Advanced Drug Metabolism MEDCH 527

Winter Quarter 2013

“Bioactivation and Idiosyncratic Drug Toxicity”

Thomas A. Baillie School of Pharmacy, UW

tbaillie@uw.edu

1

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-dependent, 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); P. T. Illing et al., Curr. Opin. Immunol., 25, 1-9 (2013)

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-dependent, 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); P. T. Illing et al., Curr. Opin. Immunol., 25, 1-9 (2013)

Why Should Minimizing Exposure to Reactive Intermediates be a Specific Objective in Drug Discovery?

•  > 40 Years of research have provided strong indirect evidence that some (but not all) reactive metabolites are toxic to the host organ (and even to distant organs in certain cases)

•  We can deduce probable structures of electrophilic intermediates, but we cannot predict which ones will be toxic since the current level of understanding of mechanistic biochemical toxicology does not allow us to do so

•  Practical solutions for industry: (1) Ignore the issue (2) Minimize formation of reactive metabolites prior to entry into development

•  To be successful, a proactive approach requires close interaction between drug

metabolism scientists and medicinal chemists such that key information on lead compounds is provided in a timely fashion

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•  How is it determined that a drug candidate is subject to metabolic activation?

•  How is the mechanism of bioactivation established? •  What medicinal chemistry strategies are available to

minimize this potential liability? S. Kumar and T. A. Baillie, in Handbook of Drug Metabolism, 2nd ed., P.G. Pearson & L. C. Wienkers, eds.,

Informa Healthcare, New York, 2009, pp. 597-618 W. Tang and A. Y. H. Lu, Drug Metab. Rev., 42, 225-249 (2010) L. Leung, A. Kalgutkar, and R. S. Obach, Drug Metab. Rev., 44, 18-33 (2011) B. K. Park et al., Nature Rev. Drug Discov., 10, 292-306 (2011)

Questions

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Detection of Electrophilic Drug Metabolites

•  Use of radiolabeled compounds – quantitative assessment of radioactivity covalently bound to cellular proteins (in vitro or in vivo)

•  Use of nucleophilic trapping agents in in vitro metabolism studies, eg GSH (many ‘soft’ electrophiles), CN- (iminium ions), semicarbazide or methoxyamine (reactive carbonyls)

•  Time-dependent P-450 inactivation studies in vitro

•  Analysis of metabolic profiles, both in vitro and in vivo - formation of some stable metabolites occurs via a reactive intermediate

D. C. Evans et al., Chem. Res. Toxicol., 17, 3-16 (2004) T. A. Baillie, Chem. Res. Toxicol., 19, 889-893 (2006)

6

Identification of Electrophilic Drug Metabolites

•  LC-MS/MS analysis of ‘trapped’ reactive intermediates is the most widely used approach; NMR of isolated adducts may be employed for more definitive assignments

•  Structural information inferred from trapping studies is used to guide

chemical modifications that minimize bioactivation liability: (1)  block site of metabolism, (2) sterically hinder metabolic site, (3) introduce electronic changes,

(4) redirect metabolism to “soft spot”, (5) replacement of offending functional group, (6) combinations thereof

(+)ve ESI-MS/MS: [MH+-75] and [MH+-129] (-)ve ESI-MS/MS: MH - m/z 272

C. M. Dieckhaus et al., Chem. Res. Toxicol., 18, 630-638 (2005)

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Medicinal Chemistry Approaches (1) Block Site of Metabolism

Y.-J. Wu et al., J. Med. Chem., 46, 3778-3781 (2003)

CYP3A4 CYP3A4

C. M. Diekhaus et al., Chem.-Biol. Interact., 142, 99-117 (2002)

TDI

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Medicinal Chemistry Approaches (2) Sterically Hinder the Site of Metabolism

Re-positioning the S atom and the phenylsulfone reduces oxidative metabolism on the thiazole sulfur

L. A. Trimble et al., Bioorg. Med. Chem. Lett., 7, 53-56 (1997) P. Roy et al., Bioorg. Med. Chem. Lett., 7, 57-62 (1997)

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Medicinal Chemistry Approaches (3) Introduce Electronic Changes – The Taranabant Story

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)

1 (3900)

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Evolution of Taranabant (CB-1 Inverse Agonist)

W. K. Hagmann, J. Med. Chem., 51: 4359-4369 (2008) 11

Medicinal Chemistry Approaches (4) Redirect Metabolism to “Soft Spot”

Reactive metabolites of thiazole ring oxidation, thiourea formation

R. S. Obach et al., Chem. Res. Toxicol., 21, 1890-1899 (2008) 12

Medicinal Chemistry Approaches (5) Replacement of Offending Functionality

3 Lead candidates

•  All showed high potency and good selectivity towards the orexin receptor •  All had acceptable animal PK

Which compound to select for development? Radiolabeled analogs prepared (3H in phenyl ring)

•  Covalent binding studies with human and rat liver microsomes •  All 3 compounds showed high binding (>1,000 pmol eq / mg protein)!

Orexin Receptor Antagonist

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In Vitro Metabolism of Orexin Antagonists

•  Major in vitro metabolites identified as products of diazepan ring cleavage

•  CYP-Mediated ring cleavage predicted to proceed via aldehyde intermediate

•  For all 3 candidates, aldehydes trapped with semicarbazide (Δm = 57 Da)

•  Does semicarbazide also block covalent binding to microsomal proteins?

Reactive aldehyde?

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Effect of Trapping Agents on Covalent Binding of Substituted Diazepans to HLMs

Trapping agent (200µM)

Compound A (10µM)

Compound B (10µM)

Compound C (10µM)

None 1526 1060 1488

Semicarbazide 1289 1104 1400

GSH 197 214 188

•  Semicarbazide does not block covalent binding, but GSH does! •  What is being trapped by GSH?

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Bioactivation of para-Fluoroaniline Derivatives

MH+ at m/z = (P + 305 – 2)

N. H. Cnubben et al., Biochem. Pharmacol., 49: 1235-1248 (1995) 16

RT: 1.11 - 27.90 SM: 7B

2 4 6 8 10 12 14 16 18 20 22 24 26 Time (min)

0 20 40 60 80

100 0

20 40 60 80

100

Rel

ativ

e A

bund

ance

13.05

20.84

23.33 15.90

27.40 23.68 17.13 25.95 22.69 11.66 14.25 3.39 3.98 18.07 5.23 6.35 10.26 1.57 9.49

11.66

4.56 11.04 18.62 23.15 3.54 13.15 17.23 2.22 7.00 5.51 14.18 25.64 27.83 21.67 9.35 19.52

NL: 4.84E6 Base Peak F: FTMS + c ESI Full ms [400.00-850.00]

NL: 9.81E4 m/z= 734.70-735.70 F: FTMS + c ESI Full ms [400.00-850.00]

TIC

XIC (m/z 735.2)

LC-MS Analysis of Products of Incubation of Lead Compound with Human Liver Microsomes Fortified with GSH

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Mass Spectrum (ESI, Full Scan) of GSH Adduct from Lead Thermo Orbitrap (30,000 resolution)

# 127-1628 RT: 8.46-13.41 AV 301 NL: 7.20E3 F: FTMS + c ESI Full ms [400.00-850.00]

560 580 600 620 640 660 680 700 720 740 760 780 800 820 840 m/z

0 10 20 30 40 50 60 70 80 90

100

Rel

ativ

e A

bund

ance

735.2696

624.2172 602.3135

701.3931 753.2600

773.2261 638.0779 591.1634 813.4719 848.4297 729.7230 676.2457 561.1630

C 3 3 H 3 9 N 1 0 O 8 S E x a c t M a s s : 7 3 5 . 2 6 7 3 NN

N

N

OH

R2

R1

O

SG

MH+ = 735

Δ = 3ppm

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Minimizing Metabolic Activation Through Structural Modification

No evidence of metabolic activation

Orexin receptor antagonist lead

C. Boss et al., ChemMedChem, 5: 1197-1214 (2010)

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Medicinal Chemistry Approaches (6) Combination of Steric and Electronic Changes

Metabolic Activation of Pyrazinone-Containing Thrombin Inhibitors

N

N

O

R 1

H N R 2 R

R. Singh et al., Chem. Res. Toxicol.,16, 198-207 (2003)

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N H

N N H

N

O

O

N V I I

N H

N N H N

F F

N

O

O N

F V I I I

N H

N N H N

F F

N C l

O

O N

F I X

N H 2

Pyrazinone-Containing Thrombin Inhibitors

21

N H

N N

N H O

O

F F

S

H N N

H H O

O

O O H

N H 2

O O 4 3 1

6 0 9

M H + = 7 3 8

N N

F

200 250 300 350 400 450 500 550 600 650 700 750

m/z

0

20

40

60

80

100 431.1

737.9 (MH+)

Rel

ativ

e In

tens

ity (%

)

m/z

(MH+ - 307)

609 (MH+ - 129)

738 MH+ = 738

MS2 Spectrum of Conjugate GSH-1 from Compound VIII

22

N

N

NH

ArNH

Ar

O

OFF

Compound VIII

N

N

NAr

NH

Ar

O

OFF

P-450

N

N

NH

ArNH

Ar

O

OFF

GSH

SG

GSH-1MH+ at m/z 738

GSH-2MH+ at m/z 738

?

GSH

Metabolic Activation of Compound VIII

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ArNH

NN

NHAr

F F O

O8

9

11

129 11 12

OHNH

O

SH

NH

OH

O

NH2

O O

aa'

b

c

de

fb

d c f

e

a

11

912

b cc

fe

aa'

a'

8

1H NMR Spectra: Conjugate GSH-2 (top), GSH (center), and Compound VIII (bottom)

“GSH-2”

GSH

VIII

24

N

NNH

O

NH

OAr

Ar

GS

9

8

12

11

GSH-2F F

c/c

11/12

2D-NOESY Spectrum (Aliphatic Region) of Conjugate GSH-2 of Compound VIII

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Proposed Mechanism for Formation of GSH Adducts of Compound VIII

ArNH

NN

NHAr

O

O

F F

ArNH

NN

NHO

O

F F

SG

ArArN

NN

NHO

O

F F

Ar

ArNH

NN

NHO

O

F F

O

ArAr

NH

NN

NHO

O

F F

SGOH

Ar

ArNH

HN

N

NHArO

O

F F

SGO

HN N

H

Ar

O

ON

N

GS

F

FAr

ArN

HN

N

NHArO

O

F F

SGHO

11

129

GSH-2

Compound VIII

GSH-1

P-450

P-450GSH

GSH

26

N H

N N H

N

O

O

N V I I

N H

N N H N

F F

N

O

O N

F V I I I

N H

N N H N

F F

N C l

O

O N

F I X

N H 2

Pyrazinone-Containing Thrombin Inhibitors

27

Key Messages

•  Reactive intermediate formation is considered an undesirable feature of any drug candidate.

•  Bioactivation potential should be fully assessed during lead optimization, and viewed in the context of overall risk assessment

•  Low-dose compounds strongly preferred over high-dose drugs (reduced “body burden” of reactive intermediates)

•  Close integration of DMPK, Medicinal Chemistry and Safety Assessment plays a key role in eliminating potentially ‘defective’ compounds before they enter development

•  Minimizing metabolic activation liabilities in drug candidates hopefully will decrease overall attrition rates in development, and thereby increase ‘probability of success’

B. K. Park et al., Nature Rev. Drug Discov., 10, 292-306 (2011)

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Risk Assessment Considerations Based on Bioactivation Potential

•  Chemical tractability of structural series? -  What potential exists to modify structure? -  Has metabolic activation been minimized relative to preceding compound?

•  Availability of existing treatments for target disease?

•  Is the prognosis disabling or life-threatening?

•  Is the anticipated clinical dose <10mg?

•  Are the metabolic clearance routes primarily non-Phase I? -  Consider studies in hepatocytes

•  Expected duration of therapy? -  Will the drug be used chronically / prophylactically?

•  What is the intended target population? - Is the drug intended for a pediatric indication?

D. C. Evans et al., Chem. Res. Toxicol., 17, 3-16 (2004) 29