Hidetaka Kamimura

Characterization of Human Organic Cation Transporter 1 (OCT1/SLC22A1)- and OCT2 (SLC22A2)-Mediated Transport of 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 Survivin Suppressant

1-(2-Methoxyethyl)-2-methyl-4,9-dioxo-3-(pyrazin-2-ylmethyl)-4,9-dihydro-1H-naphtho[2,3-d]imidazolium bromide (YM155 monobromide) is a novel small-molecule survivin suppressant that induces the down-regulation of survivin and exhibits potent antitumor activity in nude mice bearing human hormone refractory prostate carcinoma cell line PC-3. Although YM155, which has a cationic moiety in its structure, is influxed into its pharmacologically effective site (cancer cells) and one of its eliminating organs (hepatocytes) in a transporter-mediated manner, the mechanism seems to be different between the two cell types. The other eliminating organ is the kidney. In this study, the transport of [14C]YM155 was characterized by using human embryonic kidney 293 cells expressing organic cation transporter 1 (OCT1/SLC22A1), OCT2 (SLC22A2), and OCT3 (SLC22A3). YM155 inhibited the uptake of a typical substrate [3H]1-methyl-4-phenylpyridinium via OCT1, OCT2, and OCT3 with IC50 values of 23.8, 15.9, and 108 μM, respectively. The time- and saturable concentration-dependent uptake of [14C]YM155 was observed in cells expressing OCT1 and OCT2 with Km values of 22.1 and 2.67 μM, respectively, but not in cells expressing OCT3. By taking into consideration the tissue distribution and localization of each transporter, these results suggest that, in humans, YM155 is taken up from the blood into hepatocytes and proximal tubular cells via OCT1 and OCT2, respectively. The comparison of the IC50 values of OCT inhibitors and Km values for the uptake of YM155 into cells expressing OCTs with those into cancer cell lines indicated that transporter(s) other than OCT1 and OCT2 are involved in the uptake of YM155 into cancer cell lines.

Carrier-Mediated Uptake of 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 Survivin Suppressant, into Human Solid Tumor and Lymphoma Cells

1-(2-Methoxyethyl)-2-methyl-4,9-dioxo-3-(pyrazin-2-ylmethyl)-4,9-dihydro-1H-naphtho[2,3-d]imidazolium bromide (YM155 monobromide) is a novel small-molecule survivin suppressant that induces the down-regulation of survivin and exhibits potent antitumor activity in nude mice bearing the human hormone refractory prostate carcinoma cell line PC-3. In this study, radioluminographic determination of the in vivo distribution of radioactivity after administration of [14C]YM155 to PC-3-xenografted nude mice revealed a relatively high level of radioactivity in the PC-3 xenograft. Therefore, the uptake of [14C]YM155 was further characterized in vitro using PC-3, lung cancer (Calu-6 and NCI-H358), malignant melanoma (A375 and SK-MEL-5), and non-Hodgkins lymphoma (RL and Ramos) cell lines. The uptake of [14C]YM155 in these cell lines was dependent on incubation time, temperature, and drug concentration. The Michaelis-Menten constant values were similar among the seven cell lines (0.189–0.367 μM). The effects of various compounds on the uptake of [14C]YM155 were tested in PC-3, Calu-6, A375, RL, and Ramos cell lines. Of the compounds tested, the cationic transporter substrates/inhibitors (tetraethylammonium, 1-methyl-4-phenylpyridium, cimetidine, prazosin, corticosterone, verapamil, amantadine, procainamide, and N-methylnicotinamide) inhibited the uptake of [14C]YM155 to a similar extent among the five cell lines. The half-maximal inhibitory concentration values (IC50) of several compounds for the uptake of [14C]YM155 into PC-3 differed from those reported in the literature for human organic cation transporter 1 (OCT1/SLC22A1), OCT2 (SLC22A2), and OCT3 (SLC22A3). To summarize, YM155 was taken up into cancer cells in a carrier-mediated manner and with a similar affinity among all the cancer cell lines tested. An influx transporter(s) may contribute to this process.

Involvement of human organic cation transporter 1 in the hepatic uptake of 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 survivin suppressant.

1-(2-Methoxyethyl)-2-methyl-4,9-dioxo-3-(pyrazin-2-ylmethyl)-4,9-dihydro-1H-naphtho[2,3-d]imidazolium bromide (YM155 monobromide), which is a hydrophilic and cationic compound, exhibits antitumor activity in experimental human hormone refractory prostate carcinoma models. Urinary excretion was 18.3 to 28.6% of the dose in the clinical phase I study, and nonrenal elimination may be explained by the biliary excretion of YM155 in its unchanged form. Because the penetration through the sinusoidal membrane of the hepatocytes is the first step and an important part of biliary excretion, we evaluated the uptake of [14C]YM155 into human cryopreserved hepatocytes. YM155 was taken up into hepatocytes in a temperature- and concentration-dependent manner. The saturable uptake component was much higher than the nonsaturable passive diffusion component. In vitro hepatic uptake clearance was consistent with the in vivo hepatic intrinsic clearance calculated using clinical study data. Hepatic uptake of YM155 was inhibited by organic cation transporter (OCT) inhibitors, and the IC50 values for YM155 uptake were comparable to those reported for human OCT1-mediated transport. The interaction of YM155 with candidate transporter, OCT1, was also characterized using S2 cells stably expressing human OCT1 (OCT1-S2) cells. In OCT1-expressing S2 cells, YM155 inhibited the OCT1-mediated uptake of a typical OCT1 substrate, [14C]tetraethylammonium. In addition, YM155 was taken up into OCT1-S2 cells These results indicated that OCT1 was the predominant transporter for the hepatic uptake of YM155, and the transporter-mediated uptake clearance observed in vitro may account for the in vivo intrinsic hepatic clearance.

Hepatic uptake and excretion of 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.

Absorption, Distribution, and Excretion of 14C-labeled Tacrolimus (FK506) after a Single or Repeated Ocular Instillation in Rabbits

PURPOSE The aim of this study was to investigate the absorption, distribution, and excretion of radioactivity in male rabbits after a single or repeated instillation of (14)C-labeled tacrolimus (FK506) ophthalmic suspension or an intravenous (i.v.) administration of (14)C-FK506. METHODS The 0.3% (14)C-FK506 suspension was administered in single and repeated (three times, 5-min intervals) instillation studies, and 1 mg/kg of (14)C-FK506 was administered in the i.v. dose study. RESULTS Results for single and repeated instillation studies were similar. In eyeball microautoradiograms, 15 min after dosing, the level of radioactivity in the cornea was the highest, followed by conjunctiva. After 1 h, little specific distribution was detected in the corneal epithelium, stroma, or Descemets membrane. At 24 h, the level of radioactivity in the cornea decreased. Whole-body autoradiograms showed that the radioactivity was distributed to the digestive tract through the nasal meatus and esophagus and then was excreted into the feces. In the i.v. dose study, the distribution of radioactivity in whole-body autoradiographs was similar to that in quantitative tissue distribution measurements. The excretion of radioactivity in the urine and feces up to 168 h were 4.5 and 94.9%, respectively. CONCLUSIONS After the ocular instillation, FK506 is first absorbed in the cornea, conjunctiva, and nasolacrimal duct, and then the rest is distributed to digestive tract through the nasal meatus and esophagus, after which it is excreted mainly into the feces.

Formation of the Accumulative Human Metabolite and Human-Specific Glutathione Conjugate of Diclofenac in TK-NOG Chimeric Mice with Humanized Livers

3′-Hydroxy-4′-methoxydiclofenac (VI) is a human-specific metabolite known to accumulate in the plasma of patients after repeated administration of diclofenac sodium. Diclofenac also produces glutathione-conjugated metabolites, some of which are human-specific. In the present study, we investigated whether these metabolites could be generated in humanized chimeric mice produced from TK-NOG mice. After a single oral administration of diclofenac to humanized mice, the unchanged drug in plasma peaked at 0.25 hour and then declined with a half-life (t1/2) of 2.4 hours. 4′-Hydroxydiclofenac (II) and 3′-hydroxydiclofenac also peaked at 0.25 hour and were undetectable within 24 hours. However, VI peaked at 8 hours and declined with a t1/2 of 13 hours. When diclofenac was given once per day, peak and trough levels of VI reached plateau within 3 days. Studies with administration of II suggested VI was generated via II as an intermediate. Among six reported glutathione-conjugated metabolites of diclofenac, M1 (5-hydroxy-4-(glutathion-S-yl)diclofenac) to M6 (2′-(glutathion-S-yl)monoclofenac), we found three dichlorinated conjugates [M1, M2 (4′-hydroxy-3′-(glutathion-S-yl)diclofenac), and M3 (5-hydroxy-6-(glutathion-S-yl)diclofenac)], and a single monochlorinated conjugate [M4 (2′-hydroxy-3′-(glutathion-S-yl)monoclofenac) or M5 (4′-hydroxy-2′-(glutathion-S-yl)monoclofenac)], in the bile of humanized chimeric mice. M4 and M5 are positional isomers and have been previously reported as human-specific in vitro metabolites likely generated via arene oxide and quinone imine–type intermediates, respectively. The biliary monochlorinated metabolite exhibited the same mass spectrum as those of M4 and M5, and we discuss whether this conjugate corresponded to M4 or M5. Overall, humanized TK-NOG chimeric mice were considered to be a functional tool for the study of drug metabolism of diclofenac in humans.

Assessment of chimeric mice with humanized livers in new drug development: generation of pharmacokinetics, metabolism and toxicity data for selecting the final candidate compound.

Abstract 1. Chimeric mice with humanized livers are expected to be a novel tool for new drug development. This review discusses four applications where these animals can be used efficiently to collect supportive data for selecting the best compound in the final stage of drug discovery. 2. The first application is selection of the final compound based on estimated pharmacokinetic parameters in humans. Since chimeric mouse livers are highly repopulated with human hepatocytes, hepatic clearance values in vivo could be used preferentially to estimate pharmacokinetic profiles for humans. 3. The second is prediction of human-specific or disproportionate metabolites. Chimeric mice reproduce human-specific metabolites of drugs under development to conform to ICH guidance M3(R2), except for compounds that were extensively eliminated by co-existing mouse hepatocytes. 4. The third is identifying metabolites with distinct pharmacokinetic profiles in humans. Slow metabolite elimination specifically in humans increases its exposure level, but if its elimination is faster in laboratory animals, the animal exposure level might not satisfy ICH guidance M3(R2). 5. Finally, two examples of reproducing acute liver toxicity in chimeric mice are introduced. Integrated pharmacokinetics, metabolism and toxicity information are expected to assist pharmaceutical scientists in selecting the best candidate compound in new drug development.

Identification of human metabolites of YM758, a novel If channel inhibitor, and investigation of the transporter-mediated renal and hepatic excretion of these metabolites

(–)-N-{2-[(R)-3-(6,7-Dimethoxy-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)piperidino]ethyl}-4-fluorobenzamide (YM758) is a novel inhibitor of the “funny” If current channel (If channel) that is expressed in the sinus node of heart and is being developed as a treatment for stable angina and atrial fibrillation. Its metabolites were identified in human urine, plasma, and feces by radio-high-performance liquid chromatography and liquid chromatographytandem mass spectrometry analyses after oral administration of [14C]YM758. 6,7-Dimethoxy-2-[(3R)-piperidin-3-ylcarbonyl]-1,2,3,4-tetrahydroisoquinoline (YM-252124), (5R)-5-[(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)carbonyl]piperidin-2-one (YM-385459), 2-{[(3R)-1-{2-[(4-fluorobenzoyl)amino]ethyl}piperidin-3-yl]carbonyl}-7-methoxy-1,2,3,4-tetrahydroisonolin-6-yl β-d-glucopyranosiduronic acid (AS2036329), and the unchanged drug were detected as major constituents in both urine and plasma, whereas N-(4-fluorobenzoyl)glycine (YM-385461) was detected in plasma, but not in urine. The renal and hepatic uptake transporters for these metabolites were investigated by assessing their inhibitory effect on uptake activity in human (h) organic cation transporter (OCT) 1–3/rat (r) Oct1–3, human organic anion transporter (OAT) 1/rOat1, hOAT3/rOat3, and organic anion-transporting protein 1B1/1B3-expressing HEK293 cells. IC50 values of YM-252124 for 1-methyl-4-phenylpyridinium uptake via hOCT2 and rOct2 were 93.9 and 1700 μM, respectively, suggesting that this metabolite is secreted into urine via hOCT2/rOct2 and that the large difference in the inhibitory potentials between hOCT2 and rOct2 explains the species difference in the urinary excretion ratio of the radioactivity. The renal secretion of YM-385461, one derivative of p-aminohippuric acid, via hOAT1/rOat1, and hepatic uptake of YM-252124 via hOCT1/rOct1 was also expected. This kind of study was useful in investigating the relationship between the urinary/hepatic elimination and the transport activity for metabolites.1 Title page Identification of human metabolites of YM758, a novel If channel inhibitor, and investigation of the transporter-mediated renal and hepatic excretion of these metabolites Ken-ichi Umehara, Nobuaki Shirai, Takafumi Iwatsubo, Kiyoshi Noguchi, Takashi Usui, and Hidetaka Kamimura Drug Metabolism Research Laboratories, Drug Discovery Research, Astellas Pharma Inc., 1-8, Azusawa 1-chome, Itabashi-ku, Tokyo 174-8511, Japan (K.U., T.I., K.N., T.U., H.K.); Tsukuba Laboratories, Nemoto Science Co., Ltd., 6136-4, Ohnogo-machi, Joso-shi, Ibaraki 300-2521, Japan (N.S.) DMD Fast Forward. Published on May 13, 2009 as doi:10.1124/dmd.108.026294

Time-dependent Inhibitory Effects of FK1706, a Novel Non-immunosuppressive Immunophilin Ligand, on CYP3A4/5 Activity in Humans in Vivo and in Vitro

We investigated the inhibitory effects of (1R,9S,12S,13R,14S,17R,18E,21S,23S,24R,25S,27R)-1, 14-dihydroxy-12-(E)-2-[(1R,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylvinyl-23,25-dimethoxy-13,19,21,27-tetramethyl-17-(2-oxopropyl)-11,28-dioxa-4-azatricyclo [22.3.1.04.9]octacos-18-ene-2,3,10,16-tetrone (FK1706), a novel nonimmunosuppressive immunophilin ligand, on CYP3A4/5 in in vitro and in vivo settings. First, the inhibitory effects of FK1706 (preincubation dependence, inactivation rate estimation, and reversibility) were tested using human liver microsomes. Second, the effect of repeated oral doses of FK1706 (60 mg q.d. for 14 days) on the pharmacokinetics of midazolam (single oral 2-mg dose) was tested in healthy volunteers. Finally, pharmacokinetic modeling and simulation were performed. In vitro experiments showed that FK1706 inhibited CYP3A4/5 in a time-dependent and irreversible manner. The in vitro maximum inactivation rate constant (kinact) and concentration of inhibitor that gave half-maximal kinact (KI) were estimated to be 10.1 h−1 and 2050 ng/ml, respectively. In the clinical study, FK1706 produced a 2-fold increase in the area under the time-concentration curve (AUC) of midazolam. A pharmacokinetic model developed for this study, which described the time course of concentrations of both FK1706 and midazolam and incorporated CYP3A4/5 inactivation in the liver and intestine, successfully predicted the change in the pharmacokinetics of midazolam using in vitro kinact and KI values (1.66- to 2.81-fold increases in AUC predicted) and estimated the in vivo inactivation rate to be 0.00404 to 0.0318 h−1. ml/ng. In conclusion, FK1706 weakly or moderately inhibited the activity of CYP3A4/5 in vitro and vivo at the tested dose. The model developed here would be helpful in predicting drug-drug interactions and in the design of dose regimens that avoid drug-drug interactions.

Identification of human metabolites 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, and investigation of the transporter-mediated renal and hepatic excretion of these metabolites.

(–)-N-{2-[(R)-3-(6,7-Dimethoxy-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)piperidino]ethyl}-4-fluorobenzamide (YM758) is a novel inhibitor of the “funny” If current channel (If channel) that is expressed in the sinus node of heart and is being developed as a treatment for stable angina and atrial fibrillation. Its metabolites were identified in human urine, plasma, and feces by radio-high-performance liquid chromatography and liquid chromatographytandem mass spectrometry analyses after oral administration of [14C]YM758. 6,7-Dimethoxy-2-[(3R)-piperidin-3-ylcarbonyl]-1,2,3,4-tetrahydroisoquinoline (YM-252124), (5R)-5-[(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)carbonyl]piperidin-2-one (YM-385459), 2-{[(3R)-1-{2-[(4-fluorobenzoyl)amino]ethyl}piperidin-3-yl]carbonyl}-7-methoxy-1,2,3,4-tetrahydroisonolin-6-yl β-d-glucopyranosiduronic acid (AS2036329), and the unchanged drug were detected as major constituents in both urine and plasma, whereas N-(4-fluorobenzoyl)glycine (YM-385461) was detected in plasma, but not in urine. The renal and hepatic uptake transporters for these metabolites were investigated by assessing their inhibitory effect on uptake activity in human (h) organic cation transporter (OCT) 1–3/rat (r) Oct1–3, human organic anion transporter (OAT) 1/rOat1, hOAT3/rOat3, and organic anion-transporting protein 1B1/1B3-expressing HEK293 cells. IC50 values of YM-252124 for 1-methyl-4-phenylpyridinium uptake via hOCT2 and rOct2 were 93.9 and 1700 μM, respectively, suggesting that this metabolite is secreted into urine via hOCT2/rOct2 and that the large difference in the inhibitory potentials between hOCT2 and rOct2 explains the species difference in the urinary excretion ratio of the radioactivity. The renal secretion of YM-385461, one derivative of p-aminohippuric acid, via hOAT1/rOat1, and hepatic uptake of YM-252124 via hOCT1/rOct1 was also expected. This kind of study was useful in investigating the relationship between the urinary/hepatic elimination and the transport activity for metabolites.1 Title page Identification of human metabolites of YM758, a novel If channel inhibitor, and investigation of the transporter-mediated renal and hepatic excretion of these metabolites Ken-ichi Umehara, Nobuaki Shirai, Takafumi Iwatsubo, Kiyoshi Noguchi, Takashi Usui, and Hidetaka Kamimura Drug Metabolism Research Laboratories, Drug Discovery Research, Astellas Pharma Inc., 1-8, Azusawa 1-chome, Itabashi-ku, Tokyo 174-8511, Japan (K.U., T.I., K.N., T.U., H.K.); Tsukuba Laboratories, Nemoto Science Co., Ltd., 6136-4, Ohnogo-machi, Joso-shi, Ibaraki 300-2521, Japan (N.S.) DMD Fast Forward. Published on May 13, 2009 as doi:10.1124/dmd.108.026294