Preclinical assay checklist for oral small molecule candidates for drug development

The guide was created by: 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).

Assay	Target profile	Comment
Target binding affinity in cell-free assay (EC50 or IC50)	< 10 nM	May 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 nM	May not be achievable for all targets
Functional cell activity in vitro (EC50 or IC50)	< 100 nM	May not be achievable for all targets
Pharmacological activity in animal model	< 10 mg/kg	Sufficient effect to give an achievable human dose
PK/PD analysis	Dose-dependent exposure-effect correlation	PK/PD profile consistent with mechanism of action
Human tissue activity ex-vivo	< 1 uM	Consistent with in vitro potency and free concentration
Predicted human dose	Total human daily dose <100 mg	Particularly for chronic dosing
logP/D measurement	1-3
pKa measurement	< 9 (basic)	Ensure some unionised species at pH7.4
Kinetic solubility	>200 uM	Low <30 uM; Medium 30-200 uM
Thermodynamic solubility	>200 uM	Crystalline development salt form usually less soluble than kinetic solubility
Heat, pH, and humidity stability	Stability consistent with clinical development plan	Some impurities may impact stability of active principle
Crystallinity assessment	Single stable polymorph	Advanced candidates
Salt form selection	Selected from GRAS counterions	Ideally final salt form identified
Formulation selection	Precedented oral formulations	Advanced candidates
Synthetic route	Adequate for clinical scale-up, minimal heavy metal content	Minimise use of hazardous reagents and chromatographic steps
Permeability PAMPA	> 10 x 10-6 cm/s	Measures 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/s	Efflux ratio B-A / A-B > 2 may indicate active efflux
P-glycoprotein (P-gp) transporter	Not a substrate or inhibitor	P-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 stability	Stable	Some 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	~1	Ratio >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 uM	CYP450 inhibition can lead to drug-drug interactions in polypharmacy
CYP450 time-dependent induction (TDI)	>10 uM	CYP450 inhibition can lead to drug-drug interactions in polypharmacy
Pregnane X receptor (PXR) activation	>10 uM	PXR regulates the expression of metabolic enzymes and transporters
Aryl hydrocarbon receptor (AhR) activation	>10 uM	AhR regulates the expression of metabolic enzymes and transporters
Aldehyde oxidase metabolism	Not an AO substrate	Several human allelic variants are known
Metabolic clearance (microsomes)	< half liver blood flow	Depends on species
Metabolic clearance (hepatocytes)	< half liver blood flow	Includes secondary met pathways
Extraction ratio	< 0.5	<0.3 low; 0.3-0.7 med; >0.7 high
Metabolic pathways	Fate of parent and metabolites identified
Metabolite identification	Main metabolites identified	Best if human metabolites are observed in animal species used for safety studies
CYPs responsible for metabolism	Isoforms identified	Several CYPs are polymorphic in man (leading to fast / slow metabolisers)
Broad off-target screening panel	No activity < 10 uM	Depends on potency of candidate and whether drug will reach the off-target
hERG inhibition	No activity < 30 uM	hERG inhibition has significant cardiac safety implications
Nav1.5 inhibition	No activity < 10 uM	Nav1.5 blockade has significant cardiac safety implications
HepG2 cytotoxicity	No activity < 10 uM	HepG2 mito-toxicity is a surrogate for drug-induced liver injury
Phospholipidosis	Absent	Phospholipidosis may lead to inflammation and fibrosis
Phototoxicity	Absent	Usually in the UVA range (wavelength 315–400 nm)
Ames Test	Negative for parent, metabolites, intermediates	Important first screen for mutagenic potential of new chemicals and drugs
Micronucleus test (in vitro)	Negative	Required to support initial human administration
Chromosome aberration test	Negative	Dependent on other genotox results and receiving expert opinion
Mouse lymphoma assay	Negative	Detects genotoxic and clastogenic effects
Intravenous & oral pharmacokinetics:		Usually two species (used as tox species)
Tmax, Cmax, T1/2, Cl, Vd, AUC, %F	Programme specific	Depends on route of admin, target dosing. Oral %F (bioavailability) > 30%
Dose exposure linearity	Yes	Based 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 TI	Reversible or monitorable effects may be acceptable in some indications.
Cardiovascular	No adverse clinical or biochemical observations, or acceptable TI	Reversible or monitorable effects may be acceptable in some indications.
Respiratory	No adverse clinical or biochemical observations, or acceptable TI	Reversible 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 observations	No adverse clinical or biochemical observations, or acceptable TI	Reversible or monitorable effects may be acceptable in some indications.
Blood chemistry	No or minimal/reversible changes
Organ histology	No or minimal/reversible changes
No observable adverse effect level (NOAEL)	As high as possible	NOAEL 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)	>10	A lower TI may be acceptable for particular indications e.g. oncology