Aiji Miyashita

Optimized Methods for Targeted Peptide-Based Quantification of Human Uridine 5′-Diphosphate-Glucuronosyltransferases in Biological Specimens Using Liquid Chromatography–Tandem Mass Spectrometry

The aim of this study was to optimize methods for quantifying 13 uridine 5′-diphosphate-glucuronosyltransferase (UGT) isoforms (UGT1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B10, 2B15, and 2B17) in human liver, intestinal, and kidney microsomes, and in recombinant human UGT-expressing insect cell membranes (rhUGTs) by targeted peptide–based quantification using liquid chromatography–tandem mass spectrometry. Production of targeted peptides was compared by combining three denaturing agents (urea, sodium deoxycholate, and octyl glucoside) and three denaturing temperatures (37°C, 60°C, and 95°C) followed by tryptic digestion for 2–20 hours. Denaturing conditions and digestion times yielding high production efficiency varied markedly among isoforms and specimens, indicating the importance of specific optimization. Each UGT isoform was quantified using the methods found to be optimal. The expression of 10 (1A1, 1A3, 1A4, 1A6, 1A9, 2B4, 2B7, 2B10, 2B15, and 2B17), 6 (1A1, 1A3, 1A4, 1A10, 2B7, and 2B17), and 3 (1A6, 1A9, and 2B7) isoforms was detected in human liver, intestinal, and kidney microsomes, respectively, and levels were reproducible using multiple protocols. All isoforms were quantified in rhUGTs. Determining the levels of UGTs in human tissue specimens and those in rhUGTs is important for estimating the contribution of glucuronidation to body clearance based on in vitro–in vivo extrapolation.

Absorption, Metabolism and Excretion of [14C]Mirabegron (YM178), a Potent and Selective β3-Adrenoceptor Agonist, after Oral Administration to Healthy Male Volunteers

The mass balance and metabolite profiles of 2-(2-amino-1,3-thiazol-4-yl)-N-[4-(2-{[(2R)-2-hydroxy-2-phenylethyl]amino}ethyl)[U-14C]phenyl]acetamide ([14C]mirabegron, YM178), a β3-adrenoceptor agonist for the treatment of overactive bladder, were characterized in four young, healthy, fasted male subjects after a single oral dose of [14C]mirabegron (160 mg, 1.85 MBq) in a solution. [14C]Mirabegron was rapidly absorbed with a plasma tmax for mirabegron and total radioactivity of 1.0 and 2.3 h postdose, respectively. Unchanged mirabegron was the most abundant component of radioactivity, accounting for approximately 22% of circulating radioactivity in plasma. Mean recovery in urine and feces amounted to 55 and 34%, respectively. No radioactivity was detected in expired air. The main component of radioactivity in urine was unchanged mirabegron, which accounted for 45% of the excreted radioactivity. A total of 10 metabolites were found in urine. On the basis of the metabolites found in urine, major primary metabolic reactions of mirabegron were estimated to be amide hydrolysis (M5, M16, and M17), accounting for 48% of the identified metabolites in urine, followed by glucuronidation (M11, M12, M13, and M14) and N-dealkylation or oxidation of the secondary amine (M8, M9, and M15), accounting for 34 and 18% of the identified metabolites, respectively. In feces, the radioactivity was recovered almost entirely as the unchanged form. Eight of the metabolites characterized in urine were also observed in plasma. These findings indicate that mirabegron, administered as a solution, is rapidly absorbed after oral administration, circulates in plasma as the unchanged form and metabolites, and is recovered in urine and feces mainly as the unchanged form.

Identification of human cytochrome P450 isoforms and esterases involved in the metabolism of mirabegron, a potent and selective β3-adrenoceptor agonist

Human cytochrome P450 (CYP) enzymes and esterases involved in the metabolism of mirabegron, a potent and selective human β3-adrenoceptor agonist intended for the treatment of overactive bladder, were identified in in vitro studies. Incubations of mirabegron with recombinant human CYP enzymes showed significant metabolism of mirabegron by CYP2D6 and CYP3A4 only. Correlation analyses showed a significant correlation between mirabegron metabolism and testosterone 6β-hydroxylation (CYP3A4/5 marker activity). In inhibition studies using antiserum against CYP3A4, a strong inhibition (at maximum 80% inhibition) of the metabolism of mirabegron was observed, whereas the inhibitory effects of monoclonal antibodies against CYP2D6 were small (at maximum 10% inhibition). These findings suggest that CYP3A4 is the primary CYP enzyme responsible for in vitro oxidative metabolism of mirabegron, with a minor role of CYP2D6. Mirabegron hydrolysis was catalyzed in human blood, plasma and butyrylcholinesterase (BChE) solution, but not in human liver microsomes, intestinal microsomes, liver S9, intestinal S9 and recombinant acetylcholinesterase solution. Km values of mirabegron hydrolysis in human blood, plasma and BChE solution were all similar (13.4–15.2 μM). The inhibition profiles in human blood and plasma were also similar to those in BChE solution, suggesting that mirabegron hydrolysis is catalyzed by BChE.

Protein quantification of UDP-glucuronosyltransferases 1A1 and 2B7 in human liver microsomes by LC-MS/MS and correlation with glucuronidation activities

The aims of this study were to quantify absolute protein levels of uridine 5′-diphosphate-glucuronosyltransferases (UGTs) 1A1 and 2B7 in human liver microsomes (HLMs) and to investigate their correlation with marker activities. A quantification method for UGT1A1 and UGT2B7 in HLMs was developed. Unique tryptic peptides of UGT1A1 and UGT2B7 in tryptically digested HLMs were simultaneously quantified by liquid chromatography (LC) equipped with tandem mass spectrometry (MS) using corresponding stable isotope-labelled peptides as internal standards. Bovine serum albumin was used as a blank matrix for calibration curve samples. Our procedure had good digestion efficiency, sensitivity, calibration curve linearity, and reproducibility of digestion to quantification. In 16 individual HLMs, the protein levels of UGT1A1 and UGT2B7 ranged from 6.50 to 44.6 pmol/mg and 4.45 to 18.2 pmol/mg, respectively. Estradiol 3β-glucuronidation correlated strongly with the UGT1A1 level, indicating its high reliability as a reaction marker. Both morphine 3-O- and 6-O-glucuronidation significantly correlated with UGT2B7 level. However, the intercept of the linear regression clearly indicates that morphine glucuronidation was mediated by other UGT isoforms in addition to UGT2B7.

In vitro inhibition and induction of human cytochrome P450 enzymes by mirabegron, a potent and selective β3-adrenoceptor agonist

The potential for mirabegron, a β3-adrenoceptor agonist for the treatment of overactive bladder, to cause drug–drug interactions via inhibition or induction of cytochrome P450 (CYP) enzymes was investigated in vitro. Mirabegron was shown to be a time-dependent inhibitor of CYP2D6 in the presence of NADPH as the IC50 value in human liver microsomes decreased from 13 to 4.3 μM after 30-min pre-incubation. Further evaluation indicated that mirabegron may act partly as an irreversible or quasi-irreversible metabolism-dependent inhibitor of CYP2D6. Therefore, the potential of mirabegron to inhibit the metabolism of CYP2D6 substrates in vivo cannot be excluded. Mirabegron was predicted not to cause clinically significant metabolic drug–drug interactions via inhibition of CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2E1, or CYP3A4/5 because the IC50 values for these enzymes both with and without pre-incubation were >100 μM (370 times maximum human plasma concentration [Cmax]). Whereas positive controls (100 µM omeprazole and 10 µM rifampin) caused the anticipated CYP induction, the highest concentration of mirabegron (10 µM; 37 times plasma Cmax) had minimal effect on CYP1A2 and CYP3A4/5 activity, and CYP1A2 and CYP3A4 mRNA levels in freshly isolated human hepatocytes, suggesting that mirabegron is not an inducer of these enzymes.

Identification of UDP-Glucuronosyltransferases Responsible for the Glucuronidation of Darexaban, an Oral Factor Xa Inhibitor, in Human Liver and Intestine

Darexaban maleate is a novel oral direct factor Xa inhibitor, which is under development for the prevention of venous thromboembolism. Darexaban glucuronide was the major component in plasma after oral administration of darexaban to humans and is the pharmacologically active metabolite. In this study, we identified UDP-glucuronosyltransferases (UGTs) responsible for darexaban glucuronidation in human liver microsomes (HLM) and human intestinal microsomes (HIM). In HLM, the Km value for darexaban glucuronidation was >250 μM. In HIM, the reaction followed substrate inhibition kinetics, with a Km value of 27.3 μM. Among recombinant human UGTs, UGT1A9 showed the highest intrinsic clearance for darexaban glucuronidation, followed by UGT1A8, -1A10, and -1A7. All other UGT isoforms were inactive toward darexaban. The Km value of recombinant UGT1A10 for darexaban glucuronidation (34.2 μM) was comparable to that of HIM. Inhibition studies using typical UGT substrates suggested that darexaban glucuronidation in both HLM and HIM was mainly catalyzed by UGT1A8, -1A9, and -1A10. Fatty acid-free bovine serum albumin (2%) decreased the unbound Km for darexaban glucuronidation from 216 to 17.6 μM in HLM and from 35.5 to 18.3 μM in recombinant UGT1A9. Recent studies indicated that the mRNA expression level of UGT1A9 is extremely high among UGT1A7, -1A8, -1A9, and -1A10 in human liver, whereas that of UGT1A10 is highest in the intestine. Thus, the present results strongly suggest that darexaban glucuronidation is mainly catalyzed by UGT1A9 and UGT1A10 in human liver and intestine, respectively. In addition, UGT1A7, -1A8, and -1A9 play a minor role in human intestine.

Intestinal Absorption Mechanism of Mirabegron, a Potent and Selective β3-Adrenoceptor Agonist: Involvement of Human Efflux and/or Influx Transport Systems

Mirabegron, a weak-basic compound, is a potent and selective β3-adrenoceptor agonist for the treatment of overactive bladder. Mirabegron extended release formulation shows dose-dependent oral bioavailability in humans, which is likely attributable to saturation of intestinal efflux abilities leading to higher absorption with higher doses. This study evaluated the membrane permeability of mirabegron and investigated the involvement of human intestinal transport proteins in the membrane permeation of mirabegron. Transcellular transport and cellular/vesicular uptake assays were performed using Caco-2 cells and/or human intestinal efflux (P-glycoprotein [P-gp], breast cancer resistance protein [BCRP], and multidrug resistance associated protein 2 [MRP2]) and influx (peptide transporter 1 [PEPT1], OATP1A2, and OATP2B1) transporter-expressing cells, vesicles, or Xenopus laevis oocytes. The absorptive permeability coefficients of mirabegron in Caco-2 cells (1.68-1.83 × 10(-6) cm/s) at the apical and basal pH of 6.5 and 7.4, respectively, were slightly higher than those of nadolol (0.97-1.41 × 10(-6) cm/s), a low permeability reference standard, but lower than those of metoprolol and propranolol (both ranged from 8.49 to 11.6 × 10(-6) cm/s), low/high permeability boundary reference standards. Increasing buffer pH at the apical side from 5.5 to 8.0 gradually increased the absorptive permeation of mirabegron from 0.226 to 1.66 × 10(-6) cm/s, but was still less than the value in the opposite direction (11.0-14.2 × 10(-6) cm/s). The time- and concentration-dependent transport of mirabegron was observed in P-gp-expressing cells and OATP1A2-expressing oocytes with apparent Km values of 294 and 8.59 μM, respectively. In contrast, no clear BCRP-, MRP2-, PEPT1-, or OATP2B1-mediated uptake of mirabegron was observed in their expressing vesicles or cells. These findings suggest that mirabegron has low-to-moderate membrane permeability and P-gp is likely to be involved in its efflux into the lumen in the intestinal absorption process. The results also suggest that mirabegron could possibly be transported by intestinal influx transporters as well as simple diffusion.

Conclusive identification of the oxybutynin-hydrolyzing enzyme in human liver.

The aim of this study was to conclusively determine the enzyme responsible for the hydrolysis of oxybutynin in human liver. Hydrolysis in human liver microsomes (HLMs) and human liver cytosol (HLC) followed Michaelis-Menten kinetics with similar Km values. In recombinant human carboxylesterase (CES)-expressing microsomes, CES1 was much more efficient than CES2 and yielded a Km value more comparable with that found in HLMs or HLC than did CES2. A correlation analysis using a set of individual HLMs, in which both CESs acted independently showed that the hydrolysis rate of oxybutynin, correlated significantly with a CES1 marker reaction, clopidogrel hydrolysis, but not with a CES2 marker reaction, irinotecan (CPT-11) hydrolysis. Chemical inhibition studies using bis-(p-nitrophenyl) phosphate, clopidogrel, nordihydroguaiaretic acid, procainamide, physostigmine, and loperamide revealed that the effects of these compounds in HLMs, HLC, and recombinant CES1-expressing microsomes were similar, whereas those in CES2-expressing microsomes were clearly different. These results strongly suggest that CES1, rather than CES2, is the principal enzyme responsible for the hydrolysis of oxybutynin in human liver.

Absorption, metabolism and excretion of darexaban (YM150), a new direct factor Xa inhibitor in humans

1. The absorption, metabolism and excretion of darexaban (YM150), a novel oral direct factor Xa inhibitor, were investigated after a single oral administration of [14C]darexaban maleate at a dose of 60 mg in healthy male human subjects. 2. [14C]Darexaban was rapidly absorbed, with both blood and plasma concentrations peaking at approximately 0.75 h post-dose. Plasma concentrations of darexaban glucuronide (M1), the pharmacological activity of which is equipotent to darexaban in vitro, also peaked at approximately 0.75 h. 3. Similar amounts of dosed radioactivity were excreted via faeces (51.9%) and urine (46.4%) by 168 h post-dose, suggesting that at least approximately half of the administered dose is absorbed from the gastrointestinal tract. 4. M1 was the major drug-related component in plasma and urine, accounting for up to 95.8% of radioactivity in plasma. The N-oxides of M1, a mixture of two diastereomers designated as M2 and M3, were also present in plasma and urine, accounting for up to 13.2% of radioactivity in plasma. In faeces, darexaban was the major drug-related component, and N-demethyl darexaban (M5) was detected as a minor metabolite. 5. These findings suggested that, following oral administration of darexaban in humans, M1 is quickly formed during first-pass metabolism via UDP-glucuronosyltransferases, exerting its pharmacological activity in blood before being excreted into urine and faeces.

Pharmacokinetics, distribution and excretion of YM155 monobromide, a novel small‐molecule survivin suppressant, in male and pregnant or lactating female rats

YM155 monobromide is a novel small‐molecule survivin suppressant. The pharmacokinetics, distribution and excretion of YM155/[14C]YM155 were investigated using males and pregnant or lactating female rats after a single intravenous bolus administration. For the 0.1, 0.3 and 1 mg/kg YM155 doses given to male rats, increases in area under the plasma concentration–time curves were approximately proportional to the increase in the dose level. After administering [14C]YM155, radioactivity concentrations in the kidney and liver were highest among the tissues in both male and pregnant rats: e.g. 14.8‐ and 5.24‐fold, respectively, and higher than in plasma at 0.1 h after dosing to male rats. The YM155 concentrations in the brain were lowest: 25‐fold lower than in plasma. The transfer of radioactivity into fetuses was low (about 2‐fold lower than in plasma). In lactating rats, the radioactivity was transferred into milk at a level 8‐ to 21‐fold higher than for plasma. Radioactivity was primarily excreted in feces (64.0%) and urine (35.2%). The fecal excretion was considered to have occurred mainly by biliary excretion and partly by secretion across the gastrointestinal membrane from the blood to the lumen. Copyright