Table 2.

Key in vitro assays in early drug discovery

Assays	Target value	Comments
Aqueous solubility	>100 µM	Important for running in vitro assays and for in vivo delivery of drug
Log D7.4	0–3 (for BBB penetration ca 2)	A measure of lipophilicity hence movement across membranes
Microsomal stability Clint	<30 µL·min−1·mg−1 protein	Liver microsomes contain membrane bound drug metabolizing enzymes. This assay measures compound clearance and can give an idea of how fast it will be cleared out in vivo
CYP450 inhibition	>10 µM	Main enzymes in body which metabolize drugs and their inhibition can cause toxicity
Caco-2 permeability Papp	>1 × 10−6 cm−1 (asymmetry <2)	Caco-2 colon carcinoma cell line used to estimate permeability across intestinal epithelium, important for drug absorption from gut
MDR1-MDCK permeability Papp	>10 × 10−6 cm−1 (asymmetry <2)	MDCK cells transfected with the MDR1 gene, which encodes the efflux protein P glycoprotein (P-gp). An important efflux transporter in many tissues including intestine, kidney and brain, P-gp can be used to predict intestinal and brain permeability
Hep G2 hepatotoxicity	No effect at 50 × IC50 or EC50	Human HepG2 cells can act as a surrogate for effects of toxicity on human liver, an important cause of drug failure in the clinic
Cytotoxicity in suitable cell line	No effect at 50 × IC50 or EC50	Reduce the likelyhood of cellular toxicity in vivo

IC₅₀, half maximal inhibitory concentration.