Modeling approach for multiple transporters-mediated drug-drug interactions in sandwich-cultured human hepatocytes: effect of cyclosporin A on hepatic disposition of mycophenolic acid phenyl-glucuronide.

A lower exposure of mycophenolic acid (MPA) in patients receiving MPA-mofetil in combination with cyclosporin A (CsA) is thought to be due to the inhibition of enterohepatic circulation of phenyl-glucuronide of MPA (MPAG). This study aimed to evaluate the interaction of CsA with hepatic disposition of MPA and MPAG in sandwich-cultured human hepatocytes (SCHH) by a mathematical modeling approach. In addition, the inhibition of CsA for glucuronidation of MPA to MPAG was examined in human liver microsomes. Inhibitory parameters of CsA for hepatic disposition of MPAG were estimated using a non-linear mixed effect model program, NONMEM. As a result, CsA did not influence the conversion of MPA to MPAG in either SCHH or human liver microsomes. In contrast, CsA inhibited the basolateral uptake of MPAG with an estimated maximum inhibitory effect (Imax) of 32.4%. CsA also inhibited basolateral efflux and biliary excretion of MPAG formed in SCHH, and the concentration producing 50% of Imax (IC50) for biliary excretion was lower than that for basolateral efflux. Our modeling approach suggests that CsA inhibits both basolateral uptake and biliary excretion of MPAG and leads to changes in systemic exposure of MPA and MPAG in humans.

Modeling and simulation of bone mineral density response from a phase 2 study of ONO-5334, a new cathepsin K inhibitor, to support dose selection in osteoporosis.

ONO-5334, a selective inhibitor of cathepsin K, is a potential new treatment for osteoporosis. The objectives of this modeling study were to (1) develop exposure-response (E-R) models to relate ONO-5334 exposure to bone mineral density (BMD), (2) predict BMD responses to various doses of ONO-5334 for both immediate release tablet (IRT) and sustained release tablet (SRT) formulations where only BMD response after administration of IRT had been studied to date, (3) inform selection of appropriate formulation/dose using simulation for future clinical trials. A population pharmacokinetic (PK) model was developed to simultaneously analyze data for both IRT and SRT. The exposure metrics at steady state were estimated by post hoc Bayesian prediction using the final population PK model. E-R models were developed using dose-ranging data with only IRT from postmenopausal females with osteoporosis. Based on the developed model, lumbar spine and total hip BMD after administration of ONO-5334 SRT as well as IRT were simulated. The simulation results showed that ONO-5334 SRT should provide comparable BMD responses at a lower dose relative to IRT (a finding consistent with the results from a previous population PK-PD modeling study with bone resorption markers).

Mathematical modeling of the in vitro hepatic disposition of mycophenolic acid and its glucuronide in sandwich-cultured human hepatocytes.

In recent years, it has become increasingly important to test the safety of circulating metabolites of novel drugs as part of drug discovery and development programs. Accordingly, it is essential to develop suitable methods for identifying the major metabolites and their disposition in animal species and in humans. Mycophenolic acid (MPA), a selective inosine-5'-monophosphate dehydrogenase (IMPDH) inhibitor, is metabolized by glucuronidation and enterohepatic circulation of MPA-glucuronides is an important factor in the continuous systemic exposure of MPA. In humans, about 90% of the administered MPA dose is finally excreted as MPA phenyl-glucuronide (MPAG) in urine. Notably, the plasma concentration of MPAG is much higher than that of MPA. These factors suggest that, after its formation in hepatocytes, MPAG is excreted into bile and is also transported across the basolateral membrane to enter the circulation. In the present study, we performed metabolic/hepatobiliary transport studies of MPA and MPAG using sandwich-cultured human hepatocytes (SCHH) and constructed mathematical models of their hepatic disposition. We also performed vesicular transport studies to identify which human multidrug resistance-associated proteins (MRPs) are involved in the transport of MPAG from hepatocytes. MPAG was a preferred substrate for the biliary excretion transporter MRP2 and the hepatic basolateral transporters MRP3 and MRP4 in conventional and metabolic/hepatobiliary transport studies using SCHH and vesicular transport studies using human MRP-expressing membrane vesicles. The resulting mathematical model suggested that the basolateral transport plays an important role in the hepatic disposition of MPAG formed in hepatocytes. Our findings suggest that mathematical modeling of metabolic/hepatobiliary transport studies using SCH will provide useful information for determining the fate of metabolites formed in hepatocytes.

Population pharmacokinetic and pharmacodynamic modeling of different formulations of ONO-5334, cathepsin K inhibitor, in Caucasian and Japanese postmenopausal females.

ONO-5334, a selective inhibitor of cathepsin K, is a potential new treatment for osteoporosis. The objectives of this study were to (1) develop population pharmacokinetic-pharmacodynamic (PK-PD) models for ONO-5334 using dose-ascending data from healthy postmenopausal females, (2) examine comparability of PK and/or PD profile between Caucasian and Japanese, and (3) compare PK-PD profile between immediate release tablet (IRT) and sustained release tablet (SRT). The population PK-PD models were developed for each formulation for post-dose levels of bone resorption markers (serum CTX and NTX). The data were provided from 4 phase 1 studies with total of 201 Caucasian and 94 Japanese subjects. Plasma concentrations of ONO-5334 and bone resorption markers were thoroughly evaluated in those studies. An indirect response model described relationships between bone resorption markers and plasma concentrations of ONO-5334. There was no significant difference in PK and pharmacodynamic potency (IC50 ) between Caucasian and Japanese. Based on the developed model, serum CTX and NTX after administration of ONO-5334 IRT or SRT were simulated, and the results showed that ONO-5334 SRT would provide comparable PD effect on bone resorption markers with lower dose relative to IRT.

Evaluation of hepatic disposition of paroxetine using sandwich-cultured rat and human hepatocytes.

Paroxetine, a selective serotonin reuptake inhibitor, is metabolized in the liver and excreted into bile and urine as metabolites, but species differences have been observed in hepatic disposition between rats and humans. A major metabolite in rats is M1-glucuronide, whereas M1-glucuronide and M1-sulfate are found in humans. The primary excretion route of paroxetine-derived radioactivity in rats and humans is bile and urine, respectively. The aim of this study was to examine the usefulness of sandwich-cultured hepatocytes (SCH) to evaluate in vivo species differences of the hepatic disposition of paroxetine between rats and humans. The metabolite profile of [(3)H]paroxetine in SCH was similar to that in hepatocytes in suspension, and the in vitro metabolite profiles were similar to the published in vivo metabolic pathways for both species. Furthermore, the biliary excretion index (BEI) of formed M1-glucuronide in rat SCH (25.8-50.9%) was higher than that in human SCH (15.1-16.7%). The BEI of formed M1-sulfate (16.4-29.1%) was comparable to that of M1-glucuronide in human SCH, whereas the BEIs of paroxetine were negligible in SCH of both species. Moreover, M1-glucuronide was demonstrated to be a multidrug resistance-associated protein 2 substrate in both species, as determined by its uptake into ATP-binding cassette transporter-expressing membrane vesicles. SCH should prove to be useful to evaluate the processes of hepatic uptake and metabolism of parent drugs and the simultaneous examination of the biliary excretion of both parent drug and liver-derived metabolites.

Population pharmacokinetics and exposure-response relationship of a muscarinic receptor antagonist, imidafenacin.

This study was designed to update the population pharmacokinetic model and investigate the exposure-response (efficacy and safety) and concentration-QT relationships for imidafenacin, a synthetic orally active muscarinic receptor antagonist. The population pharmacokinetic model was updated using data from 90 healthy subjects and 852 patients with an overactive bladder. Plasma concentration data from nine clinical studies were used, including new data from a long-term dose escalation study. The updated population pharmacokinetic model for imidafenacin adequately described the plasma concentration profile. The results were generally consistent with those obtained from the previous population pharmacokinetic analysis, indicating that no new covariates were found to influence the pharmacokinetics of imidafenacin. Exposure-response relationships in the long-term dose escalation study were investigated using a regression analysis with efficacy and safety endpoints as dependent variables. There was no clear relationship between exposure and any endpoint. The concentration-QT relationship was also evaluated to assess whether imidafenacin prolonged the concentration-dependent QT interval. There was no clear relationship between the plasma concentration of imidafenacin and QTc, indicating that concentration-dependent QTc interval prolongation was not observed.

The prediction of human response to ONO-4641, a sphingosine 1-phosphate receptor modulator, from preclinical data based on pharmacokinetic-pharmacodynamic modeling.

The pharmacokinetic (PK) and pharmacodynamic (PD) parameters of ONO-4641 in humans were estimated using preclinical data in order to provide essential information to better design future clinical studies. The characterization of PK/PD was measured in terms of decreased lymphocyte counts in blood after administration of ONO-4641, a sphingosine 1-phosphate receptor modulator. Using a two-compartment model, human PK parameters were estimated from preclinical PK data of cynomolgus monkey and in vitro human metabolism data. To estimate human PD parameters, the relationship between lymphocyte counts and plasma concentrations of ONO-4641 in cynomolgus monkeys was determined. The relationship between lymphocyte counts and plasma concentrations of ONO-4641 was described by an indirect-response model. The indirect-response model had an I(max) value of 0.828 and an IC(50) value of 1.29 ng/ml based on the cynomolgus monkey data. These parameters were used to represent human PD parameters for the simulation of lymphocyte counts. Other human PD parameters such as input and output rate constants for lymphocytes were obtained from the literature. Based on these estimated human PK and PD parameters, human lymphocyte counts after administration of ONO-4641 were simulated. In conclusion, the simulation of human lymphocyte counts based on preclinical data led to the acquisition of useful information for designing future clinical studies.

In vitro metabolism and inhibitory effects of pranlukast in human liver microsomes.

We investigated the metabolism of pranlukast, a selective leukotriene agonist, and the potential for drug-drug interactions. Although cytochrome P450 (CYP) 3A4 appeared to be the major cytochrome P450 isoform involved in the metabolism of pranlukast, the results suggested that pranlukast metabolism was inhibited less than 50% by ketoconazole, a reversible CYP3A4 inhibitor, or by anti-CYP3A4 antibodies. Irreversible macrolide CYP3A4 inhibitors, clarithromycin, erythromycin and roxithromycin, exhibited little effect on pranlukast metabolism. On the other hand, pranlukast reversibly inhibited CYP2C8 and/or 2C9, and CYP3A4, with K(i) values of 3.9 and 4.1 micromol/l, respectively. The [I](in,max,u)/K(i) ratios were 0.004 and 0.003, respectively. The K(i) values were about 300-fold greater than the [I](in,max,u), therefore it is suggested that, at clinical doses, pranlukast will not affect the pharmacokinetics of concomitantly administered drugs that are primarily metabolized by CYP2C8 and/or 2C9 or CYP3A4.

Development and validation of an on-line two-dimensional reversed-phase liquid chromatography-tandem mass spectrometry method for the simultaneous determination of prostaglandins E(2) and F(2alpha) and 13,14-dihydro-15-keto prostaglandin F(2alpha) levels in human plasma.

We developed and validated an on-line reverse-phase two-dimensional LC/MS/MS (2D-LC/MS/MS) system for simultaneous determination of the levels of prostaglandin (PG) E(2) as well as PGF(2alpha) and its metabolite 13,14-dihydro-15-keto PGF(2alpha) (F(2alpha)-M) in human plasma. Analytes were extracted by a three-step solid-phase extraction. Samples were then analyzed by on-line 2D-LC/MS/MS with electrospray ionization in negative mode. The 2D-LC system is composed of two reverse-phase analytical columns with a trapping column linking the two analytical columns. While an acidic buffer was used for both separation dimensions, differing organic solvents were employed for each dimension: methanol for the first and acetonitrile for the second to increase resolving power. The 2D-LC/MS/MS method was highly selective and sensitive with a significantly lower limit of quantitation (0.5 pg/mL for PGE(2) and 2.5 pg/mL for PGF(2alpha) and F(2alpha)-M, respectively). Linearity of the 2D-LC/MS/MS system was demonstrated for the calibration ranges of 0.5-50 pg/mL for PGE(2) and 2.5-500 pg/mL for PGF(2alpha) and F(2alpha)-M, respectively. Acceptable precision and accuracy were obtained throughout the calibration curve ranges. This highly selective and sensitive method was successfully utilized to determine the endogenous levels of PGE(2), PGF(2alpha), and F(2alpha)-M in plasma samples from six (four male and two female) normal volunteers. The mean concentrations for each analyte were 0.755 pg/mL for PGE(2), 5.70 pg/mL for PGF(2alpha) and 9.48 pg/mL for F(2alpha)-M.

A practical synthesis and biological evaluation of 9-halogenated PGF analogues.

A series of 9-halo PGF analogues 1-2 and 5-13 were synthesized and biologically evaluated. Among the compounds, 2 was the best EP2-receptor agonist. A practical method of synthesizing 2 via the Julia olefination of an aldehyde 3 with an optically active sulfone 4, which was prepared by Sharpless asymmetric epoxidation of 15, was developed. Other 9-halogenated PGF analogues were synthesized essentially by the same procedure and evaluated. The absolute configuration of 16-OH of 2 was determined as S by the X-ray analysis of a salt consisting of a 1/1 molar ratio of 2 and L-lysine.

Development of a highly selective EP2-receptor agonist. Part 2: identification of 16-hydroxy-17,17-trimethylene 9beta-chloro PGF derivatives.

Further chemical modification of 1a and 2 was undertaken to identify a more chemically stable selective EP2-receptor agonist for development as a clinical candidate. 9beta-chloro PG analogues 4a-e and 5a, c-e were found to be potent and selective EP2-receptor agonists. Among them, the compound 4aLy, which is a chemically stabilized lysine salt of 4a, exhibited an excellent profile both in biological activities and physicochemical properties. The agonist 4aLy was found to suppress uterine motility in anesthetized pregnant rats, while PGE2 stimulated uterine motility. Structure-activity relationships (SARs) are discussed.

Development of a highly selective EP2-receptor agonist. Part 1: identification of 16-hydroxy-17,17-trimethylene PGE2 derivatives.

Design and synthesis of an EP2-receptor selective agonist began with the chemical modification of alpha- and omega-chains of butaprost 1a, which exhibits an affinity for the IP-receptor. Two series of prostaglandin (PG) analogues with a 16-hydroxy-17,17-trimethylene moiety as an omega-chain were identified. Among those tested, 4a,b,e,f,h and 6a,b,e,f,h were found to be highly selective EP2-receptor agonists. Structure-activity relationships are discussed.