Metabolic capacities for debrisoquin, sparteine, mephenytoin, nifedipine, and midazolam, which are substrates of polymorphic CYP2D6, CYP2C19, and CYP3A, have been reported to exhibit, in many cases, remarkable interindividual and ethnic differences. These ethnic differences are partly associated with genetic differences. In the case of the drug transporter ABCB1/MDR1, interindividual differences in its transporter activities toward various clinical drugs are also attributed to several ABCB1/MDR1 genetic polymorphisms. In this review, the existence and frequency of various low-activity alleles of drug metabolizing enzymes as well as populational drug metabolic capacities are compared among several different races or ethnicities. Distribution of nonsynonymous ABCB1/MDR1 SNPs and haplotype frequency in various races are summarized, with the association of nonsynonymous SNPs with large functional alterations as a rare event.
We evaluated hepatic CYP3A activity during development in male and female rats and the effect of neonatal exposure of 17β-estradiol and tamoxifen. In untreated and olive oil-treated (control) rats, hepatic CYP3A activities evaluated by erythromycin metabolism in vitro increased several-fold from age 2 to 9 weeks in males. In contrast, activity in females remained at a low and constant level from 2 to 15 weeks. Exposure of 17β-estradiol to neonates at a dose of 10 μmol/kg daily for 3 days on day 1~3 or 4~6 after birth significantly increased hepatic CYP3A activity during the developmental period in both males and females, and a greater influence was observed in females exposed during days 4~6. Pubertal exposure of 17β-estradiol (7-weeks old, 10 μmol/kg daily for 3 days) also increased hepatic CYP3A activity, but only in females. Neonatal exposure to tamoxifen (10 μmol/kg daily for 3 days) showed no appreciable effect in either males or females. In conclusion, a marked sex-difference was observed in hepatic CYP3A activity, and exposure of 17β-estradiol to neonates increased hepatic CYP3A activity during the developmental period, especially in female rats.
In an assay system using a human CYP3A4 reporter constructed with the promoter (+11 nt to -362 nt) and enhancer (-7.2 knt to -7.8 knt) regions including everted repeat separated by six nucleotides (ER-6) and direct repeat separated by three nucleotides (DR-3) motifs, the CYP3A4 transactivation was detected without overexpression of any nuclear receptors in rifampicin-treated HepG2 cells. Overexpression of human pregnane X receptor (hPXR) enhanced the transactivation. Rat CYP3A1 reporter constructed with the promoter region (+31 nt to -171 nt) including both DR-3 and ER-6 motifs was, however, not transactivated in rifampicin-treated cells, even after overexpression of hPXR. Although overexpression of retinoid X receptor alpha (RXRα) had no clear effect for both CYP3A reporters, co-expression of apolipoprotein AI regulatory protein-1 (ARP-1) with hPXR resulted in the rifampicin-induced transactivation of the CYP3A1 reporter. A truncated CYP3A4 reporter retaining the both motifs showed the rifampicin-induced transactivation by overexpression of hPXR and ARP-1, while the transactivation in hPXR-overexpressed cells was not observed. These results support the idea that a nuclear receptor other than RXRα may play a role in the CYP3A transactivation together with hPXR. The present study also suggests the involvement of a novel cis-element in the hPXR-mediated CYP3A4 transactivation.
Human CYP4B1 is a CYP4 enzyme with activity towards xenobiotics. Five alleles of human CYP4B1 have been identified in French Caucasians, but allelic variants of enzyme have not been determined in the Japanese population. To establish a rapid and sensitive means of detecting variant CYP4B1 alleles, we analyzed those of 192 Japanese individuals using denaturing HPLC (DHPLC). We then determined the optimal conditions required to detect SNPs for each PCR fragment. Analysis by DHPLC revealed the novel alleles, CYP4B1*6 (517C>T and 1033G>A) and CYP4B1*7 (AT881-882-del, 993G>A, and 1018C>T), as well as 3 known alleles. The frequencies of the CYP4B1*1, *2, *3, *4, *5, *6, and *7 alleles in 192 Japanese individuals were 0.490, 0.328, 0.154, 0, 0.016, 0.008, and 0.005, respectively. The allele frequencies among Japanese relative to those in French Caucasians for CYP4B1*1 (0.490 vs. 0.724) and CYP4B1*2 (0.328 vs. 0.147) significantly differed. Our results suggest that high throughput DHPLC can rapidly detect pharmacologically important variants in CYP genes.
The purpose of this study was to evaluate a contribution of polymorphic cytochrome P450 (CYP) 3A5 to the oxidation of diltiazem, midazolam and testosterone by liver microsomes from Japanese subjects. Twenty-seven liver samples were classified into three groups according to the CYP3A5 genotypes; CYP3A5*1/*1 (n=3), *1/*3 (n=12) and *3/*3 (n=12). The results of genotyping and immunochemical quantitation of CYP3A5 protein showed a good accordance between the CYP3A5 genotype and CYP3A5 content but not CYP3A4 content in liver microsomes. The expression levels of hepatic CYP3A5 protein ranged from 20 to 60% of the sum of CYP3A4 and CYP3A5 contents in subjects with at least one wild type allele (*1). The CYP3A5 contents correlated well with liver microsomal activities of diltiazem N-demethylation, midazolam 1′- and 4-hydroxylations and testosterone 6β-hydroxylation among subjects carrying at least one *1 allele. In addition, the correlation coefficients of CYP3A5 contents with the rates of diltiazem N-demethylation, midazolam 1′-hydroxylation and testosterone 6β- hydroxylation were higher than those of CYP3A4, although the value of CYP3A5 with the midazolam 4-hydroxylation rate was similar to that of CYP3A4. Kinetic analyses revealed a biphasic diltiazem N-demethylation in liver microsomes from subjects carrying the *1 allele. The apparent Vmax/Km values for recombinant CYP3A5 indicated the greater contributions to diltiazem N-demethylation and midazolam 1′-hydroxylation as compared with CYP3A4. These results suggest that polymorphic CYP3A5 contributes markedly to the drug oxidations, particularly diltiazem N-demethylation, midazolam 1′- hydroxylation and testosterone 6β-hydroxylation by liver microsomes from Japanese subjects.
Presystemic intestinal metabolism reduces the intestinal absorption and bioavailability of orally administered drugs. The factors affecting glucuronidation activity in Caco-2 cells seeded in Transwell (4.7 cm2) require clarification to establish an in-vitro system to assess intestinal glucuronidation metabolism for novel drug development. α-Naphthol (α-NA), a substrate for UGT1A6 in Caco-2 cells, has often been used as a model substrate for gluruonidation. α-Naphthol glucuronidation activity increased from 7 to 21 culture days after seeding in Transwell and stabilized after 21 days. The higher the passage number of Caco-2 cells, the larger the variance of glucuronidation activity, but apical pH did not significantly influence glucuronidation in the pH range of 5.5 to 7.4. When the passage number ranged from 83 to 159, Km,app was highest at passage number 130. In contrast, Vmax,app increased with the passage number. This indicates that the kinetic parameters for glucuronidation in Caco-2 cells are dependent on the passage number of the cells. These results should be useful for establishing the experimental conditions for Caco-2 cells that predict intestinal glucuronidation activity in vivo.
Pairs of forward and reverse primers and TaqMan probes specific to each human nuclear receptor were prepared. Analysis of the mRNA expression level of each target of 43 nuclear receptors in total RNA from single and pooled specimens of various human organs (liver, kidney, adrenal gland, lung, heart, brain, cerebellum, skeletal muscle, spleen, thymus, thyroid gland, prostate, testis, uterus, placenta, bone marrow, trachea, and salivary gland) was performed by real-time reverse transcription PCR using an ABI PRISM 7700 sequence detector system. The mRNA expression of 33 nuclear receptors (NR1A1, 1A2, 1B1, 1B2, 1B3, 1C1, 1C2, 1C3, 1D1, 1D2, 1F1, 1F2, 1F3, 1H2, 1H3, 1I1, 1I2, 2B1, 2B2, 2B3, 2C1, 2C2, 2F1, 2F2, 3A2, 3B1, 3C1, 3C2, 3C4, 4A1, 4A2, 4A3, and 6A1) was successfully detected in all of the tissues by this method. NR1H4, 2A1, and 3C3 mRNAs were not detectable in the heart, heart, and liver, respectively. NR5A2 mRNA was not detectable in either the brain or cerebellum. NR3A1 mRNA was not detectable in the small intestine, colon, brain, and cerebellum. NR5A1 mRNA was not detectable in the kidney, stomach, small intestine, and colon. NR1I3 mRNA was detected in the liver, kidney, stomach, small intestine, adrenal gland, lung, brain, skeletal muscle, thymus, thyroid gland, prostate, testis, placenta, and trachea. NR2A2 mRNA was detected in the liver, kidney, prostate, testis, uterus, and trachea. NR2E1 mRNA was detected in the adrenal gland, brain, cerebellum, testis, placenta, and bone marrow. NR2E3 mRNA was detected in the adrenal gland, thyroid gland, prostate, testis, uterus, trachea, and salivary gland. This study provides information concerning the tissue distribution of the mRNA expression of 43 human nuclear receptors. The mRNA expression profiles of CYP3A4, CYP3A5 and ABC-transporters are also shown. These results are valuable for establishing a nuclear receptor-mediated screening system for new chemical entities in new drug development.
Long-Evans Cinnamon (LEC) rats have an abnormality similar to that observed in Wilson's disease in humans and are therefore a good animal model for the study of Wilson's disease. LEC rats develop hereditary hepatitis and severe jaundice. Mutant animals with hyperbilirubinemia have been widely used as animal models for human diseases. Among these mutant animals, Eisai hyperbilirubinemic rats (EHBR) have defective biliary excretion of organic anions. Thus, biliary excretion of sulfobromophthalein (BSP) and urinary excretion of phenolsulfonphthalein (PSP) in LEC rats were compared with those in Long-Evans Agouti (LEA) rats. In LEC rats, the excretion of BSP, a multidrug resistance-associated protein 2 (Mrp2/Abcc2) substrate, was significantly decreased compared to that in LEA rats. It has been reported that the transport function for organic anions on the kidney is maintained in EHBR. However, the urinary excretion of PSP is impaired in LEC rats. It is possible that organic anion transporters responsible for the urinary excretion of PSP in LEA rats and EHBR are impaired in LEC rats. It is important to elucidate the relationship between organic anion secretion and Wilson's disease.
We sequenced all exons and exon-intron junctions of the CYP2B6 gene from 200 Japanese individuals. We found three novel single nucleotide polymorphisms (SNPs) (1375A>G, 1427G>A and 1454A>T) causing amino acid substitutions (Met459Val, Gly476Asp and Gln485Leu in exon 9), respectively. The detected SNP was as follows: 1) SNP, 031226Hiratsuka01; GENE NAME, CYP2B6; ACCESSION NUMBER, AC023172; LENGTH, 25 base; 5′-CAGAACTTCTCCA/GTGGCCAGCCCCG-3′. 2) SNP, 031226Hiratsuka02; GENE NAME, CYP2B6; ACCESSION NUMBER, AC023172; LENGTH, 25 base; 5′-CCCAGGAGTGTGG/ATGTGGGCAAAAT-3′. 3) SNP, 031226Hiratsuka03; GENE NAME, CYP2B6; ACCESSION NUMBER, AC023172; LENGTH, 25 base; 5′-CCCCAACATACCA/TGATCCGCTTCCT-3′.