The aim of this study is to investigate the metabolic (cytochrome P450-dependent) behaviour of pyrazolo[3,4-d]pyrimidines 1–10 dual Abl/Src kinase inhibitors. All the compounds demonstrate good metabolic stability both in human liver (HLM) and in rat liver (RLM) microsomes. Moreover, all the tested molecules undergo the same metabolic CYP-dependent reactions, namely oxidative dechlorination and N-dealkylation. These metabolic pathways are fully characterized for compound 1. In HLM, the dehalogenated metabolite accounts for about 87% of the full 1 metabolism, while the N-dealkylated metabolite accounts for 12%. Inhibition studies performed using different CYP-inhibitors indicate that the 3A family is the isoenzyme family most involved in pyrazolo[3,4-d]pyrimidine metabolism. This observation is confirmed by studies performed by using CYP3A selective substrates. Furthermore kinetic analysis performed in RLM, HLM and cDNA CYP3A4 shows that the affinity of CYPs towards compound 1 is similar in all the tested preparations (K_m = 32.7, 21.8, and 48.7 µM, respectively).

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Bosutinib is an orally active, competitive inhibitor of Src/Abl tyrosine kinases. A population pharmacokinetic model was developed using data pooled from 3 studies of patients (n = 870) with solid tumors or Philadelphia chromosome–positive leukemia. Patients (aged 18–91 y, weighing 35–221 kg) who received bosutinib 50 to 600 mg orally with food each contributed 6–9 pharmacokinetic samples. The final pharmacokinetic model was a linear two-compartment model with first-order absorption, an absorption lag-time, and dose-dependent bioavailability. Oral absorption was relatively slow, with a half-time of 1.14 h and a lag-time of 0.87 h; time to peak concentration was 5–6 h. Apparent clearance was 120 L/h. The apparent volume of the peripheral compartment was large with a slow turnover; alpha and beta half-lives were 19 h and 290 days, respectively. All parameters were estimated with acceptable precision (standard error <30%). No tested covariate (protocol, baseline demographic/clinical characteristics, or laboratory results) explained the high inter-individual variability of bosutinib pharmacokinetics. Therefore, adjusting bosutinib dose for body size (weight, surface area) would not provide benefit over fixed dosing. Using this exposure model in pharmacodynamic assessment of one study, adverse event incidence was shown to be similar in overall and bosutinib-responsive populations.

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Nilotinib potently inhibits human uridine diphosphate-glucuronosyltransferase (UGT1A1) activity, causing hyperbilirubinemia. We investigated the influence of UGT1A1 polymorphisms and nilotinib plasma trough concentrations (C₀) on nilotinib-induced hyperbilirubinemia in 34 Japanese patients with chronic myeloid leukemia (CML). The proportion of patients with hyperbilirubinemia was significantly higher among patients with the UGT1A1*6/*6 and *6/*28 genotypes (poor metabolizers) than among those with other genotypes (p = 0.004). The median time to elevation of bilirubin levels in UGT1A1 poor metabolizers was 2.0 weeks (hazard ratio, 6.11). The median time to reduction in nilotinib dose in UGT1A1 poor metabolizers was 4.0 weeks (hazard ratio, 7.52; p = 0.002). Consequently, in the maintenance phase 3 months following the initiation of nilotinib therapy, the median daily dose and C₀ of nilotinib were 350 mg/day and 372 ng/mL, respectively, in UGT1A1 poor metabolizers, and 600 mg/day and 804 ng/mL, respectively, in the other patients. Patients at increased hyperbilirubinemia risk could be identified by prospective UGT1A1 genotyping prior to nilotinib therapy. To avoid an interruption of CML treatment due to nilotinib-induced hyperbilirubinemia, it may be beneficial to reduce the initial nilotinib dose to 300–400 mg/day for UGT1A1 poor metabolizers.

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A pharmacokinetic/pharmacodynamic (PK/PD) analysis is important in antibiotic chemotherapy. Basically, the in vivo efficacy of antibiotics that exert concentration-dependent effects can be predicted using conventional PK/PD indices such as the ratio of the area under the curve to the minimum inhibitory concentration (AUC/MIC) and/or the ratio of the maximum plasma concentration to MIC (C_max/MIC), whereas that of antibiotics with time-dependent effects can be determined using the period of time for which the drug concentration exceeds the MIC (time above MIC [TAM]). However, an optimal PK/PD index remains to be established for some antibiotics. Thus, a PK/PD model which describes the PK profile and effect of an antibiotic was developed, and the results obtained from this model were interpreted to form a PK/PD index map to assess the optimal PK/PD index for the antibiotic. The findings from the map were generally consistent with clinical outcomes even for the antibiotics which proved to be exceptions to the conventional classification. For example, AUC/MIC was an optimal index for azithromycin despite its time-dependent bactericidal activity, and C_max/MIC was a poor index for arbekacin despite its concentration-dependent profile. Thus, the map would be useful for selecting the appropriate PK/PD index for an antibiotic.

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Nitrilase, which is found in plants and many types of bacteria, is known as the enzyme that catalyzes hydrolysis of a wide variety of nitrile compounds. While human nitrilase-like protein (NIT), which is a member of the nitrilase superfamily, has two distinct isozymes, NIT1 and NIT2, their function has not been well understood. In this study, we investigated whether human NIT1 and NIT2 are involved in the hydrolysis of drugs using vildagliptin as a substrate. We performed Western blot analysis using human liver samples to examine protein expression of human NIT in the liver, finding that human NIT1 and NIT2 were highly expressed in the liver cytosol. We established stable single expression systems of human NIT1 and NIT2 in HEK293 cells to clarify the contribution of human NIT to hydrolysis of vildagliptin. Although the formation of a carboxylic acid metabolite of vildagliptin (M20.7) was observed in human liver samples, M20.7 was not formed by incubating vildagliptin with HEK293 cells expressing human NIT1 or NIT2. This suggests that human NIT1 or NIT2 is not involved in the metabolism of vildagliptin. Further investigation using other drugs is needed to clarify the contribution of human NIT to drug metabolism.

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l-Phenylalanyl-Ψ[CS-N]-l-alanine (Phe-Ψ-Ala), a thiourea dipeptide, was evaluated as a probe for peptide transporter 1 (PEPT1). Uptake of Phe-Ψ-Ala in PEPT1-overexpressing HeLa cells was significantly higher than that in vector-transfected HeLa cells and the K_m value was 275 ± 32 µM. The uptake was pH-dependent, being highest at pH 6.0, and was significantly decreased in the presence of PEPT1 inhibitors [glycylsarcosine (Gly-Sar), cephalexin, valaciclovir, glycylglycine, and glycylproline]. In metabolism assay using rat intestinal mucosa, rat hepatic microsomes, and human hepatocytes, the amount of Phe-Ψ-Ala was unchanged, whereas phenylalanylalanine was extensively decomposed. The clearance, distribution volume, and half-life of intravenously administered Phe-Ψ-Ala in rats were 0.151 ± 0.008 L/h/kg, 0.235 ± 0.012 L/kg, and 1.14 ± 0.07 h, respectively. The maximum plasma concentration of orally administered Phe-Ψ-Ala (2.31 ± 0.60 µg/mL) in the presence of Gly-Sar was significantly decreased compared with that in the absence of glycylsarcosine (3.74 ± 0.44 µg/mL), suggesting that the intestinal absorption of Phe-Ψ-Ala is mediated by intestinal PEPT1. In conclusion, our results indicate that Phe-Ψ-Ala is a high-affinity, metabolically stable, non-radioactive probe for PEPT1, and it should prove useful in studies of PEPT1, e.g., for predicting drug-drug interactions mediated by PEPT1 in vitro and in vivo.

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The present study investigated the effect of calf thymus DNA (ctDNA) on human hepatic cytochrome P450s (CYP450s) in vitro. Specific substrate probes for each isoform, CYP1A2, 2C9, 2C19, 2D6 and 3A4, were incubated using pooled human liver microsomes with or without ctDNA, and liquid chromatography coupled tandem mass spectrometry (LC-MS/MS) method was developed for the analysis of probe metabolites. Enzyme kinetics parameters K_i and IC₅₀ values were estimated to determine the types and strength of inhibition. ctDNA could specifically inhibit the metabolism of CYP2C9 probe substrates, with the IC₅₀ = 0.9955 µg/ml, while it was not able to inhibit CYP1A2, CYP2C19, CYP2D6 or CYP3A4 (IC₅₀ > 100 µg/ml). The results showed that ctDNA was a potent inhibitor of CYP2C9 enzyme, and has the metabolic interaction potential with the model drugs which are metabolism substrates of CYP2C9. The inhibition mechanism study suggested ctDNA may inhibit CYP2C9 by decreasing the activity of CYP450 reductase. These findings indicated that when the medical agents catalyzed mainly by CYP2C9 were co-administered in vivo with adsorptive material in vitro, the potential inhibitory effect of ctDNA on enzyme activity and the following metabolism character changes of the former should be highly focused on.

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Altered expression of P-glycoprotein (P-gp), a drug efflux transporter expressed by brain capillary endothelial cells (BCECs), may contribute to the development of opioid analgesic tolerance, as demonstrated by cumulative evidence from research. However, the detailed mechanism by which chronic morphine treatment increases P-gp expression remains unexplained. Ezrin/radixin/moesin (ERM) are scaffold proteins that are known to regulate the plasma membrane localization of some drug transporters such as P-gp in peripheral tissues, although a few reports suggest its role in the central nervous system as well. In this study, we investigated the involvement of ERM in the development of morphine analgesic tolerance through altered P-gp expression in BCECs. Repeated treatment with morphine (10 mg/kg/day, s.c. for 5 days) decreased its analgesic effect in the tail-flick test and increased P-gp protein expression in BCECs, as determined by Western blotting. Furthermore, moesin protein expression increased in the same fraction whereas that of ezrin decreased; no change was observed in the radixin expression. Furthermore, immunoprecipitation and immunofluorescence assays revealed interaction between moesin and P-gp molecules, along with co-localization, in BCECs. In conclusion, an increase in moesin expression may contribute to the increased expression of P-gp in BCECs, leading to the development of morphine analgesic tolerance.

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ATP-binding cassette transporter, sub-family G, member 2 (ABCG2/BCRP) is a xenobiotic transporter and also regulates serum uric acid levels as a urate transporter. We have shown that the severity of ABCG2 dysfunction can be estimated by simple genotyping of two dysfunctional variants, Q126X (rs72552713) and Q141K (rs2231142). This genotyping method is widely accepted for the risk analysis of hyperuricemia/gout, but there is no report on ethnic differences in ABCG2 dysfunctions. Here, we estimated ABCG2 dysfunctions by its genotype combination (Q126X and Q141K) and compared them in three different ethnic groups (500 Japanese, 200 Caucasians and 100 African-Americans). The minor allele frequencies of Q126X and Q141K in Japanese (0.025 and 0.275, respectively) were significantly higher than those in Caucasians (0.005 and 0.085, respectively) and African-Americans (0 and 0.090, respectively). Additionally, the rates of mild, moderate and severe ABCG2 dysfunctions in Japanese (35.4%, 12.4% and 1.6%, respectively) were higher than those in Caucasians (14.0%, 2.5% and 0%, respectively) and African-Americans (14.0%, 2.0% and 0%, respectively). Because ABCG2 dysfunctional diplotypes were commonly observed in both Caucasians (16.5%) and African-Americans (16.0%), the genotyping of the two ABCG2 dysfunctional variants is useful for evaluating individual differences in the ABCG2 dysfunction which affect the pharmacokinetics of substrate drugs and hyperuricemia risk in all three ethnic groups.

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