The Experts below are selected from a list of 162977619 Experts worldwide ranked by ideXlab platform
Stephen D. Hall - One of the best experts on this subject based on the ideXlab platform.
-
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.
-
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
-
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.
-
Mechanism-Based Inactivation of CYP3A by HIV Protease Inhibitors
2004Co-Authors: C. Steven, 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 de-creases in activity as defined by maximum rate of inactivation (kinact) and inhibitor concentration that gives 50 % maximal in-activation (KI) of CYP3A by amprenavir, indinavir, lopinavir, nelfinavir, ritonavir, and saquinavir were quantified using tes-tosterone 6-hydroxylation as a marker for CYP3A activity with recombinant CYP3A4(b5), recombinant CYP3A5, and pooled human liver microsomes (HLMs). All the PIs, except indinavir, displayed inactivation with CYP3A4(b5) and HLMs. Ritonavir was the most potent (KI 0.10 and 0.17 M) and demon-strated high kinact values (0.32 and 0.40 min 1) with bot
-
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, 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.
Christopher J Gorski - One of the best experts on this subject based on the ideXlab platform.
-
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
-
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.
-
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.
-
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, 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.
-
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.
-
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.
David R Jones - One of the best experts on this subject based on the ideXlab platform.
-
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.
-
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.
-
Mechanism-Based Inactivation of CYP3A by HIV Protease Inhibitors
2004Co-Authors: C. Steven, 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 de-creases in activity as defined by maximum rate of inactivation (kinact) and inhibitor concentration that gives 50 % maximal in-activation (KI) of CYP3A by amprenavir, indinavir, lopinavir, nelfinavir, ritonavir, and saquinavir were quantified using tes-tosterone 6-hydroxylation as a marker for CYP3A activity with recombinant CYP3A4(b5), recombinant CYP3A5, and pooled human liver microsomes (HLMs). All the PIs, except indinavir, displayed inactivation with CYP3A4(b5) and HLMs. Ritonavir was the most potent (KI 0.10 and 0.17 M) and demon-strated high kinact values (0.32 and 0.40 min 1) with bot
-
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.
-
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.
Kenneth E. Thummel - One of the best experts on this subject based on the ideXlab platform.
-
identification of cyp3a7 for glyburide metabolism in human fetal livers
Biochemical Pharmacology, 2014Co-Authors: Diana L. Shuster, Justina C Calamia, Jenna L Voellinger, Mary F. Hebert, Bhagwat Prasad, Linda J Risler, Jashvant D Unadkat, Danny D Shen, Edward J Kelly, Kenneth E. ThummelAbstract:Abstract Glyburide is commonly prescribed for the treatment of gestational diabetes mellitus; however, fetal exposure to glyburide is not well understood and may have short- and long-term consequences for the health of the child. Glyburide can cross the placenta; fetal concentrations at term are nearly comparable to maternal levels. Whether or not glyburide is metabolized in the fetus and by what mechanisms has yet to be determined. In this study, we determined the kinetic parameters for glyburide depletion by CYP3A isoenzymes; characterized glyburide metabolism by human fetal liver tissues collected during the first or early second trimester of pregnancy; and identified the major enzyme responsible for glyburide metabolism in human fetal livers. CYP3A4 had the highest metabolic capacity towards glyburide, followed by CYP3A7 and CYP3A5 (Clint,u = 37.1, 13.0, and 8.7 ml/min/nmol P450, respectively). M5 was the predominant metabolite generated by CYP3A7 and human fetal liver microsomes (HFLMs) with approximately 96% relative abundance. M5 was also the dominant metabolite generated by CYP3A4, CYP3A5, and adult liver microsomes; however, M1–M4 were also present, with up to 15% relative abundance. CYP3A7 protein levels in HFLMs were highly correlated with glyburide Clint, 16α-OH DHEA formation, and 4′-OH midazolam formation. Likewise, glyburide Clint was highly correlated with 16α-OH DHEA formation. Fetal demographics as well as CYP3A5 and CYP3A7 genotype did not alter CYP3A7 protein levels or glyburide Clint. These results indicate that human fetal livers metabolize glyburide predominantly to M5 and that CYP3A7 is the major enzyme responsible for glyburide metabolism in human fetal livers.
-
pharmacogenetic determinants of human liver microsomal alfentanil metabolism and the role of cytochrome p450 3a5
Anesthesiology, 2005Co-Authors: Theresa Mariero Klees, Kenneth E. Thummel, Pamela Sheffels, Evan D. KharaschAbstract:Background: There is considerable unexplained interindividual variability in the clearance of alfentanil. Alfentanil undergoes extensive metabolism by cytochrome P4503A4 (CYP3A4). CYP3A5 is structurally similar to CYP3A4 and metabolizes most CYP3A4 substrates but is polymorphically expressed. Livers with the CYP3A5*1 allele contain higher amounts of the native CYP3A5 protein than livers homozygous for the mutant CYP3A5*3 allele. This investigation tested the hypothesis that alfentanil is a substrate for CYP3A5 and that CYP3A5 pharmacogenetic variability influences human liver alfentanil metabolism. Methods: Alfentanil metabolism to noralfentanil and N-phenylpropionamide was determined in microsomes from two groups of human livers, characterized for CYP3A4 and CYP3A5 protein content: low CYP3A5 (2.0-5.2% of total CYP3A, n = 10) and high CYP3A5 (46-76% of total CYP3A, n = 10). Mean CYP3A4 content was the same in both groups. The effects of the CYP3A inhibitors troleandomycin and ketoconazole, the latter being more potent toward CYP3A4, on alfentanil metabolism were also determined. Results: In the low versus high CYP3A5 livers, respectively, noralfentanil formation was 77 ± 31 versus 255 ± 170 pmol . min -1 . mg -1 , N-phenylpropionamide formation was 8.0 ± 3.1 versus 20.5 ± 14.0 pmol . min -1 . mg -1 , and the metabolite ratio was 9.5 ± 0.4 versus 12.7 ± 1.4 (P < 0.05 for all). There was a poor correlation between alfentanil metabolism and CYP3A4 content but an excellent correlation when CYP3A5 (i.e., total CYP3A content) was considered (r 2 = 0.81, P < 0.0001). Troleandomycin inhibited alfentanil metabolism similarly in the low and high CYP3A5 livers; ketoconazole inhibition was less in the high CYP3A5 livers. Conclusion: In microsomes from human livers expressing the CYP3A5*1 allele and containing higher amounts of CYP3A5 protein, compared with those with the CYP3A5*3 allele and little CYP3A5, there was greater alfentanil metabolism, metabolite ratios more closely resembled those for expressed CYP3A5, and inhibitors with differing CYP3A4 and CYP3A5 selectivities had effects resembling those for expressed CYP3A5. Therefore, alfentanil is metabolized by human liver microsomal CYP3A5 in addition to CYP3A4, and pharmacogenetic variability in CYP3A5 expression significantly influences human liver alfentanil metabolism in vitro. Further investigation is warranted to assess whether the CYP3A5 polymorphism is a factor in the interindividual variability of alfentanil metabolism and clearance in vivo.
-
co regulation of cyp3a4 and CYP3A5 and contribution to hepatic and intestinal midazolam metabolism
Molecular Pharmacology, 2002Co-Authors: Yvonne S Lin, Amy L S Dowling, Sean D Quigley, Federico M Farin, Jiong Zhang, Jatinder K Lamba, Erin G Schuetz, Kenneth E. ThummelAbstract:We recently demonstrated that a variant allele of CYP3A5 (CYP3A5*3) confers low CYP3A5 expression as a result of improper mRNA splicing. In this study, we further evaluated the regulation of CYP3A5 in liver and jejunal mucosa from white donors. For all tissues, high levels of CYP3A5 protein were strongly concordant with the presence of a wild-type allele of the CYP3A5 gene (CYP3A5*1). CYP3A5 represented greater than 50% of total CYP3A content in nearly all of the livers and jejuna that carried the CYP3A5*1 wild-type allele. Overall, CYP3A5 protein content accounted for 31% of the variability in hepatic midazolam hydroxylation activity. Improperly spliced mRNA (SV1-CYP3A5) was found only in tissues containing a CYP3A5*3 allele. Properly spliced CYP3A5 mRNA (wt-CYP3A5) was detected in all tissues, but the median wt-CYP3A5 mRNA was 4-fold higher in CYP3A5*1/*3 livers compared with CYP3A5*3/*3 livers. Differences in wt-CYP3A5 and CYP3A4 mRNA content explained 53 and 51% of the interliver variability in CYP3A5 and CYP3A4 content, respectively. Hepatic CYP3A4 and CYP3A5 contents were not correlated when all livers were compared. However, for CYP3A5*1/*3 livers, levels of the two proteins were strongly correlated (r = 0.93) as were wt-CYP3A5 and CYP3A4 mRNA (r = 0.76). These findings suggest that CYP3A4 and CYP3A5 genes share a common regulatory pathway for constitutive expression, possibly involving conserved elements in the 5'-flanking region.
-
inhibition of cytochrome p 450 3a cyp3a in human intestinal and liver microsomes comparison of ki values and impact of CYP3A5 expression
Drug Metabolism and Disposition, 1999Co-Authors: Megan A Gibbs, Kenneth E. Thummel, Danny D Shen, Kent L KunzeAbstract:The purpose of this study was to compare the kinetics of intestinal and hepatic cytochrome P-450 3A (CYP3A) inhibition by using microsomal midazolam 1′-hydroxylation as a marker of enzyme activity. The effect of two antifungal agents commonly implicated in CYP3A drug-drug interactions was examined. Inhibition type and affinities were determined for human liver and intestinal microsomes screened for the presence or absence of CYP3A4 and CYP3A5, as well as for cDNA-expressed CYP3A4 and CYP3A5 microsomes. Ketoconazole and fluconazole were found to be noncompetitive inhibitors of both enzymes. Ketoconazole exhibited a K i for cDNA-expressed CYP3A4 of 26.7 ± 1.71 nM, whereas the K i for cDNA expressed CYP3A5 was 109 ± 19.7 nM. Corresponding K i values for fluconazole were 9.21 ± 0.51 μM and 84.6 ± 12.9 μM. For liver and intestinal microsomes that contained only CYP3A4, the average ketoconazole K i was found to be 14.9 ± 6.7 nM and 17.0 ± 7.9 nM, respectively, whereas fluconazole yielded mean respective K i values of 10.7 ± 4.2 μM and 10.4 ± 2.9 μM. Liver and intestinal microsomes that contained an equal or greater amount of CYP3A5, in addition to CYP3A4, were less susceptible to inhibition by both ketoconazole and fluconazole. These findings suggest that there can be significant differences in the affinity of these two enzymes for inhibitors. This may further broaden interindividual variability with respect to the magnitude of in vivo drug-drug interactions. We also conclude that there is no significant difference in inhibition type and affinity of ketoconazole and fluconazole for hepatic versus intestinal CYP3A4.
-
expression of enzymatically active cyp3a4 by caco 2 cells grown on extracellular matrix coated permeable supports in the presence of 1α 25 dihydroxyvitamin d3
Molecular Pharmacology, 1997Co-Authors: Phyllissa Schmiedlinren, Kenneth E. Thummel, Jeannine M Fisher, Mary F Paine, Kenneth S LownAbstract:The human colon carcinoma cell line, Caco-2, is widely used as a model for oral absorption of xenobiotics. The usefulness of Caco-2 cells has been limited, however, because they do not express appreciable quantities of CYP3A4, the principle cytochrome P450 present in human small bowel epithelial cells. We report that treatment of Caco-2 cells with 1 alpha,25-dihydroxyvitamin D3, beginning at confluence, results in a dose- and duration-dependent increase in CYP3A4 mRNA and protein, with little apparent effect on the expression of CYP3A5 or CYP3A7. This treatment also results in increases in NADPH cytochrome P450 reductase and P-glycoprotein (the MDR1 gene product) but has no detectable effect on expression of CYP1A1, CYP2D6, cytochrome b5, liver or intestinal fatty acid binding proteins, or villin. Maximal expression of CYP3A4 requires an extracellular matrix on a permeable support and the presence of serum. In the treated cells, the intrinsic formation clearance of 1'-hydroxymidazolam (a reaction characteristically catalyzed by CYP3A enzymes) was estimated to be somewhat lower than that of human jejunal mucosa (1.14 and 3.67 ml/min/g of cells, respectively). The 1'-OH-midazolam/4-OH-midazolam product ratio produced by the cells (approximately 5.3) is comparable to, but somewhat lower than, that observed in human jejunal microsomes (7.4-15.4), which may reflect the presence of CYP3A7 in the Caco-2 cells. 25-Hydroxyvitamin D3 is less efficacious but reproduces the effects of the dihydroxy compound, whereas unhydroxylated vitamin D is without appreciable effect. These observations, together with the time course of response, suggest that the vitamin D receptor may be involved in CYP3A4 regulation. The culture model we describe should prove useful in defining the role of CYP3A4 in limiting the oral bioavailability of many xenobiotics.
