Pharmacokinetics of a single oral dose of acyclovir and valacyclovir in North American box turtles (Terrapene sp.).

Eastern box turtle (Terrapene carolina) populations have significantly declined throughout their range (Currylow et al., 2011). While a combination of factors are likely playing a role in the decline of the box turtle, disease outbreaks have been emerging across the Eastern United States in chelonians and may be important (De Voe et al., 2004; Johnson et al., 2008; Allender et al., 2011). Herpesviruses and iridoviruses are two common virus families that lead to clinical signs of upper respiratory tract disease in affected chelonians (Harper et al., 1982; Brown et al., 1999; Johnson et al., 2008; Allender et al., 2011). Acyclovir has been used anecdotally in the treatment of both viral agents in chelonians (Marschang et al., 1997; De Voe et al., 2004; Funk & Diethelm, 2006); however, to date, only a single pharmacokinetic study has been performed in a single species (Gaio et al., 2007). Acyclovir and its pro-drug valacyclovir are guanine analogue antiviral drugs (Elion, 1993). They are activated by phosphorylation of a virus-specific thymidine kinase (TK). Acyclovir uptake has been shown to be enhanced in herpesvirus-infected cells, with a 10to 30-fold greater affinity for infected cells than uninfected cells (Beutner, 1995). Once incorporated into the viral genome, relatively low levels of the drug are needed to achieve viral inhibition, and adequate intracellular concentrations can be maintained for several hours (Beutner, 1995). Thymidine kinase genes or functional TK enzymes have similarly been identified in iridoviruses (Scholz et al., 1988; Jakob et al., 2001; Coupar et al., 2005; Tsai et al., 2005). This study evaluated the pharmacokinetics of acyclovir and valacyclovir after a single oral dose in twelve North American box turtles. Animals were housed individually in Vision cages (Model #332; Vision Products Plus Inc., Canoga Park, CA, USA) that maintained a thermal gradient of 72 F to 87 F. A varied diet of fruits and vegetables was provided every other day. All activities were approved by the University of Illinois IACUC (protocol 11003). A pilot study using oral acyclovir was initially performed at doses of 40 and 80 mg ⁄kg in one animal each. After the results demonstrated low maximum observed plasma concentration (Cmax) at the doses used, a decision was made to evaluate the pharmacokinetics of oral valacyclovir at 20 and 40 mg ⁄kg in one animal each. Based on these results, eight animals were administered a single oral dose of 40 mg ⁄kg valacyclovir. Venipuncture was performed via the subcarapacial sinus for all phases of the study. For the final study, blood (up to 0.3 mL) was collected in a 3-mL syringe with a 22-ga needle at 0, 0.25, 0.5, 1, 2, 4, 8, 12, 24, 48, 72, 96, and 120 h following oral valacyclovir. Samples were placed in a lithium heparin microtainer (Becton Dickinson, Franklin Lakes, NJ, USA), centrifuged immediately, the plasma placed in a separate cryovial, and stored at )20 C. Samples were transported on dry ice to the Pharmacology Laboratory at the University of Tennessee for analysis. Plasma samples were analyzed using a reverse-phase highperformance liquid chromatography method (HPLC). The system consisted of a 2695 separations module, a 2475 fluorescence detector, and a computer equipped with Empower software (Waters, Milford, MA, USA). Valacyclovir and acyclovir were extracted from plasma samples using 1 cc HLB solid-phase extraction (SPE) cartridges. The compounds were separated on an Atlantis T3 (4.6 · 100 mm, 5 lm) column with a guard column. The mobile phase was a mixture of (A) 10 mM ammonium phosphate pH 2.9 and (B) acetonitrile (97:3). The flow rate was 1.2 mL ⁄min, and the column temperature was ambient. Fluorescence was measured at an excitation of 260 nm and an emission of 375 nm with a gain of ·100. Standard curves for plasma analysis were prepared by fortifying untreated, pooled turtle plasma with valacyclovir and acyclovir to produce a linear concentration range of 10–1500 ng ⁄mL. Average recovery for both drugs was 87%, while intraand inter-assay variabilities were <10%. The lower limit of quantification was 10 ng ⁄mL. J. vet. Pharmacol. Therap. 36, 205–208. doi: 10.1111/j.1365-2885.2012.01418.x. SHORT COMMUNICATION