Affect Drug Metabolism Research Articles

Postpartum Bleeding in Pregnant Women Receiving SSRIs/SNRIs: New Insights From a Descriptive Observational Study and an Analysis of Data from the FAERS Database

PurposeTo date, the available data on the relationship between the use of selective serotonin reuptake inhibitors (SSRIs) or the serotonin and norepinephrine reuptake inhibitor (SNRI) venlafaxine and postpartum hemorrhage (PPH) are conflicting and have not been extensively investigated, especially in terms of plasma drug concentrations. We performed data mining of antidepressant-induced PPH reported to the US Food and Drug Administration's Adverse Event Reporting System database, to assess the strength of the potential association between antidepressant pharmacotherapy and PPH in pregnant women. Concurrently, we carried out a descriptive observational population (pregnant women) analysis of the correlation between the plasma concentrations of SSRIs/SNRIs used during pregnancy and the extent of bleeding at delivery. MethodsA disproportionality analysis of individual case study reports of PPH associated with SSRIs or venlafaxine in pregnant women was performed. Reporting odds ratio was used as a measure of disproportionality analysis. Pregnant women treated with an SSRI or SNRI (venlafaxine) for depressive or anxiety disorder and who consented to plasma drug concentration monitoring at the time of delivery were recruited. Plasma drug concentration assay was performed according to validated LC-MS/MS. Based on plasma drug concentrations, patients were classified into 1 of 2 groups, in therapeutic range or below therapeutic range for the drug administered, in accordance with the Arbeitsgemeinschaft für Neuropsychopharmakologie und Pharmakopsychiatrie guideline, and correlations with blood loss were identified, with PPH defined as a blood loss of >500 mL. FindingsOnly 43 Individual Case Safety Reports (ICSRs) reported at least one SSRIs or venlafaxine as suspect drug in 14 years (database analyses). Forty-three women were enrolled in the study population (observational study). In 24 patients (55.8%) the plasma drug concentration was below the therapeutic threshold. Unexpectedly, the mean blood loss in the below-range group was significantly higher than that in the in-range group. PPH occurred in 30% of women: in 9.3% and in 20.7% of patients in the in-range and below-range groups, respectively. ImplicationsAlthough preliminary, these data indicate a rather good tolerability profile of SSRIs/SNRIs regarding postpartum bleeding. Moreover, they suggest that keeping the plasma levels of SSRIs/SNRIs low as a precautionary measure does not reduce postpartum bleeding, which was higher in the below-range group. The findings from this study suggest that the use of therapeutic drug monitoring in pregnancy, a period in which multiple variables affect drug metabolism, may allow for better treatment customization, with subsequent advantages in terms of tolerability and efficacy of treatment.

Use of pharmacogenetic data to guide individualized opioid prescribing after surgery

BackgroundDespite the current strategies aimed at avoiding opioid overprescription by implementing institutional guidelines, the use of opioids after surgical procedures remains highly variable. It is well known that opioids are activated by the cytochrome p450 CYP2D6 enzyme to exert pharmacologic effect. Individual variation in CYP2D6 activity affects drug metabolism, and genotyping can be performed to predict an individual’s ability to metabolize CYP2D6 substrates. We postulate that the pharmacogenomic identification of patients with different opioid metabolism capacity may allow for the individualization of postsurgical opioid prescription. MethodsThis study was generated by the unison of data from 2 prior initiatives taking place at our Institution. In the first study, patients undergoing 1 of 25 elective surgical procedures were prospectively identified as part of a quality initiative and surveyed by phone 21 to 35 days after hospital discharge to complete a 29-question survey regarding opioid utilization and pain experience. Additional chart abstraction was conducted to obtain prescribing data and pain scores during the hospitalization. The second study was the Mayo Clinic Right Drug, Right Dose, Right Time study protocol, in which 5 pharmacogenes, including CYP2D6, were genotyped for 1,000 Mayo Clinic Biobank participants. The goal of this study was to implement preemptive pharmacogenomics in an academic health care setting and to generate data for further pharmacogenomic research. Patients were classified by their predicted CYP2D6 activity based on their CYP2D6 genotype. ResultsOf the 2,486 patients with prospective opioid utilization data, 21 had pharmacogenetic data available and were included in the analysis. These patients were classified according to their activity as opioid metabolizers, with 10 patients (48%) classified as intermediate, 4 patients (19%) as intermediate to normal, and 7 patients (33%) as normal or extensive. Compared with the intermediate to normal and intermediate phenotypes, normal or extensive patients had the highest percentages of preoperative opioid naivety and recorded pain scores throughout the surgical experience. The percentage of unused opioids for intermediate, intermediate to normal, and normal or extensive categories was 79%, 63%, and 46%, respectively. Moreover, of the 14 patients declaring the highest level of satisfaction for their pain control after discharge, 60% belonged to intermediate, 100% to intermediate to normal, and 57% to the normal or extensive group. ConclusionThis study outlines a possible correlation between genetically controlled metabolism and opioid requirements after surgery. In this setting, an increased CYP2D6 enzymatic activity was associated to a greater opioid consumption, lesser amount of unused opioids, and a lower satisfaction level from opioid prescription.

Changes in Drug Metabolizing Enzyme Cytochrome P450 CYP3A4 Activities Due to Polymorphisms in Human Cytochrome P450 Oxidoreductase

BackgroundCytochromes P450 proteins are responsible for the metabolism of drugs and xenobiotics. All cytochromes P450s in the endoplasmic reticulum rely on P450 oxidoreductase (POR) for their catalytic activities. Previously it has been shown that mutations in POR cause metabolic disorders of steroid hormone biosynthesis but less attention has been paid to drug metabolizing P450 activities. Considering the critical role of POR in activities of drug metabolizing P450 enzymes it is likely that polymorphisms and mutations in POR also affect drug metabolism. We aimed to characterize the variations identified in nonclinical individuals to access their effects on drug metabolizing cytochrome P450 CYP3A4. The variant P284T of POR (rs72557937, NM_000941.2:c.850C>A) is exclusively present in the African population, while A503V allele of POR is found in about 25% of alleles across different populations.MethodsWT and mutant POR proteins, as well as cytochrome P450s, were expressed in recombinant form in bacteria and purified. We used liposomes to embed the P450 and POR proteins and created a functional P450 metabolic system. Metabolism of Testosterone, small molecule dyes, and tracer compounds was evaluated by radioactive ligand metabolism, fluorescent substrate metabolism and colorimetric assays using chromogenic substrates. Enzyme kinetic analysis was performed using Prizm.ResultsTo study the effect of the P284T variant on POR activity, we first measured the ability of WT and P284T variant to transfer electrons from NADPH to cytochrome c, MTT or ferricyanide. Compared to WT POR, the P284T variant had ~50% activity to reduce cytochrome c and 15 % activity to reduce MTT. However, the ferricyanide reduction activity was not affected by the P284T mutation. As compared to WT POR, both FMN and FAD binding was affected due to the P284T mutation. The FMN content of POR‐P284T was 64% as compared to WT while the FAD content was reduced by 35% suggesting that these mutations do affect FMN and FAD binding to POR. The PORvariant P284T had severe loss of CYP3A4 activity with only 22.8% residual activity as compared to WT indicating a potential impact on drug metabolism. The POR common polymorphism A503V which is found in around 25% of all alleles across different genetic populations showed increased activities of drug metabolizing enzyme CYP3A4.ConclusionsThese results indicate that effects of mutations on activities of individual cytochromes P450 can be variable and a detailed analysis of each variant with different partner proteins is necessary to determine the genotype‐phenotype correlations of POR variants accurately. These results further affirm the critical role of POR in drug metabolism and suggest that variations in POR should also be considered during the evaluation of abnormal drug‐metabolizing cytochrome P450 activities.Support or Funding InformationThis research was supported by grants to Amit V Pandey from the Swiss National Science Foundation, Novartis Foundation for Medical Biological Research and BurgerGemeinde Bern, Switzerland.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Non‐Additive Effects of Changing the Composition of the Cytochrome P450 Ensemble in Human Liver Microsomes: the Focus on CYP2E1 and Alcohol‐Drug Interactions

Recent studies on a large selection of human liver microsomes (HLM) demonstrated the lack of a straight correlation between the composition of human cytochrome P450 ensemble and the profile of drug metabolism. At the same time, there is an emerging recognition of the functional importance of physical interactions between multiple P450 species co‐localized in the microsomal membrane. Due to complex effects of these interactions any change in the content of a particular P450 enzyme may affect drug metabolism in a complex, hard‐to‐predict manner. Of particular practical importance are the functional effects of the changes in the composition of P450 ensemble induced by alcohol consumption. Although the multi‐fold increase in the content of cytochrome P450 2E1 (CYP2E1) in liver observed in both alcoholics and moderate alcohol consumers is well documented, the role of CYP2E1 in the instances of alcohol‐drug interactions is commonly considered insignificant due to a minor role of this enzyme in drug metabolism. However, the involvement of CYP2E1 in alcohol‐drug interactions may stretch far beyond its immediate effects on the metabolism of its substrates.The aim of this study is to probe the effect of increasing concentrations of CYP2E1 in HLM on the metabolism of drug substrates of other P450 species. In a search for a model system suitable for these studies we introduced a method based on incorporation of purified cytochromes P450 into HLM that allows creating the microsomal preparations with altered composition of the P450 ensemble. Incorporation of CYP2E1 into HLM results in an increase in the rate of CYP2E1‐dependent metabolism of chlorzoxazone, which is proportional to the amount of the incorporated enzyme up to its content of 0.3 nmol per mg protein (or ~50% of CYP2E1 in the total P450 content). Studies with a series of substrates of drug‐metabolizing P450 species showed that CYP2E1 incorporation also results in pronounced alterations in the metabolism of substrates of several other P450 enzymes. The most important effect is observed on CYP2C19. The changes in the effective KM and Vmax values with such probe substrates as 7‐etoxy‐4‐cyanocoumarin (CEC, the substrate of CYP2C19 and CYP1A2), 7‐dimethylamino‐4‐trifluoromethylcoumarin (coumarin‐152, the substrate of CYP2B6, CYP2C19 and CYP2C9) and N‐butyl 7‐methoxy‐4‐(aminomethyl)‐coumarin (BMAMC, the substrate of CYP1A2, CYP2C19 and CYP2D6) reveal a substantial decrease in the involvement of CYP2C19 in their metabolism. Furthermore, increase in CYP2E1 content causes an important changes in the function of CYP3A enzymes, which is revealed in a compromised homotropic cooperativity, decreased affinity and increased Vmax observed with 7‐benzyloxyquinoline, a selective substrate of CYP3A4 and CYP3A5. These observations suggest that ethanol‐dependent induction CYP2E1 may be involved in pronounced pharmacokinetic interactions of alcohol with such drug substrates of CYP2C19 and CYP3A enzymes as benzodiazepines (diazepam), tricyclic antidepressants (amitriptylline, imipramine) and anticonvulsants (phenytoin, carbamazepine).Support or Funding InformationThis research was supported by grant R21‐AA024548 from NIH.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Case‐Based Discussion Improves Medical Student Application of Pharmacogenetics Knowledge

With the advent of precision medicine, practitioners now consider patient genotypes when selecting pharmacotherapy and dosing for drugs that exhibit significant genotype‐specific morbidity. It is therefore prudent to incorporate pharmacogenetics training into undergraduate medical education. To improve student understanding of pharmacogenetics, we exposed 1st year medical students to pharmacogenetics through case‐based discussions (CBD) within a pharmacology course.The pharmacogenetics module included a short, prerecorded lecture on warfarin pharmacogenetics. During the CBD, 3 patient cases with varying indications for warfarin therapy were presented. Students in small groups discussed and answered questions relating to warfarin sensitivity, bleeding/clotting risk, warfarin dose optimization, and patient counseling. At the end of the CBD, answers were elicited using voting cards and students explained their reasoning in a large group setting. Anonymous pre‐ and post‐CBD surveys were administered to determine student satisfaction and understanding of pharmacogenetics concepts. Pre‐CBD survey results indicated that 80% of students watched the prerecorded lecture and only 20% of students agreed that they had not considered the role of genotype in drug metabolism and response prior to watching the lecture. After the CBD, 90% of students agreed that the CBD increased their awareness of how different genotypes can affect drug metabolism and response; 92% of students agreed that the CBD improved their understanding that drug response is a result of both the drug acting on its target and drug metabolism/excretion; 98% of the students agreed that the CBD effectively applied pharmacogenetics to clinical presentations; 93% agreed this session increased their confidence to anticipate and discuss the use of pharmacogenetics in clinical practice. In an open response question regarding patient factors affecting warfarin dose, 86% of students identified genotype as a major factor on the pre‐session survey vs. 76% in the post‐session survey. In comparison to the pre‐session survey, post‐session responses identifying patient demographics, BMI, medication, diet, and alcohol use as important factors in warfarin dosing increased by at least 10% and up to 50%.The clinical relevance and interactive nature of the exercise resulted in excellent student engagement. Informal polling of CBD group voting revealed that most groups answered correctly and a satisfaction survey revealed that the activity was well‐received. The pharmacogenetics exercise provided students with a foundation for understanding modifiable and non‐modifiable patient factors that affect warfarin dosing. The session's effectiveness at improving students' knowledge in pharmacogenetics was further validated by students' high performance (mean = 81%) on pharmacogenetics exam questions. We believe student comprehension was facilitated by student application of knowledge from the pre‐session recording to disparate cases of warfarin therapy.Support or Funding InformationThis study was not grant funded.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cost-effectiveness analysis of pharmacogenomics-guided clopidogrel treatment in Spanish patients undergoing percutaneous coronary intervention.

Clopidogrel is an antiplatelet drug given to patients before and after having a percutaneous coronary intervention (PCI). Genomic variants in the CYP2C19 gene are associated with variable enzyme activities affecting drug metabolism and hence, patients with reduced or increased enzymatic function have increased risk of bleeding. We conducted a cost-effectiveness analysis to compare a pharmacogenomics versus a non-pharmacogenomics-guided clopidogrel treatment for coronary artery syndrome patients undergoing PCI in the Spanish healthcare setting. A total of 549 patients diagnosed with coronary artery disease followed by PCI were recruited. Dual antiplatelet therapy was administrated to all patients from 1 to 12 months after PCI. Patients were classified into two groups: the Retrospective group was treated with clopidogrel based on the clinical routine practice and the Prospective group were initially genotyped for the presence of CYP2C19 variant alleles before treatment with those carrying more than one CYP2C19 variant alleles given prasugrel treatment. We collected data on established clinical and health outcome measures, including, per treatment arm: the percentage of patients that suffered from (a) myocardial infraction, (b) major bleeding and minor bleeding, (c) stroke, (d) the number of hospitalization days, and (e) the number of days patients spent in Intensive Care Unit. Our primary outcome measure for the cost-effectiveness analysis was Quality Adjusted Life Years (QALYs). To estimate the treatment cost for each patient, individual data on its resource used were combined with unit price data, obtained from Spanish national sources. The analysis predicts a survival of 0.9446 QALYs in the pharmacogenomicsarm and 0.9379 QALYs in the non-pharmacogenomics armwithin a 1-year horizon. The cumulative costs per patient were €2971 and €3205 for the Prospective and Retrospective groups, respectively. The main cost driver of total cost in both arms was hospitalization costs. The incremental cost-effectiveness ratio (ICER) was negative indicating that the PGx was a dominant option. Our data show that pharmacogenomics-guided clopidogrel treatment strategy may represent a cost-effective choice compared with non-pharmacogenomics-guided strategy for patients undergoing PCI.

Pharmacogenomics: Personalized medicine and its impact on breast cancer

Background: Personalized medicine in breast cancer aims to optimize treatment by classifying cancer subtypes and tailoring therapy based on individual patient profiles, including genetic and epigenetic factors. Pharmacogenomics plays a crucial role in this strategy by investigating how genetic variations affect drug metabolism and therapy response. Aim: This article reviews the impact of pharmacogenomics on personalized medicine for breast cancer, focusing on different molecular subtypes and their responses to targeted therapies. Methods: The study involved a comprehensive review of current literature, examining the molecular classification of breast cancer, the role of genetic and epigenetic factors, and advancements in pharmacogenomics. Key resources, including open-source databases and clinical trials, were analyzed to understand treatment resistance and efficacy. Results: Breast cancer is categorized into molecular subtypes such as hormone receptor-positive, HER2-positive, and triple-negative. Each subtype exhibits distinct responses to therapies. For instance, hormone receptor-positive cancers benefit from endocrine therapies, while HER2-positive cancers respond to targeted antibodies. Triple-negative breast cancer, characterized by its heterogeneity, shows varied responses to platinum-based compounds and PARP inhibitors. The study highlights the challenges of drug resistance and the potential of personalized therapies to overcome these issues.

Clinical Characteristics of Elderly Chronic Myeloid Leukemia Patients in Korea; Based on the National Health Information Database

ObjectivesAging involves progressive impairments in the functional reserve of multiple organs, which might also affect drug metabolism and pharmacokinetics. Elderly might experience more drug adverse events than younger patients. Thus, we evaluated clinical characteristics and tyrosine kinase inhibitor (TKI) prescription pattern in Korean elderly chronic myeloid leukemia (CML) patients, using National Health Information Database “NHID”.MethodsThis study included patients 70 and older who were diagnosed with Philadelphia chromosome positive CML (ICD code C92.1) from 2005 to 2013 and prescribed TKIs (which included imatinib, dasatinib, nilotinib and radotinib which were covered by the National Health Insurance Service (NHIS), currently). We excluded following cases; ①TKI was prescribed just once ② TKI was started with a higher dose than the recommended for chronic phase (imatinib, 400mg QD; dasatinib, 100mg QD; nilotinib, 300mg BID; radotinib 300mg BID). ③ interferon was treated prior to TKI ④ TKI dosage data was missing. We extracted age, sex, medical insurance premium (which is imposed by proportionate to income), prescribed medication list, and date of death from NHID. Medication possession ratio (MPR) was used for adherence. Each patients' laboratory result, disease phase (chronic phase, accelerate phase, or blastic crisis), or the reasons for TKI change or dose reduction were not available.Overall survival (OS) was calculated by Kaplan-Meier methods. Additionally, Univariate and multivariate analysis were done with Cox proportional hazard regression, with following variables using SAS; sex (male vs female), age (70 ≤age<75, 75≤age<80, age≥80) medical insurance premium (high vs low), frontline TKI (imatinib vs other TKIs), and MPR (≥0.9 vs <0.9).ResultA total of 385 patients were followed up median 52 (1-133) months until the end of 2016. The patients consisted of 54% male (n=208), median age of 75 (70-95), and 64 patients (16.6%) who were diagnosed after the age of 80. About 85% of the patients experienced imatinib. Dasatinib was prescribed to 107 patients (27.8%), nilotinib to 71 (18.4%), and radotinib to 16 (4.2%). Frontline TKI was maintained in 279 (72.5%) patients, and the others were treated with more than one TKI; 2TKIs, n=80 (20.8%); 3TKIs, n=22 (5.7%); 4TKIs n=4 (1%). Imatinib was selected as frontline TKI in 318 patients (81.8%). Median MPR to TKI was 0.89 (0.00-1.84), and 51% of patients' MPR was less than 0.9. Patients were treated median 42 (0-146) months, and 186 (48.3%) were expired during the follow up period.Among 327 patients, 42 (12.8%) started with reduced dose of imatinib, and 285 (87.2%) started with imatinib 400mg QD. After median 2 (0-118) months, full dose imatinib reduced to mean 263mg±63 in 165 patients. Only 111 patients (34%) maintained imatinib. Dasatinib 100 mg was initiated in 89.9% (n=94) of 107 patients. Thirty patients (28%) did not change dasatinib dose. Others, reduced dose to mean 57±13mg after median 3 (0-33) months. Patients who were taking nilotinib or radotinib, full dose without dose reduction, were 31 (43%) and 7 (43%) respectively. Nilotinib was reduced to mean 325±62 after median 2(0-29) months, and radotinib to mean 357±162mg after median 4 (0-43) months.The estimated OS at 5-year was 60.3% and 10-year was 24.6%. For both univariate and multivariate analysis, age and MPR associated OS significantly; Patients aged 80 and older and from 75 to 80 showed poor OS than those aged from 70 to 75 through adjusted HR 2.67[95% CI.I, 1.81-3.93] (p<0.0001) and 1.81[95% C.I., 1.29-2.53] (p=0.0006), respectively. Patients with high MPR (≥0.9) survived longer than those with low MPR (<0.9), and its adjusted HR was 2.34 [95% C.I. 1.70-3.23] (p<0.0001).ConclusionBased on NHID, 10-50% of TKIs were initiated with reduced dose in age 70 and older CML patients. Full dose was maintained only 28-43% of prescribed TKIs. Despite of this, the outcome was favorable showing that because the estimated OS at 5-year was 60.3%. Age and adherence to TKI were associated with OS in elderly CML patients. DisclosuresNo relevant conflicts of interest to declare.

Prevalence of CYP2D6*4 1934G/A polymorphism in Western Indian HIV patients.

Hepatic CYP2D6 enzyme metabolizes antiretroviral drugs (ARVs) including nevirapine. Polymorphism in CYP2D6 gene affects drug metabolism and displays distinctive phenotypes in the population. Hence, we investigated the prevalence of CYP2D6*4 1934G/A polymorphism in a total of 165 HIV patients that include 34 with and 131 without hepatotoxicity and 160 unrelated healthy controls by the PCR-RFLP method. The prevalence of CYP2D6*4 1934AA genotype was higher in total HIV patients as compared to healthy controls (1.81% vs 0.6%, OR=2.86). Similarly, CYP2D6*4 1934AA genotype was much more prevalent in HIV patients without hepatotoxicity as compared to healthy controls (2.3% vs 0.6%, OR=2.87). Likewise, CYP2D6*4 1934AA genotype was predominant in advanced HIV disease stage as compared to healthy controls (3.8% vs 0.6%, OR=6.15). CYP2D6*4 1934GA genotype was distributed higher in HIV patients taking tobacco and nevirapine as compared to non-users (23.3% vs 19.3%, OR=1.21, 21.0% vs 16.7%, OR=1.2). Likewise, CYP2D6*4 1934GA genotype was overrepresented in patients with hepatotoxicity taking alcohol+nevirapine as compared to alcohol non-users+nevirapine users (20.00% vs 16.67%, OR=1.25). Thus, there was no significant difference in genotype or allele frequencies of CYP2D6*4 1934G/A polymorphism between the patients with hepatotoxicity and those without or healthy controls.

Drug-gut microbiota interactions: implications for neuropharmacology.

The fate and activity of drugs are frequently dictated not only by the host per se but also by the microorganisms present in the gastrointestinal tract. The gut microbiome is known to, both directly and indirectly, affect drug metabolism. More evidence now hints at the effects that drugs can have on the function and composition of the gut microbiome. Both microbiota-mediated alterations in drug metabolism and drug-mediated alterations in the gut microbiome can have beneficial or detrimental effects on the host. Greater insights into the mechanisms driving these reciprocal drug-gut microbiota interactions are needed to guide the development of microbiome-targeted dietary or pharmacological interventions, which may have the potential to enhance drug efficacy or reduce drug side effects. In this review, we explore the relationship between drugs and the gut microbiome, with a specific focus on potential mechanisms underpinning the drug-mediated alterations on the gut microbiome and the potential implications for psychoactive drugs. LINKED ARTICLES: This article is part of a themed section on When Pharmacology Meets the Microbiome: New Targets for Therapeutics? To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.24/issuetoc.

Inflammation down-regulates CYP3A4-catalysed drug metabolism in hemodialysis patients

BackgroundRecent studies indicate that inflammation may also affect CYP3A4 activity. Associations of CYP3A4-mediated metabolism of quinine, with inflammatory biomarkers were investigated in patients undergoing maintenance hemodialysis (HD).MethodsA single dose of 100 mg quinine was given to 44 HD patients and the plasma concentration of quinine and its metabolite 3-OH-quinine were measured 12 h after drug intake. The ratios of quinine/3-OH-quinine and 4β-OH-cholesterol/cholesterol were used as markers of CYP3A4 activity. Inflammatory biomarkers, high-sensitive CRP (hsCRP), pentraxin 3 (PTX3) and orosomucoid were followed during 4 weeks prior to quinine administration.ResultsThe quinine/3-OH-quinine ratio correlated with median concentrations of hsCRP (Rho = 0.48; p = 0.001) and orosomucoid (Rho = 0.44; p = 0.003), and also with interleukin-6 at 12 h after drug intake (Rho = 0.43; P = 0.004) but not PTX3. In multivariate regression analysis, the correlation between CYP3A4 activity and median hsCRP remained borderline significant (p = 0.05). 4β-OH-cholesterol/cholesterol ratio correlated with quinine/3-OH-quinine (p = 0.008), but not with any of the inflammation markers.ConclusionsThe association between CYP3A4 activity and inflammatory biomarkers suggest that the activity of CYP3A4 is reduced by inflammation in HD patients. Further studies are needed to confirm this finding and to assess to what extent magnitude and duration of inflammation as well as the microbiota affect drug metabolism.

Posttranslational regulation of CYP2J2 by nitric oxide.

Nitric oxide (NO) is an essential signaling molecule in the body, regulating numerous biological processes. Beside its physiological roles, NO affects drug metabolism by modulating the activity and/or expression of cytochrome P450 enzymes. Previously, our lab showed that NO generation caused by inflammatory stimuli results in CYP2B6 degradation via the ubiquitin-proteasome pathway. In the current study, we tested the NO-mediated regulation of CYP2J2 that metabolizes arachidonic acids to bioactive epoxyeicosatrienoic acids, as well as therapeutic drugs such as astemizole and ebastine. To investigate the effects of NO on CYP2J2 expression and activity, Huh7 cells stably transduced with CYP2J2 with a C-terminal V5 tag were treated with dipropylenetriamine-NONOate (DPTA), a NO donor. The level of CYP2J2 proteins were decreased in a time- and concentration-dependent manner, and the activity was also rapidly inhibited. However, mRNA expression was not altered and the protein synthesis inhibitor cycloheximide did not attenuate DPTA-mediated downregulation of CYP2J2. Removal of DPTA from the culture media quickly restored the activity of remaining CYP2J2, and no further CYP2J2 degradation occurred. To determine the mechanism of CYP2J2 down-regulation by NO, cells were treated with DPTA in the presence or absence of protease inhibitors including proteasomal, lysosomal and calpain inhibitors. Remarkably, the down-regulation of CYP2J2 by NO was attenuated by calpeptin, a calpain inhibitor. However, other calpain inhibitors or calcium chelator show no inhibitory effects on the degradation. The proteasome inhibitor bortezomib showed small but significant restoration of CYP2J2 levels although stimulated ubiquitination of CYP2J2 was not detected. In conclusion, these data suggest that NO regulates CYP2J2 posttranslationally and NO-evoked CYP2J2 degradation undergoes ubiquitin-independent proteasomal degradation pathway unlike CYP2B6.

A Transcriptional Regulatory Network Containing Nuclear Receptors and Long Noncoding RNAs Controls Basal and Drug-Induced Expression of Cytochrome P450s in HepaRG Cells

Cytochrome P450 (P450) enzymes are responsible for metabolizing drugs. Expression of P450s can directly affect drug metabolism, resulting in various outcomes in therapeutic efficacy and adverse effects. Several nuclear receptors are transcription factors that can regulate expression of P450s at both basal and drug-induced levels. Some long noncoding RNAs (lncRNAs) near a transcription factor are found to participate in the regulatory functions of the transcription factors. The aim of this study is to determine whether there is a transcriptional regulatory network containing nuclear receptors and lncRNAs controlling both basal and drug-induced expression of P450s in HepaRG cells. Small interfering RNAs or small hairpin RNAs were applied to knock down four nuclear receptors [hepatocyte nuclear factor 1α (HNF1α), hepatocyte nuclear factor 4α (HNF4α), pregnane X receptor (PXR), and constitutive androstane receptor (CAR)] as well as two lncRNAs [HNF1α antisense RNA 1 (HNF1α-AS1) and HNF4α antisense RNA 1 (HNF4α-AS1)] in HepaRG cells with or without treatment of phenobarbital or rifampicin. Expression of eight P450 enzymes was examined in both basal and drug-induced levels. CAR and PXR mainly regulated expression of specific P450s. HNF1α and HNF4α affected expression of a wide range of P450s as well as other transcription factors. HNF1α and HNF4α controlled the expression of their neighborhood lncRNAs, HNF1α-AS1 and HNF4α-AS1, respectively. HNF1α-AS1 and HNF4α-AS1 was also involved in the regulation of P450s and transcription factors in diverse manners. Altogether, our study concludes that a transcription regulatory network containing the nuclear receptors and lncRNAs controls both basal and drug-induced expression of P450s in HepaRG cells.

Utilizing Cases in Pharmacogenetics Education of Undergraduate Medical Students

Pharmacogenetics should have an integral role in undergraduate medical education as personalized medicine is the end goal of treating individual patients effectively. Increasingly, practitioners are being asked to incorporate patient genotypes when considering therapy options, including drug choice and dosing, particularly for drugs that exhibit significant morbidity in patients with a specific genotype. To improve student comprehension of the interplay between pharmacology and genetics, we sought to expose first‐year medical students to the core concepts and practice of pharmacogenetics through a case‐based discussion (CBD) within their pharmacology course.The CBD included a short, pre‐recorded lecture (~13 min) on the pharmacogenetics of warfarin, as preparatory material. During the session, 3 mini‐cases with different indications for warfarin therapy were presented. Students in small groups answered questions related to the patient's warfarin sensitivity, bleeding/clotting risk, warfarin dose optimization and patient counseling issues. After working through the cases in groups, answers were elicited using voting cards, and students were asked to defend their answers in the large group setting. Anonymous pre and post session surveys were administered to determine student satisfaction and understanding of pharmacogenetics concepts. The session's effectiveness at improving students' knowledge in pharmacogenetics was assessed in the end of course final exam. Survey results indicated that 80% of students listened to the pre‐recorded lecture. 20% of students agreed that prior to the pharmacogenetics activity, they had not previously considered that genotypes could play a role in drug metabolism and response. 90% of students agreed or strongly agreed that the CBD increased their awareness of how different genotypes may affect drug metabolism and response and 92% of students agreed or strongly agreed that the CBD increased their awareness of the fact that drug response is due both to drugs acting on target proteins as well as drugs being metabolized and/or excreted by the body. 98% of the students agreed or strongly agreed that this CBD was effective at integrating and applying pharmacogenetics to clinical presentations and 93% agreed or strongly agreed this session increased their level of confidence to be able to anticipate and discuss how pharmacogenetics can be used in clinical care and drug prescriptions.The CBD was well‐received, and informal polling of voting responses revealed that the majority of groups came to the correct answers during the CBD. The clinical relevance and interactive nature of the exercise resulted in excellent student engagement and satisfaction. Students' high performance on pharmacogenetics exam questions (mean of 81%) revealed a clear grasp of the pharmacogenetics concepts taught in the course. We believe that the combination of the presession recording followed by the student discussion of disparate cases of warfarin treatment were responsible for student comprehension.Support or Funding InformationThis study was not supported by a grant and has been approved by FIU IRB.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Towards a System Approach to the Human Cytochrome P450 Ensemble: A New Strategy for Studying the Network of P450‐P450 Interactions in Human Liver Microsomes

The composition of the human cytochrome P450 ensemble is commonly recognized as a key factor determining the interindividual variability in drug metabolism. However, recent studies on a large selection of human liver microsomes (HLM) demonstrated the lack of a direct correlation between the composition of the cytochrome P450 ensemble and the profile of drug metabolism. At the same time, there is an emerging recognition of the functional importance of interactions between multiple cytochrome P450 species co‐localized in the microsomal membrane. A change in the content of any particular P450 enzyme alters the landscape of protein‐protein interactions and thus affects drug metabolism in a complex, hard‐to‐predict manner. Therefore, a systematic exploration of the network of P450‐P450 interactions and identification of their functional consequences represents an issue of emerging importance for revealing the interrelationships between the profile of P450 expression and the profile of drug metabolism. In a search for a model system suitable for these studies we introduced a method based on incorporation of purified cytochromes P450 into the microsomes. Here we demonstrate high potential of this approach for creating preparations of HLM with altered composition of the P450 ensemble. An important characteristic of this strategy is its adaptability for incorporating genetically or chemically modified proteins. This includes the incorporation of proteins bearing fluorescent probes and light‐activating crosslinkers. Therefore, this strategy may be used for monitoring protein‐protein interactions in HLM with a variety of biophysical techniques, as well as for searching the partners of P450 protein‐protein interactions with the technique of molecular fishing.Here we report the use of our new approach for studying the effect of enriching the HLM with ethanol‐inducible CYP2E1, one of the the most populated P450 species in human liver. Incorporation of CYP2E1 into HLM results in an increase in the rate of metabolism of CYP2E1‐specific substrates, which is proportional to the amount of the incorporated enzyme up to its content of 0.3 nmol per mg protein (or ~50% of CYP2E1 in the total P450 content). Our studies with a series of substrates of drug‐metabolizing P450s showed that CYP2E1 incorporation results in pronounced alterations in the metabolism of substrates of several other P450 species present in HLM. In particular, we observed over 2‐fold increase in the rate of metabolism and a substantial decrease in the apparent KM observed with 3‐cyano‐7‐etoxycoumarin (CEC), the substrate of CYP1A2 and CYP2C19. This finding may indicate that the increase in CYP2E1 concentration results in redirecting the main route of CEC metabolism from CYP2C19 to CYP1A2, which is characterized with lower KM and higher turnover numbers with this substrate. Similarly, increase in CYP2E1 content causes an important changes in the function of CYP3A enzymes, which is revealed in a compromised homotropic cooperativity, decreased affinity and increased Vmax, observed with 7‐benzyloxyquinoline, a selective substrate of CYP3A4 and CYP3A5.Support or Funding InformationThis research was supported by grant R01‐GM114369 from NIH.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A Transcriptional Regulatory Network containing Nuclear Receptors and Long Noncoding RNAs Controls Basal and Induced Expression of Cytochrome P450s in Liver Cells

Cytochrome P450 (P450) enzymes are responsible for biotransformation of xenobiotics, including environmental toxicants and drugs. Expression of P450s can directly affect drug metabolism, resulting in various outcome in either therapeutic efficacy or adverse effects. Nuclear receptors are a class of transcription factors that can regulate expression of P450s at both basal and drug‐induced levels. Some long non‐coding RNAs (lncRNAs) near a transcription factor are found to participate in the regulatory functions of the transcription factors in either promotion or repression manner. The aim of this study is to determine whether there is a transcriptional regulatory network containing nuclear receptors and lncRNAs controlling both basal and drug‐induced expression of P450s in liver cells. Four nuclear receptors, hepatocyte nuclear factor 1a (HNF1a), hepatocyte nuclear factor 4a (HNF4a), pregnane X receptor (PXR), and constitutive androstane receptor (CAR) as well as two lncRNAs, HNF1a‐antisense 1 (HNF1a‐AS1) and HNF4a‐antisense 1 (HNF4a‐AS1) were examined in control of basal and drug‐induced expression of 8 different P450 enzymes. Small interfering RNAs (siRNAs) were applied to knock down the nuclear receptors and lncRNAs in HepaRG cells with or without treatment of phenobarbital or rifampicin. Expression of the nuclear receptors, lncRNAs, and P450s was measured by RT‐PCR and Western blots. Knocking down of HNF1a or HNF4a affected basal expression of numerous P450s as well as CAR and PXR, and also impaired phenobarbital or rifampicin induced expression of several P450s, which may be due to decreased CAR/PXR expression. Moreover, HNF4a knockdown was able to decrease HNF1a expression and showed more potent effect than HNF1a knockdown. Knocking down of CAR or PXR only affected basal and induced expression of their target P450s without affecting expression of HNF1a or HNF4a. These data suggested the existence of a regulatory network in controlling P450 expression in an order through HNF4a to HNF1a to CAP/PXR. Expression of HNF1a‐AS1 and HNF4a‐AS1 is largely dependent on expression of their nearby coding gene HNF1a and HNF4a. Moreover, HNF4a knockdown depleted expression of both lncRNAs, but HNF1a knockdown only depleted HNF1a‐AS1 expression. Knocking down of HNF1a‐AS1 showed a similar regulation trend on the P450s compared to HNF1a or HNF4a knockdown, indicating involvement of this lncRNA in the regulation of P450s. However, knocking down of HNF4a‐AS1 showed an opposite regulation trend compared to HNF1a or HNF4a knockdown, indicating that HNF4a‐AS1 may play an inhibitory role in the HNF4a function. Altogether, out study concludes that there is a transcription regulatory network among the different nuclear receptors and lncRNAs in the regulation of both basal and drug‐induced expression of P450s in liver cells.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Metabolism of Sesamin and Drug-Sesamin Interaction

Sesamin, derived from sesame seeds, is known to have various biological effects. Since some of these effects appear to be derived from its metabolites, the elucidation of sesamin metabolism is essential to understanding the molecular mechanism of its effects. In addition, it is important to clarify drug-sesamin interactions in order to address safety concerns, as some food factors are known to affect drug metabolism. Our previous studies revealed that sesamin was sequentially metabolized by cytochrome P450 (CYP) and UDP-glucuronosyltransferase or sulfotransferase. Whereas sesamin metabolism is mainly mediated by CYP2C9 in human liver, sesamin causes a mechanism-based inhibition (MBI) of CYP2C9. However, we found that the metabolite-intermediate complex between CYP2C9 and sesamin was unstable, and the effects of sesamin appeared to be minimal. To confirm this assumption, in vivo studies using rats were conducted. After the administration of sesamin to rats for 3 d, diclofenac (an NSAID) was administered to measure the time course of plasma concentration of diclofenac. No significant differences were observed in the diclofenac Cmax, Tmax, and AUC0-24 h between the group that was administered sesamin and the group that was not. Based on these results, it could be concluded that no significant interaction occurs in people who take sesamin supplements at a standard dose.

Epigenetic Alterations Impact on Antipsychotic Treatment in Elderly Patients

Antipsychotics are commonly prescribed for the treatment of psychosis as well as behavioral and psychological symptoms of dementia (BPSD) in elderly patients. However, elderly patients often experience decreased antipsychotic efficacy and increased side effects, though the mechanisms underlying these changes with age are not clear. Although aging can affect drug metabolism and clearance through changes in renal and hepatic function, additional pharmacokinetic and pharmacodynamic changes due to aging-induced epigenetic alterations also impact processes important for antipsychotic function. Epigenetic mechanisms account for some of the altered efficacy and increased side effects seen in elderly patients. Both clinical and animal studies from our group and others have demonstrated a plausible epigenetic mechanism involving histone modifications that can adversely affect the efficacy of antipsychotics and increase their side effects in elderly patients. Hopefully, further investigation of this mechanism will benefit elderly patients who need treatment for psychosis and BPSD.

In Vitro Validation of miRNA-Mediated Gene Expression Linked to Drug Metabolism.

Pharmacogenomic approaches used to investigate how genes affect drug responses are critical for designing personalized therapies aimed at maximizing efficacy and minimizing adverse effects. Drug efficacy is often dependent on the sequence and expression levels of drug target genes or those involved in the metabolism and transport of the therapeutic agent. Expression of these genes, in turn, is negatively regulated by small noncoding miRNAs. The levels of miRNAs in bodily fluids have been studied extensively as potential diagnostic and prognostic biomarkers. Studies have shown that miRNAs regulate multiple genes and sequence homology is used to predict which genes are subject to regulation by a particular miRNA. Once a gene is identified as a potential target for an miRNA of interest, experiments are undertaken to confirm that the miRNA interacts with the target gene and can alter its level of expression and/or its activity. For example, the differential expression of miRNAs in whole blood obtained from good and poor responders to ketamine has been reported both prior to, and following treatment for complex regional pain syndrome. In this case, hsa-miR-548d-5p was significantly lower in poor responders relative to good responders. This miRNA was predicted to target UDP-glucuronyl transferase 1A1 (UGT1A1), a key drug metabolizing enzyme. Described in this unit are protocols used to confirm miR-548d-5p-mediated UGT1A1 regulation. The approaches described can be employed broadly for the validation of miRNA-mediated negative regulation of any gene. Determining miRNA-mediated regulation of enzymes and transporters affecting drug metabolism is a critical step in designing personalized therapy and for understanding the mechanisms responsible for variations in the responses to therapeutic agents. © 2017 by John Wiley & Sons, Inc.

Transcriptional Regulation of Disease‐Drug Interactions

Drug metabolism and disposition are critical in maintaining the chemical and functional homeostasis of xenobiotics and endobiotics. Accumulating evidence suggests that many hepatic and systemic diseases can affect drug metabolism and disposition by regulating the expression and/or activity of DMEs and transporters in the liver. This presentation will focus on the recent progress in describing and understanding the hepatic injury and sepsis responsive regulation of DMEs in animal models. Liver ischemia and reperfusion (I/R) will be used as a typical example of a hepatic injury model, whereas lipopolysaccharides (LPS) and cecal ligation and puncture (CLP) will be used as the sepsis models. Our results showed that liver I/R and sepsis can affect liver metabolism through the transcriptional regulation of DMEs in the liver. In the meantime, the expression and/or activity of DMEs can affect the disease manifestations of I/R and sepsis. It is hoped that understanding the disease effect on drug metabolism will facilitate the efficient and safe use of drugs in the clinic.Support or Funding InformationThis work is supported in part by the NIH grant ES023438.