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Pharmacokinetics of Ceftazidime in Loggerhead Sea Turtles (Caretta Caretta) After Single Intravenous and Intramuscular Injections

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Pharmacokinetics of Ceftazidime in Loggerhead Sea Turtles (Caretta Caretta) After Single Intravenous and Intramuscular Injections Journal o/Zoo and WildlVe Medicine 30(1): 32-35, 1999 Copyright 1999 by American Association of Zoo Veterinarians PHARMACOKINETICS OF CEFTAZIDIME IN LOGGERHEAD SEA TURTLES (CARETTA CARETTA) AFTER SINGLE INTRAVENOUS AND INTRAMUSCULAR INJECTIONS M. Andrew Stamper, D.V.M., Mark G. Papich, D.V.M., M.S., Greg A. Lewbart, M.S., V.M.D., Stuart B. May, B.A., Delta D. Plummer, and Michael K. Stoskopf, D.V.M., Ph.D. Abstract: The pharmacokinetics of ceftazidime in yearling loggerhead sea turtles (Caretta caretta) following single i.m and i.v. injections were studied. Eight juvenile 1.25 ± 0.18 kg turtles were divided into two groups. Four animals received 20 mg/kg of ceftazidime i.v. and four received the same dose i.m. Plasma ceftazidime concentrations were analyzed by reverse-phase high-performance liquid chromatography. The i.v. and i.m. administration half-lives were 20.59 ± 3.24 hr and 19.08 ± 0.77 hr, respectively. The volume of distribution was 0.42 ± 0.07 L/kg, and the systemic clearence was 0.217 ± 0.005 ml/minlkg. Ceftazidime was detected in all blood samples and its concentration exceeded the minimum inhibitory concentration for Pseudomonas for 60 hr after i.m. and i.v. injections. Key words: Caretta caretta, ceftazidime. loggerhead sea turtle. pharmacokinetics. INTRODUCTION and blood was collected for a complete blood count and serum chemistry analysis using an eosinophil Safe and effective antibacterial dosage regimens unopette (Unopette, Becton Dickinson, Rutherford, are poorly established for many reptiles. including New Jersey 07070, USA). The turtles were held sea turtles. Most are empirical and are based on individually in 190-L aquariums arranged in two extrapolations from other species. Differences in groups of four, with each group connected by a re­ antibiotic disposition between reptiles and mam­ circulating system consisting of a 118 horsepower mals and even among different species of reptiles pump (Little Giant TE5MD-HC pump®, Aquanet­ makes cross-species extrapolation risky. ics, San Diego, California 92110, USA) and a 60­ Ceftazidime, a third-generation cephalosporin, is W flow-through ultraviolet light sterilizer (Aqua­ active against such gram-negative bacteria as Vib­ netics). Two 300-W in-line heaters (Aquanetics) rio spp., Pseudomonas spp., and Aeromonas spp., kept the water temperature at approximately 24DC. which are often associated with morbidity and mor­ 7 The turtles were randomly divided into i.v. and tality of sea turtles. '6 We studied ceftazidime phar­ Lm. treatment groups of equal sizes. Each animal macokinetics following single i.v. and Lm. injec­ received a single dose of 20 mg/kg of ceftazidime tions in juvenile loggerhead sea turtles (Caretta (Tazidime®, Eli Lilly, Indianapolis, Indiana 46284, caretta) to establish effective clinical dosing re­ USA) using a 25-ga needle attached to a I-ml sy­ gimes. ringe. Intravenous doses were administered in the MATERIALS AND METHODS left cervical sinus, and Lm. doses were injected into the left deltoid muscle. Subjects and sample collection Blood collection sites alternated between right Eight yearling 1.25 :':: 0.18 kg juvenile logger­ and left cervical sinuses at 0 (predose sample), 0.5, head sea turtles were studied. Prior to the drug trial, 1.5, 3, 6, 12, 24, 48, 96, and 120 hr after injection. each animal was weighed and examined visually, Approximately 0.5 ml of blood was collected each time using a I-ml tuberculin syringe with a 26-ga needle. The syringe and needle interiors were From the Environmental Medicine Consortium, North rinsed before use with 0.1 ml of 1,000 IU/ml so­ Carolina State University, 4700 Hillsborough Street, Ra­ dium heparin solution (Elkins-Sinn, Cherry Hill, leigh, North Carolina 27606, USA (Stamper, Papich, Lew­ New Jersey 08003, USA) as an anticoagulant. bart, Stoskopf); the Department of Companion Animal Blood was placed into polyethylene microcentri­ and Special Species Medicine (Stamper, Lewbart, Stos­ fuge tubes (Fisher Scientific, Pittsburgh, Pennsyl­ kopf) and the Department of Anatomy, Physiological Sci­ vania 15219, USA), which were capped and im­ ences, and Radiology (Papich, Plummer), college of Vet­ mediately submerged in ice water. The blood was erinary Medicine 4700 Hillsborough Street, Raleigh, North Carolina 27606, USA; and North Carolina Aquar­ then centrifuged to harvest approximately 0.3 ml of ium, Pine Knoll Shores, P.O. Box 580 Atlantic Beach, plasma, which was placed in polyethylene micro­ North Carolina 28512, USA (May). Present address centrifuge tubes via micropipet. The tubes were (Stamper): New England Aquarium, Central Wharf, Bos­ capped and stored at -70°C until high-performance ton, Massachussetts 02210-3399, USA. liquid chromatography (HPLC) analysis. 32 STAMPER ET AL.-PHARMACOKINETICS OF CEFTAZlDIME IN SEA TURTLES 33 Equipment and reagents noise from blank samples was determined for each Ceftazidime in plasma was quantitated using animal and the limit of detection (LOD) and limit HPLC. The apparatus consisted of a pump (Waters of quantitation (LOQ) were established as 3 times Model 600 Solvent Delivery System, Millipore and 10 times the baseline noise, respectively. Corp., Milford, Massachusetts 01757, USA), auto­ sampler (Series 1050 Autosampler, Hewlett-Pack­ Sample preparation ard, Palo Alto, California 94304, USA), and a vari­ Plasma samples collected during the experiment able wavelength ultraviolet light detector (Series and samples prepared for quality control were pre­ 1050 VWD, Hewlett-Packard) set at a wavelength pared for HPLC injection by pipetting 300 flol of of 260 nm. Data were recorded on computer soft­ plasma into a clean glass tube. To this tube was ware (HPLC'D ChemStation, Hewlett-Packard). added 10 flol of a solution of 1.0 mg/ml cefuroxime, which served as the internal standard. To this mix­ HPLC conditions ture was added 300 flol of 0.8 M perchloric acid to Ceftazidime and the internal standard, cefuro­ precipitate plasma proteins. The tube was centri­ xime sodium, were eluted on a C-18 reverse-phase fuged at 2,000 rpm at 4°C for 10 min, and the clear column (Waters NovaPak C-18 4flo Radial Com­ supernatant was transferred to an autosampler in­ pression Cartridge Millipore Corp.) with an iso­ jection vial. Injection volume was 50 flol. cratic mobile phase consisting of 28% methanol: 72% 0.01 M acetate buffer (pH 4.2-4.5) at a flow Pharmacokinetic analysis rate of 1.5 mllmin. All solvents were filtered and Noncompartmental pharmacokinetic methods degassed prior to use. The mobile phase was peri­ were used to determine the following parameters odically sparged with a flow of helium (10 ml/min) during analysis. The retention times for ceftazidime from concentration vs. time curves for disposition and the internal standard were approximately 3.5­ of ceftazidime after i. v. and i.m. administration: 3.7 and 4.4-4.6 min, respectively. first order elimination rate constant (K,,) using pro­ portionately weighted least squares linear regres­ Preparation of stock solutions and sion of the 10g"'lJ plasma from y-axis intercept (Co), calibration standards terminal half-life (t,/2), area under the curve (AUC) Ceftazidime was obtained as ceftazidime penta­ from time zero to infinity area under the moment hydrate and the internal standard, cefuroxime, as curve (AUMC) from zero to infinity, and mean res­ cefuroxime sodium, both as USP reference stan­ idence time (MRT). From the i.v. administration, dards (U.S. Pharmacopoeia, Rockville, Maryland the following parameters were calculated: volume 20852, USA). A 1 mg/ml (calculated for the base) of distribution area method (Vd"",,), volume of dis­ stock solution was prepared for each compound in tribution at steady-state (Vd,), and total systemic distilled water. The ceftazidime stock solution was clearance (Cl).' Values for the elimination rate con­ further diluted with distilled water to prepare cali­ stant and intercept of the curves were calculated bration standards. Stock solutions were stable in with the use of a nonlinear curve-fitting program tightly capped glass vials for 48 hr in the refriger­ (Fig.P Software Corp., Durham, North Carolina ator. Blank plasma was fortified with ceftazidime 27717, USA). AUC was calculated using the trap­ and used for quality control samples and calibration ezoidal method. Percent systemic availability from standards. Calibration standards for the calibration the i.m. injection (%F) was calculated from the ra­ curve ranged from 0.5 to 100 flog/ml. Quantitation tio of AUC,mIAUC". The values for the detected was performed by calculating a ratio of the ceftaz­ maximum plasma concentration (C\lIX) and time of idime peak height (mAU) to internal standard peak maximum detected plasma concentration (~\1AX) height (mAU) and plotting on a linear graph against were the observed values. Statistical differences be­ the true concentration using least-squares linear re­ tween routes of administration were measured with gression. Fresh calibration standards were prepared Student's (-test for independent samples (SAS In­ for each day's analysis. The calibration curve was stitute, Cary, North Carolina 27513, USA). The linear with a r' value of at least 0.99, and all cali­ level for significance was a P-value :S 0.05. bration standards could be back-calculated to with­ Concentrations were compaired to minimum in­ in 15% of the true value. hibitory concentration (MlC) of a susceptible ref­ Blank samples (plasma collected prior to drug erence Pseudomonas aeruginosa (American Type administration) from each experimental
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