Sensitive and specific radioimmunoassay for fialuridine: initial assessment of pharmacokinetics after single oral doses to healthy volunteers

Abstract

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

Utility of human hepatocyte spheroids for evaluation of hepatotoxicity

“…Changes of the IC 50 values for albumin secretion were observed, except in the case of diclofenac. In particular, the IC 50 of fialuridine, a nucleoside analogue, changed dramatically from 18.1 μM at 7 days to 0.9 μM at 21 days; the latter value is close to the reported plasma concentration of fialuridine in clinical studies, 0.64 μM (Bowsher et al, 1994). Fialuridine or its metabolite is incorporated into mitochondrial DNA, leading to a decrease of mitochondrial function.…”

Antiviral activity and toxicity of fialuridine in the woodchuck model of hepatitis B virus infection

“…The serum concentration of FIAU was determined using a competitive binding radioimmunoassay based on a rabbit antibody raised against FIAU (5Ј-0-hemisuccinate conjugated to keyhole limpet hemocyanin). 17 The sensitivity (0.2 ng/mL), specificity (negligible interference from known FIAU metabolites and endogenous nucleosides), and interassay variability (5.0% to 19.7%) of this procedure have been described. 17 For determination of hepatic FIAU incorporated into cellular DNA, specimens of liver were obtained immediately following euthanasia and processed by a previously described procedure.…”

“…17 The sensitivity (0.2 ng/mL), specificity (negligible interference from known FIAU metabolites and endogenous nucleosides), and interassay variability (5.0% to 19.7%) of this procedure have been described. 17 For determination of hepatic FIAU incorporated into cellular DNA, specimens of liver were obtained immediately following euthanasia and processed by a previously described procedure. 9 Briefly, DNA was isolated from whole liver homogenate using the ASAP Genomic DNA Isolation Kit (Boehringer Mannheim, Corp., Indianapolis, IN).…”

“…DNA extracted from liver homogenate was denatured by heating to 95°C for 5 minutes, and the solution placed on ice. DNA was hydrolyzed with snake venom phosphodiesterase and bacterial AP, FIAU measured by radioimmunoassay, 17 and the results expressed as picomoles of FIAU per micromoles of thymidine determined by high-pressure liquid chromatography.…”

Fialuridine accumulates in DNA of dogs, monkeys, and rats following long-term oral administration.

“…These techniques are not amenable to assessing FIAU accumulation in DNA following long-term dosing regimens (90 days) and in larger species (dogs and monkeys) because of the large amounts of radioactivity required and the safety hazards associated with administering large quantities ofradioactivity to animals over extended periods of time. Recently, however, the development of a sensitive radioimmunoassay (RIA) for use on biological fluids (13) has made it feasible to measure concentrations of FIAU-DNA under such conditions.…”