Eiji Kashiyama

In Vitro P‐Glycoprotein Interactions and Steady‐State Pharmacokinetic Interactions Between Tolvaptan and Digoxin in Healthy Subjects

Interactions between tolvaptan and digoxin were determined in an open‐label, sequential study where 14 healthy subjects received tolvaptan 60 mg once daily (QD) on days 1 and 12 to 16 and digoxin 0.25 mg QD on days 5 to 16. Mean maximal concentrations (Cmax) and area under the curve during the dosing interval (AUCτ) for digoxin with tolvaptan (day 16) were increased 1.27‐ and 1.18‐fold compared with digoxin alone (day 11); mean renal clearance of digoxin was decreased by 59% (P < .05). Tolvaptan Cmax and AUC0–24h for a single dose with digoxin (day 12) were each increased about 10% compared with tolvaptan alone (day 1). Tolvaptan did not accumulate upon multiple dosing. After a single dose of tolvaptan (day 1, day 12), 24‐hour urine volume was about 7.5 L. As expected, after 5 days of tolvaptan, 24‐hour urine volume decreased about 20%. In vitro studies in control and MDR1‐expressing LLC‐PK1 cells indicate that tolvaptan is a substrate of P‐glycoprotein. Tolvaptan (50 μM) inhibited basolateral to apical digoxin secretion to the same extent as 30 μM verapamil; the IC50 of tolvaptan was determined to be 15.9 μM. The increase in steady‐state digoxin concentrations is likely mediated by tolvaptan inhibition of digoxin secretion.

Two Novel CYP2D6*10 Haplotypes As Possible Causes of a Poor Metabolic Phenotype in Japanese

During the course of sequencing for the CYP2D6 gene, we found a novel single nucleotide polymorphism of g.3318G>A (E383K) associated with CYP2D6*10, termed as CYP2D6*72. We also found a g.1611T>A (F120I) in the CYP2D6*49, which was previously identified as a CYP2D6*10-associated allele in an independent Japanese population. To clarify the effects of these novel CYP2D6*10 haplotypes on the functions of CYP2D6, kinetic analysis for dextromethorphan O-demethylation was performed using the Escherichia coli expression system and human liver microsomes. The Vmax/Km values for dextromethorphan O-demethylation catalyzed by recombinant CYP2D6 forms encoded by CYP2D6*10, CYP2D6*49, and CYP2D6*72 were 3.0, 0.5, and 1.3%, respectively, compared with that catalyzed by CYP2D6.1. Liver microsomes from a human subject genotyped as CYP2D6*10/*49 also showed a reduced dextromethorphan O-demethylase activity. CYP2D6.49 formed a 7-hydroxydextromethorphan, with a roughly similar Vmax/Km value to that of O-demethylation. These results suggest that these two CYP2D6*10 haplotypes are possible causes of interindividual variation in the activities and the substrate specificity of CYP2D6.

Nonclinical Pharmacokinetics of a New Nonpeptide V2 Receptor Antagonist, Tolvaptan

PurposeTo evaluate the pharmacokinetic profile of tolvaptan.MethodThe nonclinical pharmacokinetic profile of [14C]tolvaptan was evaluated in an absorption, distribution, and excretion study in rats after single oral administration. An in vitro protein binding study was also performed.ResultsThe tolvaptan-derived radioactivity was rapidly absorbed and extensively distributed to all tissues in rats. The radioactivity levels were greatest in the gastrointestinal tract and liver, though the levels in the cerebrum, cerebellum and medulla oblongata were low. The serum and tissue concentrations of radioactivity, and serum concentration of tolvaptan in male and female rats showed a marked sex difference. The radioactivity was crossed the placenta and was distributed to the fetal tissues in pregnant rats. The milk showed 1.5–15.8-fold higher radioactivity than blood in lactating rats. The radioactivity mainly excreted into the feces via the biliary route. Tolvaptan binds extensively to plasma proteins (≥97.2%) in mouse, rat, rabbit, dog and human plasma.

Expression levels of multidrug resistance-associated protein 4 (MRP4) in human leukemia and lymphoma cell lines, and the inhibitory effects of the MRP-specific inhibitor MK-571 on methotrexate distribution in rats

In the development of anti-blood cancer drugs, the chronic myelocytic leukemia (KU812), acute myelocytic leukemia (KG-1) and lymphoma (U937) cell lines are commonly used in preclinical pharmacology studies as human cancer xenograft models in mice. In the present study, mRNA expression levels of typical human ATP-binding cassette (ABC) transporters in these human blood cancer cell lines were analyzed by real-time polymerase chain reaction (RT-PCR). Based on the results, the expression level of multidrug resistance-associated protein 4 (MRP4) was found to be extremely high in KU812 cells compared with those of other transporters. Additionally, MRP4 expression levels were found to be relatively high in U937, KG-1 and a blood cell line derived from a healthy subject (RPMI 1788). In addition, to elucidate the contribution of MRP4 to the methotrexate (MTX) distribution in normal blood cells and tissues, [3H]MTX was intravenously (i.v.) administered to two groups of rats. Animals in one group received [3H]MTX only; the other group was concomitantly administered i.v. MK-571, a typical inhibitor of MRP transporters. No marked difference was observed between the two groups; the Kp values (tissue concentration/plasma concentration) of the concomitant group showed slightly higher values compared with those of the MTX alone group in erythrocytes (1.4 times, P<0.001), spleen (1.3 times, P<0.05) and thymus (1.2 times, P<0.05), respectively. Although in the present study we could not evaluate the direct involvement of MRP4 in blood cancer cells in which MRP4 expression was excessively high, these results suggest a possible functional role of MRP4 in blood cancer cells and albeit only slightly in normal blood cells/tissues.

Stereospecific and simultaneous high-performance liquid chromatographic assay of flosequinan and its metabolites in human plasma.

A high-performance liquid chromatographic method was developed for the simultaneous determination of the enantiomers of flosequinan [(+/-)-7-fluoro-1-methyl-3-methylsulphinyl-4-quinolone] and its metabolites, flosequinan sulphide and sulphone, in human plasma. These compounds were extracted from plasma with chloroform. The compounds were separated on a chiral stationary phase of cellulose tris-3,5- dimethylphenylcarbamate coated on silica gel, with a mobile phase of ethanol-methanol (22:78, v/v). Flosequinan enantiomers and flosequinan sulphone were determined by UV detection at a wavelength of 320 nm. Flosequinan sulphide was determined using fluorescence detection (excitation at 370 nm, emission at 430 nm). Standard curves were linear over the concentration range 5-10,000 ng/ml for both enantiomers and flosequinan sulphide, and 20-10,000 ng/ml for flosequinan sulphone. This method is adequate for pharmacokinetic studies of the enantiomers of flosequinan and its metabolites.

Characterization of intestinal and hepatic P450 enzymes in cynomolgus monkeys with typical substrates and inhibitors for human P450 enzymes

Cynomolgus monkeys are widely used to predict human pharmacokinetic and/or toxic profiles in the drug developmental stage. Characterization of cynomolgus monkey P450s such as the mRNA expression level, substrate specificity, and inhibitor selectivity were conducted to provide helpful information in designing monkey in vivo studies and monkey-to-human extrapolation. The expression levels of 12 monkey P450 mRNAs, which are considered to be important P450 subfamilies in drug metabolism, were investigated in the liver, small intestine (duodenum, jejunum, and ileum), and colon of individual monkeys. iIn vitro activities and intrinsic clearance values were determined in monkey intestinal and liver microsomes (MIM and MLM, respectively) using nine typical oxidative reactions for human P450s. Paclitaxel 6α-hydroxylation, diclofenac 4′-hydroxylation, and S-mephenytoin 4′-hydroxylation showed low activities in MIM and MLM. IC50 values of eight selective inhibitors of human P450s were determined in MIM and MLM. Inhibitory effects of furafylline and sulfaphenazole were weak in monkeys on phenacetin O-deethylation and diclofenac 4′-hydroxylation, respectively. These results show profiles of monkey P450s in both the intestine and liver in detail and contribute to a better understanding of the species difference in substrate specificity and inhibitor selectivity between cynomolgus monkeys and humans.

Antitubercular Agent Delamanid and Metabolites as Substrates and Inhibitors of ABC and Solute Carrier Transporters

ABSTRACT Delamanid (Deltyba, OPC-67683) is the first approved drug in a novel class of nitro-dihydro-imidazooxazoles developed for the treatment of multidrug-resistant tuberculosis. Patients with tuberculosis require treatment with multiple drugs, several of which have known drug-drug interactions. Transporters regulate drug absorption, distribution, and excretion; therefore, the inhibition of transport by one agent may alter the pharmacokinetics of another, leading to unexpected adverse events. Therefore, it is important to understand how delamanid affects transport activity. In the present study, the potencies of delamanid and its main metabolites as the substrates and inhibitors of various transporters were evaluated in vitro. Delamanid was not transported by the efflux ATP-binding cassette (ABC) transporters P-glycoprotein (P-gp; MDR1/ABCB1) and breast cancer resistance protein (BCRP/ABCG2), solute carrier (SLC) transporters, organic anion-transporting polypeptides, or organic cation transporter 1. Similarly, metabolite 1 (M1) was not a substrate for any of these transporters except P-gp. Delamanid showed no inhibitory effect on ABC transporters MDR1, BCRP, and bile salt export pump (BSEP; ABCB11), SLC transporters, or organic anion transporters. M1 and M2 inhibited P-gp- and BCRP-mediated transport but did so only at the 50% inhibitory concentrations (M1, 4.65 and 5.71 μmol/liter, respectively; M2, 7.80 and 6.02 μmol/liter, respectively), well above the corresponding maximum concentration in plasma values observed following the administration of multiple doses in clinical trials. M3 and M4 did not affect the activities of any of the transporters tested. These in vitro data suggest that delamanid is unlikely to have clinically relevant interactions with drugs for which absorption and disposition are mediated by this group of transporters.

Erratum for Sasabe et al., Antitubercular Agent Delamanid and Metabolites as Substrates and Inhibitors of ABC and Solute Carrier Transporters

Volume 60, no. 6, p. [3497–3508][1], 2016. Page 3507, left column, lines 2 and 3: “nonnucleoside reverse transcriptase inhibitors (e.g., zidovudine)” should read “nucleoside reverse transcriptase inhibitors (e.g., zidovudine).” [1]: /lookup/doi/10.1128/AAC.03049-15

Hemodialysis clearance of iosimenol, a novel iso-osmolar radiographic contrast medium.

Background Iodinated contrast media (CM) have molecular and pharmacokinetic properties likely to make them highly dialyzable. Controlled clinical studies allowing for comparisons of hemodialysis clearance between different test substances and in multiple hemodialysis filters are, however, complex and not always practically feasible. A miniaturized in vitro method was therefore developed to evaluate the dialyzability of a new CM. Purpose To evaluate hemodialysis clearance of iosimenol, a novel iso-osmolar contrast medium (CM), in a select variety of hemodialysis filters and in comparison to commercially available CM. Material and Methods Three different high-flux and one low-flux membrane were used in miniaturized dialyzers to evaluate the in vitro blood clearance of iosimenol. Commercially available CM (iodixanol and iohexol) served as control substances. In vitro dialysis parameters were then used to predict clinical hemodialysis clearances. Residual ratios of endogenous substances (inorganic phosphate, urea nitrogen, creatinine, total bilirubin, and albumin) were used as proof of reliability of the in vitro dialysis system. Results Dialyzable small endogenous molecules were readily eliminated in all membranes. The removal ratios of iosimenol were generally similar to that of iodixanol in all membranes except the high-flux polysulfone but were consistently lower than that of iohexol. The blood clearance of iosimenol during clinical hemodialysis was predicted as, on average, approximately 85 mL/min with the high-flux membranes and 47 mL/min with the low-flux membrane. Conclusion The dialyzability of iosimenol was evaluated using a newly developed in vitro dialysis system, and iosimenol was readily cleared from blood with all four tested membranes. And it is suggested that the dialysis parameters can predict clinical hemodialysis clearance of CM.