[Show abstract][Hide abstract] ABSTRACT: Acetaminophen is cleared primarily by hepatic glucuronidation. Polymorphisms in genes encoding the acetaminophen UDP-glucuronosyltransferase (UGT) enzymes could explain interindividual variability in acetaminophen glucuronidation and variable risk for liver injury following acetaminophen overdose. In this study, human liver bank samples were phenotyped for acetaminophen glucuronidation activity and genotyped for the major acetaminophen glucuronidating enzymes (UGTs 1A1, 1A6, 1A9 and 2B15). Of these, only 3 linked SNPs located in the shared UGT1A-3'UTR region (rs10929303, rs1042640, rs8330) were associated with acetaminophen glucuronidation activity, with rs8330 consistently showing higher acetaminophen glucuronidation at all acetaminophen concentrations tested. Mechanistic studies using luciferase-UGT1A-3'UTR reporters indicated that these SNPs do not alter mRNA stability or translation efficiency. However, there was evidence for allelic imbalance and a gene-dose proportional increase in the amount of exon 5a versus exon 5b containing UGT1A mRNA spliced transcripts in livers with the rs8330 variant allele. Cotransfection studies demonstrated an inhibitory effect of exon 5b containing cDNAs on acetaminophen glucuronidation by UGT1A1 and UGT1A6 cDNAs containing exon 5a. In silico analysis predicted that rs8330 creates an exon splice enhancer site that could favor exon 5a (over exon 5b) utilization during splicing. Finally, the prevalence of rs8330 was significantly lower (p=0.027, Chi-squared test) in patients who had acute liver failure from unintentional acetaminophen overdose, as compared to patients with acute liver failure from other causes, or a race/ethnicity matched population. Together these findings suggest that rs8330 is an important determinant of acetaminophen glucuronidation, and could affect an individual's risk for acetaminophen-induced liver injury.
Preview · Article · Feb 2013 · Journal of Pharmacology and Experimental Therapeutics
[Show abstract][Hide abstract] ABSTRACT: Ketoconazole is extensively used as an index inhibitor of cytochrome P450-3A (CYP3A) activity in vitro and in vivo, but the mechanism of ketoconazole inhibition of CYP3A still is not clearly established.
Inhibition of metabolite formation by ketoconazole (seven concentrations from 0.01 to 1.0 µm) was studied in human liver microsomes (n = 4) at six to seven substrate concentrations for triazolam, midazolam, and testosterone, and at two substrate concentrations for nifedipine.
Analysis of multiple data points per liver sample based on a mixed competitive-noncompetitive model yielded mean inhibition constant K(i) values in the range of 0.011 to 0.045 µm. Ketoconazole IC50 increased at higher substrate concentrations, thereby excluding pure noncompetitive inhibition. For triazolam, testosterone, and midazolam α-hydroxylation, mean values of α (indicating the 'mix' of competitive and noncompetitive inhibition) ranged from 2.1 to 6.3. However, inhibition of midazolam 4-hydroxylation was consistent with a competitive process. Determination of K(i) and α based on the relation between 50% inhibitory concentration values and substrate concentration yielded similar values. Pre-incubation of ketoconazole with microsomes before addition of substrate did not enhance inhibition, whereas inhibition by troleandomycin was significantly enhanced by pre-incubation.
Ketoconazole inhibition of triazolam α- and 4-hydroxylation, midazolam α-hydroxylation, testosterone 6β-hydroxylation, and nifedipine oxidation appeared to be a mixed competitive-noncompetitive process, with the noncompetitive component being dominant but not exclusive. Quantitative estimates of K(i) were in the low nanomolar range for all four substrates.
[Show abstract][Hide abstract] ABSTRACT: CYP2E1 is widely accepted as the sole form of cytochrome P450 responsible for alcohol-mediated increases in acetaminophen (APAP) hepatotoxicity. However, we previously found that alcohol [ethanol and isopentanol (EIP)] causes increases in APAP hepatotoxicity in Cyp2e1(-/-) mice, indicating that CYP2E1 is not essential. Here, using wild-type and Cyp2e1(-/-) mice, we investigated the relative roles of CYP2E1 and CYP3A in EIP-mediated increases in APAP hepatotoxicity. We found that EIP-mediated increases in APAP hepatotoxicity occurred at lower APAP doses in wild-type mice (300 mg/kg) than in Cyp2e1(-/-) mice (600 mg/kg). Although this result suggests that CYP2E1 has a role in the different susceptibilities of these mouse lines, our findings that EIP-mediated increases in CYP3A activities were greater in wild-type mice compared with Cyp2e1(-/-) mice raises the possibility that differential increases in CYP3A may also contribute to the greater APAP sensitivity in EIP-pretreated wild-type mice. At the time of APAP administration, which followed an 11 h withdrawal from the alcohols, alcohol-induced levels of CYP3A were sustained in both mouse lines, whereas CYP2E1 was decreased to constitutive levels in wild-type mice. The CYP3A inhibitor triacetyloleandomycin (TAO) decreased APAP hepatotoxicity in EIP-pretreated wild-type and Cyp2e1(-/-) mice. TAO treatment in vivo resulted in inhibition of microsomal CYP3A-catalyzed activity, measured in vitro, with no inhibition of CYP1A2 and CYP2E1 activities. In conclusion, these findings suggest that both CYP3A and CYP2E1 contribute to APAP hepatotoxicity in alcohol-treated mice.
No preview · Article · Jul 2007 · Drug Metabolism and Disposition
[Show abstract][Hide abstract] ABSTRACT: The pregnane X receptor (PXR) is a transcriptional regulator of xenobiotic metabolizing enzymes, including cytochrome P450 3A (CYP3A), and transporters. Pretreatment of mice and rats with inducers of CYP3A increases acetaminophen (APAP) hepatotoxicity. In untreated mice, the amount of hepatic CYP3A11 mRNA is 4-fold greater in PXR(-/-) mice compared to wild-type mice (Guo et al., 2003), a finding anticipated to increase APAP hepatotoxicity in PXR(-/-) mice. We investigated APAP hepatotoxicity in wild-type and PXR(-/-) mice in a C57BL/6 background, with APAP administered by gavage. Despite a 2.5-fold higher level of total hepatic CYP3A protein and a 3.6-fold higher level of CYP3A activity compared to wild-type mice, PXR(-/-) mice were less sensitive to APAP hepatotoxicity. Hepatic levels of CYP2E1 were identical in the two mouse lines, but hepatic CYP1A2 levels were 3-fold greater in wild-type mice compared to PXR(-/-) mice. Caffeine, an inhibitor of CYP1A2 activity and an enhancer of CYP3A activity, decreased APAP hepatotoxicity in wild-type mice. APAP uptake was 1.5-fold greater in wild-type mice compared to PXR(-/-) mice. No significant differences in the formation of APAP glucuronide and sulfate-conjugated metabolites were observed between wild-type and PXR(-/-) mice. Glutathione levels were similar in the two mouse lines and were transiently decreased to similar amounts after APAP administration. Our finding that APAP hepatotoxicity was decreased in PXR(-/-) mice indicates that PXR is an important modulator of APAP hepatotoxicity, through positive modulation of constitutive CYP1A2 expression and possibly through increased APAP absorption.
Preview · Article · Jan 2006 · Drug Metabolism and Disposition
[Show abstract][Hide abstract] ABSTRACT: The effect of atazanavir on P-glycoprotein (P-gp) expression and activity, as well as its inhibitory potency against CYP3A activity, was evaluated in vitro. Induction of P-gp activity and expression was studied using LS180V cells. P-gp inhibition was studied using both LS180V cells and Caco-2 cells. P-gp activity was assessed by measuring P-gp-mediated rhodamine 123 (Rh123) transport, and P-gp expression was determined using SDS-polyacrylamide gel electrophoresis/Western blot analysis. CYP3A inhibition was tested using triazolam hydroxylation in human liver microsomes (HLM). Extended (3-day) exposure of LS180V cells to 30 microM atazanavir caused a 2.5-fold increase in immunoreactive P-gp expression as well as a concentration-dependent decrease of intracellular Rh123 to a mean 45% (S.D. 5.2%) of control. Acute exposure (2 h) of LS180V cells to atazanavir increased intracellular Rh123 concentrations up to 300% of control at 100 microM atazanavir. At 30 microM and above, acute atazanavir exposure reversed P-gp induction caused by 3-day pretreatment with 10 microM ritonavir. P-gp inhibition was also observed in Caco-2 cells, causing an effect comparable to that observed for the known P-gp inhibitor verapamil (50% of control). In HLM, atazanavir was an inhibitor of triazolam hydroxylation, with inhibitory potency greatly increased by preincubation. IC50 values with and without preincubation were 0.31 microM (S.D. 0.13) and 5.7 microM (S.D. 4.1), respectively. Thus, atazanavir is an inhibitor and inducer of P-gp as well as a potent inhibitor of CYP3A in vitro, suggesting a potential for atazanavir to cause drug-drug interactions in vivo.
No preview · Article · Jul 2005 · Drug Metabolism and Disposition
[Show abstract][Hide abstract] ABSTRACT: UDP-glucuronosyltransferase 2B7 (UGT2B7) is involved in the glucuronidation of a wide array of clinically important drugs and endogenous compounds in humans. The aim of this study was to identify an isoform-selective probe substrate that could be used to investigate genetic and environmental influences on glucuronidation mediated by UGT2B7. Three potential probe substrates [3'-azido-3'-deoxythymidine (AZT), morphine, and codeine], were evaluated using recombinant UGTs and human liver microsomes (HLMs; n = 54). Of 11 different UGTs screened, UGT2B7 was the principal isoform mediating AZT glucuronidation, morphine-3-glucuronidation, and morphine-6-glucuronidation. Codeine was glucuronidated equally well by UGT2B4 and UGT2B7. Enzyme kinetic analysis of these activities typically showed higher apparent Km values for HLMs (pooled and individual) compared with UGT2B7. This difference was least (less than 2-fold higher Km) for AZT glucuronidation and greatest (3- to 6-fold higher Km) for codeine glucuronidation. Microsomal UGT2B7 protein content correlated well with AZT glucuronidation (rs = 0.77), to a lesser extent with morphine-3-glucuronidation (rs = 0.50) and morphine-6-glucuronidation (rs = 0.51), but very weakly with codeine glucuronidation (rs = 0.33). Livers were also genotyped for the UGT2B7*2 (H268Y) polymorphism. No effect of genotype on microsomal glucuronidation or UGT2B7 protein content was observed. In conclusion, although both AZT and morphine can serve as in vitro probe substrates for UGT2B7, AZT appears to be more selective than morphine. Codeine is not a useful UGT2B7 probe substrate because of significant glucuronidation by UGT2B4. The UGT2B7*2 polymorphism is not a determinant of glucuronidation of AZT, morphine, or codeine in HLMs.
No preview · Article · Oct 2003 · Drug Metabolism and Disposition
[Show abstract][Hide abstract] ABSTRACT: 1. The main purpose was to develop a high-performance liquid chromatography (HPLC)-based method to assay serotonin glucuronidation activity using liver microsomal fractions. Application of this method was then demonstrated by determining serotonin UDP-glucuronosyltransferase (UGT) enzyme kinetics using human liver microsomes and recombinant human UGT1A6. Interspecies differences were also evaluated using liver microsomes from 10 different mammalian species. 2. Incubation of liver microsomes with serotonin, UDP-glucuronic acid and magnesium resulted in the formation of a single product peak using HPLC with fluorescence and ultraviolet absorbance detection. This peak was confirmed as serotonin glucuronide based on sensitivity to beta-glucuronidase and by obtaining the expected mass of 352 with positive-ion mass spectrometry. 3. Following a preparative HPLC isolation, the structure of this metabolite was established as serotonin-5-O-glucuronide by (1)H-NMR spectroscopy. 4. Enzyme kinetic studies showed apparent K(m) and V(max) of 8.8 +/- 0.3 mM and 43.4 +/- 0.4 nmoles min(-1) mg(-1) protein, respectively, for human liver microsomes, and 5.9 +/- 0.2 mM and 15.8 +/- 0.2 nmoles min(-1) mg(-1), respectively, for recombinant UGT1A6. 5. The order of serotonin-UGT activities in animal liver microsomes was rat > mouse > human > cow > pig > horse > dog > rabbit > monkey > ferret. Cat livers showed no serotonin-UGT activity. Heterozygous and homozygous mutant Gunn rat livers had 40 and 13%, respectively, of the activity of the normal Wistar rat, indicating a significant contribution by a rat UGT1A isoform to serotonin glucuronidation. 6. This assay provides a novel sensitive and specific technique for the measurement of serotonin-UGT activity in vitro.
[Show abstract][Hide abstract] ABSTRACT: Investigation of human UDP-glucuronosyltransferase (UGT) isoforms has been limited by a lack of specific substrate probes. In this study serotonin was evaluated for use as a probe substrate for human UGT1A6 using recombinant human UGTs and tissue microsomes. Of the 10 commercially available recombinant UGT isoforms, only UGT1A6 catalyzed serotonin glucuronidation. Serotonin-UGT activity at 40 mM serotonin concentration varied more than 40-fold among human livers (n = 54), ranging from 0.77 to 32.9 nmol/min/mg of protein with a median activity of 7.1 nmol/min/mg of protein. Serotonin-UGT activity kinetics of representative human liver microsomes (n = 7) and pooled human kidney, intestinal and lung microsomes and recombinant human UGT1A6 typically followed one enzyme Michaelis-Menten kinetics. Serotonin glucuronidation activity in these human liver microsomes had widely varying V(max) values ranging from 0.62 to 51.3 nmol/min/mg of protein but very similar apparent K(m) values ranging from 5.2 to 8.8 mM. Pooled human kidney, intestine, and lung microsomes had V(max) values (mean +/- standard error of the estimates) of 8.8 +/- 0.4, 0.22 +/- 0.00, and 0.03 +/- 0.00 nmol/min/mg of protein (respectively) and apparent K(m) values of 6.5 +/- 0.9, 12.4 +/- 2.0, and 4.9 +/- 3.3 mM (respectively). In comparison, recombinant UGT1A6 had a V(max) of 4.5 +/- 0.1 nmol/min/mg of protein and an apparent K(m) of 5.0 +/- 0.4 mM. A highly significant correlation was found between immunoreactive UGT1A6 protein content and serotonin-UGT activity measured at 4 mM serotonin concentration in human liver microsomes (R(s) = 0.769; P < 0.001) (n = 52). In conclusion, these results indicate that serotonin is a highly selective in vitro probe substrate for human UGT1A6.
No preview · Article · Jan 2003 · Drug Metabolism and Disposition
[Show abstract][Hide abstract] ABSTRACT: (R,S)-Oxazepam is a 1,4-benzodiazepine anxiolytic drug that is metabolized primarily by hepatic glucuronidation. In previous studies, S-oxazepam (but not R-oxazepam) was shown to be polymorphically glucuronidated in humans. The aim of the present study was to identify UDP-glucuronosyltransferase (UGT) isoforms mediating R- and S-oxazepam glucuronidation in human liver, with the long term objective of elucidating the molecular genetic basis for this drug metabolism polymorphism. All available recombinant UGT isoforms were screened for R- and S-oxazepam glucuronidation activities. Enzyme kinetic parameters were then determined in representative human liver microsomes (HLMs) and in UGTs that showed significant activity. Of 12 different UGTs evaluated, only UGT2B15 showed significant S-oxazepam glucuronidation. Furthermore, the apparent K(m) for UGT2B15 (29-35 microM) was similar to values determined for HLMs (43-60 microM). In contrast, R-oxazepam was glucuronidated by UGT1A9 and UGT2B7. Although apparent K(m) values for HLMs (256-303 microM) were most similar to UGT2B7 (333 microM) rather than UGT1A9 (12 microM), intrinsic clearance values for UGT1A9 were 10 times higher than for UGT2B7. A common genetic variation results in aspartate (UGT2B15*1) or tyrosine (UGT2B15*2) at position 85 of the UGT2B15 protein. Microsomes from human embryonic kidney (HEK)-293 cells overexpressing UGT2B15*1 showed 5 times higher S-oxazepam glucuronidation activity than did UGT2B15*2 microsomes. Similar results were obtained for other substrates, including eugenol, naringenin, 4-methylumbelliferone, and androstane-3alpha-diol. In conclusion, S-oxazepam is stereoselectively glucuronidated by UGT2B15, whereas R-oxazepam is glucuronidated by multiple UGT isoforms. Allelic variation associated with the UGT2B15 gene may explain polymorphic S-oxazepam glucuronidation in humans.
Preview · Article · Dec 2002 · Drug Metabolism and Disposition
[Show abstract][Hide abstract] ABSTRACT: Aims To determine the human cytochromes mediating biotransformation of the imidazopyridine hypnotic, zolpidem, and the clinical correlates of the findings. Methods Kinetic properties of zolpidem biotransformation to its three hydroxylated metabolites were studied in vitro using human liver microsomes and heterologously expressed individual human cytochromes. Results The metabolic product termed M-3 accounted for more than 80% of net intrinsic clearance by liver microsomes in vitro. Microsomes containing human cytochromes CYP1A2, 2C9, 2C19, 2D6, and 3 A4 expressed by cDNA-transfected human lymphoblastoid cells mediated zolpidem metabolism in vitro. The kinetic profile for zolpidem metabolite formation by each individual cytochrome was combined with estimated relative abundances based on immunological quantification, yielding projected contributions to net intrinsic clearance of: 61% for 3 A4, 22% for 2C9, 14% for 1A2, and less than 3% for 2D6 and 2C19. These values were consistent with inhibitory effects of ketoconazole and sulfaphenazole on zolpidem biotransformation by liver microsomes. Ketoconazole had a 50% inhibitory concentration (IC50 ) of 0.61 μm vs formation of the M-3 metabolite of zolpidem in vitro; in a clinical study, ketoconazole coadministration reduced zolpidem oral clearance by ≈40%, somewhat less than anticipated based on the IC50 value and total plasma ketoconazole levels, but much more than predicted based on unbound plasma ketoconazole levels. Conclusions The incomplete dependence of zolpidem clearance on CYP3A activity has clinical implications for susceptibility to metabolic inhibition.
Full-text · Article · Dec 2001 · British Journal of Clinical Pharmacology
[Show abstract][Hide abstract] ABSTRACT: Interindividual variability in acetaminophen (APAP) glucuronidation may contribute to differences in susceptibility to APAP intoxication in humans. The purpose of this study was to identify the relevant UDP-glucuronosyltransferase (UGT) isoforms mediating APAP-UGT activity in human liver microsomes (HLMs). APAP-UGT activities and enzyme kinetics were determined using HLMs from 56 donors and nine recombinant human UGTs. Activities mediated by UGT1A1, UGT1A4, UGT1A9, and UGT2B7, and relative UGT1A6 protein content were quantified using 20 livers. More than 15-fold variation in liver microsomal APAP-UGT activities was observed with a distribution skewed toward lower activities. Although most UGTs could glucuronidate APAP, UGT1A1, UGT1A6, and UGT1A9 were most active. UGT1A6 was a relatively high-affinity (K(m) = 2.2 mM), low-capacity enzyme; UGT1A1 was intermediate in affinity (K(m) = 9.4 mM) and capacity; and UGT1A9 was a low-affinity (K(m) = 21 mM), high-capacity enzyme. K(m) values were similar to UGT1A1 in high- and intermediate-activity HLMs (6-10 mM) and UGT1A9 in low-activity HLMs (10-55 mM). APAP-UGT activities correlated best with propofol-UGT (r = 0.85; UGT1A9) and bilirubin-UGT (r = 0.66; UGT1A1) activities, but poorly with UGT1A6 protein (r = 0.30). A kinetic model was constructed from these data that identified UGT1A9 as the predominant APAP-UGT (>55% total activity) in HLMs over a clinically relevant APAP concentration range (50 microM-5 mM). UGT1A1 was also predicted to contribute substantially at toxic concentrations (>1 mM; >28% activity), whereas UGT1A6 was most active at relatively low concentrations (<50 microM; >29% activity).
No preview · Article · Dec 2001 · Journal of Pharmacology and Experimental Therapeutics
[Show abstract][Hide abstract] ABSTRACT: Oxidation of propofol to 4-hydroxypropofol represents a significant pathway in the metabolism of this anesthetic agent in humans. The aim of this study was to identify the principal cytochrome P-450 (CYP) isoforms mediating this biotransformation.
Propofol hydroxylation activities and enzyme kinetics were determined using human liver microsomes and cDNA-expressed CYPs. CYP-specific marker activities and CYP2B6 protein content were also quantified in hepatic microsomes for correlational analyses. Finally, inhibitory antibodies were used to ascertain the relative contribution of CYPs to propofol hydroxylation by hepatic microsomes.
Propofol hydroxylation by hepatic microsomes showed more than 19-fold variability and was most closely correlated to CYP2B6 protein content (r = 0.904), and the CYP2B6 marker activities, S-mephenytoin N-demethylation (r = 0.919) and bupropion hydroxylation (r = 0.854). High- and intermediate-activity livers demonstrated high-affinity enzyme kinetics (K(m) < 8 microm), whereas low-activity livers displayed low-affinity kinetics (K(m) > 80 microm). All of the CYPs evaluated were capable of hydroxylating propofol; however, CYP2B6 and CYP2C9 were most active. Kinetic analysis indicated that CYP2B6 is a high-affinity (K(m) = 10 +/- 2 microm; mean +/- SE of the estimate), high-capacity enzyme, whereas CYP2C9 is a low-affinity (K(m) = 41 +/- 8 microm), high-capacity enzyme. Furthermore, immunoinhibition showed a greater contribution of CYP2B6 (56 +/- 22% inhibition; mean +/- SD) compared with CYP2C isoforms (16 +/- 7% inhibition) to hepatic microsomal activity.
Cytochrome P-450 2B6, and to a lesser extent CYP2C9, contribute to the oxidative metabolism of propofol. However, CYP2B6 is the principal determinant of interindividual variability in the hydroxylation of this drug by human liver microsomes.
[Show abstract][Hide abstract] ABSTRACT: The capacity of three clinically available nonnucleoside reverse transcriptase inhibitors (NNRTIs) to inhibit the activity of human cytochromes P450 (CYPs) was studied in vitro using human liver microsomes. Delavirdine, nevirapine, and efavirenz produced negligible inhibition of phenacetin O-deethylation (CYP1A2) or dextromethorphan O-demethylation (CYP2D6). Nevirapine did not inhibit hydroxylation of tolbutamide (CYP2C9) or S-mephenytoin (CYP2C19), but these CYP isoforms were importantly inhibited by delavirdine and efavirenz. This indicates the likelihood of significantly impaired clearance of CYP2C substrate drugs (such as phenytoin, tolbutamide, and warfarin) upon initial exposure to these two NNRTIs. Delavirdine and efavirenz (but not nevirapine) also were strong inhibitors of CYP3A, consistent with clinical hazards of initial cotreatment with either of these drugs and substrates of CYP3A. The in vitro microsomal model provides relevant predictive data on probable drug interactions with NNRTIs when the mechanism is inhibition of CYP-mediated drug biotransformation. However, the model does not incorporate interactions attributable to enzyme induction.
No preview · Article · Feb 2001 · The Journal of Clinical Pharmacology
[Show abstract][Hide abstract] ABSTRACT: The in vitro biotransformation of bupropion to hydroxybupropion was studied in human liver microsomes and microsomes containing heterologously expressed human cytochromes P450 (CYP). The mean (+/-S.E.) K(m) in four human liver microsomes was 89 (+/-14) microM. In microsomes containing cDNA-expressed CYPs, hydroxybupropion formation was mediated only by CYP2B6 at 50 microM bupropion (K(m) 85 microM). A CYP2B6 inhibitory antibody produced more than 95% inhibition of bupropion hydroxylation in four human livers. Bupropion hydroxylation activity at 250 microM was highly correlated with S-mephenytoin N-demethylation activity (yielding nirvanol), another CYP2B6-mediated reaction, in a panel of 32 human livers (r = 0.94). The CYP2B6 content of 12 human livers highly correlated with bupropion hydroxylation activity (r = 0.96). Thus bupropion hydroxylation is mediated almost exclusively by CYP2B6 and can serve as an index reaction reflecting activity of this isoform. IC(50) values for inhibition of a CYP2D6 index reaction (dextromethorphan O-demethylation) by bupropion and hydroxybupropion were 58 and 74 microM, respectively. This suggests a low inhibitory potency versus CYP2D6, the clinical importance of which is not established. Since bupropion is frequently coadministered with other antidepressants, IC(50) values (microM) for inhibition of bupropion hydroxylation were determined as follows: paroxetine (1.6), fluvoxamine (6.1), sertraline (3.2), desmethylsertraline (19.9), fluoxetine (59.5), norfluoxetine (4.2), and nefazodone (25.4). Bupropion hydroxylation was only weakly inhibited by venlafaxine, O-desmethylvenlafaxine, citalopram, and desmethylcitalopram. The inhibition of bupropion hydroxylation in vitro by a number of newer antidepressants suggests the potential for clinical drug interactions.
No preview · Article · Nov 2000 · Drug Metabolism and Disposition
[Show abstract][Hide abstract] ABSTRACT: Biotransformation of triazolam to its alpha-hydroxy and 4-hydroxy metabolites by human liver microsomes in vitro was used as an index of human cytochrome P450 3A (CYP3A) activity.
The reaction was strongly inhibited by co-incubation with the viral protease inhibitors ritonavir (IC50 = 0.14 microM) and amprenavir (IC50 = 2.5 2.9 microM), and by the azole derivative ketoconazole (IC50 = 0.07 microM). Pre-incubation of microsomes with ritonavir or amprenavir increased inhibitory potency (IC50 reduced to 0.07 microM and 1.4 microM, respectively). This was not the case with ketoconazole.
Thus, ritonavir and amprenavir are highly potent mechanism-based inhibitors of human CYP3A isoforms.
No preview · Article · Jul 2000 · European Journal of Clinical Pharmacology
[Show abstract][Hide abstract] ABSTRACT: Biotransformation of citalopram (CT), a newly available selective serotonin reuptake inhibitor antidepressant, to its principal metabolite, desmethycitalopram (DCT), and the capacity of CT and DCT to inhibit human cytochromes P450, were studied in vitro.
Formation of DCT from CT was evaluated using human liver microsomes and microsomes from cDNA-transfected human lymphoblastoid cells. Cytochrome inhibition by CT and DCT in liver microsomes was studied using isoform-specific index reactions.
Formation of DCT from CT in liver microsomes had a mean apparent K(m) of 174 mumol/L. Coincubation with 1 mumol/L ketoconazole reduced reaction velocity to 46 to 58% of control values, while omeprazole, 10 mumol/L, reduced velocity to 80% of control. Quinidine produced minimal inhibition. DCT was formed from CT by heterologously expressed human P450-2D6, -2C19, -3A4. After accounting for the relative abundance of individual cytochromes, 3A4 and 2C19 were estimated to make major contributions to net reaction velocity, with a possible contribution of 2D6 at therapeutic CT concentrations. CT and DCT themselves produced negligible inhibition of 2C9, 2E1, and 3A, and only weak inhibition of 1A2, 2C19, and 2D6.
Formation of DCT from CT is mediated mainly by P450-3A4 and 2C19, with an additional contribution of 2D6. CT at therapeutic doses in humans may produce a small degree of inhibition of P450-1A2, -2C19, and -2D6, but negligible inhibition of P450-2C9, -2E1, and -3A.
Full-text · Article · Oct 1999 · Biological Psychiatry
[Show abstract][Hide abstract] ABSTRACT: To determine the human cytochromes mediating biotransformation of the imidazopyridine hypnotic, zolpidem, and the clinical correlates of the findings.
Kinetic properties of zolpidem biotransformation to its three hydroxylated metabolites were studied in vitro using human liver microsomes and heterologously expressed individual human cytochromes.
The metabolic product termed M-3 accounted for more than 80% of net intrinsic clearance by liver microsomes in vitro. Microsomes containing human cytochromes CYP1A2, 2C9, 2C19, 2D6, and 3 A4 expressed by cDNA-transfected human lymphoblastoid cells mediated zolpidem metabolism in vitro. The kinetic profile for zolpidem metabolite formation by each individual cytochrome was combined with estimated relative abundances based on immunological quantification, yielding projected contributions to net intrinsic clearance of: 61% for 3 A4, 22% for 2C9, 14% for 1A2, and less than 3% for 2D6 and 2C19. These values were consistent with inhibitory effects of ketoconazole and sulfaphenazole on zolpidem biotransformation by liver microsomes. Ketoconazole had a 50% inhibitory concentration (IC50 ) of 0.61 microm vs formation of the M-3 metabolite of zolpidem in vitro; in a clinical study, ketoconazole coadministration reduced zolpidem oral clearance by approximately 40%, somewhat less than anticipated based on the IC50 value and total plasma ketoconazole levels, but much more than predicted based on unbound plasma ketoconazole levels.
The incomplete dependence of zolpidem clearance on CYP3A activity has clinical implications for susceptibility to metabolic inhibition.
Full-text · Article · Aug 1999 · British Journal of Clinical Pharmacology
[Show abstract][Hide abstract] ABSTRACT: Pharmacokinetic drug interactions with viral protease inhibitors are of potential clinical importance. An in vitro model was applied to the quantitative identification of possible interactions of protease inhibitors with substrates of cytochrome P450-2D6. Biotransformation of desipramine (DMI) to hydroxydesipramine (OH-DMI), an index reaction used to profile activity of human cytochrome P450-2D6, was studied in vitro using human liver microsomes. Quinidine and four viral protease inhibitors currently used to treat human immunodeficiency virus infection were tested as chemical inhibitors in this system. Formation of OH-DMI from DMI was consistent with Michaelis-Menten kinetics, having a mean Km value of 11.7 microM (range: 9.9-15.3 microM). Quinidine, a highly potent and relatively selective inhibitor of P450-2D6, strongly inhibited OH-DMI formation with an apparent competitive mechanism, having a mean inhibition constant of 0.16 microM (range: 0.13-0.18 microM). All four protease inhibitors impaired OH-DMI formation; the pattern was consistent with a mixed competitive-noncompetitive mechanism. Mean inhibition constants (small numbers indicating greater inhibiting potency) were as follows: ritonavir, 4.8 microM; indinavir, 15.6 microM; saquinavir, 24.0 microM; nelfinavir, 51.9 microM. In a clinical pharmacokinetic study, coadministration of ritonavir with DMI inhibited DMI clearance by an average of 59%. The in vitro findings, together with observed plasma ritonavir concentrations, provided a reasonable quantitative forecast of this interaction, whereas estimated unbound plasma or intrahepatic ritonavir concentrations yielded poor quantitative forecasts. Thus the in vitro model correctly identifies ritonavir as a potent and clinically important inhibitor of human P450-2D6. Other protease inhibitors may also inhibit 2D6 activity in humans, but with lower potency than ritonavir.
No preview · Article · Nov 1998 · Journal of Pharmaceutical Sciences
[Show abstract][Hide abstract] ABSTRACT: The capacity of hydroxyitraconazole, an in-vivo metabolite of the antifungal agent itraconazole, to inhibit human cytochrome P4503A isoforms was evaluated in-vitro using a human microsomal model.Varying concentrations of ketoconazole, itraconazole, hydroxyitraconazole and fluconazole were incubated with human liver microsomes and triazolam (250 μM), an index compound used to profile activity of P4503A isoforms. Formation of α-OH- and 4-OH-triazolam was quantitated by high-performance liquid chromatography. Ketoconazole was the most potent inhibitor of triazolam metabolite formation (mean IC50 0.074–0.076 μM), while fluconazole was less potent (IC50 4.4–4.9 μM). Both itraconazole and hydroxy-itraconazole had similar IC50 values (0.4–0.7 μM), with potency intermediate between ketoconazole and fluconazole.Hydroxyitraconazole may contribute to impaired clearance of P4503A substrates, and consequent pharmacokinetic drug interactions, during treatment with itraconazole in man.
[Show abstract][Hide abstract] ABSTRACT: The activity of D-fenfluramine, L-fenfluramine, and phentermine as inhibitors of five human cytochromes P450 was evaluated using human liver microsomes in vitro. All three compounds produced negligible inhibition of P450-1A2, -2C9, -2E1, and -3A. Phentermine also did not inhibit P450-2D6. However, D- and L-fenfluramine significantly inhibited P450-2D6 activity as measured by dextromethorphan O-demethylation, with mean 50% inhibitory concentrations (15.1 microM) within one order of magnitude of that for fluoxetine (2.7 microM). Findings from the in vitro assay are consistent with clinical studies showing significant inhibition of desipramine clearance by coadministration of fenfluramine.
No preview · Article · Sep 1998 · Journal of Clinical Psychopharmacology