Baytril® 100 (enrofl oxacin) Injectable Now Approved for Control of Colibacillosis Associated with Escherichia coli in weaned pigs and for Intramuscular Administration in swine

Introduction

Colibacillosis associated with E. coli is a serious threat to the swine industry, aff ecting

herds worldwide. Many normal pigs can be infected with potentially pathogenic E. coli, and nursing pigs can ingest pathogens from the skin and mammary glands of dams.

Whether pigs contract colibacillosis depends on several factors: virulence of the

pathogen, resistance to infection and environmental considerations.1

Diarrhea is one of the fi rst symptoms of colibacillosis. Ingested E. coli pathogens colonize

the small intestine, producing enterotoxins that can eventually cause excessive diarrhea.

This increases exposure of other penmates and can lead to weight loss.1

Young pigs in particular may lose as much as 40% of their body weight due to fl uid loss.

Dehydration and other disease-related complications can ultimately result in death.1

For use by or on the order of a licensed veterinarian.

The following studies show that

Baytril® 100 (enrofl oxacin) Injectable is eff ective for the

control of colibacillosis in groups or pens of weaned pigs where

colibacillosis associated with E. coli has been diagnosed, and that intramuscular

administration of Baytril 100 is both 1) eff ective as measured by its

bioequivalence to subcutaneous administration and 2) safe.

Objective

The objective of this study was to evaluate the eff ectiveness

of Baytril® 100 (enrofl oxacin) Injectable for the control of

naturally acquired colibacillosis associated with E. coli in

weaned pigs.

Materials and methods

This study was placebo-controlled and incorporated

randomization and blinding as bias control measures.

Study sites

This study was conducted fi ve separate times at four

facilities:

1. LFM Quality Laboratories, Inc., Terre Haute, IN, USA

2. Midwest Veterinary Services, Inc., Oakland, NE, USA

3. LFM Quality Laboratories, Inc., Terre Haute, IN, USA (two separate studies conducted here)

4. Thomas D. Morris, Inc., Reisterstown, MD, USA

5. Veterinary Resources, Inc., Ames, IA, USA

Animals

A total of 1500 weaned pigs were sourced from U.S.

commercial swine operations and enrolled across fi ve

studies at four sites (two studies were conducted at LFM

Quality Laboratories, Inc.). Each site consisted of 60 pens of

fi ve pigs (30 pens per treatment group). Prior to allocating

pigs to respective treatment groups and pens, a natural

onset of colibacillosis was induced in the candidate pig

population at each site by commingling candidate pigs

with a cohort of 30 to 75 “seeder pigs.” Seeder pigs were

previously confi rmed positive for beta-hemolytic E. coli and exhibited clinical signs of colibacillosis at the time of

commingling.

When a minimum of 5% of the candidate pigs were

considered clinically aff ected with colibacillosis, all candidate

pigs were individually scored for clinical signs of colibacillosis.

Eligible candidate pigs clinically aff ected with colibacillosis

and subclinical pigs were randomly allocated to 60 pens

(30 pens per treatment group) of fi ve pigs per pen and

administered the appropriately assigned treatment; this day

represented Study Day 0. Enrolled “study pigs” were between 16

and 33 days of age and weighed between 4.7 and 26.5 lbs on

Study Day 0 (day of treatment administration). Seeder pigs were

not enrolled in the study.

Treatments

At each study site, 150 pigs (30 pens of fi ve pigs) were enrolled

in each treatment group. Treatments were randomly assigned

to pens within blocks of two.

1. Baytril 100 — 7.5 mg enrofl oxacin/kg body weight

administered as a single dose intramuscularly

2. Saline (placebo) control — administered once

intramuscularly at the same volume as pigs receiving

Baytril 100

Study procedures

Pigs were injected intramuscularly with their assigned

treatment once on the day of enrollment (Study Day 0). Pigs

were observed at least once after treatment administration on

Study Day 0 for adverse events. General health observations

were conducted twice daily from Study Days 1 to 6 and once

on Study Day 7.

Pigs that became moribund due to colibacillosis and pigs

that developed conditions not related to colibacillosis were

removed and euthanized. Removals attributable to colibacillosis

were included in the fi nal analysis and considered treatment

failures whereas removals not attributable to colibacillosis were

removed from the fi nal analysis and not considered treatment

failures. On Study Day 7, all remaining enrolled pigs were

clinically scored for colibacillosis and weighed.

Pigs that met the following criteria were considered clinically

aff ected with colibacillosis:

A diarrhea score ≥ 2 on a severity scale of 0 (normal) to 3

A depression score ≥ 1 on a severity scale of 0 (normal)

to 3 and/or a gauntness score ≥ 1 on a severity scale of 0

(normal) to 2

Control of colibacillosis associated with Escherichia coli

Extra-label use in food-producing animals is prohibited.

Results

Taken as an average across all study sites, the overall

mean treatment success rate was signifi cantly higher

(P = 0.035) for the Baytril® 100 (enrofl oxacin) Injectable

group compared to the saline control group. N = 30 pens

per treatment, per study site.

The average mean treatment success rate was 61.5%

and 44.7% for the Baytril 100 and saline control groups,

respectively (Figure 1).

Baytril 100 body weight gains were signifi cantly greater

(P = 0.035) than the saline control body weight gains at all

study sites. N = 30 pens per treatment, per study site.

Mea

n tr

eatm

ent s

ucc

ess

rate

61.5%61 5%

44.7%

100% –

90% -

80% -

70% -

60% -

50% -

40% -

30% -

20% -

10% -

0% -

Baytril 100 Saline

Figure 1: Mean Percentage of Treatment Success — Average Across All Study Sites

Baytril 100 Saline

BWG (lbs) — Study 1 3.15 1.61

BWG (lbs) — Study 2 5.30 4.28

BWG (lbs) — Study 3 1.25 0.27

BWG (lbs) — Study 4 1.29 0.12

BWG (lbs) — Study 5 3.12 1.77

Total Average BWG (lbs)

2.82 1.61

Table 1: Mean Body Weight Gain (BWG) of Study Pigs by Treatment and Site

Special considerations

In a commercial setting, all pigs in an aff ected barn would

normally be treated for colibacillosis because of the high

transmission rate of E. coli. However, the study design*

required blocked allocation of Baytril 100-treated and saline-

treated pens, resulting in adjacent housing of treatment

groups. This likely caused transmission of E. coli between

treatment groups. Effi cacy was likely diminished in this test

system due to the high likelihood of reinfection of Baytril

100-treated pigs by adjacently housed untreated control pigs.

Given the above, higher effi cacy outcomes may be

observed under standard commercial swine management

practices. However, even in the face of 7 days of constant

disease pressure, Baytril 100 pigs consistently outgained

saline-treated pigs across all study sites (Table 1).

Statistical evaluation

One pen was the experimental unit. The percentage

of pigs with treatment success within each pen was

compared between the two treatment groups. Treatment

was considered successful if a pig met all of the following

criteria on Study Day 7:

A diarrhea score of ≤ 1

A depression score of 0

A gauntness score of ≤ 1

A total of 299 (of 300) enrolled pens were included in the

analysis. One pen was removed from the study and analysis

due to reasons not associated with colibacillosis. Percent

success and 95% confi dence intervals were used to

summarize the results.

Taken as an average across all study sites, mean body weight

gains from Study Day 0 to 7 were 2.82 and 1.61 lbs for the

Baytril 100 and saline control groups, respectively (Table 1).

Conclusions: Based upon the results of this study, Baytril 100 proved eff ective for the control of colibacillosis associated with E. coli in weaned pigs.

*Study design was agreed upon between the study Sponsor and Regulatory Agency.

Swine intended for human consumption must not be slaughtered within 5 days of receiving a single-injection dose.

Eff ectiveness of intramuscular administration

Objective

The objective of this study was to evaluate the

eff ectiveness of intramuscular administration of

Baytril® 100 (enrofl oxacin) Injectable* as measured

by its bioequivalence to subcutaneous administration.

Materials and methods

This study incorporated randomization as a bias

control measure.

Study site

1. Bayer HealthCare AG, Animal Health Division,

Leverkusen, Germany

Animals

A total of 32 healthy, male and female, German Landrace

Hybrid fattening pigs were used in the study. Of these, 28

were included in the determination of bioequivalence. Pigs

were approximately 4 months old and ranged in weight

from 115.7 to 142.2 lbs at the start of the study.

Pigs were housed in an indoor facility with each pen

containing two pigs of the same gender. Pigs were fed once

daily and allowed unlimited access to water.

Pigs were blocked by gender and randomly assigned to

pen and treatment using a randomization table. A clinical

examination was performed on Study Day -1 to ensure pigs

were healthy and acceptable for inclusion.

Treatments

Pigs were randomized to one of three treatment groups:

1. Baytril 100* IM — 7.5 mg enrofl oxacin/kg body

weight administered as a single dose

intramuscularly (N = 14)

2. Baytril 100* SC — 7.5 mg enrofl oxacin/kg body weight

administered as a single dose subcutaneously (N = 14)

3. Untreated control — no treatment given (N = 4)

Study procedures

Pigs in the two treatment groups were administered

7.5 mg Baytril 100*/kg body weight according to their

assigned administration route (IM or SC). Blood samples

were collected from each animal at 0 (pre-dose), 0.5, 1, 2,

4, 6, 8, 10, 12, 32, 48 and 72 hours after injection. Samples

were analyzed for serum enrofl oxacin concentrations to

determine bioequivalence.†

General health observations were also conducted

from Study Day -1 through Study Day 6, and a clinical

examination was performed on Study Day 6. Body weights

were recorded on Study Day -1 and Study Day 6.

Statistical evaluation

For the individual and combined analyses, drug

pharmacokinetics was quantifi ed via the use of a non-

compartmental analysis. The results of that analysis were

used in the estimation of the 90% confi dence limits about

the ratio of treatment means.

Clinical and general health observations

At study inclusion, fi ve pigs were noted to have bilateral

conjunctivitis, one pig had small nodules in the neck

region, and one pig had swellings on all four legs. These

pigs were considered within the defi nition of healthy

and were included in the study. One pig had a temporary

protrusion of the third eyelid and one pig had an abscess

in the right neck at 6 days post-injection. No other

abnormal observations were recorded during the post-

injection period.

For use by or on the order of a licensed veterinarian.

Results

Serum enrofl oxacin concentrations obtained after IM or

SC injection of Baytril® 100 (enrofl oxacin) Injectable* were

comparable (Figure 2).

Adverse reactions

No test-related adverse events were observed during

the study.

Extra-label use in food-producing animals is prohibited.

Conclusions: Based upon the results of this study, comparable

eff ectiveness will be achieved when Baytril 100* is

administered at a dose of 7.5 mg/kg by either SC or IM

injection into the neck of swine.

* This study used Baytril® Max 10% Injectable Solution, a formulation of enrofl oxacin identical to Baytril 100.

† Ciprofl oxacin concentrations were also measured, because enrofl oxacin metabolizes into ciprofl oxacin in the body. However, it is the parent compound (enrofl oxacin) as opposed to the metabolite (ciprofl oxacin) that typically provides the most sensitive treatment comparison. Therefore, for the purpose of this study, only the enrofl oxacin concentrations were considered (Figure 2).

Figure 2: Average Concentration-Time Profi le for Enrofl oxacin After IM or SC Administration

Mea

n En

rofl o

xaci

n Se

rum

Con

cent

ratio

n (u

g/L) Mean Concentration-Time Profi le for Enrofl oxacin

After IM or SC Administration (Mean ± SD)

0 10 20 30 40 50 60 70 80

Time (hours)

IM Mean Serum Concentration (ug/L) SC Mean Serum Concentration (ug/L)

2000

1500

1000

500

0

-500

Safety of intramuscular administration

Objective

The objective of this study was to evaluate the safety of

Baytril® 100 (enrofl oxacin) Injectable when administered

by intramuscular (IM) injection in weaned pigs at 1X, 3X

and 5X body weight.

Materials and methods

This placebo-controlled study incorporated randomization

as a bias control measure.

Study site

1. Midwest Veterinary Services, Inc., Oakland, NE, USA

Animals

Forty-eight healthy, crossbred, weaned pigs (24 females

and 24 intact males) were enrolled in this study. At study

initiation (Day 0), pigs were 20 to 22 days old and weighed

between 11.2 and 20.1 lbs. Pigs were housed in an

environmentally controlled room with unlimited access to

water and feed.

Treatments

Pigs were randomized to one of four treatment groups,

with equal numbers males and females in each group:

1. Baytril 100 1X dose — 7.5 mg enrofl oxacin/kg body

weight administered intramuscularly (N = 12)

2. Baytril 100 3X dose — 22.5 mg enrofl oxacin/kg

body weight administered intramuscularly (N = 12)

3. Baytril 100 5X dose — 37.5 mg enrofl oxacin/kg body

weight administered intramuscularly (N = 12)

4. Saline (placebo) control — administered

intramuscularly at the same volume as pigs receiving

37.5 mg Baytril 100/kg body weight (N = 12)

Study procedures

Pigs were dosed with their assigned treatment in the

neck on Study Days 0, 7 and 14. A maximum of 3 mL was

injected per injection site.

Body weights were recorded on Study Days -3 (or -2), -2 (or

-1), 6, 13 and 15. Clinical evaluations (attitude, locomotion,

respiration, abdominal fi ll, hydration, injection site and

adverse events) were conducted twice daily on Study Days

1 to 6 and Study Days 8 to 13; four times daily on Study

Days 0, 7 and 14; and once on Study Day 15.

Daily feed and water consumption was also measured

and blood, urine and fecal sample evaluations conducted.

Blood was collected to measure serum enrofl oxacin levels.

On Day 15, all pigs were humanely euthanized and a

complete necropsy was conducted.

Note: In order to facilitate data collection and necropsy,

“Study Day 0” was staggered by one calendar day for half

of the study pigs. This is why measurements before Study

Day 0 are shown as taking place on two days: e.g., Study

Day -3 (or -2).

Statistical evaluation

Measurements and observations include clinical and

physical evaluations; serum enrofl oxacin levels; daily feed

and water consumption; and blood, urine and fecal

sample evaluations.

All continuous variables were analyzed using a mixed

model analysis. Statistical comparisons of treatment eff ects

and comparison of treatment by time were performed at

the 0.1 level of signifi cance. Comparison of treatment by

time by sex was performed at the 0.05 level of signifi cance.

Swine intended for human consumption must not

be slaughtered within 5 days of receiving a single-

injection dose.

Results

A wide range of factors was considered in determining the

safety of IM administration of Baytril® 100 (enrofl oxacin)

Injectable. These results are summarized below.

Body weight increases were observed in all individual

pigs and in all treatment groups. Although not statistically

signifi cant, weight gain per treatment group was inversely

proportional to dose.

Daily feed and water consumption generally increased

in all treatment groups. However, each treatment was

associated with a transient (~72 hours), dose-related

decrease in feed and water consumption.

There were no mortalities or unscheduled removals.

Mild injection site swelling was recorded in one 5X pig after

the fi rst treatment, in two 5X pigs after the second treatment,

and in eight 5X pigs following the third treatment. Swelling

resolved without intervention within 24 to 48 hours of onset

(fi rst and second treatments). There were no other clinical

observations or abnormal physical examination fi ndings

related to administration of Baytril 100.

No clinically relevant test-related diff erences in blood

hematology or coagulation, urinalysis, or fecal analysis

parameters were detected. Serum concentrations of

some enzymes were statistically signifi cantly higher

(P = 0.10) in Baytril 100-treated groups compared to the

control group. These results were not considered to be

clinically relevant to safety because all pigs remained

clinically healthy during the study (other than injection

site swelling) and the elevations were consistent with

muscle damage related to injection site injury.

The only post-mortem fi ndings attributed to

administration of Baytril 100 were injection site lesions.

The appearance and location of lesions seemed

to correspond to day/site of injection and dose volume.

Pharmacokinetic evaluation showed minor

accumulation of enrofl oxacin or its active metabolite,

ciprofl oxacin, with a once-weekly dosing regimen. In the

3X and 5X dose groups, prolonged exposure to the drug

resulted in its remaining in the system longer

than predicted.

Conclusions:

Based upon the results of this study, IM injection of Baytril 100

is safe in swine when administered at a dose of 7.5 mg

enrofl oxacin/kg body weight in the neck.

For use by or on the order of a licensed veterinarian.

Dilution schedule now available

Baytril® 100 (enrofl oxacin) Injectable may now be diluted with sterile water prior to injection in smaller pigs. The diluted

product should be used within 24 hours. Store diluted solution in amber glass bottles between 4° – 40°C (36° – 104°F).

Dilution Schedule*

Swine Weight mL of Baytril 100 mL of sterile water Number of doses

10 lb 34 mL 66 mL 100

15 lb 51 mL 49 mL 100

20 lb 68 mL 32 mL 100

25 lb 85 mL 15 mL 100

Extra-label use in food-producing animals is prohibited.

*For 1 mL dose volume from diluted solution.

1Neumann EJ, Ramirez A, Schwartz KJ, eds. (2009). Swine Disease Manual. 4th edition. Published by AASV. Information accessed via: http://vetmed.

iastate.edu/vdpam/new-vdpam-employees/food-supply-veterinary-

medicine/swine/swine-diseases/colibacillosis-ecoli-. Accessed

November 19, 2014.

©2015 Bayer HealthCare LLC, Animal Health, Shawnee Mission, Kansas 66201

Bayer, the Bayer Cross, Baytril and Right the fi rst time are registered trademarks of Bayer

BL15317

Swine intended for human consumption must not

be slaughtered within 5 days of receiving a single-injection dose.