CHARACTERIZATION OF THE RENAL TUBULAR TRANSPORT OF ZONAMPANEL, A NOVEL ␣-AMINO-3-HYDROXY-5-METHYLISOXAZOLE-4-PROPIONIC ACID RECEPTOR ANTAGONIST, BY HUMAN ORGANIC ANION TRANSPORTERS

ABSTRACT: Zonampanel monohydrate (YM872; [2,3-dioxo-7-(1H-imidazol-1-yl)-6-nitro-1,2,3,4-tetrahydro-1-quinoxalinyl]acetic acid monohydrate) is a novel ␣-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor antagonist. The major elimination route for zonampanel has been reported to be by urine via the kidneys. The purpose of this study is to elucidate the molecular mechanism of the renal excretion of zonampanel using cells stably expressing human organic anion transporters (hOAT) 1, hOAT2, hOAT3, and hOAT4, as well as human organic cation transporters The excessive synaptic release of glutamate and activation of postsynaptic glutamate receptors are considered to mediate ischemiainduced neuronal damage in stroke victims. In a wide variety of cerebral ischemia animal models, receptor antagonists against ␣-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), a glutamate receptor subtype, have been shown to be neuroprotective It has been found that the major elimination route for zonampanel monohydrate in humans and animals is by renal excretion. Zonampanel monohydrate in the body was excreted into urine mainly as the unchanged form within 2 h after completion of intravenous infusion in humans, and fecal excretion was very low (unpublished observation). In addition, renal clearance of zonampanel monohydrate was much higher than the product of the fraction unbound in plasma and the glomerular filtration rate. Therefore, it is considered that renal tubular secretion plays an important role in the renal excretion of this compound. Renal tubular secretion is accomplished through two steps of membrane transport: the uptake from blood through the basolateral mem-ABBREVIATIONS: AMPA, ␣-amino-3-hydroxy-5-methylisoxazole-4-propionic acid; OAT, organic anion transporter; OCT, organic cation transporter; hOAT, human OAT; hOCT, human OCT; PAH, para-aminohippurate; ES, estrone sulfate; D-PBS, Dulbecco's modified phosphate-buffered saline; TEA, tetraethylammonium; PG, prostaglandin, S 1 , S 2 , and S 3 , the first, second, and third segment of the proximal tubule; YM872, zonampanel monohydrate ([2,3-dioxo-7-(1H-imidazol-1-yl)-6-nitro-1,2,3,4-tetrahydro-1-quinoxalinyl]acetic acid monohydrate); YM90K, 6-(1H-imidazol-1-yl)-7-nitro-2,3(1H,4H)-quinoxalinedione monohydrochloride

Title page Involvement of Human Organic Cation Transporter 1 (OCT1) in the Hepatic Uptake of YM155 Monobromide, 1-(2-Methoxyethyl)-2-methyl-4,9-dioxo-3-(pyrazin-2-ylmethyl)- 4,9-dihydro-1H-naphtho[2,3-d]imidazolium Bromide, a Novel, Small Molecule Survivi

Abstract YM155 monobromide, 1-(2-methoxyethyl)-2-methyl-4,9-dioxo-3-(pyrazin-2-ylmethyl)-4,9-dihydro-1H-naphtho[2,3-d]imidazolium bromide, which is a hydrophilic and cationic compound, exhibits anti-tumor 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 non-renal elimination may be explained by the biliary excretion of YM155 in its unchanged form. As the penetration through the sinusoidal membrane of the hepatocytes is the first step and an important part of biliary excretion, we evaluated the uptake o

Characterization of human organic cation transporter 1 (OCT1/SLC22A1)- and OCT2 (SLC22A2)-mediated transport of 1-(2-methoxyethyl)-2-methyl-4,9dioxo-3-(pyrazin-2-ylmethyl)-4,9-dihydro-1h-naphtho[2,3-d]imidazolium bromide (YM155 monobromide), a novel small

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 two cell types. The other eliminating organ is the kidney. In this study, the transport of

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

Abstract We investigated the inhibitory effects of FK1706, a novel non-immunosuppressive immunophilin ligand, on CYP3A4/5 in in vitro and in vivo settings. First, the inhibitory effects of FK1706 (preincubation dependency, inactivation rate estimation, and reversibility) were tested using human liver microsomes. Second, the effect of repeated oral dose 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 time-dependent and irreversible manner. In vitro maximum inactivation rate constant (k inact ) and concentration of inhibitor that gave half-maximal k inact (K I ) were estimated to be 10.1 h -1 and 2,050 ng/mL, respectively. In the clinical study, FK1706 produced a two-fold increase in 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 k inact and K I values (1.66-to 2.81-fold increase 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 which avoided drug-drug interactions

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

ABSTRACT: 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 [ 14 C]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 IC 50 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, [ 14 C]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. Increasing evidence suggests that survivin, a member of the inhibitor of apoptosis protein family, is an essential regulator of cell division and a modulator of apoptotic cell death 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 small molecule that specifically inhibits survivin gene transcription and protein expression in several tumor cell lines Article, publication date, and citation information can be found at http://dmd.aspetjournals.org. doi:10.1124/dmd.109.027359. ABBREVIATIONS: YM155 monobromide, 1-(2-methoxyethyl)-2-methyl-4,9-dioxo-3-(pyrazin-2-ylmethyl)-4,9-dihydro-1H-naphtho [2,3-d] DMD 37:1856DMD 37: -1863DMD 37: , 2009 Printed in U.S.A. 1856 ( Penetration through the sinusoidal membrane of hepatocytes may represent a rate-limiting step for hepatic intrinsic clearance. Organic anion transporting polypeptide (OATP) 1B1, OATP1B3, OATP2B1, organic anion transporter 2, Na ϩ -taurocholate cotransporting polypeptide, and organic cation transporter (OCT) 1 are known to be localized on the basolateral membrane of hepatocytes and mediate the hepatic uptake of their substrates from circulating blood The effects of hepatic uptake transporters on the pharmacokinetics of their substrates have been well characterized for anionic drugs, particularly HMG CoA reductase inhibitors (statins) In this study, we elucidated the hepatic uptake mechanism of YM155, a certain amount of which is expected to be eliminated via the bile. Uptake of [ 14 C]YM155 was characterized using human cryopreserved hepatocytes and various transporter inhibitors, and the in vitro hepatic uptake clearance value obtained was compared with the in vivo hepatic intrinsic clearance estimated from a previous clinical study. Involvement of the transporter was confirmed using cells transfected to express the candidate transporter, OCT1, derived from the second portion of the proximal tubes (S2 cells). Materials and Methods Materials. [ 14 C]YM155 monobromide (specific activity, 3.05 MBq/mg; radiochemical purity, 98.9%) was synthesized at Sekisui Medical Co. Ltd. (Ibaraki, Japan). YM155 monobromide (assay value, 100.1%) was synthesized at Astellas Pharma Inc. (Tokyo, Japan). [ 14 C]TEA (specific activity, 2.035 GBq/mmol) was purchased from American Radiolabeled Chemicals, Inc. (St. Louis, MO). All other chemicals and reagents used were commercially available and of guaranteed purity. Cell Culture. OCT1-S2 cells and mock-transfected (control) cells established as described previously In Vitro Uptake Experiments. Uptake study using human cryopreserved hepatocytes. The uptake study was performed as described previously 14 C]YM155 solution to yield a final viable cell concentration of 1.0 ϫ 10 6 /ml. [ 14 C]YM155 concentrations, uptake time, and inhibitor concentrations, if any, were described in each experiment. Uptake was stopped by separating the cells from the [ 14 C]YM155 solution, after which the mixture was incubated for a designated time period. For this purpose, an aliquot of the incubation mixture was transferred to a 400-l polyethylene tube containing 50 l of NaOH (2 M) under a 100-l layer of an oil mixture. The sample tube was then centrifuged briefly. After overnight incubation in alkali to dissolve the hepatocytes, the centrifuge tube was cut into two compartments: one containing the [ 14 C]YM155 solution (component M), and the other containing the dissolved cells (component C). After the addition of 10 ml of Hionic Fluor, the amount of uptake was then determined by measuring the radioactivity in both the cells and the solution using a liquid scintillation counter. Uptake study using OCT1-S2 cells. Two days before the uptake experiments, the cells were seeded in 24-well cell culture dishes at a density of 1 ϫ 10 5 cells/well. The cells were preincubated for 10 min at 37°C in Dulbecco's phosphate-buffered saline (DPBS; pH 7.4), which consisted of 137 mM NaCl, 3 mM KCl, 8 mM NaHPO 4 , 1 mM KH 2 PO 4 , 1 mM CaCl 2 , and 0. 14 C]TEA uptake concentration (5 M) used in the IC 50 determination was less than its K m values, which was evaluated as 566.2 M in this study. After the designated period of incubation, the uptake was stopped by adding ice-cold DPBS. The cells were washed three times with DPBS and then lysed with 0.5 ml of NaOH (0.1 N). The radioactivity in the solution was measured using a liquid scintillation counter after adding 2 ml of the emulsifying scintillator Aquasol-2. The amount of cellular protein was determined separately using BCA protein assay reagent (Pierce, Rockford, IL). These cells were seeded at the same time and treated the same way as in the uptake experiments, except that DPBS containing no radiolabeled ligands or tested compounds was used. Data analysis and estimation of kinetic parameters. Uptake into human cryopreserved hepatocytes was given as the cell-to-medium ratio (C/M), which was determined as the radioactivity in component C divided by that in For on-ice incubation: where P dif is component of uptake clearance by passive diffusion, adsorption to cell surface, and adhering medium. To compare in vitro transporter-mediated hepatic uptake clearance with in vivo nonrenal clearance in humans, in vitro transporter-mediated intrinsic uptake clearance (PS uptake,in vitro ) was calculated as V max /K m . The physiological parameters used for scaling PS uptake,in vitro from "l/ min/10 6 cells" to "L/h (hr)/person" are described in 14 C]YM155 uptake via OCT1 (V) was calculated by subtracting uptake in mock cells from that in OCT1-S2 cells. In the inhibition study using hepatocytes, carrier-mediated uptake was calculated by subtracting the C/M on ice from that at 37°C. The C/M on ice used for the calculation shown in In the inhibition study using OCT1-S2 cells, mode of inhibition (competitive or noncompetitive) was determined by Lineweaver-Burk plot analysis, and the K i values were calculated from the following equation: where [I] is inhibitor concentration, and K m(ϩinhibitor) and K m(Ϫinhibitor) are the K m value of substrate compound with and without inhibitor, respectively. In Vivo Pharmacokinetic Data Analysis. The values for the fraction of unchanged YM155 excreted into urine (Fe) and total clearance calculated from the plasma YM155 concentrations (CL tot ) after continuous intravenous infusion at 1.8, 3.6, and 4.8 mg/m 2 /day to cancer patients were derived from a previous study where Q H is hepatic blood flow, with a value of 1450 ml/min/70 kg (87 l/h/70-kg person) Results Time Courses of the Uptake of [ 14 C]YM155 into Human Cryopreserved Hepatocytes. The time courses for the uptake of [ 14 C]YM155 into human cryopreserved hepatocytes are shown in Concentration-Dependent Uptake of [ 14 C]YM155 into Human Cryopreserved Hepatocytes. Concentration dependence of [ 14 C]-YM155 uptake using three different lots of human hepatocytes is shown in The K m values for YM155 among the three lots of hepatocytes were comparable and ranged from 6.13 to 9.47 M. In a phase I clinical study, the mean steady-state plasma concentration of YM155 was 21 nM at the maximum tolerated dose (4.8 mg/m 2 /day) IWAI ET AL. intrinsic clearance (V max /K m ) values (4.22-9.77 l/min/10 6 cells, or 54.7-126.5 l/h/person) were much higher than the nonsaturable component of hepatic uptake clearance (P dif value), accounting for 73 to 85% of total uptake clearance (V max /K m ϩ P dif ). Comparison of In Vitro Hepatic Uptake Clearance and in Vivo Hepatic Intrinsic Clearance. Parameters obtained by in vitro study were compared with the CL H int in vivo , which was calculated using the well stirred and dispersion models ( INVOLVEMENT OF OCT1 IN THE HEPATIC UPTAKE OF YM155 inhibited by MPP, verapamil, amantadine, procainamide, corticosterone, prazosin, and cimetidine, and moderately inhibited by TEA, probenecid, and K-strophanthin. In contrast, no marked inhibition was observed in the presence of 1-methylnicotinamide and taurocholate. Detailed concentration-dependent inhibition profiles obtained using corticosterone, MPP, prazosin, and cimetidine as inhibitors are shown in Discussion In this study, our examination of the uptake of YM155 into human cryopreserved hepatocytes and OCT1-S2 cells showed that YM155 was taken up in a carrier-mediated manner. IC 50 values of organic cations for YM155 transport were consistent with those reported for OCT1. Results from the present study suggest that the in vitro hepatic uptake clearance of YM155 may reflect in vivo clearance well in humans, and they also indicate the importance of OCT1-mediated transport in elimination of YM155 via bile. In the earlier phase I study, intravenous continuous infusion of YM155 resulted in 18.3 to 28.6% of the dose being excreted into urine in the unchanged form In the present study, the uptake of [ 14 C]YM155 was characterized using three lots of human cryopreserved hepatocytes. [ 14 C]YM155 was taken up into the hepatocytes in a time-and incubation temperature-dependent manner For YM155, nonrenal clearance can be used as a substitute parameter for the evaluation of hepatic clearance. Focusing on the human study, the fp/R B ⅐ CL H int in vivo value calculated was comparable with the Q H value, hence both the CL H int in vivo and Q H values were important factors affecting the hepatic elimination of YM155. In general, hepatic intrinsic clearance can be described as follows: CL H int in vivo ϭ PS uptake, basal ⅐ PS eff, apical ϩ CL metab PS eff, apical ϩ CL metab ϩ PS eff, basal where PS uptake,basal is the uptake clearance via the sinusoidal membrane of the hepatocytes, PS eff,apical is efflux clearance via the apical membrane into the bile, CL metab is metabolic clearance, and PS eff,basal is the efflux clearance via the sinusoidal membrane of the hepatocytes. For YM155, CL metab may be neglected, because YM155 is expected to be metabolized very little, if at all. With regard to hepatic metabolic clearance, many approaches hav