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 315400 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 |