Genotypic and phenotypic analyses of hepatitis C virus from patients treated with JTK-853 in a three-day monotherapy.

JTK-853, a palm site-binding NS5B nonnucleoside polymerase inhibitor, shows antiviral activity in vitro and in hepatitis C virus (HCV)-infected patients. Here, we report the results of genotypic and phenotypic analyses of resistant variants in 24 HCV genotype 1-infected patients who received JTK-853 (800, 1,200, or 1,600 mg twice daily or 1,200 mg three times daily) in a 3-day monotherapy. Viral resistance in NS5B was investigated using HCV RNA isolated from serum specimens from the patients. At the end of treatment (EOT) with JTK-853, the amino acid substitutions M414T (methionine [M] in position 414 at baseline was replaced with threonine [T] at EOT), C445R (cysteine [C] in position 445 at baseline was replaced with arginine [R] at EOT), Y448C/H (tyrosine [Y] in position 448 at baseline was replaced with cysteine [C] or histidine [H] at EOT), and L466F (leucine [L] in position 466 at baseline was replaced with phenylalanine [F] at EOT), which are known to be typical resistant variants of nonnucleoside polymerase inhibitors, were observed in a clonal sequencing analysis. These substitutions were also selected by a treatment with JTK-853 in vitro, and the 50% effective concentration of JTK-853 in the M414T-, C445F-, Y448H-, and L466V-harboring replicons attenuated the susceptibility by 44-, 5-, 6-, and 21-fold, respectively, compared with that in the wild-type replicon (Con1). These findings suggest that amino acid substitutions of M414T, C445R, Y448C/H, and L466F are thought to be viral resistance mutations in HCV-infected patients receiving JTK-853 in a 3-day monotherapy.

Pharmacokinetics, Food Effect, Ketoconazole Interaction, and Safety of JTK-853, a Novel Nonnucleoside HCV Polymerase Inhibitor, After Ascending Single and Multiple Doses in Healthy Subjects.

Pharmacokinetics, safety, and tolerability of JTK-853, a novel HCV polymerase inhibitor, were evaluated in single and multiple ascending dose (SAD, MAD) studies, with food- and ketoconazole-related effects on exposure to JTK-853 and its active (CYP3A4 mediated) metabolite M2. JTK-853 was safe and well tolerated in both studies. In the SAD study, at doses >1600 mg (with standard breakfast [SBF]), JTK-853 exposure did not increase further, was substantially higher (AUCinf increase: 3- to 8-fold) with SBF (vs fasted), with a moderate increase in AUCinf (approximately 1.5-fold [1600 mg]) with a high-fat breakfast (vs SBF). In the SAD study (400-1600 mg, SBF), mean effective half-life (t1/2(eff) ) of JTK-853 was 8.3 to 10.9 hours, and 20.3 to 27.3 hours in the MAD study (twice daily dosing, fed condition), with 2- to 3-fold accumulation in exposure (AUCtau ). At steady-state, AUCtau increased dose proportionally, and was approximately 2-fold higher with ketoconazole coadministration. Metabolite M2 (equipotent to JTK-853 in vitro) did not contribute significantly to parent drug exposure and decreased with increase in dose, repeated dosing, and ketoconazole coadministration. Trial simulation-based ratios (n = 1000/dose level) of trough JTK-853 plasma concentrations to the in vitro EC90 for HCV genotype 1b were assessed for dose selection in a separate proof-of-concept study in patients. The studies enabled delineation of key drug attributes for further assessments in the target population.

Induction of late airway response was involved in serum antigen-specific immunoglobulin G in rats.

The antigen-induced immediate airway response (IAR) has been considered a form of bronchoconstriction mainly provoked by histamine and leukotriene C4/D4/E4, which are released by stimulation by antigen-specific IgE. However, the pathophysiological features of the antigen-induced late airway response (LAR) are not yet fully understood. In the present study, sensitized rats were repeatedly exposed to ovalbumin (OVA) to induce IAR and LAR, and the immunological profiles of IAR and LAR were examined. The first antigen inhalation induced only IAR but not LAR. However, the second antigen inhalation 7 days after IAR induced LAR but not IAR. Tumor necrosis factor (TNF)-alpha level in BALF in LAR was significantly higher than that in IAR, although there were no differences in histamine, leukotriene C4/D4/E4, interleukin (IL)-1beta, or IL-13 levels between IAR and LAR. Serum antigen-specific IgE titer was high in both IAR and LAR, but serum antigen-specific IgG, IgG1, and IgG2a titers were dramatically high in LAR but not IAR. There were significant correlations between antigen-specific IgG, IgG1, and IgG2a titers and LAR. Interestingly, LAR could be induced in normal rats by transfer of serum from LAR rats, which exhibited high antigen-specific IgG, IgG1, and IgG2a titers. In conclusion, these findings suggest that repeated antigen inhalation converts IAR to LAR, and that LAR is a reaction triggered by antigen-specific IgG and involving TNF-alpha. This is the first study to directly suggest the involvement of antigen-specific IgG in the induction of LAR.

Pharmacological analysis of antigen-induced late airway response in rats.

The pharmacological and pathophysiological characteristics of rat antigen-induced late airway response (LAR) are not yet fully understood. In this study, the pharmacological properties of rat ovalbumin (OVA)-induced LAR and effects of the clinically used anti-asthmatic drugs salbutamol (beta2-agonist), ketotifen (antihistamine), pranlukast (anti-leukotriene C4/D4/E4), and prednisolone (steroid) were examined. In addition, a comparison was made of cell infiltration in bronchoalveolar lavage fluid (BALF) between immediate airway response (IAR) and LAR, and the edematous features of lung during LAR were also examined. Although infiltration of inflammatory cells into BALF was increased in both IAR and LAR, only the increase in eosinophils at 1, 3, and 6 h during LAR were significantly higher than those during IAR. Although beta2-agonist, antihistamine, and anti-leukotriene C4/D4/E4 exhibited no effects on rat LAR, steroid attenuated LAR and decreased eosinophil number in BALF. LAR and the percentage water content were both increased after antigen inhalation, suggesting that LAR is involved in pulmonary edema in rats. In conclusion, antigen-induced LAR was related to pulmonary edema and eosinophil infiltration rather than contraction of airway smooth muscle. This is the first comprehensive study of the profiles of rat antigen-induced LAR, and these analyses of LAR improve understanding of the diverse mechanisms underlying human asthmatic diseases.

Two pharmacological phases in antigen-induced immediate airway response in rats.

The pharmacological profiles of antigen-induced immediate airway response (IAR) in rats are not fully understood. In this study, we established an ovalbumin (OVA)-induced IAR model using noninvasive measurement in rats, and evaluated the effects of commonly used and effective antiasthmatic drugs, i.e. ketotifen (antihistamine), pranlukast (anti-leukotriene C(4)/D(4)/E(4) (LT)), seratrodast (anti-thromboxane A(2) (TXA(2))), salbutamol (beta2-agonist), and prednisolone (steroid). The rat IAR model exhibited an optimal rapid airway response, and salbutamol inhalation completely suppressed the IAR. Ketotifen inhibited only the quick phase (QP; the reaction from 3 to 6 min after challenge), while pranlukast and seratrodast suppressed only the early phase (EP; the reaction from 6 to 30 min after challenge). Prednisolone inhibited both QP and EP. Further, continuous administration of compound 48/80, which depletes connective tissue mast cells (CTMC), partially inhibited QP but not EP. In conclusion, these findings suggest that the pharmacological profiles of noninvasive rat IAR are similar to those of asthmatic patients, and that rat IAR exhibits additional, immunological diverse characteristics, i.e. QP caused by the exocytosis of mediators in CTMCs and EP mediated by LT and TXA(2), which are produced by mucosal mast cells (MMCs) and possibly by other types of cells. This is the first report about the comprehensive pharmacological profiles of rodent IAR model, and these analyses of rat IAR model may help expand our understanding of the diverse mechanisms underlying human asthmatic diseases.