Masanori Horinouchi

MDR1 genotype-related pharmacokinetics of digoxin after single oral administration in healthy Japanese subjects.

AbstractPurpose. To evaluate the MDR1 genotype frequency in the Japanese population and to study the relationship between the MDR1 genotype and the pharmacokinetics of digoxin after single oral administration in healthy subjects. Methods. The MDR1 genotype at exon 26 was determined in 114 healthy volunteers by polymerase chain reaction-restriction fragment length polymorphism. The serum concentration-time profile of digoxin was examined after single oral administration at a dose of 0.25 mg. Results. It was found that 35.1 % (40/114) of subjects were homozygous for the wild-type allele (C/C), 52.6 % (60/114) were compound heterozygotes with a mutant T-allele (C3435T) (C/T), and 12.3 % (14/114) were homozygous for the mutant allele (T/T). There was no effect of gender or age on the distribution. The serum concentration of digoxin after a single oral administration increased rapidly, attaining a steady state in all subjects; however, it was lower in the subjects harboring the T-allele. AUC0-4 h values (±SD) were 4.11 ± 0.57, 3.20 ± 0.49, and 3.27± 0.58 ng h/ml, respectively, with a significant difference between C/C and C/T or T/T. Conclusions. The serum concentration of digoxin after single oral administration was lower in the subjects harboring a mutant allele (C3435T) at exon 26 of the MDR1 gene.

Effect of the mutation (C3435T) at exon 26 of the MDR1 gene on expression level of MDR1 messenger ribonucleic acid in duodenal enterocytes of healthy Japanese subjects.

The effect of the C3435T mutation at exon 26 of the MDR1 gene on the expression levels of MDR1 messenger ribonucleic acid (mRNA) was evaluated by means of real‐time polymerase chain reaction in 51 biopsy specimens of duodenum obtained from 13 healthy Japanese subjects. The mRNA levels of MDR1 were 0.38 ± 0.15, 0.56 ± 0.14, and 1.13 ± 0.42 (mean value ± SE) in the subjects with the homozygote of wild‐type allele (C/C), compound heterozygote with mutant T allele (C/T), and the homozygote of the mutant allele (T/T), respectively, reasonably explaining the lower digoxin serum concentration after administration of a single oral dose to subjects harboring a mutant T allele. Good correlation (r = .797; P < .01) was observed between the mRNA concentrations of MDR1 and CYP3A4 in the individual biopsy specimens. This finding suggested a lower plasma concentration of the substrates for CYP3A4 in subjects harboring the C3435T mutation of the MDR1 gene.

Significant genetic linkage of MDR1 polymorphisms at positions 3435 and 2677: Functional relevance to pharmacokinetics of digoxin

The human MDR1 gene encodes MDR1, also called Pglycoprotein (P-gp), which functions in the energy-dependent export of substances from the inside of cells to the outside. MDR1 was originally isolated from resistant tumor cells as the protein responsible for conferring resistance against antitumor agents (1). Human MDR1 is also expressed in normal tissues including the mucosal cells in the small and large intestine, the epithelial cells of renal proximal tubules, the biliary canalicular side of hepatocytes, capillary endothelial cells of the brain and testis, and cells of the leukocyte lineage (2). MDR1 contributes to the limitation of drug absorption from the gastrointestinal tract, secretion of the drugs into bile and urine, and the prevention of penetration of drugs across the blood-brain barrier. Thus, MDR1 in these normal tissues defines the pharmacokinetics of many drugs, which are substrates for MDR1. In 1989, nine nucleotide differences on the human MDR1 gene were found from drug-selected multidrugresistant cultured cells (3). To date, more than 20 single nucleotide polymorphisms (SNPs) in the exonic regions have been identified (4–7). Among them, the mutation in exon 26, position 3435 (C3435T), neighboring on the ATP binding domain, has been focused due to its suppressive effect on the expression of MDR1 protein in duodenal biopsies and resultant increase of plasma concentration of digoxin under rifampin induction or at steady-state in Caucasian subjects, although this is a silent mutation (5). C3435T mutation was shown to decrease efflux of the MDR1 substrate rhodamine from CD56 natural killer cells and lower MDR1 mRNA expression in leukocytes (8). However, recent investigations have suggested that C3435T has no effect on placental MDR1 expression (7) and moreover that C3435T mutation is related to a higher level of MDR1 mRNA expression in duodenal biopsies in healthy Japanese subjects (9). The C3435T mutation has been reported to have no effect on the plasma concentration of digoxin (10) and to be lower in subjects with T/T genotype (11). As for other MDR1 substrates, the C3435T mutation has been reported to have no effect on the cyclosporin A trough concentration (12) and to be lower plasma concentration of fexofenadine (13). These investigations suggested the importance of MDR1 genotyping, especially for C3435T. However, the molecular mechanisms underlying the effects of this polymorphism remain unclear, and further investigations should be addressed to elucidate these discrepancies. The C3435T SNP has been suggested to be linked with the SNP at exon 21, position 2677 (G2677(A,T)) producing Ala893Thr and Ala893Ser, respectively (7,13), and haplotype analysis might provide a rational explanation for these discrepancies. This study was, therefore, designed to elucidate the linkage of SNPs at positions 3435, 2677, and -129 in 117 healthy Japanese subjects. Position 2677 locates in the intracellular domain between the 10th and 11th transmembrane spanning domains. Position –129 is in the promoter region, exon 1b. In addition, the effects of SNPs at positions 3435 and 2677 on the serum concentration-time profiles of digoxin after single oral administration were examined in healthy Japanese subjects.

MDR1 genotype-related duodenal absorption rate of digoxin in healthy Japanese subjects.

AbstractPurpose. Recent clinical studies suggest the importance of the MDR1 genotype at position 3435 (C3435T) in terms of pharmacokinetics, but there is still no consensus in reports on the relationship between the genotype and plasma/serum concentration-time profiles of drugs after conventional oral administration. This study was performed to elucidate the effects of C3435T on the rate of duodenal absorption of digoxin in healthy Japanese subjects. Methods. Digoxin solution was sprinkled directly over the surface of the duodenum using an endoscope, and its absorption rate was evaluated by serial monitoring of the serum concentration and by analysis of its initial 15-min increasing phase. Results. The duodenal absorption rates of digoxin were 911 ± 91 ng/min and 506 ± 76 ng/min for C/C and T/T, respectively (±SE, p = 0.007). Conclusions. The C3435T mutation of the MDR1 gene is associated with suppression of duodenal absorption of digoxin.

Effects of 12 Ca2+ antagonists on multidrug resistance, MDR1-mediated transport and MDR1 mRNA expression

The effects of 12 Ca(2+) antagonists on MDR1 were examined by two independent models: the inhibitory effect on MDR1-mediated transport of [(3)H]digoxin using MDR1-overexpressing LLC-GA5-COL150 cell monolayers and the reversal effect on cytotoxicity of vinblastine or paclitaxel using MDR1-overexpressing Hvr100-6 cells. The inhibitory effects on [(3)H]digoxin transport were assessed as the 50% inhibitory concentration during 4 h exposure, and the values were the lowest for nicardipine (4.54 microM), manidipine (4.65 microM) and benidipine (4.96 microM), followed by bepridil (10.6 microM), barnidipine (12.6 microM), efonidipine (13.0 microM), verapamil (13.2 microM) and nilvadipine (18.0 microM). The reversal effect on cytotoxicity was assessed by the 50% growth inhibitory concentration after 3 days exposure, and the resistance to vinblastine or paclitaxel in Hvr100-6 cells was reversed by manidipine, verapamil, benidipine, barnidipine, and nicardipine, in that order. Bepridil, barnidipine, efonidipine, verapamil and nilvadipine showed similar inhibitory effects on [(3)H]digoxin transport, but barnidipine and verapamil showed a stronger effect in reversal of cytotoxicity. Real-time quantitative RT-PCR assay indicated a decrease in MDR1 mRNA expression by barnidipine and verapamil. It is concluded that Ca(2+) antagonists cannot only be direct inhibitors of MDR1 but that some may at the same time act as inhibitors of expression of MDR1 via down-regulation of MDR1 mRNA.

Haloperidol is an inhibitor but not substrate for MDR1/P-glycoprotein

The involvement of the multidrug resistant transporter MDR1/P‐glycoprotein in the penetration of haloperidol into the brain and absorption in the intestine was investigated to examine its role in inter/intra‐individual variability, using the porcine kidney epithelial cell line LLC‐PK1 and its MDR1‐overexpressing transfectant, LLC‐GA5‐COL150. The inhibitory effect of haloperidol on other MDR1 substrates was also investigated in terms of the optimization of haloperidol‐based pharmacotherapy. The transepithelial transport of [3H]haloperidol did not differ between the two cell lines, and vinblastine, a typical MDR1 substrate, had no effect on the transport, suggesting that haloperidol is not a substrate for MDR1, and it is unlikely that MDR function affects haloperidol absorption and brain distribution, and thereby the response to haloperidol. However, haloperidol was found to have an inhibitory effect on the MDR1‐mediated transport of [3H]digoxin and [3H]vinblastine with an IC50 value of 7.84 ± 0.76 and 3.60 ± 0.64 μM, respectively, suggesting that the intestinal absorption, not distribution into the brain, of MDR1 substrate drugs could be altered by the co‐administration of haloperidol in the clinical setting, although further clinical studies are needed.