Development of LC-MS methods for quantitation of hepcidin and demonstration of siRNA-mediated hepcidin suppression in serum.

Abstract

A requisite step in developing a therapeutic to modulate the levels of hepcidin is the development of a quantitative method for measuring the concentration of serum hepcidin. To this end, an LC-MS method, based on selected reaction monitoring (SRM) with a triple quadrupole MS and an isotopically labeled hepcidin as internal standard, was developed to measure hepcidin in mouse and monkey sera. Initially, 40 normal cynomolgus monkeys and 40 normal mice were studied to determine the normal endogenous levels of hepcidin, and an average of 50ng/mL was found in the monkeys and 46ng/mL in the mice. Next, experiments were conducted where an siRNA, targeting hepcidin, was administered to cynomolgus monkeys, resulting in effective hepcidin reduction (inhibition rate) of 87% after 24h and 74% after 48h, demonstrating to effectively reduce serume level of hepcidin. For better sensitivity, especially for the low volumes available for mouse sera, a second LC-MS method, based on parallel reaction monitoring (PRM) using a Orbitrap MS was developed and shown to be at least 10 fold lower in detection limits (or consumption of serum volume) than the SRM approach.