Guidelines for characterization of probes to be used in animals

Chemical probes that meet the desired criteria for in vitro cell systems may or may not be suitable for use in animal models, such as rodent species that are widely employed in biomedical research. In particular, it is important that a probe can be administered so as to achieve the appropriate level of exposure that would support modulation of the desired protein target for the required duration.

In order to aid the evaluation of probes in animal models, typically rodents, the Chemical Probes Team recommends a set of measured criteria considered essential for the in vivo pharmacokinetic and pharmacodynamic characterisation of chemical probes. These can be applied to guide the choice of probe and rational selection of the dosing schedule of a compound for preclinical in vivo experiments^1,2 .

Pharmacokinetic data should be provided to inform on the plasma and target organ exposure achieved with the compound at specified doses and dosing route(s). Protein binding data should be included to enable calculation of the unbound (free) concentration of the compound, i.e. the amount of compound not bound by plasma and tissue protein.

Together with the measured in vitro potency of the compound, this information will typically enable estimation of the dose(s) of compound required to achieve unbound concentrations sufficient to interact with the compound’s intended molecular target, and for which duration³ . The likelihood of interactions with known off-targets can be similarly quantified.

Where available, inclusion of quantitative data on the modulation of specific biomarkers reporting on the activity of the intended molecular target is encouraged, as evidence of target engagement in vivo, providing a pharmacological audit trail at the measured exposures⁴ .

Guidelines for useful pharmacokinetic data to be provided (rodents¹)

Specification of the preclinical model(s) in which pharmacokinetic parameters have been measured

• Species used
• Dosing route and vehicle
• Dose quantity and schedule

Pharmacokinetic data informing on the plasma exposure of the drug and the plasma free fraction

• Elimination half-life (T_1/2 )
• Systemic clearance (CL)
• Fraction of the compound that is protein-bound (f_b )
• Maximum plasma concentration after drug administration (C_max ) and time to reach maximum plasma concentration (T_max ) for the given dose

For compounds targeting the CNS

• Ratio of drug in the brain to that in plasma (B:P)
• Ratio of drug found free in the brain (C_u,p ) defined as C _u,b /C_u,p
• Any potential impact of drug transporters (found on the rodent blood-brain-barrier) in limiting brain exposure

References:

1. Kleiman RJ, Ehlers MD. Sci Transl Med 2016 8: 320ps1. DOI: 10.1126/scitranslmed.aac9888
2. Rossanese O, Eccles S, Springer C, Swain A, Raynaud FI, Workman P, Kirkin V. Drug Discovery Today: Disease Models 2016 21: 23-32. DOI: 10.1016/j.ddmod.2017.07.002
3. Smith DA, Di L, Kerns EH. Nat Rev Drug Discov 2010 9: 929-39. DOI: 10.1038/nrd3287
4. Banerji U, Workman P. Semin Oncol 2016 43: 436-45. DOI: 10.1053/j.seminoncol.2016.06.001