Allosteric modulation of cytochrome P450 enzymes by the NADPH cytochrome P450 reductase FMN-containing domain.

NADPH-cytochrome P450 reductase delivers electrons required by heme oxygenase, squalene monooxygenase, fatty acid desaturase, and 48 human cytochrome P450 enzymes. While conformational changes supporting reductase intramolecular electron transfer are well defined, intermolecular interactions with these targets are poorly understood, in part because of their transient association. Herein the reductase FMN domain responsible for interacting with targets was fused to the N-terminus of three drug-metabolizing and two steroidogenic cytochrome P450 enzymes to increase the probability of interaction. These artificial fusion enzymes were profiled for their ability to bind their respective substrates and inhibitors and to perform catalysis supported by cumene hydroperoxide. Comparisons with the isolated P450 enzymes revealed that even the oxidized FMN domain causes substantial and diverse effects on P450 function. The FMN domain could increase, decrease, or not affect total ligand binding and/or dissociation constants depending on both P450 enzyme and ligand. As examples, FMN domain fusion has no effect on inhibitor ketoconazole binding to CYP17A1 but substantially altered CYP21A2 binding of the same compound. FMN domain fusion to CYP21A2 resulted in differential effects dependent on whether the ligand was 17α-hydroxyprogesterone versus ketoconazole. Similar enzyme-specific effects were observed on steady-state kinetics. These observations are most consistent with FMN domain interacting with the proximal P450 surface to allosterically impact P450 ligand binding and metabolism separate from electron delivery. The variety of effects on different P450 enzymes and on the same P450 with different ligands suggests intricate and differential allosteric communication between the P450 active site and its proximal reductase-binding surface.

Selective control of parasitic nematodes using bioactivated nematicides.

Parasitic nematodes are a major threat to global food security, particularly as the world amasses 10 billion people amid limited arable land1-4. Most traditional nematicides have been banned owing to poor nematode selectivity, leaving farmers with inadequate means of pest control4-12. Here we use the model nematode Caenorhabditis elegans to identify a family of selective imidazothiazole nematicides, called selectivins, that undergo cytochrome-p450-mediated bioactivation in nematodes. At low parts-per-million concentrations, selectivins perform comparably well with commercial nematicides to control root infection by Meloidogyne incognita, a highly destructive plant-parasitic nematode. Tests against numerous phylogenetically diverse non-target systems demonstrate that selectivins are more nematode-selective than most marketed nematicides. Selectivins are first-in-class bioactivated nematode controls that provide efficacy and nematode selectivity.

Inhibitory effect of 20(S)-protopanaxadiol on cytochrome P450: Potential of its pharmacokinetic interactions in vivo.

20(S)-Protopanaxadiol [20(S)-PPD] is a fully deglycosylated ginsenoside metabolite produced by the gut microbiota in the gastrointestinal tract. Although diverse pharmacological effects have been reported, information on the pharmacokinetic interactions of 20(S)-PPD with cytochrome P450s (CYPs) remains limited. Therefore, the inhibitory potential of 20(S)-PPD on CYP enzymes, which mainly contribute to drug pharmacokinetics, was investigated in this study. The inhibitory effect of 20(S)-PPD was strong for CYP3A4 and moderate for CYP2B6 in human liver microsomes. 20(S)-PPD inhibited Cyp3a and Cyp2b in mouse liver microsomes with a potency similar to that in humans. The solubility of 20(S)-PPD in the artificial intestinal fluid was close to IC50 values of Cyp3a and Cyp2b in the mouse intestine. Systemic exposure to buspirone (Cyp3a specific substrate) and bupropion (Cyp2b specific substrate) increased significantly, whereas the area under the plasma concentration-time curve (AUC) ratio of metabolite to parent drug decreased significantly when co-administered with 20(S)-PPD in mice. The pharmacokinetics of felodipine, a widely used anti-hypertensive agent metabolized mainly by Cyp3a, was also altered following 20(S)-PPD treatment in mice. In conclusion, 20(S)-PPD likely affects the in vivo metabolism of CYP3A4 or CYP2B6 substrates, suggesting a need for careful attention when concomitantly administering 20(S)-PPD with other medications. This study will broaden our understanding of ginseng and products containing precursor ginsenosides of 20(S)-PPD for safer and more efficient use in humans.

In vitro characterization of the furin inhibitor MI-1851: Albumin binding, interaction with cytochrome P450 enzymes and cytotoxicity.

The substrate-analog furin inhibitor MI-1851 can suppress the cleavage of SARS-CoV-2 spike protein and consequently produces significant antiviral effect on infected human airway epithelial cells. In this study, the interaction of inhibitor MI-1851 was examined with human serum albumin using fluorescence spectroscopy and ultrafiltration techniques. Furthermore, the impacts of MI-1851 on human microsomal hepatic cytochrome P450 (CYP) 1A2, 2C9, 2C19, 2D6 and 3A4 activities were assessed based on fluorometric assays. The inhibitory action was also examined on human recombinant CYP3A4 enzyme and on hepatocytes. In addition, microsomal stability (60 min) and cytotoxicity were tested as well. MI-1851 showed no relevant interaction with human serum albumin and was significantly depleted by human microsomes. Furthermore, it did not inhibit CYP1A2, 2C9, 2C19 and 2D6 enzymes. In human hepatocytes, CYP3A4 was significantly suppressed by MI-1851 and weak inhibition was noticed in regard to human microsomes and human recombinant CYP3A4. Finally, MI-1851 did not impair the viability and the oxidative status of primary human hepatocytes (up to 100 µM concentration). Based on these observations, furin inhibitor MI-1851 appears to be potential drug candidates in the treatment of COVID-19, due to the involvement of furin in S protein priming and thus activation of the pandemic SARS-CoV-2.

Interactions of resveratrol and its metabolites (resveratrol-3-sulfate, resveratrol-3-glucuronide, and dihydroresveratrol) with serum albumin, cytochrome P450 enzymes, and OATP transporters.

Resveratrol (RES) is a widely-known natural polyphenol which is also contained by several dietary supplements. Large doses of RES can result in high micromolar levels of its sulfate and glucuronide conjugates in the circulation, due to the high presystemic metabolism of the parent polyphenol. Pharmacokinetic interactions of RES have been extensively studied, while only limited data are available regarding its metabolites. Therefore, in the current study, we examined the interactions of resveratrol-3-sulfate (R3S), resveratrol-3-glucuronide, and dihydroresveratrol (DHR; a metabolite produced by the colon microbiota) with human serum albumin (HSA), cytochrome P450 (CYP) enzymes, and organic anion transporting polypeptides (OATP) employing in vitro models. Our results demonstrated that R3S and R3G may play a major role in the RES-induced pharmacokinetic interactions: (1) R3S can strongly displace the site I marker warfarin from HSA; (2) R3G showed similarly strong inhibitory action on CYP3A4 to RES; (3) R3S proved to be similarly strong (OATP1B1/3) or even stronger (OATP1A2 and OATP2B1) inhibitor of OATPs tested than RES, while R3G and RES showed comparable inhibitory actions on OATP2B1.

The impact of legacy and novel perfluoroalkyl substances on human cytochrome P450: An in vitro study on the inhibitory potential and underlying mechanisms.

Per- and polyfluoroalkyl substances (PFASs) are a group of synthetic compounds with a wide range of industrial applications. PFOA and PFOS have been the most extensively studied and have been associated with hepatotoxicity. Recently, the interaction with cytochrome P450 (CYP) has been proposed as a potential key molecular event leading to PFAS-induced hepatotoxicity. In the present study, we aimed to determine a structure-activity relationship between thirteen PFASs and their inhibitory potential on the activities of four CYPs (CYP2E1, CYP2D6, CYP3A4 and CYP2C19). The influence of PFASs (5-3200 µM) on CYP enzyme activities was measured using the Vivid® P450 metabolism assays. Using the same assays, Michaelis-Menten saturation curves were determined to explore the type of PFAS-induced CYP inhibition. Most PFASs were capable of inhibiting activity of the tested CYPs, as shown by their IC50 values. CYP2E1 is particularly inhibited by 3:1 FTOH, PFOA, and PFOS, whereas CYP2D6 is inhibited by PFHxS, PFHpA, PFOA, PFOS, PFNA, and PFDA. Additionally, CYP3A4 is most strongly inhibited by PFHxS, PFOA, PFOS, PFNA, and PFDA. Finally, CYP2C19 is inhibited by PFBS, PFHxS, PFHpA, PFOA, PFOS, PFNA, and PFDA. Interestingly, PFHxA and PFHxS induced an increase in CYP2E1 activity, whereas 4:2 FTOH strongly induced CYP2D6 activity. The mechanism of inhibition of CYPs by PFASs differed per CYP isoenzyme. CYP3A4 was competitively inhibited by PFBS, PFHxS, PFOS, PFNA and PFDA and non-competitively by PFOA. Additionally, CYP2C19 was competitively inhibited by PFHxA, PFOS and PFNA, whereas PFBS and PFHxS induced a mixed inhibition. Inhibition of CYP2C19 by PFHpA was atypical with an increased Vmax and a decreased Km. Finally, PFHxS competitively inhibited CYP2D6, whereas PFBS, PFOA, PFOS, PFDA and PFNA induced an atypical inhibition. Our results show that CYP inhibition by PFASs appears to be structure-dependent as well as CYP dependent. Inhibition of CYP2D6, CYP2C19 and CYP3A4 increased with increasing chain-lengths between six and nine carbons. The PFTOHs were only able to inhibit CYP2E1 and did not affect any of the other CYPS. Some PFASs remarkably induced the enzyme activity of CYPs. These results indicate that in addition to PFOA and PFOS, multiple novel PFASs may alter drug metabolism by the interference with CYPs.

In vitro metabolism of helenalin and its inhibitory effect on human cytochrome P450 activity.

Sesquiterpene lactone helenalin is used as an antiphlogistic in European and Chinese folk medicine. The pharmacological activities of helenalin have been extensively investigated, yet insufficient information exists about its metabolic properties. The objectives of the present study were (1) to investigate the in vitro NADPH-dependent metabolism of helenalin (5 and 100 µM) using human and rat liver microsomes and liver cytosol, (2) to elucidate the role of human cytochrome P450 (CYP) enzymes in its oxidative metabolism, and (3) to study the inhibition of human CYPs by helenalin. Five oxidative metabolites were detected in NADPH-dependent human and rat liver microsomal incubations, while two reduced metabolites were detected only in NADPH-dependent human microsomal and cytosolic incubations. In human liver microsomes, the main oxidative metabolite was 14-hydroxyhelenalin, and in rat liver microsomes 9-hydroxyhelenalin. The overall oxidation of helenalin was several times more efficient in rat than in human liver microsomes. In humans, CYP3A4 and CYP3A5 followed by CYP2B6 were the main enzymes responsible for the hepatic metabolism of helenalin. The extrahepatic CYP2A13 oxidized helenalin most efficiently among CYP enzymes, possessing the Km value of 0.6 µM. Helenalin inhibited CYP3A4 (IC50 = 18.7 µM) and CYP3A5 (IC50 = 62.6 µM), and acted as a mechanism-based inhibitor of CYP2A13 (IC50 = 1.1 µM, KI = 6.7 µM, and kinact = 0.58 ln(%)/min). It may be concluded that the metabolism of helenalin differs between rats and humans, in the latter its oxidation is catalyzed by hepatic CYP2B6, CYP3A4, CYP3A5, and CYP3A7, and extrahepatic CYP2A13.

Pyrrole as an Important Scaffold of Anticancer Drugs: Recent Advances.

With the significant increase of patients suffering from different types of cancer, it is evident that prompt measures in the development of novel and effective agents need to be taken. Pyrrole moiety has been found in various active compounds with anti-inflammatory, antiseptic, antibacterial, lipid-lowering and anticancer properties. Recent advances in the exploration of highly active and selective cytotoxic structures containing pyrrole motifs have shown promising data for future investigations. Accordingly, this review presents an overview of recent developments in the pyrrole derivatives as anticancer agents, with a main focus towards the key moieties required for the anti-tumor activities. Pyrrole molecules comprising prominent targeting capacities against microtubule polymerization, tyrosine kinases, cytochrome p450 family 1, histone deacetylase and bcl-2 proteins were reported. In addition, several mechanisms of action, such as apoptosis, cell cycle arrest, inhibiting kinases, angiogenesis, disruption of cell migration, modulation of nuclear receptor responsiveness and others were analyzed. Furthermore, in most of the discussed cases we provided synthesis schemes of the mentioned molecules. Overall, the utilization of pyrrole scaffold for the design and synthesis of novel anticancer drugs could be a promising approach for future investigations.

CYP450 drug inducibility in NAFLD via an in vitro hepatic model: Understanding drug-drug interactions in the fatty liver.

Drug-drug-interactions (DDIs) occur when a drug alters the metabolic rate, efficacy, and toxicity of concurrently used drugs. While almost 1 in 4 adults now use at least 3 concurrent prescription drugs in the United States, the Non-alcoholic fatty liver disease (NAFLD) prevalence has also risen over 25%. The effect of NALFD on DDIs is largely unknown. NAFLD is characterized by lipid vesicle accumulation in the liver, which can progress to severe steatohepatitis (NASH), fibrosis, cirrhosis, and hepatic carcinoma. The CYP450 enzyme family dysregulation in NAFLD, which might already alter the efficacy and toxicity of drugs, has been partially characterized. Nevertheless, the drug-induced dysregulation of CYP450 enzymes has not been studied in the fatty liver. These changes in enzymatic inducibility during NAFLD, when taking concurrent drugs, could cause unexpected fatalities through inadvertent DDIs. We have, thus, developed an in vitro model to investigate the CYP450 transcriptional regulation in NAFLD. Specifically, we cultured primary human hepatocytes in a medium containing free fatty acids, high glucose, and insulin for seven days. These cultures displayed intracellular macro-steatosis after 5 days and cytokine secretion resembling NAFLD patients. We further verified the model's dysregulation in the transcription of key CYP450 enzymes. We then exposed the NAFLD model to the drug inducers rifampicin, Omeprazole, and Phenytoin as activators of transcription factors pregnane X receptor (PXR), aryl hydrocarbon receptor (AHR) and constitutive androstane receptor (CAR), respectively. In the NAFLD model, Omeprazole maintained an expected induction of CYP1A1, however Phenytoin and Rifampicin showed elevated induction of CYP2B6 and CYP2C9 compared to healthy cultures. We, thus, conclude that the fatty liver could cause aggravated drug-drug interactions in NAFLD or NASH patients related to CYP2B6 and CYP2C9 enzymes.

Risk assessment of the inhibition of hydroxygenkwanin on human and rat cytochrome P450 by cocktail method.

Hydroxygenkwanin (HGK), a natural flavonoid extracted from the buds of Daphne genkwa Sieb.et Zucc. (Thymelaeaceae), possesses a wide range of pharmacological activities, including anti-inflammatory, antibacterial and anticancer. However, the inhibitory effect of HGK on cytochrome P450 (CYP) remains unclear. This study investigated the potential inhibitory effects of HGK on CYP1A2, 2B1/6, 2C9/11, 2D1/6, 2E1 and 3A2/4 enzymes in human and rat liver microsomes (HLMs and RLMs) by the cocktail approach. HGK exhibited no time-dependent inhibition of CYP activities in HLMs and RLMs. Enzyme inhibition kinetics indicated that HGK was not only a competitive inhibitor of human CYP1A2 and 2C9, but also competitively inhibited rat CYP1A2 and 2C11 activities, with Ki value at 0.84 ± 0.03, 8.09 ± 0.44, 2.68 ± 0.32 and 8.35 ± 0.31 µM, respectively. Further studies showed that the inhibitory effect of HGK on CYP enzymes was weaker than that of diosmetin, which may be related to the substitution of hydroxyl and methoxy in the A and B rings of the flavone skeleton. Therefore, the low Ki values of HGK for CYP1A2 and 2C may lead to potential drug-drug interactions and toxicity.

Impact of aerosols on liver xenobiotic metabolism: A comparison of two methods of exposure.

Assessment of aerosols effects on liver CYP function generally involves aqueous fractions (AF). Although easy and efficient, this method has not been optimized recently or comparatively assessed against other aerosol exposure methods. Here, we comparatively evaluated the effects of the AFs of cigarette smoke (CS) and Tobacco Heating System (THS) aerosols on CYP activity in liver spheroids. We then used these data to develop a physiological aerosol exposure system combining a multi-organs-on-a-chip, 3D lung tissues, liver spheroids, and a direct aerosol exposure system. Liver spheroids incubated with CS AF showed a dose-dependent increase in CYP1A1/1B1, CYP1A2, and CYP2B6 activity and a dose-dependent decrease in CYP2C9, CYP2D6, and CYP3A4 activity relative to untreated tissues. In our physiological exposure system, repeated CS exposure of the bronchial tissues also caused CYP1A1/1B1 and CYP1A2 induction in the bronchial tissues and liver spheroids; but the spheroids showed an increase in CYP3A4 activity and no effect on CYP2C9 or CYP2D6 activity relative to air-exposed tissues, which resembles the results reported in smokers. THS aerosol did not affect CYP activity in bronchial or liver tissues, even at 4 times higher concentrations than CS. In conclusion, our system allows us to physiologically test the effects of CS or other aerosols on lung and liver tissues cultured in the same chip circuit, thus delivering more in vivo like data.

The impact of individual human cytochrome P450 enzymes on oxidative metabolism of anticancer drug lenvatinib.

Lenvatinib, a small molecule tyrosine kinase inhibitor (TKI), exhibits good inhibitory effect in several types of carcinomas. Specifically, it is the most effective TKI used for treatment of thyroid cancer. To extend pharmacokinetics data on this anticancer agent, we aimed to identify the metabolites of lenvatinib formed during in vitro incubation of lenvatinib with human hepatic microsomes or recombinant cytochromes P450 (CYPs) by using high performance liquid chromatography and mass spectrometry. The role of CYPs in the oxidation of lenvatinib was initially investigated in hepatic microsomes using specific CYP inhibitors. CYP-catalytic activities in each microsomal sample were correlated with the amounts of lenvatinib metabolites formed by these samples. Further, human recombinant CYPs were employed in the metabolic studies. Based on our data, lenvatinib is metabolized to O-desmethyl lenvatinib, N-descyclopropyl lenvatinib and lenvatinib N-oxide. In the presence of cytochrome b5, recombinant CYP3A4 was the most efficient to form these metabolites. In addition, CYP1A1 significantly contributes to the lenvatinib metabolism. It was even more efficient in forming of O-desmethyl lenvatinib than CYP3A4 in the absence of cytochrome b5. The present study indicates that further research focused on drug-drug interactions, in particular on CYP3A4 and CYP1A1 modulators, is needed. This will pave new avenues towards TKIs-mediated personalized therapy.

Aidi injection altered the activity of CYP2D4, CYP1A2, CYP2C19, CYP3A2, CYP2E1 and CYP2C11 in normal and diethylnitrosamine-induced hepatocellular carcinoma in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Aidi injection (ADI), a traditional chinese medicine preparation, is widely used in combination with chemotherapy for the treatment of various malignant tumors, such as hepatocellular carcinoma (HCC). Studies have shown that changes in cytochrome P450 (CYP450) activity in disease states would affect the metabolism of drugs in vivo, especially liver diseases. However, the changes of Aidi injection on the activities of CYP2D4, CYP1A2, CYP2C19, CYP3A2, CYP2E1 and CYP2C11 in normal and HCC states are still unknown. AIM OF THE STUDY: The cocktail probe drugs method was used to investigate the effects of ADI on the activity of CYP2D4, CYP1A2, CYP2C19, CYP3A2, CYP2E1 and CYP2C11 in normal and HCC rats. MATERIALS AND METHODS: The HCC rats was induced by diethylnitrosamine (DEN). Then, both normal and HCC rats were randomly divided into 2 groups (n = 6). They were given saline or ADI (10 mL/kg/d, i.p) for 2 weeks, respectively. On the fifteenth day, cocktail probe mixing solution, including metoprolol (10 mg/kg), caffeine (1.0 mg/kg), omeprazole (2.0 mg/kg), midazolam (2.0 mg/kg), chlorzoxazone (4.0 mg/kg) and tolbutamide (0.5 mg/kg), was injected into tail vein of all rats in each group. The blood sample was obtained at specified time. After the protein is precipitated, six probe drugs are analyzed by ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). RESULTS: Compared with control group, the activity of CYP3A2 and CYP2E1 was significantly lower in the ADI group. Compared with the model group, the activities of CYP1A2, CYP3A2, CYP2E1, and CYP2C11 enzymes in the ADI model group were significantly reduced. Additionally, the activity of CYP2D4, CYP1A2, CYP2C19, CYP3A2, CYP2E1 and CYP2C11 enzymes in model group was significantly lower than control group. CONCLUSIONS: ADI can inhibit a lot of CYP450 enzyme, so it may reduce the dosage of chemotherapeutic drugs to reach the required plasma concentration of chemotherapeutic drugs, which is of great significance for the combination of anti-tumor chemotherapeutic drugs and is worthy of further in-depth study and clinical attention.

Translational aspects of cytochrome P450-mediated drug-drug interactions: A case study with clopidogrel.

Multimorbidity, polypharmacotherapy and drug interactions are increasingly common in the ageing population. Many drug-drug interactions (DDIs) are caused by perpetrator drugs inhibiting or inducing cytochrome P450 (CYP) enzymes, resulting in alterations of the plasma concentrations of a victim drug. DDIs can have a major negative health impact, and in the past, unrecognized DDIs have resulted in drug withdrawals from the market. Signals to investigate DDIs may emerge from a variety of sources. Nowadays, standard methods are widely available to identify and characterize the mechanisms of CYP-mediated DDIs in vitro. Clinical pharmacokinetic studies, in turn, provide experimental data on pharmacokinetic outcomes of DDIs. Physiologically based pharmacokinetic (PBPK) modelling utilizing both in vitro and in vivo data is a powerful tool to predict different DDI scenarios. Finally, epidemiological studies can provide estimates on the health outcomes of DDIs. Thus, to fully characterize the mechanisms, clinical effects and implications of CYP-mediated DDIs, translational research approaches are required. This minireview provides an overview of translational approaches to study CYP-mediated DDIs, going beyond regulatory DDI guidelines, and an illustrative case study of how the DDI potential of clopidogrel was unveiled by combining these different methods.

Flavonoid-statin interactions causing myopathy and the possible significance of OATP transport, CYP450 metabolism and mevalonate synthesis.

3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase inhibitors, statins, are a primary treatment for hyperlipidemic cardiovascular diseases which are a leading global cause of death. Statin therapy is life saving and discontinuation due to adverse events such as myotoxicity may lead to unfavourable outcomes. There is no known mechanism for statin-induced myotoxicity although it is theorized that it is due to inhibition of downstream products of the HMG-CoA pathway. It is known that drug-drug interactions with conventional medicines exacerbate the risk of statin-induced myotoxicity, though little attention has been paid to herb-drug interactions with complementary medicines. Flavonoids are a class of phytochemicals which can be purchased as high dose supplements. There is evidence that flavonoids can raise statin plasma levels, increasing the risk of statin-induced myopathy. This could be due to pharmacokinetic interactions involving hepatic cytochrome 450 (CYP450) metabolism and organic anion transporter (OATP) absorption. There is also the potential for flavonoids to directly and indirectly inhibit HMG-CoA reductase which could contraindicate statin-therapy. This review aims to discuss what is currently known about the potential for high dose flavonoids to interact with the hepatic CYP450 metabolism, OATP uptake of statins or their ability to interact with HMG-CoA reductase. Flavonoids of particular interest will be covered and the difficulties of examining herbal products will be discussed throughout.

A hypothetical skin sensitisation next generation risk assessment for coumarin in cosmetic products.

Next generation Risk Assessment (NGRA) is an exposure-led, hypothesis-driven approach which integrates new approach methodologies (NAMs) to assure safety without generating animal data. This hypothetical skin allergy risk assessment of two consumer products - face cream containing 0.1% coumarin and deodorant containing 1% coumarin - demonstrates the application of our skin allergy NGRA framework which incorporates our Skin Allergy Risk Assessment (SARA) Model. SARA uses Bayesian statistics to provide a human relevant point of departure and risk metric for a given chemical exposure based upon input data that can include both NAMs and historical in vivo studies. Regardless of whether NAM or in vivo inputs were used, the model predicted that the face cream and deodorant exposures were low and high risk respectively. Using only NAM data resulted in a minor underestimation of risk relative to in vivo. Coumarin is a predicted pro-hapten and consequently, when applying this mechanistic understanding to the selection of NAMs the discordance in relative risk could be minimized. This case study demonstrates how integrating a computational model and generating bespoke NAM data in a weight of evidence framework can build confidence in safety decision making.

Pharmacokinetic study on the interaction between succinic acid and irbesartan in rats and its potential mechanism.

CONTEXT: Succinic acid and irbesartan are commonly used drugs in cardiovascular disease treatment. The interaction might occur during their co-administration, which was still unclear. OBJECTIVE: To reveal the effect of succinic acid on the metabolism of irbesartan and its potential mechanism. MATERIALS AND METHODS: The Sprague-Dawley rats (n = 6) were treated with a single dose of 30 mg/kg irbesartan (control) or the co-administration with the pre-treatment of 200 mg/kg succinic acid for 7 d. The effect of succinic acid on the metabolic stability and the activity of CYP2C9 was evaluated in rat liver microsomes. RESULTS: Succinic acid increased the AUC (5328.71 ± 959.31 µg/L × h vs. 3340.23 ± 737.75 µg/L × h) and prolonged the half-life of irbesartan (from 12.79 ± 0.73 h to 20.59 ± 6.35 h). The Tmax (2.83 ± 0.75 h vs. 3.83 ± 1.10 h) and clearance rate (3.46 ± 1.13 L/h/kg vs. 6.91 ± 1.65 L/h/kg) of irbesartan was reduced by succinic acid. Consistently, succinic acid improved the metabolic stability (half-life from 23.32 ± 3.46 to 27.35 ± 2.15 min, intrinsic clearance rate from 59.43 ± 6.12 to 50.68 ± 5.64 µL/min/mg protein). Succinic acid was also found to inhibit the activity of CYP2C9 with the IC50 value of 13.87 µM. DISCUSSION AND CONCLUSIONS: Succinic acid increased the system exposure of irbesartan via inhibiting CYP2C9. The experiment design of this study also provides a reference for the further validation of this interaction in humans.

Plumbagin and Plumbago indica Differentially Modulated Cytochrome P450 and Transporter Profiles in BeWo and HepG2 Cells.

<b>Background and Objective:</b> The medicinal herb <i>Plumbago indica</i> (PI) and its major constituent plumbagin have reported pharmacological properties but there is a lack of information about their herb-drug interactions. The effects of methanolic (PI-MeOH) and ethanolic (PI-EtOH) crude extracts of PI and plumbagin on the expression of cytochrome P450s (<i>CYP1A2</i>, <i>CYP2E1</i> and <i>CYP3A4</i>) and transporters (<i>ABCC1</i>, <i>ABCG2</i> and <i>SLC22A11</i>) were investigated in BeWo and HepG2 cells. <b>Materials and Methods:</b> BeWo or HepG2 cells were treated with 0.5-5 µM plumbagin or 25-500 µg mL¹ of PI-MeOH or PI-EtOH for 24 hrs. Total RNA was extracted and mRNA expression of CYPs and transporters were determined using RT-qPCR. <b>Results:</b> PI and plumbagin affected mRNA expression differently in the two tested cell types. In BeWo cells, all concentrations of PI-MeOH induced <i>CYP2E1</i>, 100 and 500 µg Ml¹ PI-MeOH and PI-EtOH up-regulated <i>CYP1A2</i>, <i>CYP3A4 </i>and <i>ABCG2 </i>and 500 µg mL¹ PI-EtOH induced <i>ABCG2</i> expression. Plumbagin suppressed <i>CYP1A2</i> and induced <i>SLC22A11 </i>expression at the highest concentration, 5 µM. In HepG2 cells, 5 µM plumbagin and 500 µg Ml¹ PI-EtOH suppressed <i>CYP3A4 </i>expression and 500 µg mL¹ PI-MeOH and PI-EtOH up-regulated <i>CYP1A2</i> and <i>CYP2E1 </i>expression. <i>ABCC1</i> expression was induced by all treatments while <i>ABCG2</i> and <i>SLC22A11 </i>were induced only by 500 µg mL¹ PI-MeOH and PI-EtOH. <b>Conclusion:</b> The use of PI or plumbagin supplements in large quantities or for long periods should be carefully considered due to the risk of herbal drug interactions via modulated expression of CYPs and transporters.

Chronic treatment with asenapine affects cytochrome P450 2D (CYP2D) in rat brain and liver. Pharmacological aspects.

Neuroleptics have to be used for a long time to produce a therapeutic effect. Cytochrome P450 2D (CYP2D) enzymes mediate alternative pathways of neurotransmitter synthesis (i.e. tyramine hydroxylation to dopamine and 5-methoxytryptamine O-demethylation to serotonin), and metabolism of neurosteroids. The aim of our present study was to examine the influence of chronic treatment with the new atypical neuroleptic asenapine on CYP2D in rat brain. In parallel, liver CYP2D was investigated for comparison. Asenapine added in vitro to microsomes of control rats competitively, but weakly inhibited the activity of CYP2D (brain: Ki = 385 µM; liver: Ki = 36 µM). However, prolonged administration of asenapine (0.3 mg/kg sc. for 2 weeks) significantly diminished the activity and protein level of CYP2D in the frontal cortex, nucleus accumbens, hippocampus and cerebellum, but did not affect the enzyme in the hypothalamus, brain stem, substantia nigra and the remainder of the brain. In contrast, asenapine enhanced the enzyme activity and protein level in the striatum. In the liver, chronically administered asenapine reduced the activity and protein level of CYP2D, and the CYP2D1 mRNA level. In conclusion, prolonged administration of asenapine alters the CYP2D expression in the brain structures and in the liver. Through affecting the CYP2D activity in the brain, asenapine may modify its pharmacological effect. By increasing the CYP2D expression/activity in the striatum, asenapine may accelerate the synthesis of dopamine (via tyramine hydroxylation) and serotonin (via 5-methoxytryptamine O-demethylation), and thus alleviate extrapyramidal symptoms. By reducing the CYP2D expression/activity in other brain structures asenapine may diminish the 21-hydroxylation of neurosteroids and thus have a beneficial influence on the symptoms of schizophrenia. In the liver, by reducing the CYP2D activity, asenapine may slow the biotransformation of concomitantly administered CYP2D substrates (drugs) during continuous treatment of schizophrenia or bipolar disorders.

Transcriptomic Analyses Reveal 2 and 4 Family Members of Cytochromes P450 (CYP) Involved in LPS Inflammatory Response in Pharynx of Ciona robusta.

Cytochromes P450 (CYP) are enzymes responsible for the biotransformation of most endogenous and exogenous agents. The expression of each CYP is influenced by a unique combination of mechanisms and factors including genetic polymorphisms, induction by xenobiotics, and regulation by cytokines and hormones. In recent years, Ciona robusta, one of the closest living relatives of vertebrates, has become a model in various fields of biology, in particular for studying inflammatory response. Using an in vivo LPS exposure strategy, next-generation sequencing (NGS) and qRT-PCR combined with bioinformatics and in silico analyses, compared whole pharynx transcripts from naïve and LPS-exposed C. robusta, and we provide the first view of cytochrome genes expression and miRNA regulation in the inflammatory response induced by LPS in a hematopoietic organ. In C. robusta, cytochromes belonging to 2B,2C, 2J, 2U, 4B and 4F subfamilies were deregulated and miRNA network interactions suggest that different conserved and species-specific miRNAs are involved in post-transcriptional regulation of cytochrome genes and that there could be an interplay between specific miRNAs regulating both inflammation and cytochrome molecules in the inflammatory response in C. robusta.