Cytochrome P450 (P450) constitutes a superfamily of enzymes which activate dioxygen and carry out monooxygenation reactions of large numbers of endogenous and xenobiotic compounds. Drug metabolism is a particularly important P450 function, and, therefore, elucidating the metabolic products and pathways of drugs is essential for drug development. To explain the substrate selectivity of P450 reactions, it is necessary to understand the formation of multiple activated oxygen species to determine the type of catalyzed reactions, in addition to conducting structure analyses of P450s. Although an oxo-Fe(IV)-porphyrin-π-cation radical is regarded as an activated oxygen species in P450 reactions, a nucleophilic Fe(III)-peroxo species has also been proposed as another oxidant. In the past decade, various studies indicated that P450-catalyzed oxygenations are complex, and that a single reaction pathway cannot explain all of the experimental results. In addition, the microsomal P450 system is known to generate reactive oxygen species (ROS). However, the contribution of ROS to P450 reactions remains unclear. We recently found that singlet oxygen (1O2) was involved in both several rat liver microsomal P450 reactions and four human CYP subfamily activities, as confirmed by the ESR spin-trapping method. In this review, we describe the studies that have been conducted on the detection and characterization of ROS in P450 reactions related to drug metabolism that involve the possibility of 1O2 in the P450 catalytic cycle. Gaining an understanding of the activated oxygen species that determine the type of drug metabolism will help us to predict the important metabolites formed.
Previously, we reported that singlet oxygen (1O2) was involved in rat liver microsomal P450-dependent substrate oxygenations in such reactions as p-hydroxylation of aniline, O-deethylation of 7-ethoxycoumarin, ω- and (ω-1)-hydroxylations of lauric acid, O-demethylation of p-nitroanisole, and N-demethylation of aminopyrine. In order to confirm the generality of 1O2 involvement, we have further investigated which kinds of reactive oxygen species (ROS) are formed during P450-dependent substrate oxygenation in microsomes. We examined CYP2E1-dependent hydroxylation of p-nitrophenol in rat liver microsomes in the presence of some ROS scavengers, because CYP2E1 has been reported to predominantly generate ROS in the hepatic microsomes and to relate with the oxidative stress in the body. The addition of 1O2 quenchers, β-carotene, suppressed the hydroxylation of p-nitrophenol. Furthermore, a nonspecific P450 inhibitor, SKF525A, and a ferric chelator, deferoxamine, both suppressed the hydroxylation. No other ROS scavengers such as superoxide dismutase (SOD), catalase, or mannitol altered the reaction. 1O2 was detectable during the reaction in the microsomes as measured by an electron spin resonance (ESR) spin-trapping method when 2,2,6,6-tetramethyl-4-piperidone (TMPD) was used as a spin-trapping reagent. The 1O2 was quenched by additions of β-carotene, p-nitrophenol, and SKF525A. The reactivity of p-nitrophenol and 1O2 correlated linearly with its hydroxylation rate in the microsomes. On the basis of these results, we conclude that 1O2 contributes to the p-nitrophenol hydroxylation in rat liver microsomes, by adding a new example of 1O2 involvement in the CYP2E1-dependent substrate oxygenations.
In order to identify genetic polymorphisms and haplotype frequencies of CYP1A2 in a Japanese population, the enhancer and promoter regions, all the exons with their surrounding introns, and intron 1 were sequenced from genomic DNA from 250 Japanese subjects. Thirty-three polymorphisms were found, including 13 novel ones: 2 in the enhancer region, 5 in the exons, and 6 in the introns. The most common single nucleotide polymorphism (SNP) was -163C>A (CYP1A2*1F allele) with a 0.628 frequency. In addition to six previously reported non-synonymous SNPs, three novel ones, 125C>G (P42R, CYP1A2*15 allele, MPJ6_1A2032), 1130G>A (R377Q, *16 allele, MPJ6_1A2033), and 1367G>A (R456H, *8 allele, MPJ6_1A2019), were found with frequencies of 0.002, 0.002, and 0.004, respectively. No polymorphism was found in the known nuclear transcriptional factor-binding sites in the enhancer region. Based on linkage disequilibrium analysis, the CYP1A2 gene was analyzed as one haplotype block. Using the 33 detected polymorphisms, 14 haplotypes were unambiguously identified, and 17 haplotypes were inferred by aid of an expectation-maximization-based program. Among them, the second major haplotype CYP1A2*1L is composed of -3860G>A (*1C allele), -2467delT (*1D allele), and -163C>A (*1F allele). Network analysis suggested that relatively rare haplotypes were derived from three major haplotypes, *1A, *1M, and *1N in most cases. Our findings provide fundamental and useful information for genotyping CYP1A2 in the Japanese, and probably Asian populations.
Evodia fruit (Evodiae Fructus) is used as a herbal medicine prepared from the matured fruit of the Evodia rutaecarpa Bentham or Evodia officinalis Dode, of the Rutaceae plant family. An extract of Evodia fruit in the presence of NADPH was shown to inhibit human liver microsomal erythromycin N-demethylation activity, mediated by cytochrome P450 3A4 (CYP3A4), in a preincubation-time dependent manner. The present study was conducted to identify components of Evodia fruit extract having preincubation-time dependent inhibitory effects on CYP3A4 by analyzing human liver microsomal erythromycin N-demethylation activity. Rutaecarpine, a major component of Evodia fruit, and limonin caused the most dramatic decrease in residual CYP3A4 activity (IC50 before and after 20 min preincubation with: rutaecarpine, >100 μM and 1.4 μM; limonin, 23.5 μM and 1.8 μM, respectively). Furthermore, rutaecarpine and limonin were identified as mechanism-based inhibitors of CYP3A4 from the following observations: 1) The inhibitory effects of rutaecarpine and limonin on CYP3A4 activity were dependent on the preincubation time, 2) The inhibition required NADPH, 3) The inhibition was depressed in the presence of the competitive CYP3A4 inhibitor, ketoconazole, 4) Dialysis resulted in no recovery of CYP3A4 activity. The kinetic parameters for inactivation kinact and KI were: 0.387 min-1 and 107.7 μM for rutaecarpine, 0.266 min-1 and 23.2 μM for limonin, respectively. These results indicate that rutaecarpine and limonin are mechanism-based inhibitors of CYP3A4.
Total parenteral nutrition (TPN) is associated with cholestasis and hepatic steatosis in human infants. The present study focused on the changes in hepatic xenobiotic transporters associated with overdose of fat-free or fat-containing TPN in infant rats. Three-week-old male Sprague-Dawley rats were divided into three groups: group 1 received an oral diet, group 2 received TPN without fat, and group 3 received TPN with 20% of its calories from fat (soybean oil emulsion). After TPN administration for 4 days, both serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels, which are indicators of hepatic dysfunction, in group 2 were significantly higher (p<0.001) than those in the other groups, whereas there were no differences between groups 1 and 3 in either serum AST or ALT levels. The serum bilirubin concentration in group 2 was also markedly higher than that in the other groups. Mdr2, Bsep, Mrp2, Mrp6, Oct1, and Oat2 mRNA levels were decreased in group 2 (fat-free TPN) compared with those in group 1 (oral diet), whereas Mdr1b, Mrp1, and Mrp5 mRNA levels were increased. Specifically, the level of Mdr1b mRNA in group 2 was 16 times higher (p<0.001) than that in group 1. On the other hand, the changes in these mRNA expression levels in group 3 (fat-containing TPN) were smaller than those in group 2, and specifically, the expression levels of Mdr1b, Mrp1, Mrp5, Mrp6, and Oat2 mRNA in group 3 were not significantly different from those in group 1. The results of the present study indicate that including fat in the TPN regimen is very important in preventing the mRNA up- and down-regulation of xenobiotic transporters, which is considered to be the main factor responsible for the abnormal hepatic changes such as cholestasis associated with the excessive administration of fat-free TPN.
The results of in vitro studies indicated that ARIPIPRAZOLE, a newly developed antipsychotic, is mainly metabolized by the human cytochrome P450 isozymes CYP3A4 and CYP2D6. The objective of the present study was to investigate the influence of itraconazole (hereafter referred to as ITZ) co-administration (CYP3A4 inhibition) on the pharmacokinetics of ARIPIPRAZOLE administered to 24 healthy adult male volunteers in a fasting condition. The influence of CYP3A4 inhibition was also examined by CYP2D6 genotype. All subjects were administered a single oral dose of ARIPIPRAZOLE alone in Period I and a single oral dose of ARIPIPRAZOLE following administration of ITZ at 100 mg/day for 7 consecutive days in Period II. The pharmacokinetic parameters of ARIPIPRAZOLE and its main metabolite OPC-14857 were determined. Co-administration of ITZ increased the Cmax, AUC336 hr, and t1/2,z of ARIPIPRAZOLE and OPC-14857 by 19.4%, 48.0%, and 18.6% and by 18.6%, 38.8%, and 53.4%, respectively. By co-administration of ITZ, the CL/F of ARIPIPRAZOLE in extensive metabolizers was decreased by 26.6%, with an even greater decrease (47.3%) in intermediate metabolizers. For the co-administration period, the CL/F of ARIPIPRAZOLE in intermediate metabolizers was about half of that in extensive metabolizers. For Cmax, there was no significant difference between extensive metabolizers and intermediate metabolizers, and the percent change by co-administration of ITZ was less than 20% in both extensive metabolizers and intermediate metabolizers. For OPC-14857, the tmax in intermediate metabolizers was longer than that in extensive metabolizers, with the difference being amplified by co-administration of ITZ. The AUC336 hr showed similar increases by co-administration of ITZ in all genotypes. The urinary 6β-hydroxycortisol/cortisol concentration ratio following ITZ administration for 7 consecutive days was about half of that before the start of ITZ administration, indicating that CYP3A4 metabolic activity was inhibited by administration of ITZ. The influence of CYP3A4 inhibition on the pharmacokinetics of ARIPIPRAZOLE was not considered to be clinically significant. On the other hand, definite differences in pharmacokinetics were observed between CYP2D6 genotypes.
The absorption enhancing effect of 1-[2-(decylthio) ethyl] azacyclopentan-2-one (Pirotiodecane), on drug permeation across rabbit nasal mucosa was studied. The nasal epithelial mucosa was isolated from rabbit nasal septum and mounted in an Ussing chamber to allow for monitoring of the membrane resistance (Rm), and the permeation of fluorescein isothiocyanate-labeled dextran (FD-4, M.W. 4,400 Da). Treatment with 0.05, 0.1, and 0.2% Pirotiodecane for 60 min decreased Rm, and increased the cumulative amount of FD-4 permeated in a concentration-dependent manner, suggesting that Pirotiodecane possesses passively a disassembly of tight junction to enable the enhanced FD-4 permeation. The remarkable increase in plasma concentration of FD-4 was also observed in intranasal co-administration with 1% Pirotiodecane in rabbits. The Rm was virtually maintained after the removal of Pirotiodecane, although recovery of Rm was not seen. On the other hand, the increase in plasma concentration of FD-4 with intranasal co-administration of 1% Pirotiodecane in rabbits in vivo was not observed in FD-4 administration at 15-60 min after administration of 1% Pirotiodecane alone. It was concluded that Pirotiodecane possesses a relatively short absorption enhancing effect through nasal epithelial.
It is known that secretory transport limits the oral bioavailability of certain drugs. However, there is little information on the secretion of anionic compounds in the intestine. Phenolsulfonphthalein (PSP) and p-aminohippuric acid (PAH) have been used widely as substrates for organic anion transport systems. PAH is transported in the secretory direction in the intestine. It is possible that PSP and PAH share the same transport system at the mucosal membrane. The purpose of this study was to characterize the transport system for PSP in the intestine. In the jejunum, the serosal-to-mucosal permeation rate of PSP was significantly reduced in an ATP-depleted condition, whereas a significant difference was not observed in the ileum. Some multidrug resistance-associated protein 2 (Mrp2) inhibitors inhibited PSP permeation in the jejunum. However, pravastatin, a substrate of Mrp2, did not inhibit the PSP permeation. The jejunal secretory transport of pravastatin was significantly reduced in an ATP-depleted condition and by addition of probenecid, but PSP did not affect the jejunal permeation of pravastatin. These results suggest that PSP is secreted into the intestinal lumen by Mrp2-like transporter and that two Mrp2 substrates, PSP and pravastatin, are likely to be transported by different transport systems at the mucosal membrane.
Glucocorticoid receptor, encoded by NR3C1, is a transcriptional regulator of many drug metabolizing enzymes and anti-inflammatory molecules. In order to identify genetic variations of the NR3C1 gene, genomic DNA from 265 Japanese individuals was sequenced. Fifty genetic polymorphisms were identified, including 32 novel ones [3 were in coding exons, 17 in the introns, 4 in the 5′-untranslated region (UTR), and 8 in the 5′-flanking region]. The novel nonsynonymous variation was 420G>T (Lys140Asn), and the allele frequency was 0.004. We did not detect any nonsynonymous polymorphism reported previously in other races, including a relatively frequent SNP Asn363Ser found in Caucasians and African-Americans. Thus, ethnic differences between Japanese and other races are suggested to exist in NR3C1.
Thirteen single nucleotide polymorphisms (SNPs), including 6 novel ones, were found in exon 1 and its flanking region of UDP-glucuronosyltransferase (UGT) 1A6 from 195 Japanese subjects. Several novel SNPs were identified, including 269G>A (R90H), 279A>G (S93S), and 308C>A (S103X) in exon 1, and IVS1+109C>T, IVS1+120A>G, and IVS1+142C>T in the intron downstream of exon 1. Among these SNPs, 308C>A confers termination of translation at codon 103, resulting in the production of an immature protein that probably lacks enzymatic activity. The allele frequencies were 0.003 for all the 6 SNPs. In addition, the 3 known nonsynonymous SNPs were detected: 19T>G (S7A), 541A>G (T181A), and 552A>C (R184S) with frequencies of 0.226, 0.218, and 0.226, respectively. High linkage disequilibrium was observed among 19T>G (S7A), 315A>G (L105L), 541A>G (T181A), 552A>C (R184S), and IVS1+130G>T, as reported in Caucasian and African-American populations. Then, 11 haplotypes in UGT1A6 were estimated. The novel nonsynonymous variant, 269A or 308A, was shown to be located on the same DNA strand together with 19G, 315G, 541G, 552C, and IVS1+130T. Our results provide fundamental and useful information for genotyping UGT1A6 in the Japanese, and probably Asian populations.