Biomarkers  of  paracetamol  toxicity  

Dr  James  Dear  Edinburgh  University  

Hospital  Episode  Sta?s?cs  2010-­‐11  

Poison   Emergency  admissions  (England)  

Paracetamol   38,464  

An?depressants   16,180  

NSAIDs   9,429  

Opiates   9.135  

Benzodiazepines   8,952  

Hospital  Episode  Sta?s?cs  2010-­‐11  

Reason  for  admission   Emergency  admissions  (England)  

Paracetamol   38,464  

Fracture  of  neck  of  femur   42,616  

Conges?ve  heart  failure   37,148  

Acute  myocardial  infarc?on   26,967  

Acute  exacerba?on  of  COPD   44,969  

Na?onal  Poisons  Informa?on  Service  UK    Toxbase  use  

Na?onal  Poisons  Informa?on  Service  UK    Telephone  enquiries  

Paracetamol  

Oxida?on  by  cytochrome  P450  enzymes  –  a  minor  route  

in  therapeu?c  doses  

NAPQI  

Reacts  with  SH-­‐  group  in    

glutathione  

Paracetamol  conjugates  

Conjuga?on  –  the  major  route  of  metabolism  in  therapeu?c  dose  

NAPQI  conjugate  

Mechanism  of  paracetamol  hepatotoxicity  

Paracetamol  

In  overdose,  oxida?on  by  cytochrome  P450s  becomes  

important    

NAPQI  

Glutathione  supply  exhausted  

Paracetamol  conjugates  

Conjuga?on  –  saturated  in  overdose  

NAPQI  conjugate  

SH-­‐  

Excess  NAPQI  binds  to  SH-­‐groups  in  structural  protein  

Mechanism  of  paracetamol  hepatotoxicity  

Acetylcysteine  

Risk  assessment  •  Majority  of  pa?ents  present  soon  aaer  OD  before  liver  injury  can  be  diagnosed  using  current  tests  such  as  ALT    

•  Therefore,  use  surrogate  marker    •  Blood  paracetamol  concentra4on  

BJCP  2009  68  260  -­‐  268  

Risk  assessment  –  paracetamol  concentra?on  

Prescoc  LF,  Health  Bulle?n  1978,  204-­‐212  

ADRs  to  acetylcysteine  and  paracetamol  level  at  presenta4on    

Clin  Tox  2013  51  467-­‐472  

Risk  assessment  –  paracetamol  concentra?on  UK   USA  

Risk  assessment  –  paracetamol  concentra?on  

Prescoc  LF,  Health  Bulle?n  1978,  204-­‐212  

No  injury    without  NAC  

What  is  a  biomarker?  

•  Biomarker.  A  biomarker  is  a  biological  characteris?c  that  is  objec?vely  measured  and  evaluated  as  an  indicator  of  normal  biological  processes,  pathogenic  processes,  or  pharmacologic  response  to  therapeu?c  interven?on.    

Pregnancy  test  

•  Perfect  biomarker!  

•  Posi?ve  result  has  a  significant  impact  –  adds  real  value  •  Highly  sensi?ve/specific  for  early  diagnosis  –  accurate  (in  context)  •  Detects  a  urine  protein  not  normally  expressed  –  accessible  ?ssue  •  Urine  concentra?on  not  an  issue  –  no  normaliza?on  needed  

•  Simple  accurate  test  –  measurable  

•  Rapid  result  –  appropriate  turnaround  ?me  •  Widely  available  –  used  by  those  who  need  it  

Paracetamol  poisoning  

•  Good  model  for  biomarker  discovery  in  humans  as:  •  Young  pa?ents  •  Oaen  no  co-­‐morbidity  

•  Clearly  defined  insult  •  No  symptoms  (at  first)  

•  Loads  of  pa?ents  •  Neglected  from  research  viewpoint  

JASN 2004;15:1677-1689

Uses  of  biomarkers  Disease  severity

 

Chest  pain   ECG   Cardiac  enzymes  CK,AST,LDH  

Presen?ng    complaint   Risk  stra?fy   Detect  cell  death  

?me  

Heart  acacks…..  

Early,  diagnos?c  markers…….  

Chest  pain   ECG   Cardiac  enzymes  CK,AST,LDH  

Presen?ng    complaint   Risk  stra?fy   Detect  cell  death  

?me  

Troponin  

Heart  acacks…..  

Early,  diagnos?c  markers…….  

History  of  overdose  

Timed  paracetamol  concentra?on  

Liver  enzymes  ALT  etc  

Presen?ng    complaint   Risk  stra?fy   Detect  cell  death  

?me  

Paracetamol  OD…..  

Early,  diagnos?c  markers…….  

History  of  overdose  

Timed  paracetamol  concentra?on  

Liver  enzymes  ALT  etc  

Presen?ng    complaint   Risk  stra?fy   Detect  cell  death  

?me  

New  marker  

Paracetamol  OD…..  

Early,  diagnos?c  markers…….  

MicroRNA  -­‐  122  

Circulating microRNAs as translational biomarkers

miR-122 miR-192

miR-1

miR-218

miR-218 miR-219 miR-709

miR-146a miR-155

miR-21 miR-133a

HCC – miR-15b, 130b Kidney – miR-378 ( Wang et al, 2009, Mitchell et al, 2007, Redova et al, 2012, Liu et al,

2012

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Prostate – miR-629,650

Proc  Natl  Acad  Sci  U  S  A.  2009  106:4402-­‐7.  

How  paracetamol  could  increase  microRNA  in  circula?on  

Paracetamol        

Human  Studies  

Study  1.  Pa?ent  groups  1.  APAP  –  induced  acute  liver  injury.  Defined  as  a  sudden  

deteriora?on  in  liver  func?on  with  associated  coagulopathy  in  the  absence  of  a  history  of  chronic  liver  disease.  Clear  history  of  excess  APAP  inges?on.  n=53.  

2.  APAP  –  no  liver  injury.  Single  APAP  inges?on  in  overdose  that  required  treatment  with  acetylcysteine.  Absence  of  liver  injury  was  confirmed  by  a  normal  serum  ALT  ac?vity  (defined  as  ≤3  x  ULN).  n=6.  

3.  Non  APAP  acute  liver  injury.  Causes:  HBV,  HCV,  AFLP,  AIH,  DILI,  Ischemia,  Malignancy.  n=11.  

4.  Healthy  controls.  Age  and  sex  matched  with  APAP-­‐ALI  group.  n=25.  

5.  Chronic  kidney  disease.  Mean  urinary  protein  excre?on  rate  was  1570  ±  371  µg/min.  Mean  GFR  of  43  ±  5  ml/min/1.73m2.    n=22.  

MicroRNA  species  

•  miR-­‐122      Liver  enriched  *                              *  Increased  in  mice  

•  miR-­‐192      Liver  enriched  (also  kidney)  *  

•  miR-­‐1      Heart  enriched  

•  miR-­‐218  Brain  enriched  

•  All  normalized  to  U6  snRNA    

•  Day  1  =  day  of  entry  into  study  NOT  day  of  inges?on  

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PAP

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-APA

P A

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Hepatology.  2011  54:1767-­‐76    

Study  2.  Method  •  Pa?ents  (total  N=129)  were  recruited  from  the  Royal  Infirmary  of  Edinburgh  

(N=107)  and  the  Royal  Victoria  Infirmary,  Newcastle-­‐Upon-­‐Tyne  (N=22).    

•  Inclusion   criteria   were:   adults   with   a   clear   history   of   a   single   excess  paracetamol   inges?on   and   a   ?med   blood   paracetamol   concentra?on   that  was  judged  to  necessitate  hospital  admission  for  intravenous  acetylcysteine  therapy,  as  per  UK  guidelines  at  the  ?me  of  study  

•  Exclusion   criteria   were:   pa?ents   detained   under   the   Mental   Health   Act;  pa?ents   with   permanent   cogni?ve   impairment;   pa?ents   with   a   life-­‐threatening   illness;   unreliable   history   of   paracetamol   overdose;   pa?ents  who   take   an?coagulants   therapeu?cally   or   have   taken   an   overdose   of  an?coagulants;   and   pa?ents   who,   in   the   opinion   of   the   responsible  clinician/nurse,  were  unlikely  to  complete  the  full  course  of  acetylcysteine.  

•  All  pa?ents  completed  the  full  course  of  acetylcysteine.      

Method  

 miR-­‐122  was  measured  in  plasma  at  first  presenta?on  to  hospital  before  acetylcysteine  started  

 Primary  outcome:    Acute  liver  injury    -­‐  peak  serum  ALT  ac?vity  greater  than  3x  the  upper  limit  of  normal  (>150IU/L)  

Presenta4on  biomarkers  vs  peak  ALT  

1 10 100 1000 10000 100000

0.0001

0.001

0.01

0.1

1

10

100

1000

10000

100000

Peak ALT activity (U/l)

Pres

enta

tion

miR

-122

(Let

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orm

alis

ed)

Biomarker R2 Pearson R (95% CI)

P

miR-122 0.14 0.37 (0.21-0.52) <0.0001

Hepatology.  2013  58:777-­‐787      

Peak ALT <ULN Peak ALT >3x ULN 0.01

0.1

1

10

100

1000

10000

Pres

enta

tion

miR

-122

(Let

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orm

alis

ed)

P < 0.0001

miR-­‐122    at  presenta?on  was  elevated  in  pa?ents  who  develop  ALI  

In  pa?ents  with  a  normal  ALT    

Hepatology.  2013  58:777-­‐787      

0.00 0.25 0.50 0.75 1.00 0.00

0.25

0.50

0.75

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1 - Specificity

Sens

itivi

ty

miR-122 AUC 0.93 P < 0.0001 SENS 0.83 PPV 70.6 % NPV 97.5%

Performance  of  first  presenta?on  miR-­‐122  at  predic?ng  acute  liver  injury  In  pa?ents  with  a  normal  ALT    

Hepatology.  2013  58:777-­‐787    

Time  course  of  miR-­‐122    11053

20 30 40 50 600

100

200

300

400ALT

miR-122

Time after overdose (h)

X34

0 10 20 30 40 50 600

5

10

15

20

25

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miR-122

Time after overdose (h)

FV2

0 10 20 30 40 500.0

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Time after overdose (h)

Time  course  of  miR-­‐122    

11041

0 10 20 30 4005

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miR-122

Time after overdose (h)

11053

20 30 40 50 600

100

200

300

400ALT

miR-122

Time after overdose (h)

X34

0 10 20 30 40 50 600

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miR-122

Time after overdose (h)

FV2

0 10 20 30 40 500.0

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Time after overdose (h)

Zebrafish  

Zebrafish  in  press  

Zebrafish  

miR-­‐122  is  transla?onal  across  species  Zebrafish  in  press  

Other  microRNA?  

Mechanism  markers  

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! ! ! ! Mitochondrial  DNA  and  Enzymes   Reflect  mitochondrial  damage  

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J Hepatology 2012 J  Hepatol  2012  56:1070-­‐9    

J  Clin  Invest.  2012;122:1574-­‐83  

Method  •  Pa?ents  (total  N=129)  were  recruited  from  the  Royal  Infirmary  of  Edinburgh  

(N=107)  and  the  Royal  Victoria  Infirmary,  Newcastle-­‐Upon-­‐Tyne  (N=22).    

•  Inclusion   criteria   were:   adults   with   a   clear   history   of   a   single   excess  paracetamol   inges?on   and   a   ?med   blood   paracetamol   concentra?on   that  was  judged  to  necessitate  hospital  admission  for  intravenous  acetylcysteine  therapy,  as  per  UK  guidelines  at  the  ?me  of  study  

•  Exclusion   criteria   were:   pa?ents   detained   under   the   Mental   Health   Act;  pa?ents   with   permanent   cogni?ve   impairment;   pa?ents   with   a   life-­‐threatening   illness;   unreliable   history   of   paracetamol   overdose;   pa?ents  who   take   an?coagulants   therapeu?cally   or   have   taken   an   overdose   of  an?coagulants;   and   pa?ents   who,   in   the   opinion   of   the   responsible  clinician/nurse,  were  unlikely  to  complete  the  full  course  of  acetylcysteine.  

•  All  pa?ents  completed  the  full  course  of  acetylcysteine.      

Method  

 New  biomarkers  were  measured  in  plasma  at  first  presenta?on  to  hospital  before  acetylcysteine  started  

 Primary  outcome:  

 Acute  liver  injury    -­‐  peak  serum  ALT  ac?vity  greater  than  3x  the  upper  limit  of  normal  (>150IU/L)  

1 10 100 1000 10000 100000 0.01

0.1

1

10

100

Peak ALT activity (U/l)

Pres

enta

tion

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1 (n

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100

1000

10000

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tion

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10000

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Pres

enta

tion

necr

osis

K18

(U

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Biomarker R2 Pearson R (95% CI) P

GLDH 0.45 0.67 (0.56-0.76) <0.0001

HMGB1 0.67 0.82 (0.75-0.87) <0.0001

Apop K18 0.57 0.75 (0.67-0.82) <0.0001

Necrosis K18 0.59 0.77 (0.69-0.83) <0.0001

Presenta4on  biomarkers  vs  peak  ALT  

Hepatology  2013  58:777-­‐787    

Nec  K18   HMGB1  

Apop  K18  

1 10 100 1000 10000 100000 1

10

100

1000

10000

100000

Peak ALT activity (U/l)

Pres

enta

tion

GLD

H a

ctiv

ity

(U/l)

GLDH  

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0.75

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1 - Specificity

Sens

itivi

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0.00 0.25 0.50 0.75 1.00 0.00

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0.50

0.75

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1 - Specificity

Sens

itivi

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1 - Specificity

Sens

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ty

0.00 0.25 0.50 0.75 1.00 0.00

0.25

0.50

0.75

1.00

1 - Specificity

Sens

itivi

ty

HMGB1 AUC 0.97 P < 0.0001 SENS 0.91

Necrosis K18 AUC 0.94 P < 0.0001 SENS 0.90

Apoptosis K18 AUC 0.77 P = 0.0009 SENS 0.21

GLDH AUC 0.80 P = 0.0003 SENS 0.19

0.00 0.20 0.40 0.60 0.80 1.00 0.00

0.25

0.50

0.75

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1 - Specificity

ALT AUC 0.54 P = 0.059 SENS 0.09 Se

nsiti

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Performance  of  first  presenta?on  biomarkers  at  predic?ng  acute  liver  injury  

Apoptosis  K18  

Apo

ptosis  K18                    

(AUROC,  0.755  [0.639–0.885,  p  <  0.001]  sensi?vity,  89%;  specificity,  61%;  cutoff,  2,718)  

Day  1  single  liver  unit    Predic?ng  Death/LT  

Crit  Care  Med  2013  

Progression  biomarkers  

Day  1  UK  and  USA  liver  unit    Predic?ng  Death/LT  

J  Hepatol.  2012  56:1070-­‐79  

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Spontaneous survivors

Died / Required Liver transplant

• potential novel biomarker • evidence in man for innate immune

system • need for multi cellular systems for

prediction of human DILI

CASE  REPORT:  25  year  old  male  Single  overdose  of  35g  paracetamol  at  02:30  (?ming  supported  by  Facebook  message)  Assessed  4.5h  aaer  OD  No  risk  factors  for  hepatotoxicity.  Paracetamol  level  107  mg/L  (below  nomogram)  Normal  biochemical  evidence  of  liver  injury  Assessed  by  senior  doctor  and  not  treated  Discharged  aaer  psychiatry  review  

Represented  to  hospital  43h  aaer  OD  Lethargic  and  vomi?ng  Tender  abdomen    

Time  from  OD  (h)   4.5   43  

Paracetamol  (mg/L)   107   9  

ALT  (U/L)    (ULN  50)  

34   11314  

INR     1.0   2.1  

miR-­‐122  (/  let-­‐7d)    (ULN  5.2*)  

261          (x50)  

HMGB1  (ng/ml)  (ULN  0.9*)  

7.2            (x8)  

Necrosis  K18  (U/L)  (ULN  480*)  

4018      (x8)  

NEW  MARKERS  CORRECTLY  IDENTIFIED  

LIFE  THREATENING  HEPATOTOXICITY  MISSED  

BY  CURRENT  TESTS  *95%  predic?on  interval  –  no  liver  injury  aaer  overdose  n=82  Hepatology  2013  

CASE  REPORT:  25  year  old  male  Single  overdose  of  35g  paracetamol  at  02:30  (?ming  supported  by  Facebook  message)  Assessed  4.5h  aaer  OD  No  risk  factors  for  hepatotoxicity.  Paracetamol  level  107  mg/L  (below  nomogram)  Normal  biochemical  evidence  of  liver  injury  Assessed  by  senior  doctor  and  not  treated  Discharged  aaer  psychiatry  review  

Represented  to  hospital  43h  aaer  OD  Lethargic  and  vomi?ng  Tender  abdomen    

Time  from  OD  (h)   4.5   43  

Paracetamol  (mg/L)   107   9  

ALT  (U/L)    (ULN  50)  

34   11314  

INR     1.0   2.1  

miR-­‐122  (/  let-­‐7d)    (ULN  5.2*)  

261          (x50)  

HMGB1  (ng/ml)  (ULN  0.9*)  

7.2            (x8)  

Necrosis  K18  (U/L)  (ULN  480*)  

4018      (x8)  

NEW  MARKERS  CORRECTLY  IDENTIFIED  

LIFE  THREATENING  HEPATOTOXICITY  MISSED  

BY  CURRENT  TESTS  *95%  predic?on  interval  –  no  liver  injury  aaer  overdose  n=82  Hepatology  2013  

CASE  REPORT:  25  year  old  male  Single  overdose  of  35g  paracetamol  at  02:30  (?ming  supported  by  Facebook  message)  Assessed  4.5h  aaer  OD  No  risk  factors  for  hepatotoxicity.  Paracetamol  level  107  mg/L  (below  nomogram)  Normal  biochemical  evidence  of  liver  injury  Assessed  by  senior  doctor  and  not  treated  Discharged  aaer  psychiatry  review  

Represented  to  hospital  43h  aaer  OD  Lethargic  and  vomi?ng  Tender  abdomen    

Time  from  OD  (h)   4.5   43  

Paracetamol  (mg/L)   107   9  

ALT  (U/L)    (ULN  50)  

34   11314  

INR     1.0   2.1  

miR-­‐122  (/  let-­‐7d)    (ULN  5.2*)  

261          (x50)  

HMGB1  (ng/ml)  (ULN  0.9*)  

7.2            (x8)  

Necrosis  K18  (U/L)  (ULN  480*)  

4018      (x8)  

NEW  MARKERS  CORRECTLY  IDENTIFIED  

LIFE  THREATENING  HEPATOTOXICITY  MISSED  

BY  CURRENT  TESTS  *95%  predic?on  interval  –  no  liver  injury  aaer  overdose  n=82  Hepatology  2013  BJCP  2013  Published  online  

Biomarker  discovery  in  

model  systems    

Biomarker  qualifica?on  in  animal  models  

Proof  of  concept  in  humans  

Biomarker  qualifica?on  in  humans  

Combina?on  with  therapeu?c  in  

stra?fied  clinical  trial  

Transla?on  to  other  causes  of  DILI  in  humans  

Qualifica?on  as  tool  in  preclinical  and  clinical  drug  development  

Paracetamol  poisoning  as  model  of  DILI    

Completed   Completed   Completed  

Current    posi?on  

Markers  and  Paracetamol  Poisoning  Study  (MAPP)  

Biomarker  valida?on  study    

Recrui?ng  across  UK  

Target  1000  pa?ents  

Recruited  around  400  so  far  

Will  validate  the  markers  performance  at  hospital  front  door  

Acknowledgements  Edinburgh  

Wilna  Oosthuyzen  Bas?aan  Vliegenthart  Jonathan  Street    Machew  Bailey  Professor  Nick  Bateman  Judy  Coyle  Professor  Alasdair  Gray  Moyra  Masson  Professor  David  J  Webb  Professor  Chris  Gregory  Carl  Tucker  

CDSS  Liverpool  

Professor  Kevin  Park  Dr  Dan  Antoine  Dr  Chris  Goldring  Phillip  Starkey-­‐Lewis  Vivien  Plac  

Newcastle    Ruben  Thanacoody  Professor  Simon  Thomas  

Novar?s  Dr  Jonathan  Moggs