Cytochrome P450 Enzyme Activity Research Articles

Effects of catalpol on the activity of human liver cytochrome P450 enzymes

1. Catalpol possesses numerous pharmacological activities, and however, little data available for the effects of catalpol on the activity of human liver cytochrome P450 (CYP) enzymes.2. This study investigates the inhibitory effects of catalpol on the main human liver CYP isoforms. In this study, the inhibitory effects of catalpol on the eight human liver CYP isoforms 1A2, 2A6, 2E1, 2D6, 2C9, 2C19, 2C8 and 3A4 were investigated in human liver microsomes.3. The results indicated that catalpol could inhibit the activity of CYP3A4, CYP2E1 and CYP2C9, with IC50 values of 14.27, 22.4 and 14.69 μM, respectively, but those other CYP isoforms were not affected. Enzyme kinetic studies showed that catalpol was not only a noncompetitive inhibitor of CYP3A4, but also a competitive inhibitor of CYP2E1 and CYP2C9, with Ki values of 7.40, 10.75 and 7.37 μM, respectively. In addition, catalpol is a time-dependent inhibitor for CYP3A4, with maximum inactivation (kinact) and 50% maximum inactivation (KI) values of 0.02 min−1 and 1.86 μM, respectively.4. The in vitro studies of catalpol with CYP isoforms suggest that catalpol has the potential to cause pharmacokinetic drug interactions with other co-administered drugs metabolized by CYP3A4, CYP2E1 and CYP2C9. Further in vivo studies are needed in order to evaluate the significance of this interaction.

Pharmacokinetics behaviors of l-menthol after inhalation and intravenous injection in rats and its inhibition effects on CYP450 enzymes in rat liver microsomes

1. l-Menthol, as a kind of monocyclic terpene, is widely used in inhalation formulations, food and tobacco. The purpose of this study was to investigate the pharmacokinetic behavior of l-menthol as well as its influence on the activities of cytochrome P450 enzymes.2. The pharmacokinetic behaviors of l-menthol after inhalation (50 mg/kg) and intravenous injection (10 mg/kg) were investigated. A rat liver microsomal model was adopted to elucidate the inhibitory effect of l-menthol on CYP1A2, CYP2C11, CYP2D1/2, CYP2D4, CYP2E1 and CYP3A1 using phenacetin, tolbutamide, omeprazole, dextromethorphan, chlorzoxazone and testosterone as probe drugs, respectively.3. The plasma concentration reached the Cmax within 1.0 h (inhalation) and descended with the T1/2 of 8.53 and 6.69 h for inhalation and i.v. administration, respectively. IC50 for inhibition of l-menthol on CYP 450 enzymes were 4.35 μM for 2D4, 8.67 μM for 1A2, 13.02 μM for 3A1, 14.78 μM for 2D1/2, 234.9 μM for 2C11 and 525.4 μM for 2E1, respectively.4. The results illustrate the pharmacokinetic process of l-menthol in rats and provide information for further rational applications. l-Menthol had moderate inhibitions on CYP2D4 and 1A2, which might affect the disposition of medicines primarily dependent on these pathways.

In vitro inhibitory effects of sophocarpine on human liver cytochrome P450 enzymes

1. Sophocarpine is a biologically active component isolated from the foxtail-like sophora herb and seed that is often orally administered for the treatment of cancer and chronic bronchial asthma. However, whether sophocarpine affects the activity of human liver cytochrome P450 (CYP) enzymes remains unclear.2. In this study, the inhibitory effects of sophocarpine on the eight human liver CYP isoforms (CYP1A2, 3A4, 2A6, 2E1, 2D6, 2C9, 2C19, and 2C8) were investigated in vitro using human liver microsomes (HLMs).3. The results indicate that sophocarpine could inhibit the activity of CYP3A4 and 2C9, with the IC50 values of 12.22 and 15.96 μM, respectively, but that other CYP isoforms were not affected. Enzyme kinetic studies showed that sophocarpine is not only a noncompetitive inhibitor of CYP3A4 but also a competitive inhibitor of CYP2C9, with Ki values of 6.74 and 9.19 μM, respectively. Also, sophocarpine is a time-dependent inhibitor of CYP3A4 with Kinact/KI value of 0.082/21.54 μM−1 min−1.4. The in vitro studies of sophocarpine with CYP isoforms suggested that sophocarpine has the potential to cause pharmacokinetic drug interactions with other co-administered drugs metabolized by CYP3A4 and 2C9. Further clinical studies are needed to evaluate the significance of this interaction.

Validation of in vitro methods for human cytochrome P450 enzyme induction: Outcome of a multi-laboratory study

CYP enzyme induction is a sensitive biomarker for phenotypic metabolic competence of in vitro test systems; it is a key event associated with thyroid disruption, and a biomarker for toxicologically relevant nuclear receptor-mediated pathways. This paper summarises the results of a multi-laboratory validation study of two in vitro methods that assess the potential of chemicals to induce cytochrome P450 (CYP) enzyme activity, in particular CYP1A2, CYP2B6, and CYP3A4. The methods are based on the use of cryopreserved primary human hepatocytes (PHH) and human HepaRG cells.The validation study was coordinated by the European Union Reference Laboratory for Alternatives to Animal Testing of the European Commission's Joint Research Centre and involved a ring trial among six laboratories. The reproducibility was assessed within and between laboratories using a validation set of 13 selected chemicals (known human inducers and non-inducers) tested under blind conditions. The ability of the two methods to predict human CYP induction potential was assessed. Chemical space analysis confirmed that the selected chemicals are broadly representative of a diverse range of chemicals.The two methods were found to be reliable and relevant in vitro tools for the assessment of human CYP induction, with the HepaRG method being better suited for routine testing. Recommendations for the practical application of the two methods are proposed.

Transcriptional Repression of CYP3A4 by Increased miR-200a-3p and miR-150-5p Promotes Steatosis in vitro.

Hepatic cytochrome P450 enzyme activities correlate with non-alcoholic fatty liver disease (NAFLD) and hepatic steatosis. The decreased activity of CYP3A4, an important drug-metabolizing enzyme, is associated with the progression of NAFLD. CYP3A4 is predicted as a target gene of miR-200a-3p and miR-150-5p by MicroInspector and TargetScan algorithms analyses. Here, we found decreased CYP3A4 and increased miR-200a-3p and miR-150-5p in LO2 cells with free fatty acid (FFA)-induced steatosis. Dual-luciferase assay confirmed that both miR-200a-3p and miR-150-5p targeted the 3′-untranslated region (3′-UTR) of CYP3A4 and that such interaction was abolished by miRNA binding site mutations in 3′-UTR of CYP3A4. Using miR-200a-3p and miR-150-5p mimics and inhibitors, we further confirmed that endogenous CYP3A4 was regulated posttranscriptionally by miR-200a-3p or miR-150-5p. Moreover, miR-200a-3p and miR-150-5p inhibitors attenuated FFA-induced steatosis in LO2 cells, and such effect was dependent on CYP3Y4 expression. These results suggest that miR-200a-3p and miR-150-5p, through directly targeting 3′-UTR of CYP3A4, contribute to the development of FFA-induced steatosis.

Variability in human drug metabolizing cytochrome P450 CYP2C9, CYP2C19 and CYP3A5 activities caused by genetic variations in cytochrome P450 oxidoreductase

A broad spectrum of human diseases are caused by mutations in the NADPH cytochrome P450 oxidoreductase (POR). Cytochrome P450 proteins perform several reactions, including the metabolism of steroids, drugs, and other xenobiotics. In 2004 the first human patients with defects in POR were reported, and over 250 variations in POR are known. Information about the effects of POR variants on drug metabolizing enzymes is limited and has not received much attention. By analyzing the POR sequences from genomics databases, we identified potentially disease-causing variations and characterized these by in vitro functional studies using recombinant proteins. Proteins were expressed in bacteria and purified for activity assays. Activities of cytochrome P450 enzymes were tested in vitro using liposomes prepared with lipids into which P450 and P450 reductase proteins were embedded. Here we are reporting the effect of POR variants on drug metabolizing enzymes CYP2C9, CYP2C19, and CYP3A5 which are responsible for the metabolism of many drugs. POR Variants A115V, T142A, A281T, P284L, A287P, and Y607C inhibited activities of all P450 proteins tested. Interestingly, the POR variant Q153R showed a reduction of 20–50% activities with CYP2C9 and CYP2C19 but had a 400% increased activity with CYP3A5. The A287P is most common POR mutation found in patients of European origin, and significantly inhibited drug metabolism activities which has important consequences for monitoring and treatment of patients. In vitro, functional assays using recombinant proteins provide a useful model for establishing the metabolic effect of genetic mutations. Our results indicate that detailed knowledge about POR variants is necessary for correct diagnosis and treatment options for persons with POR deficiency and the role of changes in drug metabolism and toxicology due to variations in POR needs to be addressed.

Estimation of the infl uence of cytochrome P450 induction on carbon tetrachloride genotoxicity in vitro

Introduction. One of the stages of the pathogenesis of the toxic eff ect of carbon tetrachloride is the formation of compounds of reactive oxygen species with DNA, leading to the modifi cation of nitrogenous bases. The frequency of formation of nucleotides modifi ed by nitrogenous bases correlates with the number of single-and double-chain breaks of deoxyribonucleic acid (DNA) molecules. The initiator of the formation of active forms of oxygen and lipid peroxidation in liver cells upon receipt of carbon tetrachloride is a trichloromethyl radical formed during biotransformation by microsomal enzymes of cytochrome P450.The aim of the study was to analyze the changes in the genotoxicity of carbon tetrachloride in hepatocytes at normal and increased activity of cytochrome P450 caused by the infl uence of an inductor (sovol).Materials and methods. Evaluation of genotoxicity is performed by the method of DNA-comets aft er gavage with carbon tetrachloride culture of mouse hepatocytes МН22а in 96-well microplates without the induction of cytochrome P450 and chemical induction of cytochrome P450 by sovol. Determination of DNA content in comet tail (%), comet tail length (μm) and tail moment was performed in ImageJ 1.48. Statistical analysis of the results was performed in the program SPSS Statistics 21.Results: Experimental data on the genotoxic eff ect of carbon tetrachloride on hepatocytes of the MN–22A cell line without induction of cytochrome P450 and chemical induction of cytochrome P450 by sovol are presented. It was found that 0.5 mm solution of carbon tetrachloride in 1 hour aft er addition to the culture medium is genotoxic for hepatocytes MH–22a without the use of sovol (p<0.001). Th e lack of determined using the method of DNA-comet signs of genotoxicity of 5 mm carbon tetrachloride (p>0.05) in the culture medium, probably due to the transition of the cells into a state of parametros. Genotoxic eff ect is not detected by DNA comet aft er 3 and 24 hours of incubation of hepatocytes MN–22A with 0.5 and 5 mm solutions of carbon tetrachloride without preincubation with sovol (p>0.05), which may indicate repair of the damage. Aft er 72 hours of preliminary incubation of hepatocytes with sovol and the followin g four-hour cell priming with 2.5 mm tetrachloromethane solution, higher values of the parameters of DNA comets are observed than when seeding with tetrachloromethane without incubation with sovol (p<0.05).Conclusions: According to the results of the study 72 hours of cytochrome P450 induction by sovol increases the genotoxicity of carbon tetrachloride in vitro, compared with 24 h of inductor exposure, which may indirectly indicate a higher level of formed reactive oxygen species caused by increased activity of cytochrome P450 enzymes.

In vitro inhibitory effects of phellodendrine on human liver cytochrome P450 enzymes

1. Phellodendrine possesses numerous pharmacological activities. However, whether phellodendrine affects the activity of human liver cytochrome P450 (CYP) enzymes remains unclear.2. In this study, the inhibitory effects of phellodendrine on eight human liver CYP isoforms (i.e. 1A2, 3A4, 2A6, 2E1, 2D6, 2C9, 2C19 and 2C8) were investigated in vitro using human liver microsomes (HLMs).3. The results showed that phellodendrine inhibited the activity of CYP1A2, 3A4 and 2C9, with IC50 values of 20.56, 14.98 and 16.30 μM, respectively, but that other CYP isoforms were not affected. Enzyme kinetic studies showed that phellodendrine was not only a non-competitive inhibitor of CYP3A4, but also a competitive inhibitor of CYP1A2 and 2C9, with Ki values of 7.15, 10.52 and 7.98 μM, respectively. In addition, phellodendrine is a time-dependent inhibitor for CYP3A4 with Kinact/KI value of 0.046/11.57 μM−1 min−1.4. The in vitro studies of phellodendrine with CYP isoforms indicate that phellodendrine could inhibit the activities of CYP1A2, 3A4 and 2C9. Further clinical studies are needed to evaluate the significance of this interaction.

Derived esterase activity in Drosophila sechellia contributes to evolved octanoic acid resistance.

The dietary specialist fruit fly Drosophila sechellia has evolved resistance to the secondary defence compounds produced by the fruit of its host plant, Morinda citrifolia. The primary chemicals that contribute to lethality of M.citrifolia are the medium-chain fatty acids octanoic acid (OA) and hexanoic acid. At least five genomic regions contribute to this adaptation in D.sechellia and whereas the fine-mapped major effect locus for OA resistance on chromosome 3R has been thoroughly analysed, the remaining four genomic regions that contribute to toxin resistance remain uncharacterized. To begin to identify the genetic basis of toxin resistance in this species, we removed the function of well-known detoxification gene families to determine whether they contribute to toxin resistance. Previous work found that evolution of cytochrome P450 enzymatic activity or expression is not responsible for the OA resistance in D.sechellia. Here, we tested the role of the two other major detoxification gene families in resistance to Morinda fruit toxins - glutathione-S-transferases and esterases - through the use of the pesticide synergists diethyl maleate and tribufos that inhibit the function of these gene families. This work suggests that one or more esterase(s) contribute to evolved OA resistance in D.sechellia.

Long-term exposure to the non-steroidal anti-inflammatory drug (NSAID) naproxen causes thyroid disruption in zebrafish at environmentally relevant concentrations

The presence of trace levels of pharmaceuticals is an emerging issue impacting the aquatic ecosystem. Naproxen (NPX) is a nonsteroidal anti-inflammatory drug (NSAID) that has been frequently detected in aquatic environments worldwide. Recently, concerns regarding endocrine disruption by NSAIDs have increased; however, their effects on the thyroid system have yet to be understood. In this study, zebrafish were utilized to evaluate the thyroid-disrupting effects of NPX. After a 60-day exposure to various concentrations of NPX (0.1, 1, 10 and 100 μg/L), the body length and weight of the zebrafish were significantly decreased. The decrease of cytochrome P450 gene expression and enzyme activity might inhibit the metabolism of NPX, which might result in the significant bioconcentration in zebrafish. Thyroid hormone (TH) analysis showed that both triiodothyronine (T3) and thyroxine (T4) levels were substantially decreased. Gene transcription expressions along the hypothalamic-pituitary-thyroid (HPT) axis were also markedly affected. Significant downregulation of dio1, dio2, nis, nkx2.1, pax8, tg, tpo, trβ and ttr levels, along with the stimulation of the tshβ gene, were also observed in exposed fish compared to controls. Western blot analysis indicated that expression of the TTR protein was significantly decreased, which coincides with the results of the gene expression analysis. Collectively, our observations show that NPX increases the risk of bioconcentration and thyroid disruption in zebrafish. Given the continued increasing consumption and emission of pharmaceuticals, thyroid disruption should be considered when assessing the aquatic risk of long-term exposure to environmentally relevant concentrations of pharmaceuticals.

Cold storage of porcine hepatocyte spheroids for spheroid bioartificial liver.

Cell-based therapies for liver disease such as bioartificial liver rely on a large quantity and high quality of hepatocytes. Cold storage was previously shown to be a better way to preserve the viability and functionality of hepatocytes during transportation rather than freezing, but this was only proved at a lower density of rat hepatocytes spheroids. The purpose of this study was to optimize conditions for cold storage of high density of primary porcine hepatocyte spheroids. Porcine hepatocytes were isolated by a three-step perfusion method; hepatocyte spheroids were formed by a 24hours rocked culture technique. Hepatocyte cell density was 5×106 /mL in 1000mL spheroid forming medium. Spheroids were then maintained in rocked culture at 37°C (control condition) or cold stored at 4°C for 24, 48 or 72hours in four different cold storage solutions: histidine-tryptophan-ketoglutarate (HTK) alone; HTK+1mM deferoxamine (DEF); HTK+5mM N-acetyl-L-cysteine (NAC); and HTK+1mM DEF+5mM NAC. The viability, ammonia clearance, albumin production, gene expression, and functional activity of cytochrome P450 enzymes were measured after recovery from the cold storage. In this study, we observed that cold-induced injury was reduced by the addition of the iron chelator. Viability of HTK+DEF group hepatocyte spheroids was increased compared with other cold storage groups (P<0.05). Performance metrics of porcine hepatocyte spheroids cold stored for 24hours were similar to those in control conditions. The hepatocyte spheroids in control conditions started to lose their ability to clear ammonia while production of albumin was still active at 48 and 72hours (P<0.05). In contrast, the viability and functionality of hepatocyte spheroids including ammonia clearance and albumin secretion were preserved in HTK+DEF group at both 48- and 72-hour time points (P<0.05). The beneficial effects of HTK supplemented with DEF were more obvious after cold storage of high density of porcine hepatocyte spheroids for 72hours. The porcine hepatocyte spheroids were above the cutoff criteria for use in a spheroid-based bioartificial liver.

Essential oil components and cytochrome P450 enzymes: a review

AbstractPhase‐I metabolism mediated by cytochrome P450 (CYP) enzymes represents a major route of elimination of many drugs that can compete for the same CYP enzyme. The bioactivity of essential oils (EOs) and their flavour and fragrance constituents have been known since ancient times, and like any other physiological process in the human body the activity of CYP enzymes can also be influenced (increased and/or decreased) by these natural compounds. This review discusses the effects of EO constituents on important drug‐metabolizing CYP enzymes. Exposure to EO constituents through commonly used products via cutaneous, oral, and inhalation routes is outlined, and the impact of some important EO constituents on CYP enzymes is described in more detail. In particular, the simultaneous application of EO constituents with drugs or the excessive use of EOs and their constituents can lead to unexpected adverse effects.

Isoform‐Dependent Effects of Cytochrome P450 Oxidoreductase Polymorphisms on Drug Metabolism by Cytochrome P450 Enzymes in Dogs

Cytochrome P450 oxidoreductase (POR) is the obligate electron donor for all microsomal cytochrome P450 (CYP) enzymes. We have recently discovered three polymorphisms in the dog POR coding region resulting in one (POR‐H2 and POR‐H4) or two (POR‐H3) amino acid substitutions. In humans, POR mutations can have CYP isoform‐dependent effects on CYP enzyme activity depending on the POR amino acid residue that is substituted.1 Genotyping of 2,040 DNA samples collected from 65 different dog breeds revealed a heterogeneous distribution of POR alleles across breeds. The POR‐H2 allele was found in 19 of 65 breeds and was most common in Rottweilers and Doberman Pinschers (51% and 19% allele frequencies, respectively). The POR‐H3 allele was found in 8 of 65 breeds and was most common in Scottish Deerhounds and Greyhounds (36% and 35% allele frequencies, respectively). The POR‐H4 allele was rare, only being found in 5 of 2,040 dogs. Greyhounds have been shown to metabolize substrates of CYP2B11 (but not other CYPs including CYP2D15) much more slowly compared with mixed‐breed dogs.2 Preliminary studies identified polymorphisms in the CYP2B11 gene that only partially explained reduced CYP2B11 metabolism in Greyhounds. Consequently, we hypothesized that the newly discovered canine POR variants found in Greyhounds would reduce CYP metabolism in a CYP isoform‐dependent manner. The aim of this study was to determine the functional impact of the canine POR variants on drug metabolism by CYP2B11 and CYP2D15 in vitro. In silico molecular structure modelling predicted that POR‐H3 and POR‐H4 but not POR‐H2 would have altered function. The three POR variants and wild‐type were expressed alone or co‐expressed with CYP2B11 or CYP2D15 using an insect cell system. Enzyme kinetic experiments were conducted to determine the impact of the mutations on POR function and CYP‐catalyzed reactions. The POR variants were not significantly different from wild‐type POR in protein expression or function (cytochrome c reduction) when expressed alone. POR variants also did not significantly alter CYP2D15 protein level or catalytic activities or CYP2B11 protein level when co‐expressed. However, POR‐H3 and POR‐H4 (but not POR‐H2) significantly reduced CYP2B11 catalytic efficiency (CLint) by 37% and 69%, respectively, compared to wild‐type POR. This was due to reduced maximal velocities (Vmax) but not substrate affinities (Km). POR‐H3 and POR‐H4 appear to reduce CYP activities in a CYP isoform‐dependent manner. Further investigations are needed to determine POR variant effects on other CYP isoforms and redox partners. The POR‐H3 and POR‐H4 variants are likely to contribute to slower CYP2B11 drug metabolism in Greyhounds and possibly related sighthound breeds (such as Scottish Deerhounds), but results must be confirmed in vivo.Support or Funding InformationAmerican Kennel Club Canine Health Foundation (Grants 2242 and 2529) and the William R. Jones Endowment at Washington State University College of Veterinary Medicine.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Screening of ten cytochrome P450 enzyme activities with 12 probe substrates in human liver microsomes using cocktail incubation and liquid chromatography-tandem mass spectrometry.

Testing for potential drug interactions of new chemical entities is essential when developing a novel drug. In this study, an assay was designed to evaluate drug interactions with 10 major human cytochrome P450 (P450) enzymes incubated in liver microsomes, involving 12 probe substrates with two cocktail incubation sets used in a single liquid chromatography-tandem mass spectrometry (LC-MS/MS) run. The P450 substrate composition in each cocktail set was optimized to minimize solvent effects and mutual drug interactions among substrates as follows: cocktail A was composed of phenacetin for CYP1A2, bupropion for CYP2B6, amodiaquine for CYP2C8, diclofenac for CYP2C9, S-mephenytoin for CYP2C19, and dextromethorphan for CYP2D6; cocktail B was composed of coumarin for CYP2A6, chlorzoxazone for CYP2E1, astemizole for CYP2J2, and midazolam, nifedipine, and testosterone for CYP3A. Multiple probe substrates were used for CYP3A owing to the multiple substrate-binding sites and substrate-dependent inhibition. After incubation in human liver microsomes, each incubation mixture was pooled and all probe metabolites were simultaneously analysed in a single LC-MS/MS run. Polarity switching was used to acquire the negative-ion mode for hydroxychlorzoxazone and positive-ion mode for the remaining analytes. The method was validated by comparing the inhibition data obtained from incubation of each individual probe substrate alone and with the substrate cocktails. The half-maximal inhibitory concentration values obtained from the cocktail and individual incubations were well correlated and in agreement with previously reported values. This new method will be useful in assessing the drug interaction potential of new chemical entities during new drug development.

Rational high-throughput system for screening of high sophorolipids-producing strains of Candida bombicola

A rational high-throughput screening can significantly improve the efficiency of strain screening with high performance. In this study, based on the addition reaction of unsaturated fatty acids in the sophorolipids (SLs) and I2 molecules, a simple and rapid high-throughput detection method for SLs was established which demonstrated a correlation coefficient (R2) of 0.9106 with high-performance liquid chromatography (HPLC) method. Moreover, chlorpromazine, as a rational selecting pressure for enrichment of mutants with high cytochrome P450 enzyme activity, which was a key enzyme in the synthesis of SLs, was introduced into the high-throughput screening model. Consequently, with the aid of this effective screening system, a high-yielding mutant designated as Candida bombicola F6.5 was successfully screened out from 1500 single colonies, which presented improvements of 40.3% and 11.4% on SLs titer and yield, respectively, compared to the parent strain in a 1L bioreactor.

Influences of Corydalis decumbens on the Activities of CYP450 Enzymes in Rats with a Cocktail Approach.

Corydalis decumbens, a Traditional Chinese Medicine, has been widely used for the alternative and/or complementary therapy of hypertension, arrhythmias rheumatoid arthritis, sciatica, stroke, hemiplegia, paraplegia, and vascular embolism. The aim of this study was to determinate the potential effects of Corydalis decumbens on the five cytochrome P450 (CYP) enzyme activities (CYP1A2, CYP3A4, CYP2C9, CYP2C19, and CYP2D6) by cocktail approach. To evaluate whether concurrent use of Corydalis decumbens interferes with the effect of several prescription drugs, saline (control group) or Corydalis decumbens (XTW group) were administrated via gavage for 7 successive days. A probe cocktail solution (phenacetin, omeprazole, metoprolol, tolbutamide, and midazolam) was given 24 h after the last dose of saline or Corydalis decumbens. A specific and sensitive UHPLC–MS/MS method was validated for the determination of five substrates and their metabolites in control group and XTW group. Our results indicated that Corydalis decumbens could have inductive effects of CYP2C19 and inhibit the activities of CYP1A2 and CYP3A4. However, Corydalis decumbens had no significant influence on CYP2C9 and CYP2D6. The herb-drug interaction should require more attention by careful monitoring and appropriate drug dosing adjustments to the concurrent use of western medications which were metabolized by CYP1A2, CYP2C19, and CYP3A4 in human—Corydalis decumbens, Cytochrome P450, Cocktail, Pharmacokinetics, herb–drug interactions.

Effect of pterostilbene on in vitro drug metabolizing enzyme activity

Pterostilbene is a natural polyphenol compound found in small berries that is related to resveratrol, but has better bioavailability and a longer half-life. The purpose of this study was to assess the potential inhibitory effect of pterostilbene on in vitro drug metabolism. The effect of pterostilbene on cytochrome P450 (CYP) and UDP-glucuronosyltransferase (UGT) enzyme activities were studied using the enzyme-selective substrates amodiaquine (CYP2C8), midazolam (CYP3A4), estradiol (UGT1A1), serotonin (UGT1A6) and mycophenolic acid (UGT1A8/9/10). The IC50 value was used to express the strength of inhibition. Further, a volume per dose index (VDI) was used to estimate the potential for in vivo interactions. Pterostilbene significantly inhibited CYP2C8 and UGT1A6 activities. The IC50 (mean ± SE) values for CYP2C8 and UGT1A6 inhibition were 3.0 ± 0.4 µM and 15.1 ± 2.8 µM, respectively; the VDI exceeded the predefined threshold of 5 L/dose for both CYP2C8 and UGT1A6, suggesting a potential for interaction in vivo. Pterostilbene did not inhibit the metabolism of the other enzyme-selective substrates. The results of this study indicate that pterostilbene inhibits CYP2C8 and UTG1A6 activity in vitro and may inhibit metabolism by these enzymes in vivo. Clinical studies are warranted to evaluate the in vivo relevance of these interactions.

Alteration in Inflammatory Responses and Cytochrome P450 Expression of Porcine Jejunal Cells by Drinking Water Supplements.

The intestinal epithelium is the first determining barrier to the drugs administered per os. Cytochrome P450 (CYP) enzymes are substantial in the initial step of xenobiotic metabolism; therefore, intestinal CYP enzyme activities could be an important influencing factor of the oral utilization of xenobiotic substances. In this study, the effect of four drinking water supplements on CYP mRNA levels of porcine intestinal epithelial cells was examined. Further goal of the study is to describe the effect of these feed additives on the proinflammatory response of the LPS-treated enterocytes. The nontransformed porcine intestinal epithelial cells (IPEC-J2) were grown on six-well polyester membrane inserts. Cell cultures were treated with LPS (10 μg/ml), β-glucan (5 and 50 μg/ml), sanguinarine-containing additive (5 and 50 μg/ml), drinking water acidifier (0.1 and 1 μl/ml), and fulvic acid (25 and 250 μg/ml) for 1 hour. Cells were washed with culture medium and incubated for additional 1 h before total RNA isolation. IL-6, IL-8, TNF-α, HSP70, CYP1A1, CYP1A2, and CYP3A29 mRNA levels were measured. The LPS treatment upregulated the gene expression of IL-8 and TNF-α. The relative gene expression of IL-6 remained unchanged and TNF-α and HSP70 were downregulated after the treatment with each feed additive. CYP1A1 and CYP1A2 expressions increased after sanguinarine-containing solution, fulvic acid, and drinking water acidifier treatment. None of the treatments changed the gene expression of CYP3A29, responsible for the metabolism of the majority of drug substances used in swine industry. The feed additive substances inhibited the expression of proinflammatory mediators HSP70 and TNF-α; however, β-glucan and fulvic acid elevated the production of the chemokine IL-8 mRNA in endotoxin-treated enterocytes. All acidic supplements increased the expression of CYP1A1 gene; their constituents may serve as a ligand of CYP1A1 nuclear receptors.

Effects of infection of MRSA on the expression and activity of renal cytochrome P450s in mice.

Methicillin-resistant Staphylococcus aureus (MRSA) leads to serious infections, but it is not known whether it changes the expression of kidney drug metabolizing enzymes during infection. The mice were infected with different doses of MRSA and the oxidative stress and inflammation levels in the kidney were examined. The mRNA expression and activity of cytochrome P450 enzyme was analysed. Mice infected with high levels of MRSA showed a decrease in renal antioxidant capability and an elevated level of oxidative metabolites, which was accompanied by the release of inflammatory cytokines. The levels of interleukin 1β, tumour necrosis factor alpha, and macrophage inflammatory protein-1α were significantly increased along with the levels of nitric oxide and malondialdehyde. On day 7, mRNA expression of Cyp1a2, 2d22, and 3a11 were decreased by the high level of MRSA, but the low level of MRSA increased their expressions. Cyp2e1 mRNA expression was increased by MRSA in the kidney of mice. High dose of MRSA infection increased the oxidative stress and inflammatory response in mouse kidney, leading to the decrease in the expression of renal drug-metabolizing enzymes and no recovery within 7 days.

Probing membrane enhanced protein-protein interactions in a minimal redox complex of cytochrome-P450 and P450-reductase.

Investigating the interplay in a minimal redox complex of cytochrome-P450 and its reductase is crucial for understanding cytochrome-P450's enzymatic activity. Probing the hotspots of dynamic structural interactions using NMR revealed the engagement of loop residues from P450-reductase to be responsible for the enhanced affinity of CYP450 towards its obligate redox partner.