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PHARMACOKINETIC BEHAVIOR OF ENROFLOXACINAND ITS METABOLITE CIPROFLOXACIN IN URUTUPIT VIPERS (BOTHROPS ALTERNATUS) AFTERINTRAMUSCULAR ADMINISTRATIONAuthor(s): Samanta Waxman , D.V.M., Ph.D., Ana Paula Prados , D.V.M, JoséJulio de Lucas , D.V.M., Ph.D., Manuel Ignacio San Andrés , D.V.M., Ph.D.,Pablo Regner , D.V.M., Vanesa Costa de Oliveira , B.Sc., D.V.M., Adolfo deRoodt , D.V.M., Ph.D. and Casilda Rodríguez , D.V.M., Ph.D.Source: Journal of Zoo and Wildlife Medicine, 45(1):78-85. 2014.Published By: American Association of Zoo VeterinariansDOI: http://dx.doi.org/10.1638/2013-0131R.1URL: http://www.bioone.org/doi/full/10.1638/2013-0131R.1
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Journal of Zoo and Wildlife Medicine 45(1): 78–85, 2014
Copyright 2014 by American Association of Zoo Veterinarians
PHARMACOKINETIC BEHAVIOR OF ENROFLOXACIN AND ITS
METABOLITE CIPROFLOXACIN IN URUTU PIT VIPERS
(BOTHROPS ALTERNATUS) AFTER INTRAMUSCULAR
ADMINISTRATION
Samanta Waxman, D.V.M., Ph.D., Ana Paula Prados, D.V.M, Jose Julio de Lucas, D.V.M., Ph.D.,
Manuel Ignacio San Andres, D.V.M., Ph.D., Pablo Regner, D.V.M., Vanesa Costa de Oliveira, B.Sc.,
D.V.M., Adolfo de Roodt, D.V.M., Ph.D., and Casilda Rodrıguez, D.V.M., Ph.D.
Abstract: Enrofloxacin is widely used in veterinary medicine and is an important alternative to treating
bacterial infections, which play an important role as causes of disease and death in captive snakes. Its extralabel
use in nontraditional species has been related to its excellent pharmacokinetic and antimicrobial characteristics.
This can be demonstrated by its activity against gram-negative organisms implicated in serious infectious diseases
of reptile species with a rapid and concentration-dependent bactericidal effect and a large volume of distribution.
Pharmacokinetic parameters for enrofloxacin were investigated in seven urutu pit vipers (Bothrops alternatus),
following intramuscular injections of 10 mg/kg. The plasma concentrations of enrofloxacin and its metabolite,
ciprofloxacin, were measured using high-performance liquid chromatography. Blood samples were collected from
the ventral coccygeal veins at 0.5, 1, 2, 4, 8, 12, 24, 36, 48, 72, 96, 108, and 168 hr. The kinetic behavior was
characterized by a relatively slow absorption (time of maximal plasma concentration¼ 4.50 6 3.45 hr) with peak
plasma concentration of 4.81 6 1.12 lg/ml. The long half-life during the terminal elimination phase (t1/2k¼27.91 6
7.55 hr) of enrofloxacin after intramuscular administration, calculated in the present study, could suggest that the
antibiotic is eliminated relatively slowly and/or the presence of a slow absorption in urutu pit vipers.
Ciprofloxacin reached a peak plasma concentration of 0.35 lg/ml at 13.45 hr, and the fraction of enrofloxacin
metabolized to ciprofloxacin was 13.06%. If enrofloxacin’s minimum inhibitory concentration (MIC90) values of
0.5 lg/ml were used, the ratios AUCeþc : MIC90 (276 6 67 hr) and Cmaxeþc : MIC90 (10 6 2) reach the proposed
threshold values (125 hr and 10, respectively) for optimized efficacy and minimized resistance development when
treating infections caused by Pseudomonas. The administration of 10 mg/kg of enrofloxacin by the i.m. route
should be considered to be a judicious choice in urutu pit vipers against infections caused by microorganisms with
MIC values �0.5 lg/ml. For less susceptible bacteria, a dose increase and/or an interval reduction should be
evaluated.
Key words: Bothrops alternatus, enrofloxacin, fluoroquinolone, intramuscular, pharmacokinetic, urutu.
INTRODUCTION
Bacterial infections play an important role as
causes of disease and death in captive snakes. It is
now understood that most reptile bacterial path-
ogens are gram negative, although many of these
pathogens could be part of the host’s normal flora,
becoming pathogenic when the animal is immune
suppressed, after viral infection or stressed under
the conditions of captivity. Bacteria commonly
isolated from reptiles include Aeromonas hydroph-
ila, Klebsiella oxytoca, Morganella morganii, Provi-
dencia rettgeri, Pseudomonas aeruginosa, and Sal-
monella arizonae.1,5 Fluoroquinolones’ spectrum of
activity includes mainly gram-negative bacteria,
such as Enterobacteriaceae, although these drugs
are less active against gram-positive bacteria.
They have also shown to be effective against
some other pathogens including chlamydias, rick-
ettsias, mycobacteria, and mycoplasmas.1,18,20 Iso-
lates from different animal species usually show
minimum inhibitory concentration (MIC90) values
for fluoroquinolones �0.125 lg/ml, except for P.
aeruginosa, which can reach MIC90 values of 0.5–8
lg/ml.20,26
Enrofloxacin is a fluoroquinolone that is widely
used in veterinary medicine. Its extralabel use in
nontraditional species has been related to its
excellent pharmacokinetic and antimicrobial
characteristics. This can be demonstrated by its
activity against gram-negative organisms impli-
From the Facultad de Ciencias Veterinarias, Universi-
dad de Buenos Aires, Buenos Aires, 1427, Argentina
(Waxman, Prados, Regner); the Department of Toxicology
and Pharmacology, Facultad de Veterinaria, Universidad
Complutense de Madrid, Ciudad Universitaria, Madrid,
28040, Spain, (de Lucas, San Andres, Rodrıguez); the
Laboratorio de Toxinopatologıa, Centro de Patologıa
Experimental y Aplicada, Facultad de Medicina, Universi-
dad de Buenos Aires, Buenos Aires, 1121, Argentina (de
Roodt, Regner, Costa de Oliveira). Correspondence should
be directed to Dr. Rodrıguez ([email protected]).
78
cated in serious infectious diseases of reptile
species. These properties in general include low
MICs (except for Pseudomonas sp. and Proteus sp.),
a rapid and concentration-dependent bactericidal
effect, and a large volume of distribution.18,20,24
Also, fluoroquinolones are an important alterna-
tive to aminoglycosides, the most frequently used
antibiotics against gram-negative bacteria in rep-
tiles, as enrofloxacin is less nephrotoxic than
aminoglycosides.1
Metabolic scaling of drugs could be a practical
and useful tool in antimicrobial dosing; however,
there are many limitations that must be consid-
ered in reptiles, such as mass constant or phar-
macokinetic differences between widely divergent
species independent of metabolic rate.13,17 Also,
significant differences in the pharmacokinetic
behavior of fluoroquinolones among reptile spe-
cies have been described.7,8,10,12,14–16,22,23,26 These
considerations mark the importance of conduct-
ing studies for individual species rather than
extrapolating doses and dosing intervals from
data generated in other species, even within a
taxonomic group. The aim of this study was to
determine the pharmacokinetic behavior of enro-
floxacin after intramuscular administration in
urutu pit vipers (Bothrops alternatus), in order to
estimate pharmacokinetic/pharmacodynamic
(PK/PD) integration for the optimization of
dosage schedules in this species.
MATERIALS AND METHODS
Seven clinically healthy adult snakes (B. alter-
natus), aged 2 to 4 yr, weighing 0.67 6 0.13 kg,
were used. The animals were housed at the
National Institute for Biologics Production
INPB–ANLIS ‘‘Dr. Carlos G. Malbran,’’ Buenos
Aires, Argentina. Snakes were acclimated at 27–
298C for at least 6 mo prior to the study and
maintained at this temperature during the period
of sample collection. Animals were allocated
individually in plastic containers (50 3 45 3 25
cm) at 27–298C, with relative humidity ranging
between 70% and 75%, and fed in an appropriate
manner for the species (diet consisted of approx-
imately two to four mice every 20 days). No drugs
were administered for at least 2 mo prior to the
start of the study. Routine acceptance of daily
meals, maintenance of body weight, hematologic
control, and complete physical examination were
used as criteria for selection of healthy animals.
The injection site was monitored each time the
animals were taken out of the cages for sample
collection during the sample collection period,
once daily during the second week and 1 mo after
the intramuscular administration of enrofloxacin.
The study was approved by the Institutional
Animal Care and Use Committee.
Each animal received a 10 mg/kg single dose of
an enrofloxacin 5% injectable solution (Baytrilt,
Bayer Hispania, S. L., Sant Joan Despı, Barcelo-
na, Comunidad Autonoma de Cataluna, 08970,
Spain), into the dorsal muscles of the cranial half
of the animal. Blood samples (0.6 ml at each time
point) were collected from the ventral coccygeal
veins with a 22 ga needle attached to a 1-ml
heparinized syringe at 0.5, 1, 2, 4, 8, 12, 24, 36, 48,
72, 96, 108, and 168 hr. No anesthetics or
tranquilizers were administered for sample col-
lection. Animals had free access to water during
the entire study. Plasma was separated immedi-
ately in a refrigerated centrifuge and frozen at
�808C until analysis.
Plasma concentrations of enrofloxacin and its
active metabolite, ciprofloxacin, were simulta-
neously quantified in all samples using high-
performance liquid chromatography with an UV
detector, according to a method previously de-
scribed.2 Ofloxacin (Lot No. 038K1555, Sigma-
Aldrich Quımica, S. L., Tres Cantos, Comunidad
Autonoma de Madrid, 28760, Madrid, Spain) was
used as the internal standard (5 lg/ml). Enro-
floxacin (Lot No. 0001369030, Sigma-Aldrich
Quımica, S. L.) and ciprofloxacin (Lot No.
0001396108, Sigma-Aldrich Quımica, S. L.) were
used for the preparation of calibration standards.
The quantification limits (LOQs) of the assay
method were 0.025 and 0.1 lg/ml for enrofloxacin
and ciprofloxacin, respectively. The standard
curves were linear between 0.025 and 10 lg/ml
for enrofloxacin, and between 0.1 and 5 lg/ml for
its active metabolite ciprofloxacin. Calibration
curves had R2 . 0.99 for each day’s analysis,
intraday precision was ,6%, interday precision
was ,10%, and accuracy oscillated between 85%and 120%.
The data are expressed as arithmetic mean 6
SD. The statistical analysis was performed using
the SPSS 19.0 software package. Pharmacokinet-
ic parameters were determined by means of
noncompartmental analysis (PCNONLIN ver-
sion 4.0 program; SCI Software, Lexington,
Kentucky 40504, USA). Values calculated fol-
lowing the intramuscular administration were
area under the plasma concentration vs. time
curve (AUC), area under the first moment curve
(AUMC), mean residence time (MRT, where
MRT ¼ AUMC : AUC), terminal rate constant
(k, calculated as the slope of the terminal phase
of the plasma concentration curve that included a
WAXMAN ET AL.—ENROFLOXACIN PHARMACOKINETIC IN URUTU PIT VIPERS 79
minimum of four points), and terminal half-life
(t1/2k, where t1/2k¼0.693/k). The AUC and AUMC
were calculated using a trapezoidal rule with
extrapolation to infinity (‘). The extrapolated
area did not exceed 5.5% of the total area.
Also, the following PK/PD indices to predict
clinical success and the development of resistant
mutants were determined: peak drug concentra-
tion (Cmax) : MIC and AUC : MIC. These indices
were calculated on the basis of the MICs of 0.12,
0.2, 0.5, and 1 lg/ml according to previously
published works done with reptile species.15,26
Ciprofloxacin is described to have a more potent
antimicrobial effect than the parent drug for many
veterinary pathogens.6 For this reason, it would be
more appropriate to utilize the AUCeþc and
Cmaxeþc values obtained by analyzing both drugs
together, enrofloxacin and ciprofloxacin, in com-
puting pharmacodynamic variables, even though
this species biotransformed only a small portion
of enrofloxacin into this metabolite.
RESULTS
All animals remained in good health through-
out the acclimatization and study periods. Clinical
evaluation, including review of food intake,
responses to stimuli, and physical observation,
of all animals during and 2 mo after the experi-
ment did not demonstrate that the experiment had
not altered them after drug administration. No
signs of pain or other adverse reactions at the site
of injection were observed. Arithmetic mean
(6SD) enrofloxacin and ciprofloxacin plasma
concentration vs. time curves after intramuscular
administration are shown in Figure 1. The phar-
macokinetic parameters of both drugs subjected
to noncompartmental analysis are presented in
Table 1. In B. alternatus, ciprofloxacin comprised
13.06 6 7.51% of the total fluoroquinolone
(enrofloxacin and ciprofloxacin) AUC. PK/PD
values obtained following intramuscular adminis-
tration of enrofloxacin using different MIC values
are present in Table 2.
DISCUSSION
Intramuscular administration was selected in
this current study because parenteral injections
are preferred in larger specimens or when working
around the head and mouth of poisonous snakes
(which could be dangerous). Also, the marked
variability of the pharmacokinetic parameters
that has been observed in some reptile species,
such as green iguanas, after oral administration,16
may make the intramuscular route more suitable
for the treatment of critical infections.
In a previously published study in ball pythons
(n¼6), 39 blood samples were obtained from each
manually restrained individual over a serial col-
lection study of 120 days by cardiocentesis.11 No
clinically apparent complications were noticed in
any of the study animals after each cardiocentesis
procedure until at least 73 days. However, these
authors observed a small pericardial hematoma in
one animal and moderate collagen fibrosis and
focal thickening in epicardium with a mild infil-
trate of macrophages and heterophils in the
epicardium of all snakes.11 Because a researcher
should select a method that causes the least
amount of pain and suffering, especially if an
animal is awake and manually restrained when the
sample is collected, venipuncture was preferred
over cardiocentesis in this current study as the
method for sample collection, as suggested by
other authors.26
Recent works in turtles and pythons failed to
demonstrate a significant effect of the injection
site on the pharmacokinetics of drugs, even those
whose kinetic behavior might be affected by the
renal-portal system.9 This would make the caudal
region of the reptilian body available for the
administration of medicines. However, any possi-
ble influence of the reptile renal-portal system on
enrofloxacin’s pharmacokinetics was avoided by
injecting the drug into the cranial muscles of the
snake.
In previous studies, some signs suggesting
tissue damage following intramuscular adminis-
tration of enrofloxacin have been described. Some
studies have indicated that this problem could be
related to the injection volume (.1 ml).26 These
authors suggest that volumes .1 ml should be
injected at multiple sites, and values lower than
Figure 1. Plasma concentrations (mean 6 SD) of
enrofloxacin and its metabolite, ciprofloxacin, plotted
against time for urutu pit vipers (n ¼ 7), following
intramuscular administration of enrofloxacin at a single
dose of 5 mg/kg.
80 JOURNAL OF ZOO AND WILDLIFE MEDICINE
0.5 ml are adequate. In this current study, no signs
of local pain were observed, possibly because of
the volume administered, which varied between
0.11 and 0.12 ml.
The study was conducted at a room tempera-
ture of 27–298C, which is within the optimum
temperature range of urutu pit vipers (27–328C)
and is similar to that reported in other pharma-
cokinetic studies (Table 3). In ectothermic ani-
mals, body temperature may have a major effect
on the therapeutic levels achieved as well as the
toxicity of agents. This was taken into account
because it has been reported that, in sick snakes, it
is important to correct hypothermia and to
maintain a preferred optimum temperature zone,
not only to improve the animal’s condition, but
also to improve drug absorption and distribu-
tion.1,4
Enrofloxacin is biotransformed into ciprofloxa-
cin in many animal species; this metabolite is
more active than enrofloxacin against gram-nega-
tive pathogen microorganisms. Therefore, plasma
concentrations of ciprofloxacin may have an
additive antimicrobial effect on concurrent enro-
floxacin levels. In urutu pit vipers, ciprofloxacin
reached a peak plasma concentration of 0.35 lg/ml at 13.45 hr and the fraction of enrofloxacin
metabolized to ciprofloxacin was 13.06%. This
Table 1. Pharmacokinetic parameters of enrofloxacin and its active metabolite ciprofloxacin obtained afterintramuscular administration of enrofloxacin (10 mg/kg) in B. alternatus.a
Pharmacokinetic parameters Arithmetic mean Median Minimum Maximum SD Geometric mean
Enrofloxacin
Tmax (h) 4.50 4.00 0.50 8.00 3.45 2.97
Cmax (lg/mL) 4.81 4.61 3.64 6.42 1.12 4.71
Clast (lg/mL) 0.0522 0.0335 0.0137 0.1827 0.0589 0.0363
k (h�1) 0.0263 0.0278 0.0169 0.0333 0.0063 0.0256
T1/2k (h)b 27.91 24.94 20.84 41.03 7.55 26.39
AUCt (lg h/mL) 118.0 121.2 70.90 146.8 24.78 115.4
AUC‘ (lg h/mL) 120.0 121.5 72.44 155.1 26.10 117.2
Vz-F (L/kg) 3.56 2.79 2.47 6.84 1.57 3.34
Cl-F (L/h kg) 0.0877 0.0823 0.0645 0.1380 0.0240 0.0853
MRTt (h)b 30.39 29.22 23.20 44.86 7.06 29.24
MRT‘ (h)b 33.24 31.44 23.79 54.65 10.14 31.26
Ciprofloxacin
Tmax (h) 13.45 8.12 8.00 36.00 10.22 11.42
Cmax (lg/mL) 0.3914 0.3700 0.2000 0.6200 0.1326 0.3719
Tlast (h) 90.50 96.68 8.00 168.75 50.50 68.52
Clast (lg/mL) 0.1364 0.1200 0.0950 0.2000 0.0411 0.1314
AUCt (lg h/mL) 20.00 19.05 0.83 44.60 13.77 13.10
MRTt (h)b 36.94 41.32 5.95 60.97 17.63 21.65
Enrofloxacin and ciprofloxacin
Tmax (h) 5.29 4.00 1.00 12.00 4.11 3.84
Cmaxeþc (lg/mL) 4.98 4.56 3.95 6.57 1.11 4.88
AUCeþc (lg h/mL) 138.0 140.2 71.72 182.6 36.01 133.1
a Tmax, time of maximal plasma concentration; Cmax, peak drug concentration; Clast, last measurable plasma concentration
(168 hr); k, rate constant for decline in plasma concentration; T1/2k, terminal half-life; AUCt, area under the plasma concentration
vs. time curve from time 0 to last quantifiable time (168 hr); AUC‘, area under the plasma concentration vs. time curve from time
0 to infinity; Vz-F, apparent volume of distribution during terminal phase after intramuscular administration; Cl-F, apparent total
clearance of the drug from plasma after intramuscular administration; MRTt: mean residence time from time 0 to last time (168
hr); MRTi, mean residence time from time 0 to infinity.b Harmonic mean.
Table 2. Efficacy indices obtained after intramus-cular administration of enrofloxacin in B. alternatus.Data are presented as mean 6 SD.a
MIC90 (lg/mL ) AUCeþc/MIC Cmaxeþc/MIC
0.12 1,150 6 277.8 41.51 6 8.57
0.25 552.0 6 133.3 19.92 6 4.12
0.5 276.0 6 66.67 9.96 6 2.06
1 138.0 6 33.34 4.98 6 1.03
a MIC, minimum inhibitory concentration; AUCeþc, area
under the plasma concentration time curve from time 0 to last
quantifiable time obtained by analyzing enrofloxacin and
ciprofloxacin together; Cmaxeþc, peak concentration obtained
by analyzing enrofloxacin and ciprofloxacin together.
WAXMAN ET AL.—ENROFLOXACIN PHARMACOKINETIC IN URUTU PIT VIPERS 81
suggests that, in this species, the antimicrobial
activity of enrofloxacin could be attributable, at
least in part, to its main metabolite, ciprofloxa-
cin. Similar values of ciprofloxacin’s Cmax and
time of maximal plasma concentration have
been observed in Burmese pythons (0.35 lg/ml
and 13 hr, respectively), despite the lower dose
received (5 mg/kg) and the lower values of
enrofloxacin AUC described.26 Low levels of
conversion to ciprofloxacin (around 0.1 lg/ml)
have been described for species such as Amer-
ican alligators8 (oral administration), Savannah
monitors,10 and green iguanas16 (,LOQ). Be-
cause metabolism of enrofloxacin to ciprofloxa-
cin occurs via de-ethylation of the ethyl group
on the piperazine ring, such results may suggest
a difference in the enzymatic metabolism and/or
in the rate of hepatic metabolism among reptile
species.
The long half-life during the terminal elimi-
nation phase (t1/2k . 27 hr) of enrofloxacin after
intramuscular administration calculated in the
present study suggests that the antibiotic is
eliminated relatively slowly in urutu pit vipers.
Also, this rate of elimination could be influenced
by a slow rate of absorption in this species. The
t1/2k obtained in urutu is similar to the values of
26 hr found in green iguana16 and 19.02 hr in
estuarine crocodiles,15 higher than those de-
scribed in Indian star tortoises23 (5.1 hr) and
Burmese pythons26 (6.37 hr) and lower than the
value reported for Savanna monitors (56 hr).
The dose used in most of these studies was 5
mg/kg; however, a 10 mg/kg dose was adminis-
tered in urutu pit vipers and Savanna monitors.
An advantage of such a long half-life is that the
administration of enrofloxacin in this snake may
be performed less frequently when compared
with other species.
In snakes, only one pharmacokinetic study has
been performed on pythons.26 It can be ob-
served, when comparing the other study to this
present study, that the dose of 10 mg/kg
administered to urutu pit vipers produced
higher AUC and Cmax values and a longer
permanence than those obtained in pythons26
(area under the plasma concentration vs. time
curve from time 0 to last quantifiable time
[AUCt] ¼ 22.17 lg hr/ml; Cmax ¼ 1.66 lg/ml;
T1/2k ¼ 6.37 hr) after an intramuscular dose of 5
mg/kg. The AUCt and Cmax values obtained in
urutu pit vipers were fivefold and threefold
higher than those obtained in pythons, respec-
tively, even though they received only twice the
dose. There are many factors, such as random
Table
3.
Pharm
aco
kineticparameters
obtainedafterintramuscularadministrationofenro
floxacinin
reptiles.
a
Species
Method
Ambienttemperature
(8C)
Dose
(mg/kg)
Cmax
(lg/mL)
Tmax
(h)
t 1/2k
(h)
AUC
t
(lgh/mL)
AUC
‘
(lgh/mL)
MRT
t
(h)
MRT
‘
(h)
Croco
dylusporosus1
5HPLC
24–3
25
8.9
0.65
19.02
74.38
133.23
10.2
28.2
Gopheruspolyphem
us2
2HPLC
30
61
52.4
123.1
56.7
na
27.6
na
Geo
cheloneelegans2
3HPLC
26–3
05
3.59
0.5
5.1
na
19.9
7
Varanusex
anthem
aticu
s10
HPLC
27
10
12.47
656
na
na
na
na
Iguanaiguana16
HPLC
na
52.03
126
na
na
na
na
Pythonmolurus2
6HPLC
30
51.66
5.75
6.37
22.17
na
na
na
B.alternatus
HPLC
27–2
910
4.81
4.5
27.91
118
120
30.4
33.2
aCmax,peakdru
gco
nce
ntration;Tmax,timeofmaxim
alplasm
aco
nce
ntration;T
1/2k,term
inalhalf-life;AUCt,areaundertheplasm
aco
nce
ntrationvs.
timecurvefrom
time0to
last
quantifiable
time(168hr);AUC
‘,areaundertheplasm
aco
nce
ntrationvs.timecu
rvefrom
time0to
infinity;M
RTt:meanresidence
timefrom
time0to
last
time(168hr);na,notavailable.
82 JOURNAL OF ZOO AND WILDLIFE MEDICINE
variability, study design, sample collection, data
analysis, sample analysis, species differential
metabolism and elimination, and/or nonlinear
pharmacokinetics that could justify these find-
ings. If both studies performed in snakes were
compared, it could be observed that a similar
number of animals (urutu, n ¼ 7; python,26 n ¼ 6)
and pharmacokinetic analysis was used. There
are, however, differences in sample collection
times (urutu, at 0.5, 1, 2, 4, 8, 12, 24, 36, 48, 72,
96, 108, and 168 hr; python, at 0.5, 1, 3, 6, 12, 24,
48, 72, and 96 hr26) and different limits of
quantification were described. These factors
could also be implicated in the pharmacokinetic
differences observed between the two species.
Perhaps a nonlinear pharmacokinetic behavior
could be involved, as was previously described for
this drug in loggerhead sea turtles after oral
administration of 10 and 20 mg/kg, with values
of AUC‘ of 261 and 1799 lg hr/ml, respectively.
Also, species differences have been observed
between tortoise species.14,22,23 Although it is very
difficult to draw meaningful conclusions from
historical data and, therefore, they should be
made with extreme caution, the main differences
in the pharmacokinetic behavior between the two
snake species could support the importance of
conducting studies for individual species. The
foregoing information tends to solicit caution
when extrapolating doses and dosing intervals
from data generated in other species, even within
a taxonomic group.
Previous studies suggest that an optimum
bactericidal effect of fluoroquinolones and a
lower incidence of resistance development are
associated with values of the efficacy parameters
Cmax : MIC and AUC : MIC �10 and �125–250,respectively. These values would allow for pre-
dicting clinical success. However, these break-
points were obtained from studies in laboratory
animals (usually immunosuppressed) or people
with severe illness and, perhaps, the ratios needed
for a cure in many veterinary patients could be
lower. Thus, in immunocompetent patients, val-
ues of AUC : MIC lower than 125 (50–60) and
Cmax : MIC ratios of 3–5.5 may also likely to be
effective.20,25
It is important to coassess the PK/PD param-
eters AUC : MIC90 and Cmax : MIC90 because the
in vitro activity of enrofloxacin and ciprofloxacin
may differ by 1 or 2 log2 dilutions for some
pathogens. To the authors’ knowledge, only enro-
floxacin’s MIC value for Pseudomonas spp. (0.5
lg/ml) has been reported from snake isolates.26 If
we use enrofloxacin’s MIC90 values of 0.5 lg/ml,
the ratios AUCeþc : MIC90 (276 6 67 hr) and
Cmaxeþc : MIC90 (10 6 2) reach the proposed
threshold values (125 hr and 10, respectively) for
optimized efficacy and minimized resistance de-
velopment while treating infections caused by
microorganism with an MIC90 value �0.5 lg/ml
(Table 2). However, it is likely that the MIC90 of
Pseudomonas has increased in snake species in
recent years, as is described for fluoroquinolones
in other species where higher MIC90 values of up
to 4 and 8 lg/ml were determined for the P.
aeruginosa isolates from urinary/genital tract and
skin/ear infections, respectively.24 Therefore,
higher MIC values should be taken into account
for P. aeruginosa. In this current study, 0.53 lg/ml
is an MIC breakpoint because values of Cmax :
MIC and AUC : MIC are higher than 10 and 125,
respectively. Thus, with the administration of 10
mg/kg of enrofloxacin by the i.m. route in urutu
pit vipers, the above current recommended min-
imum values of PK/PD indices could be reached
when an MIC90 � 0.53 lg/ml is observed for an
individual-specific isolate. If an MIC90 value
.0.53 lg/ml (as described for Pseudomonas sp.
for other fluoroquinolones in companion ani-
mals24) is reached, this dose (10 mg/kg) might be
insufficient. Therefore, it should be noted that
individual clinical responses to the treatment will
vary based on the MIC90 of the specific isolate.
On the other hand, the increasing problem of
emergence of resistance under the influence of
antibiotic selection pressure has led to the
identification of PK/PD indices that best corre-
late with the prevention of antimicrobial resis-
tance. A drug concentration capable of inhibiting
the growth of the least-susceptible single-step
mutant subpopulation has been called the pre-
vention concentration (MPC). Whereas MIC
determines the susceptibility of most of the cells
of a bacterial population, MPC provides infor-
mation about the sensibility of small resistant
subpopulations. MPC is a pharmacodynamic
parameter that may be a useful tool to guide the
dosage of antibiotics in order to reduce the
emergence of bacteria with decreased antibiotic
susceptibility. Some authors have suggested that
the single pharmacodynamic index that shows
the least variation, and therefore best predicts
the prevention of resistance emergence, is
AUC : MPC.19,21 There are no data available
about MPC values of enrofloxacin in reptiles,
although it has been reported in a work with
original strains that MPC values for enrofloxacin
against P. aeruginosa were 16 times MIC.21 If the
results obtained in that study were extrapolat-
WAXMAN ET AL.—ENROFLOXACIN PHARMACOKINETIC IN URUTU PIT VIPERS 83
ed,21 an MPC value of 8 lg/ml and an AUCeþc :
MIC value of 17.25 could be attained. In a
previously published work concerning dose-re-
lated selection of fluoroquinolone resistance, an
AUC : MPC value of 35 proved to be enough to
prevent the growth of the resistant single mutant,
whereas the next-lowest tested AUC : MPC value
of 14 was insufficient.19 The low AUC : MPC
ratios of enrofloxacin against P. aeruginosa may
predict a low in vivo efficacy in preventing the
emergence of resistant P. aeruginosa strains.
Taking into account these preliminary indices,
which have not yet been established for enro-
floxacin in reptiles, these current results suggest
that enrofloxacin, at the administered dose in
snakes, would probably not avoid resistance of
wild strains of Pseudomonas spp. With the AUC
values obtained in this current study, in order to
reach an AUC : MPC value of 35, only an MPC
for sensitive isolates (MPC , 4 lg/ml) could be
attained with a dose of 10 mg/kg. Furthermore,
the concentrations needed for infections involv-
ing isolates with high MPCs are unlikely to be
achievable in vivo. Taking all these data into
account, it could be concluded that the dosage
regimen used in this current study would not
avoid the selection of resistant mutants for
subpopulations with high MPC values. And
moreover, treatment with combinations of anti-
microbials should be adopted. Nevertheless,
these results should be interpreted very carefully,
because MPC values were calculated for isolates
from mammalian species.21
Even though there is no recommended dose for
enrofloxacin in urutu pit vipers, different values
have been reported for other snake species (5 mg/
kg q. 24 hr–10 mg/kg q. 48 hr).3 Accordingly,
based on the results of this study, and with the
authors’ clinical experience and previously pub-
lished data,3,26 it is considered that the adminis-
tration of 10 mg/kg of enrofloxacin by the i.m.
route, with an interval of 48–72 hr, could be
appropriate for infectious diseases caused by
microorganisms with MIC90 , 0.53 lg/ml in this
species. However, further studies with multiple
dosing protocols must be performed in order to
assess for drug accumulation. For less susceptible
bacteria, a dose increase and/or an interval
reduction should be evaluated. Moreover, addi-
tional studies should be conducted in order to
assess possible side effects derived from an
increase in the dose.
Acknowledgments: The authors wish to thank
Mr. Mariano Dıaz-Flores for his technical assis-
tance. Special thanks to the staff of the Biblioteca
de la Facultad de Veterinaria for their invaluable
help. This study was performed as part of the
UBACyT project 20020090200230, supported by
the Secretarıa de Ciencia y Tecnica, Universidad
de Buenos Aires.
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Received for publication 13 June 2013
WAXMAN ET AL.—ENROFLOXACIN PHARMACOKINETIC IN URUTU PIT VIPERS 85
