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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

(2)

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)

(3)

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)

(4)

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

(5)

NAPQI–Mediated Protein Damage

(6)

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)

(7)

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)

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(9)

“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 Thiophene

Structural 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)

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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

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Experimental Approaches for the Study of

Experimental Approaches for the Study of

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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

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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)

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MI Complex Formation from MC4R Agonists

MI Complex CYP3A4 TDI

No MI Complex (steric hindrance to N-oxidation)

Compound 2 Compound 2

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Use of Glutathione Trapping to Guide Structural Modification

Orexin receptor antagonist lead

No evidence of metabolic activation

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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)

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Evolution of Taranabant (CB-1 Inverse Agonist)

(18)

Reactive Drug Metabolites and

Idi

ti D

T

i it

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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)

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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)

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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.

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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)

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Integrating Reactive Metabolite Studies with

In Vitro

Toxicology

Assays – An Integrated Approach to Risk Mitigation

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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

References

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