Mikio Hayashi

JTT-305, an orally active calcium-sensing receptor antagonist, stimulates transient parathyroid hormone release and bone formation in ovariectomized rats

Intermittent administration of parathyroid hormone (PTH) has a potent anabolic effect on bone in humans and animals. Calcium-sensing receptor (CaSR) antagonists stimulate endogenous PTH secretion through CaSR on the surface of parathyroid cells and thereby may be anabolic agents for osteoporosis. JTT-305 is a potent oral short-acting CaSR antagonist and transiently stimulates endogenous PTH secretion. The objective of the present study was to investigate the effects of JTT-305 on PTH secretion and bone in ovariectomized rats. Female rats, immediately after ovariectomy (OVX), were orally administered vehicle or JTT-305 (0.3, 1, or 3 mg/kg) for 12 weeks. The serum PTH concentrations were transiently elevated with increasing doses of JTT-305. In the proximal tibia, JTT-305 prevented OVX-induced decreases in both the cancellous and total bone mineral density (BMD) except for the 0.3mg/kg dose. At the 3mg/kg dose, JTT-305 increased the mineralizing surface and bone formation rate in histomorphometry. The efficacy of JTT-305 at the 3mg/kg dose on the BMD corresponded to that of exogenous rat PTH1-84 injection at doses between 3 and 10 μg/kg. In conclusion, JTT-305 stimulated endogenous transient PTH secretion and bone formation, and consequently prevented bone loss in OVX rats. These results suggest that JTT-305 is orally active and has the potential to be an anabolic agent for the treatment of osteoporosis.

Discovery of a potent and short-acting oral calcilytic with a pulsatile secretion of parathyroid hormone.

Short-acting oral calcilytics, calcium-sensing receptor (CaSR) antagonists, have been considered as alternatives for parathyroid hormone (PTH), an injectable bone anabolic drug used in the treatment of osteoporosis. Previously, we identified aminopropandiol 1, which transiently stimulated endogenous PTH secretion in rats. However, the inhibition of cytochrome P450 (CYP) 2D6 and the low bioavailability of 1 remain to be solved. Attempts to change the physicochemical properties of the highly lipophilic amine 1 by introduction of a carboxylic acid group as well as further structural modifications led to the discovery of the highly potent biphenylcarboxylic acid 15, with a markedly reduced CYP2D6 inhibition and a significantly improved bioavailability. Compound 15 evoked a rapid and transient elevation of endogenous PTH levels in rats after oral administration in a dose-dependent manner at a dose as low as 1 mg/kg. The PTH secretion pattern correlated with the pharmacokinetic profile and agreed well with that of the exogenous PTH injection which exerts a bone anabolic effect.

Antithrombotic effects of a synthetic inhibitor of activated factor X, JTV-803, in animals.

JTV-803, 4-[(2-amidino-1,2,3,4-tetrahydroisoquinolin-7-yloxy)methyl]-1-(4-pyridinyl)piperidine-4-carboxylic acid monomethanesulfonate trihydrate, at > or = 0.1 mg/kg/h inhibited the increase in plasma thrombin-antithrombin III complex in response to continuous infusion of thromboplastin in rats. JTV-803 inhibited thrombus formation in an arteriovenous shunt model by intravenous infusion at > or = 0.3 mg/kg/h and prolonged the occlusion time of photochemically induced arterial thrombus in the middle cerebral artery at >1.5 mg/kg/0.5 h. Activated partial thromboplastin time was prolonged at 10 mg/kg/h. Intravenous administration of JTV-803 prolonged bleeding time at 30 mg/kg/h, a dose 10-100 times higher than the dose that inhibited thrombus formation. Compared with thrombin inhibitor, JTV-803 had less of an effect on the bleeding time. In the arteriovenous shunt model in cynomolgus monkey, JTV-803 prolonged the occlusion time when administered by continuous infusion at 0.3 mg/kg/h or orally at 10 mg/kg. These results suggest that the human factor Xa inhibitor JTV-803 is an orally active anticoagulant that does not affect bleeding time and is useful for the prevention of thrombus.

JTS-653 Blocks Afferent Nerve Firing and Attenuates Bladder Overactivity Without Affecting Normal Voiding Function

PURPOSE We evaluated the role of TRPV1 in bladder overactivity based on afferent nerve firing and urodynamic parameters using the selective TRPV1 antagonist JTS-653. MATERIALS AND METHODS We evaluated the effects of JTS-653 on the increased pelvic nerve discharge and intravesical pressure induced by intravesical infusion of 100 μM capsaicin in anesthetized rats. The effects of JTS-653 on the urodynamic parameters of bladder overactivity induced by intravesical infusion of 30 nM resiniferatoxin or 0.2% acetic acid, or on normal bladder activity were evaluated by cystometry in conscious rats. The effects of JTS-653 on carbachol induced contraction were investigated using bladder muscle strips. RESULTS JTS-653 significantly suppressed the capsaicin induced increase in nerve discharge and intravesical pressure. Intravesical infusion of resiniferatoxin or acetic acid decreased the intercontraction interval and voided volume. JTS-653 significantly increased the intercontraction interval and voided volume in rats with resiniferatoxin or acetic acid induced bladder overactivity without affecting maximal voiding pressure. The antimuscarinic agent propiverine significantly decreased maximal voiding pressure but did not affect the intercontraction interval or voided volume in rats with acetic acid induced bladder overactivity. In normal rats JTS-653 showed no significant effects on the intercontraction interval, voided volume or maximal voiding pressure. JTS-653 did not affect carbachol induced contraction of the bladder muscle. CONCLUSIONS Our findings suggest that TRPV1 is involved in bladder overactivity via afferent nerve activation but it is not associated with normal voiding function. A TRPV1 antagonist would be a useful drug for bladder overactivity with a different pharmacological profile than antimuscarinic agents.

Pharmacological Characterization of (3S)-3-(Hydroxymethyl)-4-(5-Methylpyridin-2-yl)-N-[6-(2,2,2-Trifluoroethoxy)pyridin-3-yl]-3,4-Dihydro-2H-Benzo[b][1,4]Oxazine-8-Carboxamide (JTS-653), a Novel Transient Receptor Potential Vanilloid 1 Antagonist

Transient receptor potential vanilloid 1 (TRPV1) activation in peripheral sensory nerve is known to be associated with various pain-related diseases, thus TRPV1 has been the focus as a target for drug discovery. In this study, we characterized the pharmacological profiles of (3S)-3-(hydroxymethyl)-4-(5-methylpyridin-2-yl)-N-[6-(2,2,2-trifluoroethoxy)pyridin-3-yl]-3,4-dihydro-2H-benzo[b][1,4]oxazine-8-carboxamide (JTS-653), a novel TRPV1 antagonist. JTS-653 displaced [3H]resiniferatoxin binding to human and rat TRPV1. JTS-653 competitively antagonized the capsaicin-induced activation of human TRPV1 with pA2 values of 10.1. JTS-653 also inhibited proton-induced activation of human and rat TRPV1 with IC50 values of 0.320 and 0.347 nM, respectively. Electrophysiological studies indicated that JTS-653 blocked heat-induced inward currents in rat TRPV1 with IC50 values of 1.4 nM. JTS-653 showed weak or no inhibitory effects on other TRP channels, receptors, and enzymes. JTS-653 significantly prevented capsaicin-induced mechanical hyperalgesia at 1 mg/kg p.o. and attenuated carrageenan-induced mechanical hyperalgesia at 0.3 mg/kg p.o. JTS-653 significantly attenuated carrageenan-induced thermal hyperalgesia at 0.1 mg/kg p.o. and fully reversed at 0.3 mg/kg p.o. without affecting the volume of the carrageenan-treated paw. JTS-653 showed a transient increase of body temperature at 0.3 mg/kg p.o. These results indicated that JTS-653 is a highly potent and selective TRPV1 antagonist in vitro and in vivo and suggested that JTS-653 is one of the most potent TRPV1 antagonists. The profiles of JTS-653, high potency in vivo and transient hyperthermia, seem to be associated with polymodal inhibition of TRPV1 activation.

A novel JAK inhibitor JTE-052 reduces skin inflammation and ameliorates chronic dermatitis in rodent models: Comparison with conventional therapeutic agents

Janus kinases (JAKs) are required for several inflammatory cytokine signalling pathways and are implicated in the pathogenesis of chronic dermatitis, including atopic dermatitis and psoriasis. JAK inhibitors are therefore promising therapeutic candidates for chronic dermatitis. In this study, we evaluated the effects of the novel JAK inhibitor JTE‐052 on inflammatory responses associated with chronic dermatitis, and compared its profile with those of conventional therapeutic agents in rodent models of chronic dermatitis. JTE‐052 inhibited the Th1‐, Th2‐ and Th17‐type inflammatory responses of human T cells and mast cells in vitro. Oral administration of JTE‐052 inhibited skin inflammation in hapten‐induced chronic dermatitis in mice, associated with reduced levels of inflammatory cytokines in the skin and immunoglobulin (Ig) E in serum. In contrast, although ciclosporin partly inhibited skin inflammation, it did not reduce interleukin (IL)‐4 production in skin, and enhanced IgE production in serum. Oral administration of JTE‐052 also inhibited skin inflammation in mouse models of atopic dermatitis and psoriasis induced by a mite extract, thymic stromal lymphopoietin or IL‐23. The maximal efficacy of JTE‐052 in these dermatitis models was superior to the conventional therapeutic agents, ciclosporin and methotrexate. Topical application of JTE‐052 ointment ameliorated hapten‐induced chronic dermatitis in rats more effectively than tacrolimus ointment. Furthermore, JTE‐052 ointment did not cause the thinning of normal skin associated with topical corticosteroids. These results indicate that JTE‐052 is a promising candidate as an anti‐inflammatory drug for various types of chronic dermatitis, with a distinctly different profile from conventional therapy following either oral or topical application.

A Refined Culture System for Human Induced Pluripotent Stem Cell-Derived Intestinal Epithelial Organoids

Summary Gut epithelial organoids are routinely used to investigate intestinal biology; however, current culture methods are not amenable to genetic manipulation, and it is difficult to generate sufficient numbers for high-throughput studies. Here, we present an improved culture system of human induced pluripotent stem cell (iPSC)-derived intestinal organoids involving four methodological advances. (1) We adopted a lentiviral vector to readily establish and optimize conditioned medium for human intestinal organoid culture. (2) We obtained intestinal organoids from human iPSCs more efficiently by supplementing WNT3A and fibroblast growth factor 2 to induce differentiation into definitive endoderm. (3) Using 2D culture, followed by re-establishment of organoids, we achieved an efficient transduction of exogenous genes in organoids. (4) We investigated suspension organoid culture without scaffolds for easier harvesting and assays. These techniques enable us to develop, maintain, and expand intestinal organoids readily and quickly at low cost, facilitating high-throughput screening of pathogenic factors and candidate treatments for gastrointestinal diseases.

Inhibitory effect of JTV-803, a new cyclic guanidine derivative, on factor Xa in vitro and in vivo

JTV-803, 4-[(2-amidino-1,2,3,4-tetrahydroisoquinolin-7-yloxy)methyl]-1-(4-pyridinyl)piperidine-4-carboxylic acid monomethanesulfonate trihydrate showed a competitive inhibitory effect on human factor Xa, with a K(i) value of 0.019 microM. This compound was 100 times more selective in inhibiting human factor Xa as compared to its inhibitory activity against thrombin, plasmin, and trypsin. JTV-803 was also examined for its inhibitory effect on activated factor Xa obtained from plasma of various animal species. JTV-803 exerted a potent inhibitory effect on human factor Xa (IC(50): 0.081 microM). JTV-803 prolonged activated partial thromboplastin time and prothrombin time in a dose-dependent manner. Oral anticoagulant efficacy of JTV-803 was examined ex vivo for its inhibition of human factor Xa in cynomolgus monkeys. JTV-803 produced more than 20% inhibition of human factor Xa for 8 h. Taken together, the results indicate JTV-803 is a long-acting oral anticoagulant which exerts its effect via specific inhibition of human factor Xa.

Reciprocal Inflammatory Signaling Between Intestinal Epithelial Cells and Adipocytes in the Absence of Immune Cells

Visceral fat accumulation as observed in Crohns disease and obesity is linked to chronic gut inflammation, suggesting that accumulation of gut adipocytes can trigger local inflammatory signaling. However, direct interactions between intestinal epithelial cells (IECs) and adipocytes have not been investigated, in part because IEC physiology is difficult to replicate in culture. In this study, we originally prepared intact, polarized, and cytokine responsive IEC monolayers from primary or induced pluripotent stem cell-derived intestinal organoids by simple and repeatable methods. When these physiological IECs were co-cultured with differentiated adipocytes in Transwell, pro-inflammatory genes were induced in both cell types, suggesting reciprocal inflammatory activation in the absence of immunocompetent cells. These inflammatory responses were blocked by nuclear factor-κB or signal transducer and activator of transcription 3 inhibition and by anti-tumor necrosis factor- or anti-interleukin-6-neutralizing antibodies. Our results highlight the utility of these monolayers for investigating IEC biology. Furthermore, this system recapitulates the intestinal epithelium–mesenteric fat signals that potentially trigger or worsen inflammatory disorders such as Crohns disease and obesity-related enterocolitis.

Effects of activated factor X inhibitor, JTV-803, in a pig model of hemodialysis

JTV-803 (4-[(2-amidino-1,2,3,4-tetrahydroisoquinolin-7-yloxy)methyl]-1-(4-pyridinyl)piperidine-4-carboxylic acid monomethanesulfonate trihydrate), a specific inhibitor of factor Xa, was evaluated in a pig hemodialysis model with ligation of renal arteries. In this model, JTV-803 administered into the dialysis circuit showed an anticoagulant effect (prolongation of dialysis time) at doses of 0.3 mg/kg + 0.3, 1.0 and 3.0 mg/kg/h. The prolongation of dialysis time in the 0.3 mg/kg + 1.0 mg/kg/h JTV-803 group was comparable to that in the 15 U/kg + 7.5 U/kg/h heparin group. The plasma concentrations of JTV-803 following 0.3 mg/kg + 1.0 mg/kg/h infusion of the drug into the dialysis circuit was 500 ng/ml or less, and prothrombin time was 1.5-fold or less than the pretreatment value. JTV-803 was removed by passing the blood through a dialyzer, resulting in a clearance rate of 53.4-81.8%. After the end of dialysis, plasma concentrations of JTV-803 decreased rapidly with time. These results suggest that human factor Xa inhibitor JTV-803 may have good potential as an antithrombotic agent during hemodialysis, with lower likelihood of bleeding after the end of dialysis.