Major P450s Research Articles

Evaluation of machine learning models for cytochrome P450 3A4, 2D6, and 2C9 inhibition.

Cytochrome P450 (CYP) enzymes are involved in the metabolism of approximately 75% of marketed drugs. Inhibition of the major drug-metabolizing P450s could alter drug metabolism and lead to undesirable drug-drug interactions. Therefore, it is of great significance to explore the inhibition of P450s in drug discovery. Currently, machine learning including deep learning algorithms has been widely used for constructing in silico models for the prediction of P450 inhibition. These models exhibited varying predictive performance depending on the use of machine learning algorithms and molecular representations. This leads to the difficulty in the selection of appropriate models for practical use. In this study, we systematically evaluated the conventional machine learning and deep learning models for three major P450 enzymes, CYP3A4, CYP2D6, and CYP2C9 from several perspectives, such as algorithms, molecular representation, and data partitioning strategies. Our results showed that the XGBoost and CatBoost algorithms coupled with the combined fingerprint/physicochemical descriptor features exhibited the best performance with Area Under Curve (AUC) of 0.92, while the deep learning models were generally inferior to the conventional machine learning models (average AUC reached 0.89) on the same test sets. We also found that data volume and sampling strategy had a minor effect on model performance. We anticipate that these results are helpful for the selection of molecular representations and machine learning/deep learning algorithms in the P450 model construction and the future model development of P450 inhibition.

Machine Learning Models to Predict Cytochrome P450 2B6 Inhibitors and Substrates.

Cytochrome P450 2B6 (CYP2B6) is responsible for the metabolism of ∼7% of marketed drugs. The in vitro drug interaction studies guidance for industry issued by the FDA stipulates that drug sponsors need to evaluate whether the investigated drugs interact with the major drug-metabolizing P450s including CYP2B6. Therefore, there has been greater attention to the development of predictive models for CYP2B6 inhibitors and substrates. In this study, conventional machine learning and deep learning models were developed to predict CYP2B6 inhibitors and substrates. Our results showed that the best CYP2B6 inhibitor model yielded the AUC values of 0.95 and 0.75 with the 10-fold cross-validation and the test set, respectively, and the best CYP2B6 substrate model produced the AUC values of 0.93 and 0.90 with the 10-fold cross-validation and the test set, respectively. The generalization ability of the CYP2B6 inhibitor and substrate models was assessed by using the external validation sets. Several significant substructural fragments relevant to CYP2B6 inhibitors and substrates were detected via frequency substructure analysis and information gain. In addition, the applicability domain of the models was defined by employing a nonparametric method based on the probability density distribution. We anticipate that our results would be useful for the prediction of potential CYP2B6 inhibitors and substrates in the early stage of drug discovery.

A n injured liver caused by overdose of acetaminophen has altered expression and activities of P450 enzymes and associated drug efficacy during liver recovery and regeneration

Acetaminophen (APAP)-induced liver injury (AILI) is the leading cause of acute liver failure in the United States. Alterations on therapeutic efficacy and adverse drug reactions (ADRs) in patients with AILI are not fully investigated. Our previous study showed that AILI could result in dose-dependent and immediate decreases in expression and activities of major hepatic drug-metabolizing cytochrome P450s (P450s) enzymes in mice. However, liver will go through regeneration after AILI. How P450-mediated drug metabolism alters during liver regeneration is unclear. The aim of the study was to investigate the alterations in expression and activities of major P450 enzymes and associated drug efficacy and ADRs during liver regeneration after AILI. Adult male C57BL6/J mice were treated with various doses of APAP at 0 (no damage control), 200 (mild damage and recoverable), 400 (severe damage but recoverable), and 600 mg/kg (life-threatening damage and unrecoverable). The mice were sacrificed at 24, 48, 72, and 96 hours after the APAP treatment. Severity of liver damage was detected by plasma levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and miR122, with further confirmation by H & E staining in liver tissue sections. The expression and activities of CYP1A2, 2B10, 2C29, 2E1, and 3A11 were measured by RT-PCR (mRNAs) and MS/MS quantification of probe drugs, respectively. Sedation efficacy of midazolam was monitored at different time points after APAP treatment at different doses. The levels of ALT and AST significantly increased at 24 hours after APAP treatment at 200 and 400 mg/kg but dropped back to normal levels at 72 and 96 hours. No mice treated with 600 mg/kg APAP could survive to 72 hours. The magnitude of liver damage was further confirmed by H & E staining. The major examined P450 enzymes showed significant decreases at 24 hours in gene expression and activity levels and recovered back to normal with the doses of 200 and 400, but not 600 mg/kg of APAP. The activities of examined major P450s are correlated with the levels of ALT, AST, and mir122. The alterations of CYP3A11 resulted in correlated changes in sedation efficacy as reflected by sleeping times with midazolam treatment. These results indicated that P450 expression and function could recover after mild and even severe, but not lethal AILI. Overall, these results suggest that ALT, AST, and miR122 are potential biomarkers for predicting how alterations in P450 activities can justify dose selection of other drugs for desired efficacy. Further studies are needed to confirm these findings in human patient populations with AILI and to understand the underlying molecular mechanisms mediating recovery of hepatic P450s during AILI.

Oxygen Surrogate Systems for Supporting Human Drug-Metabolizing Cytochrome P450 Enzymes.

Oxygen surrogates (OSs) have been used to support cytochrome P450 (P450) enzymes for diverse purposes in drug metabolism research, including reaction phenotyping, mechanistic and inhibition studies, studies of redox partner interactions, and to avoid the need for NADPH or a redox partner. They also have been used in engineering P450s for more cost-effective, NADPH-independent biocatalysis. However, despite their broad application, little is known of the preference of individual P450s for different OSs or the substrate dependence of OS-supported activity. Furthermore, the biocatalytic potential of OSs other than cumene hydroperoxide (CuOOH) and hydrogen peroxide (H2O2) is yet to be explored. Here, we investigated the ability of the major human drug-metabolizing P450s, namely CYP3A4, CYP2C9, CYP2C19, CYP2D6, and CYP1A2, to use the following OSs: H2O2, tert-butyl hydroperoxide (tert-BuOOH), CuOOH, (diacetoxyiodo)benzene, and bis(trifluoroacetoxy)iodobenzene. Overall, CuOOH and tert-BuOOH were found to be the most effective at supporting these P450s. However, the ability of P450s to be supported by OSs effectively was also found to be highly dependent on the substrate used. This suggests that the choice of OS should be tailored to both the P450 and the substrate under investigation, underscoring the need to employ screening methods that reflect the activity toward the substrate of interest to the end application. SIGNIFICANCE STATEMENT: Cytochrome P450 (P450) enzymes can be supported by different oxygen surrogates (OSs), avoiding the need for a redox partner and costly NADPH. However, few data exist comparing relative activity with different OSs and substrates. This study shows that the choice of OS used to support the major drug-metabolizing P450s influences their relative activity and regioselectivity in a substrate-specific fashion and provides a model for the more efficient use of P450s for metabolite biosynthesis.

Assay of Endocannabinoid Oxidation by Cytochrome P450.

Cytochrome P450 enzymes are a large family of heme-containing proteins that have important functions in the biotransformation of xenobiotics, including pharmacologic and environmental agents, as well as of endogenously produced chemicals with broad structural and functional diversity. Anandamide and 2-arachidonoylglycerol (2-AG) are substrates for P450s expressed in multiple tissues, leading to the production of a diverse set of mono- and di-oxygenated metabolites. This chapter describes tools and methods that have been used to identify major endocannabinoid-metabolizing P450s and their corresponding products, by using subcellular tissue fractions, cultured cells, and purified recombinant enzymes in a reconstituted system.

Upcyte Human Hepatocytes: a Potent In Vitro Tool for the Prediction of Hepatic Clearance of Metabolically Stable Compounds.

In vitro models based on primary human hepatocytes (PHH) have been advanced for clearance (CL) prediction of metabolically stable compounds, representing state-of-the-art assay systems for drug discovery and development. Yet, limited cell availability and large interindividual variability of metabolic profiles remain shortcomings of PHH. Upcyte human hepatocytes (UHH) represent a novel hepatic cell system derived from PHH, exhibiting proliferative capacity for approximately 35 population doublings. UHH from three donors were evaluated during culture for up to 18 days, investigating relative mRNA expression and in situ enzyme activity of cytochrome P450s (P450s), UDP-glucuronosyltransferases, and sulfotransferases. Furthermore, UHH were used for predicting hepatic CL of 21 marketed low to intermediate CL drugs. In a typical experiment, expansion from 3.9 × 10(6) up to 8.5 × 10(7) cells was achieved during subculture. When maintained at confluence, transcripts of major P450s were expressed at donor-specific levels with sustained activities for the majority of isoforms, showing generally low CYP1A2 and high CYP2B6 activity levels. For donor 151-03, CL prediction based on depletion experiments resulted in an average fold error of 2.0, and 80% of compounds being predicted within twofold to in vivo CL for a subset of 10 low CL drugs. UHH showed sustained and consistent activity of drug-metabolizing enzymes (DME), resulting in highly reproducible CL prediction performance. In conclusion, UHH show promising potential as alternative to PHH for standardized in vitro applications in discovery research based on their stable, hepatocyte-like DME phenotype and virtually unlimited cell availability.

Sulfaphenazole and α-Naphthoflavone Attenuate the Metabolism of the Synthetic Cannabinoids JWH-018 and AM2201 Found in K2/Spice

"K2" or "Spice" is an emerging drug of abuse that is laced with psychoactive synthetic cannabinoids JWH-018 and AM2201. Previous studies have identified hydroxylated (OH) and carboxylated (COOH) species as primary human metabolites, and kinetic studies have implicated CYP2C9 and -1A2 as major hepatic P450s involved in JWH-018 and AM2201 oxidation. The present study extends these findings by testing the hypothesis that CYP2C9- and 1A2-selective chemical inhibitors, sulfaphenazole (SFZ) and α-naphthoflavone (ANF), block oxidation of JWH-018 and AM2201 in human liver microsomes (HLM). A concentration-dependent inhibition of JWH-018 and AM2201 oxidation was observed in the presence of increasing concentration of SFZ (0.5 - 50 μM) and ANF (0.1 - 5.0 μM). No metabolic inhibition was observed with omeprazole, quinidine, and ketoconazole. The results presented herein further demonstrate the importance of CYP2C9- and 1A2-mediated oxidation of JWH-018 and AM2201 and the likelihood of adverse toxicity in populations with polymorphic alleles of these enzymes.

Dietary Regulation of Mouse Intestinal P450 Expression and Drug Metabolism

To determine the impact of dietary changes on intestinal P450 expression and drug metabolism, we studied wild‐type (WT) and intestinal epithelium (IE)‐specific P450 reductase (CPR) knockout (IE‐Cpr‐null) mice, fed either with a regular chow or with a synthetic diet devoid of phytochemicals. A change from regular chow to the synthetic diet diminished expression of CYP1A, 2B, 2C, and 3A in the small intestine (SI), and of CYP2B, 2C, and 3A in liver, of both WT and IE‐Cpr‐null mice; it also decreased microsomal midazolam‐hydroxylase activity in vitro and first‐pass clearance of midazolam in vivo. However, compensatory increase in SI P450 expression still occurred in IE‐Cpr‐null, compared to WT, mice, on the synthetic diet. The dietary change, but not Cpr deletion, caused large decreases in bile acid (BA) levels in plasma, liver, SI, and intestinal content. Furthermore, the diet change in WT mice led to increases in SI expression of FGF15, an enterohepatic regulator of hepatic BA synthesis, and a decrease in hepatic expression of CYP7A1, a key enzyme in BA synthesis. These results highlight the vital role of diet in maintaining adequate expression of major drug‐metabolizing P450s in the digestive tract, and suggest potential involvement of FGF15 and BA signaling in the regulatory mechanisms. (Supported in part by NIH grant GM082978)

In Vitro Assessment of Drug-Drug Interaction Potential of Boceprevir Associated with Drug Metabolizing Enzymes and Transporters

The inhibitory effect of boceprevir (BOC), an inhibitor of hepatitis C virus nonstructural protein 3 protease was evaluated in vitro against a panel of drug-metabolizing enzymes and transporters. BOC, a known substrate for cytochrome P450 (P450) CYP3A and aldo-ketoreductases, was a reversible time-dependent inhibitor (k(inact) = 0.12 minute(-1), K(I) = 6.1 µM) of CYP3A4/5 but not an inhibitor of other major P450s, nor of UDP-glucuronosyltransferases 1A1 and 2B7. BOC showed weak to no inhibition of breast cancer resistance protein (BCRP), P-glycoprotein (Pgp), or multidrug resistance protein 2. It was a moderate inhibitor of organic anion transporting polypeptide (OATP) 1B1 and 1B3, with an IC(50) of 18 and 4.9 µM, respectively. In human hepatocytes, BOC inhibited CYP3A-mediated metabolism of midazolam, OATP1B-mediated hepatic uptake of pitavastatin, and both the uptake and metabolism of atorvastatin. The inhibitory potency of BOC was lower than known inhibitors of CYP3A (ketoconazole), OATP1B (rifampin), or both (telaprevir). BOC was a substrate for Pgp and BCRP but not for OATP1B1, OATP1B3, OATP2B1, organic cation transporter, or sodium/taurocholate cotransporting peptide. Overall, our data suggest that BOC has the potential to cause pharmacokinetic interactions via inhibition of CYP3A and CYP3A/OATP1B interplay, with the interaction magnitude lower than those observed with known potent inhibitors. Conversely, pharmacokinetic interactions of BOC, either as a perpetrator or victim, via other major P450s and transporters tested are less likely to be of clinical significance. The results from clinical drug-drug interaction studies conducted thus far are generally supportive of these conclusions.

Dietary Regulation of Mouse Intestinal P450 Expression and Drug Metabolism

The study was originally designed to test the hypothesis that the compensatory increase in intestinal P450 (cytochrome P450) expression in the intestinal epithelium-specific P450 reductase (CPR) knockout (IE-Cpr-null) mice was attributable to decreased metabolism of putative P450 inducers present in the diet. Thus, we determined the impact of a dietary change from regular rodent chow to a synthetic diet devoid of phytochemicals on the expression of P450 enzymes in the small intestine (SI) and liver of wild-type (WT) and IE-Cpr-null mice. The dietary change diminished expression of CYP1A, 2B, 2C, and 3A in SI and CYP2B, 2C, and 3A in liver of both WT and IE-Cpr-null mice. However, the compensatory increase in SI P450 expression still occurred in IE-Cpr-null, compared with WT, mice, on the synthetic diet. The diet change-induced decrease in P450 expression was accompanied by decreases in microsomal midazolam-hydroxylase activity in vitro and first-pass clearance of midazolam in vivo in WT mice. Further studies showed that the dietary change, but not Cpr deletion, caused large decreases in bile acid (BA) levels in plasma, liver, SI, and intestinal content and that treatment of WT mice on the synthetic diet with GW4064, a farnesoid-X-receptor agonist, restored the levels of CYP3A expression in both liver and SI to those seen in mice fed with regular chow. Taken together, these results highlight the vital role of diet in maintaining adequate expression of major drug-metabolizing P450s and their associated drug-metabolizing activities in the digestive tract and suggest potential involvement of BA signaling in the regulatory mechanisms.

Yeast as a Humanized Model Organism for Biotransformation-Related Toxicity

High drug attrition rates due to toxicity, the controversy of experimental animal usage, and the EU REACH regulation demanding toxicity profiles of a high number of chemicals demonstrate the need for new, in vitro toxicity models with high predictivity and throughput. Metabolism by cytochrome P450s (P450s) is one of the main causes of drug toxicity. As some of these enzymes are highly polymorphic leading to large differences is metabolic capacity, isotype-specific test systems are needed. In this review, we will discuss the use of yeast expressing (mammalian) P450s as a powerful, additional model system in drug safety. We will discuss the various cellular model systems for bioactivation-related toxicity and subsequently describe the properties of yeast as a model system, including the endogenous bioactivation enzymes present, the heterologous expression of (mammalian) P450s and the application of yeasts expressing heterologous P450s and/or other biotransformation enzymes in toxicity studies. All major human drug-metabolizing P450s have been successfully expressed in yeast and various mutagenicity tests have been performed with these humanized yeast strains. The few examples of non-mutagenic toxicity studies with these strains and of the combination of P450s with phase II or other human enzymes show the potential of yeast as a model system in metabolism-related toxicity studies. The wide variety of genome-wide screens available in yeast, combined with its well-annotated genome, also facilitate follow-up studies on the genes involved in toxicity. Unless indicated otherwise "yeast" will refer to baker's yeast Saccharomyces cerevisiae.

Cytochrome P450-mediated oxidative metabolism of abused synthetic cannabinoids found in K2/Spice: identification of novel cannabinoid receptor ligands.

Abuse of synthetic cannabinoids (SCs), such as [1-naphthalenyl-(1-pentyl-1H-indol-3-yl]-methanone (JWH-018) and [1-(5-fluoropentyl)-1H-indol-3-yl]-1-naphthalenyl-methanone (AM2201), is increasing at an alarming rate. Although very little is known about the metabolism and toxicology of these popular designer drugs, mass spectrometric analysis of human urine specimens after JWH-018 and AM2201 exposure identified monohydroxylated and carboxylated derivatives as major metabolites. The present study extends these initial findings by testing the hypothesis that JWH-018 and its fluorinated counterpart AM2201 are subject to cytochrome P450 (P450)-mediated oxidation, forming potent hydroxylated metabolites that retain significant affinity and activity at the cannabinoid 1 (CB(1)) receptor. Kinetic analysis using human liver microsomes and recombinant human protein identified CYP2C9 and CYP1A2 as major P450s involved in the oxidation of the JWH-018 and AM2201. In vitro metabolite formation mirrored human urinary metabolic profiles, and each of the primary enzymes exhibited high affinity (K(m) = 0.81-7.3 μM) and low to high reaction velocities (V(max) = 0.0053-2.7 nmol of product · min(-1) · nmol protein(-1)). The contribution of CYP2C19, 2D6, 2E1, and 3A4 in the hepatic metabolic clearance of these synthetic cannabinoids was minimal (f(m) = <0.2). In vitro studies demonstrated that the primary metabolites produced in humans display high affinity and intrinsic activity at the CB(1) receptor, which was attenuated by the CB(1) receptor antagonist (6aR,10aR)-3-(1-methanesulfonylamino-4-hexyn-6-yl)-6a,7,10,10a-tetrahydro-6,6,9-trimethyl-6H-dibenzo[b,d]pyran (O-2050). Results from the present study provide critical, missing data related to potential toxicological properties of "K2" parent compounds and their human metabolites, including mechanism(s) of action at cannabinoid receptors.

D2-Dopaminergic Receptor-Linked Pathways: Critical Regulators of CYP3A, CYP2C, and CYP2D

Various hormonal and monoaminergic systems play determinant roles in the regulation of several cytochromes P450 (P450s) in the liver. Growth hormone (GH), prolactin, and insulin are involved in P450 regulation, and their release is under dopaminergic control. This study focused on the role of D₂-dopaminergic systems in the regulation of the major drug-metabolizing P450s, i.e., CYP3A, CYP2C, and CYP2D. Blockade of D₂-dopaminergic receptors with either sulpiride (SULP) or 4-(4-chlorophenyl)-1-(1H-indol-3-ylmethyl)piperidin-4-ol (L-741,626) markedly down-regulated CYP3A1/2, CYP2C11, and CYP2D1 expression in rat liver. This suppressive effect appeared to be mediated by the insulin/phosphatidylinositol 3-kinase/Akt/FOXO1 signaling pathway. Furthermore, inactivation of the GH/STAT5b signaling pathway appeared to play a role in D₂-dopaminergic receptor-mediated down-regulating effects on these P450s. SULP suppressed plasma GH levels, with subsequently reduced activation of STAT5b, which is the major GH pulse-activated transcription factor and has up-regulating effects on various P450s in hepatic tissue. Levels of prolactin, which exerts down-regulating control on P450s, were increased by SULP, which may contribute to SULP-mediated effects. Finally, it appears that SULP-induced inactivation of the cAMP/protein kinase A/cAMP-response element-binding protein signaling pathway, which is a critical regulator of pregnane X receptor and hepatocyte nuclear factor 1α, and inactivation of the c-Jun N-terminal kinase contribute to SULP-induced down-regulation of the aforementioned P450s. Taken together, the present data provide evidence that drugs acting as D₂-dopaminergic receptor antagonists might interfere with several major signaling pathways involved in the regulation of CYP3A, CYP2C, and CYP2D, which are critical enzymes in drug metabolism, thus affecting the effectiveness of the majority of prescribed drugs and the toxicity and carcinogenic potency of a plethora of toxicants and carcinogens.

Cytochrome P450 Dysregulations in Thioacetamide-Induced Liver Cirrhosis in Rats and the Counteracting Effects of Hepatoprotective Agents

Dysregulations of cytochromes P450 (P450s) under liver injury have been extensively studied. However, little is known about the possible reversing effects of hepatoprotective agents, the understanding of which is of great importance in guiding clinical dosage adjustment for patients with liver injury. This study aims to investigate the dysregulation patterns of major P450s in thioacetamide (TAA)-induced liver cirrhosis in rats and the potential counteracting effects of hepatoprotective agents schisandra lignans extract (SLE) and dimethyl diphenyl bicarboxylate (DDB). TAA intoxications for 6 weeks induced apparent liver injury and dramatically reduced the hepatic protein expressions of CYP1A2, CYP2C6, CYP2E1, and CYP3A2 to 18, 71, 30, and 21% of that in the normal control, respectively. Both SLE and DDB treatments could significantly reverse the TAA-induced loss of P450 protein levels, which may be ascribed to their hepatoprotective effects and direct P450-inducing effects that have been confirmed in healthy rats. However, the recovery of enzyme activities of most P450s by SLE and DDB treatment was less evident than that for the protein expression levels. TAA exhibited NADPH-, time-, and concentration-dependent inactivating effects on all of the four major P450 isozymes; both DDB and GSH showed little effects on counteracting such an inactivation efficacy. These findings provided a good explanation on the disproportional effects of hepatoprotective agents in recovering the protein levels and enzyme activities of TAA-induced dysregulated P450s.

Predictions of Cytochrome P450-Mediated Drug-Drug Interactions Using Cryopreserved Human Hepatocytes: Comparison of Plasma and Protein-Free Media Incubation Conditions

Cryopreserved human hepatocytes suspended in human plasma (HHSHP) have previously provided accurate CYP3A drug-drug interaction (DDI) predictions from a single IC(50) that captures both reversible and time-dependent inhibition. The goal of this study was to compare the accuracy of DDI predictions by a protein-free human hepatocyte system combined with the fraction unbound in plasma for inhibitor(s) with those obtained with protein-containing incubations. Seventeen CYP3A, CYP2C9, or CYP2D6 inhibitors were incubated with hepatocytes in human plasma or hepatocyte maintenance medium (HMM) for 20 min over a range of concentrations after which midazolam 1'-hydroxylation, diclofenac 4'-hydroxylation or (R)-bufuralol 1'-hydroxylation were used to quantify the corresponding cytochrome P450 (P450) catalytic activities. Two methods were used to predict the human exposure ratio of the victim drug in the presence and absence of inhibitor. The HMM K(i, app) values were combined with the free average systemic plasma concentration ("free [I] with HMM K(i, app)") and the plasma K(i, app) values were combined with the total average systemic plasma concentration ("total [I] with plasma K(i, app)"). Of 63 clinical DDI studies, the total [I] with plasma K(i, app) method predicted 89% of cases within 2-fold of the reported interaction whereas the free [I] with HMM K(i, app) method predicted only 59%. There was a general underprediction by the free [I] with HMM K(i, app) method, which is consistent with an underestimation of in vitro inhibition potency in this system. In conclusion, the HHSHP system proved to be a simple, accurate predictor of DDIs for three major P450s and superior to the protein-free approach.

Can piperonyl butoxide enhance the efficacy of pyrethroids against pyrethroid‐resistant Aedes aegypti?

Pyrethroid resistance can be considered the main threat to the continued control of many mosquito vectors of disease. Piperonyl butoxide (PBO) has been used as a synergist to help increase the efficacy of certain insecticides. This enhancement stems from its ability to inhibit two major metabolic enzyme systems, P450s and non-specific esterases, and to enhance cuticular penetration of the insecticide. To compare the mortality of a characterized resistant Aedes aegypti strain, Nha Trang, from Vietnam and the susceptible laboratory strain Bora Bora on netting with the pyrethroid deltamethrin (DM) alone and in combination with PBO. Resistance mechanisms were characterized using molecular and bioassay techniques; standard PCR was used to test for the kdr target site mutation. Potential genes conferring metabolic resistance to DM were identified with microarray analysis using the Ae. aegypti 'detox chip'. These data were analysed alongside results from WHO susceptibility tests. P450, CYP9J32, was significantly overexpressed in the DM-resistant strain compared with the susceptible Bora Bora strain. Another five genes involved with oxidative stress responses in mosquitoes were also significantly overexpressed. The Nha Trang strain was homozygous for two kdr mutations. WHO cone bioassays were used to investigate mortality with incorporated DM-treated nets with and without PBO. PBO used in combination with DM resulted in higher mortality than DM alone. Synergists may have an important role to play in the future design of vector control products in an era when alternatives to pyrethroids are scarce.

Effects of a Commonly Occurring Genetic Polymorphism of Human CYP3A4 (I118V) on the Metabolism of Anandamide

The endocannabinoid system plays an important role in numerous physiological processes including mood, appetite, and pain sensation. A critical compound in maintaining cannabinoid tone is the endocannabinoid anandamide (AEA). We have recently shown that AEA is metabolized by several different human cytochromes P450 (P450) to form a number of metabolites, one of which exhibits increased biological activity. CYP3A4, one of the major P450s involved in the metabolism of AEA, produces four major metabolites. One of these metabolites, 5,6-epoxyeicosatrienoic acid ethanolamide (5,6-EET-EA), exhibits a much higher affinity than AEA for the cannabinoid 2 receptor (CB-2), which leads to a marked decrease in intracellular cAMP levels in cells expressing CB-2. There are multiple human alleles of CYP3A4, and the CYP3A4.4 allele has been shown to exhibit a significant decrease in activity. Recombinant CYP3A4*4 was expressed in Escherichia coli and was demonstrated to produce 60% less 6-hydroxytestosterone than the wild-type (WT) 3A4 in a reconstituted system. The metabolism of AEA by the WT and the CYP3A4.4 variant was investigated. The mutant produced 60% less of the four EET-EA metabolites than the WT. The mutant also produced a new peak on liquid chromatography-mass spectrometry not seen with the WT, which corresponded to 19-hydroxyeicosatetraenoic acid-ethanolamide. In addition, the mutant produces four novel peaks at m/z 380, which correspond to the addition of two oxygen atoms, possibly to form a peroxide bond. These data indicate that individuals expressing the CYP3A4.4 allele may exhibit significant variations in the metabolism of AEA as well as any other compounds resembling AEA.

Effect of Hepatitis C Virus Infection on the mRNA Expression of Drug Transporters and Cytochrome P450 Enzymes in Chimeric Mice with Humanized Liver

The expression of drug transporters and metabolizing enzymes is a primary determinant of drug disposition. Chimeric mice with humanized liver, including PXB mice, are an available model that is permissive to the in vivo infection of hepatitis C virus (HCV), thus being a promising tool for investigational studies in development of new antiviral molecules. To investigate the potential of HCV infection to alter the pharmacokinetics of small molecule antiviral therapeutic agents in PXB mice, we have comprehensively determined the mRNA expression profiles of human ATP-binding cassette (ABC) transporters, solute carrier (SLC) transporters, and cytochrome P450 (P450) enzymes in the livers of these mice under noninfected and HCV-infected conditions. Infection of PXB mice with HCV resulted in an increase in the mRNA expression levels of a series of interferon-stimulated genes in the liver. For the majority of genes involved in drug disposition, minor differences in the mRNA expression of ABC and SLC transporters as well as P450s between the noninfected and HCV-infected groups were observed. The exceptions were statistically significantly higher expression of multidrug resistance-associated protein 4 and organic anion-transporting polypeptide 2B1 and lower expression of organic cation transporter 1 and CYP2D6 in HCV-infected mice. Furthermore, the enzymatic activities of the major human P450s were, in general, comparable in the two experimental groups. These data suggest that the pharmacokinetic properties of small molecule antiviral therapies in HCV-infected PXB mice are likely to be similar to those in noninfected PXB mice. However, caution is needed in the translation of this relationship to HCV-infected patients as the PXB mouse model does not accurately reflect the pathology of patients with chronic HCV infection.

Abstract 5667: Identification of chemopreventive agents against dibenzo[a,l]pyrene-induced DNA adducts and potential mechanisms

Abstract Intervention with compounds of natural origin with little or no known toxicity is considered an effective means to prevent cancer development. We describe here a microsomal activation system in combination with 32P-postlabeling adduct assay to identify efficacious phytochemicals against the potent lung and breast carcinogen, dibenzo[a,l]pyrene (DBP). Briefly, salmon testes (st)-DNA was incubated with test agents (150 µM), DBP (10 µM), β-naphthoflavone-induced rat liver microsomes, co-factors in Tris. HCl (50 mM, pH 7.4). After incubation for 60 min at 37°C, the reaction was terminated. Analysis of the purified DNA by 32P-postlabeling showed essentially the same spectra of multiple adducts in all samples. However, quantitative differences were observed. Of nine phytochemicals tested, several compounds almost completely abolished the DNA adduct formation, some showed modest inhibition, while others were ineffective: resveratrol (95% reduction), delphinidin (94%), tanshinone I, (84%) tanshinone IIA (78%), diindolylmethane (73%), indole-3-carbinol (49%) and cyanidin (46%) and withaferin A (33%); cucurbitacin B showed insignificant reduction. All adducts were found to be equally inhibited, suggesting that activity of both CYP1A1 and 1B1, the major cytochromes P450s involved in the metabolism of DBP, were similarly affected. Oltipraz included as positive control, as expected, showed over 80-90% inhibition. To determine if the adduct inhibition occurred due to scavenging of electrophilic metabolites of DBP, st-DNA was incubated with the most effective test agents in the presence of anti-dibenzo[a,l]pyrene diolepoxide (anti-DBPDE). Analysis of the resultant adducts showed that only delphinidin, cyanidin and resveratrol showed modest inhibition (30-45%); other compounds were ineffective. Ellagic acid, which is known to scavenge the DBP electrophilic metabolite, almost completely abolished the adduct formation. Together, our data suggest that resveratrol, delphinidin, tanshinones and diindolylmethane are potent inhibitors of microsomal DBP-DNA adduct formation. While, the adduct inhibition for delphinidin and resveratrol may occur, in part, due to scavenging of the electrophilic metabolite of DBP, both these agents, tanshinones and diindolylmethane may also elicit their activity by inhibiting the cytochromes P450 activity. In summary, resveratrol, delphinidin, tanshinones and diindolylmethane, all of which are consumed either in fruits and vegetables or via folklore medicine (tanshinones) may be useful in the prevention of DBP-mediated lung and mammary tumorigenesis (Supported from the USPHS grants CA-118114, ES-011564 and Agnes Brown Duggan Endowment). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5667.

In Vitro Characterization of Sarizotan Metabolism: Hepatic Clearance, Identification and Characterization of Metabolites, Drug-Metabolizing Enzyme Identification, and Evaluation of Cytochrome P450 Inhibition

In vitro biotransformation studies of sarizotan using human liver microsomes (HLM) showed aromatic and aliphatic monohydroxylation and dealkylation. Recombinant cytochromes P450 (P450) together with P450-selective inhibitors in HLM/hepatocyte cultures were used to evaluate the relative contribution of different P450s and revealed major involvement of CYP3A4, CYP2C9, CYP2C8, and CYP1A2 in sarizotan metabolism. The apparent K(m, u) and V(max) of sarizotan clearance, as investigated in HLM, were 9 microM and 3280 pmol/mg/min, predicting in vivo hepatic clearance of 0.94 l/h, which indicates that sarizotan is a low-clearance compound in humans and suggests nonsaturable metabolism at the targeted plasma concentration (< or =1 microM). This finding is confirmed by the reported human clearance (CL/F of 3.6-4.4 l/h) and by the dose-linear area under the curve increase observed with doses up to 25 mg. The inhibitory effect of sarizotan toward six major P450s was evaluated using P450-specific marker reactions in pooled HLM. K(i, u) values of sarizotan against CYP2C8, CYP2C19, and CYP3A4 were >10 microM, whereas those against CYP2D6 and CYP1A2 were 0.43 and 8.7 microM, respectively. Based on the estimates of sarizotan concentrations at the enzyme active sites, no clinically significant drug-drug interactions (DDIs) due to P450 inhibition are expected. This result has been confirmed in human DDI studies in which no inhibition of five major P450s was observed in terms of marker metabolite formation.