Cytochrome P450 Isoforms Research Articles

Synthesis of mono Cytochrome P450 in a modified CHO-CPR cell-free protein production platform

Cytochromes P450 (CYPs) are a group of monooxygenases that can be found in almost all kinds of organisms. For CYPs to receive electrons from co-substrate NADPH, the activity of NADPH-Cytochrome-P450-oxidoreductase (CPR) is required as well. In humans, CYPs are an integral part of liver-based phase-1 biotransformation, which is essential for the metabolization of multiple xenobiotics and drugs. Consequently, CYPs are important players during drug development and therefore these enzymes are implemented in diverse screening applications. For these applications it is usually advantageous to use mono CYP microsomes containing only the CYP of interest. The generation of mono-CYP containing mammalian cells and vesicles is difficult since endogenous CYPs are present in many cell types that contain the necessary co-factors. By obtaining translationally active lysates from a modified CHO-CPR cell line, it is now possible to generate mono CYPs in a cell-free protein synthesis process in a straightforward manner. As a proof of principle, the synthesis of active human CYPs from three different CYP450 gene families (CYP1A2, CYP2B6 and CYP3A4), which are of outstanding interest in industry and academia was demonstrated. Luciferase based activity assays confirm the activity of the produced CYPs and enable the individual adaptation of the synthesis process for efficient cell-free enzyme production. Furthermore, they allow for substrate and inhibitor screenings not only for wild-type CYPs but also for mutants and further CYP isoforms and variants. As an example, the turnover of selected CYP substrates by cell-free synthesized CYPs was demonstrated via an indirect luciferase assay-based screening setup.

Pharmacokinetics of asundexian with combined CYP3A and P-gp inhibitors and an inducer: Target in vitro and in vivo studies.

Asundexian is an oral, direct and reversible inhibitor of activated factor XI (FXIa) in development for the treatment of thromboembolic events. This article summarizes results from preclinical and clinical studies, including identification of enzymes involved in asundexian pharmacokinetics, and evaluation of potential target drug-drug interactions. In vitro studies investigated the substrate characteristics of asundexian towards several cytochrome P450 (CYP) isoforms, hydrolytic enzymes and drug transporters. Inhibition of the amide hydrolysis of asundexian was investigated in vitro for several relevant drugs. Phase 1 studies in healthy male participants investigated the pharmacokinetics (PK) of asundexian upon co-administration of combined inhibitors or an inducer of P-gp and CYP3A4 (itraconazole, verapamil or carbamazepine, respectively, or the moderate CYP3A4 inhibitor fluconazole). The pharmacodynamic (PD) markers are activated partial thromboplastin time and FXIa inhibition. Asundexian was predominantly metabolized via carboxylesterase 1 and, to a lesser extent, via CYP3A4 and is a substrate of P-gp. The asundexian area under the plasma concentration-time curve (AUC) increased by 103% and 75.6% upon combined inhibition of P-gp and strong or moderate inhibition of CYP3A4, respectively, but was unaffected by moderate CYP3A4 inhibition. Combined P-gp and CYP3A4 induction by carbamazepine decreased asundexian AUC by 44.4%. PD is concentration-dependent, thus no differences in maximum responses and recovery commensurate with PK effect(s) were observed. Adverse events were mild and asundexian was well tolerated. The presented studies confirmed that CYP3A4 and P-gp contribute to asundexian metabolism and excretion. Observed effects were in line with data from a previous mass balance study.

Relevance of pharmacogenetic analyses and therapeutic drug monitoring of antidepressants for an individualized treatment of peripartum psychopathology.

Psychiatric disorders burden the peripartum period, often requiring psychopharmacological treatment, including antidepressants. Efficacy and tolerability of antidepressants are influenced by the physiological changes of the peripartum and individual metabolic profiles, which in turn can be modified by pregnancy. The objective of this study is to assess the relationship between antidepressants' pharmacokinetic profiles during pregnancy and individual metabolic profiles, along with the efficacy of the treatment. In total 87 outpatients with diagnoses of bipolar disorder, major depression, anxiety, obsessive-compulsive disorder and post-traumatic stress disorder who required antidepressant treatment during pregnancy were recruited. Genotyping analysis of hepatic cytochrome P450 (CYPs) individual isoforms was performed. Antidepressants' blood concentrations and psychometric assessments were collected at five time points. Antidepressants' cord blood concentrations were assessed at birth. Sertraline showed greater stability in plasma concentrations and a lower placental penetrance index. Most of the antidepressants' concentrations below the therapeutic range were found in women with an extensive/ultrarapid metabolic profile. Antidepressants mainly metabolized by CYP2C19 were less frequently below the therapeutic range compared with antidepressants metabolized by CYP2D6. Pregnancy modulates cytochrome activity and drugs' pharmacokinetics. Genotyping analysis of CYPs isoforms and therapeutic drug monitoring might be used to guide clinicians in a well-tolerated treatment of psychiatric symptoms in pregnant women.

Deciphering the Role of Fatty Acid-Metabolizing CYP4F11 in Lung Cancer and Its Potential As a Drug Target.

Lung cancer is the leading cause of cancer deaths worldwide. We found that the cytochrome P450 isoform CYP4F11 is significantly overexpressed in patients with lung squamous cell carcinoma. CYP4F11 is a fatty acid ω-hydroxylase and catalyzes the production of the lipid mediator 20-hydroxyeicosatetraenoic acid (20-HETE) from arachidonic acid. 20-HETE promotes cell proliferation and migration in cancer. Inhibition of 20-HETE-generating cytochrome P450 enzymes has been implicated as novel cancer therapy for more than a decade. However, the exact role of CYP4F11 and its potential as drug target for lung cancer therapy has not been established yet. Thus, we performed a transient knockdown of CYP4F11 in the lung cancer cell line NCI-H460. Knockdown of CYP4F11 significantly inhibits lung cancer cell proliferation and migration while the 20-HETE production is significantly reduced. For biochemical characterization of CYP4F11-inhibitor interactions, we generated recombinant human CYP4F11. Spectroscopic ligand binding assays were conducted to evaluate CYP4F11 binding to the unselective CYP4A/F inhibitor HET0016. HET0016 shows high affinity to recombinant CYP4F11 and inhibits CYP4F11-mediated 20-HETE production in vitro with a nanomolar IC 50 Cross evaluation of HET0016 in NCI-H460 cells shows that lung cancer cell proliferation is significantly reduced together with 20-HETE production. However, HET0016 also displays antiproliferative effects that are not 20-HETE mediated. Future studies aim to establish the role of CYP4F11 in lung cancer and the underlying mechanism and investigate the potential of CYP4F11 as a therapeutic target for lung cancer. SIGNIFICANCE STATEMENT: Lung cancer is a deadly cancer with limited treatment options. Cytochrome P450 4F11 (CYP4F11) is significantly upregulated in lung squamous cell carcinoma. Knockdown of CYP4F11 in a lung cancer cell line significantly attenuates cell proliferation and migration with reduced production of the lipid mediator 20-hydroxyeicosatetraenoic acid (20-HETE). Studies with the unselective inhibitor HET0016 show a high inhibitory potency of CYP4F11-mediated 20-HETE production using recombinant enzyme. Overall, our studies demonstrate the potential of targeting CYP4F11 for new transformative lung cancer treatment.

Predicting routes of phase I and II metabolism based on quantum mechanics and machine learning

Unexpected metabolism could lead to the failure of many late-stage drug candidates or even the withdrawal of approved drugs. Thus, it is critical to predict and study the dominant routes of metabolism in the early stages of research. We describe the development and validation of a ‘WhichEnzyme’ model that accurately predicts the enzyme families most likely to be responsible for a drug-like molecule’s metabolism. Furthermore, we combine this model with our previously published regioselectivity models for Cytochromes P450, Aldehyde Oxidases, Flavin-containing Monooxygenases, UDP-glucuronosyltransferases and Sulfotransferases – the most important Phase I and Phase II drug metabolising enzymes – and a ‘WhichP450’ model that predicts the Cytochrome P450 isoform(s) responsible for a compound’s metabolism. The regioselectivity models are based on a mechanistic understanding of these enzymes’ actions and use quantum mechanical simulations with machine learning methods to accurately predict sites of metabolism and the resulting metabolites. We train heuristics based on the outputs of the ‘WhichEnzyme’, ‘WhichP450’, and regioselectivity models to determine the most likely routes of metabolism and metabolites to be observed experimentally. Finally, we demonstrate that this combination delivers high sensitivity in identifying experimentally reported metabolites and higher precision than other methods for predicting in vivo metabolite profiles.

The discovery of aryl-2-nitroethyl triamino pyrimidines as anti-Trypanosoma brucei agents

Pteridine reductase 1 (PTR1) is a catalytic protein belonging to the folate metabolic pathway in Trypanosmatidic parasites. PTR1 is a known target for the medicinal chemistry development of antiparasitic agents against Trypanosomiasis and Leishmaniasis. In previous studies, new nitro derivatives were elaborated as PTR1 inhibitors. The compounds showing a diamino-pyrimidine core structure were previously developed but they showed limited efficacy. Therefore, a new class of phenyl-, heteroaryl- and benzyloxy-nitro derivatives based on the 2-nitroethyl-2,4,6-triaminopyrimidine scaffold were designed and tested. The compounds were assayed for their ability to inhibit T. brucei and L. major PTR1 enzymes and for their antiparasitic activity towards T. brucei and L. infantum parasites. To understand the structure-activity relationships of the compounds against TbPTR1, the X-ray crystallographic structure of the 2,4,6-triaminopyrimidine (TAP) was obtained and molecular modelling studies were performed. As a next step, only the most effective compounds against T. brucei were then tested against the amastigote cellular stage of T. cruzi, searching for a broad-spectrum antiprotozoal agent. An early ADME-Tox profile evaluation was performed. The early toxicity profile of this class of compounds was investigated by measuring their inhibition of hERG and five cytochrome P450 isoforms (CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4), cytotoxicity towards A549 cells and mitochondrial toxicity. Pharmacokinetic studies (SNAP-PK) were performed on selected compounds using hydroxypropyl-β-cyclodextrins (50 % w/v) to preliminarily study their plasma concentration when administered per os at a dose of 20 mg/kg. Compound 1p, showed the best pharmacodynamic and pharmacokinetic properties, can be considered a good candidate for further bioavailability and efficacy studies.

The roles of CYP2C19 and CYP3A4 in the invitro metabolism of β-eudesmol in human liver: Reaction phenotyping and enzyme kinetics.

β-eudesmol is a major bioactive component of Atractylodes lancea (AL). AL has been developed as the capsule formulation of standardized AL extract for treating cholangiocarcinoma (CCA). However, the complex constituents of herbal products increase the risk of adverse drug interactions. β-eudesmol has demonstrated inhibitory effects on rCYP2C19 and rCYP3A4 in the previous research. This study aimed to identify the cytochrome P450 (CYP) isoforms responsible for the metabolism of β-eudesmol and determine the enzyme kinetic parameters and the metabolic stability of β-eudesmol metabolism in the microsomal system. Reaction phenotyping using human recombinant CYPs (rCYPs) and selective chemical inhibitors of CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 was performed, and enzyme kinetics and metabolic stability were investigated using human liver microsome (HLM). The results suggest that CYP2C19 and CYP3A4 play significant roles in β-eudesmol metabolism. The disappearance half-life (t1/2 ) and intrinsic clearance (CLint ) of β-eudesmol were 17.09 min and 0.20 mL/min·mg protein, respectively. Enzyme kinetic analysis revealed the Michaelis-Menten constant (Km ) and maximum velocity (Vmax ) of 16.76 μM and 3.35 nmol/min·mg protein, respectively. As a component of AL, β-eudesmol, as a substrate and inhibitor of CYP2C19 and CYP3A4, has a high potential for drug-drug interactions when AL is co-administered with other herbs or conventional medicines.

Prediction of Cytochrome P450 Inhibition Using a Deep Learning Approach and Substructure Pattern Recognition.

Cytochrome P450 (CYP) is a family of enzymes that are responsible for about 75% of all metabolic reactions. Among them, CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 participate in the metabolism of most drugs and mediate many adverse drug reactions. Therefore, it is necessary to estimate the chemical inhibition of Cytochrome P450 enzymes in drug discovery and the food industry. In the past few decades, many computational models have been reported, and some provided good performance. However, there are still several issues that should be resolved for these models, such as single isoform, models with unbalanced performance, lack of structural characteristics analysis, and poor availability. In the present study, the deep learning models based on python using the Keras framework and TensorFlow were developed for the chemical inhibition of each CYP isoform. These models were established based on a large data set containing 85715 compounds extracted from the PubChem bioassay database. On external validation, the models provided good AUC values with 0.97, 0.94, 0.94, 0.96, and 0.94 for CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4, respectively. The models can be freely accessed on the Web server named CYPi-DNNpredictor (cypi.sapredictor.cn), and the codes for the model were made open source in the Supporting Information. In addition, we also analyzed the structural characteristics of chemicals with CYP450 inhibition and detected the structural alerts (SAs), which should be responsible for the inhibition. The SAs were also made available online, named CYPi-SAdetector (cypisa.sapredictor.cn). The models can be used as a powerful tool for the prediction of CYP450 inhibitors, and the SAs should provide useful information for the mechanisms of Cytochrome P450 inhibition.

Panax ginseng C.A. Mey. as a potential raw material in the treatment of negative effects of alcohol consumption

An alcohol hangover is a complex set of adverse symptoms that occur after excessive consumption of alcoholic beverages. Hangovers are not solely caused by ethanol, but mainly by its toxic metabolites, such as acetaldehyde and acetic acid. Most of the negative symptoms occur when the concentration of alcohol in the blood decreases after drinking. Given that a large percentage of people consume alcohol and experience negative effects from its consumption, potential supplementation to help eliminate these adverse effects has been proposed. One promising ingredient is ginseng, which has been used in Eastern culture and traditional Chinese medicine for over 2,000 years. The aim of this study was to present the potential of Panax ginseng C.A. Mey. as an aid for the adverse effects of alcohol consumption and its protective effect on liver function. The literature analysis was based on data indexed in medical databases PubMed, Scopus, and Medline since 2010, using keywords such as "Panax ginseng," "hangover," "alcohol consumption," and "metabolism," connected by the logical conjuction “and.” The analysis confirmed the effectiveness of P. ginseng in the treatment and prevention of hangovers. Preparations containing P. ginseng can support ethanol metabolism by inducing enzymes such as alcohol dehydrogenase, aldehyde dehydrogenase, and the cytochrome P450 isoform 2E1 system. An additional advantage of using P. ginseng may be its hepatoprotective effect.

Updates on the Interactions of Herbs Constituents with Cytochrome P450 Drug Metabolizing Enzymes

Abstract: Cytochrome P450 enzymes are the main drug-metabolizing enzyme, and their activity is influenced by numerous factors, including several plants’ constituents. Herbal products mediated induction or inhibition of cytochrome P450 leads to a change in drug metabolism and consequently, drug bioavailability, safety, therapeutic efficacy, and herb-drug interaction. Since herbal preparations are commonly used either alone or concomitantly with conventional drugs, understanding the interactions of plant constituents with cytochrome P450 is crucial. Therefore, this review highlighted plants with significant inhibition, induction, or modulatory /contradictory effects on human and animal cytochrome P450 enzymes. An extensive literature search was conducted on PubMed, ScienceDirect, and Google Scholar databases to identify relevant literature. About sixty medicinal plants with significant interaction with cytochrome P450 isoforms were summarized with their common uses and affected cytochrome P450 enzyme isoforms. Besides, representative examples of plant constituents and other factors contributing to plant-induced cytochrome P450 modulation and possible herb-drug interactions were also discussed in this review.

A Comparative GC/MS Analysis of Citrus Essential Oils: Unveiling the Potential Benefits of Herb-Drug Interactions in Preventing Paracetamol-Induced Hepatotoxicity.

Our study aimed to test the potential of Citrus oils in protecting against paracetamol (PAR)-induced hepatotoxicity. The essential oils of Pineapple sweet orange (OO), Murcott mandarin (MO), Red grapefruit (GO), and Oval kumquat (KO) were investigated using gas chromatography coupled with mass spectrometry (GC/MS). Twenty-seven compounds were identified, with monoterpene hydrocarbons being abundant class. d-Limonene had the highest percentage (92.98 %, 92.82 %, 89.75 %, and 94.46 % in OO, MO, GO, and KO, respectively). Hierarchical cluster analysis (HCA) and principal components analysis (PCA) revealed that octanal, linalool, germacrene D, and d-limonene were the principal discriminatory metabolites that segregated the samples into three distinct clusters. In vitro antioxidant capacities were ranged from 1.2-12.27, 1.79-5.91, and 235.05-585.28 μM Trolox eq/mg oil for 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic (ABTS), ferric-reducing antioxidant power (FRAP), and oxygen radical absorbance capacity (ORAC), respectively. In vivo, citrus oils exhibited a significant reduction in alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), and nitric oxide (NO). Additionally, there was an increase in glutathione reductase (GSH), and the liver architecture was nearly normal. Molecular docking revealed that d-limonene exhibited a good inhibitory interaction with cytochrome P450 (CYP450) isoforms 1A2, 3A4, and 2E1, with binding energies of -6.17, -4.51, and -5.61 kcal/mol, respectively.

A comprehensive evaluation in clinic and physiologically-based pharmacokinetic modeling and simulation to confirm lack of cytochrome P450-mediated drug-drug interaction potential for pomotrelvir.

Pomotrelvir is a new chemical entity and potent direct-acting antiviral inhibitor of the main protease of coronaviruses. Here the cytochrome P450 (CYP)-mediated drug-drug interaction (DDI) potential of pomotrelvir was evaluated for major CYP isoforms, starting with in vitro assays followed by the basic static model assessment. The identified CYP3A4-mediated potential DDIs were evaluated clinically at a supratherapeutic dose of 1050 mg twice daily (b.i.d.) of pomotrelvir, including pomotrelvir coadministration with ritonavir (strong inhibitor of CYP3A4) or midazolam (sensitive substrate of CYP3A4). Furthermore, a physiologically-based pharmacokinetic (PBPK) model was developed within the Simcyp Population-based Simulator using in vitro and in vivo information and validated with available human pharmacokinetic (PK) data. The PBPK model was simulated to assess the DDI potential for CYP isoforms that pomotrelvir has shown a weak to moderate DDI in vitro and for CYP3A4 at the therapeutic dose of 700 mg b.i.d. To support the use of pomotrelvir in women of childbearing potential, the impact of pomotrelvir on the exposure of the representative oral hormonal contraceptive drugs ethinyl estradiol and levonorgestrel was assessed using the PBPK model. The overall assessment suggested weak inhibition of pomotrelvir on CYP3A4 and minimal impact of a strong CYP3A4 inducer or inhibitor on pomotrelvir PK. Therefore, pomotrelvir is not anticipated to have clinically meaningful DDIs at the clinical dose. These comprehensive in vitro, in clinic, and in silico efforts indicate that the DDI potential of pomotrelvir is minimal, so excluding patients on concomitant medicines in clinical studies would not be required.

Evaluation of the impact of vindoline, an active components of Catharanthus roseus, on rat hepatic cytochrome P450 enzymes by using a cocktail of probe drugs.

The objection of this study was to investigate the effects of vindoline(VDL) on the cytochrome P450 (CYP 450) isoforms (CYP1A2, 2B, 2C11, 2D1 and 3A) in rats. Firstly, the rats were randomly divided into VDL pretreatment group and blank group, each group had six rats. VDL pretreatment group was administrated VDL (20 mg·kg-1) by oral gavage for fifteen days consecutively, and the equivalent CMC-Na solution without VDL was given to the blank group by gavage. Secondly, a cocktail of caffeine, bupropion, diclofenac, dextromethorphan and midazolam was then administered on the sixteenth day. Finally, blood samples were collected at the specified time point, and the plasma concentration of the probe drug was determined by UHPLC-QTOF-MS/MS. The effects of VDL on the activity of these CYP enzymes in rats were evaluated by pharmacokinetic parameters. VDL pretreatment group compared with the blank group, accelerated the metabolism of diclofenac, and weakened the metabolism of caffeine. These results suggested that VDL could induce the activity of CYP2C11, and inhibits the activity of CYP1A2, but had no significant effects on CYP2B, CYP2D1 and CYP3A. The results in this study can provide beneficial information for the later clinical application of VDL.

Taming Food-Drug Interaction Risk: Potential Inhibitory Effects of Citrus Juices on Cytochrome Liver Enzymes Can Safeguard the Liver from Overdose Paracetamol-Induced Hepatotoxicity.

Paracetamol overdose is the leading cause of drug-induced hepatotoxicity worldwide. Because of N-acetyl cysteine's limited therapeutic efficacy and safety, searching for alternative therapeutic substitutes is necessary. This study investigated four citrus juices: Citrus sinensis L. Osbeck var. Pineapple (pineapple sweet orange), Citrus reticulata Blanco × Citrus sinensis L. Osbeck (Murcott mandarin), Citrus paradisi Macfadyen var. Ruby Red (red grapefruit), and Fortunella margarita Swingle (oval kumquat) to improve the herbal therapy against paracetamol-induced liver toxicity. UHPLC-QTOF-MS/MS profiling of the investigated samples resulted in the identification of about 40 metabolites belonging to different phytochemical classes. Phenolic compounds were the most abundant, with the total content ranked from 609.18 to 1093.26 μg gallic acid equivalent (GAE)/mL juice. The multivariate data analysis revealed that phloretin 3',5'-di-C-glucoside, narirutin, naringin, hesperidin, 2-O-rhamnosyl-swertisin, fortunellin (acacetin-7-O-neohesperidoside), sinensetin, nobiletin, and tangeretin represented the crucial discriminatory metabolites that segregated the analyzed samples. Nevertheless, the antioxidant activity of the samples was 1135.91-2913.92 μM Trolox eq/mL juice, 718.95-3749.47 μM Trolox eq/mL juice, and 2304.74-4390.32 μM Trolox eq/mL juice, as revealed from 2,2'-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid, ferric-reducing antioxidant power, and oxygen radical absorbance capacity, respectively. The in vivo paracetamol-induced hepatotoxicity model in rats was established and assessed by measuring the levels of hepatic enzymes and antioxidant biomarkers. Interestingly, the concomitant administration of citrus juices with a toxic dose of paracetamol effectively recovered the liver injury, as confirmed by normal sections of hepatocytes. This action could be due to the interactions between the major identified metabolites (hesperidin, hesperetin, phloretin 3',5'-di-C-glucoside, fortunellin, poncirin, nobiletin, apigenin-6,8-digalactoside, 6',7'-dihydroxybergamottin, naringenin, and naringin) and cytochrome P450 isoforms (CYP3A4, CYP2E1, and CYP1A2), as revealed from the molecular docking study. The most promising compounds in the three docking processes were hesperidin, fortunellin, poncirin, and naringin. Finally, a desirable food-drug interaction was achieved in our research to overcome paracetamol overdose-induced hepatotoxicity.

A 3D-printed micro-perfused culture device with embedded 3D fibrous scaffold for enhanced biomimicry

Additive manufacturing has rapidly revolutionized the medical sectors since it is a versatile, cost-effective, assembly free technique with the ability to replicate geometrically complicated features. Some of the widely reported applications include the printing of scaffolds, implants, or microfluidic devices. In this study, a 3D-printed micro-perfused culture (MPC) device embedded with a nanofibrous scaffold was designed to create an integrated micro-perfused 3D cell culture environment for living cells. The addition of 3D fibrous scaffold onto the microfluidic chip was to provide a more physiologically relevant microenvironment for cell culture studies. Stereolithography was adopted in this study as this technique obviates excessive preassembly and bonding steps, which would otherwise be needed in conventional microfluidic fabrication. Huh7.5 hepatocellular carcinoma cells were used as model cells for this platform since liver cells experience similar perfused microenvironment. Preliminary cell studies revealed that gene expressions of albumin (ALB) and cytochrome P450 isoform (CYP3A7) were found to be significantly upregulated on the 3D-printed MPC device as compared to the static counterpart. Taken together, the 3D-printed MPC device is shown to be a physiologically relevant platform for the maintenance of liver cells. The device and printing technique developed in this study is highly versatile and tailorable to mimic local in vivo microenvironment needs of various tissues, which could be studied in future.  

Reducing Hepatotoxicity Mechanism of Radix Wikstroemia Indica by Processing with “Sweat Soaking Method” Using UPLC-MS/MS and a Cocktail Probe Substrate

Background: Radix Wikstroemia indica is a traditional Chinese medicine (TCM) used as anti-inflammatory and anti-tumor drug. However, it has serious hepatotoxicity, "Sweat soaking method" processed could effectively decrease its hepatotoxicity. Objective: The objective of this study is to study the effects of Radix Wikstroemia indica on six kinds of cytochrome P450(CYP450) isozymes of rat liver microsomes before and after processing, and to study the mechanism of Radix Wikstroemia indica processed by the "Sweat soaking method" to reduce liver toxicity in rats. Methods: In this study, the effects of Radix Wikstroemia indica and processed Radix Wikstroemia indica on the six main CYP450 isoforms (2E1, 1A2, 2C6, 2D1, 2C11, and 3A1) were investigated in vitro. Using a cocktail probe of CYP450 isoform-specific substrates and their metabolites, we carried out in vitro enzymatic studies in liver microsomal incubation systems via UPLC-MS/MS. Results: The results showed that the established UPLC-MS/MS method was precise and reliable. Compared with the blank group, the activities of six enzymes in the RWI and PRWI groups were higher than those in the blank group. At the same dose, the enzyme activities of CYP2E1, CYP1A2, CYP2C6, CYP2C11, and CYP3A1 increased with the increase in dose, and the enzyme activities of the RWI group were higher than those of the PRWI group. The enzyme activities of CYP2E1 and CYP1A2 in the Radix Wikstroemia indica group were significantly increased compared with the blank group, CYP3A1 in the RWI high-dose group was higher than that in the blank group and PRWI group with statistical differences (p<0.05 or p<0.01). Conclusion: The processed Radix Wikstroemia indica could reduce liver injury, and its detoxication mechanism might be related to the decrease in enzyme activity of CYP1A2, CYP2E1 and CYP3A1.

Application to Butterbur Products of a Suggested Daily Intake-Based Safety Evaluation of Individual Herbal Supplements with Cytochrome P450 Expression as a Major Index.

The present paper first proposes a method for ensuring the safety of commercial herbal supplements, termed the suggested daily intake-based safety evaluation (SDI-based safety evaluation). This new method was inspired as a backward analog of the acceptable daily intake (ADI) derivation from the no observed adverse effect level (NOAEL), the basis of food additive risk analysis; namely, rats are dosed with individual herbal supplement products at the SDI for human use multiplied by 100 (the usual uncertainty factor value) per body weight for 8 d. The primary endpoint is the sign of adverse effects on liver, especially gene expression of cytochrome P450 (CYP) isoforms. The proposed method was then applied to three butterbur (Petasites hybridus) products without pyrrolizidine alkaloids but lacking clear safety information. Results showed that two oily products markedly enhanced the mRNA expression of CYP2B (>10-fold) and moderately enhanced that of CYP3A1 (<4-fold) with liver enlargement. These products also caused the renal accumulation of alpha 2-microglobulin. One powdery product showed no significant effect on liver and kidney. The large difference in effects of products was due to the difference in chemical composition revealed by liquid chromatography-mass spectroscopy. The oily and the powdery products required attention in terms of safety and effectiveness, respectively. Finally, the results from the SDI-based safety evaluation of butterbur and other herbal supplement products were grouped into four categories and cautionary notes were discussed. The SDI-based safety evaluation of their products by herbal supplement operators would contribute to safe and secure use by consumers.

Sex- and lifestyle-related factors are associated with altered hepatic CYP protein levels in people diagnosed with mental disorders.

In this study, we used human postmortem tissue to investigate hepatic protein expression levels of CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 by LC‒MS/MS in a population of people suffering from mental disorders (n=171). We report hepatic protein levels of these six CYP isoforms in 171 individuals in total, and define a focused population dataset of 116 individuals after excluding 55 samples due to low Microsomal Protein Per Gram of Liver (MPPGL) yield. Postmortem decay was most likely the reason for the low MPPGL yield in the 55 samples. In the focused population, we found women to have significantly higher protein levels of CYP3A4 than men in addition to decreased CYP3A4 protein levels among obese individuals. Furthermore, MPPGL was negatively correlated with body mass index (BMI). An increase in CYP1A2 protein levels was observed among smokers and increased CYP2E1 protein levels were observed among individuals with a history of alcohol abuse. Finally, individuals who received phenobarbital (CYP3A4 inducer) had significantly higher CYP3A4 levels. In conclusion, lifestyle-related factors prevalent among people suffering from mental disorders are associated with altered CYP protein levels which may alter drug metabolism and affect the efficacy of commonly prescribed drugs. Furthermore, this investigation demonstrates that postmortem hepatic tissue can be used to study how lifestyle and effectors affect hepatic CYP-levels in a large cohort of patients. Significance Statement Using a large number of postmortem hepatic tissue specimens (n=116) originating from the autopsy of individuals diagnosed with mental disorders, we were able to show that hepatic CYP-levels were affected by alcohol, smoking, BMI, and sex and that MPPGL was affected by BMI. Theses lifestyle-related changes may alter drug metabolism and affect the efficacy of commonly prescribed drugs. It is a novel approach to use a large postmortem cohort to investigate how lifestyle and effectors affect hepatic CYP-levels.

Resistance to Resveratrol Treatment in Experimental PTSD Is Associated with Abnormalities in Hepatic Metabolism of Glucocorticoids.

Glucocorticoids are metabolized by the CYP3A isoform of cytochrome P450 and by 11-β-hydroxysteroid dehydrogenase type 1 (11β-HSD-1). Experimental data suggest that post-traumatic stress disorder (PTSD) is associated with an increase in hepatic 11β-HSD-1 activity and a concomitant decrease in hepatic CYP3A activity. Trans-resveratrol, a natural polyphenol, has been extensively studied for its antipsychiatric properties. Recently, protective effects of trans-resveratrol were found in relation to PTSD. Treatment of PTSD rats with trans-resveratrol allowed the rats to be divided into two phenotypes. The first phenotype is treatment-sensitive rats (TSR), and the second phenotype is treatment-resistant rats (TRRs). In TSR rats, trans-resveratrol ameliorated anxiety-like behavior and reversed plasma corticosterone concentration abnormalities. In contrast, in TRR rats, trans-resveratrol aggravated anxiety-like behavior and decreased plasma corticosterone concentration. In TSR rats, hepatic 11β-HSD-1 activity was suppressed, with a concomitant increase in CYP3A activity. In TRR rats, the activities of both enzymes were suppressed. Thus, the resistance of PTSD rats to trans-resveratrol treatment is associated with abnormalities in hepatic metabolism of glucocorticoids. The free energy of binding of resveratrol, cortisol, and corticosterone to the human CYP3A protein was determined using the molecular mechanics Poisson-Boltzmann surface area approach, indicating that resveratrol could affect CYP3A activity.

Development and validation of a proteomic correlation profiling technique to detect and identify enzymes involved in metabolism of drugs of concern.

It is important to be able to detect previously unknown and unsuspected enzymes involved in drug metabolism, in order to predict the variation of pharmacological or toxicological effect caused by pharmacokinetic variance. We investigated the use of proteomic correlation profiling (PCP) as a technique to identify the enzymes involved in metabolism of drugs of concern. By evaluating the metabolic activities of each enzyme (including isoforms of cytochrome P450, uridine 5' diphospho‑glucuronosyltransferase, and hydrolases, plus aldehyde oxidase and carbonyl reductase) on their typical substrates using a panel of human liver samples, we were able to show the validity of PCP for this purpose. Pearson or Spearman's rank correlation coefficient and P values were calculated for the association between the protein abundance profile of each protein and the metabolic rate profile of each typical substrate. For the 18 enzymatic activities examined, 13 of the enzymes reported to be responsible for the reactions had correlation coefficients higher than 0.7, and were ranked first to third. For the remaining five activities, the responsible enzymes had correlation coefficients lower than 0.7 and lower rankings. The reasons for this were diverse, including confounding resulting from low protein abundance ratios, artificially high correlations of other enzymes due to limited sample numbers, the presence of inactive enzyme forms, and genetic polymorphisms. Overall, PCP was able to identify the majority of responsible drug-metabolizing enzymes across several enzyme classes (oxidoreductase, transferase, hydrolase); use of this methodology could allow more timely and accurate identification of unknown drug-metabolizing enzymes. Significance Statement Proteomic correlation profiling using samples from individual human donors was proven to be a useful methodology for the identification of enzymes responsible for drug-metabolism. This methodology could accelerate the identification of unknown drug-metabolizing enzymes in the future.