Cytochrome P450 3A Family Research Articles

Arachidonic Acid Metabolism by Cyp4A Modulates Caveolae-Mediated Transcytotic LPS Transport in Mouse Jejunum

Arachidonic acid (AA) is metabolized not only by cyclooxygenases and lipoxygenases, but also by cytochrome P-450 4A family (Cyp4A) which is an androgen-driven enzyme that produces 20-hydroxyeicosatetraenoic acid (20-HETE). 20-HETE is an endogenous ligand for GPR75, which is highly associated with pathogenesis of obesity. Since metabolic endotoxemia is implicated in the induction of obesity, and the gut lumen is the primary source of endotoxin (lipopolysaccharides [LPS]), we hypothesized that AA metabolism may affect LPS entry through the small intestine. Furthermore, we have found that increased LPS transport during long-chain fatty acid (LCFA) absorption is abolished in caveolin-1 (Cav1) knockout (KO) jejunum, suggesting the involvement of caveolae-mediated endocytosis. We thus examined the effects of AA on LPS transport in murine jejunum. FITC-LPS (10 μg/ml) or FD4 (0.1 mM) was applied to the mucosal (m) bath of Ussing chambered muscle-stripped jejunum from male C57Bl6 wild type (WT) and Cav1 KO mice with measurement of the serosal (s) appearance of FITC-LPS or FD4 and transepithelial electrical resistance (TER). We examined the effects of the luminal application (10 mM) of oleic acid (OA), AA, eicosapentaenoic acid (EPA), or docosahexaenoic (DHA) with taurocholic acid (0.1 mM) on these parameters with or without the lipid raft inhibitor methyl-β-cyclodextrin (MβCD, 0.1 mM) or Cyp4A inhibitor HET0016 (10 μM). In another study, male and female WT and Cav1 KO mice were fed a standard diet (SD), or a diet enriched in saturated LCFA fat and sugar (LCD) for 6 wks. Luminal addition of OA and AA, but not EPA nor DHA, increased LPS m-to-s movement in WT jejunal mucosa. AA also increased FD4 m-to-s movement with no effect on TER. AA-induced LPS and FD4 movements were inhibited by luminal application of MβCD or HET0016, suggesting the involvement of lipid rafts and Cyp4A activity. Luminal AA had no effect on LPS and FD4 m-to-s movement in KO jejunum. LPS and FD4 m-to-s movement were also increased by luminal application of 20-HETE (0.3 and 1 μM). Immunostaining revealed that GPR75 was expressed on the plasma membranes of jejunal villous but not crypt cells. LCD induced obesity in WT, not in KO, with increased blood glucose levels, perigonadal fat weight, and liver weight, with changes more evident in male WT than in female WT. LCD increased LPS m-to-s movement in WT male jejunum, not in KO jejunum, which was inhibited by luminal addition of MβCD or HET0016. These results suggest that luminal AA increases 20-HETE production by Cyp4A, then activates Cav1-mediated endocytosis which in turn enhances LPS transport in the small intestine, possibly via GPR75 activation. The AA-Cyp4A-20-HETE-GPR75 system may be a new therapeutic target for metabolic endotoxemia associated with diet-induced obesity, especially in males. VA Merit Review and DoD grants. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Culturing of primary bovine mammary epithelial cells and validation of biotransformation capacity in experiments with enrofloxacin

Many drugs and toxic compounds are subjected to disposition and metabolism in bovine mammary epithelial cells (bMECs). For rapid investigation of different compounds and their possible interactions, validated in vitro models are needed. Therefore, the first objective of described experiments was to develop the techniques for cell isolation, purification and culturing of bMECs. The second objective was the application of these cell cultures in a well-known substrate for one of the major biotransformation enzymes in epithelial cells. To this end, the metabolism of enrofloxacin (ENR) into its active metabolite ciprofloxacin (CPR), was studied. This conversion is known to be catalysed by enzymes of the cytochrome P4501A and P4503A family. The expression profile of these enzymes shows a close correlation with cellular ABC-efflux transporters. Primary bMECs were isolated from healthy udders of lactating cows (n=5 animals). mRNA levels of α-casein, b-lactoferrin and cyclophilin B were determined as markers of cell identity of purity of the cultures. Subsequently, bMECs cultures were incubated with ENR (10 µM). Concentrations of ENR and its main metabolite CPR in the medium and in the cells were determined by HPLC-FL analysis. Gene expression of CYP1A1, CYP1A2 and CYP3A4, bovine ABCG2 was detected by qRT-PCR. Results showed that ENR penetrated into bMECs and was converted to CPR. CPR was excreted in the medium suggesting participation of ABCG2 in fluoroquinolone efflux. In conclusion, the data showed that the established bMEC cultures expressed major CYP450 enzymes as well as the most relevant efflux transport ABGG2. This model should be further validated and can serve as an interesting model for further studies on site-specific drug/toxin metabolism and transport in the bovine mammary gland.

A proposed simple screening method to determine relative contributions of CYP3A4 and CYP3A5 to drug metabolism in vitro

Abstract Purpose The cytochrome P450 (CYP) 3A family of enzymes metabolize the majority of clinically used drugs. CYP3A4 and CYP3A5 are the two major CYP3A isoforms, but exhinbit different substrate specificity. The aim of this study was to establish a simple screening method to determine the relative contributions of CYP3A4 and CYP3A5 to drug metabolism in vitro. Methods A screening method was developed based on competitive inhibition using luciferin-PPXE (L-PPXE), a luminogenic CYP3A substrate. CYP3cide, tacrolimus, and midazolam were selected as standard compounds metabolized by CYP3A4 or CYP3A5. Nine clinically-used drugs were evaluated for their abilities to inhibit luminescence resulting from L-PPXE metabolism. Appropriate reaction conditions for the screening method were determined using recombinant CYP3A4 and CYP3A5. Results A significant decrease in luminescence resulting from L-PPXE metabolism by CYP3A4 and CYP3A5 was observed only for drugs reported to be metabolized by CYP3As. The substrate specificities of CYP3A4 or CYP3A5 for the proposed CYP3A substrates using our screening method were consistent with those of previous reports or available drug information from pharmaceutical companies. The reaction volume for this method was 50 μL, and the time required for the entire procedure was 70 min. Furthermore, this screening can be performed using a single tube with minimal training. Conclusions Through the establishment of our screening method in the present study, we are sure it is useful to determine the relative contributions of CYP3A4 and CYP3A5 to drug metabolism in vitro.

Effect of miR-433-3p and miR-883b-5p on murine CYP 3A family enzymes in AML12 cells.

Here we searched for microRNAs that could interact with cytochrome P450 (CYP) enzymes in silico, and then investigated their effects on Cyp gene expressions using the cultured mouse liver cell line AML12. Among the mouse Cyp3a genes, some miRNAs were found to interact with Cyp3a11, 13, 16, and 44 by the in silico analysis using the miRWalk2.0 database. In addition to this software, which included twelve miRNA target prediction algorithms, we also applied our in-house-developed Excel VBA algorithm to obtain predictions more efficiently. Finally, two miRNAs, miR-433-3p and miR-883b-5p, were extracted as candidates that interact with Cyp3a genes. To evaluate the effects of these miRNAs on Cyp3a gene expression, we first examined whether they actually interacted with the Cyp3a 3'-untranslated region (3'-UTR) using a luciferase assay system in AML12 cells. We then evaluated whether the expression of each miRNA affected the expression of Cyp3a mRNAs and their transcribed proteins. We found that the transiently expressed miRNAs significantly reduced the reporter activity of the Cyp3a 3'-UTR site in AML12 cells. In addition, the mRNA and protein expressions of the corresponding Cyp3as were significantly decreased in the miRNA-treated AML12 cells. Using cultured cells, we clearly demonstrated that miR-433-3p and miR-883b-5p, which were identified by in silico prediction, actually bind to Cyp3a mRNAs and regulate Cyp gene expressions.

CYP3A genes and the association between prenatal methylmercury exposure and neurodevelopment

BackgroundResults on the association between prenatal exposure to methylmercury (MeHg) and child neuropsychological development are heterogeneous. Underlying genetic differences across study populations could contribute to this varied response to MeHg. Studies in Drosophila have identified the cytochrome p450 3A (CYP3A) family as candidate MeHg susceptibility genes. ObjectivesWe evaluated whether genetic variation in CYP3A genes influences the association between prenatal exposure to MeHg and child neuropsychological development. MethodsThe study population included 2639 children from three birth cohort studies: two subcohorts in Seychelles (SCDS) (n=1160, 20 and 30months of age, studied during the years 2001–2012), two subcohorts from Spain (INMA) (n=625, 14months of age, 2003–2009), and two subcohorts from Italy and Greece (PHIME) (n=854, 18months of age, 2006–2011). Total mercury, as a surrogate of MeHg, was analyzed in maternal hair and/or cord blood samples. Neuropsychological development was evaluated using Bayley Scales of Infant Development (BSID). Three functional polymorphisms in the CYP3A family were analyzed: rs2257401 (CYP3A7), rs776746 (CYP3A5), and rs2740574 (CYP3A4). ResultsThere was no association between CYP3A polymorphisms and cord mercury concentrations. The scores for the BSID mental scale improved with increasing cord blood mercury concentrations for carriers of the most active alleles (β[95% CI]:=2.9[1.53,4.27] for CYP3A7 rs2257401 GG+GC, 2.51[1.04,3.98] for CYP3A5 rs776746 AA+AG and 2.31[0.12,4.50] for CYP3A4 rs2740574 GG+AG). This association was near the null for CYP3A7 CC, CYP3A5 GG and CYP3A4 AA genotypes. The interaction between the CYP3A genes and total mercury was significant (p<0.05) in European cohorts only. ConclusionsOur results suggest that the polymorphisms in CYP3A genes may modify the response to dietary MeHg exposure during early life development.

Ortho-Methylarylamines as Time-Dependent Inhibitors of Cytochrome P450 1A1 Enzyme.

Members of the cytochrome P450 1A family metabolize many procarcinogens such as polycyclicaromatic hydrocarbons and heterocyclic amines. Inactivation of these enzymes is a prerequisite for cancer prevention and treatment in certain cases. Mechanism-based inhibition (time and co-factor dependent) is an effective method for the inactivation of these enzymes. Our recent study on emodin analogs revealed an anthraquinone with ortho-methylarylamine moiety that exhibited timedependent inhibition of P450 enzymes 1A1 and 1A2. To determine whether the amino group or the methyl group or both were responsible for the time-dependent inhibition of these enzymes, a set of eleven compounds containing the orthomethylarylamine moiety were identified through a database search, and studied for the inhibition of the P450 enzymes 1A1, 1A2, 2A6 and 2B1. Our earlier studies on carbazole derivatives provided us with highly selective P450 1A2 inhibitors. Glycine scanning studies were performed on the docked proteinligand complexes of compounds 1-20 in order to understand the contribution of different protein residues towards the ligand binding. Four compounds were found to cause selective time-dependent inhibition of P450 1A1 with KI values ranging from 0.24 to 8.25 mM. These compounds exhibited only direct inhibition of P450 1A2. Molecular modeling studies of these molecules indicated that the shapes of the molecules, their binding modes, and the methyl substituent in close proximity (4.5-5.7 Å) to the heme-Fe all contributed to their selective time-dependent inhibition activity on P450 1A1. Glycine scanning studies for P450 1A1 indicated that ligand interaction with Phe123 was the strongest binding contributor and similar studies for P450 1A2 indicated that ligand interactions with the phenylalanine residues 226 and 260 were the largest binding contributors. Four compounds have been identified that exhibit selective time-dependent inhibition of P450 1A1. Modeling studies have indicated that the proximity of the aromatic methyl group to the heme-Fe could be the main contributor for time-dependent inhibition. Future studies will focus on the confirmation of the involvement of the aromatic methyl group in enzyme inactivation.

Navigating CYP1A Induction and Arylhydrocarbon Receptor Agonism in Drug Discovery. A Case History with S1P1 Agonists.

This article describes the finding of substantial upregulation of mRNA and enzymes of the cytochrome P450 1A family during a lead optimization campaign for small molecule S1P1 agonists. Fold changes in mRNA up to 10,000-fold for CYP1A1 in vivo in rat and cynomolgus monkey and up to 45-fold for CYP1A1 and CYP1A2 in vitro in rat and human hepatocytes were observed. Challenges observed with correlating induction in vitro and induction in vivo resulted in the implementation of a short, 4 day in vivo screening study in the rat which successfully identified noninducers. Subtle structure-activity relationships in this series of S1P1 agonists are described extending beyond planarity and lipophilicity, and the impact and considerations of AhR and CYP1A induction in the context of drug development are discussed.

Three common pathways of nephrotoxicity induced by halogenated alkenes.

Glutathione-dependent bioactivation is a common pathway in nephrotoxicity caused by haloalkanes and haloalkenes. Glutathione conjugation forms the link between halogenated hydrocarbons, based on the formation of an episulfonium ion (vicinal halomethanes) or a cysteine conjugate (haloalkenes). Herein, we review the metabolic pathways underlying the nephrotoxic effects of the three well-known haloalkenes trichloroethylene, tetrachloroethylene, and hexachloro-1:3-butadiene to emphasize the role of cysteine-conjugate β-lyase and the oxidative metabolism in renal toxicity. Activation by cysteine-conjugate β-lyase is the best-characterized mechanism causing toxicity due to haloalkene treatment in experimental models. However, the severity of toxicity differs considerably, with S-(1,2,2-trichlorovinyl)-L-cysteine being more toxic than S-(1,2-dichlorovinyl)-L-cysteine, which is in turn more toxic than S-(1,2,3,4,4-pentachloro-1:3-butadienyl)-L-cysteine. Moreover, two oxidative pathways involving cysteine S-conjugates (mediated by flavin-containing monooxigenase 3) and N-acetyl-L-cysteine conjugates (mediated by cytochrome P-450 3A) form derived sulfoxides, which represent alternative metabolites with toxic effects. In vitro and in vivo studies showed that sulfoxide metabolites are more toxic than cysteine-conjugate derivates. The cytochrome P-450 3A family, on the other hand, is sex specific, and its expression has only been reported in adult male rats and rabbits. In summary, haloalkenes are highly nephrotoxic in vivo and in vitro and their toxicity mechanisms are well documented experimentally. However, little information is available on their toxicity in humans, except for the carcinogenic effects established for high exposure levels of trichloroethylene and tetrachloroethylene.

Defining molecular sensors to assess long-term effects of pesticides on carcinogenesis.

The abundance of dioxins and dioxin-like pollutants has massively increased in the environment due to human activity. These chemicals are particularly persistent and accumulate in the food chain, which raises major concerns regarding long-term exposure to human health. Most dioxin-like pollutants activate the aryl hydrocarbon receptor (AhR) transcription factor, which regulates xenobiotic metabolism enzymes that belong to the cytochrome P450 1A family (that includes CYP1A1 and CYP1B1). Importantly, a crosstalk exists between estrogen receptor α (ERα) and AhR. More specifically, ERα represses the expression of the CYP1A1 gene, which encodes an enzyme that converts 17β-estradiol into 2-hydroxyestradiol. However, (ERα) does not repress the CYP1B1 gene, which encodes an enzyme that converts 17β-estradiol into 4-hydroxyestradiol, one of the most genotoxic estrogen metabolites. In this review, we discuss how chronic exposure to xenobiotic chemicals, such as pesticides, might affect the expression of genes regulated by the AhR–ERα crosstalk. Here, we focus on recent advances in the understanding of molecular mechanisms that mediate this crosstalk repression, and particularly on how ERα represses the AhR target gene CYP1A1, and could subsequently promote breast cancer. Finally, we propose that genes implicated in this crosstalk could constitute important biomarkers to assess long-term effects of pesticides on human health.

Clinical and Genetic Determinants of Tacrolimus Dose During Induction and Stable Phase of Therapy.

Background Tacrolimus is a calcineurin inhibitor widely used in patients undergoing solid organ transplantation. However, tacrolimus therapy is hindered by highly variable dose requirements in patients associated with toxicity and efficacy (prevention of rejection). Metabolism of tacrolimus via the cytochrome P450 (CYP) 3A family accounts for a significant portion of tacrolimus dosing requirements. Currently, there is paucity of data regarding predictors of tacrolimus dosing, particularly during the initiation (induction) phase of therapy. Thus, we assessed the differential impact of CYP3A genetic polymorphisms on tacrolimus dose and plasma exposure during induction and maintenance phases of therapy. Methods We enrolled 167 renal transplant patients treated with tacrolimus (as bid Prograf) who were at least 3 months post-transplant. Tacrolimus trough concentrations and dose adjustments during induction (0 - 3 months) and stable phase (after 3 months) were retrospectively analyzed. Results The mean tacrolimus dose required during both induction and stable phase was significantly greater in CYP3A5 expressors (CYP3A5*1/*1 and *1/*3) compared to non-expressors (CYP3A5*3/*3; P<0.0001). Importantly, dose-normalized trough concentrations were significantly lower for CYP3A5 expressors (P<0.0001). In addition, dose-normalized concentrations appeared to be consistent throughout the treatment periods, indicating that intrinsic metabolism of tacrolimus does not vary between induction and stable phase of therapy. Regression analysis indicated that CYP3A5*3 and CYP3A4*22 genotype, hemoglobin, and weight were significant predictors of induction dose, accounting for 30% of the variation, while CYP3A5*3 genotype, months post transplant, and age were significant predictors of stable dose, accounting for 42% of the variation. Other significant variables with less impact on tacrolimus dose include POR*28, ABCB1 3435C>T, PXR -25385C>T, and creatinine clearance. Conclusions We demonstrated that CYP3A5*3 genotype is the major determinant of tacrolimus dose variability during induction and stable therapy. Genetic variation in CYP3A4 (*22) was identified as yet unrecognized significant contributor to tacrolimus dose. These findings have the potential to individualize tacrolimus dosing, reduce time to target concentration, improve efficacy, and minimize toxicities. DISCLOSURES:Muirhead, N.: Grant/Research Support, Astella. Kim, R.: Grant/Research Support, Astella.

Combined effect of donor cytochrome P450 3A family polypeptide 5 and recipient cytochrome P450 3A family polypeptide 4 polymorphisms on the individual dosage regimen of tacrolimus in Chinese liver transplant patients

Objective To investigate the effect of donor cytochrome P450 3A family polypeptide 5 (CYP3A5) and recipient cytochrome P450 3A family polypeptide 4 (CYP3A4) gene polymorphisms on the concentration/dose (C/D) ratio of tacrolimus in Chinese liver transplant patients.Methods Ninety liver transplant recipients treated with tacrolimus and their co-respective donors were enrolled in this study.Tacrolimus dosage and trough blood concentration were determined at 1 st month after liver transplantation.Polymerase chain reaction (PCR) amplification and DNA sequencing were applied to detect CYP3A5 rs776746 and CYP3A4 rs12333983 polymorphisms for all subjects.The relationship between gene polymorphisms and tacrolimus C/D ratios was analyzed.Results Both donor CYP3A5 rs776746 and recipient CYP3A4 rs12333983 polymorphisms were highly correlated with the tacrolimus C/D ratios at first month after liver transplantation.No linkage disequilibrium between donor CYP3A5 rs776746 and recipient CYP3A4 rs12333983 polymorphisms was observed (D' =0.017,r2 ＜ 0.001).The combined effect of these two polymorphisms demonstrated that there was significant difference in tacrolimus C/D ratio among groups.The trend of tacrolimus C/D ratios was highly significant at all investigated time points:extensive metabolizers (n =22) ＜ intermediate metabolizers (n =44) ＜ poor metabolizers (n =24).Conclusion Combined detection of donor CYP3A5 and recipient CYP3A4 genotypes may be performed prospectively to help individualize tacrolimus dose regimen for liver transplant patients. Key words: Liver transplantation; Tacrolimus ; Cytochrome P450 ; Gene polymorphism

Association of donor cytochrome P450 3A family polypeptide 5 and interleulin-18 polymorphisms with the individual dosage regimen of tacrolimns in chinese liver transplantation recipients

Objective To investigate the effect of cytochrome P450 3A family polypeptide 5 (CYP3A5) and interleukin-18 (IL-18) polymorphisms on the concentration/dose ratio of tacrolimus in Chinese liver transplant patients.Methods Donor CYP3A5 polymorphisms at rs776746 and IL-18 polymorphisms at rs5744247 were assessed by polymerase chain reaction (PCR) amplification and DNA sequencing for 84 liver transplant patients.The relationship between gene polymorphism and tacrolimus concentration/dose ralio was analyzed.Results The tacrolimus C/D ratios(ng.ml-1 ·mg-1 · kg-1) of the recipients with donors who were IL-18 rs5744247 GG + GC (163.1 ± 100.8) genotype were higher than those with donors who were CC genotype (116.7 ± 78.2,P ＜ 0.001).The tacrolimus C/D ratios of the recipients with donors who were CYP3A5 expressors and had IL-18 rs5744247 GG + GC genotype (133.4 ± 65.3) were higher than those with donors who were CYP3A5 expressors and had IL-18 rs5744247 CC genotype (88.3 ± 39.9,P ＜ 0.05).Conclusion Determination of donor CYP3A5 and IL-18 genotype may be performed prospectively to help individualizing tacrolimus dose regimen for liver transplant patients. Key words: Liver transplantation; Tacrolimus; Cytochrome P450 3A family polypeptide 5; Interleukin-18 ; Polymorphism

Fucoxanthin Attenuates Rifampin-Induced Cytochrome P450 3A4 (CYP3A4) and Multiple Drug Resistance 1 (MDR1) Gene Expression Through Pregnane X Receptor (PXR)-Mediated Pathways in Human Hepatoma HepG2 and Colon Adenocarcinoma LS174T Cells

Pregnane X receptor (PXR) has been reported to regulate the expression of drug-metabolizing enzymes, such as the cytochrome P450 3A (CYP3A) family and transporters, such as multiple drug resistance 1 (MDR1). Fucoxanthin, the major carotenoid in brown sea algae, is a putative chemopreventive agent. In this study, we determined whether fucoxanthin could overcome drug resistance through attenuation of rifampin-induced CYP3A4 and MDR1 gene expression by PXR-mediated pathways in HepG2 hepatoma cells. We found that fucoxanthin (1–10 μM) significantly attenuated rifampin (20 μM)-induced CYP3A4, MDR1 mRNA and CYP3A4 protein expression at 24 h of incubation. Mechanistically, fucoxanthin strongly attenuated the PXR-mediated CYP3A4 promoter activity in HepG2 cells. In addition, fucoxanthin attenuated constitutive androstane receptor (CAR)- and rPXR-mediated CYP3A4 promoter activity in this cell line. Using the mammalian two-hybrid assay, we found that fucoxanthin significantly decreased the interaction between PXR and SRC-1, a PXR co-activator. Thus, fucoxanthin can decrease rifampin-induced CYP3A4 and MDR1 expression through attenuation of PXR-mediated CYP3A4 promoter activation and interaction between PXR and co-activator. These findings could lead to potentially important new therapeutic and dietary approaches to reduce the frequency of adverse drug reactions.

MRNA expression profiles of P450 3A enzymes in the liver and small intestine of the domestic pig

The cytochrome P450 (P450) 3A family is considered to be the most important and abundantly expressed P450 subfamily in mammals. The mRNA expression levels of four P450 3A enzymes in porcine liver and small intestine were investigated using real-time reverse transcriptase-polymerase chain reaction (RT-PCR). The expression of P450 3A mRNAs (P450 3A pool) was higher in the liver than that in the small intestine. In the small intestine, the P450 3A mRNAs were gradually decreased from the duodenum to the ileum. P450 3A29 and P450 3A22 were predominantly expressed both in liver and small intestine tissues with larger ratios in the P450 3A pool than the other P450 3A enzymes. These results demonstrate that P450 3A29 and P450 3A22 probably serve as the major P450 3A contributors for both the hepatic and intestinal P450 3A pool. This work provides a deeper comprehension of the contribution of P450 3A enzymes to xenobiotic metabolism in pigs.

Evaluation of in vitro PXR-based assays and in silico modeling approaches for understanding the binding of a structurally diverse set of drugs to PXR

The pregnane X-receptor (PXR) is a promiscuous nuclear receptor primarily responsible for the induction of genes from the cytochrome P450 3A family. In this study, we used a previously described PXR/SRC tethered protein to establish two in vitro assays for identifying PXR ligands: automated ligand identification system (ALIS) and temperature-dependent circular dichroism (TdCD). Kd values determined by ALIS and TdCD showed good correlations with the EC50 values determined by a PXR luciferase reporter-gene assay for 37 marketed drugs. The same set of compounds was modeled into the PXR ligand-binding domain that takes into consideration the structural variations of five published X-ray structures of PXR–ligand complexes. Major findings from our in silico analysis are as follows. First, the primary determinants for non-binders of PXR are molecular size and shape of the compounds. Low molecular weight (MW < 300) compounds were in general found to be non-binders, and those molecules that do not match the shape of the PXR ligand-binding site may also act as a non-binder. Secondly, the favorable hydrophobic interactions, mostly through aromatic π–π interactions, and the presence of suitable hydrogen bond(s) between the compounds and PXR are attributes of strong binders. Thirdly, the structures of the PXR binding domain possess the flexibility that accommodates structurally diverse compounds, while some of the strong binders may also adapt flexible conformations for fitting into the binding site. The results from this study provide a molecular basis for future efforts in reducing/abolishing the PXR-dependent CYP3A4 induction liability.

The Inhaled Glucocorticoid Fluticasone Propionate Efficiently Inactivates Cytochrome P450 3A5, a Predominant Lung P450 Enzyme

Inhaled glucocorticoid (GC) therapy is a vital part of the management of chronic asthma. GCs are metabolized by members of the cytochrome P450 3A family in both liver and lung, but the enzymes are differentially expressed. Selective inhibition of one or more P450 3A enzymes could substantially modify target and systemic concentrations of GCs. In this study, we have evaluated the mechanism-based inactivation of P450 3A4, 3A5, and 3A7 enzymes by GCs. Among the five major inhaled GCs approved for clinical use in the United States, fluticasone propionate (FLT) was the most potent mechanism-based inactivator of P450 3A5, the predominant P450 enzyme in the lung. FLT inactivated P450 3A5 in a time- and concentration-dependent manner with K(I), k(inact), and partition ratio of 16 muM, 0.027 min(-1), and 3, respectively. In contrast, FLT minimally inactivated P450 3A4 and did not inactivate 3A7, even with a concentration of 100 muM. The inactivation of P450 3A5 by FLT was irreversible because dialysis did not restore enzyme activity. In addition, the exogenous nucleophilic scavenger GSH did not attenuate inactivation. The prosthetic heme of P450 3A5 was not modified by FLT. The loss of P450 3A5 activity in lung cells could substantially decrease the metabolism of FLT, which would increase the effective FLT concentration at its target site, the respiratory epithelium. Also, inactivation of lung P450 3A5 could increase the absorption of inhaled FLT, which could lead to high systemic concentrations and adverse effects, such as life-threatening adrenal crises or cataracts that have been documented in children receiving high doses of inhaled GCs.

Differential expression of CYP3A12 and CYP3A26 mRNAs in canine liver and intestine

The cytochrome P450 (CYP) 3A family is often considered the most important CYP subfamily with regard to drug metabolism in people. Certainly, CYP3A4 contributes to poor oral bioavailability of a number of drugs, and a tremendous amount of effort has been made in attempting to find an appropriate model system to predict the oral bioavailability of candidate drugs. The dog is a species widely used as a preclinical model for the evaluation of drug safety and pharmacokinetics. Compared with other species, little information is available on the tissue distribution of CYP enzymes. The purpose of this study was to determine the level of messenger RNA (mRNA) expression of the canine CYP3A subfamily (CYP3A12 and CYP3A26) in the liver and duodenum. Overall, expression of CYP3A mRNA was greater in the liver than in the duodenum. Hepatic expression of CYP3A26 was greater than CYP3A12 in all dogs, with CYP3A26 comprising 75.2% of the hepatic mRNA CYP3A pool. Conversely, duodenal expression of CYP3A12 was greater than CYP3A26 in all dogs, with CYP3A12 comprising 99.8% of the duodenal mRNA CYP3A pool. In summary, these results represent an important step toward the systematic comparison of human and canine CYP3A enzymes, particularly in relation to oral bioavailability of substrate drugs.

Why study the cytochrome P4503A (CYP3A) family?

Why study the cytochrome P4503A (CYP3A) family?

Down-Regulation of Pregnane X Receptor Contributes to Cell Growth Inhibition and Apoptosis by Anticancer Agents in Endometrial Cancer Cells

Recent studies have revealed that pregnane X receptor (PXR) can function as a master regulator to control the expression of drug-metabolizing enzymes, cytochrome P450 3A (CYP3A) family, and members of the drug transporter family, including multiple drug resistance 1 (MDR1). We demonstrated previously that steroid/xenobiotic metabolism by tumor tissue through the PXR-CYP3A pathway might play an important role in endometrial cancer and that PXR ligands enhance PXR-mediated transcription in a ligand- and promoter-dependent fashion, leading to differential regulation of individual PXR targets, especially CYP3A4 and MDR1. In this study, we investigated the potential contribution of PXR down-regulation by RNA interference toward the augmentation of drug sensitivity and the overcoming of drug resistance. We observed the protein levels of both CYP3A4 and MDR1 in PXR small interfering RNA (siRNA)-transfected cells were not increased in the presence of PXR ligands, paclitaxel, cisplatin, estradiol, or medroxyprogesterone acetate (MPA) compared with control siRNA-transfected cells. There was no PXR-mediated transactivation or augmentation of transcription by coactivators in the presence of these ligands. We then found that PXR down-regulation caused a significant increase in cell growth inhibition and enhancement of apoptosis in the presence of the anticancer agents, paclitaxel, cisplatin, and MPA. Finally, we demonstrated that PXR overexpression caused a significant decrease in cell growth inhibition and inhibited apoptosis in the presence of paclitaxel or cisplatin. These data suggest that PXR down-regulation could be a novel therapeutic approach for the augmentation of sensitivity to anticancer agents, or to overcome resistance to them, in the treatment of endometrial cancer.

Immunosuppression in Pediatric Liver and Intestinal Transplantation: A Closer Look at the Arsenal

Immunosuppression in Pediatric Liver and Intestinal Transplantation: A Closer Look at the Arsenal