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Stephen D. Hall - One of the best experts on this subject based on the ideXlab platform.
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effect of cyp3a5 expression on vincristine metabolism with human liver microsomes
Journal of Pharmacology and Experimental Therapeutics, 2007Co-Authors: Jennifer B Dennison, David R Jones, Jamie L Renbarger, Stephen D. HallAbstract:Vincristine is preferentially metabolized to a secondary amine, M1, by CYP3A5 with a 9- to 14-fold higher intrinsic clearance than CYP3A4 using cDNA-expressed enzymes. The genetically polymorphic expression of CYP3A5 may contribute to interindividual variability in vincristine efficacy and toxicity. The current study quantifies the contribution of cytochromes P450 (P450s), including CYP3A4 and CYP3A5, to vincristine metabolism with a bank of human liver microsomes (HLMs). M1 was the major metabolite formed with HLMs, and selective chemical inhibition of P450s confirmed that CYP3A was the major metabolizing subfamily. The liver tissues were genotyped for low expression alleles, CYP3A5*3,*6, and *7, and the HLMs were phenotyped for CYP3A4 and CYP3A5 expression by Western blot. Testosterone 6beta-hydroxylation and itraconazole hydroxylation were used to quantify CYP3A4 activity in the HLMs. For each CYP3A5 high expresser (n=10), the rate of M1 formation from vincristine due to CYP3A5 was quantified by subtracting the CYP3A4 contribution as determined by linear regression with CYP3A5*3/*3 samples. For CYP3A5 high expressers, the contribution of CYP3A5 to the metabolism of vincristine was 54 to 95% of the total activity, and the rate of M1 formation mediated by CYP3A5 correlated with CYP3A5 protein content (r2=0.95). Selective inhibition of CYP3A4 demonstrated that the M1 formation rate with CYP3A5 high expressers was differentially inhibited based on CYP3A4 activity. Using median values, the estimated hepatic clearances were 5-fold higher for CYP3A5 high expressers than low expressers. We conclude that polymorphic expression of CYP3A5 may be a major determinant in the P450-mediated clearance of vincristine.
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inhibition of human intestinal wall metabolism by macrolide antibiotics effect of clarithromycin on cytochrome p450 3a4 5 activity and expression
Clinical Pharmacology & Therapeutics, 2005Co-Authors: Amar Pinto, Dhanashri Kolwankar, Suthat Liangpunsakul, Million Arefayene, Christopher J Gorski, Mitchell A. Hamman, Naga Chalasani, Ying-hong Wang, Todd C. Skaar, Stephen D. HallAbstract:Background Clarithromycin increases both hepatic and intestinal availability of the selective cytochrome P450 (CYP) 3A probe midazolam. This study was designed to identify determinants of variability in the extent of intestinal wall CYP3A inhibition by clarithromycin, such as CYP3A5 genotype, and the mechanism of inhibition. Methods Ten healthy volunteers received 500 mg oral clarithromycin twice a day for 7 days. Before and after administration of clarithromycin, small-bowel mucosal biopsy specimens were obtained endoscopically. Intestinal CYP3A activity was determined from the rate of 1′-hydroxymidazolam and 4-hydroxymidazolam formation by incubation of small-bowel homogenate with midazolam (25 Μmol/L) and NADPH for 5 minutes. Intestinal CYP3A4 and CYP3A5 messenger ribonucleic acid was quantified by real-time reverse transcriptase-polymerase chain reaction. Intestinal CYP3A4 and CYP3A5 protein concentrations were determined by immunoblotting. Serum and homogenate concentrations of midazolam, clarithromycin, and metabolites were determined by liquid chromatography-mass spectrometry. CYP3A5 genotype was determined by real-time polymerase chain reaction. Results The formation of 1′-hydroxymidazolam (1.36 ± 0.46 pmol · min−1 · mg−1 at baseline versus 0.35 ± 0.16 pmol · min−1 · mg−1 after administration) and 4-hydroxymidazolam (0.39 ± 0.12 pmol · min−1 · mg−1 at baseline versus 0.12 ± 0.05 pmol · min−1 · mg−1 after administration) was significantly (P < .001) reduced after clarithromycin administration. Clarithromycin administration did not result in a significant change in intestinal CYP3A4 and CYP3A5 messenger ribonucleic acid and protein expression. All subjects had detectable serum clarithromycin concentrations after 7 days of clarithromycin (3.71 ± 2.43 Μmol/L). The mean concentration of clarithromycin in the intestinal biopsy homogenate was 1.2 ± 0.7 nmol/L (range, 0.42–2.39 nmol/L). Compared with CYP3A5 nonexpressers, subjects with at least 1 CYP3A5*1 allele (CYP3A5 expressers) had greater inhibition of intestinal CYP3A activity after treatment with clarithromycin. There was a strong linear relationship between the decrease in intestinal CYP3A activity and baseline catalytic activity (R2 = 0.9). Conclusion Baseline intestinal activity of CYP3A4 was a key determinant of variability of the inhibitory effect of clarithromycin among individuals. CYP3A5*1 alleles were associated with greater baseline intestinal CYP3A activity and, therefore, greater extent of inhibition. The primary in vivo mechanism was not rapidly reversible competitive or irreversible inhibition but was likely formation of metabolic intermediate complexes. Clinical Pharmacology & Therapeutics (2005) 77, 178–188; doi: 10.1016/j.clpt.2004.10.002
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mechanism based inactivation of cyp3a by hiv protease inhibitors
Journal of Pharmacology and Experimental Therapeutics, 2005Co-Authors: Steven C Ernest, Stephen D. Hall, David R JonesAbstract:Human immunodeficiency virus (HIV) protease inhibitors (PIs) are inhibitors of CYP3A enzymes, but the mechanism is poorly defined. In this study, time- and concentration-dependent decreases in activity as defined by maximum rate of inactivation ( k inact ) and inhibitor concentration that gives 50% maximal inactivation ( K I ) of CYP3A by amprenavir, indinavir, lopinavir, nelfinavir, ritonavir, and saquinavir were quantified using testosterone 6β-hydroxylation as a marker for CYP3A activity with recombinant CYP3A4(+ b 5 ), recombinant CYP3A5, and pooled human liver microsomes (HLMs). All the PIs, except indinavir, displayed inactivation with CYP3A4(+ b 5 ) and HLMs. Ritonavir was the most potent ( K I = 0.10 and 0.17 μM) and demonstrated high k inact values (0.32 and 0.40 min -1 ) with both CYP3A4(+ b 5 ) and HLMs. Ritonavir was not significantly depleted by high-affinity binding with CYP3A4(+ b 5 ) and confirmed that estimation of reversible inhibition was confounded with irreversible inhibition. For CYP3A5, nelfinavir exhibited the highest k inact (0.47 min -1 ), but ritonavir was the most potent ( K I = 0.12 μM). Saquinavir and indinavir did not show time- and concentration-dependent decreases in activity with CYP3A5. Spectrophototmetrically determined metabolic intermediate complex formation was observed for all of the PIs with CYP3A4(+ b 5 ), except for lopinavir and saquinavir. The addition of nucleophilic and free aldehyde trapping agents and free iron and reactive oxygen species scavengers did not prevent inactivation of CYP3A4(+ b 5 ) by ritonavir, amprenavir, or nelfinavir, but glutathione decreased the inactivation by saquinavir (17%) and catalase decreased the inactivation by lopinavir (39%). In conclusion, all the PIs exhibited mechanism-based inactivation, and predictions of the extent and time course of drug interactions with PIs could be underestimated if based solely on reversible inhibition.
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bimodal distribution of renal cytochrome p450 3a activity in humans
Molecular Pharmacology, 1996Co-Authors: Barbara D Haehner, S K Janardan, Steven A Wrighton, Mark Vandenbranden, Paul B Watkins, J. C. Gorski, Stephen D. HallAbstract:It has been proposed that excessive intrarenal conversion of cortisol to 6 beta-hydroxycortisol by CYP3A may mediate increased tubular reabsorption of sodium, leading to a state of mild volume expansion and the clinical phenotype of salt-sensitive hypertension. Therefore, we characterized CYP3A activity in a bank of microsomes from human kidneys using the formation of 1'-hydroxymidazolam (1'-OHM) as a prototypical CYP3A-catalyzed reaction. Maximal rates of metabolite formation occurred at midazolam concentrations of 12.5-50 microM; higher concentrations resulted in dramatic substrate inhibition. At 12.5 microM midazolam, 4 of 27 kidneys exhibited relatively high mean +/- standard deviation 1'-OHM formation rate (184.0 +/- 14.4 pmol/hr/mg) compared with the remaining 23 samples, which had a mean formation rate of (10.1 +/- 6.4 pmol/hr/mg). Triacetyloleandomycin and anti-CYP3A antibody inhibited midazolam hydroxylation by 53% and 57%, respectively. The correlation between CYP3A5 content, determined through immunoblotting, and 1'-OHM formation rate was high (r2 = 0.84, 24 experiments). The expressions of mRNA corresponding to CYP3A3, CYP3A4, CYP3A5, and CYP3A7 were determined through polymerase chain reaction with specific oligonucleotides as primers. All kidneys examined (25 experiments) expressed CYP3A5 protein and contained the corresponding CYP3A5 mRNA. CYP3A4 mRNA was detected in 40% of the kidney samples, and 70% of those that contained detectable CYP3A4 mRNA also expressed detectable levels of the corresponding protein. Therefore, in contrast to hepatic tissue, in which CYP3A4 is universally expressed, CYP3A5 is the ubiquitously expressed member of the CYP3A family in renal tissue. The distribution of enzyme activity and protein content suggests bimodality and may represent induction of CYP3A5 in a select population and/or a genetically determined organ-specific pattern of expression.
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regioselective biotransformation of midazolam by members of the human cytochrome p450 3a cyp3a subfamily
Biochemical Pharmacology, 1994Co-Authors: Stephen D. Hall, Christopher J Gorski, David R Jones, Mark VandenbrandenAbstract:Abstract The capabilities of cytochrome P4503A4 (CYP3A4), CYP3A5, and fetal hepatic microsomes containing CYP3A7 to metabolize midazolam were investigated using human hepatic microsomes and purified CYP3A4 and CYP3A5. Under initial rate conditions and high substrate concentration (400 μM midazolam), variability among eighteen human liver microsomal samples was 30- and 16- fold for 1′- and 4-hydroxylation of midazolam, respectively. Exclusion of two samples isolated from patients previously administered barbiturates reduced the inter-individual variability to 10.5- and 6.0-fold for 1′- and 4-hydroxylation, respectively. Six fetal hepatic microsomal samples showed 10-fold variation in both 1′-hydroxymidazolam and 4-hydroxymidazolam formation rates. The rates of formation of 4-hydroxymidazolam and 1′-hydroxymidazolam from midazolam by adult samples containing only CYP3A4 and by fetal liver samples were highly correlated ( r 2 =0.99 and 0.97, P r 2 =0.95 and 0.92, respectively), 6β-hydroxylate testosterone ( r 2 =0.96 and 0.96, respectively), and the CYP3A4 content of the samples ( r 2 =0.89 and 0.86, respectively). Microsomal samples containing CYP3A5 in addition to CYP3A4 exhibited a significantly greater ration of 1′-hydroxymidazolam to 4-hydroxymidazolan compared with samples containing only CYP3A4 or CYP3A7 (P b 5 , and NADPH-cytochrome P450 reductase, and an NADPH-regenerating system displayed a 2-fold greater rate of 1′-hydroxymidazolam formation and a similar rate of 4-hydroxymidazolam formation compared with a reconstituted system with CYP3A4. In conclusion, CYP3A4, CYP3A5, and fetal microsomes containing CYP3A7 catalyze 1′- and 4-hydroxylation of midazolam with the ratio of these metabolites indicative of the CYP3A form.
Tetsuya Kamataki - One of the best experts on this subject based on the ideXlab platform.
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ethnic differences between japanese and caucasians in the expression levels of mrnas for cyp3a4 cyp3a5 and CYP3A7 lack of co regulation of the expression of cyp3a in japanese livers
Xenobiotica, 2005Co-Authors: Satoshi Yamaori, Hiroshi Yamazaki, Shunsuke Iwano, Kazuma Kiyotani, Kenji Matsumura, Tetsuya Saito, Andrew Parkinson, Kazuko Nakagawa, Tetsuya KamatakiAbstract:Using a newly developed real-time reverse transcriptase-polymerase chain reaction method, mRNAs were quantitated for CYP3A4, CYP3A5 and CYP3A7 in adult livers from 24 Japanese and 24 Caucasian subjects to elucidate the potential ethnic differences in the expression levels of human cytochrome P450 (CYP) 3As. The expression level of CYP3A4 mRNA in Japanese livers (n = 24) was approximately three times higher than that in Caucasian livers (n = 24, p < 0.001). The mean level of CYP3A5 mRNA was approximately twice higher in Japanese (n = 9) than in Caucasians (n = 5) heterozygous for the CYP3A5 *1 allele (p = 0.057). The CYP3A7 mRNA level was twice higher in Japanese (n = 24) than in Caucasians (n = 22) carrying the CYP3A7 *1A/ *1A genotype (p = 0.042). The level of CYP3A4 mRNA did not correlate with those of CYP3A5 (r = 0.044, n = 24) or CYP3A7 (r = 0.21, n = 24) mRNAs in Japanese livers in contrast to co-regulatory expression of CYP3A4, CYP3A5 and CYP3A7 in Caucasian livers. The results indicate that there are ethnic differences in the expression levels of adult liver CYP3A mRNAs between Japanese and Caucasians, and that the mechanism(s) regulating the hepatic CYP3A expression may be different between these ethnic groups.
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effects of cytochrome b5 on drug oxidation activities of human cytochrome p450 cyp 3as similarity of cyp3a5 with cyp3a4 but not CYP3A7
Biochemical Pharmacology, 2003Co-Authors: Satoshi Yamaori, Hiroshi Yamazaki, Akihiro Suzuki, Ayako Yamada, Hirofumi Tani, Tamio Kamidate, Kenichi Fujita, Tetsuya KamatakiAbstract:Abstract Effects of cytochrome b 5 ( b 5 ) on catalytic activities of human cytochrome P450 (CYP) 3A5, CYP3A4, and CYP3A7 coexpressed with human NADPH-cytochrome P450 reductase in Escherichia coli membranes were investigated using 14 substrates. The activities of CYP3A5 were enhanced by addition of b 5 in approximately one third of the substrates employed in this study. Such enhancement by b 5 was roughly similar to that of CYP3A4, while the activities of CYP3A7 were not enhanced by b 5 with any substrates employed. V max values for midazolam 1′-hydroxylation and amitriptyline N -demethylation by CYP3A5 were increased about twice by addition of b 5 , which was also seen with CYP3A4, although the extent of the effects of b 5 on S 50 ( K m ) and Hill coefficient differed dependent on substrates used. In contrast, b 5 did not alter any of these kinetic parameters of CYP3A7. The effects of b 5 on kinetic parameters of CYP3A5 were similar to those of CYP3A4 but not CYP3A7. These results suggest that roles of b 5 in drug oxidation activities of CYP3A5 and CYP3A4 are different from those of CYP3A7.
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development of bacterial expression system with high yield of CYP3A7 a human fetus specific form of cytochrome p450
Biochemical and Biophysical Research Communications, 2000Co-Authors: Eri Inoue, Yoshiki Takahashi, Yoshio Imai, Tetsuya KamatakiAbstract:Abstract In an E. coli expression system for human cytochrome P450 3A7 (CYP3A7), holo-CYP3A7 was not expressed as judged by CO-difference spectra, although apo-CYP3A7 was clearly detected by Western blot analysis. Unlike CYP3A7, CYP3A4 was expressed efficiently as a hemoprotein in E. coli transformed with a CYP3A4 expression plasmid. To achieve the high yield of the holo-CYP3A7 in E. coli, we examined a causal residue(s) preventing the expression of the holo-CYP3A7 using the chimeric gene of CYP3A4 with CYP3A7. It was found that the region between residues 405 and 503 of CYP3A7 was responsible for the prevention of the holo-CYP3A7 expression in E. coli. Among amino acids examined, substitution of Thr at position 485 in CYP3A7 with Pro, which is at the corresponding position of CYP3A4, resulted in an increase in the amount of holo-CYP3A7. The Thr residue was adjacent to the heme-binding region of CYP3A7. Thus, it appeared that the incorporation of heme into CYP3A7 was possibly affected by this particular amino acid residue. Moreover, holo-CYP3A7 was expressed efficiently when CYP3A7 was co-expressed with molecular chaperone GroEL, known to assist the correct folding of unfolded proteins. Dehydroepiandrosterone 16α-hydroxylation was catalyzed by CYP3A7 expressed in the presence of GroEL.
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Fetus-specific CYP3A7 and Adult-specific CYP3A4 Expressed in Chinese Hamster CHL Cells Have Similar Capacity to Activate Carcinogenic Mycotoxins
Cancer research, 1995Co-Authors: Hisashi Hashimoto, Tetsuya Nakagawa, Tsuyoshi Yokoi, Minoru Sawada, Susumu Itoh, Tetsuya KamatakiAbstract:Abstract To assess whether CYP3A4 and CYP3A7 have a similar capacity to activate carcinogenic mycotoxins, we established cell lines stably expressing human CYP3A4 and CYP3A7, which are adult- and fetal-specific forms of cytochrome P450 in human livers, respectively. Each cDNA was introduced into CR-119 cells which had been established by introducing guinea pig NADPH-cytochrome P450 reductase cDNA into Chinese hamster lung cells. The cell lines (4-line and 7-line) stably expressed the mRNA and the protein corresponding to CYP3A4 and CYP3A7, respectively. The concentration-response for aflatoxin B1 (AFB1) cytotoxicity in 4-line and 7-line, respectively, was compared. 4–10 and 7–40 cells were approximately 17- and 20 times more sensitive to AFB1 than the parental CR-119 cells, respectively. In addition, the sensitivities to AFB1 of both 4–10 and 7–40 cells were enhanced approximately seven times by the addition of 10 µm α-naphthoflavone, a known activator of CYP3A enzyme, while the sensitivities were suppressed approximately four times by the addition of 100 µm troleandomycin, which forms a metabolite intermediate complex with CYP3A enzyme. Moreover, both cell lines showed approximately 10 and 2 times higher sensitivity to sterigmatocystin and aflatoxin G1 than CR-119 cells, respectively. These results indicate that CYP3A4 and CYP3A7 have essentially similar capacities to activate AFB1, sterigmatocystin, and aflatoxin G1 to produce toxic metabolites.
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the possible functions of human fetus specific cytochrome p450 CYP3A7 using transgenic mice carrying CYP3A7cdna
Drug Metabolism and Pharmacokinetics, 1995Co-Authors: L I Yong, Tsuyoshi Yokoi, Ryuji Kitamura, Makoto Sasaki, Masae Gunji, Motoya Katsuki, Tetsuya KamatakiAbstract:Six lines of mice transgenic for the human fetus-specific CYP3A7 have been established using microinjection method. The CYP3A7 transgene was found to be integrated into mouse genome randomly with different copy numbers, ranging from 1 to several hundreds. Northern blot analysis revealed that the transgene expressed in livers only in line M10, whereas all except line M10 expressed the transgene in kidneys. In line M10 mice, increases of weight were observed in the liver, kidney, and uterus while the testis was found to decrease in weight, comparing to their non-transgenic littermates. In addition, serum total testosterone was significantly elevated in line M10 mice, indicating the involvement of CYP3A7 in the metabolism of steroid hormones.The CYP3A7 expressed in the transgenic mice activated AFB1 in vivo resulting in significant increases of DNA damage in the livers and kidneys in line M10 and M2 mice, respectively. The data of midazolam metabolism using line M 10 mice also revealed that CYP3A7 mainly catalyzed hydroxylation at 1' position, similar to CYP3A4 which is an adult-specific isozyme in the CYP3A subfamily.
David R Jones - One of the best experts on this subject based on the ideXlab platform.
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effect of cyp3a5 expression on vincristine metabolism with human liver microsomes
Journal of Pharmacology and Experimental Therapeutics, 2007Co-Authors: Jennifer B Dennison, David R Jones, Jamie L Renbarger, Stephen D. HallAbstract:Vincristine is preferentially metabolized to a secondary amine, M1, by CYP3A5 with a 9- to 14-fold higher intrinsic clearance than CYP3A4 using cDNA-expressed enzymes. The genetically polymorphic expression of CYP3A5 may contribute to interindividual variability in vincristine efficacy and toxicity. The current study quantifies the contribution of cytochromes P450 (P450s), including CYP3A4 and CYP3A5, to vincristine metabolism with a bank of human liver microsomes (HLMs). M1 was the major metabolite formed with HLMs, and selective chemical inhibition of P450s confirmed that CYP3A was the major metabolizing subfamily. The liver tissues were genotyped for low expression alleles, CYP3A5*3,*6, and *7, and the HLMs were phenotyped for CYP3A4 and CYP3A5 expression by Western blot. Testosterone 6beta-hydroxylation and itraconazole hydroxylation were used to quantify CYP3A4 activity in the HLMs. For each CYP3A5 high expresser (n=10), the rate of M1 formation from vincristine due to CYP3A5 was quantified by subtracting the CYP3A4 contribution as determined by linear regression with CYP3A5*3/*3 samples. For CYP3A5 high expressers, the contribution of CYP3A5 to the metabolism of vincristine was 54 to 95% of the total activity, and the rate of M1 formation mediated by CYP3A5 correlated with CYP3A5 protein content (r2=0.95). Selective inhibition of CYP3A4 demonstrated that the M1 formation rate with CYP3A5 high expressers was differentially inhibited based on CYP3A4 activity. Using median values, the estimated hepatic clearances were 5-fold higher for CYP3A5 high expressers than low expressers. We conclude that polymorphic expression of CYP3A5 may be a major determinant in the P450-mediated clearance of vincristine.
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mechanism based inactivation of cyp3a by hiv protease inhibitors
Journal of Pharmacology and Experimental Therapeutics, 2005Co-Authors: Steven C Ernest, Stephen D. Hall, David R JonesAbstract:Human immunodeficiency virus (HIV) protease inhibitors (PIs) are inhibitors of CYP3A enzymes, but the mechanism is poorly defined. In this study, time- and concentration-dependent decreases in activity as defined by maximum rate of inactivation ( k inact ) and inhibitor concentration that gives 50% maximal inactivation ( K I ) of CYP3A by amprenavir, indinavir, lopinavir, nelfinavir, ritonavir, and saquinavir were quantified using testosterone 6β-hydroxylation as a marker for CYP3A activity with recombinant CYP3A4(+ b 5 ), recombinant CYP3A5, and pooled human liver microsomes (HLMs). All the PIs, except indinavir, displayed inactivation with CYP3A4(+ b 5 ) and HLMs. Ritonavir was the most potent ( K I = 0.10 and 0.17 μM) and demonstrated high k inact values (0.32 and 0.40 min -1 ) with both CYP3A4(+ b 5 ) and HLMs. Ritonavir was not significantly depleted by high-affinity binding with CYP3A4(+ b 5 ) and confirmed that estimation of reversible inhibition was confounded with irreversible inhibition. For CYP3A5, nelfinavir exhibited the highest k inact (0.47 min -1 ), but ritonavir was the most potent ( K I = 0.12 μM). Saquinavir and indinavir did not show time- and concentration-dependent decreases in activity with CYP3A5. Spectrophototmetrically determined metabolic intermediate complex formation was observed for all of the PIs with CYP3A4(+ b 5 ), except for lopinavir and saquinavir. The addition of nucleophilic and free aldehyde trapping agents and free iron and reactive oxygen species scavengers did not prevent inactivation of CYP3A4(+ b 5 ) by ritonavir, amprenavir, or nelfinavir, but glutathione decreased the inactivation by saquinavir (17%) and catalase decreased the inactivation by lopinavir (39%). In conclusion, all the PIs exhibited mechanism-based inactivation, and predictions of the extent and time course of drug interactions with PIs could be underestimated if based solely on reversible inhibition.
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comparative metabolic capabilities of cyp3a4 cyp3a5 and CYP3A7
Drug Metabolism and Disposition, 2002Co-Authors: Andrew J Williams, David R Jones, Barbara J Ring, Varon E Cantrell, James A Eckstein, Kenneth J RuterboriesAbstract:The human cytochromes P450 (P450) CYP3A contribute to the biotransformation of 50% of oxidatively metabolized drugs. The predominant hepatic form is CYP3A4, but recent evidence indicates that CYP3A5 contributes more significantly to the total liver CYP3A than was originally thought. CYP3A7 is the major fetal form and is rarely expressed in adults. To compare the metabolic capabilities of CYP3A forms for 10 substrates, incubations were performed using a consistent molar ratio (1:7:9) of recombinant CYP3A, P450 reductase, and cytochrome b5. A wide range of substrate concentrations was examined to determine the best fit to kinetic models for metabolite formation. In general, K(m) or S(50) values for the substrates were 3 to 4 times lower for CYP3A4 than for CYP3A5 or CYP3A7. For a more direct comparison of these P450 forms, clearance to the metabolites was determined as a linear relationship of rate of metabolite formation for the lowest substrate concentrations examined. The clearance for 1'-hydroxy midazolam formation at low substrate concentrations was similar for CYP3A4 and CYP3A5. For CYP3A5 versus CYP3A4, clearance values at low substrate concentrations were 2 to 20 times lower for the other biotransformations. The clearance values for CYP3A7-catalyzed metabolite formation at low substrate concentrations were substantially lower than for CYP3A4 or CYP3A5, except for clarithromycin, 4-OH triazolam, and N-desmethyl diltiazem (CYP3A5 - CYP3A7). The CYP3A forms demonstrated regioselective differences in some of the biotransformations. These results demonstrate an equal or reduced metabolic capability for CYP3A5 compared with CYP3A4 and a significantly lower capability for CYP3A7.
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regioselective biotransformation of midazolam by members of the human cytochrome p450 3a cyp3a subfamily
Biochemical Pharmacology, 1994Co-Authors: Stephen D. Hall, Christopher J Gorski, David R Jones, Mark VandenbrandenAbstract:Abstract The capabilities of cytochrome P4503A4 (CYP3A4), CYP3A5, and fetal hepatic microsomes containing CYP3A7 to metabolize midazolam were investigated using human hepatic microsomes and purified CYP3A4 and CYP3A5. Under initial rate conditions and high substrate concentration (400 μM midazolam), variability among eighteen human liver microsomal samples was 30- and 16- fold for 1′- and 4-hydroxylation of midazolam, respectively. Exclusion of two samples isolated from patients previously administered barbiturates reduced the inter-individual variability to 10.5- and 6.0-fold for 1′- and 4-hydroxylation, respectively. Six fetal hepatic microsomal samples showed 10-fold variation in both 1′-hydroxymidazolam and 4-hydroxymidazolam formation rates. The rates of formation of 4-hydroxymidazolam and 1′-hydroxymidazolam from midazolam by adult samples containing only CYP3A4 and by fetal liver samples were highly correlated ( r 2 =0.99 and 0.97, P r 2 =0.95 and 0.92, respectively), 6β-hydroxylate testosterone ( r 2 =0.96 and 0.96, respectively), and the CYP3A4 content of the samples ( r 2 =0.89 and 0.86, respectively). Microsomal samples containing CYP3A5 in addition to CYP3A4 exhibited a significantly greater ration of 1′-hydroxymidazolam to 4-hydroxymidazolan compared with samples containing only CYP3A4 or CYP3A7 (P b 5 , and NADPH-cytochrome P450 reductase, and an NADPH-regenerating system displayed a 2-fold greater rate of 1′-hydroxymidazolam formation and a similar rate of 4-hydroxymidazolam formation compared with a reconstituted system with CYP3A4. In conclusion, CYP3A4, CYP3A5, and fetal microsomes containing CYP3A7 catalyze 1′- and 4-hydroxylation of midazolam with the ratio of these metabolites indicative of the CYP3A form.
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regioselective biotransformation of midazolam by members of the human cytochrome p450 3a cyp3a subfamily
Biochemical Pharmacology, 1994Co-Authors: Stephen D. Hall, Christopher J Gorski, David R Jones, Mark VandenbrandenAbstract:The capabilities of cytochrome P4503A4 (CYP3A4), CYP3A5, and fetal hepatic microsomes containing CYP3A7 to metabolize midazolam were investigated using human hepatic microsomes and purified CYP3A4 and CYP3A5. Under initial rate conditions and high substrate concentration (400 microM midazolam), variability among eighteen human liver microsomal samples was 30- and 16- fold for 1'- and 4-hydroxylation of midazolam, respectively. Exclusion of two samples isolated from patients previously administered barbiturates reduced the inter-individual variability to 10.5- and 6.0-fold for 1'- and 4-hydroxylation, respectively. Six fetal hepatic microsomal samples showed 10-fold variation in both 1'-hydroxymidazolam and 4-hydroxymidazolam formation rates. The rates of formation of 4-hydroxymidazolam and 1'-hydroxymidazolam from midazolam by adult samples containing only CYP3A4 and by fetal liver samples were highly correlated (r2 = 0.99 and 0.97, P < 0.01, respectively). The rates of formation of 1'-hydroxymidazolam and 4-hydroxymidazolam from midazolam (400 microM) by adult samples that contained only CYP3A4 were correlated significantly (P < 0.01) with the ability of the samples to N-demethylate erythromycin (r2 = 0.95 and 0.92, respectively). 6 beta-hydroxylate testosterone (r2 = 0.96 and 0.96, respectively), and the CYP3A4 content of the samples (r2 = 0.89 and 0.86, respectively). Microsomal samples containing CYP3A5 in addition to CYP3A4 exhibited a significantly greater ratio of 1'-hydroxymidazolam to 4-hydroxymidazolam compared with samples containing only CYP3A4 or CYP3A7 (P < 0.001). Purified CYP3A5 in a reconstituted system, consisting of dilauroylphosphatidylcholine, cytochrome b5, and NADPH-cytochrome P450 reductase, and an NADPH-regenerating system displayed a 2-fold greater rate of 1'-hydroxymidazolam formation and a similar rate of 4-hydroxymidazolam formation compared with a reconstituted system with CYP3A4. In conclusion, CYP3A4, CYP3A5, and fetal microsomes containing CYP3A7 catalyze 1'- and 4-hydroxylation of midazolam with the ratio of these metabolites indicative of the CYP3A form.
Christopher J Gorski - One of the best experts on this subject based on the ideXlab platform.
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inhibition of human intestinal wall metabolism by macrolide antibiotics effect of clarithromycin on cytochrome p450 3a4 5 activity and expression
Clinical Pharmacology & Therapeutics, 2005Co-Authors: Amar Pinto, Dhanashri Kolwankar, Suthat Liangpunsakul, Million Arefayene, Christopher J Gorski, Mitchell A. Hamman, Naga Chalasani, Ying-hong Wang, Todd C. Skaar, Stephen D. HallAbstract:Background Clarithromycin increases both hepatic and intestinal availability of the selective cytochrome P450 (CYP) 3A probe midazolam. This study was designed to identify determinants of variability in the extent of intestinal wall CYP3A inhibition by clarithromycin, such as CYP3A5 genotype, and the mechanism of inhibition. Methods Ten healthy volunteers received 500 mg oral clarithromycin twice a day for 7 days. Before and after administration of clarithromycin, small-bowel mucosal biopsy specimens were obtained endoscopically. Intestinal CYP3A activity was determined from the rate of 1′-hydroxymidazolam and 4-hydroxymidazolam formation by incubation of small-bowel homogenate with midazolam (25 Μmol/L) and NADPH for 5 minutes. Intestinal CYP3A4 and CYP3A5 messenger ribonucleic acid was quantified by real-time reverse transcriptase-polymerase chain reaction. Intestinal CYP3A4 and CYP3A5 protein concentrations were determined by immunoblotting. Serum and homogenate concentrations of midazolam, clarithromycin, and metabolites were determined by liquid chromatography-mass spectrometry. CYP3A5 genotype was determined by real-time polymerase chain reaction. Results The formation of 1′-hydroxymidazolam (1.36 ± 0.46 pmol · min−1 · mg−1 at baseline versus 0.35 ± 0.16 pmol · min−1 · mg−1 after administration) and 4-hydroxymidazolam (0.39 ± 0.12 pmol · min−1 · mg−1 at baseline versus 0.12 ± 0.05 pmol · min−1 · mg−1 after administration) was significantly (P < .001) reduced after clarithromycin administration. Clarithromycin administration did not result in a significant change in intestinal CYP3A4 and CYP3A5 messenger ribonucleic acid and protein expression. All subjects had detectable serum clarithromycin concentrations after 7 days of clarithromycin (3.71 ± 2.43 Μmol/L). The mean concentration of clarithromycin in the intestinal biopsy homogenate was 1.2 ± 0.7 nmol/L (range, 0.42–2.39 nmol/L). Compared with CYP3A5 nonexpressers, subjects with at least 1 CYP3A5*1 allele (CYP3A5 expressers) had greater inhibition of intestinal CYP3A activity after treatment with clarithromycin. There was a strong linear relationship between the decrease in intestinal CYP3A activity and baseline catalytic activity (R2 = 0.9). Conclusion Baseline intestinal activity of CYP3A4 was a key determinant of variability of the inhibitory effect of clarithromycin among individuals. CYP3A5*1 alleles were associated with greater baseline intestinal CYP3A activity and, therefore, greater extent of inhibition. The primary in vivo mechanism was not rapidly reversible competitive or irreversible inhibition but was likely formation of metabolic intermediate complexes. Clinical Pharmacology & Therapeutics (2005) 77, 178–188; doi: 10.1016/j.clpt.2004.10.002
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regioselective biotransformation of midazolam by members of the human cytochrome p450 3a cyp3a subfamily
Biochemical Pharmacology, 1994Co-Authors: Stephen D. Hall, Christopher J Gorski, David R Jones, Mark VandenbrandenAbstract:Abstract The capabilities of cytochrome P4503A4 (CYP3A4), CYP3A5, and fetal hepatic microsomes containing CYP3A7 to metabolize midazolam were investigated using human hepatic microsomes and purified CYP3A4 and CYP3A5. Under initial rate conditions and high substrate concentration (400 μM midazolam), variability among eighteen human liver microsomal samples was 30- and 16- fold for 1′- and 4-hydroxylation of midazolam, respectively. Exclusion of two samples isolated from patients previously administered barbiturates reduced the inter-individual variability to 10.5- and 6.0-fold for 1′- and 4-hydroxylation, respectively. Six fetal hepatic microsomal samples showed 10-fold variation in both 1′-hydroxymidazolam and 4-hydroxymidazolam formation rates. The rates of formation of 4-hydroxymidazolam and 1′-hydroxymidazolam from midazolam by adult samples containing only CYP3A4 and by fetal liver samples were highly correlated ( r 2 =0.99 and 0.97, P r 2 =0.95 and 0.92, respectively), 6β-hydroxylate testosterone ( r 2 =0.96 and 0.96, respectively), and the CYP3A4 content of the samples ( r 2 =0.89 and 0.86, respectively). Microsomal samples containing CYP3A5 in addition to CYP3A4 exhibited a significantly greater ration of 1′-hydroxymidazolam to 4-hydroxymidazolan compared with samples containing only CYP3A4 or CYP3A7 (P b 5 , and NADPH-cytochrome P450 reductase, and an NADPH-regenerating system displayed a 2-fold greater rate of 1′-hydroxymidazolam formation and a similar rate of 4-hydroxymidazolam formation compared with a reconstituted system with CYP3A4. In conclusion, CYP3A4, CYP3A5, and fetal microsomes containing CYP3A7 catalyze 1′- and 4-hydroxylation of midazolam with the ratio of these metabolites indicative of the CYP3A form.
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regioselective biotransformation of midazolam by members of the human cytochrome p450 3a cyp3a subfamily
Biochemical Pharmacology, 1994Co-Authors: Stephen D. Hall, Christopher J Gorski, David R Jones, Mark VandenbrandenAbstract:The capabilities of cytochrome P4503A4 (CYP3A4), CYP3A5, and fetal hepatic microsomes containing CYP3A7 to metabolize midazolam were investigated using human hepatic microsomes and purified CYP3A4 and CYP3A5. Under initial rate conditions and high substrate concentration (400 microM midazolam), variability among eighteen human liver microsomal samples was 30- and 16- fold for 1'- and 4-hydroxylation of midazolam, respectively. Exclusion of two samples isolated from patients previously administered barbiturates reduced the inter-individual variability to 10.5- and 6.0-fold for 1'- and 4-hydroxylation, respectively. Six fetal hepatic microsomal samples showed 10-fold variation in both 1'-hydroxymidazolam and 4-hydroxymidazolam formation rates. The rates of formation of 4-hydroxymidazolam and 1'-hydroxymidazolam from midazolam by adult samples containing only CYP3A4 and by fetal liver samples were highly correlated (r2 = 0.99 and 0.97, P < 0.01, respectively). The rates of formation of 1'-hydroxymidazolam and 4-hydroxymidazolam from midazolam (400 microM) by adult samples that contained only CYP3A4 were correlated significantly (P < 0.01) with the ability of the samples to N-demethylate erythromycin (r2 = 0.95 and 0.92, respectively). 6 beta-hydroxylate testosterone (r2 = 0.96 and 0.96, respectively), and the CYP3A4 content of the samples (r2 = 0.89 and 0.86, respectively). Microsomal samples containing CYP3A5 in addition to CYP3A4 exhibited a significantly greater ratio of 1'-hydroxymidazolam to 4-hydroxymidazolam compared with samples containing only CYP3A4 or CYP3A7 (P < 0.001). Purified CYP3A5 in a reconstituted system, consisting of dilauroylphosphatidylcholine, cytochrome b5, and NADPH-cytochrome P450 reductase, and an NADPH-regenerating system displayed a 2-fold greater rate of 1'-hydroxymidazolam formation and a similar rate of 4-hydroxymidazolam formation compared with a reconstituted system with CYP3A4. In conclusion, CYP3A4, CYP3A5, and fetal microsomes containing CYP3A7 catalyze 1'- and 4-hydroxylation of midazolam with the ratio of these metabolites indicative of the CYP3A form.
Mark Vandenbranden - One of the best experts on this subject based on the ideXlab platform.
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bimodal distribution of renal cytochrome p450 3a activity in humans
Molecular Pharmacology, 1996Co-Authors: Barbara D Haehner, S K Janardan, Steven A Wrighton, Mark Vandenbranden, Paul B Watkins, J. C. Gorski, Stephen D. HallAbstract:It has been proposed that excessive intrarenal conversion of cortisol to 6 beta-hydroxycortisol by CYP3A may mediate increased tubular reabsorption of sodium, leading to a state of mild volume expansion and the clinical phenotype of salt-sensitive hypertension. Therefore, we characterized CYP3A activity in a bank of microsomes from human kidneys using the formation of 1'-hydroxymidazolam (1'-OHM) as a prototypical CYP3A-catalyzed reaction. Maximal rates of metabolite formation occurred at midazolam concentrations of 12.5-50 microM; higher concentrations resulted in dramatic substrate inhibition. At 12.5 microM midazolam, 4 of 27 kidneys exhibited relatively high mean +/- standard deviation 1'-OHM formation rate (184.0 +/- 14.4 pmol/hr/mg) compared with the remaining 23 samples, which had a mean formation rate of (10.1 +/- 6.4 pmol/hr/mg). Triacetyloleandomycin and anti-CYP3A antibody inhibited midazolam hydroxylation by 53% and 57%, respectively. The correlation between CYP3A5 content, determined through immunoblotting, and 1'-OHM formation rate was high (r2 = 0.84, 24 experiments). The expressions of mRNA corresponding to CYP3A3, CYP3A4, CYP3A5, and CYP3A7 were determined through polymerase chain reaction with specific oligonucleotides as primers. All kidneys examined (25 experiments) expressed CYP3A5 protein and contained the corresponding CYP3A5 mRNA. CYP3A4 mRNA was detected in 40% of the kidney samples, and 70% of those that contained detectable CYP3A4 mRNA also expressed detectable levels of the corresponding protein. Therefore, in contrast to hepatic tissue, in which CYP3A4 is universally expressed, CYP3A5 is the ubiquitously expressed member of the CYP3A family in renal tissue. The distribution of enzyme activity and protein content suggests bimodality and may represent induction of CYP3A5 in a select population and/or a genetically determined organ-specific pattern of expression.
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regioselective biotransformation of midazolam by members of the human cytochrome p450 3a cyp3a subfamily
Biochemical Pharmacology, 1994Co-Authors: Stephen D. Hall, Christopher J Gorski, David R Jones, Mark VandenbrandenAbstract:Abstract The capabilities of cytochrome P4503A4 (CYP3A4), CYP3A5, and fetal hepatic microsomes containing CYP3A7 to metabolize midazolam were investigated using human hepatic microsomes and purified CYP3A4 and CYP3A5. Under initial rate conditions and high substrate concentration (400 μM midazolam), variability among eighteen human liver microsomal samples was 30- and 16- fold for 1′- and 4-hydroxylation of midazolam, respectively. Exclusion of two samples isolated from patients previously administered barbiturates reduced the inter-individual variability to 10.5- and 6.0-fold for 1′- and 4-hydroxylation, respectively. Six fetal hepatic microsomal samples showed 10-fold variation in both 1′-hydroxymidazolam and 4-hydroxymidazolam formation rates. The rates of formation of 4-hydroxymidazolam and 1′-hydroxymidazolam from midazolam by adult samples containing only CYP3A4 and by fetal liver samples were highly correlated ( r 2 =0.99 and 0.97, P r 2 =0.95 and 0.92, respectively), 6β-hydroxylate testosterone ( r 2 =0.96 and 0.96, respectively), and the CYP3A4 content of the samples ( r 2 =0.89 and 0.86, respectively). Microsomal samples containing CYP3A5 in addition to CYP3A4 exhibited a significantly greater ration of 1′-hydroxymidazolam to 4-hydroxymidazolan compared with samples containing only CYP3A4 or CYP3A7 (P b 5 , and NADPH-cytochrome P450 reductase, and an NADPH-regenerating system displayed a 2-fold greater rate of 1′-hydroxymidazolam formation and a similar rate of 4-hydroxymidazolam formation compared with a reconstituted system with CYP3A4. In conclusion, CYP3A4, CYP3A5, and fetal microsomes containing CYP3A7 catalyze 1′- and 4-hydroxylation of midazolam with the ratio of these metabolites indicative of the CYP3A form.
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regioselective biotransformation of midazolam by members of the human cytochrome p450 3a cyp3a subfamily
Biochemical Pharmacology, 1994Co-Authors: Stephen D. Hall, Christopher J Gorski, David R Jones, Mark VandenbrandenAbstract:The capabilities of cytochrome P4503A4 (CYP3A4), CYP3A5, and fetal hepatic microsomes containing CYP3A7 to metabolize midazolam were investigated using human hepatic microsomes and purified CYP3A4 and CYP3A5. Under initial rate conditions and high substrate concentration (400 microM midazolam), variability among eighteen human liver microsomal samples was 30- and 16- fold for 1'- and 4-hydroxylation of midazolam, respectively. Exclusion of two samples isolated from patients previously administered barbiturates reduced the inter-individual variability to 10.5- and 6.0-fold for 1'- and 4-hydroxylation, respectively. Six fetal hepatic microsomal samples showed 10-fold variation in both 1'-hydroxymidazolam and 4-hydroxymidazolam formation rates. The rates of formation of 4-hydroxymidazolam and 1'-hydroxymidazolam from midazolam by adult samples containing only CYP3A4 and by fetal liver samples were highly correlated (r2 = 0.99 and 0.97, P < 0.01, respectively). The rates of formation of 1'-hydroxymidazolam and 4-hydroxymidazolam from midazolam (400 microM) by adult samples that contained only CYP3A4 were correlated significantly (P < 0.01) with the ability of the samples to N-demethylate erythromycin (r2 = 0.95 and 0.92, respectively). 6 beta-hydroxylate testosterone (r2 = 0.96 and 0.96, respectively), and the CYP3A4 content of the samples (r2 = 0.89 and 0.86, respectively). Microsomal samples containing CYP3A5 in addition to CYP3A4 exhibited a significantly greater ratio of 1'-hydroxymidazolam to 4-hydroxymidazolam compared with samples containing only CYP3A4 or CYP3A7 (P < 0.001). Purified CYP3A5 in a reconstituted system, consisting of dilauroylphosphatidylcholine, cytochrome b5, and NADPH-cytochrome P450 reductase, and an NADPH-regenerating system displayed a 2-fold greater rate of 1'-hydroxymidazolam formation and a similar rate of 4-hydroxymidazolam formation compared with a reconstituted system with CYP3A4. In conclusion, CYP3A4, CYP3A5, and fetal microsomes containing CYP3A7 catalyze 1'- and 4-hydroxylation of midazolam with the ratio of these metabolites indicative of the CYP3A form.
