Species differences in the pharmacokinetics of KW-7158 [(2S)-(+)-3,3,3-Trifluoro-2-hydroxy-2-methyl-N-(5,5,10-trioxo-4,10-dihydrothieno[3,2-c][1]benzothiepin-9-yl)propanamide]: formation of hydrolyzed metabolite in human and animals.

Species differences in the pharmacokinetics of KW-7158 [(2S)-(+)-3,3,3-Trifluoro-2-hydroxy-2-methyl-N-(5,5,10-trioxo-4,10-dihydrothieno[3,2-c][1]benzothiepin-9-yl)propanamide] were studied in in vivo and in vitro experiments. The exposure ratio of hydrolyzed metabolite (M2, primary metabolite in human plasma)/KW-7158 was higher than the ratio of thiophen-to-furan converted metabolite (M1)/KW-7158 in human subjects after oral administration, but the mouse, rat and dog studies gave opposite results. M2 was produced in the highest amount by the 9000g supernatant of small intestine, followed by that of liver and kidney in human subjects. After correction for protein contents, the results obtained suggested that the small intestine plays a major role in the metabolism to M2 for the first pass effect after oral administration of KW-7158. The formation of M2 was independent of the presence of NADPH and was inhibited by various esterase inhibitors. These observations suggested that the predominant enzymes or isozymes involved in the formation of M2 are esterases, which differ between humans and animals. Such differences may be one of the reasons for the species differences in the pharmacokinetics of KW-7158 between humans and animals.

The generation of rat dorsal root ganglion cell lines to identify the target of KW-7158, a novel treatment for overactive bladder.

KW-7158 is a drug candidate for the treatment of overactive bladder. Although pharmacological studies have suggested that it suppresses afferent nerve conduction, its molecular target is unknown. We herein report the establishment of dorsal root ganglion (DRG) cell lines useful for identification of the target of this compound. First, we confirmed that the target exists in rat primary DRG by [(3)H]KW-7158 binding. To establish DRG cell lines, we used DRG from transgenic rats harboring the temperature-sensitive large T-antigen. The immortalized cells were initially screened for their expression of neuronal markers, and 72 positive clones were obtained (designated as TRD cells). Next, in order to select TRD cells expressing the target of KW-7158, we measured the binding affinity and amount of the binding sites present in each clone. Most clones expressed two binding sites, one with low affinity and one with high affinity. Differential binding of KW-7158 derivatives to each site revealed that the high affinity site is pharmacologically relevant. Therefore, we successfully identified "TRD-10" which express the largest amount of the high affinity site. These cell lines will therefore be useful tools to identify the target of KW-7158.

Orally active CCR5 antagonists as anti-HIV-1 agents: synthesis and biological activity of 1-benzothiepine 1,1-dioxide and 1-benzazepine derivatives containing a tertiary amine moiety.

The search for orally active CCR5 antagonists was performed by chemical modification of the 1-benzothiepine 1,1-dioxide 3 and 1-benzazepine 4 lead compounds containing a tertiary amine moiety. Replacement of methyl group with a 2-(C(2-4) alkoxy)ethoxy group at the 4-position on the 7-phenyl group of the 1-benzothiepine ring resulted in both enhanced activity and significant improvement in the pharmacokinetic properties upon oral administration in rats. Introduction of C(2-4) alkyl, phenyl or (hetero)arylmethyl groups as the 1-substituent on the 1-benzazepine ring together with the 2-(butoxy)ethoxy group led to further increase of activity. Among the 1-benzazepine derivatives, the isobutyl (6i), benzyl (6o) or 1-methylpyrazol-4-ylmethyl (6s) compounds were found to exhibit highly potent inhibitory effects, equivalent to the injectable CCR5 antagonist 1, in the HIV-1 envelope-mediated membrane fusion assay. In particular, compound 6s showed the most potent CCR5 antagonistic activity (IC(50)=2.7 nM) and inhibitory effect (IC(50)=1.2 nM) on membrane fusion, together with good pharmacokinetic properties in rats. The synthesis of 1-benzothiepine 1,1-dioxide and 1-benzazepine derivatives and their biological activity are described.

The pharmacological properties of Y-23684, a benzodiazepine receptor partial agonist.

1. The pharmacological properties of a benzodiazepine receptor (BZR) partial agonist, Y-23684 were investigated in comparison with those of diazepam, a conventional BZR full agonist. 2. Y-23684 and diazepam showed high and selective affinity for the BZR with Ki values of 41 and 5.8 nM, respectively. 3. In contrast to diazepam, variability was noted in the anticonvulsive potency of Y-23684 depending on convulsants (bicuculline, pentylenetetrazol and maximal electrical shock). Y-23684 produced the most potent protective effect against bicuculline in rats and mice with ED50S of 1.3 and 1.2 mg kg-1, respectively. 4. In rat conflict models (Geller-Seifter and water-lick tests), Y-23684 produced an antipunishment action at doses 2-4 times lower than diazepam. In contrast to diazepam, Y-23684 did not affect unpunished responding up to 50 mg kg-1 in the Geller-Seifter test. 5. In other rat models of anxiety (social interaction and elevated plus-maze tests), Y-23684 was as efficacious as and ten fold more potent than diazepam. In a mouse model of anxiety (exploration (light/dark box) test), Y-23684 was as efficacious and two fold less potent as diazepam. In these paradigms, Y-23684 showed a selective anxiolytic profile over a wide dose-range without loss of efficacy and sedative action. 6. The impairment of motor coordination (rotarod) and potentiation of CNS depressants (ethanol and hexobarbitone) by Y-23684 was much weaker than that of diazepam. 7. These results suggest that Y-23684 would be a potent and selective anxiolytic agent in man with less side-effects than conventional BZ-anxiolytics.

[Interaction of CN-100, a novel non-steroidal anti-inflammatory drug, on biopolymers].

Interaction of CN-100, a novel non-steroidal anti-inflammatory drug, with biopolymers were investigated. In collagen induced rat platelet aggregation, the inhibitory effect of CN-100 was almost equipotent as indomethacin (IM) but less potent than that of pranoprofen (PP). The effect of CN-100 on rat platelet aggregation induced by arachidonic acid (AA) was less potent than that of IM and PP. CN-100 inhibited rat platelet functions, serotonin release and malondialdehyde formation, induced by collagen more potently than those induced by AA. In heat-induced rat erythrocyte lysis and Ca2(+)-induced liposome aggregation, the inhibitory effect of CN-100 was less potent than IM but more than those of PP. CN-100 was inhibited with heat denaturation of BSA, and the effect was more potent than IM and PP. The metachromagy based on the binding of an azodye, HABA, to BSA was potentiated weakly by CN-100, but IM had no effect on it. CN-100 and IM increased the fluorescence of the binding of dansyl amide (site I probe) to BSA. These results support that there is considerable interaction between CN-100 and membrane protein, and this effect influences the membrane to increase its stability.

Pharmacological investigations of the new antiinflammatory agent 2-(10,11-dihydro-10-oxodibenzo[b,f]thiepin-2-yl)propionic acid. 5th communication: antiplatelet effect of the drug and antiinflammatory effect of its main metabolite.

Since nonsteroidal antiinflammatory drugs (NSAID) usually have an antiplatelet effect, the inhibitory effect of 2-(10,11-dihydro-10-oxodibenzo[b,f]thiepin-2-yl) propionic acid (CN-100), which exerts a potent antiinflammatory effect, was compared with those of reference drugs, indometacin and pranoprofen, in this study. Indometacin at 10(-5) mol/l inhibited completely (100%) rat and rabbit platelet aggregation induced by collagen and arachidonic acid. Pranoprofen at 10(-5) mol/l also entirely inhibited rat platelet aggregation induced by the two aggregators, but an about 10 times higher concentration was required to produce 100% inhibition of rabbit platelet aggregation. CN-100 at 10(-5) mol/l exerted 100% inhibition of rat platelet aggregation induced by collagen, whereas more than 10(-4) mol/l was needed to exhibit 100% inhibition of aggregation induced by arachidonic acid and ADP. The inhibitory activity of CN-100 on aggregation of rat platelets ex vivo was weaker than those of reference NSAID, i.e., the antiplatelet effect of CN-100 was found to be weak. The main metabolite of CN-100 also had a weak antiplatelet effect, and its antiinflammatory effect on carrageenin edema and UV erythema was apparently weaker than that of CN-100. The inhibitory effect of the metabolite on endotoxin diarrhea was weak. The ulcerogenic effect of the metabolite on gastric mucosa was similar to that of CN-100, but the effect rarely seemed to be a clinical problem because it was basically weak.