Drug induced hepatotoxicity Hepatotoxicity implies chemical-driven liver damage. Liver injury may be produced by a large variety of chemical substances The liver plays a central role in transforming and clearing chemicals and is susceptible to the toxicity from these agents Certain medicinal agents, when taken in overdoses & sometimes even when introduced within therapeutic ranges may injure the liver It might not be the drug that cause hepatotoxicity but its metabolite might The herbal drugs and agents can also cause the liver injury

1. Drug induced hepatotoxicity and
its regulatory implications
Chander K Negi
M.S (Pharm)
chandernegi09@gmail.com
Department of Pharmacology and Toxicology
National Institute of Pharmaceutical Education and Research (NIPER)
Sector-67, S.A.S. Nagar, Mohali, Punjab-160062

2. Physiological function of liver
2
Formation and
secretion of bile
Metabolism and
micronutrients
storage
Detoxification &
inactivation of
various substances
(toxin, drug)
Synthesis of
plasma proteins
(albumin, clotting
factor)
Jaeschke H, Gores GJ, Cederbaum AI, Hinson JA, Pessayre D, Lemasters JJ (2002). Toxicological sciences : an official journal of the
Society of Toxicology 65(2): 166-176.

3. Drug induced hepatotoxicity
3
Hepatotoxicity implies chemical-driven liver
damage. Liver injury may be produced by a
large variety of chemical substances
The liver plays a central role in transforming
and clearing chemicals and is susceptible to the
toxicity from these agents
Certain medicinal agents, when taken in
overdoses & sometimes even when introduced
within therapeutic ranges may injure the liver
It might not be the drug that cause
hepatotoxicity but its metabolite might
The herbal drugs and agents can also cause the
liver injury
Jaeschke H, Gores GJ, Cederbaum AI, Hinson JA, Pessayre D, Lemasters JJ (2002). Toxicological sciences : an official journal of the
Society of Toxicology 65(2): 166-176.

4. Biotransformation of drug
4
Friedman, S.L., McQuaid K.R., 2003, Current Diagnosis and Treatment in
Gastroenterology, USA: The McGraw-Hills, Comp.

5. Types of drug induced hepatotoxicity
Intrinsic Idiosyncratic
Incidence More common all
individuals
Less common 1%
Predictability Predicted Unpredicted
Dose related Dose dependent Dose independent
Latency period Short latency period Variable latency period
weeks or months
Type of injury Usually necrosis Necrosis or apoptosis
Associated Acute liver failure Rash, fever, eosinophilia
Examples Acetaminophen Isoniazid
5Roth RA, Ganey PE (2010). The Journal of pharmacology and experimental therapeutics332(3): 692-697

6. 3 Step model of drug induced hepatotoxicity
Direct cell stress, direct mitochondria
impairment and specific immune reactions
Direct and Death receptor mediated
pathways leading to MPT
Apoptosis and necrosis
6
Russmann S, Kullak-Ublick GA, Grattagliano I (2009). Current medicinal chemistry16(23): 3041-3053.

7. 1. Initial mechanism of toxicity: Direct cell stress, direct mitochondria
impairment and specific immune reactions.
If the drug metabolite acts as a hapten it binds covalently with the liver protein, alter the protein and
that protein is recognized as neo-antigen, the altered protein (Hapten) then be perceived as foreign
by the immune system resulting in an autoimmune attack on hepatocellular constituents and cyp450
enzymes also
Damage mitochondrial DNA, interfere with the replication process, causes the opening of MPT
Inhibits the MRC, cause ATP depletion and increase in ROS, inhibits β-oxidation, cause steatosis,
Reactive metabolites depletes GSH, covalently binding to proteins, enzymes, lipids, nucleic acid
and other cell structures or induce lipid peroxidation
7Russmann S, Kullak-Ublick GA, Grattagliano I (2009). Current medicinal chemistry16(23): 3041-3053.

8. 2. Direct and Death receptor mediated pathways leading to MPT
Extrinsic pathway is the indirect
death receptor mediated pathway
If the initial event is a specific
immune reaction, the hapten
activate the release of TNFα, and
FasL from Kupffer cells (hepatic
and cytotoxic T-cells) TNFα, and
FasL bind to intracellular death
receptors, TNF and Fas receptor
associated death domain proteins
will subsequently activate
initiator caspase 8
Intrinsic pathway direct
pathway
Several intracellular stress
activates the proapoptotic
factors as Bax, Bak, Bad and
inhibits the anti-apoptotic
factors as Bcl2, BclxL proteins
of the Bcl-2 family then
activates MPT
8
Russmann S, Kullak-Ublick GA, Grattagliano I (2009). Current medicinal chemistry16(23): 3041-3053.

9. Apoptotic signalling pathway Intrinsic and Extrinsic
9Slee EA, Adrain C, Martin SJ (2001). The Journal of biological chemistry276(10): 7320-7326

10. 3. Apoptosis and necrosis
MPT allows the influx of protons through the
inner mitochondrial membrane which stops
mitochondrial ATP synthesis
Mitochondrial matrix expansion and
mitochondrial outer membrane permeabilization
,release of cytochrome C
Cytochrome C binds to the apaf-1 apoptotic
protease activating factor and procaspase 9 to
form apoptosome which activates pro caspases
9 & effector caspase 3,6,7, causes apoptosis
Active process requires ATP, occur only if MPT
is not rapidly occurred in mitochondria
Necrosis is developed in case of initial injury
which causes rapid MPT in all mitochondria or
rapid severe mitochondrial ATP depletion.
10Holt MP, Ju C (2006). The AAPS journal8(1): E48-54

11. Cellular mechanism of drug hepatotoxicity
11Kaplowitz N (2005). Nature reviews. Drug discovery4(6): 489-499.

12. Mechanism of drug induced hepatotoxicity
12
Russmann S, Kullak-Ublick GA, Grattagliano I (2009). Current medicinal chemistry16(23): 3041-3053.

13. Hepatotoxic drugs and their effects on mitochondria
Drug MPTP opening Direct inhibition of
mitochondrial FAO
OXPHOS
uncoupling
Direct
inhibition of
MRC
mtDNA
depletion or
damage
Acetaminophen + + + +
Amiodarone + + + +
Buprenorphine + + +
Diclofenac + + + +
Didanosine +
Disulfiram + +
Ibuprofen + +
Nimesulide + +
Panadiplon +
Perhexiline + + +
Pirprofen +
Salicylic acid + + +
Stavudine +
Tacrine + +
Tamoxifen + + + +
Tetracycline & derivatives +
Troglitazone + + +
Valprovic acid + +
Zidovudine +
13
Begriche K, Massart J, Robin MA, Borgne-Sanchez A, Fromenty B (2011). Journal of hepatology54(4): 773-794.

14. List of drugs withdrawn or severely limited because of hepatotoxicity
Drug Chemical Class Discontinued Country
TienilicAcid
(Ticrynafen)
Loop Diuretics 1982 Germany, France, UK, US,
Others
Benoxaprofen (Oraflex) NSAIDS 1982 Germany, Spain, UK, US
Pirprofen NSAIDS 1990 France, Germany, Spain
Tolrestat
(Alredase)
Aldose Reductase Inhibitor 1996 Argentina, Canada, Italy,
Tolcapone (Tasmar) Catechol-O-Methyl Transferas
(COMT)
1998 European Union, Canada,
Australia
Bromfenac (Duract) NSAIDS June 22, 1998 US
Trovafloxacin (Trovan) Fluoroquinolones Antibiotics June 1999 European Union, US
Troglitazone (Rezulin) Antidiabetic And
Anti-inflammatory
March 21 2000 US, Germany
Ximelagatran
(Exanta)
Anticoagulant 2006 Germany
Sitaxentan Endothilin receptor antagonist 2010 Germany
Tacrine (Cognex) Anticholinesterase 2013 US *
14
Labbe G, Pessayre D, Fromenty B (2008).Fundamental & clinical pharmacology22(4): 335-353.
* http://www.medicinenet.com/tacrine/page2.htm

15. List of marketed drugs capable of inducing hepatotoxicity caused by
mitochondrial dysfunction, which have received warnings from drug
agencies
Drug Chemical class Regulatory action
Felbamate Anticonvulsant Restricted use
Pemoline CNS stimulant Restricted use
Acetaminophen Analgesic Warnings
Leflunomide Immunomodulator Restricted use
Nefazodone Antipsychotic Warnings
Nevirapine Antiviral Warnings
Pyrazinamide Antituberculosis Warnings
Rifampin Antituberculosis Warnings
Terbinafine Antifungal Warnings
Valproic acid Anticonvulsant Warnings
Zafirlukast Asthma Warnings
15
Maddrey WC (2005). Journal of clinical gastroenterology39(4 Suppl 2): S83-89

16. Streams of evidence for chemical toxicity assessment in
clinical and environmental health sciences
• New chemical entity
• In vitro and in vivo toxicity testing
• Human experimental studies
• Enter market place and clinic
• Post exposure observation
16
Ballet F (1997). Journal of hepatology26 Suppl 2: 26-36.

17. Parallelogram Approach to Characterize Toxicity
17Ballet F (1997). Journal of hepatology26 Suppl 2: 26-36.

18. In vitro cytotoxicity screening assay
Several toxicity parameters are assessed over a wide range of compound
concentration
 LDH leakage Lactate dehydrogenase leakage
 MTT reduction colorimetric assay
 ATP contents
Thus providing the IC50% (The half maximal inhibitory concentration) value
IC50% is the measure of the effectiveness of a substance in inhibiting a
specific biological or biochemical function.
18
Numerous models are used for in vitro hepatotoxicity studies, Isolated
perfused liver
 Hepatocytes
 Liver slices
 Primary cultures of hepatocytes
 Cell lines
Dambach DM, Andrews BA, Moulin F (2005). Toxicologic pathology33(1): 17-26.

19. In vivo hepatotoxicity studies
• Toxicity testing in two animal species Rats and Dogs another species may be
chosen If the compound is not bioavailable in the rat and dog
• The in vivo toxicity studies includes the following
• A rat acute toxicity study
• A rat repeated dose toxicity study
• A dog rising-dose tolerance study
Acute toxicity study Repeated toxicity study Rising dose tolerance study
Administer compound in a single
dose up to that which induces
lethality not exceeding 2000 mg/kg
Small number of animals (5/sex
per dose) are dosed daily
Determine plasma drug level
Small number of dogs (1- 2/sex)
are treated by an
“incrementation” process
Monitor clinical signs as bodyweight,
food consumption
Clinical signs, bodyweight, food
consumption, clinical pathology
coagulation time, bilirubin,
transaminases and alkaline
phosphatases) necropsy with
histopathological examination
Same as in case of rat but no
necropsy.
19
Dambach DM, Andrews BA, Moulin F (2005). Toxicologic pathology33(1): 17-26.

20. Conclusion
• Drug-induced hepatotoxicity is a significant clinical problem.
It is also a problem with major economic impact as the most
frequent cause of post–marketing withdrawal of new
medications
• Current preclinical test systems for hepatotoxicity are
inadequate, reflecting our limited understanding of
mechanisms of drug toxicity, particularly the
“hypersensitivity” or “idiosyncratic” types of reactions
• The main challenge is to be able to detect drug induced
mitochondrial dysfunction during preclinical studies
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