LY309887, a reduced analogue of
folic acid, is a potent inhibitor of
glycinamide ribonucleotide formyltransferase and possesses a broad spectrum of antitumor activity. During preclinical studies using supplementation with oral
folic acid, this second-generation inhibitor displayed both the desired safety profile and the pharmacology to warrant clinical investigation. A sensitive analytical method was needed to assess the pharmacokinetics of
LY309887 due to the low doses planned for Phase I studies and the potential for low concentrations in plasma long after i.v. administration. We therefore undertook the development of a competitive RIA. A highly specific antiserum was raised in rabbits following immunization with
LY309887 coupled to BSA. A RIA tracer was prepared by radioiodination of compound 389753, the adduct of
LY309887 with
p-tyramine. We developed a competitive-binding RIA procedure and used superparamagnetic particles coated with goat antirabbit
IgG as a method for separating the bound and free forms of
LY309887. The RIA is sensitive (0.5 ng/ml in serum and 25 ng/ml in urine), specific (negligible interference from endogenous folates), and reproducible (interassay 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 metastatic
cancer and dogs. The sensitivity of the RIA permitted the demonstration that serum concentrations of
LY309887 decline in a multiexponential manner with a prolonged terminal elimination phase. We conclude that the RIA is a valid method for quantifying
LY309887 in
biological fluids.