SUMMARYPurpose: 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. ]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 lM). 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 dosedependent 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.

We have determined and compared the promoter, coding, and intronic sequences of the urate oxidase (Uox) gene of various primate species. Although we confirm the previous observation that the inactivation of the gene in the clade of the human and the great apes results from a single CGA to TGA nonsense mutation in exon 2, we find that the inactivation in the gibbon lineage results from an independent nonsense mutation at a different CGA codon in exon 2 or from either one-base deletion in exon 3 or one-base insertion in exon 5, contrary to the previous claim that the cause is a 13-bp deletion in exon 2. We also find that compared with other organisms, the primate functional Uox gene is exceptional in terms of usage of CGA codons which are prone to TGA nonsense mutations. Nevertheless, we demonstrate rather strong selective constraint against nonsynonymous sites of the functional Uox gene and argue that this observation is consistent with the fact that the Uox gene is unique in the genome and evolutionarily conserved not only among animals but also among eukaryotes. Another finding that there are a few substitutions in the cis-acting element or CAAT-box (or both) of primate functional Uox genes may explain the lowered transcriptional activity. We suggest that although the inactivation of the hominoid Uox gene was caused by independent nonsense or frameshift mutations, the gene has taken a two-step deterioration process, first in the promoter and second in the coding region during primate evolution. It is also argued that the high concentration of uric acid in the blood of humans and nonhuman primates has developed molecular coevolution with the xanthine oxidoreductase in purine metabolism. However, it remains to be answered whether loss of Uox activity in hominoids is related to protection from oxidative damage and the prolonged life span.

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-17b-glucuronide (E 2 G), 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 K i values (mM) 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 K i values were within 2.8-fold of those obtained using E 2 G 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 K i 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 E 2 G 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 substratedependent K i variation.

The phylogenetic relationship among primates, ferungulates (artiodactyls + cetaceans + perissodactyls + carnivores), and rodents was examined using proteins encoded by the H strand of mtDNA, with marsupials and monotremes as the outgroup. Trees estimated from individual proteins were compared in detail with the tree estimated from all 12 proteins (either concatenated or summing up log-likelihood scores for each gene). Although the overall evidence strongly suggests ((primates, ferungulates), rodents), the ND1 data clearly support another tree, ((primates, rodents), ferungulates). To clarify whether this contradiction is due to (1) a stochastic (sampling) error; (2) minor model-based errors (e.g., ignoring site rate variability), or (3) convergent and parallel evolution (specifically between either primates and rodents or ferungulates and the outgroup), the ND1 genes from many additional species of primates, rodents, other eutherian orders, and the outgroup (marsupials + monotremes) were sequenced. The phylogenetic analyses were extensive and aimed to eliminate the following artifacts as possible causes of the aberrant result: base composition biases, unequal site substitution rates, or the cumulative effects of both. Neither more sophisticated evolutionary analyses nor the addition of species changed the previous conclusion. That is, the statistical support for grouping rodents and primates to the exclusion of all other taxa fluctuates upward or downward in quite a tight range centered near 95% confidence. These results and a site-by-site examination of the sequences clearly suggest that convergent or parallel evolution has occurred in ND1 between primates and rodents and/or between ferungulates and the outgroup. While the primate/rodent grouping is strange, ND1 also throws some interesting light on the relationships of some eutherian orders, marsupials, and montremes. In these parts of the tree, ND1 shows no apparent tendency for unexplained convergences.

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