Cytochrome P450 3A5 Expression Research Articles

CYP3A5 Polymorphisms Leading to Tacrolimus Toxicity Following an Adult Renal Transplant.

Tacrolimus is one of the calcineurin inhibitors used for maintaining immuno-suppression in thoracic and abdominal transplantations including heart, lung, liver, intestine, pancreas, and renal transplants. It has a narrow therapeutic window requiring therapeutic drug monitoring (TDM). Genetic polymorphism in the expression of cytochrome P3A5 enzyme plays a significant role in the bioavailability of tacrolimus in patients, leading to toxicity or rejection. In this case, we studied a renal transplant patient who received a standard dose of tacrolimus and experienced toxicity related to the poor expression of cytochrome P450 3A5 (CYP3A5), which required the withholding of tacrolimus and cutting the dose for several days with more frequent TDM. Similar cases have been reported before, yet there is no consensus on the appropriate dosage. The projected cost of additional TDM and hospitalization significantly exceeds the one-time cost of genetic CYP3A5 testing. In high-risk renal transplant recipients, pharmacogenetic testing must be considered to cut the time to limit TDM, prevent extended hospitalization, and reduce the total cost of transplantation.

Yeast β-glucan Increases Etoposide Sensitivity in Lung Cancer Cell Line A549 by Suppressing Nuclear Factor Erythroid 2-Related Factor 2 via the Noncanonical Nuclear Factor Kappa B Pathway.

Etoposide is regarded as one of the main standard cytotoxic drugs for lung cancer. However, mutations in Kelch-like ECH-associated protein 1 (Keap1), the main regulator of nuclear factor erythroid 2-related factor 2 (Nrf2), are often detected in lung cancer and lead to chemoresistance. Since the aberrant activation of Nrf2 enhances drug resistance, the suppression of the Nrf2 pathway is a promising therapeutic strategy for lung cancer. We herein used the human lung adenocarcinoma cell line A549 because it harbors a Keap1 loss-of-function mutation. A treatment with β-glucan, a major component of the fungal cell wall, reduced Nrf2 protein levels; downregulated the expression of cytochrome P450 3A5, UDP glucuronosyltransferase 1A1, and multidrug resistance protein 1; and increased etoposide sensitivity in A549 cells. Furthermore, the ephrin type-A receptor 2 (EphA2) receptor was important for the recognition and biologic activity of β-glucan in A549 cells. EphA2 signaling includes nuclear factor kappa B (NF-κB), signal transducer and activator of transcription 3 (STAT3), and p38 mitogen-activated protein kinase (MAPK). However, treatment of cells with stattic (STAT3 inhibitor) or SB203580 (p38 MAPK inhibitor) did not diminish the effects of β-glucan. In contrast, knockdown of v-rel reticuloendotheliosis viral oncogene homolog B (RelB) abolished the effects of β-glucan, suggesting the involvement of the noncanonical NF-κB pathway. The β-glucan effects were also attenuated by the knockdown of WD40 Repeat protein 23 (WDR23). The β-glucan treatment and RelB overexpression induced the expression of Cullin-4A (CUL4A), which increased WDR23 ligase activity and promoted the subsequent depletion of Nrf2. These results revealed a novel property of β-glucan as a resistance-modifying agent in addition to its widely reported immunomodulatory effects for lung cancer therapy via the EphA2-RelB-CUL4A-Nrf2 axis. SIGNIFICANCE STATEMENT: Chemotherapeutic resistance remains a major obstacle in cancer therapy despite extensive efforts to elucidate the underlying molecular mechanisms and overcome multidrug resistance. The present study revealed a novel resistance-modifying property of β-glucan, thereby expanding our knowledge on the beneficial roles of β-glucan and providing an alternative strategy to prevent drug resistance by cancer. The present results provide evidence for the involvement of a novel mode of NF-κB and Nrf2 crosstalk in the drug resistance phenotype.

Temporary decrease in tacrolimus clearance in cytochrome P450 3A5 non-expressors early after living donor kidney transplantation: Effect of interleukin 6-induced suppression of the cytochrome P450 3A gene.

Tacrolimus is important for immunosuppression in kidney transplantation. In this historical cohort and in vitro study, we evaluated the changes in tacrolimus pharmacokinetics early after living donor kidney transplantation and the effects of interleukin (IL)-6 on cytochrome P450 3A4 (CYP3A4) and cytochrome P450 3A5 (CYP3A5) expression. In the historical cohort study, 22 patients who met the inclusion criteria were classified into CYP3A5 expressors and non-expressors (n=16 and 6, respectively). The blood tacrolimus concentration per dose ratio (C/D) temporarily increased post-kidney transplantation on days 3-4 only in CYP3A5 non-expressors. The effects of IL-6 on CYP3A4 and CYP3A5 expression were also investigated in vitro using HepG2 and Caco-2 cells. IL-6 induced a significant concentration- and time-dependent decrease in CYP3A4 and CYP3A5 expression in both cells. The mean CYP3A4 expression level at 12hours after IL-6 exposure (% of 0hour) was 44.0 and 62.6 in HepG2 and Caco-2 cells, respectively, whereas the CYP3A5 expression level was 30.7 and 52.4, respectively. We hypothesize that CYP3A5 non-expressors might exhibit a temporary decrease in the oral clearance of tacrolimus via an increase in serum IL-6 concentrations early after kidney transplantation. These results may help develop strategies to improve kidney transplant outcome.

CYP3A5 polymorphisms and their effects on tacrolimus exposure in an ethnically diverse South African renal transplant population.

Tacrolimus forms the cornerstone for immunosuppression in solid-organ transplantation. It has a narrow therapeutic window with wide inter- and intra-patient variability (IPV). Cytochrome P-450 3A5 (CYP3A5) is the main enzyme involved in tacrolimus metabolism, and rs776746A>G is the most frequently studied polymorphism in the CYP3A5 gene. The rs776746A>G (i.e. CYP3A5*3) single-nucleotide polymorphism in CYP3A5 alters tacrolimus predose trough concentration (C0) and may also affect IPV, which may lead to immune- and/or drug-mediated allograft injury. CYP3A5*3 may result in absent (*3/*3), partial (*1/*3) or normal (*1/*1) CYP3A5 expression. The effect of CYP3A5*3 on tacrolimus exposure and variability has not been examined in South African (SA) transplant recipients. To determine the frequencies and effect of CYP3A5 and adenosine triphosphate-binding cassette subfamily B member 1 (ABCB1) polymorphisms on tacrolimus C0/dose ratios in different ethnic groups attending a tertiary renal transplant clinic in SA, and other factors that may explain inter- and IPV in tacrolimus C0. All consenting stable renal transplant recipients on tacrolimus at the Livingstone Hospital Renal Unit in Port Elizabeth, SA, were included. Tacrolimus concentrations were obtained using a microparticle enzyme immunoassay method (ARCHITECT analyser, Abbott Laboratories). Polymerase chain reaction/restriction fragment length polymorphism was used to genotype for CYP3A5*3 and *6 allelic variants. There were 43 participants (35% black African, 44% mixed ancestry and 21% white), with a mean age of 44.5 years, median duration post-transplant of 47 months and median (interquartile range) creatinine and estimated glomerular filtration rate levels of 118(92- 140) µmol/L and 62 (49 - 76) mL/min at study inclusion. The mean tacrolimus C0 in the study was 6.7 ng/mL, with no difference across the different ethnic groups. However, the mean total daily dose of tacrolimus required was 9.1 mg (0.12 mg/kg), 7.2 mg (0.09 mg/kg) and 4.3 mg (0.06 mg/kg) in black, mixed-ancestry and white patients, respectively (p=0.017). The frequencies for CYP3A5 expressors (i.e. CYP3A5*1/*1 + CYP3A5*1/*3 genotypes) were 72%, 100%, 76% and 12% for all patients combined and black, mixed-ancestry and white patients, respectively. The frequencies for CYP3A5 non-expressors (i.e. CYP3A5*3/*3 genotypes) were 0%, 24% and 88% among the black, mixed-ancestry and white patients, respectively. None of the patients carried the CYP3A5*6 allele. CYP3A5*1/*1 and CYP3A5*1/*3 genotype carriers required a two-fold increase in dose compared with the non-expressor genotype carriers, CYP3A5*3/*3 (p<0.05). CYP3A5*3/*3 carriers also demonstrated higher IPV than CYP3A5*1/*1 and *1/*3 carriers (18.1% v. 14.2%; p=0.125). Compared with global transplant populations, SA renal transplant recipients demonstrated a very high rate of CYP3A5 expression, with a significant impact on tacrolimus pharmacokinetics. Genetic variation in CYP3A5 expression affects tacrolimus dosing requirements, and knowing the CYP3A5 genotype of transplant patients may allow better dose prediction compared with current standard dosing recommendations in a multi-ethnic population. Overall, black African patients required higher doses of tacrolimus than their white counterparts. While further prospective studies are needed to better evaluate dosing algorithms, it would appear that the starting dose of tacrolimus should be higher in black and mixed-race patients.

Abstract B77: CYP3A5 mediates basal and acquired therapy resistance in different subtypes of pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a very aggressive disease with poor prognosis. Treatment with gemcitabine, the FOLFIRINOX scheme or nab-paclitaxel offer only a modest increase in overall survival. For a number of other carcinomas, tumor subtypes have been uncovered that allow the use of targeted therapies. Although subtypes of PDAC were described, this malignancy is clinically still treated as a single disease. We established patient-derived models representing the full spectrum of previously identified quasi-mesenchymal (QM-PDA), classical and exocrine-like PDAC subtypes, and identified two markers—HNF1A and KRT81—that enable stratification of tumors into different subtypes by immunohistochemistry. Patients bearing tumors of these subtypes show significant differences in overall survival and their tumors differ in drug sensitivity, with the exocrine-like subtype being resistant to tyrosine kinase inhibitors and paclitaxel. The xenobiotic enzyme, cytochrome P450 3A5 (CYP3A5), metabolizes these compounds in tumors of the exocrine-like subtype, and pharmacological or short hairpin RNA (shRNA)-mediated CYP3A5 inhibition sensitizes tumor cells to these drugs. Additionally, retrospective analysis of a large patient cohort confirmed that CYP3A5 is predominantly found in those patient tumors classified as exocrine-like. Whereas the hepatocyte nuclear factor 4, alpha (HNF4A) controls basal expression of CYP3A5, drug-induced CYP3A5 upregulation is mediated by the nuclear receptor NR1I2. Interfering with these regulatory mechanisms may provide an alternative approach to suppress the CYP3A5 pathway. CYP3A5 also contributes to acquired drug resistance in QM-PDA and classical PDAC, and is highly expressed in several additional malignancies. These findings designate CYP3A5 as predictor of therapy response and as a tumor cell-autonomous detoxification mechanism that must be overcome to prevent drug resistance. Citation Format: Elisa M. Noll, Christian Eisen, Albrecht Stenzinger, Elisa Espinet, Alexander Muckenhuber, Corinna Klein, Vanessa Vogel, Bernd Klaus, Wiebke Nadler, Christoph Rösli, Christian Lutz, Michael Kulke, Jan Engelhardt, Franziska Zickgraf, Octavio Espinosa, Matthias Schlesner, Xiaoqi Jiang, Annette Kopp-Schneider, Peter Neuhaus, Marcus Bahra, Bruno V. Sinn, Roland Eils, Nathalia A. Giese, Thilo Hackert, Oliver Strobel, Jens Werner, Markus W. Büchler, Wilko Weichert, Andreas Trumpp, Martin R. Sprick.{Authors}. CYP3A5 mediates basal and acquired therapy resistance in different subtypes of pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr B77.

Abstract LB-120: CYP3A5 mediates basal and acquired therapy resistance in different subtypes of pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive disease with poor prognosis. Treatment with gemcitabine, the FOLFIRINOX scheme or nab-paclitaxel offer only a modest increase in overall survival. For a number of other carcinomas, tumor subtypes have been uncovered that allow the use of targeted therapies. Although subtypes of PDAC were described, this malignancy is clinically still treated as a single disease. We established patient-derived models representing the full spectrum of previously identified quasi-mesenchymal (QM-PDA), classical and exocrine-like PDAC subtypes, and identified two markers—HNF1A and KRT81—that enable stratification of tumors into different subtypes by immunohistochemistry. Patients bearing tumors of these subtypes show significant differences in overall survival and their tumors differ in drug sensitivity, with the exocrine-like subtype being resistant to tyrosine kinase inhibitors and paclitaxel. The xenobiotic enzyme, cytochrome P450 3A5 (CYP3A5), metabolizes these compounds in tumor cells of the exocrine-like subtype, and pharmacological or short hairpin RNA (shRNA)-mediated CYP3A5 inhibition sensitizes tumor cells to these drugs. Additionally, retrospective analysis of a large patient cohort confirmed that CYP3A5 is predominantly found in those patient tumors classified as exocrine-like (Noll, Eisen et al., Nature Medicine (2016) accepted). Whereas the hepatocyte nuclear factor 4, alpha (HNF4A) controls basal expression of CYP3A5, drug-induced CYP3A5 upregulation is mediated by the nuclear receptor NR1I2. Interfering with these regulatory mechanisms may provide an alternative approach to suppress the CYP3A5 mediated resistance pathway. CYP3A5 also contributes to acquired drug resistance in QM-PDA and classical PDAC in vitro and in vivo. Finally, CYP3A5 is highly expressed in several additional malignancies including hepatocellular and cervical carcinomas raising the possibility that the CYP3A5 resistance mechanism is operational in a variety of human cancers. These findings designate CYP3A5 as predictor of therapy response and as a tumor cell-autonomous detoxification mechanism that must be overcome to prevent drug resistance. Citation Format: Elisa M. Noll, Christian Eisen, Albrecht Stenzinger, Elisa Espinet, Alexander Muckenhuber, Corinna Klein, Vanessa Vogel, Bernd Klaus, Wiebke Nadler, Christoph Rösli, Christian Lutz, Michael Kulke, Jan Engelhardt, Franziska Zickgraf, Octavio Espinosa, Matthias Schlesner, Xiaoqi Jiang, Annette Kopp-Schneider, Peter Neuhaus, Marcus Bahra, Bruno Sinn, Roland Eils, Nathalia Giese, Thilo Hackert, Oliver Strobel, Jens Werner, Markus W. Büchler, Wilko Weichert, Andreas Trumpp, Martin R. Sprick. CYP3A5 mediates basal and acquired therapy resistance in different subtypes of pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-120.

Abstract IA22: CYP3A5 mediates resistance to small molecule inhibitors in a subtype of pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDAC) is clinically still treated as a single disease. We have generated patient-derived models representing the recently identified quasi-mesenchymal, classical and exocrine-like PDAC subtypes, and report a two-marker set facilitating patient stratification by immunohistochemistry. The subtypes show significant differences in overall survival and drug sensitivity, with the exocrine-like subtype being resistant to the tyrosine kinase inhibitors erlotinib, dasatinib, as well as the chemotherapeutic paclitaxel. Highly expressed cytochrome P450 3A5 (CYP3A5) actively metabolizes these compounds in the exocrine-like subtype, and pharmacological or shRNA-mediated CYP3A5 inhibition sensitizes tumor cells in vivo. Additionally, we investigated the transcriptional network underlying the subtype-specific CYP3A5 expression. Hence, these data show that exocrine-like PDAC adopts a highly effective detoxification mechanism akin to that of hepatocytes. High expression of CYP3A5 in other tumor entities suggests this pathway as an important target to overcome drug resistance and to predict response to therapy with small molecule drugs. Citation Format: Elisa M. Noll, Elisa M. Noll, Christian Eisen, Christian Eisen, Elisa Espinet, Elisa Espinet, Albrecht Stenzinger, Wilko Weichert, Martin R. Sprick, Andreas Trumpp, Andreas Trumpp. CYP3A5 mediates resistance to small molecule inhibitors in a subtype of pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr IA22.

CYP3A5 mediates basal and acquired therapy resistance in different subtypes of pancreatic ductal adenocarcinoma.

Although subtypes of pancreatic ductal adenocarcinoma (PDAC) were described, this malignancy is clinically still treated as a single disease. Here, we present patient-derived models representing the full spectrum of previously identified quasi-mesenchymal (QM-PDA), classical and exocrine-like PDAC subtypes, and identify two markers—HNF1A and KRT81—that enable stratification of tumors into different subtypes by immunohistochemistry. Individuals bearing tumors of these subtypes show significant differences in overall survival and their tumors differ in drug sensitivity, with the exocrine-like subtype being resistant to tyrosine kinase inhibitors and paclitaxel. Cytochrome P450 3A5 (CYP3A5) metabolizes these compounds in tumors of the exocrine-like subtype, and pharmacological or shRNA-mediated CYP3A5 inhibition sensitizes tumor cells to these drugs. Whereas hepatocyte nuclear factor 4 alpha (HNF4A) controls basal expression of CYP3A5, drug-induced CYP3A5 upregulation is mediated by the nuclear receptor NR1I2. CYP3A5 also contributes to acquired drug resistance in QM-PDA and classical PDAC, and is highly expressed in several additional malignancies. These findings designate CYP3A5 as predictor of therapy response and as a tumor cell-autonomous detoxification mechanism that must be overcome to prevent drug resistance.

Renal drug metabolism in humans: the potential for drug–endobiotic interactions involving cytochrome P450 (CYP) and UDP‐glucuronosyltransferase (UGT)

Although knowledge of human renal cytochrome P450 (CYP) and UDP-glucuronosyltransferase (UGT) enzymes and their role in xenobiotic and endobiotic metabolism is limited compared with hepatic drug and chemical metabolism, accumulating evidence indicates that human kidney has significant metabolic capacity. Of the drug metabolizing P450s in families 1 to 3, there is definitive evidence for only CYP 2B6 and 3A5 expression in human kidney. CYP 1A1, 1A2, 1B1, 2A6, 2C19, 2D6 and 2E1 are not expressed in human kidney, while data for CYP 2C8, 2C9 and 3A4 expression are equivocal. It is further known that several P450 enzymes involved in the metabolism of arachidonic acid and eicosanoids are expressed in human kidney, CYP 4A11, 4F2, 4F8, 4F11 and 4F12. With the current limited evidence of drug substrates for human renal P450s drug-endobiotic interactions arising from inhibition of renal P450s, particularly effects on arachidonic acid metabolism, appear unlikely. With respect to the UGTs, 1A5, 1A6, 1A7, 1A9, 2B4, 2B7 and 2B17 are expressed in human kidney, whereas UGT 1A1, 1A3, 1A4, 1A8, 1A10, 2B10, 2B11 and 2B15 are not. The most abundantly expressed renal UGTs are 1A9 and 2B7, which play a significant role in the glucuronidation of drugs, arachidonic acid, prostaglandins, leukotrienes and P450 derived arachidonic acid metabolites. Modulation by drug substrates (e.g. NSAIDs) of the intrarenal activity of UGT1A9 and UGT2B7 has the potential to perturb the metabolism of renal mediators including aldosterone, prostaglandins and 20-hydroxyeicosatetraenoic acid, thus disrupting renal homeostasis.

Cytochrome P450 3A5 Genotype Is Associated with Early Post-Operative Tacrolimus Serum Levels in Lung Transplant Patients

Purpose Tacrolimus use in lung transplantation is complicated by variable pharmacokinetics. Cytochrome P450 3A5 (CYP3A5) is one enzyme involved in tacrolimus metabolism. Due to aberrant mRNA splicing, CYP3A5 expression is decreased in major allele homozygotes at the rs776746 locus. This SNP has been associated with differing dose requirements after one or more months of therapy, but its importance has not been studied immediately after transplantation, when initiation of the proper dose may be the most critical. We hypothesized that CYP3A5 genotype at rs776746 would be associated with tacrolimus time-within-therapeutic range (TTR) in the early post-transplant period. Methods and Materials We performed a retrospective cohort study of patients undergoing lung or heart-lung transplantation at a single center. Genotype was determined using a cardiovascular gene centric SNP array. Tacrolimus TTR was calculated as the percentage of total tacrolimus trough levels that were therapeutic (8-12 ng/mL) within 14 days of transplantation or prior to hospital discharge. The association between genotype and TTR was tested using the Wilcoxon rank-sum test. Results Genotype and tacrolimus troughs were available in 213 patients, 198 (93%) of whom had at least four trough levels. Due to large population differences in allele frequency by race, analysis was restricted to the 171 (86%) Caucasian recipients. 150 (88%) patients were major allele homozygotes, 21 (12%) were heterozygotes, and there were no minor allele homozygotes. The median number of tacrolimus trough levels was 11 in both groups (p=0.58). Homozygotes had a median TTR of 35.7%, compared with a median TTR of 22.2% in heterozygotes (p=0.005). Conclusions Major allele homozygotes at rs776746 in CYP3A5 have higher TTR in the early post-lung transplant period compared to heterozygotes at this locus. In the future, trials to evaluate the clinical impact of initial post-lung transplant tacrolimus dosing based on genotype may be warranted.

Molecular diversity and population structure at the Cytochrome P450 3A5 gene in Africa

BackgroundCytochrome P450 3A5 (CYP3A5) is an enzyme involved in the metabolism of many therapeutic drugs. CYP3A5 expression levels vary between individuals and populations, and this contributes to adverse clinical outcomes. Variable expression is largely attributed to four alleles, CYP3A5*1 (expresser allele); CYP3A5*3 (rs776746), CYP3A5*6 (rs10264272) and CYP3A5*7 (rs41303343) (low/non-expresser alleles). Little is known about CYP3A5 variability in Africa, a region with considerable genetic diversity. Here we used a multi-disciplinary approach to characterize CYP3A5 variation in geographically and ethnically diverse populations from in and around Africa, and infer the evolutionary processes that have shaped patterns of diversity in this gene. We genotyped 2538 individuals from 36 diverse populations in and around Africa for common low/non-expresser CYP3A5 alleles, and re-sequenced the CYP3A5 gene in five Ethiopian ethnic groups. We estimated the ages of low/non-expresser CYP3A5 alleles using a linked microsatellite and assuming a step-wise mutation model of evolution. Finally, we examined a hypothesis that CYP3A5 is important in salt retention adaptation by performing correlations with ecological data relating to aridity for the present day, 10,000 and 50,000 years ago.ResultsWe estimate that ~43% of individuals within our African dataset express CYP3A5, which is lower than previous independent estimates for the region. We found significant intra-African variability in CYP3A5 expression phenotypes. Within Africa the highest frequencies of high-activity alleles were observed in equatorial and Niger-Congo speaking populations. Ethiopian allele frequencies were intermediate between those of other sub-Saharan African and non-African groups. Re-sequencing of CYP3A5 identified few additional variants likely to affect CYP3A5 expression. We estimate the ages of CYP3A5*3 as ~76,400 years and CYP3A5*6 as ~218,400 years. Finally we report that global CYP3A5 expression levels correlated significantly with aridity measures for 10,000 [Spearmann’s Rho= −0.465, p=0.004] and 50,000 years ago [Spearmann’s Rho= −0.379, p=0.02].ConclusionsSignificant intra-African diversity at the CYP3A5 gene is likely to contribute to multiple pharmacogenetic profiles across the continent. Significant correlations between CYP3A5 expression phenotypes and aridity data are consistent with a hypothesis that the enzyme is important in salt-retention adaptation.

Association Between Tacrolimus Concentration and Genetic Polymorphisms of CYP3A5 and ABCB1 During the Early Stage After Liver Transplant in an Iranian Population

Tacrolimus is widely used as an immunosuppressive drug in liver transplant recipients with a narrow therapeutic range and variable individualized pharmacokinetics. Tacrolimus is a substrate of cytochrome P-450 3A enzyme and the drug transporter, P-glycoprotein. We determined the genotypic frequencies of cytochrome P-4503A5 (rs776746), and ABCB1 (rs1045642), single nucleotide polymorphisms in a population of 100 Iranian liver transplant patients, and investigated the influence of the above-mentioned single nucleotide polymorphisms on tacrolimus concentrations. At 7 and 30 days after transplant, tacrolimus dosages (mg/kg/d), trough blood levels (T0), and dose-adjusted concentrations (concentration/dosage ratio) were determined. Polymerase chain reaction, followed by restriction fragment length polymorphism analysis, was used for genotyping cytochrome P-4503A5*3 [6986A>G] as well as ABCB1 [3435C>T]. Ninety-five percent of the population showed a cytochrome P-4503A5*3/*3 genotype. ABCB13435TT genotype was observed in 33 cases (33%); whereas 51 cases (51%) carried 3435CT, and 16 cases (16%) carried 3435CC. With regard to the ABCB1 and cytochrome P-4503A5, they showed no influence on tacrolimus dosing requirements at 1 week or 1 month after transplant. No association of any genetic variant with the acute rejection rate was found. Finally, as the liver donor genotype influences tacrolimus pharmacokinetics with regard to expression of cytochrome P-4503A5, far more than the genotype of the recipient; therefore, it should be considered before recommending any personal immunosuppressive treatment based on pharmacogenetics.

Expression of CYP3A5 and P-glycoprotein in Renal Allografts With Histological Signs of Calcineurin Inhibitor Nephrotoxicity

Susceptibility to calcineurin inhibitor nephrotoxicity (CNIT) after solid organ transplantation could be related to an interindividual variability in renal expression and function of the metabolizing cytochrome P450 3A5 (CYP3A5) isoenzyme and of the multidrug efflux transporter P-glycoprotein (P-gp, ABCB1). We compared renal expression of CYP3A5 and P-gp, measured by immunohistochemistry, in 32 renal allograft biopsies with de novo arteriolar hyalinosis as a sign of CNIT with a control group, consisting of normal protocol allograft biopsies (n=50) and protocol biopsies demonstrating alloimmune injury (n=21). In addition, we studied the association between renal expression and donor and recipient single-nucleotide polymorphisms CYP3A5 A6986G (rs776746), ABCB1 C3435T (rs1045642), and G2677T (rs2032582). CYP3A5 positivity at the brushborder of the proximal tubules was present in 47% of CNIT and 14% of control biopsies (P<0.01). In contrast, brushborder staining for CYP3A5 in distal tubules was present in 10% of CNIT and 39% of control biopsies (P<0.01). No significant association between tubular cell P-gp expression and CNIT was detected. The presence of genetic polymorphisms CYP3A5 A6986G and ABCB1 C3435T and G2677T in donors and recipients was not predictive of the renal expression profile of these molecules. Based on retrospectively collected data of 103 renal transplant recipients receiving tacrolimus in combination with mycophenolate mofetil and corticosteroids, we found that renal expression and localization of CYP3A5 but not P-gp is associated with the occurrence of CNIT. Common genetic polymorphisms in these proteins did not influence their expression profile as measured by immunohistochemistry.

The impact of CYP3A5*3 on risk and prognosis in childhood acute lymphoblastic leukemia

Acute lymphoblastic leukemia (ALL) is the most common cancer in childhood; however, little is known of the molecular etiology and environmental exposures causing the disease. Cytochrome P450 3A5 (CYP3A5) plays a crucial role in the catalytic oxidation of endogenous metabolites and toxic substances, including chemotherapeutic agents. The aim of this study was to investigate the role of a single-nucleotide polymorphism (CYP3A5*3 6986A>G), which renders low enzyme activity, in the risk of developing ALL and in the outcome for children with ALL. Six hundred and sixteen childhood patients with ALL and 203 controls were genotyped by allelic discrimination. Individuals with the A allele had a 64% increased risk of developing childhood ALL (odds ratio = 1.64; 95% CI, 1.009-2.657). In general, event-free survival (EFS) did not differ in relation to CYP3A5 genotype. However, for patients with T-ALL, presence of the A allele was associated with better prognosis (EFS = 94.1%), while patients with the low-activity GG genotype only had an EFS of 61.5% (P = 0.015). Thus, for patients with T-ALL having no A allele and therefore low expression of CYP3A5, the risk of experiencing an event was almost eight times higher compared to those having at least one A allele (P = 0.045, hazard ratio = 7.749; 95% CI, 1.044-57.52). This study shows that genetics may play a role in the risk of developing childhood ALL and indicates that improved treatment stratification of childhood patients with ALL may require addition of host genetic information.

A Systematic Review of the Effect of CYP3A5 Genotype on the Apparent Oral Clearance of Tacrolimus in Renal Transplant Recipients

Tacrolimus is a commonly used immunosuppressive agent in renal transplantation. Therapeutic drug monitoring of tacrolimus is recommended because it demonstrates wide pharmacokinetic interpatient variability. Part of that variability may be the result of metabolism by cytochrome P450 3A5 (CYP3A5), which is only expressed in some adult individuals. The expression of CYP3A5 has been linked to the CYP3A5 genotype, in which individuals with one or more wild-type allele (CYP3A5*1) are considered CYP3A5 expressors, and individuals homozygous for the mutant allele CYP3A5*3 are considered nonexpressors. An association has been established between CYP3A5 genotype (expressors versus nonexpressors) and tacrolimus dose requirements to achieve target concentrations. Tacrolimus pharmacokinetic variability is based on bioavailability and systemic clearance, which are represented by apparent oral clearance. The focus of this review was to use a systematic method to investigate whether the CYP3A5 genotype has an effect on the apparent oral clearance of tacrolimus in renal transplant recipients. A total of five studies were identified that reported apparent oral clearance in CYP3A5 expressors and CYP3A5 nonexpressors. The weighted mean apparent oral clearance was found to be 48% lower in CYP3A5 nonexpressors than CYP3A5 expressors (range, 26%-65%). This difference in apparent oral clearance could be used in future studies to guide initial dosing strategies of tacrolimus in renal transplant recipients based on genotype.

Expression and Induction of Cytochrome P450 Isoenzymes in Human Skin Equivalents

Organotypic skin models are frequently used for a wide range of applications and latterly also for dermatotoxicological studies. To evaluate their practicability for the investigation of xenobiotic metabolism in human skin we compared three types of organotypic skin models, acquired by purchase from different manufacturers, to a self-constructed in-house model with regard to cytochrome P450 (CYP) isoenzyme expression on mRNA and protein level and the inducibility of these enzymes by aryl hydrocarbon receptor ligands. To induce enzyme activity, models were treated with benzanthracene, liquor carbonis detergens, pix lithanthracis or dimethyl sulfoxide as a solvent control. RNA was isolated by phenol-chloroform extraction and purified. Gene expression patterns were studied by cDNA microarray analysis. Microarray data were confirmed by real-time PCR. For quality control of the models and to detect and localize enzyme expression, immunofluorescence staining was performed with antibodies against CYPs and structure proteins. The immunofluorescence staining demonstrated the regular structure of our models. We could provide evidence for the expression of CYP types 1A1, 1B1, 2E1, 2C and 3A5 in organotypic skin models. The expression of CYP1A1 and CYP1B1 was highly inducible by treatment with liquor carbonis detergens. The proof of the expression and inducibility of CYP enzymes in organotypic skin models suggests that skin equivalents are a valuable tool that can emulate CYP-dependent metabolism of drugs and other xenobiotics in human skin.

Apports récents de la pharmacologie des traitements immunosuppresseurs utilisés en transplantation d’organe

Individualization of immunosuppressive drugs may be helped by the use of therapeutic drug monitoring determining the best unique time point for concentration to be controlled or using limited sample strategy to perform abbreviated area under the curve (AUC). Pharmacogenetic analyzes the role of enzymes genetic polymorphisms on pharmacokinetic characteristics. Recent studies have demonstrated that cyclosporine concentration obtained 2 hours after oral intake is the best single point. However, a recent analysis of published data did not sustain the clinical benefit of this approach. Regarding mycophenolate mofetil, the targeted AUC has been defined between 30 and 60 mg/h/l. Clinical results are contradictory and it is not possible for the time being to give a clear answer to its clinical relevance. For tacrolimus, it has been demonstrated that the daily dose needed to achieve adequate trough levels are correlated with cytochrome P450 3A5 expression and therefore the presence of *1 allele. A prospective study has been conducted to demonstrate the clinical consequences of this pharmacogenetic approach.

RT-PCR-based cytochrome P450 expression profile of oral tissue samples1

To analyse the expression of individual forms of cytochrome P450 (CYP) at the mRNA level in five homogenized oral buccal tissue samples from four individuals with or without oral malignancy. Individual forms of CYPs were studied by reverse transcriptase-polymerase chain reaction (RT-PCR), using specific primers for CYPs 2B6, 2C, 2D6, 2E1, 3A3/4 and 3A5, and oral CYP expressions were compared with CYP expression in liver tissue. Consistent expression of CYPs 2C, 2E1 and 3A5 was observed in oral buccal tissue at mRNA level. These particular CYPs have possible roles in the protection of the body against orally ingested xenobiotics as well as influence the bioavailability of therapeutic compounds.

Cytochrome P450 3A5 expression in the kidneys of patients with calcineurin inhibitor nephrotoxicity

Nephrotoxicity secondary to calcineurin inhibitors is common in renal transplant recipients, occurring in 76-94% of patients. The role of drug transporters (P-glycoprotein) and drug metabolizing enzymes (cytochrome P450) as predisposing factors toward nephrotoxicity or its prevention has not been thoroughly examined. The objective of this study was to analyse cytochrome P450 3A5 (CYP3A5) expression in kidneys of solid organ recipients by immunohistochemistry to determine if there is an association between expression of this enzyme and calcineurin inhibitor toxicity. Transplant recipients were compared with a control group. Apical tubular plasma membrane staining for CYP3A5 was present in 62% of study and 100% of control biopsies (P=0.0012). Proximal and distal tubular nuclear staining intensity was similar between groups. Cytoplasmic staining in both the proximal (2.1+/-0.9 vs 1.4+/-0.9) and distal (2.8+/-0.5 vs 1.8+/-1.1) tubules was greater in the control vs study population specimens, respectively (P=0.0093 and P=0.0005, respectively). Regression models that controlled for use of CYP3A inhibiting and inducing medications, age, gender, race and glomerular filtration rate did not predict differences between study groups with regard to staining locations and intensity, except for the cytoplasm of the distal tubule, where intensity of staining was significantly lower in the study group (0.9+/-0.3; P=0.002). This study showed decreased expression of CYP3A5 in nephrotoxic biopsies as compared with a control group. Our data suggest that the relationship between reduced presence of CYP3A5 in the kidney tubules and nephrotoxicity should be further explored to elucidate the role of this enzyme in mediating toxicity.

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.