Pharmacokinetics of cefovecin sodium after subcutaneous administration to Hermann’s tortoises (Testudo hermanni) Giordano Nardini, DMV, PhD; Andrea Barbarossa, DMV, PhD; Andrea Dall’Occo, DMV; Nicola Di Girolamo, DMV; Petra Cagnardi, DMV, PhD; William Magnone, DMV; Mattia Bielli, DMV; Paola Roncada, MS; Anna Zaghini, MS
Objective—To determine the pharmacokinetics of cefovecin sodium after SC administration to Hermann’s tortoises (Testudo hermanni). Animals—23 healthy adult Hermann’s tortoises (15 males and 8 females). Procedures—Cefovecin (8.0 mg/kg) was injected once in the subcutis of the neck region of Hermann’s tortoises, and blood samples were obtained at predetermined time points. Plasma cefovecin concentrations were measured via ultraperformance liquid chromatography coupled to tandem mass spectrometry, and pharmacokinetic parameters were calculated with a noncompartmental model. Plasma protein concentration was quantified, and the percentage of cefovecin bound to protein was estimated with a centrifugation technique. Results—Cefovecin was absorbed rapidly, reaching maximum plasma concentrations between 35 minutes and 2 hours after administration, with the exception of 1 group, in which it was reached after 4 hours. The mean ± SD time to maximum concentration was 1.22 ± 1.14 hours; area under the concentration-time curve was 220.35 ± 36.18 h•µg/mL The mean protein-bound fraction of cefovecin ranged from 41.3% to 47.5%. No adverse effects were observed. Conclusions and Clinical Relevance—Administration of a single dose of cefovecin SC appeared to be well-tolerated in this population of tortoises. Results of pharmacokinetic analysis indicated that the 2-week dosing interval suggested for dogs and cats cannot be considered effective in tortoises; however, further research is needed to determine therapeutic concentrations of the drug and appropriate dose ranges. (Am J Vet Res 2014;75:918–923)
H
ermann’s tortoises (Testudo hermanni) are frequently kept as pets. The small size (carapace rarely > 210 mm in length in wild populations),1 herbivorous habits, and resilience make these tortoises an exceptional garden inhabitant. Although Hermann’s tortoises are listed by the International Union for Conservation of Nature2 as a near-threatened species, the number of such tortoises in the wild continues to decrease. Antimicrobial treatment is often necessary in chelonian medicine,3 but only a few studies have been perReceived January 30, 2014. Accepted June 6, 2014. From the Clinica Veterinaria Modena Sud, Piazza dei Tintori 1, 41057 Spilamberto, MO, Italy (Nardini); the Department of Veterinary Medical Sciences, University of Bologna, 40064 Ozzano dell’Emilia, BO, Italy (Barbarossa, Dall’Occo, Roncada, Zaghini); the Clinica per Animali Esotici, Centro Veterinario Specialistico, Via Sandro Giovannini 53, 00137 Roma, Italy (Di Girolamo); the Università degli Studi di Milano, Dip. Scienze Veterinarie per la Salute, la Produzione Animale e la Sicurezza Alimentare, 20133 Milano, Italy (Cagnardi); the Parco Natura Viva, Garda Zoological Park, Loc. Figara 40, 37012 Bussolengo, VR, Italy (Magnone); and the Ambulatorio Veterinario, Viale Buonarroti 20, 28100 Novara, Italy (Bielli). The authors declare no financial conflicts of interest. The authors thank Dr. Paola Boretto for providing the pure cefovecin standard. Address correspondence to Dr. Barbarossa ([email protected]). 918
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AUC0–last AUMC0–last Cmax MIC Tmax
ABBREVIATIONS
Area under the concentrationtime curve from time 0 to last measurable drug concentration Area under the first moment curve from time 0 to last measurable drug concentration Maximum (peak) drug concentration Minimum inhibitory concentration Time to reach maximum (peak) drug concentration
formed to investigate the pharmacokinetics and pharmacodynamics of antimicrobials in these animals. Most studies have been performed with marine turtles,4–12 with few focusing on freshwater13,14 and terrestrial chelonians.15–17 Consequently, to establish antimicrobial treatment protocols, veterinary practitioners often have to rely on anecdotal reports18 for information instead of proper pharmacokinetics studies. Cefovecin sodium is a semisynthetic third-generation cephalosporin registered for veterinary use in dogs and cats in the United States and in Europe. Cephalosporins act by interfering with bacterial cell wall synthesis and are often used in treatment of reptiles.19,20 In AJVR, Vol 75, No. 10, October 2014
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dogs and cats, cefovecin is a long-acting antimicrobial, and a single SC injection can provide up to 14 days of treatment.21,22 In dogs, cefovecin (8.0 mg/kg) had a mean elimination half-life of 5.5 days after SC administration, whereas that in cats (following administration with the same dose and route) was 6.9 days.21,22 Following SC or IV administration, cefovecin has been described in a noncompartmental model characterized by a rapid initial distribution phase, followed by a slower elimination phase.21,22 The use of a long-acting, broad-spectrum antimicrobial would be of particular interest for clinicians working with chelonians. The metabolism of reptiles differs from that of mammals in several aspects.23 In addition to variations in energy requirements,24 drug efficacy and metabolism may be substantially different from those in mammals.25,26 Therefore, it may be inappropriate to prescribe medical treatment for reptiles by extrapolating doses from research performed in other animal species. The purpose of the study reported here was to determine the pharmacokinetics of cefovecin in healthy Hermann’s tortoises and to assess whether the therapeutic protocol used in dogs and cats could be applicable to chelonians.
Tortoises’ clinical conditions were monitored throughout the experiment, including possible variations in the amount of food consumed and feces produced. Experimental procedures were approved by the Italian Ministry of Health, in accordance with European regulations.28
Materials and Methods
Injection and sample collection—The day before beginning the experiment, an IV catheter was aseptically placed in a jugular vein of each tortoise. On day 0, a commercially available cefovecin sodium formulationa was reconstituted with the associated solvent (to a concentration of 80 mg/mL) and delivered by a single 0.1 mL/kg (8.0 mg of cefovecin as the sodium salt/kg) injection with a 20-gauge needle in the subcutis of the pectoral region contralateral to the catheter site. Blood samples were collected into lithium heparin–containing tubes prior to administration (baseline), at 35 minutes, 1, 2, 4, 8, 12, and 24 hours during day 1 (the first day after treatment), and then every 24 hours on days 3, 4, 5, 6, 8, 10, 12, and 14. Catheters were flushed with 0.5 mL of a calcium heparin solution after sample collection. To create a pool of approximately 1 mL of blood for each group, 0.3 to 0.4 mL/tortoise/time point was obtained. Samples were centrifuged within 30 minutes after collection. Plasma was collected and stored in microtubes at –20°C until assayed.
Animals—Twenty-three healthy adult Hermann’s tortoises (15 males and 8 females; body weight, 0.735 to 2.300 kg) were included in the study. The tortoises were obtained from the Parco Natura Viva Garda Zoological Park, Bussolengo, Verona, Italy, and were identified by writing a unique number on each carapace with an aqueous-based marker. Health status was determined on the basis of clinical history, physical examination, and results of a CBC and serum biochemical analysis for each animal. The tortoises were divided in 8 same-sex groups (7 groups of 3 tortoises and 1 group of 2 tortoises). Each group was placed in a vivarium and acclimatized for 7 days prior to the start of the study. Each vivarium was equipped with a fluorescent UVBemitting lamp and an infrared lamp as heat source. The fluorescent bulb was oriented diagonally with the end of the longitudinal axis 21 cm above the floor of the vivarium. The infrared lamp was oriented so that it heated the same portion of the vivarium that was exposed to UVB radiation via the fluorescent lamp. For each vivarium, light and heat were provided for 12 continuous hours each day (8:00 AM to 8:00 PM).27 Shelters were provided in each vivarium so that tortoises could easily regulate their exposure to UVB radiation and heat. Environmental temperature was maintained at 22 ± 1°C during the day (8:00 AM to 8:00 PM) and at 19 ± 1°C during the night (8:00 PM to 8:00 AM). A thermostat was used in basking zones to prevent temperatures from exceeding 35°C. Substrates in the enclosures consisted of natural soil and a layer of fir bark and coconut fiber a few centimeters thick. Relative humidity was recorded by an analogical hygrometer and ranged between 65% and 75%. The tortoises were fed a mixed diet composed of vegetables suitable for human consumption and spontaneously growing vegetation. Greens and water were provided to the tortoises ad libitum.
Sample preparation and testing—For optimization and validation of the analytical method as well as preparation of matrix-matched calibration curves during each day of analysis, a pure cefovecin standardb was used. Cephalexinc was used as an internal standard. Standard stock solutions of cefovecin and cephalexin were each prepared at a concentration of 2 mg/mL by dissolving the pure substances in flasks with ultrapure water. For spiking purposes, cefovecin standard solutions were prepared at different concentrations, obtaining a specific dilution for each point of the calibration curve; this allowed adding a fixed amount of standard solution to each sample. An internal standard working solution was obtained by diluting cephalexin stock solution in ultrapure water at a concentration of 0.01 mg/mL, and 10 µL of the working solution was added to each sample. All standard solutions were stored in glassware at 5 ± 3°C in darkness until use. Plasma samples were thawed at room temperature (approx 23°C) and mixed by vortexing. A 100-µL aliquot was transferred into a self-capping 1-mL microcentrifuge tube, and 10 µL of internal standard solution (cephalexin-water solution at a concentration of 10 µg/mL) and 10 µL of water were added. Samples were mixed, 400 µL of acetonitrile was added, and the mixture was vortexed again for 10 seconds and centrifuged for 10 minutes at 3,400 X g. After centrifugation, 200 µL of supernatant was transferred into another microcentrifuge tube containing 800 µL of 0.3% formic acid solution and 2mM ammonium acetate in water and vortexed again. The contents were transferred into a glass vial, and 5 µL was injected in the ultraperformance liquid chromatography–tandem mass spectrometry system. Similarly, to prepare the calibration curves, 10 µL of the specific standard solution was added to 100 µL of plasma to obtain the follow-
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ing cefovecin concentrations: 0, 0.01, 0.05, 0.25, 1, 5, 10, 25, and 50 µg/mL. Quality control samples were prepared at cefovecin concentrations of 0.05, 1 and 25 µg/mL. After adding cefovecin and cephalexin working solutions, samples were vortexed for 10 seconds and allowed to stand for 10 minutes at room temperature before extraction. Analysis was performed with an ultraperformance liquid chromatography binary pumpd equipped with a 50 X 2.1-mm (1.7 µm particle size) C18 reversedphase columne and interfaced with a tandem mass spectrometer.f The mobile phase consisted of 0.3% formic acid and 2mM of ammonium acetate in water (solvent A) and 0.3% formic acid in acetonitrile (solvent B), at a flow rate of 0.5 mL/min. The gradient used was 95% solvent A and 5% solvent B from time 0 to 0.5 minutes, switched to 90% solvent B over 1.0 minute and held for 0.5 minutes, then returned to 95% solvent A and 5% solvent B over 1.0 minute and held for 1.0 additional minute to equilibrate the column before sample injection. The mass spectrometer interface was operated in positive electrospray ionization mode with capillary voltage at 4.00 kV. Analysis was performed in multiple reaction monitoring mode, observing 2 transitions for cefovecin (cone voltage = 24 V; m/z, 453.6→240.9 at 14 eV of collision energy and 453.6→125.1 at 55 eV) and 2 transitions for cephalexin (cone voltage = 15 V; m/z, 347.7→157.9 at 9 eV and 347.70→173.9 at 14 eV). Method validation—The developed method was validated in accordance with current European guidelines29 by evaluation of specificity, linearity, trueness, and precision. Plasma samples collected from tortoises that had not been treated with cefovecin or cephalexin were used for the validation. Specificity was evaluated by injection of 10 blank plasma samples extracted and analyzed following the described procedure, to assess presence or absence of potential interfering compounds with the same retention times as cefovecin and cephalexin. A 9-point matrix-matched calibration curve was prepared each day of analysis with blank plasma samples fortified at various concentrations (0 to 50 µg/mL) to assess the method’s linearity; peak area ratios between cefovecin and the internal standard were plotted against their concentrations, and the resultant coefficient of determination (R2) was always > 0.99. The calibration curve was also used to calculate the limit of quantification of cefovecin in plasma, defined as the concentration providing a chromatographic signal with a signal-to-noise ratio of 10, which was 0.015 µg/mL. Because no certified reference material was available, accuracy (the combination of trueness and precision) was evaluated through the injection of 3 replicates of plasma samples spiked at 3 concentrations of cefovecin (0.05, 1.0, and 25.0 µg/mL) prepared on 2 days. Trueness, expressed as relative difference between the mean value measured and the spiked concentration, fell within the range set in the European guidelines.29 Precision was acceptable, with results always within the range of accepted values and with a maximum relative SD to the mean (coefficient of variation) of 15%. 920
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Pharmacokinetic analysis—Pharmacokinetics parameters were deduced from plasma concentration-time data by use of softwareg that allowed both compartmental and noncompartmental analyses of experimental data. All data points were weighted by the inverse square of the fitted value. A noncompartmental analysis was carried out on cefovecin plasma concentrations. The terminal elimination half-life was calculated as ln2/λz, where λz represents the first-order rate constant of the terminal portion of the curve. Harmonic means and pseudo-SDs were calculated for half-life with a jackknife technique.30 The AUMC0–last and AUC0–last were calculated via the trapezoidal method. Mean residence time to the last detectable drug concentration (MRTlast) was determined from the following equation31: MRTlast = AUMC0–last/AUC0–last
The Cmax and Tmax of cefovecin in plasma were obtained from the experimentally observed data. Plasma protein binding analysis—After measuring total protein and albumin concentrations in samples before spiking with cefovecin, estimates of plasma protein binding were determined as follows: aliquots (450 µL) of a pool of tortoise plasma thawed at room temperature were fortified with 50 µL of cefovecin stock solutions at various concentrations to obtain final concentrations of 1, 5, and 10 µg/mL, prepared in triplicate (9 samples in total); 3 additional blank aliquots (plasma without cefovecin added) were also prepared. The samples were vortexed and incubated at 30°C for 60 minutes, then transferred to centrifugal filter unitsh and centrifuged at 14,000 X g for 10 minutes. For each sample, 100 µL of the ultrafiltrates was collected and, after spiking each blank to obtain 1, 5, or 10 µg of cefovecin/mL, processed as previously described for cefovecin extraction and quantification. The bound fraction was then calculated at each concentration as follows: % protein binding = ([total concentration – unbound concentration] / total concentration) X 100%
where total concentration is the amount of drug measured in the blank sample fortified after centrifugation and the unbound concentration is the mean of the drug concentrations found in the 3 samples spiked at the beginning of the experiment. Results No evidence of change in food consumption or fecal production was observed in any tortoises during the study. All animals appeared to be in good clinical condition, with stable activity levels and good adaption to group housing and to the temperature gradients of the vivaria. No gross changes were observed at the cefovecin injection sites. Cefovecin was detected at measurable concentrations (> 0.015 µg/mL) in all groups for 3 days, then it was measurable only in some groups in the following 3 days and remained below the limit of quantification in all groups after 1 week (Figure 1). A degree of variability was observed among treatment groups, as confirmed by relative SD at each time point (between 19.1% and AJVR, Vol 75, No. 10, October 2014
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The Cmax of cefovecin (27.37 ± 4.43 µg/mL) was considerably lower than those reported for dogs and cats (121 ± 51 and 141 ± 12 µg/mL, respectively),21,22 various species of nonhuman primates (which ranged from 42 ± 9 µg/ mL to 93.5 ± 6.61 µg/mL),33–35 and ferrets (41.05 ± 3.87 µg/mL),i which had all received a single 8.0 mg/kg injection of cefovecin SC (the same dose administered to tortoises in the present study). According to data obtained by Thesuen et al,32 Cmax values of cefovecin in green iguanas and female Lohmann hens after SC administration at 10 mg/kg were similar or even lower (35 ± 12 µg/mL for iguanas and 6 ± 2 µg/mL for hens) than those found in our study. These obserFigure 1—Mean plasma concentrations of cefovecin sodium following administration vations are supported by differences in of a single dose (8.0 mg/kg, SC) to 23 healthy adult Hermann’s tortoises (Testudo her0–last reported for the manni). The inset graph shows cefovecin concentrations over the first 24 hours after AUMC0–last and AUC various species,21,22,33–35,i and for tortoises administration. Error bars indicate SD. in the present study. The mean Tmax Table 1—Pharmacokinetics parameters derived via noncompartin tortoises of the present study (1.22 ± 1.14 hours) mental analysis following administration of a single dose of cefoveshowed a certain variability, which might have been cin sodium (8.0 mg/kg, SC) to 23 healthy adult Hermann’s tortoises. partially influenced by differences in terms of sex and body weight among the groups and was longer than Parameter Range Estimate that in iguanas (0.33 hours) and hen (0.28 hours)32 but Cmax (µg/mL) 20.20–33.52 27.37 ± 4.43 similar to the value reported for primates (0.5 to 2.83 Tmax (h) 0.58–4.00 1.22 ± 1.14 AUC0–last (h•µg/mL) 155.79–249.22 220.35 ± 36.18 hours).33–35 In all of these species, Tmax was generalAUMC0–last (h•µg/mL) 1,036.46–2,745.18 2,003.62 ± 542.33 ly shorter than in cats and dogs (2.0 ± 2.0 hours and t1/2λz (h) 12.35–123.80 20.78 ± 12.62* 6.2 ± 3.0 hours, respectively)21,22; however, it must be MRTlast (h) 6.65–11.28 9.03 ± 1.79 considered that the Tmax is influenced by sample colEstimates are presented as mean ± SD unless otherwise indicated. lection times. In tortoises, the absorption of cefovecin *Harmonic mean ± pseudo-SD. was rapid, and the decrease in circulating concentraMRTlast = Mean residence time to the last detectable tion after Cmax was prolonged in time, suggesting a concentration of the drug. t1/2λz = Elimination half-life. theoretical plateau during the first 4 hours. Although with relevant fluctuations (probably related to group variability) the elimination half-life in tortoises in this 26.9%). The pharmacokinetic parameters are reported study (20.78 ± 12.62 hours) was much lower than in (Table 1). After SC administration, cefovecin reached cats (166 ± 18 hours)22 and dogs (133 ± 16 hours),21 Cmax in plasma between 35 minutes and 2 hours (with but higher than in hens (0.87 ± 0.27 hours), green the exception of 1 group, in which it was reached after iguanas, (3.90 hours),32 and nonhuman primates (re4 hours), with similar values (mean ± SD, 24.30 ± 6.19 ported values ranged from 4.95 ± 1.47 to 9.17 ± 1.84 µg/mL at 35 minutes, 24.92 ± 4.88 µg/mL at 1 hour, hours, depending on the species).33–35 The MRTlast oband 23.00 ± 5.97 µg/mL at 2 hours). The mean Tmax served in our study (9.03 ± 1.79 hours) was similar to was 1.22 ± 1.14 hours. those reported for some nonhuman primates (10.5 ± Cefovecin was not highly bound to plasma pro1.3 hours and 6.12 ± 0.887 hours in cynomolgus mateins. Mean tortoise plasma total protein and albumin caques and olive baboons, respectively)34 but considerconcentrations measured in samples before spiking ably shorter than in dogs (165 to 200 hours)21 and cats with cefovecin were 5.3 and 2.1 g/dL, respectively The (215 to 256 hours).22 In the studies21,22,34 of nonhuman mean bound fraction was 47.5% at a cefovecin concenprimates, dogs, and cats, this parameter was evaluated tration of 1 µg/mL, 41.3% at 5 µg/mL, and 42.5% at 10 only following IV administration. µg/mL, with all values in the range of 37.9% to 50.2%. On the basis of these outcomes, a difference in behavior of cefovecin in Hermann’s tortoises versus that Discussion in dogs and cats can be hypothesized. Results of the experiment conducted in the present study suggested that The analytic method used in this study was found the percentage of protein-bound drug in tortoise plasma to be suitable for pharmacokinetics studies, which re(mean values from 41.3% to 47.5% at various cefovecin quire reliable performance with a wide range of drug concentrations) is somewhat lower than that reported in concentrations. The noncompartmental analysis was dogs (96.0% to 98.7%)21 and cats (99.5% to 99.8%)22; suitable to describe the pharmacokinetic properties moreover, mean plasma total protein and albumin conof cefovecin in Hermann’s tortoises, similar to the obcentrations measured in samples from tortoises before servations in mammals and in other nonmammalian species.21,22,32–34 spiking with cefovecin (5.3 and 2.1 g/dL, respectively) AJVR, Vol 75, No. 10, October 2014
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were lower than concentrations described for dogs (6.0 to 7.8 and 2.5 to 3.7 g/dL) and cats (6.0 to 7.5 and 2.4 to 3.8 g/dL).36 It is known that for β-lactam antimicrobials there is not a linear correlation between drug concentration and bactericidal activity. Therefore, for this class of time-dependent antimicrobials, it is more important to consider the time the concentration exceeds the MIC, which has been shown through in vivo studies to be the pharmacokinetic-pharmacodynamic parameter that correlates best with their therapeutic efficacy.37–40 To evaluate the concentrations at which cefovecin can be considered effective and the duration of effect, MIC of the drug for common bacterial pathogens of the species of interest must be considered. However, since no tortoise-specific data on the MIC of cefovecin for common pathogens is available, it is not presently possible to suggest a therapeutically effective administration interval. Currently, only a few reports on the use of cefovecin in exotic animals have been published. This is likely attributable, in part, to the relatively recent introduction of the drug in veterinary medicine. Some reports have highlighted relevant differences in cefovecin pharmacokinetics among various animal species. In dogs and cats, a single-dose SC administration at 8.0 mg/kg can effectively maintain the therapeutic effect on skin and soft tissue (through the maintenance of an effective threshold concentration) for a long period, allowing a 14-day dose interval.21,22 This therapeutic approach seems unsuitable for other species that have faster cefovecin elimination; according to the results obtained in other pharmacokinetics studies, daily administration is suggested in nonhuman primates33–35 and, similarly, a 2- to 3-day maximum dose interval has been recommended in ferrets,i with a 12-hour to 2-day interval suggested for green iguanas.32 Clearly, when considered from the practical standpoint and for animal well-being, a long duration of effect requiring less frequent drug administration is beneficial because it contributes to reduction of patient stress and helps to optimize owner compliance at the same time.41–43 Especially in nondomestic and wild or feral animals, stress must be considered a very important and limiting factor because these types of animals can be more susceptible to stress caused by repeated handling and restraint.44 In light of the results of the present pharmacokinetics study, and particularly on the basis of the plasma elimination half-life, it seems that the dosing interval of cefovecin described for dogs and cats (14 days)21,22 cannot be considered valid for tortoises. To our knowledge, our study represents the first research to investigate the pharmacokinetics of cefovecin in Hermann’s tortoises after SC administration at the dose recommended for use in dogs and cats (8.0 mg/kg) and also to evaluate plasma protein binding to cefovecin for this species. With regard to the tolerability of the drug, no apparent adverse reactions (local or systemic) were observed in the study population during the experiments. Finally, it would be interesting, once MIC data for pathogens common to tortoises are available, to evaluate the time the concentration exceeds the MIC values in this species to propose reasonable administration intervals. 922
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Convenia, Pfizer Animal Health, New York, NY. Cefovecin standard, Pfizer Animal Health, New York, NY. Fluka, Sigma-Aldrich, St Louis, Mo. Acquity UPLC, Waters Corp, Milford, Mass. Acquity BEH, Waters Corp, Milford, Mass. Quattro Premier XE, Waters Corp, Milford, Mass. WinNonLin Prof 6.1, Pharsight Corp, St Louis, Mo. Amicon Ultra 0.5 mL 30,000 NMWL, Millipore, Billerica, Mass. Montesinos A, Diez-Delgado I, Gilabert JA, et al. Pharmacokinetics of cefovecin (Convenia) by subcutaneous route in ferrets (Mustela putorius furo)(abstr), in Proceedings. Int Conf Dis Zoo Wild Anim 2010;231–232.
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30. Lam FC, Hung CT, Perrier DG. Estimation of variance for harmonic mean half-lives. J Pharm Sci 1985;74:229–231. 31. Gibaldi M, Perrier D. Pharmacokinetics. 2nd ed. New York: Marcel Dekker, 1982. 32. Thuesen LR, Bertelsen MF, Brimer L, et al. Selected pharmacokinetic parameters for Cefovecin in hens and green iguanas. J Vet Pharmacol Ther 2009;32:613–617. 33. Bakker J, Thuesen LR, Braskamp G, et al. Single subcutaneous dosing of cefovecin in rhesus monkeys (Macaca mulatta): a pharmacokinetic study. J Vet Pharmacol Ther 2011;34:464–468. 34. Raabe BM, Lovaglio J, Grover GS, et al. Pharmacokinetics of cefovecin in cynomolgus macaques (Macaca fascicularis), olive baboons (Papio anubis), and rhesus macaques (Macaca mulatta). J Am Assoc Lab Anim Sci 2011;50:389–395. 35. Papp R, Popovic A, Kelly N, et al. Pharmacokinetics of cefovecin in squirrel monkey (Saimiri sciureus), rhesus macaques (Macaca mulatta), and cynomolgus macaques (Macaca fascicularis). J Am Assoc Lab Anim Sci 2010;49:805–808. 36. Johnson MC. Immunologic and plasma protein disorders. In: Willard MD, Tvedten H, eds. Small animal clinical diagnosis by laboratory methods. 5th ed. St Louis: Saunders Elsevier, 2012;278–293. 37. Craig WA, Ebert SC. Killing and regrowth of bacteria in vitro: a review. Scand J Infect Dis Suppl 1990;74:63–70. 38. Craig WA. Interrelationship between pharmacokinetics and pharmacodynamics in determining dosage regimens for broad-spectrum cephalosporins. Diagn Microbiol Infect Dis 1995;22:89–96. 39. Craig WA. Pharmacokinetic/pharmacodynamic parameters: rationale for antibacterial dosing of mice and men. Clin Infect Dis 1998;26:1–10. 40. Vogelman B, Gudmundsson S, Leggett J, et al. Correlation of antimicrobial pharmacokinetic parameters with therapeutic efficacy in an animal model. J Infect Dis 1988;158:831–847. 41. Barter LS, Watson ADJ, Maddison JE. Owner compliance with short term antimicrobial medication in dogs. Aust Vet J 1996;74:277–280. 42. Cazzola M, Matera MG, Donner CF. Pharmacokinetics and pharmacodynamics of newer oral cephalosporins: implications for treatment of community-acquired lower respiratory tract infections. Clin Drug Investig 1998;16:335–346. 43. Six R, Cherni J, Chesebrough R, et al. Efficacy and safety of cefovecin in treating bacterial folliculitis, abscesses, or infected wounds in dogs. J Am Vet Med Assoc 2008;233:433–439. 44. Fowler ME. Restraint and anesthesia in zoo animal practice. J Am Vet Med Assoc 1974;164:706–711.
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Objective—To determine the pharmacokinetics of cefovecin sodium after SC administration to Hermann’s tortoises (Testudo hermanni). Animals—23 healthy adult Hermann’s tortoises (15 males and 8 females). Procedures—Cefovecin (8.0 mg/kg) was injected once in the subcutis of the neck region of Hermann’s tortoises, and blood samples were obtained at predetermined time points. Plasma cefovecin concentrations were measured via ultraperformance liquid chromatography coupled to tandem mass spectrometry, and pharmacokinetic parameters were calculated with a noncompartmental model. Plasma protein concentration was quantified, and the percentage of cefovecin bound to protein was estimated with a centrifugation technique. Results—Cefovecin was absorbed rapidly, reaching maximum plasma concentrations between 35 minutes and 2 hours after administration, with the exception of 1 group, in which it was reached after 4 hours. The mean ± SD time to maximum concentration was 1.22 ± 1.14 hours; area under the concentration-time curve was 220.35 ± 36.18 h•µg/mL The mean protein-bound fraction of cefovecin ranged from 41.3% to 47.5%. No adverse effects were observed. Conclusions and Clinical Relevance—Administration of a single dose of cefovecin SC appeared to be well-tolerated in this population of tortoises. Results of pharmacokinetic analysis indicated that the 2-week dosing interval suggested for dogs and cats cannot be considered effective in tortoises; however, further research is needed to determine therapeutic concentrations of the drug and appropriate dose ranges. (Am J Vet Res 2014;75:918–923)
H
ermann’s tortoises (Testudo hermanni) are frequently kept as pets. The small size (carapace rarely > 210 mm in length in wild populations),1 herbivorous habits, and resilience make these tortoises an exceptional garden inhabitant. Although Hermann’s tortoises are listed by the International Union for Conservation of Nature2 as a near-threatened species, the number of such tortoises in the wild continues to decrease. Antimicrobial treatment is often necessary in chelonian medicine,3 but only a few studies have been perReceived January 30, 2014. Accepted June 6, 2014. From the Clinica Veterinaria Modena Sud, Piazza dei Tintori 1, 41057 Spilamberto, MO, Italy (Nardini); the Department of Veterinary Medical Sciences, University of Bologna, 40064 Ozzano dell’Emilia, BO, Italy (Barbarossa, Dall’Occo, Roncada, Zaghini); the Clinica per Animali Esotici, Centro Veterinario Specialistico, Via Sandro Giovannini 53, 00137 Roma, Italy (Di Girolamo); the Università degli Studi di Milano, Dip. Scienze Veterinarie per la Salute, la Produzione Animale e la Sicurezza Alimentare, 20133 Milano, Italy (Cagnardi); the Parco Natura Viva, Garda Zoological Park, Loc. Figara 40, 37012 Bussolengo, VR, Italy (Magnone); and the Ambulatorio Veterinario, Viale Buonarroti 20, 28100 Novara, Italy (Bielli). The authors declare no financial conflicts of interest. The authors thank Dr. Paola Boretto for providing the pure cefovecin standard. Address correspondence to Dr. Barbarossa ([email protected]). 918
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AUC0–last AUMC0–last Cmax MIC Tmax
ABBREVIATIONS
Area under the concentrationtime curve from time 0 to last measurable drug concentration Area under the first moment curve from time 0 to last measurable drug concentration Maximum (peak) drug concentration Minimum inhibitory concentration Time to reach maximum (peak) drug concentration
formed to investigate the pharmacokinetics and pharmacodynamics of antimicrobials in these animals. Most studies have been performed with marine turtles,4–12 with few focusing on freshwater13,14 and terrestrial chelonians.15–17 Consequently, to establish antimicrobial treatment protocols, veterinary practitioners often have to rely on anecdotal reports18 for information instead of proper pharmacokinetics studies. Cefovecin sodium is a semisynthetic third-generation cephalosporin registered for veterinary use in dogs and cats in the United States and in Europe. Cephalosporins act by interfering with bacterial cell wall synthesis and are often used in treatment of reptiles.19,20 In AJVR, Vol 75, No. 10, October 2014
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dogs and cats, cefovecin is a long-acting antimicrobial, and a single SC injection can provide up to 14 days of treatment.21,22 In dogs, cefovecin (8.0 mg/kg) had a mean elimination half-life of 5.5 days after SC administration, whereas that in cats (following administration with the same dose and route) was 6.9 days.21,22 Following SC or IV administration, cefovecin has been described in a noncompartmental model characterized by a rapid initial distribution phase, followed by a slower elimination phase.21,22 The use of a long-acting, broad-spectrum antimicrobial would be of particular interest for clinicians working with chelonians. The metabolism of reptiles differs from that of mammals in several aspects.23 In addition to variations in energy requirements,24 drug efficacy and metabolism may be substantially different from those in mammals.25,26 Therefore, it may be inappropriate to prescribe medical treatment for reptiles by extrapolating doses from research performed in other animal species. The purpose of the study reported here was to determine the pharmacokinetics of cefovecin in healthy Hermann’s tortoises and to assess whether the therapeutic protocol used in dogs and cats could be applicable to chelonians.
Tortoises’ clinical conditions were monitored throughout the experiment, including possible variations in the amount of food consumed and feces produced. Experimental procedures were approved by the Italian Ministry of Health, in accordance with European regulations.28
Materials and Methods
Injection and sample collection—The day before beginning the experiment, an IV catheter was aseptically placed in a jugular vein of each tortoise. On day 0, a commercially available cefovecin sodium formulationa was reconstituted with the associated solvent (to a concentration of 80 mg/mL) and delivered by a single 0.1 mL/kg (8.0 mg of cefovecin as the sodium salt/kg) injection with a 20-gauge needle in the subcutis of the pectoral region contralateral to the catheter site. Blood samples were collected into lithium heparin–containing tubes prior to administration (baseline), at 35 minutes, 1, 2, 4, 8, 12, and 24 hours during day 1 (the first day after treatment), and then every 24 hours on days 3, 4, 5, 6, 8, 10, 12, and 14. Catheters were flushed with 0.5 mL of a calcium heparin solution after sample collection. To create a pool of approximately 1 mL of blood for each group, 0.3 to 0.4 mL/tortoise/time point was obtained. Samples were centrifuged within 30 minutes after collection. Plasma was collected and stored in microtubes at –20°C until assayed.
Animals—Twenty-three healthy adult Hermann’s tortoises (15 males and 8 females; body weight, 0.735 to 2.300 kg) were included in the study. The tortoises were obtained from the Parco Natura Viva Garda Zoological Park, Bussolengo, Verona, Italy, and were identified by writing a unique number on each carapace with an aqueous-based marker. Health status was determined on the basis of clinical history, physical examination, and results of a CBC and serum biochemical analysis for each animal. The tortoises were divided in 8 same-sex groups (7 groups of 3 tortoises and 1 group of 2 tortoises). Each group was placed in a vivarium and acclimatized for 7 days prior to the start of the study. Each vivarium was equipped with a fluorescent UVBemitting lamp and an infrared lamp as heat source. The fluorescent bulb was oriented diagonally with the end of the longitudinal axis 21 cm above the floor of the vivarium. The infrared lamp was oriented so that it heated the same portion of the vivarium that was exposed to UVB radiation via the fluorescent lamp. For each vivarium, light and heat were provided for 12 continuous hours each day (8:00 AM to 8:00 PM).27 Shelters were provided in each vivarium so that tortoises could easily regulate their exposure to UVB radiation and heat. Environmental temperature was maintained at 22 ± 1°C during the day (8:00 AM to 8:00 PM) and at 19 ± 1°C during the night (8:00 PM to 8:00 AM). A thermostat was used in basking zones to prevent temperatures from exceeding 35°C. Substrates in the enclosures consisted of natural soil and a layer of fir bark and coconut fiber a few centimeters thick. Relative humidity was recorded by an analogical hygrometer and ranged between 65% and 75%. The tortoises were fed a mixed diet composed of vegetables suitable for human consumption and spontaneously growing vegetation. Greens and water were provided to the tortoises ad libitum.
Sample preparation and testing—For optimization and validation of the analytical method as well as preparation of matrix-matched calibration curves during each day of analysis, a pure cefovecin standardb was used. Cephalexinc was used as an internal standard. Standard stock solutions of cefovecin and cephalexin were each prepared at a concentration of 2 mg/mL by dissolving the pure substances in flasks with ultrapure water. For spiking purposes, cefovecin standard solutions were prepared at different concentrations, obtaining a specific dilution for each point of the calibration curve; this allowed adding a fixed amount of standard solution to each sample. An internal standard working solution was obtained by diluting cephalexin stock solution in ultrapure water at a concentration of 0.01 mg/mL, and 10 µL of the working solution was added to each sample. All standard solutions were stored in glassware at 5 ± 3°C in darkness until use. Plasma samples were thawed at room temperature (approx 23°C) and mixed by vortexing. A 100-µL aliquot was transferred into a self-capping 1-mL microcentrifuge tube, and 10 µL of internal standard solution (cephalexin-water solution at a concentration of 10 µg/mL) and 10 µL of water were added. Samples were mixed, 400 µL of acetonitrile was added, and the mixture was vortexed again for 10 seconds and centrifuged for 10 minutes at 3,400 X g. After centrifugation, 200 µL of supernatant was transferred into another microcentrifuge tube containing 800 µL of 0.3% formic acid solution and 2mM ammonium acetate in water and vortexed again. The contents were transferred into a glass vial, and 5 µL was injected in the ultraperformance liquid chromatography–tandem mass spectrometry system. Similarly, to prepare the calibration curves, 10 µL of the specific standard solution was added to 100 µL of plasma to obtain the follow-
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ing cefovecin concentrations: 0, 0.01, 0.05, 0.25, 1, 5, 10, 25, and 50 µg/mL. Quality control samples were prepared at cefovecin concentrations of 0.05, 1 and 25 µg/mL. After adding cefovecin and cephalexin working solutions, samples were vortexed for 10 seconds and allowed to stand for 10 minutes at room temperature before extraction. Analysis was performed with an ultraperformance liquid chromatography binary pumpd equipped with a 50 X 2.1-mm (1.7 µm particle size) C18 reversedphase columne and interfaced with a tandem mass spectrometer.f The mobile phase consisted of 0.3% formic acid and 2mM of ammonium acetate in water (solvent A) and 0.3% formic acid in acetonitrile (solvent B), at a flow rate of 0.5 mL/min. The gradient used was 95% solvent A and 5% solvent B from time 0 to 0.5 minutes, switched to 90% solvent B over 1.0 minute and held for 0.5 minutes, then returned to 95% solvent A and 5% solvent B over 1.0 minute and held for 1.0 additional minute to equilibrate the column before sample injection. The mass spectrometer interface was operated in positive electrospray ionization mode with capillary voltage at 4.00 kV. Analysis was performed in multiple reaction monitoring mode, observing 2 transitions for cefovecin (cone voltage = 24 V; m/z, 453.6→240.9 at 14 eV of collision energy and 453.6→125.1 at 55 eV) and 2 transitions for cephalexin (cone voltage = 15 V; m/z, 347.7→157.9 at 9 eV and 347.70→173.9 at 14 eV). Method validation—The developed method was validated in accordance with current European guidelines29 by evaluation of specificity, linearity, trueness, and precision. Plasma samples collected from tortoises that had not been treated with cefovecin or cephalexin were used for the validation. Specificity was evaluated by injection of 10 blank plasma samples extracted and analyzed following the described procedure, to assess presence or absence of potential interfering compounds with the same retention times as cefovecin and cephalexin. A 9-point matrix-matched calibration curve was prepared each day of analysis with blank plasma samples fortified at various concentrations (0 to 50 µg/mL) to assess the method’s linearity; peak area ratios between cefovecin and the internal standard were plotted against their concentrations, and the resultant coefficient of determination (R2) was always > 0.99. The calibration curve was also used to calculate the limit of quantification of cefovecin in plasma, defined as the concentration providing a chromatographic signal with a signal-to-noise ratio of 10, which was 0.015 µg/mL. Because no certified reference material was available, accuracy (the combination of trueness and precision) was evaluated through the injection of 3 replicates of plasma samples spiked at 3 concentrations of cefovecin (0.05, 1.0, and 25.0 µg/mL) prepared on 2 days. Trueness, expressed as relative difference between the mean value measured and the spiked concentration, fell within the range set in the European guidelines.29 Precision was acceptable, with results always within the range of accepted values and with a maximum relative SD to the mean (coefficient of variation) of 15%. 920
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Pharmacokinetic analysis—Pharmacokinetics parameters were deduced from plasma concentration-time data by use of softwareg that allowed both compartmental and noncompartmental analyses of experimental data. All data points were weighted by the inverse square of the fitted value. A noncompartmental analysis was carried out on cefovecin plasma concentrations. The terminal elimination half-life was calculated as ln2/λz, where λz represents the first-order rate constant of the terminal portion of the curve. Harmonic means and pseudo-SDs were calculated for half-life with a jackknife technique.30 The AUMC0–last and AUC0–last were calculated via the trapezoidal method. Mean residence time to the last detectable drug concentration (MRTlast) was determined from the following equation31: MRTlast = AUMC0–last/AUC0–last
The Cmax and Tmax of cefovecin in plasma were obtained from the experimentally observed data. Plasma protein binding analysis—After measuring total protein and albumin concentrations in samples before spiking with cefovecin, estimates of plasma protein binding were determined as follows: aliquots (450 µL) of a pool of tortoise plasma thawed at room temperature were fortified with 50 µL of cefovecin stock solutions at various concentrations to obtain final concentrations of 1, 5, and 10 µg/mL, prepared in triplicate (9 samples in total); 3 additional blank aliquots (plasma without cefovecin added) were also prepared. The samples were vortexed and incubated at 30°C for 60 minutes, then transferred to centrifugal filter unitsh and centrifuged at 14,000 X g for 10 minutes. For each sample, 100 µL of the ultrafiltrates was collected and, after spiking each blank to obtain 1, 5, or 10 µg of cefovecin/mL, processed as previously described for cefovecin extraction and quantification. The bound fraction was then calculated at each concentration as follows: % protein binding = ([total concentration – unbound concentration] / total concentration) X 100%
where total concentration is the amount of drug measured in the blank sample fortified after centrifugation and the unbound concentration is the mean of the drug concentrations found in the 3 samples spiked at the beginning of the experiment. Results No evidence of change in food consumption or fecal production was observed in any tortoises during the study. All animals appeared to be in good clinical condition, with stable activity levels and good adaption to group housing and to the temperature gradients of the vivaria. No gross changes were observed at the cefovecin injection sites. Cefovecin was detected at measurable concentrations (> 0.015 µg/mL) in all groups for 3 days, then it was measurable only in some groups in the following 3 days and remained below the limit of quantification in all groups after 1 week (Figure 1). A degree of variability was observed among treatment groups, as confirmed by relative SD at each time point (between 19.1% and AJVR, Vol 75, No. 10, October 2014
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The Cmax of cefovecin (27.37 ± 4.43 µg/mL) was considerably lower than those reported for dogs and cats (121 ± 51 and 141 ± 12 µg/mL, respectively),21,22 various species of nonhuman primates (which ranged from 42 ± 9 µg/ mL to 93.5 ± 6.61 µg/mL),33–35 and ferrets (41.05 ± 3.87 µg/mL),i which had all received a single 8.0 mg/kg injection of cefovecin SC (the same dose administered to tortoises in the present study). According to data obtained by Thesuen et al,32 Cmax values of cefovecin in green iguanas and female Lohmann hens after SC administration at 10 mg/kg were similar or even lower (35 ± 12 µg/mL for iguanas and 6 ± 2 µg/mL for hens) than those found in our study. These obserFigure 1—Mean plasma concentrations of cefovecin sodium following administration vations are supported by differences in of a single dose (8.0 mg/kg, SC) to 23 healthy adult Hermann’s tortoises (Testudo her0–last reported for the manni). The inset graph shows cefovecin concentrations over the first 24 hours after AUMC0–last and AUC various species,21,22,33–35,i and for tortoises administration. Error bars indicate SD. in the present study. The mean Tmax Table 1—Pharmacokinetics parameters derived via noncompartin tortoises of the present study (1.22 ± 1.14 hours) mental analysis following administration of a single dose of cefoveshowed a certain variability, which might have been cin sodium (8.0 mg/kg, SC) to 23 healthy adult Hermann’s tortoises. partially influenced by differences in terms of sex and body weight among the groups and was longer than Parameter Range Estimate that in iguanas (0.33 hours) and hen (0.28 hours)32 but Cmax (µg/mL) 20.20–33.52 27.37 ± 4.43 similar to the value reported for primates (0.5 to 2.83 Tmax (h) 0.58–4.00 1.22 ± 1.14 AUC0–last (h•µg/mL) 155.79–249.22 220.35 ± 36.18 hours).33–35 In all of these species, Tmax was generalAUMC0–last (h•µg/mL) 1,036.46–2,745.18 2,003.62 ± 542.33 ly shorter than in cats and dogs (2.0 ± 2.0 hours and t1/2λz (h) 12.35–123.80 20.78 ± 12.62* 6.2 ± 3.0 hours, respectively)21,22; however, it must be MRTlast (h) 6.65–11.28 9.03 ± 1.79 considered that the Tmax is influenced by sample colEstimates are presented as mean ± SD unless otherwise indicated. lection times. In tortoises, the absorption of cefovecin *Harmonic mean ± pseudo-SD. was rapid, and the decrease in circulating concentraMRTlast = Mean residence time to the last detectable tion after Cmax was prolonged in time, suggesting a concentration of the drug. t1/2λz = Elimination half-life. theoretical plateau during the first 4 hours. Although with relevant fluctuations (probably related to group variability) the elimination half-life in tortoises in this 26.9%). The pharmacokinetic parameters are reported study (20.78 ± 12.62 hours) was much lower than in (Table 1). After SC administration, cefovecin reached cats (166 ± 18 hours)22 and dogs (133 ± 16 hours),21 Cmax in plasma between 35 minutes and 2 hours (with but higher than in hens (0.87 ± 0.27 hours), green the exception of 1 group, in which it was reached after iguanas, (3.90 hours),32 and nonhuman primates (re4 hours), with similar values (mean ± SD, 24.30 ± 6.19 ported values ranged from 4.95 ± 1.47 to 9.17 ± 1.84 µg/mL at 35 minutes, 24.92 ± 4.88 µg/mL at 1 hour, hours, depending on the species).33–35 The MRTlast oband 23.00 ± 5.97 µg/mL at 2 hours). The mean Tmax served in our study (9.03 ± 1.79 hours) was similar to was 1.22 ± 1.14 hours. those reported for some nonhuman primates (10.5 ± Cefovecin was not highly bound to plasma pro1.3 hours and 6.12 ± 0.887 hours in cynomolgus mateins. Mean tortoise plasma total protein and albumin caques and olive baboons, respectively)34 but considerconcentrations measured in samples before spiking ably shorter than in dogs (165 to 200 hours)21 and cats with cefovecin were 5.3 and 2.1 g/dL, respectively The (215 to 256 hours).22 In the studies21,22,34 of nonhuman mean bound fraction was 47.5% at a cefovecin concenprimates, dogs, and cats, this parameter was evaluated tration of 1 µg/mL, 41.3% at 5 µg/mL, and 42.5% at 10 only following IV administration. µg/mL, with all values in the range of 37.9% to 50.2%. On the basis of these outcomes, a difference in behavior of cefovecin in Hermann’s tortoises versus that Discussion in dogs and cats can be hypothesized. Results of the experiment conducted in the present study suggested that The analytic method used in this study was found the percentage of protein-bound drug in tortoise plasma to be suitable for pharmacokinetics studies, which re(mean values from 41.3% to 47.5% at various cefovecin quire reliable performance with a wide range of drug concentrations) is somewhat lower than that reported in concentrations. The noncompartmental analysis was dogs (96.0% to 98.7%)21 and cats (99.5% to 99.8%)22; suitable to describe the pharmacokinetic properties moreover, mean plasma total protein and albumin conof cefovecin in Hermann’s tortoises, similar to the obcentrations measured in samples from tortoises before servations in mammals and in other nonmammalian species.21,22,32–34 spiking with cefovecin (5.3 and 2.1 g/dL, respectively) AJVR, Vol 75, No. 10, October 2014
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were lower than concentrations described for dogs (6.0 to 7.8 and 2.5 to 3.7 g/dL) and cats (6.0 to 7.5 and 2.4 to 3.8 g/dL).36 It is known that for β-lactam antimicrobials there is not a linear correlation between drug concentration and bactericidal activity. Therefore, for this class of time-dependent antimicrobials, it is more important to consider the time the concentration exceeds the MIC, which has been shown through in vivo studies to be the pharmacokinetic-pharmacodynamic parameter that correlates best with their therapeutic efficacy.37–40 To evaluate the concentrations at which cefovecin can be considered effective and the duration of effect, MIC of the drug for common bacterial pathogens of the species of interest must be considered. However, since no tortoise-specific data on the MIC of cefovecin for common pathogens is available, it is not presently possible to suggest a therapeutically effective administration interval. Currently, only a few reports on the use of cefovecin in exotic animals have been published. This is likely attributable, in part, to the relatively recent introduction of the drug in veterinary medicine. Some reports have highlighted relevant differences in cefovecin pharmacokinetics among various animal species. In dogs and cats, a single-dose SC administration at 8.0 mg/kg can effectively maintain the therapeutic effect on skin and soft tissue (through the maintenance of an effective threshold concentration) for a long period, allowing a 14-day dose interval.21,22 This therapeutic approach seems unsuitable for other species that have faster cefovecin elimination; according to the results obtained in other pharmacokinetics studies, daily administration is suggested in nonhuman primates33–35 and, similarly, a 2- to 3-day maximum dose interval has been recommended in ferrets,i with a 12-hour to 2-day interval suggested for green iguanas.32 Clearly, when considered from the practical standpoint and for animal well-being, a long duration of effect requiring less frequent drug administration is beneficial because it contributes to reduction of patient stress and helps to optimize owner compliance at the same time.41–43 Especially in nondomestic and wild or feral animals, stress must be considered a very important and limiting factor because these types of animals can be more susceptible to stress caused by repeated handling and restraint.44 In light of the results of the present pharmacokinetics study, and particularly on the basis of the plasma elimination half-life, it seems that the dosing interval of cefovecin described for dogs and cats (14 days)21,22 cannot be considered valid for tortoises. To our knowledge, our study represents the first research to investigate the pharmacokinetics of cefovecin in Hermann’s tortoises after SC administration at the dose recommended for use in dogs and cats (8.0 mg/kg) and also to evaluate plasma protein binding to cefovecin for this species. With regard to the tolerability of the drug, no apparent adverse reactions (local or systemic) were observed in the study population during the experiments. Finally, it would be interesting, once MIC data for pathogens common to tortoises are available, to evaluate the time the concentration exceeds the MIC values in this species to propose reasonable administration intervals. 922
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