Preclinical assay checklist for oral small molecule candidates for drug development

The guide was a collaboration between Tim Ritchie of Zerlavanz Consulting Ltd; Rick Cousins of Cinnabar Consulting Ltd; Simon Macdonald of RGDscience Ltd; Richard Hatley of RGDscience Ltd.

Discovering oral small molecule clinical candidates which balance all the necessary attributes is a challenging, multidisciplinary enterprise.  Provided here are guidelines (not rules!) for properties a molecule should ideally have alongside standard assays and should be considered.  The list is not comprehensive but maybe useful as an aide memoire.  The suggested cut-offs are those that would apply most of the time to most projects, but each project is unique and the project team may need to set its own values dependent on the particular biological target and therapeutic context.  It is unlikely that any clinical candidate will meet all these criteria and the project team will need to make judgements as to what compromises are acceptable whilst also building in mitigation plans as required.

Useful papers and webpages with more information about the assays can be accessed by clicking on the words in the first column.  Comments are welcome.  Please contact any of the authors via the Contact Us page or through Linked-In profiles (see above).

AssayTarget profileComment
Target binding affinity in cell-free assay (EC50 or IC50)< 10 nMMay not be achievable for all targets
Selectivity over related receptors> 30 fold> 100 fold selective over off-targets
Cell-free functional target affinity (EC50 or IC50)< 10 nMMay not be achievable for all targets
Functional cell activity in vitro (EC50 or IC50)< 100 nMMay not be achievable for all targets
Pharmacological activity in animal model< 10 mg/kgSufficient effect to give an achievable human dose
PK/PD analysisDose-dependent exposure-effect correlationPK/PD profile consistent with mechanism of action
Human tissue activity ex-vivo< 1 uMConsistent with in vitro potency and free concentration
Predicted human doseTotal human daily dose <100 mgParticularly for chronic dosing
logP/D measurement1-3
pKa measurement< 9 (basic)Ensure some unionised species at pH7.4
Kinetic solubility>200 uMLow <30 uM; Medium 30-200 uM
Thermodynamic solubility>200 uMCrystalline development salt form usually less soluble than kinetic solubility
Heat, pH, and humidity stabilityStability consistent with clinical development planSome impurities may impact stability of active principle
Crystallinity assessmentSingle stable polymorphAdvanced candidates
Salt form selectionSelected from GRAS counterionsIdeally final salt form identified
Formulation selectionPrecedented oral formulationsAdvanced candidates
Synthetic routeAdequate for clinical scale-up, minimal heavy metal contentMinimise use of hazardous reagents and chromatographic steps
Permeability PAMPA> 10 x 10-6 cm/sMeasures passive permeability only
Permeability Caco-2 A-B> 5 x 10-6 cm/s ( = 50 nm/s)Efflux ratio B-A / A-B > 2 may indicate active efflux
Permeability MDCK-MDR1 A-B> 5 x 10-6 cm/sEfflux ratio B-A / A-B > 2 may indicate active efflux
P-glycoprotein (P-gp) transporterNot a substrate or inhibitorP-gp efflux may limit GI, CNS, and cell penetration
Breast Cancer Resistance Protein (BCRP)Not a substrate or inhibitor
Multidrug and Toxin Extrusion protein 1 (MATE1)Not a substrate or inhibitor
Organic anion transporting polypeptide (OATP1B1)Not a substrate or inhibitor
Organic anion transporting polypeptide (OATP1B3)Not a substrate or inhibitor
Multidrug Resistance Protein 2 (MRP2)Not a substrate or inhibitor
Plasma stabilityStableSome instability may be desirable in some cases to reduce systemic exposure
Plasma protein binding< 98%Depends on PK parameters and target potency of molecule
Blood/plasma ratio~1Ratio >1 suggests binding to red blood cells
Brain/blood ratio>0.5 (CNS projects); <<1 (peripheral projects)Values >1 may indicate potential for brain accumulation
Kp,uu (concentration ratio of unbound drug in brain to blood)> 0.3 and <1.0< 0.3 = limited access to brain; >1.0 potential for accumulation
CYP450 phenotyping>10 uM (particularly 3A4 and 2D6)Several CYPs are polymorphic in man (leading to fast / slow metabolisers)
CYP450 time-dependent inhibition (TDI)>10 uMCYP450 inhibition can lead to drug-drug interactions in polypharmacy
CYP450 time-dependent induction (TDI)>10 uMCYP450 inhibition can lead to drug-drug interactions in polypharmacy
Pregnane X receptor (PXR) activation>10 uMPXR regulates the expression of metabolic enzymes and transporters
Aryl hydrocarbon receptor (AhR) activation>10 uMAhR regulates the expression of metabolic enzymes and transporters
Aldehyde oxidase metabolismNot an AO substrateSeveral human allelic variants are known
Metabolic clearance (microsomes)< half liver blood flowDepends on species
Metabolic clearance (hepatocytes)< half liver blood flowIncludes secondary met pathways
Extraction ratio< 0.5<0.3 low; 0.3-0.7 med; >0.7 high
Metabolic pathwaysFate of parent and metabolites identified
Metabolite identificationMain metabolites identifiedBest if human metabolites are observed in animal species used for safety studies
CYPs responsible for metabolismIsoforms identifiedSeveral CYPs are polymorphic in man (leading to fast / slow metabolisers)
Broad off-target screening panelNo activity < 10 uMDepends on potency of candidate and whether drug will reach the off-target
hERG inhibitionNo activity < 30 uMhERG inhibition has significant cardiac safety implications
Nav1.5 inhibitionNo activity < 10 uMNav1.5 blockade has significant cardiac safety implications
HepG2 cytotoxicityNo activity < 10 uMHepG2 mito-toxicity is a surrogate for drug-induced liver injury
PhospholipidosisAbsentPhospholipidosis may lead to inflammation and fibrosis
PhototoxicityAbsentUsually in the UVA range (wavelength 315–400 nm)
Ames TestNegative for parent, metabolites, intermediatesImportant first screen for mutagenic potential of new chemicals and drugs
Micronucleus test (in vitro)NegativeRequired to support initial human administration
Chromosome aberration testNegativeDependent on other genotox results and receiving expert opinion
Mouse lymphoma assayNegativeDetects genotoxic and clastogenic effects
Intravenous & oral pharmacokinetics:Usually two species (used as tox species)
Tmax, Cmax, T1/2, Cl, Vd, AUC, %FProgramme specificDepends on route of admin, target dosing. Oral %F (bioavailability) > 30%
Dose exposure linearityYesBased on exposure (AUC) as well as dose
Safety pharmacology:FDA requirement for advanced candidates
Central nervous system (CNS)No adverse clinical or biochemical observations, or acceptable TIReversible or monitorable effects may be acceptable in some indications.
CardiovascularNo adverse clinical or biochemical observations, or acceptable TIReversible or monitorable effects may be acceptable in some indications.
RespiratoryNo adverse clinical or biochemical observations, or acceptable TIReversible or monitorable effects may be acceptable in some indications.
1-, 2-, or 4-week toxicology in vivo:Usually two species (rodent and non-rodent); matches planned dosing in human
Clinical observationsNo adverse clinical or biochemical observations, or acceptable TIReversible or monitorable effects may be acceptable in some indications.
Blood chemistryNo or minimal/reversible changes
Organ histologyNo or minimal/reversible changes
No observable adverse effect level (NOAEL)As high as possibleNOAEL should preferably be higher than the minimum anticipated biological effect level (MABEL)
Lowest observable adverse effect level (LOAEL)As high as possible
Therapeutic Index (TI)>10A lower TI may be acceptable for particular indications e.g. oncology
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