Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental carcinogens and metabolized by a variety of xenobiotic-metabolizing enzymes such as cytochrome P450 (P450 or CYP), epoxide hydrolase, glutathione transferase, UDP-glucuronosyltransferase, sulfotransferase, NAD(P)H quinone oxidoreductase 1, and aldo-keto reductase. These enzymes mainly participate in the conversion of PAHs to more polar and water-soluble metabolites, and the resultant metabolites are readily excreted from the body. However, during the course of metabolism, a variety of unstable and reactive intermediates of PAHs are formed, and these metabolites attack DNA, causing cell toxicity and transformation. P450s and epoxide hydrolase convert PAHs to proximate carcinogenic metabolites, PAH-diols, and these products are further metabolized by P450s to ultimate carcinogenic metabolites, PAH diol-epoxides, or by aldo-keto reductase to reactive PAH o-quinones. PAHs are also activated by P450 and peroxidases to reactive radical cations that bind covalently to DNA. The oxygenated and reactive metabolites of PAHs are usually converted to more polar and detoxified products by phase II enzymes. Inter-individual differences exist in levels of expression and catalytic activities of a variety of enzymes that activate and/or detoxify PAHs in various organs of humans and these phenomena are thought to be critical in understanding the basis of individual differences in response to PAHs. Factors affecting such variations include induction and inhibition of enzymes by diverse chemicals and, more importantly, genetic polymorphisms of enzymes in humans.
Coumarin 7-hydroxylation (COH), which is catalyzed almost solely by human CYP2A6 and mouse CYP2A5, shows large differences in activity (humans»mice) and inhibitor specificity between mice and humans. To differentiate human and mouse liver functions of chimeric mice (CM1, CM2 and CM3) prepared with hepatocytes from 3 donors, the microsomal COH activities were measured with and without benzaldehyde and undecanoic γ-lactone as a specific inhibitor of human CYP2A6 and mice CYP2A5, respectively. The replacement % to human hepatocytes designated as replacement index (RI) was calculated from human specific cytokeratin 8/18 expression in the liver section. The COH activities correlated well with RIs in CM2 (R2=0.98) and CM3 (R2=0.94), except CM1 whose genotype of donor is CYP2A6*4/*4. However, the COH activities expressed as % of donor activities were not always coincident with RIs, and the inhibition pattern of CM2 and CM3 was human-type after RI exceeded approximately 50%. Subsequently, our attempts to use % of COH activities or inhibition patterns as an accurate functional replacement index were unsuccessful. Since the detection of human CYP2A6 protein in the liver and the steep increase of human albumin (hAlb) levels in the blood were begun from almost RI=50% similarly to the changes of inhibition pattern, RI=50% is the turning point for chimeric mice to have humanized liver function.
The genetic polymorphism of CYP2C19 was examined in three Southeast Asian populations. This study was conducted in 774 Thais, 127 Burmeses and 131 Karens. Genomic DNA was extracted from leucocytes and analyzed by the PCR-RFLP technique. Genotype analysis revealed that the allele frequencies of CYP2C19*1, CYP2C19*2 and CYP2C19*3 in the Thais were 0.68, 0.29 and 0.03, respectively, and those of the Burmese population were 0.66, 0.30 and 0.04, respectively. For Karens, the minority ethnic in Mynmar, the allele frequencies of CYP2C19*1, CYP2C19*2 and CYP2C19*3 were 0.71, 0.28 and 0.01, respectively. The prevalence of PM estimated from genotype data among these three ethnic populations were 9.2%, 11.0%, and 8.4%, respectively. The PM phenotype and the frequencies of CYP2C19 defective alleles, particularly CYP2C19*3 among these three Southeast Asian ethnics appeared to be lower than other Asian populations. Lower prevalence of CYP2C19 PM suggests that these ethnics may have different capacity to metabolize drugs that are substrates of CYP2C19. Certain drug dosage regiments should be considered differently for Asian populations.
The plasma concentration profile of the antidiabetic agent tolbutamide was investigated in glycerol-induced acute renal failure (ARF) rats receiving or not receiving peritoneal dialysis (PD) to assess the impact of performing dialysis on tolbutamide pharmacokinetics. It was revealed that the plasma concentration of tolbutamide was decreased by 23.4% by performing PD in ARF rats, while it was not changed by PD in normal rats. The decrease in the plasma concentration of tolbutamide was nearly proportional to the increase in its volume of distribution. To clarify the mechanisms responsible for the decreased tolbutamide concentration caused by PD, the plasma protein binding of tolbutamide was examined in normal and ARF rats. The plasma unbound fraction of tolbutamide was higher in ARF rats than in normal rats, and the dissociation constants were 3.5±0.7 and 5.5±0.2 μg/mL in normal and ARF rats, respectively. These results indicated that the unbound fraction of tolbutamide was increased in ARF rats because of its protein binding being suppressed. It is therefore likely that since a measurable amount of tolbutamide can distribute in peritoneal dialysate in ARF rats, but not in normal rats, the plasma concentration of tolbutamide was decreased by performing PD only in ARF rats. These findings suggest that diabetes medication with tolbutamide should be carefully performed in patients receiving dialysis treatment.
The mRNA induction of various transporters by rifampicin (Rif), dexamethasone (Dex) and omeprazole (Ome) was investigated in primary cultures of cryopreserved human and rat hepatocytes. Analysis was performed by quantitative real-time RT-PCR using primers and TaqMan probes. In primary cultures of human hepatocytes, mRNA levels of MDR and MRP1 were increased by about 1.5 fold and 1.3 fold, respectively, by exposure to Rif at 2 to 50 μM as compared with 0.1% DMSO-treated controls. MRP2 mRNA levels in the same human hepatocytes were significantly increased by 1.2 to 1.8 fold by exposure to Rif at 50 μM as compared with controls. In primary cultures of rat hepatocytes, Mdr1a and Mdr1b mRNA levels were not increased or only slightly increased at 24 hr by exposure to any of the inducers at 2, 10 or 50 μM. Mrp2 mRNA levels in the same rat hepatocytes were significantly increased by 7 to 45 fold by exposure to Dex at 2 μM as compared with controls. Based on the species differences observed in the present study, primary cultures of cryopreserved hepatocytes from both the human and rat should be useful in preclinical drug development for evaluating candidate drugs for transporter induction.
Both influx and efflux transporters are thought to be involved in the intestinal absorption of fexofenadine. The present study examined the influx transporter-mediated intestinal absorption of fexofenadine in rats, focusing on the role of rat oatp3 (Oatp1a5). The intestinal permeability of fexofenadine was evaluated by means of the Ussing chamber method in the presence of a P-glycoprotein inhibitor to block efflux transport. The permeability of fexofenadine from the mucosal to the serosal side was higher than that from the serosal side to the mucosal side. Transport of fexofenadine was saturable, and was significantly decreased by an organic anion transporting polypeptide (oatp) inhibitor. Furthermore, uptake of fexofenadine by Xenopus oocytes expressing rat oatp3 was significantly greater than that by water-injected oocytes, and the affinity of oatp3 for fexofenadine (Km) was about 60 μM, which is comparable with the value obtained by the Ussing chamber method using rat intestinal tissues. These results indicate that oatp3 plays a role as an influx transporter in the intestinal absorption of fexofenadine in rats.
Hydroxysteroid sulfotransferase catalyzing bile acid sulfation plays an essential role in protection against lithocholic acid (LCA)-induced liver toxicity. Hepatic levels of Sult2a is up to 8-fold higher in farnesoid X receptor-null mice than in the wild-type mice. Thus, the influence of FXR ligand (chenodeoxycholic acid (CDCA) and LCA) feeding on hepatic Sult2a expression was examined in FXR-null and wild-type mice. Hepatic Sult2a protein content was elevated in FXR-null and wild-type mice fed a LCA (1% and 0.5%) diet. Treatment with 0.5% CDCA diet decreased hepatic Sult2a to 20% of the control in wild-type mice, but increased the content in FXR-null mice. Liver Sult2a1 (St2a4) mRNA levels were reduced to 26% in wild-type mice after feeding of a CDCA diet, while no decrease was observed on Sult2a1 mRNA levels in FXR-null mice after CDCA feeding. A significant inverse relationship (r2=0.523) was found between hepatic Sult2a protein content and small heterodimer partner (SHP) mRNA level. PCN-mediated increase in Sult2a protein levels were attenuated by CDCA feeding in wild-type mice, but not in FXR-null mice. Human SULT2A1 protein and mRNA levels were decreased in HepG2 cells treated with the FXR agonists, CDCA or GW4064 in dose-dependent manners, although SHP mRNA levels were increased. These results suggest that SULT2A is negatively regulated through CDCA-mediated FXR activation in mice and humans.
The object of this analysis was to develop a population pharmacokinetic model of micafungin, a new anti-fungal agent of the echinocandin class, to optimize dosing in Japanese patients with fungal infections. Population pharmacokinetics parameters were determined using NONMEM based on pharmacokinetic data from 198 subjects in seven clinical studies, comprising four phase I, two phase II and one pediatric phase III study. The healthy subjects received intravenous infusion of 2.5-150 mg micafungin. Adult and pediatric patients, age range of 8 month to 15 yeras old, were received 25-150 mg and 1-6 mg/kg daily, respectively. A total of 1825 micafungin plasma samples were available for this analysis. Two-compartment pharmacokinetic model was adopted. The clearance of micafungin was influenced by body weight in children and platelet counts (PLT). However the PLT accounted for less than 20% of the variation of micafungin clearance in Japanese subjects. In conclusions, body weight is the primary covariate factor in pediatric patients. The dose adjustment by body weight would be required only pediatric patients for the micafungin therapy in Japanese patients with fungal infection.
In this study, the entire coding sequence and the exon-intron junctions of the thiopurine S-methyltransferase (TPMT) gene from 200 Japanese individuals were screened for mutation. Three novel single nucleotide polymorphisms (SNPs) were identified-106G>A in exon 3 (Gly36Ser, *20 allele), 967A>G in 3′-untranslated region, and -87C>T in intron 8. The allele frequencies were 0.003 for 106G>A, 0.003 for 967A>G, and 0.010 for IVS8 -87C>T. In addition, the three known SNPs, 474T>C (Ile158Ile), 719A>G (Tyr240Cys, *3C allele), and IVS4 +35C>T were detected at frequencies of 0.299, 0.010, and 0.421, respectively.
Thirty-nine single nucleotide variations, including 16 novel ones, were found in the 5′ promoter region, all of the exons and their surrounding introns of HNF4A in 74 Japanese type II diabetic patients. The following novel variations were identified (based on the amino acid numbering of splicing variant 2): -208G>C in the 5′ promoter region; 1154C>T (A385V) and 1193T>C (M398T) in the coding exons; 1580G>A, 1852G>T, 2180C>T, 2190G>A, and 2362_2380delAAGAATGGTGTGGGAGAGG in the 3′-untranslated region, and IVS1+231G>A, IVS2-83C>T, IVS3+50C>T, IVS3-54delC, IVS5+173_176delTTAG, IVS5-181_-180delAT, IVS8-106A>G, and IVS9-151A>C in the introns. The allele frequencies were 0.311 for 2362_2380delAAGAATGGTGTGGGAGAGG, 0.054 for 1580G>A, 0.047 for 1852G>T, 0.020 for IVS1+231G>A, 0.014 for IVS9-151A>C, and 0.007 for the other 11 variations. In addition, one known nonsynonymous single nucleotide polymorphism, 416C>T (T139I), was detected at a 0.007 frequency. Based on the linkage disequilibrium profiles, the region analyzed was divided into three blocks. Haplotype analysis determined/inferred 10, 16, and 12 haplotypes for block 1, 2, and 3, respectively. Our results on HNF4A variations and haplotypes would be useful for pharmacogenetic studies in Japanese.