Takafumi Iwatsubo

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.

Functional involvement of organic cation transporter1 (OCT1/Oct1) in the hepatic uptake of organic cations in humans and rats

The contribution of organic cation transporters to the saturable component in the hepatic uptake of 1-methyl-4-phenylpyridinium (MPP), tetraethylammonium (TEA), cimetidine, and metformin was examined by the use of human/rat organic cation transporter (hOCT1/rOct1)-expressing cells and human/rat hepatocytes. Transfection of rOct1 resulted in a considerable increase in the uptake of metformin, whereas that of hOCT1 resulted in only a slight increase. All test compounds (MPP, TEA, cimetidine, and metformin) accumulated in human and rat hepatocytes in a carrier-mediated manner. The Km values for the uptake of MPP, TEA, cimetidine, and metformin into human and rat hepatocytes were comparable with those into hOCT1 and rOct1-expressing cells, respectively. In addition, the relative uptake activities, which were obtained by normalizing the intrinsic uptake clearances of TEA, cimetidine, and metformin against those values of MPP in human and rat hepatocytes, were similar with the uptake activities in hOCT1 and rOct1, respectively. These results suggest that the saturable component in the hepatic uptake of these cationic compounds may be mediated mainly by hOCT1/rOct1; therefore, it is meaningful to evaluate the saturable uptake profile of cationic compounds by the liver using both hOCT1/rOct1-expressing cells and human/rat hepatocytes.

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.

Comparison of the kinetic characteristics of inhibitory effects exerted by biguanides and H2-blockers on human and rat organic cation transporter-mediated transport: Insight into the development of drug candidates

In this study, the comparison of the transport of substrates (1-methyl-4-phenylpydinium (MPP) and tetraethyl ammonium (TEA)) and the inhibition potency of the inhibitors (biguanides and H2-blockers) for human and rat organic cation transporters (hOCTs and rOcts), and the inhibition type of inhibitors for these transporters were investigated using HEK293 cells that stably express hOCT/rOct. The concentration-dependent uptake of [3H]-MPP and [14C]-TEA by hOCT1-3/rOct1-3 had Km values similar to those in the literature. It was also deduced that MPP and TEA are competitive inhibitors for hOCT1-2/rOct1-2. The Ki values for phenformin inhibition of [3H]-MPP and [14C]-TEA uptake by hOCT1-3/rOct1-3 were lower than that for metformin. The [3H]-MPP uptake by hOCT1/rOct1 and hOCT3/rOct3 was inhibited by famotidine and ranitidine whereas that by hOCT2/rOct2 was not. The inhibitory potency of cimetidine for hOCT1-2 was very weak. In most cases, the differences in the Vmax/Km values of substrates and the Ki values of inhibitors between hOCT and rOct were minor. The acquisition of information on OCT/Oct mediated-transport and/or inhibition such as that presented in this report is very useful for further understanding of certain aspects of uptake, distribution, and excretion for drug candidates.

Utility of P-Glycoprotein and Organic Cation Transporter 1 Double-Transfected LLC-PK1 Cells for Studying the Interaction of YM155 Monobromide, Novel Small-Molecule Survivin Suppressant, with P-Glycoprotein

1-(2-Methoxyethyl)-2-methyl-4,9-dioxo-3-(pyrazin-2-ylmethyl)-4,9-dihydro-1H-naphtho[2,3-d]imidazolium bromide (YM155 monobromide), a novel small molecule that downregulates survivin and exhibits potent antitumor activity, is hydrophilic and cationic. Although previous studies have shown that influx transporters play important roles in the uptake of YM155 into hepatocytes and possibly into cancer cells, efflux transporters have yet to be investigated. In this study, we assessed the interaction of YM155 with P-glycoprotein [multidrug resistance 1 (MDR1)/ATP-binding cassette B1] using two kinds of transcellular transport systems: Caco-2 and MDR1-expressing LLC-PK1 cells (LLC-MDR1). We also used a newly established LLC-OCT1/MDR1 cell line, which expresses basal YM155 uptake transporter organic cation transporter1 (OCT1) and apical MDR1. Direct interaction between YM155 and MDR1 and other efflux transporters was evaluated using transporter-expressing membrane vesicles. A bidirectional transporter assay using Caco-2 and LLC-MDR1 cells showed low permeability and no vectorial transport of YM155, suggesting that YM155 is not a substrate of MDR1. However, vectorial transport across LLC-OCT1/MDR1 cells was identified, which was inhibited by the MDR1 inhibitor cyclosporine A, clearly indicating that YM155 is in fact a substrate of MDR1. Insufficient expression of basal uptake transporter of YM155 in Caco-2 and LLC-MDR1 might have confounded conclusions regarding YM155 and MDR1. Using the transporter-expressing vesicles, MDR1-mediated transport was most significantly involved in YM155 transport among the efflux transporters examined. In conclusion, these findings suggest that YM155 is a substrate of MDR1, and that MDR1 may play an important role in the pharmacokinetics of YM155. Transcellular assays lacking basal uptake transporters may be inaccurate in the assessment of hydrophilic compounds that have poor membrane permeability by passive diffusion.

Effect of cationic drugs on the transporting activity of human and rat OCT/Oct 1–3 in vitro and implications for drug–drug interactions

The inhibitory effects of cationic drugs (β-adrenoreceptor antagonists, calcium (Ca)-channel blocker, If channel inhibitor, antiarrhythmic drugs, and antibacterial drugs) that inhibit 1-methyl-4-phenylpyridinium (MPP) and/or metformin uptake into hOCT1–3/rOct1–3-expressing cells and human/rat hepatocytes were investigated in this study. The drug–drug interaction (DDI) potential of these drugs for the hOCT/rOct-mediated hepatic/renal uptake process was also assessed. The IC50 values of cardiovascular drugs, including an If channel inhibitor with a new mechanism of action, were greater for hOCT2/rOct2 than those for hOCT1/rOct1 or hOCT3/rOct3. No species differences in these values were observed between hOCTs and rOcts. As for hOCT2-mediated uptake, the IC50 values of quinidine and the If channel inhibitor for metformin uptake were lower than those for MPP uptake. However, previous clinical studies found that the IC50 values of these drugs for hOCT1/rOct1 and hOCT2/rOct2 were much greater than their unbound plasma concentrations, which suggests that the DDIs of these cationic compounds may not be related to hOCT/rOct-mediated hepatic/renal uptake pathways. In addition, investigation of the luminal transporters of cationic compounds in the kidney, as well as the in vitro DDI potential of their inhibitors, is important for the clarification of cationic compound DDIs in humans.

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.

Pharmacokinetics and Tissue Distribution of Tacrolimus (FK506) After a Single or Repeated Ocular Instillation in Rabbits

PURPOSE The aim of this study was to investigate the ocular distribution of tacrolimus (FK506) and absorption into the systemic circulation after a single or repeated topical instillation of FK506 ophthalmic suspension in male New Zealand white rabbits. METHODS In the single instillation study (group 1), 29.1-34.8 microL of a 0.1, 0.3, and 1% suspension was administered to each of the 15 rabbits. In the repeated instillation study (group 2), 27.1-39.5 microL of a 0.3% suspension was administered to 27 rabbits q.i.d. (i.e., at 3-h intervals) for 14 days. In the intravenous (i.v.) dose study (group 3), 1 mg/kg of FK506 was administered to 3 rabbits. The amount of FK506 was measured by using a competitive enzyme immunoassay. RESULTS The results for single and repeated instillation studies were similar. In the single instillation study, blood T(max) after an instillation of the 0.1, 0.3, and 1% suspensions (at 0.8, 1.0, and 1.0 hours) did not differ significantly among these doses. One (1) h after an instillation of the 1% suspension, ocular tissue concentrations, except the retina/choroid, vitreous body, and lens, were higher than the blood concentration (C(max): 2.7 ng/mL). In particular, concentrations in the conjunctiva, cornea, iris, and anterior sclera were much higher than the blood concentration (148, 900, 120, and 145 ng/g tissue). In the repeated instillation study, concentrations in the blood and ocular tissues (except the lens) reached a steady state by the 7th day. In the i.v. dose study, AUC(0-24h) and T(1/2) were 1643 ng h/mL and 18.5 h, respectively. CONCLUSIONS The high-level distribution of FK506 was observed in the conjunctiva, which is desirable because the conjunctiva is the target tissue for pharmacologic effect (i.e., efficacy).

Hepatic Uptake and Excretion of (–)-N-{2-[(R)-3-(6,7-Dimethoxy-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)piperidino]ethyl}-4-fluorobenzamide (YM758), a Novel If Channel Inhibitor, in Rats and Humans

(–)-N-{2-[(R)-3-(6,7-Dimethoxy-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)piperidino]ethyl}-4-fluorobenzamide (YM758), a novel “funny” If current channel (If channel) inhibitor, is being developed as a treatment for stable angina and atrial fibrillation. The hepatic uptake/excretion of YM758 was clarified using transporter-expressing mammalian cells and hepatocytes mainly in humans and partly in rats. cDNA-expressing human embryonic kidney 293 cells were used to determine that YM758 was greatly taken up via organic anion-transporting polypeptide (OATP) 1B1 and slightly via human organic cation transporter (hOCT) 1/rat organic cation transporter 1 but not via OATP1B3. In addition, the uptake of 17β-estradiol-d-17β-glucuronide via OATP1B1 was inhibited in the presence of YM758, whereas that via OATP1B3 was not. In contrast, time-dependent uptake of YM758 into rat/human hepatocytes at 37°C was observed, as was concentration-dependent uptake into human hepatocytes (Km value of 87.9 μM). This saturable uptake of YM758 into human hepatocytes was inhibited in the presence of quinidine (an inhibitor for OATP1B1) but not cimetidine (an inhibitor for the hOCT family). Moreover, the permeation clearance ratios for the transcellular transport of YM758 across multidrug resistance (MDR) 1-expressing LLC-PK1 cells were extensively higher than those across LLC-PK1 cells, which indicate that MDR1-mediated transport is one of the possible pathways through which YM758 may be excreted into the bile. These results indicate that YM758 is taken up into hepatocytes mainly via OATP1B1, but not via hOCT1, and is excreted into the bile via MDR1 in humans; however, passive diffusion or an unknown uptake/excretion mechanism could be at work in the hepatocytes. This study is the first to clarify the saturable hepatic uptake and/or the excretion mechanism by the If channel inhibitor.