1

Metabolic Activation and Idiosycratic Metabolic Activation and Idiosycratic Drug Toxicity: By Avoiding Structural Drug Toxicity: By Avoiding Structural

Alerts, Do We Mitigate Risks?Alerts, Do We Mitigate Risks?

Amit S. Kalgutkar, Ph.D.Pfizer Global Research and Development

Groton, CT 06340, USA

2

Cause(s) of Attrition in Drug DiscoveryCause(s) of Attrition in Drug Discovery

• In the early 90’s, major cause of attrition was poor pharmacokinetics1,2

– Largely resolved via involvement of DM/PK groups at early stages of drug discovery (Exploratory/Lead development/candidate-seeking)

• Of late: lack of efficacy (achieving POM for novel targets) and drug safety are the leading causes of candidate attrition– Pharmacology tactics to counterbalance attrition

• Better understanding of pharmacological targets • Incorporation of translational pharmacology (PK/PD, disease

biomarkers, etc)• Probe concept (exploratory INDs, etc)

– Tactics to counterbalance safety-related attrition arising from IADRs• ?

1Roberts SA (2003) Drug metabolism and pharmacokinetics in drug discovery. Curr Opin Drug Discov Devel. 6(1):66-80. 2Kola I and Landis J (2004) Can the pharmaceutical industry reduce attrition rates? Nat Rev Drug Discov. 3(8):711-5

3

Safety-Related Attrition: Adverse Drug Reactions (ADRs)Safety-Related Attrition: Adverse Drug Reactions (ADRs)

• ADRs Contribute to patient morbidity and mortality– One of the most common causes for drug recalls or black box warning labels

• Of a total of 548 drugs approved in the period from 1975-1999, 45 drugs (8.2%) acquired 1 or more black box warnings, 16 (2.9%) were withdrawn from the market

• ADR Classification – Type A ADRs: ~ 80% of ADRs fall in this category

• Type A ADRs can be predicted from known drug pharmacology (e.g. Hemorrhage with anticoagulants)

• Dose dependent – can be reversed with dose reduction• Generally identified in preclinical species (animal models of pharmacology)

– Type B (Bizarre) or Idiosyncratic ADRs (e.g., hepatotoxicity, skin rashes, aplastic anaemia, agranulocytosis) – e.g., black box warning for “sulfonamides”

• Unrelated to primary pharmacology • Dose independent (with the exception of some drugs) – can occur at any dose within the

therapeutic range• Temporal relationship - Symptoms subsides after cessation of treatment; rapid onset upon

re-challenge• Can be severe - maybe fatal - most common cause for drug withdrawal• Cannot be predicted from traditional toxicological studies in animals• Rare - frequency of occurrence - 1 in 10,000 to 1 in 100,000

– Normally not observed until phase III or post launch

4

Associating a Functional Group with Adverse Drug ReactionsAssociating a Functional Group with Adverse Drug Reactions – Sulfonamides have achieved notoriety with respect to

hypersensitivity (e.g., skin rashes)

See - Kalgutkar, A. S., Jones, R. and Sawant, A. ”Sulfonamide as an Essential Functional Group in Drug Design” In Metabolism, Pharmacokinetics and Toxicity of Functional Groups: Impact of the Building Blocks of Medicinal Chemistry on ADMET (Royal Society of Chemistry), [Dennis A. Smith, Editor], 2010, Chapter 5, pp. 210 – 273.

5

BLACK BOX WARNING FATALITIES ASSOCIATED WITH THE ADMINISTRATION OF SULFONAMIDES, ALTHOUGH RARE,HAVE OCCURRED DUE TO SEVERE REACTIONS, INCLUDING STEVENS-JOHNSON SYNDROME, TOXIC EPIDERMAL NECROLYSIS, FULMINANT HEPATIC NECROSIS, AGRANULOCYTOSIS, APLASTIC ANEMIA, AND OTHER BLOOD DYSCRASIAS. SULFONAMIDES, INCLUDING SULFONAMIDE CONTAINING PRODUCTS SUCH AS TRIMETHOPRIM/SULFAMETHOXAZOLE, SHOULD BE DISCONTINUED AT THE FIRSTAPPEARANCE OF SKIN RASH OR ANY SIGN OF ADVERSE REACTION. In rare instances, a skin rash may be followed by a more severe reaction, such as Stevens-Johnson syndrome, toxic epidermal necrolysis, hepatic necrosis, and serious blood disorder (see PRECAUTIONS ). Clinical signs, such as rash, sore throat, fever, arthralgia, pallor, purpura, or jaundice may be earlyindications of serious reactions.

H2N

SNH

O O

NO

CH3

H3CO OCH3

OCH3

N

N

NH2

NH2

Sulfamethoxazole Trimethoprim

(Bactrim®)

6

7

The Concept of Xenobiotic Bioactivation to The Concept of Xenobiotic Bioactivation to Reactive Metabolites (RMs)Reactive Metabolites (RMs)

Origins in the field of chemical carcinogenicityOrigins in the field of chemical carcinogenicityAmes Test for genotoxicity has S-9/NADPH-dependent bioactivation arm; required for FDA submissions

• RM “covalently adducts” to DNA resulting in genotoxic responseRM “covalently adducts” to DNA resulting in genotoxic response– Fungal mycotoxin aflatoxin B1 (AFB1) – established hepatocarcinogen†

• Exposure occurs primarily through ingestion of mold-contaminated foods (e.g., corn and peanuts)

• Rate-limiting step is P450-catalyzed RM formationRate-limiting step is P450-catalyzed RM formation

O

O

H

H

OCH3

O

O

AFB1

O

O

H

H

OCH3

O

O

P450

O

Reaction with DNA-base(s) Guanine - (DNA adducts)Reaction with glutathioneReacttion with water

†Guengerich FP, Johnson WW, Shimada T, Ueng YF, Yamazaki H and Langouet S (1998) Mutat. Res. 402:121-128. §Miller EC and Miller JA (1947) Cancer Res. 7:468-480.

Furan epoxide Furan epoxide (A reactive metabolite)(A reactive metabolite)

8

RMs and CYP Isozyme InactivationRMs and CYP Isozyme Inactivation• RM “covalently adducts” to metabolizing enzymes (e.g., RM “covalently adducts” to metabolizing enzymes (e.g.,

cytochrome P450) responsible for its formationcytochrome P450) responsible for its formation– Leads to enzyme inactivation and:Leads to enzyme inactivation and:

• Non-linear PK if P450 enzyme is also primarily responsible for clearance• Drug-drug interactions (DDIs) (Atorvastatin/Grapefruit juice)

– Furanocoumarins, bergamottin and 6’,7’-dihydroxybergamottin, the abundant Furanocoumarins, bergamottin and 6’,7’-dihydroxybergamottin, the abundant constituents of GFJ, are mechanism-based inactivators of P4503A4 constituents of GFJ, are mechanism-based inactivators of P4503A4

O

OO

R

R = H2C R =

OH

OH

H2C

P4503A4

O

OO

R

O

P4503A4 Inactivation

He K, Iyer KR, Hayes RN, Sinz MW, Woolf TF and Hollenberg PF (1998) Chem. Res. Toxicol. 11:252-259.Kent UM, Lin HL, Noon KR, Harris DL and Hollenberg PF (2006) J. Pharmacol. Exp. Ther. 318:992-1005.

9

Toxic Drug Metabolites – Toxic Drug Metabolites – Acetaminophen as an Acetaminophen as an ExampleExample

• Brodie et al. (National Institutes of Health) first to demonstrate:– Bioactivation of acetaminophen and covalent binding to liver tissue

• Nelson et al. elucidated the mechanism of acetaminophen bioactivation (involving a 2 electron oxidation to a reactive quinone-imine intermediate)– “Gold standard” of human and animal hepatotoxicity assessments

Dose-dependent (> 1 gm/day) hepatotoxin Depletes GSH upon toxic overdose Covalent binding to > 30 hepatic proteins liver toxicity can be observed in animals N-Acetylcysteine as antidote

HN

OH

CH3

O

Acetaminophen

P450

N

O

CH3

O

NAPQI

GSH

HN

OH

CH3

O

S

NH

NH

O

COOH

O

NH2

COOH

GSH = glutathione – endogenous antioxidant (~ 10 mM concn. in mammals)14C-APAP covalent binding to microsomes prevented by GSH; confirms the protective role of the thiol

Reactive quinone-imineReactive quinone-imine

10

Drugs Associated With IADRsDrugs Associated With IADRsDrugs Withdrawn

Aclcofenac (antiinflammatory) Hepatitis, rashAlpidem (anxiolytic) Hepatitis (fatal)Amodiaquine (antimalarial) Hepatitis, agranulocytosisAmineptine (antidepressant) Hepatitis, cutaneous ADRsBenoxaprofen (antiinflammatory) Hepatitis, cutaneous ADRsBromfenac (antiinflammatory) Hepatitis (fatal)Carbutamide (antidiabetic) Bone marrow toxicityIbufenac (antiinflammatory) Hepatitis (fatal)Iproniazid (antidepressant) Hepatitis (fatal)Metiamide (antiulcer) Bone marrow toxicityNomifensine (antidepressant) Hepatitis (fatal), anaemiaPractolol (antiarrhythmic) Severe cutaneous ADRsRemoxipride (antipsychotic) Aplastic anaemiaSudoxicam (antiinflammatory) Hepatitis (fatal)Tienilic Acid (diuretic) Hepatitis (fatal)Tolrestat (antidiabetic) Hepatitis (fatal)Troglitazone (antidiabetic) Hepatitis (fatal)Zomepirac (antiinflammatory) Hepatitis, cutaneous ADRs

Marketed DrugsAbacavir (antiretroviral) Cutaneous ADRsAcetaminophen (analgesic) Hepatitis (fatal)Captopril (antihypertensive) Cutaneous ADRs, agranulocytosisCarbamazepine (anticonvulsant) Hepatitis, agranulocytosisClozapine (antipsychotic) AgranulocytosisCyclophosphamide (anticancer) Agranulocytosis, cutaneous ADRsDapsone (antibacterial) Agranulocytosis, cutaneous ADRs, aplastic anaemiaDiclofenac (antiinflammatory) HepatitisFelbamate (anticonvulsant) Hepatitis (fatal), aplastic anaemia (fatal), severe restriction in useFurosemide (diurectic) Agranulocytosis, cutaneous ADRs, aplastic anaemiaHalothane (anesthetic) HepatitisImipramine (antidepressant) HepatitisIndomethacin (antiinflammatory) Hepatitis

Isoniazid (antibacterial) Hepatitis (can be fatal)Phenytoin (anticonvulsant) Agranulocytosis, cutaneous ADRsProcainamide (antiarrhythmic) Hepatitis, agranulocytosisSulfamethoxazole (antibacterial) Agranulocytosis, aplastic anaemiaTerbinafine (antifungal) Hepatitis, cutaneous ADRsTiclopidine (antithrombotic) Agranulocytosis, aplastic anaemiaTolcapone (antiparkinsons) Hepatitis (fatal),severe restriction in useTrazodone (antidepressant) HepatitisTrimethoprim (antibacterial) Agranulocytosis, aplastic anaemia, cutaneous ADRsThalidomide (immunomodulator) TeratogenicityValproic acid (anticonvulsant) Hepatitis (fatal), teratogenicity

Temp. Withdrawnor Withdrawn in other Countries

Aminopyrine (analgesic) AgranulocytosisNefazodone (antidepressant) Hepatitis (> 200 deaths)Trovan (antibacterial) HepatitisZileuton (antiasthma) Hepatitis

For many drugs associated with IADRs, circumstantial evidence suggests a link with RM formationFor many drugs associated with IADRs, circumstantial evidence suggests a link with RM formationStructure-toxicity relationships – evident and present a compelling case against RM positivesStructure-toxicity relationships – evident and present a compelling case against RM positives

11

Structure-Toxicity Relationships – Example 1Structure-Toxicity Relationships – Example 1

Enol-carboxamide-containing NSAIDs

SudoxicamHepatotoxic (acute liver failure)Withdrawn from Phase III trials

Meloxicam

“Clean” drug

Piroxicam

“Clean” drug

SN

OH

CH3

O O

NH

O

S

N

SN

OH

CH3

O O

NH

O

S

NCH3

SN

OH

CH3

O O

NH

O

N

12

Rationalizing the Differences in Toxicological Profile Through Rationalizing the Differences in Toxicological Profile Through Differences in MetabolismDifferences in Metabolism

SN

OH

CH3

O O

NH

O

S

N

P450

SN

OH

CH3

O O

NH

O

S

N O

SN

OH

CH3

O O

NH

O

S

N

OH

OH

SN

OH

CH3

O O

NH

O NH2

S

Epoxide Thiourea

OH

OHThioureas are toxic substances –Can oxidize proteins, glutathione, etc

SN

OH

CH3

O O

NH

O

S

NCH3

P450

SN

OH

CH3

O O

NH

O

S

N OH

Sudoxicam

Piroxicam

Principal metabolism in humans is hydroxylation on methyl Principal metabolism in humans is hydroxylation on methyl Very minimal thiazole ring opening Very minimal thiazole ring opening

Obach, R. S., Kalgutkar, A. S., Ryder T. and Walker, G. W. “In Vitro Metabolism and Covalent Binding of Enol-Carboxamide Derivatives and Anti-inflammatory Agents Sudoxicam and Meloxicam: Insights into the Hepatotoxicity of Sudoxicam.” Chem. Res. Toxicol. 2008, 21, 1890-1899.

13

Structure-Toxicity Relationships – Example 2Structure-Toxicity Relationships – Example 2

Antipsychotic agentsAntipsychotic agents

NH

NClN

NCH3

S

NN

NO

OH

O

NN

NCH3

Cl

ClozapineAgranulocytosis /Hepatotoxicity (Black box warning – requires intensive monitoring)

Quetiapine (Seroquel®)

Commericial blockbuster

Loxapine

“Clean” drug

Liu ZC, Uetrecht JP (1995) Clozapine is oxidized by activated human neutrophils to a reactive nitrenium ion that irreversibly binds to the cells. J. Pharmacol. Exp. Ther. 275:1476-1483.Gardner I, Leeder JS, Chin T, Zahid N, Uetrecht JP (1995) A comparison of the covalent binding of clozapine and olanzapine to human neutrophils in vitro and in vivo. Mol. Pharmacol. 53:999-1008.

14

Rationalizing the Differences in Toxicological Profile Rationalizing the Differences in Toxicological Profile Through Differences in MetabolismThrough Differences in Metabolism

NH

NClN

NCH3

Clozapine

P450 or

Peroxidase

N

NClN

NCH3

Electrophilic iminium species

GSH NH

NClN

NCH3

S

NH

HN

O

COOH

O

H2N

COOH

Bioactivation of clozapine catalyzed by peroxidases in neutrophilsBioactivation of clozapine catalyzed by peroxidases in neutrophils Reactive metabolite responsible for covalent binding to neutrophilsReactive metabolite responsible for covalent binding to neutrophils

O

NN

NCH3

Loxapine

ClS

NN

N

Quetiapine

O

OH

Quetiapine and loxapine cannot form electrophilic iminium like clozapine doesQuetiapine and loxapine cannot form electrophilic iminium like clozapine does

Uetrecht J, Zahid N, Tehim A, Fu JM, Rakhit S. (1997) Structural features associated with reactive metabolite formation in clozapine analogues. Chem. Biol. Interact. 104:117-129.

15

RM Detection – Electrophile TrappingRM Detection – Electrophile Trapping • Reactive metabolites (Reactive metabolites (with the exception of acyl glucuronideswith the exception of acyl glucuronides) are ) are

unstableunstable– Need derivatization techniques for indirect characterization

• RM trapping with exogenous nucleophilesRM trapping with exogenous nucleophiles – Can be used with diverse metabolism vectors

• Liver microsomes, S-9, hepatocytes, etc• Glutathione, , N-acetylcysteine (soft nucleophiles)

– Traps soft electrophiles (e.g., Michael acceptors — quinones)• Methoxylamine, semicarbazide, cyanide (hard nucleophiles)

– Traps hard electrophiles (e.g., aldehydes, iminium ion)– LC-MS/MS and/or NMR methodology for structure elucidation of

conjugateHS

NH

NH

O

COOH

O

H2N COOH

Glutathione

CN

Cyanide

CH3ONH2

Amines

NH2

HNH2N

O

16

RM Detection – Covalent BindingRM Detection – Covalent Binding

• Limited to availability of radiolabeled drug candidateLimited to availability of radiolabeled drug candidate– May not be suitable in early discovery

• In VitroIn Vitro covalent binding can be assessed with covalent binding can be assessed with diverse metabolism vectorsdiverse metabolism vectors– Effect of competing/detoxicating drug metabolizing enzymes on

covalent binding can also be examined

• In vivoIn vivo covalent binding can be assessed in covalent binding can be assessed in preclinical speciespreclinical species

• Covalent binding data is quantitative Covalent binding data is quantitative – No information on nature of proteins modifiedNo information on nature of proteins modified

17

Utility of RM Detection Tools in Drug Discovery - Utility of RM Detection Tools in Drug Discovery - Identifying the Metabolic Basis for MutagenicityIdentifying the Metabolic Basis for Mutagenicity

N

N

N

HN

OCl

CP-809,101

Selective and potent 5-HTSelective and potent 5-HT2C2C Agonist Agonist • Excellent in vivo pharmacology for weight reduction• Excellent predicted human pharmacokinetics•Potential as an anti-obesity agent

• Mutagenic in Salmonella Ames assay Mutagenic in Salmonella Ames assay • Requires Ariclor Rat S-9/NADPH Requires Ariclor Rat S-9/NADPH

• Suggests DNA-Reactive Metabolites FormedSuggests DNA-Reactive Metabolites Formed• Compound dropped from developmentCompound dropped from development

Need to elucidate mutagenic mechanism(s) for design of follow-on candidates ___ Available tools: [14C]-CP-809,101, RM traps (GSH, CH3ONH2, etc)

GOAL

Kalgutkar, A. S., Dalvie, D., Aubrecht, J., Smith, E., Coffing, S. et al. Genotoxicity of 2-(3-Chlorobenzyloxy)-6-piperazinyl)pyrazine. A Novel 5-HT2C Receptor Agonist for the Treatment of Obesity: Role of Metabolic Activation. Drug Metab. Dispos. 2007, 35, 848-858.

No toxicophore / Structural alert present; clean in DEREK assessment

18

NADPH-Dependent Covalent Binding to Calf-NADPH-Dependent Covalent Binding to Calf-Thymus DNA by [Thymus DNA by [1414C]-CP-809101C]-CP-809101

TABLE 1

S-9/NADPH-dependent covalent binding of [14C]-CP-809,10 to calf-thymus DNA

Incubation Test I

Mean DPMa/20 g DNA

Test II

Mean DPMa/20 g DNA

Vehicle (DMSO) 35 (34, 35) 23 (24, 24, 22)

CP-809101 (0.5 M) - S9 51 (54, 47) 52 (48, 48, 59)

CP-809101 (0.5 M) + incomplete S-9 40 (39,41) 42 (42, 41, 43)

CP-809101 (0.5 M) + complete S-9 105 (104, 106) 124 (126, 121)

CP-809101 (5.0 M) + complete S-9 495 (490,501) 462 (472, 451)

- S-9, without metabolic activation; + incomplete S-9 (-NADPH); complete S-9 (+ NADPH) NADPH was used in test I and NADPH regenerating system was used in test II. a mean DPM represents average from two to three separate experiments.

NADPH-dependent covalent binding to DNA suggests NADPH-dependent covalent binding to DNA suggests P450-mediated bioactivation to DNA-reactive metabolite(s)P450-mediated bioactivation to DNA-reactive metabolite(s)

N

N

N

HN

OCl

CP-809,101

*

*

19

Deciphering CP-809101 Bioactivation PathwaysDeciphering CP-809101 Bioactivation Pathways

Covalent binding to DNA significantly attenuated in the presence of CH3ONH2 and GSH

Cl

O

N

N N

NH

CP-809,101

Cl

O

HOCl

CH2

O

Quinone-methideO

N

HN N

NH

S-9 / NADPH

GSH

Cl

S

HO

NH

OHN COOH

OCOOH

NH2

S-9 / NADPH O

N

N N

NH

OHO

N

N N

NH2

O

CH3ONH2

Cl

O

N

N N

NH2

NOCH3

S-9 / NADPH

O

N

N N

NOH

O

N

N N

NO

CN O

N

N N

NOH

CN

Aldehyde

Nitrone

20

Metabolic soft spots(Minimal ring opening)

Cannot form quinone-methide

Non-mutagenicin Ames Assay

HN

N N

N

O

F

F

HN

N N

N

O

F

F

PrimaryPharmacologyMaintained

PK AttributesMaintained

Rational Chemical Modifications to Circumvent Rational Chemical Modifications to Circumvent MutagenicityMutagenicity

Kalgutkar, A. S., Bauman, J. N., McClure, K. F.; Aubrecht, J. Cortina, S. R. and Paralkar, J. “Biochemical Basis for Differences in Metabolism-Dependent Genotoxicity by Two Diazinylpiperazine-Based 5-HT2C Receptor Agonists.” Bioorg. Med. Chem. Lett. 2009, 19, 1559-1563.

21

RM Trapping and/or Covalent Binding Studies in Drug DiscoveryRM Trapping and/or Covalent Binding Studies in Drug DiscoveryIdentifying Intrinsically Electrophilic Compounds - Identifying Intrinsically Electrophilic Compounds - Influencing Scaffold Influencing Scaffold

DesignDesign

SAR Studies in a early discovery program

Compound Compound 11 identified as meeting desired criteria for primary identified as meeting desired criteria for primary in vitroin vitro pharmacology pharmacology andand progressed for further profiling (e.g., in vitro ADME, metabolism studies, etc) as progressed for further profiling (e.g., in vitro ADME, metabolism studies, etc) as part of lead optimization efforts part of lead optimization efforts

OR

NN

CN

ON

CH3(1)

22

Glutathione Trapping Studies on Glutathione Trapping Studies on 11

0

50

1000

50

100

Inte

nsi

ty

23.96

21.7618.36

19.81

15.94 23.92

21.7518.36 19.8215.26

1

M1-1M2-1

M3-1

M5-1M4-1

1

0

50

10023.99

15.98M4-1 1

0

50

100

15.98M4-1 1

24.02

0

50

100 16.05

24.07

M4-11

0

50

100

23.95

M4-11

15.95

Time (min)14 16 18 20 22 24 26

(A)

(B)

(C)

(D)

(E)

(F)

M3-1 M2-1M1-1

HLM + NADPH

HLM + NADPH + GSH

HLM – NADPH + GSH

GSH in buffer

Cytosol + GSH

GST + GSH

Kalgutkar AS, Sharma R, Walker GS et al. Unpublished data

23

Mass Spectra of Mass Spectra of M4-M4-1 and 1 and M5-1M5-1Intensity

m/z

150 200 250 300 350 400 450 500 550 600 6500

20

40

60

80

100439.1021

310.0600

423.1437

298.0600

509.1800212.1276 563.1909594.2327552.1862469.1495

489.1540364.0707257.0761

228.0434638.2099

M4-1

150 200 250 300 350 400 450 500 550 600 6500

20

40

60

80

100636.2067

423.1436

552.1854439.1019

298.0599525.1748

579.1861228.1229281.0338

489.1560193.8677 340.8997 396.3724

M5-1

N N

O

CN

RS

HN

NHO

COOH

O

COOH

H2N

M4-1 (Exact mass + H+ = 638.2238)

N N

O

CN

RS

HN

NHO

COOH

O

COOH

H2N

M5-1 (Exact mass + H+ = 654.2182)

OH

(- H2O)

24

Additional Confirmation of Adduct Structure using Additional Confirmation of Adduct Structure using NMRNMR

COSY

HMBC

N N

C

N

S

HN

HN O

OH

O

O

NH2

HO

O

Oa

d

f

g

kji

h

ecb

l

6 5

4

2

7

R

Position Group (1H) ppma (13C) ppma

a C? O 170.4

b CH 3.30 52.9 c CH2 1.88 26.8 d CH2 2.34 31.5 e C? O 171.8 f NH 8.71 g CH 4.61 51.6 h C? O 169.1 i NH 8.09 j CH2 3.52 42.5 k C? O 171.4 l CH2 3.40,3.92 31.3 2 CH 8.85 159.8 4 C 167.3 5 C 91.5 6 C 173.4 7 CN 112.4 8 CH 5.52 74.3 9 CH 2.04 32.4 10 & 12 CH2 3.22,3.33,4.05, 4.13 42.0 13 CH 2.04 32.4 14 & 15 CH2 3.71,3.97 70.4 16 C? O 154.4 17 CH 4.78 67.3 18 & 19 CH2 1.18 21.4

a Proton chemical shifts were measured relative to DMSO-d6 signal at 2.50 ppm. b Carbon chemical shifts were obtained from the g-HSQC and g-HMBC spectra and measured relative to the DMSO-d6 signal at 39.5 ppm.

25

The Cyanide Group in The Cyanide Group in 11 is Essential for Nucleophilic is Essential for Nucleophilic Displacement by GSHDisplacement by GSH

Cyanide substituent required for nucleophilic displacement by glutathione

Conclusions – GSH adduct formation does not require “bioactivation”4-Aryloxy-5-cyanopyrimidines can function as potential affinity labels (protein alkylation) or cause GSH depletion

Cyano replacements in the current scaffold avoidedCyano replacements in the current scaffold avoided

N N

R

C

ON

CH3

GSHN N

R

C

O

GS

N

N

CH3

Meisenheimer Complex

N N

R

C

O

GS

N

N

CH3

N

N N

R

C

S

N

HN

OHN

COOH

O

NH2

COOH

OHN

CH3

N N

R

CH3

ON

CH3

NO REACTION

N N

R

CH3

SHN

OHN

COOH

O

NH2

COOH

GSH

26

Eliminating Toxicity Risks in Drug Discovery SettingEliminating Toxicity Risks in Drug Discovery Setting

• Structure-toxicity analyses teaches us that avoiding RM formation Structure-toxicity analyses teaches us that avoiding RM formation with drug candidates represents with drug candidates represents oneone potential solution to potential solution to preventing drug toxicity preventing drug toxicity

• Avoid chemical functionalities known to be Avoid chemical functionalities known to be susceptible to reactive metabolitessusceptible to reactive metabolites– Tall order but avoids risk Tall order but avoids risk

27

Examples of Functional Groups Susceptible to RM FormationExamples of Functional Groups Susceptible to RM Formation

Anilines (masked anilines)p-AminophenolsNitrobenzenesHydrazines (phenylhydrazines)BenzylaminesCatecholsCyclopropylamines1,2,3,6-Tetrahydopyridines2-Halopyridines and pyrimidinesHaloalkanesUnsubstituted alkenesAcetylenesImidesFormamidesSulfonylureasThioureasMethylenedioxy groups

Reduced aromatic thiols5-Hydroxy(or methoxy) indoles3-MethylindolesUnsubstitued furansUnsubstitued thiophenesUnsubstitued thiazolesUnsubstitued oxazolesThiazolidinedionesFatty acids (medium to long chain)Carboxylic acidsHydroxylaminesHydroxamic acidsMichael AcceptorsHydroquinonesBromobenzeneBENZENE !!!!!

Kalgutkar AS, Gardner I, Obach RS et al. (2005) Curr Drug Metab, 6, 161-225.

N

F

HN

O

OHOH

COOH

RR = H, OH

Lipitor®

28

And What about the False Negatives?And What about the False Negatives?

O

OH2N

O O

NH2

I really don’t see a “ugly” looking structure hereI really don’t see a “ugly” looking structure here

I checked for glutathione conjugates in HLM and human I checked for glutathione conjugates in HLM and human hepatocytes and saw nonehepatocytes and saw none

I assessed covalent binding to HLM and human hepatocytesI assessed covalent binding to HLM and human hepatocytesand saw nothing of significanceand saw nothing of significance

Within a year of its release in 1993• 34 cases of aplastic anemia resulting in 13 deaths (Incidence rate 1:4800 – 1:37000) • 23 cases of hepatotoxicity resulting in 5 deaths (Incidence rate 1:18000 – 1:25000

Black box warning (severe restriction in use)• ~ 12,000 patients estimated to be on drug

But this is the anti-convulsant felbamate (Daily Dose > 3000 mg)But this is the anti-convulsant felbamate (Daily Dose > 3000 mg)

Leone AM, Kao LM, McMillian MK et al. Evaluation of Felbamate and Other Antiepileptic Drug Toxicity Potential Based on Hepatic Protein Covalent Binding and Gene Expression. Chem. Res. Toxicol. 2007, 20:600-608.Obach, R.S., Kalgutkar, A. S., Soglia, J. R. and Zhao, S. X. “Can In Vitro Metabolism-Dependent Covalent Binding Data in Liver Microsomes Distinguish Hepatotoxic from Non-hepatotoxic Drugs? An Analysis of Eighteen Drugs with Consideration of Intrinsic Clearance and Daily Dose.” Chem. Res. Toxicol. 2008, 21, 1814-1822.Bauman, J., Kelly, J., Tripathy, S., Zhao, S., Lam, W., Kalgutkar, A. S. and Obach, R. S. “Can In Vitro Metabolism-Dependent Covalent Binding Data Distinguish Hepatotoxic from Non-Hepatotoxic Drugs? An Analysis Using Human Hepatocytes and Liver S-9 Fraction.” Chem. Res. Toxicol. 2009, 22, 332-340.

29

In VivoIn Vivo Observations on Felbamate Observations on Felbamate Conversion to RMs in HumansConversion to RMs in Humans

O

O NH2

O

O NH2

Felbamate

CO2, NH3

HO

O NH2

O

O

O NH2

O

AmidaseALD

OO

SG

CO2, NH3

GSH

R

S

NHCOCH3HOOC

R = CH2OH or CO2H

metabolites in human urine

2-Phenylpropenal

A heavy duty electrophile

Thompson CD, Barthen MT, Hopper DW, Miller TA, Quigg M, Hudspeth C, Montouris G et al. (1999) Quantification of patient urine samples of felbamate and three metabolites: acid carbamate and two mercapturic acids. Epilepsia 40:769-776.Diekhaus CM, Thompson CD, Roller SG, Macdonald TL (2002) Mechanisms of idiosyncratic drug reactions: the case of felbamate. Chem. Biol. Interact. 142:99-117.

30

And What about the False Negatives?And What about the False Negatives?H2N NOH

HN

O N

O HNO

O Et

Ximelagatran

H2N NOH

HN

O N

O HNOH

O

N-Hydroxymelagatran

H2Oe

H2N NH

HN

O N

O HNOH

O

Melagatran

No obvious structural alertNo obvious structural alertNo evidence of glutathione conjugate formation No evidence of glutathione conjugate formation in vitroin vitro or or in vivoin vivoPrimary metabolic pathways in humans – ester hydrolysis, reduction to active drug “Melagatran”Primary metabolic pathways in humans – ester hydrolysis, reduction to active drug “Melagatran”

Ximelagatran (Exanta®), the first orally active thrombin inhibitor (anticoagulant) was Ximelagatran (Exanta®), the first orally active thrombin inhibitor (anticoagulant) was withdrawn due to several cases of hepatotoxicitywithdrawn due to several cases of hepatotoxicity• Daily dose 20 – 60 mg BID Daily dose 20 – 60 mg BID • Short term use (< 12 days) in humans did not indicate hepatotoxic potentialShort term use (< 12 days) in humans did not indicate hepatotoxic potential• Long term use (> 35 days) in human showed elevated hepatic enzyme levels in 0.5% of patientsLong term use (> 35 days) in human showed elevated hepatic enzyme levels in 0.5% of patients

• Withdrawal triggered from severe liver damage in a patientWithdrawal triggered from severe liver damage in a patient• Immune component demonstrated upon pharmacogenomic analysisImmune component demonstrated upon pharmacogenomic analysis

Testa L, Bhindi R, Agostoni P, Abbate A, Zoccai GG, van Gaal WJ. The direct thrombin inhibitor ximelagatran/melagatran: a systemic review on clinical applications and an evidence based assessment of risk benefit profile. Expert Opinion in Drug Safety 2007;6:397-406.

31

Furthermore, How do we Handle the False Positives?Furthermore, How do we Handle the False Positives?

Paroxetine (Paxil®)Paroxetine (Paxil®)

NH

N

S CH3

N

NCH3

F

NH

O

O

O

Olanzapine (Zyprexa®)Olanzapine (Zyprexa®)

OH

S

OH

O

ON

Raloxifene (Evista®)Raloxifene (Evista®)

O

O

N

N

N

N

H2N

O

O

Prazosin (Minipress®)Prazosin (Minipress®)

Cl

Cl

N

N

OHN O

Aripiprazole (Abilify®)Aripiprazole (Abilify®)

RM = Catechol /quinone

RM = Furan Ring OpeningRM = quinone imine

RM = iminium

RM = quinone

Adding insult to injury, some are commercial blockbustersAdding insult to injury, some are commercial blockbusters

Cl

N

O OH

S

Clopidogrel (Plavix®)Clopidogrel (Plavix®)RM = Thiophene ring opening

RM required for efficacy !!RM required for efficacy !!

GSH conjugate/covalent binding demonstrated for all compoundsGSH conjugate/covalent binding demonstrated for all compounds

32

RM Detoxication as a Mitigating Factor for IADRsRM Detoxication as a Mitigating Factor for IADRsThe case of paroxetineThe case of paroxetine

O

O O

NH

F

CYP2D6

O

HO

HO O

O

O

O

H3CO

HO O

HO

H3CO

+

COMT

Detoxication

Covalent Binding to Hepatic Tissue

O

HO

HOGS

O

HO

HOGS

SG

GSH

N

S

O

O

HN

COOHDetoxication

The case of RaloxifeneThe case of Raloxifene

SHO

O

OH

O

N

O

Raloxifene

P4503A4

SO

O

O GSH

SHO

O

OH

SG

HOO

HO O

HO2C

OH

S

+UGT SHO

O

OGlu

Detoxication

Bioactivation

33

Dose Size as a Mitigating Factor for IADR Potential of Dose Size as a Mitigating Factor for IADR Potential of New Drug CandidatesNew Drug Candidates

Atypical anti-schizophrenia agentsAtypical anti-schizophrenia agents

Agranulocytosis in 2% of patientsDaily Dose = 300 mg

Safe and Successful Drug Sales > US $ 2 billionForms GSH conjugates via the iminium ion in a manner similar to clozapineCovalent binding to proteinsOnly 3 cases of agranulocytosis

Higher than recommended doseDaily Dose = 10 mg

NH

NClN

NCH3

NH

N

S CH3

N

NCH3

Clozapine

Olanzapine

There are many examples of two structurally related drugs that possess a common structural alert prone to

bioactivation, but the one administered at the lower dose is much safer than the one given at a higher dose

34

Bioactivation Data Needs to be Placed in Proper Context Bioactivation Data Needs to be Placed in Proper Context — — Risk/Benefit Assessments (Qualifying Considerations)Risk/Benefit Assessments (Qualifying Considerations)

• Nature of the medical need– Life-threatening disease / unmet medical need– First in class

• Target population– Underlying disease state (immune-compromised patients)

• Is the drug candidate intended to provide proof of a novel mechanism?

• What % of clearance mechanism involves bioactivation– Existence of detoxication pathways; renal excretion, etc

• What is the daily dose of the drug?– IADRs are rare for drugs dosed below 20 mg QD

35

For an account of a discovery strategy for dealing with RM For an account of a discovery strategy for dealing with RM

positive compound(s) as a drug candidate, please see:positive compound(s) as a drug candidate, please see: Kalgutkar, A. S., Griffith, D. A., Ryder, T., Sun, H., Miao, Z., Bauman, J. N., Didiuk, M. T., Frederick, K. S., Zhao, S. X., Prakash, C., Soglia, J. R., et al. Discovery Tactics to Mitigate Toxicity Risks Due to Reactive Metabolite Formation with 2-(2-Hydroxyaryl)-5-(trifluoromethyl)pyrido[4,3-d]pyrimidin-4(3H)-one Derivatives, Potent Calcium-Sensing Receptor Antagonists and Clinical Candidate(s) for the Treatment of Osteoporosis.” Chem. Res. Toxicol. 2010, In Press.

N

N

N

CF3 O

HO

Me

1

RM negative

CaSR IC50 = 41 nM

N

N

N

CF3 O

N

HO

Me

11CaSR IC50 = 8 nM

F

F

N

N

N

CF3 O

N

F3C

Me

13CaSR IC50 = 64 nM

HLM CLb = 12 mL/min/kg HLM CLb = 7.4 mL/min/kg HLM CLb = 6.0 mL/min/kg

X

HO

X = CH, N

CYPX

HO

OH

CYPX

O

O

GSHor

GSH-EE

X

HO

OH

S NH

ONH

COOR

O

NH2HO2C

GSH: R = HGSH-EE: R = CH2CH3Reactive metabolite (RM) formation pathway

RM positive RM positive

Predicted Human Dose = 45 mg QD Predicted Human Dose = 10 mg QD

(GSH-EE conjugate peak area = 166810) (GSH-EE conjugate peak area = 3000)