A RADIOIMMUNOASSAY FOR LY315902, AN ANALOG OF GLUCAGON-LIKE INSULINOTROPIC PEPTIDE, AND ITS APPLICATION IN THE STUDY OF CANINE PHARMACOKINETICS

Glucagon-like insulinotropic peptide (GLP-1) and its analogs are of in terest because of their therapeutic potential in type II diabetes, LY3 15902 is a GLP-1-(737)-OH analog with a modified N-terminus (IP7), an octanoic acid (C8) acylated on the lysine residue at position 34, and a substitution with arginine at position 26. We developed a sensitive and specific radioimmunoassay (RIA) for the determination of immunorea ctive LY315902 in the plasma of animals. A homobifunctional crosslinke r was used to couple the nonacylated form of LY315902 [IP7-R26-GLP-1-( 7-37)-OH] to carrier proteins to enhance its immunogenicity. Following immunization, animal antisera were screened by RIA for the presence o f LY315902 antibodies. One rabbit produced a high-affinity antiserum t hat display insignificant cross-reactivity against two forms of native GLP-1 and possible major metabolites of LY315902. In this RIA method, plasma samples were combined with radioiodinated LY315902 and rabbit anti-IP7-R26-GLP-1-(7-37)-OH serum, and then incubated overnight at ro om temperature. The bound forms of LY315902 were separated by polyethy lene glycol assisted second antibody precipitation, The sensitivity of the assay was estimated to be 19 pM, Inter-assay precision (%CV) and accuracy (recovery) for quality control samples in dog plasma ranged f rom 8.0% to 14.7% and 92.8% to 107.3%, respectively. By applying this assay to measure plasma concentrations of immunoreactive LY315902 in d ogs following twice daily subcutaneous injections of LY315902, we dete rmined that the plasma half-life of LY315902 is significantly longer t han that of native GLP-1-(737)-OH. We concluded that the structural mo difications which were made to produce LY315902 prolonged its plasma h alf-life. The extended plasma half-life of LY315902 correlated well wi th its prolonged pharmacology in dogs.