The formation of estrogen-like tamoxifen metabolites and their influence on enzyme activity and gene expression of ADME genes.

Tamoxifen, a standard therapy for breast cancer, is metabolized to compounds with anti-estrogenic as well as estrogen-like action at the estrogen receptor. Little is known about the formation of estrogen-like metabolites and their biological impact. Thus, we characterized the estrogen-like metabolites tamoxifen bisphenol and metabolite E for their metabolic pathway and their influence on cytochrome P450 activity and ADME gene expression. The formation of tamoxifen bisphenol and metabolite E was studied in human liver microsomes and Supersomes™. Cellular metabolism and impact on CYP enzymes was analyzed in upcyte® hepatocytes. The influence of 5 µM of tamoxifen, anti-estrogenic and estrogen-like metabolites on CYP activity was measured by HPLC MS/MS and on ADME gene expression using RT-PCR analyses. Metabolite E was formed from tamoxifen by CYP2C19, 3A and 1A2 and from desmethyltamoxifen by CYP2D6, 1A2 and 3A. Tamoxifen bisphenol was mainly formed from (E)- and (Z)-metabolite E by CYP2B6 and CYP2C19, respectively. Regarding phase II metabolism, UGT2B7, 1A8 and 1A3 showed highest activity in glucuronidation of tamoxifen bisphenol and metabolite E. Anti-estrogenic metabolites (Z)-4-hydroxytamoxifen, (Z)-endoxifen and (Z)-norendoxifen inhibited the activity of CYP2C enzymes while tamoxifen bisphenol consistently induced CYPs similar to rifampicin and phenobarbital. On the transcript level, highest induction up to 5.6-fold was observed for CYP3A4 by tamoxifen, (Z)-4-hydroxytamoxifen, tamoxifen bisphenol and (E)-metabolite E. Estrogen-like tamoxifen metabolites are formed in CYP-dependent reactions and are further metabolized by glucuronidation. The induction of CYP activity by tamoxifen bisphenol and the inhibition of CYP2C enzymes by anti-estrogenic metabolites may lead to drug-drug-interactions.

Highly sensitive simultaneous quantification of estrogenic tamoxifen metabolites and steroid hormones by LC-MS/MS.

Tamoxifen is a mainstay in the treatment of estrogen receptor-positive breast cancer and is metabolized to more than 30 different compounds. Little is known about in vivo concentrations of estrogenic metabolites E-metabolite E, Z-metabolite E, and bisphenol and their relevance for tamoxifen efficacy. Therefore, we developed a highly sensitive HPLC-ESI-MS/MS quantification method for tamoxifen metabolites bisphenol, E-metabolite E, and Z-metabolite E as well as for the sex steroid hormones estradiol, estrone, testosterone, androstenedione, and progesterone. Plasma samples were subjected to protein precipitation followed by solid phase extraction. Upon derivatization with 3-[(N-succinimide-1-yl)oxycarbonyl]-1-methylpyridinium iodide, all analytes were separated on a sub-2-µm column with a gradient of acetonitrile in water with 0.1 % of formic acid. Analytes were detected on a triple-quadrupole mass spectrometer with positive electrospray ionization in the multiple reaction monitoring mode. Our method demonstrated high sensitivity, accuracy, and precision. The lower limits of quantification were 12, 8, and 25 pM for bisphenol, E-metabolite E, and Z-metabolite E, respectively, and 4 pM for estradiol and estrogen, 50 pM for testosterone and androstenedione, and 25 pM for progesterone. The method was applied to plasma samples of postmenopausal patients taken at baseline and under tamoxifen therapy. Graphical Abstract Sample preparation and derivatization for highly sensitive quantification of estrogenic tamoxifen metabolites and steroid hormones by HPLC-MS/MS.

Cross-Ancestry Genome-Wide Association Study Defines the Extended CYP2D6 Locus as the Principal Genetic Determinant of Endoxifen Plasma Concentrations.

The therapeutic efficacy of tamoxifen is predominantly mediated by its active metabolites 4-hydroxy-tamoxifen and endoxifen, whose formation is catalyzed by the polymorphic cytochrome P450 2D6 (CYP2D6). Yet, known CYP2D6 polymorphisms only partially determine metabolite concentrations in vivo. We performed the first cross-ancestry genome-wide association study with well-characterized patients of European, Middle-Eastern, and Asian descent (n = 497) to identify genetic factors impacting active and parent metabolite formation. Genome-wide significant variants were functionally evaluated in an independent liver cohort (n = 149) and in silico. Metabolite prediction models were validated in two independent European breast cancer cohorts (n = 287, n = 189). Within a single 1-megabase (Mb) region of chromosome 22q13 encompassing the CYP2D6 gene, 589 variants were significantly associated with tamoxifen metabolite concentrations, particularly endoxifen and metabolic ratio (MR) endoxifen/N-desmethyltamoxifen (minimal P = 5.4E-35 and 2.5E-65, respectively). Previously suggested other loci were not confirmed. Functional analyses revealed 66% of associated, mostly intergenic variants to be significantly correlated with hepatic CYP2D6 activity or expression (ρ = 0.35 to -0.52), and six hotspot regions in the extended 22q13 locus impacting gene regulatory function. Machine learning models based on hotspot variants (n = 12) plus CYP2D6 activity score (AS) increased the explained variability (~ 9%) compared with AS alone, explaining up to 49% (median R2 ) and 72% of the variability in endoxifen and MR endoxifen/N-desmethyltamoxifen, respectively. Our findings suggest that the extended CYP2D6 locus at 22q13 is the principal genetic determinant of endoxifen plasma concentration. Long-distance haplotypes connecting CYP2D6 with adjacent regulatory sites and nongenetic factors may account for the unexplained portion of variability.

Susceptibility to amoxicillin-clavulanate-induced liver injury is influenced by multiple HLA class I and II alleles.

BACKGROUND & AIMS: Drug-induced liver injury (DILI), especially from antimicrobial agents, is an important cause of serious liver disease. Amoxicillin-clavulanate (AC) is a leading cause of idiosyncratic DILI, but little is understood about genetic susceptibility to this adverse reaction. METHODS: We performed a genome-wide association study using 822,927 single nucleotide polymorphism (SNP) markers from 201 White European and US cases of DILI following AC administration (AC-DILI) and 532 population controls, matched for genetic background. RESULTS: AC-DILI was associated with many loci in the major histocompatibility complex. The strongest effect was with an HLA class II SNP (rs9274407, P=4.8×10(-14)), which correlated with rs3135388, a tag SNP of HLA-DRB1*1501-DQB1*0602 that was previously associated with AC-DILI. Conditioned on rs3135388, rs9274407 is still significant (P=1.1×10(-4)). An independent association was observed in the class I region (rs2523822, P=1.8×10(-10)), related to HLA-A*0201. The most significant class I and II SNPs showed statistical interaction (P=.0015). High-resolution HLA genotyping (177 cases and 219 controls) confirmed associations of HLA-A*0201 (P=2×10(-6)) and HLA-DQB1*0602 (P=5×10(-10)) and their interaction (P=.005). Additional, population-dependent effects were observed in HLA alleles with nominal significance. In an analysis of autoimmune-related genes, rs2476601 in the gene PTPN22 was associated (P=1.3×10(-4)). CONCLUSIONS: Class I and II HLA genotypes affect susceptibility to AC-DILI, indicating the importance of the adaptive immune response in pathogenesis. The HLA genotypes identified will be useful in studies of the pathogenesis of AC-DILI but have limited utility as predictive or diagnostic biomarkers because of the low positive predictive values.

Thiopurine methyltransferase genetics is not a major risk factor for secondary malignant neoplasms after treatment of childhood acute lymphoblastic leukemia on Berlin-Frankfurt-Münster protocols.

Thiopurine methyltransferase (TPMT)is involved in the metabolism of thiopurines such as 6-mercaptopurine and 6-thioguanine. TPMT activity is significantly altered by genetics, and heterozygous and even more homozygous variant people reveal substiantially decreased TPMT activity. Treatment for childhood acute lymphoblastic leukemia (ALL) regularly includes the use of thiopurine drugs. Importantly, childhood ALL patients with low TPMT activity have been considered to be at increased risk of developing therapy-associated acute myeloid leukemia and brain tumors. In the present study, we genotyped 105 of 129 patients who developed a secondary malignant neoplasm after ALL treatment on 7 consecutive German Berlin-Frankfurt-Münster trials for all functionally relevant TPMT variants. Frequencies of TPMT variants were similarly distributed in secondary malignant neoplasm patients and the overall ALL patient population of 814 patients. Thus, TPMT does not play a major role in the etiology of secondary malignant neoplasm after treatment for childhood ALL, according to Berlin-Frankfurt-Münster strategies.

Effect of CYP2B6, ABCB1, and CYP3A5 polymorphisms on efavirenz pharmacokinetics and treatment response: an AIDS Clinical Trials Group study.

In AIDS Clinical Trials Group protocols 384, A5095, and A5097s, we characterized relationships between 22 polymorphisms in CYP2B6, ABCB1, and CYP3A5; plasma efavirenz exposure; and/or treatment responses. A stepwise logistic regression procedure selected polymorphisms associated with reduced drug clearance adjusted for body mass index and the composite CYP2B6 516/983 genotype. Relationships between selected polymorphisms and treatment responses were characterized by competing risk methodology. Association analyses involved 821 individuals (317 for pharmacokinetics and 643 for treatment response). Models that included CYP2B6 516/983 genotype best predicted pharmacokinetics. Slow-metabolizer genotypes were associated with increased central nervous system events among white participants and decreased virologic failure among black participants.

Characterization of cytochrome P450 (CYP) 2D6 drugs as substrates of human organic cation transporters and multidrug and toxin extrusion proteins.

BACKGROUND AND PURPOSE: The metabolic activity of cytochrome P450 (CYP) 2D6 is highly variable and CYP2D6 genotypes insufficiently explain the extensive and intermediate metabolic phenotypes, limiting the prediction of drug response plus adverse drug reactions. Since CYP2D6 prototypic substrates are positively charged, the aim of this study was to evaluate the organic cation transporters (OCTs) and multidrug and toxin extrusion proteins (MATEs) as potential contributors to the variability of CYP2D6 hydroxylation of debrisoquine, dextromethorphan, diphenhydramine, perhexiline and sparteine. EXPERIMENTAL APPROACH: OCT1/SLC22A1-, OCT2/SLC22A2-, OCT3/SLC22A3-, MATE1/SLC47A1-, and MATE2K/SLC47A2-overexpressing cell lines were used to investigate the transport of the selected drugs. Individuals from a study cohort, well defined with respect to CYP2D6 genotype and sparteine pharmacokinetics, were genotyped for the common OCT1 variants rs12208357 (OCT1-R61C), rs34130495 (OCT1-G401S), rs202220802 (OCT1-Met420del), rs34059508 (OCT1-G465R), OCT2 variant rs316019 (OCT2-A270S) and MATE1 variant rs2289669. Sparteine pharmacokinetics was stratified according to CYP2D6 and OCT1, OCT2 or MATE1 genotype. KEY RESULTS: OCTs and MATE1 transport sparteine and debrisoquine with high affinity in vitro, but OCT- and MATE1-dependent transport of dextromethorphan, diphenhydramine and perhexiline was not detected. Sparteine and debrisoquine transport depends on OCT1 genotype; however, sparteine pharmacokinetics is independent from OCT1 genotype. CONCLUSIONS AND IMPLICATIONS: Some drugs that are substrates of CYP2D6 are also substrates of OCTs and MATE1, suggesting overlapping specificities. Variability in sparteine hydroxylation in extensive and intermediate metabolizers cannot be explained by OCT1 genetic variants indicating presence of other factors. Dose-dependent toxicities of dextromethorphan, diphenhydramine and perhexiline appear to be independent from OCTs and MATEs.

Regulation of CYP3A4 by pregnane X receptor: The role of nuclear receptors competing for response element binding.

Induction of the major drug metabolizing enzyme CYP3A4 by xenobiotics contributes to the pronounced interindividual variability of its expression and often results in clinically relevant drug-drug interactions. It is mainly mediated by PXR, which regulates CYP3A4 expression by binding to several specific elements in the 5' upstream regulatory region of the gene. Induction itself shows a marked interindividual variability, whose underlying determinants are only partly understood. In this study, we investigated the role of nuclear receptor binding to PXR response elements in CYP3A4, as a potential non-genetic mechanism contributing to interindividual variability of induction. By in vitro DNA binding experiments, we showed that several nuclear receptors bind efficiently to the proximal promoter ER6 and distal xenobiotic-responsive enhancer module DR3 motifs. TRalpha1, TRbeta1, COUP-TFI, and COUP-TFII further demonstrated dose-dependent repression of PXR-mediated CYP3A4 enhancer/promoter reporter activity in transient transfection in the presence and absence of the PXR inducer rifampin, while VDR showed this effect only in the absence of treatment. By combining functional in vitro characterization with hepatic expression analysis, we predict that TRalpha1, TRbeta1, COUP-TFI, and COUP-TFII show a strong potential for the repression of PXR-mediated activation of CYP3A4 in vivo. In summary, our results demonstrate that nuclear receptor binding to PXR response elements interferes with PXR-mediated expression and induction of CYP3A4 and thereby contributes to the interindividual variability of induction.

Automated extraction of DNA and RNA from a single formalin-fixed paraffin-embedded tissue section for analysis of both single-nucleotide polymorphisms and mRNA expression.

BACKGROUND: There is an increasing need for the identification of both DNA and RNA biomarkers from pathodiagnostic formalin-fixed paraffin-embedded (FFPE) tissue samples for the exploration of individualized therapy strategies in cancer. We investigated a fully automated, xylene-free nucleic acid extraction method for the simultaneous analysis of RNA and DNA biomarkers related to breast cancer. METHODS: We copurified both RNA and DNA from a single 10-µm section of 210 paired samples of FFPE tumor and adjacent normal tissues (1-25 years of archival time) using a fully automated extraction method. Half of the eluate was DNase I digested for mRNA expression analysis performed by using reverse-transcription quantitative PCR for the genes estrogen receptor 1 (ESR1), progesterone receptor (PGR), v-erb-b2 erythroblastic leukemia viral oncogene homolog 2, neuro/glioblastoma derived oncogene homolog (avian) (ERBB2), epoxide hydrolase 1 (EPHX1), baculoviral IAP repeat-containing 5 (BIRC5), matrix metallopeptidase 7 (MMP7), vascular endothelial growth factor A (VEGFA), and topoisomerase (DNA) II alpha 170kDa (TOP2A). The remaining undigested aliquot was used for the analysis of 7 single-nucleotide polymorphisms (SNPs) by MALDI-TOF mass spectrometry. RESULTS: In 208 of 210 samples (99.0%) the protocol yielded robust quantification-cycle values for both RNA and DNA normalization. Expression of the 8 breast cancer genes was detected in 81%-100% of tumor tissues and 21%-100% of normal tissues. The 7 SNPs were successfully genotyped in 91%-97% of tumor and 94%-97% of normal tissues. Allele concordance between tumor and normal tissue was 98.9%-99.5%. CONCLUSIONS: This fully automated process allowed an efficient simultaneous extraction of both RNA and DNA from a single FFPE section and subsequent dual analysis of selected genes. High gene expression and genotyping detection rates demonstrate the feasibility of molecular profiling from limited archival patient samples.

Pharmacogenomics of tamoxifen therapy.

BACKGROUND: Tamoxifen is a standard endocrine therapy for the prevention and treatment of steroid hormone receptor-positive breast cancer. CONTENT: Tamoxifen requires enzymatic activation by cytochrome P450 (CYP) enzymes for the formation of active metabolites 4-hydroxytamoxifen and endoxifen. As compared with the parent drug, both metabolites have an approximately 100-fold greater affinity for the estrogen receptor and the ability to inhibit cell proliferation. The polymorphic CYP2D6 is the key enzyme in this biotransformation, and recent mechanistic, pharmacologic, and clinical evidence suggests that genetic variants and drug interaction by CYP2D6 inhibitors influence the plasma concentrations of active tamoxifen metabolites and the outcomes of tamoxifen-treated patients. In particular, nonfunctional (poor metabolizer) and severely impaired (intermediate metabolizer) CYP2D6 alleles are associated with higher recurrence rates. SUMMARY: Accordingly, CYP2D6 (cytochrome P450, family 2, subfamily D, polypeptide 6) genotyping before treatment to predict metabolizer status may open new avenues for individualizing endocrine treatment, with the maximum benefit being expected for extensive metabolizers. Moreover, strong CYP2D6 inhibitors such as the selective serotonin reuptake inhibitors paroxetine and fluoxetine, which are used to treat hot flashes, should be avoided because they severely impair formation of the active metabolites.

Association between CYP2D6 polymorphisms and outcomes among women with early stage breast cancer treated with tamoxifen.

CONTEXT: The growth inhibitory effect of tamoxifen, which is used for the treatment of hormone receptor-positive breast cancer, is mediated by its metabolites, 4-hydroxytamoxifen and endoxifen. The formation of active metabolites is catalyzed by the polymorphic cytochrome P450 2D6 (CYP2D6) enzyme. OBJECTIVE: To determine whether CYP2D6 variation is associated with clinical outcomes in women receiving adjuvant tamoxifen. DESIGN, SETTING, AND PATIENTS: Retrospective analysis of German and US cohorts of patients treated with adjuvant tamoxifen for early stage breast cancer. The 1325 patients had diagnoses between 1986 and 2005 of stage I through III breast cancer and were mainly postmenopausal (95.4%). Last follow-up was in December 2008; inclusion criteria were hormone receptor positivity, no metastatic disease at diagnosis, adjuvant tamoxifen therapy, and no chemotherapy. DNA from tumor tissue or blood was genotyped for CYP2D6 variants associated with reduced (*10, *41) or absent (*3, *4, *5) enzyme activity. Women were classified as having an extensive (n=609), heterozygous extensive/intermediate (n=637), or poor (n=79) CYP2D6 metabolism. MAIN OUTCOME MEASURES: Time to recurrence, event-free survival, disease-free survival, and overall survival. RESULTS: Median follow-up was 6.3 years. At 9 years of follow-up, the recurrence rates were 14.9% for extensive metabolizers, 20.9% for heterozygous extensive/intermediate metabolizers, and 29.0% for poor metabolizers, and all-cause mortality rates were 16.7%, 18.0%, and 22.8%, respectively. Compared with extensive metabolizers, there was a significantly increased risk of recurrence for heterozygous extensive/intermediate metabolizers (time to recurrence adjusted hazard ratio [HR], 1.40; 95% confidence interval [CI], 1.04-1.90) and for poor metabolizers (time to recurrence HR, 1.90; 95% CI, 1.10-3.28). Compared with extensive metabolizers, those with decreased CYP2D6 activity (heterozygous extensive/intermediate and poor metabolism) had worse event-free survival (HR, 1.33; 95% CI, 1.06-1.68) and disease-free survival (HR, 1.29; 95% CI, 1.03-1.61), but there was no significant difference in overall survival (HR, 1.15; 95% CI, 0.88-1.51). CONCLUSION: Among women with breast cancer treated with tamoxifen, there was an association between CYP2D6 variation and clinical outcomes, such that the presence of 2 functional CYP2D6 alleles was associated with better clinical outcomes and the presence of nonfunctional or reduced-function alleles with worse outcomes.

TGFbeta2 and TbetaRII are valid molecular biomarkers for the antiproliferative effects of tamoxifen and tamoxifen metabolites in breast cancer cells.

Response to treatment with the antiestrogen tamoxifen is variable and at least partially due to its highly complex metabolism. Tamoxifen is transformed by polymorphic and inducible cytochrome P450 enzymes to a large number of metabolites with varying biological activities. The estrogen receptor dependent growth inhibitory effect of antiestrogens is mediated by activation of antiproliferative Transforming Growth Factor beta (TGFbeta) signal transduction pathways. The aim of the present study was to establish if TGFbeta2 or TGFbeta receptor II (TbetaRII), could be used as markers to assess the pharmacological potency of tamoxifen and its metabolites. Consequently, we analyzed the growth inhibitory effect of tamoxifen and its major metabolites and explored whether it correlated with their capacity to induce TGFbeta2 and TbetaRII expression. Human breast cancer cells (MCF-7 and T47D) were treated with tamoxifen and tamoxifen metabolites and mRNA expression of TGFbeta2 and TbetaRII was analyzed by quantitative RT-PCR. Only two metabolites 4-hydroxytamoxifen and N-desmethyl-4-hydroxytamoxifen had significant antiproliferative activity and were able to induce TGFbeta2 and TbetaRII. Plasma concentrations of these metabolites are usually very low in patients. However, even minor growth inhibitory effects at concentrations which are below the limit of quantification in plasma samples resulted in clearly discernible effects on expression of TGFbeta2 and TbetaRII. Taken together, our data demonstrate that TGFbeta2 and TbetaRII are very specific and sensitive biomarkers for the antiestrogenic activity of tamoxifen metabolites in breast cancer.

Highly multiplexed genotyping of thiopurine s-methyltransferase variants using MALD-TOF mass spectrometry: reliable genotyping in different ethnic groups.

BACKGROUND: To avoid severe hematotoxicity in patients, determination of the TPMT (thiopurine S-methyltransferase) genotype before commencing thiopurine therapy has become accepted. METHODS: We used MALDI-TOF mass spectrometry (MS) based on Sequenom iPLEX technology to develop novel multiplex assays for comprehensive testing of TPMT. Two assays, a 15-plex and a 7-plex assay, consisting of multiplex PCR, shrimp alkaline phosphatase treatment, primer extension, and MALDI-TOF MS analysis, allow detection of all currently known functionally relevant 24 TPMT alleles (TPMT*2 to *18, *20 to *23). Previously identified variant DNA samples and newly constructed synthetic templates were used as quality controls. RESULTS: Assay evaluation performed on a panel of 586 genomic DNA samples previously genotyped by other methods (denaturing HPLC, sequencing) resulted in 100% agreement. Analyses of the distribution of TPMT alleles in 116 samples from a Ghanaian population revealed a TPMT*8 allele frequency of 3.4%. In a Korean population of 118 unrelated individuals, we found a TPMT*6 allele frequency of 1.3%. CONCLUSIONS: The newly developed multiplex MALDI-TOF MS assay allows efficient genotyping for all currently known functional TPMT variants. To achieve the most accurate prediction of TPMT phenotype, molecular diagnosis of TPMT should include all these variants.

Increased absorption of digoxin from the human jejunum due to inhibition of intestinal transporter-mediated efflux.

BACKGROUND AND OBJECTIVES: The contribution of transport in the small intestine by the apically located efflux pump P-glycoprotein to variable drug absorption in humans is still poorly understood. We therefore investigated whether inhibition of intestinal P-glycoprotein-mediated efflux by quinidine leads to increased absorption of the P-glycoprotein substrate digoxin. METHODS: Using a multilumen perfusion catheter, we investigated the impact of P-glycoprotein inhibition on absorption of two compounds: the P-glycoprotein substrate digoxin and the marker for passive transcellular absorption antipyrine. Two 20cm adjacent jejunal segments were isolated with the multilumen perfusion catheter in seven healthy subjects. Unlabelled and deuterated digoxin and antipyrine, respectively, were simultaneously infused into either of the intestinal segments. One of the segments was additionally perfused with the P-glycoprotein inhibitor quinidine. Intestinal perfusates were collected for 3 hours, and drug concentrations were determined in the intestinal perfusates, plasma and urine. RESULTS: Quinidine did not affect the disposition of antipyrine. In contrast, coadministration of quinidine into one jejunal segment caused a considerable increase in the amount of digoxin absorbed from this segment compared with the absorption from the other quinidine-free segment (22.3 +/- 8.9% vs 55.8 +/- 21.2% of the dose; p < 0.05). Accordingly, the area under the plasma concentration-time curve and the maximum plasma concentration of digoxin were considerably higher when luminal quinidine was coadministered (p < 0.05 and p < 0.001, respectively). Differences in digoxin absorption from the two intestinal segments were also reflected by pronounced differences in urinary digoxin elimination (5.5 +/- 3.3% vs 19.2 +/- 8.1% of the dose; p < 0.01). CONCLUSIONS: P-glycoprotein inhibition in enterocytes increases systemic exposure of orally administered drugs that are P-glycoprotein substrates. These data highlight the importance of the small intestine as an active barrier against xenobiotics.

Functional analysis of the polymorphism -211C>T in the regulatory region of the human ABCC3 gene.

The multidrug resistance protein 3 (MRP3/gene symbol: ABCC3) is an ATP-dependent efflux pump mediating the transport of endogenous glucuronides and conjugated drug metabolites across cell membranes. In humans the hepatic expression of ABCC3 mRNA seems to be influenced by the polymorphism C>T at the position -211 in the promoter of the ABCC3 gene. The aim of this study was to investigate the possible mechanisms of how this SNP influences the MRP3 expression. Promoter luciferase reporter gene constructs representing 0.5, 1.1, 4.4, and 8.1 kb upstream of the translational start site were cloned with cytosine or thymine at position -211 and transfected into HepG2, Caco-2, and LS174T cells. Reporter gene activity was dependent on the length of the promoter sequence but interestingly not on the nucleotide at position -211. Cotransfection with FTF cDNA (Fetoprotein Transcription Factor) binding to elements near the -211 polymorphism increased promoter activity in all constructs except the 0.5 kb fragment also independently of the -211 SNP. Taken together, we did not find any influence of the -211C>T ABCC3 promoter polymorphism on either the basal or the FTF induced reporter gene activity. Whether other tissue specific mechanisms reveal an impact of this SNP on the in vivo regulation of MRP3 remains to be determined.

Improved Prediction of Endoxifen Metabolism by CYP2D6 Genotype in Breast Cancer Patients Treated with Tamoxifen.

Purpose: Prediction of impaired tamoxifen (TAM) to endoxifen metabolism may be relevant to improve breast cancer treatment, e.g., via TAM dose increase. The polymorphic cytochrome P450 2D6 (CYP2D6) strongly determines an individual's capacity for endoxifen formation, however, CYP2D6 phenotype assignments inferred from genotype widely differ between studies. Thus, we modeled plasma endoxifen predictability depending on variable CYP2D6 genotype groupings. Methods: CYP2D6 diplotype and metabolite plasma concentrations were assessed in 908 pre- and post-menopausal estrogen receptor (ER)-positive, TAM treated early breast cancer patients of Caucasian (N = 678), Middle-Eastern Arab (N = 77), and Asian (N = 153) origin. Robust coefficients of determination (R2) were estimated for endoxifen (E) or metabolic ratio endoxifen/desmethyl-TAM (E/DMT) as dependent and different CYP2D6 phenotype assignments as independent variables. Allele activity scores (ASs) were modified with respect to a reduced ∗10 allele activity. Predictability of endoxifen plasma concentrations above the clinical threshold of 5.9 ng/mL was investigated by receiver operating characteristic (ROC) analysis. Results: CYP2D6 diplotypes (N = 898) were strongly associated with E and E/DMT independent of age (P < 10-15). Across all ethnicities, 68-82% inter-patient variability of E/DMT was explained by CYP2D6 diplotype, while plasma endoxifen was predictable by 39-58%. The previously used codeine specific phenotype classification showed worse prediction for both endpoints particularly in Asians (median R2< 20%; P < 10-9). Downgrading of ∗10 activity slightly improved the explanatory value of metabolizer phenotype (P < 0.002). Endoxifen plasma concentrations above the clinical threshold of 5.9 ng/mL were achieved in 82.3% of patients and were predictable (96% sensitivity, 57% specificity) by CYP2D6 diplotypes with AS > 0.5, i.e., omitting PM/PM and PM/IM patients. Conclusion: The CYP2D6 explanatory power for active drug level assessment is maximized by TAM-specific phenotype assignments while a genotype cutoff that separates PM/PM and PM/IM from the remaining patients may improve clinical benefit via increased endoxifen concentrations.

Three novel thiopurine S-methyltransferase allelic variants (TPMT*20, *21, *22) - association with decreased enzyme function.

The genetic polymorphism of the thiopurine S-methyltransferase, TPMT, comprises at least 21 alleles causing three distinct drug metabolism phenotypes termed normal/high, intermediate, and deficient methylators. In consequence, adverse drug reactions may occur if standard doses of thiopurines are applied routinely. Genetic prediction of the methylator phenotype as a basis for dose selection requires the extensive knowledge of single nucleotide polymorphisms occurring naturally in the population. Here we describe three novel missense variants in the TPMT gene which were associated with an intermediate red blood cell TPMT activity in three Caucasians. The following alleles were designated: TPMT*20 (c.712A>G), *21 (c.205C>G), and *22 (c.488G>C). No further genetic variations in remaining coding regions as well as the 5'flanking region of TPMT were identified. These sequence variants are present in highly conserved nucleotide positions of the TPMT gene throughout various mammalian species and in zebra fish, and are predicted to be intolerant when the functional consequences of variations were analyzed using SIFT (Sorting Intolerant From Tolerant) algorithm. In Caucasians the occurrence of these genetic variants appears to be extremely rare since none of these alleles were identified in a randomly selected control population of 1048 individuals.

Transcriptional profiling of genes induced in the livers of patients treated with carbamazepine.

BACKGROUND: The antiepileptic drug carbamazepine (CBZ) is a potent inducer of human drug metabolism, resulting in serious interactions with many commonly prescribed drugs. The molecular mechanisms underlying this response are not well understood, however, and the spectrum of CBZ-inducible genes in human liver has not been thoroughly investigated. METHODS: The availability of liver ribonucleic acid from 2 epileptic patients treated with CBZ and from 7 control subjects enabled us to study the global induction response of drug-metabolizing enzymes, drug transporters, and nuclear receptors in vivo. RESULTS: Using expression profiling, we identified 64 significantly up-regulated transcripts but only 1 significantly down-regulated transcript (SLC22A5). We confirmed the induction of several genes that previously have been shown to be inducible by drugs in vitro, including multiple cytochrome P450 (CYP) genes in the CYP1A, CYP2A, CYP2B, CYP2C, and CYP3A subfamilies, as well as glutathione S-transferase A1, uridine diphosphate-glucuronosyltransferase 1As, the drug transporter ABCC2, and the nuclear receptors CAR (constitutive androstane receptor) and PXR (pregnane X receptor). Moreover, we identified a number of additional genes not previously known to be induced by CBZ, including CYP39A1, sulfotransferase 1A1, glutathione S-transferase Z1, and the drug transporters SLCO1A2, ABCG2, and ABCB7, as well as the glucocorticoid and aldosterone receptors. In transactivation studies in CV-1 cells, we demonstrated that both CBZ and its major metabolite, CBZ-10,11-epoxide, activate the nuclear receptor PXR in a concentration-dependent fashion and at therapeutic concentrations with 50% inhibitory concentration values of approximately 50 micromol/L. CONCLUSIONS: CBZ is a potent inducer of a broad spectrum of drug-metabolizing enzymes and drug transporters in the human liver, and these effects are mediated at least in part by activation of PXR.