Risk Assessment of Reactive Metabolites in Drug Discovery: A Focus on Acyl Glucuronides

and Acyl-CoA Thioesters

The Delaware Valley Drug Metabolism Discussion Group 2017 Rozman Symposium

HUMAN BIOTRANSFORMATION: THROUGH THE MIST

Langhorne, PA, June 13, 2017

Mark Grillo, PhD Drug Metabolism & Pharmacokinetics

MyoKardia South San Francisco, CA mgrillo@myokardia.com

2

Commercialization Process Overview

Therapeutic Area/Disease

Opportunity Assessment

Lead Selection &

Pre-clinical Testing

Product Strategy

Development

Market Readiness &

Regulatory Approval

Launch / Post-Launch

Lifecycle Management

Discovery Lead

Optimization Pre-clinical Phase 1 Phase 2 Phase 3 Filing Launch

Post-

Launch

Hit-to-

Lead Screen

Drug Discovery & Development

Discovery Lead Optimization Hit-to-Lead Screen

Target Selection & Validation Lead compound Selection

Early DDI Assessment

Enzyme induction PXR activation

CYP inhibition

Optimize Pharmacokinetics

Liver microsomal stability

Membrane permeability

Efflux/transport assays

Plasma stability

Rat PK

Protein binding

Blood to plasma ratio

Characterize Preclinical Candidate

Intrinsic clearance

Excretion balance

Definitive metabolite ID

Higher species PK

Human PK prediction

Assess DDI Potential

Competitive CYP IC50

Time-dependent CYP inhibition

Hepatocyte induction

CYP reaction phenotyping

Detection and Assessment of Chemically-Reactive Metabolites

Preclinical Pharmacokinetics & Drug Metabolism Assays

• Toxicity is a frequent cause of drug withdrawal from the market.

• Reactive drug metabolites may mediate liver injury via covalent modification of biological macromolecules.

• Reactive metabolites of carboxylic acid-containing drugs, acyl glucuronides and acyl-CoA thioesters, might contribute to idiosyncratic drug toxicity.

• Underlying mechanisms not clear. • Believed to be immune-mediated.

Circumstantial Evidence Links Reactive Metabolites to Adverse Drug Reactions

Liver Injury

3

Drugs Withdrawn and Associated with Idiosyncratic Drug Toxicity Alcofenac (anti-inflammatory) Hepatitis, rash Alpidem (anxiolytic) Hepatitis (fatal) Amodiaquine (antimalarial) Hepatitis, agranulocytosis Amineptine (antidepressant) Hepatitis, cutaneous ADRs Benoxaprofen (anti-inflammatory) Hepatitis, cutaneous ADRs Bromfenac (anti-inflammatory) Hepatitis (fatal) Carbutamide (antidiabetic) Bone marrow toxicity Ibufenac (anti-inflammatory) Hepatitis (fatal) Iproniazid (antidepressant) Hepatitis (fatal)

Metiamide (antiulcer) Bone marrow toxicity Nomifensine (antidepressant) Hepatitis (fatal), anemia Practolol (antiarrhythmic) Severe cutaneous ADRs Remoxipride (antipsychotic) Aplastic anaemia Sudoxicam (anti-inflammatory) Hepatitis (fatal) Tienilic Acid (diuretic) Hepatitis (fatal) Tolrestat (antidiabetic) Hepatitis (fatal) Troglitazone (antidiabetic) Hepatitis (fatal) Zomepirac (anti-inflammatory) Hepatitis, cutaneous ADRs

Kalgutkar and Soglia (2005) Exp. Opin. Drug Metab. Toxicol. 1, 91-141.

In many cases, metabolic activation reactive metabolites leading to covalent binding to protein demonstrated in vitro and/or in vivo.

4 Carboxylic acid drugs

Danger Hypothesis Illustration for Immune-mediated Idiosyncratic Hepatotoxicity

Kaplowitz (2005) Idiosyncratic drug hepatotoxicity. Nature Reviews 4, 489-499. 5

Assessing Formation of / Exposure to Reactive Drug Metabolites

• Formation of adducts with nucleophiles

• In vitro “trapping” studies with glutathione (GSH) or CN-, and others

• In vivo metabolic profiling studies (e.g., GSH-adducts in bile, NAC-adducts in urine)

• Covalent binding studies with radiolabeled drug

• Definitive

• Measures “total” burden of protein bound-drug residue

• Helpful compliment to trapping studies

Park et al. (2011) Nature Rev. Drug Discov., 10:292-306. Evans et al. (2004) Chem. Res. Toxicol., 17:3-16.

• Trapping studies with glutathione and covalent binding studies with radiolabel carboxylic acid-containing drugs to examine the importance of acyl glucuronides vs. acyl-CoA thioesters as reactive intermediates in vitro and in vivo.

6

Kalgutkar (2009) Chem. Biodiver. 6, 2115-36; Lammert et al., Hepatology 47, (2008)

Relationship between daily-dose of oral medications and idiosyncratic drug-induced liver injury (DILI)

Drugs dosed at <10 mg/day extremely rare to lead to idiosyncratic drug toxicity (whether or not these drugs are prone to metabolic activation).

7

Assessing the Potential for Risk of Idiosyncratic Drug Toxicity Caused by Candidate Drugs (AstraZeneca, 2012)

Drugs that demonstrated high covalent binding (CVB) burden (>1 mg/day, Zones 3 and 4) correlated with many of the toxic compounds.

Thompson et al. (2012) Chem. Res. Toxicol. 25: 1616-1632.

CVB Burden (#9) Ibufenac 24 (#32) Ibuprofen 5.2 (#20) Diclofenac 1.8 (#29) Caffeine <0.23

Recommendation: <1 mg/day exposure (CVB burden) to reactive metabolites

fcvb= (CVB•0.26 mg)/(turnover•4000 pmol) CVB burden = fcvb•dose

8

Cross-species Liver Microsomes Metabolite Profile: LC/UV detection Identification of GSH-adduct as Major Metabolite in HLM fortified with NADPH and GSH

RT: 6.88 - 22.09 SM: 11G

7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

Time (min)

-4000

-3000

-2000

-1000

0

1000

2000

uA

U

-4000

-3000

-2000

-1000

0

1000

2000

uA

U

-4000

-3000

-2000

-1000

0

1000

2000

uA

U

-4000

-3000

-2000

-1000

0

1000

2000

uA

U

NL:5.38E4

nm=280.0-280.0 PDA 3509rlm+n+g

NL:5.94E4

nm=280.0-280.0 PDA 3509dlm+n+g

NL:5.16E4

nm=280.0-280.0 PDA 3509clm+n+g

NL:5.92E4

nm=280.0-280.0 PDA 3509hlm+n+g

GSH-adduct UV 280 nm Rat

UV 280 nm Dog

UV 280 nm Cyno monkey

UV 280 nm

Human

• GSH-adduct major metabolite formed in HLM (1.5 % relative peak area to parent, ~50% of total metabolite peak area)

• Decision to perform reactive metabolite/toxicity risk assessment prior to advancement.

hydroxylated metabolite metabolite

ndr

ndr, not drug related

Drug

9 Unpublished observations

In c u b a tio n T im e (h )

Co

va

len

t B

ind

ing

to

Pro

tein

(pm

ol

eq

./m

g/p

rote

in)

0 1 2 3 4

0

2 5

5 0

7 5

1 0 0

1 2 5

1 5 0

1 7 5

R a d io la b e led

T e s t C o m p o u n d

[1 4

C ]D ic lo fe n a c

[1 4

C ]D ic lo fe n a c ( lite ra tu re )

In c u b a tio n T im e (h )

Co

va

len

t B

ind

ing

to

Pro

tein

(pm

ol

eq

./m

g/p

rote

in)

0 1 2 3 4

0

1 0

2 0

3 0

4 0

5 0

6 0

7 0

8 0

9 0

1 0 0

1 1 0 R a d io la b e led

T e s t C o m p o u n d

(0 % tu rn o v e r )

[1 4

C ]D ic lo fe n a c

(8 7 .5 % tu rn o v e r )

[1 4

C ]D ic lo fe n a c ( lite ra tu re )

Covalent Binding (CVB) of Radiolabeled Test Drug and [14C]Diclofenac to Protein in Human Hepatocytes in Suspension:

Prediction of “CVB Burden” in Human Covalent Binding Metabolic Stability

CompoundCVB (pmol/mg

protein)Incubation

time (h)Turnover

(%)fcvb

Daily Dose (mg)

CVB Burden (mg)

Test Compound24.0 ± 2.6 1 0 ± 3.0 0.174*

80†13.9

34.8 ± 4.8 2 0 ± 2.9 0.252* 20.249.8 ± 8.3 4 0 ± 4.3 0.361* 28.8

Diclofenac78.9 ± 2.6 1 68.6 ± 2.8 0.008

2001.67

109. ± 8.3 2 78.9 ± 1.1 0.010 2.01151.6 ± 7.5 4 87.6 ± 0.5 0.013 2.51

Diclofenac (Thompson, et al.)

140.8 2 100 0.0092 200 1.84

Substrate concentration (10 µM), incubation volume (2.5 mL); *no loss of drug detected (assume 1% turnover); †Preliminary prediction of drug dose in human based on rat PK/PD and simple allometric scaling (4-species) for human PK.

10

Metabolites in Safety Testing (MIST) (FDA, 2008) Acyl Glucuronides

https://www.fda.gov/downloads/Drugs/.../Guidances/ucm079266.pdf

Ref. 5 Faed, EM, 1984, Properties of Acyl Glucuronides. Implications for Studies of the Pharmacokinetics and Metabolism of Acidic Drugs, Drug Metab Rev, 15: 1213–1249.

11

Partial list of carboxylic-acid containing drugs withdrawn from clinical use and/or also associated with allergic reactions

Withdrawn Allergic reactions

Fung et al. (2001) Drug Information Journal, 35, 293-317. Stepan et al. (2011) Chem Res Toxicol, 24, 1345-1410.

Tolmetin

OH

OC l

O

OH

NH2

O

B r O

OH

O

O H

O

N

H3C

C l

OC H3

O

OH

O

O

S

C l C l

O H

OO

N

C l

C H3

H

OH

OO

N

F

C H3

H

OH

O

C H3

HN

O

OH

O

H3CH

OH

ONHC l

C l

OH

O

H3CH

O

OH

O

N

C H3

O

H3C

O

OH

O

C H3

C H3C l

OH

O

SN

OO

OH

O

HO

F

F

OH

O

Alclofenac

Bromfenac

Ibufenac

Zomepirac

Tienilic Acid

Benoxaprofen

Flunoxaprofen

Indoprofen

Ibuprofen

Diclofenac

Fenoprofen

Clofibric Acid

Probenecid

Diflunisal

Valproic Acid

12

Drug Acyl Glucuronide: Instability (Primarily by Acyl Migration)

• Incubation of 1-O-β-drug-acyl glucuronide in buffer (pH 7.4, 37 oC) to determine degradation rate/ degradation half-life. •LC-MS/MS analysis: Xue et al. “Optimization to eliminate interference of migration isomers for measuring 1-O-β-acyl glucuronide with out extensive chromatographic separation”. Rapid Commun. Mass Spectrom. 2008; 22: 109–120.

Time = 0 minpH 7.4, 37oC

1-O-b-isomer

Time = 20 minpH 7.4, 37oC

*

1-O-b-isomer

*

*

*Acyl migration isomers

0 5 1 0 1 5 2 0 2 5 3 0

0

2 0

4 0

6 0

8 0

1 0 0

t1 /2 ~ 1 0 m in

In c u b a tio n T im e (m in )

% R

em

ain

ing

Rel

ativ

e In

ten

sity

(%

)

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

Retention Time (min)

10

20

30

40

50

60

70

80

90

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 170

10

20

30

40

50

60

70

80

90

0

100

100

*

*

13

Mechanisms for A) the transacylation of protein nucleophiles by 1-β-O-acyl-linked glucuronides and B) for stable adduct formation with lysine-residues on proteins by

reaction with open-chain acyl glucuronide migration isomers.

14

Predictability of Covalent Binding of Acidic Drugs in Man

0.0 0.5 1.0 1.5 2.00

1

2

3

4

5

6

7

8

TolmetinZomepirac

(R )-Fenoprofen

(R )-Carprofen

(S )-Carprofen

(S )-Fenoprofen

Furosemide

(R,S )-Beclobric Acid

Degradation Rate Constant (k, 1/hrs)

Co

vale

nt

Bin

din

g (

mo

le/m

ole

pro

tein

x e

-3)

Beclobric Acid

Cl

O

C3H

C3HO H

OFurosemide

OHN

O

O

N2H

S

Cl

O HO

H

C3

H

H

Cl

NO

OHCarprofen Fenoprofen

TolmetinZomepirac

C 3HO

C3HN

O

OH

OH

C3H

O

OH

C3H

C 3HO

ClN

O

OH

0.0 0.5 1.0 1.5 2.00

1

2

3

4

5

6

7

8

TolmetinZomepirac

(R )-Fenoprofen

(R )-Carprofen

(S )-Carprofen

(S )-Fenoprofen

Furosemide

(R,S )-Beclobric Acid

Degradation Rate Constant (k, 1/hrs)

Co

vale

nt

Bin

din

g (

mo

le/m

ole

pro

tein

x e

-3)

Beclobric Acid

Cl

O

C3H

C3HO H

OFurosemide

OHN

O

O

N2H

S

Cl

O HO

H

C3

H

H

Cl

NO

OHCarprofen

0.0 0.5 1.0 1.5 2.00

1

2

3

4

5

6

7

8

TolmetinZomepirac

(R )-Fenoprofen

(R )-Carprofen

(S )-Carprofen

(S )-Fenoprofen

Furosemide

(R,S )-Beclobric Acid

Degradation Rate Constant (k, 1/hrs)

Co

vale

nt

Bin

din

g (

mo

le/m

ole

pro

tein

x e

-3)

Beclobric Acid

Cl

O

C3H

C3HO H

OFurosemide

OHN

O

O

N2H

S

Cl

O HO

H

C3

H

H

Cl

NO

OHCarprofen Fenoprofen

TolmetinZomepirac

C 3HO

C3HN

O

OH

OH

C3H

O

OH

C3H

C 3HO

ClN

O

OH

“Increasing the degree of substitution at the alpha-carboxy-carbon correlates with increasing stability (decreasing reactivity with protein) of the acyl glucuronide.

Benet et al. (1993) Life Sci, 53:141-6

Smith et al. (1986) J Clin. Invest. 77:934-39

15

• “The data provide conclusive evidence for the covalent binding of tolmetin glucuronide to HSA by Schiff-base formation with lysine ε-amino groups. In all of the identified tolmetin-containing peptides, the glucuronic acid moiety was present.” Ding et al., Proc. Natl. Acad. Sci. 90 (1993).

• “Our results establish that the binding of these reactive metabolites to nucleophilic sites of proteins occur via two different mechanisms: one involving imine (Schiff base) formation and the other involving nucleophilic displacement of glucuronic acid.” Ding et al., Drug Metab Dispos., 23 (1994).

•Zomepirac Zia-Amirhosseini et al. (1995) Biochem. J.

311:431-435.

•Benoxaprofen Qiu et al. (1998) Drug Metab. Dispos. 26:246-56.

•Diclofenac Hammond et al. (2014) J Pharmacol. Exper.

Therap. 350:387-402.

•Fevipiprant Pearson et al. (April, 2017) DMD Fast Forward

16

•The classification value of the degradation half-life which separated the safe drugs from the withdrawn drugs was

calculated to be 3.6 h.

“The KPB system was considered to be the best for evaluating the stability of AGs, and the classification value of the half-life in KPB serves as a useful key predictor for the IDT risk.”

Predictability of Idiosyncratic Drug Toxicity Risk for Carboxylic Acid-Containing Drugs Based on the Chemical Stability of Acyl Glucuronide

Sawamura et al. (2010) Drug Metab. Dispos., 38: 1857-1864. 17

Covalent Binding: Bioactivation of Valproic acid (VPA)

•Microvesicular steatosis proposed to be due to irreversible inhibition of fatty acid metabolism leading to a build-up of lipids.

•Covalent binding studies in rat hepatocytes showed that inhibition of P450 or Glucuronidation had no effect on covalent binding to protein, whereas inhibition of acyl-CoA formation led to a 90% decrease in covalent binding to protein.

•VPA-acyl-CoA implicated in CB to protein

(UW) Porubek et al. (1989) Drug Metab Dispos. 2: 123-130. 18

Proposed Metabolism Routes of Carboxylic Acid-Containing Drugs to Reactive Derivatives and Non-Toxic Products

1. Acyl-CoA Formation 2. Acyl Glucuronidation 3. Acyl Migration 4. Conjugation Reactions 5. Ring-Chain Tautomerism 6. Schiff-Base Formation With Proteins

4 Conjugation Reactions:

Glycine Amides Taurine Amides Carnitine Esters Hybrid Triglycerides Choline Esters Fatty Acylation?

Potential Toxicity

3 3 2 - , - , 4 - O - A c y l Glucuronides

1

Acyl-CoA Thioester

Covalent Binding to Protein

6

5

Open-Chain Aldehydes

- G

2

1 - O A c y l l u cu r o n i d e

Excretion in urine, bile, detection in

plasma

Remains intracellular, not excreted, doesn’t circulate in plasma

19

Acyl-CoA Synthetase-mediated Formation of S-acyl-CoA Thioesters Leading to the Transacylation of GSH and Protein nucleophiles

Adverse Drug Reactions?

Detoxification

Detoxification?

20

Acyl-CoA Thioesters Shown to Acylate Proteins In Vitro (early reports)

21

Nafenopin-SCoA

Yamashita A. et al. (1995) Acyl-CoA binding and acylation of UDP-glucuronosyl-transferase isoforms of rat liver: their effect on enzyme activity. Biochem. J . 312:301-308.

Arachidonyl-SCoA

S allustio, B. C . (2000) In vitro covalent binding of nafenopin-C oA to humanliver proteins. Toxicol. App. Pharmacol. 163:176-182.

Palmitoyl-SCoA

Duncan, J. A. and Gilman, A. G. (1996) Autoacylation of G protein alpha subunits.J . Biol. Chem. 271:23594-23600.

Clofibryl-SCoA

Grillo, M.P. and Benet L.Z. (2002) Studies on the reactivity of clofibryl-S-acyl-CoA thioester with glutathione in vitro. Drug Metab. Dispos. 30:55-62.

Salicyl-SCoA

Tishler and Goldman (1970) Properties and reactions of salicyl-Coenzyme A. Biochem. Pharmacol. 19:143-150.

Enantioselective Covalent Binding Studies with a Model Profen, 2-Phenylpropionic Acid (2-PPA), in Rat Hepatocytes

(UCSF) Li et al. (2002) Chem. Res. Toxicol. 15: 1480-1487. 22

Enantioselective Covalent Binding Studies with 2-Phenylpropionic Acid in vitro in Rat Hepatocytes

•Covalent binding to protein correlates closely with acyl-CoA formation but not acyl glucuronidation. •Corresponding studies in vivo in

rat showed CB to liver protein in favor of the acyl-CoA thioester.

Li et al., JPET, 305, 250-256 (2003)

23 (UCSF) Li et al. (2002) Chem. Res. Toxicol. 15: 1480-1487.

[14C]Phenylacetic Acid: Reversible Covalent Binding to Protein in Rat Hepatocytes

SNH

NH

OP

OP

O O

H OH

H3C CH3

O

OH

O

OHO

O

H

H

O OH

H

H

N

NN

N

NH2

P

OH

OHO

O

O

OH

O

NH

O

OH

Phenylacetic Acid (PAA)

Phenylacetyl-S-Acyl-CoA (PA-CoA)

Phenylacetylglycine (PA-gly)

Protein adducts

Acyl-CoA Synthetase

ATP, CoASH

Transacylation of protein nucleophiles

Glycine conjugation

(Amgen) Grillo and Lohr (2009) Covalent binding of phenylacetic acid to protein in rat hepatocytes. Drug Metab. Dispos. 1073-82.

97

KDa

200

66

45

31

21

116

3329

KDa

• PAA-acyl-CoA formation in rat hepatocytes leads to the highly selective, but reversible,

covalent binding to hepatocyte proteins, but not to the transacylation of glutathione.

24

0

20

40

60

80

10

0

12

0

14

0

16

0

18

0

0

50

100

150

200

250

300

350 PA-CoA

0.00.10.20.30.40.50.60.70.80.91.01.11.21.31.4

Covalent Binding

Incubation Time (min)

Co

va

len

t B

ind

ing

(pm

ol e

qu

iva

len

ts/m

g p

rote

in)

[PA

-Co

A],

M

Ibuprofen, Ibufenac, Zomepirac, Tolmetin, Suprofen, Tienilic acid, Fenclozic acid all formed acyl glucuronides, but did not lead to covalent binding to liver microsomal protein.

Ibuprofen, Ibufenac, Fenclozic acid, and Tolmetin → Acyl-CoA-mediated covalent binding

Suprofen and Tienilic acid → P450-mediated covalent binding

Darnel et al. (2015) Chem. Res. Toxicol. 28: 886-896.

In vitro covalent binding/HLM

25

Metabolism of Nicotinic Acid

Stern (2007) The role of nicotinic acid metabolites in flushing and hepatotoxicity. J. Clin. Lipidology 1: 191-193.

http://lpi.oregonstate.edu/mic/vitamins/niacin

Flushing and hepatotoxicity are important adverse effects of nicotinic acid.

26

Liver Dysfunction Cases of severe hepatic toxicity, including fulminant hepatic necrosis.

Dosing: • Recommended

dietary allowance = 12-16 mg/day.

• Tolerable upper intake level = 35 mg/day (to avoid flushing).

•Hepatotoxicity observed at 500 mg/day and higher.

In Vitro Covalent Binding Studies with [14C]Nicotinic Acid and [14C]Diclofenac in Human Hepatocytes

fcvb= (CVB•0.26 mg)/(turnover•4000 pmol) CVB burden = fcvb*dose

Unpublished observations *Thompson et al. (2012) Chem. Res. Toxicol. 25: 1616-1632. 27

0 1 2 3 4

0

5 0

1 0 0

1 5 0

2 0 0

2 5 0

0

2 0

4 0

6 0

8 0

1 0 0

[1 4

C ]-N ic o tin ic a c id (1 0 M ) a n d [1 4

C ]D ic lo fe n a c (1 0 M )

T im e -d e p e n d e n t C o v a le n t B in d in g to P ro te in in In c u b a tio n s w ith

H u m a n H e p a to c y te s

(1 m ill io n v ia b le c e lls /m L ) in S u s p e n s io n (W ill ia m s M e d ia E )

In c u b a tio n T im e (h )

Co

va

len

t B

ind

ing

(pm

ol/

mg

pro

tein

)

D ic lo fe n a c

C o v a le n t B in d in g

D ic lo fe n a c

M e ta b o lism

% R

em

ain

ing

N ic o t in ic A c id

C o v a le n t B in d in g

N ic o t in ic A c id

M e ta b o lism

CompoundCVB (pmol/mg

protein)Incubation

Time (h)Turnover fcvb

Daily Dose (mg)

CBV Burden (mg)

Nicotinic Acid

83 2 0.35 0.015500

7.7158 4 0.52 0.020 9.9

Diclofenac114 2 0.95 0.008

2001.6

*140 *2 *1.0 *0.0092 *1.8

Acyl-CoAs reported to be 20- to 100-fold more reactive toward GSH than the corresponding acyl glucuronides in buffer (pH 7.4, 37°C)

Grillo (2011) Current Drug Metab. 12:229-244. 28

• Ibuprofen 20-fold

• Clofibric acid 40-fold

• 2-Phenylproprionic 70-fold

• Naproxen 100-fold

Acyl-S-CoA Reactivity with Glutathione: Influenced by both Steric and Electronic Effects

Salicylic (ortho-hydroxy-benzoic)

Phenoxy- acetic

Skonberg et al.(2008) Metabolic Activation of Carboxylic Acid Drugs. Expert Opin. Drug Metab. Toxicol. 4: 425-438.

•Increasing the degree of substitution at the alpha-carboxy-carbon correlates with increasing stability (decreasing reactivity with GSH) of the acyl-CoA.

29

Reactivity of S-Acyl-Glutathione Thioesters (0.1 mM) with N-Acetylcysteine (NAC, 1 mM) at 25oC and pH 7.4

(UCSF) Grillo and Benet (2002) Drug Metab Dispos. 30: 55-62.

• Increasing the degree of substitution at the alpha-carboxy-carbon correlates with increasing stability (decreasing reactivity with NAC) of the

acyl-SG.

30

•Proposal that Ibuprofen-acyl-CoA thioester, and not ibuprofen acyl glucuronide, mediates the transacylation of GSH in rat hepatocytes.

Enantioselective Studies with (R)- and (S)-Ibuprofen/Hepatocytes

(Pharmacia/Pfizer) Grillo and Hua (2008) Chem. Res. Toxicol. 21:1749-1759. 31

Enantioselective Bioactivation of Ibuprofen by Acyl-CoA Formation Favoring the R-Ibuprofen Isomer in Rat Hepatocytes

Acyl-CoA Formation Acyl-SG Formation

(Pharmacia/Pfizer) Grillo and Hua (2008) Chem. Res. Toxicol. 21:1749-1759. 32

Carboxylic Acids Bioactivated by Acyl-CoA Formation Leading to S-Acyl-Glutathione Adducts

Grillo (2011) Current Drug Metab. 12:229-244. 33

Ibuprofen

Diclofenac

Flunoxaprofen

Benoxaprofen

Zomepirac

Tolmetin

Mefenamic Acid

Cholic Acid analogues

In vivo (Advil, 800 mg)

(Amgen) Grillo et al. (2013) Interaction of Ƴ-Glutamyltranspeptidase with Ibuprofen-S-Acyl-glutathione in vitro and in vivo in human. Drug Metab. Dispos. 41:111-121. 34

In Vivo Formation of Ibuprofen-N-Acyl-Cysteine in Rats dosed with Pseudoracemic Deuterated D3-(R)- and D0-(S)-Ibuprofen

LC-MS/MS Analysis of Rat Urine Extracts

(Pharmacia/Pfizer) Grillo and Hua (2008) Unpublished observations

D3-(R)-Ibuprofen

H

D3C O

OH

H3-(S)-Ibuprofen

H

H3C O

OH

Ibuprofen-N-Acyl-Cysteine

OH

HD3C

O

HN

O

SH

35

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Retention Time (min)

0

20

40

60

80100

020

40

60

80100

020

40

60

80100

Re

lati

ve

Ab

un

da

nc

e

020

40

60

80

100

310 > 161

313 > 164

313 > 164

310 > 161

A) Ibuprofen Psuedoracemate Dosed Rat Urine Extract

B) Ibuprofen Psuedoracemate Dosed Rat Urine Extract

C) D3-(R)-Ibuprofen-N-Acyl-Cystiene Synthetic Standard

D) D0-(R,S)-Ibuprofen-N-Acyl-Cystiene Synthetic Standard

Benoxaprofen: Mechanisms of Benoxaprofen-induced Cholestasis

•A number of cases of hepatotoxicity in elderly patients led to the withdrawal of the drug from the market.

•“…the history of this drug and its adverse effects is an important chapter in the annals of hepatotoxicity.”

•Hepatotoxicity observed mostly in elderly females.

• “Morphologic features consisted of severe hepatic cholestasis accompanied by slight to moderate necrosis. No evidence for immune-based hypersensitivity”.

•Bioactivation by P450 not observed.

•Metabolic idiosyncratic reaction proposed.

•Proposed to be due to insoluble metabolite (“acyl glucuronide”) excreted in bile leading to blockage of bile flow, cholestasis.

Boelsterli et al. (1995) Critical. Rev. Toxicol. 25:207-235. Boelsterli (2007) Mechanistic Toxicology 2nd ed. CRC Press.

Benoxaprofen glucuronide

Insoluble metabolite?

36

Benoxaprofen: Mechanisms of Benoxaprofen-induced Cholestasis

• Incubation of benoxaprofen-S-acyl-glutathione with bovine gamma-glutamyltransferase (Ƴ-GT) leads to precipitate formation in vitro.

• Identity of precipitate not yet known.

•Ƴ-GT is located in the bile canaliculus membrane.

•Species differences in hepatic Ƴ-GT levels. -Rat low, human high.

•Ƴ-GT levels increase with age.

•Proposal that insoluble precipitate formed from the Ƴ-GT-mediated breakdown of benoxaprofen-acyl-SG may play a role in hepatic cholestasis.

Unpublished observations, Hinchman and Ballatori (1990) Glutathione-degrading capacities of liver and kidney in different species. Biochem. Pharmacol. 40: 1131-1135.

Benoxaprofen-S-acyl

Glutathione

Benoxaprofen-S-acyl

Glutathione

Insoluble precipitate?

Ƴ-GT

37

7th International Conference on Early Toxicity Screening ETS-2008, Seattle, WA Metabolism-Based Toxicity in Drug Development Role of Drug Metabolism in Drug-induced Liver Toxicity (Sid Nelson, University of Washington; Seattle, WA) Drug-induced liver injury is one of the most common reasons for failure of drugs in development, and for either removal of drugs from the market or "black-box" warnings on marketed drugs. This presentation will focus on the role of reactive metabolites as a major cause of drug-induced liver injury, on structure-toxicity relationships, and on methods to assess risk from reactive metabolites.

“The role of acyl glucuronides in carboxylic acid drug toxicity may have been oversold.”

38

Summary

The chemical reactivity, covalent binding, and toxicity of acyl glucuronides has been widely-studied, although a clear link has not been established.

Acyl-CoA thioesters are substantially more reactive than their corresponding acyl glucuronides, and increasing evidence demonstrates that acyl-CoA thioesters contribute more importantly than acyl glucuronides to the covalent modification of hepatic protein in vitro and in vivo.

The importance of chemically-reactive acyl-CoA metabolites in drug-induced hepatotoxicity may be underestimated.

39

Acknowledgements UW Seattle

Tom Baillie , PhD, DSc Dave Porubek, PhD

UC San Francisco Chunze Li, PhD

Leslie Z. Benet, PhD

MyoKardia Jim Driscoll

Pharmacia/Pfizer PDM Amgen PKDM

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