Drug induced hepatotoxicity and its regulatory implications
Chander K Negi
M.S (Pharm)
Department of Pharmacology and Toxicology
National Institute of Pharmaceutical Education and Research (NIPER)
Sector-67, S.A.S. Nagar, Mohali, Punjab-160062
Physiological function of liver
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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.
Drug induced hepatotoxicity
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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.
Biotransformation of drug
4Friedman, S.L., McQuaid K.R., 2003, Current Diagnosis and Treatment in Gastroenterology, USA: The McGraw-Hills, Comp.
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
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
6Russmann S, Kullak-Ublick GA, Grattagliano I (2009). Current medicinal chemistry16(23): 3041-3053.
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.
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
8Russmann S, Kullak-Ublick GA, Grattagliano I (2009). Current medicinal chemistry16(23): 3041-3053.
Apoptotic signalling pathway Intrinsic and Extrinsic
9Slee EA, Adrain C, Martin SJ (2001). The Journal of biological chemistry276(10): 7320-7326
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 CCytochrome 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 apoptosisActive process requires ATP, occur only if MPT is not rapidly occurred in mitochondriaNecrosis 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
Cellular mechanism of drug hepatotoxicity
11Kaplowitz N (2005). Nature reviews. Drug discovery4(6): 489-499.
Mechanism of drug induced hepatotoxicity
12Russmann S, Kullak-Ublick GA, Grattagliano I (2009). Current medicinal chemistry16(23): 3041-3053.
Hepatotoxic drugs and their effects on mitochondria Drug MPTP opening Direct inhibition of
mitochondrial FAOOXPHOS 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 +
13Begriche K, Massart J, Robin MA, Borgne-Sanchez A, Fromenty B (2011). Journal of hepatology54(4): 773-794.
List of drugs withdrawn or severely limited because of hepatotoxicity Drug Chemical Class Discontinued CountryTienilicAcid (Ticrynafen)
Loop Diuretics 1982 Germany, France, UK, US, Others
Benoxaprofen (Oraflex) NSAIDS 1982 Germany, Spain, UK, US
Pirprofen NSAIDS 1990 France, Germany, SpainTolrestat(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 USTrovafloxacin (Trovan) Fluoroquinolones Antibiotics June 1999 European Union, USTroglitazone (Rezulin) Antidiabetic And
Anti-inflammatoryMarch 21 2000 US, Germany
Ximelagatran(Exanta)
Anticoagulant 2006 Germany
Sitaxentan Endothilin receptor antagonist 2010
Germany
Tacrine (Cognex) Anticholinesterase 2013 US *
14Labbe G, Pessayre D, Fromenty B (2008).Fundamental & clinical pharmacology22(4): 335-353.
* http://www.medicinenet.com/tacrine/page2.htm
List of marketed drugs capable of inducing hepatotoxicity caused by mitochondrial dysfunction, which have received warnings from drug agencies
Drug Chemical class Regulatory actionFelbamate 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
15Maddrey WC (2005). Journal of clinical gastroenterology39(4 Suppl 2): S83-89
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
16Ballet F (1997). Journal of hepatology26 Suppl 2: 26-36.
Parallelogram Approach to Characterize Toxicity
17Ballet F (1997). Journal of hepatology26 Suppl 2: 26-36.
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) valueIC50% is the measure of the effectiveness of a substance in inhibiting a specific biological or biochemical function.
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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.
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.
19Dambach DM, Andrews BA, Moulin F (2005). Toxicologic pathology33(1): 17-26.
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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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