Acta Pharmaceutica Sinica B 2018;8(5):721–732

Chinese Pharmaceutical Association

Institute of Materia Medica, Chinese Academy of Medical Sciences

Acta Pharmaceutica Sinica B

www.elsevier.com/locate/apsb
www.sciencedirect.com

REVIEW

Drug metabolism in drug discovery

and development
Zhoupeng Zhanga,n, Wei Tangb

a
Department of Pharmacokinetics, Pharmacodynamics and Drug Metabolism, Merck Sharp & Dohme Corp., West Point, PA 19486, USA
b
Shanghai ChemPartners, Shanghai 201203, China

Received 27 February 2018; received in revised form 25 March 2018; accepted 2 April 2018

KEY WORDS Abstract Drug metabolism as a discipline plays an important role in drug discovery and development
Bioactivation;
and the effects of drug metabolism on pharmacokinetics (PK), pharmacodynamics (PD), and safety should
Drug discovery and be carefully considered. This communication provides an overview of common strategies in the area of
development; drug metabolism for improving PK/PD and safety profiles of drug candidates; these include, but are not
Drug metabolism; limited to, collaboration with medicinal chemists on structure–activity relationships (SAR) to overcome
Metabolite; high clearance, using deuterium replacement to further optimize a lead, prodrug approaches to circumvent
Pharmacokinetics; formulation and delivery difficulties, and addressing issues such as species differences in metabolism,
Pharmacodynamics; drug–drug interactions (DDI) and formation of reactive metabolites.
Safety;
Toxicity & 2018 Chinese Pharmaceutical Association and Institute of Materia Medica, Chinese Academy of Medical
Sciences. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND
license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

n
Corresponding author. Tel.: +1 215 652 6255.
E-mail address: [email protected] (Zhoupeng Zhang).
Peer review under responsibility of Institute of Materia Medica, Chinese Academy of Medical Sciences and Chinese Pharmaceutical Association.

https://doi.org/10.1016/j.apsb.2018.04.003
2211-3835 & 2018 Chinese Pharmaceutical Association and Institute of Materia Medica, Chinese Academy of Medical Sciences. Production and hosting by
Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
722 Zhoupeng Zhang, Wei Tang

1. Introduction particular functional group (soft spot) in metabolizing enzyme

systems. One of common approaches to address the metabolic soft
Drug discovery and development is a time-consuming and costly spot issue is to use bioisosteres to replace those identified soft
process. Based on the study by the Tufts Center for the Study of spots. Bioisosteres are substituents or groups that have chemical or
Drug Development (2016)1, on average, it takes more than 10 physical similarities and related molecular shapes and may
years and over $2.6 billion to develop a new drug. One of the produce roughly similar biological properties7. For example, in
biggest challenges for the pharmaceutical research community is some cases, when a benzylic methyl group is identified as a
therefore to execute an optimal process for drug discovery metabolic soft spot, a fluorine or a chlorine atom, or a -CF3 group,
and development. Rational drug design represents an approach could be used to replace the benzylic methyl group.
to expedite such a process with efficiency as one of the In cases of structure–metabolism relationship studies, blocking
primary objectives, combining the latest science and technology a metabolic soft spot is one of the approaches to lower intrinsic
to advance medicines rapidly from laboratory bench side to clearance which may lead to lower total clearance and, assuming
hospital bed side. no change in volume of distribution, longer half-life for modified
The disposition of a drug in the body involves absorption, molecules. For example, zileuton (1, Fig. 1) is a 5-lipoxygenase
distribution, metabolism, and excretion (ADME). ADME is an (5-LO) inhibitor used for the maintenance treatment of asthma in
important component in the drug design process, which studies the human. Zileuton showed half-lives of 0.4 h in cynomolgus
fate of a drug molecule after administration. It is a complex monkey, and 2.4 h in human in vivo8. The major metabolism
process involving transporters and metabolizing enzymes with pathway for zileuton in cynomolgus monkey and human is
physiological consequences on pharmacological and toxicological glucuronidation at the N-hydroxyurea moiety to afford metabolite
effects, and can play a major role in drug design for identifying 2 (Fig. 1) with subsequent urinary excretion9. Structure–activity
better drug molecules in a more efficient way. Metabolism of relationship (SAR) studies indicated that the N-hydroxyurea
drugs in the body is a complex biotransformation process where portion is a pharmacophore required for activity. Thus, structural
drugs are structurally modified to different molecules (metabolites) modification on zileuton to minimize the glucuronidation could
by various metabolizing enzymes. Studies on drug metabolism are only be focused on the linker and the benzothiophene portions of
key processes to optimize lead compounds for optimal PK/PD zileuton (3, Fig. 1). Compound 4, which resulted from the removal
properties, to identify new chemical entities based on the finding of the methyl group from the linkage group of zileuton showed a
of active metabolites, to minimize potential safety liabilities due to 6-fold increase in the intrinsic clearance (Clint) in cynomolgus
formation of reactive or toxic metabolites, and to compare monkey liver microsomes as well as a 2-fold decrease in the half-
preclinical metabolism in animals with humans for ensuring life and 4-fold increase in plasma clearance in cynomolgus
potential adequate coverage of human metabolites in animals monkey in vivo8. This suggests that the steric hindrance of the
and for supporting human dose prediction, etc.2 This review neighboring methyl group from the linker of zileuton indeed
focuses on the study of drug metabolism as a discipline for its diminished the glucuronidation at the N-hydroxyurea portion. SAR
roles in optimizing pharmacokinetics (PK), pharmacodynamics studies on the linker portion indicated that compounds with the
(PD), and safety profiles of drug candidates in drug discovery and acetylene linkage generally had lower rates of glucuronidation in
development. The impact of protein binding and transporter on PK cynomolgus monkey liver microsomes as well as longer half-lives
and PD properties of drug candidates are beyond the scope of this and lower plasma clearances in cynomolgus monkey in vivo8. SAR
review. studies on the benzothiophene portion indicated that the use of a
simple thiophene ring in place of benzothiophene portion could
significantly reduce the glucuronidation rate8. Finally, ABT-761
2. Improving PK and PD properties (5, Fig. 1) with a single furan ring and an acetylene linker was
identified as a second generation 5-LO inhibitor which showed
2.1. Metabolic soft spot 412-fold and 429-fold lower rate of glucuronidation in cyno-
molgus monkey and human liver microsomes than that of zileuton,
Drugs need to reach the site(s) of action to elicit their pharmaco- respectively. Accordingly, ABT-761 showed 440-fold and 429-
logical effects after administration into the body. However, if fold longer half lives in cynomolgus monkey and human in vivo
drugs show inferior PK properties, e.g., high clearance, short half- than that of zileuton, respectively8. Consequently, ABT-761
life (t1/2) and low bioavailability following oral dosing, it is likely showed a significant increase in efficacy relative to zileuton at
that their PD effects will be sub-optimal. In vitro metabolism the same dose10. Thanks to the increased potency and improved
studies in human and animal tissue (e.g., liver) preparations and/or PK properties, ABT-761 requires only once-daily dosing in
in vivo metabolism studies in animals are useful approaches to human, compared to the multiple daily dosing regimen for
identify major metabolism pathways (“soft spots”) of drugs3. It is zileuton10. This study demonstrated that blocking a metabolism
known that the benzylic C–H bond, the allylic methyl and the soft spot indeed could improve PK properties of a new chemical
O-, N-, S-methyl groups are the mostly preferred metabolic soft entity (NCE) while maintaining the same or producing better
spots when these groups are not sterically hindered, subjecting to pharmacological activity.
P450 mediated metabolism4. Because of the nature of the enzyme- In studies of 4-substituted methoxybenzoyl-aryl-thiozoles as
catalyzed hydroxylation reactions, the chemo- and regiospecicity novel anti-cancer agents, 4-(3,4,5-trimethoxybenzoyl)-2-phe-
of substrate oxidation as well as the rate of metabolism is largely nylthiazole SMART-H (6, Fig. 1) was identified as a lead with
determined by the intrinsic reactivity of the substrate sites that are potent inhibition activity against tubulin polymerization and
accessible to the ferryl oxidizing species in the P450-substrate cancer cell growth11. However, SMART-H showed high meta-
complex5,6. Thus, the tendency to be metabolic soft spots will be bolic instability in human, dog, rat and mouse liver microsomes
depending on the intrinsic reactivity of the functional groups and with in vitro half-lives ranging from o5 to 30 min11. Metabolite
the substrate specificity of the particular molecule bearing this profiling of SMART-H in human liver microsomes indicated
Drug metabolism in drug discovery and development 723

Figure 1 Structures of zileuton (1), SMART-H (6), 7-ethoxycoumarin (11) and their analogs or metabolites.

that the main metabolism pathway was the reduction of the

ketone functional group. The O-demethylation of the methoxy
groups of SMART-H was the second most prevalent metabo-
lism pathway. Thus, the ketone and the methoxy groups of
SMART-H were considered as metabolic soft spots. When the
ketone group was replaced with an oxime group for SMART-
173A (7) and a hydrazide group for SMART-176A (8, Fig. 1),
SMART-173A and SMART-176A demonstrated an increase in
metabolic stability in human liver microsomes by 3-fold and
2-fold, respectively. SMART-173A partially retained the antic-
Figure 2 Schematic presentation for oxidation of the C-H bond at
ancer activity with the average IC50 value of 143 nmol/L against
the α-carbon of the ethoxy group of 7-ethoxycoumarin (A) and for
four prostate tumor cell lines (compared to IC50 of 44 nmol/L of
oxidation of the C-H bond at the 6-position of [D2]-7-ethoxycoumarin
SMART-H), while SMART-176A significantly lost the antic-
(B) at the active site of a cytochrome P450 enzyme.
ancer activity with the average IC50 value of 1250 nmol/L11.
With the removal of the most labile ketone functional group of
SMART-H, SMART-329 (9, Fig. 1) increased the metabolic an initial binding of a drug to the active site of the enzyme,
stability by 2-fold11. However, SMART-329 completely lost followed by catalytic turnover of a drug to a metabolite through
anticancer activity, suggesting that the ketone functional group a so called P450 catalytic cycle12. Whereas the structure–
is critical for anticancer activity. When the second soft spot metabolism relationship of drugs involving CYPs is complex
(three methoxy groups) of SMART-H was replaced with and not completely understood, it is generally accepted that
fluorine atoms, the resulting SMART-213 (10, Fig. 1) showed major drug-metabolizing CYP enzymes, e.g., CYP3A, have a
little change in metabolic stability and no anticancer activity11. large active site and prefer lipophilic substrates. The binding
This suggests that the methoxy groups might also contribute to and orientation of a substrate are often determined by hydro-
anticancer activity. Thus, it is possible that structural modifica- phobic and steric interactions of the substrate with the specific
tion on or around the metabolic soft spots of a lead compound amino acids at the active site of a CYP enzyme, although
could negatively affect the biological activity. infrequently substrates interact with CYP via hydrogen bonding
or ionic interactions5,12–16. These hydrophobic and steric inter-
actions mainly depend on the lipophilicity (e.g, logP) and steric
2.2. Metabolic switching and deuterium replacement structures of the substrates17,18. Subsequent catalytic turnover
will then oxidize a C-H bond of the substrate to a C-OH bond to
Many small molecule drugs are metabolized by cytochrome give a specific metabolite. One of the common approaches to
P450 (CYP) enzymes in the body. CYPs, predominantly slow down or block the metabolism on a specific site of a
residing in the endoplasmic reticulum of hepatocytes, are a molecule is to use a substituent, (e.g., a bioisostere, a bulky
class of enzymes which catalyze a range of oxidative and group, a halogen or a deuterium atom) to replace the H-atom of
reductive biotransformation, including carbon hydroxylation, the C-H bond of the molecule or to place a bulky group in a
heteroatom oxidation, bond oxidation, hydrocarbon desatura- neighboring position to reduce or block the accessibility of the
tion, and halocarbon dehalogenation, etc.4 CYP-catalyzed iron-bound oxidizing species of the P450 enzyme. Once the
biotransformation of drugs to metabolites generally involves molecule is bound within the active site, the specific site of
724 Zhoupeng Zhang, Wei Tang

Figure 3 Structures of compound 16, tetrabenazine (18) and their analogs or metabolites.

metabolism in the substrate where the structural modification an approach to utilize the so-called “isotope effect” when
occurs, is then largely determined by the intrinsic reactivity of designing new bioactive molecules. Because the carbon-deuter-
the sites on the molecule that are accessible to the iron-bound ium bond is more difficult to break than the carbon-hydrogen
oxidizing species in the active site of the CYP. Due to the bond, the deuterated molecule may have reduced metabolism on
nature of the active site(s) of CYP enzymes (e.g., CYP 3A4) the carbon atom where the deuterium atom is attached, poten-
with a large space binding pocket, blockage of the metabolically tially lowering the in vitro and in vivo clearance or altering the
labile site (or soft spot) of a lead molecule using one of the metabolism. This was exemplified by the effort to identify new
above mentioned substituents may result in a change of the mGlu3-selective and CNS-penetrant negative allosteric modula-
binding and orientation of the newly synthesized molecule, tors20. In SAR studies, compound 16 (Fig. 3) was identified as a
switching the original metabolism site to a different C-H bond lead with good biological activity. However, compound 16 was
and resulting in the formation of other metabolite(s) to a greater metabolically unstable in human and rat liver microsomes with a
extent, as compared to that of the lead compound. Because CYP calculated hepatic clearance of 18.9 mL/min/kg in human and
enzymes may have multiple binding sites, it is also possible that 54.1 mL/min/kg in rats. The major metabolic pathway (soft spot)
the blockage of a soft spot of a lead molecule via structural for 16 was the CYP-mediated O-demethylation. This soft spot
modification may cause the binding of the new molecule to the issue could not be fixed through traditional electronic or steric
alternative binding site of the metabolizing enzyme, resulting in perturbations due to extremely shallow allosteric ligand SAR20.
metabolism on a different site of new molecule. This metabolic However, by replacing the hydrogen atoms of the -OCH3 group
switching phenomenon is not uncommon. For example, of 16 with the deuterium atoms (17, Fig. 3), the calculated
7-ethoxycoumarin (11, Fig. 1) was extensively metabolized to hepatic clearance in rats decreased from 54.1 mL/min/kg for
7-hydroxycoumarin (12) with a minor metabolite 6-hydroxy-7- compound 16 to 35.9 mL/min/kg for 17. Correspondingly,
ethoxycoumarin (13) through a CYP1A1-mediated oxidations the plasma clearance in rats after IV dosing decreased from
in rat19. Mechanistically, the C-H bond at the α-carbon of the 5.2 mL/min/kg for 16 to 2.9 mL/min/kg for 1720. The observed
ethoxy group of 7-ethoxycoumarin 11 is initially oxidized to reduced deuterium effect might be due to the possible metabolic
give an unstable hemiacetal intermediate, which subsequently switching for 17. This deuterium replacement approach resulted
loses an acetaldehyde to give the metabolite 7-hydroxycoumarin in the discovery of a new compound which warranted further
(12). [D2]-7-Ethoxycoumarin (14), resulting from the replace- in vitro and in vivo biological testing20.
ment of two hydrogen atoms with deuterium atoms at the Another example involves tetrabenazine (18, Fig. 3), where its
α-carbon of the ethoxy group of 7-ethoxycoumarin 11, showed deuterated version represents a new drug form with much
a 2-fold reduction of the formation of 7-hydroxycoumarin (12) improved safety profiles. Tetrabenazine is a marketed drug for
in rat liver microsomes. However, [D2]-7-ethoxycoumarin (14) treatment of chorea associated with Huntington's disease and is
showed a 5-fold increase in the formation of 6-hydroxy-[D2]-7- extensively metabolized to form an active metabolite 19 (Fig. 3),
ethoxycoumarinin (15) in rat liver microsomes, resulting in which then is further metabolized to active metabolites 20 and 21
almost no change of total metabolic stability, compared to (Fig. 3) by the polymorphic enzyme CYP2D621. However, the
7-ethoxycoumarin (11)19. This result suggests that the oxidation observed clinical adverse effects, e.g., sedation, somnolence,
of the C-H bond at the α-carbon of the ethoxy group of fatigue and insomnia might be associated with not only the high
7-ethoxycoumarin 11 (Fig. 2A) was switched to the oxidation Cmax of the drug itself but also with the variable levels of active
of the C-H bond at 6 position of [D2]-7-ethoxycoumarin 14 metabolites in patients due to CYP2D6 polymorphism21. Thus, it
(Fig. 2B). Thus, addressing the metabolically labile sites (or soft is required to individualize and slowly titrate dosage over several
spots) of a lead molecule is a systematic SAR process, where weeks to identify a dose that reduces chorea while still being well
multiple factors, including lipophilicity, stereochemistry, and tolerated. SD-809 (22, Fig. 3) from Auspex Pharmaceuticals is a
intrinsic reactivity should be considered in the drug design deuterated version of tetrabenazine with an attempt to alter the
process. O-demethylation reaction22. SD-809 showed a longer half-life and
Replacement of hydrogen atoms with deuterium atoms to higher exposure with a slight Cmax increase relative to tetrabena-
block a metabolic soft spot or to alter the route of metabolism is zine in humans22. This may allow patients to take a much lower
Drug metabolism in drug discovery and development 725

are probably the enzymes responsible for the final hydrolysis

of the monoester intermediate 25 to the active drug tenofovir
26 (Scheme 1) 26.
After administration to the body, drugs will be cleared through
metabolism and/or excretion in intact form. The latter process
sometimes involves active transport, which is beyond the scope
of this manuscript. In a majority of cases, sites of metabolism are
unpredictable and metabolites could have no pharmacological
activity or have activity less than, equivalent to, or more than that
of the parent molecules. Metabolites with similar or better
pharmacological activity are commonly considered as active
metabolites. The conversion of drugs to active metabolites is
distinct from the conversion of prodrugs to active drugs in the
following aspects. In the case of the conversion of drugs to active
metabolites, drugs and active metabolites are pharmacologically
active. Metabolism (biotransformation) of drugs is enzymatic and
the sites of metabolism are not predictable. However, the
conversion of the pharmacologically inactive prodrugs to active
drugs can be either an enzymatic or chemical process, and is
designed with intended purposes. The unpredictability of the
formation of metabolites through metabolism of a drug in the
Scheme 1 Proposed mechanism for the formation of active drug
body offers an opportunity for identifying active metabolites as
tenofovir (26) from the prodrug tenoforvir disoproxil (23).
NCEs (new drugs) or new structural templates for further
optimization in drug discovery. A hint of the presence of active
dose of SD-809 to achieve a similar exposure with a lower Cmax
metabolites may come from a lack of PK–PD correlation or a
than tetrabenazine. Thus, if the observed adverse effects in humans
lack of in vivo efficacy–in vitro potency correlation of a drug
associated with administration of tetrabenazine are Cmax-driven,
molecule. For example, losartan (27, Fig. 4) is used as an
SD-809 may provide an improved safety profile. Auspex Pharma-
angiotensin II receptor antagonist for treatment of hypertension
ceuticals announced that SD-809 showed strong efficacy and
in human. The alcohol functional group of losartan is oxidized to
safety results in its phase 3 clinical registration trial studies in its
the carboxylic acid group to afford the metabolite EXP3174
news release on December 16, 201423. FDA approved SD-809
(28, Fig. 4) in the body27. EXP3174 is one of the major
(Austedo) for the treatment of chorea associated with Huntington's
circulating metabolites in human. In vitro studies showed that
disease on April 3, 2017. SD-809 (Austedo) is indeed the first
EXP3174 has an IC50 of 0.2 nmol/L against the angiotensin II
deuterated product approved by the FDA for human use.
receptor, compared to 4 nmol/L for losartan. By considering the
in vitro potency, plasma exposure and the free fraction in plasma,
2.3. Prodrugs or active metabolites as new drug candidates it was estimated that the active metabolite EXP3174 may have
contributed approximately 14-times the activity in vitro–in vivo
Prodrugs are a class of drugs administered in a pharmacolo- losartan itself27,28, suggesting that further in vitro and in vivo
gically inactive form which is enzymatically or chemically studies on EXP3174 is warranted.
transformed to a pharmacologically active form in vivo. This is In some cases, active metabolites have been developed as
a traditional strategy to chemically modify pharmacologically new drugs. For example, amitriptyline (29, Fig. 4) is a widely
active molecules to overcome problems associated with used drug for treatment of mental disorders, including depres-
absorption barrier, route of administration, metabolism, excre- sion and anxiety. Amitriptyline is metabolized by CYP2D6,
tion, toxicology, as well as to have a site-selective delivery. 3A4 and 2C19 to a demethylated metabolite 30 (Fig. 4)29,30.
Prodrugs commonly have functional groups of esters, amides, This metabolite 30 is a more potent and selective norepinephr-
phosphates, carbonates, or carbamates which are easily ine reuptake inhibitor with Ki of 4.4 nmol/L, compared to
cleaved enzymatically or chemically in the body. One of the 22.4 nmol/L for amitriptyline, against norepinephrine transpor-
common usages of prodrugs is to mask the polar or ionizable ter. Consequently, metabolite 30 underwent development and
functional groups of active molecules to improve oral bioa- eventually became a marketed drug, nortriptyline (Fig. 4).
vailability. For example, tenofovir (26, Scheme 1) is a
nucleotide inhibitor of reverse transcriptase, a crucial viral
enzyme in human immunodeficiency virus 1 (HIV-1) infec- 3. Improving safety profile of drug candidates
tions. Because of the high hydrophilicity of the phosphonic
acid group, tenofovir exhibited very low oral bioavailability in 3.1. Species difference in metabolism of drugs
human (o5%), which limited its use as a drug 24. SAR studies
on bis-carbonate esters of tenofovir led to the discovery of the Drugs are converted to various metabolites by metabolizing
prodrug tenofovir disoproxil (23, Scheme 1) with oral bioa- enzymes in the body, and some metabolites may lead to
vailability of 39% in human24,25. Mechanistically, tenofovir toxicological consequences. In vitro metabolism studies of drug
disoproxil is initially hydrolyzed to the intermediate 24 candidates should be initially conducted to compare the similarity
enzymatically (carboxyesterases) or chemically, followed by of metabolism fate of drug candidates between humans and animal
spontaneous loss of CO2 and formaldehyde to afford the species, and these in vitro results need to be further confirmed in
monoester intermediate 25 (Scheme 1) 26. Phosphodiesterases animals in vivo. Animals with similar metabolism fate to human
726 Zhoupeng Zhang, Wei Tang

Figure 4 Structures of losartan (27), amitriptyline (29), GDC-0834 (31), codeine (33), metoprolol (35), compound 37 and their analogs or
metabolites.

would be selected as safety species in the hope that any major 0834 were below the limit of quantitation (o1 ng/mL)32. However,
metabolite(s) formed in human will be present in animals to a substantial amount of metabolite 32 was detected with Cmax and
similar extent in preclinical safety assessment studies31. In addi- AUC of 142 ng/mL (238 nmol/L) and 837 h·ng/mL (1404 nmol/L·h)
tion, the similarity of in vitro and in vivo metabolism of drug at a dose of 35 mg, and 390 ng/mL (654 nmol/L) and 2448 h·ng/mL
candidates in animals would provide a supporting evidence for us (4105 nmol/L·h) at a dose of 105 mg, respectively. Anilines or
to use the in vitro–in vivo extrapolation (IVIVE) to predict human aromatic amines are a known class of compounds which have a
PK properties. For example, (R)-N-(3-(6-(4-(1,4-dimethyl-3-oxo- potential to cause methaemoglobinemia, agranulocytosis, aplastic
piperazin-2-yl)phenylamino)-4-methyl-5-oxo-4,5-dihydropyr- anaemia, hepatotoxicity, skin hypersensitivity and increased risk of
azin-2-yl)-2-methylphenyl)-4,5,6,7-tetrahydrobenzo[b]thiophene- mutagenicity in animals and humans33. Mechanistically, the aniline
2-carboxamide GDC-0834 (31, Fig. 4) is a potent and selective nitrogen could be oxidized to hydroxylamine, nitroso, nitro and
inhibitor of Bruton's tyrosine kinase (BTK) for potential treatment related species. These metabolites or intermediates are either chemi-
of rheumatoid arthritis32. In vitro metabolism studies in hepato- cally reactive or could be further metabolized to reactive metabolites
cytes indicated that GDC-0834 was extensively metabolized in toward proteins and DNAs. Redox cycling between oxidized aniline
human (80% turnover), moderately metabolized in mouse (56% species (e.g., nitroso and nitro) leads to formation of reactive oxygen
turnover) and cynomolgus monkey (53% turnover) and relatively species (e.g., hydrogen peroxide, superoxide, etc.) which may cause
stable in rat (20% turnover) and dog (17% turnover) after 3-h oxidative stress to cells33. Covalent binding to proteins or DNAs as
incubations. The major hydrolyzed aniline-like metabolite 32 well as oxidative stress in living cells might be one of the possible
(Fig. 4) detected in hepatocyte incubations accounted for about causes for the aniline-associated side effects or toxicities. Due to the
62% (human), 9% (mouse), 4% (cynomolgus monkey), 9% (rat) high exposure of the aniline-like metabolite 32 in human with a
and 12% (dog) of GDC-083432. The amount of metabolite 32 was possible safety liability for the aniline structural alert, the clinical
2-fold more than that of GDC-0834 in human hepatocytes at 3 h, development of GDC-0834 was terminated32. This case study
while the amount of GDC-0834 was the major drug-related suggests that it is critical to consider potential impacts of species
component in hepatocytes of mouse, cynomolgus monkey, rat differences in metabolism of drugs and the potential coverage of
and dog. In vivo studies indicated that metabolite 32 was minor (in major human metabolites in animals should be carefully assessed in
rat and cynomolgus monkey) to moderate (in dog), and GDC-0834 drug design. From a regulatory perspective, it is strongly encouraged
was the major drug-related component in plasma after oral to identify circulating metabolite(s) as early as possible in clinical
administration of GDC-0834 to cynomolgus monkey, rat and trials. This will ensure that the exposure of human metabolite(s) is
dog32. If this in vitro–in vivo correlation for 32 in animals was to covered in animals in preclinical safety assessment studies.
hold true for human, then metabolite 32 might be a circulating
metabolite whose exposure might be much higher than that of
GDC-0834. Additional studies indicated that GDC-0834 was 3.2. Minimizing polymorphic drug metabolizing enzyme-related
stable in intestinal S9 fractions, urine, blood and simulated gastric risk and drug–drug interaction potential
fluid. These data suggest that the conversion of GDC-0834 to
metabolite 32 occurs mainly in the liver. Subsequent human PK Most drugs are mainly cleared from the body through
prediction using IVIVE and allometric scaling methods provided a metabolizing enzyme-mediated processes34. Some metaboliz-
wide range of human blood clearance (5.4–19 mL/min/kg) with ing enzymes (e.g., CYP2D6, 2C9 and 2C19, etc.) are poly-
low confidence32. This significant difference in the major route of morphic in nature and exist in two or more variant forms with
metabolism in human and animals, and low confidence of human different enzymatic activity in different individuals. Poor
PK prediction, prompted the investigators to conduct a single-dose metabolizers (individuals with low or no enzymatic activity)
study in healthy volunteers to assess quickly the human PK of could have much higher drug exposure than extensive meta-
GDC-0834. At the doses of 35 and 105 mg, concentrations of GDC- bolizers (individuals with high enzymatic activity) when a
Drug metabolism in drug discovery and development 727

Scheme 2 Formation of a drug-protein adduct through a bioactivation process where a drug is metabolized to a reactive intermediate which can
subsequently bind to a protein.

given dose of a drug is administered. In the case of prodrugs

where metabolizing enzymes are required to transform pro-
drugs to active drugs, the concentrations of active drugs could
be lower in poor metabolizers than that in normal patients,
resulting in less optimal therapeutic effects. Thus, drug
candidates which are mainly metabolized by a polymorphic
enzyme could have different pharmacological and toxicologi-
cal effects among poor metabolizers and extensive metaboli-
zers. The opioid analgesic drug codeine 33 (Fig. 4) is a good
example of this. Codeine is a prodrug that only weakly binds
to the opioid receptors for analgesic effect. Polymorphic
enzyme CYP2D6 catalyzes the O-demethylation of codeine
to the active drug morphine 34 (Fig. 4) which has 200-fold
greater affinity than codeine. Poor metabolizer patients with
low CYP2D6 activities have lower levels of morphine, result-
ing in less optimal analgesic effect. However, ultrarapid
metabolizer patients with higher CYP2D6 activities can have Figure 5 Structures of commonly used trapping agents in vitro.
much higher levels of morphine. It was reported that respira-
tory depression and death occurred in children who received Some drugs could be inhibitors or inducers of metabolizing
codeine following tonsillectomy and/or adenoidectomy and enzymes. If a drug is an inhibitor of a metabolizing enzyme for
had evidence of being ultra-rapid metabolizers of codeine due another drug, when these two drugs are co-administered to the
to a CYP2D6 polymorphism35. To avoid treatment complica- body, the exposure of the other drug could be higher than
tions in patients who are either ultrarapid or poor metabolizers, expected, resulting in a potential safety issue if the safety margin
opioids that are not metabolized by CYP2D6 may be used of that drug is limited. If a drug is an inducer of a metabolizing
(e.g., morphine, oxymorphone, buprenorphine, fentanyl, enzyme for another drug, when the two drugs are co-administered,
methadone, hydromorphone), alongside non-opioids, depend- the exposure of another drug could be lower than expected,
ing upon the type of pain being treated36 . resulting in potential suboptimal pharmacological effects on the
In SAR studies, structural modification of a lead compound body. This phenomenon where the systemic exposure of a drug is
which is mainly metabolized by a polymorphic enzyme could lead affected by co-administration of another drug is commonly
to the discovery of a new drug candidate which is mainly referred to as a drug–drug interaction (DDI). Thus, it is desirable
metabolized by multiple enzymes. For example, metoprolol
to minimize DDI potential in the drug design process. In cases
(35, Fig. 4), a selective β1 receptor blocker for treatment of high
where a drug candidate has been found to have a high DDI
blood pressure, is mainly metabolized by CYP2D6 in human liver
potential with other drugs, development is generally terminated
microsomes37. In humans, metroprolol demonstrated low oral
and new drug candidates with lower DDI potential may need to be
bioavailability and a relatively short duration of action, due to
identified. For example, in an effort to develop potent α7 nicotinic
fast hepatic metabolism to an O-demethylated metabolite, account-
acetylcholine receptor (α7 nAChR) partial agonists for potential
ing for 65% of the total metabolism of metoprolol, with
α-hydroxylation and N-demethylation accounting for 10% each2. treatment of several neurological and psychiatric disorders includ-
To reduce the major O-demethylation pathway, a bulky cyclopro- ing cognitive dysfunction, (R)-3′-(5-chlorothiophen-2-yl)spiro
pyl group was used to replace the methyl group of metoprolol to [1-azabicyclo[2.2.2]octane-3,5′-oxazolidin]-2′-one (37, Fig. 4)
give betaxolol (36, Fig. 4), which showed much slower metabo- was identified as a lead with a high affinity (Ki ¼ 9 nmol/L) toward
lism. Betaxolol is metabolized by both CYP2D6 and CYP1A2, the α7 nAChR38. However, compound 37 also exhibited potent
while CYP2D6 accounted for only 40% of metabolism in human2. inhibition toward CYP2D6 with an IC50 of 2.0 mol/L. By contrast,
Indeed, a clinical study examining metoprolol polymorphic compound 38 (Fig. 4), which was derived from the replacement of
metabolism by CYP2D6 showed a 100-fold variation for both the 5-chlorothiophene ring with a benzene ring, showed minimal
plasma concentrations of metoprolol and its metabolite α-hydroxy inhibition toward CYP2D6 (IC50430 mol/L), suggesting that the
metoprolol in patients with cardiovascular diseases2. It is expected CYP2D6 inhibition issue might be addressed by structural
that betaxolol (36, Fig. 4) should possess much lower CYP2D6- modification around the 5-chlorothiophene ring of 3738. After an
dependent polymorphism-related risk in human. This example intensive SAR investigation, a novel and potent α7 nAChR partial
illustrates that reaction phenotyping followed by structural opti- agonist (R)-3′-(3-methylbenzo[b]thiophen-5-yl)spiro-[1-azabicyclo
mization can result in a molecule with a minimized polymorphic [2.2.2]-octane-3,5′-oxazolidin]-2′-one (39, Fig. 4) with a high
metabolizing enzyme-related risk. affinity (Ki ¼ 3 nmol/L) toward the α7 nAChR was discovered,
728 Zhoupeng Zhang, Wei Tang

Scheme 3 Proposed mechanism for the bioactivation of [3H]40.

which possessed minimal inhibition toward CYP2D6 (IC50 The reaction rates and selectivity of electrophiles and nucleophiles
430 μmol/L)38. are mainly dependent upon comparable states of “hardness”40. For
example, a soft electrophile such as the α,β-unsaturated ketone can
react predominantly with a soft nucleophile such as the thiol group
3.3. Minimizing toxicity potential associated with bioactivation
of glutathione (GSH). Similarly, a hard electrophile such as the
methyl carbonium ion formed from dimethyl nitrosamine will react
Having an acceptable safety profile is one of the most
important requirements for an NCE to become a successful with hard nucleophiles such as the nitrogen atoms of purine/
drug. However, in some cases, development of drugs is pyrimidine bases in DNA. It is worthwhile mentioning that some
terminated due to preclinical or clinical observations of electrophiles may react with both soft and hard nucleophiles. For
toxicity. Drug-induced liver injury (DILI) and genotoxicity example, styrene oxide is generally considered as a soft electro-
are among the most commonly observed toxicities. There are phile. It can react with either GSH (a soft nucleophile) to form
many possible causes leading to such preclinical and clinical GSH adducts41 or react with one of the endocyclic nitrogen atoms
toxicity. One of the causes is thought to be metabolism-related of guanine in DNA (a hard nucleophile) to form 7-alkylguanine
bioactivation39 . In some cases, metabolism may convert drugs adducts42.
to chemically reactive metabolites/intermediates. Due to the Electrophilic reactive metabolites, in general, are highly
nature of their high electrophilicity, those reactive metabolites unstable, and readily react with nucleophilic macromolecules
may react with components of cellular proteins, DNA, or even (proteins, DNA etc.) in biological systems. Due to their highly
the metabolizing enzymes (which catalyze the formation of reactive nature, these metabolites are often short-lived and rarely
reactive metabolites) to form corresponding drug-protein detectable per se even using the state-of-art modern instrumenta-
adducts, drug-DNA adducts, etc. (Scheme 2). Even though tion. One approach to deduce the structures of reactive metabolites
there is no clear correlation between formation of drug-protein is via in vitro trapping studies43,44. In such a practice, one of the
adducts/drug-DNA adducts and DILI/genetoxicity, it is nucleophilic trapping agents of GSH, N-acetylcysteine (NAC),
believed that bioactivation processes may disrupt normal potassium cyanide or semicarbazide (Fig. 5) is added in incuba-
cellular functions or trigger sequential immune responses tions of drugs with liver microsomes or recombinant metabolizing
and may represent one of the possible causes leading to the enzymes. If drugs are incubated in hepatocytes, no additional GSH
observed DILI or genetoxicity. In drug design, it is prudent to is needed due to the fact that concentration of the endogenous
design/select compounds which possess a low propensity to GSH in hepatocytes is high enough for trapping purposes.
form reactive metabolites for further development. Structures of corresponding adducts can be detected and char-
Electrophilic reactive metabolites formed from bioactivation of acterized by LC–MS and/or NMR. Based on the structures of
drugs could be roughly grouped into two categories: soft electro- those adducts, we can postulate the structures of unstable reactive
philes and hard electrophiles. Based on the hard and soft (Lewis) metabolites. Interested readers may refer to additional reviews43,44.
acids and bases theory (HSAB), hard electrophiles have either a It is believed that covalent protein binding of reactive metabo-
high positive charge density or a formal positive charge at the lites formed through a bioactivation process is one of the possible
electrophilic center40. Conversely, soft electrophiles have a lower causes leading to DILI signals in animals and human. To quantify
positive charge density. Hard nucleophiles have high electronega- covalent protein binding of drugs in biological systems, 3H- or
14
tivity and low polarization of valence electrons, whereas soft C- labeled drugs are required. This is a very resource consuming
nucleophiles have low electronegativity and are more polarizable. process and has been a subject of other review articles39,43,44. In
Drug metabolism in drug discovery and development 729

Scheme 4 Proposed mechanism for the bioactivation of [3H]46.

cases where the 3H- or 14C-labeled drugs are not available, several (10 µmol/L) showed covalent protein values of 461 pmol-equiv/mg
approaches were attempted to provide semi-quantitative measure- protein in human liver microsomes and 48 pmol-equiv/mg protein
ment of bioactivation potential of drugs in biological system. One in human hepatocytes, when [3H]46 (10 µmol/L) was incubated in
of the approaches is to use commercially available radiolabeled human liver microsomes (1 mg proteins/mL) for 45 min or in
trapping agents (e.g., [35S]cysteine or [14C]sodium cyanide, etc.) in human hepatocytes (1×106 cells/mL) for 60 min48. These in vitro
trapping studies with unlabeled test compounds in biological covalent protein binding values of [3H]46 were much lower than
systems. Then, the relative amount of the radioactive peaks of that of [3H]4047. In vitro metabolism studies of [3H]46 identified a
the drug adducts could be used to rank compounds with similar hydroquine metabolite 46 and a biphenyl hydroquinone metabolite
structures45,46. Dansyl glutathione (dGSH, Fig. 5), a fluorescent- 50 (Scheme 4)48. Metabolites 48 and 50 could be formed from
tagged GSH derivative, is one of the other trapping agents used for reduction of corresponding reactive quinone intermediates 47 and
semi quantification of bioactivation potential of test compounds47. 49, respectively. Additional in vitro trapping studied identified a
The drug-dGSH adducts could be detected using both the NAC adduct 51 with the structure confirmed by LC–MS/MS and
fluorescence spectroscopy detection and LC–MS. Structural infor- NMR48. However, no cyano adduct was detected in trapping
mation of the drug-dGSH adducts obtained in LC–MS is used to studies in the presence of potassium cyanide. These results suggest
postulate structures of reactive metabolites formed in the biologi- that the replacement of the piperidine group of 40 with a
cal systems. At the same time, the relative amount of the dGSH pyrrolidine group did block the pathway for the formation of the
adducts could be quantified by fluorescent detection. This reactive iminium ion 4448. However, the pathways leading to the
approach also could be used to rank compounds with similar formation of the reactive quinone metabolites 47 and 49 still
structures. existed, which might be responsible for the observed covalent
In our previous effort to develop a selective estrogen receptor protein binding in human liver microsomes and hepatocytes48.
modulator (SERM) with an estrogen receptor-α (ER-α) subtype These studies indicate that understanding of bioactivation mechan-
selectivity for potential treatment of osteoporosis, (2S,3R)-3-(4- ism could help chemists to better design molecules with low
hydroxyphenyl)-2-[4-(2-piperidin-1-ylethoxy)phenyl]-2,3-dihydro- propensity toward bioactivation and eventually to find drug
1,4-benzoxathiin-6-ol 40 (Scheme 3) was identified as a lead with candidates with lower risk of drug metabolism-induced toxicity.
potent and selective ER-α antagonist activities in vitro and in vivo In some cases where bioactivation is believed to likely be one
in animals48. In vitro covalent protein binding values of [3H]40 of the causes for observed genetoxicity, trapping studies of drugs
were 1106 pmol-equiv/mg protein in human liver microsomes, and with DNA or DNA bases might be performed to elucidate the
170 pmol-equiv/mg protein in human hepatocytes, when [3H]40 structures of reactive metabolites formed in biological systems. For
(10 µmol/L) was incubated in human liver microsomes (1 mg example, the above mentioned new lead compound 46 showed
protein/mL) for 45 min or in human hepatocytes (1×106 cells/mL) genetoxicity in chromosomal aberration assay in Chinese hamster
for 60 min48. In vitro metabolism studies identified a quinone ovary (CHO) cells in vitro and micronucleus induction assay in
metabolite 41 and a hydroquinone metabolite 43 which might be mouse bone marrow in vivo. Subsequent in vitro trapping studies
formed from reduction of the reactive para-quinone metabolite 42. using DNA bases indicated that up to five adenine adducts were
Subsequent in vitro trapping studies identified a bis-cyano adduct detected in incubations of 46 with human and monkey liver
45, indicating the formation of reactive iminium ion species 4448. microsomes or recombinant human CPY3A449. Based on the LC–
These results suggest that compound 40 is subject to bioactivation MS/MS and NMR data, the major adenine adduct 50 has a
through pathways involving at least reactive quinone species 41 cyclized structure (Scheme 5). This further confirmed the existence
and 42 and iminium species 44 (Scheme 3). Subsequently, of the reactive ring-opened para-quinone intermediate 47 as
(2S,3R)-(þ)-3-(3-hydroxyphenyl)-2-[4-(2-pyrrolidin-1-ylethoxy) discussed above. A single cell gel electrophoresis assay (Comet
phenyl]-2,3-dihydro-1,4-benzoxathiin-6-ol (46, Scheme 4) was assay) in human hepatocytes further indicated that 46 caused DNA
synthesized in the effort to minimize formation of reactive damage in a dose-dependent manner49. It is possible that bioacti-
metabolites48. In vitro covalent protein binding values of [3H]46 vation of 46 may be related to the observed genetoxicity.
730 Zhoupeng Zhang, Wei Tang

Scheme 5 Proposed mechanism for the formation of the adenine adduct 52 through bioactivation processes of 46 in the presence of a trapping
agent adenine.

It is worth mentioning that there might be several pathways reactive intermediates to the amino acid residues in the specific
affecting the fate of the reactive intermediates formed in biological regions of the CYP enzyme. In a hypothetical extreme case where
systems. Scheme 6 is a schematic presentation for multiple the intermediate is highly reactive, a majority of the reactive
possible pathways involving a CYP-catalyzed formation of a intermediate formed at the active site may react with amino acid
reactive epoxide intermediate. Binding of a drug to the active site residues in situ to form drug-CYP adducts. Thus, only a small
of a CYP enzyme, followed by the assistance of cytochrome P450 amount of reactive intermediate escapes from the active site to the
reductase and cytochrome b5, triggers the formation of an biological system (Scheme 6, pathway D) to be trapped by a
oxidative oxygen species associated with a CYP enzyme trapping agent (e.g., GSH) to form drug-GSH adducts (Scheme 6,
(Scheme 6, pathway A). The oxygen species oxidizes the drug pathway E). Unfortunately, due to the complexity of the drug-CYP
molecule in its proximity to form a reactive epoxide intermediate adducts, the extent of the formation of these drug-protein adducts
at the active site (Scheme 6, pathway B). This reactive epoxide cannot be easily assessed in bioanalysis of samples from trapping
intermediate may react with amino acid residues of a CYP enzyme studies. In this case, the amount of the drug-adducts with the
to form drug-CYP (drug-protein) adducts (Scheme 6, pathway C). trapping agents detected from semi-quantitative approaches is
The formation of the drug-CYP adducts may or may not inversely proportional to the actual amount of drug-protein
demonstrate a time-dependent inactivation of the catalytic activity adducts. In another case where the reactive epoxide intermediate
of this CYP enzyme, depending on the covalent binding of the is stable enough, a portion of this reactive intermediate can escape
from the CYP active site (Scheme 6, pathway D). This epoxide
intermediate can either be hydrated by epoxide hydrolase, fol-
lowed by dehydration to give a mono-oxygenated metabolite
(Scheme 6, pathway F) or react with surrounding proteins,
including CYP enzymes, to form drug-protein adducts
(Scheme 6, pathway G). In the presence of a trapping agent
(e.g., GSH), this reactive epoxide intermediate can also react with
the trapping agent to form the drug-GSH adducts (Scheme 6,
pathway E). In this case, pathways E, F and G are competitive in
nature, and the amount of the detected drug-GSH adduct depends
on the partitioning among these pathways. The nature of the
partitioning among these pathways will be compound-dependent
and cannot be easily predicted. In addition, the mono-oxygenated
metabolite, formed through the epoxide hydrolase Pathway F, may
also be formed through a CYP-mediated direct hydrogen abstrac-
tion, followed by oxygen rebounding mechanism. This makes it
impossible to use the formation of this metabolite formed from the
epoxide intermediate as an additional measure for assessing the
bioactivation potential of a compound. It is possible that one
compound with less amount of GSH adducts may have higher
bioactivation potential to form more drug-protein adducts than the
other compound with more GSH adducts. Thus, caution needs to
be taken when a comparison of bioactivation potential is used to
rank compounds based on semi-quantification or the mass spectro-
Scheme 6 Schematic presentation of the processes for bioactivation metry responses of drug adducts with trapping agents as a relative
of a drug catalyzed by a cytochrome P450 enzyme. A: binding of a percentage of total drug-related components.
drug to P450 active site; B: formation of reactive intermediate at active Even though the bioactivation-mediated covalent binding of a
site; C: binding of reactive intermediate to P450; D: release of reactive reactive metabolite to proteins of human and animals may have a
intermediate from active site; E: formation of a drug-GSH adduct; F: potential to cause toxicity, a specific group of drugs, called covalent
hydration of reactive intermediate; G: binding of reactive intermediate drugs, indeed efficiently utilize the covalent mechanism toward its
to proteins in biological system. biological targets for action50. Clopidogrel, lansoprazole and
Drug metabolism in drug discovery and development 731

esomeprazole are among the marketed covalent drugs50. In devel- 6. Zhang Z, Sibbesen O, Johnson RA, De Montellano PR. The substrate
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