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Identification of reactive metabolites in
early drug discoveryPhil Butler Ph.D. Senior Research Scientist, Cyprotex
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Overview
- Impact of reactive metabolites on drug safety/discovery
- Assessing reactive metabolite formation
- High throughput methods of trapping reactive metabolites
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- Most frequently formed via oxidation reactions with P450 enzymes
being predominantly involved in the catalysis.
- Specific chemical substituents (structural alerts/toxicophores) mayundergo oxidative metabolism leading to reactive metaboliteformation.
- Not solely restricted to phase I metabolic pathways. Phase IImetabolism (e.g. glucuronidation) may lead to reactive metabolitegeneration.
Reactive metabolite formation
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Bioactivation
Reactivemetabolites
DRUGPhase I/II
Stable metabolites
Excretion
Toxicity
Covalent modification of cellularmacromolecules
Altered cellular function
Bioinactivation
Defencemechanisms
Reactive metabolite formation
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Adverse drug reactions (ADRs)
- Any unwanted effect of a drug aside from its expected therapeuticactions.
- Major cause of patient morbidity and mortality.
- Major impediment to process of drug development.
- Drug withdrawal from market?
A. W. Asscher et al., (1995) Bmj. 311, 1003-1006J. Lazarou et al., (2005) Jama. 279, 1200-1205
M. Pirmohamed et al., (1998) Bmj. 316, 1295-1298S. Michelson & K. Joho, (2000) Curr Opin Mol Ther. 2, 651-654
In 1998 >$20 billion was spent on identification and development of drugs.>20% spent on screening methods and toxicity tests.
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Type A (augmented):
- predictable- dose-dependent- exaggeration of pharmacology of drug
B. K. Park et al., (1998) Chem Res Toxicol. 11, 969-988
Classification of ADRs
Type C (chemical):
- predictable from chemical structure- e.g. acetaminophen
Type B (idiosyncratic):- unpredictable- more frequently life-threatening
- less common- seemingly dose-independent
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- Liver is common target due to its major role in metabolism ofxenobiotics.
- >800 drugs have been implicated in causing hepatic injury orhepatotoxicity.
- Drug-induced liver injury is most frequent reason for removal of an
approved drug from the market.
- Drug-induced liver injury accounts for more than 50% of cases ofacute liver failure in USA.
M. Dossing & J. Sonne, (1993) Drug Saf. 9, 441-449
W. M. Lee, (2003) Semin LiverDis. 23, 217-226
Effect of ADRs on the liver
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Drugs
withdrawn for
hepatotoxicity:
5 of 6 havereactive
metabolites
BenoxaprofenIproniazidNefazodoneTienilic acidTroglitazone
Bromfenac (notdetermined)
Drugs with Black Box
warnings for
hepatotoxicity:
8 of 15 have reactivemetabolites
Dacarbazine, DantroleneFelbamate, FlutamideIsoniazid, KetoconazoleTolcapone, Valproic acid
Reactive metabolites notreported for :
Acitretin, Bosentan,Gemtuzumab,Ozogamicin, Naltrexone,Nevirapine, Pemoline,Trovafloxacin
Drugs with a warning
of precaution for
hepatotoxicity:
AcetaminophenCarbamazepineClozapine, DiclofenacDisulfiram, HalothaneLeflunomide,Methyldopa, RifampinTacrine, TamoxifenTerbinafine, TiclopidineZileuton
Drugs associated
with hepatotoxicity
& never approved
in US:
Alpidem, AmineptineAmodiaquineCinchophenDihydralazineDilevaolo, EbrotidineGlafenine, IbufenacIsoxanine,Niperotidien
Perhexiline,PirprofenTilbroquinol
62% involve metabolism
and reactive products
J. L.Walgren et al., (2005) Crit. Rev. Toxicol. 35, 325-361
Impact of reactive metaboliteson drug safety
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- Elucidation of the role of a particular reactive metabolite in ADRs is difficult.
- Unable to predict the potential of a new drug to cause ADRs.
Prevent reactive metabolite formation
Improve drug safety Reduce chance of ADRs
Reactive metabolites
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- Chemical manipulation.- Avoiding structural alerts/toxicophores.
- Avoid structure-based risk- Greater scholarship regarding potential metabolic routes anddownstream consequences.
Improving drug safety
- Screening out metabolic risk- Covalent binding studies- Reactive metabolite screens
- Greater efficacy.- Lower dose.
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- Chemical manipulation.- Avoiding structural alerts/toxicophores.
- Avoid structure-based risk- Greater scholarship regarding potential metabolic routes and
downstream consequences.
Improving drug safety
- Screening out metabolic risk- Covalent binding studies- Reactive metabolite screens
- Greater efficacy.- Lower dose.
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Y.-J. Wu et al., (2003) J Med Chem. 46, 3778-3781
Potassium channel opener
Equipotent potassium channel opener
Irreversible CYP3A4 inhibitor
No CYP3A4 inhibition
O
NH
N
O
O
NH
F F
N
O
Designing around metabolic risk
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- Chemical manipulation.- Avoiding structural alerts/toxicophores.
- Avoid structure-based risk- Greater scholarship regarding potential metabolic routes anddownstream consequences.
Improving drug safety
- Screening out metabolic risk- Covalent binding studies- Reactive metabolite screens
- Greater efficacy.
- Lower dose.
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- High doses commonly associated with adverse events.
- However, exposure/biologically effective dose (e.g. AUC) is far better indicator ofactual amount of a drug that a patient is exposed to due to ADME/protein
binding etc.
Reactive metabolite formation in vitro.1 % incidence of agranulocytosis.Dose: 300 mg/day.
0.1-0.5 % glucuronidation.
Reactive metabolite formation in vitro.No in vivo manifestation.Dose: 10 mg/day.
21-25 % glucuronidation.
J. M. Alvir & J. A. Lieberman, (1994) J Clin Psychiatry. 55, 137-138I. Gardner et al., (1998) Mol Pharmacol. 53, 991-1008
J. P. Uetrecht, (2000) CurrDrug Metab. 1, 133-141
Cl
NH
N
N
N
CH3
NH
N
N
N
CH3
CH3
Role of dose in drug-induced toxicity
Clozapine Olanzapine
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- Chemical manipulation.- Avoiding structural alerts/toxicophores.
- Avoid structure-based risk- Greater scholarship regarding potential metabolic routes anddownstream consequences.
Improving drug safety
- Screening out metabolic risk
- Covalent binding studies
- Reactive metabolite screens
- Greater efficacy.- Lower dose.
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- Greater chemical flexibility.
- Less quantitative, higher-throughput screening.
- Rank ordering of compounds
Later stage screening may be more quantitative, detailed and definitive, but
what options do you have when detecting a reactive metabolite at a later
stage?
Screening for reactive metabolites in early drugdiscovery
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Radiolabeled Studies
covalent binding of radiolabeled compound (10M)
in vitro using liver preparations (HLM and RLM; 1mg/ml; NADPH)
in vivo studies dose to rat (20mg/kg)
assess adducts to liver and plasma proteins
D.C. Evans et al., (2004) Chem. Res. Toxicol. 17, 3-16
Merck approach for assessingreactive metabolite formation
Biomarker Studies
covalent binding to a model nucleophile (e.g. glutathione (GSH), cyanide)
uses liver microsomes
trap and characterise reactive metabolites
stable adducts formed
surrogate marker of covalent binding potential
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D.C. Evans et al., (2004) Chem. Res. Toxicol. 17, 3-16
Merck approach for assessingreactive metabolite formation
Radiolabeled Studies
>50pmol eq/mg protein?
- Potential structural modification?
- Availability of existing treatments?
- Daily dose
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Radiolabeled Method
Advantages
- In vitro studies - species differences can be explored
- In vivo studies - related to safety studies
- extrahepatic bioactivation
Advantages and disadvantages ofradiolabeled method
Disadvantages
- Requires radiolabeled compound
- Not suitable for early stage studies
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Biomarker Method
Disadvantages
- No single small molecule serves as universal surrogate- May require follow up study in hepatocytes if positive in microsomes
Advantages and disadvantagesof biomarker method
Advantages
-Amenable to HTS and early stage studies
- Characterisation by LC-MS/MS
- Prioritisation of compounds for radiolabeling
- Indirect information on structure of reactive species
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- Most commonly used biomarker for covalent binding
- Endogenous tripeptide thiol.
- Serves several functions including:- Detoxifying electrophiles- Maintaining essential thiol status of proteins- Modulating critical cellular processes
- Soft nucleophile forms conjugates either spontaneously orenzymatically in reactions catalyzed by GSH S-transferases (GSTs).
- Found in most cells but particularly abundant in liver (5mM).
Physiological role of GSH
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-To clarify the relationship between in vitro formation rate of GSHconjugates and covalent binding to protein.
N. Masubuchi et al., (2007) Chem. Res. Toxicol. 20, 455-464
1- Tienilic acid2- Furosemide3- Clozapine4- Imipramine5- Acetaminophen6- Indomethacin7- Diclofenac8- Carbamazepine
Relationship between covalent binding and GSHconjugate formation
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Microsomal incubation + NADPH + GSH
Detection by LC-MS/MS
Monitoring for a constant neutral loss (CNL) of 129(loss of glutamic acid of GSH)
Independent of drug structure
Amenable to HTS
CNL of 129 is not exclusive for GSH adducts
- Issues with sensitivity and selectivity
- False positives
HS
HN
O
NH2
COOHO
NH
HOOC
Standard method for analysis of GSH conjugates
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Exact mass neutral loss of 129.046 Da.- Endogenous structures in biological matrices may have samenominal mass of 129 Da but less likely to have same exact mass
- Excludes false positives
Improving selectivity
Negative ion MS/MS affords common fragment at m/z 272- Not all GSH conjugates may undergo CNL 129.- MS/MS of GSH adducts in negative mode mostly fragments ofGSH- Precursor ion scan of m/z 272 & positive ion CNL 129?
J. Castro-Perez et al., (2005) Rapid Commun Mass Spectrom. 19, 798C. M. Dieckhaus et al., (2005) Chem Res Toxicol. 18, 630
Sensitivity?
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Better MS signal intensity
- Improves detection of reactive metabolites
Enables use of lower substrate concentration
- Reduces compound solubility issues
- Minimises chance of enzyme saturation
- Decreases compound use
J. R. Soglia et al., (2004)J Pharm Biomed Anal. 36
, 105-116
Use of GSH ethyl ester as reactive metabolitetrapping agent
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J. Gan et al., (2005) Chem Res Toxicol. 18, 896-903
- Synthesised fluorescent trapping agent .
- Comparative chemical reactivity vs GSH.
- NOT a co-factor for GST-mediated adduct formation.
- Some separation issues from dGSH itself (30 min HPLC gradient).
- No characteristic loss of 129.
- Issues with compounds that causefluorescent interference.
Use of dansyl GSH as a trapping agent
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Semi-quantitative method for determining reactive metabolite levels usingquaternary ammonium GSH analogue (QA-GSH)
- Fixed positive charge significantly increased limit of detection.
- Equalized MS response from equimolar amounts of different GSHconjugates.
- m/z of QA-GSH conjugate determined for M+ or MH2+ ion.
- Response factor for IS with same charge state determined (peakarea/conc.)
- Peak area of QA-GSH conjugate/response factor of IS providessemi-quantification.
J. R. Soglia et al., (2006) Chem Res Toxicol. 19, 480-490
Quantifying conjugation?
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GSH
Drug
Reactive metabolite
GSH conjugate (MH++305) Triply labeled GSH adduct (MH++308)
GSH*
Z. Yan & G. W. Caldwell, (2004)Anal Chem. 76, 6835-6847Z. Yan et al., (2005) Drug Metab Dispos. 33, 706-713Z. Yan et al., (2005) Rapid Commun Mass Spectrom. 19, 3322-3330
NH
O
HS
NH
O
NH2
COOH
CH2HOOC
13
13
15
NH
O
HS
NH
O
NH2
COOH
CH2HOOC
13
13
15NH
O
HS
NH
O
NH2
COOH
CH2HOOC
Use of stable-isotope labeled GSH in high-throughput screenings of reactive metabolites
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GSH Triply-labeled GSH
1:1 mixture
Microsomal incubation
Solid-phase extraction Neutral loss scanning(129 Da)
M-SG*M-SG
Z. Yan & G. W. Caldwell, (2004)Anal Chem. 76, 6835-6847Z. Yan et al., (2005) Drug Metab Dispos. 33, 706-713Z. Yan et al., (2005) Rapid Commun Mass Spectrom. 19, 3322-3330
3 Da
Use of stable-isotope labeled GSH in high-throughput screenings of reactive metabolites
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- Unique MS signature of isotopic doublet that differs in mass by 3 Da.
- Isotopic doublet exhibits approximately same intensity.
- Halogenated compounds (e.g. diclofenac, bromobenzene) form > 1doublet providing further confirmation of conjugation to GSH.
- High confidence by subsequent MS/MS analysis of neutral losses of 75and 129 Da for GSH adducts and 78 and 129 Da for isotopic GSH*
adducts.
- Weak intensity of [MH+129] and [MH+75]?- Supporting data Rt and peak area ratio of isotopic adducts.
Z. Yan & G. W. Caldwell, (2004)Anal Chem. 76, 6835-6847Z. Yan et al., (2005) Drug Metab Dispos. 33, 706-713
Z. Yan et al., (2005) Rapid Commun Mass Spectrom. 19, 3322-3330A. Mutlib et al., (2005) Rapid Commun Mass Spectrom. 19, 3482-3492
Use of stable-isotope labeled GSH in high-throughput screenings of reactive metabolites
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Positive controls
BromobenzeneCarbamazepineClozapinep-cresolDiclofenac-estradiol17--ethynylestradiolFelbamate4-hydroxyestrone3-methylindoleOmeprazolePhenacetin
DextromethorphanFluoxetineKetoconazoleMidazolamTerfenadineTestosteroneTolbutamide
Negative controls
Felbamate requires both esterase and aldehyde dehydrogenase.
Z. Yan & G. W. Caldwell, (2004)Anal Chem. 76, 6835-6847Z. Yan et al., (2005) Drug Metab Dispos. 33, 706-713Z. Yan et al., (2005) Rapid Commun Mass Spectrom. 19, 3322-3330
Use of stable-isotope labeled GSH in high-throughput screenings of reactive metabolites
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Trapping Agents Functional Group
Glutathione (ester), mercaptoethanol,cysteine
Quinones, enones
Cyanide Iminium ionsSemicarbazideMethoxylamine
Aldehydes
Lysine Imides, aryl halides
Lysine + cysteine Furan, epoxide
TEMPO
(Tetramethylpiperidin-N-oxyl)
Free radical trap
Trapping soft and hard reactive metabolites
Standard screens may detect soft electrophiles such as quinones,quinone imines, epoxides etc
Detection of hard electrophiles may require separate trappingexperiments
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Z. Yan et al., (2007)Anal Chem. 79, 4206-4214
Trapping soft and hard reactive metabolites
- Glycine of GSH replaced by lysine residue.
- Isotopic analogue used for stable isotope trapping experiments
- All adducts undergo CNL of 129 Da.
- Both natural and labeled agents added to incubations distinct isotopicdoublet with mass difference of 8 Da.
-Glu Cys 13C6-15N2-Lys
-129 DaS-R N=R
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- Not all reactive intermediates form adducts with GSH.
- Presence of GSH adduct in vitro does not mean pathwaypredominates in vivo.
- Risk of false negatives enzymes involved in bioactivation.
- Covalent binding does not always result in toxicity.
- Absence of GSH adduct does not guarantee safety.
- Liability screens.
Interpreting GSH conjugation screens
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Points to consider
- Quantification of reactive metabolite formation?
- In vitro to in vivo relationship?
- What does it mean?
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LaterStage
< 50 pmol eq/mg
Advance compound
> 50 pmol eq/mg
Qualifying considerations
Suggested screening strategy forreactive metabolite assessment
Designing out metabolic weaknesses?
Prioritization of drug candidates
Radiolabeling for covalent binding
Assess bioactivation potential
Optimize lead compounds
Early
Stage
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