In vitro pharmacological profiling is increasingly being used earlier in the drug discovery process to identify undesirable off-target activity profiles that could hinder or halt the development of candidate drugs or even lead to market withdrawal if discovered after a drug is approved. Here, for the first time, the rationale, strategies and methodologies for in vitro pharmacological profiling at four major pharmaceutical companies (AstraZeneca, GlaxoSmithKline, Novartis and Pfizer) are presented and illustrated with examples of their impact on the drug discovery process. We hope that this will enable other companies and academic institutions to benefit from this knowledge and consider joining us in our collaborative knowledge sharing.
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Stevens, J. L. Future of toxicology — mechanisms of toxicity and drug safety: where do we go from here? Chem. Res. Toxicol. 19, 1393–1401 (2006).
Redfern, W. S. et al. Safety pharmacology — a progressive approach. Fundam. Clin. Pharmacol. 16, 161–173 (2002).
Smith, D. A. & Schmid, E. F. Drug withdrawals and the lessons within. Curr. Opin. Drug Discov. Devel. 9, 38–46 (2006).
European Medicines Agency (EMA). ICH Topic S7A: Safety pharmacology studies for human pharmaceuticals. CPMP/ICH/539/00. EMA website [online], (2001).
European Medicines Agency (EMA). ICH Topic S7B: The nonclinical evaluation of the potential for delayed ventricular repolarization (QT interval prolongation) by human pharmaceuticals. CPMP/ICH/423/02. EMA website [online], (2005).
European Medicines Agency (EMA). ICH Topic M3 (R2): Non-clinical safety studies for the conduct of human clinical trials and marketing authorization for pharmaceuticals. CPMP/ICH/286/95. EMA website [online], (2009).
Sanguinetti, M. C., Jiang, C., Curran, M. E. & Keating, M. T. A mechanistic link between an inherited and an acquired cardiac arrhythmia: HERG encodes the IKr potassium channel. Cell 81, 299–307 (1995).
Redfern, W. S. et al. Relationships between preclinical cardiac electrophysiology, clinical QT interval prolongation and torsade de pointes for a broad range of drugs: evidence for a provisional safety margin in drug development. Cardiovasc. Res. 58, 32–45 (2003).
European Medicines Agency (EMA). Guideline on the non-clinical investigation of the dependence potential of medicinal products. EMEA/CHMP/SWP/94227/2004. EMA website [online], (2006).
Rothman, R. B. et al. Evidence for possible involvement of 5-HT2B receptors in the cardiac valvulopathy associated with fenfluramine and other serotonergic medications. Circulation 102, 2836–2841 (2000).
Huang, X. P. et al. Parallel functional activity profiling reveals valvulopathogens are potent 5-hydroxytryptamine2B receptor agonists: implications for drug safety assessment. Mol. Pharmacol. 76, 710–722 (2009).
Whitebread, S., Hamon, J., Bojanic, D. & Urban, L. In vitro safety pharmacology profiling: an essential tool for drug development. Drug Discov. Today 10, 1421–1433 (2005).
Bowes, J. et al. in The Process of New Drug Discovery and Development 2nd edn (eds Smith, C. G. & O'Donnell, J. T.) 103–134 (Informa Healthcare, 2006).
Laverty, H. G. et al. How can we improve our understanding of cardiovascular safety liabilities to develop safer medicines? Br. J. Pharmacol. 163, 675–693 (2011).
Hamon, J. & Whitebread, S. in Hit and Lead Profiling (eds Faller, B. & Urban, L.) 273–295 (Wiley VCH, 2009).
Spence, S., Anderson, C., Cukierski, M. & Patrick, D. Teratogenic effects of the endothelin receptor antagonist L-753,037 in the rat. Reprod. Toxicol. 13, 15–29 (1999).
Hulme, E. C., Birdsall, N. J. M. & Buckley, N. J. Muscarinic receptor subtypes. Annu. Rev. Pharmacol. Toxicol. 30, 633–673 (1990).
Gerretsen, P. & Pollock, B. G. Drugs with anticholinergic properties: a current perspective on use and safety. Expert Opin. Drug Saf. 10, 751–765 (2011).
Terstappen, G. C., Roncarati, R., Dunlop, J. & Peri, R. Screening technologies for ion channel drug discovery. Future Med. Chem. 2, 691–695 (2010).
Force, T. & Kolaja, K. L. Cardiotoxicity of kinase inhibitors: the prediction and translation of preclinical models to clinical outcomes. Nature Rev. Drug Discov. 10, 111–126 (2011).
Mellor, H. R., Bell, A. R., Valentin, J.-P. & Roberts, R. R. A. Cardiotoxicity associated with targeting kinase pathways in cancer. Toxicol. Sci. 120, 14–32 (2011).
Bi, K., Lebakken, C. S. & Vogel, K. W. Transformation of in vitro tools for kinase profiling: keeping an eye over the off-target liabilities. Expert Opin. Drug Discov. 6, 701–712 (2011).
Gilchrist, A. (ed.) GPCR Molecular Pharmacology and Drug Targeting: Shifting Paradigms and New Directions (Wiley, 2010).
Bridgland-Taylor, M. H. et al. Optimisation and validation of a medium-throughput electrophysiology-based hERG assay using IonWorks HT. J. Pharmacol. Toxicol. Methods 54, 189–199 (2006).
Harmer, A. R. et al. Optimisation and validation of a medium-throughput electrophysiology-based hNav1.5 assay using IonWorks™. J. Pharmacol. Toxicol. Methods 57, 30–41 (2008).
Hamon, J. et al. In vitro safety pharmacology profiling: what else beyond hERG? Future Med. Chem. 1, 645–665 (2009).
Migeon, J. in Polypharmacology in Drug Discovery (ed. Peters, J.-U. ) 111–132 (Wiley, 2012).
Valentin, J.-P. & Hammond, T. J. Safety and secondary pharmacology: successes, threats, challenges and opportunities. J. Pharmacol. Toxicol. Methods 58, 77–87 (2008).
Heath, B. M., et al. Translation of flecainide- and mexiletine-induced cardiac sodium channel inhibition and ventricular conduction slowing from nonclinical models to clinical. J. Pharmacol. Toxicol. Methods 63, 258–268 (2011).
Lazzara, R. Antiarrhythmic drugs and torsade de pointes. Eur. Heart J. 14 (Suppl. H), 88–92 (1993).
Hamon, J. et al. In vitro safety pharmacology profiling. Eur. Pharmaceut. Rev. 2006, 60–63 (2006).
Leeson, P. D. & Springthorpe, B. The influence of drug-like concepts on decision-making in medicinal chemistry. Nature Rev. Drug Discov. 6, 881–890 (2007).
Azzaoui, K. et al. Modeling promiscuity based on in vitro safety pharmacology profiling data. ChemMedChem 2, 874–880 (2007).
Hughes, J. D. et al. Physiochemical drug properties associated with in vivo toxicological outcomes. Bioorg. Med. Chem. Lett. 18, 4872–4875 (2008).
Peters, J.-U. et al. Can we discover pharmacological promiscuity early in the drug discovery process? Drug Discov. Today 17, 325–335 (2012).
Peters, J.-U., Schnider, P., Mattei, P. & Kansy, M. Pharmacological promiscuity: dependence on compound properties and target specificity in a set of recent Roche compounds. ChemMedChem 4, 680–686 (2009).
Fryer, R. M. et al. Mitigation of off-target adrenergic binding and effects on cardiovascular function in the discovery of novel ribosomal S6 kinase 2 inhibitors. J. Pharmacol. Exp. Ther. 340, 492–500 (2012).
Gintant, G. An evaluation of hERG current assay performance: translating preclinical safety studies to clinical QT prolongation. Pharmacol. Ther. 129, 109–119 (2011).
Harmer, A. R., Valentin, J.-P. & Pollard, C. E. On the relationship between block of the cardiac Na+ channel and drug-induced prolongation of the QRS complex. Br. J. Pharmacol. 164, 260–273 (2011).
O'Connor, E. C., Chapman, K., Butler, P. & Mead, A. N. The predictive validity of the rat self-administration model for abuse liability. Neurosci. Biobehav. Rev. 35, 912–938 (2011).
Overington, J. P., Al-Lazikani, B. & Hopkins, A. L. How many drug targets are there? Nature Rev. Drug Discov. 5, 993–996 (2006).
Taboureau, O. & Jørgensen, F. S. In silico predictions of hERG channel blockers in drug discovery: from ligand-based and target-based approaches to systems chemical biology. Comb. Chem. High Throughput Screen. 14, 375–387 (2011).
Marchant, C. A., Briggs, K. A. & Long, A. In silico tools for sharing data and knowledge on toxicity and metabolism: Derek for Windows, Meteor, and Vitic. Toxicol. Mech. Methods 18, 177–187 (2008).
Ekins, S., Mestres, J. & Testa, B. In silico pharmacology for drug discovery: applications to targets and beyond. Br. J. Pharmacol. 152, 21–37 (2007).
Bender, A. et al. Analysis of pharmacology data and the prediction of adverse drug reactions and off-target effects from chemical structure. ChemMedChem 2, 861–873 (2007).
Nigsch, F. et al. Computational methods for early predictive safety assessment from biological and chemical data. Expert Opin. Drug Metab. Toxicol. 7, 1497–1511 (2011).
Lounkine, E. et al. Large scale prediction and testing of drug activity on side-effect targets. Nature 486, 361–367 (2012).
Vargas, H. M. et al. Scientific review and recommendations on preclinical cardiovascular safety evaluation of biologics. J. Pharmacol. Toxicol. Methods 58, 72–76 (2008).
Mattes, W. B. & Walker, E. G. Translational toxicology and the work of the predictive safety testing consortium. Clin. Pharmacol. Ther. 85, 327–330 (2009).
Knudsen, T. B. et al. Activity profiles of 309 ToxCast™ chemicals evaluated across 292 biochemical targets. Toxicology 282, 1–15 (2011).
Wasserman, A. M. & Bajorath, J. BindingDB and ChEMBL: online compound databases for drug discovery. Expert Opin. Drug Discov. 6, 683–687 (2011).
Mirams, G. R. et al. Simulation of multiple ion channel block provides improved early prediction of drug molecules' clinical torsadogenic risk. Cardiovasc. Res. 91, 53–61 (2011).
Orchard, S. et al. Minimum information about a bioactive entity (MIABE). Nature Rev. Drug Discov. 10, 661–669 (2011).
Gintant, G. A., Gallacher, D. J. & Pugsley, M. K. The 'overly-sensitive' heart: sodium channel block and QRS interval prolongation. Br. J. Pharmacol. 164, 254–259 (2011).
Pfeufer, A. et al. Genome-wide association study of PR interval. Nature Genet. 42, 153–161 (2010).
Erdemli, G. et al. Cardiac safety implications of hNav1.5 blockade and a framework for pre-clinical evaluation. Front. Pharmacol. 3, 1–9 (2012).
Benarroch, E. E. Adenosine and its receptors: Multiple modulatory functions and potential therapeutic targets for neurologic disease. Neurology 70, 231–236 (2008).
Michelotti, G. A., Price, D. T. & Schwinn, D. A. α1-adrenergic receptor regulation: basic science and clinical implications. Pharmacol. Ther. 88, 281–309 (2000).
Philipp, M., Brede, M. & Hein, L. Physiological significance of α2-adrenergic receptor subtype diversity: one receptor is not enough. Am. J. Physiol. Regul. Integr. Comp. Physiol. 283, R287–R295 (2002).
Lohse, M. J., Engelhardt, S. & Eschenhagen, T. What is the role of β-adrenergic signaling in heart failure? Circ. Res. 93, 896–906 (2003).
Cazzola, M., Matera, M. G. & Donner, C. F. Inhaled β2-adrenoceptor agonists: cardiovascular safety in patients with obstructive lung disease. Drugs 65, 1595–1610 (2005).
Le Foll, B., Gorelick, D. A. & Goldberg, S. R. The future of endocannabinoid-oriented clinical research after CB1 antagonists. Psychopharmacology (Berl.) 205, 171–174 (2009).
Basu, S. & Dittel, B. N. Unraveling the complexities of cannabinoid receptor 2 (CB2) immune regulation in health and disease. Immunol. Res. 51, 26–38 (2011).
Dufresne, M., Seva, C. & Fourmy, D. Cholecystokinin and gastrin receptors. Physiol. Rev. 86, 805–847 (2006).
Peacock, L. & Gerlach, J. Aberrant behavioral effects of a dopamine D1 receptor antagonist and agonist in monkeys: evidence of uncharted dopamine D1 receptor actions. Biol. Psychiatry 50, 501–509 (2001).
Emilien, G. et al. Dopamine receptors — physiological understanding to therapeutic intervention potential. Pharmacol. Ther. 84, 133–156 (1999).
Palmer, M. J. Endothelin receptor antagonists: status and learning 20 years on. Prog. Med. Chem. 47, 203–237 (2009).
Walsh, G. M. Emerging safety issues regarding long-term usage of H1 receptor antagonists. Expert Opin. Drug Saf. 1, 225–235 (2002).
Hattori, Y. Cardiac histamine receptors: their pharmacological consequences and signal transduction pathways. Methods Find. Exp. Clin. Pharmacol. 21, 123–131 (1999).
Barron, B. A. Cardiac opioids. Proc. Soc. Exp. Biol. Med. 224, 1–7 (2000).
Walsh, S. L. et al. Enadoline, a selective κ opioid agonist: comparison with butorphanol and hydromorphone in humans. Psychopharmacology (Berl.) 157, 151–162 (2001).
Trescot, A. M., Datta, S. & Lee, M. Opioid pharmacology. Pain Physician 11 (Suppl. 2), S133–S153 (2008).
Medina, A. et al. Effects of central muscarinic-1 receptor stimulation on blood pressure regulation. Hypertension 29, 828–834 (1997).
Jooste, E., Klafter, F., Hirshman, C. A. & Emala, C. W. A mechanism for rapacuronium-induced bronchospasm: M2 muscarinic receptor antagonism. Anesthesiology 98, 906–911 (2003).
Krejsa, C. M. et al. Predicting ADME properties and side effects: the BioPrint approach. Curr. Opin. Drug Discov. Dev. 6, 470–480 (2003).
Lacivita, E., Leopoldo, M., Berardi, F. & Perrone, R. 5-HT1A receptor, an old target for new therapeutic agents. Curr. Top. Med. Chem. 8, 1024–1034 (2008).
Van de Kar, L. D. et al. ICV injection of the serotonin 5-HT1B agonist CP-93,129 increases the secretion of ACTH, prolactin, and renin and increases blood pressure by nonserotonergic mechanisms. Pharmacol. Biochem. Behav. 48, 429–436 (1994).
Sun-Edelstein, C., Tepper, S. J. & Shapiro, R. E. Drug-induced serotonin syndrome: a review. Expert Opin. Drug Saf. 7, 587–596 (2008).
Roth, B. L. Drugs and valvular heart disease. N. Engl. J. Med. 356, 6–9 (2007).
Barrett, L. K., Singer, M. & Clapp, L. H. Vasopressin: mechanisms of action on the vasculature in health and in septic shock. Crit. Care Med. 35, 33–40 (2007).
Kalamida, D. et al. Muscle and neuronal nicotinic acetylcholine receptors. FEBS J. 274, 3799–3845 (2007).
Splawski, I. et al. Cav1.2 calcium channel dysfunction causes a multisystem disorder including arrhythmia and autism. Cell 119, 19–31 (2004).
Lader, M. Effectiveness of benzodiazepines: do they work or not? Expert Rev. Neurother. 8, 1189–1191 (2008).
Curran, M. E. et al. A molecular basis for cardiac arrhythmia: HERG mutations cause long QT syndrome. Cell 80, 795–803 (1995).
Towart, R. et al. Blockade of the IKs potassium channel: An overlooked cardiovascular liability in drug safety screening? J. Pharmacol. Tox. Methods 60, 1–10 (2009).
Murray, J. B. Phencyclidine (PCP): a dangerous drug, but useful in schizophrenia research. J. Psychol. 136, 319–327 (2002).
Goodin, S. & Cunningham, R. 5-HT3-receptor antagonists for the treatment of nausea and vomiting: a reappraisal of their side-effect profile. Oncologist 7, 424–436 (2002).
Smits, J. P. P. et al. Cardiac sodium channels and inherited electrophysiologic disorders: a pharmacogenetic overview. Exp. Opin. Pharmacother. 9, 537–549 (2008).
Moretto, A. Experimental and clinical toxicology of anticholinesterase agents. Toxicol. Lett. 102–103, 509–513 (1998).
Süleyman, H., Demircan, B. & Karagöz, Y. Anti-inflammatory and side effects of cyclooxygenase inhibitors. Pharmacol. Rep. 59, 247–258 (2007).
Grosser, T., Fries, S. & FitzGerald, G. A. Biological basis for the cardiovascular consequences of COX-2 inhibition: therapeutic challenges and opportunities. J. Clin. Invest. 116, 4–15 (2006).
Youdim, M. B. & Weinstock, M. Therapeutic applications of selective and non-selective inhibitors of monoamine oxidase A and B that do not cause significant tyramine potentiation. Neurotoxicology 25, 243–250 (2004).
Aguirre, S. A. et al. Cardiovascular effects in rats following exposure to a receptor tyrosine kinase inhibitor. Toxicol. Pathol. 38, 416–428 (2010).
Absallem, E., Kasparian, C., Haddour, G., Boissel, J. P. & Nony, P. Phosphodiesterase III inhibitors for heart failure. Cochrane Database Syst. Rev. 2005, CD002230 (2005).
Giembycz, M. A. Can the anti-inflammatory potential of PDE4 inhibitors be realized: guarded optimism or wishful thinking? Br. J. Pharmacol. 155, 288–290 (2008).
Spina, D. PDE4 inhibitors: current status. Br. J. Pharmacol. 155, 308–315 (2008).
Goldman, F. D. et al. Defective expression of p56lck in an infant with severe combined immunodeficiency. J. Clin. Invest. 102, 421–429 (1998).
Bannon, M. J. The dopamine transporter: role in neurotoxicity and human disease. Toxicol. Appl. Pharmacol. 204, 355–360 (2005).
Mayer, A. F. et al. Influences of norepinephrine transporter function on the distribution of sympathetic activity in humans. Hypertension 48, 120–126 (2006).
Stahl, S. M. Mechanism of action of serotonin selective reuptake inhibitors: serotonin receptors and pathways mediate therapeutic effects and side effects. J. Affect. Disord. 51, 215–235 (1998).
Mooradian, A. D., Morley, J. E. & Korenman, S. G. Biological actions of androgens. Endocr. Rev. 8, 1–28 (1987).
Davison, S. L. & Bell, R. Androgen physiology. Semin. Reprod. Med. 24, 71–77 (2006).
McMaster, A. & Ray, D. W. Drug insight: selective agonists and antagonists of the glucocorticoid receptor. Nature Clin. Pract. Endocrinol. Metab. 4, 91–101 (2008).
Muller, P. Y. & Milton, M. N. The determination and interpretation of the therapeutic index in drug development. Nature Rev. Drug Discov. 11, 751–761 (2012).
The authors thank the following individuals for their valuable discussions: J.-P. Valentin and C. Pollard from AstraZeneca; L. Urban, P. Muller and G. Erdemli from Novartis; N. McMahon and J. Louttit from GlaxoSmithKline; and A. Mead from Pfizer.
The authors declare no competing financial interests.
Concentration required to elicit a 50% response in an in vitro assay. IC50 refers to an inhibitory response (the half maximal inhibitory concentration) and EC50 refers to an effect (the effector concentration for half-maximum response), usually an activation or stimulation. AC50 is a collective term used for any activity.
- Adverse drug reactions
(ADRs). Any noxious, unintended and undesired effects of a drug, occurring at doses used in humans for prophylaxis, diagnosis or therapy. These exclude therapeutic failures, intentional and accidental poisoning and drug abuse.
The concentration of an agonist that is required to produce 50% of the maximum response of that agonist.
- Free Cmax
The fraction of the Cmax (peak total plasma concentration of a drug at a certain dose) that is not bound to plasma proteins. The percentage of the bound drug is determined separately and the Cmax is corrected accordingly.
The half maximal inhibitory concentration, or the concentration of an inhibitor that is required for 50% inhibition of the maximum control response in a biochemical or cellular assay.
- K i
Inhibition constant; can be derived from the IC50 (half maximal inhibitory concentration) if the concentration of ligand or substrate and its dissociation or Michaelis constant is known. Should be used in preference to IC50 for binding assays.
- Safety margins
Ratios of an AC50 (concentration required to elicit a 50% response in an in vitro assay) — or the inhibition constant Ki — of a drug at a target known to mediate specific adverse drug reactions (ADRs) and the therapeutic free plasma concentration. The latter can be directly determined in preclinical or clinical studies, or estimated from models. The AC50 is taken from the most relevant assay available for that target. Safety margins should be used as early as possible in the preclinical phase to continually assess the risk of an ADR occurring in the clinic.
The ratio of the AC50 (concentration required to elicit a 50% response in an in vitro assay) — or the inhibition constant Ki if available — of a drug at any target that is known or suspected to mediate an adverse drug reaction, and the primary (therapeutic) target.
- Therapeutic free plasma concentration
The concentration of a compound in the plasma following a therapeutic dose. Often quoted as the maximum exposure.
- Therapeutic index
In a drug development setting: the quantitative ratio of the exposure level at the chosen safety end point divided by the exposure level at the chosen efficacy end point, typically the ratio of the highest exposure to the drug that results in no toxicity over that which produces the desired efficacy. This term is often used incorrectly to describe the safety margin.
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