Differences In Drug-metabolizing Enzymes Research Articles

Ontogeny of Small Intestinal Drug Transporters and Metabolizing Enzymes Based on Targeted Quantitative Proteomics.

Most drugs are administered to children orally. An information gap remains on the protein abundance of small intestinal drug-metabolizing enzymes (DMEs) and drug transporters (DTs) across the pediatric age range, which hinders precision dosing in children. To explore age-related differences in DMEs and DTs, surgical leftover intestinal tissues from pediatric and adult jejunum and ileum were collected and analyzed by targeted quantitative proteomics for apical sodium-bile acid transporter, breast cancer resistance protein (BCRP), monocarboxylate transporter 1 (MCT1), multidrug resistance protein 1 (MDR1), multidrug resistance-associated protein (MRP) 2, MRP3, organic anion-transporting polypeptide 2B1, organic cation transporter 1, peptide transporter 1 (PEPT1), CYP2C19, CYP3A4, CYP3A5, UDP glucuronosyltransferase (UGT) 1A1, UGT1A10, and UGT2B7. Samples from 58 children (48 ileums, 10 jejunums, age range: 8 weeks to 17 years) and 16 adults (8 ileums, 8 jejunums) were analyzed. When comparing age groups, BCRP, MDR1, PEPT1, and UGT1A1 abundance was significantly higher in adult ileum as compared with the pediatric ileum. Jejunal BCRP, MRP2, UGT1A1, and CYP3A4 abundance was higher in the adults compared with children 0-2 years of age. Examining the data on a continuous age scale showed that PEPT1 and UGT1A1 abundance was significantly higher, whereas MCT1 and UGT2B7 abundance was lower in adult ileum as compared with the pediatric ileum. Our data contribute to the deeper understanding of the ontogeny of small intestinal drug-metabolizing enzymes and drug transporters and shows DME-, DT-, and intestinal location-specific, age-related changes. SIGNIFICANCE STATEMENT: This is the first study that describes the ontogeny of small intestinal DTs and DMEs in human using liquid chromatography with tandem mass spectrometry-based targeted quantitative proteomics. The current analysis provides a detailed picture about the maturation of DT and DME abundances in the human jejunum and ileum. The presented results supply age-related DT and DME abundance data for building more accurate PBPK models that serve to support safer and more efficient drug dosing regimens for the pediatric population.

Pharmacokinetic modeling of R and S‐Methadone and their metabolites to study the effects of various covariates in post‐operative children

Methadone is a synthetic opioid used as an analgesic and for the treatment of opioid abuse disorder. The analgesic dose in the pediatric population is not well‐defined. The pharmacokinetics (PKs) of methadone is highly variable due to the variability in alpha‐1 acid glycoprotein (AAG) and genotypic differences in drug‐metabolizing enzymes. Additionally, the R and S enantiomers of methadone have unique PK and pharmacodynamic properties. This study aims to describe the PKs of R and S methadone and its metabolite 2‐ethylidene‐1,5‐dimethyl‐3,3‐diphenylpyrrolidine (EDDP) in pediatric surgical patients and to identify sources of inter‐ and intra‐individual variability. Children aged 8–17.9 years undergoing orthopedic surgeries received intravenous methadone 0.1 mg/kg intra‐operatively followed by oral methadone 0.1 mg/kg postoperatively every 12 h. Pharmacokinetics of R and S methadone and EDDP were determined using liquid chromatography tandem mass spectrometry assays and the data were modeled using nonlinear mixed‐effects modeling in NONMEM. R and S methadone PKs were well‐described by two‐compartment disposition models with first‐order absorption and elimination. EDDP metabolites were described by one compartment disposition models with first order elimination. Clearance of both R and S methadone were allometrically scaled by bodyweight. CYP2B6 phenotype was a determinant of the clearance of both the enantiomers in an additive gene model. The intronic CYP3A4 single‐nucleotide polymorphism (SNP) rs2246709 was associated with decreased clearance of R and S methadone. Concentrations of AAG and the SNP of AAG rs17650 independently increased the volume of distribution of both the enantiomers. The knowledge of these important covariates will aid in the optimal dosing of methadone in children.

Changes and sex- and age-related differences in the expression of drug metabolizing enzymes in a KRAS-mutant mouse model of lung cancer.

BackgroundThis study aimed to systematically profile the alterations and sex- and age-related differences in the drug metabolizing enzymes (DMEs) in a KRAS-mutant mouse model of lung cancer (KRAS mice).MethodologyIn this study, the LC-MS/MS approach and a probe substrate method were used to detect the alterations in 21 isoforms of DMEs, as well as the enzymatic activities of five isoforms, respectively. Western blotting was applied to study the protein expression of four related receptors.ResultsThe proteins contents of CYP2C29 and CYP3A11, were significantly downregulated in the livers of male KRAS mice at 26 weeks (3.7- and 4.4-fold, respectively, p < 0.05). SULT1A1 and SULT1D1 were upregulated by 1.8- to 7.0- fold at 20 (p = 0.015 and 0.017, respectively) and 26 weeks (p = 0.055 and 0.031, respectively). There were positive correlations between protein expression and enzyme activity for CYP2E1, UGT1A9, SULT1A1 and SULT1D1 (r2 ≥ 0.5, p < 0.001). Western blotting analysis revealed the downregulation of AHR, FXR and PPARα protein expression in male KRAS mice at 26 weeks. For sex-related differences, CYP2E1 was male-predominant and UGT1A2 was female-predominant in the kidney. UGT1A1 and UGT1A5 expression was female-predominant, whereas UGT2B1 exhibited male-predominant expression in liver tissue. For the tissue distribution of DMEs, 21 subtypes of DMEs were all expressed in liver tissue. In the intestine, the expression levels of CYP2C29, CYP27A1, UGT1A2, 1A5, 1A6a, 1A9, 2B1, 2B5 and 2B36 were under the limitation of quantification. The subtypes of CYP7A1, 1B1, 2E1 and UGT1A1, 2A3, 2B34 were detected in kidney tissue.ConclusionsThis study, for the first time, unveils the variations and sex- and age-related differences in DMEs in C57 BL/6 (WT) mice and KRAS mice.

DNA methylation determines the regulation of pregnane X receptor on CYP3A4 expression.

The expression and activity of CYP3A4 vary among individuals. With the development of epigenetics, it is now possible to elucidate interindividual differences in drug-metabolizing enzymes. Here, we aimed to explore the potential relationship between DNA methylation and CYP3A4 expression. We analyzed the effect of a DNA methylation inhibitor, 5-aza-2-deoxycytidine, on pregnane X receptor (PXR) and CYP3A4 expression in HepG2 cells. In addition, pCpGL-CYP3A4-promoter and pCpGL-CYP3A4-enhancer plus promoter plasmids were constructed, methylated, and transfected. We found that treatment with 5-aza-2-deoxycytidine significantly increased the expression of PXR and CYP3A4 in a concentration- and time-dependent manner. In addition, CYP3A4 expression was significantly enhanced by overexpressing PXR via transfection of pSG5-PXR plasmids. Methylation of CYP3A4 enhancer inhibited CYP3A4 transcriptional activity mediated through PXR and inhibited the binding of PXR and CYP3A4 promoter. We also observed that when the promoter and enhancer of CYP3A4 were methylated, CYP3A4 expression did not increase after treatment with rifampicin. In conclusion, the investigation demonstrates that DNA methylation of CYP3A4 enhancer significantly inhibits CYP3A4 expression, mediated through PXR, which is not influenced by rifampicin.

Development and Validation of a Rapid and Reliable Real-Time PCR Method for CYP3A5 Genotyping

A prominent example for inter-individual differences in drug-metabolizing enzymes is the cytochrome P450 family. These monooxygenases comprise enzymes responsible for metabolism of about 90% of common medications. CYP3A5 and CYP3A4 account for 50% of hepatic cytochrome P450 and conversion of about half of all their substrates. CYP3A5 is the predominant extra-hepatic CYP enzyme and shows varying inter-individual expression attributable to genetic variations in the corresponding gene. CYP3A5*2 and *3 are the most common among Caucasian populations. Among CYP3A5 substrates are cyclosporine and tacrolimus prescribed after organ transplantations. A high incidence of nephrotoxic events after administering these drugs is related to low expression of the CYP3A5 enzyme. A fast and reliable genotyping method for the CYP3A5 gene would help avoid unwanted adverse drug reactions. Blood samples from 143 Caucasian subjects were genotyped by means of a real-time PCR multiplex assay testing the two CYP3A5 variations, CYP3A5S*2 and *3. This assay was validated against RFLP-PCR. Both mutations examined could be found in the study. No sample was homozygous for CYP3A5*2, but 2 out of 143 showed heterozygosity (allele frequency: 0.7%). For the CYP3A5*3 variant 17 samples were heterozygous and 115 were homozygous (allele frequency 86.4%). The multiplex real-time PCR yields shorter hands-on time and reduced cost compared to RFLP-PCR. Establishment of a multiplex real-time PCR has been successful as could be proven by correctly identifying the desired mutations CYP3A5*2 and CYP3A5*3 against a standard reference method.

Obstetric therapeutics-how pharmacogenetics may inform drug therapy for pregnant women in the future.

Most pregnant women take prescription medications. There are many factors related to pregnancy that make finding the most effective and safe dose of a therapeutic drug difficult for providers. Genetic differences in drug-metabolizing enzymes, transporters, and receptors may also account for some of the differences in drug response. Single-nucleotide polymorphisms in drug-metabolizing enzymes, such as the cytochrome P450 families, have been studied in relation to drugs used in pregnancy. Combining clinical characteristics, physiologic parameters in pregnancy, and possibly pharmacogenetic models may allow for providers to individualize pharmacotherapy in pregnancy and get to the most effective and safe dose of medication more quickly than in the current practice model. This article discusses these issues along with helpful Web sites and references for providers.

Humanized Transgenic Mouse Models for Drug Metabolism and Pharmacokinetic Research

Extrapolation of the metabolic, pharmacokinetic and toxicological data obtained from animals to humans is not always straightforward, given the remarkable species difference in drug metabolism that is due in large part to the differences in drug-metabolizing enzymes between animals and humans. Furthermore, genetic variations in drug-metabolizing enzymes may significantly alter pharmacokinetics, drug efficacy and safety. Thus, humanized transgenic mouse lines, in which the human drug-metabolizing enzymes are expressed in mouse tissues in the presence or absence of mouse orthologues, have been developed to address such challenges. These humanized transgenic mice are valuable animal models in understanding the significance of specific human drug-metabolizing enzymes in drug clearance and pharmacokinetics, as well as in predicting potential drug-drug interactions and chemical toxicity in humans. This review, therefore, aims to summarize the development and application of some humanized transgenic mouse models expressing human drug-metabolizing enzymes. The limitations of these genetically modified mouse models are also discussed.

Genetic Polymorphisms in CYP3A5 and MDR1 Genes and Their Correlations With Plasma Levels of Tacrolimus and Cyclosporine in Renal Transplant Recipients

Immunosuppressive drugs, such as tacrolimus (FK506) and cyclosporine (CsA), play an essential role in graft survival, preventing rejection. Large interindividual differences in drug-metabolizing enzymes as well as in drug transporters make the task of reaching the optimal concentrations difficult. The bioavailability of CsA and FK506 seems to be associated with the cytocrhome P450 IIIA ( CYP3A) gene. It has also been described that the Multi Drug Resistance 1 ( MDR1) gene that encodes for polyglycoprotein-P (P-gp) may influence the metabolizing action of FK506 and CsA. Therefore, we sought, to correlate single nucleotide polymorphisms (SNPs) in the CYP3A and MDR1 genes with the concentrations of FK506 and CsA. For this purpose we analyzed 2 groups of renal transplant recipients by sequencing: one receiving a CsA immunosuppressive regime, and other, an FK506-immunosuppression. This study showed that subjects in the FK506 group who had encoded the 1236C>T substitution in the MDR1 gene displayed 44.4% higher drug concentrations compared with (“wild-type”) individuals. Individuals carrying the 2677G>T,A mutation showed FK506 concentrations that were 44.7% higher than the wild-type individuals. Concerning the CsA group, individuals carrying the 22915A>C substitution displayed CsA concentrations 52.1% higher than wild-type individuals.

Pharmacogenomic progress in individualized dosing of key drugs for cancer patients

Determining the correct dosage for the majority of traditional chemotherapeutic agents presents a challenge because most drugs have a narrow therapeutic index, which results in a fine balance between doses that cause significant drug toxicity and loss of efficacy. Dosing calculations for most agents use the patient's body surface area, a method that correlates poorly with drug pharmacokinetics. Genetic differences in drug-metabolizing enzymes are being evaluated in an effort to explain the pharmacokinetic and pharmacodynamic variability seen with many chemotherapeutic agents. Elucidation of the underlying reasons for this variability will enable individualization of therapy to minimize toxicity and maximize efficacy, and thus improve control over the narrow therapeutic index of these agents. Such investigations have led to Clinical Pharmacology FDA Subcommittee recommendations for changes to drug package instructions. This Review discusses the current limitations of body-surface-area-based dosing, examples of successful pharmacogenomic investigations that have used drug-metabolizing enzymes to decrease drug toxicity and/or improve efficacy, and the future promises of pharmacogenomic-directed pharmacotherapy.

Do Sex and Ethnicity Influence Drug Pharmacokinetics in Solid Organ Transplantation?

A number of reports have identified differences in drug pharmacokinetics and pharmacodynamics between males and females, and between patients of different ethnicity. These differences are observed in both solid organ transplant recipients and in nontransplant patients. This review summarizes the pharmacokinetic differences thought to be dependent on sex and ethnicity noted with immunosuppressive agents commonly used in transplantation. This manuscript also touches on how these differences may translate into observed drug interactions. Furthermore, potential factors underlying the sex and ethnicity differences are evaluated. Current evidence points to differences in drug-metabolizing enzymes, transporters, cytokines, and environmental influences to account for the observed heterogeneity in drug response. Many of these factors seem to be under genetic control, and thus areas for further research are suggested.

NAT2 and GSTM1 DNA polymorphisms: increased GSTM1 (active) genotypes in endometriosis

Objective: Although there is evidence of genetic predisposition and environmental factors contributing to the overall incidence of endometriosis, causative gene(s) and mechanism remain elusive. Given that endometriosis displays features similar to cancer and given that heritable differences in drug-metabolizing enzymes (DMEs) often influence cancer development and/or progression, we sought to determine whether a genotype association exists between polymorphic DME genes and endometriosis. Because dioxin and dioxin-like compounds have been implicated as factors involved in the development of endometriosis, two polymorphic DMEs that are induced by dioxin—N-acetyltransferase 2 (NAT2) and Glutathione S Transferase M1 (GSTM1)—were selected for analysis.

Heritability and molecular genetic studies of endometriosis.

Endometriosis is a common disease defined as the growth of endometrial tissue outside the uterine cavity that often results in a vast array of gynaecological problems including dyspareunia, dysmenorrhoea, pelvic pain and infertility. Despite the increasing evidence that supports a genetic component to this common gynaecological condition, the basic aetiology and pathogenesis of endometriosis remain unknown. It is likely that endometriosis is a common polygenic/multifactorial disease caused by an interaction between multiple genes as well as the environment. Such conditions do not have a clear Mendelian pattern of inheritance. Recent molecular cytogenetic studies on endometriotic tissue and an established endometriosis-derived cell line provide novel evidence that acquired chromosome-specific alterations may be involved in endometriosis, possibly reflecting clonal expansion of chromosomally abnormal cells. Molecular DNA studies examining the role of loss of heterozygosity in endometriotic lesions has identified candidate tumour suppressor gene loci, including 5q, 6q, 9p, 11q and 22q, that may play a role in the malignant transformation of endometriotic implants to endometrioid ovarian cancers. Evidence of mutations in the tumour suppressor PTEN gene in the endometrioid subtype of epithelial ovarian cancer further suggests that somatic genetic alterations represent early events in the transformation of benign endometriotic cells. Genetic factors are also likely to influence individual susceptibility to endometriosis. There is now evidence that heritable allelic differences in drug-metabolizing enzymes play an important role in the development of endometriosis. Further studies are warranted to identify major susceptibility gene(s) and the mechanism involved in endometriosis to assist in the development of better methods for early detection, diagnosis and prevention.

GENETIC EPIDEMIOLOGY OF ENVIRONMENTAL TOXICITY AND CANCER SUSCEPTIBILITY: HUMAN ALLELIC POLYMORPHISMS IN DRUG-METABOLIZING ENZYME GENES, THEIR FUNCTIONAL IMPORTANCE, AND NOMENCLATURE ISSUES

Pharmacogenetics is the study of idiosyncratic drug responses that have an hereditary basis and usually reflect differences in drug-metabolizing enzymes (DMEs) and the receptors that control DME levels. The purpose of this review is to provide a brief overview of recent findings concerning more than a dozen clinically important polymorphisms and to emphasize the need to standardize the nomenclature of these alleles in each polymorphism, as quickly as possible. This nomen- clature system should be consistent with the Human Gene Nomenclature Guide- lines.Because DMEs have existed before divergence of prokaryotes and eukaryotes more than 2 billion years ago, it is clear that DME genes first must have evolved for critical life functions and that, more recently in animals, DME genes expanded to include the role of detoxification of dietary products, evolving plant metabolites, and, of course, pharmaceutical drugs. Many human DME polymorphisms are relevant to clinical problems in that they represent the basis of risk factors in the development of cancer, toxicity, and other diseases associated with drug, chemical, or dietary exposure. The study of the relationship among human genetic polymorphisms, cancer susceptibility, toxicity, and environmental exposure is a new and exciting area of research—which will undoubtedly have increasingly important implications for risk assessment and the prevention, early diagnosis, and intervention of clinical disease.

Strain differences in in vitro rat hepatocyte unscheduled DNA synthesis (UDS): Effect of UV is independent of strain while increased sensitivity is apparent using Fischer-344 instead of Sprague-Dawley rats

The unscheduled DNA synthesis (UDS) assay measures DNA repair following in vitro treatment of rat primary hepatocytes. This report compares the UDS response of primary hepatocytes from 2 widely used rat strains, the Fischer-344 (F344) and Sprague-Dawley (SD) strains. Ultraviolet (UV) light and 5 known genotoxic chemicals were evaluated in each strain in parallel experiments. The chemicals tested were 2-acetylaminofluorene (2-AAF), 4-aminobiphenyl (4-AB), benzidine, dimethylnitrosamine (DMN) and N-propyl- N′-nitro- N-nitrosoguanidine (PNNG). Four of these compounds (2-AAF, 4-AB, benzidine and DMN) require metabolic activation. Benzidine and PNNG were both negative using SD rat hepatocytes, but were weakly positive using F344 rat hepatocytes. In the first of 2 experiments, 4-AB was inconclusive in SD hepatocytes, but strongly positive in F344 cells. In the second experiment, 4-AB was positive in hepatocytes from both strains. 2-AAF was more strongly positive in F344 cells than in SD cells. DMN and UV light induced positive dose responses with little or no differences between strains. It is concluded that hepatocytes from F344 rats may be more sensitive, qualitatively and quantitatively, than hepatocytes from SD rats as indicators of UDS. This difference is not due to intrinsic differences in DNA repair mechanisms but is probably due to differences in drug-metabolizing enzymes between these strain. Thus, for routine screening, F344 rats are preferable for measurement of the in vitro UDS-inducing potential of compounds.

The multiple-indicator dilution technique for characterization of normal and retrograde flow in once-through rat liver perfusions.

The technique of normal and retrograde rat liver perfusion has been widely used to probe zonal differences in drug-metabolizing activities. The validity of this approach mandates the same tissue spaces being accessed by substrates during both normal and retrograde perfusions. Using the multiple-indicator dilution technique, we presently examine the extent to which retrograde perfusion alters the spaces accessible to noneliminated references. A bolus dose of 51Cr-labeled red blood cells, 125I-albumin, 14C-sucrose and 3H2O was injected into the portal (normal) or hepatic (retrograde) vein of rat livers perfused at 10 ml per min per liver. The outflow perfusate was serially collected over 220 sec to characterize the transit times and the distribution spaces of the labels. During retrograde perfusion, red blood cells, albumin and sucrose profiles peaked later and lower than during normal perfusion, whereas the water curves were similar. The transit times of red blood cells, albumin and sucrose were longer (p less than 0.005), whereas those for water did not change. Consequently, retrograde flow resulted in significantly larger sinusoidal blood volumes (45%), albumin Disse space (42%) and sucrose Disse space (25%) than during normal flow, whereas the distribution spaces for total and intracellular water remained unaltered. The distension of the vascular tree was confirmed by electron microscopy, by which occasional isolated foci of widened intercellular recesses and spaces of Disse were observed. Cellular ultrastructure was otherwise unchanged, and there was no difference found between normal and retrograde perfusion for bile flow rates, AST release, perfusion pressure, oxygen consumption and metabolic removal of ethanol, a substrate with flow-limited distribution, which equilibrates rapidly with cell water (hepatic extraction ratios were virtually identical: normal vs. retrograde, 0.50 vs. 0.48 at 6 to 7.4 mM input concentration). These findings suggest that the functional and metabolic capacities of the liver remain unperturbed during retrograde perfusion, rendering the technique suitable for the investigation of zonal differences in drug-metabolizing enzymes.