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

Abstract

Accumulation of the antiviral nucleoside analogue fraluridine (FIAU; 1-(2'-deoxy-2'-fluoro-P-D-arab- In part because of the lactic acidosis observed in the clinical trials, it has been proposed that FIAU may selectively affect mitochondrial function. Depletion of muscle mitochondrial DNA (mtDNA) has been observed with dideoxycytidine (ddC) (6) and 3'-azido-3'-deoxythymidine (AZT) (7) and has been suggested as a possible mechanism for the delayed toxicity of FIAU. Unlike AZT and ddC, however, FIAU has a free 3'-OH, which in theory permits internal incorporation into full-length DNA. Acute studies have shown that FIAU is incorporated into the DNA of (10). While the biological consequences of FIAU incorporation into DNA remain unclear, in vitro studies with 9-Darabinofuranosyl-2-fluoroadenine (F-ara-A) (11) and 1-Darabinofuranosylcytosine (araC) (12) have correlated toxicity with incorporation into genomic DNA, suggesting that incorporation of FIAU into genomic DNA could also lead to cytotoxicity. Since clinical deterioration only occurred after long durations of exposure, it could be hypothesized that not simply incorporation but net accumulation of FIAU in DNA could be critical to the delayed toxicity of FIAU. Therefore, studies were conducted to measure FIAU accumulation in DNA of rats, monkeys, and dogs following long-term oral administration.In the past, measuring FIAU incorporation into DNA was done by using standard radiolabeling techniques and only in acute pulse-dose studies (9, 10). 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. MATERIALS AND METHODSAll DNA samples were obtained from animals used in studies conducted in the Toxicology Division of Eli Lilly and Company. In this manuscript, full descriptions of all toxicology studies have not been provided; complete reports will be published elsewhere. Only information necessary to the interpretation of FIAU-DNA incorporation has been provided. Since toxicological evaluation was the primary purpose of these studies, the doses for each species vary and are

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

“…19,42 Comparable decreases in hepatic mtDNA were observed in monkeys, rats, and dogs treated with FIAU for 30, 70, and 90 days, respectively, but toxicity similar to that seen in humans and woodchucks was not observed. 43 Incorporation of FIAU into cellular DNA of humans 42 and the woodchucks described in the current study may have been important in determining FIAU toxicity, but no toxicity was reported in dogs 9 with comparable levels of FIAU integrated into cellular DNA or in rats 9,47 with levels of integrated FIAU that were 10 times higher than the levels formed in humans and woodchucks. Based on these observations, neither integration of FIAU into cellular DNA 9 nor decreased abundance of mtDNA can be considered sufficient to explain the toxicity of FIAU.…”

“…[44][45][46] In studies of monkeys, rats, and dogs treated with FIAU, significant integration of FIAU was observed into cellular DNA in all three species. 9 In rats, the liver appeared to be a minor target for FIAU integration after 1 day, but after 77 days of FIAU treatment, the FIAU content of hepatic DNA was higher than the DNA of any other tissue. 47 Rats treated with 255 or 510 mg/kg of FIAU for 70 to 110 days had levels of FIAU incorporated into hepatic DNA that were 10-fold higher 9,47 than that observed in woodchucks in the present study.…”

“…9 In rats, the liver appeared to be a minor target for FIAU integration after 1 day, but after 77 days of FIAU treatment, the FIAU content of hepatic DNA was higher than the DNA of any other tissue. 47 Rats treated with 255 or 510 mg/kg of FIAU for 70 to 110 days had levels of FIAU incorporated into hepatic DNA that were 10-fold higher 9,47 than that observed in woodchucks in the present study. Dogs treated for 90 days with doses comparable with those used in woodchucks had levels of FIAU integrated into hepatic DNA that were similar to the woodchucks of this study.…”

“…Dogs treated for 90 days with doses comparable with those used in woodchucks had levels of FIAU integrated into hepatic DNA that were similar to the woodchucks of this study. 9 FIAU toxicity in humans and woodchucks was associated with marked decreases in hepatic mtDNA. 19,42 Comparable decreases in hepatic mtDNA were observed in monkeys, rats, and dogs treated with FIAU for 30, 70, and 90 days, respectively, but toxicity similar to that seen in humans and woodchucks was not observed.…”

“…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.…”