Hiromi Endo

S-oxidation of S-methyl-esonarimod by flavin-containing monooxygenases in human liver microsomes

1. Studies using human liver microsomes and recombinant human cytochrome P450 (CYP) and flavin-containing monooxygenase (FMO) were performed to identify the enzymes responsible for the metabolism of S-methyl-esonarimod (M2), an active metabolite of esonarimod (KE-298, a novel antirheumatic drug). 2. S-oxidative activities of M2 significantly correlated with those of methyl p-tolyl sulfide, a specific substrate of FMOs, as tested using 10 different human liver microsomes (r2 = 0.539, p<0.05). Thermal treatment of microsomes reduced the S-oxidative activity in the absence of the NADPH-generating system at 45°C for 5 min. However, methimazole, a known competitive substrate of FMOs, was a weak inhibitor of the S-oxidation in liver microsomes. 3. Recombinant human FMO1 and FMO5 produced M3 in greater quantities than recombinant human FMO3. The S-oxidation of M2 by recombinant human FMO5 was not appreciably inhibited in the presence of methimazole. In contrast, methimazole was effective in suppressing the catalytic activity of recombinant human FMO1 and FMO3. 4. The apparent Km (Km app) for the S-oxidation of M2 in human recombinant FMO5 (2.71 μM) was similar to that obtained using human liver microsomes (2.43 μM). 5. The present results suggest that the S-oxidation of S-methyl esonarimod reflects FMO5 activity in the human liver because the recombinant FMO5 data match well with the human liver microsomal experiments.

Analgesic Effect of the Newly Developed S(+)-Flurbiprofen Plaster on Inflammatory Pain in a Rat Adjuvant-Induced Arthritis Model

Preclinical Research

Effects of Lansoprazole and Amoxicillin on Uptake of [14C]Clarithromycin into Gastric Tissue in Rats

ABSTRACT Triple therapy consisting of clarithromycin (CLR), lansoprazole (LPZ), and amoxicillin (AMZ) is effective as eradication therapy for patients with peptic ulcer disease and Helicobacter pylori infection. We evaluated the effects of LPZ and AMZ on the uptake of [14C]CLR into the gastric tissue of rats. After administration of [14C]CLR alone or in combination with LPZ and AMZ, the distributions of [14C]CLR in the main organs and gastrointestinal tissues were compared. LPZ and AMZ had no effect on the distribution of [14C]CLR in any tissue except gastric tissue. The concentration of radioactivity in gastric tissue was several times higher when [14C]CLR was administered orally together with LPZ than when it was administered alone. The gastric emptying of [14C]CLR became smaller in the case of the coadministration of LPZ. AMZ had no apparent influence on the disposition of [14C]CLR. After the intravenous administration of [14C]CLR, no effects of drug coadministration were evident. In vitro uptake of [14C]CLR into gastric tissue was enhanced in the case of a high-pH environment. The uptake was not influenced by the concurrent presence of LPZ and AMZ. These results suggest that the penetration of [14C]CLR possibly depends on elevated gastric pH, as gastric acid secretion was inhibited by LPZ, and this may be a primary factor in explaining why the concentration of [14C]CLR at the target site, gastric tissue, was enhanced by the coadministration of LPZ.

Mechanistic studies on metabolic chiral inversion of 4-(4-methylphenyl)-2-methylthiomethyl-4-oxobutanoic acid (KE-748), an active metabolite of the new anti-rheumatic agent 2-acetylthiomethyl-4-(4-methylphenyl)-4-oxobutanoic acid (KE-298), in rats.

The chiral inversion properties of 4-(4-methylphenyl)-2-methylthiomethyl-4-oxobutanoic acid (KE-748), an active metabolite of 2-acetylthiomethyl-4-(4-methylphenyl)-4-oxobutanoic acid (KE-298), were compared with those of ibuprofen in rats. After administration of R(-)-[2 alpha-2H]KE-748, S(+)-KE-748 was present in the rat plasma, and the deuterium atoms of the S(+)-enantiomer were almost all replaced by hydrogen atoms. After administration of S(+)-[2 alpha-2H]KE-748, the deuterium content of S(+)-KE-748 in the plasma remained intact. In the in vitro study, using a cell-free system and rat liver homogenates, the chiral inversion of ibuprofen was apparent when both CoA and ATP were present; however, KE-748 was not inverted. In the study on isolated rat hepatocytes, the unidirectional chiral inversion from R(-)-to S(+)-enantiomer was observed for both ibuprofen and KE-748. When R(-)-ibuprofen was incubated with medium and long chain fatty acids (carbon chain length C6 to C16), using isolated hepatocytes, the chiral inversion decreased significantly. On the other hand, when R(-)-KE-748 was incubated with short and medium chain fatty acids (carbon chain length C3 to C8), chiral inversion was inhibited markedly. To induce hepatic microsomal long chain fatty acid CoA ligase, rats were treated with clofibric acid (CF rats). In both in vitro and in vivo experiments on CF rats, chiral inversion from R(-)-to S(+)-ibuprofen was enhanced significantly compared with that in controls, whereas the enhancement was not observed in the case of R(-)-KE-748. There was no influence of benzoic acid, a typical substrate on medium chain fatty acid CoA ligase in the mitochondrial matrix, on chiral inversion of R(-)-ibuprofen, using, isolated hepatocytes. In contrast, the chiral inversion from R(-)-to S(+)-KE-748 was strongly inhibited in the presence of benzoic acid. These results indicate that chiral inversion of R(-)-KE-748 may proceed via formation of the CoA-thioester intermediate with loss of the 2 alpha-methine proton, in a manner similar to that seem with R(-)-ibuprofen. However, the enzymes needed to form CoA-thioester of R(-)-KE-748 differ from those for R(-)-ibuprofen.

Stereoselective pharmacokinetics of [14C]-labeled KE-298, a new anti-rheumatic drug, in rats.

The stereoselective pharmacokinetics of two enantiomers of [14C]-labeled KE-298 [2-acetylthiomethyl-4-(4-methylphenyl)-4-oxobutanonic acid] were investigated in rats. The blood levels of radioactivity after the oral administration of (+)-(S)-[14C]KE-298 were higher than that for (-)-(R)-[14C]KE-298; the AUC of the former was approximately twice that of the latter. No significant stereoselectivity was observed in absorption rate. The tissue/ plasma level ratios at 30 min after oral administration of (-)-(R)-[14C]KE-298 in the liver and kidney, the major metabolic and/or excretory organs, were 2 to 3 times higher than those for (+)-(S)-[14C]KE-298. Neither was evidence of stereoselectivity found in the excretion of radioactivity. During incubation with isolated rat hepatocytes in vitro, the metabolic rates of KE-298 enantiomers were not significantly different. Plasma protein binding 20 min after the oral administration of (+)-(S)-[14C]KE-298 and (-)-(R)-[14C]KE-298 was 99.3% and 97.0% respectively. Comparing the unbound fraction, (-)-(R)-[14C]KE-298 was approximately 4 times higher than (+)-(S)-[14C]KE-298. In order to make clear the relationship between stereoselective pharmacokinetics and protein binding for [14C]KE-298, the comparative pharmacokinetics of (+)-(S)-[14C]KE-298 and (-)-(R)-[14C]KE-298 were investigated in analbuminemic rats. In these animals, no evidence of stereoselectivity was found for either blood level-time profiles or plasma protein binding. These results revealed that the stereoselective pharmacokinetics of KE-298 in rats might be due to enantiomeric differences in binding to plasma albumin.

Stereoselective disposition and chiral inversion of KE‐298, a new antirheumatic drug, in rats

The present study was an attempt to elucidate the relationship between stereoselective pharmacokinetics and protein binding of KE-298 and its active metabolites, deacetyl-KE-298 (M-1) and S-methyl-KE-298 (M-2). Metabolic chiral inversion was also investigated. The levels of unchanged KE-298 in plasma after oral administration of (+)-(S)-KE-298 to rats were lower than those of (-)-(R)-KE-298, whereas the levels of M-1 and M-2 after administration of (+)-(S)-KE-298 were higher than after (-)-(R)-KE-298. In vitro, rat plasma protein binding of (+)-(S)-KE-298 was lower than that of (-)-(R)-KE-298. In contrast, the binding of (+)-(S)-M-1 and (+)-(S)-M-2 was higher than that of (-)-(R)-M-1 and (-)-(R)-M-2. Displacement studies revealed that the (+)-(S) and (-)-(R)- enantiomers of KE-298 and their metabolites bound to the warfarin binding site on rat serum albumin. These results suggested that the stereoselective plasma levels in KE-298 and its metabolites were closely related to enantiomeric differences in protein binding attributed to quantitative differences in binding to albumin rather than to the different binding sites. Unidirectional chiral inversion was detected after oral administration of either (-)-(R)-KE-298 or (-)-(R)-M-2 to rats both yielding (+)-(S)-M-2.

Impact of Humidity on In Vitro Human Skin Permeation Experiments for Predicting In Vivo Permeability.

In vitro skin permeation studies have been commonly conducted to predict in vivo permeability for the development of transdermal therapeutic systems (TTSs). We clarified the impact of humidity on in vitro human skin permeation of two TTSs having different breathability and then elucidated the predictability of in vivo permeability based on in vitro experimental data. Nicotinell(®) TTS(®) 20 and Frandol(®) tape 40mg were used as model TTSs in this study. The in vitro human skin permeation experiments were conducted under humidity levels similar to those used in clinical trials (approximately 50%) as well as under higher humidity levels (approximately 95%). The skin permeability values of drugs at 95% humidity were higher than those at 50% humidity. The time profiles of the human plasma concentrations after TTS application fitted well with the clinical data when predicted based on the in vitro permeation parameters at 50% humidity. On the other hand, those profiles predicted based on the parameters at 95% humidity were overestimated. The impact of humidity was higher for the more breathable TTS; Frandol(®) tape 40mg. These results show that in vitro human skin permeation experiments should be investigated under realistic clinical humidity levels especially for breathable TTSs.

Effects of clarithromycin and amoxicillin on gastric emptying in rats.

ABSTRACT The effects of oral administration of clarithromycin (CLR), amoxicillin (AMX), and lansoprazole (LPZ) on gastric emptying in rats were investigated by a glass powder method and a phenol red method. By both test methods, no significant effects on gastric emptying were observed when CLR, AMX, or LPZ was administered alone or when the three drugs were administered concomitantly. The levels of gastrointestinal absorption of [14C]CLR and [14C]AMX were measured. Four hours after injection of [14C]CLR or [14C]AMX into the stomach and duodenum loops of rats, 86.63 and 1.27% of the original amount of [14C]CLR administered were recovered in the contents of the stomach and duodenum loops, respectively, and 80.01 and 55.88% of the original amount of [14C]AMX administered were recovered in the contents of the stomach and duodenum loops, respectively.

In vitro evaluation of potential drug interactions mediated by cytochrome P450 and transporters for luseogliflozin, an SGLT2 inhibitor

Abstract 1. We evaluated potential in vitro drug interactions of luseogliflozin, a sodium-glucose cotransporter 2 (SGLT2) inhibitor, mediated by CYP inhibition, CYP induction and drug transporters using human liver microsomes, primary hepatocytes and recombinant cells-expressing efflux or uptake transporters, respectively. 2. Human CYP inhibition studies indicated that luseogliflozin was a weak inhibitor for CYP2C19 with an IC50 value of 58.3 μM, whereas it was not an inhibitor of the other eight major isoforms that were tested. The exposure of primary hepatocytes to luseogliflozin for 72 hrs weakly induced CYP3A4 at a concentration of 10 μM, whereas it did not induce CYP1A2 or CYP2B6 at concentrations of 0.1–10 μM. 3. An in vitro transport study suggested that luseogliflozin is a substrate for human P-glycoprotein (P-gp), but not for breast cancer resistance protein (BCRP), organic anion transporting polypeptide (OATP) 1B1 and OATP1B3, organic anion transporter (OAT) 1 and OAT3, or organic cation transporter (OCT) 2. Luseogliflozin weakly inhibited OATP1B3 with an IC50 value of 93.1 μM, but those for other transporters are greater than 100 μM. 4. Based on the therapeutic plasma concentration of the drug, clinically relevant drug interactions are unlikely to occur between luseogliflozin and coadministered drugs mediated by CYPs and/or transporters.

Formation of a disulfide protein conjugate of the SH-group-containing metabolite (M-I) of esonarimod (KE-298) and its elimination in rats

The reactivity of the thiol moiety of the active main metabolite (M‐I) of esonarimod (kE‐298), a novel anti‐rheumatic agent, was investigated in rats. After repeated oral administration of 14C‐kE‐298, the radioactivity decreased rapidly and no tendency towards accumulation was found, in marked contrast to other common SH‐group‐containing drugs. At 30 min after intravenous administration of 14C‐M‐I to rats, the concentration of the 14C‐M‐I plasma protein conjugate in plasma was extremely low at 0.143 nmol mL−1 (0.66% of total plasma radioactivity). The 14C‐M‐I plasma protein conjugate that formed in rat plasma was mixed disulfide with plasma protein. After intravenous administration of synthetic 14C‐M‐I plasma protein conjugate to rats, the radioactivity in plasma decreased rapidly, with the terminal half‐life at 6.90 h. In‐vitro, the 14C‐M‐I plasma protein conjugate was readily dissociated by the endogenous thiol compounds, cysteine and glutathione. These results suggest that the reactivity of the thiol moiety of M‐I is extremely low. Furthermore, the 14C‐M‐I plasma protein conjugate decreased rapidly in‐vivo, which would be related to interaction with endogenous thiol compounds. These properties of M‐I are principally responsible for the zero accumulation in rat tissues. kE‐298 could therefore be expected to have reduced adverse effects compared with other SH‐group‐containing anti‐rheumatic drugs.