Biomedical subjects

K E Thummel

Publications and source records attributed to K E Thummel.

At least 37 records · Page 2Linked to original sources

PubMed1999

Effect of inhibitor depletion on inhibitory potency: tight binding inhibition of CYP3A by clotrimazole.

The purpose of this work was to evaluate the effect of mutual unbound inhibitor and unbound enzyme depletion on the potency of three antifungal cytochrome P-450 (CYP)3A inhibitors with over 1000-fold range in enzyme affinity. Incubations were performed with human liver microsomal protein concentrations that varied from 25 to 1000 microg/ml. The effect of each inhibitor was evaluated using midazolam as a CYP3A probe. Clotrimazole was found to be a tight binding inhibitor of CYP3A with a Ki of 250 pM. Analysis of percent inhibition data by stepwise linear regression for the matrix of inhibitor and enzyme concentrations used showed that protein concentrations predicted the percent inhibition by clotrimazole (r2 = 0.60, p <.001). When clotrimazole concentrations were added to the model, the r2 improved to 0.81, p =.003. Clotrimazole concentrations alone were not a significant predictor of percent inhibition (r2 = 0. 21, p =.08). For ketoconazole, protein concentrations provided a weak prediction of the percent inhibition (r2 = 0.39, p =.006). Conversely, ketoconazole concentrations alone were a good predictor of percent inhibition (r2 = 0.55, p <.001). In contrast to results with clotrimazole and ketoconazole, percent inhibition by fluconazole was not dependent on protein concentrations (r2 = 0.06, p =.39). We conclude that microsomal inhibitory potency can be affected by incubation conditions that deplete the unbound concentration of inhibitor available to the enzyme. This may introduce serious error into a quantitative prediction of an in vivo drug-drug interaction based on an in vitro derived Ki value.

Anti-Infective Agents↗

PubMed1999

Lack of single-dose disulfiram effects on cytochrome P-450 2C9, 2C19, 2D6, and 3A4 activities: evidence for specificity toward P-450 2E1.

Disulfiram and its primary metabolite diethyldithiocarbamate are effective mechanism-based inhibitors of cytochrome P-450 2E1 (CYP2E1)1 in vitro. Single-dose disulfiram diminishes CYP2E1 activity in vivo and has been used to identify CYP2E1 participation in human drug metabolism and prevent CYP2E1-mediated toxification. Specificity of single-dose disulfiram toward CYP2E1 in vivo, however, remains unknown. This investigation determined single-dose disulfiram effects on human CYP 2C9, 2C19, 2D6, and 3A4 activities in vivo. In four randomized crossover experiments, volunteers received isoform-selective probes (oral tolbutamide, mephenytoin, dextromethorphan, or i.v. midazolam) on two occasions, 10 h after oral disulfiram or after no pretreatment (controls). Plasma and/or urine parent and/or metabolite concentrations were measured by HPLC or gas chromatography-mass spectrometry. CYP2C9, 2C19, 2D6, and 3A4 activities were determined from the tolbutamide metabolic ratio, 4'-hydroxymephenytoin excretion, and dextromethorphan/dextrorphan ratios in urine and midazolam systemic clearance, respectively. Midazolam clearance (670 +/- 190 versus 700 +/- 240 ml/min, disulfiram versus controls), dextromethorphan/dextrorphan metabolic ratio (0.013 +/- 0.033 versus 0.015 +/- 0.035), 4'-hydroxymephenytoin excretion (122 +/- 22 versus 128 +/- 25 micromol), and tolbutamide metabolite excretion (577 +/- 157 versus 610 +/- 208 micromol) were not significantly altered by disulfiram pretreatment, although the tolbutamide metabolic ratio was slightly diminished after disulfiram (60 +/- 17 versus 81 +/- 40, p <.05). Results show that single-dose disulfiram does not cause clinically significant inhibition of human CYP2C9, 2C19, 2D6, and 3A4 activities in vivo. When single-dose disulfiram is used as an in vivo probe for P-450, inhibition of drug metabolism suggests selective involvement of CYP2E1. Single-dose disulfiram should not cause untoward drug interactions from inhibition of other P-450 isoforms.

Adult↗

PubMed1999

Duration of cytochrome P-450 2E1 (CYP2E1) inhibition and estimation of functional CYP2E1 enzyme half-life after single-dose disulfiram administration in humans.

Disulfiram (DSF) is a mechanism-based inhibitor of cytochrome P-450 2E1 (CYP2E1), resulting in loss of CYP2E1 protein and activity, which may be useful in preventing CYP2E1-mediated xenobiotic toxicity. The duration of inhibition after a single DSF dose is, however, unknown. The purpose of this investigation was to determine this duration, and CYP2E1 formation and degradation rates, in humans. Oral chlorzoxazone (CLZ) was used as the selective in vivo probe for CYP2E1. Healthy subjects received CLZ to determine baseline CYP2E1 activity (CLZ plasma clearance and 6-hydroxychlorzoxazone fractional metabolic clearance). One week later, DSF (500 mg orally) was administered at bedtime, and CLZ administered the following morning and 3, 6, 8, 10, and 13 days after DSF. A terminal DSF metabolite, 2-thiothiazolidine-4 carboxylic acid, was also measured in each 24-h urine sample. The mean CLZ clearance and 6-hydroxychlorzoxazone fractional metabolic clearance on the first day declined to 10.2 and 5.5% of baseline values, indicating rapid and profound CYP2E1 inhibition. CYP2E1 activity returned to half that of control on day 3, and to baseline values on day 8. Assuming zero-order synthesis and first-order degradation, the in vivo CYP2E1 synthesis rate and degradation half-life was estimated to be 11 +/- 5 nmol/h and 50 +/- 19 h, respectively. Significant amounts of 2-thiothiazolidine-4 carboxylic acid were present only on day 1, suggesting that the return of in vivo CYP2E1 activity was not caused by inhibitor washout, but by enzyme resynthesis. Results regarding CYP2E1 disposition may be useful for modeling the effects of CYP2E1 inducers and inhibitors. For prevention of CYP2E1-mediated bioactivation, depending on protoxicant disposition, a second DSF dose might be necessary to completely prevent toxicity.

Chlorzoxazone↗

PubMed1999

Molecular and physical mechanisms of first-pass extraction.

This is a report of a symposium held at the March 1997 meeting of the American Society for Pharmacology and Therapeutics in San Diego. Our understanding of the events that control first-pass drug elimination in humans has increased tremendously by two sequential discoveries. First, cytochrome P-450s 3A4 and 5 are expressed at high concentrations in both hepatocytes and upper intestinal enterocytes, and therefore limit the systemic availability of many drugs. Second, P-glycoprotein is expressed at the lumenal surface of the intestinal epithelium and therefore also acts to oppose the absorption of unchanged drug. The following discussion brings together our current understandings of these interrelated phenomena to aid a more complete picture of how they may contribute both qualitatively and quantitatively to first-pass elimination.

Animals↗

PubMed1999

Inhibition of cytochrome P-450 3A (CYP3A) in human intestinal and liver microsomes: comparison of Ki values and impact of CYP3A5 expression.

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 Ki for cDNA-expressed CYP3A4 of 26. 7 +/- 1.71 nM, whereas the Ki for cDNA expressed CYP3A5 was 109 +/- 19.7 nM. Corresponding Ki values for fluconazole were 9.21 +/- 0.51 microM and 84.6 +/- 12.9 microM. For liver and intestinal microsomes that contained only CYP3A4, the average ketoconazole Ki was found to be 14.9 +/- 6.7 nM and 17.0 +/- 7.9 nM, respectively, whereas fluconazole yielded mean respective Ki values of 10.7 +/- 4.2 microM and 10.4 +/- 2.9 microM. 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.

Algorithms↗

PubMed1998

Single-dose disulfiram does not inhibit CYP2A6 activity.

BACKGROUND: Disulfiram and its primary metabolite diethyldithiocarbamate are effective mechanism-based inhibitors of human liver cytochrome P450 2E1 (CYP2E1) in vitro. A single dose of disulfiram, which significantly diminishes human P450 2E1 activity in vivo, has been used to investigate the role of CYP2E1 in human drug metabolism and to prevent CYP2E1-mediated biotransformation. Nevertheless, the specificity of single-dose disulfiram toward human CYP2E1 in vivo is unknown. Because diethyldithiocarbamate also inhibits human liver CYP2A6 in vitro, this investigation explored the effect of single-dose disulfiram on human CYP2A6 activity in vivo. METHODS: CYP2A6 activity was assessed by the 7-hydroxylation of coumarin, which is catalyzed selectively by CYP2A6. Ten healthy volunteers received 50 mg oral coumarin on two occasions in a randomized crossover design, approximately 10 hours after 500 mg oral disulfiram was administered or after no pretreatment (control group). Plasma and urine 7-hydroxycoumarin and plasma coumarin concentrations were determined by HPLC. RESULTS: The area under the plasma 7-hydroxycoumarin versus time curve (2.69 +/- 0.90 micrograms.hr/ml) was not decreased after disulfiram pretreatment (3.33 +/- 0.93 micrograms.hr/ml). Furthermore, maximum plasma concentration (Cmax) of 7-hydroxycoumarin (1.4 +/- 0.5 versus 1.8 +/- 0.6 micrograms/ml) and time to reach Cmax (1.0 +/- 0.2 and 1.0 +/- 0.4 hour) were unchanged by disulfiram pretreatment. Urinary 7-hydroxycoumarin excretion over a 24-hour period (38.9 +/- 10.8 mg) was also undiminished by disulfiram pretreatment (45.2 +/- 6.6 mg). CONCLUSIONS: Single-dose disulfiram does not inhibit human CYP2A6 activity in vivo. When single-dose disulfiram is used as an in vivo probe for P450, inhibition of drug metabolism suggests involvement of CYP2E1 but not CYP2A6.

Administration, Oral↗

PubMed1998

In vitro and in vivo drug interactions involving human CYP3A.

Cytochrome P4503A (CYP3A) is importantly involved in the metabolism of many chemically diverse drugs administered to humans. Moreover, its localization in high amounts both in the small intestinal epithelium and liver makes it a major contributor to presystemic elimination following oral drug administration. Drug interactions involving enzyme inhibition or induction are common following the coadministration of two or more CYP3A substrates. Studies using in vitro preparations are useful in identifying such potential interactions and possibly permitting extrapolation of in vitro findings to the likely in vivo situation. Even if accurate quantitative predictions cannot be made, several classes of drugs can be expected to result in a drug interaction based on clinical experience. In many instances, the extent of such drug interactions is sufficiently pronounced to contraindicate the therapeutic use of the involved drugs.

Aryl Hydrocarbon Hydroxylases↗

PubMed1997

Influence of stiripentol on cytochrome P450-mediated metabolic pathways in humans: in vitro and in vivo comparison and calculation of in vivo inhibition constants.

OBJECTIVE: The spectrum of cytochrome P450 inhibition of stiripentol, a new anticonvulsant, was characterized in vitro and in vivo. METHODS: Stiripentol was incubated in vitro with (R)-warfarin, coumarin, (S)-warfarin, (S)-mephenytoin, bufuralol, p-nitrophenol, and carbamazepine as probes for CYPs 1A2, 2A6, 2C9, 2C19, 2D6, 2E1, and 3A4, respectively. Caffeine demethylation and the 6 beta-hydroxycortisol/cortisol ratio were monitored in vivo before and after 14 days of treatment with stiripentol as measures of CYP1A2 and CYP3A4 activity, and dextromethorphan O- and N-demethylation were used to measure CYP2D6 and CYP3A4 activity, respectively. In vivo inhibition constants for CYP3A4 were calculated with use of data that previously documented the interaction between stripentol and carbamazepine. RESULTS: In vitro, stiripentol inhibited CYPs 1A2, 2C9, 2C19, 2D6, and 3A4, with inhibition constant values at or slightly higher than therapeutic (total) concentrations of stiripentol, but it did not inhibit CYPs 2A6 and 2E1 even at tenfold therapeutic concentrations. In vivo inhibition of caffeine demethylation and dextromethorphan N-demethylation were consistent with inhibition of CYP1A2 and CYP3A4, respectively. The 6 beta-hydroxycortisol/cortisol ratio did not provide a reliable index of CYP3A4 inhibition. Inhibition of CYP2D6-mediated O-demethylation was not observed in vivo. With use of carbamazepine, in vivo inhibition constants for CYP3A4 ranged between 12 and 35 mumol/L, whereas the corresponding in vitro value was 80 mumol/L. CONCLUSIONS: Stiripentol appears to inhibit several CYP450 enzymes in vitro and in vivo. In vivo inhibition constants show that stiripentol inhibition of CYP3A4 is linearly related to plasma concentration in patients with epilepsy.

Adult↗

PubMed1997

Influence of polymorphic N-acetyltransferase phenotype on the inhibition and induction of acetaminophen bioactivation with long-term isoniazid.

OBJECTIVE: To determine in patients receiving isoniazid prophylaxis whether an increase in the CYP2E1 dependent formation clearance of acetaminophen (paracetamol) to N-acetyl-p-benzoquinone imine (NAPQI) occurs during a normal 24-hour isoniazid dose interval and whether the interaction is dependent on acetylation status. METHODS: Acetaminophen elimination kinetics were determined on four different occasions. Ten subjects were assigned to receive acetaminophen either simultaneously with the 8 am dose of isoniazid or 12 hours after the isoniazid dose. One week later, on the last day of isoniazid therapy, subjects received acetaminophen at the alternate time of day. The control phase acetaminophen administrations were repeated 1 and 2 weeks later, following the initial randomization. Isoniazid acetylation (NAT2) genotype was determined by analysis of genomic DNA obtained from peripheral blood leukocytes. RESULTS: The mean NAPQI formation clearance was inhibited 57% when acetaminophen and isoniazid were coadministered but was unchanged compared with time-matched control when acetaminophen was given 12 hours after the isoniazid dose. However, when data from subjects was segregated according to isoniazid (INH) acetylation phenotype, the mean ratio of NAPQI formation clearances (+INH/-INH) with 8 PM acetaminophen was significantly higher for fast acetylators compared with slow acetylators (1.36 versus 0.68; p = 0.006). CONCLUSIONS: Fast metabolizers of isoniazid appeared to clear the inducer or inhibitor from the active site of CYP2E1 more rapidly, which resulted in an increased formation of NAPQI 12 hours after the isoniazid dose. In contrast, formation of NAPQI for slow isoniazid metabolizers remained inhibited.

Acetaminophen↗

PubMed1997

The role of cytochrome P450 3A4 in alfentanil clearance. Implications for interindividual variability in disposition and perioperative drug interactions.

BACKGROUND: There is considerable unexplained variability in alfentanil pharmacokinetics, particularly systemic clearance. Alfentanil is extensively metabolized in vivo, and thus systemic clearance depends on hepatic biotransformation. Cytochrome P450 3A4 was previously shown to be the predominant P450 isoform responsible for human liver microsomal alfentanil metabolism in vitro. This investigation tested the hypothesis that P450 3A4 is responsible for human alfentanil metabolism and clearance in vivo. METHODS: Nine healthy male volunteers who provided institutionally approved written informed consent were studied in a three-way randomized crossover design. Each subject received alfentanil (20 micrograms/kg given intravenously) 30 min after midazolam (1 mg injected intravenously) on three occasions: control; high P450 3A4 activity (rifampin induction); and low P450 3A4 activity (selective inhibition by troleandomycin). Midazolam is a validated selective in vivo probe for P450 3A4 activity. Venous blood was sampled for 24 h and plasma concentrations of midazolam and alfentanil and their primary metabolites 1'-hydroxymidazolam and noralfentanil were measured by gas chromatography-mass spectrometry. Pharmacokinetic parameters were determined by two-stage analysis using both noncompartmental and three-compartment models. RESULTS: Plasma alfentanil concentration-time profiles depended significantly on P450 3A4 activity. Alfentanil noncompartmental clearance was 5.3 +/- 2.3, 14.6 +/- 3.8, and 1.1 +/- 0.5 ml.kg-1.min-1, and elimination half-life was 58 +/- 13, 35 +/- 7, and 630 +/- 374 min, respectively, in participants with normal (controls), high (rifampin), and low (troleandomycin) P450 3A4 activity (means +/- SD; P < 0.05 compared with controls). Multicompartmental modeling suggested a time-dependent inhibition-resynthesis model for troleandomycin effects on P450 3A4 activity, characterized as k10(t) = k10[1-phi e-alpha(t-tzero)], where k10(t) is the apparent time-dependent rate constant, k10 is the uninhibited rate constant, phi is the fraction of P450 3A4 inhibited, and alpha is the apparent P450 3A4 reactivation rate. Alfentanil clearance was calculated as V1 k10 for controls and men receiving rifampin, and as V1.average k10(t) for men receiving troleandomycin. This clearance was 4.9 +/- 2.1, 13.2 +/- 3.6, and 1.5 +/- 0.8 ml.kg-1.min-1, respectively, in controls and in men receiving rifampin or troleandomycin. There was a significant correlation (r = 0.97, P < 0.001) between alfentanil systemic clearance and P450 3A4 activity. CONCLUSIONS: Modulation of P450 3A4 activity by rifampin and troleandomycin significantly altered alfentanil clearance and disposition. These results strongly suggest that P450 3A4 is the major isoform of P450 responsible for clinical alfentanil metabolism and clearance. This observation, combined with the known population variability in P450 3A4 activity, provides a mechanistic explanation for the interindividual variability in alfentanil disposition. Furthermore, known susceptibility of human P450 3A4 activity to induction and inhibition provides a conceptual framework for understanding and predicting clinical alfentanil drug interactions. Finally, human liver microsomal alfentanil metabolism in vitro is confirmed as an excellent model for human alfentanil metabolism in vivo.

Adult↗

PubMed1997

Expression of enzymatically active CYP3A4 by Caco-2 cells grown on extracellular matrix-coated permeable supports in the presence of 1alpha,25-dihydroxyvitamin D3.

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.

Caco-2 Cells↗

PubMed1997

Cytochrome P450 2E1 is the principal catalyst of human oxidative halothane metabolism in vitro.

The volatile anesthetic halothane undergoes substantial biotransformation generating metabolites that mediate hepatotoxicity. Aerobically, halothane undergoes cytochrome P450-catalyzed oxidation to trifluoroacetic acid (TFA), bromide and a reactive intermediate that can acetylate liver proteins. These protein neo-antigens stimulate an immune reaction that mediates severe hepatic necrosis ("halothane hepatitis"). This investigation identified the human P450 isoform(s) that catalyze oxidative halothane metabolism. Halothane oxidation by human liver microsomes was assessed by TFA and bromide formation. Eadie-Hofstee plots of TFA and bromide formation were both nonlinear, suggesting the participation of multiple P450s. Microsomal TFA and bromide formation were inhibited 45 to 66% and 21 to 26%, respectively, by the P450 2A6 inhibitors 8-methoxypsoralen and coumarin, 84 to 90% by the P450 2E1 inhibitor 4-methylpyrazole and 55% by diethyldithiocarbamate, an inhibitor of both P450 2A6 and 2E1. Selective inhibitors of P450s 1A, 2B6, 2C9/10, 2D6 and 3A4 did not affect halothane oxidation. At saturating halothane concentrations (2.4 vol%) only cDNA-expressed P450 2A6 and 2B6 catalyzed significant rates of TFA and bromide formation, and P450 2E1 catalyzed comparatively minimal oxidation. Conversely, at subsaturating halothane concentrations (0.30 vol%), metabolism by P450 2E1 exceeded that by P450 2A6. Among a panel of human liver microsomes, there were significant linear correlations between halothane oxidation and P450 2A6 activity and protein content at saturating halothane concentrations (2.4 vol%), and a significant correlation between metabolite formation and P450 2E1 activity (but not P450 2A6 activity) at subsaturating concentrations (0.12 vol%). These experiments suggested P450 2A6 and 2E1 as the predominant catalysts at saturating and subsaturating halothane concentrations, respectively. Further kinetic analysis using cDNA-expressed P450 and liver microsomes clearly demonstrated that P450 2E1 is the high affinity/low capacity isoform (Km = 0.030-0.053 vol%) and P450 2A6 is the low affinity/high capacity isoform (Km = 0.77-1.2 vol%). Evidence was also obtained for substrate inhibition of P450 2E1. The in vitro clearance estimates (Vmax/Km) for microsomal P450 2E1 (4.3-5.7 ml/min/g) were substantially greater than those for microsomal P450 2A6 (0.12-0.21). These clearances, as well as rates of apparent halothane oxidation predicted from kinetic parameters in conjunction with plasma halothane concentrations measured during clinical anesthesia in humans, demonstrated that both P450 2E1 and P450 2A6 participate in human halothane metabolism, and that P450 2E1 is the predominant catalytic isoform.

Anesthetics, Inhalation↗

PubMed1997

Fentanyl metabolism by human hepatic and intestinal cytochrome P450 3A4: implications for interindividual variability in disposition, efficacy, and drug interactions.

The synthetic opioid fentanyl undergoes extensive metabolism in humans. Systemic elimination occurs primarily by hepatic metabolism. When administered as a lozenge for oral transmucosal absorption, swallowed fentanyl is subject to first pass metabolism in the liver and possibly small intestine. Little is known, however, about the identity and formation of human fentanyl metabolites. This investigation identified routes of human liver microsomal fentanyl metabolism and their relative importance, tested the hypothesis that fentanyl is metabolized by human duodenal microsomes, and identified the predominantly responsible cytochrome P450 isoforms. A GC/MS assay using deuterated internal standards was developed for fentanyl metabolites. Piperidine N-dealkylation to norfentanyl was the predominant pathway of liver microsomal metabolism. Amide hydrolysis to despropionylfentanyl and alkyl hydroxylation to hydroxyfentanyl were comparatively minor pathways. Hydroxynorfentanyl was identified as a minor, secondary metabolite arising from N-dealkylation of hydroxyfentanyl. Liver microsomal norfentanyl formation was significantly inhibited by the mechanism-based P450 3A4 inhibitor troleandomycin and the P450 3A4 substrate and competitive inhibitor midazolam, and was significantly correlated with P450 3A4 protein content and catalytic activity. Of six expressed human P450 isoforms (P450s 1A2, 2B6, 2C9, 2D6, 2E1, and 3A4), only P450 3A4 exhibited significant fentanyl dealkylation to norfentanyl. These results indicate the predominant role of P450 3A4 in the primary route of hepatic fentanyl metabolism. Human duodenal microsomes also catalyzed fentanyl metabolism to norfentanyl; the average rate was approximately half that of hepatic metabolism. Rates of duodenal norfentanyl formation were diminished by troleandomycin and midazolam, and were significantly correlated with P450 3A4 activity, suggesting a prominent role for P450 3A4. These results demonstrate that human intestinal as well as liver microsomes catalyze fentanyl metabolism, and N-dealkylation by P450 3A4 is the predominant route in both organs. The fraction of fentanyl lozenge that is swallowed likely undergoes significant intestinal, as well as hepatic, first-pass metabolism. Intestinal and hepatic first-pass metabolism, as well as systemic metabolism, may be subject to individual variability in P450 3A4 expression and to drug interactions involving P450 3A4.

Analgesics, Opioid↗

PubMed1997

Mechanism of cytochrome P450 activation by caffeine and 7,8-benzoflavone in rat liver microsomes.

Caffeine and 7,8-benzoflavone activate CYP3A2 in rat liver microsomes. Both activators appear to enhance enzyme activity by an increase in Vmax and to a lesser extent a decrease in Km. Additive effect studies demonstrated that the two activators oppose one another's effect. Electron transfer steps in the cytochrome P450 cycle are involved in the mechanism of cytochrome P450 activation, as indicated by the lack of effect of caffeine or 7,8-benzoflavone on cumene hydroperoxide-supported oxidation of acetaminophen by cytochrome P450. The involvement of cytochrome b5 in the formation of N-acetyl-p-benzoquinone imine (NAPQI) was implicated through a synergistic effect of NADH on the NADPH-supported reaction. Anti-cytochrome b5, but not anti-cytochrome P450 reductase IgG, diminished the activation effect of caffeine on NAPQI formation. Neither antibody altered the effect of 7,8-benzoflavone on NAPQI formation. The impairment of NAPQI formation by cytochrome b5 antibody suggests that cytochrome P450 activation by caffeine but not 7,8-benzoflavone is mediated in part through enhancement of the transfer of the second electron to cytochrome P450 from cytochrome b5.

Animals↗

PubMed1997

Pharmacokinetic consequences of induction of CYP2E1 by ligand stabilization.

Models of the time course of the effect of P450 induction on substrate clearance have previously only considered induction through enhanced synthesis of protein. Induction of CYP2E1 does not always conform to this model, in that many chemicals induce the enzyme through stabilization of the protein apparently by binding to the active site. While such binding protects the enzyme from degradation, it also results in competitive inhibition of substrate clearance. We present a model based on experimental studies of chemical induction of CYP2E1 by ligand stabilization through which this mechanism of induction can be translated into its pharmacokinetic consequence with regard to clearance of substrate. CYP2E1 is considered to be localized in two pools: Pool 1 at which two mechanisms of degradation, fast and slow, operate and pool 2, at which only the slower mechanism operates. Binding of substrate to enzyme in pool 1 stabilizes it from degradation by the fast process, leaving only the slow process. Ligand stabilization therefore results in induction of CYP2E1 as enzyme accumulates as a consequence of unchanged synthesis. Binding of ligand to the active site results in competitive inhibition of the clearance of substrate. Model-based computer simulations show that the time course of interaction between inhibitor/inducer and substrate can be predicted from knowledge of I/Ki and S/Km and the synthesis and degradation kinetics of CYP2E1. The simulations demonstrate further that as long as inhibitor/inducer administration is not interrupted, the clearance of substrate will always be less than the value observed at low concentration of substrate even if the substrate concentration is raised to displace inhibitor/inducer from the active site. On the other hand, the degree of inhibition of clearance is less than would be seen if induction had not taken place. Clearance of substrate is observed to rise above the value observed in the absence of the inhibitor/inducer only after the inhibitor/inducer concentration declines low enough for substrate to gain access to the active site of the enzyme. The model-based simulations agree with reports of the interaction between isoniazid and acetaminophen in humans.

Acetaminophen↗

PubMed1997

Mechanisms of enhanced oral availability of CYP3A4 substrates by grapefruit constituents. Decreased enterocyte CYP3A4 concentration and mechanism-based inactivation by furanocoumarins.

Grapefruit juice increases the oral availability of a variety of CYP3A4 substrates. It has been shown that recurrent grapefruit juice ingestion results in a loss of CYP3A4 from the small bowel epithelium. We now show that the reduction in intestinal CYP3A4 concentration is rapid; a 47% decrease occurred in a healthy volunteer within 4 hr after consuming grapefruit juice. To identify the specific components of the juice responsible for this effect, we used a recently developed Caco-2 cell culture model of human intestinal epithelium that expresses catalytically active CYP3A4. We found that grapefruit oil and two furanocoumarin constituents (6', 7'-dihydroxybergamottin and a closely related dimer) caused a dose-dependent fall in CYP3A4 catalytic activity and immunoreactive CYP3A4 concentration. The effect was selective in that concentrations of CYP1A1 and CYP2D6 did not fall, consistent with previous results obtained in vivo. Assays of various juices confirmed that 6',7'-dihydroxybergamottin is the major furanocoumarin present and, although its concentration varies significantly among types and brands of grapefruit juice, it is consistently present in concentrations exceeding the IC50 (1 microM) for loss of midazolam 1'-hydroxylase activity determined in the Caco-2 cells. Studies with recombinant CYP3A4 revealed that 6', 7'-dihydroxybergamottin is a mechanism-based inactivator, which supports the idea that loss of CYP3A4 results from accelerated degradation of the enzyme. We conclude that the effect of grapefruit juice on oral availability of CYP3A4 substrates can be largely accounted for by the presence of 6',7'-dihydroxybergamottin although other furanocoumarins probably also contribute.

Biological Availability↗

PubMed1997

Characterization of interintestinal and intraintestinal variations in human CYP3A-dependent metabolism.

Cytochrome P450 3A (CYP3A) metabolizes a diverse array of clinically important drugs. For some of these (e.g., cyclosporine, verapamil, midazolam), CYP3A in the intestinal mucosa contributes to their extensive and variable first-pass extraction. To further characterize this phenomenon, we measured CYP3A content and catalytic activity toward the probe substrate midazolam in mucosa isolated from duodenal, jejunal and ileal sections of 20 human donor intestines. For comparison, the same measurements were performed for 20 human donor livers, eight of which were obtained from the same donors as eight of the intestines. Excellent correlations existed between homogenate and microsomal CYP3A content for the three intestinal regions. Median microsomal CYP3A content was greatest in the duodenum and lowest in the ileum (31 vs. 17 pmol/mg of protein). With respect to midazolam 1'-hydroxylation kinetics, the median Km for each intestinal region was similar to the median hepatic Km, approximately 4 microM. In contrast, the median Vmax decreased from liver to duodenum to jejunum to ileum (850 vs. 644 vs. 426 vs. 68 pmol/min/mg). Intrinsic clearance (Vmax/Km) followed a similar trend for the intestinal regions; median duodenal intrinsic clearance was comparable to hepatic intrinsic clearance (157 and 200 microl/min/mg, respectively). Vmax correlated with CYP3A content for all tissues except the ileum. Duodenal and jejunal Vmax and CYP3A content varied by >30-fold among donors. Microsomes prepared from every other 1-foot section of six intestines were also analyzed for CYP3A as well as for two coenzymes. In general, CYP3A activity, CYP3A content and CYP reductase activity rose slightly from duodenum to middle jejunum and then declined to distal jejunum and ileum. Cytochrome b5 content and cytochrome b5 reductase activity varied little throughout the intestinal tract. Regional intrinsic midazolam 1'-hydroxylation clearance was greatest for the jejunum, followed by the duodenum and ileum (144, 50 and 19 ml/min, respectively). Collectively, these results demonstrate that the upper small intestine serves as the major site for intestinal CYP3A-mediated first-pass metabolism and provides a rationale for interindividual differences in oral bioavailability for some CYP3A substrates.

Aryl Hydrocarbon Hydroxylases↗

PubMed1996

Baculovirus expression and purification of human and rat cytochrome P450 2E1.

High-level expression of human and rat cytochrome P450 2E1 (CYP2E1) was achieved using a baculovirus expression system. A full length cDNA encoding human CYP2E1 was cloned from a human liver cDNA library and sequenced using the dideoxy sequencing method. Insect cells were infected with the homologous recombinant baculoviruses containing the human and rat CYP2E1 cDNAs, respectively. The infected cells were harvested at a time when 450-nm peak intensities were at a maximal level and there was no 420-nm peak observed in the reduced CO difference spectrum. Both human and rat CYP2E1 were then purified to electrophoretic homogeneity by a relatively rapid and efficient procedure. The specific contents of the purified human and rat CYP2E1 were 13.8 and 17.0 nmol/mg protein, respectively. The lambda(max) of the reduced CO difference spectra of both purified rat and human CYP2E1 was found to be 451.5 nm. When the purified rat and human CYP2E1 were reconstituted with purified rat NADPH-P450 reductase and human cytochrome b5, they were able to metabolize several known CYP2E1 substrates: chlorzoxazone, p-nitrophenol, acetaminophen, and carbon tetrachloride. Interestingly, cytochrome b5 markedly stimulated the CYP2E1-mediated two-electron oxidation of the first three substrates, while it had almost no effect on the presumed one-electron reduction of carbon tetrachloride.

Animals↗