Send Orders for Reprints to [email protected] Current Medicinal Chemistry, 2015, 22, 465-489 465 Gauging Reactive Metabolites in Drug-Induced Toxicity

Marsha R. Eno and Michael D. Cameron*

Scripps Research Institute – Scripps Florida, Department of Molecular Therapeutics, 130 Scripps Way, Jupi- ter, FL 33458, USA

Abstract: Over the past decades, it has become abundantly clear that enzymes evolved to detoxify and elimi- nate foreign chemicals from the body, occasionally generate highly reactive metabolites which have toxico- logical implications. To decrease the probability of late clinical failure or market withdrawal, there has been an increased prioritization on understanding key metabolic processes that might cause drug interactions or toxicities. Significant advances have been made in the detection of reactive metabolites and in understanding the structure activity relationship. It is now widely accepted that compounds with certain functional groups such as ani- lines, quinones, hydrazines, thiophenes, furans, acylpropionic acids, and alkynes have a much greater associated risk to- wards formation of reactive metabolites than compounds that do not contain such “structural alerts”. Detection of reactive metabolites is usually done with in vitro assays, which have become more sensitive with advances in mass spectrometry. As an increasingly large number of compounds that form reactive metabolites have been identified, much of the focus has shifted from detection to evaluation of toxicological implication. While there is a disproportionate number of compounds metabolized to reactive metabolites that are associated with drug-induced hepatotoxicity and serious skin toxicities such as toxic endothelial necrolysis and Steven’s Johnson syndrome, attempts to predict toxicity based on in vitro testing have been discouraging. In this review we attempt to summarize the experimental options available to evaluate reactive me- tabolites. Keywords: Bioactivation, cytochrome P450, hepatotoxicity, reactive metabolites.

INTRODUCTION manuscripts that discuss relevant factors or retrospectively evaluate data for use in making risk-based assessments [1, 8- Experiments designed to detect the formation of reactive 10]. metabolites have historically been done retrospectively to understand the toxicological mechanisms of compounds What are Reactive Metabolites? shown to have associated human toxicities. More recently, reactive metabolite screening has worked its way into the The term reactive metabolite is not universally defined, metabolic and safety profiling of compounds in the drug but generally indicates a short lived metabolite capable of discovery process [1-5]. This brings us to the central focus of chemically modifying biomolecules formed through the me- this review. Upon being informed that a compound or a se- tabolism of a “non-reactive” parent compound. Cases where ries of compounds is metabolized to a reactive metabolite, the parent compound is inherently reactive or is converted to what additional in vitro or in vivo experiments might be per- a reactive form by facile chemical means such as encounter- formed to predict the toxicological risk while making deci- ing the low pH environment of the stomach are not usually sions to advance or deprioritize compounds? termed reactive metabolites even though they might be asso- ciated with similar toxicities. Hepatic toxicity is the leading cause for market with- drawal of approved drugs [6]. Additionally, the typical rate Why are Reactive Metabolites Generated and from an of acute hepatic failure (hepatic toxicity leading to death or Evolutionary Standpoint Why are they Tolerated? liver transplant) for withdrawn drugs was one case per ten- Because of the excessive number of exogenous chemicals thousand patients [7]. Most compounds that fail in clinical that we come into contact with, it is not possible to have spe- development, obtain black box warnings, or are withdrawn cialized enzymes to safely eliminate each foreign compound. due to hepatic toxicity may have had no cases of hepatic One option to deal with the array of threats would be with an failure during the clinical development and the majority of arrangement similar to the immune system where multiple clinical trial patients would have shown no markers indicat- ing even mild hepatic toxicity. While the decision making layers of defense combine to make the innate and adaptive immune system. This provides protection against common trees for individual companies are living documents with pathogens and permits tailored responses to new threats continual updates, several companies have published through the incorporation of a hyper variable domain that

allows the binding epitope of antibodies to rapidly mutate in *Address correspondence to this author at the Scripps Research Institute – response to newly encountered pathogens. Scripps Florida, Department of Molecular Therapeutics, 130 Scripps Way, Jupiter, FL 33458, USA; Tel: 561-228-2223; Fax: 561-228-3081; Whereas biological pathogens are built upon twenty E-mail: [email protected] standard amino acids with similar stereochemistry, only

1875-533X/15 $58.00+.00 © 2015 Bentham Science Publishers 466 Current Medicinal Chemistry, 2015, Vol. 22, No. 4 Eno and Cameron slightly expanded by non-standard and modified amino ac- a test set of compounds comprised exclusively of molecules ids, the plethora of xenobiotics encountered are structurally that were deemed “drug-like” and had sufficiently clean pre- diverse and make an adaptive response implausible. A frac- clinical toxicity profiles to progress into human trials. Com- tion of compounds can be excreted unchanged via filtration pounds that can be clearly identified as toxins from in vitro in the kidney or export into bile whereas others, particularly or animal studies, e.g. aflatoxin b1 which is bioactivated by lipophilic compounds would otherwise accumulate in bio- P450 to form a reactive epoxide leading to necrosis, cirrho- logical membranes and must be modified prior to elimina- sis, and carcinoma of the liver with lethal doses in most spe- tion. While there are several families of enzymes responsible cies below 1 mg/kg would not be included in the analysis for “chemical decontamination”, we will focus on the cyto- [26, 27]. Compounds identified as toxic in preclinical species chrome P450 enzymes as they are the most prominent of would either be excluded from progressing to subsequent these systems. human clinical trials, or the dose would be reduced to levels where significant toxicity was not expected. This leads to the The human genome encodes for fifty-seven P450 pro- possibility that drugs known to form reactive metabolites but teins. P450s are most prevalent in the liver where their heme not showing hepatic toxicity may comprise a sub-group of group is responsible for the distinctive color of liver. A ma- compounds with reduced toxicity profiles. jority of the P450 enzymes are minimally expressed or cata- lyze reactions involved in such cellular processes as steroid What Protection Does the Liver have from Reactive Me- and prostaglandin biosynthesis; however, approximately a tabolites? dozen P450s are exceptionally non-specific and are capable of oxidizing thousands of substrates [11-14]. This high de- The liver resides between the gut and general circulation. gree of non-specificity is absolutely essential to deal with the Foreign substances, including toxins and bacteria, that are wide range of xenobiotics encountered in the natural envi- ingested are typically absorbed in the small intestine where ronment. The substrate promiscuity of P450s is achieved they enter the portal vein and are directed to the liver before through harnessing the oxidative potential of molecular oxy- proceeding to the rest of the body. Because of its location gen to generate a powerful oxidant, the porphyrin iron (IV) and function, the liver is exposed to higher levels of toxins oxo species, which has sufficient potential to oxidize most than most other organs. One of the key reasons that serious organic molecules [15-17]. These same organic molecules hepatic events are rare is that the liver has well-orchestrated could have been degraded directly through the three electron safeguards. reduction of molecular oxygen to form the hydroxyl radical, One of the key regulators in response to electrophiles is an exceptionally strong oxidant; however, this mechanism Nrf2. In its “resting” state, Nrf2 is retained in the cytoplasm cannot be controlled and would lead to excessive cellular by binding to Keap1. When bound to Keap1, Nrf2 is toxicity. P450s “sequester” and control access to the oxida- ubiquinated and rapidly turned over with a cellular half-life tive species, thus decreasing the oxidation of unintended of approximately 15 minutes [28]. In the presence of ele- biomolecules. Because the P450 oxidative species is so vated levels of electrophiles, the sulfhydryl groups of the strong, substrates can often be oxidized at more than one exposed cysteines on Keap1 are covalently modified result- position and the metabolite profile has as much to do with ing in a decrease in the binding affinity for Nrf2 [29, 30]. the presentation of the molecule to the activated oxygen as it Nrf2 translocates to the nucleus and binds to the antioxidant does the “easiest” site of oxidation based on redox potential response element and promotes the transcription of numer- [18-21]. This often leads to the catalysis of multiple metabo- ous cytoprotective genes [31, 32]. These include but are not lites from a single P450 enzyme or sequential metabolism limited to: NAD(P)H dehydrogenase which can reduce qui- giving rise to secondary and tertiary metabolites. nones, quinone-imines, and semi-quinones; aldo-keto reduc- Depending on the nature of the substrate, oxidations cata- tase, which reduces reactive ketones and aldehydes; sul- lyzed by P450 can generate electron deficient metabolites fotransferases, glutathione S-transferases and glutathione which in turn oxidize cellular components. There are certain reductase, proteins to increase glutathione synthesis, heme chemical moieties that are more prone to the generation of oxygenase, ferritin and metallothioneins to reduce heavy reactive metabolites and there have been several excellent metal toxicity and redox cycling, and efflux transporters to reviews that have discussed “structural alerts” and correlated increase the flow of xenobiotics into the bile [33-35]. these to the formation of reactive metabolites and/or hepato- Endogenous levels of proteins that function to protect toxicity [22-25]. against electrophiles are elevated in the liver over their con- centration in other tissues. The most abundant proteins in the Are Reactive Metabolites Always Toxic? liver, the organ with the highest concentration of P450s, are glutathione-S-transferase enzymes, which have a detoxifica- Clinically severe toxicity is observed in only a portion of tion role by “quenching” electrophiles generated by P450s compounds that have been shown to generate reactive me- through coupling them with glutathione [36-39]. tabolites in vitro. This is one of the key shortcomings in the ability to understand the importance of reactive metabolites. The liver has the largest population of tissue resident It should be pointed out that these studies are built upon bi- immune cells with approximately 25% of the non- ased data sets as they do not comprise a random sampling of hepatocytes in the liver being macrophages or lymphocytes molecules positive or negative for reactive metabolite forma- [40]. In addition to its resident macrophages, liver has the tion. Literature references where the extent of reactive me- ability to rapidly recruit additional neutrophils, monocytes, tabolite formation was indexed to toxicity were done primar- and lymphocytes in response to tissue injury. There is sig- ily with drugs or drug candidates. This biases the analysis to nificant literature dealing with the role of immune cells in Reactive Metabolites – Experimental Evaluation Current Medicinal Chemistry, 2015, Vol. 22, No. 4 467 drug-induced liver injury. Earlier literature reported contra- treatment to minimize cellular damage caused by NAPQI dictory conclusions, but more recent reports have elucidated [79, 80], acetaminophen-induced deaths would be even a dual role of many immune cells. While they can release higher. Many of the overdoses are associated with co- cytokines and pro-inflammatory mediators which can lead to formulations, particularly with prescription pain killers increased tissue damage, they also release factors involved in where acetaminophen is mixed with oxycodone (Percocet) or cell survival and tissue repair [41-43]. For additional reading hydrocodone (Vicodin) [78, 81, 82]. The FDA has recently on the function of immune cells in drug-induced hepatotox- restricted the amount of acetaminophen that can be used in icity see the indicated references [44-48]. The high hepatic these pain management co-formulations. Vicodin, the most macrophage population allows damaged cells to be safely prescribed medication in America with over 130 million pre- cleared, reducing necrotic cell death and the accumulation of scriptions in 2010, has recently been reformulated to de- scar tissue/cirrhosis. crease the acetaminophen dose from 750 mg to 300 mg while retaining hydrocodone dosage. The liver is an immunologically privileged organ and the default response to antigens is immune tolerance [49-52]. While select individuals may have greater or lower sensi- Despite the propensity for hepatic immune tolerance, there tivity to acetaminophen associated toxicities, if dosed high are several compounds where toxicity has clear genetic links enough, 100% of individuals would be expected to experi- implicating an immune reaction. Immune-mediated hepato- ence hepatic damage leading to liver failure. NAPQI is elec- toxicity will be briefly discussed later in this review. tron deficient and capable of reacting with multiple bio- molecules which gives rise to an array of cellular damage. Are Reactive Metabolites Associated with Dose- Papers have been published suggesting that the reactive me- Dependent or Idiosyncratic Toxicity? tabolite formed by acetaminophen overdose causes toxicity via changing the cellular concentration of glutathione [83, One of the challenges of evaluating metabolism-induced 84], changing the redox potential of the cell, covalently toxicity is that it may exhibit features of acute, chronic or modifying key cellular proteins [69, 85-87], causing mito- mixed toxicities. Observed toxicity is a function of the prop- chondrial damage [88-91] and endoplasmic reticulum stress erties of the compound combined with the susceptibility of [92]. All of these are likely correct and combine to amplify the individual [53, 54]. Compounds that are acutely toxic the toxicity that would be observed from any one pathway. typically exhibit a dose-dependent increase in toxicity. Reac- While acetaminophen is certainly acutely toxic, it also shares tive metabolites have been implicated in the toxicity of nu- some similarities to an idiosyncratic toxin. While observed in merous drugs or environmental pollutants and may manifest very few patients, a subset of the population is hypersensi- through multiple mechanisms including endoplasmic reticu- tive to acetaminophen and exhibit significant toxicity even lum [55] or mitochondrial stress [56, 57], chromosomal when taking the drug at recommended doses. This is likely damage [58-62] or change in cellular redox potential [63-65]. due to an immunological response to NAPQI-conjugated Reactive metabolite formation has also been implicated proteins. Acetaminophen specific IgE antibodies and aceta- in idiosyncratic hepatic or dermal drug-induced toxicity. By minophen positive skin tests have been detected in hypersen- definition, an idiosyncratic toxin demonstrates toxicity that is sitive patients [93, 94]. peculiar to the individual. Idiosyncratic toxins display rare Because of the large number of people that safely take and unpredictable toxicity which is not strictly dose- acetaminophen every year, it is frequently brought up in dis- dependent. Information explaining why the majority of pa- cussions about reactive metabolites as the reason why com- tients taking these drugs are subjected to similar levels of reactive metabolite, yet only a small percentage of patients pounds that covalently modify proteins can be safe drugs. develop significant toxicity is a source of active investiga- This argument is tenuous. The FDA has recently expanded tion. The best supported hypothesis involves an immune the warning labels and restricted acetaminophen doses due to response and this is discussed in greater detail later in this the danger of liver toxicity [95-104]. It is highly likely that review. acetaminophen would not gain approval if it was submitted for regulatory approval today. Benchmarking against such a The Acetaminophen Paradox – Safe Drug or Bad Actor compound is exceedingly risky. The most studied compound to form a reactive metabo- What Assays are Available to Evaluate Reactive Metabo- lite is acetaminophen (paracetamol). Acetaminophen gener- lite Formation? ally exhibits dose-dependent toxicity, it is toxic across ani- mal species, rapidly causes high levels of hepatic damage, A range of methods to identify compounds that generate and the toxic metabolite is known [66-70]. Acetaminophen is reactive metabolites and try to predict future risk of serious oxidized to an electrophilic metabolite N-acetyl-p- drug-induced toxicity are discussed in the remainder of this benzoquinone imine (NAPQI) by multiple hepatic P450 [67, review. They are listed in Table 1 and where they might be 71-77]. Acetaminophen toxicity is the leading cause of acute incorporated in the drug discovery and development timeline liver failure in the Western World and factors associated is depicted in Fig. (2). with acute acetaminophen-induced toxicity are well known and universal. While acetaminophen is widely regarded as IN VITRO METHODS being safe, a 2006 study by the FDA – Office of Drug Safety Radiolabel Incorporation/Covalent Adduction estimated that in the United States alone there were 56,000 emergency room visits and 458 deaths from acute liver fail- The easiest assay to understand, and paradoxically the ure caused by acetaminophen [78]. Without N-acetylcysteine most controversial method for detection of reactive 468 Current Medicinal Chemistry, 2015, Vol. 22, No. 4 Eno and Cameron

Table 1. Methods to evaluate reactive metabolites.

Method Pro Con

• simple • requires radiolabeled compound In vitro – microsomal • potential to identify reactive secondary or tertiary metabo- • P450-centric 1 radiolabel incorporation/ lites • lack of detoxification pathways covalent adduction • quantitative • many compounds that bind extensively are safe drugs

In vitro – reactive • established methodology • unknown significance 2 metabolite trapping • amenable to moderate throughput • usually not quantitative

• simple • correlation with toxicity is modest In vitro – P450 time- 3 • data is already collected to predict pharmacokinetic drug- • TDI mechanism may be independent of reactive metabo- dependent inhibition drug interactions lite formation

In vitro – genotoxicity • mechanism of genotoxicity may not involve a reactive 4 • data is already required for IND package (Ames test) metabolite

• data may eventually help identify a fingerprint or profile • methodology needs improved In vitro/in vivo – pro- 5 for compounds that have higher probabilities of clinical • generally not quantitative teomics toxicity • minimal historical data

In vivo – micronucleous • mechanism of genotoxicity may not involve a reactive 6 • part of the preclinical safety package assay metabolite

• requires radiolabeled (or positron-emitting) compound • identifies accumulation in tissues In vivo – whole body • does not distinguish between the parent drug and metabo- • pharmacokinetics, transporters, and cellular detoxification 7 radiolabel incorporation lites pathways are accounted for or PET • requires knowledge of the metabolic pathway to assure • quantitative that the label is not lost

• possible with urine, feces, and/or bile • uncertainty if the detected adduct is from reaction with In vivo – glutathione and 8 • structural information can be obtained via mass spectro- cellular glutathione pools, protein cysteines, or glutathione mercapturate adducts metry experiments to identify the likely reactive metabolite S-transferase

In vivo – markers of • identifies hepatic injury • elevation of transaminases have been observed without 9 hepatocellular injury • biliary vs hepatocellular injury can be identified progression to significant hepatic injury (ALT, ALP, bilirubin) • plasma samples are sufficient

• usually performed retrospectively • association may be complicated if multiple HLA are in- In vivo – HLA associa- 10 • may identify at-risk populations volved tion • unclear if HLA association would be useful for mild he- patic injury

• labor intensive • only approximately half of patients with serious immune- In vivo – lymphocyte mediated hepatic or cutaneous injury are positive in LTT test 11 • only a blood draw is required proliferation assay • uncertain if evaluation of patients with mild ALT elevation vs control patients would predict increased risk of significant toxicity

In vivo – patch/skin • may harm patient 12 • simple to administer hypersensitivity test • compounds that are irritants may be difficult to test

In vivo – eosinophilia/ • observed in a small percentage of patients 13 • demonstrates immune-mediated toxicity DRESS • eosonophils may be elevated by non-related alergens

• mechanism is not certain • non-invasive patient sampling In vivo – re-activation of • unclear if the method would have predictive value for later 14 • high percentage of positive samples among patients that virus toxicity if used with asymptomatic patients during clinical experience severe immune mediated toxicity trials

In vivo – autoantibodies • high throughput ELISA based assays for autoantibody 15 or drug-protein antibod- • utility in prediction of severe toxicity is unknown detection could be developed ies Reactive Metabolites – Experimental Evaluation Current Medicinal Chemistry, 2015, Vol. 22, No. 4 469 metabolite formation, is the radiolabel incorporation assay. studies [1]. This correlation improved when the levels of This was extensively published by researchers from Merck thiol adduct formation and daily dose were incorporated into and they used the methodology for many years [9, 105-108]. the analysis. Radiolabeled drug is incubated with a high concentration of It would be of interest if data from individual patients en- liver microsomes and NADPH for an extended time period. rolled in extended dosing studies were made available where The protein is precipitated, extensively washed, and the hepatic function is tracked via weekly or bi-weekly blood amount of radioactivity covalently incorporated into the mi- chemistry analysis. Correlation of ALT elevation during the crosomal protein fraction is determined [109, 110]. The as- first few days of treatment (acute toxicity) and 3-10 weeks say casts a wide net and is typically able to identify com- after starting the drug (common for immune-mediated toxic- pounds oxidized to reactive metabolites by microsomal en- ity) could then be evaluated in context with binding data and zymes even if the ultimate reactive metabolite is a tertiary or administered dose. quaternary metabolite due to the long incubation time. Modifications to this general study design have been In Vitro Chemical Trapping Experiments used for compounds that are not primarily metabolized in the While there are several compounds that have been util- liver. For instance, the generation of reactive clozapine me- ized as trapping agents for detection of reactive metabolites, tabolites by neutrophils has been demonstrated through the isolation of neutrophils and incubation with radiolabeled some of the most common are glutathione [1, 113-116], N- acetylcysteine [117-120], cyanide [121-124], semicarbazide clozapine [111]. [125], methoxylamine [126, 127], N-acetyllysine [128, 129], Two major objections are raised against this method. The and N-acetyltyrosine [130-132]. Glutathione and N- first centers on experimental set-up due to the lack of detoxi- acetylcysteine are used to trap soft electrophiles such as qui- fication pathways in the assay and the bias towards P450 nones. N-acetyllysine and cyanide are used to trap harder catalyzed reactions through the use of microsomes. The sec- electrophiles. Semicarbizide and methoxylamine [133] are ond frequently raised objection to the radiolabel incorpora- primarily used to trap reactive aldehydes. N-acetyltyrosine tion/covalent adduction assay is that the correlation between can dimerize with some free radicals. In a typical experi- the amount of radiolabel incorporated (pMoles/mg protein) ment, human liver microsomes or human hepatocytes are and observed toxicity in human patients or rodents is poor incubated in the presence and absence of trapping agent. [112]. While a few detection methods have been utilized to detect While it is doubtful many people would argue that cova- trapped metabolites, mass spectrometry is the most common. lent modification of proteins by reactive metabolites is bene- Glutathione covalently binds to electrophiles and can be ficial, there are people that argue that the lack of correlation detected using multiple scan types including positive ion between covalent binding and observed toxicity justify not mode with a neutral loss scan following loss of 129 Da, cor- using reactive metabolite formation within the criteria for responding to the collision-induced fragmentation/loss of candidate selection. An internal review by Merck in 2009 glutamate. An alternative method using negative ion mode found that a quarter of lead optimization projects had issues looks for a fragment ion of 272 Da corresponding to break- with covalent binding of reactive metabolites and that it took ing the thioether bond [132, 134-136]. The negative ion ap- on average 6 months to redesign the molecules to dial out proach is generally more sensitive and gives fewer false reactive metabolite formation while retaining potency and in positives, but does not provide information regarding the vivo efficacy [112]. As part of their evaluation of the benefit position of glutathione binding to the reactive metabolite in drug safety verses the huge investment in redesigning because the negative charge resides on glutathione. Modern molecules, Merck took 100 internal drug candidates with mass spectrometers can rapidly switch between negative and known covalent binding and tested hepatic toxicity in rat. No positive ion mode and it is now possible to run the experi- correlation was observed between binding levels in liver ment in both modes using a single injection [135]. microsomes and toxicity in rat. This study was limited in scope, with the experimental methodology restricted to Glutathione and N-acetylcysteine are not suitable for evaluation of acute toxicity (primarily necrosis). Chronic and trapping reactive aldehydes. N-acetylysine is sometimes immune-mediated toxicity were not examined. Additionally, used, but is not ideal because the generated imine is reversi- because of the long standing practice by Merck to minimize ble. Semicarbazide and methoxylamine are soluble, mini- covalent binding, few compounds that extensively bound mally inhibit P450 enzymes, and react with aldehydes to proteins were included in the evaluation. form stable products. Reaction with semicarbazide and methoxylamine typically increase the mass by 57 and 29 Da, A perspective piece written by scientists from GlaxoS- respectively [125-127]. Other hydrazine containing mole- mithKline examined covalent binding data along with cules such as phenylhydrazine or 2, 4-dinitrophenyl- chemical trapping and P450 inactivation data for over 200 hydrazine can also be used depending on the specialized marketed drugs [8]. Their data demonstrated trends between needs of the researcher. observed hepatotoxicity in human patients and in vitro measurements of reactive metabolite formation when the Cyanide adducts can often be detected as a neutral loss of assay results were combined with information about daily 27 Da corresponding to the collision-induced loss of the cya- dose. Similar results were reported from a team at Bristol- nide group. The use of a 1:1 ratio of stable labeled cyanide Myers Squibb where 50 approved drugs were evaluated. (12C14N:13C15N) gives a characteristic doublet that can help Observed hepatotoxicity marginally correlated with detection differentiate true cyanide adducts from other components of reactive metabolites, assessed by glutathione trapping within the reaction that can generate a 27 Da neutral loss 470 Current Medicinal Chemistry, 2015, Vol. 22, No. 4 Eno and Cameron

[123, 132, 137]. Because of the high sensitivity of modern strate access channel and thus restricts access of other sub- mass spectrometers, care needs to be taken to avoid false strates. Several compounds have been identified that cova- positives. In our experience we are generally distrustful of lently modify the reactive site of P450s. Some of these are results from cyanide trapping experiments without the inclu- complemented by mutational studies where the adducted sion of significant controls. We have seen many compounds amino acid was mutated and the enzyme was shown to no where non-enzyme catalyzed cyanide adducts can clearly be longer be inactivated [147-152]. Covalent modification is identified by LC-MS/MS. Control samples +/- NADPH are usually associated with, but does not necessitate enzyme not sufficient and parallel samples +/- NADPH, samples inactivation. Work from the labs of Hollenberg and Halpert with cyanide added after stopping the reaction with organic demonstrated covalent modification of CYP2B4 which re- solvent, heat inactivated enzyme controls, and compound + sulted in reduction, but not elimination of enzymatic function cyanide in buffer or media should be included and carried [153, 154]. The observed time-dependent decrease in enzy- through all sample processing steps to assure the detected matic activity was caused by a metabolite of t- cyanide adduct is enzymatically generated. butylphenylacetylene covalently binding to threonine 302 of helix I in the active site. As the percentage of covalently Inactivation of P450 modified threonine 302 increased, the rate of enzymatic ac- tivity decreased but did not go to zero. This is an interesting Because many reactive metabolites are generated within observation which may extend to other P450s and time- the active site of a cytochrome P450, it follows that cyto- dependent inhibitors. chrome P450s are a likely target. Tienilic acid was removed from the market three years after gaining regulatory ap- Whereas covalent modification of P450 has a high prob- proval. Tienilic acid is a mechanism-based inhibitor of ability of causing pharmacokinetic drug-drug interactions, it CYP2C9 and in some patients that experienced severe he- is unclear how significant this is in terms of hepatic toxicity. patic toxicity anti-CYP2C9 antibodies were detected [138]. There appear to be at least a subset of compounds where A GlaxoSmithKline perspective piece presented an analysis reactive metabolites can induce an immune response to of P450 time-dependent inhibition for 200 marketed drugs P450, e.g. tienilic acid where anti-CYP2C9 antibodies were and showed a moderate correlation with hepatic toxicity [8]. detected in patient serum [138] or with halothane [155] or Compounds that demonstrate time-dependent P450 inhibi- disulfiram [156] where anti-CYP2E1 and anti-CYP1A2 anti- tion should be considered as potentially generating a reactive bodies have been reported. Reactive metabolites that inacti- metabolite capable of oxidizing or covalently modifying vate P450s can often exit the active site where they can react cellular biomolecules until experiments are conducted to with non-P450 proteins leading to the potential for toxicity. determine otherwise. Heme Modification For most of the known P450 inactivators, there is not a 1:1 ratio of inactivated enzyme per metabolic event [139- Alkylation or cleaving the porphyrin ring of the heme can 146]. Instead, individual P450 proteins may be able to oxi- result in P450 inactivation. Several compounds including dize the inhibitor tens or hundreds of times before becoming mibefradil and methadone have been shown to directly mod- inactivated. This phenomena results in the inhibition having ify the P450 heme [157-159]. The pyrrole nitrogens, vinyl, a time-dependence where the observed inhibition increases and propionate groups of the heme have been reported to be with extended incubation time. While P450s catalyze reac- oxidized [160-164]. Significant work in this area has been tions at varying rates depending on the individual enzyme done by the Correia lab where they have shown that P450 and substrate, all are relatively slow with typical rates rang- heme destruction can sometimes be reversed in cells by re- ing between 0.1 to 50 substrates oxidized per second. In a constitution with fresh heme. In cases where the heme be- theoretical reaction where a P450 metabolizes an inhibitory comes covalently cross linked to the protein or when there is compound once per second with one in every 200 metabolic insufficient heme to reconstitute the P450 the protein is rap- cycles leading to enzyme inactivation, the enzymatic half- idly degraded via ubiquitin-dependent proteasomal degrada- life would be 2.3 minutes. Because of the time-dependence, tion [165-169]. Using in vitro assays, test compounds can be incubated with recombinantly expressed enzyme and de- remaining enzymatic activity can be evaluated after various struction of the heme can be monitored using the heme ab- incubation times, e.g. 0, 1, 3, 5, 10, and 15 minutes, and the sorption spectrum in a process sometimes referred to as rate of enzyme inactivation can be calculated. Compounds “heme bleaching”, or by quantitating the heme by LC-UV or where the inactivation efficiency approaches 1:1 are more LC-MS [152, 157, 162, 170, 171]. It is best to use prepara- difficult to detect by measuring time-dependent inhibition tions that have minimal cytochrome b5 to avoid confusion because enzyme inactivation is completed so quickly. with the b5 heme which will not be lost during P450 inactiva- Time-dependent P450 inhibition is typically tested using tion. Hepatic toxicity specifically due to heme destruction is in vitro experiments with liver microsomes or recombinant not expected, but molecules that cause heme destruction may P450. Compounds that show time-dependent inhibition fall also react with amino acids within the P450 active site, or es- into at least four categories, two of which are related to reac- cape the active site and react with other cellular biomolecules. tive metabolites, but all have clinical significance in terms of drug-drug interactions. These are discussed below: MI Complex Enzyme Modification Metabolite-Intermediate (MI) complexes exhibit time- dependent decreases in enzyme activity, but do not irreversi- The generated reactive metabolite can oxidize key amino bly modify the P450. An MI complex can be considered a acids or covalently bind within the P450 active site or sub- metabolite “trapped” within the active site and often involve Reactive Metabolites – Experimental Evaluation Current Medicinal Chemistry, 2015, Vol. 22, No. 4 471 reactive nitroso or carbine intermediates [172-178]. The proteins for better analysis and there are promising new af- compound is coordinated to the heme iron and experiences a finity chemistries that can be utilized. Traditional in gel di- stable geometry which greatly decreases the rate of release. gestion methods have successfully identified several protein The coordination of the substrate to the heme iron results in targets of electrophilic drugs. For instance, with the advent characteristic spectral changes causing an increase in absor- of proteomics in late 1990’s, 23 liver proteins modified by bance at approximately 455 nm [179]. For many compounds acetaminophen were determined using Two-dimensional the MI complex can be reversed through the addition of po- (2D) gel-based proteomic approaches [200]. By using tassium ferricyanide [178, 180, 181]. While the enzyme is autoradiography the location of adducted proteins were re- not truly modified and activity is restored upon removal of vealed and the adducted protein spots were excised from the the trapped intermediate, this does not imply MI complexes gel and digested with trypsin. Peptides were then analyzed are benign. Numerous compounds that form MI complexes by MALDI-MS to identify proteins. The 2-D gel methods with CYP3A4 have been shown to cause clinically signifi- based on autoradiography is dependent on the specific activ- cant drug-drug interactions, e.g. erythromycin [182-187], ity of the radiolabeled drug and is biased towards proteins nicardipine [188-190], and troleandomycin [177, 191, 192]. that are abundantly expressed [199]. A variation that does Erythromycin is a weak inhibitor of CYP3A4, but during the not use radioactivity is the use of antibodies directed against N-demethylation of erythromycin, a nitroso intermediate is adducted protein (2-D Western Blotting). Labeled secondary formed which ligates the heme iron and forms a stable com- antibodies can then be used to identify bands that contain plex which prevents further catalysis by the enzyme. adducted protein. Whereas formation of an MI-complex can cause significant As an alternative to using electrophoresis to separate pro- drug-drug interactions, it is unlikely that this would directly teins, complex samples can be enriched through the incorpo- cause increased toxicity. ration of affinity tags such as biotin into the molecule. The Inhibitory Metabolite molecule proposed to form a reactive metabolite is synthe- sized with a biotin tag and when the mixture of adducted and Time dependent inhibition can be observed in an in vitro unadducted proteins are passed through a streptavidin or setting due to the generation of a metabolite which is a avidin column, the biotin labeled adducted proteins are cap- stronger inhibitor than the parent compound. For example, tured [201-203]. Elution of these proteins followed by diges- both fluoxetine and its demethylated metabolite norfluoxet- tion enriches the sample for adducted peptides. Alternatively ine potently inhibit CYP2D6 with similar observed IC50 val- the proteins can be digested prior to loading them onto the ues. However, norfluoxetine inhibits CYP3A4 approximately affinity column [199]. The incorporation of fluorescent or 4- to 10-fold more potently than fluoxetine [193-195]. This biotin tags into the test molecule can prevent metabolism or gives the appearance of fluoxetine having weak CYP3A4 alter the metabolite profile and prevent the formation of the time-dependent inhibition as fluoxetine is converted to nor- reactive metabolite even if the tag is spatially removed from fluoxetine during the incubation. Such a mechanism would the portion of the molecule that is responsible for the forma- not be expected to directly influence hepatotoxicity. tion of the reactive metabolite. If the target protein can be expressed recombinantly, Assessing Covalent Binding of Reactive Drug Metabolites analysis can be simplified. For example, upon incubation of by Proteomics recombinant CYP3A4 with raloxifene, a single raloxifene Electrophilic drug metabolites are capable of forming was found to bind by examining the mass shift of intact 3A4 stable covalent protein adducts [196, 197]. This remains a using MADLI-TOF whole protein mass spectrometry. De- poorly understood cause of drug toxicity. Even though in spite observing a mass shift indicative of the binding of a vitro trapping experiments help in the identification of reac- single raloxifene, upon enzymatic digestion of the adducted tive metabolites, they do not help in determining the proteins 3A4, followed by tandem MS, two sites of adduction were that are modified. Sometimes short-lived reactive metabo- elucidated for raloxifene, Cys 239 and Tyr 75 [204, 205]. lites can react with nucleophilic residues present in the en- Using similar techniques, 17-R-ethynylestradiol was shown zyme’s active site leading to enzyme inactivation, but they to modify Ser360 in both P450 2B1 and 2B6 [206]. may be too reactive to diffuse out of the enzyme active site, Pähler et al. have described a targeted proteomic ap- thus precluding detection using trapping techniques [198]. proach to enrich and identify peptides modified by reactive The identification of proteins modified by reactive metabo- drug metabolites of 2-amino-pyrimidine obtained from rat lites allows information to be obtained at the structural level liver microsomal proteins [198]. Their procedure involved and can aid in understanding the role between covalent bind- the use of an equimolar mixture of non- and radioisotope- ing and drug-induced toxicity. Similar patterns in protein labeled (14C) substrate. The protein mixture was resolved by modifications among reactive metabolite species may help in 1D SDS-PAGE and protein bands were visualized by predicting the outcome of toxicity with greater specificity. autoradiography, and in-gel digested with trypsin. Peptides were then separated by strong cation exchange (SCX) chro- The analysis of proteins modified by reactive metabolites matography and radioactivity containing fractions analyzed is an analytical challenge due to the complexity of protein by nanoLC-MS/MS. samples. Since protein targets are diverse and adducted pro- teins represent only a small fraction of all the proteins pre- Non-enriched shotgun proteomics can be used to identify sent in the cell, this scenario was rightly described as a “nee- proteins in a complex mixture with minimal specific enrich- dle in a haystack problem” by Liebler et al. [199]. Detection ment. The protein mixture is digested to peptides, followed of target proteins is aided if they can be enriched from other by their analysis by LC-MS-MS to generate tandem MS-MS 472 Current Medicinal Chemistry, 2015, Vol. 22, No. 4 Eno and Cameron spectra [205, 207-210]. By searching the MS-MS spectra in patients. This represents a significant challenge for discern- protein databases. Adducted peptides can be detected ing those compounds with higher probabilities of causing through spectral matching [211-214]. serious toxicity from those that may cause mild hepatic is- sues that do not progress to more serious toxicity. Currently Genotoxicity studies can be designed to evaluate multiple factors that cor- relate with serious adverse events, but none have 100% pre- The role of metabolites in genotoxicity has long been ap- dictive value. Fig. (1) presents a map of physiological steps preciated. Genotoxicity is typically evaluated using at least associated with chemical-induced immune-mediated toxicity. one and usually a battery of in vitro assays as a preliminary Most of the common experimental observations used to evaluation and followed up with in vivo evaluation during evaluate reactive metabolites focus on the first few steps, but the preclinical toxicology package in IND enabling studies. interruptions at multiple stages along the path would be ex- One of the most common in vitro assays is the Ames test pected to mitigate toxicity. Experimental observations are which evaluates the mutagenic potential of compounds in likely to provide parameters to incorporate in risk assess- engineered Salmonella typhimurium bacterial cultures [215- ment, but based upon current understanding, the predictive 218]. Bacteria are modified to be deficient in histidine bio- value is not sufficient to provide a definitive answer on the synthesis and must be cultured in histidine augmented media. likelihood of an individual compound to cause rare idiosyn- Strains can be modified to identify compounds that initiate cratic toxicity prior to exposure of high numbers of patients. point mutations or frame shifts that re-instate the ability to synthesize histidine. In practice, the bacteria are plated at a low density in media containing a pH indicator with suffi- cient histidine to allow for minimal growth. Metabolic proc- esses acidify the media and wells containing revertant cells continue to grow causing further acidification of the media. Due to the presence of the pH indicator, revertant wells can be identified by simple visual inspection. Compounds that cause higher than background rates of inversion are consid- ered potential mutagens. Hepatic S9 (the soluble fraction from liver homogenate after centrifugation at 9,000 x g) can be added to the Ames test to improve predictions by incorporation of a metabolism component [219]. Known carcinogens such as benzo[a]pyrene are positive only upon the addition of S9 [220, 221]. Benzo[a]pyrene does not directly modify DNA. CYP1A1 catalyzes the formation of an epoxide which is hydrolyzed to form a diol [222-224]. The diol metabolite is further metabolized to benzo[a]pyrene-7,8-dihydrodiol-9,10- epoxide which covalently binds DNA. Human S9 or induced rat S9 microsomes are used. Rats can be treated with Aroclor 1254 for two to three days prior to harvesting the liver and preparation of S9. The specific content of many of the drug metabolism enzymes is greatly increased in Aroclor 1254 induced S9 [225, 226]. Fig. (1). Immune-mediated drug-induced toxicity. IN VIVO METHODS Idiosyncratic toxins are reported to poorly replicate in There are several assays that can be utilized to examine animal models. However, this may be an issue with having in vivo generation of reactive metabolites and try to gauge too grand of expectations. It is not practical to screen thou- their toxicity risk. Based on the results from in vitro experi- sands of mice to see if a compound causes significant hepatic ments, additional in vivo experiments may be warranted. It is damage in a small fraction of the mice. Designing a mouse important to have realistic expectations when evaluating in model to evaluate compounds that are toxic in a minority of vivo data. With the current state of knowledge, experiments humans that demonstrates toxicity in a majority of mice to verify observed in vitro reactive metabolites are possible without generating false positives while replicating injury through several mechanisms. Utilization of this data to pre- with identical histopathology is unreasonable. Perhaps a dict the toxicological implication is more difficult. The focus more realistic expectation would be for the mouse model to will be on rodent and human clinical studies. Other mam- demonstrate that a compound binds to proteins, or that it mals including primates can be similarly used. depletes hepatic glutathione levels, or that the mouse gener- ates antibodies against protein-drug adducts or anti-self anti- Among FDA approved drugs that are associated with bodies at statistically higher levels in the drug treated group idiosyncratic hepatic toxicity, rates of drug-induced hepatic over vehicle control. failure are frequently one in ten-thousand to one in a few hundred thousand [227-231]. Even among drugs that were Most of the procedures detailed in the remainder of this withdrawn due to their risk of hepatotoxicity the typical inci- review can be done in both human patients and in animal dence rate of hepatic failure was one case per ten-thousand models. Reactive Metabolites – Experimental Evaluation Current Medicinal Chemistry, 2015, Vol. 22, No. 4 473

NDA/ Post Lead Candidate Lead Identification IND Enabling Phase 1 Trials Phase 2 Trials Phase 3 Trials market Optimization Selection Surveillance In vitro – microsomal radiolabel incorporation/covalent adduction In vitro – reactive metabolite trapping In vitro – P450 TDI In vitro – genotoxicity (Ames test) In vitro/in vivo – proteomics micronucleus micronucleus assay (man) assay (animal) In vivo – whole body radiolabel Radiolabel/PET incorporation or PET(animal) (man) In vivo – glutathione and In vivo – glutathione and mercapturate adducts (animal) mercapturate adducts (man) In vivo – markers of hepatocellular injury (ALT, ALP, bilirubin) In vivo – HLA association In vivo – lymphocyte proliferation assay In vivo – patch/skin hypersensitivity test In vivo – eosinophilia/ DRESS In vivo – re-activation of virus In vivo – autoantibodies or drug-protein antibodies Fig. (2). Timeframe for various methods to evaluate reactive metabolites.

In vivo Genotoxicity liver sample to evaluate adduct levels in tissue [251]. In vivo, glutathione conjugated drugs are typically further metabo- As part of the preclinical toxicology evaluation, in vivo lized and detected as mercapturic acid adducts [252-254]. genotoxicity is evaluated using a micronucleus assay [232- The c-peptide linkage between glutamate and cysteine is 240]. During cell division, the parent cell should result in resistant to normal proteases which make glutathione adducts two daughter cells each containing identical copies of DNA. relatively stable in most tissue and allows for the direct de- In the presence of compounds or reactive metabolites that tection of intact drug adducts. However, GSH-adducts can be can cause chromosomal damage, it is possible to have one of enzymatically degraded by the enzyme -glutamyl trans- the daughter cells lacking a part or all of a chromosome. The ferase which removes the glutamate followed by further missing chromosomal fragment can develop a nuclear mem- processing by cellular dipeptidases which remove glycine. brane where some cells appear to have one large and one The amine group of the cysteine is then acetylated to form a small nucleus (the micronuclei). Comparison of the percent- mercapturate. The tissue with the highest expression level of age of micronucleus positive cells in the treated vs. control these enzymes is the kidney and in many cases the glu- groups provide a readout of genotoxicity. The assay is typi- tathione conjugate is exported from the liver then processed cally done with erythrocytes in the peripheral blood or bone in the kidney where it can be excreted in urine or be re- marrow [241-245]. Erythrocytes are anucleated which sim- absorbed by the liver and excreted in the bile [253]. Detec- plifies detection because the micronuclei are not extruded as tion of mercapturate adducts in bile or feces does not neces- efficiently. sarily indicate inter organ transport, especially in rodent where expression levels of -glutamyl transferase and cellu- In vivo Binding Studies lar dipeptidases in the bile duct are sufficient to facilitate the Experimental options focusing on binding are quite simi- degradation of GSH-adducts [253, 254]. Detection of mer- lar to those used in vitro, but incorporate factors such as capturates using mass spectrometry indicates that the com- pharmacokinetics and tissue levels which are not easily rep- pound was metabolized to a nucleophilic metabolite, but may licated in vitro. Similarly, the in vivo systems have a full not indicate a high level of protein modification [255]. The complement of detoxification pathways such as antioxidants, mercapturate could have been derived from the direct reac- glutathione S-transferase, superoxide dismutase/catalase, tion with glutathione from cellular glutathione pools, enzy- glutathione peroxidase, epoxide hydrolase, etc. Alternative matically catalyzed by glutathione S-transferase, or even methodologies to evaluate covalent modification are pre- derived from adducted proteins where cysteine is covalently sented. Similar to the in vitro systems, radiolabel incorpora- modified. If the reactive electrophile is a substrate for glu- tion is the most frequently utilized methodology [87, 246, tathione S-transferase it is possible that cellular levels of the 247]. electrophile can be minimized and associated toxicities re- duced [256]. Detection of mercapturates indicates the pres- Detection of Glutathione or Mercapturate Adducts ence of an electrophile capable of reacting with cysteine, but is not a direct indicator of toxicity. Positive results from glutathione adduction studies in vi- tro can be easily followed up with in vivo studies using pre- Whole-Body Radiography clinical species or humans. This is typically done through obtaining samples of urine and either bile or feces from Quantitative Whole Body Autoradiography (qWBA) is dosed rats or humans and comparing to vehicle dosed con- an imaging technique used to provide information on the trols [248-250]. In rodent studies it is also possible to get a localization of radiolabeled compounds in lab animals [257- 474 Current Medicinal Chemistry, 2015, Vol. 22, No. 4 Eno and Cameron

260]. It is usually performed on only advanced safety- graphic techniques due to its broad dynamic range and sensi- assessment candidates during the preclinical drug develop- tivity, and its ability to provide qWBA data in days versus ment stage, and involves imaging of the whole-body, and/or weeks to months [263-266]. Some of the shortcomings of specific tissues or organs. The data can provide important this method are that these experiments require the synthesis information about tissue pharmacokinetics, tissue distribu- of radiolabeled drug, which can be practically challenging. tion, metabolism, drug-drug interactions, elimination, and Thus this approach is not amenable for early discovery pro- clearance and is especially helpful in pinpointing the site of jects where the best-in-class compound is likely to change compound retention [261]. In these experiments, animals are quickly. Another disadvantage is that this technique provides 14 125 3 given a single dose of radioactive compound ( C, I, H) and data on the concentration of radioactivity only, and it is not may be visualized live or euthanized at different time points. possible to determine if the radioactivity represents parent Autoradiograph results show the localization of the radiola- drug, metabolites and/or degradation products without fur- beled drug at different times and the amount of radioactivity ther evaluation. from tissues is quantitated by comparative dosimetry. IN VIVO qWBA studies can also provide tissue samples for analy- TOXICITY BASED STUDIES sis of reactive metabolites that may bind covalently to pro- The literature regarding severe drug-induced hepatic tox- teins in various tissues or organs over time [257]. The use of icity can be confusing. Part of this is due to differences in the whole-body sections and/or residual frozen tissue can supply intended audiences. Publications targeting clinicians have samples for bio analysis using radio-HPLC and mass spec- often advised caution about over-interpreting measures of troscopy to identify the adduct and modified proteins. For hepatic toxicity such as increases in serum transaminases example, drugs like amodiaquin (withdrawn) and clozapine [267-271]. This is primarily an underlying argument of prob- (black box warning), which cause severe and life-threatening ability. Most patients that demonstrate mild or moderate in- agranulocytosis, have been evaluated by qWBA and showed creased serum transaminases in the first few weeks after ini- retention of radioactivity in the bone marrow and spleen up tiation of treatment revert to normal levels through tolerance to 168 hours after administration, Fig. (3) [262]. If the reten- or unknown adaptation mechanisms with continued expo- tion of these radiolabeled compounds is correlated with its sure. For example, the drug tacrine is commonly associated covalent binding data, qWBA can be used for toxological with mild hepatic injury that can cause significant increases risk assessment to estimate body burden in larger species or in plasma ALT levels three to six weeks after the initiation of humans. treatment, often 5- to 10-fold the upper limit of normal. However, severe hepatic injury caused by tacrine is exceed- ingly rare and for most patients ALT levels return to normal by twelve weeks [268]. If clinicians withdrew treatment with the first observation of elevated ALT, they may be denying their patient beneficial treatment. On the opposite spectrum are publications targeting drug discovery scientists where compounds such as troglitazone are highlighted. Elevated ALT levels in troglitazone patients are both lower and less frequent than what is seen with tacrine, but in the three years between the approval of troglitazone and its removal from the market, at least 94 cases of troglitazone-induced liver failure were reported to the FDA [272]. The relative prob- ability of liver failure for an individual patient will be quite low for a compound exhibiting idiosyncratic hepatic toxicity, but the overall incidence may be too high to remain on the market [227, 273-276].

Measures of In Vivo Hepatic Damage During or upon completion of multi-day dosing studies, it is common to collect blood which can be evaluated for markers of hepatic damage. Numerous markers have been Fig. (3). Whole-body radiographs demonstrating compound reten- historically utilized to evaluate liver damage, with the four tion in the liver and bone marrow 72 and 128 hours after a single most common readouts being bilirubin, aspartate transa- 3mg/kg oral dose of radiolabeled amodiaquine (A) and clozapine minase (AST), alanine transaminase (ALT), and alkaline (B) [31], reprinted with permission. phosphatase (ALP). The liver is not comprised of a single cell type nor is it uniform in its architecture. A hepatologist One of the main advantages of this technique is that it can detail numerous classifications to describe liver injury, provides a comprehensive visual of radiolabel distribution in but for most individuals there are four major categories to organs and tissues. Unlike other techniques such as tissue consider: hepatocellular, cholestatic, infiltrative, and auto- dissection/liquid scintillation counting where all tissue sam- immune [269, 270, 275, 277-281]. ples are not routinely tested, qWBA provides the distribution of radioactivity in all organs and tissues of an intact animal Hepatocellular injury is a primary injury to the hepato- carcass. Phosphor-imaging technique has replaced photo- cytes. In cases where hepatocyte membrane integrity is lost Reactive Metabolites – Experimental Evaluation Current Medicinal Chemistry, 2015, Vol. 22, No. 4 475 such as necrotic cell death, plasma levels of transaminases trate the liver and collect bile excreted from hepatocytes. increase. There is little alkaline phosphatase increase in he- Alkaline phosphatase derived from bone and bile duct can be patocellular injury. Bilirubin increases may be small or unde- differentiated based upon the reduced thermal stability of tectable, but are elevated as the extent of hepatic injury pro- bone derived alkaline phosphatase [303]. Alkaline phospha- gresses. Bilirubin is cleared from the blood through conjuga- tase levels naturally increase in pregnant woman and do not tion and excreted from the hepatocyte into the bile. There is necessarily imply liver dysfunction. excess hepatic capacity to carry out this function so increases Elevated bilirubin levels (jaundice) are often associated in bilirubin are not seen with mild hepatocellular injury. Dis- with damage to hepatic biliary excretion [275, 277, 282, eases that primarily affect hepatocytes, such as viral hepati- 302]. Bilirubin is not released from the liver, but is from the tis, will cause disproportionate elevations of ALT and AST breakdown of heme containing proteins, particularly hemo- compared with alkaline phosphatase or bilirubin [229, 279, globin and myoglobin. In the liver, bilirubin is conjugated by 282]. glucuronidation and sulfation and then exported into the bile Cholestatic hepatic injury, sometimes referred to as ob- [304-309]. This creates an important caveat in the evaluation structive liver injury, is primarily a bile duct injury [279, of liver damage: increases in total bilirubin levels are not 282, 283]. This may be due to gall stones, tumors obstructing necessarily indicative of liver dysfunction and can be due to bile flow, or due to significant inflammation or necrosis of increased hemolysis. Elevated plasma concentration of con- the cells forming the bile canaliculi which causes leakage of jugated serum bilirubin (not total) implies liver dis- alkaline phosphatase. In addition to increases in alkaline ease/dysfunction [310, 311]. However, most commercial phosphatase in the plasma, cholestatic injury is also associ- blood chemistry analyzers provide tBIL for “total bilirubin”. ated with elevated bilirubin. This is not surprising that block- When the two are differentiated, the term “direct bilirubin” is ing biliary excretion leads to a buildup of bilirubin because used to refer to conjugated bilirubin and “indirect bilirubin” conjugated bilirubin is exported to the bile. for unconjugated. Bilirubin should not be present in the urine of a healthy patient, but conjugated bilirubin may be present Infiltrative hepatic injury is characterized by the liver be- in urine when biliary export is impaired leading to elevation ing invaded or replaced by non-hepatic substances. This is of conjugated bilirubin in the blood. commonly neoplasm (an abnormal growth or division of cells which may be benign or malignant) or amyloid [284- Clinical Implications 287]. Infiltrative hepatic injury is not usually associated with reactive metabolites or drug-induced toxicity but has a simi- In most cases where increases in ALT are detected, this lar increase in alkaline phosphatase as seen with cholestatic indicates damage to hepatocytes. However, the liver is an injury. However, bilirubin changes tend to be reduced. Imag- amazingly resilient organ and mild damage is rarely associ- ing studies or liver biopsy may be required to unambiguously ated with a significant toxicological event. In clinical trials, diagnose infiltrative hepatic injury along with its cause. ALT is commonly measured and compared to the control Autoimmune mechanisms can have a hepatocellular pat- group [271, 312-315]. A typical threshold of significance is tern if hepatocyte damage is involved (autoimmune hepatitis) 3-times the upper limit of normal (ULN). Higher levels of 5- or cholestatic if the immune system targets the biliary duct and 10-times ULN are often used to indicate more significant (primary biliary cirrhosis) [288-291]. Immune-mediated he- hepatic damage. patotoxicity is discussed in greater detail in a separate sec- Almost all compounds that cause hepatic failure also had tion. an associated increase in the percentage of patients with ALT elevation in clinical trials. However, the opposite (increased Measureable Hepatic Markers ALT indicating imminent severe toxicity) is not always true. Elevated transaminases are indicative of hepatocellular Notable compounds that cause ALT elevations but are rarely injury. Both ALT and AST are at high concentrations in the associated with severe adverse events are tacrine, aspirin, liver cytosol. In cases where hepatocyte membrane integrity heparin, and statins [7, 271, 316-318]. Hy Zimmerman pro- is lost, they can be detected at elevated levels in the blood. vided evidence that increases in transaminases were most AST is not favored as a primary readout of liver function predictive of serious hepatic toxicity when they were associ- because it is also present at high levels in muscle leading to ated with a concomitant measure of altered liver function fluctuations in AST for benign reasons such as muscle mass (measured as a greater than 2-fold increase in serum biliru- and exercise [292-297]. Both transaminases require the pros- bin). This observation has been refined and is sometimes thetic group pyridoxal-phosphate which is the active form of referred to as Hy’s Law [319-326]. Unfortunately, by the vitamin B6 [298-300]. Vitamin B6 deficiency is rare, but time hepatocellular injury is severe enough to lead to greater would be expected to decrease activity and mask the eleva- than 2-fold increase in serum bilirubin there is approximately tion of plasma transaminase levels [298]. Other interactions a ten percent chance the patient will progress to liver failure have been noted with chemical inhibitors. For example, resulting in death or liver transplant. This represents a seri- isoniazid associated hepatic damage is largely masked be- ous deficiency in our current knowledge base. Earlier bio- cause isoniazid inhibits ALT [301]. markers that describe the loss of liver function/capacity prior to reaching 2-times increase in bilirubin concentration may Alkaline phosphatase is a hydrolase enzyme which cata- provide the opportunity for earlier intervention without the lyzes the removal of phosphate from numerous compounds. associated risk of patient mortality. It is primarily elevated in the bile duct, kidney, placenta, and bone [302, 303]. Liver levels have also been reported to be Summary of Hy’s Law: A drug that causes serious hepatic elevated but this may be due to bile canaliculi which infil- injury will generally: 476 Current Medicinal Chemistry, 2015, Vol. 22, No. 4 Eno and Cameron

 Show an increased incidence in elevated plasma transa- experienced flucloxacillin-induced liver failure and a com- minases (>3xULN) than the control parison was made to a control group of similar ethnicity.  Lead to total bilirubin >2xULN in a small percentage of Sixty-three of seventy-four liver injury patients had a HLA- patients without initial elevation of serum ALP indicating B*5701 genotype compared to four of sixty-four controls. the increase in bilirubin is due to a decrease in hepatocel- The researchers concluded that the odds of a patient with lular function and not due to obstruction of bile flow. HLA-B*5701 genotype experiencing severe liver injury Complicating factors such as hepatitis, underlying liver from taking flucloxacillin were 80.6-times higher than for disease and co-medications should be factored in, as patients with alternative genetic HLA profiles [339]. While should the presence of increased red blood cell lysis as the 80-fold increase in risk is exceptionally large, there is this would increase bilirubin levels independent of liver still under a one percent chance for an individual to experi- function. ence severe flucloxacillin-induced liver injury even if they do have the HLA-B*5701 genotype. Obviously there are In the FDA issued guidance for evaluation of drug- additional factors involved in distinguishing what patients induced liver injury, Hy’s law is prominently featured and will and will not experience toxicity. used to estimate the expected incidence of serious hepatic events [7, 327]. Other studies have demonstrated an immune component in serious drug-induced skin toxicity from compounds that In the clinical trials of a hypothetical compound where are known to generate reactive metabolites or otherwise co- 6,000 total patients are dosed for extended periods and five valently modify proteins [340]. The risk of moderate carba- Hy’s Law cases are observed (individuals with >3xALT and mazepine hypersensitivity increased 12-fold and severe Ste- 2xbillirubin) the estimated rate of hepatic failure would be vens–Johnson syndrome or toxic epidermal necrolysis in- 1/12,000. This is based on 5/6,000 (1/1,200) patients exceed- creased 26-fold for patients with HLA-A*3101 [341]. Addi- ing Hy’s law and an estimate that 10% of Hy’s law cases tional studies associating antigen presentation and drug tox- proceed to hepatic failure. When there is an increase in the icity have been published. A partial list includes: lapatinib overall rate of >3xALT but no patients with associated bili- [334], abacavir [342], nevirapine [343], and lumiracoxib rubin increases a different calculation is used. Evaluation of [344]. Results from numerous association studies were the same hypothetical 6,000 patients with an observed in- evaluated to see if there were common features in peptide crease in the percentage of treated patients with >3xALT, but binding between those HLA that have been associated with assuming no Hy’s Law cases are observed, the estimated rate drug-induced toxicities and those that have not. Researchers of hepatic failure would be less than 1/20,000. This is be- found that within HLA class I alleles those that were associ- cause in the absence of findings the calculation is based upon ated with toxicity were actually less similar than the control a 95% confidence level. Using the “rule of 3”, three times set of HLA analyzed [345]. Similarity between HLA class II more samples are required to say with 95% confidence that alleles associated with drug-induced toxicity were only the lack of an observed case was not due to statistical slightly more similar than control [345]. This implies that chance. If the rate of hepatic failure were to equal 1/20,000 antigen presentation is either not due to a common subset of then 1/2,000 patients would be expected to be Hy’s Law presented antigens or that there are additional factors that we cases, and three times that number (6000 patients) would be do not yet understand. needed to say with 95% confidence that the expected rate of hepatic failure is less than 1/20,000 patients [7]. Despite not understanding all of the specifics leading to immune-mediated toxicity, patient benefit is starting to be Immune-Mediated Toxicity realized. Two of the strongest HLA associations are with abacavir hypersensitivity (HLA-B*5701) and severe cutane- Immune-mediated toxicity has been implicated with nu- ous injury due to carbamazepine in the Han Chinese and merous compounds and almost all of these compounds have Thai populations (HLA-B*1502) [346, 347]. Through ge- been shown to generate reactive metabolites [328-335]. The netic pre-testing, reduced rates of toxicity have been evidence for immune involvement is strong and includes a achieved for both drugs [335]. One of the major obstacles to delayed onset of toxicity with rapid recurrence upon re- implementing such tests on a wider scale is the role of eth- challenge, detection of drug specific antibodies and drug nicity. Whereas HLA-B*1502 increases the chance of seri- responsive T-cells, macrophage recruitment to the site of ous cutaneous injury by over 100-fold in the Han Chinese, toxicity, positive skin hypersensitivity tests, and positive the increased risk of carbamazepine-induced toxicity due to correlations between patient susceptibility to drug-induced HLA-B*1502 is minimal among Caucasian populations toxicity and human leukocyte antigen (HLA, the protein that [348, 349]. Understanding if there are both primary and sec- binds proteolytic peptides and presents them to T-cells) ondary drivers for toxicity and having the ability to appropri- [333-337]. The ability to predict what compounds will and ately weight their significance would be a major step for- will not elicit immune-mediated toxicity or to predict the ward. frequency of severe events prior to entering the clinic is not Genome-wide association studies have been valuable in currently validated. Severe immune-mediated toxicities identifying increased-risk populations for several drugs, but where the patient fails to gain tolerance are not common, but this is a retrospective analysis [350]. Patient injury is re- can be particularly severe and have high rates of fatality. quired in order to have patient pools that are positive and The rate of drug-induced cholestatic liver disease with negative for toxicity. The ideal situation would be to identify flucloxacillin is approximately 1/10,000 [338]. A genome- populations at increased risk prior to entering the clinic or as wide association study was conducted with patients that had soon as possible after human patients are exposed. There are Reactive Metabolites – Experimental Evaluation Current Medicinal Chemistry, 2015, Vol. 22, No. 4 477 additional tests that have demonstrated drug-induced im- relevant concentrations. However, small molecule-antibody mune-mediated toxicity and statistically significant differ- interactions are possible at much lower concentrations and ences have been demonstrated between patients that experi- have even been utilized to establish analytical diagnostics. enced toxicity and untreated controls, but even with the best Commercial ELISA kits are available from abcam tests currently available, not all patients in the toxicity group (ab20768), Pierce (PC10-242.6), Beckman Coulter (Emit give positive results. In some cases, patients that have been 2000 procainamide assay), and other providers for quantita- treated but show no observable toxicity have come back tion of free procainamide in patient plasma. These are based positive for an immune response. It is unclear if detection of on monoclonal antibodies that were generated using a pro- an immunological response in non-symptomatic groups fore- cainamide-KLH immunogen through covalently attaching tells an increased likelihood for future toxicity or what per- the amine group of procainamide to acidic amino acids of centage of patients would experience serious adverse events. KLH. Through the fusion with myeloma cells, monoclonal antibodies were obtained and expanded in the presence of Lymphocyte Transformation Test (LTT) free procainamide. Evaluation of lymphocyte proliferation in response to The percentage of lymphocytes that sufficiently recog- drug challenge is much more common in Europe and Japan nize free drug is likely different for individual drugs and for than in the United States. Blood collected from patients that individual patients. This is likely the reason for the wide experience potential immune-mediated drug-induced toxicity range in responders in LTT evaluation. is processed to isolate lymphocytes which are evaluated im- When evaluating increasing numbers of patients, LTT mediately or frozen. Lymphocytes are then cultured in the clearly demonstrates increases in the rate of positive re- presence or absence of the suspected drug and lymphocyte sponses for both cutaneous and hepatic immune-mediated proliferation is evaluated [351, 352]. A common readout of drug-induced toxicity [354-362]. However, the rate of posi- proliferation is to measure radioactive thymidine incorpora- tive responders can fluctuate greatly and the severity of tox- tion. Generally, stimulation greater than two-fold is consid- icity is not necessarily tied to a positive LTT. The drug ered a positive response. Phytohemagglutinin can be used as lamotrigine is associated with severe cases of cutaneous in- a positive control to demonstrate that induction of prolifera- jury including Stevens-Johnson syndrome (SJS) and toxic tion is possible. epidermal necrolysis (TEN) [360, 363]. Using LTT, lamo- The LTT test is built upon the natural propensity for acti- trigine-naive control patients were compared to lamotrigine vated lymphocytes to increase their proliferation in the pres- treated patients without observable adverse reaction, with ence of the specific antigen that they are primed to recog- mild epidermal eruption, active SJS/TEN, and patients re- nize. In the LTT assay, lymphocytes are incubated with the cently recovered from SJS/TEN. Positive LTT did not trend parent drug and not with drug-protein or drug-peptide conju- with the severity of toxicity; however, positive tests were gates. It is generally accepted that a small molecule cannot detected for seven of twenty nine patients that had a reaction directly illicit an immune response leading to the generation and in zero of twelve lamotrigine-naive controls. Interest- of specific antibodies. Instead, the molecule is presented as a ingly, one of six patients that had been exposed to lamo- larger complex, typically through a covalently modified pep- trigine but showed no reaction had a positive test [364]. tide or protein (hapten hypothesis). The reverse is not true. In a test of 95 suspected cases of drug-induced liver in- An antibody (at least polyclonal antibodies), originated in jury associated with 33 different drugs, 25/95 of the liver response to haptenized proteins, do have the potential to di- injury patients had a positive LTT, whereas positive tests rectly bind free small molecules and their metabolites. were obtained for 0/106 healthy controls. When the prosta- Anti-clozapine antibodies were raised by trapping the re- glandin inhibitor indomethacin was added to the LTT cell active clozapine metabolite with N-acetycysteine (NAC). media to inhibit decreased lymphocyte proliferation due to The NAC was then used to link clozapine to Keyhole limpet excreted prostaglandins, the percentage of positives in the hemocyanin (KLH) and used for antibody generation [353]. LTT increased to 53/95 [365]. Serum was tested and shown to recognize clozapine conju- Patch/Skin Hypersensitivity Test gated proteins. Of importance to the LTT assay is that anti- body binding could be inhibited by clozapine, its close ana- Positive skin hypersensitivity tests have been observed log olanzapine, or clozapine-NAC which was the actual anti- for several drugs including acetaminophen. The patch test gen used for antibody generation. Binding was not inhibited applies drug to the skin and tests if the dermal dendritic cells when structurally unrelated compounds were tested [353]. (Langerhans cells), or other lymphocytes that migrate to the Similarly, antigen binding of antibodies generated against area are capable of responding to the presented antigen. hydroxyfluperlapine, the metabolite of fluperlapine, could be Patch tests have been utilized to demonstrate drug hypersen- inhibited by both fluperlapine and hydroxyfluperlapine sitivity [366, 367]. In contrast to the LTT assay which offers [111]. The importance of these findings is that activated absolute safety due to being an in vitro evaluation, the patch lymphocytes, which recognize antigens through surface test has the potential for complications. While the FDA does bound antibodies, would be expected to behave similarly and not have firm regulations regarding the use of the patch test may react with the parent drug even if the actual antigen was to evaluate hypersensitivity, they do specifically advise a metabolite bound to protein. against its use with abacavir, citing concerns of patient safety. Instead of using the patch test to evaluate sensitive vs. The previously described interactions with fluperlapine non-sensitive individuals, they recommend testing for the and clozapine all utilized 20-100 M drug to inhibit antibody HLA-B*5701 allele and caution against abacavir use in such binding and it could be argued that these are not clinically patients. 478 Current Medicinal Chemistry, 2015, Vol. 22, No. 4 Eno and Cameron

(http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/psn/tr These include antinuclear antibody (ANA, also called anti- anscript.cfm?show=84#3). histone antibody), anti-Smooth muscle antibody (ASMA), liver/kidney microsomal antibody (LKM-1, LKM-2, LKM- Elevation of Eosinophils 3), anti-soluble liver antigen (SLA), anti-actin antibody (AAA), and anti-mitochondrial antibody (AMA) [47, 138, Through the collection of patient blood samples, blood 155, 156, 328, 387-390]. ASMA and AAA have been re- counts can be performed to evaluate eosinophil count. Eosi- ported to associate with disease state in autoimmune hepati- nophils are a portion of the circulating leukocyte population tis [391, 392]. Increased levels of autoantibodies have been and elevations are often associated with allergic reaction. reported in cases of chronic drug-induced liver injury rela- Eosinophil count is not causal with respect to the suspected tive to those seen for acute injury [388]. Autoantibodies are treatment drug and can be elevated by certain parasites or likely detectable as a marker of chronic drug-induced injury non-related allergens [368, 369]. and may have little to do with the drug being presented as an Eosinophilia is often referred to as Drug Reaction with antigen or specifically altering antigen processing. Patients Eosinophilia and Systemic Symptoms (DRESS). DRESS with suspected drug-induced liver injury taking three bio- onset is typically three to eight weeks after drug initiation logical anti-TNF- agents, infliximab (n = 26), etanercept (n and is frequently associated with fever and skin eruption = 4), and adalimumab (n = 4) were evaluated for autoanti- [370]. Some of the drugs most frequently observed in bodies and 67 percent of the patients tested positive for anti- DRESS such as allopurinol, phenytoin, carbamazepine and nuclear and/or smooth muscle antibodies. 88 percent of pa- dapsone are associated with other immune-mediated drug- tients testing positive for ANA or ASMA had clear features induced toxicities and have been shown to generate reactive of autoimmunity [393]. metabolites [369]. DRESS has been reported to positively In contrast to the before mentioned autoantibodies, anti- correlate with viral reactivation. P450 antibodies appear to be in direct response to hapten formation [155, 156, 390]. The drug tienilic acid was found Re-Activation of Viruses to illicit the formation of anti-CYP2C9 antibodies. This cor- Viral reactivation has been utilized in Japan more so than relates with tienilic acid metabolism as it is oxidized by in other countries. An increase in herpes virus (HHV-6) was CYP2C9 and is a potent mechanism based inactivator of 2C9 shown to be strongly associated with drug-hypersensitivity activity [138]. Similar to tienilic acid, anti-CYP2E1 antibod- reactions, but other viruses have also been implicated [371- ies were detected in 25 of 56 patients diagnosed with halo- 385]. The largest volume of data is available for reactions thane-induced hepatitis [155] and anti-CYP1A2 antibodies with anti-convulsants. The mechanism of the association is have been found associated with disulfiram [156]. A recent largely still hypothetical and this may explain why it has not paper reports the detection of both anti-isoniazid and anti- been adopted in a wider setting. The simplicity of a rapid P450 antibodies in a patient with isoniazid-induced liver PCR based analysis along with no additional patient risk is failure [328]. Where these accounts appear to have mecha- quite attractive. HHV-6 reactivation has a short delay from nistic utility, all deal with the appearance of anti-P450 anti- the initial signs of hypersensitivity [386] and differing results bodies in patients diagnosed with severe drug-induced inju- are reported for minor hypersensitivity vs. more severe reac- ries by comparing them to control patients. It would be of tions. While HHV-6 has been most widely studied, great benefit to know if less severely injured patients would reactivation of three other herpes viruses: HHV-7, Epstein- have had detectable anti-P450 antibodies in there sera. Barr virus (EBV or HHV-4) and cytomegalovirus (HHV-5) Whereas viral infection or other causes of liver injury may have also been reported. increase the potential for detectable levels of anti-self anti- bodies, anti-P450 antibodies would be expected to correlate In one report HHV-6, HHV-7 or Epstein-Barr virus were to metabolic activation. It is unknown if evaluation of anti- reactivated in 76% of patients with DRESS associated with P450 antibodies during clinical development of compounds taking carbamazepine, allopurinol, or sulfamethoxazole. In demonstrating time-dependent inhibition would be benefi- all patients, circulating CD8+ T lymphocytes were activated cial. For example, would evaluation of anti-CYP2C9 anti- with elevated secretion of cytokines [368]. bodies during the development of tienilic acid (approved by Both the mechanism of increase and the predictive value the FDA in 1979 and withdrawn in 1982) have shown differ- of viral reactivation are unknown. While reactivation may ences between the tienilic acid vs control groups? track well for DRESS, it is unclear if the association is as strong with other drug-induced toxicities or if there is a CONCLUDING REMARKS threshold effect, e.g. is viral reactivation only seen with ad- Because of the low incidence of severe drug-induced tox- vanced toxicity. An ideal marker would be capable of identi- fying potentially toxic compounds early in clinical develop- icity, it remains a difficult challenge to identify idiosyncratic hepatic toxins during clinical development. The ability to ment and not be limited to classifying the mechanism of ob- identify reactive metabolites has outpaced the ability to as- served toxicity. The value of determining levels of HHV-6 or certain the toxicological implication. There is currently no Epstein-Barr virus after 4-8 weeks dosing in treated vs. con- way to unequivocally provide answers to questions about the trol patients during clinical trials is uncertain. ultimate toxicity of an individual preclinical compound that Anti-Self Antibodies is metabolized to a reactive metabolite. Prevention of reac- tive metabolite formation or decreasing the body-burden Several antibodies have been associated with autoim- through lowering the dose have been demonstrated to reduce mune diseases including drug-induced autoimmune diseases. the risk of idiosyncratic toxicity based on statistical analysis, Reactive Metabolites – Experimental Evaluation Current Medicinal Chemistry, 2015, Vol. 22, No. 4 479 but there are numerous outlier drugs that have been benefi- [4] Uetrecht, J., Screening for the potential of a drug candidate to cause cial to patients despite being associated with high body bur- idiosyncratic drug reactions. Drug discovery today, 2003, 8, (18), 832-837. dens of reactive metabolites. There is strong evidence that [5] Chen, W.G.; Zhang, C.; Avery, M.J.; Fouda, H.G., Reactive the immune system plays a prominent role in many chronic metabolite screen for reducing candidate attrition in drug drug-induced toxicities. To date, most studies have been discovery. Advances in experimental medicine and biology, 2001, retrospective to evaluate the mechanism of toxicity but much 500, 521-524. of the methodology could be adopted at an earlier stage to [6] Smith, D.A.; Obach, R.S., Metabolites and safety: What are the concerns, and how should we address them? Chemical research in evaluate the potential for immune-mediated drug-induced toxicology, 2006, 19, (12), 1570-1579. toxicity during clinical development. Evaluation of the po- [7] FDA. Administration, F.a.D., Ed.: http://www.fda.gov/Drugs/ tential for compounds to be recognized as an antigen, acti- GuidanceComplianceRegulatoryInformation/Guidances/default.ht vate an immune response and influence tolerance will in- m, 2009. [8] Reese, M.; Sakatis, M.; Ambroso, J.; Harrell, A.; Yang, E.; Chen, crease the predictive value of early studies where reduced L.; Taylor, M.; Baines, I.; Zhu, L.; Ayrton, A.; Clarke, S., An numbers of patients are tested and decrease the number of integrated reactive metabolite evaluation approach to assess and compounds that are eventually removed from market or fail reduce safety risk during drug discovery and development. Chem. late in clinical development due to hepatic toxicity. Biol. Interact., 2011, 192, (1-2), 60-64. [9] Evans, D.C.; Watt, A.P.; Nicoll-Griffith, D.A.; Baillie, T.A., Drug- protein adducts: an industry perspective on minimizing the LIST OF ABBREVIATIONS potential for drug bioactivation in drug discovery and development. Chem. Res. Toxicol., 2004, 17, (1), 3-16. ALT = Alanine transaminase [10] Antoine, D.J.; Williams, D.P.; Park, B.K., Understanding the role DRESS = Drug reaction with eosinophilia and systemic of reactive metabolites in drug-induced hepatotoxicity: state of the science. Expert Opin. Drug Met., 2008, 4, (11), 1415-1427. symptoms [11] Bu, H.Z., A literature review of enzyme kinetic parameters for HLA = Human leukocyte antigen CYP3A4-mediated metabolic reactions of 113 drugs in human liver microsomes: structure-kinetics relationship assessment. Curr. Drug IND = Investigational new drug Metab., 2006, 7, (3), 231-249. [12] Niwa, T.; Murayama, N.; Emoto, C.; Yamazaki, H., Comparison of Keap1 = Kelch-like ECH-associated protein 1 kinetic parameters for drug oxidation rates and substrate inhibition potential mediated by cytochrome P450 3A4 and 3A5. Curr. Drug KLH = Keyhole limpet hemocyanin Metab., 2008, 9, (1), 20-33. LDH = Lactate dehydrogenase [13] Wilkinson, G.R., Cytochrome P4503A (CYP3A) metabolism: prediction of in vivo activity in humans. J. Pharmacokinet LTT = Lymphocyte transformation test Biopharm., 1996, 24, (5), 475-490. [14] Thummel, K.E.; Wilkinson, G.R., In vitro and in vivo drug NAC = N-acetycysteine interactions involving human CYP3A. Annu. Rev. Pharmacol. Toxicol., 1998, 38, 389-430. Nrf2 = Nuclear factor erythroid 2-related factor [15] Ogliaro, F.; de Visser, S.P.; Cohen, S.; Sharma, P.K.; Shaik, S., Searching for the second oxidant in the catalytic cycle of P450 = Cytochrome P450 cytochrome P450: a theoretical investigation of the iron(III)- qWBA = Quantitative whole body autoradiography. hydroperoxo species and its epoxidation pathways. Journal of the American Chemical Society, 2002, 124, (11), 2806-2817. SJS = Stevens-Johnson syndrome [16] Ortiz de Montellano, P.R., Cytochrome P-450 catalysis: radical intermediates and dehydrogenation reactions. Trends Pharmacol. TEN = Toxic epidermal necrolysis Sci., 1989, 10, (9), 354-359. [17] Popescu, D.L.; Vrabel, M.; Brausam, A.; Madsen, P.; Lente, G.; CONFLICT OF INTEREST Fabian, I.; Ryabov, A.D.; van Eldik, R.; Collins, T.J., Thermodynamic, electrochemical, high-pressure kinetic, and The Authors report no financial conflicts of interest. mechanistic studies of the formation of oxo Fe(IV)-TAML species Salaries were partially paid for by NIH grant in water. Inorganic chemistry, 2010, 49, (24), 11439-11448. [18] Cameron, M.D.; Wen, B.; Roberts, A.G.; Atkins, W.M.; Campbell, 1R21DK091630 and P01DA033622 to MDC. A.P.; Nelson, S.D., Cooperative binding of acetaminophen and caffeine within the P450 3A4 active site. Chem. Res. Toxicol., ACKNOWLEDGEMENTS 2007, 20, (10), 1434-1441. [19] Cameron, M.D.; Wen, B.; Allen, K.E.; Roberts, A.G.; Schuman, Declared none. 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Received: December 16, 2013 Revised: May 14, 2014 Accepted: May 21, 2014