Inhibition Of Cytochrome P450s Research Articles

Fenoxaprop-P-ethyl, mesosulfuron-ethyl, and isoproturon resistance status in Beckmannia syzigachne from wheat fields across Anhui Province, China

Severe infestations of American sloughgrass (Beckmannia syzigachne (Steud.) Fernald) in wheat fields throughout Anhui Province, China, pose a significant threat to local agricultural production. This study aims to evaluate the susceptibility of 37 B. syzigachne populations collected from diverse wheat fields in Anhui Province to three commonly used herbicides: fenoxaprop-P-ethyl, mesosulfuron-ethyl, and isoproturon. Single-dose testing revealed that out of the 37 populations, 31, 26, and 11 populations had either evolved or were evolving resistance to fenoxaprop-P-ethyl, mesosulfuron-ethyl, and isoproturon, respectively. Among them, 25 populations displayed concurrent resistance to both fenoxaprop-P-ethyl and mesosulfuron-ethyl, while eight exhibited resistance to all three tested herbicides. Whole-plant bioassays confirmed that approximately 84% of the fenoxaprop-P-ethyl-resistant populations manifested high-level resistance (resistance index (RI) ≥10); 62% of the mesosulfuron-ethyl-resistant populations and 82% of the isoproturon-resistant populations exhibited low- to moderate-level resistance (2 ≤ RI <10). Three distinct target-site mutations were identified in 27% of fenoxaprop-P-ethyl-resistant populations, with no known resistance mutations detected in the remaining herbicide-resistant populations. The inhibition of cytochrome P450s (P450s) and/or glutathione S-transferases (GSTs) substantially increased susceptibility in the majority of resistant populations lacking mutations at the herbicide target site. In conclusion, resistance to fenoxaprop-P-ethyl and mesosulfuron-ethyl was widespread in B. syzigachne within Anhui Province's wheat fields, while resistance to isoproturon was rapidly evolving due to its escalating usage. Target-site mutations were present in approximately one-third of fenoxaprop-P-ethyl-resistant populations, and alternative mechanisms involving P450s and/or GSTs could explain the resistance observed in most of the remaining populations.

Selection for insecticide resistance can promote Plasmodium falciparum infection in Anopheles.

Insecticide resistance is under strong selective pressure in Anopheles mosquitoes due to widespread usage of insecticides in vector control strategies. Resistance mechanisms likely cause changes that profoundly affect mosquito physiology, yet it remains poorly understood how selective pressures imposed by insecticides may alter the ability of the mosquito to host and transmit a Plasmodium infection. From pyrethroid-resistant field-derived Anopheles gambiae s.l. mosquitoes, we established resistant (RES) and susceptible (SUS) colonies by either selection for, or loss of insecticide resistance. We show increased oocyst intensity and growth rate as well as increased sporozoite prevalence and intensity in RES compared to SUS females infected with Plasmodium falciparum. The increase in infection intensity in RES females was not associated with the presence of the kdrL1014F mutation and was not impacted by inhibition of Cytochrome P450s. The lipid transporter lipophorin (Lp), which was upregulated in RES compared to SUS, was at least partly implicated in the increased intensity of P. falciparum but not directly involved in the insecticide resistance phenotype. Interestingly, we observed that although P. falciparum infections were not affected when RES females were exposed to permethrin, these females had decreased lipid abundance in the fat body following exposure, pointing to a possible role for lipid mobilization in response to damage caused by insecticide challenge. The finding that selection for insecticide resistance can increase P. falciparum infection intensities and growth rate reinforces the need to assess the overall impact on malaria transmission dynamics caused by selective pressures mosquitoes experience during repeated insecticide challenge.

Detoxification Cytochrome P450s (CYPs) in Families 1-3 Produce Functional Oxylipins from Polyunsaturated Fatty Acids.

This manuscript reviews the CYP-mediated production of oxylipins and the current known function of these diverse set of oxylipins with emphasis on the detoxification CYPs in families 1-3. Our knowledge of oxylipin function has greatly increased over the past 3-7 years with new theories on stability and function. This includes a significant amount of new information on oxylipins produced from linoleic acid (LA) and the omega-3 PUFA-derived oxylipins such as α-linolenic acid (ALA), docosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA). However, there is still a lack of knowledge regarding the primary CYP responsible for producing specific oxylipins, and a lack of mechanistic insight for some clinical associations between outcomes and oxylipin levels. In addition, the role of CYPs in the production of oxylipins as signaling molecules for obesity, energy utilization, and development have increased greatly with potential interactions between diet, endocrinology, and pharmacology/toxicology due to nuclear receptor mediated CYP induction, CYP inhibition, and receptor interactions/crosstalk. The potential for diet-diet and diet-drug/chemical interactions is high given that these promiscuous CYPs metabolize a plethora of different endogenous and exogenous chemicals.

Potential Effects of Ibuprofen, Remdesivir and Omeprazole on Dexamethasone Metabolism in Control Sprague Dawley Male Rat Liver Microsomes (Drugs Often Used Together Alongside COVID-19 Treatment).

The role of individual cytochrome P450 (CYPs) responsible for the drug metabolism can be determined through their chemical inhibition. During the pandemic, dexamethasone and remdesivir with omeprazole were used for the treatment of COVID-19, while Ibuprofen was taken to treat the symptoms of fever and headache. This study aimed to examine the potency of ibuprofen remdesivir, and omeprazole as inhibitors of cytochrome P450s using rat liver microsomes in vitro. Dexamethasone a corticosteroid, sometimes used to reduce the body’s immune response in the treatment of COVID-19, was used as a probe substrate and the three inhibitors were added to the incubation system at different concentrations and analysed by a validated High Performance Liquid Chromatography (HPLC) method. The CYP3A2 isoenzyme is responsible for dexamethasone metabolism in vitro. The results showed that ibuprofen acts as a non-competitive inhibitor for CYP3A2 activity with Ki = 224.981 ± 1.854 µM and IC50 = 230.552 ± 2.020 µM, although remdesivir showed a mixed inhibition pattern with a Ki = 22.504 ± 0.008 µM and IC50 = 45.007 ± 0.016 µM. Additionally, omeprazole uncompetitively inhibits dexamethasone metabolism by the CYP3A2 enzyme activity with a Ki = 39.175 ± 0.230 µM and IC50 = 78.351 ± 0.460 µM. These results suggest that the tested inhibitors would not exert a significant effect on the CYP3A2 isoenzyme responsible for the co-administered dexamethasone drug’s metabolism in vivo.

Inhibition of Cytochrome P450s by Strobilanthes crispus Sub-Fraction (F3): Implication for Herb-Drug Interaction.

Strobilanthes crispus Blume sub-fraction (F3) has been reported to be cytotoxic against cancer cells and to cause murine mammary tumor regression. Potential utilization of F3 as an adjuvant in breast cancer treatment to alleviate chemotherapeutic drug resistance is currently hampered by potential cytochrome P450 (CYP)-mediated herb-drug interactions (HDIs). The current study assessed the inhibitory potency of F3 towards five CYP enzymes involved in tamoxifen metabolism. Potential CYP inhibition by F3 was first determined using fluorescence assays, using known CYP inhibitors as reference. To further ascertain the inhibitory potency and mode of inhibition, high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) analysis of specific metabolites of a CYP probe substrate was conducted. The half-maximal inhibitory concentration (IC50) values indicate that F3 exhibited relatively weak inhibition on CYP2B6, CYP2C19, CYP2D6, and CYP3A4. Highest susceptibility to inhibition by F3 was observed for CYP2C9, where the IC50 value from fluorescence-based assay was 35-fold higher than control. Further analysis by HPLC-MS/MS revealed relatively weak mixed-type inhibition of F3 on CYP2C9, as indicated by IC50 and inhibition constant (KI) values. The risk of clinically significant CYP2C9 inhibition by F3 was then predicted based on the attained KI value and the presumed amount of F3 absorbed from S. crispus leaves following consumption. The calculated maximum plasma concentration to inhibition constant Cmax/KI) ratio suggests that F3 consumption could potentially result in clinically significant drug interactions with medications metabolized by CYP2C9. Taken together, the results revealed a low probability of inhibition by F3 on CYP enzymes involved in tamoxifen metabolism. However, further in vivo investigation is necessary for potential F3 interaction with CYP2C9. The utility of a preliminary in vitro approach in the assessment of potential HDI was demonstrated in this study.

Enhanced metabolism and target gene overexpression confer resistance against acetolactate synthase-inhibiting herbicides in Bromus sterilis.

Intensive application of acetolactate synthase (ALS)-inhibiting herbicides has resulted in herbicide-resistance in many weeds, including Bromus sterilis. The present study was conducted to identify the mechanisms conferring resistance to ALS-inhibiting herbicides in a Bromus sterilis biotype. Dose-response studies revealed the resistant biotype to be 288 times less sensitive to pyroxsulam than the susceptible biotype. Furthermore, experiment with a single-dose, proved this biotype was also cross-resistant to propoxycarbazone, iodosulfuron plus mesosulfuron and sulfosulfuron. Prior treatment with malathion, a known inhibitor of cytochrome P450s, reduced the level of resistance to pyroxsulam. No mutations were detected from the partial ALS gene sequencing. Flow cytometry and chromosome counting rejected ploidy level variation between the susceptible and resistant biotypes. Relative copy number variation ruled out gene amplification. Quantitative real-time polymerase chain reaction (PCR) detected a significant difference in ALS gene expression between the susceptible and resistant biotypes. Target gene overexpression and enhanced metabolism by cytochrome P450s are likely mechanisms of resistance to pyroxsulam in Bromus sterilis. The current findings highlight the need to monitor additional brome populations for herbicide resistance in Europe and endorse the need for alternate herbicides in integrated weed management to delay the possible evolution of herbicide resistance in these species. © 2020 Society of Chemical Industry.

Association of CYP1A1 and CYP1B1 inhibition in in vitro assays with drug-induced liver injury.

Drug-induced liver injury (DILI) is one of the major causes for the discontinuation of drug development and withdrawal of drugs from the market. Since it is known that reactive metabolite formation and being substrates or inhibitors of cytochrome P450s (P450s) are associated with DILI, we systematically investigated the association between human P450 inhibition and DILI. The inhibitory activity of 266 DILI-positive drugs (DILI drugs) and 92 DILI-negative drugs (no-DILI drugs), which were selected from Liver Toxicity Knowledge Base (US Food and Drug Administration), against 8 human P450 forms was assessed using recombinant enzymes and luminescent substrates, and the threshold values showing the highest balanced accuracy for DILI discrimination were determined for each P450 enzyme using receiver operating characteristic analyses. The results showed that among the P450s tested, CYP1A1 and CYP1B1 were inhibited by DILI drugs more than no-DILI drugs with a statistical significance. We found that 91% of drugs that showed inhibitory activity greater than the threshold values against CYP1A1 or CYP1B1 were DILI drugs. The results of internal 5-fold cross-validation confirmed the usefulness of CYP1A1 and CYP1B1 inhibition data for the threshold-based discrimination of DILI drugs. Although the contribution of these P450s to drug metabolism in the liver is considered minimal, our present findings suggest that the assessment of CYP1A1 and CYP1B1 inhibition is useful for screening DILI risk of drug candidates at the early stage of drug development.

Mechanism of acequinocyl resistance and cross-resistance to bifenazate in the two-spotted spider mite, Tetranychus urticae (Acari: Tetranychidae)

Acequinocyl and bifenazate are acaricides that inhibit mitochondrial respiratory chain complex III. To analyze acequinocyl resistance mechanisms and cross-resistance risk to bifenazate, we conducted laboratory selection with acequinocyl to a Japanese field population of Tetranychus urticae Koch (Acari: Tetranychidae). Then we characterized resistance traits by examining synergism and maternal inheritance and by DNA sequencing analysis of the target site. Laboratory selection for acequinocyl resistance resulted in 19.8–38.1-fold increases in the resistance factor against a susceptible strain of T. urticae. Selection for bifenazate resistance likewise resulted in a 15.9–37.5-fold increase in the resistance factor. Acequinocyl resistance showed maternal inheritance and strong synergism with piperonyl butoxide (PBO), a cytochrome P450s inhibitor. Target site (mitochondrial cytochrome b) sequencing demonstrated the substitution of an amino acid residue, G126S. These results indicate that target site mutation and detoxification by cytochrome P450 play essential roles in acequinocyl resistance. In contrast, maternal effects were not clearly detected in bifenazate susceptibility of F1 females from reciprocal crosses between acequinocyl-resistant and -susceptible strains. Therefore, G126S is unlikely to be a major resistance factor for decreased bifenazate susceptibility in the acequinocyl-resistant strain. Significant synergistic effects of PBO indicate that cytochrome P450 genes selected by acequinocyl likely confer cross-resistance against bifenazate.

Exploring the Chemical Space of Cytochrome P450 Inhibitors Using Integrated Physicochemical Parameters, Drug Efficiency Metrics and Decision Tree Models

The cytochrome P450s (CYPs) play a central role in the metabolism of various endogenous and exogenous compounds including drugs. CYPs are vulnerable to inhibition and induction which can lead to adverse drug reactions. Therefore, insights into the underlying mechanism of CYP450 inhibition and the estimation of overall CYP inhibitor properties might serve as valuable tools during the early phases of drug discovery. Herein, we present a large data set of inhibitors against five major metabolic CYPs (CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4) for the evaluation of important physicochemical properties and ligand efficiency metrics to define property trends across various activity levels (active, efficient and inactive). Decision tree models for CYP inhibition were developed with an accuracy &gt;90% for both the training set and 10-folds cross validation. Overall, molecular weight (MW), hydrogen bond acceptors/donors (HBA/HBD) and lipophilicity (clogP/logPo/w) represent important physicochemical descriptors for CYP450 inhibitors. However, highly efficient CYP inhibitors show mean MW, HBA, HBD and logP values between 294.18–482.40,5.0–8.2,1–7.29 and 1.68–2.57, respectively. Our results might help in optimization of toxicological profiles associated with new chemical entities (NCEs), through a better understanding of inhibitor properties leading to CYP-mediated interactions.

Metabolic Activation of Myristicin and Its Role in Cellular Toxicity.

Myristicin is widely distributed in spices and medicinal plants. The aim of this study was to explore the role of metabolic activation of myristicin in its potential toxicity through a metabolomic approach. The myristicin- N-acetylcysteine adduct was identified by comparing the metabolic maps of myristicin and 1'-hydroxymyristicin. The supplement of N-acetylcysteine could protect against the cytotoxicity of myristicin and 1'-hydroxymyristicin in primary mouse hepatocytes. When the depletion of intracellular N-acetylcysteine was pretreated with diethyl maleate in hepatocytes, the cytotoxicity induced by myristicin and 1'-hydroxymyristicin was deteriorated. It suggested that the N-acetylcysteine adduct resulting from myristicin bioactivation was closely associated with myristicin toxicity. Screening of human recombinant cytochrome P450s (CYPs) and treatment with CYP inhibitors revealed that CYP1A1 was mainly involved in the formation of 1'-hydroxymyristicin. Collectively, this study provided a global view of myristicin metabolism and identified the N-acetylcysteine adduct resulting from myristicin bioactivation, which could be used for understanding the mechanism of myristicin toxicity.

Molecular evidence for the inhibition of cytochrome p450s and cholinesterases in ticks by the repellent DEET

For more than 50 years DEET (N,N-Diethyl-m-toluamide) has been considered the gold standard of repellents. It is applied to the skin or clothing to deter mosquitoes and other blood-sucking invertebrate pests from approaching and/or settling, and ultimately it provides temporary protection from bites. Despite rampant global use, surprisingly little is understood about DEET’s mode of action and the molecular targets of the active ingredient. Furthermore, the theories into its mechanism for repellency are largely based off fruit fly and mosquito research. Since ticks possess a unique sensory structure, the Haller’s organ, the specific genes and pathways associated with DEET avoidance may differ from insects. In these studies, we collected American dog ticks (Dermacentor variabilis) from four natural populations within Manitoba, Canada. We first carried out behavior assays, which showed DEET effectively repelled the ticks. RNA sequencing revealed that DEET caused a rapid and substantial reduction in the abundance of transcripts encoding cytochrome P450 and acetylcholinesterase genes, which gradually recovered over the 24 h time course. Finally, enzymatic kinetics provided functional support for DEET’s role as an effective inhibitor of P450 s. While many facets of its mode of action remain to be worked out, our study provides valuable insights into the molecular underpinnings of DEET’s repellence in ticks.

Benzo(a)pyrene in Cigarette Smoke Enhances HIV-1 Replication through NF-\u03baB Activation via CYP-Mediated Oxidative Stress Pathway

Smoking aggravates HIV-1 pathogenesis and leads to decreased responses to antiretroviral therapy. In this study, we aim to find a molecular mechanism that would explain smoking-induced HIV-1 replication. Benzo(a)pyrene (BaP), a major carcinogen in cigarette, requires metabolic activation through cytochrome P450s (CYPs) to exert its toxic effects. We hypothesized that CYP-mediated BaP metabolism generates reactive oxygen species (ROS), and the resultant oxidative stress aggravates HIV-1 replication. As expected, we observed ~3 to 4-fold increase in HIV-1 replication in U1 cells and human primary macrophages after chronic BaP exposure. We also observed ~30-fold increase in the expression of CYP1A1 at mRNA level, ~2.5-fold increase in its enzymatic activity as well as elevated ROS and cytotoxicity in U1 cells. The knock-down of the CYP1A1 gene using siRNA and treatment with selective CYP inhibitors and antioxidants significantly reduced HIV-1 replication. Further, we observed a nuclear translocation of NF-κB subunits (p50 and p65) after chronic BaP exposure, which was reduced by treatment with siRNA and antioxidants/CYP inhibitors. Suppression of NF-κB pathway using specific NF-κB inhibitors also significantly reduced HIV-1 replication. Altogether, our results suggest that BaP enhances HIV-1 replication in macrophages by a CYP-mediated oxidative stress pathway followed by the NF-κB pathway.

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.

In vitro inhibitory mechanisms and molecular docking of 1′-S-1′-acetoxychavicol acetate on human cytochrome P450 enzymes

BackgroundThe compound, 1′-S-1′-acetoxychavicol acetate (ACA), isolated from the rhizomes of a Malaysian ethno-medicinal plant, Alpinia conchigera Griff. (Zingiberaceae), was previously shown to have potential in vivo antitumour activities. In the development of a new drug entity, potential interactions of the compound with the cytochrome P450 superfamily metabolizing enzymes need to be ascertain. PurposeThe concomitant use of therapeutic drugs may cause potential drug-drug interactions by decreasing or increasing plasma levels of the administered drugs, leading to a suboptimal clinical efficacy or a higher risk of toxicity. Thus, evaluating the inhibitory potential of a new chemical entity, and to clarify the mechanism of inhibition and kinetics in the various CYP enzymes is an important step to predict drug-drug interactions. Study designThis study was designed to assess the potential inhibitory effects of Alpinia conchigera Griff. rhizomes extract and its active constituent, ACA, on nine c-DNA expressed human cytochrome P450s (CYPs) enzymes using fluorescent CYP inhibition assay. Methods/ResultsThe half maximal inhibitory concentration (IC50) of Alpinia conchigera Griff. rhizomes extract and ACA was determined for CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C19, CYP2D6, CYP2E1, CYP3A4 and CYP3A5. A. conchigera extract only moderately inhibits on CYP3A4 (IC50 = 6.76 ± 1.88µg/ml) whereas ACA moderately inhibits the activities of CYP1A2 (IC50 = 4.50 ± 0.10µM), CYP2D6 (IC50 = 7.50 ± 0.17µM) and CYP3A4 (IC50 = 9.50 ± 0.57µM) while other isoenzymes are weakly inhibited. In addition, mechanism-based inhibition studies reveal that CYP1A2 and CYP3A4 exhibited non-mechanism based inhibition whereas CYP2D6 showed mechanism-based inhibition. Lineweaver-Burk plots depict that ACA competitively inhibited both CYP1A2 and CYP3A4, with a Ki values of 2.36 ± 0.03 µM and 5.55 ± 0.06µM, respectively, and mixed inhibition towards CYP2D6 with a Ki value of 4.50 ± 0.08µM. Further, molecular docking studies show that ACA is bound to a few key amino acid residues in the active sites of CYP1A2 and CYP3A4, while one amino residue of CYP2D6 through predominantly Pi-Pi interactions. ConclusionOverall, ACA may demonstrate drug-drug interactions when co-administered with other therapeutic drugs that are metabolized by CYP1A2, CYP2D6 or CYP3A4 enzymes. Further in vivo studies, however, are needed to evaluate the clinical significance of these interactions.

In vitro metabolism of exemestane by hepatic cytochrome P450s: impact of nonsynonymous polymorphisms on formation of the active metabolite 17β-dihydroexemestane.

Exemestane (EXE) is an endocrine therapy commonly used by postmenopausal women with hormone‐responsive breast cancer due to its potency in inhibiting aromatase‐catalyzed estrogen synthesis. Preliminary in vitro studies sought to identify phase I EXE metabolites and hepatic cytochrome P450s (CYP450s) that participate in EXE biotransformation. Phase I metabolites were identified by incubating EXE with HEK293‐overexpressed CYP450s. CYP450s 1A2, 2C8, 2C9, 2C19, 2D6, 3A4, and 3A5 produce 17β‐dihydroexemestane (17β‐DHE), an active major metabolite, as well as two inactive metabolites. 17β‐DHE formation in pooled human liver microsomes subjected to isoform‐specific CYP450 inhibition was also monitored using tandem mass spectrometry. 17β‐DHE production in human liver microsomes was unaffected by isoform‐specific inhibition of CYP450s 2A6, 2B6, and 2E1 but decreased 12–39% following inhibition of drug‐metabolizing enzymes from CYP450 subfamilies 1A, 2C, 2D, and 3A. These results suggest that redundancy exists in the EXE metabolic pathway with multiple hepatic CYP450s catalyzing 17β‐DHE formation in vitro. To further expand the knowledge of phase I EXE metabolism, the impact of CYP450 genetic variation on 17β‐DHE formation was assessed via enzyme kinetic parameters. Affinity for EXE substrate and enzyme catalytic velocity were calculated for hepatic wild‐type CYP450s and their common nonsynonymous variants by monitoring the reduction of EXE to 17β‐DHE. Several functional polymorphisms in xenobiotic‐metabolizing CYP450s 1A2, 2C8, 2C9, and 2D6 resulted in deviant enzymatic activity relative to wild‐type enzyme. Thus, it is possible that functional polymorphisms in EXE‐metabolizing CYP450s contribute to inter‐individual variability in patient outcomes by mediating overall exposure to the drug and its active metabolite, 17β‐DHE.

β-Naphthoflavone-Induced Mitochondrial Respiratory Damage in Cyp1 Knockout Mouse and in Cell Culture Systems: Attenuation by Resveratrol Treatment.

A number of xenobiotic-inducible cytochrome P450s (CYPs) are now known to be localized in the mitochondrial compartment, though their pharmacological or toxicological roles remain unclear. Here, we show that BNF treatment markedly inhibits liver mitochondrial O2 consumption rate (OCR), ADP-dependent OCR, and also reserve OCR, in wild-type mice but not in Cyp1a1/1a2(−/−) double knockout mice. BNF treatment markedly affected mitochondrial complex I and complex IV activities and also attenuated mitochondrial gene expression. Furthermore, under in vitro conditions, BNF treatment induced cellular ROS production, which was inhibited by mitochondria-targeted antioxidant Mito-CP and CYP inhibitor proadefin, suggesting that most of the ROS production was intramitochondrial and probably involved the catalytic activity of mitochondrial CYP1 enzymes. Interestingly, our results also show that the AHR antagonist resveratrol, markedly attenuated BNF-induced liver mitochondrial defects in wild-type mice, confirming the role of AHR and AHR-regulated CYP1 genes in eliciting mitochondrial dysfunction. These results are consistent with reduced BNF-induced mitochondrial toxicity in Cyp1a1/1a2(−/−) mice and elevated ROS production in COS cells stably expressing CYP1A1. We propose that increased mitochondrial ROS production and respiratory dysfunction are part of xenobiotic toxicity. Resveratrol, a chemopreventive agent, renders protection against BNF-induced toxicity.

CYP2D6 and CYP2A6 biotransform dietary tyrosol into hydroxytyrosol

The dietary phenol tyrosol has been reported to be endogenously transformed into hydroxytyrosol, a potent antioxidant with multiple health benefits. In this work, we evaluated whether tyrosine hydroxylase (TH) and cytochrome P450s (CYPs) catalyzed this process. To assess TH involvement, Wistar rats were treated with α-methyl-L-tyrosine and tyrosol. Tyrosol was converted into hydroxytyrosol whilst α-methyl-L-tyrosine did not inhibit the biotransformation. The role of CYP was assessed in human liver microsomes (HLM) and tyrosol-to-hydroxytyrosol conversion was observed. Screening with selective enzymatic CYP inhibitors identified CYP2A6 as the major isoform involved in this process. Studies with baculosomes further demonstrated that CYP2D6 and CYP3A4 could transform tyrosol into hydroxytyrosol. Experiments using human genotyped livers showed an interindividual variability in hydroxytyrosol formation and supported findings that CYP2D6 and CYP2A6 mediated this reaction. The dietary health benefits of tyrosol-containing foods remain to be evaluated in light of CYP pharmacogenetics.

Evaluation and Quantitative Prediction of Renal Transporter-Mediated Drug-Drug Interactions.

With numerous drugs cleared renally, inhibition of uptake transporters localized on the basolateral membrane of renal proximal tubule cells, eg, organic anion transporters (OATs) and organic cation transporters (OCTs), may lead to clinically meaningful drug-drug interactions (DDIs). Additionally, clinical evidence for the possible involvement of efflux transporters, such as P-glycoprotein (P-gp) and multidrug and toxin extrusion protein 1/2-K (MATE1/2-K), in the renal DDIs is emerging. Herein, we review recent progress regarding mechanistic understanding of transporter-mediated renal DDIs as well as the quantitative predictability of renal DDIs using static and physiologically based pharmacokinetic (PBPK) models. Generally, clinical DDI data suggest that the magnitude of plasma exposure changes attributable to renal DDIs is less than 2-fold, unlike the DDIs associated with inhibition of cytochrome P-450s and/or hepatic uptake transporters. It is concluded that although there is a need for risk assessment early in drug development, current available data imply that safety concerns related to the renal DDIs are generally low. Nevertheless, consideration must be given to the therapeutic index of the victim drug and potential risk in a specific patient population (eg, renal impairment). Finally, in vitro transporter data and clinical pharmacokinetic parameters obtained from the first-in-human studies have proven useful in support of quantitative prediction of DDIs associated with inhibition of renal secretory transporters, OATs or OCTs.

Inhibitory and inductive effects of Phikud Navakot extract on human cytochrome P450

Effects of the hydroethanolic extract of Phikud Navakot (PN), a Thai traditional remedy, on human cytochrome P450s (CYPs) were investigated in vitro. Selective substrates of CYPs were used to investigate the effects and kinetics of PN on CYP inhibition using human liver microsomes. Primary human hepatocytes were used to assess the inductive effects of PN on CYP enzyme activities and protein expressions. The results showed that PN inhibited the activities of CYP1A2, CYP2C9, CYP2D6, and CYP3A4 with half maximal inhibitory concentration (IC50) values of 13, 62, 67, and 88 μg/mL, respectively. Meanwhile, it had no effect on the activities of CYP2C19 and CYP2E1 (IC50 > 1 mg/mL). PN exhibited competitive inhibition of CYP1A2 (Ki = 34 μg/mL), mixed type inhibition of CYP2C9 and CYP2D6 (Ki = 80 and 12 μg/mL, respectively), and uncompetitive inhibition of CYP3A4 (Ki = 150 μg/mL). PN did not have an inductive effect on CYP1A2, CYP2C9, CYP2C19 and CYP3A4 in primary human hepatocytes, which is an advantageous characteristic of the extract. However the extract may cause herb–drug interactions via inhibition of CYP1A2, CYP2C9, CYP2D6 and CYP3A4, and precautions should be taken when PN is coadministered with drugs that are metabolized by these CYP enzymes.

An improved substrate cocktail for assessing direct inhibition and time-dependent inhibition of multiple cytochrome P450s.

The substrate cocktail is frequently used to evaluate cytochrome P450 (CYP) enzyme-mediated drug interactions and potential interactions among the probe substrates. Here, we re-optimized the substrate cocktail method to increase the reliability and accuracy of screening for candidate compounds and expanded the method from a direct CYP inhibition assay to a time-dependent inhibition (TDI) assay. In the reaction mixtures containing human liver microsome (0.1 mg/mL), both the concentrations of a substrate cocktail (phenacetin for 1A2, coumarin for 2A6, bupropion for 2B6, diclofenac for 2C9, dextromethorphan for 2D6, and testosterone for 3A4) and the incubation time were optimized. Metabolites of the substrate probes were simultaneously analyzed by multiple-reaction monitoring (MRM) using a routine LC/MS/MS. Direct CYP inhibition was validated using 7 inhibitors (α-naphthoflavone, tranylcypromine, ticlopidine, fluconazole, quinidine, ketoconazole and 1-ABT). The time-dependent inhibition was partially validated with 5 inhibitors (ketoconazole, verapamil, quinidine, paroxetine and 1-ABT). The inhibition curve profiles and IC50 values of 7 CYP inhibitors were approximate when a single substrate and the substrate cocktail were tested, and were consistent with the previously reported values. Similar results were obtained in the IC50 shifts of 5 inhibitors when a single substrate and the substrate cocktail were tested in the TDI assay. The 6-in-1 substrate cocktail (for 1A2, 2A6, 2B6, 2C9, 2D6 and 3A) is reliable for assessing CYP inhibition and time-dependent inhibition of drug candidates.