The role of CYP2B6 in human xenobiotic metabolism.
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Aryl Hydrocarbon Hydroxylases↗
Biomedical subjects
S A Wrighton
Publications and source records attributed to S A Wrighton.
First-pass midazolam metabolism catalyzed by 1alpha,25-dihydroxy vitamin D3-modified Caco-2 cell monolayers.
Cytochrome P-450 (CYP) 3A4 accounts for approximately 50% of all P-450s found in the small intestine (Paine et al., 1997) and contributes to the extensive and variable first-pass extraction of drugs such as cyclosporine and saquinavir. We recently demonstrated that CYP3A4 expression in a differentiated Caco-2 subclone is increased when cell monolayers are treated with 1alpha,25-dihydroxy-vitamin-D3 (Schmiedlin-Ren et al., 1997). This improved metabolic capacity permits the in vitro modeling of first-pass intestinal metabolic kinetics. Midazolam (MDZ) 1'-hydroxylation was used as a specific probe for CYP3A-mediated metabolism in modified Caco-2 monolayers. Caco-2 cells were grown to confluence on laminin-coated culture inserts, and then for two additional weeks in the presence of 1alpha,25-dihydroxy vitamin-D3. Cell monolayers were subsequently exposed to MDZ for varying lengths of time and concentrations. The amount of MDZ in the monolayer increased rapidly after apical drug administration, reaching a pseudo steady state within 6 min. The cellular uptake rate was considerably slower after a basolateral dose. By either route of administration, the rate of 1'-hydroxymidazolam formation was stable and linear for 2 h. Under basolateral sink conditions and low apical MDZ dosing concentration (1-8 microM), the first-pass extraction ratio was found to be approximately 15%. Higher dosing concentrations led to saturation of the hydroxylation reaction and reduction in the extraction ratio. The modified Caco-2 cell monolayer is an excellent model for studying drug absorption and first-pass intestinal metabolic kinetic processes. In this system, the selective CYP3A probe MDZ was rapidly absorbed, yet extensively metabolized, as is observed in vivo.
Biotransformation↗
Midazolam metabolism by modified Caco-2 monolayers: effects of extracellular protein binding.
It has been suggested that the binding of a drug to plasma proteins will influence the intestinal extraction efficiency when drug is delivered to the mucosal epithelium via either the gut lumen or vasculature. We evaluated this hypothesis using cytochrome P-450 (CYP)3A4-expressing Caco-2 monolayers as a model for the intestinal epithelial barrier and midazolam as a CYP3A-specific enzyme probe. The rate of 1'-hydroxylation was measured following apical or basolateral midazolam administration to monolayers incubated in the presence or absence of 4 g/dl of human serum albumin (HSA) in the basolateral compartment medium. The midazolam-free fraction in culture medium containing HSA was 3.3%. Inclusion of HSA in the basolateral medium decreased peak intracellular midazolam accumulation after an apical midazolam dose (3 microM) by 35% and reduced the 1'-hydroxymidazolam formation rate by approximately 20%. Because of the accelerated diffusion of midazolam through the cell monolayer and into the basolateral compartment, there was a 61% reduction in the first-pass metabolic extraction ratio: 13.3 +/- 0. 12% for control versus 5.2 +/- 1% with HSA. Compared with control, addition of HSA resulted in a 91% decrease in the peak intracellular midazolam level and a 86% decrease in the rate of 1'-hydroxylation after the administration of midazolam into basolateral medium. These findings suggest that, in vivo, binding of a drug to plasma proteins will impact both first-pass and systemic intestinal midazolam extraction efficiency. Furthermore, the effect will be more pronounced for a drug that is delivered to mucosal enterocytes by way of arterial blood, compared with oral drug delivery.
Algorithms↗
Three- and four-dimensional quantitative structure activity relationship analyses of cytochrome P-450 3A4 inhibitors.
The program Catalyst was used to build three-dimensional quantitative structure activity relationship (3D-QSAR) pharmacophore models of the structural features common to competitive-type inhibitors of cytochrome P-450 (CYP) 3A4. These were compared with 3D- and four-dimensional (4D)-QSAR partial least-squares (PLS) models built using molecular surface-weighted holistic invariant molecular (MS-WHIM) descriptors for size and shape of the inhibitor. The Catalyst pharmacophore model generated from multiple conformers of competitive inhibitors of CYP3A4-mediated midazolam 1'-hydroxylation (n = 14) yielded a high correlation of observed and predicted Ki values of r = 0.91. Similarly, PLS MS-WHIM was used to produce 3D- and 4D-QSARs for this data set and produced models that were statistically predictable after cross-validation. Two additional Catalyst pharmacophores were constructed from literature Ki values (n = 32) derived from the inhibition of CYP3A-mediated cyclosporin A metabolism and IC50 data (n = 22) from the inhibition of CYP3A4-mediated quinine 3-hydroxylation. These Catalyst pharmacophores illustrated correlations of observed and predicted inhibition for CYP3A4 of r = 0.77 and 0.92, respectively. The corresponding 4D-QSARs generated by PLS MS-WHIM for these data sets were of comparable quality as judged by cross-validation. Both Ki pharmacophores generated with Catalyst were also validated by predicting the Ki(apparent) values of a test set of eight CYP3A4 inhibitors not included in either model. In seven of eight cases, the residuals of the predicted Ki(apparent) values were within 1 log unit of the observed values. The 3D- and 4D-QSAR models produced in this study suggest the utility of future in silico prediction of CYP3A4-mediated drug-drug interactions.
Catalysis↗
Selectivity of the multidrug resistance modulator, LY335979, for P-glycoprotein and effect on cytochrome P-450 activities.
Overexpression of ATP-dependent drug efflux pumps, P-glycoprotein (Pgp) or multidrug resistance-associated protein (MRP), confers multidrug resistance to tumor cells. Modulators of multidrug resistance block the action of these pumps, thereby sensitizing cells to oncolytics. A potent Pgp modulator is LY335979, which fully sensitizes Pgp-expressing cells at 0.1 microM in cytotoxicity assays and for which Pgp has an affinity of 59 nM. The present study examines its effect on MRP1-mediated drug resistance and cytochrome P-450 (CYP) activity and its ability to serve as a Pgp substrate. Drug resistance was examined with HL60/ADR and MRP1-transfected HeLa-T5 cells. Drug cytotoxicity was unaffected by 1 microM LY335979; leukotriene C4 uptake into HeLa-T5 membrane vesicles was unaffected. Because the substrate specificity of Pgp and CYP3A overlap, the effect of LY335979 on the 1'-hydroxylation of midazolam by CYP3A in human liver microsomes was examined. The apparent K(i) was 3.8 microM, approximately 60-fold higher than the affinity of Pgp for LY335979. The modulator's effect on Pgp was evaluated with Pgp-overexpressing CEM/vinblastine (VLB)(100) and parental CCRF-CEM cells. Both cell lines accumulated [(3)H]LY335979 equally well and did not efflux [(3)H]LY335979 during a 3-h incubation, indicating that it is not a substrate of Pgp. Equilibrium-binding studies with CEM/VLB(100) plasma membranes and [(3)H]LY335979 showed that Pgp had a K(d) of 73 nM, which is in good agreement with the previously determined K(i) value. Thus, LY335979 is an extremely potent Pgp, and not MRP1 or MRP2, modulator and has a significantly lower affinity for CYP3A than for Pgp.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
The use of human hepatocyte cultures to study the induction of cytochrome P-450.
We have previously reported that paclitaxel (Taxol) is a potent inducer of cytochrome P-450 (CYP) 3A protein and CYP3A mRNA in human hepatocyte cultures. Here we report that Taxol increased CYP3A-dependent testosterone 6beta-hydroxylation in intact hepatocytes. This effect was concentration-dependent, with maximal increase in enzyme activity being observed at 10 microM Taxol. Treatment of hepatocyte cultures with concentrations of Taxol higher than 10 microM caused a dose-dependent decrease in testosterone 6beta-hydroxylase activity, amount of CYP3A protein, and total protein synthesis. The maximal CYP3A activity detected after treatment with Taxol or rifampicin was similar in six separate human hepatocyte cultures, suggesting that the cultures have achieved a limit of maximally inducible CYP3A. The fold increase in enzyme activity, however, was different and was inversely related to the level of expression in untreated hepatocytes, with the greatest increases being observed in the hepatocytes that expressed the lowest basal level of CYP3A. Pretreatment of hepatocytes with triacetyloleandomycin resulted in a 90% inhibition of testosterone 6beta-hydroxylase activity. Our results demonstrate the use of human hepatocyte cultures to investigate the induction of cytochrome P-450 by xenobiotics in intact cells and stress the importance of large dose-response studies as well as the need to assess toxicity in these investigations. The response to inducers of CYP3A activity were very consistent among different hepatocyte donors. Absolute values of testosterone 6beta-hydroxylase activity did not vary more than 2- and 5-fold in induced and untreated hepatocytes, respectively.
Antibiotics, Antitubercular↗
Three-dimensional-quantitative structure activity relationship analysis of cytochrome P-450 3A4 substrates.
To gain a better understanding of the active site of cytochrome P-450 (CYP) 3A4, a three-dimensional-quantitative structure activity relationship model was constructed using the structures and K(m (apparent)) values of 38 substrates of human liver microsomal CYP3A4. This pharmacophore was built using the program Catalyst and consisted of four features: two hydrogen bond acceptors, one hydrogen bond donor, and one hydrophobic region. The pharmacophore demonstrated a fit value (r) of observed and expected K(m(apparent)) value of 0.67. The validity of the CYP3A4 substrate model was tested by twice permuting (randomizing) the activity values and substrate structures. The results of this validation procedure indicated that the original model was a significant representation of the features required of CYP3A4 substrates. The second validation method used the Catalyst model to predict the K(m(apparent)) values of a test set of structurally diverse substrates for CYP3A4 not included in the 38 molecules used to build the model. Two fitting algorithms included in this software were examined: fast fit and best fit. The fast fitting method resulted in predictions for all 12 substrates that were within 1 log unit for the residual [i.e., the difference between predicted and observed K(m(apparent))]. In contrast, the best fit algorithm poorly predicted the K(m (apparent)) values (i.e., residual >1 log unit) of 4 of 12 substrates. These poor fits with the best fit function suggest that the fast fit method within Catalyst is more representative of the observed K(m(apparent)) values for CYP3A4 substrates and enables good in silico prediction of this activity. A Catalyst common features pharmacophore was also constructed from three molecules known to activate their own metabolism included in the 38 molecules of the initial CYP3A4 model. This demonstrated that activators of CYP3A4 possess multiple hydrophobic regions that might correspond with a region in the active site away from the metabolic site.
Catalysis↗
Flavin-containing monooxygenase-mediated N-oxidation of the M(1)-muscarinic agonist xanomeline.
The involvement of flavin-containing monooxygenases (FMOs) in the formation of xanomeline N-oxide was examined in various human and rat tissues. Expressed FMOs formed xanomeline N-oxide at a significantly greater rate than did expressed cytochromes P-450. Consistent with the involvement of FMO in the formation of xanomeline N-oxide in human liver, human kidney, rat liver, and rat kidney microsomes, this biotransformation was sensitive to heat treatment, increased at pH 8.3, and inhibited by methimazole. The latter two characteristics were effected to a lesser extent in human kidney, rat liver, and rat kidney microsomes than were observed in human liver microsomes, suggesting the involvement of a different FMO family member in this reaction in these tissues. As additional proof of the involvement of FMO in the formation of xanomeline N-oxide, the formation of this metabolite by a characterized human liver microsomal bank correlated with FMO activity. The FMO forming xanomeline N-oxide by human kidney microsomes exhibited a 20-fold lower K(M) (average K(M) = 5.5 microM) than that observed by the FMO present in human liver microsomes (average K(M) of 107 microM). The involvement of an FMO in the formation of xanomeline N-oxide in rat lung could not be unequivocally demonstrated. These data and those in the literature suggest that the increased prevalence of N-oxidized metabolites of xanomeline after s.c. dosing as compared with oral dosing may be due to differences in the affinity of various FMO family members for xanomeline or to differences in exposure to xanomeline that these enzymes receive under different dosing regimens.
Animals↗
Three and four dimensional-quantitative structure activity relationship (3D/4D-QSAR) analyses of CYP2D6 inhibitors.
Three- and four-dimensional quantitative structure activity relationship (3D/4D-QSAR) pharmacophore models of competitive inhibitors of CYP2D6 were constructed using data from our laboratory or the literature. The 3D-QSAR pharmacophore models of the common structural features of CYP2D6 inhibitors were built using the program Catalyst (Molecular Simulations, San Diego, CA, USA). These 3D-QSAR models were compared with 3D and 4D-QSAR partial least squares (PLS) models which were constructed using molecular surface-weighted holistic invariant molecular (MS-WHIM) descriptors of size and shape of inhibitors. The first Catalyst model was generated from multiple conformers of competitive inhibitors (n = 20) of CYP2D6 mediated bufurolol 1'-hydroxylation. This model demonstrated a correlation of observed and predicted Ki (apparent) values of r = 0.75. A second Catalyst model was constructed from literature derived Ki (apparent) values (n = 31) for the inhibition of CYP2D6. This model provided a correlation of observed and predicted inhibition for CYP2D6 of r = 0.91. Both Catalyst Ki pharmacophores were then validated by predicting the Ki (apparent) of a test set of known CYP2D6 inhibitors (n = 15). Ten out of 15 of these Ki (apparent) values were predicted to be within one log residual of the observed value using our CYP2D6 inhibitor model, while the literature model predicted nine out of 15 values. Similarly, 3D- and 4D-QSARs derived from PLS MS-WHIM for our dataset yielded predictable models as assessed using cross-validation. The corresponding cross-validated PLS MS-WHIM model for the literature dataset yielded a comparable 3D-QSAR and improved 4D-QSAR value. Such computational models will aid in future prediction of drug-drug interactions.
Cytochrome P-450 CYP2D6 Inhibitors↗
Three-dimensional quantitative structure activity relationship analyses of substrates for CYP2B6.
To begin to build an understanding of the interactions of CYP2B6 with substrates, two different 3-dimensional quantitative structure activity relationship (3D-QSAR) models were constructed using 16 substrates of B-lymphoblastoid expressed CYP2B6. A pharmacophore model was built using the program Catalyst, which was compared with a partial least-squares (PLS) model using molecular surface-weighted holistic invariant molecular (MS-WHIM) descriptors. The Catalyst model yielded a 3-dimensional model of the common structural features of CYP2B6 substrates, whereas PLS MS-WHIM generated a model based on statistical analyses of molecular descriptors for size and shape of the substrate. The pharmacophore model obtained with Catalyst consisted of three hydrophobes and one hydrogen bond acceptor region. The cross-validated PLS MS-WHIM model gave a good q2 value of 0.607. Size, positive electrostatic potential, hydrogen bonding acceptor capacity, and hydrophobicity were found to be the most relevant descriptors for the model. These models were then used to predict the Km (apparent) values of a test set of structurally diverse substrates for CYP2B6 not included in the model building, specifically lidocaine, amitriptyline, bupropion, arteether, and verapamil. Overall, both 3D-QSAR methods yielded satisfactory Km (apparent) value predictions for the majority of the molecules in the test set. However, PLS MS-WHIM was unable to reliably predict the Km (apparent) value for verapamil, whereas Catalyst did not predict the Km (apparent) value for lidocaine. In both of these cases the residual of the Km (apparent) value was greater than one log unit. The strengths and limitations of both of these 3D-QSAR approaches are discussed.
Aryl Hydrocarbon Hydroxylases↗
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↗
Induction of cytochrome P4503A by taxol in primary cultures of human hepatocytes.
In primary cultures of human hepatocytes, paclitaxel (Taxol), at pharmacological concentrations, was demonstrated to induce immunoreactive cytochrome P4503A (CYP3A). The magnitude of the inductive response of the hepatocytes to Taxol varied in five separate cultures. In general, exposure to increasing concentrations of Taxol (0.2 to 10 microM) resulted in increases in immunoreactive CYP3A. In four of the cultures, treatment of hepatocytes with the lowest concentration of Taxol tested (0.2 microM) resulted in approximately two-fold increases in CYP3A. In the other culture, however, a six-fold increase in CYP3A was observed at 0.2 microM. Taxol was almost as effective as rifampicin in inducing CYP3A in two of the cultures, but less effective than rifampicin in two other cultures. CYP3A4 mRNA was increased by Taxol. Increases in CYP3A4 mRNA correlated with increases in the levels of immunoreactive CYP3A. These results demonstrate that Taxol is a potent inducer of CYP3A in human hepatocytes. The clinical significance of these findings is discussed.
Adolescent↗
Liver dihydropyrimidine dehydrogenase activity in human, cynomolgus monkey, rhesus monkey, dog, rat and mouse.
Interspecies differences in dihydropyrimidine dehydrogenase (DPD), the initial and rate-limiting enzyme in pyrimidine degradation, were assessed in cytosol from livers isolated from human, monkey, dog, rat, and mouse. Hepatic DPD activity was measured by an HPLC assay with on-line radioactivity detection, using 14C-5-fluorouracil as a substrate. Activity was highly variable within each species and significant interspecies differences in liver DPD activity were observed. The order of activity was mouse > rat > human > dog > or = cynomolgus monkey > rhesus monkey. These data suggest that careful selection must be made when choosing in vivo models of human DPD for the preclinical development of novel fluoropyrimidine anticancer agents and DPD inhibitors.
Animals↗
Human cytochrome P450 3A (CYP3A) mediated midazolam metabolism: the effect of assay conditions and regioselective stimulation by alpha-naphthoflavone, terfenadine and testosterone.
The effect of ionic strength, assay constituents, alpha-naphthoflavone (aNF), terfenadine and testosterone on human CYP3A mediated midazolam (MDZ) 1'-hydroxylation (MDZ 1'-OH) and 4-hydroxylation (MDZ 4-OH) in vitro was examined. Increasing concentration of Tris-HCl (Tris) and sodium phosphate (PO4) buffers differentially affected MDZ 1'-OH and MDZ 4-OH formation rates and had a different effect on MDZ metabolism mediated by microsomes containing CYP3A4 versus CYP3A4 and CYP3A5. MDZ metabolism was not affected by PO4 buffer concentration when cumene hydroperoxide (CUOOH) was used as the source of reactive oxygen. Interestingly, the ammonium ion present in the solution of glucose 6-phosphate dehydrogenase was found to inhibit MDZ metabolism. The addition of MgCl2 up to 50 mM and CaCl2 (5-30 mM) had no affect or inhibited MDZ metabolism, respectively. Formation of MDZ 1'-OH by microsomes from adult and fetal liver and expressed CYP3A4 was regioselectively stimulated by aNF (10 microM). In human hepatocytes, aNF stimulated MDZ 1'-OH formation (up to 100%). Terfenadine (20 microM) regioselectively stimulated MDZ 1'-OH formation in Tris (1-200 mM) and PO4 (1-10 mM) buffers by up to 159%. Surprisingly, with expressed CYP3A4, terfenadine (20 microM) inhibited MDZ 1'-OH formation. Terfenadine (20 microM) had little effect on MDZ 1'-OH formation by fetal liver microsomes. Testosterone (10 and 100 microM) regioselectively stimulated (up to 269%) MDZ 4-OH formation by adult liver microsomes and expressed CYP3A4. Testosterone (100 microM) inhibited (> 40%) MDZ 1'-OH and MDZ 4-OH formation by fetal liver microsomes. With adult liver microsomes, aNF and terfenadine had little effect on the Km for MDZ 1'-OH formation. However, the Km for MDZ 4-OH formation was decreased (up to 94%) by 100 microM testosterone. In the presence of CUOOH, no stimulation of MDZ metabolism was observed by aNF, terfenadine or testosterone in adult liver microsomes. These studies indicate that because assay conditions can substantially alter the catalytic activity of CYP3A, caution should be exerted when extrapolating results between in vitro and in vivo, and when results from different laboratories are compared. Further, these results suggest that the stimulation of CYP3A4 may also occur in vivo and, consequently, may have clinical importance.
Adult↗
Liquid chromatography/nuclear magnetic resonance spectroscopy and liquid chromatography/mass spectrometry identification of novel metabolites of the multidrug resistance modulator LY335979 in rat bile and human liver microsomal incubations.
Compound LY335979 is a P-glycoprotein inhibitor currently entering phase I clinical trials for potential reversal of multidrug resistance to cancer chemotherapy. In early exploratory studies, LY335979 was found to be rapidly transformed in incubations with liver microsomes from rats, dogs, monkeys, and humans. Although the parent compound was completely metabolized, no prominent metabolite peaks were observed. One peak did appear early in the time course, but it did not increase over time. In another preliminary experiment, rats were treated iv with [3H]LY335979 (prepared for pharmacology studies), and urine and bile fractions were collected. Analysis of the urine by reverse-phase HPLC with UV and radioactivity detection revealed that almost all of the material eluted with the solvent front. More than half the radioactivity in bile was accounted for by two peaks eluting earlier than the parent compound (the rest eluted at the solvent front). With both bile and the incubations with microsomes, initial attempts to isolate metabolites were not successful. There was also evidence in both systems of products derived from cleavage of LY335979 (by both further metabolism and degradation). LC/NMR was thus used to analyze materials directly in their respective matrices. An N-oxide metabolite (LY389551) formed by oxidation of the quinoline nitrogen was identified in the microsomal incubations; in bile, three glucuronide metabolites were identified, all of which were conjugates of products formed by oxidation of the quinoline ring of LY335979. There have been few reports in the literature of LC/NMR analysis of bile, which is a more complex matrix than either urine or microsomal suspensions. However, the HPLC techniques developed in this work for the HPLC/UV and LC/MS analyses of LY335979 metabolites in the microsomal matrix and in bile proved readily adaptable for LC/NMR. Using a 500-MHz instrument, basic 1H NMR spectra could be obtained in 2-3 hr with approximately 100 ng of material in the LC/NMR microprobe. With approximately 1.5 microg of material injected onto the column, 1H-1H correlation spectroscopy spectra could be acquired overnight. Along with LC/MS data, the LC/NMR technique facilitated direct identification of a number of metabolites of LY335979 at a point at which their identification by traditional methods would not have been pursued.
Animals↗
Further characterization of the expression in liver and catalytic activity of CYP2B6.
Previous studies in this laboratory have determined the lack of specificity of several antibody and substrate probes of CYP2B6. The goals of the current study were to examine the expression of CYP2B6 in a bank of human liver microsome (HLM) samples using a new specific monoclonal antibody (MAb 49-10-20) and to further characterize the substrate specificity of CYP2B6. A 100-fold variability in expression of immunodetectable CYP2B6 was demonstrated in a bank of 19 HLM samples (0.7 pmol/mg protein to 71. 1 pmol/mg protein) using MAb 49-10-20. CYP2B6 levels were found to significantly (P < .0001) correlate with S-mephenytoin N-demethylation to nirvanol (r2 = 0.89), 7-hydroxy-4-trifluoromethylcoumarin formation (r2 = 0.81) and several markers of CYP3A levels and activity. The relationships between nirvanol formation and CYP3A levels or activity were found to depend on two HLM samples. Km (apparent) values were generated for benzyloxyresorufin O-deethylation (1.3 microM), benzphetamine N-demethylation (93.4 microM), 3-cyano 7-ethoxycoumarin O-deethylation (71.3 microM), midazolam 1'-hydroxylation (46.1 microM) and 4-chloromethyl-7-ethoxycoumarin O-deethylation (33.7 microM) using expressed CYP2B6. Testosterone 16beta-hydroxylation by expressed CYP2B6 resulted in atypical kinetics characteristic of substrate activation. The data best fit the Hill equation with a Km (apparent) of 50.5 microM and an n of 1.3 (n = number of sites bound by activator). In conclusion, the highly specific MAb 49-10-20 was used to provide further confirmation that S-mephenytoin N-demethylation to nirvanol is a CYP2B6 selective probe. Finally, some, but not all substrates of CYP2B6 demonstrate autoactivation.
Antibodies, Monoclonal↗
Alterations of the catalytic activities of drug-metabolizing enzymes in cultures of human liver slices.
Precision-cut human liver slices are an important tool for defining the metabolism and hepatotoxicity of drug candidates early in development. Because of the frequent use of this in vitro tool, a knowledge of the catalytic activities of the drug-metabolizing enzymes during human liver slice culture is necessary. Therefore, marker catalytic activities for various cytochrome P450 (P450 or CYP) forms, as well as phase II activities (glucuronidation and sulfation of 7-hydroxycoumarin), were measured in slices from three different human livers during 96 hr in culture. Standard viability measures were found to be stable from 8 to 24 hr and then declined to 96 hr. Catalytic activities measured for the P450s were ethoxyresorufin O-deethylase (CYP1A2), coumarin 7-hydroxylase (CYP2A6), (S)-mephenytoin N-demethylase (CYP2B6), diclofenac 4'-hydroxylase (CYP2C9), (S)-mephenytoin 4'-hydroxylase (CYP2C19), bufuralol 1'-hydroxylase (CYP2D6), chlorzoxazone 6-hydroxylase (CYP2E1), and midazolam 1'-hydroxylase (CYP3A). The P450 activities decreased by approximately 20% by 4 hr and by at least 65% by 24 hr and were not measurable by 96 hr. In contrast to the phase I activities, 7-hydroxycoumarin glucuronosyltransferase activity was increased at the 8-hr time point by approximately 100% and then decreased to approximately initial values by 96 hr. The 7-hydroxycoumarin sulfotransferase activity of the slices decreased significantly more slowly than did the P450 activities. In conclusion, using conventional methods of liver slice preparation and culture, most of the metabolic capabilities of human liver slices are rapidly lost with time. Therefore, the development of culture methods for human liver slices that can improve the preservation of the drug-metabolizing capabilities may be required.
Catalysis↗
Autoactivation and activation of the cytochrome P450s.
OBJECTIVE: In order to reliably predict in vivo pharmacokinetic parameters from in vitro data, we must thoroughly understand the systems we currently use to determine enzyme kinetic parameters. There have been a number of reports of atypical Michaelis-Menten kinetics for cytochrome- (CYP) P4503A mediated metabolism in vitro but little discussion of its clinical relevance. In this manuscript, we examined the scope of CYP autoactivation and confirmed that CYP1A2 demonstrates atypical Michaelis-Menten kinetics in vitro. MATERIALS: Human liver microsomes, baculovirus-expressed CYP1A2, CYP1A2 in the RECO format, and E. coli expressed CYP1A2 were utilized. METHODS: Enzyme kinetics were performed using the various human CYP1A2 sources and ethoxyresorufin O-deethylation as a prototypical biotransformation. The data were fit to various models of enzyme kinetics. In some cases the data best fit the Hill equation, which was used to empirically model allosteric-type autoactivation kinetics. RESULTS: RECO CYP1A2 and E. coli expressed CYPIA2 both demonstrated autoactivation kinetics for ethoxyresorufin O-deethylation. When the data were fit to the Hill equation, n (the slope factor) was found to be 1.4 and 1.8 for RECO and E. coli expressed CYP1A2, respectively. Human liver microsomal and insect expressed sources of CYP1A2 illustrated classical Michaelis-Menten kinetics for the O-deethylation of ethoxyresorufin. CONCLUSION: Data generated in the current study and previous work suggest many CYPs, not only CYP3A, appear to behave as allosteric enzymes. We would argue that this is not necessarily a classical allosteric mechanism because n is frequently a non-integer. This autoactivation appears to be a function of several factors including substrate physicochemical characteristics, specific interactions of the substrates (activators) with the enzyme active site, and presence of other enzyme modulators. These factors interact to increase the catalytic activity of CYP and thus the complexity of predicting enzyme kinetic parameters or drug interactions.
Cytochrome P-450 CYP1A1↗