Prediction of Human Pharmacokinetic Profiles of the Antituberculosis Drug Delamanid from Nonclinical Data: Potential Therapeutic Value against Extrapulmonary Tuberculosis.

Delamanid has been studied extensively and approved for the treatment of pulmonary multidrug-resistant tuberculosis; however, its potential in the treatment of extrapulmonary tuberculosis remains unknown. We previously reported that, in rats, delamanid was broadly distributed to various tissues in addition to the lungs. In this study, we simulated human plasma concentration-time courses (pharmacokinetic profile) of delamanid, which has a unique property of metabolism by albumin, using two different approaches (steady-state concentration of plasma-mean residence time [Css-MRT] and physiologically based pharmacokinetic [PBPK] modeling). In Css-MRT, allometric scaling predicted the distribution volume at steady state based on data from mice, rats, and dogs. Total clearance was predicted by in vitro-in vivo extrapolation using a scaled albumin amount. A simulated human pharmacokinetic profile using a combination of human-predicted Css and MRT was almost identical to the observed profile after single oral administration, which suggests that the pharmacokinetic profile of delamanid could be predicted by allometric scaling from these animals and metabolic capacity in vitro. The PBPK model was constructed on the assumption that delamanid was metabolized by albumin in circulating plasma and tissues, to which the simulated pharmacokinetic profile was consistent. Moreover, the PBPK modeling approach demonstrated that the simulated concentrations of delamanid at steady state in the lung, brain, liver, and heart were higher than the in vivo effective concentration for Mycobacterium tuberculosis. These results indicate that delamanid may achieve similar concentrations in various organs to that of the lung and may have the potential to treat extrapulmonary tuberculosis.

Feature importance of machine learning prediction models shows structurally active part and important physicochemical features in drug design.

The objective of this study was to obtain the indicators of physicochemical parameters and structurally active sites to design new chemical entities with desirable pharmacokinetic profiles by investigating the process by which machine learning prediction models arrive at their decisions, which are called explainable artificial intelligence. First, we developed the prediction models for metabolic stability, CYP inhibition, and P-gp and BCRP substrate recognition using 265 physicochemical parameters for designing the molecular structures. Four important parameters, including the well-known indicator h_logD, are common in some in vitro studies; as such, these can be used to optimize compounds simultaneously to address multiple pharmacokinetic concerns. Next, we developed machine learning models that had been programmed to show structurally active sites. Many types of machine learning models were developed using the results of in vitro metabolic stability study of around 30000 in-house compounds. The metabolic sites of in-house compounds predicted using some prediction models matched experimentally identified metabolically active sites, with a ratio of number of metabolic sites (predicted/actual) of over 90%. These models can be applied to several screening projects. These two approaches can be employed for obtaining lead compounds with desirable pharmacokinetic profiles efficiently.

Predicting drug metabolism and pharmacokinetics features of in-house compounds by a hybrid machine-learning model.

We constructed machine learning-based pharmacokinetic prediction models with very high performance. The models were trained on 26138 and 16613 compounds involved in metabolic stability and cytochrome P450 inhibition, respectively. Because the compound features largely differed between the publicly available and in-house compounds, the models learned on the public data could not predict the in-house compounds, suggesting that outside of a certain applicability domain (AD), the prediction results are unreliable and can mislead the design of novel compounds. To exclude the uncertain prediction results, we constructed another machine learning model that determines whether the newly designed compound is inside or outside the AD. The AD was evaluated multi-dimensionally with some explanatory variables: The structural similarities and the probability obtained from the pharmacokinetic prediction model. The accuracy of predicting metabolic stability was 79.9% on the test set, increasing significantly to 93.6% after excluding the low-reliability compounds. The model properly classified the reliability of the compounds. After learning on the in-house compounds, the reliability model classified almost all (90%) of the public compounds as low reliability, indicating that the AD was properly determined by the model.

Pharmacokinetics and metabolism of brexpiprazole, a novel serotonin-dopamine activity modulator and its main metabolite in rat, monkey and human.

The pharmacokinetics of brexpiprazole were investigated in the in vitro and in vivo.The total body clearance of brexpiprazole in rat and monkey was 2.32 and 0.326 L/h/kg, respectively, after intravenous administration, and oral availability was 13.6% and 31.0%, respectively. Dose-dependent exposures were observed at dose ranges between 1-30 mg/kg in the rat and 0.1-3 mg/kg in the monkey.Brexpiprazole distributed widely to body tissues, and Vd,z were 2.81 and 1.82 L/kg in rat and monkey, respectively. The serum protein binding of brexpiprazole was 99% or more in animals and human. Uniform distribution character among the species was suggested by a traditional animal scale-up method.A common main metabolite, DM-3411 was found in animals and humans in the metabolic reactions with the liver S9 fraction. CYP3A4 and CYP2D6 were predominantly involved in the metabolism.The affinity of DM-3411 for D2 receptors was lower than that of brexpiprazole, and neither DM-3411 nor any metabolites with affinity other than M3 were detected in the brain, demonstrating that brexpiprazole is only involved in the pharmacological effects.Overall, brexpiprazole has a simple pharmacokinetic profile with good metabolic stability, linear kinetics, and no remarkable species differences with regard to metabolism and tissue distribution.

In vitro evaluations for pharmacokinetic drug-drug interactions of a novel serotonin-dopamine activity modulator, brexpiprazole.

Brexpiprazole, a serotonin-dopamine activity modulator, is indicated for the treatment of schizophrenia and also adjunctive therapy to antidepressants for the treatment of Major Depressive Disorder. To determine the drug-drug interaction risk for cytochrome P450, and SLC and ABC transporters, brexpiprazole and its metabolite, DM-3411 were assessed in this in vitro investigation.Brexpiprazole exhibited weak inhibitory effects (IC50 >13 µmol/L) on CYP2C9, CYP2C19, CYP2D6 and CYP3A4 activities, but had moderate inhibitor activity on CYP2B6 (IC50 8.19 µmol/L). The ratio of systemic unbound concentration (3.8 nmol/L) to the Ki value was sufficiently low. DM-3411 had comparable inhibitory potentials with brexpiprazole only for CYP2D6 and CYP3A4. The mRNA expressions of CYP1A2, CYP2B6 and CYP3A4 were not changed by the exposure of brexpiprazole to human hepatocytes.Brexpiprazole and DM-3411 exhibited weak or no inhibitory effects for hepatic and renal transporters (OATPs, OATs, OCTs, MATE1, and BSEP), except for MATE-2K (0.156 µmol/L of DM-3411), even for which the ratio to systemic unbound concentration (5.3 nmol/L) was sufficiently low.Brexpiprazole effected the functions of P-gp and BCRP with IC50 values of 6.31 and 1.16 µmol/L, respectively, however, the pharmacokinetic alteration was not observed in the clinical concomitant study on P-gp and BCRP substrates.These in vitro data suggest that brexpiprazole is unlikely to cause clinically relevant drug interactions resulting from the effects on CYPs or transporters mediating the absorption, metabolism, and/or disposition of co-administered drugs.

Antitubercular Agent Delamanid and Metabolites as Substrates and Inhibitors of ABC and Solute Carrier Transporters.

Delamanid (Deltyba, OPC-67683) is the first approved drug in a novel class of nitro-dihydro-imidazooxazoles developed for the treatment of multidrug-resistant tuberculosis. Patients with tuberculosis require treatment with multiple drugs, several of which have known drug-drug interactions. Transporters regulate drug absorption, distribution, and excretion; therefore, the inhibition of transport by one agent may alter the pharmacokinetics of another, leading to unexpected adverse events. Therefore, it is important to understand how delamanid affects transport activity. In the present study, the potencies of delamanid and its main metabolites as the substrates and inhibitors of various transporters were evaluated in vitro Delamanid was not transported by the efflux ATP-binding cassette (ABC) transporters P-glycoprotein (P-gp; MDR1/ABCB1) and breast cancer resistance protein (BCRP/ABCG2), solute carrier (SLC) transporters, organic anion-transporting polypeptides, or organic cation transporter 1. Similarly, metabolite 1 (M1) was not a substrate for any of these transporters except P-gp. Delamanid showed no inhibitory effect on ABC transporters MDR1, BCRP, and bile salt export pump (BSEP; ABCB11), SLC transporters, or organic anion transporters. M1 and M2 inhibited P-gp- and BCRP-mediated transport but did so only at the 50% inhibitory concentrations (M1, 4.65 and 5.71 µmol/liter, respectively; M2, 7.80 and 6.02 µmol/liter, respectively), well above the corresponding maximum concentration in plasma values observed following the administration of multiple doses in clinical trials. M3 and M4 did not affect the activities of any of the transporters tested. These in vitro data suggest that delamanid is unlikely to have clinically relevant interactions with drugs for which absorption and disposition are mediated by this group of transporters.

Hemodialysis clearance of iosimenol, a novel iso-osmolar radiographic contrast medium.

BACKGROUND: Iodinated contrast media (CM) have molecular and pharmacokinetic properties likely to make them highly dialyzable. Controlled clinical studies allowing for comparisons of hemodialysis clearance between different test substances and in multiple hemodialysis filters are, however, complex and not always practically feasible. A miniaturized in vitro method was therefore developed to evaluate the dialyzability of a new CM. PURPOSE: To evaluate hemodialysis clearance of iosimenol, a novel iso-osmolar contrast medium (CM), in a select variety of hemodialysis filters and in comparison to commercially available CM. MATERIAL AND METHODS: Three different high-flux and one low-flux membrane were used in miniaturized dialyzers to evaluate the in vitro blood clearance of iosimenol. Commercially available CM (iodixanol and iohexol) served as control substances. In vitro dialysis parameters were then used to predict clinical hemodialysis clearances. Residual ratios of endogenous substances (inorganic phosphate, urea nitrogen, creatinine, total bilirubin, and albumin) were used as proof of reliability of the in vitro dialysis system. RESULTS: Dialyzable small endogenous molecules were readily eliminated in all membranes. The removal ratios of iosimenol were generally similar to that of iodixanol in all membranes except the high-flux polysulfone but were consistently lower than that of iohexol. The blood clearance of iosimenol during clinical hemodialysis was predicted as, on average, approximately 85 mL/min with the high-flux membranes and 47 mL/min with the low-flux membrane. CONCLUSION: The dialyzability of iosimenol was evaluated using a newly developed in vitro dialysis system, and iosimenol was readily cleared from blood with all four tested membranes. And it is suggested that the dialysis parameters can predict clinical hemodialysis clearance of CM.

Comparison of p450 enzymes between cynomolgus monkeys and humans: p450 identities, protein contents, kinetic parameters, and potential for inhibitory profiles.

Cynomolgus monkeys are used to predict human pharmacokinetic and/or toxic profiles in the drug developmental stage. Cynomolgus P450s exhibit a high degree of identity (more than 90%) in both cDNA and amino acid sequences with corresponding human P450s. CYP3A protein predominantly exists in cynomolgus monkey liver microsomes, followed by CYP2A, CYP2C, CYP2B6, CYP2E1, and CYP2D. There are many similarities of metabolic properties in cytochrome P450s between cynomolgus monkeys and humans, but the species differences between cynomolgus monkey and human P450s are clearly present in substrate specificity and inhibitor selectivity. Diclofenac 4'-hydroxylation (DFOH) in monkey liver and intestinal microsomes shows much lower activities compared with those in human liver and intestinal microsomes. Sulfaphenazole strongly inhibits DFOH in human liver microsomes, but does not effectively inhibit DFOH in monkey liver and intestinal microsomes. Cynomolgus CYP2C19 exhibits higher activity for DFOH than cynomolgus CYP2C9 although this reaction is a marker reaction of human CYP2C9. On the other hand, cynomolgus CYP2C76 orthologue is not expressed in humans and shows 70-72% identity in amino acid sequences of human CYP2C subfamilies. Cynomolgus CYP2C76 metabolizes non-CYP2C substrates, 7-ethoxyresorufin (human CYP1A substrate) and bufuralol (human CYP2D6 substrate). In addition, cynomolgus CYP3A4 and CYP3A5 also exhibits wider substrate selectivity toward human CYP2D6 and CYP2E1 substrates. These enzymes may be responsible for species difference in drug metabolism between cynomolgus monkeys and humans. The comparative data presented here can be helpful for designing in vivo metabolic assays using cynomolgus monkeys in terms of substrate specificity and inhibitor selectivity.

Expression levels of multidrug resistance-associated protein 4 (MRP4) in human leukemia and lymphoma cell lines, and the inhibitory effects of the MRP-specific inhibitor MK-571 on methotrexate distribution in rats.

In the development of anti-blood cancer drugs, the chronic myelocytic leukemia (KU812), acute myelocytic leukemia (KG-1) and lymphoma (U937) cell lines are commonly used in preclinical pharmacology studies as human cancer xenograft models in mice. In the present study, mRNA expression levels of typical human ATP-binding cassette (ABC) transporters in these human blood cancer cell lines were analyzed by real-time polymerase chain reaction (RT-PCR). Based on the results, the expression level of multidrug resistance-associated protein 4 (MRP4) was found to be extremely high in KU812 cells compared with those of other transporters. Additionally, MRP4 expression levels were found to be relatively high in U937, KG-1 and a blood cell line derived from a healthy subject (RPMI 1788). In addition, to elucidate the contribution of MRP4 to the methotrexate (MTX) distribution in normal blood cells and tissues, [(3)H]MTX was intravenously (i.v.) administered to two groups of rats. Animals in one group received [(3)H]MTX only; the other group was concomitantly administered i.v. MK-571, a typical inhibitor of MRP transporters. No marked difference was observed between the two groups; the Kp values (tissue concentration/plasma concentration) of the concomitant group showed slightly higher values compared with those of the MTX alone group in erythrocytes (1.4 times, P<0.001), spleen (1.3 times, P<0.05) and thymus (1.2 times, P<0.05), respectively. Although in the present study we could not evaluate the direct involvement of MRP4 in blood cancer cells in which MRP4 expression was excessively high, these results suggest a possible functional role of MRP4 in blood cancer cells and albeit only slightly in normal blood cells/tissues.

Characterization of intestinal and hepatic P450 enzymes in cynomolgus monkeys with typical substrates and inhibitors for human P450 enzymes.

Cynomolgus monkeys are widely used to predict human pharmacokinetic and/or toxic profiles in the drug developmental stage. Characterization of cynomolgus monkey P450s such as the mRNA expression level, substrate specificity, and inhibitor selectivity were conducted to provide helpful information in designing monkey in vivo studies and monkey-to-human extrapolation. The expression levels of 12 monkey P450 mRNAs, which are considered to be important P450 subfamilies in drug metabolism, were investigated in the liver, small intestine (duodenum, jejunum, and ileum), and colon of individual monkeys. 3. In vitro activities and intrinsic clearance values were determined in monkey intestinal and liver microsomes (MIM and MLM, respectively) using nine typical oxidative reactions for human P450s. Paclitaxel 6α-hydroxylation, diclofenac 4'-hydroxylation, and S-mephenytoin 4'-hydroxylation showed low activities in MIM and MLM. IC50 values of eight selective inhibitors of human P450s were determined in MIM and MLM. Inhibitory effects of furafylline and sulfaphenazole were weak in monkeys on phenacetin O-deethylation and diclofenac 4'-hydroxylation, respectively. These results show profiles of monkey P450s in both the intestine and liver in detail and contribute to a better understanding of the species difference in substrate specificity and inhibitor selectivity between cynomolgus monkeys and humans.

Nonclinical pharmacokinetics of a new nonpeptide V2 receptor antagonist, tolvaptan.

PURPOSE: To evaluate the pharmacokinetic profile of tolvaptan. METHOD: The nonclinical pharmacokinetic profile of [(14)C]tolvaptan was evaluated in an absorption, distribution, and excretion study in rats after single oral administration. An in vitro protein binding study was also performed. RESULTS: The tolvaptan-derived radioactivity was rapidly absorbed and extensively distributed to all tissues in rats. The radioactivity levels were greatest in the gastrointestinal tract and liver, though the levels in the cerebrum, cerebellum and medulla oblongata were low. The serum and tissue concentrations of radioactivity, and serum concentration of tolvaptan in male and female rats showed a marked sex difference. The radioactivity was crossed the placenta and was distributed to the fetal tissues in pregnant rats. The milk showed 1.5-15.8-fold higher radioactivity than blood in lactating rats. The radioactivity mainly excreted into the feces via the biliary route. Tolvaptan binds extensively to plasma proteins (≥ 97.2%) in mouse, rat, rabbit, dog and human plasma.

In vitro P-glycoprotein interactions and steady-state pharmacokinetic interactions between tolvaptan and digoxin in healthy subjects.

Interactions between tolvaptan and digoxin were determined in an open-label, sequential study where 14 healthy subjects received tolvaptan 60 mg once daily (QD) on days 1 and 12 to 16 and digoxin 0.25 mg QD on days 5 to 16. Mean maximal concentrations (C(max)) and area under the curve during the dosing interval (AUC(τ)) for digoxin with tolvaptan (day 16) were increased 1.27- and 1.18-fold compared with digoxin alone (day 11); mean renal clearance of digoxin was decreased by 59% (P < .05). Tolvaptan C(max) and AUC(0-24h) for a single dose with digoxin (day 12) were each increased about 10% compared with tolvaptan alone (day 1). Tolvaptan did not accumulate upon multiple dosing. After a single dose of tolvaptan (day 1, day 12), 24-hour urine volume was about 7.5 L. As expected, after 5 days of tolvaptan, 24-hour urine volume decreased about 20%. In vitro studies in control and MDR1-expressing LLC-PK1 cells indicate that tolvaptan is a substrate of P-glycoprotein. Tolvaptan (50 µM) inhibited basolateral to apical digoxin secretion to the same extent as 30 µM verapamil; the IC50 of tolvaptan was determined to be 15.9 µM. The increase in steady-state digoxin concentrations is likely mediated by tolvaptan inhibition of digoxin secretion.

Two novel CYP2D6*10 haplotypes as possible causes of a poor metabolic phenotype in Japanese.

During the course of sequencing for the CYP2D6 gene, we found a novel single nucleotide polymorphism of g.3318G>A (E383K) associated with CYP2D6*10, termed as CYP2D6*72. We also found a g.1611T>A (F120I) in the CYP2D6*49, which was previously identified as a CYP2D6*10-associated allele in an independent Japanese population. To clarify the effects of these novel CYP2D6*10 haplotypes on the functions of CYP2D6, kinetic analysis for dextromethorphan O-demethylation was performed using the Escherichia coli expression system and human liver microsomes. The V(max)/K(m) values for dextromethorphan O-demethylation catalyzed by recombinant CYP2D6 forms encoded by CYP2D6*10, CYP2D6*49, and CYP2D6*72 were 3.0, 0.5, and 1.3%, respectively, compared with that catalyzed by CYP2D6.1. Liver microsomes from a human subject genotyped as CYP2D6*10/*49 also showed a reduced dextromethorphan O-demethylase activity. CYP2D6.49 formed a 7-hydroxydextromethorphan, with a roughly similar V(max)/K(m) value to that of O-demethylation. These results suggest that these two CYP2D6*10 haplotypes are possible causes of interindividual variation in the activities and the substrate specificity of CYP2D6.

High performance liquid chromatographic methods for the determination of aripiprazole with ultraviolet detection in rat plasma and brain: application to the pharmacokinetic study.

High performance liquid chromatographic (HPLC) methods were validated for the determination of aripiprazole (OPC-14597, Abilify) in rat plasma and brain. Separation was by Nova-pak phenyl column; flow rate, 1.0 ml/min; mobile phase, acetonitrile-methanol-20 mM sodium sulfate-acetic acid (27:25:48:1, v/v/v/v); UV detection at 254 nm. Reproducibility in plasma and brain showed excellent precision (within 7.8 and 10.6%) and accuracy (96.0-102.4% and 99.0-108.7%) with calibration curve ranges 10.0-2000 ng/ml and 30.0-6000 ng/g, respectively. Validated HPLC methods were successfully applied to pharmacokinetic study of aripiprazole in rats, demonstrating brain concentrations after oral administration five times higher than plasma concentrations.

Functional characterization of single nucleotide polymorphisms with amino acid substitution in CYP1A2, CYP2A6, and CYP2B6 found in the Japanese population.

As a part of the studies conducted by the Pharma SNPs Consortium (PSC), the enzyme activities of CYP1A2, CYP2A6 and CYP2B6 variants with altered amino acids as a result of single nucleotide polymorphisms (SNPs) found among the Japanese population were analyzed under a unified protocol using the same lots of reagents by the laboratories participating in the PSC. Mutations in CYP1A2, CYP2A6 and CYP2B6 were introduced by site-directed mutagenesis and the wild type and mutated CYP molecules were expressed in Escherichia coli. The expressed cytochrome P450s were purified and the enzyme activities were measured in reconstitution systems. CYP1A2 and CYP1A2Gln478His did not show any differences in 7-ethoxyresorufin O-deethylase activity. CYP2A6 and CYP2A6Glu419Asp metabolized coumarin to form 7-hydroxycoumarin in a similar manner, whereas CYP2A6Ile471Thr showed low activity compared to the wild-type CYP2A6. CYP2B6, CYP2B6Pro167Ala and CYP2B6Arg487Cys showed the same activity for 7-ethoxy-4-triflouromethyl-coumarin O-deethylation. However, CYP2B6Gln172His was roughly twice as active as CYP2B6 and the other CYP2B6 variants for 7-ethoxy-4-triflouromethylcoumarin O-deethylation activity. Although higher inter- and intra-laboratory variations were observed for the calculated Km and V(max) values because the studies were conducted in several different laboratories, the degree of variations was reduced by the increased number of analyses and the adoption of a simple analysis system.