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 [email protected] CommercializationDrug Process Discovery Overview & Development

Therapeutic Area/Disease Lead Selection & Product Strategy Market Readiness & Launch / Post-Launch Opportunity Assessment Pre-clinical Testing Development Regulatory Approval Lifecycle Management

Hit-to- Lead Post- Discovery Screen Pre-clinical Phase 1 Phase 2 Phase 3 Filing Launch Lead Optimization Launch

Preclinical Pharmacokinetics & Drug Metabolism Assays Target Selection & Validation Lead compound Selection Discovery Screen Hit-to-Lead Lead Optimization Optimize Pharmacokinetics Characterize Preclinical Candidate Liver microsomal stability Intrinsic clearance Membrane permeability Excretion balance Efflux/transport assays Definitive metabolite ID Plasma stability Higher species PK Rat PK Human PK prediction Protein binding Assess DDI Potential Blood to plasma ratio Competitive CYP IC50 Early DDI Assessment Time-dependent CYP inhibition Enzyme induction PXR activation Hepatocyte induction CYP inhibition CYP reaction phenotyping

Detection and Assessment of Chemically-Reactive Metabolites

2 Circumstantial Evidence Links Reactive Metabolites to Adverse Drug Reactions

• 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. Liver • Underlying mechanisms not clear. Injury

3 • Believed to be immune-mediated. Drugs Withdrawn and Associated with Idiosyncratic Drug Toxicity Alcofenac (anti-inflammatory) Metiamide (antiulcer) Hepatitis, rash Bone marrow toxicity Alpidem (anxiolytic) Nomifensine (antidepressant) Hepatitis (fatal) Hepatitis (fatal), anemia Amodiaquine (antimalarial) Practolol (antiarrhythmic) Hepatitis, agranulocytosis Severe cutaneous ADRs Amineptine (antidepressant) Remoxipride (antipsychotic) Hepatitis, cutaneous ADRs Aplastic anaemia Benoxaprofen (anti-inflammatory) Sudoxicam (anti-inflammatory) Hepatitis, cutaneous ADRs Hepatitis (fatal) Bromfenac (anti-inflammatory) Tienilic Acid (diuretic) Hepatitis (fatal) Hepatitis (fatal) Carbutamide (antidiabetic) Tolrestat (antidiabetic) Bone marrow toxicity Hepatitis (fatal) Ibufenac (anti-inflammatory) Troglitazone (antidiabetic) Hepatitis (fatal) Hepatitis (fatal) Iproniazid (antidepressant) Zomepirac (anti-inflammatory) Hepatitis (fatal) Hepatitis, cutaneous ADRs  In many cases, metabolic activation reactive metabolites leading to covalent binding to protein demonstrated in vitro and/or in vivo.

4 Carboxylic acid drugs Kalgutkar and Soglia (2005) Exp. Opin. Drug Metab. Toxicol. 1, 91-141. Danger Hypothesis Illustration for Immune-mediated Idiosyncratic Hepatotoxicity

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

• 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 Park et al. (2011) Nature Rev. Drug Discov., 10:292-306. Evans et al. (2004) Chem. Res. Toxicol., 17:3-16. 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 Kalgutkar (2009) Chem. Biodiver. 6, 2115-36; Lammert et al., Hepatology 47, (2008) 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.

CVB Burden f cvb= (CVB•0.26 mg)/(turnover•4000 pmol) (#9) Ibufenac 24 CVB burden = fcvb•dose (#32) Ibuprofen 5.2 (#20) Diclofenac 1.8 Recommendation: <1 mg/day exposure (#29) Caffeine <0.23 (CVB burden) to reactive metabolites

8 Thompson et al. (2012) Chem. Res. Toxicol. 25: 1616-1632. 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 Drug NL: 2000 UV 280 nm GSH-adduct 5.38E4 1000 nm=280.0- Rat 280.0 PDA 0 hydroxylated 3509rlm+n+g -1000 metabolite uAU metabolite -2000 ndr -3000 -4000 NL: 2000 UV 280 nm 5.94E4 nm=280.0- 1000 280.0 PDA 0 Dog 3509dlm+n+ g -1000 uAU -2000 -3000 -4000

NL: 2000 5.16E4 1000 nm=280.0- UV 280 nm 280.0 PDA 0 3509clm+n+ Cyno monkey g

-1000 uAU -2000 -3000 -4000 NL: 2000 5.92E4 UV 280 nm nm=280.0- 1000 280.0 PDA 0 3509hlm+n+ Human g -1000 uAU -2000 -3000 -4000 ndr, not drug related

7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 Time (min) • 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. 9 Unpublished observations 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 1 7 5 1 1 0 R a d io la b e le d T e st C o m p o u n d

1 4 1 0 0

n n i [ C ]D ic lo fe n a c

1 5 0 i (0 % tu rn o v e r)

e

e )

t 1 4 )

t 9 0

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

o

n

o

i

r

i

r

e

e P

t 1 2 5 8 0

P

t

o

o

o

r

o

r

t t

7 0

p

p

/

/

g g

g 1 0 0

g

n

n i

i 6 0

m

d

m

d

/

/

.

n

.

n

i i

q 5 0

7 5 q

B

B

e

e

t

l t

l 4 0

n

o

n o

e R a d io la b e le d

e

l l

m 5 0 m

a 3 0 a

p T e st C o m p o u n d

p

v

(

v

(

o o

2 0 1 4 C 2 5 C [ C ]D ic lo fe n a c 1 0 (8 7 .5 % tu rn o v e r) 1 4 0 0 [ C ]D ic lo fe n a c (lite ra tu re ) 0 1 2 3 4 0 1 2 3 4 In c u b a tio n T im e (h ) In c u b a tio n T im e (h ) CVB (pmol/mg Incubation Turnover Daily CVB Burden Compound f protein) time (h) (%) cvb Dose (mg) (mg) 24.0 ± 2.6 1 0 ± 3.0 0.174* 13.9 Test Compound 34.8 ± 4.8 2 0 ± 2.9 0.252* 80† 20.2 49.8 ± 8.3 4 0 ± 4.3 0.361* 28.8 78.9 ± 2.6 1 68.6 ± 2.8 0.008 1.67 Diclofenac 109. ± 8.3 2 78.9 ± 1.1 0.010 200 2.01 151.6 ± 7.5 4 87.6 ± 0.5 0.013 2.51 Diclofenac 140.8 2 100 0.0092 200 1.84 (Thompson, et al.) 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

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 https://www.fda.gov/downloads/Drugs/.../Guidances/ucm079266.pdf Partial list of carboxylic-acid containing drugs withdrawn from clinical use and/or also associated with allergic reactions Withdrawn Allergic reactions O C l C l O O C l O OH O OH OH OH C l O O C H3 O H C H3 S H3C Ibuprofen Clofibric Acid Alclofenac Tienilic Acid O OH O C l Br NH2O C l O O OH OH C l NH O N N S H OH O C H3 O Bromfenac Benoxaprofen Probenecid Diclofenac O O F HO OH O O OH OH O N OH H F O C H3 H Ibufenac H C Flunoxaprofen 3 O Fenoprofen Diflunisal C H O O O 3 F O N OH OH O C H3 O OH C l N H N OH H3C C H3 H 3C Indoprofen Zomepirac Tolmetin Valproic Acid

Fung et al. (2001) Drug Information Journal, 35, 293-317. 12 Stepan et al. (2011) Chem Res Toxicol, 24, 1345-1410. Drug Acyl Glucuronide: Instability (Primarily by Acyl Migration)

100 •Incubation of 1-O-β- 90 1 0 0 drug-acyl glucuronide 80 o 8 0

g in buffer (pH 7.4, 37 C)

Time = 0 min n 70 i

b n 1-O- - i t ~ 1 0 m in 6 0 1 /2 60 o a to determine pH 7.4, 37 C m

isomer e

R 4 0

50 degradation rate/ % 40 2 0 degradation half-life. 30 0 20 0 5 1 0 1 5 2 0 2 5 3 0 In c u b a tio n T im e (m in ) •LC-MS/MS analysis: 10 0 Xue et al. 100 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 “Optimization to 90 1-O-b- * eliminate interference 80 isomer

Relative Intensity (%) (%) Intensity Relative 70 Time = 20 min of migration isomers 60 o for measuring 1-O-β- pH 7.4, 37 C ** 50 acyl glucuronide with 40 out extensive 30 *Acyl migration isomers 20 chromatographic ** 10 separation”. Rapid 0 Commun. Mass 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Retention Time (min) Spectrom. 2008; 22: 109–120. 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 O O O CH3 O CH3 N OH Tolmetin N OH Zomepirac Cl H3 C

H3C

H O N O OH Carprofen Fenoprofen OH O Cl H H H3 C H 3 C

H C 3 O H O O H O H C H 3 N Furosemide Beclobric Acid O O O S H N O Cl 2 Cl

“Increasing the degree of 8

3) 3) 3)

- - substitution at the alpha- - 7 carboxy-carbon correlates 6 Tolmetin with increasing stability Zomepirac (decreasing reactivity with 5

protein) of the acyl 4

glucuronide. 3

Benet et al. (1993) Life Sci, 53:141-6 (R)-Fenoprofen 2 (S)-Fenoprofen (R)-Carprofen

1 (S)-Carprofen

Covalent Binding (mole/mole protein x x e e protein protein (mole/mole (mole/mole Binding Binding Covalent Covalent Covalent Binding (mole/mole protein x e protein (mole/mole Binding Covalent Furosemide 0 0.0 0.5 1.0 1.5 2.0 Smith et al. (1986) J Clin. Invest. 77:934-39 (R,S )-Beclobric Acid

15 Degradation Rate Constant (k, 1/hrs) • “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 Predictability of Idiosyncratic Drug Toxicity Risk for Carboxylic Acid-Containing Drugs Based on the Chemical Stability of Acyl Glucuronide

•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.”

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

18 (UW) Porubek et al. (1989) Drug Metab Dispos. 2: 123-130. Proposed Metabolism Routes of Carboxylic Acid-Containing Drugs to Reactive Derivatives and Non-Toxic Products

Remains intracellular, not excreted, doesn’t Excretion in urine, circulate in plasma bile, detection in plasma 1 2

Conjugation 4 3 2 - , 3 - , 4 - O - A c y l Reactions: Glucuronides

Glycine Amides Taurine Amides Acyl-CoA Thioester 5 Carnitine Esters Hybrid Triglycerides 1 - O - A c y l G l u cu r o n i d e Choline Esters Covalent Binding 6 Fatty Acylation? Open-Chain to Protein Aldehydes

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

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

Detoxification?

Adverse Drug Reactions?

Detoxification 20 Acyl-CoA Thioesters Shown to Acylate Proteins In Vitro (early reports)

Salicyl-SCoA

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

Arachidonyl-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.

Palmitoyl-SCoA

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

Nafenopin-SCoA

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

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

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

350 PA-CoA 1.4 OH Covalent Binding 1.3 300 1.2 O 1.1

250 1.0 [PA-CoA], Phenylacetic Acid (PAA) 0.9 200 0.8 Acyl-CoA ATP, CoASH NH2 0.7 Synthetase

N 150 0.6  N CH M O O H3C 3 OH OH 0.5 S O O O N N N P P H H N CovalentBinding 100 0.4 H H O H OH O O 0.3 O H H Phenylacetyl-S-Acyl-CoA O OH 50 0.2 (PA-CoA) equivalents/mgprotein) (pmol 0.1 Transacylation of protein O P OH Glycine 0 0.0

nucleophiles conjugation OH

0

40 60 80

O 20

100 120 140 160 180 Protein NH Incubation Time (min) adducts OH O Phenylacetylglycine (PA-gly)

KDa 200 116 97 • PAA-acyl-CoA formation in rat hepatocytes 66 leads to the highly selective, but reversible, 45 KDa covalent binding to hepatocyte proteins, but 31 33 29

not to the transacylation of glutathione. 21

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

 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

25 Darnel et al. (2015) Chem. Res. Toxicol. 28: 886-896. Metabolism of Nicotinic Acid

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.

 Flushing and hepatotoxicity are important adverse effects of nicotinic acid.

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

26 http://lpi.oregonstate.edu/mic/vitamins/niacin In Vitro Covalent Binding Studies with [14C]Nicotinic Acid and [14C]Diclofenac in Human Hepatocytes [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 illio n v ia b le c e lls /m L ) in S u s p e n s io n (W illia m s M e d ia E ) 2 5 0 1 0 0

D ic lo fe n a c C o v a le n t B in d in g ) 2 0 0 8 0

%

g

n

i n

i D ic lo fe n a c e

R t d M e ta b o lism

e

o

n

r i

1 5 0 6 0 m

p

B

a

t

i g

n n i

m N ic o tin ic A c id e

n

/ l l 1 0 0 4 0 g

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

o

v m

o N ic o tin ic A c id

p C ( 5 0 2 0 M e ta b o lism

0 0 0 1 2 3 4 fcvb= (CVB•0.26 mg)/(turnover•4000 pmol) In c u b a tio n T im e (h ) CVB burden = fcvb*dose CVB (pmol/mg Incubation Daily Dose CBV Burden Compound Turnover f protein) Time (h) cvb (mg) (mg) Nicotinic 83 2 0.35 0.015 7.7 500 Acid 158 4 0.52 0.020 9.9 114 2 0.95 0.008 1.6 Diclofenac 200 *140 *2 *1.0 *0.0092 *1.8 Unpublished observations 27 *Thompson et al. (2012) Chem. Res. Toxicol. 25: 1616-1632. 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)

• Ibuprofen 20-fold

• Clofibric acid 40-fold

• 2-Phenylproprionic 70-fold

• Naproxen 100-fold

28 Grillo (2011) Current Drug Metab. 12:229-244. Acyl-S-CoA Reactivity with Glutathione: Influenced by both Steric and Electronic Effects

Phenoxy- acetic

Salicylic (ortho-hydroxy-benzoic)

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

29 Skonberg et al.(2008) Metabolic Activation of Carboxylic Acid Drugs. Expert Opin. Drug Metab. Toxicol. 4: 425-438. Reactivity of S-Acyl-Glutathione Thioesters (0.1 mM) with N-Acetylcysteine (NAC, 1 mM) at 25oC and pH 7.4

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

30 (UCSF) Grillo and Benet (2002) Drug Metab Dispos. 30: 55-62. Enantioselective Studies with (R)- and (S)-Ibuprofen/Hepatocytes

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

31 (Pharmacia/Pfizer) Grillo and Hua (2008) Chem. Res. Toxicol. 21:1749-1759. Enantioselective Bioactivation of Ibuprofen by Acyl-CoA Formation Favoring the R-Ibuprofen Isomer in Rat Hepatocytes Acyl-CoA Formation Acyl-SG Formation

32 (Pharmacia/Pfizer) Grillo and Hua (2008) Chem. Res. Toxicol. 21:1749-1759. Carboxylic Acids Bioactivated by Acyl-CoA Formation Leading to S-Acyl-Glutathione Adducts

Zomepirac Ibuprofen Mefenamic Acid

Tolmetin Flunoxaprofen Cholic Acid analogues

Diclofenac Benoxaprofen

33 Grillo (2011) Current Drug Metab. 12:229-244. In vivo (Advil, 800 mg)

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

A) Ibuprofen Psuedoracemate Dosed Rat Urine Extract 100 80 310 > 161 60 D3C O 40 H 20 OH 0 D -(R)-Ibuprofen 100 B) Ibuprofen Psuedoracemate Dosed Rat Urine Extract 3 80 313 > 164 60 40 20 0 100 80 C) D3-(R)-Ibuprofen-N-Acyl-Cystiene Synthetic Standard 313 > 164 H3C O 60 H Relative Abundance 40 OH 20 0 H3-(S)-Ibuprofen 100 80 D) D0-(R,S)-Ibuprofen-N-Acyl-Cystiene Synthetic Standard 310 > 161 60 40 SH 20 O OH 0 N H 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 H O D3C Retention Time (min) Ibuprofen-N-Acyl-Cysteine

35 (Pharmacia/Pfizer) Grillo and Hua (2008) Unpublished observations 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. Benoxaprofen glucuronide •Proposed to be due to insoluble metabolite (“acyl glucuronide”) excreted in bile leading to blockage of bile flow, cholestasis.

Insoluble metabolite? Boelsterli et al. (1995) Critical. Rev. Toxicol. 25:207-235. 36 Boelsterli (2007) Mechanistic Toxicology 2nd ed. CRC Press. 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. Benoxaprofen -Rat low, human high.

-S-acyl Glutathione •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. Benoxaprofen -S-acyl Glutathione

Ƴ-GT

Insoluble Unpublished observations, Hinchman and Ballatori (1990) Glutathione-degrading precipitate? capacities of liver and kidney in different species. Biochem. 37 Pharmacol. 40: 1131-1135.

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

40