Identification of reactive metabolites in

early drug discovery

Phil Butler Ph.D. Senior Research Scientist, Cyprotex
- Impact of reactive metabolites on drug safety/discovery

- Assessing reactive metabolite formation

- High throughput methods of trapping reactive metabolites

Overview
- Most frequently formed via oxidation reactions with P450 enzymes
being predominantly involved in the catalysis.

- Specific chemical substituents (structural alerts/toxicophores) may

undergo oxidative metabolism leading to reactive metabolite
formation.

- Not solely restricted to phase I metabolic pathways. Phase II

metabolism (e.g. glucuronidation) may lead to reactive metabolite
generation.

Reactive metabolite formation

 DRUG
Bioactivation Phase I/II

Reactive Bioinactivation
Stable metabolites
metabolites Defence
mechanisms

Covalent modification of cellular

macromolecules Excretion

Altered cellular function

Toxicity

Reactive metabolite formation

 - Any unwanted effect of a drug aside from its expected therapeutic
actions.

- Major cause of patient morbidity and mortality.

- Major impediment to process of drug development.

- Drug withdrawal from market?

In 1998 >$20 billion was spent on identification and development of drugs.

>20% spent on screening methods and toxicity tests.

Adverse drug reactions (ADRs)

A. W. Asscher et al., (1995) Bmj. 311, 1003-1006
J. Lazarou et al., (2005) Jama. 279, 1200-1205
M. Pirmohamed et al., (1998) Bmj. 316, 1295-1298
S. Michelson & K. Joho, (2000) Curr Opin Mol Ther. 2, 651-654
Type A (augmented):
- predictable
- dose-dependent
- exaggeration of pharmacology of drug

Type B (idiosyncratic):
- unpredictable
- more frequently life-threatening
- less common
- seemingly dose-independent

Type C (chemical):
- predictable from chemical structure
- e.g. acetaminophen

Classification of ADRs

B. K. Park et al., (1998) Chem Res Toxicol. 11, 969-988

- Liver is common target due to its major role in metabolism of
xenobiotics.

- >800 drugs have been implicated in causing hepatic injury or

hepatotoxicity.

- 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 of

acute liver failure in USA.

Effect of ADRs on the liver

M. Dossing & J. Sonne, (1993) Drug Saf. 9, 441-449

W. M. Lee, (2003) Semin Liver Dis. 23, 217-226
 Drugs Drugs with Black Box
withdrawn for warnings for Drugs associated
hepatotoxicity: hepatotoxicity: Drugs with a warning with hepatotoxicity
5 of 6 have 8 of 15 have reactive of precaution for & never approved
reactive metabolites hepatotoxicity: in US:
metabolites
Dacarbazine, Dantrolene Acetaminophen Alpidem, Amineptine
Benoxaprofen Felbamate, Flutamide Carbamazepine Amodiaquine
Iproniazid Isoniazid, Ketoconazole Clozapine, Diclofenac Cinchophen
Nefazodone Tolcapone, Valproic acid Disulfiram, Halothane Dihydralazine
Tienilic acid Leflunomide, Dilevaolo, Ebrotidine
Troglitazone Reactive metabolites not Methyldopa, Rifampin Glafenine, Ibufenac
reported for : Tacrine, Tamoxifen Isoxanine,
Bromfenac (not Terbinafine, Ticlopidine Niperotidien
determined) Acitretin, Bosentan, Zileuton Perhexiline,
Gemtuzumab, Pirprofen
Ozogamicin, Naltrexone, Tilbroquinol
Nevirapine, Pemoline,
Trovafloxacin

62% involve metabolism

and reactive products

Impact of reactive metabolites

on drug safety

J. L.Walgren et al., (2005) Crit. Rev. Toxicol. 35, 325-361

 - 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
- Chemical manipulation.
- Avoiding structural alerts/toxicophores.
- Avoid structure-based risk
- Greater scholarship regarding potential metabolic routes and
downstream consequences.

- Greater efficacy.
- Lower dose.

- Screening out metabolic risk

- Covalent binding studies
- Reactive metabolite screens

Improving drug safety

- Chemical manipulation.
- Avoiding structural alerts/toxicophores.
- Avoid structure-based risk
- Greater scholarship regarding potential metabolic routes and
downstream consequences.

- Greater efficacy.
- Lower dose.

- Screening out metabolic risk

- Covalent binding studies
- Reactive metabolite screens

Improving drug safety

 O O
N
NH

Irreversible CYP3A4 inhibitor

Potassium channel opener

O O
N
NH

F F

Equipotent potassium channel opener No CYP3A4 inhibition

Designing around metabolic risk

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

- Chemical manipulation.
- Avoiding structural alerts/toxicophores.
- Avoid structure-based risk
- Greater scholarship regarding potential metabolic routes and
downstream consequences.

- Greater efficacy.
- Lower dose.

- Screening out metabolic risk

- Covalent binding studies
- Reactive metabolite screens

Improving drug safety

- High doses commonly associated with adverse events.

- However, exposure/biologically effective dose (e.g. AUC) is far better indicator of

actual amount of a drug that a patient is exposed to – due to ADME/protein
binding etc.

CH 3 CH3
N N

N N
N N
Cl

NH NH S CH 3

Clozapine Olanzapine

Reactive metabolite formation in vitro. Reactive metabolite formation in vitro.

1 % incidence of agranulocytosis. No in vivo manifestation.
Dose: 300 mg/day. Dose: 10 mg/day.
0.1-0.5 % glucuronidation. 21-25 % glucuronidation.

Role of dose in drug-induced toxicity

J. M. Alvir & J. A. Lieberman, (1994) J Clin Psychiatry. 55, 137-138
I. Gardner et al., (1998) Mol Pharmacol. 53, 991-1008
J. P. Uetrecht, (2000) Curr Drug Metab. 1, 133-141
- Chemical manipulation.
- Avoiding structural alerts/toxicophores.
- Avoid structure-based risk
- Greater scholarship regarding potential metabolic routes and
downstream consequences.

- Greater efficacy.
- Lower dose.

- Screening out metabolic risk

- Covalent binding studies
- Reactive metabolite screens

Improving drug safety

 - 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 drug

discovery
Radiolabeled Studies
 covalent binding of radiolabeled compound (10µM)
 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

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

Merck approach for assessing

reactive metabolite formation
D.C. Evans et al., (2004) Chem. Res. Toxicol. 17 , 3-16
Radiolabeled Studies

>50pmol eq/mg protein?

Qualifying considerations
- Potential structural modification?

- Availability of existing treatments?

- Daily dose <10 mg?

- Metabolic clearance routes?

- Duration of therapy?

- Intended target population?

Merck approach for assessing

reactive metabolite formation
D.C. Evans et al., (2004) Chem. Res. Toxicol. 17 , 3-16
Radiolabeled Method
Advantages
- In vitro studies - species differences can be explored
- In vivo studies - related to safety studies
- extrahepatic bioactivation

Disadvantages
- Requires radiolabeled compound
- Not suitable for early stage studies

Advantages and disadvantages of

radiolabeled method
Biomarker Method
Disadvantages
- No single small molecule serves as universal surrogate
- May require follow up study in hepatocytes if positive in microsomes

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

Advantages and disadvantages

of biomarker method
- 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 or

enzymatically in reactions catalyzed by GSH S-transferases (GSTs).

- Found in most cells but particularly abundant in liver (5mM).

Physiological role of GSH

-To clarify the relationship between in vitro formation rate of GSH
conjugates and covalent binding to protein.

1- Tienilic acid
2- Furosemide
3- Clozapine
4- Imipramine
5- Acetaminophen
6- Indomethacin
7- Diclofenac
8- Carbamazepine

Relationship between covalent binding and GSH

conjugate formation
N. Masubuchi et al., (2007) Chem. Res. Toxicol. 20, 455-464
 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 O COOH
H
N
HOOC N NH2
H O
HS

Standard method for analysis of GSH conjugates

Exact mass neutral loss of 129.046 Da.
- Endogenous structures in biological matrices may have same
nominal mass of 129 Da but less likely to have same exact mass
- Excludes false positives

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 of
GSH
- Precursor ion scan of m/z 272 & positive ion CNL 129?

Sensitivity?

Improving selectivity
J. Castro-Perez et al., (2005) Rapid Commun Mass Spectrom. 19, 798
C. M. Dieckhaus et al., (2005) Chem Res Toxicol. 18, 630
  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

Use of GSH ethyl ester as reactive metabolite

trapping agent

J. R. Soglia et al., (2004) J Pharm Biomed Anal. 36, 105-116

 Compound GSH conjugate GSH-EE conjugate
Acetaminophen + +
Clozapine + +
Amodiaquine - +
Diclofenac + +
Rosiglitazone + +
Indomethacin - -
Sulfamethoxazole - +
Carbamazepine - +
Felbamate - +
Pioglitazone - +
Imipramine - +
Valproic acid - -

Use of GSH ethyl ester as reactive metabolite

trapping agent

J. R. Soglia et al., (2004) J Pharm Biomed Anal. 36, 105-116

- 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 cause

fluorescent interference.

Use of dansyl GSH as a trapping agent

J. Gan et al., (2005) Chem Res Toxicol. 18, 896-903

Semi-quantitative method for determining reactive metabolite levels using
quaternary ammonium GSH analogue (QA-GSH)

- Fixed positive charge significantly increased limit of detection.

- Equalized MS response from equimolar amounts of different GSH

conjugates.

- m/z of QA-GSH conjugate determined for M+ or MH2+ ion.

- Response factor for IS with same charge state determined (peak

area/conc.)

- Peak area of QA-GSH conjugate/response factor of IS provides

semi-quantification.

Quantifying conjugation?
J. R. Soglia et al., (2006) Chem Res Toxicol. 19, 480-490
 Drug

Reactive metabolite

GSH GSH*

O COOH O COOH
13
CH2 NH 13 CH2 15 NH
HOOC NH NH2 HOOC NH NH2
O O
HS HS

GSH conjugate (MH++305) Triply labeled GSH adduct (MH++308)

Use of stable-isotope labeled GSH in high-

throughput screenings of reactive metabolites
Z. Yan & G. W. Caldwell, (2004) Anal Chem. 76, 6835-6847
Z. Yan et al., (2005) Drug Metab Dispos. 33, 706-713
Z. Yan et al., (2005) Rapid Commun Mass Spectrom. 19, 3322-3330
 GSH Triply-labeled GSH

1:1 mixture

Microsomal incubation

Solid-phase extraction Neutral loss scanning

(129 Da)

M-SG M-SG*
3 Da

Use of stable-isotope labeled GSH in high-

throughput screenings of reactive metabolites
Z. Yan & G. W. Caldwell, (2004) Anal Chem. 76, 6835-6847
Z. Yan et al., (2005) Drug Metab Dispos. 33, 706-713
Z. Yan et al., (2005) Rapid Commun Mass Spectrom. 19, 3322-3330
- 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 > 1

doublet – providing further confirmation of conjugation to GSH.

- High confidence by subsequent MS/MS analysis of neutral losses of 75

and 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.

Use of stable-isotope labeled GSH in high-

throughput screenings of reactive metabolites
Z. Yan & G. W. Caldwell, (2004) Anal Chem. 76, 6835-6847
Z. Yan et al., (2005) Drug Metab Dispos. 33, 706-713
Z. Yan et al., (2005) Rapid Commun Mass Spectrom. 19, 3322-3330
A. Mutlib et al., (2005) Rapid Commun Mass Spectrom. 19, 3482-3492
 Positive controls Negative controls
Bromobenzene Dextromethorphan
Carbamazepine Fluoxetine
Clozapine Ketoconazole
p-cresol Midazolam
Diclofenac Terfenadine
β-estradiol Testosterone
17-α-ethynylestradiol Tolbutamide
Felbamate
4-hydroxyestrone
3-methylindole
Omeprazole
Phenacetin

Felbamate – requires both esterase and aldehyde dehydrogenase.

Use of stable-isotope labeled GSH in high-

throughput screenings of reactive metabolites
Z. Yan & G. W. Caldwell, (2004) Anal Chem. 76, 6835-6847
Z. Yan et al., (2005) Drug Metab Dispos. 33, 706-713
Z. Yan et al., (2005) Rapid Commun Mass Spectrom. 19, 3322-3330
Standard screens may detect “soft” electrophiles such as quinones,
quinone imines, epoxides etc

Detection of “hard” electrophiles may require separate trapping

experiments

Trapping Agents Functional Group

Glutathione (ester), mercaptoethanol, Quinones, enones
cysteine
Cyanide Iminium ions
Semicarbazide Aldehydes
Methoxylamine
Lysine Imides, aryl halides
Lysine + cysteine Furan, epoxide
TEMPO Free radical trap
(Tetramethylpiperidin-N-oxyl)

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 isotopic

doublet with mass difference of 8 Da.

γ-Glu Cys C6-15N2-Lys

13

-129 Da
S-R N=R

Trapping “soft” and “hard” reactive metabolites

Z. Yan et al., (2007) Anal Chem. 79, 4206-4214

- Not all reactive intermediates form adducts with GSH.

- Presence of GSH adduct in vitro does not mean pathway

predominates 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

- Quantification of reactive metabolite formation?

- In vitro to in vivo relationship?

- What does it mean?

Points to consider
 Designing out ‘metabolic weaknesses’?

Assess bioactivation potential Early

Stage
Optimize lead compounds

Prioritization of drug candidates

Radiolabeling for covalent binding

Later
Stage
> 50 pmol eq/mg < 50 pmol eq/mg

Qualifying considerations Advance compound

Suggested screening strategy for

reactive metabolite assessment
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