Hepatic CYP3A Research Articles

Undifferentiated HepaRG cells show reduced sensitivity to the toxic effects of M8OI through a combination of CYP3A7-mediated oxidation and a reduced reliance on mitochondrial function

The methylimidazolium ionic liquid M8OI (1-octyl-3-methylimidazolium chloride, also known as [C8mim]Cl) has been detected in the environment and may represent a hazard trigger for the autoimmune liver disease primary biliary cholangitis, based in part on studies using a rat liver progenitor cell. The effect of M8OI on an equivalent human liver progenitor (undifferentiated HepaRG cells; u-HepaRG) was therefore examined. u-HepaRG cells were less sensitive (>20-fold) to the toxic effects of M8OI. The relative insensitivity of u-HepaRG cells to M8OI was in part, associated with a detoxification by monooxygenation via CYP3A7 followed by further oxidation to a carboxylic acid. Expression of CYP3A7 - in contrast to the related adult hepatic CYP3A4 and CYP3A5 forms - was confirmed in u-HepaRG cells. However, blocking M8OI metabolism with ketoconazole only partly sensitized u-HepaRG cells. Despite similar proliferation rates, u-HepaRG cells consumed around 75% less oxygen than B-13 cells, reflective of reduced dependence on mitochondrial activity (Crabtree effect). Replacing glucose with galactose, resulted in an increase in u-HepaRG cell sensitivity to M8OI, near similar to that seen in B-13 cells. u-HepaRG cells therefore show reduced sensitivity to the toxic effects of M8OI through a combination of metabolic detoxification and their reduced reliance on mitochondrial function.

AP-1 and SP1 trans-activate the expression of hepatic CYP1A1 and CYP2A6 in the bioactivation of AFB1 in chicken.

Dietary exposure to aflatoxin B1 (AFB1) is harmful to the health and performance of domestic animals. The hepatic cytochrome P450s (CYPs), CYP1A1 and CYP2A6, are the primary enzymes responsible for the bioactivation of AFB1 to the highly toxic exo-AFB1-8,9-epoxide (AFBO) in chicks. However, the transcriptional regulation mechanism of these CYP genes in the liver of chicks in AFB1 metabolism remains unknown. Dual-luciferase reporter assay, bioinformatics and site-directed mutation results indicated that specificity protein 1 (SP1) and activator protein-1 (AP-1) motifs were located in the core region -1,063/-948, -606/-541 of the CYP1A1 promoter as well as -636/-595, -503/-462, -147/-1 of the CYP2A6 promoter. Furthermore, overexpression and decoy oligodeoxynucleotide technologies demonstrated that SP1 and AP-1 were pivotal transcriptional activators regulating the promoter activity of CYP1A1 and CYP2A6. Moreover, bioactivation of AFB1 to AFBO could be increased by upregulation of CYP1A1 and CYP2A6 expression, which was trans-activated owing to the upregulalion of AP-1, rather than SP1, stimulated by AFB1-induced reactive oxygen species. Additionally, nano-selenium could reduce ROS, downregulate AP-1 expression and then decrease the expression of CYP1A1 and CYP2A6, thus alleviating the toxicity of AFB1. In conclusion, AP-1 and SP1 played important roles in the transactivation of CYP1A1 and CYP2A6 expression and further bioactivated AFB1 to AFBO in chicken liver, which could provide novel targets for the remediation of aflatoxicosis in chicks.

Physiologically-based pharmacokinetic modelling guided dose evaluations of nirmatrelvir/ritonavir in renal impairment for the management of COVID-19.

We aimed to address factors contributing to the pharmacokinetic changes of nirmatrelvir/ritonavir in renal impaired (RI) patients and recommend dosing adjustment via a physiologically-based pharmacokinetic (PBPK) modelling approach. A PBPK model of nirmatrelvir/ritonavir was developed via Simcyp® Simulator. Sensitivity analysis of the influence of hepatic CYP3A4 intrinsic clearance and abundance, as well as hepatic non-CYP3A4 metabolism (other human liver microsomes [HLM] CLint) was performed to evaluate the effects of RI on oral clearance of nirmatrelvir. Other HLM CLint, the most sensitive parameter, was adjusted, and the simulated plasma concentration profiles of nirmatrelvir in severe RI subjects were within the therapeutic index of 292-10 000 ng/mL for dosing regimens of loading doses of 300/100 mg followed by 150/100 mg or 75/100 mg twice daily of nirmatrelvir/ritonavir. Considering that nirmatrelvir is available as a 150 mg tablet, we recommend 300/100 mg followed by 150/100 mg twice daily as the dosing regimen to be investigated in severe RI.

PBTK model-based analysis of CYP3A4 induction and the toxicokinetics of the pyrrolizidine alkaloid retrorsine in man

Cytochrome P450 (CYP)3A4 induction by drugs and pesticides plays a critical role in the enhancement of pyrrolizidine alkaloid (PA) toxicity as it leads to increased formation of hepatotoxic dehydro-PA metabolites. Addressing the need for a quantitative analysis of this interaction, we developed a physiologically-based toxicokinetic (PBTK) model. Specifically, the model describes the impact of the well-characterized CYP3A4 inducer rifampicin on the kinetics of retrorsine, which is a prototypic PA and contaminant in herbal teas. Based on consumption data, the kinetics after daily intake of retrorsine were simulated with concomitant rifampicin treatment. Strongest impact on retrorsine kinetics (plasma AUC24\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$_{24}$$\\end{document} and Cmax\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$C_{{\ ext {max}}}$$\\end{document} reduced to 67% and 74% compared to the rifampicin-free reference) was predicted directly after withdrawal of rifampicin. At this time point, the competitive inhibitory effect of rifampicin stopped, while CYP3A4 induction was still near its maximum. Due to the impacted metabolism kinetics, the cumulative formation of intestinal retrorsine CYP3A4 metabolites increased to 254% (from 10 to 25 nmol), while the cumulative formation of hepatic CYP3A4 metabolites was not affected (57 nmol). Return to baseline PA toxicokinetics was predicted 14 days after stop of a 14-day rifampicin treatment. In conclusion, the PBTK model showed to be a promising tool to assess the dynamic interplay of enzyme induction and toxification pathways.

In Vitro to In Vivo Scalars for Drug Clearance in Nonalcoholic Fatty Liver and Steatohepatitis.

In vitro-in vivo extrapolation (IVIVE) allows prediction of clinical outcomes across populations from in vitro data using specific scalars tailored to the biologic characteristics of each population. This study experimentally determined scalars for patients with varying degrees of nonalcoholic fatty liver disease (NAFLD), ranging from fatty liver to nonalcoholic steatohepatitis (NASH) and cirrhosis. Microsomal, S9, and cytosol fractions were extracted from 36 histologically normal and 66 NAFLD livers (27 nonalcoholic fatty liver [NAFL], 13 NASH, and 26 NASH with cirrhosis). Corrected microsomal protein per gram liver (MPPGL) progressively decreased with disease severity (26.8, 27.4, and 24.3 mg/g in NAFL, NASH, and NASH/cirrhosis, respectively, compared with 35.6 mg/g in normal livers; ANOVA, P < 0.001). Homogenate, S9, and cytosolic protein showed a consistent trend of decline in NASH/cirrhosis relative to normal control (post-hoc t test, P < 0.05). No differences across the groups were observed in homogenate, S9, cytosolic, and microsomal protein content in matched kidney samples. MPPGL-based scalars that combine protein content with liver size revealed that the reduction in MPPGL in NAFL and NASH was compensated by the reported increase in liver size (relative scalar ratios of 0.96 and 0.99, respectively), which was not the case with NASH/cirrhosis (ratio of 0.63), compared with healthy control. Physiologically based pharmacokinetics-informed global sensitivity analysis of the relative contribution of IVIVE scalars (hepatic CYP3A4 abundance, MPPGL, and liver size) to variability in exposure (area under the curve) to three CYP3A substrates (alprazolam, midazolam, and ibrutinib) revealed enzyme abundance as the most significant parameter, followed by MPPGL, whereas liver volume was the least impactful factor. SIGNIFICANCE STATEMENT: Nonalcoholic fatty liver disease-specific scalars necessary for extrapolation from in vitro systems to liver tissue are lacking. These are required in clearance prediction and dose selection in nonalcoholic fatty liver and steatohepatitis populations. Previously reported disease-driven changes have focused on cirrhosis, with no data on the initial stages of liver disease. The authors obtained experimental values for microsomal, cytosolic, and S9 fractions and assessed the relative impact of microsomal scalars on predicted exposure to substrate drugs using physiologically based pharmacokinetics.

FXR-FGF19 signaling in the gut–liver axis is dysregulated in patients with cirrhosis and correlates with impaired intestinal defence

Background and aimsExperimental studies linked dysfunctional Farnesoid X receptor (FXR)-fibroblast growth factor 19 (FGF19) signaling to liver disease. This study investigated key intersections of the FXR-FGF19 pathway along the gut–liver axis and their link to disease severity in patients with cirrhosis.MethodsPatients with cirrhosis undergoing hepatic venous pressure gradient measurement (cohort-I n = 107, including n = 53 with concomitant liver biopsy; n = 5 healthy controls) or colonoscopy with ileum biopsy (cohort-II n = 37; n = 6 controls) were included. Hepatic and intestinal gene expression reflecting FXR activation and intestinal barrier integrity was assessed. Systemic bile acid (BA) and FGF19 levels were measured.ResultsSystemic BA and FGF19 levels correlated significantly (r = 0.461; p < 0.001) and increased with cirrhosis severity. Hepatic SHP expression decreased in patients with cirrhosis (vs. controls; p < 0.001), indicating reduced FXR activation in the liver. Systemic FGF19 (r = −0.512, p < 0.001) and BA (r = −0.487, p < 0.001) levels correlated negatively with hepatic CYP7A1, but not SHP or CYP8B1 expression, suggesting impaired feedback signaling in the liver. In the ileum, expression of FXR, SHP and FGF19 decreased in patients with cirrhosis, and interestingly, intestinal FGF19 expression was not linked to systemic FGF19 levels. Intestinal zonula occludens-1, occludin, and alpha-5-defensin expression in the ileum correlated with SHP and decreased in patients with decompensated cirrhosis as compared to controls.ConclusionsFXR-FGF19 signaling is dysregulated at essential molecular intersections along the gut–liver axis in patients with cirrhosis. Decreased FXR activation in the ileum mucosa was linked to reduced expression of intestinal barrier proteins. These human data call for further mechanistic research on interventions targeting the FXR-FGF19 pathway in patients with cirrhosis.Clinical trial number: NCT03267615Graphical abstractPhysiology of enterohepatic FXR-FGF19 signaling and its regulation in patients with advanced chronic liver disease (ACLD). (FXR) farnesoid X receptor; (FGF19) fibroblast growth factor 19; (BA) bile acids; (c/dACLD) compensated/decompensated advanced chronic liver disease; (FXR) farnesoid X receptor; (SHP) small heterodimer partner; (OST-α/-β) organic solute transporter subunit alpha/beta; (CYP7A1) cholesterol 7 alpha-hydroxylase; (NTCP) Na+-taurocholate cotransporting polypeptide; (CYP8B1) sterol 12-alpha-hydroxylase; (HVPG) hepatic venous pressure gradient; (TJ) tight junctions; (AMP) antimicrobial peptides; (ASBT) Apical Sodium Dependent Bile Acid Transporter; (ZO 1) zonula occludens-1; (OCLN) occluding; (DEFA5) alpha-5-defensin.

Investigation of the Expression of CYP3A4 in Diabetic Rats in Xenobiotic Metabolism.

This study investigated the impact of a high-fat diet streptozotocin (STZ)-induced diabetes and dapagliflozin treatment on hepatic protein expression of CYP3A4. In our study, 34 male Sprague-Dawley rats were randomly divided into four groups: Control, high-fat diet and STZ-induced diabetes, dapagliflozin-treated control, and dapagliflozin-treated diabetes. In the microsomes obtained from the livers of these rats, the protein expression levels of CYP3A4 were determined by Western blotting. Hepatic CYP3A4 protein expression levels in the control group treated with dapagliflozin were significantly decreased compared with those in the control group. In addition, hepatic CYP3A4 protein expression levels were decreased in dapagliflozin-treated diabetic Sprague-Dawley rats compared with those in both control and diabetic group rats, but the difference between the groups was not statistically significant. According to these two results, the use of dapagliflozin inhibited hepatic CYP3A4 protein expression.

Evaluation of probiotic bifidobacteria strains from Iranian traditional dairy products for their anti-hyperlipidemic potential.

This study investigated the therapeutic potential of probiotic bifidobacteria, isolated from Iranian fermented dairy products, in a hyperlipidemic animal model. Bifidobacterium strains were extracted from traditional dairy samples and screened using physiological and phenotypic examinations, 16S rRNA analysis, and probiotic properties such as tolerance to gastrointestinal juice, antimicrobial activity, and antibiotic susceptibility. The ability of the screened bifidobacteria to reduce serum and liver lipids in vivo was tested using male Wistar rats. Six strains of bifidobacteria were isolated from traditional Iranian fermented dairy. These strains showed promising in vitro activity in lowering triglyceride and cholesterol, tolerance to simulated gastrointestinal juice, the ability to adhere to Caco-2 cells, acceptable antibiotic susceptibility, and a broad spectrum of antibacterial activity. The diet supplemented with isolated bifidobacteria significantly reduced serum total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), liver tissue lipid levels, and hepatic enzymes in animals when compared to a high-fat diet without strains (p < 0.01). Additionally, the potential probiotic-supplemented diet significantly increased bile acid excretion in the feces and upregulated hepatic CYP7A1 expression levels (p < 0.05), while NPC1L1, ACAT2, and MTP gene expressions in small intestinal cells were downregulated (p < 0.05). Bifidobacteria isolated from Iranian traditional dairy showed potential for use in the production of fermented foods that have hypolipemic activity in the host.

Maribavir: Mechanism of action, clinical, and translational science.

Maribavir is an oral benzimidazole riboside for treatment of post-transplant cytomegalovirus (CMV) infection/disease that is refractory to prior antiviral treatment (with or without resistance). Through competitive inhibition of adenosine triphosphate, maribavir prevents the phosphorylation actions of UL97 to inhibit CMV DNA replication, encapsidation, and nuclear egress. Maribavir is active against CMV strains with viral DNA polymerase mutations that confer resistance to other CMV antivirals. After oral administration, maribavir is rapidly and highly absorbed (fraction absorbed >90%). The approved dose of 400 mg twice daily (b.i.d.) achieves a steady-state area under the curve per dosing interval of 128 h*μg/mL and trough concentration of 4.90 μg/mL (13.0 μM). Maribavir is highly bound to human plasma proteins (98%) with a small apparent volume of distribution of 27.3 L. Maribavir is primarily cleared by hepatic CYP3A4 metabolism; its major metabolite, VP44669 (pharmacologically inactive), is excreted in the urine and feces. There is no clinically relevant impact on maribavir pharmacokinetics by age, sex, race/ethnicity, body weight, transplant type, or hepatic/renal impairment status. In phase II dose-ranging studies, maribavir showed similar rates of CMV viral clearance across 400, 800, or 1200 mg b.i.d. groups, ranging from 62.5-70% in study 202 (NCT01611974) and 74-83% in study 203 (EudraCT 2010-024247-32). In the phase III SOLSTICE trial (NCT02931539), maribavir 400 mg b.i.d. demonstrated superior CMV viremia clearance at week 8 versus investigator-assigned treatments, with lower treatment discontinuation rates. Dysgeusia, nausea, vomiting, and diarrhea were commonly experienced adverse events among patients treated with maribavir in clinical trials.

Acetaminophen: for Its Mechanism of Analgesic Action in Vivo Using Molecular Docking

Paracetamol is the most widely used over-the-counter drug in world. The mechanism of action of its analgesic and antipyretic effect by relying on the association of activation of dopamine D2 receptors through two mechanisms of action. A new metabolic pathway involving the generation of an active metabolite p-aminophenol By being metabolized in the liver by acylamidase/Ndeacetylase, and Metabolism of paracetamol to C_6 H_7 NO_2 by its hepatic metabolism CYP2A6. This paper describes the experimental Data that showed the involvement of these two metabolic pathways in analgesic and antipyretic action of paracetamol and its relationship with dopamine D2 receptors “It also explains how new targets and systems play an important role in the action of paracetamol” Finally, treatment strategies for pain and Antipyretic

Vincristine Disposition and Neurotoxicity Are Unchanged in Humanized CYP3A5 Mice.

Previous studies have suggested that the incidence of vincristine-induced peripheral neuropathy (VIPN) is potentially linked with cytochrome P450 (CYP)3A5, a polymorphic enzyme that metabolizes vincristine in vitro, and with concurrent use of azole antifungals such as ketoconazole. The assumed mechanism for these interactions is through modulation of CYP3A-mediated metabolism, leading to decreased vincristine clearance and increased susceptibility to VIPN. Given the controversy surrounding the contribution of these mechanisms, we directly tested these hypotheses in genetically engineered mouse models with a deficiency of the entire murine Cyp3a locus [Cyp3a(-/-) mice] and in humanized transgenic animals with hepatic expression of functional and nonfunctional human CYP3A5 variants. Compared with wild-type mice, the systemic exposure to vincristine was increased by only 1.15-fold (95% confidence interval, 0.84-1.58) in Cyp3a(-/-) mice, suggesting that the clearance of vincristine in mice is largely independent of hepatic Cyp3a function. In line with these observations, we found that Cyp3a deficiency or pretreatment with the CYP3A inhibitors ketoconazole or nilotinib did not influence the severity and time course of VIPN and that exposure to vincristine was not substantially altered in humanized CYP3A5*3 mice or humanized CYP3A5*1 mice compared with Cyp3a(-/-) mice. Our study suggests that the contribution of CYP3A5-mediated metabolism to vincristine elimination and the associated drug-drug interaction potential is limited and that plasma levels of vincristine are unlikely to be strongly predictive of VIPN. SIGNIFICANCE STATEMENT: The current study suggests that CYP3A5 genotype status does not substantially influence vincristine disposition and neurotoxicity in translationally relevant murine models. These findings raise concerns about the causality of previously reported relationships between variant CYP3A5 genotypes or concomitant azole use with the incidence of vincristine neurotoxicity.

Divergence in aerobic capacity influences hepatic and systemic metabolic adaptations to bile acid sequestrant and short-term high-fat/sucrose feeding in rats.

High versus low aerobic capacity significantly impacts the risk for metabolic diseases. Rats selectively bred for high or low intrinsic aerobic capacity differently modify hepatic bile acid metabolism in response to high-fat diets (HFDs). Here we tested if a bile acid sequestrant would alter hepatic and whole body metabolism differently in rats with high and low aerobic capacity fed a 1-wk HFD. Male rats (8 mo of age) that were artificially selected to be high (HCR) and low-capacity runners (LCR) with divergent intrinsic aerobic capacities were transitioned from a low-fat diet (LFD, 10% fat) to an HFD (45% fat) with or without a bile acid sequestrant (BA-Seq, 2% cholestyramine resin) for 7 days while maintained in an indirect calorimetry system. HFD + BA-Seq increased fecal excretion of lipids and bile acids and prevented weight and fat mass gain in both strains. Interestingly, HCR rats had increased adaptability to enhance fecal bile acid and lipid loss, resulting in more significant energy loss than their LCR counterpart. In addition, BA-Seq induced a greater expression of hepatic CYP7A1 gene expression, the rate-limiting enzyme of bile acid synthesis in HCR rats both on HFD and HFD + BA-Seq diets. HCR displayed a more significant reduction of RQ in response to HFD than LCR, but HFD + BA-Seq lowered RQ in both groups compared with HFD alone, demonstrating a pronounced impact on metabolic flexibility. In conclusion, BA-Seq provides uniform metabolic benefits for metabolic flexibility and adiposity, but rats with higher aerobic capacity display adaptability for hepatic bile acid metabolism.NEW & NOTEWORTHY The administration of bile acid sequestrant (BA-Seq) has uniform metabolic benefits in terms of metabolic flexibility and adiposity in rats with high and low aerobic capacity. However, rats with higher aerobic capacity demonstrate greater adaptability in hepatic bile acid metabolism, resulting in increased fecal bile acid and lipid loss, as well as enhanced fecal energy loss.

QiShenYiQi pill inhibits atherosclerosis by promoting TTC39B-LXR mediated reverse cholesterol transport in liver

BackgroundTetranucleotide repeat domain protein 39B (TTC39B) was found to combine with ubiquitin ligase E3, and promote the ubiquitination modification of liver X receptor (LXR), which led to the inhibition of reverse cholesterol transport and development of atherosclerosis. QiShenYiQi pill (QSYQ) is a modern Chinese patent drug for treating ischemic cardiovascular diseases, the underlying mechanism is found to promote the expression of LXR-α/ ATP-binding cassette transporter G5 (ABCG5) in the liver of atherosclerotic mice. PurposeThe aim of this study is to investigate the effect of QSYQ on TTC39B-LXR mediated reverse cholesterol transport in atherosclerotic mice. Study design and methodsMale apolipoprotein E gene knockout mice (7 weeks old) were fed with high-fat diet and treated with low dose of QSYQ (QSYQ-l, 0.3 g/kg·d), high dose of QSYQ (QSYQ-H, 1.2 g/kg·d) and LXR-α agonist (LXR-A, GW3965 10 mg/kg·d) for 8 weeks. C57BL/6 J mice were fed with normal diet and used as negative control. Oil red O staining, HE staining, ELISA, RNA sequencing, western blot, immunohistochemistry, RT-PCR, cell culture and RNA interference were performed to analyze the effect of QSYQ on atherosclerosis. ResultsHE staining showed that QSYQ reduced the atherosclerotic lesion significantly when compared to the control group. ELISA measurement showed that QSYQ decreased serum VLDL and increased serum ApoA1. Oil Red O staining showed that QSYQ reduced the lipid content of liver and protect liver function. Comparative transcriptome RNA-sequence of liver showed that DEGs after QSYQ treatment enriched in high-density lipoprotein particle, ubiquitin ligase complex, bile secretion, etc. Immunohistochemical staining and western blot proved that QSYQ increased the protein expression of hepatic SR-B1, LXR-α, LXR-β, CYP7A1 and ABCG5. Targeted inhibiting Ttc39b gene in vitro further established that QSYQ inhibited the gene expression of Ttc39b, increased the protein expression of SR-B1, LXR-α/β, CYP7A1 and ABCG5 in rat hepatocyte. ConclusionOur results demonstrated the new anti-atherosclerotic mechanism of QSYQ by targeting TTC39B-LXR mediated reverse cholesterol transport in liver. QSYQ not only promoted reverse cholesterol transport, but also improved fatty liver and protected liver function.

Pharmacokinetic and pharmacodynamic drug-drug interaction of Nomilin with atorvastatin in hyperlipidemic mice

Atorvastatin (Atv) is widely used to lower cholesterol levels and treat hyperlipidemia in clinical application. Nomilin (Nom) is a kind of limonoids, which is found and isolated from the citrus herbs of Rutaceae family, which are widely used as patent medicines, functional foods, and nutritional supplements in many countries. In previous studies, Nom has the effect of anti-obesity and curing other metabolic diseases. Nevertheless, in recent years, the drug-drug interaction (DDI) caused by the administration of drugs with synergistic effects have raised worldwide concerns. To investigate the DDI of Nom and Atv in vivo, the pharmacokinetic studies were performed with using C57BL/6 mice. The plasma concentrations of Nom and Atv were measured after oral administration of different drug combinations by a simple and sensitive UHPLC-MS/MS method. The experimental mice were randomly divided into five groups, including control group, model group, administered Nom individually group, administered Atv individually group and co-administered of Nom and Atv group. The lipid levels including total cholesterol (TC), triglycerides (TG), high density lipoproteins-cholesterol (HDL-C), low density lipoproteins-cholesterol (LDL-C) were measured for pharmacodynamic study. The hepatic microsomal Cytochrome P450 (CYP1A2, CYP2E1 and CYP3A11) activities were probed using cocktail assay. The gene and protein expressions of CYP3A11 were detected via qPCR and Western blot method. The results shown that the area under the plasma concentration-time curve (AUC) of Atv in administered Atv individually group was 69.30 ± 15.45 ng/mL × h, while that of combined Nom with Atv group was 42.37 ± 10.15 ng/mL × h (p＜0.05). The degree of reduction in lipid levels of mice treated with co-administration of Atv and Nom was less than that of mice treated with Atv alone. In addition, Nom could cause an increased hepatic microsomal CYP3A11 activity significantly, and induce the gene levels and protein expressions of CYP3A11 elevated in mice livers. In conclusion, Nom could up-regulate CYP3A11 activity, thereby impacting on the pharmacokinetic profile and pharmacodynamic effect of Atv. The findings provide more insight for the use risk of these two drugs to treat hyperlipidemia diseases.

CRY1/2 regulate rhythmic CYP2A5 in mouse liver through repression of E4BP4

CYP2A5, an enzyme responsible for metabolism of diverse drugs, displays circadian rhythms in its expression and activity. However, the underlying mechanisms are not fully established. Here we aimed to investigate a potential role of CRY1/2 (circadian clock modulators) in circadian regulation of hepatic CYP2A5. Regulatory effects of CRY1/2 on CYP2A5 were determined using Cry1-null and Cry2-null mice, and validated using AML-12, Hepa1-6 and HepG2 cells. CYP2A5 activities both in vivo and in vitro were assessed using coumarin 7-hydroxylation as a probe reaction. mRNA and protein levels were detected by qPCR and western blotting, respectively. Regulatory mechanism was studied using a combination of luciferase reporter assays, chromatin immunoprecipitation (ChIP) and co-immunoprecipitation (Co-IP). We found that ablation of Cry1 or Cry2 in mice reduced hepatic CYP2A5 expression (at both mRNA and protein levels) and blunted its diurnal rhythms. Consistently, these knockouts showed decreased CYP2A5 activity (characterised by coumarin 7-hydroxylation) and a loss of its time-dependency, as well as exacerbated coumarin-induced hepatotoxicity. Cell-based assays confirmed that CRY1/2 positively regulated CYP2A5 expression and rhythms. Based on combined luciferase reporter, ChIP and Co-IP assays, we unraveled that CRY1/2 interacted with E4BP4 protein to repress its inhibitory effect on Cyp2a5 transcription and expression. In conclusion, CRY1/2 regulate rhythmic CYP2A5 in mouse liver through repression of E4BP4. These findings advance our understanding of circadian regulation of drug metabolism and pharmacokinetics.

Activation of CYP3A by ABC phenomenon potentiates the hepatocellular carcinoma-targeting therapeutic effects of PEGylated anticancer prodrug liposomes.

Reduced enzyme activity in hepatocellular carcinoma (HCC) and poor targeting limit the application of enzyme-activating prodrugs, which is also detrimental to the effective treatment of HCC. Here, we investigated whether accelerated blood clearance (ABC) phenomenon occurs in HCC models following repeated injections of PEGylated liposomes (PEG-L), thus inducing prodrugs accumulation and activation in the liver and exerting highly effective and low-toxicity therapeutic effects on HCC. Firstly, PEGylated liposomal cyclophosphamide (PEG-CP-L) was prepared by solvent injection and characterized. Importantly, pre-injection of PEG-L induced ABC phenomenon and activation of CYP3A in both HCC rats and HCC mice by investigating the effects of repeated injections of PEG-L on pharmacokinetics and tissue distribution. Next, the efficacy and toxicity of repeated injections of PEG-L in HCC mice were examined, and our data indicate that repeated injections are administered in a manner that significantly enhances the antitumor effect compared to controls with little or no toxicity to other organs. To further reveal the pharmacokinetic mechanism of PEG-L repeated administration for the treatment of HCC, the protein expression of hepatic CYP3A and the concentration of CP in the liver and spleen of HCC mice by inhibiting CYP3A were analyzed. These results revealed that inducing CYP3A to accelerate the rapid conversion of prodrugs that accumulate significantly in the liver is a key mechanism for the treatment of HCC with repeated injections of PEG-L. Collectively, this work taps into the application potential of the ABC phenomenon and provides new insights into the clinical application of PEGylated nanoformulations. Significance Statement This study revealed that repeated injections of PEG-L could induce the ABC phenomenon characterized by hepatic accumulation and CYP3A activation based on HCC rats and HCC mice. Furthermore, it was verified that induction of the ABC phenomenon dependent on hepatic accumulation and CYP3A activation could enhance the anti-hepatocellular carcinoma effects of PEGylated anticancer prodrugs in HCC mice. This elucidated the relevant pharmacokinetic mechanisms and unearths new clues for solving the clinical application of PEGylated nanoparticles.

Consideration of Nevirapine Analogs To Reduce Metabolically Linked Hepatotoxicity: A Cautionary Tale of the Deuteration Approach.

Idiosyncratic drug reactions (IDRs) in their most deleterious form can lead to serious medical complications and potentially fatal events. Nevirapine (NVP), still widely used in developing countries for combinatorial antiretroviral and prophylactic therapies against HIV infection, represents a prototypical example of IDRs causing severe skin rashes and hepatotoxicity. Complex metabolic pathways accompanied by production of multiple reactive metabolites often complicate our understanding of IDR's origin. While assessment of NVP analogs has helped characterize the pathways involved in IDRs for NVP, which are largely driven by metabolism at the 12-methyl position, it has yet to be investigated if some of these analogs could be valuable replacement drugs with reduced reactive metabolite properties and drug-drug interaction (DDI) risks. Here, we evaluated a set of eight NVP analogs, including the deuterated 12-d3-NVP and two NVP metabolites, for their efficacy and inhibitory potencies against HIV reverse transcriptase (HIV-RT). A subset of three analogs, demonstrating >85% inhibition for HIV-RT, was further assessed for their hepatic CYP induction-driven DDI risks. This led to a closer investigation of the inactivation properties of 12-d3-NVP for hepatic CYP3A4 and a comparison of its propensity in generating reactive metabolite species. The metabolic shift triggered with 12-d3-NVP, increasing formation of the 2-hydroxy and glutathione metabolites, emphasized the importance of the dynamic balance between induction and metabolism-dependent inactivation of CYP3A4 and its impact on clearance of NVP during treatment. Unfortunately, the strategy of incorporating deuterium to reduce NVP metabolism and production of the electrophile species elicited opposite results, illustrating the great challenges involved in tackling IDRs through deuteration.

B-337 Case Report: Fatal Opioid Overdose Induced by Over-the-Counter Cold Medication Diphenhydramine

Abstract Background Accidental drug-related deaths frequently arise following ingestion of multiple co-intoxicants, including opioid analgesics. Hydrocodone, the most prescribed opioid for pain management, is metabolized by hepatic enzymes CYP2D6 and CYP3A4 to the pharmacologically active metabolite hydromorphone and the inactive metabolite norhydrocodone, respectively. As a result, inhibition of CYP2D6 by other pharmacologic agents can impair the analgesic properties of hydrocodone and prolong its half-life in circulation. One such example is diphenhydramine, which is a potent inhibitor of CYP2D6 activity. This case highlights a drug-drug interaction likely resulting in an accidental fatal opioid overdose. Case Presentation A 58-year-old female had longstanding prescriptions for hydrocodone/acetaminophen (Norco® 10/325), alprazolam, carisoprodol, and diltiazem. The decedent had recently taken an over-the-counter cold medication, diphenhydramine (Benadryl), shortly preceding death. A toxicology report (NMS labs, Horsham, PA) from a post-mortem femoral blood sample detected the presence of the following therapeutic compounds by High Performance Liquid Chromatography/Time of Flight-Mass Spectrometry (LC/TOF-MS) analysis: acetaminophen (14 μg/mL), hydrocodone (410 ng/mL), dihydrocodeine (hydrocodol, 24 ng/mL), alprazolam (42 ng/mL), carisoprodol (88 ng/mL), diltiazem (110 ng/mL), and diphenhydramine (150 ng/mL). Hydromorphone, the active metabolite of hydrocodone, was not detected (&amp;lt;2 ng/mL). With the exception of hydrocodone, which was present at fatal concentrations, all other compounds were within therapeutic range. Together, these observations are strongly suggestive of a drug-drug interaction leading to impaired hydrocodone metabolism. Conclusions This case report highlights a fatal opioid overdose as a result of CYP2D6 inhibition by diphenhydramine, a readily available over-the-counter medication. Post-mortem toxicological analysis revealed hydrocodone was present in lethal concentrations, whereas its pharmacologically active metabolite, hydromorphone, was undetectable at time of autopsy. Further, the patient was prescribed a formulation of hydrocodone in combination with acetaminophen, which was present at therapeutic concentrations despite the two drugs having similar elimination half-lives. These toxicological findings suggest administration of diphenhydramine precipitated a deadly accumulation of hydrocodone in the deceased. Overall, this case report underscores the importance of understanding and preventing harmful drug-drug interactions in patients taking opioid medications for pain management.

Physiologically-based pharmacokinetic modeling for investigating the effect of simeprevir on concomitant drugs and an endogenous biomarker of OATP1B.

The orally available anti-hepatitis C virus (HCV) drug simeprevir exhibits nonlinear pharmacokinetics at the clinical doses due to saturation of cytochrome P450 (CYP) 3A4 metabolism and organic anion transporting peptide (OATP) 1B mediated hepatic uptake. Additionally, simeprevir increases exposures of concomitant drugs by CYP3A4 and OATP1B inhibition. The objective of this study was to develop physiologically-based pharmacokinetic (PBPK) models that could describe drug-drug interactions (DDIs) of simeprevir with concomitant drugs via CYP3A4 and OATP1B inhibition, and also to capture the effects on coproporphyrin-I (CP-I), an endogenous biomarker of OATP1B. PBPK modeling estimated unbound simeprevir inhibitory constant (Ki ) of 2.89 μM against CYP3A4 in the DDI results between simeprevir and midazolam in healthy volunteers. Then, we analyzed the DDIs between simeprevir and atorvastatin, a dual substrate of CYP3A4 and OATP1B, in healthy volunteers, and unbound Ki against OATP1B was estimated to be 0.00347 μM. Finally, we analyzed the increase in the blood level of CP-I by simeprevir to verify the Ki,OATP1B . Because CP-I was measured in subjects with HCV with various hepatic fibrosis state, Monte Carlo simulation was performed to involve the decreases in expression levels of hepatic CYP3A4 and OATP1B and their interindividual variabilities. The PBPK modeling coupled with Monte Carlo simulation using the Ki,OATP1B value obtained from atorvastatin study reasonably recovered the observed relationship between CP-I and simeprevir blood levels. In conclusion, the simeprevir PBPK model developed in this study can quantitatively describe the increase in exposures of concomitant drugs and an endogenous biomarker via inhibition of CYP3A4 and OATP1B.

Investigating Tacrolimus Disposition in Paediatric Patients with a Physiologically Based Pharmacokinetic Model Incorporating CYP3A4 Ontogeny, Mechanistic Absorption and Red Blood Cell Binding.

Tacrolimus is a crucial immunosuppressant for organ transplant patients, requiring therapeutic drug monitoring due to its variable exposure after oral intake. Physiologically based pharmacokinetic (PBPK) modelling has provided insights into tacrolimus disposition in adults but has limited application in paediatrics. This study investigated age dependency in tacrolimus exposure at the levels of absorption, metabolism, and distribution. Based on the literature data, a PBPK model was developed to predict tacrolimus exposure in adults after intravenous and oral administration. This model was then extrapolated to the paediatric population, using a unique reference dataset of kidney transplant patients. Selecting adequate ontogeny profiles for hepatic and intestinal CYP3A4 appeared critical to using the model in children. The best model performance was achieved by using the Upreti ontogeny in both the liver and intestines. To mechanistically evaluate the impact of absorption on tacrolimus exposure, biorelevant in vitro solubility and dissolution data were obtained. A relatively fast and complete release of tacrolimus from its amorphous formulation was observed when mimicking adult or paediatric dissolution conditions (dose, fluid volume). In both the adult and paediatric PBPK models, the in vitro dissolution profiles could be adequately substituted by diffusion-layer-based dissolution modelling. At the level of distribution, sensitivity analysis suggested that differences in blood plasma partitioning of tacrolimus may contribute to the variability in exposure in paediatric patients.