Cytochrome P450 Isozymes Research Articles

A novel calcimimetic agent, evocalcet (MT-4580/KHK7580), suppresses the parathyroid cell function with little effect on the gastrointestinal tract or CYP isozymes in vivo and in vitro.

Cinacalcet hydrochloride (cinacalcet), an oral calcimimetic agent has been widely used for the management of secondary hyperparathyroidism (SHPT) in chronic kidney disease (CKD). In sharp contrast to vitamin D receptor activators, cinacalcet suppresses SHPT without inducing hypercalcemia or hyperphosphatemia. Nevertheless, some patients remain refractory to SHPT with this agent, as the dose cannot be sufficiently increased due to gastrointestinal symptoms. In order to resolve this issue, we have developed a newly synthesized calcimimetic agent, evocalcet (MT-4580/KHK7580). In a rat model of CKD induced by 5/6 nephrectomy, oral administration of evocalcet efficiently suppressed the secretion of parathyroid hormone (PTH). With regard to the gastro-intestinal effects, cinacalcet induced a significant delay in gastric emptying in rats, while evocalcet did no marked effects on it. Evocalcet also demonstrated the less induction of emesis compared to cinacalcet in common marmosets. The pharmacological effects of evocalcet were observed at lower doses because of its higher bioavailability than cinacalcet, which may have contributed to the reduced GI tract symptoms. In addition, evocalcet showed no substantial direct inhibition of any CYP isozymes in in vitro liver microsome assay, suggesting a better profile in drug interactions than cinacalcet that inhibits cytochrome P450 (CYP) 2D6. These findings suggest that evocalcet can be a better alternative to cinacalcet, an oral calcimimetic agent, with a wider safety margin.

Characterization of the Preclinical Pharmacology of the New 2-Aminomethylphenol, JPC-3210, for Malaria Treatment and Prevention.

The new 2-aminomethylphenol, JPC-3210, has potent in vitro antimalarial activity against multidrug-resistant Plasmodium falciparum lines, low cytotoxicity, and high in vivo efficacy against murine malaria. Here we report on the pharmacokinetics of JPC-3210 in mice and monkeys and the results of in vitro screening assays, including the inhibition of cytochrome P450 (CYP450) isozymes. In mice, JPC-3210 was rapidly absorbed and had an extensive tissue distribution, with a brain tissue-to-plasma concentration ratio of about 5.4. JPC-3210 had a lengthy plasma elimination half-life of about 4.5 days in mice and 11.8 days in monkeys. JPC-3210 exhibited linear single-oral-dose pharmacokinetics across the dose range of 5 to 40 mg/kg of body weight with high oral bioavailability (∼86%) in mice. Systemic blood exposure of JPC-3210 was 16.6% higher in P. berghei-infected mice than in healthy mice. In vitro studies with mice and human hepatocytes revealed little metabolism and the high metabolic stability of JPC-3210. The abundance of human metabolites from oxidation and glucuronidation was 2.0% and 2.5%, respectively. CYP450 studies in human liver microsomes showed JPC-3210 to be an inhibitor of CYP2D6 and, to a lesser extent, CYP3A4 isozymes, suggesting the possibility of a metabolic drug-drug interaction with drugs that are metabolized by these isozymes. In vitro studies showed that JPC-3210 is highly protein bound to human plasma (97%). These desirable pharmacological findings of a lengthy blood elimination half-life, high oral bioavailability, and low metabolism as well as high in vivo potency have led the Medicines for Malaria Venture to select JPC-3210 (MMV892646) for further advanced preclinical development.

Characterized in Vitro Metabolism Kinetics of Alkyl Organophosphate Esters in Fish Liver and Intestinal Microsomes.

Tris(2-butoxyethyl) phosphate (TBOEP) and tris( n-butyl) phosphate (TNBP) are the most commonly used alkyl organophosphate esters (alkyl-OPEs), and they increasingly accumulate in organisms and create potential health hazards. This study examined the metabolism of TNBP and TBOEP in Carassius carassius liver and intestinal microsomes and the production of their corresponding monohydroxylated and dealkylated metabolites. After 140 min of incubation with fish liver microsomes, the rapid depletion of TNBP and TBOEP were both best fitted to the Michaelis-Menten model (at administrated concentrations ranging from 0.5 to 200 μM), with a CLint (intrinsic clearance) of 3.1 and 3.9 μL·min-1·mg-1 protein, respectively. But no significant ( P > 0.05) biotransformation was observed for these compounds in intestinal microsomes at any administrated concentrations. In fish liver microsomes assay, bis(2-butoxyethyl) hydroxyethyl phosphate (BBOEHEP) and bis(2-butoxyethyl) 3-hydroxyl-2-butoxyethyl phosphate (3-OH-TBOEP) were the most abundant metabolites of TBOEP, and dibutyl-3-hydroxybutyl phosphate (3-OH-TNBP) was the predominant metabolite of TNBP. Similarly, the apparent Vmax values (maximum metabolic rate) of BBOEHEP and 3-OH-TNBP were also respectively highest among those of other metabolites. Further inhibition studies were conducted to identify the specific cytochrome P450 (CYP450) isozymes involved in the metabolism of TNBP and TBOEP in liver microsomes. It was confirmed that CYP3A4 and CYP1Awere the significant CYP450 isoforms catalyzing the metabolism of TNBP and TBOEP in fish liver microsomes. Overall, this study emphasized the importance of hydroxylated metabolites as biomarkers for alkyl-OPEs exposure, and further research is needed to validate the in vivo formation and toxicological implications of these metabolites.

Shikonin upregulates the expression of drug-metabolizing enzymes and drug transporters in primary rat hepatocytes

Ethnopharmacological relevanceShikonin, a naphthoquinone pigment abundant in the root of the Chinese herb Lithospermum erythrorhizon, has been widely used to treat inflammatory diseases for thousands of years. Whether shikonin changes drug metabolism remains unclear. Aim of the studyWe investigated whether shikonin modulates the expression of hepatic drug-metabolizing enzymes and transporters as well as the possible mechanisms of this action. Materials and methodsPrimary hepatocytes isolated from Sprague-Dawley rats were treated with 0–2 μM shikonin and the protein and mRNA levels of drug-metabolizing enzymes and transporters as well as the activation of aryl hydrocarbon receptor (AhR) and NF-E2-related factor 2 (Nrf2) were determined. ResultsShikonin dose-dependently increased the protein and RNA expression of phase I enzymes, i.e., cytochrome P450 (CYP) 1A1/2, CYP3A2, CYP2D1, and CYP2C6; phase II enzymes, i.e., glutathione S-transferase (GST), NADP(H) quinone oxidoreductase 1 (NQO1), and UDP glucuronosyltransferase 1A1; and phase III drug transporters, i.e., P-glycoprotein, multidrug resistance-associated protein 2/3, organic anion transporting polypeptide (OATP) 1B1, and OATP2B1. Immunoblot analysis and EMSA revealed that shikonin increased AhR and Nrf2 nuclear contents and DNA binding activity. AhR and Nrf2 knockdown by siRNA attenuated the ability of shikonin to induce drug-metabolizing enzyme expression. In addition, shikonin increased p38, JNK, and ERK1/2 phosphorylation, and inhibitors of the respective kinases inhibited shikonin-induced Nrf2 nuclear translocation. ConclusionsShikonin effectively upregulates the transcription of CYP isozymes, phase II detoxification enzymes, and phase III membrane transporters and this function is at least partially through activation of AhR and Nrf2. Moreover, Nrf2 activation is dependent on mitogen-activated protein kinases.

Methods for determining of cytochrome P450 isozymes functional activity

The review is dedicated to modern methods and technologies for determining of cytochrome P450 isozymes functional activity, such as absorbance and fluorescent spectroscopy, electron paramagnetic resonance (EPR), nuclear magnetic resonance (NMR), Raman, Mossbauer, and X-ray spectroscopy, surface plasmon resonance (SPR), atomic force microscopy (AFM). Methods of molecular genetic analysis were reviewed from personalized medicine point of view. The use of chromate-mass-spectrometric methods for cytochrome P450-dependent catalytic reactions' products was discussed. The review covers modern electrochemical systems based on cytochrome P450 isozymes for their catalytic activity analysis, their use in practice and further development perspectives for experimental pharmacology, biotechnology and translational medicine.

<b>Population Profile of Cytochrome P450 Isozymes in the Small Intestine of Japanese </b>

<b>Population Profile of Cytochrome P450 Isozymes in the Small Intestine of Japanese </b>

Development of a multitask deep learning QSAR model using data from individual cytochrome P450 isozymes

Development of a multitask deep learning QSAR model using data from individual cytochrome P450 isozymes

Human cytochrome P450 kinetic studies on six N-2-methoxybenzyl (NBOMe)-derived new psychoactive substances using the substrate depletion approach

A huge number of new chemical derivatives of known drugs of abuse, so-called new psychoactive substances (NPS), are sold and consumed without prior preclinical and clinical testing. For assessing the elimination behaviors, determination of the kinetic constants Km and Vmax of the cytochrome P450 (CYP) isoforms involved in the hepatic metabolism of NPS could help to predict their contributions to hepatic clearance, drug–drug interactions and polymorphisms. Therefore, the aims of the present study were to determine the Km and Vmax values for CYP isoforms using the substrate depletion approach for the six N-2-methoxybenzyl (NBOMe)-derived NPS 25B-NBOMe, 25C-NBOMe, 25I-NBOMe, 3,4-DMA-NBOMe, 4-EA-NBOMe, and 4-MMA-NBOMe. Furthermore, the contributions of each CYP isozyme to the hepatic net clearance were elucidated using the relative activity factor approach. Several CYPs including CYP1A2, CYP2B6, CYP2C19, CYP2D6, and CYP3A4 were identified to be involved in the metabolism of the investigated compounds. The determined Km values ranged from 0.010 μM (CYP2D6, 4-MMA-NBOMe) to 13 μM (CYP2B6, 4-EA-NBOMe). All NBOMes were good substrates of CYP2C19 and CYP2D6 resulting in very low Km values in the nanomolar range. The main contributors to hepatic net clearance were CYP2D6 for 25B-NBOMe (69%), 25C-NBOMe (83%), 25I-NBOMe (61%), 3,4-DMA-NBOMe (89%) as well as for 4-EA-NBOMe (62%) and CYP2C19 for 4-MMA-NBOMe (64%). As more than one isoform was involved in the particular steps, the risk of harm associated with drug–drug interactions might be considered low. However, in cases where substances with high contributions from polymorphically expressed CYP2C19 and CYP2D6 are encountered, inter-individual variations in metabolism and excretion cannot be excluded.

Metabolic Pathway of Icotinib In Vitro: The Differential Roles of CYP3A4, CYP3A5, and CYP1A2 on Potential Pharmacokinetic Drug-Drug Interaction

Icotinib is the first self-developed small molecule drug in China for targeted therapy of non–small cell lung cancer. To date, systematic studies on the pharmacokinetic drug-drug interaction of icotinib were limited. By identifying metabolite generated in human liver microsomes and revealing the contributions of major cytochromes P450 (CYPs) in the formation of major metabolites, the aim of the present work was to understand the mechanisms underlying pharmacokinetic and pharmacological variability in clinic. A liquid chromatography/UV/high-resolution mass spectrometer method was developed to characterize the icotinib metabolites. The formation of 6 major metabolites was studied in recombinant CYP isozymes and human liver microsomes with specific inhibitors to identify the CYPs responsible for icotinib metabolism. The metabolic pathways observed in vitro are consistent with those observed in human. Results demonstrated that the metabolites are predominantly catalyzed by CYP3A4 (77%∼87%), with a moderate contribution from CYP3A5 (5%∼15%) and CYP1A2 (3.7%∼7.5%). The contribution of CYP2C8, 2C9, 2C19, and 2D6 is insignificant. Based on our observations, to minimize drug-drug interaction risk in clinic, coprescription of icotinib with strong CYP3A inhibitors or inducers must be weighed. CYP1A2, a highly inducible enzyme in the smoking population, may also represent a determinant of pharmacokinetic and pharmacological variability of icotinib, especially in lung cancer patients with smoking history.

Development of Guanfacine Extended-Release Dosing Strategies in Children and Adolescents with ADHD Using a Physiologically Based Pharmacokinetic Model to Predict Drug-Drug Interactions with Moderate CYP3A4 Inhibitors or Inducers.

BackgroundGuanfacine extended-release (GXR) is an orally administered, non-stimulant treatment for children and adolescents with attention-deficit/hyperactivity disorder (ADHD) and is primarily metabolized by the 3A4 isozyme of cytochrome P450 (CYP3A4). The results of clinical pharmacokinetic (PK) studies indicate that guanfacine is sensitive to drug–drug interactions (DDIs) perpetrated by strong inhibitors and inducers of CYP3A4.ObjectiveThe aim was to provide guidance on the possible requirement for GXR dose adjustment in children and adolescents with ADHD by predicting DDIs following co-administration with moderate CYP3A4 inhibitors and inducers.MethodsA physiologically based PK model for GXR orally administered to healthy adults was developed based on physicochemical, in vitro and clinical PK data. The model was validated using clinical PK data for co-administration of GXR with ketoconazole (strong CYP3A4 inhibitor) or rifampicin (strong CYP3A4 inducer).ResultsModel predictions indicated that co-administration of GXR with the moderate CYP3A4 inhibitors erythromycin 500 mg three times a day or fluconazole 200 mg daily (q.d.) increased the guanfacine area under the plasma concentration–time curve (AUC) by 2.31-fold or 1.98-fold, respectively, compared with GXR monotherapy. The moderate CYP3A4 inducer efavirenz 400 mg or 600 mg q.d. was predicted to reduce guanfacine AUC to 58 or 33% of its value for GXR monotherapy, respectively.ConclusionWithout the requirement for additional clinical studies, the following GXR dose recommendations were developed and approved for US labeling for use in children and adolescents with ADHD: (1) decrease GXR to 50% of the usual target dose when it is co-administered with strong or moderate CYP3A4 inhibitors; (2) consider titrating GXR up to double the usual target dose over 1–2 weeks when it is co-administered with strong or moderate CYP3A4 inducers.

Long-enduring primary hepatocyte-based co-cultures improve prediction of hepatotoxicity

The failure of drug candidates during clinical trials and post-marketing withdrawal due to Drug Induced Liver Injury (DILI), results in significant late-stage attrition in the pharmaceutical industry. Animal studies have proven insufficient to definitively predict DILI in the clinic, therefore a variety of in vitro models are being tested in an effort to improve prediction of human hepatotoxicity. The model system described here consists of cryopreserved primary rat, dog or human hepatocytes co-cultured together with a fibroblast cell line, which aids in the hepatocytes' maintenance of more in vivo-like characteristics compared to traditional hepatic mono-cultures, including long term viability and retention of activity of cytochrome P450 isozymes. Cell viability was assessed by measurement of ATP following treatment with 29 compounds having known hepatotoxic liabilities. Hμrelrat™, Hμreldog™, and Hμrelhuman™ hepatic co-cultures were treated for 24h, or under repeat-dosing for 7 or 13days, and compared to rat and human hepatic mono-cultures following single-dose exposure for 24h. The results allowed for a comparison of cytotoxicity, species-specific responses and the effect of repeat compound exposure on the prediction of hepatotoxic potential in each model. Results show that the co-culture model had greater sensitivity compared to that of the hepatic mono-cultures. In addition, “time-based ratios” were determined by dividing the compounds' 24-hour TC50/Cmax values by TC50/Cmax values measured after dosing for either 7 or 13days. The results suggest that this approach may serve as a useful adjunct to traditional measurements of hepatotoxicity, improving the predictive value of early screening studies.

Effects of Paederia foetida and its Bioactive Phytochemical Constituent Lupeol on Hepatic Phase I Drug Metabolism

There are many possible complications associated with the concomitant use of herbs and medications, but limited information is available on herb-herb or herb-drug interactions. Paederia foetida Linn. (family: Rubiaceae) is utilized in the Indian traditional medicine. It exhibits various pharmacological properties, such as antidiabetic, analgesic, anti-inflammatory, antimicrobial, hepatoprotective, anthelmintic, antiulcer and antioxidant activities. The purpose of the present work was to investigate the inhibitory potential of the P. foetida ethanolic extract and its bioactive constituent lupeol on hepatic phase I drug metabolizing enzymes. The high performance thin layer chromatography was performed for qualitative analysis of various extracts of P. foetida. The effects of P. foetida extract on rat liver microsomes (RLMs) and individual cytochrome P-450 (CYP) isozymes (CYP3A4 and CYP2D6) were investigated using CYP450-carbon monoxide complex assay and fluorescence microplate assay, respectively. The ethanolic extract and lupeol (both at a concentration of 100 μg/mL) showed 45±3.3 and 44±3.8% inhibition of rat liver microsomes, respectively, which were significantly less than that of known inhibitor ketoconazole (74±5.4% inhibition at 100 μg/mL). The 50% inhibitory concentrations (IC50) of ethanolic extract on CYP3A4 and CYP2D6 were 78±2.3 and 82±3.1 μg/mL, respectively, whereas its major bioactive constituent lupeol has IC50 values of 83±2.0 and 84±2.6 μg/mL for CYP3A4 and CYP2D6, respectively. The results were of lesser magnitude compared to known inhibitors, ketoconazole and quinidine, respectively. The current study revealed that P. foetida has less inhibitory potential in comparison to that of known inhibitors, ketoconazole and quinidine, on two major drug metabolizing isozymes, CYP3A4 and CYP2D6. Thus, the use of P. foetida as a complementary or alternative medicine may be safe in regard to herb-drug interactions.

Effects of clarithromycin on the pharmacokinetics of evogliptin in healthy volunteers.

Evogliptin (DA-1229), a novel dipeptidyl peptidase (DPP)-4 inhibitor with high potency and selectivity, was approved in Korea for the treatment of type 2 diabetes. Preclinical studies suggest that it is metabolized by cytochrome (CYP) P450 isozymes. Based on these findings, a clinical study was designed to investigate the pharmacokinetic (PK) interaction of evogliptin with a CYP inhibitor, clarithromycin. An open-label, two-phase, crossover study was conducted with 12 healthy subjects. On day 1, a single dose of evogliptin 5mg was administered alone to assess the reference PK profile of evogliptin. On day 10, after a 2-day pretreatment with clarithromycin, evogliptin 5mg was administered again to evaluate the effect of CYP inhibition on the PK profile of evogliptin. Administration of clarithromycin continued until day 14. Blood sampling in the reference and test phases was performed until 96 and 168hours after dosing, respectively for PK assays. Eleven of the 12 subjects completed the study, and their data were analysed. In the presence of clarithromycin, exposure to evogliptin increased without any serious adverse events and the geometric mean peak plasma concentration (Cmax ) and area under the concentration-time curve from time 0 extrapolated to infinity (AUC0-∞ ) of evogliptin increased by 116.5% and 89.6%, respectively. Administration of clarithromycin significantly increased exposure to evogliptin in healthy subjects.

Time-dependent Inhibition of CYP2C8 and CYP2C19 by Hedera helix Extracts, A Traditional Respiratory Herbal Medicine.

The extract of Hedera helix L. (Araliaceae), a well-known folk medicine, has been popularly used to treat respiratory problems, worldwide. It is very likely that this herbal extract is taken in combination with conventional drugs. The present study aimed to evaluate the effects of H. helix extract on cytochrome P450 (CYP) enzyme-mediated metabolism to predict the potential for herb–drug interactions. A cocktail probe assay was used to measure the inhibitory effect of CYP. H. helix extracts were incubated with pooled human liver microsomes or CYP isozymes with CYP-specific substrates, and the formation of specific metabolites was investigated to measure the inhibitory effects. H. helix showed significant inhibitory effects on CYP2C8, CYP2C19 and CYP2D6 in a concentration-dependent manner. In recombinant CYP2C8, CYP2C19 and CYP2D6 isozymes, the IC50 values of the extract were 0.08 ± 0.01, 0.58 ± 0.03 and 6.72 ± 0.22 mg/mL, respectively. Further investigation showed that H. helix extract has a positive time-dependent inhibition property on both CYP2C8 and CYP2C19 with IC50 shift value of 2.77 ± 0.12 and 6.31 ± 0.25, respectively. Based on this in vitro investigation, consumption of herbal medicines or dietary supplements containing H. helix extracts requires careful attention to avoid any CYP-based interactions.

Accumulation properties of polychlorinated biphenyl congeners in Yusho patients and prediction of their cytochrome P450-dependent metabolism by in silico analysis.

In what has become known as the Yusho incident, thousands of people in western Japan were poisoned by the accidental ingestion of rice bran oil contaminated with polychlorinated biphenyls (PCBs) and various dioxins and dioxin-like compounds. In this study, we investigated the accumulation patterns of 69 PCB congeners in the blood of Yusho patients in comparison with those of non-exposed controls. The blood samples were collected at medical check-ups in 2004 and 2005. To compare the patterns of PCB congeners, we calculated the concentration ratio of each congener relative to the 2,2',4,4',5,5'-hexaCB (CB153) concentration. The concentration ratios of tetra- and penta-chlorinated congeners in the blood of Yusho patients were significantly lower than those of controls. To examine the cytochrome P450 (CYP)-dependent metabolic potential of the 2,3',4,4'5-pentaCB (CB118), CB153, and 2,3,3',4,4'5-hexaCB (CB156) congeners, we conducted PCB-CYP (CYP1A1, CYP1A2, CYP2A6, and CYP2B6) docking simulation by in silico analysis. The docking models showed that human CYP1A1, CYP2A6, and CYP2B6 isozymes have the potential to metabolize CB118 and CB153. On the other hand, it was inferred that CB156 is difficult to be metabolized by these four CYP isozymes. These results indicate that CYP1 and CYP2 isozymes may be involved in the characteristic accumulation patterns of PCB congeners in the blood of Yusho patients.

Alignment-Based Prediction of Sites of Metabolism.

Prediction of metabolically labile atom positions in a molecule (sites of metabolism) is a key component of the simulation of xenobiotic metabolism as a whole, providing crucial information for the development of safe and effective drugs. In 2008, an exploratory study was published in which sites of metabolism were derived based on molecular shape- and chemical feature-based alignment to a molecule whose site of metabolism (SoM) had been determined by experiments. We present a detailed analysis of the breadth of applicability of alignment-based SoM prediction, including transfer of the approach from a structure- to ligand-based method and extension of the applicability of the models from cytochrome P450 2C9 to all cytochrome P450 isozymes involved in drug metabolism. We evaluate the effect of molecular similarity of the query and reference molecules on the ability of this approach to accurately predict SoMs. In addition, we combine the alignment-based method with a leading chemical reactivity model to take reactivity into account. The combined model yielded superior performance in comparison to the alignment-based approach and the reactivity models with an average area under the receiver operating characteristic curve of 0.85 in cross-validation experiments. In particular, early enrichment was improved, as evidenced by higher BEDROC scores (mean BEDROC = 0.59 for α = 20.0, mean BEDROC = 0.73 for α = 80.5).

Personalized medicine in internal medicine

Personalized medicine - a new direction in medicine, which is based on the study of various biomarkers and the use of new methods of molecular analysis (primarily evaluating the activity of isoenzymes of cytochrome P450), allowing individualized approach to the selection of both the drugs and the selection of the dosing regimen for the purpose of maximize the effectiveness and safety of pharmacotherapy. This personalized medicine is to change the development and use of preventive and curative interventions. Genetic polymorphism isozymes of cytochrome P450 may determine the individual activity of a particular isozyme, and thus, to predict the clinical effectiveness, and in some cases, the risk of adverse reactions. The article is an example of the use of information on the activity of cytochrome P450 in clinical practice in matters of diagnosis, treatment and prevention of diseases. The scheme of the five best-known activity of isoenzymes of cytochrome P450 is shown.

Effects of 6-paradol, an unsaturated ketone from gingers, on cytochrome P450-mediated drug metabolism

Paradols are unsaturated ketones produced by biotransformation of shogaols in gingers. Among them, 6-paradol has been investigated as a new drug candidate due to its anti-inflammatory, apoptotic, and neuroprotective activities. In this study, the inhibitory effects of 6-paradol on the activities of cytochrome P450 (CYP) enzymes were investigated with human liver microsomes and recombinant CYP isozymes. 6-Paradol showed concentration-dependent inhibitory effects on CYP1A2, CYP2B6, CYP2C8, CYP2C9, and CYP2C19 isozymes, with IC50 values ranging from 3.8 to 21.4µM in recombinant CYP isozymes. However, the inhibition was not potentiated following pre-incubation, indicating that 6-paradol is not a mechanism-based inhibitor. These results suggest that pharmacokinetic drug-drug interactions might occur with 6-paradol, which must be considered in the process of new drug development.

Effect of clinical doses of Realgar-Indigo Naturalis formula and large-dose of realgar on CYP450s of rat liver

To investigate the effect of clinical dose of Realgar-Indigo Naturais formula (RIF) and large-dose of Realgar on main drug-metabolizing enzymes CYP450s of rat liver, as well as its regulatory effect on mRNA expression. Wistar rats were administrated orally with tested drugs for 14 days. A Cocktail method combined with HPLC-MS/MS was used in the determination of 4 cytochrome P450 isozymes (CYP1A2, CYP2B, CYP3A and CYP2C) in liver of the rats, and the mRNA expression levels of the above subtypes were detected by real-time fluorescent quantitative PCR. The results showed that RIF can significantly induce CYP1A2 and CYP2B enzyme activity, and inhibit CYP3A enzyme activity. This result was consistent with the mRNA expression. However, its single compound showed weaker or even contrary phenomenon. Different doses of Realgar also showed significant inconsistencies on CYP450 enzymes activity and mRNA expression. These phenomena may be relevant with RIF compatibility synergies or toxicity reduction. The results can also prompt drug interactions when RIF is combined with other medicines in application.

Cytochrome P450 reaction phenotyping and inhibition and induction studies of pinostrobin in human liver microsomes and hepatocytes.

Pinostrobin (PI, 5-hydroxy-7-methoxyflavanone) is a natural flavonoid known for its rich pharmacological activities. The objective of this study was to identify the human liver cytochrome P450 (CYP450) isoenzymes involved in the metabolism of PI. A single hydoxylated metabolite was obtained from PI after an incubation with pooled human liver microsomes (HLMs). The relative contributions of different CYP450s were evaluated using CYP450-selective inhibitors in HLMs and recombinant human CYP450 enzymes, and the results revealed the major involvement of CYP1A2, CYP2C9 and CYP2E1 in PI metabolism. We also evaluated the ability of PI to inhibit and induce human cytochrome P450 enzymes in vitro. High-performance liquid chromatography and liquid chromatography-tandem mass spectrometry analytical techniques were used to estimate the enzymatic activities of seven drug-metabolizing CYP450 isozymes in vitro. In HLMs, PI did not inhibit CYP 1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 or CYP3A4 (IC50 >100 μm). In the induction studies, PI had minimal effects on CYP1A2, CYP2B6and CYP3A4 activity. Based on these results, PI would not be expected to cause clinically significant CYP450 inhibition or induction.