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Israel Journal of Veterinary Medicine  Vol. 70 (1)  March 2015 45 Enrofoxacin in Tortoises
SHORT COMMUNICATION
Fluoroquinolone antibiotics belong to a group of synthetic
antimicrobials that are widely used in veterinary medicine.
Teir spectrum of activity includes Gram positive, Gram neg-
ative and Mycoplasma species responsible for a vast array of
pulmonary, urinary and digestive infections (1). Enrofoxacin
is a prototypical fuoroquinolone, showing treatment efcacy
for the major bacterial conditions in several animal species
(2, 3). Te enhanced efcacy demonstrated by enrofoxacin
is due to the formation of an active metabolite, ciprofoxacin
which exhibits potency and spectrum of activity similar
to that of the parental drug (1). Te pharmacokinetics of
enrofoxacin following various routes of administration have
been investigated in diferent species of turtles and tortoises
with plasma concentrations of enrofoxacin and ciprofoxacin
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).
Te treatment of bacterial infections should be based on
Pharmacokinetics of Enrofoxacin and its Metabolite Ciprofoxacin
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, Te 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
ABSTRACT
Enrofoxacin belongs to the fuoroquinolone class of antibiotics. It is commonly used in a variety of reptile
species due to its wide spectrum of efcacy, partly due to its formation of an active metabolite ciprofoxacin.
Enrofoxacin shows wide disposition variability among all species resulting in large diferences in the
plasma concentrations of both enrofoxacin and ciprofoxacin. Te aim of this study was to evaluate the
pharmacokinetics of enrofoxacin and ciprofoxacin after a single intracoelomic injection of 10 mg/kg of
enrofoxacin 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) forescence method. Plasma concentrations of enrofoxacin were quantifable
in all subjects for up to 240 h, while ciprofoxacin was detected in all subjects up to 120 h. Te C
max
(s) of
enrofoxacin and ciprofoxacin were 8614 ± 1116 ηg/mL obtained at 2.19 h and 605 ± 43 ηg/mL obtained
at 4.23 h, respectively. Te values of C
max
/MIC ratio and AUC
0-24
/MIC ratio of enrofoxacin 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 enrofoxacin via the intracoelomic route in Hermann’s tortoises produced optimal pharmacodynamic
parameters.
Keywords: Enrofoxacin; Ciprofoxacin; Pharmacokinetics; Intracoelomic Administration;
Testudo hermanni Tortoises.
Israel Journal of Veterinary Medicine  Vol. 70 (1)  March 2015 Salvadori, M. 46
a rational scientifc approach. Te 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 signifcant increase in antimi-
crobial resistance (1). Terefore, several PK/PD indices such
as C
max
/MIC and AUC
0-24
/MIC have been included in the
present study to evaluate the clinical efcacy of enrofoxacin.
Te aim of this study was to evaluate the PK of enrofoxa-
cin and its metabolite ciprofoxacin in Testudo hermanni after
a single intracoelomic injection of 10 mg/kg enrofoxacin, and
to establish if 10 mg/kg is an optimal dosage for treatment
of diferent bacterial infections.
Nine tortoises of undetermined age, including both sexes
(fve males and four females), with a body weight range from
0.4 to 2.95 kg, were used. Te 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 foor 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. Tese 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. Te
study protocol was approved by the University of Pisa’s ethics
committee for animal welfare (CEASA) and transmitted to
the Italian Ministry of Health.
Enrofoxacin 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. Te dose used in the present study
was selected based on previous studies in turtles (3, 5, 8). Te
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). Tese 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
enrofoxacin administration. Although subcarapacial blood
collection could be considered suboptimal because of po-
tential lymph contamination, enrofoxacin has been reported
to be equally distributed in blood and lymph (7), and thus
the pharmacokinetic data were not expected to be afected
by sampling method. Te 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. Te analytical
method was based on a previous method using high perfor-
mance liquid chromatography (HPLC) with a fuorescence
detector (5). Pharmacokinetic analysis of enrofoxacin and
ciprofoxacin was performed using WinNonlin 5.3.1 software
program according to a non-compartmental model.
No adverse efects 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
efects 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 efects.
Blood levels of enrofoxacin were quantifed in all
subjects up to 240 h following injection. Blood levels of
ciprofoxacin were detected in all subjects up to 120 h. Te
semi-logarithmic blood concentration vs. time average curves
for enrofoxacin and ciprofoxacin are reported in Figure 1.
Te pharmacokinetic parameters are reported in Table 1.
Te mean maximum blood concentration of enrofoxacin
(C
max
8614.64 ± 1116.36 ηg/mL) was reached at 2.19 h. Tis
value, if normalized for the dose, was within the range of peak
concentrations shown in previous studies on intramuscular
injection of 5 mg/kg enrofoxacin in Gopher tortoises (2.4 µg/
mL) (4) and Indian star tortoises (3.59 µg/mL) (11). Similar
trends were noticed also in the ciprofoxacin concentration.
Te C
max
of ciprofoxacin was 605.16 ± 43.04 ηg/mL attained
at 4.23 h. Tis value was comparable to that shown in Indian
star tortoises (0.35 µg/mL) after intramuscular injection of
5 mg/kg enrofoxacin, 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
enrofoxacin (5). Te values of apparent terminal half-life
(T
1/2
λz) of enrofoxacin and ciprofoxacin were 37.00 ± 11.97 h
Research Articles
Israel Journal of Veterinary Medicine  Vol. 70 (1)  March 2015 47 Enrofoxacin in Tortoises
and 49.06 ± 5.82 h, respectively. T
1/2
λz of enrofoxacin was
longer than that reported in earlier studies: Gopher tortoises
(23.1 h) (4) and Indian star tortoises (5.1 h) (11). Tis dif-
ference might be due to the diferent 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
enrofoxacin in yellow-bellied slider turtles (5) has shown that
intracoelomic administration signifcantly increased the drug
T
1/2
λz 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).
Te reported MICs of enrofoxacin 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). C
max
/MIC ratio > 10 and AUC
0-24
/
MIC ratio of 100 and 125, are required for fuoroquinolones
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 C
max
/MIC ratio of enrofoxacin was 17.23 and
the average AUC
0-24
/MIC ratio was higher (132.78) than
the required safety value. In contrast, C
max
/MIC ratio and
AUC
0-24
/MIC ratio of ciprofoxacin were below the target
ranges. Tese results could be due to the limited extent
to which ciprofoxacin is produced in reptiles (<15%), as
compared to mammals (35%) (14). Tis fnding is in line
with the low contribution of ciprofoxacin shown in reptiles
(5,8). It has been postulated that this minimal presence of
ciprofoxacin could be due to the slow metabolism of turtles
and tortoises. In fact, cytochrome P450 3A, the enzyme that
metabolizes enrofoxacin to ciprofoxacin, has been found to
be poorly expressed in reptiles and fsh (15-16).
In conclusion, the plasma concentrations of enrofoxacin
achieved in this study after intracoelomic administration
of 10 mg/kg enrofoxacin are adequate to reach the target
end points associated with efcacy of fuoroquinolones in
tortoises (Testudo hermanni).
CONFLI CT OF I NTEREST STATEMENT
None of the authors of this paper have a fnancial or personal
relationship with other people or organizations that could inap-
propriately infuence or bias the content of the paper.
ACKNOWLEDGMENTS
Tis 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 enrofoxacin
(-○-) and ciprofoxacin (-●-) vs time curves following intracoelomic
injection of enrofoxacin (10 mg/kg) in tortoises (n=9). Bars represent
the standard deviations.
Table 1: Pharmacokinetic parameters of enrofoxacin and ciprofoxacin
after 10 mg/kg enrofoxacin intracoelomic injection in tortoises
(Testudo hermani) (n=9)
Enrofoxacin Ciprofoxacin
Parameter Units Mean SD Mean SD
r
2
0.99 ± 0.01 0.97 ± 0.01
λz 1/hr 0.02 ± 0.03 0.01 ± 0.01
T
1/2
λz
hr 37.00 ± 11.97 49.06 ± 5.82
T
max
hr 2.19 ± 0.58 4.23 ± 0.93
C
max
ng/mL 8614 ± 1116 605.16 ± 43.04
AUC
0-24
hr*ng/mL 66388 ± 4647 7952 ± 318
AUC
0-∞
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
AUMC
0-∞
hr*hr*ng/mL 3383764 ± 42011 412688 ± 58959
MRT
0-∞
hr 33.13 ± 1.69 32.15 ± 1.28
r
2
= correlation coefcient.
λz = terminal phase rate constant.
T
1/2
λz = terminal half-life.
T
max
= time of peak.
C
max
= peak plasma
concentration.
Vz/F = apparent volume of
distribution.
CL/F = apparent clearance.
AUC
0-24
= area under the
plasma concentration-time
from 0-24 h curve.
AUC
0-∞
= area under the plasma
concentration-time from 0 h to
infnity curve.
AUMC
0-∞
= area under the frst
moment curve.
MRT
0-∞
= mean resident time.
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Israel Journal of Veterinary Medicine  Vol. 70 (1)  March 2015 Salvadori, M. 48
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Research Articles