Physiologically-based pharmacokinetic modeling to predict drug-drug interaction of enzalutamide with combined P-gp and CYP3A substrates.

Enzalutamide is known to strongly induce cytochrome P450 3A4 (CYP3A4). Furthermore, enzalutamide showed induction and inhibition of P-glycoprotein (P-gp) in in vitro studies. A clinical drug-drug interaction (DDI) study between enzalutamide and digoxin, a typical P-gp substrate, suggested enzalutamide has weak inhibitory effect on P-gp substrates. Direct oral anticoagulants (DOACs), such as apixaban and rivaroxaban, are dual substrates of CYP3A4 and P-gp, and hence it is recommended to avoid co-administration of these DOACs with combined P-gp and strong CYP3A inducers. Enzalutamide's net effect on P-gp and CYP3A for apixaban and rivaroxaban plasma exposures is of interest to physicians who treat patients for venous thromboembolism with prostate cancer. Accordingly, a physiologically-based pharmacokinetic (PBPK) analysis was performed to predict the magnitude of DDI on apixaban and rivaroxaban exposures in the presence of 160 mg once-daily dosing of enzalutamide. The PBPK models of enzalutamide and M2, a major metabolite of enzalutamide which also has potential to induce CYP3A and P-gp and inhibit P-gp, were developed and verified as perpetrators of CYP3A-and P-gp-mediated interaction. Simulation results predicted a 31% decrease in AUC and no change in Cmax for apixaban and a 45% decrease in AUC and a 25% decrease in Cmax for rivaroxaban when 160 mg multiple doses of enzalutamide were co-administered. In summary, enzalutamide is considered to decrease apixaban and rivaroxaban exposure through the combined effects of CYP3A induction and net P-gp inhibition. Concurrent use of these drugs warrants careful monitoring for efficacy and safety.

Physiologically-based pharmacokinetic modeling for mirabegron: a multi-elimination pathway mediated by cytochrome P450 3A4, uridine 5'-diphosphate-glucuronosyltransferase 2B7, and butyrylcholinesterase.

This was the first study to construct a physiologically-based pharmacokinetic (PBPK) model for mirabegron which incorporates the overall elimination pathways of metabolism by cytochrome P450 (CYP) 3A4, uridine 5'-diphosphate-glucuronosyltransferase (UGT) 2B7, and butyrylcholinesterase (BChE) and renal excretion. The objective was to assess the risk of drug-drug interactions (DDIs) by estimating the contribution of each elimination pathway and simulating the magnitude of the DDIs with UGT2B7 inhibitors. A PBPK model for mirabegron was constructed to reproduce the plasma concentration-time curves from a phase 1 study and the magnitude of the DDI with ketoconazole taking into account the overall elimination pathways. The PBPK model was subsequently verified using data from other DDI studies. The constructed PBPK model estimated the contribution for each elimination pathway: 44% and 29% for CYP3A4 and UGT2B7 in the liver, 1.6% for UGT2B7 in the kidney, 3.2% for BChE in plasma, and 22% for renal excretion. Co-administration of probenecid (an UGT2B7 inhibitor) or fluconazole (an UGT2B7 and CYP3A4 inhibitor) was predicted to increase area under the curve for mirabegron to 115% or 174%, respectively. In conclusion, PBPK modeling and simulation revealed a low DDI risk for mirabegron following co-administration with BChE or UGT2B7 inhibitors.

Application of a physiologically based pharmacokinetic model for the prediction of mirabegron plasma concentrations in a population with severe renal impairment.

We previously verified a physiologically based pharmacokinetic (PBPK) model for mirabegron in healthy subjects using the Simcyp Simulator by incorporating data on the inhibitory effect on cytochrome P450 (CYP) 2D6 and a multi-elimination pathway mediated by CYP3A4, uridine 5'-diphosphate-glucuronosyltransferase (UGT) 2B7 and butyrylcholinesterase (BChE). The aim of this study was to use this PBPK model to assess the magnitude of drug-drug interactions (DDIs) in an elderly population with severe renal impairment (sRI), which has not been evaluated in clinical trials. We first determined the system parameters, and meta-analyses of literature data suggested that the abundance of UGT2B7 and the BChE activity in an elderly population with sRI was almost equivalent to and 20% lower than that in healthy young subjects, respectively. Other parameters, such as the CYP3A4 abundance, for an sRI population were used according to those built into the Simcyp Simulator. Second, we confirmed that the PBPK model reproduced the plasma concentration-time profile for mirabegron in an sRI population (simulated area under the plasma concentration-time curve (AUC) was within 1.5-times that of the observed value). Finally, we applied the PBPK model to simulate DDIs in an sRI population. The PBPK model predicted that the AUC for mirabegron with itraconazole (a CYP3A4 inhibitor) was 4.12-times that in healthy elderly subjects administered mirabegron alone, and predicted that the proportional change in AUC for desipramine (a CYP2D6 substrate) with mirabegron was greater than that in healthy subjects. In conclusion, the PBPK model was verified for the purpose of DDI assessment in an elderly population with sRI.

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.

Preclinical Characterization of ASP2713, a Novel Igß and FcγRIIB Cross-Linking Antibody, for Prediction of Human Pharmacokinetics and Clinically Effective Dose.

Previously, we reported the fundamental pharmacological characteristics of a novel Igß and Fc gamma receptor IIB cross-linking antibody, ASP2713, as a new treatment option for systemic lupus erythematosus. The aims of the present study were to investigate ASP2713's characteristics with regard to pharmacological effect, pharmacokinetics (PK), and receptor occupancy, and to predict its human PK and clinically effective dose. The relationship between the concentration and receptor occupancy of ASP2713 for Igß of B cell receptors was examined using whole blood B cells. Calculated EC50 values in cynomolgus monkeys and healthy volunteers were 0.35 and 0.058 µg/mL, respectively. Dose-dependent inhibition of anti-tetanus toxoid (TTx) antibody production, PK, and receptor occupancy of ASP2713 in TTx-sensitized cynomolgus monkeys suggested a minimally effective dose of 1 mg/kg by single intravenous (IV) administration. Scaling-up of monkey PK parameters to humans by allometric scaling predicted a clinically effective dose of 0.4 mg/kg IV administration at 4-week intervals to maintain a trough concentration in humans which achieved the same receptor occupancy expected at the effective dose in monkeys. This study aids in understanding the characteristics of ASP2713 and can be used as a basis for clinical dose setting.

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.

Bottom-up modeling and simulation of tacrolimus clearance: prospective investigation of blood cell distribution, sex and CYP3A5 expression as covariates and assessment of study power.

The objectives were to investigate the ability of population-based in vitro-in vivo extrapolation (IVIVE) to reproduce the influence of haematocrit on the clearance of tacrolimus, observed previously, and to assess the power of clinical studies to detect the effects of covariates on the clearance of tacrolimus. A population-based pharmacokinetic simulator (Simcyp) was used to simulate tacrolimus clearance from in vitro metabolism data and demographic characteristics of Japanese liver transplant patients (JLTs). The relationship between haematocrit and dose-to-concentration (D/C) ratio was validated using seven JLTs, whose highly variable haematocrit and D/C ratio were previously analysed. This validation was used as a surrogate for establishing 'interindividual' variability and to assess the power of clinical studies to discern the effect of haematocrit, sex and CYP3A5 genotype on tacrolimus clearance in a virtual JLT population. The relationship between haematocrit and D/C ratio was reproducible by Simcyp and corresponded well to those observed in seven JLTs. The number of JLTs required to detect the influence of CYP3A5 genotype and sex were estimated to be about 50 and > 600, respectively, which was consistent with the results of previous population pharmacokinetic studies for tacrolimus. In conclusion, population-based IVIVE is considered to be a useful approach to assess the influence of covariates a priori before conducting clinical studies. This is also helpful with study design and assessment of the statistical power of clinical studies involving population-based pharmacokinetics to detect the effects of covariates.

Virtual clinical trial simulations for a novel KRASG12C inhibitor (ASP2453) in non-small cell lung cancer.

KRAS is a small GTPase family protein that relays extracellular growth signals to cell nucleus. KRASG12C mutations lead to constitutive proliferation signaling and are prevalent across human cancers. ASP2453 is a novel, highly potent, and selective inhibitor of KRASG12C . Although preclinical data suggested impressive efficacy, it remains unclear whether ASP2453 will show more favorable clinical response compared to more advanced competitors, such as AMG 510. Here, we developed a quantitative systems pharmacology (QSP) model linking KRAS signaling to tumor growth in patients with non-small cell lung cancer. The model was parameterized using in vitro ERK1/2 phosphorylation and in vivo xenograft data for ASP2453. Publicly disclosed clinical data for AMG 510 were used to generate a virtual population, and tumor size changes in response to ASP2453 and AMG 510 were simulated. The QSP model predicted ASP2453 exhibits greater clinical response than AMG 510, supporting potential differentiation and critical thinking for clinical trials.

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 [(14)C]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 [(3)H]1-methyl-4-phenylpyridinium via OCT1, OCT2, and OCT3 with IC(50) values of 23.8, 15.9, and 108 microM, respectively. The time- and saturable concentration-dependent uptake of [(14)C]YM155 was observed in cells expressing OCT1 and OCT2 with K(m) values of 22.1 and 2.67 microM, 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 IC(50) values of OCT inhibitors and K(m) 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.

Time-dependent 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[22.3.1.0(4.9)]octacos-18-ene-2,3,10,16-tetrone (FK1706), a novel nonimmunosuppressive 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 [22.3.1.0(4.9)]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 (k(inact)) and concentration of inhibitor that gave half-maximal k(inact) (K(I)) 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 k(inact) and K(I) 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) x 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.

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 [(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.

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 [(14)C]YM155 to PC-3-xenografted nude mice revealed a relatively high level of radioactivity in the PC-3 xenograft. Therefore, the uptake of [(14)C]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-Hodgkin's lymphoma (RL and Ramos) cell lines. The uptake of [(14)C]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 microM). The effects of various compounds on the uptake of [(14)C]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 [(14)C]YM155 to a similar extent among the five cell lines. The half-maximal inhibitory concentration values (IC(50)) of several compounds for the uptake of [(14)C]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.

Role of organic anion transporters in the pharmacokinetics of zonampanel, an alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate receptor antagonist, in rats.

Zonampanel monohydrate ([2,3-dioxo-7-(1H-imidazol-1-yl)-6-nitro-1,2,3,4-tetrahydro-1-quinoxalinyl] acetic acid monohydrate, YM872) is a novel alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor antagonist. In humans, almost all administered zonampanel is excreted in the urine unchanged. Furthermore, zonampanel is transported by human organic anion transporter (OAT) 1, and OAT3 but not by OAT2, suggesting the contribution of OATs to renal excretion. In rats also, zonampanel is predominantly eliminated via urine but partly also via bile as the unchanged form. In this study, the molecular mechanism of the excretion of zonampanel was elucidated using cells expressing rat Oat1, Oat2, and Oat3. Furthermore, zonampanel (15 mg/kg) was given i.v. to rats with or without probenecid (50 mg/kg) or cimetidine (40 mg/kg), and pharmacokinetic parameters were compared. Zonampanel inhibited the uptake of typical substrates by Oat1, Oat2, and Oat3 with inhibition constant (K(i)) values of 7.02 to 10.4 microM. A time- and saturable concentration-dependent increase in [14C]zonampanel uptake was observed in these cells [Michaelis-Menten constant (K(m)) values: 13.4 to 53.6 microM]. Probenecid and cimetidine inhibited [14C]zonampanel uptake by Oats. In in vivo experiments, probenecid and cimetidine decreased intrinsic clearance for both the renal secretion and biliary excretion of zonampanel. Considering the tissue distribution and localization of each transporter, these results suggest that in rats zonampanel is taken up from the blood into proximal tubular cells via Oat1 and Oat3 and, unlike the case in humans, also into hepatocytes via Oat2 and Oat3. The interspecies differences in the excretion of zonampanel between rats and humans may thus be explained by those in the substrate selectivity and tissue distribution of OATs.

Interactions of tyrosine kinase inhibitors with organic cation transporters and multidrug and toxic compound extrusion proteins.

The drug-drug interaction (DDI) potential of tyrosine kinase inhibitors (TKI) as interacting drugs via transporter inhibition has not been fully assessed. Here, we estimated the half maximal inhibitory concentration (IC(50)) values for 8 small-molecule TKIs (imatinib, dasatinib, nilotinib, gefitinib, erlotinib, sunitinib, lapatinib, and sorafenib) on [(14)C]metformin transport by human organic cation transporters (OCT), OCT1, OCT2, and OCT3, and multidrug and toxic compound extrusion (MATE) proteins, MATE1 and MATE2-K, using human embryonic kidney cells stably expressing these transporters. We then compared the estimated IC(50) values to the maximum clinical concentrations of unbound TKIs in plasma (unbound C(max,sys,p)). Results showed that imatinib, nilotinib, gefitinib, and erlotinib exerted selectively potent inhibitory effects, with unbound C(max,sys,p)/IC(50) values ≥0.1, on MATE1, OCT3, MATE2-K, and OCT1, respectively. In comparison to the common form of OCT1, the OCT1 polymorphism, M420del, was more sensitive to drug inhibition by erlotinib. Major metabolites of several TKIs showed IC(50) values similar to those for unchanged TKIs. Taken together, these findings suggest the potential of clinical transporter-mediated DDIs between specific TKIs and OCTs and MATEs, which may affect the disposition, efficacy, and toxicity of metformin and other drugs that are substrates of these transporters. The study provides the basis for further clinical DDI studies with TKIs.

Liquid chromatography-electrospray tandem mass spectrometric assay suitable for quantitation of YM155, a novel small-molecule survivin suppressant, in dog plasma.

This paper describes a sensitive and selective liquid chromatography with tandem mass spectrometry (LC-MS/MS) method for determination of the novel survivin suppressant YM155, 1-(2-methoxyethyl)-2-methyl-4,9-dioxo-3-(pyrazin-2-ylmethyl)-4,9-dihydro-1H-naphtho[2,3-d]imidazolium, which is developed for the treatment of solid tumors. This method uses a liquid-liquid extraction from 0.25 mL of dog plasma. LC separation was carried out on a Genesis Silica column (50 mm x 3.0 mm i.d.) at a flow-rate of 0.5 mL/min. Compounds were eluted using a mobile phase of 5 mm ammonium acetate and 0.1% formic acid in water-0.1% formic acid in acetonitrile, 17:83 (v/v). MS/MS detection was carried out with an MDS-Sciex API3000 triple quadrupole mass spectrometer in positive electrospray ionization mode. The standard curve was linear from 0.05 to 50 ng/mL (r > or = 0.9968). The lower limit of quantitation was 0.05 ng/mL. Good intra- and inter-day assay precision (within 7.4% RSD) and accuracy (within +/-12.3%) were obtained. The extraction recovery was 66.2%. The method was successfully applied to preclinical pharmacokinetic studies in dogs.

Characterization of the renal tubular transport of zonampanel, a novel alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor antagonist, by human organic anion transporters.

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 alpha-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 (hOCT) 1 and hOCT2. Another AMPA receptor antagonist, YM90K [6-(1H-imidazol-1-yl)-7-nitro-2,3(1H,4H)-quinoxalinedione monohydrochloride], a decarboxymethylated form of zonampanel, was also used for comparing the substrate specificity. Zonampanel inhibited the uptake of prototypical organic anion substrates, [14C]para-aminohippurate in hOAT1 and [3H]estrone sulfate in hOAT3 and hOAT4, in a competitive manner. A time- and concentration-dependent increase in [14C]zonampanel uptake was observed in cells expressing hOAT1, hOAT3, and hOAT4. The Km values of zonampanel uptake by hOAT1, hOAT3, and hOAT4 were 1.4, 7.7, and 215 microM, respectively. Considering the localization of each transporter, results suggest that zonampanel is taken up via hOAT1 and hOAT3 from the blood into proximal tubular cells and then effluxed into the lumen via hOAT4. Probenecid and cimetidine competitively inhibited [14C]zonampanel uptake by the hOATs (hOAT1, hOAT3, and hOAT4 for probenecid; hOAT3 for cimetidine). YM90K inhibited the uptake of the prototypical substrate via hOAT3 competitively, but the uptake via hOAT1 noncompetitively. These findings suggest that the prototypical organic anion substrates (para-aminohippurate and estrone sulfate), cimetidine, probenecid, and zonampanel share binding specificity in each hOAT, whereas YM90K does not in hOAT1, possibly due to it being the decarboxymethylated form.

Application of LC-NMR for characterization of rat urinary metabolites of zonampanel monohydrate (YM872).

Zonampanel monohydrate (YM872) has a potent and selective antagonistic effect on the glutamate receptor subtype, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor. Metabolic fingerprinting in rat urine after a single intravenous administration of (14)C-labeled YM872 ((14)C-YM872) revealed the presence of two metabolites, R1 and R2. The two metabolites were semi-purified by preparative HPLC from rat urine after a single intravenous administration of non-labeled YM872, and their structures were elucidated by various instrumental analyses involving LC-NMR. The results showed that R1 and R2 have a hydroxyamino group and an amino group at the C-7 position of the quinoxalinedione skeleton, respectively. Therefore, the proposed metabolic pathway of YM872 in rats involves the reduction of the nitro group to a hydroxyamino group and then subsequent reduction to an amino group.