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Sample records for 3a4 cyp 3a4

  1. Interactions between CYP3A4 and Dietary Polyphenols

    PubMed Central

    Basheer, Loai; Kerem, Zohar

    2015-01-01

    The human cytochrome P450 enzymes (P450s) catalyze oxidative reactions of a broad spectrum of substrates and play a critical role in the metabolism of xenobiotics, such as drugs and dietary compounds. CYP3A4 is known to be the main enzyme involved in the metabolism of drugs and most other xenobiotics. Dietary compounds, of which polyphenolics are the most studied, have been shown to interact with CYP3A4 and alter its expression and activity. Traditionally, the liver was considered the prime site of CYP3A-mediated first-pass metabolic extraction, but in vitro and in vivo studies now suggest that the small intestine can be of equal or even greater importance for the metabolism of polyphenolics and drugs. Recent studies have pointed to the role of gut microbiota in the metabolic fate of polyphenolics in human, suggesting their involvement in the complex interactions between dietary polyphenols and CYP3A4. Last but not least, all the above suggests that coadministration of drugs and foods that are rich in polyphenols is expected to stimulate undesirable clinical consequences. This review focuses on interactions between dietary polyphenols and CYP3A4 as they relate to structural considerations, food-drug interactions, and potential negative consequences of interactions between CYP3A4 and polyphenols. PMID:26180597

  2. Interactions between CYP3A4 and Dietary Polyphenols.

    PubMed

    Basheer, Loai; Kerem, Zohar

    2015-01-01

    The human cytochrome P450 enzymes (P450s) catalyze oxidative reactions of a broad spectrum of substrates and play a critical role in the metabolism of xenobiotics, such as drugs and dietary compounds. CYP3A4 is known to be the main enzyme involved in the metabolism of drugs and most other xenobiotics. Dietary compounds, of which polyphenolics are the most studied, have been shown to interact with CYP3A4 and alter its expression and activity. Traditionally, the liver was considered the prime site of CYP3A-mediated first-pass metabolic extraction, but in vitro and in vivo studies now suggest that the small intestine can be of equal or even greater importance for the metabolism of polyphenolics and drugs. Recent studies have pointed to the role of gut microbiota in the metabolic fate of polyphenolics in human, suggesting their involvement in the complex interactions between dietary polyphenols and CYP3A4. Last but not least, all the above suggests that coadministration of drugs and foods that are rich in polyphenols is expected to stimulate undesirable clinical consequences. This review focuses on interactions between dietary polyphenols and CYP3A4 as they relate to structural considerations, food-drug interactions, and potential negative consequences of interactions between CYP3A4 and polyphenols. PMID:26180597

  3. Comparison of Paeoniflorin and Albiflorin on Human CYP3A4 and CYP2D6

    PubMed Central

    Gao, Li-Na; Zhang, Ye; Cui, Yuan-Lu; Akinyi, Olunga Mary

    2015-01-01

    Peony (Paeonia lactiflora Pall-) is a plant medicine and a functional food ingredient with wide application for more than 2000 years. It can be coadministrated with many other drugs, composed of traditional Chinese medicine compound such as shaoyao-gancao decoction. In order to explore the efficacy and safety of peony, effects of paeoniflorin and albiflorin (the principal components of peony) on cytochrome P450 (CYP) 3A4 and CYP2D6 were analyzed in human hepatoma HepG2 cells and evaluated from the level of recombinant CYP enzymes in vitro. The findings indicated that albiflorin possessed stronger regulation on the mRNA expression of CYP3A4 and CYP2D6 than paeoniflorin. For the protein level of CYP3A4, albiflorin showed significant induction or inhibition with the concentration increasing from 10−7 M to 10−5 M, but no remarkable variation was observed in paeoniflorin-treated group. Enzyme activity assay implied that both paeoniflorin and albiflorin could regulate CYP3A4 and CYP2D6 with varying degrees. The results showed that albiflorin should be given more attention because it may play a vital role on the overall efficacy of peony. The whole behavior of both paeoniflorin and albiflorin should be focused on ensuring the rationality and effectiveness of clinical application. PMID:26089940

  4. Comparison of Paeoniflorin and Albiflorin on Human CYP3A4 and CYP2D6.

    PubMed

    Gao, Li-Na; Zhang, Ye; Cui, Yuan-Lu; Akinyi, Olunga Mary

    2015-01-01

    Peony (Paeonia lactiflora Pall-) is a plant medicine and a functional food ingredient with wide application for more than 2000 years. It can be coadministrated with many other drugs, composed of traditional Chinese medicine compound such as shaoyao-gancao decoction. In order to explore the efficacy and safety of peony, effects of paeoniflorin and albiflorin (the principal components of peony) on cytochrome P450 (CYP) 3A4 and CYP2D6 were analyzed in human hepatoma HepG2 cells and evaluated from the level of recombinant CYP enzymes in vitro. The findings indicated that albiflorin possessed stronger regulation on the mRNA expression of CYP3A4 and CYP2D6 than paeoniflorin. For the protein level of CYP3A4, albiflorin showed significant induction or inhibition with the concentration increasing from 10(-7) M to 10(-5) M, but no remarkable variation was observed in paeoniflorin-treated group. Enzyme activity assay implied that both paeoniflorin and albiflorin could regulate CYP3A4 and CYP2D6 with varying degrees. The results showed that albiflorin should be given more attention because it may play a vital role on the overall efficacy of peony. The whole behavior of both paeoniflorin and albiflorin should be focused on ensuring the rationality and effectiveness of clinical application. PMID:26089940

  5. Inhibitory Effects of Vegetable Juices on CYP3A4 Activity in Recombinant CYP3A4 and LS180 Cells.

    PubMed

    Tsujimoto, Masayuki; Uchida, Tomoe; Kozakai, Hiroyuki; Yamamoto, Saori; Minegaki, Tetsuya; Nishiguchi, Kohshi

    2016-01-01

    It is thought that eating habits induces individual variation in intestinal absorption and metabolism of drugs. The objective of this research was to clarify the influence of vegetables juices on CYP3A4 activity, which is an important enzyme in intestine. Five vegetables juices (VJ-o, Kagome Original(®); VJ-g, Kagome 30 kinds of vegetables and fruits(®); VJ-p, Kagome Purple vegetables(®); VJ-r, Kagome Sweet Tomato(®); and VJ-y, Kagome Fruity Salada(®); KAGOME Co., Ltd., Aichi, Japan) were centrifuged (1630×g, 10 min) and filtered using filter paper and 0.45-µm membrane filters. In this study, recombinant CYP3A4 and LS180 cells were used for the evaluation of CYP3A4 activity. The metabolisms to 6β-hydroxytestosterone by recombinant CYP3A4 were significantly inhibited by VJ-o, VJ-g, and VJ-y in a preincubation time-dependent manner, and CYP3A4 activity in LS180 cells were significantly inhibited by VJ-o and VJ-y. These results show that the difference in ingestion volume of vegetable juices and vegetables might partially induce individual difference in intestinal drug metabolism.

  6. Inhibitory Effects of Vegetable Juices on CYP3A4 Activity in Recombinant CYP3A4 and LS180 Cells.

    PubMed

    Tsujimoto, Masayuki; Uchida, Tomoe; Kozakai, Hiroyuki; Yamamoto, Saori; Minegaki, Tetsuya; Nishiguchi, Kohshi

    2016-01-01

    It is thought that eating habits induces individual variation in intestinal absorption and metabolism of drugs. The objective of this research was to clarify the influence of vegetables juices on CYP3A4 activity, which is an important enzyme in intestine. Five vegetables juices (VJ-o, Kagome Original(®); VJ-g, Kagome 30 kinds of vegetables and fruits(®); VJ-p, Kagome Purple vegetables(®); VJ-r, Kagome Sweet Tomato(®); and VJ-y, Kagome Fruity Salada(®); KAGOME Co., Ltd., Aichi, Japan) were centrifuged (1630×g, 10 min) and filtered using filter paper and 0.45-µm membrane filters. In this study, recombinant CYP3A4 and LS180 cells were used for the evaluation of CYP3A4 activity. The metabolisms to 6β-hydroxytestosterone by recombinant CYP3A4 were significantly inhibited by VJ-o, VJ-g, and VJ-y in a preincubation time-dependent manner, and CYP3A4 activity in LS180 cells were significantly inhibited by VJ-o and VJ-y. These results show that the difference in ingestion volume of vegetable juices and vegetables might partially induce individual difference in intestinal drug metabolism. PMID:27582329

  7. Cytochrome P450 3A4 and CYP3A5-Catalyzed Bioactivation of Lapatinib.

    PubMed

    Towles, Joanna K; Clark, Rebecca N; Wahlin, Michelle D; Uttamsingh, Vinita; Rettie, Allan E; Jackson, Klarissa D

    2016-10-01

    Metabolic activation of the dual-tyrosine kinase inhibitor lapatinib by cytochromes CYP3A4 and CYP3A5 has been implicated in lapatinib-induced idiosyncratic hepatotoxicity; however, the relative enzyme contributions have not been established. The objective of this study was to examine the roles of CYP3A4 and CYP3A5 in lapatinib bioactivation leading to a reactive, potentially toxic quinoneimine. Reaction phenotyping experiments were performed using individual human recombinant P450 enzymes and P450-selective chemical inhibitors. Lapatinib metabolites and quinoneimine-glutathione (GSH) adducts were analyzed using liquid chromatography-tandem mass spectrometry. A screen of cDNA-expressed P450s confirmed that CYP3A4 and CYP3A5 are the primary enzymes responsible for quinoneimine-GSH adduct formation using lapatinib or O-dealkylated lapatinib as the substrate. The mean kinetic parameters (Km and kcat) of lapatinib O-dealkylation revealed that CYP3A4 was 5.2-fold more efficient than CYP3A5 at lapatinib O-dealkylation (CYP3A4 kcat/Km = 6.8 μM(-1) min(-1) versus CYP3A5 kcat/Km = 1.3 μM(-1) min(-1)). Kinetic analysis of GSH adduct formation indicated that CYP3A4 was also 4-fold more efficient at quinoneimine-GSH adduct formation as measured by kcat (maximum relative GSH adduct levels)/Km (CYP3A4 = 0.0082 vs. CYP3A5 = 0.0021). In human liver microsomal (HLM) incubations, CYP3A4-selective inhibitors SR-9186 and CYP3cide reduced formation of GSH adducts by 78% and 72%, respectively, compared with >90% inhibition by the pan-CYP3A inhibitor ketoconazole. The 16%-22% difference between CYP3A- and CYP3A4-selective inhibition indicates the involvement of remaining CYP3A5 activity in generating reactive metabolites from lapatinib in pooled HLMs. Collectively, these findings support the conclusion that both CYP3A4 and CYP3A5 are quantitatively important contributors to lapatinib bioactivation. PMID:27450182

  8. A comparative study of CYP3A4 polymorphisms in Mexican Amerindian and Mestizo populations.

    PubMed

    Reyes-Hernández, Octavio D; Lares-Asseff, Ismael; Sosa-Macias, Martha; Vega, Libia; Albores, Arnulfo; Elizondo, Guillermo

    2008-01-01

    Cytochrome P-450 3A4 (CYP3A4) contributes to the metabolism of approximately half the drugs in clinical use today. The aim of the present study was to determine the frequency of the CYP3A4*1B, *2, *4, *5, and *18 alleles amongst both Tepehuan Amerindians, a native group that has inhabited northern Mexico for thousands of years, and Mestizo Mexicans, and to compare the data with those of other populations. Genotyping experiments revealed that 8.8 and 8.0% of the Mestizo and Tepehuano subjects, respectively, carried the CYP3A4*1B allele. Only one Mestizo subject was heterozygous for the CYP3A4*2 variant, while CYP3A4*4, *5 and *18 allelic variants were not detected in either group. On the other hand, the frequencies of the CYP3A4*1B variant in Mestizos and Tepehuanos were similar to those reported for Caucasians, but different from those observed for African and Asian populations.

  9. Characterization of the genetic variation present in CYP3A4 in three South African populations.

    PubMed

    Drögemöller, Britt; Plummer, Marieth; Korkie, Lundi; Agenbag, Gloudi; Dunaiski, Anke; Niehaus, Dana; Koen, Liezl; Gebhardt, Stefan; Schneider, Nicol; Olckers, Antonel; Wright, Galen; Warnich, Louise

    2013-01-01

    The CYP3A4 enzyme is the most abundant human cytochrome P450 (CYP) and is regarded as the most important enzyme involved in drug metabolism. Inter-individual and inter-population variability in gene expression and enzyme activity are thought to be influenced, in part, by genetic variation. Although Southern African individuals have been shown to exhibit the highest levels of genetic diversity, they have been under-represented in pharmacogenetic research to date. Therefore, the aim of this study was to identify genetic variation within CYP3A4 in three South African population groups comprising of 29 Khoisan, 65 Xhosa and 65 Mixed Ancestry (MA) individuals. To identify known and novel CYP3A4 variants, 15 individuals were randomly selected from each of the population groups for bi-directional Sanger sequencing of ~600 bp of the 5'-upstream region and all thirteen exons including flanking intronic regions. Genetic variants detected were genotyped in the rest of the cohort. In total, 24 SNPs were detected, including CYP3A4(*)12, CYP3A4(*)15, and the reportedly functional CYP3A4(*)1B promoter polymorphism, as well as two novel non-synonymous variants. These putatively functional variants, p.R162W and p.Q200H, were present in two of the three populations and all three populations, respectively, and in silico analysis predicted that the former would damage the protein product. Furthermore, the three populations were shown to exhibit distinct genetic profiles. These results confirm that South African populations show unique patterns of variation in the genes encoding xenobiotic metabolizing enzymes. This research suggests that population-specific genetic profiles for CYP3A4 and other drug metabolizing genes would be essential to make full use of pharmacogenetics in Southern Africa. Further investigation is needed to determine if the identified genetic variants influence CYP3A4 metabolism phenotype in these populations. PMID:23423246

  10. CYP3A4 drug interactions: correlation of 10 in vitro probe substrates

    PubMed Central

    Kenworthy, K E; Bloomer, J C; Clarke, S E; Houston, J B

    1999-01-01

    Aims Many substrates of cytochrome P450 (CYP) 3A4 are used for in vitro investigations of drug metabolism and potential drug–drug interactions. The aim of the present study was to determine the relationship between 10 commonly used CYP3A4 probes using modifiers with a range of inhibitory potency. Methods The effects of 34 compounds on CYP3A4-mediated metabolism were investigated in a recombinant CYP3A4 expression system. Inhibition of erythromycin, dextromethorphan and diazepam N-demethylation, testosterone 6β-hydroxylation, midazolam 1-hydroxylation, triazolam 4-hydroxylation, nifedipine oxidation, cyclosporin oxidation, terfenadine C-hydroxylation and N-dealkylation and benzyloxyresorufin O-dealkylation was evaluated at the apparent Km or S50 (for substrates showing sigmoidicity) value for each substrate and at an inhibitor concentration of 30 μm. Results While all CYP3A4 probe substrates demonstrate some degree of similarity, examination of the coefficients of determination, together with difference and cluster analysis highlighted that seven substrates can be categorized into two distinct substrate groups. Erythromycin, cyclosporin and testosterone form the most closely related group and dextromethorphan, diazepam, midazolam and triazolam form a second group. Terfenadine can be equally well placed in either group, while nifedipine shows a distinctly different relationship. Benzyloxyresorufin shows the weakest correlation with all the other CYP3A4 probes. Modifiers that caused negligible inhibition or potent inhibition are generally comparable in all assays, however, the greatest variability is apparent with compounds causing, on average, intermediate inhibition. Modifiers of this type may cause substantial inhibition, no effect or even activation depending on the substrate employed. Conclusions It is recommended that multiple CYP3A4 probes, representing each substrate group, are used for the in vitro assessment of CYP3A4-mediated drug interactions. PMID

  11. High frequency and founder effect of the CYP3A4*20 loss-of-function allele in the Spanish population classifies CYP3A4 as a polymorphic enzyme.

    PubMed

    Apellániz-Ruiz, M; Inglada-Pérez, L; Naranjo, M E G; Sánchez, L; Mancikova, V; Currás-Freixes, M; de Cubas, A A; Comino-Méndez, I; Triki, S; Rebai, A; Rasool, M; Moya, G; Grazina, M; Opocher, G; Cascón, A; Taboada-Echalar, P; Ingelman-Sundberg, M; Carracedo, A; Robledo, M; Llerena, A; Rodríguez-Antona, C

    2015-06-01

    Cytochrome P450 3A4 (CYP3A4) is a key drug-metabolizing enzyme. Loss-of-function variants have been reported as rare events, and the first demonstration of a CYP3A4 protein lacking functional activity is caused by CYP3A4*20 allele. Here we characterized the world distribution and origin of CYP3A4*20 mutation. CYP3A4*20 was determined in more than 4000 individuals representing different populations, and haplotype analysis was performed using CYP3A polymorphisms and microsatellite markers. CYP3A4*20 allele was present in 1.2% of the Spanish population (up to 3.8% in specific regions), and all CYP3A4*20 carriers had a common haplotype. This is compatible with a Spanish founder effect and classifies CYP3A4 as a polymorphic enzyme. This constitutes the first description of a CYP3A4 loss-of-function variant with high frequency in a population. CYP3A4*20 results together with the key role of CYP3A4 in drug metabolism support screening for rare CYP3A4 functional alleles among subjects with adverse drug events in certain populations. PMID:25348618

  12. Aloe vera juice: IC₅₀ and dual mechanistic inhibition of CYP3A4 and CYP2D6.

    PubMed

    Djuv, Ane; Nilsen, Odd Georg

    2012-03-01

    The aim of this study was to evaluate the inhibitory potency (IC₅₀ values) of ethanol extracts of two commercially available aloe vera juice (AVJ) products, on CYP3A4 and CYP2D6 activities in vitro and to determine if such inhibitions could be mechanism-based. Recombinant human CYP3A4 and CYP2D6 enzymes were used and the activities were expressed by the metabolism of testosterone and dextromethorphan with ketoconazole and quinidine as positive inhibitor controls, respectively. The formed metabolites were quantified by validated HPLC techniques. Time- and NADPH- dependent inhibition assays were performed to evaluate a possible mechanism-based inhibition. One of the AVJ extracts showed about twice the inhibitory potency towards both CYP enzymes over the other with IC₅₀ values of 8.35 ± 0.72 and 12.5 ± 2.1 mg/mL for CYP3A4 and CYP2D6, respectively. The AVJ was found to exert both CYP mediated and non-CYP mediated inhibition of both CYP3A4 and CYP2D6. This dual mechanistic inhibition, however, seems to be governed by different mechanisms for CYP3A4 and CYP2D6. Estimated IC₅₀ inhibition values indicate no major interference of AVJ with drug metabolism in man, but the dual mechanistic inhibition of both enzymes might be of clinical significance.

  13. Single-Walled Carbon Nanotubes Inhibit the Cytochrome P450 Enzyme, CYP3A4

    NASA Astrophysics Data System (ADS)

    El-Sayed, Ramy; Bhattacharya, Kunal; Gu, Zonglin; Yang, Zaixing; Weber, Jeffrey K.; Li, Hu; Leifer, Klaus; Zhao, Yichen; Toprak, Muhammet S.; Zhou, Ruhong; Fadeel, Bengt

    2016-02-01

    We report a detailed computational and experimental study of the interaction of single-walled carbon nanotubes (SWCNTs) with the drug-metabolizing cytochrome P450 enzyme, CYP3A4. Dose-dependent inhibition of CYP3A4-mediated conversion of the model compound, testosterone, to its major metabolite, 6β-hydroxy testosterone was noted. Evidence for a direct interaction between SWCNTs and CYP3A4 was also provided. The inhibition of enzyme activity was alleviated when SWCNTs were pre-coated with bovine serum albumin. Furthermore, covalent functionalization of SWCNTs with polyethylene glycol (PEG) chains mitigated the inhibition of CYP3A4 enzymatic activity. Molecular dynamics simulations suggested that inhibition of the catalytic activity of CYP3A4 is mainly due to blocking of the exit channel for substrates/products through a complex binding mechanism. This work suggests that SWCNTs could interfere with metabolism of drugs and other xenobiotics and provides a molecular mechanism for this toxicity. Our study also suggests means to reduce this toxicity, eg., by surface modification.

  14. Single-Walled Carbon Nanotubes Inhibit the Cytochrome P450 Enzyme, CYP3A4

    PubMed Central

    El-Sayed, Ramy; Bhattacharya, Kunal; Gu, Zonglin; Yang, Zaixing; Weber, Jeffrey K.; Li, Hu; Leifer, Klaus; Zhao, Yichen; Toprak, Muhammet S.; Zhou, Ruhong; Fadeel, Bengt

    2016-01-01

    We report a detailed computational and experimental study of the interaction of single-walled carbon nanotubes (SWCNTs) with the drug-metabolizing cytochrome P450 enzyme, CYP3A4. Dose-dependent inhibition of CYP3A4-mediated conversion of the model compound, testosterone, to its major metabolite, 6β-hydroxy testosterone was noted. Evidence for a direct interaction between SWCNTs and CYP3A4 was also provided. The inhibition of enzyme activity was alleviated when SWCNTs were pre-coated with bovine serum albumin. Furthermore, covalent functionalization of SWCNTs with polyethylene glycol (PEG) chains mitigated the inhibition of CYP3A4 enzymatic activity. Molecular dynamics simulations suggested that inhibition of the catalytic activity of CYP3A4 is mainly due to blocking of the exit channel for substrates/products through a complex binding mechanism. This work suggests that SWCNTs could interfere with metabolism of drugs and other xenobiotics and provides a molecular mechanism for this toxicity. Our study also suggests means to reduce this toxicity, eg., by surface modification. PMID:26899743

  15. Hemopericardium with tamponade following rivaroxaban administration and its attenuation by CYP3A4 inhibitors

    PubMed Central

    Menendez, Denisse

    2016-01-01

    Novel oral anticoagulants including the factor Xa inhibitor rivaroxaban are important alternatives to warfarin for the prevention of thromboembolic stroke in patients with nonvalvular atrial fibrillation. The pharmacology and metabolism of these agents differ from those of the vitamin K antagonists used over the decades preceding their introduction. We present a case of spontaneous hemopericardium and cardiac tamponade following administration of rivaroxaban. A review of the patient's medications revealed a total of seven agents known to be metabolized through cytochrome P450 3A4 (CYP3A4), the major pathway for rivaroxaban metabolism. While most physicians are familiar with recommendations to monitor renal function in patients prescribed rivaroxaban, we suspect that many fail to evaluate possible interactions with other agents having CYP3A4 inhibitory or inducer activity. PMID:27695181

  16. Hemopericardium with tamponade following rivaroxaban administration and its attenuation by CYP3A4 inhibitors

    PubMed Central

    Menendez, Denisse

    2016-01-01

    Novel oral anticoagulants including the factor Xa inhibitor rivaroxaban are important alternatives to warfarin for the prevention of thromboembolic stroke in patients with nonvalvular atrial fibrillation. The pharmacology and metabolism of these agents differ from those of the vitamin K antagonists used over the decades preceding their introduction. We present a case of spontaneous hemopericardium and cardiac tamponade following administration of rivaroxaban. A review of the patient's medications revealed a total of seven agents known to be metabolized through cytochrome P450 3A4 (CYP3A4), the major pathway for rivaroxaban metabolism. While most physicians are familiar with recommendations to monitor renal function in patients prescribed rivaroxaban, we suspect that many fail to evaluate possible interactions with other agents having CYP3A4 inhibitory or inducer activity.

  17. Polychlorinated biphenyl (PCB) induction of CYP3A4 enzyme activity in healthy Faroese adults

    SciTech Connect

    Petersen, Maria Skaalum Halling, Jonrit; Damkier, Per; Nielsen, Flemming; Grandjean, Philippe; Weihe, Pal; Brosen, Kim

    2007-10-15

    The CYP3A4 enzyme is, along with other cytochrome P450 enzymes, involved in the metabolism of environmental pollutants and is highly inducible by these substances. A commercial polychlorinated biphenyl (PCB) mixture, 1,1,1,-trichloro-2-(o-chlorophenyl), 2-(p'-chlorophenyl)ethane (o,p'-DDT) and 1,1,-dichloro-2,2-bis (p-chlorophenyl)ethene (p,p'-DDE) are known to induce CYP3A4 activity through activation of nuclear receptors, such as the pregnane X receptor. However, this induction of CYP3A4 has not yet been investigated in humans. Thus, the aim of the study was to determine the variability of the CYP3A4 phenotype in regard to increased concentrations of PCBs and other persistent organohalogen pollutants (POPs) in healthy Faroese adults. In 310 randomly selected Faroese residents aged 18-60 years, the CYP3A4 activity was determined based on the urinary 6{beta}-hydroxycortisol/cortisol (6{beta}-OHC/FC) ratio. POP exposures were assessed by measuring their concentrations in serum lipid. The results showed a unimodal distribution of the 6{beta}-OHC/FC ratio with values ranging from 0.58 to 27.38. Women had a slightly higher 6{beta}-OHC/FC ratio than men (p = 0.07). Confounder-adjusted multiple regression analysis showed significant associations between 6{beta}-OHC/FC ratios and {sigma}PCB, PCB-TEQ and p,p'-DDE, o,p'-DDT and HCB, respectively, but the associations were statistically significant for men only.

  18. In vitro metabolism of piperaquine is primarily mediated by CYP3A4

    PubMed Central

    Lee, Tina Ming-Na; Huang, Liusheng; Johnson, Marla K.; Lizak, Patricia; Kroetz, Deanna; Aweeka, Francesca; Parikh, Sunil

    2016-01-01

    Piperaquine (PQ) is part of a first-line treatment regimen for Plasmodium falciparum malaria recommended by the World Health Organization (WHO). We aimed to determine the major metabolic pathway(s) of PQ in vitro. A reliable, validated tandem mass spectrometry method was developed. Concentrations of PQ were measured after incubation with both human liver microsomes (HLMs) and expressed cytochrome P450 enzymes (P450s). In pooled HLMs, incubations with an initial PQ concentration of 0.3 µM resulted in a 34.8 ± 4.9% loss of substrate over 60 min, corresponding to a turnover rate of 0.009 min−1 (r2 = 0.9223). Miconazole, at nonspecific P450 inhibitory concentrations, resulted in almost complete inhibition of PQ metabolism. The greatest inhibition was demonstrated with selective CYP3A4 (100%) and CYP2C8 (66%) inhibitors. Using a mixture of recombinant P450 enzymes, turnover for PQ metabolism was estimated as 0.0099 min−1; recombinant CYP3A4 had a higher metabolic rate (0.017 min−1) than recombinant CYP2C8 (p < .0001). Inhibition of CYP3A4-mediated PQ loss was greatest using the selective inhibitor ketoconazole (9.1 ± 3.5% loss with ketoconazole vs 60.7 ± 5.9% with no inhibitor, p < .0001). In summary, the extent of inhibition of in vitro metabolism with ketoconazole (83%) denotes that PQ appears to be primarily catalyzed by CYP3A4. Further studies to support these findings through the identification and characterization of PQ metabolites are planned. PMID:22671777

  19. Prediction of CYP3A4 enzyme activity using haplotype tag SNPs in African Americans.

    PubMed

    Perera, M A; Thirumaran, R K; Cox, N J; Hanauer, S; Das, S; Brimer-Cline, C; Lamba, V; Schuetz, E G; Ratain, M J; Di Rienzo, A

    2009-02-01

    The CYP3A locus encodes hepatic enzymes that metabolize many clinically used drugs. However, there is marked interindividual variability in enzyme expression and clearance of drugs metabolized by these enzymes. We utilized comparative genomics and computational prediction of transcriptional factor binding sites to evaluate regions within CYP3A that were most likely to contribute to this variation. We then used a haplotype tagging single-nucleotide polymorphisms (htSNPs) approach to evaluate the entire locus with the fewest number of maximally informative SNPs. We investigated the association between these htSNPs and in vivo CYP3A enzyme activity using a single-point IV midazolam clearance assay. We found associations between the midazolam phenotype and age, diagnosis of hypertension and one htSNP (141689) located upstream of CYP3A4. 141689 lies near the xenobiotic responsive enhancer module (XREM) regulatory region of CYP3A4. Cell-based studies show increased transcriptional activation with the minor allele at 141689, in agreement with the in vivo association study findings. This study marks the first systematic evaluation of coding and noncoding variation that may contribute to CYP3A phenotypic variability.

  20. Endosulfan induces CYP2B6 and CYP3A4 by activating the pregnane X receptor

    SciTech Connect

    Casabar, Richard C.T.; Das, Parikshit C.; DeKrey, Gregory K.; Gardiner, Catherine S.; Cao Yan; Rose, Randy L.; Wallace, Andrew D.

    2010-06-15

    Endosulfan is an organochlorine pesticide commonly used in agriculture. Endosulfan has affects on vertebrate xenobiotic metabolism pathways that may be mediated, in part, by its ability to activate the pregnane X receptor (PXR) and/or the constitutive androstane receptor (CAR) which can elevate expression of cytochrome P450 (CYP) enzymes. This study examined the dose-dependency and receptor specificity of CYP induction in vitro and in vivo. The HepG2 cell line was transiently transfected with CYP2B6- and CYP3A4-luciferase promoter reporter plasmids along with human PXR (hPXR) or hCAR expression vectors. In the presence of hPXR, endosulfan-alpha exposure caused significant induction of CYP2B6 (16-fold) and CYP3A4 (11-fold) promoter activities over control at 10 {mu}M. The metabolite endosulfan sulfate also induced CYP2B6 (12-fold) and CYP3A4 (6-fold) promoter activities over control at 10 {mu}M. In the presence of hCAR-3, endosulfan-alpha induced CYP2B6 (2-fold) promoter activity at 10 {mu}M, but not at lower concentrations. These data indicate that endosulfan-alpha significantly activates hPXR strongly and hCAR weakly. Using western blot analysis of human hepatocytes, the lowest concentrations at which CYP2B6 and CYP3A4 protein levels were found to be significantly elevated by endosulfan-alpha were 1.0 {mu}M and 10 {mu}M, respectively. In mPXR-null/hPXR-transgenic mice, endosulfan-alpha exposure (2.5 mg/kg/day) caused a significant reduction of tribromoethanol-induced sleep times by approximately 50%, whereas no significant change in sleep times was observed in PXR-null mice. These data support the role of endosulfan-alpha as a strong activator of PXR and inducer of CYP2B6 and CYP3A4, which may impact metabolism of CYP2B6 or CYP3A4 substrates.

  1. Substrate-specific modulation of CYP3A4 activity by genetic variants of cytochrome P450 oxidoreductase (POR)

    PubMed Central

    Agrawal, Vishal; Choi, Ji Ha; Giacomini, Kathleen M.; Miller, Walter L.

    2010-01-01

    Objectives CYP3A4 receives electrons from P450 oxidoreductase (POR) to metabolize about 50% of clinically used drugs. There is substantial inter-individual variation in CYP3A4 catalytic activity that is not explained by CYP3A4 genetic variants. CYP3A4 is flexible and distensible, permitting it to accommodate substrates varying in shape and size. To elucidate mechanisms of variability in CYP3A4 catalysis, we examined the effects of genetic variants of POR, and explored the possibility that substrate-induced conformational changes in CYP3A4 differentially affect the ability of POR variants to support catalysis. Methods We expressed human CYP3A4 and four POR variants (Q153R, A287P, R457H, A503V) in bacteria, reconstituted them in vitro and measured the Michaelis constant and maximum velocity with testosterone, midazolam, quinidine and erythromycin as substrates. Results POR A287P and R457H had low activity with all substrates; Q153R had 76–94% of wild type (WT) activity with midazolam and erythromycin, but 129–150% activity with testosterone and quinidine. The A503V polymorphism reduced CYP3A4 activity to 61–77% of wild type with testosterone and midazolam, but had nearly wild type activity with quinidine and erythromycin. Conclusion POR variants affect CYP3A4 activities. The impact of a POR variant on catalysis by CYP3A4 is substrate-specific, probably due to substrate-induced conformational changes in CYP3A4. PMID:20697309

  2. Reductive metabolism of oxymatrine is catalyzed by microsomal CYP3A4

    PubMed Central

    Liu, Wenqin; Shi, Jian; Zhu, Lijun; Dong, Lingna; Luo, Feifei; Zhao, Min; Wang, Ying; Hu, Ming; Lu, Linlin; Liu, Zhongqiu

    2015-01-01

    Oxymatrine (OMT) is a pharmacologically active primary quinolizidine alkaloid with various beneficial and toxic effects. It is confirmed that, after oral administration, OMT could be transformed to the more toxic metabolite matrine (MT), and this process may be through the reduction reaction, but the study on the characteristics of this transformation is limited. The aim of this study was to investigate the characteristics of this transformation of OMT in the human liver microsomes (HLMs) and human intestinal microsomes (HIMs) and the cytochrome P450 (CYP) isoforms involved in this transformation. The current studies demonstrated that OMT could be metabolized to MT rapidly in HLMs and HIMs and CYP3A4 greatly contributed to this transformation. All HLMs, HIMs, and CYP3A4 isoform mediated reduction reaction followed typical biphasic kinetic model, and Km, Vmax, and CL were significant higher in HLMs than those in HIMs. Importantly, different oxygen contents could significantly affect the metabolism of OMT, and with the oxygen content decreased, the formation of metabolite was increased, suggesting this transformation was very likely a reduction reaction. Results of this in vitro study elucidated the metabolic pathways and characteristics of metabolism of OMT to MT and would provide a theoretical basis and guidance for the safe application of OMT. PMID:26586934

  3. Diindolylmethane, a naturally occurring compound, induces CYP3A4 and MDR1 gene expression by activating human PXR

    PubMed Central

    Pondugula, Satyanarayana R.; Flannery, Patrick C.; Abbott, Kodye L.; Coleman, Elaine S.; Mani, Sridhar; Samuel, Temesgen; Xie, Wen

    2015-01-01

    Activation of human pregnane X receptor (hPXR)-regulated expression of cytochrome P450 3A4 (CYP3A4) and multidrug resistance protein 1 (MDR1) plays an important role in mediating adverse drug interactions. Given the common use of natural products as part of adjunct human health behavior, there is a growing concern about natural products for their potential to induce undesired drug interactions through the activation of hPXR-regulated CYP3A4 and MDR1. Here, we studied whether 3,3′-diindolylmethane (DIM), a natural health supplement, could induce hPXR-mediated regulation of CYP3A4 and MDR1 in human hepatocytes and intestinal cells. DIM, at its physiologically relevant concentrations, not only induced hPXR transactivation of CYP3A4 promoter activity but also induced gene expression of CYP3A4 and MDR1. DIM decreased intracellular accumulation of MDR1 substrate rhodamine 123, suggesting that DIM induces the functional expression of MDR1. Pharmacologic inhibition or genetic knockdown of hPXR resulted in attenuation of DIM induced CYP3A4 and MDR1 gene expression, suggesting that DIM induces CYP3A4 and MDR1 in an hPXR-dependent manner. Together, these results support our conclusion that DIM induces hPXR-regulated CYP3A4 and MDR1 gene expression. The inductive effects of DIM on CYP3A4 and MDR1 expression caution the use of DIM in conjunction with other medications metabolized and transported via CYP3A4 and MDR1, respectively. PMID:25542144

  4. Augmented Inhibition of CYP3A4 in Human Primary Hepatocytes by Ritonavir Solid Drug Nanoparticles.

    PubMed

    Martin, Philip; Giardiello, Marco; McDonald, Tom O; Smith, Darren; Siccardi, Marco; Rannard, Steven P; Owen, Andrew

    2015-10-01

    Ritonavir is a protease inhibitor utilized primarily as a pharmaco-enhancer with concomitantly administered antiviral drugs including other protease inhibitors. However, poor tolerance, serious side effects, and toxicities associated with drug-drug interactions are common during exposure to ritonavir. The aim of this work was to investigate the impact of nanoformulation on ritonavir pharmacological properties. Emulsion-templated freeze-drying techniques were used to generate ritonavir (10 wt %) solid drug nanoparticle formulations. A total of 68 ritonavir formulations containing various mixtures of excipients were assessed for inhibition of CYP3A4 in baculosomes and primary human hepatocytes. Accumulation and cytotoxicity were assessed in HepG2 (hepatocytes), Caco-2 (intestinal), THP-1 (monocytes), A-THP-1 (macrophage), and CEM (lymphocytes). Transcellular permeation across Caco-2 cells was also assessed. From 68 solid drug nanoparticle formulations tested, 50 (73.5%) for baculosome and 44 (64.7%) for human primary hepatocytes exhibited enhanced CYP3A4 inhibition relative to an aqueous ritonavir solution. Sixty-one (89.7%) and 49 (72%) solid drug nanoformulations had higher apical to basal permeation across Caco-2 cells than aqueous solution of ritonavir after 60 and 120 min, respectively. No significant difference in cellular accumulation was observed for any solid drug nanoparticle for any cell type compared to aqueous ritonavir. However, incubation with the vast majority of solid drug nanoparticle formulations resulted in lower cytotoxicity of ritonavir than detected with an aqueous solution. These data provide in vitro proof of concept for improved inhibition of CYP3A4 by ritonavir through formation of solid drug nanoparticles. Nanodispersions also showed enhanced permeability across Caco-2 cells lower cytotoxicity across hepatic, intestinal, and immune cell types compared to an aqueous solution of ritonavir.

  5. Oxidase uncoupling in heme monooxygenases: Human cytochrome P450 CYP3A4 in Nanodiscs

    SciTech Connect

    Grinkova, Yelena V.; Denisov, Ilia G.; McLean, Mark A.; Sligar, Stephen G.

    2013-01-25

    Highlights: ► Substantial reducing equivalents are lost in human P450 CYP3A4 via an oxidase channel. ► Substrate binding has a pronounced effect on uncoupling in cytochrome P450. ► Anionic phospholipids improve the overall coupling in CYP3A4 Nanodiscs. -- Abstract: The normal reaction mechanism of cytochrome P450 operates by utilizing two reducing equivalents to reduce atmospheric dioxygen, producing one molecule of water and an oxygenated product in an overall stoichiometry of 2 electrons:1 dioxygen:1 product. However, three alternate unproductive pathways exist where the intermediate iron–oxygen states in the catalytic cycle can yield reduced oxygen products without substrate metabolism. The first involves release of superoxide from the oxygenated intermediate while the second occurs after input of the second reducing equivalent. Superoxide rapidly dismutates and hence both processes produce hydrogen peroxide that can be cytotoxic to the organism. In both cases, the formation of hydrogen peroxide involves the same overall stoichiometry as oxygenases catalysis. The key step in the catalytic cycle of cytochrome P450 involves scission of the oxygen–oxygen bond of atmospheric dioxygen to produce a higher valent iron-oxo state termed “Compound I”. This intermediate initiates a radical reaction in the oxygenase pathway but also can uptake two additional reducing equivalents from reduced pyridine nucleotide (NADPH) and the flavoprotein reductase to produce a second molecule of water. This non-productive decay of Compound I thus yields an overall oxygen to NADPH ratio of 1:2 and does not produce hydrocarbon oxidation. This water uncoupling reaction provides one of a limited means to study the reactivity of the critical Compound I intermediate in P450 catalysis. We measured simultaneously the rates of NADPH and oxygen consumption as a function of substrate concentration during the steady-state hydroxylation of testosterone catalyzed by human P450 CYP3A4

  6. Time-dependent inhibition of CYP3A4 by gallic acid in human liver microsomes and recombinant systems.

    PubMed

    Pu, Qiang-Hong; Shi, Liang; Yu, Chao

    2015-03-01

    1.Gallic acid is a main polyphenol in various fruits and plants. Inhibitory characteristics of gallic acid on CYP3A4 were still unclear. The objective of this work is hence to investigate inhibitory characteristics of gallic acid on CYP3A4 using testosterone as the probe substrate in human liver microsomes (HLMs) and recombinant CYP3A4 (rCYP3A4) systems. 2.Gallic acid caused concentration-dependent loss of CYP3A4 activity with IC50 values of 615.2 μM and 669.5 μM in HLM and rCYP3A4 systems, respectively. IC50-shift experiments showed that pre-incubation with gallic acid in the absence of NADPH contributed to 12- or 14-fold reduction of IC50 in HLM and rCYP3A4 systems, respectively, supporting a time-dependent inhibition. In HLM, time-dependent inactivation variables KI and Kinact were 485.8 μM and 0.05 min(-1), respectively. 3.Compared with the presence of NADPH, pre-incubation of gallic acid in the absence of NADPH markedly increased its inhibitory effects in HLM and rCYP3A4 systems. Those results indicate that CYP3A4 inactivation by gallic acid was independent on NADPH and was mainly mediated its oxidative products. 4.In conclusion, we showed that gallic acid weakly and time-dependently inactivated CYP3A4 via its oxidative products.

  7. A Fibroblast Growth Factor 21-Pregnane X Receptor Pathway Downregulates Hepatic CYP3A4 in Nonalcoholic Fatty Liver Disease.

    PubMed

    Woolsey, Sarah J; Beaton, Melanie D; Mansell, Sara E; Leon-Ponte, Matilde; Yu, Janice; Pin, Christopher L; Adams, Paul C; Kim, Richard B; Tirona, Rommel G

    2016-10-01

    Nonalcoholic fatty liver disease (NAFLD) alters drug response. We previously reported that NAFLD is associated with reduced in vivo CYP3A drug-metabolism activity and hepatic CYP3A4 expression in humans as well as mouse and human hepatoma models of the disease. Here, we investigated the role of the lipid- and glucose-modulating hormone fibroblast growth factor 21 (FGF21) in the molecular mechanism regulating CYP3A4 expression in NAFLD. In human subjects, mouse and cellular NAFLD models with lower CYP3A4 expression, circulating FGF21, or hepatic FGF21 mRNA levels were elevated. Administration of recombinant FGF21 or transient hepatic overexpression of FGF21 resulted in reduced liver CYP3A4 luciferase reporter activity in mice and decreased CYP3A4 mRNA expression and activity in cultured Huh7 hepatoma cells. Blocking canonical FGF21 signaling by pharmacological inhibition of MEK1 kinase in Huh7 cells caused de-repression of CYP3A4 mRNA expression with FGF21 treatment. Mice with high-fat diet-induced simple hepatic steatosis and lipid-loaded Huh7 cells had reduced nuclear localization of the pregnane X receptor (PXR), a key transcriptional regulator of CYP3A4 Furthermore, decreased nuclear PXR was observed in mouse liver and Huh7 cells after FGF21 treatment or FGF21 overexpression. Decreased PXR binding to the CYP3A4 proximal promoter was found in FGF21-treated Huh7 cells. An FGF21-PXR signaling pathway may be involved in decreased hepatic CYP3A4 metabolic activity in NAFLD. PMID:27482056

  8. A Fibroblast Growth Factor 21-Pregnane X Receptor Pathway Downregulates Hepatic CYP3A4 in Nonalcoholic Fatty Liver Disease.

    PubMed

    Woolsey, Sarah J; Beaton, Melanie D; Mansell, Sara E; Leon-Ponte, Matilde; Yu, Janice; Pin, Christopher L; Adams, Paul C; Kim, Richard B; Tirona, Rommel G

    2016-10-01

    Nonalcoholic fatty liver disease (NAFLD) alters drug response. We previously reported that NAFLD is associated with reduced in vivo CYP3A drug-metabolism activity and hepatic CYP3A4 expression in humans as well as mouse and human hepatoma models of the disease. Here, we investigated the role of the lipid- and glucose-modulating hormone fibroblast growth factor 21 (FGF21) in the molecular mechanism regulating CYP3A4 expression in NAFLD. In human subjects, mouse and cellular NAFLD models with lower CYP3A4 expression, circulating FGF21, or hepatic FGF21 mRNA levels were elevated. Administration of recombinant FGF21 or transient hepatic overexpression of FGF21 resulted in reduced liver CYP3A4 luciferase reporter activity in mice and decreased CYP3A4 mRNA expression and activity in cultured Huh7 hepatoma cells. Blocking canonical FGF21 signaling by pharmacological inhibition of MEK1 kinase in Huh7 cells caused de-repression of CYP3A4 mRNA expression with FGF21 treatment. Mice with high-fat diet-induced simple hepatic steatosis and lipid-loaded Huh7 cells had reduced nuclear localization of the pregnane X receptor (PXR), a key transcriptional regulator of CYP3A4 Furthermore, decreased nuclear PXR was observed in mouse liver and Huh7 cells after FGF21 treatment or FGF21 overexpression. Decreased PXR binding to the CYP3A4 proximal promoter was found in FGF21-treated Huh7 cells. An FGF21-PXR signaling pathway may be involved in decreased hepatic CYP3A4 metabolic activity in NAFLD.

  9. CYP3A4*1B polymorphism and cancer risk: a HuGE review and meta-analysis.

    PubMed

    Zhou, Li-Ping; Yao, Fan; Luan, Hong; Wang, Yin-Ling; Dong, Xi-Hua; Zhou, Wen-Wen; Wang, Qi-Hui

    2013-04-01

    CYP450 3A4 (CYP3A4), encoded by the CYP3A4 gene, is a major enzyme catalyzing the metabolism of both endogenous and exogenous agents that may play a role in the etiology of carcinogenesis. Several potentially functional polymorphisms of the CYP3A4 gene have been implicated in cancer risk, but individually published studies have shown inconclusive results. The aim of this Human Genome Epidemiology (HuGE) review and meta-analysis was to investigate the association between CYP3A4*1B (rs2740574 A > G) polymorphism and cancer risk. Eleven studies were included with a total of 3,810 cancer patients and 3,173 healthy controls. We found that the G allele and GG genotype of CYP3A4*1B polymorphism were associated with increased risk of cancers using the fixed effects model (allele model: odds ratio (OR) = 1.24, 95 %CI: 1.09-1.42, P = 0.001; recessive model: OR = 1.77, 95 %CI: 1.30-2.41, P < 0.001; homozygous model: OR = 1.72, 95 %CI: 1.19-2.47, P = 0.004). Subgroup analyses by cancer type showed that the G allele and G carrier (AG + GG) of CYP3A4*1B polymorphism had significant associations with increased risk of prostate cancer, but not with breast cancer, leukemia, or other cancers. With further subgroup analysis based on different ethnicities, the results indicated that the GG genotype of CYP3A4*1B polymorphism might increase the risk of cancer among African populations. However, similar associations were not observed among Caucasian and Asian populations. Results from the current meta-analysis indicate that the G allele and GG genotype of CYP3A4*1B polymorphism might be associated with increased cancer risk, especially for prostate cancer among African populations.

  10. A simultaneous assessment of CYP3A4 metabolism and induction in the DPX-2 cell line.

    PubMed

    Trubetskoy, Olga; Marks, Bryan; Zielinski, Thomas; Yueh, Mei-Fei; Raucy, Judy

    2005-03-04

    The DPX-2 cell line, a derivative of HepG2 cells, harbors human PXR and a luciferase-linked CYP3A4 promoter. These cells were used in a panel of cell-based assays for a parallel assessment of CYP3A4 induction, metabolism, and inhibition at the cellular level. CYP3A4 induction in the DPX-2 cell line by various agents was monitored in 96-well plates by a luciferase-based transcriptional activation assay. Of the prototypical CYP3A4 inducers examined, all exhibited elevated luciferase activity in DPX-2 cells. CYP3A4 enzyme activity in noninduced and rifampicin-induced DPX-2 cells was also assessed using Vivid fluorogenic substrates. Significantly elevated CYP3A4 activity levels (2.8-fold +/- 0.2-fold above DMSO-treated cells) were found in DPX-2 cells after 48 hours of exposure to rifampicin, but were undetectable in parental HepG2 cells. Rifampicin-induced activity levels were found to be suitable for assessing the inhibitory potential of new chemical entities in downstream CYP3A4 inhibition assays. The elevated CYP3A4 activity was inhibited 85% by 10 microM ketoconazole. In addition, a cytotoxicity assay to correct for possible toxic effects of compounds at the cellular level was applied. The comparative data obtained with a combination of the above assays suggests that the application of several independent in vitro technologies used in DPX-2 cells is the best possible strategy for the assessment of the complex phenomena of CYP3A4 induction and inhibition.

  11. Trainable structure-activity relationship model for virtual screening of CYP3A4 inhibition.

    PubMed

    Didziapetris, Remigijus; Dapkunas, Justas; Sazonovas, Andrius; Japertas, Pranas

    2010-11-01

    A new structure-activity relationship model predicting the probability for a compound to inhibit human cytochrome P450 3A4 has been developed using data for >800 compounds from various literature sources and tested on PubChem screening data. Novel GALAS (Global, Adjusted Locally According to Similarity) modeling methodology has been used, which is a combination of baseline global QSAR model and local similarity based corrections. GALAS modeling method allows forecasting the reliability of prediction thus defining the model applicability domain. For compounds within this domain the statistical results of the final model approach the data consistency between experimental data from literature and PubChem datasets with the overall accuracy of 89%. However, the original model is applicable only for less than a half of PubChem database. Since the similarity correction procedure of GALAS modeling method allows straightforward model training, the possibility to expand the applicability domain has been investigated. Experimental data from PubChem dataset served as an example of in-house high-throughput screening data. The model successfully adapted itself to both data classified using the same and different IC₅₀ threshold compared with the training set. In addition, adjustment of the CYP3A4 inhibition model to compounds with a novel chemical scaffold has been demonstrated. The reported GALAS model is proposed as a useful tool for virtual screening of compounds for possible drug-drug interactions even prior to the actual synthesis. PMID:20814717

  12. Intestinal CYP3A4 protects against lithocholic acid-induced hepatotoxicity in intestine-specific VDR-deficient mice.

    PubMed

    Cheng, Jie; Fang, Zhong-Ze; Kim, Jung-Hwan; Krausz, Kristopher W; Tanaka, Naoki; Chiang, John Y L; Gonzalez, Frank J

    2014-03-01

    Vitamin D receptor (VDR) mediates vitamin D signaling involved in bone metabolism, cellular growth and differentiation, cardiovascular function, and bile acid regulation. Mice with an intestine-specific disruption of VDR (Vdr(ΔIEpC)) have abnormal body size, colon structure, and imbalance of bile acid metabolism. Lithocholic acid (LCA), a secondary bile acid that activates VDR, is among the most toxic of the bile acids that when overaccumulated in the liver causes hepatotoxicity. Because cytochrome P450 3A4 (CYP3A4) is a target gene of VDR-involved bile acid metabolism, the role of CYP3A4 in VDR biology and bile acid metabolism was investigated. The CYP3A4 gene was inserted into Vdr(ΔIEpC) mice to produce the Vdr(ΔIEpC)/3A4 line. LCA was administered to control, transgenic-CYP3A4, Vdr(ΔIEpC), and Vdr(ΔIEpC)/3A4 mice, and hepatic toxicity and bile acid levels in the liver, intestine, bile, and urine were measured. VDR deficiency in the intestine of the Vdr(ΔIEpC) mice exacerbates LCA-induced hepatotoxicity manifested by increased necrosis and inflammation, due in part to over-accumulation of hepatic bile acids including taurocholic acid and taurodeoxycholic acid. Intestinal expression of CYP3A4 in the Vdr(ΔIEpC)/3A4 mouse line reduces LCA-induced hepatotoxicity through elevation of LCA metabolism and detoxification, and suppression of bile acid transporter expression in the small intestine. This study reveals that intestinal CYP3A4 protects against LCA hepatotoxicity.

  13. Evaluation of CYP3A4 inhibition and hepatotoxicity using DMSO-treated human hepatoma HuH-7 cells.

    PubMed

    Liu, Yitong; Flynn, Thomas J; Xia, Menghang; Wiesenfeld, Paddy L; Ferguson, Martine S

    2015-10-01

    A human hepatoma cell line (HuH-7) was evaluated as a metabolically competent cell model to investigate cytochrome P450 3A4 (CYP3A4) inhibition, induction, and hepatotoxicity. First, CYP3A4 gene expression and activity were determined in HuH-7 cells under three culture conditions: 1-week culture, 3-week culture, or 1 % dimethyl sulfoxide (DMSO) treatment. HuH-7 cells treated with DMSO for 2 weeks after confluence expressed the highest CYP3A4 gene expression and activity compared to the other two culture conditions. Furthermore, CYP3A4 activity in DMSO-treated HuH-7 cells was compared to that in a human hepatoma cell line (HepG2/C3A) and human bipotent progenitor cell line (HepaRG), which yielded the following ranking: HepaRG > DMSO-treated HuH-7 > HepG2/C3A cells. The effects of three known CYP3A4 inhibitors were evaluated using DMSO-treated HuH-7 cells. CYP3A4 enzyme inhibition in HuH-7 cells was further compared to human recombinant CYP3A4, indicating similar potency for reversible inhibitors (IC 50 within 2.5-fold), but different potency for the irreversible inhibitor. Next, induction of CYP3A4 activity was compared between DMSO-treated HuH-7 and HepaRG cells using two known inducers. DMSO-treated HuH-7 cells yielded minimal CYP3A4 induction compared to that in the HepaRG cells after 48-h treatments. Finally, the cytotoxicity of five known hepatotoxicants was evaluated in DMSO-treated HuH-7, HepG2/C3A, and HepaRG cells, and significant differences in cytotoxic sensitivity were observed. Overall, DMSO-treated HuH-7 cells are a valuable model for medium- or high-throughput screening of chemicals for CYP3A4 inhibition and hepatotoxicity.

  14. Evaluation of CYP3A4 inhibition and hepatotoxicity using DMSO-treated human hepatoma HuH-7 cells

    PubMed Central

    Liu, Yitong; Flynn, Thomas J.; Xia, Menghang; Wiesenfeld, Paddy L.; Ferguson, Martine S.

    2016-01-01

    A human hepatoma cell line (HuH-7) was evaluated as a metabolically competent cell model to investigate cytochrome P450 3A4 (CYP3A4) inhibition, induction, and hepatotoxicity. First, CYP3A4 gene expression and activity were determined in HuH-7 cells under three culture conditions: 1-week culture, 3-week culture, or 1% dimethyl sulfoxide (DMSO) treatment. HuH-7 cells treated with DMSO for 2 weeks after confluence expressed the highest CYP3A4 gene expression and activity compared to the other two culture conditions. Furthermore, CYP3A4 activity in DMSO-treated HuH-7 cells was compared to that in a human hepatoma cell line (HepG2/C3A) and human bipotent progenitor cell line (HepaRG), which yielded the following ranking: HepaRG > DMSO-treated HuH-7 >> HepG2/C3A cells. The effects of three known CYP3A4 inhibitors were evaluated using DMSO-treated HuH-7 cells. CYP3A4 enzyme inhibition in HuH-7 cells was further compared to human recombinant CYP3A4, indicating similar potency for reversible inhibitors (IC50 within 2.5 fold), but different potency for the irreversible inhibitor. Next, induction of CYP3A4 activity was compared between DMSO-treated HuH-7 and HepaRG cells using two known inducers. DMSO-treated HuH-7 cells yielded minimal CYP3A4 induction compared to that in the HepaRG cells after 48-h treatments. Finally, the cytotoxicity of five known hepatotoxicants was evaluated in DMSO-treated HuH-7 cells, HepG2/C3A, and HepaRG cells, and significant differences in cytotoxic sensitivity were observed. Overall, DMSO-treated HuH-7 cells are a valuable model for medium- or high-throughput screening of chemicals for CYP3A4 inhibition and hepatotoxicity. PMID:26377104

  15. Effect of Methamphetamine on Spectral Binding, Ligand Docking and Metabolism of Anti-HIV Drugs with CYP3A4

    PubMed Central

    Ande, Anusha; Wang, Lei; Vaidya, Naveen K.; Li, Weihua; Kumar, Santosh; Kumar, Anil

    2016-01-01

    Cytochrome P450 3A4 (CYP3A4) is the major drug metabolic enzyme, and is involved in the metabolism of antiretroviral drugs, especially protease inhibitors (PIs). This study was undertaken to examine the effect of methamphetamine on the binding and metabolism of PIs with CYP3A4. We showed that methamphetamine exhibits a type I spectral change upon binding to CYP3A4 with δAmax and KD of 0.016±0.001 and 204±18 μM, respectively. Methamphetamine-CYP3A4 docking showed that methamphetamine binds to the heme of CYP3A4 in two modes, both leading to N-demethylation. We then studied the effect of methamphetamine binding on PIs with CYP3A4. Our results showed that methamphetamine alters spectral binding of nelfinavir but not the other type I PIs (lopinavir, atazanavir, tipranavir). The change in spectral binding for nelfinavir was observed at both δAmax (0.004±0.0003 vs. 0.0068±0.0001) and KD (1.42±0.36 vs.2.93±0.08 μM) levels. We further tested effect of methamphetamine on binding of 2 type II PIs; ritonavir and indinavir. Our results showed that methamphetamine alters the ritonavir binding to CYP3A4 by decreasing both the δAmax (0.0038±0.0003 vs. 0.0055±0.0003) and KD (0.043±0.0001 vs. 0.065±0.001 nM), while indinavir showed only reduced KD in presence of methamphetamine (0.086±0.01 vs. 0.174±0.03 nM). Furthermore, LC-MS/MS studies in high CYP3A4 human liver microsomes showed a decrease in the formation of hydroxy ritonavir in the presence of methamphetamine. Finally, CYP3A4 docking with lopinavir and ritonavir in the absence and presence of methamphetamine showed that methamphetamine alters the docking of ritonavir, which is consistent with the results obtained from spectral binding and metabolism studies. Overall, our results demonstrated differential effects of methamphetamine on the binding and metabolism of PIs with CYP3A4. These findings have clinical implication in terms of drug dose adjustment of antiretroviral medication, especially with ritonavir

  16. Effect of Methamphetamine on Spectral Binding, Ligand Docking and Metabolism of Anti-HIV Drugs with CYP3A4.

    PubMed

    Nookala, Anantha R; Li, Junhao; Ande, Anusha; Wang, Lei; Vaidya, Naveen K; Li, Weihua; Kumar, Santosh; Kumar, Anil

    2016-01-01

    Cytochrome P450 3A4 (CYP3A4) is the major drug metabolic enzyme, and is involved in the metabolism of antiretroviral drugs, especially protease inhibitors (PIs). This study was undertaken to examine the effect of methamphetamine on the binding and metabolism of PIs with CYP3A4. We showed that methamphetamine exhibits a type I spectral change upon binding to CYP3A4 with δAmax and KD of 0.016±0.001 and 204±18 μM, respectively. Methamphetamine-CYP3A4 docking showed that methamphetamine binds to the heme of CYP3A4 in two modes, both leading to N-demethylation. We then studied the effect of methamphetamine binding on PIs with CYP3A4. Our results showed that methamphetamine alters spectral binding of nelfinavir but not the other type I PIs (lopinavir, atazanavir, tipranavir). The change in spectral binding for nelfinavir was observed at both δAmax (0.004±0.0003 vs. 0.0068±0.0001) and KD (1.42±0.36 vs.2.93±0.08 μM) levels. We further tested effect of methamphetamine on binding of 2 type II PIs; ritonavir and indinavir. Our results showed that methamphetamine alters the ritonavir binding to CYP3A4 by decreasing both the δAmax (0.0038±0.0003 vs. 0.0055±0.0003) and KD (0.043±0.0001 vs. 0.065±0.001 nM), while indinavir showed only reduced KD in presence of methamphetamine (0.086±0.01 vs. 0.174±0.03 nM). Furthermore, LC-MS/MS studies in high CYP3A4 human liver microsomes showed a decrease in the formation of hydroxy ritonavir in the presence of methamphetamine. Finally, CYP3A4 docking with lopinavir and ritonavir in the absence and presence of methamphetamine showed that methamphetamine alters the docking of ritonavir, which is consistent with the results obtained from spectral binding and metabolism studies. Overall, our results demonstrated differential effects of methamphetamine on the binding and metabolism of PIs with CYP3A4. These findings have clinical implication in terms of drug dose adjustment of antiretroviral medication, especially with ritonavir

  17. Arsenite and its metabolites, MMA{sup III} and DMA{sup III}, modify CYP3A4, PXR and RXR alpha expression in the small intestine of CYP3A4 transgenic mice

    SciTech Connect

    Medina-Diaz, I.M.; Estrada-Muniz, E.; Reyes-Hernandez, O.D.; Ramirez, P.; Vega, L.; Elizondo, G.

    2009-09-01

    Arsenic is an environmental pollutant that has been associated with an increased risk for the development of cancer and several other diseases through alterations of cellular homeostasis and hepatic function. Cytochrome P450 (P450) modification may be one of the factors contributing to these disorders. Several reports have established that exposure to arsenite modifies P450 expression by decreasing or increasing mRNA and protein levels. Cytochrome P450 3A4 (CYP3A4), the predominant P450 expressed in the human liver and intestines, which is regulated mainly by the Pregnane X Receptor-Retinoid X Receptor alpha (PXR-RXR alpha) heterodimer, contributes to the metabolism of approximately half the drugs in clinical use today. The present study investigates the effect of sodium arsenite and its metabolites monomethylarsonous acid (MMA{sup III}) and dimethylarsinous acid (DMA{sup III}) on CYP3A4, PXR, and RXR alpha expression in the small intestine of CYP3A4 transgenic mice. Sodium arsenite treatment increases mRNA, protein and CYP3A4 activity in a dose-dependent manner. However, the increase in protein expression was not as marked as compared to the increase in mRNA levels. Arsenite treatment induces the accumulation of Ub-protein conjugates, indicating that the activation of this mechanism may explain the differences observed between the mRNA and protein expression of CYP3A4 induction. Treatment with 0.05 mg/kg of DMA{sup III} induces CYP3A4 in a similar way, while treatment with 0.05 mg/kg of MMA{sup III} increases mostly mRNA, and to a lesser degree, CYP3A4 activity. Sodium arsenite and both its metabolites increase PXR mRNA, while only DMA{sup III} induces RXR alpha expression. Overall, these results suggest that sodium arsenite and its metabolites induce CYP3A4 expression by increasing PXR expression in the small intestine of CYP3A4 transgenic mice.

  18. Pivotal Role of P450-P450 Interactions in CYP3A4 Allostery: the Case of α-Naphthoflavone

    PubMed Central

    Davydov, Dmitri R.; Davydova, Nadezhda Y.; Sineva, Elena V.; Kufareva, Irina; Halpert, James R.

    2014-01-01

    SYNOPSIS We investigated the relationship between oligomerization of cytochrome P450 3A4 (CYP3A4) and its response to α-naphthoflavone (ANF), a prototypical heterotropic activator. Addition of ANF resulted in over a two-fold increase in the rate of CYP3A4-dependent debenzylation of 7-benzyloxy-4-(trifluoromethyl)coumarin (7-BFC) in human liver microsomes (HLM) but failed to produce activation in BD Supersomes™ or Baculosomes® containing recombinant CYP3A4 and NADPH-cytochrome P450 reductase (CPR). However, incorporation of purified CYP3A4 into Supersomes containing only recombinant CPR reproduced the behavior observed with HLM. The activation in this system was dependent on the surface density of the enzyme. While no activation was detectable at a lipid:P450 (L/P) ratio ≥ 750, it reached 225% at an L/P ratio of 140. To explore the relationship between this effect and CYP3A4 oligomerization we probed P450-P450 interactions with a new technique based on luminescence resonance energy transfer (LRET). The amplitude of LRET in mixed oligomers of the heme protein labeled with donor and acceptor fluorophores exhibited a sigmoidal dependence on the surface density of CYP3A4 in Supersomes. Addition of ANF eliminated this sigmoidal character and increased the degree of oligomerization at low enzyme concentrations. Therefore, the mechanisms of CYP3A4 allostery with ANF involve effector-dependent modulation of P450-P450 interactions. PMID:23651100

  19. Altered CYP2C9 Activity Following Modulation of CYP3A4 Levels in Human Hepatocytes: an Example of Protein-Protein Interactions

    PubMed Central

    Tweedie, Donald J.; Chan, Tom S.; Tracy, Timothy S.

    2014-01-01

    Cytochrome P450 (P450) protein-protein interactions resulting in modulation of enzyme activities have been well documented using recombinant isoforms. This interaction has been less clearly demonstrated in a more physiologic in vitro system such as human hepatocytes. As an expansion of earlier work (Subramanian et al., 2010), in which recombinant CYP2C9 activity decreased with increasing levels of CYP3A4, the current study modulated CYP3A4 content in human hepatocytes to determine the impact on CYP2C9. Modulation of CYP3A4 levels in situ was enabled by the use of a long-term human hepatocyte culture model (HepatoPac) shown to retain phenotypic hepatocyte function over a number of weeks. The extended period of culture allowed time for knockdown of CYP3A4 protein by small interfering RNA (siRNA) with subsequent recovery, as well as upregulation through induction with a recovery period. CYP3A4 gene silencing resulted in a 60% decrease in CYP3A4 activity and protein levels with a concomitant 74% increase in CYP2C9 activity, with no change in CYP2C9 mRNA levels. Upon removal of siRNA, both CYP2C9 and CYP3A4 activities returned to pre-knockdown levels. Importantly, modulation of CYP3A4 protein levels had no impact on cytochrome P450 reductase activities or levels. However, the possibility for competition for limiting reductase cannot be ruled out. Interestingly, lowering CYP3A4 levels also increased UDP-glucuronosyltransferase 2B7 activity. These studies clearly demonstrate that alterations in CYP3A4 levels can modulate CYP2C9 activity in situ and suggest that further studies are warranted to evaluate the possible clinical consequences of these findings. PMID:25157098

  20. Different effects of proton pump inhibitors and famotidine on the clopidogrel metabolic activation by recombinant CYP2B6, CYP2C19 and CYP3A4.

    PubMed

    Ohbuchi, Masato; Noguchi, Kiyoshi; Kawamura, Akio; Usui, Takashi

    2012-07-01

    Inhibitory potential of proton pump inhibitors (PPIs) and famotidine, an H(2) receptor antagonist, on the metabolic activation of clopidogrel was evaluated using recombinant CYP2B6, CYP2C19 and CYP3A4. Formation of the active metabolite from an intermediate metabolite, 2-oxo-clopidogrel, was investigated by liquid chromatography-tandem mass spectrometry and three peaks corresponding to the pharmacologically active metabolite and its stereoisomers were detected. Omeprazole potently inhibited clopidogrel activation by CYP2C19 with an IC(50) of 12.8 μmol/L and more weakly inhibited that by CYP2B6 and CYP3A4. IC(50) of omeprazole for CYP2C19 and CYP3A4 was decreased about two- and three-fold, respectively, by 30-min preincubation with NADPH. Lansoprazole, esomeprazole, pantoprazole, rabeprazole and rabeprazole thioether, a major metabolite, also inhibited metabolic activation by CYP2C19, with an IC(50) of 4.3, 8.9, 48.3, 36.2 and 30.5 μmol/L, respectively. In contrast, famotidine showed no more than 20% inhibition of clopidogrel activation by CYP2B6, CYP2C19 and CYP3A4 at up to 100 μmol/L and had no time-dependent CYP2C19 and CYP3A4 inhibition. These results provide direct evidence that PPIs inhibit clopidogrel metabolic activation and suggest that CYP2C19 inhibition is the main cause of drug-drug interaction between clopidogrel and omeprazole. Famotidine is considered as a safe anti-acid agent for patients taking clopidogrel. PMID:22313038

  1. Investigating the binding interactions of the anti-Alzheimer's drug donepezil with CYP3A4 and P-glycoprotein.

    PubMed

    McEneny-King, Alanna; Edginton, Andrea N; Rao, Praveen P N

    2015-01-15

    The anti-Alzheimer's agent donepezil is known to bind to the hepatic enzyme CYP3A4, but its relationship with the efflux transporter P-glycoprotein (P-gp) is not as well elucidated. We conducted in vitro inhibition studies of donepezil using human recombinant CYP3A4 and P-gp. These studies show that donepezil is a weak inhibitor of CYP3A4 (IC50=54.68±1.00μM) whereas the reference agent ketoconazole exhibited potent inhibition (CYP3A4 IC50=0.20±0.01μM). P-gp inhibition studies indicate that donepezil exhibits better inhibition relative to CYP3A4 (P-gp EC50=34.85±4.63μM) although it was less potent compared to ketoconazole (P-gp EC50=9.74±1.23μM). At higher concentrations, donepezil exhibited significant inhibition of CYP3A4 (69%, 84% and 87% inhibition at 100, 250 and 500μM, respectively). This indicates its potential to cause drug-drug interactions with other CYP3A4 substrates upon co-administration; however, this scenario is unlikely in vivo due to the low therapeutic concentrations of donepezil. Similarly, donepezil co-administration with P-gp substrates or inhibitors is unlikely to result in beneficial or adverse drug interactions. The molecular docking studies show that the 5,6-dimethoxyindan-1-one moiety of donepezil was oriented closer to the heme center in CYP3A4 whereas in the P-gp binding site, the protonated benzylpiperidine pharmacophore of donepezil played a major role in its binding ability. Energy parameters indicate that donepezil complex with both CYP3A4 and P-gp was less stable (CDOCKER energies=-15.05 and -4.91kcal/mol, respectively) compared to the ketoconazole-CYP3A4 and P-gp complex (CDOCKER energies=-41.89 and -20.03kcal/mol, respectively).

  2. Oral intake of curcumin markedly activated CYP 3A4: in vivo and ex-vivo studies.

    PubMed

    Hsieh, Yow-Wen; Huang, Ching-Ya; Yang, Shih-Ying; Peng, Yu-Hsuan; Yu, Chung-Ping; Chao, Pei-Dawn Lee; Hou, Yu-Chi

    2014-10-10

    Curcumin, a specific secondary metabolite of Curcuma species, has potentials for a variety of beneficial health effects. It is nowadays used as a dietary supplement. Everolimus (EVL) is an immunosuppressant indicated for allograft rejection and cancer therapy, but with narrow therapeutic window. EVL is a substrate of P-glycoprotein (P-gp) and cytochrome P450 3A4 (CYP3A4). This study investigated the effect of coadministration of curcumin on the pharmacokinetics of EVL in rats and the underlying mechanisms. EVL (0.5 mg/kg) was orally administered without and with 50 and 100 mg/kg of curcumin, respectively, in rats. Blood samples were collected at specific time points and EVL concentrations in blood were determined by QMS immunoassay. The underlying mechanisms were evaluated using cell model and recombinant CYP 3A4 isozyme. The results indicated that 50 and 100 mg/kg of curcumin significantly decreased the AUC0-540 of EVL by 70.6% and 71.5%, respectively, and both dosages reduced the Cmax of EVL by 76.7%. Mechanism studies revealed that CYP3A4 was markedly activated by curcumin metabolites, which apparently overrode the inhibition effects of curcumin on P-gp. In conclusion, oral intake of curcumin significantly decreased the bioavailability of EVL, a probe substrate of P-gp/CYP 3A4, mainly through marked activation on CYP 3A4.

  3. Expression of CYP3A4 and CYP3A7 in Human Foetal Tissues and its Correlation with Nuclear Receptors.

    PubMed

    Betts, Stina; Björkhem-Bergman, Linda; Rane, Anders; Ekström, Lena

    2015-10-01

    Previous reports have suggested that the nuclear receptors vitamin D receptor (VDR), peroxisome proliferator-activated receptor α (PPARα), pregnane X receptor (PXR) and constitutive androstane receptor (CAR) are involved in the regulation of the drug-metabolizing enzyme cytochrome P450 (CYP) 3A4 expression in adults. The aim of this study was to investigate the gene expression of CYP3A4 and the foetal CYP3A7 in human foetal tissues and their relation to gene expression and genetic variations in the nuclear receptors VDR, PPARα, PXR and CAR. We determined the relative expression of CYP3A4 and CYP3A7 and these nuclear receptors in foetal livers, intestines and adrenals, using quantitative PCR. In addition, the expression of these enzymes was also analysed in adult liver. There was a high interindividual variability in CYP3A4 and CYP3A7, 49 times and 326 times, respectively. Both CYP3A4 and CYP3A7 had the highest expression in the liver. There were significant correlations (p < 0.001) between the nuclear receptors studied and the expression of CYP3A4 and CYP3A7 in foetal liver, as well as the expression of CYP3A4 in foetal intestine. Polymorphisms in the VDR gene, rs1544410 and rs1523130 (TaqI), in the PXR gene, rs1523130, and in the PPARα gene, rs4253728, were not correlated with CYP3A4 or CYP3A7 expression. However, C-homozygous individuals of the TaqI VDR polymorphism had 60% lower VDR gene expression (p < 0.05), than individuals carrying one or two T alleles. In conclusion, differences in the expression of nuclear receptors might determine the variability in CYP3A4 and CYP3A7 expression observed in foetal liver.

  4. Rifampicin-activated human pregnane X receptor and CYP3A4 induction enhance acetaminophen-induced toxicity.

    PubMed

    Cheng, Jie; Ma, Xiaochao; Krausz, Kristopher W; Idle, Jeffrey R; Gonzalez, Frank J

    2009-08-01

    Acetaminophen (APAP) is safe at therapeutic levels but causes hepatotoxicity via N-acetyl-p-benzoquinone imine-induced oxidative stress upon overdose. To determine the effect of human (h) pregnane X receptor (PXR) activation and CYP3A4 induction on APAP-induced hepatotoxicity, mice humanized for PXR and CYP3A4 (TgCYP3A4/hPXR) were treated with APAP and rifampicin. Human PXR activation and CYP3A4 induction enhanced APAP-induced hepatotoxicity as revealed by hepatic alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities elevated in serum, and hepatic necrosis after coadministration of rifampicin and APAP, compared with APAP administration alone. In contrast, hPXR mice, wild-type mice, and Pxr-null mice exhibited significantly lower ALT/AST levels compared with TgCYP3A4/hPXR mice after APAP administration. Toxicity was coincident with depletion of hepatic glutathione and increased production of hydrogen peroxide, suggesting increased oxidative stress upon hPXR activation. Moreover, mRNA analysis demonstrated that CYP3A4 and other PXR target genes were significantly induced by rifampicin treatment. Urinary metabolomic analysis indicated that cysteine-APAP and its metabolite S-(5-acetylamino-2-hydroxyphenyl)mercaptopyruvic acid were the major contributors to the toxic phenotype. Quantification of plasma APAP metabolites indicated that the APAP dimer formed coincident with increased oxidative stress. In addition, serum metabolomics revealed reduction of lysophosphatidylcholine in the APAP-treated groups. These findings demonstrated that human PXR is involved in regulation of APAP-induced toxicity through CYP3A4-mediated hepatic metabolism of APAP in the presence of PXR ligands.

  5. Milk thistle's active components silybin and isosilybin: novel inhibitors of PXR-mediated CYP3A4 induction.

    PubMed

    Mooiman, Kim D; Maas-Bakker, Roel F; Moret, Ed E; Beijnen, Jos H; Schellens, Jan H M; Meijerman, Irma

    2013-08-01

    Because cancer is often treated with combination therapy, unexpected pharmacological effects can occur because of drug-drug interactions. Several drugs are able to cause upregulation or downregulation of drug transporters or cytochrome P450 enzymes, particularly CYP3A4. Induction of CYP3A4 may result in decreased plasma levels and therapeutic efficacy of anticancer drugs. Since the pregnane X receptor (PXR) is one of the major transcriptional regulators of CYP3A4, PXR antagonists can possibly prevent CYP3A4 induction. Currently, a limited number of PXR antagonists are available. Some of these antagonists, such as sulphoraphane and coumestrol, belong to the so-called complementary and alternative medicines (CAM). Therefore, the aim was to determine the potential of selected CAM (β-carotene, Echinacea purpurea, garlic, Ginkgo biloba, ginseng, grape seed, green tea, milk thistle, saw palmetto, valerian, St. John's Wort, and vitamins B6, B12, and C) to inhibit PXR-mediated CYP3A4 induction at the transcriptional level, using a reporter gene assay and a real-time polymerase chain reaction assay in LS180 colon adenocarcinoma cells. Furthermore, computational molecular docking and a LanthaScreen time-resolved fluorescence resonance energy transfer (TR-FRET) PXR competitive binding assay were performed to explore whether the inhibiting CAM components interact with PXR. The results demonstrated that milk thistle is a strong inhibitor of PXR-mediated CYP3A4 induction. The components of milk thistle responsible for this effect were identified as silybin and isosilybin. Furthermore, computational molecular docking revealed a strong interaction between both silybin and isosilybin and PXR, which was confirmed in the TR-FRET PXR assay. In conclusion, silybin and isosilybin might be suitable candidates to design potent PXR antagonists to prevent drug-drug interactions via CYP3A4 in cancer patients.

  6. Co-medication of statins and CYP3A4 inhibitors before and after introduction of new reimbursement policy

    PubMed Central

    Devold, Helene M; Molden, Espen; Skurtveit, Svetlana; Furu, Kari

    2009-01-01

    AIMS To assess the prevalence of co-medication of statins and CYP3A4 inhibitors before and after introduction of a new Norwegian reimbursement policy, which states that all patients should be prescribed simvastatin as first-line lipid-lowering therapy. METHODS Data from patients receiving simvastatin, lovastatin, pravastatin, fluvastatin or atorvastatin in 2004 and 2006, including co-medication of potent CYP3A4 inhibitors, were retrieved from the Norwegian Prescription Database covering the total population of Norway. Key measurements were prevalence of continuous statin use (two or more prescriptions on one statin) and proportions of different statin types among all patients and those co-medicated with CYP3A4 inhibitors. RESULTS In 2004, 5.9% (n = 272 342) of the Norwegian population received two or more prescriptions on one statin compared with 7.0% (n = 324 267) in 2006. The relative number of simvastatin users increased from 39.7% (n = 112 122) in 2004 to 63.1% (n = 226 672) in 2006. A parallel increase was observed within the subpopulation co-medicated with statins and CYP3A4 inhibitors, i.e. from 42.9% (n = 7706) in 2004 to 63.6% (n = 13 367) in 2006. For all other statins the number of overall users decreased to a similar extent to those co-medicated with CYP3A4 inhibitors. CONCLUSIONS In both 2004 and 2006, the choice of statin type did not depend on whether the patient used a CYP3A4 inhibitor or not. Considering the pronounced interaction potential of simvastatin with CYP3A4 inhibitors, a negative influence of the new policy on overall statin safety seems likely. PMID:19220274

  7. Amlodipine metabolism in human liver microsomes and roles of CYP3A4/5 in the dihydropyridine dehydrogenation.

    PubMed

    Zhu, Yanlin; Wang, Fen; Li, Quan; Zhu, Mingshe; Du, Alicia; Tang, Wei; Chen, Weiqing

    2014-02-01

    Amlodipine is a commonly prescribed calcium channel blocker for the treatment of hypertension and ischemic heart disease. The drug is slowly cleared in humans primarily via dehydrogenation of its dihydropyridine moiety to a pyridine derivative (M9). Results from clinical drug-drug interaction studies suggest that CYP3A4/5 mediate metabolism of amlodipine. However, attempts to identify a role of CYP3A5 in amlodipine metabolism in humans based on its pharmacokinetic differences between CYP3A5 expressers and nonexpressers failed. Objectives of this study were to determine the metabolite profile of amlodipine (a racemic mixture and S-isomer) in human liver microsomes (HLM), and to identify the cytochrome P450 (P450) enzyme(s) involved in the M9 formation. Liquid chromatography/mass spectrometry analysis showed that amlodipine was mainly converted to M9 in HLM incubation. M9 underwent further O-demethylation, O-dealkylation, and oxidative deamination to various pyridine derivatives. This observation is consistent with amlodipine metabolism in humans. Incubations of amlodipine with HLM in the presence of selective P450 inhibitors showed that both ketoconazole (an inhibitor of CYP3A4/5) and CYP3cide (an inhibitor of CYP3A4) completely blocked the M9 formation, whereas chemical inhibitors of other P450 enzymes had little effect. Furthermore, metabolism of amlodipine in expressed human P450 enzymes showed that only CYP3A4 had significant activity in amlodipine dehydrogenation. Metabolite profiles and P450 reaction phenotyping data of a racemic mixture and S-isomer of amlodipine were very similar. The results from this study suggest that CYP3A4, rather than CYP3A5, plays a key role in metabolic clearance of amlodipine in humans. PMID:24301608

  8. Establishment of In Silico Prediction Models for CYP3A4 and CYP2B6 Induction in Human Hepatocytes by Multiple Regression Analysis Using Azole Compounds.

    PubMed

    Nagai, Mika; Konno, Yoshihiro; Satsukawa, Masahiro; Yamashita, Shinji; Yoshinari, Kouichi

    2016-08-01

    Drug-drug interactions (DDIs) via cytochrome P450 (P450) induction are one clinical problem leading to increased risk of adverse effects and the need for dosage adjustments and additional therapeutic monitoring. In silico models for predicting P450 induction are useful for avoiding DDI risk. In this study, we have established regression models for CYP3A4 and CYP2B6 induction in human hepatocytes using several physicochemical parameters for a set of azole compounds with different P450 induction as characteristics as model compounds. To obtain a well-correlated regression model, the compounds for CYP3A4 or CYP2B6 induction were independently selected from the tested azole compounds using principal component analysis with fold-induction data. Both of the multiple linear regression models obtained for CYP3A4 and CYP2B6 induction are represented by different sets of physicochemical parameters. The adjusted coefficients of determination for these models were of 0.8 and 0.9, respectively. The fold-induction of the validation compounds, another set of 12 azole-containing compounds, were predicted within twofold limits for both CYP3A4 and CYP2B6. The concordance for the prediction of CYP3A4 induction was 87% with another validation set, 23 marketed drugs. However, the prediction of CYP2B6 induction tended to be overestimated for these marketed drugs. The regression models show that lipophilicity mostly contributes to CYP3A4 induction, whereas not only the lipophilicity but also the molecular polarity is important for CYP2B6 induction. Our regression models, especially that for CYP3A4 induction, might provide useful methods to avoid potent CYP3A4 or CYP2B6 inducers during the lead optimization stage without performing induction assays in human hepatocytes.

  9. Regulation of CYP3A4 by pregnane X receptor: The role of nuclear receptors competing for response element binding

    SciTech Connect

    Istrate, Monica A.; Nussler, Andreas K.; Eichelbaum, Michel; Burk, Oliver

    2010-03-19

    Induction of the major drug metabolizing enzyme CYP3A4 by xenobiotics contributes to the pronounced interindividual variability of its expression and often results in clinically relevant drug-drug interactions. It is mainly mediated by PXR, which regulates CYP3A4 expression by binding to several specific elements in the 5' upstream regulatory region of the gene. Induction itself shows a marked interindividual variability, whose underlying determinants are only partly understood. In this study, we investigated the role of nuclear receptor binding to PXR response elements in CYP3A4, as a potential non-genetic mechanism contributing to interindividual variability of induction. By in vitro DNA binding experiments, we showed that several nuclear receptors bind efficiently to the proximal promoter ER6 and distal xenobiotic-responsive enhancer module DR3 motifs. TR{alpha}1, TR{beta}1, COUP-TFI, and COUP-TFII further demonstrated dose-dependent repression of PXR-mediated CYP3A4 enhancer/promoter reporter activity in transient transfection in the presence and absence of the PXR inducer rifampin, while VDR showed this effect only in the absence of treatment. By combining functional in vitro characterization with hepatic expression analysis, we predict that TR{alpha}1, TR{beta}1, COUP-TFI, and COUP-TFII show a strong potential for the repression of PXR-mediated activation of CYP3A4 in vivo. In summary, our results demonstrate that nuclear receptor binding to PXR response elements interferes with PXR-mediated expression and induction of CYP3A4 and thereby contributes to the interindividual variability of induction.

  10. Effect of myricetin on cytochrome P450 isoforms CYP1A2, CYP2C9 and CYP3A4 in rats.

    PubMed

    Guo, Yu-Jin; Zheng, Shuang-Li

    2014-04-01

    Myricetin is one of the main ingredients of Chinese bayberry, which is used as a traditional medicine. The purpose of this study was to find out whether myricetin influences the rat cytochrome P450 (CYP) enzymes (CYP1A2, CYP2C9 and CYP3A4) by using cocktail probe drugs in vivo. A cocktail solution at a dose of 5 mL/kg, which contained phenacetin (20 mg/kg), tolbutamide (5 mg/kg) and midazolam (10 mg/kg), was orally administered to rats treated for 14 days with myricetin. Blood samples were collected at a series of time-points and the concentrations of probe drugs in plasma were determined by HPLC-MS/MS. The corresponding pharmacokinetic parameters were calculated by the software of DAS 2.0. Our study showed that treatment with multiple doses of myricetin had no effects on rat CYP1A2. However, CYP2C9 and CYP3A4 enzyme activities were inhibited after multiple doses of myricetin. Therefore, caution is needed when myricetin is co-administered with CYP2C9 or CYP3A4 substrates, which may result in herb-drug interactions.

  11. Systematic and quantitative assessment of the effect of chronic kidney disease on CYP2D6 and CYP3A4/5

    PubMed Central

    Yoshida, K; Sun, B; Zhang, L; Zhao, P; Abernethy, DR; Nolin, TD; Rostami‐Hodjegan, A; Zineh, I

    2016-01-01

    Recent reviews suggest that chronic kidney disease (CKD) can affect the pharmacokinetics of nonrenally eliminated drugs, but the impact of CKD on individual elimination pathways has not been systematically evaluated. In this study we developed a comprehensive dataset of the effect of CKD on the pharmacokinetics of CYP2D6‐ and CYP3A4/5‐metabolized drugs. Drugs for evaluation were selected based on clinical drug–drug interaction (CYP3A4/5 and CYP2D6) and pharmacogenetic (CYP2D6) studies. Information from dedicated CKD studies was available for 13 and 18 of the CYP2D6 and CYP3A4/5 model drugs, respectively. Analysis of these data suggested that CYP2D6‐mediated clearance is generally decreased in parallel with the severity of CKD. There was no apparent relationship between the severity of CKD and CYP3A4/5‐mediated clearance. The observed elimination‐route dependency in CKD effects between CYP2D6 and CYP3A4/5 may inform the need to conduct clinical CKD studies with nonrenally eliminated drugs for optimal use of drugs in patients with CKD. PMID:26800425

  12. Priapism Induced by Boceprevir-CYP3A4 Inhibition and α-Adrenergic Blockade: Case Report

    PubMed Central

    Hammond, Kyle P.; Nielsen, Craig; Linnebur, Sunny A.; Langness, Jacob A.; Ray, Graham; Maroni, Paul; Kiser, Jennifer J.

    2014-01-01

    A 44-year-old white man presented to the emergency department with a 3-day history of priapism requiring a surgically performed distal penile shunt. A drug–drug interaction is the suspected cause whereby CYP3A4 inhibition by boceprevir led to increased exposures of doxazosin, tamsulosin, and/or quetiapine, resulting in additional α-adrenergic blockade. PMID:24092799

  13. Priapism induced by boceprevir-CYP3A4 inhibition and α-adrenergic blockade: case report.

    PubMed

    Hammond, Kyle P; Nielsen, Craig; Linnebur, Sunny A; Langness, Jacob A; Ray, Graham; Maroni, Paul; Kiser, Jennifer J

    2014-01-01

    A 44-year-old white man presented to the emergency department with a 3-day history of priapism requiring a surgically performed distal penile shunt. A drug-drug interaction is the suspected cause whereby CYP3A4 inhibition by boceprevir led to increased exposures of doxazosin, tamsulosin, and/or quetiapine, resulting in additional α-adrenergic blockade.

  14. Effects of Commonly Used Excipients on the Expression of CYP3A4 in Colon and Liver Cells

    PubMed Central

    Tompkins, Leslie; Lynch, Caitlin; Haidar, Sam; Polli, James; Wang, Hongbing

    2013-01-01

    Purpose The objective of this investigation was to assess whether common pharmaceutical excipients regulate the expression of drug-metabolizing enzymes in human colon and liver cells. Methods Nineteen commonly used excipients were evaluated using a panel of experiments including cell-based human PXR activation assays, real-time RT-PCR assays for CYP3A4 mRNA expression, and immunoblot analysis of CYP3A4 protein expression in immortalized human liver cells (HepG2 and Fa2N4), human primary hepatocytes, and the intestinal LS174T cell models. Results No excipient activated human PXR or practically induced CYP3A4. However, three excipients (polysorbate 80, pregelatinized starch, and hydroxypropyl methylcellulose) tended to decrease mRNA and protein expression across experimental models. Conclusion This study represents the first investigation of the potential role of excipients in the expression of drug-metabolizing enzymes. Findings imply that some excipients may hold potential for excipient-drug interactions by repression of CYP3A4 expression. PMID:20503067

  15. Immunohistochemical Markers of CYP3A4 and CYP3A7: A New Tool Towards Personalized Pharmacotherapy of Hepatocellular Carcinoma

    PubMed Central

    Fanni, D.; Manchia, M.; Lai, F.; Gerosa, C.; Ambu, R.; Faa, G.

    2016-01-01

    Hepatocellular carcinoma (HCC) represents a major global health problem, since more than 90% of primary liver cancers worldwide are HCC. Most cases of HCC are secondary to viral hepatitis infection (hepatitis B or C), alcoholism and cirrhosis. Sorafenib, an oral tyrosine kinase inhibitor that suppresses tumor proliferation and angiogenesis, emerged as the first effective systemic treatment for HCC after 30 years of research, and is currently the standard-of-care for patients with advanced HCC. Sorafenib is metabolized by cytochrome P450 (CYP450), particularly from the 3A4 isoform, producing two main metabolites: the N-oxide and the N-hydroxymethyl metabolite. We studied 11 HCC sample showing the presence of CYP3A4 and CYP3A7 in most of the samples analysed. Specifically, the immunoreactivity of CYP3A4 was stronger and more widespread than that of CYP3A7. The CYP3A4 immunoreactivity was observed in surrounding hepatocytes in 8 out of 11 cases; while the CYP3A7 immunostaining was found in normal liver cells, in 7 out of 11 cases. These results suggest the existence of a marked inter-individual variability regarding the presence of the isoforms of CYP3A. In addition, since sorafenib is metabolized by CYP3A4, but not by CYP3A7, an overexpression of CYP3A4 may lead to an increase in the degradation of the drug and then to clinical ineffectiveness. These results might implicate the necessity of an individualized approach in the treatment of HCC as positivity to CYP3A4 in HCC liver samples might predict a scarce response to sorafenib. PMID:27349315

  16. In Silico Prediction of Cytochrome P450-Drug Interaction: QSARs for CYP3A4 and CYP2C9

    PubMed Central

    Nembri, Serena; Grisoni, Francesca; Consonni, Viviana; Todeschini, Roberto

    2016-01-01

    Cytochromes P450 (CYP) are the main actors in the oxidation of xenobiotics and play a crucial role in drug safety, persistence, bioactivation, and drug-drug/food-drug interaction. This work aims to develop Quantitative Structure-Activity Relationship (QSAR) models to predict the drug interaction with two of the most important CYP isoforms, namely 2C9 and 3A4. The presented models are calibrated on 9122 drug-like compounds, using three different modelling approaches and two types of molecular description (classical molecular descriptors and binary fingerprints). For each isoform, three classification models are presented, based on a different approach and with different advantages: (1) a very simple and interpretable classification tree; (2) a local (k-Nearest Neighbor) model based classical descriptors and; (3) a model based on a recently proposed local classifier (N-Nearest Neighbor) on binary fingerprints. The salient features of the work are (1) the thorough model validation and the applicability domain assessment; (2) the descriptor interpretation, which highlighted the crucial aspects of P450-drug interaction; and (3) the consensus aggregation of models, which largely increased the prediction accuracy. PMID:27294921

  17. [CYP2D6, CYP3A5, and CYP3A4 gene polymorphism in Russian, Tatar, and Bashkir populations].

    PubMed

    Mustafina, O E; Tuktarova, I A; Karimov, D D; Somova, R sh; Nasibullin, T R

    2015-01-01

    The allele and genotype frequency distribution at polymorphic loci rs3892097 (184G>A) of CYP2D6 gene, rs776746 (6986A>G) of the CYP3A5 gene and rs2740574 (-392A>G) of the CYP3A4 gene in Russians, Tatars, and Bashkirs was examined. Samples were taken from residents of Bashkortostan Republic (1240 men and women aged from 20 to 109 years and consisted of 443 Russians, 517 Tatars, and 280 Bashkirs). Allele identification was conducted using PCR-RFLP or PCR with TaqMan probes. The "nonfunctional" allele rs3892097*A of the CYP2D6 gene was detected in populations of Russians, Tatars, and Bashkirs in 17.2, 9.5, and 7.1% cases, respectively. The rs776746*G allele of the CYP3A5 gene encoding the CYP3A5 isoenzyme with decreased activity was revealed with a frequency of 94.6% in populations of Russians, 94.3% in the Tatar population, and 91.5% in the Bashkir population. The share of the minor allele rs2740574*G of the CYP3A4 was 4.0% in populations of Russians, 0.5% in the Tatar population, and 0.9% in the Bashkir population. It has been previously shown that the rs3892097*A, rs776746*G, and rs2740574*G allele frequencies vary significantly in different world populations. Since allele variants of CYP2D6, CYP3A5, and CYP3A4 genes can play essential role in interindividual and in interethnic differences in the metabolism of many therapeutic agents, the obtained results could be used in the prognosis of pharmacotherapy efficacy in populations of Russians, Tatars, and Bashkirs.

  18. Effect of CYP2C19 and CYP3A4 gene polymorphisms on the efficacy of bortezomib-based regimens in patients with multiple myeloma

    PubMed Central

    ZHOU, WEIWEI; AN, GUANGYU; JIAN, YUAN; GUO, HUAN; CHEN, WENMING

    2015-01-01

    Bortezomib is used to treat patients with multiple myeloma. It is primarily metabolized by the enzyme cytochrome P450 (CYP). Variations in the capacity of bortezomib metabolism affect the treatment outcomes and the side-effects experienced by patients. In the present study, polymorphisms in the CYP3A4 and CYP2C19 genes were analyzed by polymerase chain reaction in 56 newly-diagnosed patients with multiple myeloma. The polymorphisms analyzed included the c.681G>A, c.636G>A and c.-806C>T polymorphisms of CYP2C19. The CYP3A4 gene was sequenced after amplification and was classified into normal and mutant types. Associations between the metabolizer genotypes of CYP3A4 and CYP2C19, the therapeutic efficacy of bortezomib-based regimens, and the occurrence of peripheral neuropathy were studied. The results identified no significant differences in gender, serum β2 microglobulin, creatinine, blood albumin, isotypes, and the Durie-Salmon and International Staging System stages between the CYP2C19 poor + intermediate metabolizer types and the extensive + ultrarapid metabolizer types. In addition, it was revealed that the CYP2C19 and CYP3A4 phenotypes did not affect the efficacy of bortezomib-based regimens, nor were they correlated with peripheral neuropathy. Additional large-scale studies are required in order to evaluate the role of CYP enzymes in bortezomib treatments for patients with multiple myeloma. PMID:26622646

  19. Inhibition of CYP2C19 and CYP3A4 by Omeprazole Metabolites and Their Contribution to Drug-Drug Interactions

    PubMed Central

    Shirasaka, Yoshiyuki; Sager, Jennifer E.; Lutz, Justin D.; Davis, Connie

    2013-01-01

    The aim of this study was to evaluate the contribution of metabolites to drug-drug interactions (DDI) using the inhibition of CYP2C19 and CYP3A4 by omeprazole and its metabolites as a model. Of the metabolites identified in vivo, 5-hydroxyomeprazole, 5′-O-desmethylomeprazole, omeprazole sulfone, and carboxyomeprazole had a metabolite to parent area under the plasma concentration–time curve (AUCm/AUCp) ratio ≥ 0.25 when either total or unbound concentrations were measured after a single 20-mg dose of omeprazole in a cocktail. All of the metabolites inhibited CYP2C19 and CYP3A4 reversibly. In addition omeprazole, omeprazole sulfone, and 5′-O-desmethylomeprazole were time dependent inhibitors (TDI) of CYP2C19, whereas omeprazole and 5′-O-desmethylomeprazole were found to be TDIs of CYP3A4. The in vitro inhibition constants and in vivo plasma concentrations were used to evaluate whether characterization of the metabolites affected DDI risk assessment. Identifying omeprazole as a TDI of both CYP2C19 and CYP3A4 was the most important factor in DDI risk assessment. Consideration of reversible inhibition by omeprazole and its metabolites would not identify DDI risk with CYP3A4, and with CYP2C19, reversible inhibition values would only identify DDI risk if the metabolites were included in the assessment. On the basis of inactivation data, CYP2C19 and CYP3A4 inhibition by omeprazole would be sufficient to identify risk, but metabolites were predicted to contribute 30–63% to the in vivo hepatic interactions. Therefore, consideration of metabolites may be important in quantitative predictions of in vivo DDIs. The results of this study show that, although metabolites contribute to in vivo DDIs, their relative abundance in circulation or logP values do not predict their contribution to in vivo DDI risk. PMID:23620487

  20. Effects of capsicine on rat cytochrome P450 isoforms CYP1A2, CYP2C19, and CYP3A4.

    PubMed

    Zhu, Hui-dan; Gu, Ni; Wang, Meng; Kong, Hong-ru; Zhou, Meng-tao

    2015-01-01

    Due to the frequent consumption of capsaicin (CAP) and its current therapeutic application, the correct assessment of this compound is important from a public health standpoint. The purpose of this study was to find out whether CAP affects rat cytochrome P450 (CYP) enzymes (CYP1A2, CYP2C19, and CYP3A4) by using cocktail probe drugs in vivo. A cocktail solution at a dose of 5 mL/kg, which contained phenacetin (15 mg/kg), omeprazole (15 mg/kg), and midazolam (10 mg/kg), was given orally to rats treated for 7 d with oral administration of CAP. Blood samples were collected at a series of time-points and the concentrations of probe drugs in plasma were determined by HPLC-MS. The results showed that treatment with multiple doses of CAP had no significant effect on rat CYP1A2. However, CAP had a significant inhibitory effect on CYP2C19 and an inductive effect on CYP3A4. Therefore, caution is needed when CAP is co-administered with some CYP substrates clinically because of potential drug-CAP interactions.

  1. Effect of Curcuma longa on CYP2D6- and CYP3A4-mediated metabolism of dextromethorphan in human liver microsomes and healthy human subjects.

    PubMed

    Al-Jenoobi, Fahad Ibrahim; Al-Thukair, Areej A; Alam, Mohd Aftab; Abbas, Fawkeya A; Al-Mohizea, Abdullah M; Alkharfy, Khalid M; Al-Suwayeh, Saleh A

    2015-03-01

    Effect of Curcuma longa rhizome powder and its ethanolic extract on CYP2D6 and CYP3A4 metabolic activity was investigated in vitro using human liver microsomes and clinically in healthy human subjects. Dextromethorphan (DEX) was used as common probe for CYP2D6 and CYP3A4 enzymes. Metabolic activity of CYP2D6 and CYP3A4 was evaluated through in vitro study; where microsomes were incubated with NADPH in presence and absence of Curcuma extract. In clinical study phase-I, six healthy human subjects received a single dose (30 mg) of DEX syrup, and in phase-II DEX syrup was administered with Curcuma powder. The enzyme CYP2D6 and CYP3A4 mediated O- and N-demethylation of dextromethorphan into dextrorphan (DOR) and 3-methoxymorphinan (3-MM), respectively. Curcuma extract significantly inhibited the formation of DOR and 3-MM, in a dose-dependent and linear fashion. The 100 μg/ml dose of curcuma extract produced highest inhibition, which was about 70 % for DOR and 80 % for 3-MM. Curcuma significantly increases the urine metabolic ratio of DEX/DOR but the change in DEX/3-MM ratio was statistically insignificant. Present findings suggested that curcuma significantly inhibits the activity of CYP2D6 in in vitro as well as in vivo; which indicates that curcuma has potential to interact with CYP2D6 substrates.

  2. Investigation of CYP3A4 and CYP2D6 Interactions of Withania somnifera and Centella asiatica in Human Liver Microsomes.

    PubMed

    Savai, Jay; Varghese, Alice; Pandita, Nancy; Chintamaneni, Meena

    2015-05-01

    Withania somnifera is commonly used as a rejuvenator, whereas Centella asiatica is well known for its anxiolytic and nootropic effects. The present study aims at investigating the effect of crude extracts and principal phytoconstituents of both the medicinal plants with CYP3A4 and CYP2D6 enzyme activity in human liver microsomes (HLM). Phytoconstituents were quantified in the crude extracts of both the medicinal plants using reverse phase HPLC. Crude extracts and phytoconstituents of W. somnifera showed no significant interaction with both CYP3A4 and CYP2D6 enzymes in HLM. Of the crude extracts of C. asiatica screened in vitro, methanolic extract showed potent noncompetitive inhibition of only CYP3A4 enzyme (Ki-64.36 ± 1.82 µg/mL), whereas ethanol solution extract showed potent noncompetitive inhibition of only CYP2D6 enzyme (Ki-36.3 ± 0.44 µg/mL). The flavonoids, quercetin, and kaempferol showed potent (IC50 values less than 100 μM) inhibition of CYP3A4 activity, whereas quercetin alone showed potent inhibition of CYP2D6 activity in HLM. Because methanolic extract of C. asiatica showed a relatively high percentage content of quercetin and kaempferol than ethanol solution extract, the inhibitory effect of methanolic extract on CYP3A4 enzyme activity could be attributed to the flavonoids. Thus, co-administration of the alcoholic extracts of C. asiatica with drugs that are substrates of CYP3A4 and CYP2D6 enzymes may lead to undesirable herb-drug interactions in humans.

  3. Modulation of human cytochrome P450 3A4 (CYP3A4) and P-glycoprotein (P-gp) in Caco-2 cell monolayers by selected commercial-source milk thistle and goldenseal products.

    PubMed

    Budzinski, Jason W; Trudeau, Vance L; Drouin, Cathy E; Panahi, Mitra; Arnason, J Thor; Foster, Brian C

    2007-09-01

    In this study, we used an in vitro Caco-2 cell monolayer model to evaluate aqueous extracts of commercial-source goldenseal (Hydrastis canadensis) and milk thistle (Silybum marianum) capsule formulations, their marker phytochemicals (berberine and silibinin, respectively), as well as dillapiol, vinblastine, and the HIV protease inhibitor saquinavir for their ability to modulate CYP3A4 and ABCB1 expression after short-term exposure (48 h). Both upregulation and downregulation of CYP3A4 expression was observed with extracts of varying concentrations of the two natural health products (NHPs). CYP3A4 was highly responsive in our system, showing a strong dose-dependent modulation by the CYP3A4 inhibitor dillapiol (upregulation) and the milk thistle flavonolignan silibinin (downregulation). ABCB1 was largely unresponsive in this cellular model and appears to be of little value as a biomarker under our experimental conditions. Therefore, the modulation of CYP3A4 gene expression can serve as an important marker for the in vitro assessment of NHP-drug interactions.

  4. Structural Optimization of Ghrelin Receptor Inverse Agonists to Improve Lipophilicity and Avoid Mechanism-Based CYP3A4 Inactivation.

    PubMed

    Takahashi, Bitoku; Funami, Hideaki; Shibata, Makoto; Maruoka, Hiroshi; Koyama, Makoto; Kanki, Satomi; Muto, Tsuyoshi

    2015-01-01

    Structural optimization of 2-aminonicotinamide derivatives as ghrelin receptor inverse agonists is reported. So as to avoid mechanism-based inactivation (MBI) of CYP3A4, 1,3-benzodioxol ring of the lead compound was modified. Improvement of the main activity and lipophilicity was achieved simultaneously, leading to compound 18a, which showed high lipophilic ligand efficiency (LLE) and low MBI activity. PMID:26423040

  5. Echinacea purpurea up-regulates CYP1A2, CYP3A4 and MDR1 gene expression by activation of pregnane X receptor pathway

    PubMed Central

    Awortwe, Charles; Manda, Vamshi K.; Avonto, Cristina; Khan, Shabana I.; Khan, Ikhlas A.; Walker, Larry A.; Bouic, Patrick J.; Rosenkranz, Bernd

    2015-01-01

    This study investigated the mechanism underlying Echinacea-mediated induction of CYP1A2, CYP3A4 and MDR1 in terms of human pregnane X receptor (PXR) activation. Crude extracts and fractions of Echinacea purpurea were tested for PXR activation in HepG2 cells by a reporter gene assay. Quantitative real-time PCR was carried out to determine their effects on CYP1A2 and CYP3A4 mRNA expressions. Capsules and fractions were risk ranked as high, intermediate and remote risk of drug-metabolizing enzymes induction based on EC50 values determined for respective CYPs. Fractions F1, F2 and capsule (2660) strongly activated PXR with 5-, 4- and 3.5-fold increase in activity, respectively. Echinacea preparations potentiated up-regulation of CYP1A2, CYP3A4 and MDR1 via PXR activation. Thus E. purpurea preparations cause herb–drug interaction by up-regulating CYP1A2, CYP3A4 and P-gp via PXR activation. PMID:25377539

  6. The CYP3A4 inhibitor intraconazole does not affect the pharmacokinetics of a new calcium-sensitizing drug levosimendan.

    PubMed

    Antila, S; Honkanen, T; Lehtonen, L; Neuvonen, P J

    1998-08-01

    Itraconazole is a potent inhibitor of CYP3A4 isoenzyme and it can cause clinically significant interactions with some other drugs. Levosimendan is a new calcium-sensitizing drug intended for congestive heart failure. We aimed to study possible interactions of itraconazole with levosimendan in healthy volunteers. Twelve healthy male volunteers were included into a randomized, double-blind, two-phase crossover study. A wash-out period of 4 weeks was held between the phases. The subjects were given orally itraconazole 200 mg or placebo daily for 5 days. On the fifth day, they received a single oral dose of 2 mg of levosimendan. Levosimendan plasma concentrations were determined up to 12 hours and ECG, heart rate, and blood pressure followed-up to 8 hours after intake of levosimendan. Itraconazole had no significant effects on the pharmacokinetic parameters of levosimendan. Neither were there any differences in heart rate, PQ-, QTc- or QRS intervals between the placebo and itraconazole phases. The systolic blood pressure was decreased slightly more (p < 0.05) during the itraconazole phase than during the placebo phase. In conclusion, because the potent CYP3A4 inhibitor itraconazole had no significant pharmacokinetic interaction with levosimendan, interactions with CYP3A4 inhibitor, and oral levosimendan are unlikely.

  7. The CYP3A4*22 C>T single nucleotide polymorphism is associated with reduced midazolam and tacrolimus clearance in stable renal allograft recipients.

    PubMed

    de Jonge, H; Elens, L; de Loor, H; van Schaik, R H; Kuypers, D R J

    2015-04-01

    Tacrolimus, a dual substrate of CYP3A4 and CYP3A5 has a narrow therapeutic index and is characterized by high between-subject variability in oral bioavailability. This study investigated the effects of the recently described CYP3A4*22 intron 6 C>T single nucleotide polymorphism on in vivo CYP3A4 activity as measured by midazolam (MDZ) clearance and tacrolimus pharmacokinetics in two cohorts of renal allograft recipients, taking into account the CYP3A5*1/*3 genotype and other determinants of drug disposition. In CYP3A5 non-expressers, the presence of one CYP3A4*22T-allele was associated with a 31.7-33.6% reduction in MDZ apparent oral clearance, reflecting reduced in vivo CYP3A4 activity. In addition, at ⩾12 months after transplantation, steady-state clearance of tacrolimus was 36.8% decreased compared with homozygous CYP3A4*22CC-wild type patients, leading to 50% lower dose requirements. Both concurrent observations in stable renal allograft recipients are consistent with a reduced in vivo CYP3A4 activity for the CYP3A4*22T-allele.

  8. Individual and combined associations of genetic variants in CYP3A4, CYP3A5, and SLCO1B1 with simvastatin and simvastatin acid plasma concentrations

    PubMed Central

    Luzum, Jasmine A.; Theusch, Elizabeth; Taylor, Kent D.; Wang, Ann; Sadee, Wolfgang; Binkley, Philip F.; Krauss, Ronald M.; Medina, Marisa W.; Kitzmiller, Joseph P.

    2015-01-01

    Our objective was to evaluate the associations of genetic variants affecting simvastatin (SV) and simvastatin acid (SVA) metabolism (CYP3A4*22 and CYP3A5*3) and transport (SLCO1B1 T521C) with 12-hour plasma SV and SVA concentrations. The variants were genotyped, and concentrations were quantified by HPLC-MS/MS in 646 participants of the Cholesterol and Pharmacogenetics clinical trial of 40 mg/day SV for 6 weeks. The genetic variants were tested for association with 12-hour plasma SV, SVA, or the SVA/SV ratio using general linear models. CYP3A5*3 was not significantly associated with 12-hour plasma SV or SVA concentration. CYP3A4*1/*22 participants had 58% higher 12-hour plasma SV concentration compared to CYP3A4*1/*1 participants (p=0.006). SLCO1B1 521T/C and 521C/C participants had 71% (p<0.001) and 248% (p<0.001) higher 12-hour plasma SVA compared to SLCO1B1 521T/T participants, respectively. CYP3A4 and SLCO1B1 genotypes combined categorized participants into low (<1), intermediate (≈1), and high (>1) SVA/SV ratio groups (p=0.001). In conclusion, CYP3A4*22 and SLCO1B1 521C were significantly associated with increased plasma 12-hour concentrations of SV and SVA, respectively. CYP3A5*3 was not significantly associated with 12-hour plasma SV or SVA concentrations. The combination of CYP3A4*22 and SLCO1B1 521C was significantly associated with SVA/SV ratio, which may translate into different clinical SV risk/benefit profiles. PMID:26164721

  9. Substrate-dependent modulation of CYP3A4 catalytic activity: analysis of 27 test compounds with four fluorometric substrates.

    PubMed

    Stresser, D M; Blanchard, A P; Turner, S D; Erve, J C; Dandeneau, A A; Miller, V P; Crespi, C L

    2000-12-01

    Inhibition of cytochrome P450 catalytic activity is a principal mechanism for pharmacokinetic drug-drug interactions. Rapid, in vitro testing for cytochrome P450 inhibition potential is part of the current paradigm for identifying drug candidates likely to give such interactions. We have explored the extent that qualitative and quantitative inhibition parameters are dependent on the cytochrome P450 (CYP) 3A4 probe substrate. Inhibition potential (e.g., IC(50) values from 8-point inhibition curves) or activation potential for most compounds varied dramatically depending on the fluorometric probe substrates for CYP3A4 [benzyloxyresorufin (BzRes), 7-benzyloxy-4-trifluoromethylcoumarin (BFC), 7-benzyloxyquinoline (BQ), and dibenzylfluorescein (DBF)]. For 21 compounds that were primarily inhibitors, the range of IC(50) values for the four substrates varied from 2.1- to 195-fold with an average of 29-fold. While the rank order of sensitivity among the fluorometric substrates varied among the individual inhibitors, on average, BFC dealkylation was the most sensitive to inhibition, while BQ dealkylation was least sensitive. Partial inhibition was observed with BzRes and BQ but not for BFC and DBF. BzRes was more prone to activation, whereas dramatic changes in IC(50) values were observed when the BQ concentration was below the S(50). Three different correlation analyses indicated that IC(50) values with BFC, BQ, and DBF correlated well with each other, whereas the response with BzRes correlated more weakly with the other substrates. One of these correlation analyses was extended to the percent inhibition of 10 microM inhibitor with the standard CYP3A4 probe substrates testosterone, midazolam, and nifedipine. In this analysis the responses with BQ, BFC and DBF correlated well with testosterone and midazolam but more poorly with nifedipine. In the aggregate, BFC and DBF appear more suitable as an initial screen for CYP3A4 inhibition. However, the substrate-dependent effects

  10. Drug membrane transporters and CYP3A4 are affected by hypericin, hyperforin or aristoforin in colon adenocarcinoma cells.

    PubMed

    Šemeláková, M; Jendželovský, R; Fedoročko, P

    2016-07-01

    Our previous results have shown that the combination of hypericin-mediated photodynamic therapy (HY-PDT) at sub-optimal dose with hyperforin (HP) (compounds of Hypericum sp.), or its stable derivative aristoforin (AR) stimulates generation of reactive oxygen species (ROS) leading to antitumour activity. This enhanced oxidative stress evoked the need for an explanation for HY accumulation in colon cancer cells pretreated with HP or AR. Generally, the therapeutic efficacy of chemotherapeutics is limited by drug resistance related to the overexpression of drug efflux transporters in tumour cells. Therefore, the impact of non-activated hypericin (HY), HY-PDT, HP and AR on cell membrane transporter systems (Multidrug resistance-associated protein 1-MRP1/ABCC1, Multidrug resistance-associated protein 2-MRP2/ABCC2, Breast cancer resistance protein - BCRP/ABCG2, P-glycoprotein-P-gp/ABCC1) and cytochrome P450 3A4 (CYP3A4) was evaluated. The different effects of the three compounds on their expression, protein level and activity was determined under specific PDT light (T0+, T6+) or dark conditions (T0- T6-). We found that HP or AR treatment affected the protein levels of MRP2 and P-gp, whereas HP decreased MRP2 and P-gp expression mostly in the T0+ and T6+ conditions, while AR decreased MRP2 in T0- and T6+. Moreover, HY-PDT treatment induced the expression of MRP1. Our data demonstrate that HP or AR treatment in light or dark PDT conditions had an inhibitory effect on the activity of individual membrane transport proteins and significantly decreased CYP3A4 activity in HT-29 cells. We found that HP or AR significantly affected intracellular accumulation of HY in HT-29 colon adenocarcinoma cells. These results suggest that HY, HP and AR might affect the efficiency of anti-cancer drugs, through interaction with membrane transporters and CYP3A4. PMID:27261575

  11. Influence of Various Polymorphic Variants of Cytochrome P450 Oxidoreductase (POR) on Drug Metabolic Activity of CYP3A4 and CYP2B6

    PubMed Central

    Naranmandura, Hua; Zeng, Su; Chen, Shu Qing

    2012-01-01

    Cytochrome P450 oxidoreductase (POR) is known as the sole electron donor in the metabolism of drugs by cytochrome P450 (CYP) enzymes in human. However, little is known about the effect of polymorphic variants of POR on drug metabolic activities of CYP3A4 and CYP2B6. In order to better understand the mechanism of the activity of CYPs affected by polymorphic variants of POR, six full-length mutants of POR (e.g., Y181D, A287P, K49N, A115V, S244C and G413S) were designed and then co-expressed with CYP3A4 and CYP2B6 in the baculovirus-Sf9 insect cells to determine their kinetic parameters. Surprisingly, both mutants, Y181D and A287P in POR completely inhibited the CYP3A4 activity with testosterone, while the catalytic activity of CYP2B6 with bupropion was reduced to approximately ∼70% of wild-type activity by Y181D and A287P mutations. In addition, the mutant K49N of POR increased the CLint (Vmax/Km) of CYP3A4 up to more than 31% of wild-type, while it reduced the catalytic efficiency of CYP2B6 to 74% of wild-type. Moreover, CLint values of CYP3A4-POR (A115V, G413S) were increased up to 36% and 65% of wild-type respectively. However, there were no appreciable effects observed by the remaining two mutants of POR (i.e., A115V and G413S) on activities of CYP2B6. In conclusion, the extent to which the catalytic activities of CYP were altered did not only depend on the specific POR mutations but also on the isoforms of different CYP redox partners. Thereby, we proposed that the POR-mutant patients should be carefully monitored for the activity of CYP3A4 and CYP2B6 on the prescribed medication. PMID:22719896

  12. Indirubin, a component of Ban-Lan-Gen, activates CYP3A4 gene transcription through the human pregnane X receptor.

    PubMed

    Kumagai, Takeshi; Aratsu, Yusuke; Sugawara, Ryosuke; Sasaki, Takamitsu; Miyairi, Shinichi; Nagata, Kiyoshi

    2016-04-01

    Ban-Lan-Gen is the common name for the dried roots of indigo plants, including Polygonum tinctorium, Isatis indigotica, Isatis tinctoria, and Strobilanthes cusia. Ban-Lan-Gen is frequently used as an anti-inflammatory and an anti-viral for the treatment of hepatitis, influenza, and various types of inflammation. One of the cytochrome P450 (CYP) enzymes, CYP3A4, is responsible for the metabolism of a wide variety of xenobiotics, including an estimated 60% of all clinically used drugs. In this study, we investigated the effect of Ban-Lan-Gen on the transcriptional activation of the CYP3A4 gene. Ban-Lan-Gen extract increased CYP3A4 gene reporter activity in a dose-dependent manner. Indirubin, one of the biologically active ingredients in the Ban-Lan-Gen, also dose-dependently increased CYP3A4 gene reporter activity. Expression of short hairpin RNA for the human pregnane X receptor (hPXR-shRNA) inhibited CYP3A4 gene reporter activity, and overexpression of human PXR increased indirubin- and rifampicin-induced CYP3A4 gene reporter activity. Furthermore, indirubin induced CYP3A4 mRNA expression in HepG2 cells. Taken together, these results indicate that indirubin, a component of Ban-Lan-Gen, activated CYP3A4 gene transcription through the activation of the human PXR. PMID:26987505

  13. Indirubin, a component of Ban-Lan-Gen, activates CYP3A4 gene transcription through the human pregnane X receptor.

    PubMed

    Kumagai, Takeshi; Aratsu, Yusuke; Sugawara, Ryosuke; Sasaki, Takamitsu; Miyairi, Shinichi; Nagata, Kiyoshi

    2016-04-01

    Ban-Lan-Gen is the common name for the dried roots of indigo plants, including Polygonum tinctorium, Isatis indigotica, Isatis tinctoria, and Strobilanthes cusia. Ban-Lan-Gen is frequently used as an anti-inflammatory and an anti-viral for the treatment of hepatitis, influenza, and various types of inflammation. One of the cytochrome P450 (CYP) enzymes, CYP3A4, is responsible for the metabolism of a wide variety of xenobiotics, including an estimated 60% of all clinically used drugs. In this study, we investigated the effect of Ban-Lan-Gen on the transcriptional activation of the CYP3A4 gene. Ban-Lan-Gen extract increased CYP3A4 gene reporter activity in a dose-dependent manner. Indirubin, one of the biologically active ingredients in the Ban-Lan-Gen, also dose-dependently increased CYP3A4 gene reporter activity. Expression of short hairpin RNA for the human pregnane X receptor (hPXR-shRNA) inhibited CYP3A4 gene reporter activity, and overexpression of human PXR increased indirubin- and rifampicin-induced CYP3A4 gene reporter activity. Furthermore, indirubin induced CYP3A4 mRNA expression in HepG2 cells. Taken together, these results indicate that indirubin, a component of Ban-Lan-Gen, activated CYP3A4 gene transcription through the activation of the human PXR.

  14. Interactions of endosulfan and methoxychlor involving CYP3A4 and CYP2B6 in human HepaRG cells.

    PubMed

    Savary, Camille C; Jossé, Rozenn; Bruyère, Arnaud; Guillet, Fabrice; Robin, Marie-Anne; Guillouzo, André

    2014-08-01

    Humans are usually exposed to several pesticides simultaneously; consequently, combined actions between pesticides themselves or between pesticides and other chemicals need to be addressed in the risk assessment. Many pesticides are efficient activators of pregnane X receptor (PXR) and/or constitutive androstane receptor (CAR), two major nuclear receptors that are also activated by other substrates. In the present work, we searched for interactions between endosulfan and methoxychlor, two organochlorine pesticides whose major routes of metabolism involve CAR- and PXR-regulated CYP3A4 and CYP2B6, and whose mechanisms of action in humans remain poorly understood. For this purpose, HepaRG cells were treated with both pesticides separately or in mixture for 24 hours or 2 weeks at concentrations relevant to human exposure levels. In combination they exerted synergistic cytotoxic effects. Whatever the duration of treatment, both compounds increased CYP3A4 and CYP2B6 mRNA levels while differently affecting their corresponding activities. Endosulfan exerted a direct reversible inhibition of CYP3A4 activity that was confirmed in human liver microsomes. By contrast, methoxychlor induced this activity. The effects of the mixture on CYP3A4 activity were equal to the sum of those of each individual compound, suggesting an additive effect of each pesticide. Despite CYP2B6 activity being unchanged and increased with endosulfan and methoxychlor, respectively, no change was observed with their mixture, supporting an antagonistic effect. Altogether, our data suggest that CAR and PXR activators endosulfan and methoxychlor can interact together and with other exogenous substrates in human hepatocytes. Their effects on CYP3A4 and CYP2B6 activities could have important consequences if extrapolated to the in vivo situation.

  15. A novel in vitro approach for simultaneous evaluation of CYP3A4 inhibition and kinetic aqueous solubility.

    PubMed

    Pérez, José; Díaz, Caridad; Asensio, Francisco; Palafox, Alexandra; Genilloud, Olga; Vicente, Francisca

    2015-02-01

    In the early stages of the drug discovery process, evaluation of the drug metabolism and physicochemical properties of new chemical entities is crucial to prioritize those candidates displaying a better profile for further development. In terms of metabolism, drug-drug interactions mediated through CYP450 inhibition are a significant safety concern, and therefore the effect of new candidate drugs on CYP450 activity should be screened early. In the initial stages of drug discovery, when physicochemical properties such as aqueous solubility have not been optimized yet, there might be a large number of candidate compounds showing artificially low CYP450 inhibition, and consequently potential drug-drug interaction toxicity might be overlooked. In this work, we present a novel in vitro approach for simultaneous evaluation of CYP3A4 inhibition potential and kinetic aqueous solubility (NIVA-CYPI-KS). This new methodology is based on fluorogenic CYP450 activities and turbidimetric measurements for compound solubility, and it provides a significant improvement in the use of resources and a better understanding of CYP450 inhibition data.

  16. Polymorphisms in the cytochrome P450 genes CYP1A2, CYP1B1, CYP3A4, CYP3A5, CYP11A1, CYP17A1, CYP19A1 and colorectal cancer risk

    PubMed Central

    Bethke, Lara; Webb, Emily; Sellick, Gabrielle; Rudd, Matthew; Penegar, Stephen; Withey, Laura; Qureshi, Mobshra; Houlston, Richard

    2007-01-01

    Background Cytochrome P450 (CYP) enzymes have the potential to affect colorectal cancer (CRC) risk by determining the genotoxic impact of exogenous carcinogens and levels of sex hormones. Methods To investigate if common variants of CYP1A2, CYP1B1, CYP3A4, CYP3A5, CYP11A1, CYP17A1 and CYP19A1 influence CRC risk we genotyped 2,575 CRC cases and 2,707 controls for 20 single nucleotide polymorphisms (SNPs) that have not previously been shown to have functional consequence within these genes. Results There was a suggestion of increased risk, albeit insignificant after correction for multiple testing, of CRC for individuals homozygous for CYP1B1 rs162558 and heterozygous for CYP1A2 rs2069522 (odds ratio [OR] = 1.36, 95% confidence interval [CI]: 1.03–1.80 and OR = 1.34, 95% CI: 1.00–1.79 respectively). Conclusion This study provides some support for polymorphic variation in CYP1A2 and CYP1B1 playing a role in CRC susceptibility. PMID:17615053

  17. Kidney transplant recipients carrying the CYP3A4*22 allelic variant have reduced tacrolimus clearance and often reach supratherapeutic tacrolimus concentrations.

    PubMed

    Pallet, N; Jannot, A-S; El Bahri, M; Etienne, I; Buchler, M; de Ligny, B H; Choukroun, G; Colosio, C; Thierry, A; Vigneau, C; Moulin, B; Le Meur, Y; Heng, A-E; Subra, J-F; Legendre, C; Beaune, P; Alberti, C; Loriot, M A; Thervet, E

    2015-03-01

    CYP3A4*22 is an allelic variant of the cytochrome P450 3A4 associated with a decreased activity. Carriers of this polymorphism may require reduced tacrolimus (Tac) doses to reach the target residual concentrations (Co). We tested this hypothesis in a population of kidney transplant recipients extracted from a multicenter, prospective and randomized study. Among the 186 kidney transplant recipients included, 9.3% (18 patients) were heterozygous for the CYP3A4*22 genotype and none were homozygous (allele frequency of 4.8%). Ten days after transplantation (3 days after starting treatment with Tac), 11% of the CYP3A4*22 carriers were within the target range of Tac Co (10-15 ng/mL), whereas among the CYP3A4*1/*1 carriers, 40% were within the target range (p = 0.02, OR = 0.19 [0.03; 0.69]). The mean Tac Co at day 10 in the CYP3A4*1/*22 group was 23.5 ng/mL (16.6-30.9) compared with 15.1 ng/mL (14-16.3) in the CYP3A4*1/*1 group, p < 0.001. The Tac Co/dose significantly depended on the CYP3A4 genotype during the follow-up (random effects model, p < 0.001) with the corresponding equivalent dose for patients heterozygous for CYP3A4*22 being 0.67 [0.54; 0.84] times the dose for CYP3A4*1/*1 carriers. In conclusion, the CYP3A4*22 allelic variant is associated with a significantly altered Tac metabolism and carriers of this polymorphism often reach supratherapeutic concentrations. PMID:25588704

  18. Fentanyl Enhances Hepatotoxicity of Paclitaxel via Inhibition of CYP3A4 and ABCB1 Transport Activity in Mice.

    PubMed

    Xie, Jing-Dun; Huang, Yang; Chen, Dong-Tai; Pan, Jia-Hao; Bi, Bing-Tian; Feng, Kun-Yao; Huang, Wan; Zeng, Wei-An

    2015-01-01

    Fentanyl, a potent opioid analgesic that is used to treat cancer pain, is commonly administered with paclitaxel in advanced tumors. However, the effect of fentanyl on the hepatotoxicity of paclitaxel and its potential mechanism of action is not well studied. The purpose of this study was to investigate the effect of fentanyl on the hepatotoxicity of paclitaxel and its potential mechanisms of action. Pharmacokinetic parameters of paclitaxel were tested using reversed phase high-performance liquid chromatography (RP-HPLC). Aspartate transaminase (AST), alanine aminotransferase (ALT), and mouse liver histopathology were examined. Moreover, the cytotoxicity of anti-carcinogens was examined using 1-(4, 5-dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT), and the intracellular accumulation of doxorubicin and rhodamine 123 was detected by flow cytometry. Furthermore, the expression of ABCB1 and the activity of ABCB1 ATPase and CYP3A4 were also examined. In this study, the co-administration of fentanyl and paclitaxel prolonged the half-life (t1/2) of paclitaxel from 1.455 hours to 2.344 hours and decreased the clearance (CL) from 10.997 ml/h to 7.014 ml/h in mice. Fentanyl significantly increased the levels of ALT in mice to 88.2 U/L, which is more than 2-fold higher than the level detected in the control group, and it increased the histological damage in mouse livers. Furthermore, fentanyl enhanced the cytotoxicity of anti-carcinogens that are ABCB1 substrates and increased the accumulation of doxorubicin and rhodamine 123. Additionally, fentanyl stimulated ABCB1 ATPase activity and inhibited CYP3A4 activity in the liver microsomes of mice. Our study indicates that the obvious hepatotoxicity during this co-administration was due to the inhibition of CYP3A4 activity and ABCB1 transport activity. These findings suggested that the accumulation-induced hepatotoxicity of paclitaxel when it is combined with fentanyl should be avoided.

  19. Mechanism of Drug-Drug Interactions Mediated by Human Cytochrome P450 CYP3A4 Monomer

    PubMed Central

    Denisov, Ilia G.; Grinkova, Yelena V.; Baylon, Javier L.; Tajkhorshid, Emad; Sligar, Stephen G.

    2016-01-01

    Using Nanodiscs, we quantitate the heterotropic interaction between two different drugs mediated by monomeric CYP3A4 incorporated into a native-like membrane environment. The mechanism of this interaction is deciphered by global analysis of multiple turnover experiments performed under identical conditions using the pure substrates progesterone (PGS) and carbamazepine (CBZ) and their mixtures. Activation of CBZ epoxidation and simultaneous inhibition of PGS hydroxylation are measured and quantitated through differences in their respective affinities towards both a remote allosteric site and the productive catalytic site near the heme iron. Preferred binding of PGS at the allosteric site and higher preference of CBZ binding at the productive site give rise to a non-trivial drug-drug interaction. Molecular dynamics simulations indicate functionally important conformational changes caused by PGS binding at the allosteric site and by two CBZ molecules positioned inside the substrate binding pocket. Structural changes involving Phe-213, Phe-219, and Phe-241 are suggested to be responsible for the observed synergetic effects and positive allosteric interactions between these two substrates. Such a mechanism is likely of general relevance to the mutual heterotropic effects caused by biologically active compounds which exhibit different patterns of interaction with the distinct allosteric and productive sites of CYP3A4, as well as other xenobiotic metabolizing cytochromes P450 that are also involved in drug-drug interactions. Importantly, this work demonstrates that a monomeric CYP3A4 can display the full spectrum of activation and cooperative effects that are observed in hepatic membranes. PMID:25777547

  20. The independent contribution of miRNAs to the missing heritability in CYP3A4/5 functionality and the metabolism of atorvastatin

    PubMed Central

    Liu, Ju-E; Ren, Bin; Tang, Lan; Tang, Qian-Jie; Liu, Xiao-Ying; Li, Xin; Bai, Xue; Zhong, Wan-Ping; Meng, Jin-Xiu; Lin, Hao-Ming; Wu, Hong; Chen, Ji-Yan; Zhong, Shi-Long

    2016-01-01

    To evaluate the independent contribution of miRNAs to the missing heritability in CYP3A4/5 functionality and atorvastatin metabolism, the relationships among three levels of factors, namely (1) clinical characteristics, CYP3A4/5 genotypes, and miRNAs, (2) CYP3A4 and CYP3A5 mRNAs, and (3) CYP3A activity, as well as their individual impacts on atorvastatin metabolism, were assessed in 55 human liver tissues. MiR-27b, miR-206, and CYP3A4 mRNA respectively accounted for 20.0%, 5.8%, and 9.5% of the interindividual variations in CYP3A activity. MiR-142 was an independent contributor to the expressions of CYP3A4 mRNA (partial R2 = 0.12, P = 0.002) and CYP3A5 mRNA (partial R2 = 0.09, P = 0.005) but not CYP3A activity or atorvastatin metabolism. CYP3A activity was a unique independent predictor of variability of atorvastatin metabolism, explaining the majority of the variance in reduction of atorvastatin (60.0%) and formation of ortho-hydroxy atorvastatin (78.8%) and para-hydroxy atorvastatin (83.9%). MiR-27b and miR-206 were found to repress CYP3A4 gene expression and CYP3A activity by directly binding to CYP3A4 3′-UTR, while miR-142 was found to indirectly repress CYP3A activity. Our study indicates that miRNAs play significant roles in bridging the gap between epigenetic effects and missing heritability in CYP3A functionality. PMID:27211076

  1. The independent contribution of miRNAs to the missing heritability in CYP3A4/5 functionality and the metabolism of atorvastatin.

    PubMed

    Liu, Ju-E; Ren, Bin; Tang, Lan; Tang, Qian-Jie; Liu, Xiao-Ying; Li, Xin; Bai, Xue; Zhong, Wan-Ping; Meng, Jin-Xiu; Lin, Hao-Ming; Wu, Hong; Chen, Ji-Yan; Zhong, Shi-Long

    2016-01-01

    To evaluate the independent contribution of miRNAs to the missing heritability in CYP3A4/5 functionality and atorvastatin metabolism, the relationships among three levels of factors, namely (1) clinical characteristics, CYP3A4/5 genotypes, and miRNAs, (2) CYP3A4 and CYP3A5 mRNAs, and (3) CYP3A activity, as well as their individual impacts on atorvastatin metabolism, were assessed in 55 human liver tissues. MiR-27b, miR-206, and CYP3A4 mRNA respectively accounted for 20.0%, 5.8%, and 9.5% of the interindividual variations in CYP3A activity. MiR-142 was an independent contributor to the expressions of CYP3A4 mRNA (partial R(2) = 0.12, P = 0.002) and CYP3A5 mRNA (partial R(2) = 0.09, P = 0.005) but not CYP3A activity or atorvastatin metabolism. CYP3A activity was a unique independent predictor of variability of atorvastatin metabolism, explaining the majority of the variance in reduction of atorvastatin (60.0%) and formation of ortho-hydroxy atorvastatin (78.8%) and para-hydroxy atorvastatin (83.9%). MiR-27b and miR-206 were found to repress CYP3A4 gene expression and CYP3A activity by directly binding to CYP3A4 3'-UTR, while miR-142 was found to indirectly repress CYP3A activity. Our study indicates that miRNAs play significant roles in bridging the gap between epigenetic effects and missing heritability in CYP3A functionality. PMID:27211076

  2. Pharmacogenetics in American Indian Populations: Analysis of CYP2D6, CYP3A4, CYP3A5, and CYP2C9 in the Confederated Salish and Kootenai Tribes

    PubMed Central

    Fohner, Alison; Muzquiz, LeeAnna I.; Austin, Melissa A.; Gaedigk, Andrea; Gordon, Adam; Thornton, Timothy; Rieder, Mark J.; Pershouse, Mark A.; Putnam, Elizabeth A.; Howlett, Kevin; Beatty, Patrick; Thummel, Kenneth E.; Woodahl, Erica L.

    2014-01-01

    Objectives Cytochrome P450 enzymes play a dominant role in drug elimination and variation in these genes is a major source of interindividual differences in drug response. Little is known, however, about pharmacogenetic variation in American Indian and Alaska Native (AI/AN) populations. We have developed a partnership with the Confederated Salish and Kootenai Tribes (CSKT) in northwestern Montana to address this knowledge gap. Methods We resequenced CYP2D6 in 187 CSKT subjects and CYP3A4, CYP3A5, and CYP2C9 in 94 CSKT subjects. Results We identified 67 variants in CYP2D6, 15 in CYP3A4, 10 in CYP3A5, and 41 in CYP2C9. The most common CYP2D6 alleles were CYP2D6*4 and *41 (20.86 and 11.23%, respectively). CYP2D6*3, *5, *6, *9, *10, *17, *28, *33, *35, *49, *1xN, *2xN, and *4xN frequencies were less than 2%. CYP3A5*3, CYP3A4*1G, and *1B were detected with frequencies of 92.47, 26.81, and 2.20%, respectively. Allelic variation in CYP2C9 was low: CYP2C9*2 (5.17%) and *3 (2.69%). In general, allele frequencies in CYP2D6, CYP2C9 and CYP3A5 were similar to those observed in European Americans. There was, however, a marked divergence in CYP3A4 for the CYP3A4*1G allele. We also observed low levels of linkage between CYP3A4*1G and CYP3A5*1 in the CSKT. The combination of nonfunctional CYP3A5*3 and putative reduced function CYP3A4*1G alleles may predict diminished clearance of CYP3A substrates. Conclusions These results highlight the importance of conducting pharmacogenomic research in AI/AN populations and demonstrate that extrapolation from other populations is not appropriate. This information could help to optimize drug therapy for the CSKT population. PMID:23778323

  3. Dual effects of ketoconazole cis-enantiomers on CYP3A4 in human hepatocytes and HepG2 Cells.

    PubMed

    Novotná, Aneta; Krasulová, Kristýna; Bartoňková, Iveta; Korhoňová, Martina; Bachleda, Petr; Anzenbacher, Pavel; Dvořák, Zdeněk

    2014-01-01

    Antifungal drug ketoconazole causes severe drug-drug interactions by influencing gene expression and catalytic activity of major drug-metabolizing enzyme cytochrome P450 CYP3A4. Ketoconazole is administered in the form of racemic mixture of two cis-enantiomers, i.e. (+)-ketoconazole and (-)-ketoconazole. Many enantiopure drugs were introduced to human pharmacotherapy in last two decades. In the current paper, we have examined the effects of ketoconazole cis-enantiomers on the expression of CYP3A4 in human hepatocytes and HepG2 cells and on catalytic activity of CYP3A4 in human liver microsomes. We show that both ketoconazole enantiomers induce CYP3A4 mRNA and protein in human hepatocytes and HepG2 cells. Gene reporter assays revealed partial agonist activity of ketoconazole enantiomers towards pregnane X receptor PXR. Catalytic activity of CYP3A4/5 towards two prototypic substrates of CYP3A enzymes, testosterone and midazolam, was determined in presence of both (+)-ketoconazole and (-)-ketoconazole in human liver microsomes. Overall, both ketoconazole cis-enantiomers induced CYP3A4 in human cells and inhibited CYP3A4 in human liver microsomes. While interaction of ketoconazole with PXR and induction of CYP3A4 did not display enantiospecific pattern, inhibition of CYP3A4 catalytic activity by ketoconazole differed for ketoconazole cis-enantiomers ((+)-ketoconazole IC₅₀ 1.69 µM, Ki 0.92 µM for testosterone, IC₅₀ 1.46 µM, Ki 2.52 µM for midazolam; (-)-ketoconazole IC₅₀ 0.90 µM, Ki 0.17 µM for testosterone, IC₅₀ 1.04 µM, Ki 1.51 µM for midazolam).

  4. Comparative inhibitory potential of selected dietary bioactive polyphenols, phytosterols on CYP3A4 and CYP2D6 with fluorometric high-throughput screening.

    PubMed

    Vijayakumar, Thangavel Mahalingam; Kumar, Ramasamy Mohan; Agrawal, Aruna; Dubey, Govind Prasad; Ilango, Kaliappan

    2015-07-01

    Cytochrome P450 (CYP450) inhibition by the bioactive molecules of dietary supplements or herbal products leading to greater potential for toxicity of co-administered drugs. The present study was aimed to compare the inhibitory potential of selected common dietary bioactive molecules (Gallic acid, Ellagic acid, β-Sitosterol, Stigmasterol, Quercetin and Rutin) on CYP3A4 and CYP2D6 to assess safety through its inhibitory potency and to predict interaction potential with co-administered drugs. CYP450-CO complex assay was carried out for all the selected dietary bioactive molecules in isolated rat microsomes. CYP450 concentration of the rat liver microsome was found to be 0.474 nmol/mg protein, quercetin in DMSO has shown maximum inhibition on CYP450 (51.02 ± 1.24 %) but less when compared with positive control (79.02 ± 1.61 %). In high throughput fluorometric assay, IC50 value of quercetin (49.08 ± 1.02-54.36 ± 0.85 μg/ml) and gallic acid (78.46 ± 1.32-83.84 ± 1.06 μg/ml) was lower than other bioactive compounds on CYP3A4 and CYP2D6 respectively but it was higher than positive controls (06.28 ± 1.76-07.74 ± 1.32 μg/ml). In comparison of in vitro inhibitory potential on CYP3A4 and CYP2D6, consumption of food or herbal or dietary supplements containing quercetin and gallic acid without any limitation should be carefully considered when narrow therapeutic drugs are administered together. PMID:26139922

  5. Transport and uptake of clausenamide enantiomers in CYP3A4-transfected Caco-2 cells: An insight into the efflux-metabolism alliance.

    PubMed

    Hua, Fang; Shi, Mei-jun; Zhu, Xiao-lu; Li, Meng; Wang, Hong-xu; Yu, Xiao-ming; Li, Yan; Zhu, Chuan-jiang

    2015-11-01

    The present study developed a CYP3A4-expressed Caco-2 monolayer model at which effects of the efflux-metabolism alliance on the transport and uptake of clausenamide (CLA) enantiomers as CYP3A4 substrates were investigated. The apparent permeability coefficients (Papp) of (-) and (+)CLA were higher in the absorptive direction than those in the secretory direction with efflux ratios (ER) of 0.709±0.411 and 0.867±0.250 (×10(-6)cm/s), respectively. Their bidirectional transports were significantly reduced by 75.6-87.5% after treatment with verapamil (a P-glycoprotein inhibitor) that increased the rate of metabolism by CYP3A4, whereas the CYP3A4 inhibitor ketoconazole treatment markedly enhanced the basolateral to apical flux of (-) and (+)CLA with ERs being 2.934±1.432 and 1.877±0.148(×10(-6)cm/s) respectively. These changes could be blocked by the duel CYP3A4/P-glycoprotein inhibitor cyclosporine A, consequently, Papp values for CLA enantiomers in both directions were significantly greater than those obtained by using verapamil or ketoconazole, and their ERs were similar to those following (-) or (+)-isomer treatment alone. Furthermore, the uptake of (-)CLA was more than that of (+)CLA in the transfected cells. Incubation with ketoconazole decreased the intracellular concentrations of the two enantiomers. This effect disappeared in the presence of a CYP3A4 inducer dexamethasone. These results indicated that CYP3A4 could influence P-gp efflux, transport and uptake of CLA enantiomers as CYP3A4 substrates and that a duel inhibition to CYP3A4/ P-glycoprotein could enhance their absorption and bioavailability, which provides new insight into the efflux-metabolism alliance and will benefit the clinical pharmacology of (-)CLA as a candidate drug for treatment of Alzheimer's disease. PMID:26301745

  6. Transport and uptake of clausenamide enantiomers in CYP3A4-transfected Caco-2 cells: An insight into the efflux-metabolism alliance.

    PubMed

    Hua, Fang; Shi, Mei-jun; Zhu, Xiao-lu; Li, Meng; Wang, Hong-xu; Yu, Xiao-ming; Li, Yan; Zhu, Chuan-jiang

    2015-11-01

    The present study developed a CYP3A4-expressed Caco-2 monolayer model at which effects of the efflux-metabolism alliance on the transport and uptake of clausenamide (CLA) enantiomers as CYP3A4 substrates were investigated. The apparent permeability coefficients (Papp) of (-) and (+)CLA were higher in the absorptive direction than those in the secretory direction with efflux ratios (ER) of 0.709±0.411 and 0.867±0.250 (×10(-6)cm/s), respectively. Their bidirectional transports were significantly reduced by 75.6-87.5% after treatment with verapamil (a P-glycoprotein inhibitor) that increased the rate of metabolism by CYP3A4, whereas the CYP3A4 inhibitor ketoconazole treatment markedly enhanced the basolateral to apical flux of (-) and (+)CLA with ERs being 2.934±1.432 and 1.877±0.148(×10(-6)cm/s) respectively. These changes could be blocked by the duel CYP3A4/P-glycoprotein inhibitor cyclosporine A, consequently, Papp values for CLA enantiomers in both directions were significantly greater than those obtained by using verapamil or ketoconazole, and their ERs were similar to those following (-) or (+)-isomer treatment alone. Furthermore, the uptake of (-)CLA was more than that of (+)CLA in the transfected cells. Incubation with ketoconazole decreased the intracellular concentrations of the two enantiomers. This effect disappeared in the presence of a CYP3A4 inducer dexamethasone. These results indicated that CYP3A4 could influence P-gp efflux, transport and uptake of CLA enantiomers as CYP3A4 substrates and that a duel inhibition to CYP3A4/ P-glycoprotein could enhance their absorption and bioavailability, which provides new insight into the efflux-metabolism alliance and will benefit the clinical pharmacology of (-)CLA as a candidate drug for treatment of Alzheimer's disease.

  7. Fentanyl Enhances Hepatotoxicity of Paclitaxel via Inhibition of CYP3A4 and ABCB1 Transport Activity in Mice

    PubMed Central

    Pan, Jia-Hao; Bi, Bing-Tian; Feng, Kun-Yao; Huang, Wan; Zeng, Wei-An

    2015-01-01

    Fentanyl, a potent opioid analgesic that is used to treat cancer pain, is commonly administered with paclitaxel in advanced tumors. However, the effect of fentanyl on the hepatotoxicity of paclitaxel and its potential mechanism of action is not well studied. The purpose of this study was to investigate the effect of fentanyl on the hepatotoxicity of paclitaxel and its potential mechanisms of action. Pharmacokinetic parameters of paclitaxel were tested using reversed phase high-performance liquid chromatography (RP-HPLC). Aspartate transaminase (AST), alanine aminotransferase (ALT), and mouse liver histopathology were examined. Moreover, the cytotoxicity of anti-carcinogens was examined using 1-(4, 5-dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT), and the intracellular accumulation of doxorubicin and rhodamine 123 was detected by flow cytometry. Furthermore, the expression of ABCB1 and the activity of ABCB1 ATPase and CYP3A4 were also examined. In this study, the co-administration of fentanyl and paclitaxel prolonged the half-life (t1/2) of paclitaxel from 1.455 hours to 2.344 hours and decreased the clearance (CL) from 10.997 ml/h to 7.014 ml/h in mice. Fentanyl significantly increased the levels of ALT in mice to 88.2 U/L, which is more than 2-fold higher than the level detected in the control group, and it increased the histological damage in mouse livers. Furthermore, fentanyl enhanced the cytotoxicity of anti-carcinogens that are ABCB1 substrates and increased the accumulation of doxorubicin and rhodamine 123. Additionally, fentanyl stimulated ABCB1 ATPase activity and inhibited CYP3A4 activity in the liver microsomes of mice. Our study indicates that the obvious hepatotoxicity during this co-administration was due to the inhibition of CYP3A4 activity and ABCB1 transport activity. These findings suggested that the accumulation-induced hepatotoxicity of paclitaxel when it is combined with fentanyl should be avoided. PMID:26633878

  8. GW4064, an Agonist of Farnesoid X Receptor, Represses CYP3A4 Expression in Human Hepatocytes by Inducing Small Heterodimer Partner Expression

    PubMed Central

    Zhang, Shu; Pan, Xian

    2015-01-01

    Farnesoid X receptor (FXR) functions as a regulator of bile acid and lipid homeostasis and is recognized as a promising therapeutic target for metabolic diseases. The biologic function of FXR is mediated in part by a small heterodimer partner (SHP); ligand-activated FXR enhances SHP expression, and SHP in turn represses the activity of multiple transcription factors. This study aimed to investigate the effect of FXR activation on expression of the major drug-metabolizing enzyme CYP3A4. The effects of 3-(2,6-dichlorophenyl)-4-(3′-carboxy-2-chlorostilben-4-yl)oxymethyl-5-isopropylisoxazole (GW4064), a synthetic agonist of FXR, on the expression and activity of CYP3A4 were examined in primary human hepatocytes by using quantitative real-time polymerase chain reaction and S9 phenotyping. In human hepatocytes, treatment of GW4064 (1 μM) for 48 hours resulted in a 75% decrease in CYP3A4 mRNA expression and a 25% decrease in CYP3A4 activity, accompanied by ∼3-fold increase in SHP mRNA expression. In HepG2 cells, SHP repressed transactivation of CYP3A4 promoter by pregnane X receptor (PXR), constitutive androstane receptor (CAR), and glucocorticoid receptor. Interestingly, GW4064 did not repress expression of CYP2B6, another target gene of PXR and CAR; GW4064 enhanced CYP2B6 promoter activity. In conclusion, GW4064 represses CYP3A4 expression in human hepatocytes, potentially through upregulation of SHP expression and subsequent repression of CYP3A4 promoter activity. Clinically significant drug-drug interaction involving FXR agonists and CYP3A4 substrates may occur. PMID:25725071

  9. GW4064, an agonist of farnesoid X receptor, represses CYP3A4 expression in human hepatocytes by inducing small heterodimer partner expression.

    PubMed

    Zhang, Shu; Pan, Xian; Jeong, Hyunyoung

    2015-05-01

    Farnesoid X receptor (FXR) functions as a regulator of bile acid and lipid homeostasis and is recognized as a promising therapeutic target for metabolic diseases. The biologic function of FXR is mediated in part by a small heterodimer partner (SHP); ligand-activated FXR enhances SHP expression, and SHP in turn represses the activity of multiple transcription factors. This study aimed to investigate the effect of FXR activation on expression of the major drug-metabolizing enzyme CYP3A4. The effects of 3-(2,6-dichlorophenyl)-4-(3'-carboxy-2-chlorostilben-4-yl)oxymethyl-5-isopropylisoxazole (GW4064), a synthetic agonist of FXR, on the expression and activity of CYP3A4 were examined in primary human hepatocytes by using quantitative real-time polymerase chain reaction and S9 phenotyping. In human hepatocytes, treatment of GW4064 (1 μM) for 48 hours resulted in a 75% decrease in CYP3A4 mRNA expression and a 25% decrease in CYP3A4 activity, accompanied by ∼3-fold increase in SHP mRNA expression. In HepG2 cells, SHP repressed transactivation of CYP3A4 promoter by pregnane X receptor (PXR), constitutive androstane receptor (CAR), and glucocorticoid receptor. Interestingly, GW4064 did not repress expression of CYP2B6, another target gene of PXR and CAR; GW4064 enhanced CYP2B6 promoter activity. In conclusion, GW4064 represses CYP3A4 expression in human hepatocytes, potentially through upregulation of SHP expression and subsequent repression of CYP3A4 promoter activity. Clinically significant drug-drug interaction involving FXR agonists and CYP3A4 substrates may occur.

  10. Reduction in hepatic drug metabolizing CYP3A4 activities caused by P450 oxidoreductase mutations identified in patients with disordered steroid metabolism

    SciTech Connect

    Flueck, Christa E.; Mullis, Primus E.; Pandey, Amit V.

    2010-10-08

    Research highlights: {yields} Cytochrome P450 3A4 (CYP3A4), metabolizes 50% of drugs in clinical use and requires NADPH-P450 reductase (POR). {yields} Mutations in human POR cause congenital adrenal hyperplasia from diminished activities of steroid metabolizing P450s. {yields} We are reporting that mutations in POR may reduce CYP3A4 activity. {yields} POR mutants Y181D, A457H, Y459H, V492E and R616X lost 99%, while A287P, C569Y and V608F lost 60-85% CYP3A4 activity. {yields} Reduction of CYP3A4 activity may cause increased risk of drug toxicities/adverse drug reactions in patients with POR mutations. -- Abstract: Cytochrome P450 3A4 (CYP3A4), the major P450 present in human liver metabolizes approximately half the drugs in clinical use and requires electrons supplied from NADPH through NADPH-P450 reductase (POR, CPR). Mutations in human POR cause a rare form of congenital adrenal hyperplasia from diminished activities of steroid metabolizing P450s. In this study we examined the effect of mutations in POR on CYP3A4 activity. We used purified preparations of wild type and mutant human POR and in vitro reconstitution with purified CYP3A4 to perform kinetic studies. We are reporting that mutations in POR identified in patients with disordered steroidogenesis/Antley-Bixler syndrome (ABS) may reduce CYP3A4 activity, potentially affecting drug metabolism in individuals carrying mutant POR alleles. POR mutants Y181D, A457H, Y459H, V492E and R616X had more than 99% loss of CYP3A4 activity, while POR mutations A287P, C569Y and V608F lost 60-85% activity. Loss of CYP3A4 activity may result in increased risk of drug toxicities and adverse drug reactions in patients with POR mutations.

  11. Racial Differences in CYP3A4 Genotype and Survival Among Men Treated on Radiation Therapy Oncology Group (RTOG) 9202: A Phase III Randomized Trial

    SciTech Connect

    Roach, Mack Silvio, Michelle de; Rebbick, Timothy; Grignon, David; Rotman, Marvin; Wolkov, Harvey; Fisher, Barbara; Hanks, Gerald; Shipley, William U.; Pollack, Alan; Sandler, Howard; Watkins-Bruner, Deborah Ph.D.

    2007-09-01

    Purpose: Inherited genotypes may explain the inferior outcomes of African American (AA) men with prostate cancer. To understand how variation in CYP3A4 correlated with outcomes, a retrospective examination of the CYP3A4*1B genotype was performed on men treated with Radiation Therapy Oncology Group (RTOG) 92-02. Methods and Materials: From 1,514 cases, we evaluated 56 (28.4%) of 197 AA and 54 (4.3%) of 1,274 European American (EA) patients. All patients received goserelin and flutamide for 2 months before and during RT (STAD-RT) {+-} 24 months of goserelin (long-term androgen deprivation plus radiation [LTAD-RT]). Events studied included overall survival and biochemical progression using American Society for Therapeutic Radiology and Oncology consensus guidelines. Results: There were no differences in outcome in patients in with or without CYP3A4 data. There was an association between race and CYP3A4 polymorphisms with 75% of EAs having the Wild Type compared to only 25% of AA men (p <0.0001). There was no association between CYP3A4 classification or race and survival or progression. Conclusions: The samples analyzed support previously reported observations about the distribution of CYP3A4*1B genotype by race, but race was not associated with poorer outcome. However, patient numbers were limited, and selection bias cannot be completely ruled out.

  12. CYP3A4-transfected Caco-2 cells as a tool for understanding biochemical absorption barriers: studies with sirolimus and midazolam.

    PubMed

    Cummins, Carolyn L; Jacobsen, Wolfgang; Christians, Uwe; Benet, Leslie Z

    2004-01-01

    CYP3A4-transfected Caco-2 cells were used as an in vitro system to predict the importance of drug metabolism and transport on overall drug absorption. We examined the transport and metabolism of two drugs; midazolam, an anesthetic agent and CYP3A4 substrate, and sirolimus, an immunosuppressant and a dual CYP3A4/P-glycoprotein (P-gp) substrate, in the presence of cyclosporine (CsA, a CYP3A4/P-gp inhibitor) or N-[4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)-ethyl]-phenyl]-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamine (GG918) (an inhibitor of P-gp and not CYP3A4). All major CYP3A4 metabolites were formed in the cells (1-OH > 4-OH midazolam and 39-O-desmethyl > 12-OH > 11-OH sirolimus), consistent with results from human liver microsomes. There was no bidirectional transport of midazolam across CYP3A4-transfected Caco-2 cells, whereas there was a 2.5-fold net efflux of sirolimus (1 microM) that disappeared in the presence of CsA or GG918. No change in the absorption rate or extraction ratio (ER) for midazolam was observed when P-gp was inhibited with GG918. Addition of GG918 had a modest impact on the absorption rate and ER for sirolimus (increased 58% and decreased 25%, respectively), whereas a 6.1-fold increase in the absorption rate and a 75% decrease in the ER were found when sirolimus was combined with CsA. Although both midazolam and sirolimus metabolites were preferentially excreted to the apical compartment, only sirolimus metabolites were transported by P-gp as determined from inhibition studies with GG918. Using CYP3A4-transfected Caco-2 cells we determined that, in contrast to P-gp, CYP3A4 is the major factor limiting sirolimus absorption. The integration of CYP3A4 and P-gp into a combined in vitro system was critical to unveil the relative importance of each biochemical barrier. PMID:14569063

  13. Size and surface modification of amorphous silica particles determine their effects on the activity of human CYP3A4 in vitro

    NASA Astrophysics Data System (ADS)

    Imai, Shunji; Yoshioka, Yasuo; Morishita, Yuki; Yoshida, Tokuyuki; Uji, Miyuki; Nagano, Kazuya; Mukai, Yohei; Kamada, Haruhiko; Tsunoda, Shin-ichi; Higashisaka, Kazuma; Tsutsumi, Yasuo

    2014-12-01

    Because of their useful chemical and physical properties, nanomaterials are widely used around the world - for example, as additives in food and medicines - and such uses are expected to become more prevalent in the future. Therefore, collecting information about the effects of nanomaterials on metabolic enzymes is important. Here, we examined the effects of amorphous silica particles with various sizes and surface modifications on cytochrome P450 3A4 (CYP3A4) activity by means of two different in vitro assays. Silica nanoparticles with diameters of 30 and 70 nm (nSP30 and nSP70, respectively) tended to inhibit CYP3A4 activity in human liver microsomes (HLMs), but the inhibitory activity of both types of nanoparticles was decreased by carboxyl modification. In contrast, amine-modified nSP70 activated CYP3A4 activity. In HepG2 cells, nSP30 inhibited CYP3A4 activity more strongly than the larger silica particles did. Taken together, these results suggest that the size and surface characteristics of the silica particles determined their effects on CYP3A4 activity and that it may be possible to develop silica particles that do not have undesirable effects on metabolic enzymes by altering their size and surface characteristics.

  14. Frog intestinal perfusion to evaluate drug permeability: application to p-gp and cyp3a4 substrates

    PubMed Central

    Yerasi, Neelima; Vurimindi, Himabindu; Devarakonda, Krishna

    2015-01-01

    To evaluate the reliability of using in situ frog intestinal perfusion technique for permeability assessment of carrier transported drugs which are also substrates for CYP enzymes. Single Pass Intestinal Perfusion (SPIP) studies were performed in frogs of the species Rana tigrina using established method for rats with some modifications after inducing anesthesia. Effective permeability coefficient (Peff) of losartan and midazolam was calculated in the presence and absence of inhibitors using the parallel-tube model. Peff of losartan when perfused alone was found to be 0.427 ± 0.27 × 10-4cm/s and when it was co-perfused with inhibitors, significant change in Peff was observed. Peff of midazolam when perfused alone was found to be 2.03 ± 0.07 × 10-4cm/s and when it was co-perfused with inhibitors, no significant change in Peff was observed. Comparison of Peff calculated in frog with that of other available models and also humans suggested that the Peff-values are comparable and reflected well with human intestinal permeability. It is possible to determine the Peff-value for compounds which are dual substrates of P-glycoprotein and CYP3A4 using in situ frog intestinal perfusion technique. The calculated Peff-values correlated well with reported Peff-values of probe drugs. comparison of the Peff-value of losartan obtained with that of reported human’s Peff and Caco 2 cell data, and comparison of the Peff-value of midazolam with that of reported rat’s Peff, we could conclude that SPIP from model can be reliably used in preclinical studies for permeability estimation. This model may represent a valuable alternative to the low speed and high cost of conventional animal models (typically rodents) for the assessment of intestinal permeability. PMID:26236236

  15. The Effect of microRNAs in the Regulation of Human CYP3A4: a Systematic Study using a Mathematical Model

    NASA Astrophysics Data System (ADS)

    Wei, Zhiyun; Jiang, Songshan; Zhang, Yiting; Wang, Xiaofei; Peng, Xueling; Meng, Chunjie; Liu, Yichen; Wang, Honglian; Guo, Luo; Qin, Shengying; He, Lin; Shao, Fengmin; Zhang, Lirong; Xing, Qinghe

    2014-03-01

    CYP3A4 metabolizes more than 50% of the drugs on the market. The large inter-individual differences of CYP3A4 expression may contribute to the variability of human drug responses. Post-transcriptional regulation of CYP3A4 is poorly understood, whereas transcriptional regulation has been studied much more thoroughly. In this study, we used multiple software programs to predict miRNAs that might bind to CYP3A4 and identified 112 potentially functional miRNAs. Then a luciferase reporter system was used to assess the effect of the overexpression of each potentially functional miRNA in HEK 293T cells. Fourteen miRNAs that significantly decreased reporter activity were measured in human liver samples (N = 27) as candidate miRNAs. To establish a more effective way to analyze in vivo data for miRNA candidates, the relationship between functional miRNA and target mRNA was modeled mathematically. Taking advantage of this model, we found that hsa-miR-577, hsa-miR-1, hsa-miR-532-3p and hsa-miR-627 could significantly downregulate the translation efficiency of CYP3A4 mRNA in liver. This study used in silico, in vitro and in vivo methods to progressively screen functional miRNAs for CYP3A4 and to enhance our understanding of molecular events underlying the large inter-individual differences of CYP3A4 expression in human populations.

  16. Content of CYP3A4 inhibitors, naringin, naringenin and bergapten in grapefruit and grapefruit juice products.

    PubMed

    Ho, P C; Saville, D J; Coville, P F; Wanwimolruk, S

    2000-04-01

    The flavonoids, naringin and naringenin and the furanocoumarin, bergapten (5-methoxypsoralen), were detected in some fresh grapefruit and commercial grapefruit juices but were not detected in other fruit juices tested (orange; orange with apple base; dark grape; orange and mango with apple base; orange, peach, passion fruit juice). The contents of these three grapefruit constituents in commercial juice and fresh grapefruit varied from brand to brand and also from lot to lot. Juice was prepared from the fresh fruit via different methods (by hand, squeezer or blender). The naringin content, after hand-squeeze, ranged from 115 to 384 mg/l. With hand-squeeze juice production, bergapten was not detected (less than 0.5 mg/l) in two varieties of grapefruit, and naringenin was usually not in detectable levels (less than 2 mg/l) in three varieties. All three constituents were present in New Zealand grapefruit preparations (including juice by hand-squeeze) and different lots showed variation in content (1.5-, 2.3- and 4.7-fold for naringin, naringenin and bergapten, respectively). Differences in the concentrations of these three constituents, which have potential for drug interaction, may contribute to the variability in pharmacokinetics of CYP3A4 drugs and some contradictory results of drug interaction studies with grapefruit juice. PMID:10812937

  17. Effects of Panax notoginseng saponins on the activities of CYP1A2, CYP2C9, CYP2D6 and CYP3A4 in rats in vivo.

    PubMed

    Liu, Rui; Qin, Mengnan; Hang, Pengzhou; Liu, Yan; Zhang, Zhiren; Liu, Gaofeng

    2012-08-01

    The aim of this study was to assess the influence of the Panax notoginseng saponins (PNS) on the activities of the drug-metabolizing enzymes cytochrome P450 (CYP450) 1A2, 2 C9, 2D6 and 3A4 in rats. The activities of CYP1A2, 2 C9, 2D6 and 3A4 were measured using specific probe drugs. After pretreatment for 1 week with PNS or physiological saline (control group), probe drugs caffeine (10 mg/kg; CYP1A2 activity), tolbutamide (15 mg/kg; CYP2C9 activity), metoprolol (20 mg/kg; CYP2D6 activity) and dapsone (10 mg/kg; CYP3A4 activity) were administered to rats by intraperitoneal injection. The blood was then collected at different times for ultra performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) analysis. The data showed that PNS exhibited an induction effect on CYP1A2 by decreasing caffeine C(max) (36.3%, p < 0.01) and AUC(0-∞) (22.77%, p < 0.05) and increasing CL/F (27.03%, p < 0.05) compared with those of the control group. Western blot analysis was used to detect the effect of PNS on the protein level of CYP1A2, and the results showed that PNS could upregulate the protein expression of CYP1A2. However, no significant changes in CYP2C9, 2D6 or 3A4 activities were observed. In conclusion, the results indicate that PNS could induce CYP1A2, which may affect the disposition of medicines primarily dependent on the CYP1A2 pathway. Our work may be the basis of related herb-drug interactions in the clinic.

  18. Effects of the CYP3A4*1B Genetic Polymorphism on the Pharmacokinetics of Tacrolimus in Adult Renal Transplant Recipients: A Meta-Analysis

    PubMed Central

    Shi, Wei-Long; Tang, Hui-Lin; Zhai, Suo-Di

    2015-01-01

    Background and Objective The association between the CYP3A4*1B single nucleotide polymorphism (SNP) and tacrolimus pharmacokinetics in different studies is controversial. Therefore, a meta-analysis was employed to evaluate the correlation between the CYP3A4*1B genetic polymorphism and tacrolimus pharmacokinetics at different post-transplantation times in adult renal transplant recipients. Methods Studies evaluating the CYP3A4*1B genetic polymorphism and tacrolimus pharmacokinetics were retrieved through a systematical search of Embase, PubMed, the Cochrane Library, ClinicalTrials.gov and three Chinese literature databases (up to Sept. 2014). The pharmacokinetic parameters (weight-adjusted tacrolimus daily dose and tacrolimus trough concentration/weight-adjusted tacrolimus daily dose ratio) were extracted, and the meta-analysis was performed using Stata 12.1. Results Seven studies (involving 1182 adult renal transplant recipients) were included in this meta-analysis. For the weight-adjusted tacrolimus daily dose, in all included renal transplant recipients (European & Indian populations), CYP3A4*1/*1 recipients required a significantly lower weight-adjusted tacrolimus daily dose than did CYP3A4*1B carriers at 7 days (WMD -0.048; 95% CI -0.083 ~ -0.014), 6 months (WMD -0.058; 95% CI -0.081 ~ -0.036) and 12 months (WMD - 0.061; 95% CI -0.096 ~ -0.027) post-transplantation. In light of the heterogeneity, the analysis was repeated after removing the only study in an Indian population, and CYP3A4*1/*1 European recipients (mostly Caucasian) required a lower weight-adjusted tacrolimus daily dose within the first year post-transplantation. The tacrolimus trough concentration/weight-adjusted tacrolimus daily dose ratio (C0/Dose ratio) was significantly higher in CYP3A4*1/*1 recipients than in CYP3A4*1B carriers at 6 months (WMD 52.588; 95% CI 22.387 ~ 82.789) and 12 months (WMD 62.219; 95% CI 14.218 ~ 110.221) post-transplantation. When the only study in an Indian population

  19. Monoester-Diterpene Aconitum Alkaloid Metabolism in Human Liver Microsomes: Predominant Role of CYP3A4 and CYP3A5

    PubMed Central

    Ye, Ling; Yang, Xiao-Shan; Lu, Lin-lin; Chen, Wei-Ying; Zeng, Shan; Yan, Tong-Meng; Dong, Ling-Na; Peng, Xiao-Juan; Shi, Jian; Liu, Zhong-Qiu

    2013-01-01

    Aconitum, widely used to treat rheumatoid arthritis for thousands of years, is a toxic herb that can frequently cause fatal cardiac poisoning. Aconitum toxicity could be decreased by properly hydrolyzing diester-diterpene alkaloids into monoester-diterpene alkaloids. Monoester-diterpene alkaloids, including benzoylaconine (BAC), benzoylmesaconine (BMA), and benzoylhypaconine (BHA), are the primary active and toxic constituents of processed Aconitum. Cytochrome P450 (CYP) enzymes protect the human body by functioning as the defense line that limits the invasion of toxicants. Our purposes were to identify the CYP metabolites of BAC, BMA, and BHA in human liver microsomes and to distinguish which isozymes are responsible for their metabolism through the use of chemical inhibitors, monoclonal antibodies, and cDNA-expressed CYP enzyme. High-resolution mass spectrometry was used to characterize the metabolites. A total of 7, 8, and 9 metabolites were detected for BAC, BMA, and BHA, respectively. The main metabolic pathways were demethylation, dehydrogenation, demethylation-dehydrogenation, hydroxylation and didemethylation, which produced less toxic metabolites by decomposing the group responsible for the toxicity of the parent compound. Taken together, the results of the chemical inhibitors, monoclonal antibodies, and cDNA-expressed CYP enzymes experiments demonstrated that CYP3A4 and CYP3A5 have essential functions in the metabolism of BAC, BMA, and BHA. PMID:23864901

  20. CYP1A1 induction and CYP3A4 inhibition by the fungicide imazalil in the human intestinal Caco-2 cells-comparison with other conazole pesticides.

    PubMed

    Sergent, Thérèse; Dupont, Isabelle; Jassogne, Coralie; Ribonnet, Laurence; van der Heiden, Edwige; Scippo, Marie-Louise; Muller, Marc; McAlister, Dan; Pussemier, Luc; Larondelle, Yvan; Schneider, Yves-Jacques

    2009-02-10

    Imazalil (IMA) is a widely used imidazole-antifungal pesticide and, therefore, a food contaminant. This compound is also used as a drug (enilconazole). As intestine is the first site of exposure to ingested drugs and pollutants, we have investigated the effects of IMA, at realistic intestinal concentrations, on xenobiotic-metabolizing enzymes and efflux pumps by using Caco-2 cells, as a validated in vitro model of the human intestinal absorptive epithelium. For comparison, other conazole fungicides, i.e. ketoconazole, propiconazole and tebuconazole, were also studied. IMA induced cytochrome P450 (CYP) 1A1 activity to the same extent as benzo(a)pyrene (B(a)P) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), in a dose- and time-dependent manner. Cell-free aryl hydrocarbon receptor (AhR) binding assay and reporter gene assay suggested that IMA is not an AhR-ligand, implying that IMA-mediated induction should involve an AhR-independent pathway. Moreover, IMA strongly inhibited the CYP3A4 activity in 1,25-vitamin D(3)-induced Caco-2 cells. The other fungicides had weak or nil effects on CYP activities. Study of the apical efflux pump activities revealed that ketoconazole inhibited both P-glycoprotein (Pgp) and multidrug resistance-associated protein 2 (MRP-2) or breast cancer resistance protein (BCRP), whereas IMA and other fungicides did not. Our results imply that coingestion of IMA-contaminated food and CYP3A4- or CYP1A1-metabolizable drugs or chemicals could lead to drug bioavailability modulation or toxicological interactions, with possible adverse effects for human health.

  1. Prilocaine- and lidocaine-induced methemoglobinemia is caused by human carboxylesterase-, CYP2E1-, and CYP3A4-mediated metabolic activation.

    PubMed

    Higuchi, Ryota; Fukami, Tatsuki; Nakajima, Miki; Yokoi, Tsuyoshi

    2013-06-01

    Prilocaine and lidocaine are classified as amide-type local anesthetics for which serious adverse effects include methemoglobinemia. Although the hydrolyzed metabolites of prilocaine (o-toluidine) and lidocaine (2,6-xylidine) have been suspected to induce methemoglobinemia, the metabolic enzymes that are involved remain uncharacterized. In the present study, we aimed to identify the human enzymes that are responsible for prilocaine- and lidocaine-induced methemoglobinemia. Our experiments revealed that prilocaine was hydrolyzed by recombinant human carboxylesterase (CES) 1A and CES2, whereas lidocaine was hydrolyzed by only human CES1A. When the parent compounds (prilocaine and lidocaine) were incubated with human liver microsomes (HLM), methemoglobin (Met-Hb) formation was lower than when the hydrolyzed metabolites were incubated with HLM. In addition, Met-Hb formation when prilocaine and o-toluidine were incubated with HLM was higher than that when lidocaine and 2,6-xylidine were incubated with HLM. Incubation with diisopropyl fluorophosphate and bis-(4-nitrophenyl) phosphate, which are general inhibitors of CES, significantly decreased Met-Hb formation when prilocaine and lidocaine were incubated with HLM. An anti-CYP3A4 antibody further decreased the residual formation of Met-Hb. Met-Hb formation after the incubation of o-toluidine and 2,6-xylidine with HLM was only markedly decreased by incubation with an anti-CYP2E1 antibody. o-Toluidine and 2,6-xylidine were further metabolized by CYP2E1 to 4- and 6-hydroxy-o-toluidine and 4-hydroxy-2,6-xylidine, respectively, and these metabolites were shown to more efficiently induce Met-Hb formation than the parent compounds. Collectively, we found that the metabolites produced by human CES-, CYP2E1-, and CYP3A4-mediated metabolism were involved in prilocaine- and lidocaine-induced methemoglobinemia.

  2. Mechanistic insight from in silico pharmacokinetic experiments: roles of P-glycoprotein, Cyp3A4 enzymes, and microenvironments.

    PubMed

    Lam, Tai Ning; Hunt, C Anthony

    2010-02-01

    Saquinavir exhibits paradoxical transport across modified Caco-2 cell monolayers (doi: 10.1124/jpet.103.056390) expressing P-glycoprotein and Cyp3A4. The data implicate complicated intracellular transport mechanisms. Drawing on recent discrete event modeling and simulation advances, we built an in silico analog of the confluent, asymmetric cell monolayer used in the cited work. We call it in silico experimental Caco-2 (cell monolayer) culture (ISECC). Concrete, working, hypothesized spatial mechanisms were implemented. Validation was achieved when in silico experimental results met similarity measure (SM) expectations that targeted 16 wet-lab experimental conditions. Initial mechanistic hypotheses turned out to be necessary parts of a more complicated explanation. We progressed through four stages of an iterative refinement and validation protocol that enabled and facilitated discovery of plausible, new mechanistic details. The process exercised abductive reasoning, a primary means of scientific knowledge creation and creative cognition. The ISECC that survived the most stringent SM challenge produced transport data that was statistically indistinguishable from referent wet-lab observations. It required a 7:1 ratio of apical transporters to metabolizing enzymes, a 97% reduction of efflux activity by an inhibitor, a biased distribution of metabolizing enzymes, heterogeneous intracellular spaces, and restrictions on intracellular drug movement. Experimenting on synthetic analogs such as ISECC provides a former unavailable means of discovering new mechanistic details and testing their plausibility. The approach thus provides a powerful new expansion of the scientific method: an independent, scientific means to challenge, explore, better understand, and improve any inductive mechanism and, importantly, the assumptions on which it rests. PMID:19864617

  3. The effect of induction of CYP3A4 by St John's wort on ambrisentan plasma pharmacokinetics in volunteers of known CYP2C19 genotype.

    PubMed

    Markert, Christoph; Kastner, Ida Maria; Hellwig, Regina; Kalafut, Peter; Schweizer, Yvonne; Hoffmann, Michael Marcus; Burhenne, Jürgen; Weiss, Johanna; Mikus, Gerd; Haefeli, Walter Emil

    2015-05-01

    To evaluate the impact of CYP2C19 polymorphisms on ambrisentan exposure and to assess its modification by St. John's wort (SJW), 20 healthy volunteers (10 CYP2C19 extensive, four poor and six ultrarapid metabolizers) received therapeutic doses of ambrisentan (5 mg qd po) for 20 days and concomitantly SJW (300 mg tid po) for the last 10 days. To quantify changes of CYP3A4 activity, midazolam (3 mg po) as a probe drug was used. Ambrisentan pharmacokinetics was assessed on days 1, 10 and 20, and midazolam pharmacokinetics before and on days 1, 10, 17 and 20. At steady state, ambrisentan exposure was similar in extensive and ultrarapid metabolizers but 43% larger in poor metabolizers (p < 0.01). In all volunteers, SJW reduced ambrisentan exposure and the relative change (17-26%) was similar in all genotype groups. The extent of this interaction did not correlate with the changes in CYP3A activity (midazolam clearance) (rs = 0.23, p = 0.34). Ambrisentan had no effect on midazolam pharmacokinetics. In conclusion, SJW significantly reduced exposure with ambrisentan irrespective of the CYP2C19 genotype. The extent of this interaction was small and thus likely without clinical relevance.

  4. In silico and in vitro screening for inhibition of cytochrome P450 CYP3A4 by comedications commonly used by patients with cancer.

    PubMed

    Marechal, Jean-Didier; Yu, Jinglei; Brown, Simon; Kapelioukh, Iouri; Rankin, Elaine M; Wolf, C Roland; Roberts, Gordon C K; Paine, Mark J I; Sutcliffe, Michael J

    2006-04-01

    Cytochrome P450 3A4 (CYP3A4) is the major enzyme responsible for phase I drug metabolism of many anticancer agents. It is also a major route for metabolism of many drugs used by patients to treat the symptoms caused by cancer and its treatment as well as their other illnesses, for example, cardiovascular disease. To assess the ability to inhibit CYP3A4 of drugs most commonly used by our patients during cancer therapy, we have made in silico predictions based on the crystal structures of CYP3A4. From this set of 33 common comedicated drugs, 10 were predicted to be inhibitors of CYP3A4, with the antidiarrheal drug loperamide predicted to be the most potent. There was significant correlation (r(2) = 0.75-0.66) between predicted affinity and our measured IC(50) values, and loperamide was confirmed as a potent inhibitor (IC(50) of 0.050 +/- 0.006 microM). Active site docking studies predicted an orientation of loperamide consistent with formation of the major (N-demethylated) metabolite, where it interacts with the phenylalanine cluster and Arg-212 and Glu-374; experimental evidence for the latter interaction comes from the approximately 12-fold increase in K(M) for loperamide observed for the Glu-374-Gln mutant. The commonly prescribed drugs loperamide, amitriptyline, diltiazem, domperidone, lansoprazole, omeprazole, and simvastatin were identified by our in silico and in vitro screens as relatively potent inhibitors of CYP3A4 that have the potential to interact with cytotoxic agents to cause adverse effects, highlighting the likelihood of drug-drug interactions affecting chemotherapy treatment.

  5. Demethylation of neferine in human liver microsomes and formation of quinone methide metabolites mediated by CYP3A4 accentuates its cytotoxicity.

    PubMed

    Shen, Qi; Zuo, Minjuan; Ma, Li; Tian, Ye; Wang, Lu; Jiang, Huidi; Zhou, Quan; Zhou, Hui; Yu, Lushan; Zeng, Su

    2014-12-01

    Neferine is a bisbenzylisoquinoline alkaloid isolated from the seed embryos of Nelumbonucifera Gaertn (Lotus) with various potent pharmacological effects. Recently, neferine has attracted attention for its anti-tumor activities. Our study explored its metabolism and cytotoxicity mechanism. Approaches using chemical inhibitors and recombinant human enzymes to characterize the involved enzymes and kinetic studies indicated that the demethylation of neferine by cytochrome P450 (CYP) 2D6 and CYP3A4 fitted a biphasic kinetic profile. Glutathione (GSH) was used as a trapping agent to identify reactive metabolites of neferine, and four novel GSH conjugates were detected with [M+H](+) ions at m/z 902.4, 916.2, 916.1, and 930.4. Based on its structure containing para-methylene phenol and results from a product ion scan, GSH tends to conjugate with C9' after undergoing oxidative metabolism to form the binding site predominated by CYP3A4. Furthermore, the addition of recombinant human GSTA1, GSTT1, and GSTP1 had little effect on the production of the GSH conjugates. In a 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide assay, combined with the GSH modulators l-buthionine sulfoximine or N-acetyl-l-cysteine, neferine treatment of MDCK-hCYP3A4 and HepG2 cells revealed that CYP3A4 expression and cellular GSH content could cause an EC50 shift. Metabolic activation mediated by CYP3A4 and GSH depletion significantly enhanced neferine-induced cytotoxicity. PMID:25451576

  6. Relevance of in vitro and clinical data for predicting CYP3A4-mediated herb-drug interactions in cancer patients.

    PubMed

    Goey, Andrew K L; Mooiman, Kim D; Beijnen, Jos H; Schellens, Jan H M; Meijerman, Irma

    2013-11-01

    The use of complementary and alternative medicines (CAM) by cancer patients is increasing. Concomitant use of CAM and anticancer drugs could lead to serious safety issues in patients. CAM have the potential to cause pharmacokinetic interactions with anticancer drugs, leading to either increased or decreased plasma levels of anticancer drugs. This could result in unexpected toxicities or a reduced efficacy. Significant pharmacokinetic interactions have already been shown between St. John's Wort (SJW) and the anticancer drugs imatinib and irinotecan. Most pharmacokinetic CAM-drug interactions, involve drug metabolizing cytochrome P450 (CYP) enzymes, in particular CYP3A4. The effect of CAM on CYP3A4 activity and expression can be assessed in vitro. However, no data have been reported yet regarding the relevance of these in vitro data for the prediction of CAM-anticancer drug interactions in clinical practice. To address this issue, a literature research was performed to evaluate the relevance of in vitro data to predict clinical effects of CAM frequently used by cancer patients: SJW, milk thistle, garlic and Panax ginseng (P. ginseng). Furthermore, in clinical studies the sensitive CYP3A4 substrate probe midazolam is often used to determine pharmacokinetic interactions. Results of these clinical studies with midazolam are used to predict pharmacokinetic interactions with other drugs metabolized by CYP3A4. Therefore, this review also explored whether clinical trials with midazolam are useful to predict clinical pharmacokinetic CAM-anticancer drug interactions. In vitro data of SJW have shown CYP3A4 inhibition after short-term exposure and induction after long-term exposure. In clinical studies using midazolam or anticancer drugs (irinotecan and imatinib) as known CYP3A4 substrates in combination with SJW, decreased plasma levels of these drugs were observed, which was expected as a consequence of CYP3A4 induction. For garlic, no effect on CYP3A4 has been shown in vitro

  7. Lack of effect of brivanib on the pharmacokinetics of midazolam, a CYP3A4 substrate, administered intravenously and orally in healthy participants.

    PubMed

    Syed, Shariq; Clemens, Pamela L; Lathers, Deanne; Kollia, Georgia; Dhar, Arindam; Walters, Ian; Masson, Eric

    2012-06-01

    Brivanib alaninate is the orally available prodrug of brivanib, a dual inhibitor of fibroblast growth factor and vascular endothelial growth factor signaling pathways that is under therapeutic investigation for various malignancies. Brivanib alaninate inhibits CYP3A4 in vitro, and thus there is potential for drug-drug interaction with CYP3A4 substrates, such as midazolam. The present study evaluated pharmacokinetic parameters and safety/tolerability upon coadministration of brivanib alaninate and midazolam. Healthy participants received intravenous (IV) or oral midazolam with and without oral brivanib alaninate. Blood samples for pharmacokinetic analysis were collected up to 12 hours after midazolam and up to 48 hours after brivanib alaninate. Twenty-four participants were administered study drugs; 21 completed the trial. No clinically relevant effect of brivanib alaninate on the overall exposure to midazolam following IV or oral administration was observed. Orally administered brivanib alaninate was generally well tolerated in the presence of IV or oral midazolam. The lack of a pharmacokinetic interaction between brivanib and midazolam indicates that brivanib alaninate does not influence either intestinal or hepatic CYP3A4 and confirms that brivanib alaninate may be safely coadministered with midazolam and other CYP3A4 substrates. PMID:21659627

  8. Evaluation of calibration curve-based approaches to predict clinical inducers and noninducers of CYP3A4 with plated human hepatocytes.

    PubMed

    Zhang, J George; Ho, Thuy; Callendrello, Alanna L; Clark, Robert J; Santone, Elizabeth A; Kinsman, Sarah; Xiao, Deqing; Fox, Lisa G; Einolf, Heidi J; Stresser, David M

    2014-09-01

    Cytochrome P450 (P450) induction is often considered a liability in drug development. Using calibration curve-based approaches, we assessed the induction parameters R3 (a term indicating the amount of P450 induction in the liver, expressed as a ratio between 0 and 1), relative induction score, Cmax/EC50, and area under the curve (AUC)/F2 (the concentration causing 2-fold increase from baseline of the dose-response curve), derived from concentration-response curves of CYP3A4 mRNA and enzyme activity data in vitro, as predictors of CYP3A4 induction potential in vivo. Plated cryopreserved human hepatocytes from three donors were treated with 20 test compounds, including several clinical inducers and noninducers of CYP3A4. After the 2-day treatment, CYP3A4 mRNA levels and testosterone 6β-hydroxylase activity were determined by real-time reverse transcription polymerase chain reaction and liquid chromatography-tandem mass spectrometry analysis, respectively. Our results demonstrated a strong and predictive relationship between the extent of midazolam AUC change in humans and the various parameters calculated from both CYP3A4 mRNA and enzyme activity. The relationships exhibited with non-midazolam in vivo probes, in aggregate, were unsatisfactory. In general, the models yielded better fits when unbound rather than total plasma Cmax was used to calculate the induction parameters, as evidenced by higher R(2) and lower root mean square error (RMSE) and geometric mean fold error. With midazolam, the R3 cut-off value of 0.9, as suggested by US Food and Drug Administration guidance, effectively categorized strong inducers but was less effective in classifying midrange or weak inducers. This study supports the use of calibration curves generated from in vitro mRNA induction response curves to predict CYP3A4 induction potential in human. With the caveat that most compounds evaluated here were not strong inhibitors of enzyme activity, testosterone 6β-hydroxylase activity was

  9. The use of isomeric testosterone dimers to explore allosteric effects in substrate binding to cytochrome P450 CYP3A4.

    PubMed

    Denisov, Ilia G; Mak, Piotr J; Grinkova, Yelena V; Bastien, Dominic; Bérubé, Gervais; Sligar, Stephen G; Kincaid, James R

    2016-05-01

    Cytochrome P450 CYP3A4 is the main drug-metabolizing enzyme in the human liver, being responsible for oxidation of 50% of all pharmaceuticals metabolized by human P450 enzymes. Possessing a large substrate binding pocket, it can simultaneously bind several substrate molecules and often exhibits a complex pattern of drug-drug interactions. In order to better understand structural and functional aspects of binding of multiple substrate molecules to CYP3A4 we used resonance Raman and UV-VIS spectroscopy to document the effects of binding of synthetic testosterone dimers of different configurations, cis-TST2 and trans-TST2. We directly demonstrate that the binding of two steroid molecules, which can assume multiple possible configurations inside the substrate binding pocket of monomeric CYP3A4, can lead to active site structural changes that affect functional properties. Using resonance Raman spectroscopy, we have documented perturbations in the ferric and Fe-CO states by these substrates, and compared these results with effects caused by binding of monomeric TST. While the binding of trans-TST2 yields results similar to those obtained with monomeric TST, the binding of cis-TST2 is much tighter and results in significantly more pronounced conformational changes of the porphyrin side chains and Fe-CO unit. In addition, binding of an additional monomeric TST molecule in the remote allosteric site significantly improves binding affinity and the overall spin shift for CYP3A4 with trans-TST2 dimer bound inside the substrate binding pocket. This result provides the first direct evidence for an allosteric effect of the peripheral binding site at the protein-membrane interface on the functional properties of CYP3A4. PMID:26774838

  10. Role of cytochrome B5 in modulating peroxide-supported cyp3a4 activity: evidence for a conformational transition and cytochrome P450 heterogeneity.

    PubMed

    Kumar, Santosh; Davydov, Dmitri R; Halpert, James R

    2005-08-01

    The role of cytochrome b(5) (b(5)) in the alpha-naphthoflavone (alpha-NF)-mediated inhibition of H(2)O(2)-supported 7-benzyloxyquinoline (7-BQ) debenzylation by heterologously expressed and purified cytochrome P450 3A4 (CYP3A4) was studied. Although alpha-NF showed negligible effect in an NADPH-dependent reconstituted system, inhibition of 7-BQ oxidation was observed in the H(2)O(2) system. Analysis of the effect of various constituents of a standard reconstituted system on H(2)O(2)-supported activity showed that b(5) alone resulted in a 2.5-fold increase in the k(cat) value and reversed the inhibitory effect of alpha-NF. In addition, titration with b(5) suggested that only 65% of the CYP3A4 participated in the interaction with b(5), consistent with cytochrome P450 (P450) heterogeneity. Study of the influence of b(5) on the kinetics of H(2)O(2)-dependent destruction of the P450 heme moiety suggested two distinct conformers of CYP3A4 with different sensitivity to heme loss. In the absence of b(5), 66% of the wild-type enzyme was bleached in the fast phase, whereas the addition of b(5) decreased the fraction of the fast phase to 16%. Finally, to locate amino acid residues that might influence b(5) action, several active site mutants were tested. Substitution of Ser-119, Ile-301, Ala-305, Ile-369, or Ala-370 with the larger Phe or Trp decreased or even abolished the activation by b(5). Ser-119 is in the B'-C loop, a predicted b(5)-P450 interaction site, and Ile-301 and Ala-305 are closest to the heme. In conclusion, the interaction of b(5) with P450 apparently leads to a conformational transition, which results in redistribution of the CYP3A4 pool. PMID:15870379

  11. 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

    PubMed Central

    Liu, Cheng-Ling; Lim, Yun-Ping; Hu, Miao-Lin

    2012-01-01

    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. PMID:22363234

  12. Effects of Radix Astragali and Radix Rehmanniae, the components of an anti-diabetic foot ulcer herbal formula, on metabolism of model CYP1A2, CYP2C9, CYP2D6, CYP2E1 and CYP3A4 probe substrates in pooled human liver microsomes and specific CYP isoforms.

    PubMed

    Or, Penelope M Y; Lam, Francis F Y; Kwan, Y W; Cho, C H; Lau, C P; Yu, H; Lin, G; Lau, Clara B S; Fung, K P; Leung, P C; Yeung, John H K

    2012-04-15

    The present study investigated the effects of Radix Astragali (RA) and Radix Rehmanniae (RR), the major components of an anti-diabetic foot ulcer herbal formula (NF3), on the metabolism of model probe substrates of human CYP isoforms, CYP1A2, CYP2C9, CYP2D6, CYP2E1 and CYP3A4, which are important in the metabolism of a variety of xenobiotics. The effects of RA or RR on human CYP1A2 (phenacetin O-deethylase), CYP2C9 (tolbutamide 4-hydroxylase), CYP2D6 (dextromethorphan O-demethylase), CYP2E1 (chlorzoxazone 6-hydroxylase) and CYP3A4 (testosterone 6β-hydroxylase) activities were investigated using pooled human liver microsomes. NF3 competitively inhibited activities of CYP2C9 (IC(50)=0.98mg/ml) and CYP3A4 (IC(50)=0.76mg/ml), with K(i) of 0.67 and 1.0mg/ml, respectively. With specific human CYP2C9 and CYP3A4 isoforms, NF3 competitively inhibited activities of CYP2C9 (IC(50)=0.86mg/ml) and CYP3A4 (IC(50)=0.88mg/ml), with K(i) of 0.57 and 1.6mg/ml, respectively. Studies on RA or RR individually showed that RR was more important in the metabolic interaction with the model CYP probe substrates. RR dose-dependently inhibited the testosterone 6β-hydroxylation (K(i)=0.33mg/ml) while RA showed only minimal metabolic interaction potential with the model CYP probe substrates studied. This study showed that RR and the NF3 formula are metabolized mainly by CYP2C9 and/or CYP3A4, but weakly by CYP1A2, CYP2D6 and CYP2E1. The relatively high K(i) values of NF3 (for CYP2C9 and CYP3A4 metabolism) and RR (for CYP3A4 metabolism) would suggest a low potential for NF3 to cause herb-drug interaction involving these CYP isoforms.

  13. Sex differences in the clearance of CYP3A4 substrates: exploring possible reasons for the substrate dependency and lack of consensus.

    PubMed

    Chetty, Manoranjenni; Mattison, Donald; Rostami-Hodjegan, Amin

    2012-07-01

    Sex differences in the clearance of substrates of Cytochrome P4503A (CYP3A4) have been reported frequently although there has been no consensus on reasons for variation in observations amongst drugs which are seemingly all dependent on this enzyme for their metabolism. Moreover, these observations could not be replicated in all studies even when investigating the same drugs. Differing study designs and inadequate power to identify the sex differences may explain the conflicting reports. The aim of the current study was to use in vitro data on a number of CYP3A4 substrates to develop mechanistic population pharmacokinetic models which are capable of integrating various attributes of drugs and estimating the statistical power of in vivo studies designed to discern sex differences in the clearance of CYP3A4 substrates. Midazolam, triazolam, alprazolam, nifedipine and zolpidem were selected as test substrates. These compounds are predominantly metabolized by CYP3A4, unaffected by p-glycoprotein and have abundant clinical studies which can be used for validation purposes. Simulated apparent clearance, obtained by use of the Simcyp® Population-based Simulator and in vitro in vivo extrapolation (IVIVE) techniques, was compared in males and females after correcting for weight (CL/wt) in 1560 trials. Results suggested that about 105 subjects per study are required for an 80% probability of identifying a higher CL/wt in females with alprazolam, while the corresponding numbers for a similar power were 120, about 150 and 300 for nifedipine, triazolam and oral midazolam, respectively. The results were consistent with outcomes in published clinical studies and support the view that many of the published studies have inadequate power to detect these sex differences in drug clearance, thereby contributing to the lack of consensus on this subject.

  14. Investigation of drug-drug interactions caused by human pregnane X receptor-mediated induction of CYP3A4 and CYP2C subfamilies in chimeric mice with a humanized liver.

    PubMed

    Hasegawa, Maki; Tahara, Harunobu; Inoue, Ryo; Kakuni, Masakazu; Tateno, Chise; Ushiki, Junko

    2012-03-01

    The induction of cytochrome P450 (P450) enzymes is one of the risk factors for drug-drug interactions (DDIs). To date, the human pregnane X receptor (PXR)-mediated CYP3A4 induction has been well studied. In addition to CYP3A4, the expression of CYP2C subfamily is also regulated by PXR, and the DDIs caused by the induction of CYP2C enzymes have been reported to have a major clinical impact. The purpose of the present study was to investigate whether chimeric mice with a humanized liver (PXB mice) can be a suitable animal model for investigating the PXR-mediated induction of CYP2C subfamily, together with CYP3A4. We evaluated the inductive effect of rifampicin (RIF), a typical human PXR ligand, on the plasma exposure to the four P450 substrate drugs (triazolam/CYP3A4, pioglitazone/CYP2C8, (S)-warfarin/CYP2C9, and (S)-(-)-mephenytoin/CYP2C19) by cassette dosing in PXB mice. The induction of several drug-metabolizing enzymes and transporters in the liver was also examined by measuring the enzyme activity and mRNA expression levels. Significant reductions in the exposure to triazolam, pioglitazone, and (S)-(-)-mephenytoin, but not to (S)-warfarin, were observed. In contrast to the in vivo results, all the four P450 isoforms, including CYP2C9, were elevated by RIF treatment. The discrepancy in the (S)-warfarin results between in vivo and in vitro studies may be attributed to the relatively small contribution of CYP2C9 to (S)-warfarin elimination in the PXB mice used in this study. In summary, PXB mice are a useful animal model to examine DDIs caused by PXR-mediated induction of CYP2C and CYP3A4. PMID:22126990

  15. Towards a Best Practice Approach in PBPK Modeling: Case Example of Developing a Unified Efavirenz Model Accounting for Induction of CYPs 3A4 and 2B6.

    PubMed

    Ke, A; Barter, Z; Rowland-Yeo, K; Almond, L

    2016-07-01

    In this study, we present efavirenz physiologically based pharmacokinetic (PBPK) model development as an example of our best practice approach that uses a stepwise approach to verify the different components of the model. First, a PBPK model for efavirenz incorporating in vitro and clinical pharmacokinetic (PK) data was developed to predict exposure following multiple dosing (600 mg q.d.). Alfentanil i.v. and p.o. drug-drug interaction (DDI) studies were utilized to evaluate and refine the CYP3A4 induction component in the liver and gut. Next, independent DDI studies with substrates of CYP3A4 (maraviroc, atazanavir, and clarithromycin) and CYP2B6 (bupropion) verified the induction components of the model (area under the curve [AUC] ratios within 1.0-1.7-fold of observed). Finally, the model was refined to incorporate the fractional contribution of enzymes, including CYP2B6, propagating autoinduction into the model (Racc 1.7 vs. 1.7 observed). This validated mechanistic model can now be applied in clinical pharmacology studies to prospectively assess both the victim and perpetrator DDI potential of efavirenz. PMID:27435752

  16. Towards a Best Practice Approach in PBPK Modeling: Case Example of Developing a Unified Efavirenz Model Accounting for Induction of CYPs 3A4 and 2B6

    PubMed Central

    Ke, A; Barter, Z; Rowland‐Yeo, K

    2016-01-01

    In this study, we present efavirenz physiologically based pharmacokinetic (PBPK) model development as an example of our best practice approach that uses a stepwise approach to verify the different components of the model. First, a PBPK model for efavirenz incorporating in vitro and clinical pharmacokinetic (PK) data was developed to predict exposure following multiple dosing (600 mg q.d.). Alfentanil i.v. and p.o. drug‐drug interaction (DDI) studies were utilized to evaluate and refine the CYP3A4 induction component in the liver and gut. Next, independent DDI studies with substrates of CYP3A4 (maraviroc, atazanavir, and clarithromycin) and CYP2B6 (bupropion) verified the induction components of the model (area under the curve [AUC] ratios within 1.0–1.7‐fold of observed). Finally, the model was refined to incorporate the fractional contribution of enzymes, including CYP2B6, propagating autoinduction into the model (Racc 1.7 vs. 1.7 observed). This validated mechanistic model can now be applied in clinical pharmacology studies to prospectively assess both the victim and perpetrator DDI potential of efavirenz. PMID:27435752

  17. Towards a Best Practice Approach in PBPK Modeling: Case Example of Developing a Unified Efavirenz Model Accounting for Induction of CYPs 3A4 and 2B6.

    PubMed

    Ke, A; Barter, Z; Rowland-Yeo, K; Almond, L

    2016-07-01

    In this study, we present efavirenz physiologically based pharmacokinetic (PBPK) model development as an example of our best practice approach that uses a stepwise approach to verify the different components of the model. First, a PBPK model for efavirenz incorporating in vitro and clinical pharmacokinetic (PK) data was developed to predict exposure following multiple dosing (600 mg q.d.). Alfentanil i.v. and p.o. drug-drug interaction (DDI) studies were utilized to evaluate and refine the CYP3A4 induction component in the liver and gut. Next, independent DDI studies with substrates of CYP3A4 (maraviroc, atazanavir, and clarithromycin) and CYP2B6 (bupropion) verified the induction components of the model (area under the curve [AUC] ratios within 1.0-1.7-fold of observed). Finally, the model was refined to incorporate the fractional contribution of enzymes, including CYP2B6, propagating autoinduction into the model (Racc 1.7 vs. 1.7 observed). This validated mechanistic model can now be applied in clinical pharmacology studies to prospectively assess both the victim and perpetrator DDI potential of efavirenz.

  18. Sulforaphane- and phenethyl isothiocyanate-induced inhibition of aflatoxin B1-mediated genotoxicity in human hepatocytes: role of GSTM1 genotype and CYP3A4 gene expression.

    PubMed

    Gross-Steinmeyer, Kerstin; Stapleton, Patricia L; Tracy, Julia H; Bammler, Theo K; Strom, Stephen C; Eaton, David L

    2010-08-01

    Primary cultures of human hepatocytes were used to investigate whether the dietary isothiocyanates, sulforaphane (SFN), and phenethyl isothiocyanate (PEITC) can reduce DNA adduct formation of the hepatocarcinogen aflatoxin B(1) (AFB). Following 48 h of pretreatment, 10 and 50 microM SFN greatly decreased AFB-DNA adduct levels, whereas 25muM PEITC decreased AFB-DNA adducts in some but not all hepatocyte preparations. Microarray and quantitative reverse transcriptase (RT)-PCR analyses of gene expression in SFN and PEITC-treated hepatocytes demonstrated that SFN greatly decreased cytochrome P450 (CYP) 3A4 mRNA but did not induce the expression of either glutathione S-transferase (GST) M1 or GSTT1. The protective effects of SFN required pretreatment; cotreatment of hepatocytes with SFN and AFB in the absence of pretreatment had no effect on AFB-DNA adduct formation. When AFB-DNA adduct formation was evaluated by GST genotype, the presence of one or two functional alleles of GSTM1 was associated with a 75% reduction in AFB-DNA adducts, compared with GSTM1 null. In conclusion, these results demonstrate that the inhibition of AFB-DNA adduct formation by SFN is dependent on changes in gene expression rather than direct inhibition of catalytic activity. Transcriptional repression of genes involved in AFB bioactivation (CYP3A4 and CYP1A2), but not transcriptional activation of GSTs, may be responsible for the protective effects of SFN. Although GSTM1 expression was not induced by SFN, the presence of a functional GSTM1 allele can afford substantial protection against AFB-DNA damage in human liver. The downregulation of CYP3A4 by SFN may have important implications for drug interactions. PMID:20442190

  19. In Silico Predictions of Drug - Drug Interactions Caused by CYP1A2, 2C9 and 3A4 Inhibition - a Comparative Study of Virtual Screening Performance.

    PubMed

    Kaserer, Teresa; Höferl, Martina; Müller, Klara; Elmer, Sebastian; Ganzera, Markus; Jäger, Walter; Schuster, Daniela

    2015-06-01

    The cytochrome P450 (CYP) superfamily represents the major enzyme class responsible for the metabolism of exogenous compounds. Investigation of clearance pathways is therefore an integral part in early drug development, as any alteration of metabolic enzymes may markedly influence the toxicological profile and efficacy of novel compounds. In silico methods are widely applied in drug development to complement experimental approaches. Several different tools are available for that purpose, however, for CYP enzymes they have only been applied retrospectively so far. Within this study, pharmacophore- and shape-based models and a docking protocol were generated for the prediction of CYP1A2, 2C9, and 3A4 inhibition. All theoretically validated models, the validated docking workflow, and additional external bioactivity profiling tools were applied independently and in parallel to predict the CYP inhibition of 29 compounds from synthetic and natural origin. After subsequent experimental assessment of the in silico predictions, we analyzed and compared the prospective performance of all methods, thereby defining the suitability of the applied techniques for CYP enzymes. We observed quite substantial differences in the performances of the applied tools, suggesting that the rational selection of that virtual screening method that proved to perform best can largely improve the success rates when it comes to CYP inhibition prediction. PMID:27490388

  20. Homotropic cooperativity of monomeric cytochrome P450 3A4

    SciTech Connect

    Baas, Bradley J.; Denisov, Ilia G.; Sligar, Stephen G.

    2010-11-16

    Mechanistic studies of mammalian cytochrome P450s are often obscured by the phase heterogeneity of solubilized preparations of membrane enzymes. The various protein-protein aggregation states of microsomes, detergent solubilized cytochrome or a family of aqueous multimeric complexes can effect measured substrate binding events as well as subsequent steps in the reaction cycle. In addition, these P450 monooxygenases are normally found in a membrane environment and the bilayer composition and dynamics can also effect these catalytic steps. Here, we describe the structural and functional characterization of a homogeneous monomeric population of cytochrome P450 3A4 (CYP 3A4) in a soluble nanoscale membrane bilayer, or Nanodisc [Nano Lett. 2 (2002) 853]. Cytochrome P450 3A4:Nanodisc assemblies were formed and purified to yield a 1:1 ratio of CYP 3A4 to Nanodisc. Solution small angle X-ray scattering was used to structurally characterize this monomeric CYP 3A4 in the membrane bilayer. The purified CYP 3A4:Nanodiscs showed a heretofore undescribed high level of homotropic cooperativity in the binding of testosterone. Soluble CYP 3A4:Nanodisc retains its known function and shows prototypic hydroxylation of testosterone when driven by hydrogen peroxide. This represents the first functional characterization of a true monomeric preparation of cytochrome P450 monooxygenase in a phospholipid bilayer and elucidates new properties of the monomeric form.

  1. A food contaminant ochratoxin A suppresses pregnane X receptor (PXR)-mediated CYP3A4 induction in primary cultures of human hepatocytes.

    PubMed

    Doricakova, Aneta; Vrzal, Radim

    2015-11-01

    Ochratoxin A (OCHA) is a mycotoxin, which can be found in food such as coffee, wine, cereals, meat, nuts. Since it is absorbed via gastrointestinal tract, it is reasonable to anticipate that the liver will be the first organ to which OCHA comes into the contact before systemic circulation. Many xenobiotics are metabolically modified after the passage of the liver to biologically more active substances, sometimes with more harmful activity. Promoting own metabolism is often achieved via transcriptional regulation of biotransformation enzymes through ligand-activated transcription factors. Pregnane X receptor (PXR) belongs to such a group of regulators and it was demonstrated to be activated by many compounds of synthetic as well as natural origin. Our intention was to investigate if OCHA is capable of activating the PXR with consequent induction of PXR-regulated CYP3A4 gene. We found that OCHA does not activate PXR but displays antagonist-like behavior when combined with rifampicin (RIF) in gene reporter assay in human embryonal kidney cells (Hek293T). It was very weak inducer of CYP3A4 mRNA in primary cultures of human hepatocytes and it antagonized RIF-mediated CYP3A4 induction of mRNA as well as protein. In addition, it caused the decline of PXR protein as well as mRNA which was faster than that with actinomycin D, a transcription inhibitor. Since we found that OCHA induced the expression of miR-148a, which was described to regulate PXR expression, we conclude that antagonist-like behavior of OCHA is not due to the antagonism itself but due to the downregulation of PXR gene expression. Herein we provide important findings which bring a piece of puzzle into the understanding of mechanism of toxic action of ochratoxin A.

  2. A food contaminant ochratoxin A suppresses pregnane X receptor (PXR)-mediated CYP3A4 induction in primary cultures of human hepatocytes.

    PubMed

    Doricakova, Aneta; Vrzal, Radim

    2015-11-01

    Ochratoxin A (OCHA) is a mycotoxin, which can be found in food such as coffee, wine, cereals, meat, nuts. Since it is absorbed via gastrointestinal tract, it is reasonable to anticipate that the liver will be the first organ to which OCHA comes into the contact before systemic circulation. Many xenobiotics are metabolically modified after the passage of the liver to biologically more active substances, sometimes with more harmful activity. Promoting own metabolism is often achieved via transcriptional regulation of biotransformation enzymes through ligand-activated transcription factors. Pregnane X receptor (PXR) belongs to such a group of regulators and it was demonstrated to be activated by many compounds of synthetic as well as natural origin. Our intention was to investigate if OCHA is capable of activating the PXR with consequent induction of PXR-regulated CYP3A4 gene. We found that OCHA does not activate PXR but displays antagonist-like behavior when combined with rifampicin (RIF) in gene reporter assay in human embryonal kidney cells (Hek293T). It was very weak inducer of CYP3A4 mRNA in primary cultures of human hepatocytes and it antagonized RIF-mediated CYP3A4 induction of mRNA as well as protein. In addition, it caused the decline of PXR protein as well as mRNA which was faster than that with actinomycin D, a transcription inhibitor. Since we found that OCHA induced the expression of miR-148a, which was described to regulate PXR expression, we conclude that antagonist-like behavior of OCHA is not due to the antagonism itself but due to the downregulation of PXR gene expression. Herein we provide important findings which bring a piece of puzzle into the understanding of mechanism of toxic action of ochratoxin A. PMID:26341324

  3. Stereoselective Metabolism of α-, β-, and γ-Hexabromocyclododecanes (HBCDs) by Human Liver Microsomes and CYP3A4.

    PubMed

    Erratico, Claudio; Zheng, Xiaobo; van den Eede, Nele; Tomy, Gregg; Covaci, Adrian

    2016-08-01

    This is the first study investigating the in vitro metabolism of α-, β-, and γ-hexabromocyclododecane (HBCD) stereoisomers in humans and providing semiquantitative metabolism data. Human liver microsomes were incubated with individual racemic mixtures and with individual stereoisomers of α-, β-, and γ-HBCDs, the hydroxylated metabolites formed were analyzed by liquid chromatography-tandem mass spectrometry, and the value of the intrinsic in vitro clearance (Clint,vitro) was calculated. Several mono- and dihydroxylated metabolites of α-, β-, and γ-HBCDs were formed, with mono-OH-HBCDs being the major metabolites. No stereoisomerization of any of the six α-, β-, and γ-HBCD isomers catalyzed by cytochrome P450 (CYP) enzymes occurred. The value of Clint,vitro of α-HBCDs was significantly lower than that of β-HBCDs, which, in turn, was significantly lower than that of γ-HBCDs (p < 0.05). Such differences were explained by the significantly lower values of Clint,vitro of each α-HBCD stereoisomer than those of the β- and γ-HBCD stereoisomers. In addition, significantly lower values of Clint,vitro of the (-) over the (+)α- and β-HBCD stereoisomers, but not γ-HBCDs, were obtained. Our data offer a possible explanation of the enrichment of α-HBCDs over β- and γ-HBCDs on the one hand and, on the other hand, of (-)α-HBCDs over (+)α-HBCDs previously reported in human samples. It also offers information about the mechanism resulting in such enrichments, the stereoisomer-selective metabolism of α-, β-, and γ-HBCDs catalyzed by CYPs with the lack of stereoisomerization.

  4. Effects of strong CYP2D6 and 3A4 inhibitors, paroxetine and ketoconazole, on the pharmacokinetics and cardiovascular safety of tamsulosin

    PubMed Central

    Troost, Joachim; Tatami, Shinji; Tsuda, Yasuhiro; Mattheus, Michaela; Mehlburger, Ludwig; Wein, Martina; Michel, Martin C

    2011-01-01

    AIM To determine the effect of the strong CYP2D6 inhibitor paroxetine and strong CYP3A4 inhibitor ketoconazole on the pharmacokinetics and safety (orthostatic challenge) of tamsulosin. METHODS Two open-label, randomized, two-way crossover studies were conducted in healthy male volunteers (extensive CYP2D6 metabolizers). RESULTS Co-administration of multiple oral doses of 20 mg paroxetine once daily with a single oral dose of the 0.4 mg tamsulosin HCl capsule increased the adjusted geometric mean (gMean) values of Cmax and AUC(0,∞) of tamsulosin by factors of 1.34 (90% CI 1.21, 1.49) and 1.64 (90% CI 1.44, 1.85), respectively, and increased the terminal half-life (t1/2) of tamsulosin HCl from 11.4 h to 15.3 h. Co-administration of multiple oral doses of 400 mg ketoconazole once dailywith a single oral dose of the 0.4 mg tamsulosin increased the gMean values of Cmax and AUC(0,∞) of tamsulosin by a factor of 2.20 (90% CI 1.96, 2.45) and 2.80 (90% CI 2.56, 3.07), respectively. The terminal half-life was slightly increased from 10.5 h to 11.8 h. These pharmacokinetic changes were not accompanied by clinically significant alterations of haemodynamic responses during orthostatic stress testing. CONCLUSION The exposure to tamsulosin is increased upon co-administration of strong CYP2D6 inhibitors and even more so of strong 3A4 inhibitors, but neither PK alteration was accompanied by clinically significant haemodynamic changes during orthostatic stress testing. PMID:21496064

  5. Identification of rifampin-inducible P450IIIA4 (CYP3A4) in human small bowel enterocytes.

    PubMed Central

    Kolars, J C; Schmiedlin-Ren, P; Schuetz, J D; Fang, C; Watkins, P B

    1992-01-01

    Enzymes within the P450IIIA (CYP3A) subfamily appear to account for significant "first pass" metabolism of some drugs in the intestine. To identify which of the known P450IIIA genes are expressed in intestine, enterocyte RNA was hybridized on Northern blots with synthetic oligonucleotides complementary to hypervariable regions of hepatic P450IIIA4, P450IIIA5, and P450IIIA7 cDNAs. Hybridization was detected only with the P450IIIA4-specific oligonucleotide. The identity of the hybridizing mRNA was confirmed to be P450IIIA4 by direct sequencing of a DNA fragment amplified from enterocyte cDNA by the polymerase chain reaction. To determine if enterocyte P450IIIA4 is inducible, biopsies of small bowel mucosa were obtained from five volunteers before and after they received 7d of treatment with rifampin, a known inducer of P450IIIA4 in liver. Rifampin treatment resulted in a five- or eightfold mean increase (P < 0.05) in the biopsy concentration of P450IIIA4 mRNA when normalized for content of sucrase isomaltase or intestinal fatty acid binding protein mRNAs, respectively. Rifampin also induced P450IIIA immunoreactive protein in enterocytes in each of the subjects, as judged by immunohistochemistry, and resulted in a 10-fold increase in P450IIIA4-specific catalytic activity (erythromycin N-demethylation) in the one patient studied. Our identification of inducible P450IIIA4 in enterocytes may in part account for drug interactions characteristic of P450IIIA4 substrates and suggests a strategy for controlling entry into the body of a major class of xenobiotics. Images PMID:1430211

  6. Predicting the "First dose in children" of CYP3A-metabolized drugs: Evaluation of scaling approaches and insights into the CYP3A7-CYP3A4 switch at young ages.

    PubMed

    Strougo, Ashley; Yassen, Ashraf; Monnereau, Claire; Danhof, Meindert; Freijer, Jan

    2014-09-01

    First-dose-in-children relies on the prediction of clearance from adults for which little information is available on the accuracy of the scaling-approaches applied. For CYP3A-metabolized compounds, scaling of clearance is further challenged by different isoforms and by the CYP3A7 to CYP3A4 switch at young ages. This investigation aimed to evaluate the accuracy of two frequently used scaling approaches and to gain insights into the ontogeny of CYP3A. Hence, a literature database was compiled containing 203 clearance values from term-neonates to adults for 18 CYP3A-metabolized compounds. The clearances in adults were scaled to children using (i) allometric scaling plus maturation function and (ii) a mechanistic approach based on the well-stirred model. Three maturation functions were separately evaluated. In children >3 months, all approaches were interchangeable heeding the maturation function applied and biases were mostly observed in children <3 months. The results from a sensitivity analysis indicate that these biases are possibly caused by disregarding the CYP3A7 activity which could account for up to 86% of the metabolism in term-neonates. Only the mechanistic approach using an overall-CYP3A maturation function led to unbiased predictions of clearances across all ages. The current investigation adds to the predictions of the first-dose-in-children of compounds (partially) metabolized by CYP3A.

  7. Polymorphism of CYP3A4 and ABCB1 genes increase the risk of neuropathy in breast cancer patients treated with paclitaxel and docetaxel

    PubMed Central

    Kus, Tulay; Aktas, Gokmen; Kalender, Mehmet Emin; Demiryurek, Abdullah Tuncay; Ulasli, Mustafa; Oztuzcu, Serdar; Sevinc, Alper; Kul, Seval; Camci, Celaletdin

    2016-01-01

    Background Interindividual variability of pharmacogenetics may account for unpredictable neurotoxicities of taxanes. Methods From March 2011 to June 2015, female patients with operable breast cancer who had received docetaxel- or paclitaxel-containing adjuvant chemotherapy were included in this study. All patients were treated with single-agent paclitaxel intravenously (IV) 175 mg/m2 every 3 weeks for four cycles, or IV 80 mg/m2 weekly for 12 cycles, and IV 100 mg/m2 docetaxel for four cycles as adjuvant treatment. We evaluated the relationship between neurotoxicity of taxanes and single-nucleotide polymorphisms of ABCB1, CYP3A4, ERCC1, ERCC2, FGFR4, TP53, ERBB2, and CYP2C8 genes. Taxane-induced neurotoxicity during the treatment was evaluated according to the National Cancer Institute Common Toxicity Criteria version 4.03 prior to each cycle. Chi-squared tests were used to compare the two groups, and multivariate binary logistic regression models were used for determining possible risk factors of neuropathy. Results Pharmacogenetic analysis was performed in 219 females. ABCB1 3435 TT genotype had significantly higher risk for grade ≥2 neurotoxicity (odds ratio [OR]: 2.759, 95% confidence interval [CI]: 1.172–6.493, P: 0.017) compared to TC and CC genotype, and also CYP3A4 392 AA and AG genotype had significantly higher risk for grade ≥2 neurotoxicity (OR: 2.259, 95% CI: 1.033–4.941, P: 0.038) compared to GG genotype. For FDGF4 gene with AG and GG genotype, OR was 1.879 (95% CI: 1.001–3.525, P: 0.048) compared to AA genotype with regard to any grade of neuropathy risk. We could not find any other association of other genotypes with neurotoxicity grades. Conclusion ABCB1 3435 TT genotype and CYP3A4 392 AA/AG genotypes may be used as predictors of neurotoxicity during taxane chemotherapy. PMID:27574448

  8. Unexpected contribution of cytochrome P450 enzymes CYP11B2 and CYP21, as well as CYP3A4 in xenobiotic androgen elimination - insights from metandienone metabolism.

    PubMed

    Parr, Maria Kristina; Zöllner, Andy; Fusshöller, Gregor; Opfermann, Georg; Schlörer, Nils; Zorio, Mirela; Bureik, Matthias; Schänzer, Wilhelm

    2012-09-18

    The metabolism of a variety of anabolic steroids frequently misused for doping purposes has been investigated in the last years. This research mainly focused on main and long-term metabolites suitable for detection, but detailed clearance mechanisms have rarely been elucidated. Recent studies on metandienone focused on the identification of 17β-hydroxymethyl-17α-methyl-18-norandrosta-1,4,13-trien-3-one (20βOH-NorMD) as long-term metabolite, however, the metabolic pathway of its generation remained unclear. Metandienone and its Wagner-Meerwein rearrangement product 17,17-dimethyl-18-norandrosta-1,4,13-trien-3-one (NorMD) were hydroxylated by different human cytochrome P450 enzymes (CYPs). Some of their hydroxylation products were chemically synthesized and characterized by mass spectrometry to allow for their trace detection in urine samples. Following oral administration of metandienone or NorMD in one human volunteer each the post administration urines were checked for the presence of those hydroxylated metabolites using GC-MS/MS analysis. The human mitochondrial steroid hydroxylating enzymes CYP11B1 and CYP11B2 were capable to metabolize metandienone leading to the formation of 11β-hydroxymetandienone and 18-hydroxymetandienone. Following Wagner-Meerwein rearrangement, the resulting products could be assigned to 20βOH-NorMD and 11βOH-NorMD. The contribution of CYP11B1 and CYP11B2 in human metabolism of metandienone was confirmed by analysis of post-administration samples of metandienone and NorMD. Combined with the results from a previous study, enzymatic pathways were identified that involve CYP21 and CYP3A4 in the hydroxylation of NorMD, while CYP21, CYP3A4 and CYP11B2 take part in 20βOH-NorMD generation from MD. The current study represents a valuable contribution to the elucidation of clearance mechanisms of anabolic steroids and also indicates that mainly non-liver CYPs seem to be involved in these processes. PMID:22885098

  9. Identification of stable and reactive metabolite(s) of nelfinavir in human liver microsomes and rCYP3A4.

    PubMed

    Jhajra, Shalu; Singh, Saranjit

    2016-01-25

    The present study was performed to detect trace level stable and reactive metabolites of nelfinavir in human liver microsomes and rCYP3A4. Initially, chromatographic and MS parameters were optimized and fragmentation pattern of the drug was delineated. The structures of metabolites were then elucidated by comparison of their MS/MS fragmentation patterns with the drug. A total of thirty nine stable metabolites were formed, of which twelve were established to be monohydroxylated, eighteen dihydroxy, two dehydrogenated, and one each a diquinone, keto, carboxylic, N-deacylated, dealkylated, oxo and dehydro monohydroxyl metabolite. Previously, a biotransformation product with hydroxylation at tert-butyl group of nelfinavir is reported as an active metabolite of the drug. In our case, ortho-diquinone and N-oxide metabolites were detected, which are known to be reactive in nature. However, these metabolites did not show any interaction with nucleophiles, possibly due to steric hindrance at the site of interface.

  10. Identification of nicotinamide phosphoribosyltransferase (NAMPT) inhibitors with no evidence of CYP3A4 time-dependent inhibition and improved aqueous solubility.

    PubMed

    Zak, Mark; Liederer, Bianca M; Sampath, Deepak; Yuen, Po-Wai; Bair, Kenneth W; Baumeister, Timm; Buckmelter, Alexandre J; Clodfelter, Karl H; Cheng, Eric; Crocker, Lisa; Fu, Bang; Han, Bingsong; Li, Guangkun; Ho, Yen-Ching; Lin, Jian; Liu, Xiongcai; Ly, Justin; O'Brien, Thomas; Reynolds, Dominic J; Skelton, Nicholas; Smith, Chase C; Tay, Suzanne; Wang, Weiru; Wang, Zhongguo; Xiao, Yang; Zhang, Lei; Zhao, Guiling; Zheng, Xiaozhang; Dragovich, Peter S

    2015-02-01

    Herein we report the optimization efforts to ameliorate the potent CYP3A4 time-dependent inhibition (TDI) and low aqueous solubility exhibited by a previously identified lead compound from our NAMPT inhibitor program (1, GNE-617). Metabolite identification studies pinpointed the imidazopyridine moiety present in 1 as the likely source of the TDI signal, and replacement with other bicyclic systems was found to reduce or eliminate the TDI finding. A strategy of reducing the number of aromatic rings and/or lowering cLogD7.4 was then employed to significantly improve aqueous solubility. These efforts culminated in the discovery of 42, a compound with no evidence of TDI, improved aqueous solubility, and robust efficacy in tumor xenograft studies.

  11. In vitro Effects of Four Native Brazilian Medicinal Plants in CYP3A4 mRNA Gene Expression, Glutathione Levels, and P-Glycoprotein Activity.

    PubMed

    Mazzari, Andre L D A; Milton, Flora; Frangos, Samantha; Carvalho, Ana C B; Silveira, Dâmaris; de Assis Rocha Neves, Francisco; Prieto, Jose M

    2016-01-01

    Erythrina mulungu Benth. (Fabaceae), Cordia verbenacea A. DC. (Boraginaceae), Solanum paniculatum L. (Solanaceae) and Lippia sidoides Cham. (Verbenaceae) are medicinal plant species native to Brazil shortlisted by the Brazilian National Health System for future clinical use. However, nothing is known about their effects in metabolic and transporter proteins, which could potentially lead to herb-drug interactions (HDI). In this work, we assess non-toxic concentrations (100 μg/mL) of the plant infusions for their in vitro ability to modulate CYP3A4 mRNA gene expression and intracellular glutathione levels in HepG2 cells, as well as P-glycoprotein (P-gp) activity in vincristine-resistant Caco-2 cells (Caco-2 VCR). Their mechanisms of action were further studied by measuring the activation of human pregnane X receptor (hPXR) in transiently co-transfected HeLa cells and the inhibition of γ-glutamyl transferase (GGT) in HepG2 cells. Our results show that P-gp activity was not affected in any case and that only Solanum paniculatum was able to significantly change CYP3A4 mRNA gene expression (twofold decrease, p < 0.05), this being correlated with an antagonist effect upon hPXR (EC50 = 0.38 mg/mL). Total intracellular glutathione levels were significantly depleted by exposure to Solanum paniculatum (-44%, p < 0.001), Lippia sidoides (-12%, p < 0.05) and Cordia verbenacea (-47%, p < 0.001). The latter plant extract was able to decrease GGT activity (-48%, p < 0.01). In conclusion, this preclinical study shows that the administration of some of these herbal medicines may be able to cause disturbances to metabolic mechanisms in vitro. Although Erythrina mulungu appears safe in our tests, active pharmacovigilance is recommended for the other three species, especially in the case of Solanum paniculatum. PMID:27594838

  12. Construction of Metabolism Prediction Models for CYP450 3A4, 2D6, and 2C9 Based on Microsomal Metabolic Reaction System

    PubMed Central

    He, Shuai-Bing; Li, Man-Man; Zhang, Bai-Xia; Ye, Xiao-Tong; Du, Ran-Feng; Wang, Yun; Qiao, Yan-Jiang

    2016-01-01

    During the past decades, there have been continuous attempts in the prediction of metabolism mediated by cytochrome P450s (CYP450s) 3A4, 2D6, and 2C9. However, it has indeed remained a huge challenge to accurately predict the metabolism of xenobiotics mediated by these enzymes. To address this issue, microsomal metabolic reaction system (MMRS)—a novel concept, which integrates information about site of metabolism (SOM) and enzyme—was introduced. By incorporating the use of multiple feature selection (FS) techniques (ChiSquared (CHI), InfoGain (IG), GainRatio (GR), Relief) and hybrid classification procedures (Kstar, Bayes (BN), K-nearest neighbours (IBK), C4.5 decision tree (J48), RandomForest (RF), Support vector machines (SVM), AdaBoostM1, Bagging), metabolism prediction models were established based on metabolism data released by Sheridan et al. Four major biotransformations, including aliphatic C-hydroxylation, aromatic C-hydroxylation, N-dealkylation and O-dealkylation, were involved. For validation, the overall accuracies of all four biotransformations exceeded 0.95. For receiver operating characteristic (ROC) analysis, each of these models gave a significant area under curve (AUC) value >0.98. In addition, an external test was performed based on dataset published previously. As a result, 87.7% of the potential SOMs were correctly identified by our four models. In summary, four MMRS-based models were established, which can be used to predict the metabolism mediated by CYP3A4, 2D6, and 2C9 with high accuracy. PMID:27735849

  13. In vitro Effects of Four Native Brazilian Medicinal Plants in CYP3A4 mRNA Gene Expression, Glutathione Levels, and P-Glycoprotein Activity.

    PubMed

    Mazzari, Andre L D A; Milton, Flora; Frangos, Samantha; Carvalho, Ana C B; Silveira, Dâmaris; de Assis Rocha Neves, Francisco; Prieto, Jose M

    2016-01-01

    Erythrina mulungu Benth. (Fabaceae), Cordia verbenacea A. DC. (Boraginaceae), Solanum paniculatum L. (Solanaceae) and Lippia sidoides Cham. (Verbenaceae) are medicinal plant species native to Brazil shortlisted by the Brazilian National Health System for future clinical use. However, nothing is known about their effects in metabolic and transporter proteins, which could potentially lead to herb-drug interactions (HDI). In this work, we assess non-toxic concentrations (100 μg/mL) of the plant infusions for their in vitro ability to modulate CYP3A4 mRNA gene expression and intracellular glutathione levels in HepG2 cells, as well as P-glycoprotein (P-gp) activity in vincristine-resistant Caco-2 cells (Caco-2 VCR). Their mechanisms of action were further studied by measuring the activation of human pregnane X receptor (hPXR) in transiently co-transfected HeLa cells and the inhibition of γ-glutamyl transferase (GGT) in HepG2 cells. Our results show that P-gp activity was not affected in any case and that only Solanum paniculatum was able to significantly change CYP3A4 mRNA gene expression (twofold decrease, p < 0.05), this being correlated with an antagonist effect upon hPXR (EC50 = 0.38 mg/mL). Total intracellular glutathione levels were significantly depleted by exposure to Solanum paniculatum (-44%, p < 0.001), Lippia sidoides (-12%, p < 0.05) and Cordia verbenacea (-47%, p < 0.001). The latter plant extract was able to decrease GGT activity (-48%, p < 0.01). In conclusion, this preclinical study shows that the administration of some of these herbal medicines may be able to cause disturbances to metabolic mechanisms in vitro. Although Erythrina mulungu appears safe in our tests, active pharmacovigilance is recommended for the other three species, especially in the case of Solanum paniculatum.

  14. In vitro Effects of Four Native Brazilian Medicinal Plants in CYP3A4 mRNA Gene Expression, Glutathione Levels, and P-Glycoprotein Activity

    PubMed Central

    Mazzari, Andre L. D. A.; Milton, Flora; Frangos, Samantha; Carvalho, Ana C. B.; Silveira, Dâmaris; de Assis Rocha Neves, Francisco; Prieto, Jose M.

    2016-01-01

    Erythrina mulungu Benth. (Fabaceae), Cordia verbenacea A. DC. (Boraginaceae), Solanum paniculatum L. (Solanaceae) and Lippia sidoides Cham. (Verbenaceae) are medicinal plant species native to Brazil shortlisted by the Brazilian National Health System for future clinical use. However, nothing is known about their effects in metabolic and transporter proteins, which could potentially lead to herb-drug interactions (HDI). In this work, we assess non-toxic concentrations (100 μg/mL) of the plant infusions for their in vitro ability to modulate CYP3A4 mRNA gene expression and intracellular glutathione levels in HepG2 cells, as well as P-glycoprotein (P-gp) activity in vincristine-resistant Caco-2 cells (Caco-2 VCR). Their mechanisms of action were further studied by measuring the activation of human pregnane X receptor (hPXR) in transiently co-transfected HeLa cells and the inhibition of γ-glutamyl transferase (GGT) in HepG2 cells. Our results show that P-gp activity was not affected in any case and that only Solanum paniculatum was able to significantly change CYP3A4 mRNA gene expression (twofold decrease, p < 0.05), this being correlated with an antagonist effect upon hPXR (EC50 = 0.38 mg/mL). Total intracellular glutathione levels were significantly depleted by exposure to Solanum paniculatum (-44%, p < 0.001), Lippia sidoides (-12%, p < 0.05) and Cordia verbenacea (-47%, p < 0.001). The latter plant extract was able to decrease GGT activity (-48%, p < 0.01). In conclusion, this preclinical study shows that the administration of some of these herbal medicines may be able to cause disturbances to metabolic mechanisms in vitro. Although Erythrina mulungu appears safe in our tests, active pharmacovigilance is recommended for the other three species, especially in the case of Solanum paniculatum.

  15. In vitro Effects of Four Native Brazilian Medicinal Plants in CYP3A4 mRNA Gene Expression, Glutathione Levels, and P-Glycoprotein Activity

    PubMed Central

    Mazzari, Andre L. D. A.; Milton, Flora; Frangos, Samantha; Carvalho, Ana C. B.; Silveira, Dâmaris; de Assis Rocha Neves, Francisco; Prieto, Jose M.

    2016-01-01

    Erythrina mulungu Benth. (Fabaceae), Cordia verbenacea A. DC. (Boraginaceae), Solanum paniculatum L. (Solanaceae) and Lippia sidoides Cham. (Verbenaceae) are medicinal plant species native to Brazil shortlisted by the Brazilian National Health System for future clinical use. However, nothing is known about their effects in metabolic and transporter proteins, which could potentially lead to herb-drug interactions (HDI). In this work, we assess non-toxic concentrations (100 μg/mL) of the plant infusions for their in vitro ability to modulate CYP3A4 mRNA gene expression and intracellular glutathione levels in HepG2 cells, as well as P-glycoprotein (P-gp) activity in vincristine-resistant Caco-2 cells (Caco-2 VCR). Their mechanisms of action were further studied by measuring the activation of human pregnane X receptor (hPXR) in transiently co-transfected HeLa cells and the inhibition of γ-glutamyl transferase (GGT) in HepG2 cells. Our results show that P-gp activity was not affected in any case and that only Solanum paniculatum was able to significantly change CYP3A4 mRNA gene expression (twofold decrease, p < 0.05), this being correlated with an antagonist effect upon hPXR (EC50 = 0.38 mg/mL). Total intracellular glutathione levels were significantly depleted by exposure to Solanum paniculatum (-44%, p < 0.001), Lippia sidoides (-12%, p < 0.05) and Cordia verbenacea (-47%, p < 0.001). The latter plant extract was able to decrease GGT activity (-48%, p < 0.01). In conclusion, this preclinical study shows that the administration of some of these herbal medicines may be able to cause disturbances to metabolic mechanisms in vitro. Although Erythrina mulungu appears safe in our tests, active pharmacovigilance is recommended for the other three species, especially in the case of Solanum paniculatum. PMID:27594838

  16. Assessment of a Candidate Marker Constituent Predictive of a Dietary Substance–Drug Interaction: Case Study with Grapefruit Juice and CYP3A4 Drug Substrates

    PubMed Central

    Ainslie, Garrett R.; Wolf, Kristina K.; Li, Yingxin; Connolly, Elizabeth A.; Scarlett, Yolanda V.; Hull, J. Heyward

    2014-01-01

    Dietary substances, including herbal products and citrus juices, can perpetrate interactions with conventional medications. Regulatory guidances for dietary substance–drug interaction assessment are lacking. This deficiency is due in part to challenges unique to dietary substances, a lack of requisite human-derived data, and limited jurisdiction. An in vitro–in vivo extrapolation (IVIVE) approach to help address some of these hurdles was evaluated using the exemplar dietary substance grapefruit juice (GFJ), the candidate marker constituent 6′,7′-dihydroxybergamottin (DHB), and the purported victim drug loperamide. First, the GFJ-loperamide interaction was assessed in 16 healthy volunteers. Loperamide (16 mg) was administered with 240 ml of water or GFJ; plasma was collected from 0 to 72 hours. Relative to water, GFJ increased the geometric mean loperamide area under the plasma concentration–time curve (AUC) significantly (1.7-fold). Second, the mechanism-based inhibition kinetics for DHB were recovered using human intestinal microsomes and the index CYP3A4 reaction, loperamide N-desmethylation (KI [concentration needed to achieve one-half kinact], 5.0 ± 0.9 µM; kinact [maximum inactivation rate constant], 0.38 ± 0.02 minute−1). These parameters were incorporated into a mechanistic static model, which predicted a 1.6-fold increase in loperamide AUC. Third, the successful IVIVE prompted further application to 15 previously reported GFJ-drug interaction studies selected according to predefined criteria. Twelve of the interactions were predicted to within the 25% predefined criterion. Results suggest that DHB could be used to predict the CYP3A4-mediated effect of GFJ. This time- and cost-effective IVIVE approach could be applied to other dietary substance–drug interactions to help prioritize new and existing drugs for more advanced (dynamic) modeling and simulation and clinical assessment. PMID:25253884

  17. The isolation of minor-occurring furanocoumarins in grapefruit and analysis of their inhibition of cyp 3a4 and p-glycoprotein transport of talinolol from caco-2 cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The effects of grapefruit juice on the disposition of certain prescription drugs in humans have been mainly attributed to the inhibition of intestinal cytochrome P450 3A4 (CYP 3A4) by linear furanocoumarins. A number of the main furanocoumarins in grapefruit juice have been identified and analyzed ...

  18. An evaluation of the CYP2D6 and CYP3A4 inhibition potential of metoprolol metabolites and their contribution to drug-drug and drug-herb interaction by LC-ESI/MS/MS.

    PubMed

    Borkar, Roshan M; Bhandi, Murali Mohan; Dubey, Ajay P; Ganga Reddy, V; Komirishetty, Prashanth; Nandekar, Prajwal P; Sangamwar, Abhay T; Kamal, Ahmed; Banerjee, Sanjay K; Srinivas, R

    2016-10-01

    The aim of the present study was to evaluate the contribution of metabolites to drug-drug interaction and drug-herb interaction using the inhibition of CYP2D6 and CYP3A4 by metoprolol (MET) and its metabolites. The peak concentrations of unbound plasma concentration of MET, α-hydroxy metoprolol (HM), O-desmethyl metoprolol (ODM) and N-desisopropyl metoprolol (DIM) were 90.37 ± 2.69, 33.32 ± 1.92, 16.93 ± 1.70 and 7.96 ± 0.94 ng/mL, respectively. The metabolites identified, HM and ODM, had a ratio of metabolic area under the concentration-time curve (AUC) to parent AUC of ≥0.25 when either total or unbound concentration of metabolite was considered. In vitro CYP2D6 and CYP3A4 inhibition by MET, HM and ODM study revealed that MET, HM and ODM were not inhibitors of CYP3A4-catalyzed midazolam metabolism and CYP2D6-catalyzed dextromethorphan metabolism. However, DIM only met the criteria of >10% of the total drug related material and <25% of the parent using unbound concentrations. If CYP inhibition testing is solely based on metabolite exposure, DIM metabolite would probably not be considered. However, the present study has demonstrated that DIM contributes significantly to in vitro drug-drug interaction. Copyright © 2016 John Wiley & Sons, Ltd.

  19. Modeling chemical interaction profiles: I. Spectral data-activity relationship and structure-activity relationship models for inhibitors and non-inhibitors of cytochrome P450 CYP3A4 and CYP2D6 isozymes.

    PubMed

    McPhail, Brooks; Tie, Yunfeng; Hong, Huixiao; Pearce, Bruce A; Schnackenberg, Laura K; Ge, Weigong; Valerio, Luis G; Fuscoe, James C; Tong, Weida; Buzatu, Dan A; Wilkes, Jon G; Fowler, Bruce A; Demchuk, Eugene; Beger, Richard D

    2012-03-15

    An interagency collaboration was established to model chemical interactions that may cause adverse health effects when an exposure to a mixture of chemicals occurs. Many of these chemicals--drugs, pesticides, and environmental pollutants--interact at the level of metabolic biotransformations mediated by cytochrome P450 (CYP) enzymes. In the present work, spectral data-activity relationship (SDAR) and structure-activity relationship (SAR) approaches were used to develop machine-learning classifiers of inhibitors and non-inhibitors of the CYP3A4 and CYP2D6 isozymes. The models were built upon 602 reference pharmaceutical compounds whose interactions have been deduced from clinical data, and 100 additional chemicals that were used to evaluate model performance in an external validation (EV) test. SDAR is an innovative modeling approach that relies on discriminant analysis applied to binned nuclear magnetic resonance (NMR) spectral descriptors. In the present work, both 1D ¹³C and 1D ¹⁵N-NMR spectra were used together in a novel implementation of the SDAR technique. It was found that increasing the binning size of 1D ¹³C-NMR and ¹⁵N-NMR spectra caused an increase in the tenfold cross-validation (CV) performance in terms of both the rate of correct classification and sensitivity. The results of SDAR modeling were verified using SAR. For SAR modeling, a decision forest approach involving from 6 to 17 Mold2 descriptors in a tree was used. Average rates of correct classification of SDAR and SAR models in a hundred CV tests were 60% and 61% for CYP3A4, and 62% and 70% for CYP2D6, respectively. The rates of correct classification of SDAR and SAR models in the EV test were 73% and 86% for CYP3A4, and 76% and 90% for CYP2D6, respectively. Thus, both SDAR and SAR methods demonstrated a comparable performance in modeling a large set of structurally diverse data. Based on unique NMR structural descriptors, the new SDAR modeling method complements the existing SAR

  20. Building Structure Feature-based Models for Predicting Isoform-specific Human Cytochrome P-450 (hCYP 3A4, 2D6 and 2C9) Inhibition Assay Results in ToxCast

    EPA Science Inventory

    EPA’s ToxCast project is using high-throughput screening (HTS) to profile and prioritize chemicals for further testing. ToxCast Phase I evaluated 309 unique chemicals, the majority pesticide actives, in over 500 HTS assays. These included 3 human cytochrome P450 (hCYP3A4, hCYP2...

  1. Development and validation of a LC-MS/MS method for the in vitro analysis of 1-hydroxymidazolam in human liver microsomes: application for determining CYP3A4 inhibition in complex matrix mixtures.

    PubMed

    Mooiman, K D; Maas-Bakker, R F; Rosing, H; Beijnen, J H; Schellens, J H M; Meijerman, I

    2013-09-01

    Complementary and alternative medicines (CAM) can affect the pharmacokinetics of anticancer drugs by interacting with the metabolizing enzyme cytochrome P450 (CYP) 3A4. To evaluate changes in the activity of CYP3A4 in patients, levels of 1-hydroxymidazolam in plasma are often determined with liquid chromatography-quadrupole mass spectrometry (LC-MS/MS). However, validated LC-MS/MS methods to determine in vitro CYP3A4 inhibition in human liver microsomes are scarce and not optimized for evaluating CYP3A4 inhibition by CAM. The latter is necessary because CAM are often complex mixtures of numerous compounds that can interfere with the selective measurement of 1-hydroxymidazolam. Therefore, the aim was to validate and optimize an LC-MS/MS method for the adequate determination of CYP3A4 inhibition by CAM in human liver microsomes. After incubation of human liver microsomes with midazolam, liquid-liquid extraction with tert-butyl methyl ether was applied and dried samples were reconstituted in 50% methanol. These samples were injected onto a reversed-phase chromatography consisting of a Zorbax Extend-C18 column (2.1 × 150 mm, 5.0 µm particle size), connected to a triple quadrupole mass spectrometer with electrospray ionization. The described LC-MS/MS method was validated over linear range of 1.0-500 nm for 1-hydroxymidazolam. The results revealed good inter-assay accuracy (≥85% and ≤115%) and within-day and between-day precisions (coefficient of variation ≤ 4.43%). Furthermore, the applicability of this assay for the determination of CYP3A4 inhibition in complex matrix mixtures was successfully demonstrated in an in vitro experiment in which CYP3A4 inhibition by known CAM (β-carotene, green tea, milk thistle and St. John's wort) was determined.

  2. RS-Predictor: A new tool for predicting sites of cytochrome P450-mediated metabolism applied to CYP 3A4

    PubMed Central

    Zaretzki, Jed; Bergeron, Charles; Rydberg, Patrik; Huang, Tao-wei; Bennett, Kristin P.; Breneman, Curt M.

    2011-01-01

    This article describes RegioSelectivity-Predictor (RS-Predictor), a new in silico method for generating predictive models of P450-mediated metabolism for drug-like compounds. Within this method, potential sites of metabolism (SOMs) are represented as “metabolophores”: A concept that describes the hierarchical combination of topological and quantum chemical descriptors needed to represent the reactivity of potential metabolic reaction sites. RS-Predictor modeling involves the use of metabolophore descriptors together with multiple-instance ranking (MIRank) to generate an optimized descriptor weight vector that encodes regioselectivity trends across all cases in a training set. The resulting pathway-independent,i isozyme-specific regioselectivity model may be used to predict potential metabolic liabilities. In the present work, cross-validated RS-Predictor models were generated for a set of 394 substrates of CYP 3A4 as a proof-of-principle for the method. Rank aggregation was then employed to merge independently generated predictions for each substrate into a single consensus prediction. The resulting consensus RS-Predictor models were shown to reliably identify at least one observed site of metabolism in the top two rank-positions on 78% of the substrates. Comparisons between RS-Predictor and previously described regioselectivity prediction methods reveal new insights into how in silico metabolite prediction methods should be compared. PMID:21528931

  3. Milk thistle, a herbal supplement, decreases the activity of CYP3A4 and uridine diphosphoglucuronosyl transferase in human hepatocyte cultures.

    PubMed

    Venkataramanan, R; Ramachandran, V; Komoroski, B J; Zhang, S; Schiff, P L; Strom, S C

    2000-11-01

    Milk thistle extract is one of the most commonly used nontraditional therapies, particularly in Germany. Milk thistle is known to contain a number of flavonolignans. We evaluated the effect of silymarin, on the activity of various hepatic drug-metabolizing enzymes in human hepatocyte cultures. Treatment with silymarin (0.1 and 0.25 mM) significantly reduced the activity of CYP3A4 enzyme (by 50 and 100%, respectively) as determined by the formation of 6-beta-hydroxy testosterone and the activity of uridine diphosphoglucuronosyl transferase (UGT1A6/9) (by 65 and 100%, respectively) as measured by the formation of 4-methylumbelliferone glucuronide. Silymarin (0.5 mM) also significantly decreased mitochondrial respiration as determined by MTT reduction in human hepatocytes. These observations point to the potential of silymarin to impair hepatic metabolism of certain coadministered drugs in humans. Indiscriminate use of herbal products may lead to altered pharmacokinetics of certain drugs and may result in increased toxicity of certain drugs.

  4. Associations of CYP3A4, NR1I2, CYP2C19 and P2RY12 polymorphisms with clopidogrel resistance in Chinese patients with ischemic stroke

    PubMed Central

    Liu, Rui; Zhou, Zi-yi; Chen, Yi-bei; Li, Jia-li; Yu, Wei-bang; Chen, Xin-meng; Zhao, Min; Zhao, Yuan-qi; Cai, Ye-feng; Jin, Jing; Huang, Min

    2016-01-01

    Aim: There is a high incidence of the antiplatelet drug clopidogrel resistance (CR) in Asian populations. Because clopidogrel is a prodrug, polymorphisms of genes encoding the enzymes involved in its biotransformation may be the primary influential factors. The goal of this study was to investigate the associations of polymorphisms of CYP3A4, NR1I2, CYP2C19 and P2RY12 genes with CR in Chinese patients with ischemic stroke. Methods: A total of 191 patients with ischemic stroke were enrolled. The patients were treated with clopidogrel for at least 5 days. Platelet function was measured by light transmission aggregometry. The SNPs NR1I2 (rs13059232), CYP3A4*1G (rs2242480), CYP2C19*2 (rs4244285) and P2RY12 (rs2046934) were genotyped. Results: The CR rate in this population was 36%. The CYP2C19*2 variant was a risk factor for CR (*2/*2+wt/*2 vs wt/wt, OR: 2.366, 95% CI: 1.180–4.741, P=0.014), whereas the CYP3A4*1G variant had a protective effect on CR (*1/*1 vs *1G/*1G+*1/*1G, OR: 2.360, 95% CI: 1.247–4.468, P=0.008). The NR1I2 (rs13059232) polymorphism was moderately associated with CR (CC vs TT+TC, OR: 0.533, 95% CI: 0.286–0.991, P=0.046). The C allele in P2RY12 (rs2046934) was predicted to be a protective factor for CR (CC+TC vs TT, OR: 0.407, 95% CI: 0.191–0.867, P=0.018). In addition, an association was found between hypertension and CR (P=0.022). Conclusion: The individuals with both the CYP2C19*2 allele and hypertension are at high risk of CR during anti-thrombosis therapy. The CYP3A4*1G allele, P2RY12 (rs2046934) C allele and NR1I2 (rs13059232) CC genotype may be protective factors for CR. The associated SNPs studied may be useful to predict clopidogrel resistance in Chinese patients with ischemic stroke. PMID:27133299

  5. Amine-free melanin-concentrating hormone receptor 1 antagonists: Novel 1-(1H-benzimidazol-6-yl)pyridin-2(1H)-one derivatives and design to avoid CYP3A4 time-dependent inhibition.

    PubMed

    Igawa, Hideyuki; Takahashi, Masashi; Shirasaki, Mikio; Kakegawa, Keiko; Kina, Asato; Ikoma, Minoru; Aida, Jumpei; Yasuma, Tsuneo; Okuda, Shoki; Kawata, Yayoi; Noguchi, Toshihiro; Yamamoto, Syunsuke; Fujioka, Yasushi; Kundu, Mrinalkanti; Khamrai, Uttam; Nakayama, Masaharu; Nagisa, Yasutaka; Kasai, Shizuo; Maekawa, Tsuyoshi

    2016-06-01

    Melanin-concentrating hormone (MCH) is an attractive target for antiobesity agents, and numerous drug discovery programs are dedicated to finding small-molecule MCH receptor 1 (MCHR1) antagonists. We recently reported novel pyridine-2(1H)-ones as aliphatic amine-free MCHR1 antagonists that structurally featured an imidazo[1,2-a]pyridine-based bicyclic motif. To investigate imidazopyridine variants with lower basicity and less potential to inhibit cytochrome P450 3A4 (CYP3A4), we designed pyridine-2(1H)-ones bearing various less basic bicyclic motifs. Among these, a lead compound 6a bearing a 1H-benzimidazole motif showed comparable binding affinity to MCHR1 to the corresponding imidazopyridine derivative 1. Optimization of 6a afforded a series of potent thiophene derivatives (6q-u); however, most of these were found to cause time-dependent inhibition (TDI) of CYP3A4. As bioactivation of thiophenes to form sulfoxide or epoxide species was considered to be a major cause of CYP3A4 TDI, we introduced electron withdrawing groups on the thiophene and found that a CF3 group on the ring or a Cl adjacent to the sulfur atom helped prevent CYP3A4 TDI. Consequently, 4-[(5-chlorothiophen-2-yl)methoxy]-1-(2-cyclopropyl-1-methyl-1H-benzimidazol-6-yl)pyridin-2(1H)-one (6s) was identified as a potent MCHR1 antagonist without the risk of CYP3A4 TDI, which exhibited a promising safety profile including low CYP3A4 inhibition and exerted significant antiobesity effects in diet-induced obese F344 rats. PMID:27112449

  6. Amine-free melanin-concentrating hormone receptor 1 antagonists: Novel 1-(1H-benzimidazol-6-yl)pyridin-2(1H)-one derivatives and design to avoid CYP3A4 time-dependent inhibition.

    PubMed

    Igawa, Hideyuki; Takahashi, Masashi; Shirasaki, Mikio; Kakegawa, Keiko; Kina, Asato; Ikoma, Minoru; Aida, Jumpei; Yasuma, Tsuneo; Okuda, Shoki; Kawata, Yayoi; Noguchi, Toshihiro; Yamamoto, Syunsuke; Fujioka, Yasushi; Kundu, Mrinalkanti; Khamrai, Uttam; Nakayama, Masaharu; Nagisa, Yasutaka; Kasai, Shizuo; Maekawa, Tsuyoshi

    2016-06-01

    Melanin-concentrating hormone (MCH) is an attractive target for antiobesity agents, and numerous drug discovery programs are dedicated to finding small-molecule MCH receptor 1 (MCHR1) antagonists. We recently reported novel pyridine-2(1H)-ones as aliphatic amine-free MCHR1 antagonists that structurally featured an imidazo[1,2-a]pyridine-based bicyclic motif. To investigate imidazopyridine variants with lower basicity and less potential to inhibit cytochrome P450 3A4 (CYP3A4), we designed pyridine-2(1H)-ones bearing various less basic bicyclic motifs. Among these, a lead compound 6a bearing a 1H-benzimidazole motif showed comparable binding affinity to MCHR1 to the corresponding imidazopyridine derivative 1. Optimization of 6a afforded a series of potent thiophene derivatives (6q-u); however, most of these were found to cause time-dependent inhibition (TDI) of CYP3A4. As bioactivation of thiophenes to form sulfoxide or epoxide species was considered to be a major cause of CYP3A4 TDI, we introduced electron withdrawing groups on the thiophene and found that a CF3 group on the ring or a Cl adjacent to the sulfur atom helped prevent CYP3A4 TDI. Consequently, 4-[(5-chlorothiophen-2-yl)methoxy]-1-(2-cyclopropyl-1-methyl-1H-benzimidazol-6-yl)pyridin-2(1H)-one (6s) was identified as a potent MCHR1 antagonist without the risk of CYP3A4 TDI, which exhibited a promising safety profile including low CYP3A4 inhibition and exerted significant antiobesity effects in diet-induced obese F344 rats.

  7. In vitro inhibition of cytochrome P450 3A4 by Aronia melanocarpa constituents.

    PubMed

    Bräunlich, Marie; Christensen, Hege; Johannesen, Siri; Slimestad, Rune; Wangensteen, Helle; Malterud, Karl E; Barsett, Hilde

    2013-01-01

    Extracts, subfractions, isolated anthocyanins and procyanidins, and two phenolic acids from aronia [Aronia melanocarpa] were investigated for their CYP3A4 inhibitory effects, using midazolam as the probe substrate and recombinant insect cell microsomes expressing CYP3A4 as the enzyme source. Procyanidin B5 was a considerably stronger CYP3A4 inhibitor in vitro than the isomeric procyanidin B2 and comparable to bergamottin, a known CYP3A4 inhibitor from grapefruit juice. The inhibitory activity of proanthocyanidin-containing fractions was correlated to the degree of polymerization. Among the anthocyanins, cyanidin 3-arabinoside showed stronger CYP3A4 inhibition than cyanidin 3-galactoside and cyanidin 3-glucoside. Thus, the ability to inhibit CYP3A4 in vitro seems to be influenced by the sugar unit linked to the anthocyanidin.

  8. Hyperconjugation with lone pair of morpholine nitrogen stabilizes transition state for phenyl hydroxylation in CYP3A4 metabolism of ( S)- N-[1-(3-morpholin-4-yl phenyl) ethyl]-3-phenylacrylamide

    NASA Astrophysics Data System (ADS)

    Shaikh, Abdul Rajjak; Broclawik, Ewa; Ismael, Mohamed; Tsuboi, Hideyuki; Koyama, Michihisa; Kubo, Momoji; Del Carpio, Carlos A.; Miyamoto, Akira

    2006-02-01

    Using quantum chemical modelling we describe a novel effect in the mechanism of CYP3A4 metabolism for the arene substrate with o-substituent yielding a lone pair donation to conjugate π system; this will compensate for the loss of aromaticity on formation of the tetrahedral complex and lower the rate-determining energy barrier.

  9. Modeling chemical interaction profiles: II. Molecular docking, spectral data-activity relationship, and structure-activity relationship models for potent and weak inhibitors of cytochrome P450 CYP3A4 isozyme.

    PubMed

    Tie, Yunfeng; McPhail, Brooks; Hong, Huixiao; Pearce, Bruce A; Schnackenberg, Laura K; Ge, Weigong; Buzatu, Dan A; Wilkes, Jon G; Fuscoe, James C; Tong, Weida; Fowler, Bruce A; Beger, Richard D; Demchuk, Eugene

    2012-03-15

    Polypharmacy increasingly has become a topic of public health concern, particularly as the U.S. population ages. Drug labels often contain insufficient information to enable the clinician to safely use multiple drugs. Because many of the drugs are bio-transformed by cytochrome P450 (CYP) enzymes, inhibition of CYP activity has long been associated with potentially adverse health effects. In an attempt to reduce the uncertainty pertaining to CYP-mediated drug-drug/chemical interactions, an interagency collaborative group developed a consensus approach to prioritizing information concerning CYP inhibition. The consensus involved computational molecular docking, spectral data-activity relationship (SDAR), and structure-activity relationship (SAR) models that addressed the clinical potency of CYP inhibition. The models were built upon chemicals that were categorized as either potent or weak inhibitors of the CYP3A4 isozyme. The categorization was carried out using information from clinical trials because currently available in vitro high-throughput screening data were not fully representative of the in vivo potency of inhibition. During categorization it was found that compounds, which break the Lipinski rule of five by molecular weight, were about twice more likely to be inhibitors of CYP3A4 compared to those, which obey the rule. Similarly, among inhibitors that break the rule, potent inhibitors were 2-3 times more frequent. The molecular docking classification relied on logistic regression, by which the docking scores from different docking algorithms, CYP3A4 three-dimensional structures, and binding sites on them were combined in a unified probabilistic model. The SDAR models employed a multiple linear regression approach applied to binned 1D ¹³C-NMR and 1D ¹⁵N-NMR spectral descriptors. Structure-based and physical-chemical descriptors were used as the basis for developing SAR models by the decision forest method. Thirty-three potent inhibitors and 88 weak

  10. UNDERSTANDING THE MECHANISM OF CYTOCHROME P450 3A4: RECENT ADVANCES AND REMAINING PROBLEMS

    PubMed Central

    Sevrioukova, Irina F.; Poulos, Thomas L.

    2013-01-01

    Cytochromes P450 (CYPs) represent a diverse group of heme-thiolate proteins found in almost all organisms. CYPs share a common protein fold but differ in substrate selectivity and catalyze a wide variety of monooxygenation reactions via activation of molecular oxygen. Among 57 human P450s, the 3A4 isoform (CYP3A4) is the most abundant and the most important because it metabolizes the majority of the administered drugs. A remarkable feature of CYP3A4 is its extreme promiscuity in substrate specificity and cooperative substrate binding, which often leads to undesirable drug-drug interactions and toxic side effects. Owing to its importance in drug development and therapy, CYP3A4 has been the most extensively studied mammalian P450. In this review we provide an overview on recent progress and remaining problems in the CYP3A4 research. PMID:23018626

  11. In Silico Predictions and In Vivo Results of Drug-Drug Interactions by Ketoconazole and Verapamil on AZD1305, a Combined Ion Channel Blocker and a Sensitive CYP3A4 Substrate.

    PubMed

    Johansson, Susanne; Löfberg, Boel; Aunes, Maria; Lunde, Helen; Frison, Lars; Edvardsson, Nils; Cullberg, Marie

    2016-09-01

    The objectives were to estimate and compare, in silico and in vivo, the effects of a strong and a moderate CYP3A4 inhibitor on AZD1305 pharmacokinetics. In silico, simulations were performed with the computer software Simcyp, and the predicted outcome was compared with the results observed in healthy male subjects. In silico, the geometric mean plasma exposure of AZD1305 + ketoconazole showed a 7.1-fold higher AUC and a 4.4-fold higher Cmax compared with AZD1305 alone. Coadministration with verapamil gave a 1.9-fold higher AUC and a 1.7-fold higher Cmax compared with AZD1305 alone. In vivo, the plasma exposure of AZD1305 + ketoconazole showed a 7.7-fold higher AUC and a 4.8 -fold higher Cmax compared with AZD1305 alone. Coadministration with verapamil gave a 2.2-fold higher AUC and a 2.0-fold higher Cmax compared with AZD1305 alone. The mean maximum QTcF increase from baseline was 407, 487, and 437 milliseconds for AZD1305, alone and in combination with verapamil or ketoconazole, respectively. Simcyp predicted the effects of ketoconazole and verapamil on the sensitive CYP3A4 substrate AZD1305 pharmacokinetics well. Both the in vivo study and the Simcyp predictions suggest a contraindication for strong CYP3A4 inhibitors and AZD1305 when given in combination. PMID:27627192

  12. Current Approaches for Investigating and Predicting Cytochrome P450 3A4-Ligand Interactions

    PubMed Central

    Poulos, Thomas L.

    2015-01-01

    Cytochrome P450 3A4 (CYP3A4) is the major and most important drug-metabolizing enzyme in humans that oxidizes and clears over a half of all administered pharmaceuticals. This is possible because CYP3A4 is promiscuous with respect to substrate binding and has the ability to catalyze diverse oxidative chemistries in addition to traditional hydroxylation reactions. Furthermore, CYP3A4 binds and oxidizes a number of substrates in a cooperative manner and can be both induced and inactivated by drugs. In vivo, CYP3A4 inhibition could lead to undesired drug-drug interactions and drug toxicity, a major reason for late-stage clinical failures and withdrawal of marketed pharmaceuticals. Owing to its central role in drug metabolism, many aspects of CYP3A4 catalysis have been extensively studied by various techniques. Here, we give an overview of experimental and theoretical methods currently used for investigation and prediction of CYP3A4-ligand interactions, a defining factor in drug metabolism, with an emphasis on the problems addressed and conclusions derived from the studies. PMID:26002732

  13. Fungal lactone ring opening of 6', 7'-dihydroxybergamottin diminishes cytochrome P450 3A4 inhibitory activity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Furanocoumarins (FCs) are a class of aromatic compounds in grapefruit that inhibit human intestinal cytochrome P450 3A4 (CYP3A4). Since fungi metabolize polycyclic aromatic hydrocarbons, we hypothesized that certain fungi might also metabolize FCs into forms that may be inactive as CYP3A4 inhibitors...

  14. Quantitative Prediction of Regioselectivity Toward Cytochrome P450/3A4 Using Machine Learning Approaches.

    PubMed

    Hasegawa, Kiyoshi; Koyama, Michio; Funatsu, Kimito

    2010-03-15

    In the drug discovery process, it is important to know the properties of both drug candidates and their metabolites. Fast and precise prediction of metabolites is essential. However, it has been difficult to predict metabolites because of the complexity of the mechanism of cytochrome P450/3A4 (CYP 3A4), which is the main metabolite enzyme of drugs. In this study, we focus on the regioselectivity of CYP 3A4, i.e., the selectivity of metabolic sites. We have developed a model to predict the regioselectivity of drug candidates by using machine learning (ML) approaches.

  15. Mechanism-Based Inactivation of Human Cytochrome P450 3A4 by Two Piperazine-Containing Compounds

    PubMed Central

    Bolles, Amanda K.; Fujiwara, Rina; Briggs, Erran D.; Nomeir, Amin A.

    2014-01-01

    Human cytochrome P450 3A4 (CYP3A4) is responsible for the metabolism of more than half of pharmaceutic drugs, and inactivation of CYP3A4 can lead to adverse drug-drug interactions. The substituted imidazole compounds 5-fluoro-2-[4-[(2-phenyl-1H-imidazol-5-yl)methyl]-1-piperazinyl]pyrimidine (SCH 66712) and 1-[(2-ethyl-4-methyl-1H-imidazol-5-yl)methyl]-4-[4-(trifluoromethyl)-2-pyridinyl]piperazine (EMTPP) have been previously identified as mechanism-based inactivators (MBI) of CYP2D6. The present study shows that both SCH 66712 and EMTPP are also MBIs of CYP3A4. Inhibition of CYP3A4 by SCH 66712 and EMTPP was determined to be concentration, time, and NADPH dependent. In addition, inactivation of CYP3A4 by SCH 66712 was shown to be unaffected by the presence of electrophile scavengers. SCH 66712 displays type I binding to CYP3A4 with a spectral binding constant (Ks) of 42.9 ± 2.9 µM. The partition ratios for SCH 66712 and EMTPP were 11 and 94, respectively. Whole protein mass spectrum analysis revealed 1:1 binding stoichiometry of SCH 66712 and EMTPP to CYP3A4 and a mass increase consistent with adduction by the inactivators without addition of oxygen. Heme adduction was not apparent. Multiple mono-oxygenation products with each inactivator were observed; no other products were apparent. These are the first MBIs to be shown to be potent inactivators of both CYP2D6 and CYP3A4. PMID:25273356

  16. Mechanism-based inactivation of human cytochrome P450 3A4 by two piperazine-containing compounds.

    PubMed

    Bolles, Amanda K; Fujiwara, Rina; Briggs, Erran D; Nomeir, Amin A; Furge, Laura Lowe

    2014-12-01

    Human cytochrome P450 3A4 (CYP3A4) is responsible for the metabolism of more than half of pharmaceutic drugs, and inactivation of CYP3A4 can lead to adverse drug-drug interactions. The substituted imidazole compounds 5-fluoro-2-[4-[(2-phenyl-1H-imidazol-5-yl)methyl]-1-piperazinyl]pyrimidine (SCH 66712) and 1-[(2-ethyl-4-methyl-1H-imidazol-5-yl)methyl]-4-[4-(trifluoromethyl)-2-pyridinyl]piperazine (EMTPP) have been previously identified as mechanism-based inactivators (MBI) of CYP2D6. The present study shows that both SCH 66712 and EMTPP are also MBIs of CYP3A4. Inhibition of CYP3A4 by SCH 66712 and EMTPP was determined to be concentration, time, and NADPH dependent. In addition, inactivation of CYP3A4 by SCH 66712 was shown to be unaffected by the presence of electrophile scavengers. SCH 66712 displays type I binding to CYP3A4 with a spectral binding constant (Ks) of 42.9 ± 2.9 µM. The partition ratios for SCH 66712 and EMTPP were 11 and 94, respectively. Whole protein mass spectrum analysis revealed 1:1 binding stoichiometry of SCH 66712 and EMTPP to CYP3A4 and a mass increase consistent with adduction by the inactivators without addition of oxygen. Heme adduction was not apparent. Multiple mono-oxygenation products with each inactivator were observed; no other products were apparent. These are the first MBIs to be shown to be potent inactivators of both CYP2D6 and CYP3A4. PMID:25273356

  17. Gomisin A is a Novel Isoform-Specific Probe for the Selective Sensing of Human Cytochrome P450 3A4 in Liver Microsomes and Living Cells.

    PubMed

    Wu, Jing-Jing; Ge, Guang-Bo; He, Yu-Qi; Wang, Ping; Dai, Zi-Ru; Ning, Jing; Hu, Liang-Hai; Yang, Ling

    2016-01-01

    Nearly half of prescription medicines are metabolized by human cytochrome P450 (CYP) 3A. CYP3A4 and 3A5 are two major isoforms of human CYP3A and share most of the substrate spectrum. A very limited previous study distinguished the activity of CYP3A4 and CYP3A5, identifying the challenge in predicting CYP3A-mediated drug clearance and drug-drug interaction. In the present study, we introduced gomisin A (GA) with a dibenzocyclooctadiene skeleton as a novel selective probe of CYP3A4. The major metabolite of GA was fully characterized as 8-hydroxylated GA by LC-MS and NMR. CYP3A4 was assigned as the predominant isozyme involved in GA 8-hydroxylation by reaction phenotyping assays, chemical inhibition assays, and correlation studies. GA 8-hydroxylation in both recombinant human CYP3A4 and human liver microsomes followed classic Michaelis-Menten kinetics. The intrinsic clearance values indicated that CYP3A4 contributed 12.8-fold more than CYP3A5 to GA 8-hydroxylation. Molecular docking studies indicated different hydrogen bonds and π-π interactions between CYP3A4 and CYP3A5, which might result in the different catalytic activity for GA 8-hydroxylation. Furthermore, GA exhibited a stronger inhibitory activity towards CYP3A4 than CYP3A5, which further suggested a preferred selectivity of CYP3A4 for the transformation of GA. More importantly, GA has been successfully applied to selectively monitor the modulation of CYP3A4 activities by the inducer rifampin in hepG2 cells, which is consistent with the level change of CYP3A4 mRNA expression. In summary, our results suggested that GA could be used as a novel probe for the selective sensing of CYP3A4 in tissue and cell preparations.

  18. The effect of interferon-{alpha} on the expression of cytochrome P450 3A4 in human hepatoma cells

    SciTech Connect

    Flaman, Anathea S.; Gravel, Caroline; Hashem, Anwar M.; Tocchi, Monika; Li Xuguang

    2011-06-01

    Interferon {alpha} (IFN{alpha}) is used to treat malignancies and chronic viral infections. It has been found to decrease the rate of drug metabolism by acting on cytochrome P450 enzymes, but no studies have investigated the consequences of IFN{alpha} treatment on the CYP3A4 isoform, responsible for the metabolism of a majority of drugs. In this study, we have examined the effect of IFN{alpha} on CYP3A4 catalytic activity and expression in human hepatoma cells. We found that IFN{alpha} inhibits CYP3A4 activity and rapidly down-regulates the expression of CYP3A4, independent of de novo protein synthesis. Pharmacologic inhibitors and a dominant-negative mutant expression plasmid were used to dissect the molecular pathway required for CYP3A4 suppression, revealing roles for Jak1 and Stat1 and eliminating the involvement of the p38 mitogen-activated and extracellular regulated kinases. Treatment of hepatoma cells with IFN{alpha} did not affect the nuclear localization or relative abundance of Sp1 and Sp3 transcription factors, suggesting that the suppression of CYP3A4 by IFN{alpha} does not result from inhibitory Sp3 out-competing Sp1. To our knowledge, this is the first report that IFN{alpha} down-regulates CYP3A4 expression largely through the JAK-STAT pathway. Since IFN{alpha} suppresses CYP3A4 expression, caution is warranted when IFN{alpha} is administered in combination with CYP3A4 substrates to avoid the occurrence of adverse drug interactions.

  19. Inactivation of Human Cytochrome P450 3A4 and 3A5 by Dronedarone and N-Desbutyl Dronedarone.

    PubMed

    Hong, Yanjun; Chia, Yvonne Mei Fen; Yeo, Ray Hng; Venkatesan, Gopalakrishnan; Koh, Siew Kwan; Chai, Christina Li Lin; Zhou, Lei; Kojodjojo, Pipin; Chan, Eric Chun Yong

    2016-01-01

    Dronedarone is an antiarrhythmic agent approved in 2009 for the treatment of atrial fibrillation. An in-house preliminary study demonstrated that dronedarone inhibits cytochrome P450 (CYP) 3A4 and 3A5 in a time-dependent manner. This study aimed to investigate the inactivation of CYP450 by dronedarone. We demonstrated for the first time that both dronedarone and its main metabolite N-desbutyl dronedarone (NDBD) inactivate CYP3A4 and CYP3A5 in a time-, concentration-, and NADPH-dependent manner. For the inactivation of CYP3A4, the inactivator concentration at the half-maximum rate of inactivation and inactivation rate constant at an infinite inactivator concentration are 0.87 µM and 0.039 minute(-1), respectively, for dronedarone, and 6.24 µM and 0.099 minute(-1), respectively, for NDBD. For CYP3A5 inactivation, the inactivator concentration at the half-maximum rate of inactivation and inactivation rate constant at an infinite inactivator concentration are 2.19 µM and 0.0056 minute(-1) for dronedarone and 5.45 µM and 0.056 minute(-1) for NDBD. The partition ratios for the inactivation of CYP3A4 and CYP3A5 by dronedarone are 51.1 and 32.2, and the partition ratios for the inactivation of CYP3A4 and CYP3A5 by NDBD are 35.3 and 36.6. Testosterone protected both CYP3A4 and CYP3A5 from inactivation by dronedarone and NDBD. Although the presence of Soret peak confirmed the formation of a quasi-irreversible metabolite-intermediate complex between dronedarone/NDBD and CYP3A4/CYP3A5, partial recovery of enzyme activity by potassium ferricyanide illuminated an alternative irreversible mechanism-based inactivation (MBI). MBI of CYP3A4 and CYP3A5 was further supported by the discovery of glutathione adducts derived from the quinone oxime intermediates of dronedarone and NDBD. In conclusion, dronedarone and NDBD inactivate CYP3A4 and CYP3A5 via unique dual mechanisms of MBI and formation of the metabolite-intermediate complex. Our novel findings contribute new knowledge for

  20. Metabolism of risperidone to 9-hydroxyrisperidone by human cytochromes P450 2D6 and 3A4.

    PubMed

    Fang, J; Bourin, M; Baker, G B

    1999-02-01

    Risperidone is a relatively new antipsychotic drug that has been reported to improve both the positive and the negative symptoms of schizophrenia and produces relatively few extrapyramidal side effects at low doses. Formation of 9-hydroxyrisperidone, an active metabolite, is the most important metabolic pathway of risperidone in human. In the present study, in vitro metabolism of risperidone (100 microM) was investigated using the recombinant human cytochrome P450 (CYP) enzymes CYP1A1, CYP1A2, CYP2C8, CYP2C9-arg144, CYP2C9-cys144, CYP2C19, CYP2D6, CYP3A4 and CYP3A5 supplemented with an NADPH-generating system. 9-Hydroxyrisperidone was determined by a new HPLC method with an Hypersil CN column and a UV detector. Of these enzymes, CYPs 2D6, 3A4 and 3A5 were found to be the ones capable of metabolising risperidone to 9-hydroxyrisperidone, with activities of 7.5, 0.4 and 0.2 pmol pmol(-1) CYP min(-1), respectively. A correlation study using a panel of human liver microsomes showed that the formation of 9-hydroxyrisperidone is highly correlated with CYP2D6 and 3A activities. Thus, both CYP2D6 and 3A4 are involved in the 9-hydroxylation of risperidone at the concentration of risperidone used in this study. This observation is confirmed by the findings that both quinidine (inhibitor of CYP2D6) and ketoconazole (inhibitor of CYP3A4) can inhibit the formation of 9-hydroxyrisperidone. Furthermore, inducers of CYP can significantly increase the formation of 9-hydroxyrisperidone in rat. The formation of 9-hydroxyrisperidone is highly correlated with testosterone 6beta-hydroxylase activities, suggesting that inducible CYP3A contributes significantly to the metabolism of risperidone in rat.

  1. Study Liver Cytochrome P450 3A4 Inhibition and Hepatotoxicity Using DMSO-Differentiated HuH-7 Cells.

    PubMed

    Liu, Yitong

    2016-01-01

    Metabolically competent, inexpensive, and robust in vitro cell models are needed for studying liver drug-metabolizing enzymes and hepatotoxicity. Human hepatoma HuH-7 cells develop into a differentiated in vitro model resembling primary human hepatocytes after a 2-week dimethyl sulfoxide (DMSO) treatment. DMSO-treated HuH-7 cells express elevated cytochrome P450 3A4 (CYP3A4) enzyme gene expression and activity compared to untreated HuH-7 cells. This cell model could be used to study CYP3A4 inhibition by reversible and time-dependent inhibitors, including drugs, food-related substances, and environmental chemicals. The DMSO-treated HuH-7 model is also a suitable tool for investigating hepatotoxicity. This chapter describes a detailed methodology for developing DMSO-treated HuH-7 cells, which are subsequently used for CYP3A4 inhibition and hepatotoxicity studies. PMID:27518624

  2. A Large-Scale Allosteric Transition in Cytochrome P450 3A4 Revealed by Luminescence Resonance Energy Transfer (LRET)

    PubMed Central

    Sineva, Elena V.; Rumfeldt, Jessica A. O.; Halpert, James R.; Davydov, Dmitri R.

    2013-01-01

    Effector-induced allosteric transitions in cytochrome P450 3A4 (CYP3A4) were investigated by luminescence resonance energy transfer (LRET) between two SH-reactive probes attached to various pairs of distantly located cysteine residues, namely the double-cysteine mutants CYP3A4(C64/C468), CYP3A4(C377/C468) and CYP3A4(C64/C121). Successive equimolar labeling of these proteins with the phosphorescent probe erythrosine iodoacetamide (donor) and the near-infrared fluorophore DY-731 maleimide (acceptor) allowed us to establish donor/acceptor pairs sensitive to conformational motions. The interactions of all three double-labeled mutants with the allosteric activators α-naphthoflavone and testosterone resulted in an increase in the distance between the probes. A similar effect was elicited by cholesterol. These changes in distance vary from 1.3 to 8.5 Å, depending on the position of the donor/acceptor pair and the nature of the effector. In contrast, the changes in the interprobe distance caused by such substrates as bromocriptine or 1-pyrenebutanol were only marginal. Our results provide a decisive support to the paradigm of allosteric modulation of CYP3A4 and indicate that the conformational transition caused by allosteric effectors increases the spatial separation between the beta-domain of the enzyme (bearing residues Cys64 and Cys377) and the alpha-domain, where Cys121 and Cys468 are located. PMID:24376769

  3. In Silico Docking of Ligands to Drug Oxidation Enzymes Cytochrome P450 3A4 and Cytochrome P450 1A2.

    NASA Astrophysics Data System (ADS)

    Smith, David; Guglielmon, Jonathan; Glenn, Marsch; Peter, Guengerich F.

    2009-03-01

    Cytochrome P450 3A4 (CYP3A4) and Cytochrome P450 1A2 (CYP1A2) oxidize most drugs in humans. Protein modeling toolkits from OpenEye Scientific Software were used to examine the interaction of drug substrates with CYP3A4 and CYP1A2. Conformers and partial atomic charges were generated for each drug molecule. User-defined volumes were defined around CYP3A4 and CYP1A2 active sites. Ligands were docked assuming protein and substrates as rigid bodies. To assess rigid docking accuracy, x-ray diffraction coordinates of CYP3A4-erythromycin and CYP3A4-metyrapone complexes were obtained. Rigid re-docking of erythromycin and metyrapone into CYP3A4 yielded poses similar to the crystal structures. Rigid docking revealed two other energetically-favorable CYP3A4-metyrapone poses. The best poses were obtained by using all the Open Eye scoring functions. Optimization of protein-ligand interactions within 5-10 Angstroms of the docked ligand was then performed using the Merck Molecular Force Field in which the protein was assumed to be flexible and the ligand to be rigid. Nearby protein residues pulled slightly closer to the substrate, reducing the volume of the active site.

  4. Concurrent cooperativity and substrate inhibition in the epoxidation of carbamazepine by cytochrome P450 3A4 active site mutants inspired by molecular dynamics simulations.

    PubMed

    Müller, Christian S; Knehans, Tim; Davydov, Dmitri R; Bounds, Patricia L; von Mandach, Ursula; Halpert, James R; Caflisch, Amedeo; Koppenol, Willem H

    2015-01-27

    Cytochrome P450 3A4 (CYP3A4) is the major human P450 responsible for the metabolism of carbamazepine (CBZ). To explore the mechanisms of interactions of CYP3A4 with this anticonvulsive drug, we carried out multiple molecular dynamics (MD) simulations, starting with the complex of CYP3A4 manually docked with CBZ. On the basis of these simulations, we engineered CYP3A4 mutants I369F, I369L, A370V, and A370L, in which the productive binding orientation was expected to be stabilized, thus leading to increased turnover of CBZ to the 10,11-epoxide product. In addition, we generated CYP3A4 mutant S119A as a control construct with putative destabilization of the productive binding pose. Evaluation of the kinetics profiles of CBZ epoxidation demonstrate that CYP3A4-containing bacterial membranes (bactosomes) as well as purified CYP3A4 (wild-type and mutants I369L/F) exhibit substrate inhibition in reconstituted systems. In contrast, mutants S119A and A370V/L exhibit S-shaped profiles that are indicative of homotropic cooperativity. MD simulations with two to four CBZ molecules provide evidence that the substrate-binding pocket of CYP3A4 can accommodate more than one molecule of CBZ. Analysis of the kinetics profiles of CBZ metabolism with a model that combines the formalism of the Hill equation with an allowance for substrate inhibition demonstrates that the mechanism of interactions of CBZ with CYP3A4 involves multiple substrate-binding events (most likely three). Despite the retention of the multisite binding mechanism in the mutants, functional manifestations reveal an exquisite sensitivity to even minor structural changes in the binding pocket that are introduced by conservative substitutions such as I369F, I369L, and A370V.

  5. Effects of mace and nutmeg on human cytochrome P450 3A4 and 2C9 activity.

    PubMed

    Kimura, Yuka; Ito, Hideyuki; Hatano, Tsutomu

    2010-01-01

    Pharmacokinetic or pharmacodynamic interactions between herbal medicines or food constituents and drugs have been studied as crucial factors determining therapeutic efficacy and outcome. Most of these interactions are attributed to inhibition or induction of activity of cytochrome P450 (CYP) metabolic enzymes. Inhibition or induction of CYP enzymes by beverages, including grapefruit, pomegranate, or cranberry juice, has been well documented. Because spices are a common daily dietary component, other studies have reported inhibition of CYP activity by spices or their constituents/derivatives. However, a systematic evaluation of various spices has not been performed. In this study, we investigated effects of 55 spices on CYP3A4 and CYP2C9 activity. Cinnamon, black or white pepper, ginger, mace, and nutmeg significantly inhibited CYP3A4 or CYP2C9 activity. Furthermore, bioassay-guided fractionation of mace (Myristica fragrans) led to isolation and structural characterization of a new furan derivative (1) along with other 16 known compounds, including an acylphenol, neolignans, and phenylpropanoids. Among these isolates, (1S,2R)-1-acetoxy-2-(4-allyl-2,6-dimethoxyphenoxy)-1-(3,4-dimethoxyphenyl)propane (9) exhibited the most potent CYP2C9 inhibitory activity with an IC₅₀ value comparable to that of sulfaphenazole, a CYP2C9 inhibitor. Compound 9 competitively inhibited CYP2C9-mediated 4'-hydroxylation of diclofenac. The inhibitory constant (K(i)) of 9 was determined to be 0.037 µM. Compound 9 was found to be 14-fold more potent than was sulfaphenazole.

  6. [Protein-protein interactions of cytochromes P450 3A4 and 3A5 with their intermediate redox partners cytochromes b5].

    PubMed

    Gnedenko, O V; Ivanov, A S; Iablokov, E O; Usanov, S A; Mukha, D V; Sergeev, G V; Kuzikov, A V; Moskaleva, N E; Bulko, T V; Shumiantseva, V V; Archakov, A I

    2014-01-01

    Molecular interactions between proteins redox partners (cytochromes P450 3A4, 3A5 and cytochrome b5) within the monooxygenase system, which is known to be involved in drug biotransformation, were investigated. Human cytochromes P450 3A4 and 3A5 (CYP3A4 and CYP3A5) form complexes with various cytochromes b5: the microsomal (b5mc) and mitochondrial (b5om) forms of this protein, as well as with 2 "chimeric" proteins, b5(om-mc), b5(mc-om). Kinetic constants and equilibrium dissociation constants were determined by the SPR biosensor. Essential distinction between CYP3A4 and CYP3A5 was only observed upon their interactions with cytochrome b5om. Electroanalytical characteristics of electrodes with immobilized hemoproteins were obtained. The electrochemical analysis of CYP3A4, CYP3A5, b5mc, b5om, b5(om-mc), and b5(mc-om) immobilized on screen printed graphite electrodes modified with membranous matrix revealed that these proteins have very close reduction potentials -0.435 - -0.350 V (vs. Ag/AgCl). Cytochrome b5mc was shown to be capable of stimulating the electrocatalytic activity of CYP3A4 to testosterone.

  7. Cytochrome P450 2D6 and 3A4 enzyme inhibition by amine stimulants in dietary supplements.

    PubMed

    Liu, Yitong; Santillo, Michael F

    2016-01-01

    A number of dietary supplements used for weight loss and athletic performance enhancement have been recently shown to contain a variety of stimulants, for which there is a lack of pharmacological and toxicological information. One concern for these emerging compounds is their potential to inhibit metabolic enzymes in the liver such as cytochromes P450 (CYP), which can lead to unexpected interactions among dietary supplements, drugs, and other xenobiotics. In this study, inhibition of human recombinant CYP2D6 and CYP3A4 by 27 amine stimulants associated with dietary supplements and their analogs was evaluated by luminescence assays. The strongest CYP2D6 inhibitors were coclaurine (IC50  = 0.14 ± 0.01 μM) and N-benzylphenethylamine (IC50  = 0.7 ± 0.2 μM), followed by several other relatively strong inhibitors (IC50 , 2-12 μM) including β-methylphenethylamine, N,β-dimethylphenethylamine (phenpromethamine), 1,3-dimethylamylamine (DMAA), N,α-diethylphenethylamine, higenamine (norcoclaurine) and N,N-diethylphenethylamine. Only nine compounds inhibited CYP3A4 by 20-55% at 100 μM. Results of this study illustrate that several amine stimulants associated with dietary supplements inhibit CYP2D6 and CYP3A4 in vitro, and these compounds may participate in adverse drug-dietary supplement interactions in vivo. Copyright © 2015 John Wiley & Sons, Ltd.

  8. A neural network based virtual screening of cytochrome P450 3A4 inhibitors.

    PubMed

    Molnar, László; Keseru, György M

    2002-02-11

    A virtual screening test to identify potential CP450 3A4 inhibitors has been developed. Molecular structures of inhibitors and non-inhibitors available in the Genetest database were represented using 2D Unity fingerprints and a feedforward neural network was trained to classify molecules regarding their inhibitory activity. Validation tests revealed that our neural net recognizes at least 89% of 3A4 inhibitors and suggest using this methodology in our virtual screening protocol.

  9. Differential Interactions of Cytochrome P450 3A5 and 3A4 with Chemotherapeutic Agent-Vincristine: A Comparative Molecular Dynamics Study.

    PubMed

    Saba, Nikhat; Bhuyan, Rajabrata; Nandy, Suman Kumar; Seal, Alpana

    2015-01-01

    The chemotherapeutic agent vincristine, used for treatment of acute lymphoblastic leukemia is metabolized preferentially by polymorphic cytochrome P450 3A5 (CYP3A5) with higher clearance rate than cytochrome P450 3A4 (CYP3A4). As a result, CYP3A5 expressers have a reduced amount of vincristine-induced peripheral neuropathy than non-expressers. We modeled the structure of CYP3A5 and its interaction with vincristine, compared with CYP3A4-vincristine complex using molecular docking and simulation studies. This relative study helped us to understand the molecular mechanisms behind the interaction at the atomic level through interaction energy, binding free energy, hydrogen bond and solvent accessible surface area analysis - giving an insight into the binding mode and the main residues involved in this particular interaction. Our results show that the interacting groups get closer in CYP3A5-vincristine complex due to different orientation of vincristine. This leads to higher binding affinity of vincristine towards CYP3A5 compared to CYP3A4 and explains the preferential metabolism of vincristine by CYP3A5. We believe that, the results of the current study will be helpful for future studies on structure-based drug design in this area.

  10. Peripheral ligand-binding site in cytochrome P450 3A4 located with fluorescence resonance energy transfer (FRET).

    PubMed

    Davydov, Dmitri R; Rumfeldt, Jessica A O; Sineva, Elena V; Fernando, Harshica; Davydova, Nadezhda Y; Halpert, James R

    2012-02-24

    The mechanisms of ligand binding and allostery in the major human drug-metabolizing enzyme cytochrome P450 3A4 (CYP3A4) were explored with fluorescence resonance energy transfer (FRET) using a laser dye, fluorol-7GA (F7GA), as a model substrate. Incorporation into the enzyme of a thiol-reactive FRET probe, pyrene iodoacetamide, allowed us to monitor the binding by FRET from the pyrene donor to the F7GA acceptor. Cooperativity of the interactions detected by FRET indicates that the enzyme possesses at least two F7GA-binding sites that have different FRET efficiencies and are therefore widely separated. To probe spatial localization of these sites, we studied FRET in a series of mutants bearing pyrene iodoacetamide at different positions, and we measured the distances from each of the sites to the donor. Our results demonstrate the presence of a high affinity binding site at the enzyme periphery. Analysis of the set of measured distances complemented with molecular modeling and docking allowed us to pinpoint the most probable peripheral site. It is located in the vicinity of residues 217-220, similar to the position of the progesterone molecule bound at the distal surface of the CYP3A4 in a prior x-ray crystal structure. Peripheral binding of F7GA causes a substantial spin shift and serves as a prerequisite for the binding in the active site. This is the first indication of functionally important ligand binding outside of the active site in cytochromes P450. The findings strongly suggest that the mechanisms of CYP3A4 cooperativity involve a conformational transition triggered by an allosteric ligand.

  11. Spectroscopic studies and molecular docking on the interaction of organotin antitumor compound bis[2,4-difluoro-N-(hydroxy-⟨κ⟩O)benzamidato-⟨κ⟩O]diphenyltin(IV) with human cytochrome P450 3A4 protease

    NASA Astrophysics Data System (ADS)

    Wei, Ying; Niu, Lin; Liu, Xinxin; Zhou, Hongyan; Dong, Hongzhou; Kong, Depeng; Li, Yunlan; Li, Qingshan

    2016-06-01

    A novel organotin DFDPT was synthesized and characterized by elemental analysis, IR, 1H, 13C, 119Sn, NMR techniques,etc. In order to investigate profoundly the relationship between DFDPT with human CYP3A4 proteaset and anticancer molecular mechanism of DFDPT, the intercalative mode of binding of DFDPT with CYP3A4 under physiological conditions were comprehensively evaluated using steady state, synchronous, three-dimensional fluorescence spectroscopy,circular dichroism and molecular docking. Fluorescence emission data showed that CYP3A4 fluorescence affected by DFDPT was a static quenching procedure, which implied that DFDPT-CYP3A4 complex had been formed. Apparent binding constants Kb of CYP3A4 with compound at 298 and 310 K were 2.51 × 107 and 3.09 × 105, respectively. The binding sites number n was 1.64 and 1.22, respectively. The thermodynamic parameters ΔH and ΔS of the DFDPT-CYP3A4 complex were negative, which indicated that their interaction was driven mainly by hydrogen bonding and van der Waals force. The binding of DFDPT-CYP3A4 was spontaneous process in which ΔG was negative. The synchronous results showed DFDPT induced conformational changes of CYP3A4 protein. Three-dimensional fluorescence and circular dichroism spectra results also revealed conformation of CYP3A4 protein had been possible changed in the presence of DFDPT. Molecular docking was used to study the interaction orientation between DFDPT and CYP3A4 protease. The results indicated that DFDPT interacted with a panel of amino acids in the active sites of CYP3A4 protein mainly through formation of hydrogen bond. Furthermore, the predicted binding mode of DFDPT into CYP3A4 appeared to adopt an orientation with interactions among Arg105, Ser119 and Thr309.

  12. Spectral Resolution of a Second Binding Site for Nile Red on Cytochrome P450 3A4

    PubMed Central

    Nath, Abhinav; Fernández, Cristina; Lampe, Jed N.; Atkins, William M.

    2008-01-01

    Nile Red is sequentially metabolized by Cytochrome P4503A4 to the N-monoethyl and N-desethyl products, which typifies the metabolism of many amine-containing drugs. Sequential metabolism of a single substrate results in complex kinetics that confound predictive models of drug clearance. As a fluorescent model for drugs which undergo sequential metabolism, Nile Red provides the opportunity to monitor drug-CYP interactions wherein the fluorescent properties of Nile Red could, in principle, be exploited to determine individual rate and equilibrium constants for the individual reactions. Previously, it was shown that Nile Red binds at the active site and fluoresces (KD = 50 nM) with maximum emission at ~620 nm, but it was unclear whether a red-shifted emission, at ~660 nm, consisted of only free Nile Red or Nile Red bound at a second site on the protein. Here, equilibrium binding studies, including ‘reverse titrations’ spanning low ratios of CYP3A4/Nile Red, indicate two binding sites for Nile Red with a contribution to the ‘red emission’ greater than can be accounted for by free Nile Red. Singular value decomposition affords basis spectra for both spectral components and fits well to the experimentally determined concentration dependence of Nile Red emission. In addition, the red spectral component, with an apparent KD = 2.2 µM, is selectively eliminated by titration with the known allosteric effectors of CYP3A4, α-napthoflavone and testosterone. Furthermore, the double mutant L2311F/D214E, previously demonstrated to perturb a peripheral allosteric site, lacks the red-emitting Nile Red binding site, but retains the blue-emitting site. Together these data indicate that a second Nile Red site competes with other effectors of CYP3A4 at a site that results in Nile Red emission at 660 nm. PMID:18395506

  13. Prediction of small-molecule binding to cytochrome P450 3A4: flexible docking combined with multidimensional QSAR.

    PubMed

    Lill, Markus A; Dobler, Max; Vedani, Angelo

    2006-01-01

    The inhibition of cytochrome P450 3A4 (CYP3A4) by small molecules is a major mechanism associated with undesired drug-drug interactions, which are responsible for a substantial number of late-stage failures in the pharmaceutical drug-development process. For a quantitative prediction of associated pharmacokinetic parameters, a computational model was developed that allows prediction of the inhibitory potential of 48 structurally diverse molecules. Based on the experimental structure of CYP3A4, possible binding modes were first sampled by using automated docking (Yeti software) taking protein flexibility into account. The results are consistent with both X-ray crystallographic data and data from metabolic studies. Next, an ensemble of energetically favorable orientations was composed into a 4D dataset for use as input for a multidimensional QSAR technique (Raptor software). A dual-shell binding-site model that allows an explicit induced fit was then generated by using hydrophobicity scoring and hydrogen-bond propensity. The simulation reached a cross-validated r2 value of 0.825 and a predictive r2 value of 0.659. On average, the predicted binding affinity of the training ligands deviates by a factor of 2.7 from the experiment; those of the test set deviate by a factor of 3.8 in Ki.

  14. [Protein-protein interactions of cytochromes P450 3A4 and 3A5 with their intermediate redox partners cytochromes b5].

    PubMed

    Gnedenko, O V; Ivanov, A S; Yablokov, E O; Usanov, S A; Mukha, D V; Sergeev, G V; Kuzikov, A V; Bulko, T V; Moskaleva, N E; Shumyantseva, V V; Archakov, A I

    2015-01-01

    Molecular interactions between proteins redox partners (cytochromes Р450 3А4, 3А5 and cytochrome b5) within the monooxygenase system, which is known to be involved in drug biotransformation, were investigated. Human cytochromes Р450 3А4 and 3А5 (CYP3A4 and CYP3A5) form complexes with various cytochromes b5: the microsomal (b5mc) and mitochondrial (b5om) forms of this protein, as well as with 2 "chimeric" proteins, b5(om-mc), b5(mc-om). Kinetic constants and equilibrium dissociation constants were determined by the SPR biosensor. Essential distinction between CYP3A4 and CYP3A5 was only observed upon their interactions with cytochrome b5om. Electroanalytical characteristics of electrodes with immobilized hemoproteins were obtained. The electrochemical analysis of CYP3A4, CYP3A5, b5mc, b5om, b5(om-mc), and b5(mc-om) immobilized on screen printed graphite electrodes modified with membranous matrix revealed that these proteins have very close reduction potentials -0.435  -0.350 V (vs. Ag/AgCl). Cytochrome b5mc was shown to be capable of stimulating the electrocatalytic activity of CYP3A4 in the presence of its substrate testosterone.

  15. Inhibition on human liver cytochrome P450 3A4 by constituents of fennel (Foeniculum vulgare): identification and characterization of a mechanism-based inactivator.

    PubMed

    Subehan; Zaidi, Syed F H; Kadota, Shigetoshi; Tezuka, Yasuhiro

    2007-12-12

    Fennel, a seed of Foeniculum vulgare, is used as a culinary spice and traditional medicine. The methanolic extract of fennel showed a characteristic of mechanism-based inactivation on erythromycin N-demethylation mediated by human liver microsomal cytochrome P450 3A4 (CYP3A4). The present study was conducted to identify the fennel constituent having the inhibition. Thirteen compounds have been isolated from a methanol extract of fennel and tested for their inhibition on CYP3A4. Among them, 5-methoxypsoralen (5-MOP) showed the strongest inhibition with an IC50 value of 18.3 microM and a mixed type of inhibition. In addition, with the preincubation time of 20 min only 5-MOP showed preincubation time dependency; the IC50 value decreased from 18.3 microM with a preincubation time of 0 min to 4.6 microM with a preincubation time of 20 min. Further investigation on 5-MOP showed the characteristics of time-dependent inhibition, requirement of NADPH, lack of protecting effect of nucleophiles, and recovery of CYP3A4 activity by the competitive inhibitor. This result suggests that the inhibitory activity of CYP3A4 by 5-MOP was a mechanism-based inactivation. The kinetic parameter for mechanism-based inactivation was characterized by a KI value of 15.0 microM and a kinact value of 0.098 min(-1).

  16. Mechanism of interactions of α-naphthoflavone with cytochrome P450 3A4 explored with an engineered enzyme bearing a fluorescent probe†

    PubMed Central

    Tsalkova, Tamara N.; Davydova, Nadezhda Y.; Halpert, James R.; Davydov, Dmitri R.

    2008-01-01

    Design of a partially cysteine-depleted C98S/C239S/C377S/C468A cytochrome P450 3A4 mutant designated CYP3A4(C58,C64) allowed site-directed incorporation of thiol-reactive fluorescent probes into α-helix A‥ The site of modification was identified as Cys-64 with the help of CYP3A4(C58) and CYP3A4(C64), each bearing only one accessible cysteine. Changes in the fluorescence of CYP3A4(C58,C64) labeled with 6-bromoacetyl-2-dimethylaminonaphthalene (BADAN), 7-diethylamino-3-(4’-maleimidylphenyl)-4-methylcoumarin (CPM), or monobromobimane (mBBr) were used to study the interactions with bromocriptine (BCT), 1-pyrenebutanol (1-PB), testosterone (TST), and α-naphthoflavone (ANF). Of these substrates only ANF has a specific effect, causing a considerable decrease in fluorescence intensity of BADAN and CPM and increasing the fluorescence of mBBr. This ANF-binding event in the case of BADAN-modified enzyme is characterized by an S50 of 18.2 ± 0.7, compared with the value of 2.2 ± 0.3 for the ANF-induced spin transition, thus revealing an additional low affinity binding site. Studies of the effect of TST, 1-PB, and BCT on the interactions of ANF monitored by changes in fluorescence of CYP3A4(C58,C64)-BADAN or by the ANF-induced spin transition revealed no competition by these substrates. Investigation of the kinetics of fluorescence increase upon H2O2-dependent heme depletion suggests that labeled CYP3A4(C58,C64) is represented by two conformers, one of which has the fluorescence of the BADAN and CPM labels completely quenched, presumably by photoinduced electron transfer from the neighboring Trp-72 and/or Tyr-68 residues. The binding of ANF to the newly discovered binding site appears to affect the interactions of the label with the above residue(s), thus modulating the fraction of the fluorescent conformer. PMID:17198380

  17. Risk of adverse events among older adults following co-prescription of clarithromycin and statins not metabolized by cytochrome P450 3A4

    PubMed Central

    Li, Daniel Q.; Kim, Richard; McArthur, Eric; Fleet, Jamie L.; Bailey, David G.; Juurlink, David; Shariff, Salimah Z.; Gomes, Tara; Mamdani, Muhammad; Gandhi, Sonja; Dixon, Stephanie; Garg, Amit X.

    2015-01-01

    Background: The cytochrome P450 3A4 (CYP3A4) inhibitor clarithromycin may also inhibit liver-specific organic anion–transporting polypeptides (OATP1B1 and OATP1B3). We studied whether concurrent use of clarithromycin and a statin not metabolized by CYP3A4 was associated with an increased frequency of serious adverse events. Methods: Using large health care databases, we studied a population-based cohort of older adults (mean age 74 years) who were taking a statin not metabolized by CYP3A4 (rosuvastatin [76% of prescriptions], pravastatin [21%] or fluvastatin [3%]) between 2002 and 2013 and were newly prescribed clarithromycin (n = 51 523) or azithromycin (n = 52 518), the latter an antibiotic that inhibits neither CYP3A4 nor OATP1B1 and OATP1B3. Outcomes were hospital admission with a diagnostic code for rhabdomyolysis, acute kidney injury or hyperkalemia, and all-cause mortality. All outcomes were assessed within 30 days after co-prescription. Results: Compared with the control group, patients co-prescribed clarithromycin and a statin not metabolized by CYP3A4 were at increased risk of hospital admission with acute kidney injury (adjusted relative risk [RR] 1.65, 95% confidence interval [CI] 1.31 to 2.09), admission with hyperkalemia (adjusted RR 2.17, 95% CI 1.22 to 3.86) and all-cause mortality (adjusted RR 1.43, 95% CI 1.15 to 1.76). The adjusted RR for admission with rhabdomyolysis was 2.27 (95% CI 0.86 to 5.96). The absolute increase in risk for each outcome was small and likely below 1%, even after we considered the insensitivity of some hospital database codes. Interpretation: Among older adults taking a statin not metabolized by CYP3A4, co-prescription of clarithromycin versus azithromycin was associated with a modest but statistically significant increase in the 30-day absolute risk of adverse outcomes. PMID:25534598

  18. A Mechanism-Based Model for the Prediction of the Metabolic Sites of Steroids Mediated by Cytochrome P450 3A4.

    PubMed

    Dai, Zi-Ru; Ai, Chun-Zhi; Ge, Guang-Bo; He, Yu-Qi; Wu, Jing-Jing; Wang, Jia-Yue; Man, Hui-Zi; Jia, Yan; Yang, Ling

    2015-06-30

    Early prediction of xenobiotic metabolism is essential for drug discovery and development. As the most important human drug-metabolizing enzyme, cytochrome P450 3A4 has a large active cavity and metabolizes a broad spectrum of substrates. The poor substrate specificity of CYP3A4 makes it a huge challenge to predict the metabolic site(s) on its substrates. This study aimed to develop a mechanism-based prediction model based on two key parameters, including the binding conformation and the reaction activity of ligands, which could reveal the process of real metabolic reaction(s) and the site(s) of modification. The newly established model was applied to predict the metabolic site(s) of steroids; a class of CYP3A4-preferred substrates. 38 steroids and 12 non-steroids were randomly divided into training and test sets. Two major metabolic reactions, including aliphatic hydroxylation and N-dealkylation, were involved in this study. At least one of the top three predicted metabolic sites was validated by the experimental data. The overall accuracy for the training and test were 82.14% and 86.36%, respectively. In summary, a mechanism-based prediction model was established for the first time, which could be used to predict the metabolic site(s) of CYP3A4 on steroids with high predictive accuracy.

  19. Piperine activates human pregnane X receptor to induce the expression of cytochrome P450 3A4 and multidrug resistance protein 1

    PubMed Central

    Wang, Yue-Ming; Lin, Wenwei; Chai, Sergio C.; Wu, Jing; Ong, Su Sien; Schuetz, Erin G.; Chen, Taosheng

    2013-01-01

    Activation of the pregnane X receptor (PXR) and subsequently its target genes, including those encoding drug transporters and metabolizing enzymes, while playing substantial roles in xenobiotics detoxification, might cause undesired drug-drug interactions. Recently, an increased awareness has been given to dietary components for potential induction of diet-drug interactions through activation of PXR. Here, we studied, whether piperine (PIP), a major component extracted from the widely-used daily spice black pepper, could induce PXR-mediated expression of cytochrome P450 3A4 (CYP3A4) and multidrug resistance protein 1 (MDR1). Our results showed that PIP activated human PXR (hPXR)-mediated CYP3A4 and MDR1 expression in human hepatocytes, intestine cells, and a mouse model; PIP activated hPXR by recruiting its coactivator SRC-1 in both cellular and cell-free systems; PIP bound to the hPXR ligand binding domain in a competitive ligand binding assay in vitro. The dichotomous effects of PIP on induction of CYP3A4 and MDR1 expression observed here and inhibition of their activity reported elsewhere challenges the potential use of PIP as a bioavailability enhancer and suggests that cautions should be taken for PIP consumption during drug treatment in patients, particularly those who favor daily pepper spice or rely on certain pepper remedies. PMID:23707768

  20. A Mechanism-Based Model for the Prediction of the Metabolic Sites of Steroids Mediated by Cytochrome P450 3A4

    PubMed Central

    Dai, Zi-Ru; Ai, Chun-Zhi; Ge, Guang-Bo; He, Yu-Qi; Wu, Jing-Jing; Wang, Jia-Yue; Man, Hui-Zi; Jia, Yan; Yang, Ling

    2015-01-01

    Early prediction of xenobiotic metabolism is essential for drug discovery and development. As the most important human drug-metabolizing enzyme, cytochrome P450 3A4 has a large active cavity and metabolizes a broad spectrum of substrates. The poor substrate specificity of CYP3A4 makes it a huge challenge to predict the metabolic site(s) on its substrates. This study aimed to develop a mechanism-based prediction model based on two key parameters, including the binding conformation and the reaction activity of ligands, which could reveal the process of real metabolic reaction(s) and the site(s) of modification. The newly established model was applied to predict the metabolic site(s) of steroids; a class of CYP3A4-preferred substrates. 38 steroids and 12 non-steroids were randomly divided into training and test sets. Two major metabolic reactions, including aliphatic hydroxylation and N-dealkylation, were involved in this study. At least one of the top three predicted metabolic sites was validated by the experimental data. The overall accuracy for the training and test were 82.14% and 86.36%, respectively. In summary, a mechanism-based prediction model was established for the first time, which could be used to predict the metabolic site(s) of CYP3A4 on steroids with high predictive accuracy. PMID:26133240

  1. Piperine activates human pregnane X receptor to induce the expression of cytochrome P450 3A4 and multidrug resistance protein 1

    SciTech Connect

    Wang, Yue-Ming; Lin, Wenwei; Chai, Sergio C.; Wu, Jing; Ong, Su Sien; Schuetz, Erin G.; Chen, Taosheng

    2013-10-01

    Activation of the pregnane X receptor (PXR) and subsequently its target genes, including those encoding drug transporters and metabolizing enzymes, while playing substantial roles in xenobiotic detoxification, might cause undesired drug-drug interactions. Recently, an increased awareness has been given to dietary components for potential induction of diet–drug interactions through activation of PXR. Here, we studied, whether piperine (PIP), a major component extracted from the widely-used daily spice black pepper, could induce PXR-mediated expression of cytochrome P450 3A4 (CYP3A4) and multidrug resistance protein 1 (MDR1). Our results showed that PIP activated human PXR (hPXR)-mediated CYP3A4 and MDR1 expression in human hepatocytes, intestine cells, and a mouse model; PIP activated hPXR by recruiting its coactivator SRC-1 in both cellular and cell-free systems; PIP bound to the hPXR ligand binding domain in a competitive ligand binding assay in vitro. The dichotomous effects of PIP on induction of CYP3A4 and MDR1 expression observed here and inhibition of their activity reported elsewhere challenges the potential use of PIP as a bioavailability enhancer and suggests that caution should be taken in PIP consumption during drug treatment in patients, particularly those who favor daily pepper spice or rely on certain pepper remedies. - Highlights: • Piperine induces PXR-mediated CYP3A4 and MDR1 expression. • Piperine activates PXR by binding to PXR and recruiting coactivator SRC-1. • Piperine induces PXR activation in vivo. • Caution should be taken in piperine consumption during drug treatment.

  2. Pyrethroid insecticides: isoform-dependent hydrolysis, induction of cytochrome P450 3A4 and evidence on the involvement of the pregnane X receptor.

    PubMed

    Yang, Dongfang; Wang, Xiliang; Chen, Yi-Tzai; Deng, Ruitang; Yan, Bingfang

    2009-05-15

    Pyrethroids account for more than one-third of the insecticides currently marketed in the world. In mammals, these insecticides undergo extensive metabolism by carboxylesterases and cytochrome P450s (CYPs). In addition, some pyrethroids are found to induce the expression of CYPs. The aim of this study was to determine whether pyrethroids induce carboxylesterases and CYP3A4, and whether the induction is correlated inversely with their hydrolysis. Human liver microsomes were pooled and tested for the hydrolysis of 11 pyrethroids. All pyrethroids were hydrolyzed by the pooled microsomes, but the hydrolytic rates varied by as many as 14 fold. Some pyrethroids such as bioresmethrin were preferably hydrolyzed by carboxylesterase HCE1, whereas others such as bifenthrin preferably by HCE2. In primary human hepatocytes, all pyrethroids except tetramethrin significantly induced CYP3A4. In contrast, insignificant changes were detected on the expression of carboxylesterases. The induction of CYP3A4 was confirmed in multiple cell lines including HepG2, Hop92 and LS180. Overall, the magnitude of the induction was correlated inversely with the rates of hydrolysis, but positively with the activation of the pregnane X receptor (PXR). Transfection of a carboxylesterase markedly decreased the activation of PXR, and the decrease was in agreement with carboxylesterase-based preference for hydrolysis. In addition, human PXR variants as well as rat PXR differed from human PXR (wild-type) in responding to certain pyrethroids (e.g., lambda-cyhalothrin), suggesting that induction of PXR target genes by these pyrethroids varies depending on polymorphic variants and the PXR species identity.

  3. Pyrethroid insecticides: Isoform-dependent hydrolysis, induction of cytochrome P450 3A4 and evidence on the involvement of the pregnane X receptor

    SciTech Connect

    Yang Dongfang; Wang Xiliang; Chen Yitzai; Deng Ruitang; Yan Bingfang

    2009-05-15

    Pyrethroids account for more than one-third of the insecticides currently marketed in the world. In mammals, these insecticides undergo extensive metabolism by carboxylesterases and cytochrome P450s (CYPs). In addition, some pyrethroids are found to induce the expression of CYPs. The aim of this study was to determine whether pyrethroids induce carboxylesterases and CYP3A4, and whether the induction is correlated inversely with their hydrolysis. Human liver microsomes were pooled and tested for the hydrolysis of 11 pyrethroids. All pyrethroids were hydrolyzed by the pooled microsomes, but the hydrolytic rates varied by as many as 14 fold. Some pyrethroids such as bioresmethrin were preferably hydrolyzed by carboxylesterase HCE1, whereas others such as bifenthrin preferably by HCE2. In primary human hepatocytes, all pyrethroids except tetramethrin significantly induced CYP3A4. In contrast, insignificant changes were detected on the expression of carboxylesterases. The induction of CYP3A4 was confirmed in multiple cell lines including HepG2, Hop92 and LS180. Overall, the magnitude of the induction was correlated inversely with the rates of hydrolysis, but positively with the activation of the pregnane X receptor (PXR). Transfection of a carboxylesterase markedly decreased the activation of PXR, and the decrease was in agreement with carboxylesterase-based preference for hydrolysis. In addition, human PXR variants as well as rat PXR differed from human PXR (wild-type) in responding to certain pyrethroids (e.g., lambda-cyhalothrin), suggesting that induction of PXR target genes by these pyrethroids varies depending on polymorphic variants and the PXR species identity.

  4. Thiazide-like diuretic drug metolazone activates human pregnane X receptor to induce cytochrome 3A4 and multidrug-resistance protein 1

    PubMed Central

    Banerjee, Monimoy; Chen, Taosheng

    2014-01-01

    Human pregnane X receptor (hPXR) regulates the expression of drug-metabolizing enzyme cytochrome P450 3A4 (CYP3A4) and drug transporters such as multidrug-resistance protein 1 (MDR1). PXR can be modulated by small molecules, including Federal Drug Administration (FDA)–approved drugs, thus altering drug metabolism and causing drug-drug interactions. To determine the role of FDA-approved drugs in PXR-mediated regulation of drug metabolism and clearance, we screened 1481 FDA-approved small-molecule drugs by using a luciferase reporter assay in HEK293T cells and identified the diuretic drug metolazone as an activator of PXR. Our data showed that metolazone activated hPXR-mediated expression of CYP3A4 and MDR1 in human hepatocytes and intestine cells and increased CYP3A4 promoter activity in various cell lines. Mammalian two-hybrid assays showed that hPXR recruits its co-activator SRC-1 upon metolazone binding in HepG2 cells, explaining the mechanism of hPXR activation. To understand the role of other commonly-used diuretics in PXR activation and the structure-activity relationship of metolazone, thiazide and non-thiazide diuretics drugs were also tested but only metolazone activates PXR. To understand the molecular mechanism, docking studies and mutational analysis were carried out and showed that metolazone binds in the ligand-binding pocket and interacts with mostly hydrophobic amino acid residues. This is the first report showing that metolazone activates PXR. Because activation of hPXR might cause drug-drug interactions, metolazone should be used with caution for drug treatment in patients undergoing combination therapy. PMID:25181459

  5. Thiazide-like diuretic drug metolazone activates human pregnane X receptor to induce cytochrome 3A4 and multidrug-resistance protein 1.

    PubMed

    Banerjee, Monimoy; Chen, Taosheng

    2014-11-15

    Human pregnane X receptor (hPXR) regulates the expression of drug-metabolizing enzyme cytochrome P450 3A4 (CYP3A4) and drug transporters such as multidrug-resistance protein 1 (MDR1). PXR can be modulated by small molecules, including Federal Drug Administration (FDA)-approved drugs, thus altering drug metabolism and causing drug-drug interactions. To determine the role of FDA-approved drugs in PXR-mediated regulation of drug metabolism and clearance, we screened 1481 FDA-approved small-molecule drugs by using a luciferase reporter assay in HEK293T cells and identified the diuretic drug metolazone as an activator of hPXR. Our data showed that metolazone activated hPXR-mediated expression of CYP3A4 and MDR1 in human hepatocytes and intestine cells and increased CYP3A4 promoter activity in various cell lines. Mammalian two-hybrid assays showed that hPXR recruits its co-activator SRC-1 upon metolazone binding in HepG2 cells, explaining the mechanism of hPXR activation. To understand the role of other commonly-used diuretics in hPXR activation and the structure-activity relationship of metolazone, thiazide and non-thiazide diuretics drugs were also tested but only metolazone activates hPXR. To understand the molecular mechanism, docking studies and mutational analysis were carried out and showed that metolazone binds in the ligand-binding pocket and interacts with mostly hydrophobic amino acid residues. This is the first report showing that metolazone activates hPXR. Because activation of hPXR might cause drug-drug interactions, metolazone should be used with caution for drug treatment in patients undergoing combination therapy.

  6. Biotransformations of 6',7'-dihydroxybergamottin and 6',7'-epoxybergamottin by the citrus-pathogenic fungi diminish cytochrome P450 3A4 inhibitory activity.

    PubMed

    Myung, Kyung; Manthey, John A; Narciso, Jan A

    2012-03-15

    Penicillium digitatum, as well as five other citrus pathogenic species, (Penicillium ulaiense Link, Geotrichum citri Link, Botrytis cinerea P. Micheli ex Pers., Lasiodiplodia theobromae (Pat.) Griffon & Maubl., and Phomopsis citri (teleomorph Diaporthe citri)) were observed to convert 6',7'-epoxybergamottin (1) into 6',7'-dihydroxybergamottin (2), bergaptol (3), and an opened lactone ring metabolite 6,7-furano-5-(6',7'-dihydroxy geranyloxy)-2-hydroxy-hydrocoumaric acid (4). Metabolism of 2 by these fungi also proceeded to 4. The structure of 4 was established by high resolution mass spectrometry and (1)H and (13)C NMR techniques. The inhibitory activity of 4 towards human intestinal cytochrome P450 3A4 (CYP3A4) was greatly decreased (IC(50) >172.0 μM) compared to 2 (IC(50)=0.81 μM). PMID:22342630

  7. A Cytochrome P450 3A4 Biosensor Based on Generation 4.0 PAMAM Dendrimers for the Detection of Caffeine

    PubMed Central

    Müller, Michael; Agarwal, Neha; Kim, Jungtae

    2016-01-01

    Cytochromes P450 (CYP, P450) are a large family of heme-active-site proteins involved in many catalytic processes, including steroidogenesis. In humans, four primary enzymes are involved in the metabolism of almost all xenobiotics. Among these enzymes, CYP3A4 is responsible for the inactivation of the majority of used drugs which makes this enzyme an interesting target for many fields of research, especially pharmaceutical research. Since the late 1970s, attempts have been made to construct and develop electrochemical sensors for the determination of substrates. This paper is concerned with the establishment of such a CYP3A4-containing biosensor. The sensor was constructed by adsorption of alternating layers of sub-nanometer gold particle-modified PAMAM (poly-amido-amine) dendrimers of generation 4.0, along with the enzyme by a layer-by-layer assembly technique. Atomic force microscopy (AFM), quartz crystal microbalance (QCM), and Fourier-transformed infrared spectroscopy (FTIR) were employed to elucidate the sensor assembly. Additionally, the biosensor was tested by cyclic voltammetry using caffeine as a substrate. PMID:27548239

  8. A Cytochrome P450 3A4 Biosensor Based on Generation 4.0 PAMAM Dendrimers for the Detection of Caffeine.

    PubMed

    Müller, Michael; Agarwal, Neha; Kim, Jungtae

    2016-01-01

    Cytochromes P450 (CYP, P450) are a large family of heme-active-site proteins involved in many catalytic processes, including steroidogenesis. In humans, four primary enzymes are involved in the metabolism of almost all xenobiotics. Among these enzymes, CYP3A4 is responsible for the inactivation of the majority of used drugs which makes this enzyme an interesting target for many fields of research, especially pharmaceutical research. Since the late 1970s, attempts have been made to construct and develop electrochemical sensors for the determination of substrates. This paper is concerned with the establishment of such a CYP3A4-containing biosensor. The sensor was constructed by adsorption of alternating layers of sub-nanometer gold particle-modified PAMAM (poly-amido-amine) dendrimers of generation 4.0, along with the enzyme by a layer-by-layer assembly technique. Atomic force microscopy (AFM), quartz crystal microbalance (QCM), and Fourier-transformed infrared spectroscopy (FTIR) were employed to elucidate the sensor assembly. Additionally, the biosensor was tested by cyclic voltammetry using caffeine as a substrate. PMID:27548239

  9. Development and validation of an enzyme-linked immunosorbent assay for the quantification of cytochrome 3A4 in human liver microsomes.

    PubMed

    De Bock, Lies; Colin, Pieter; Boussery, Koen; Van Bocxlaer, Jan

    2012-09-15

    Little is known about the influence of hepatic pathologies on cytochrome P450 (CYP) mediated drug metabolism in children. The determination of the abundance of the different isoforms in pediatric microsomes may provide valuable information on the mechanisms of possible changes in activity. Until now, western blotting was mostly used for abundance measurements, but this technique only provides semi-quantitative data. Therefore, this study aimed to develop and validate an indirect ELISA for the quantification of the most important CYP isoform, CYP3A4, in human liver microsomes, using commercially available reagents. Samples, calibrators and validation samples were diluted to a final concentration of 10 μg microsomal protein/ml. A polyclonal antibody raised against the full length human protein was used as primary antibody; horseradish peroxidase conjugated secondary antibodies for detection. The assay was validated for sensitivity, working range and calibration, accuracy and precision. Amounts of CYP3A4 between 2 and 300 pmol/mg microsomal protein could be quantified with a 5-parameter logistics function with 1/x weighting factor. Coefficients of variation of intra and inter assay variability were between 9.54 and 13.98% (16.34% at LLOQ), and between 10.51 and 14.55% (19.44% at LLOQ), respectively. The relative error (%RE) varied between -5.96 and 6.68% (11.53% at LLOQ), and the total error between 11.93 and 21.23% (30.97% at LLOQ). The cross-reactivity of the method with human CYP2E1 showed to have no significant effect on the accuracy of the results. Successful analysis of five samples from an ongoing study demonstrated the usefulness of the method.

  10. A Combined Molecular Docking/Dynamics Approach to Probe the Binding Mode of Cancer Drugs with Cytochrome P450 3A4.

    PubMed

    Panneerselvam, Suresh; Yesudhas, Dhanusha; Durai, Prasannavenkatesh; Anwar, Muhammad Ayaz; Gosu, Vijayakumar; Choi, Sangdun

    2015-08-14

    Cytarabine, daunorubicin, doxorubicin and vincristine are clinically used for combinatorial therapies of cancers in different combinations. However, the knowledge about the interaction of these drugs with the metabolizing enzyme cytochrome P450 is limited. Therefore, we utilized computational methods to predict and assess the drug-binding modes. In this study, we performed docking, MD simulations and free energy landscape analysis to understand the drug-enzyme interactions, protein domain motions and the most populated free energy minimum conformations of the docked protein-drug complexes, respectively. The outcome of docking and MD simulations predicted the productive, as well as the non-productive binding modes of the selected drugs. Based on these interaction studies, we observed that S119, R212 and R372 are the major drug-binding residues in CYP3A4. The molecular mechanics Poisson-Boltzmann surface area analysis revealed the dominance of hydrophobic forces in the CYP3A4-drug association. Further analyses predicted the residues that may contain favorable drug-specific interactions. The probable binding modes of the cancer drugs from this study may extend the knowledge of the protein-drug interaction and pave the way to design analogs with reduced toxicity. In addition, they also provide valuable insights into the metabolism of the cancer drugs.

  11. Norcocaine and N-hydroxynorcocaine formation in human liver microsomes: role of cytochrome P-450 3A4.

    PubMed

    LeDuc, B W; Sinclair, P R; Shuster, L; Sinclair, J F; Evans, J E; Greenblatt, D J

    1993-05-01

    Cocaine was metabolized to norcocaine by microsomes prepared from lymphoblastoid cells expressing transfected human P-450 3A4. The specific activities of norcocaine formation by microsomes prepared from three human liver samples correlated with the amount of P-450 3A immunoreactive protein detected by immunoblot. Triacetyloleandomycin, a specific inhibitor of P-450 3A isoforms, inhibited formation of norcocaine from cocaine, but not formation of N-hydroxynorcocaine from norcocaine. The chemical identity of the norcocaine and N-hydroxynorcocaine produced by human liver microsomes was established by combination of gas chromatography and mass spectrometry. Thus, human P-450 3A4 is a cocaine demethylase, and P-450 isoforms of the 3A family are responsible for the majority of norcocaine production by human hepatic microsomes.

  12. Clopidogrel Has No Clinically Meaningful Effect on the Pharmacokinetics of the Organic Anion Transporting Polypeptide 1B1 and Cytochrome P450 3A4 Substrate Simvastatin.

    PubMed

    Itkonen, Matti K; Tornio, Aleksi; Neuvonen, Mikko; Neuvonen, Pertti J; Niemi, Mikko; Backman, Janne T

    2015-11-01

    Simvastatin and clopidogrel are commonly used together in the treatment of cardiovascular diseases. Organic anion transporting polypeptide (OATP) 1B1 activity markedly affects the hepatic uptake of simvastatin acid, whereas both simvastatin and simvastatin acid are sensitive to changes in cytochrome P450 3A4 activity. Clopidogrel and its metabolites inhibit OATP1B1 and CYP3A4 in vitro. We studied the effect of clopidogrel on the pharmacokinetics of simvastatin in a randomized crossover study. Twelve healthy volunteers ingested either a dose of placebo (control) or 300 mg of clopidogrel on day 1 and 75 mg on days 2 and 3. Simvastatin 40 mg was administered 1 hour after placebo and after clopidogrel on days 1 and 3. Plasma drug concentrations were measured for up to 12 hours. Clopidogrel 300 mg (day 1) increased the concentrations of simvastatin and simvastatin acid during the absorption phase. After clopidogrel 300 mg, the area under the concentration time curve (AUC) of simvastatin from 0 to 2 hours was 156% (P = 0.02) and its AUC(0-12 hours) was 132% (P = 0.08) of that during placebo, whereas the AUC(0-2 hours) and the AUC(0-12 hours) of simvastatin acid were 148% (P = 0.04) and 112% (P = 0.52) of control. Clopidogrel 75 mg (day 3) had no significant effect on the pharmacokinetic variables of simvastatin or simvastatin acid compared with placebo. The effect of clopidogrel seemed independent of the SLCO1B1 c.521T>C genotype. In conclusion, as clopidogrel did not have significant effects on the total exposure to simvastatin or simvastatin acid, clopidogrel does not seem to inhibit OATP1B1 or CYP3A4 to a clinically relevant extent.

  13. Linkage and association of haplotypes at the APOA1/C3/A4/A5 genecluster to familial combined hyperlipidemia

    SciTech Connect

    Eichenbaum-Voline, Sophie; Olivier, Michael; Jones, Emma L.; Naoumova, Rossitza P.; Jones, Bethan; Gau, Brian; Seed, Mary; Betteridge,D. John; Galton, David J.; Rubin, Edward M.; Scott, James; Shoulders,Carol C.; Pennacchio, Len A.

    2002-09-15

    Combined hyperlipidemia (CHL) is a common disorder of lipidmetabolism that leads to an increased risk of cardiovascular disease. Thelipid profile of CHL is characterised by high levels of atherogeniclipoproteins and low levels of high-density-lipoprotein-cholesterol.Apolipoprotein (APO) A5 is a newly discovered gene involved in lipidmetabolism located within 30kbp of the APOA1/C3/A4 gene cluster. Previousstudies have indicated that sequence variants in this cluster areassociated with increased plasma lipid levels. To establish whethervariation at the APOA5 gene contributes to the transmission of CHL, weperformed linkage and linkage disequilibrium (LD) tests on a large cohortof families (n=128) with familial CHL (FCHL). The linkage data producedevidence for linkage of the APOA1/C3/A4/A5 genomic interval to FCHL (NPL= 1.7, P = 0.042). The LD studies substantiated these data. Twoindependent rare alleles, APOA5c.56G and APOC3c.386G of this gene clusterwere over-transmitted in FCHL (P = 0.004 and 0.007, respectively), andthis was associated with a reduced transmission of the most commonAPOA1/C3/A4/A5 haplotype (frequency 0.4425) to affected subjects (P =0.013). The APOA5c.56G allele was associated with increased plasmatriglyceride levels in FCHL probands, whereas the second, andindependent, APOC3c.386G allele was associated with increased plasmatriglyceride levels in FCHL pedigree founders. Thus, this allele (or anallele in LD) may mark a quantitative trait associated with FCHL, as wellas representing a disease susceptibility locus for the condition. Thisstudy establishes that sequence variation in the APOA1/C3/A4/A5 genecluster contributes to the transmission of FCHL in a substantialproportion of affected families, and that these sequence variants mayalso contribute to the lipid abnormalities of the metabolic syndrome,which is present in up to 40 percent of persons with cardiovasculardisease.

  14. ALTERATION IN CYTOCHROME P450 3A4 ACTIVITY AS MEASURED BY A URINE CORTISOL ASSAY IN HIV-1-INFECTED PREGNANT WOMEN AND RELATIONSHIP TO ANTIRETROVIRAL PHARMACOKINETICS

    PubMed Central

    Aweeka, Francesca T.; Hu, Chengcheng; Huang, Liusheng; Best, Brookie M.; Stek, Alice; Lizak, Patricia; Burchett, Sandra K.; Read, Jennifer S.; Watts, Heather; Mirochnick, Mark; Capparelli, Edmund V.

    2014-01-01

    Objectives Pregnancy results in physiological changes altering the pharmacokinetics of drugs metabolized by cytochrome p450 3A4. The urinary ratio of 6-β hydroxycortisol to cortisol (6βHF:F) is a marker of CYP3A4 induction. We sought to evaluate its change in antiretroviral (ARV) treated HIV-1-infected women and to relate this change to ARV pharmacokinetics. Methods Women receiving various ARV had pharmacokinetic evaluations during third trimester pregnancy (>30 weeks) and postpartum with determination of 6βHF:F carried out on the same days. Wilcoxon signed rank test compared the ratio antepartum to postpartum. The relationship between the change in ratio to the change in pharmacokinetics was done using Kendall’s tau. Results 6βHF:F ratios were available for 107 women antepartum with 54 having postpartum values. The ratio was higher antepartum (p=0.033) [median comparison 1.35 (95% CI: 1.01, 1.81]. For 71 women taking a protease inhibitor (PI), the antepartum versus postpartum 6βHF:F comparison was marginally significant (p=0.058). When relating the change in the 6βHF:F ratio to the change in the dose-adjusted ARV AUC antepartum to postpartum, the 35 subjects in the LPV/r arms demonstrated an inverse relationship (p=0.125), albeit this correlation did not reach statistical significance. Conclusions A 35% increase in the urinary 6βHF:F ratio was measured during late pregnancy compared to postpartum, indicating CYP3A induction occurs during pregnancy. The trend to an inverse relationship between the change in the 6βHF:F ratio and the change in the LPV AUC antepartum versus postpartum suggests CYP3A induction may be one mechanism behind altered LPV exposure during pregnancy. PMID:25407158

  15. Minor furanocoumarins and coumarins in grapefruit peel oil as inhibitors of human cytochrome P450 3A4.

    PubMed

    César, Thaïs B; Manthey, John A; Myung, Kyung

    2009-09-01

    A new cyclic acetal (1) of marmin (6',7'-dihydroxy-7-geranyloxycoumarin), two new cyclic acetals (5, 6) of 6',7'-dihydroxybergamottin, and the known compounds marmin (2), 7-geranyloxycoumarin (3), bergamottin (4), and 6',7'-dihydroxybergamottin (7) were isolated from grapefruit peel oil. All compounds were tested for inhibitory activity against intestinal cytochrome P450 3A4, an enzyme involved in the "grapefruit/drug" interactions in humans. Coumarins (1-3) exhibited negligible inhibitory activity, while the furanocoumarins (4-7) showed potent in vitro inhibitory activity with IC(50) values of 2.42, 0.13, 0.27, and 1.58 microM, respectively. PMID:19689106

  16. Combined application of plasma mutagenesis and gene engineering leads to 5-oxomilbemycins A3/A4 as main components from Streptomyces bingchenggensis.

    PubMed

    Wang, Hai-Yan; Zhang, Ji; Zhang, Yue-Jing; Zhang, Bo; Liu, Chong-Xi; He, Hai-Rong; Wang, Xiang-Jing; Xiang, Wen-Sheng

    2014-12-01

    Milbemycin oxime has been commercialized as effective anthelmintics in the fields of animal health, agriculture, and human infections. Currently, milbemycin oxime is synthesized by a two-step chemical reaction, which involves the ketonization of milbemycins A3/A4 to yield the intermediates 5-oxomilbemycins A3/A4 using CrO3 as catalyst. Due to the low efficiency and environmental unfriendliness of the ketonization of milbemycins A3/A4, it is imperative to develop alternative strategies to produce 5-oxomilbemycins A3/A4. In this study, the atmospheric and room temperature plasma (ARTP) mutation system was first employed to treat milbemycin-producing strain Streptomyces bingchenggensis, and a mutant strain BC-120-4 producing milbemycins A3, A4, B2, and B3 as main components was obtained, which favors the construction of genetically engineered strains producing 5-oxomilbemycins. Importantly, the milbemycins A3/A4 yield of BC-120-4 reached 3,890 ± 52 g/l, which was approximately two times higher than that of the initial strain BC-109-6 (1,326 ± 37 g/l). The subsequent interruption of the gene milF encoding a C5-ketoreductase responsible for the ketonization of milbemycins led to strain BCJ60 (∆milF) with the production of 5-oxomilbemycins A3/A4 and the elimination of milbemycins A3, A4, B2, and B3. The high 5-oxomilbemycins A3/A4 yield (3,470 ± 147 g/l) and genetic stability of BCJ60 implied the potential use in industry to prepare 5-oxomilbemycins A3/A4 for the semisynthesis of milbemycins oxime. PMID:25081559

  17. Identification of cytochrome P450 3A4 modification site with reactive metabolite using linear ion trap-Fourier transform mass spectrometry.

    PubMed

    Yukinaga, Hideo; Takami, Tomonori; Shioyama, Sho-Hei; Tozuka, Zenzaburo; Masumoto, Hiroshi; Okazaki, Osamu; Sudo, Ken-Ichi

    2007-10-01

    Covalent binding of reactive metabolites to cytochrome P450s (P450s) often causes their mechanism-based inactivation (MBI), resulting in drug-drug interactions or toxicity. The detection and identification of the P450 sites to which reactive metabolites bind would elucidate MBI mechanisms. We describe a proteomic approach using nano-LC/linear ion trap-Fourier transform ion cyclotron resonance (FTICR) mass spectrometry to characterize the binding of a reactive metabolite of raloxifene, which is a known P450 3A4 inhibitor, to the P450 3A4 isozyme. LTQ-FT analyses revealed that the metabolic reaction of raloxifene in a reconstituted P450 3A4 system formed a reactive metabolite adduct to P450 3A4 apoprotein, accompanied by a mass shift of 471 Da relative to intact P450 3A4 apoprotein. The reaction mixtures were digested with trypsin, and then the tryptic digests were analyzed by nano-LC-MS/MS. This technique revealed that VWGFYDGQQPVLAITDPDMIK (position 71-91) was a tryptic peptide modified by the reactive metabolite derived from raloxifene. The site of adduction with the reactive metabolite was further postulated to be the nucleophilic OH group of Tyr-75 of P450 3A4. A proteomic approach using LTQ-FT can yield direct information on the P450 3A4 modification site without radiolabeled compounds. In addition, this information can elucidate mechanisms involved in the covalent binding of reactive metabolites and the inactivation of P450 3A4. PMID:17867646

  18. Functional analysis of Pid3-A4, an ortholog of rice blast resistance gene Pid3 revealed by allele mining in common wild rice.

    PubMed

    Lv, Qiming; Xu, Xiao; Shang, Junjun; Jiang, Guanghuai; Pang, Zhiqian; Zhou, Zhuangzhi; Wang, Jing; Liu, Ya; Li, Ting; Li, Xiaobing; Xu, Jichen; Cheng, Zhukuan; Zhao, Xianfeng; Li, Shigui; Zhu, Lihuang

    2013-06-01

    The rice blast resistance gene Pid3 encodes a nucleotide-binding-site leucine-rich repeat (NBS-LRR) protein. This gene was cloned from the rice 'Digu' (indica) by performing a genome-wide comparison of the NBS-LRR gene family between two genome-sequenced varieties, '9311' (indica) and 'Nipponbare' (japonica). In this study, we performed functional analysis of Pid3-A4, an ortholog of Pid3 revealed by allele mining in the common wild rice A4 (Oryza rufipogon). The predicted protein encoded by Pid3-A4 shares 99.03% sequence identity with Pid3, with only nine amino-acid substitutions. In wild rice plants, Pid3-A4 is constitutively expressed, and its expression is not induced by Magnaporthe oryzae isolate Zhong-10-8-14 infection. Importantly, in transgenic plants, Pid3-A4, as compared with Pid3, displays a distinct resistance spectrum to a set of M. oryzae isolates, including those that prevail in the rice fields of Sichuan Province. Therefore, Pid3-A4 should be quite useful for the breeding of rice blast resistance, especially in southwestern China.

  19. Inhibition of CYP3A4 and CYP1A2 b Aegle marmelos and its constituents

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aegle marmelos (bael) is a popular tree in India and other Southeast Asian countries. The fruit is usually consumed as dried, fresh or juice and is reported to have a high nutritional value and many perceived health benefits. Despite of the edible nature and therapeutic properties of A. marmelos, no...

  20. The APOA1/C3/A4/A5 cluster and markers of allostatic load in the Boston Puerto Rican Health Study

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The APOA1/C3/A4/A5 cluster encodes key regulators of plasma lipids. Interactions between dietary factors and single nucleotide polymorphisms (SNPs) in the cluster have been reported. Allostatic load, or physiological dysregulation in response to stress, has been implicated in shaping health disparit...

  1. Lack of electron transfer from cytochrome b5 in stimulation of catalytic activities of cytochrome P450 3A4. Characterization of a reconstituted cytochrome P450 3A4/NADPH-cytochrome P450 reductase system and studies with apo-cytochrome b5.

    PubMed

    Yamazaki, H; Johnson, W W; Ueng, Y F; Shimada, T; Guengerich, F P

    1996-11-01

    Many catalytic activities of cytochrome P450 (P450) 3A4, the major human liver P450 enzyme, require cytochrome b5 (b5) for optimal rates. The stimulatory effect of b5 on P450 reactions has generally been thought to be due to transfer of electrons from ferrous b5 to the ferrous P450-O2-substrate complex. We found that apo-b5, devoid of heme, could substitute for b5 in stimulating two prototypic activities, testosterone 6beta hydroxylation and nifedipine oxidation. The stimulatory effect was not seen with albumin, hemoglobin, catalase, or cytochrome c. Apo-b5 could not substitute for b5 in a testosterone 6beta hydroxylation system composed of NADH-b5 reductase and P450 3A4. Rates of electron transfer from NADPH-P450 reductase to ferric P450 3A4 were too slow (<2 min-1) to support testosterone 6beta hydroxylation ( approximately 14 min-1) unless b5 or apo-b5 was present, when rates of approximately 700 min-1 were measured. The oxidation-reduction potential (Em,7) of the ferric/ferrous couple of P450 3A4 was unchanged ( approximately -310 mV) under different conditions in which the kinetics of reduction were altered by the addition of substrate and/or apo-b5. Rapid reduction of P450 3A4 required substrate and a preformed complex of P450 3A4, NADPH-P450 reductase, and b5; the rates of binding of the proteins to each other were 2-3 orders of magnitude less than reduction rates. We conclude that b5 can facilitate some P450 3A4-catalyzed oxidations by complexing with P450 3A4 and enhancing its reduction by NADPH-P450 reductase, without directly transferring electrons to P450. PMID:8910324

  2. Effects of variations in the APOA1/C3/A4/A5 gene cluster on different parameters of postprandial lipid metabolism in healthy young men.

    PubMed

    Delgado-Lista, Javier; Perez-Jimenez, Francisco; Ruano, Juan; Perez-Martinez, Pablo; Fuentes, Francisco; Criado-Garcia, Juan; Parnell, Laurence D; Garcia-Rios, Antonio; Ordovas, Jose M; Lopez-Miranda, Jose

    2010-01-01

    The APOA1/C3/A4/A5 gene cluster encodes important regulators of fasting lipids, but the majority of lipid metabolism takes place in the postprandial state and knowledge about gene regulation in this state is scarce. With the aim of characterizing possible regulators of lipid metabolism, we studied the effects of nine single nucleotide polymorphisms (SNPs) during postprandial lipid metabolism. Eighty-eight healthy young men were genotyped for APOA1 -2630 (rs613808), APOA1 -2803 (rs2727784), APOA1 -3012 (rs11216158), APOC3 -640 (rs2542052), APOC3 -2886 (rs2542051), APOC3 G34G (rs4520), APOA4 N147S (rs5104), APOA4 T29T (rs5092), and A4A5_inter (rs1263177) and were fed a saturated fatty acid-rich meal (1g fat/kg of weight with 60% fat, 15% protein and 25% carbohydrate). Serial blood samples were extracted for 11 h after the meal. Total cholesterol and fractions [HDL-cholesterol, LDL-cholesterol, trifacylglycerols (TGs) in plasma, TG-rich lipoproteins (TRLs) (large TRLs and small TRLs), apolipoprotein A-I and apolipoprotein B] were determined. APOA1 -2803 homozygotes for the minor allele and A4A5_inter carriers showed a limited degree of postprandial lipemia. Carriers of the rare alleles of APOA4 N147S and APOA4 T29T had lower APOA1 plasma concentration during this state. APOC3 -640 was associated with altered TG kinetics but not its magnitude. We have identified new associations between SNPs in the APOA1/C3/A4/A5 gene cluster and altered postprandial lipid metabolism.

  3. Haplotypes in the APOA1-C3-A4-A5 gene cluster affect plasma lipids in both humans and baboons.

    PubMed

    Wang, Qian-fei; Liu, Xin; O'Connell, Jeff; Peng, Ze; Krauss, Ronald M; Rainwater, David L; VandeBerg, John L; Rubin, Edward M; Cheng, Jan-Fang; Pennacchio, Len A

    2004-05-15

    Genetic studies in non-human primates serve as a potential strategy for identifying genomic intervals where polymorphisms impact upon human disease-related phenotypes. It remains unclear, however, whether independently arising polymorphisms in orthologous regions of non-human primates leads to similar variation in a quantitative trait found in both species. To explore this paradigm, we studied a baboon apolipoprotein gene cluster (APOA1/C3/A4/A5) for which the human gene orthologs have well-established roles in influencing plasma HDL-cholesterol and triglyceride concentrations. Our extensive polymorphism analysis of this 68 kb gene cluster in 96 pedigreed baboons identified several haplotype blocks each with limited diversity, consistent with haplotype findings in humans. To determine whether baboons, like humans, also have particular haplotypes associated with lipid phenotypes, we genotyped 634 well-characterized baboons using 16 haplotype tagging SNPs. Genetic analysis of single SNPs, as well as haplotypes, revealed an association of APOA5 and APOC3 variants with HDL-cholesterol and triglyceride concentrations, respectively. Thus, independent variation in orthologous genomic intervals does associate with similar quantitative lipid traits in both species, supporting the possibility of uncovering human quantitative trait loci genes in a highly controlled non-human primate model.

  4. Haplotypes in the APOA1-C3-A4-A5 gene cluster affect plasma lipids in both humans and baboons

    SciTech Connect

    Wang, Qian-fei; Liu, Xin; O'Connell, Jeff; Peng, Ze; Krauss, Ronald M.; Rainwater, David L.; VandeBerg, John L.; Rubin, Edward M.; Cheng, Jan-Fang; Pennacchio, Len A.

    2003-09-15

    Genetic studies in non-human primates serve as a potential strategy for identifying genomic intervals where polymorphisms impact upon human disease-related phenotypes. It remains unclear, however, whether independently arising polymorphisms in orthologous regions of non-human primates leads to similar variation in a quantitative trait found in both species. To explore this paradigm, we studied a baboon apolipoprotein gene cluster (APOA1/C3/A4/A5) for which the human gene orthologs have well established roles in influencing plasma HDL-cholesterol and triglyceride concentrations. Our extensive polymorphism analysis of this 68 kb gene cluster in 96 pedigreed baboons identified several haplotype blocks each with limited diversity, consistent with haplotype findings in humans. To determine whether baboons, like humans, also have particular haplotypes associated with lipid phenotypes, we genotyped 634 well characterized baboons using 16 haplotype tagging SNPs. Genetic analysis of single SNPs, as well as haplotypes, revealed an association of APOA5 and APOC3 variants with HDL cholesterol and triglyceride concentrations, respectively. Thus, independent variation in orthologous genomic intervals does associate with similar quantitative lipid traits in both species, supporting the possibility of uncovering human QTL genes in a highly controlled non-human primate model.

  5. Molecular and crystal structures of some novel derivatives of 3-aryl-7-arylidene-3,3a,4,5,6,7-hexahydroindazoles

    SciTech Connect

    Abakumov, V. V.; Shishkina, S. V.; Zubatyuk, R. I.; Gella, I. M.; Pivnenko, N. S.; Kutulya, L. A. Shishkin, O. V.

    2007-03-15

    The stereochemical aspects of the interaction of 2,6-bis(arylidene)-cyclohexanone and 2,6-bis(arylidene)-3-methylcyclohexanone with arylhydrazine (aryl is phenyl or 4-nitrophenyl) and methylhydrazine are investigated using X-ray diffraction analysis. The molecular structure of the 3-aryl-7-arylidene-3,3a,4,5,6,7-hexahydroindazoles synthesized is determined by X-ray diffraction analysis for the first time. It is established that the stereochemistry of the products of the interaction between the cyclohexanone derivatives and arylhydrazines depends on the electronic nature of the substituent in the aryl group. Two regioisomeric products with different positions of the methyl group in the cyclohexane ring with respect to the arylidene fragment are synthesized by the reaction of 2,6-bis(4-methoxybenzylidene)-3-methylcyclohexanone with methylhydrazine. The influence of the substituents at the nitrogen atom of the pyrazoline ring on the intramolecular electronic interactions and the geometry of the heterocycle in the compounds under investigation is discussed.

  6. CROSS-SPECIES COMPARISON OF CONAZOLE FUNGICIDE METABOLITES USING RAT AND RAINBOW TROUT (ONCHORHYNCHUS MYKISS) HEPATIC MICROSOMES AND PURIFIED HUMAN CYTOCHROME P450 3A4

    EPA Science Inventory

    Conazoles represent a unique class of azole-containing fungicides that are widely used in both pharmaceutical and agriculture applications. The antifungal property of conazoles occurs via complexation with cytochrome P450 monooxygenases (CYP) responsible for mediating fungal cell...

  7. Differential inhibition of cytochromes P450 3A4 and 3A5 by the newly synthesized coumarin derivatives 7-coumarin propargyl ether and 7-(4-trifluoromethyl)coumarin propargyl ether.

    PubMed

    Sridar, Chitra; Kent, Ute M; Noon, Kate; McCall, Alecia; Alworth, Bill; Foroozesh, Maryam; Hollenberg, Paul F

    2008-11-01

    The abilities of 7-coumarin propargyl ether (CPE) and 7-(4-trifluoromethyl)coumarin propargyl ether (TFCPE) to act as mechanism-based inactivators of P450 3A4 and 3A5 in the reconstituted system have been investigated using 7-benzyloxy-4-(trifluoromethyl)coumarin (BFC) and testosterone as probes. CPE inhibited the BFC O-debenzylation activity of P450 3A4 in a time-, concentration-, and NADPH-dependent manner characteristic of a mechanism-based inactivator with a half-maximal inactivation (K(I)) of 112 microM, a maximal rate of inactivation (k(inact)) of 0.05 min(-1), and a t(1/2) of 13.9 min. Similarly, TFCPE inhibited the BFC O-debenzylation activity of P450 3A4 in a time-, concentration-, and NADPH-dependent manner with a K(I) of 14 microM, a k(inact) of 0.04 min(-1), and a t(1/2) of 16.5 min. Parallel losses of P450 3A4 enzymatic activity and heme were observed with both compounds as measured by high-performance liquid chromatography and reduced CO spectra. Interestingly, neither compound inhibited the BFC O-debenzylation activity of P450 3A5. Reactive intermediates of CPE and TFCPE formed by P450 3A4 were trapped with glutathione, and the resulting adducts were identified using tandem mass spectral analysis. Metabolism studies using TFCPE resulted in the identification of a single metabolite that is formed by P450 3A4 but not by P450 3A5 and that may play a role in the mechanism-based inactivation.

  8. Generation of in-silico cytochrome P450 1A2, 2C9, 2C19, 2D6, and 3A4 inhibition QSAR models.

    PubMed

    Gleeson, M Paul; Davis, Andrew M; Chohan, Kamaldeep K; Paine, Stuart W; Boyer, Scott; Gavaghan, Claire L; Arnby, Catrin Hasselgren; Kankkonen, Cecilia; Albertson, Nan

    2007-01-01

    In-silico models were generated to predict the extent of inhibition of cytochrome P450 isoenzymes using a set of relatively interpretable descriptors in conjunction with partial least squares (PLS) and regression trees (RT). The former was chosen due to the conservative nature of the resultant models built and the latter to more effectively account for any non-linearity between dependent and independent variables. All models are statistically significant and agree with the known SAR and they could be used as a guide to P450 liability through a classification based on the continuous pIC50 prediction given by the model. A compound is classified as having either a high or low P450 liability if the predicted pIC(50) is at least one root mean square error (RMSE) from the high/low pIC(50) cut-off of 5. If predicted within an RMSE of the cut-off we cannot be confident a compound will be experimentally low or high so an indeterminate classification is given. Hybrid models using bulk descriptors and fragmental descriptors do significantly better in modeling CYP450 inhibition, than bulk property QSAR descriptors alone.

  9. Fragrance material review on (3aalpha,4alpha,6alpha,7alpha,7aalpha)-3a,4,5,6,7,7a-hexahydro-3-methyl-5-methylene-4,7-methano-1H-inden-6-yl acetate.

    PubMed

    Bhatia, S P; Jones, L; Letizia, C S; Api, A M

    2008-12-01

    A toxicologic and dermatologic review of (3aalpha,4alpha,6alpha,7alpha,7aalpha)-3a,4,5,6,7,7a-hexahydro-3-methyl-5-methylene-4,7-methano-1H-inden-6-yl acetate when used as a fragrance ingredient is presented.

  10. 40 CFR 721.10710 - 4, 7-Methano-1H-indene, 3a, 4, 7, 7a-tetrahydro-, polymer with 2-methyl-1, 3-butadiene and 5-(1...

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 31 2014-07-01 2014-07-01 false 4, 7-Methano-1H-indene, 3a, 4, 7, 7a-tetrahydro-, polymer with 2-methyl-1, 3-butadiene and 5-(1-methylethenyl)bicyclo hept-2-ene. 721.10710 Section 721.10710 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) TOXIC SUBSTANCES CONTROL ACT SIGNIFICANT NEW USES...

  11. Development of a new fluorescent probe: 1,3,5,7-tetramethyl-8-(4'-aminophenyl)-4,4-difluoro-4-bora-3a,4a-diaza-s-indacence for the determination of trace nitrite.

    PubMed

    Li, Mengling; Wang, Hong; Zhang, Xian; Zhang, Hua-Shan

    2004-03-01

    A new fluorescent probe, 1,3,5,7-tetramethyl-8-(4'-aminophenyl)-4,4-difluoro-4-bora-3a,4a-diaza-s-indacence (TMABODIPY) has been developed for the determination of trace nitrite in terms of the reaction of nitrite with TMABODIPY first in acidic solution and then in alkaline solution to form diazotate, a stable and highly fluorescent reagent. The method offered the advantage of specificity, sensitivity and simplicity. The linear calibration range for nitrite was 8-300 nmol l-1s with a 3 sigma detection limit of 0.65 nmol l-1. The proposed method has been applied to monitor the trace nitrite in drinking water and vegetable without extraction.

  12. Oral delivery of paclitaxel nanocrystal (PNC) with a dual Pgp-CYP3A4 inhibitor: preparation, characterization and antitumor activity.

    PubMed

    Patel, Ketan; Patil, Anand; Mehta, Miten; Gota, Vikram; Vavia, Pradeep

    2014-09-10

    Several molecular inheritances have severely restrained the peroral delivery of taxanes. The main objective of the present investigation was to develop a paclitaxel (PTX) formulation which can circumvent the hurdles of its extremely poor solubility and permeability, Pgp efflux and high pre-systemic metabolism. Positively charged PTX nanocrystals of 209 nm were prepared by sonoprecipitation with high pressure homogenization technique, wherein an arginine based surfactant was explored as a stabilizer. The BET surface area analysis revealed that the surface area of PNC was 8.53 m(2)/gm, reflecting significant rise in surface area with nanonization of PTX. The DSC and XRD pattern suggested that the PTX is in the form of the most stable dihydrate crystal. The PNC showed very rapid dissolution profile compared to plain PTX in both sinks and non-sink conditions. Clarithromycin (CLM) was evaluated as a better alternative to cyclosporin A in improving PTX permeability. The PNC-CLM showed remarkable enhancement of 453% in relative bioavailability along with maintaining the therapeutic concentration of PTX for 8h. Efficacy data in B16 F10 melanoma tumor bearing mice showed substantial reduction in tumor volume and improvement in percentage survival compared to the control group.

  13. CROSS-SPECIES COMPARISON OF CONAZOLE FUNGICIDE METABOLITES USING RAT AND RAINBOW TROUT (ONCHLORHYNCHUS MYKISS) HEPATIC MICROSOMES AND PURIFIED HUMAN CYP 3A4

    EPA Science Inventory

    Ecological risk assessment frequently relies on cross-species extrapolation to predict acute toxicity from chemical exposures. A major concern for environmental risk characterization is the degree of uncertainty in assessing xenobiotic biotansformation processes. Although inheren...

  14. THE PUTATIVE HIGH ACTIVITY VARIANT CYP3A4*1B PREDICTS THE ONSET OF PUBERTY IN YOUNG GIRLS. (R825816)

    EPA Science Inventory

    The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

  15. Electron impact mass spectral fragmentation of 3a,5-disubstituted 1, 3-diphenyl-3a,4,5,6-tetra-hydro-3H-1,2,4-triazolo[4,3-a][1, 5]benzo-diazepines.

    PubMed

    Xu, J; Zhang, Q; Wang, C

    2000-01-01

    The mass spectrometric behaviour of six 3a,5-disubstituted 1, 3-diphenyl-3a,4,5,6-tetrahydro-3H-1,2,4-triazolo[4,3-a][1, 5]benzodiazepines has been studied with the aid of mass-analyzed ion kinetic energy spectrometry and accurate mass measurements under electron impact ionization. All compounds show a tendency to eliminate (substituted) styrene molecules, aryl radicals, arylmethyl radicals or phenylnitrene (PhN:). All of the resulting fragment ions, except [M - PhN:](+.), could further undergo a reverse [2 + 3] cycloaddition. The [M - PhN:](+.) ions could further lose styrene derivatives and undergo a ring enlargement rearrangement. The molecular ions also show a tendency to eliminate a phenyl radical, and the [M - Ph](+) ions could eliminate styrene derivatives. The [M - R(1)CH = CH(2)](+.) ions could further lose NH(2) to yield stable tetracyclic 1,3-diphenyl-1,2,4-triazolo[4,3-d]phenanthridine ions, which could further lose benzonitrile, or undergo a reverse [2 + 3] cycloaddition. The molecular ions could also undergo a reverse [2 + 3] cycloaddition to produce N-phenylbenzonitrile imine ions and 2, 4-disubstituted 2,3-dihydro-1H-1,5-benzodiazepine ions, whose further fragmentations were also investigated.

  16. Synthesis, crystal structure analysis, spectral characterization, quantum chemical calculations, antioxidant and antimicrobial activity of 3-(4-chlorophenyl)-3a,4,7,7a-tetrahydro-4,7-methanobenzo[d]isoxazole

    NASA Astrophysics Data System (ADS)

    Eryılmaz, Serpil; Gül, Melek; İnkaya, Ersin; İdil, Önder; Özdemir, Namık

    2016-10-01

    In this paper, 3-(4-chlorophenyl)-3a,4,7,7a-tetrahydro-4,7-methanobenzo[d]isoxazole was synthesized via 1,3 dipolar cycloaddition, characterized by spectroscopic analysis such as FT-IR, 1H NMR, 13C NMR, UV-Vis, LC-MS/MS, Elemental Analysis, and X-ray Single Crystal diffraction technique. The Density Functional Theory (DFT/B3LYP) method with 6-311G(d,p) basis set in the ground state was applied for quantum chemical calculations and molecular geometric parameters of the compound were compared with the X-ray analysis results. FT-IR, NMR and UV-Vis spectral analysis were analysed to determine the compliance with the vibrational frequencies, 1H NMR and 13C NMR chemical shifts and absorption wavelength values. The frontier molecular orbitals (FMOs), some global reactivity descriptors, molecular electrostatic potential (MEP), thermodynamic properties, non-linear optical (NLO) behaviour of the compound were examined with the same method in gas phase, theoretically. Moreover, antioxidant activity was determined with three different methods - DPPH radical scavenging, reducing and metal chelating, antimicrobial activity were carried out with Gram positive, Gram negative and Eukaryote for the title compound.

  17. Chemical synthesis, pharmacological evaluation and in silico analysis of new 2,3,3a,4,5,6-hexahydrocyclopenta[c]pyrazole derivatives as potential anti-mitotic agents.

    PubMed

    Minu, Maninder; Singh, Deepti; Mahaddalkar, Tejashree; Lopus, Manu; Winter, Philip; Ayoub, Ahmed T; Missiaen, Kristal; Tilli, Tatiana Martins; Pasdar, Manijeh; Tuszynski, Jack

    2016-08-15

    We have synthesized new, biologically active mono- and di-substituted 2,3,3a,4,5,6-hexahydrocyclopenta[c]pyrazole derivatives bearing electron withdrawing groups and electron donating groups. These derivative structures were characterized by their spectral and analytical data. The newly synthesized hexahydropyrazole analogues were evaluated for their in vitro anticancer activity against breast and lung cancer cell lines using a cytotoxicity bioassay. To understand their mechanism of action, tubulin binding assays were performed which pointed to their binding to microtubules in a mode similar to but not identical to colchicine, as evidenced by their KD value evaluation. Computational docking studies also suggested binding near the colchicine binding site on tubulin. These results were further confirmed by colchicine-binding assays on the most active compounds, which indicated that they bound to tubulin near but not at the colchicine site. The moderate cytotoxic effects of these compounds may be due to the presence of electron donating groups on the para-position of the phenyl ring, along with the hexahydropyrazole core nucleus. The observed anti-cancer activity based on inhibition of microtubule formation may be helpful in designing more potent compounds with a hexahydropyrazole moiety. PMID:27449957

  18. The effects of azole-based heme oxygenase inhibitors on rat cytochromes P450 2E1 and 3A1/2 and human cytochromes P450 3A4 and 2D6.

    PubMed

    Hum, Maaike; McLaughlin, Brian E; Roman, Gheorghe; Vlahakis, Jason Z; Szarek, Walter A; Nakatsu, Kanji

    2010-09-01

    Heme oxygenases (HOs) catalyze the degradation of heme to biliverdin, carbon monoxide (CO), and free iron. The two major isoforms, HO-1 (inducible) and HO-2 (constitutive), are involved in a variety of physiological functions, including inflammation, apoptosis, neuromodulation, and vascular regulation. Major tools used in exploring these actions have been metalloporphyrin analogs of heme that inhibit the HOs. However, these tools are limited by their lack of selectivity; they affect other heme-dependent enzymes, such as cytochromes P450 (P450s), soluble guanylyl cyclase (sGC), and nitric-oxide synthase (NOS). Our laboratory has successfully synthesized a number of nonporphyrin azole-based HO inhibitors (QC-xx) that had little or no effect on sGC and NOS activity. However, their effects on various P450 isoforms have yet to be fully elucidated. To determine the effects of the QC-xx inhibitors on P450 enzyme activity, microsomal preparations of two rat P450 isoforms (2E1 and 3A1/3A2) and two human P450 supersome isoforms (3A4 and 2D6) were incubated with varying concentrations of HO inhibitor, and the activity was determined by spectrophotometric or fluorometric analysis. Results indicated that some QC compounds demonstrated little to no inhibition of the P450s, whereas others did inhibit these P450 isoforms. Four structural regions of QC-xx were analyzed, leading to the identification of structures that confer a decreased effect on both rat and human P450 isoforms studied while maintaining an inhibitory effect on the HOs.

  19. Three-dimensional quantitative structure-activity relationship analysis for human pregnane X receptor for the prediction of CYP3A4 induction in human hepatocytes: structure-based comparative molecular field analysis.

    PubMed

    Handa, Koichi; Nakagome, Izumi; Yamaotsu, Noriyuki; Gouda, Hiroaki; Hirono, Shuichi

    2015-01-01

    The pregnane X receptor [PXR (NR1I2)] induces the expression of xenobiotic metabolic genes and transporter genes. In this study, we aimed to establish a computational method for quantifying the enzyme-inducing potencies of different compounds via their ability to activate PXR, for the application in drug discovery and development. To achieve this purpose, we developed a three-dimensional quantitative structure-activity relationship (3D-QSAR) model using comparative molecular field analysis (CoMFA) for predicting enzyme-inducing potencies, based on computer-ligand docking to multiple PXR protein structures sampled from the trajectory of a molecular dynamics simulation. Molecular mechanics-generalized born/surface area scores representing the ligand-protein-binding free energies were calculated for each ligand. As a result, the predicted enzyme-inducing potencies for compounds generated by the CoMFA model were in good agreement with the experimental values. Finally, we concluded that this 3D-QSAR model has the potential to predict the enzyme-inducing potencies of novel compounds with high precision and therefore has valuable applications in the early stages of the drug discovery process.

  20. [Effect of Fuzheng Huayu recipe on CYP450 isozymes in normal and liver fibrosis rats].

    PubMed

    Zheng, Tian-hui; Liu, Wei; Li, Shu-ping; Yang, Tao; Wang, Chang-hong; Liu, Cheng-hai

    2015-03-01

    To study the effect of Fuzheng Huayu recipe (FZHY) on five types of isozymes of cytochrome P450 (CYP450) of normal and liver fibrosis rats by using the cocktail probe method. Dimethylnitrosamine ( DMN) was injected to induce the liver fibrosis model. After the tail vein injection with Cocktail probe solutions prepared with five CYP450s probe substrates (phenacetin-CYP1A2, omeprazole-CYP2C9, tolbutamide-CYP2C19, dextromethorphan-CYP2D6, midazolam-CYP3A4), the plasma concentrations of the five probe substrates were determined by LC-MS/MS, and the pharmacokinetic parameters were calculated by PK solutions 2. After the oral administration with FZHY, normal rats given phenacetin, omeprazole, tolbutamide and dextromethorphan showed increase in AUC(0-t) and decrease in CL to varying degrees, indicating that FZHY obviously inhibited the activities of CYP1A2, CYP2C9, CYP2C19 and CYP2D6 in normal rats, but with no obvious effect on the activity of CYP3A4. After the oral administration with FZHY, liver fibrosis rats treated with CYP2C9 showed the significant increase in AUC(0-t) and significant decrease in Vd, hut with no obvious changes in the pharmacokinetic parameters of other four types of prove substances, suggesting that FZHY could significantly inhibit the activity of CYP2C9 in rats but had no effect on the activities of CYP1A2, CYP2C19, CYP2D6 and CYP3A4. The changes in the activity of CYP450 isozymes in liver fibrosis rats may be the reason for FZHY's different effects on CYP450 isozymes in normal and liver fibrosis rats. PMID:26226765

  1. Evaluation of CYP2B6 Induction and Prediction of Clinical Drug-Drug Interactions: Considerations from the IQ Consortium Induction Working Group-An Industry Perspective.

    PubMed

    Fahmi, Odette A; Shebley, Mohamad; Palamanda, Jairam; Sinz, Michael W; Ramsden, Diane; Einolf, Heidi J; Chen, Liangfu; Wang, Hongbing

    2016-10-01

    Drug-drug interactions (DDIs) due to CYP2B6 induction have recently gained prominence and clinical induction risk assessment is recommended by regulatory agencies. This work aimed to evaluate the potency of CYP2B6 versus CYP3A4 induction in vitro and from clinical studies and to assess the predictability of efavirenz versus bupropion as clinical probe substrates of CYP2B6 induction. The analysis indicates that the magnitude of CYP3A4 induction was higher than CYP2B6 both in vitro and in vivo. The magnitude of DDIs caused by induction could not be predicted for bupropion with static or dynamic models. On the other hand, the relative induction score, net effect, and physiologically based pharmacokinetics SimCYP models using efavirenz resulted in improved DDI predictions. Although bupropion and efavirenz have been used and are recommended by regulatory agencies as clinical CYP2B6 probe substrates for DDI studies, CYP3A4 contributes to the metabolism of both probes and is induced by all reference CYP2B6 inducers. Therefore, caution must be taken when interpreting clinical induction results because of the lack of selectivity of these probes. Although in vitro-in vivo extrapolation for efavirenz performed better than bupropion, interpretation of the clinical change in exposure is confounded by the coinduction of CYP2B6 and CYP3A4, as well as the increased contribution of CYP3A4 to efavirenz metabolism under induced conditions. Current methods and probe substrates preclude accurate prediction of CYP2B6 induction. Identification of a sensitive and selective clinical substrate for CYP2B6 (fraction metabolized > 0.9) is needed to improve in vitro-in vivo extrapolation for characterizing the potential for CYP2B6-mediated DDIs. Alternative strategies and a framework for evaluating the CYP2B6 induction risk are proposed.

  2. CYP isoform specificity toward drug metabolism: analysis using common feature hypothesis.

    PubMed

    Ramesh, M; Bharatam, Prasad V

    2012-02-01

    Three dimensional pharmacophoric maps were generated for each isoforms of CYP2C9, CYP2D6 and CYP3A4 separately using independent training sets consist of highly potent substrates (seven substrates for each isoform). HipHop module of CATALYST software was used in the generation of pharmacophore models. The best pharmacophore model was chosen out of the several models on the basis of (i) highest ranking score, (ii) better fit value among training set, (iii) capability to screen substrates from data set and (iv) efficiency to identify the isoform specificity. The individual pharmacophore models (CYP2C9-hypo1, CYP2D6-hypo1 and CYP3A4-hypo1) are characterized by the pharmacophoric features XZDH, RPZH and XYZHH for the CYP2C9, CYP2D6 and CYP3A4 respectively. Each of the chosen models was validated by using data sets of CYP substrates. This comparative study of CYP substrates demonstrates the importance of acidic character along with HBD and HBAl features for CYP2C9, basic character with ring aromatic features for CYP2D6 and hydrophobic features for CYP3A4. Acidity, basicity and hydrophobicity features arising from the functional groups of the substrates are also responsible for demonstrating CYP isoform specificity. Hence, these chemical features are incorporated in the decision tree along with pharmacophore maps. Finally, a decision tree based on chemical features and pharmacophore features was generated to identify the isoform specificity of novel query molecule toward the three isoforms. PMID:21562823

  3. CYP isoform specificity toward drug metabolism: analysis using common feature hypothesis.

    PubMed

    Ramesh, M; Bharatam, Prasad V

    2012-02-01

    Three dimensional pharmacophoric maps were generated for each isoforms of CYP2C9, CYP2D6 and CYP3A4 separately using independent training sets consist of highly potent substrates (seven substrates for each isoform). HipHop module of CATALYST software was used in the generation of pharmacophore models. The best pharmacophore model was chosen out of the several models on the basis of (i) highest ranking score, (ii) better fit value among training set, (iii) capability to screen substrates from data set and (iv) efficiency to identify the isoform specificity. The individual pharmacophore models (CYP2C9-hypo1, CYP2D6-hypo1 and CYP3A4-hypo1) are characterized by the pharmacophoric features XZDH, RPZH and XYZHH for the CYP2C9, CYP2D6 and CYP3A4 respectively. Each of the chosen models was validated by using data sets of CYP substrates. This comparative study of CYP substrates demonstrates the importance of acidic character along with HBD and HBAl features for CYP2C9, basic character with ring aromatic features for CYP2D6 and hydrophobic features for CYP3A4. Acidity, basicity and hydrophobicity features arising from the functional groups of the substrates are also responsible for demonstrating CYP isoform specificity. Hence, these chemical features are incorporated in the decision tree along with pharmacophore maps. Finally, a decision tree based on chemical features and pharmacophore features was generated to identify the isoform specificity of novel query molecule toward the three isoforms.

  4. Immunochemical quantification of cynomolgus CYP2J2, CYP4A and CYP4F enzymes in liver and small intestine.

    PubMed

    Uehara, Shotaro; Murayama, Norie; Nakanishi, Yasuharu; Nakamura, Chika; Hashizume, Takanori; Zeldin, Darryl C; Yamazaki, Hiroshi; Uno, Yasuhiro

    2015-02-01

    1. An increasing number of studies have indicated the roles of CYP4 proteins in drug metabolism; however, CYP4 expression has not been measured in cynomolgus monkeys, an important animal species for drug metabolism studies. 2. In this study, cynomolgus CYP4A11, CYP4F2/3, CYP4F11 and CYP4F12, along with CYP2J2, were immunoquantified using selective antibodies in 28 livers and 35 small intestines, and their content was compared with CYP1A, CYP2A, CYP2B6, CYP2C9/19, CYP2D, CYP2E1, CYP3A4 and CYP3A5, previously quantified. 3. In livers, CYP2J2, CYP4A11, CYP4F2/3, CYP4F11 and CYP4F12, varied 1.3- to 4.3-fold, represented 11.2, 14.4, 8.0, 2.7 and 0.3% of total immunoquantified CYP1-4 proteins, respectively. 4. In small intestines, CYP2J2, CYP4F2/3, CYP4F11 and CYP4F12, varied 2.4- to 9.7-fold, represented 6.9, 36.4, 2.4 and 9.3% of total immunoquantified CYP1-4 proteins, respectively, making CYP4F the most abundant P450 subfamily in small intestines. CYP4A11 was under the detection limit in all of the samples analyzed. 5. Significant correlations were found in liver for CYP4A11 with lauric acid 11-/12-hydroxylation and for CYP4F2/3 and CYP4F11 with astemizole hydroxylation. 6. This study revealed the relatively abundant contents of cynomolgus CYP2J2, CYP4A11 and CYP4Fs in liver and/or small intestine, suggesting their potential roles for the metabolism of xenobitotics and endogenous substrates.

  5. Immunochemical quantification of cynomolgus CYP2J2, CYP4A and CYP4F enzymes in liver and small intestine.

    PubMed

    Uehara, Shotaro; Murayama, Norie; Nakanishi, Yasuharu; Nakamura, Chika; Hashizume, Takanori; Zeldin, Darryl C; Yamazaki, Hiroshi; Uno, Yasuhiro

    2015-02-01

    1. An increasing number of studies have indicated the roles of CYP4 proteins in drug metabolism; however, CYP4 expression has not been measured in cynomolgus monkeys, an important animal species for drug metabolism studies. 2. In this study, cynomolgus CYP4A11, CYP4F2/3, CYP4F11 and CYP4F12, along with CYP2J2, were immunoquantified using selective antibodies in 28 livers and 35 small intestines, and their content was compared with CYP1A, CYP2A, CYP2B6, CYP2C9/19, CYP2D, CYP2E1, CYP3A4 and CYP3A5, previously quantified. 3. In livers, CYP2J2, CYP4A11, CYP4F2/3, CYP4F11 and CYP4F12, varied 1.3- to 4.3-fold, represented 11.2, 14.4, 8.0, 2.7 and 0.3% of total immunoquantified CYP1-4 proteins, respectively. 4. In small intestines, CYP2J2, CYP4F2/3, CYP4F11 and CYP4F12, varied 2.4- to 9.7-fold, represented 6.9, 36.4, 2.4 and 9.3% of total immunoquantified CYP1-4 proteins, respectively, making CYP4F the most abundant P450 subfamily in small intestines. CYP4A11 was under the detection limit in all of the samples analyzed. 5. Significant correlations were found in liver for CYP4A11 with lauric acid 11-/12-hydroxylation and for CYP4F2/3 and CYP4F11 with astemizole hydroxylation. 6. This study revealed the relatively abundant contents of cynomolgus CYP2J2, CYP4A11 and CYP4Fs in liver and/or small intestine, suggesting their potential roles for the metabolism of xenobitotics and endogenous substrates. PMID:25138712

  6. PXR-Mediated Upregulation of CYP3A Expression by Herb Compound Praeruptorin C from Peucedanum praeruptorum Dunn

    PubMed Central

    Huang, Ling; Wu, Qian; Li, Yu-Hua; Wang, Yi-Tao; Bi, Hui-Chang

    2013-01-01

    We recently reported that Praeruptorin C effectively transactivated the mRNA, protein expression, and catalytic activity of CYP3A4 via the CAR-mediated pathway, but whether and how PC could affect the expression and catalytic activity of CYP3A4 via PXR pathway remains unknown. Therefore, in this study, the effect of PC on the CYP3A gene expression was investigated in mice primary hepatocytes after knockdown of PXR by transient transfection of PXR siRNA, and the gene expression, protein expression, and catalytic activity of CYP3A4 in the LS174T cells with PXR overexpression were determined by real-time PCR, western blot analysis, and LC-MS/MS-based CYP3A4 substrate assay, respectively. We found that the level of CYP3a11 gene expression in mouse primary hepatocytes was significantly increased by praeruptorin C, but such an induction was suppressed after knockdown of pregnane X receptor by its siRNA. In PXR-overexpressed LS174T cells, PC significantly enhanced CYP3A4 mRNA, protein expression, and functional activity through PXR-mediated pathway; conversely, no such increase was found in the untransfected cells. These findings suggest that PC can significantly upregulate CYP3A level via the PXR-mediated pathway, and this should be taken into consideration to predict any potential herb-drug interactions between PC, Qianhu, and the other coadministered drugs. PMID:24379885

  7. Future Trends in the Pharmacogenomics of Brain Disorders and Dementia: Influence of APOE and CYP2D6 Variants

    PubMed Central

    Cacabelos, Ramón; Fernández-Novoa, Lucía; Martínez-Bouza, Rocío; McKay, Adam; Carril, Juan C.; Lombardi, Valter; Corzo, Lola; Carrera, Iván; Tellado, Iván; Nebril, Laura; Alcaraz, Margarita; Rodríguez, Susana; Casas, Ángela; Couceiro, Verónica; Álvarez, Antón

    2010-01-01

    About 80% of functional genes in the human genome are expressed in the brain and over 1,200 different genes have been associated with the pathogenesis of CNS disorders and dementia. Pharmacogenetic studies of psychotropic drug response have focused on determining the relationship between variations in specific candidate genes and the positive and adverse effects of drug treatment. Approximately, 18% of neuroleptics are substrates of CYP1A2 enzymes, 40% of CYP2D6, and 23% of CYP3A4; 24% of antidepressants are substrates of CYP1A2 enzymes, 5% of CYP2B6, 38% of CYP2C19, 85% of CYP2D6, and 38% of CYP3A4; 7% of benzodiazepines are substrates of CYP2C19 enzymes, 20% of CYP2D6, and 95% of CYP3A4. 10-20% of Western populations are defective in genes of the CYP superfamily; and the pharmacogenomic response of psychotropic drugs also depends on genetic variants associated with dementia. Prospective studies with anti-dementia drugs or with multifactorial strategies have revealed that the therapeutic response to conventional drugs in Alzheimer’s disease is genotype-specific. The disease-modifying effects (cognitive performance, biomarker modification) of therapeutic intervention are APOE-dependent, with APOE-4 carriers acting as the worst responders (APOE-3/3 > APOE-3/4 > APOE-4/4). APOE-CYP2D6 interactions also influence the therapeutic outcome in patients with dementia.

  8. Use of fluorescence-activated flow cytometry to determine membrane lipid peroxidation during hypothermic liquid storage and freeze-thawing of viable boar sperm loaded with 4, 4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid.

    PubMed

    Guthrie, H D; Welch, G R

    2007-06-01

    Part of the reduction in boar sperm motility and fertility associated with hypothermic liquid storage and cryopreservation may be due to membrane lipid peroxidation. Lipid peroxidation was monitored by the shift from red to green fluorescence emission of the lipophilic probe 4, 4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid, C(11)BODIPY(581/591) (BODIPY), as measured by fluorescence-activated flow cytometry in live sperm (negative for propidium iodide). Experiments were conducted with Percoll-washed sperm to determine the specificity of BODIPY oxidation in the presence of different reactive oxygen species generators and metal chelators. Compared with no FeSO(4) and Na ascorbate, the combination of FeSO(4) and Na ascorbate (FeAc) increased (P < 0.01) the percentage of sperm containing oxidized BODIPY from 70% and increased (P < 0.05) BOD-IPY fluorescence intensity/cell by 5- to 10-fold after a 30-min incubation. Motility was depressed (P < 0.05) after exposure to FeAc, but viability was not affected. Of the reactive oxygen species generators tested, BODIPY oxidation was specific for FeAc, because menadione and H(2)O(2) had little or no effect. The oxidization of hydroethidine to ethidium was specific for menadione and H(2)O(2); FeAc had no effect. The presence of the metal chelators EDTA or deferoxamine mesylate at 3 and 9 muM inhibited FeAc-induced BODIPY oxidation and maintained motility. Experiments were conducted to determine the effect of liquid storage at 17 degrees C for 1 and 5 d and the effect of freeze-thawing on basal and FeAc-induced BODIPY oxidation. Basal BODIPY oxidation (no FeAc) was low in liquid stored and thawed viable sperm (1.3 and 3.4%, respectively). Although the incidence of basal or spontaneous membrane lipid peroxidation was low during liquid storage and after freeze-thawing, viable boar sperm were susceptible to FeAc-induced lipid peroxidation. PMID:17296775

  9. Drug-drug Interaction between Losartan and Paclitaxel in Human Liver Microsomes with Different CYP2C8 Genotypes.

    PubMed

    Mukai, Yuji; Senda, Asuna; Toda, Takaki; Hayakawa, Toru; Eliasson, Erik; Rane, Anders; Inotsume, Nobuo

    2015-06-01

    The cytochrome P450 (CYP) 2C8*3 allele is associated with reduced metabolic activity of paclitaxel. This study was aimed to investigate the inhibitory effect of losartan on paclitaxel metabolism in human liver microsomes (HLMs) and to determine the impact of the CYP2C8*3 polymorphism. HLMs that contained the CYP2C8*1 homozygote (HL60) or CYP2C8*3 heterozygote (HL54) genotype were used for the inhibition study. Losartan, at a concentration of 50 μmol/L, significantly inhibited paclitaxel metabolism by 29% and 57% in the HL60 (p < 0.001) and HL54 (p < 0.01), respectively. When using HL60, losartan and the CYP3A4-selective inhibitors, erythromycin and ketoconazole, caused a greater inhibition of the paclitaxel metabolism than quercetin, a CYP2C8-selective inhibitor. This demonstrated that the paclitaxel metabolism was mainly catalysed by CYP3A4 in HL60. There were no significant differences found for the inhibitory effects caused by the four inhibitors of the paclitaxel metabolism in HL54, indicating that both CYP2C8 and CYP3A4 play important roles in paclitaxel metabolism in HL54. These findings suggest that 50 μmol/L of losartan inhibits both CYP2C8 and CYP3A4 in HLMs. In summary, losartan inhibited paclitaxel metabolism, with concentrations over 50 μmol/L in HLMs. The CYP2C8*3 allele carriers are likely susceptible to the interactions of losartan and CYP3A4 inhibitors to paclitaxel metabolism.

  10. Enzyme kinetic study of a new cardioprotective agent, KR-32570 using human liver microsomes and recombinant CYP isoforms.

    PubMed

    Kim, Hyojin; Seo, Kyung-Ah; Kim, Hyunmi; Lee, Hye Suk; Lee, Choong-Hwan; Shin, Jae-Gook; Liu, Kwang-Hyeon

    2007-04-01

    KR-32570 (5-(2-Methoxy-5-chlorophenyl)furan-2-ylcarbonyl)guanidine) is a new cardioprotective agent for preventing ischemia-reperfusion injury. Human liver microsomal incubation of KR-32570 in the presence of NADPH resulted in the formation of two metabolites, hydroxy-KR-32570 and O-desmethyl-KR-32570. In this study, a kinetic analysis of the metabolism of two metabolites from KR-32570 was performed in human liver microsomes, and recombinant CYP1A2, and CYP3A4. The metabolism for hydroxy- and O-desmethyl-KR-32570 formation from KR-32570 by human liver microsomes was best described by a Michaelis-Menten equation and a Hill equation, respectively. The Cl(int) values of hydroxy- and O-desmethyl-KR-32570 formation were similar to each other (0.03 vs 0.04 microL/min/pmol CYP, respectively). CYP3A4 mediated the formation of hydroxy-KR-32570 from KR-32570 with Cl(int) = 0.24 microL/min/pmol CYP3A4. The intrinsic clearance for O-desmethyl-KR-32570 formation by CYP1A2 was 0.83 AL/min/pmol CYP1A2. These findings suggest that CYP3A4 and CYP1A2 enzymes are major enzymes contributing to the metabolism of KR-32570.

  11. Cross-sectional study of hepatic CYP1A and CYP3A enzymes in hybrid striped bass, channel catfish and Nile tilapia following oxytetracycline treatment.

    PubMed

    Topic Popovic, N; Howell, T; Babish, J G; Bowser, P R

    2012-04-01

    Terramycin for Fish® (oxytetracycline, OTC) is one of three approved drugs for therapeutic treatment of fish in the United States. Nothing is known, however, of the effects of this therapeutic on drug metabolizing enzymes in fish post-treatment. The main purpose of the study was to examine whether the fish CYP1A and CYP3A enzymes would cross-react with antibodies to known mammalian cytochrome P-450 forms (CYP1A1 and CYP3A). Observational feeding studies of OTC effects were conducted in hybrid striped bass, channel catfish and Nile tilapia. Oxytetracycline was mixed into the feed to achieve a daily dose of 82.8 mg per kg body weight at a feeding rate of 1% body weight per day. Hepatic microsomes of each fish were prepared and Western blotting of CYP1A1 and CYP3A4 and enzyme assays of CYP1A2 and CYP3A4 were performed prior to OTC treatment and on post-treatment days 1, 6, 11 and 21. Both goat anti-rat CYP1A1 and rabbit anti-human CYP3A4 showed good cross-reactivity with all three species in this study. All three species exhibited distinct perturbations in one or more of the variables examined on day 1 post-treatment. Immediately following the 10-day medication period, relative liver weight (RLW) of hybrid striped bass was increased 44% and remained elevated through post-treatment day 21. Increased CYP3A4 enzyme activity and protein abundance were noted in channel catfish and Nile tilapia, respectively. This observational approach demonstrated species differences both in control activities and in the timing and extent of hepatic responses to OTC. The unique perturbations of hepatic CYP450 enzymes in different fish species to OTC treatment observed in this study may have relevance for the use of additional antibiotics or other therapeutics used in aquaculture. PMID:21458012

  12. Cross-sectional study of hepatic CYP1A and CYP3A enzymes in hybrid striped bass, channel catfish and Nile tilapia following oxytetracycline treatment.

    PubMed

    Topic Popovic, N; Howell, T; Babish, J G; Bowser, P R

    2012-04-01

    Terramycin for Fish® (oxytetracycline, OTC) is one of three approved drugs for therapeutic treatment of fish in the United States. Nothing is known, however, of the effects of this therapeutic on drug metabolizing enzymes in fish post-treatment. The main purpose of the study was to examine whether the fish CYP1A and CYP3A enzymes would cross-react with antibodies to known mammalian cytochrome P-450 forms (CYP1A1 and CYP3A). Observational feeding studies of OTC effects were conducted in hybrid striped bass, channel catfish and Nile tilapia. Oxytetracycline was mixed into the feed to achieve a daily dose of 82.8 mg per kg body weight at a feeding rate of 1% body weight per day. Hepatic microsomes of each fish were prepared and Western blotting of CYP1A1 and CYP3A4 and enzyme assays of CYP1A2 and CYP3A4 were performed prior to OTC treatment and on post-treatment days 1, 6, 11 and 21. Both goat anti-rat CYP1A1 and rabbit anti-human CYP3A4 showed good cross-reactivity with all three species in this study. All three species exhibited distinct perturbations in one or more of the variables examined on day 1 post-treatment. Immediately following the 10-day medication period, relative liver weight (RLW) of hybrid striped bass was increased 44% and remained elevated through post-treatment day 21. Increased CYP3A4 enzyme activity and protein abundance were noted in channel catfish and Nile tilapia, respectively. This observational approach demonstrated species differences both in control activities and in the timing and extent of hepatic responses to OTC. The unique perturbations of hepatic CYP450 enzymes in different fish species to OTC treatment observed in this study may have relevance for the use of additional antibiotics or other therapeutics used in aquaculture.

  13. Characterization of the active site properties of CYP4F12.

    PubMed

    Eksterowicz, John; Rock, Dan A; Rock, Brooke M; Wienkers, Larry C; Foti, Robert S

    2014-10-01

    Cytochrome P450 4F12 is a drug-metabolizing enzyme that is primarily expressed in the liver, kidney, colon, small intestine, and heart. The properties of CYP4F12 that may impart an increased catalytic selectivity (decreased promiscuity) were explored through in vitro metabolite elucidation, kinetic isotope effect experiments, and computational modeling of the CYP4F12 active site. By using astemizole as a probe substrate for CYP4F12 and CYP3A4, it was observed that although CYP4F12 favored astemizole O-demethylation as the primary route of metabolism, CYP3A4 was capable of metabolizing astemizole at multiple sites on the molecule. Deuteration of astemizole at the site of O-demethylation resulted in an isotope effect of 7.1 as well as an 8.3-fold decrease in the rate of clearance for astemizole by CYP4F12. Conversely, although an isotope effect of 3.8 was observed for the formation of the O-desmethyl metabolite when deuterated astemizole was metabolized by CYP3A4, there was no decrease in the clearance of astemizole. Development of a homology model of CYP4F12 based on the crystal structure of cytochrome P450 BM3 predicted an active site volume for CYP4F12 that was approximately 76% of the active site volume of CYP3A4. As predicted, multiple favorable binding orientations were available for astemizole docked into the active site of CYP3A4, but only a single binding orientation with the site of O-demethylation oriented toward the heme was identified for CYP4F12. Overall, it appears that although CYP4F12 may be capable of binding similar ligands to other cytochrome P450 enzymes such as CYP3A4, the ability to achieve catalytically favorable orientations may be inherently more difficult because of the increased steric constraints of the CYP4F12 active site. PMID:25074871

  14. Eletriptan metabolism by human hepatic CYP450 enzymes and transport by human P-glycoprotein.

    PubMed

    Evans, David C; O'Connor, Desmond; Lake, Brian G; Evers, Raymond; Allen, Christopher; Hargreaves, Richard

    2003-07-01

    "Reaction phenotyping" studies were performed with eletriptan (ETT) to determine its propensity to interact with coadministered medications. Its ability to serve as a substrate for human P-glycoprotein (P-gp) was also investigated since a central mechanism of action has been proposed for this "triptan" class of drug. In studies with a characterized bank of human liver microsome preparations, a good correlation (r2 = 0.932) was obtained between formation of N-desmethyl eletriptan (DETT) and CYP3A4-catalyzed testosterone 6 beta-hydroxylation. DETT was selected to be monitored in our studies since it represents a significant ETT metabolite in humans, circulating at concentrations 10 to 20% of those observed for parent drug. ETT was metabolized to DETT by recombinant CYP2D6 (rCYP2D6) and rCYP3A4, and to a lesser extent by rCYP2C9 and rCYP2C19. The metabolism of ETT to DETT in human liver microsomes was markedly inhibited by troleandomycin, erythromycin, miconazole, and an inhibitory antibody to CYP3A4, but not by inhibitors of other major P450 enzymes. ETT had little inhibitory effect on any of the P450 enzymes investigated. ETT was determined to be a good substrate for human P-gp in vitro. In bidirectional transport studies across LLC-MDR1 and LLC-Mdr1a cell monolayers, ETT had a BA/AB transport ratio in the range 9 to 11. This finding had significance in vivo since brain exposure to ETT was reduced 40-fold in Mdr1a+/+ relative to Mdr1a-/- mice. ETT metabolism to DETT is therefore catalyzed primarily by CYP3A4, and plasma concentrations are expected to be increased when coadministered with inhibitors of CYP3A4 and P-gp activity. PMID:12814962

  15. Troglitazone quinone formation catalyzed by human and rat CYP3A: an atypical CYP oxidation reaction.

    PubMed

    He, K; Woolf, T F; Kindt, E K; Fielder, A E; Talaat, R E

    2001-07-15

    Oxidative ring opening of troglitazone (TGZ)(1) a thiazolidine 2,4-dione derivative used for the treatment of type II diabetes mellitus, leads to the formation of a quinone metabolite. The formation of TGZ quinone was shown to be NADPH dependent and to require active microsomal enzymes. Quinone formation was not affected by co-incubation with catalase or sodium azide and was partially inhibited (25%) by superoxide dismutase (SOD). Kinetic analysis of TGZ quinone formation in human liver microsomes implied single enzyme involvement. CYP3A isoforms were characterized as the primary enzymes involved in quinone formation by several lines of evidence including: (a) troleandomycin and ketoconazole almost completely inhibited microsomal quinone formation when SOD was present, whereas other CYP inhibitors had minimal effects (<20%); (b) TGZ quinone formation was highly correlated with regard to both contents (r(2): 0.9374) and activities (r(2): 0.7951) of CYP3A4 in human liver microsomes (HLM); (c) baculovirus insect cell-expressed human CYP3A4 was able to catalyze TGZ quinone formation at a higher capacity (V(max)/K(m)) than other human CYPs with the relative contribution of CYP3A4 in HLM estimated to be 20-fold higher than that of other CYPs; (d) TGZ quinone formation was increased by 350% in liver microsomes from rats pretreated with dexamethasone (DEX); and (e) plasma concentrations of TGZ quinone were increased by 260-680% in rats pretreated with DEX. The chemical nature of the quinone metabolite suggests an atypical CYP reaction consistent with a one-electron oxidation mechanism where an intermediate phenoxy radical combines with ferryl oxygen to subsequently form the quinone metabolite.

  16. Evaluation of 89 compounds for identification of substrates for cynomolgus monkey CYP2C76, a new bupropion/nifedipine oxidase.

    PubMed

    Hosaka, Shinya; Murayama, Norie; Satsukawa, Masahiro; Shimizu, Makiko; Uehara, Shotaro; Fujino, Hideki; Iwasaki, Kazuhide; Iwano, Shunsuke; Uno, Yasuhiro; Yamazaki, Hiroshi

    2015-01-01

    Cynomolgus monkeys are widely used in preclinical studies during drug development because of their evolutionary closeness to humans, including their cytochrome P450s (P450s). Most cynomolgus monkey P450s are almost identical (≥90%) to human P450s; however, CYP2C76 has low sequence identity (approximately 80%) to any human CYP2Cs. Although CYP2C76 has no ortholog in humans and is partly responsible for species differences in drug metabolism between cynomolgus monkeys and humans, a broad evaluation of potential substrates for CYP2C76 has not yet been conducted. In this study, a screening of 89 marketed compounds, including human CYP2C and non-CYP2C substrates or inhibitors, was conducted to find potential CYP2C76 substrates. Among the compounds screened, 19 chemicals were identified as substrates for CYP2C76, including substrates for human CYP1A2 (7-ethoxyresorufin), CYP2B6 (bupropion), CYP2D6 (dextromethorphan), and CYP3A4/5 (dextromethorphan and nifedipine), and inhibitors for CYP2B6 (sertraline, clopidogrel, and ticlopidine), CYP2C8 (quercetin), CYP2C19 (ticlopidine and nootkatone), and CYP3A4/5 (troleandomycin). CYP2C76 metabolized a wide variety of the compounds with diverse structures. Among them, bupropion and nifedipine showed high selectivity to CYP2C76. As for nifedipine, CYP2C76 formed methylhydroxylated nifedipine, which was not produced by monkey CYP2C9, CYP2C19, or CYP3A4, as identified by mass spectrometry and estimated by a molecular docking simulation. This unique oxidative metabolite formation of nifedipine could be one of the selective marker reactions of CYP2C76 among the major CYP2Cs and CYP3As tested. These results suggest that monkey CYP2C76 contributes to bupropion hydroxylation and formation of different nifedipine oxidative metabolites as a result of its relatively large substrate cavity.

  17. Evaluation of 89 compounds for identification of substrates for cynomolgus monkey CYP2C76, a new bupropion/nifedipine oxidase.

    PubMed

    Hosaka, Shinya; Murayama, Norie; Satsukawa, Masahiro; Shimizu, Makiko; Uehara, Shotaro; Fujino, Hideki; Iwasaki, Kazuhide; Iwano, Shunsuke; Uno, Yasuhiro; Yamazaki, Hiroshi

    2015-01-01

    Cynomolgus monkeys are widely used in preclinical studies during drug development because of their evolutionary closeness to humans, including their cytochrome P450s (P450s). Most cynomolgus monkey P450s are almost identical (≥90%) to human P450s; however, CYP2C76 has low sequence identity (approximately 80%) to any human CYP2Cs. Although CYP2C76 has no ortholog in humans and is partly responsible for species differences in drug metabolism between cynomolgus monkeys and humans, a broad evaluation of potential substrates for CYP2C76 has not yet been conducted. In this study, a screening of 89 marketed compounds, including human CYP2C and non-CYP2C substrates or inhibitors, was conducted to find potential CYP2C76 substrates. Among the compounds screened, 19 chemicals were identified as substrates for CYP2C76, including substrates for human CYP1A2 (7-ethoxyresorufin), CYP2B6 (bupropion), CYP2D6 (dextromethorphan), and CYP3A4/5 (dextromethorphan and nifedipine), and inhibitors for CYP2B6 (sertraline, clopidogrel, and ticlopidine), CYP2C8 (quercetin), CYP2C19 (ticlopidine and nootkatone), and CYP3A4/5 (troleandomycin). CYP2C76 metabolized a wide variety of the compounds with diverse structures. Among them, bupropion and nifedipine showed high selectivity to CYP2C76. As for nifedipine, CYP2C76 formed methylhydroxylated nifedipine, which was not produced by monkey CYP2C9, CYP2C19, or CYP3A4, as identified by mass spectrometry and estimated by a molecular docking simulation. This unique oxidative metabolite formation of nifedipine could be one of the selective marker reactions of CYP2C76 among the major CYP2Cs and CYP3As tested. These results suggest that monkey CYP2C76 contributes to bupropion hydroxylation and formation of different nifedipine oxidative metabolites as a result of its relatively large substrate cavity. PMID:25318994

  18. CYP2D6 and CYP2A6 biotransform dietary tyrosol into hydroxytyrosol.

    PubMed

    Rodríguez-Morató, Jose; Robledo, Patricia; Tanner, Julie-Anne; Boronat, Anna; Pérez-Mañá, Clara; Oliver Chen, C-Y; Tyndale, Rachel F; de la Torre, Rafael

    2017-02-15

    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. PMID:27664690

  19. In vitro inhibition and induction of human liver cytochrome P450 enzymes by gentiopicroside: potent effect on CYP2A6.

    PubMed

    Deng, Yating; Wang, Lu; Yang, Yong; Sun, Wenji; Xie, Renming; Liu, Xueying; Wang, Qingwei

    2013-01-01

    Gentiopicroside (GE), a naturally occurring iridoid glycoside, has been developed into a Novel Traditional Chinese Drug named gentiopicroside injection, and it was approved for the treatment of acute jaundice and chronic active hepatitis by SFDA. However, the inhibitory and inducible effects of GE on the activity of cytochrome P450 (CYP450) are unclear. The purpose of this study was to evaluate the ability of GE to inhibit and induce human cytochrome P450 enzymes in vitro. In human liver microsomes, GE inhibited CYP2A6 and CYP2E1 in a concentration-dependent manner, with IC₅₀ values of 21.8 µg/ml and 594 µg/ml, respectively, and the IC₅₀ of CYP2A6 was close to the C(max) value observed clinically. GE was a non-competitive inhibitor of CYP2A6 at lower concentrations and a competitive inhibitor at higher concentrations. GE did not produce inhibition of CYP2C9, CYP2D6, CYP1A2 or CYP3A4 activities. However, a significant increase of CYP1A2 and CYP3A4 activity was observed at high concentrations. In cultured human hepatocytes no significant induction of CYP1A2, CYP3A4 or CYP2B6 was observed. Given these results, the in vivo potential inhibition of GE on CYP2A6 deserves further investigation, and it seems that the hepatoprotective effect of GE is irrelevant to its effect on P450s.

  20. 17 CFR 270.3a-4 - Status of investment advisory programs.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... requirement under section 5 of the Securities Act of 1933 with respect to programs that are organized and... presumption about a program that is not organized and operated in the manner contemplated by the section. (a... on the management of the account. (2)(i) At the opening of the account, the sponsor or another...

  1. [Role of antioxidants in electro catalytic activity of cytochrome P450 3A4].

    PubMed

    Shumiantseva, V V; Makhova, A A; Bulko, T V; Shikh, E V; Kukes, V G; Usanov, S A; Archakov, A I

    2014-01-01

    The electrochemical analysis of cytochrome Р450 3А4 catalytic activity has shown that vitamins C, A and Е influence on electron transfer and Fe3+/Fe2+ reduction process of cytochrome Р450 3А4. These data allow to assume possibility of cross effects and interference of vitamins-antioxidants with drugs metabolised by cytochrome Р450 3А4, at carrying out of complex therapy. This class of vitamins shows antioxidant properties that lead to increase of the cathodic current corresponding to heme reduction of this functionally significant haemoprotein. Ascorbic acid of 0.028-0.56 mM concentration stimulates cathodic peak (an electrochemical signal) of cytochrome Р450 3А4. At the presence of diclofenac (Voltaren) - a typical substrate of cytochrome Р450 3А4 - the increase growth of a catalytic current testifying to an electrocatalysis and stimulating action of ascorbic acid is observed. In the presence of vitamins A and Е also is registered dose-dependent (in a range of 10-100 M) increase in a catalytic current of cytochrome Р450 3А4: the maximum increase corresponds to 229 ± 20% for 100 M of vitamin A, and 162±10% for 100 M of vitamin E. Vitamin E in the presence of P450's inhibitor itraconazole doesn't give essential increase in a reductive current, unlike retinol (vitamin A). This effect can manifest substrate properties of tocopherol (vitamin E). The electrochemical approach for the analysis of catalytic activity of cytochrome Р450 3А4 and studies of influence of biologically active compounds on an electrocatalysis is the sensitive and effective sensor approach, allowing to use low concentration of protein on an electrode (till 10-15 mol/electrode), to carry out the analysis without participation of protein redox partners, and to reveal drug-drug or drug-vitamins interaction in pre-clinical experiments.

  2. Modulation of CYPs, P-gp, and PXR by Eschscholzia californica (California Poppy) and Its Alkaloids.

    PubMed

    Manda, Vamshi K; Ibrahim, Mohamed A; Dale, Olivia R; Kumarihamy, Mallika; Cutler, Stephen J; Khan, Ikhlas A; Walker, Larry A; Muhammad, Ilias; Khan, Shabana I

    2016-04-01

    Eschscholzia californica, a native US plant, is traditionally used as a sedative, analgesic, and anxiolytic herb. With the rapid rise in the use of herbal supplements together with over-the-counter and prescription drugs, the risk for potential herb-drug interactions is also increasing. Most of the clinically relevant pharmacokinetic drug interactions occur due to modulation of cytochrome P450 enzymes (CYPs), P-glycoprotein, and the pregnane X receptor by concomitantly used herbs. This study aimed to determine the effects of an EtOH extract, aqueous extract (tea), basic CHCl3 fractions, and isolated major alkaloids, namely protopine (1), escholtzine (2), allocryptopine (3), and californidine (4), of E. californica on the activity of cytochrome P450s, P-glycoprotein and the pregnane X receptor. The EtOH extract and fractions showed strong time-dependent inhibition of CYP 3A4, CYP 2C9, and CYP 2C19, and reversible inhibition of CYP 2D6. Among the alkaloids, escholtzine (2) and allocryptopine (3) exhibited time-dependent inhibition of CYP 3A4, CYP 2C9, and CYP 2C19 (IC50 shift ratio > 2), while protopine (1) and allocryptopine (3) showed reversible inhibition of CYP 2D6 enzyme. A significant activation of the pregnane X receptor (> 2-fold) was observed with the EtOH extract, basic CHCl3 fraction, and alkaloids (except protopine), which resulted into an increased expression of mRNA and the activity of CYP 3A4 and CYP 1A2. The expression of P-glycoprotein was unaffected. However, aqueous extract (tea) and its main alkaloid californidine (4) did not affect cytochrome P450s, P-glycoprotein, or the pregnane X receptor. This data suggests that EtOH extract of E. californica and its major alkaloids have a potential of causing interactions with drugs that are metabolized by cytochrome P450s, while the tea seems to be safer. PMID:27054913

  3. CYP450 Enzyme-Mediated Metabolism of TCAS and Its Inhibitory and Induced Effects on Metabolized Enzymes in Vitro.

    PubMed

    Shen, Guolin; Wang, Cheng; Zhou, Lili; Li, Lei; Chen, Huiming; Yu, Wenlian; Li, Haishan

    2015-09-02

    In this study, we investigated the enzymes catalyzing the phase I metabolism of thiacalixarene (TCAS) based on in vitro system including cDNA-expressed P450 enzymes, human liver microsomes plus inhibitors and monoclonal antibodies. In addition, the inhibitory potential of TCAS on major CYP450 drug metabolizing enzymes (CYP1A2, CYP2C9, CYP2B6, CYP2D6 and CYP3A4) was assessed. The results showed that CYP1A2 and CYP2C9 mediated TCAS hydroxylation. IC50 values for TCAS in rat and human liver microsomes were greater than 50 µM, and it demonstrated a weak inhibition of rat and human CYP450 enzymes. Finally, sandwiched hepatocytes were used to evaluate the induction of CYP1A and CYP3A to define the function of TCAS in vivo. The results showed that incubation of TCAS at different concentrations for 72 h failed to induce CYP1A and CYP3A. However, incubation of the cells with 50 and 100 µM TCAS caused a profound decrease in the activities of CYP1A and CYP3A, which was probably due to cytotoxic effects, suggesting that exposure to TCAS might be a health concern.

  4. CYP450 Enzyme-Mediated Metabolism of TCAS and Its Inhibitory and Induced Effects on Metabolized Enzymes in Vitro

    PubMed Central

    Shen, Guolin; Wang, Cheng; Zhou, Lili; Li, Lei; Chen, Huiming; Yu, Wenlian; Li, Haishan

    2015-01-01

    In this study, we investigated the enzymes catalyzing the phaseⅠmetabolism of thiacalixarene (TCAS) based on in vitro system including cDNA-expressed P450 enzymes, human liver microsomes plus inhibitors and monoclonal antibodies. In addition, the inhibitory potential of TCAS on major CYP450 drug metabolizing enzymes (CYP1A2, CYP2C9, CYP2B6, CYP2D6 and CYP3A4) was assessed. The results showed that CYP1A2 and CYP2C9 mediated TCAS hydroxylation. IC50 values for TCAS in rat and human liver microsomes were greater than 50 µM, and it demonstrated a weak inhibition of rat and human CYP450 enzymes. Finally, sandwiched hepatocytes were used to evaluate the induction of CYP1A and CYP3A to define the function of TCAS in vivo. The results showed that incubation of TCAS at different concentrations for 72 h failed to induce CYP1A and CYP3A. However, incubation of the cells with 50 and 100 µM TCAS caused a profound decrease in the activities of CYP1A and CYP3A, which was probably due to cytotoxic effects, suggesting that exposure to TCAS might be a health concern. PMID:26404338

  5. Bioactive components of Glycyrrhiza uralensis mediate drug functions and properties through regulation of CYP450 enzymes.

    PubMed

    Chen, Hao; Zhang, Xiaomei; Feng, Yifan; Rui, Wen; Shi, Zhongfeng; Wu, Lirong

    2014-09-01

    Glycyrrhiza uralensis (G. uralensis) is a common medicinal plant that has mainly been used to modulate the pharmaceutical activity of herbal medicines. Although G. uralensis has been shown to affect the expression and activity of the key metabolic enzyme cytochrome P450 (CYP450), the detailed mechanism of this process has yet to be elucidated. The present study aimed to elucidate the effects of bioactive components of G. uralensis on different isoforms of CYP450 and determine the ability of these components to modulate drug properties. In the present study, mRNA levels of CYP1A2, CYP2D6, CYP2E1, and CYP3A4 were investigated by quantitative polymerase chain reaction (qPCR) in HepG2 cells following treatment with the major bioactive compounds of G. uralensis. The activity of CYP450 enzymes was investigated in human liver microsomes using the cocktail probe drug method, and the metabolites of specific probes were detected by UPLC‑MS/MS. The effects of G. uralensis on CYP450 were assessed using bioinformatics network analysis. Several compounds from G. uralensis had various effects on the expression and activity of multiple CYP450 isoforms. The majority of the compounds analysed the inhibited expression of CYP2D6 and CYP3A4. Several CYP isoforms were differentially modulated depending on the specific compound and dose tested. In conclusion, the present study suggested that G. uralensis influenced the expression and activity of CYP450 enzymes. Therefore, caution should be taken when G. uralensis is co‑administered with drugs that are known to be metabolized by CYP450. This study contributed to the knowledge of the mechanisms by which this medicinal plant, commonly known as licorice, modulates drug efficacy.

  6. Effect of Ethanol on the Metabolic Characteristics of HIV-1 Integrase Inhibitor Elvitegravir and Elvitegravir/Cobicistat with CYP3A: An Analysis Using a Newly Developed LC-MS/MS Method.

    PubMed

    Midde, Narasimha M; Rahman, Mohammad A; Rathi, Chetan; Li, Junhao; Meibohm, Bernd; Li, Weihua; Kumar, Santosh

    2016-01-01

    Elvitegravir (EVG), an integrase inhibitor for the treatment HIV infection, is increasingly becoming the part of first-line antiretroviral therapy (ART) regimen. EVG is mainly metabolized through cytochrome P450 (CYP) 3A4. Previously, we have shown that ethanol alters ART-CYP3A4 interactions with protease inhibitors thereby altering their metabolisms. However, as EVG is a fairly new class of drug, its kinetic characteristics and the effect of ethanol on EVG-CYPP3A4 interaction is poorly understood. In this study, we characterized EVG and cobicistat (COBI)-boosted EVG metabolism in human microsomes followed by ethanol-EVG, ethanol-COBI-EVG interaction with CYP3A. First, we developed and validated a simple, sensitive, and robust liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of EVG in the human liver microsomes. The lower limit of quantification for the drug was at 0.003 μM (1.34 ng/ml). Extraction yield, matrix effects, drug stability, and calibration curves for the proposed method were validated according to the FDA guidelines. Time dependent kinetics data showed that 20mM ethanol decreases the apparent half-life of EVG degradation by ~50% compared to EVG alone. Our substrate kinetic results revealed that ethanol mildly decreases the catalytic efficiency for EVG metabolism. Inhibition studies demonstrated that EVG inhibits CYP3A4, and 20 mM ethanol causes a decrease in the IC50 of EVG. However, in the presence of COBI we were unable to determine these parameters effectively because COBI, being a strong inhibitor of CYP3A4, blocked the EVG/ethanol-CYP3A4 interactions. Docking studies predicted a shift of EVG or COBI binding to the active site of CYP3A4 in the presence of ethanol. Taken together, these results suggest that ethanol interacts with microsomal CYP3A and alters EVG-CYP3A4 interaction thereby altering EVG metabolism and inhibition of CYP3A4 by EVG. This finding has clinical significance because alcohol use is

  7. Effect of Ethanol on the Metabolic Characteristics of HIV-1 Integrase Inhibitor Elvitegravir and Elvitegravir/Cobicistat with CYP3A: An Analysis Using a Newly Developed LC-MS/MS Method.

    PubMed

    Midde, Narasimha M; Rahman, Mohammad A; Rathi, Chetan; Li, Junhao; Meibohm, Bernd; Li, Weihua; Kumar, Santosh

    2016-01-01

    Elvitegravir (EVG), an integrase inhibitor for the treatment HIV infection, is increasingly becoming the part of first-line antiretroviral therapy (ART) regimen. EVG is mainly metabolized through cytochrome P450 (CYP) 3A4. Previously, we have shown that ethanol alters ART-CYP3A4 interactions with protease inhibitors thereby altering their metabolisms. However, as EVG is a fairly new class of drug, its kinetic characteristics and the effect of ethanol on EVG-CYPP3A4 interaction is poorly understood. In this study, we characterized EVG and cobicistat (COBI)-boosted EVG metabolism in human microsomes followed by ethanol-EVG, ethanol-COBI-EVG interaction with CYP3A. First, we developed and validated a simple, sensitive, and robust liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of EVG in the human liver microsomes. The lower limit of quantification for the drug was at 0.003 μM (1.34 ng/ml). Extraction yield, matrix effects, drug stability, and calibration curves for the proposed method were validated according to the FDA guidelines. Time dependent kinetics data showed that 20mM ethanol decreases the apparent half-life of EVG degradation by ~50% compared to EVG alone. Our substrate kinetic results revealed that ethanol mildly decreases the catalytic efficiency for EVG metabolism. Inhibition studies demonstrated that EVG inhibits CYP3A4, and 20 mM ethanol causes a decrease in the IC50 of EVG. However, in the presence of COBI we were unable to determine these parameters effectively because COBI, being a strong inhibitor of CYP3A4, blocked the EVG/ethanol-CYP3A4 interactions. Docking studies predicted a shift of EVG or COBI binding to the active site of CYP3A4 in the presence of ethanol. Taken together, these results suggest that ethanol interacts with microsomal CYP3A and alters EVG-CYP3A4 interaction thereby altering EVG metabolism and inhibition of CYP3A4 by EVG. This finding has clinical significance because alcohol use is

  8. Effects of capsaicin and dihydrocapsaicin on human and rat liver microsomal CYP450 enzyme activities in vitro and in vivo.

    PubMed

    Zhang, Qing-Hao; Hu, Jin-Ping; Wang, Bao-Lian; Li, Yan

    2012-01-01

    Capsaicin and dihydrocapsaicin, the two most abundant members of capsaicinoids in chili peppers, are widely used as food additives and for other purposes. In this study, we examined the inhibitory potentials of capsaicin and dihydrocapsaicin against CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4/5 activities in human liver microsomes. The effects of these two capsaicinoids on CYP450 enzymes were also evaluated in vivo in rats. The results demonstrated that capsaicin and dihydrocapsaicin moderately inhibited five isozymes (IC₅₀) values ranging from 4.4 to 61.8 μM), with the exception of CYP2E1 (IC₅₀ > 200 μM). Both capsaicinoids exhibited competitive, mixed, and noncompetitive inhibition on these isozymes (K (i) = 3.1 ± 0.5 - 78.6 ± 8.4 μM). Time-dependent inhibition of CYP3A4/5 by capsaicin was found. After multiple administrations of capsaicin and dihydrocapsaicin (1, 4, and 10 mg/kg) to rats, chlorzoxazone 6-hydroxylase activity and the expression of CYP2E1 were increased in liver microsomes. Our findings indicated that the possibility of food-drug interactions mediated by capsaicin and dihydrocapsaicin could not be excluded, and provided the useful information for evaluating the anticarcinogenic potentials of these two capsaicinoids. PMID:22375877

  9. Cytochrome P450 CYP3A in marsupials: cloning and characterisation of the second identified CYP3A subfamily member, isoform 3A78 from koala (Phascolarctos cinereus).

    PubMed

    El-Merhibi, Adaweyah; Ngo, Suong N T; Crittenden, Tamara A; Marchant, Ceilidh L; Stupans, Ieva; McKinnon, Ross A

    2011-11-01

    Cytochromes P450 (CYPs) are critically important in the oxidative metabolism of a diverse array of xenobiotics and endogenous substrates. Previously, we cloned and characterised the CYP2C, CYP4A, and CYP4B gene subfamilies from marsupials and demonstrated important species-differences in both activity and tissue expression of these CYP enzymes. Recently, we isolated the Eastern grey kangaroo CYP3A70. Here we have cloned and characterised the second identified member of marsupial CYP3A gene subfamily, CYP3A78 from the koala (Phascolarctos cinereus). In addition, we have examined the gender-differences in microsomal erythromycin N-demethylation activity (a CYP3A marker) and CYP3A protein expression across test marsupial species. Significant differences in hepatic erythromycin N-demethylation activity were observed between male and female koalas, with the activity detected in female koalas being 2.5-fold higher compared to that in male koalas (p<0.01). No gender-differences were observed in tammar wallaby or Eastern grey kangaroo. Immunoblot analysis utilising anti-human CYP3A4 antibody detected immunoreactive proteins in liver microsomes from all test male and female marsupials including the koala, tammar wallaby, and Eastern grey kangaroo, with no gender-differences detected across test marsupials. A 1610 bp koala hepatic CYP3A complete cDNA, designated CYP3A78, was cloned by reverse transcription-polymerase chain reaction approaches. It displays 64% nucleotide and 57% amino acid sequence identity to the Eastern grey kangaroo CYP3A70. The CYP3A78 cDNA encodes a protein of 515 amino acids, shares approximately 68% nucleotide and 56% amino acid sequence identity to human CYP3A4, and displays high sequence similarity to other published mammalian CYP3As from human, monkey, cow, pig, dog, rat, rabbit, mouse, hamster, and guinea pig. Collectively, this study provides primary molecular data regarding koala hepatic CYP3A78 gene and enables further functional analyses of CYP

  10. In vitro inhibition of the cytochrome P450 (CYP450) system by the antiplatelet drug ticlopidine: potent effect on CYP2C19 and CYP2D6

    PubMed Central

    Ko, Jae Wook; Desta, Zeruesenay; Soukhova, Nadia V; Tracy, Timothy; Flockhart, David A

    2000-01-01

    Aims To examine the potency of ticlopidine (TCL) as an inhibitor of cytochrome P450s (CYP450s) in vitro using human liver microsomes (HLMs) and recombinant human CYP450s. Methods Isoform-specific substrate probes of CYP1A2, 2C19, 2C9, 2D6, 2E1 and 3A4 were incubated in HLMs or recombinant CYPs with or without TCL. Preliminary data were generated to simulate an appropriate range of substrate and inhibitor concentrations to construct Dixon plots. In order to estimate accurately inhibition constants (Ki values) of TCL and determine the type of inhibition, data from experiments with three different HLMs for each isoform were fitted to relevant nonlinear regression enzyme inhibition models by WinNonlin. Results TCL was a potent, competitive inhibitor of CYP2C19 (Ki = 1.2 ± 0.5 µm) and of CYP2D6 (Ki = 3.4 ± 0.3 µm). These Ki values fell within the therapeutic steady-state plasma concentrations of TCL (1–3 µm). TCL was also a moderate inhibitor of CYP1A2 (Ki = 49 ± 19 µm) and a weak inhibitor of CYP2C9 (Ki > 75 µm), but its effect on the activities of CYP2E1 (Ki = 584 ± 48 µm) and CYP3A (> 1000 µm) was marginal. Conclusions TCL appears to be a broad-spectrum inhibitor of the CYP isoforms, but clinically significant adverse drug interactions are most likely with drugs that are substrates of CYP2C19 or CYP2D6. PMID:10759690

  11. The tyrosine kinase inhibitor nilotinib selectively inhibits CYP2C8 activities in human liver microsomes.

    PubMed

    Kim, Min-Jung; Lee, Jae-Won; Oh, Kyung-Suk; Choi, Chang-Soo; Kim, Kwang Hee; Han, Won Seok; Yoon, Chang-No; Chung, Eun Sook; Kim, Dong-Hyun; Shin, Jae-Gook

    2013-01-01

    The tyrosine kinase inhibitor nilotinib was examined for its inhibition of cytochrome P450s (CYPs) in human liver microsomes and in human CYPs expressed in a baculovirus-insect cell system. Nilotinib demonstrated preferential inhibition of CYP2C8-mediated paclitaxel 6α-hydroxylation, rosiglitazone hydroxylation and amodiaquine N-deethylation in human liver microsomes, with IC₅₀ values of 0.4, 7.5 and 0.7 µM, respectively. The IC₅₀ value of nilotinib for paclitaxel 6α-hydroxylation was 20-fold lower than that of the other five tyrosine-kinase inhibitors tested. Nilotinib appears to display competitive inhibition against paclitaxel 6α-hydroxylation and amodiaquine N-deethylation, with estimated mean Ki values of 0.90 and 0.15 µM in human liver microsomes and 0.10 and 0.61 µM in recombinant human CYP2C8, respectively. These results are consistent with those of molecular docking simulations, where paclitaxel could not access the CYP2C8 catalytic site in the presence of nilotinib, but the binding of midazolam, a substrate of CYP3A4, to the catalytic site of CYP3A4 was not affected by nilotinib. The demonstrated inhibitory activity of nilotinib against CYP2C8 at concentrations less than those observed in patients who received nilotinib therapy is of potential clinical relevance and further in vivo exploration is warranted.

  12. Chlorogenic acid prevents acetaminophen-induced liver injury: the involvement of CYP450 metabolic enzymes and some antioxidant signals.

    PubMed

    Pang, Chun; Sheng, Yu-chen; Jiang, Ping; Wei, Hai; Ji, Li-li

    2015-07-01

    Chlorogenic acid (CGA), a polyphenolic compound, is abundant in fruits, dietary vegetables, and some medicinal herbs. This study investigated the prevention of CGA against acetaminophen (AP)-induced hepatotoxicity and its engaged mechanisms. CGA reversed the decreased cell viability induced by AP in L-02 cells in vitro. In addition, CGA reduced the AP-induced increased serum levels of alanine/aspartate aminotransferase (ALT/AST) in vivo. The effect of CGA on cytochrome P450 (CYP) enzymatic (CYP2E1, CYP1A2, and CYP3A4) activities showed that CGA caused very little inhibition on CYP2E1 and CYP1A2 enzymatic activities, but not CYP3A4. The measurement of liver malondialdehyde (MDA), reactive oxygen species (ROS), and glutathione (GSH) levels showed that CGA prevented AP-induced liver oxidative stress injury. Further, CGA increased the AP-induced decreased mRNA expression of peroxiredoxin (Prx) 1, 2, 3, 5, 6, epoxide hydrolase (Ephx) 2, and polymerase (RNA) II (DNA directed) polypeptide K (Polr2k), and nuclear factor erythroid-2-related factor 2 (Nrf2). In summary, CGA ameliorates the AP-induced liver injury probably by slightly inhibiting CYP2E1 and CYP1A2 enzymatic properties. In addition, cellular important antioxidant signals such as Prx1, 2, 3, 5, 6, Ephx2, Polr2k, and Nrf2 also contributed to the protection of CGA against AP-induced oxidative stress injury. PMID:26160718

  13. Chlorogenic acid prevents acetaminophen-induced liver injury: the involvement of CYP450 metabolic enzymes and some antioxidant signals*

    PubMed Central

    Pang, Chun; Sheng, Yu-chen; Jiang, Ping; Wei, Hai; Ji, Li-li

    2015-01-01

    Chlorogenic acid (CGA), a polyphenolic compound, is abundant in fruits, dietary vegetables, and some medicinal herbs. This study investigated the prevention of CGA against acetaminophen (AP)-induced hepatotoxicity and its engaged mechanisms. CGA reversed the decreased cell viability induced by AP in L-02 cells in vitro. In addition, CGA reduced the AP-induced increased serum levels of alanine/aspartate aminotransferase (ALT/AST) in vivo. The effect of CGA on cytochrome P450 (CYP) enzymatic (CYP2E1, CYP1A2, and CYP3A4) activities showed that CGA caused very little inhibition on CYP2E1 and CYP1A2 enzymatic activities, but not CYP3A4. The measurement of liver malondialdehyde (MDA), reactive oxygen species (ROS), and glutathione (GSH) levels showed that CGA prevented AP-induced liver oxidative stress injury. Further, CGA increased the AP-induced decreased mRNA expression of peroxiredoxin (Prx) 1, 2, 3, 5, 6, epoxide hydrolase (Ephx) 2, and polymerase (RNA) II (DNA directed) polypeptide K (Polr2k), and nuclear factor erythroid-2-related factor 2 (Nrf2). In summary, CGA ameliorates the AP-induced liver injury probably by slightly inhibiting CYP2E1 and CYP1A2 enzymatic properties. In addition, cellular important antioxidant signals such as Prx1, 2, 3, 5, 6, Ephx2, Polr2k, and Nrf2 also contributed to the protection of CGA against AP-induced oxidative stress injury. PMID:26160718

  14. Physiologically Based Pharmacokinetic Predictions of Tramadol Exposure Throughout Pediatric Life: an Analysis of the Different Clearance Contributors with Emphasis on CYP2D6 Maturation.

    PubMed

    T'jollyn, Huybrecht; Snoeys, Jan; Vermeulen, An; Michelet, Robin; Cuyckens, Filip; Mannens, Geert; Van Peer, Achiel; Annaert, Pieter; Allegaert, Karel; Van Bocxlaer, Jan; Boussery, Koen

    2015-11-01

    This paper focuses on the retrospective evaluation of physiologically based pharmacokinetic (PBPK) techniques used to mechanistically predict clearance throughout pediatric life. An intravenous tramadol retrograde PBPK model was set up in Simcyp® using adult clearance values, qualified for CYP2D6, CYP3A4, CYP2B6, and renal contributions. Subsequently, the model was evaluated for mechanistic prediction of total, CYP2D6-related, and renal clearance predictions in very early life. In two in vitro pediatric human liver microsomal (HLM) batches (1 and 3 months), O-desmethyltramadol and N-desmethyltramadol formation rates were compared with CYP2D6 and CYP3A4 activity, respectively. O-desmethyltramadol formation was mediated only by CYP2D6, while N-desmethyltramadol was mediated in part by CYP3A4. Additionally, the clearance maturation of the PBPK model predictions was compared to two in vivo maturation models (Hill and exponential) based on plasma concentration data, and to clearance estimations from a WinNonlin® fit of plasma concentration and urinary excretion data. Maturation of renal and CYP2D6 clearance is captured well in the PBPK model predictions, but total tramadol clearance is underpredicted. The most pronounced underprediction of total and CYP2D6-mediated clearance was observed in the age range of 2-13 years. In conclusion, the PBPK technique showed to be a powerful mechanistic tool capable of predicting maturation of CYP2D6 and renal tramadol clearance in early infancy, although some underprediction occurs between 2 and 13 years for total and CYP2D6-mediated tramadol clearance. PMID:26209290

  15. Cytochrome P450 CYP3A in marsupials: cloning and identification of the first CYP3A subfamily member, isoform 3A70 from Eastern gray kangaroo (Macropus giganteus).

    PubMed

    El-Merhibi, Adaweyah; Ngo, Suong N T; Marchant, Ceilidh L; Height, Tamara A; Stupans, Ieva; McKinnon, Ross A

    2012-09-15

    Australian marsupials are unique fauna that have evolved and adapted to unique environments and thus it is likely that their detoxification systems differ considerably from those of well-studied eutherian mammals. Knowledge of these processes in marsupials is therefore vital to understanding the consequences of exposure to xenobiotics. Cytochromes P450 (CYPs) are critically important in the oxidative metabolism of a diverse array of both xenobiotics and endogenous substrates. In this study we have cloned and characterized CYP3A70, the first identified member of the CYP3A gene subfamily from Eastern gray kangaroo (Macropus giganteus). A 1665 base pair kangaroo hepatic CYP3A complete cDNA, designated CYP3A70, was cloned by reverse transcription-polymerase chain reaction approaches, which encodes a protein of 506 amino acids. The CYP3A70 cDNA shares approximately 71% nucleotide and 65% amino acid sequence homology to human CYP3A4 and displays high sequence similarity to other published mammalian CYP3As from human, monkey, cow, pig, dog, rat, rabbit, mouse, hamster, and guinea pig. Transfection of the CYP3A70 cDNAs into 293T cells resulted in stable cell lines expressing a CYP3A immuno-reactive protein that was recognized by a goat anti-human CYP3A4 polyclonal antibody. The anti-human CYP3A4 antibody also detected immunoreactive proteins in liver microsomes from all test marsupials, including the kangaroo, koala, wallaby, and wombat, with multiple CYP3A immunoreactive bands observed in kangaroo and wallaby tissues. Relatively, very low CYP catalytic activity was detected for the kangaroo CYP3A70 cDNA-expressed proteins (19.6 relative luminescent units/μg protein), which may be due to low protein expression levels. Collectively, this study provides primary molecular data regarding the Eastern kangaroo hepatic CYP3A70 gene and enables further functional analyses of CYP3A enzymes in marsupials.

  16. Effect of Radix Sophorae Flavescentis on activity of CYP450 isoforms in rats

    PubMed Central

    Chen, Lianguo; Cai, Jinzhang; Wang, Shuanghu; Hu, Lufeng; Yang, Xuezhi

    2015-01-01

    Kushen (Radix Sophorae Flavescentis) is the dried roots of Sophora Flavescens Ait, alkaloids and flavonoids are the main active constituents of Radix Sophorae Flavescentis. The influence of Radix Sophorae Flavescentis on the activities of CYP450 isoforms CYP2B6, CYP2C19, CYP1A2, CYP2C9, CYP3A4 and CYP2D6 were evaluated by cocktail method. The rats were randomly divided into Radix Sophorae Flavescentis group and control group. The Radix Sophorae Flavescentis group rats were given 5 g/kg Radix Sophorae Flavescentis decoction by intragastric administration. The six probe drugs (bupropion, omeprazole, phenacetin, tolbutamide, midazolam and metroprolol) were given to rats through intragastric administration, and the plasma concentration were determined by UPLC-MS/MS. The result of Radix Sophorae Flavescentis group compared to control group, there were statistical pharmacokinetics difference for omeprazole, phenacetin, tolbutamide and metroprolol. It indicated that the Radix Sophorae Flavescentis may induce the activities of CYP2D6, and inhibit of CYP2C19, CYP1A2 and CYP2C9 of rats. As other drugs are always used after Radix Sophorae Flavescentis, interactions between other drugs and Radix Sophorae Flavescentis undertake the risk of either diminished efficacy or adverse effects. This may give advising for reasonable drug use after Radix Sophorae Flavescentis. PMID:26885078

  17. Coexpression of CPR from various origins enhances biotransformation activity of human CYPs in S. pombe.

    PubMed

    Neunzig, Ina; Widjaja, Maria; Peters, Frank T; Maurer, Hans H; Hehn, Alain; Bourgaud, Frédéric; Bureik, Matthias

    2013-08-01

    Cytochrome P450 enzymes (CYPs or P450s) are the most important enzymes involved in the phase I metabolism of drugs (and other xenobiotics) in humans, and the corresponding drug metabolites are needed as reference substances for their structural confirmation and for pharmacological or toxicological characterization. We have previously shown that biotechnological synthesis of such metabolites is feasible by whole-cell biotransformation with human CYPs recombinantly expressed in the fission yeast Schizosaccharomyces pombe. It was the aim of this study to compare the activity of seven human microsomal CYPs (CYP2C9, CYP2D6, CYP3A4, CYP3A5, CYP3A7, CYP17, and CYP21) upon coexpression with NADPH-cytochrome P450 oxidoreductases (CPRs) from various origins, namely, human CPR (hCPR) and its homologues from fission yeast (ccr1) and the bishop's weed Ammi majus (AmCPR), respectively. For this purpose, 28 recombinant strains were needed, with five of them having been constructed previously and 23 strains being newly constructed. Bioconversion experiments showed that coexpression of a CPR does not only influence the reaction rate but, in some cases, also exerts an influence on the metabolite pattern. For CYP3A enzymes, coexpression of hCPR yielded the best results, while for another two, hCPR was equally helpful as ccr1 (both CYP17 and CYP21) or AmCPR (CYP17 only), respectively. Interestingly, CYP2D6 displayed its highest activity when coexpressed with ccr1 and CYP2C9 with AmCPR. These results corroborate the view of CPR as a well-suited bio-brick in synthetic biology for the construction of artificial enzyme complexes.

  18. Insights into drug metabolism by cytochromes P450 from modelling studies of CYP2D6-drug interactions.

    PubMed

    Maréchal, J-D; Kemp, C A; Roberts, G C K; Paine, M J I; Wolf, C R; Sutcliffe, M J

    2008-03-01

    The cytochromes P450 (CYPs) comprise a vast superfamily of enzymes found in virtually all life forms. In mammals, xenobiotic metabolizing CYPs provide crucial protection from the effects of exposure to a wide variety of chemicals, including environmental toxins and therapeutic drugs. Ideally, the information on the possible metabolism by CYPs required during drug development would be obtained from crystal structures of all the CYPs of interest. For some years only crystal structures of distantly related bacterial CYPs were available and homology modelling techniques were used to bridge the gap and produce structural models of human CYPs, and thereby obtain useful functional information. A significant step forward in the reliability of these models came seven years ago with the first crystal structure of a mammalian CYP, rabbit CYP2C5, followed by the structures of six human enzymes, CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2D6 and CYP3A4, and a second rabbit enzyme, CYP2B4. In this review we describe as a case study the evolution of a CYP2D6 model, leading to the validation of the model as an in silico tool for predicting binding and metabolism. This work has led directly to the successful design of CYP2D6 mutants with novel activity-including creating a testosterone hydroxylase, converting quinidine from inhibitor to substrate, creating a diclofenac hydroxylase and creating a dextromethorphan O-demethylase. Our modelling-derived hypothesis-driven integrated interdisciplinary studies have given key insight into the molecular determinants of CYP2D6 and other important drug metabolizing enzymes. PMID:18026129

  19. Cytochrome P450 CYP3A in human renal cell cancer

    PubMed Central

    Murray, G I; McFadyen, M C E; Mitchell, R T; Cheung, Y-L; Kerr, A C; Melvin, W T

    1999-01-01

    Renal cell cancer is the main malignant tumour of the kidney and has an increasing incidence. This type of tumour has a poor prognosis and shows intrinsic resistance to several anti-cancer drugs. The CYP3A P450 family, which consists of three closely related forms, is involved in the oxidative activation and deactivation of a variety of carcinogens and several anti-cancer drugs. In this study the presence and cellular localization of CYP3A has been investigated using a combination of immunohistochemistry, immunoblotting and reverse transcriptase polymerase chain reaction (RT-PCR) in renal cell cancer and corresponding normal kidney. CYP3A was consistently expressed in both renal call cancer and in normal kidney. In renal cell cancer, CYP3A was localized to tumour cells and in normal kidney the predominant cellular localization of CYP3A was to proximal tubular epithelial cells. RT-PCR showed that both CYP3A5 mRNA and CYP3A7 mRNA were consistently present in both tumour and normal samples, while CYP3A4 mRNA was present in 65% of tumours and 90% of normal samples. This study indicates that individual members of the CYP3A family are expressed in renal cell cancer. The presence of CYP3A in renal cell cancer might be important in the metabolic potentiation as well as the detoxification of chemotherapeutic agents used to renal cancer. © 1999 Cancer Research Campaign PMID:10206301

  20. Inhibitory effects of phthalimide derivatives on the activity of the hepatic cytochrome P450 monooxygenases CYP2C9 and CYP2C19.

    PubMed

    Kolukisaoglu, Üner; Wendler, Christian; Goerdes, Dirk; Diener, Annette; Thurow, Kerstin

    2010-12-01

    Affecting hepatic cytochrome (CYP) activity is one of the major concerns in drug-drug interaction. Thus the testing of drug candidates on their impact on these enzymes is an essential step in early drug discovery. We tested a collection of 480 in-house phthalimide derivatives against different CYP450s using a high throughput inhibition assay. In initial tests with the isoform CYP2C19 about 57.5% of the tested phthalimide derivatives showed significantly enhanced inhibitory effects against this enzyme. In addition similar patterns of phthalimide inhibition for CYP2C9 and CYP2C19 were found, whereas the unrelated isoforms CYP2D6 and CYP3A4 were not specifically affected. Also less than 10% of randomly chosen substances inhibited CYP2C9. Analyses of structure-function relationships revealed that the substituent at the nitrogen atom in the isoindole ring is of crucial impact for the activity of CYP2C9/19.

  1. Peroxisome proliferator-activated receptor alpha, PPARα, directly regulates transcription of cytochrome P450 CYP2C8

    PubMed Central

    Thomas, Maria; Winter, Stefan; Klumpp, Britta; Turpeinen, Miia; Klein, Kathrin; Schwab, Matthias; Zanger, Ulrich M.

    2015-01-01

    The cytochrome P450, CYP2C8, metabolizes more than 60 clinically used drugs as well as endogenous substances including retinoic acid and arachidonic acid. However, predictive factors for interindividual variability in the efficacy and toxicity of CYP2C8 drug substrates are essentially lacking. Recently we demonstrated that peroxisome proliferator-activated receptor alpha (PPARα), a nuclear receptor primarily involved in control of lipid and energy homeostasis directly regulates the transcription of CYP3A4. Here we investigated the potential regulation of CYP2C8 by PPARα. Two linked intronic SNPs in PPARα (rs4253728, rs4823613) previously associated with hepatic CYP3A4 status showed significant association with CYP2C8 protein level in human liver samples (N = 150). Furthermore, siRNA-mediated knock-down of PPARα in HepaRG human hepatocyte cells resulted in up to ∼60 and ∼50% downregulation of CYP2C8 mRNA and activity, while treatment with the PPARα agonist WY14,643 lead to an induction by >150 and >100%, respectively. Using chromatin immunoprecipitation scanning assay we identified a specific upstream gene region that is occupied in vivo by PPARα. Electromobility shift assay demonstrated direct binding of PPARα to a DR-1 motif located at positions –2762/–2775 bp upstream of the CYP2C8 transcription start site. We further validated the functional activity of this element using luciferase reporter gene assays in HuH7 cells. Moreover, based on our previous studies we demonstrated that WNT/β-catenin acts as a functional inhibitor of PPARα-mediated inducibility of CYP2C8 expression. In conclusion, our data suggest direct involvement of PPARα in both constitutive and inducible regulation of CYP2C8 expression in human liver, which is further modulated by WNT/β-catenin pathway. PPARA gene polymorphism could have a modest influence on CYP2C8 phenotype. PMID:26582990

  2. Homology model of human retinoic acid metabolising enzyme cytochrome P450 26A1 (CYP26A1): active site architecture and ligand binding.

    PubMed

    Gomaa, Mohamed Sayed; Yee, Sook Wah; Milbourne, Ceri Elizabeth; Barbera, Maria Chiara; Simons, Claire; Brancale, Andrea

    2006-08-01

    Homology models of cytochrome P450 RA1 (CYP26A1) were constructed using three human P450 structures, CYP2C8, CYP2C9 and CYP3A4 as templates for the model building. Using MOE software the lowest energy CYP26A1 model was then assessed for stereochemical quality and side chain environment. Further active site optimisation of the CYP26A1 model built using the CYP3A4 template was performed by molecular dynamics to generate a final CYP26A1 model. The natural substrate, all-trans-retinoic acid (atRA), and inhibitor R 15866, were docked into the model allowing further validation of the active site architecture. Using the docking studies structurally and functionally important residues were identified with subsequent characterisation of secondary structure. Multiple hydrophobic interactions, including the side chains of TRP112, PHE299, PHE222, PHE84, PHE374 and PRO371, are important for binding of atRA and R115866. Additional hydrogen bonding interactions were noted as follows: atRA-- C==O of the atRA carboxylate group and ARG86; R115866--benzothiazole nitrogen and the backbone NH of SER115.

  3. Identifying a selective substrate and inhibitor pair for the evaluation of CYP2J2 activity.

    PubMed

    Lee, Caroline A; Jones, J P; Katayama, Jonathan; Kaspera, Rüdiger; Jiang, Ying; Freiwald, Sascha; Smith, Evan; Walker, Gregory S; Totah, Rheem A

    2012-05-01

    CYP2J2, an arachidonic acid epoxygenase, is recognized for its role in the first-pass metabolism of astemizole and ebastine. To fully assess the role of CYP2J2 in drug metabolism, a selective substrate and potent specific chemical inhibitor are essential. In this study, we report amiodarone 4-hydoxylation as a specific CYP2J2-catalyzed reaction with no CYP3A4, or other drug-metabolizing enzyme, involvement. Amiodarone 4-hydroxylation enabled the determination of liver relative activity factor and intersystem extrapolation factor for CYP2J2. Amiodarone 4-hydroxylation correlated with astemizole O-demethylation but not with CYP2J2 protein content in a sample of human liver microsomes. To identify a specific CYP2J2 inhibitor, 138 drugs were screened using terfenadine and astemizole as probe substrates with recombinant CYP2J2. Forty-two drugs inhibited CYP2J2 activity by ≥50% at 30 μM, but inhibition was substrate-dependent. Of these, danazol was a potent inhibitor of both hydroxylation of terfenadine (IC(50) = 77 nM) and O-demethylation of astemizole (K(i) = 20 nM), and inhibition was mostly competitive. Danazol inhibited CYP2C9, CYP2C8, and CYP2D6 with IC(50) values of 1.44, 1.95, and 2.74 μM, respectively. Amiodarone or astemizole were included in a seven-probe cocktail for cytochrome P450 (P450) drug-interaction screening potential, and astemizole demonstrated a better profile because it did not appreciably interact with other P450 probes. Thus, danazol, amiodarone, and astemizole will facilitate the ability to determine the metabolic role of CYP2J2 in hepatic and extrahepatic tissues. PMID:22328583

  4. The influence of standardized Valeriana officinalis extract on the CYP3A1 gene expression by nuclear receptors in in vivo model.

    PubMed

    Bogacz, Anna; Mrozikiewicz, Przemyslaw M; Karasiewicz, Monika; Bartkowiak-Wieczorek, Joanna; Majchrzycki, Marian; Mikolajczak, Przemyslaw L; Ozarowski, Marcin; Grzeskowiak, Edmund

    2014-01-01

    Valeriana officinalis is one of the most popular medicinal plants commonly used as a sedative and sleep aid. It is suggested that its pharmacologically active compounds derived from the root may modulate the CYP3A4 gene expression by activation of pregnane X receptor (PXR) or constitutive androstane receptor (CAR) and lead to pharmacokinetic herb-drug interactions. The aim of the study was to determine the influence of valerian on the expression level of CYP3A1 (homologue to human CYP3A4) as well as nuclear receptors PXR, CAR, RXR, GR, and HNF-4α. Male Wistar rats were given standardized valerian extract (300 mg/kg/day, p.o.) for 3 and 10 days. The expression in liver tissue was analyzed by using real-time PCR. Our result showed a decrease of CYP3A1 expression level by 35% (P = 0.248) and 37% (P < 0.001), respectively. Moreover, Valeriana exhibited statistically significant reduction in RXR (approximately 28%) only after 3-day treatment. We also demonstrated a decrease in the amount HNF-4α by 22% (P = 0.005) and 32% (P = 0.012), respectively. In case of CAR, the increase of expression level by 46% (P = 0.023) was noted. These findings suggest that Valeriana officinalis extract can decrease the CYP3A4 expression and therefore may lead to interactions with synthetic drugs metabolized by this enzyme. PMID:25302309

  5. [Interaction of butylphthalide with rat and human liver CYP450 isoenzymes].

    PubMed

    Zhao, Qian; Hu, Jin-ping; Jiang, Ji; Li, Yan; Hu, Pei

    2015-05-01

    The work aims to study the drug metabolizing enzymes involved in the metabolism of butylphthalide and evaluate the induction and inhibition activities of butylphthalide on CYP450 isoenzymes by using in vitro (liver microsome incubation system of rats and human) and in vivo (CYP induced model of rats) method. Butylphthalide was incubated with selective inhibitors of CYP450, and its metabolic rate was determined to identify the metabolizing isoenzymes of NBP in rat (normal and induced rats) and human liver microsomes. The in vitro inhibition effect of butylphthalide on 6 main liver microsomal CYP450 isoenzymes was evaluated by using probe drugs; the induction and inhibition activities in vivo of butylphthalide on CYP450 isoenzymes were evaluated by NBP ig dosing (160 mg x kg(-1)) and iv dosing (20 mg x kg(-1)) in rats. After adding the specific inhibitors of CYP2C11, 2E1 and 3A 1/2 for rat, CYP2C19, 2E1 and 3A4/5 for human, the metabolism of NBP in rat and human liver microsomes were reduced 38.8%, 86.2%, 78.4% and 51.0%, 92.0%, 58.9% of control, respectively. The metabolic rates of NBP in CYP2E1 and 3A 1/2 induced rat liver microsomes were increased 25.5% and 68.9%. High concentration of NBP (≥ 200 μmol x L(-1), in vitro) could inhibit the activities of CYP1A2, 2C6, 2C11 and 2D2 in rats, and high concentration of NBP ( ≥ 15 μmol x L(-1), in vitro) could inhibit the activity of CYP2C19 in human. All the results indicated that NBP should be mainly metabolized by CYP2E1, 2C11 and 3A 1/2 in rats and CYP2E1, 2C19 and 3A4/5 in human. High concentration of NBP could inhibit human CYP2C19 in vitro. No significant induction/inhibition effects of NBP were observed on rat liver CYP450 isoforms after ig 160 mg x kg(-1) NBP or iv 20 mg x kg(-1) NBP. PMID:26234133

  6. Metabolic Pathways of Inhaled Glucocorticoids by the CYP3A Enzymes

    PubMed Central

    Moore, Chad D.; Roberts, Jessica K.; Orton, Christopher R.; Murai, Takahiro; Fidler, Trevor P.; Reilly, Christopher A.; Ward, Robert M.

    2013-01-01

    Asthma is one of the most prevalent diseases in the world, for which the mainstay treatment has been inhaled glucocorticoids (GCs). Despite the widespread use of these drugs, approximately 30% of asthma sufferers exhibit some degree of steroid insensitivity or are refractory to inhaled GCs. One hypothesis to explain this phenomenon is interpatient variability in the clearance of these compounds. The objective of this research is to determine how metabolism of GCs by the CYP3A family of enzymes could affect their effectiveness in asthmatic patients. In this work, the metabolism of four frequently prescribed inhaled GCs, triamcinolone acetonide, flunisolide, budesonide, and fluticasone propionate, by the CYP3A family of enzymes was studied to identify differences in their rates of clearance and to identify their metabolites. Both interenzyme and interdrug variability in rates of metabolism and metabolic fate were observed. CYP3A4 was the most efficient metabolic catalyst for all the compounds, and CYP3A7 had the slowest rates. CYP3A5, which is particularly relevant to GC metabolism in the lungs, was also shown to efficiently metabolize triamcinolone acetonide, budesonide, and fluticasone propionate. In contrast, flunisolide was only metabolized via CYP3A4, with no significant turnover by CYP3A5 or CYP3A7. Common metabolites included 6β-hydroxylation and Δ6-dehydrogenation for triamcinolone acetonide, budesonide, and flunisolide. The structure of Δ6-flunisolide was unambiguously established by NMR analysis. Metabolism also occurred on the D-ring substituents, including the 21-carboxy metabolites for triamcinolone acetonide and flunisolide. The novel metabolite 21-nortriamcinolone acetonide was also identified by liquid chromatography–mass spectrometry and NMR analysis. PMID:23143891

  7. Metabolic pathways of inhaled glucocorticoids by the CYP3A enzymes.

    PubMed

    Moore, Chad D; Roberts, Jessica K; Orton, Christopher R; Murai, Takahiro; Fidler, Trevor P; Reilly, Christopher A; Ward, Robert M; Yost, Garold S

    2013-02-01

    Asthma is one of the most prevalent diseases in the world, for which the mainstay treatment has been inhaled glucocorticoids (GCs). Despite the widespread use of these drugs, approximately 30% of asthma sufferers exhibit some degree of steroid insensitivity or are refractory to inhaled GCs. One hypothesis to explain this phenomenon is interpatient variability in the clearance of these compounds. The objective of this research is to determine how metabolism of GCs by the CYP3A family of enzymes could affect their effectiveness in asthmatic patients. In this work, the metabolism of four frequently prescribed inhaled GCs, triamcinolone acetonide, flunisolide, budesonide, and fluticasone propionate, by the CYP3A family of enzymes was studied to identify differences in their rates of clearance and to identify their metabolites. Both interenzyme and interdrug variability in rates of metabolism and metabolic fate were observed. CYP3A4 was the most efficient metabolic catalyst for all the compounds, and CYP3A7 had the slowest rates. CYP3A5, which is particularly relevant to GC metabolism in the lungs, was also shown to efficiently metabolize triamcinolone acetonide, budesonide, and fluticasone propionate. In contrast, flunisolide was only metabolized via CYP3A4, with no significant turnover by CYP3A5 or CYP3A7. Common metabolites included 6β-hydroxylation and Δ(6)-dehydrogenation for triamcinolone acetonide, budesonide, and flunisolide. The structure of Δ(6)-flunisolide was unambiguously established by NMR analysis. Metabolism also occurred on the D-ring substituents, including the 21-carboxy metabolites for triamcinolone acetonide and flunisolide. The novel metabolite 21-nortriamcinolone acetonide was also identified by liquid chromatography-mass spectrometry and NMR analysis. PMID:23143891

  8. 17 CFR 240.3a4-1 - Associated persons of an issuer deemed not to be brokers.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... reclassification of securities of the issuer, a merger or consolidation or a similar plan of acquisition involving... investment company (or registered separate account); an insurance company; a bank; a savings and loan association; a trust company or similar institution supervised by a state or federal banking authority; or...

  9. Minor furanocoumarins and coumarins in grapefruit peel oil as inhibitors of human cytochrome P450 3A4

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A novel cyclic acetal of 6',7'-dihydroxy-7-geranyloxycoumarin (marmin) and two novel cyclic acetals of 6',7'-dihydroxybergamottin were isolated from nonvolatile residues of grapefruit peel oil. Other known compounds, marmin, 7-geranyloxycoumarin, bergamottin, and 6',7'-dihydroxybergamottin were also...

  10. CYP2C subfamily, primarily CYP2C9, catalyses the enantioselective demethylation of the endocrine disruptor pesticide methoxychlor in human liver microsomes: use of inhibitory monoclonal antibodies in P450 identification.

    PubMed

    Hu, Y; Krausz, K; Gelboin, H V; Kupfer, D

    2004-02-01

    1. The endocrine disruptor pesticide methoxychlor undergoes O-demethylation by mammalian liver microsomes forming chiral mono-phenolic (1,1,1-trichloro-2-(4-hydroxyphenyl)-2-(4-methoxyphenyl)ethane, i.e. mono-OH-M) and achiral bis-phenolic oestrogenic metabolites. Human liver microsomes (HLM) generated primarily the S-mono-OH-M. 2. Inhibitory monoclonal antibodies (MAb) identified those P450s catalysing the enantioselective O-demethylation of methoxychlor. In HLM, O-demethylation was inhibited by MAb anti-2C9 (30-40%), diminishing the per cent of S-mono-OH-M from about 80 to 55-60%. MAb anti-CYP1A2, 2A6, 2B6, 2C8, 2C19, 2D6 and 3A4 did not affect the demethylation rate in HLM. Nevertheless, MAb anti-CYP1A2 decreased the formation of R-mono-OH-M from 21-23 to 10-17%, indicating that CYP1A2 exhibits a role in generating the R-enantiomer. 3. Among cDNA-expressed human P450s (supersomes), CYP2C19 was the most active in demethylation, but in HLM, CYP2C19 appeared inactive (no inhibition by MAb anti-CYP2C19). There was a substantial difference in the per cent inhibition of demethylation by MAb anti-CYP2C9 and anti-rat CYP2C (MAb inhibiting all human CYP2C forms) and in altering the enantioselectivity, suggesting that demethylation by combined CYP2C8, 2C18 and 2C19 was significant (20-30%). 4. Polymorphism of methoxychlor demethylation was examined with supersomes and HLM-expressing CYP2C9 allelic variants. CYP2C9*1 and 2C9*2 were highly active; however, CYP2C9*3 appeared inactive.

  11. Update on allele nomenclature for human cytochromes P450 and the Human Cytochrome P450 Allele (CYP-allele) Nomenclature Database.

    PubMed

    Sim, Sarah C; Ingelman-Sundberg, Magnus

    2013-01-01

    Interindividual variability in xenobiotic metabolism and drug response is extensive and genetic factors play an important role in this variation. A majority of clinically used drugs are substrates for the cytochrome P450 (CYP) enzyme system and interindividual variability in expression and function of these enzymes is a major factor for explaining individual susceptibility for adverse drug reactions and drug response. Because of the existence of many polymorphic CYP genes, for many of which the number of allelic variants is continually increasing, a universal and official nomenclature system is important. Since 1999, all functionally relevant polymorphic CYP alleles are named and published on the Human Cytochrome P450 Allele (CYP-allele) Nomenclature Web site (http://www.cypalleles.ki.se). Currently, the database covers nomenclature of more than 660 alleles in a total of 30 genes that includes 29 CYPs as well as the cytochrome P450 oxidoreductase (POR) gene. On the CYP-allele Web site, each gene has its own Webpage, which lists the alleles with their nucleotide changes, their functional consequences, and links to publications identifying or characterizing the alleles. CYP2D6, CYP2C9, CYP2C19, and CYP3A4 are the most important CYPs in terms of drug metabolism, which is also reflected in their corresponding highest number of Webpage hits at the CYP-allele Web site.The main advantage of the CYP-allele database is that it offers a rapid online publication of CYP-alleles and their effects and provides an overview of peer-reviewed data to the scientific community. Here, we provide an update of the CYP-allele database and the associated nomenclature.

  12. Metabolic activation of clopidogrel: in vitro data provide conflicting evidence for the contributions of CYP2C19 and PON1.

    PubMed

    Polasek, Thomas M; Doogue, Matthew P; Miners, John O

    2011-12-01

    The recent report that clopidogrel efficacy may be more dependent on paraoxonase-1 (PON1) than on cytochrome P450 2C19 (CYP2C19) activity raises questions about the roles of these and other enzymes in clopidogrel activation. To provide insight into the emerging PON1 versus CYP2C19 debate, this commentary summarizes the clinical evidence on the pharmacokinetic determinants of clopidogrel efficacy. We then review the in vitro studies investigating the enzymes involved in clopidogrel activation, and comment on their strengths and limitations. There is agreement amongst in vitro studies regarding the involvement of CYP1A2 and CYP2B6 in the metabolism of clopidogrel to 2-oxo-clopidogrel. However, the evidence for other CYP enzymes in the first activation step (e.g. CYP2C19 and CYP3A4) is inconsistent and dependent on the in vitro test system and laboratory. All major drug metabolizing CYP enzymes are capable of converting 2-oxo-clopidogrel to sulfenic acid intermediates that subsequently form the active thiol metabolite. However, the extent of CYP involvement in this second step has been challenged, and new evidence suggests that CYP-independent hydrolytic cleavage of the thioester bond may be more important than oxidative metabolism.

  13. CYP450 phenotyping and metabolite identification of quinine by accurate mass UPLC-MS analysis: a possible metabolic link to blackwater fever

    PubMed Central

    2013-01-01

    Background The naturally occurring alkaloid drug, quinine is commonly used for the treatment of severe malaria. Despite centuries of use, its metabolism is still not fully understood, and may play a role in the haemolytic disorders associated with the drug. Methods Incubations of quinine with CYPs 1A2, 2C9, 2C19, 2D6, and 3A4 were conducted, and the metabolites were characterized by accurate mass UPLC-MSE analysis. Reactive oxygen species generation was also measured in human erythrocytes incubated in the presence of quinine with and without microsomes. Results The metabolites 3-hydroxyquinine, 2’-oxoquininone, and O-desmethylquinine were observed after incubation with CYPs 3A4 (3-hydroxyquinine and 2’-oxoquininone) and 2D6 (O-desmethylquinine). In addition, multiple hydroxylations were observed both on the quinoline core and the quinuclidine ring system. Of the five primary abundance CYPs tested, 3A4, 2D6, 2C9, and 2C19 all demonstrated activity toward quinine, while 1A2 did not. Further, quinine produced robust dose-dependent oxidative stress in human erythrocytes in the presence of microsomes. Conclusions Taken in context, these data suggest a CYP-mediated link between quinine metabolism and the poorly understood haemolytic condition known as blackwater fever, often associated with quinine ingestion. PMID:23800033

  14. Inhibition of Major Drug Metabolizing CYPs by Common Herbal Medicines used by HIV/AIDS Patients in Africa– Implications for Herb-Drug Interactions

    PubMed Central

    Awortwe, Charles; Bouic, Patrick J.; Masimirembwa, Collen M.; Rosenkranz, Bernd

    2015-01-01

    The purpose of this study was to evaluate the potential risk of common herbal medicines used by HIV-infected patients in Africa for herb-drug interactions (HDI). High throughput screening assays consisting of recombinant Cytochrome P450 enzymes (CYPs) and fluorescent probes, and parallel artificial membrane permeability assays (PAMPA) were used. The potential of herbal medicines to cause HDI was ranked according to FDA guidelines for reversible inhibition and categorization of time dependent inhibition was based on the normalized ratio. CYPs 1A2 and 3A4 were most inhibited by the herbal extracts. H. hemerocallidea (IC50 = 0.63 μg/mL and 58 μg/mL) and E. purpurea (IC50 = 20 μg/mL and 12 μg/mL) were the potent inhibitors of CYPs 1A2 and 3A4 respectively. L. frutescens and H. hemerocallidea showed clear time dependent inhibition on CYP3A4. Furthermore, the inhibitory effect of both H. hemerocallidea and L. frutescens before and after PAMPA were identical. The results indicate potential HDI of H. hemerocallidea, L. frutescens and E. purpurea with substrates of the affected enzymes if maximum in vivo concentration is achieved. PMID:24475926

  15. The enhanced atorvastatin hepatotoxicity in diabetic rats was partly attributed to the upregulated hepatic Cyp3a and SLCO1B1.

    PubMed

    Shu, Nan; Hu, Mengyue; Ling, Zhaoli; Liu, Peihua; Wang, Fan; Xu, Ping; Zhong, Zeyu; Sun, Binbin; Zhang, Mian; Li, Feng; Xie, Qiushi; Liu, Xiaodong; Liu, Li

    2016-01-01

    Liver injury is a common adverse effect of atorvastatin. This study aimed to investigate atorvastatin-induced hepatotoxicity in diabetic rats induced by high-fat diet combined with streptozotocin. The results showed that 40 mg/kg atorvastatin was lethal to diabetic rats, whose mean survival time was 6.2 days. Severe liver injury also occurred in diabetic rats treated with 10 mg/kg and 20 mg/kg atorvastatin. The in vitro results indicated that atorvastatin cytotoxicity in hepatocytes of diabetic rats was more severe than normal and high-fat diet feeding rats. Expressions and activities of hepatic Cyp3a and SLCO1B1 were increased in diabetic rats, which were highly correlated with hepatotoxicity. Antioxidants (glutathione and N-Acetylcysteine), Cyp3a inhibitor ketoconazole and SLCO1B1 inhibitor gemfibrozil suppressed cytotoxicity and ROS formation in primary hepatocytes of diabetic rats. In HepG2 cells, up-regulations of CYP3A4 and SLCO1B1 potentiated hepatotoxicity and ROS generation, whereas knockdowns of CYP3A4 and SLCO1B1 as well as CYP3A4/SLCO1B1 inhibitions showed the opposite effects. Phenobarbital pretreatment was used to induce hepatic Cyp3a and SLCO1B1 in rats. Phenobarbital aggravated atorvastatin-induced hepatotoxicity, while decreased plasma exposure of atorvastatin. All these findings demonstrated that the upregulations of hepatic Cyp3a and SLCO1B1 in diabetic rats potentiated atorvastatin-induced hepatotoxicity via increasing ROS formation.

  16. The enhanced atorvastatin hepatotoxicity in diabetic rats was partly attributed to the upregulated hepatic Cyp3a and SLCO1B1.

    PubMed

    Shu, Nan; Hu, Mengyue; Ling, Zhaoli; Liu, Peihua; Wang, Fan; Xu, Ping; Zhong, Zeyu; Sun, Binbin; Zhang, Mian; Li, Feng; Xie, Qiushi; Liu, Xiaodong; Liu, Li

    2016-01-01

    Liver injury is a common adverse effect of atorvastatin. This study aimed to investigate atorvastatin-induced hepatotoxicity in diabetic rats induced by high-fat diet combined with streptozotocin. The results showed that 40 mg/kg atorvastatin was lethal to diabetic rats, whose mean survival time was 6.2 days. Severe liver injury also occurred in diabetic rats treated with 10 mg/kg and 20 mg/kg atorvastatin. The in vitro results indicated that atorvastatin cytotoxicity in hepatocytes of diabetic rats was more severe than normal and high-fat diet feeding rats. Expressions and activities of hepatic Cyp3a and SLCO1B1 were increased in diabetic rats, which were highly correlated with hepatotoxicity. Antioxidants (glutathione and N-Acetylcysteine), Cyp3a inhibitor ketoconazole and SLCO1B1 inhibitor gemfibrozil suppressed cytotoxicity and ROS formation in primary hepatocytes of diabetic rats. In HepG2 cells, up-regulations of CYP3A4 and SLCO1B1 potentiated hepatotoxicity and ROS generation, whereas knockdowns of CYP3A4 and SLCO1B1 as well as CYP3A4/SLCO1B1 inhibitions showed the opposite effects. Phenobarbital pretreatment was used to induce hepatic Cyp3a and SLCO1B1 in rats. Phenobarbital aggravated atorvastatin-induced hepatotoxicity, while decreased plasma exposure of atorvastatin. All these findings demonstrated that the upregulations of hepatic Cyp3a and SLCO1B1 in diabetic rats potentiated atorvastatin-induced hepatotoxicity via increasing ROS formation. PMID:27624558

  17. The enhanced atorvastatin hepatotoxicity in diabetic rats was partly attributed to the upregulated hepatic Cyp3a and SLCO1B1

    PubMed Central

    Shu, Nan; Hu, Mengyue; Ling, Zhaoli; Liu, Peihua; Wang, Fan; Xu, Ping; Zhong, Zeyu; Sun, Binbin; Zhang, Mian; Li, Feng; Xie, Qiushi; Liu, Xiaodong; Liu, Li

    2016-01-01

    Liver injury is a common adverse effect of atorvastatin. This study aimed to investigate atorvastatin-induced hepatotoxicity in diabetic rats induced by high-fat diet combined with streptozotocin. The results showed that 40 mg/kg atorvastatin was lethal to diabetic rats, whose mean survival time was 6.2 days. Severe liver injury also occurred in diabetic rats treated with 10 mg/kg and 20 mg/kg atorvastatin. The in vitro results indicated that atorvastatin cytotoxicity in hepatocytes of diabetic rats was more severe than normal and high-fat diet feeding rats. Expressions and activities of hepatic Cyp3a and SLCO1B1 were increased in diabetic rats, which were highly correlated with hepatotoxicity. Antioxidants (glutathione and N-Acetylcysteine), Cyp3a inhibitor ketoconazole and SLCO1B1 inhibitor gemfibrozil suppressed cytotoxicity and ROS formation in primary hepatocytes of diabetic rats. In HepG2 cells, up-regulations of CYP3A4 and SLCO1B1 potentiated hepatotoxicity and ROS generation, whereas knockdowns of CYP3A4 and SLCO1B1 as well as CYP3A4/SLCO1B1 inhibitions showed the opposite effects. Phenobarbital pretreatment was used to induce hepatic Cyp3a and SLCO1B1 in rats. Phenobarbital aggravated atorvastatin-induced hepatotoxicity, while decreased plasma exposure of atorvastatin. All these findings demonstrated that the upregulations of hepatic Cyp3a and SLCO1B1 in diabetic rats potentiated atorvastatin-induced hepatotoxicity via increasing ROS formation. PMID:27624558

  18. Therapeutic drug monitoring of voriconazole: a case report of multiple drug interactions in a patient with an increased CYP2C19 activity

    PubMed Central

    2014-01-01

    Background Voriconazole is metabolized by cytochrome P450 (CYP) 2C19 and CYP 3A4. Drug-drug interactions and genetic polymorphisms modulate their activities. Case presentation A 35-year old African female patient with resistant HIV and a cerebral mass of unknown origin was treated with voriconazole for a suspicion of disseminated Aspergillosis infection. Voriconazole trough concentrations (C0) were within target range while the patient was under esomeprazole, a CYP2C19 inhibitor. Phenotyping showed decreased CYP2C19 activity, whereas genotyping showed a variant allele associated with increased enzyme activity. The patient was switched to ranitidine because of the introduction of atazanavir. CYP3A4 inhibition by atazanavir combined with uninhibited CYP2C19 activity resulted in subtherapeutic voriconazole C0. The reintroduction of esomeprazole allowed restoring voriconazole C0 back to target range. Conclusion The integration of drug-drug interactions and pharmacogenetics data is crucial to interpret drug concentrations correctly, thus preventing suboptimal exposure to voriconazole. PMID:25120580

  19. Bioactivation of 4-ipomeanol by CYP4B1: adduct characterization and evidence for an enedial intermediate.

    PubMed

    Baer, Brian R; Rettie, Allan E; Henne, Kirk R

    2005-05-01

    4-Ipomeanol (IPO) is a pneumotoxin that is bioactivated to a reactive intermediate that binds to DNA and other cellular macromolecules. Despite over 30 years of research in this area, detailed structural information on the nature of the IPO reactive intermediate is still lacking. In the present study, we reacted IPO with rabbit CYP4B1 in the presence of exogenous nucleophiles and analyzed the products by liquid chromatography/electrospray ionization-mass spectrometry. Coincubation of IPO and rabbit CYP4B1 with glutathione gave rise to multiple products due likely to the presence of both sulfur and nitrogen nucleophiles in the same trapping molecule. Reaction mixtures containing equimolar N-acetyl cysteine (NAC) and N-acetyl lysine (NAL) provided a major NADPH- and CYP4B1-dependent product. A combination of high-resolution mass spectrometry and two-dimensional NMR analysis following large-scale isolation of the biologically derived material provided evidence for an N-substituted cysteinyl pyrrole derivative of IPO, analogous to that characterized previously in model chemical studies conducted with cis-2-butene-1,4-dial. Purified native rabbit lung CYP4B1 and purified recombinant rabbit CYP4B1 produced the trapped NAC/NAL-IPO pyrrole adduct at rates of 600-700 nmol/nmol P450/30 min. A panel of 14 commercially available recombinant human CYPs was also studied, and substantial rates of IPO bioactivation (>100 nmol/nmol/30 min) were observed with CYP1A2, CYP2C19, CYP2D6, and CYP3A4. These studies provide evidence for the formation of an enedial reactive intermediate during CYP-mediated IPO bioactivation, identify multiple human liver P450s capable of IPO bioactivation, and demonstrate that the same reactive intermediate is formed by both rabbit CYP4B1 and human P450s. PMID:15892579

  20. Downregulation of Mouse Hepatic CYP3A Protein by 3-Methylcholanthrene Does Not Require Cytochrome P450-Dependent Metabolism

    PubMed Central

    Lee, Chunja; Ding, Xinxin

    2013-01-01

    The aryl hydrocarbon receptor (AHR)–dependent induction of cytochromes P450 (P450) such as CYP1A1 by 3-methylcholanthrene (MC) and related polycyclic aromatic hydrocarbons is well characterized. We reported previously that MC treatment triggers a pronounced downregulation, particularly at the protein level, of mouse hepatic Cyp3a11, a counterpart of the key human drug-metabolizing enzyme CYP3A4. To determine whether this effect of MC requires hepatic microsomal P450 activity, we studied liver Cpr-null (LCN) mice with hepatocyte-specific conditional deletion of the NADPH-cytochrome P450 oxidoreductase gene. In vehicle-treated animals, basal levels of CYP3A11 mRNA and CYP3A protein immunoreactivity were elevated by approximately 9-fold in LCN mice compared with wild-type (WT) mice, whereas CYP3A catalytic activity was profoundly compromised in LCN mice. MC treatment caused suppression of CYP3A11 mRNA, CYP3A protein immunoreactivity, and CYP3A catalytic activity in WT mice, and the MC effects at the mRNA and protein levels were maintained in LCN mice. Flavin-containing monooxygenase-3 (Fmo3) induction by MC was suggested previously to occur via an AHR-dependent mechanism requiring conversion of the parent compound to DNA-damaging reactive metabolites; however, hepatic FMO3 mRNA levels were dramatically increased by MC in both WT and LCN mice. MC did not function as a mechanism-based inactivator of CYP3A enzymes in hepatic microsomes prepared from untreated WT mice, under conditions in which 1-aminobenzotriazole caused marked NADPH-dependent loss of total P450 content and CYP3A catalytic activity. These results indicate that MC downregulates mouse hepatic CYP3A protein via a pretranslational mechanism that does not require hepatic microsomal P450-dependent activity. PMID:23846873

  1. The Investigational Drug VT-1129 Is a Highly Potent Inhibitor of Cryptococcus Species CYP51 but Only Weakly Inhibits the Human Enzyme.

    PubMed

    Warrilow, Andrew G S; Parker, Josie E; Price, Claire L; Nes, W David; Garvey, Edward P; Hoekstra, William J; Schotzinger, Robert J; Kelly, Diane E; Kelly, Steven L

    2016-08-01

    Cryptococcosis is a life-threatening disease often associated with HIV infection. Three Cryptococcus species CYP51 enzymes were purified and catalyzed the 14α-demethylation of lanosterol, eburicol, and obtusifoliol. The investigational agent VT-1129 bound tightly to all three CYP51 proteins (dissociation constant [Kd] range, 14 to 25 nM) with affinities similar to those of fluconazole, voriconazole, itraconazole, clotrimazole, and ketoconazole (Kd range, 4 to 52 nM), whereas VT-1129 bound weakly to human CYP51 (Kd, 4.53 μM). VT-1129 was as effective as conventional triazole antifungal drugs at inhibiting cryptococcal CYP51 activity (50% inhibitory concentration [IC50] range, 0.14 to 0.20 μM), while it only weakly inhibited human CYP51 activity (IC50, ∼600 μM). Furthermore, VT-1129 weakly inhibited human CYP2C9, CYP2C19, and CYP3A4, suggesting a low drug-drug interaction potential. Finally, the cellular mode of action for VT-1129 was confirmed to be CYP51 inhibition, resulting in the depletion of ergosterol and ergosta-7-enol and the accumulation of eburicol, obtusifolione, and lanosterol/obtusifoliol in the cell membranes. PMID:27161631

  2. 3,4-methylenedioxymethamphetamine (MDMA) interacts with therapeutic drugs on CYP3A by inhibition of pregnane X receptor (PXR) activation and catalytic enzyme inhibition.

    PubMed

    Antolino-Lobo, Irene; Meulenbelt, Jan; Nijmeijer, Sandra M; Maas-Bakker, Roel F; Meijerman, Irma; van den Berg, Martin; van Duursen, Majorie B M

    2011-05-30

    Metabolism of MDMA (3,4-methylenedioxymethamphetamine, Ecstasy) by the major hepatic drug-metabolizing enzyme cytochrome P450 3A (CYP3A), plays an important role in MDMA-induced liver toxicity. In the present study, we investigated interactions between MDMA and several therapeutic and recreational drugs on CYP3A and its regulator pregnane X receptor (PXR), using a human PXR-mediated CYP3A4-reporter gene assay, rat primary hepatocytes and microsomes. MDMA significantly inhibited hPXR-mediated CYP3A4-reporter gene expression induced by the human PXR activator rifampicin (IC₅₀ 1.26 ± 0.36 mM) or the therapeutic drugs paroxetine, fluoxetine, clozapine, diazepam and risperidone. All these drugs concentration-dependently inhibited CYP3A activity in rat liver microsomes, but in combination with MDMA this inhibition became more efficient for clozapine and risperidone. In rat primary hepatocytes that were pretreated with or without the rodent PXR activator pregnenolone 16alpha-carbonitrile (PCN), MDMA inhibited CYP3A catalytic activity with IC₅₀ values of 0.06 ± 0.12 and 0.09 ± 0.13 mM MDMA, respectively. This decrease appeared to be due to decreased activation of PXR and subsequent decreased CYP3A gene expression, and catalytic inhibition of CYP3A activity. These data suggest that in situations of repeated MDMA use in combination with other (therapeutic) drugs, adverse drug-drug interactions through interactions with PXR and/or CYP3A cannot be excluded.

  3. Pharmacogenomics of CYP3A: considerations for HIV treatment

    PubMed Central

    Lakhman, Sukhwinder S; Ma, Qing

    2009-01-01

    The understanding of the cytochrome P450 3A SNP in antiretroviral therapy is important, because it is highly inducible, extremely polymorphic and metabolizes many of the drugs that are key components of highly active antiretroviral therapy regimens. This enzyme is prolific and promiscuous towards drug and xenobiotic substrate selection and it is also unpredictable among individuals, having a 5- to 20-fold variability in its ability to contribute to drug clearance. The importance of human CYP3A pharmacogenetics is also gaining attention in other established areas of pharmacotherapy as it may contribute to the goal of predicting efficacy and/or toxicity, specifically with the discovery of null allele CYP3A4*20. This review summarizes the current understanding, implications of genetic variation in the CYP3A enzymes, the central role of CYP3A in linking human genetics, the pharmacokinetics and resulting pharmacodynamic responses to certain antiretroviral drugs, and their eventual place in applied clinical pharmacotherapy. PMID:19663676

  4. Assessment of competitive and mechanism-based inhibition by clarithromycin: use of domperidone as a CYP3A probe-drug substrate and various enzymatic sources including a new cell-based assay with freshly isolated human hepatocytes.

    PubMed

    Michaud, Veronique; Turgeon, Jacques

    2010-04-01

    Clarithromycin is involved in a large number of clinically relevant drug-drug interactions. Discrepancies are observed between the magnitude of drug interactions predicted from in vitro competitive inhibition studies and changes observed clinically in the plasma levels of affected CYP3A substrates. The formation of metabolic-intermediate complexes has been proposed to explain these differences. The objectives of our study were: 1) to determine the competitive inhibition potency of clarithromycin on the metabolism of domperidone as a CYP3A probe drug using human recombinant CYP3A4 and CYP3A5 isoenzymes, human liver microsomes and cultured human hepatocytes; 2) to establish the modulatory role of cytochrome b5 on the competitive inhibition potency of clarithromycin; 3) to demonstrate the clarithromycin-induced formation of CYP450 metabolic-intermediate complexes in human liver microsomes; and 4) to determine the extent of CYP3A inhibition due to metabolic-intermediate complex formation using human liver microsomes and cultured human hepatocytes. At high concentrations (100 µM), clarithromycin had weak competitive inhibition potency towards CYP3A4 and CYP3A5. Inhibition potency was further decreased by the addition of cytochrome b5 (9-19%). Clarithromycin-induced metabolic-intermediate complexes were revealed by spectrophotometry analysis using human liver microsomes while time- and concentration-dependent mechanism-based inhibitions were quantified using isolated hepatocytes. These results indicate that mechanism-based but not competitive inhibition of CYP3As is the major underlying mechanism of drug-drug interactions observed clinically with clarithromycin. Drug interactions between clarithromycin and several CYP3A substrates are predicted to be insidious; the risk of severe adverse events should increase over time and persist for a few days after cessation of the drug.

  5. Effect of tamoxifen on the enzymatic activity of human cytochrome CYP2B6.

    PubMed

    Sridar, Chitra; Kent, Ute M; Notley, Lisa M; Gillam, Elizabeth M J; Hollenberg, Paul F

    2002-06-01

    Tamoxifen is primarily used in the treatment of breast cancer. It has been approved as a chemopreventive agent for individuals at high risk for this disease. Tamoxifen is metabolized to a number of different products by cytochrome P450 enzymes. The effect of tamoxifen on the enzymatic activity of bacterially expressed human cytochrome CYP2B6 in a reconstituted system has been investigated. The 7-ethoxy-4-(trifluoromethyl)coumarin O-deethylation activity of purified CYP2B6 was inactivated by tamoxifen in a time- and concentration-dependent manner. Enzymatic activity was lost only in samples that were incubated with both tamoxifen and NADPH. The inactivation was characterized by a K(I) of 0.9 microM, a k(inact) of 0.02 min(-1), and a t(1/2) of 34 min. The loss in the 7-ethoxy-4-(trifluoromethyl)coumarin O-deethylation activity did not result in a similar percentage loss in the reduced carbon monoxide spectrum, suggesting that the heme moiety was not the major site of modification. The activity of CYP2B6 was not recovered after removal of free tamoxifen using spin column gel filtration. The loss in activity seemed to be due to a modification of the CYP2B6 and not reductase because adding fresh reductase back to the inactivated samples did not restore enzymatic activity. A reconstituted system containing purified CYP2B6, NADPH-reductase, and NADPH-generating system was found to catalyze tamoxifen metabolism to 4-OH-tamoxifen, 4'-OH-tamoxifen, and N-desmethyl-tamoxifen as analyzed by high-performance liquid chromatography analysis. Preliminary studies showed that tamoxifen had no effect on the activities of CYP1B1 and CYP3A4, whereas CYP2D6 and CYP2C9 exhibited a 25% loss in enzymatic activity. PMID:12023523

  6. Rational development of 4-aminopyridyl-based inhibitors targeting Trypanosoma cruzi CYP51 as anti-Chagas agents

    PubMed Central

    Choi, Jun Yong; Calvet, Claudia M.; Gunatilleke, Shamila S.; Ruiz, Claudia; Cameron, Michael D.; McKerrow, James H.; Podust, Larissa M.; Roush, William R.

    2013-01-01

    A new series of 4-aminopyridyl-based lead inhibitors targeting Trypanosoma cruzi CYP51 (TcCYP51) has been developed using structure-based drug design as well as structure-property relationship (SPR) analyses. The screening hit starting point, LP10 (KD ≤ 42 nM; EC50 of 0.65 µM), has been optimized to give the potential leads 14t, 27i, 27q, 27r, and 27t, that have low nanomolar binding affinity to TcCYP51 and significant activity against T. cruzi amastigotes cultured in human myoblasts (EC50 = 14–18 nM for 27i and 27r). Many of the optimized compounds have improved microsome stability, and most are selective against human CYPs 1A2, 2D6 and 3A4 (<50% inhibition at 1 µM). A rationale for the improvement of microsome stability and selectivity of inhibitors against human metabolic CYP enzymes is presented. In addition, the binding mode of 14t with the T. brucei CYP51 (TbCYP51) ortholog has been characterized by x-ray structure analysis. PMID:24079662

  7. The correlation between CYP2D6 isoenzyme activity and haloperidol efficacy and safety profile in patients with alcohol addiction during the exacerbation of the addiction

    PubMed Central

    Sychev, Dmitry Alekseevich; Zastrozhin, Mikhail Sergeevich; Smirnov, Valery Valerieevich; Grishina, Elena Anatolievna; Savchenko, Ludmila Mikhailovna; Bryun, Evgeny Alekseevich

    2016-01-01

    Background Today, it is proved that isoenzymes CYP2D6 and CYP3A4 are involved in metabolism of haloperidol. In our previous investigation, we found a medium correlation between the efficacy and safety of haloperidol and the activity of CYP3A4 in patients with alcohol abuse. Objective The aim of this study was to evaluate the correlation between the activity of CYP2D6 and the efficacy and safety of haloperidol in patients with diagnosed alcohol abuse. Methods The study involved 70 men (average age: 40.83±9.92 years) with alcohol addiction. A series of psychometric scales were used in the research. The activity of CYP2D6 was evaluated by high-performance liquid chromatography with mass spectrometry using the ratio of 6-hydroxy-1,2,3,4-tetrahydro-beta-carboline to pinoline. Genotyping of CYP2D6 (1846G>A) was performed using real-time polymerase chain reaction. Results According to results of correlation analysis, statistically significant values of Spearman correlation coefficient (rs) between the activity of CYP2D6 and the difference of points in psychometric scale were obtained in patients receiving haloperidol in injection form (Sheehan Clinical Anxiety Rating Scale =−0.721 [P<0.001] and Udvald for Kliniske Undersogelser Side Effect Rating Scale =0.692 [P<0.001]) and in those receiving haloperidol in tablet form (Covi Anxiety Scale =−0.851 [P<0.001] and Udvald for Kliniske Undersogelser Side Effect Rating Scale =0.797 [P<0.001]). Conclusion This study demonstrated the correlations between the activity of CYP2D6 isozyme and the efficacy and safety of haloperidol in patients with alcohol addiction. PMID:27695358

  8. The correlation between CYP2D6 isoenzyme activity and haloperidol efficacy and safety profile in patients with alcohol addiction during the exacerbation of the addiction

    PubMed Central

    Sychev, Dmitry Alekseevich; Zastrozhin, Mikhail Sergeevich; Smirnov, Valery Valerieevich; Grishina, Elena Anatolievna; Savchenko, Ludmila Mikhailovna; Bryun, Evgeny Alekseevich

    2016-01-01

    Background Today, it is proved that isoenzymes CYP2D6 and CYP3A4 are involved in metabolism of haloperidol. In our previous investigation, we found a medium correlation between the efficacy and safety of haloperidol and the activity of CYP3A4 in patients with alcohol abuse. Objective The aim of this study was to evaluate the correlation between the activity of CYP2D6 and the efficacy and safety of haloperidol in patients with diagnosed alcohol abuse. Methods The study involved 70 men (average age: 40.83±9.92 years) with alcohol addiction. A series of psychometric scales were used in the research. The activity of CYP2D6 was evaluated by high-performance liquid chromatography with mass spectrometry using the ratio of 6-hydroxy-1,2,3,4-tetrahydro-beta-carboline to pinoline. Genotyping of CYP2D6 (1846G>A) was performed using real-time polymerase chain reaction. Results According to results of correlation analysis, statistically significant values of Spearman correlation coefficient (rs) between the activity of CYP2D6 and the difference of points in psychometric scale were obtained in patients receiving haloperidol in injection form (Sheehan Clinical Anxiety Rating Scale =−0.721 [P<0.001] and Udvald for Kliniske Undersogelser Side Effect Rating Scale =0.692 [P<0.001]) and in those receiving haloperidol in tablet form (Covi Anxiety Scale =−0.851 [P<0.001] and Udvald for Kliniske Undersogelser Side Effect Rating Scale =0.797 [P<0.001]). Conclusion This study demonstrated the correlations between the activity of CYP2D6 isozyme and the efficacy and safety of haloperidol in patients with alcohol addiction.

  9. Evidence of CYP3A Allosterism In Vivo: Analysis of Fluconazole and Midazolam Interaction

    PubMed Central

    Yang, Jing; Atkins, William M.; Isoherranen, Nina; Paine, Mary F.; Thummel, Kenneth E.

    2013-01-01

    The allosteric effect of fluconazole (effector) on the formation of 1’-hydroxymidazolam (1’-OH-MDZ) and 4-hydroxymidazolam (4-OH-MDZ) from the CYP3A4/5 substrate, midazolam (MDZ), was examined in healthy volunteers. Following pre-treatment of fluconazole, AUC4-OH/AUCMDZ increased 35–62%, while AUC1’-OH/AUCMDZ decreased 5–37%; AUC1’-OH/AUC4-OH ratio decreased 46–58% by fluconazole and had no association with CYP3A5 genotype. 1’-OH-MDZ formation in vitro was more susceptible than 4-OH-MDZ formation to inhibition by fluconazole. Fluconazole decreased the intrinsic formation clearance ratio of 1’-OH-MDZ/4-OH-MDZ to an extent that was quantitatively comparable to in vivo observations. The elimination clearance of midazolam metabolites appeared unaffected by fluconazole. This study demonstrated that fluconazole alters midazolam product formation both in vivo and in vitro in a manner consistent with an allosteric interaction. The 1'-OH-MDZ/4-OH-MDZ ratio may serve as a biomarker of such interactions between midazolam, CYP3A4/5 and other putative effectors. PMID:22048224

  10. Modulation of CYP3a expression and activity in mice models of type 1 and type 2 diabetes

    PubMed Central

    Patoine, Dany; Petit, Michaël; Pilote, Sylvie; Picard, Frédéric; Drolet, Benoit; Simard, Chantale

    2014-01-01

    CYP3A4, the most abundant cytochrome P450 enzyme in the human liver and small intestine, is responsible for the metabolism of about 50% of all marketed drugs. Numerous pathophysiological factors, such as diabetes and obesity, were shown to affect CYP3A activity. Evidences suggest that drug disposition is altered in type 1 (T1D) and type 2 diabetes (T2D). The objective was to evaluate the effect of T1D and T2D on hepatic and intestinal CYP3a drug-metabolizing activity/expression in mice. Hepatic and intestinal microsomes were prepared from streptozotocin-induced T1D, db/db T2D and control mice. Domperidone was selected as a probe substrate for CYP3a and formation of five of its metabolites was evaluated using high performance liquid chromatography. Hepatic CYP3a protein and mRNA expression were assessed by Western blot and reverse-transcription quantitative polymerase chain reaction respectively. Hepatic microsomal CYP3a activity was significantly increased in both T1D and T2D groups versus control group. Intestinal CYP3a activity was also significantly increased in both T1D and T2D groups. Moreover, significant increases of both hepatic CYP3a mRNAs and protein expression were observed in both T1D and T2D groups versus control group. Additional experiments with testosterone further validated the increased activity of CYP3a under the effect of both T1D and T2D. Although differences exist in the pathophysiological insults associated with T1D and T2D, our results suggest that these two distinct diseases may have the same modulating effect on the regulation of CYP3a, ultimately leading to variability in drug response, ranging from lack of effect to life-threatening toxicity. PMID:25505621

  11. The impact of CYP2C8 polymorphism and grapefruit juice on the pharmacokinetics of repaglinide

    PubMed Central

    Bidstrup, Tanja Busk; Damkier, Per; Olsen, Anette Kristensen; Ekblom, Marianne; Karlsson, Anders; Brøsen, Kim

    2006-01-01

    Aims The primary aim of the study was to investigate the possible effect of the CYP2C8 3 allele and of grapefruit juice on the pharmacokinetics of repaglinide. Furthermore, the impact of a single dose of grapefruit juice on the pharmacokinetics of repaglinide in relation to dose. Methods Thirty-six healthy male subjects, genotyped for CYP2C8 3 (11 genotyped as CYP2C8 1/ 3, one as CYP2C8 3/ 3 and 24 as CYP2C8 1/ 1), participated in a randomized, cross-over trial. In the two phases, the subjects drank 300 mL water or 300 mL grapefruit juice, in randomized order, 2 h before administration of a single dose of either 0.25 mg or 2 mg repaglinide. Results Neither the mean AUC0−∞ (geometric mean ratio: 1.01; 95% CI: 0.93–1.1, P = 0.88) nor the mean Cmax (geometric mean ratio: 1.05; 95% CI: 0.94–1.2, P = 0.35) of repaglinide were statistically significantly different in the group carrying the CYP2C8 3 mutant allele compared with wild-types. Grapefruit juice caused a 19% decrease in the geometric mean ratio of the 3-hydroxyquinidine to quinidine ratio (difference: 0.81; 95% CI: 0.75–0.87, P < 0.0001), which was used as an index of CYP3A4 activity, and an increase in the mean AUC0−∞ of repaglinide (geometric mean ratio: 1.13; 95% CI: 1.04–1.2, P = 0.0048), but had no statistically significant effect on the t1/2. There was no statistically significant difference in blood glucose concentration in subjects who had or had not ingested grapefruit juice. The effect was more pronounced at the low dose of repaglinide (0.25 mg) than at the therapeutic dose of 2 mg. Conclusions The pharmacokinetics of repaglinide in subjects carrying the CYP2C8*3 mutant allele did not differ significantly from those in the wild-types. Grapefruit juice increased the bioavailability of repaglinide, suggesting significant intestinal elimination of the drug which was assumed to be primarily mediated by CYP3A4 in the gut. PMID:16390351

  12. Effects of the differentiated keratinocyte phenotype on expression levels of CYP1-4 family genes in human skin cells

    SciTech Connect

    Du Liping; Neis, Mark M.; Ladd, Patricia A.; Yost, Garold S.; Keeney, Diane S. . E-mail: diane.keeney@vanderbilt.edu

    2006-06-01

    Epoxyeicosatrienoic acids produced by mouse CYP2B19 have been implicated in mechanisms regulating epidermal cornification (Ladd, P.A., Du, L., Capdevila, J.H., Mernaugh, R., Keeney, D.S., 2003. Epoxyeicosatrienoic acids activate transglutaminases in situ and induce cornification of epidermal keratinocytes. J. Biol. Chem. 278, 35184-35192). In this study, we aimed to identify CYPs that are up-regulated during keratinocyte differentiation and potentially responsible for epoxyeicosatrienoic acid formation in human skin. The cellular differentiation state of human epidermal cell cultures was manipulated to resemble the basal, spinous, and granular cell phenotypes in vivo. Changes in CYP mRNA levels were measured as a function of differentiation state for a panel of 15 CYPs that included known and putative arachidonate monooxygenases. Quantitative real-time PCR analyses showed that all of the CYPs were expressed in differentiating epidermal cell cultures and in human epidermis, with the exception of CYP2B6, which was poorly expressed in vitro. Six CYPs were strongly up-regulated at Day 6 and Day 8 of in vitro differentiation (CYP4B1, 2W1, 2C18, 3A4, 2C19, 2C9); the increase in mRNA levels ranged from 27- to 356-fold. Only CYP2U1 mRNA levels decreased (6-fold change) during cellular differentiation. Six CYPs showed little variation (<2-fold change) in mRNA levels during in vitro differentiation (CYP2S1, 2J2, 1B1, 1A1, 2E1, 2D6). No single CYP was identifiable as being a functional counterpart to CYP2B19 in mouse skin since none qualified as being mainly responsible for epidermal epoxyeicosatrienoic acid formation. Rather, the data suggest that epoxyeicosatrienoic acids in human skin are formed by several CYPs expressed in different cell layers of the epidermis. This would predict that CYP-derived eicosanoids have different functions in different epidermal cell layers.

  13. A high throughput screening assay to screen for CYP2E1 metabolism and inhibition using a fluorogenic vivid p450 substrate.

    PubMed

    Marks, Bryan D; Smith, Ronald W; Braun, Heidi A; Goossens, Tony A; Christenson, Marie; Ozers, Mary S; Lebakken, Connie S; Trubetskoy, Olga V

    2002-11-01

    Large-scale screening of multiple compound libraries and combinatorial libraries for pharmacological activity is one of the novel approaches of the modern drug discovery process. The application of isozyme-specific high-throughput screening (HTS) assays for characterizing the interactions of potential drug candidates with major human drug-metabolizing cytochrome p450 enzymes (p450s) is newly becoming an essential part of this process. Fluorescence-based HTS assays have been successfully employed for in vitro assessment of drug-drug interactions and enzyme inhibition with several p450 isoforms, including CYP3A4, CYP2D6, CYP2C9, and CYP2C19. Here we describe a fluorescence-based HTS assay for detecting drug metabolism and inhibition with human CYP2E1. CYP2E1 plays an important role in the metabolism of several drugs, many solvents, and toxins and therefore has been repeatedly linked to numerous pathologies, including cancer, liver and kidney toxicity, diabetes, and alcoholism. The assay is based on the ability of a drug to compete with the fluorogenic Vivid CYP2E1 Blue Substrate for CYP2E1 metabolism and thus enables rapid screening of lead molecules for their inhibitory potential. We have used this assay to screen a panel of drugs and compounds for their effects on CYP2E1 metabolism and inhibition. Our results demonstrate the assay's usefulness in identifying CYP2E1 substrates and inhibitors and in enabling in-depth characterization of their interactions with the CYP2E1 isozyme. We also present detailed characteristics of the assay, including its dynamic range and Z'-factor values, which indicate that this robust assay is well suited for kinetic and inhibition studies in HTS formats.

  14. Fipronil induces CYP isoforms and cytotoxicity in human hepatocytes.

    PubMed

    Das, Parikshit C; Cao, Yan; Cherrington, Nathan; Hodgson, Ernest; Rose, Randy L

    2006-12-15

    Recent studies have demonstrated the potential of pesticides to either inhibit or induce xenobiotic metabolizing enzymes in humans. Exposure of human hepatocytes to doses of fipronil (5-amino-1-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4-[(trifluoromethyl) sulfinyl]-1H-pyrazole-3-carbonitrile) ranging from 0.1 to 25 microM resulted in a dose dependent increase in CYP1A1 mRNA expression (3.5 to approximately 55-fold) as measured by the branched DNA assay. In a similar manner, CYP3A4 mRNA expression was also induced (10-30-fold), although at the higher doses induction returned to near control levels. CYP2B6 and 3A5 were also induced by fipronil, although at lower levels (2-3-fold). Confirmation of bDNA results were sought through western blotting and/or enzyme activity assays. Western blots using CYP3A4 antibody demonstrated a dose responsive increase from 0.5 to 1 microM followed by decreasing responses at higher concentrations. Similar increases and decreases were observed in CYP3A4-specific activity levels as measured using 6beta-hydroxytestosterone formation following incubation with testosterone. Likewise, activity levels for a CYP1A1-specific substrate, luciferin CEE, demonstrated that CYP1A1 enzyme activities were maximally induced by 1 microM fipronil followed by dramatically declining activity measurements at 10 and 25 microM. Cytotoxic effects of fipronil and fipronil sulfone were examined using the adenylate kinase and the trypan blue exclusion assays in HepG2 cells and human hepatocytes. The results indicate both that HepG2 cells and primary human hepatocytes are sensitive to the cytotoxic effects of fipronil. The maximum induction of adenylate kinase was ca. 3-fold greater than the respective controls in HepG2 and 6-10-fold in the case of primary hepatocytes. A significant time- and dose-dependent induction of adenylate kinase activity in HepG2 cells was noted from 0.1 to 12.5 microM fipronil followed by decreasing activities at 25 and 50 microM. For

  15. CYP3A Specifically Catalyzes 1β-Hydroxylation of Deoxycholic Acid: Characterization and Enzymatic Synthesis of a Potential Novel Urinary Biomarker for CYP3A Activity.

    PubMed

    Hayes, Martin A; Li, Xue-Qing; Grönberg, Gunnar; Diczfalusy, Ulf; Andersson, Tommy B

    2016-09-01

    The endogenous bile acid metabolite 1β-hydroxy-deoxycholic acid (1β-OH-DCA) excreted in human urine may be used as a sensitive CYP3A biomarker in drug development reflecting in vivo CYP3A activity. An efficient and stereospecific enzymatic synthesis of 1β-OH-DCA was developed using a Bacillus megaterium (BM3) cytochrome P450 (P450) mutant, and its structure was confirmed by nuclear magnetic resonance (NMR) spectroscopy. A [(2)H4]-labeled analog of 1β-OH-DCA was also prepared. The major hydroxylated metabolite of deoxycholic acid (DCA) in human liver microsomal incubations was identified as 1β-OH-DCA by comparison with the synthesized reference analyzed by UPLC-HRMS. Its formation was strongly inhibited by CYP3A inhibitor ketoconazole. Screening of 21 recombinant human cytochrome P450 (P450) enzymes showed that, with the exception of extrahepatic CYP46A1, the most abundant liver P450 subfamily CYP3A, including CYP3A4, 3A5, and 3A7, specifically catalyzed 1β-OH-DCA formation. This indicated that 1β-hydroxylation of DCA may be a useful marker reaction for CYP3A activity in vitro. The metabolic pathways of DCA and 1β-OH-DCA in human hepatocytes were predominantly via glycine and, to a lesser extent, via taurine and sulfate conjugation. The potential utility of 1β-hydroxylation of DCA as a urinary CYP3A biomarker was illustrated by comparing the ratio of 1β-OH-DCA:DCA in a pooled spot urine sample from six healthy control subjects to a sample from one patient treated with carbamazepine, a potent CYP3A inducer; 1β-OH-DCA:DCA was considerably higher in the patient versus controls (ratio 2.8 vs. 0.4). Our results highlight the potential of 1β-OH-DCA as a urinary biomarker in clinical CYP3A DDI studies. PMID:27402728

  16. Identification and characterization of reactive metabolites in myristicin-mediated mechanism-based inhibition of CYP1A2.

    PubMed

    Yang, Ai-Hong; He, Xin; Chen, Jun-Xiu; He, Li-Na; Jin, Chun-Huan; Wang, Li-Li; Zhang, Fang-Liang; An, Li-Jun

    2015-07-25

    Myristicin belongs to the methylenedioxyphenyl or allyl-benzene family of compounds, which are found widely in plants of the Umbelliferae family, such as parsley and carrot. Myristicin is also the major active component in the essential oils of mace and nutmeg. However, this compound can cause adverse reactions, particularly when taken inappropriately or in overdoses. One important source of toxicity of natural products arises from their metabolic biotransformations into reactive metabolites. Myristicin contains a methylenedioxyphenyl substructure, and this specific structural feature may allow compounds to cause a mechanism-based inhibition of cytochrome P450 enzymes and produce reactive metabolites. Therefore, the aim of this work was to identify whether the role of myristicin in CYP enzyme inhibition is mechanism-based inhibition and to gain further information regarding the structure of the resulting reactive metabolites. CYP cocktail assays showed that myristicin most significantly inhibits CYP1A2 among five CYP enzymes (CYP1A2, CYP2D6, CYP2E1, CYP3A4 and CYP2C19) from human liver microsomes. The 3.21-fold IC50 shift value of CYP1A2 indicates that myristicin may be a mechanism-based inhibitor of CYP1A2. Next, reduced glutathione was shown to block the inhibition of CYP1A2, indicating that myristicin utilized a mechanism-based inhibition. Phase I metabolism assays identified two metabolites, 5-allyl-1-methoxy-2,3-dihydroxybenzene (M1) and 1'-hydroxymyristicin or 2',3'-epoxy-myristicin (M2). Reduced glutathione capturing assays captured the glutathione-M1 adduct, and the reactive metabolites were identified using UPLC-MS(2) as a quinone and its tautomer. Thus, it was concluded that myristicin is a mechanism-based inhibitor of CYP1A2, and the reactive metabolites are quinone tautomers. Additionally, the cleavage process of the glutathione-M1 adduct was analyzed in further detail. This study provides additional information on the metabolic mechanism of myristicin

  17. Guanfu base A, an antiarrhythmic alkaloid of Aconitum coreanum, Is a CYP2D6 inhibitor of human, monkey, and dog isoforms.

    PubMed

    Sun, Jianguo; Peng, Ying; Wu, Hui; Zhang, Xueyuan; Zhong, Yunxi; Xiao, Yanan; Zhang, Fengyi; Qi, Huanhuan; Shang, Lili; Zhu, Jianping; Sun, Yue; Liu, Ke; Liu, Jinghan; A, Jiye; Ho, Rodney J Y; Wang, Guangji

    2015-05-01

    Guanfu base A (GFA) is a novel heterocyclic antiarrhythmic drug isolated from Aconitum coreanum (Lèvl.) rapaics and is currently in a phase IV clinical trial in China. However, no study has investigated the influence of GFA on cytochrome P450 (P450) drug metabolism. We characterized the potency and specificity of GFA CYP2D inhibition based on dextromethorphan O-demethylation, a CYP2D6 probe substrate of activity in human, mouse, rat, dog, and monkey liver microsomes. In addition, (+)-bufuralol 1'-hydroxylation was used as a CYP2D6 probe for the recombinant form (rCYP2D6), 2D1 (rCYP2D1), and 2D2 (rCYP2D2) activities. Results show that GFA is a potent noncompetitive inhibitor of CYP2D6, with inhibition constant Ki = 1.20 ± 0.33 μM in human liver microsomes (HLMs) and Ki = 0.37 ± 0.16 μM for the human recombinant form (rCYP2D6). GFA is also a potent competitive inhibitor of CYP2D in monkey (Ki = 0.38 ± 0.12 μM) and dog (Ki = 2.4 ± 1.3 μM) microsomes. However, GFA has no inhibitory activity on mouse or rat CYP2Ds. GFA did not exhibit any inhibition activity on human recombinant CYP1A2, 2A6, 2C8, 2C19, 3A4, or 3A5, but showed slight inhibition of 2B6 and 2E1. Preincubation of HLMs and rCYP2D6 resulted in the inactivation of the enzyme, which was attenuated by GFA or quinidine. Beagle dogs treated intravenously with dextromethorphan (2 mg/ml) after pretreatment with GFA injection showed reduced CYP2D metabolic activity, with the Cmax of dextrorphan being one-third that of the saline-treated group and area under the plasma concentration-time curve half that of the saline-treated group. This study suggests that GFA is a specific CYP2D6 inhibitor that might play a role in CYP2D6 medicated drug-drug interaction.

  18. P-glycoprotein, CYP3A, and Plasma Carboxylesterase Determine Brain Disposition and Oral Availability of the Novel Taxane Cabazitaxel (Jevtana) in Mice.

    PubMed

    Tang, Seng Chuan; Kort, Anita; Cheung, Ka Lei; Rosing, Hilde; Fukami, Tatsuki; Durmus, Selvi; Wagenaar, Els; Hendrikx, Jeroen J M A; Nakajima, Miki; van Vlijmen, Bart J M; Beijnen, Jos H; Schinkel, Alfred H

    2015-10-01

    We aimed to clarify the roles of the multidrug-detoxifying proteins ABCB1, ABCG2, ABCC2, and CYP3A in oral availability and brain accumulation of cabazitaxel, a taxane developed for improved therapy of docetaxel-resistant prostate cancer. Cabazitaxel pharmacokinetics were studied in Abcb1a/1b, Abcg2, Abcc2, Cyp3a, and combination knockout mice. We found that human ABCB1, but not ABCG2, transported cabazitaxel in vitro. Upon oral cabazitaxel administration, total plasma levels were greatly increased due to binding to plasma carboxylesterase Ces1c, which is highly upregulated in several knockout strains. Ces1c inhibition and in vivo hepatic Ces1c knockdown reversed these effects. Correcting for Ces1c effects, Abcb1a/1b, Abcg2, and Abcc2 did not restrict cabazitaxel oral availability, whereas Abcb1a/1b, but not Abcg2, dramatically reduced cabazitaxel brain accumulation (>10-fold). Coadministration of the ABCB1 inhibitor elacridar completely reversed this brain accumulation effect. After correction for Ces1c effects, Cyp3a knockout mice demonstrated a strong (six-fold) increase in cabazitaxel oral availability, which was completely reversed by transgenic human CYP3A4 in intestine and liver. Cabazitaxel markedly inhibited mouse Ces1c, but human CES1 and CES2 only weakly. Ces1c upregulation can thus complicate preclinical cabazitaxel studies. In summary, ABCB1 limits cabazitaxel brain accumulation and therefore potentially therapeutic efficacy against (micro)metastases or primary tumors positioned wholly or partly behind a functional blood-brain barrier. This can be reversed with elacridar coadministration, and similar effects may apply to ABCB1-expressing tumors. CYP3A4 profoundly reduces the oral availability of cabazitaxel. This may potentially be greatly improved by coadministering ritonavir or other CYP3A inhibitors, suggesting the option of patient-friendly oral cabazitaxel therapy.

  19. Relationship between Genotypes Sult1a2 and Cyp2d6 and Tamoxifen Metabolism in Breast Cancer Patients

    PubMed Central

    Fernández-Santander, Ana; Gaibar, María; Novillo, Apolonia; Romero-Lorca, Alicia; Rubio, Margarita; Chicharro, Luis Miguel; Tejerina, Armando; Bandrés, Fernando

    2013-01-01

    Tamoxifen is a pro-drug widely used in breast cancer patients to prevent tumor recurrence. Prior work has revealed a role of cytochrome and sulfotransferase enzymes in tamoxifen metabolism. In this descriptive study, correlations were examined between concentrations of tamoxifen metabolites and genotypes for CYP2D6, CYP3A4, CYP3A5, SULT1A1, SULT1A2 and SULT1E1 in 135 patients with estrogen receptor-positive breast cancer. Patients were genotyped using the Roche-AmpliChip® CYP450 Test, and Real-Time and conventional PCR-RFLP. Plasma tamoxifen, 4-hydroxy-tamoxifen, N-desmethyl-tamoxifen, endoxifen and tamoxifen-N-oxide were isolated and quantified using a high-pressure liquid chromatography-tandem mass spectrometry system. Significantly higher endoxifen levels were detected in patients with the wt/wt CYP2D6 compared to the v/v CYP2D6 genotype (p<0.001). No differences were detected in the remaining tamoxifen metabolites among CYP2D6 genotypes. Patients featuring the SULT1A2*2 and SULT1A2*3 alleles showed significantly higher plasma levels of 4-hydroxy-tamoxifen and endoxifen (p = 0.025 and p = 0.006, respectively), as likely substrates of the SULT1A2 enzyme. Our observations indicate that besides the CYP2D6 genotype leading to tamoxifen conversion to potent hydroxylated metabolites in a manner consistent with a gene-dose effect, SULT1A2 also seems to play a role in maintaining optimal levels of both 4-hydroxy-tamoxifen and endoxifen. PMID:23922954

  20. CYP2B6 Variants and Plasma Efavirenz Concentrations during Antiretroviral Therapy in Port-au-Prince, Haiti

    PubMed Central

    Leger, Paul; Dillingham, Rebecca; Beauharnais, Carole Anne; Kashuba, Angela D. M.; Rezk, Naser L.; Fitzgerald, Daniel W.; Pape, Jean William; Haas, David W.

    2009-01-01

    Background Polymorphisms in CYP2B6 are known to predict increased steady-state plasma concentrations of efavirenz. We characterized relationships between genetic polymorphisms and plasma efavirenz concentrations among 45 Haitians who initiated antiretroviral therapy in Port-au-Prince. Methods An observational study characterized relationships between clinical factors, pharmacokinetics, and treatment response among antiretroviral-naïve patients initiating once-daily efavirenz plus twice-daily AZT/3TC. Plasma drug concentrations were determined at weeks 2 and 4. Drug doses were directly observed by field workers or designated family members. We retrospectively characterized relationships between efavirenz concentrations and 50 single nucleotide polymorphisms in CYP2B6, and several polymorphisms in CYP2A6, CYP3A4, CYP3A5 and ABCB1. Results Plasma for efavirenz assay was obtained 13.9 ±1.6 hours (mean ± SD) post-dose. As expected, CYP2B6 516G→T was associated with increased plasma efavirenz concentrations (Spearman’s rho=0.71, P<0.0001), as were 10 polymorphisms in linkage disequilibrium with 516G→T. Distinct CYP2B6 polymorphisms were associated with decreased plasma efavirenz concentrations (greatest absolute rho=0.48, P=0.0008). Associations were replicated by results from a recent pharmacokinetic study involving 34 healthy, HIV-negative African Americans. Conclusions Relatively frequent CYP2B6 polymorphisms may predict decreased plasma efavirenz exposure in patients of African descent. If replicated in other cohorts, the implications of these novel associations for treatment response warrant further study. PMID:19659438

  1. Isolation and identification of intestinal CYP3A inhibitors from cranberry (Vaccinium macrocarpon) using human intestinal microsomes.

    PubMed

    Kim, Eunkyung; Sy-Cordero, Arlene; Graf, Tyler N; Brantley, Scott J; Paine, Mary F; Oberlies, Nicholas H

    2011-02-01

    Cranberry juice is used routinely, especially among women and the elderly, to prevent and treat urinary tract infections. These individuals are likely to be taking medications concomitantly with cranberry juice, leading to concern about potential drug-dietary substance interactions, particularly in the intestine, which, along with the liver, is rich in expression of the prominent drug metabolizing enzyme, cytochrome P450 3A (CYP3A). Using a systematic in vitro-in vivo approach, a cranberry juice product was identified recently that elicited a pharmacokinetic interaction with the CYP3A probe substrate midazolam in 16 healthy volunteers. Relative to water, cranberry juice inhibited intestinal first-pass midazolam metabolism. In vitro studies were initiated to identify potential enteric CYP3A inhibitors from cranberry via a bioactivity-directed fractionation approach involving dried whole cranberry [Vaccinium macrocarpon Ait. (Ericaceae)], midazolam, and human intestinal microsomes (HIM). Three triterpenes (maslinic acid, corosolic acid, and ursolic acid) were isolated. The inhibitory potency (IC(50)) of maslinic acid, corosolic acid, and ursolic acid was 7.4, 8.8, and < 10 µM, respectively, using HIM as the enzyme source and 2.8, 4.3, and < 10 µM, respectively, using recombinant CYP3A4 as the enzyme source. These in vitro inhibitory potencies, which are within the range of those reported for two CYP3A inhibitory components in grapefruit juice, suggest that these triterpenes may have contributed to the midazolam-cranberry juice interaction observed in the clinical study.

  2. The role of human cytochrome P450 enzymes in the formation of 2-hydroxymetronidazole: CYP2A6 is the high affinity (low Km) catalyst.

    PubMed

    Pearce, Robin E; Cohen-Wolkowiez, Michael; Sampson, Mario R; Kearns, Gregory L

    2013-09-01

    Despite metronidazole's widespread clinical use since the 1960s, the specific enzymes involved in its biotransformation have not been previously identified. Hence, in vitro studies were conducted to identify and characterize the cytochrome P450 enzymes involved in the formation of the major metabolite, 2-hydroxymetronidazole. Formation of 2-hydroxymetronidazole in human liver microsomes was consistent with biphasic, Michaelis-Menten kinetics. Although several cDNA-expressed P450 enzymes catalyzed 2-hydroxymetronidazole formation at a supratherapeutic concentration of metronidazole (2000 μM), at a "therapeutic concentration" of 100 μM only CYPs 2A6, 3A4, 3A5, and 3A7 catalyzed metronidazole 2-hydroxylation at rates substantially greater than control vector, and CYP2A6 catalyzed 2-hydroxymetronidazole formation at rates 6-fold higher than the next most active enzyme. Kinetic studies with these recombinant enzymes revealed that CYP2A6 has a Km = 289 μM which is comparable to the Km for the high-affinity (low-Km) enzyme in human liver microsomes, whereas the Km values for the CYP3A enzymes corresponded with the low-affinity (high-Km) component. The sample-to-sample variation in 2-hydroxymetronidazole formation correlated significantly with CYP2A6 activity (r ≥ 0.970, P < 0.001) at substrate concentrations of 100 and 300 μM. Selective chemical inhibitors of CYP2A6 inhibited metronidazole 2-hydroxylation in a concentration-dependent manner and inhibitory antibodies against CYP2A6 virtually eliminated metronidazole 2-hydroxylation (>99%). Chemical and antibody inhibitors of other P450 enzymes had little or no effect on metronidazole 2-hydroxylation. These results suggest that CYP2A6 is the primary catalyst responsible for the 2-hydroxylation of metronidazole, a reaction that may function as a marker of CYP2A6 activity both in vitro and in vivo.

  3. Methyl 6-eth-oxy-3-phenyl-3a,4-dihydro-3H-chromeno[4,3-c]isoxazole-3a-car-boxylate.

    PubMed

    Suresh, G; Srinivasan, J; Bakthadoss, M; Aravindhan, S

    2013-02-01

    In the title compound, C(20)H(19)NO(5), the dihedral angle between the mean plane of the pyran ring (which has a half-chair conformation) and the benzene ring of the chromeno ring system is 7.21 (7)°. The dihedral angle between the mean plane of the chromeno ring system and the isoxazole ring is 21.78 (6)°, while the isoxazole ring forms a dihedral angle of 72.60 (8)° with the attached phenyl ring. In the crystal, mol-ecules are linked via pairs of C-H⋯O hydrogen bonds, forming inversion dimers with an R(2) (2)(10) ring motif. These dimers are linked via C-H⋯N hydrogen bonds, forming chains along [001].

  4. Biotransformations of 6',7'-dihydroxybergamottin and 6',7'-epoxybergamottin by the citrus-pathogenic fungi diminish cytochrome P450 3A4 inhibitory activity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Penicillium digitatum, as well as five other citrus pathogenic species, (P. ulaiense Link, Geotrichum citri Link, Botrytis cinerea P. Micheli ex Pers., Lasiodiplodia theobromae (Pat.)Griffon & Maubl. and Phomopsis citri (teleomorph Diaporthe citri)) was observed to convert 6',7'-epoxybergamottin (1)...

  5. Biotransformation of BDE-47 to Potentially Toxic Metabolites Is Predominantly Mediated by Human CYP2B6

    PubMed Central

    Feo, Maria Luisa; Gross, Michael S.; McGarrigle, Barbara P.; Eljarrat, Ethel; Barceló, Damià; Olson, James R.

    2012-01-01

    Background: Previous studies have indicated that cytochrome P450s (CYPs) are involved in the metabolism of polybrominated diphenyl ether (PBDE) flame retardants in humans, resulting in the formation of hydroxylated PBDEs (OH-PBDEs) that are potentially more toxic than the parent PBDEs. However, the specific enzymes responsible for the formation of OH-PBDEs are unknown. Objectives: The purposes of this study were to characterize the in vitro metabolism of 2,2´,4,4´-tetrabromodiphenyl ether (BDE-47) by human liver microsomes (HLM) and recombinant human CYPs, and to identify the CYP(s) that are active in the oxidative metabolism of BDE-47. Methods: Recombinant human CYPs (CYP1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4) were incubated with BDE-47 (20 µM), and the metabolites were measured and characterized using gas chromatography with tandem mass spectrometry (GC-MS/MS). For kinetic studies, CYP2B6 and pooled human liver microsomes (HLMs) were incubated with BDE-47 (0–60 µM). Results: CYP2B6 was the predominant CYP capable of forming six OH-BDEs, including 3-OH-BDE-47, 5-OH-BDE-47, 6-OH-BDE-47, 4-OH-BDE-42, 4´-OH-BDE-49, and a metabolite tentatively identified as 2´-OH-BDE-66. On the basis of full-scan GC-MS analysis, we hypothesized the formation of two other metabolites: di-OH-tetra-BDE and di-OH-tetrabrominated dioxin. In kinetic studies of BDE-47 metabolism by CYP2B6 and pooled HLMs, we found Km values ranging from 3.8 to 6.4 µM and 7.0 to 11.4 µM, respectively, indicating the high affinity toward the formation of OH-BDEs. Conclusion: Our findings support a predominant role of CYP2B6 in the metabolism of BDE-47 to potentially toxic metabolites, including a hypothesized di-OH-tetrabrominated dioxin metabolite. These results will assist future epidemiological studies investigating the potential of PBDEs and their metabolites to produce neurobehavioral/neurodevelopmental disorders. PMID:23249762

  6. The potential for CYP2D6 inhibition screening using a novel scintillation proximity assay-based approach.

    PubMed

    Delaporte, E; Slaughter, D E; Egan, M A; Gatto, G J; Santos, A; Shelley, J; Price, E; Howells, L; Dean, D C; Rodrigues, A D

    2001-08-01

    High throughput inhibition screens for human cytochrome P450s (CYPs) are being used in preclinical drug metabolism to support drug discovery programs. The versatility of scintillation proximity assay (SPA) technology has enabled the development of a homogeneous high throughput assay for cytochrome P450 2D6 (CYP2D6) inhibition screen using [O-methyl-(14)C]dextromethorphan as substrate. The basis of the assay was the trapping of the O-demethylation product, [(14)C]HCHO, on SPA beads. Enzyme kinetics parameters V(max) and apparent K(m), determined using pooled human liver microsomes and microsomes from baculovirus cells coexpressing human CYP2D6 and NADPH-cytochrome P450 reductase, were 245 pmol [(14)C]HCHO/min/mg protein and 11 microM, and 27 pmol [(14)C]HCHO/min/pmol and 1.6 microM, respectively. In incubations containing either pooled microsomes or recombinant CYP2D6, [(14)C]dextromethorphan O-demethylase activity was inhibited in the presence of quinidine (IC(50) = 1.0 microM and 20 nM, respectively). By comparison, inhibitors selective for other CYP isoforms were relatively weak (IC(50) > 25 microM). In agreement, a selective CYP2D6 inhibitory monoclonal antibody caused greater than 90% inhibition of [(14)C]dextromethorphan O-demethylase activity in human liver microsomes, whereas CYP2C9/19- and CYP3A4/5-selective antibodies elicited a minimal inhibitory effect. SPA-based [(14)C]dextromethorphan O-demethylase activity was also shown to correlate (r(2) = 0.6) with dextromethorphan O-demethylase measured by high-performance liquid chromatography in a bank of human liver microsomes (N = 15 different organ donors). In a series of known CYP2D6 inhibitors/substrates, the SPA-based assay resolved potent inhibitors (IC(50) < 2 microM) from weak inhibitors (IC(50) >or= 20 microM). It is concluded that the SPA-based assay described herein is suitable for CYP2D6 inhibition screening using either native human liver microsomes or cDNA-expressed CYP2D6. PMID:11689122

  7. A Systematic Approach to Evaluate Herb-Drug Interaction Mechanisms: Investigation of Milk Thistle Extracts and Eight Isolated Constituents as CYP3A Inhibitors

    PubMed Central

    Brantley, Scott J.; Graf, Tyler N.; Oberlies, Nicholas H.

    2013-01-01

    Despite increasing recognition of potential untoward interactions between herbal products and conventional medications, a standard system for prospective assessment of these interactions remains elusive. This information gap was addressed by evaluating the drug interaction liability of the model herbal product milk thistle (Silybum marianum) with the CYP3A probe substrate midazolam. The inhibitory effects of commercially available milk thistle extracts and isolated constituents on midazolam 1′-hydroxylation were screened using human liver and intestinal microsomes. Relative to vehicle, the extract silymarin and constituents silybin A, isosilybin A, isosilybin B, and silychristin at 100 μM demonstrated >50% inhibition of CYP3A activity with at least one microsomal preparation, prompting IC50 determination. The IC50s for isosilybin B and silychristin were ∼60 and 90 μM, respectively, whereas those for the remaining constituents were >100 μM. Extracts and constituents that contained the 1,4-dioxane moiety demonstrated a >1.5-fold shift in IC50 when tested as potential mechanism-based inhibitors. The semipurified extract, silibinin, and the two associated constituents (silybin A and silybin B) demonstrated mechanism-based inhibition of recombinant CYP3A4 (KI, ∼100 μM; kinact, ∼0.20 min−1) but not microsomal CYP3A activity. The maximum predicted increases in midazolam area under the curve using the static mechanistic equation and recombinant CYP3A4 data were 1.75-fold, which may necessitate clinical assessment. Evaluation of the interaction liability of single herbal product constituents, in addition to commercially available extracts, will enable elucidation of mechanisms underlying potential clinically significant herb-drug interactions. Application of this framework to other herbal products would permit predictions of herb-drug interactions and assist in prioritizing clinical evaluation. PMID:23801821

  8. A systematic approach to evaluate herb-drug interaction mechanisms: investigation of milk thistle extracts and eight isolated constituents as CYP3A inhibitors.

    PubMed

    Brantley, Scott J; Graf, Tyler N; Oberlies, Nicholas H; Paine, Mary F

    2013-09-01

    Despite increasing recognition of potential untoward interactions between herbal products and conventional medications, a standard system for prospective assessment of these interactions remains elusive. This information gap was addressed by evaluating the drug interaction liability of the model herbal product milk thistle (Silybum marianum) with the CYP3A probe substrate midazolam. The inhibitory effects of commercially available milk thistle extracts and isolated constituents on midazolam 1'-hydroxylation were screened using human liver and intestinal microsomes. Relative to vehicle, the extract silymarin and constituents silybin A, isosilybin A, isosilybin B, and silychristin at 100 μM demonstrated >50% inhibition of CYP3A activity with at least one microsomal preparation, prompting IC50 determination. The IC50s for isosilybin B and silychristin were ∼60 and 90 μM, respectively, whereas those for the remaining constituents were >100 μM. Extracts and constituents that contained the 1,4-dioxane moiety demonstrated a >1.5-fold shift in IC50 when tested as potential mechanism-based inhibitors. The semipurified extract, silibinin, and the two associated constituents (silybin A and silybin B) demonstrated mechanism-based inhibition of recombinant CYP3A4 (KI, ∼100 μM; kinact, ∼0.20 min(-1)) but not microsomal CYP3A activity. The maximum predicted increases in midazolam area under the curve using the static mechanistic equation and recombinant CYP3A4 data were 1.75-fold, which may necessitate clinical assessment. Evaluation of the interaction liability of single herbal product constituents, in addition to commercially available extracts, will enable elucidation of mechanisms underlying potential clinically significant herb-drug interactions. Application of this framework to other herbal products would permit predictions of herb-drug interactions and assist in prioritizing clinical evaluation.

  9. Recommendations on the Development of a Bioanalytical Assay for 4β-Hydroxycholesterol, an Emerging Endogenous Biomarker of CYP3A Activity.

    PubMed

    Aubry, Anne-Françoise; Dean, Brian; Diczfalusy, Ulf; Goodenough, Angela; Iffland, André; McLeod, James; Weng, Naidong; Yang, Ziping

    2016-09-01

    The availability of reliable assays for measuring 4β-hydroxycholesterol (4β-HC), a CYP3A metabolite of cholesterol, is an important step in qualifying this endogenous moiety as a biomarker of CYP3A activity. Liquid and gas chromatographic methods with mass spectrometric detection have been developed with varying sensitivities, with or without derivatization. Care must be taken to chromatographically resolve 4β-HC from the multiple isobaric cholesterol oxidation products present in plasma, including 4α-hydroxycholesterol (4α-HC). Plasma concentrations of 4β-HC are low in humans (10-60 ng/ml), lower than many other cholesterol metabolites and far less than cholesterol itself. Stability of 4β-HC has been established for at least 12 months at -20°C in plasma samples obtained with a typical clinical workflow. Oxidation of plasma cholesterol during storage produces both 4β-HC and 4α-HC, and 4α-HC may be used as assessment of sample quality. As 4β-HC concentrations over time in untreated individuals have low intra-individual variability, assay precision and reproducibility are the key assay attributes in assessing CYP3A4 induction, and potentially inhibition. Assessment of CYP3A4/5 activity with 4β-HC relies on the differences between pre- and post-dose concentrations, in which each subject acts as their own control. To reduce analytical variability, samples from a single subject should be analyzed together to facilitate interpretation of study results. As an endogenous biomarker, 4β-HC offers the opportunity for less invasive assessment of CYP3A induction potential of new drugs during clinical development. PMID:27350147

  10. CYP3A variation and the evolution of salt-sensitivity variants.

    PubMed

    Thompson, E E; Kuttab-Boulos, H; Witonsky, D; Yang, L; Roe, B A; Di Rienzo, A

    2004-12-01

    Members of the cytochrome P450 3A subfamily catalyze the metabolism of endogenous substrates, environmental carcinogens, and clinically important exogenous compounds, such as prescription drugs and therapeutic agents. In particular, the CYP3A4 and CYP3A5 genes play an especially important role in pharmacogenetics, since they metabolize >50% of the drugs on the market. However, known genetic variants at these two loci are not sufficient to account for the observed phenotypic variability in drug response. We used a comparative genomics approach to identify conserved coding and noncoding regions at these genes and resequenced them in three ethnically diverse human populations. We show that remarkable interpopulation differences exist with regard to frequency spectrum and haplotype structure. The non-African samples are characterized by a marked excess of rare variants and the presence of a homogeneous group of long-range haplotypes at high frequency. The CYP3A5*1/*3 polymorphism, which is likely to influence salt and water retention and risk for salt-sensitive hypertension, was genotyped in >1,000 individuals from 52 worldwide population samples. The results reveal an unusual geographic pattern whereby the CYP3A5*3 frequency shows extreme variation across human populations and is significantly correlated with distance from the equator. Furthermore, we show that an unlinked variant, AGT M235T, previously implicated in hypertension and pre-eclampsia, exhibits a similar geographic distribution and is significantly correlated in frequency with CYP3A5*1/*3. Taken together, these results suggest that variants that influence salt homeostasis were the targets of a shared selective pressure that resulted from an environmental variable correlated with latitude.

  11. RS-Predictor models augmented with SMARTCyp reactivities: Robust metabolic regioselectivity predictions for nine CYP isozymes

    PubMed Central

    Zaretzki, Jed; Rydberg, Patrik; Bergeron, Charles; Bennett, Kristin P.; Olsen, Lars

    2012-01-01

    RS-Predictor is a tool for creating pathway-independent, isozyme-specific site of metabolism (SOM) prediction models using any set of known cytochrome P450 substrates and metabolites. Until now, the RS-Predictor method was only trained and validated on CYP 3A4 data, but in the present study we report on the versatility the RS-Predictor modeling paradigm by creating and testing regioselectivity models for substrates of the nine most important CYP isozymes. Through curation of source literature, we have assembled 680 substrates distributed among CYPs 1A2, 2A6, 2B6, 2C19, 2C8, 2C9, 2D6, 2E1 and 3A4, which we believe is the largest publicly accessible collection of P450 ligands and metabolites ever released. A comprehensive investigation into the importance of different descriptor classes for predicting the regioselectivity of each isozyme is made through the generation of multiple independent RS-Predictor models for each set of isozyme substrates. Two of these models include a DFT reactivity descriptor derived from SMARTCyp. Optimal combinations of RS-Predictor and SMARTCyp are shown to have stronger performance than either method alone, while also exceeding the accuracy of the commercial regioselectivity prediction methods distributed by StarDrop and Schrödinger, correctly identifying a large proportion of the metabolites in each substrate set within the top two rank-positions: 1A2(83.0%), 2A6(85.7%), 2B6(82.1%), 2C19(86.2%), 2C8(83.8%), 2C9(84.5%), 2D6(85.9%), 2E1(82.8%), 3A4(82.3%) and merged(86.0%). Comprehensive datamining of each substrate set and careful statistical analyses of the predictions made by the different models revealed new insights into molecular features that control metabolic regioselectivity and enable accurate prospective prediction of likely SOMs. PMID:22524152

  12. Mechanism-based inactivation of CYP450 enzymes: a case study of lapatinib

    PubMed Central

    Ho, Han Kiat; Chan, Chun Yip; Hardy, Klarissa D; Chan, Eric Chun Yong

    2015-01-01

    Mechanism-based inactivation (MBI) of CYP450 enzymes is a unique form of inhibition in which the enzymatic machinery of the victim is responsible for generation of the reactive metabolite. This precondition sets up a time-dependency for the inactivation process, a hallmark feature that characterizes all MBI. Yet, MBI itself is a complex biochemical phenomenon that operates in different modes, namely covalent binding to apoprotein, covalent binding of the porphyrin group, and also complexation of the catalytic iron. Using lapatinib as a recent example of toxicological interest, we present an example of a mixed-function MBI that can confound clinical drug-drug interactions manifestation. Lapatinib exhibits both covalent binding to the apoprotein, and formation of a metabolite-intermediate (MI) complex in an enzyme-selective manner (CYP3A4 versus CYP3A5), each with different reactive metabolites. The clinical implication of this effect is also contingent upon genetic polymorphisms of the enzyme involved as well as the co-administration of other substrates, inhibitors or inducers, culminating in drug-drug interactions. This understanding recapitulates the importance of applying isoform-specific mechanistic investigations to develop customized strategies to manage such outcomes. PMID:25639891

  13. Mechanism-based inactivation of CYP450 enzymes: a case study of lapatinib.

    PubMed

    Ho, Han Kiat; Chan, James Chun Yip; Hardy, Klarissa D; Chan, Eric Chun Yong

    2015-02-01

    Mechanism-based inactivation (MBI) of CYP450 enzymes is a unique form of inhibition in which the enzymatic machinery of the victim is responsible for generation of the reactive metabolite. This precondition sets up a time-dependency for the inactivation process, a hallmark feature that characterizes all MBI. Yet, MBI itself is a complex biochemical phenomenon that operates in different modes, namely, covalent binding to apoprotein, covalent binding of the porphyrin group and also complexation of the catalytic iron. Using lapatinib as a recent example of toxicological interest, we present an example of a mixed-function MBI that can confound clinical drug-drug interactions manifestation. Lapatinib exhibits both covalent binding to the apoprotein and formation of a metabolite-intermediate complex in an enzyme-selective manner (CYP3A4 versus CYP3A5), each with different reactive metabolites. The clinical implication of this effect is also contingent upon genetic polymorphisms of the enzyme involved as well as the co-administration of other substrates, inhibitors or inducers, culminating in drug-drug interactions. This understanding recapitulates the importance of applying isoform-specific mechanistic investigations to develop customized strategies to manage such outcomes. PMID:25639891

  14. Epoxidation of the methamphetamine pyrolysis product, trans-phenylpropene, to trans-phenylpropylene oxide by CYP enzymes and stereoselective glutathione adduct formation

    SciTech Connect

    Sanga, Madhu; Younis, Islam R.; Tirumalai, Padma S.; Bland, Tina M.; Banaszewska, Monica; Konat, Gregory W.; Tracy, Timothy S.; Gannett, Peter M.; Callery, Patrick S. . E-mail: pcallery@hsc.wvu.edu

    2006-03-01

    Pyrolytic products of smoked methamphetamine hydrochloride are well established. Among the various degradation products formed, trans-phenylpropene (trans-{beta}-methylstyrene) is structurally similar to styrene analogues known to be bioactivated by CYP enzymes. In human liver microsomes, trans-phenylpropene was converted to the epoxide trans-phenylpropylene oxide (trans-2-methyl-3-phenyloxirane) and cinnamyl alcohol. Incubation of trans-phenylpropene with microsomes in the presence of enzyme-specific P450 enzyme inhibitors indicated the involvement of CYP2E1, CYP1A2, and CYP3A4 enzymes. Both (R,R)-phenylpropylene oxide and (S,S)-phenylpropylene oxide were formed in human liver microsomal preparations. Enantiomers of trans-phenylpropylene oxide were stereoselectively and regioselectively conjugated in a Phase II drug metabolism reaction catalyzed by human liver cytosolic enzymes consisting of conjugation with glutathione. The structure of the phenylpropylene oxide-glutathione adduct is consistent with nucleophilic ring-opening by attack at the benzylic carbon. Exposure of cultured C6 glial cells to (S,S)-phenylpropylene oxide produced a cytotoxic response in a concentration-dependent manner based on cell degeneration and death.

  15. Schisandrol B protects against acetaminophen-induced hepatotoxicity by inhibition of CYP-mediated bioactivation and regulation of liver regeneration.

    PubMed

    Jiang, Yiming; Fan, Xiaomei; Wang, Ying; Chen, Pan; Zeng, Hang; Tan, Huasen; Gonzalez, Frank J; Huang, Min; Bi, Huichang

    2015-01-01

    Acetaminophen (APAP) overdose is the most frequent cause of drug-induced acute liver failure. Schisandra sphenanthera is a traditional hepato-protective Chinese medicine and Schisandrol B (SolB) is one of its major active constituents. In this study, the protective effect of SolB against APAP-induced acute hepatotoxicity in mice and the involved mechanisms were investigated. Morphological and biochemical assessments clearly demonstrated a protective effect of SolB against APAP-induced liver injury. SolB pretreatment significantly attenuated the increases in alanine aminotransferase and aspartate aminotransferase activity, and prevented elevated hepatic malondialdehyde formation and the depletion of mitochondrial glutathione (GSH) in a dose-dependent manner. SolB also dramatically altered APAP metabolic activation by inhibiting the activities of CYP2E1 and CYP3A11, which was evidenced by significant inhibition of the formation of the oxidized APAP metabolite NAPQI-GSH. A molecular docking model also predicted that SolB had potential to interact with the CYP2E1 and CYP3A4 active sites. In addition, SolB abrogated APAP-induced activation of p53 and p21, and increased expression of liver regeneration and antiapoptotic-related proteins such as cyclin D1 (CCND1), PCNA, and BCL-2. This study demonstrated that SolB exhibited a significant protective effect toward APAP-induced liver injury, potentially through inhibition of CYP-mediated APAP bioactivation and regulation of the p53, p21, CCND1, PCNA, and BCL-2 to promote liver regeneration. PMID:25319358

  16. Schisandrol B protects against acetaminophen-induced hepatotoxicity by inhibition of CYP-mediated bioactivation and regulation of liver regeneration.

    PubMed

    Jiang, Yiming; Fan, Xiaomei; Wang, Ying; Chen, Pan; Zeng, Hang; Tan, Huasen; Gonzalez, Frank J; Huang, Min; Bi, Huichang

    2015-01-01

    Acetaminophen (APAP) overdose is the most frequent cause of drug-induced acute liver failure. Schisandra sphenanthera is a traditional hepato-protective Chinese medicine and Schisandrol B (SolB) is one of its major active constituents. In this study, the protective effect of SolB against APAP-induced acute hepatotoxicity in mice and the involved mechanisms were investigated. Morphological and biochemical assessments clearly demonstrated a protective effect of SolB against APAP-induced liver injury. SolB pretreatment significantly attenuated the increases in alanine aminotransferase and aspartate aminotransferase activity, and prevented elevated hepatic malondialdehyde formation and the depletion of mitochondrial glutathione (GSH) in a dose-dependent manner. SolB also dramatically altered APAP metabolic activation by inhibiting the activities of CYP2E1 and CYP3A11, which was evidenced by significant inhibition of the formation of the oxidized APAP metabolite NAPQI-GSH. A molecular docking model also predicted that SolB had potential to interact with the CYP2E1 and CYP3A4 active sites. In addition, SolB abrogated APAP-induced activation of p53 and p21, and increased expression of liver regeneration and antiapoptotic-related proteins such as cyclin D1 (CCND1), PCNA, and BCL-2. This study demonstrated that SolB exhibited a significant protective effect toward APAP-induced liver injury, potentially through inhibition of CYP-mediated APAP bioactivation and regulation of the p53, p21, CCND1, PCNA, and BCL-2 to promote liver regeneration.

  17. CYP63A2, a catalytically versatile fungal P450 monooxygenase capable of oxidizing higher-molecular-weight polycyclic aromatic hydrocarbons, alkylphenols, and alkanes.

    PubMed

    Syed, Khajamohiddin; Porollo, Aleksey; Lam, Ying Wai; Grimmett, Paul E; Yadav, Jagjit S

    2013-04-01

    Cytochrome P450 monooxygenases (P450s) are known to oxidize hydrocarbons, albeit with limited substrate specificity across classes of these compounds. Here we report a P450 monooxygenase (CYP63A2) from the model ligninolytic white rot fungus Phanerochaete chrysosporium that was found to possess a broad oxidizing capability toward structurally diverse hydrocarbons belonging to mutagenic/carcinogenic fused-ring higher-molecular-weight polycyclic aromatic hydrocarbons (HMW-PAHs), endocrine-disrupting long-chain alkylphenols (APs), and crude oil aliphatic hydrocarbon n-alkanes. A homology-based three-dimensional (3D) model revealed the presence of an extraordinarily large active-site cavity in CYP63A2 compared to the mammalian PAH-oxidizing (CYP3A4, CYP1A2, and CYP1B1) and bacterial aliphatic-hydrocarbon-oxidizing (CYP101D and CYP102A1) P450s. This structural feature in conjunction with ligand docking simulations suggested potential versatility of the enzyme. Experimental characterization using recombinantly expressed CYP63A2 revealed its ability to oxidize HMW-PAHs of various ring sizes, including 4 rings (pyrene and fluoranthene), 5 rings [benzo(a)pyrene], and 6 rings [benzo(ghi)perylene], with the highest enzymatic activity being toward the 5-ring PAH followed by the 4-ring and 6-ring PAHs, in that order. Recombinant CYP63A2 activity yielded monohydroxylated PAH metabolites. The enzyme was found to also act as an alkane ω-hydroxylase that oxidized n-alkanes with various chain lengths (C9 to C12 and C15 to C19), as well as alkyl side chains (C3 to C9) in alkylphenols (APs). CYP63A2 showed preferential oxidation of long-chain APs and alkanes. To our knowledge, this is the first P450 identified from any of the biological kingdoms that possesses such broad substrate specificity toward structurally diverse xenobiotics (PAHs, APs, and alkanes), making it a potent enzyme biocatalyst candidate to handle mixed pollution (e.g., crude oil spills).

  18. The effect of ethinyloestradiol and levonorgestrel on the CYP2C19-mediated metabolism of omeprazole in healthy female subjects

    PubMed Central

    Palovaara, Sanna; Tybring, Gunnel; Laine, Kari

    2003-01-01

    Aims To study the effect of an oral contraceptive (OC) formulation containing ethinyloestradiol and levonorgestrel (LNG) (combination OC) or LNG alone on the CYP2C19-mediated hydroxylation of omeprazole in healthy females. Methods This was an open crossover study with three phases. In phase one, 10 healthy females received a single 40-mg dose of omeprazole. Thereafter the subjects received in a random order either 40 µg ethinyloestradiol and 75 µg LNG or 60 µg LNG alone once daily for 10 days. On day 10, 1 h after the last OC dose, subjects received a single 40-mg oral dose of omeprazole. The plasma concentrations of omeprazole, 5′-hydroxyomeprazole and omeprazole sulphone were determined for up to 8 h. Results The use of combination OC increased the area under the curve (AUC) of omeprazole by 38% [95% confidence interval (CI) − 3.8, 80; P = 0.040] and caused a 48% increase (95% CI 28, 68) in the AUC ratio of omeprazole/5-hydroxyomeprazole. LNG alone did not effect the 5′-hydroxylation of omeprazole. Neither of the OC preparations seemed to have an inhibitory effect on the formation of omeprazole sulphone by CYP3A4. Conclusions Oral contraceptives containing ethinyloestradiol but not those containing only LNG decrease CYP2C19 activity. PMID:12895199

  19. Evaluation of first-pass cytochrome P4503A (CYP3A) and P-glycoprotein activities using felodipine and hesperetin in combination in Wistar rats and everted rat gut sacs in vitro.

    PubMed

    Sridhar, V; Surya Sandeep, M; Ravindra Babu, P; Naveen Babu, K

    2014-05-01

    The effects of hesperetin on the pharmacokinetics and the role of P-glycoprotein (P-gp) in the transport of felodipine were investigated in rats and in vitro. Felodipine was administered orally (10 mg/kg) without or with hesperetin (25, 50 and 100 mg/kg) to rats for 15 consecutive days. Blood samples were collected at different time intervals on 1(st) day in single dose pharmacokinetic study (SDS) and on 15(th) day in multiple dose pharmacokinetic study (MDS). The area under the plasma concentration-time curve (AUC0-∞ ) and the peak plasma concentration (Cmax ) of felodipine were dose-dependently increased in SDS and MDS with hesperetin compared to control ( p < 0.001). The half-life (t1/2 ) and mean residence time was longer than the control group in both studies. The role of P-gp determined using everted rat gut sacs in vitro by incubating felodipine with or without hesperetin and verapamil (typical P-gp and CYP3A4 inhibitor). The in vitro experiments revealed that the verapamil and hesperetin increased the intestinal absorption of felodipine (p < 0.01). Concurrent use of hesperetin dramatically altered the pharmacokinetics of felodipine leading to an increase in systemic exposure. The likely mechanism is inhibition of CYP3A4-mediated first-pass metabolism and P-gp in the intestine and the liver.

  20. Renal drug metabolism in humans: the potential for drug–endobiotic interactions involving cytochrome P450 (CYP) and UDP-glucuronosyltransferase (UGT)

    PubMed Central

    Knights, Kathleen M; Rowland, Andrew; Miners, John O

    2013-01-01

    Although knowledge of human renal cytochrome P450 (CYP) and UDP-glucuronosyltransferase (UGT) enzymes and their role in xenobiotic and endobiotic metabolism is limited compared with hepatic drug and chemical metabolism, accumulating evidence indicates that human kidney has significant metabolic capacity. Of the drug metabolizing P450s in families 1 to 3, there is definitive evidence for only CYP 2B6 and 3A5 expression in human kidney. CYP 1A1, 1A2, 1B1, 2A6, 2C19, 2D6 and 2E1 are not expressed in human kidney, while data for CYP 2C8, 2C9 and 3A4 expression are equivocal. It is further known that several P450 enzymes involved in the metabolism of arachidonic acid and eicosanoids are expressed in human kidney, CYP 4A11, 4F2, 4F8, 4F11 and 4F12. With the current limited evidence of drug substrates for human renal P450s drug–endobiotic interactions arising from inhibition of renal P450s, particularly effects on arachidonic acid metabolism, appear unlikely. With respect to the UGTs, 1A5, 1A6, 1A7, 1A9, 2B4, 2B7 and 2B17 are expressed in human kidney, whereas UGT 1A1, 1A3, 1A4, 1A8, 1A10, 2B10, 2B11 and 2B15 are not. The most abundantly expressed renal UGTs are 1A9 and 2B7, which play a significant role in the glucuronidation of drugs, arachidonic acid, prostaglandins, leukotrienes and P450 derived arachidonic acid metabolites. Modulation by drug substrates (e.g. NSAIDs) of the intrarenal activity of UGT1A9 and UGT2B7 has the potential to perturb the metabolism of renal mediators including aldosterone, prostaglandins and 20-hydroxyeicosatetraenoic acid, thus disrupting renal homeostasis. PMID:23362865

  1. Renal drug metabolism in humans: the potential for drug-endobiotic interactions involving cytochrome P450 (CYP) and UDP-glucuronosyltransferase (UGT).

    PubMed

    Knights, Kathleen M; Rowland, Andrew; Miners, John O

    2013-10-01

    Although knowledge of human renal cytochrome P450 (CYP) and UDP-glucuronosyltransferase (UGT) enzymes and their role in xenobiotic and endobiotic metabolism is limited compared with hepatic drug and chemical metabolism, accumulating evidence indicates that human kidney has significant metabolic capacity. Of the drug metabolizing P450s in families 1 to 3, there is definitive evidence for only CYP 2B6 and 3A5 expression in human kidney. CYP 1A1, 1A2, 1B1, 2A6, 2C19, 2D6 and 2E1 are not expressed in human kidney, while data for CYP 2C8, 2C9 and 3A4 expression are equivocal. It is further known that several P450 enzymes involved in the metabolism of arachidonic acid and eicosanoids are expressed in human kidney, CYP 4A11, 4F2, 4F8, 4F11 and 4F12. With the current limited evidence of drug substrates for human renal P450s drug-endobiotic interactions arising from inhibition of renal P450s, particularly effects on arachidonic acid metabolism, appear unlikely. With respect to the UGTs, 1A5, 1A6, 1A7, 1A9, 2B4, 2B7 and 2B17 are expressed in human kidney, whereas UGT 1A1, 1A3, 1A4, 1A8, 1A10, 2B10, 2B11 and 2B15 are not. The most abundantly expressed renal UGTs are 1A9 and 2B7, which play a significant role in the glucuronidation of drugs, arachidonic acid, prostaglandins, leukotrienes and P450 derived arachidonic acid metabolites. Modulation by drug substrates (e.g. NSAIDs) of the intrarenal activity of UGT1A9 and UGT2B7 has the potential to perturb the metabolism of renal mediators including aldosterone, prostaglandins and 20-hydroxyeicosatetraenoic acid, thus disrupting renal homeostasis. PMID:23362865

  2. Fukinolic Acid Derivatives and Triterpene Glycosides from Black Cohosh Inhibit CYP Isozymes, but are Not Cytotoxic to Hep-G2 Cells In Vitro

    PubMed Central

    Huang, Yue; Jiang, Bei; Nuntanakorn, Paiboon; Kennelly, Edward J.; Shord, Stacy; Lawal, Temitope O.; Mahady, G.B.

    2013-01-01

    Black cohosh (Actaea racemosa L. [syn. Cimifuga racemosa L.]) extracts (BCE) are marketed worldwide for the management of menopausal symptoms. However, recently more than 75 cases of hepatotoxicity associated with black cohosh ingestion have been reported. While these cases have not been fully substantiated for causality, the data suggest that herb-drug interactions may be involved rather than a direct hepatotoxic event. This work describes the in vitro inhibition of four CYP450 enzymes (1A2, 2D6, 2C9, 3A4) by black cohosh extracts and identifies the active inhibitory constituents. Ethanol extracts (75 and 80% ethanol) and a 40% isopropanol extract induced a concentration-dependent inhibition of all CYP450 isozyme activities, with median inhibitory concentrations (IC50) ranging from 21.9 μg/ml to 65.0 μg/ml. Isolation of the active chemical constituents, showed that the triterpene glycosides were weakly active (IC50 25-100 μM), while fukinolic acid and cimicifugic acids A and B strongly inhibited all CYP isozymes (IC50 1.8-12.6 μM). None of the extracts inhibited the growth of Hep-G2 cells in concentrations up to 50 μg/ml. These data suggest that BCEs are not directly hepatotoxic, but may have the potential to induce herb-drug interactions, which may in turn explain the rare cases of hepatotoxicity observed in women using multiple medications and dietary supplements, including black cohosh. PMID:20406160

  3. Inactivation of CYP2A6 by the Dietary Phenylpropanoid trans-Cinnamic Aldehyde (Cinnamaldehyde) and Estimation of Interactions with Nicotine and Letrozole.

    PubMed

    Chan, Jeannine; Oshiro, Tyler; Thomas, Sarah; Higa, Allyson; Black, Stephen; Todorovic, Aleksandar; Elbarbry, Fawzy; Harrelson, John P

    2016-04-01

    Human exposure to trans-cinnamic aldehyde [t-CA; cinnamaldehyde; cinnamal; (E)-3-phenylprop-2-enal] is common through diet and through the use of cinnamon powder for diabetes and to provide flavor and scent in commercial products. We evaluated the likelihood of t-CA to influence metabolism by inhibition of P450 enzymes. IC50 values from recombinant enzymes indicated that an interaction is most probable for CYP2A6 (IC50 = 6.1 µM). t-CA was 10.5-fold more selective for human CYP2A6 than for CYP2E1; IC50 values for P450s 1A2, 2B6, 2C9, 2C19, 2D6, and 3A4 were 15.8-fold higher or more. t-CA is a type I ligand for CYP2A6 (KS = 14.9 µM). Inhibition of CYP2A6 by t-CA was metabolism-dependent; inhibition required NADPH and increased with time. Glutathione lessened the extent of inhibition modestly and statistically significantly. The carbon monoxide binding spectrum was dramatically diminished after exposure to NADPH and t-CA, suggesting degradation of the heme or CYP2A6 apoprotein. Using a static model and mechanism-based inhibition parameters (K(I) = 18.0 µM; k(inact) = 0.056 minute(-1)), changes in the area under the concentration-time curve (AUC) for nicotine and letrozole were predicted in the presence of t-CA (0.1 and 1 µM). The AUC fold-change ranged from 1.1 to 3.6. In summary, t-CA is a potential source of pharmacokinetic variability for CYP2A6 substrates due to metabolism-dependent inhibition, especially in scenarios when exposure to t-CA is elevated due to high dietary exposure, or when cinnamon is used as a treatment of specific disease states (e.g., diabetes). PMID:26851241

  4. Estradiol-17β, and its CYP450- and COMT-Derived Metabolites Stimulate Proliferation in Uterine Artery ECs: Role of ER-α vs. ER-β

    PubMed Central

    Jobe, Sheikh O.; Ramadoss, Jayanth; Koch, Jill M.; Jiang, Yizhou; Zheng, Jing; Magness, Ronald R

    2010-01-01

    Estradiol-17β and its metabolites which are sequentially synthesized by cytochrome P450s (CYP450s) and catechol-O-methyltransferase (COMT) to form 2 and 4-Hydroxyestradiol (2-OHE2 and 4-OHE2) and 2- and 4-Methoxestradiol (2-ME2, and 4-ME2) are elevated during pregnancy. We investigated whether CYP450s and COMT are expressed in uterine artery endothelial cells (UAECs) and if E2β and its metabolites modulate cell proliferation via ER-α and/or ER-β and play roles in physiologic uterine angiogenesis during pregnancy. Cultured ovine UAECs from pregnant (P-UAECs) and nonpregnant (NP-UAECs) ewes were treated with 0.1-100 nmol/L of E2β, 2-OHE2, 4-OHE2, 2-ME2, and 4-ME2. ER-α or ER-β specificity was tested using ICI 182,780, ER-α-specific MPP, ER-β –specific PHTPP antagonists and their respective agonists ER-α-specific PPT and ER-β –specific DPN. Angiogenesis was evaluated using BrdU Proliferation Assay. Utilizing confocal microscopy and Western analyses to determine enzyme location and levels, we observed CYP1A1, CYP1A2, CYP1B1, CYP3A4 and COMT expression in UAECs; however, expressions were similar between NP-UAECs and P-UAECs. E2β, 2-OHE2, 4-OHE2, and 4-ME2 treatments concentration-dependently stimulated proliferation in P-UAECs, but not NP-UAECs; 2-ME2 did not stimulate proliferation in either cell type. Proliferative responses of P-UAECs to E2β were solely mediated by ER-β, whereas responses to E2β metabolites were neither ER-α nor ER-β mediated. We demonstrate an important vascular role for E2β, its CYP450- and COMT-derived metabolites and ER-β in uterine angiogenesis regulation during pregnancy that may be dysfunctional in preeclampsia and other cardiovascular disorders. PMID:20212268

  5. Physiologically based pharmacokinetic modeling for sequential metabolism: effect of CYP2C19 genetic polymorphism on clopidogrel and clopidogrel active metabolite pharmacokinetics.

    PubMed

    Djebli, Nassim; Fabre, David; Boulenc, Xavier; Fabre, Gérard; Sultan, Eric; Hurbin, Fabrice

    2015-04-01

    Clopidogrel is a prodrug that needs to be converted to its active metabolite (clopi-H4) in two sequential cytochrome P450 (P450)-dependent steps. In the present study, a dynamic physiologically based pharmacokinetic (PBPK) model was developed in Simcyp for clopidogrel and clopi-H4 using a specific sequential metabolite module in four populations with phenotypically different CYP2C19 activity (poor, intermediate, extensive, and ultrarapid metabolizers) receiving a loading dose of 300 mg followed by a maintenance dose of 75 mg. This model was validated using several approaches. First, a comparison of predicted-to-observed area under the curve (AUC)0-24 obtained from a randomized crossover study conducted in four balanced CYP2C19-phenotype metabolizer groups was performed using a visual predictive check method. Second, the interindividual and intertrial variability (on the basis of AUC0-24 comparisons) between the predicted trials and the observed trial of individuals, for each phenotypic group, were compared. Finally, a further validation, on the basis of drug-drug-interaction prediction, was performed by comparing observed values of clopidogrel and clopi-H4 with or without dronedarone (moderate CYP3A4 inhibitor) coadministration using a previously developed and validated physiologically based PBPK dronedarone model. The PBPK model was well validated for both clopidogrel and its active metabolite clopi-H4, in each CYP2C19-phenotypic group, whatever the treatment period (300-mg loading dose and 75-mg last maintenance dose). This is the first study proposing a full dynamic PBPK model able to accurately predict simultaneously the pharmacokinetics of the parent drug and of its primary and secondary metabolites in populations with genetically different activity for a metabolizing enzyme.

  6. (3-Methyl-3a,4,7,7a-tetra­hydro-5H-4,7-methano­isoxazolo[4,5-d][1,2]oxazin-5-yl)(phen­yl)methanone

    PubMed Central

    Lough, Alan J.; Nagireddy, Jaipal R.; Tam, William

    2014-01-01

    The title compound, C14H14N2O3, is the exo isomer with a syn arrangement of two O atoms in the isoxazole and oxazine rings. The dihedral angle between the isoxazole and phenyl rings is 60.38 (4)°. In the crystal, weak C—H⋯O hydrogen bonds link the mol­ecules, forming a three-dimensional network. The isoxazole O atom is an acceptor for three of these hydrogen bonds. PMID:24860351

  7. 5,8-Dimeth­oxy-3,9-dimethyl-3a,4,9,9a-tetra­hydro-4,9-ep­oxy­naphtho­[2,3-d]isoxazole

    PubMed Central

    Lough, Alan J.; Nagireddy, Jaipal R.; Tam, William

    2014-01-01

    The title compound, C15H17NO4, is the exo isomer with a syn arrangement of the O atom in the isoxazole ring to the methyl group of the bicyclic alkene. The dihedral angle between the isoxazole ring and the benzene ring is 7.42 (9)°. In the crystal, weak C—H⋯O hydrogen bonds link mol­ecules, forming a three-dimensional network. The isoxazole O atom is an acceptor for both weak hydrogen bonds. PMID:24860352

  8. 8-Chloro­methyl-5-(2,5-dioxooxolan-3-yl)-3,3a,4,5-tetra­hydro-1H-naphtho­[1,2-c]furan-1,3-dione

    PubMed Central

    Guo, Y. Z.; Song, Y. Z.; Liu, J. G.; Yang, S. Y.

    2013-01-01

    The title compound, C17H13ClO6, is an asymmetric alicyclic dianhydride containing a chloro­methyl-substituted tetra­hydro­naphthalene moiety. The cyclo­hexene ring in the tetra­hydro­naphthalene moiety exhibits an envelope conformation with the tertiary C atom as the flap The dihedral angle between the two anhydride rings is 79.96 (6)°, while those between the benzene ring and the non-fused and fused anhydride rings are 71.03 (5) and 42.57 (7)°, respectively. In the crystal, mol­ecules are connected by weak C—H⋯O inter­actions, forming a three-dimensional supramolecular structure. PMID:24046610

  9. PROCEEDINGS: 1991 INTERNATIONAL CONFERENCE ON MUNICIPAL WASTE COMBUSTION - VOLUME 1. SESSIONS P, 0, 1A, 2A, 3A, 4A, 6A, 6B, 9C, AND 10B

    EPA Science Inventory

    The three-volumes document 82 presentations by authors from 15 countries at the Second International Conference on Municipal Waste Combustion (MWC) in Tampa, Florida, April 16-19, 1991. The Conference fostered the exchange of current information on research concerning MWC, ash di...

  10. 4-(4-Bromo-phen-yl)-2,3,3a,4,5,11c-hexa-hydro-benzo[f]furo[3,2-c]quinoline.

    PubMed

    Wu, Nan; Zhang, Rongli; Li, Xinnian; Xu, Xin; Xu, Zhou

    2011-09-01

    In the title compound, C(21)H(18)BrNO, both heterocyclic rings, viz. the hydro-pyridine ring and the adjacent hydro-furan ring, adopt envelope conformations. These two heterocycles make a dihedral angle of 37.3 (1)°. The dihedral angle between the hydro-pyridine and benzene rings is 69.6 (1)°. In the crystal, adjacent mol-ecules are linked by pairs of inter-molecular C-H⋯O hydrogen bonds, forming centrosymmetric dimers. PMID:22065710

  11. Effects of co-treatment with sulforaphane and autophagy modulators on uridine 5′-diphospho-glucuronosyltransferase 1A isoforms and cytochrome P450 3A4 expression in Caco-2 human colon cancer cells

    PubMed Central

    WANG, MIN; ZHU, JING-YU; CHEN, SHUO; QING, YING; WU, DONG; LIN, YING-MIN; LUO, JI-ZHUANG; HAN, WEI; LI, YAN-QING

    2014-01-01

    Sulforaphane (SFN), which is highly enriched in cruciferous vegetables, has been investigated for its cancer chemopreventive properties and ability to induce autophagy. Uridine 5′-diphospho (UDP)-glucuronosyltransferase (UGT)1A induction is one of the mechanisms that is responsible for the cancer chemopreventive activity of SFN. The current study demonstrates that rapamycin may enhance the chemopreventive effects of SFN on Caco-2 cells; this may be partially attributed to nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2)- and human pregnane X receptor (hPXR)-mediated UGT1A1, UGT1A8 and UGT1A10 induction. These results indicate that targeting autophagy modulation may be a promising strategy for increasing the chemopreventive effects of SFN in cases of colon cancer. PMID:25364403

  12. Crystal structure of methyl 3-(3-fluoro-phen-yl)-1-methyl-1,3a,4,9b-tetra-hydro-3H-thio-chromeno[4,3-c]isoxazole-3a-carboxyl-ate.

    PubMed

    Savithri, M P; Suresh, M; Raghunathan, R; Vimala, G; SubbiahPandi, A

    2015-08-01

    In the title compound, C19H18FNO3S, the five-membered oxazolidine ring adopts an envelope conformation with the methine C atom of the fused bond as the flap. Its mean plane is oriented at a dihedral angle of 50.38 (1)° with respect to the fluoro-phenyl ring. The six-membered thio-pyran ring has a half-chair conformation and its mean plane is almost coplanar with the fused benzene ring, making a dihedral angle of 4.94 (10)°. The two aromatic rings are inclined to one another by 85.96 (11)°, and the mean planes of the oxazolidine and thio-pyran rings are inclined to one another by 57.64 (12)°. In the crystal, mol-ecules are linked by C-H⋯π inter-actions, forming a three-dimensional structure. PMID:26396818

  13. Effects of co-treatment with sulforaphane and autophagy modulators on uridine 5'-diphospho-glucuronosyltransferase 1A isoforms and cytochrome P450 3A4 expression in Caco-2 human colon cancer cells.

    PubMed

    Wang, Min; Zhu, Jing-Yu; Chen, Shuo; Qing, Ying; Wu, Dong; Lin, Ying-Min; Luo, Ji-Zhuang; Han, Wei; Li, Yan-Qing

    2014-12-01

    Sulforaphane (SFN), which is highly enriched in cruciferous vegetables, has been investigated for its cancer chemopreventive properties and ability to induce autophagy. Uridine 5'-diphospho (UDP)-glucuronosyltransferase (UGT)1A induction is one of the mechanisms that is responsible for the cancer chemopreventive activity of SFN. The current study demonstrates that rapamycin may enhance the chemopreventive effects of SFN on Caco-2 cells; this may be partially attributed to nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2)- and human pregnane X receptor (hPXR)-mediated UGT1A1, UGT1A8 and UGT1A10 induction. These results indicate that targeting autophagy modulation may be a promising strategy for increasing the chemopreventive effects of SFN in cases of colon cancer. PMID:25364403

  14. Analysis of genetic variations in CYP2C9, CYP2C19, CYP2D6 and CYP3A5 genes using oligonucleotide microarray

    PubMed Central

    Dong, Yuanyuan; Xiao, Huasheng; Wang, Qi; Zhang, Chunxiu; Liu, Xiuming; Yao, Na; Sheng, Haihui; Li, Haiyan

    2015-01-01

    The cytochrome P450 enzymes play a critical role in the metabolism of many commonly prescribed drugs. Among them, the most important enzymes are highly polymorphic CYP2C9, CYP2C19, CYP2D6 and CYP3A5, which are responsible for about 40% of the metabolism of clinical used drugs. Here we developed a novel CYP450 oligonucleotide microarray that allow for detection of 32 known variations of CYP genes from a single multiplex reaction, including 19 polymorphisms of CYP2D6 gene, 8 polymorphisms of CYP2C9 gene, 4 polymorphisms of CYP2C19 gene and 1 polymorphism of CYP3A5 gene. 229 genomic DNA samples from unrelated Han subjects were analyzed. The microarray results showed to have high call rate and accuracy according to concordance with genotypes identified by independent bidirectional sequencing. Furthermore, we found that the major CYP2C9, CYP2C19, CYP2D6 and CYP3A5 alleles in Chinese Han population were CYP2C9*3 (allelic frequency of 10.7%), CYP2C9*2 (20.31%), CYP2C19*2 (5.68%), CYP2D6*10 (58.52%), CYP2D6*2 (13.76) and CYP3A5*3 (70.69%). With flexible DNA preparation, the microarray can significantly facilitates the process of detecting genetics variations in CYP2C9, CYP2C19, CYP2D6 and CYP3A5 gene and provide safe and effective therapy for individual patients. PMID:26770516

  15. In vitro inhibitory effects of asiaticoside and madecassoside on human cytochrome P450.

    PubMed

    Winitthana, T; Niwattisaiwong, N; Patarapanich, C; Tantisira, M H; Lawanprasert, S

    2011-06-01

    The inhibitory effects and types of inhibition of asiaticoside and madecassoside on human CYPs were studied in vitro using recombinant human CYPs. The median inhibitory concentrations (IC50) of asiaticoside and madecassoside were determined for CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4. Asiaticoside inhibited CYP2C19 (IC50 = 412.68 ± 15.44 μM) and CYP3A4 (IC50=343.35 ± 29.35 μM). Madecassoside also inhibited CYP2C19 (IC50 = 539.04 ± 14.18 μM) and CYP3A4 (IC50 = 453.32 ± 39.33 μM). Asiaticoside and madecassoside had no effect on the activities of CYP1A2, CYP2C9 and CYP2D6 and CYP2E1. Assessment of mechanism-based inhibition and the type of inhibition were performed for asiaticoside and madecassoside with CYP2C19 and CYP3A4. These results suggested that madecassoside is a mechanism-based inhibitor of CYP2C19 and CYP3A4. Assessment of mechanism-based inhibition by asiaticoside was limited by its low solubility. Asiaticoside exhibited non-competitive inhibition of CYP2C19 (Ki=385.24 ± 8.75 μM) and CYP3A4 (Ki = 535.93 ± 18.99 μM). Madecassoside also showed non-competitive inhibition of CYP2C19 (Ki = 109.62 ± 6.14 μM) and CYP3A4 (Ki = 456.84 ± 16.43 μM). These results suggest that asiaticoside and madecassoside could cause drug-drug interactions via inhibition of CYP2C19 and CYP3A4. An in vivo study is needed to examine this further.

  16. CYP21-catalyzed production of the long-term urinary metandienone metabolite 17beta-hydroxymethyl-17 alpha-methyl-18-norandrosta-1,4,13-trien-3-one: a contribution to the fight against doping.

    PubMed

    Zöllner, Andy; Parr, Maria Kristina; Drăgan, Călin-Aurel; Dräs, Stefan; Schlörer, Nils; Peters, Frank T; Maurer, Hans H; Schänzer, Wilhelm; Bureik, Matthias

    2010-01-01

    Anabolic-androgenic steroids are some of the most frequently misused drugs in human sports. Recently, a previously unknown urinary metabolite of metandienone, 17beta-hydroxymethyl-17 alpha-methyl-18-norandrosta-1,4,13-trien-3-one (20OH-NorMD), was discovered via LC-MS/MS and GC-MS. This metabolite was reported to be detected in urine samples up to 19 days after administration of metandienone. However, so far it was not possible to obtain purified reference material of this metabolite and to confirm its structure via NMR. Eleven recombinant strains of the fission yeast Schizosaccharomyces pombe that express different human hepatic or steroidogenic cytochrome P450 enzymes were screened for production of this metabolite in a whole-cell biotransformation reaction. 17,17-Dimethyl-18-norandrosta-1,4,13-trien-3-one, chemically derived from metandienone, was used as substrate for the bioconversion, because it could be converted to the final product in a single hydroxylation step. The obtained results demonstrate that CYP21 and to a lesser extent also CYP3A4 expressing strains can catalyze this steroid hydroxylation. Subsequent 5 l-scale fermentation resulted in the production and purification of 10 mg of metabolite and its unequivocal structure determination via NMR. The synthesis of this urinary metandienone metabolite via S. pombe-based whole-cell biotransformation now allows its use as a reference substance in doping control assays. PMID:19919183

  17. CYP2S1: A short review

    SciTech Connect

    Saarikoski, Sirkku T. . E-mail: sirkku.saarikoski@ktl.fi; Rivera, Steven P.; Hankinson, Oliver; Husgafvel-Pursiainen, Kirsti

    2005-09-01

    A new member of the cytochrome P450 superfamily, CYP2S1, has recently been identified in human and mouse. In this paper, we review the data currently available for CYP2S1. The human CYP2S1 gene is located in chromosome 19q13.2 within a cluster including CYP2 family members CYP2A6, CYP2A13, CYP2B6, and CYP2F1. These genes also show the highest homology to the human CYP2S1. The gene has recently been found to harbor genetic polymorphism. CYP2S1 is inducible by dioxin, the induction being mediated by the Aryl Hydrocarbon Receptor (AHR) and Aryl Hydrocarbon Nuclear Translocator (ARNT) in a manner typical for CYP1 family members. In line with this, CYP2S1 has been shown to be inducible by coal tar, an abundant source of PAHs, and it was recently reported to metabolize naphthalene. This points to the involvement of CYP2S1 in the metabolism of toxic and carcinogenic compounds, similar to other dioxin-inducible CYPs. CYP2S1 is expressed in epithelial cells of a wide variety of extrahepatic tissues. The highest expression levels have been observed in the epithelial tissues frequently exposed to xenobiotics, e.g., the respiratory, gastrointestinal, and urinary tracts, and in the skin. The observed ubiquitous tissue distribution, as well as the expression of CYP2S1 throughout embryogenesis suggest that CYP2S1 is likely to metabolize important endogenous substrates; thus far, retinoic acid has been identified. In conclusion, CYP2S1 exhibits many features of interest for human health and thus warrants further investigation.

  18. Study to Evaluate the Effect of Rifampicin, Ketoconazole, and Omeprazole on the Pharmacokinetics of Sativex

    ClinicalTrials.gov

    2013-06-13

    Evaluation of Pharmacokinetics of Sativex in the Absence and Presence of a Known Inducer of CYP3A4; Evaluation of Pharmacokinetics of Sativex in the Absence and Presence of a Potent Inhibitor of CYP3A4; Evaluation of Pharmacokinetics of Sativex in the Absence and Presence of a CYP2C19 Inhibitor.

  19. Cocktail approach for in vivo phenotyping of 5 major CYP450 isoenzymes: development of an effective sampling, extraction, and analytical procedure and pilot study with comparative genotyping.

    PubMed

    Wohlfarth, Ariane; Naue, Jana; Lutz-Bonengel, Sabine; Dresen, Sebastian; Auwärter, Volker

    2012-08-01

    In this study, the authors developed a phenotyping method for CYP1A2, 2C9, 2C19, 2D6, and 3A4 using a cocktail of 100 mg caffeine, 125 mg tolbutamide, 20 mg omeprazole, 30 mg dextromethorphan, and 2 mg midazolam. A simple sampling scheme was established collecting 3 blood samples at 0, 4, and 24 hours followed by solid-phase extraction and liquid chromatography/tandem mass spectrometry analysis. After addition of 8 deuterated internal standards and extraction, the analytes were separated using gradient elution with ammonium acetate and methanol. Data acquisition was performed on a triple quadrupole linear ion trap mass spectrometer in multiple-reaction monitoring mode with positive electrospray ionization. The assay was validated according to international guidelines: limits of quantification (LOQs) were between 0.25 and 1.0 ng/mL for all analytes, except for paraxanthine and caffeine (20 ng/mL). Extraction efficiencies ranged between 77% and 103% and matrix effects between 23% and 95%; precision and accuracy data fulfilled accepted criteria. Calibration curves from LOQ to 1000 ng/mL were established for undiluted and 1:10 diluted plasma (r > 0.998). The method was tested in a pilot study with 14 volunteers. Additional genotyping of the probands generally demonstrated good accordance with the measured phenotyping indices but also disclosed certain contradictory results.

  20. A comparative study of the CYP450 inhibition potential of marketed drugs using two fluorescence based assay platforms routinely used in the pharmaceutical industry.

    PubMed

    Kajbaf, Mahmud; Longhi, Raffaele; Montanari, Dino; Vinco, Federica; Rigo, Monica; Fontana, Stefano; Read, Kevin D

    2011-01-01

    Semi-automated high throughput screening for the inhibition of major human cytochrome P450 enzymes (1A2, 2C9, 2C19, 2D6 and 3A4) expressed in Escherichia Coli (Cypex bactosomes) or human lymphoblastoid cells (Gentest cDNA microsomes) using fluorescent probes has been evaluated using 68 marketed drugs. In general lower IC50 values were obtained with Cypex bactosomes compared with Gentest cDNA microsomes. This could be due to use of higherconcentration of protein and also the lower activity of Gentest cDNA microsomes. Notably, when compared with in vivo clinical drug-drug interactions (cDDIs) gathered from clinical studies reported in the scientific literature Cypex bactosome data was better at predicting in vivo cDDI. Consequently, from the data obtained in this comparative study, a fluorescence based assay using Cypex bactosomes is more suitable as a front-line screen for the prediction of potential downstream CYP450 driven cDDIs.

  1. Inhibition of human cytochrome P450 enzymes by the natural hepatotoxin safrole.

    PubMed

    Ueng, Yune-Fang; Hsieh, Chih-Hang; Don, Ming-Jaw

    2005-05-01

    The hepatotoxin, safrole is a methylenedioxy phenyl compound, found in sassafras oil and certain other essential oils. Recombinant cytochrome P450 (CYP, P450) and human liver microsomes were studied to investigate the selective inhibitory effects of safrole on human P450 enzymes and the mechanisms of action. Using Escherichia coli-expressed human P450, our results demonstrated that safrole was a non-selective inhibitor of CYP1A2, CYP2A6, CYP2D6, CYP2E1, and CYP3A4 in the IC(50) order CYP2E1 < CYP1A2 < CYP2A6 < CYP3A4 < CYP2D6. Safrole strongly inhibited CYP1A2, CYP2A6, and CYP2E1 activities with IC(50) values less than 20 microM. Safrole caused competitive, non-competitive, and non-competitive inhibition of CYP1A2, CYP2A6 and CYP2E1 activities, respectively. The inhibitor constants were in the order CYP1A2 < CYP2E1 < CYP2A6. In human liver microsomes, 50 microM safrole strongly inhibited 7-ethoxyresorufin O-deethylation, coumarin hydroxylation, and chlorzoxazone hydroxylation activities. These results revealed that safrole was a potent inhibitor of human CYP1A2, CYP2A6, and CYP2E1. With relatively less potency, CYP2D6 and CYP3A4 were also inhibited.

  2. Global variation in CYP2C8–CYP2C9 functional haplotypes

    PubMed Central

    Speed, William C; Kang, Soonmo Peter; Tuck, David P; Harris, Lyndsay N; Kidd, Kenneth K

    2009-01-01

    We have studied the global frequency distributions of 10 single nucleotide polymorphisms (SNPs) across 132 kb of CYP2C8 and CYP2C9 in ∼2500 individuals representing 45 populations. Five of the SNPs were in noncoding sequences; the other five involved the more common missense variants (four in CYP2C8, one in CYP2C9) that change amino acids in the gene products. One haplotype containing two CYP2C8 coding variants and one CYP2C9 coding variant reaches an average frequency of 10% in Europe; a set of haplotypes with a different CYP2C8 coding variant reaches 17% in Africa. In both cases these haplotypes are found in other regions of the world at <1%. This considerable geographic variation in haplotype frequencies impacts the interpretation of CYP2C8/CYP2C9 association studies, and has pharmacogenomic implications for drug interactions. PMID:19381162

  3. In vitro evaluation of cytochrome P450 induction and the inhibition potential of mitragynine, a stimulant alkaloid.

    PubMed

    Lim, Ee Lin; Seah, Tiong Chai; Koe, Xue Fen; Wahab, Habibah Abdul; Adenan, Mohd Ilham; Jamil, Mohd Fadzly Amar; Majid, Mohamed Isa Abdul; Tan, Mei Lan

    2013-03-01

    CYP450 enzymes are key determinants in drug toxicities, reduced pharmacological effect and adverse drug reactions. Mitragynine, an euphoric compound was evaluated for its effects on the expression of mRNAs encoding CYP1A2, CYP2D6 and CYP3A4 and protein expression and resultant enzymatic activity. The mRNA and protein expression of CYP450 isoforms were carried out using an optimized multiplex qRT-PCR assay and Western blot analysis. CYP1A2 and CYP3A4 enzyme activities were evaluated using P450-Glo™ assays. The effects of mitragynine on human CYP3A4 protein expression were determined using an optimized hCYP3A4-HepG2 cell-based assay. An in silico computational method to predict the binding conformation of mitragynine to the active site of the CYP3A4 enzyme was performed and further validated using in vitro CYP3A4 inhibition assays. Mitragynine was found to induce mRNA and protein expression of CYP1A2. For the highest concentration of 25 μM, induction of mRNA was approximately 70% that of the positive control and was consistent with the increased CYP1A2 enzymatic activity. Thus, mitragynine is a significant in vitro CYP1A2 inducer. However, it appeared to be a weak CYP3A4 inducer at the transcriptional level and a weak CYP3A4 enzyme inhibitor. It is therefore, unlikely to have any significant clinical effects on CYP3A4 activity. PMID:23274770

  4. Prediction of Drug-Drug Interactions Arising from CYP3A induction Using a Physiologically Based Dynamic Model.

    PubMed

    Almond, Lisa M; Mukadam, Sophie; Gardner, Iain; Okialda, Krystle; Wong, Susan; Hatley, Oliver; Tay, Suzanne; Rowland-Yeo, Karen; Jamei, Masoud; Rostami-Hodjegan, Amin; Kenny, Jane R

    2016-06-01

    Using physiologically based pharmacokinetic modeling, we predicted the magnitude of drug-drug interactions (DDIs) for studies with rifampicin and seven CYP3A4 probe substrates administered i.v. (10 studies) or orally (19 studies). The results showed a tendency to underpredict the DDI magnitude when the victim drug was administered orally. Possible sources of inaccuracy were investigated systematically to determine the most appropriate model refinement. When the maximal fold induction (Indmax) for rifampicin was increased (from 8 to 16) in both the liver and the gut, or when the Indmax was increased in the gut but not in liver, there was a decrease in bias and increased precision compared with the base model (Indmax = 8) [geometric mean fold error (GMFE) 2.12 vs. 1.48 and 1.77, respectively]. Induction parameters (mRNA and activity), determined for rifampicin, carbamazepine, phenytoin, and phenobarbital in hepatocytes from four donors, were then used to evaluate use of the refined rifampicin model for calibration. Calibration of mRNA and activity data for other inducers using the refined rifampicin model led to more accurate DDI predictions compared with the initial model (activity GMFE 1.49 vs. 1.68; mRNA GMFE 1.35 vs. 1.46), suggesting that robust in vivo reference values can be used to overcome interdonor and laboratory-to-laboratory variability. Use of uncalibrated data also performed well (GMFE 1.39 and 1.44 for activity and mRNA). As a result of experimental variability (i.e., in donors and protocols), it is prudent to fully characterize in vitro induction with prototypical inducers to give an understanding of how that particular system extrapolates to the in vivo situation when using an uncalibrated approach. PMID:27026679

  5. Prediction of Drug-Drug Interactions Arising from CYP3A induction Using a Physiologically Based Dynamic Model

    PubMed Central

    Mukadam, Sophie; Gardner, Iain; Okialda, Krystle; Wong, Susan; Hatley, Oliver; Tay, Suzanne; Rowland-Yeo, Karen; Jamei, Masoud; Rostami-Hodjegan, Amin; Kenny, Jane R.

    2016-01-01

    Using physiologically based pharmacokinetic modeling, we predicted the magnitude of drug-drug interactions (DDIs) for studies with rifampicin and seven CYP3A4 probe substrates administered i.v. (10 studies) or orally (19 studies). The results showed a tendency to underpredict the DDI magnitude when the victim drug was administered orally. Possible sources of inaccuracy were investigated systematically to determine the most appropriate model refinement. When the maximal fold induction (Indmax) for rifampicin was increased (from 8 to 16) in both the liver and the gut, or when the Indmax was increased in the gut but not in liver, there was a decrease in bias and increased precision compared with the base model (Indmax = 8) [geometric mean fold error (GMFE) 2.12 vs. 1.48 and 1.77, respectively]. Induction parameters (mRNA and activity), determined for rifampicin, carbamazepine, phenytoin, and phenobarbital in hepatocytes from four donors, were then used to evaluate use of the refined rifampicin model for calibration. Calibration of mRNA and activity data for other inducers using the refined rifampicin model led to more accurate DDI predictions compared with the initial model (activity GMFE 1.49 vs. 1.68; mRNA GMFE 1.35 vs. 1.46), suggesting that robust in vivo reference values can be used to overcome interdonor and laboratory-to-laboratory variability. Use of uncalibrated data also performed well (GMFE 1.39 and 1.44 for activity and mRNA). As a result of experimental variability (i.e., in donors and protocols), it is prudent to fully characterize in vitro induction with prototypical inducers to give an understanding of how that particular system extrapolates to the in vivo situation when using an uncalibrated approach. PMID:27026679

  6. Identification of novel substrates for human cytochrome P450 2J2.

    PubMed

    Lee, Caroline A; Neul, David; Clouser-Roche, Andrea; Dalvie, Deepak; Wester, Michael R; Jiang, Ying; Jones, J P; Freiwald, Sascha; Zientek, Michael; Totah, Rheem A

    2010-02-01

    Several antihistamine drugs including terfenadine, ebastine, and astemizole have been identified as substrates for CYP2J2. The overall importance of this enzyme in drug metabolism has not been fully explored. In this study, 139 marketed therapeutic agents and compounds were screened as potential CYP2J2 substrates. Eight novel substrates were identified that vary in size and overall topology from relatively rigid structures (amiodarone) to larger complex structures (cyclosporine). The substrates displayed in vitro intrinsic clearance values ranging from 0.06 to 3.98 mul/min/pmol CYP2J2. Substrates identified for CYP2J2 are also metabolized by CYP3A4. Extracted ion chromatograms of metabolites observed for albendazole, amiodarone, astemizole, thioridazine, mesoridazine, and danazol showed marked differences in the regioselectivity of CYP2J2 and CYP3A4. CYP3A4 commonly metabolized compounds at multiple sites, whereas CYP2J2 metabolism was more restrictive and limited, in general, to a single site for large compounds. Although the CYP2J2 active site can accommodate large substrates, it may be more narrow than CYP3A4, limiting metabolism to moieties that can extend closer toward the active heme iron. For albendazole, CYP2J2 forms a unique metabolite compared with CYP3A4. Albendazole and amiodarone were evaluated in various in vitro systems including recombinant CYP2J2 and CYP3A4, pooled human liver microsomes (HLM), and human intestinal microsomes (HIM). The Michaelis-Menten-derived intrinsic clearance of N-desethyl amiodarone was 4.6 greater in HLM than in HIM and 17-fold greater in recombinant CYP3A4 than in recombinant CYP2J2. The resulting data suggest that CYP2J2 may be an unrecognized participant in first-pass metabolism, but its contribution is minor relative to that of CYP3A4. PMID:19923256

  7. Pungent ginger components modulates human cytochrome P450 enzymes in vitro

    PubMed Central

    Li, Mian; Chen, Pei-zhan; Yue, Qing-xi; Li, Jing-quan; Chu, Rui-ai; Zhang, Wei; Wang, Hui

    2013-01-01

    Aim: Ginger rhizome is used worldwide as a spicy flavor agent. This study was designed to explore the potential effects of pungent ginger components, 6-, 8-, and 10-gingerol, on human cytochrome P450 (CYP450) enzymes that are responsible for the metabolism of many prescription drugs. Methods: The activities of human CYP2C9, CYP2C19, CYP2D6, and CYP3A4 were analyzed using Vivid P450 assay kits. The mRNA expression of CYP3A4 in human hepatocellular carcinoma cell line HepG2 was measured using quantitative real-time PCR assay. Results: All three gingerols potently inhibited CYP2C9 activity, exerted moderate inhibition on CYP2C19 and CYP3A4, and weak inhibion on CYP2D6. 8-Gingerol was the most potent in inhibition of P450 enzymes with IC50 values of 6.8, 12.5, 8.7, and 42.7 μmol/L for CYP2C9, CYP2C19, CYP3A4, and CYP2D6, respectively. By comparing the effects of gingerols on CYP3A4 with three different fluorescent substrate probes, it was demonstrated that the inhibition of gingerols on CYP3A4 had no substrate-dependence. In HepG2 cells, 8-gingerol and 10-gingerol inhibited, but 6-gingerol induced mRNA expression of CYP3A4. Conclusion: 6-, 8-, and 10-gingerol suppress human cytochrome P450 activity, while 8- and 10-gingerol inhibit CYP3A4 expression. The results may have an implication for the use of ginger or ginger products when combined with therapeutic drugs that are metabolized by cytochrome P450 enzymes. PMID:23770984

  8. Simultaneous in vivo phenotyping of CYP enzymes.

    PubMed

    Ghassabian, Sussan; Murray, Michael

    2013-01-01

    As major determinants of the duration of drug action the CYP enzymes strongly influence drug efficacy and toxicity. In vivo phenotyping for CYP activities using cocktails of well-tolerated CYP-specific substrates may be valuable in the development of personalized medicine protocols, particularly for drugs that have significant toxicity profiles. However, the use of the cocktail approach in the clinic is dependent on the rapid provision of patient-specific information to the clinician. Here we describe the application of liquid chromatography-tandem mass spectrometry (LC-MS-MS) for the simultaneous phenotyping of five major drug-metabolizing CYPs in patients within a 5-min assay.

  9. Combination Analysis in Genetic Polymorphisms of Drug-Metabolizing Enzymes CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A5 in the Japanese Population

    PubMed Central

    Ota, Tomoko; Kamada, Yuka; Hayashida, Mariko; Iwao-Koizumi, Kyoko; Murata, Shigenori; Kinoshita, Kenji

    2015-01-01

    The Cytochrome P450 is the major enzyme involved in drug metabolism. CYP enzymes are responsible for the metabolism of most clinically used drugs. Individual variability in CYP activity is one important factor that contributes to drug therapy failure. We have developed a new straightforward TaqMan PCR genotyping assay to investigate the prevalence of the most common allelic variants of polymorphic CYP enzymes CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A5 in the Japanese population. Moreover, we focused on the combination of each genotype for clinical treatment. The genotype analysis identified a total of 139 out of 483 genotype combinations of five genes in the 1,003 Japanese subjects. According to our results, most of subjects seemed to require dose modification during clinical treatment. In the near future, modifications should be considered based on the individual patient genotype of each treatment. PMID:25552922

  10. Vitamin D Enhances the Efficacy of Irinotecan through miR-627-Mediated Inhibition of Intratumoral Drug Metabolism.

    PubMed

    Sun, Meiyan; Zhang, Qunshu; Yang, Xiaoyu; Qian, Steven Y; Guo, Bin

    2016-09-01

    Cytochrome P450 enzyme CYP3A4 is an important drug-metabolizing enzyme, and high levels of tumoral expression of CYP3A4 are linked to drug resistance. We investigated the function of vitamin D-regulated miR-627 in intratumoral CYP3A4 suppression and its role in enhancing the efficacy of chemotherapy. We found that miR-627 targets CYP3A4 and suppresses CYP3A4 expression in colon cancer cell lines. Furthermore, calcitriol (the active form of vitamin D) suppressed CYP3A4 expression by activating miR-627. As a result, calcitriol inhibited CYP3A4-mediated metabolism of irinotecan (a topoisomerase I inhibitor) in cancer cells. We show that calcitriol enhanced the efficacy of irinotecan in growth inhibition and apoptosis induction. When miR-627 is inhibited, calcitriol fails to enhance the activity of irinotecan. In addition, overexpression of miR-627 or siRNA knockdown of CYP3A4 enhanced the efficacy of irinotecan in growth inhibition and apoptosis induction. In contrast, overexpression of CYP3A4 abolished the effects of calcitriol on the activity of irinotecan. Using a nude mouse xenograft model, we demonstrated that calcitriol inhibited CYP3A4 and enhanced the in vivo antitumor activity of irinotecan without causing side effects. Our study identified a novel target for improving cancer therapy, i.e., modulating the intratumoral CYP3A4-mediated drug metabolism with vitamin D. This strategy could enhance the therapeutic efficacy without eliciting the side effects. Mol Cancer Ther; 15(9); 2086-95. ©2016 AACR. PMID:27458137

  11. Thiomethylstilbenes as inhibitors of CYP1A1, CYP1A2 and CYP1B1 activities.

    PubMed

    Mikstacka, Renata; Baer-Dubowska, Wanda; Wieczorek, Marcin; Sobiak, Stanislaw

    2008-06-01

    Resveratrol (3,5,4'-trihydroxy-trans-stilbene) is a natural stilbene derivative occurring in grapes, peanuts and red wine. Its chemopreventive action has been established in studies on animal models. Recently, numerous classes of compounds with stilbene backbone have been investigated for their biological activity concerning cancer prevention; e. g. resveratrol methyl ethers appeared to be specific and potent inhibitors of cytochromes P450 (CYP) family 1 involved in the activation of procarcinogens. Since the replacement of the 4'-hydroxyl with a thiomethyl group is supposed to reduce toxicity of stilbene derivatives, the purpose of this study was the synthesis and evaluation of a series of 4-thiomethyl-trans-stilbene derivatives differing in a number and position of additional methoxy groups. Their inhibitory potency toward human recombinant CYPs: CYP1A1, CYP1A2 and CYP1B1 have been studied and compared with the effect of resveratrol and its analogues. Among compounds tested, 2-methoxy-4'-thiomethyl-trans-stilbene and 3-methoxy-4'-thiomethyl-trans-stilbene demonstrated the most potent and selective inhibitory effect on CYP1A1 and CYP1B1 activities. The results of our study indicate that modification of stilbene derivatives with thiomethyl group may influence the selectivity and inhibitory potency of these compounds toward P450 isozymes. Thus, it should be considered in developing new chemopreventive agents based on their mechanism of action.

  12. Characterization of the cytochrome P450 enzymes involved in the metabolism of a new cardioprotective agent KR-33028.

    PubMed

    Kim, Hyojin; Yoon, Yune-Jung; Kim, Hyunmi; Kang, Suil; Cheon, Hyae Gyeong; Yoo, Sung-Eun; Shin, Jae-Gook; Liu, Kwang-Hyeon

    2006-10-10

    KR-33028 (N-[4-cyano-benzo[b]thiophene-2-carbonyl]guanidine) is a new cardioprotective agent for preventing ischemia-reperfusion injury. This study was performed to characterize the cytochrome P450 (CYP) enzymes that are involved in the metabolism of KR-33028. Hydroxylation (5-hydroxy- and 7-hydroxy-KR-33028) is major pathways for the metabolism of KR-33028 in human liver microsomes. Among the nine c-DNA expressed CYP isoforms tested, KR-33028 was 5-hydroxylated by CYP3A4 and 7-hydroxylated by CYP1A2, CYP3A4, and CYP2C19. These findings were supported by the combination of chemical inhibition studies in human liver microsomes and correlation analysis. Furafylline and ketoconazole potently inhibited hydroxylation of KR-33028 in human liver microsomes. Correlation analysis between the known CYP enzyme activities and the rates of the formation of 5-hydroxy- and 7-hydroxy-KR-33028 in the 16 human liver microsomes has showed significant correlations with CYP3A4-mediated midazolam 1'-hydroxylation and CYP1A2-mediated phenacetin O-deethylation, respectively. A 7-hydroxy-KR-33028 formation is also weakly correlated with CYP3A4-mediated midazolam 1'-hydroxylation. The kinetics of the major biotransformation of KR-33028 were studied: CYP3A4 mediated the formation of 5-hydroxy-KR-33028 from KR-33028 with Cl(int)=0.22microl/min/pmol CYP. The intrinsic clearance for 7-hydroxy-KR-33028 formation by CYP1A2, CYP2C19, and CYP3A4 were 0.26, 0.19, and 0.03microl/min/pmol CYP, respectively. Taken together, these results provide evidence that CYP3A4 and CYP1A2 are the major isoforms responsible for the hydroxy metabolites formation from KR-33028.

  13. Dynamics of CYP51: implications for function and inhibitor design

    PubMed Central

    Yu, Xiaofeng; Cojocaru, Vlad; Mustafa, Ghulam; Salo-Ahen, Outi M. H.; Lepesheva, Galina I.; Wade, Rebecca C.

    2015-01-01

    Sterol 14α-demethylase (cytochrome P450 family 51 (CYP51)) is an essential enzyme occurring in all biological kingdoms. In eukaryotes, it is located in the membrane of the endoplasmic reticulum. Selective inhibitors of trypanosomal CYP51s that do not affect the human CYP51 have been discovered in vitro and found to cure acute and chronic mouse Chagas disease without severe side effects in vivo. Crystal structures indicate that CYP51 may be more rigid than most CYPs, and it has been proposed that this property may facilitate antiparasitic drug design. Therefore, to investigate the dynamics of trypanosomal CYP51, we built a model of membrane-bound Trypanosoma brucei CYP51 and then performed molecular dynamics simulations of T. brucei CYP51 in membrane-bound and soluble forms. We compared the dynamics of T. brucei CYP51 with those of human CYP51, CYP2C9, and CYP2E1. In the simulations, the CYP51s display low mobility in the buried active site although overall mobility is similar in all the CYPs studied. The simulations suggest that in CYP51, pathway 2f serves as the major ligand access tunnel, and both pathways 2f (leading to membrane) and S (leading to solvent) can serve as ligand egress tunnels. Compared with the other CYPs, the residues at the entrance of the ligand access tunnels in CYP51 have higher mobility that may be necessary to facilitate the passage of its large sterol ligands. The water (W) tunnel is accessible to solvent during most of the simulations of CYP51, but its width is affected by the conformations of the heme's two propionate groups. These differ from those observed in the other CYPs studied because of differences in their hydrogen-bonding network. Our simulations give insights into the dynamics of CYP51 that complement the available experimental data and have implications for drug design against CYP51 enzymes. PMID:25601796

  14. VX-509 (Decernotinib)-Mediated CYP3A Time-Dependent Inhibition: An Aldehyde Oxidase Metabolite as a Perpetrator of Drug-Drug Interactions.

    PubMed

    Zetterberg, Craig; Maltais, Francois; Laitinen, Leena; Liao, Shengkai; Tsao, Hong; Chakilam, Ananthsrinivas; Hariparsad, Niresh

    2016-08-01

    (R)-2-((2-(1H-pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-4-yl)amino)-2-methyl-N-(2,2,2-trifluoroethyl)butanamide (VX-509, decernotinib) is an oral Janus kinase 3 inhibitor that has been studied in patients with rheumatoid arthritis. Patients with rheumatoid arthritis often receive multiple medications, such as statins and steroids, to manage the signs and symptoms of comorbidities, which increases the chances of drug-drug interactions (DDIs). Mechanism-based inhibition is a subset of time-dependent inhibition (TDI) and occurs when a molecule forms a reactive metabolite which irreversibly binds and inactivates drug-metabolizing enzymes, potentially increasing the systemic load to toxic concentrations. Traditionally, perpetrating compounds are screened using human liver microsomes (HLMs); however, this system may be inadequate when the precipitant is activated by a non-cytochrome P450 (P450)-mediated pathway. Even though studies assessing competitive inhibition and TDI using HLM suggested a low risk for CYP3A4-mediated DDI in the clinic, VX-509 increased the area under the curve of midazolam, atorvastatin, and methyl-prednisolone by approximately 12.0-, 2.7-, and 4.3-fold, respectively. Metabolite identification studies using human liver cytosol indicated that VX-509 is converted to an oxidative metabolite, which is the perpetrator of the DDIs observed in the clinic. As opposed to HLM, hepatocytes contain the full complement of drug-metabolizing enzymes and transporters and can be used to assess TDI arising from non-P450-mediated metabolic pathways. In the current study, we highlight the role of aldehyde oxidase in the formation of the hydroxyl-metabolite of VX-509, which is involved in clinically significant TDI-based DDIs and represents an additional example in which a system-dependent prediction of TDI would be evident.

  15. VX-509 (Decernotinib)-Mediated CYP3A Time-Dependent Inhibition: An Aldehyde Oxidase Metabolite as a Perpetrator of Drug-Drug Interactions.

    PubMed

    Zetterberg, Craig; Maltais, Francois; Laitinen, Leena; Liao, Shengkai; Tsao, Hong; Chakilam, Ananthsrinivas; Hariparsad, Niresh

    2016-08-01

    (R)-2-((2-(1H-pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-4-yl)amino)-2-methyl-N-(2,2,2-trifluoroethyl)butanamide (VX-509, decernotinib) is an oral Janus kinase 3 inhibitor that has been studied in patients with rheumatoid arthritis. Patients with rheumatoid arthritis often receive multiple medications, such as statins and steroids, to manage the signs and symptoms of comorbidities, which increases the chances of drug-drug interactions (DDIs). Mechanism-based inhibition is a subset of time-dependent inhibition (TDI) and occurs when a molecule forms a reactive metabolite which irreversibly binds and inactivates drug-metabolizing enzymes, potentially increasing the systemic load to toxic concentrations. Traditionally, perpetrating compounds are screened using human liver microsomes (HLMs); however, this system may be inadequate when the precipitant is activated by a non-cytochrome P450 (P450)-mediated pathway. Even though studies assessing competitive inhibition and TDI using HLM suggested a low risk for CYP3A4-mediated DDI in the clinic, VX-509 increased the area under the curve of midazolam, atorvastatin, and methyl-prednisolone by approximately 12.0-, 2.7-, and 4.3-fold, respectively. Metabolite identification studies using human liver cytosol indicated that VX-509 is converted to an oxidative metabolite, which is the perpetrator of the DDIs observed in the clinic. As opposed to HLM, hepatocytes contain the full complement of drug-metabolizing enzymes and transporters and can be used to assess TDI arising from non-P450-mediated metabolic pathways. In the current study, we highlight the role of aldehyde oxidase in the formation of the hydroxyl-metabolite of VX-509, which is involved in clinically significant TDI-based DDIs and represents an additional example in which a system-dependent prediction of TDI would be evident. PMID:27298338

  16. Analysis of CYP21A1P and the duplicated CYP21A2 genes.

    PubMed

    Tsai, Li-Ping; Lee, Hsien-Hsiung

    2012-09-10

    The RCCX module on chromosome 6p21.3 has 3 possible forms: monomodular, bimodular, and trimodular. Chromosomes with 4 RCCX modules are very rare. In the monomodule, most of the CYP21A1P genes do not exist. However, haplotypes of the RCCX module with more than one CYP21A2 gene were observed. Obviously, the gene located downstream of the XA gene can possibly include the CYP21A2 as well as the CYP21A1P gene.

  17. Crystal structure and absolute configuration of (3aS,4S,5R,7aR)-2,2,7-trimethyl-3a,4,5,7a-tetra­hydro-1,3-benzodioxole-4,5-diol

    PubMed Central

    Macías, Mario A.; Suescun, Leopoldo; Pandolfi, Enrique; Schapiro, Valeria; Tibhe, Gaurao D.; Mombrú, Álvaro W.

    2015-01-01

    The absolute configuration of the title compound, C10H16O4, determined as 3aS,4S,5R,7aR on the basis of the synthetic pathway, was confirmed by X-ray diffraction. The mol­ecule contains a five- and a six-membered ring that adopt twisted and envelope conformations, respectively. The dihedral angle between the mean planes of the rings is 76.80 (11)° as a result of their cis-fusion. In the crystal, mol­ecules are linked by two pairs of O—H⋯O hydrogen bonds, forming chains along [010]. These chains are further connected by weaker C—H⋯O inter­actions along [100], creating (001) sheets that inter­act only by weak van der Waals forces. PMID:26396837

  18. N-[3a-(4-Bromo-phen-yl)-8b-hy-droxy-6,8-dimeth-oxy-3-phenyl-2,3,3a,8b-tetra-hydro-1H-cyclo-penta-[b]benzofuran-1-yl]formamide monohydrate.

    PubMed

    Aubert, Emmanuel; Thuaud, Frédéric; Ribeiro, Nigel; Désaubry, Laurent; Espinosa, Enrique

    2013-01-01

    In the title compound, C26H24BrNO5·H2O, a synthetic analogue of natural flavagline, the cyclo-pentane ring adopts an envelope conformation (the flap atom bearing the phenyl group) and the vicinal phenyl and bromo-phenyl groups are slightly shifted relative to each other [CPh-C-C-CPhBr = 36.3 (2)°]. Intra-molecular N-H⋯O and C-H⋯O hydrogen bonds form S(5) motifs. In the crystal, the organic and the water mol-ecules are linked by an O-H⋯O hydrogen bond. Pairs of organic and water mol-ecules, located about inversion centers, inter-act through O-H⋯O hydrogen bonds, forming R4(4)(20) and R4(4)(26) motifs, which together lead to C2(2)(9) motifs. The crystal packing is also characterized by N-H⋯O and C-H⋯O hydrogen bonds between neighbouring organic mol-ecules, forming R2(2)(10) and R2(2)(18) motifs, respectively. PMID:23476436

  19. Crystal structure and computational study of 3,4-dihy­droxy-3-hy­droxy­methyl-9-methyl-6-methyl­idene-3a,4,5,6,6a,9,9a,9b-octa­hydro­azuleno[4,5-b]furan-2,8(3H,7H)-dione

    PubMed Central

    Çelik, Ísmail; Akkurt, Mehmet; Akşit, Hüseyin; Erenler, Ramazan; García-Granda, Santiago

    2015-01-01

    In the mol­ecule of title compound, C15H20O6, also known as cynarinin A, the cyclo­pentane ring having twist conformation and a γ-lactone ring assuming an envelope conformation are trans- and cis-fused, respectively, to a cyclo­heptane ring adopting a twist-chair conformation. In the crystal, O—H⋯O hydrogen bonds link neighbouring mol­ecules, forming a three-dimensional network. Theoretical calculations of the mol­ecular structure using the CNDO approximation and MOPAC PM3 geometry optimization are in satisfactory agreement with the results of the X-ray structure analysis. PMID:26870396

  20. Crystal structure of 2-[(3aS,6R)-3,3,6-trimethyl-3,3a,4,5,6,7-hexa-hydro-2H-indazol-2-yl]thia-zol-4(5H)-one.

    PubMed

    Ousidi, Abdellah N'ait; Itto, My Youssef Ait; Auhmani, Aziz; Riahi, Abdelkhalek; Daran, Jean-Claude; Abdelwahed, Auhmani

    2016-03-01

    The title compound, C13H19N3OS, is a new thia-zolidin-4-one derivative prepared and isolated as the pure (3aS,6R)-diastereisomer from (R)-thio-semicarbazone pulegone. It crystallized with two independent mol-ecules (A and B) in the asymmetric unit. The compound is composed of a hexhydro-indazole ring system (viz. a five-membered di-hydro-pyrazole ring fused to a cyclo-hexyl ring) with a thia-zole-4-one ring system attached to one of the pyrazole N atoms (at position 2). The overall geometry of the two mol-ecules differs slightly, with the mean planes of the pyrazole and thia-zole rings being inclined to one another by 10.4 (1)° in mol-ecule A and 0.9 (1)° in mol-ecule B. In the crystal, the A and B mol-ecules are linked via C-H⋯O hydrogen bonds, forming slabs parallel to the ab plane. There are C-H⋯π inter-actions present within the layers, and between the layers, so forming a three-dimensional structure. PMID:27006801

  1. Crystal structure of 2-[(3aS,6R)-3,3,6-trimethyl-3,3a,4,5,6,7-hexa­hydro-2H-indazol-2-yl]thia­zol-4(5H)-one

    PubMed Central

    Ousidi, Abdellah N’ait; Itto, My Youssef Ait; Auhmani, Aziz; Riahi, Abdelkhalek; Daran, Jean-Claude; Abdelwahed, Auhmani

    2016-01-01

    The title compound, C13H19N3OS, is a new thia­zolidin-4-one derivative prepared and isolated as the pure (3aS,6R)-diastereisomer from (R)-thio­semicarbazone pulegone. It crystallized with two independent mol­ecules (A and B) in the asymmetric unit. The compound is composed of a hexhydro­indazole ring system (viz. a five-membered di­hydro­pyrazole ring fused to a cyclo­hexyl ring) with a thia­zole-4-one ring system attached to one of the pyrazole N atoms (at position 2). The overall geometry of the two mol­ecules differs slightly, with the mean planes of the pyrazole and thia­zole rings being inclined to one another by 10.4 (1)° in mol­ecule A and 0.9 (1)° in mol­ecule B. In the crystal, the A and B mol­ecules are linked via C—H⋯O hydrogen bonds, forming slabs parallel to the ab plane. There are C—H⋯π inter­actions present within the layers, and between the layers, so forming a three-dimensional structure. PMID:27006801

  2. Strong synergistic induction of CYP1A1 expression by andrographolide plus typical CYP1A inducers in mouse hepatocytes

    SciTech Connect

    Jaruchotikamol, Atika; Jarukamjorn, Kanokwan Sirisangtrakul, Wanna; Sakuma, Tsutomu; Kawasaki, Yuki; Nemoto, Nobuo

    2007-10-15

    The effects of andrographolide, the major diterpenoid constituent of Andrographis paniculata, on the expression of cytochrome P450 superfamily 1 members, including CYP1A1, CYP1A2, and CYP1B1, as well as on aryl hydrocarbon receptor (AhR) expression in primary cultures of mouse hepatocytes were investigated in comparison with the effects of typical CYP1A inducers, including benz[a]anthracene, {beta}-naphthoflavone, and 2,3,7,8-tetrachlorodibenzo-p-dioxin. Andrographolide significantly induced the expression of CYP1A1 and CYP1A2 mRNAs in a concentration-dependent manner, as did the typical CYP1A inducers, but did not induce that of CYP1B1 or AhR. Interestingly, andrographolide plus the typical CYP1A inducers synergistically induced CYP1A1 expression, and the synergism was blocked by an AhR antagonist, resveratrol. The CYP1A1 enzyme activity showed a similar pattern of induction. This is the first report that shows that andrographolide has a potency to induce CYP1A1 enzyme and indicates that andrographolide could be a very useful compound for investigating the regulatory mechanism of the CYP1A1 induction pathway. In addition, our findings suggest preparing advice for rational administration of A. paniculata, according to its ability to induce CYP1A1 expression.

  3. Predicting drug metabolism by CYP1A1, CYP1A2, and CYP1B1: insights from MetaSite, molecular docking and quantum chemical calculations.

    PubMed

    Pragyan, Preeti; Kesharwani, Siddharth S; Nandekar, Prajwal P; Rathod, Vijay; Sangamwar, Abhay T

    2014-11-01

    Recently, CYP1 enzymes are documented for selective metabolism of anticancer leads in cancer prevention and/or progression. Elucidation of specificity of substrates/inhibitors of CYP1 isoforms plays a vital role in design of more selective and potent anticancer leads. However, an area of concern is the broad range of substrate specificities and planar nature of substrates with limited dataset which makes it difficult to predict their site of metabolism (SOM) accurately. In the present study, various models for prediction of site of metabolism in case of CYP1A1, CYP1A2, and CYP1B1 substrates were developed using MetaSite, molecular docking, and quantum chemical descriptors. The predictive accuracy of MetaSite, molecular docking, and quantum chemical descriptors in identifying experimental site of metabolism was analyzed at three levels; top rank, top three ranks, and top five ranks. Two quantum chemical descriptors, chemical hardness and local nucleophilicity are proposed for the prediction of CYP-mediated SOM for the first time. The predictive accuracy shown by chemical hardness at top three ranks was 83.3, 85.7, and 84.6 % for CYP1A1, CYP1A2 and CYP1B1, respectively, whereas local nucleophilicity gave poor predictions of 50, 42.8, and 46.2 %, respectively. The predictability of chemical hardness descriptor outperformed at all three levels of ranks for CYP1A1, CYP1A2, and CYP1B1. Hence, we propose chemical hardness as an useful quantum chemical descriptor for prediction of metabolically vulnerable prints in CYP1A1, CYP1A2, and CYP1B1 mediated metabolism and support the optimization efforts in drug discovery and development programs.

  4. Troglitazone thiol adduct formation in human liver microsomes: enzyme kinetics and reaction phenotyping.

    PubMed

    Gan, Jinping; Qu, Qinling; He, Bing; Shyu, Wen C; Rodrigues, A David; He, Kan

    2008-08-01

    Troglitazone (TGZ) induced hepatotoxicity has been linked to cytochrome P450 (CYP)-catalyzed reactive metabolite formation. Therefore, the kinetics and CYP specificity of reactive metabolite formation were studied using dansyl glutathione (dGSH) as a trapping agent after incubation of TGZ with human liver microsomes (HLM) and recombinant human CYP proteins. CYP2C8 exhibited the highest rate of TGZ adduct (TGZ-dGS) formation, followed by CYP3A4, CYP3A5, and CYP2C19. The involvement of CYP2C8 and CYP3A4 was confirmed with CYP form-selective chemical inhibitors. The impact of TGZ concentration on the rate of TGZ-dGS formation was also evaluated. In this instance, two distinctly different profiles were observed with recombinant CYP3A4 and CYP2C8. It is concluded that both CYP3A4/5 and CYP2C8 play a major role in the formation of TGZ adduct in HLM. However, the contribution of these CYPs varies depending on their relative expression and the concentration of TGZ. PMID:19356091

  5. Allele and genotype frequencies of CYP2C9, CYP2C19 and CYP2D6 in an Italian population.

    PubMed

    Scordo, Maria Gabriella; Caputi, Achille P; D'Arrigo, Concetta; Fava, Giuseppina; Spina, Edoardo

    2004-08-01

    The polymorphic cytochrome P450 isoenzymes (CYPs) 2C9, 2C19 and 2D6 metabolise many important drugs, as well as other xenobiotics. Their polymorphism gives rise to important interindividual and interethnic variability in the metabolism and disposition of several therapeutic agents and may cause differences in the clinical response to these drugs. In this study, we determined the genotype profile of a random Italian population in order to compare the CYP2C9, CYP2C19 and CYP2D6 allele frequencies among Italians with previous findings in other Caucasian populations. Frequencies for the major CYP2C9, CYP2C19 and CYP2D6 mutated alleles and genotypes have been evaluated in 360 unrelated healthy Italian volunteers (210 males and 150 females, aged 19-52 years). Genotyping has been carried out on peripheral leukocytes DNA by molecular biology techniques (PCR, RFLP, long-PCR). CYP2C9, CYP2C19 and CYP2D6 allele and genotype frequencies resulted in equilibrium with the Hardy-Weinberg equation. One hundred and fourteen subjects (31.7%) carried one and 23 subjects (6.4%) carried two CYP2C9 mutated alleles. Sixty-eight (18.9%) volunteers were found to be heterozygous and six (1.7%) homozygous for the CYP2C19*2, while no CYP2C19*3 was detected in the evaluated population. Volunteers could be divided into four CYP2D6 genotypes groups: 192 subjects (53.3%) with no mutated alleles (homozygous extensive metabolisers, EM), 126 (35.0%) with one mutated allele (heterozygous EM), 12 (3.4%) with two mutated alleles (poor metabolisers, PM) and 30 (8.3%) with extracopies of a functional gene (ultrarapid metabolisers, UM). Frequencies of both CYP2C9 and CYP2C19 allelic variants, as well as CYP2D6 detrimental alleles, in Italian subjects were similar to those of other Caucasian populations. Conversely, the prevalence of CYP2D6 gene duplication among Italians resulted very high, confirming the higher frequency of CYP2D6 UM in the Mediterranean area compared to Northern Europe. PMID:15177309

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

    PubMed

    Chiangsom, Abhiruj; Lawanprasert, Somsong; Oda, Shingo; Kulthong, Kornphimol; Luechapudiporn, Rataya; Yokoi, Tsuyoshi; Maniratanachote, Rawiwan

    2016-06-01

    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. PMID:27212065

  7. CYP2D6*36 gene arrangements within the cyp2d6 locus: association of CYP2D6*36 with poor metabolizer status.

    PubMed

    Gaedigk, Andrea; Bradford, L Dianne; Alander, Sarah W; Leeder, J Steven

    2006-04-01

    Unexplained cases of CYP2D6 genotype/phenotype discordance continue to be discovered. In previous studies, several African Americans with a poor metabolizer phenotype carried the reduced function CYP2D6*10 allele in combination with a nonfunctional allele. We pursued the possibility that these alleles harbor either a known sequence variation (i.e., CYP2D6*36 carrying a gene conversion in exon 9 along the CYP2D6*10-defining 100C>T single-nucleotide polymorphism) or novel sequences variation(s). Discordant cases were evaluated by long-range polymerase chain reaction (PCR) to test for gene rearrangement events, and a 6.6-kilobase pair PCR product encompassing the CYP2D6 gene was cloned and entirely sequenced. Thereafter, allele frequencies were determined in different study populations comprising whites, African Americans, and Asians. Analyses covering the CYP2D7 to 2D6 gene region established that CYP2D6*36 did not only exist as a gene duplication (CYP2D6*36x2) or in tandem with *10 (CYP2D6*36+*10), as previously reported, but also by itself. This "single" CYP2D6*36 allele was found in nine African Americans and one Asian, but was absent in the whites tested. Ultimately, the presence of CYP2D6*36 resolved genotype/phenotype discordance in three cases. We also discovered an exon 9 conversion-positive CYP2D6*4 gene in a duplication arrangement (CYP2D6*4Nx2) and a CYP2D6*4 allele lacking 100C>T (CYP2D6*4M) in two white subjects. The discovery of an allele that carries only one CYP2D6*36 gene copy provides unequivocal evidence that both CYP2D6*36 and *36x2 are associated with a poor metabolizer phenotype. Given a combined frequency of between 0.5 and 3% in African Americans and Asians, genotyping for CYP2D6*36 should improve the accuracy of genotype-based phenotype prediction in these populations.

  8. Captan impairs CYP-catalyzed drug metabolism in the mouse.

    PubMed

    Paolini, M; Barillari, J; Trespidi, S; Valgimigli, L; Pedulli, G F; Cantelli-Forti, G

    1999-11-30

    To investigate whether the fungicide captan impairs CYP-catalyzed drug metabolism in murine liver, kidney and lung, the modulation of the regio- and stereo-selective hydroxylation of testosterone, including 6beta-(CYP3A), 6alpha-(CYP2A1 and CYP2B1) and 16alpha-(CYP2B9) oxidations was studied. Specific substrates as probes for different CYP isoforms such as p-nitrophenol (CYP2E1), pentoxyresorufin (CYP2B1), ethoxyresorufin (CYP1A1), aminopyrine (CYP3A), phenacetin and methoxyresorufin (CYP1A2), and ethoxycoumarin (mixed) were also considered. Daily doses of captan (7.5 or 15 mg/kg b.w., i.p.) were administered to different groups of Swiss Albino CD1 mice of both sexes for 1 or 3 consecutive days. While a single dose of this fungicide did not affect CYP-machinery, repeated treatment significantly impaired the microsomal metabolism; in the liver, for example, a general inactivating effect was observed, with the sole exception of testosterone 2alpha-hydroxylase activity which was induced up to 8.6-fold in males. In vitro studies showed that the mechanism-based inhibition was related to captan metabolites rather than the parental compound. In the kidney, both CYP3A- and CYP1A2-linked monooxygenases were significantly induced (2-fold) by this pesticide. Accelerated phenacetin and methoxyresorufin metabolism (CYP1A2) was also observed in the lung. Data on CYP3A (kidney) and CYP1A2 (kidney and lung) induction were corroborated by Western immunoblotting using rabbit polyclonal anti-CYP3A1/2 and CYP1A1/2 antibodies. By means of electron spin resonance (EPR) spectrometry coupled to a spin-trapping technique, it was found that the recorded induction generates a large amounts of the anion radical superoxide (O*2-) either in kidney or lung microsomes. These findings suggest that alterations in CYP-associated activities by captan exposure may result in impaired (endogenous) metabolism as well as of coadministered drugs with significant implications for their disposition. The

  9. Screening of Drug Metabolizing Enzymes for the Ginsenoside Compound K In Vitro: An Efficient Anti-Cancer Substance Originating from Panax Ginseng

    PubMed Central

    Lin, Xiu-Xian; Peng, Shi-Fang; Xiao, Mei-Fang; Huang, Wei-Hua; Wang, Yi-Cheng; Peng, Jing-Bo; Zhang, Wei; Ouyang, Dong-Sheng; Chen, Yao

    2016-01-01

    Ginsenoside compound K (CK), a rare ginsenoside originating from Panax Ginseng, has been found to possess unique pharmacological activities specifically as anti-cancers. However, the role of cytochrome P450s (CYPs) in the metabolism of CK is unclear. In this study, we screened the CYPs for the metabolism of CK in vitro using human liver microsomes (HLMs) or human recombinant CYPs. The results showed that CK inhibited the enzyme activities of CYP2C9 and CYP3A4 in the HLMs. The Km and Vmax values of CK were 84.20±21.92 μM and 0.28±0.04 nmol/mg protein/min, respectively, for the HLMs; 34.63±10.48 μM and 0.45±0.05 nmol/nmol P450/min, respectively, for CYP2C9; and 27.03±5.04 μM and 0.68±0.04 nmol/nmol P450/min, respectively, for CYP3A4. The IC50 values were 16.00 μM and 9.83 μM, and Ki values were 14.92 μM and 11.42μM for CYP2C9 and CYP3A4, respectively. Other human CYP isoforms, including CYP1A2, CYP2A6, CYP2D6, CYP2E1, and CYP2C19, showed minimal or no effect on CK metabolism. The results suggested that CK was a substrate and also inhibitors for both CYP2C9 and CYP3A4. Patients using CK in combination with therapeutic drugs that are substrates of CYP2C9 and CYP3A4 for different reasons should be careful, although the inhibiting potency of CK is much poorer than that of enzyme-specific inhibitors. PMID:26845774

  10. Screening of Drug Metabolizing Enzymes for the Ginsenoside Compound K In Vitro: An Efficient Anti-Cancer Substance Originating from Panax Ginseng.

    PubMed

    Xiao, Jian; Chen, Dan; Lin, Xiu-Xian; Peng, Shi-Fang; Xiao, Mei-Fang; Huang, Wei-Hua; Wang, Yi-Cheng; Peng, Jing-Bo; Zhang, Wei; Ouyang, Dong-Sheng; Chen, Yao

    2016-01-01

    Ginsenoside compound K (CK), a rare ginsenoside originating from Panax Ginseng, has been found to possess unique pharmacological activities specifically as anti-cancers. However, the role of cytochrome P450s (CYPs) in the metabolism of CK is unclear. In this study, we screened the CYPs for the metabolism of CK in vitro using human liver microsomes (HLMs) or human recombinant CYPs. The results showed that CK inhibited the enzyme activities of CYP2C9 and CYP3A4 in the HLMs. The Km and Vmax values of CK were 84.20±21.92 μM and 0.28±0.04 nmol/mg protein/min, respectively, for the HLMs; 34.63±10.48 μM and 0.45±0.05 nmol/nmol P450/min, respectively, for CYP2C9; and 27.03±5.04 μM and 0.68±0.04 nmol/nmol P450/min, respectively, for CYP3A4. The IC50 values were 16.00 μM and 9.83 μM, and Ki values were 14.92 μM and 11.42μM for CYP2C9 and CYP3A4, respectively. Other human CYP isoforms, including CYP1A2, CYP2A6, CYP2D6, CYP2E1, and CYP2C19, showed minimal or no effect on CK metabolism. The results suggested that CK was a substrate and also inhibitors for both CYP2C9 and CYP3A4. Patients using CK in combination with therapeutic drugs that are substrates of CYP2C9 and CYP3A4 for different reasons should be careful, although the inhibiting potency of CK is much poorer than that of enzyme-specific inhibitors.

  11. CYP2D7 Sequence Variation Interferes with TaqMan CYP2D6*15 and *35 Genotyping

    PubMed Central

    Riffel, Amanda K.; Dehghani, Mehdi; Hartshorne, Toinette; Floyd, Kristen C.; Leeder, J. Steven; Rosenblatt, Kevin P.; Gaedigk, Andrea

    2016-01-01

    TaqMan™ genotyping assays are widely used to genotype CYP2D6, which encodes a major drug metabolizing enzyme. Assay design for CYP2D6 can be challenging owing to the presence of two pseudogenes, CYP2D7 and CYP2D8, structural and copy number variation and numerous single nucleotide polymorphisms (SNPs) some of which reflect the wild-type sequence of the CYP2D7 pseudogene. The aim of this study was to identify the mechanism causing false-positive CYP2D6*15 calls and remediate those by redesigning and validating alternative TaqMan genotype assays. Among 13,866 DNA samples genotyped by the CompanionDx® lab on the OpenArray platform, 70 samples were identified as heterozygotes for 137Tins, the key SNP of CYP2D6*15. However, only 15 samples were confirmed when tested with the Luminex xTAG CYP2D6 Kit and sequencing of CYP2D6-specific long range (XL)-PCR products. Genotype and gene resequencing of CYP2D6 and CYP2D7-specific XL-PCR products revealed a CC>GT dinucleotide SNP in exon 1 of CYP2D7 that reverts the sequence to CYP2D6 and allows a TaqMan assay PCR primer to bind. Because CYP2D7 also carries a Tins, a false-positive mutation signal is generated. This CYP2D7 SNP was also responsible for generating false-positive signals for rs769258 (CYP2D6*35) which is also located in exon 1. Although alternative CYP2D6*15 and *35 assays resolved the issue, we discovered a novel CYP2D6*15 subvariant in one sample that carries additional SNPs preventing detection with the alternate assay. The frequency of CYP2D6*15 was 0.1% in this ethnically diverse U.S. population sample. In addition, we also discovered linkage between the CYP2D7 CC>GT dinucleotide SNP and the 77G>A (rs28371696) SNP of CYP2D6*43. The frequency of this tentatively functional allele was 0.2%. Taken together, these findings emphasize that regardless of how careful genotyping assays are designed and evaluated before being commercially marketed, rare or unknown SNPs underneath primer and/or probe regions can impact

  12. CYP2D7 Sequence Variation Interferes with TaqMan CYP2D6 (*) 15 and (*) 35 Genotyping.

    PubMed

    Riffel, Amanda K; Dehghani, Mehdi; Hartshorne, Toinette; Floyd, Kristen C; Leeder, J Steven; Rosenblatt, Kevin P; Gaedigk, Andrea

    2015-01-01

    TaqMan™ genotyping assays are widely used to genotype CYP2D6, which encodes a major drug metabolizing enzyme. Assay design for CYP2D6 can be challenging owing to the presence of two pseudogenes, CYP2D7 and CYP2D8, structural and copy number variation and numerous single nucleotide polymorphisms (SNPs) some of which reflect the wild-type sequence of the CYP2D7 pseudogene. The aim of this study was to identify the mechanism causing false-positive CYP2D6 (*) 15 calls and remediate those by redesigning and validating alternative TaqMan genotype assays. Among 13,866 DNA samples genotyped by the CompanionDx® lab on the OpenArray platform, 70 samples were identified as heterozygotes for 137Tins, the key SNP of CYP2D6 (*) 15. However, only 15 samples were confirmed when tested with the Luminex xTAG CYP2D6 Kit and sequencing of CYP2D6-specific long range (XL)-PCR products. Genotype and gene resequencing of CYP2D6 and CYP2D7-specific XL-PCR products revealed a CC>GT dinucleotide SNP in exon 1 of CYP2D7 that reverts the sequence to CYP2D6 and allows a TaqMan assay PCR primer to bind. Because CYP2D7 also carries a Tins, a false-positive mutation signal is generated. This CYP2D7 SNP was also responsible for generating false-positive signals for rs769258 (CYP2D6 (*) 35) which is also located in exon 1. Although alternative CYP2D6 (*) 15 and (*) 35 assays resolved the issue, we discovered a novel CYP2D6 (*) 15 subvariant in one sample that carries additional SNPs preventing detection with the alternate assay. The frequency of CYP2D6 (*) 15 was 0.1% in this ethnically diverse U.S. population sample. In addition, we also discovered linkage between the CYP2D7 CC>GT dinucleotide SNP and the 77G>A (rs28371696) SNP of CYP2D6 (*) 43. The frequency of this tentatively functional allele was 0.2%. Taken together, these findings emphasize that regardless of how careful genotyping assays are designed and evaluated before being commercially marketed, rare or unknown SNPs underneath primer

  13. Reversible inhibition of three important human liver cytochrome p450 enzymes by tiliroside.

    PubMed

    Sun, Dong-Xue; Lu, Jin-Cai; Fang, Zhong-Ze; Zhang, Yan-Yan; Cao, Yun-Feng; Mao, Yu-Xi; Zhu, Liang-Liang; Yin, Jun; Yang, Ling

    2010-11-01

    Tiliroside, an active flavonoid extensively found in many medicinal plants including Helichrysum italicum, Geranium mexicanum and Helianthemum glomeratum, has been demonstrated to exert multiple biological effects including antiinflammatory, antimicrobial, antioxidant and antitumor activities. Cytochrome P450 (CYP) enzymes play an important role in the Phase I oxidation metabolism of a wide range of xenobiotics and inhibition of CYP isoforms might influence the elimination of drugs and induce serious adverse drug response. The inhibition of seven CYP isoforms (CYP3A4, CYP1A2, CYP2A6, CYP2D6, CYP2C9, CYP2C8 and CYP2E1) by tiliroside was investigated using in vitro human liver microsomal incubation assays. The results showed that tiliroside strongly inhibited the activity of CYP3A4 (IC(50) = 9.0 ± 1.7 μm), CYP2C8 (IC(50) = 12.1 ± 0.9 μm) and CYP2C9 (IC(50) = 10.2 ± 0.9 μm) with other CYP isoforms negligibly influenced. Further kinetic analysis showed that inhibition of these three CYP isoforms by tiliroside is best fit to a competitive way. The K(i) value was calculated to be 5.5 μm, 3.3 μm, 9.4 μm for CYP3A4, CYP2C9 and CYP2C8, respectively. The relatively low K(i) values suggested that tiliroside might induce drug-drug interactions with many clinically used drugs which are mainly metabolized by these three CYP isoforms. Therefore, attention should be given to the probable drug-drug interaction between tiliroside-containing herbs and substrates of CYP3A4, CYP2C9 and CYP2C8.

  14. Chimeric CYP21A1P/CYP21A2 genes identified in Czech patients with congenital adrenal hyperplasia.

    PubMed

    Vrzalová, Zuzana; Hrubá, Zuzana; Hrabincová, Eva Sťahlová; Vrábelová, Slávka; Votava, Felix; Koloušková, Stanislava; Fajkusová, Lenka

    2011-01-01

    Congenital adrenal hyperplasia (CAH) comprises a group of autosomal recessive disorders caused by an enzymatic deficiency which impairs the biosynthesis of cortisol and, in the majority of severe cases, also the biosynthesis of aldosterone. Approximately 95% of all CAH cases are caused by mutations in the steroid 21-hydroxylase gene (CYP21A2). The CYP21A2 gene and its inactive pseudogene (CYP21A1P) are located within the HLA class III region of the major histocompatibility complex (MHC) locus on chromosome 6p21.3. In this study, we describe chimeric CYP21A1P/CYP21A2 genes detected in our patients with 21-hydroxylase deficiency (21OHD). Chimeric CYP21A1P/CYP21A2 genes were present in 171 out of 508 mutated CYP21A2 alleles (33.8%). We detected four types of chimeric CYP21A1P/CYP21A2 genes: three of them have been described previously as CH-1, CH-3, CH-4, and one type is novel. The novel chimeric gene, termed CH-7, was detected in 21.4% of the mutant alleles. Possible causes of CYP21A1P/CYP21A2 formation are associated with 1) high recombination rate in the MHC locus, 2) high recombination rate between highly homologous genes and pseudogenes in the CYP21 gene area, and 3) the existence of chi-like sequences and repetitive minisatellite consensus sequences in CYP21A2 and CYP21A1P which play a role in promoting genetic recombination.

  15. CYP79F1 and CYP79F2 have distinct functions in the biosynthesis of aliphatic glucosinolates in Arabidopsis.

    PubMed

    Chen, Sixue; Glawischnig, Erich; Jørgensen, Kirsten; Naur, Peter; Jørgensen, Bodil; Olsen, Carl-Erik; Hansen, Carsten H; Rasmussen, Hasse; Pickett, John A; Halkier, Barbara A

    2003-03-01

    Cytochromes P450 of the CYP79 family catalyze the conversion of amino acids to oximes in the biosynthesis of glucosinolates, a group of natural plant products known to be involved in plant defense and as a source of flavor compounds, cancer-preventing agents and bioherbicides. We report a detailed biochemical analysis of the substrate specificity and kinetics of CYP79F1 and CYP79F2, two cytochromes P450 involved in the biosynthesis of aliphatic glucosinolates in Arabidopsis thaliana. Using recombinant CYP79F1 and CYP79F2 expressed in Escherichia coli and Saccharomyces cerevisiae, respectively, we show that CYP79F1 metabolizes mono- to hexahomomethionine, resulting in both short- and long-chain aliphatic glucosinolates. In contrast, CYP79F2 exclusively metabolizes long-chain elongated penta- and hexahomomethionines. CYP79F1 and CYP79F2 are spatially and developmentally regulated, with different gene expression patterns. CYP79F2 is highly expressed in hypocotyl and roots, whereas CYP79F1 is strongly expressed in cotyledons, rosette leaves, stems, and siliques. A transposon-tagged CYP79F1 knockout mutant completely lacks short-chain aliphatic glucosinolates, but has an increased level of long-chain aliphatic glucosinolates, especially in leaves and seeds. The level of long-chain aliphatic glucosinolates in a transposon-tagged CYP79F2 knockout mutant is substantially reduced, whereas the level of short-chain aliphatic glucosinolates is not affected. Biochemical characterization of CYP79F1 and CYP79F2, and gene expression analysis, combined with glucosinolate profiling of knockout mutants demonstrate the functional role of these enzymes. This provides valuable insights into the metabolic network leading to the biosynthesis of aliphatic glucosinolates, and into metabolic engineering of altered aliphatic glucosinolate profiles to improve nutritional value and pest resistance. PMID:12609033

  16. Analysis of the CYP21A1P pseudogene: indication of mutational diversity and CYP21A2-like and duplicated CYP21A2 genes.

    PubMed

    Tsai, Li-Ping; Cheng, Ching-Feng; Chuang, Shu-Hua; Lee, Hsien-Hsiung

    2011-06-15

    The CYP21A1P gene downstream of the XA gene, carrying 15 deteriorated mutations, is a nonfunctional pseudogene that shares 98% nucleotide sequence homology with CYP21A2 located on chromosome 6p21.3. However, these mutations in the CYP21A1P gene are not totally involved in each individual. From our analysis of 100 healthy ethnic Chinese (i.e., Taiwanese) (n=200 chromosomes) using the polymerase chain reaction (PCR) products combined with an amplification-created restriction site (ACRS) method and DNA sequencing, we found that approximately 10% of CYP21A1P alleles (n=195 chromosomes) presented the CYP21A2 sequence; frequencies of P30, V281, Q318, and R356 in that locus were approximately 24%, 21%, 11%, and 34%, respectively, and approximately 90% of the CYP21A1P alleles had 15 mutated loci. In addition, approximately 2.5% (n=5 chromosomes) showed four haplotypes of the 3.7-kb TaqI-produced fragment of the CYP21A2-like gene and one duplicated CYP21A2 gene. We conclude that the pseudogene of the CYP21A1P mutation presents diverse variants. Moreover, the existence of the CYP21A2-like gene is more abundant than that of the duplicated CYP21A2 gene downstream of the XA gene and could not be distinguished from the CYP21A2-TNXB gene; thus, it may be misdiagnosed by previously established methods for congenital adrenal hyperplasia caused by a 21-hydroxylase deficiency.

  17. CYP2E1 and Oxidative Liver Injury by Alcohol

    PubMed Central

    Lu, Yongke; Cederbaum, Arthur I.

    2008-01-01

    Ethanol-induced oxidative stress appears to play a major role in mechanisms by which ethanol causes liver injury. Many pathways have been suggested to contribute to the ability of ethanol to induce a state of oxidative stress. One central pathway appears to be the induction of cytochrome P450 2E1 (CYP2E1) by ethanol. CYP2E1 metabolizes and activates many toxicological substrates, including ethanol, to more reactive, toxic products. Levels of CYP2E1 are elevated under a variety of physiological and pathophysiological conditions, and after acute and chronic alcohol treatment. CYP2E1 is also an effective generator of reactive oxygen species such as the superoxide anion radical and hydrogen peroxide, and in the presence of iron catalysts, produces powerful oxidants such as the hydroxyl radical. This Review Article summarizes some of the biochemical and toxicological properties of CYP2E1, and briefly describes the use of cell lines developed to constitutively express CYP2E1 in assessing the actions of CYP2E1. Possible therapeutic implications for treatment of alcoholic liver injury by inhibition of CYP2E1 or CYP2E1-dependent oxidative stress will be discussed, followed by some future directions which may help to understand the actions of CYP2E1 and its role in alcoholic liver injury. PMID:18078827

  18. Identification of Cytochrome P450 ( CYP) genes in Zhikong scallop ( Chlamys farreri)

    NASA Astrophysics Data System (ADS)

    Guo, Huihui; Bao, Zhenmin; Du, Huixia; Zhang, Lingling; Wang, Shi; Sun, Luyang; Mou, Xiaoyu; Hu, Xiaoli

    2013-03-01

    Cytochrome P450 ( CYP) superfamily is one of the membership largest and function most diverse protein superfamily recogniozed among living beings. Members of this superfamily were further assigned to different families and subfamilies based on their amino acid similarities. According to their phylogenetic relationships, the CYP genes which likely diverged from common ancestor gene and may share common functions were grouped into one clan. Widely distributing scallops are a group of the most conspicuous bivalve; however the studies on their CYP is acarce. In this study, we searched the genome and expressed sequence tags of Zhikong scallop ( Chlamys farreri) for CYP genes. In total, 88 non-redundant CYP were identified, which were homed in 13 CYPs gene families. Phylogenetic analysis divided these genes into 4 CYP clans. As in deuterostomes, Clan 2 was the largest, which contained 33 genes belonging to CYP1, CYP2, CYP17 and CYP356 families. Clan 3 contgained 19 genes belonging to CYP3, CYP5 and CYP30 families. Clan 4 contained 23 genes, all belonging to CYP4 family. The mitochondrial CYP clan contained 9 genes belonging to CYP10 and CYP24 families. In comparison, protostomes ( C. farreri, D. pluex, D. melanogaster) contained more CYP genes than deuterostomes ( S. purpuratus and vertebrates) in Clan 2 but less genes in Clan 3 and Clan 4. Our findings will aid to deciphering CYP function and evolution in scallops and bivalves.

  19. Effect of Natural Polyphenols on CYP Metabolism: Implications for Diseases.

    PubMed

    Korobkova, Ekaterina A

    2015-07-20

    Cytochromes P450 (CYPs) are a large group of hemeproteins located on mitochondrial membranes or the endoplasmic reticulum. They play a crucial role in the metabolism of endogenous and exogenous molecules. The activity of CYP is associated with a number of factors including redox potential, protein conformation, the accessibility of the active site by substrates, and others. This activity may be potentially modulated by a variety of small molecules. Extensive experimental data collected over the past decade point at the active role of natural polyphenols in modulating the catalytic activity of CYP. Polyphenols are widespread micronutrients present in human diets of plant origin and in medicinal herbs. These compounds may alter the activity of CYP either via direct interactions with the enzymes or by affecting CYP gene expression. The polyphenol-CYP interactions may significantly alter the pharmacokinetics of drugs and thus influence the effectiveness of chemical therapies used in the treatment of different types of cancers, diabetes, obesity, and cardiovascular diseases (CVD). CYPs are involved in the oxidation and activation of external carcinogenic agents, in which case the inhibition of the CYP activity is beneficial for health. CYPs also support detoxification processes. In this case, it is the upregulation of CYP genes that would be favorable for the organism. A CYP enzyme aromatase catalyzes the formation of estrone and estradiol from their precursors. CYPs also catalyze multiple reactions leading to the oxidation of estrogen. Estrogen signaling and oxidative metabolism of estrogen are associated with the development of cancer. Thus, polyphenol-mediated modulation of the CYP's activity also plays a vital role in estrogen carcinogenesis. The aim of the present review is to summarize the data collected over the last five to six years on the following topics: (1) the mechanisms of the interactions of CYP with food constituents that occur via the direct binding of

  20. Effect of Natural Polyphenols on CYP Metabolism: Implications for Diseases.

    PubMed

    Korobkova, Ekaterina A

    2015-07-20

    Cytochromes P450 (CYPs) are a large group of hemeproteins located on mitochondrial membranes or the endoplasmic reticulum. They play a crucial role in the metabolism of endogenous and exogenous molecules. The activity of CYP is associated with a number of factors including redox potential, protein conformation, the accessibility of the active site by substrates, and others. This activity may be potentially modulated by a variety of small molecules. Extensive experimental data collected over the past decade point at the active role of natural polyphenols in modulating the catalytic activity of CYP. Polyphenols are widespread micronutrients present in human diets of plant origin and in medicinal herbs. These compounds may alter the activity of CYP either via direct interactions with the enzymes or by affecting CYP gene expression. The polyphenol-CYP interactions may significantly alter the pharmacokinetics of drugs and thus influence the effectiveness of chemical therapies used in the treatment of different types of cancers, diabetes, obesity, and cardiovascular diseases (CVD). CYPs are involved in the oxidation and activation of external carcinogenic agents, in which case the inhibition of the CYP activity is beneficial for health. CYPs also support detoxification processes. In this case, it is the upregulation of CYP genes that would be favorable for the organism. A CYP enzyme aromatase catalyzes the formation of estrone and estradiol from their precursors. CYPs also catalyze multiple reactions leading to the oxidation of estrogen. Estrogen signaling and oxidative metabolism of estrogen are associated with the development of cancer. Thus, polyphenol-mediated modulation of the CYP's activity also plays a vital role in estrogen carcinogenesis. The aim of the present review is to summarize the data collected over the last five to six years on the following topics: (1) the mechanisms of the interactions of CYP with food constituents that occur via the direct binding of

  1. Identification and Expression of Multiple CYP1-like and CYP3-like Genes in the Bivalve Mollusk Mytilus edulis

    PubMed Central

    Zanette, Juliano; Jenny, Matthew J.; Goldstone, Jared V.; Parente, Thiago; Woodin, Bruce R.; Bainy, Afonso C. D.; Stegeman, John J.

    2013-01-01

    Various sequencing projects over the last several years have aided the discovery of previously uncharacterized invertebrate sequences, including new cytochrome P450 genes (CYPs). Here we present data on the identification and characterization of two CYP1-like and three CYP3-like genes from the bivalve mollusk Mytilus edulis, and assess their potential as biomarkers based on their responses to several known vertebrate aryl hydrocarbon receptor (AHR) agonists. Quantitative real-time PCR was used to measure CYP transcript levels in digestive gland, labial palps, adductor muscle, gill, foot, and different regions of the mantle. Levels of both CYP1-like genes were highest in digestive gland, whereas labial palps had the highest expression levels of the three CYP3-like genes followed by digestive gland and outer margin of the mantle. Mussels were exposed by injection to the AHR agonists, β-naphthoflavone (BNF; 25 μg.g−1), 3,3′,4,4′,5-polychlorinated biphenyl (PCB126; 2 μg.g−1), or 6-formylindolo[3,2-b]carbazole (FICZ; 0.1 μg.g−1), or to Aroclor 1254 (a mixture of PCBs; 50 μg.g−1) for 24 hours, followed by CYP expression analysis. There was no statistically significant change in expression of either of the CYP1-like genes after exposure to the various AHR agonists. The CYP3-like-1 gene was significantly up-regulated by BNF in gill tissues and the CYP3-like-2 gene was up-regulated in digestive gland by PCB126 and in gill tissue by BNF. These results suggest that distinct mechanisms of CYP gene activation could be present in M. edulis, although the importance of the CYP1-like and CYP3-like genes for xenobiotic and endogenous lipids biotransformation requires additional investigation. PMID:23277104

  2. Design synthesis and evaluation of the inhibitory selectivity of novel trans-resveratrol analogues on human recombinant CYP1A1 CYP1A2 and CYP1B1

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A series of trans-stilbene derivatives containing 4’-thiomethyl substituent were synthesized and evaluated for inhibitory activities on human recombinant cytochrome P450(s): CYP1A1, CYP1A2, and CYP1B1. CYP1A2-related metabolism of stilbene derivatives was estimated by using NADPH oxidation assay. A...

  3. Effect of butylated hydroxytoluene, curcumin, propyl gallate and thiabendazole on cytochrome P450 forms in cultured human hepatocytes.

    PubMed

    Price, R J; Scott, M P; Giddings, A M; Walters, D G; Stierum, R H; Meredith, C; Lake, B G

    2008-06-01

    1. The objective of this study was to investigate the effects of four food chemicals, namely butylated hydroxytoluene (BHT), curcumin (CC), propyl gallate (PG) and thiabendazole (TB), on cytochrome P450 (CYP) forms in cultured human hepatocytes. 2. Treatment of human hepatocytes for 72 h with 2-200 microM TB produced concentration-dependent increases in CYP1A2, CYP2B6 and CYP3A4 mRNA levels, whereas treatment with BHT increased CYP2B6 and CYP3A4 mRNA levels. CYP1A2, CYP2B6 and CYP3A4 mRNA levels were induced around 48-, 21- and 9-fold, respectively, by 200 microM TB, with CYP2B6 and CYP 3A4 mRNA levels being induced around 12- and 7-fold, respectively, by 200 microM BHT. 3. In contrast, the treatment of human hepatocytes for 72 h with PG and CC had little or no effect on CYP mRNA levels. 4. The treatment of human hepatocytes with TB also induced CYP1A-dependent 7-ethoxyresorufin O-deethylase activity, whereas BHT induced CYP3A-dependent testosterone 6beta-hydroxylase activity. 5. In summary, the results demonstrate that TB is a mixed inducer of CYP forms in human hepatocytes inducing CYP1A, CYP2B and CYP3A forms, whereas BHT is an inducer of CYP2B and CYP3A forms.

  4. Identification of human liver cytochrome P450 enzymes involved in the metabolism of SCH 530348 (Vorapaxar), a potent oral thrombin protease-activated receptor 1 antagonist.

    PubMed

    Ghosal, Anima; Lu, Xiaowen; Penner, Natalia; Gao, Lan; Ramanathan, Ragu; Chowdhury, Swapan K; Kishnani, Narendra S; Alton, Kevin B

    2011-01-01

    Vorapaxar (SCH 530348), a potent oral thrombin protease-activated receptor 1 antagonist, is being developed as an antiplatelet agent for patients with established vascular disease. The objective of this study was to identify the human liver cytochrome P450 (P450) enzyme(s) responsible for the metabolism of SCH 530348. Human liver microsomes metabolized SCH 530348 to M19, an amine metabolite formed via carbamate cleavage, and M20 (monohydroxy-SCH 530348). Recombinant human CYP3A4 exhibited the most activity (11.5% profiled radioactivity) for the formation of M19, followed by markedly less substrate conversion with CYP1A1 and CYP2C19. Trace levels of M19, a major excreted human metabolite, were detected with CYP1A2, CYP3A5, and CYP4F3A. Formation of M19 by human liver microsomes was inhibited 89% by ketoconazole (IC(50), 0.73 μM), 34% by tranylcypromine, and 89% by anti-CYP3A4 monoclonal antibody. There was a significant correlation between the rate of M19 formation and midazolam 1'-hydroxylation (r = 0.75) or M19 formation and testosterone 6β-hydroxylation (r = 0.92). The results of screening, inhibition, and correlation studies confirmed that CYP3A4 is the major P450 enzyme responsible for M19 formation from SCH 530348. In contrast, formation of M20, a major circulating human metabolite at steady state, was primarily catalyzed by CYP3A4 and CYP2J2. M20 is pharmacologically equipotent to SCH 530348, whereas M19 is an inactive metabolite. Formation of M20 by human liver microsomes was inhibited 89% by ketoconazole, 75% by astemizole (a CYP2J2 inhibitor), and 43% by CYP3A4 monoclonal antibody. These results suggest that CYP3A4 and CYP2J2 are both involved in the formation of M20 metabolite. PMID:20926621

  5. Interactions among Cytochromes P450 in Microsomal Membranes

    PubMed Central

    Davydov, Dmitri R.; Davydova, Nadezhda Y.; Sineva, Elena V.; Halpert, James R.

    2015-01-01

    The body of evidence of physiologically relevant P450-P450 interactions in microsomal membranes continues to grow. Here we probe oligomerization of human CYP3A4, CYP3A5, and CYP2E1 in microsomal membranes. Using a technique based on luminescence resonance energy transfer, we demonstrate that all three proteins are subject to a concentration-dependent equilibrium between the monomeric and oligomeric states. We also observed the formation of mixed oligomers in CYP3A4/CYP3A5, CYP3A4/CYP2E1, and CYP3A5/CYP2E1 pairs and demonstrated that the association of either CYP3A4 or CYP3A5 with CYP2E1 causes activation of the latter enzyme. Earlier we hypothesized that the intersubunit interface in CYP3A4 oligomers is similar to that observed in the crystallographic dimers of some microsomal drug-metabolizing cytochromes P450 (Davydov, D. R., Davydova, N. Y., Sineva, E. V., Kufareva, I., and Halpert, J. R. (2013) Pivotal role of P450-P450 interactions in CYP3A4 allostery: the case of α-naphthoflavone. Biochem. J. 453, 219–230). Here we report the results of intermolecular cross-linking of CYP3A4 oligomers with thiol-reactive bifunctional reagents as well as the luminescence resonance energy transfer measurements of interprobe distances in the oligomers of labeled CYP3A4 single-cysteine mutants. The results provide compelling support for the physiological relevance of the dimer-specific peripheral ligand-binding site observed in certain CYP3A4 structures. According to our interpretation, these results reveal an important general mechanism that regulates the activity and substrate specificity of the cytochrome P450 ensemble through interactions between multiple P450 species. As a result of P450-P450 cross-talk, the catalytic properties of the cytochrome P450 ensemble cannot be predicted by simple summation of the properties of the individual P450 species. PMID:25533469

  6. Role of CYP3A in isoniazid metabolism in vivo

    PubMed Central

    Liu, Ke; Li, Feng; Lu, Jie; Gao, Zhiwei; Klaassen, Curtis D.; Ma, Xiaochao

    2014-01-01

    Summary Isoniazid (INH), a first-line drug for tuberculosis control, frequently causes liver injury. Multiple previous reports suggest that CYP3A is involved in INH metabolism, bioactivation and hepatotoxicity, although direct evidence is unavailable. In the current study, wild-type and Cyp3a-null mice were used to determine the potential role of Cyp3a in INH metabolism in vivo. Compared to wild-type mice, there were no significant differences in the pharmacokinetic profiles of INH and acetyl-isoniazid in Cyp3a-null mice after an oral administration of 50 mg/kg INH. With the same treatment, distribution of INH and its major metabolites was similar in the liver of wild-type and Cyp3a-null mice. A reactive metabolite of INH was trapped by N-α-acetyl-L-lysine in mouse liver microsomes, but Cyp3a does not contribute to this bioactivation pathway. In addition, no liver injury was observed in wild-type and Cyp3a-null mice treated with 60 or 120 mg/kg INH. In summary, Cyp3a has no effect on systemic pharmacokinetics of INH in mice. Further studies are needed to determine whether and how exactly CYP3A is involved in INH bioactivation and hepatotoxicity. PMID:24172716

  7. Sequence analysis of novel CYP4 transcripts from Mytilus galloprovincialis.

    PubMed

    Ravlić, Sanda; Žučko, Jurica; Tanković, Mirta Smodlaka; Fafanđel, Maja; Bihari, Nevenka

    2015-07-01

    Cytochrome P450 enzymes (CYPs) are essential components of cellular detoxification system. We identified and characterized seven new cytochrome P450 gene transcript clusters in the populations of bivalve mollusc Mytilus galloprovincialis from three different locations. The phylogenetic analysis identified all transcripts as clusters within the CYP4 branch. Identified clusters, each comprising a number of transcript variants, were designated CYP4Y1, Y2, Y3, Y4, Y5, Y6 and Y7. Transcript clusters CYP4Y2 and Y7, and CYP4Y5 and Y6 showed site specificity, while the transcript clusters CYP4Y1, Y3 and Y4 were present at all investigated locations. The comparison of transcripts deduced amino acid sequences with CYP4s from vertebrate and invertebrate species showed high conservation of the residues and domains essential to the putative function of the enzyme, as terminal ω-hydroxylation and prostaglandin hydroxylation. Our results suggest the great expansion of the CYP4Y cDNAs indicative of CYP4 proteins in the mussel M. galloprovincialis presumably as a response to different environmental conditions.

  8. High-throughput screening assays for the assessment of CYP2C9*1, CYP2C9*2, and CYP2C9*3 metabolism using fluorogenic Vivid substrates.

    PubMed

    Marks, Bryan D; Thompson, David V; Goossens, Tony A; Trubetskoy, Olga V

    2004-08-01

    CYP2C9 is a genetically polymorphic human cytochrome P450 isozyme involved in the oxidative metabolism of many drugs, including nonsteroidal anti-inflammatory compounds. Individuals genotyped heterozygous or homozygous for CYP2C9 allelic variants have demonstrated altered metabolism of some drugs primarily metabolized by CYP2C9. The ability to expand screening of CYP2C9 allelic variants to a larger set of drugs and pharmaceutical agents would contribute to a better understanding of the significance of CYP2C9 polymorphisms in the population and to predictions of possible outcomes. The authors report the development of an in vitro fluorescence-based assay employing recombinant CYP2C9 variants (CYP2C9*1, CYP2C9*2, and CYP2C9*3) and fluorogenic Vivid(R) CYP2C9 substrates to explore the effects of CYP2C9 polymorphisms on drug metabolism, using drugs primarily metabolized by CYP2C9. Several chemically diverse fluorogenic substrates (Vivid(R) CYP2C9 blue, green, and red substrates) were used as prototypic probes to obtain in vitro CYP2C9 metabolic rates and kinetic parameters, such as apparent K(m), V(max), and V(max)/K(m) ratios for each allelic variant. In addition, a diverse panel of drugs was screened as assay modifiers with CYP2C9*1, CYP2C9*2, CYP2C9*3, and the fluorogenic Vivid(R) CYP2C9 substrates. The inhibitory potential of this large group of chemically diverse drugs and compounds has been assessed on the basis of their ability to compete with Vivid(R) CYP2C9 substrates in fluorescent reporter assays, thus providing a sensitive and quick assessment of polymorphism-dependent changes in CYP2C9 metabolism.

  9. In vitro metabolism of nobiletin, a polymethoxy-flavonoid, by human liver microsomes and cytochrome P450.

    PubMed

    Koga, Nobuyuki; Ohta, Chiho; Kato, Yoshihisa; Haraguchi, Koichi; Endo, Tetsuya; Ogawa, Kazunori; Ohta, Hideaki; Yano, Masamichi

    2011-11-01

    Cytochrome P450 enzymes (CYPs) in the liver metabolize drugs prior to excretion, with different enzymes acting at different molecular motifs. At present, the human CYPs responsible for the metabolism of the flavonoid, nobiletin (NBL), are unidentified. We investigated which enzymes were involved using human liver microsomes and 12 cDNA-expressed human CYPs. Human liver microsomes metabolized NBL to three mono-demethylated metabolites (4'-OH-, 7-OH- and 6-OH-NBL) with a relative ratio of 1:4.1:0.5, respectively, by aerobic incubation with nicotinamide adenine dinucleotide phosphate (NADPH). Of 12 human CYPs, CYP1A1, CYP1A2 and CYP1B1 showed high activity for the formation of 4'-OH-NBL. CYP3A4 catalyzed the formation of 7-OH-NBL with the highest activity and of 6-OH-NBL with lower activity. CYP3A5 also catalyzed the formation of both metabolites but considerably more slowly than CYP3A4. In contrast, seven CYPs (CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP2E1) were inactive for NBL. Both ketoconazole and troleandomycin (CYP3A inhibitors) almost completely inhibited the formation of 7-OH- and 6-OH-NBL. Similarly, α-naphthoflavone (CYP1A1 inhibitor) and furafylline (CYP1A2 inhibitor) significantly decreased the formation of 4'-OH-NBL. These results suggest that CYP1A2 and CYP3A4 are the key enzymes in human liver mediating the oxidative demethylation of NBL in the B-ring and A-ring, respectively.

  10. Functional characterization of CYP2D6 enhancer polymorphisms

    PubMed Central

    Wang, Danxin; Papp, Audrey C.; Sun, Xiaochun

    2015-01-01

    CYP2D6 metabolizes nearly 25% of clinically used drugs. Genetic polymorphisms cause large inter-individual variability in CYP2D6 enzyme activity and are currently used as biomarker to predict CYP2D6 metabolizer phenotype. Previously, we had identified a region 115 kb downstream of CYP2D6 as enhancer for CYP2D6, containing two completely linked single nucleotide polymorphisms (SNPs), rs133333 and rs5758550, associated with enhanced transcription. However, the enhancer effect on CYP2D6 expression, and the causative variant, remained to be ascertained. To characterize the CYP2D6 enhancer element, we applied chromatin c