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

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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