Organic Anion-Transporting Polypeptide (OATP)-Mediated Drug-Drug Interaction Study between Rosuvastatin and Cyclosporine A in Chimeric Mice with Humanized Liver.

The influence of transporters on the pharmacokinetics of drugs is being increasingly recognized, and DDIs via transporters may be a risk factor for adverse events. Cyclosporine A, a strong OATP inhibitor, has been reported to increase the systemic exposure of rosuvastatin, an OATP substrate, by 7.1-fold in clinical studies. PXB mice are chimeric mice with humanized livers that are highly repopulated with human hepatocytes and have been widely used for drug discovery in drug metabolism and pharmacokinetics studies. In the present study, we examined in vivo and in vitro DDIs between rosuvastatin and cyclosporine A in PXB mice and fresh human hepatocytes (PXB cells) obtained from PXB mice. We initially investigated the active transport of rosuvastatin into PXB cells, and found concentration-dependent uptake with a Michaelis-Menten constant value of 4.0 µmol/l and a Vmax value of 4.63 pmol/min per 106 cells. Cyclosporine A inhibited the uptake of rosuvastatin with an IC50 value of 0.21 µmol/l. We then examined in vivo DDIs, and the exposure of orally administered rosuvastatin increased by 3.3-fold and 11-fold in PXB mice pretreated with 10 and 50 mg/kg cyclosporine A, whereas it increased by 2.5-fold and 6.2-fold when rosuvastatin was administered intravenously, in studies that were conducted for considering gastrointestinal DDIs. The liver-to-blood concentration ratio of rosuvastatin was dose-dependently decreased by pretreatment with cyclosporine A in PXB mice and SCID mice. Observed DDIs in vivo were considered to be reasonable based on the estimated concentrations of cyclosporine A at the inlet to the liver and in the liver tissues of both mice. In conclusion, our results indicate that PXB mice might be a useful tool for predicting human OATP-mediated DDIs in drug discovery, and its limitation due to the differences of gastrointestinal condition from human should also be considered.

Piperidine derivatives as nonprostanoid IP receptor agonists 2.

We searched for a strong and selective nonprostanoid IP agonist bearing piperidine and benzanilide moieties. Through optimization of substituents on the benzanilide moiety, the crucial part of the agonist, 43 (2-((1-(2-(N-(4-tolyl)benzo[d][1,3]dioxole-5-carboxamido)ethyl)piperidin-4-yl)oxy)acetic acid monohydrate monohydrochloride) was discovered and exhibited strong platelet aggregation inhibition (IC50=21nM) and 100-fold selectivity for IP receptor over other PG receptors. The systemic exposure level and bioavailability after oral administration of 43 were also good in dog.

Analysis of gene expression for microminipig liver transcriptomes using parallel long-read technology and short-read sequencing.

The microminipig is one of the smallest minipigs that has emerged as a possible experimental animal model, because it shares many anatomical and/or physiological similarities with humans, including the coronary artery distribution in the heart, the digestive physiology, the kidney size and its structure, and so on. However, information on gene expression profiles, including those on drug-metabolizing phase I and II enzymes, in the microminipig is limited. Therefore, the aim of the present study was to identify transcripts in microminipig livers and to determine gene expression profiles. De novo assembly and expression analyses of microminipig transcripts were conducted with liver samples from three male and three female microminipigs using parallel long-read and short-read sequencing technologies. After unique sequences had been automatically aligned by assembling software, the mean contig length of 50843 transcripts was 707 bp. The expression profiles of cytochrome P450 (P450) 1A2, 2C, 2E1 and 3A genes in livers in microminipigs were similar to those in humans. Liver carboxylesterase (CES) precursor, liver CES-like, UDP-glucuronosyltransferase (UGT) 2C1-like, amine sulfotransferase (SULT)-like, N-acetyltransferases (NAT8) and glutathione S-transferase (GST) A2 genes, which are relatively unknown genes in pigs and/or humans, were expressed strongly. Furthermore, no significant gender differences were observed in the gene expression profiles of phase I enzymes, whereas UGT2B17, SULT1E1, SULT2A1, amine SULT-like, NAT8 and GSTT4 genes were different between males and females among phase II enzyme genes under the present sample conditions. These results provide a foundation for mechanistic studies and the use of microminipigs as model animals for drug development in the future. Copyright © 2016 John Wiley & Sons, Ltd.

Piperidine derivatives as nonprostanoid IP receptor agonists.

The discovery of a new class of nonprostanoid prostaglandin I2 receptor (IP receptor) agonists is reported. Among them, the unique piperidine derivative 31b (2-((1-(2-(N-(4-tolyl)benzamido)ethyl)piperidin-4-yl)oxy)acetic acid) was a good IP receptor agonist and was 50-fold more selective for the human IP receptor than for other human prostanoid receptors. This compound showed good pharmacokinetic properties in dog.

Indolylpiperidine derivatives as potent and selective α1B adrenoceptor antagonists.

A series of novel indolylpiperidine derivatives were synthesized, and their pharmacological profiles were assessed at rat α1A and α1B adrenoceptors through in vitro binding studies. Compound 12 (2-(3-(4-(6-fluoro-1H-indol-3-yl)piperidin-1-yl)propyl)-1,2,3,4-tetrahydroisoquinoline) was a potent α1B adrenoceptor antagonist (Ki=0.61 nM) and was about 40-fold more selective for the α1B adrenoceptor than for the α1A adrenoceptor. In addition, useful structure-activity relationship information was acquired for further improving selectivity for the α1B adrenoceptor.

Identification of alpha-substituted acylamines as novel, potent, and orally active mGluR5 negative allosteric modulators.

This Letter describes the identification of a series of novel non-acetylenic mGluR5 negative allosteric modulators based on the alpha-substituted acylamine structure. An initial structure-activity relationship study suggested that (R)-19b and (R)-19j might have good in vitro activity. When administered orally, these compounds were found to have an anxiolytic-like effect in a mouse model of stress-induced hyperthermia.

Discovery of potent α1L-adrenoceptor agonists: Design and synthesis of bicyclic derivatives.

We aimed to create a novel and potent α(1L)-adrenoceptor agonist because such agonists are possible drug candidates for stress urinary incontinence. We used ligand-based drug design and evaluated the α(1L)-adrenoceptor agonist activity of the designed compounds. Among them, tetrahydroquinoline derivative 50 showed the most potent activity (ratio of noradrenaline half maximal effective concentration, 0.0028) and effectively induced contraction of rat bladder neck.

The discovery of potent glycine transporter type-2 inhibitors: design and synthesis of phenoxymethylbenzamide derivatives.

We describe the discovery of phenoxymethylbenzamide derivatives as a novel class of glycine transporter type-2 (GlyT-2) inhibitors. We found hit compound 1 (human GlyT-2, IC50=4040 nM) in our library and converted its 1-(1-(naphthalen-2-ylmethyl)piperidin-4-yl)pyrrolidin-3-yl group to an 1-(N,N-dimethylaminopropyl)piperidyl group and its tert-butyl group to a trifluoromethyl group to obtain N-(1-(3-(dimethylamino)propyl)piperidin-4-yl)-4-((4-(trifluoromethyl)phenoxy)methyl)benzamide (20). Compound 20 showed good inhibitory activity against human GlyT-2 (IC50=15.3 nM) and exhibited anti-allodynia effects in a mouse neuropathic pain model.

Pharmacologically distinct phenotypes of α1B -adrenoceptors: variation in binding and functional affinities for antagonists.

BACKGROUND AND PURPOSE: The pharmacological properties of particular receptors have recently been suggested to vary under different conditions. We compared the pharmacological properties of the α1B -adrenoceptor subtype in various tissue preparations and under various conditions. EXPERIMENTAL APPROACH: [(3) H]-prazosin binding to α1B -adrenoceptors in rat liver (segments, dispersed hepatocytes and homogenates) was assessed and the pharmacological profiles were compared with the functional and binding profiles in rat carotid artery and recombinant α1B -adrenoceptors. KEY RESULTS: In association and saturation-binding experiments with rat liver, binding affinity for [(3) H]-prazosin varied significantly between preparations (KD value approximately ten times higher in segments than in homogenates). The binding profile for various drugs in liver segments also deviated from the representative α1B -adrenoceptor profile observed in liver homogenates and recombinant receptors. L-765,314 and ALS-77, selective antagonists of α1B -adrenoceptors, showed high binding and antagonist affinities in liver homogenates and recombinant α1B -adrenoceptors. However, binding affinities for both ligands in the segments of rat liver and carotid artery were 10 times lower, and the antagonist potencies in α1B -adrenoceptor-mediated contractions of carotid artery were more than 100 times lower than the representative α1B -adrenoceptor profile. CONCLUSIONS AND IMPLICATIONS: In contrast to the consistent profile of recombinant α1B -adrenoceptors, the pharmacological profile of native α1B -adrenoceptors of rat liver and carotid artery varied markedly under various receptor environments, showing significantly different binding properties between intact tissues and homogenates, and dissociation between functional and binding affinities. In addition to conventional 'subtype' characterization, 'phenotype' pharmacology must be considered in native receptor evaluations in vivo and in future pharmacotherapy.

TRK-380, a novel selective human ß3-adrenoceptor agonist, ameliorates formalin-induced pollakiuria in rats and carbachol-induced bladder contraction in dogs.

OBJECTIVE: To investigate the effects of TRK-380, a selective ß3-adrenoceptor (ß3-AR) agonist, on voiding behavior in rats with pollakiuria and on carbachol (CCh)-induced bladder contraction in dogs. METHODS: The voiding behavior of female Sprague Dawley rats was recorded continuously with a balance. Rats were intravesically pretreated with 2.5% formalin under isoflurane anesthesia. The next day, the effect of TRK-380 (7.5-30 mg/kg, orally) or tolterodine, an antimuscarinic drug (3.75-15 mg/kg, orally), on the voiding frequency was evaluated. In another experiment, male beagle dogs were anesthetized with pentobarbital, CCh (3 µg/kg, intravenously) was administered to them, and the effect of TRK-380 (0.1 or 0.3 µg/kg/minute, intravenously infusion) on CCh-induced bladder contraction was evaluated. RESULTS: Rats treated with formalin showed a significant increase in the voiding frequency compared with the sham group, and the increase in it was significantly and dose-dependently suppressed by TRK-380 at doses of ≥15 mg/kg. In contrast, tolterodine did not lead to a significant change in the voiding frequency even at the highest dose. In dogs, CCh-induced bladder contraction was dose-dependently suppressed by TRK-380; the plasma concentration required for 30% suppression of the CCh-induced bladder contraction (30% relaxation) was 4.90 ng/mL. CONCLUSION: This study indicated that TRK-380 ameliorated pollakiuria, which was resistant to an antimuscarinic drug, and that it also suppressed the bladder contraction induced by cholinergic stimulation in dogs, whose bladder relaxation is known to be predominantly mediated by ß3-ARs, as in humans. These data strengthen the therapeutic potential of ß3-AR for the treatment of overactive bladder.

Discovery of potent transient receptor potential vanilloid 1 antagonists: design and synthesis of phenoxyacetamide derivatives.

We aimed to discover a novel type of transient receptor potential vanilloid 1 (TRPV1) antagonist because such antagonists are possible drug candidates for treating various disorders. We modified the structure of hit compound 7 (human TRPV1 IC50=411 nM) and converted its pyrrolidino group to a (hydroxyethyl)methylamino group, which substantially improved inhibitory activity (15d; human TRPV1 IC50=33 nM). In addition, 15d ameliorated bladder overactivity in rats in vivo.

Synthesis of novel IP agonists via N-aminoethyl cyclic amines prepared by decarboxylative ring-opening reactions.

An efficient synthesis of a highly potent and selective IP (PGI(2) receptor) agonist that is not structurally analogous to PGI(2) is described. This synthesis is accomplished through the following key steps: Nucleophilic ring-opening of 3-(4-chlorophenyl)-oxazolidin-2-one prepared by a one-pot procedure with 4-piperidinol and selective O-alkylation of 1-(2-(4-chlorophenylamino)ethyl)piperidin-4-ol. The obtained compound is a potent and selective IP agonist displaying a long duration of action.

Pharmacological effect of TRK-380, a novel selective human ß3-adrenoceptor agonist, on mammalian detrusor strips.

OBJECTIVE: To clarify the potential of TRK-380 as a drug for overactive bladder in humans by evaluating the agonistic activities for human ß-adrenergic receptors (ß-ARs) and the relaxing effects on isolated detrusor strips. METHODS: The agonistic activities for human ß-ARs were evaluated in SK-N-MC cells (for human ß(3)-ARs) and Chinese hamster ovary cells expressing human ß(1)- or human ß(2)-ARs using the cyclic adenosine monophosphate accumulation assay. The relaxing effects on the resting tension in isolated detrusor strips from humans, monkeys, dogs, and rats and on carbachol- or KCl-induced contractions in human detrusor strips were evaluated. RESULTS: In the cyclic adenosine monophosphate accumulation assay, the agonistic activity of TRK-380 for human ß(3)-ARs was potent and equivalent to that of the potent nonselective ß-AR agonist isoproterenol and superior to that of selective ß(3)-AR agonists, such as BRL-37344 and CL316,243. TRK-380 showed no agonistic activity for human ß(1)-ARs and a weak agonistic effect on human ß(2)-ARs. In isolated detrusor strips, the concentration-dependent relaxing effects of TRK-380 on the resting tension were equivalent to those of isoproterenol in humans, monkeys, and dogs but weaker than the effects in rats. The selective ß(3)-AR antagonist SR59230A shifted the concentration-response curve in a concentration-dependent manner to TRK-380 for the resting tension of human detrusor strips to the right. TRK-380 had a concentration-dependent relaxing effect on the contractile responses to carbachol and KCl in human detrusor strips. CONCLUSION: TRK-380 was a potent and selective human ß(3)-AR agonist, and the isolated human detrusor relaxation was mainly mediated by activation of the ß(3)-AR. Consequently, TRK-380 might be a promising compound for the treatment of overactive bladder.

Efficient synthesis of a novel m-phenylene derivative as a selective EP4 agonist inducing follicular growth and maturation in the ovary.

An efficient and straightforward synthesis of a novel m-phenylene derivative has been developed. The optically pure dibromo compound was selected as a starting material. Through a protocol involving the Prins reaction and two steps of the Horner-Wadsworth-Emmons reaction, the basic skeleton was constructed with appropriate alpha and omega side chains. The compound proved to be a highly selective EP(4) agonist and a possible drug candidate for maturation of the uterine cervix.

Design and synthesis of selective alpha1B adrenoceptor antagonists.

A series of novel indolylpiperidine derivatives were synthesized and assessed for their pharmacological profiles at alpha1 adrenoceptor subtypes by in vitro binding studies at rat alpha1A and alpha1B receptors. Compound 11 was a potent (Ki=0.63 nM) and selective (approximately 30-fold more selective for the alpha1B receptor than for the alpha1A receptor) alpha1B adrenoceptor antagonist.