reached a plateau around 4-6 weeks. Serum total FKN rose simultaneously. Five (18%) patients developed anti-E6011 antibodies during the study. Overall, clinical response and clinical remission were observed at Week 12 in 40% (10/25) and 16% (4/25) of active CD patients, respectively. CONCLUSION: E6011 was well-tolerated and might be effective in CD patients. These findings need to be clarified in a randomized controlled study.

Comparison of the Predictability of Human Hepatic Clearance for Organic Anion Transporting Polypeptide Substrate Drugs Between Different In Vitro-In Vivo Extrapolation Approaches.

Prediction of human pharmacokinetic profiles of drug candidates is an essential step toward first-in-human studies. However, it remains difficult to quantitatively predict hepatic clearance, particularly when hepatic uptake is mediated by transporter(s). Using 15 organic anion transporting polypeptide (OATP) substrate drugs, we tested 3 in vitro-in vivo extrapolation (IVIVE) approaches to predict overall hepatic intrinsic clearance in vivo (CLint,all,vivo). IVIVE approaches involved metabolic intrinsic clearance in human liver microsomes (CLint,met) with or without hepatocyte-to-buffer maximum unbound concentration ratio (Kp,uu,max) correction and uptake intrinsic clearance at 37°C (PSinf,37°C) in human hepatocyte suspensions. Kp,uu,max and PSinf,37°C values were determined in 2 hepatocyte batches, and all tested compounds showed temperature-dependent uptake, consistent with the fact of transporter-mediated uptake. CLint,met substantially underestimated CLint,all,vivo. By multiplying CLint,met by Kp,uu,max values, the prediction performance was much improved; however, in vitro-in vivo correlation was poor. Among the IVIVE approaches, PSinf,37°C showed the most robust correlation with CLint,all,vivo, and one of the hepatocyte batches could predict CLint,all,vivo with a minimal empirical scaling factor. These results suggested IVIVE with hepatic uptake clearance determined in hepatocyte suspensions as one of the most practical approaches for predicting CLint,all,vivo of OATP substrate drugs.

Investigation of utility of cerebrospinal fluid drug concentration as a surrogate for interstitial fluid concentration using microdialysis coupled with cisternal cerebrospinal fluid sampling in wild-type and Mdr1a(-/-) rats.

In drug discovery, the cerebrospinal fluid (CSF) drug concentration (CCSF) has been used as a surrogate for the interstitial fluid (ISF) concentration (CISF). However, the CCSF-to-CISF gradient suggested for P-glycoprotein (P-gp) substrates in rodents causes uncertainty in CISF estimations and subsequent pharmacokinetic-pharmacodynamic analyses. To evaluate the utility of CCSF as a surrogate for CISF, this study directly compared the CCSF with the CISF of 12 compounds, including P-gp substrates, under steady-state conditions in wild-type and Mdr1a(-/-) rats using microdialysis coupled with cisternal CSF sampling. In wild-type rats, the ISF-to-unbound plasma (Kp,uu,ISF) and CSF-to-unbound plasma (Kp,uu,CSF) concentration ratios of the P-gp substrates, except for metoclopramide, were lower than those of the non-P-gp substrates, and the Kp,uu,CSF values were within or close to 3-fold of the Kp,uu,ISF values for all the compounds examined. The Kp,uu,CSF values of the selected P-gp substrates increased in Mdr1a(-/-) rats with a similar magnitude to the Kp,uu,ISF values, resulting in the Kp,uu,CSF-to-Kp,uu,ISF ratios being unchanged. These results suggested that P-gp-mediated active efflux at the blood-brain barrier is a major determinant not only for CISF, but also for CCSF, and that CCSF can be used as a surrogate for CISF even for P-gp substrates in rats.

Investigation of Fluorescein Derivatives as Substrates of Organic Anion Transporting Polypeptide (OATP) 1B1 To Develop Sensitive Fluorescence-Based OATP1B1 Inhibition Assays.

Organic anion transporting polypeptide (OATP) 1B1 plays an important role in the hepatic uptake of various drugs. Because OATP1B1 is a site of drug-drug interactions (DDIs), evaluating the inhibitory potential of drug candidates on OATP1B1 is required during drug development. For establishing a highly sensitive, high-throughput fluorescence-based OATP1B1 inhibition assay system, the present study focused on fluorescein (FL) and its derivatives and evaluated their uptake via OATP1B1 as well as OATP1B3 and OATP2B1 using the transporter-expressing human embryonic kidney 293 cells. We identified 2',7'-dichlorofluorescein (DCF), 4',5'-dibromofluorescein (DBF), and Oregon green (OG) as good OATP1B1 substrates with Km values of 5.29, 4.16, and 54.1 µM and Vmax values of 87.9, 48.1, and 187 pmol/min/mg protein, respectively. In addition to FL, fluo-3, and 8-fluorescein-cAMP, OG, and DBF were identified as OATP1B3 substrates. FL, OG, DCF, and DBF were identified as OATP2B1 substrates. Among the FL derivatives, DCF displayed the highest OATP1B1-mediated uptake. The Ki values of 14 compounds on OATP1B1 determined with DCF as a probe exhibited good agreement with those obtained using [(3)H]estradiol-17ß-glucuronide (E2G) as a substrate, whereas [(3)H]estrone-3-sulfate and [(3)H]sulfobromophthalein yielded higher Ki values for all inhibitors than DCF. Mutually competitive inhibition observed between DCF and E2G suggested that they share the same binding site on OATP1B1. Therefore, DCF as well as E2G can be used as sensitive probes for in vitro OATP1B1 inhibition assays, which will help mitigate the risk of false-negative DDI predictions potentially caused by substrate-dependent Ki variations.

HPLC with fluorescence detection assay of perampanel, a novel AMPA receptor antagonist, in human plasma for clinical pharmacokinetic studies.

Perampanel (Fycompa®), a novel α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist, is registered for the adjunctive treatment of patients (aged ≥12 years) with refractory partial-onset seizures. To support therapeutic drug monitoring, a simple high-performance liquid chromatography (HPLC) assay with fluorescence detection was developed to determine perampanel concentrations in human plasma and validated to support clinical trials. Human plasma samples (1.0 mL) were processed by liquid extraction using diethyl ether, followed by chromatographic separation on a YMC Pack Pro C18 column (150 × 4.6 mm i.d., 5 µm) with isocratic elution of acetonitrile-water-acetic acid-sodium acetate (840:560:3:1.8, v/v/v/w) at a flow rate of 1.0 mL/min. Column eluent was monitored at excitation and emission wavelengths of 290 and 430 nm, respectively. The assay was linear (range 1.0-500 ng/mL) and this could be extended to 25 µg/mL by 50-fold dilution integrity. No endogenous peaks were detected in the elution of analytes in drug-free blank human plasma from six individuals and no interference was observed with co-medications tested. Intra- and inter-batch reproducibility studies demonstrated accuracy and precision within the acceptance criteria of bioanalytical guidelines. Validation data demonstrated that our assay is simple, selective, reproducible and suitable for therapeutic drug monitoring of perampanel.

High-performance liquid chromatography-tandem mass spectrometry method for the determination of perampanel, a novel α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor antagonist in human plasma.

Perampanel (Fycompa(®)) is a novel α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist registered for the adjunctive treatment of patients (≥12 years) with refractory partial onset seizures. In order to support clinical trials, as well as therapeutic drug monitoring, a sensitive bioanalytical method for the determination of perampanel concentrations in human plasma was established and validated using liquid chromatography with tandem mass spectrometry. Perampanel and an internal standard were extracted from human plasma (100 µL) by liquid extraction using methyl t-butyl ether, then evaporated and reconstituted. The chromatographic separation was conducted on a C8 column with isocratic elution at a flow rate of 0.2 mL/min. The established method showed linearity in the range 0.25-200 ng/mL with correlation coefficients of >0.99 that could be extended 10-fold as validated by dilution integrity analyses. No significant endogenous peaks were detected in the elution of analytes in blank human plasma and no significant matrix effect was observed. The intra- and inter-batch reproducibility analyses demonstrated accuracy and precision within the acceptance criteria. To check the impact of anti-epileptic drugs on the perampanel assay, accuracy, precision, and specificity were assessed in the presence of 14 anti-epileptic drugs. No anti-epileptic drugs at clinically relevant levels showed a significant impact on the perampanel assay.

Investigation of the impact of substrate selection on in vitro organic anion transporting polypeptide 1B1 inhibition profiles for the prediction of drug-drug interactions.

The risk assessment of organic anion transporting polypeptide (OATP) 1B1-mediated drug-drug interactions (DDIs) is an indispensable part of drug development. We previously reported that in vitro inhibitory potencies of several inhibitors on OATP1B1 depend on the substrates when prototypical substrates, estradiol-17ß-glucuronide (E2G), estrone-3-sulfate, and sulfobromophthalein were used as test substrates. The purpose of this study was to comprehensively investigate this substrate-dependent inhibition of OATP1B1 using clinically relevant OATP1B1 inhibitors and substrate drugs. Effects of cyclosporine A (CsA), rifampin, and gemfibrozil on OATP1B1-mediated uptake of 12 substrate drugs were examined in OATP1B1-expressing human embryonic kidney 293 cells. The Ki values (µM) for CsA varied from 0.0771 to 0.486 (6.3-fold), for rifampin from 0.358 to 1.23 (3.4-fold), and for gemfibrozil from 9.65 to 252 (26-fold). Except for the inhibition of torasemide uptake by CsA and that of nateglinide uptake by gemfibrozil, the Ki values were within 2.8-fold of those obtained using E2G as a substrate. Preincubation potentiated the inhibitory effect of CsA on OATP1B1 with similar magnitude regardless of the substrates. R values calculated based on a static model showed some variation depending on the Ki values determined with various substrates, and such variability could have an impact on the DDI predictions particularly for a weak-to-moderate inhibitor (gemfibrozil). OATP1B1 substrate drugs except for torasemide and nateglinide, or E2G as a surrogate, is recommended as an in vitro probe in the inhibition experiments, which will help mitigate the risk of false-negative DDI predictions potentially caused by substrate-dependent Ki variation.

Design, synthesis, and structure-activity relationships of a series of novel N-aryl-2-phenylcyclopropanecarboxamide that are potent and orally active orexin receptor antagonists.

Herein we describe the design, synthesis, and structure-activity relationships (SARs) of a novel phenylcyclopropane series represented by 7 and 33 b as antagonists of orexin 1 and orexin 2 receptors. With 4 serving as the initial lead for the development of orexin antagonists, exploration of SAR resulted in improved binding affinity for orexin 1 and orexin 2 receptors. Among the synthesized compounds, 33 b ((-)-N-(5-cyanopyridin-2-yl)-2-[(3,4-dimethoxyphenyl)oxymethyl]-2-phenylcyclopropanecarboxamide) exhibited potent in vitro activity and oral efficacy in animal sleep measurement experiments. The results of our study suggest that compound 33 b may serve as a valuable template for the development of new orexin receptor antagonists.

Utility of cerebrospinal fluid drug concentration as a surrogate for unbound brain concentration in nonhuman primates.

In central nervous system drug discovery, cerebrospinal fluid (CSF) drug concentration (C(CSF)) has been widely used as a surrogate for unbound brain concentrations (C(u,brain)). However, previous rodent studies demonstrated that when drugs undergo active efflux by transporters, such as P-glycoprotein (P-gp), at the blood-brain barrier, the C(CSF) overestimates the corresponding C(u,brain). To investigate the utility of C(CSF) as a surrogate for interstitial fluid (ISF) concentration (C(ISF)) in nonhuman primates, this study simultaneously determined the C(CSF) and C(ISF) of 12 compounds, including P-gp substrates, under steady-state conditions in cynomolgus monkeys using intracerebral microdialysis coupled with cisternal CSF sampling. Unbound plasma concentrations of non- or weak P-gp substrates were within 2.2-fold of the C(ISF) or C(CSF), whereas typical P-gp substrates (risperidone, verapamil, desloratadine, and quinidine) showed ISF-to-plasma unbound (K(p,uu,ISF)) and CSF-to-plasma unbound concentration ratios (K(p,uu,CSF)) that were appreciably lower than unity. Although the K(p,uu,CSF) of quinidine, verapamil, and desloratadine showed a trend of overestimating the K(p,uu,ISF), K(p,uu,CSF) showed a good agreement with K(p,uu,ISF) within 3-fold variations for all compounds examined. C(u,brain) of some basic compounds, as determined using brain homogenates, overestimated the C(ISF) and C(CSF). Therefore, C(CSF) could be used as a surrogate for C(ISF) in nonhuman primates.

Genetic analysis of group composition and relatedness in white-headed langurs.

We collected fecal samples of white-headed langurs from 3 of the 4 remaining habitat fragments (Fa, Fb and CZ) located in southwestern Guangxi, China in Nov 2005, and used 5 microsatellite loci and the SRY gene to assess the relatedness between 46 langurs within and between groups. We observed 2 forms of group structure: one-male/multi-female groups (OMGs) and all-male groups (AMGs). One AMG in Fa was composed of 2 generations, included a father, 2 sons and 1 unrelated male, and all OMGs in all 3 habitats included 1 resident male, several adult females and offspring. Of the 21 identified father-offspring cases, the resident male fathered 20 (95%) and the non-resident male sired 1 (5%), suggesting that adult males had overwhelming priority of access to females as the resident male in an OMG, while the non-resident male may also have the opportunity to adopt surreptitious mating strategies.

A small-animal pharmacokinetic/pharmacodynamic PET study of central serotonin 1A receptor occupancy by a potential therapeutic agent for overactive bladder.

Serotonin 1A (5-HT1A) receptors have been mechanistically implicated in micturition control, and there has been a need for an appropriate biomarker surrogating the potency of a provisional drug acting on this receptor system for developing a new therapeutic approach to overactive bladder (OAB). Here, we analyzed the occupancy of 5-HT1A receptors in living Sprague-Dawley rat brains by a novel candidate drug for OAB, E2110, using positron emission tomography (PET) imaging, and assessed the utility of a receptor occupancy (RO) assay to establish a pharmacodynamic index translatable between animals and humans. The plasma concentrations inducing 50% RO (EC50) estimated by both direct and effect compartment models were in good agreement. Dose-dependent therapeutic effects of E2110 on dysregulated micturition in different rat models of pollakiuria were also consistently explained by achievement of 5-HT1A RO by E2110 in a certain range (≥ 60%). Plasma drug concentrations inducing this RO range and EC50 would accordingly be objective indices in comparing pharmacokinetics-RO relationships between rats and humans. These findings support the utility of PET RO and plasma pharmacokinetic assays with the aid of adequate mathematical models in determining the in vivo characteristics of a drug acting on 5-HT1A receptors and thereby counteracting OAB.

Substrate-dependent inhibition of organic anion transporting polypeptide 1B1: comparative analysis with prototypical probe substrates estradiol-17ß-glucuronide, estrone-3-sulfate, and sulfobromophthalein.

Organic anion transporting polypeptide (OATP) 1B1 plays an important role in the hepatic uptake of many drugs, and the evaluation of OATP1B1-mediated drug-drug interactions (DDIs) is emphasized in the latest DDI (draft) guidance documents from U.S. and E.U. regulatory agencies. It has been suggested that some OATP1B1 inhibitors show a discrepancy in their inhibitory potential, depending on the substrates used in the cell-based assay. In this study, inhibitory effects of 14 compounds on the OATP1B1-mediated uptake of the prototypical substrates [³H]estradiol-17ß-glucuronide (E2G), [³H]estrone-3-sulfate (E1S), and [³H]sulfobromophthalein (BSP) were studied in OATP1B1-transfected cells. Inhibitory potencies of tested compounds varied depending on the substrates. Ritonavir, gemfibrozil, and erythromycin caused remarkable substrate-dependent inhibition with up to 117-, 14-, and 13-fold difference in their IC50 values, respectively. Also, the clinically relevant OATP inhibitors rifampin and cyclosporin A exhibited up to 12- and 6-fold variation in their IC50 values, respectively. Regardless of the inhibitors tested, the most potent OATP1B1 inhibition was observed when [³H]E2G was used as a substrate. Mutual inhibition studies of OATP1B1 indicated that E2G and E1S competitively inhibited each other, whereas BSP noncompetitively inhibited E2G uptake. In addition, BSP inhibited E1S in a competitive manner, but E1S caused an atypical kinetics on BSP uptake. This study showed substrate-dependent inhibition of OATP1B1 and demonstrated that E2G was the most sensitive in vitro OATP1B1 probe substrate among three substrates tested. This will give us an insight into the assessment of clinically relevant OATP1B1-mediated DDI in vitro with minimum potential of false-negative prediction.

Species difference in the mechanism of nonlinear pharmacokinetics of E2074, a novel sodium channel inhibitor, in rats, dogs, and monkeys.

New chemical entities often exhibit nonlinear pharmacokinetics (PK) profiles in experimental animals. However, the number of studies that have focused on species differences in nonlinear PK is very limited; thus, the aim of this study was to clarify the mechanism of the nonlinear PK of E2074 (2-[(2R)-2-fluoro-3-{(3r)-[(3-fluorobenzyl)oxy]-8-azabicyclo[3.2.1]oct-8-yl}propyl]-4,5-dimethyl-2,4-dihydro-3H-1,2,4-triazol-3-one), a novel sodium channel inhibitor, in rats, dogs, and monkeys. Nonlinear PK profiles with more than dose-proportional increases of Cmax and area under the plasma concentration curve were observed in all species after oral administration. The Michaelis-Menten constant (Km) values of hepatic microsomal metabolism were 7.23 and 0.41 µM in rats and dogs in vitro, respectively, which were lower than the unbound maximum plasma concentrations after oral administration in vivo, indicating that the nonlinear PK in rats and dogs was attributable to the saturation of hepatic metabolism. However, we do not believe that the saturation of hepatic metabolism was the mechanism of nonlinearity in monkeys because of the high Km value (42.44 µM) observed in liver microsomes. Intestinal metabolism was observed in monkey intestinal microsomes but not in rats and dogs, and the nonlinear PK in monkeys was diminished by inhibition of intestinal metabolism with a concomitant oral dose of ketoconazole. These results suggest that saturation of the intestinal metabolism is the potential mechanism of nonlinearity in monkeys. P-glycoprotein was not involved in the nonlinear PK profiles in any species. In conclusion, the mechanism of the nonlinear PK of E2074 is species dependent, with the saturation of hepatic metabolism in rats and dogs and that of intestinal metabolism in monkeys being the primary cause.

Discovery of 2-(2-oxo-1-phenyl-5-pyridin-2-yl-1,2-dihydropyridin-3-yl)benzonitrile (perampanel): a novel, noncompetitive α-amino-3-hydroxy-5-methyl-4-isoxazolepropanoic acid (AMPA) receptor antagonist.

Dysfunction of glutamatergic neurotransmission has been implicated in the pathogenesis of epilepsy and numerous other neurological diseases. Here we describe the discovery of a series of 1,3,5-triaryl-1H-pyridin-2-one derivatives as noncompetitive antagonists of AMPA-type ionotropic glutamate receptors. The structure-activity relationships for this series of compounds were investigated by manipulating individual aromatic rings located at positions 1, 3, and 5 of the pyridone ring. This culminated in the discovery of 2-(2-oxo-1-phenyl-5-pyridin-2-yl-1,2-dihydropyridin-3-yl)benzonitrile (perampanel, 6), a novel, noncompetitive AMPA receptor antagonist that showed potent activity in an in vitro AMPA-induced Ca2+ influx assay (IC50=60 nM) and in an in vivo AMPA-induced seizure model (minimum effective dose of 2 mg/kg po). Perampanel is currently in regulatory submission for partial-onset seizures associated with epilepsy.

Perampanel: a novel, orally active, noncompetitive AMPA-receptor antagonist that reduces seizure activity in rodent models of epilepsy.

PURPOSE: To assess the pharmacology of perampanel and its antiseizure activity in preclinical models. Perampanel [2-(2-oxo-1-phenyl-5-pyridin-2-yl-1,2-dihydropyridin-3-yl) benzonitrile] is a novel, orally active, prospective antiepileptic agent currently in development for refractory partial-onset seizures. METHODS: Perampanel pharmacology was assessed by examining changes in intracellular free Ca(2+) ion concentration ([Ca(2+) ](i) ) in primary rat cortical neurones, and [(3) H]perampanel binding to rat forebrain membranes. Antiseizure activity of orally administered perampanel was examined in amygdala-kindled rats and in mice exhibiting audiogenic, maximal electroshock (MES)-induced, pentylenetetrazole (PTZ) -induced, or 6 Hz-induced seizures. KEY FINDINGS: In cultured rat cortical neurones, perampanel inhibited α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-induced increases in [Ca(2+) ](i) (IC(50) 93 nm vs. 2 µm AMPA). Perampanel had a minimal effect on N-methyl-d-aspartate (NMDA)-induced increases in [Ca(2+) ](i) , and only at a high concentration (30 µm). [(3) H]Perampanel binding to rat forebrain membranes was not significantly displaced by glutamate or AMPA but was displaced by the noncompetitive AMPA receptor antagonists CP465022 (K(i) 11.2 ± 0.8 nm) and GYKI52466 (K(i) 12.4 ± 1 µm). In mice, perampanel showed protective effects against audiogenic, MES-induced, and PTZ-induced seizures (ED(50) s 0.47, 1.6, and 0.94 mg/kg, respectively). Perampanel also inhibited 6 Hz electroshock-induced seizures when administered alone or in combination with other antiepileptic drugs (AEDs). In amygdala-kindled rats, perampanel significantly increased afterdischarge threshold (p<0.05 vs. vehicle), and significantly reduced motor seizure duration, afterdischarge duration, and seizure severity recorded at 50% higher intensity than afterdischarge threshold current (p<0.05 for all measures vs. vehicle). Perampanel caused dose-dependent motor impairment in both mice (TD(50) 1.8 mg/kg) and rats (TD(50) 9.14 mg/kg), as determined by rotarod tests. In mice, the protective index (TD(50) in rotarod test/ED(50) in seizure test) was 1.1, 3.8, and 1.9 for MES-induced, audiogenic, and PTZ-induced seizures, respectively. In rat, dog, and monkey, perampanel had a half-life of 1.67, 5.34, and 7.55 h and bioavailability of 46.1%, 53.5%, and 74.5%, respectively. SIGNIFICANCE: These data suggest that perampanel is an orally active, noncompetitive, selective AMPA receptor antagonist with potential as a broad spectrum antiepileptic agent.

Lineage-specific duplication and loss of pepsinogen genes in hominoid evolution.

Fourteen different pepsinogen-A cDNAs and one pepsinogen-C cDNA have been cloned from gastric mucosa of the orangutan, Pongo pygmaeus. Encoded pepsinogens A were classified into two groups, i.e., types A1 and A2, which are different in acidic character. The occurrence of 9 and 5 alleles of A1 and A2 genes (at least 5 and 3 loci), respectively was anticipated. Respective orthologous genes are present in the chimpanzee genome although their copy numbers are much smaller than those of the orangutan genes. Only A1 genes are present in the human probably due to the loss of the A2 gene. Molecular phylogenetic analyses showed that A1 and A2 genes diverged before the speciation of great hominoids. Further reduplications of respective genes occurred several times in the orangutan lineage, with much higher frequencies than those occurred in the chimpanzee and human lineages. The rates of non-synonymous substitutions were higher than those of synonymous ones in the lineage of A2 genes, implying the contribution of the positive selection on the encoded enzymes. Several sites of pepsin moieties were indeed found to be under positive selection, and most of them locate on the surface of the molecule, being involved in the conformational flexibility. Deduced from the known genomic structures of pepsinogen-A genes of primates and other mammals, the duplication/loss were frequent during their evolution. The extreme multiplication in the orangutan might be advantageous for digestion of herbaceous foods due to the increase in the level of enzymes in stomach and the diversification of enzyme specificity.

Relic of ancient recombinations in gibbon ABO blood group genes deciphered through phylogenetic network analysis.

The primate ABO blood group gene encodes a glycosyl transferase (either A or B type), and is known to have large coalescence times among the allelic lineages in human. We determined nucleotide sequences of ca. 2.2kb of this gene for 23 individuals of three gibbon species (agile gibbon, white-handed gibbon, and siamang), and observed a total of 24 haplotypes. We found relics of five ancient intragenic recombinations, occurred during ca. 2-7 million years ago, through a phylogenetic network analysis. The coalescence time between A and B alleles estimate precede the divergence (ca. 8MYA) of siamang and common gibbon lineages. This establishes the coexistence of divergent allelic lineages of the ABO blood group gene for a long period in the ancestral gibbon species, and strengthens the non-neutral evolution for this gene.

Relatedness in wild chimpanzees: influence of paternity, male philopatry, and demographic factors.

In chimpanzees (Pan troglodytes), high-ranking males are expected to have high reproductive success and females typically emigrate upon reaching maturity. Although high average relatedness among males in the same social groups has been assumed, previous reports have indicated that relatedness among males is not necessarily significantly higher than that among females. The paternity of 11 offspring and the relatedness of 50 individuals in the M group of chimpanzees at Mahale Mountains National Park, Tanzania, were investigated using DNA analyses. We determined the fathers of 10 offspring. Two different alpha males sired a total of five offspring, whereas the other males had low reproductive success. The proportion of paternal half-sibling pairs among the 10 offspring was 15.6%. The average relatedness among mature males was significantly higher than that among mature females. The existence of an old male and the long tenure of one alpha male may have contributed to this significant difference. The average dyadic relatedness among mature natal individuals was significantly higher than that in natal-immigrant pairs in which the individuals came from different groups. The average relatedness among immigrant females was similar to that in pairs of natal and immigrant females, suggesting that the immigrants came from various groups. Thus, female transfer acts to maintain low average relatedness within the group. A comparison of our results to those from other study sites suggests that although the average relatedness among adult males does not reach the level of half-siblings, under some circumstances it can exceed the relatedness of females.