A variant 2677A allele of the MDR1 gene affects fexofenadine disposition

BACKGROUND AND OBJECTIVES: There have been considerable disagreements regarding the influence of MDR1 (ABCB1) polymorphisms on the disposition of P-glycoprotein (P-gp) substrates. We speculated that the unknown function of the A allele of exon 21 G2677T/A (Ala893Ser/Thr) provides one of the reasons for the contradictory results. This study was performed to clarify the effects of major MDR1 gene polymorphisms, including a variant A allele in exon 21, on fexofenadine pharmacokinetics. METHODS: We investigated the occurrence of 3 high-frequency single-nucleotide polymorphisms (SNPs) in exons 12 (C1236T), 21 (G2677T/A), and 26 (C3435T) of the MDR1 gene in 232 healthy Koreans, using a polymerase chain reaction-restriction fragment length polymorphism method, and performed haplotype analysis on these 3 SNPs. A single oral dose of 180 mg fexofenadine hydrochloride was administered to 33 healthy Korean male volunteers, who were divided into 6 groups based on the MDR1 genotype for the G2677T/A polymorphism in exon 21 and the C3435T polymorphism in exon 26. RESULTS: A strong linkage disequilibrium was observed among the 3 SNPs. The frequencies of the 4 major haplotypes, 1236C-2677A-3435C, C-G-C, T-G-C, and T-T-T, were 16.4%, 18.6%, 21.6%, and 32.2%, respectively. Fexofenadine disposition varied considerably among the groups. In the 2677AA/3435CC genotype group (n=3), the values of area under the concentration-time curve from time 0 to 24 hours [AUC(0-24)] were significantly lower (P=.014) than those of the other 5 genotype groups (GG/CC, GT/CT, TT/TT, GA/CC, and TA/CT). As compared with the 2677GG/3435CC subjects, the AUC(0-24) values were 17% lower in the 2677AA/3435CC subjects and 47% higher in the 2677TT/3435TT subjects (GG/CC versus AA/CC versus TT/TT, 4017 +/- 1137 ng . h/mL versus 3315 +/- 958 ng . h/mL versus 5934 +/- 2,064 ng . h/mL; P=.018). By stratification for genotypes at position 3435, homozygous 3435TT subjects were found to have significantly higher AUC(0-24) (P=.024) and maximum plasma concentration (P=.040) values than CC subjects [AUC(0-24), 5934 +/- 2064 ng . h/mL versus 3998 +/- 1241 ng . h/mL; maximum plasma concentration, 958 +/- 408 ng/mL versus 673 +/- 242 ng/mL]. CONCLUSIONS: The plasma concentrations of fexofenadine after a single oral administration were lower in 2677AA/3435CC subjects than in subjects with the other 5 genotype combinations of the SNPs of G2677T/A and C3435T. These findings confirm the importance of analyzing MDR1 haplotypes and provide a plausible explanation for the conflicting results regarding the effect of MDR1 polymorphisms on the disposition of P-gp substrates.     

Role of human MDR1 gene polymorphism in bioavailability and interaction of digoxin,a substrate of P-glycoprotein

OBJECTIVE: Our objective was to quantitate the contribution of the genetic polymorphism of the human MDR1 gene to the bioavailability and interaction profiles of digoxin, a substrate of P-glycoprotein. METHODS: The pharmacokinetics of digoxin was studied in 15 healthy volunteers, who were divided into 3 groups (n = 5 each) on the basis of genotyping for the MDR1 gene, in a 4-dose study after single doses of digoxin alone (0.5 mg orally and intravenously) and coadministered with clarithromycin (400 mg orally for 8 days). The dose of digoxin was reduced during the clarithromycin phase (0.25 mg orally and intravenously). RESULTS: The bioavailability of digoxin in G/G2677C/C3435, G/T2677C/T3435, and T/T2677T/T3435 subjects were 67.6% +/- 4.3%, 80.9% +/- 8.9%, and 87.1% +/- 8.4%, respectively, and the difference between G/G2677C/C3435 and T/T2677T/T3435 subjects was statistically significant (P <.05). The MDR1 variants were also associated with differences in disposition kinetics of digoxin, with the renal clearance being almost 32% lower in T/T2677T/T3435 subjects (1.9 +/- 0.1 mL/min per kilogram) than G/G2677C/C3435 subjects (2.8 +/- 0.3 mL/min per kilogram), and G/T2677C/T3435 subjects having an intermediate value (2.1 +/- 0.6 mL/min per kilogram). Coadministration of clarithromycin did not consistently affect digoxin clearance or renal clearance. However, a significant increase in digoxin bioavailability was observed in G/G2677C/C3435 subjects (67.6% +/- 4.3% versus 85.4% +/- 6.1%; P <.05) but not in the other 2 genotype groups. CONCLUSION: The allelic variants in the human MDR1 gene are likely to be associated with altered absorption and/or disposition profiles of digoxin and P-glycoprotein-mediated drug interaction     

Influence of functional polymorphisms of the MDR1 gene on vincristine pharmacokinetics in childhood acute lymphoblastic leukemia

OBJECTIVE: Our objective was to investigate the effect of single nucleotide polymorphisms (SNPs) in the P-glycoprotein MDR1 gene on vincristine pharmacokinetics and side effects in childhood acute lymphoblastic leukemia. METHODS: From 52 of 70 children who participated in a previous study on vincristine pharmacokinetics, patient material was available for investigation of the MDR1 genetic variants. The SNPs C3435T and G2677T were determined by use of polymerase chain reaction-restriction fragment length polymorphism. Vincristine side effects were scored retrospectively from patient records. RESULTS: No association was observed between C3435T or G2677T and vincristine pharmacokinetic variables. When haplotypes were assigned, haplotype 1/1 carriers (3435C/2677G) showed a longer elimination half-life than noncarriers (1156 versus 805 minutes, P =.038). In contrast, haplotype 1/2 carriers (3435T/2677G) had a shorter elimination half-life than noncarriers (805 versus 1180 minutes, P =.044). However, this significance was lost after Bonferroni correction for multiple testing. The haplotypes did not affect the other pharmacokinetic parameters, such as clearance and area under the concentration-time curve, suggesting that the observed effect on elimination half-life is of very limited relevance. Moreover, SNPs in the MDR1 gene did not identify patients with an increased risk for vincristine-induced constipation. CONCLUSION: The genetic variants in the MDR1 gene alone cannot explain the large variability in vincristine pharmacokinetics.     

Detection of the four sequence variations of MDR1 gene using TaqMan MGB probe based real-time PCR and haplotype analysis in healthy Japanese subjects

OBJECTIVES: P-glycoprotein (P-gp) is significant from the viewpoint of pharmacokinetics/pharmacodynamics (PK/PD). MDR1 gene encodes P-gp and has a wide variety of SNPs. As the SNPs may be one of the factors that induce pharmacogenetic individual difference, haplotype analysis is necessary to evaluate the PK/PD. DESIGN AND METHODS: The SNPs of the detected MDR1 were -129T>C, 325G>A, 2677G>T/A, and 3435C>T. For the analysis of linkage disequilibrium (LD) and haplotype analysis, and for the reconstruction of the haplotype pair, ARLEQUIN and PHASE were employed. RESULTS: The result of the chi(2) test detected significant LD between -129 and 2677, -129 and 3435, and 2677 and 3435. There were 9 haplotypes: T-G-C, T-T-C, C-T-C, T-A-C, C-A-C, T-G-T, T-T-T, C-G-T, and C-T-T. CONCLUSIONS: LD was found among the positions -129, 2677 and 3435. As a result, 9 haplotypes exists in the Japanese population. These results suggest that it would be necessary to give consideration to haplotype for the purpose of evaluating the PK/PD of the drugs transported by P-gp.     

Modulation of steady-state kinetics of digoxin by haplotypes of the P-glycoprotein MDR1 gene

OBJECTIVE: We investigated the effect of polymorphisms in the P-glycoprotein (P-gp) MDR1 gene on steady-state pharmacokinetics of digoxin in Caucasians. According to earlier data, homozygous TT of the exon 26 complementary deoxyribonucleic acid (cDNA) 3435C>T polymorphism was associated with low P-gp expression in the human intestine. METHODS: Eight healthy male homozygous carriers of the wild-type exon-26 3435C>T (CC), 8 heterozygous subjects (CT), and 8 homozygous mutant (TT) subjects were selected. Seven further MDR1 polymorphisms were determined. Digoxin was administered orally twice daily on the first two study days; on days 3 to 5, 0.25 mg was given in the morning. On day 5, kinetic parameters were analyzed for genotype-phenotype and haplotype-phenotype relationships. RESULTS: The area under the plasma concentration-time curve from time zero to 4 hours [AUC(0-4)] (P =.042) and C(max) (P =.043) values of digoxin were higher in subjects with the 3435TT genotype than in those with the 3435CC. No influence of other single nucleotide polymorphisms (SNPs) on digoxin parameters was detected. Comparison of genotypes deduced from SNPs 2677G>T (exon 21) and 3435C>T revealed significant differences for AUC(0-4) (P =.034) and C(max) (P =.039), which were substantiated by haplotype analysis. Haplotype 12 (2677G/3435T), which had a frequency of 13.3% in a randomly drawn Caucasian sample (n = 687), was associated (Mann-Whitney test) with higher AUC(0-4) values (P =.009) than were found in noncarriers (mean +/- SD, 5.7 +/- 0.9 microg. h/L [n = 7] versus 4.8 +/- 0.9 microg. h/L [n = 17]). Haplotype 11 (2677G/3435C) had lower AUC(0-4) values (P =.013) compared with those of noncarriers (mean +/- SD, 4.7 +/- 0.9 microg. h/L [n = 16] versus 5.6 +/- 0.9 microg. h/L [n = 8]). Results of haplotype analysis match data of other MDR1 studies. CONCLUSION: Haplotype 12 codes for high values of AUC(0-4) and C(max) of orally administered digoxin. Analysis of MDR1 haplotypes is superior to unphased SNP analysis to predict MDR1 phenotype.     

No effect of MDR1 C3435T variant on loperamide disposition and central nervous system effects

BACKGROUND: The MDR1 gene encodes the efflux transporter P-glycoprotein, which is highly expressed in the small intestine and in the blood-brain barrier. A major function of P-glycoprotein is to limit the absorption and central nervous system exposure of numerous xenobiotics. A genetic polymorphism in the MDR1 gene (C3435T) has been associated with changes in the intestinal expression level and function of P-glycoprotein. The aim of this study was to investigate the effect of this polymorphism on disposition and brain entry of the P-glycoprotein substrate loperamide. METHODS: Healthy white volunteers were genotyped for the MDR1 C3435T polymorphism, and a 16-mg oral dose of loperamide was administered to 8 subjects with the 3435TT genotype and 8 subjects with the 3435CC genotype. Plasma levels of loperamide were determined by liquid chromatography-tandem mass spectrometry. Loperamide-induced respiratory depression was detected as the ventilatory response to carbon dioxide and was used as a measure of central nervous system side effects. RESULTS: We found no significant difference in loperamide pharmacokinetics between individuals homozygous for the C and the T alleles in position 3435 of MDR1, as follows: peak plasma drug concentration, 3164 +/- 1053 pg/mL and 3021 +/- 984 pg/mL; area under the concentration-time curve from 0 to 8 hours, 14414 +/- 4756 pg. h/mL and 14923 +/- 6466 pg. h/mL; and time to peak plasma drug concentration, 3.9 +/- 1.4 hours and 3.9 +/- 2.6 hours for the MDR1 3435CC and 3435TT genotypes, respectively (P >.05, for all parameters). Hypercapnic ventilatory response changed only minimally after ingestion of loperamide (the coefficient of variation during the 0- to 8-hour period was 21% +/- 14% for the sample population), and there was no MDR1 3435 genotype-related effect on respiratory response. Carriers of the 2 major MDR1 haplotypes, MDR1*1 and MDR1*13, did not differ in their response to loperamide. CONCLUSION: There was no association between the MDR1 C3435T variation and plasma levels or central nervous system effects of the P-glycoprotein substrate loperamide in a white study population. The MDR1 haplotype structure was quite variable and supports the use of haplotypes instead of single nucleotide polymorphisms in determining clinical consequences of genetic variation.     

MDR1 gene polymorphisms are associated with neuropsychiatric adverse effects of mefloquine

BACKGROUND: Mefloquine, a drug used for treatment and prophylaxis of malaria, is known for its neuropsychiatric adverse effects. We hypothesized that neuropsychiatric adverse effects of mefloquine are associated with polymorphisms in the MDR1/ABCB1 gene that encodes for the efflux pump P-glycoprotein. METHODS: The association between MDR1 C1236T, G2677T, and C3435T single-nucleotide polymorphisms and the occurrence of neuropsychiatric adverse effects was examined in a prospective cohort study of 89 healthy white travelers taking mefloquine. RESULTS: Of the subjects, 27 (28%) reported neuropsychiatric adverse effects, women significantly more frequently than men. Allele frequencies of the C1236T, G2677T, and C3435T polymorphisms were similar to those found in other white populations, and there was no significant association between any of the individual polymorphisms and neuropsychiatric adverse effects. However, women with the 1236TT, 2677TT, and 3435TT genotypes had a higher risk of neuropsychiatric adverse effects than the reference groups of women with heterozygous and homozygous CC or GG genotypes, with odds ratios of 6.3 (95% confidence interval [CI], 1.1-36.9), 10.5 (95% CI, 1.1-100.6), and 5.4 (95% CI, 1.1-30.0), respectively. The association for women homozygous for the 1236-2677-3435 TTT haplotype was even stronger (P = .004) than the effect of any of the individual polymorphisms. No associations with mefloquine blood levels were observed. CONCLUSION: In this study the MDR1 1236TT, 2677TT, and 3435TT genotypes, along with the 1236-2677-3435 TTT haplotype, were associated with neuropsychiatric adverse effects of mefloquine in women. MDR1 polymorphisms may play an important role in predicting the occurrence of neuropsychiatric adverse effects of mefloquine, particularly in female travelers.     

The effects of the human MDR1 genotype on the expression of duodenal P-glycoprotein and disposition of the probe drug talinolol

BACKGROUND AND OBJECTIVES: A single-nucleotide polymorphism (SNP) of the human multidrug-resistance gene in wobble position of exon 26 reportedly predicts expression and function of P-glycoprotein in human enterocytes and lymphocytes. Several other allelic variants of MDR1 have been identified, some of which lead to amino acid exchange with as yet unknown functional relevance. METHODS: In healthy white volunteers, we investigated the influence of the hereditary variants C3435T in exon 26 and G2677T/A (Ala893Ser/Thr) in exon 21 and the influence of 7 frequent or putative functional SNPs on duodenal MDR1 messenger ribonucleic acid (n = 32) and immunoreactive P-glycoprotein (n = 37) expression. Moreover, the disposition of the probe drug talinolol was evaluated in 55 subjects after oral administration (100 mg) and in 23 subjects after intravenous administration(30 mg). RESULTS: Duodenal MDR1 messenger ribonucleic acid and P-glycoprotein, as assessed by real-time polymerase chain reaction (TaqMan) and immunostaining, were not influenced by any MDR1 polymorphism studied. Talinolol disposition was not affected by the exon 26 mutation C3435T. In carriers of the TT/TA variants of G2677T/A, the area under the serum concentration-time curve values of oral talinolol were slightly but significantly elevated compared with those in carriers of at least 1 wild-type allele (P <.05, Kruskal-Wallis test; P =.014, Mann-Whitney U test). However, multiple comparisons with combinations of putative functional SNPs did not confirm a significant influence of the MDR1 genotype on talinolol disposition. CONCLUSIONS: We did not identify any influence of MDR1 genotypes on duodenal expression of P-glycoprotein and disposition of talinolol in humans.     

The single nucleotide polymorphism and haplotype analysis of MDR1 in Jiangsu Han population of China

The aim of this study was to perform the frequency distribution of MDR1 gene SNPs and haplotypes of Jiangsu Han population in China. A total of 225 Jiangsu Han unrelated volunteers were enrolled and genotyped by PCR-ASP method at three loci: C1236 T (rs1128503), G2677 T/A (rs2032582) and C3435 T (rs1045642). In total, C and T were found at locus 1236, with the frequency of 35% and 65%, respectively. The most frequent allele at locus 2677 was G with the frequency of 44%, followed by T (41%) and A (15%). At locus 3435, C was more common (60%) than T (40%). The most common haplotype at loci 1236-2677-3435 was T-T-T (31.84%), at loci 1236-2677 was T-T (37.68%), at loci 2677-3435 was G-C (39.06%), and at loci 1236-3435 was T-T (34.28%). The haplotype linkage disequilibrium study found that all three loci were in linkage disequilibrium, such as T-T at loci 1236-2677, T-T at loci 2677-3435 and C-C at loci 1236-3435 (P < 0.01). The dendrogram study indicated that the distribution of MDR1 SNPs in Jiangsu Han population were close to Japan and Malay populations and far away from European countries. These findings could shade new lights in population genetics and anthropology studies of Han-Chinese. It also provides basic data for research on MDR1 gene polymorphism, disease association and drug resistance study.     

Influence of functional haplotypes in the drug transporter gene ABCB1 on central nervous system drug distribution in humans

BACKGROUND AND OBJECTIVE: Single nucleotide polymorphisms in the human multidrug-resistance gene ABCB1 have been reported to be associated with altered expression and function of P-glycoprotein, an efflux transporter, expressed at the blood-brain barrier. To test whether certain ABCB1 haplotypes contribute to interindividual differences in central nervous system drug distribution, brain distribution of a model P-glycoprotein substrate, the calcium channel inhibitor verapamil, was measured by positron emission tomography (PET) in 2 groups of healthy volunteers. METHODS: Ten homozygous carriers (cases) of the TTT haplotype (3435T, 1236T, and 2677T) and 10 controls homozygous for the wild-type CGC haplotype (3435C, 2677G, and 1236C) were administered a mean intravenous bolus of 412 +/- 114 MBq carbon 11-labeled verapamil containing less than 15 nmol of unlabeled verapamil. PET imaging of brain tissue and venous blood sampling were performed for 1 hour after dosing. RESULTS: As a measure of brain penetration, the ratio of PET area under the time-radioactivity curve (AUC) to plasma AUC was calculated from time-radioactivity curves, with a mean ratio of 1.1 +/- 0.3 (SD) (95% confidence interval, 0.9-1.3) for cases and 1.1 +/- 0.2 (95% confidence interval, 0.9-1.2) for controls, respectively (P = .96). Mean brain AUC values were 31.2 +/- 3.9 and 35.7 +/- 5.7 for the TTT and CGC haplotype, respectively (P = .11). Plasma AUCs were not significantly different. CONCLUSION: No difference in the brain distribution of [(11)C]verapamil could be detected in healthy volunteers differing in ABCB1 haplotypes.     

CYP3A5 and MDR1 genetic polymorphisms and cyclosporine pharmacokinetics after renal transplantation

BACKGROUND: The immunosuppressive drug cyclosporine (INN, ciclosporin), whose pharmacokinetic characteristics vary greatly among individuals, is a substrate for cytochrome P450 (CYP) 3A and P-glycoprotein, the product of the multidrug resistance 1 (MDR1) gene. Some of the single nucleotide polymorphisms (SNPs) in these genes are associated with deficient protein expression and reduced in vivo activity. We postulated that, in renal transplant recipients, these SNPs could be associated with interindividual variations in cyclosporine pharmacokinetics. PURPOSE: In 106 renal transplant patients, we evaluated retrospectively the effects of 4 MDR1 SNPs [T-129C, C1236T, G2677(T,A), and C3435T] and of the CYP3A5*1/*3 SNP on cyclosporine pharmacokinetic parameters and exposure indices. RESULTS: The CYP3A5*1 allele was present in 8.5% of patients. The MDR1 C1236T, G2677(T,A), and C3435T SNPs were frequent (17.9%, 18.9%, and 33%, respectively, for the variant homozygous genotype) and exhibited incomplete linkage disequilibrium. None of the cyclosporine pharmacokinetic parameters were associated with the CYP3A5 genetic polymorphism. Patients with the wild-type genotype in MDR1 C1236T SNP had slightly but significantly lower dose-adjusted peak drug concentrations (-16%) (P <.02) and dose-adjusted area under the concentration-time curve (AUC) values over the first 4 hours (-14%) (P <.05) as compared with mutated allele carriers. Haplotype analysis including MDR1 C1236T, G2677(T,A), and C3435T SNPs showed no significant association between haplotypes and cyclosporine pharmacokinetics or systemic exposure, although there was a nonsignificant trend toward higher dose-adjusted AUC values over the first 4 hours and AUC over the 12-hour administration interval for the T-T-T haplotype. CONCLUSION: The presence of the CYP3A5 SNP does not explain the high variability of cyclosporine pharmacokinetics in stable renal transplant patients. Despite the weak association found for the MDR1 C1236T SNP, MDR1 SNPs are unlikely to be useful for cyclosporine dose optimization in clinical practice.     

MDR1 genotype is associated with hepatic cytochrome P450 3A4 basal and induction phenotype

OBJECTIVES: Variant cytochrome P450 (CYP) 3A4 alleles cannot explain human variation in CYP3A4 expression. This study investigated whether common single-nucleotide polymorphisms (SNPs) in multidrug resistance 1 (MDR1), encoding P-glycoprotein, or the pregnane X receptor (PXR) were associated with basal or inducible CYP3A4 expression. METHODS: MDR1 G2677T and C3435T SNPs and a PXR 6-base pair (bp) deletion were genotyped in the deoxyribonucleic acid from 144 human livers in 3 cohorts each phenotyped for basal or rifampin (INN, rifampicin)-inducible hepatic CYP3A4 expression (or both) and in 57 human small bowel biopsy specimens from 3 cohorts each phenotyped for either basal or rifampin-induced CYP3A4 expression (or both). RESULTS: Hepatic CYP3A4 expression/function was significantly higher in persons homozygous for the MDR1 2677T (Ser893) allele compared with persons homozygous for 2677G (Ala893) in all 3 hepatic cohorts. For example, homozygous MDR1 2677 TT livers had higher midazolam hydroxylase activity than homozygous 2677 GG livers (1831 +/- 1336 pmol x min(-1) x mg protein(-1) versus 1060 +/- 552 pmol x min(-1) x mg protein(-1), P = .03). In 2 of the 3 groups the association was observed in men but not in women. For example, homozygous MDR1 2677 TT male hepatocytes had significantly higher testosterone 6beta-hydroxylase activity compared with homozygous 2677 GG livers (0.120 +/- 0.06 pmol x min(-1) x mg protein(-1) versus 0.069 +/- 0.04 pmol x min(-1) x mg protein(-1), P = .0002). Conversely, rifampin induction of testosterone 6beta-hydroxylation in primary human hepatocytes was significantly higher in persons homozygous for 2677G (12.0 +/- 5.7-fold) compared with MDR1 homozygous TT carriers (7.3 +/- 4.6-fold) (P = .01). Suggestive evidence for higher CYP3A4 expression in MDR1 2677T carriers was also observed in human intestines. CYP3A4 expression was also related to a 6-bp deletion in PXR in 2 of the liver cohorts. Two-factor ANOVA analysis revealed a significant interaction between the MDR1 2677 SNP and the PXR 6-bp deletion influencing CYP3A4 expression (P = .007). CONCLUSIONS: Individuals homozygous for the MDR1 2677T allele show enhanced constitutive CYP3A4 expression in the liver and intestine, as compared with those homozygous for the MDR1 2677G allele, particularly in men. Conversely, the magnitude of CYP3A4 induction by rifampin is greater in persons with the MDR1 2677G allele and is inversely related to baseline CYP3A4 expression. MDR1 likely influences basal CYP3A4 expression by limiting the intracellular concentration of an endogenous regulator. MDR1 genotype may be a useful predictor of basal CYP3A4 and the extent of some CYP3A4-mediated drug interactions in humans. Moreover, the influence of MDR1 genotype on CYP3A4 expression adds additional complexity to determining the relative contribution of the MDR1 alleles to the disposition of substrates shared by CYP3A4 and MDR1/P-glycoprotein.     

Identification of functionally variant MDR1 alleles among European Americans and African Americans

MDR1 (P-glycoprotein) is an important factor in the disposition of many drugs, and the involved processes often exhibit considerable interindividual variability that may be genetically determined. Single-strand conformational polymorphism analysis and direct sequencing of exonic MDR1 deoxyribonucleic acid from 37 healthy European American and 23 healthy African American subjects identified 10 single nucleotide polymorphisms (SNPs), including 6 nonsynonymous variants, occurring in various allelic combinations. Population frequencies of the 15 identified alleles varied according to racial background. Two synonymous SNPs (C1236T in exon 12 and C3435T in exon 26) and a nonsynonymous SNP (G2677T, Ala893Ser) in exon 21 were found to be linked (MDR1*2 ) and occurred in 62% of European Americans and 13% of African Americans. In vitro expression of MDR1 encoding Ala893 (MDR1*1 ) or a site-directed Ser893 mutation (MDR1*2 ) indicated enhanced efflux of digoxin by cells expressing the MDR1-Ser893 variant. In vivo functional relevance of this SNP was assessed with the known P-glycoprotein drug substrate fexofenadine as a probe of the transporter's activity. In humans, MDR1*1 and MDR1*2 variants were associated with differences in fexofenadine levels, consistent with the in vitro data, with the area under the plasma level-time curve being almost 40% greater in the *1/*1 genotype compared with the *2/*2 and the *1/*2 heterozygotes having an intermediate value, suggesting enhanced in vivo P-glycoprotein activity among subjects with the MDR1*2 allele. Thus allelic variation in MDR1 is more common than previously recognized and involves multiple SNPs whose allelic frequencies vary between populations, and some of these SNPs are associated with altered P-glycoprotein function.     

多药耐药基因C3435T与G2677T/A单核苷酸多态性检测方法的探讨

目的 建立简便、快速检测多药耐药基因(MDR1)C3435T与G2677T/A单核苷酸多态性(SNPs)的方法 .方法 针对MDR1 C3435T分别设计相对的两对引物-聚合酶链反应(PCR-CTPP)、序列特异性聚合酶链反应(PCR-SSP)及DNA测序方法 的引物,针对MDR1 G2677T/A分别设计PCR-SSP和DNA测序方法 的引物,优化PCR反应条件.将PCR-CTPP和PCR-SSP方法 的基因分型结果 与DNA测序结果 进行比对,确定准确性.在优化条件下,分别对50例健康体检者的外周血白细胞DNA进行MDR1 C3435T和C2677T/A基因型分析.结果 通过条件优化,PCR-CIPP、PCR-SSP方法 可快速的清晰区分MDR1 C3435T与G2677T/A的基因型,结果 与DNA测序方法 相符合.50例健康体检个体MDR1 C3435T与G2677T/A的基因型分布均符合Hardy-Weinberg平衡(P0.05).MDR1 C3435T PCR-CTPP结合G2677T/A PCR-SSP的检测方法 为最佳选择.结论PCR-CTPP、PCR-SSP方法 可简单、准确、经济、快速地检测MDR1 C3435T、G2677T/A SNPs,具有临床应用价值.     

Simultaneous analysis of MDR1 C3435T, G2677T/A, and C1236T genotypes by multiplexed mutagenically separated PCR.

P-glycoprotein (PGP) encoded by the multi-drug-resistance 1 (MDR1) gene is a member of the ATP-binding cassette (ABC) transporter family, drug-transporting proteins involved in the bioavailability and pharmacokinetics of various drugs. Several single nucleotide polymorphisms (SNPs) in the MDR1 gene have been identified so far that may influence PGP expression levels and function. Thus, genotyping for MDR1 polymorphisms and determining specific haplotypes may become an important tool in predicting individual susceptibility to developing drug resistance. We developed a new multiplexed allele-specific PCR method based on the principle of mutagenically separated PCR (MS-PCR) for rapid and reliable simultaneous genotyping of the C3435T polymorphism in exon 26 of the MDR1 gene and two additional SNPs (G2677T/A in exon 21 and C1236T in exon 12), which are in linkage disequilibrium with MDR1 C3435T. The accuracy and reliability of this method was confirmed by sequencing the respective regions in the MDR1 gene. This newly developed MDR1 MS-PCR will facilitate fast, accurate and economic analysis of MDR1 genotypes and will provide important information in optimizing individual therapeutic approaches.     

The effect of ABCB1 polymorphism on the pharmacokinetics of saquinavir alone and in combination with ritonavir

This genotype panel study investigated the effect of ABCB1 polymorphism in exon 26 (C3435T), exon 21 (G2677T/A), and exon 12 (C1236T) on saquinavir pharmacokinetics and on the expression and activity of P-glycoprotein (P-gp) in peripheral blood monocytic cells (PBMCs). One hundred and fifty healthy volunteers were genotyped to identify 15 TT3435 and 15 CC3435 individuals. In these individuals, saquinavir pharmacokinetics were assessed after administration of a single oral dose of saquinavir 1,000 mg and saquinavir/ritonavir 1,000/100 mg. PBMC P-gp expression and activity were assessed in 15 and 19 subjects. The co-administration of ritonavir on study day 2 caused a significant increase in saquinavir exposure, in both TT3435 and CC3435 individuals. No correlation was observed between the ABCB1 C3435T, G2677T/A, and C1236T polymorphisms, separately and in haplotypes, with saquinavir pharmacokinetics, administered with or without ritonavir and with PBMC P-gp expression and activity. In conclusion, ABCB1 polymorphism has no pronounced effect on saquinavir exposure.     

Frequency of single nucleotide polymorphisms in the P-glycoprotein drug transporter MDR1 gene in white subjects

BACKGROUND: P-glycoprotein, the gene product of MDR1, confers multidrug resistance against antineoplastic agents but also plays an important role in the bioavailability of common drugs in medical treatment. Various polymorphisms in the MDR1 gene were recently identified. A silent mutation in exon 26 (C3435T) was correlated with intestinal P-glycoprotein expression and oral bioavailability of digoxin. OBJECTIVE: We wanted to establish easy-to-use and cost-effective genotyping assays for the major known MDR1 single nucleotide polymorphisms and study the allelic frequency distribution of the single nucleotide polymorphisms in a large sample of volunteers. METHODS: In this study, the distribution of the major MDR1 alleles was determined in 461 white volunteers with the use of polymerase chain reaction and restriction fragment length polymorphism. RESULTS: Five amino acid exchanges were found with allelic frequencies of 11.2% for Asn21Asp and 5.5% for Ser400Asn. Strikingly, in exon 21 three variants were discovered at the same locus: 2677G (56.4%), 2677T (41.6%), and 2677A (1.9%), coding for 893Ala, Ser, or Thr. A novel missense Gln1107Pro mutation was found in two cases (0.2%). The highest frequencies were observed for intronic and silent polymorphisms; C3435T occurred in 53.9% of the subjects heterozygously, and 28.6% of individuals were homozygous carriers of 3435T/T with functionally restrained P-glycoprotein. CONCLUSION: This study provides the first analysis of MDR1 variant genotype distribution in a large sample of white subjects. It gives a basis for large-scale clinical investigations on the functional role of MDR1 allelic variants for bioavailability of a substantial number of drugs.     

CYP3A4 and MDR1 alleles in a Portuguese population.

Polymorphisms in cytochrome P450 CYP3A4 and multidrug resistance (MDR) 1 genes coding for the important drug-metabolising CYP3A4 and the ATP-binding cassette (ABC) transporter P-glycoprotein (Pgp) are poorly documented in the Portuguese population. In this study we have determined the frequencies of CYP3A4 and MDR1 alleles in Portuguese Caucasians. Both genes were simultaneously analysed as these genes are known to be frequently co-induced and their products to show a pronounced overlap of substrates. CYP3A4 A-392G (CYP3A4*1B), T673C (CYP3A4*2) and MDR1 T-129C, G2677T and C3435T single nucleotide polymorphisms (SNPs) were analysed in 100 individuals from the southern region of the country. We observed a frequency of 4.0% for CYP3A4*1B, not significantly different from that reported on other Caucasian European populations. CYP3A4*2 was found at an allele frequency of 4.5%, constituting the first report of the presence of this allele outside the Finnish population. Significant differences were found concerning the MDR1 C3435T SNP frequency (64.5%) compared with other European populations, while no differences were found concerning G2677T (47.5%) or T-129C (5%) SNPs. Linkage between the C3435T and G2677T SNPs was observed, although not as evidently as documented in other Caucasian populations. No preferential associations were detected between CYP3A4 and MDR1 alleles.     

Impact of P-glycoprotein on clopidogrel absorption

OBJECTIVE: The antiplatelet activity of clopidogrel is characterized by considerable interindividual differences. Variable intestinal absorption is suggested to contribute to the inconsistencies in response. We tested the hypothesis that the intestinal efflux transporter P-glycoprotein (P-gp) limits the oral bioavailability of clopidogrel and that variance in the MDR1 gene encoding P-gp predicts absorption variability. METHODS AND RESULTS: P-gp-mediated transport of clopidogrel was assessed by transflux, influx, and efflux experiments by use of Caco-2 cells. Inhibition of P-gp activity by different modulators increased the absorptive clopidogrel flux across Caco-2 monolayers from 0.51+/-0.19 pmol/cm2 (mean+/-SD) at baseline by a maximum of 5- to 9-fold (P<.001) and the intracellular accumulation from 0.99+/-0.11 pmol/mg protein by a maximum of 2.5-fold (P<.001) in response to 1-micromol/L clopidogrel and decreased clopidogrel efflux to the level of passive diffusion. In 60 patients with coronary artery disease who underwent percutaneous coronary intervention, the peak plasma concentration (Cmax) and the total area under the plasma concentration-time curve (AUC) of clopidogrel and its active metabolite after a single oral loading dose of 300, 600, or 900 mg were tested for correlation with the MDR1 genotype. In the 300-mg and 600-mg groups (but not in the 900-mg group) Cmax and AUC values were lower in subjects homozygous for the MDR1 3435T variant compared with subjects with the 3435C/T and 3435C/C genotypes. After the 600-mg loading dose, Cmax values (mean+/-SD) of clopidogrel and its active metabolite in 3435T/T carriers were 13.3+/-5.2 ng/mL and 2.5+/-1.2 ng/mL, respectively, compared with 49.7+/-41.6 ng/mL (P=.001) and 6.6+/-3.6 ng/mL (P=.011), respectively, in 3435C/T and 3435C/C carriers; AUC values were 1502+/-463 ng/mLxmin for clopidogrel and 209+/-99 ng/mL x min for its active metabolite in 3435T/T carriers compared with 7057+/-5443 ng/mLxmin (P=.0006) and 744+/-541 ng/mLxmin (P=.011), respectively, in 3435C/T and 3435C/C carriers. CONCLUSIONS: Clopidogrel absorption and thereby active metabolite formation are diminished by P-gp-mediated efflux and are influenced by the MDR1 C3435T genotype.     

乳腺癌MDR1基因多态性与紫杉类药物化疗血液毒副反应的关系

目的:探讨MDR1基因多态性与乳腺癌紫杉类药物为基础化疗毒副反应间的关系,为临床个体化药物治疗提供信息。方法:筛选93例汉族女性乳腺癌患者,利用PCR-RFLP技术检测其外周血MDR1 C3435T和G2677T/A基因型。结果:在本组病例中,白细胞和中性粒细胞减少症(Ⅲ～Ⅳ度)的发生频率相对较高,分别为27.2%和25%。MDR1 C3435T各基因型患者间中性粒细胞减少反应差异显著,CC型发生频率为5%,低于CT和TT型(26.3%和46.7%;χ2=8.075,P=0.018;95%CI0.017～0.022);未发现G2677T/A多态性与血液毒性的关联。结论:MDR1 3435T等位基因携带者在紫杉类药物治疗后发生中性粒细胞减少症的风险可能较大。