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Michel Eichelbaum

Publications and source records attributed to Michel Eichelbaum.

At least 37 records · Page 2Linked to original sources
PubMed2005

A natural CYP2B6 TATA box polymorphism (-82T--> C) leading to enhanced transcription and relocation of the transcriptional start site.

We investigated the impact of promoter polymorphisms on transcription of the human CYP2B6 gene. In total, 98 DNA samples from white persons from a previously characterized liver bank were sequenced throughout 2.3 kilobases of upstream sequence and haplotype structures were determined using additional coding sequence information. HepG2 cells and primary rat and human hepatocytes were transfected with luciferase reporter gene constructs driven by 2033 base pairs (bp) of the most frequent promoter variants. The novel haplotype *22 (-1848C--> A, -801G--> T, -750T--> C, and -82T--> C) showed 3- to 9-fold enhanced transcriptional activity in all transfected cells. Constructs containing single mutations surprisingly revealed -82T--> C, predicted to disrupt a putative TATA box, to be alone responsible for this effect. In silico analysis and electrophoretic mobility shift assay demonstrated conversion of the putative TATA box into a functional CCAAT/enhancer-binding protein binding site. Analysis of transcriptional start sites showed the mutant promoter to be transcribed from a start site located approximately 30 bp downstream of the wild-type start site, consistent with the use of a noncanonical TATA box at -55 bp. Median CYP2B6 mRNA expression and bupropion hydroxylase activity as a selective marker of CYP2B6 catalytic activity were approximately 2-fold higher in livers genotyped -82TC as in those genotyped -82TT (20.4 versus 9.8 arbitrary units, p = 0.007, and 201.8 versus 106.7 pmol/mg/min, p = 0.042, respectively). This promoter polymorphism thus contributes to CYP2B6 functional variability and represents a novel mechanism by which mutations can enhance transcription. Furthermore, a detailed interspecies comparison of CYP2B promoters and transcriptional start sites provided novel insights into evolutionary relationships.

Animals↗
PubMed2005

Polymorphic organic anion transporting polypeptide 1B1 is a major determinant of repaglinide pharmacokinetics.

BACKGROUND AND OBJECTIVE: A large interindividual variability exists in the plasma concentrations of repaglinide. Our aim was to investigate possible associations between the pharmacokinetics of repaglinide and single nucleotide polymorphisms (SNPs) in the genes encoding for the drug transporters organic anion transporting polypeptide 1B1 (OATP1B1) (SLCO1B1 ) and P-glycoprotein ( MDR1 , ABCB1 ) and the drug-metabolizing enzymes cytochrome P450 (CYP) 2C8 and CYP3A5. METHODS: A total of 56 healthy volunteers ingested a single 0.25-mg dose of repaglinide. Plasma repaglinide and blood glucose concentrations were measured for up to 7 hours. All subjects were genotyped for the -11187G>A and 521T>C SNPs in SLCO1B1 and the 3435C>T and 2677G>T/A SNPs in ABCB1 , as well as for the CYP2C8*3 (416G>A, 1196A>G), CYP2C8*4 (792C>G), and CYP3A5*3 (6986A>G) alleles. RESULTS: The area under the plasma concentration-time curve from time 0 to infinity [AUC(0-infinity)] and peak concentration in plasma (Cmax) of repaglinide varied 16.9-fold and 10.7-fold, respectively, between individual subjects. Multiple regression analyses indicated that the SLCO1B1 521T>C SNP and the CYP2C8*3 allele were independent predictors of the AUC(0-infinity) and Cmax of repaglinide (adjusted multiple R2 = 45% and 36%, respectively). In subjects with the SLCO1B1 521CC genotype, the AUC(0-infinity) of repaglinide was 107% and 188% higher, respectively, than in subjects with the SLCO1B1 521TC or 521TT (reference) genotype (P < .0001). In subjects with the CYP2C8*1/*3 genotype, the AUC(0-infinity) and Cmax of repaglinide were 48% and 44% lower, respectively, than in those with the CYP2C8*1/*1 genotype (P < .05). The pharmacokinetics of repaglinide was not associated with the studied ABCB1 SNPs or the CYP3A5*3 allele. The elimination half-life of repaglinide was not associated with any SNP. Only the SLCO1B1 -11187GA genotype was significantly associated with an enhanced effect of repaglinide on blood glucose. CONCLUSIONS: Genetic polymorphism in SLCO1B1 is a major determinant of interindividual variability in the pharmacokinetics of repaglinide. The effect of SLCO1B1 polymorphism on the pharmacokinetics of repaglinide may be clinically important.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2005

Impact of CYP2D6 genotype on adverse effects during treatment with metoprolol: a prospective clinical study.

OBJECTIVE: Our objective was to study the impact of the cytochrome P450 (CYP) 2D6 polymorphism on the tolerability of metoprolol in a real-life primary care setting. The adverse effects studied comprised effects related to the central nervous system, cardiovascular effects, and sexual dysfunction. METHODS: Patients in whom treatment with metoprolol was considered were enrolled into this prospective, 6-week multicenter study. The dosage of metoprolol was determined on an individual basis and could be freely adjusted on clinical grounds. The indication for treatment was hypertension in about 90% of cases. Systolic and diastolic blood pressure, resting heart rate, and plasma metoprolol and alpha-hydroxymetoprolol concentrations were measured. CYP2D6 genotyping covered alleles *3 to *10 and *41 and the duplications. Possible adverse effects of metoprolol were systematically assessed over a 6-week period by means of standardized rating scales and questionnaires. RESULTS: The final study population comprised 121 evaluable patients (all white patients); among them, there were 5 ultrarapid metabolizers (UMs) (4.1%), 91 extensive metabolizers (EMs) (75%), 21 intermediate metabolizers (IMs) (17%), and 4 poor metabolizers (PMs) (3.3%). Plasma metoprolol concentrations normalized for the daily dose and metoprolol/alpha-hydroxymetoprolol ratios at steady state were markedly influenced by CYP2D6 genotype and displayed a gene-dose effect. The median of the dose-normalized metoprolol concentration was 0.0088 ng/mL, 0.047 ng/mL, 0.34 ng/mL, and 1.34 ng/mL among UMs, EMs, IMs, and PMs, respectively (P<.0001). There was no significant association between CYP2D6 genotype-derived phenotype (EMs and UMs combined versus PMs and IMs combined) and adverse effects during treatment with metoprolol. There was a tendency toward a more frequent occurrence of cold extremities in the PM plus IM group as compared with the EM plus UM group (16.0% versus 4.2%, P=.056; relative risk, 3.8 [95% confidence interval, 1.03--14.3]). CONCLUSIONS: CYP2D6 genotype-derived phenotype was not significantly associated with a propensity for adverse effects to develop during treatment with metoprolol. However, the results concerning tolerability of metoprolol in PMs were inconclusive because of the small number of PMs enrolled.

Adrenergic beta-1 Receptor Antagonists↗
PubMed2005

6-thioguanosine diphosphate and triphosphate levels in red blood cells and response to azathioprine therapy in Crohn's disease.

BACKGROUND & AIMS: Azathioprine is the gold standard for immunosuppressive therapy in Crohn's disease (CD) and its molecular mechanism of action is caused by the metabolite 6-thioguanosine triphosphate (TGTP). In this study we assessed the impact of TGTP levels for monitoring of azathioprine therapy. METHODS: A novel, highly sensitive assay was established to measure levels of TGTP and its precursors 6-thioguanosine monophosphates and 6-thioguanosine diphosphates (TGDP) in red blood cells from 50 CD patients. The results were correlated with clinical outcome. RESULTS: TGTP levels could be quantified in 47 patients and a subgroup of these patients showed significantly high levels of TGDP. 6-thioguanine nucleotide (6-TGN) levels showed a significant correlation with TGDP plus TGTP concentrations, suggesting that active TGTP and its inactive precursor TGDP are the main metabolites within 6-TGN. Patients with 6-TGN levels higher than 100 pmol/8x10(8) red blood cells showed better response rates, on average, than patients with lower 6-TGN levels. The subgroup of patients with higher 6-TGN and increased TGDP levels showed a worse outcome with lower response rates, more flares, and higher infliximab demand than patients with high 6-TGN, low TGDP, and predominantly detectable TGTP levels. CONCLUSIONS: This study shows that quantification of TGTP levels can be used to monitor azathioprine therapy in inflammatory bowel disease patients. Furthermore, the data suggest that TGDP levels of more than 15% of total 6-TGN levels may be a useful surrogate parameter to predict poor response in a subgroup of azathioprine-treated patients.

Adult↗
PubMed2005

Acute effects of pravastatin on cholesterol synthesis are associated with SLCO1B1 (encoding OATP1B1) haplotype *17.

OBJECTIVE: The aim was to investigate whether polymorphisms in the SLCO1B1 gene, encoding the hepatic uptake transporter OATP1B1, influence the short-term effects of pravastatin on cholesterol synthesis. METHODS: We determined plasma concentrations of lathosterol and cholesterol up to 12 h after intake of a single dose of 40 mg pravastatin in 41 healthy Caucasian subjects, in whom SLCO1B1 single nucleotide polymorphisms (SNP; 521T>C and -11187G>A) and haplotypes (*15B and *17) had been previously shown to be associated with considerably elevated plasma pravastatin levels. RESULTS: The effects of pravastatin on plasma lathosterol concentration and lathosterol to cholesterol concentration ratio, which are established markers of the rate of cholesterol synthesis in vivo, were significantly smaller among the three heterozygous carriers of the SLCO1B1 *17 haplotype (containing the -11187G>A, 388A>G and 521T>C SNPs) as compared with non-carriers. Significant inverse relationships were found between pravastatin area under the concentration-time curve (AUC) values and effects of pravastatin on lathosterol and lathosterol to cholesterol ratio among the whole study population. CONCLUSION: These results suggest that uptake of pravastatin into hepatocytes is impaired in carriers of the SLCO1B1 haplotype *17, resulting in higher plasma pravastatin concentrations but lower concentrations of pravastatin in hepatocytes and thereby in a smaller inhibitory effect on cholesterol synthesis. The cholesterol-lowering response to pravastatin may be impaired in carriers of the *17 haplotype.

Anticholesteremic Agents↗
PubMed2005

Genetic variability of CYP2B6 in populations of African and Asian origin: allele frequencies, novel functional variants, and possible implications for anti-HIV therapy with efavirenz.

The present study investigated CYP2B6 genetic variability by sequencing genomic DNA samples of African-American, Ghanaian, Taiwanese, Japanese and Korean subjects throughout all exons and exon-intron boundaries. The most common nonsynonymous single nucleotide polymorphisms (SNPs) were 15631G > T (Q172H) and 18053A > G (K262R, together defining allele 2B6*6), both of which had frequencies close to 50% in Ghanaians and 30% in African-Americans. These SNPs have recently been shown to affect efavirenz pharmacokinetics and response in HIV patients. Eight new missense mutations (76A > T [T26S], 83A > G [D28G], 85C > A, 86G > C [both R29T], 15618C>T [T168I], 18038G > A [D257N], 21034C > T [R336C], 21498C > A [P428T]), three new silent mutations and two new intronic SNPs defining six novel alleles (*17A and B, *18, *19, *20, *21) were identified. Heterologous expression in COS-1 cells revealed pronounced reduction in expression and/or bupropion hydroxylase activity for variants T168I, D257N, R336C and P428T, whereas the triple mutant 2B6.17 (T26S, D28G, R29T) appeared to be functionally normal. These data extend the CYP2B6 knowledge base and should be particularly relevant for anti-HIV-therapy with efavirenz.

Acquired Immunodeficiency Syndrome↗
PubMed2005

A role for constitutive androstane receptor in the regulation of human intestinal MDR1 expression.

MDR1/P-glycoprotein is an efflux transporter determining the absorption and presystemic elimination of many xenobiotics in the gut. Thus, interindividual differences in MDR1 expression may affect the efficacy of drug treatment. The expression of MDR1 is partially controlled by the pregnane X receptor (PXR), which mediates induction by many xenobiotics. Since it has been described that the nuclear receptors PXR and constitutive androstane receptor (CAR) can bind to the same binding sites, we investigated the role of CAR in the regulation of MDR1 gene expression. We demonstrate here by gel shift and transfection experiments that CAR binds to distinct nuclear receptor response elements in the -7.8 kbp enhancer of MDR1 and transactivates MDR1 expression through DR4 motifs to which the receptor binds as a heterodimer with RXR or as a monomer, respectively. Expression of the endogenous MDR1 gene is elevated in cells stably expressing CAR, thus arguing for the functional relevance of CAR-dependent activation of MDR1 . The physiological relevance of the regulation of MDR1 by CAR is further suggested by correlation of the expression of CAR and MDR1 in the human small intestine. In summary, our data suggest that CAR plays a role in the regulation of intestinal MDR1 expression.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2005

GSTP1 and MDR1 genotypes and central nervous system relapse in childhood acute lymphoblastic leukemia.

The probability of event-free survival of childhood acute lymphoblastic leukemia (ALL) approaches 80% or more with the use of modern multiagent chemotherapeutic regimens. One major contribution to this success has been reduction of the rate of central nervous system (CNS) relapses to less than 5%. However, heterogeneity is observed with regard to the incidence of CNS relapse in homogenously treated patient populations. One potential explanation for this heterogeneity is variation in the genetic background of these populations. Glutathione S-transferase P1 and P-glycoprotein are implicated in resistance to a variety of chemotherapeutic agents and have been localized to the blood-brain barrier. In a matched case-control study, we investigated the associations between CNS relapse in childhood ALL and the presence of phenotypically relevant single nucleotide polymorphisms within the GSTP1 (codon 105 and 114) and MDR1 genes (ABCB1; coding for Pgp; exon 26, C3435T). Significant reductions in risk of CNS relapse were observed for patients homozygous for the GSTP1 Val105 allele as well as for patients with the MDR1 3435T/T or C/T genotype. For both genotypes, the effect was restricted to patients at intermediate or high risk of treatment failure. These results suggested a modulating role for host genetic variation in the development of CNS relapse in childhood ALL treated according to Berlin-Frankfurt-Münster protocols.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2005

CYP3A5 genotype is associated with diagnosis of hypertension in elderly patients: data from the DEBATE Study.

OBJECTIVE: The aim of this study was to address the presently controversial question of whether cytochrome P450 (CYP) 3A5 polymorphism is associated with hypertension. METHOD: We studied 373 elderly (age > or =75 years) Finnish (Caucasian) patients from the ongoing DEBATE (Drugs and Evidence Based Medicine in the Elderly) trial. The patients were classified into those with a history of hypertension (n = 229) and those without a history of hypertension (n = 144) on the basis of a detailed questionnaire on each patient's medical history and an interview. The patients were genotyped for the CYP3A5 6986A/G single nucleotide polymorphism (SNP) [CYP3A5*1/*3 alleles]. RESULTS: The proportion of individuals with the CYP3A5*1/*3 genotype, i.e. CYP3A5 expressors, was significantly higher among patients with a diagnosis of hypertension than among patients without (18.3% vs 9.0%, p = 0.016). The corresponding odds ratio was 2.26 (95% CI 1.17, 4.38). The allele and genotype frequencies for the two control SNPs, ABCB1 (MDR1) 3435C/T and SLCO1B1 521T/C, did not differ between the two groups. CONCLUSION: This work lends support to the theory that the polymorphic CYP3A5 enzyme may be involved in regulation of blood pressure. The possible role of CYP3A5 as a genetic contributor to hypertension susceptibility warrants further study.

Aged↗
PubMed2004

Inhibition of human CYP2B6 by N,N',N''-triethylenethiophosphoramide is irreversible and mechanism-based.

The chemotherapeutic agent N,N',N''-triethylenethiophosphoramide (thioTEPA) is frequently used in high-dose chemotherapy regimens including cyclophosphamide. Previous studies demonstrated partial inhibition by thioTEPA of the cytochrome P4502B6 (CYP2B6)-catalyzed 4-hydroxylation of cyclophosphamide, which is required for its bioactivation. The aim of our study was to investigate the detailed mechanism of CYP2B6 inhibition by thioTEPA. Using human liver microsomes and recombinant P450 enzymes we confirmed potent inhibition of CYP2B6 enzyme activity determined with bupropion as substrate. ThioTEPA was found to inhibit CYP2B6 activity in a time- and concentration-dependent manner. The loss of CYP2B6 activity was NADPH-dependent and could not be restored by extensive dialysis. The maximal rates of inactivation (K(inact)) were 0.16 min(-1) in human liver microsomes and 0.17 min(-1) in membrane preparations expressing recombinant CYP2B6. The half-maximal inactivator concentrations (K(I)) were 3.8 microM in human liver microsomes and 2.2 microM in recombinant CYP2B6. Inhibition was attenuated by the presence of alternative active site ligands but not by nucleophilic trapping agents or reactive oxygen scavengers, further supporting mechanism-based action. Inactivated CYP2B6 did not lose its ability to form a CO-reduced complex suggesting a modification of the apoprotein, which is common for sulfur-containing compounds. Pharmacokinetic consequences of irreversible inactivation are more complicated than those of reversible inactivation, because the drug's own metabolism can be affected and drug interactions will not only depend on dose but also on duration and frequency of application. These findings contribute to better understanding of drug interactions with thioTEPA.

Aryl Hydrocarbon Hydroxylases↗
PubMed2004

Characterisation of cerivastatin as a P-glycoprotein substrate: studies in P-glycoprotein-expressing cell monolayers and mdr1a/b knock-out mice.

The aim of this study was to characterise the role of the efflux transporter P-glycoprotein in the disposition of cerivastatin. We investigated directional transport characteristics of [14C]cerivastatin across cell monolayers expressing P-glycoprotein (Caco-2 and L-MDR1) and disposition of cerivastatin in mice with disrupted mdr1a and mdr1b genes. The mice were given orally 1 mg/kg cerivastatin and plasma and tissue samples for analysis of cerivastatin were obtained 10, 20, or 30 min after drug administration. Four knock-out mice and four wild-type mice were studied at each time point. In addition, the hypothesis that gemfibrozil-mediated inhibition of P-glycoprotein contributes to the interaction between gemfibrozil and cerivastatin was tested in Caco-2 cells. The apparent permeability coefficient (P(app)) value for the basal-to-apical transport of cerivastatin in Caco-2 and L-MDR1 cell monolayers was 2.4 times (P<0.001) and 3.8 times (P<0.001) as high as the apical-to-basal P(app) value respectively. The P-glycoprotein inhibitor PSC-833 (1 microM) inhibited the net basal-to-apical transport of cerivastatin in Caco-2 monolayers by 35% (P<0.01) and the MRP inhibitor MK-571 (10 microM) by 50% (P<0.01). At concentrations up to 250 microM, gemfibrozil showed no significant effects on the net transport of cerivastatin in Caco-2 cells. The concentration of cerivastatin in the brain at 30 min was 3.1 times higher in the knock-out mice than in the wild-type mice (P<0.05). The brain-to-plasma cerivastatin concentration ratio at 20 min and 30 min was 2.1 (P<0.05) and 3.6 times (P<0.05) higher respectively in the knock-out animals compared with the wild-type animals. Collectively, these results indicate that cerivastatin is a P-glycoprotein substrate, although other transporters probably contribute to cerivastatin transport in humans. As several statins are P-glycoprotein substrates, beneficial as well as adverse effects of the statins might be affected by interindividual differences in P-glycoprotein expression or function caused by, e.g., the MDR1 polymorphism.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed2004

The induction of cytochrome P450 3A5 (CYP3A5) in the human liver and intestine is mediated by the xenobiotic sensors pregnane X receptor (PXR) and constitutively activated receptor (CAR).

Induction of cytochrome P450 3A (CYP3A) by xenobiotics may lead to clinically relevant drug interactions. In contrast with other CYP3A family members, studies on the inducibility of CYP3A5 indicate conflicting results. We report the induction of CYP3A5 mRNA in 13 of 16 hepatocyte preparations exposed to rifampin. Furthermore, induction of CYP3A5 mRNA was observed in intestinal biopsies in three of eight probands following exposure to the antibiotic. The highest absolute levels of CYP3A5 transcripts were found following rifampin treatment in hepatocytes and intestines from carriers of CYP3A5*1 alleles. Elucidation of the mechanism involved in CYP3A5 induction revealed that constitutively activated receptor (CAR) and pregnane X receptor (PXR) transactivated the CYP3A5 promoter (-688 to +49) and that the transactivation was dependent on an everted repeat separated by 6 bp (ER6-dependent). Treatment with the prototypical PXR ligand rifampin led to a 2-fold induction of the CYP3A5 promoter activity. In agreement with these observations, PXR and CAR bound specifically to the ER6 motif. Hepatic expression of PXR correlated with that of CYP3A5 mRNA levels in a bank of liver samples. Taken together, studies here revealed the presence of a functional ER6 motif in the CYP3A5 promoter located -100 bp upstream from the transcription start site, suggesting that CYP3A5 is inducible by mechanisms similar to those involved in CYP3A4 induction. Enhanced expression of CYP3A5 caused by exposure to inducers may phenocopy the effects of the high expression allele CYP3A5*1. In this manner, induction of CYP3A5 may contribute to the overall importance of this P450 in drug metabolism and drug interactions.

Alleles↗
PubMed2004

Multiple novel nonsynonymous CYP2B6 gene polymorphisms in Caucasians: demonstration of phenotypic null alleles.

The human microsomal cytochrome P450, CYP2B6, is involved in the biotransformation of several clinically important drugs. By complete sequence analysis of the human CYP2B6 gene coding regions in selected Caucasian DNA samples, we identified the five novel missense mutations 62A>T (Q21L in exon 1), 136A>G (M46V in exon 1), 12820G>A (G99E in exon 2), 13076G>A (R140Q in exon 3), and 21388T>A (I391N in exon 8). The recently described but functionally uncharacterized variant 13072A>G (K139E) was also observed. Haplotype analysis indicated the presence of at least six novel alleles that code for the protein variants CYP2B6.10 (Q21L, R22C), CYP2B6.11 (M46V), CYP2B6.12 (G99E), CYP2B6.13 (K139E, Q172H, K262R), CYP2B6.14 (R140Q), and CYP2B6.15 (I391N). Heterologous expression in COS-1 cells revealed comparable levels of CYP2B6 apoprotein and bupropion hydroxylase activity for CYP2B6.1 (wild type) and CYP2B6.10, whereas all other variants exhibited reduced expression and/or function. The three amino acid changes M46V, G99E, and I391N resulted in almost unmeasurable (M46V) or undetectable (G99E and I391N) enzyme activity, despite the presence of residual protein. The K139E change led to completely abolished protein expression; as a consequence, no function was detected. Expression in insect cells by recombinant baculoviruses confirmed these results and demonstrated the virtual absence of incorporated heme in these protein variants. The collective allele frequency of the four very low or null activity variants M46V, G99E, K139E, and I391N was 2.6% in a Caucasian study population. These data provide further insight into the genetic variability of CYP2B6 and demonstrate the existence of phenotypic null alleles in this gene.

Alleles↗
PubMed2004

Alternative splicing affects the function and tissue-specific expression of the human constitutive androstane receptor.

BACKGROUND: The constitutive androstane receptor (CAR) plays a key role in the control of drug metabolism and transport by mediating the phenobarbital-type induction of many phase I and II drug metabolizing enzymes and drug transporters. RESULTS: We identified transcripts generated by four different alternative splicing events in the human CAR gene. Two of the corresponding ligand binding domain isoforms demonstrated novel functional properties: First, CAR(SV3), which is encoded by a transcript containing an lengthened exon 7, differentially transactivated target gene promoters. Second, CAR(SV2), which results from the use of an alternative 3' splice site lengthening exon 8, showed ligand-dependent instead of constitutive interaction with coactivators. Furthermore, alternatively spliced transcripts demonstrated a tissue-specific expression pattern. In most tissues, only transcripts generated by alternative splicing within exon 9 were expressed. The encoded variant demonstrated a loss-of-function phenotype. Correct splicing of exon 8 to exon 9 is restricted to only a few tissues, among them liver and small intestine for which CAR function has been demonstrated, and is associated with the induction of CAR expression during differentiation of intestinal cells. CONCLUSION: Due to their specific activities, CAR variant proteins SV2 and SV3 may modulate the activity of reference CAR(SV1). Furthermore, we propose that transcriptional activation and regulation of splicing of exon 9 may be coupled to ensure appropriate tissue- and differentiation state-specific expression of transcripts encoding functional CAR protein. Altogether, alternative splicing seems to be of utmost importance for the regulation of CAR expression and function.

Journal Article↗
PubMed2004

Cytochrome P450 3A4 and P-glycoprotein expression in human small intestinal enterocytes and hepatocytes: a comparative analysis in paired tissue specimens.

OBJECTIVES: Our objectives were to determine the content of cytochrome P450 (CYP) 3A4, CYP3A5, and P-glycoprotein and to measure CYP3A4-dependent catalytic activity in paired human small intestinal and liver specimens. METHODS: Samples of duodenum or proximal jejunum and liver wedge biopsy specimens were obtained from 15 patients undergoing a gastrointestinal operation. Enterocytes were isolated from the intestinal samples. The contents of CYP3A4, CYP3A5, and P-glycoprotein and CYP3A4-mediated catalytic activities were determined in homogenized enterocyte and liver samples. RESULTS: The CYP3A4 protein content was about 3 times (P <.01) and the P-glycoprotein content about 7 times (P <.0001) higher in the enterocyte homogenates than in the liver homogenates. CYP3A5 protein was detected in all samples, but the levels were too low in most cases to allow quantification. The 2 cases with a quantifiable hepatic CYP3A5 content had the CYP3A5*1/*3 genotype; all other cases were homozygous for the CYP3A5*3 allele. No intraindividual correlations between the intestine and liver with respect to CYP3A4 content, P-glycoprotein content, or the measured catalytic activities were present. Values for the maximum rate of metabolism (V(max)) of verapamil N-dealkylation (formation of D-617) and N-demethylation (formation of norverapamil) activities correlated with the CYP3A4 protein content in both organs. CONCLUSIONS: This work demonstrated a much higher content of both CYP3A4 protein and P-glycoprotein in enterocytes isolated from human duodenal or jejunal mucosa than in paired specimens of liver tissue. These results lend support to the view that biotransformation in the gut wall substantially contributes to the overall first-pass metabolism of many CYP3A4 substrates. Furthermore, the high content of P-glycoprotein on the apical surface of enterocytes supports the theory that this efflux transporter may act in concert with CYP3A4 to limit oral drug bioavailability. Finally, these results indicate that neither CYP3A4 nor MDR1 (P-glycoprotein) is coordinately regulated in the liver and intestine.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2004

A novel intronic mutation, 2988G>A, with high predictivity for impaired function of cytochrome P450 2D6 in white subjects.

BACKGROUND: Individuals with the cytochrome P450 (CYP) 2D6 intermediate metabolizer (IM) phenotype have low residual enzyme activity and compose about 10% to 15% of white populations. Their identification is clinically relevant but remains unsatisfactory because of incomplete characterization of the major allele involved, termed CYP2D6*41 (-1584C, R296C, S486T). METHODS: To search for novel mutations, resequencing of the entire CYP2D6 gene was performed in selected individuals. Genotype-phenotype correlation analysis was done in a population sample of 308 white subjects phenotyped with sparteine and previously genotyped for all major alleles. RESULTS: A total of 16 novel polymorphic positions were identified, of which 7 were located within 2.4 kilobases of previously uncharacterized 2D7-2D6 intergenic sequence and 9 were located within intronic regions. The novel mutation 2988G>A in intron 6 appeared to be specifically associated with the IM phenotype. Further analysis in the population sample demonstrated that 2988G>A was strongly linked to allele *41 but not to any other alleles including *1, *2, *2xN, *4, *6, *7, *8, *9, *10, and *35. The overall frequency of the novel polymorphism was 8.4% in the normal white population. Compared with conventionally determined *41, 2988G>A was shown to have improved predictivity for the IM phenotype. With 2988G>A being taken into account, alleles *1, *2, and *35 (-1584G, V11M, R296C, S486T) were found to be phenotypically equivalent. CONCLUSIONS: CYP2D6 genotyping can be considerably simplified by using 2988G>A as a marker for *41 and by omitting genotyping for the functionally equivalent alleles *2 and *35.

Adult↗
PubMed2004

Impact of concentration and rate of intraluminal drug delivery on absorption and gut wall metabolism of verapamil in humans.

BACKGROUND AND AIMS: In humans gut wall metabolism can be quantitatively as important as hepatic drug metabolism in limiting the systemic exposure to drugs after oral administration. However, it has been proposed that the role of gut wall metabolism might be overemphasized, because high luminal drug concentrations would lead to a saturation of gut wall metabolism. Therefore we investigated the impact of concentration and rate of intraluminal drug delivery on absorption (F(abs)) and gastrointestinal extraction (E(GI)) of a luminally administered cytochrome P450 (CYP) 3A4 substrate (verapamil) using a multilumen perfusion catheter in combination with a stable isotope technique. METHODS: Two 20-cm-long, adjacent jejunal segments were isolated with the multilumen perfusion catheter in 7 subjects. In this study 80 mg of unlabeled verapamil (d0-verapamil 15 min) was infused into one segment over a 15-minute period, 80 mg of 3-fold deuterated verapamil (d3-verapamil 240 min) was administered over a 240-minute period into the other segment, and simultaneously, 5 mg of 7-fold deuterated verapamil (d7-verapamil) was injected intravenously over a 15-minute period. RESULTS: The rate of intraluminal drug delivery had only a modest effect on bioavailability of the verapamil isotopes (after correction for F abs ) (F/F abs d3-verapamil 240 min versus d0-verapamil 15 min, 0.24 +/- 0.10 versus 0.20 +/- 0.09; P <.05). Accordingly, the E GI value for d3-verapamil 240 min was 0.50 +/- 0.18 compared with 0.59 +/- 0.14 for d0 -verapamil 15 min ( P <.05). In vivo, E GI (d0-verapamil 15 min ) correlated strongly with E GI (d3-verapamil 240 min ) (r = 0.94, P <.005). Moreover, intrinsic clearance of CYP3A4-mediated verapamil metabolism in homogenates of simultaneously collected shed enterocytes correlated with in vivo E GI of d0-verapamil 15 min /d3-verapamil 240 min (r = 0.62, P =.03). CONCLUSIONS: Substantial gut wall metabolism of verapamil occurs in humans and can be predicted from ex vivo data by use of shed enterocytes. The different intraluminal concentrations and rates of intraluminal drug delivery did not lead to a pronounced saturation of intestinal drug metabolism.

Adult↗