The first step after knowing that a compound is efficacious on a target is the in vitro characterization of the ADME properties
A good preclinical candidate needs to be soluble in all relevant efficacy and in vitro ADME assays, and additionally able to permeate cell membranes. These physicochemical properties are evaluated using kinetic solubility, log D and PAMPA assays. After that, and considering the chemical structure, stability assays are established.
At IRBM, we customize the screening cascade for each chemical series, avoiding “box ticking” but ensuring a smooth transition to in vivo studies in preclinical species.
Initial stability testing is conducted using SGF (simulated gastric fluid), SIF (simulated intestinal fluid), plasma, and blood. This is particularly critical if labile groups, such as esters or amides are present in the molecule.
Metabolic stability is assessed using microsomes from selected species (typically human microsomes, and microsomes from the species used in the efficacy model), or other subcellular fractions, if appropriate. We have successfully extrapolated intrinsic clearance with microsomes even for drugs subject to conjugative metabolism.
Hepatocyte stability screening is included if metabolism does not mainly involve endoplasmic reticulum enzymes. Hepatocytes are used in suspension, or plated when longer incubation times are required.
All the stability assays are detected using liquid chromatography-high-resolution OrbitrapTM mass spectrometry (LC-HRMS). HRMS data are acquired using quantitative/qualitative analysis. This allows for the quantification of parent compound disappearance, while identifying the main metabolic pathways.
We have implemented novel approaches to assess stability of therapeutic peptides in plasma/blood and in subcutaneous tissue, that can be fully integrated on peptide discovery programs.
Ultra-high performance liquid-chromatography coupled to HRMS and fast fraction collection (FC) (≤ 1 s/fraction) followed by microplate scintillation counting (MSC) is used for profiling radiolabelled metabolites in different tissues.
Distribution assays such as plasma protein binding (PPB), binding to microsomes or tissue homogenates and blood/plasma partition, are also available, with a variety of protocols for different throughputs.
Assessment of brain penetration can be performed using an in vitro human blood-brain barrier model. Cytochrome P450 inhibition and induction potential is also determined using optimized protocols.