Managing the challenge of chemically reactive metabolites in drug ...
Risk Assessment of Reactive Metabolites in Drug Discovery ...
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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]
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
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Drugs Withdrawn and Associated with Idiosyncratic Drug Toxicity Alcofenac (anti-inflammatory) Hepatitis, rash Alpidem (anxiolytic) Hepatitis (fatal) Amodiaquine (antimalarial)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.
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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).
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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
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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.
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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.
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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
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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.
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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
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• “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?
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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
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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.”
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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.
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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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