2nd international veraflox® symposium
TRANSCRIPT
2nd InternationalVeraflox® Symposium
29 – 30 November 2012 Rome, Italy
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2nd InternationalVeraflox® Symposium
29 – 30 November 2012 Rome, Italy
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contentSIntroduction Dr. R. Ebert (D) / Dr. J. Mottet (D) ............................ 04
Pharmacology of pradofloxacin: a novel third-generation fluoroquinolone Prof. Dr. P. Lees (UK) ........................................ 06
What does Mutant Prevention Concentration (MPC) mean and how does it apply to Veraflox®? Prof. Dr. J. M. Blondeau (CAN) ....... 20
Tissue concentrations in canine pyoderma: does it reach high enough directly in affected skin? Dr. C. Restrepo (USA) ..........28
Veraflox® in bacterial pyoderma: how well does it work? Prof. Dr. R. S. Mueller (D) .......................... 48
Susceptibility of canine and feline bacterial pathogens to pradofloxacin and comparison with other fluoroquinolones approved for companion animals Prof. Dr. S. Schwarz (D) ............. 54
Safety and convenience of Veraflox® – the art and science of tailoring therapy for cats and dogs Dr. J. Olsen (USA) ............... 64
Anaerobic activity and killing: how effective is Veraflox® really? Prof. Dr. P. Silley (UK) ................. 72 Veraflox® and its role in canine periodontal infections Dr. Dr. P. Fahrenkrug (D) ............................................. 82
Tissue concentrations: what about penetration to the site of infection? Dr. G. Hauschild (D) ................................. 90
Veraflox® in feline respiratory tract infections: is it a good choice? Prof. Dr. M. R. Lappin (USA) ........................... 92
Veraflox® in canine urinary tract infections: efficacy under field conditions Dr. B. Stephan (D) ......................... 96
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IntroductIon
There is growing consensus in the veterinary community that veterinary antibiotics are
an essential weapon in the fight against bacterial infections in companion animals. How
ever, very few innovative antibiotics have been approved in the last two decades. Consist
ent with its commitment for innovation, Bayer presented its latest antibiotic development
during the 1st International Veraflox® Symposium in 2006. After going through a long
regulatory process, Veraflox® was successfully approved by the European Commission in
2011.
Veraflox® is a unique molecule with significant microbiological and clinical advantages, in
cluding an extended spectrum with enhanced bacteriological cure in key infections of dogs
and cats. Also, its flavoured oral suspension allows for treating feline bacterial infections
more conveniently.
Antibacterial resistance is an increasing concern and it may threaten the longterm utility
of veterinary antibiotics. Bayer is committed to prudent use of antibiotics, and thanks to its
traditional presence in the antibiotic field, it gained a great degree of understanding resist
ance. This helped to design a molecule which – compared to other veterinary fluoroquino
lones – has the lowest Mutant Prevention Concentration (MPC), thus allowing for reducing
the likelihood for resistance induction in natural infections at therapeutic dosing regimens.
This 2nd International Veraflox® Symposium is a great opportunity to meet colleagues and
experts from different continents and countries, and to share the latest research findings
and clinical experiences. We are confident that this highquality information will also be of
benefit for our fourlegged patients. Rome, the eternal city, the city of the Caesars, per
fectly suits as host for this event, as it has been a historic melting pot from which many
important cultural and scientific milestones emerged.
We thank the distinguished researchers and lecturers for their efforts to provide cutting
edge information in their manuscripts and lectures, as well as for contributing to raise the
bar of Veraflox® knowledge. Special thanks also go to Prof. Dr. Jolle Kirpensteijn, who kind
ly accepted to moderate a scientific journey which we trust will be enriching for all of us.
Dr. Ralf Ebert Dr. Jose Mottet
Global Brand Management CAP Global Veterinary Services CAP
Bayer Animal Health Bayer Animal Health
04 | 05
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IntroductionFluoroquinolones are synthetic antimicrobial drugs. Sixteen years have elapsed since publica
tion of a review on fluoroquinolones used in veterinary medicine (Brown, 1996). The reader is
referred to this review and the chapter of Papich and Riviere (2009) for excellent descriptions
of the chemistry, pharmacological properties and therapeutic indications of drugs of this class.
Pradofloxacin is a novel fluoroquinolone, recommended for oral administration in the treatment
of a range of infections in the dog and cat. It is licensed for use in two formulations, flavoured
tablets (Veraflox® 15 mg tablet for small dogs and cats, Veraflox® 60 mg and 120 mg tablet for
dogs) and a 2.5 % oral suspension (Veraflox® 25 mg/ml oral suspension) for feline use. In the
latter formulation, pradofloxacin is bound to an ion exchange resin, this ensures avoidance of
its bitter taste and good palatability. Within the upper gastrointestinal tract, at low pH values,
pradofloxacin is rapidly released from the resin. The recommended once daily dose rates are
3 mg/kg (tablet formulation) and 5 mg/kg (oral suspension).
Chemical structures and physicochemical propertiesThe earlier fluoroquinolones in veterinary and human use are amphoteric molecules that can
be protonated at the carboxyl and tertiary amine groups. For example, for enrofloxacin, the
pKa for the carboxyl group is 6.0 and for the amine it is 8.8, so that at physiological pH it exists
in zwitterion form, with charged anionic and cationic groups. The carboxyl group (position 3)
and ketone group (position 4) are required for antibacterial activity. The fluorine atom at posi
tion 6 extends the Gramnegative and Grampositive activity spectrum, increases potency
and enhances penetration of bacterial cells (Figure1). The piperazine group in enrofloxacin at
pos ition 7 broadens the spectrum to include pseudomonads. Substitution at position 8 in
creases the Grampositive spectrum and also extends activity to anaerobes.
Pradofloxacin is a brownishyellow crystalline compound of molecular mass 396.42. It is a
thirdgeneration fluoroquinolone, related to the human drugs, travafloxacin, grepafloxacin,
gati floxacin, gemifloxacin and moxifloxacin. pKa values for the molecule’s two acid dissociation
constants are 5.5 and 8.8. It is relatively stable in neutral and acid, but not alkaline, solutions.
Pradofloxacin is an 8cyanofluoroquinolone, containing two centres of asymmetry (Figure 1).
The chemical name is 8cyano1cyclopropyl7([S,S]2,8diazabicyclo(4,3,0)non8y1)6
Pharmacology of pradofloxacin: a novel third-generation fluoro-quinoloneProf. Dr. Peter Lees
Emeritus Professor of Veterinary Pharmacology, The Royal Veterinary College, London University, UK
2nd International Veraflox® Symposium | 29 – 30 Nov 2012, Rome
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fluoro1,4dihydro4oxo3quinoline carboxylic acid. It differs
structurally from enrofloxacin in possessing an electronwith
drawing cyano group at position C8, in place of hydrogen, and
an S,Spyrrolidinopiperidine group replacing an ethylpiperazine
moiety at C7 (Figure1). Pradofloxacin is the pure SS isomer.
The enhanced potency of pradofloxacin is attributable to sub
stitutions in C7 and C8 positions in the molecule (Himmler et
al., 2002; Wetzstein and Hallenbach, 2004; 2011). The syn
thetic pathway has been described by Himmler et al. (2002).
Peter Lees
Prof. Dr. Peter Lees is a pharmacologist with
inter ests and expertise in basic and applied
veterinary aspects of the discipline. He has
a working knowledge of all fields of pharma
cology and some aspects of toxicology. His
research interests have spanned the fields
of renal pharmacology, the pharmacology
of drugs acting on the C.N.S., inflammation,
antiinflammatory drugs, cartilage biology
and antimicrobial chemotherapy. His current
principal research interests are in the fields of
antimicrobial and antiinflammatory drugs.
His work involves PK and PD interrelation
ships of antimicrobial and antiinflammatory
drugs of the NSAID class. Investigations are
conducted in vitro, ex vivo and in vivo, in
cluding use of the principles of PKPD inte
gration and PKPD modelling in the design
of dosage schedules for clinical use. Most
recently, he has investigated the population
PK of antimicrobial drugs in cattle.
Figure 1 Enrofloxacin
Figure 2 Pradofloxacin
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Pharmacodynamics of pradofloxacinFor a full discussion of the microbiological properties of pradofloxacin, see the papers of
Blondeau, Schwarz and Silley in this Symposium.
Molecular mechanism of action
The activity of pradofloxacin is due to inhibition of replication at two bacterial enzyme sites,
subunit A of topoisomerase II (DNA gyrase) and topoisomerase IV (Körber et al., 2002). The
former introduces negative superhelical twists in the bacterial DNA double helix ahead of the
replication fork. This catalyses the separation of daughter chromosomes, essential for initia
tion of DNA replication. Topoisomerase IV is principally involved in decatenation, the unlinking
of replicated daughter chromosomes. Other fluoroquinolones in veterinary use may also act at
both sites. However, the enzyme primarily targeted varies with bacterial species, one en zyme
generally being targeted preferentially. Thus, for earlier fluoroquinolones, DNA gyrase and
topoisomerase IV are the primary and secondary targets, respectively, of Gramnegative
bacteria, and target preference is reversed in Grampositive organisms (Peterson, 2001;
Drlica and Malik, 2003). In comparison with earlier generation veterinary fluoroquinolones,
prado floxacin targets both enzymes with increased affinity (Wetzstein et al., 2005a, b).
Although these authors identified topoisomerase IV as the primary and topoisomerase II
as the secondary target for pradofloxacin in Staphylococcus aureus, pradofloxacin had a
16fold higher affinity for the secondary target compared to ciprofloxacin. The consequence of
inhibition of topo isomerases II and IV is stabilisation of DNA double strand breaks in covalent
enzymeDNA complexes, and this results in inhibition of DNA replication and chromosome
regeneration, respectively.
Lewin et al. (1991) defined bactericidal mechanisms A, B, B1, and C for fluoroquinolones as
follows: mechanism A requires both cell division and protein synthesis; B requires neither;
B1 requires cell division; and C requires protein synthesis. Körber et al. (2002) compared
the mechanism of the killing actions of pradofloxacin, enrofloxacin, marbofloxacin and cipro
floxacin against susceptible strains of E. coli, Staph. aureus and Staph. pseudintermedius,
and also singlestep and doublestepresistant mutants of E. coli and Staph. aureus. All
drugs exerted mechanism A against all strains. However, only pradofloxacin was effective
in killing wildtype strains by mechanism B, indicating inhibitory activity, even in the absence
of both protein synthesis and cell division, and consequently exerting actions in vitro under
conditions which may occur in vivo.
Spectrum of activity
Pradofloxacin retains the broad spectrum of activity of first and secondgeneration fluoro
quinolones against Gramnegative bacteria (bacilli and cocci). In addition, it possesses an
extend ed spectrum against Grampositive and anaerobic bacteria, and also Mycoplasma
species and the intracellular organisms, Rickettsia spp. and Mycobacterium spp. (Abraham et
al., 2002a, b; de Jong and Bleckmann, 2003; de Jong et al., 2004; Silley et al., 2007; Stephan
et al., 2003, 2005, 2008; Wetzstein and Ochtrop, 2002).
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Potency and type of killing action
MIC90 values for pradofloxacin against feline and canine pathogens are presented for EU,
Germany and USA isolates in Table 1. There are some differences, depending on geographic
al locations. For three of nine bacterial species, MIC90 was higher for German isolates.
Silley et al. (2005) compared MBC and MIC values for five strains of each of ten bacterial
species. MIC and MBC were equal for 38 %, and MBC was one dilution (18 %), two dilutions
(22 %), three dilutions (16 %) and four dilutions (6 %) greater than MIC. Based on timekill
studies, pradofloxacin exerted a concentrationdependent killing action against all strains of
all species, both aerobes and anaerobes. This was indicated by its rapid killing action and
reduction in bacterial count of 5 log10 CFU/ml or greater. There was also absence of regrowth
at 48 h with concentrations as low as 0.125 µg/ml (Silley et al., 2012).
A recent study confirmed the significantly lower MICs of pradofloxacin compared to other
fluoroquinolones (ciprofloxacin, enrofloxacin, marbofloxacin, ibafloxacin, orbifloxacin and di
floxacin) for canine and feline isolates of Staph. pseudintermedius, E. coli and P. multocida
(Schink et al., 2012).
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Prof. dr. P. Lees | Pharmacology of pradofloxacin: a novel third-generation fluoroquinolone
Table 1 Pradofloxacin MIC90 values (µg/ml) against feline and canine isolates from the EU1, Germany2 and USA3
Species EU Germany USA
Bordetella bronchiseptica 0.254 0.25 0.25
Escherichia coli 0.125 2.0 0.03
Klebsiella pneumoniae 0.0625 0.25 0.06
Pasteurella spp. ≤ 0.016 0.0156 0.015
Proteus spp. 0.5 4.07 0.25
Pseudomonas aeruginosa 0.5 2.0 > 2.0
Salmonella spp. not tested 0.015 0.03
Staphylococcus pseudintermedius 0.125 0.06 0.06
Staphylococcus spp. 0.25 0.58 0.12
Streptococcus canis 0.125 not tested 0.129
1 Data from Pridmore et al. (2005) 2 Data from de Jong et al. (2004) 3 Data from Abraham et al. (2002a) 4 Bordetella spp. 5 Klebsiella spp. 6 Pasteurella multocida 7 Proteus mirabilis 8 Staphylococcus aureus 9 Streptococcus spp.
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Post-antibiotic effect
Wetzstein (2003) reported on the in vitro postantibiotic effect (PAE) of pradofloxacin. Values
for strains of E. coli, Staph. aureus and Staph. pseudintermedius were 2.3, 2.4 and 2.8 h,
respectively, after previous exposure for 2 h to concentrations similar to the mutant prevention
concentration (MPC). When these three species were exposed to concentrations correspond
ing to 0.5 x MICs of pradofloxacin (that is subMIC PAE), after exposure to the high concen
trations used to determine PAE, the periods of growth inhibition, compared to controls were
7.2, 9.0 and 6.1 h. In addition, exposure to pradofloxacin at subMIC concentrations in the
absence of initial exposure to a higher concentration also partially inhibited bacterial growth.
Wetzstein (2008) confirmed a pronounced PAE subMIC effect of pradofloxacin in highdensity
bacterial populations. Relatively long PAE and subMIC PAE effects are typical of drugs with a
concentrationdependent killing action.
Comparative potency studies and structure activity relationships
Ganiere et al. (2005) reported MIC50 and MIC90 values for 18 antimicrobial drugs against
50 strains of Staph. pseudintermedius isolated from canine pyoderma cases in 2002. Prado
floxacin was the most potent; MIC50 and MIC90 values were 0.032 and 0.063 µg/ml, respec
tively. Corresponding values were for enrofloxacin 0.125 and 0.5 µg/ml, and for marbofloxacin
0.25 and 0.5 µg/ml.
Himmler et al. (2002) determined MIC values for pradofloxacin in comparison with six other
fluoroquinolones in veterinary use (danofloxacin, difloxacin, enrofloxacin, marbofloxacin, orbi
floxacin and sarafloxacin) and two references drugs, ciprofloxacin and moxifloxacin. For four
E. coli strains, two Staph. aureus strains and two Staph. pseudintermedius strains, prado
floxacin had lower MIC values compared with all veterinary drugs and equal or greater poten
cy to the two reference drugs. Greater potency of pradofloxacin than other fluoroquinolones
against feline and canine pathogens was also reported by Abraham et al. (2002a, b), de Jong
and Bleckmann (2003), de Jong et al. (2004) and Silley et al. (2007).
Structure activity relationships of pradofloxacin and related compounds were evaluated by
Wetzstein and Hallenbach (2004) against Staph. aureus and E. coli. The strains investigated
included wildtype and fluoroquinoloneresistant mutant strains with differing enzyme struc
tures for gyrase A or topoisomerase IVA or both. For all strains, pradofloxacin (the S,S isomer)
was more potent (lower MICs) by a factor of 2 to 8 than the R,R isomer. The S,S isomer was
also more potent than enrofloxacin and 8cyanoenrofloxacin against all strains and more
potent than decyano8Hpradofloxacin against several strains. These data indicate that
the potency of pradofloxacin is dependent on both the amino (SSpyrrolidinopiperidine) and
cyano moieties.
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pH dependency of antimicrobial activity
KörberIrrgang et al. (2009) investigated the pH dependency of the action of pradofloxacin
against E. coli and Staph. aureus using 2 reference strains and 12 clinical isolates of each
species. Against E. coli, pradofloxacin had highest potency (that is lowest MIC) at alkaline
pHs; pH 8 = 7.3 > 6 > 5. For Staph. aureus, the potency order was pH 7.3 > 8 = 6 > 5.
The potency of pradofloxacin, under differing pH conditions, was compared to 4 analogs
with substituents of H, Cl, F and OCH3 in place of the CN grouping in position 8 of the prado
floxacin molecule (KörberIrrgang et al., 2009). The higher MICs for the H and OCH3 analogs
established that the CN grouping was essential for high potency at neutral and slightly acidic
pH values against E. coli. For Staph. aureus, the halogenated (Cl or F) substituents provided
compounds with greater potency than pradofloxacin at some pH values. However, at slightly
acidic pHs, pradofloxacin was the second most active of the five compounds.
Pharmacokinetics of pradofloxacin
Pharmacokinetic parameters and variables
The pharmacokinetic profiles of pradofloxacin in the dog and cat after intravenous (Table 2)
and oral tablet (Table 3) dosing are characterised by fairly rapid clearance, high volume of
distribution (three to six times body water volume), rapid attainment of Cmax and high bioavail
ability (70 to 105 %) (Fraatz et al., 2002; Fraatz, 2006). These profiles are very similar to
those reported for other fluoroquinolones (ciprofloxacin, difloxacin, enrofloxacin, levofloxacin,
marbo floxacin and orbifloxacin) as summarised by Papich and Riviere (2009).
10 | 11
Table 2 Pharmacokinetics of pradofloxacin in the dog and cat after intravenous dosing (mean values)*
Parameter (units) Dog (beagle) Cat
Whole body clearance (l/h/kg) 0.24 0.28
Renal clearance (l/h/kg) – 0.06
Volume of distribution (l/kg) 2.20 4.50
Elimination halflife (h) 6.60 10.0
* Data from Fraatz et al. (2003a) and Fraatz (2006)
Prof. dr. P. Lees | Pharmacology of pradofloxacin: a novel third-generation fluoroquinolone
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The administration of higher doses of pradofloxacin than those clinically recommended was
associated with linear pharmacokinetics in the dog (Fraatz et al. 2003b). This was indicated
by dose normalisation of AUC024h values.
Accumulation
With repeat administration of recommended doses of pradofloxacin at 24 h intervals, accumu
lation is minimal; the accumulation index for tablets in the dog = 1.01:1 (Fraatz, 2003b) and for
the oral suspension in the cat = 1.13:1 (Daube et al., 2006).
Plasma protein binding
Pradofloxacin binding to plasma proteins in vitro was independent of total concentration
over the concentration range 150 to 1,500 ng/ml. For free drug concentration, mean values
rang ed from 63.4 % to 64.2 % (dog) and 68.6 % to 71.2 % (cat) (Bregante et al., 2003). The
thera peutic significance of protein binding is that only “free” drug is microbiologically active
(Zeitlinger et al., 2004).
Extravascular distribution
Other authors in this Symposium will present data on the distribution of pradofloxacin to skin
(Restrepo) and other tissues (Hauschild). Hartmann et al. (2008) compared the distribution of
pradofloxacin in serum, saliva and tear fluid in the cat. The ready penetration of pradofloxacin
was indicated by the pharmacokinetic variables reported in Table 4.
2nd International Veraflox® Symposium | 29 – 30 Nov 2012, Rome
Table 3 Pharmacokinetics of pradofloxacin in the dog and cat after oral dosing with tablets (mean values)*
Variable (units) Dog (beagle) Cat
Cmax (µg/ml) 1.20 1.19
Tmax (h) 2.1 0.5 – 1.0
AUC024 h (mg·h/l) – 4.96
F (%) 105 70
* Data from Fraatz et al. (2003a) and Fraatz (2006)
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The terminal half-life of pradofloxacin was longer and Cmax was markedly higher in both fluids
compared to serum, whilst AUCs were similar for the three fluids. The authors proposed that
the high peak concentrations in both fluids may be attributable to an active transport mech-
anism. Although active transport has not been reported for pradofloxacin, other investigators
have demonstrated active secretion of ciprofloxacin across human intestinal (Caco-2) cells
(Griffiths et al., 1993, 1994; Cavet et al., 1997). In addition, several groups have shown that
fluoroquinolones are substrates of the ATP-binding ABC transporters, including the multidrug
resistance protein 1 (MDR1) a P-glycoprotein (P-gp) and the multidrug resistance-associated
proteins 1 and 2 (MRP1 and 2). Hartmann et al. (2008) suggested that P-gp/MDR1, which
is expressed in the respiratory tract, may be responsible for secretion of pradofloxacin into
tear fluid and saliva. However, not all fluoroquinolones are substrates for this transporter, so
that any role in relation to pradofloxacin transport remains to be elucidated. Regardless of the
transport mechanism, Hartmann et al. (2008) proposed that the distribution of pradofloxacin
was favourable for the treatment of upper respiratory tract and conjunctival infections in cats
caused by organisms such as Chlamydophila felis (Greene, 2006).
Metabolism and excretion
In both dog and cat the major excretion products of pradofloxacin are unchanged drug and
glucuronide conjugate. As a percentage of administered dose, 40 % and 10 % are excreted
in urine as parent drug plus glucuronide in the dog and cat, respectively (European Public
Assessment Report, EMA/142130/2011). It is assumed that only parent drug possesses anti-
microbial activity, as glucuronides of most drugs are polar and poorly lipid-soluble molecules,
which do not readily penetrate cell membranes, including cell walls and cell membranes of
bacterial cells.
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Table 4 Concentration of pradofloxacin in biological fluids (mean values, n = 6) of the cat after oral dosing of a suspension at a dose rate of 5 mg/kg*
Variable (units)Fluid
Serum Saliva Tear fluid
Cmax (µg/ml) 1.09 6.33 13.41
t½ (h) 2.95 18.03 16.36
MRT (h) 5.12 16.77 3.30
AUC0-24 h (µg·h/ml) 5.32 6.77 7.23
* Data from Hartmann et al. (2008)
Prof. Dr. P. Lees | Pharmacology of pradofloxacin: a novel third-generation fluoroquinolone
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Therapeutic uses of pradofloxacin and the polyanna phenomenonOther contributors to this Symposium (Mueller, Restrepo, Lappin, Stephan, Fahrenkrug) pre
sent data on the therapeutic uses and clinical efficacy of pradofloxacin for several clinical
diseases of the dog and cat. The data from several investigations have indicated its non
inferiority in all studies and superiority to other drugs in some instances, when administered
at manufacturers’ recommended dose rates. These findings are not considered here but it
is of interest, from therapeutic and pharmacological perspectives, to note one example of
the Polyanna phenomenon (Marchant et al., 1992). The latter term is used to describe the
circumstance in which a relatively high clinical cure rate is associated with a lower, even poor,
bacteriological cure rate. Stephan et al. (2006) compared pradofloxacin tablets (dogs receiv
ing 3 mg/kg once daily) with amoxicillin/clavulanic acid tablets (dogs receiving 12.5 mg/kg
twice daily) for periods of 7–21 days). The treated conditions were cystitis (77 % of dogs in
both groups) and prostatitis (23 % of dogs in both groups). The main organisms isolated were
E. coli (n = 139), Staph. pseudintermedius (n = 28), Pseudomonas spp. (n = 24) and Proteus
mirabilis (n = 22). Differences between treatments were significant for bacteriological but not
for clinical cure rates (Table 5).
These data are consistent with the fact that the activity of amoxicillin/clavulanic acid is weak
or absent against some pathogens e. g. Pseudomonas spp.
PK-PD integrationThe integration of pharmacokinetic and pharmacodynamic data provides, in most circum
stances, the most appropriate approach to determining dosing regimens of antimicrobial
drugs for subsequent evaluation in disease models and clinical trials. As fluoroquinolones,
against most if not all susceptible pathogens, kill bacteria by a concentrationdependent killing
action, the PKPD parameters widely used to predict effective doses are Cmax/MIC and AUC/
2nd International Veraflox® Symposium | 29 – 30 Nov 2012, Rome
Table 5 Efficacy of pradofloxacin and amoxicillin/clavulanic acid in the treatment of canine cystitis and prostatitis*
Response (%)Pradofloxacin
(n = 85)
Amoxicillin/ clavulanic acid
(n = 77)
Significance between
treatments
Reduction in total clinical score 96.8 93.4 NS
Clinical cure rate 89.3 89.9 NS
Bacteriological cure rate 85.3 48.0 p = 0.002
* Data from Stephan et al. (2006): NS = nonsignificant
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MIC ratios, where Cmax and AUC refer to plasma or serum free drug concentrations. The op
timal ratios are both drug and bacterial species specific (Aliabadi and Lees, 2001, 2002; Sidhu
et al., 2010). Proposed numerical targets for fluoroquinolones are Cmax / MIC ≥ 10 and AUC024 h /
MIC ≥ 125 h for Gramnegative bacteria (Drusano et al., 1998), Cmax / MIC ≥ 10 and AUC024 h /
MIC ≥ 40 h for Grampositive bacteria (Andes and Craig, 2002) and AUC024 h / MIC ≥ 7.5 h
for anaerobes (Noel et al., 2005). These values provide general guidance only and lower or
higher numerical values may apply for individual drugs against bacteria of all classes.
In vivo pharmacokinetic and in vitro MIC data indicate that pradofloxacin, at clinically recom
mended dose rates, meets most of these targets for species against which activity is claimed.
Table 6 presents data for Cmax / MIC90 and AUC / MIC90 ratios for pradofloxacin for large num
bers (n = 173 to 1,097) of field isolates of several bacterial species.
14 | 15
Table 6a PK / PD ratios for pathogens in dogs after oral administration of pradofloxacin tablets (3 mg/kg): (Cmax = 1.01 µg/ml and AUC024 h = 8.19 µg·h/ml)
OrganismNumber
of strainsMIC90
(µg/ml)Cmax / MIC90*
AUC0-24h / MIC90*(h)
Staph. pseudintermedius 1,097 0.062 16.4 132
E. coli 173 0.062 16.4 132
Porphyromonas spp. 310 0.125 8.19 65.5
Prevotella spp. 320 0.25 3.78 32.8
All anaerobes 630 0.25 3.78 32.8
Table 6b PK/ PD ratios for pathogens in cats after administration of pradofloxacin suspension (5 mg/kg): (Cmax = 1.45 µg/ml and AUC024 h = 6.21 µg·h/ml)
OrganismNumber
of strainsMIC90s
(µg/ml)Cmax / MIC90
AUC0-24 h / MIC90
(h)
Staph. pseudintermedius 184 0.125 11.7 49.7
E. coli 135 0.031 46.9 200
P. multocida 323 0.016 90.4 388
MIC90 = MIC90 for the susceptible part of the population only, where the distributions are bimodal or multimodal.
* All ratios based on free drug concentration, comprising 0.63 and 0.69 fraction of total concentrations of pradofloxacin in the dog
and cat, respectively. Data from Silley (personal communications).
Prof. dr. P. Lees | Pharmacology of pradofloxacin: a novel third-generation fluoroquinolone
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Kresken et al. (2007) developed an in vitro one compartment pharmacokinetic model of in
fection, based on Staph. pseudintermedius, to compare PKPD of pradofloxacin and marbo
floxacin. The model simulated freedrug concentrations in dogs provided by single oral doses
of pradofloxacin (3 mg/kg) and marbofloxacin (2 mg/kg). Against 3 clinical isolates of the test
organism, pradofloxacin provided 4fold higher Cmax:MIC ratios and 3.3 to 3.5fold higher
AUC24h:MIC ratios than marbofloxacin. The reductions in bacterial count at 24 h were corre
spondingly greater; differences between the two drugs in log10 CFU/ml reductions were 1.0,
1.6 and 2.6 for the three strains investigated.
Summary and conclusionsThe pharmacokinetic profile (clearance, distribution volume, elimination halflife, bioavail
ability and tissue distribution) of pradofloxacin in the dog and cat is broadly similar to other
fluoro quinolones licensed for use in these species. However, there are significant pharmaco
dynamic (microbiological) differences, in that pradofloxacin is more potent (lower MICs, MBCs
and MPCs) and possesses a broader spectrum of activity, which includes clinically important
anaerobes. Moreover, it possesses high activity against topoisomerase II as well as topo
isomerase IV.
Tbe selection of an appropriate dosage for fluoroquinolones, to maximise the level of bacterial
kill and minimise the emergence of resistance, should be based on integration of pharmaco
kinetic with pharmacodynamic data. Thus an effective oncedaily dose is provided by the
general equation:
Cl x AUC24h / MIC x MIC90
Dose = ______________________
F x fu
where Cl = whole body clearance
F = bioavailability
fu = fraction of serum drug concentration not bound to protein
MIC90 = MIC for 90 % of strains of a given organism
AUC24h / MIC = ratio of area under curve of drug concentration in serum
to MIC for an individual strain determined experimentally
AUC24h/MIC can be defined for differing levels of bacterial kill e. g. bacteriostatic, bactericidal
and eradication of organisms. As discussed in this review, AUC24h / MIC values of pradofloxa
cin provided by clinically recommended dose rates are achieved or exceeded for a wide range
of Grampositive, Gramnegative and anaerobic organisms.
2nd International Veraflox® Symposium | 29 – 30 Nov 2012, Rome
Veraflox_2012_Proceedings_fa.indd 16 20.11.12 11:02
References
01 | Abraham J, Ewert K, de Jong A. Comparative in vitro activity against selected pathogens from the US, In Program and Abstracts of the 42nd ICAAC, American Society of Microbiology, San Diego, CA, 2002a, p.189.
02 | Abraham K, Ewert K, de Jong A. Pradofloxacin: comparative in vitro activity against selected pathogens, in Proceedings of the 42nd ICAAC, American Society of Microbiology, San Diego, CA, 2002b, pp. 15–16.
03 | Aliabadi FS, Lees P. Pharmacokinetics and pharmacodynamics of danofloxacin in serum and tissue fluids of goats following intravenous and intramuscular administration. Am J Vet Res 2001; 62:1979–1989.
04 | Aliabadi FS, Lees P. Pharmacokinetics and pharmacokinetic/pharmacodynamic integration of marbofloxacin in calf serum, exudates and transudate. J Vet Pharmacol Ther 2002; 25:161–174.
05 | Andes D, Craig WA. Animal model pharmacokinetics and pharmacodynamics: a critical review. Int J Antimicrob Agents 2002; 19:261–268.
06 | Bregante MA, de Jong A, Calvo A, Hernandez E, Rey R, Garcia MA. Protein binding of pradofloxacin, a novel 8cyanofluoroquinolone, in dog and cat plasma. J Vet Pharmacol Ther 2003; 26(1):87–88.
07 | Brown SA. Fluoroquinolones in animal health. J Vet Pharmacol Ther 1996; 19:1–14.
08 | Cavet ME, West M, Simmons NL. Fluoroquinolone (ciprofloxacin) secretion by human intestinal epithelial (Caco2) cells. Br J Pharmacol 1997; 121:1567–1578.
09 | Daube G, Krebber R, Greife HA (2006). Pharmacokinetic properties of pradofloxacin administered as an oral suspension to cats. J Vet Pharmacol Ther 2006; 29(1):266–267.
10 | de Jong, A, Bleckmann I. Comparative activity of pradofloxacin against clinical canine and feline strains of Germany. Program and Abstracts of the 43rd ICAAC, American Society of Microbiology, Chicago, IL, 2003, p. 223.
11 | de Jong A., Stephan B, Friederichs S. Bacterial activity of pradofloxacin against canine and feline pathogens isolated from clinical cases. 2nd International Conference AAVM, Ottowa, Canada, 2004.
12 | Drlica K, Malik M (2003). Fluoroquinolones: action and resistance. Curr Top Med Chem 2003; 3:249–282.
13 | Drusano GL, Labro MT, Cars O, Mendes P, Shah P, Sorgel F, Weber W. Pharmacokinetics and pharmacodynamics of fluoroquinolones. Clin Microbiol Infect 1998; 4(2):2S27–2S41.
14 | Fraatz K, Heinen K, Krebber R, Edingloh M, Heinen E. Serum pharmacokinetics of pradofloxacin in dogs after multiple oral administrations at various dosages. Proceedings of the 42nd ICAAC, American Society of Microbiology, San Diego, CA, 2002, p. 189.
15 | Fraatz K, Krebber R, Edingloh M, Heinen E. Oral bioavailability of pradofloxacin tablets and renal drug excretion in dogs. J Vet Pharmacol Ther 2003a; 26(1):88–89.
16 | Fraatz K, Heinen K, Krebber R, Edingloh M. Skin concentrations and serum pharmacokinetics of pradofloxacin in dogs after multiple oral administrations at four different dosages. J Vet Pharmacol Ther 2003b; 26(1):89.
17 | Fraatz K. Serum pharmacokinetics of pradofloxacin after oral administration to cats. J Vet Pharmacol Ther 2006; 29(1):266.
18 | Ganiere JP, Medaille C, Mangion C. Antimicrobial drug susceptibility of Staphylococcus intermedius clinical isolates from canine pyoderma. J Vet Med 2005; 52:25–31.
19 | Greene CE. Chlamydial infections. In: Infectious Diseases of the Dog and Cat, 3rd edn., Greene CE (ed.), pp. 245–252. Saunders Elsevier Inc., St. Louis, 2006.
20 | Griffiths NM, Hirst BH, Simmons NL. Active secretion of the fluoroquinolone ciprofloxacin by human intestinal epithelial Caco2 cell layers. Br J Pharmacol 1993; 108:575–576.
21 | Griffiths, NM, Hirst BH, Simmons NL. Active intestinal secretion of the fluoroquinolone antibacterials ciprofloxacin, norfloxacin and pefloxacin; a common secretory pathway? J Pharmacol Exper Ther 1994; 269:496–502.
22 | Hartmann A, Krebber R, Daube G, Hartmann K. Pharmacokinetics of pradofloxacin and doxycycline in serum, saliva and tear fluid of cats after oral administration. J Vet Pharmacol Ther 2008; 31:87–94.
23 | Himmler T, Hallenbach W, Marhold A, Pirro F, Wetzstein H, Bartel S. Synthesis and in vitro activity of pradofloxacin, a novel 8cyanofluoroquinolone. Program and Abstracts of the 42nd ICAAC, American Society of Microbiology, San Diego, CA, 2002, p. 188.
24 | KörberIrrgang B, Kresken M, Wetzstein HG. pHdependence of the activity of pradofloxacin and four structural analogs against Escherichia coli and Staphylococcus aureus. 49th ICAAC, American Society for Microbiology, 2009, p. 180.
16 | 17
Prof. dr. P. Lees | Pharmacology of pradofloxacin: a novel third-generation fluoroquinolone
Veraflox_2012_Proceedings_fa.indd 17 20.11.12 11:02
25 | Körber B, Luhmer E, Wetzstein H, Heisig P. Bactericidal mechanisms of pradofloxacin, a novel 8-cyanofluoro-quinolone. Program and Abstracts of the 42nd ICAAC, American Society of Microbiology, San Diego, CA, 2002, p. 188.
26 | Kresken M, Bagel S, Körber-Irrgang B, Wetzstein HG. Comparative study on the pharmacodynamics of prado-floxacin (PRA) and marbofloxacin (MAR against Staphylococcus intermedius in an in vitro pharmacokinetic model of infection (VPM). 47th ICAAC, American Society of Microbiology, Chicago, IL, 2007, p. 4
27 | Lewin CS, Howard BM, Smith JT. Protein and RNA-synthesis-independent bactericidal activity of ciprofloxacin that involves the A unit of DNA gyrase. J Med Microbiol 1991; 34:19–22.
28 | Marchant CD, Carlin SA, Johnson CE, Shurin PA (1992). Measuring the comparative efficacy of antibacterial agents for acute otitis media: The “Pollyanna phenomenon”. J Pediatr 1992; 120:72–77.
29 | Noel AR, Bowker KE, MacGowan AP. Pharmacodynamics of moxifloxacin against anaerobes studied in an in vitro pharmacokinetic model. Antimicrob Agents Chemother 2005; 49:4234–4239.
30 | Papich M, Riviere J. Fluoroquinolone Antimicrobial Drugs. In: Veterinary Pharmacology and Therapeutics, 9th edn., Riviere J, Papich M (eds), Wiley-Blackwell, Ames, IA, USA, 2009, pp. 983–1012.
31 | Peterson LR (2001). Quinolone molecular structure-activity relationships: what we have learned about improv-ing antimicrobial activity. Clin Infect Dis 2001; 33(3):180–186.
32 | Pridmore A, Stephan B, Greife HA (2005). In vitro activity of pradofloxacin against clinical isolates from Euro-pean field studies. ASM 105th General Meeting, 2005, p. 617.
33 | Schink AK, Kadlec K, Hauschild T, Brenner-Michael G, Dörner JC, Ludwig C, Werckenthin C, Hehnen HR, Stephan B, Schwarz S. Susceptibility of canine and feline bacterial pathogens to pradofloxacin and compari-son with other fluoroquinolones approved for companion animals. Vet Microbiol 2012.
34 | Sidhu PK, Landoni MF, Aliabadi FS, Lees P. Pharmacokinetic and pharmacodynamic modelling of marbofloxa-cin administered alone and in combination with tolfenamic acid in goats. Vet J 2010; 184:219–229.
35 | Silley P, Stephan B, Greife HA, Pridmore A. Bactericidal activity of pradofloxacin (PRA) against aerobic and anaerobic bacteria. ASM 105th General Meeting, 2005, pp. 617–618.
36 | Silley P, Stephan B, Greife HA, Pridmore A. Comparative activity of pradofloxacin against anaerobic bacteria isolated from dogs and cats. J Antimicrob Chemother 2007; 60:999–1003.
37 | Silley P, Stephan B, Greife HA, Pridmore A. Bactericidal properties of pradofloxacin against veterinary patho-gens, Vet Microbiol 2012, 157:106–111.
38 | Stephan B, Pridmore A, Silley P. In vitro activity of pradofloxacin and metronidazole against anaerobic bacteria from dogs and cats. Program and Abstracts of the 43rd ICCAC, American Society of Microbiology, Chicago, IL, 2003, p. 223.
39 | Stephan B, Hellmann K, Adler K, Greife HA. Clinical efficacy of pradofloxacin in the treatment of feline upper respiratory tract infections. 45th ICAAC Abstracts, American Society of Microbiology, 2005, p. 184.
40 | Stephan B, Friederichs S, Pridmore A, Roy O, Edingloh M, Greife HA. Treatment of canine cystitis and prosta-titis with pradofloxacin: clinical and microbiological results. J Vet Pharmacol Ther 2006; 29(1):61–88.
41 | Stephan B, Greife HA, Pridmore A, Silley P. Activity of pradofloxacin against Porphyromonas and Prevotella spp. implicated in periodontal disease in dogs: Susceptibility test data from a European multicenter study. Antimicrob Agents Chemother 2008; 52:2149–2155.
42 | Wetzstein HG, Ochtrop S. Bactericidal activity of pradofloxacin (PRA) at concentrations ranging from MBCs up to selected mutant prevention concentrations (MPCs) and serum levels. 42nd ICAAC Abstracts, American Society of Microbiology, 2002, p. 189.
43 | Wetzstein HG (2003). In vitro postantibiotic effects of pradofloxacin in Escherichia coli and Staphylococcus aureus are greatly exceeded at sub-MIC drug concentrations. 43rd ICAAC Abstracts, American Society of Microbiology, 2003, p. 223.
44 | Wetzstein HG, Hallenbach W. Relative contributions of the C-7 amine and C-8 cyano substituents to the antibacterial potency of pradofloxacin. 104th General Meeting, American Society for Microbiology, 2004, pp. 672–673.
45 | Wetzstein HG, Heisig A, Heisig P. Target preference of pradofloxacin (PRA) in Staphylococcus aureus (Sa). 45th ICAAC Abstracts, American Society of Microbiology, 2005a, p. 152.
46 | Wetzstein HG, Heisig A, Heisig P. Target preference of pradofloxacin in Staphyloccocus aureus. Proceedings of the 45th ICAAC, American Society of Microbiology, 2005b, p. 80.
2nd International Veraflox® Symposium | 29 – 30 Nov 2012, Rome
Veraflox_2012_Proceedings_fa.indd 18 20.11.12 15:05
47 | Wetzstein HG. Pradofloxacin causes a pronounced postantibiotic subMIC effect in high density bacterial populations. 108th General Meeting American Society for Microbiology, Boston, MA, 2008, Abstract Z033.
48 | Wetzstein HG, Hallenbach W. Tuning of antibacterial activity of a cyclopropyl fluoroquinolone by variation of the substituent at position C8. J Antimicrob Chemother 2011; 66:2801–2808.
49 | Zeitlinger MA, Sauermann R, Traunmüller F, Georgopoulos A, Müller M, Joukhadar C. Impact of plasma protein binding on antimicrobial activity using timekilling curves. J Antimicrob Chemother 2004; 54:876–880.
18 | 19
Prof. dr. P. Lees | Pharmacology of pradofloxacin: a novel third-generation fluoroquinolone
Veraflox_2012_Proceedings_fa.indd 19 20.11.12 11:02
20 | 21
Fluoroquinolones are antibacterial, antimicrobial agents with a long history of clinical use in
both human and veterinary medicine. Such drugs are considered safe and efficacious when
used for the approved indications and at recommended dosages. Most quinolone compounds
have antibacterial spectrums to include Grampositive (Staphylococcus spp., Streptococcus
spp.) and Gramnegative (Enterobacteriaceae, Vibroniaceae, Pasteurella spp. and fastidious
Gramnegative bacilli including Haemophilus spp. and others) bacteria. Some quino lones also
have activity against anaerobic organisms. Quinolones are not all uniform in their in vitro activi
ty against key Grampositive pathogens nor against Pseudomonas aeruginosa and other afer
mentative Gramnegative bacilli. In vitro activity is based on the measurement of the minimum
inhibitory concentration (MIC) and then considering this value along with drug pharmacology
and established breakpoints. A breakpoint is a drug concentration value that determines the
susceptibility or resistance of an organism to a particular antibiotic based on the measured
MIC value. If the MIC is at or below the susceptibility breakpoint, the organism is considered
susceptible; for an MIC at or above the resistance breakpoint, the organism is considered
resistant.
Quinolones exert their antibacterial activity by inhibiting two enzymes critical for DNA repli
cation. These enzymes include DNA gyrase (topoisomerase II) and topoisomerase IV. Quino
lones such as enrofloxacin, marbofloxacin and orbifloxacin preferentially target one of these
two targets; in general, topoisomerase IV is the primary target in Grampositive bacteria,
where as DNA gyrase is the primary target in Gramnegative bacteria, however, exceptions
occur. Pradofloxacin is the newest fluoroquinolone to be approved for veterinary use in com
panion animals (dogs and cats) and is unique in that it simultaneously targets both DNA
gyrase and topoisomerase IV in both Grampositive and negative bacteria.1 One value of
dual targeting fluoroquinolones relates to the likelihood for resistance as compared to drugs
that preferentially target one of the two targets. Additionally, pradofloxacin has in vitro activity
against anaerobic organisms, and clinical outcome data support its use for indications where
anaerobic organisms are potentially problematic (i. e., periodontal infections).
The mutant prevention concentration (MPC) was initially described in 1999 by Dong et al.2 In
its simplest definition, the MPC is the drug concentration necessary to block the growth of the
least susceptible cell present in highdensity bacterial populations such as those seen dur
What does Mutant Prevention concentration (MPc) mean and how does it apply to Veraflox®?Prof. Dr. Joseph M. Blondeau
Departments of Laboratory Medicine (Clinical Microbiology), Royal University Hospital and the Saskatoon Health Region; Departments of Pathology, Microbiology and Immunology and Ophthalmology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada
2nd International Veraflox® Symposium | 29 – 30 Nov 2012, Rome
Veraflox_2012_Proceedings_fa.indd 20 20.11.12 11:02
Joseph M. Blondeau
ing infection. The concept of MPC arose out of the realisation
that during acute infection, bacterial burdens present at the
site of infection could exceed 107 colonyforming units (CFU).
From one report in the human infectious diseases literature, it
was reported that during acute pneumonia with the pathogen
Streptococcus pneumoniae, the total bacterial burden may ex
ceed 1012 bacteria.3 Other reports suggest bacterial burdens
at or above 107 CFU/millilitre or per gram of tissue in patients
with meningitis or with chronic lung disease with an acute bac
terial exacerbation.4, 5 Spontaneous bacterial mutants confer
ring drug resistance or reduced susceptibility (increased MIC
level) may be present in bacterial populations between 107–109
CFU.6 As such, in infected patients with high bacterial burdens,
spontaneous mutants conferring drug resistance may be pre
sent. The measurement of MPC was designed around this re
alisation and as such is based on the testing of ≥109 CFU of
bacteria against varying drug concentrations. The lowest drug
concentration preventing growth is the MPC. The measurement
of MPC differs from MIC testing in that 105 CFU/ml are tested
in the MIC assay. The mutant selection window (MSW) is de
fined as the drug concentration range between the meas ured
MIC and MPC values. It has been previously argued that the
wider this drug concentration range (wide window) the great
er the risk for resistance selection than in scenarios where the
MSW has a narrow drug concentration range (narrow window).
Dur ing drug therapy, therapeutic drug concentrations exceed
ing the MSW would be expected to have a low likelihood for
resistance selection, whereas drug concentrations falling within
the MSW (above the MIC drug concentration but below the
MPC drug concentration) selectively amplify the mutant cells;
the longer the drug concentration remains within the MSW,
the greater the likelihood for resistance selection7. Therapeutic
drug concentrations in the MSW eliminate susceptible bacteria
inhibited by the MIC drug concentration but allow for the proli
feration of mutant cells in the presence of the drug as the drug
concentration is below the MPC value – the drug concentra
tion necessary to block mutant growth. Dosing to exceed MPC
values and the MSW is suggested to be a strategy for mini
mising resistance selection from susceptible bacterial popula
tions.8–11
Prof. Dr. Blondeau (MSc, PhD, RSM (CCM),
SM (AAM), SM (ASCP), FCCP) is a Clinical
Microbiologist and Head of Clinical Micro
biology at Royal University Hospital (Sas
katoon Health Region) and the University of
Saskatchewan in Saskatoon, Saskatche
wan, Can ada. He is also the current Interim
Head of the Departments of Pathology and
Laboratory Medicine and holds appoint
ments as an Associate Professor of Patho
logy, Adjunct Professor of Microbiology and
Immunology and Clinical Associate Profes
sor of Ophthalmology. Dr. Blondeau’s main
research interests are in the area of antimi
crobial agents and antimicrobial resistance,
clinical microbiology and clinical outcomes
associated with antimicrobial therapy.
To date, he has published in excess of 140
peerreviewed manuscripts, more than 200
abstracts at international meetings and 5
books.
Dr. Blondeau has been twice nominated for a
University of Saskatchewan Student‘s Union
teaching award. He was also the University
of Saskatchewan, College of Medicine no
minee for the Henry Friesen Award and Lec
ture.
Veraflox_2012_Proceedings_fa.indd 21 20.11.12 11:02
Fluoroquinolones targeting one intracellular target (DNA gyrase or topoisomerase IV) need
only a single mutation in the gene that encodes for that target to elevate the MIC to the drug
and the increased MIC value may be high enough such that the organism is considered resist
ant. Clinical cases from human medicine have documented such findings and the selection of
resistant bacteria to the treatment drug has been associated with clinical failure.12 For a dual
targeting fluoroquinolone, the frequency with which a bacterial cell (from a susceptible bac
terial population) containing two simultaneous mutations would be expected to occur is very
small. The explanation is based on simple mathematics; if the frequency of a single mutational
event (conferring drug resistance) was reported to be 1 x 107 (1 mutant cell for every 107 bac
teria), then a double mutant could be argued to occur only rarely – 1 x 107 x 1 x 107 (or some
1014 bacterial cells) – from a susceptible population. As such, it has been previously argued
that dual targeting fluoroquinolones would have a lower propensity to select for resistance
than would single targeting agents.6, 13–15 Pradofloxacin as a dual targeting fluoroquinolone
would be expected to have a low propensity to select for resistance based on the explanation
above.
MIC and MPC measurements have been completed with pradofloxacin against key com
panion animal pathogens such as E. coli and Staphylococcus pseudintermedius and Sta
phylococcus aureus. Wetzstein et al.1 compared MIC values for pradofloxacin and other
quinolones against a standard laboratory strain of E. coli (American Type Culture Collection
(ATCC) strain #8739) and reported the following values; pradofloxacin 0.015 – 0.03 µg/ml,
enrofloxacin 0.03 – 0.06 µg/ml, marbofloxacin 0.03 µg/ml, danofloxacin 0.06 µg/ml and orbi
floxacin 0.125 µg/ml. By comparison, MPC values were 0.2 – 0.25 µg/ml, 0.3 – 0.35 µg/ml,
0.25 – 0.3 µg/ml, 0.5 – 0.55 µg/ml and 1 – 1.25 µg/ml. While pradofloxacin has the lowest
MPC values of the compounds summarised, other compounds such as enrofloxacin and
marbo floxacin also had low MPC values that would be within therapeutic drug concentrations
when considering drug pharmacology. In contrast, the differences were more striking when
MIC and MPC values were determined for the aforementioned compounds tested against
S. aureus ATCC 6538. MIC values for pradofloxacin were 0.03 – 0.06 µg/ml as compared to
0.06 – 0.125 µg/ml for enrofloxacin, 0.25 – 0.5 µg/ml for marbofloxacin, 0.125 – 0.25 µg/ml for
danofloxacin and 0.5 µg/ml for orbifloxacin. A greater difference was seen for the measured
MPC values; 0.5 – 0.6 µg/ml for pradofloxacin, 3 – 3.5 µg/ml for enrofloxacin, 3 – 3.5 µg/ml of
marbofloxacin, 10 – 11 µg/ml for danofloxacin and 8 – 9 µg/ml for orbifloxacin. As such, prado
floxacin had substantially lower MPC values than the other quinolones tested. Wetzstein et
al. also went on to test pradofloxacin and other quinolones against other strains of E. coli,
S. aureus and S. pseudintermedius and found similar results – i. e., lowest MPC results for
pradofloxacin.
The in vitro activity of pradofloxacin against key anaerobic bacteria was determined. Silley
et al. (2007) reported MIC90 values for pradofloxacin, enrofloxacin, difloxacin, ibafloxacin and
marbofloxacin against several anaerobic genus of bacteria.16 For Clostridium species (spp.),
the MIC90 value for pradofloxacin was 0.5 µg/ml as compared to 2 – 8 µg/ml for the other
2nd International Veraflox® Symposium | 29 – 30 Nov 2012, Rome
Veraflox_2012_Proceedings_fa.indd 22 20.11.12 11:02
agents tested. Against Bacteroides spp., Fusobacterium spp. and Prevotella spp., MIC90
val ues for pradofloxacin were 1 µg/ml as compared to 4 – 32 µg/ml, 16 – 64 µg/ml and
4 – 16 µg/ml, respectively, for the other agents tested. Against Porphyromonas spp., Sporo
musa spp. and Propionibacterium spp., MIC90 values were 0.062 – 0.25 µg/ml for prado floxa
cin and these values were lower than for the other agents tested. When all strains (n = 141)
were considered together, no strain had an MIC to pradofloxacin > 2 µg/ml and the MIC90
value was 1 µg/ml. The MIC90 values for the other agents tested against all strains were as
follows: enrofloxacin 16 µg/ml, difloxacin 16 µg/ml, ibafloxacin 16 µg/ml and marbofloxacin
8 µg/ml. For 310 strains of Porphyromonas spp. tested against pradofloxacin and metronida
zole, MIC90 values were 0.125 µg/ml and 0.25 µg/ml, respectively; against 320 strains of Pre
votella spp., MIC90 values were 0.25 µg/ml and 0.5 µg/ml, respectively. The Porphyromonas
spp. and Prevotella spp. isolates were collected from canine clinical cases from 6 European
countries.17 To date, published MPC values have not been determined for pradofloxacin or
other veterinary quinolones against anaerobic organisms.
Quinolones are concentrationdependent antibacterial agents and as such two pharmaco
dynamic/pharmacokinetic (PK/PD) parameters define their activity; maximum serum (Cmax)/
MIC ratio and area under the curve (AUC)/MIC ratio. Previously published literature from hu
man medicine suggests a Cmax/MIC ratio of 8 – 12 or higher and an AUC/MIC ratio of >100
were desirable for a favourable clinical outcome and minimisation of resistance.18 Others have
argued that the AUC/MIC ratio of > 100 was necessary for Gramnegative pathogens while a
value of 30 to 50 was necessary for Grampositive pathogens (studies primarily with Strepto
coccus pneumoniae).19 At least one clinical study suggests a higher AUC/MIC ratio is bene
ficial for Grampositive pathogens as well. File et al. studied human patients with chronic
lung diseases and that had infectious exacerbations.20 For patients treated with an agent for
which the AUC/MIC was < 100, these patients were statistically more likely to go on to devel
op pneumonia than patients treated with agents where the AUC/MIC was > 100. That study
suggests a clinical benefit in the higher AUC/MIC. One study has suggested from in vitro
investigations with ciprofloxacin and E. coli that an AUC/MPC ratio of ≥ 22 was necessary for
resistance prevention.
Figures 1– 2 show the serum drug concentration for pradofloxacin in dogs along with MIC and
MPC values for E. coli and S. pseudintermedius, respectively; Figures 3 – 4 are for cats and the
aforementioned organisms, respectively. Considering drug pharmacology of pradofloxacin in
dogs and cats, the Cmax drug concentration is ~1.45 µg/ml in dogs (Figure 1) as compared to
~2.1 µg/ml (Figure 3) in cats. Blondeau 2009 reported MIC90 values for pradofloxacin against
E. coli and S. pseudintermedius to be 0.016 µg/ml and 0.063 µg/ml, respectively; MPC90 val
ues were 0.125 µg/ml and 0.125 µg/ml. The Cmax/MIC ratio in dogs for E. coli and S. pseud
intermedius are 90.6 and 23.0; in cats 131.3 and 33.3, respectively. The Cmax/MPC ratio in
dogs for E. coli and S. pseudintermedius are 11.6 and 11.6; for cats 16.8 and 16.8. For dogs,
the AUC/MIC for E. coli and S. pseudintermedius are 806.3 and 204.8; in cats 525 and 133.3,
respectively. The AUC/MPC ratios in dogs for E. coli and S. pseudintermedius were 103.2 and
103.2; in cats 67.2 and 67.2, respectively.
Prof. dr. J.M. Blondeau | What does Mutant Prevention Concentration (MPC)
mean and how does it apply to Veraflox®
22 | 23
Veraflox_2012_Proceedings_fa.indd 23 20.11.12 11:02
As previously stated, pradofloxacin is a dual targeting quinolone. This along with the PK/PD
profiles for pradofloxacin in dogs and cats when considering E. coli and S. pseudintermedius
and the values considered here suggest a drug with a low potential for resistance selection
by considering the MPC model. Additionally, serum drug concentrations exceed the mutant
selection window for prolonged periods over the dose also reducing the likelihood for resist
ance selection. Pradofloxacin represents an important addition to veterinary medicine for the
treatment of infections. Clinical efficacy along with a reduced likelihood for resistance selection
are desirable characteristics.
2nd International Veraflox® Symposium | 29 – 30 Nov 2012, Rome
Figure 1 Favorable MPC profile against E. coli with clinical use in dogs.
1.8
1.5
1.2
0.9
0.6
0.3
0.0
Mea
n se
rum
conc
entr
atio
n (µ
g/m
l)
0 2 4 6 8 10 12 14 16 18 20 22 24
MPC90 – 0.125 µg/mlMIC90 – 0.016 µg/ml
3 mg/kg
Cmax/MIC: 90.6Cmax/MPC: 11.6AUC/MIC: 806.3AUC/MPC: 103.2
Sampling time (hours)
MSW
Figure 2 Favorable MPC profile against S. pseudintermedius with clinical use in dogs.
1.8
1.5
1.2
0.9
0.6
0.3
0.0
Mea
n se
rum
conc
entr
atio
n (µ
g/m
l)
0 2 4 6 8 10 12 14 16 18 20 22 24
MPC90 – 0.125 µg/mlMIC90 – 0.063 µg/ml
3 mg/kg
Cmax/MIC: 23.0Cmax/MPC: 11.6AUC/MIC: 204.8AUC/MPC: 103.2
Sampling time (hours)
MSW
Veraflox_2012_Proceedings_fa.indd 24 20.11.12 11:02
References
01 | Wetzstein HG. Comparative mutant prevention concentrations of pradofloxacin and other veterinary fluoroquinolones indicate differing potentials in preventing selection of resistance. Antimicrob Agents Chemother 2005; 49(10):4166–4173.
02 | Dong Y, Zhao X, Domagala J, Drlica K. Effect of fluoroquinolone concentration on selection of resistant mutants of Mycobacterium bovis BCG and Staphylococcus aureus. Antimicrob Agents Chemother 1999; 43:1756–1758.
03 | Frisch AW, Tripp JT, Barrett CD Jr, Pidgeon BE. The specific polysaccharide content of pneumonic lungs. J Exp Med 1942; 76(6):505–510.
04 | Bingen E, LambertZechovsky N, MarianiKurkdjian P et al. Bacterial counts in cerebrospinal fluid of children with meningitis. Eur J Clin Microbiol Infect Dis 1990; 9:278–281.
Prof. dr. J.M. Blondeau | What does Mutant Prevention Concentration (MPC)
mean and how does it apply to Veraflox®
24 | 25
Figure 3 Favorable MPC profile against E. coli with clinical use in cats.
2.5
2.0
1.5
1.0
0.5
0.0
Mea
n se
rum
conc
entr
atio
n (µ
g/m
l)
0 2 4 6 8 10 12 14 16 18 20 22 24
MPC90 – 0.125 µg/mlMIC90 – 0.016 µg/ml
5 mg/kg
Cmax/MIC: 131.3Cmax/MPC: 16.8AUC/MIC: 525.0AUC/MPC: 67.2
Sampling time (hours)
MSW
Figure 4 Favorable MPC profile against S. pseudintermedius with clinical use in cats.
2.5
2.0
1.5
1.0
0.5
0.0
Mea
n se
rum
conc
entr
atio
n (µ
g/m
l)
0 2 4 6 8 10 12 14 16 18 20 22 24
MPC90 – 0.125 µg/mlMIC90 – 0.063 µg/ml
5 mg/kg
Cmax/MIC: 33.3Cmax/MPC: 16.8AUC/MIC: 133.3AUC/MPC: 67.2
Sampling time (hours)
MSW
Veraflox_2012_Proceedings_fa.indd 25 20.11.12 11:02
2nd International Veraflox® Symposium | 29 – 30 Nov 2012, Rome
05 | Fagon J, Chastre J, Trouillet JL et al. Characterization of distal bronchial microflora during acute exacerbation of chronic bronchitis. Use of the protected specimen brush technique in 54 mechanically ventilated patients. Am Rev Respir Dis 1990; 142(5):1004–1008.
06 | Blondeau JM. Clinical utility of the new fluoroquinolones for treating respiratory and urinary tract infections. Expert Opin Investig Drugs 2001; 10(2):213–237.
07 | Croisier D, Etienne M, Bergoin E et al. Mutant selection window in levofloxacin and moxifloxacin treatments of experimental pneumococcal pneumonia in a rabbit model of human therapy. Antimicrob Agents Chemother 2004; 48(5):1699–1707.
08 | Drlica K. The mutant selection window and antimicrobial resistance. J Antimicrob Chemother 2003; 52:11–17.
09 | Drlica K. Controlling Antibioitc Resistance: strategies based on the mutant selection window. In Reemergence of established pathogens in the 21st century, Drlica F (ed.), Plenum publishers, New York 2003; pp. 295–331.
10 | Drlica K, Zhao X. Mutant selection window hypothesis updated. Clin Infect Dis 2007; 44:681–688.
11 | Blondeau JM. New concepts in antimicrobial susceptibility testing: the mutant prevention concentration and mutant selection window approach. Vet Dermatol 2009; 20:383–396.
12 | Davidson RJ, Cavalcanti R, Brunton JL et al. Resistance to levofloxacin and failure of treatment of pneumococcal pneumonia. N Engl J Med 2002; 346(10):747–750.
13 | Hansen G, Metzler KL, Drlica K, Blondeau JM. Mutant prevention concentration of gemifloxacin for clinical isolates of Streptococcus pneumoniae. Antimicrob Agents Chemother 2003; 47(1):440–441.
14 | Blondeau JM, Hansen G, Metzler KL, Hedlin P. The role of PK/PD parameters to avoid selection and increase of resistance: mutant prevention concentration. J Chemother 2004; 16(3):1–19.
15 | Hesje C, Tillotson GS, Blondeau JM. MICs, MPCs and PK/PDs: A match (sometimes) made in hosts. Exp Rev Resp Med 2007; 1(1):7–16.
16 | Silley P, Stephan B, Greife HA, Pridmore A. Comparative activity of pradofloxacin against anaerobic bacteria isolated from dogs and cats. J Antimicrob Chemother 2007; 60:999–1003.
17 | Stephan B, Greife HA, Pridmore A, Silley P. Activity of pradofloxacin against Porphyromonas and Prevotella spp. implicated in periodontal disease in dogs: susceptibility test data from a European Multicenter study. Antimicrob Agents Chemother 2008; 52(6):2149–2155.
18 | Schentag JJ, Gilliland KK, Paladino JA. What have we learned from pharmacokinetic and pharmacodynamic theories? Clin Infect Dis 2001; 32(1):39–46.
19 | Drusano GL, Preston SL, Owens RC, Ambrose PG Jr. Fluoroquinolone pharmacodynamics (Correspondence). Clin Infect Dis 2001; 33:2091–2092.
20 | File TM Jr, Monte SV, Schentag JJ et al. A disease model discriptive of progression between chronic obstructive pulmonary disease exacerbations and communityacquired pneumonia: roles for underlying lung disease and the pharmacokinetics/pharmacodynamics of the antibiotic. Int J Antimicrob Agents 2009; 33:58–64.
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26 | 27
Prof. dr. J.M. Blondeau | What does Mutant Prevention Concentration (MPC)
mean and how does it apply to Veraflox®
Veraflox_2012_Proceedings_fa.indd 27 20.11.12 11:02
28 | 29
IntroductionBacterial pyoderma is one of the top causes of canine skin disease in small animal prac
tice.1, 2, 43 Classification of pyoderma is typically based on depth of infection (superficial versus
deep) and commonly develops secondary to underlying causes.2 Secondary infections may
result from hypersensitivities, endocrinopathies, ectoparasite infestation, or immunological
conditions.2– 8, 43 Lesions indicative of superficial pyoderma include follicular pustules, papules,
epidermal collarettes, and alopecia.2, 9 Clinical signs indicative of deep pyoderma such as fu
runculosis and cellulitis are clinically evident as nodules, fistulae, scarring, and/or hemorrhagic
bullae.2, 9, 10 Staphylococcus (S.) pseudintermedius is the most common pathogen isolated in
canine pyoderma.1, 11, 12, 43 In addition to S. pseudintermedius, chronic deep pyoderma may
be associated with other pathogens such as Pseudomonas aeruginosa, Escherichia coli,
Proteus spp., Bacteroides spp., Peptostreptococcus spp., and Fusobacterium spp.1, 2, 13, 14, 43
Treatment of both generalized superficial and deep pyoderma involves administration of sys
temic antibiotics for 2 to 3 weeks beyond clinical resolution of lesions, which may require
4 to 12 weeks of initial therapy.1, 2, 15 – 17, 43 However, treatment failures are reported due to ca
nine skin pathogens increasingly exhibiting resistance to many classes of antibiotics, including
fluoroquinolones, βlactams, macrolides, and sulfonamides.16 – 21, 43
Pradofloxacin (PRA) is a novel thirdgeneration fluoroquinolone specifically developed for vet
erinary medicine with enhanced in vitro activity against a wide range of Grampositive, Gram
negative, and anaerobic veterinary pathogens.22 – 24 Pradofloxacin has already demonstrated
efficacy in the treatment of canine pyoderma and wound infections in clinical trials in Europe.10
For example, one study reported that clinical remission was obtained in 86 % of dogs with
deep pyoderma. The mean treatment duration was 34 days at an oral dose of 3 mg/kg.10
The bioavailability of orally administered PRA is approximately 100 % in dogs.26 Following
an oral dose of 3 mg/kg, a maximum serum concentration (Cmax) of 1.26 µg/ml is reached in
2.1 hours (Tmax).27 The minimum inhibitory concentration (MIC) of PRA for 90 % of S. inter
medius isolates (MIC90) is 0.06 µg/ml, which is at least two to four times more active than other
veterinary fluoroquinolones.22 In dogs, PRA has low in vitro plasma protein binding (29 % to
37 %),28 similar to enrofloxacin; this is important, because free drug concentrations often cor
relate well with antibacterial activity.29 Furthermore, PRA has been shown to have higher con
2nd International Veraflox® Symposium | 29 – 30 Nov 2012, Rome
tissue concentrations in canine pyoderma: does it reach high enough directly in skin?
Dr. Christina Restrepo
Veterinary Dermatology Center, Maitland, Florida, USA
Veraflox_2012_Proceedings_fa.indd 28 20.11.12 11:02
christina restrepo
centrations in the skin than in serum.27 Tissue concentra tion
(compared to serum concen trations alone) is likely a better indi
cator for predicting potential efficacy of a drug.43 Prado floxacin
is well tolerated and possesses a wide margin of safety when
used according to dosage recommendations. The occurrence
of adverse reactions was low in all of the clinical trials perform
ed to date.10, 26, 27, 30, 43 Previously reported ad verse effects for
other veterinary fluoroquinolones include gas trointestinal dis
turbances (e. g., nausea, transient vomiting, diarrhoea, or mild
changes in feces); how ever, these adverse effects usual ly only
occur at higher doses, are not serious, and do not require dis
continuation of therapy.10, 31, 32, 43 – 44
Pradofloxacin is indicated for the treatment of skin, soft tis
sue, respiratory, and urinary tract infections associated with
suscept ible Grampositive, anaerobic, and Gramnegative
bac terial organisms.10, 17, 21, 23, 30, 33, 43 The purpose of this study
included 1) determining the clinical efficacy of PRA tablets in
the treatment of naturally occurring superficial and deep pyo
derma in dogs in an open study design and 2) determining
the concentration of PRA in serum and skin in dogs with and
without pyoderma. We hypothesized that the concentration of
PRA is greater in diseased tissue versus normal canine skin
tissue.
Materials and methods
Dog selection Group I
As previously described, between June 1, 2004, and Novem
ber 1, 2006, 20 privately owned adult dogs of any breed,
weight, or sex that were referred to the dermatology service
at the Veterinary Medical Teaching Hospital, University of Cali
forniaDavis, for clinical signs consistent with superficial and/
or deep pyoderma were enrolled in the study.43 The diagnosis
was based on clinical, cytological, and histopathological fea
tures of pyoderma as described.2, 9 The study was approved
by the university’s Animal Care and Use Committee, and all
owners signed a consent agreement.43
Dr. Christina Restrepo graduated in 2003
from the University of Florida College of Ve
terinary Medicine. After graduation, Dr. Re
strepo completed a oneyear internship in
small animal medicine and surgery at the re
nowned Animal Medical Center in New York
City. She then practiced small animal medi
cine and surgery in Miami, Florida where she
gained further experience in dermatologic
conditions and utilized her fluent Spanish
language skills. This led to her acceptance
of a dermatology residency position at the
University of California at Davis, in 2005.
During the residency program, Dr. Restrepo
received the American College of Veterinary
Dermatology Resident Research Award for
her clinical research regarding the antibiotic
Pradofloxacin. She additionally presented
her research in Germany for the European
College of Veterinary Dermatology.
In 2009, Dr. Restrepo reached the culmi
nation of extensive training and became a
boardcertified Diplomate of the American
College of Veterinary Dermatology. She con
tinued to practice in California until returning
home to Florida, and joining Veterinary Der
matology Center in 2011.
Dr. Restrepo enjoys lecturing to local and
national audiences regarding the field of
dermatology.
Veraflox_2012_Proceedings_fa.indd 29 20.11.12 11:02
Dog selection Group II
During the same time frame, 10 clinically normal dogs (defined as having no known underlying
dermatological or metabolic disease), were randomly enrolled into Group II. These dogs were
all owned by various veterinary students and teaching hospital employees who volunteered
their pet for study purposes.
Inclusion and exclusion criteria
Dogs were excluded from the study if, within 14 days prior to enrollment in the study, they
had received systemic or topical antibiotic therapy, systemic or topical antifungal therapy,
oral antihistamines, or killed bacterial products used to stimulate the immune system (e. g.,
Staphage Lysate). Oral and/or topical glucocorticoid therapy was not allowed for at least
4 weeks prior to the study and 6 weeks prior to the study if longacting injectable gluco corti
coids (e. g., methylprednisolone acetate, triamcinolone acetonide) were administered. Dogs
younger than 12 months (small/medium breeds) or 18 months of age (large/giant breeds),
breed ing animals, and pregnant/lactating females were excluded from the study because of the
risk of fluoroquinolones causing an arthropathy in young, rapidly growing animals.34 Concurrent
medications allowed during the study included heartworm and fleapreventive products,
topical flea treatments, prescription diets, nonsteroidal antiinflammatory drugs, vitamin/
mineral or fatty acid supplements, and vaccines. Medications (e. g., thyroid supplements,
cardio vascular medications, etc.) to control underlying medical conditions were permitted.
Shampoos with coatconditioning or hypoallergenic properties were allowed during the study.
German shepherd dogs with pyoderma and dogs with demodicosis and any other underlying
etiology of pyoderma were included in the study.43
Underlying etiology of the pyoderma was diagnosed by use of the abovelisted standard
diagnostic methods. Atopic dermatitis was diagnosed by criteria established by Willemse35
and Prelaud et al.36 A hypoallergenic diet trial was used to diagnose food hypersensitivity.5
Identification of the organisms and response to ectoparasite treatment were used to diag
nose sarcoptic mange, demodicosis, and flea allergy dermatitis.37 – 39 Hypothyroidism was
diag nosed via detection of low total thyroxine (T4), free thyroxine, and a high concentration
of thyroxinestimulating hormone,40 compared to reference ranges (1.0 to 3.6 µg/dl, 1.0 to
3.5 ng/ml, and 0 to 0.6 ng/ml, respectively) established at the veterinary microbiology labo
ratory at the University of CaliforniaDavis Veterinary Medical Teaching Hospital.43 Adequate
control of hypothyroidism was defined as having a T4 (4 to 6 hours postpill) at the upper
end of the reference range40, 41 determined within the 3month period prior to presentation.
Underlying diseases were defined as diseases that, when treated successfully in combination
with oral antibiotics, resulted in resolution of the pyoderma or a decrease in the relapse rate
of the pyoderma. Included dogs were prescribed an approximate dose of 3 mg/kg PRA PO
q 24 hours for 28 days in cases of superficial pyoderma, and for 42 days in cases of deep
pyoderma.43 Dogs were returned for followup assessment of clinical progress at 28 days for
superficial pyoderma and at 21 and 42 days for deep pyoderma.43
2nd International Veraflox® Symposium | 29 – 30 Nov 2012, Rome
Veraflox_2012_Proceedings_fa.indd 30 20.11.12 11:02
Experimental protocol Group I
On initial examination (Day 0), prior to administration of oral PRA tablets, all dogs had the
following diagnostics performed:
a. the dogs’ general and dermatological histories were obtained; these included previous
episodes of pyoderma, preexisting conditions, concurrent therapies, and antimicrobial
treatment within the previous year
b. complete dermatological examination was performed, and specific clinical signs or le
sions indicative of pyoderma were documented. The affected body sites were recorded
on the dorsal and ventral views of a schematic dog
c. CBC, chemistry profile performed
d. digital photographs obtained of patient and skin lesions
e. acetate tape cytology of lesional skin (e. g., pustules, papules, crusts, epidermal collaret
tes, nodules, fistulae); 4 sites
f. one 6mm skin punch biopsy of lesional skin submitted to the histopathology service to
confirm superficial versus deep pyoderma
g. aerobic culture and antimicrobial sensitivity collected using a sterile, dry swab rolled over
one epidermal collarette12 (in cases presenting with superficial pyoderma)
h. one 6mm skin biopsy obtained using aseptic technique and submitted for aerobic and
anaerobic tissue culture (if the dog had clinical signs of deep pyoderma). Susceptibility
was measured by MIC determination at the microbiology laboratory at the University of
CaliforniaDavis Veterinary Medical Teaching Hospital by use of broth microdilution tech
niques in accordance with the CLSI. Additionally, S. pseudintermedius isolates were
sent to Microbial Research Incorporated (Fort Collins) for PRA MIC testing (6 months
after comple tion of the study protocol for retrospective analysis, as PRA MIC testing
was not available during the study) by use of broth microdilution techniques in ac
cordance with the CLSI document M31A2.The bacterial isolates were frozen and stored
at – 80 °C in a sterile vial containing porous beads (Prolab Diagnostics, Austin).
i. serum sample (3.5 mls whole blood collected in red top tube and serum separated) col
lected for HPLC analysis to serve as negative control
j. clients were sent home with instructions to administer the prescribed approximate dose
of 3 mg/kg PRA PO q 24 hours in the morning on all days except on the day of the sec
ond visit, when they must not administer the PRA. Clients were instructed to not feed the
pet after 10 pm the night before the second visit
k. the second visit was scheduled on any day between 3 – 6 days of receiving PRA daily
Second visit [Day 3 – 6]: pre-administration of PRA
a. acetate tape cytology of lesional skin (4 sites)
b. one 6mm punch biopsy from lesional skin was obtained for HPLC analysis of PRA;
sample labeled t0 L
c. one 6mm punch biopsy from nonlesional skin was obtained for HPLC of PRA;
sample labeled t0 NL
d. serum sample was obtained for HPLC analysis and labeled as TROUGH (8 – 14 hours
fast); sample labeled t0 Serum
dr. c. restrepo | Tissue concentrations in canine pyoderma: does it reach highly enough
directly in affected skin?
30 | 31
Veraflox_2012_Proceedings_fa.indd 31 20.11.12 11:02
e. prescribed dose of PRA was administered orally by the investigator
f. sideeffects as reported by owner recorded
Second visit [Day 3 – 6]: 2 hours post-administration of PRA
a. one 6mm punch biopsy from lesional skin was obtained for HPLC analysis of PRA;
sample labeled t2 L
b. one 6mm punch biopsy from nonlesional skin was obtained for HPLC of PRA; sample
labeled t2 NL
c. serum sample was obtained for HPLC analysis and labeled as PEAK (2 hours additional
fast); sample labeled t2 Serum
Second visit [Day 3 – 6]: 4 hours post-administration of PRA
a. one 6mm punch biopsy from lesional skin was obtained for HPLC analysis of PRA;
sample labeled t4 L
b. one 6mm punch biopsy from nonlesional skin was obtained for HPLC of PRA; sample
labeled t4 NL
c. serum sample was obtained for HPLC analysis and labeled as PEAK (4 hours additional
fast); sample labeled t4 Serum
d. 28 – 42 days of PRA prescribed for superficial and deep pyoderma, respectively
Third visit [Day 28 – 42 of PRA]
a. record clinical response in terms of resolution of pyoderma; performed at Day 28 for
dogs with superficial pyoderma, and at Day 42 for dogs with deep pyoderma
b. acetate tape cytology of lesional skin (4 sites)
c. CBC and chemistry profile performed
d. digital photographs obtained of patient
e. if needed, 3 additional weeks of PRA prescribed for dogs deep pyoderma
Experimental protocol Group II
On initial presentation (Day 0), prior to administration of oral PRA tablets, all dogs had the
following diagnostics performed:
a. complete blood count (CBC) and chemistry profile were performed
b. digital photographs obtained of patient
c. skin cytology (4 sites) obtained to ensure normal skin
d. serum sample (3.5 mls whole blood collected in red top tube and serum separated) col
lected for HPLC analysis to serve as negative control
e. one 6mm punch biopsy of skin (dorsum) submitted for histopathology to confirm nor
mal skin and assess presence of leukocytes
f. clients were sent home with instructions to administer the prescribed approximate dose
of 3 mg/kg PRA PO q 24 hours in the morning on all days except on the day of the sec
ond visit, when they must not administer the PRA. Clients were instructed to not feed the
pet after 10 pm the night before the second visit
g. the second visit was scheduled on any day between 3 – 6 days of receiving PRA daily
2nd International Veraflox® Symposium | 29 – 30 Nov 2012, Rome
Veraflox_2012_Proceedings_fa.indd 32 20.11.12 11:02
Second visit [Day 3 – 6]: Pre-administration of PRA
a. skin cytology (4 sites) obtained to ensure normal skin
b. one 6mm punch biopsy from skin (dorsum) was obtained for HPLC analysis of PRA;
sample labeled t0 Skin
c. serum sample was obtained for HPLC analysis and labeled as TROUGH (8 – 14 hours
fast); sample labeled t0 Serum
d. prescribed dose of PRA was administered orally by the investigator
e. sideeffects as reported by owner recorded
Second visit [Day 3 – 6]: 2 hours post-administration of PRA
a. one 6mm punch biopsy from skin (dorsum) was obtained for HPLC analysis of PRA;
sample labeled t2 Skin
b. serum sample was obtained for HPLC analysis and labeled as PEAK (2 hours additional
fast); sample labeled t2 Serum
Second visit [Day 3 – 6]: 4 hours post-administration of PRA
a. one 6mm punch biopsy from skin (dorsum) was obtained for HPLC analysis of PRA;
sample labeled t4 Skin
b. serum sample was obtained for HPLC analysis and labeled as PEAK (4 hour additional
fast); sample labeled t4 Serum
Assay of pradofloxacin
Upon collection, all tissue biopsy samples collected for histopathology were placed in formalin
and submitted to the university histopathology service. Upon collection, all tissue biopsy sam
ples collected for HPLC analysis were placed in preweighed Oring screw cap polypropylene
1.5ml tubes, and then placed in – 80 °C until analyzed by HPLC. All serum samples for HPLC
analysis were stored in plastic capped 12 x 75 polystyrene containers and placed in – 80 °C
until analyzed by HPLC.
Serum and skin samples were assayed using a highperformance liquid system equipped with
fluorescence detection. One hundred microliters (µl) of serum was diluted with 400 µl of 2 %
tetrabutylammonium hydrogen sulfate [TBAHS] in water. After addition of 500 µl of acetonitrile
to precipitate protein, the cloudy solution was vortexed and incubated at room temperature
for 30 minutes. Following centrifugation at 13,000 x g, 400 µl of the supernatant was removed
and diluted with 800 µl of 2 % TBAHS. A 6mm skin biopsy weighing approximately 40 mg,
from which the hair and underlying subdermis had been removed, was homogenized in 0.8 ml
of 2 % TBAHS and acetonitrile (1:1) in a Brinkman Polytron on a setting of 6 for 3 x 5 seconds.
The homogenate was incubated at room temperature for 30 minutes and then centri fuged
at 13,000 x g for 2 minutes 400 µl of supernatant was removed and added to 800 µl of 2 %
TBAHS solution.
100 µl of a skin or serum sample was analyzed using a 4.6 mm x 25 mm 5 µm C18 equilibrat
ed in 2 % TBAHS and 17.5 % acetonitrile using isocratic elution. Under these conditions, PRA
32 | 33
dr. c. restrepo | Tissue concentrations in canine pyoderma: does it reach highly enough
directly in affected skin?
Veraflox_2012_Proceedings_fa.indd 33 20.11.12 11:02
eluted between 5 and 6 minutes. The fluorescence detector had an excitation wavelength of
289 nm and an emission detection set at 427 nm. The limit of detection under these conditions
was 0.25 ng.
Treatment evaluation
At each followup visit, adverse effects associated with PRA administration were recorded,
a complete dermatological examination was performed, and clinical efficacy was assessed
based on appearance of lesions. Clinical resolution of lesions was defined as the total dis
appearance of all lesions, including erythema and scaling. Efficacy was considered excellent
if > 75 % of lesions were resolving, good if 50 % to 75 % of lesions were resolved, and a fail
ure if < 50 % of lesions had resolved. An additional 14 to 28 days of PRA were prescribed
accordingly. If a dog was considered to have failed treatment, PRA was discontinued, and the
bacterial skin culture and skin biopsy were repeated.43
2nd International Veraflox® Symposium | 29 – 30 Nov 2012, Rome
Table 1 Dogs with deep pyoderma: participant characteristics and treatment outcome
Dog Breed Sex*Age (y)
Wt (kg)
Underlying disease
Clinical response
at 3 weeks
Clinical response
at 6 weeks
1New
foundlandM 8 51 AD, Demodicosis
Good; started
wDemodex txExcellent
2Australian cattle dog
FS 4 26 FAD ExcellentComplete resolution
3 Labrador F 8 41.8Allergic
hypersensitivityGood Good
4 Labrador MC 6 49.4 HTM, AD GoodExcellent; started T4
supplementation
5 Labrador FS 3 34.6Allergic
hypersensitivityGood
Flare of allergy & pyoderma
6Australian shepherd
MC 6 26
Pyogranulomatous deep dermatitis and
panniculitis
Good Good
7Am Staff
terrFS 5 23.5
DTM, Demodex, AD
Good Excellent
8Am Staff
terrM 5 40.4
Severe deep dermal furuncu
losis, actinic keratosis, allergic hypersensitivity
Good Good
* M = male; MC = male castrated; F = female; FS = female spayed
Abbreviations: AD = atopic dermatitis; FAD = flea allergy dermatitis; HTM = hypothyroidism;
DTM = dermatophytosis; Am Staff terr = American Staffordshire terrier; tx = treatment; T4 = thyroid hormone; Wt = weight
Veraflox_2012_Proceedings_fa.indd 34 20.11.12 11:02
Results
Group I study participants
Twenty dogs (eight dogs with a deep pyoderma and twelve dogs with a superficial pyo derma)
completed the study, as previously published.43 Population characteristics are shown in
Tables 1 and 2. Thirteen dogs were male (eight castrated), and seven were female (five spay
ed). Ages ranged from 2 to 11 years (mean 5.9 years). Body weights on Day 0 of treat
ment ranged from 8.2 to 51 kg (mean 33.1 kg). All dogs were presented for chronic, recurrent
pyoderma that was previously unresponsive to treatment with various systemic antibiotics.
Seventeen dogs (i. e., all dogs except case nos. 2, 12, and 17) had received oral antibiotics
within the 12month period prior to enrollment in this study. Case no. 17 had intermittently
received a topical otic medication containing gentamicin sulfate, betamethasone valerate, and
clotrimazole (Otomax; ScheringPlough Animal Health Corp), in both ears during the 2year
34 | 35
Table 2 Dogs with superficial pyoderma: participant characteristics and treatment outcome
Dog Breed Sex*Age (y)
Wt (kg)
Underlying disease
Treatment outcome
at 4 weeks
9Golden retriever
M 8 39.7Allergic
hypersensitivity, HTMComplete resolution
10English pointer
MC 7 25Actinic keratosis, HTM,
furunculosisExcellent
11 Terrier mix MC 3 8.2Allergic hypersensitivity,
calcinosis cutis, furunculosis
Excellent
12Labrador
mixFS 8 19.6 Allergic
hypersensitivityExcellent
13Rough collie
M 11 32Allergic
hypersensitivityGood
14 Boxer MC 3 31.4 Demodicosis Excellent
15 Labrador FS 2 34.4Allergic
hypersensitivityExcellent
16Rough collie
F 3 29 Allergic hypersensitivity
Complete resolution
17 Labrador MC 5 42.1Allergic
hypersensitivityExcellent
18 Dalmatian M 9 33.2Allergic
hypersensitivityExcellent
19 Malamute MC 7 42.3 HTM, alopexia X Excellent
20Golden retriever
MC 7 31.9Allergic
hypersensitivityExcellent
* M = male; MC = male castrated; F = female; FS = female spayed
Abbreviations: HTM = hypothyroidism; Wt = weight
dr. c. restrepo | Tissue concentrations in canine pyoderma: does it reach highly enough
directly in affected skin?
Veraflox_2012_Proceedings_fa.indd 35 20.11.12 11:02
period prior to parti cipating in this study. Three dogs (case nos. 9, 10, and 19) had preexisting
hypothyroidism that was adequately controlled on thyroid supplement ation. Case no. 4 was
diagnosed with hypothyroidism and started on thyroid supplementation after completion of
the PRA study protocol (i. e., 42 days after initiation of PRA).
Underlying diseases diagnosed in included dogs have also been summarized in Tables 1 and
3, as previously published.43 Allergic hypersensitivity dermatitis (i.e., flea allergy, food allergy,
and/or atopic dermatitis) was diagnosed in ten (50 %) of the twenty dogs. An additional six of
the remaining ten dogs had allergic dermatitis with a concurrent underlying disease (e. g., hy
pothyroidism, demodicosis, actinic keratosis); thus, sixteen of the twenty dogs had underlying
allergic dermatitis with or without concurrent conditions.43
Group I histopathology
All dogs had histopathological evidence of pyoderma on the basis of established criteria.9
Disease was further classified as deep pyoderma if evidence of deep folliculitis (at the level of
isthmus and below), furunculosis, or cellulitis was present on skin biopsy specimens.9 Accord
ing to histological criteria, eight dogs were categorized as having deep pyoderma.43 Case
no. 1 had demodicosis and atopic dermatitis. Case no. 6 had severe, chronic, pyogranulo
matous deep dermatitis and panniculitis (acidfast stain was negative; the broadspectrum
immunohistochemical marker for bacterial or fungal organisms (bacillus Calmette–Guerin) was
negative; Brown and Brenn (B & B) Gram stain and Periodic acidSchiff (PAS) stain for bacterial
and fungal organisms, respectively, were also negative). Case no. 7 had intralesional dermato
phyte endospores and hyphae. Case no. 8 had allergic dermatitis, mild actinic keratosis, and
severe, nodular, superficial and deep dermatitis with furunculosis (special stains including
B & B and PAS were negative for bacterial and fungal organisms, respectively). Twelve dogs
met the histological criteria for superficial pyoderma and included case no. 14 with demodi
cosis; case no. 10 with actinic keratosis, hypothyroidism, and focal furunculosis; and case
no. 11 with allergic dermatitis, folliculitis, focal furunculosis, and calcinosis cutis. The latter two
dogs were initially enrolled in the protocol for dogs with superficial pyoderma according to
clinical lesions (despite the later discovery of focal lesions of deep pyoderma on histopatho
logical examination).43
Group I skin cytology
All 20 dogs had initial skin cytology results consistent with bacterial pyoderma. After PRA
treatment, all dogs had reduction or complete resolution of bacterial counts. Skin cytology
results, as previously published, are shown in Table 3.43
Group I bacterial culture and antimicrobial susceptibility
Deep pyoderma dogs (case nos. 1– 8)
Anaerobic tissue cultures were negative for all dogs except case no. 3, in which small num
bers of Peptostreptococcus anaerobius were cultured (susceptibility panel not performed).
Staphylococcus pseudintermedius was isolated in pure culture from case nos. 1, 2, 4, and 5.
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Veraflox_2012_Proceedings_fa.indd 36 20.11.12 11:02
36 | 37
Table 3 Group 1: skin cytology results for all dogs
Dog Day 0* Day 21* Day 42*
1 TNTC rods/cocci 2 – 5 cocci/rods 1 – 5 cocci
2 11 – 20 cocci 1 – 5 cocci negative
3 20 cocci, TNTC PMNs 11 – 20 cocci 1 – 5 cocci
4 TNTC cocci 6 –10 cocci 1 – 5 cocci
5 11 – 20 cocci 1 – 5 cocci 1 – 5 cocci
6 TNTC cocci / PMNs 20 cocci, PMNs 6 –10 cocci
7 6 – 10 cocci 6 – 10 cocci negative
8 TNTC cocci / PMNs 11 – 20 cocci 6 – 20 cocci
Dog Day 0* Day 28*
9 11 – 20 cocci negative
10 6 – 10 cocci negative
11 TNTC cocci, 6 – 10 rods 1 – 5 cocci
12 6 – 10 cocci 1 – 5 cocci
13 1 – 5 cocci negative
14 6 – 10 cocci 6 – 10 cocci
15 6 – 10 cocci 1 – 5 cocci
16 6 – 10 cocci negative
17 6 – 10 cocci 1 – 10 cocci
18 6 – 10 cocci 1 – 5 cocci
19 6 – 10 cocci 1 – 5 cocci
20 6 – 10 cocci 1 – 2 cocci
* M = male; MC = male castrated; F = female; FS = female spayed
Abbreviations: HTM = hypothyroidism
Dr. C. Restrepo | Tissue concentrations in canine pyoderma: does it reach highly enough
directly in affected skin?
Veraflox_2012_Proceedings_fa.indd 37 20.11.12 15:02
2nd International Veraflox® Symposium | 29 – 30 Nov 2012, Rome
AntibioticDog
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Dog 8†
Amikacin S S S S S S –– ––
Amoxicillin/ clavulanic acid
S S S S S S –– ––
Cefazolin S S S S S S –– ––
Ceftiofur S S S S S S –– ––
Ceftizoxime S S S S S S –– ––
Chloramphenicol S S S S S S –– ––
Erythromycin S S S S S R –– ––
Clindamycin NA NA NA I S NA –– ––
Gentamicin S S S S S S –– ––
Oxacillin + 2 % NaCl S S S S S S –– ––
Penicillin S S S S S R –– ––
Rifampin S S S S S S –– ––
Tetracycline S S S S S R –– ––
Trimethoprim/ sulphamethoxazole
S S S S S S –– ––
Cefpodoxime NA NA NA S S S –– ––
Imipenem NA NA NA S S S –– ––
Enrofloxacin S S S S S S –– ––
Marbofloxacin NA NA NA S S S –– ––
Orbifloxacin NA NA NA I S I –– ––
Pradofloxacin‡ 0.06 0.12 NA 0.12 0.12 0.06 –– ––
Table 4 Antibiotic susceptibility* of S. pseudintermedius for dogs with deep pyoderma
* = R = resistant; S = susceptible; I = intermediate; NA = testing not performed;
† = no bacterial growth; ‡ = PRA MIC results ug/ml for S. pseudintermedius
Veraflox_2012_Proceedings_fa.indd 38 20.11.12 15:02
In addition to S. pseudintermedius, case nos. 3 and 6 had very small numbers of Streptococ
cus canis growth (sensitivity panel not performed). Case nos. 7 and 8 had no aerobic bacterial
growth from tissue culture. Thus, S. pseudintermedius was isolated from six of eight dogs.
Details of the culture results (including PRA MIC results) obtained on Day 0, as previously pub
lished, are shown in Table 4.43
Superficial pyoderma dogs (case nos. 9 – 20).
Staphylococcus pseudintermedius was isolated in pure culture from nine of twelve dogs (case
nos. 9 to 17). Methicillinresistant, coagulasenegative Staphylococcus spp. was isolated in
small numbers from case no. 18. Pseudomonas aeruginosa was cultured from an intact pus
tule in case no. 19. Case no. 20 had a negative bacterial culture.
Details of the culture results (including PRA MIC results) obtained on Day 0 of the study, as
previously published, are shown in Table 5.43
Group I clinical results
Pradofloxacin dosages were calculated and rounded up to the nearest tablet size; this re sult
ed in a PRA dosage range of 3.0 to 4.6 mg/kg, with a mean dosage of 3.7 mg/kg. Of the eight
dogs diagnosed with deep pyoderma, one had an excellent response, and seven had a good
response after 21 days of PRA administration. The dog with the excellent response at 21 days
went on to have complete resolution by 42 days. Of the other seven dogs, three had excellent
responses, and three had good responses after 42 days of PRA administration. The last dog
had a flare of allergic dermatitis and subsequent pyoderma diagnosed 42 days after initiating
PRA administration. Treatment outcomes for dogs with deep pyoderma are listed in Table 1.43
Of the twelve dogs diagnosed with superficial pyoderma, two had complete resolution, nine
had excellent response, and one had a good response after 28 days of PRA adminis tration. An
excellent clinical response was obtained within 28 days of treatment for case no. 17 de spite
the reported in vitro resistance to enrofloxacin, marbofloxacin, ciprofloxacin, and orbifloxacin.
An excellent clinical response was also achieved in case no. 18 with methicillinresistant,
coagulasenegative Staphylococcus spp. (enrofloxacin and orbifloxacinsuscept ible) and in
case no. 19 with multiantibioticresistant P. aeruginosa (enrofloxacinsusceptible; orbifloxacin
intermediate susceptibility) within 28 days of treatment. Treatment outcomes for dogs with
superficial pyoderma, as previously published, are listed in Table 2.43
Group II study participants
Five dogs were male (four castrated), and five were spayed females. Ages ranged from 2 to
13 years (mean 7.3 years). Body weights on initial exam (Day 0) ranged from 11.6 kg to 38 kg
(mean 24.6 kg). PRA dosage ranged from 2.9 to 5 mg/kg (mean 3.6 mg/kg).
Group II histopathology
All dogs had histopathologically normal skin on the basis of established criteria.9
38 | 39
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Veraflox_2012_Proceedings_fa.indd 39 20.11.12 11:02
2nd International Veraflox® Symposium | 29 – 30 Nov 2012, Rome
Table 5 Antibiotic susceptibility for dogs with superficial pyoderma
AntibioticDog
9 –11*Dog 12*
Dog 13 /14*
Dog 15*
Dog 16*
Dog 17*
Dog 18†
Dog19‡
Dog20§
Amikacin S S S S S S S S ––
Amoxi/Clav S S S S S S R R ––
Cefazolin S S S S S S R R ––
Ceftiofur S S S S S S R R ––
Ceftizoxime S S S S S S R I ––
Chloramphenicol S S S S S S S R ––
Erythromycin S S S S S R R R ––
Clindamycin NA NA S S NA R S R ––
Gentamicin S S S S S S S S ––
Oxacillin + 2 % NaCl S S S S S S R R ––
Penicillin S R S S R R R R ––
Rifampin S S S S S S S R ––
Tetracycline S S R R R R R R ––
Doxycycline NA NA NA NA NA S NA NA ––
Trimethoprim/ sulphamethoxazole
S S S S S R S R ––
Cefpodoxime S S S S NA S R R ––
Imipenem S S S S NA S R S ––
Enrofloxacin S S S S S R S S ––
Marbofloxacin S S S S NA R S S ––
Orbifloxacin NA NA NA NA NA R S I ––
Ciprofloxacin NA NA NA NA NA R NA NA ––
Pradofloxacin# 0.12 0.12 4.0 0.12 0.12 4.0 –– –– ––
* = Susceptibility results for S. pseudintermedius; † = susceptibility results for methicillin-resistant coagulase-negative
Staphylococcus spp.; ‡ = susceptibility results for Pseudomonas aeruginosa; § = no bacterial growth;
# = PRA MIC results µg/ml for S. pseudintermedius
Veraflox_2012_Proceedings_fa.indd 40 20.11.12 15:02
40 | 41
Group II CBC and chemistry profiles
On initial exam, all dogs had values within normal reference range.
Group II skin cytology
Bacteria were not found on cytol ogy in all normal dogs.
Group I and II: adverse effects
All adverse events reported by the owners were mild. One dog had one episode of vomiting
on the second day of PRA administration along with 2 days of diarrhoea. Treatment was not
required, and these signs resolved completely by the fourth day of PRA administration. On
the second day of PRA administration, one dog started having soft stools at the end of an
initially normal bowel movement. This condition resolved upon completion of the PRA trial. A
third dog reportedly had feces that were occasionally lighter in color during PRA treatment.43
Group I and II: PRA concentration in serum
Mean concentration of PRA in serum of Group I and Group II dogs is summarized in Table 6.
After 3 – 6 days of PRA administration, results at all three time points for both groups was
equivalent. The mean peak serum concentration occurred at 2 hours postpill administration
in both groups. These levels were sustained through the 4hour timepoint, in both groups.
Data for certain time points and individual patients was lost during processing of samples.
Therefore, data were reported when available, as listed in Table 6. See Graph 1 and 2 for
summary of mean values.
Group I and II: PRA concentration in skin
Mean concentration of PRA in skin of dogs in Group I and Group II is summarized in Table 7.
PRA concentration in skin affected with pyoderma (i. e., lesional skin) at 2 and 4 hours post PRA
administration was approximately double the concentration of nonlesional skin in dogs with
pyoderma and approximately three times the concentration of skin in normal dogs (Group II).
For all groups, the mean peak skin concentration also occurred at 2 hours post PRA admin
istration and remained consistently elevated at the 4hour postpill time point. The standard
deviations were characteristic of variables in clinical trials such as body fat distribution. See
Graph 1 and 2 for summary of mean values.
Table 6 Pradofloxacin serum concentration (mean)
Group Iµg/ml (mean)Pyoderma dogs
Data available in no. of patients
Group II µg/ml (mean)Normal dogs
Data available in no. of patients
T0 = 0.46 ± 0.34 13/20 T0 = 0.44 ± 0.26 10/10
T2 = 2.07 ± 0.99 12/20 T2 = 2.09 ± 0.67 10/10
T4 = 1.92 ± 0.84 13/20 T4 = 1.82 ± 0.66 10/10
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Veraflox_2012_Proceedings_fa.indd 41 20.11.12 11:02
2nd International Veraflox® Symposium | 29 – 30 Nov 2012, Rome
DiscussionIn this study, all twenty dogs diagnosed with either superficial or deep pyoderma had good,
excellent, or complete responses from 21 to 42 days after initiating PRA. The criteria cho
sen for classification of good, excellent, or complete response were markedly stringent and
not consistent with criteria used in clinical practice. However, the investigators chose these
criteria in order to reduce potential bias and reduce the likelihood of overestimating clinical
efficacy. In clinical practice, patients with mild to moderate postinflammatory scaling (seen
clinically as scaling on the skin after treatment of superficial pyoderma) after 4 weeks of treat
ment are considered to have complete resolution of pyoderma. Therefore, the patients in
this study classified as having excellent response would have been considered to have had
complete resolution of pyoderma, by general clinical practice standards. Clinically, the in
vestigators observed rapid response to PRA treatment. In several cases, by the time of the
first visit (3 – 6 days of PRA administration), pyoderma lesions had greatly improved and few
were visible. Owners frequently remarked how rapidly their dog was responding to treatment.
Within 28 days of treatment, nine of twelve dogs diagnosed with super ficial pyoderma had
excellent responses, and two of the twelve dogs had complete resolution. Recurrence of the
superficial pyoderma within 14 days after cessation of oral antibiotic therapy with PRA was
not seen in any of the twelve dogs. While twelve dogs included in this study had superficial
pyoderma based on histopathological evaluation, two of these dogs also had focal evidence
of moderate (case no. 10) to severe (case no. 11) furunculosis. Both of these dogs had an
excellent clinical response within 28 days of PRA treatment but required an additional 14 to
28 days of treatment with PRA for complete resolution of the deep pyoderma. Case no. 13
had complete resolution of truncal lesions at 28 days of PRA treatment; however, the dog
was only classified as having a good response because of a persistent abdominal fold inter
trigo. Abdominal fold intertrigo (also known as skin fold dermatitis) cannot be resolved with
systemic antibiotics. Correction of the anatomical defect (e. g., excess abdominal fold from
obesity, as in case no. 13) is necessary for a cure. If correction of the anatomical defect is
unattainable, longterm topical treatments will be required to remove surface organisms and
Table 7 Pradofloxacin skin concentration (mean)
Group I Pyoderma Lesional skin µg/g (mean)
Data avail-able in no. of pa-tients
Group I Pyoderma Non-lesional skin µg/g (mean)
Data avail-able in no. of pa-tients
Group II Normal skin µg/g (mean)
Data avail-able in no. of pa-tients
T0 = 1.14 ± 1.03 13/20 T0 = 0.79 ± 0.91 11/20 T0 = 0.96 ± 1.28 10/10
T2 = 5.02 ± 3.32 11/20 T2 = 2.34 ± 1.29 12/20 T2 = 1.49 ± 1.19 10/10
T4 = 4.56 ± 2.81 13/20 T4 = 2.26 ± 2.03 13/20 T4 = 1.47 ± 1.36 10/10
Veraflox_2012_Proceedings_fa.indd 42 20.11.12 11:02
42 | 43
the entrapped debris.2 Of the eight dogs with deep pyoderma, only one dog (case no. 2) had
an underlying disease (flea allergy dermatitis) that was fully controlled and therefore allowed
complete resolution of the deep pyoderma within 42 days of initiating PRA therapy. Case no. 6
had deep pyoderma secondary to a primary immunemediated inflammatory process (nodular
dermatitis and panniculitis). After 42 days of PRA treatment alone, the pyoderma substantially
improved as evidenced by marked reduction in lesion size and lesional exudate. A complete
resolution of the skin disease was only achieved in case no. 6 when immunosuppressive
ther apy was initiated. Case no. 8 had a 3year history of allergic pruritus in addition to large cu
taneous furuncles that would rupture periodically. Histopathological examination in this case
revealed deep dermal furunculosis, chronic granulation tissue and scarring, and evidence
of actinic keratosis.These confounding factors impeded complete resolution of skin disease
with oral antibiotics alone. Nonetheless, while the underlying skin disease was unable to be
re solved, the secondary pyoderma showed a good response to PRA therapy.43
In clinical practice, it is important to remember that the use of fluoroquinolones only be con
sidered in cases where canine pyoderma has been refractory to appropriate “firstline” anti
biotics. Fluoroquinolones are most useful in the management of recurrent pyoderma and in
chronic, deep pyoderma cases with extensive scar tissue.32 The clinical outcomes of these
cases highlight the importance of determining and treating underlying causes of superficial
and deep pyoderma in order to achieve complete resolution of pyoderma.1, 2 Furthermore,
S. pseudintermedius has been shown to adhere to corneo cytes preferentially in dogs with
atopic dermatitis.42 In this study,43 culture of S. pseudintermedius from fifteen (75 %) of
twenty dogs with superficial and deep pyoderma is in agreement with results of previously
report ed studies,10 – 12 indicating that this organism is the most common canine skin pathogen.
Interestingly, case no. 17 cultured positive for a methicillinsensi tive, fluoroquinoloneresistant
S. pseudintermedius. This dog had an excellent clinical response to PRA at 28 days. It is
well known that in vitro susceptibility may not always correlate with in vivo clinical response.
Graph 2 Group II (normal dogs): serum and skin [PRA]
Serum concentrations Skin concentrations
Trough 2 hrs 4 hrs
3.0
2.5
2.0
1.5
1.0
0.5
0.0
µg/m
l Graph 1 Group I (pyoderma dogs): serum and skin [PRA]
Patient serum Nonlesional skin Skin lesion
Trough 2 hrs 4 hrs
8
7
6
5
4
3
2
1
0
µg/m
l
dr. c. restrepo | Tissue concentrations in canine pyoderma: does it reach highly enough
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Veraflox_2012_Proceedings_fa.indd 43 20.11.12 11:02
Figure 1 Histopathology of normal dog skin in Group II dogs
2nd International Veraflox® Symposium | 29 – 30 Nov 2012, Rome
This may be due to individual
variation regarding drug concen
tration, metabolism, absorp
tion, and in vivo variance of the
bac terium itself and its re sponse
to the drug. Case nos. 18 and
19 had methicillinresist ant, co
agu laseneg a tive Sta phy lo coc
cus spp. and P. aeruginosa, re
spectively. Both of these dogs
also had excellent clinical re
sponse to PRA within 28 days of
treatment.43
The PRA serum concentration
in both groups, as evaluated by
HPLC, was similar for all time
points assessed. Interestingly,
the 4hour postpill PRA con
centration remains similar to the
2hour peak concentration. The
most notable difference between
the groups was the marked in
crease in PRA concentration of
lesional skin in dogs with pyo
derma. The peak PRA concen
tration in lesional skin in dogs
with pyoder ma was approxi
mately double the peak concen
tration of nonlesional skin in dogs
with pyoderma as well as peak
serum concentration of all dogs;
and triple the peak concentration
of normal skin (Group II). These
results confirm our hypothesis
that diseased tissue confers a
marked increase in PRA fluoro
quinolone concen tration.
Figure 2 Histopathology of lesional skin in Group I dogs
Figure 3 Closeup of Figure 2: pyoderma affected skin with high white blood cell count
Veraflox_2012_Proceedings_fa.indd 44 20.11.12 11:02
44 | 45
ConclusionBased on results of this study, at a mean dosage of 3.7 mg/kg PO q 24 hours, PRA exceed
ed therapeutic tissue concentrations in dogs with pyoderma as early as 2 hours post drug
administration. These results support previous data showing high concentrations of fluoro
quinolones in chronic inflammation.44 Higher concentrations of PRA in lesional skin (see Figure 2)
support active uptake of PRA by inflammatory cells (see Figure 3). The majority of dogs de
monstrated complete to excellent clinical efficacy within 3 – 6 weeks for superficial and deep
pyoderma, respectively.43 PRA is a safe and highly efficacious treatment for canine superficial
and deep pyoderma, regardless of underlying skin condition.43 This is in agreement with the
results of a recently published study.10
Thirdgeneration fluoroquinolones such as PRA have enhanced activity against Grampositive
bacteria relative to first and secondgeneration compounds, which differentiates PRA from
earliergeneration fluoroquinolone compounds used in veterinary medicine. The efficacy of
many antimicrobial agents is being threatened by a global increase in the number of resistant
bacterial pathogens. Therefore, thirdgeneration fluoroquinolones, such as PRA, clearly have
important utility in veterinary medicine as singledrug therapy for conditions caused by aero
bic/anaerobic infections.
References
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03 | White SD, Ceragioli KL, Stewart LJ, et al. Cutaneous markers of canine hyperadrenocorticism. Compend Contin Educ 1989; 11:446–465.
04 | Ihrke PJ. Bacterial infections of the skin. In: Infectious Diseases of the Dog and Cat. Greene CE (ed.), W.B. Saunders, Philadelphia, 1998, pp. 541–547.
05 | White S. Food allergy in dogs. Compend Contin Educ 1998; 20:261–269.
06 | DeBoer DJ, Marsella R. The ACVD task force on canine atopic dermatitis (XII): the relationship of cutaneous infections to the pathogenesis and clinical course of canine atopic dermatitis. Vet Immunol Immunopathol 2001; 81:239–249.
07 | Gulikers K, Panciera D. Influence of various medications on canine thyroid function. Compend Contin Educ Pract Vet 2002; 24:511–523.
08 | Barriga OO, alKhalidi NW, Martin S, et al. Evidence of immunosuppression by Demodex canis. Vet Immunol Immunopathol 1992; 32:37–46.
09 | Gross TL, Ihrke PJ, Walder EJ, et al. Skin Diseases of the Dog and Cat: Clinical and Histopathologic Diagnosis. 2nd edn., Blackwell Sciences Ltd., Ames, 2005.
10 | Mueller RS, Stephan B. Pradofloxacin in the treatment of canine deep pyoderma: a multicenter, blinded, randomized parallel trial. Vet Dermatol 2007; 18:144–151.
11 | Petersen AD, Walker RD, Bowman MM, et al. Frequency of isolation and antimicrobial susceptibility patterns of Staphylococcus intermedius and Pseudomonas aeruginosa isolates from canine skin and ear samples over a 6year period (1992–1997). J Am Anim Hosp Assoc 2002; 38:407–413.
12 | White SD, Brown AE, Chapman PL, et al. Evaluation of aerobic bacteriologic culture of epidermal collarette specimens in dogs with superficial pyoderma. J Am Vet Med Assoc 2005; 226:904–908.
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13 | Hillier A, Alcorn JR, Cole LK, et al. Pyoderma caused by Pseudomonas aeruginosa infection in dogs: 20 cases. Vet Dermatol 2006; 17:432–439.
14 | Jang SS, Breher JE, Dabaco LA, et al. Organisms isolated from dogs and cats with anaerobic infections and susceptibility to selected antimicrobial agents. J Am Vet Med Assoc 1997; 210:1610–1614.
15 | White SD. Systemic treatment of bacterial skin infections of dogs and cats. Vet Dermatol 1996; 7:133–144.
16 | Carlotti DN. New trends in systemic antibiotic therapy of bacterial skin disease in dogs. Compend Contin Educ 1996; 18:40–47.
17 | Dowling PM. Antimicrobial therapy of skin and ear infections. Can Vet J 1996; 37:695–699.
18 | Jazic E, Coyner KS, Loeffler DG, et al. An evaluation of the clinical, cytological, infectious and histopathological features of feline acne. Vet Dermatol 2006; 17:134–140.
19 | Patel A, Lloyd DH, Lamport AI. Antimicrobial resistance of feline staphylococci in southeastern England. Vet Dermatol 1999; 10:257–261.
20 | Morris DO, Rook KA, Shofer FS, et al. Screening of Staphylococcus aureus, Staphylococcus intermedius, and Staphylococcus schleiferi isolates obtained from small companion animals for antimicrobial resistance: a retrospective review of 749 isolates (2003–2004). Vet Dermatol 2006; 17:332–337.
21 | Ganiere JP, Medaille C, Mangion C. Antimicrobial drug susceptibility of Staphylococcus intermedius clinical isolates from canine pyoderma. J Vet Med B Infect Dis Vet Public Health 2005; 52:25–31.
22 | Abraham J, Morris D, Griffeth G, et al. Screening of healthy cats and cats with inflammatory skin disease for colonization of the skin by methicillinresistant coagulasepositive staphylococci and Staphylococcus schleiferi ssp. schleiferi. 22nd Proceedings North American Veterinary Dermatology Forum, Kauai, HI, 2007; p. 173.
23 | Silley P, Stephan B, Greife HA, et al. Comparative activity of pradofloxacin against anaerobic bacteria isolated from dogs and cats. J Antimicrob Chemother 2007; 60:999–1003.
24 | Pridmore A, Stephan B, Greife HA, et al. In vitro activity of pradofloxacin against clinical isolates from European field studies. 105th Annual General Meeting American Society of Microbiology, Atlanta, GA, 2005.
25 | Fraatz K, Krebber R, Edingloh M, et al. Communications: oral bioavailability of pradofloxacin tablets and renal drug excretion in dogs. J Vet Pharmacol Ther 2003; 26:88–89.
26 | Fraatz K, Heinen K, Krebber R, et al. Communications: skin concentrations and serum pharmacokinetics of pradofloxacin in dogs after multiple oral administrations at four different dosages. J Vet Pharmacol Ther 2003; 26:89.
27 | Bregante MA, De Jong A, Calvo A, et al. Communications: protein binding of pradofloxacin, a novel 8cyanofluoroquinolone, in dog and cat plasma. J Vet Pharmacol Therap 2003; 26:87–88.
28 | Wright DH, Brown GH, Peterson ML, et al. Application of fluoroquinolone pharmacodynamics. J Antimicrob Chemother 2000; 46:669–683.
29 | Litster A, Moss S, Honnery M, et al. Clinical efficacy and palatability of pradofloxacin 2.5 % oral suspension for the treatment of bacterial lower urinary tract infections in cats. J Vet Intern Med 2007; 21:990–995.
30 | Paradis M, Abbey L, Baker B, et al. Evaluation of the clinical efficacy of marbofloxacin (Zeniquin) tablets for the treatment of canine pyoderma: an open clinical trial. Vet Dermatol 2001; 12:163–169.
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32 | Spindel ME, Lappin MR, Bachman D, et al. Pradofloxacin for the treatment of suspected feline bacterial rhinitis. J Vet Intern Med 2007; 21:627–628.
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35 | Prelaud P, Guagere E, Alhaidari Z. Reevaluation of diagnostic criteria of canine atopic dermatitis. Rev Med Vet 1998; 149:1057–1064.
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37 | Mueller RS. Treatment protocols for demodicosis: an evidencebased review. Vet Dermatol 2004; 15:75–89.
38 | Shanks DJ, McTier TL, Behan S, et al. The efficacy of selamectin in the treatment of naturally acquired infestations of Sarcoptes scabiei on dogs. Vet Parasitol 2000; 91:269–281.
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40 | Ferguson D. Thyroid hormone replacement therapy. In: Current Veterinary Therapy IX. Small Animal Practice. Kirk R (ed.), W.B. Saunders, Philadelphia, 1986, pp. 1018–1025.
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Introduction
Bacterial infection is one of the most common skin complaints in small animal practice. Skin
infections are divided into surface, superficial, and deep infections. Surface infections include
pyotraumatic dermatitis and intertrigo. Pyotraumatic dermatitis (“hot spot”, acute moist der
ma titis) is a superficial, ulcerative inflammatory process. It is most commonly due to allergies
(most hot spots are due to flea bite hypersensitivity), but ectoparasites, foreign bodies, psy
choses, and painful musculoskeletal disorders may also be involved. Swimming can be a
triggering factor in dogs with dense coat in summer. Severe, large lesions can be produced
within hours. Intertrigo or skin fold dermatitis is produced by minor trauma and friction to skin
caused by anatomic defects in certain breeds. The irritation and poor air circulation in com
bination with moisture due to excretions such as tears, sweat, sebum, saliva, and urine favor
skin maceration and bacterial growth. Both of these conditions may be dealt with adequately
by topical antimicrobial therapy and treatment of the underlying disease. Superficial bacterial
folliculitis (SBF) is a common infection confined to the superficial portion of the follicle. In
dogs, SBF is caused mainly by S. intermedius (Figure 1) and it can be pruritic or nonpruritic.
Allergies are a common underlying cause of SPF. Other potential underlying causes include
Veraflox® in bacterial pyoderma – how well does it work?Prof. Dr. Ralf S. Mueller
Center for Clinical Veterinary Medicine, Ludwig Maximilian University Munich, Germany
Figure 1 Superficial bacterial folliculitis in a bull terrier.
Figure 2 Pyoderma on the ventrum of a dachshund with alopecia, erythema, papules, plaques, and crusts.
2nd International Veraflox® Symposium | 29 – 30 Nov 2012, Rome
Veraflox_2012_Proceedings_fa.indd 48 20.11.12 11:02
ralf S. Mueller
endocrine diseases such as hypothyroidism or hyperadreno
corticism, immunodeficiencies, chronic immunosuppression
(steroid therapy!), demodicosis or chemotherapy. Identification
and treatment of the underlying disease in addition to the anti
microbial therapy are essential for longterm remission (details
on searches for underlying diseases are described elsewhere1
and will not be discussed here).
The primary feature of bacterial folliculitis is an inflammatory
pustule with a hair shaft protruding from the center. The most
important differential diagnoses for follicular pustules include
demodicosis and dermatophytosis (even though bacterial folli
culitis is by far the most common). Papules, crusts, epidermal
collarettes, hyperpigmentation and excoriation, alopecia and
target lesions, or bull’seyes may be seen (Figure 2). Epidermal
collarettes suggest any bullous or pustular dermatosis. The
papular, truncal form of SBF in shortcoated dogs can be mis
diagnosed as urticaria because all you see clinically are the
erect hair shafts resembling wheals. Inflamed lesions may be
misdiagnosed as dermatophytosis (because the hairs are lost
in a circle). Noninflammatory SBF with hair loss as the pre
dominant feature may resemble an endocrine imbalance espe
cially in the sharpei. If the follicles rupture, deep furunculosis
may develop (Figure 3).
Prof. Dr. Ralf S. Mueller graduated in Mu
nich/Germany, completed his doctoral the
sis in 1987, and worked in several large and
small animal practices before completing a
residency in veterinary dermatology at the
University of California/Davis in 1992. In
1992 he moved to Melbourne/Australia to
work with his partner and wife Dr. Sonya
Bettenay. Together, they created the first,
purposebuilt specialist practice in Australia.
During that time, Dr. Mueller was concur
rently consulting and teaching at the Veteri
nary Teaching Hospital/University of Sydney.
Ralf and Sonya established (and continue to
conduct) the Distance Education Program
in Veterinary Dermatology of the Postgradu
ate Foundation in Veterinary Science of the
University of Sydney. In 1999, Ralf became
Assistant Professor in Veterinary Derma tol
ogy at the College of Veterinary Medicine
and Biomedical Sciences/Colorado State
University. In 2004, he accepted a position
as chief of the veterinary dermatology ser
vice at the University of Munich/Germany.
He has published over 150 studies, articles,
book chapters, and books, and given sever
al hundred seminars, lectures and talks in
Australasia, Europe and North America.
Figure 3 Furunculosis on the dorsal paw in a Labrador due to bacterial infection.
Veraflox_2012_Proceedings_fa.indd 49 20.11.12 11:02
In cats, bacterial infections (except abscesses) are less common. The skin lesions reported
include erythema, fistulae, papules, pustules, or clinically nonlesional pruritus, and routine
cytology of these lesions with impression smears is recommended. Coagulasepositive sta
phylococci such as S. aureus and S. intermedius have been isolated. Diagnosis is made by
stained smears, culture, and skin biopsy.
All of these infections rely on cytology and clinical presentation for the diagnosis; in practice,
a trial therapy with antibiotics was also often performed. However, in light of the increasing
occurrence of multiresistant bacteria involved in skin infections, it is more sensible to base the
use of antibiotics on bacterial culture and sensitivity.
Diagnostic approach and its relevance for therapeutic decisions
The diagnosis of bacterial pyoderma is made by a combination of clinical findings and results
of cutaneous cytology. Clinical findings as described above cannot be diagnostic as diseases
such as dermatophytosis, demodi
cosis, or pemphigus foliaceus (to
name a few) can mimic bacterial
skin infections and look clinically
perfectly similar. Cutaneous cytolo
gy alone is diagnostic of bacterial
pyoderma, when inflammatory cells
with intracellular bacteria (Figure 4)
are visualized micro scopically, as
an active immune response against
bacteria indicates that those organ
isms are pathogens rather than cu
taneous resident bacteria. When
inflam matory cells and intracellular
bacteria are seen with only mild
clinical signs such as cutaneous
erythema and greasiness, topical therapy may suffice, but when they are seen in association
with papules, plaques, furuncles, or deep abscesses, systemic antibiotics should be part of
the therapeutic protocol and a bacterial culture and sensitivity is indicated to choose the right
antibiotic.
When extracellular bacteria are present, interpretation becomes more difficult. Bacteria in
association with inflammatory cells may be secondary to other inflammatory diseases or may
indicate a vigorous immune response against a skin infection. However, a large number of
those bacteria make an infection more likely. Again the clinical presentation determines further
treatment. When there are papules, plaques, furuncles, or deep abscesses and the dog‘s
Figure 4 Neutrophils with intracellular cocci on cytology of canine pyoderma.
2nd International Veraflox® Symposium | 29 – 30 Nov 2012, Rome
Veraflox_2012_Proceedings_fa.indd 50 20.11.12 11:02
general wellbeing is compromised, systemic antibiotics are indicated based on a bacterial
culture and sensitivity. When the clinical signs are mild and the dog shows no systemic clinical
signs, only topical therapy may be needed.
If only bacterial organisms without
inflammatory cells are detectable
on cytology (Figure 5), the bacte
ria may be benign resident organ
isms or may be pathogens. What
number of bacterial organisms on
cytology indicates true bacterial
overgrowth is a matter of debate
and depends on the site of sam
pling (lips, feet, or perianal area
have more resident bacteria than
back or chest) and individual pa
tient. The clinician must make this
decision based on clinical signs
and personal experience. If only mild erythema or pruritus is present, exclusive topical therapy
may be attempted. If however clinical signs are severe, oral antibiotics based on culture and
sensitivity will often benefit the patient and lead to quicker resolution.
In addition to the number of bacterial organisms, cytology can provide information about the
type of bacteria predominating. If rodshaped bacteria are seen almost exclusively and culture
reveals a mix of Staphylococcus pseudintermedius and Escherichia (E.) coli with different
sensitivity patterns, an antibiotic effective against E. coli should be chosen. If cocci were pre
dominating on cytology, the same culture result may lead to a decision for a different antibiotic
effective against the staphylococci, as Gramnegative organisms have been shown to „be in
there for a ride“ and disappear with successful treatment of the cocci.
Not all owners may be willing to pay for a culture and sensitivity. However, with recurrent or
treatmentresistant infections as well as with rodshaped organisms on cytology, great effort
should be invested in convincing owners to agree to a culture, as this may save time and
money in the long run.
Pradofloxacin in the treatment of bacterial pyoderma
The special aspects of pradofloxacin have been covered in other lectures of this symposium.
The advantages in regard to microbial kill in vitro and the low mutant prevention concentra
tions were presented and are a major reason for the recommendation for pradofloxacin as
the fluoroquinolone of choice in our clinic. How well does pradofloxacin perform in clinical
practice?
Prof. dr. r.S. Mueller | Veraflox® in bacterial pyoderma – how well does it work?
50 | 51
Figure 5 Cocci adhering to canine corneocytes on cytology of a dog with bacterial overgrowth.
Veraflox_2012_Proceedings_fa.indd 51 20.11.12 11:02
A couple of clinical studies evaluating pradofloxacin for skin disease have been published. The
first study looked at pradofloxacin in a multicentered, randomized, blinded study and com
pared it with amoxicillin/clavulanic acid.2 Dogs were included based on a positive bacterial
culture in association with clinical signs of deep pyoderma such as furunculosis, hemorrhagic
bullae, and cellulitis; and randomly treated with either pradofloxacin at 3 mg/kg/day orally
(n = 56) or amoxicillin/clavulanic acid (n = 51). There was no difference in remission rates (86 %
versus 73 %) and times to clinical remission (49 days and 37 days, respectively), but dogs
treated with pradofloxacin had a significantly lower rate of recurrences in the first two weeks
after cessation of therapy (p < 0.01, 0 % versus 11 %), indicating a more complete microbial kill
by pradofloxacin. In a recent systematic review, this was considered fair evidence for a high
efficacy of pradofloxacin in canine deep pyoderma.3 A more recent, smaller case series of
20 dogs with superficial and deep pyodermas based on clinical examination and bacteri
al cultures also showed an excellent to good clinical response within 3 to 6 weeks for all
20 dogs, when using pradofloxacin at 3.7 mg / kg / day orally.4 Further studies looking at bac
terial skin infections treated with pradofloxacin are planned or ongoing. In our clinic, we use
prado floxacin based on culture and sensitivity results as our fluoroquinolone of choice and
have been happy with results, particularly in dogs with severe and deep pyoderma. Our clini
cal impres sion certainly corresponds to the results seen in the above cited studies.
References
01 | Scott DW, Miller WH, Griffin CE. Muller‘s & Kirk‘s Small Animal Dermatology. W. B. Saunders, Philadelphia, 2001.
02 | Mueller RS, Stephan B. Pradofloxacin in the treatment of canine deep pyoderma: a multicentred, blinded, randomized parallel trial. Vet Dermatol 2007; 18:144 –151.
03 | Summers JF, Brodbelt DC, Forsythe PJ, et al. The effectiveness of systemic antimicrobial treatment in canine superficial and deep pyoderma: a systematic review. Vet Dermatol 2012; 23:305–e361.
04 | Restrepo C, Ihrke PJ, White SD, et al. Evaluation of the clinical efficacy of pradofloxacin tablets for the treatment of canine pyoderma. J Am Anim Hosp Assoc 2010; 46:301–311.
2nd International Veraflox® Symposium | 29 – 30 Nov 2012, Rome
Veraflox_2012_Proceedings_fa.indd 52 20.11.12 11:02
Prof. dr. r.S. Mueller | Veraflox® in bacterial pyoderma – how well does it work?
52 | 53
Veraflox_2012_Proceedings_fa.indd 53 20.11.12 11:02
54 | 55
2nd International Veraflox® Symposium | 29 – 30 Nov 2012, Rome
IntroductionPradofloxacin is an 8cyanofluoroquinolone which has been approved in April 2011 for the
treat ment of bacterial infections in dogs and cats. The indications in dogs include the treat
ment of (i) wound infections as well as superficial and deep pyoderma caused by susceptible
strains of the Staphylococcus intermedius group (including Staphylococcus pseudinterme
dius), (ii) acute urinary tract infections caused by susceptible strains of Escherichia coli and the
S. intermedius group (including S. pseudintermedius), and (iii) adjunctive treatment of severe
infections of the gingiva and periodontal tissues caused by susceptible strains of anaerobic
organisms, for example Porphyromonas spp. and Prevotella spp. (Stephan et al., 2008). The
indications in cats comprise (i) wound infections and abscesses caused by susceptible strains
of the S. intermedius group (including S. pseudintermedius) and Pasteurella multocida, and (ii)
acute infections of the upper respiratory tract caused by susceptible strains of P. multocida,
E. coli and the S. intermedius group (including S. pseudintermedius).
The aim of this study was to gain insight into the pradofloxacin MIC distributions of bacterial
pathogens from infections of the respiratory tract, skin and ear, the urinary/genital tract as well
as the gastrointestinal tract of dogs and cats.
Material and methods
For this, 761 bacterial pathogens from defined infections of dogs and cats collected in the
BfTGermVet monitoring study 2004–2006 (Schwarz et al., 2007a) were tested for their
susceptibility to the novel fluoroquinolone pradofloxacin. This included 34 Staphylococcus
aureus and 177 S. pseudintermedius (Schwarz et al., 2007b), 190 βhaemolytic streptococci
(Schwarz et al., 2007c), 91 P. multocida and 42 Bordetella bronchiseptica (Schwarz et al.,
2007d) as well as 227 E. coli (Grobbel et al., 2007).
Susceptibility testing followed the recommendations given in the CLSI document M31–A3
(CLSI, 2008). E. coli ATCC®25922 and S. aureus ATCC®29213 served as quality control
strains. For the comparison of pradofloxacin with other fluoroquinolones, custommade
microtitre plates (MCS Diagnostics, Swalmen, The Netherlands) containing cipro floxacin,
Susceptibility of canine and feline bac-terial pathogens to pradofloxacin and comparison with other fluoro quinolones approved for companion animalsProf. Dr. Stefan SchwarzSchink AK1, Kadlec K1, Hauschild T1, Brenner Michael G1, Dörner JC2, Ludwig C2, Werckenthin C3, Hehnen HR2, Stephan B2, Schwarz S1
1 Institute of Farm Animal Genetics, FriedrichLoefflerInstitut (FLI), NeustadtMariensee, Germany2 Bayer HealthCare AG, Animal Health GmbH, Leverkusen, Germany3 Niedersächsisches Landesamt für Verbraucherschutz und Lebensmittelsicherheit (LAVES), Lebensmittel und Veterinärinstitut Oldenburg,
Oldenburg, Germany
Veraflox_2012_Proceedings_fa.indd 54 20.11.12 11:02
Stefan Schwarz
enrofloxacin, marbofloxacin, orbifloxacin, difloxacin and iba
floxacin were used. The E. coli and S. pseudintermedius iso
lates with elevated pradofloxacin MICs were investigated by
PCRdirected amplification and subsequent sequence analysis
of the quinolone resistance determining regions (QRDRs) of the
target genes.
Results
All bacteria tested (S. aureus, S. pseud inter medius, E. coli,
βhaemolytic streptococci, P. multocida and B. bronchisep
tica) exhibited low pradofloxacin MIC50 and MIC90 values of
≤ 0.25 µg/ml (Table 1).
Only six (3.4 %) of the 177 S. pseudintermedius and 12 (5.3 %)
of the 227 E. coli isolates showed pradofloxacin MICs of
≥ 2 µg/ml. Analysis of the quinolone resistance determining
regions (QRDRs) of the target genes identified double muta
tions in GyrA that resulted in amino acid exchanges S83L +
D87N or S83L + D87Y and single or double mutations in ParC
that resulted in amino acid exchanges S80I or S80I + E84G in
all 12 E. coli isolates. The six S. pseudintermedius isolates ex
hibited amino acid exchanges S84L or E88K in GyrA and S80I
in GrlA (Table 2).
Comparative analysis of the MICs of pradofloxacin and the
MICs determined for enrofloxacin and its main metabolite
cipro floxacin, but also marbofloxacin, orbifloxacin, difloxa
cin, and ibafloxacin was conducted for the target pathogens
S. pseudintermedius, E. coli and P. multocida. This compari son
showed that the MICs of pradofloxacin were up to six dou bling
dilutions (64fold) lower than those of the other fluoro quinolones.
The pradofloxacin MICs were significantly lower than those
of the other tested fluoroquinolones (Tables 3a–c). Statistical
analyses showed that pradofloxacin is more active in vitro
than the comparator fluoroquinolones against the three target
pathogens. The p values for the median differences (log2)
are significant or highly significant for S. pseudintermedius
(p < 0.0001), for E. coli (p < 0.0001– 0.001) and for P. multocida
(p < 0.0001– 0.0004).
Career
Friedrich-Loeffler-Institut (FLI)
since 01/2008
Head of the Research Unit “Molecular
Micro biology & Antimicrobial Resistance”,
Institute of Farm Animal Genetics, Neustadt
Mariensee
Federal Agricultural Research Centre
(FAL)
06/2001 – 12/2007
Head of the Department ”Product and
Process Quality, Environment”, Institute for
Animal Breeding, NeustadtMariensee
01/1998 – 05/2001
Head of the Research Unit “Molecular
Microbiology and Diagnostics”, Institute for
Animal Science and Animal Behaviour, Celle
10/1992 – 12/1997
Head of the Research Unit “Diagnostics”
in the Institute for Small Animal Research,
Celle
Justus-Liebig University Gießen
04/1988 – 09/1992
Postdoctoral Research Fellow in the Institute
of Bacteriology and Immunology
02/1987 – 03/1988
Postdoctoral Research Fellow in the Institute
of Virology
Research Interests
Antibiotic resistance in Grampositive /
Gram negative bacteria;
Structure and regulation of antibiotic re
sist ance genes;
Molecular genetics and epidemiology of
plasmids, transposons, gene cassettes and
integrons;
Monitoring of antimicrobial susceptibility;
Susceptibility testing methods;
Molecular typing of bacteria
Veraflox_2012_Proceedings_fa.indd 55 20.11.12 11:02
2nd International Veraflox® Symposium | 29 – 30 Nov 2012, Rome
Table 1 MIC50 and MIC90 values of the 761 canine and feline bacterial pathogens
Bacterial species Infections of theAnimal origin
nMIC50
(µg/ml)MIC90
(µg/ml)
S. aureus respiratory tract Σ 12 0.06 0.12dog 4cat 8
skin and ear Σ 22 0.06 0.12
dog 16cat 6
S. pseudintermedius respiratory tract Σ 45 0.06 0.12dog 32cat 13
skin and ear Σ 74 0.06 0.06
dog 68cat 6
urinary/genital tract Σ 58 0.06 0.12
dog 58cat 0
E. coli respiratory tract Σ 28 0.015 0.03
dog 17cat 11
urinary/genital tract Σ 99 0.015 0.25
dog 73cat 26
gastrointestinal tract Σ 100 0.015 0.03
dog 57cat 43
P. multocida respiratory tract Σ 71 ≤0.008 0.015
dog 15cat 56
skin and ear Σ 20 ≤0.008 0.015
dog 6cat 14
B. bronchiseptica respiratory tract Σ 42 0.25 0.25
dog 34cat 8
βhaemolytic streptococci respiratory tract Σ 21 0.12 0.12
dog 16cat 5
skin and ear Σ 79 0.12 0.25
dog 73cat 6
urinary/genital tract Σ 90 0.12 0.25
dog 84cat 6
Veraflox_2012_Proceedings_fa.indd 56 20.11.12 11:02
Prof. dr. S. Schwarz | Susceptibility of canine and feline bacterial pathogens to pradofloxacin and
comparison with other fluoroquinolones approved for companion animals
56 | 57
Table 2 Mutations in the QRDR regions of target genes among isolates with pradofloxacin MICs of ≥ 2 µg/ml
Bacterial speciesPradofloxacin MIC (µg/ml)
Amino acid exhanges in the QRDR regions of
GyrA ParC GyrA GrlA
E. coli 8S83L, D87Y
S80I, E84G
n.a. n.a.
4S83L, D87N
S80I n.a. n.a.
4S83L, D87N
S80I n.a. n.a.
8S83L, D87N
S80I n.a. n.a.
8S83L, D87N
S80I n.a. n.a.
8S83L, D87N
S80I n.a. n.a.
8S83L, D87N
S80I n.a. n.a.
4S83L, D87N
S80I n.a. n.a.
4S83L, D87N
S80I n.a. n.a.
2S83L, D87N
S80I n.a. n.a.
8S83L, D87N
S80I n.a. n.a.
4S83L, D87N
S80I n.a. n.a.
S. pseudintermedius 2 n.a. n.a. S84L S80I
2 n.a. n.a. S84L S80I
4 n.a. n.a. E88K S80I
2 n.a. n.a. E88K S80I
2 n.a. n.a. S84L S80I
2 n.a. n.a. E88K S80I
n.a. = not applicable
Veraflox_2012_Proceedings_fa.indd 57 20.11.12 11:02
2nd International Veraflox® Symposium | 29 – 30 Nov 2012, Rome
Table 3a Comparative analysis of MICs of pradofloxacin and other fluoroquinolones among 177 canine/feline S. pseudintermedius (from respiratory tract infections (n = 45), skin/ear infections (n = 74), and urinary/genital tract infections (n = 58). The horizontal blue bar indicates the pradofloxacin MIC value
MIC µg/ml Ci En Ma Ib Or Di Ci En Ma Ib Or Di Ci En Ma Ib Or Di
≥ 32
16
8
4 1 2
2 1
1 1 1 4 1 5 3 3 1 9 3
0.5 28 4 7 1 8 93 46 7 1 10 17 21
0.25 11 2 37 1 14 30 70 28 90 4 1 49 15 14 14 2 1
0.12 31 27 5 23 1 8 21 67 86 1 9 23
0.06 17 19 1 8 1
≤ 0.03 1 1
≤ 0.03 µg/mlpradofloxacin (n = 43)
0.06 µg/mlpradofloxacin (n = 98)
0.12 µg/mlpradofloxacin (n = 27)
MIC µg/ml Ci En Ma Ib Or Di Ci En Ma Ib Or Di Ci En Ma Ib Or Di
≥ 32 5 3 2 1 3 5 1 1 1 1
16 2 3 4 2 1
8 1
4 1
2 1 1
1 2 2 3 1 1 2
0.5 1 1
0.25 2
0.12
0.06
≤ 0.03
0.25 µg/mlpradofloxacin (n = 3)
2 µg/mlpradofloxacin (n = 5)
4 µg/mlpradofloxacin (n = 1)
Veraflox_2012_Proceedings_fa.indd 58 20.11.12 11:02
58 | 59
Table 3b Comparative analysis of MICs of pradofloxacin and other fluoroquinolones among 127 canine/feline E. coli isolates from urinary/genital tract infections (n = 99) and respiratory tract infections (n = 28)
MIC µg/ml
Ci En Ma Ib Or Di Ci En Ma Ib Or Di Ci En Ma Ib Or Di
≥ 32
16
8
4
2
1
0.5 1
0.25 26 28 6
0.12 6 38 35 33 9 30 36
0.06 3 5 1 2 1 3 27 31 10 2 2 2
0.03 1 3 1 31 49 49 5 3 34 27 35
0.015 5 6 5 34 15 16 4 1 1
≤ 0.008 1 1 1 1
≤ 0.008 µg/mlpradofloxacin (n = 6)
0.015 µg/mlpradofloxacin (n = 67)
0.03 µg/mlpradofloxacin (n = 38)
MIC µg/ml
Ci En Ma Ib Or Di Ci En Ma Ib Or Di Ci En Ma Ib Or Di
≥ 32 1 1 1
16 1
8 1 1
4 2
2 1 1 2 2 2
1 1
0.5 2 2 1 1 1 2 2 2 2
0.25 1 1 1
0.12 1 1 1 1
0.06 1 1
0.03 1
0.015
≤ 0.008
0.06 µg/mlpradofloxacin (n = 4)
0.12 µg/mlpradofloxacin (n = 2)
2 µg/mlpradofloxacin (n = 1)
Continuation of Table 3b see next page
Prof. dr. S. Schwarz | Susceptibility of canine and feline bacterial pathogens to pradofloxacin and
comparison with other fluoroquinolones approved for companion animals
Veraflox_2012_Proceedings_fa.indd 59 20.11.12 11:02
2nd International Veraflox® Symposium | 29 – 30 Nov 2012, Rome
MIC µg/ml Ci En Ma Ib Or Di Ci En Ma Ib Or Di
≥ 32 1 1 4 4 4 5 5 2 5 5 5
16 2 2 1 3
8 1 1 3
4
2
1
0.5
0.25
0.12
0.06
0.03
0.015
≤ 0.008
4 µg/mlpradofloxacin (n = 4)
8 µg/mlpradofloxacin (n = 5)
continuation of Table 3b
Table 3c Comparative analysis of MICs of pradofloxacin and other fluoroquinolones among 48 P. multocida isolates from respiratory tract infections (n = 28) and skin/ear infections (n = 20)
MIC µg/ml Ci En Ma Ib Or Di Ci En Ma Ib Or Di Ci En Ma Ib Or Di
≥ 32
16
8
4
2
1
0.5
0.25
0.12 2 2
0.06 3 1 8 10 2 3 2 1 1
0.03 1 13 3 13 23 4 10 11 9 7 6 3 1 1 1 1
0.015 14 24 15 21 14 5 11 6 2 5 1 1 1 1 1
≤ 0.008 14 3 4 1 1 1
≤ 0.008 µg/mlpradofloxacin (n = 28)
0.015 µg/mlpradofloxacin (n = 16)
0.03 µg/mlpradofloxacin (n = 4)
Veraflox_2012_Proceedings_fa.indd 60 20.11.12 11:02
Discussion and conclusionThe present study provides for the first time MIC distributions of a large number of canine and
feline bacterial pathogens from respiratory tract infections, skin and ear infections, urinary/
genital tract infections and gastrointestinal tract infections.
Only a small number of S. pseudintermedius and E. coli isolates with pradofloxacin MICs of
≥ 2 µg/ml was detected. The analysis of the corresponding E. coli and S. pseudintermedius
isolates identified mutations in the target genes which resulted in single or double amino acid
exchanges at positions previously described to be involved in fluoroquinolone resistance in
E. coli (Hopkins et al., 2005) and S. pseudintermedius (Descloux et al., 2008).
The statistical comparison of the pradofloxacin MICs with those of other fluoroquinolones
showed that the pradofloxacin MICs were significantly lower. The most pronounced differ
ences in the MICs were seen between pradofloxacin and difloxacin/orbifloxacin in S. pseud
intermedius and between pradofloxacin and ibafloxacin in E. coli.
The data presented in this study may provide valuable information for the generation of clinical
breakpoints (Bywater et al., 2006; Simjee et al., 2008). Moreover, as the bacterial pathogens
investigated in this study have been collected prior to the approval of pradofloxacin, this data
set might represent a baseline to assess potential changes of the pradofloxacin susceptibility
in the postapproval period.
AcknowledgementsThe authors thank Mike Schiwek, Vera Nöding, Roswitha Becker and Kerstin Meyer for expert
technical assistance.
FundingThis study was funded by Bayer HealthCare Animal Health. AnneKathrin Schink was sup
port ed by a scholarship of the H. Wilhelm Schaumann foundation.
60 | 61
Prof. dr. S. Schwarz | Susceptibility of canine and feline bacterial pathogens to pradofloxacin and
comparison with other fluoroquinolones approved for companion animals
Veraflox_2012_Proceedings_fa.indd 61 20.11.12 11:02
2nd International Veraflox® Symposium | 29 – 30 Nov 2012, Rome
References
01 | Bywater R, Silley P, Simjee S. Antimicrobial breakpointsdefinitions and conflicting requirements. Vet Microbiol 2006; 118:158–159.
02 | Clinical and Laboratory Standards Institute, 2008. Performance standards for antimicrobial disk and dilution susceptibility test for bacteria isolated from animals; approved standard – third edition (ISBN Number: 156238659X). CLSI document M31– A3. Clinical and Laboratory Standards Institute, Wayne, PA, USA.
03 | Descloux S, Rossano A, Perreten V. Characterization of new staphylococcal cassette chromosome mec (SCCmec) and topoisomerase genes in fluoroquinolone and methicillinresistant Staphylococcus pseudintermedius. J Clin Microbiol 2008; 46:1818–1823.
04 | Grobbel M, LübkeBecker A, Alešík E, Schwarz S, Wallmann J, Werckenthin C, Wieler LH. Antimicrobial sus ceptibility of Escherichia coli from swine, horses, dogs and cats as determined in the BfTGermVet monitoring program 2004–2006. Berl Münch Tierärztl Wochenschr 2007; 120:391–401.
05 | Hopkins KL, Davies RH, Threlfall EJ. Mechanisms of quinolone resistance in Escherichia coli and Salmonella: recent developments. Int J Antimicrob Agents 2005; 2:358–373.
06 | Schwarz S, Alešík E, Grobbel M, LübkeBecker A, Wallmann J, Werckenthin C, Wieler LH. The BfTGermVet monitoring program – aims and basics. Berl Münch Tierärztl Wochenschr 2007a; 120:357–362.
07 | Schwarz S, Alešík E, Werckenthin C, Grobbel M, LübkeBecker A, Wieler LH, Wallmann J. Antimicrobial sus ceptibility of coagulasepositive and coagulasevariable staphylococci from various indications of swine, dogs and cats as determined in the BfTGermVet monitoring program 2004–2006. Berl Münch Tierärztl Wochenschr 2007b; 120:372–379.
08 | Schwarz S, Alešík E, Grobbel M, LübkeBecker A, Werckenthin C, Wieler LH, Wallmann J. Antimicrobial sus ceptibility of streptococci from various indications of swine, horses, dogs and cats as determined in the BfTGermVet monitoring program 2004–2006. Berl Münch Tierärztl Wochenschr 2007c; 120:380–390.
09 | Schwarz S, Alešík E, Grobbel M, LübkeBecker A, Werckenthin C, Wieler LH, Wallmann J. Antimicrobial sus ceptibility of Pasteurella multocida and Bordetella bronchiseptica from dogs and cats as determined in the BfTGermVet monitoring program 2004–2006. Berl Münch Tierärztl Wochenschr 2007d; 120:423–430.
10 | Simjee S, Silley P, Werling HO, Bywater R. Potential confusion regarding the term ‘resistance’ in epidemiological surveys. J Antimicrob Chemother 2008; 61:228–229.
11 | Stephan B, Greife HA, Pridmore A, Silley P. Activity of pradofloxacin against Porphyromonas and Prevotella spp. implicated in periodontal disease in dogs: susceptibility test data from a European multicenter study. Antimicrob Agents Chemother 2008; 52:2149–2155.
The data presented in this extended abstract are taken from Schink A-K, Kadlec K, Hauschild T, Brenner Michael G, Dörner JC, Ludwig C, Werckenthin C, Hehnen HR, Stephan B, Schwarz S. Susceptibility of canine and feline bacterial pathogens to pradofloxacin and comparison with other fluoroquinolones approved for companion animals. Vet Microbiol 2012 Aug 8. [Epub ahead of print] http://dx.doi.org/10.1016/j.vetmic.2012.08.001
Veraflox_2012_Proceedings_fa.indd 62 20.11.12 11:02
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Prof. dr. S. Schwarz | Susceptibility of canine and feline bacterial pathogens to pradofloxacin and
comparison with other fluoroquinolones approved for companion animals
Veraflox_2012_Proceedings_fa.indd 63 20.11.12 11:02
Introduction
Pradofloxacin is a new generation fluoroquinolone specifically developed for animal health that
has demonstrated excellent clinical efficacy and unique advantages over previous veterinary
quinolones, including enhanced spectrum of antibacterial activity and decreased potential for
the selection of resistant organisms.1, 2 Equally important to clinical efficacy in the consider a
tion of antimicrobial selection in practice are the safety profile and the availability of formula
tions that can be conveniently and reliably administered to companion animals by pet owners.
This latter point is especially relevant for feline patients, where drug administration and com
pliance present a greater challenge, and has led to increased focus on formulation technology
and development of formulations tailored to these species.
Safety profileThe safety of Veraflox® tablets and oral suspension in dogs and cats has been extensively
evaluated in target animal safety studies and clinical field safety and efficacy trials, as well as
ongoing monitoring of postapproval experience in Europe.
Target animal safety studies using doses of one (1 x), three (3 x) and five times (5 x) the re
commended label dose for treatment duration of at least 91 days in dogs and 21 days in
cats demonstrated that Veraflox® tablets and Veraflox® oral suspension are well tolerated at
the recommended doses with an adequate margin of safety under clinical conditions. The
parameters monitored for the evaluation of safety included: body weight, food consump
tion, clinical observations, haematology, clinical chemistry, urinalysis, physical examinations
(monthly), vision and pupillary reflex examinations (weekly), ophthalmological examinations,
gross pathology and histopathology.
Additional laboratory studies were performed to assess the safety of pradofloxacin with marked
overdosing in dogs. The minimum tested dose in these studies was 12 mg/kg, 4 times (4 x)
the recommended therapeutic dose of 3 mg/kg body weight and went up to 120 mg/kg.
Statistically significant decreases in the absolute neutrophil count, red blood cell count and
platelet count could be observed starting at 12 x overdosing (36 mg/kg); haematological in
dices were generally unaffected up to 9 x overdosing (27 mg/kg). These parameters returned
to pretreatment levels after the end of treatment. These studies indicated that haematological
changes can occur at extremely elevated dosages and that these changes are reversible.
Safety and convenience of Veraflox® – the art and science of tailoring therapy for cats and dogs
Dr. Joy Olsen
Bayer HealthCare, USA
2nd International Veraflox® Symposium | 29 – 30 Nov 2012, Rome
64 | 65
Veraflox_2012_Proceedings_fa.indd 64 20.11.12 11:02
A number of studies investigated the influence of pradofloxacin
on cartilage of young growing dogs at different dosages and
for variable lengths of treatment (ranging from 5.15 mg/kg for
91 days to 100 mg/kg once). Treatment with elevated doses,
e. g., 5.15 mg/kg for 13 weeks and after 2 weeks at 10 mg/kg,
resulted in detection of articular lesions in some of the juvenile
dogs. Gross lesions included increases in synovial fluid and
surface changes in some joints. Histopathology gave evidence
of treatmentrelated findings in the form of primary degenera
tive changes of the articular cartilage of multiple joints con
sisting of blisters or erosions originating from the interme diate
zone of the articular cartilage. These effects consistent with
chondrotoxicity are characteristic of all veterinary fluoroquino
lones in dogs and an appropriate contraindication is included
in the European Veraflox® tablet summary of product charac
teristics, indicating it should not be used in dogs of less than
12 months of age for the majority of breeds and in giant breeds
less than 18 months.
The safety of Veraflox® in young kittens was specifically inves
tigated in a study evaluating immature cats treated with the
oral suspension at 0, 5, 15, and 25 mg/kg body weight once
daily for 21 consecutive days (n = 8 animals per group). The
kittens were approximately six weeks of age at study start and
evaluated parameters included monitoring of body weights,
clinical signs, haematology and serum chemistry, ophthal
mological examinations and histopathological evaluation of
ocular tis sues and joints. The ophthalmological examinations
and histo pathology of ocular tissues were evaluated for the
presence of retino toxicity, an effect that has been reported in
associa tion with elevated dosages of fluoroquinolones in cats.3
Recent research suggests that this may be related to amino
acid chang es specific to cats in the ABCG2 transport protein,
resulting in functional defects that decrease efficacy as a drug
transporter.4 A further objective of the study in kittens was to
investigate potential for chondrotoxic ity, a wellknown effect of
fluoroquinolone treatment in juvenile canines.
No treatmentrelated effects in the evaluated parameters were
observed in young kittens for any of the doses tested, including
ocular and chondral histopathology. In conclusion, Veraflox®
oral suspension was demonstrated to be safe in kittens as
Joy olsen
Dr. Joy Olsen is currently the manager of the
Veterinary Technical Services group in the
western half of the United States within the
Animal Health division of Bayer HealthCare.
She previously held the position of Global
Veterinary Services Manager with Bayer
HealthCare in the international headquart
ers for Animal Health located in Monheim,
Germany, with primary focus in the areas of
antiinfectives and pharmacologicals. She is
a 1990 graduate of Kansas State University
College of Veterinary Medicine. Prior to join
ing Bayer, Dr. Olsen practiced small animal
medicine in San Francisco, California, for
several years and held an assistant profes
sorship in small animal anatomy at Kansas
State University. She worked as a profes
sional services veterinarian for Bayer Animal
Health in the United States prior to transfer
ring to the global headquarters in Germany
in 2002, and returning in 2012. Her ongoing
areas of interest include companion animal
internal medicine and pharmacol ogy. Among
Dr. Olsen’s professional affiliations are the
American Veterinary Association, the Amer
ican Association of Feline Practitioners, the
International Society of Feline Medicine, the
Veterinary Cancer Society and the Inter
national Society of Companion Animal Infec
tious Diseases.
Veraflox_2012_Proceedings_fa.indd 65 20.11.12 11:02
young as 6 weeks of age, with no adverse ocular effects or effects on articular cartilage, even
at up to 25 mg/kg daily (five times the recommended dosage in Europe) for three consecutive
weeks, providing a safe and convenient alternative for treatment of young cats.
Ocular safety evaluations
In addition to general target animal safety studies and field efficacy and safety trials in adult
cats, the effects of pradofloxacin on feline ocular parameters have been extensively eval u ated.5
These investigations employed routine ophthalmological examinations (slitlamp microscopy
and indirect ophthalmoscopy), electroretinography (ERG), histopathological ex amination of
tissues (light and electron microscopy) and optical coherence tomography (OCT), a newer
methodology not previously employed in veterinary studies.
Optical coherence tomography (OCT) is a novel transpupillary imaging technology that pro
vides a method to noninvasively assess retinal morphology and thickness of the nerve fiber
layer in vivo.6 OCT has been described as “optical ultrasound”, using light waves instead of
sound waves to obtain images of tissues at a resolution equivalent to a lowpower micro
scope. By use of an optical beam directed into the eye, the portions of light reflecting from
subsurface features are collected and optical coherence interferometry is used to record
the optical path of photons and diffusely scattered light that would otherwise obscure the
image. In this way, highresolution crosssectional images of the retina are produced and
the anatomic layers within the retina can be differentiated and retinal thickness measured.
OCT has attracted interest and become an established technique in the human medical
community because it provides tissue morphology imagery at a much higher resolution (bet ter
than 10 µm) than other imaging technologies, such as magnetic resonance imaging (MRI) and
ultrasound. It has been applied in other medical areas such as cardiology, however thus far
OCT has had the largest clinical impact in human ophthalmology.
The ocular investigations with pradofloxacin were designed to evaluate the effects of high
doses of pradofloxacin on the feline retina. Two groups of cats were treated orally with
prado floxacin pure drug substance in gelatin capsules at either 30 mg/kg (n = 10 cats) or
50 mg/kg body weight (n = 14 cats) for 23 consecutive days. Nine cats served as untreated
controls and another seven cats as positive controls that received enrofloxacin at a dose
of 30 mg/kg body weight. The parameters monitored included general health assessment
determin ed by twicedaily clinical examinations, body weight, haematology, and clinical chem
istry. Evaluation of ocular parameters was conducted with weekly ophthalmological examina
tions, examinations of retinal morphology via optical coherence tomography with the Stratus
III OCT (Carl Zeiss, Germany) and electroretinography, including baseline determination prior
to treatment. The ERG protocol was developed and performed based on International Society
for Clinical Electrophysiology of Vision (ISCEV) standards.7 Gross pathology and histo pathol
ogy, including light and electron microscopy, of the retina were performed following comple
tion of the treatment period.
2nd International Veraflox® Symposium | 29 – 30 Nov 2012, Rome
Veraflox_2012_Proceedings_fa.indd 66 20.11.12 11:02
Clinical signs observed in pradofloxacintreated animals consisted of slight weight loss in a few
male cats at 50 mg/kg, vomiting and salivation of some cats of both groups, and diarrhoea in
two animals. There was a nonsignificant dosedependent decrease of leukocytes at doses of
30 mg/kg and above in males, and at 50 mg/kg body weight in females. There were no signs
of retinal electrophysiological dysfunction in pradofloxacintreated cats as monitored by ERG.
OCT evaluations throughout the study revealed that retinal thickness in cats on both elevated
dosages of pradofloxacin remained constant over the treatment period. Additionally, histo
logical examinations of tissues (light microscopy, immunohistochemistry, electron microscopy)
at the conclusion of the treatments showed no indication of induced changes in the control
group and in the animals treated with 30 mg/kg and 50 mg/kg pradofloxacin. In the highdose
enrofloxacintreated group (30 mg/kg, 6 times label dosage), clinical signs evident of sys
temic toxicity and weight loss were observed, and changes in retinal function and morphol
ogy were evidenced on electroretinography and OCT, respectively, as well as histopathology.
These find ings were consistent to previous studies demonstrating evidence of retinal toxicity
at mark edly elevated dosages associated with systemic toxicity.8 In contrast, ocular and
retinal toxicity were absent in all pradofloxacintreated cats as investigated by ophthalmo
scopy, optical coherence tomography, electroretinography, and histopathological evaluation
of tissues via light and electron microscopy.
The above investigations thoroughly demonstrate that pradofloxacin at very high doses
(50 mg/kg) in cats, for relatively long treatment periods (3 weeks), does not induce any gen
eral ocular changes or specific damage to the retina. As described previously, no changes
in the retina were observed in a study in 6weekold kittens with the oral suspension at a
dose of up to 25 mg/kg for three weeks.
Clinical safety
The safety of pradofloxacin in cats was further evaluated in clinical field studies in Europe
assessing both efficacy and tolerability. Three controlled, randomized, blinded multicentre
clinical field studies were performed, one with Veraflox® tablets for treatment of feline acute
upper respiratory tract infections and two with Veraflox® oral suspension for treatment of
feline acute upper respiratory tract infections and wound infections and abscesses. In total
474 cats were treated with pradofloxacin at recommended dosages and for the normal
duration of treatment for each indication.
Veraflox® administered by tablet or oral suspension was demonstrated to be welltolerated in
cats and the majority of adverse events reported were limited to mild, transient gastrointestinal
signs (vomiting, diarrhoea) in isolated cases (Table 1). The incidence of events was compa
rable to that observed with the control product.
The safety of Veraflox® tablets under field conditions in dogs was evaluated in controlled,
blind ed, randomized studies in Europe assessing both clinical efficacy and safety. A total of
dr. J. olsen | Safety and convenience of Veraflox® – the art and science of tailoring
therapy for cats and dogs
66 | 67
Veraflox_2012_Proceedings_fa.indd 67 20.11.12 11:02
2nd International Veraflox® Symposium | 29 – 30 Nov 2012, Rome
750 clientowned dogs were enrolled in different multicentre studies on wound infections,
pyoderma, periodontal disease and urinary tract infections; of these, 395 of the dogs were
treated with Veraflox® tablets at the recommended dose of 3 mg/kg. Pradofloxacin was
demonstrated to be welltolerated in dogs, with the majority of events reported limited to mild,
transient gastrointestinal signs (vomiting, diarrhoea) in isolated cases (Table 2).
Table 1 Safety of Veraflox® in feline clinical field trials – reported adverse events
Adverse event
Tablet Suspension Total
No. Cats % No. Cats % No. Cats %
Diarrhoea 6 8.6 6 1.5 12 2.5
Vomiting 2 2.9 4 1.0 6 1.3
Salivation 2 2.9 0 0 2 0.4
Anorexia 1 1.4 1 0.25 2 0.4
Polydipsia 0 0 1 0.25 1 0.2
Apathy 1 1.4 0 0 1 0.2
Total no. of cats: 474; (70 tablet, 404 suspension)
Table 2 Safety of Veraflox® tablets in canine clinical field trials – reported adverse events
Adverse event Number of dogs %
Diarrhoea 17 4.3
Vomiting 13 3.3
Polydipsia 5 1.3
Tiredness/sleepiness 5 1.3
Salivation 3 0.75
Polyuria 3 0.75
Decreased appetite 1 0.25
Anorexia 1 0.25
Constipation 1 0.25
Weakness 1 0.25
Blood in faeces 1 0.25
Total no. of dogs treated: 395
Veraflox_2012_Proceedings_fa.indd 68 20.11.12 11:02
68 | 69
Post-approval experience
Postapproval pharmacovigilance monitoring in the EU has identified a very low incidence
of reported adverse events with both Veraflox® tablets and Veraflox® oral suspension. The
majority of reported events involve gastrointestinal symptoms, consistent with observations
from clinical field studies, and the incidence based on pharmacovigilance reports and estimat
ed treatments has been classified as extremely rare.
In summary, the safety and tolerability of Veraflox® tablets and Veraflox® 25 mg/ml oral sus
pension when used as recommended has been demonstrated in laboratory evaluations and
clinical field experience.
Formulation mattersPet owners’ busy lifestyles and the increasing popularity of cats as animal companions have
increased focus on the development of novel drug formulations that ease administration and
enhance the compliance of therapy for these species. Cats are special in a number of ways,
including their food preferences and feeding behaviours and acceptance, or lack thereof, to
handling and medication. In addition to being obligate carnivores, research in recent years has
identified that felines lack a functional sweet taste receptor as the result of an unexpressed
pseudogene in one of the proteins composing the receptor,9 further elucidating their selective
preferences.
Responding to the high need for more convenient formulations for feline patients, an oral liquid
formulation of pradofloxacin was specifically designed and developed to be wellaccepted by
cats. Veraflox® oral suspension contains a patented composition of a novel ion exchange resin
reversibly bound to the active drug substance pradofloxacin. The loaded ion exchange resin
masks the bitter flavour of the drug particles, allowing them to pass undetected by the taste
receptors of the cat. Following administration of the oral suspension, pradofloxacin is released
from the ion exchanger in the acidic environment of the stomach and rapidly absorbed. Due
to differences in bioavailability, the oral suspension formulation is dosed higher than the tablet
formulation (3 mg/kg), with a label dosage in Europe of 5 mg/kg body weight daily. At this
dosage, peak serum concentrations (Cmax) of approximately 2.1 µg/ml are reached within
1 hour following administration (Table 3).10
Table 3 Serum pharmacokinetic profile of pradofloxacin oral suspension at 5 mg/kg
SpeciesCmax
(µg/ml)Tmax
(h)t1/2
(h)AUC0-24 h
(µg*h/ml)AUCinf
(µg*h/ml)MRT
(h)F
(%)
cat 2.2 0.9 9.8 8.5 9.6 8.8 > 60
Cmax maximum concentration Tmax time of maximum concentration t1/2 half-life AUC0-24 h area under the concentration vs. time curve (24-h interval)AUCinf area under the concentration vs. time curve (unlimited)
MRT mean residence time F bioavailability
dr. J. olsen | Safety and convenience of Veraflox® – the art and science of tailoring
therapy for cats and dogs
Veraflox_2012_Proceedings_fa.indd 69 20.11.12 11:02
2nd International Veraflox® Symposium | 29 – 30 Nov 2012, Rome
The palatability of Veraflox® oral suspension was evaluated in a controlled study in a group of
40 healthy adult cats of various breeds. A highly palatable vitamin paste, Nutriplus Cat paste
(Virbac), was used as a positive control in this investigation, and cats in both groups were
administered product on 3 consecutive days. With each adminis tration, an acceptance score
was given by the administrator ranging from 1 (being most unacceptable) to 5 (administered
without any difficulties).
The mean administration score over all three applications for the Veraflox® oral suspension
was 4.1, which compared favourably to the vitamin paste administration score of 4.7 (Figure 1).
Clinical studies and post
approval experience have
underscored the favour able
acceptance of Vera flox®
in feline patients suf fering
from bacterial infections,
providing a highly effective
option that opti mizes com
pliance of therapy.11
The palatability of Vera flox®
tab lets in dogs was eval
uated with scor ing during
multi cen tre field stud ies as
sessing the efficacy and safety of Veraflox® tablets in clientown ed dogs. Investigators in each
of the different field studies assessed the accept ability of the product according to a 4point
scale (very good, good, poor, very poor) and assessments were compared between treatment
groups. Veraflox® tablets were shown to be very well accepted, with acceptance being as
sessed as ‘good’ to ‘very good’ in 92 % of dogs with periodontal disease, 96 % and 100 % of
dogs with pyoderma and wound infections, and 100 % of dogs treated for urinary tract infec
tions (Table 4). The acceptance in these studies was compared to Synulox® (amoxicillincla
vulanic acid) or Antirobe®
(clindamycin) and Veraflox®
tablets were found to be at
least as pal atable as the
other tested compounds
(no statistical differences).
Table 4 Palatability of Veraflox® tablets in canine field trials
Clinical field study Palatability (%)
Canine wound infections 100
Canine urinary tract infections 100
Canine pyoderma 96.3
Canine periodontal disease 92.2
5
4.5
4
3.5
3
2.5
2
1.5
1Veraflox® oral suspension NutriPlus Cat®
Figure 1 Acceptance scoring of Veraflox® oral suspension.
Veraflox_2012_Proceedings_fa.indd 70 20.11.12 11:02
70 | 71
SummaryThe safety of Veraflox® tablets and oral suspension in dogs and cats has been established
in target animal safety studies and clinical field trials. Specific safety evaluations in young
kittens indicate that Veraflox® oral suspension is safe in cats as young as 6 weeks of age.
Additionally, pradofloxacin was demonstrated to have no effects on feline retinal morphology
and function, even at doses of up to 50 mg/kg body weight daily for 3 consecutive weeks.
The availability of a wellaccepted oral suspension specifically developed for cats offers a
safe, highly effective and convenient option for the management of bacterial infections for
approved indications in feline patients.
References
01 | Wetzstein HG. Comparative mutant prevention concentrations of pradofloxacin and other veterinary fluoroquinolones indicate differing potentials in preventing selection of resistance. Antimicrob Agents Chemother 2007; 49:4166–4173.
02 | Silley P, Stephan B, Greife HA, Pridmore A. Comparative activity of pradofloxacin against anaerobic bacteria isolated from dogs and cats. J Antimicrob Chemother 2007; 60(5):999–1003.
03 | Wiebe V, Hamilton P. Fluoroquinoloneinduced retinal degeneration in cats. J Am Vet Med Assoc 2002; 221(11): 1568–1571.
04 | Ramirez CJ, Minch JD, Gay JM, Lahmers SM, Guerra DJ, Haldorson GJ, Schneider T, Mealey KL. Molecular genetic basis for fluoroquinoloneinduced retinal degeneration in cats. Pharmacogenet Genom 2010; 21(2):66–75.
05 | Messias A, Gekeler F, Wegener A, Dietz K, Kohler K, Zrenner E. Retinal safety of a new fluoroquinolone, pradofloxacin, in cats: assessment with electroretinography. Doc Ophthalmol 2008; 116:177–191.
06 | Jaffe GJ, Caprioli J. Optical coherence tomography to detect and manage retinal disease and glaucoma. Am J Ophthalmol 2004; 137(1):156–169.
07 | Marmor MF, Zrenner E. Standard for clinical electroretinography (1999 update). International Society for Clinical Electrophysiology of Vision. Doc Ophthalmol 1998; 97:143–156.
08 | Ford MM, Dubielzig RR, Giuliano EA, et al. Ocular and systemic manifestations after oral administration of a high dose of enrofloxacin in cats. Am J Vet Res 2007; 68:190–202.
09 | Li X, Li W, Wang H, Bayley DL, Cao J, Reed DR, Bachamonov AA, Huang L, LegrandDefretin V, Beauchamp GK, Brand JG. Cats lack a sweet taste receptor. J Nutr 2006;136(7):1932–1934.
10 | Daube G, Krebber R, Greife HA. Pharmacokinetic properties of pradofloxacin administered as an oral suspension to cats. J Vet Pharmacol Therap 2006; 1:266–267.
11 | Litster A, Moss S, Honnery M, Rees B, Edingloh M, Trott D. Clinical efficacy and palatability of pradofloxacin 2.5 % oral suspension for the treatment of bacterial lower urinary tract infections in cats. J Vet Intern Med 2007; 21:990–995.
dr. J. olsen | Safety and convenience of Veraflox® – the art and science of tailoring
therapy for cats and dogs
Veraflox_2012_Proceedings_fa.indd 71 20.11.12 11:02
Introduction
Quinolones are among the most potent antibacterial agents developed. The spectrum of
ac tiv ity of the older quinolones was essentially against Enterobacteriaceae whereas the
newer fluoroquinolones have a wider spectrum including activity against many Gramnegative
species (bacilli and cocci), some Grampositive species, intracellular organisms (Rickettsia
spp. and Mycobacterium spp.) and Mycoplasma spp. Thirdgeneration fluoroquinolones such
as moxifloxacin have enhanced activity against Grampositive bacteria relative to first and
secondgeneration compounds and good activity against anaerobes (Hawkey, 2003).
Pradofloxacin, exclusively developed for use in veterinary medicine, is a thirdgeneration
fluoroquinolone, and therefore can be expected to show more activity against Grampositive
organisms and anaer obes than the secondgeneration compounds enrofloxacin and marbo
floxacin.
Activity against anaerobic organisms
Initial MIC data on a range of anaerobic bacterial pathogens isolated from oral infections, ab
scesses and wound infections and also from faecal flora of dogs and cats are detailed in Table 1,
demonstrating the broad spectrum of activity. In all cases, the protocols used to determine
MICs were standardised in accordance with Clinical Laboratory Standards Institute (CLSI)
methodology, although guidelines were not available for all the tested bacterial groupings, and
in such cases, testing principles in keeping with the guidelines were followed. Table 1 only
includes bacterial groups for which data are available for more than 5 isolates.
The activity of pradofloxacin against the above mentioned isolates has been compared to
other veterinary fluoroquinolones and has been reported (Silley et al., 2007). A total of 141
strains isolated from dogs (94) and cats (47), all of which were from the UK and obtained from
animals that had not received antimicrobial agents for at least 3 months prior to sampling
were screened against pradofloxacin, marbofloxacin, enrofloxacin, difloxacin, and ibafloxacin,
according to standardised agar dilution methodology. Pradofloxacin exerted the greatest anti
bacterial activity followed by marbofloxacin, enrofloxacin, difloxacin, and ibafloxacin. Based
on the distinctly lower MIC50, MIC90, and mode MIC values, pradofloxacin exhibited a higher
in vitro activity than any of the comparator fluoroquinolones. The respective susceptibility
Anaerobic activity and killing: how effective is Veraflox® really?Prof. Dr. Peter Silley
MB Consult Limited and University of Bradford, Great Britain
2nd International Veraflox® Symposium | 29 – 30 Nov 2012, Rome
72 | 73
Veraflox_2012_Proceedings_fa.indd 72 20.11.12 11:02
distributions are shown in Figure 1. The enhanced activity of
pradofloxacin relative to the other tested compounds is con
sistent with what would be expected from a thirdgeneration
fluoroquinolone. Figure 1 clearly demonstrates that the mode
MIC for pradofloxacin was 0.25 µg/ml compared with 1 µg/
ml for marbofloxacin, 2 µg/ml for enrofloxacin and difloxacin
and 4 µg/ml for ibafloxacin. The data also show that all iso
lates were sus ceptible to pradofloxacin at 2 µg/ml, whereas the
MIC range extended to 32 µg/ml for difloxacin and 64 µg/ml for
marbo floxacin, enrofloxacin, and ibafloxacin.
Whilst this data addresses the general antianaerobe activity of
pradofloxacin, there is inevit able more interest in activity against
anaerobes implicated in periodontal disease. Prado floxa cin is
indicated as adjunctive treatment to mechanical or surgical
periodontal therapy in the treatment of severe infections of the
gingiva and periodontal tissues caused by susceptible strains
of anaerobic organisms, for example Porphyromonas spp. and
Prevotella spp.
Peter Silley
Prof. Dr. Peter Silley is a microbiologist with
career that has been spent in academia and
pharma, work ing in human and veterinary
medicine. MB Consult was formed in 1999
to handle the increasing demand for micro
biology consult ancy work and coexisted
alongside a successful CRO that had been
built up by Peter; since the mid 2000s Peter
has just been involved with the MB Consult
business. He also is Professor of Applied Mi
crobiology at the University of Bradford, UK,
a member of CLSI Veterinary Antimicrobial
Susceptibility Testing SubCommittee and
serves as a Member of the Scientific Adviso
ry Board of the US Healthy People, Healthy
Animals Healthy Planet program as well as a
number of editorial boards. Peter has exten
sive experience of regulatory systems with
regard to microbiological requirements for
successful registration of antimicrobial com
pounds and feed additives. Working in Euro
pe and the USA as well as Japan, Australia,
Canada, and Brazil gives him a valuable in
sight into how to meet respective worldwide
regulatory requirements. With significant ex
perience of public health and infectious dis
ease, the antimicrobial resistance issues
and involvement in risk analysis Peter is able
to provide expertise on the right approach to
address safety and efficacy issues in today‘s
onerous regulatory climate. For further infor
mation please see: www.mbconsult.coma
Table 1 Pradofloxacin spectrum of activity against anaerobic organisms
Bacterial Genus (n) nMIC Range
(µg/ml)
Clostridium spp. 32 0.062 – 2
Bacteroides spp. 29 0.062 – 1
Fusobacterium spp. 22 0.031 – 2
Prevotella spp.1 20 _< 0.016 – 1
Porphyromonas spp.1 6 0.062 – 0.5
Sporomusa spp. 6 _< 0.016 – 1
Propionibacterium spp. 5 0.125 – 1
All strains 141 _< 0.016 – 2
1 = SPC organism
Veraflox_2012_Proceedings_fa.indd 73 20.11.12 11:02
Periodontal disease is a chronic, multifactorial disease
of the tissues supporting the teeth, and the significance
of micro organisms in the development of all types of pe
riodontal disease is indisputable. It has been estimated
that approximately 80 % of dogs and cats demonstrate
some degree of periodontal disease by 4 years of age
(Harvey and Emily, 1993). For humans and dogs, the
dental practitioner has relied heavily upon mechanical
debridement in combating periodontal infections (Har
vey, 1998). There is evidence, however, that additional
strategies, including the use of antimicrobials, are nec
essary to effectively combat periodontal infection (Har
vey, 1998; Pattison, 1996; Stambaugh et al., 1981).
For periodontal antimicrobial therapy to be effective, it
must at minimum be able to target and effectively con
trol microorganisms capable of destroying periodontal
connective tissue. It has been established for a number
of years that the absence of blackpigmented anaero
bic indicator bacterial species, such as Porphyromonas
spp. and Prevotella spp., was a better predictor of ces
sation of further loss of attachment than the presence of
these species was for further disease progression (Har
vey, 1998). On this basis, it has been concluded that
anti microbial therapy can be of great use in the treat
ment of periodontal disease (Rodenburg et al., 1990).
Consequently, data have been generated by sampling
canine periodontal pockets by experienced veterinari
ans in France, Germany, Italy, Poland, Sweden, and the
United Kingdom. Sterile endodontic paper points were
used for collection of periodontal pocket samples and
then placed in transport medium and transferred un
opened into an anaerobic workstation in a central labo
ratory. In total, 310 strains of Porphyromonas spp. and
320 strains of Prevotella spp. were isolated and identi
fied. The Porphyromonas strains identified to the species
level were P. circumdentaria / P. endodontalis (n = 126),
P. levii (n = 49), P. asaccharolytica (n = 39), P. macacae
(n = 39), P. salivosa (n = 33), and P. gingi valis (n = 24).
The Prevotella strains were P. heparinolytica (n = 77),
P. corporis (n = 34), P. nigrescens (n = 26), P. oris (n = 9),
P. disiens (n = 8), P. intermedia (n = 6), P. oralis (n = 6),
P. denticola (n = 4), P. loescheii (n = 3), P. oulorum (n = 2),
Figure 1 MIC distribution of anaerobic bacteria from dogs and cats (n = 141) for (a) pradofloxacin, (b) marbofloxacin, (c) enrofloxacin, (d) difloxacin and (e) ibafloxacin.
0.02 0.03 0.06 0.13 0.25 0.5 1 2 4 8 16 32 64
50
40
30
20
10
0
MIC (µg/l)
n
a) Pradofloxacin
0.02 0.03 0.06 0.13 0.25 0.5 1 2 4 8 16 32 64
50
40
30
20
10
0
MIC (µg/l)
n
c) Enrofloxacin
0.02 0.03 0.06 0.13 0.25 0.5 1 2 4 8 16 32 64
50
40
30
20
10
0
MIC (µg/l)
n
e) Ibafloxacin
0.02 0.03 0.06 0.13 0.25 0.5 1 2 4 8 16 32 64
50
40
30
20
10
0
MIC (µg/l)
n
b) Marbofloxacin
0.02 0.03 0.06 0.13 0.25 0.5 1 2 4 8 16 32 64
50
40
30
20
10
0
MIC (µg/l)
n
d) Difloxacin
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Veraflox_2012_Proceedings_fa.indd 74 20.11.12 11:02
P. buccae (n = 2), and P. zoogleoformans (n = 1). There were a number of Prevotella isolates for
which species names could not be defined; these were referred to as Prevotella spp. (n = 142).
There was good geographic distribution of the isolates (France, 26.8 %; Poland, 23.9 %; Ger
many, 18.3 %; Sweden, 13.7 %; Italy, 11.3 %; and the United Kingdom, 6 %). Within any one
country, there was an almost equal distribution between the two genera, although in Germany,
strains of Prevotella spp. made up only 41.7 %, whereas in Italy, they predominated (64.8 %)
relative to the Porphyromonas strains. Clearly, both of these bacterial groups are implicated
in periodontal disease.
All isolates were tested against pradofloxacin and metronidazole and MICs of both com
pounds were determined using the agar dilution methodology described by the Clinical
and Laboratory Standards Institute in complete accordance with the procedures detailed in
74 | 75
Prof. dr. P. Silley | Anaerobic activity and killing: how effective is Veraflox® really?
Figure 2 MIC distribution for pradofloxacin of 310 Porphyromonas (a) and 320 Prevotella (b) strains iso lated from cases of periodontal disease in dogs from a European multicenter study (from Stephan et al., 2008)
0.002 0.004 0.008 0.016 0.03 0.06 0.125 0.25 0.5 1 2 4 8 16 32 64 128 256
180
160
140
120
100
80
60
40
20
0
MIC (µg/ml)
n
a) Porphyromonas spp. (n = 310)
0.002 0.004 0.008 0.016 0.03 0.06 0.125 0.25 0.5 1 2 4 8 16 32 64 128 256
140
120
100
80
60
40
20
0
MIC (µg/ml)
n
b) Prevotella spp. (n = 320)
Veraflox_2012_Proceedings_fa.indd 75 20.11.12 11:02
Figure 3 Antibacterial kill kinetics of pradofloxacin against Porphyromonas gingivalis (canine periodontal disease isolate). Error bars indicate ±1 standard deviation, values are the mean of 3 replicates.
Untreated control PRA 0.062 µg/ml PRA 0.125 µg/ml PRA 0.25 µg/ml
0 4 8 12 16 20 24 28 32 36 40 44 48
8
7
6
5
4
3
2
1
0
Via
ble
coun
t (lo
g C
FU/m
l)
document M11A6, using Brucella blood agar supplemented with hemin and vitamin K, with
incubation for up to 48 hours. Bacteroides fragilis ATCC 25285 and Eubacterium lentum
ATCC 43055 were used as quality control organisms.
The summary MIC data are presented by country in Table 2, from which it can be seen that
there are no differences in pradofloxacin susceptibility between the different countries. It is
for this reason that the overall susceptibility distributions for the two genera are presented in
Figure 2, which clearly demonstrates that both genera are equally susceptible to pradofloxa
cin and exhibit the same wildtype distribution. On the basis of this distribution, there are no
strains obviously carrying resistance determinants, and the respective populations are clearly
fully susceptible to pradofloxacin.
The metronidazole MIC data were similarly consistent for all countries and for both of the
tested genera, however, there were strains that were outside the wildtype distribution. Three
Prevotella strains had intermediate metronidazole susceptibilities (MICs of 16 µg / ml), while
one Prevotella and one Porphyromonas strain were fully metronidazoleresistant (MICs of
128 µg / ml and 256 µg / ml, respectively). This data have been published by Stephan et al.
(2008) who were the first to show that a veterinary fluoroquinolone, i. e., pradofloxacin, has the
potential to be used to treat anaerobes implicated in periodontal disease.
2nd International Veraflox® Symposium | 29 – 30 Nov 2012, Rome
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Rate of kill
The kinetics of killing is highly relevant to predicting in vivo activity of an antimicrobial com
pound. The data are presented in Figures 3 and 4 and were reported by Silley et al. (2012).
The bactericidal activity against the anaerobes, Porphyromonas gingivalis and Prevotella cor
poris were marked, of particular relevance was the complete absence of grow back even at
48 hours and at pradofloxacin concentrations as low as 0.125 µg / ml, clearly exceeded in the
target animal.
From the pharmacodynamic point of view, the MIC approach provides only limited information
on the kinetics of drug action. However, kill kinetic approaches and subsequent pharmaco
kineticpharmacodynamic analysis may provide more meaningful information about the inter
action between bacteria and antiinfectives because these approaches describe this inter
action in a more dimensional way by a dynamic integration of concentration and time and,
hence, use the complete available information (Silley et al., 2012).
There is unpublished evidence that the high antianaerobe activity of pradofloxacin is attribut
able to the cyano group at position C8; further studies will need to be completed to fully
elucidate this hypothesis.
76 | 77
Prof. dr. P. Silley | Anaerobic activity and killing: how effective is Veraflox® really?
Figure 4 Antibacterial kill kinetics of pradofloxacin against Prevotella corporis (canine periodontal disease isolate). Error bars indicate ±1 standard deviation, values are the mean of 3 replicates.
0 4 8 12 16 20 24 28 32 36 40 44 48
10
9
8
7
6
5
4
3
2
1
0
Untreated control PRA 0.062 µg/ml PRA 0.125 µg/ml PRA 0.25 µg/ml
Via
ble
coun
t (lo
g C
FU/m
l)
Veraflox_2012_Proceedings_fa.indd 77 20.11.12 11:02
Table 2 Susceptibilities of oral anaerobes by genus and country and results for all strains combined
Country(ies)
Value for*: Porphyromonas spp.
MIC (µg/ml)
No. of isolates
PRA MTZ
50 % 90 % Range 50 % 90 % Range
France 87 0.062 0.125 _< 0.016 – 0.25 0.125 0.5 _< 0.016 – 0.5
Poland 72 0.062 0.125 _< 0.016 – 0.25 0.062 0.25 _< 0.016 – 0.5
Germany 67 0.062 0.125 0.03 – 0.25 0.125 0.25 0.03 – 0.25
Sweden 43 0.062 0.125 _< 0.016 – 0.25 0.125 0.25 _< 0.016 – 256
Italy 25 0.062 0.25 0.03 – 0.25 0.25 0.25 0.03 – 0.5
United Kingdom 16 0.062 0.25 0.03 – 0.5 0.062 0.125 _< 0.016 – 0.25
All 310 0.062 0.125 _< 0.016 – 0.5 0.125 0.25 _< 0.016 – 256
Country(ies)
Value for*: Prevotella spp.
MIC (µg/ml)
No. of isolates
PRA MTZ
50 % 90 % Range 50 % 90 % Range
France 82 0.062 0.25 _< 0.016 – 1 0.25 0.5 _< 0.016 – 1
Poland 79 0.062 0.25 _< 0.016 – 0.5 0.25 0.5 _< 0.016 – 16
Germany 48 0.062 0.25 _< 0.016 – 1 0.125 0.25 _< 0.016 – 2
Sweden 43 0.062 0.25 _< 0.016 – 0.5 0.25 0.5 _< 0.016 – 128
Italy 46 0.125 0.25 0.03 – 1 0.25 0.5 0.03 – 2
United Kingdom 22 0.062 0.25 0.03 – 0.5 0.125 0.5 _< 0.016 – 2
All 320 0.062 0.25 _< 0.016 – 1 0.25 0.5 _< 0.016 – 128
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Veraflox_2012_Proceedings_fa.indd 78 20.11.12 11:02
78 | 79
Prof. dr. P. Silley | Anaerobic activity and killing: how effective is Veraflox® really?
Conclusions
Pradofloxacin is highly active against anaerobes and shows rapid killing of bacteria implicated
in periodontal disease; the rate of kill studies indicate that these bacterial populations do not
recover from the bactericidal activity of pradofloxacin.
References
01 | Blondeau JM. A review of the comparative in vitro activities of 12 antimicrobial agents, with a focus on five new “respiratory quinolones”. J Antimicrob Chemother 1999; 43(Suppl B):1–11.
02 | Cambau E, Bordon F, Collatz E, Gutmann L. Novel gyrA point mutation in a strain of Escherichia coli resistant to fluoroquinolones but not to nalidixic acid. Antimicrob Agents Chemother 1993; 37:1247–1252.
03 | Cutler, C W, Kulmar J R, Genco C A. Pathogenic strategies of the oral anaerobe Porphyromonas gingivalis. Trends Microbiol 1995; 3:45–50.
04 | GautierBouchardon AV, Reinhardt AK, Kobisch M, Kempf I. In vitro development of resistance to enrofloxacin, erythromycin, tylosin, tiamulin and oxytetracycline in Mycoplasma gallisepticum, Mycoplasma iowae and Myco plasma synoviae. Vet Microbiol 2002; 88:47–58.
05 | Hannan PCT, Windsor GD, de Jong A, Schmeer N, Stegemann M. Comparative susceptibilities of various animalpathogenic mycoplasmas to fluoroquinolones. Antimicrob Agents Chemother 1997; 41:2037–2040.
06 | Hardham J, Dreier K, Wong J, Sfintescu CR, Evans T. Pigmentedanaerobic bacteria associated with canine periodontitis. Vet Microbiol 2005; 106:119–128.
07 | Harvey C E. Periodontal disease in dogs. Etiopathogenesis, prevalence, and significance. Vet Clin N Am Small Anim Pract 1998; 28:1111–1128.
08 | Harvey CE, Emily PP. Small animal dentistry, MosbyYear Books, St. Louis, MO, 1993, p. 104.
Country(ies)
Value for*: All strains
MIC (µg/ml)
No. of isolates
PRA MTZ
50 % 90 % Range 50 % 90 % Range
France 169 0.062 0.25 _< 0.016 – 1 0.125 0.5 _< 0.016 – 1
Poland 151 0.062 0.25 _< 0.016 – 0.5 0.125 0.5 _< 0.016 – 16
Germany 115 0.062 0.125 _< 0.016 – 1 0.125 0.25 _< 0.016 – 2
Sweden 86 0.062 0.25 _< 0.016 – 0.5 0.125 0.5 _< 0.016 – 256
Italy 71 0.062 0.25 _< 0.016 – 1 0.25 0.5 0.03 – 2
United Kingdom 38 0.062 0.25 0.03 – 0.5 0.125 0.5 _< 0.016 – 2
All 630 0.062 0.25 _< 0.016 – 1 0.125 0.5 _< 0.016 – 256
* PRA, pradofloxacin; MTZ, metronidazole
Veraflox_2012_Proceedings_fa.indd 79 20.11.12 11:02
2nd International Veraflox® Symposium | 29 – 30 Nov 2012, Rome
09 | Hawkey PM. Mechanisms of quinolone action and microbial response. J Antimicrob Chemother 2003; 51(1):29–35.
10 | Hirsh DC. Selected bacterial infections: anaerobes. In: JF Prescott, JD Baggot, RD Walker (eds), Anti microbial therapy in veterinary medicine. Iowa State University Press, Ames, IA, 2000, pp. 458–460.
11 | Lesher GY, Froelich EJ, Gruett MD, Bailey JH, Brundage RP. 1,8Naphthyridine derivatives: a new class of chemotherapeutic agents. J Med Pharmaceut Chem 1962; 5:1063–1065.
12 | Loesche WJ, Grossman NS. Periodontal disease as a specific albeit chronic infection: diagnosis and treatment. Clin Microbiol Rev 2001; 14:727–732.
13 | Malik M, Hussain S, Drlica K. Effect of anaerobic growth on quinolone lethality with Escherichia coli. Anti microb Agent Chemother 2007; 51:28–34.
14 | Miller BR and Harvey CE. Compliance with oral hygiene recommendations following periodontal treatment in clientowned dogs. J Vet Dent 1994; 11:18–19.
15 | Mueller M, de la Peña A, Derendorf H. Issues in pharmacokinetics and pharmacodynamics of antiinfective agents: kill curves versus MIC. Antimicrob Agent Chemother 2004; 48:369–377.
16 | Nielsen D, Walser C, Kodan GK. Chaney RD, Yonkers T, VerSteeg JD, Elfring G, Slots J. Effects of treatment with clindamycin hydrochloride on progression of canine periodontal disease after ultrasonic scaling. Vet Ther 2000; 1:150–158.
17 | Page RC. Vaccination and periodontitis: myth or reality. J Int Acad Periodontol 2000; 2:31–43.
18 | Page RC, Houston LS. Prospects for vaccination against plaquerelated oral diseases. In: Newman HN, Wilson M (eds), Dental plaque revisited: oral biofilms in health and disease. BioLine, Cardiff, United Kingdom 1999; 563–585.
19 | Pattison AM. The use of hand instruments in supportive periodontal treatment. Periodontol 2000 1996; 12: 71–89.
20 | Rodenburg JP, Van Winkelhoff AJ, Winkel EG, Goené RJ, Abbas F,, Graff J. Occurrence of Bacteroides gingivalis, Bacteroides intermedius and Actinobacillus actinomycetemcomitans in severe periodontitis in relation to age and treatment history. J Clin Periodontol 1999; 17:392–399.
21 | Rolain JM, Stuhl L, Maurin M, Raoult D. Evaluation of antibiotic susceptibilities of three rickettsial species including Rickettsia felis by a quantitative PCR DNA assay. Antimicrob Agents Chemother 2002; 46:2747–2751.
22 | Silley P, Stephan B, Greife HA, Pridmore A. Comparative activity of pradofloxacin against anaerobic bacteria isolated from dogs and cats. J Antimicrob Chemother 2007; 60:999–1003.
23 | Silley P, Stephan B, Greife HA, Pridmore A. Bactericidal properties of pradofloxacin against veterinary pathogens. Vet Microbiol 2012; 157:106–111.
24 | Slots J, Jorgensen MG. Effective, safe, practical and affordable periodontal antimicrobial therapy: where are we going, and are we there yet? Periodontol 2000 2002; 28:298–312.
25 | Stambaugh RV, Dragoo M, Smith DM, Carasali L. The limits of subgingival scaling. Int J Periodontics Restorative Dent 1985; 1:30–41.
26 | Stephan B, Greife HA, Pridmore A, Silley P. Activity of pradofloxacin against Porphyromonas and Prevotella spp. Implicated in periodontal disease in dogs: susceptibility test data from a European multicenter study. Antimicrob Agents Chemother 2008; 52:2149–2155.
27 | Walker RD. Fluoroquinolones. In: “Antimicrobial Therapy in Veterinary Medicine” 3rd edn, Prescott JF, Baggot JD, Walker RD (eds), Iowa State University Press, Iowa, USA, 2000, Chapter 15, pp. 315–338.
28 | Wetzstein HG, Hallenbach W. Relative contributions of the C7 amine and the C8 cyano substituents to the antibacterial potency of pradofloxacin. In: Proceedings of the 104th Annual General Meeting, 2004, American Society for Microbiology, Abstract Z026, pp. 672–673.
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Veraflox_2012_Proceedings_fa.indd 81 20.11.12 11:02
82 | 83
Periodontal disease (etiology and definition)
Periodontal disease is caused by a number of factors. The most common are lack of oral
hygiene or nutritional problems. Domestic animals nowadays are generally fed prepared food
and have no chance to clean their teeth and gums through catching or tearing apart their
prey. Thus, plaque or calculus tends to build up rapidly, unless home dental care is performed.
Calculus builds up more easily on teeth which are badly positioned, have enamel defects, or
trap food. Plaque and calculus contain massive numbers of bacteria, and lead to gingivitis
and/or infection. It should be noted, however, that the amount of calculus on the teeth is not
necessarily related to the degree of periodontal disease. There can often be large amounts of
calculus found on the teeth with minimal gingivitis. Conversely, there can be severe gingivitis
and periodontitis with little or no calculus build-up.
Periodontal disease is classified as simple gingivitis, chronic periodontitis, and other diseases
of the periodontium. Gingivitis is limited to gingival inflammation with no bone resorption.
It is the initial stage of periodontal disease and is reversible. Many, but not necessarily all,
cases progress to periodontitis. Periodontitis is a chronic disease characterized by gingival
inflammation, periodontal pocket formation, bleeding and suppuration from the pocket, tooth
mobility, alveolar bone resorption and, finally, tooth loss. Periodontitis is the result of progres-
sion of the inflammatory process from the gingiva to deeper structures of the periodontium.
Consequences of the disease are resorption of alveolar bone and loss of attachment, followed
by formation of true periodontal pockets. Some cases of periodontitis may progress to acute
periodontal abscesses. Most forms of gingivitis and periodontitis are caused primarily by bac-
teria that colonize the gingival crevice and attach to tooth surfaces.
Often periodontal disease is long-standing, especially in many geriatric patients. It should be
stressed that this chronic reservoir of infection may eventually spread systemically to other
parts of the body, passing easily through the gingival tissues into the bloodstream. Chronic
bacterial endocarditis, nephritis, hepatitis and pneumonia can result. The sooner these pa-
tients are treated the better it is. There is usually more chance of losing a geriatric patient to
these diseases than with the anaesthetic.
For a clearer overview, a few related definitions:
Plaque – a soft, sticky, white material that adheres to a pellicle (acellular membrane) covering
the tooth surface and is comprised mostly of bacteria, salivary glycoproteins, extracellular
2nd International Veraflox® Symposium | 29 – 30 Nov 2012, Rome
Veraflox® and its role in canine periodontal infectionsDr. Dr. Peter Fahrenkrug
VetDent Academy, Quickborn, Germany
Veraflox_2012_Proceedings_fa.indd 82 20.11.12 15:02
Peter Fahrenkrug
poly saccharide-like glucans and fructans, and exfoliated epi-
thelial cells. The metabolic by-products diffuse into the gingival
margin epithelium, causing inflammation of the gingiva (gingi-
vitis) and stimulating leucocyte movement into the epithelium.
Calculus (tartar) – a hard, creamy to brown substance formed
on top of the plaque, caused by mineralization of plaque, cal-
cification of necrotic micro-organisms and continuous growth
through more plaque deposition and mineralization. Minerali-
zation occurs by the precipitation of calcium phosphate and
calcium carbonate. Normally found supragingivally and mostly
near the salivary ducts, i.e., on the lingual surfaces of the lower
front teeth and on the buccal surfaces of the upper fourth pre-
molars and first molars.
Concretion (concrement) – concretion is the name given to
the subgingival calculus. It is harder, develops more slowly, and
adheres more tightly to the cementum than calculus does to
the enamel. It is usually dark brown to green, from blood cell
pigments, and is formed by mineralization the sulcular fluid, un-
like calculus which incorporates saliva. It is composed of 80 %
of inorganic material, calcium phosphate, calcium carbonate,
and magnesium phosphate integrated into a mesh of hydroxy-
lapatite. The remaining 20 % of organic material includes
keratin, mucopolysaccharides, amino acids and mucin.
Gingivitis – inflammation of the gingival margin which does
not affect the deeper parts of the periodontium, although it can
progress to ulcerative gingivitis. Symptoms may be swelling,
bleeding, possible lymph node involvement, possible fever and
generalized illness. Surveys indicate that about 80 % of cats
and dogs have some degree of gingivitis, stressing the need
for home dental care.
Progressive marginal gingivitis – is a progression of chron-
ic gingivitis. One sees a progressive loss of attachment and
regression from the tooth, and a steadily deepening pocket
which can be probed with a periodontal probe. The base of the
pocket becomes lined with granulation tissue and concretion,
leading to further infection. This vicious cycle progressively de-
stroys the alveolar bone until the tooth becomes loose and is
eventually lost, often with fistulas developing.
Dr. Dr. Peter Fahrenkrug graduated from the
Hanover Veterinary Faculty in 1977, got his
Dr. med. vet. there in 1978, and graduated
from the Medical University in Hamburg as
Dentist and Dr. med. dent. in 1982.
Working as a human dentist from 1982 until
1994, he worked clinically also with animals
of all species and did scientific reasearch in
veterinary dentistry.
He received numerous specialist titles: Fel-
low, Academy of Veterinary Dentistry (USA),
Dipl. EVDC, board certified Spec. in VetDent
and Equine VetDent, Fachtierarzt (Germany).
Today he works as self-employed veterinary
dentist with a focus on teaching at the Ha-
nover Veterinary Faculty as well as on semi-
nars in Germany and abroad.
Regular lectures (more than 800) on vete-
rinary dentistry in Germany (BPT-Seminars)
and abroad; regular presentations and lec-
tures at domestic and international con-
ferences.
68 publications in german and international
scientific journals and books.
Education and support of veterinarians in
practice organisation and practice manage-
ment.
He is active member of various national
and international veterinary organizations,
nu mer ous board positions.
He sees clinical cases in two small animal
and an equine clinic in the Hamburg area on
a regular basis.
Veraflox_2012_Proceedings_fa.indd 83 20.11.12 15:02
Gingival recession – is the regression of the gingival margin away from (apically) its normal
position. It is usually caused by chronic gingivitis, periodontal disease or trauma. The decision
as to whether to treat it is dependant largely on the amount of remaining attached gingiva, the
degree of periodontal involvement and its location. Surgical corrections vary depending on
these and other factors, although often in small animals more frequent professional prophy-
laxis and good home care give better long-term results.
Treatment of periodontal diseaseThe first line of defence is perfect prophylaxis. Hand instruments (scalers, currettes, explorers,
etc.) or mechanical instruments (sonic or ultrasonic scalers and roto-pros) are used to clean
the teeth of all traces of plaque and calculus. It is especially important to remove the plaque
and calculus from the gingival crevice, or subgingal pocket, and to measure the depth of the
subgingival pockets of every tooth, with a periodontal probe. Normal pocket depth should be
no more than 1 – 3 mm. Although any gingival recession has to be taken into consideration,
pockets 4 mm or deeper usually indicate periodontal disease and should be marked on the
dental chart, and the condition treated appropriately. Bleeding from pockets is generally un-
avoidable and should not be used as a reason to discontinue the treatment. After removal of
the plaque and calculus, the teeth should be polished with a rubber prophy cup and medium
grit pumice to inhibit the build-up of further plaque. The owner should be instructed to check
regularly for further signs of plaque build-up, and start brushing his pet‘s teeth. The single
most important factor in preventing the recurrence of periodontal disease is regular home
dental care, just as in humans. Most animals will allow their teeth to be cleaned with a small
animal toothbrush and a special animal toothpaste.
Periodontal surgery is performed to eliminate or reduce pockets, remove diseased sub-
gingival tissue and correct unfavourable gingival contours. The procedures that can be used
include gingival curettage gingivoplasty, gingivectomy, gingival flap operations (includ ing open
gingival flap with subgingival curettage, reverse bevel flap, modified Widman flap), mucogingi-
val surgery (including frenectomy, lateral sliding flap, apically repositioned flap, coronally repo-
sitioned flap, free gingival grafts), osteoplasty, bone graft and furcation involvement treatment.
Although all of these procedures can be used in veterinary dentistry, the most commonly used
one, apart from gingival curettage, is gingivectomy.
Gingivectomy is the removal of gingival tissue, usually with a scalpel, electrosurgery unit or
fine scissors.
Gingivectomy is used to:
– remove excessive, inflamed, infected or hyperplastic gingiva.
– remove epulis growth and papillomas
– restore the physiologic gingival contour
– improve oral hygiene by removing all pockets or pseudopockets. The remaining gingiva
should be self cleaning. This breaks the vicious cycle of inflammation and bone loss.
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Microbiology of periodontal diseaseThe initial colonisation of the dental pellicle is mainly done by Streptococcus spp. and Actino
myces spp.These bacteria synthesise extracellular polysaccharides, to which further bacteria,
e.g., staphylococci, coliforms, lactobacilli, and many other species, are able to adhere. With
extension of the supragingival plaque into the gingival sulcus, aerobes consume the available
oxygen, thereby creating a low redox potential particularly at the bottom of the gingival sulcus.
These environmental conditions favour the growth of anaerobic organisms. As the disease
progresses, deeper periodontal pockets develop with heavy accumulation of bacteria that
further lower the oxygen levels. Anaerobes take over and constitute approximately 95 % of the
subgingival flora in periodontitis.
During this development, there is also a shift from the predominantly nonmotile Grampositive
flora found in the supragingival plaque and the healthy gingival sulcus to a flora of Gramnega
tive motile anaerobic rods found in periodontal pockets (Eisenberg et al., 1991). As a general
rule, one can say, that in periodontal health the flora is composed of 85 % Grampositive and
15 % Gramnegative bacteria, whereas in periodontal disease this ratio is reversed to 80 %
Gramnegatives and 20 % Grampositives. This change can occur within two weeks when
plaque is allowed to accumulate (Colmery and Frost, 1986). Harvey et al. (1995) studied this
period of transition, in that they took subgingival plaque samples from dogs with severe gingi
vitis that had not developed periodontitis around the sampled teeth. They found 41 % Gram
positive bacteria and 59 % Gramnegative bacteria, showing that the shift from Grampositive
to Gramnegative flora is well on its way in severe gingivitis. The shift of the bacterial flora in
periodontitis could also be demonstrated by Isogai et al. (1988) for dogs and is a wellknown
fact in human dentistry. Hence, the isolation of mainly Gramnegative rods from dental pock
ets can be viewed as an indicator of periodontal disease.
It is known from human dentistry, that certain periodontal pathogens like Actinobacillus actino
mycetemcomitans, Porphyromonas gingivalis and spirochetes are able to invade periodontal
tissues, where they contribute to the destructive inflammatory process. Similar invasiveness
of periodontal pathogens is also assumed in dogs (Sarkiala and Harvey, 1993; Hennet 1995b,
Nieves et al., 1997).
Periodontal disease is caused by plaque, which is a highly complex bacterial flora. Harvey et
al. (1995), in their detailed investigations of the bacterial flora of subgingival plaque, identi
fied as many as 60 different aerobic and anaerobic bacterial species or groups. Many of
the plaque bacteria can initiate gingivitis if they are present in high numbers, and it is this
com plexity of the bacterial plaque that has lead to the assumption that periodontal disease
is caused by an overwhelming unspecific bacterial load in the gingival sulcus, the socalled
nonspecific plaque hypothesis. Indeed, the bacterial volume and mix required to produce
dis ease probably varies greatly from animal to animal and perhaps from site to site in the
animal (Harvey, 1998).
dr. dr. P. Fahrenkrug | Veraflox® and its role in canine periodontal infections
84 | 85
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More recently, an increasing amount of information has become available that periodontal
disease is caused by a more specific bacterial flora, which resulted in the specific plaque hy
pothesis. The general conclusion is that tissuedestructive effects do not develop until Gram
negative anaerobic rods are present in large numbers and that blackpigmented anaerobic
bacilli (BPAB) are the main offenders (Harvey, 1998). In humans, the BPAB Porphyromonas
gingivalis and Prevotella intermedia are now firmly implicated as periodontal pathogens, and
there is an increasing amount of evidence that Porphyromonas spp., particularly P. gingivalis,
are implicated in canine periodontal disease (Harvey, 1998). It should be noted here that the
canine biotype of P. gingivalis should now be referred to as a different new species Porphyro
monas gulae (Fournier et al., 2001). Further new Porphyromonas spp. have only been isolat
ed from dogs and cats so far. These are Porphyromonas canoris, Porphyromonas salivosa,
Porphyromonas cangingivalis, Porphyromonas cansulci, Porphyromonas crevioricanis and
Porphyromonas gingivicanis (Harvey, 1998). In the study of Harvey et al. (1995), Porphyromo
nas spp. and Prevotella spp. were the most frequently isolated anaerobes from subgingival
plaque of dogs. Further anaerobes isolated from dogs were Peptostreptococcus spp., Clos
tridium spp., Eubacterium spp., Propionibacterium spp., Fusobacterium spp., Bacteroides
spp., Veillonella spp., Mobiluncus spp., and many unidentifiable Gramnegative anaerobic
rods (Harvey et al., 1995; Hennet, 1995b). This shows the great complexity of the anaerobic
bacterial flora in periodontal disease.
It has long been known that spirochetes also make up a high proportion of bacteria in plaque
from sites with periodontitis. However, as spirochetes are extremely difficult to culture (Hennet
and Harvey, 1991; Harvey, 1998), the knowledge on the role of these bacteria in periodontal
disease is scarce. Treponema denticola and Treponema socranskii have been detected in
plaque samples from dogs with the aid of monoclonal antibodies (Riviere et al., 1996). They
were present in higher proportion in deep periodontal pockets, suggesting an involvement in
periodontal disease.
However, the specific plaque hypothesis does not mean that we are dealing with a classi
cal bacterial infection. The interactions between Porphyromonas spp., spirochetes and other
anaerobic species are probably still very complex and none of the bacterial species would
induce periodontal disease on its own. Indeed, Koch’s postulate that a specific organism is
isolated from disease, reproduces disease in healthy animals (the healthy gingival sulcus in
this case) and can be reisolated from the induced disease, cannot be proven for periodontal
disease. The reason is that the site of disease is an external surface (the crown and later the
root of the tooth), which is constantly exposed to a great variety of bacterial species (Harvey,
1998). Hence, cultures from periodontal pockets routinely result in multiple and inconsistent
isolations.
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Use of antibiotics• Inmoreseverecasesofgingivitisandperiodontaldisease,antibiotic therapy is recom
mend ed 3 – 5 days before the procedure, because of the possibility of bacterial seeding
from scaling and curettage, and 5 – 10 days postoperatively. Broadspectrum antibiotics
that have proved successful against aerobes and anaerobes in these cases are tetracycline
(except under 6 months of age or pregnant), amoxicillin potentiated with clavulanic acid,
clindamycin HCl (especially good if there is concurrent osteomyelitis), enrofloxacin, and
against anaerobes and flagellates, metronidazole.
• Whilethesedrugshavebeenwidelyusedinperiodontaltherapy,athoroughstudyabout
the use of pradofloxacin has shown a complete, broad spectrum of activity against all
relevant putative periodontal pathogens, which, in contrast to other systemic products
registered for the indication periodontal disease, also includes Gramnegative aerobic bac
teria.
While the pre and postoperative use of antibiotics is not recommended in healthy animals
with potent immunological defence mechanisms suffering from light to moderate stages of
periodontal disease, antibiotics are very effective in severe cases and in all cases which in
clude also surgical interventions (gingivectomies, extractions of unsalvageable teeth, etc.).
Given circa 3 days preoperatively, the general conditions of the patients are in most cases
healthier compared to nonantibiotic cases.
A huge amount of plaque is already destroyed at the date of the surgery and thus the patho
logical influence of their toxins.
This results in healthier, less swollen gingivae, which makes all periodontal treatments by far
easier. Since the amount of bacteria and toxins transported in the bloodstream (bacteriaemia)
is reduced, the general health status (heart valve affections!) is, sometimes dramatically, better
compared to nontreated patients.
This results in a by far safer full anaesthetic protocol. Postoperatively, wound healing and
recovery is faster and better.
Some authors prefer an antibiotical therapy on the day of the surgery, e.g., as an injection prior
to the administration of sedatives/narcotics.
It is the author‘s opinion and welldocumented longterm clinical experience that the ‘3day
priorprotocol‘ is by far more effective, since the overall general cardiovascular condition and
the amount of disease in all of the periodontal tissues, soft or hard, is more favourable for any
intra and postoperative procedures and healing.
The rational use of antibiotics saves lifes.
dr. dr. P. Fahrenkrug | Veraflox® and its role in canine periodontal infections
86 | 87
Veraflox_2012_Proceedings_fa.indd 87 20.11.12 11:02
Conclusions
• Periodontaldiseaseaffectsmostdogsofmorethanfiveyearsofage.Itisadiseaseofthe
individual tooth rather than a generalised disease of the complete dentition. Physiological
and pathological pockets can be found at the same tooth at the same time.
• Periodontaldiseaseisasevereinflammationofperiodontaltissuescausedbydentalplaque
resulting in progressive destruction of the periodontium and ultimate loss of the affected
tooth.
• Periodontal disease is not caused by one or a few defined bacterial species, but is a
complex polymicrobial infection that interacts with complex host defence mechanisms.
It is characterised by a change from healthy periodontal flora mainly composed of Gram
positive nonmotile cocci to the flora of periodontal disease dominated by aggressive an
aerobic Gramnegative motile rods. Porphyromonas spp., Prevotella spp. and spirochetes
are likely to be implicated in periodontal disease of dogs.
• Periodontal diseasecanhave systemic consequences suchas cardiovascular disease,
endocarditis, pneumonia, stroke, as well as renal and hepatic disorders, all mediated via
bacteraemia or LPS and cytokines being released into the bloodstream.
• There isnocurative treatmentofperiodontaldisease.However,progressionofdisease
can be prevented by suitable periodontal treatment. Achievable objectives of periodontal
therapy are reduction of inflammatory processes, moderate regain of attachment and sta
bilisation of a healthy periodontal flora.
• Mechanicalperiodontaltherapyisthefirstlinetreatmentofperiodontaldisease.
• Undercertainconditions,broadspectrumantimicrobialsarean importantpartofperio
dontal therapy.
• Antimicrobialsinperiodontaldiseaseshouldbeusedasanadjuncttomechanicalcleaning.
• Pradofloxacin has a complete, broad spectrum of activity against all relevant putative
perio dontal pathogens, which, in contrast to other systemic products registered for the
indication periodontal disease, also includes Gramnegative aerobic bacteria.
• Anexploratorystudyandclinicalfieldstudywithpradofloxacinhaveutilisedappropriate
designs and assessed relevant periodontal parameters.
• Pradofloxacinexertedbeneficialeffectsonthe importantclinicalperiodontalparameters
pocket depth, loss of attachment and bleeding on probing. General clinical signs were
alleviated. Pradofloxacin was able to reestablish and stabilise healthy periodontal flora
over prolonged periods of time and to reduce the total subgingival anaerobic count.
• Pradofloxacinwasclinicallyequivalent,butmicrobiologicallysuperiortoStomorgyl® (metro
nidazole + spiramycin) and Antirobe® (clindamycin hydrochloride), both established and
leading products in the treatment of periodontal disease.
• Thefavourableclinicalandmicrobiologicalpropertiesofpradofloxacinmakeitapromising
alternative for the adjunctive antimicrobial therapy of periodontal disease in dogs.
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ReferencesList of references upon request (Fahrenkrugvetdent@tonline.de)
dr. dr. P. Fahrenkrug | Veraflox® and its role in canine periodontal infections
88 | 89
Veraflox_2012_Proceedings_fa.indd 89 20.11.12 11:02
2nd International Veraflox® Symposium | 29 – 30 Nov 2012, Rome
tissue concentrations: what about penetration to the site of infection?Prof. Dr. Gregor Hauschild
Klinik und Poliklinik für Allgemeine Orthopädie und Tumororthopädie – Experimentelle Orthopädie, Universitätsklinikum Münster, Germany
90 | 91
Since the majority of bacterial infections are extracellular, optimisation of the antimicrobial drug
concentration at the site of infection, i. e., in the interstitial fluid (ISF), is important to reach a
therapeutic effect. Pradofloxacin is a newly developed 8cyanofluoroquinolone with enhanc
ed activity against Grampositive organisms and anaerobes to treat canine and feline bacterial
infections with enhanced activity of its unbound fraction at the site of infection. The purpose
of this crossover study was to measure the unbound drug concentration of pradofloxa cin in
the ISF using ultrafiltration and to compare the kinetics of pradofloxacin in serum, ISF and
tissue using enrofloxacin as reference. Under oral administration of enrofloxacin (5 mg/kg)
and pradofloxacin (3 mg/kg and 6 mg/kg, respectively) for six days each, serum collec
tion and ultrafiltration in regular intervals over a period of 24 h were performed, starting on
day 5, followed by tissue sampling at the end of the third dosing protocol (pradofloxacin
6 mg/kg). Pharmakokinetic values for enrofloxacin were similar to those of other groups
confirming study design and methods. Peak concentrations of pradofloxacin (3 mg/kg) were
1.55 ± 0.31 µg/ml in the ISF and 1.85 ± 0.23 µg/ml in serum and for pradofloxacin (6 mg/kg)
2.71 ± 0.81 µg/kg in the ISF and 2.77 ± 0.64 µg/kg in serum; both without a statistical differ
ence between ISF and serum.
Concentration values of ISF and tissue, calculated for 0.5 and 1 h after administration, were
statistically different for cartilage, kidney (0.5 h after administration) and liver but not for bone,
fat, cerebrospinal fluid, muscle, kidney (1 h after administration) and skin. The comparison of
serum and tissue values showed statistical differences 0.5 h after administration only for car
tilage. 1 h after administration this was true for bone, fat, cerebrospinal fluid, muscle and skin.
PK/PD ratios exceeded the target values to reach for most of the clinical relevant bacterial
strains with a MIC of 0.125 µg/ml using the standard dosage of 3 mg/kg pradofloxacin. For
the mode MIC (0.25 µg/ml), only the higher dosage (6 mg/kg) exceeded the target values.
Despite some technical shortcomings, the ultrafiltration approach in context of this study
appears to be the most sensitive sampling technique to estimate pharmacokinetic values of
pradofloxacin at the infection site followed by serum analysis which slightly under or over
estimates the actual values in the interstitial fluid.
Veraflox_2012_Proceedings_fa.indd 90 20.11.12 11:02
gregor Hauschild
Prof. Dr. Gregor Hauschild graduated in
1997 from the University of Hanover (Ve
terinary Medicine). After graduation, Dr.
Hauschild completed a doctoral thesis on
interlock ing nails in dogs at the same Univer
sity. He practiced veterinary surgery in a pri
vate clin ic in Neuss (Germany) before com
pleting a postdoc in Experimental Surgery
at the University of Hanover in 2002. From
2004 to 2006, Dr. Hauschild was appointed
Junior Professor of Tissue Engineering at
the same university. In 2006, Dr. Hauschild
join ed the University of Münster (Department
of Exper imental Surgery) where he obtained
a postdoctoral lecture qualification, and in
2008, he became Head of the Department
of Experimental Orthopedics. He is currently
working as veterinary surgeon (surgery) in
the LESIA center for veterinary medicine in
Düsseldorf (Germany).
Dr. Hauschild is reviewer for Veterinary and
Comparative Orthopaedics and Traumatol
ogy and for Tierärztliche Praxis.
Veraflox_2012_Proceedings_fa.indd 91 20.11.12 11:02
92 | 93
IntroductionPradofloxacin (Veraflox®, Bayer Animal Health, Leverkusen, Germany) is an 8cyanofluoro
quinolone that exerts its primary bactericidal effects by interaction with enzymes responsible
for major DNA functions (Wetzstein, 2005; Silley, 2012). Efficacy of pradofloxacin has been
eval uated against firststep fluoroquinoloneresistant strains of Escherichia coli and Staphylo
coccus aureus suggesting that at appropriate doses, pradofloxacin may have potential in
treating and limiting antimicrobialresistant bacteria. Pradofloxacin has a great affinity for two
different targets within bacterial DNA which may account for its decreased resistance profile
(Wetzstein, 2005; Heisig, 2006; Stephan, 2006; Silley, 2006, 2012). In addition, prado floxacin
has an enhanced spectrum against Mycoplasma species and anaerobic bacteria compared
to other fluoroquinolones.
Although fluoroquinolones like enrofloxacin and orbifloxacin have been linked to retinal de
generation in several species, particularly cats, the safety margin of pradofloxacin in cats
appears very high. Pradofloxacin at 6 or 10 times the recommended dosage showed no evi
dence of retinal toxicity (Messias, 2008). In addition, Veraflox® for cats is a suspension and so
is unlikely to be associated with esophageal irritation like some tablets and capsules (German,
2005; Beatty, 2006).
Recently, pradofloxacin for use in cats has been approved for several indications in some
countries. The formulation is very well tolerated by cats making it potentially much easier for
owners to administer when compared to tablets or capsules. In the past, our laboratory has
been involved in different research or clinical studies in cats of which some will be summarized
in these proceedings.
Veraflox® for feline bacterial respiratory infectionsWhile feline bacterial upper respiratory tract infections are believed to primarily be induced by
feline herpesvirus 1 and feline calicivirus infections, bacterial infections commonly occur sec
ondarily when normal flora that colonize the nasal cavities invade damaged tissues (Quimby
and Lappin, 2009; Quimby and Lappin, 2010). Staphylococcus spp., Streptococcus spp.,
Pasteurella spp., Escherichia coli, and anaerobes can induce secondary bacterial infections
after the primary insult damages the epithelium. Bordetella bronchiseptica, Mycoplasma spp.,
2nd International Veraflox® Symposium | 29 – 30 Nov 2012, Rome
Veraflox® in feline respiratory tract infections: is it a good choice?Prof. Dr. Michael R. Lappin
College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, Colorado, USA
Veraflox_2012_Proceedings_fa.indd 92 20.11.12 11:02
Michael r. Lappin
Chlamydia felis, Pasteurella multocida, and Streptococcus ca
nis are the bacteria most commonly indicated as being primary
pathogens in this syndrome and as such can induce clinical
illness without other concurrent problems or viral infections.
Prim ary or secondary bacterial infections cannot be distin
guished based on clinical signs because the resultant muco
purulent discharges are similar no matter what the cause. In
addition, use of bacterial culture and antibiotic sensitivity has
little clinical benefit because the large numbers of bacteria that
colonize the nasal cavity make it difficult to determine which
cultured organisms are associated with disease.
Our study of cats with upper respiratory disease complex had
two major objectives; to identify organisms associated with
feline rhinitis in a natural setting and to compare the efficacy
and safety of pradofloxacin and amoxicillin for the treatment of
suspected bacterial rhinitis in cats residing in a humane society
in NorthCentral Colorado (Spindel, 2008).
40 humane society cats with suspected bacterial upper res
piratory infections were studied. Nasal discharges were col
lected for performance of infectious disease diagnostic tests
prior to random placement into one of three treatment groups.
Cats were administered amoxicillin at 22 mg/kg q 12 h,
prado floxacin at 5 mg/kg q 24 h, or pradofloxacin at 10 mg/
kg q 24 h; all drugs were administered by mouth. Cats fail ing
to initially respond to either pradofloxacin protocol were
crossed to the amoxicillin protocol and cats that failed amoxi
cillin were crossed to one of the two pradofloxacin protocols.
The organisms most frequently isolated or amplified by poly
merase chain reaction assays (PCR) pretreatment were feline
herpesvirus1 (75 %), Mycoplasma species (62.5 %), Borde
tella species (47.5 %), Staphylococcus species (12.5 %), and
Streptococcus species (10.0 %).
The initial treatment was amoxicillin for 15 cats, pradofloxa
cin at 5 mg/kg for 13 cats, and pradofloxacin at 10 mg/kg for
12 cats. Of the amoxicillintreated cats, clinical signs re solved
in 10 cats (66.7 %) and five cats were switched to prado
After graduating from Oklahoma State Uni
versity in 1981, Prof. Dr. Lappin com plet ed
a rotat ing internship in small animal medici
ne and surgery at the University of Georgia.
After two years in a small animal practice in
Los Angeles, he returned to the University of
Georgia where he completed a small animal
internal medicine residency and a PhD in
Parasitology. Dr. Lappin was boardcertified
by the American College of Veterinary
Internal Medicine in 1987. He is current
ly Professor of Small Animal Inter nal Medi
cine at the College of Veteri nary Medicine
and Bio medical Sci ences at Colorado State
University. Dr. Lappin has written over 200
primary research manuscripts and book
chapters. His principal areas of inter est are
prevention of infectious diseases, the up
per respiratory disease complex, infectious
causes of fever, infectious causes of diar
rhoea, and zoonoses of cats. Dr. Lappin
is on the editorial board of Feline Medicine
and Surgery and Compendium for Continu
ing Education for the Practicing Veterinarian
and is the editor of the textbook Feline Inter
nal Medicine Secrets. He has received the
Beecham Research Award and the Norden
Distinguished Teaching Award. Dr. Lappin
is the Kenneth W. Smith Professor in Small
Animal Clinical Veterinary Medicine at Co
lorado State University and is currently the
Assistant Department Head for Research.
Dr. Lappin is the director of the “Center for
Companion Animal Studies.” He was se
lected to receive the European Society of
Feline Medicine International Award 2008
for Outstanding Contribution to Feline Me
dicine, the Winn Feline Research Award in
2009, and was named an Oklahoma State
University Distinguished Professor in 2010.
Veraflox_2012_Proceedings_fa.indd 93 20.11.12 11:02
floxacin (10 mg/kg for one cat and 5 mg/kg for four cats) after which clinical signs resolved in
four. Of the pradofloxacintreated cats (5 mg/kg), clinical signs resolved in 10 cats (76.9 %),
and three cats were switched to amoxicillin after which clinical signs resolved in all three.
Of the pradofloxacintreated cats (10 mg/kg), clinical signs resolved in 11 cats (91.7 %) and
one cat was switched to amoxicillin after which clinical signs resolved. Overall, 73.7 % of
amoxicillintreated cats resolved and 83.3 % of pradofloxacintreated cats resolved. However,
differences in response rates between groups were not statistically different (p = 0.2919), po
tentially because of the relatively small sample size. Drug toxicity was not noted and all cats
were reported to tolerate the administration of the drug. We concluded in the manuscript that
pradofloxacin can be a safe, efficacious therapy for some cats with suspected bacterial upper
respiratory infections (Spindel, 2008).
SummaryOur research group has found pradofloxacin to be clinically effective when administered to
clinically ill, naturally infected cats with suspected bacterial upper respiratory infections. Sever
al potential advantages to pradofloxacin compared to previously used therapies have been
recognized. We have found the prado floxacin protocols we have studied to be well tolerated
by cats and sideeffects have not been noted.
References01 | Beatty JA, Swift N, Foster DJ, Barrs VR. Suspected clindamycinassociated oesophageal injury in cats: five
cases. J Feline Med Surg 2006; 8:412–419.
02 | Dowers KL, Olver C, Radecki SV, et al. Use of enrofloxacin for treatment of largeform Haemobartonella felis in experimentally infected cats. J Am Vet Med Assoc 2002; 221:250–253.
03 | Dowers KL, Tasker S, Radecki SV, Lappin MR. Use of pradofloxacin to treat experimentally induced Mycoplasma hemofelis infection in cats. Am J Vet Res 2009; 70:105–111.
04 | German AJ, Cannon MJ, Dye C, Booth MJ, Pearson GR, Reay CA, GruffyddJones TJ. Oesophageal strictures in cats associated with doxycycline therapy. J Feline Med Surg 2005; 7:33–41.
05 | Hartmann AD, Helps CR, Lappin MR, et al. Efficacy of pradofloxacin in cats with feline upper respiratory tract disease due to Chlamydophila felis or Mycoplasma infections. J Vet Intern Med 2008; 22:44–52.
06 | Heisig P. Bacterial resistance to antibiotics: the exceptional case of the fluoroquinolones (abstract). In: 1st International Veraflox Symposium, Berlin, 2006, pp. 10–11.
07 | Messias A, Gekeler F, Wegener A, Dietz K, Kohler K, Zrenner E. Retinal safety of a new fluoroquinolone, pradofloxacin, in cats: assessment with electroretinography. Doc Ophthalmol 2008; 116:177–191.
08 | Quimby J, Lappin MR. Feline focus: update on feline upper respiratory diseases: introduction and diagnostics. Compend Contin Educ Vet 2009; 31:554–564.
09 | Quimby J, Lappin MR. Update on feline upper respiratory diseases: conditionspecific recommendations. Compend Contin Educ Vet 2010; 32:E1–E10.
10 | Silley P. Pradofloxacin in vitro: more than just MIC data (abstract). In: 1st International Veraflox Symposium, Berlin, 2006, pp. 8–9.
11 | Silley P, Stephan B, Greife HA, Pridmore A. Comparative activity of pradofloxacin against anaerobic bacteria isolated from dogs and cats. J Antimicrob Chemother 2007; 60:999–1003.
12 | Silley P, Stephan B, Greife HA, Pridmore A. Bactericidal properties of pradofloxacin against veterinary pathogens. Vet Microbiol 2012; 157:106–111.
13 | Spindel ME, Veir JK, Radecki SV, Lappin MR. Evaluation of pradofloxacin for the treatment of feline rhinitis. J Feline Med Surg 2008; 10:472–479.
2nd International Veraflox® Symposium | 29 – 30 Nov 2012, Rome
Veraflox_2012_Proceedings_fa.indd 94 20.11.12 11:02
14 | Stephan B. Summary of clinical efficacy and palatability of Veraflox® (abstract). In: 1st International Veraflox Symposium, Berlin, 2006, pp. 32–33.
15 | Westfall DS, Jensen WA, Reagan WJ, et al. Inoculation of two genotypes of Hemobartonella felis (California and Ohio variants) to induce infection in cats and the response to treatment with azithromycin. Am J Vet Res 2001; 62:687–691.
16 | Wetzstein HG. Comparative mutant prevention concentrations of pradofloxacin and other veterinary fluoroquinolones indicate differing potentials in preventing selection of resistance. Antimicrob Agents Chemother 2005; 49:4166–4173.
Prof. dr. M.r. Lappin | Veraflox® in feline respiratory tract infections: is it a good choice?
94 | 95
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96 | 97
Introduction
Bacterial urinary tract infections (UTI) are a common disease in dogs. Female spayed
dogs have an increased risk of developing UTI, and in intact male dogs, cystitis and pro
statitis are often found in parallel. Several predisposing factors of canine UTI have been
de scribed, such as bladder incontinence in female spayed dogs, incomplete bladder
emptying, anatomical abnormalities of the vulva, urolithiasis, dilute urine, kidney diseases, dia
betes mellitus, Cushing’s disease, catheterisation, immunosuppressive therapy or glucocorti
coids. Escherichia coli is by far the most prevalent causative bacterium of canine UTI.
Bacterial UTI are treated with appropriate antimicrobial drugs. Since April 2011, Veraflox®
tab lets (active substance pradofloxacin) have been approved in the EU for the treatment of
acute urinary tract infections in dogs caused by susceptible strains of Escherichia coli and the
Staphylococcus intermedius group (incl. S. pseudinter medius). A clinical field study was con
ducted in France, Germany and Belgium according to VICH GCP in order to prove efficacy
and safety of Veraflox® in this indication under normal practice conditions.
Materials and methods
162 dogs with clinical signs of UTI were included in the study, 85 of which were treat ed with
Vera flox® and 77 with the control prod uct amoxicillin/clavulanic acid (A/C). In the Vera flox®
group, 65 dogs presented with cystitis and 20 with prostatitis. Of the control animals,
58 showed cystitis, 17 prostatitis and 2 upper UTI. The percentage of bacteriologically pos
itive dogs was 52 % in the Veraflox® and 56 % in the A/C group. Veraflox® tablets were admin
istered at a dose of 3 mg/kg body weight once daily. The control group was treat ed with A/C
tablets at a dose of 12.5 mg/kg body weight (10 mg amoxicillin, 2.5 mg clavulanic acid) twice
daily. Treatment duration was 7– 21 consecutive days in both groups. Clinical cure, bacterio
logical cure and the reduction of the total clinical score (TCS) were determin ed seven days
after the end of treatment. Clinical and bacteriological cure were compared between the two
treatment groups using the Chisquare test. The reduction of the TCS was analysed by two
way ANOVA for repeated measures. The statistical analyses were based on the total number
of included UTI cases per group, the analysis of bacteriological cure included bacteriologically
positive animals, only. Descriptive statistics were used for analysis of the subpopulations of
dogs suf fering from cystitis or prostatitis. Further parameters assessed were the percentage
of improv ed animals (TCS reduc ed by > 50 %), treatment failures and relapses as well as the
investigators’ assessment of efficacy and palat ability, mean time to cure and ad verse events.
Veraflox® in canine urinary tract infections: efficacy under field conditionsDr. Bernd Stephan, Dr. Gert Daube, Dr. Carolin Ludwig
Bayer Animal Health GmbH, Leverkusen, Germany
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Bernd Stephan
Dr. Bernd Stephan graduated from Hanover
Veterinary School in 1992. Having worked
on resistance in Eimeria spp. of chickens,
he obtained his Doctorate in Veterinary
Medi cine from Hanover Veterinary School in
1995. In 1996 he joined Bayer Animal Health
as postdoctorate in poultry science. His
main fields of work were avian competitive
exclusion, antimicrobial therapy and moni
toring of GCP field studies. In March 1997,
Bernd was seconded to Microbial Develop
ments Ltd, Malvern, UK. As Assistant Tech
nical Manager he was responsible for vali
dation of in vivo test systems and research
studies in the field of competitive exclusion.
From 1998–2000 he assumed the position
of the Technical Manager at Microbial De
velopments Ltd where he was responsible
for product quality control, ISO 9001 and
GMP implementation. He also continued
his research interest in competitive exclu
sion and gained experience in aerobic and
anaerobic microbiology. From 2001–2008,
Bernd was responsible for the microbio
logical and clinical development of Veraflox®
tablets for dogs and cats and Veraflox® oral
suspension for cats. In 2008, Bernd joined
the Regulatory Affairs department where
he coordinated the submission of the Ver
aflox® tablets and oral suspension dossiers.
Since April 2009, Bernd has been heading
the Antiinfectives Research department of
Bayer Animal Health.
MICs of prado floxacin against bacteria isolated from urine
samples were determined by agar dilution according to CLSI
methodology. MIC50, MIC90, and geometric mean MIC (GMIC)
were calculat ed.
Results
The study results are summarised in Table 1. The MIC values of
the isolated bacteria are presented in Table 2.
* p = 0.002; ** cases assessed as very good and good;
BP = bacteriologically positive
Table 1 Results of the canine UTI field study
Parameter
Result (%)
Veraflox® Amoxicillin/ Clavulanic
acid
Clinical cure (BP cases) 92.3 81.8
Clinical cure (all cases) 89.3 83.9
Bacteriological cure 85.3* 48.0
Improved cases 2.6 9.1
Treatment failures 5.1 9.1
Relapse rate 11.9 14.8
Reduction TCS 96.8 93.4
Clinical cure cystitis 93.8 91.4
Bact. cure cystitis 88.5 52.4
Clinical cure prostatitis 80.0 76.5
Bact. cure prostatitis 75.0 50.0
Inv. ass. efficacy** 97.7 95.3
Inv. ass. palatability** 100.0 97.7
Veraflox_2012_Proceedings_fa.indd 97 20.11.12 11:03
The mean time to cure was 9 days in the Veraflox® and 10 days in the A/C group. Mild
and transient gastrointestinal symptoms (diarrhoea, vomiting, salivation), tiredness and
poly dipsia/polyuria were observed at low frequencies in both treatment groups.
Discussion and conclusions
An appropriate antimicrobial drug for treatment of UTI should have an activity spectrum
that covers all relevant bacterial UTI pathogens, reach sufficiently high concentrations in the
organs of the urinary tract and cross the bloodprostate barrier in sufficient amounts. The
novel fluoroquinolone pradofloxacin has enhanced activity against Grampositive and an
aerobic bacteria while retaining full activity against Gramnegative bacteria. Hence, the full
spectrum of UTI pathogens is covered. Furthermore, a study of Boothe (2006) showed that
high concentrations of active are reached in the urine (76 µg/ml), kidney (5.3 µg/g), bladder
wall (5.1 µg/g) and prostate (2.6 µg/g). Given all this, Veraflox® tablets provide the veterinary
prac titioner with a valuable new alternative for the treatment of UTI in dogs. Indeed, Veraflox®
tablets showed excellent efficacy and were safe in the treatment of canine UTI under field
conditions. Using clinical endpoints only, it is difficult to detect differences in efficacy between
older and newer antimicrobials. However, such differences are more likely to be detected
if also a bacterial endpoint is used. This was demonstrated in the UTI field study, in which
pradofloxacin was clinically equivalent but microbiologically superior to A/C. Hence, the high
in vitro activity of pradofloxacin against relevant UTI pathogens translates into superior micro
biological cure in the field.
Table 2 Susceptibility of UTI pathogens to pradofloxacin
Bacterial SpeciesMIC (µg/ml)
n MIC50 MIC90 GMIC
Gram-positive
Staphylococcus pseudintermedius 28 0.03 0.125 0.046
Streptococcus spp. 13 0.125 0.25 0.101
Enterococcus faecalis 10 0.25 0.5 0.330
Gram-negative
Escherichia coli 139 0.03 0.06 0.040
Pseudomonas spp. 24 0.5 1 0.631
Proteus spp. 22 0.25 0.25 0.213
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References
01 | Boothe DM. Tissue concentrations of pradofloxacin. Presented at the First International Veraflox Symposium, Berlin, March 2006.
dr. B. Stephan | Veraflox in canine urinary tract infections: efficacy under field conditions
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Notes
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Notes
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