Dealing with reactive metabolites

Dominic Williams(dom@liv.ac.uk)

The University of LiverpoolU.K.

Lee AASLD, 2009; Verma & Kaplowitz 2009

Drug-Induced Liver Injury (DILI)

Leading cause of acute liver failureo Paracetamol 46% of all caseso 12% other drugs

High morbidity & mortalityo 20% survival without transplant

DILI causes: o attrition due to preclinical toxicity o attrition due to toxicity in man in late clinical trialso failed drug registrationo drug withdrawal post-licensingo cautionary and restrictive labelling

DRUG Cellularaccumulation Toxicity

Chemicallyreactive

metabolites

Stablemetabolites

Excretion

Phase I/II/III bioactivation

bioinactivation

Drug Disposition: Pharmacological & Toxicological

DRUG Cellularaccumulation Toxicity

Chemicallyreactive

metabolites

InhibitionOf

P450s

Excretion

Phase I/II/III bioactivation

bioinactivation

Consequences of bioactivation

• heme complex• protein alkylation

DRUG Cellularaccumulation Toxicity

Carcinogenicity

Necrosis

Apoptosis

Hypersensitivity

Chemically reactive

metabolites

Stablemetabolites

Excretion

Phase I/II/III Chemical Stress

Modification of:

• nucleic acid• enzyme• transporter• signalling protein• receptor• random autologous protein

bioactivation

bioinactivation

Consequences of bioactivation

DRUG Cellularaccumulation Toxicity ??

Chemically reactive

metabolites

Stablemetabolites

Excretion

bioactivation

bioinactivation

Phase I/II/III

furanthiophenealiphatic aminearomatic amineepoxidequinonequinoneiminecarbocationacyl halidehydroxylamineallylic alcoholacyl glucuronide

PHARMACOLOGICAL EFFECT

ADVERSEEFFECT

CHEMICAL STRUCTURE

Consequences of bioactivation:Toxicophores (structural alerts)

Focus on Drug-Induced Liver Injury

DRUGMETABOLITE

REACTIVE METABOLITE

SM

EC

CLEARANCE

inhibition of biliary efflux

Intrahepaticcholestasis

CLEARANCE

hypersensitivityimmunoallergic toxicity

mitochondrialysosome

phospholipidosismicrovesicular steatosishepatocyte apoptosishepatocyte necrosis

organelle impairmentbioaccumulation

Influx & efflux transporters

Patient-specific factorsDrug

Drug-specific factors

Drug absorption & dispositione.g. hepatic uptake

Chemical Insult in livere.g. reactive metabolite-mediated

Biological response in target celle.g. cell toxicity, stress response

Biological response in tissuee.g. cytokine release, immune cell response

1. 2. 3. 4.

Screening opportunity

Outcome:pre-clinical species vs man

Protectione.g. stress response

AmplificationInnate & adaptive

immunity

Tolerance & adaptation

Toxicity

vs

DILI – a consequence of multiple steps

Hepatotoxic drugs in man: Withdrawn or black-box warning label

Drugs withdrawn for hepatotoxicitydrug date dose

(mg/day)reactive products

cincopher 1930 300 noiproniazid 1959 25-150 yespipamazinc 1969 15 nofenclozic acid 1970 300 yesoxyphenisatin 1973 50 nonialamide 1974 200 yestienilic acid 1980 250-500 yesbenoxaprofen 1982 300-600 yesnomifensine 1986 125 yeschlomezanone 1996 600 nobromfenac 1998 25-50 yestroglitazone 2000 400 yesnefazodone 2004 200 yespemoline 2005 38-110 no

Drugs with black warnings for hepatotoxicity*drug dose (mg/day) reactive productsacitretin 25-50 no

bosentan 125-250 no

dacarbazine 140-315 yes

dantrolene 300-400 yes

felbamate 1200 yes

flutamide 750 yes

gemtuzumab 9 mg.m-3 yes (?)

isoniazid 300 yes

ketoconazole 200 yes

naltrexone 50 no

nevirapine 200 yes

tolcapone 300 yes

trovafloxacin 100-500 no

valproic acid 1000-2400 Yes (10/14 = 71%)

*Definition: a black box warning is the strongest type of warning that the FDA can require for a drug and is generally reserved for warning prescribers about adverse drug reactions that can cause serious injury or death. An issue here is the benefit/risk ratio.

Hepatotoxic drugs in man: Withdrawn or black-box warning label

Drugs withdrawn for hepatotoxicitydrug date dose

(mg/day)reactive products

cincopher 1930 300 noiproniazid 1959 25-150 yespipamazinc 1969 15 nofenclozic acid 1970 300 yes

oxyphenisatin 1973 50 nonialamide 1974 200 yestienilic acid 1980 250-500 yesbenoxaprofen 1982 300-600 yesnomifensine 1986 125 yeschlomezanone 1996 600 nobromfenac 1998 25-50 yestroglitazone 2000 400 yesnefazodone 2004 200 yespemoline 2005 38-110 no

Drugs with black warnings for hepatotoxicity*drug dose (mg/day) reactive productsacitretin 25-50 no

bosentan 125-250 no

dacarbazine 140-315 yesdantrolene 300-400 yesfelbamate 1200 yesflutamide 750 yesgemtuzumab 9 mg.m-3 yes (?)isoniazid 300 yesketoconazole 200 yesnaltrexone 50 no

nevirapine 200 yestolcapone 300 yestrovafloxacin 100-500 no

valproic acid 1000-2400 Yes (10/14 = 71%)

*Definition: a black box warning is the strongest type of warning that the FDA can require for a drug and is generally reserved for warning prescribers about adverse drug reactions that can cause serious injury or death. An issue here is the benefit/risk ratio.

2011; 10: 1-15

Ideal working relationship betweenchemistry & drug metabolism

detoxication bioactivation cell defence apoptosis necrosis

innate immunity adaptive immunity

REQUIRE NEW TEST SYSTEMS

&BIOMARKERS

Bioactivation

DetoxicationGLUCURONIDE

SULPHATE

GSH

COVALENT BINDING TOXICITY

Overdose

NCOCH3

O

NHCOCH3

OH

NHCOCH3

OH

SG

Acetaminophen (APAP; paracetamol)

• Recommended dose - 4g. Toxic dose >4g

• Most common form DILI in US & UK

• 400-500 deaths/yr, 70-100,000 hospital visits/yr

• Centrilobular damage

• Concern over chronic administration

• Treatment with N-acetylcysteine

• Cannot design out toxicity

NCOCH3

O

NHCOCH3

OH

NHCOCH3

OH

NHCOCH3

OH

NHCOCH3

OH

GSH depletionOxidative StressCovalent Binding

protein damage

Mildinjury

Pro:IFNgFasLTNF

HMGB-1

Anti:IL-10IL-6

IL-13

Innate immune responseNK/NK T cellsLymphocytes

Neutrophils (?)*Kupffer cells

Infiltrating Macrophages

SeverityOf

injury

Inflammation--- + ++

Yee et al., 2007; Masson et al., 2008; Holt et al., 2008; *Williams et al., 2010

Injury Progression: a multicellular event

Keratin-18 FragmentIs cleaved by active caspases

Fragment released into serum

Apoptotic Cell

Serum Biomarkerof Apoptosis

Keratin-18 Full lengthPassively released into serum

HMGB1Passively released into serum

‘Functional’Activates innate immune cells

Necrotic Cell

Serum Biomarkerof Necrosis

Injury Progression: serum reporters

Acetylated HMGB1‘HMGB-1-cytokine’ Secreted into serum

Activated innate immune cell

Serum Biomarkerof Innate immune

cell activation

TLR2TLR4RAGETLR9

DILI Models: Of mice…. mechanistic marker of necrosis

Release of HMGB- 1 due to necrosis

Many cell types

injury

Necrosis

Histopathology -HMGB-1 -ALT +++

APAP (530mg/kg)

Time course

Mouse

Histopathology +++HMGB1 +++ALT +

Histopathology +++HMGB1 ++ALT -

1h 3h 5h 24h

Histopathology -HMGB1 -ALT -

Time (Hrs)

Seru

m H

MGB

1 (n

g/m

l) Serum ALT (U/l)

*

***

HEPATOTOXICITY0 5 10 15 20 25 30

0

50

100

150

200

250

0

1000

2000

3000

4000

5000

6000

***

**

**

*

*

Histopathology -HMGB-1 -ALT +++

APAP (530mg/kg)

Time course

Mouse

Histopathology +++HMGB1 +++ALT +

Histopathology +++HMGB1 ++ALT -

1h 3h 5h 24h

Histopathology -HMGB1 -ALT -

Time (Hrs)

Seru

m H

MGB

1 (n

g/m

l) Serum ALT (U/l)

*

***

HEPATOTOXICITY0 5 10 15 20 25 30

0

50

100

150

200

250

0

1000

2000

3000

4000

5000

6000

***

**

**

*

*

Male CD-1 micedosed APAP 530 mg/kg

ZVAD.fmk

0

200

400

600

800

1000* *

Frag

men

ted

K18

(pm

ol/m

l)

controlZVAD.fmk

APAPAPAP &

ZVAD.fmk

0

50

100

150

200

250

300

350***

*** *

HMGB

1 (n

g/m

l)

0

1000

2000

3000

4000

5000***

*** *

Seru

m A

LT (U

/l)

Keratin-18 ALTHMGB-1

CV

PT

10XPath score 3.4 ± 0.5

Paracetamol 530mg/kg; 5hr; mouse

Path score 2.2 ± 0.4

CV

PT

10X

controlZVAD.fmk APAP

APAP &

ZVAD.fmk controlZVAD.fmk APAP

APAP &

ZVAD.fmk

DILI Models: Of mice…. Caspase inhibition enhances necrosis

HMGB1 – a possible biomarker for necrosis and inflammation

K182-185K180K177

K172

K173

COOHH2N

NuclearLocalisation

sequence

Cytokinedomain

HMGB1HMGB1

NHCOCH3

OH

ACETYLHMGB1

HMGB1

- HMGB1 - 42 lysine- 8 modified residues within the nuclear localisation sequence-Lysine acetylation directs for active release to act as a cytokine

TLR9

RAGE

TLR4 INNATE IMMUNE SYSTEM

CELLS

INFLAMMATION?

K182-185K180K177

K172

K173

COOHH2N

NuclearLocalisation

sequence

Cytokinedomain

HMGB1HMGB1

NHCOCH3

OH

ACETYLHMGB1

HMGB1

- HMGB1 - 42 lysine- 8 modified residues within the nuclear localisation sequence-Lysine acetylation directs for active release to act as a cytokine

Hyper-acetylated HMGB1 (Fold Inc)

0 5 10 15 20 25

0

10

20

30

40

0

2

4

6

8

10

APAP (530mg/kg) - hrHypo

-ace

tyla

ted

HMGB

1 (F

old

Inc)

**

******

**

***

***

*

**

HMGB1 – a possible biomarker for necrosis and inflammation

Translation..........Prof Hartmut Jaeschke (KU) & Dr James Dear (Edin)

BIOBANK:2 independent US/UK cohortsTESTS: 78 patients CONTROL 1: 6 O.D. patients (No ALT increase) 20 patients (longitudinal; 2-14 day) CONTROL 2: Age matched healthy volunteers

25 single samples6 longitudinal (7 days)

Hyper-acetylated HMGB1

Fold Increase0 5 10 15 20 25

0

10

20

30

40

0

2

4

6

8

10

HoursAPAP (530mg/kg)

HMGB

1 Fo

ld In

crea

se

**

******

**

******

**

*

Of mice................... ................... and men

0 1 2 3 4 5 6

010002000300040005000

010203040506070

Day

ALT

(U/l

)

Total & Acetylated

HMGB1 (ng/m

l)

APAP overdose patient

DILI Models: ….& men APAP clinical biomarker analysis

0 2 4 6 8 10 120

2000400060008000

1000012000

0

200

400

600

800

1000

Day

Tota

l K18

& ca

spas

e-cle

aved

K18

(U/L

)

0 2 4 6 8 10 120

5

10

15

20

25

0

200

400

600

800

1000

Day

Tota

l HM

GB1

(ng/

ml) ALT

(U/L)

ALT(U

/L)

Patient #6 (female 45)<10mg/ml APAP at 1st sample+ve salicylates

Total K18Caspase-cleaved K18 ALT

Total HMGB1ALT

Recovered without transplant

DILI Models: ….& (wo)men APAP clinical biomarker analysis

R2=0.58P<0.0001

R2=0.60P<0.0001

0 5000 10000 15000 200000

25

50

75

100

125

Serum ALT activity (U/l)

Tota

l HM

GB1

(ng/

ml)

0 5000 10000 15000 200000

50000

100000

150000

200000

250000

300000

350000

Serum ALT activity (U/l)

Nec

rosis

K18

(U/l

)

0 50 100 150 2000

25

50

75

100

125

Prothrombin time (sec)

Tota

l HM

GB1

(ng/

ml)

0 50 100 150 2000

50000

100000

150000

200000

250000

300000

350000

Nec

rosis

K18

(U/l

)

Prothrombin time (sec)

R2=0.62P<0.0001

R2=0.71P<0.0001

DILI Models: ….& men APAP clinical biomarker analysis

78 cross sectional samples from patients admitted to the Units

Combined UK & US patient data

ALT activity does NOT reflect prognosis:

Does HMGB1 / K18 forms reflect survival?

DILI Models: ….& men APAP clinical biomarker analysis

Translation..........

Hyper-acetylated HMGB1

Fold Increase

0 5 10 15 20 25

0

10

20

30

40

0

2

4

6

8

10

HoursAPAP (530mg/kg)

HMGB

1 Fo

ld In

crea

se

**

******

**

******

**

*

Of mice................... ................... and men

0 1 2 3 4 5 6

010002000300040005000

010203040506070

Day

ALT

(U/l

)

Total & Acetylated

HMGB1 (ng/m

l)

APAP overdose patient

Biphasic time course – two distinct origins of HMGB1 / biological processes, process similar in man to animals

Acetylated HMGB1 – a biomarker of immune cell activation

Clinical APAP overdose

Healthy v

olunteers

APAP-N

o ALI

APAP-ALI

0.001

0.01

0.1

1

10

100

Acet

ylat

ed H

MGB

1 (n

g/m

l)

All patientsBlind analyses

HMGB1

HMGB1

ACETYLHMGB1

HMGB1

Necrotic cells

Activated innateimmune cells

Acetylated HMGB1 – outcome prediction

Health

y voluntee

rs

APAP-No ALI

APAP-ALI

0.001

0.01

0.1

1

10

100

Ace

tyla

ted

HM

GB

1 (n

g/m

l)

Spontaneous survivors

Died / Required Liver transplant

Clinical APAP overdose

Acetylated HMGB1 – outcome prediction

Health

y voluntee

rs

APAP-N

o ALI

APAP-ALI

0.001

0.01

0.1

1

10

100

Ace

tyla

ted

HM

GB

1 (n

g/m

l)

0.00 0.25 0.50 0.75 1.000.00

0.25

0.50

0.75

1.00

1 - Specificity

Sens

itivi

ty

ROC curve – survival vs Death / Liver transplant

Acetylated HMGB1ALT

Clinical APAP overdose

Clinical situation reflects basic animal research models Biomarker fingerprint More sensitive than ALT (t1/2 & presence)

Necrosis was major form of cell death in all cases

Basic & Clinical scientific evidence for APAP induced apoptosis New therapeutic approaches Prognostic capability ?

DILI Models: Of mice & men Can HMGB-1 predict patient survival?

Managing CRM’s in drug discovery

CB should be regarded as a marker of bioactivation and not toxicity

In vitro studies of drug metabolism cannot predict risk and can only identify potential hazards

Not all idiosyncratic drug toxicity involves either the immune system or CRMs; removing metabolic liability is not a universal panacea for ADRs

Removing potential liability associated with a particular bioactivation pathway without evaluation of toxicity: can never be able to measure the success of the process.

Improved biomarkers are required for adaptation and toxicity

Occurrence, Frequency& Severity of

Drug Hepatotoxicity+= f1

Chemistryof drug f2

Biology of individual

Acknowledgements

Liverpool

Dan AntoineRoz JenkinsAnja KiparVal TilstonNeil KitteringhamJames MaggsNeil FrenchKevin Park

Collaborators

KUMCHartmut JaeschkeMitch McGillDave WilliamsMatt SharpeAZIna Schuppe-KoistinenTim HammondEdinburghJames Dear