18 October 2022

The importance of mitochondrial toxicity assessment in drug development

Mitochondrial toxicity is responsible for a variety of toxic effects, seen by clinicians as adverse reactions in humans. Cardiotoxicity and hepatotoxicity are the main two reasons for the withdrawal of drugs from the market. A comprehensive knowledge of the root cause of such incidents allows for a solid preclinical safety evaluation and testing strategy that will yield candidate drugs with a low risk of serious issues.


Billev Pharma East can assist you in the toxicity risk assessment of your molecules, during the R&D, registration, and postmarketing phase.


Mitochondria are the metabolic centers of eukaryotic cells, harbouring the fatty acid oxidation, tricarboxylic acid cycle, oxidative phosphorylation, ATP synthesis, and many other crucial processes. Moreover, they also play an important role in intracellular homeostasis, controlling the calcium equilibrium, the redox equilibrium, hormonal signalling, and apoptosis (1). Mitochondrial toxicants are compounds that disrupt the normal mitochondrial function and consequently cause damage by disrupting the above-mentioned mechanisms (2).


The pharmaceutical industry brings thousands of compounds to the market as drugs, only to see them withdrawn due to liver failure or cardiotoxicity, associated to mitochondrial toxicity. Some examples are presented in Table 1.


Mitochondrial toxicity is especially important in relation to cells that utilise high levels of energy, such as cardiomyocytes, hepatocytes, and myocytes.  Several drugs are known to cause mitochondrial damage, and affect their morphology, structure, and their quality control pathways (3). For example, NSAIDs cause mitochondrial swelling, which can be manifested in humans as hypertension, arrhythmias, thrombosis, and myocardial infarction (7, 8). Ethanol is also a mitochondrial toxicant, causing disruptions in mitochondrial quality control systems (9).


Table 1: Examples of rugs, withdrawn due to mitochondrial toxicity issues (3, 4, 5, 6)

Product Year launched Year withdrawn Adverse effect Mitochondrial toxicity
Isoprenaline 1949 1992 Severe cardiac arrhythmias Inhibition of oxidative phosphorylation, mPTP opening

Swelling of mitochondria, disappearance of the cristae

Nifedipine 1975 1996 Hypertension, angina pectoris, myocardial infarction Inhibition of ATP synthase
Rosiglitazone 1999 2011 Chronic heart failure, myocardial infarction Inhibition of ETC

Increase in mitochondrial oxidative stress

Impairment of mitochondrial bioenergetics

Uncoupling of oxidative phosphorilation

Clozapine 1972 1975 Cardiomyopathy, myocardial infarction, prolonged QT Inhibition of ETC

Increase in ROS formation, GSH depletion, mitochondrial dysfunction, and swelling

Prenylamine 1960 1989 QT prolongation, sudden cardiac death, ventricular tachycardia Inhibition of FAO
Fenfluramine 1973 1997 Valvular heart disease Mitochondrial fragmentation
Celecoxib 2003 2011 Serious (and potentially fatal) cardiovascular thrombotic events Decrease in mitochondrial complex IV activity and induces oxidative stress
Etoricoxib 2002 2007 Serious (and potentially fatal) cardiovascular thrombotic events Inhibition of oxidative phosphorilation
Thioridazine 1959 2000 QT prolongation, TdP, sudden cardiac death mPTP opening

MMP collapse

Trovafloxacin 1997 1999 Fatal liver damage Peroxynitrite stress

Decreases mitochondrial aconitase-2 activity in Sod2+/−

Cerivastatin 1990 2001 Rhabdomyolysis and kidney failure Uncoupling of oxidative phosphorylation

Dysfunction of complex I

It is therefore imperative to have an adequate risk assessment plan in place, both in the R&D and the registration phase of the drug. The currently established preclinical models for assessing mitochondrial toxicity are not optimal, especially not in the scope of drug-induced cardiotoxicity assays. Two main ICH guidelines (ICH S7B and ICH E14) determine the assessment of cardiotoxicity. However, as the QT prolongation study, as determined by ICH E14, is not a robust surrogate for the overall arrhythmogenic potential, many drugs are being mistakenly discarded in the development phase. As a response, ICH published an updated guideline in 2020, combining Questions and Answers to both implicated guidelines. The new proposed strategy (CiPA; comprehensive in vitro proarrhythmia assay) evaluates many repolarization-related currents, electrophysiology in silico, and subsequent validation in human induced pluripotent stem cells. However, mitochondrial toxicity testing is still not included in the routine cardiotoxicity assessment, despite the fact that many cardiotoxic effects are directly attributable to mitochondrial dysfunction.


Another important aspect in the evaluation is the so-called “hidden cardiotoxicity”, this is the toxicity of drugs that only manifests itself in the diseased heart. Drugs are not routinely tested in preclinical models of cardiovascular disease, and this may be one of the main causes of the relatively high rate of cardiotoxicity issues with many medicinal products.


In a recent review by Tang (2022), a novel approach to preclinical assessment for drug-induced mitochondrial toxicity has been proposed. The methodology uses in vitro cardiomyocyte models and proposes to include mitochondrial endpoints in cardiotoxicity assays (morphology, oxygen consumption rate, ATP level, redox homeostasis, MMP). The mitochondrial toxicity assays are performed in the preclinical phase, alongside the established in vitro hERG/Ikr assay and in vivo QT assay. The inclusion of such testing in the integrated risk assessment yields a low-risk candidate drug that may be safely tested in early phase clinical trials.


(1)   Nunnari, J.; Suomalainen, A. Mitochondria: In sickness and in health. Cell 2012, 148, 1145–1159.

(2)   Varga, Z.V.; Ferdinandy, P.; Liaudet, L.; Pacher, P. Drug-induced mitochondrial dysfunction and cardiotoxicity. Am. J. Physiol. Heart Circ. Physiol. 2015, 309, H1453–H1467.

(3)   Tang X, Wang Z, Hu S, Zhou B. Assessing Drug-Induced Mitochondrial Toxicity in Cardiomyocytes: Implications for Preclinical Cardiac Safety Evaluation. Pharmaceutics. 2022 Jun 21;14(7):1313. doi: 10.3390/pharmaceutics14071313. PMID: 35890211; PMCID: PMC9319223.

(4)   Avram VF, Chamkha I, Åsander-Frostner E, Ehinger JK, Timar RZ, Hansson MJ, Muntean DM, Elmér E. Cell-Permeable Succinate Rescues Mitochondrial Respiration in Cellular Models of Statin Toxicity. Int J Mol Sci. 2021 Jan 3;22(1):424. doi: 10.3390/ijms22010424. PMID: 33401621; PMCID: PMC7796258.

(5)   Will Y, Dykens J. Mitochondrial toxicity assessment in industry–a decade of technology development and insight. Expert Opin Drug Metab Toxicol. 2014 Aug;10(8):1061-7. doi: 10.1517/17425255.2014.939628. PMID: 25023361.

(6)   Hsiao CJ, Younis H, Boelsterli UA. Trovafloxacin, a fluoroquinolone antibiotic with hepatotoxic potential, causes mitochondrial peroxynitrite stress in a mouse model of underlying mitochondrial dysfunction. Chem Biol Interact. 2010 Oct 6;188(1):204-13. doi: 10.1016/j.cbi.2010.07.017. Epub 2010 Jul 23. PMID: 20655887.

(7)   Salimi, A.; Neshat, M.R.; Naserzadeh, P.; Pourahmad, J. Mitochondrial Permeability Transition Pore Sealing Agents and Antioxidants Protect Oxidative Stress and Mitochondrial Dysfunction Induced by Naproxen, Diclofenac and Celecoxib. Drug Res. 2019, 69, 598–605.

(8)   Khezri, S.; Atashbar, S.; Azizian, S.; Shaikhgermchi, Z.; Kurdpour, P.; Salimi, A. Calcitriol Reduces Adverse Effects of Diclofenac on Mitochondrial Function in Isolated Rat Heart Mitochondria. Drug Res. 2020, 70, 317–324