Emre M. Isin

In vitro approach to assess the potential for risk of idiosyncratic adverse reactions caused by candidate drugs.

Idiosyncratic adverse drug reactions (IADRs) in humans can result in a broad range of clinically significant toxicities leading to attrition during drug development as well as postlicensing withdrawal or labeling. IADRs arise from both drug and patient related mechanisms and risk factors. Drug related risk factors, resulting from parent compound or metabolites, may involve multiple contributory mechanisms including organelle toxicity, effects related to compound disposition, and/or immune activation. In the current study, we evaluate an in vitro approach, which explored both cellular effects and covalent binding (CVB) to assess IADR risks for drug candidates using 36 drugs which caused different patterns and severities of IADRs in humans. The cellular effects were tested in an in vitro Panel of five assays which quantified (1) toxicity to THLE cells (SV40 T-antigen-immortalized human liver epithelial cells), which do not express P450s, (2) toxicity to a THLE cell line which selectively expresses P450 3A4, (3) cytotoxicity in HepG2 cells in glucose and galactose media, which is indicative of mitochondrial injury, (4) inhibition of the human bile salt export pump, BSEP, and (5) inhibition of the rat multidrug resistance associated protein 2, Mrp2. In addition, the CVB Burden was estimated by determining the CVB of radiolabeled compound to human hepatocytes and factoring in both the maximum prescribed daily dose and the fraction of metabolism leading to CVB. Combining the aggregated results from the in vitro Panel assays with the CVB Burden data discriminated, with high specificity (78%) and sensitivity (100%), between 27 drugs, which had severe or marked IADR concern, and 9 drugs, which had low IADR concern, we propose that this integrated approach has the potential to enable selection of drug candidates with reduced propensity to cause IADRs in humans.

Kinetics and Thermodynamics of Ligand Binding by Cytochrome P450 3A4

Cytochrome P450 (P450) 3A4, the major catalyst involved in human drug oxidation, displays substrate- and reaction-dependent homotropic and heterotropic cooperative behavior. Although several models have been proposed, these mainly rely on steady-state kinetics and do not provide information on the contribution of the individual steps of P450 catalytic cycle to the observed cooperativity. In this work, we focused on the kinetics of substrate binding, and the fluorescent properties of bromocriptine and α-naphthoflavone allowed analysis of an initial ligand-P450 3A4 interaction that does not cause a perturbation of the heme spectrum. The binding stoichiometry for bromocriptine was determined to be unity using isothermal titration calorimetry and equilibrium dialysis methods, suggesting that the ligand bound to the peripheral site during the initial encounter dissociates subsequently. A three-step substrate binding model is proposed, based on absorbance and fluorescence stopped-flow kinetic data and equilibrium binding data obtained with bromocriptine, and evaluated using kinetic modeling. The results are consistent with the substrate molecule binding at a site peripheral to the active site and subsequently moving toward the active site to bind to the heme and resulting in a low to high spin iron shift. The last step is attributed to a conformational change in the enzyme active site. The later steps of binding were shown to have rate constants comparable with the subsequent steps of the catalytic cycle. The P450 3A4 binding process is more complex than a two-state system, and the overlap of rates of some of the events with subsequent steps is proposed to underlie the observed cooperativity.

Use of radiolabeled compounds in drug metabolism and pharmacokinetic studies.

As part of the drug discovery and development process, it is important to understand the fate of the drug candidate in humans and the relevance of the animal species used for preclinical toxicity and pharmacodynamic studies. Therefore, various in vitro and in vivo studies are conducted during the different stages of the drug development process to elucidate the absorption, distribution, metabolism, and excretion properties of the drug candidate. Although state-of-the-art LC/MS techniques are commonly employed for these studies, radiolabeled molecules are still frequently required for the quantification of metabolites and to assess the retention and excretion of all drug related material without relying on structural information and MS ionization properties. In this perspective, we describe the activities of Isotope Chemistry at AstraZeneca and give a brief overview of different commonly used approaches for the preparation of (14)C- and (3)H-labeled drug candidates. Also various drug metabolism and pharmacokinetic studies utilizing radiolabeled drug candidates are presented with in-house examples where relevant. Finally, we outline strategic changes to our use of radiolabeled compounds in drug metabolism and pharmacokinetic studies, with an emphasis on delaying of in vivo studies employing radiolabeled drug molecules.

Risk assessment and mitigation strategies for reactive metabolites in drug discovery and development.

Drug toxicity is a leading cause of attrition of candidate drugs during drug development as well as of withdrawal of drugs post-licensing due to adverse drug reactions in man. These adverse drug reactions cause a broad range of clinically severe conditions including both highly reproducible and dose dependent toxicities as well as relatively infrequent and idiosyncratic adverse events. The underlying risk factors can be split into two groups: (1) drug-related and (2) patient-related. The drug-related risk factors include metabolic factors that determine the propensity of a molecule to form toxic reactive metabolites (RMs), and the RM and non-RM mediated mechanisms which cause cell and tissue injury. Patient related risk factors may vary markedly between individuals, and encompass genetic and non-genetic processes, e.g. environmental, that influence the disposition of drugs and their metabolites, the nature of the adverse responses elicited and the resulting biological consequences. We describe a new strategy, which builds upon the strategies used currently within numerous pharmaceutical companies to avoid and minimize RM formation during drug discovery, and that is intended to reduce the likelihood that candidate drugs will cause toxicity in the human population. The new strategy addresses drug-related safety hazards, but not patient-related risk factors. A common target organ of toxicity is the liver and to decrease the likelihood that candidate drugs will cause liver toxicity (both non-idiosyncratic and idiosyncratic), we propose use of an in vitro Hepatic Liability Panel alongside in vitro methods for the detection of RMs. This will enable design and selection of compounds in discovery that have reduced propensity to cause liver toxicity. In vitro Hepatic Liability is assessed using toxicity assays that quantify: CYP 450 dependent and CYP 450 independent cell toxicity; mitochondrial impairment; and inhibition of the Bile Salt Export Pump. Prior to progression into development, a Hepatotoxicity Hazard Matrix combines data from the Hepatic Liability Panel with the Estimated RM Body Burden. The latter is defined as the level of covalent binding of radiolabelled drug to human hepatocyte proteins in vitro adjusted for the predicted human dose. We exemplify the potential value of this approach by consideration of the thiazolidinedione class of drugs.

Multiple Sequential Steps Involved in the Binding of Inhibitors to Cytochrome P450 3A4

Cytochrome P450 (P450) 3A4 is an extensively studied human enzyme involved in the metabolism of >50% of drugs. The mechanism of the observed homotropic and heterotropic cooperativity in P450 3A4-catalyzed oxidations is not well understood, and together with the cooperative behavior, a detailed understanding of interaction of drug inhibitors with P450 3A4 is important in predicting clinical drug-drug interactions. The interactions of P450 3A4 with several structurally diverse inhibitors were investigated using both kinetic and thermodynamic approaches to resolve the steps involved in binding of these ligands. The results of pre-steady-state absorbance and fluorescence experiments demonstrate that inhibitor binding is clearly a multistep process, even more complex than the binding of substrates. Based on spectrophotometric equilibrium binding titrations as well as isothermal titration calorimetry experiments, the stoichiometry of binding appears to be 1:1 in the concentration ranges studied. Using a sequential-mixing stopped-flow approach, we were also able to show that the observed multiphasic binding kinetics is the result of sequential events as opposed to the existence of multiple enzyme populations in dynamic equilibrium that interact with ligands at different rates. We propose a three-step minimal model for inhibitor binding, developed with kinetic simulations, consistent with our previously reported model for the binding of substrates, although it is possible that even more steps are involved.

Cooperativity in oxidation reactions catalyzed by cytochrome P450 1A2: highly cooperative pyrene hydroxylation and multiphasic kinetics of ligand binding.

Rabbit liver cytochrome P450 (P450) 1A2 was found to catalyze the 5,6-epoxidation of α-naphthoflavone (αNF), 1-hydroxylation of pyrene, and the subsequent 6-, 8-, and other hydroxylations of 1-hydroxy (OH) pyrene. Plots of steady-state rates of product formation versus substrate concentration were hyperbolic for αNF epoxidation but highly cooperative (Hill n coefficients of 2-4) for pyrene and 1-OH pyrene hydroxylation. When any of the three substrates (αNF, pyrene, 1-OH pyrene) were mixed with ferric P450 1A2 using stopped-flow methods, the changes in the heme Soret spectra were relatively slow and multiphasic. Changes in the fluorescence of all of the substrates were much faster, consistent with rapid initial binding to P450 1A2 in a manner that does not change the heme spectrum. For binding of pyrene to ferrous P450 1A2, the course of the spectra revealed sequential changes in opposite directions, consistent with P450 1A2 being involved in a series of transitions to explain the kinetic multiphasicity as opposed to multiple, slowly interconverting populations of enzyme undergoing the same event at different rates. Models of rabbit P450 1A2 based on a published crystal structure of a human P450 1A2-αNF complex show active site space for only one αNF or for two pyrenes. The spectral changes observed for binding and hydroxylation of pyrene and 1-OH pyrene could be fit to a kinetic model in which hydroxylation occurs only when two substrates are bound. Elements of this mechanism may be relevant to other cases of P450 cooperativity.

Reactive Metabolites: Current and Emerging Risk and Hazard Assessments.

Although idiosyncratic adverse drug reactions are rare, they are still a major concern to patient safety. Reactive metabolites are widely accepted as playing a pivotal role in the pathogenesis of idiosyncratic adverse drug reactions. While there are today well established strategies for the risk assessment of stable metabolites within the pharmaceutical industry, there is still no consensus on reactive metabolite risk assessment strategies. This is due to the complexity of the mechanisms of these toxicities as well as the difficulty in identifying and quantifying short-lived reactive intermediates such as reactive metabolites. In this review, reactive metabolite risk and hazard assessment approaches are discussed, and their pros and cons highlighted. We also discuss the nature of idiosyncratic adverse drug reactions, using acetaminophen and nefazodone to exemplify the complexity of the underlying mechanisms of reactive metabolite mediated hepatotoxicity. One of the key gaps moving forward is our understanding of and ability to predict the contribution of immune activation in idiosyncratic adverse drug reactions. Sections are included on the clinical phenotypes of immune mediated idiosyncratic adverse drug reactions and on the present understanding of immune activation by reactive metabolites. The advances being made in microphysiological systems have a great potential to transform our ability to risk assess reactive metabolites, and an overview of the key components of these systems is presented. Finally, the potential impact of systems pharmacology approaches in reactive metabolite risk assessments is highlighted.

Design of Small Molecule Inhibitors of Acetyl-Coa Carboxylase 1 and 2 Showing Reduction of Hepatic Malonyl-Coa Levels in Vivo in Obese Zucker Rats.

Inhibition of acetyl-CoA carboxylases has the potential for modulating long chain fatty acid biosynthesis and mitochondrial fatty acid oxidation. Hybridization of weak inhibitors of ACC2 provided a novel, moderately potent but lipophilic series. Optimization led to compounds 33 and 37, which exhibit potent inhibition of human ACC2, 10-fold selectivity over inhibition of human ACC1, good physical and in vitro ADME properties and good bioavailability. X-ray crystallography has shown this series binding in the CT-domain of ACC2 and revealed two key hydrogen bonding interactions. Both 33 and 37 lower levels of hepatic malonyl-CoA in vivo in obese Zucker rats.

Cooperativity of cytochrome P450 1A2: Interactions of 1,4-phenylene diisocyanide and 1-isopropoxy-4-nitrobenzene

Homotropic cooperativity of 1-alkoxy-4-nitrobenzene substrates and also their heterotropic cooperative binding interactions with the iron ligand 1,4-phenylene diisocyanide (Ph(NC)2) had been demonstrated previously with rabbit cytochrome P450 (P450) 1A2 [G.P. Miller, F.P. Guengerich, Biochemistry 40 (2001) 7262-7272]. Multiphasic kinetics were observed for the binding of Ph(NC)2 to both ferric and ferrous P450 1A2, including relatively slow steps. Ph(NC)2 induced an apparently rapid change in the circular dichroism spectrum, consistent with a structural change, but had no effect on tryptophan fluorescence. Ph(NC)2 binds the P450 iron in both the ferric and ferrous forms; ferric P450 1A2 was reduced rapidly in the absence of added ligands, and the rate was attenuated when Ph(NC)2 was bound. No oxidation products of Ph(NC)2 were detected. Docking studies with a rabbit P450 1A2 homology model based on the published structure of a human P450 1A2.alpha-naphthoflavone (alphaNF) complex indicated adequate room for a complex with either two 1-isopropoxy-4-nitrobenzene molecules or a combination of one 1-isopropoxy-4-nitrobenzene and one Ph(NC)2; in the case of alphaNF no space for an extra ligand was available. The patterns of homotropic cooperativity seen with 1-alkoxy-4-nitrobenzenes (biphasic plots of v vs. S) differ from those seen with polycyclic hydrocarbons (positive cooperativity), suggesting that only with the latter does the ligand interaction produce improved catalysis. Consistent with this view, Ph(NC)2 inhibited the oxidation of 1-isopropoxy-4-nitrobenzene and other substrates.

Bioactivation Pathways of the Cannabinoid Receptor 1 Antagonist Rimonabant

In the present work, the characterization of the biotransformation and bioactivation pathways of the cannabinoid receptor 1 antagonist rimonabant (Acomplia) is described. Rimonabant was approved in Europe in 2006 for the treatment of obesity but was withdrawn in 2008 because of a significant drug-related risk of serious psychiatric disorders. The aim of the present work is to characterize the biotransformation and potential bioactivation pathways of rimonabant in vitro in human and rat liver microsomes. The observation of a major iminium ion metabolite led us to perform reactive metabolite trapping, covalent binding to proteins, and time-dependent inhibition of cytochrome P450 3A4 studies. The major biotransformation pathways were oxidative dehydrogenation of the piperidinyl ring to an iminium ion, hydroxylation of the 3 position of the piperidinyl ring, and cleavage of the amide linkage. In coincubations with potassium cyanide, three cyanide adducts were detected. A high level of covalent binding of rimonabant in human liver microsomes was observed (920 pmol equivalents/mg protein). In coincubations with potassium cyanide and methoxylamine, the covalent binding was reduced by approximately 40 and 30%, respectively, whereas GSH had no significant effect on covalent binding levels. Rimonabant was also found to inhibit cytochrome P450 3A4 irreversibly in a time-dependent manner. In view of these findings, it is noteworthy that, to date, no toxicity findings related to the formation of reactive metabolites from rimonabant have been reported.