Dan A. Rock

CYP2C9 Inhibition: Impact of Probe Selection and Pharmacogenetics on in Vitro Inhibition Profiles

Drug-drug interactions may cause serious adverse events in the clinical setting, and the cytochromes P450 are the enzyme system most often implicated in these interactions. Cytochrome P450 2C is the second most abundant subfamily of cytochrome P450 enzymes and is responsible for metabolism of almost 20% of currently marketed drugs. The most abundant isoform of this subfamily is CYP2C9, which is the major clearance pathway for the low therapeutic index drugs warfarin and phenytoin. Considering the importance of CYP2C9 to drug-drug interactions, the in vitro-in vivo extrapolation of drug-drug interactions for CYP2C9 may be confounded by the presence of polymorphic variants and the possibility of multiple binding regions within the CYP2C9 active site, leading to the potential for genotype- and substrate-dependent inhibition. To address the issues of genotype-dependent enzyme inhibition as well as probe substrate correlations, the inhibitory potency (Ki) of 28 effector molecules was assessed with five commonly used probes of CYP2C9 in both the CYP2C9.1 and CYP2C9.3 proteins. The inhibition of CYP2C9.1 and CYP2C9.3 by the battery of inhibitors with five substrate probes demonstrated differential inhibition potency not only between the two genotypes but also across substrate probes. Furthermore, the substrate probes fell into three distinct classes depending on genotype, suggesting that multiple probes may be needed to fully assess inhibition of CYP2C9 in vitro. Thus, both genotype and choice of probe substrate must be considered when attempting to predict potential CYP2C9 drug-drug interactions from in vitro data.

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.

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 EC50 values that differed by isoenzyme. Some EC50 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 EC50 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.

Enzyme Source Effects on CYP2C9 Kinetics and Inhibition

When choosing a recombinant cytochrome P450 (P450) enzyme system for in vitro studies, it is critical to understand the strengths, limitations, and applicability of the enzyme system to the study design. Although literature kinetic data may be available to assist in enzyme system selection, comparison of data from separate laboratories is often confounded by differences in experimental conditions and bioanalytical techniques. We measured the Michaelis-Menten kinetic parameters for four CYP2C9 substrates (diclofenac, (S)-warfarin, tolbutamide, and (S)-flurbiprofen) using four recombinant CYP2C9 enzyme systems (Supersomes, Baculosomes, RECO system, and in-house purified, reconstituted enzyme) to determine whether the enzyme systems exhibited kinetic differences in metabolic product formation rates under uniform experimental conditions. The purified, reconstituted enzyme systems exhibited higher Km values, reduced substrate affinity, and lower calculated intrinsic clearance values compared with baculovirus microsomal preparations. Six- to 25-fold differences in predicted intrinsic clearance values were calculated for each substrate depending on the enzyme system-substrate combination. Results suggest that P450 reductase interactions with the CYP2C9 protein and varying ratios of CYP2C9/P450 reductase in the enzyme preparations may play a role in these observed differences. In addition, when (S)-flurbiprofen was used as a substrate probe to determine CYP2C9 inhibition with a set of 12 inhibitors, decreased inhibition potency was observed across 11 of those inhibitors in the RECO purified, reconstituted enzyme compared with the Supersomes baculovirus microsomal preparation and pooled human liver microsomes. Considering these differences, consistent use of an enzyme source is an important component in producing comparable and reproducible kinetics and inhibition data with CYP2C9.

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-β-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.

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.

A method for determining two substrates binding in the same active site of cytochrome P450BM3: an explanation of high energy ω product formation

A number of enzymes from the cytochrome P450 family show atypical (non-Michaelis-Menten) kinetic behavior resulting from substrate activation, inhibition, partial inhibition, biphasic saturation, or autoactivation. Herein, we provide a technique that can identify multiple substrate occupancy in the same active site of a P450 as a result of an altered kinetic profile. Using an isotope effect on product ratios confirms that the enzyme-substrate (ES) complex responsible for omega hydroxylation of palmitic acid (palmitate) is in rapid equilibrium with the ES complex that leads to omega-1 hydroxylation of palmitate. Co-incubation of a second substrate, lauric acid (laurate), results in a change in the ratio of omega to omega-1 hydroxylated palmitate. Furthermore, an isotope effect on palmitate is observed when deuterated laurate is co-incubated with non-deuterated palmitate. These results are only consistent with both substrates being in the same active site simultaneously. This mode of binding explains how the F87A mutant of P450BM3 is able to produce the omega alcohol, a product that arises from the high-energy primary radical.

Metabolism and related human risk factors for hepatic damage by usnic acid containing nutritional supplements

Usnic acid is a component of nutritional supplements promoted for weight loss that have been associated with liver-related adverse events including mild hepatic toxicity, chemical hepatitis, and liver failure requiring transplant. To determine if metabolism factors might have had a role in defining individual susceptibility to hepatotoxicity, in vitro metabolism studies were undertaken using human plasma, hepatocytes, and liver subcellular fractions. Usnic acid was metabolized to form three monohydroxylated metabolites and two regio-isomeric glucuronide conjugates of the parent drug. Oxidative metabolism was mainly by cytochrome P450 (CYP) 1A2 and glucuronidation was carried out by uridine diphosphate-glucuronosyltransferase (UGT) 1A1 and UGT1A3. In human hepatocytes, usnic acid at 20 µM was not an inducer of CYP1A2, CYP2B6, or CYP3A4 relative to positive controls omeprazole, phenobarbital, and rifampicin, respectively. Usnic acid was a relatively weak inhibitor of CYP2D6 and a potent inhibitor of CYP2C19 (the concentration eliciting 50% inhibition (IC50) = 9 nM) and CYP2C9 (IC50 = 94 nM), with less potent inhibition of CYP2C8 (IC50 = 1.9 µM) and CYP2C18 (IC50 = 6.3 µM). Pre-incubation of microsomes with usnic acid did not afford any evidence of time-dependent inhibition of CYP2C19, although evidence of slight time-dependent inhibition of CYP2C9 (KI = 2.79 µM and Kinact = 0.022 min−1) was obtained. In vitro data were used with SimCYPRto model potential drug interactions. Based on usnic acid doses in case reports of 450 mg to >1 g day−1, these in vitro data indicate that usnic acid has significant potential to interact with other medications. Individual characteristics such as CYP1A induction status, co-administration of CYP1A2 inhibitors, UGT1A1 polymorphisms, and related hyperbilirubinaemias, or co-administration of low therapeutic index CYP2C substrates could work alone or in consort with other idiosyncrasy risk factors to increase the risk of adverse events and/or hepatotoxicity. Thus, usnic acid in nutritional supplements might be involved as both victim and/or perpetrator in clinically significant drug–drug interactions.

Off-Target Platelet Activation in Macaques Unique to a Therapeutic Monoclonal Antibody

AMG X, a human neutralizing monoclonal antibody (mAb) against a soluble human protein, caused thrombocytopenia, platelet activation, reduced mean arterial pressure, and transient loss of consciousness in cynomolgus monkeys after first intravenous administration. In vitro, AMG X induced activation in platelets from macaque species but not from humans or baboons. Other similar mAbs against the same pharmacological target failed to induce these in vivo and in vitro effects. In addition, the target protein was known to not be expressed on platelets, suggesting that platelet activation occurred through an off-target mechanism. AMG X bound directly to cynomolgus platelets and required both the Fab and Fc portion of the mAb for platelet activation. Binding to platelets was inhibited by preincubation of AMG X with its pharmacological target or with anti-human Fc antibodies or by preincubation of platelets with AMG X F(ab′)2 or human immunoglobulin (IVIG). AMG X F(ab′)2 did not activate platelets. Thus, platelet activation required both recognition/binding of a platelet ligand with the Fab domain and interaction of platelet Fc receptors (i.e., FcγRIIa) with the Fc domain. These findings reflect the complexity of the mechanism of action of mAbs and the increasing awareness of potential for unintended effects in preclinical species.

Intact mass analysis of monoclonal antibodies by capillary electrophoresis-Mass spectrometry.

Characterization of monoclonal antibody (mAb) therapeutics by intact mass analysis provides important information on sequence integrity and post-translational modifications. In order to obtain domain specific information, monoclonal antibodies are reduced to heavy and light chain components or enzymatically digested into smaller portions or peptides. Liquid chromatography (LC) is widely used for separation of the antibody fragments in line with mass spectrometry (MS) for characterization. Capillary electrophoresis (CE) is an analytical technique with high separation efficiency, high sensitivity, and minimal inter-run sample carryover. Combining the resolving power of CE with electrospray ionization (ESI) MS has great potential in regards to accurate mass characterization of protein therapeutics and has been a long sought-after approach. However, the intrinsic technical difficulty in coupling CE to MS has hindered the broad application of CE-MS across the biopharmaceutical industry. Recently, a CE-MS interface has been developed [1] and commercialized. Herein, we report implementation of this technology for coupling CE to an Agilent time-of-flight (TOF) mass spectrometer. CE-MS provides an attractive complement to LC-MS for separation and intact mass determination of mAbs and antibody-based therapeutics.