Cytochrome P450 Substrates Research Articles

Impact of Interferons and Biological Drug Inhibitors of IL-2 and IL-6 on Small-Molecule Drug Metabolism Through the Cytochrome P450 System.

Assess the impact of interferons and interleukin (IL)-2 and IL-6 inhibitors on cytochrome P450 (CYP) drug metabolism in human subjects. PubMed search from 1980 to March 31, 2021, limited to human subjects and English language via search strategy: (biological drug names) [AND] (cytochrome [OR] CYP metabolism). Narrative review of human studies assessing biological drugs in select classes that affect CYP drug metabolism. Exogenous interferons suppress CYP1A2 (theophylline, caffeine, antipyrone) clearance by 20% to 49% in patients; have minimal impact on CYP3A4 (midazolam and dapsone), CYP2C9 (tolbutamide), or CYP2C19 (mephenytoin) metabolism; and increase CYP2D6 (debrisoquine, dextromethorphan) metabolism. Biological IL-2 inhibitors (basiliximab, daclizumab) have no effect on metabolism via CYP1A2 (caffeine), CYP2C9 (s-warfarin), CYP2C19 (omeprazole), CYP2D6 (dextromethorphan), and CYP3A4 (midazolam, tacrolimus) but may enhance CYP3A4 (cyclosporin) metabolism over time. IL-6 inhibitors (sirukumab, tocilizumab, sarilumab) significantly enhance metabolism via CYP2C9 (s-warfarin), CYP2C19 (omeprazole), and CYP3A4 (simvastatin, midazolam) and reduce metabolism via CYP1A2 (caffeine). Patients using interferons, IL-2, or IL-6 blocking drugs at steady state with CYP substrates could have altered drug metabolism and experience adverse events. With interferons and biological anti-inflammatory drugs, some isoenzymes will be inhibited, whereas others will be enhanced, and the magnitude of the effect can sometimes be significant. In clinical practice, clinicians may consider these metabolic changes as an additive effect to a patient's entire disease and medication profile when determining risk/benefit of treatment. Interferon therapy or inflammatory suppression via IL-2 or IL-6 can alter steady-state concentrations of CYP-metabolized small-molecule drugs.

Effect of Wnt5a on drug resistance in estrogen receptor-positive breast cancer

BackgroundPreviously, we reported that Wnt5a-positive breast cancer can be classified as estrogen receptor (ER)-positive breast cancer; its prognosis is worse than that of Wnt5a-negative breast cancer. This study aimed to investigate the mechanisms underlying the poor prognosis in Wnt5a-positive breast cancer patients.MethodsIn total, 151 consecutive ER-positive breast cancer patients who underwent resection between January 2011 and February 2014 were enrolled. DNA microarray and pathway analyses were conducted using MCF-7 cells stably expressing Wnt5a [MCF-7/Wnt5a (+)]. Based on the outcomes, cell viability/drug sensitivity assays, and mutation analysis were performed using cell cultures and breast cancer tissues. The relationship between Wnt5a and the PI3K–AKT–mTOR signaling pathway was also examined.ResultsThe relapse-free survival rate in patients with Wnt5a-positive breast cancer was significantly lower than that in patients with Wnt5a-negative breast cancer (P = 0.047). DNA microarray data suggest that only the cytochrome P450 (CYP) pathway was significantly upregulated in MCF-7/Wnt5a (+) cells (P = 0.0440). Additionally, MCF-7/Wnt5a (+) cells displayed reduced sensitivity to the metabolic substrates of CYP, tamoxifen (P < 0.001), paclitaxel (P < 0.001), and cyclophosphamide (P < 0.001). Of note, PIK3CA mutations were not associated with the expression of Wnt5a in breast cancer tissue and culture cells.ConclusionsIn ER-positive breast cancer, Wnt5a upregulates the CYP metabolic pathway and suppresses tamoxifen, paclitaxel, and cyclophosphamide resistance, all of the three, standard treatment methods for ER-positive breast cancer. Wnt5a is thus potentially involved in the poor prognosis of ER-positive breast cancer independently of the PI3K–AKT–mTOR signaling pathway.

CyProduct: A Software Tool for Accurately Predicting the Byproducts of Human Cytochrome P450 Metabolism.

In silico metabolism prediction is a cheminformatic task of autonomously predicting the set of metabolic byproducts produced from a specified molecule and a set of enzymes or reactions. Here, we describe a novel machine learned in silico cytochrome P450 (CYP450) metabolism prediction suite, called CyProduct, that accurately predicts metabolic byproducts for a specified molecule and a human CYP450 isoform. It includes three modules: (1) CypReact, a tool that predicts if the query compound reacts with a given CYP450 enzyme, (2) CypBoM, a tool that accurately predicts the "bond site" of the reaction (i.e., which specific bonds within the query molecule react with the CYP isoform), and (3) MetaboGen, a tool that generates the metabolic byproducts based on CypBoM's bond-site prediction. CyProduct predicts metabolic biotransformation products for each of the nine most important human CYP450 enzymes. CypBoM uses an important new concept called "bond of metabolism" (BoM), which extends the traditional "site of metabolism" (SoM) by specifying the information about the set of chemical bonds that is modified or formed in a metabolic reaction (rather than the specific atom). We created a BoM database for 1845 CYP450-mediated Phase I reactions, then used this to train the CypBoM Predictor to predict the reactive bond locations on substrate molecules. CypBoM Predictor's cross-validated Jaccard score for reactive bond prediction ranged from 0.380 to 0.452 over the nine CYP450 enzymes. Over variants of a test set of 68 known CYP450 substrates and 30 nonreactants, CyProduct outperformed the other packages, including ADMET Predictor, BioTransformer, and GLORY, by an average of 200% (with respect to Jaccard score) in terms of predicting metabolites. The CyProduct suite and the data sets are freely available at https://bitbucket.org/wishartlab/cyproduct/src/master/.

Design of New Schiff-Base Copper(II) Complexes: Synthesis, Crystal Structures, DFT Study, and Binding Potency toward Cytochrome P450 3A4

We report the synthesis and crystal structures of three new copper(II) Schiff-base complexes. The complexes have been characterized by elemental analysis and Fourier transform infrared (FT-IR) and UV–visible spectroscopies. The X-ray diffraction (XRD) analysis reveals that complexes 1 and 3 crystallize in a monoclinic space group C2/c and 2 in a triclinic space group P1̅, each adopting a square planar geometry around the metal center. We use a density functional theory method to explore the quantum chemical properties of these complexes. The calculation proceeds with the three-dimensional (3D) crystal structure characterization of the complexes in which the calculated IR and UV–vis values are comparable to the experimental results. Charge distribution and molecular orbital analyses enabled quantum chemical property prediction of these complexes. We study the drug-likeness properties and binding potentials of the synthesized complexes. The in silico outcome showed that they could serve as permeability-glycoprotein (P-gp) and different cytochrome P450 substrates. Our calculations showed that the complexes significantly bind to cytochrome P450 3A4.

Drug metabolism in severe chronic obstructive pulmonary disease: A phenotyping cocktail study.

To evaluate the effect of severe chronic obstructive pulmonary disease (COPD) on drug metabolism by comparing the pharmacokinetics of patients with severe COPD with healthy volunteers and using the modified Inje drug cocktail. This was a single-centre pharmacokinetic study with 12 healthy participants and 7 participants with GOLD D COPD. Midazolam 1mg, dextromethorphan 30 mg, losartan 25 mg, omeprazole 20 mg, caffeine 130 mg and paracetamol 1000 mg were simultaneously administered and intensive pharmacokinetic sampling was conducted over 8hours. Drug metabolism by CYP3A4, CYP2D6, CYP2C9, CYP2C19, CYP1A2, UGT1A6 and UGT1A9 in participants with COPD were compared with phenotypes in healthy controls. The oral clearance (95% confidence interval) in participants with COPD relative to controls was: midazolam 63% (60-67%); dextromethorphan 72% (40-103%); losartan 53% (52-55%); omeprazole 35% (31-39%); caffeine 52% (50-53%); and paracetamol 73% (72-74%). There was a 5-fold increase in AUC for omeprazole and approximately 2-fold increases for caffeine, losartan, dextromethorphan, and midazolam. The AUC of paracetamol, which is mostly glucuronidated, was increased by about 60%. Severe COPD is associated with a clinically significant reduction in oral drug clearance. This may be greater for cytochrome P450 substrates than for glucuronidated drugs. This supports reduced starting doses when prescribing for patients with severe COPD.

A comprehensive characterization of novel CYP-BM3 homolog (CYP-BA) from Bacillus aryabhattai

Functionalizing C-H bond poses one of the most significant challenges for chemists providing them with very few substrate-specific synthetic routes. Despite being incredibly plastic in their enzymatic ability, they are confined with deficient enzymatic action and limited explicitness of the substrates. In this study, we have endeavored to characterize novel cytochrome P450 from Bacillus aryabhattai (CYP-BA), a homolog of CYP P450-BM3, by taking interdisciplinary approaches. We conducted structure and sequence comparison to understand the conservation pattern for active site residues, conserved fold, evolutionary relationships among others. Molecular dynamics simulations were performed to understand the dynamic nature and interaction with the substrates. CYP-BA was successfully cloned, purified, and characterized. The enzyme's stability toward various physicochemical parameters was evaluated by UV–vis spectroscopy and Circular Dichroism (CD) spectroscopy. Various saturated fatty acids being the natural cytochrome P450 substrates were evaluated as catalytic efficiency of substrate oxidation by CYP-BA. The binding affinity of these natural substrates was monitored against CYP-BA by isothermal titration calorimetry (ITC). The catalytic performance of CYP-BA was satisfactory enough to proceed to the next step, that is, engineering to expand the substrate range to include polycyclic aromatic hydrocarbons (PAH). This is the first evidence of cloning, purifying and characterizing a novel homolog of CYP-BM3 to enable a better understanding of this novel biocatalyst and to provide a platform toward expanding its catalytic process through enzyme engineering.

The interactions between COVID-19 drugs and psychotropic agents

Coronavirus disease (COVID-19) is a systemic infection targeting multiple organs. Interstitial pneumonia is the landmark feature of this condition. Severe acute respiratory symptoms requiring intensive care support arises for about one out of twenty symptomatic cases. Aminochinolone, antiviral, antibiotic, corticoid, anticoagulant and immunobiological drugs are used, mostly to treat symptoms. Only remdesivir exhibiting weak antiviral activity is approved for COVID-19. Psychotropic medications may interact with medical treatments for COVID-19. The aim of this presentation is to highlight pharmacokinetic and pharmacodynamic drug-drug interactions to be expected for medical treatments of COVID-19. Remdesivir and favipiravir exhibit hepatotoxic properties which may be enhanced under combinations with tricyclic antidepressants or agomelatine. Favipiravir, hydroxychloroquine, chloroquine, azithromycin, lopinavir/ritonavir have QT interval prolongation potential and must be considered for combinations with antidepressant and antipsychotic drugs. For hydroxychloroquine, hypoglycemic activity may give rise to endocrine disturbances. Pharmacokinetic drug-drug interactions can be expected for lopinavir/ritonavir which inhibit cytochrome P-450 (CYP) 3A4 and induce CYP2C9 and CYP2C19. Combinations with psychotropic drugs that are substrates of these enzymes (victim drugs) will affect drug concentrations in blood and lead to supra- or subtherapeutic levels. Moreover, it must be assumed that the COVID-19 infection is associated with an enhanced production of cytokines which has a known impact on CYP enzyme activities. Though studies on interactions between psychotropic medications and medical treatments for COVID-19 are lacking, multiple drug interactions can be predicted and expected considering the side effect profiles and CYP inhibitory, inducing and substrate properties of combined drugs.DisclosureNo significant relationships.

Exploring the potential of functional polymer-lipid hybrid nanoparticles for enhanced oral delivery of paclitaxel

Most biopharmaceutics classification system (BCS) class IV drugs, with poor solubility and inferior permeability, are also substrates of P-glycoprotein (P-gp) and cytochrome P450 (CYP450), leading to their low oral bioavailability. The objective of this study is to explore the potential of using functional polymer-lipid hybrid nanoparticles (PLHNs) to enhance the oral absorption of BCS IV drugs. In this paper, taking paclitaxel (PTX) as a drug model, PTX-loaded PLHNs were prepared by a self-assembly method. Chitosan was selected to modify the PLHN to enhance its mucoadhesion and stability. Three P-gp inhibitors (D-α-tocopherol polyethylene glycol 1000 succinate, pluronic P123 and SolutolⓇ HS15) were incorporated into selected PLHNs, and a CYP450 inhibitor (the extract of VBRB, BC0) was utilized to jointly promote the drug absorption. Properties of all the PLHNs were characterized systemically, including particle size, zeta potential, encapsulation efficiency, morphology, stability, in vitro drug release, mucoadhesion, in situ intestinal permeability and in vivo systemic exposure. It was found mucoadhesion of the CS-modified PLHNs was the strongest among all the formulations tested, with absolute bioavailability 21.95%. P-gp and CYP450 inhibitors incorporation further improved the oral bioavailability of PTX to 42.60%, 8-fold increase compared with that of PTX itself (4.75%). Taken together, our study might shed light on constructing multifunctional PLHNs based on drug delivery barriers for better oral absorption, especially for BCS IV drugs.

A pharmacovigilance study of pharmacokinetic drug interactions using a translational informatics discovery approach.

While the pharmacokinetic (PK) mechanisms for many drug interactions (DDIs) have been established, pharmacovigilance studies related to these PK DDIs are limited. Using a large surveillance database, a translational informatics approach can systematically screen adverse drug events (ADEs) for many DDIs with known PK mechanisms. We collected a set of substrates and inhibitors related to the cytochrome P450 (CYP) isoforms, as recommended by the United States Food and Drug Administration (FDA) and Drug Interactions Flockhart table™. The FDA's Adverse Events Reporting System (FAERS) was used to obtain ADE reports from 2004 to 2018. The substrate and inhibitor information were used to form PK DDI pairs for each of the CYP isoforms and Medical Dictionary for Regulatory Activities (MedDRA) preferred terms used for ADEs in FAERS. A shrinkage observed-to-expected ratio (Ω) analysis was performed to screen for potential PK DDI and ADE associations. We identified 149 CYP substrates and 62 CYP inhibitors from the FDA and Flockhart tables. Using FAERS data, only those DDI-ADE associations were considered that met the disproportionality threshold of Ω>0 for a CYP substrate when paired with at least two inhibitors. In total, 590 ADEs were associated with 2085 PK DDI pairs and 38 individual substrates, with ADEs overlapping across different CYP substrates. More importantly, we were able to find clinical and experimental evidence for the paclitaxel-clopidogrel interaction associated with peripheral neuropathy in our study. In this study, we utilized a translational informatics approach to discover potentially novel CYP-related substrate-inhibitor and ADE associations using FAERS. Future clinical, population-based and experimental studies are needed to confirm our findings.

Pharmacokinetics of the CYP3A4 and CYP2B6 Inducer Carbamazepine and Its Drug-Drug Interaction Potential: A Physiologically Based Pharmacokinetic Modeling Approach.

The anticonvulsant carbamazepine is frequently used in the long-term therapy of epilepsy and is a known substrate and inducer of cytochrome P450 (CYP) 3A4 and CYP2B6. Carbamazepine induces the metabolism of various drugs (including its own); on the other hand, its metabolism can be affected by various CYP inhibitors and inducers. The aim of this work was to develop a physiologically based pharmacokinetic (PBPK) parent−metabolite model of carbamazepine and its metabolite carbamazepine-10,11-epoxide, including carbamazepine autoinduction, to be applied for drug–drug interaction (DDI) prediction. The model was developed in PK-Sim, using a total of 92 plasma concentration−time profiles (dosing range 50–800 mg), as well as fractions excreted unchanged in urine measurements. The carbamazepine model applies metabolism by CYP3A4 and CYP2C8 to produce carbamazepine-10,11-epoxide, metabolism by CYP2B6 and UDP-glucuronosyltransferase (UGT) 2B7 and glomerular filtration. The carbamazepine-10,11-epoxide model applies metabolism by epoxide hydroxylase 1 (EPHX1) and glomerular filtration. Good DDI performance was demonstrated by the prediction of carbamazepine DDIs with alprazolam, bupropion, erythromycin, efavirenz and simvastatin, where 14/15 DDI AUClast ratios and 11/15 DDI Cmax ratios were within the prediction success limits proposed by Guest et al. The thoroughly evaluated model will be freely available in the Open Systems Pharmacology model repository.

Abstract PS19-23: Effect of Wnt5a on drug resistance in estrogen receptor-positive breast cancer

Abstract Purpose: We previously showed that Wnt5a-positive breast cancer belongs to a subgroup of estrogen receptor (ER)-positive breast cancers and its prognosis is worse than that of Wnt5a-negative breast cancer. In this study, we aimed to investigate the molecular mechanisms underlying the poor prognosis of patients with Wnt5a-positive breast cancer.Methods: A total of 151 patients with ER-positive invasive breast cancer were recruited for this study between January 2011 and February 2014. The association between Wnt5a expression and recurrence rate was examined. To identify the pathways associated with Wnt5a-positive breast cancer, we established a Wnt5a-expressing cell line (MCF-7/Wnt5a cells) and conducted DNA microarray analysis of MCF-7/Wnt5a cells. We also performed pathway analysis associated with Wnt5a expression, and evaluated the effects of Wnt5a in vitro using MCF-7/Wnt5a cells.Results: Data showed poorer relapse-free survival of patients with Wnt5a-positive breast cancer (P = 0.047). The median length of follow-up was 6.08 years (range, 0.027 to 8.47 years) for all patients. According to DNA microarray data, only the cytochrome P450 (CYP) pathway was significantly upregulated and related with Wnt5a (P = 0.0440). Moreover, MCF-7/Wnt5a cells were less sensitive to tamoxifen and paclitaxel, which are metabolic substrates of CYP (P &amp;lt; 0.05). Although the PI3K-AKT-mTOR signaling pathway is involved in the poor prognosis of ER-positive breast cancer, it was not associated with Wnt5a.Conclusions: In ER-positive breast cancer, Wnt5a expression upregulated the CYP metabolic pathway and decreased the sensitivity to tamoxifen and paclitaxel, the standard treatment options for ER-positive breast cancer. Citation Format: Ai Amioka, Takayuki Kadoya, Satoshi Sueoka, Yoshie Kobayashi, Shinsuke Sasada, Akiko Emi, Norio Masumoto, Masaoki Ito, Morihito Okada. Effect of Wnt5a on drug resistance in estrogen receptor-positive breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS19-23.

Impact of body size on first-line Helicobacter pylori eradication success using vonoprazan and amoxicillin dual therapy.

As a first-line therapy for Helicobacter pylori, dual therapy with vonoprazan and amoxicillin (VA-dual) provides an eradication rate similar to that of vonoprazan-based triple therapy. As the factors associated with the eradication rate of H. pylori with VA-dual are unknown,we investigated them in this study. Overall, 163 patients diagnosed with H. pylori infection received VA-dual (vonoprazan 20mg twice daily and amoxicillin 750mg twice daily for 7 d). The association between successful H. pylori eradication and the following patient clinical factors was analyzed: sex, age, height, weight, body surface area (BSA), body mass index (BMI), history of early gastric carcinoma and peptic ulcer, comorbidity of cirrhosis, alcohol consumption habit, smoking habit, common use of proton pump inhibitors, and concomitant use of drugs that are substratesof cytochrome P450 (CYP) 3A4. The association between post-eradication adverse events and clinical factors was analyzed retrospectively. Successful H. pylori eradication was associated with a lower BSA (eradication rate: 90.8% in patients with BSA <1.723 vs. 79.6% in those with BSA ≥1.723; p=0.045). The incidence of adverse events was higher in women than in men (adverse events: 40.0% in women vs. 19.4% in men; p=0.004). Successful H. pylori eradication with VA-dual was associated with the small body size of patients. This therapy may have to be adjusted per body size.

Impact of Acute Inflammation on Cytochromes P450 Activity Assessed by the Geneva Cocktail

Cytochromes P450 (CYP) are subject to important interindividual variability in their activity due to genetic and environmental factors and some diseases. Limited human data support the idea that inflammation downregulates CYP activities. Our study aimed to evaluate the impact of orthopedic surgery (acute inflammation model) on the activity of six human CYP. This prospective observational study was conducted in 30 patients who underwent elective hip surgery at the Geneva University Hospitals in Switzerland. The Geneva phenotyping cocktail containing caffeine, bupropion, flurbiprofen, omeprazole, dextromethorphan, and midazolam as probe drugs respectively assessing CYP1A2, 2B6, 2C9, 2C19, 2D6, and 3A activities was administered orally before surgery, day 1 (D1) and 3 (D3) postsurgery and at discharge. Capillary blood samples were collected 2 hours after cocktail intake to assess metabolic ratios (MRs). Serum inflammatory markers (CRP, IL‐6, IL‐1β, TNF‐α, and IFN‐γ) were also measured in blood. CYP1A2 MRs decreased by 53% (P < 0.0001) between baseline and the nadir at D1. CYP2C19 and CYP3A activities (MRs) decreased by 57% (P = 0.0002) and 61% (P < 0.0001), respectively, with the nadir at D3. CYP2B6 and CYP2C9 MRs increased by 120% (P < 0.0001) and 79% (P = 0.018), respectively, and peaked at D1. Surgery did not have a significant impact on CYP2D6 MR. Hip surgery was a good acute inflammation model as CRP, IL‐6, and TNF‐α peak levels were reached between D1 and day 2 (D2). Acute inflammation modulated CYP activity in an isoform‐specific manner, with different magnitudes and kinetics. Acute inflammation may thus have a clinically relevant impact on the pharmacokinetics of these CYP substrates.

Dose adjustment of venetoclax when co-administered with posaconazole: clinical drug-drug interaction predictions using a PBPK approach.

Venetoclax, a targeted anticancer agent approved for the treatment of chronic lymphocytic leukemia and acute myeloid leukemia, is a substrate of cytochrome P450 (CYP) 3A enzyme (CYP3A4). Posaconazole, commonly used to prevent invasive fungal infections in neutropenic patients with hematological malignancies, potently inhibits CYP3A4. The purpose of this evaluation was to predict venetoclax exposures following co-administration of posaconazole at doses not previously studied clinically. Two physiologically based pharmacokinetic (PBPK) models were developed for posaconazole based on published parameters, one for an oral suspension and another for delayed released tablets. Parameter optimization, guided by sensitivity analyses, was conducted such that the models could replicate clinical exposures of posaconazole and drug-drug interactions with sensitive CYP3A substrates including venetoclax. The clinically verified posaconazole PBPK models were then utilized to predict DDI with a previously published venetoclax PBPK model at clinically relevant dosing scenarios. The posaconazole PBPK models predicted posaconazole exposure and DDI related fold changes with acceptable prediction errors for both posaconazole formulations. The model predicted exposures of venetoclax, when co-administered with a 300mg QD dose of delayed release tablets of posaconazole, were in concordance with observed data. Increasing the posaconazole dose to 500mg QD increased venetoclax exposures by about 12% relative to 300mg QD, which were still within the venetoclax safe exposure range. The posaconazole PBPK models were developed and clinically verified. Predictions using the robust PBPK model confirmed the venetoclax label recommendation of 70mg in the presence of posaconazole at doses up to 500mg QD.

APPROACHES TO INCREASING THE EFFICIENCY OF THE ELECTROCATALYSIS OF CYTOCHROME P450 3A4

An electrochemical system based on cytochrome P450 and riboflavin has been developed, which makes it possible to increase the yield of the product in a model system using cytochrome P450 substrate - erythromycin. Achieved an increase in the catalytic activity of cytochrome P450 by 28-36%.

Cytochrome P450 Expression and Chemical Metabolic Activity before Full Liver Development in Zebrafish.

Zebrafish are used widely in biomedical, toxicological, and developmental research, but information on their xenobiotic metabolism is limited. Here, we characterized the expression of 14 xenobiotic cytochrome P450 (CYP) subtypes in whole embryos and larvae of zebrafish (4 to 144 h post-fertilization (hpf)) and the metabolic activities of several representative human CYP substrates. The 14 CYPs showed various changes in expression patterns during development. Many CYP transcripts abruptly increased at about 96 hpf, when the hepatic outgrowth progresses; however, the expression of some cyp1s (1b1, 1c1, 1c2, 1d1) and cyp2r1 peaked at 48 or 72 hpf, before full liver development. Whole-mount in situ hybridization revealed cyp2y3, 2r1, and 3a65 transcripts in larvae at 55 hpf after exposure to rifampicin, phenobarbital, or 2,3,7,8-tetrachlorodibenzo-p-dioxin from 30 hpf onward. Marked conversions of diclofenac to 4′-hydroxydiclofenac and 5-hydroxydiclofenac, and of caffeine to 1,7-dimethylxanthine, were detected as early as 24 or 50 hpf. The rate of metabolism to 4’-hydroxydiclofenac was more marked at 48 and 72 hpf than at 120 hpf, after the liver had become almost fully developed. These findings reveal the expression of various CYPs involved in chemical metabolism in developing zebrafish, even before full liver development.

Addition of Phenylmethylsulfonyl Fluoride Increases the Working Lifetime of the Trout Liver S9 Substrate Depletion Assay, Resulting in Improved Detection of Low Intrinsic Clearance Rates.

The activity of a trout liver S9 substrate depletion assay has been shown to decline over time, presumably due to proteolytic degradation of biotransformation enzymes. To address this problem, assay performance was evaluated following the addition of phenylmethylsulfonyl fluoride (PMSF) or a general-purpose protease inhibitor cocktail to liver homogenization buffers and/or S9 reaction mixtures. Addition of PMSF to liver homogenization buffers and/or S9 reaction mixtures had little or no effect on clearance of phenanthrene, a model cytochrome P450 substrate, in short-term (25 or 30 min) depletion experiments but resulted in significant improvements in retention of this initial activity over time. The protease inhibitor cocktail strongly inhibited initial activity when added to homogenization buffers or reaction mixtures. Taking into consideration potential effects on liver carboxylesterases, the treatment approach determined to be optimal was addition of 10 µM PMSF to the S9 reaction mixture. Addition of 10 µM PMSF to the mixture resulted in significantly higher rates of phenanthrene clearance in 2-h incubations relative to those obtained in the absence of PMSF and a 6-fold increase in the working lifetime of the preparation. The results of a statistical power analysis suggest that by increasing the working lifetime of the assay, addition of PMSF to the reaction mixture could result in substantially improved detection of low in vitro clearance rates when compared to current practice. These findings demonstrate the value of adding PMSF to the trout S9 preparation and may have broad implications for use of this assay to support chemical bioaccumulation assessments for fish. Environ Toxicol Chem 2021;40:148-161. © 2020 SETAC. This article has been contributed to by US Government employees and their work is in the public domain in the USA.

Clinical evaluation of drug-drug interactions between the cytochrome P450 substrates selexipag and clopidogrel in Japanese volunteers.

The strong cytochrome P450 (CYP) 2C8 inhibitor gemfibrozil has been demonstrated to increase the area under the plasma concentration-time curve from 0 to infinity (AUC0-∞ ) of ACT-333679, an active metabolite of selexipag, by 11-fold. Similarly to gemfibrozil, the CYP2C8 inhibitor clopidogrel increased ACT-333679 concentration by 1.9-fold after a single loading dose (300 mg once daily) and 2.7-fold after repeated treatment with the maintenance dose (75 mg once daily) in Europeans. However, the effects of clopidogrel on the pharmacokinetics of selexipag and ACT-333679 have not been fully elucidated in the Japanese population. We investigated the effect of clopidogrel on the pharmacokinetics of selexipag and ACT-333679 in 14 healthy Japanese volunteers. The concomitant administration of clopidogrel with selexipag did not influence the maximum concentration and AUC0-∞ of selexipag, whereas it significantly increased AUC0-∞ of ACT-333679 by approximately 1.90-fold (90% confidence interval 1.69-2.14) without changing the maximum concentration. When selexipag was administered 1 day after clopidogrel was discontinued, the increase in AUC0-∞ of ACT-333679 was 1.37-fold (90% confidence interval 0.93-2.02), suggesting that, although the inhibitory effect of clopidogrel on CYP2C8 was reduced, it persisted for at least 1 day after withdrawal. Our results demonstrated the impact of clopidogrel on the pharmacokinetics of selexipag and its active metabolite and suggested that selexipag should be carefully prescribed with clopidogrel with dose adjustment or reducing the dosing frequency in Japanese clinical settings.

Modifications pharmacocinétiques liées à une infection aiguë. Exemples issus de la pandémie de SARS-CoV-2

Afin de personnaliser la prise en charge médicamenteuse, il est important d’identifier les facteurs de variabilité pharmacocinétique, en particulier des médicaments à marge thérapeutique étroite pour lesquels un suivi thérapeutique pharmacologique est recommandé. L’inflammation, réaction de protection contre les infections et blessures aiguës, contribue aux variabilités intra- et interindividuelles de l’exposition aux médicaments en modulant l’activité d’enzymes et de transporteurs impliqués dans le métabolisme des médicaments, et en modifiant leur liaison aux protéines plasmatiques. Pour mieux comprendre la variabilité d’exposition aux médicaments, il est important de connaître l’impact de l’inflammation sur le métabolisme des médicaments et les conséquences cliniques. Ce travail présente d’abord les mécanismes moléculaires par lesquels l’inflammation peut inhiber les enzymes du métabolisme des médicaments, puis rapporte les conséquences de la survenue d’événements inflammatoires infectieux sur l’exposition aux médicaments à marge thérapeutique étroite et substrats des cytochromes P450, ainsi que les conséquences cliniques qui en découlent. Enfin, dans le contexte de la pandémie de l’infection au SARS-CoV-2 (COVID-19), pathologie associée à une inflammation aiguë importante, nous avons rapporté des exemples de modifications pharmacocinétiques de médicaments à marge thérapeutique étroite observées chez des patients COVID-19.

In Vitro to In Vivo Extrapolation of Metabolic Clearance for UGT Substrates Using Short-Term Suspension and Long-Term Co-cultured Human Hepatocytes.

The use of micro-patterned co-cultured hepatocytes for human hepatic clearance predictions has previously been demonstrated using drugs metabolized by cytochrome P450 enzymes. The present study evaluates the in vitro to in vivo extrapolation (IVIVE) performance for UDP-glucuronosyltransferase (UGT) substrates. In vitro intrinsic clearances for 13 drugs mainly cleared by UGTs were determined using HepatoPac and suspended hepatocytes. The in vivo intrinsic clearance was predicted from in vitro intrinsic clearance and compared with weighted mean in vivo intrinsic clearance estimated from several clinical studies. A conventional scaling methodology accounting for protein binding in plasma and incubation medium was used for the IVIVE assuming that only free drug is accessible at the site of metabolism. The in vivo hepatic intrinsic clearance was predicted within threefold error for six and nine out of thirteen drugs using suspended hepatocytes and HepatoPac, respectively. A reduced under-estimation of hepatic intrinsic clearance was observed in the average fold error (AFE) in HepatoPac (AFE, 0.69) compared with the suspended hepatocytes (AFE, 0.37). The current study shows reasonable performance of hepatic clearance prediction of drugs mainly metabolized by UGT enzymes using HepatoPac with a similar under-prediction bias as obtained in the reported IVIVEs for cytochrome P450 substrates. This study provides a validation of the approach for drugs cleared via UGT conjugation mechanisms and discusses potential causes for outlier behavior considering pharmacokinetic or physicochemical properties.