Biological Evaluation of Compounds

Performed by Sue Charman's laboratory at the CDCO at Monash University.

Two compounds, OSM-S-106 and OSM-S-111, which possessed favourable activity have been assessed in assays designed to probe metabolic stability and measure kinetic stability.

Assay Details (provided by Karen White at Centre for Drug Candidate Optimisation, Monash University, 17th July 2013)

General method for assessing kinetic solubility

DMSO solutions of each test compound are spiked (at a 1:100 dilution) into aqueous test media (pH 2 and 6.5) in a 96-well plate. The final concentrations of compound in the test media typically span the range of 1.6 – 100 µg/mL. Plates are allowed to stand for
30 minutes at room temperature, after which the extent of compound precipitation is measured by nephelometry. The kinetic solubility limit is determined by the compound concentration at which precipitation becomes evident.

 General method for metabolism studies conducted in hepatic microsomes

A solution of each test compound (and quality control compounds) prepared in 50% acetonitrile/water is spiked into microsomal matrix (microsomes suspended in phosphate buffer) and incubated at 37°C. The reaction is initiated by the addition of NADPH (the cofactor for CYP450-mediated metabolism) and then the reaction is quenched at various time points over a 60 minute incubation period by the addition of ice-cold acetonitrile. Additional control samples are also included to monitor for potential compound degradation in the absence of cofactor. Quenched samples are analysed by UPLC-MS to determine the extent and rate of loss of parent compound. The depletion rate constant for each compound is then used to calculate a degradation half-life and in vitro intrinsic clearance.

Metabolic Data:

received 21st June 2013

Kinetic Solubility Data:

received 1st July 2013

Glutathione Trapping Study for OSM-S-35 

Results

Conclusion

We’ve run the glutathione trapping study for OSM-S-35 in the presence of metabolic activation (i.e. human microsomes). We also included a search for other potential metabolites formed. A number of metabolites were detected, mainly oxygenated species (mono, bis and tri-oxygenated metabolites) with the predominant metabolite (based on peak area and assuming similar response factors for each metabolite) arising from likely hydroxylation of the pyrrole substituted benzene. In the presence of GSH-EE, adducts were observed both in the presence and absence of metabolic activation. The results are summarised in the attached file. Please let me or Sue know if you have any questions or comments