Pharmacokinetic Behaviour of Enrofloxacin after Single Intramuscular Dosage in American Black Vultures (Coragyps atratus)

Abstract

The aim of the study was to investigate the intramuscular pharmacokinetics of enrofloxacin in black vultures (Coragyps atratus). The pharmacokinetics of a single intramuscular dose (10 mg/kg) of enrofloxacin was studied in six vultures. Plasma concentrations of enrofloxacin and its active metabolite, ciprofloxacin, were determined by high-performance liquid chromatography (HPLCuv). Pharmacokinetic parameters were estimated using non-compartmental and compartmental analysis. After intramuscular administration, enrofloxacin showed a rapid and complete absorption, reaching a Cmax value of 3.26 ± 0.23 μg/mL at 1.75 ± 0.53 h. A long terminal half-life of 19.58 h has been observed. Using previously published MIC values to perform a PK/PD analysis, cumulative fraction responses obtained after Monte Carlo simulation for AUC/MIC > 30, 50 and 125 were 72.93%, 72.34% and 30.86% for E. coli and 89.29%, 88.89% and 58.57% for Mycoplasma synoviae, respectively. Cumulative fraction responses obtained for Cmax/MIC index were 33.93% and 40.18% for E. coli and M. synoviae, respectively. The intramuscular administration of 10 mg/kg could be appropriate to treat infectious diseases caused by gram-positive bacteria with MIC value lower than 1 µg/mL; however, although enrofloxacin showed a slow elimination in black vultures, plasma concentrations were insufficient to reach the gram-negative stablished breakpoints.

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Article
Pharmacokinetic Behaviour of Enrofloxacin after Single
Intramuscular Dosage in American Black Vultures
(Coragyps atratus)
Samanta Waxman 1,2,*, JoséJulio de Lucas 3, Guillermo Wiemeyer 1,4,5, Laura Torres Bianchini 1,4,
Manuel Ignacio San Andrés3and Casilda Rodríguez 3


Citation: Waxman, S.; de Lucas, J.J.;
Wiemeyer, G.; Torres Bianchini, L.;
San Andrés, M.I.; Rodríguez, C.
Pharmacokinetic Behaviour of
Enrofloxacin after Single
Intramuscular Dosage in American
Black Vultures (Coragyps atratus).
Antibiotics 2021,10, 957. https://
doi.org/10.3390/ antibiotics10080957
Academic Editor: Abdelwahab Omri
Received: 20 July 2021
Accepted: 6 August 2021
Published: 9 August 2021
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
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iations.
Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
1Facultad de Ciencias Veterinarias, Universidad de Buenos Aires, Chorroarin 280,
Buenos Aires 1427, Argentina; gwiemeyer@fvet.uba.ar (G.W.); laura.wallaby@gmail.com (L.T.B.)
2Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires 1425, Argentina
3Department of Pharmacology and Toxicology, Veterinary Faculty, Universidad Complutense de Madrid,
Av. Puerta de Hierro s/n, 28040 Madrid, Spain; delucas@vet.ucm.es (J.J.d.L.);
misanand@vet.ucm.es (M.I.S.A.); rodfermc@vet.ucm.es (C.R.)
4Jardín Zoológico de la Ciudad de Buenos Aires, R. de la India 3000, Buenos Aires 1425, Argentina
5Fundación Caburé-í, Sucre 2842, Buenos Aires 1428, Argentina
*Correspondence: waxman@fvet.uba.ar; Tel.: +54-11-52872109
Abstract:
The aim of the study was to investigate the intramuscular pharmacokinetics of enrofloxacin
in black vultures (Coragyps atratus). The pharmacokinetics of a single intramuscular dose (10 mg/kg)
of enrofloxacin was studied in six vultures. Plasma concentrations of enrofloxacin and its active
metabolite, ciprofloxacin, were determined by high-performance liquid chromatography (HPLCuv).
Pharmacokinetic parameters were estimated using non-compartmental and compartmental analysis.
After intramuscular administration, enrofloxacin showed a rapid and complete absorption, reaching
a Cmax value of 3.26
±
0.23
µ
g/mL at 1.75
±
0.53 h. A long terminal half-life of 19.58 h has been
observed. Using previously published MIC values to perform a PK/PD analysis, cumulative fraction
responses obtained after Monte Carlo simulation for AUC/MIC > 30, 50 and 125 were 72.93%,
72.34% and 30.86% for E. coli and 89.29%, 88.89% and 58.57% for Mycoplasma synoviae, respectively.
Cumulative fraction responses obtained for Cmax/MIC index were 33.93% and 40.18% for E. coli
and M. synoviae, respectively. The intramuscular administration of 10 mg/kg could be appropriate
to treat infectious diseases caused by gram-positive bacteria with MIC value lower than 1
µ
g/mL;
however, although enrofloxacin showed a slow elimination in black vultures, plasma concentrations
were insufficient to reach the gram-negative stablished breakpoints.
Keywords: black vultures; enrofloxacin; pharmacokinetic; PK/PD; Monte Carlo simulation
1. Introduction
Black vultures, as obligate scavengers, are of great value for their ecosystems by remov-
ing carrion and facilitating the flow within the food chain. This species is distributed along
the American continent, being widely represented, not only in South and Central America,
but also in the south of North America [
1
,
2
]. Infectious diseases are a common issue in
these New World vultures; they are susceptible to Pasteurella multocida and sepsis involving
other gram-negative bacteria, for which a broad-spectrum therapy with, for example, en-
rofloxacin, is indicated [
3
,
4
]. This drug is a fluoroquinolone with concentration-dependent
bactericidal effect, good activity against gram-negative organisms, some activity against
gram-positive organisms, minimal activity against anaerobic bacteria, but occasional ac-
tivity against Chlamydia spp., Mycoplasma spp., and Mycobacterium spp. Enrofloxacin may
be considered a good choice in cases of pododermatitis, showing evidence of persistent
or deep infection [
5
]. This drug is very frequently used in the treatment of infectious
diseases of vultures admitted in wildlife rehabilitation centres by the intramuscular (im)
Antibiotics 2021,10, 957. https://doi.org/10.3390/antibiotics10080957 https://www.mdpi.com/journal/antibiotics

Antibiotics 2021,10, 957 2 of 7
route. This route could represent an interesting alternative to oral administration, in as
much as anorexia, regurgitation and emesis have been observed in these species frequently
after oral administration of enrofloxacin [
6
]. Different pharmacokinetic behaviour has been
observed in vultures compared to other birds due to a slow elimination of drugs, such
as fluroquinolones [
7
,
8
] or diclofenac [
9
]; however, empirical dosage, recommended for
other species, is used for black vultures [
10
]. Dose extrapolation by linear, metabolic, or
allometric scaling has limitations and, ideally, dosage recommendations should be based
on species-specific pharmacokinetic and pharmacodynamic studies [
11
,
12
]. Intramuscular
administration of enrofloxacin is a practical route of administration, however, studies
reporting the intramuscular behaviour of enrofloxacin in birds of prey are scarce [13].
Currently, fluoroquinolones are included into highest priority critically important
antimicrobials in human medicine, since they are one of few available therapies for serious
Salmonella spp. and E. coli infections [
14
]. Additionally, they are included in Category B
(Restrict) of the categorization of antibiotic used in animals. They should be considered
only when there are no antibiotics in Categories Caution or Prudence (C or D) that could
be clinically effective and their use should be based on antimicrobial susceptibility testing,
whenever possible [15].
For these reasons, a rational use of this drug, based on pharmacological data is
necessary. The aim of our study was to investigate the intramuscular pharmacokinetics
of enrofloxacin in black vultures (Coragyps atratus) and to perform a PK/PD analysis by
Monte Carlo simulation using previously published MIC values against isolates obtained
from avian species, in order to evaluate the probability of a successful clinical outcome for
infections caused by such microorganisms.
2. Results
The individual concentration vs time curves, the final model fits, and the confidence
limits (95%) of the predictive check for model evaluation are shown in Figure 1. Measures
of central tendency and variability of the pharmacokinetic parameters obtained after
non-compartmental and compartmental analysis are shown in Table 1.
Antibiotics 2021, 10, x FOR PEER REVIEW 3 of 8
Figure 1. Plasma concentrations vs time profiles of enrofloxacin after a single intramuscular dose of
10 mg/kg in Coragyps atratus (n = 6). Solid lines show the final model fits. Shaded grey represents
the confidence intervals of 95% of the predictive check for model evaluation after Monte Carlo sim-
ulation. Observed values of each bird are shown in greyscale.
Table 1. Pharmacokinetic parameters obtained after enrofloxacin intramuscular administration (10 mg/kg) in black vul-
tures (Coragyps atratus) (n = 6).
Pharmacokinetic Paramete
r
Mean Median Geometric Mean Standard Deviation Range CV (%)
Non-Compartmental
Tmax (h) 1.75 2.08 1.67 0.53 1.05 30.3
Cmax (μg/mL) 3.26 3.25 3.25 0.23 0.73 7.1
T1/2λ (h) 1 19.58 19.51 19.45 1.74 5.32 8.9
AUCt (μg·h/mL) 60.41 63.39 60.00 7.49 17.89 12.4
MRTt (h)1 17.00 16.97 16.96 0.96 2.90 5.6
Tlast (h) 48.62 48.66 48.62 0.20 0.45 0.4
Clast (μg/mL) 0.55 0.58 0.55 0.10 0.24 18.2
Monocompartmental
AUCinf (μg·h/mL) 75.79 80.44 75.05 11.2 26.97 14.8
T1/2K01 (h) 1 0.24 0.25 0.23 0.07 0.18 29.2
T1/2K10 (h) 1 18.16 18.56 17.99 1.86 4.96 10.2
Tmax (h) 1.53 1.62 1.49 0.37 0.9 24.2
Cmax (μg/mL) 2.72 2.72 2.72 0.18 0.49 6.61
1 Harmonic mean.
Figure 1.
Plasma concentrations vs time profiles of enrofloxacin after a single intramuscular dose of
10 mg/kg in Coragyps atratus (n= 6). Solid lines show the final model fits. Shaded grey represents the
confidence intervals of 95% of the predictive check for model evaluation after Monte Carlo simulation.
Observed values of each bird are shown in greyscale.

Antibiotics 2021,10, 957 3 of 7
Table 1.
Pharmacokinetic parameters obtained after enrofloxacin intramuscular administration (10 mg/kg) in black vultures
(Coragyps atratus) (n= 6).
Pharmacokinetic Parameter Mean Median Geometric Mean Standard Deviation Range CV (%)
Non-Compartmental
Tmax (h) 1.75 2.08 1.67 0.53 1.05 30.3
Cmax (µg/mL) 3.26 3.25 3.25 0.23 0.73 7.1
T1/2λ(h) 119.58 19.51 19.45 1.74 5.32 8.9
AUCt(µg·h/mL) 60.41 63.39 60.00 7.49 17.89 12.4
MRTt(h) 117.00 16.97 16.96 0.96 2.90 5.6
Tlast (h) 48.62 48.66 48.62 0.20 0.45 0.4
Clast (µg/mL) 0.55 0.58 0.55 0.10 0.24 18.2
Monocompartmental
AUCinf (µg·h/mL) 75.79 80.44 75.05 11.2 26.97 14.8
T1/2K01 (h) 10.24 0.25 0.23 0.07 0.18 29.2
T1/2K10 (h) 118.16 18.56 17.99 1.86 4.96 10.2
Tmax (h) 1.53 1.62 1.49 0.37 0.9 24.2
Cmax (µg/mL) 2.72 2.72 2.72 0.18 0.49 6.61
1Harmonic mean.
Data were best fitted to a monocompartmental model. Monte Carlo simulation of
target attainment for a simulated black vulture population after intramuscular adminis-
tration of enrofloxacin (10 mg/kg) is shown in Table 2for AUC/MIC
≥
30, 50 and 125
and Cmax/MIC
≥
10 using Escherichia coli and Mycoplasma synoviae MIC values, re-
spectively. Cumulative fraction responses (CFR) obtained after Monte Carlo simulation
for
AUC/MIC > 30
, 50 and 125 were 72.93%, 72.34% and 30.86% for E. coli and 89.29%,
88.89% and 58.58% for M. synoviae, respectively. Cumulative fraction responses obtained
for Cmax/MIC index were 33.93% and 40.18% for E. coli and M. synoviae, respectively.
Table 2.
Monte Carlo simulation (n= 10000 subjects) of target attainment for a simulated black vulture population after
intramuscular administration of enrofloxacin at 10 mg/kg for MIC distribution of E. coli and M. synoviae previously
published [16,17].
PK/PD Index PTA (%) with an E. coli MIC
0.008 0.016 0.03 0.06
0.125
0.25 0.5 1 2 4 8 16
AUC/MIC = 125 100 100 100 100 100
50.12
0.01 0 0 0 0 0
CFR (%) 30.86
AUC/MIC = 50 100 100 100 100 100 100 100 100 0.15 0 0 0
CFR (%) 72.34
AUC/MIC = 30 100 100 100 100 100 100 100 100
69.71
0 0 0
CFR (%) 72.93
Cmax/MIC > 10 100 100 100 100 100
94.31
0 0 0 0 0 0
CFR (%) 33.93
PTA (%) with an M. synoviae MIC
0.1 0.25 0.5 1 2.5 10
AUC/MIC = 125 100 100
50.87
0 0 0
CFR (%) 58.57
AUC/MIC = 50 100 100 100 100 0 0
CFR (%) 88.89
AUC/MIC = 30 100 100 100 100 7.27 0
CFR (%) 89.29
Cmax/MIC > 10 100 87.2 0 0 0 0
CFR (%) 40.18

Antibiotics 2021,10, 957 4 of 7
3. Discussion
After intramuscular administration, enrofloxacin showed a rapid and complete ab-
sorption, reaching high Cmax and AUC values. Taking into account AUC values from
previously published data on intravenously administered enrofloxacin to black vultures [
8
],
a bioavailability value of 90% is estimated. This finding is in agreement with values de-
scribed in other avian species, which oscillated between 90% and 99% [
13
,
18
–
21
]. The
lowest bioavailability was observed in red-tailed hawk [
13
], rheas [
22
], and southern
crested caracaras [
23
]. Very low values of ciprofloxacin plasma concentrations were found
in black vultures. After intramuscular administration, ciprofloxacin was detected in only
three birds, and only one showed detectable values between 2 to 8 h, which oscillated
between 0.051 to 0.064
µ
g/mL. For these reasons, pharmacokinetic analysis of the active
metabolite was not performed.
Compared to other species, black vultures present the highest AUC. The obtained
AUC value for vultures is higher to that found for owls (15 mg/kg = 65.3
µ
g h/mL) [
13
],
but almost two folds those found in houbara bustard or chicken [
18
,
19
]. The lowest AUC
was found in ratites (ostriches 5 mg/kg = 1
µ
g h/mL, rheas,
15 mg/kg = 4.18 µg h/mL
, os-
triches 15 mg/kg = 6.24
µ
g h/mL) [
21
,
22
,
24
]. If AUC is corrected by the administered dose
(AUC/D), in general, birds of prey show high values, black vultures showing the highest
one (vultures 7.39; great horned owls 4.35, red tailed hawks 3.60; caracaras
3.44 kg h/L
),
while ratites show very low values [
22
–
24
]. These findings could be related to a slow
enrofloxacin clearance and a low extraction ratio observed in vultures after intravenous
administration [
8
]. This behaviour could also be related to a long enrofloxacin permanence
in black vultures, since elimination half-life showed values higher than 18 h. Compared to
other avian species, we observed that vultures present the longest body permanence, with
an elimination half-life almost double that observed in chickens (10.6 h) [
19
], and is almost
20 times longer than in ostriches [21].
These high Cmax and AUC values could be of clinical relevance for a concentration
dependent-antimicrobial as enrofloxacin. However, when the probability analysis using a
Monte Carlo simulation and previously published pharmacodynamic values is performed,
the pharmacokinetic profile does not seem to be enough to reach a clinical outcome. Most
experts agree that a Cmax/MIC ratio around 8 to 10 or an AUC/MIC ratio greater than
100 or 125 are associated with clinical success. For this reason, those breakpoints have
been taken into account in our study. The probability of target attainment (PTA) findings
revealed that the empirical enrofloxacin dose could be appropriate for patients with
E. coli
or M. synoviae infections with MIC values of 0.125 or 0.25, respectively, when a target cut-off
of AUC/MIC > 125 and Cmax/MIC > 10 is to be achieved. However, it has been stated
that infections caused by gram-positive bacteria can be successfully treated even when
AUC/MIC ratios are lower than 100. Thus, for this kind of infections, a ratio of 50 has
been proposed as acceptable [
25
]. Moreover, Ambrose et al. (2001) [
26
] have shown that a
successful clinical outcome can occur even at AUC/MIC ratios around 30 for community-
acquired pneumococcal pneumonia. If 30 and 50 cut-off targets were used, PTA > 90% can
be obtained for MIC values ≤1µg/mL.
The cumulative fraction of response pertains to the success probability for a treatment
without clinical susceptibility of the isolated pathogen. Data from the assessment of CFR
(%) for the enrofloxacin dose evaluated in black vultures show that CFR reached <90%
for E. coli and M. synoviae at an AUC/MIC of 125 h. The obtained CFR values are very
low, barely exceeding 50% for this breakpoint, although this species has shown a slower
enrofloxacin elimination and higher Cmax and AUC values than those found in all the
previously studied avian species. The estimated values have been slightly better for
stablished breakpoints of 30 and 50; especially to M. synoviae, being very close to 90%.
4. Materials and Methods
The experiment was performed in adult healthy black vultures (Coragyps atratus,
n= 6
),
weighing 1.8–2.2 kg, housed at Buenos Aires Zoological Garden, Argentina. Complete

Antibiotics 2021,10, 957 5 of 7
physical examination, haematological analysis, maintenance of body weight and routine
acceptance of daily meals were used as criteria for selection of healthy animals. No drugs
were administered for at least two months prior to the start of the experiment. Vultures
were housed in captivity, fed in an appropriate manner for the species, and had access to
water ad libitum.
A commercial 5% enrofloxacin injectable solution (Baytril, Bayer, Argentina) was
used. Ofloxacin, enrofloxacin and ciprofloxacin analytical standards were purchased from
Sigma-Aldrich (Sigma-Aldrich, Madrid, Spain). Stock standard solutions were prepared
from the reference standards, dissolved in 0.1 N formic acid in water, and stored at
−
80
◦
C.
Ofloxacin (Sigma-Aldrich, Madrid, Spain) was used as internal standard.
A single 10 mg/kg im administration of enrofloxacin was performed through a
24 G
catheter placed in the left ulnar vein. Blood samples (0.6 mL at each time point) were
collected from the medial tarsal vein with a 27 G needle attached to a 1 mL heparinized
syringe at 0, 10, 15, 35 min, 1, 2, 4, 6, 8, 11, 24, 29, 34, and 48 h after im administration.
For each bird, the total sample volume did not exceed 10% of the blood volume of the
animal. Plasma was separated immediately in a refrigerated centrifuge and frozen at
−80 ◦C until analysed.
Sample processing and drug detection methodologies for both enrofloxacin and
ciprofloxacin were slightly modified from a previously published method [
23
]. Enrofloxacin
was quantified using high performance liquid chromatography (HPLC/u.v.: Spectra Sys-
temThermo Separation Products Inc., Madrid, Spain) where the separation was accom-
plished using an ion-pairing reverse-phase column (PR C-18 5
µ
m 150
×
4.6 mm). No
chromatography interferences were observed in the retention time of the analytes. The
limit of quantification (LOQ) was 0.025
µ
g/mL for enrofloxacin and 0.05
µ
g/mL for
ciprofloxacin, and the calibration curve was linear up to 5
µ
g/mL (R2 > 0.99 for both
drugs). LOQ were determined by the lowest point on a linear calibration curve that was
within precision and accuracy acceptance criteria. Intraday precision was <8% and inter-
day precision was <12%. Accuracy ranged between 82–120% and 88–113%, for enrofloxacin
and ciprofloxacin, respectively.
Plasma concentrations of enrofloxacin after im administration were subjected to com-
partmental and non-compartmental analysis using the software package PCnonlinV4.0
(Statistical Consultants Inc., Lexintong, MA, USA). The non-compartmental pharmacoki-
netic parameters, determined for each individual animal, were: the observed Cmax, Tmax,
Clast and Tlast; area under the plasma concentration vs time curve (AUC) [AUC values
were calculated using trapezoidal rule from time 0 to the last concentration time point
(AUCt)], mean residence time [MRT, where MRT = AUMC/AUC; calculated from time 0 to
the last concentration time point (MRTt)], elimination rate constant (
λ
, calculated as the
slope of the terminal phase of the plasma concentration curve that included a minimum
of four points) and terminal half-life (t1/2
λ
, where t1/2
λ
= 0.693/
λ
). As the percentage
ratio 100
×
AUCt/AUCinf exceeds 80% in each subject, these AUCinf values were not
considered for the analysis [
27
]. The monocompartmental parameters were: Estimated
Cmax and Tmax; AUC calculated to infinite (AUCinf), absorption (T
1/2K01
) and elimination
(T1/2K10) half-lives.
Pharmacokinetic concentration-time profiles of enrofloxacin administered by the im
route were plotted under single dose condition by Monte Carlo simulation, based on the
descriptive statistical data obtained for pharmacokinetic parameters (subjects = 100). AUC
and Cmax parameters obtained from this simulated curves and previously published
MIC values from E. coli strains [
16
] and M. synoviae [
17
] were used to perform a PK/PD
analysis based on Monte Carlo simulation (subjects = 10,000). A log-normal distribution
was assumed for PK parameters. Monte Carlo simulations were conducted using Oracle
Crystal 174 Ball V.11.1.1.0.00 software (Oracle Corporation, Redwood Shores, CA, USA).
The PK/PD index and pharmacodynamic target associated with the efficacy of enrofloxacin
were Cmax/MIC > 10 and AUC/MIC > 30, 50 and 125 [
28
]. The probability of target
attainment, defined as the probability of the dose regimen to achieve a determined PK/PD

Antibiotics 2021,10, 957 6 of 7
endpoint for each MIC value and the probability of a dose regimen to achieve a determined
PK/PD endpoint taking into account the entire MIC distribution of the tested bacterial
population (cumulative fraction of response; CFR), was calculated for the proposed dose
regimen of enrofloxacin. PTA and CFR values > 90% were considered adequate [29].
5. Conclusions
In conclusion, the im enrofloxacin administration of 10 mg/kg in Coragyps atratus
could be appropriate to treat infectious diseases caused by gram-positive bacteria with MIC
values lower than 1
µ
g/mL. However, although enrofloxacin showed a slow elimination in
black vultures, the observed plasma concentrations were insufficient to reach the gram-
negative stablished breakpoints. However, it should be taken into account that, as there
is no data available for birds of prey, the calculations were performed using MIC values
against poultry isolates, which are probably higher than those corresponding to wild birds’
populations. Further studies, evaluating higher doses and its adverse reactions, should
be performed.
Author Contributions:
Conceptualization, S.W., M.I.S.A. and C.R.; formal analysis, J.J.d.L.; funding
acquisition, S.W.; investigation, S.W., J.J.d.L., G.W. and L.T.B.; methodology, S.W., G.W. and L.T.B.;
project administration, S.W.; resources, M.I.S.A.; supervision, S.W., J.J.d.L., M.I.S.A. and C.R.; valida-
tion, S.W., J.J.d.L. and C.R.; visualization, M.I.S.A. and C.R.; writing—original draft, S.W. and C.R.;
writing—review and editing, S.W., J.J.d.L., G.W., L.T.B., M.I.S.A. and C.R. All authors have read and
agreed to the published version of the manuscript.
Funding:
This study was performed as part of an UBACyT Project, Secretaría de Ciencia y Técnica,
Universidad de Buenos Aires.
Institutional Review Board Statement:
The study was conducted according to the guidelines of the
Declaration of Helsinki, and approved by the Institutional Animal Care and Use Committee of the
Veterinary Sciences School, University of Buenos Aires, Argentina (authorization reference number
2008/19).
Data Availability Statement:
The data presented in this study are available on request from the
corresponding author.
Acknowledgments:
Special thanks to M.A. Rivolta from Buenos Aires Zoological Garden. Thanks
to the staff of the Biblioteca de la Facultad de Veterinaria (BUCM), especially Mar Sanz, Cristina
Canchado and Carmen Muñoz Serrano, for their invaluable help, Mariano Díaz for his technical
assistance and Santiago Cano for his advice.
Conflicts of Interest: The authors declare no conflict of interest.
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