Mitigation Strategies for Reactive Intermediates in
Drug Discovery
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 USA Seattle, WA, USA tbaillie@uw.edu
Chemically Reactive Drug Metabolites
Role in Liver Toxicity
Role in Liver Toxicity
Cancer Res., , 7, 468-480 (1947), ( )
J. Pharmacol. Exp. Ther., 187, 185-194 (1973)
Bioactivation and Liver Toxicity
A
t
i
h
(P
t
l)
Acetaminophen (Paracetamol)
(Quinone imine)
D C Dahlinet al Proc Natl Acad Sci USA 81 1327 1331 (1984) D. C. Dahlinet al., Proc. Natl. Acad. Sci. USA, 81, 1327-1331 (1984)
NAPQI–Mediated Activation of Nrf2
Nrf2
ARE Cell defence genes
Nrf2 GSH depletion
Adduct formation Protein oxidation
Glutamate Cysteine Ligase Glutathione transferases
Keap1
Nrf2
NAD(P)H quinone oxidoreductase Haem oxygenase Glucuronyl transferase
Catalase
Proteosomal proteolysis
NAPQI–Mediated Protein Damage
Acetaminophen–Induced Hepatotoxicity
f
Nrf2
Nrf2
ARE Cell defence genes GSH depletion Adduct formation Protein oxidation Nrf2
Glutamate Cysteine Ligase
Glutathione transferases Proteasomal t l i Keap1 Nrf2 Glutathione transferases NAD(P)H quinone oxidoreductase
Haem oxygenase Glucuronyl transferase
Catalase proteolysis
N f2 D f P i d
Increasing dose of acetaminophen
Nrf2 Defence Protein damage
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 Implications
g
p
Target Organ Toxicity
Note Metabolism can often Note: Metabolism can often introduce / expose –OH / –NH functionalities
Oxidative Damage (DNA, Proteins)
“Quinoid” Precursors as Structural Alerts
Cl SG O N HN HN Cl Cl O CYP O N N NHR O GSH O N HN NHR O SG I l N N H N N O N H N F CYP R1N N N NHR2 GSH R1HN N N NHR2 SG Isoxazole H O H F F F R1N NHR2 O 1 2 O Pyrazinone Cl Cl Cl N Cl NH H N Ar H3C CN O N Cl N N Ar H3C CN O N Cl NH H N Ar H3C CN O GS CYP GSH Pyridine HN NH2 O N NH2 O CYP GSH HN NH2 O S HN N H COAr H2N O S N N COAr H2N O CYP GSH S HN N H COAr H2N O SG ThiopheneStructural alerts must be supplemented by experimental data! S. D. Nelson, Adv. Exp. Med. Biol., 500; 33-43 (2001); A. S. Kalgutkaret al., Curr. Drug Metab., 6: 161-225 (2005)
Reactive Drug Metabolites – Lessons Learned
• Some, but not all, reactive drug metabolites appear to be responsible for the toxicity of their parent compounds
HN O
HN O
• High dose drugs (> 50-100 mg/day) that generate reactive
OH
OH
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
Experimental Approaches for the Study of
Experimental Approaches for the Study of
Assessing Formation of / Exposure to
Reactive Drug Metabolites
Reactive Drug Metabolites
(A) Observation of time-dependent P450 inhibition
in vitro
(A) Observation of time 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)
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 studiesHelpful complement to trapping studies
Timing: Late Discovery / Early Lead Optimization
Goal: Structural re-design to minimize exposure to reactive metabolites
Elimination of CYP3A4 Time-Dependent Inhibition (TDI)
Compound 1
Compound 1 formed a Metabolic Intermediate (MI) complex with CYP3A4 due to oxidation on the –NH2 group
with CYP3A4 due to oxidation on the NH2 group
In Compound 2, steric hindrance prevented –NH2 oxidation, MI complex formation, and CYP3A4 inhibition
14 W. Tang et al., Xenobiotica, 38, 1437-1451 (2008)
MI Complex Formation from MC4R Agonists
MI Complex CYP3A4 TDI
No MI Complex (steric hindrance to N-oxidation)
Compound 2 Compound 2
Use of Glutathione Trapping to Guide Structural Modification
Orexin receptor antagonist lead
No evidence of metabolic activation
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)
Reactive Drug Metabolites and
Idi
ti D
T
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 (A) Chemistry of drug and its interaction with biological systems
- On- and off-target pharmacology
- Metabolic activation of accessible toxicophore (eg acetaminophen) Normally dose dependent predictable reproducible in animals - Normally dose-dependent, predictable, reproducible in animals
(B) Phenotype and genotype of patient
- Not related to pharmacology of drugNot 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)
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. Griloet al., Toxicol. Lett., 224, 416-423 (2014)
ADRs:
Reaction Frequency
vs.
Allele Frequency
Abacavir Hypersensitivity HLA-B*5701 0.08
Allele frequency Allele
Reaction frequency
0.05-0.08
Flucloxacillin Hepatotoxicity HLA-B*5701 0.08
0 000085 0.000085
Carbamazepine SJS HLA-B*1502 0.08
0.0001 (Chinese)
C b i H i i i HLA A*3101 0 06
Carbamazepine Hypersensitivity HLA-A*3101 0.06
0.029 (Japanese)
Carbamazepine Hypersensitivity HLA-A*3010 0.02 0.05 (Caucasians)
Lumiracoxib Hepatotoxicity HLA-DQA1 0.34
0 025 *0102
0.025 *0102
Ximelagatran Hepatotoxicity HLA-DRB1 0.16
0.06-0.13 *0701
Mallal, 2008; Kindmarket al., 2008; Daly et al., 2009; Chung et al., 2004; Williams et al., 2004; Levine et al., 2004; Kamaliet 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 formationEvidence of reactive metabolite formation In vitro trapping studies (GSH, CN-)
Covalent binding to proteins
Estimated reactive metabolite body burden in humans (>10 mg/day) Estimated reactive metabolite body burden in humans (>10 mg/day)
• In vitro time-dependent inhibition of CYP enzymes
>5 fold shift in IC >5-fold shift in IC50
Implications for drug-drug interactions
• In vitroIn vitro inhibition of hepatic efflux transportersinhibition of hepatic efflux transporters
BSEP Mrp2
S. Verma and N. Kaplowitz, Gut, 58, 1555-1564 (2009); A. F. Stepanet 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
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
complex series of factors related to the host and the environment, as well as p , the reactive intermediate
• While “avoidance” strategies continue to be pursued during the lead • While 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 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
pharmaceuticals p