Enrofloxacin belongs to the fluoroquinolone class of antibiotics. It is commonly used in a variety of reptile species due to its wide spectrum of efficacy, partly due to its formation of an active metabolite ciprofloxacin. Enrofloxacin shows wide disposition variability among all species resulting in large differences in the plasma concentrations of both enrofloxacin and ciprofloxacin. The aim of this study was to evaluate the pharmacokinetics of enrofloxacin and ciprofloxacin after a single intracoelomic injection of 10 mg/kg of enrofloxacin in 9 tortoises (Testudo hermanni). Blood samples were collected at 0, 0.5, 2, 4, 10, 24, 48, 72, 96, 120, 144, 168, 192, 216, 240 and 264 h and analyzed using a validated high performance liquid chromatography (HPLC) florescence method. Plasma concentrations of enrofloxacin were quantifiable in all subjects for up to 240 h, while ciprofloxacin was detected in all subjects up to 120 h. The C (s) of enrofloxacin and ciprofloxacin were 8614 ± 1116 ηg/mL obtained at 2.19 h and 605 ± 43 ηg/mL obtained at 4.23 h, respectively. The values of C/MIC ratio and AUC/MIC ratio of enrofloxacin with a MIC value of 0.5 μg/mL were 17.23 and 132.78, respectively. In conclusion, an administration of 10 mg/kg of enrofloxacin via the intracoelomic route in Hermann’s tortoises produced optimal pharmacodynamic parameters

De Vito, V.

Giorgi, M.

Owen, H.

Salvadori, M.

University of Queensland eSpace

Israel Journal of Veterinary Medicine  Vol. 70 (1)  March 2015 45 Enrofloxacin in Tortoises
SHORT COMMUNICATION
Fluoroquinolone antibiotics belong to a group of synthetic 
antimicrobials that are widely used in veterinary medicine. 
Their spectrum of activity includes Gram positive, Gram neg-
ative and Mycoplasma species responsible for a vast array of 
pulmonary, urinary and digestive infections (1). Enrofloxacin 
is a prototypical fluoroquinolone, showing treatment efficacy 
for the major bacterial conditions in several animal species 
(2, 3). The enhanced efficacy demonstrated by enrofloxacin 
is due to the formation of an active metabolite, ciprofloxacin 
which exhibits potency and spectrum of activity similar 
to that of the parental drug (1). The pharmacokinetics of 
enrofloxacin following various routes of administration have 
been investigated in different species of turtles and tortoises 
with plasma concentrations of enrofloxacin and ciprofloxacin 
showing wide disposition variability among the species (3-
5). Considering tortoise species are more closely related to 
one another than to other orders of animals, this underlines 
the importance of conducting pharmacokinetic studies for 
individual species rather than extrapolating doses from data 
generated in other reptile species (6).
The treatment of bacterial infections should be based on 
Pharmacokinetics of Enrofloxacin and its Metabolite Ciprofloxacin 
after Intracoelomic administration in Tortoises (Testudo hermanni)
Salvadori, M.,1 De Vito, V.,2 Owen, H.3 and Giorgi, M.2*
1 ExoticVet Veterinary Center, Via U. Dini 157, San Giuliano Terme, Pisa, Italy.
2 Department of Veterinary Sciences, University of Pisa, Via Livornese (lato monte) 1, San Piero a Grado, 56122 Pisa, Italy.
3 School of Veterinary Science, The University of Queensland, Gatton Campus, Gatton, Queensland 4343, Australia.
* Corresponding Author: Prof. Mario Giorgi, Chem D., Spec. Pharmacol., Department of Veterinary Sciences, University of Pisa, Via Livornese (lato monte), 
San Piero a Grado (Pisa), Italy. Email: mario.giorgi@unipi.it
ABST RACT
Enrofloxacin belongs to the fluoroquinolone class of antibiotics. It is commonly used in a variety of reptile 
species due to its wide spectrum of efficacy, partly due to its formation of an active metabolite ciprofloxacin. 
Enrofloxacin shows wide disposition variability among all species resulting in large differences in the 
plasma concentrations of both enrofloxacin and ciprofloxacin. The aim of this study was to evaluate the 
pharmacokinetics of enrofloxacin and ciprofloxacin after a single intracoelomic injection of 10 mg/kg of 
enrofloxacin in 9 tortoises (Testudo hermanni). Blood samples were collected at 0, 0.5, 2, 4, 10, 24, 48, 
72, 96, 120, 144, 168, 192, 216, 240 and 264 h and analyzed using a validated high performance liquid 
chromatography (HPLC) florescence method. Plasma concentrations of enrofloxacin were quantifiable 
in all subjects for up to 240 h, while ciprofloxacin was detected in all subjects up to 120 h. The Cmax (s) of 
enrofloxacin and ciprofloxacin were 8614 ± 1116 ηg/mL obtained at 2.19 h and 605 ± 43 ηg/mL obtained 
at 4.23 h, respectively. The values of Cmax/MIC ratio and AUC0-24/MIC ratio of enrofloxacin with a MIC 
value of 0.5 µg/mL were 17.23 and 132.78, respectively. In conclusion, an administration of 10 mg/kg 
of enrofloxacin via the intracoelomic route in Hermann’s tortoises produced optimal pharmacodynamic 
parameters.
Keywords: Enrofloxacin; Ciprofloxacin; Pharmacokinetics; Intracoelomic Administration; 
Testudo hermanni Tortoises.
Israel Journal of Veterinary Medicine  Vol. 70 (1)  March 2015Salvadori, M.46
a rational scientific approach. The most common pharma-
cokinetic/pharmacodynamic (PK/PD) approach for anti-
microbial agents uses plasma concentration as the PK input 
value and minimum inhibitory concentration (MIC) as the 
PD input value (7). Fluoroquinolones are considered to be 
well-tolerated drugs in both humans and animals, however, 
their intensive use has led to a significant increase in antimi-
crobial resistance (1). Therefore, several PK/PD indices such 
as Cmax/MIC and AUC0-24/MIC have been included in the 
present study to evaluate the clinical efficacy of enrofloxacin.
The aim of this study was to evaluate the PK of enrofloxa-
cin and its metabolite ciprofloxacin in Testudo hermanni after 
a single intracoelomic injection of 10 mg/kg enrofloxacin, and 
to establish if 10 mg/kg is an optimal dosage for treatment 
of different bacterial infections.
Nine tortoises of undetermined age, including both sexes 
(five males and four females), with a body weight range from 
0.4 to 2.95 kg, were used. The tortoises were housed indoors, 
divided equally into three glass containers, with access to 
indirect sun light and heat lamps (UVB 5%). Animals were 
maintained at 30 to 33° C with 250-W infrared heat lamps 
suspended 0.5 m above the floor to allow turtles to regulate 
their own body temperature. Tortoises were conditioned 
for a 2-week period prior the commencement of the study.
Tortoises were judged to be in good health based on physi-
cal examinations, normal activity, and routine acceptance of 
food. These observations were made by specialized veterinary 
personnel. All the tortoises were fed a mixture of vegetables, 
and given access to fresh water ad libitum.
Animal care and handling was performed according to 
the provision of the EC council Directive 86/609 EEC. The 
study protocol was approved by the University of Pisa’s ethics 
committee for animal welfare (CEASA) and transmitted to 
the Italian Ministry of Health.
Enrofloxacin as the commercial injectable solution 
(Enrovet® 25mg/mL, Bio98, Milan Italy), was diluted with 
saline to 10 mg/mL and given as a 10 mg/kg bolus by intra-
coelomic injection in the left prefemoral fossa using a sterile 
22-gauge, 3.75-cm needle. The dose used in the present study 
was selected based on previous studies in turtles (3, 5, 8). The 
drug was diluted because a previous study demonstrated that 
a 10 mg/kg intracoelomic injection of 10 mg/mL in yellow 
bellied slider turtles did not cause local irritation and soft 
tissue necrosis (5). These changes did occur when the same 
dose was used at higher concentrations (25 mg/mL) (9-10). 
Blood samples (0.5 mL or 0.25 mL in subjects greater than 
or less than 0.5 kg body weight, respectively) were collected 
from the subcarapacial venipuncture site at 0, 0.5, 2, 4, 10, 
24, 48, 72, 96, 120, 144, 168, 192, 216, 240 and 264 h after 
enrofloxacin administration. Although subcarapacial blood 
collection could be considered suboptimal because of po-
tential lymph contamination, enrofloxacin has been reported 
to be equally distributed in blood and lymph (7), and thus 
the pharmacokinetic data were not expected to be affected 
by sampling method. The blood samples were immediately 
transferred to tubes containing heparin, centrifuged and 
stored at -20° C until they were analyzed. Sample analysis 
was completed within 30 days of collection. The analytical 
method was based on a previous method using high perfor-
mance liquid chromatography (HPLC) with a fluorescence 
detector (5). Pharmacokinetic analysis of enrofloxacin and 
ciprofloxacin was performed using WinNonlin 5.3.1 software 
program according to a non-compartmental model.
No adverse effects at the point of injection and no be-
havioural or health alterations were observed in the animals 
during or after the study. Some transient, self-resolving side 
effects such as uncoordinated movements were noticed in an 
earlier study (5) in yellow bellied slider turtles. Species dif-
ferences might have triggered this distinction in side effects.
Blood levels of enrofloxacin were quantified in all 
subjects up to 240 h following injection. Blood levels of 
ciprofloxacin were detected in all subjects up to 120 h. The 
semi-logarithmic blood concentration vs. time average curves 
for enrofloxacin and ciprofloxacin are reported in Figure 1. 
The pharmacokinetic parameters are reported in Table 1.
The mean maximum blood concentration of enrofloxacin 
(Cmax 8614.64 ± 1116.36 ηg/mL) was reached at 2.19 h. This 
value, if normalized for the dose, was within the range of peak 
concentrations shown in previous studies on intramuscular 
injection of 5 mg/kg enrofloxacin in Gopher tortoises (2.4 µg/
mL) (4) and Indian star tortoises (3.59 µg/mL) (11). Similar 
trends were noticed also in the ciprofloxacin concentration. 
The Cmax of ciprofloxacin was 605.16 ± 43.04 ηg/mL attained 
at 4.23 h. This value was comparable to that shown in Indian 
star tortoises (0.35 µg/mL) after intramuscular injection of 
5 mg/kg enrofloxacin, if normalized for the dose (11), but 
higher than that shown in yellow-bellied slider turtles (0.32 
µg/mL) following an intracoelomic injection of 10 mg/kg 
enrofloxacin (5). The values of apparent terminal half-life 
(T1/2λz) of enrofloxacin and ciprofloxacin were 37.00 ± 11.97 h 
Research Articles
Israel Journal of Veterinary Medicine  Vol. 70 (1)  March 2015 47 Enrofloxacin in Tortoises
and 49.06 ± 5.82 h, respectively. T1/2λz of enrofloxacin was 
longer than that reported in earlier studies: Gopher tortoises 
(23.1 h) (4) and Indian star tortoises (5.1 h) (11). This dif-
ference might be due to the different routes of administra-
tion (intracoelomic vs. intramuscular) and the previously 
mentioned wide variability in pharmacokinetic parameters 
among tortoise species. In agreement with this speculation, a 
recent study involving a 10 mg/kg intracoelomic injection of 
enrofloxacin in yellow-bellied slider turtles (5) has shown that 
intracoelomic administration significantly increased the drug 
T1/2λz compared to the same parameter after intramuscular 
and oral administration in red-eared slider turtles (8) and after 
oral administration in Loggerhead sea turtles (3).
The reported MICs of enrofloxacin for most susceptible 
Gram negative, Gram positive bacteria and Mycoplasma 
isolated from domestic animals were < 0.1 µg/mL, with some 
additional moderately susceptible isolates having MICs of 
0.125-0.5 µg/mL (4). Cmax/MIC ratio > 10 and AUC0-24/
MIC ratio of 100 and 125, are required for fluoroquinolones 
to have antibiotic activity and to limit the development of 
bacterial resistance, respectively (7, 12-13). In the present 
study, considering a bacterium with a MIC value of 0.5 µg/
mL, the Cmax/MIC ratio of enrofloxacin was 17.23 and 
the average AUC0-24/MIC ratio was higher (132.78) than 
the required safety value. In contrast, Cmax/MIC ratio and 
AUC0-24/MIC ratio of ciprofloxacin were below the target 
ranges. These results could be due to the limited extent 
to which ciprofloxacin is produced in reptiles (<15%), as 
compared to mammals (35%) (14). This finding is in line 
with the low contribution of ciprofloxacin shown in reptiles 
(5,8). It has been postulated that this minimal presence of 
ciprofloxacin could be due to the slow metabolism of turtles 
and tortoises. In fact, cytochrome P450 3A, the enzyme that 
metabolizes enrofloxacin to ciprofloxacin, has been found to 
be poorly expressed in reptiles and fish (15-16).
In conclusion, the plasma concentrations of enrofloxacin 
achieved in this study after intracoelomic administration 
of 10 mg/kg enrofloxacin are adequate to reach the target 
end points associated with efficacy of fluoroquinolones in 
tortoises (Testudo hermanni).
CONFLICT OF INTEREST STATEMENT
None of the authors of this paper have a financial or personal 
relationship with other people or organizations that could inap-
propriately influence or bias the content of the paper.
ACKNOWLEDGMENTS
This work was supported by funds of University of Pisa (Athenaeum 
ex 60%). Any external funding did not support the preparation of 
manuscript.
Figure 1: Mean semi-logarithm plasma concentrations of enrofloxacin 
(-○-) and ciprofloxacin (-●-) vs time curves following intracoelomic 
injection of enrofloxacin (10 mg/kg) in tortoises (n=9). Bars represent 
the standard deviations.
Table 1: Pharmacokinetic parameters of enrofloxacin and ciprofloxacin 
after 10 mg/kg enrofloxacin intracoelomic injection in tortoises 
(Testudo hermani) (n=9)
Enrofloxacin Ciprofloxacin
Parameter Units Mean SD Mean SD
r2 0.99 ± 0.01 0.97 ± 0.01
λz 1/hr 0.02 ± 0.03 0.01 ± 0.01
T1/2λz hr 37.00 ± 11.97 49.06 ± 5.82
Tmax hr 2.19 ± 0.58 4.23 ± 0.93
Cmax ng/mL 8614 ± 1116 605.16 ± 43.04
AUC0-24 hr*ng/mL 66388 ± 4647 7952 ± 318
AUC0-∞ hr*ng/mL 102123 ± 9476 12835 ± 1244
Vz/F mL/kg 5227 ± 926 55140 ± 443
CL/F mL/hr/kg 97.92 ± 19.98 779.08 ± 88.17
AUMC0-∞ hr*hr*ng/mL 3383764 ± 42011 412688 ± 58959
MRT0-∞ hr 33.13 ± 1.69 32.15 ± 1.28
r2 = correlation coefficient.
λz = terminal phase rate constant.
T1/2λz = terminal half-life.
Tmax = time of peak.
Cmax = peak plasma 
concentration.
Vz/F = apparent volume of 
distribution.
CL/F = apparent clearance.
AUC0-24 = area under the 
plasma concentration-time 
from 0-24 h curve.
AUC0-∞ = area under the plasma 
concentration-time from 0 h to 
infinity curve.
AUMC0-∞ = area under the first 
moment curve.
MRT0-∞ = mean resident time.
Research Articles
Israel Journal of Veterinary Medicine  Vol. 70 (1)  March 2015Salvadori, M.48
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Research Articles


Pharmacokinetics of enrofloxacin and its metabolite ciprofloxacin after intracoelomic administration in Tortoises (Testudo hermanni)

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Pharmacokinetics of enrofloxacin and its metabolite ciprofloxacin after intracoelomic administration in Tortoises (Testudo hermanni)

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