Human CYP2B6 produces oxylipins from polyunsaturated fatty acids and reduces diet-induced obesity.

Multiple factors in addition to over consumption lead to obesity and non-alcoholic fatty liver disease (NAFLD) in the United States and worldwide. CYP2B6 is the only human detoxification CYP whose loss is associated with obesity, and Cyp2b-null mice show greater diet-induced obesity with increased steatosis than wildtype mice. However, a putative mechanism has not been determined. LC-MS/MS revealed that CYP2B6 metabolizes PUFAs, with a preference for metabolism of ALA to 9-HOTrE and to a lesser extent 13-HOTrE with a preference for metabolism of PUFAs at the 9- and 13-positions. To further study the role of CYP2B6 in vivo, humanized-CYP2B6-transgenic (hCYP2B6-Tg) and Cyp2b-null mice were fed a 60% high-fat diet for 16 weeks. Compared to Cyp2b-null mice, hCYP2B6-Tg mice showed reduced weight gain and metabolic disease as measured by glucose tolerance tests, however hCYP2B6-Tg male mice showed increased liver triglycerides. Serum and liver oxylipin metabolite concentrations increased in male hCYP2B6-Tg mice, while only serum oxylipins increased in female hCYP2B6-Tg mice with the greatest increases in LA oxylipins metabolized at the 9 and 13-positions. Several of these oxylipins, specifically 9-HODE, 9-HOTrE, and 13-oxoODE, are PPAR agonists. RNA-seq data also demonstrated sexually dimorphic changes in gene expression related to nuclear receptor signaling, especially CAR > PPAR with qPCR suggesting PPARγ signaling is more likely than PPARα signaling in male mice. Overall, our data indicates that CYP2B6 is an anti-obesity enzyme, but probably to a lesser extent than murine Cyp2b's. Therefore, the inhibition of CYP2B6 by xenobiotics or dietary fats can exacerbate obesity and metabolic disease potentially through disrupted PUFA metabolism and the production of key lipid metabolites.

Artemisia extracts differ from artemisinin effects on human hepatic CYP450s 2B6 and 3A4 in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE: The Chinese medicinal herb, Artemisia annua L., has been used for >2,000 yr as traditional tea infusions to treat a variety of infectious diseases including malaria, and its use is spreading globally (along with A. afra Jacq. ex Willd.) mainly through grassroots efforts. AIM OF THE STUDY: Artemisinin is more bioavailable delivered from the plant, Artemisia annua L. than the pure drug, but little is known about how delivery via a hot water infusion (tea) alters induction of hepatic CYP2B6 and CYP3A4 that metabolize artemisinin. MATERIALS AND METHODS: HepaRG cells were treated with 10 µM artemisinin or rifampicin (positive control), and teas (10 g/L) of A. annua SAM, and A. afra SEN and MAL with 1.6, 0.05 and 0 mg/g DW artemisinin in the leaves, respectively; qPCR and Western blots were used to measure CYP2B6 and CYP3A4 responses. Enzymatic activity of these P450s was measured using human liver microsomes and P450-Glo assays. RESULTS: All teas inhibited activity of CYP2B6 and CYP3A4. Artemisinin and the high artemisinin-containing tea infusion (SAM) induced CYP2B6 and CYP3A4 transcription, but artemisinin-deficient teas, MAL and SEN, did not. Artemisinin increased CYP2B6 and CYP3A4 protein levels, but none of the three teas did, indicating a post-transcription inhibition by all three teas. CONCLUSIONS: This study showed that Artemisia teas inhibit activity and artemisinin autoinduction of CYP2B6 and CYP3A4 post transcription, a response likely the effect of other phytochemicals in these teas. Results are important for understanding Artemisia tea posology.

Bulbine natalensis (currently Bulbine latifolia) and select bulbine knipholones modulate the activity of AhR, CYP1A2, CYP2B6, and P-gp.

Bulbine natalensis, an emerging medicinal herb on the global market with androgenic properties, is often formulated in dietary supplements that promote perceived sexual enhancement. However, to date, comprehensive safety studies of B. natalensis are lacking, particularly those related to its herb-drug interaction potential. The purpose of this study was to assess the inductive and inhibitory effects of extracts and pure compounds of B. natalensis on human cytochrome P-450 isozymes in vitro. Our findings demonstrated that both water and methanolic extracts of B. natalensis as well as knipholone, bulbine-knipholone, and 6'-O-methylknipholone dose-dependently increased mRNA expression encoded by CYP2B6, CYP1A2, and ABCB1 genes. Functional analyses showed that water (60 to 2.20 µg/mL) and methanolic (30 to 3.75 µg/mL) extracts and knipholones (10 to 0.33 µM) increased CYP2B6 and CYP1A2 activity in a dose-dependent manner. Additionally, water extract (60 µg/mL), methanolic extract (30 µg/mL), and knipholone (10 µM) caused activation of the aryl hydrocarbon receptor up to 11.1 ± 0.7, 8.9 ± 0.6, and 7.1 ± 2.0-fold, respectively. Furthermore, inhibition studies revealed that methanolic extract attenuated the activity of metabolically active CYP1A2 (IC50, 22.6 ± 0.4 µg/mL) and CYP2B6 (IC50, 34.2 ± 6.6 µg/mL) proteins, whereas water extracts had no inhibitory effect on either isoform. These findings suggest that chronic consumption of B. natalensis may affect normal homeostasis of select CYPs with subsequent risks for HDIs when concomitantly ingested with conventional medications that are substrates of CYP2B6 and CYP1A2. However, more in-depth translational studies are required to validate our current findings and their clinical relevance.

Oxypeucedanin is a Mechanism-based Inactivator of CYP2B6 and CYP2D6.

BACKGROUND: Herbal medicine Angelica dahurica is widely employed for the treatment of rheumatism and pain relief in China. Oxypeucedanin is a major component in the herb. OBJECTIVES: The objectives of this study are aimed at the investigation of mechanism-based inactivation of CYP2B6 and CYP2D6 by oxypeucedanin, characterization of the reactive metabolites associated with the enzyme inactivation, and identification of the P450s participating in the bioactivation of oxypeucedanin. METHODS: Oxypeucedanin was incubated with liver microsomes or recombinant CYPs2B6 and 2D6 under designed conditions, and the enzyme activities were measured by monitoring the generation of the corresponding products. The resulting reactive intermediates were trapped with GSH and analyzed by LC-MS/MS. RESULTS: Microsomal incubation with oxypeucedanin induced a time-, concentration-, and NADPH-dependent inhibition of CYPs2B6 and 2D6 with kinetic values of KI/kinact 1.82 µM/0.07 min-1 (CYP2B6) and 8.47 µM/0.044 min-1 (CYP2D6), respectively. Ticlopidine and quinidine attenuated the observed time-dependent enzyme inhibitions. An epoxide and/or γ-ketoenal intermediate(s) derived from oxypeucedanin was/were trapped in microsomal incubations. CYP3A4 was the primary enzyme involved in the bioactivation of oxypeucedanin. CONCLUSION: Oxypeucedanin was a mechanism-based inactivator of CYP2B6 and CYP2D6. An epoxide and/or γ- ketoenal intermediate(s) may be responsible for the inactivation of the two enzymes.

Investigation of CYP2B6, 3A4 and ß-esterase interactions of Withania somnifera (L.) dunal in human liver microsomes and HepG2 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Withania somnifera (L.) Dunal (Solanaceae) is a traditional herb, used in African indigenous systems of medicine for the treatment of various diseases (including HIV/AIDS and tuberculosis). The relevance of clinically significant interactions of Withania with ARVs and anti-TB drugs needs to be investigated. AIM OF THE STUDY: This study evaluated the effects of its roots on cytochromes P450 (CYPs) 2B6, 3A4, and rifampicin metabolism pathway, using methanol, ethanol, aqueous, and ethyl acetate solvent extractions. MATERIALS AND METHODS: The extracts were tested on human liver microsomes (HLM) for CYP inhibition, mRNA expression in HepG2 cells for CYP induction. Biochemical qualitative tests and LC-MS/MS methodology were used to determine active phytoconstituents. RESULTS: The methanolic and ethyl acetate extracts inhibited CYP2B6 with IC50s 79.16 and 57.96 µg/ml respectively, while none of the extracts had any effect on rifampicin metabolism or showed time-dependant inhibition (TDI). All extracts were moderate inducers of CYP3A4; the aqueous extract exhibited 38%-fold shift induction of CYP3A4 compared to the control. The methanolic extract had the lowest CTC50 (50% of cytotoxicity inhibition) (67.13 ± 0.83 µg/ml). LC-MS/MS-PDA full scans were consistent with the presence of flavone salvigenin (m/z 327), alkaloid isopelletierine (m/z 133), steroidal lactone 2,3-dihydrowithaferin-A (m/z 472), and other withanolides including withaperuvin I (m/z 533), withaferin derivative (m/z 567), some of these compounds likely being responsible for the observed CYP2B6 inhibition and CYP3A4 induction. The putative gastrointestinal tract (GIT) concentration for the active extracts was 1800 µg/ml and the hepatic circulation concentrations were estimated at about 220 µg/ml and 13.5 µg/ml for the methanolic and ethyl acetate extracts, respectively. The extrapolated in vivo percentage of inhibition was at 85% for the methanolic extract against CYP2B6. CONCLUSIONS: The findings reported in this study suggest that W. somnifera extracts have the potential of causing clinically significant herb-drug interactions (HDI) as moderate inducer of CYP3A4 and inhibitor of CYP2B6 metabolism pathway (methanol and ethyl acetate extracts).

In vitro effects of 95% khat ethanol extract (KEE) on human recombinant cytochrome P450 (CYP)1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C19, CYP2E1, CYP2J2 and CYP3A5.

OBJECTIVES: Khat, a natural amphetamine-like psychostimulant plant, are widely consumed globally. Concurrent intake of khat and xenobiotics may lead to herb-drug interactions and adverse drug reactions (ADRs). This study is a continuation of our previous study, targeted to evaluate the in vitro inhibitory effects of khat ethanol extract (KEE) on human cytochrome (CYP) 1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C19, CYP2E1, CYP2J2, and CYP3A5, major human drug metabolizing enzymes. METHODS: In vitro fluorescence enzyme assays were employed to assess CYPs inhibition with the presence and absence of various KEE concentrations. RESULTS: KEE reversibly inhibited CYP2A6, CYP2B6, CYP2C8, CYP2C19, CYP2E1, CYP2J2 and CYP3A5 but not CYP1A2 with IC50 values of 25.5, 99, 4.5, 21, 27, 17, and 10 µg/mL respectively. No irreversible inhibition of KEE on all the eight CYPs were identified. The Ki values of CYP2A6, CYP2B6, CYP2C8, CYP2C19, CYP2E1, CYP2J2 and CYP3A5 were 20.9, 85, 4.8, 18.3, 59.3, 3, and 21.7 µg/mL, respectively. KEE inhibited CYP2B6 via competitive or mixed inhibition; CYP2E1 via un-competitive or mixed inhibition; while CYP2A6, CYP2C8, CYP2C19, CYP2J2 and CYP3A5 via non-competitive or mixed inhibition. CONCLUSIONS: Caution should be taken by khat users who are on medications metabolized by CYP2A6, CYP2B6, CYP2C8, CYP2C19, CYP2E1, CYP2J2, and CYP3A5.

Dietary Squalene Induces Cytochromes Cyp2b10 and Cyp2c55 Independently of Sex, Dose, and Diet in Several Mouse Models.

SCOPE: To investigate the effects of squalene, the main hydrocarbon present in extra virgin olive oil, on liver transcriptome in different animal models and to test the influence of sex on this action and its relationship with hepatic lipids. METHODS AND RESULTS: To this purpose, male C57BL/6J Apoe-deficient mice are fed a purified Western diet with or without squalene during 11 weeks and hepatic squalene content is assessed, so are hepatic lipids and lipid droplets. Hepatic transcriptomic changes are studied and confirmed by RT-qPCR. Dietary characteristics and influence of squalene doses are tested in Apoe-deficient on purified chow diets with or without squalene. These diets are also given to Apoa1 and wild-type mice on C57BL/6J background and to C57BL/6J xOla129 Apoe-deficient mice. Squalene supplementation increases its hepatic content without differences among sexes and hormonal status. The Cyp2b10 and Cyp2c55 gene expressions are significantly up-regulated by the squalene intake in all models, with independence of sex, sexual hormones, dietary fat content, genetic background and dose, and in Apoe-deficient mice consuming extra-virgin olive oil. CONCLUSION: Hepatic squalene increases the expression of these cytochromes and their changes in virgin olive oil diets may be due to their squalene content.

Analysis of hiPSCs differentiation toward hepatocyte-like cells upon extended exposition to oncostatin.

The capability to produce and maintain functional human adult hepatocytes remains one of the major challenges for the use of in-vitro models toward liver cell therapy and industrial drug-screening applications. Among the suggested strategies to solve this issue, the use of human-induced pluripotent stem cells (hiPSCs), differentiated toward hepatocyte-like cells (HLCs) is promising. In this work, we propose a 31-day long protocol, that includes a final 14-day long phase of oncostatin treatment, as opposed to a 7-day treatment which led to the formation of a hepatic tissue functional for CYP1A2, CYP2B6, CYP2C8, CYP2D6, and CYP3A4. The production of albumin, as well as bile acid metabolism and transport, were also detected. Transcriptome profile comparisons and liver transcription factors (TFs) motif dynamics revealed increased expression of typical hepatic markers such as HNF1A and of important metabolic markers like PPARA. The performed analysis has allowed for the extraction of potential targets and pathways which would allow enhanced hepatic maturation in-vitro. From this investigation, NRF1 and SP3 appeared as transcription factors of importance. Complex epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) patterns were also observed during the differentiation process. Moreover, whole transcriptome analysis highlighted a response typical of the one observed in liver regeneration and hepatocyte proliferation. While a complete maturation of hepatocytes was yet to be obtained, the results presented in this work provide new insights into the process of liver development and highlight potential targets aimed to improve in-vitro liver regeneration.

NADPH-Independent Inactivation of CYP2B6 and NADPH-Dependent Inactivation of CYP3A4/5 by PBD: Potential Implication for Assessing Covalent Modulators for Time-Dependent Inhibition.

Pyrrolo[2,1-c][1,4]benzodiazepine dimer (PBD) has shown broad antitumor properties and potential as a therapeutic agent for cancers. During a routine drug-drug interaction assessment, it was found that PBD is a reversible inhibitor of CYP2C8 (IC50 = 1.1 µM) but not CYP1A2, 2B6, 2C9, 2C19, 2D6, or 3A4/5. Additionally, PBD is a classic time-dependent inhibition (TDI) of CYP3A4/5, with >30-fold shift in IC50 after a preincubation with NADPH. All other CYPs tested did not show evidence for TDI, but potent inhibition of CYP2B6 (IC50 = 1.5 µM) was observed after a preincubation with or without (w/wo) NADPH, which was an unexpected observation given the fact that no inhibition was observed in the direct inhibition assay. No other CYP isoforms were susceptible to this apparent non-NADPH-dependent inhibition, suggesting that PBD may selectively inactivate CYP2B6 without metabolic activation. The washing of the human liver microsome pellet after incubation with PBD did not fully recover CYP2B6 activity, indicating that PBD is covalently bound to CYP2B6, leading to inactivation of the enzyme. To further investigate the mechanism of NADPH-independent inhibition, the IC50 shift was determined for several PBD analogs, and it was found that the compounds without both reactive imines did not show NADPH-independent inhibition of CYP2B6, implying that NADPH-independent inactivation was likely caused by direct covalent binding of PBD to the enzyme in a highly structure-specific manner. These data clearly highlight the need to assess direct and time-dependent inhibition w/wo NADPH to adequately characterize the in vitro CYP inhibitory properties of drug candidates with reactive moieties. SIGNIFICANCE STATEMENT: We described a very unique in vitro CYP inhibition profile of pyrrolo[2,1-c][1,4]benzodiazepine dimer as a potent reversible CYP2C8 inhibitor, an NADPH-dependent CYP3A4/5 time-dependent inhibition (TDI) inhibitor, and an NADPH-independent CYP2B6 TDI inhibitor, and inhibition of CYPs occurs through three distinct mechanisms: reversible drug-enzyme binding, enzyme inactivation via bioactivation, and enzyme inactivation by covalent binding via chemical reactions. Our results suggest that, for compounds with reactive functional moieties, false positives can be reported when the conventional TDI assay is utilized.

Differential Effects of Components in Artemisia annua Extract on the Induction of Drug-Metabolizing Enzyme Expression Mediated by Nuclear Receptors.

Artemisia annua tea is a popular dosage form used to treat and prevent malaria in some developing countries. However, repeated drinking leads to an obviously decreased efficacy, which may be related to the induction of metabolizing enzymes by artemisinin. In the present study, the ability of different components in A. annua to activate the pregnane X receptor and constitutive androstane receptor was evaluated by the dual luciferase reporter gene system. The changes in mRNA and protein expression of CYP3A4 and CYP2B6 were determined by quantitative real-time PCR and Western blotting. Results showed that in the pregnane X receptor-mediated CYP3A4 reporter gene system, chrysosplenetin and arteannuin B exhibited a weak induction effect on pregnane X receptor wt, while arteannuin A had a strong induction effect on pregnane X receptor wt and pregnane X receptor 370 and a weak induction effect on pregnane X receptor 163. In the pregnane X receptor-mediated CYP2B6 reporter gene system, arteannuin A had a moderate induction effect on pregnane X receptor wt and pregnane X receptor 379, and a weak induction effect on pregnane X receptor 403, while arteannuin B had a weak induction effect on pregnane X receptor wt and pregnane X receptor 379. Arteannuin A had a strong induction effect on constitutive androstane receptor 3 in constitutive androstane receptor-mediated CYP3A4/2B6 reporter gene systems, while arteannuin B showed a weak induction effect on constitutive androstane receptor 3 in the constitutive androstane receptor-mediated CYP2B6 reporter gene system. The mRNA and protein expressions of CYP3A4 and CYP2B6 were increased when the pregnane X receptor or constitutive androstane receptor was activated. Various components present in A. annua differentially affect the activities of pregnane X receptor isoforms and the constitutive androstane receptor, which indicates the possibility of a drug-drug interaction. This partly explains the decline in efficacy after repeated drinking of A. annua tea.

Evaluation of Cytochrome P450 Selectivity for Hydralazine as an Aldehyde Oxidase Inhibitor for Reaction Phenotyping.

Hydralazine has been reported as a selective mechanism-based inactivator of aldehyde oxidase (AO) and it is widely used in the pharmaceutical industry for reaction phenotyping to estimate fraction metabolized by AO and to identify AO substrates. In this study, however, hydralazine was found to inhibit CYP1A2, 2B6, 2D6, and 3A in human suspension hepatocytes under reaction phenotyping assay conditions, at concentrations that chemically knocked out most of the AO activities (≥50 µM). Furthermore, hydralazine is a time-dependent inhibitor of CYP1A2. Based on these findings, precautions need to be taken when using hydralazine as an AO inhibitor for in vitro studies because fraction metabolized by AO is likely to be overestimated and the likelihood of false positives in identifying AO substrates increases.

Essential oils of culinary herbs and spices activate PXR and induce CYP3A4 in human intestinal and hepatic in vitro models.

Essential oils (EOs) are extensively used in food industry, gastronomy and alternative medicine. They are multicomponent mixtures of bioactive compounds; hence, their potential for food-drug interactions is substantial. In this study, we investigated the effects of 31 EOs of culinary herbs and spices on the transcriptional activity of pregnane X receptor (PXR) and expression of cytochrome P450 3A4 (CYP3A4), using human intestinal and hepatic in vitro models. All tested EOs activated PXR in intestinal LS180 cells transiently transfected with PXR, as revealed by a reporter gene assay. Consistently, all EOs induced CYP3A4 mRNA expression in PXR-transfected LS180 cells, primary human hepatocytes and wild-type hepatic progenitor HepaRG cells. EO-mediated induction of CYP3A4 mRNA expression was nullified in PXR-knock out HepaRG cells, suggesting the involvement of PXR in these effects. Collectively, we showed that EOs of culinary herbs and spices might be common activators of PXR and inducers of CYP3A4 at doses present in foods, thereby, they might have a potential for food-drug interactions. Follow-up studies are warranted to identify the bioactive constituents in the tested EOs.

Enzyme Kinetics and Molecular Docking Studies on Cytochrome 2B6, 2C19, 2E1, and 3A4 Activities by Sauchinone.

Sauchinone, an active lignan isolated from the aerial parts of Saururus chinensis (Saururaceae), exhibits anti-inflammatory, anti-obesity, anti-hyperglycemic, and anti-hepatic steatosis effects. As herb-drug interaction (HDI) through cytochrome P450s (CYPs)-mediated metabolism limits clinical application of herbs and drugs in combination, this study sought to explore the enzyme kinetics of sauchinone towards CYP inhibition in in vitro human liver microsomes (HLMs) and in vivo mice studies and computational molecular docking analysis. In in vitro HLMs, sauchinone reversibly inhibited CYP2B6, 2C19, 2E1, and 3A4 activities in non-competitive modes, showing inhibition constant (Ki) values of 14.3, 16.8, 41.7, and 6.84 µM, respectively. Also, sauchinone time-dependently inhibited CYP2B6, 2E1 and 3A4 activities in vitro HLMs. Molecular docking study showed that sauchinone could be bound to a few key amino acid residues in the active site of CYP2B6, 2C19, 2E1, and 3A4. When sibutramine, clopidogrel, or chlorzoxazone was co-administered with sauchinone to mice, the systemic exposure of each drug was increased compared to that without sauchinone, because sauchinone reduced the metabolic clearance of each drug. In conclusion, when sauchinone was co-treated with drugs metabolized via CYP2B6, 2C19, 2E1, or 3A4, sauchinone-drug interactions occurred because sauchinone inhibited the CYP-mediated metabolic activities.

Modulation of pregnane X receptor (PXR) and constitutive androstane receptor (CAR) activation by ursolic acid (UA) attenuates rifampin-isoniazid cytotoxicity.

BACKGROUND: Interactions between transcriptional inducers of cytochrome P450 (CYP450) enzymes and therapeutic drugs may be prevented by antagonizing the activation of a nuclear receptor (NR), pregnane X receptor (PXR, NR1I2), thus improving therapeutic efficacy. PURPOSE: In the present study, we aim to identify that ursolic acid (UA), a widely distributed pentacyclic triterpene, may act as an effective antagonist of PXR and its sister NR receptor, constitutive androstane receptor (CAR, NR1I3). METHODS: The hepatocellular carcinoma cell line, HepG2, was used to evaluate the promoter activity of PXR and CAR target genes, CYP3A4 and CYP2B6, respectively. Catalytic activities, mRNA, and protein expression of CYP3A4 and CYP2B6 were evaluated in a differentiated HepaRG cell line. Coregulation of PXR with coregulators on CYP3A4 promoter response elements was also been characterized. RESULTS: Transient transfection assays showed that UA effectively attenuated CYP3A4 and CYP2B6 promoter activities mediated by rifampin (RIF, human PXR agonist) and CITCO (human CAR agonist). These inhibitory effects were well correlated with the expression and catalytic activities of CYP3A4 and CYP2B6. Furthermore, the interaction of co-regulators with PXR and the transcriptional complexes in the CYP3A4 promoter activity and CYP3A4 promoter xenobiotic response element (everted repeat 6, ER6), respectively, were disrupted in the presence of UA. UA showed an antagonistic effect against PXR, and reversed the cytotoxic effects of isoniazid (INH) induced by RIF. Taken together, these results show that UA inhibits the transactivation effects of PXR and CAR, and reduces the expression and function of CYP3A4 and CYP2B6. CONCLUSION: The present study suggests that UA could be a powerful agent for reducing potentially dangerous interactions between transcriptional inducers of CYP enzymes and therapeutic drugs.

Inhibitory Effects of Garcinia cambogia Extract on CYP2B6 Enzyme Activity.

This study assessed the inhibitory effects of Garcinia cambogia extract on the cytochrome P450 enzymes in vitro. G. cambogia extract was incubated with cytochrome P450 isozyme-specific substrates in human liver microsomes and recombinant CYP2B6 isozyme, and the formation of the marker metabolites was measured to investigate the inhibitory potential on cytochrome P450 enzyme activities. The results showed that G. cambogia extract has significant inhibitory effects on CYP2B6 activity in a concentration-dependent manner. Furthermore, the inhibition was potentiated following preincubation with NADPH, indicating that G. cambogia extract is a time-dependent inhibitor of CYP2B6. Meanwhile, hydroxycitric acid, the major bioactive ingredient of G. cambogia extract, did not exhibit significant inhibition effects on cytochrome P450 enzyme activities. G. cambogia extract could modulate the pharmacokinetics of CYP2B6 substrate drugs and lead to interactions with those drugs. Therefore, caution may be required with respect to concomitant intake of dietary supplements containing G. cambogia extract with CYP2B6 substrates.

The Use of Human Liver Cell Model and Cytochrome P450 Substrate-Inhibitor Panel for Studies of Dasatinib and Warfarin Interactions.

The possibility of interactions between warfarin and dasatinib and their interactions with other drugs metabolized by cytochrome P450 isoform CYP3A4 was demonstrated using a previously created cytochrome P450 substrate-inhibitor panel for preclinical in vitro studies of drug biotransformation on a 3D histotypical microfluidic cell model of human liver (liver-on-a-chip technology). Dasatinib and warfarin are inhibitors of CYP2C19 isoform and hence, can interfere the drugs metabolized by this isoform. Our findings are in line with the data obtained on primary culture of human hepatocytes and suggest that the model can be used in preclinical in vitro studies of drugs.

Strong Induction of Cytochrome P450 1A/3A, But not P450 2B, in Cultured Hepatocytes from Common Marmosets and Cynomolgus Monkeys by Typical Human P450 Inducing Agents.

BACKGROUND: Common marmosets (Callithrix jacchus) and cynomolgus monkeys (Macaca fascicularis) are used as non-human primate models in preclinical studies for drug development. OBJECTIVE: The assessment of P450 induction in hepatocytes from marmosets and cynomolgus monkeys was performed using typical P450 inducers. METHODS: Induction of cytochrome P450 1-4 family enzymes was analyzed in two lots of cultured hepatocytes from common marmosets and cynomolgus monkeys after 24-h treatment with typical human P450 inducing agents by real-time reverse transcription-polymerase chain reaction. RESULTS: Marmoset P450 3A4 mRNA and P450 2C8/2C19 mRNA in hepatocytes were strongly (>10- fold) and weakly (>2) induced by rifampicin, respectively. Marmoset 1A1 and 1A2 mRNA were induced strongly (>200-fold) by ß-naphthoflavone and omeprazole. Marmoset P450 2B6 mRNA was induced (~5-fold) by a constitutive androstane receptor agonist, but not by phenobarbital. Cynomolgus monkey P450 3A4 mRNA and P450 1A1 mRNA in cultured hepatocytes were also induced by rifampicin and omeprazole, respectively, but P450 2B6 mRNA was not induced by phenobarbital. CONCLUSION: These results indicate that P450 1A/3A induction by typical human P450 inducers in hepatocytes from marmosets and/or cynomolgus monkeys are similar to those of humans (except for P450 2B induction by phenobarbital in humans), suggesting that marmosets and cynomolgus monkeys might be suitable models for evaluating the drug interactions in preclinical studies.

Inhibition of CYP2B6 by Medicinal Plant Extracts: Implication for Use of Efavirenz and Nevirapine-Based Highly Active Anti-Retroviral Therapy (HAART) in Resource-Limited Settings.

Highly active antiretroviral therapy (HAART) has greatly improved health parameters of HIV infected individuals. However, there are several challenges associated with the chronic nature of HAART administration. For populations in health transition, dual use of medicinal plant extracts and conventional medicine poses a significant challenge. There is need to evaluate interactions between commonly used medicinal plant extracts and antiretroviral drugs used against HIV/AIDS. Efavirenz (EFV) and nevirapine (NVP) are the major components of HAART both metabolized by CYP2B6, an enzyme that can potentially be inhibited or induced by compounds found in medicinal plant extracts. The purpose of this study was to evaluate the effects of extracts of selected commonly used medicinal plants on CYP2B6 enzyme activity. Recombinant human CYP2B6 was used to evaluate inhibition, allowing the assessment of herb-drug interactions (HDI) of medicinal plants Hyptis suaveolens, Myrothamnus flabellifolius, Launaea taraxacifolia, Boerhavia diffusa and Newbouldia laevis. The potential of these medicinal extracts to cause HDI was ranked accordingly for reversible inhibition and also classified as potential time-dependent inhibitor (TDI) candidates. The most potent inhibitor for CYP2B6 was Hyptis suaveolens extract (IC50 = 19.09 ± 1.16 µg/mL), followed by Myrothamnus flabellifolius extract (IC50 = 23.66 ± 4.86 µg/mL), Launaea taraxacifolia extract (IC50 = 33.87 ± 1.54 µg/mL), and Boerhavia diffusa extract (IC50 = 34.93 ± 1.06 µg/mL). Newbouldia laevis extract, however, exhibited weak inhibitory effects (IC50 = 100 ± 8.71 µg/mL) on CYP2B6. Launaea taraxacifolia exhibited a TDI (3.17) effect on CYP2B6 and showed a high concentration of known CYP450 inhibitory phenolic compounds, chlorogenic acid and caffeic acid. The implication for these observations is that drugs that are metabolized by CYP2B6 when co-administered with these herbal medicines and when adequate amounts of the extracts reach the liver, there is a high likelihood of standard doses affecting drug plasma concentrations which could lead to toxicity.

Regulation of drug-metabolizing enzymes and efflux transporters by Astragali radix decoction and its main bioactive compounds: Implication for clinical drug-drug interactions.

ETHNOPHARMACOLOGICAL RELEVANCE: Astragali radix ("Huang Qi" in Chinese, HQ) is a well-known traditional Chinese herbal medicine that possesses various biological functions. Astragaloside IV (AS-IV), calycosin (CS), and formononetin (FMNT) are the three main bioactive compounds of HQ that are responsible for its pharmacological activities and therapeutic efficacy. AIM OF THE STUDY: This study aims to investigate the effects of HQ, AS-IV, CS, and FMNT on major human drug-metabolizing enzymes (DMEs), including CYP3A4, CYP2B6, CYP2E1, UGT1A, UGT1A6, SULT1A1, and SULT1A3, as well as efflux transporters (ETs), including P-gp, MRP2, BCRP, MRP1, and MRP3, by using HepG2 cell line. Results would provide beneficial information for the proper clinical application of HQ. MATERIALS AND METHODS: HepG2 cells were treated with HQ, AS-IV, CS, and FMNT for 96h. Cell viability was examined by MTT assay. The protein and mRNA levels of DMEs and ETs were measured using Western blot and real-time PCR, respectively. RESULTS: Compared with the control group, HQ considerably increased the expression levels of CYP3A4, CYP2B6, CYP2E1, UGT1A, P-gp, MRP2, BCRP, and MRP3 in a dose-dependent manner. Inversely, HQ significantly decreased the protein levels of UGT1A6, SULT1A1, and MRP1. Exposure to AS-IV induced the protein levels of UGT1A, P-gp, MRP1, and MRP3, but produced inhibitory effects on CYP3A4, CYP2B6, and BCRP. The expression levels of CYP3A4, UGT1A, SULT1A1, P-gp, MRP2, and MRP3 were remarkably increased in the CS-treated cells, whereas the protein levels of SULT1A3 and BCRP were decreased. FMNT treatment induced the protein levels towards CYP3A4, CYP2B6, UGT1A, P-gp, MRP1, MRP2, and MRP3, but inhibited the expression of CYP2E1, SULT1A1, and SULT1A3. CONCLUSIONS: HQ and its main bioactive compounds, including AS-IV, CS, and FMNT significantly regulated the expression of the major DMEs and ETs. HQ produced stronger regulations (induction or inhibition) on DMEs and ETs than AS-IV, CS, or FMNT alone. The results indicate that potential drug-drug interactions might exist when the tested drugs, specifically HQ, are co-administered with other substrate drugs that are metabolized or transported via the studied DMEs or ETs. This study provides beneficial information for appropriate use of HQ for clinical therapy.

Mechanism-based inactivation of cytochrome P450 2B6 by isopsoralen.

1. Isopsoralen (IPRN) is a major component in many traditional medicinal herbs widely used in Asian countries. The objective of the present study was to investigate the inhibitory effect of IPRN on cytochrome P450 2B6 (CYP2B6) and the mechanism involved in the enzyme inactivation. 2. Pre-incubation of CYP2B6 with IPRN resulted in a time- and concentration-dependent enzyme activity loss. The values of K(I) and k(inact) were found to be 7.89 µM and 0.067 min(-1), respectively. Ticlopidine exhibited protective effect on the IPRN-induced enzyme inactivation. The estimated partition ratio of the inactivation was 122. The GSH trapping experiments indicate that an epoxide and/or γ-ketoenal intermediate were/was generated in IPRN-fortified microsomal incubations. The synthetic work verified the formation of the reactive intermediate(s). Additionally, CYPs2E1, 2C19, 2B6 and 1A2 were found to be the major enzymes participating in the bioactivation of IPRN. 3. IPRN was characterized as a mechanism-based inactivator of CYP2B6. An IPRN-derived furanoepoxide and/or γ-ketoenal intermediate(s) were/was generated and may be responsible for the inactivation of CYP2B6.