Preclinical in vivo Neurotoxicity Studies of Drug Candidates

Neurotoxic effects are one of the common reasons for discontinuation of preclinical and/or clinical studies. Preclinical evaluation of neurotoxic effects is complicated due to a wide range of manifestations and degrees of severity. Current experimental approaches to neurotoxicity assessment are cumbersome, laborious and not adapted enough for preclinical studies in the early stages of drug development. The aim of the study was to review existing approaches to experimental assessment of neurotoxic potential of new drugs and to discuss the need for and feasibility of developing and using integrated rapid neurotoxicity tests for early assessment of a pharmacological project’s potential. The authors reviewed scientific literature and guidance documents and analysed current approaches to chemical compound neurotoxicity assessment in laboratory animals. The paper analyses the main issues of neurotoxicity assessment for new drugs and compares Irwin tests with the functional observation battery. It analyses issues related to assessment of drugs’ effects on the development and maturation of central nervous system functions at pre- and postnatal stages. It was determined that the current practice is not sufficient for assessment of potential adverse effects on cognitive functions. The authors assessed factors affecting cognitive functions of rodents during studies. The “Acute suppression of the exploratory and orientation response” and “Extrapolation escape task” tests were proposed for validation as potential rapid tests for detection of an array of organic and functional neurotoxic disorders at early stages of preclinical studies.

neurotoxicity, preclinical studies, Irwin tests, functional observational battery, cognitive studies, mice, rats

1. Kerbrat A, Ferré JC, Fillatre P, Ronziere T, Vannier S, CarsinNicol B, et al. Acute neurologic disorder from an inhibitor of fatty acid amide hydrolase. N Engl J Med. 2016;375(18):1717–25. https://doi.org/10.1056/NEJMoa1604221

2. Valentin JP, Hammond T. Safety and secondary pharmacology: successes, threats, challenges and opportunities. J Pharmacol Toxicol Methods. 2008;58(2):77–87. https://doi.org/10.1016/j.vascn.2008.05.007

3. Cook D, Brown D, Alexander R, March R, Morgan P, Satterthwaite M, Pangalos MN. Lessons learned from the fate of AstraZeneca’s drug pipeline: a five-dimensional framework. Nat Rev Drug Discov. 2014;13(6):419–31. https://doi.org/10.1038/nrd4309

4. Walker AL, Imam SZ, Roberts RA. Drug discovery and development: Biomarkers of neurotoxicity and neurodegeneration. Exp Biol Med (Maywood). 2018;243(13):1037–45. https://doi.org/10.1177/1535370218801309

5. Costa LG. Overview of neurotoxicology. Curr Protoc Toxicol. 2017;74(1):11.1.1–11.1.11. https://doi.org/10.1002/cptx.36

6. Fang H, Harris SC, Liu Z, Zhou G, Zhang G, Xu J, et al. FDA drug labeling: rich resources to facilitate precision medicine, drug safety, and regulatory science. Drug Discov Today. 2016;21(10):1566–70. https://doi.org/10.1016/j.drudis.2016.06.006

7. Pugsley MK, Authier S, Curtis MJ. Principles of safety pharmacology. Br J Pharmacol. 2008;154(7):1382–99. https://doi.org/10.1038/bjp.2008.280

8. Irwin S. Comprehensive observational assessment: Ia. A systematic, quantitative procedure for assessing the behavioral and physiologic state of the mouse. Psychopharmacologia. 1968;13(3):222–57. https://doi.org/10.1007/bf00401402

9. Moser VC. Screening approaches to neurotoxicity: A functional observational battery. J Am Coll Toxicol. 1989;8(1):85–93. https://doi.org/10.3109%2F10915818909009095

10. Moser VC, McCormick JP, Creason JP, MacPhail RC. Comparison of chlordimeform and carbaryl using a functional observational battery. Fundam Appl Toxicol. 1988;11(2):189–206. https://doi.org/10.1016/0272-0590(88)90144-3

11. Moser VC, MacPhail RC. Neurobehavioral effect of triadimefon, a triazole fungicide, in male and female rats. Neurotoxicol Teratol. 1989;11(3):285–93. https://doi.org/10.1016/0892-0362(89)90071-8

12. Ewart L, Milne A, Adkins D, Benjamin A, Bialecki R, Chen Y, et al. A multi-site comparison of in vivo safety pharmacology studies conducted to support ICH S7A & B regulatory submissions. J Pharmacol Toxicol Methods. 2013;68(1):30–43. https://doi.org/10.1016/j.vascn.2013.04.008

13. Gauvin DV, Yoder JD, Holdsworth DL, Harter ML, May JR, Cotey N, et al. The standardized functional observational battery: Its intrinsic value remains in the instrument of measure: the rat. J Pharmacol Toxicol Methods. 2016;82:90–108. https://doi.org/10.1016/j.vascn.2016.08.001

14. Mathiasen JR, Moser VC. The Irwin test and functional observational battery (FOB) for assessing the effects of compounds on behavior, physiology, and safety pharmacology in rodents. Curr Protoc Pharmacol. 2018;83(1):e43. https://doi.org/10.1002/cpph.43

15. Redfern WS, Dymond A, Strang I, Storey S, Grant C, Marks L, et al. The functional observational battery and modified Irwin test as global neurobehavioral assessments in the rat: Pharmacological validation data and a comparison of methods. J Pharmacol Toxicol Methods. 2019;98:106591. https://doi.org/10.1016/j.vascn.2019.106591

16. Roux S, Sablé E, Porsolt RD. Primary observation (Irwin) test in rodents for assessing acute toxicity of a test agent and its effects on behavior and physiological function. Curr Protoc Pharmacol. 2005;Chapter 10:Unit 10.10. https://doi.org/10.1002/0471141755.ph1010s27

17. Moser VC. Functional assays for neurotoxicity testing. Toxicol Pathol. 2011;39(1):36–45. https://doi.org/10.1177/0192623310385255

18. Gauvin DV, Zimmermann ZJ. FOB vs modified Irwin: What are we doing? J Pharmacol Toxicol Methods. 2019;97:24–8. https://doi.org/10.1016/j.vascn.2019.02.008

19. Moser VC. Applications of a neurobehavioral screening battery. Int J Toxicol. 1991;10(6):661–9. https://doi.org/10.3109/10915819109078658

20. Mead AN, Amouzadeh HR, Chapman K, Ewart L, Giarola A, Jackson SJ, et al. Assessing the predictive value of the rodent neurofunctional assessment for commonly reported adverse events in phase I clinical trials. Regul Toxicol Pharmacol. 2016;80:348–57. https://doi.org/10.1016/j.yrtph.2016.05.002

21. Fonck C, Easter A, Pietras MR, Bialecki RA. CNS adverse effects: From functional observation battery/irwin tests to electrophysiology. Handb Exp Pharmacol. 2015;229:83–113. https://doi.org/10.1007/978-3-662-46943-9_4

22. Lynch JJ 3rd, Castagné V, Moser PC, Mittelstadt SW. Comparison of methods for the assessment of locomotor activity in rodent safety pharmacology studies. J Pharmacol Toxicol Methods. 2011;64(1):74–80. https://doi.org/10.1016/j.vascn.2011.03.003

23. Himmel HM, Delaunois A, Deurinck M, Dinklo T, Eriksson Faelker TM, Habermann C, et al. Variability of non-clinical behavioral CNS safety assessment: An intercompany comparison. J Pharmacol Toxicol Methods. 2019;99:106571. https://doi.org/10.1016/j.vascn.2019.03.002

24. Bessalova EYu. Methods of researching the behavior of rats in an open field. Neyronauki = Neuroscience. 2011;7(2):106–9 (In Russ.)

25. Górska AM, Kamińska K, Wawrzczak-Bargieła A, Costa G, Morelli M, Przewlocki R, et al. Neurochemical and neurotoxic effects of MDMA (Ecstasy) and Caffeine after chronic combined administration in mice. Neurotox Res. 2018;33(3):532–48. https://doi.org/10.1007/s12640017-9831-9

26. Umezawa M, Onoda A, Korshunova I, Jensen A, Koponen IK, Jensen KA, et al. Maternal inhalation of carbon black nanoparticles induces neurodevelopmental changes in mouse offspring. Part Fibre Toxicol. 2018;15(1):36. https://doi.org/10.1186/s12989-018-0272-2

27. Walsh RN, Cummins RA. The open-field test: a critical review. Psychol Bull. 1976;83(3):482–504. PMID: 17582919

28. Stanford SC. The open field test: reinventing the wheel. J Psychopharmacol. 2007;21(2):134–5. https://doi.org/10.1177/0269881107073199

29. Blizard DA, Takahashi A, Galsworthy MJ, Martin B, Koide T. Test standardization in behavioural neuroscience: a response to Stanford. J Psychopharmacol. 2007;21(2):136–9. https://doi.org/10.1177/0269881107074513

30. Bondy SC, Campbell A. Developmental neurotoxicology. J Neurosci Res. 2005;81(5):605–12. https://doi.org/10.1002/jnr.20589

31. Ornoy A. Valproic acid in pregnancy: How much are we endangering the embryo and fetus? Reprod Toxicol. 2009;28(1):1–10. https://doi.org/10.1016/j.reprotox.2009.02.014

32. Veroniki AA, Rios P, Cogo E, Straus SE, Finkelstein Y, Kealey R, et al. Comparative safety of antiepileptic drugs for neurological development in children exposed during pregnancy and breast feeding: a systematic review and network meta-analysis. BMJ Open. 2017;7(7):e017248. https://doi.org/10.1136/bmjopen-2017-017248

33. Bauer AZ, Kriebel D, Herbert MR, Bornehag CG, Swan SH. Prenatal paracetamol exposure and child neurodevelopment: A review. Horm Behav. 2018;101:125–47. https://doi.org/10.1016/j.yhbeh.2018.01.003

34. Healy D, Le Noury J, Mangin D. Links between serotonin reuptake inhibition during pregnancy and neurodevelopmental delay/ spectrum disorders: A systematic review of epidemiological and physiological evidence. Int J Risk Saf Med. 2016;28(3):125–41. https://doi.org/10.3233/JRS-160726

35. Sie SD, Wennink JM, van Driel JJ, Winkel AGW, Boer K, Casteelen G, van Weissenbruch MM. Maternal use of SSRIs, SNRIs and NaSSAs: Practical recommendations during pregnancy and lactation. Arch Dis Child Fetal Neonatal Ed. 2012;97(6):F472–6 [Erratum. Arch Dis Child Fetal Neonatal Ed. 2013;98(2):F180]. https://doi.org/10.1136/archdischild-2011-214239

36. Manson JE. Prenatal exposure to sex steroid hormones and behavioral/cognitive outcomes. Metabolism. 2008;57(Suppl 2):S16–21. https://doi.org/10.1016/j.metabol.2008.07.010

37. Grandjean P, Landrigan PJ. Developmental neurotoxicity of industrial chemicals. Lancet. 2006;368(9553):2167–78. https://doi.org/10.1016/S0140-6736(06)69665-7

38. Grandjean P, Landrigan PJ. Neurobehavioural effects of developmental toxicity. Lancet Neurol. 2014;13(3):330–8. https://doi.org/10.1016/S1474-4422(13)70278-3

39. Liu L, Zhang D, Rodzinka-Pasko JK, Li YM. Environmental risk factors for autism spectrum disorders. Nervenarzt. 2016;87 (Suppl 2):55–61. https://doi.org/10.1007/s00115-016-0172-3

40. London Z, Albers JW. Toxic neuropathies associated with pharmaceutic and industrial agents. Neurol Clin. 2007;25(1):257–76. https://doi.org/10.1016/j.ncl.2006.10.001

41. Adityanjee, Munshi KR, Thampy A. The syndrome of irreversible lithium-effectuated neurotoxicity. Clin Neuropharmacol. 2005;28(1):38–49. https://doi.org/10.1097/01.wnf.0000150871.52253.b7

42. Vlisides P, Xie Z. Neurotoxicity of general anesthetics: an update. Curr Pharm Des. 2012;18(38):6232–40. https://doi.org/10.2174/138161212803832344

43. Lebedev IV, Pleskacheva MG, Anokhin KV. C57BL/6 mice open field behavior qualitatively depends on arena size. Zhurnal vysshey nervnoy deyatelnosti im. I.P. Pavlova = I.P. Pavlov Journal of Higher Nervous Activity. 2012;62(4):485–96 (In Russ.)

44. Suter L, Babiss LE, Wheeldon EB. Toxicogenomics in predictive toxicology in drug development. Chem Biol. 2004;11(2):161–71. https://doi.org/10.1016/j.chembiol.2004.02.003

45. Vorhees CV, Williams MT. Morris water maze: Procedures for assessing spatial and related forms of learning and memory. Nat Protoc. 2006;1(2):848–58. https://doi.org/10.1038/nprot.2006.116

46. Rosenfeld CS, Ferguson SA. Barnes maze testing strategies with small and large rodent models. J Vis Exp. 2014;(84):e51194. https://doi.org/10.3791/51194

47. Leger M, Quiedeville A, Bouet V, Haelewyn B, Boulouard M, Schumann-Bard P, Fleret T. Object recognition test in mice. Nat Protoc. 2013;8(12):2531–7. https://doi.org/10.1038/nprot.2013.155

48. Bevins RA, Besheer J. Object recognition in rats and mice: a onetrial non-matching-to-sample learning task to study “recognition memory”. Nat Protoc. 2006;1(3):1306–11. https://doi.org/10.1038/nprot.2006.205

49. Daldrup T, Remmes J, Lesting J, Gaburro S, Fendt M, Meuth P, et al. Expression of freezing and fear-potentiated startle during sustained fear in mice. Genes Brain Behav. 2015;14(3):281–91. https://doi.org/10.1111/gbb.12211

50. Alamed J, Wilcock DM, Diamond DM, Gordon MN, Morgan D. Two-day radial-arm water maze learning and memory task; robust resolution of amyloid-related memory deficits in transgenic mice. Nat Protoc. 2006;1(4):1671–9. https://doi.org/10.1038/nprot.2006.275

51. Hölter SM, Garrett L, Einicke J, Sperling B, Dirscherl P, Zimprich A, et al. Assessing cognition in mice. Curr Protoc Mouse Biol. 2015;5(4):331–58. https://doi.org/10.1002/9780470942390.mo150068

52. Ostrovskaya RU, Gudasheva TA. Rapid inhibition of exploratory movements as a test of nootropic activity. Bull Exp Biol Med. 1991;111(5):644–7 https://doi.org/10.1007/BF00841006

53. Bondarenko NA. The study of the possibility of the formation of targeted behavior in rats from the one test in the test Extrapolation disposal. In: Kharitonov AN, ed. Evolutionary and comparative psychology in Russia: traditions and prospects. Moscow: Institut psikhologii RAN; 2013. P. 122–30 (In Russ.)

54. Andreeva AK, Dubina DSh, Feldman BV, Gorshkov DA, Galimzyanova AKh. Influence of erythropoetin on psychomotor activity. Estestvennye nauki = Natural Sciences. 2009;(4):87–9 (In Russ.)

55. Bagmetova VV, Borodkina LE, Tyurenkov IN, Berestovitskaya VM, Vasilieva OS. A comparative experimental study of the nootropic properties of the GABA analogue of phenibut and its methyl ester. Fundamentalnye issledovaniya = Fundamental Research. 2011;10(3):467–71 (In Russ.)

56. Gaynetdinov AR, Fesenko ZS, Khismatullina ZR. Behavioral changes in heterozygous rats by gene knockout of the dopamine transporter (DAT). Biomeditsina = Biomedicine. 2020;16(1):82–8 (In Russ.) https://doi.org/10.33647/2074-5982-16-1-82-88

57. Solomina AS, Shreder ED, Kolik LG, Durnev AD. Study of fabomotizole effect on behavioral disorders in rat offsprings exposed to tobacco smoke and ethanol. Farmakokinetika i farmakodinamika = Pharmacokinetics and Pharmacodynamics. 2018;(2):3–11 (In Russ.) https://doi.org/10.24411/2587-7836-2018-10008

58. Whishaw IQ, Tomie JA. Of mice and mazes: Similarities between mice and rats on dry land but not water mazes. Physiol Behav. 1996;60(5):1191–7. https://doi.org/10.1016/s0031-9384(96)00176-x

59. Frick KM, Stillner ET, Berger-Sweeney J. Mice are not little rats: Species differences in a one-day water maze task. Neuroreport. 2000;11(16):3461–5. https://doi.org/10.1097/00001756-20001109000013

60. Yoshida M, Goto K, Watanabe S. Task-dependent strain difference of spatial learning in C57BL/6N and BALB/c mice. Physiol Behav. 2001;73(1–2):37–42. https://doi.org/10.1016/s0031-9384(01)00419-x

61. Grissom EM, Hawley WR, Hodges KS, Fawcett-Patel JM, Dohanich GP. Biological sex influences learning strategy preference and muscarinic receptor binding in specific brain regions of prepubertal rats. Hippocampus. 2013;23(4):313–22. https://doi.org/10.1002/hipo.22085

62. Hawley WR, Grissom EM, Barratt HE, Conrad TS, Dohanich GP. The effects of biological sex and gonadal hormones on learning strategy in adult rats. Physiol Behav. 2012;105(4):1014–20. https://doi.org/10.1016/j.physbeh.2011.11.021

63. Maguire EA, Burgess N, Donnett JG, Frackowiak RS, Frith CD, O’Keefe J. Knowing where and getting there: a human navigation network. Science. 1998;280(5365):921–4. https://doi.org/10.1126/science.280.5365.921

64. Parrella E, Maxim T, Maialetti F, Zhang L, Wan J, Wei M, et al. Protein restriction cycles reduce IGF-1 and phosphorylated Tau, and improve behavioral performance in an Alzheimer’s disease mouse model. Aging Cell. 2013;12(2):257–68. https://doi.org/10.1111/acel.12049

65. Méndez-López M, Méndez M, Arias J, Arias JL. Effects of a high protein diet on cognition and brain metabolism in cirrhotic rats. Physiol Behav. 2015;149:220–8. https://doi.org/10.1016/j.physbeh.2015.05.038

66. Roberts AJ, Hedlund PB. The 5-HT(7) receptor in learning and memory. Hippocampus. 2012;22(4):762–71. https://doi.org/10.1002/hipo.20938

67. Olvera-Cortés E, Pérez-Vega MI, Barajas-López G, Del Angel-Meza AR, González-Burgos I, Feria-Velasco A. Place learning impairment in chronically tryptophan-restricted rats. Nutr Neurosci. 1998;1(3):223–35. https://doi.org/10.1080/1028415X.1998.11747232

68. Yagi S, Galea LAM. Sex differences in hippocampal cognition and neurogenesis. Neuropsychopharmacology. 2019;44(1):200–13. https://doi.org/10.1038/s41386-018-0208-4

69. Barron AM, Tokunaga M, Zhang MR, Ji B, Suhara T, Higuchi M. Assessment of neuroinflammation in a mouse model of obesity and β-amyloidosis using PET. J Neuroinflammation. 2016;13(1):221. https://doi.org/10.1186/s12974-016-0700-x

70. Ledreux A, Wang X, Schultzberg M, Granholm AC, Freeman LR. Detrimental effects of a high fat/high cholesterol diet on memory and hippocampal markers in aged rats. Behav Brain Res. 2016;312:294–304. https://doi.org/10.1016/j.bbr.2016.06.012