Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Review Article
  • Published:

The application of discovery toxicology and pathology towards the design of safer pharmaceutical lead candidates

Nature Reviews Drug Discovery volume 6, pages 636–649 (2007)Cite this article

Key Points

  • Toxicity is a primary cause of compound attrition at all stages of drug discovery and development. Approximately 70% of all toxicity-related attrition occurs preclinically, and most of these findings are predictive of human toxicities.

  • An acceptable preclinical safety margin is a key to selecting the most viable development candidates. The strategic and early application of standard toxicology and pathology approaches, together with new technologies, will identify most preclinical toxicities. This has the potential to lower toxicity-related attrition, reduce resources expended on compounds with an unacceptable safety profile and deliver molecules into development with a better chance of reaching first-in-human studies and eventually becoming marketed pharmaceutical agents.

  • Together with the ability to discard compounds with an unacceptable safety profile earlier, identification of toxicity during discovery provides an opportunity to engage an active synthetic chemistry effort in order to understand the structure–toxicity relationship and minimize or circumvent safety liabilities.

  • Preclinical toxicities can be divided into three broad categories: primary pharmacology, secondary pharmacology and chemically mediated toxicity. Each of these toxicities is managed differently.

  • Key information such as safety margin, reversibility, and the existence of appropriate biomarkers is often best determined using repeat-dose in vivo toxicology studies. New molecular technologies, divorced from the more traditional toxicology methodologies, may do little to advance compounds into the clinic. In particular, efforts to apply new technologies towards the prediction of idiosyncratic toxicity may be doomed to failure, as the resulting assays would be virtually impossible to validate, and may only serve to generate untestable safety concerns or uninterpretable assay results around otherwise promising drug candidates.

  • Safety assessment is often given too little attention during drug discovery, despite the fact that safety is the primary focus of preclinical development and the early phases of clinical development. Prospective pharmaceutical partners pay close attention to the safety of potential in-licensed compounds when performing due diligence. Failure to identify toxicity early leaves one with only the option of attempting to manage the toxicity, or discontinue the development of the compound after significant time and expense.

Abstract

Toxicity is a leading cause of attrition at all stages of the drug development process. The majority of safety-related attrition occurs preclinically, suggesting that approaches to identify 'predictable' preclinical safety liabilities earlier in the drug development process could lead to the design and/or selection of better drug candidates that have increased probabilities of becoming marketed drugs. In this Review, we discuss how the early application of preclinical safety assessment — both new molecular technologies as well as more established approaches such as standard repeat-dose rodent toxicology studies — can identify predictable safety issues earlier in the testing paradigm. The earlier identification of dose-limiting toxicities will provide chemists and toxicologists the opportunity to characterize the dose-limiting toxicities, determine structure–toxicity relationships and minimize or circumvent adverse safety liabilities.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: The discovery testing scheme.
Figure 2: Toxicity classification.
Figure 3: Toxicology profiling in discovery.
Figure 4: Toxicology issue management decision tree.

Similar content being viewed by others

References

  1. Reichert, J. M. Trends in development and approval times for new therapeutics in the United States. Nature Rev. Drug Discov. 2, 695–702 (2003).

    CAS  Google Scholar 

  2. Pharmaceutical Research and Manufacturers of America (PhRMA). Pharmaceutical Industry Profile 2005. (PhRMA, Washington DC, 2005). The PhRMA industry profile report contains a wealth of information on industry trends.

  3. Booth, B. & Zemmel, R. Prospects for productivity. Nature Rev. Drug Discov. 3, 451–456 (2004).

    CAS  Google Scholar 

  4. Centre for Medicines Research (CMR) International. 2005 Global R&D Performance Metrics Programme: Industry Success Rates Report. (CMR International, Epsom, 2005).

  5. DiMasi, J. A., Hansen, R. W. & Grabowski, H. G. The price of innovation: new estimates of drug development costs. J. Health Econ. 22, 151–185 (2003).

    PubMed  Google Scholar 

  6. Prentis, R. A., Lis, Y. & Walker, S. R. Pharmaceutical innovation by the seven UK-owned pharmaceutical companies (1964–1985). Br. J. Clin. Pharmacol. 25, 387–396 (1988).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Kola, I. & Landis, J. Can the pharmaceutical industry reduce attrition rates? Nature Rev. Drug Discov. 3, 711–715 (2004). A review detailing the sources of attrition in the pharmaceutical industry.

    CAS  Google Scholar 

  8. Feng, W. Y. Mass spectrometry in drug discovery: a current review. Curr. Drug Discov. Technol. 1, 295–312 (2004).

    CAS  PubMed  Google Scholar 

  9. Alden, C. L. et al. The pathologist and toxicologist in pharmaceutical product discovery. Toxicol. Pathol. 27, 104–106 (1999).

    CAS  PubMed  Google Scholar 

  10. Olson, H. et al. Concordance of the toxicity of pharmaceuticals in humans and in animals. Regul. Toxicol. Pharmacol. 32, 56–67 (2000). An excellent report, sponsored by the International Life Sciences Institute, detailing the predictivity of preclinical studies for human toxicity.

    CAS  PubMed  Google Scholar 

  11. Greaves, P., Williams, A. & Eve, M. First dose of potential new medicines to humans: how animals help. Nature Rev. Drug Discov. 3, 226–236 (2004).

    CAS  Google Scholar 

  12. Booth, B. L., Lennon, D. J. & McCafferty, E. J. Improving the pharma research pipeline. McKinsey Q. 4, 9–11 (2004).

    Google Scholar 

  13. Boston Consulting Group. in Parexel's Pharmaceutical R&D Statistical Sourcebook 2002/2003. (ed. Mathieu, M. P.) (Parexel Int Corp., Waltham, Massacheusetts, 2002).

  14. US Food and Drug Administration (FDA). Innovation or Stagnation? Challenge and opportunity on the critical path to new medicinal products. FDA web site[online], (2004).

  15. Serabian, M. A. & Pilaro, A. M. Safety assessment of biotechnology-derived pharmaceuticals: ICH and beyond. Toxicol. Pathol. 27, 27–31 (1999).

    CAS  PubMed  Google Scholar 

  16. Medjitna, T. D., Stadler, C., Bruckner, L., Griot, C. & Ottiger, H. P. DNA vaccines: safety aspect assessment and regulation. Dev. Biol. 126, 261–270 (2006).

    CAS  Google Scholar 

  17. Ames, B. N., McCann, J. & Yamasaki, E. Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test. Mutat. Res. 31, 347–364 (1975). Although now more than 30 years old, the Ames reverse-mutation assay remains a gold standard prospective toxicology assay that predicts a toxicity that would otherwise be missed until significant time, money and effort were lost on the development of a compound.

    CAS  PubMed  Google Scholar 

  18. Green, M. H. L. & Muriel, W. J. Mutagen testing using trp+ reversion in Escherichia coli. Mutat. Res. 38, 3–32 (1976).

    CAS  PubMed  Google Scholar 

  19. Maron, D. M. & Ames B . Revised methods for the Salmonella mutagenicity test Mutat. Res. 113, 173–215 (1983).

    CAS  PubMed  Google Scholar 

  20. Galloway, S. M. et al. Report from working group on in vitro tests for chromosomal aberrations. Mutat. Res. 312, 241–261 (1994).

    CAS  PubMed  Google Scholar 

  21. Tennant, R. W. et al. Prediction of chemical carcinogenicity in rodents from in vitro genetic toxicity assays. Science 236, 933–941 (1987).

    CAS  PubMed  Google Scholar 

  22. Kirkland, D., Aardema, M., Henderson, L. & Muller, L. Evaluation of the ability of a battery of three in vitro genotoxicity tests to discriminate rodent carcinogens and non-carcinogens I. Sensitivity, specificity and relative predictivity. Mutat. Res. 584, 1–256 (2005).

    CAS  PubMed  Google Scholar 

  23. Cote, C., Blaise, C., Delisle, C. E., Meighen, E. A. & Hansen, P. D. A miniaturized Ames mutagenicity assay employing bioluminescent strains of Salmonella typhimurium. Mutat. Res. 345, 137–146 (1995).

    CAS  PubMed  Google Scholar 

  24. Green, M. H., Muriel, W. J. & Bridges, B. A. Use of a simplified fluctuation test to detect low levels of mutagens. Mutat. Res. 38, 33–42 (1976).

    CAS  PubMed  Google Scholar 

  25. Van Gompel, J. et al. An assessment of the utility of the yeast GreenScreen assay in pharmaceutical screening. Mutagenesis 20, 449–454 (2005).

    CAS  PubMed  Google Scholar 

  26. Quillardet, P. & Hofnung, M. The SOS chromotest: a review. Mutat. Res. 297, 235–279 (1993).

    CAS  PubMed  Google Scholar 

  27. Verschaeve, L. et al. VITOTOX bacterial genotoxicity and toxicity test for the rapid screening of chemicals. Environ. Mol. Mutagen. 33, 240–248 (1999).

    CAS  PubMed  Google Scholar 

  28. Romagna, F. & Staniforth, C. D. The automated bone marrow micronucleus test. Mutat. Res. 213, 91–104 (1989).

    CAS  PubMed  Google Scholar 

  29. Dertinger, S. D., Torous, D. K. & Tometsko, K. R. Flow cytometric analysis of micronucleated reticulocytes in mouse bone marrow. Mutat. Res. 390, 257–262 (1997).

    CAS  PubMed  Google Scholar 

  30. Styles, J. A., Clark, H., Festing, M. F. & Rew, D. A. Automation of mouse micronucleus genotoxicity assay by laser scanning cytometry. Cytometry 44, 153–155 (2001).

    CAS  PubMed  Google Scholar 

  31. Kirpnick, Z. et al. Yeast DEL assay detects clastogens. Mutat. Res. 582, 116–34 (2005).

    CAS  PubMed  Google Scholar 

  32. Shuga, J., Zhang, J., Samson, L. D., Lodish, H. F. & Griffith, L. G. In vitro erythropoiesis from bone marrow-derived progenitors provides a physiological assay for toxic and mutagenic compounds. Proc. Natl. Acad. Sci. USA 104, 8737–8742 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Albertini, S. et al. Suppression of mutagenic activity of a series of 5HT2c receptor agonists by the incorporation of a gem-dimethyl group: SAR using the Ames test and a DNA unwinding assay. Mutagenesis 13, 397–403 (1998).

    CAS  PubMed  Google Scholar 

  34. Hu, T. et al. Identification of a gene expression profile that discriminates indirect-acting genotoxins from direct-acting genotoxins. Mutat. Res. 549, 5–27 (2004).

    CAS  PubMed  Google Scholar 

  35. Newton, R. K., Aardema, M. & Aubrecht, J. The utility of DNA microarrays for characterizing genotoxicity. Environ. Health Perspect. 112, 420–422 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Yap, Y. G. & Camm, A. J. Arrhythmogenic mechanisms of non-sedating antihistamines. Clin. Exp. Allergy 29 (Suppl. 3), 174–181 (1999).

    CAS  PubMed  Google Scholar 

  37. Hoffmann, P. & Warner, B. Are hERG channel inhibition and QT interval prolongation all there is in drug-induced torsadogenesis? A review of emerging trends. J. Pharmacol. Toxicol. Methods 53, 87–105 (2006).

    CAS  PubMed  Google Scholar 

  38. De Ponti, F., Poluzzi, E. & Montanaro, N. QT-interval prolongation by non-cardiac drugs: lessons to be learned from recent experience. Eur. J. Clin. Pharmacol. 56, 1–18 (2000).

    CAS  PubMed  Google Scholar 

  39. Murphy, S. M. et al. Evaluation of functional and binding assays in cells expressing either recombinant or endogenous hERG channel. J. Pharmacol. Toxicol. Methods 52, 42–55 (2006).

    Google Scholar 

  40. Chaudhary, K. W. et al. Evaluation of the rubidium efflux assay for preclinical identification of HERG blockade. Assay Drug Dev. Technol. 4, 73–82 (2006).

    CAS  PubMed  Google Scholar 

  41. Matasi, J. J. et al. 2-(2-Furanyl)-7-phenyl[1,2,4]triazolo[1,5-c]pyrimidin-5-amine analogs as adenosine A2A antagonists: the successful reduction of hERG activity. Part 2. Bioorg. Med. Chem. Lett. 15, 3675–3678 (2005).

    CAS  PubMed  Google Scholar 

  42. Friesen, R. W. et al. Optimization of a tertiary alcohol series of phosphodiesterase-4 (PDE4) inhibitors: structure–activity relationship related to PDE4 inhibition and human ether-a-go-go related gene potassium channel binding affinity. J. Med. Chem. 46, 2413–2426 (2003).

    CAS  PubMed  Google Scholar 

  43. Wienkers, L. C. & Heath, T. G. Predicting in vivo drug interactions from in vitro drug discovery data. Nature Rev. Drug Discov. 4, 825–833 (2005).

    CAS  Google Scholar 

  44. Hollo, Z., Homolya, L., Davis, C. W. & Sarkadi, B. Calcein accumulation as a fluorometric functional assay of the multidrug transporter. Biochim. Biophys. Acta 1191, 384–388 (1994).

    CAS  PubMed  Google Scholar 

  45. Ashwell, M. A. et al. The design, preparation and SAR of novel small molecule sodium (Na(+)) channel blockers. Bioorg. Med. Chem. Lett. 14, 2025–2030 (2004).

    CAS  PubMed  Google Scholar 

  46. Macdonald, G. J. et al. Design and synthesis of trans-3-(2-(4-((3-(3-(5-methyl-1,2,4-oxadiazolyl))-phenyl)carboxamido)cyclohexyl)ethyl)-7-methylsulfonyl-2,3,4, 5-tetrahydro-1H-3-benzazepine (SB-414796): a potent and selective dopamine D3 receptor antagonist. J. Med. Chem. 46, 4952–4964 (2003).

    CAS  PubMed  Google Scholar 

  47. Revesz, L. et al. SAR of 4-hydroxypiperidine and hydroxyalkyl substituted heterocycles as novel p38 map kinase inhibitors. Bioorg. Med. Chem. Lett. 10, 1261–1264 (2000).

    CAS  PubMed  Google Scholar 

  48. Wang, S. et al. Design and synthesis of new templates derived from pyrrolopyrimidine as selective multidrug-resistance-associated protein inhibitors in multidrug resistance. J. Med. Chem. 47, 1339–1350 (2004).

    CAS  PubMed  Google Scholar 

  49. Baillie, T. A. et al. Drug metabolites in safety testing. Toxicol. Appl. Pharmacol. 182, 188–196 (2002).

    CAS  PubMed  Google Scholar 

  50. Vignati, L. et al. An in vitro approach to detect metabolite toxicity due to CYP3A4-dependent bioactivation of xenobiotics. Toxicology 216, 154–167 (2005).

    CAS  PubMed  Google Scholar 

  51. Walgren, J. L., Mitchell, M. D. & Thompson, D. C. Role of metabolism in drug-induced idiosyncratic hepatotoxicity. Crit. Rev. Toxicol. 35, 325–361 (2005). An exhaustive overview of the role of metabolism in idiosyncratic liver toxicity of marketed and withdrawn drugs.

    CAS  PubMed  Google Scholar 

  52. Park, K., Williams, D. P., Naisbitt, D. J., Kitteringham, N. R. & Pirmohamed, M. Investigation of toxic metabolites during drug development. Toxicol. Appl. Pharmacol. 207 (Suppl. 2), 425–434 (2005).

    PubMed  Google Scholar 

  53. Evans, D. C., Watt, A. P., Nicoll-Griffith, D. A. & Baillie, T. A. Drug–protein adducts: an industry perspective on minimizing the potential for drug bioactivation in drug discovery and development. Chem. Res. Toxicol. 17, 3–16 (2004). A review describing suggested criteria by which compounds with reactive metabolites may be managed.

    CAS  PubMed  Google Scholar 

  54. Chen, Q. et al. Cytochrome P450 3A4-mediated bioactivation of raloxifene: irreversible enzyme inhibition and thiol adduct formation. Chem. Res. Toxicol. 15, 907–914 (2002).

    CAS  PubMed  Google Scholar 

  55. Baillie, T. A. Future of toxicology-metabolic activation and drug design: challenges and opportunities in chemical toxicology. Chem. Res. Toxicol. 19, 889–893 (2006).

    CAS  PubMed  Google Scholar 

  56. Mesfin, G. M. et al. Pathogenesis of cardiovascular alterations in dogs treated with minoxidil. Toxicol. Pathol. 17, 164–181 (1989).

    CAS  PubMed  Google Scholar 

  57. Mesfin, G. M., Robinson, F. G., Higgins, M. J., Zhong, W. Z. & DuCharme, D. W. The pharmacologic basis of the cardiovascular toxicity of minoxidil in the dog. Toxicol. Pathol. 23, 498–506 (1995).

    CAS  PubMed  Google Scholar 

  58. Mesfin, G. M., Higgins, M. J., Robinson, F. G. & Zhong, W. Z. Relationship between serum concentrations, hemodynamic effects, and cardiovascular lesions in dogs treated with minoxidil. Toxicol. Appl. Pharmacol. 140, 337–344 (1996).

    CAS  PubMed  Google Scholar 

  59. Kerns, W. et al. (Expert Working Group on Drug-Induced Vascular Injury). Drug-induced vascular injury-a quest for biomarkers. Toxicol. Appl. Pharmacol. 203, 62–87 (2005). An excellent overview of research demonstrating the pharmacological nature of the role of haemodynamic effects in vascular injury.

    CAS  PubMed  Google Scholar 

  60. Gonzalez, F. J. Recent update on the PPARα-null mouse. Biochimie 79, 139–144 (1997).

    CAS  PubMed  Google Scholar 

  61. Park, B. K., Pirmohamed, M. & Kitteringham, N. R. Role of drug disposition in drug hypersensitivity: a chemical, molecular, and clinical perspective. Chem. Res. Toxicol. 11, 969–988 (1998). An interesting review of drug hypersensitivity, including a useful system of characterizing clinical adverse drug reactions.

    CAS  PubMed  Google Scholar 

  62. Edwards, I. R. & Aronson, J. K. Adverse drug reactions: definitions, diagnosis, and management. Lancet 356, 1255–1259 (2000).

    CAS  PubMed  Google Scholar 

  63. Roth, R. A., Luyendyk, J. P., Maddox, J. F. & Ganey, P. E. Inflammation and drug idiosyncrasy — is there a connection? J. Pharmacol. Exp. Ther. 307, 1–8 (2003).

    CAS  PubMed  Google Scholar 

  64. Tafazoli, S., Spehar, D. D. & O'Brien, P. J. Oxidative stress mediated idiosyncratic drug toxicity. Drug Metab. Rev. 37, 311–325 (2005).

    CAS  PubMed  Google Scholar 

  65. Waring, J. F. et al. Microarray analysis of lipopolysaccharide potentiation of trovafloxacin-induced liver injury in rats suggests a role for proinflammatory chemokines and neutrophils. J. Pharmacol. Exp. Ther. 316, 1080–1087 (2006).

    CAS  PubMed  Google Scholar 

  66. Elcombe, C. R. et al. Prediction of rodent nongenotoxic carcinogenesis: evaluation of biochemical and tissue changes in rodents following exposure to nine nongenotoxic NTP carcinogens. Environ. Health Perspect. 110, 363–375 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  67. Kramer, J. A. et al. Acute molecular markers of rodent hepatic carcinogenesis identified by transcription profiling. Chem. Res. Toxicol. 17, 463–470 (2004).

    CAS  PubMed  Google Scholar 

  68. Tabacova, S. Mode of action: angiotensin-converting enzyme inhibition — developmental effects associated with exposure to ACE inhibitors. Crit. Rev. Toxicol. 35, 747–755 (2005).

    CAS  PubMed  Google Scholar 

  69. Valdes, G., Marinovic, D., Falcon, C., Chuaqui, R. & Duarte, I. Placental alterations, intrauterine growth retardation and teratogenicity associated with enalapril use in pregnant rats. Biol. Neonate 61, 124–130 (1992).

    CAS  PubMed  Google Scholar 

  70. Hilgers, K. F., Reddi, V., Krege, J. H., Smithies, O. & Gomez, R. A. Aberrant renal vascular morphology and renin expression in mutant mice lacking angiotensin-converting enzyme. Hypertension 29, 216–221 (1997).

    CAS  PubMed  Google Scholar 

  71. Matsushima, N. et al. Platelet inhibitory activity and pharmacokinetics of prasugrel (CS-747) a novel thienopyridine P2Y12 inhibitor: A multiple-dose study in healthy humans. Platelets 17, 218–226 (2006).

    CAS  PubMed  Google Scholar 

  72. Orford, J. L. et al. Safety and efficacy of aspirin, clopidogrel, and warfarin after coronary stent placement in patients with an indication for anticoagulation. Am. Heart J. 147, 463–467 (2004).

    CAS  PubMed  Google Scholar 

  73. Daykin, H. J., Sturgeon, S. A., Jones, C. & Wright, C. E. Arterial antithrombotic effects of aspirin, heparin, enoxaparin and clopidogrel alone, or in combination, in the rat. Thromb. Res. 118, 755–762 (2006).

    CAS  PubMed  Google Scholar 

  74. Sugidachi, A., Asai, F., Ogawa, T., Inoue, T. & Koike, H. The in vivo pharmacological profile of CS-747, a novel antiplatelet agent with platelet ADP receptor antagonist properties. Br. J. Pharmacol. 129, 1439–1446 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  75. Elg, M., Gustafsson, D. & Carlsson, S. Antithrombotic effects and bleeding time of thrombin inhibitors and warfarin in the rat. Thromb. Res. 94, 187–197 (1999).

    CAS  PubMed  Google Scholar 

  76. Schumacher, W. A., Heran, C. L. & Steinbacher, T. E. Low-molecular-weight heparin (fragmin) and thrombin active-site inhibitor (argatroban) compared in experimental arterial and venous thrombosis and bleeding time. J. Cardiovasc. Pharmacol. 28, 19–25 (1996).

    CAS  PubMed  Google Scholar 

  77. Connolly, H. M. et al. Valvular heart disease associated with fenfluramine-phentermine. N. Engl. J. Med. 337, 581–588 (1997).

    CAS  PubMed  Google Scholar 

  78. Miller, K. J. Serotonin 5-HT2c receptor agonists: potential for the treatment of obesity. Mol. Interv. 5, 282–291 (2005).

    CAS  PubMed  Google Scholar 

  79. Fitzgerald, L. W. et al. Possible role of valvular serotonin 5-HT(2B) receptors in the cardiopathy associated with fenfluramine. Mol. Pharmacol. 57, 75–81 (2000).

    CAS  PubMed  Google Scholar 

  80. Rothman, R. B. et al. Evidence for possible involvement of 5-HT(2B) receptors in the cardiac valvulopathy associated with fenfluramine and other serotonergic medications. Circulation 102, 2836–2841 (2000).

    CAS  PubMed  Google Scholar 

  81. Smith, B. M., Thomsen, W. J. & Grottick, A. J. The potential use of selective 5-HT2C agonists in treating obesity. Expert Opin. Investig. Drugs. 15, 257–266 (2006).

    CAS  PubMed  Google Scholar 

  82. Silverstein, F. E. et al. Gastrointestinal toxicity with celecoxib vs nonsteroidal anti-inflammatory drugs for osteoarthritis and rheumatoid arthritis: the CLASS study: a randomized controlled trial. Celecoxib Long-term Arthritis Safety Study. JAMA 284, 1247–1255 (2000).

    CAS  PubMed  Google Scholar 

  83. Bombardier, C. et al. Comparison of upper gastrointestinal toxicity of rofecoxib and naproxen in patients with rheumatoid arthritis. VIGOR Study Group. N. Engl. J. Med. 343, 1520–1528 (2000).

    CAS  PubMed  Google Scholar 

  84. Reasor, M. J. & Kacew, S. Drug-induced phospholipidosis: are there functional consequences? Exp. Biol. Med. 226, 825–830 (2001).

    CAS  Google Scholar 

  85. Reasor, M. J., Hastings, K. L. & Ulrich, R. G. Drug-induced phospholipidosis: issues and future directions. Expert Opin. Drug Saf. 5, 567–583 (2006).

    CAS  PubMed  Google Scholar 

  86. Foroozesh, M. et al. Aryl acetylenes as mechanism-based inhibitors of cytochrome P450-dependent monooxygenase enzymes. Chem. Res. Toxicol. 10, 91–102 (1997).

    CAS  PubMed  Google Scholar 

  87. Helvig, C. et al. Suicide inactivation of cytochrome P450 by midchain and terminal acetylenes. A mechanistic study of inactivation of a plant lauric acid ω-hydroxylase. J. Biol. Chem. 272, 414–421 (1997).

    CAS  PubMed  Google Scholar 

  88. Zhou, S. et al. Mechanism-based inhibition of cytochrome P450 3A4 by therapeutic drugs. Clin. Pharmacokinet. 44, 279–304 (2005).

    CAS  PubMed  Google Scholar 

  89. Wolfe, A., Shimer, G. H. Jr & Meehan, T. Polycyclic aromatic hydrocarbons physically intercalate into duplex regions of denatured DNA. Biochemistry 26, 6392–6396 (1987).

    CAS  PubMed  Google Scholar 

  90. Topliss, J. G. Utilization of operational schemes for analog synthesis in drug design. J. Med. Chem. 15, 1006–1011 (1972).

    CAS  PubMed  Google Scholar 

  91. Renkiewicz, R. et al. Broad-spectrum matrix-metalloproteinase inhibitor marimastat-induced musculoskeletal side effects in rats. Arthritis Rheum. 48, 1742–1749 (2003).

    CAS  PubMed  Google Scholar 

  92. Kramer, J. A. et al. Transcription profiling distinguishes dose-dependent effects in the livers of rats treated with clofibrate. Toxicol. Pathol. 31, 417–431 (2003).

    CAS  PubMed  Google Scholar 

  93. Elrick, M. M. et al. Differential display in rat livers treated for 13 weeks with phenobarbital implicates a role for metabolic and oxidative stress in nongenotoxic carcinogenicity. Toxicol. Pathol. 33, 118–126 (2005).

    CAS  PubMed  Google Scholar 

  94. Amin, R. P. et al. Identification of putative gene based markers of renal toxicity. Environ. Health Perspect. 112, 465–479 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  95. Bendele, A. et al. Animal models of arthritis: relevance to human disease. Toxicol. Pathol. 27, 134–142 (1999).

    CAS  PubMed  Google Scholar 

  96. March, T. H., Green, F. H., Hahn, F. F. & Nikula, K. J. Animal models of emphysema and their relevance to studies of particle-induced disease. Inhal. Toxicol. 12 (Suppl. 4), 155–187 (2000).

    CAS  PubMed  Google Scholar 

  97. Nikula, K. J. & Green, F. H. Animal models of chronic bronchitis and their relevance to studies of particle-induced disease. Inhal. Toxicol. 12 (Suppl. 4), 123–153 (2000).

    CAS  PubMed  Google Scholar 

  98. Heyen, J. R., et al. Structural, functional, and molecular characterization of the SHHF model of heart failure. Am. J. Physiol. Heart Circ. Physiol. 283, H1775–H1784 (2002). An excellent example of the integration of histopathology and molecular end points to understand the predictivity of a preclinical model of human disease.

    CAS  PubMed  Google Scholar 

  99. March, T. H., Barr, E. B., Finch, G. L., Nikula, K. J. & Seagrave, J. C. Effects of concurrent ozone exposure on the pathogenesis of cigarette smoke-induced emphysema in B6C3F1 mice. Inhal. Toxicol. 14, 1187–1213 (2002).

    CAS  PubMed  Google Scholar 

  100. Weinreb, D. B., Aguinaldo, J. G., Feig, J. E., Fisher, E. A. & Fayad, Z. A. Non-invasive MRI of mouse models of atherosclerosis. NMR Biomed. 20, 256–264 (2007).

    PubMed  Google Scholar 

  101. Joner, M. et al. Pioglitazone inhibits in-stent restenosis in atherosclerotic rabbits by targeting transforming growth factor-β and MCP-1. Arterioscler. Thromb. Vasc. Biol. 27, 182–189 (2007).

    CAS  PubMed  Google Scholar 

  102. Nachtigal P. et al. MDOC and atorvastatin have potential antiinflammatory effects in vascular endothelium of apoE−/− mouse model of atherosclerosis. Life Sci. 78, 1983–1989 (2006).

    CAS  PubMed  Google Scholar 

  103. Jaakkola, K. et al. In vivo detection of vascular adhesion protein-1 in experimental inflammation. Am. J. Pathol. 157, 463–471 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  104. Hicks, S. M. et al. Immunohistochemical analysis of Clara cell secretory protein expression in a transgenic model of mouse lung carcinogenesis. Toxicology. 187, 217–228 (2003).

    CAS  PubMed  Google Scholar 

  105. Blomme, E. A. et al. Selective cyclooxygenase-2 inhibition does not affect the healing of cutaneous full-thickness incisional wounds in SKH-1 mice. Br. J. Dermatol. 148, 211–223 (2003).

    CAS  PubMed  Google Scholar 

  106. Blasi, E. R. et al. Aldosterone/salt induces renal inflammation and fibrosis in hypertensive rats. Kidney Int. 63, 1791–1800 (2003).

    CAS  PubMed  Google Scholar 

  107. Khan, K. N. et al. Interspecies differences in renal localization of cyclooxygenase isoforms: implications in nonsteroidal anti-inflammatory drug-related nephrotoxicity. Toxicol. Pathol. 26, 612–620 (1998).

    CAS  PubMed  Google Scholar 

  108. Sellers, R. S., Senese, P. B. & Khan, K. N. Interspecies differences in the nephrotoxic response to cyclooxygenase inhibition. Drug. Chem. Toxicol. 27, 111–122 (2004).

    CAS  PubMed  Google Scholar 

  109. Jones, M. B. et al. Proteomic analysis and identification of new biomarkers and therapeutic targets for invasive ovarian cancer. Proteomics 2, 76–84 (2002).

    CAS  PubMed  Google Scholar 

  110. Koop, R. Combinatorial biomarkers: From early toxicology assays to patient population profiling. Drug Discov. Today. 10, 781–788 (2005).

    CAS  PubMed  Google Scholar 

  111. Gundert-Remy, U. et al. Molecular approaches to the identification of biomarkers of exposure and effect — report of an expert meeting organized by COST Action B15. Toxicol. Lett. 156, 227–240 (2005).

    CAS  PubMed  Google Scholar 

  112. Thukral, S. K. et al. Prediction of nephrotoxicant action and identification of candidate toxicity-related biomarkers. Toxicol. Pathol. 33, 343–355 (2005).

    CAS  PubMed  Google Scholar 

  113. Petricoin, E. F. et al. Toxicoproteomics: serum proteomic pattern diagnostics for early detection of drug induced cardiac toxicities and cardioprotection. Toxicol. Pathol. 32 (Suppl. 1), 122–130 (2004).

    CAS  PubMed  Google Scholar 

  114. Searfoss, G. H. et al. Adipsin, a biomarker of gastrointestinal toxicity mediated by a functional γ-secretase inhibitor. J. Biol. Chem. 278, 46107–46116 (2003).

    CAS  PubMed  Google Scholar 

  115. Moody, D. E., Reddy, J. K., Lake, B. G., Popp, J. A. & Reese, D. H. Peroxisome proliferation and nongenotoxic carcinogenesis: commentary on a symposium. Fundam. Appl. Toxicol. 16, 233–248 (1991).

    CAS  PubMed  Google Scholar 

  116. Tugwood, J. D., Aldridge, T. C., Lambe, K. G., Macdonald, N. & Woodyatt, N. J. Peroxisome proliferator-activated receptor-α and the pleiotropic responses to peroxisome proliferators. Arch. Toxicol. Suppl. 20, 377–386 (1998).

    CAS  PubMed  Google Scholar 

  117. Woodyatt, N. J., Lambe, K. G., Myers, K. A., Tugwood, J. D. & Roberts, R. A. The peroxisome proliferator (PP) response element upstream of the human acyl CoA oxidase gene is inactive among a sample human population: significance for species differences in response to PPs. Carcinogenesis 20, 369–372 (1999).

    CAS  PubMed  Google Scholar 

  118. Cattley, R. C. et al. Do peroxisome proliferating compounds pose a hepato-carcinogenic hazard to humans? Regul. Toxicol. Pharmacol. 27, 47–60 (1998).

    CAS  PubMed  Google Scholar 

  119. McClain, R. M., Posch, R. C., Bosakowski, T. & Armstrong, J. M. Studies on the mode of action for thyroid gland tumor promotion in rats by phenobarbital. Toxicol. Appl. Pharmacol. 94, 254–265 (1988).

    CAS  PubMed  Google Scholar 

  120. McClain, R. M. Thyroid gland neoplasia: non-genotoxic mechanisms. Toxicol. Lett. 64–65, 397–408 (1992).

    Google Scholar 

  121. Williams, E. D. Mechanisms and pathogenesis of thyroid cancer in animals and man. Mutat. Res. 333, 123–129 (1995).

    CAS  PubMed  Google Scholar 

  122. Davila, J. C. et al. Use and application of stem cells in toxicology. Toxicol. Sci. 79, 214–223 (2004).

    CAS  PubMed  Google Scholar 

  123. Gross, C. J. & Kramer, J. A. The role of investigative molecular toxicology in early stage drug development. Expert Opin. Drug Saf. 2, 147–159 (2003).

    CAS  PubMed  Google Scholar 

  124. Walgren, J. L. & Thompson, D. C. Application of proteomic technologies in the drug development process. Toxicol. Lett. 149, 377–385 (2004).

    CAS  PubMed  Google Scholar 

  125. Griffin, J. L. & Bollard, M. E. Metabonomics: its potential as a tool in toxicology for safety assessment and data integration. Curr. Drug Metab. 5, 389–398 (2004).

    CAS  PubMed  Google Scholar 

  126. Heijne, W. H., Kienhuis, A. S., van Ommen, B., Stierum, R. H. & Groten, J. P. Systems toxicology: applications of toxicogenomics, transcriptomics, proteomics and metabolomics in toxicology. Expert Rev. Proteomics 2, 767–780 (2005).

    CAS  PubMed  Google Scholar 

  127. Ulrich, R. & Friend, S. H. Toxicogenomics and drug discovery: will new technologies help us produce better drugs? Nature Rev. Drug Discov. 1, 84–88 (2002). An outstanding overview of the promise of toxicogenomics for mechanistic toxicology studies.

    CAS  Google Scholar 

  128. Kramer, J. A. & Kolaja, K. L. Toxicogenomics: an opportunity to optimise drug development and safety evaluation. Expert Opin. Drug Saf. 1, 275–286 (2002).

    CAS  PubMed  Google Scholar 

  129. Rodi, C. P. et al. Revolution through genomics in investigative and discovery toxicology. Toxicol. Pathol. 27, 107–10 (1999).

    CAS  PubMed  Google Scholar 

  130. Gomez-Brouchet A. et al. Critical role for sphingosine kinase-1 in regulating survival of neuroblastoma cells exposed to amyloid b peptide. Mol. Pharmacol. 23 May 2007 (doi:10.1124/mol.106.033738).

    CAS  PubMed  Google Scholar 

  131. Tateno, C. et al. Near completely humanized liver in mice shows human-type metabolic responses to drugs. Am. J. Pathol. 165, 901–912 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  132. van Kreijl, C. F. et al. Xpa and Xpa/p53+/− knockout mice: overview of available data. Toxicol. Pathol. 29 (Suppl. 1), 117–127 (2001).

    CAS  PubMed  Google Scholar 

  133. Tennant, R. W., Stasiewicz, S., Eastin, W. C., Mennear, J. H. & Spalding, J. W. The Tg. AC (v-Ha-ras) transgenic mouse: nature of the model. Toxicol. Pathol. 29 (Suppl. 1), 51–59 (2001).

    CAS  PubMed  Google Scholar 

  134. Storer, R. D. et al. P53+/− hemizygous knockout mouse: overview of available data. Toxicol. Pathol. 29 (Suppl. 1), 30–50 (2001).

    CAS  PubMed  Google Scholar 

  135. Bolon, B. Genetically engineered animals in drug discovery and development: a maturing resource for toxicologic research. Basic Clin. Pharmacol. Toxicol. 95, 154–161 (2004).

    CAS  PubMed  Google Scholar 

  136. Vadolas, J. et al. Humanized β-thalassemia mouse model containing the common IVSI-110 splicing mutation. J. Biol. Chem. 281, 7399–7405 (2006).

    CAS  PubMed  Google Scholar 

  137. McMillian, M. K. et al. Nile Red binding to HepG2 cells: an improved assay for in vitro studies of hepatosteatosis. In Vitr. Mol. Toxicol. 14, 177–190 (2001).

    CAS  PubMed  Google Scholar 

  138. Gum, R. J. et al. Analysis of two matrix metalloproteinase inhibitors and their metabolites for induction of phospholipidosis in rat and human hepatocytes. Biochem. Pharmacol. 62, 1661–1673 (2001).

    CAS  PubMed  Google Scholar 

  139. Morelli, J. K. et al. Validation of an in vitro screen for phospholipidosis using a high-content biology platform. Cell. Biol. Toxicol. 22, 15–27 (2006).

    CAS  PubMed  Google Scholar 

  140. Portera-Cailliau, C., Sung, C. H., Nathans, J. & Adler, R. Apoptotic photoreceptor cell death in mouse models of retinitis pigmentosa. Proc. Natl Acad. Sci. USA 91, 974–978 (1994).

    CAS  PubMed  PubMed Central  Google Scholar 

  141. Fairbairn, D. W., Olive, P. L. & O'Neill, K. L. The comet assay: a comprehensive review. Mutat. Res. 339, 37–59 (1995).

    CAS  PubMed  Google Scholar 

  142. Vermes, I., Haanen, C., Steffens-Nakken, H. & Reutelingsperger, C. A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labeled Annexin V. J. Immunol. Methods 184, 39–51 (1995).

    CAS  PubMed  Google Scholar 

  143. Gurtu, V., Kain, S. R. & Zhang, G. Fluorometric and colorimetric detection of caspase activity associated with apoptosis. Anal. Biochem. 251, 98–102 (1997).

    CAS  PubMed  Google Scholar 

  144. Hyslop, P. A. & Sklar, L. A. A quantitative fluorimetric assay for the determination of oxidant production by polymorphonuclear leukocytes: its use in the simultaneous fluorimetric assay of cellular activation processes. Anal. Biochem. 141, 280–286 (1984).

    CAS  PubMed  Google Scholar 

  145. Capaldi, R. A., Murray, J., Byrne, L., Janes, M. S. & Marusich, M. F. Immunological approaches to the characterization and diagnosis of mitochondrial disease. Mitochondrion 4, 417–426 (2004).

    CAS  PubMed  Google Scholar 

  146. Letteron. P., Sutton, A., Mansouri, A., Fromenty, B. & Pessayre, D. Inhibition of microsomal triglyceride transfer protein: another mechanism for drug-induced steatosis in mice. Hepatology 38, 133–140 (2003).

    CAS  PubMed  Google Scholar 

  147. Sun, M. et al. Synthesis and SAR of 5-amino- and 5-(aminomethyl)benzofuran histamine H3 receptor antagonists with improved potency. J. Med. Chem. 48, 6482–6490 (2005).

    CAS  PubMed  Google Scholar 

  148. Needleman, P. & Isakson, P. C. The discovery and function of COX-2. J. Rheumatol. 24 (Suppl. 49), 6–8 (1997).

    Google Scholar 

  149. Masferrer, J. L., Seibert, K., Zweifel, B. & Needleman, P. Endogenous glucocorticoids regulate an inducible cyclooxygenase enzyme. Proc. Natl Acad. Sci. USA 89, 3917–3921 (1992).

    CAS  PubMed  PubMed Central  Google Scholar 

  150. Menard, J. The 45-year story of the development of an anti-aldosterone more specific than spironolactone. Mol. Cell. Endocrinol. 217, 45–52 (2004). An interesting review of the research required to demonstrate the secondary pharmacological nature of a dose-limiting adverse effect of spironolactone, and the effort to develop a drug sparing those effects.

    CAS  PubMed  Google Scholar 

  151. Sica, D. A. Pharmacokinetics and pharmacodynamics of mineralocorticoid blocking agents and their effects on potassium homeostasis. Heart Fail. Rev. 10, 23–29 (2005).

    CAS  PubMed  Google Scholar 

  152. Manning, G., Whyte, D. B., Martinez, R., Hunter, T. & Sudarsanam, S. The protein kinase complement of the human genome. Science 298, 1912–1934 (2002).

    CAS  PubMed  Google Scholar 

  153. Ridings, J. E. & Baldwin, J. A. A qualitative assessment of developmental toxicity within a series of structurally related dopamine mimetics. Toxicology 76, 197–207 (1992).

    CAS  PubMed  Google Scholar 

  154. Nomanbhoy, T. K., Rosenblum, J., Aban, A. & Burbaum, J. J. Inhibitor focusing: direct selection of drug targets from proteomes using activity-based probes. Assay Drug Dev. Technol. 1, 137–146 (2003).

    CAS  PubMed  Google Scholar 

  155. McKenney, J. M. Pharmacologic options for aggressive low-density lipoprotein cholesterol lowering: benefits versus risks. Am. J. Cardiol. 96, 60E–66E (2005).

    PubMed  Google Scholar 

  156. Paoletti, R., Corsini, A. & Bellosta, S. Pharmacological interactions of statins. Atheroscler. Suppl. 3, 35–40 (2002).

    CAS  PubMed  Google Scholar 

  157. Evans, M. & Rees, A. Effects of HMG-CoA reductase inhibitors on skeletal muscle: are all statins the same? Drug Saf. 25, 649–663 (2002).

    CAS  PubMed  Google Scholar 

  158. Ucar, M., Mjorndal, T. & Dahlqvist, R. HMG-CoA reductase inhibitors and myotoxicity. Drug Saf. 22, 441–457 (2000).

    CAS  PubMed  Google Scholar 

  159. Zambrowicz, B. P. & Sands, A. T. Knockouts model the 100 best-selling drugs — will they model the next 100? Nature Rev. Drug Discov. 2, 38–51 (2003).

    CAS  Google Scholar 

  160. Hopkins, A. L. & Groom, C. R. The druggable genome. Nature Rev. Drug Discov. 1, 727–730 (2002).

    CAS  Google Scholar 

  161. Pennie, W., Pettit S. D. & Lord, P. G. Toxicogenomics in risk assessment: an overview of an HESI collaborative research program. Environ. Health Perspect. 112, 417–419 (2004).

    PubMed  PubMed Central  Google Scholar 

  162. Petruska, J. M. et al. Cardiovascular effects after inhalation of large doses of albuterol dry powder in rats, monkeys, and dogs: a species comparison. Fundam. Appl. Toxicol. 40, 52–62 (1997).

    CAS  PubMed  Google Scholar 

  163. Broadhead, C. L. et al. Prospects for reducing and refining the use of dogs in the regulatory toxicity testing of pharmaceuticals. Hum. Exp. Toxicol. 19, 440–447 (2002).

    Google Scholar 

  164. Lipinski, C. A., Lombardo, F., Dominy, B. W. & Feeney, P. J. Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Adv. Drug Deliv. Rev. 46, 3–25 (1997).

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Drug Metabolism and Pharmacokinetics, Lexicon Pharmaceuticals Inc., 8800 Technology Forest Place, The Woodlands, 77381, Texas, USA

    Jeffrey A. Kramer

  2. Seventh Wave Laboratories, Suite 209, 743 Spirit 40 Park Drive, Chesterfield, 63005, Missouri, USA

    John E. Sagartz

  3. Drug Safety Research and Development, Pfizer Inc., 700 Chesterfield Parkway West T1A, Chesterfield, 63017, Missouri, USA

    Dale L. Morris

Authors
  1. Jeffrey A. Kramer
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. John E. Sagartz
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Dale L. Morris
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jeffrey A. Kramer.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Related links

Related links

FURTHER INFORMATION

Lexicon Pharmaceuticals

Pfizer

Seventh Wave Laboratories

Glossary

Development-limiting toxicity

A toxicity that is either irreversible or unmonitorable, has an unacceptable safety margin or therapeutic index, or would negatively affect sales, patient compliance, competitive advantage or marketability.

Structure–toxicity relationship

(STR). An assessment of the structural features that determine the occurence and/or severity of a particular toxicity.

Safety margin

A preclinical indication of the safety of a compound that represents the ratio of a maximum safe exposure divided by an efficacious exposure.

Dose-limiting toxicity

Any toxicity that limits the ability to continue escalating the dose.

Signal generation

A study intended to identify the dose-limiting safety liability of a compound or drug target.

Ames assay

A bacterial 'reverse mutation' mutagenicity assay that is designed to identify frame-shift and base-pair-substitution point mutations. A good laboratory practice Ames assay is required before first-in-human studies.

Micronucleus assay

An assay that identifies chromosomal aberrations, visible as an extra staining material in metaphase/anaphase cells. Both an in vitro and in vivo chromosomal aberration assay are required before first-in-human studies.

Mutagenicity

This is DNA damage that is considered to be predictive of carcinogenicity.

Clastogenicity

This is chromosome breakage, a form of mutagenesis that is considered to be predictive of carcinogenicity.

Freedom to operate

The ability to synthesize new molecular entities in a chemical space that has not been previously described in relevant existing patents.

Patch-clamp assay

An assay that uses a microelectrode to study the activity of ion channels in single cells.

Reactive metabolite

A chemically reactive metabolite that binds covalently to cellular proteins.

Electrophilic metabolite

A reactive metabolite characterized by an affinity to form covalent modifications with endogenous nucleophiles.

Idiosyncratic toxicity

A toxicity that occurs rarely (with a frequency that is typically less than 1 in 1000) and unpredictably among the population.

Therapeutic index

A clinical indication of the safety of a compound determined by dividing the exposure at which dose-limiting clinical adverse effects are first observed by the exposure at which efficacy is achieved.

NOAEL

No observable adverse effect level is the highest exposure at which no adverse effects are observed.

Exaggerated pharmacology

Toxicity that is due to excessive modulation of the activity of the primary pharmacological target beyond the point necessary for efficacy.

Primary pharmacology

Also referred to as target-based toxicity, this is toxicity that is caused by a modulation of the primary pharmacological target.

Secondary pharmacology

Toxicity caused by a lack of specificity for the primary target resulting in a molecule crossing over onto and modulating the activity of a secondary, often structurally and/or evolutionarily related target.

Chemically mediated toxicity

A toxicity that is due to the physical and chemical properties of a particular chemical or an entire chemical class.

Black box warning

The most serious safety warning required on a pharmaceutical label, indicative of a significant risk of a serious or even life-threatening adverse drug reaction.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Kramer, J., Sagartz, J. & Morris, D. The application of discovery toxicology and pathology towards the design of safer pharmaceutical lead candidates. Nat Rev Drug Discov 6, 636–649 (2007). https://doi.org/10.1038/nrd2378

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1038/nrd2378

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing