AU1277497A - Oral veterinary fluoroquinolone antibacterial composition for extended durati on of therapy and method of treating - Google Patents

Description

TITLE OF THE INVENTION

ORAL VETERINARY FLUOROQUINOLONE ANTIBACTERIAL COMPOSITION FOR EXTENDED DURATION OF THERAPY AND METHOD OF TREATING

BACKGROUND OF THE INVENTION Field of the Invention:

The present invention concerns an oral veterinary composition, containing the fluoroquinolone antimicrobial agent difloxacin or a pharmaceutically acceptable salt thereof. When administered orally to a ruminating animal (e.g. , a cow or a feedlot calf) , the present veterinary composition surprisingly provides (1) ready and extensive absorption of the fluoroquinolone from the rumen into the circulating blood and (2) an unexpectedly extended duration of therapeutic antimicrobial blood level.

Discussion of the Background:

It is commonly believed that orally administered antimicrobials are not readily absorbed from the gastrointestinal tract of ruminating animals. Typically, the drug will be poorly absorbed, and will show blood levels of questionable therapeutic efficacy. Typically, the blood levels of an orally-administered antimicrobial are substantially lower than that of a dose administered parenterally . It is believed that fluoroquinolones are biodegraded in the rumen of adult ruminating animals, resulting in less active ingredient available for absorption.

The present Inventors have discovered that orally administered difloxacin is well-absorbed into a ruminant's blood and persists over a surprisingly long period of time, and at a level nearly equal the same dose of the drug administered parenterally. These are both unexpected and beneficial effects.

SUMMARY OF THE INVENTION Accordingly, one object: of the present invention is to provide a novel veterinary composition providing a blood concentration of a fluoroquinolone antimicrobial nearly equal to the concentration provided by the same dose of the drug administered parenterally.

A further object of the present invention is to provide a novel veterinary composition which provides an effective concentration of antimicrobial agent in the blood stream of a ruminating or pre-ruminating (including a milk-fed non- ruminating) ruminant over an extended length of time.

A further object of the present invention is to provide a simple and effective method for treating bacterial infections in ruminants.

These and other objects, which will become apparent during the following detailed description of the preferred embodiments are provided by an oral veterinary composition, comprising an effective amount of difloxacin or a pharmacologically acceptable salt thereof, in a pharmacologically acceptable, orally acceptable carrier, and a method of treating bacterial infection in a ruminant, comprising the step of orally administering the present veterinary composition to a ruminant in need thereof.

BRIEF DESCRIPTION OF THE DRAWINGS A more complete appreciation of the invention and many of the attendant advantages thereof will be readily obtained as the same becomes better understood by reference to the following detailed description when considered in connection with the accompanying drawing, wherein:

Fig. 1 is a graph showing mean plasma difloxacin concentrations in ruminating cattle after administration of difloxacin (5 mg/kg of body weight) per os (in a capsule) and by gavage (in a suspension);

Fig. 2 is a graph showing the concentration of difloxacin (in μg/mL) against time (in h) for two examples of the present oral veterinary bolus (5 and 10 mg/kg);

Fig. 3 is a graph showing average plasma difloxacin concentrations in ruminating cattle after administration of a difloxacin bolus (5 mg/kg or 10 mg/kg of body weight) as a function of time, with the IC„₀ levels against Haemophilus somnus , Pasteurella multocida and Pasteurella hemolytica provided for reference; and Fig. 4 is a graph plotting the average plasma difloxacin concentration (in μg/mL) in pre-ru inating ruminants as a function of time (in h) for two examples of the present oral veterinary composition (5 and 10 mg/kg) , with the MIC₉₀ levels against H . somnus , P . mul tocida and P. hemolytica provided for reference.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention concerns an oral veterinary composition containing a fluoroquinolone (preferably difloxacin) or a pharmaceutically acceptable salt thereof.

In the present invention, a veterinary composition providing an extended duration of therapeutic antimicrobial blood levels is one formulated in a manner which provides an effective concentration of a fluoroquinolone, or pharmacologically acceptable salt thereof, for a length of time sufficiently long to effectively treat a microbial (e.g., bacterial) infection. In a preferred embodiment, the present veterinary composition provides a concentration of the fluoroquinolone (or a pharmacologically acceptable salt thereof) in the ruminant which exceeds the MIC₉₀ level for a given bacterium or bacteria for a length of time of at least 24 hours, and more preferably at least 48 hours, thus enabling a daily or less frequent dosing regimen.

The present invention also concerns a method of treating a bacterial infection in a ruminant which may comprise, consist essentially of, or consist of, orally administering an effective amount of the present composition to a ruminant in need thereof .

In the context of the present application, a "non- ruminating" or "pre-ruminating" ruminant is considered to be an animal still nursing or otherwise ingesting little or no roughage (e.g., a "calf" feeding on milk). The rumen is not designed to digest milk, and as a result, the non-ruminating or nursing animal is able to "tunnel" milk directly into its stomach, thus bypassing the rumen. The rumen of a ruminant is^: induced to function as a result of feeding grasses, roughages or concentrates (e.g., hay, alfalfa, corn, etc.) to the animal. The development of a functioning rumen also depends upon other factors, such as the physiological development of the particular animal. Typically, however, feeding roughages or concentrates to a nursing animal of from 3 to 8 months of age will result in the onset of normal physiological functions of the rumen (i.e., the ability to digest cellulosic materials) .

In the present application, the phrase "consisting essentially of" refers to (a) one or more additional components in the composition which do not materially affect the properties of the composition, and/or (b) one or more additional steps in the merhod which do not materially affect the beneficial effects of the method. Fluoroquinolone antimicrobial agents, including difloxacin, pharmaceutically acceptable salts thereof, pharmaceutical compositions containing the same and general methods of treating bacterial infections (including infections in non- ruminant animals) are disclosed in U.S. Patent Nos. 4,730,000 and 5,145,853, the entireties of which are incorporated herein by reference. These antimicrobial agents and salts possess a wide spectrum of antimicrobial activity, including activity against gram-positive and gram-negative microorganisms, such as Staphylococcus , Lactobacillus , Streptococcus, Sarcina, Escherichia, Enter -ob acte , Klebsiella, Pseudomonas, Acinetobacter, Proteus, Citrobacter, Nisseria, Bacillus, Bacteroides, Peptococcus, Clostridium, Salmonella, Shigella, Serratia, Haemophilus, Pasteurella, Brucella , and others.

Pharmaceutically acceptable salts include, e.g., salts obtained by reacting a fluoroquinolone with a mineral acid such as hydrochloric acid, sulfuric acid, sodium hydrogen sulfate, phosphoric acid, boric acid, nitric acid, etc.; an alkyl- or arylsulfonic acid such as ethanesulfonic acid, ethanesulfonic acid, laurylsulfonic acid, benzenesulfonic acid, toluenesulfonic acid, etc. ; a carboxylic acid or fatty acid such as acetic acid, propionic acid, valeric acid, oxalic acid, aleic acid, succinic acid, fumaric acid, benzoic acid, tartaric acid, citric acid, lactic acid, oleic acid, palmitic acid, stearic acid, lauric acid, glucoheptonic acid, etc. A "free base equivalent weight" of a pharmaceutically acceptable salt of a fluoroquinolone refers to the corresponding weight of the salt which provides the equivalent weight of free base fluoroquinolone. For example, a composition containing 1.09 g of difloxacin hydrochloride provides a "free base equivalent weight" of 1.00 g of difloxacin.

The present veterinary composition also comprises a pharmaceutically acceptable, orally acceptable carrier. A carrier is "orally acceptable" if it can be administered to a ruminant orally, and can be digested by the ruminant. Orally acceptable carriers include conventional solid and liquid carriers, excipients and/or diluents which are digestible in ruminants which can be sterilized, if desired or necessary.

Solid dosage forms for oral administration include capsules, tablets, pills, powders, granules, effervescing granules, gels, pastes, troches and pastilles. In such solid dosage forms, the active compound is admixed with at least one conventional inert diluent such as cellulose, silica, sucrose, lactose, starch or modified starch. Such dosage forms can also comprise, as in normal practice, additional substances other than inert diluents, e.g., conventional lubricating agents such as magnesium stearate. In the case of capsules, tablets and pills, the dosage forms may also comprise conventional buffering agents. Tablets and pills can additionally be prepared with conventional enteric coatings.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs containing conventional inert diluents, such as water. Besides such inert diluents, the present composition may also include adjuvants, such as wetting agents, emulsifying and suspending agents, and sweetening, flavoring and perfuming agents.

In a preferred embodiment, the present veterinary composition is in the form of a bolus. In the field of veterinary medicine, a "bolus" typically refers to a large pill (e.g. > 5 grams) . In the present application, a "bolus" refers to a solid ready-to-swallow pharmaceutical preparation, which may be administered orally and which may have extended duration of therapeutic antimicrobial blood levels properties.

In a preferred form, the present bolus contains a sufficient amount of fluoroquinolone (more preferably, difloxacin) or a pharmaceutically acceptable salt thereof to provide an effective dosage of the antimicrobial in the ruminant to which the bolus is administered, even if only administered once daily or less frequently to the ruminant. Thus, the present bolus preferably contains at least 100 mg (for example, for administration to a pre-ruminating or non- ruminating ruminant), more preferably at least 1,000 mg (for example, for administration to a ruminating ruminant) , and even more preferably at least 1,500 mg of the fluoroquinolone (preferably difloxacin) or a free base equivalent weight of a pharmaceutically acceptable salt thereof. From a practical perspective, however, the present bolus may contain, for example, a maximum of 10,000 mg of difloxacin (preferably at most 5,000 mg) or a free base equivalent weight of a pharmaceutically acceptable salt thereof.

For example, for a 250 kg animal, a preferred formulation may include the following ingredients, in the quantities indicated:

Ingredient Amount per Bolus Preferred Quantity

Difloxacin HC1 1000-5000 mg 1700 mg

Binder 1000-10,000 mg 3000 mg

Disintegrant 100-1000 mg 400 mg

Lubricant 50-500 mg 200 mg

Glidant 100-1000 mg 200 mg

Bulking Agent 10,000-21,000 mg q.s. ad 23.0 g

The proportions of the ingredients may be modified to give compositions with different dosage strengths, without affecting the properties of the composition.

Although the present invention is not limited to single- dosage administration of the composition, it is preferred that the present method consist of orally administering an effective amount of the composition in a single dosage to a ruminant in need thereof. However, the present method may consist essentially of or consist of orally administering the present composition to a ruminant in need thereof as needed to maintain an effective blood level in the ruminant for a length of time sufficient to treat the microbial infection.

The ruminants to whom the present composition is administered include animals of the genera Bos (cows) , Ovis (sheep) , Capra (goats) , Lama (llamas) and Bison (buffalo) , preferably Bos . Particularly preferred ruminants include members of the species Bos taurus , Bos gaurus , Bos javanicus, Bos mutus grunniens, Bos primigenius , Capra hircus, Ovis ammon, Ovis aries, Ovis musimon, Ovis orientalis , Lama guanico and Bison bison , most preferably Bos taurus .

In one embodiment, the present method comprises administering the composition to a ruminant in need thereof with the aid of a "balling gun." A balling gun is a tulip-shaped device comprising a tube with one concave, bell- shaped end and a plunger at the opposite end. The composition is placed in the bell end of the balling gun, the bell end of the balling gun is placed into or down the throat of the ruminant, and the plunger is depressed, thus releasing the composition into the throat or esophagus of the ruminant.

Other substances, such as a feed or second active agent (e.g., a nutrient such as a vitamin, essential amino acid, etc. ; a second pharmaceutically active compound such as a second antibacterial compound, an antiviral agent or other antimicrobial agent; etc.), may be co-administered with the present composition. A "feed" refers to a commercially available product digestible in ruminants, sold for the purpose of feeding ruminants, and also includes those substances such as hay, grasses, concentrates, etc., grown and/or prepared at the location where the ruminants are kept (e.g. , farm) for the purpose of feeding the same to the ruminants. Thus, the present method may further comprise, consist essentially of, or consist of co-administering a feed or second active agent to a ruminant in need thereof.

It is remarkable that the difloxacin hydrochloride is absorbed when administered to an animal having a functioning rumen. Typically, absorption of any drug from the rumen is surprising in view of the fact that the rumen typically contains a large amount of material (e.g., in cows, up to 50 gallons) . The material in a functioning rumen is mostly cellulosic in nature, and is able to chemically or physically bind drugs. Rumen microflora are also able to inactivate, bind or chemically alter orally administered drugs. Therefore, it is even more unexpected that the absorption of the drug can be improved by formulating it into an oral composition (particularly a solid composition, and more particularly a bolus) .

Another surprising result provided by the present invention is the "extended duration of therapeutic antimicrobial blood levels" properties that the present composition exhibits in ruminating and non-ruminating ruminants. The present composition is able to provide an effective dose of fluoroquinolone antimicrobial over quite an extended period of time (preferably at least 48 hours in a ruminating ruminant, and at least 72 hours in a non-ruminating or pre-ruminating ruminant) . Based on results known to the present Inventors prior to the present invention, the length of time during which an effective concentration of a fluoroquinolone antimicrobial is maintained in the blood stream of both ruminating and non-ruminating ruminants by the present composition is unexpected.

Other features of the invention will become apparent in the course of the following descriptions of exemplary embodiments which are given for illustration of the invention and are not intended to be limiting thereof.

EXPERIMENT 1 Experiment 1 tested the plasma concentration of a fluoroquinolone antimicrobial (difloxacin) as a function of time, provided by a solid composition administered to ruminating steers in accordance with two different techniques.

MATERIALS AND METHODS: STUDY DESIGN - PHARMACOKINETICS ;

TEST MATERIALS:

(1) Difloxacin HC1 oral suspension; Concentration as free base difloxacin = 50 mg/ml

(2) Difloxacin capsule; Concentration as free base difloxacin = 91.64%

TEST ANIMALS:

Ten 12- to 18-month-old steers having an average body weight of approximately 250-300 kg were initially obtained, and eight of the steers were studied. Steers had not been administered drugs or biological agents that could have interfered with the study of the effects of difloxacin. No concomitant medication was administered during the study.

The steers were placed in a single pen, and started on a diet of ad libitum grower ration. Water was offered free choice. The animals were acclimated 7 days prior to the start of the study. Physical examinations were performed on each steer before the start of the study to ensure that they had no clinical or subclinical disease. The steers were observed daily during the test period.

TEST ARTICLE ADMINISTRATION:

Difloxacin oral suspension was administered at 5 mg base activity/kg as a gavage using standard techniques. The difloxacin capsule was administered at 5 mg base activity/kg using standard techniques. Day 0 weights were determined and dosages were based on those weights. Gelatin capsules were prepared at the time of dosing.

BLOOD SAMPLING:

Approximately 10 mL blood samples were collected into EDTA glass evacuated tubes by venipuncture of the left or right jugular veins. Blood samples were harvested at post- injection hours 0, 0.083, 0.25, 0.5, 0.75, 1, 2, 4, 6, 8, 10, 12, 24, 48, and 72.

Following collection, blood samples were chilled on ice. Blood samples were centrifuged at 1500 x G for 10 minutes, within one hour of collection. The plasma was pipetted into duplicate vials which were sealed and stored at < -20°C until analysis.

PHARMACOKINETIC ANALYSIS:

The determination of the best fit compartmenta1 model and initial estimates of the model dependent pharmacokinetic parameters of A,, A-,, apparent K, and apparent K₂ (Baggot , Principles of Drug Disposition in Domestic Animals , W.B. Saunders Co., Philadelphia (1977)) were made using RSTRIP (Fox et al. RSTRIP , MicroMath, Inc. (1986)). The procedure used to determine the best fit compartment model involved computing the sum of the squares of the deviations: The set of parameters obtained that minimized F was chosen as the best estimate. The compart ental model utilized is represented by the general equation:

C„t = A_te^" A-,e^"

where A, and A-, are the time zero plasma drug concentrations, K, is the apparent rate constant of absorption and K₂ is the apparent rate constant of elimination. Respective half-lives were computed from the relationship t_1/2 = 0.693 /rate constant. Confidence intervals for the half-lives were computed as described by Bartoszynski et al, Construction of best confidence intervals for half-lives, Journal of Veterinary Pharmacology and Therapeutics , 13, 1-6 (1990)).

The plasma concentration-time data were also analyzed using statistical moments (Gibaldi et al, Pharmacokinetics , Marcel Dekker Inc., New York (1982)). The system moment mean residence time (MRT) was determined from the equation MRT = AUMC/AUC, where AUMC Ls the area under the curve of a plot of the product of time and plasma drug concentration-time, from 0 to infinity, and AUC is the area under the concentration-time curve from zero to infinity. The AUC and AUMC were calculated using the method described by Dunne et al. Estimation of noncompartmenta] parameters: a technical note. Journal of Pharmacokinetics and Biophar aceuticals , 17, 131-137 (1989)). Mean plasma difloxacin and concentrations at steady-state (^C _SS,_AV) were estimated from the relationship:

^C _SS._AV ⁼ AUC/dosing interval

For these calculations, it was assumed that the dosing interval was 24 h. Maximum plasma difloxacin concentrations (^cm_a) / plasma concentrations at 24 hours (C₂₄) and times to maximum concentration (t^ were determined from the mean values of difloxacin concentration-time curves using four independent observations per post dosing time. All routine statistical methods were described by Snedecor et al, Statistical Methods , Iowa State University Press, Ames, Iowa (1982) .

RESULTS AND DISCUSSION:

Mean plasma difloxacin concentrations in ruminating cattle after administration (5 mg difloxacin/kg of body weight) of a difloxacin capsule per os or suspension by gavage for all sampling periods are presented in Table 1. These values are depicted graphically in Figure 1. The mean plasma pharmacokinetic parameters calculated from these data are summarized in Table 2.

After oral administration of a single dose (5 mg/kg of body weight) of difloxacin suspension, the absorption of difloxacin in ruminating cattle was rapid. The mean peak plas a concentration was 0.968 μg/mL, occurring 4.667 hours after dosing. The AUC after gavage administration was 25.362 μg-h/mL. The elimination half-life was 18.722 hours. The calculated steady-state concentration based on 24 hour interval dosing was 0.942 μg/mL.

After oral administration of a single dose (5 mg/kg of body weight) of a difloxacin capsule, the absorption of difloxacin in ruminating cattle was not as rapid as the suspension and mean peak plasma concentration was 0.525 μg/mL and occurred 8.0 hours after dosing. The AUC after gavage administration was 20.797 μg-h/mL. The elimination half-life was 21.636 hours. The calculated steady-state concentration based on 24 hour interval dosing was 0.764 μg/mL.

No adverse effects were observed after the administration of either difloxacin formulation.

Table 1: Mean plasma difloxacin concentrations in ruminating cattle after administration of a difloxacin capsule per os or suspension by gavage (5 mg difloxacin/kg body weight)

Postdosing Time Suspension Capsule [hr] Mean* Mean

[μg/mL] [μg/mLl

0 <.01 <.01

0.083 0.048 <.01

0.25 0.220 0.014

0.5 0.219 0.028

0.75 0.284 0.043

1 0.428 0.142

2 0.700 0.264

4 0.738 0.455

6 0.927 0.454

8 0.724 0.500

10 0.694 0.455

12 0.585 0.405

24 0.521 0.411*

48 0.061 0.165

72 0.030 0.030

* Value (s) from three animals.

Table 2: Mean plasma pharmacokinetic parameters for difloxacin in ruminating cattle after administration (5 mg difloxacin/kg body weight) of a difloxacin capsule per os or suspension by gavage

Parameter Units Suspension* Capsule

Compartmental Model

Ax μg/mL -1.093 -0.777

Apparent K_t h ' 0.631 0.259

Apparent t ( ) _t h 1.177 3.008

A₂ μg/mL 1.093 0.777

Apparent K₂ h ^l 0.04 0.033

Apparent t( )₂ h 18.722 21.636

Noncompartmental Model c₂₄ μg/mL 0.521 0.411 c μg/mL 0.942 0.764

^max h 4.667 8.000

MRT h 28.709 35.554

AUC (0-t) μg ^• h/mL 22.618 18.332

AUC (O-∞) μg ^• h/mL 25.362 20.797

AUMC μg ^• h²/mL 727.56 756.23

* Data from three animals.

The compartmental model is represented by the general equation:

A,e-^f A,e where h_± and A₂ refer to time zero plasma drug concentrations; K_: is the apparent rate constant of absorption; K₂ is the apparent rate constant of elimination; t( )₁ is the apparent half-life of absorption; and t( )₂ is the apparent half-life of elimination.

The noncompartmental model is based on observations and computations from means of 4 independent sampling units at each of 14 postdosing time periods. C_max is the maximum plasma concentration observed; C₂₄ is the plasma drug concentration at 24 hours after dosing; C_{S3 av} is the average drug concentration in plasma during a dosing interval at steady state on administering the stated dose every 24 hours; t_max is the observed time of peak plasma concentration; MRT refers to the mean residence time; AUC (0-t) is the area under the tissue drug concentration-time curve from zero to time t. AUC ( 0-∞) is the area under the tissue drug concentration-time curve from zero to infinity. AUMC refers to the area under the curve of a plot of the product of time and plasma drug concentration-time.

The results of this experiment demonstrate that solid compositions (particularly capsules and boluses) of fluoroquinolone antimicrobials or pharmaceutically acceptable salts thereof (particularly difloxacin or an HCl salt thereof) provide surprising absorption and extended duration of therapeutic antimicrobial blood levels properties when administered orally to ruminating ruminants. EXPERIMENT 2

PHARMACOKINETICS AND TISSUE RESIDUE DEPLETION OF DIFLOXACIN ORAL BOLUS IN FEEDLOT CATTLE

SUMMARY:

Experiment 2 determined the plasma pharmacokinetics and tissue residue depletion in ruminating steers following a single administration of a difloxacin bolus (at a dosage of either 5 or 10 mg free base equivalent/kg body weight) .

SAFETY:

There were no adverse affects observed after the administration of the oral difloxacin- HCl boluses.

TEST MATERIALS:

Tl and T2 : Difloxacin -HCl bolus, 1134.0 mg base equivalent/bolus (5 mg/kg); T3 and T4 : Difloxacin -HCl bolus, 2268.0 mg base equivalent/bolus (10 mg/kg).

STUDY DESIGN:

TEST ANIMALS:

Twelve 4- to 8-month-old ruminating steers having an average body weight of approximately 227 kg were studied. Steers had not been administered drugs or biological agents that could have interfered with the study of the effects of difloxacin. No concomitant medication was administered during the study.

Steers were acclimated to the test environment 7 days prior to the start of the study. Physical examinations were performed on each steer before the start of the study to ensure that they had no clinical or subclinical disease. The steers were placed in individual pens and started on a diet of free choice hay. Water was offered free choice.

The steers were observed daily. All steers were sacrificed at the termination of the study.

TEST ARTICLE ADMINISTRATION:

The 12 steers were randomly allocated to treatment. On study day 0, difloxacin was administered orally via a bolus at 5.0 (Tl, T2) or 10.0 (T3 , T4) mg base equivalent/kg of body weight. Boluses were shaved, as necessary, to more accurately dose the animal at the desired levels, based on the animal's body weight. BLOOD SAMPLING:

Blood samples were collected in the same manner and volume as in Experiment 1, but at time 0 and at 0.5, 1, 2, 4, 8, 12, 24, 48, 72, 96, 120, 144, 168, 192, 216, 240, 264, 288, 312, 336, 504 and 672 hours after test article administration. Following collection, blood samples were treated and stored in the same manner as in Experiment 1.

SCHEDULE :

Day Activity ( ies)

-7 Cattle received

0 All cattle dosed perorally

0-14, 21, 28 Cattle bled 0-28: Daily observations made

PHARMACOKINETIC RESULTS AND DISCUSSION:

Pharmacokinetic analysis was performed as in Experiment 1.

Mean plasma difloxacin concentrations in steers following a single oral administration of a difloxacin bolus (5 and 10 mg base equivalent/kg body weight) are presented in Table 3. These values are depicted graphically in Figure 2. The mean plasma pharmacokinetic parameters calculated from these data are summarized in Table 4.

After oral administration of 5 mg base equivalent/kg body weight of difloxacin HCl, the absorption of difloxacin was rapid and sustained. Mean peak plasma concentration was 0.682 μg/mL, occurring 17.3 hours after dosing. The AUC (0-t) after oral administration was 24.378 μg-h/mL. The elimination half- life was 17.210 hours. The calculated steady-state concentration based on 24 hour interval dosing was 1.064 μg/mL.

After oral administration of 10 mg base equivalent/kg body weight of difloxacin HCl, the absorption of difloxacin was rapid and sustained. Mean peak plasma concentration was 1.166 μg/mL, occurring 10.0 hours after dosing. The AUC (0-t) after oral administration was 30.848 μg-h/mL. The elimination half-life was 14.170 hours. The calculated steady-state concentration based on 24 hour interval dosing was 1.297 μg/mL.

Table 3 : Mean plasma difloxacin concentrations in calves receiving a single 5 mg/kg or 10 mg/kg oral dose

Tl or T2 T3 or T4

Postdosing Time BHR551-C BHR551-A (hr) Mean (μg/mL) Mean (μg/mL)

0 <0. 010 <0. 010

0. 0. 201 0. 318

1 0. 220 0. 373

2 0. 272 0. 528

4 0. 345 0. 714

8 0. 494 0. 996

12 0. ,587 1. 057

24 0. ,558 0. 473

48 0. ,160 0. ,201

72 0. ,087 0. ,112

96 0. ,049* 0. ,073***

120 0. .023** 0. , 064****

144 <0, .010 <0. .010

168 <0. .010 <0, .010

192 <0. .010 <0, .010

216 <0 .010 <0, .010

240 <0 .010 <0. .010

264 <0 .010 <0 .010

288 <0 .010 <0 .010

312 <0 .010 <0 .010

336 <0 .010 <0 .010

504 <0 .010 <0 .010

672 <0 .010 <0 .010

* Mean of three animals ** Value from one animal *** Mean of five animals **** Mean of four animals

Table 4 : Mean plasma pharmacokinetic parameters for difloxacin in calves receiving a single 5 mg/kg or 10 mg/kg oral dosage

The results of this experiment demonstrate that solid compositions (particularly boluses) of fluoroquinolone antimicrobials or pharmaceutically acceptable salts thereof (particularly difloxacin or an HCl salt thereof) provide surprising absorption and extended duration of therapeutic antimicrobial blood levels properties when orally administered to ruminating ruminants . EXPERIMENT 3

PHARMACOKINETICS OF A DIFLOXACIN ORAL BOLUS IN FEEDLOT CATTLE

SUMMARY:

Experiment 3 determined the plasma pharmacokinetics in ruminating steers in a feedlot environment following a single administration of a difloxacin bolus (at a dosage of either 5 or 10 mg free base equivalent/kg body weight) .

TEST MATERIALS:

Tl: Difloxacin -HCl bolus, 1277.0 mg base equivalent/bolus (5 mg/kg); T2 : Difloxacin -HCl bolus, 2518.0 mg base equivalent/bolus (10 mg/kg) .

STUDY DESIGN:

TEST ANIMALS:

Twelve 5- to 9-month-old ruminating steers having an average body weight of approximately 212 kg were studied. Steers had not been administered drugs or biological agents that could have interfered with the study of the effects of difloxacin. No concomitant medication was administered during the study . Ξteers were acclimated to the test environment 7 days prior to the start of the study. Physical examinations were performed on each steer before the start of the study to ensure that they had no clinical or subclinical disease. The steers were group-housed with approximately 2.3 feet of bunk space and 276.6 square feet per head. The steers had water and a basal ration ad libitum . The steers had fully functioning rumens. The steers were observed daily throughout the test period.

TEST ARTICLE ADMINISTRATION:

The 12 steers were randomly allocated to treatment. On study day 0, difloxacin was administered orally via a bolus at 5.0 (Tl) or 10.0 (T2) mg base equivalent/kg of body weight. Boluses were shaved, as necessary, to more accurately dose the animal at the desired levels, based on the animal's body weight.

BLOOD SAMPLING:

Blood samples were collected in the same manner and volume as in Experiment 1, but at time 0 and at 1, 2, 4, 6, 9, 11, 13, 15, 18, 20, 24, 36, 48, 72, 120 and 168 hours after test article administration. Following collection, blood samples were treated and stored in the same manner as in Experiment 1. Cattle plasma samples were analyzed for difloxacin using a protein precipitation and ultrafiltration process followed by high performance liquid chromatography (HPLC) with fluorescence detection. An internal standard (A-56681.1 hydrochloride, obtained from Abbott Laboratories) was added to the plasma followed by a large volume of base and then a small volume of acid. After centrifugation to precipitate proteins, the supernatant was placed above an ultrafiltration membrane and centrifuged again. The ultrafiltrate was injected into the liquid chromatograph.

SCHEDULE :

Day Activity (ies)

-7 Potential study cattle selected (approximately

15) ; Physical examinations given; Steers identified

-7 - 0 Steers acclimated

-1 Body weight measured, physical examinations given; Dosages prepared; Scales for boluses and for steer body weight validated; Animals randomized

0 All cattle dosed perorally

0 to 7 Cattle bled

PHARMACOKINETIC RESULTS AND DISCUSSION:

Pharmacokinetic analysis was performed as in Experiment 1.

Mean plasma difloxacin concentrations in steers following a single oral administration of a difloxacin bolus (5 and 10 mg base equivalent/kg body weight) are presented in Table 5 and are depicted graphically in Figure 3. The mean plasma pharmacokinetic parameters calculated from these data are summarized in Table 6.

After oral administration of 5 mg base equivalent/kg body weight of difloxacin HCl, the absorption of difloxacin was rapid and sustained, resulting in persistently high plasma difloxacin concentrations. Mean peak plasma concentration was 0.35 μg/mL, occurring - 11 hours after dosing. The AUC₍₀._168hl (area under the plasma difloxacin concentration versus time curve from time 0 to 168 hours) after oral administration was 13.89 μg-h/mL. The apparent elimination half-life was 21.44 hours. The calculated steady-state concentration based on 24 hour interval dosing was 0.578 μg/mL.

After oral administration of 10 mg base equivalent/kg body weight of difloxacin HCl, the absorption of difloxacin was rapid and sustained, resulting in persistently high plasma difloxacin concentrations. Mean peak plasma concentration was 0.77 μg/mL, occurring - 11 hours after dosing. The AUC₍₀._168h, after oral administration was 28.71 μg-h/mL. The apparent elimination half-life was - 19.39 hours. The calculated steady-state concentration based on 24 hour interval dosing was 1.196 μg/mL.

The compartmental model is represented by the equation given in Experiment 1 above, and the noncompartmental model is based on observations and computations from means of 6 independent sampling units at each of the 17 post-dosing time periods. In Table 6, "AUC" was calculated using the trapezoidal method from time 0-168 hours, and the C._ax is the observed C_max. The "half life" was calculated using RSTRIP II exponential stripping and parameter estimation software (produced by MICROMATH'^M Scientific Software, Salt Lake City, Utah) .

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0 w

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(D t<

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izz^βim jiDd ZZ ετt> /86 OΛV 9-month-old steers

Table 6: Pharmacokinetic parameter summary for difloxacin in 5- to receiving a single 5 mg/kg or 10 mg/kg oral dosage

P multocida and P. hemolytica H. somnus Cmax/MIC90 AUC/MIC90 Cmax/MIC90 AUC/MIC90

Ratio Ratioj Ratio Ratio

Animal Dose AUC Cmax Half Lite I R

^' ϊ 54^" 5476

2057 3 2θ^"~ ϊ ϊ 408 773 500 1369 0 38 155 5602

1470 3 23 11671 768 500 1401 0 39

2224 3 46 14679 166 70^6 756 500 1762 0 42

3206 2 33 11200 1 12 5376 771 500 1344 0 28

1926 3 1 7 11613 152 557-i 769 500 1394 0 38

1981 779 500 10.63 0 26_ 2 8858 104 4252

2144 2^~1913- 1Ϊ5^~72 141 55 5^

Average 1389^{" "}θ^'35

2689 5 91 26188 283 125 70

761 1000 3143 0 71

1682 6 9-i 26892 o Jo 129 08 760 1000 3227 0 83

17.26 5 99 21767 287 104 48 754 1000 2612 0 72

2132 6 50 26042 312 125 00 770 1000 3125 0 78

1425 7 85 23492 377 1 12 76 767 1000 2819 0 94

1979 5 25 19179 2.52 92.06 777 1000 2302 0 63_

19 39 6 41 239 26 08 1 14.85^"

Average 28 71 ^"0 77

AUC = trapezoidal rule 0 to time t (t= 168 hours) MIC90 = 0 12 for P multocida & P hemolytⁱca MIC90 = 0 25 for H somnus

The results of this experiment demonstrate that solid compositions (particularly boluses) of fluoroquinolone antimicrobial agents or pharmaceutically acceptable salts thereof (particularly difloxacin or an HCl salt thereof) provide surprising absorption and extended duration of therapeutic antimicrobial blood level properties when orally administered to ruminating animals.

EXPERIMENT 4

PHARMACOKINETICS OF A DIFLOXACIN ORAL BOLUS IN NON-RUMINATING VEAL CALVES

SUMMARY:

Experiment 4 determined the plasma pharmacokinetics in non-ruminating, non-neonate "veal" calves following a single administration of a difloxacin bolus (at a dosage of either 5 or 10 mg free base equivalent/kg body weight) .

TEST MATERIALS:

Tl: Difloxaci -HCl bolus, 226.8 mg base equivalent/bolus (5 mg/kg); T2 : Difloxacin- HCl bolus, 453.6 mg base equivalent/bolus (10 mg/kg) .

STUDY DESIGN:

TEST ANIMALS:

Six 3-week to 1-month-old veal calves having an average body weight of approximately 51.2 kg were studied. The animals had not been administered drugs or biological agents that could have interfered with the effects of difloxacin. No concomitant medication was administered during the study.

Calves were acclimated to the test environment 7 days prior to the start of the study. Physical examinations were performed on each calf before the start of the study to ensure that they had no clinical or subclinical disease. The calves were individually housed, with approximately 48 square feet per head. The calves were given water ad libitum and were fed a commercial milk replacer, according to label directions. The calves did not have functioning rumens . The calves were observed daily throughout the test period.

TEST ARTICLE ADMINISTRATION:

The six calves were randomly allocated to treatment. On study day 0, difloxacin was administered orally via a bolus at 5.0 (Tl) or 10.0 (T2) mg based equivalent/kg of body weight. Boluses were shaved, as necessary, to more accurately dose the animal at the desired levels based on the animal's body weight. BLOOD SAMPLING:

Blood samples were collected in the same manner and volume as in Experiment 1, but at time 0 and at 1, 4, 7, 10, 12, 15, 18, 21, 24 and 36 hours and at 2, 3, 4, 5, 6, 7 and 14 days after test article administration. Following collection, blood samples were treated and stored in the same manner as in Experiment 1.

Calf plasma samples were analyzed for difloxacin in the same manner as Experiment 3, and pharmacokinetic analysis was conducted in the same manner as Experiment 1. The average difloxacin plasma levels after oral administration of either bolus are presented graphically in Fig. 3. The MIC₉₀ levels of the bacteria H . somnus (0.25 μg/mL), P. multocida and P. hemolytica (both 0.12 μg/mL) are presented in the graph for reference.

SCHEDULE:

Day Activity ( ies

-14 Calves acquired, vaccinated, given physical examinations and identified; status of passive transfer assessed

-7 Calves weighed and randomized

-1 Calf body weight determined, physical examinations given

0 All calves dosed perorally

0-14 Samples collected and daily observations made PHARMACOKINETIC RESULTS AND DISCUSSION:

Pharmacokinetic analysis was performed as in Experiment 1.

A raw data summary and the mean plasma difloxacin concentrations in calves following a single oral administration of a difloxacin bolus (5 and 10 mg base equivalent/kg body weight) are presented in Table 7 and depicted graphically in Figure 4. The mean plasma pharmacokinetic parameters calculated from these data are summarized in Table 8.

After oral administration of 5 mg base equivalent/kg body weight of difloxacin HCl, the absorption of difloxacin was rapid and sustained, resulting in persistently high plasma difloxacin concentrations. Mean peak plasma concentration was 0.92 μg/mL, occurring - 15.33 hours after dosing. The AUC₍₀._168hl (area under the plasma difloxacin concentration versus time curve from time 0 to 168 hours) after oral administration was 62.30 μg-h/mL. The apparent elimination half-life was 36.09 hours. The calculated steady-state concentration based on 24 hour interval dosing was 2.596 μg/mL.

After oral administration of 10 mg base equivalent/kg body weight of difloxacin HCl, the absorption of difloxacin was rapid and sustained, resulting in persistently high plasma difloxacin concentrations. Mean peak plasma concentration was 2.03 μg/mL, occurring - 16.33 hours after dosing. The AUC,₀._16ah) after oral administration was 106.32 μg-h/mL. The apparent elimination half-life was - 24.36 hours. The calculated steady-state concentration based on 24 hour interval dosing was 4.43 μg/mL.

The compartmental model is represented by the equation given in Experiment 1 above, and the noncompartmental model is based on observations and computations from means of 3 independent sampling units at each of 18 postdosing time periods. In Table 8, "AUC," C_railx and "half life" are determined as described in Experiment 3. C.,_ax/MIC_go and AUC/MIC₉₀ ratios and average values are presented in Table 7 for each of the animals tested, with reference to the above- described bacteria.

The results of this experiment demonstrate that solid compositions (particularly boluses) of difloxacin or an HCl salt thereof provide surprising extended duration of therapeutic antimicrobial blood level properties when orally administered to pre-ruminating or non-ruminating ruminants.

Table 7: Raw data summary and mean plasma difloxacin concentrations in veal calves receiving a single 5 mg/kg or 10 mg/kg oral dose

Cattle Identification, levels converted to ug/mL

5 mg/kg I 10 mg/kg Average ■ Average

Time 201 206 207 I 203 205 202 ■ 5 mg/kg ! 10 mg/kg

0 000 000 000' 000 000 000 000 000

1 032 048 006 06^ 067 048 029 060 a 106 078 039 161 172 112 07 149

7 156 069 045 196 211 147 090 185

10 121 071 049 160 168 161 080 163

12 120 071 052¹ 157 169 189 081 172

15 122 079 061 169 185 215 087 190

18 123 076 071 165 176 222 090 188

21 126 077 073 163 213 232 092 203

2<i 112 076 072 143 190 230 087 188

36 095 055 062 081 134 168 071 128

48 068 051 055 050 119 122 058 097

72 031 026 033 030 046 047 030 041

96 015 014 015 013 020 030 0151 021

120 006 008 015 006 008 013 010 009

144 003 005 010 002 004 006 006! 004

168 002 003 005 001 002 004 003 003

336 000 001 000 000 000 000 000 000

Table 8: Pharmacokinetic parameter summary for difloxacin in veal calves receiving a single 5 mg/kg or 10 mg/kg oral dosage

Pharmacokinetic Parameter Summary

Obviously, numerous modifications and variations of the present invention are possible in light of the above teachings. It is therefore to be understood that within the scope of the appended claims, the invention may be practiced otherwise than as specifically described herein.

Claims (22)

CLAIMS :

1. A veterinary composition, comprising an amount of a fluoroquinolone or a pharmacologically acceptable salt thereof effective to treat a microbial infection in a ruminating or pre-ruminating ruminant in a pharmacologically acceptable, orally acceptable inert carrier.

2. The veterinary composition of Claim 1, in a solid dosage form selected from the group consisting of a capsule, a tablet, a pill, a powder, a granule, an effervescing granule, a gel, a paste, a troche and a pastille.

3. The veterinary composition of Claim 1, in the form of an oral bolus.

4. The veterinary composition of Claim 1, wherein said fluoroquinolone is difloxacin.

5. The veterinary composition of Claim 1, formulated in a manner which provides an effective therapeutic antimicrobial blood level of said fluoroquinolone or pharmacologically acceptable salt thereof for a length of time sufficiently long to effectively treat said bacterial infection.

6. The veterinary composition of Claim 5, wherein said length of time is at least 24 hours.

7. The veterinary composition of Claim 5, wherein said length of time is at least 48 hours.

8. The veterinary composition of Claim 1, containing at least 1 gram of said fluoroquinolone or a free-base equivalent of said pharmacologically acceptable salt.

9. The veterinary composition of Claim 8, containing at least 1.5 grams of said fluoroquinolone or a free-base equivalent of said pharmacologically acceptable salt.

10. The veterinary composition of Claim 1, wherein said veterinary composition is in a liquid dosage form selected from the group consisting of an emulsion, a solution, a suspension, a syrup and an elixir.

11. A method of treating microbial infection in a ruminating or pre-ruminating ruminant, comprising orally administering the veterinary composition of Claim 1 to a ruminating or pre-ruminating ruminant in need thereof.

12. The method of Claim 11, consisting essentially of orally administering said veterinary composition as needed to maintain an effective blood level in said ruminant for a length of time sufficient to treat the microbial infection.

13. The method of Claim 12 , further consisting essentially of co-administering a second active agent to said ruminant.

14. The method of Claim 11, wherein said ruminant is a member of a genus selected from the group consisting of Bos , Ovis , Capra , Lama and Bison .

15. The method of Claim 14, wherein said ruminant is a member of the genus Bos .

16. The method of Claim 15, wherein said ruminant is a member of the species Bos taurus .

17. The method of Claim 11, wherein said microbial infection is caused by at least one gram-positive or gram- negative microorganism selected from the group consisting of Staphylococcus , Lactobacillus, Streptococcus , Sarcina, Escherichia, Enterobacter , Klebsiella, Pseudo onas, Acinetobacter , Proteus, Citrobacter, Nisseria, Bacillus, Bacteroides, Peptococcus, Clostridium, Salmonella, Shigella, Serratia, Uaemophiluε, Pasteurella and Brucella.

18. The method of Claim 11, wherein said fluoroquinolone is difloxacin.

19. The method of Claim 11, consisting of orally administering said veterinary composition once daily or less frequently to said ruminant.

20. A method of treating bacterial infection in a ruminating or pre-ruminating ruminant, comprising orally administering the veterinary composition of Claim 5 to a ruminating or pre-ruminating ruminant in need thereof.

21. The method of Claim 20, wherein said veterinary composition is in a solid dosage form selected from the group consisting of a capsule, a tablet, a pill, a powder, a granule, an effervescing granule, a gel, a paste, a troche and a pastille.

22. The method of Claim 20, wherein said veterinary composition is in the form of an oral bolus .