P450 Enzyme Activity Research Articles

Exploring Interactions between Primary Hepatocytes and Non-Parenchymal Cells on Physiological and Pathological Liver Stiffness.

Simple SummaryChronic liver disease is characterized by progressive hepatic fibrosis leading to the formation of cirrhosis irrespective of the etiology with no effective treatment currently available. Liver stiffness (LS) is currently the best clinical predictor of this fibrosis progression irrespective of the cause of the disease. However, it is not well understood how does LS regulate the critical hepatocytes–non parenchymal cell interactions. We here present, to the best of our knowledge, the first analyses of the impact of physiological and pathological stiffness on hepatocytes–non parenchymal cell interaction. Our findings indicate the role of stiffness in regulating the hepatocytes interactions with NPCs necessary for maintenance of hepatocytes function.Chronic liver disease is characterized by progressive hepatic fibrosis leading to the formation of cirrhosis irrespective of the etiology with no effective treatment currently available. Liver stiffness (LS) is currently the best clinical predictor of this fibrosis progression irrespective of the etiology. LS and hepatocytes-nonparenchymal cells (NPC) interactions are two variables known to be important in regulating hepatic function during liver fibrosis, but little is known about the interplay of these cues. Here, we use polydimethyl siloxane (PDMS) based substrates with tunable mechanical properties to study how cell–cell interaction and stiffness regulates hepatocytes function. Specifically, primary rat hepatocytes were cocultured with NIH-3T3 fibroblasts on soft (2 kPa) and stiff substrates that recreates physiologic (2 kPa) and cirrhotic liver stiffness (55 kPa). Urea synthesis by primary hepatocytes depended on the presence of fibroblast and was independent of the substrate stiffness. However, albumin synthesis and Cytochrome P450 enzyme activity increased in hepatocytes on soft substrates and when in coculture with a fibroblast. Western blot analysis of hepatic markers, E-cadherin, confirmed that hepatocytes on soft substrates in coculture promoted better maintenance of the hepatic phenotype. These findings indicate the role of stiffness in regulating the hepatocytes interactions with NPCs necessary for maintenance of hepatocytes function.

A n injured liver caused by overdose of acetaminophen has altered expression and activities of P450 enzymes and associated drug efficacy during liver recovery and regeneration

Acetaminophen (APAP)-induced liver injury (AILI) is the leading cause of acute liver failure in the United States. Alterations on therapeutic efficacy and adverse drug reactions (ADRs) in patients with AILI are not fully investigated. Our previous study showed that AILI could result in dose-dependent and immediate decreases in expression and activities of major hepatic drug-metabolizing cytochrome P450s (P450s) enzymes in mice. However, liver will go through regeneration after AILI. How P450-mediated drug metabolism alters during liver regeneration is unclear. The aim of the study was to investigate the alterations in expression and activities of major P450 enzymes and associated drug efficacy and ADRs during liver regeneration after AILI. Adult male C57BL6/J mice were treated with various doses of APAP at 0 (no damage control), 200 (mild damage and recoverable), 400 (severe damage but recoverable), and 600 mg/kg (life-threatening damage and unrecoverable). The mice were sacrificed at 24, 48, 72, and 96 hours after the APAP treatment. Severity of liver damage was detected by plasma levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and miR122, with further confirmation by H & E staining in liver tissue sections. The expression and activities of CYP1A2, 2B10, 2C29, 2E1, and 3A11 were measured by RT-PCR (mRNAs) and MS/MS quantification of probe drugs, respectively. Sedation efficacy of midazolam was monitored at different time points after APAP treatment at different doses. The levels of ALT and AST significantly increased at 24 hours after APAP treatment at 200 and 400 mg/kg but dropped back to normal levels at 72 and 96 hours. No mice treated with 600 mg/kg APAP could survive to 72 hours. The magnitude of liver damage was further confirmed by H & E staining. The major examined P450 enzymes showed significant decreases at 24 hours in gene expression and activity levels and recovered back to normal with the doses of 200 and 400, but not 600 mg/kg of APAP. The activities of examined major P450s are correlated with the levels of ALT, AST, and mir122. The alterations of CYP3A11 resulted in correlated changes in sedation efficacy as reflected by sleeping times with midazolam treatment. These results indicated that P450 expression and function could recover after mild and even severe, but not lethal AILI. Overall, these results suggest that ALT, AST, and miR122 are potential biomarkers for predicting how alterations in P450 activities can justify dose selection of other drugs for desired efficacy. Further studies are needed to confirm these findings in human patient populations with AILI and to understand the underlying molecular mechanisms mediating recovery of hepatic P450s during AILI.

Photocatalytic activity of hybrid P450 Enzymes

The Cytochromes P450 are heme-thiolate enzymes that carry out selective oxyfunctionalization of a wide range of substrates using molecular dioxygen and two reducing equivalents provided by redox partner enzymes. Alternative approaches to activate P450 enzymes have received recent attention in an attempt to circumvent the use of redox partners and cofactors. In particular, the use of light-harvesting units has proven useful in triggering P450 activity upon visible light-excitation. Our laboratory has taken advantage of the inorganic Ru(II)-diimine photosensitizers and their unique excited properties to harness P450 synthetic potential. Their covalent attachment to non-native single cysteine residues has enabled rapid electron injection into the heme domain of several P450 enzymes leading to high photocatalytic activity and coupling efficiency. Recently, we also developed a bimolecular system to activate P450 with peroxygenase activity. The crystal structure of the most efficient hybrid enzyme reveals that the photosensitizer is ideally positioned to deliver electrons to the heme active site utilizing the natural electron transfer pathway. A combination of rational and directed evolution approaches are currently employed to optimize the biocatalyst photocatalytic activity and expand the scope of the light-driven P450 activity. In addition, the tunability and derivatization of the inorganic complexes have promoted the formation of dimeric structures and heterogeneous biocatalysts and provided insights into the rate-limiting steps in the light-driven hybrid P450 enzyme.

Metabolic study of ginsenoside Rg3 and glimepiride in type 2 diabetic rats by liquid chromatography coupled with quadrupole‐Orbitrap mass spectrometry

RationaleGinsenoside Rg3 and glimepiride have been applied to treat type 2 diabetes (T2DM) because of their good hypoglycemic effects. In this study, the effects of ginsenoside Rg3 acting synergistically with glimepiride were investigated in liver microsomes from rats with type 2 diabetes.MethodsAn in vitro incubation system with normal rat liver microsomes (RLM) and type 2 diabetic rat liver microsomes (TRLM) was developed. The system also included two experimental groups consisting of RLM and TRLM pretreated with ginsenoside Rg3 and glimepiride (named the RLMR and TRLMR groups, respectively). The metabolism in the different groups was analyzed by ultra‐performance liquid chromatography coupled with quadrupole‐orbitrap mass spectrometry (UPLC/Q‐Orbitrap MS).ResultsThe results showed that the concentration of glimepiride increased in RLM and TRLM after treatment with ginsenoside Rg3. Five metabolites (M1–M5) of glimepiride were found, and they were named 3N‐hydroxyglimepiride, hydroxyglimepiride, 1,2‐epoxy ether‐3‐hydroxyglimepiride, 1N‐hydroxyglimepiride and 1N,2C,S,O,O‐epoxy ether‐3‐hydroxyglimepiride. The metabolite of ginsenoside Rg3 was ginsenoside Rh2.ConclusionsAn in vitro incubation system with RLM and TRLM was developed. The system revealed pathways that produce glimepiride metabolites. Ginsenoside Rg3 may inhibit the activity of cytochrome P450 enzymes in vitro. The present study showed that ginsenoside Rg3 and glimepiride may be combined for the treatment of T2DM.

Sertraline concentrations in pregnant women are steady and the drug transfer to their infants is low

PurposeSertraline, a selective serotonin reuptake inhibitor (SSRI), is one of the most commonly used antidepressant during pregnancy. Plasma sertraline concentrations vary markedly between individuals, partly explained by variability in hepatic drug metabolizing cytochrome P450-enzyme activity. Our purpose was to study the variability in the plasma concentrations in pregnant women and the passage to their infants.MethodPregnant women with moderate untreated depression were recruited in 2016–2019 in Stockholm Region and randomized to treatment with sertraline or placebo. All received Internet-based cognitive behavior therapy as non-medical treatment. Sertraline plasma concentrations were measured around pregnancy weeks 21 and 30, at delivery, 1-month postpartum, in cord blood and at 48 h of age in the infant. The clinical course of the infants was followed.ResultsNine mothers and 7 infants were included in the analysis. Median dose-adjusted sertraline concentration in second trimester was 0.15(ng/mL) /(mg/day), in third trimester and at delivery 0.19 and 1-month postpartum 0.25, with a 67% relative difference between second trimester and postpartum. The interindividual variation was 10-fold. Median concentrations in the infants were 33% and 25% of their mothers’, measured in cord blood, and infant plasma, respectively. Only mild and transient adverse effects were seen on the infants.ConclusionPlacental passage of sertraline to the infant is low. However, the interindividual variation in maternal concentrations during pregnancy is huge, why therapeutic drug monitoring might assist in finding the poor metabolizers at risk for adversity and increase the safety of the treatment.Trial registrationThe trial was registered at clinicaltrials.gov July 9, 2014 with TRN: NCT02185547.

Exposure of Helicoverpa armigera Larvae to Plant Volatile Organic Compounds Induces Cytochrome P450 Monooxygenases and Enhances Larval Tolerance to the Insecticide Methomyl.

Simple SummaryUpon insect herbivory plants release herbivore-induced plant volatiles (HIPVs) to elicit either direct antiherbivore defenses by intoxicating the herbivore or indirect defenses by attracting natural enemies. Insect herbivores use sophisticated detoxification systems, including cytochrome P450 monooxygenases (P450s), to cope with plant defensive chemicals and insecticides. Many behaviors of insects are manipulated to a large extent by olfaction. However, few studies concern the role of olfaction in insect adaptation to xenobiotics. In this study we show that olfactory exposure of Helicoverpa armigera, a devastating pest of many important crop plants that has developed high levels of resistance to various insecticides, to four plant volatile compounds limonene, nerolidol, 2-heptanone and cis-3-hexenyl acetate dramatically increases larval tolerance to carbamate insecticide methomyl. Inhibition of P450s by piperonyl butoxide (PBO), a well-known P450 inhibitor, compromises the effects of volatile exposure. Exposure to the four volatiles upregulates multiple detoxification systems, including cytochrome P450s. Our findings indicate that induction of insecticide-detoxifying P450s by plant volatiles prior to actually inflicting plant damage may confer insect larvae more time to pre-activate insect detoxification systems prior to the ingestion or contact of insecticides, leading to increased larval tolerance to insecticides.Plants release an array of volatile chemicals into the air to communicate with other organisms in the environment. Insect attack triggers emission of herbivore-induced plant volatiles (HIPVs). How insect herbivores use these odors to plan their detoxification systems is vital for insect adaptation to environmental xenobiotics. Here we show that the larvae of Helicoverpa armigera (Hübner), a broadly polyphagous lepidopteran herbivore, have the capacity to use plant volatiles as cues to upregulate multiple detoxification systems, including cytochrome P450 monooxygenases (P450s), for detoxification of insecticides. Olfactory exposure of the fifth instars to two terpene volatiles limonene and nerolidol, and two green-leaf volatiles 2-heptanone and cis-3-hexenyl acetate significantly reduced larval susceptibility to the insecticide methomyl. However, larval pretreatment with piperonyl butoxide (PBO), a known P450 inhibitor, neutralized the effects of volatile exposure. Furthermore, larval exposure to the four plant volatiles enhanced activities of P450 enzymes in midguts and fatbodies, and upregulated expression of CYP6B2, CYP6B6 and CYP6B7, P450s involved in detoxification of the insecticide. Larval exposure to 2-heptanone and limonene volatiles also enhanced activities of glutathione-s-transferase and carboxylesterase. Our findings suggest that olfactory exposure to HIPVs enhances larval insecticide tolerance via induction of detoxification P450s.

Sodium selenite-carbon dots nanocomposites enhance acaricidal activity of fenpropathrin: Mechanism and application.

As an essential trace element, selenium can be used to protect crops from pests, while, in nature, most crops cannot accumulate enough selenium from the soil to reach the effective dose for pest control. In this study, carbon dots modified with arginine in nano-scale was prepared and characterized, then, it was combined with sodium selenite to form selenium-carbon dots (Se-CDs). Function evaluation of Se-CDs showed that it could increase the absorption of selenium in plant leaves, promote the control efficiency of fenpropathrin, and protect plant from damage caused by Tetranychus cinnabarinus. In addition, we found that expressions of P450 genes and activity of P450 enzyme both decreased in selenium treated mites. In vivo, the acaricidal activity of fenpropathrin increased significantly when one of the P450 genes, CYP389B1, was silenced, and the recombinant protein of CYP389B1 could metabolize fenpropathrin in vitro. The results suggested that inhibiting the expression of P450 gene and repressing the detoxification of T. cinnabarinus was the molecular mechanism that how selenium promoted the acaricidal activity of fenpropathrin. The application of Se-CDs in the field will decrease the use of chemicals acaricides, reduce chemical residues, and ensure the safety of agricultural products.

Functional Analysis of Induced Human Ballooned Hepatocytes in a Cell Sheet-Based Three Dimensional Model.

Ballooned hepatocytes (BH) are akey histological hallmark of nonalcoholic steatohepatitis (NASH), yet their consequences for liver-specific functions are unknown. In our previous study, an experimental model of human induced-BHs (iBH) has been successfully developed based on cell sheet technology. This study aimed to determine the functions of iBHs in the primary human hepatocyte/normal human dermal fibroblast (PHH/NHDF) co-culture cell sheets. Normal hepatocytes in the PHH/3T3-J2 co-culture cell sheets were set as a control, since 3T3-J2 murine embryonic fibroblasts have exhibited previously long term maintenance of PHH functions. It was found that, albumin secretion was not affected in iBHs, but urea synthesis as well as cytochrome P450 enzyme (CYP) activities including CYP1A2 and CYP3A4, were significantly reduced in iBHs. Besides, loss of bile canaliculi was observed in iBHs. These findings are consistent with clinical studies of human NASH. In addition, PHH/NHDF cell sheets demonstrated twofold higher TGF-β1 secretion compared with PHH/3T3-J2 cell sheets. Furthermore, treatment with a TGF-β inhibitor and a semi-synthetic bile acid analogue (obeticholic acid, phase 3 trial of NASH therapy) ameliorated the histological appearance of established iBHs. In summary, this study demonstrates the priority of iBHs in recapitulating not only histology but also clinically relevant hepatic dysfunctions in human NASH and suggests TGF-β and bile acid related signal pathway may play important roles in the formation of iBHs.

Human Family 1-4 cytochrome P450 enzymes involved in the metabolic activation of xenobiotic and physiological chemicals: an update.

This is an overview of the metabolic activation of drugs, natural products, physiological compounds, and general chemicals by the catalytic activity of cytochrome P450 enzymes belonging to Families 1-4. The data were collected from > 5152 references. The total number of data entries of reactions catalyzed by P450s Families 1-4 was 7696 of which 1121 (~ 15%) were defined as bioactivation reactions of different degrees. The data were divided into groups of General Chemicals, Drugs, Natural Products, and Physiological Compounds, presented in tabular form. The metabolism and bioactivation of selected examples of each group are discussed. In most of the cases, the metabolites are directly toxic chemicals reacting with cell macromolecules, but in some cases the metabolites formed are not direct toxicants but participate as substrates in succeeding metabolic reactions (e.g., conjugation reactions), the products of which are final toxicants. We identified a high level of activation for three groups of compounds (General Chemicals, Drugs, and Natural Products) yielding activated metabolites and the generally low participation of Physiological Compounds in bioactivation reactions. In the group of General Chemicals, P450 enzymes 1A1, 1A2, and 1B1 dominate in the formation of activated metabolites.Drugs are mostly activated by the enzyme P450 3A4, and Natural Products by P450s 1A2, 2E1, and 3A4. Physiological Compounds showed no clearly dominant enzyme, but the highest numbers of activations are attributed to P450 1A, 1B1, and 3A enzymes. The results thus show, perhaps not surprisingly, that Physiological Compounds are infrequent substrates in bioactivation reactions catalyzed by P450 enzyme Families 1-4, with the exception of estrogens and arachidonic acid. The results thus provide information on the enzymes that activate specific groups of chemicals to toxic metabolites.

Evaluation of drug interaction potential of zanubrutinib with cocktail probes representative of CYP3A4, CYP2C9, CYP2C19, P-gp and BCRP.

AimThis study aims to assess the potential effects of zanubrutinib on the activity of cytochrome P450 (CYP) enzymes and drug transporter proteins using a cocktail probe approach.MethodsPatients received single oral doses of probe drugs alone and after at least 8 days of treatment with zanubrutinib 160 mg twice daily in a single‐sequence study in 18 healthy male volunteers. Simultaneous doses of 10 mg warfarin (CYP2C9) and 2 mg midazolam (CYP3A) were administered on Day 1 and Day 14, 0.25 mg digoxin (P‐glycoprotein [P‐gp]) and 10 mg rosuvastatin (breast cancer resistance protein [BCRP]) on Day 3 and Day 16, and 20 mg omeprazole (CYP2C19) on Day 5 and Day 18. Pharmacokinetic (PK) parameters were estimated from samples obtained up to 12 h post dose for zanubrutinib; 24 h for digoxin, omeprazole and midazolam; 48 h for rosuvastatin; and 144 h for warfarin.ResultsThe ratios (%) of geometric least squares means (90% confidence intervals) for the area under the concentration–time curve from time zero to the last quantifiable concentration in the presence/absence of zanubrutinib were 99.80% (97.41–102.2%) for S‐warfarin; 52.52% (48.49–56.88%) for midazolam; 111.3% (103.8–119.3%) for digoxin; 89.45% (78.73–101.6%) for rosuvastatin; and 63.52% (57.40–70.30%) for omeprazole. Similar effects were observed for maximum plasma concentrations.ConclusionsZanubrutinib 320 mg total daily dose had minimal or no effect on the activity of CYP2C9, BCRP and P‐gp, but decreased the systemic exposure of CYP3A and CYP2C19 substrates (mean reduction <50%).

Predominance of Metabolic Resistance in a Six-Way-Resistant Palmer Amaranth (Amaranthus palmeri) Population.

Evolution of multiple herbicide resistance in Palmer amaranth across the United States is a serious challenge for its management. Recently, a Palmer amaranth population (KCTR; Kansas Conservation Tillage Resistant) from a long-term conservation tillage research project in Kansas, United States, was found uncontrolled by several commonly used herbicides. Importantly, this field did not have a history of repeated use of some of the herbicides for which the KCTR Palmer amaranth population showed lack of control. The objectives of this study were to confirm the evolution of multiple resistances and determine possible mechanism(s) of resistance in KCTR Palmer amaranth plants. In response to post-emergence application, 28–100% of KCTR Palmer amaranth survived field recommended rates of 2,4-D, ALS-, PS II-, EPSPS-, PPO-, HPPD-inhibitor herbicides, or tank- or pre-mixture of PS II- and HPPD-inhibitor herbicides, confirming evolution of six-way resistance in this Palmer amaranth population. However, this population was found susceptible to the PS I- and glutamine synthetase inhibitor herbicides. Chlorsulfuron-, imazethapyr-, and atrazine-resistant plants did not show any previously reported mutation in ALS and psbA genes, the target sites of these herbicides, respectively. However, the survivors of glyphosate treatment showed amplification of EPSPS gene (up to 88 copies). The KCTR plants pretreated with cytochrome P450 or GST inhibitors along with atrazine, 2,4-D, lactofen, or mesotrione had significantly less biomass accumulation than those treated with herbicides alone. Plants treated with P450 inhibitor followed by imazethapyr showed moderate reduction of biomass in KCTR which was statistically similar to a susceptible Palmer amaranth population treated with imazethapyr. These results suggest predominance of metabolic resistance possibly mediated by cytochrome P450 and GST enzyme activity that may have predisposed the KCTR Palmer amaranth population to evolve resistance to multiple herbicides. This is the first report of evolution of six-way resistance in a single Palmer amaranth population. Appropriate management strategies, including integration of cultural, and mechanical, and herbicide mixtures, are warranted to control such Palmer amaranth populations.

Verification of a cocktail approach for quantitative drug–drug interaction assessment: a comparative analysis between the results of a single drug and a cocktail drug

A cocktail approach is a method to comprehensively evaluate the activity of cytochrome P450 enzymes (CYPs) by co-administering multiple CYP substrates. This is the first report that compares the results from a cocktail study to a single substrate separate administration study (single study) with concomitant administration of CYP inducers/inhibitors. The validity of a cocktail study for use as a quantitative drug-drug interactions (DDIs) assessment was evaluated. We administered a cocktail drug (caffeine, losartan, omeprazole, dextromethorphan, midazolam) with rifampicin, cimetidine or fluvoxamine. A comparative analysis was performed between the results of a cocktail study and single studies. The results of single studies were obtained from a literature review and the trials of single substrate separate administration. A strong positive correlation of the AUC ratio of all drugs between single studies and the cocktail study was obtained. The ratio of AUC change of 12 combinations converged to 0.82–1.09, and 2 combinations ranged between 0.74–1.32. The differences in the degree of interaction between the single studies and cocktail study are acceptable to evaluate DDIs for almost all combinations. Our results indicate that a cocktail study is an adequate and quantitative evaluation method for DDIs.

Anthraquinones inhibit cytochromes P450 enzyme activity in silico and in vitro.

Anthraquinones exhibit various pharmacological activities (e.g., antioxidant and laxative) and are commonly found in consumer products including foods, dietary supplements, drugs, and traditional medicines. Despite their widespread use, there are limited data available on their toxicokinetic properties. Cytochrome P450 enzymes (CYPs) in the liver play major roles in metabolizing exogenous chemicals (e.g., pharmaceuticals, food ingredients, and environmental pollutants) and endogenous biomolecules (e.g., steroid hormones and cholesterol). Inhibition of CYP activities may lead to serious interactions among these compounds. Here, in silico (quantitative structure-activity relationship modeling) and in vitro (human recombinant enzymes and liver microsomes) methods were used to identify inhibitors of five major CYP isoforms (CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4) among 22 anthraquinones. First, in silico prediction and in vitro human recombinant enzyme assays were conducted for all compounds, and results showed that most of the anthraquinones were potent CYP1A2 inhibitors. Second, five selected anthraquinones (emodin, aloe-emodin, rhein, purpurin, and rubiadin) were further evaluated in human liver microsomes. Finally, plasma concentrations of the five anthraquinones in animal and humans were identified in the literature and compared to their in vitro inhibition potency (IC50 values) towards CYP activities. Emodin, rhein, and aloe-emodin inhibited activities of multiple CYPs in human liver microsomes and potential in vivo inhibition may occur due to their high plasma concentrations. These in silico and in vitro results enabled rapid identification of potential CYP inhibitors and prioritized future in-depth studies.

Inhibition and Induction by Poziotinib of Different Rat Cytochrome P450 Enzymes In Vivo and in an In Vitro Cocktail Method.

Poziotinib is an orally active, irreversible, pan-HER tyrosine kinase inhibitor used to treat non-small cell lung cancer, breast cancer, and gastric cancer. Poziotinib is currently under clinical investigation, and understanding its drug-drug interactions is extremely important for its future development and clinical application. The cocktail method is most suitable for evaluating the activity of cytochrome P450 enzymes (CYPs). As poziotinib is partially metabolized by CYPs, cocktail probes are used to study the interaction between drugs metabolized by each CYP subtype. Midazolam, bupropion, dextromethorphan, tolbutamide, chlorzoxazone, phenacetin, and their metabolites were used to examine the effects of poziotinib on the activity of cyp1a2, 2b1, 2d1, 2c11, 2e1, and 3a1/2, respectively. The in vitro experiment was carried out by using rat liver microsomes (RLMs), whereas the in vivo experiment involved the comparison of the pharmacokinetic parameters of the probes after co-administration with poziotinib to rats to those of control rats treated with only probes. UPLC-MS/MS was used to detect the probes and their metabolites in rat plasma and rat liver microsomes. The in vitro results revealed that the half-maximal inhibitory concentration values of bupropion and tolbutamide in RLMs were 8.79 and 20.17 μM, respectively, indicating that poziotinib showed varying degrees of inhibition toward cyp2b1 and cyp2c11. Poziotinib was a competitive inhibitor of cyp2b1 and cyp2c11, with Ki values of 16.18 and 17.66 μM, respectively. No time- or concentration-dependence of inhibition by poziotinib was observed toward cyp2b1 and cyp2c11 in RLMs. Additionally, no obvious inhibitory effects were observed on the activity of cyp1a2, cyp2d1, cyp2e1, and cyp3a1/2. In vivo analysis revealed that bupropion, tolbutamide, phenacetin, and chlorzoxazone showed significantly different pharmacokinetic parameters after administration (p < 0.05); there was no significant difference in the pharmacokinetic parameters of dextromethorphan and midazolam. These results show that poziotinib inhibited cyp2b1 and cyp2c11, but induced cyp1a2 and cyp2e1 in rats. Thus, poziotinib inhibited cyp2b1 and cyp2c11 activity in rats, suggesting the possibility of interactions between poziotinib and these CYP substrates and the need for caution when combining them in clinical settings.

Effects of dapagliflozin in combination with insulin on cytochrome P450 activities in a diabetes type 1 rat model.

Previous studies reported that diabetes alters the activities of hepatic cytochrome P450 (CYP) enzymes, which, in turn, affects the disposition of some drugs. We herein examined and compared the effects of the combination of dapagliflozin with a low insulin dose, a full dose of insulin alone, and dapagliflozin alone for 3 and 8 weeks on CYP activities in a diabetes type 1 rat model. We induced type 1 diabetes in rats using a single intraperitoneal injection of 60 mg/kg streptozotocin (STZ). Daily treatment with the full dose of insulin alone, dapagliflozin alone, or dapagliflozin in combination with a low dose of insulin was then initiated. STZ-induced rats developed marked hyperglycemia and altered CYP2E activities. Dapagliflozin in combination with a low dose of insulin stabilized hyperglycemia and CYP1A, 2D, 2E and 3A activities. However, dapagliflozin alone did not improve blood glucose levels or CYP activities. These results suggest that the effects of dapagliflozin in combination with a low dose of insulin are similar to those of a full dose of insulin, and stabilize CYP activities in type 1 diabetes.

Effect of resveratrol, carnitin, quercetin and aromatic amino acids on the xenobiotic metabolising and antioxidant enzymes in the liver during obesity in rats with different genotypes

The use of minor biologically active substances (BAS) in specialized products is one of the promising areas in the diet therapy for obesity and other alimentary-dependent diseases (metabolic syndrome, type 2 diabetes mellitus, etc.). The effects of the BAS in patients are often ambiguous, depending on a number of factors, one of which is the state of the organism enzyme systems (enzymes of xenobiotic metabolism and antioxidant defense), the patient's genotype, and many others. The aim was to study the effect of BAS [quercetin (Q), L-carnitine (L-Car), resveratrol (Res), aromatic amino acids tyrosine (Tyr) and tryptophan (Trp)] on the activities of phase I and II of xenobiotic-metabolising enzymes and antioxidant enzymes in rats using various in vivo models of obesity and with impaired dopamine transport. Material and methods. The activities of cytochrome P450 enzymes (CYP1A1 and CYP3A), glutathione transferase (GT), UDPglucuronosyltransferase (UDP-GT), hemoxygenase-1 (HO-1), and quinone reductase (QR) were determined by fluorimetric, spectrophotometric methods and HPLC in microsomes and cytosolic fraction of rat's liver. We used rats of outbred Wistar line, Zucker ZF line with hereditarily determined obesity and DAT-KO line with a knockout of the DAT dopamine transporter gene, which for 62 days were fed a standard balanced control or high-carbohydrate high-fat diet (30% fat by weight and 20% fructose solution instead of water) supplemented with BAS, such as Q, Res, L-Car, Tyr and Trp in doses 50, 25, 300, 1250 and 250 mg/kg of body weight respectively. Results and discussion. The presence of a DAT knockout led to a small but statistically significant decrease in the activity of GT in the liver in both homozygous and heterozygous animals. The CYP1A1 activity was significantly decreased in all carriers of the DAT knockout gene, while HO-1 activity, on the contrary, was increased, independently of the composition of the diet used. In Zucker ZF rats of all groups, in comparison with Wistar rats fed the corresponding diets, the activities of GT, UDP-GT, CYP1A1, CYP3A and QR were significantly reduced in terms of the total protein content. HO-1 activity was reduced in Zucker ZF rats in comparison with Wistar rats to a lesser extent, however, the addition of Q significantly influenced the difference between the two lines. Trp consumption led to a significant increase in GT activity in Wistar rats. In DAT-KO homozygotes this effect was insignificant, while in heterozygotes it was absent. Similarly, consumption of Trp resulted in a significant increase in CYP1A1 activity only in Wistar rats, but not in DAT-KO rats. The activity of UDP-GT under Trp intake increased only in DAT heterozygotes. The genotype significantly influenced the response of QR activity to Trp consumption, but in an ambiguous way - there was an increase in activity in heterozygotes and a decrease in homozygotes for DAT knockout. CYP1A1 activity was significantly increased in rats treated with Tyr. Conclusion. The data obtained indicate that the effect of various dietary supplements used in the treatment of obesity and metabolic syndrome on the xenobiotic-metabolising enzymes and antioxidant enzymes can have a different nature and direction depending on the genotype and the level of spontaneous physical activity and energy expenditure determined by it, which should be taken into account when approaches to personalized diet therapy of alimentary-dependent diseases are developing.

Pogostone inhibits the activity of CYP3A4, 2C9, and 2E1 in vitro

Context Pogostone possesses various pharmacological activities, which makes it widely used in the clinic. Its effect on the activity of cytochrome P450 enzymes (CYP450s) could guide its clinical combination. Objective To investigate the effect of pogostone on the activity of human CYP450s. Materials and methods The effect of pogostone on the activity of CYP450s was evaluated in human liver microsomes (HLMs) compared with blank HLMs (negative control) and specific inhibitors (positive control). The corresponding parameters were obtained with 0–100 μM pogostone and various concentrations of substrates. Results Pogostone was found to inhibit the activity of CYP3A4, 2C9, and 2E1 with the IC50 values of 11.41, 12.11, and 14.90 μM, respectively. The inhibition of CYP3A4 by pogostone was revealed to be performed in a non-competitive and time-dependent manner with the Ki value of 5.69 μM and the KI/Kinact value of 5.86/0.056/(μM/min). For the inhibition of CYP2C9 and 2E1, pogostone acted as a competitive inhibitor with the Ki value of 6.46 and 7.67 μM and was not affected by the incubation time. Discussion and conclusions The inhibitory effect of pogostone on the activity of CYP3A4, 2C9, and 2E1 has been disclosed in this study, implying the potential risk during the co-administration of pogostone and drugs metabolized by these CYP450s. The study design provides a reference for further in vivo investigations to validate the potential interaction.

In vitro study on the effect of peucedanol on the activity of cytochrome P450 enzymes

Context Peucedanol is a major extract of Peucedanum japonicum Thunb. (Apiaceae) roots, which is a commonly used herb in paediatrics. Its interaction with cytochrome P450 enzymes (CYP450s) would lead to adverse effects or even failure of therapy. Objective The interaction between peucedanol and CYP450s was investigated. Materials and methods Peucedanol (0, 2.5, 5, 10, 25, 50, and 100 μM) was incubated with eight human liver CYP isoforms (CYP1A2, 2A6, 3A4, 2C8, 2C9, 2C19, 2D6, and 2E1), in pooled human liver microsomes (HLMs) for 30 min with specific inhibitors as positive controls and untreated HLMs as negative controls. The enzyme kinetics and time-dependent study (0, 5, 10, 15, and 30 min) were performed to obtain corresponding parameters in vitro. Results Peucedanol significantly inhibited the activity of CYP1A2, 2D6, and 3A4 in a dose-dependent manner with IC50 values of 6.03, 13.57, and 7.58 μM, respectively. Peucedanol served as a non-competitive inhibitor of CYP3A4 with a Ki value of 4.07 μM and a competitive inhibitor of CYP1A2 and 2D6 with a Ki values of 3.39 and 6.77 μM, respectively. Moreover, the inhibition of CYP3A4 was time-dependent with the Ki /Kinact value of 5.44/0.046 min/μM. Discussion and conclusions In vitro inhibitory effect of peucedanol on the activity of CYP1A2, 2A6, and 3A4 was reported in this study. As these CYPs are involved in the metabolism of various drugs, these results implied potential drug-drug interactions between peucedanol and drugs metabolized by CYP1A2, 2D6, and 3A4, which needs further in vivo validation.

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.

Spectroscopic studies of the interaction between phosphorus heterocycles and cytochrome P450

P450 fatty acid decarboxylase OleT from Staphylococcus aureus (OleTSA) is a novel cytochrome P450 enzyme that catalyzes the oxidative decarboxylation of fatty acids to yield primarily terminal alkenes and CO2 or minor α- and β-hydroxylated fatty acids as side-products. In this work, the interactions between a series of cycloalkyl phosphorus heterocycles (CPHs) and OleTSA were investigated in detail by fluorescence titration experiment, ultraviolet–visible (UV–vis) and 31P NMR spectroscopies. Fluorescence titration experiment results clearly showed that a dynamic quenching occurred when CPH-6, a representative CPHs, interacted with OleTSA with a binding constant value of 15.2 × 104 M−1 at 293 K. The thermodynamic parameters (ΔH, ΔS and ΔG) showed that the hydrogen bond and van der Waals force played major roles in the interaction between OleTSA and CPHs. The UV–vis and 31P NMR studies indicated the penetration of CPH-6 into the interior environment of OleTSA, which greatly affects the enzymatic activity of OleTSA. Therefore, our study revealed an effective way to use phosphorus heterocyclic compounds to modulate the activity of cytochrome P450 enzymes.