Fialuridine (FIAU) is a halogen-substituted analog of thymidine that was undergoing clinical investigation as a drug for the treatment of chronic hepatitis B viral infection. However, clinical trials of FIAU were terminated after adverse events occurred following chronic oral administration. Prior to the termination of clinical trials, a sensitive assay was needed for the measurement of FIAU because of the anticipated low dose administered to patients. We therefore undertook the development of a radioimmunoassay (RIA). A specific antiserum was raised in rabbits following immunization with a 5'-O-hemisuccinate analog of FIAU coupled to keyhole limpet hemocyanin. Radiolabeled FIAU was synthesized by a destannylation procedure by using sodium [125j]iodide. We developed a competitive-binding procedure and used precipitation with polyethylene glycol as the method for separating the bound and free forms of FIAU. The RIA is sensitive (0.2 ng/ml), specific (negligible interference from known metabolites and endogenous nucleosides), and reproducible (interassay coeflicients of variation range from 5 to 19.7% for serum controls). We used the RIA to assess the pharmacokinetics of FIAU in healthy adult volunteers following administration of a single 5-mg oral dose. The sensitivity of the RIA permitted the detection of a prolonged elimination phase for FIAU in healthy volunteers and dogs, with mean elimination half-lives of 29.3 and 35.3 h, respectively. We conclude the RIA is a valid method for the quantification of FIAU in biological fluids.Fialuridine (FIAU), 1-(2'-deoxy-2'-fluoro-13-D-arabinofuranosyl)-5-iodouracil (Fig. 1, compound 2), is one of a series of 2'-fluoro-substituted arabinosyl pyrimidine nucleosides that have demonstrated potent antiviral activities against a number of clinically important viruses, including hepatitis B virus (HBV) (7,11,28,47). Analogs of FIAU have been shown to inhibit viral replication in the woodchuck and duck models of HBV infection (18,19). Although the mechanism of the anti-HBV activity is not well understood, evidence suggests that the triphosphate analog of FIAU is a potent inhibitor of HBV DNA polymerase activity (17,23,43). During early clinical investigation FIAU showed much promise as an anti-HBV drug because it markedly reduced the level of HBV DNA in the serum of patients with chronic hepatitis B (20). However, clinical trials were terminated after adverse events occurred following oral administration of FIAU (0.1 and 0.25 mg/kg of body weight per day) for more than 2 months (24, 29). The mechanism of the unexpected delayed toxicity is unresolved.Prior to the termination of clinical trials an assay was needed to quantify FIAU in biological fluids. However, the anticipated low therapeutic dose in patients necessitated the development of a highly sensitive analytical method. The criterion of sensitivity precluded the use of an existing UV-based high-pressure liquid chromatographic (HPLC) method for FIAU because its

Glucagon-like insulinotropic peptide (GLP-1) and its analogs are of interest because of their therapeutic potential in type II diabetes. LY315902 is a GLP-1-(7-37)-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 immunoreactive LY315902 in the plasma of animals. A homobifunctional cross-linker 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 of LY315902 antibodies. One rabbit produced a high-affinity antiserum that 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 room temperature. The bound forms of LY315902 were separated by polyethylene 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 from 8.0% to 14.7% and 92.8% to 107.3%, respectively. By applying this assay to measure plasma concentrations of immunoreactive LY315902 in dogs following twice daily subcutaneous injections of LY315902, we determined that the plasma half-life of LY315902 is significantly longer than that of native GLP-1-(7-37)-OH. We concluded that the structural modifications which were made to produce LY315902 prolonged its plasma half-life. The extended plasma half-life of LY315902 correlated well with its prolonged pharmacology in dogs.