Evaluation of Marbofloxacin in Beagle Dogs After Oral Dosing: Preclinical Safety Evaluation and Comparative Pharmacokinetics of Two Different Tablets

Abstract

The current study evaluates a tested marbofloxacin tablet (MBT) (Petsen), in terms of bioavailability and pharmacokinetics (PK) in a comparison of the commercialized and standard tablet (Marbocyl) in beagle dogs. Four different bacterial species were selected for the determination of the minimal inhibitory concentration (MIC) against marbofloxacin (MBF). Target animal safety studies were conducted with a wide spectrum of dosages of Petsen. Pharmacokinetics and bioavailability of Petsen were observed after the oral administration of a recommended dosage of 2 mg/kg. The MIC90 of MBF against Staphylococcus aureus, Escherichia coli, Pasteurella multocida, and Streptococcus were 2.00, 4.00, 0.25, and 0.50 μg/ml, respectively. These results showed that the MBT has an expected antimicrobial activity in vitro. The main parameters of t1/2β, Clb, AUC0−∞, Cmax, and Ke were 22.14 h, 0.15 L/h, 13.27 μg.h/ml, 0.95 μg/ml, 0.09 h⁻¹, and 16.47 h, 0.14 L/h, 14.10 μg.h/ml, 0.97 μg/ml, 0.11 h⁻¹ after the orally administrated Petsen and Marbocyl, while no biologically significant changes and toxicological significance have been found by their comparison. These findings indicate that the Petsen had a slow elimination, high bioavailability and kinetically similar to the commercialized Marbocyl. Furthermore, no statistically significant differences were distinguished on the continuous gradient dosages of 2, 6, and 10 mg/kg in the term of the clinical presentation. The present study results displayed that the tested MBT (Petsen) was safe, with limited toxicity, which was similar to the commercialized tablet (Marbocyl), could provide an alternative MBT as a veterinary medicine in beagle dogs.

Full text

ORIGINAL RESEARCH
published: 10 April 2018
doi: 10.3389/fphar.2018.00306
Frontiers in Pharmacology | www.frontiersin.org 1April 2018 | Volume 9 | Article 306
Edited by:
Yurong Lai,
Gilead (United States), United States
Reviewed by:
Constantin Mircioiu,
Carol Davila University of Medicine
and Pharmacy, Romania
James Whiteford McBlane,
Medicines and Healthcare Products
Regulatory Agency, MHR,
United Kingdom
*Correspondence:
Jiyue Cao
caojiyue2@163.com
Qigai He
he628@mail.hzau.edu.cn
Specialty section:
This article was submitted to
Drug Metabolism and Transport,
a section of the journal
Frontiers in Pharmacology
Received: 17 January 2018
Accepted: 16 March 2018
Published: 10 April 2018
Citation:
Lei Z, Liu Q, Yang B, Khaliq H,
Ahmed S, Fan B, Cao J and He Q
(2018) Evaluation of Marbofloxacin in
Beagle Dogs After Oral Dosing:
Preclinical Safety Evaluation and
Comparative Pharmacokinetics of Two
Different Tablets.
Front. Pharmacol. 9:306.
doi: 10.3389/fphar.2018.00306
Evaluation of Marbofloxacin in
Beagle Dogs After Oral Dosing:
Preclinical Safety Evaluation and
Comparative Pharmacokinetics of
Two Different Tablets
Zhixin Lei 1,2,3 , Qianying Liu 1,2,3 , Bing Yang1, 2, Haseeb Khaliq 2, Saeed Ahmed 2,3 ,
Bowen Fan 1,2 , Jiyue Cao 2,3
*and Qigai He 1
*
1State Key Laboratory of Agriculture Microbiology, College of Veterinary Medicine, Huazhong Agriculture University, Wuhan,
China, 2Department of Veterinary Pharmacology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan,
China, 3National Reference Laboratory of Veterinary Drug Residues and MAO Key Laboratory for Detection of Veterinary
Drug Residues, Huazhong Agriculture University, Wuhan, China
The current study evaluates a tested marbofloxacin tablet (MBT) (Petsen), in terms of
bioavailability and pharmacokinetics (PK) in a comparison of the commercialized and
standard tablet (Marbocyl) in beagle dogs. Four different bacterial species were selected
for the determination of the minimal inhibitory concentration (MIC) against marbofloxacin
(MBF). Target animal safety studies were conducted with a wide spectrum of dosages
of Petsen. Pharmacokinetics and bioavailability of Petsen were observed after the oral
administration of a recommended dosage of 2 mg/kg. The MIC90 of MBF against
Staphylococcus aureus, Escherichia coli, Pasteurella multocida, and Streptococcus were
2.00, 4.00, 0.25, and 0.50 µg/ml, respectively. These results showed that the MBT has
an expected antimicrobial activity in vitro. The main parameters of t1/2β, Clb, AUC0−∞,
Cmax, and Kewere 22.14 h, 0.15 L/h, 13.27 µg.h/ml, 0.95 µg/ml, 0.09 h−1, and 16.47 h,
0.14 L/h, 14.10 µg.h/ml, 0.97 µg/ml, 0.11 h−1after the orally administrated Petsen and
Marbocyl, while no biologically significant changes and toxicological significance have
been found by their comparison. These findings indicate that the Petsen had a slow
elimination, high bioavailability and kinetically similar to the commercialized Marbocyl.
Furthermore, no statistically significant differences were distinguished on the continuous
gradient dosages of 2, 6, and 10 mg/kg in the term of the clinical presentation. The
present study results displayed that the tested MBT (Petsen) was safe, with limited
toxicity, which was similar to the commercialized tablet (Marbocyl), could provide an
alternative MBT as a veterinary medicine in beagle dogs.
Keywords: fluoroquinolones, marbofloxacin, pharmacokinetics, Beagle dogs, bioavailability, toxicity
BACKGROUND
Marbofloxacin (MBF), belongs to the third-generation synthetic fluoroquinolone antibiotic
formulated especially for the veterinary field. Due to its wide range of bactericidal activity, MBF is
mostly used against Mycoplasma, Gram-negative, and some of the Gram-positive pathogens (Sidhu
et al., 2011; Tohamy and El-Gendy, 2013). It is administered orally or parenterally for the treatment

Lei et al. Safety Pharmacokinetics of Marbofloxacin Tablets
of gastrointestinal and respiratory diseases in pigs and cattle,
and has a high bioavailability, near to 100% (Committee for
Veterinary Medicinal Products, 2009a; Ding et al., 2010; Tohamy
and El-Gendy, 2013; Shan et al., 2014). Due to its broad
spectrum it is efficient against canine pathogenic bacteria such
as: Streptococcus spp., Proteus spp., Staphylococcus spp., and
Escherichia coli, and is permitted for the treatment of pet animals
at a dosage level of 2.0 mg/kg body weight (b.w.) once a daily, by
an oral administration (Soussy et al., 1989; Unmack, 1990; Spreng
et al., 1995; Thomas et al., 1997; Paradis et al., 2001).
In a 13-week repeat-dose study with an oral dosage of 1, 4, and
40 mg/kg b.w. MBF in adult dogs, testicular tubular atrophy was
observed in only one of the dogs given a dose of 40 mg/kg b.w.;
no effects were observed at doses of below than 40 mg/kg. These
findings propose that MBF has a low toxicity and a broad dose
range (Committee for Veterinary Medicinal Products, 2009b).
Additionally, adverse reactions are rarely described in veterinary
clinical trials in which MBF has been evaluated (Cotard et al.,
1995; Carlotti et al., 1998, 1999; Frazier et al., 2000). Further,
MBF has been demonstrated to be a safe for the use in dogs even
if used at three times the recommended dose, continuously for
3 months (Bousquetmelou et al., 1997). Inappetence decreased
activity, and vomiting were the most commonly observed mild
signs. However, there are no available studies of intensive doses
from 4 to 40 mg/kg b.w., and a few safety evaluations regarding
the toxicity in dogs administered marbofloxacin tablet (MBT).
The pharmacokinetics (PK) actions of MBF have been studied
in various animals such as cows, goats, sheep, pigs, cats, and
dogs (Waxman et al., 2001; Schneider et al., 2004; Albarellos
et al., 2005; Ding et al., 2010; Sidhu et al., 2010a,b, 2011); these
studies showed that MBF was widely and rapidly distributed
in tissue, with a high bio-distribution in the peripheral tissue
and plasma, and showing nearly 100% bioavailability. However,
few studies on the PK of MBF in dogs were studied and the
previous reports have revealed that MBF has the PK features
such as good absorption after oral/parenteral supplementation,
higher amounts in tissue than plasma, and weak binding to the
plasmatic proteins (<10%) (Schneider et al., 1996; Haritova et al.,
2006; Andraud et al., 2011; Sun et al., 2015). MBF is widely
distributed throughout the animal’s body, which can result in 1.6
Abbreviations: MBF, Marbofloxacin (the active ingredient in marbofloxacin
powder); MBT, marbofloxacin tablet; PK, pharmacokinetics; EMA, European
Medicines Agency; HPLC, high performance liquid chromatography; TSB, tryptic
soy broth, TSA, tryptic soy agar; MIC, minimal inhibitory concentration;
CLSI, Clinical and Laboratory Standards; OECD, Economic Cooperation and
Development; FDA, Food and Drug Administration; RBC, red blood cell
count; HGB, hemoglobin concentration; WBC, white blood cell count; HCT,
hematocrit; PLT, blood platelet count; Urine analysis included ketone bodies; GLU,
glucose; BIL, bilirubin; URO, urobilin; BLD, occult blood; PRO, protein; NIT,
nitrite; ALP, alkaline phosphatase; AST, aspartate aminotransferase; ALT, alanine
aminotransferase; ALB, albumin; TP, total protein; GLU, glutamate; BUN, blood
urea nitrogen; CHOL, cholesterol; CREA, creatinine; CK, creatine kinase; TG,
triglyceride; TBIL, total bilirubin; K, potassium; Na, sodium; Ca, calcium; CI,
chloride; P, inorganic phosphorus; LLOD, the lower limit of detection; LLOQ,
the lower limit of quantitation; Ke, elimination rate of constant; t1/2β, half-life of
elimination; Clb, the total body clearance; MRT, mean residence time; AUC0−∞,
area under curve from 0 to ∞;Tmax , time to the concentration peak; Cmax, the
concentration in the peak; F, biovailability; Petsen (the test marbofloxacin tablet);
Marbocyl (the reference marbofloxacin tablet).
times more drug concentration in skin comparing to the plasma
of dogs. Moreover, MBF plasma concentrations can sustain above
the minimal inhibitory concentration (MIC) (>24 h) longer than
the dose density (Schneider et al., 1996). As MBT is a new
formulation for treatment in pets, few PK properties are available
in previous reports. In the previous plasma PK study, 1.25 and
2.5 mg MBTs (Marbocyl) were performed in beagle dogs (MBT,
FDA, Marbocyl), but the recommended dosage (2 mg/kg) by
EMA was used in this study. Compared with the Marbocyl, the
MBT (Petsen) in this study was compared the toxicity and PK
data like bioequivalence in dogs.
Bioequivalence studies support complementary applications
in the formulation, route of administration, or manufacturing
process that may affect bioavailability (Ozdemir and Yildirim,
2006; Zaid et al., 2017). The criteria for bioequivalence are
formulated by their respective organizations, and there are
relevant guidelines from regulatory bodies in Europe and the
United States. According to guidelines two products are tested
in order to prove that active ingredients are available at the
site of drug action, following similar assimilation rate and
extent (Listed, 1998; Rockville, 2000; Chen et al., 2001; U. S.
Food and Drug Administration, 2003; Davit et al., 2016). The
similarity is defined by acceptable limits of differences between
the pharmacokinetic parameters of compared products (Alp,
2009; Galgatte, 2014; Kaushal et al., 2016). In other words,
demonstrating bioequivalence between two drug products means
that the same rate and extent of absorption of the active
components is assured. Mathematical characterization is: “A
tested drug T is bioequivalent with a reference drug R if the
90% confidence interval (CI) for ratios of means µof main
pharmacokinetic parameters - area under the curve (AUC) and
maximum concentrations Cmax are included in the 0.8–1.25
interval” (Gherghiceanu et al., 2016). In fact the quantitative,
statistical rule implies that even products with different half time
of adsorption and halftime of elimination can be bioequivalent.
In this study, we wished to compare Petsen, a product in
development which contains MBF, with Marbocyl, which is a
marketed veterinary product, in China, containing MBF. Petsen
has been developed for veterinary use in treating cats and dogs:
both are presented as tablets with 20 mg MBF. Moreover, the drug
content and key excipients were similar to the referenced MBT
(Marbocyl). In this study, the aim was to assess the evaluation of
MBF in beagle dogs after oral dosing, including the preclinical
safety evaluation and comparative PK of two different tablets
the tested Petsen and the reference formulation Marbocyl. The
findings of this study could provide an alternative product for use
of MBT in veterinary medicine.
MATERIALS AND METHODS
Chemicals and Bacteria
The standard substance MBF (100.3% purity, NO. 201104005)
was formulated and supplied by Wuhan Huishen Biotechnology
Co., Ltd., The MBT (Petsen) (20 mg/tablet; NO.20110505)
containing 71.4% MBF per tablet, were formed and supplied by
Wuhan Longyu Biotechnology Co., Ltd. The all of the chemical
agents used for this analysis were of high-performance liquid
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Lei et al. Safety Pharmacokinetics of Marbofloxacin Tablets
chromatography (HPLC) grade, and other organic solvents were
of analytical grade. Marbocyl (20 mg/tablet) provided by the
French company Vetoquionol S.A. The standard substance MBF
was prepared under sterile conditions by addition of a 2%
sterilized aqueous solution of acetic acid into a sterile injectable
solution, with the concentration of 20 mg/mL by the authors
in this study. To test the susceptibility of bacteria to MBF,
each isolate was sub-cultured at least three times in tryptic
soy broth (TSB) and tryptic soy agar (TSA; Qingdao Hai Bo
Biological Technology Co., Ltd., Shangdong, China) containing
5% newborn calf serum (Zhejiang Tianhang Biotechnology Co.,
Ltd., Zhejiang, China).
Bacteria Strain Isolation
Four kinds of bacteria (Staphylococcus aureus, E. coli, Pasteurella
multocida, and Streptococcus) with 50 isolates were selected to
determine the MIC of MBF. Each kind of bacteria including
50 strains was isolated from beagle dogs from various Chinese
provinces including Hubei, Anhui, Jiangxi, Guangzhou, and
Sichuan between 2016 to 2017 years. E. coli ATCC 25922 strain
was selected to be used as a reference isolate for antibiotic
susceptibility determination. Before testing the MIC, each of
isolate was sub-cultured at least three times in TSB or TSA.
Animals
Thirty-six healthy male and female beagle dogs, weighing
between 8.0 and 10.0 kg, were selected from the Center of
Laboratory Animals of Hubei Province (Wuhan, PR China) and
were prepared for PK studies. The animals (dogs) were housed
separately in cages under a 12 h light/dark cycle and were offered
ad lib food and water during this experiment.
Animals did not receive any antimicrobial treatment for 14
days before the experiments. These animals were deemed to be
normal and clinically healthy after having a regular body checkup
and were thus used for this experiment (Lei et al., 2017c).
The study was ratified by Ethical Committee of Huazhong
Agricultural University, Faculty of Veterinary Medicine. All the
experiments involving animals were accompanied in accordance
with the Guide for the Care and Use of Laboratory Animals
of Hubei Provincial Laboratory Animal Public Service Center
(permit number SYXK 2013-0044).
Antimicrobial Susceptibility Testing
Determination of MBF susceptibility against the four kinds of
bacteria was executed using agar dilution technique, according
to Clinical and Laboratory Standards (CLSI) guidelines in the
previously described study (Lei et al., 2017b,c). Strains (2–4 µl,
about 108CFU/ml) were administrated onto TSA agar plates
having newborn calf serum, with two-fold serial dilutions of
MBF (0.0625–32 µg/ml). When the MIC values of isolates were
over 32 µg/ml, the MBF concentrations in TSA were expanded
for detecting. Strain plates were incubated at 37◦C for 48 h.
MICs were identified as the lowest concentrations of drug that
caused the growth inhibition. The MIC-value of E. coli (ATCC
25922) to chloramphenicol was used to verify the results of the
susceptibility testing.
TARGET ANIMAL SAFETY EVALUATION
Experimental Design
Twenty-four beagle dogs (50% males) weighing 8–10 kg and
aged 12–14 months old, were selected for inclusion in this
study. Dogs were randomly assigned to four groups according to
Petsen dose administration. According to the Guiding Principles
of Veterinary Drug Research and Development Technology of
China, and the Food and Drug Administration (FDA) (Kux,
2011; Shuren, 2012), the dogs in each group were orally
administered 0, 2, 6, or 10 mg/kg of Petsen daily, respectively,
for 40 continuous days. All dogs in each group were anesthetized
with pentobarbital sodium and euthanized at 22–24 h after
their last dose. Dogs in the control and high dose groups
were selected to investigate the change in visceral organs after
day 40. This study complied with the Technical Guidelines
of Veterinary Drug research and Good Laboratory Practice
Regulations of China (Good Laboratory Practice Regulations of
China, 2012).
Clinical Observations
Throughout the study, we observed Beagle dogs at least two-
times/day to determine the mortality, morbidity, severity, and
duration of any behavior change, evidence of toxicity, as well as
to observe the general appearance and abnormalities. Detailed
animal health examinations, including temperature, body weight,
and food consumption were performed on each animal on day 0,
14, and at the termination of the study (day 40).
Hematology Analysis
Blood and urine were collected from all dogs in each group, for
hematological and urine analysis, which was by use of a Coulter
HmX Hematology Analyzer (Beckman Coulter Inc., Fullerton,
CA, USA) and a UA-66 Urine Analyzer (Shanghai TianChen
Technology Inc., China). Blood and urine were collected on day
0, 14, and 40. Hematological evaluations included hemoglobin
concentration (HGB), red blood cell count (RBC), white blood
cell count (WBC), hematocrit (HCT), and blood platelet count
(PLT). Urine analysis included ketone bodies (KET), glucose
(GLU), pH, bilirubin (BIL), urobilin (URO), occult blood (BLD),
protein (PRO), nitrite (NIT).
Serum Biochemistry
Serum biochemistry was performed with a Synchron Clinical
System CX4 (Beckman Coulter, Brea, CA USA) under the
manufacturer’s guidelines (Beijing Leadman Biochemistry
Technology Co. Ltd., Bejing, China). The serum biochemistry
evaluations included aspartate aminotransferase (AST), alanine
aminotransferase (ALT), alkaline phosphatase (ALP), albumin
(ALB), total protein (TP), glutamate (GLU), cholesterol (CHOL),
blood urea nitrogen (BUN), creatinine (CREA), triglyceride
(TG), creatine kinase (CK), total bilirubin (TBIL), potassium
(K), sodium (Na), chloride (CI), inorganic phosphorus (P), and
calcium (Ca).
Histopathological Examinations
The main organs of each animal, including heart, liver, spleen,
lungs, and kidneys, were weighed separately. Organ weight/100 g
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Lei et al. Safety Pharmacokinetics of Marbofloxacin Tablets
body weight was determined on the basis of fasted animal’s
body weight. The tissues from these organs were kept in 10%
neutral buffered formalin until testing. Histopathological study
was conducted with routine paraffin-embedding method and
sections of 5 µm thickness stained with hematoxylin-eosin were
observed with light microscopy to evaluate morphology.
Pharmacokinetics and Bioequivalence
Study
Experimental Design
A crossover design was used. Twelve beagle dogs were divided
into two groups, with half males and females in each group.
One group received a single oral administration of Marbocyl,
while the other group received oral administration of Petsen
by gavage; the dosage for both groups was 2 mg/kg. After a
14-day washout period, dogs in the two groups were given the
alternate treatment, either Marbocyl or Petsen at 2 mg/kg. After
another 14-day washout period, 6 beagle dogs were selected
from these 12 and given a single i.v. injection of an aqueous
solution of the base form of MBF at the same dose. Blood
samples were collected at predetermined times as follows: 0,
10, 30, and 45 min, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 12, 16, 24, 36,
48, 60, and 72 h following the administration of the three drug
formulations.
Blood samples (2.0 mL) were collected by injecting a 7-gage
needle into the forelimb cephalic vein or the hind leg saphenous
vein and letting the blood drop into a 5 mL heparinized centrifuge
tube. Samples obtained were centrifuged at 3,000 rpm/min for
about 15 min. The plasma was immediately removed and stored
at −20◦C until analyzed.
Plasma Treatment and HPLC Conditions
Plasma samples were thawed to room temperature and MBF in
the plasma was extracted. A 0.2 mL plasma sample was placed
into a 5 mL polypropylene centrifuge tube; 2 mL chloroform
extractant was added. This blend was horizontally vortexed
for 5 min and later centrifuged for 6 min at 12,000 rpm. The
separated lower layer was shifted into 10 mL centrifuge tube and
desiccated at 45◦C under a nitrogen stream. The residue was re-
dissolved in 200 µL of a 2% aqueous solution of acetic acid. This
aqueous solution was centrifuged for 5 min at 5,000 rpm and the
supernatant was collected to be analyzed.
Agilent 1100 series equipment was used as the HPLC system,
with the variable wavelength indicator (Agilent 1100, G1314-
60086). The MBF drug detection was performed at 295 nm using
an ultraviolet detector. An automatic injection of 25 µL was
measured on an Agilent ZORBAX Extend-C18 stainless steel
column (250 ×4.6 mm, 5 µm). The mobile phase was acetonitrile
(A) and 1% formic acid aqueous solution (B) (75:25, v/v) with a
flow rate of 1.0 mL/min.
HPLC Method Validation
This method was confirmed for plasma, and a standard
calibration curve was prepared with plasma concentrations of
5, 0.5, 0.05, and 0.02 µg/mL. Linearity was determined by the
standard curve and the precision, accuracy, and recovery were
calculated between the standard substance and treated MBF in
plasma. The lower limit of detection (LLOD) and the lower
limit of quantitation (LLOQ) of MBF were defined as the drug
concentrations ensuing in a peak height of three-times, and
ten-times, the signal noise, respectively.
Pharmacokinetic and Bioequivalence Analysis
The pharmacokinetic examination was executed by WinNonlin
software. Theoretical curves and experimental data were plotted
semi-logarithmical and examined with the naked eye. Selection
of the best model was performed using Akaike’s Information
Criterion (Sandulovici et al., 2009). PK parameters were
determined for each individual animal, and the routes of
administration were compared.
STATISTICAL ANALYSIS
Analysis of variance (ANOVA) was applied to compare the
pharmacokinetic parameters of the formulations on the test
preparation with the reference ones (Pfaller et al., 2010;
Government of Canada HC, 2014). Tmax association was
achieved with a Wilcoxon signed rank test. Parametric 90%
CIs of the mean of test/reference ratios of AUC0−∞ and Cmax
were calculated using the residual variance of ANOVA with the
assumption of a multiplicative model. Confidence intervals were
measured by SPSS analysis (IBM, USA).
MIC90 was calculated using SPSS software, and statistical
analyzes were performed using Student’s t-tests, for between-
group comparisons of the parameters. The p<0.05 was
considered to indicate a statistically significant difference.
RESULTS
MIC Distributions of the Four Kinds of
Bacteria
The MIC distributions of MBF to the four kinds of bacteria
are presented in Figure 1. The values of MIC ranged from
0.03 to 4.00 µg/ml, except for E. coli, which ranged from
0.25 to 16.00 µg/ml. The values of MIC90 of MBF against
S. aureus, E. coli, P. multocida, and Streptococcus were 2.00, 4.00,
0.25, and 0.50 µg/ml, respectively. These findings indicated that
these four kinds bacteria were sensitive to MBF, according to
the CLSI M100-S23 guide document. Moreover, these results
revealed that MBF had the excepted antimicrobial activity
in vitro. MBF displayed a concentration-dependent killing
action.
Target Animal Safety Evaluation
Clinical Signs and Mortality
In the MBF group, all the dogs survived, and no significant
differences in coat condition, behavior, or mental condition
were observed, in comparison with the control group. Body
temperatures from the dogs in the low (2 mg/kg), middle (6
mg/kg), and high (10 mg/kg) dose groups were similar to each
other, and ranged from 38.05 to 38.26◦C; see Figure 2. Moreover,
there were non-significant changes in feed consumption (14.72–
16.54 kg) and body weight (8.96–10.44 kg) between the high,
middle, low and dose groups and the control group (p>0.05)
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FIGURE 1 | The MIC of marbofloxacin (Petsen) in four kinds of bacteria. (A) Represented MIC distribution of Staphylococcus aureus,(B) represented MIC distribution
of Escherichia coli,(C) represented MIC distribution of Pasteurella multocida,(D) represented MIC distribution of Streptococcus.
FIGURE 2 | The mean of temperature and body weight in the 40 days feeding
study. (A) Represented the mean of temperature.
at day 1, day 14, and day 40 of this study; see Supplemental
Table 1.
Hematological Examination
At day 1, 14, and 40, HGB, RBC, WBC, HCT, and PLT were
tested; the results are presented in Table 1. There were no
significant differences (p>0.05) in these indicators between
the low, middle, and high dose groups and the control group
(p>0.05). However, PLT was decreased and WBC was increased
in the 6 and 10 mg/kg treatment groups (Table 1).
Serum Biochemical Analysis
The results of serum biochemical analysis were non-significant
(p>0.05) between the low, middle, high dose treatment groups,
and control group excluding TBL, ALT, Na+, and AST which
were significantly decreased, and ALP and BUN which were
significantly raised (P<0.05). These findings can be found in
Table 2. However, these were not biologically significant changes
and not biologically significant changes and these values did not
fall outside the reference ranges.
Histopathological and Organ Examination
At 22 h after the last dose, the relative weights of the main organs
(liver, heart, spleen, lungs, and kidneys) were calculated and
are shown in Table 3. As compared to the control group, there
were no significant differences in the low, middle, and high dose
treatment groups. There were no histopathological findings in
the organs examined. Articular cartilage from control dogs and
dogs given 10 mg/kg MBF was investigated under microscopic
examination; no differences were seen (see Figure 3).
HPLC Method Validation
The plasma limit of detection (LLOD) and limit of quantitation
(LLOQ) of MBF was 0.02 and 0.05 µg/mL, respectively; see
Figure 4. The proposed method of HPLC was suitable for MBF
quantification in plasma. The recovery of MBF in plasma samples
was higher than 85%. The intra-assay coefficients of variation
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Lei et al. Safety Pharmacokinetics of Marbofloxacin Tablets
TABLE 1 | Hematology parameters of beagle dogs on the day 0, 14, and 40 (Mean ±SD) after orally administration Petsen.
Parameters Control 2 mg/kg 6 mg/kg 10 mg/kg
Day 0 Day 14 Day 40 Day 0 Day 14 Day 40 Day 0 Day 14 Day 40 Day 0 Day 14 Day 40
HGB (g/L) 163 ±4.3 158.5 ±4.6 162.8 ±4.3 158 ±4.1 159.2 ±6.9 167.3 ±4.9 162 ±3.6 162.3 ±5.2 168.2 ±7.4 156 ±9.9 155.3 ±4.3 160.2 ±5.2
RBC (1012/L) 6.9 ±0.4 6.6 ±0.1 6.9 ±0.4 6.8 ±0.2 6.6 ±0.2 6.4 ±0.2 6.9 ±0.2 6.6 ±0.1 6.9 ±0.6 7.1 ±0.4 6.4 ±0.2 6.5 ±0.4
WBC (109/L) 10.9 ±0.4 10.3 ±0.2 10.2 ±0.3 10.4 ±0.4 10.2 ±0.7 10.2 ±0.3 10.8 ±0.3 10.3 ±0.2 10.8 ±0.5 10.9 ±0.5 10.9 ±0.5 10.4 ±0.4
HCT (%) 46.5 ±2.4 44.5 ±1.7 46.5 ±2.4 46.0 ±1.9 46.8 ±3.6 47.4 ±1.3 48.9 ±1.3 47.4 ±1.2 47.1 ±1.8 48.3 ±3.8 45.5 ±4.1 47.5 ±3.5
PLT (109/L) 334 ±25.7 337.5 ±19.5 333.7 ±25.7 361 ±10.1 291.8 ±59.2 343.8 ±54.3 338 ±33.8 293.7 ±21.8 296.9 ±35.5 330 ±34.4 289.3 ±45.4 292.7 ±40.2
TABLE 2 | Serum biochemical analysis of beagle dogs on the day 0, 14, and 40 (Mean ±SD) after orally administration Petsen.
Parameters Control 2 mg/kg 6 mg/kg 10 mg/kg
Day 0 Day 14 Day 40 Day 0 Day14 Day 40 Day 0 Day 14 Day 40 Day0 Day 14 Day 40
TC (mmol/L) 5.0 ±0.1 5.0 ±0.1 4.9 ±0.1 4.6 ±0.2 4.5 ±0.1 4.7 ±0.3 4.6 ±0.3 4.3 ±0.3 4.7 ±0.2 47.3 ±0.4 4.7 ±0.5 4.9 ±0.4
GLU (mmol/L) 4.5 ±0.2 4.6 ±0.2 4.5 ±0.1 4.6 ±0.3 4.4 ±0.3 4.7 ±0.2 4.6 ±0.4 4.8 ±0.4 4.4 ±0.2 4.5 ±0.3 4.8 ±0.5 4.6 ±0.3
Cr (µmol/L) 93.8 ±0.9 93.5 ±0.9 93.8 ±0.6 92.9 ±2.6 95.5 ±6.0 94.5 ±3.6 94.8 ±3.7 96.5 ±6.7 94.0 ±2.8 93.9 ±2.5 92.9 ±3.9 93.5 ±3.5
TBL (µmol/L) 1.2 ±0.2 1.2 ±0.2 1.1 ±0.1 1 ±0.1 0.6 ±0.1* 0.4 ±0.2* 1 ±0.2 0.5 ±0.1* 0.5 ±0.2* 1.4 ±0.2 0.6 ±0.2* 0.5 ±0.2*
ALT (U/L) 36 ±2.0 35.9 ±2.0 35.1 ±2.1 36.3 ±4.2 34.7 ±3.1 31.8 ±2.9* 35 ±2.8 28.4 ±3.5* 21.9 ±1.6* 34.2 ±2.7 30.9 ±1.8* 22.7 ±2.0*
AST (U/L) 23.2 ±1.8 24.7 ±2.6 24.5 ±2.0 24.6 ±2.9 23.6 ±2.3 24.0 ±2.7 21.7 ±1.4 19.7 ±3.1* 20.1 ±1.9 23.7 ±2.3 20.2 ±1.4* 17.4 ±2.3*
ALP (U/L) 92.8 ±2.7 93.3 ±2.4 92.7 ±1.7 91.3 ±3.4 91.5 ±2.5 106 ±3.1* 91.2 ±3.0 109.9 ±6.9* 105 ±2.0* 106.4 ±7.9* 115.4 ±6.5* 115 ±2.0*
TP (g/L) 59.2 ±1.1 59 ±0.8 58.7 ±0.9 57.2 ±0.8 58.9 ±4.3 59.8 ±5.7 57.6 ±1.3 55.9 ±1.9 55.6 ±2.8 59.6 ±0.8 54.4 ±2.2 57.1 ±0.9
ALB (g/L) 28.8 ±0.8 29.4 ±0.8 28.6 ±0.6 28.6 ±0.6 29.2 ±1.6 31.1 ±2.4 29.1 ±0.5 31.2 ±0.8 32.1 ±1.3 28.1 ±1.4 29.5 ±1.1 30.3 ±1.4
BUN (mmol/L) 3.3 ±0.1 3.3 ±0.1 3.3 ±0.1 3.4 ±0.3 3.1 ±0.8 4.3 ±0.8 3.3 ±0.5 3.1 ±0.8 4.1 ±0.8 3.4 ±0.3 3.4 ±0.3 4.2 ±0.6
K+(mmol/L) 4.9 ±0.1 4.9 ±0.1 4.9 ±0.1 5.0 ±0.10 4.7 ±0.1 5.0 ±0.1 5.1 ±0.2 5.1 ±0.3 5.1 ±0.2 5.1 ±0.3 4.8 ±0.1 5.0 ±0.1
Na+(mmol/L) 150.0 ±1.6 147.5 ±1.6 149.9 ±1.4 148.4 ±2.0 143.3 ±2.7 149.6 ±1.3 149.6 ±1.1 143.8 ±1.7* 150.5 ±1.5 150.9 ±1.9 143.5 ±2.1* 150.8 ±1.4
Cl−(mmol/L) 102.7 ±0.5 102.5 ±0.8 102.8 ±0.6 103.2 ±1.0 103.1 ±1.21 103.3 ±1.1 103.2 ±1.4 102.9 ±0.6 103.6 ±1.5 103.7 ±0.8 102.1 ±1.3 104.1 ±0.7
Ca++ (mmol/L) 2.3 ±0.07 2.3 ±0.07 2.3 ±0.1 2.4 ±0.1 2.3 ±0.2 2.4 ±0.1 2.4 ±0.1 2.2 ±0.2 2.4 ±0.1 2.3 ±0.1 2.3 ±0.2 2.35 ±0.1
*Present significant difference P <0.05.
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Lei et al. Safety Pharmacokinetics of Marbofloxacin Tablets
TABLE 3 | Relative weight of main organ in beagle dogs.
Organs Control (%)2 mg/kg (%) 6 mg/kg (%) 10 mg/kg (%)
Heart 0.894 ±0.014 0.885 ±0.026 0.890 ±0.034 0.895 ±0.016
Liver 3.055 ±0.052 3.093 ±0.071 3.124 ±0.094 3.113 ±0.081
Spleen 0.293 ±0.021 0.286 ±0.032 0.298 ±0.044 0.296 ±0.012
Lung 0.850 ±0.029 0.837 ±0.044 0.851 ±0.036 0.841 ±0.024
Kidney 0.503 ±0.034 0.515 ±0.019 0.512 ±0.014 0.505 ±0.022
for 0.02, 0.05, 0.50, and 5.00 µg/mL were <4.54%, and the
inter-assay coefficients of variation for 0.02, 0.05, 0.50, and
5.00 µg/ml were 3.29, 2.07, and 1.33%, respectively. The typical
regression equation was y=40.737x– 2.2772, R2=0.996. The
chromatogram is shown in Figures 4A–C; the blank is shown in
Figure 4A, the lower limit of quantification (LLOQ) is shown in
Figure 4B, and the measured plasma samples 16 h after oral and
i.v. administration are shown in Figures 4C,D.
Pharmacokinetics Analysis
The theoretical compartmental theoretical concentration-time
profiles by non-linear regression equation analysis of MBF
concentration-time profiles after oral Petsen, Marbocyl and
i.v. MBF administrations are presented in Figure 5. After
orally administrated Petsen, the theoretical compartmental
theoretical concentration-time profiles of plasma were analyzed
in accordance with an absorbing two-compartment open model;
after i.v. administrated MBF, the concentration-time profile of
plasma was analyzed in accordance with the non-compartment
analysis. The main PK parameters of these two administration
methods are shown in Table 4; these parameters were determined
with WinNonlin software. The main parameters t1/2or t1/2β, Clb,
AUC0−∞,Cmax, and Kewere 13.78 h, 0.14 L/h, 13.69 µg.h/ml,
unavailable value and 0.053 h−1after intravenous administrated
MBF, 22.14 h, 0.15 L/h, 13.27 µg.h/ml, 0.95 µg/ml, and 0.09 h−1
after orally administrated Petsen, and 16.47 h, 0.14 L/h, 14.10
µg.h/ml, 0.97 µg/ml, and 0.11 h−1after orally administrated
Marbocyl. Moreover, the bioavailability values of Petsen and
Marbocyl after oral administration were 97.11 and 101.70%,
respectively.
Bioequivalence Analysis
The mean ±SD of MBF concentrations-time profiles are
presented in Figure 5 after oral two formulations, and the main
descriptive PK parameters, are reported in Table 5.
Bioequivalence Analysis
Log-transformed Cmax, AUC0−∞, and untransformed Tmax of
the test formulation (Petsen) were compared with the reference
one (Marbocyl) for a bioavailability study with ANOVA analysis
and 90% CI. It showed a significant difference in that Tmax of
Petsen (1.46 h) was longer than Marbocyl (1.10 h) in Table 5.
This point might indicate the bioequivalence between Petsen
and Marbocyl was not the same. This might be caused by
a small magnitude and biological difference. However, no
statistically significative differences were observed for Cmax or
AUC0−∞ in Table 5. The relative bioavailability of the test
product compared to the reference one was 94.11 ±10.28%
(Table 5).
The two one-sided Ttests estimated the ratios mean of
log-transformed Cmax, AUC0−∞, and 90% CI on the test to
reference formulations. Obtained values were 99.3, 99.2, and
91.9–107.2%, 92.0–102.1%, all in the range of 80–125% within
the bioequivalence acceptance range (Table 6). These results
demonstrated that Petsen was bioequivalent to the reference
product (Marbocyl) in dogs.
DISCUSSION
Compared with the previously published reports and the
PK profiles of Marbocyl, Petsen has also several advantages,
including long-action, sustained release, and convenient
administration to pets (Yang and Hu, 2006; Ghimire et al., 2007;
Walther et al., 2014). In the present study, we performed a
comprehensive toxicological evaluation of Petsen by conducting
animal safety studies in beagle dogs. At the doses tested, Petsen
was shown to be safe. In addition, this study also revealed the
antibacterial activity of MBF from Petsen against four kinds of
common pathogenic bacteria in vitro, as well as pharmacokinetic
characteristics of MBF after administration of Petsen tablets
in vivo.
In the safety study, no Petsen-related effects were observed
in beagle dogs administered Petsen, in terms of mortality,
morbidity, organ weight, body weight, total autopsy results,
or microscopic manifestations in organ and histopathological
examination. There were minimal differences in weight gain
and food consumption among the control, low and middle dose
groups, but an effect was seen at the highest dose used, as shown
in Figure 2 and Supplemental Table 1. Moreover, there were also
no treatment-related lesions based on the histopathology and
examination of organs, as seen in Figure 3. It is known that
administration of fluoroquinolones in animals and humans can
cause toxicity such as gastrointestinal disturbances, anaphylaxis,
hepatic and renal function injury, and in particular, articular
cartilage lesions (Ball, 2000; Robinson et al., 2005; Thompson,
2007). Our study found no lesions in the articular cartilage
among the three Petsen treatment groups (Supplemental Table 2).
PLT was decreased and WBC was increased in the 6 and 10
mg/kg treatment groups on day 14 and 40 (Table 1). Further
TBL, ALT, and AST were slightly decreased while ALP and
BUN were slightly increased in the 6 and 10 mg/kg treatment
groups, compared with the control group (Table 2). Moreover,
there seemed to be a fall in Na across all groups at day 14th day
compared to the control group. Although these indices presented
significant differences in the test group compared to the control
group on the 14 and 40th day (P<0.05), these were not
biologically significant changes; these values did not fall outside
the reference ranges. Therefore, the 10 mg/kg b.w. the dose
was regarded as the no observed adverse effect level (NOAEL)
of Petsen in the current study. In a previous 13-week repeat-
dose study, beagle dogs were given daily oral doses of 1, 4, and
40 mg/kg b.w. MBF in gelatin capsules. The typical quinolone-
induced changes were observed at 40 mg/kg b.w. in the articular
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Lei et al. Safety Pharmacokinetics of Marbofloxacin Tablets
FIGURE 3 | Microphotographs of articular cartilage in control and high dose treatment groups (10 mg/kg). (A) Represented control group, (B) represented high dose
treatment group (10 mg/kg).
FIGURE 4 | The HPLC method for MBF quantification in plasma. (A) Blank plasma sample, (B) plasma sample at the LLOQ of 0.05 µg/ml, (C) plasma sample after
oral administration of Petsen at the point of 16 h, (D) plasma sample after i.v. administration of MBF at the point of 16h. MBF at the peak time of 6.3min.
cartilage, and other toxic symptoms such as testicular tubular
atrophy and spermatic granuloma also occurred in one dog
at this dose. The recommended NOAEL was 4 mg/kg b.w.
(Committee for Veterinary Medicinal Products, 2009a). Another
study reported that no substance-related effects were found in
immature dogs after being given doses of up to 6 mg/kg b.w. for
13 weeks, and the recommended NOAEL of MBF was 6 mg/kg
b.w. (Committee for Veterinary Medicinal Products, 2009b).
Moreover, in a two-generation study of rats fed diets containing
10, 70, and 500 mg/kg b.w., overt signs of toxicity such as
impaired male fertility, reductions in implantation rate, litter size,
and pup weight, as well as increased pup mortality were observed
at doses of 10 and 500 mg/kg b.w. Therefore, the recommended
NOAEL in rats was 10 mg/kg b.w. (Committee for Veterinary
Medicinal Products, 2009b). In the present study, no significant
toxicological effects were found up to 10 mg/kg in beagle dogs,
and the NOAEL of Petsen was suggested to be 10 mg/kg. This
Petsen dose is higher than the previously described report (4
mg/kg b.w.) in beagle dogs, but equal to the dose in rats. The
difference for this might arises because of the decision to have
dose of 4 and 40 mg/kg in the former study. This study provided
a higher dose of NOAEL of MBF, which could be regarded as a
reference in the future study.
Four kinds of bacteria with 50 strains were selected for
MIC determination of Petsen. The MIC90 of these four kinds
of bacteria (S. aureus, E. coli, P. multocida, Streptococcus) was
2.00, 4.00, 0.25, and 0.50 µg/ml, respectively (Figure 1). All of
the MIC90 values were lower than 4 µg/ml, and the MIC90 of
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Lei et al. Safety Pharmacokinetics of Marbofloxacin Tablets
FIGURE 5 | The curves of MBF concentration-time in plasma at a dose of 2 mg/kg after i.v. administrated (A) MBF, (B) oral Petsen, and (C) Marbocyl, respectively.
MBF in plasma was determined at 0.17, 0.5, 0.75, 1, 2, 2.5, 3, 4, 6, 8, 12, 16, 24, 36, 48, 60, and 72 h.
TABLE 4 | Main PK parameters after oral and i.v. administration in beagle dogs
(n=12).
Parameters Mean ±SD (n=12)
MBF (i.v) Petsen (orally) Marbocyl (orally)
Ke(h−1) 0.053 ±0.004 0.09 ±0.01 0.11 ±0.05
t1/2(h) 13.78 ±1.21 – –
t1/2β(h) – 22.14 ±2.41 16.47 ±2.18
Clb(L/h) 0.14 ±0.08 0.15 ±0.09 0.14 ±0.04
MRT (h) 13.70 ±1.48 21.73 ±1.88 21.41 ±3.36
AUC0−∞ (µg.h/ml) 13.69 ±1.31 13.27 ±1.48 14.10 ±2.18
Tmax (h) – 1.46 ±0.12 1.10 ±0.38
Cmax (µg/ml) – 0.95 ±0.14 0.97 ±0.18
F(%) – 97.11 ±4.87 101.70%±5.12
Ke, elimination rate constant; t1/2 and t1/2β, half-life of elimination under non-
compartmental and compartmental models; Clb, total body clearance; MRT, mean
residence time; AUC0−∞, area under curve from 0 to ∞; Tmax , time to the concentration
peak; Cmax, the concentration in the peak; F, bioavailability.
P. multocida and Streptococcus was lower than 1. Streptococcus
was the most susceptible to Petsen. It had been reported that
the MICs of MBF against the isolates of E. coli,Streptococcus,
and S. aureus, isolated from pigs in China, were in the range of
0.13–0.25 µg/ml; other studies have also reported E. coli strains
resistant to MBF whose MICs ranged from 8 to 32 µg/ml (Pellet
et al., 2006; Ding et al., 2010; Andraud et al., 2011; Ferran
et al., 2013). Our findings are similar to these previous reports,
suggesting that these four kinds of bacteria are sensitive to Petsen,
according to the CLSI M100-S23 guide document. MICs of
three E. coli were up to 8 and 16 µg/ml. For the susceptibility
breakpoint evaluation of E. coli to MBF, a previous study had
TABLE 5 | Pharmacokinetic parameters for the Test Formulation (Petsen) and
Reference Formulation (Marbocyl), p-value, and relative fraction.
Parameters Unit Petsen Marbocyl ANOVA F(%)
AUC0−∞ µg.h/mL 13.27 ±1.48 14.10 ±2.18 0.313 94.11 ±10.28
Cmax µg/mL 0.95 ±0.14 0.97 ±0.18 0.874
Tmax H 1.46 ±0.12 1.10 ±0.38 >0.05*
F, represent relative bioavailability.
*Wilcoxon test.
suggested that MIC >8µg/ml was categorized as resistant
(Andraud et al., 2011). However, based on the calculated MIC90
of E. coli to MBF (4 µg/ml), the MBF concentrations were focused
on the intestinal tract, the site of infection with E. coli, and the
Cmax was 11.28 µg/ml in intestinal tract which was much higher
than the MIC90 in the previously published report by Lei et al.
(2017a). Therefore, our results suggest that Petsen will result in
concentrations of MBF active against E. coli.
Following i.v. injection, the elimination half-life (t1/2) of MBF
(13.78 h; as shown in Table 4) was much longer in beagle dogs
than in broilers (5.26 h) and buzzards (4.11 h) (Garcia-Montijano
et al., 2001; García-Montijano et al., 2003; Anadón et al., 2002;
Haritova et al., 2006). Moreover, the value of t1/2after i.v.
administration of MBF was similar (13.78 h) to a previous report
(Yohannes et al., 2015). However, after oral administration of
Petsen at a dose of 2 mg/kg, the t1/2β(22.14 h) value was higher
than that in beagles (7.51 h) after intramuscular injection (i.m.)
of MBF in the study by Yohannes (Yohannes et al., 2015), and
also higher than that after i.v. administration in the current study.
This difference was probably related to the continued absorption
of MBF from the oral administration site in the period of the
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Lei et al. Safety Pharmacokinetics of Marbofloxacin Tablets
TABLE 6 | Two-one sided T-test and 90% confidence interval.
Parameters Petsen Marbocyl 90% CI Ratio
(T/R) (%)
Acceptable
range (%)
AUC0−∞ 13.27 ±1.48 14.10 ±2.18 92.0–102.1 99.2 80–125
Cmax 0.95 ±0.14 0.97 ±0.18 91.9–107.2 99.3 80–125
*Present significant difference P <0.05.
elimination phase, thereby prolonging the elimination phase
time of MBF. Petsen showed high bioavailability, close to 100%
(97.11%), after oral administration in beagle dogs (see Table 4).
As the bactericidal activity of MBF was concentration-dependent,
the high absorption and bioavailability could contribute to
the bactericidal activity of MBF in vivo. The bioavailability of
Petsen in beagle dogs in this study was comparable with other
species, such as sheep, goats, and pigs, and was similar to that
previously reported in beagle dogs; the bioavailability in all
these animals has been shown to be close to 100% (Schneider
et al., 1996; Waxman et al., 2001; Ding et al., 2010; Sidhu
et al., 2010a,b; Marín et al., 2013). The high bioavailability may
contribute to the prolonged elimination half-life after oral or
i.m. administration; this may have induced a higher AUC. The
Cmax (1.10 µg/ml) of Petsen achieved in this study (Table 4)
was higher than the MIC90 of P. multocida and Streptococcus,
and was also higher than other breakpoints of fluoroquinolones
recommended against susceptible bacteria, based on the CLSI
M100-S19 guide document. The Cmax (1.10 µg/ml) in this study
was similar to that in pigs (1.03 µg/ml) (Ding et al., 2010;
Marín et al., 2013). Cmax obtained from orally administered
MBF (1.10 µg/ml) was lower than that obtained from i.m.
administration in pigs (1.81 µg/ml), as reported by Ding (Ding
et al., 2010). Further, Cmax in this study was lower than that
reported by Yohannes (1.76 µg/ml) (Table 4).
For the bioequivalence trial of these two MBT preparations,
and to perform a statistical comparison, AUC0−∞,Cmax, and
Tmax were chosen. When there are no statistically significant
differences in these indices, bioequivalence is considered to have
been shown (V˘
at˘
a¸sescu et al., 2011; Marchidanu et al., 2013).
In our findings, the three indices in Table 5 were revealed were
non-significant between test (Petsen) and reference formulations
(Marbocyl) (p<0.05). The AUC0−144h and AUC0−∞ ,Cmax
outcomes showing 90% of CI were inside the CIs (80–125%) set
by the all guidelines. Therefore, these findings proved that the
MBT-test product (Petsen) was bioequivalent to the reference one
(Marbocyl).
As a tablet, oral administration of Petsen is recommended
for pets. Thus, these results reveal that Petsen has high
plasma concentration, wide distribution, and high bioavailability
in beagle dogs, which supports its use as an alternative to
Marbocyl.
CONCLUSION
The results of this study revealed that, as a new formulation,
Petsen has low toxicity in target animals (beagle dogs),
antibacterial activity in vitro, and a pharmacokinetic profile in
terms of high plasma concentration, wide distribution, long
action, and bioequivalent which was similar to the reference one
(Marbocyl). This study also provided a reasonable theoretical
foundation for veterinary clinical application. Petsen might
be conveniently and widely used for pets in veterinary clinic
practice.
AUTHOR CONTRIBUTIONS
JC: Conceived the study; QL and ZL: Designed the experiments.
ZL, BF, and BY: Performed the experiments; ZL: Wrote the
manuscript; QH, SA, HK, and JC: Improved the language. All
authors reviewed the manuscript.
FUNDING
This work was partly supported by the China Agricultural
Research System (CARS-36).
ACKNOWLEDGMENTS
We thank Huazhong Agricultural University for permission to
conduct this research.
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found
online at: https://www.frontiersin.org/articles/10.3389/fphar.
2018.00306/full#supplementary-material
REFERENCES
Albarellos, G. A., Montoya, L., and Landoni, M. F. (2005). Pharmacokinetics of
marbofloxacin after single intravenous and repeat oral administration to cats.
Vet. J. 170, 222–229. doi: 10.1016/j.tvjl.2004.05.011
Alp, H. (2009). The Evaluation and Importance of Bioequivalence in
Veterinary Medicine. Atatürk Üniversitesi Veteriner Bilimleri Dergisi,
Diyarbakir.
Anadón, A., Martínez-Larra-aga, M. R., Díaz, M. J., Martínez, M. A., Frejo, M. T.,
Martínez, M., et al. (2002). Pharmacokinetic characteristics and tissue residues
for marbofloxacin and its metabolite N-desmethyl-marbofloxacin in broiler
chickens. Am. J. Vet. Res. 63, 927–933. doi: 10.2460/ajvr.2002.63.927
Andraud, M., Chauvin, C., Sanders, P., and Laurentie, M. (2011).
Pharmacodynamic modeling of in vitro activity of marbofloxacin against
Escherichia coli strains. Antimicrob. Agents Chemother. 55, 756–761.
doi: 10.1128/AAC.00865-10
Ball, P. (2000). “Fluoroquinolone safety and tolerability,” in First International
Moxifloxacin Symposium, ed L. Mandell (Berlin; Heidelberg: Springer)
138–143.
Bousquetmelou, A., Cester, C. C., Serthelon, J. P., and Gruet, P. (1997).
Pharmacodynamic Study of the Potential Epileptic Effect of Combination
Therapy with Marbofloxacin-tolfenamic Acid in the Dog. Food and
Agriculture Organization of the United Nation; FAO of the UN,
Toulouse.
Carlotti, D. N., Guaguere, E., Koch, H. J., Guiral, V., and Thomas, E. (1998).
Marbofloxacin for the Systemic Treatment of Pseudomonas spp. Suppurative
Otitis Externa in the Dog. Food and Agriculture Organization of the United
Nations; FAO of the UN, Carbon-Blanc.
Frontiers in Pharmacology | www.frontiersin.org 10 April 2018 | Volume 9 | Article 306

Lei et al. Safety Pharmacokinetics of Marbofloxacin Tablets
Carlotti, D. N., Guaguere, E., Pin, D., Jasmin, P., Thomas, E., and
Guiral, V. (1999). Therapy of difficult cases of canine pyoderma with
marbofloxacin: a report of 39 dogs. J. Small Anim. Pract. 40, 265–270.
doi: 10.1111/j.1748-5827.1999.tb03077.x
Chen, M. L., Shah, V., Patnaik, R., Adams, W., Hussain, A., Conner, D., et al.
(2001). Bioavailability and bioequivalence: an FDA regulatory overview. Pharm.
Res. 18, 1645–1650. doi: 10.1023/A:1013319408893
Committee for Veterinary Medicinal Products (2009a). Committee for Veterinary
Medicinal Products Marbofloxacin Summary Report (1). Available online
at: http://www.ema.europa.eu/docs/en_GB/document_library/Maximum_
Residue_Limits_-_Report/2009/11/WC500014864.pdf
Committee for Veterinary Medicinal Products (2009b). Committee For Veterinary
Medicinal Products Marbofloxacin Summary Report (2). http://www.ema.
europa.eu/docs/en_GB/document_library/Maximum_Residue_Limits_-_
Report/2009/11/WC500014865.pdf
Cotard, J. P., Gruet, P., Pechereaut, D., Moreaus, P., Pages, J. P., and, Thomas, E.,
et al. (1995). Comparative studv of marbofloxacin and amoxicillin- clavulanic
acid in the treatment of urinarv tract infection; in dogs. J. Small Anim. Pract.
36, 349–353. doi: 10.1111/j.1748-5827.1995.tb02948.x
Davit, B. M., Kanfer, I., Yu, C. T., and Cardot, J. M. (2016). BCS biowaivers:
similarities and differences among, EMA, FDA, and WHO requirements. AAPS
J. 18, 612–618. doi: 10.1208/s12248-016-9877-2
Ding, H., Li, Y., Chen, Z., Rizwan-Ul-Haq, M., and Zeng, Z. (2010). Plasma
and tissue cage fluid pharmacokinetics of marbofloxacin after intravenous,
intramuscular, and oral single-dose application in pigs. J. Vet. Pharmacol.
Therapeut. 33, 507–510. doi: 10.1111/j.1365-2885.2010.01164.x
Ferran, A. A., Bibbal, D., Pellet, T., Laurentie, M., Gicquel-Bruneau, M.,
Sanders, P., et al. (2013). Pharmacokinetic/pharmacodynamic assessment
of the effects of parenteral administration of a fluoroquinolone on the
intestinal microbiota: comparison of bactericidal activity at the gut versus
the systemic level in a pig model. Int. J. Antimicrob. Agents. 42, 429–435.
doi: 10.1016/j.ijantimicag.2013.07.008
Frazier, D. L., Thompson, L., Trettien, A., and Evans, E. I. (2000). Comparison
of fluoroquinolone pharmacokinetic parameters after treatment with
marbofloxacin, enrofloxacin, and difloxacin in dogs. J. Vet. Pharmacol.
Therapeut. 23, 293–302. doi: 10.1046/j.1365-2885.2000.00285.x
Galgatte, U. (2014). Study on requirements of bioequivalence for registration
of pharmaceutical products in India, South Africa and Australia. Saudi
Pharmaceut. J. 22, 391–402. doi: 10.1016/j.jsps.2013.05.001
García-Montijano, M., González, F., Waxman, S., Sánchez, C., Lucas, J. J. D.,
Andrés, M. S., et al. (2003). Pharmacokinetics of marbofloxacin after oral
administration to Eurasian buzzards (Buteo buteo). J. Avian Med. Surg. 17,
185–190. doi: 10.1647/2001-033
Garcia-Montijano, M., Waxman, S., Sánchez, C., Quetglas, J., San Andrés, M.
I., González, F., et al. (2001). The disposition of marbofloxacin in Eurasian
buzzards (Buteo buteo) after intravenous administration. J. Vet. Pharmacol.
Therapeut. 24, 155–157. doi: 10.1046/j.1365-2885.2001.00327.x
Gherghiceanu, F., Sandulovici, R., Prasacu, I., Anuta, V., and Mircioiu, C. (2016).
Bioequivalence implies therapeutic equivalence. I. biostatistical approach.
Farmacia 64, 823–827.
Ghimire, M., Mcinnes, F. J., Watson, D. G., Mullen, A. B., and Stevens, H. N.
(2007). In-vitro/in-vivo correlation of pulsatile drug release from press-coated
tablet formulations: a pharmacoscintigraphic study in the beagle dog. Eur. J.
Pharmaceut. Biopharmaceut. 67, 515–523. doi: 10.1016/j.ejpb.2007.03.002
Good Laboratory Practice Regulations of China (2012). Technical Guidelines of
Veterinary Drug Research and Good Laboratory Practice Regulations of China.
ISBN:978-7-122-13394-6.
Government of Canada HC, Health Products, and Food Branch VDD. (2014).
Government of Canada HC, Health Products, and Food Branch VDD. VICH
Guideline 52: Bioequivalence - Blood Level Bioequivalence Study (Step 4) – 2014
Health Canada Consultation Notice, Ottawa, ON.
Haritova, A. M., Rusenova, N. V., Parvanov, P. R., Lashev, L. D., and Fink-
Gremmels, J. (2006). Integration of pharmacokinetic and pharmacodynamic
indices of marbofloxacin in turkeys. Antimicrob. Agents Chemother. 50,
3779–3785. doi: 10.1128/AAC.00711-05
Kaushal, N., Singh, S. K., Gulati, M., Vaidya, Y., and Kaushik, M. (2016).
Study of regulatory requirements for the conduct of bioequivalence studies
in US, Europe, Canada, India, ASEAN and SADC countries: impact
on generic drug substitution. J. Appl. Pharmaceut. Sci. 6, 206–222.
doi: 10.7324/JAPS.2016.60430
Kux, L. (2011). Guidance for Industry on Target Animal Safety and Effectiveness
Protocol Development and Submission; Availability. Federal Register.
Lei, Z., Cao, J., and He, Q. (2017a). Pharmacokinetic and Pharmacodynamic
Evaluation of Marbofloxacin and PK/PD Modelling against E scherichia coli in
Pigs. Front. Pharmacol. 8:542. doi: 10.3389/fphar.2017.00542
Lei, Z., Liu, Q., Yang, B., Ahmed, S., Xiong, J., Song, T., et al. (2017b). Evaluation
of bioequivalence of two long-acting 20% oxytetracycline formulations in pigs.
Frontiers Vet. Sci. 4:61. doi: 10.3389/fvets.2017.00061
Lei, Z., Liu, Q., Yang, B., Xiong, J., Li, K., Ahmed, S., et al. (2017c). Clinical
efficacy and residue depletion of 10% enrofloxacin enteric-coated granules in
pigs. Front. Pharmacol. 8:294 doi: 10.3389/fphar.2017.00294
Listed, N. (1998). Bioavailability and bioequivalence requirements; abbreviated
applications; proposed revisions–FDA. Proposed rule. Federal Register. 63,
64222–64228.
Marchidanu, D., Raducanu, N., Miron, D. S., Radulescu, F. S. R., Anuta, V.,
Mircioiu, I., et al. (2013). Comparative pharmacokinetics of rifampicin and 25-
desacetyl rifampicin in healthy volunteers after single oral dose administration.
61, 398–410.
Marín, P., Alamo, L. F., Escudero, E., Fernándezvarón, E., Hernandis, V., and
Cárceles, C. M. (2013). Pharmacokinetics of marbofloxacin in rabbit after
intravenous, intramuscular, and subcutaneous administration. Res. Vet. Sci. 94,
698–700. doi: 10.1016/j.rvsc.2013.01.013
Ozdemir, N., and Yildirim, M. (2006). Bioequivalence study of two long-
acting oxytetracycline formulations in sheep. Vet. Res. Commun. 30:929.
doi: 10.1007/s11259-006-3235-2
Paradis, M., Abbey, L., Baker, B., Coyne, M., Hannigan, M., Joffe, D., et al.
(2001). Evaluation of the clinical efficacy of marbofloxacin (Zeniquin) tablets
for the treatment of canine pyoderma: an open clinical trial. Vet. Dermatol. 12,
163–169. doi: 10.1046/j.1365-3164.2001.00195.x
Pellet, T., Gicquel-Bruneau, M., Sanders, P., and Laurentie, M. (2006). Comparison
of faecal and optimal growth conditions on in vitro pharmacodynamic
activity of marbofloxacin against Escherichia coli.Res. Vet. Sci. 80, 324–335.
doi: 10.1016/j.rvsc.2005.07.001
Pfaller, M. A., Andes, D., Diekema, D. J., Espinel-Ingroff, A., Sheehan, D., and
Testing CSfAS. (2010). Wild-type MIC distributions, epidemiological cutoff
values and species-specific clinical breakpoints for fluconazole and Candida:
time for harmonization of CLSI and EUCAST broth microdilution methods.
Drug Resist. Updat. 13, 180–195. doi: 10.1016/j.drup.2010.09.002
Robinson, A. A., Belden, J. B., and Lydy, M. J. (2005). Toxicity of fluoroquinolone
antibiotics to aquatic organisms. Environ. Toxicol. Chem. 24, 423–430.
doi: 10.1897/04-210R.1
Rockville, M. (2000). FDA Guidance for Industry a) Bioavailability and
Bioequivalence Studies for Orally Administered Drug Products-General
Considerations. Food and Drug Administration, Washington, DC.
Sandulovici, R., Prasacu, I., Mircioiu, C., Voicu, V., Medvedovici, A., and
Anuta, V. (2009). Mathematical and phenomenological criteria in selection
of pharmacokinetic model for M1 metabolite of pentoxyphylline. Farmacia
57, 235–246. Available online at: https://pdfs.semanticscholar.org/c122/
0703a36f87c548062f86c1a8bb18ff707ee2.pdf
Schneider, M., Thomas, V., Boisrame, B., and Deleforge, J. (1996).
Pharmacokinetics of marbofloxacin in dogs after oral and parenteral
administration. J. Veterin. Pharmacol. Therapeut. 19, 56–61.
doi: 10.1111/j.1365-2885.1996.tb00009.x
Schneider, M., Vallé, M., Woehrlé, F., and Boisramé, B. (2004). Pharmacokinetics of
marbofloxacin in lactating cows after repeated intramuscular administrations
and pharmacodynamics against mastitis isolated strains. J. Dairy Sci. 87,
202–211. doi: 10.3168/jds.S0022-0302(04)73159-8
Shan, Q., Wang, J., Yang, F., Ding, H., Liang, C., Lv, Z., et al. (2014).
Pharmacokinetic/pharmacodynamic relationship of marbofloxacin against
Pasteurella multocida in a tissue-cage model in yellow cattle. J. Veter.
Pharmacol. Therapeut. 37, 222–230. doi: 10.1111/jvp.12078
Shuren, J. (2012). Guidance for Industry on Development of Target Animal Safety
and Effectiveness Data to Support Approval of Non-Steroidal Anti-Inflammatory
Drugs for Use in Animals Availability, Rockville.
Sidhu, P. K., Landoni, M. F., Aliabadi, F. S., and Lees, P. (2010a). Pharmacokinetic
and pharmacodynamic modelling of marbofloxacin administered alone
Frontiers in Pharmacology | www.frontiersin.org 11 April 2018 | Volume 9 | Article 306

Lei et al. Safety Pharmacokinetics of Marbofloxacin Tablets
and in combination with tolfenamic acid in goats. Vet. J. 184, 219–229.
doi: 10.1016/j.tvjl.2009.02.009
Sidhu, P. K., Landoni, M. F., Aliabadi, F. S., and Lees, P. (2010b). PK-PD
integration and modeling of marbofloxacin in sheep. Res. Vet. Sci. 88, 134–141.
doi: 10.1016/j.rvsc.2009.05.013
Sidhu, P. K., Landoni, M. F., Aliabadi, M. H., Toutain, P. L., and Lees, P.
(2011). Pharmacokinetic and pharmacodynamic modelling of marbofloxacin
administered alone and in combination with tolfenamic acid in calves. J. Veter.
Pharmacol. Therapeut. 34, 376–387. doi: 10.1111/j.1365-2885.2010.01247.x
Soussy, C. J., Leclercq, R., Deforges, L., and Duval, J. (1989). In vitro antibacterial
activity of a new fluoroquinolone, fleroxacin as a function of the sensitivity or
resistance to nalidixic acid and to pefloxacin. Pathol. Biol. 37, 364–369.
Spreng, M., Deleforge, J., Thomas, V., Boisramé, B., and Drugeon, H. (1995).
Antibacterial activity of marbofloxacin. A new fluoroquinolone for veterinary
use against canine and feline isolates. J. Veterin. Pharmacol. Therapeut. 18,
284–289. doi: 10.1111/j.1365-2885.1995.tb00592.x
Sun, J., Xiao, X., Huang, R. J., Yang, T., Chen, Y., Fang, X. et al. (2015).
In vitro dynamic pharmacokinetic/pharamco-dynamic (PK/PD) study and
COPD of Marbofloxacin against Haemophilus parasuis.BMC Vet. Res. 11:293.
doi: 10.1186/s12917-015-0604-5
Thomas, V. M., Guillardeau, L. D., Thomas, E. R., and Boisramé, B.
(1997). Update on the sensitivity of recent European canine and
feline pathogens to marbofloxacin. Veterin. Quart. 19(Suppl. 1), 52–53.
doi: 10.1080/01652176.1997.9694812
Thompson, A. M. (2007). Ocular toxicity of fluoroquinolones. Clin. Exp.
Ophthalmol. 35, 566–577. doi: 10.1111/j.1442-9071.2007.01552.x
Tohamy, M. A., and El-Gendy, A. A. M. (2013). Some pharmacokinetic aspects
and bioavailability of marbofloxacin in foals. J. Basic Appl. Sci. 2, 46–50.
doi: 10.1016/j.bjbas.2013.09.007
Unmack, J. (1990). The fluoroquinolone antibacterial agents. J. R. Austral. Nurs.
Federat. 19:28.
U. S. Food and Drug Administration. (2003). Bioavailability and Bioequivalence
Studies for Orally Administered Drug Products—General Considerations. U. S.
Food and Drug Administration. Available online at: https://www.ipqpubs.com/
wp-content/uploads/2014/04/BABEOld.pdf
V˘
at˘
a¸sescu, A., Enache, F., Mircioiu, C., Miron, D. S., and Sandulovici, R. (2011).
Failure of statistical methods to prove bioequivalence of meloxicam drug
products. I. parametric methods. Farmacia 59, 161–171.
Walther, F. M., Allan, M. J., Roepke, R. K., and Nuernberger, M. C.
(2014). Safety of fluralaner chewable tablets (Bravecto), a novel systemic
antiparasitic drug, in dogs after oral administration. Parasit. Vectors 7:87.
doi: 10.1186/1756-3305-7-87
Waxman, S., Rodríguez, C., González, F., De Vicente, M. L., Andrés, M. I. S.,
and Andrés, M. D. S. (2001). Pharmacokinetic behavior of marbofloxacin
after intravenous and intramuscular administrations in adult goats. J.
Vet. Pharmacol. Therapeut. 24, 375–378. doi: 10.1046/j.1365-2885.2001.
00357.x
Yang, Q., and Hu, Q. (2006). Pharmacokinetics and bioavailability of acyclovir
sustained-release tablets in dogs. Eur. J. Drug Metab. Pharmacokinet. 31, 17–20.
doi: 10.1007/BF03190637
Yohannes, S., Awji, E. G., Lee, S. J., and Park, S. C. (2015). Pharmacokinetics
and pharmacokinetic/pharmacodynamic integration of marbofloxacin after
intravenous and intramuscular administration in beagle dogs. Xenobiotica 45,
264–269. doi: 10.3109/00498254.2014.969794
Zaid, A. N., Mousa, A., Jaradat, N., and Bustami, R. (2017). Lornoxicam
immediate-release tablets: formulation and bioequivalence study in healthy
mediterranean volunteers using a validated LC-MS/MS method. Clin.
Pharmacol. Drug Dev. 6, 564–569. doi: 10.1002/cpdd.333
Conflict of Interest Statement: The authors declare that the research was
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