Mechanistic Multilayer Quantitative Model for Nonlinear Pharmacokinetics, Target Occupancy and Pharmacodynamics (PK/TO/PD) Relationship of D-Amino Acid Oxidase Inhibitor, TAK-831 in Mice.

PURPOSE: TAK-831 is a highly selective and potent inhibitor of D-amino acid oxidase (DAAO) currently under clinical development for schizophrenia. In this study, a mechanistic multilayer quantitative model that parsimoniously connects pharmacokinetics (PK), target occupancy (TO) and D-serine concentrations as a pharmacodynamic (PD) readout was established in mice. METHODS: PK, TO and PD time-profiles were obtained in mice and analyzed by mechanistic binding kinetics model connected with an indirect response model in a step wise fashion. Brain distribution was investigated to elucidate a possible mechanism driving the hysteresis between PK and TO. RESULTS: The observed nonlinear PK/TO/PD relationship was well captured by mechanistic modeling framework within a wide dose range of TAK-831 in mice. Remarkably different brain distribution was observed between target and reference regions, suggesting that the target-mediated slow binding kinetics rather than slow penetration through the blood brain barrier caused the observed distinct kinetics between PK and TO. CONCLUSION: A quantitative mechanistic model for concentration- and time-dependent nonlinear PK/TO/PD relationship was established for TAK-831 in mice with accounting for possible rate-determining process. The established mechanistic modeling framework will provide a quantitative means for multilayer biomarker-assisted clinical development in multiple central nervous system indications.

Impact of P-Glycoprotein on Intestinal Absorption of an Inhibitor of Apoptosis Protein Antagonist in Rats: Mechanisms of Nonlinear Pharmacokinetics and Food Effects.

PURPOSE: This study was designed to investigate the effects of P-glycoprotein (P-gp) expressed in the intestine on the nonlinear pharmacokinetics (PK) of T-3256336, an inhibitor of apoptosis protein inhibitor, and food effects on its bioavailability in rats. METHODS: To investigate the factors that contribute to nonlinear PK of T-3256336 in the intestine and liver, rats double-cannulated in the portal vein and femoral artery (PS rats) were used. FaFg (Fa, absorption ratio; Fg, intestinal availability) and hepatic availability (Fh) were simultaneously evaluated based on the difference between the portal and systemic blood area under the concentration-time curve (AUC). Elacridar was used as a P-gp inhibitor to assess the impact of P-gp on the intestinal absorption. RESULTS: After oral administration of T-3256336 to PS rats at 3 and 30 mg/kg, FaFg value increased with dose escalation, whereas Fh value was nearly constant. Moreover, co-administration of elacridar resulted in a 5-fold increase in the FaFg value at 3 mg/kg. The AUC value of T-3256336 under fed conditions was 3-fold lower than that under fasted conditions. This food effect on the oral bioavailability (BA) was reduced by concomitant administration of elacridar. CONCLUSION: P-gp expressed in the intestine would cause nonlinear PK and a food effect on BA of T-3256336 in rats.

Influence of body composition on disposition of the highly fat distributed compound as analysed by physiologically based pharmacokinetic (PBPK) modeling and simulation.

A recent study suggested that the pharmacokinetics (PK) of highly fat distributed compounds can be affected by acute changes in the volume of adipose tissue. The present study investigates possible influences of body composition on the disposition of the highly lipophilic compound TAK-357 in two rat strains. Physiologically based PK (PBPK) modeling and simulation was applied on single and multiple dose PK data of TAK-357 in obese Wistar fatty rats and Wistar lean rats having approximately 45% and 13% body fat, respectively. The observed effects of an elevated fat mass in Wistar fatty rats on the plasma concentrations appeared to be partly compensated for by other differences between the two rat strains. A decrease in the tissue to blood partition coefficients under high body fat conditions was identified as another factor contributing to the difference in PK. A higher lipid content in the plasma in high body fat animals may result in relatively lower tissue to blood partition coefficients. PBPK-based simulations indicate that the plasma concentrations of lipophilic compounds in high body fat conditions can differ by up to two-times at steady-state. This confirms that there is only a small impact of body composition change on the plasma concentration of highly lipophilic drugs and that the need for therapeutic dose adjustments may be limited.

Impact of acute fat mobilisation on the pharmacokinetics of the highly fat distributed compound TAK-357, investigated by physiologically based pharmacokinetic (PBPK) modeling and simulation.

In a dog toxicokinetic study, an unusual plasma concentration increase of the highly lipophilic compound TAK-357 was observed 2 weeks after termination of a 2-week repeated dosing in one dog with acute body weight loss. The present study investigates the cause of this increase. A physiologically based pharmacokinetic (PBPK) model was constructed using the rat and dog pharmacokinetic data. Using the constructed model, the TAK-357 concentration profile in the case of body weight change was simulated. The PBPK model-derived simulation suggested that redistribution from adipose tissues to plasma due to a loss of body fat caused the observed concentration increase of TAK-357 in dog plasma. The analysis demonstrates that the disposition of a highly lipophilic and fat-distributed compound can be affected by acute changes in adipose tissue mass. PBPK modeling and simulation proved to be efficient tools for the quantitative hypothesis testing of apparently atypical PK phenomena resulting from acute physiological changes.

Disposition and metabolism of TAK-438 (vonoprazan fumarate), a novel potassium-competitive acid blocker, in rats and dogs.

1. Following oral administration of [14C]TAK-438, the radioactivity was rapidly absorbed in rats and dogs. The apparent absorption of the radioactivity was high in both species. 2. After oral administration of [14C]TAK-438 to rats, the radioactivity in most tissues reached the maximum at 1-hour post-dose. By 168-hour post-dose, the concentrations of the radioactivity were at very low levels in nearly all the tissues. In addition, TAK-438F was the major component in the stomach, whereas TAK-438F was the minor component in the plasma and other tissues. High accumulation of TAK-438F in the stomach was observed after oral and intravenous administration. 3. TAK-438F was a minor component in the plasma and excreta in both species. Its oxidative metabolite (M-I) and the glucuronide of a secondary metabolite formed by non-oxidative metabolism of M-I (M-II-G) were the major components in the rat and dog plasma, respectively. The glucuronide of M-I (M-I-G) and M-II-G were the major components in the rat bile and dog urine, respectively, and most components in feces were other unidentified metabolites. 4. The administered radioactive dose was almost completely recovered. The major route of excretion of the drug-derived radioactivity was via the feces in rats and urine in dogs.

In vitro metabolism of TAK-438, vonoprazan fumarate, a novel potassium-competitive acid blocker.

1. TAK-438, vonoprazan fumarate, is a novel orally active potassium-competitive acid blocker, developed as an antisecretory drug. In this study, we investigated the in vitro metabolism of 14C-labeled TAK-438. In human hepatocytes, M-I, M-II, M-III and M-IV-Sul were mainly formed, and these were also detected in clinical studies. N-demethylated TAK-438 was also formed as an in vitro specific metabolite. Furthermore, CYP3A4 mainly contributed to the metabolism of TAK-438 to M-I, M-III, and N-demethylated TAK-438, and CYP2B6, CYP2C19 and CYP2D6 partly catalyzed the metabolism of TAK-438. The sulfate conjugation by SULT2A1 also contributed to the metabolism of TAK-438 to form TAK-438 N-sulfate, and CYP2C9 mediated the formation of M-IV-Sul from TAK-438 N-sulfate. The metabolite M-IV, which could be another possible intermediate in the formation of M-IV-Sul, was not observed as a primary metabolite of TAK-438 in any of the in vitro studies. 2. In conclusion, TAK-438 was primarily metabolized by multiple metabolizing enzymes including CYP3A4, CYP2B6, CYP2C19, CYP2D6, and a non-CYP enzyme SULT2A1, and the influence of the CYP2C19 genotype status on gastric acid suppression post TAK-438 dosing could be small. The multiple metabolic pathways could also minimize the effects of co-administrated CYP inhibitors or inducers on the pharmacokinetics of TAK-438.

Risk assessment of drug-drug interactions using hepatocytes suspended in serum during the drug discovery process.

1. This study optimized the reported approach for the prediction of drug-drug interactions (DDIs) using hepatocytes suspended in serum (HHSS) and provided a practical usage of HHSS in the early and late phases of drug discovery. 2. First, the IC50 was determined using HHSS and evaluated as a qualitative index for DDI risks in the early phase. A retrospective study on clinical DDI cases revealed that inhibitors with IC50 < 100 µmol/L caused clinical DDIs while those with IC50 > 100 µmol/L showed weak or no potential for DDIs. Meanwhile, a pragmatic cutoff value could not be determined using previously reported Ki values of recombinant human cytochrome P450s. 3. Second, for a more substantial DDI risk assessment in the later phase, quantitative predictions of clinical DDI based on a static model were attempted by optimizing the most appropriate inhibitor concentration ([I]). The use of hepatic input plasma concentrations as a surrogate for [I] achieved the most successful predictions of the magnitude of increase in the AUC (within a 2-fold range of the observed values for 93.8% of inhibitors). 4. Through this study, we proposed the practical application of HHSS for an effective workflow to explore and profile candidates with less DDI liability.

Retrospective analysis of P-glycoprotein-mediated drug-drug interactions at the blood-brain barrier in humans.

To date, the in vitro-in vivo correlation (IVIVC) of P-glycoprotein (P-gp)-mediated drug-drug interaction (DDI) at the blood-brain barrier (BBB) in rats indicated that the cutoff value to significantly affect the brain penetration of digoxin was [I,unbound/Ki] of 1, where I,unbound is the unbound plasma concentration of P-gp inhibitors. On the basis of the IVIVC in rats, we speculated that clinically used P-gp inhibitors do not cause DDI at the human BBB, because none of the compounds studied was [I,unbound/Ki]>1 at therapeutic doses. Recently, positron emission tomography studies with P-gp substrates, such as [(11)C]verapamil, [(11)C]N-desmethyl loperamide, and [(11)C]loperamide, together with potent P-gp inhibitors, have indicated that increases in the influx rate constant for brain entry were observed in humans. Therefore, we aimed to retrospectively analyze the results of P-gp-mediated DDIs with in vitro P-gp inhibition assays and to confirm the appropriate cutoff value. In vitro P-gp inhibition assays using verapamil, N-desmethyl loperamide, and loperamide as P-gp probe substrates were performed in human multidrug resistance protein 1-expressing LLC-PK1 cells. The efflux ratios decreased in the presence of P-gp inhibitors, and the Ki of tariquidar was 10 nmol/L, regardless of probe substrates. Taking the in vitro Ki and unbound plasma concentrations in clinical DDI studies together, the criterion [I,unbound/Ki] of 1 was an appropriate cutoff limit to observe significant P-gp-mediated DDI at the BBB in humans. On the other hand, no significant DDI was observed in cases in which [I,unbound/Ki] was less than 0.1. This criterion was comparable to the previous IVIVC result in rats.

Tetrahydro-naphthols as orally available TRPV1 inhibitors.

Starting from a naphthol-based lead series with low oral bioavailability, we have identified potent TRPV1 antagonists with oral bioavailability in rats. These compounds emerged from SAR studies aimed at replacing the lead's phenol structure whilst maintaining potency. Compound rac-6a is an orally available TRPV1 antagonist with single-digit nanomolar activity. The enantiomers show a low eudismic ratio at the receptor level.

Evaluation of cytochrome P450-mediated drug-drug interactions based on the strategies recommended by regulatory authorities.

Herein, we aimed to evaluate the recently proposed risk assessment strategies of a cytochrome P450 (CYP) mediated drug-drug interaction (DDI) according to the European Medicines Evaluation Agency (EMEA) draft guideline, and discuss the differences between this guideline and the Food and Drug Administration (FDA) draft guidance. A retrospective study on reported 35 clinical DDI cases revealed that the EMEA assessment successfully predicts moderate-to-strong DDIs, i.e. drugs that cause more than 2-fold increase in the area under the curve in the presence and absence of CYP inhibitor (AUC(i)/AUC); however, EMEA tends to overlook weak DDIs with AUC(i)/AUC ≤ 2 to > 1.25. For CYP3A4 inhibitors, even clinically insignificant DDIs were overemphasized if the intestinal DDI is considered. The differences between unbound fraction in plasma and microsomes account for the discrepancies in DDI risk assessment results between EMEA and FDA assessments. Comparing two assessment results for CYP2D6 and CYP2C9 inhibitors, the FDA assessment suggested potential DDI risks for sulphinpyrazone and amitriptyline, while the EMEA assessment indicated no potential risk for these drugs. Through a retrospective study, we showed practical differences in the DDI assessment strategies of EMEA and FDA and suggested improvements in their current strategies.

In vivo biliary clearance should be predicted by intrinsic biliary clearance in sandwich-cultured hepatocytes.

It has been reported that in vivo biliary clearance can be predicted using sandwich-cultured rat and human hepatocytes. The predicted apparent biliary clearance (CL(bile, app)) from sandwich- cultured rat hepatocytes (SCRH) based on medium concentrations correlates to in vivo CL(bile, app) based on plasma concentrations of angiotensin II receptor blockers (ARBs), HMG-CoA reductase inhibitors (statins), ß-lactam antibiotics, and topotecan. However, the predicted biliary clearance from SCRH was 7- to 300-fold lower than in vivo biliary clearance. We speculated that the process of biliary excretion might not have been evaluated using sandwich-cultured hepatocytes. To evaluate this issue, intrinsic biliary clearance (CL(bile, int)) based on intracellular compound concentrations was evaluated to investigate the in vitro-in vivo correlation of this process among ARBs, statins, ß-lactam antibiotics, and topotecan. Intrinsic biliary clearance in SCRH correlated to in vivo values obtained by constant intravenous infusion of six compounds, but not rosuvastatin and cefmetazole, to rats. Moreover, differences between SCRH and in vivo CL(bile, int) (0.7-6-fold) were much smaller than those of CL(bile, app) (7-300-fold). Therefore, in vivo CL(bile, int) is more accurately reflected using SCRH than CL(bile, app). In conclusion, to predict in vivo biliary clearance more accurately, CL(bile, int) should be evaluated instead of CL(bile, app) between SCRH and in vivo.

Development of a quantification method for digoxin, a typical P-glycoprotein probe in clinical and non-clinical studies, using high performance liquid chromatography-tandem mass spectrometry: the usefulness of negative ionization mode to avoid competitive adduct-ion formation.

Highly sensitive and accurate liquid chromatography-tandem mass spectrometry (LC/MS/MS) methods have been developed and validated for measuring digoxin (DGX), a typical P-glycoprotein probe, in human plasma, rat plasma, and rat brain. We extracted DGX and deuterium-labeled DGX (as internal standard) from sample fluids under basic conditions using acetonitrile and sodium chloride-saturated 0.1 mol/L sodium hydroxide. The upper organic layer was diluted with distilled water, and the resulting solution was injected into an LC/MS/MS system in negative ionization mode. Chromatographic separation was achieved on a C(18)-ODS column in the gradient mobile phase, which comprised 0.05% (w/v) ammonium carbonate (pH 9.0) and methanol at a flow rate of 0.7 mL/min. Regardless of the type of biological matrix, intra-day and inter-day validation tests demonstrated good linearity of calibration curves within ranges of 0.1-10 ng/mL for plasma and 0.5-50 ng/g for rat brain and gave excellent accuracy and precision of quality control samples at 4 concentration levels. Unlike existing methods, our approach uses negative ionization to avoid competitive adduct formation of DGX. Our method showed higher sensitivity and wider applicability to various types of biological matrices than existing methods. Our method will support clinical and preclinical investigation of in vivo P-glycoprotein functionality using DGX.

Characterization and evaluation of miconazole salts and cocrystals for improved physicochemical properties.

Miconazole salts and cocrystals were studied to improve the physicochemical properties of miconazole. Maleate, hemifumarate, and hemisuccinate were prepared and characterized by powder X-ray diffractometry, differential scanning calorimetry, and single crystal X-ray diffractometry. The intrinsic dissolution rate and stability of each miconazole crystal form were compared to those of freebase and nitrate to evaluate the optimal crystal form. Crystal structure analysis indicated that maleate was a salt formed by proton transfer from the acid to the imidazole group of miconazole. Hemifumarate and hemisuccinate were determined to be cocrystals formed by hydrogen bonding between the acids and the base in their crystal lattices. Intrinsic dissolution tests showed that the formation of salts and cocrystals improved the dissolution rate of miconazole. Stability tests of preliminary formulations prepared with each crystal form indicated that maleate and hemifumarate were unstable at 80°C and generated a specific degraded product, i.e., a Michael adduct, between miconazole and the acids. Hemisuccinate had a superior intrinsic dissolution rate and stability, and is thus considered a promising crystal form of miconazole.

Establishment of in vitro P-glycoprotein inhibition assay and its exclusion criteria to assess the risk of drug-drug interaction at the drug discovery stage.

The decision tree to determine whether the P-glycoprotein (P-gp)/multidrug resistance protein 1 (MDR1)-mediated drug-drug interaction (DDI) study is recommended has been proposed by the International Transporter Consortium. We, therefore, designed an in vitro P-gp inhibition assay and determined the appropriate risk criteria for P-gp-mediated DDI at the drug discovery stage. Effects of P-gp inhibitors on digoxin transport across a monolayer of MDR1-expressing cells were examined. The IC(50) (half-maximal inhibitory concentration) values generated from the efflux ratio (ER) were smaller than those generated from basolateral-to-apical directional apparent permeability. The difference in IC(50) values was kinetically described in a compartment model analysis. This analysis indicated that ER is a highly sensitive parameter that can be used for the degree of P-gp inhibition. Considering IC(50) values and the increase in digoxin exposure in clinical DDI studies, the risk criteria of [I(2)]/IC(50) = 30 ([I(2)], theoretically maximal gastrointestinal concentration) was the optimal cutoff value to predict a clinically relevant DDI. We also investigated whether the IC(50) value itself is applicable to assess the DDI risk. In conclusion, compounds with IC(50) values less than 2 µM exhibit high risk for P-gp-mediated DDIs. However, compounds with IC(50) values greater than or equal to 2 µM are inconclusive because clinical doses should be considered for the precise DDI risk assessment.

Naphthol derivatives as TRPV1 inhibitors for the treatment of urinary incontinence.

We have identified naphthol derivatives as inhibitors of the vanilloid receptor TRPV1 by high throughput screening. The initial lead showed high clearance in rats and has been optimized by enhancing the acidity of the phenol group. Compound 6b has reduced clearance, improved potency and is active in rat cystometry models of urinary incontinence after intravenous administration.

Quantitative investigation of the impact of P-glycoprotein inhibition on drug transport across blood-brain barrier in rats.

The magnitude of P-glycoprotein [(P-gp)/multidrug resistance protein 1 (MDR1)]-mediated drug-drug interaction (DDI) at the blood-brain barrier (BBB) in rats was estimated by in vitro-in vivo correlation (IVIVC). In in vitro studies, rat Mdr1a-expressing LLC-PK1 cells were examined for the evaluation of P-gp inhibitory activity using digoxin as a P-gp probe substrate. The in vitro K(i) value was calculated using a modified corrected flux ratio that reflects the P-gp function. In in vivo studies, digoxin with or without P-gp inhibitors was administered to rats by constant intravenous infusion to evaluate the effect of P-gp inhibition on digoxin transport to the brain under steady-state conditions. In the presence of elacridar, the brain-to-plasma concentration ratio (K(p,brain)) of digoxin was approximately 14 times the control value. However, no significant change in the K(p,brain) was observed in the presence of clinically used P-gp inhibitors, with the exception of cyclosporine A. A positive correlation was found between the in vivo K(p,brain) of digoxin and [I(,unbound)/K(i)] (where I(,unbound) is the unbound plasma concentration of P-gp inhibitors). Compounds with [I(,unbound)/K(i)] values of >1 increased K(p,brain) of digoxin in rats. In summary, we used a quantitative approach to evaluate the impact of P-gp-mediated DDI at the rat BBB. We successfully established the IVIVC, which indicated the potential DDI in the presence of potent P-gp inhibitors. On the basis of the IVIVC in rats and K(i) values in human MDR1, we speculated that clinically used P-gp inhibitors do not cause DDI at the human BBB, because none of the compounds studied showed [I(,unbound)/K(i)] values of >1 at therapeutic doses.

High-throughput cocrystal slurry screening by use of in situ Raman microscopy and multi-well plate.

Cocrystal has attracted much attention in order to improve poor physicochemical properties, since cocrystal former crystallize with the ionic drugs as well as nonionic drugs. Cocrystal screening was usually conducted by crystallization, slurry and co-grinding techniques, however sensitivity, cost and time for screening were limited because of issues such as dissociation of cocrystal during crystallization and cost and time required for slurry and co-grinding methods. To overcome these issues, novel high-throughput cocrystal slurry screening was developed by using in situ Raman microscope and a multi-well plate. Cocrystal screening of indomethacin was conducted with 46 cocrystal formers and potential cocrystals were prepared on a large scale for the characterization with powder X-ray diffractometry, thermal analysis, and Raman microscopy and (1)H NMR spectroscopy. Compared with the characterization of scale-up cocrystals, the cocrystal screening indicated that indomethacin structured novel cocrystals with D/L-mandelic acid, nicotinamide, lactamide and benzamide which was not obtained in the screening with crystallization technique previously reported. In addition, the screening provided not only information of cocrystal formation within a day but also information of equilibrium of cocrystal formation and polymorphic transformation in one screening. Information obtained in this screening allows effective solid form selection by saving cost and time for the development.

A 96-well plate assay for CYP4503A induction using cryopreserved human hepatocytes.

A reliable and practical CYP3A induction assay with cryopreserved human hepatocytes in a 96-well format was developed. Various 96-well plates with different basement membrane were evaluated using prototypical inducers, rifampicin, phenytoin, and carbamazepine. Thin-layer (TL) Matrigel was found to yield the highest basal and induced levels of CYP3A activity as determined by testosterone 6ß-hydroxylation. Concentration-dependent CYP3A induction of rifampicin was reproducible with the EC(50) values of 0.36 ± 0.28 µM from four batches of human hepatocytes using the 96-well plate with TL Matrigel. The rank order of induction potency for nine inducers or noninducers at a concentration of 10 µM were well comparable among the multiple donors, by expressing the results as percentage of change compared with the positive control, 10 µM rifampicin. Cotreatment of avasimibe or efavirenz with 10 µM rifampicin was found to reduce CYP3A activities induced by rifampicin at a lower rate than treatment with rifampicin alone, whereas treatment with phenobarbital and carbamazepine had no effect. From a comparison of induced CYP3A activities and gene expression levels, there were compounds that would cause induction of CYP3A4 mRNA but not activity, presumably due to their inhibitory effect on CYP3A activity. The cotreatment assay of test compound with rifampicin allows us to exclude the false-negative results caused by the cytotoxicity and/or the mechanism-based inactivation, when the drug candidate's ability for CYP3A induction is evaluating the enzyme activity. This 96-well plate assay, which is robust, reproducible, and convenient, has demonstrated the paramount applicability to the early drug discovery stage.