P450 Enzyme Activity Research Articles

Development and Validation of an HPLC-UV Method for the Quantification of 4'-Hydroxydiclofenac Using Salicylic Acid: Future Applications for Measurement of In Vitro Drug-Drug Interaction in Rat Liver Microsomes.

Salicylic acid is a key compound in nonsteroidal anti-inflammatory drugs that has been recently used for preventing the risk of hospitalization and death among COVID-19 patients and in preventing colorectal cancer (CRC) by suppressing two key proteins. Understanding drug–drug interaction pathways prevent the occurrence of adverse drug reactions in clinical trials. Drug–drug interactions can result in the variation of the pharmacodynamics and pharmacokinetic of the drug. Inhibition of the Cytochrome P450 enzyme activity leads to the withdrawal of the drug from the market. The aim of this paper was to develop and validate an HPLC-UV method for the quantification of 4′-hydroxydiclofenac as a CYP2C9 metabolite using salicylic acid as an inhibitor in rat liver microsomes. A CYP2C9 assay was developed and validated on the reversed phase C18 column (SUPELCO 25 cm × 4.6 mm × 5 µm) using a low-pressure gradient elution programming at T = 30 °C, a wavelength of 282 nm, and a flow rate of 1 mL/min. 4′-hydroxydiclofenac demonstrated a good linearity (R2 > 0.99), good reproducibility, low detection, and quantitation limit, and the inter and intra-day precision met the ICH guidelines (<15%). 4′-hydroxydiclofenac was stable for three days and showed an acceptable accuracy and recovery (80–120%) within the ICH guidelines in a rat liver microsome sample. This method will be beneficial for future applications of the in vitro inhibitory effect of salicylic acid on the CYP2C9 enzyme activity in rat microsomes and the in vivo administration of salicylic acid in clinical trials.

Liver Cirrhosis Affects the Pharmacokinetics of the Six Substrates of the BaselPhenotyping Cocktail Differently.

BackgroundActivities of hepatic cytochrome P450 enzymes (CYPs) are relevant for hepatic clearance of drugs and known to be decreased in patients with liver cirrhosis. Several studies have reported the effect of liver cirrhosis on CYP activity, but the results are partially conflicting and for some CYPs lacking.ObjectiveIn this study, we aimed to investigate the CYP activity in patients with liver cirrhosis with different Child stages (A-C) using the Basel phenotyping cocktail approach.MethodsWe assessed the pharmacokinetics of the six compounds and their CYP-specific metabolites of the Basel phenotyping cocktail (CYP1A2: caffeine, CYP2B6: efavirenz, CYP2C9: flurbiprofen, CYP2C19: omeprazole, CYP2D6: metoprolol, CYP3A: midazolam) in patients with liver cirrhosis (n = 16 Child A cirrhosis, n = 15 Child B cirrhosis, n = 5 Child C cirrhosis) and matched control subjects (n = 12).ResultsWhile liver cirrhosis only marginally affected the pharmacokinetics of the low to moderate extraction drugs efavirenz and flurbiprofen, the elimination rate of caffeine was reduced by 51% in patients with Child C cirrhosis. For the moderate to high extraction drugs omeprazole, metoprolol, and midazolam, liver cirrhosis decreased the elimination rate by 75%, 37%, and 60%, respectively, increased exposure, and decreased the apparent systemic clearance (clearance/bioavailability). In patients with Child C cirrhosis, the metabolic ratio (ratio of the area under the plasma concentration–time curve from 0 to 24 h of the metabolite to the parent compound), a marker for CYP activity, decreased by 66%, 47%, 92%, 73%, and 43% for paraxanthine/caffeine (CYP1A2), 8-hydroxyefavirenz/efavirenz (CYP2B6), 5-hydroxyomeprazole/omeprazole (CYP2C19), α-hydroxymetoprolol/metoprolol (CYP2D6), and 1′-hydroxymidazolam/midazolam (CYP3A), respectively. In comparison, the metabolic ratio 4-hydroxyflurbiprofen/flurbiprofen (CYP2C9) remained unchanged.ConclusionsLiver cirrhosis affects the activity of CYP isoforms differently. This variability must be considered for dose adjustment of drugs in patients with liver cirrhosis.Clinical Trial RegistrationNCT03337945.Supplementary InformationThe online version contains supplementary material available at 10.1007/s40262-022-01119-0.

Development of human pluripotent stem cell-derived hepatic organoids as an alternative model for drug safety assessment

Human in vitro hepatic models that faithfully recapitulate liver function are essential for successful basic and translational research. A limitation of current in vitro models, which are extensively used for drug discovery and toxicity testing, is the loss of drug metabolic function due to the low expression and activity of cytochrome P450 (CYP450) enzymes. Here, we aimed to generate human pluripotent stem cell-derived hepatic organoids (hHOs) with a high drug metabolic ability. We established a two-step protocol to produce hHOs from human pluripotent stem cells for long-term expansion and drug testing. Fully differentiated hHOs had multicellular composition and exhibited cellular polarity and hepatobiliary structures. They also displayed remarkable CYP450 activity and recapitulated the metabolic clearance, CYP450-mediated drug toxicity, and metabolism. Furthermore, hHOs successfully modeled Wilson's disease in terms of Cu metabolism, drug responses, and diagnostic marker expression and secretion. In conclusion, hHOs exhibit high capacity for drug testing and disease modeling. Hence, this hepatic model system provides an advanced tool for studying hepatic drug metabolism and diseases.

In Vitro Investigation on the Effect of Dendrobine on the Activity of Cytochrome P450 Enzymes.

Dendrobine is the major active ingredient of Dendrobium nobile, Dendrobium chrysotoxum, and Dendrobium fimbriatum, all of which are used in traditional Chinese medicine owing to their antitumor and anti-inflammation activities. Hence, investigation on the interaction of dendrobine with cytochrome P450 enzymes could provide a reference for the clinical application of Dendrobium. The effects of dendrobine on cytochrome P450 enzymes activities were investigated in the presence of 0, 2.5, 5, 10, 25, 50, and 100 µM dendrobine in pooled human liver microsomes. The specific inhibitors were employed as the positive control and the blank groups were set as the negative control. The Lineweaver-Burk plots were plotted to characterize the specific inhibition model and obtain the kinetic parameters. The study reveals that dendrobine significantly inhibited the activity of CYP3A4, 2C19, and 2D6 with IC50 values of 12.72, 10.84, and 15.47 µM, respectively. Moreover, the inhibition of CYP3A4 was found to be noncompetitive (Ki = 6.41 µM) and time dependent (KI = 2.541 µM-1, Kinact = 0.0452 min-1), while the inhibition of CYP2C19 and 2D6 was found to be competitive with the Ki values of 5.22 and 7.78 µM, respectively, and showed no time-dependent trends. The in vitro inhibitory effect of dendrobine implies the potential drug-drug interaction between dendrobine and CYP3A4-, 2C9-, and 2D6-metabolized drugs. Nonetheless, these findings need further in vivo validation.

Evaluation of the effects of four types of tea on the activity of cytochrome P450 enzymes with a probe cocktail and HPLC-MS/MS.

BackgroundTea, the world’s second most popular drink, is an essential part of some people’s lives. Thus, this study aimed to explore potential tea-drug interactions with a view to promoting the rational administration of drugs.MethodsA specific and sensitive approach involving high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) and a probe cocktail was established and validated to evaluate the inhibitory effects of four teas on five cytochrome P450 (CYP450) enzymes in rats. Metoprolol tartrate (MT), omeprazole (OMP), phenacetin (PNT), tolbutamide (TOL), and testosterone (T) were selected as the probe drugs for CYP2D6, CYP2C19, CYP1A2, CYP2C6, and CYP3A1/2, respectively, and were simultaneously quantified in the multiple reaction monitoring (MRM) mode with positive electrospray ionization (ESI+) in a single 12-min run.ResultsThe extraction recoveries, matrix effect values, as well as intra/interday accuracy and precision met the determination standards. CYP1A2 and CYP2C6 were strongly inhibited by green tea, and CYP2C6 was also strongly inhibited by Pu’er tea. Ti Kuan Yin tea had a weak inhibitory effect, and black tea had only a slight inhibitory effect, on CYP1A2. Furthermore, the four types of tea did not have significantly altered the activity of CYP2D6, CYP2C19, and CYP3A1/2 in vitro.ConclusionsThe method used in the present study was successfully applied to assess the inhibitory effects of aqueous extracts of four types of tea on CYP450 isoforms in vitro. The results suggest that different types of tea have different effects on drug metabolism.

Imidacloprid-induced pathophysiological damage in the midgut of Locusta migratoria (Orthoptera: Acrididae) in the field.

Neonicotinoids are modern insecticides widely used in agriculture worldwide. Their impact on target (nervous system) and non-target (midgut) tissues has been well studied in beneficial insects including honeybees under controlled conditions. However, their detailed effects on pest insects on the field are missing to date. Here, we have studied the effects of the neonicotinoid imidacloprid on the midgut of the pest insect Locusta migratoria caught in the field. We found that in the midgut of imidacloprid-exposed locusts the activity of enzymes involved in reactive oxygen metabolism was perturbed. By contrast, the activity of P450 enzymes that have been shown to be activated in a detoxification response and that were also reported to produce reactive oxygen species was elevated. Probably as a consequence, markers of oxidative stress including protein carbonylation and lipid peroxidation accumulated in midgut samples of these locusts. Histological analyses revealed that their midgut epithelium is disorganized and that the brush border of the epithelial cells is markedly reduced. Indeed, microvilli are significantly shorter, misshapen and possibly non-functional in imidacloprid-treated locusts. We hypothesize that imidacloprid induces oxidative stress in the locust midgut, thereby changing the shape of midgut epithelial cells and probably in turn compromising their physiological function. Presumably, these effects reduce the survival rate of imidacloprid-treated locusts and the damage they cause in the field.

Role of hesperidin and fresh orange juice in altering the bioavailability of beta-blocker, metoprolol tartrate. An in vivo model

The aim of my study was to find the effect of co-administrating orange juice and hesperidin on the bioavailability of metoprolol tartrate in rabbits. Metoprolol tartrate (10 mg/kg) was given orally to rabbits with hesperidin (10 mg/kg) and with orange juice (6 mL/kg) separately. The plasma concentrations of metoprolol tartrate were determined using reverse phase-high performance liquid chromatography at 1.5, 1, 2, 4, and 6 h, and the pharmacokinetic parameters were studied. In comparison to the control group, the AUC of metoprolol tartrate was increased significantly by 68.32% with hesperidin while orange juice substantially reduced the AUC by 37.08%. However, no significant change was observed in Tmax , Kel , and Vd in both groups. The relative bioavailability of metoprolol tartrate with hesperidin was 168.3% as compared to orange juice, that is, 62.9%. The present study revealed that the concurrent intake of hesperidin with metoprolol tartrate increased its bioavailability while orange juice administration suppressed its bioavailability. The change in bioavailability of metoprolol tartrate might be due to alterations in the activity of cytochrome P450 enzymes involved in the metabolism of metoprolol tartrate. However, the exact mechanism is still not known. These interactions may be of clinical significance.

Selective Serotonin Reuptake Inhibitor Pharmacokinetics During Pregnancy: Clinical and Research Implications.

Pregnancy and associated physiologic changes affect the pharmacokinetics of many medications, including selective serotonin reuptake inhibitors—the first-line pharmacologic interventions for depressive and anxiety disorders. During pregnancy, SSRIs exhibit extensive pharmacokinetic variability that may influence their tolerability and efficacy. Specifically, compared to non-pregnant women, the activity of cytochrome P450 (CYP) enzymes that metabolize SSRIs drastically changes (e.g., decreased CYP2C19 activity and increased CYP2D6 activity). This perspective examines the impact of pharmacokinetic genes—related to CYP activity on SSRI pharmacokinetics during pregnancy. Through a simulation-based approach, plasma concentrations for SSRIs metabolized primarily by CYP2C19 (e.g., escitalopram) and CYP2D6 (e.g., fluoxetine) are examined and the implications for dosing and future research are discussed.

Mechanistic Insights into Stereospecific Antifungal Activity of Chiral Fungicide Prothioconazole against Fusarium oxysporum F. sp. cubense.

As a typical triazole fungicide, prothioconazole (Pro) has been used extensively due to its broad spectrum and high efficiency. However, as a racemic mixture of two enantiomers (R-Pro and S-Pro), the enantiomer-specific outcomes on the bioactivity have not been fully elucidated. Here, we investigate how chirality affects the activity and mechanism of action of Pro enantiomers on Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4), the notorious virulent strain causing Fusarium wilt of banana (FWB). The Pro enantiomers were evaluated in vivo and in vitro with the aid of three bioassay methods for their fungicidal activities against TR4 and the results suggested that the fungicidal activities of Pro enantiomers are stereoselective in a dose-dependent manner with R-Pro making a major contribution to the treatment outcomes. We found that R-Pro led to more severe morphological changes and impairment in membrane integrity than S-Pro. R-Pro also led to the increase of more MDA contents and the reduction of more SOD and CAT activities compared with the control and S-Pro groups. Furthermore, the expression of Cytochrome P450 14α-sterol demethylases (CYP51), the target for triazole fungicides, was significantly increased upon treatment with R-Pro rather than S-Pro, at both transcriptional and translational levels; so were the activities of the Cytochrome P450 enzymes. In addition, surface plasmon resonance (SPR) and molecular docking illuminated the stereoselective interactions between the Pro enantiomers and CYP51 of TR4 at the target site, and R-Pro showed a better binding affinity with CYP51 than S-Pro. These results suggested an enantioselective mechanism of Pro against TR4, which may rely on the enantioselective damages to the fungal cell membrane and the enantiospecific CYP51 binding affinity. Taken together, our study shed some light on the mechanisms underlying the differential activities of the Pro enantiomers against TR4 and demonstrated that Pro can be used as a potential candidate in the treatment of FWB.

Abstract P1-08-02: Cytochrome P450 reductase gene, POR, associated with paclitaxel induced peripheral neuropathy in patients of European ancestry from the adjuvant breast cancer trial, ECOG-ACRIN E5103

Abstract Introduction: Paclitaxel induced peripheral neuropathy (TIPN) is one of the most important survivorship issues of cancer patients with the potential to substantially impact quality of life. TIPN can lead to dose reductions, early cessation, and has the potential to compromise treatment efficacy. We previously reported both rare (SBF2) and common germline (FCAMR) predictors for TIPN in patients of African descent through whole-exome sequencing (WES) and a genome-wide association study (GWAS), respectively, in the adjuvant, randomized phase III breast cancer trial, E5103. These predictors are now being evaluated in the prospective ECOG-ACRIN trial EAZ171 (NCT04001829). We have also previously reported our evaluation of common germline variants for patients of European descent through GWAS in E5103 with validation in another adjuvant breast cancer trial, E1199. Herein, we evaluated for rare variants that were associated with risk of TIPN in patients of European ancestry from WES in E5103. Methods: Gene-based rare variant analyses using SKAT were performed to compare select cases of severe TIPN (n=172) and controls without any evidence of TIPN (n=170) derived from E5103. Cases were defined as those with at least grade 3 TIPN and controls were optimally. matched by demographic and risk factors and had no reported evidence for TIPN of any grade. Germline WES was conducted using Ion Proton™ Sequencers from Life Technologies with an average &amp;gt;100X coverage of the exome. Rare variants with a minor allele frequency &amp;lt;3% predicted to be deleterious by protein prediction programs were retained for the analysis. 7,278 genes passed quality control and were included in the analysis, setting the threshold for significance at a p-value &amp;lt; 6.87 ×10-6 after Bonferroni correction. Results: Cytochrome P450 oxidoreductase (POR) was significantly associated with an increased risk of grade 3+ TIPN (p =1.82 ×10-6). Six variants predicted to be deleterious in POR were identified in the study population. Paclitaxel is predominately metabolized by cytochrome P450 (CYP) 2C8 and 3A4. A functional cytochrome P450 oxidoreductase is required for the catalytic activities of all 57 CYP family members to metabolize drugs, xenobiotics, and steroid hormones. Conclusion: Rare variants in cytochrome P450 oxidoreductase predicted an increased risk of severe TIPN in patients receiving paclitaxel. These rare variants in POR may modulate the catalytic activities of P450 enzymes that are responsible for paclitaxel metabolism and have a striking biological rationale as contributors to TIPN. Work is ongoing to validate these findings mechanistically and to identify strategies to nullify the impact of these deleterious variants. Citation Format: Fei Shen, Guanglong Jiang, Laura Gardner, Gloria Xue, Santosh Philips, Reynold Ly, Osei Wilberforce, Xi Wu, Erica Cantor, Chau Dang, Donald Northfelt, Todd Skaar, Kathy D. Miller, George Sledge, Bryan P Schneider. Cytochrome P450 reductase gene, POR, associated with paclitaxel induced peripheral neuropathy in patients of European ancestry from the adjuvant breast cancer trial, ECOG-ACRIN E5103 [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-08-02.

A Target-Site Mutation Confers Cross-Resistance to ALS-Inhibiting Herbicides in Erigeron sumatrensis from Brazil.

Cases of weed resistant to herbicides have changed the dynamics of agricultural areas in Brazil, and in recent years, Erigeron species have caused major problems to farmers in the country, mainly in relation to the ineffectiveness of herbicide treatments used. The objective of this study was to confirm the cross-resistance to ALS inhibitors in populations of Erigeron sumatrensis as well as to investigate the existence of mutations in the site of action of ALS-inhibiting herbicides. To do this, 30 populations collected in the 2016/2017 crop season were grown in a greenhouse. Dose–response (chlorimuron-ethyl and cloransulam-methyl), inhibition of cytochrome P-450 with malathion, and ALS gene sequencing experiments were carried out in the F1 generations of two fleabane populations. The results proved the cross-resistance to chlorimuron-ethyl and cloransulam-methyl herbicides applied in the post-emergence of the resistant population of E. sumatrensis. The higher activity of P450 enzymes is unlikely responsible for the resistance of the population studied. The resistance mechanism found in R was the target site mutation Pro197Ser at the ALS gene. This is the first study in Brazil to identify a target-site change as a survival mechanism in E. sumatrensis for the resistance to ALS-inhibiting herbicides.

Metagenomic ecotoxicity assessment of trace difenoconazole on freshwater microbial community

Difenoconazole, a typical triazole fungicide, inhibits the activity of cytochrome P450 enzyme in fungi, and is extensively used in protecting fruits, vegetables, and cereal crops. However, reports elucidating the effects of difenoconazole on aquatic microbial communities are limited. Our study showed that difenoconazole promoted microalgae growth at concentrations ranging from 0.1 to 5 μg/L, which was similar with its environmental residual concentrations. Metagenomic analysis revealed that the aquatic microbial structure could self-regulate to cope with difenoconazole-induced stress by accumulating bacteria exhibiting pollutant degrading abilities. In the short-term, several functional pathways related to xenobiotic biodegradation and analysis were upregulated to provide ability for aquatic microbial community to process xenobiotic stress. Moreover, most disturbed ecological functions were recovered due to the redundancy of microbial communities after prolonged exposure. Furthermore, the risks associated with the dissemination of antibiotic resistance genes were enhanced by difenoconazole in the short-term. Overall, our study contributes to a comprehensive understanding of the difenoconazole-induced ecological impacts and the behavior of aquatic microbial communities that are coping with xenobiotic stress.

Evaluation of tolerance to λ-cyhalothrin and response of detoxification enzymes in silkworms reared on artificial diet

A representative silkworm rearing mode of Ⅰ-Ⅲ instars reared on artificial diet and Ⅳ-Ⅴ instars reared on fresh mulberry leaves has been recognized in some sericultural areas of China. Under this rearing mode, silkworms are prone to be poisoned by pesticide residues on mulberry leaves at the Ⅳ and Ⅴ instar stages. As one of the most widely applied insecticides, λ-cyhalothrin was used to study the insecticide tolerance of silkworm reared on artificial diet (referred as the AD group). Our results showed that the newly ecdysized Ⅳ instar larvae in the AD group were less tolerant to λ-cyhalothrin compared to the mulberry leaves reared group (referred as the ML group). After continuous exposure to trace λ-cyhalothrin, the weight gain and the survival rate of silkworms were significantly lower in the AD group than those in the ML group, even though compensatory growth was observed in the control of the AD group. Histopathology and ultrastructure of fat body showed that λ-cyhalothrin induced more severe cell injuries in the AD group, such as shrunken nucleus, dilatation of endoplasmic reticulum, and mitochondrial swelling. The transcription levels of detoxification related genes (CYP4M5, CYP6AB4, CarE2, CarE5, GSTe1 and GSTe3) and the enzyme activities of P450s, CarEs and GSTs were inducible by trace λ-cyhalothrin in a time-specific manner, and the data showed that the response of P450 enzyme activity was retarded in the AD group, indicating a potential reason for a higher sensitivity to λ-cyhalothrin. Our results provided a new clue for the study of the relationship between feed nutrition and detoxification ability, and also provided an important reference for the development of modern silkworm rearing mode.

Blocking hepatocarcinogenesis by a cytochrome P450 family member with female-preferential expression

ObjectsThe incidence of hepatocellular carcinoma (HCC) shows an obvious male dominance in rodents and humans. We aimed to identify the key autosomal liver-specific sex-related genes and investigate their roles in...

Time of Day-Specific Changes in Metabolic Detoxification and Insecticide Tolerance in the House Fly, Musca domestica L.

Both insects and mammals all exhibit a daily fluctuation of susceptibility to chemicals at different times of the day. However, this phenomenon has not been further studied in the house fly (Musca domestica L.) and a better understanding of the house fly on chronobiology should be useful for controlling this widespread disease vector. Here we explored diel time-of-day variations in insecticide susceptibility, enzyme activities, and xenobiotic-metabolizing enzyme gene expressions. The house fly was most tolerant to beta-cypermethrin in the late photophase at Zeitgeber time (ZT) 8 and 12 [i.e., 8 and 12 h after light is present in the light-dark cycle (LD)]. The activities of cytochrome P450, GST, and CarE enzymes were determined in the house flies collected at various time, indicating that rhythms occur in P450 and CarE activities. Subsequently, we observed diel rhythmic expression levels of detoxifying genes, and CYP6D1 and MdαE7 displayed similar expression patterns with enzyme activities in LD conditions, respectively. No diel rhythm was observed for CYP6D3 expression. These data demonstrated a diel rhythm of metabolic detoxification enzymes and insecticide susceptibility in M. domestica. In the future, the time-of-day insecticide efficacy could be considered into the management of the house fly.

Efficient O-demethylation of lignin monoaromatics using the peroxygenase activity of cytochrome P450 enzymes.

A crucial reaction in harnessing renewable carbon from lignin is O-demethylation. We demonstrate the selective O-demethylation of syringol and guaiacol using different cytochrome P450 enzymes. These can efficiently use hydrogen peroxide which, when compared to nicotinamide cofactor-dependent monooxygenases and synthetic methods, allows for cheap and clean O-demethylation of lignin-derived aromatics.

Expression and functional activity of cytochrome P450 enzymes in human hepatocytes with sustainable reproducibility for in vitro phenotyping studies.

Primary human hepatocytes are an essential in vitro tool for evaluating drug metabolism, drug-drug interactions, and hepatotoxicity. This model is considered as the gold standard in matter of DMPK studies in both industrial and academic research. The primary human hepatocytes are used either in suspension or in monolayer, as fresh or frozen cells. However, the use of this model is limited due to the lack of availability, rapid loss of functionality, high cost as well as the variable hepatocyte plating efficiencies in culture and the limited stock of hepatocytes derived from the same origin. Chimeric TK-NOG mice with humanized livers (humanized liver mice) are an attractive platform for drug metabolism and toxicity, which were produced by transplanting human hepatocytes into immunodeficient mice with injured livers. Here, we show that, using humanized mouse liver, in vivo human hepatocyte repopulation was over ~100-fold enabling the continuous and abundant use of human hepatocytes of the same origin and improving their plateability. In our latest cell preparations, hepatocytes isolated from humanized liver mice (Hu-Liver cells) exhibited high purity (ratio of HLA-positive cells: 92±3%), good viability (75±12%), and yield (1.0×108 cells/mouse). Human hepatic drug metabolizing enzymes, transporters, and nuclear receptors genes were expressed in humanized mouse liver. Drug-metabolizing activities in Hu-Liver cells were comparable to or higher than those in primary human hepatocytes. An extensive P450-dependent human drug metabolism was observed in Hu-Liver cells. CYP1A2, CYP2B6, and CYP3A4/5 activities/mRNA in Hu-Liver cells were induced by the hepatocyte exposure to typical human P450 inducers, omeprazole, phenobarbital, and rifampicin, respectively. Finally, Human albumin secretion and CYP3A-mediated drug oxidation activity were maintained over 4-weeks. Altogether, the expression level of pharmacokinetics-related genes, enzyme activity, human-typed drug metabolism, and inducibility of P450 in Hu-Liver cells make from humanized mouse liver a relevant and robust model for in vitro preclinical studies, including drug metabolism, pharmacokinetics, and toxicology studies.

Substrate-dependent Inhibition of Hypericin on Human Carboxylesterase 2: Implications for Herb-drug Combination

Hypericin is the main active ingredient of St. John's wort, a Chinese herb commonly used for treating depression. Previous studies shown that hypericin can strongly inhibit human cytochrome P450 (CYP) enzyme activities; however, its potential interactions that inhibit human carboxylesterases 2 (hCE2) are unclear. This study aimed to investigate the inhibitory effect of hypericin on hCE2. The inhibition mechanism of hypericin on hCE2 was studied by using N-(2-butyl-1,3-dioxo-2,3-dihydro- 1H-phenalen-6-yl)-2-chloroacetamide (NCEN). The type of inhibition of hypericin on hCE2 and the corresponding inhibition constant (Ki) value were determined. The inhibition of hypericin on hCE2 in living cells was discussed. The risk of herb-drug interactions (HDI) of hypericin in vivo was predicted by estimating the area under the drug concentration-time curve (AUC) in the presence or absence of hypericin. To understand the inhibition mechanism of hypericin on the activity of hCE2 in-depth, molecular docking was performed. The half-maximal inhibitory concentration (IC50) values of hypericin against the hydrolysis of NCEN and irinotecan (CPT-11) were calculated to be 26.59 μM and 112.8 μM, respectively. Hypericin inhibited the hydrolysis of NCEN and CPT-11. Their Ki values were estimated as 10.53 μM and 81.77 μM, respectively. Moreover, hypericin distinctly suppressed hCE2 activity in living cells. In addition, the AUC of hCE2 metabolic drugs with metabolic sites similar to NCEN was estimated to increase by up to 5 % in the presence of hypericin. More importantly, the exposure of CPT-11 in the intestinal epithelium was predicted to increase by 2 % - 69 % following the oral coadministration of hypericin. Further, molecular simulations indicated that hypericin could strongly interact with ASP98, PHE307, and ARG355 to form four hydrogen bonds within hCE2. These findings regarding the combination of hypericin-containing herbs and drugs metabolized by hCE2 are of considerable clinical significance.

Agrimoniin inhibits the activity of CYP1A2, 2D6, and 3A4 in human liver microsomes

The effect of agrimoniin on the activity of cytochrome P450 (CYP450) enzymes would induce drug-drug interaction, which leads to adverse effects or even failure of therapy. Agrimoniin was incubated with the specific substrates of eight human liver CYP isoforms in pooled human liver microsomes. The enzyme kinetics and time-dependent study were performed to obtain kinetic parameters and characteristics in vitro. Agrimoniin significantly inhibited the activity of CYP1A2, 2D6, and 3A4 in a dose-dependent manner with IC50 values of 6.26, 9.35, and 8.30 μM, respectively. Agrimoniin served as a non-competitive inhibitor of CYP3A4 and a competitive inhibitor of CYP1A2 and 2D6. Moreover, the incubation time also significantly affected the inhibition of CYP3A4. In vitro inhibitory effect of agrimoniin on the activity of CYP1A2, 2A6, and 3A4 was reported in this study. The potential drug-drug interactions between agrimoniin and drugs metabolised by CYP1A2, 2D6, and 3A4 should be paid special attention.

Genomic comparisons between hepatocarcinogenic and non-hepatocarcinogenic organophosphate insecticides in the mouse liver

Short-term biomarkers of toxicity have an increasingly important role in the screening and prioritization of new chemicals. In this study, we examined early indicators of liver toxicity for three reference organophosphate (OP) chemicals, which are among the most widely used insecticides in the world. The OP methidathion was previously shown to increase the incidence of liver toxicity, including hepatocellular tumors, in male mice. To provide insights into the adverse outcome pathway (AOP) that underlies these tumors, effects of methidathion in the male mouse liver were examined after 7 and 28 day exposures and compared to those of two other OPs that either do not increase (fenthion) or possibly suppress liver cancer (parathion) in mice. None of the chemicals caused increases in liver weight/body weight or histopathological changes in the liver. Parathion decreased liver cell proliferation after 7 and 28 days while the other chemicals had no effects. There was no evidence for hepatotoxicity in any of the treatment groups. Full-genome microarray analysis of the livers from the 7 and 28 day treatments demonstrated that methidathion and fenthion regulated a large number of overlapping genes, while parathion regulated a unique set of genes. Examination of cytochrome P450 enzyme activities and use of predictive gene expression biomarkers found no consistent evidence for activation of AhR, CAR, PXR, or PPARα. Parathion suppressed the male-specific gene expression pattern through STAT5b, similar to genetic and dietary conditions that decrease liver tumor incidence in mice. Overall, these findings indicate that methidathion causes liver cancer by a mechanism that does not involve common mechanisms of liver cancer induction.