PHARMACOKINETICS OF CEFOVECIN (CONVENIA®) IN WHITE BAMBOO SHARKS (CHILOSCYLLIUM PLAGIOSUM) AND ATLANTIC HORSESHOE CRABS (LIMULUS POLYPHEMUS) Author(s): James C. Steeil, D.V.M., Juergen Schumacher, Dr. med. vet., Dipl. A.C.Z.M., Dipl. E.C.Z.M. (Herpetology), Robert H. George, D.V.M., Frank Bulman, B.S., Katherine Baine, D.V.M., and Sherry Cox, M.S., Ph.D. Source: Journal of Zoo and Wildlife Medicine, 45(2):389-392. 2014. Published By: American Association of Zoo Veterinarians DOI: http://dx.doi.org/10.1638/2013-0061R2.1 URL: http://www.bioone.org/doi/full/10.1638/2013-0061R2.1
BioOne (www.bioone.org) is a nonprofit, online aggregation of core research in the biological, ecological, and environmental sciences. BioOne provides a sustainable online platform for over 170 journals and books published by nonprofit societies, associations, museums, institutions, and presses. Your use of this PDF, the BioOne Web site, and all posted and associated content indicates your acceptance of BioOne’s Terms of Use, available at www.bioone.org/page/ terms_of_use. Usage of BioOne content is strictly limited to personal, educational, and non-commercial use. Commercial inquiries or rights and permissions requests should be directed to the individual publisher as copyright holder.
BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, research libraries, and research funders in the common goal of maximizing access to critical research.
Journal of Zoo and Wildlife Medicine 45(2): 389–392, 2014 Copyright 2014 by American Association of Zoo Veterinarians
PHARMACOKINETICS OF CEFOVECIN (CONVENIAt) IN WHITE BAMBOO SHARKS (CHILOSCYLLIUM PLAGIOSUM) AND ATLANTIC HORSESHOE CRABS (LIMULUS POLYPHEMUS) James C. Steeil, D.V.M., Juergen Schumacher, Dr. med. vet., Dipl. A.C.Z.M., Dipl. E.C.Z.M. (Herpetology), Robert H. George, D.V.M., Frank Bulman, B.S., Katherine Baine, D.V.M., and Sherry Cox, M.S., Ph.D.
Abstract: Cefovecin was administered to six healthy adult white bamboo sharks (Chiloscyllium plagiosum) and six healthy adult Atlantic horseshoe crabs (Limulus polyphemus) to determine its pharmacokinetics in these species. A single dose of cefovecin at 8 mg/kg was administered subcutaneously in the epaxial region of the bamboo sharks and in the proximal articulation of the lateral leg of the horseshoe crabs. Blood and hemolymph samples were collected at various time points from bamboo sharks and Atlantic horseshoe crabs. High performance liquid chromatography was performed to determine plasma levels of cefovecin. The terminal halflife of cefovecin in Atlantic horseshoe crabs was 37.70 6 9.04 hr and in white bamboo sharks was 2.02 6 4.62 hr. Cefovecin concentrations were detected for 4 days in white bamboo sharks and for 14 days in Atlantic horseshoe crabs. No adverse effects associated with cefovecin administration were seen in either species. Key words: Cefovecin, Chiloscyllium plagiosum, horseshoe crab, Limulus polyphemus, pharmacokinetics, white bamboo shark.
BRIEF COMMUNICATION Aquatic animals are routinely administered antimicrobial agents for the treatment of bacterial infections and are given orally, as baths, or as injections.3 Injectable antibiotics are the preferred choice for some aquatic species where individual care is desired. Disadvantages of injectable antibiotics may include inflammation at the administration site, patient stress due to handling, and increased morbidity associated with repeated restraint. The preferred injectable antibiotic for aquatic animal species should be safe, effective, and have a long dosing interval. Pharmacokinetic evaluation of antimicrobial drugs in aquatic species is essential for proper treatment of bacterial disease. The long-acting injectable cephalosporin, cefovecin, is an effective way of administering medications in a variety of domestic and nondomestic species.1,2,4,5 Long-acting antibiotics allow for a reliable release of therapeutic levels over a set From the Departments of Small Animal Clinical Sciences (Steeil, Schumacher, Baine) and Biomedical and Diagnostic Sciences (Cox), College of Veterinary Medicine, The University of Tennessee, Knoxville, Tennessee 37996, USA; and Ripley’s Aquarium of the Smokies (George, Bulman), Gatlinburg, Tennessee 37738, USA. Present address (Steeil): Smithsonian National Zoological Park, Department of Animal Health, 3001 Connecticut Avenue, Washington D.C. 20013-7012, USA. Correspondence should be directed to Dr. Steeil ([email protected]).
period of time, resulting in a reduction of stress associated with daily drug administration and a more-appropriate medication compliance by patients.10 Cefovecin (Conveniat) is a third-generation, long-acting injectable cephalosporin labeled for use in domestic dogs with pyoderma, caused by Staphylococcus intermedius and Streptococcus canis, and in domestic cats with wounds or abscesses caused by Pasteurella multocida.6 The pharmacokinetic profile of cefovecin is characterized by a long elimination half-life in dogs (5 days), and effective concentrations for specific pathogens can be maintained in tissue fluid for 14 days or longer.9 The present study determined protein binding and the pharmacokinetic data of a single, subcutaneous dose of cefovecin in white bamboo sharks (Chiloscyllium plagiosum) and Atlantic horseshoe crabs (Limulus polyphemus). Six adult white bamboo sharks (C. plagiosum) (weight 1.12–1.82 kg) and six adult Atlantic horseshoe crabs (L. polyphemus) (weight 1.14– 2.02 kg) were used in this study. Sharks were fed a combination of clams, squid, capelin, and herring every 2 days. The horseshoe crabs were fed a combination of clams, squid, and a variety of other fish every 3–4 days. Animals were determined to be healthy based on normal visual and physical examination findings. Horseshoe crabs were kept in an 1,890-L round tank and white bamboo sharks were housed in a 37,800-L round tank. Temperature, pH, and salinity were tested daily, ammonia and nitrite were tested weekly,
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dissolved oxygen saturation, dissolved oxygen, and alkalinity were tested bimonthly, and nitrates were tested monthly. Parameters were kept within the following ranges: Temperature (22.0–25.08C), pH (7.88–8.14), salinity (30–35 ppt), ammonia (0.00–0.054 mg/L), nitrite (0.006–0.078 ppm), nitrate (72–300 ppm), dissolved oxygen saturation (95.9–97.1%), dissolved oxygen (6.36–7.88 ppm), and alkalinity (134–220 ppm). Protein binding studies were performed by use of a micropartition device. Four replicates of identical concentration were prepared by spiking pooled horseshoe crab hemolymph and white bamboo shark plasma with a stock solution of cefovecin prepared from the pure reference standard. The replicates were incubated in a water bath (378C and 248C for 30 min). Five-hundred microliters from each replicate were placed into the reservoir of each of four micropartition devices. The devices were centrifuged at 1,500 g for 30 min, and approximately 200 ll of ultrafiltrate were collected in a filtrate cup. Solid phase extraction of 100 ll of the ultrafiltrate was performed prior to analysis by use of high performance liquid chromatography (HPLC). The resulting concentrations represented the unbound fraction. A second set of four replicates of the same concentrations was analyzed (100 ll/ replicate), except the micropartition step was omitted. These resulting concentrations represented the total (bound and unbound) fraction. The standard curve used to determine protein concentrations was prepared by use of 100 ll of spiked pooled horseshoe crab hemolymph and bamboo shark plasma. Percentage of the bound fraction was calculated with the equation: Percentage of protein binding ¼
ðtotal concentration unbound concentrationÞ total concentration 3 100:
To determine sampling intervals following a single dose of subcutaneous cefovecin (8 mg/kg), a pilot study was performed using two white bamboo sharks and two Atlantic horseshoe crabs. Cefovecin was administered subcutaneously in the epaxial region of the bamboo sharks and in the proximal articulation of the lateral leg of the horseshoe crabs. Based on results of the pilot study, cefovecin was administered to an additional four horseshoe crabs and four white bamboo sharks. Blood samples were collected at 0, 15, 30, and 60 min and at 1, 2, 3, 4, 5, 6, and 7 days from white bamboo sharks. Hemolymph samples
were collected at 0, 15, 30, and 60 min and at 1, 6, 9, 12, 14, and 18 days from horseshoe crabs. Blood was collected from the ventral tail hemoarch of the white bamboo sharks and hemolymph from the cardiac sinus of the horseshoe crabs.7 Blood and hemolymph samples were placed in lithium heparin tubes (BD Vacutainert, Becton Dickinson, Franklin Lakes, New Jersey 07417 USA), centrifuged for 5 min, and the plasma–hemolymph was collected and stored in microtainer tubes at 708C.1 Analysis of cefovecin in plasma and hemolymph samples was conducted by reversed phase HPLC. The system consisted of a 2695 separations module and a 2487 ultraviolet detector (Waters, Milford, Massachusetts 01757, USA). Separation was attained on a Waters XBridge C8 4.6 3 250 mm (3.5 lm) with a 3.5 lm XBridge guard column. The mobile phase was a mixture of (A) 10 mM ammonium acetate pH 3.5 and (B) acetonitrile. The mixture was pumped at a starting gradient of 89% A and 11% B, and was adjusted to 77% A and 23% B over 22 min and then back to initial conditions over 3 min. The drug was quantified using UV detection at 280 nm with a flow rate of 0.85 ml/min. Cefovecin was extracted from plasma and hemolymph samples using a solid phase extraction method. Samples were thawed and vortexed, 100 ll were transferred to a test tube, and 25 ll of internal standard (1 lg/ml ceftiofur) were added. Four-hundred microliters of methanol : water (15 : 85) and 25 ll of 10% phosphoric acid was added; tubes were vortexed and allowed to stand for 5 min and then were centrifuged at 1,000 g for 10 min. The supernatant was passed through a prewet Oasis HLB extraction column (Waters, Milford, Massachusetts 01757, USA). Samples were eluted with 3% ammonium hydroxide in methanol which was evaporated to dryness under a steady stream of nitrogen gas. Samples were reconstituted in 225 ll of mobile phase and 100 ll were injected into the HPLC system. Standard curves for plasma–hemolymph analysis were prepared by spiking untreated plasma– hemolymph with cefovecin, which produced a linear concentration range of 0.5–200 lg/ml. Spiked standards were treated exactly as plasma– hemolymph samples. Average recovery was 96% for cefovecin, and the lower limit of quantification was 0.1 lg/ml. For cefovecin, intra-assay variability ranged from 4.7% to 5.8% and inter-assay variability ranged from 6.0% to 8.9%, respectively.
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STEEIL ET AL.—CEFOVECIN PHARMACOKINETICS IN AQUATIC ANIMALS
Table 1. Pharmacokinetic parameters of a single subcutaneous injection of cefovecin in Atlantic horseshoe crabs (Limulus polyphemus) and white bamboo sharks (Chiloscyllium plagiosum). Pharmacokinetic parametera
Atlantic horseshoe crab
Terminal half-lifeb (hr) Elimination rate constant, kz (1/hr) Tmax (hr) Cmax (lg/ml)
37.70 0.02 0.62 26.01
6 6 6 6
9.04 0.005 0.31 3.84
White bamboo shark
2.02 0.34 0.37 52.08
6 6 6 6
4.62 0.43 0.14 16.03
Harmonic mean; not applicable Elimination rate constant (kz); terminal half-life (t½); maximum plasma concentration (Cmax); time to maximum plasma concentration (Tmax); area under the plasma concentration time curve (AUC0-‘) from time 0 to infinity; mean residence time (MRT). b a
Protein binding for the horseshoe crabs was 5% at 378C and 4% at 248C and for the white bamboo sharks was 33% at 378C and 1% at 248C. Estimated values for selected pharmacokinetic parameters of cefovecin in Atlantic horseshoe crabs and white bamboo sharks are presented in Table 1. The terminal half-life of cefovecin in Atlantic horseshoe crabs was 37.70 6 9.04 hr and in white bamboo sharks was 2.02 6 4.62 hr. The hemolymph and plasma profiles of cefovecin in Atlantic horseshoe crabs and white bamboo sharks are presented in Figures 1 and 2, respectively. Hemolymph and plasma levels were detected until day 14 in Atlantic horseshoe crabs and until day 4 in white bamboo sharks. No morbidity or mortality associated with cefovecin administration was seen in horseshoe crabs or bamboo sharks during the study period. Pharmacokinetic data of cefovecin can be highly variable in nondomestic species.2,4,10 It has been shown in domestic animals that cefovecin is highly protein bound.10 Results of protein binding assays performed in the Atlantic horseshoe crab and the white bamboo shark indicated that these
species were potentially not good candidates for cefovecin administration. Differences in body temperature may explain why the protein binding was variable and low when compared to mammalian species. Protein binding percentages decreased in both species at the lower water temperature; however, water temperature may not accurately represent actual body temperature. The half-life of cefovecin in the white bamboo sharks was 2.02 6 4.62 hr, indicating that most of the drug would be eliminated in this species within the first 24 hr. Bacterial pathogens in domestic animals, such as Escherichia coli or Staphylococcus aureus, normally have a mean inhibitory concentration of 1 lg/ml. With the low protein binding (33%) and the short half-life (2 hr), effective plasma concentrations of cefovecin in the white bamboo shark for known minimum inhibitory concentration values in terrestrial mammals would not be present past 24 hr. Results of this study showed that hemolymph and plasma levels of cefovecin were present until 340 hr in horseshoe crabs and 75 hr in white bamboo sharks, respectively. Further studies are
Figure 1. Mean 6 significant figures (SF) hemolymph concentrations of cefovecin in Atlantic horseshoe crabs (Limulus polyphemus) after administration of cefovecin at a dose of 8 mg/kg in the open hemolymph space of the leg.
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Figure 2. Mean 6 SF plasma concentrations of cefovecin in white bamboo sharks (Chiloscyllium plagiosum) after subcutaneous administration of cefovecin at a dose of 8 mg/kg.
indicated to determine the pharmacokinetics of cefovecin in teleost fish, which may have higher metabolic rates than do Atlantic horseshoe crabs and white bamboo sharks. Acknowledgments: The authors thank Ms. Amanda Gudgel and the aquarists at Ripley’s Aquarium of the Smokies for their technical assistance and Pfizer Animal Health for providing the Convenia (cefovecin) used in this study. This study was funded by the Faculty Education Advancement and Research Fund, College of Veterinary Medicine, The University of Tennessee.
LITERATURE CITED 1. Bertelsen MF, Thuesen LR, Bakker J, Hebel C, Grøndahl C, Brimer L, Skaanild MT. Limitations and usages of cefovecin in zoological practice. In: Proc Int Conf Dis Zoo Wild Anim; 2010. p. 140–141. 2. Garcı´a-Pa´rraga D, Gilbert J, Valls M, Ros-Rodrı´´ lvaro T, Rojo-Solı´s C, Encinas T. New guez J, A findings in the pharmacokinetics of cefovecin (Convenia) in marine mammals, In: Proc AAZV 43rd An Conf; 2011. p. 32–33. 3. Noga EJ. Treatment guidelines. In: Noga EJ (ed.). Fish disease diagnosis and treatment. 1st ed. St. Louis (MO): Mosby Inc.; 1996. p. 253–271. 4. Papp R, Popovic A, Kelly N, Tschirret-Guth R. Pharmacokinetics of cefovecin in squirrel monkeys
(Saimiri sciureus), rhesus macaques (Macaca mulatta), and cynomolgus monkeys (Macaca fascicularis). J Am Assoc Lab Anim. Sc. 2010;49:805–808. 5. Schrader GM, Whiteside DP, Slater OM, Black SR. Conservative management of pyothorax in an Amur tiger (Panthera tigris altaica). J Zoo Wildl Med. 2012;43:425–429. 6. Six R, Cleaver DM, Lindeman CJ, Cherni J, Chesebrough R, Papp G, Skogerboe TL, Weigel DJ, Boucher JF, Stegemann MR. Effectiveness and safety of cefovecin sodium, an extended spectrum injectable cephalosporin, in the treatment of cats with abscesses and infected wounds. J Am Vet Med Assoc. 2009;234: 81–87. 7. Smith SA. Horseshoe crabs. In: Lewbart GA (ed). Invertebrate medicine. 2nd ed. Hoboken (NJ): John Wiley & Sons Inc.; 2012. p. 173–185. 8. Stegemann MR, Passmore CA, Sherington J, Lindeman CJ, Papp G, Weigel DJ, Skogerboe TL. Antimicrobial activity and spectrum of cefovecin, a new extended-spectrum cephalosporin, against pathogens collected from dogs and cats in Europe and North America. Anti Microb. Agents. 2006;40:2286–2292. 9. Stegemann M, Sherington RJ, Blanchflower S. Pharmacokinetics and pharmacodynamics of cefovecin in dogs. J Vet Pharmacol Ther. 2006;29:501–511. 10. Thuesen LR, Bertelsen MF, Brimer L, Skaanild MT. Selected pharmacokinetic parameters for cefovecin in hens and green iguanas. J Vet. Pharmacol Ther. 2009;32:613–617. Received for publication 2 April 2013
BioOne (www.bioone.org) is a nonprofit, online aggregation of core research in the biological, ecological, and environmental sciences. BioOne provides a sustainable online platform for over 170 journals and books published by nonprofit societies, associations, museums, institutions, and presses. Your use of this PDF, the BioOne Web site, and all posted and associated content indicates your acceptance of BioOne’s Terms of Use, available at www.bioone.org/page/ terms_of_use. Usage of BioOne content is strictly limited to personal, educational, and non-commercial use. Commercial inquiries or rights and permissions requests should be directed to the individual publisher as copyright holder.
BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, research libraries, and research funders in the common goal of maximizing access to critical research.
Journal of Zoo and Wildlife Medicine 45(2): 389–392, 2014 Copyright 2014 by American Association of Zoo Veterinarians
PHARMACOKINETICS OF CEFOVECIN (CONVENIAt) IN WHITE BAMBOO SHARKS (CHILOSCYLLIUM PLAGIOSUM) AND ATLANTIC HORSESHOE CRABS (LIMULUS POLYPHEMUS) James C. Steeil, D.V.M., Juergen Schumacher, Dr. med. vet., Dipl. A.C.Z.M., Dipl. E.C.Z.M. (Herpetology), Robert H. George, D.V.M., Frank Bulman, B.S., Katherine Baine, D.V.M., and Sherry Cox, M.S., Ph.D.
Abstract: Cefovecin was administered to six healthy adult white bamboo sharks (Chiloscyllium plagiosum) and six healthy adult Atlantic horseshoe crabs (Limulus polyphemus) to determine its pharmacokinetics in these species. A single dose of cefovecin at 8 mg/kg was administered subcutaneously in the epaxial region of the bamboo sharks and in the proximal articulation of the lateral leg of the horseshoe crabs. Blood and hemolymph samples were collected at various time points from bamboo sharks and Atlantic horseshoe crabs. High performance liquid chromatography was performed to determine plasma levels of cefovecin. The terminal halflife of cefovecin in Atlantic horseshoe crabs was 37.70 6 9.04 hr and in white bamboo sharks was 2.02 6 4.62 hr. Cefovecin concentrations were detected for 4 days in white bamboo sharks and for 14 days in Atlantic horseshoe crabs. No adverse effects associated with cefovecin administration were seen in either species. Key words: Cefovecin, Chiloscyllium plagiosum, horseshoe crab, Limulus polyphemus, pharmacokinetics, white bamboo shark.
BRIEF COMMUNICATION Aquatic animals are routinely administered antimicrobial agents for the treatment of bacterial infections and are given orally, as baths, or as injections.3 Injectable antibiotics are the preferred choice for some aquatic species where individual care is desired. Disadvantages of injectable antibiotics may include inflammation at the administration site, patient stress due to handling, and increased morbidity associated with repeated restraint. The preferred injectable antibiotic for aquatic animal species should be safe, effective, and have a long dosing interval. Pharmacokinetic evaluation of antimicrobial drugs in aquatic species is essential for proper treatment of bacterial disease. The long-acting injectable cephalosporin, cefovecin, is an effective way of administering medications in a variety of domestic and nondomestic species.1,2,4,5 Long-acting antibiotics allow for a reliable release of therapeutic levels over a set From the Departments of Small Animal Clinical Sciences (Steeil, Schumacher, Baine) and Biomedical and Diagnostic Sciences (Cox), College of Veterinary Medicine, The University of Tennessee, Knoxville, Tennessee 37996, USA; and Ripley’s Aquarium of the Smokies (George, Bulman), Gatlinburg, Tennessee 37738, USA. Present address (Steeil): Smithsonian National Zoological Park, Department of Animal Health, 3001 Connecticut Avenue, Washington D.C. 20013-7012, USA. Correspondence should be directed to Dr. Steeil ([email protected]).
period of time, resulting in a reduction of stress associated with daily drug administration and a more-appropriate medication compliance by patients.10 Cefovecin (Conveniat) is a third-generation, long-acting injectable cephalosporin labeled for use in domestic dogs with pyoderma, caused by Staphylococcus intermedius and Streptococcus canis, and in domestic cats with wounds or abscesses caused by Pasteurella multocida.6 The pharmacokinetic profile of cefovecin is characterized by a long elimination half-life in dogs (5 days), and effective concentrations for specific pathogens can be maintained in tissue fluid for 14 days or longer.9 The present study determined protein binding and the pharmacokinetic data of a single, subcutaneous dose of cefovecin in white bamboo sharks (Chiloscyllium plagiosum) and Atlantic horseshoe crabs (Limulus polyphemus). Six adult white bamboo sharks (C. plagiosum) (weight 1.12–1.82 kg) and six adult Atlantic horseshoe crabs (L. polyphemus) (weight 1.14– 2.02 kg) were used in this study. Sharks were fed a combination of clams, squid, capelin, and herring every 2 days. The horseshoe crabs were fed a combination of clams, squid, and a variety of other fish every 3–4 days. Animals were determined to be healthy based on normal visual and physical examination findings. Horseshoe crabs were kept in an 1,890-L round tank and white bamboo sharks were housed in a 37,800-L round tank. Temperature, pH, and salinity were tested daily, ammonia and nitrite were tested weekly,
389
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JOURNAL OF ZOO AND WILDLIFE MEDICINE
dissolved oxygen saturation, dissolved oxygen, and alkalinity were tested bimonthly, and nitrates were tested monthly. Parameters were kept within the following ranges: Temperature (22.0–25.08C), pH (7.88–8.14), salinity (30–35 ppt), ammonia (0.00–0.054 mg/L), nitrite (0.006–0.078 ppm), nitrate (72–300 ppm), dissolved oxygen saturation (95.9–97.1%), dissolved oxygen (6.36–7.88 ppm), and alkalinity (134–220 ppm). Protein binding studies were performed by use of a micropartition device. Four replicates of identical concentration were prepared by spiking pooled horseshoe crab hemolymph and white bamboo shark plasma with a stock solution of cefovecin prepared from the pure reference standard. The replicates were incubated in a water bath (378C and 248C for 30 min). Five-hundred microliters from each replicate were placed into the reservoir of each of four micropartition devices. The devices were centrifuged at 1,500 g for 30 min, and approximately 200 ll of ultrafiltrate were collected in a filtrate cup. Solid phase extraction of 100 ll of the ultrafiltrate was performed prior to analysis by use of high performance liquid chromatography (HPLC). The resulting concentrations represented the unbound fraction. A second set of four replicates of the same concentrations was analyzed (100 ll/ replicate), except the micropartition step was omitted. These resulting concentrations represented the total (bound and unbound) fraction. The standard curve used to determine protein concentrations was prepared by use of 100 ll of spiked pooled horseshoe crab hemolymph and bamboo shark plasma. Percentage of the bound fraction was calculated with the equation: Percentage of protein binding ¼
ðtotal concentration unbound concentrationÞ total concentration 3 100:
To determine sampling intervals following a single dose of subcutaneous cefovecin (8 mg/kg), a pilot study was performed using two white bamboo sharks and two Atlantic horseshoe crabs. Cefovecin was administered subcutaneously in the epaxial region of the bamboo sharks and in the proximal articulation of the lateral leg of the horseshoe crabs. Based on results of the pilot study, cefovecin was administered to an additional four horseshoe crabs and four white bamboo sharks. Blood samples were collected at 0, 15, 30, and 60 min and at 1, 2, 3, 4, 5, 6, and 7 days from white bamboo sharks. Hemolymph samples
were collected at 0, 15, 30, and 60 min and at 1, 6, 9, 12, 14, and 18 days from horseshoe crabs. Blood was collected from the ventral tail hemoarch of the white bamboo sharks and hemolymph from the cardiac sinus of the horseshoe crabs.7 Blood and hemolymph samples were placed in lithium heparin tubes (BD Vacutainert, Becton Dickinson, Franklin Lakes, New Jersey 07417 USA), centrifuged for 5 min, and the plasma–hemolymph was collected and stored in microtainer tubes at 708C.1 Analysis of cefovecin in plasma and hemolymph samples was conducted by reversed phase HPLC. The system consisted of a 2695 separations module and a 2487 ultraviolet detector (Waters, Milford, Massachusetts 01757, USA). Separation was attained on a Waters XBridge C8 4.6 3 250 mm (3.5 lm) with a 3.5 lm XBridge guard column. The mobile phase was a mixture of (A) 10 mM ammonium acetate pH 3.5 and (B) acetonitrile. The mixture was pumped at a starting gradient of 89% A and 11% B, and was adjusted to 77% A and 23% B over 22 min and then back to initial conditions over 3 min. The drug was quantified using UV detection at 280 nm with a flow rate of 0.85 ml/min. Cefovecin was extracted from plasma and hemolymph samples using a solid phase extraction method. Samples were thawed and vortexed, 100 ll were transferred to a test tube, and 25 ll of internal standard (1 lg/ml ceftiofur) were added. Four-hundred microliters of methanol : water (15 : 85) and 25 ll of 10% phosphoric acid was added; tubes were vortexed and allowed to stand for 5 min and then were centrifuged at 1,000 g for 10 min. The supernatant was passed through a prewet Oasis HLB extraction column (Waters, Milford, Massachusetts 01757, USA). Samples were eluted with 3% ammonium hydroxide in methanol which was evaporated to dryness under a steady stream of nitrogen gas. Samples were reconstituted in 225 ll of mobile phase and 100 ll were injected into the HPLC system. Standard curves for plasma–hemolymph analysis were prepared by spiking untreated plasma– hemolymph with cefovecin, which produced a linear concentration range of 0.5–200 lg/ml. Spiked standards were treated exactly as plasma– hemolymph samples. Average recovery was 96% for cefovecin, and the lower limit of quantification was 0.1 lg/ml. For cefovecin, intra-assay variability ranged from 4.7% to 5.8% and inter-assay variability ranged from 6.0% to 8.9%, respectively.
391
STEEIL ET AL.—CEFOVECIN PHARMACOKINETICS IN AQUATIC ANIMALS
Table 1. Pharmacokinetic parameters of a single subcutaneous injection of cefovecin in Atlantic horseshoe crabs (Limulus polyphemus) and white bamboo sharks (Chiloscyllium plagiosum). Pharmacokinetic parametera
Atlantic horseshoe crab
Terminal half-lifeb (hr) Elimination rate constant, kz (1/hr) Tmax (hr) Cmax (lg/ml)
37.70 0.02 0.62 26.01
6 6 6 6
9.04 0.005 0.31 3.84
White bamboo shark
2.02 0.34 0.37 52.08
6 6 6 6
4.62 0.43 0.14 16.03
Harmonic mean; not applicable Elimination rate constant (kz); terminal half-life (t½); maximum plasma concentration (Cmax); time to maximum plasma concentration (Tmax); area under the plasma concentration time curve (AUC0-‘) from time 0 to infinity; mean residence time (MRT). b a
Protein binding for the horseshoe crabs was 5% at 378C and 4% at 248C and for the white bamboo sharks was 33% at 378C and 1% at 248C. Estimated values for selected pharmacokinetic parameters of cefovecin in Atlantic horseshoe crabs and white bamboo sharks are presented in Table 1. The terminal half-life of cefovecin in Atlantic horseshoe crabs was 37.70 6 9.04 hr and in white bamboo sharks was 2.02 6 4.62 hr. The hemolymph and plasma profiles of cefovecin in Atlantic horseshoe crabs and white bamboo sharks are presented in Figures 1 and 2, respectively. Hemolymph and plasma levels were detected until day 14 in Atlantic horseshoe crabs and until day 4 in white bamboo sharks. No morbidity or mortality associated with cefovecin administration was seen in horseshoe crabs or bamboo sharks during the study period. Pharmacokinetic data of cefovecin can be highly variable in nondomestic species.2,4,10 It has been shown in domestic animals that cefovecin is highly protein bound.10 Results of protein binding assays performed in the Atlantic horseshoe crab and the white bamboo shark indicated that these
species were potentially not good candidates for cefovecin administration. Differences in body temperature may explain why the protein binding was variable and low when compared to mammalian species. Protein binding percentages decreased in both species at the lower water temperature; however, water temperature may not accurately represent actual body temperature. The half-life of cefovecin in the white bamboo sharks was 2.02 6 4.62 hr, indicating that most of the drug would be eliminated in this species within the first 24 hr. Bacterial pathogens in domestic animals, such as Escherichia coli or Staphylococcus aureus, normally have a mean inhibitory concentration of 1 lg/ml. With the low protein binding (33%) and the short half-life (2 hr), effective plasma concentrations of cefovecin in the white bamboo shark for known minimum inhibitory concentration values in terrestrial mammals would not be present past 24 hr. Results of this study showed that hemolymph and plasma levels of cefovecin were present until 340 hr in horseshoe crabs and 75 hr in white bamboo sharks, respectively. Further studies are
Figure 1. Mean 6 significant figures (SF) hemolymph concentrations of cefovecin in Atlantic horseshoe crabs (Limulus polyphemus) after administration of cefovecin at a dose of 8 mg/kg in the open hemolymph space of the leg.
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JOURNAL OF ZOO AND WILDLIFE MEDICINE
Figure 2. Mean 6 SF plasma concentrations of cefovecin in white bamboo sharks (Chiloscyllium plagiosum) after subcutaneous administration of cefovecin at a dose of 8 mg/kg.
indicated to determine the pharmacokinetics of cefovecin in teleost fish, which may have higher metabolic rates than do Atlantic horseshoe crabs and white bamboo sharks. Acknowledgments: The authors thank Ms. Amanda Gudgel and the aquarists at Ripley’s Aquarium of the Smokies for their technical assistance and Pfizer Animal Health for providing the Convenia (cefovecin) used in this study. This study was funded by the Faculty Education Advancement and Research Fund, College of Veterinary Medicine, The University of Tennessee.
LITERATURE CITED 1. Bertelsen MF, Thuesen LR, Bakker J, Hebel C, Grøndahl C, Brimer L, Skaanild MT. Limitations and usages of cefovecin in zoological practice. In: Proc Int Conf Dis Zoo Wild Anim; 2010. p. 140–141. 2. Garcı´a-Pa´rraga D, Gilbert J, Valls M, Ros-Rodrı´´ lvaro T, Rojo-Solı´s C, Encinas T. New guez J, A findings in the pharmacokinetics of cefovecin (Convenia) in marine mammals, In: Proc AAZV 43rd An Conf; 2011. p. 32–33. 3. Noga EJ. Treatment guidelines. In: Noga EJ (ed.). Fish disease diagnosis and treatment. 1st ed. St. Louis (MO): Mosby Inc.; 1996. p. 253–271. 4. Papp R, Popovic A, Kelly N, Tschirret-Guth R. Pharmacokinetics of cefovecin in squirrel monkeys
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