Male C3H/HeN mice were shaved under isoflurane anesthesia on the lower back using an electric shaver. Only mice in the telogen phase (pink skin) were used in studies. Twenty microliters of test articles at various concentrations in propylene glycol/ethanol (30:70, v/v) or the vehicle control was topically applied to the shaved lower back of the mice to cover an area of approximately 1 cm2 (20uL/cm2). Ten mice were used in each experimental group. The treatment regimen was twice daily (BID) application for 4 weeks, 5 days/week from Monday to Friday. Local irritation was recorded before each application and hair growth scores were recorded every other day.

The competitive radio-ligand binding analysis was performed on human AR extracts from transfected baculovirus/Sf9 cells in the presence or absence of differing concentrations of test agent and a fixed concentration of 3H-dihydrotestosterone (3H-DHT) as tracer. Progressively decreasing concentrations of compounds are incubated in the presence of human AR extracts, hydroxylapatite and, 1 nM 3H-DHT for 1 h at 4 °C. Subsequently, the binding reaction mixtures are washed three times to completely remove excess unbound 3H-DHT. AR bound 3H-DHT levels are determined in the presence of compounds and compared to levels of receptor specific binding when no competitor is present. Compound binding affinity to the human AR is expressed as the concentration of compound at which 50% of the maximum specific binding is inhibited (IC50). PF-998425 binds to AR and displaces radioligand with an IC50 of 37 nM.