Abstract

In the present study, the involvement of cytochrome P450 enzyme(s) in the primary metabolism of laquinimod, a new orally active immunomodulator, has been investigated in human liver microsomes. Hydroxylated and dealkylated metabolites were formed. The metabolite formation exhibited single enzyme Michaelis-Menten kinetics with apparent K_M in the range of 0.09 to 1.9 mM and V_max from 22 to 120 pmol/mg/min. A strong correlation between the formation rate of metabolites and 6β-hydroxylation of testosterone was obtained within a panel of liver microsomes from 15 individuals (r² = 0.6 to 0.94). Moreover, ketoconazole and troleandomycin, specific inhibitors of CYP3A4 metabolism, demonstrated a significant inhibition of laquinimod metabolism. Furthermore, in incubations with recombinant CYP3A4, all the primary metabolites were formed. In vitro interaction studies with CYP3A4 substrates and possible concomitant medication demonstrated that laquinimod inhibits the metabolism of ethinyl estradiol with an IC₅₀ value of about 150 μM, which is high above the plasma level of laquinimod after clinically relevant doses. Ketoconazole, troleandomycin, erythromycin, prednisolone, and ethinyl estradiol inhibited the metabolism of laquinimod, and IC₅₀ values of 0.2, 11, 24, 87, and 235 μM, respectively, were calculated. In conclusion, the present study demonstrates that laquinimod is a low affinity substrate for CYP3A4 in human liver microsomes. The likelihood for in vivo effects of laquinimod on the metabolism of other CYP3A4 substrates is minor. However, inhibitory effects on the metabolism of laquinimod by potent and specific inhibitors of CYP3A4, such as ketoconazole, are anticipated and should be considered in the continued clinical program for laquinimod.