Human Liver Microsomal Enzymes Research Articles

Probe substrates assay estimates the effect of polyphyllin H on the activity of cytochrome P450 enzymes in human liver microsomes.

Cytochrome P450 enzymes (CYPs) play a crucial role in phase I metabolic reactions. The activity of CYPs would affect therapeutic efficacy and may even induce toxicity. Given the complex components of traditional Chinese medicine, it is important to understand the effect of active ingredients on CYPs activity to guide their prescription. This study aimed to evaluate the effect of polyphyllin H on the activity of CYPs major isoforms providing a reference for the clinical prescription of polyphyllin H and its source herbs. The effects of polyphyllin H were evaluated in pooled human liver microsomes using probe substrates of CYP1A2, 2A6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4 to determine their activities. The Lineweaver-Burk was used to model the inhibition, and a time-dependent inhibition experiment was performed to understand the characteristics of the inhibition. Polyphyllin H significantly suppressed the activity of CYP1A2, 2D6, and 3A4 with IC50 values of 6.44, 13.88, and 4.52 μM, respectively. The inhibition of CYP1A2 and 2D6 was best fitted with a competitive model, yielding the inhibition constant (Ki) values of 3.18 and 6.77 μM, respectively. The inhibition of CYP3A4 was fitted with the non-competitive model with the Ki value of 2.38 μM. Moreover, the inhibition of CYP3A4 was revealed to be time-dependent with the inhibition parameters inhibition constant (KI) and inactivation rate constant (Kinact) values of 2.26 μM-1 and 0.045 min-1. Polyphyllin H acted as a competitive inhibitor of CYP1A2 and 2D6 and a non-competitive and time-dependent inhibitor of CYP3A4.

Inhibition of soluble epoxide hydrolase by natural isothiocyanates

GoalThe long-term goal of our research is to develop safe and effective soluble epoxide hydrolase (sEH) inhibitors. The objective of this study is to evaluate the potency and selectivity of six natural isothiocyanates (ITCs) as sEH inhibitors. MethodsMolecular docking was used to model likely interactions between the ligands and receptors. The sEH inhibitory activity was tested using a validated fluorescence-based assay and PHOME as a substrate. To evaluate their selectivity as sEH inhibitors, the inhibitory potential of the ITCs was determined on microsomal epoxide hydrolase (mEH) and cytochrome P450 (CYP) enzymes in human liver microsomes. Probe substrates such as styrene oxide (mEH substrate) and established substrates for CYP2A6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 were used in this study. The metabolites of these substrates were analyzed using validated LC-MS/MS and HPLC-UV assays. ResultsMolecular Docking revealed significant differences in binding site preference among the ITCs in silico and pointed to important interactions between the ligands and the catalytic residues of the sEH enzyme. In vitro, the ITCs showed varying degrees of sEH inhibition, but sulforaphane (SFN) and phenyl isothiocyanate (PITC) were the most potent inhibitors with IC50 values of 3.65 and 7.5 μM, respectively. mEH was not significantly inhibited by any of the ITCs. Erucin and iberin were the only ITCs that did not inhibit the activity of any of the tested CYP enzymes. ConclusionOur results demonstrate that natural ITCs have the potential to offer safe, selective, and potent sEH inhibition.

Effect of tubeimoside I on the activity of cytochrome P450 enzymes in human liver microsomes

This study assessed the effect of tubeimoside I on CYP1A2, 2A6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4 to reveal the potential of tubeimoside I to induce drug-drug interaction. The evaluation of cytochromes P450 enzyme (CYP) activity was performed in pooled human liver microsomes with probing substrates of CYP1A2, 2A6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4. Typical inhibitors were employed as positive controls and the effect of 0, 2.5, 5, 10, 25, 50, and 100 μM tubeimoside I was investigated. The activity of CYP2D6, 2E1, and 3A4 was significantly inhibited by tubeimoside I with the IC50 values of 10.34, 11.58, and 9.74 μM, respectively. The inhibition of CYP2D6 and 2E1 was competitive with the Ki value of 5.66 and 5.29 μM, respectively. While the inhibition of CYP3A4 was non-competitive with the Ki value of 4.87 μM. Moreover, the inhibition of CYP3A4 was time-dependent with the KI and K inact values of 0.635 μM−1 and 0.0373 min−1, respectively. Tubeimoside I served as a competitive inhibitor of CYP2D6 and 2E1 exerting weak inhibition and a non-competitive inhibitor of CYP3A4 exerting moderate inhibition.

A simple LC-MS/MS method for the simultaneous quantification of drug metabolic enzymes

ObjectiveThe aim of this study is to develop a LC-MS/MS method for the quantitation of seven cytochrome P450 (CYP450) enzymes. MethodsA high-performance liquid chromatography-tandem mass spectrometry method was developed using multiple reaction monitoring mode with positive electrospray ionization. The method was validated with selectivity, linearity, stability, accuracy and precious. In addition, the abundance of seven CYP450 enzymes in human liver microsomes and CYP3A4 in placenta were determined using the current method. ResultsThe linear range for CYP1A2, CYP2B6 and CYP2C8 was 0.036–3.6 nM and for CYP2C9, CYP2C19, CYP2D6 and CYP3A4 was 0.090–9.0 nM. No interference was found between the blank matrix and each specific peptides. The accuracy and precious results were in accord with the requirement of analytical methods for biological samples in Chinese Pharmacopoeia. In addition, the peptides were stable under current stability conditions. The content of CYP3A4 in placenta and the seven CYP450 enzymes in human liver microsomes were accurately quantified. ConclusionThe developed method is sensitive and specific and can be applied to the quantification of enzymes abundance in different human derived samples like placenta and liver microsomes.

A New Approach Method to Study Thyroid Hormone Disruption: Optimization and Standardization of an Assay to Assess the Inhibition of DIO1 Enzyme in Human Liver Microsomes

A New Approach Method to Study Thyroid Hormone Disruption: Optimization and Standardization of an Assay to Assess the Inhibition of DIO1 Enzyme in Human Liver Microsomes

Effects of Astragaloside IV on the Pharmacokinetics of Metoprolol in Rats and its Mechanism.

Astragaloside IV (AST) and metoprolol are often used together to treat cardiovascular diseases, while the herb-drug interaction (HDI) between them is still unclear. This study investigates the effect of AST on the pharmacokinetics of metoprolol in rats and its mechanism to predict the HDI. First, IC50 value of AST on nine CYP450 enzymes in human liver microsomes (HLMs) was determined by the cocktail method. We explored the effect of AST on the pharmacokinetics of metoprolol (metabolized by CYP2D6) in vivo. Twelve male SD rats were equally divided into two groups, with or without pretreatment of AST (3 mg/kg/day) for 7 days, and they received metoprolol (27 mg/kg) by oral administration. Blood samples were determined using HPLC. Finally, the mechanism of AST was explored. AST exhibited a moderate inhibitory effect on CYP2D6 with IC50 value of 32.28 μM. The pharmacokinetic parameters of metoprolol were significantly altered by AST with the increase of AUC0-∞ (538.81 ± 51.41 to 1088.34 ± 86.46 μg*min/mL, P<0.05) and Cmax (6.21 ± 0.56 to 8.34 ± 0.87 μg/ml, P<0.05). The investigation of the mechanism showed AST to be an irreversible inhibitor of CYP2D6 with KI value of 2.9 μM and Kinact of 0.018 min-1, respectively. AST was found to increase the plasma exposure of metoprolol in rats. AST reduced the metabolism of metoprolol by inhibiting CYP2D6 activity. The HDI might enhance when metoprolol and AST will be applied in combination.

In vitro study on the effect of leonurine hydrochloride on the enzyme activity of cytochrome P450 enzymes in human liver microsomes

Leonurine hydrochloride (LH) is derived from an ingredient of Leonurus japonicus Houtt which is widely used for diseases in women. The influence of LH on the activity of cytochrome P450 (CYPs) enzymes was investigated in this study. The effect of LH on CYPs enzyme activities were studied using the enzyme-selective substrates phenacetin (1A2), coumarin (2A6), diclofenac (2C9), S-mephenytoin (2C19), paclitaxel (2C8), dextromethorphan (2D6), chlorzoxazone (2E1) and testosterone (3A4). The IC50 value was calculated to express the strength of inhibition. The inhibition of CYPs was fitted with competitive or non-competitive inhibition models and corresponding parameters were also obtained. LH exerted inhibitory effects on the activity of CYP1A2, 2D6, and 3A4 with the IC50 values of 18.05, 15.13, and 20.09 μM, respectively. The obtained results showed that LH inhibited the activity of CYP1A2 and CYP2D6 via competitive manners (Ki = 8.667 μM and Ki = 7.805 μM, respectively), while LH attenuated the CYP3A4 activity via a non-competitive manner (Ki = 9.507 μM). Moreover, LH showed time-dependent inhibition on CYP3A4 with the KI/Kinact value of 4.31/0.044 min−1·μM−1. The inhibition of CYP1A2, CYP2D6, and CYP3A4 by LH, demonstrated in vitro, indicated the potential herb–drug interaction. Therefore, pharmacokinetic interactions involving LH and CYP1A2 or CYP2D6 or CYP1A2 substrates are likely to occur.

Influence of Proteome Profiles and Intracellular Drug Exposure on Differences in CYP Activity in Donor-Matched Human Liver Microsomes and Hepatocytes.

Human liver microsomes (HLM) and human hepatocytes (HH) are important in vitro systems for studies of intrinsic drug clearance (CLint) in the liver. However, the CLint values are often in disagreement for these two systems. Here, we investigated these differences in a side-by-side comparison of drug metabolism in HLM and HH prepared from 15 matched donors. Protein expression and intracellular unbound drug concentration (Kpuu) effects on the CLint were investigated for five prototypical probe substrates (bupropion–CYP2B6, diclofenac–CYP2C9, omeprazole–CYP2C19, bufuralol–CYP2D6, and midazolam–CYP3A4). The samples were donor-matched to compensate for inter-individual variability but still showed systematic differences in CLint. Global proteomics analysis outlined differences in HLM from HH and homogenates of human liver (HL), indicating variable enrichment of ER-localized cytochrome P450 (CYP) enzymes in the HLM preparation. This suggests that the HLM may not equally and accurately capture metabolic capacity for all CYPs. Scaling CLint with CYP amounts and Kpuu could only partly explain the discordance in absolute values of CLint for the five substrates. Nevertheless, scaling with CYP amounts improved the agreement in rank order for the majority of the substrates. Other factors, such as contribution of additional enzymes and variability in the proportions of active and inactive CYP enzymes in HLM and HH, may have to be considered to avoid the use of empirical scaling factors for prediction of drug metabolism.

Species Difference of Asarinin Metabolism in vitro and its Effect on the Activity of Cytochrome P450 Enzymes

Aim: To investigate the metabolic behaviors of asarinin in different species and found animal models with metabolic pathways similar to those in humans and to study the effect (inhibition or induction) between asarinin and cytochrome P450 enzymes (CYP450s). Materials and Methods: The interspecies difference in asarinin metabolism was studied using liver microsomes (LMs) from humans and six mammals including rabbits, mini pigs, dogs, monkeys, rats, and mice. The potential drug–drug interactions (DDIs) between asarinin and cytochrome P450 enzymes was studied by incubating with six CYP enzymes in human liver microsomes (HLMs). Results: The metabolic profiles revealed substantially different metabolism of asarinin among the seven species. Moreover, the potential DDIs between asarinin and cytochrome P450 enzymes were studied in vitro by incubating with six CYP enzymes in HLMs, it was found that asarinin inhibited CYP2C9, CYP3A4, and CYP2E1 activity. Conclusion: The interspecies comparison can help find other species that have similar metabolic pathways to humans. There were potential DDIs between asarinin and some drugs metabolized by CYP2C9, CYP2E1, and CYP3A4.

Evaluation of the pharmacokinetic drug-drug interaction potential of iohexol, a renal filtration marker.

Carboplatin dose is calculated based on kidney function, commonly estimated with imperfect creatinine-based formulae. Iohexol is used to measure glomerular filtration rate (GFR) and allows calculation of a more appropriate carboplatin dose. To address potential concerns that iohexol administered during a course of chemotherapy impacts that therapy, we performed in vitro and in vivo pharmacokinetic drug-drug interaction evaluations of iohexol. Carboplatin was administered IV to female mice at 60mg/kg with or without iohexol at 300mg/kg. Plasma ultrafiltrate, kidney and bone marrow platinum was quantitated by atomic absorption spectrophotometry. Paclitaxel microsomal and gemcitabine cytosolic metabolism as well as metabolism of CYP and UGT probes was assessed with and without iohexol at 300µg/mL by LC-MS/MS. In vivo carboplatin exposure was not significantly affected by iohexol co-administration (platinum AUC combination vs alone: plasma ultrafiltrate 1,791 vs 1920µg/mL min; kidney 8367 vs 9757µg/g min; bone marrow 12.7 vs 12.7µg/mg-protein min). Paclitaxel microsomal metabolism was not impacted (combination vs alone: 6-α-OH-paclitaxel 38.3 versus 39.4ng/mL/60min; 3-p-OH-paclitaxel 26.2 versus 27.7ng/mL/60min). Gemcitabine human cytosolic elimination was not impacted (AUC combination vs gemcitabine alone: dFdU 24.1 versus 23.7µg/mL/30min). Iohexol displayed no relevant inhibition of the CYP and UGT enzymes in human liver microsomes. Iohexol is unlikely to affect the clinical pharmacokinetics of carboplatin, paclitaxel, gemcitabine, or other agents used in combination with carboplatin treatment. Measuring GFR with iohexol to better dose carboplatin is unlikely to alter the safety or efficacy of chemotherapy through pharmacokinetic drug-drug interactions.

Inhibition of Major Cytochrome P450 Enzyme Activities in Human Liver Microsomes by 9‐a and WES‐1, two Novel Carbonic Anhydrase Inhibitors.

Both 9‐a (sulphonamide derivative) and WES‐1 (coumarin derivative) were designed and synthesized as potential selective Carbonic Anhydrase Inhibitors, and are currently being tested for anti‐cancer activity. This study was undertaken to investigate their potential inhibitory effects on the major Cytochrome P450 (CYP) drug metabolizing enzymes in human liver microsomes using specific CYP probe substrates and liquid chromatography‐tandem mass spectrometry. 9a potently inhibited CYP2E1‐catalyzed chlorzoxazone‐6′‐hydrolylation and CYP2C9‐catalyzed tolbutamide‐4′‐hydrolylation with IC50 values of 0.05 and 0.7 μM, respectively. CYP2A6‐catalyzed coumarin‐7′‐hydroxylation and CYP3A4‐catalyzed testosterone‐6β‐hydroxylation were moderately inhibited by 9‐a with IC50 values of 2.7 and 12 μM, respectively. The rest of the tested CYP enzymes were weakly or negligibly inhibited by 9‐a. WES‐1, on the other hand, demonstrated weak inhibitory effects on CYP2C19‐mediated [S]‐mephenytoin‐4′‐hydrolylation, CYP2C9 activity, and CYP2D6‐catalyzed Dextromethorphan dealkylation with IC50 values of 39, 42.5, and 57.5 μM, respectively. This in‐vitro data indicates that more in vivo studies should be conducted to examine the potential pharmacokinetic drug‐drug interactions with 9‐a due to its potent and moderate inhibition of CYP2A6, CYP2C9, CYP2E1, and CYP3A4.Inhibitory effect of WES‐1 on the major CYP enzymes in human liver microsomesFigure 1Inhibitory effect of 9a on the major CYP enzymes in human liver microsomesFigure 2

Fatal intoxication of a regular drug user following N-ethyl-hexedrone and ADB-FUBINACA consumption

In Hungary, N-ethyl-hexedrone (NEH) was the most frequently seized stimulant designer drug in 2017, while among synthetic cannabinoids ADB-FUBINACA and AB-FUBINACA were the most popular. Symptoms of intoxication by these substances are well known but less is known about the pathology of overdose-related death.NEH-induced fatal intoxication has not been described in the literature and knowledge surrounding the particular circumstances of death could be useful better public education of risk and more adequate treatment of overdose patients. In this report, we characterize the case of a 23-year-old male regular drug user who died a few hours after NEH and ADB-FUBINACA consumption. His medical history showed arrhythmia in childhood, and some seizures. Autopsy found he had a BMI of 42.9, a hypertrophic and dilated heart, severe atherosclerosis of the valves, coronaries and the arteries, and edema of the internal organs. Histology confirmed those findings. Postmortem blood levels of NEH were 285 ng/ml, along with 0.08 ng/ml ADB-FUBINACA and five ADB-FUBINACA metabolites. Based on the blood concentrations measured in suspected drug users (≤83.9 ng/ml) we hypothesize that NEH intoxication was the cause of death in this case, with heart disease being a co-factor and that the synthetic cannabinoid effect might have been accompaniment.This case also offered the opportunity to identify the metabolites of ADB-FUBINACA in the blood. We identified metabolites in the post-mortem blood by comparing them to human liver microsomal enzyme metabolites in vitro. Three major and two minor metabolites were found in the blood, of which two could only be derived from ADB-FUBINACA, as opposed to other cannabinoids.The case highlights the importance of the complex analysis of drug related deaths by medico-legal autopsy, histopathology and toxicology.

Insights into Nanoarchitectual Structures to Enhance the Catalysis Properties of Human Liver Microsomes

Building a nanoarchitectual structure using 1-Pyrenylbutylamine and functionalized multiwalled carbon nanotubes (MWNT) will create an abundance of aminated sites on an electrode for electrostatic interactions binding Human Liver Microsomes (HLM). This increase in secondary interactions will allow for a higher concentration of HLM to bind to the electrode creating a higher catalytic current and increased stability of the bioprocesses involved. Using inexpensive HLM on this nanostructure with applied potential, without NADPH as an electron source, could prove to be a viable bioreactor for swift drug metabolism and inhibition assays. Using cyclic voltammetry the research confirms that High Purity Graphite (HPG) electrodes structured with aminated MWNTs in conjunction with pi-pi stacking of 1-Pyrenylbutylamine with adsorbed HLM increases the catalytic current over 4x that of HLM directly adsorbed on HPG. This nanostructural design demonstrates an almost 2x higher catalytic current using HLM over costly recombinant created CYP enzymes. The formal potential of -0.46 indicates that the CYP reductase is functioning in the HLM and the first to receive the electron transfer as published previously in this lab with recombinant enzymes. Long term impedances stability tests demonstrate that the carbon electrode with this nanostructure is more stable than a costly gold electrode. Using HPLC to measure the hydroxylation of diclofenac to 4’-hydroxydiclofenac HLM outperforms the recombinant enzymes also. Inhibition of the CYP2C9 enzyme in HLM by the competitive inhibitor sulphenazole shown by HPLC indicates that CYP2C9 is responsible for the reduction of diclofenac. This nanostructure design makes improvements over the plain electrode, and could prove to be a feasible method upon to build an inexpensive platform to examine newly developing drugs.

Stereoselective Oxidation Kinetics of Deoxycholate in Recombinant and Microsomal CYP3A Enzymes: Deoxycholate 19-Hydroxylation Is an In Vitro Marker of CYP3A7 Activity.

The primary bile acids (BAs) synthesized from cholesterol in the liver are converted to secondary BAs by gut microbiota. It was recently disclosed that the major secondary BA, deoxycholate (DCA) species, is stereoselectively oxidized to tertiary BAs exclusively by CYP3A enzymes. This work subsequently investigated the in vitro oxidation kinetics of DCA at C-1β, C-3β, C-4β, C-5β, C-6α, C-6β, and C-19 in recombinant CYP3A enzymes and naive enzymes in human liver microsomes (HLMs). The stereoselective oxidation of DCA fit well with Hill kinetics at 1-300 μM in both recombinant CYP3A enzymes and pooled HLMs. With no contributions or trace contributions from CYP3A5, CYP3A7 favors oxidation at C-19, C-4β, C-6α, C-3β, and C-1β, whereas CYP3A4 favors the oxidation at C-5β and C-6β compared with each other. Correlation between DCA oxidation and testosterone 6β-hydroxylation in 14 adult single-donor HLMs provided proof-of-concept evidence that DCA 19-hydroxylation is an in vitro marker reaction for CYP3A7 activity, whereas oxidation at other sites represents mixed indicators for CYP3A4 and CYP3A7 activities. Deactivation caused by DCA-induced cytochrome P450-cytochrome P420 conversion, as shown by the spectral titrations of isolated CYP3A proteins, was observed when DCA levels were near or higher than the critical micelle concentration (about 1500 μM). Unlike CYP3A4, CYP3A7 showed abnormally elevated activities at 500 and 750 μM, which might be associated with an altered affinity for DCA multimers. The disclosed kinetic and functional roles of CYP3A isoforms in disposing of the gut bacteria-derived DCA may help in understanding the structural and functional mechanisms of CYP3A.

Label-free absolute protein quantification with data-independent acquisition

Despite data-independent acquisition (DIA) has been increasingly used for relative protein quantification, DIA-based label-free absolute quantification method has not been fully established. Here we present a novel DIA method using the TPA algorithm (DIA-TPA) for the absolute quantification of protein expressions in human liver microsomal and S9 samples. To validate this method, both data-dependent acquisition (DDA) and DIA experiments were conducted on 36 individual human liver microsome and S9 samples. The MS2-based DIA-TPA was able to quantify approximately twice as many proteins as the MS1-based DDA-TPA method, whereas protein concentrations determined by the two approaches were comparable. To evaluate the accuracy of the DIA-TPA method, we absolutely quantified carboxylesterase 1 concentrations in human liver S9 fractions using an established SILAC internal standard-based proteomic assay; the SILAC results were consistent with those obtained from DIA-TPA analysis. Finally, we employed a unique algorithm in DIA-TPA to distribute the MS signals from shared peptides to individual proteins or isoforms and successfully applied the method to the absolute quantification of several drug-metabolizing enzymes in human liver microsomes. In sum, the DIA-TPA method not only can absolutely quantify entire proteomes and specific proteins, but also has the capability quantifying proteins with shared peptides. SignificanceData independent acquisition (DIA) has emerged as a powerful approach for relative protein quantification at the whole proteome level. However, DIA-based label-free absolute protein quantification (APQ) method has not been fully established. In the present study, we present a novel DIA-based label-free APQ approach, named DIA-TPA, with the capability absolutely quantifying proteins with shared peptides. The method was validated by comparing the quantification results of DIA-TPA with that obtained from stable isotope-labeled internal standard-based proteomic assays.

An Assessment of the In Vitro Inhibition of Cytochrome P450 Enzymes, UDP-Glucuronosyltransferases, and Transporters by Phosphodiester- or Phosphorothioate-Linked Oligonucleotides.

Oligonucleotides represent an expanding class of pharmacotherapeutics in development for various indications. Typically, oligonucleotides are developed with phosphorothioate linkages for the improvement of biologic stability; however, limited data are available on the potential of these molecules to cause drug-drug interactions (DDIs). In this study, four nontherapeutic oligonucleotides with either a phosphodiester or phosphorothioate linkage and partial sequences towards glutathione peroxidase or β-actin (PD-GP and PD-Ac or PT-GP and PT-Ac, respectively) were evaluated in vitro for their potential to inhibit cytochrome P450 (P450) enzymes and UGP-glucuronosyltransferases (UGTs) in both human liver microsomes (HLMs) and cryopreserved human hepatocytes (CHHs) and to inhibit select transporters in expression systems. PD-GP and PD-Ac had little to no inhibitory effect on any P450 or UGT enzymes in HLMs and CHHs, except for PD-Ac in HLMs for CYP2C19 (IC50 = 29 μM). Conversely, PT-GP and PT-Ac caused direct inhibition of almost all P450 and UGT enzymes, with CYP1A2 (IC50 values of 0.8-4.2 μM), CYP2C8 (IC50 values of 1.1-12 μM), and UGT1A1 (IC50 values of 4.5-5.4 μM) inhibited to the greatest extent. There was evidence of possible time-dependent inhibition (TDI) of P450 enzymes with PT-GP and PT-Ac for CYP2B6, CYP2C8, CYP2C19, CYP2C9, CYP2D6, and CYP3A4/5; however, this TDI was reversible. In contrast to HLMs, there was little to no direct P450 inhibition by any oligonucleotide in CHHs [except for PD-Ac with CYP2C19 (IC50 = 36 μM) and TDI by PT-GP with CYP2C8], demonstrating test system-dependent outcomes. Inhibition was observed for the organic anion uptake transporters, including organic anion-transporting polypeptide OATP1B1 and OATP1B3, organic anion transporters OAT1 and OAT3, and organic cation transporter OCT2 (IC50 values of 12-29 μM), but not OCT1 or the efflux transporters breast cancer resistance protein and P-glycoprotein by the phosphorothioate oligonucleotides.

Inhibition of cytochrome P450 enzymes by thymoquinone in human liver microsomes

The aim of the present study was to investigate the potential effect of thymoquinone (TQ) on the metabolic activity of four major drug metabolizing enzymes in human liver microsomes, namely cytochrome P450 (CYP) 1A2, CYP2C9, CYP2D6 and CYP3A4. The inhibition of CYP enzymatic activities by TQ was evaluated by incubating typical substrates (phenacetin for CYP1A2, tolbutamide for CYP2C9, dextromethorphan for CYP2D6, and testosterone for CYP3A4) with human liver microsomes and NADPH in the absence or presence of TQ (1, 10 and 100 µM). The respective metabolite of the substrate that was formed was measured by HPLC. Results of the presented study presented that the metabolic activities of all the investigated CYP enzymes, viz. CYP1A2, CYP2C9, CYP2D6 and CYP3A4, were inhibited by TQ. At 1 µM TQ, CYP2C9 enzyme activity was maximally inhibited by 46.35%, followed by CYP2D6 (20.26%) > CYP1A2 (13.52%) > CYP3A4 (12.82%). However, at 10 µM TQ, CYP2C9 enzyme activity was maximally inhibited by 69.69%, followed by CYP3A4 (23.59%) > CYP1A2 (23.51%) > CYP2D6 (11.42%). At 100 µM TQ, CYP1A2 enzyme activity was maximally inhibited by 81.92%, followed by CYP3A4 (79.24%) > CYP2C9 (69.22%) > CYP2D6 (28.18%). The IC50 (mean ± SE) values for CYP1A2, CYP2C9, CYP2D6 and CYP3A4 inhibition were 26.5 ± 2.9 µM, 0.5 ± 0.4 µM, >500 µM and 25.2 ± 3.1 µM, respectively. These findings suggest that there is a high probability of drug interactions resulting from the co-administration of TQ or herbs containing TQ with drugs that are metabolized by the CYP enzymes, particularly CYP2C9.

Use of SWATH-MS to relatively quantify the expression of hepatic drug metabolizing enzymes in human liver microsomes using a label-free approach

Use of SWATH-MS to relatively quantify the expression of hepatic drug metabolizing enzymes in human liver microsomes using a label-free approach

Multiplex and Label-Free Relative Quantification Approach for Studying Protein Abundance of Drug Metabolizing Enzymes in Human Liver Microsomes Using SWATH-MS.

We describe a sequential windowed acquisition of all theoretical fragment ion mass spectra (SWATH-MS) based method for label-free, simultaneous, relative quantification of drug metabolism enzymes in human liver microsomes (HLM; n = 78). In-solution tryptic digestion was aided by a pressure cycling method, which allowed a 90 min incubation time, a significant reduction over classical protocols (12-18 h). Digested peptides were separated on an Acquity UHPLC Peptide BEH C18 column using a 60 min gradient method at a flow rate of 0.100 mL/min. The quadrupole-time-of-flight mass spectrometer (ESI-QTOFMS) was operated in positive electrospray ionization mode, and data were acquired by data-dependent acquisition (DDA) and SWATH-MSALL mode. A pooled HLM sample was used as a quality control to evaluate variability in digestion and quantification among different batches, and inter-batch %CV for various proteins was between 3.1 and 7.8%. Spectral library generated from the DDA data identified 1855 distinct proteins and 25 681 distinct peptides at a 1% global false discovery rate (FDR). SWATH data were queried and analyzed for 10 major cytochrome P450 (CYP) enzymes using Skyline, a targeted data extraction software. Further, correlation analysis was performed between functional activity, protein, and mRNA expression for ten CYP enzymes. Pearson correlation coefficient (r) between protein and activity for CYPs ranged from 0.314 (CYP2C19) to 0.767 (CYP2A6). A strong correlation was found between CYP3A4 and CYP3A5 abundance and activity determined using midazolam and testosterone (r > 0.600, p < 0.001). Protein-to-activity correlation was moderate (r > 0.400-0.600, p < 0.001) for CYP1A2, CYP2A6, CYP2B6, CYP2C9, and CYP2E1 and significant but poor (r < 0.400, p < 0.05) for CYP2C8, CYP2C19, and CYP2D6. The findings suggest the suitability of SWATH-MS based method as a valuable and relatively fast analytical technique for relative quantification of proteins in complex biological samples. We also show that protein abundance is a better surrogate than mRNA to predict the activity of CYP activity.

Novel approach for evaluating pharmaceuticals toxicity using daphnia model: Analysis of the mode of cytochrome P450-generated metabolite action after acetaminophen exposure

An efficient screening assay was developed and validated for simultaneous assessment of compound-mediated inhibition of six major human cytochrome P450 (CYP) enzymes. This method employed a cocktail of six probe substrates (i.e., phenacetin, amodiaquine, diclofenac, S-mephenytoin, dextromethorphan and midazolam for CYP1A2, 2C8, 2C9, 2C19, 2D6 and 3A4, respectively) as well as individual prototypical inhibitors of the six CYP enzymes in human liver microsomes under optimized incubation conditions. The corresponding marker metabolites (i.e., acetaminophen, N-desethylamodiaquine, 4-OH-diclofenac, 4-OH-S-mephenytoin, dextrorphan and 1-OH-midazolam) in the incubates were quantified using LC–MS/MS methods either by an internal standard (IS) calibration curve or a simplified analyte-to-IS peak area ratio approach. The results showed that the IC50 values determined by the cocktail approach were in good agreement with those obtained by the individual substrate approach as well as those reported in the literature. Besides, no remarkable difference was observed between the two quantification approaches. In conclusion, this new cocktail assay can be used for reliable screening of compound-mediated CYP inhibition.