Mitigation Strategies for Reactive Intermediates in
Drug Discovery
New Perspectives in DMPK: Informing Drug Discovery
Royal Society of Chemistry, London
February 10-11, 2014
Thomas A. Baillie
School of Pharmacy
University of Washington
S ttl WA,
Seattle,
WA USA
[email protected]
Chemically Reactive Drug Metabolites
Role in Liver Toxicity
Cancer Res.,, 7,, 468-480 ((1947))
J. Pharmacol. Exp. Ther., 187, 185-194 (1973)
2
Bioactivation and Liver Toxicity
A t i
Acetaminophen
h (P
(Paracetamol)
t
l)
(Quinone imine)
D C
D.
C. Dahlin et al
al., Proc
Proc. Natl.
Natl Acad.
Acad Sci
Sci. USA
USA, 81,
81 1327-1331
1327 1331 (1984)
I. M. Copple et al., Hepatology, 48, 1292-1301 (2008)
NAPQI–Mediated Activation of Nrf2
Nrf2
Nrf2
ARE
Cell defence genes
GSH depletion
Adduct formation
Protein oxidation
Glutamate Cysteine Ligase
Glutathione transferases
NAD(P)H quinone oxidoreductase
Haem oxygenase
Glucuronyl transferase
Catalase
Nrf2
Keap1
Proteosomal
proteolysis
A. V. Stachulski et al., Med. Res. Rev., 33, 985-1080 (2013)
NAPQI–Mediated Protein Damage
A. V. Stachulski et al., Med. Res. Rev., 33, 985-1080 (2013)
Acetaminophen–Induced Hepatotoxicity
Nrf2
GSH depletion
Adduct
formation
Protein
oxidation
Nrf2
Keap1
Nrf2
ARE
Cell defence
f
genes
Glutamate Cysteine Ligase
Glutathione transferases
NAD(P)H quinone oxidoreductase
Haem oxygenase
Glucuronyl transferase
Catalase
Proteasomal
proteolysis
t l i
N f2 Defence
Nrf2
D f
Increasing dose of acetaminophen
P
Protein
i damage
d
N. Kaplowitz, Nat. Rev. Drug Discov., 4: 489-499 (2005); D. P. Williams, Toxicology 226: 1-11 (2006)
A. V. Stachulski et al., Med. Res. Rev., 33, 985-1080 (2013)
P450-Mediated Quinoid Formation
Toxicological
g
Implications
p
Target Organ
Toxicity
Note: Metabolism can often
Note
introduce / expose –OH / –NH
functionalities
Oxidative Damage
(DNA, Proteins)
“Quinoid” Precursors as Structural Alerts
“Quinoid” Precursors as Structural Alerts
N
Cl
O
N
SG
O
CYP
O
N
GSH
O
O
O
N
HN
HN
HN
NHR
Cl
NHR
I
Isoxazole
l
SG
N
N
O
N
N
F F H
N
H
O
N
CYP
N
N
R1N
N
GSH
NHR2
O
O
F
N
R1HN
NHR2
Pyrazinone
Cl
Cl
H
N
CN
O
NH
N
H3C
Cl
N
CYP
N
N
H3C
Ar
CN
O
H
N
GSH GS
N
Ar
CN
O
NH
H3C
Ar
Pyridine
O
HN
O
H2N
O
O
NH2
CYP
N
O
S
N COAr
H
NH2
H2N
GSH
O
S
S
N COAr
NH2
HN
H2N
SG
N COAr
H
Thiophene
Structural alerts must be supplemented by experimental data!
S. D. Nelson, Adv. Exp. Med. Biol., 500; 33-43 (2001); A. S. Kalgutkar et al., Curr. Drug Metab., 6: 161-225 (2005)
Reactive Drug Metabolites – Lessons Learned
O
• Some, but not all, reactive drug metabolites appear to be
responsible for the toxicity of their parent compounds
O
HN
HN
OH
OH
• High dose drugs (> 50-100 mg/day) that generate reactive
metabolites have the poorest safety record (high “body
burden” of reactive metabolites?)
Bromfenac
• In most cases, the protein targets of reactive metabolites, and their
toxicological relevance, are not yet known – need for biomarkers
• Currently, it is not possible to predict whether a certain reactive metabolite will
elicit a toxic response in vivo, although structural alerts can be helpful
• Practical risk mitigation strategy is to decrease exposure to reactive
metabolites through structural re-design
B. K. Park et al., Nature Rev. Drug Discov., 10, 292-306 (2011); A. V. Stachulski et al., Med. Res. Rev., 33, 985-1080 (2013)
Experimental Approaches for the Study of
Reactive Drug Metabolites
Assessing Formation of / Exposure to
Reactive Drug Metabolites
(A) Observation of time
time-dependent
dependent P450 inhibition in vitro
- Implications for clinical drug-drug interactions
(B) Formation of adducts with nucleophiles
In vitro “trapping” experiments with GSH or CN- (or radiolabeled counterparts)
In vivo metabolic profiling studies (eg GSH conjugates in bile)
- Invaluable in enabling rational structural re-design
(C) Covalent binding studies with radiolabeled drug
- Measures “total” burden of protein-bound drug residue
- Helpful complement to trapping studies
Timing: Late Discovery / Early Lead Optimization
Goal: Structural re-design to minimize exposure to reactive metabolites
8
Elimination of CYP3A4 Time-Dependent Inhibition (TDI)
Compound 1
Compound 1 formed a Metabolic Intermediate (MI) complex
with CYP3A4 due to oxidation on the –NH
NH2 group
In Compound 2, steric hindrance prevented –NH2 oxidation,
MI complex formation, and CYP3A4 inhibition
Compound 2
W. Tang et al., Xenobiotica, 38, 1437-1451 (2008)
14
MI Complex Formation from MC4R Agonists
MI Complex
CYP3A4 TDI
No MI Complex (steric hindrance to N-oxidation)
Compound 2
W. Tang et al., Xenobiotica, 38, 1437-1451 (2008)
Use of Glutathione Trapping to Guide Structural Modification
Orexin receptor
antagonist lead
No evidence of
metabolic activation
C. Boss et al., ChemMedChem, 5: 1197-1214 (2010) 17
Covalent Binding as an Index of Metabolic Activation:
The Discovery of Merck’s CB-1 Inverse Agonist, Taranabant
1 (3900)
G. A. Doss and T. A. Baillie, Drug Metab. Rev., 38, 641-649 (2006)
K. Samuel et al., J. Mass Spectrom., 38, 211-221 (2003)
Evolution of Taranabant (CB-1 Inverse Agonist)
W. K. Hagmann, J. Med. Chem., 51: 4359-4369 (2008)
Reactive Drug Metabolites and
Idi
Idiosyncratic
ti Drug
D
T
Toxicity
i it
Idiosyncratic Drug Toxicity
Susceptibility to adverse drug reactions (ADRs) is a function of:
(A) Chemistry of drug and its interaction with biological systems
- On- and off-target pharmacology
- Metabolic activation of accessible toxicophore (eg acetaminophen)
- Normally dose
dose-dependent,
dependent predictable
predictable, reproducible in animals
(B) Phenotype and genotype of patient
- Not related to pharmacology of drug
- Rare, no clear dose-response relationship, unpredictable, often not
reproduced in animals (“idiosyncratic”)
“Idiosyncratic” drug reactions can result from the sequence:
• Metabolic activation of parent
• Covalent modification of proteins ((“chemical stress”))
• Presentation (in susceptible individuals) of adducted proteins to T-cells
via specific HLA proteins
• Immune-mediated ADRs (often involving liver, skin, or circulatory system)
G. P. Aithal and A. K. Daly, Nature Genetics 42: 650-651 (2010)
Metabolism-Dependent Abacavir Hypersensitivity
J. S. Walsh et al., Chem.-Biol. Interact., 142, 135-154 (2002); A. K. Daly and C. P. Day, Drug Metab. Rev., 44, 116-126 (2012)
C. C. Bell et al., Chem. Res. Toxicol., 26, 1064-1072 (2013); N. M. Grilo et al., Toxicol. Lett., 224, 416-423 (2014)
ADRs: Reaction Frequency vs. Allele Frequency
Reaction frequency
Allele
Allele frequency
Abacavir
Hypersensitivity
HLA-B*5701
0.08
Flucloxacillin
0.05-0.08
Hepatotoxicity
HLA-B*5701
0.08
0.000085
0
000085
Carbamazepine SJS
HLA-B*1502
0.08
0.0001
C b
Carbamazepine
i
H
Hypersensitivity
ii i
(Chinese)
HLA
HLA-A*3101
A*3101
0.06
0 06
0.029
Carbamazepine Hypersensitivity
(Japanese)
HLA-A*3010
0.02
Lumiracoxib
0.05
Hepatotoxicity
(Caucasians)
HLA-DQA1
0.34
Ximelagatran
0.025
0 025
Hepatotoxicity
*0102
HLA-DRB1
0.16
0.06-0.13
*0701
Mallal, 2008; Kindmark et al., 2008; Daly et al., 2009; Chung et al., 2004; Williams et al., 2004;
Levine et al., 2004; Kamali et al., 2009; McCormack et al., 2011.
Risk Factors for Drug-Induced Toxicity
• High clinical dose (>50 mg/day)
• Structural alert(s) for bioactivation
• Evidence of reactive metabolite formation
In vitro trapping studies (GSH, CN-)
Covalent binding to proteins
Estimated reactive metabolite body burden in humans (>10 mg/day)
• In vitro time-dependent inhibition of CYP enzymes
>5-fold
>5
fold shift in IC50
Implications for drug-drug interactions
• In vitro inhibition of hepatic efflux transporters
BSEP
Mrp2
S. Verma and N. Kaplowitz, Gut, 58, 1555-1564 (2009); A. F. Stepan et al., Chem. Res. Toxicol., 24, 1345-1410 (2011)
S. Tujios and R. J. Fontana, Nature Rev. Gastroenterol. Hepatol., 8, 202-211 (2011)
Integrating Reactive Metabolite Studies with In Vitro Toxicology
Assays – An Integrated Approach to Risk Mitigation
R. A. Thompson et al., Chem. Res. Toxicol., 25, 1616−1632 (2012)
Conclusions
• There is compelling evidence that chemically reactive metabolites can
mediate the serious adverse reactions to drugs and other foreign compounds
• However, the frequency and severity of such ADRs will depend upon a
p
series of factors related to the host and the environment,, as well as
complex
the reactive intermediate
• While “avoidance”
avoidance strategies continue to be pursued during the lead
optimization stage of drug discovery, integrated reactive metabolite hazard
assessment strategies are now emerging, based on considerations of
reactive metabolite body burden in conjunction with in vitro toxicity markers
and preclinical in vivo safety testing
• The formation of reactive metabolites needs to be viewed as only one
component of overall risk assessment in the development of new
p
pharmaceuticals
B. K. Park et al., Nature Rev. Drug Discov., 10, 292-306 (2011); A. V. Stachulski et al., Med. Res. Rev., 33, 985-1080 (2013)