Mechanistic Studies of Cytochrome P450 3A4 Time-Dependent Inhibition Using Two Cysteine-Targeting Electrophiles.

Experiments designed to identify the mechanism of cytochrome P450 inactivation are critical to drug discovery. Small molecules irreversibly inhibit P450 enzymatic activity via two primary mechanisms: apoprotein adduct formation or heme modification. Understanding the interplay between chemical structures of reactive electrophiles and the impact on CYP3A4 structure and function can ultimately provide insights into drug design to minimize P450 inactivation. In a previous study, raloxifene and N-(1-pyrene) iodoacetamide (PIA) alkylated CYP3A4 in vitro; however, only raloxifene influenced enzyme activity. Here, two alkylating agents with cysteine selectivity, PIA and pyrene maleimide (PM), were used to investigate this apparent compound-dependent disconnect between CYP3A4 protein alkylation and activity loss. The compound's effect on 1) enzymatic activity, 2) carbon monoxide (CO) binding capacity, 3) intact heme content, and 4) protein conformation were measured. Results showed that PM had a large time-dependent loss of enzyme activity, whereas PIA did not. The differential effect on enzymatic activity between PM and PIA was mirrored in the CO binding data. Despite disruption of CO binding, neither compound affected the heme concentrations, inferring there was no destruction or alkylation of the heme. Lastly, differential scanning fluorescence showed PM-treated CYP3A4 caused a shift in the onset temperature required to induce protein aggregation, which was not observed for CYP3A4 treated with PIA. In conclusion, alkylation of CYP3A4 apoprotein can have a variable impact on catalytic activity, CO binding, and protein conformation that may be compound-dependent. These results highlight the need for careful interpretation of experimental results aimed at characterizing the nature of P450 enzyme inactivation. SIGNIFICANCE STATEMENT: Understanding the mechanism of CYP3A4 time-dependent inhibition is critical to drug discovery. In this study, we use two cysteine-targeting electrophiles to probe how subtle variation in inhibitor structure may impact the mechanism of CYP3A4 time-dependent inhibition and confound interpretation of traditional diagnostic experiments. Ultimately, this simplified system was used to reveal insights into CYP3A4 biochemical behavior. The insights may have implications that aid in understanding the susceptibility of CYP enzymes to the effects of electrophilic intermediates generated via bioactivation.

Characterization of ritonavir-mediated inactivation of cytochrome P450 3A4.

Ritonavir is a human immunodeficiency virus (HIV) protease inhibitor and an inhibitor of cytochrome P450 3A4, the major human hepatic drug-metabolizing enzyme. Given the potent inhibition of CYP3A4 by ritonavir, subtherapeutic doses of ritonavir are used to increase plasma concentrations of other HIV drugs oxidized by CYP3A4, thereby extending their clinical efficacy. However, the mechanism of inhibition of CYP3A4 by ritonavir remains unclear. To date, data suggests multiple types of inhibition by ritonavir, including mechanism-based inactivation by metabolic-intermediate complex formation, competitive inhibition, irreversible type II coordination to the heme iron, and more recently heme destruction. The results presented here demonstrate that inhibition of CYP3A4 by ritonavir occurs by CYP3A4-mediated activation and subsequent formation of a covalent bond to the apoprotein. Incubations of [(3)H]ritonavir with reconstituted CYP3A4 and human liver microsomes resulted in a covalent binding stoichiometry equal to 0.93 ± 0.04 moles of ritonavir bound per mole of inactivated CYP3A4. The metabolism of [(3)H]ritonavir by CYP3A4 leads to the formation of a covalent adduct specifically to CYP3A4, confirmed by radiometric liquid chromatography-trace and whole-protein mass spectrometry. Tryptic digestion of the CYP3A4-[(3)H]ritonavir incubations exhibited an adducted peptide (255-RM K: ESRLEDTQKHR-268) associated with a radiochromatic peak and a mass consistent with ritonavir plus 16 Da, in agreement with the whole-protein mass spectrometry. Additionally, nucleophilic trapping agents and scavengers of free oxygen species did not prevent inactivation of CYP3A4 by ritonavir. In conclusion, ritonavir exhibited potent time-dependent inactivation of CYP3A, with the mechanism of inactivation occurring though a covalent bond to Lys257 of the CYP3A4 apoprotein.

Characterization of the active site properties of CYP4F12.

Cytochrome P450 4F12 is a drug-metabolizing enzyme that is primarily expressed in the liver, kidney, colon, small intestine, and heart. The properties of CYP4F12 that may impart an increased catalytic selectivity (decreased promiscuity) were explored through in vitro metabolite elucidation, kinetic isotope effect experiments, and computational modeling of the CYP4F12 active site. By using astemizole as a probe substrate for CYP4F12 and CYP3A4, it was observed that although CYP4F12 favored astemizole O-demethylation as the primary route of metabolism, CYP3A4 was capable of metabolizing astemizole at multiple sites on the molecule. Deuteration of astemizole at the site of O-demethylation resulted in an isotope effect of 7.1 as well as an 8.3-fold decrease in the rate of clearance for astemizole by CYP4F12. Conversely, although an isotope effect of 3.8 was observed for the formation of the O-desmethyl metabolite when deuterated astemizole was metabolized by CYP3A4, there was no decrease in the clearance of astemizole. Development of a homology model of CYP4F12 based on the crystal structure of cytochrome P450 BM3 predicted an active site volume for CYP4F12 that was approximately 76% of the active site volume of CYP3A4. As predicted, multiple favorable binding orientations were available for astemizole docked into the active site of CYP3A4, but only a single binding orientation with the site of O-demethylation oriented toward the heme was identified for CYP4F12. Overall, it appears that although CYP4F12 may be capable of binding similar ligands to other cytochrome P450 enzymes such as CYP3A4, the ability to achieve catalytically favorable orientations may be inherently more difficult because of the increased steric constraints of the CYP4F12 active site.

Evaluation of near infrared fluorescent labeling of monoclonal antibodies as a tool for tissue distribution.

The pharmacokinetic (PK) behavior of monoclonal antibodies (mAbs) is influenced by target-mediated drug disposition, off-target effects, antidrug antibody-mediated clearance, and interaction with fragment-crystallizable domain (Fc) receptors such as neonatal Fc receptor. All of these interactions hold the potential to impact mAb biodistribution. Near infrared (NIR) fluorescent probes offer an approach complementary to radionuclides to characterize drug disposition. Notably, the use of FDA-approved IRDye800 (IR800; LI-COR, Lincoln, NE) as a protein-labeling agent in preclinical work holds the potential for quantitative tissue analysis. Here, we tested the utility of the IR800 dye as a quantitative mAb tracer during pharmacokinetic analysis in both plasma and tissues using a model mouse monoclonal IgG1 (8C2) labeled with ≤1.5 molecules of IR800. The plasma PK parameters derived from a mixture of IR800-8C2 and 8C2 dosed intravenously to C57BL/6 mice at 8 mg/kg exhibited a large discrepancy in exposure depending on the method of quantitation [CLplasma = 8.4 ml/d per kilogram (NIR fluorescence detection) versus 2.5 ml/d per kilogram (enzyme-linked immunosorbent assay)]. The disagreement between measurements suggests that the PK of 8C2 is altered by addition of the IR800 dye. Additionally, direct fluorescence analysis of homogenized tissues revealed several large differences in IR800-8C2 tissue uptake when compared with a previously published study using [(125)I]8C2, most notably an over 4-fold increase in liver concentration. Finally, the utility of IR800 in combination with whole body imaging was examined by comparison of IR800-8C2 levels observed in animal sagittal cross-sections to those measured in homogenized tissues. Our results represent the first PK analysis in both mouse plasma and tissues of an IR800-mAb conjugate and suggest that mAb disposition is significantly altered by IR800 conjugation to 8C2.

Evaluation of UGT protein interactions in human hepatocytes: effect of siRNA down regulation of UGT1A9 and UGT2B7 on propofol glucuronidation in human hepatocytes.

Previous experiments performed in recombinant systems have suggested that protein-protein interactions occur between the UGTs and may play a significant role in modulating enzyme activity. However, evidence of UGT protein-protein interactions either in vivo or in more physiologically relevant in vitro systems has yet to be demonstrated. In this study, we examined oligomerization and its ability to affect glucuronidation in plated human hepatocytes. siRNA down regulation experiments and activity studies were used to examine changes in metabolite formation of one UGT isoform due to down regulation of a second UGT isoform. Selective siRNA directed towards UGT1A9 or UGT2B7 resulted in significant and selective decreases in their respective mRNA levels. As expected, the metabolism of the UGT1A9 substrate propofol decreased with UGT1A9 down regulation. Interestingly, UGT1A9 activity, but not UGT1A9 mRNA expression, was also diminished when UGT2B7 expression was selectively inhibited, implying potential interactions between the two isoforms. Minor changes to UGT1A4, UGT2B4 and UGT2B7 activity were also observed when UGT1A9 expression was selectively down regulated. To our knowledge, this represents the first piece of evidence that UGT protein-protein interactions occur in human hepatocytes and suggests that expression levels of UGT2B7 may directly impact the glucuronidation activity of selective UGT1A9 substrates.

Cytochrome p450 architecture and cysteine nucleophile placement impact raloxifene-mediated mechanism-based inactivation.

The propensity for cytochrome P450 (P450) enzymes to bioactivate xenobiotics is governed by the inherent chemistry of the xenobiotic itself and the active site architecture of the P450 enzyme(s). Accessible nucleophiles in the active site or egress channels of the P450 enzyme have the potential of sequestering reactive metabolites through covalent modification, thereby limiting their exposure to other proteins. Raloxifene, a drug known to undergo CYP3A-mediated reactive metabolite formation and time-dependent inhibition in vitro, was used to explore the potential for bioactivation and enzyme inactivation of additional P450 enzymes (CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A5). Every P450 tested except CYP2E1 was capable of raloxifene bioactivation, based on glutathione adduct formation. However, raloxifene-mediated time-dependent inhibition only occurred in CYP2C8 and CYP3A4. Comparable inactivation kinetics were achieved with K(I) and k(inact) values of 0.26 µM and 0.10 min(-1) and 0.81 µM and 0.20 min(-1) for CYP2C8 and CYP3A4, respectively. Proteolytic digests of CYP2C8 and CYP3A4 Supersomes revealed adducts to Cys225 and Cys239 for CYP2C8 and CYP3A4, respectively. For each P450 enzyme, proposed substrate/metabolite access channels were mapped and active site cysteines were identified, which revealed that only CYP2C8 and CYP3A4 possess accessible cysteine residues near the active site cavities, a result consistent with the observed kinetics. The combined data suggest that the extent of bioactivation across P450 enzymes does not correlate with P450 inactivation. In addition, multiple factors contribute to the ability of reactive metabolites to form apo-adducts with P450 enzymes.

Prediction of CYP2D6 drug interactions from in vitro data: evidence for substrate-dependent inhibition.

Predicting the magnitude of potential drug-drug interactions is important for underwriting patient safety in the clinical setting. Substrate-dependent inhibition of cytochrome P450 enzymes may confound extrapolation of in vitro results to the in vivo situation. However, the potential for substrate-dependent inhibition with CYP2D6 has not been well characterized. The inhibition profiles of 20 known inhibitors of CYP2D6 were characterized in vitro against four clinically relevant CYP2D6 substrates (desipramine, dextromethorphan, metoprolol, and thioridazine) and bufuralol. Dextromethorphan exhibited the highest sensitivity to in vitro inhibition, whereas metoprolol was the least sensitive. In addition, when metoprolol was the substrate, inhibitors with structurally constrained amino moieties (clozapine, debrisoquine, harmine, quinidine, and yohimbine) exhibited at least a 5-fold decrease in inhibition potency when results were compared with those for dextromethorphan. Atypical inhibition kinetics were observed for these and other inhibitor-substrate pairings. In silico docking studies suggested that interactions with Glu216 and an adjacent hydrophobic binding pocket may influence substrate sensitivity and inhibition potency for CYP2D6. The in vivo sensitivities of the clinically relevant CYP2D6 substrates desipramine, dextromethorphan, and metoprolol were determined on the basis of literature drug-drug interaction (DDI) outcomes. Similar to the in vitro results, dextromethorphan exhibited the highest sensitivity to CYP2D6 inhibition in vivo. Finally, the magnitude of in vivo CYP2D6 DDIs caused by quinidine was predicted using desipramine, dextromethorphan, and metoprolol. Comparisons of the predictions with literature results indicated that the marked decrease in inhibition potency observed for the metoprolol-quinidine interaction in vitro translated to the in vivo situation.

Impact of organic solvents on cytochrome P450 probe reactions: filling the gap with (S)-Warfarin and midazolam hydroxylation.

(S)-Warfarin 7-hydroxylation and midazolam 1'-hydroxylation are among the preferred probe substrate reactions for CYP2C9 and CYP3A4/5, respectively. The impact of solvents on enzyme activity, kinetic parameters, and predicted in vivo hepatic clearance (Cl(H)) associated with each reaction has not been evaluated. The effects of increasing concentrations [0.1-2% (v/v)] of six organic solvents (acetonitrile, methanol, ethanol, dimethyl sulfoxide, acetone, isopropanol) were first tested on each reaction using human liver microsomes (HLMs), human intestinal microsomes (midazolam 1'-hydroxylation only), and recombinant enzymes. Across enzyme sources, relative to water, acetonitrile and methanol had the least inhibitory effect on (S)-warfarin 7-hydroxylation (0-58 and 9-96%, respectively); acetonitrile, methanol, and ethanol had the least inhibitory effect on midazolam 1'-hydroxylation (0-29, 0-22, and 0-20%, respectively). Using HLMs, both acetonitrile and methanol (0.1-2%) decreased the V(max) (32-60 and 24-65%, respectively) whereas methanol (2%) increased the K(m) (100%) of (S)-warfarin-hydroxylation. (S)-Warfarin Cl(H) was underpredicted by 21-65% (acetonitrile) and 13-84% (methanol). Acetonitrile, methanol, and ethanol had minimal to modest impact on both the kinetics of midazolam 1'-hydroxylation (10-24%) and predicted midazolam Cl(H) (2-20%). In conclusion, either acetonitrile or methanol at ≤0.1% is recommended as the primary organic solvent for the (S)-warfarin 7-hydroxylation reaction; acetonitrile is preferred if higher solvent concentrations are required. Acetonitrile, methanol, and ethanol at ≤2% are recommended as primary organic solvents for the midazolam 1'-hydroxylation reaction. This information should facilitate optimization of experimental conditions and improve the interpretation and accuracy of in vitro-in vivo predictions involving these two preferred cytochrome P450 probe substrate reactions.

In vitro characterization of the drug-drug interaction potential of catabolites of antibody-maytansinoid conjugates.

The in vitro characterization of the inhibition potential of four representative maytansinoid species observed upon hepatic and/or tumor in vivo processing of antibody-maytansine conjugates (AMCs) with cleavable and noncleavable linkers is reported. We investigated the free maytansinoid species N(2')-deacetyl-N(2')-(3-mercapto-1-oxopropyl)-maytansine (DM1), (S)-methyl-DM1, and N(2')-deacetyl-N(2')-(4-mercapto-4-methyl-1-oxopentyl)-maytansine (DM4) as representative cleavable linker catabolites and Lysine-N(ε)-N-succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate-DM1 (Lys-MCC-DM1) as the representative noncleavable linker catabolite. Studies with recombinant human cytochromes P450 (P450s) indicate CYP2D6, CYP3A4, and CYP3A5 are the primary isoforms responsible for the oxidative metabolism of DM1, (S)-methyl-DM1, and DM4. Lys-MCC-DM1 was not metabolized by any of the P450 isoforms studied. DM1 was shown to be a reversible inhibitor of CYP2C8 (K(i) = 11 ± 3 µM) and CYP2D6 (K(i) = 14 ± 2 µM). Lys-MCC-DM1 and (S)-methyl-DM1 showed no reversible or time-dependent inactivation of any of the P450s studied. DM1 and DM4 inactivated CYP3A from human liver microsomes with K(i)/k(inact) values of 4.8 ± 0.9 µM/0.035 ± 0.002 min(-1) and 3.3 ± 0.2 µM/0.114 ± 0.002 min(-1), respectively. DM1 and DM4 inactivated recombinant CYP3A4 with K(i)/k(inact) values of 3.4 ± 1.0 µM/0.058 ± 0.005 min(-1) and 1.4 ± 0.3 µM/0.117 ± 0.006 min(-1), respectively. Because of instability in plasma, further characterization of the DM1 and DM4 intramolecular and intermolecular disulfide conjugates observed in vivo is required before an accurate drug-drug interaction (DDI) prediction can be made. AMCs with noncleavable thioether-linked DM1 as the cytotoxic agent are predicted to have no potential for a DDI with any of the major human P450s studied.

Predicting the drug interaction potential of AMG 853, a dual antagonist of the D-prostanoid and chemoattractant receptor-homologous molecule expressed on T helper 2 cells receptors.

2-(4-(4-(tert-Butylcarbamoyl)-2-(2-chloro-4-cyclopropylphenylsulfonamido)phenoxy)-5-chloro-2-fluorophenyl)acetic acid (AMG 853) is an orally bioavailable and potent dual antagonist of the D-prostanoid and chemoattractant receptor-homologous molecule expressed on T helper 2 cells receptors. The drug interaction potential of AMG 853, both as a victim and a perpetrator, was investigated using in vitro, in silico, and in vivo methodologies. Experiments in human liver microsomes (HLM) and recombinant enzymes identified CYP2C8, CYP2J2, and CYP3A as well as multiple UDP-glucuronosyltransferase isoforms as being responsible for the metabolic clearance of AMG 853. With use of HLM and selective probe substrates, both AMG 853 and its acyl glucuronide metabolite (M1) were shown to be inhibitors of CYP2C8. AMG 853 and M1 did not inhibit any of the other cytochrome P450 isoforms tested, and AMG 853 exhibited minimal enzyme induction properties in human hepatocytes cultures. In light of the in vitro findings, modeling and simulation approaches were used to examine the potential for ketoconazole (a CYP3A inhibitor) to inhibit the metabolism of AMG 853 as well as for AMG 853 to inhibit the metabolism of paclitaxel, rosiglitazone, and montelukast, commonly used substrates of CYP2C8. A weak and clinically insignificant drug interaction (area under the drug concentration-time curve (AUC)(i)/AUC <2) was predicted between ketoconazole and AMG 853. No drug interactions were predicted for AMG 853 and paclitaxel, rosiglitazone, or montelukast. Finally, administration of AMG 853 to healthy human subjects in clinical trials in the presence or absence of ketoconazole confirmed that AMG 853 is unlikely to be involved in clinically significant drug interactions.

Evaluation of CYP2C8 inhibition in vitro: utility of montelukast as a selective CYP2C8 probe substrate.

Understanding the potential for cytochrome P450 (P450)-mediated drug-drug interactions is a critical step in the drug discovery process. Although in vitro studies with CYP3A4, CYP2C9, and CYP2C19 have suggested the presence of multiple binding regions within the P450 active site based on probe substrate-dependent inhibition profiles, similar studies have not been performed with CYP2C8. The ability to understand CYP2C8 probe substrate sensitivity will enable appropriate in vitro and in vivo probe selection. To characterize the potential for probe substrate-dependent inhibition with CYP2C8, the inhibition potency of 22 known inhibitors of CYP2C8 were measured in vitro using four clinically relevant CYP2C8 probe substrates (montelukast, paclitaxel, repaglinide, and rosiglitazone) and amodiaquine. Repaglinide exhibited the highest sensitivity to inhibition in vitro. In vitro phenotyping indicated that montelukast is an appropriate probe for CYP2C8 inhibition studies. The in vivo sensitivities of the CYP2C8 probe substrates cerivastatin, fluvastatin, montelukast, pioglitazone, and rosiglitazone were determined in relation to repaglinide on the basis of clinical drug-drug interaction (DDI) data. Repaglinide exhibited the highest sensitivity in vivo, followed by cerivastatin, montelukast, and pioglitazone. Finally, the magnitude of in vivo CYP2C8 DDI caused by gemfibrozil-1-O-ß-glucuronide was predicted. Comparisons of the predictions with clinical data coupled with the potential liabilities of other CYP2C8 probes suggest that montelukast is an appropriate CYP2C8 probe substrate to use for the in vivo situation.

Effects of interleukin-6 (IL-6) and an anti-IL-6 monoclonal antibody on drug-metabolizing enzymes in human hepatocyte culture.

The cytokine-mediated suppression of hepatic drug-metabolizing enzymes by inflammatory disease and the relief of this suppression by successful disease treatment have recently become an issue in the development of drug interaction labels for new biological products. This study examined the effects of the inflammatory cytokine interleukin-6 (IL-6) on drug-metabolizing enzymes in human hepatocyte culture and the abrogation of these effects by a monoclonal antibody directed against IL-6. Treatment of human hepatocytes with IL-6 (n = 9 donors) revealed pan-suppression of mRNA of 10 major cytochrome P450 isoenzymes, but with EC(50) values that differed by isoenzyme. Some EC(50) values were above the range of clinically relevant serum concentrations of IL-6. Marker activities for CYP1A2 and CYP3A4 enzyme were similarly suppressed by IL-6 in both freshly isolated and cryopreserved hepatocytes. IL-6 suppressed induction of CYP1A2 enzyme activity by omeprazole and CYP3A4 enzyme activity by rifampicin but only at supraphysiological concentrations of IL-6. Glycosylated and nonglycosylated IL-6 did not significantly differ in their ability to suppress CYP1A2 and CYP3A4 enzyme activity. A monoclonal antibody directed against IL-6 abolished or partially blocked IL-6-mediated suppression of CYP1A2 and CYP3A4 enzyme activity, respectively. These data indicate that experimentation with IL-6 and anti-IL-6 monoclonal antibodies in human hepatocyte primary culture can quantitatively measure cytochrome P450 suppression and desuppression and determine EC(50) values for IL-6 against individual cytochrome P450 isoenzymes. However, the complex biology of inflammatory disease may not allow for quantitative in vitro-in vivo extrapolation of these simple in vitro data.

Mechanism-based inactivation of cytochrome P450 3A4 by mibefradil through heme destruction.

Mibefradil (Posicor) was developed as a calcium channel blocker for the treatment of chronic hypertension. The compound was withdrawn from the market in 1998 because of the potential for rhabdomyolysis, renal failure, or bradycardia when it was coadministered with other drugs. Mibefradil has previously been shown to be a potent reversible (IC(50) = 0.3-2 µM) and mechanism-based (K(i) = 2.3 µM; k(inact) = 0.4 min(-1)) inhibitor of CYP3A4-catalyzed statin metabolism. At present, the mechanism of CYP3A4 inactivation by mibefradil is not known. Mechanism-based inactivation experiments and spectral studies were used to examine the mechanism of CYP3A4 inactivation by mibefradil and its major metabolite, des-methoxyacetyl mibefradil (Ro 40-5966), in vitro. Both mibefradil and Ro 40-5966 were shown to exhibit type I binding characteristics (K(s) = 0.69 ± 0.06 and 1.39 ± 0.04 µM, respectively) toward CYP3A4. Complete K(i)/k(inact) experiments were performed, revealing a rapid and irreversible decrease in CYP3A4-catalyzed 1'-hydroxymidazolam formation. Approximately 70% of CYP3A4 activity was lost in the first minute of incubation with mibefradil, and inactivation was nonlinear after 2 min. Ro 40-5966 also resulted in time-dependent inhibition of CYP3A4, albeit to a lesser extent than mibefradil. The decrease in CYP3A4 activity in the presence of mibefradil and NADPH was subsequently shown to have a good correlation with the time-dependent loss of CO binding, which, coupled with the lack of stable heme and/or apoprotein adducts, suggests heme destruction as the mechanism of inactivation of CYP3A4 by mibefradil.

Formation of raloxifene homo-dimer in CYP3A4, evidence for multi-substrate binding in a single catalytically competent P450 active site.

Raloxifene is a polyaromatic compound which has been reported to form radicals when incubated with horseradish peroxidase resulting in formation of a homo-dimer product. Polyaromatic phenols have also been reported to undergo oxidation by P450 enzymes to form reactive intermediates, presumably through the formation of phenoxy radical species. Recently, we observed that a raloxifene homo-dimer was formed in vitro when incubated with CYP3A4. In response to this finding, a series of experiments were designed to determine whether the observed raloxifene homo-dimer was formed via solution phase chemistry similar to that previously documented with horseradish peroxidase or if generation of the homo-dimer occurred within the P450 active site. To this end, a series of experiments were carried out to determine the structure of the CYP3A4 generated raloxifene homo-dimer using analytical techniques including: high resolution MS, NMR and H/D exchange. In addition, a variety of in vitro techniques were applied to characterize the mechanism responsible for formation of the raloxifene homo-dimer. Collectively, the results of these experiments suggest that unlike the homo-dimer formed by peroxidase enzymes, raloxifene homo-dimer formation mediated by CYP3A4 is a consequence of two raloxifene molecules binding simultaneously within the active site of a catalytically competent P450 enzyme.

Multienzyme Kinetics and Sequential Metabolism.

Enzymes are the catalysts of biological systems and are extremely efficient. A typical enzyme accelerates the rate of a reaction by factors of at least a million compared to the rate of the same reaction in the absence of the enzyme. In contrast to traditional catalytic enzymes, the family of cytochrome P450 (CYPs) enzymes are catalytically promiscuous and thus they possess remarkable versatility in substrates. The great diversity of reactions catalyzed by CYP enzymes appear to be based on two unique properties of these heme proteins, the ability of their iron to exist under multiple oxidation states with different reactivities and a flexible active site that can accommodate a wide variety of substrates. Herein, is a discussion of two distinct type of kinetics observed with CYP enzymes. The first example is of CYP complex kinetic profiles when multiple CYP enzymes form the sample product. The second is sequential metabolism, in other words, the formation of multiple products from one CYP enzyme. Given the degree of CYP enzyme promiscuity, it is hardly surprising that there is also a high degree of complex kinetic profiles generated during the catalytic cycle.

In vitro modulation of cytochrome P450 reductase supported indoleamine 2,3-dioxygenase activity by allosteric effectors cytochrome b(5) and methylene blue.

Indoleamine 2,3-dioxygenase (IDO) is a heme-containing dioxygenase involved in the degradation of several indoleamine derivatives and has been indicated as an immunosuppressive. IDO is an attractive target for therapeutic intervention in diseases which are known to capitalize on immune suppression, including cancer, HIV, and inflammatory diseases. Conventionally, IDO activity is measured through chemical reduction by the addition of ascorbate and methylene blue. Identification of potential coenzymes involved in the reduction of IDO in vivo should improve in vitro reconstitution systems used to identify potential IDO inhibitors. In this study we show that NADPH-cytochrome P450 reductase (CPR) is capable of supporting IDO activity in vitro and that oxidation of l-Trp follows substrate inhibition kinetics (k(cat) = 0.89 +/- 0.04 s(-1), K(m) = 0.72 +/- 0.15 microM, and K(i) = 9.4 +/- 2.0 microM). Addition of cytochrome b(5) to CPR-supported l-Trp incubations results in modulation from substrate inhibition to sigmoidal kinetics (k(cat) = 1.7 +/- 0.3 s(-1), K(m) = 1.5 +/- 0.9 microM, and K(i) = 1.9 +/- 0.3). CPR-supported d-Trp oxidations (+/-cytochrome b(5)) exhibit Michaelis-Menten kinetics. Addition of methylene blue (minus ascorbate) to CPR-supported reactions resulted in inhibition of d-Trp turnover and modulation of l-Trp kinetics from allosteric to Michaelis-Menten with a concurrent decrease in substrate affinity for IDO. Our data indicate that CPR is capable of supporting IDO activity in vitro and oxidation of tryptophan by IDO displays substrate stereochemistry dependent atypical kinetics which can be modulated by the addition of cytochrome b(5).

Selection of alternative CYP3A4 probe substrates for clinical drug interaction studies using in vitro data and in vivo simulation.

Understanding the potential for cytochrome P450-mediated drug-drug interactions (DDIs) is a critical step in the drug discovery process. DDIs of CYP3A4 are of particular importance because of the number of marketed drugs that are cleared by this enzyme. In response to studies that suggested the presence of several binding regions within the CYP3A4 active site, multiple probe substrates are often used for in vitro CYP3A4 DDI studies, including midazolam (the clinical standard), felodipine/nifedipine, and testosterone. However, the design of clinical CYP3A4 DDI studies may be confounded for cases such as 1-(2-hydroxy-2-methylpropyl)-N-[5-(7-methoxyquinolin-4-yloxy)pyridin-2-yl]-5-methyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazole-4-carboxamide (AMG 458), with which testosterone is predicted to exhibit a clinically relevant DDI whereas midazolam and felodipine/nifedipine are not. To develop an appropriate path forward for such clinical DDI studies, the inhibition potency of 20 known inhibitors of CYP3A4 were measured in vitro using 8 clinically relevant CYP3A4 probe substrates and testosterone. Hierarchical clustering suggested four probe substrate clusters: testosterone; felodipine; midazolam, buspirone, quinidine, and sildenafil; and simvastatin, budesonide, and fluticasone. The in vivo sensitivities of six clinically relevant CYP3A4 probe substrates (buspirone, cyclosporine, nifedipine, quinidine, sildenafil, and simvastatin) were determined in relation to midazolam from literature DDI data. Buspirone, sildenafil, and simvastatin exhibited similar or greater sensitivity than midazolam to CYP3A4 inhibition in vivo. Finally, Simcyp was used to predict the in vivo magnitude of CYP3A4 DDIs caused by AMG 458 using midazolam, sildenafil, simvastatin, and testosterone as probe substrates.

In vitro inhibition of multiple cytochrome P450 isoforms by xanthone derivatives from mangosteen extract.

Mangosteen is a xanthone-containing fruit found in Southeast Asia for which health claims include maintaining healthy immune and gastrointestinal systems to slowing the progression of tumor growth and neurodegenerative diseases. Previous studies have identified multiple xanthones in the pericarp of the mangosteen fruit. The aim of the current study was to assess the drug inhibition potential of mangosteen in vitro as well as the cytochrome P450 (P450) enzymes responsible for the metabolism of its individual components. The various xanthone derivatives were found to be both substrates and inhibitors for multiple P450 isoforms. Aqueous extracts of the mangosteen pericarp were analyzed for xanthone content as well as inhibition potency. Finally, in vivo plasma concentrations of alpha-mangostin, the most abundant xanthone derivative found in mangosteen, were predicted using Simcyp and found to be well above their respective in vitro K(i) values for CYP2C8 and CYP2C9.

Assessment of the metabolism and intrinsic reactivity of a novel catechol metabolite.

PH-302 ( 1) demonstrates potent inhibitory activity against the inducible form of nitric oxide synthase (iNOS). The primary metabolite of PH-302 is a catechol ( 2) resulting from oxidative demethylenation of the methylenedioxyphenyl moiety by cytochrome P450 3A4. Concerns regarding subsequent two-electron oxidation of 2 to an electrophilic quinone species and the potential for resulting toxicity prompted additional studies to examine the reactivity and metabolic fate of this metabolite. Contrary to literature reports of catechol reactivity, 2 appeared to be resistant to quinone formation in human liver microsomal incubations, as determined by the lack of detectable glutathione (GSH) adducts and no covalent binding to microsomal proteins. In addition, 2 showed no evidence of depletion of intracellular glutathione or cytotoxicity at concentrations up to 1 mM in primary human and rat hepatocytes. In the presence of tyrosinase, spectral evidence indicated that 2 was oxidized to the ortho-quinone, and upon incubation in the presence of GSH, two conjugates were detected and characterized by LC/MS/MS. Lastly, the metabolic pathways of 2 were investigated in rat and human hepatocytes and found to be similar, proceeding via glucuronidation, sulfation, and methylation of the catechol. Collectively, these studies demonstrate that 2 appears to be resistant to further oxidation to quinone in liver microsomes, as well as spontaneous redox cycling, while the formation of phase II metabolites in hepatocytes suggests that multiple detoxication pathways may provide added protection against toxicity in the liver.

Predicting in vivo drug interactions from in vitro drug discovery data.

In vitro screening for drugs that inhibit cytochrome P450 enzymes is well established as a means for predicting potential metabolism-mediated drug interactions in vivo. Given that these predictions are based on enzyme kinetic parameters observed from in vitro experiments, the miscalculation of the inhibitory potency of a compound can lead to an inaccurate prediction of an in vivo drug interaction, potentially precluding a safe drug from advancing in development or allowing a potent inhibitor to 'slip' into the patient population. Here, we describe the principles underlying the generation of in vitro drug metabolism data and highlight commonly encountered uncertainties and sources of bias and error that can affect extrapolation of drug-drug interaction information to the clinical setting.