2nd international veraflox® symposium

2nd InternationalVeraflox® Symposium

29 – 30 November 2012 Rome, Italy

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Veraflox_Proceedings_2012_cover_fa.indd 1 16.01.13 11:14

2nd InternationalVeraflox® Symposium

29 – 30 November 2012 Rome, Italy

Veraflox_Proceedings_2012_cover_fa.indd 2 16.01.13 11:14

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

Veraflox_2012_Proceedings_fa.indd 3 20.11.12 11:02

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


06 | 07

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


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


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).



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).

08 | 09

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.


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.



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)



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.


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)


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.

12 | 13

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


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/


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


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).



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.



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



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.



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



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



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.


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



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


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.


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



MSW


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.



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



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



MSW



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.


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.


26 | 27




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


tissue concentrations in canine pyoderma: does it reach high enough directly in skin?

Dr. Christina Restrepo

Veterinary Dermatology Center, Maitland, Florida, USA


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.


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



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


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



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


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



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


d. prescribed dose of PRA was administered orally by the investigator

e. sideeffects as reported by owner recorded


a. one 6mm punch biopsy from skin (dorsum) was obtained for HPLC analysis of PRA;


b. serum sample was obtained for HPLC analysis and labeled as PEAK (2 hours additional



a. one 6mm punch biopsy from skin (dorsum) was obtained for HPLC analysis of PRA;


b. serum sample was obtained for HPLC analysis and labeled as PEAK (4 hour additional


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




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


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


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


16Rough collie

F 3 29 Allergic hypersensitivity

Complete resolution

17 Labrador MC 5 42.1Allergic


18 Dalmatian M 9 33.2Allergic


19 Malamute MC 7 42.3 HTM, alopexia X Excellent

20Golden retriever

MC 7 31.9Allergic



Abbreviations: HTM = hypothyroidism; Wt = weight




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.



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


11 TNTC cocci, 6 – 10 rods 1 – 5 cocci

12 6 – 10 cocci 1 – 5 cocci


14 6 – 10 cocci 6 – 10 cocci

15 6 – 10 cocci 1 – 5 cocci


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


Abbreviations: HTM = hypothyroidism

Dr. C. Restrepo | Tissue concentrations in canine pyoderma: does it reach highly enough




AntibioticDog

1Dog

2Dog

3Dog

4Dog

5Dog

6Dog 7†

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


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





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


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





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)


Group II Normal skin µg/g (mean)


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


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




Figure 1 Histopathology of normal dog skin in Group II dogs


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


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

01 | Ihrke PJ. Bacterial skin disease in the dog: a guide to canine pyoderma. Trenton: Veterinary Learning Systems, 1996.

02 | Scott D, Miller W, Griffin C. Muller and Kirk’s Small Animal Dermatology. 6th edn., W.B. Saunders, Philadelphia, 2001.

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.




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.

31 | Ihrke PJ, Papich MG, DeManuelle TC. The use of fluoroquinolones in veterinary dermatology. Vet Dermatol 1999; 10:193–204.

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.

33 | Gough A, Kasali O, Sigler R, et al. Quinolone arthropathy – acute toxicity to immature articular cartilage. Toxicol Pathol 1992; 20:436–449.

34 | Willemse A. Atopic skin disease: a review and a reconsideration of diagnostic criteria. J Small Anim Pract 1986; 27:771–778.

35 | Prelaud P, Guagere E, Alhaidari Z. Reevaluation of diagnostic criteria of canine atopic dermatitis. Rev Med Vet 1998; 149:1057–1064.

36 | Carlotti DN, Jacobs DE. Therapy, control and prevention of flea allergy dermatitis in dogs and cats. Vet Dermatol 2000; 11:83–98.

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.

39 | Finora K, Greco D. Hypothyroidism and myxedema coma. Compend Contin Educ 2007; 29:19–32.



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.

41 | McEwan NA, Mellor D, Kalna G. Adherence by Staphylococcus intermedius to canine corneocytes: a preliminary study comparing noninflamed and inflamed atopic canine skin. Vet Dermatol 2006; 17:151–154.

42 | Restrepo C, 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.

43 | DeManuelle T, Ihrke P, Brandt C, et al. Determination of skin concentrations of enrofloxacin in dogs with pyoderma. Am J Vet Res 1998; 59:1599–1604.

46 | 47




48 | 49

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.



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.


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.



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.


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.



Prof. dr. r.S. Mueller | Veraflox® in bacterial pyoderma – how well does it work?

52 | 53


54 | 55


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


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



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


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


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


dog 73cat 6


dog 84cat 6


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



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)



≥ 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


2 µg/mlpradofloxacin (n = 5)



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




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




Continuation of Table 3b see next page





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



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)


≥ 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





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





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


62 | 63




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


64 | 65


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.


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.



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



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


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





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.


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.




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


72 | 73


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


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



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)


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.



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


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)


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



78 | 79


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



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.


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.



80 | 81


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


Veraflox® and its role in canine periodontal infectionsDr. Dr. Peter Fahrenkrug

VetDent Academy, Quickborn, Germany


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.


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.



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


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.



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.


86 | 87


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.



ReferencesList of references upon request (Fahrenkrugvetdent@tonline.de)


88 | 89



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.


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.


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.,


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


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.


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.

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Prof. dr. M.r. Lappin | Veraflox® in feline respiratory tract infections: is it a good choice?

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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



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


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



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



2nd international veraflox® symposium

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