A SENSITIVE AND SPECIFIC RADIOIMMUNOASSAY FOR LY309887, A POTENT INHIBITOR OF GLYCINAMIDE RIBONUCLEOTIDE FORMYLTRANSFERASE

LY309887, a reduced analogue of folic acid, is a potent inhibitor of g lycinamide ribonucleotide formyltransferase and possesses a broad spec trum of antitumor activity. During preclinical studies using supplemen tation with oral folic acid, this second-generation inhibitor displaye d both the desired safety profile and the pharmacology to warrant clin ical investigation. A sensitive analytical method was needed to assess the pharmacokinetics of LY309887 due to the low doses planned for Pha se I studies and the potential for low concentrations in plasma long a fter i.v. administration. We therefore undertook the development of a competitive RIA. A highly specific antiserum was raised in rabbits fol lowing immunization with LY309887 coupled to BSA. A RIA tracer was pre pared by radioiodination of compound 389753, the adduct of LY309887 wi th p-tyramine. We developed a competitive-binding RIA procedure and us ed superparamagnetic particles coated with goat antirabbit IgG as a me thod for separating the bound and free forms of LY309887. The RTA is s ensitive (0.5 ng/ml in serum and 25 ng/ml in urine), specific (negligi ble interference from endogenous folates), and reproducible (interassa y coefficients of variation ranging from 8.1 to 15.4% and 7.6 to 8.3% for serum and urine controls, respectively), We used the RIA to assess the i.v. pharmacokinetics of LY309887 in both patients with metastati c cancer and dogs. The sensitivity of the RIA permitted the demonstrat ion that serum concentrations of LY309887 decline in a multiexponentia l manner with a prolonged terminal elimination phase, We conclude that the RIA is a valid method for quantifying LY309887 in biological flui ds.