Microsoft word - crt_evans_metabolic activation_14oct03_final
Drug-Protein Adducts: An Industry Perspective on
Minimizing the Potential for Drug Bioactivation in Drug
Discovery and Development
David C. Evans†*, Alan P. Watt‡,
Deborah A. Nicoll-Griffith§ and Thomas A. Baillie††
†Drug Metabolism, Merck & Co., Inc., Rahway, NJ, USA.
‡Medicinal Chemistry, Drug Metabolism Section, Merck Sharp & Dohme, Terlings
Park, United Kingdom
§Medicinal Chemistry, Merck Frosst Center for Therapeutic Research, Kirkland,
††Drug Metabolism, Merck & Co., Inc., West Point, PA, USA
David C. Evans, Ph.D.,Drug Metabolism,Merck & Co.,126 East Lincoln Avenue,Rahway, NJ 07065
Running title: Drug bioactivation and covalent binding to proteins
It is generally accepted that there is neither a well defined nor consistent link between the
formation of drug-protein adducts and organ toxicity. Since the potential does exist,
however, for these processes to be causally related, the general strategy at Merck
Research Laboratories has been to minimize reactive metabolite formation to the extent
possible by appropriate structural modification during the lead optimization stage. This
requires a flexible approach to defining bioactivation issues in a variety of metabolism
vectors, and typically involves the initial use of small molecule trapping agents to define
the potential for bioactivation. At some point, however, there is a requirement to
synthesize a radiolabeled tracer and to undertake covalent binding studies in vitro,
usually in liver microsomal (and sometimes hepatocyte) preparations from preclinical
species and human, and also in vivo, typically in the rat. This Perspective article serves
to provide one pragmatic approach to addressing the issue of bioactivation from an
industry viewpoint based on protocols adopted by Merck Research Laboratories. The
availability of a dedicated Labeled Compound Synthesis group, coupled to a close
working relationship between Drug Metabolism and Medicinal Chemistry, represents a
framework within which this perspective becomes viable; the overall aim being to bring
The concept that toxicities can stem from drug bioactivation in vivo continues to
be problematic for pharmaceutical researchers, inasmuch as while the detection of a
bioactivation process is relatively straightforward, the downstream consequences of this
process remain indeterminable. This is due to the relatively little progress which has
been made in understanding the molecular events which occur following drug
haptenization of a protein. Since experimental tools are available to investigate drug
bioactivation processes, and medicinal chemists are well versed in the art of optimizing
structure-activity relationships, it is natural to assume that this latter skill set also can be
exploited to prepare drugs which do not undergo metabolic activation. This goal has
been the credo at Merck & Co., as one element of attempting to bring safe drugs to
The concept that small organic molecules can undergo bioactivation in vivo, and
that the resulting electrophiles can adduct to biological macromolecules and subsequently
elicit organ toxicity, has its origins in studies performed during the early 1930’s to mid
1950’s. Such investigations included the work of Fieser (1
) who investigated the
hepatotoxicity of polycyclic hydrocarbons, and that of Miller and Miller (2, 3
studied the hepatotoxic effects of p
-dimethylaminoazobenzene in the rat and reported that
aminoazo dyes become tightly bound to the protein constituents of liver tissues.
These early studies laid the foundation for the "Covalent Binding Theory" of
xenobiotic induced liver and lung toxicity, which emerged during the 1970’s and 1980’s
through a series of investigations performed at the National Institutes of Health by
Brodie, Mitchell, Gillette and Boyd (4-8
) who examined the bioactivation of a wide
variety of small organic molecules and correlated this process with organ toxicity. The
molecules investigated in these early studies, and in more recent work, include 4-
), acetaminophen (9
), halothane (10
), vesnarinone (11
), isoniazid (12, 13
), clozapine (15, 16
), and bromobenzene (17-19
). Many of these
investigations shared the common experimental approach of using enzyme inducers and
inhibitors to modulate the extent of bioactivation which, in turn, influenced the degree of
covalent binding and tissue damage observed in vivo; the dogma postulated being that the
processes of bioactivation, covalent binding and tissue damage were intrinsically linked.
The above dogma was challenged by several investigators during the 1980’s. For
example, Tirmenstein and Nelson (20
) reported that despite normalizing the dose level of
acetaminophen (APAP) and its regioisomer, 3′-hydroxyacetanilide, to provide
comparable levels of covalent binding in vivo in the mouse (~ 1.3 - 1.6 nmol drug
equivalents/mg liver homogenate protein), APAP was hepatotoxic, but 3′-
hydroxyacetanilide was not. From findings such as these, it was proposed that it was the
adduction of "critical proteins", namely proteins required for specific cell function and
viability, which was important for organ toxicity. While there are increasing numbers of
reports of the identities of proteins which are modified by reactive drug metabolites (18,
), few have attempted to distinguish "critical" from "non critical" proteins, or have
investigated whether there are species differences in the types of proteins adducted. The
absence of such information has essentially limited our ability to predict a priori
bioactivation, and the accompanying covalent binding of drug-related material to proteins
will, or will not, result in organ toxicity in preclinical species or human. Moreover, even
if a drug candidate that is subject to metabolic activation were to fail to cause organ
damage in a conventional preclinical safety assessment program, the concern remains that
the drug-protein adducts formed in humans may have the potential to act as haptens and
elicit an immune-mediated adverse event (24
). This represents a significant concern for
the pharmaceutical industry since it is generally acknowledged that no animal model
exists for human immune-mediated toxicities. Thus, conventional preclinical safety
studies may fail to identify those drug candidates which have the potential to elicit a
hypersensitivity reaction in humans.
II. Covalent Binding Risk Assessment in Drug Discovery
In light of our inability to predict which reactive metabolites will be toxic, and
which will not, the default position for Merck has been to minimize reactive intermediate
formation to the extent possible by appropriate structure modification during the lead
optimization stage. Initially, efforts are made to identify reactive intermediates by the
use of chemical trapping agents, such as reduced glutathione (25
) or cyanide (26, 27
form stable adducts that are amenable to characterization by liquid chromatography-
tandem mass spectrometry (LC-MS/MS) and/or NMR spectroscopy. These experiments,
normally conducted in liver microsomal preparations, provide valuable indirect
information on the identity of the original electrophile. Parallel efforts may involve the
conduct of covalent binding studies, although these require the early availability of
radiolabeled lead candidate compounds.
Regardless of the specific approach, there needs to be a readiness to identify the
mechanism by which bioactivation occurs and a willingness to abrogate the potential
liability through iterative structural modifications. Indeed, understanding the mechanism
of metabolic activation is critically important if medicinal chemists are to minimize this
process through rational changes in structure. It is these two phases, namely that of
understanding the mechanism of bioactivation and applying chemical intervention to
minimize bioactivation, which highlights the importance of a close working relationship
between Drug Metabolism and Medicinal Chemistry from the point of project inception.
This proactive approach to addressing bioactivation is justified when considering the
issue of patient safety and the economic risks in pursuing drugs which do covalently
modify proteins. These risks are somewhat compounded by the typically low incidence
of idiosyncratic reactions to drugs in the patient population (Table 1) which can result in
safety issues being identified only after the patient population has become relatively large
(Phase III clinical trials and beyond); a time when the economic commitment to drug
development already has been considerable.
For those compounds which possess the requisite characteristics of a development
candidate (high degree of efficacy and potency in animal models, acceptable
pharmacokinetics in preclinical species, appropriate physico-chemical properties, etc), a
radiolabeled analog of the compound (preferably 14C-labeled) is prepared and in vitro
covalent binding experiments are performed in liver preparations from animals and
humans. Hepatic microsomes are employed to assess metabolic activation catalyzed
primarily by oxidative enzymes, while the use of freshly isolated hepatocytes may serve
to reveal metabolic activation processes that depend upon the presence of a full
complement of cellular enzyme systems. Covalent binding studies also are carried out in
intact rats, where the level of adducts to both liver and plasma proteins is determined at
appropriate intervals after administration of a standard oral dose of the test compound
Having determined the level of covalent binding of a drug candidate, the question
then becomes, “How much apparent covalent binding is acceptable in deciding whether
to advance a drug candidate into development?” Merck’s approach to this question has
been to take the levels of covalent adducts typically found in the livers of animals given a
dose of a prototypic hepatotoxin (e.g. APAP, bromobenzene, furosemide or 4-ipomeanol)
(Table 2) associated with the expression of hepatic necrosis (approximately 1 nmol drug
equivalents/mg protein), and to reduce this figure by 20-fold to give a conservative target
‘threshold’ value for in vivo covalent binding of 50 pmol drug equivalents/mg total liver
protein. This value also corresponds to a level of radioactivity that is approximately 10-
times background under normal conditions, and thus provides a suitable dynamic range
for measurement of covalently bound drug-protein adducts. It should be emphasized that
the figure of 50 pmol drug equivalents/mg protein is viewed as a target
upper limit, and
not as an absolute threshold above which a compound would not be advanced into
development, since it is recognized that other factors must be taken into consideration in
arriving at this decision. In this regard, it is important to point out that several drugs
which are known to undergo bioactivation and have associated clinical adverse events
(AEs) are still marketed. This fact provides some initial insight into how we should view
covalent binding data in the context of risk assessment. The AEs for such drugs include
agranulocytosis (amodiaquine & clozapine), hepatotoxicity (diclofenac), hepatic failure
(bicalutamide), and Stevens-Johnson syndrome (lamotrigine). Clearly, in using these
agents, the risks have to be weighed against the benefits, and a number of factors taken
into consideration. In the context of new drug discovery and development, these factors,
which can be regarded as "qualifying considerations" for the purposes of this text (Figure
1), include the intended clinical use of the drug candidate (is it an unmet medical need or
are current therapies inadequate?), the severity of the indication (is the prognosis
disabling or life-threatening?), the dosing regimen (will the drug be used acutely, or
chronically/prophylactically?), and the intended clinical population (will the drug be used
for a pediatric indication where the tolerance for toxicity will be less?).
When assessing covalent binding data in liver microsomes in vitro, the degree to
which drug clearance is dependent on Phase 2 enzymes also should be borne in mind.
For instance, a compound providing levels of covalent binding in liver microsomes of >
50 pmol drug equivalents/mg, but which is metabolized almost exclusively in hepatocyte
preparations or in vivo by Phase 2 enzymes, where covalent binding is < 50 pmol drug
equivalents/mg protein, would still be considered viable. Also worthy of consideration is
the projected therapeutic dose. Uetrecht has reported (28
) that drugs given at a daily dose
of 10 mg or less are rarely, if ever, associated with a significant degree of idiosyncratic
drug reactions. From the foregoing considerations, it is apparent that the decision process
regarding the weight to place on covalent binding data is not digital but multifactorial.
Thus, the propensity for metabolic activation should be viewed as one of several potential
liabilities which need to be taken into account when making programatic decisions on a
As an example of the application of the above criteria, it is instructive to consider
ethinylestradiol (EE), the usual estrogenic component of the oral contraceptive pill.
While the daily oral dose is very low (35 µg), hence satisfying at least one of the risk-
benefit considerations, EE produces high levels of covalent binding in vitro (1.2 nmol
drug equivalents/mg protein following incubation of 250 µM EE for 20 min in rat liver
microsomes) (Table 2). Understanding routes of metabolism (the compound is
eliminated predominantly by sulfation and glucuronidation, with the gut making a
significant contribution to this process) (29, 30
), and the mechanism of bioactivation (in
the absence of cytosolic catechol O-methyl transferase, a reactive o
-quinone will be
formed), an informed decision can be made not to view Phase 1-mediated metabolic
Another practical issue that needs to be taken into account, namely that of
appropriate location of the radiolabel, is perhaps obvious but is critical if false negative
results are to be avoided. This is best exemplified in the case of isoniazid where, had the
primary metabolite acetylisoniazid been labeled in the pyridine moiety (as opposed to the
acetyl group) for covalent binding studies, no radioactivity would have become
associated irreversibly with liver proteins (Table 2). This example highlights the
potential need to undertake covalent binding studies with more than one radiolabeled
form of the compound-of-interest, often prompted by a better understanding of the
metabolic disposition of the drug candidate.
The process of covalent binding risk assessment in drug discovery can be
summarized as attempting to minimize reactive metabolite formation to the extent
possible by appropriate structure modification during the lead optimization stage. Studies
performed in vitro (e.g
. liver microsomes) allow species differences in covalent binding
to be explored and an insight gained into the mechanism of metabolic activation. Studies
performed in vivo (typically in the rat) provide information on the levels of covalent
binding which will occur during evaluation in safety studies, and also provide an
opportunity to assess the degree of metabolic activation in tissues other than the liver.
The decision tree employed at Merck (Figure 1), along with the risk-benefit (qualifying)
considerations, provide a rationale for compound evaluation; target covalent binding
values of < 50 pmol drug equivalents/mg protein both in vitro and in vivo are desirable,
although values in excess of this figure in vitro and/or in vivo may be acceptable under
III. Standardization of Covalent Binding Methods
Since covalent binding data have the potential to influence the fate of a program
lead compound, it is essential that these data become available at an early stage when
there is time to optimize the lead structure or to explore alternative chemical templates.
If covalent binding data from different research sites are to be viewed consistently, it is
imperative that data be derived under standardized conditions and that assay conditions
be validated accordingly. At Merck, several research sites participate in drug discovery
efforts, and therefore an inter-site validation of a covalent binding method, based on the
use of rat and human liver microsomes, was undertaken.
In brief, the method employed involves the incubation of 10 µM drug substrate
with rat or human liver microsomes (RLM, HLM; 1mg protein/ml) (± NADPH) for a 1 hr
incubation period (total final volume of 200 µl), at which point the reaction is stopped
and protein precipitated by the addition of acetone (800 µl). Precipitated protein is
collected using a Brandel cell/membrane harvester and the protein washed with 80%
aqueous methanol (1.5 liter per 96 well plate; 30 ml per filter). Individual filter discs are
punched from the Brandel harvester mat, the protein solubilized, and aliquots taken for
protein assay and determination of radioactive content by liquid scintillation counting.
In addition to evaluating test compounds, two control compounds also are routinely
investigated. The first of these is used by all sites and allows the performance of the
assay in different laboratories, and on different sites, to be monitored. The second
control is program specific, and allows the Project Team to monitor the impact of
Medicinal Chemistry intervention on improving the stability of subsequent lead
compounds towards metabolic activation.
In the context of standardizing methodology for assessing the covalent binding of
drugs to liver microsomal protein in vitro, compounds were selected on the basis of a
well characterized bioactivation process. Knowledge of the enzyme system(s)
responsible for metabolic activation also was desirable. The compounds chosen covered
a wide range of covalent binding values (~50 to >2500 pmol drug equivalents/mg) and
included diclofenac, imipramine, L-746530 and MRL-A (Figure 2). Four Merck sites
were involved in this validation process wherein four compounds were radiolabeled and
rat and human liver microsomes prepared on one site were shipped to the remaining three
sites for evaluation in a standard covalent binding protocol. The percentage coefficient of
variations (CVs) obtained for this assay generally were < 20%, although there were some
exceptions where CVs were as high as 40% (Tables 3 & 4).
Covalent binding studies in pooled hepatocyte preparations using the Brandel
tissue harvester method also have been undertaken (10 µM drug, 2 hr incubation period
with 1 x 106 cells/ml, N = 3 preparations, Brandel extraction). Due to the inherent
variability of covalent binding values originating from the use of individual preparations
of freshly isolated hepatocytes, or even from different batches of pooled cryopreserved
hepatocytes, data from these studies are not necessarily considered to be on the critical
path for decision making purposes. Rather, these data are viewed in the broader context
of better defining the metabolic disposition of the compound, the mechanism of
bioactivation, and the level of covalent binding observed in all experimental systems,
both in vitro and in vivo. For information, however, the mean values (with CVs in
parenthesis) of covalent binding obtained for the compounds under investigation in
pooled (N=5) cryopreserved human hepatocytes were: 6 (17%) pmol drug equivalents/mg
for imipramine, 81 (8%) pmol drug equivalents/mg for MRL-A, 31 (24%) pmol drug
equivalents/mg for diclofenac, and ~359 (15%) pmol drug equivalents/mg for L-746530.
Studies performed in vivo also have been standardized. Rats were dosed orally at
20 mg/kg and animals exsanguinated at 2, 6 and 24 hr (N=3 animals per time point), their
livers removed, and the samples stored frozen until analysis. This in vivo assay may, in
addition, be performed at a lower dose of 2 mg/kg to evaluate the impact of dose on the
extent of metabolic activation. For guidance, approximately 20 µCi of 14C labeled
compound or 40 µCi of 3H labeled compound typically is dosed to a 300 g rat. The dose
levels of 2 and 20 mg/kg were chosen arbitrarily to reflect a typical “low” and “high”
dose. It should be noted that the use of a tritium tracer carries with it the potential to
underestimate levels of covalent binding, either through loss by exchange (generation of
tritiated water) or through metabolism (a concern which also applies, albeit to a lesser
extent, to a 14C tracer). Metabolism studies usually provide insights into the potential for
such tracer loss, and investigators need to be prepared to undertake covalent binding and
drug disposition studies with an alternative tracer, should the need arise.
It should be clear also from the foregoing discussion that our approach to
assessing metabolic activation makes no effort to scale values of covalent binding
determined in vitro (liver microsomes or hepatocytes) to those determined in vivo.
Rather, our experiments are designed to highlight the potential for metabolic activation in
several metabolic systems so that a structure based resolution of this finding can be
instigated. In this regard, our choice of relatively high drug concentrations, 10 µM for in
vitro and up to 20 mg/kg for in vivo studies, reflect our desire to balance maximizing
analytical sensitivity with standardizing protocols.
IV Characteristics of Compounds Investigated
Diclofenac typically is prescribed to patients at 50 mg tid and can undergo both
CYP3A4- and CYP2C9-mediated bioactivation processes (31-33
). Following incubation
of 1 mM diclofenac with human liver microsomes, protein adduct formation was reported
to be mediated by CYP3A4 (33
) since the initial product of CYP3A4 catalysis, 5-
hydroxydiclofenac, is subject to further oxidation to a reactive p
intermediate. In support of this contention, an N-acetylcysteine adduct of 5-
hydroxydiclofenac was identified in urine from human subjects dosed with diclofenac
). On the other hand, Tang et al. (32
) have reported that an isomeric p
benzoquinoneimine, derived from 4′-hydroxydiclofenac, is formed by CYP2C9 in vitro at
more clinically relevant concentrations (< 50 µM), whereas the 5-hydroxylation of
diclofenac, in agreement with Shen et al. (33
), becomes a significant pathway in vitro at
higher concentrations (> 100 µM). Indeed, CYP2C9 has been shown to mediate the
formation of the putative quinoneimine which can be trapped with reduced glutathione to
form 4′-hydroxy-3′-(glutathion-S-yl)diclofenac (32
). Bougie et al
) have also shown
that the product of CYP2C9-mediated oxidation and acyl glucuronidation, namely the 4′-
hydroxydiclofenac acyl glucuronide metabolite, had an apparent role in diclofenac-
Imipramine is used in the dosage range of 75 to 225 mg per day and has been
associated with abnormalities in liver function which tend to occur during the second
month of treatment (31, 36, 37
). Imipramine 2-hydroxylation is a major route of
metabolic clearance in humans (31
), and is catalyzed almost exclusively by CYP2D6
). By analogy with rat (39
), it is proposed that imipramine undergoes metabolic
activation in humans to an arene oxide which covalently modifies proteins.
L-746530 undergoes CYP3A-mediated bioactivation (Section V). Following
oxidation on the dioxobicyclo portion of the molecule at the methylene position alpha to
the ring oxygen, an aldehyde is formed which can be trapped in vitro using
semicarbazide. Alternatively, the aldehyde can form a Schiff base adduct to proteins in
vitro resulting in levels of covalent binding of ~ 2 nmol drug equivalents/mg (Tables 3 &
4). The furan moiety of L-746530 also can undergo metabolic activation, as described in
Section V for its close structural analog, L-739010.
The levels of covalent binding following incubation of 10 µM diclofenac with rat
or human liver microsomes (~290 and ~57 pmol drug equivalents/mg) highlight a
quantitative species differences, binding being higher in rat liver microsomes. Had
diclofenac been a development candidate under the current Merck paradigm, efforts
would have been made to explain this species difference in bioactivation, with the goal of
more effectively assessing the potential risk to humans. The covalent binding values
obtained for imipramine (~ 460 and ~130 pmol drug equivalents/mg in rat and human
liver microsomes) would have warranted a similar discussion. The values for covalent
binding of L-746530 and MRL-A (> 1500 pmol drug equivalents/mg) are sufficiently
high that it is unlikely that these compounds would be advanced today as drug
V. Biomarkers of Metabolic Activation
In some instances, reactive intermediates can form adducts with small molecule
trapping agents. Characterization of these adducts using LC-MS/MS and NMR
techniques can provide indirect information on the structure of the reactive species from
which they are derived, thereby defining a potential bioactivation mechanism and hence a
rationale on which to base a chemical intervention strategy. The use of trapping agents
also provides a means by which a relatively large number of compounds can be evaluated
rapidly, thereby allowing prioritization of compounds for radiolabeling prior to classical
determination of covalent binding. A caveat to using a trapping agent is that there
probably is no single small molecule which can serve as a universal surrogate for a
complex protein macromolecule; nevertheless, several traps have found widespread
utility and are discussed below. Also, certain reactive metabolites are so reactive that
they are presumed to be unable to escape from their site of formation, or react rapidly
with water of the medium, and therefore are not efficiently trapped. Products of
hydrolysis can, however, yield information about their precursor reactive intermediates.
A good example is the trifluoroacetic acid metabolite of halothane which is excreted in
the urine of patients receiving this agent and is formed, in part, by the hydrolysis of the
reactive trifluoroacyl halide intermediate (31
Reduced glutathione (GSH), an abundant physiological nucleophile by virtue of
its cysteine sulfhydryl group, captures reactive xenobiotic metabolites to form S
substituted adducts. Once GSH stores are depleted, however, cellular proteins may be
adducted, and it is believed that in certain cases cellular function is compromised and
organ toxicity can ensue (40
). Hence, GSH serves as a natural trapping agent for
chemically reactive metabolites and has been used extensively in vitro to study a broad
range of reactive intermediates including quinoneimines (acetaminophen), nitrenium ions
(clozapine), arene oxides (carbamazepine), quinones (estrogens), imine methides (3-
methylindole), and Michael acceptors (valproic acid metabolites), for which references
Typically, in vitro experiments are conducted at 0.2 - 5 mM GSH concentration.
Glutathione adducts can be analyzed by LC-MS/MS using either the full scan mode to
search for anticipated conjugates, or by constant neutral loss scanning for 129 Da (γ-
glutamyl moiety) to detect all GSH-related species (Figure 3). However, it should be
recognized that some GSH adducts are not stable and are subject to chemical
degradation/rearrangement or enzymatic degradation (e.g., catalyzed by γ-
glutamyltranspeptidase; SectionVI A), thereby escaping detection. In certain cases, N
acetyl cysteine (NAC) can be used in place of GSH as a nucleophilic trap, although this
agent may be less efficient than GSH if the conjugation reaction in question is catalyzed
(even in part) by glutathione-S-transferase enzymes.
B. Potassium Cyanide
The cyanide anion (CN-) is a “hard” nucleophile that can be used to trap certain
electrophilic drug metabolites. An example is presented in Figure 4, in which 1 mM
KCN (a mixture of CN and 13C15N at 1:1 ratio) was used as the trapping agent. The
detection of cyano adducts by LC-MS was greatly facilitated by the presence of
prominent isotopic “doublets” that differed in mass by 2 Da (mono-adducts) or 4 Da (bis-
adducts); further, the MS/MS spectra of these adducts were characterized by a neutral
loss of 27/29 Da (HCN/H13C15N). This technique has its origins in the work of Gorrod
and co-workers (26, 27
), and has been highlighted recently in a comprehensive review of
bioactivation reactions of nitrogen-containing xenobiotics (41
C. Methoxylamine and Semicarbazide
Both methoxylamine and semicarbazide can form a Schiff base with aldehydes, a
process mimicking reactions between aldehyde metabolites with lysine residues on
proteins. Typical conditions require the addition of 5 mM of either trapping agent to the
incubation mixture followed by LC-MS/MS analysis. This approach was taken for both
L-739010 and L-746530 (42, 43
) and is presented in Figure 5.
VI. Bioactivation - ‘Structure Alerts’ Past and Present
Over the last few decades, considerable literature has accumulated on the subject
of chemical induced toxicity, a representative selection of which is summarized in Table
5. The chemicals listed share the common feature of being able to form a reactive
intermediate capable of alkylating proteins. Table 5 also contains two recent examples
from Merck Research Laboratories (L-754394 and L-739010) where metabolic activation
issues were identified and simple modifications made to the parent structures to produce
drug candidates with markedly reduced bioactivation potential.
A. Piperazine Bioactivation
MRL-A, a 3-acyl-N1-methylpiperazine derivative (Figure 6), was found to
undergo CYP3A4-mediated piperazine bioactivation (44
). A mechanism for this process
was proposed whereby formation of the end-product metabolites M1 and M2 involved a
six electron oxidation of the piperazine ring, attack by GSH, and hydrolysis of the
glutamic acid residue to afford a cysteinylglycine conjugate of the piperazinone. Attack
(aminolysis) by the cysteinyl amine moiety resulted in opening of the piperazinone ring
leading to a thiazolidine thioaminal intermediate which, in turn, underwent ring closure to
the imidazoline products observed. The structures of adducts M1 and M2, elucidated by
1H-NMR and high resolution mass spectrometry, were consistent with this sequence of
events. In keeping with the proposed mechanism, it was found that while MRL-A
alkylated proteins extensively, simple alkyl substitutions (methyl, isopropyl) alpha to the
N1-methyl functionality afforded derivatives which did not undergo metabolic activation
and therefore were more attractive as drug candidates.
B. Pyrazinone Bioactivation
When a substituted pyrazinone derivative (MRL-B, Figure 7) was incubated with
liver microsomal preparations from rats and humans fortified with GSH, two isomeric
GSH conjugates (GSH-1 and GSH-2) were detected using LC-MS/MS (45
benzylic alcohol, resulting from hydroxylation of MRL-B at the 6-methyl position, also
was observed. The proposed origins of GSH-1 and GSH-2 pointed to two distinct,
cytochrome P-450-mediated metabolic activation processes. Thus, it was proposed that
MRL-B had undergone a two-electron oxidation, either directly or via
dehydration of the
hydroxymethyl metabolite, to generate an electrophilic imine-methide. Capture of this
intermediate by GSH afforded adduct GSH-1. In the competing activation process, it was
proposed that oxidative attack of the pyrazinone ring generated an unstable epoxide
intermediate, which was attacked regiospecifically by GSH to afford a cyclic
carbinolamide. Tautomeric ring-opening of the latter species to the acyclic methyl
ketone, followed by syn/anti
isomerization of this substituted amidine, would lead to the
formation of a new ring system which, following elimination of the elements of water,
would generate the stable imidazole derivative, GSH-2. Subsequent studies performed in
the rat in vivo using radiolabeled MRL-B indicated that this compound gave rise to
appreciable levels of covalent adducts to plasma and liver proteins. However,
replacement of the 6-methyl group by chlorine, which prevented formation of the imine
methide intermediate and rendered this heterocycle less susceptible to epoxidation, led to
an analog whose potential for reactive metabolite formation and protein alkylation was
C. Bioactivation of a Series of Aryloxy Derivatives
In one of our drug discovery programs at Merck, sites of metabolic activation
within a new structural series were identified rapidly using online LC-MSn on an ion trap
mass spectrometer (46
). Following incubation of MRL-C (Table 6) with rat or human
liver microsomes, reactive intermediates were trapped as their corresponding glutathione
conjugates. Mass spectral characterization of these conjugates allowed potential sites and
mechanisms of bioactivation for MRL-C to be proposed. These studies, coupled to
informed structural modification of the series to block sites susceptible to metabolic
activation, resulted in a significant reduction in the propensity of this compound class to
label liver microsomal proteins in vitro (Table 6).
One strategy to reduce metabolic activation of MRL-C was to explore aromatic
fluorine substitution as a means of blocking sites of oxidative metabolism, the aim being
to take advantage of the electron-withdrawing effects of fluorine to reduce the π-electron
density of the aromatic ring and hence render this moiety less susceptible to cytochrome
P-450 catalyzed metabolism (47, 48
). In addition, the carbon-fluorine bond is stronger
than the corresponding carbon-hydrogen bond, and thus is less prone to oxidative
cleavage. Unfortunately, fluorinated derivatives such as MRL-D (Table 6) all underwent
extensive metabolic activation that was accompanied by oxidative dehalogenation and
formation of GSH adducts. Therefore, an alternative strategy was pursued whereby a
pyridine ring was incorporated as an isosteric replacement for the phenyl ring; this
modification resulted in reduced levels of metabolic activation of MRL-E. Despite this
improvement, MRL-E remained sub-optimal and provided levels of covalent binding
well in excess of the target threshold value of 50 pmol drug equivalents/mg. Further
improvements were made via chlorination (MRL-F) or trifluoromethylation (MRL-G) of
the pyridine, the latter substitution resulting in a compound which failed to produce GSH
adducts in either rat or human liver microsomal incubations. Furthermore, the levels of
covalent binding to liver microsomal protein determined for MRL-G were significantly
lower than those obtained in any other members of the series (Table 6). Further structural
refinements in other parts of the molecule (data not shown) provided levels of covalent
binding which were <50 pmol drug equivalents/mg in both rat and human liver
microsomal preparations, while retaining desirable pharmacokinetic and
VII. Concluding remarks
Despite 70 years of research, it is still not possible to accurately predict the
potential for toxicity of a compound which has been shown to undergo metabolic
activation. However, considerable advances have been made in the areas of analytical
methodology (mass spectrometry and NMR spectroscopy) and labeled compound
synthesis. As a result, it has become easier to identify reactive intermediates, by using
either small molecule trapping agents or by undertaking covalent binding studies using
radiolabeled compounds, and these approaches have now become a routine part of
candidate drug evaluation at Merck Research Laboratories. Importantly, these
investigations are undertaken at an early stage, during the discovery phase, when a
mechanistic understanding of a bioactivation problem enables Medicinal Chemistry to
provide a structure-based solution. Clearly there is a need to standardize methods for
evaluating the potential for a drug candidate to under-go bioactivation in order to ensure
consistency of data and uniformity in the decision making process. Future advances in
the areas of immunology, genetics and proteomics may well provide a more coherent
relationship between the characteristics of a drug-protein adduct and a toxicity outcome.
Indeed, an important problem to address in the field of toxicology has been the
characterization of protein targets of reactive electrophiles; in this regard, the work of
Badghisi and Liebler offers new LC-MS/MS / bioinformatic based approaches to
mapping protein modifications at the level of amino acid sequence and should prove of
value in such studies (49
). Our current practice of stabilizing drug candidates against
metabolic activation hopefully will contribute to bringing equally effective, but safer
The authors would like to thank Dr. Maria Beconi, Mr. Stephen Day, Ms. Ann
Mao, Ms. Rebecca White, and Dr. Timothy Schulz-Utermohl for their contributions to
the global covalent binding assay development and validation project at Merck Research
Laboratories. Our thanks go also to Drs. Ronald Franklin, Ralph Stearns, Wei Tang,
Sanjeev Kumar, and Mr. Randy Miller, for valuable discussions during the preparation of
this manuscript. For the preparation of radiolabeled compounds our thanks go to Drs.
David Melillo, Dennis Dean, Matthew Braun, Frank Tang, Chad Elmore, Eric Soli, Allen
Jones, Daniel Dubé and Mr. Wensheng Liu.
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Electrospray ionization mass spectrometric analysis of intact cytochrome P450:
Identification of tienilic acid adducts to P450 2C9. Biochemistry 38
Table 1 Incidence of clinical toxicity for drugs withdrawn from use or are still marketed
Marketed / Withdrawn
Table 3 Covalent binding of 10 µM imipramine, MRL-A, diclofenac, and L-746530following incubation with rat liver microsomes
Covalent binding, pmol drug equiv/mg proteina
a Values are means (N=3) with %CV in parenthesis.
Table 4 Covalent binding of 10 µM imipramine, MRL-A, diclofenac, and L-746530following incubation with human liver microsomes
Covalent binding, pmol drug equiv/mg proteina
a Values are means (N=3) with %CV in parenthesis.
Table 5 Examples of compounds with toxicities which are known to undergo metabolicactivation
Aryl oxidation to either an epoxide or a quinone
Drug induced hypersensitivity, teratogenic.
Lung, but covalent binding higher in liver and kidney.
Carcinogenicity (Breast, liver, endometrial, kidney)
Jaundice accompanied by elevated liver enzymes
Furan epoxidation, ring opening to yield an aldehyde
Formation of an acyl glucuronide conjugate
Hepatotoxicity. Withdrawn in 1998 – 6 deaths
Hepatotoxic in humans following N-acetylation and
Agranulocytosis - nitrenium ion implicated
Irreversible binding of radioactivity to human and rat liver microsomal
protein upon incubation with a series of tritiated analogs (MRL-C though MRL-G) at a10 µM concentration for 1 hr in the presence of an NADPH-regenerating system. Theirreversible binding was assessed both in the absence and presence of 5 mM GSH.
Covalent binding to liver microsomal protein
a Parallel experiments demonstrated that covalent binding of radioactivity to microsomal proteinat time zero and in the absence of an NADPH-regenerating system was <5 pmol drugequivalents/mg protein incubation in all cases.
b For all compounds, the tritium label was incorporated at the same position within the functional
Decision tree for assessing the suitability of lead compounds for
development based on metabolic activation considerations. (As indicated
in the text, a liability with regard to metabolic activation is viewed as only
one of a number of criteria in the selection process).
Compounds used for the inter-site evaluation of covalent binding in vitro
Proposed mechanism of bioactivation of acetaminophen and tapping of the
electrophilic intermediate by glutathione. The origin of the characteristic
neutral loss of 129 Da upon collisional activation of the MH+ species of
the glutathione conjugate in a tandem mass spectrometer is as indicated.
Use of cyanide to trap an iminium ion intermediate.
Bioactivation of L-739010 or L-746530 (Fig. 2) and trapping of an
aldehyde intermediate using (A) semicarbazide, and (B) methoxylamine.
Novel bioactivation of a 3-acyl-N1-methyl piperazine derivative. The
structures shown in brackets represent proposed intermediates which were
Novel bioactivation of a substituted pyrazinone derivative (45
shown in brackets represent proposed intermediates which were not
In vitro: Human and Rat LM (10 µM, 1 hr) and/or
In vivo: Rat (20 mg/kg P.O.; 2, 6, 24 hr, liver & plasma)
½ Chemical tractability of structural series?
- What potential exists to modify structure?- Evidence of informed chemical intervention?- Has metabolic activation been minimized relative to the preceding compound(s)?
½ Is the prognosis disabling or life-threatening?
½ Is the anticipated clinical daily dose < 10 mg?
½ Are the metabolic clearance routes predominantly non-Phase 1?
- Will the drug will be used chronically/prophylactically?
½ What is the intended target population?
- Is the drug intended for a pediatric indication?
Feeling better – Lifestyle management for chronic mental disorders In this module we have learned about three risk factors associated with poor physical health: overweight, lack of physical activity and smoking. All three factors are more common in patients with chronic mental disorders than in the general population and may be associated with a tangible reduction of life expectancy.
Research Network Social Epidemiology, Population Health and Violence (SEPHV) Research Network Description The SEPHV The research network (group) Social Epidemiology, Population Health and Violence (SEPHV) aims at advancing research on the determinants of health inequalities and determinants of interpersonal violence globally. In addition it aims to promote research on int