Development of simultaneous quantification method of loteprednol etabonate (LE) and its acidic metabolites,and analysis of LE metabolism in rat

1. Loteprednol etabonate (LE) is a soft corticosteroid with two labile ester bonds at 17α- and 17β-positions. Its corticosteroidal activity disappears upon hydrolysis of either ester bond. Hydrolysis of both ester bonds produces the inactive metabolite, Δ¹-cortienic acid (Δ¹-CA).

2. The simple high-performance liquid chromatography method using acetic acid gradient was developed for the simultaneous determination of LE and its acidic metabolites.

3. LE was hydrolyzed in rat plasma with a half-life of 9?min. However, LE hydrolysis was undetectable in rat liver and intestine. LE hydrolysis in rat plasma was completely inhibited by paraoxon and bis(p-nitrophenyl) phosphate, thus identifying carboxylesterase as the LE hydrolase. Rat plasma carboxylesterase had a K_m of 6.7?μM for LE.

4. In contrast to the disappearance rate of LE in rat plasma, the formation rate of 17α-monoester and Δ¹-CA was markedly low, and a main hydrolysate of LE was not detected in rat plasma.

5. The metabolism of LE proceeded via different pathways in human and rat plasma. LE was slowly hydrolyzed by paraoxonase in human plasma to 17α-monoester with a half-life of 12?h, but by carboxylesterase in rat plasma to yield undetectable products, presumed to include the unstable 17β-monoester.