Title of Invention	A COMPOSITION CONTAINING FLUNIXIN AND AN ANTIBIOTIC OF FORMULA I
Abstract	The present invention relates to a novel fonnulation combining a non-steroidal anti-inflammatory drug (NSAID) such as flunixin, v"ith a fluorinated chloramphenicol or thiamphenicol derivative antibiotic such as florfenicol as disclosed. Methods for using such fonnulations in the treatment and prevention of infectious diseases of bovines and swine, including bovine respiratory disease and swine respiratory disease, are also disclosed.

Title of Invention

A COMPOSITION CONTAINING FLUNIXIN AND AN ANTIBIOTIC OF FORMULA I

Abstract

The present invention relates to a novel fonnulation combining a non-steroidal anti-inflammatory drug (NSAID) such as flunixin, v"ith a fluorinated chloramphenicol or thiamphenicol derivative antibiotic such as florfenicol as disclosed. Methods for using such fonnulations in the treatment and prevention of infectious diseases of bovines and swine, including bovine respiratory disease and swine respiratory disease, are also disclosed.

Full Text	COMPOSITIONS AND METHOD FOR TREATING INFECTION IN CATTLE AND SWINE This application claims fcenefi: of priority to U.S. Provisional Patent Application 60/382,015. riled May 20, 2302. The invention relates to compositions and methods fortne trea;ment of infections in animals. More particularly, the invention relates to a composition containing both an antibiotic and a non-steroidal anti-inflammatory drug for use in the treatment of infections in animals such as cattle and swine. Ail references cited herein are hereby incorporated in their entirety by reference. Bovine respiratory disease (BRD) occurs in both dairy and beef cattle and is one of the leading causes of economic loss to the cattle industry throughout the world. These economic losses are due to excessive mortality, reducec weight gains as weil as treatment and prevention costs. BRD is often referred to as the "bovine respiratory diseases complex" cue to the multifactorial ettoicgy. The cost of death losses due to respiratory diseases vary around the world. Death losses in the U.S. are estimated to approach $1 billion annually. Losses in various European countries range from S75 to S120 million. Cattle with clinical or sub-clinical BRD do not gain weight or produce milk as well as healthy animals. Beef cattle with BRD gain less weight, have reduced feed efficiency and often produce a lower grade carcass at slaughter. Perino LJM Apley MM Bovine Respiratory Disease, in CURRENT VETERINARY THERAPY 4 (FOOD ANIMAL PRACTICE), 4TH ED. 446-455 (Howard J.L, Smith RA, eds., 1999). A direct correlation between pulmonary lesions observed at slaughter and reduced weight gains has been established in cattle with sub-clinical infections. Whittem T.E. at a/., J. Am. Vet Med. Assoc, 209:814-818 (1996). In addition to the production losses associated with mortality and morbidity, significant costs are associated with the treatment of BRD due to the costs of various therapeutic agents and the labor required to administer these agents, along with the ex:ra labor to isolate and observe these animals. The pathogenesis cf BRD is thought to te due to the interaction of environmental and physiological stresses coupled with infectious agents. Mannheimia (Pasteurella) haemolyiica, Pasieurella multocida and Haemophilus somnus are considered part of the normal flora of the bovine upper respiratory tract. When environmental and physiological stress factors reduce the natural resistance and inhibit the pulmonary defense mechanisms these organisms proliferate and colonize the lower respiratory tract. In addition, various bovine viruses such as infectious bovine rhinotracheitis virus (ISRV), bovine viral diarrhea virus (BVDV), bovine respiratory syncytial virus (BRSV) and parainfluenza 3 virus (PI-3) are known to have immunosuppressive effects in the lung. Similarly, swine respirator/ disease (SRD) also has a multifactorial etiology. Bacterial infections caused by P. multocida, H. parasuis, Bordetella bronchiseptica, Actinobacillus pleuropneumonias, Streptococcus suis, Salmonella cholerasuis and Mycoplasma sp. can result in respiratory disease in swine, resulting in significant economic losses. Stresses such as crowding, mixing and moving of pigs and transient viral infections can contribute to the intensification of the disease. These organisms can stimulate an excessive inflammatory process in the lungs by producing various toxins that stimulate the release of various cytokines, which up-regulate the inflammatory process. M. haemolytica, considered the most virulent of these organisms, also produces a leukotoxin that inhibits phagocytosis by leukocytes thus further enhancing its ability to colonize the lower respiratory tract This process often results in a bacterial bronchopneumonia. The pulmonary damage that resuits in death or morbidity is due to the excessive inflammatory response to the invading pathogens. Damage to host tissues occur as neutrophils, pulmonary alveolar macrophages and natural killer cells destroy infected ceils. As cell membranes are damaged, arachidonic acid is released. Arachidonic acid is the substrate for the formation of various prostaglandins and other eiccsanoids. The release of these biological active substances is critical to driving the inflammatory response that results in pulmonary lesions. Mcsier DA, Vet Clin. North An. Food Animal Prac, 13:-S3-493 (19G7). In general, therapy for BRD shouic be directed at achieving the following aoals: 1. Controlling the infection - In animals where the infectious process is halted early, the need for repeat treatment is significantly reduced (see Apley IV!.D. & Fajt V.R., Vet Clin. North Am. Food Anim. Prac, 14:291-313 (1998). The selection of the appropriate antimicrobial compound should be based on the antimicrobial sensitivity of the organism involved, the levels of the antimicrobial agent in the respirator/ tract, ease of acministration, the potential for injection site tissue damage and a dosing regime that minimizes the pain and stress associated with treatment. 2. Minimize the pulmonary damage - As the level of inflammation and subsequent pulmonary damage increases, the probability of repeat therapy increases and the rate of weight gain decreases. Lekeux P., Bovine Practitioner, 29:71-75 (1995); Scott P.R., J. Dairy ScL, 76(2):414-420 (1993). 3. Reduce pyrexia - Controlling the infection and reducing the inflammation will reduce the pyrexia (fever) thus increasing the potential for recovery. The feeling of well-being that accompanies the reduction of pyrexia may also improve the intake of nutrients by suppressing inappetence associated with disease and pyrexia. For years antimicrobial therapy has been the mainstay of BRD therapy. There are many effective microbial agents currently available for the treatment of BRD. NUFLOR, an injectable formulation of the broad spectrum antibiotic florfenicol, has emerged as one of the leading antibiotics on a global basis. It is indicated for the treatment and control cf BRD associated with M. hasmolytica, P. muitocida and H. somnus as well as for the prevention of respiratory cisease in cattie at high risk of developing BRD associated with these bacteria. NUFLOR is also indicated for the treatment of bovine interdigitai phlegmon (footrot. acute interdigital necobacillosis, infectious pctiodermatitis) associated with Fuscbacierium necrophorum and Bacieroides melsninogenicus. NUFLOR may be administered sjbcutaneously as well as intramuscularly. The pathogenesis of BRD involves the development of a significant inflammatory process in the lungs and the subsequent development of pulmonary lesions, often leading to pulmonary consolidation. The degree of this inflammatory process can determine whether the disease results in mortality, a chronic "poor doer' or the animal recovers uneventfully. Various anti-inflammatory agents have been investigated regarding their ability to reduce the pyrexia, lung consolidation and weight loss associated with BRD. The use of corticosteroids is generally contraindicated as ancillary therapy for BRD due to their ability to cause serious immunosuppression. The use of nonsteroidal anti-inflammatory drugs (NSAIDs) in conjunction with antibiotics, however, has been shown to be of benefit in the treatment of bovine respiratory disease. The nonsteroidal, anti-inflammatory agent flunixin meglumine has been demonstrated to be effective in rapidly reducing pyrexia associated with BRD. Fiunixin has also been demonstrated to reduce pulmonary consolidation and the need for re-treatment with antibiotics. Fiunixin meglumine is the active ingredient in FINADYNE and BANAMINE (both available from Schering-Plough Animal Health Corporation, Union, NJ). It has emerged as one of the leading NSAIDs for adjunctive therapy of BRD. Fiunixin meglumine has been studied extensively in regard to its use in conjunction with antibiotics for the treatment of BRD. While it is widely used for this indication, it has not been used in combination in the same formulation with florfenicol because the primary route of administration of fiunixin is intravenous and florfenicol is administered intramuscularly or subcutaneously. Moreover, florfenicol formulations have been designed to provide prolonged biood leveis of the antibiotic and fiunixin would not be expeoiec to have adequate oicavaiiabiiir/ in such formulations. In addition, there have beer, concerns as to whether ficrfsnicol and fiunixin would be compatible in such formulations. Fiunixin meglumine has been used in conjunction with oxyretracyciine, and products containing born fiunixin mealumine and cxvtetracvcline are commercially available in Europe. However, such combination products require once per day administration for 3 to 5 days. Furthermore, resistance to the antibiotic oxytetracycline has become commonplace in regard to bacterial pathogens, including those commonly associated with BRD. Accordingly, there is a need for conveniently administered, stable compositions that can control and prevent the infection and minimize the inflammation associated with bovine respiratory disease and other infectious diseases, while minimizing the pain and stress to the animal associated with treatment and the potential for injection site tissue damage. Summary of the Invention The present invention fulfills this need by providing improved compositions and methods for the treatment of bovine respiratory disease and other infections of cattle and swine. The present invention relates to a composition for the treatment of microbial infection in an animal comprising fiunixin or one of its pharmaceutical^ acceptable salts and a compound of Formula I: F I o YH2 °Z li I : R—CHNT--C C-^n V A V x wherein R is a member selected from the group consisting of methyl or ethyl or a hatocenated derivative thereof, dihalocenodeuteriomethvl, 1-halcceno-1-deuterioethyl, 1,2-dihalogeno-1-deuterioethyl, azidomethyl and methylsulfonylmethyi; each of X and X' is a member independently selected from the group consisting of N02) SO2R1, SOR^ SR1( SONH2, S02NH2l SONHR1r S02NHRi, CORi, OR1, Ri, CNf halogen, hydrogen, phenyl, and phenyl substituted by halogen, N02, Ri, P02R1f CONHRi, NHR1f NRiR2, CONRiR2, OCORi, or ORi, wherein each of R-\ and R2 is a member independently selected from the group consisting of methyl, ethyl, n-propyl, isopropyl, butyl, t-butyl, isobutyl and phenyl; and Z is hydrogen or an acyl group of a hydrocarboncarboxylic acid having up to 16 carbon atoms or an acyl group of an aminohydrocarboncarboxylic acid having up to 12 carbon atoms; and the pharmaceutical^ acceptable salts of said acyl groups. in a preferred embodiment, the composition for the treatment of microbial infection in an animal comprises (a) florfenicol; (b) fiunixin or one of its pharmaceutical^ acceptable salts; and (c) from about 5% to about 80% of an aprotic polar solvent. ' The present invention also relates to a method of Treating a microbial infection in an animal comprising the s;ep of subcutaneousiy sdm.nisrering to an animal in need cf such treatment a therapeutically effective amount or" a composition comprising flunixin or one of i:s pharmaceutical^ accepiaoie salts and a compound of Formula i. In a preferred embodiment, the microbial infection is seiecied from the group consisting of bovine respiratory disease, swine respiratory disease, footrot, acu:e mastitis, pinkeye, metritis and enteritis. The present invention also relates to a method of preventing a microbial infection in an animal susceptible to such an infection comprising the step of subcutaneously administering to an animal susceptible to such an infection a prophylactic amount of a composition comprising flunixin or one cf its pharmaceutical acceptable salts and a compound of Formula I. In a preferred embodiment, the microbial infection is bovine respiratory disease. Brief Description of the Drawings Figure 1 is a graph showing the mean plasma concentration (+/- SD) of florfenicol vs. time after a single subcutaneous injection of NUFLOR (florfenicol) or a composition of the present invention containing both florfenicol and flunixin meglumine. Figure 2 is a graph showing the mean plasma concentration of flunixin vs. time after parenteral administration of 3ANAMINE/FINADYNE flunixin meglumine or a flarfenicoi-flunixin meglumine combination composition of the present invention. Figure 3 is a graph showing the effect of flunixin within a florfenicol-fiunixin meglumine combination composition of the present invention. Figure 4 is a graph shewing the effect of treatment with a 'Icrfenicoi-flur.ixin meglumine cemfcinatien composition of the present invention on antipyretic response in cattle with ERD. Figure 5 is a graph snowing the effect of treatment with a flcrfenicol-flur.ixin meglumine combination composition of the present invention on lung consolidation score. Figure 6 is a graph showing the effect of treatment with a florfenicol-flunixin meglumine combination composition of the present invention on the ciinical success rate. Detailed Description of the Invention When flunixin is administered subcutaneously in a combination formulation with an antibiotic, its bioavailability is significantly reduced. One would expect that this would result in less clinical efficacy when administered in this manner compared to simultaneous administration by the intravenous route. It has surprisingly been found, however, that, despite the reduced bioavailability of subcutaneously administered flunixin meglumine, the same dose of flunixin meglumine when administered in certain formulations containing a fiuorinated chloramphenicol orthiamphenicol analog/derivative antibiotic, is as clinically efficacious as the same amount of flunixin meglumine administered intravenously in conjunction with a separate subcutaneous injection of the antibiotic. As used herein, the following terms, unless otherwise indicated, shall be understood to have the following meanings: "Acyl" means an H-C(O)-, alkyl-C(O)-, alkenyl-C(O)-, alkynyl-C(O)-, cycioalkyl-C(O)-, cycioalkenyi-C(O)-, or cycloalkynyl-C(O)- group in which the various groups are as previously described. The bond to the parent moiety is through the carbonyl. Preferred acyls contain a lower afkyl. Non-limiting examples of suitable acyi groups induce formyi, acetyl, prcpanoyl, 2-methyicrcpanoyl. butanoyi and cyciohexancyl. "Alky1" means an aiiphaiic hydrccaroon group, which may be straight or branched, comprising from 1 to about 20 carbon aioms in the chain. Preferred alkyi groups contain from 1 to about 12 carbon atoms in the chain. More preferred alkyi groups contain from 1 to about 5 carbon atoms in the chain. Branched means that one or more lower alkyi groups, such as methyl, ethyi or propyl, are attached to a linear aikyl chain. "Lower alkyi" means a group having from 1 to about 6 carbon atoms in the chain, which may be straight or branched. The term "substituted aikyl" means that the aikyl group may be substituted by one or more substituents which may be the same or different. "Aryl" means an aromatic monocyclic or multicyclic ring system comprising about 6 to about 14 carbon atoms, preferably about 6 to about 10 carbon atoms. The aryl group can be optionally substituted with one or more "ring system substituents/ which may be the same or different, and are as defined herein. " Alkoxy" means an aikyl-O- group in which the aikyl group is as previously described. Non-limiting examples of suitable alkoxy groups include methoxy, ethoxy, isopropoxy, and n-butoxy. The bond to the parent moiety is through the ether oxygen. "Azido" refers to an -N3 group. "Halo" and "halogeno" mean fluoro, chforo, bromo, or iodo groups. Preferred are fluoro, chloro or bromo, and more preferred are fluoro and chloro. "Halogen" means fluorine, chlorine, bromine, or iodine. Preferred are fluorine, chlorine or bromine, and more preferred are fluorine and chlorine. "Haloalkyi" and "halogenoalkyi" mean an aikyl group as denned above wherein one or more hydrogen atoms on the alkyi is replaced by a halo group defined above. "Ring system substituer.t" means a substituent attached tc ar aromatic or non-aromatic ring system which, for example, replaces an availacie hycrogen en the ring system. Ring system substituents may be the same or different. The term "optionally substituted" means optional substitution with the specified croups, radicals or moieties. As used herein, the term "composition" is intended to encompass a product comprising the specified ingredients in the specified amounts, as weil as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts. An "effective amount" is the dose required to alleviate a particular symptom of an infection or disease or to protect an animal against infections or disease. As used herein, the term "bovine" refers to animals of the genus Bos, such as cattle. The term "bovid" refers to animals in the family Bovidae, which includes hoofed, hollow-homed ruminants such as cattle, sheep, goats, buffaloes, oxen, etc. As used herein, the term "swine" refers to animals of the family Suidae, which includes pigs, boars, warthogs, etc. Flunixin meglumine is currently approved globally for use in the treatment of BRD. It has become a mainstay of veterinary practice for the treatment of inflammatory conditions. As the veterinarian often administers NSAID therapy with an antibiotic, development of a combination flunixin-antibiotic injectable product would be desirable. Flunixin meglumine is commercially available from, e.g., ISP (Wayne, NJ), or may be made according to methods known in the art, e.g., the methods described in U.S. Patent Nos. 3,337,570, 3,478,040 and 3,339,344. Fluorine-containing analogs of antibiotics chloramphenicol and thiamphenicol have been shown to have antibiotic activity, both against organisms sensitive to and resistant to chloramphenicol and thiamphenicol. See Schafer, T.W. et a/., "Novel Fluorine-Containing Analogs of Chloramphenicol and Thiampnenicoi: Antibacterial and Biological Properties," in CURRENT CHEMOTHERAPY AND INFECTIOUS DISEASE PROCEEDINGS OF Tr£ ': 1~~ !CC AND THE 19"* ICAAC AMER.'CAN SOCIETY OF M.CROSICLCGY I960, 444-446. Exanoies of such compounds, and methods for their manufacture, are described arc claimed in U.S. Patent No. 4.235,892. The medical profession has become increasingly concerned about the transference of bacierial resistance to humans when antibiotics useful in treating humans are administered to livestock. Because the chloramphenicol group of antibiotics is infrequently usee now to treat humans, its derivatives are particularly appropriate for veterinary use. Of particular interest are the 3-fluorc, 3-decxy derivatives. Tne invention provides novel compositions for the treatment of infectious diseases such as bovine respiratory disease in livestock. These compositions are formulations comprising flunixin in combination with certain chloramphenicol derivatives. Initial testing of these formulations demonstrated relatively low serum levels of flunixin in cattle following administration of a single dose. In subsequent clinical trials, the formulations of the present invention demonstrated an unexpected high degree of efficacy when compared to a negative control group as well as a group receiving florfenicol and flunixin concurrently but in separate formulations. The compositions of the present inventicn comprise flunixin meglumine and at least one antibiotic of Formula I: FORMULA I F I 0 ?H2 ?Z II I : R— CHN----C C-^H —X1 wherein R is a member selected from the group consisting of methyl c erhyl or a halogenated derivative thereof, dihalogencceuteriomethyl. 1-nalocer.c-i-deuterioethvi, 1.2-dihaloceno-1-deu:erioet.nvl, azidcmeihvl and methylsulfcnyirnethyf; each cf X and XT is a member independently selected from the croup consisting cf \'02l S02R-r SOR1( SRi, SONH2l S02NH2, SONHRi, SC2NHR1. CORi, ORL Fv, CN, halogen, hydrogen, prenyl, and phenyl substituted by halogen, NO,, R1t OR1f P02R1t CONHRL NHRi, NRiR2. CONRiR2 or OCOR1f wherein each of R-i and R2is a member independently selected from the group consisting of methyl, ethyl, n-propyl, isopropyl, butyl, t-butyl, isobutyl and phenyl; and 2 is hydrogen or an acyl group of a hydrocarboncarboxylic acid (preferably a hydrocarbondicarboxyiic acic} having up to 16 carbon atoms or an acyl group cf an amino- hydrocarboncarboxylic acid having up to 12 carbon atoms; and the pharmaceutical^ acceptable salts of said acyl groups. Included among the halogenated groups contemplated for the moiety R in Formula I are the mono-, di- and tri-fiuoro, the mono-, di- and tri-chloro-, the mono-and di-bromo-, and the iodo-methyl groups as well as the mono- and di-fluoro-, the mono- and di-chloro-, the mono- and di-bromo-, and the iodo-ethyl groups wherein the halogen substituents are preferably on the carbon alpha to the carbonyl function. Also included are mixed dihalogenoalkyl groups in which both halogens are preferably bonded to the carbon alpha to the carbonyl groups, e.g., groups such as fluorochloro-, fiuorobromo-, and chlorobromo-methyl and -ethyl, as well as trihalogen-methyl groups such as dichlorofluoro- and difluorochloromethyL Also included among the compounds of Formula I are the ester derivatives, e.g. 1-hydrocarboncarboxyiates of Formula I wherein Z is an acyl group of a hydrocarboncarboxylic acid having up to 15 carbon atoms that may be saturated, unsaturated, straight chain or branched chain, aliphatic, cyclic, cyciic-aiiphatic, aromatic, aryl-aliphatic, or alkyl-aromatic and may be substituted by hydroxy, alkoxy containing irorr, 1 to 5 carbon aioms, carboxyl, NO2, NHRi, NP^RZ: SR1r SOR-,. cr halogen, wherein R1 and R: are as defined aoove. Other ar.tibacterially active ester derivatives of Formula i are these wherein Z is an acyl croup of an amino acid containing up to 12 carbon atcms that may be saturated, unsaturated, straight chain, branched chain or cyclic, that may contain aromatic grccps and that may be substituted by hydroxy! groups. Preferred ester derivatives induce those derived from dibasic hydrocarboncarboxylates, e.g. the 1-succinate and 1-palmitate esters, which provide water soluble, pharmaceutical^ acceptable caticnic salts, e.g. the sodium or potassium salts as well as salts with amine, e.g. trimethylamine. Also preferred are ester derivatives of amino acids that provide water soluble, pharmaceutical^ acceptable acic addition salts with minerai or organic acids, e.g. the hydrochloric, or sulfuric acid, or succinic acid addition salts. As used herein the term "pharmaceutically acceptable salts" thus includes salts wherein the acidic hydrogen in the dibasic hydrocarboncarboxylate esters of this invention is replaced with a cation (e.g. sodium D-(threo)-l-p-nitrophenyl-2-dichloroacetamido-3-fluoro-1 -propyl hemisuccinate) as well as salts wherein the acidic hydrogen forms an acid addition salt with an amine (e.g. D-(threo)-1-p-nitrophenyl-2-dichloroacetamido-3-fluoro-1-propyl hemisuccinate N-trimethylamine salt). Also included are the acid addition salts formed between mineral or organic acids and the amine in the amino acid esters of the compounds of Formula ! (e.g. D-(threo)-1-p-nitrophenyl-2-dichloroacetamido-3-fIuoro-1-propyl giycinate hydrochloride). Among the pharmaceutically acceptable cationic salts of the dibasic hydrocarboncarboxylate esters included in Formula I are salts of alkaii and alkaline earth metais (e.g., sodium, potassium, calcium, aluminum) and salts with an amine such as triaikyiamines, procaine, dibenzylamine, N-benzyl-beta-phenethylamine, N,N'-dibenzyletnylenediamine. N-(lower:aikylpiperidines (e.g. N-strypiperidine), sod N-methyi c ucamine. Preferably R is a halogsnated derivative of methyl or ethyl, Z is a hydrogen, X is phenyl, CCRi or S02Ri, Ri is methyl, and X' is hydrogen. Mcs: preferably R is CHCI2orCHF2. A preferred antibiotic compound ;s florfenicol (D-(tnreo)-l-p-nethylsulfonyl phenyl-2-dicnlcroacetamido-3-fluoro-1-propanol). Another preferred antibiotic compound is D-(threo)-1-p-methylsulfonyl phenyl-2-difluoroacetanr.idc-3-fluoro-1-propanoi. Processes for the manufacture of these preferred antibiotic compounds, and intermediates useful in such processes, are described in U.S. Patent Nos. 4,311,857; 4,562,918; 4,973,750; 4,876,252; 5,227,494; 4,743,700: 5,567,844; 5,105,009; 5,382,673; 5,352.832; and 5.563,361. Formulation efforts on the combination product were directed at maintaining the desirable pharmacokinetic profile of florfenicol allowing for one-snot administration to cattle with BRD. A formulation containing 300 mg/mL of florfenicol and 16.5 mg/mL of flunixin, was developed and administered at a dose of 40 mg/kg of florfenicol, and a dose of 2.2 mg/kg of flunixin meglumine. The data presented demonstrated that the bioavailability of florfenicol is unaltered (see Figure 1). Moreover, the flunixin component acted in a clinically equivalent fashion to that of FINADYNE. In a clinical study in which that formulation was compared to the same formulation with twice as much flunixin and NUFLOR alone, the benefit of flunixin therapy was clearly evident. But no incremental benefit of a higher flunixin dose was apparent (see Figure 3). Clinical studies reconfirmed the weil-established benefits of flunixin in the treatment of bovine respiratory disease in terms of incremental improvement in clinical response, antipyretic response and decreased lung consolidation. Superiority to antibiotic use alone was evident particularly in the critical first 24 hours after diagnosis and treatment (see Figures 4, 5 and 6). In adc::;on re grearer cenvenience and ease of use, it is believed that a single daily s-': cutaneous administration of a combination product in accordance with the present invention will promote humane animal care by recucing the number of injections needed to treat animais and providing more rapid relief of disease symp:cms. By reducing the number of injections, manpower costs also may be significantly reduced. in the formulations of the present invention, the concentration of flunixin typically is from about 1 to about 10% by weight, with the preferred level being at from abcut 1.5% to about 3.5%, and an even more preferred level being at least about 1.65% by weight. The concentration of florfenicol or other antibiotic typically is from about 10% to about 50% w/v, with the preferred level between about 20% and about 40% w/v, even more preferred being at least about 30% w/v. The remaining portion of the formulations of the present invention is a pharmaceutical^ acceptable carrier comprising at least one solvent. The pharmaceutical^ acceptable carrier comprises from about 40% to about 80% of the formulation. Florfenicoi is generally soluble in aprotic polar solvents such as a pyrrolidone solvent, or NtN-dimethylacetamitie, N.N-dimethylformamide, DMSO, acetone or glycerol formal. Preferred pyrrolidone solvents are N-methyl-2-pyrrolidone and 2-pyrrolidone. Accordingly, such an aprotic polar solvent (or a combination of such solvents) is preferred for use in formulations of the present invention that contain florfenicol or similar antibiotics. Preferably such a solvent is present at about 5% to about 80% by weight of the formuiation. More preferably such a solvent is present at about 10% to about 35% of the formulation. Other pharmaceutical^ acceptable solvents may be present in the formulations of the present invention. Suitable solvents include water, ethanol, isopropanol, 1,2-propanediol, glycerin, benzyl alcohol, dimethyiisosorbide, triacetin, glycol ethers, propylene glycol and polyethylene glycol (PEG). Particularly preferred solvents induce PEG having an average molecular weicr.: ce?*veen about 200 and abcu; 400, triacetin. cimethyiiscsorbice, ethanoi, and water, and combinations thereof. These solvents may comprise from 0% :o areut 75% of the formulaticn. Preferably they comprise from about 15% to about 5C%. More preferably they comprise from about 40% to about 55% of the formulation. The addition of one or more of such additional solvents may be desirable :o reduce the viscosity of the formulation in order to provide a product with workable syringeabiiity. Examples of solvents particularly useful for adjusting the viscosity of the formulations of the present invention include water, ethanoi, isocropanol, propylene glycol, dimethylisosorbide and triacetin, and combinations thereof. Other inert ingredients can be added to the present composition, as desired. Such ingredients include preservatives, chelating agents, antioxidants and stabilizers. Exemplary preservatives include methyl p-hydroxybenzcate (methylparaben) and propyl p-hydroxybenzoate (propylparaben). Exemplary chelating agents include edetate sodium. Exemplary antioxidants include butylated hydroxyaniscle and sodium monothiogiycerol. In order to prevent degradation of any of the active ingredients in the formulations of the present invention, the addition of at least one stabilizer has been found to be advantageous. Citric acid is a preferred stabilizer. In order to prepare the composition of the present invention, the vehicle(s) or a portion of the vehicle(s), are added to the compounding vessel, followed by the remaining excipients and the actives. The mixture is mixed until all solids are dissolved. Additional solvent to bring the composition to final volume may be added if needed. Additives, such as those listed above, may also be included in the vessel and mixed into the formulation (the order of addition is not critical). The compositions according to the present invention will generally be administered to cattle at from about 1 mg to about 100 mg of the antibacterial per kilogram of body weight per day, and from about 0.5 mg to about 5 mg of flunixin meglumine oer kilogram of body weigh: per day. Preferably the compositions oi the present invention will be administered to bovines at from abcu: 20 rr,c to aoout 50 mg of the antibacterial per kilogram of body weight. More preferably the dose will be about 40 ma/kg of the antibacterial. Preferably the compositions of the present invention will be administerec at from about 1 mg to about 3 mg flunixin meglumine per kilogram of body weight. The compositions according to the present invention will generally be administered to swine at a dose of from 15 ma to about 100 mc of the antibacterial per kilogram of body weight per day, and from about 0.5 mg to about 5 mg of flunixin meglumine per kilogram of body weight per day. Preferably the compositions of the present invention will be administered to swine at from about 20 mg to about 50 mg of the antibacterial per kilogram of body weight and about 1 mg to about 2 mg of flunixin meglumine per kg of body weight. The compositions may be administered once daily or divided into multiple doses. Often only one dose will be sufficient to treat the infection. In some circumstances one dose followed by a second dose 48 hours later will be required to treat the animal. The precise dose will depend on the stage and severity of the infection, the susceptibility of the infecting organism to the composition, and the individual characteristics of the animal species being treated, as will be appreciated by one of ordinary skill in the art. The compositions according to the present invention are particularly useful for cattle and other bovids, swine, and other large mammals. In.addition to the treatment of bovine respiratory disease, the compositions of this invention are also suitable for the treatment of infectious diseases associated with inflammation such as swine respiratory disease, footrot, acute masliiis, pinkeye (infectious keratoconjunctivitis), acute pneumonia, metritis and enteritis. The dosage regimen for treatment of such diseases would be as described above. Mastitis is a complex disease thai occurs in lactating females, and is 01 particular economic importance in dairy cows and goats. Several orthogenic agents may be involved, including Staphylococcus aureus, E. coil, and Streptococcus. The acute form of mastitis has a sudden onset, tre udder is enlarged, hot to the touch and tender; usually the affected animal will have a fever. If net treated promptly, the udder may be permanently damaged arc milk production decreased or lost. Currently, acute mastitis is treated with antibiotics, antiinflammatories and oxytocin. The use of the formulations of the present invention would be an improvement over presently known methods of treating mastitis because it would combine one of the most effective of the standard treatment combinations into a single, conveniently administered formulation. Pinkeye is an acute infectious disease of cattle, sheep and ether animals that is characterized by inflammation of the tissues of the eye, characterized by inflammation of the tissues of the eye, accompanied by nasal discharge, lacrimation and copious ocular discharge. Affected animals may display extreme discomfort, resulting in a drop in miik production; in extreme cases permanent blindness occurs. The disease, which is caused by Moraxella bovis in cattle, is widespread, especially among range and feedlot cattle, and is of great economic importance to the cattle industry. Currently, pinkeye is treated by administration of various antibiotics. The use of the formulations of the present invention would be an improvement over presently known methods of treating pinkeye because it would provide good antibiotic therapy along with an NSAID to reduce the ocular inflammation. Footrot (interdigital phlegmon) is an acute infection of the interdigital space that occurs throughout the world in both zeei and dairy cattle. Fusobacterium necrophorum is the major cause of footrot, although other organisms, including Bacteraices me'aninogenicus, can be involved. : he maicr symptoms include pain, severe lameness, fever, anorexia, and recuced miik prcductior. Currently, footrct is treated by antibiotic therapy; recommenced therapy can involve treatment for up to .five days. The use of the formulations c: t.ne present inventicn for the treatment of footrot would be an improvement over presently known treatments because it would provide the proven efficacy of florfenicol (with fewer administrations), along with an NSAID to reduce the inflammation caused by footrot and make the animal feel better. The compositions of the present invention are also useful for the prevention of these diseases in animals at high risk of developing those diseases. For example, the presently-claimed compositions can be administered to cattle at high risk of developing bovine respiratory disease at the same dosages recommended for treatment of bovine respiratory disease. The present invention is more particularly described in the following examples which are intended as illustrative only since numerous modifications and variations therein will be apparent to those skilled in the art. EXAMPLES EXAMPLE 1 Florenicol/'-lunixin Combination Formulations The N-methyl-2-pyrrolidone, propylene glycol and approximately 90% of the PEG 300 required were charged to the compounding vessel and mixed well. The florfenicol and flunixin meglumine were added with mixing until all solids were dissolved. The volume was adjusted with the remaining PEG 300, EXAMPLE 2 Pharmacokinetics of Florfenicol in Combination Product Formulation 1A of Example 1 (SCH 529752) was assessed in a pilot pharmacokinetic study involving 12 cattle in which 6 received florfenicol (NUFLOR) subcutaneously and 6 received Formulation 1A subcutaneousiy. A dose of 40 mg/kg of florfenicol and a dose of 2.2 mg/kg of flunixin was used. The results are shown in Figure 1. This preliminary study provided good evidence that the pharmacokinetics of florfenicol, the formulation of the present invention, are very similar to that provided by the already-marketed product, NUFLOR. EXAMPLE 5 Pharmacokinetics of Fiunixin in Combination Product In a second study, the pharmacokinetic profile of the seccr.c active component in Formulation 1A, flunixin, v.;as examined. The pharmacokinetic profile of flunixin was evaluated following administration to cattle as the approved single entity flunixin meglumine product, FiNADYNE, and as the combination product Formulation 1 A. This study was conducted in four phases using six cattle (3 males, 3 femaies). All animals received FINADYNE intravenously (IV) in the first phase. This was followed, after a washout period, by the administration of FiNADYNE intramuscularly (IM) to 3 cows and FiNADYNE subcutaneously (SQ) to 3 cows. In the next phase the animals were crossed over and received FiNADYNE IM or SQ. In the final phase, all six cattle received the flunixin/florfenicol combination product (Formulation 1A). All animals received fiunixin at a dose of 2.2 mg/kg, regardless of the formulation or route of administration. Blood samples for determination of fiunixin concentration were collected at 0, 0.17,0.5,0.75, 1,2,3,4,6,3, 12 and 24 hours after dosing. The results are summarized in Table 1 below and in Ficure 2. At the final phase of the study, the 5 cattle were given Formulation 1A at a dose of 10 mL per 75 kg SQ (equivalent to 40 mg/kg of florfenicol and 2.2 mg/kg of flunixin). The kinetics of flunixin when administered as Formulation 1A are different from the kinetics of flunixin when administered as FINADYNE. As s^zh, the clinical dcse response of Formulation 1A were £s:eriained in a subsequent study (described beow). EXA.VPLE4 Clinical Response of Cattle to r!unixin in Combination Product As the study'of Example 3 demonstrated that the bioavailability of flunixin from the combination formulation (Formication 1A) was lower than the approved single-entity product, FINADYNE, a clinical study was undertaken to compare the clinical response of cattle to flunixin when administered at two different concentrations of flunixin within combination formulations (Formulation 1A and Formulation 13). Ninety-six (96) head of cattle demonstrating classic symptoms of bovine respiratory disease were randomly allocated to treatment with one of the following three treatments at a dose of 20 mL/150 i Tacle 2 All three treatments were administered subcutaneously once at 40 mg/kg BW of florfenicol (20 mL/150 kg BW). Formulation 1A co-delivered flunixin at a dose of 2.2 mg/kg BW. Formulation 1B co-delivered flunixin at a dose of 4.4 mg/kg BW. Rectal temperature was assessed Et various times throughout the study. Results are shown in Figure 3. The antipyretic response was similar in both treatment groups receiving combination rormulations and superior to NUFLOR at all timepoints. i: was concluded that :here was no incremental benefit :c increasinc the concentration of flunixin with in the combination formulation. EXAMPLE 5 Dose Confirmation Study for Flunixin The optimal dose of flunixin determined in the study of Example 4 was used in a cose confirmation study. Per this study, one-hundred seventy-five (175) beef feeder calves exhibiting clinical signs of acute BRDf were selected. Enrollment was limited to cattle demonstrating the following clinical signs: • Clinical illness index score of 2 (moderately ill) or 3 (severely iii) • At least two respiratory characteristics scored as abnormal (polypnea, dyspnea, coughing, mucopurulent nasal discharge) • Pyrexia >104.5°F Upon enrollment the cattle were randomly allocated to one of 3 treatment groups: Table 3 The objective of this study was to compare the treatment response to a combination formulation of the present invention relative to an approved positive control (FiNADYNE and NUFLOR, each given by their approved route of administration) and negative (NUFLOR aione) control. Efficacy was assessed by scoring of clinical symptoms, rectal temperature and total lung consoiidation. After enrollment and treatment en Day 0, the calves hac the.: -ectal temperatures taken at approximately 2, z anc 10 hours post-treatment. Calves were then clinically scored crce daily on Days 1, 2, 3 and 4; rectal temperatures were taken once daily after the animal's morning observation. Treatment success was assessed on Study Day 4. An anirr.ai was classified as a treatment success if the animal had a rectal temperature of £ ir3.5°F, a clinical illness index score Lastly, on Day 4 the calves were humanely euthanized for necropsy and evaluation of pneumonic lesions/lung consolidation. Percent lung consolidation was calculated based en visual and palpation scores of 0, 1, 5, 10, 23, 30, 40, 50, 60, 70, 80, 90 or 100% assigned to each of the eight lung lobes. Tne overall lung consolidation is then calculated using a standard formula (see Shaw, et a/., DISEASES OF SWINE, 8th Ed. 914 (ISU Press, 1999). Results from this study are summarized in Figures 4, 5 and 5. The addition of flunixin to treatment resulted in a more rapid decrease in rectal temperature (p The addition of flunixin to treatment resulted in numerically less lung consolidation (p=0.08 [Formulation 1A vs. NUFLOR], p=0.1 [NUFLOR + FINADYNE vs. NUFLOR]). The response is the same whether the flunixin is administered in a separate syringe and given IV (FINADYNE) or co-administered within a subcutaneous injection of Formulation 1A (see Figure 5). As shc.vn in Figure 6. the addition of flunixin numerically improved the rs:e of success (p=C.1 [Formulation 1A vs. NUFLOR], p=0.3 [NUFLOR - -INADYNE vs. NUFLOR1). EXAMPLE 6 Florfericol/Hunixin Ccnbinstion Formulations The NMP ordimethylacetamide, citric acid, propylene glycol, and approximately 90% of the PEG 300 or glycerol formal required for each formulation were charged to the compounding vessel and mixed well. The florfenicol and flunixin meglumine were added with mixing until all solids were dissolved. The volume was adjusted with the remaining PEG 300 or glycerol formal. EXAMPLE 7 Florfenicol/Flunixin Combination Formulations The Ni\;p and a portion of the polyethylene glycol 200 are charged to the compounding vessel. Rorfenicol, methyl p-hydroxybenzoate and prtrpyi p-hycroxybenzoate are added to :he vessel and mixed untii dissolved. Water is added, followed by the fiunixin meglumine, and mixed until soiic's zre dissolved. Q.S. to final volume with polyethylene glycol 200 if needed. Although certain presently preferred embodiments of the invention have been described herein, it will be apparent to those skilled in the art to which the invention pertains that variations and modifications of the described embodiments may be made without departing from the spirit and scope of the invention. Accordingly, ft is intended that the invention be limited only to the extent required by the appended claims and the applicable rules of lav/. WE CLAIM: 1. A composition for the treatment of microbial infection in an animal comprising flunixin or one of its pnarmaceutically acceptaoie salts and a compound of Formula I: FORMULA I wherein R is a member selected from the group consisting of methyl or ethyl or a halogenated derivative thereof, dihalogenodeuteriomethyl, t-haiogeno-1-deuterioethyi, 1,2-dihalogeno-1-deuterioe:hyl, azidomethyi and methyisulfonylmethyl; each of X and X' is a member independently selected from the group consisting of N02, SO2R1, SOR1f SRi, SONH2) S02NH2, SONHR1f SO2NHR1, CORi, ORi, Ri, CN, halogen, hydrogen, phenyl, and phenyl substituted by halogen, N02, Rif PO2R1, CONHR1f NHR1( NR^, CONRiR2, OCORi, or OR1f wherein each of Ri and R2 is a member independently selected from the group consisting of methyl, ethyl, n-propyl, isoprcpyl butyl, t-butyl, isobutyl and phenyl; and Z is hydrogen or an acyl group of a hydrocarboncarboxylic acid having up to 15 carbon atoms or an acyl group of an aminohydrocarboncarboxylic acid having up to 12 carbon atoms; and the pnarmaceutically acceptable salts of said acyl groups. The composition of Claim 1, wherein R is a halogenatec derivative of methyl or eth'.i. The composition of Claim 2, wherein R is CHCI2 or CHF:. The ccmpcsition of Claim 3, wherein Z is hydrogen, X is SC:Ri or phenyl, and X' is hydrogen. The composition of Claim 4, wherein Ri is methyi. The composition of Claim 1, further comprising an aproiic polar solvent, wherein the aprotic polar solvent is selected from the group consisting of a pyrrolicone solvent, N,iN-dimethyiacetamide, NtN-dimethylfcrmamide, DMSO, acetone and glycerol formal and combinations thereof. The composition of Claim 5, wherein the aprotic polar solvent is a pyrrolicone solvent and the pyrrciidone solvent is N-methyl-2-pyrrolidone or 2-pyrrolidone. The composition of Claim 5 or 7, wherein the aprotic polar solvent is present in an amount of about 5% to about 80%. The composition of Claim 6 comprising: a) from about 10% to about 50% w/v florfenicol; b) from about 1% to about 10% w/v flunixin meglumine; c) from about 25% to about 60% of the aprotic solvent. The composition of Claim 6, further comprising a second solvent, wherein the second solvent is selected from the group consisting of water, propylene glycol, polyethylene glycol, triacetin, polyethylene glycol having an average molecular weight between 200 and 400, dimethyl-isosorbide, ethanol, iscprocanol, glycerin, 1,2-propanediol, glycol ethers, benzyl alcohol, and comcinations thereof. The composition cf Claim 6. further comprising a stabilizer, wherein the stabilizer is citric acid. A method of treating a disease selected from the group consisting of bovine respiraiory disease, swine respira:on/ disease, footrot, acute mastitis, pinkeye, metritis and enteritis in an animal comprising the step of subcutaneously administering to an animal in need of such treatment a therapeutically effective amount of the composition of Claim 1 or 6. A subcutaneous composition for the treatment of microbial infection in an animal comprising; a) about 300 mg/ml of florfenicol; b) about 16.5 mg/ml of flunixin or one of its pharmaceutical^ acceptable salts; and c) from about 5% to about 80% cf an aprotic polar solvent, wherein said subcutaneous composition exhibits with respect to flunixin a Cmax of about 1500 ng/mL, a Tmax of about 1 hour and an area under the curve of about 6 to about 7 when administered subcutaneously to bovids at a flunixin dose of about 2.2 mg/kg. The composition of Claim 13, wherein the aprotic polar solvent is selected from the group consisting of a pyrrclidone solvent, N,N-dimethylacetamide, N,N-dimethylformamide, DMSO, acetone and glycerol formal and combinations thereof. The composition of Claim 14, wherein the aprotic polar solvent is a pyrroiicone solvent and the pyrrolidone solvent is N-methy!-2-pyrolidone or 2-pyrro.idone. The composition of Claim 13, wherein the sprotic polar solvent is present in an amount of about 5% to about 50%. The conoosition of Claim 13 comcrisina: a) from about 10% to about 50% w/v florfenicol; b) from about 1% to about 10% w/v flunixin meglumine; c) from about 25% to about 60% of the aprotic solvent. The composition of Claim 13, further comprising a second solvent, wherein the seoond solvent is selected from the group consisting of water, propylene glycol, polyethylene glycol, triacetin, polyethylene glycol having an average molecular weight between 200 and 400, dimethyl-isosorbide, ethanol, isopropanol, glycerin, 1,2-propaneciol, glycol ethers, benzyl alcohol, and combinations thereof. The composition of Claim 13, further comprising a stabilizer, wherein the stabilizer is citric acid. A method of treating a disease selected from the group consisting of bovine respiratory disease, swine respiratory disease, footrot, acute mastitis, pinkeye, metritis and enteritis in an animal comprising the step of subcutaneously administering to an animal in need of such treatment a therapeutically effective amount of the composition of Claim 13. The method of Claim 12 or 20, wherein treatment results in reduction in a reduction in rectal temperature for the animal. The method of Claim 21, wherein the reduction in rectal temperature is a reduction of at least 3°F durina the first 24 hours of treatment. The method of Claim 22, wherein the reduction in rectal temperature is a reduction of at least 3CF during the first 6 hours of treatment. The method of Claim 12 or 20, wherein treatment results in a reduction in lung consolidation score for the animal, and 42 wherein the reduction in lung consolidation score is to a level of about 14. A subcutaneous comoosition for the treatment of microbial infection in an animal comprising: about 300 mg/ml of florfenicol; about 16.5 mg/mi of flunixin or one of its pharmaceutical^ acceptable salts; from about 5% to about 80% of an aprotic polar solvent, and citric acid; wherein said subcutaneous composition exhibits with respect to flunixin a C max of about 1500 ng/mL, a Tmax of about 1 hour and an area under the curve of about 6 to about 7 when administered subcutaneously to bovids at a flunixin dose of about 2.2 mg/kg. The composition of Claim 25, wherein the aprotic polar solvent is selected from the group consisting of a pyrroiidone solvent, N,N-dimethylacetamide, N,N-dimethylformamide, DMSO: acetone and glycerol formal and combinations thereof. The composition of Claim 26, wherein the aprotic polar solvent is a pyrolicone solver.: and the pyrroiicone solvent is N-meihyl-I-pyrrolidcne or 2-pyrrziidone. The composition of Claim 25 comprising: d) from about 10% to about 50% v//v florfenicol; e) from about 1% to about 10% w/v flunixin meglumine; f) from about 25% to about 60% of the aprotic solvent. The composition of Claim 25, further comprising a second solvent, wherein the second solvent is selected from the group consisting of water, propylene glycol, polyethylene glycol, triacetin, polyethylene glycol having an average molecular weight between 200 and 400, dimethyl-isosorbide. ethanol, isopropanoi, glycerin, 1,2-propanediol, glycol ethers, benzyl alcohol, and combinations thereof. A method of treating a disease selected from the group consisting of bovine respiratory disease, swine respiratory disease, footrot, acute mastitis, pinkeye, metritis and enteritis in an animal comprising the step of subcutaneously administering to an animal in need of such treatment a therapeutically effective amount of the composition of Claim 25. 31. A composition for the treatment of microbial infection in an animal substantially as herein described with reference to the accompanying drawings.

Full Text

COMPOSITIONS AND METHOD FOR TREATING INFECTION
IN CATTLE AND SWINE
This application claims fcenefi: of priority to U.S. Provisional Patent Application 60/382,015. riled May 20, 2302.
The invention relates to compositions and methods fortne trea;ment of infections in animals. More particularly, the invention relates to a composition containing both an antibiotic and a non-steroidal anti-inflammatory drug for use in the treatment of infections in animals such as cattle and swine.
Ail references cited herein are hereby incorporated in their entirety by reference.
Bovine respiratory disease (BRD) occurs in both dairy and beef cattle and is one of the leading causes of economic loss to the cattle industry throughout the world. These economic losses are due to excessive mortality, reducec weight gains as weil as treatment and prevention costs. BRD is often referred to as the "bovine respiratory diseases complex" cue to the multifactorial ettoicgy.
The cost of death losses due to respiratory diseases vary around the world. Death losses in the U.S. are estimated to approach $1 billion annually. Losses in various European countries range from S75 to S120 million. Cattle with clinical or sub-clinical BRD do not gain weight or produce milk as well as healthy animals. Beef cattle with BRD gain less weight, have reduced feed efficiency and often produce a lower grade carcass at slaughter. Perino LJM Apley MM Bovine Respiratory Disease, in CURRENT VETERINARY THERAPY 4 (FOOD ANIMAL PRACTICE), 4TH ED. 446-455 (Howard J.L, Smith RA, eds., 1999). A direct correlation between pulmonary lesions observed at slaughter and reduced weight gains has been established in cattle with sub-clinical infections. Whittem T.E. at a/., J. Am. Vet Med. Assoc, 209:814-818 (1996).
In addition to the production losses associated with mortality and morbidity, significant costs are associated with the treatment of BRD due to the costs of

various therapeutic agents and the labor required to administer these agents, along with the ex:ra labor to isolate and observe these animals.
The pathogenesis cf BRD is thought to te due to the interaction of environmental and physiological stresses coupled with infectious agents. Mannheimia (Pasteurella) haemolyiica, Pasieurella multocida and Haemophilus somnus are considered part of the normal flora of the bovine upper respiratory tract. When environmental and physiological stress factors reduce the natural resistance and inhibit the pulmonary defense mechanisms these organisms proliferate and colonize the lower respiratory tract. In addition, various bovine viruses such as infectious bovine rhinotracheitis virus (ISRV), bovine viral diarrhea virus (BVDV), bovine respiratory syncytial virus (BRSV) and parainfluenza 3 virus (PI-3) are known to have immunosuppressive effects in the lung.
Similarly, swine respirator/ disease (SRD) also has a multifactorial etiology. Bacterial infections caused by P. multocida, H. parasuis, Bordetella bronchiseptica, Actinobacillus pleuropneumonias, Streptococcus suis, Salmonella cholerasuis and Mycoplasma sp. can result in respiratory disease in swine, resulting in significant economic losses. Stresses such as crowding, mixing and moving of pigs and transient viral infections can contribute to the intensification of the disease.
These organisms can stimulate an excessive inflammatory process in the lungs by producing various toxins that stimulate the release of various cytokines, which up-regulate the inflammatory process. M. haemolytica, considered the most virulent of these organisms, also produces a leukotoxin that inhibits phagocytosis by leukocytes thus further enhancing its ability to colonize the lower respiratory tract This process often results in a bacterial bronchopneumonia.
The pulmonary damage that resuits in death or morbidity is due to the excessive inflammatory response to the invading pathogens. Damage to host tissues occur as neutrophils, pulmonary alveolar macrophages and natural killer cells destroy infected ceils. As cell membranes are damaged, arachidonic acid is

released. Arachidonic acid is the substrate for the formation of various prostaglandins and other eiccsanoids. The release of these biological active substances is critical to driving the inflammatory response that results in pulmonary lesions. Mcsier DA, Vet Clin. North An. Food Animal Prac, 13:-S3-493 (19G7).
In general, therapy for BRD shouic be directed at achieving the following aoals:
1. Controlling the infection - In animals where the infectious process is halted early, the need for repeat treatment is significantly reduced (see Apley IV!.D. & Fajt V.R., Vet Clin. North Am. Food Anim. Prac, 14:291-313 (1998). The selection of the appropriate antimicrobial compound should be based on the antimicrobial sensitivity of the organism involved, the levels of the antimicrobial agent in the respirator/ tract, ease of acministration, the potential for injection site tissue damage and a dosing regime that minimizes the pain and stress associated with treatment.
2. Minimize the pulmonary damage - As the level of inflammation and subsequent pulmonary damage increases, the probability of repeat therapy increases and the rate of weight gain decreases. Lekeux P., Bovine Practitioner, 29:71-75 (1995); Scott P.R., J. Dairy ScL, 76(2):414-420 (1993).
3. Reduce pyrexia - Controlling the infection and reducing the inflammation will reduce the pyrexia (fever) thus increasing the potential for recovery. The feeling of well-being that accompanies the reduction of pyrexia may also improve the intake of nutrients by suppressing inappetence associated with disease and pyrexia.
For years antimicrobial therapy has been the mainstay of BRD therapy. There are many effective microbial agents currently available for the treatment of BRD. NUFLOR, an injectable formulation of the broad spectrum antibiotic florfenicol, has emerged as one of the leading antibiotics on a global basis. It is indicated for the treatment and control cf BRD associated with M. hasmolytica, P.

muitocida and H. somnus as well as for the prevention of respiratory cisease in cattie at high risk of developing BRD associated with these bacteria. NUFLOR is also indicated for the treatment of bovine interdigitai phlegmon (footrot. acute interdigital necobacillosis, infectious pctiodermatitis) associated with Fuscbacierium necrophorum and Bacieroides melsninogenicus. NUFLOR may be administered sjbcutaneously as well as intramuscularly.
The pathogenesis of BRD involves the development of a significant inflammatory process in the lungs and the subsequent development of pulmonary lesions, often leading to pulmonary consolidation. The degree of this inflammatory process can determine whether the disease results in mortality, a chronic "poor doer' or the animal recovers uneventfully. Various anti-inflammatory agents have been investigated regarding their ability to reduce the pyrexia, lung consolidation and weight loss associated with BRD.
The use of corticosteroids is generally contraindicated as ancillary therapy for BRD due to their ability to cause serious immunosuppression. The use of nonsteroidal anti-inflammatory drugs (NSAIDs) in conjunction with antibiotics, however, has been shown to be of benefit in the treatment of bovine respiratory disease. The nonsteroidal, anti-inflammatory agent flunixin meglumine has been demonstrated to be effective in rapidly reducing pyrexia associated with BRD. Fiunixin has also been demonstrated to reduce pulmonary consolidation and the need for re-treatment with antibiotics.
Fiunixin meglumine is the active ingredient in FINADYNE and BANAMINE (both available from Schering-Plough Animal Health Corporation, Union, NJ). It has emerged as one of the leading NSAIDs for adjunctive therapy of BRD.
Fiunixin meglumine has been studied extensively in regard to its use in conjunction with antibiotics for the treatment of BRD. While it is widely used for this indication, it has not been used in combination in the same formulation with florfenicol because the primary route of administration of fiunixin is intravenous and florfenicol is administered intramuscularly or subcutaneously. Moreover, florfenicol

formulations have been designed to provide prolonged biood leveis of the antibiotic and fiunixin would not be expeoiec to have adequate oicavaiiabiiir/ in such formulations. In addition, there have beer, concerns as to whether ficrfsnicol and fiunixin would be compatible in such formulations.
Fiunixin meglumine has been used in conjunction with oxyretracyciine, and products containing born fiunixin mealumine and cxvtetracvcline are commercially available in Europe. However, such combination products require once per day administration for 3 to 5 days. Furthermore, resistance to the antibiotic oxytetracycline has become commonplace in regard to bacterial pathogens, including those commonly associated with BRD.
Accordingly, there is a need for conveniently administered, stable compositions that can control and prevent the infection and minimize the inflammation associated with bovine respiratory disease and other infectious diseases, while minimizing the pain and stress to the animal associated with treatment and the potential for injection site tissue damage.
Summary of the Invention
The present invention fulfills this need by providing improved compositions and methods for the treatment of bovine respiratory disease and other infections of cattle and swine.
The present invention relates to a composition for the treatment of microbial infection in an animal comprising fiunixin or one of its pharmaceutical^ acceptable salts and a compound of Formula I:

F I
o YH2 °Z
li I :
R—CHNT--C C-^n
V
A
V
x
wherein R is a member selected from the group consisting of methyl or ethyl or a hatocenated derivative thereof, dihalocenodeuteriomethvl, 1-halcceno-1-deuterioethyl, 1,2-dihalogeno-1-deuterioethyl, azidomethyl and methylsulfonylmethyi;
each of X and X' is a member independently selected from the group consisting of N02) SO2R1, SOR^ SR1( SONH2, S02NH2l SONHR1r S02NHRi, CORi, OR1, Ri, CNf halogen, hydrogen, phenyl, and phenyl substituted by halogen, N02, Ri, P02R1f CONHRi, NHR1f NRiR2, CONRiR2, OCORi, or ORi, wherein each of R-\ and R2 is a member independently selected from the group consisting of methyl, ethyl, n-propyl, isopropyl, butyl, t-butyl, isobutyl and phenyl;
and Z is hydrogen or an acyl group of a hydrocarboncarboxylic acid having up to 16 carbon atoms or an acyl group of an aminohydrocarboncarboxylic acid having up to 12 carbon atoms; and the pharmaceutical^ acceptable salts of said acyl groups.
in a preferred embodiment, the composition for the treatment of microbial infection in an animal comprises (a) florfenicol; (b) fiunixin or one of its
pharmaceutical^ acceptable salts; and (c) from about 5% to about 80% of an aprotic polar solvent.

' The present invention also relates to a method of Treating a microbial
infection in an animal comprising the s;ep of subcutaneousiy sdm.nisrering to an animal in need cf such treatment a therapeutically effective amount or" a composition comprising flunixin or one of i:s pharmaceutical^ accepiaoie salts and a compound of Formula i.
In a preferred embodiment, the microbial infection is seiecied from the group consisting of bovine respiratory disease, swine respiratory disease, footrot, acu:e mastitis, pinkeye, metritis and enteritis.
The present invention also relates to a method of preventing a microbial infection in an animal susceptible to such an infection comprising the step of subcutaneously administering to an animal susceptible to such an infection a prophylactic amount of a composition comprising flunixin or one cf its pharmaceutical acceptable salts and a compound of Formula I.
In a preferred embodiment, the microbial infection is bovine respiratory disease.
Brief Description of the Drawings
Figure 1 is a graph showing the mean plasma concentration (+/- SD) of florfenicol vs. time after a single subcutaneous injection of NUFLOR (florfenicol) or a composition of the present invention containing both florfenicol and flunixin meglumine.
Figure 2 is a graph showing the mean plasma concentration of flunixin vs. time after parenteral administration of 3ANAMINE/FINADYNE flunixin meglumine
or a flarfenicoi-flunixin meglumine combination composition of the present invention.
Figure 3 is a graph showing the effect of flunixin within a florfenicol-fiunixin meglumine combination composition of the present invention.

Figure 4 is a graph shewing the effect of treatment with a 'Icrfenicoi-flur.ixin meglumine cemfcinatien composition of the present invention on antipyretic response in cattle with ERD.
Figure 5 is a graph snowing the effect of treatment with a flcrfenicol-flur.ixin meglumine combination composition of the present invention on lung consolidation score.
Figure 6 is a graph showing the effect of treatment with a florfenicol-flunixin meglumine combination composition of the present invention on the ciinical success rate.
Detailed Description of the Invention
When flunixin is administered subcutaneously in a combination formulation with an antibiotic, its bioavailability is significantly reduced. One would expect that this would result in less clinical efficacy when administered in this manner compared to simultaneous administration by the intravenous route.
It has surprisingly been found, however, that, despite the reduced bioavailability of subcutaneously administered flunixin meglumine, the same dose of flunixin meglumine when administered in certain formulations containing a fiuorinated chloramphenicol orthiamphenicol analog/derivative antibiotic, is as clinically efficacious as the same amount of flunixin meglumine administered intravenously in conjunction with a separate subcutaneous injection of the antibiotic.
As used herein, the following terms, unless otherwise indicated, shall be understood to have the following meanings:
"Acyl" means an H-C(O)-, alkyl-C(O)-, alkenyl-C(O)-, alkynyl-C(O)-, cycioalkyl-C(O)-, cycioalkenyi-C(O)-, or cycloalkynyl-C(O)- group in which the various groups are as previously described. The bond to the parent moiety is through the carbonyl. Preferred acyls contain a lower afkyl. Non-limiting examples

of suitable acyi groups induce formyi, acetyl, prcpanoyl, 2-methyicrcpanoyl. butanoyi and cyciohexancyl.
"Alky1" means an aiiphaiic hydrccaroon group, which may be straight or branched, comprising from 1 to about 20 carbon aioms in the chain. Preferred alkyi groups contain from 1 to about 12 carbon atoms in the chain. More preferred alkyi groups contain from 1 to about 5 carbon atoms in the chain. Branched means that one or more lower alkyi groups, such as methyl, ethyi or propyl, are attached to a linear aikyl chain. "Lower alkyi" means a group having from 1 to about 6 carbon atoms in the chain, which may be straight or branched. The term "substituted aikyl" means that the aikyl group may be substituted by one or more substituents which may be the same or different.
"Aryl" means an aromatic monocyclic or multicyclic ring system comprising about 6 to about 14 carbon atoms, preferably about 6 to about 10 carbon atoms. The aryl group can be optionally substituted with one or more "ring system substituents/ which may be the same or different, and are as defined herein.
" Alkoxy" means an aikyl-O- group in which the aikyl group is as previously described. Non-limiting examples of suitable alkoxy groups include methoxy, ethoxy, isopropoxy, and n-butoxy. The bond to the parent moiety is through the ether oxygen.
"Azido" refers to an -N3 group.
"Halo" and "halogeno" mean fluoro, chforo, bromo, or iodo groups. Preferred are fluoro, chloro or bromo, and more preferred are fluoro and chloro.
"Halogen" means fluorine, chlorine, bromine, or iodine. Preferred are fluorine, chlorine or bromine, and more preferred are fluorine and chlorine.
"Haloalkyi" and "halogenoalkyi" mean an aikyl group as denned above wherein one or more hydrogen atoms on the alkyi is replaced by a halo group defined above.

"Ring system substituer.t" means a substituent attached tc ar aromatic or non-aromatic ring system which, for example, replaces an availacie hycrogen en the ring system. Ring system substituents may be the same or different.
The term "optionally substituted" means optional substitution with the specified croups, radicals or moieties.
As used herein, the term "composition" is intended to encompass a product comprising the specified ingredients in the specified amounts, as weil as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts.
An "effective amount" is the dose required to alleviate a particular symptom of an infection or disease or to protect an animal against infections or disease.
As used herein, the term "bovine" refers to animals of the genus Bos, such as cattle. The term "bovid" refers to animals in the family Bovidae, which includes hoofed, hollow-homed ruminants such as cattle, sheep, goats, buffaloes, oxen, etc. As used herein, the term "swine" refers to animals of the family Suidae, which includes pigs, boars, warthogs, etc.
Flunixin meglumine is currently approved globally for use in the treatment of BRD. It has become a mainstay of veterinary practice for the treatment of inflammatory conditions. As the veterinarian often administers NSAID therapy with an antibiotic, development of a combination flunixin-antibiotic injectable product would be desirable. Flunixin meglumine is commercially available from, e.g., ISP (Wayne, NJ), or may be made according to methods known in the art, e.g., the methods described in U.S. Patent Nos. 3,337,570, 3,478,040 and 3,339,344.
Fluorine-containing analogs of antibiotics chloramphenicol and thiamphenicol have been shown to have antibiotic activity, both against organisms sensitive to and resistant to chloramphenicol and thiamphenicol. See Schafer, T.W. et a/., "Novel Fluorine-Containing Analogs of Chloramphenicol and

Thiampnenicoi: Antibacterial and Biological Properties," in CURRENT
CHEMOTHERAPY AND INFECTIOUS DISEASE PROCEEDINGS OF Tr£ ': 1~~ !CC AND THE 19"*
ICAAC AMER.'CAN SOCIETY OF M.CROSICLCGY I960, 444-446. Exanoies of such compounds, and methods for their manufacture, are described arc claimed in U.S. Patent No. 4.235,892. The medical profession has become increasingly concerned about the transference of bacierial resistance to humans when antibiotics useful in treating humans are administered to livestock. Because the chloramphenicol group of antibiotics is infrequently usee now to treat humans, its derivatives are particularly appropriate for veterinary use. Of particular interest are the 3-fluorc, 3-decxy derivatives.
Tne invention provides novel compositions for the treatment of infectious diseases such as bovine respiratory disease in livestock. These compositions are formulations comprising flunixin in combination with certain chloramphenicol derivatives. Initial testing of these formulations demonstrated relatively low serum levels of flunixin in cattle following administration of a single dose. In subsequent clinical trials, the formulations of the present invention demonstrated an unexpected high degree of efficacy when compared to a negative control group as well as a group receiving florfenicol and flunixin concurrently but in separate formulations. The compositions of the present inventicn comprise flunixin meglumine and at least one antibiotic of Formula I:
FORMULA I
F I
0 ?H2 ?Z
II I :
R— CHN----C C-^H
—X1

wherein R is a member selected from the group consisting of methyl c erhyl or a halogenated derivative thereof, dihalogencceuteriomethyl. 1-nalocer.c-i-deuterioethvi, 1.2-dihaloceno-1-deu:erioet.nvl, azidcmeihvl and methylsulfcnyirnethyf;
each cf X and XT is a member independently selected from the croup consisting cf \'02l S02R-r SOR1( SRi, SONH2l S02NH2, SONHRi, SC2NHR1. CORi, ORL Fv, CN, halogen, hydrogen, prenyl, and phenyl substituted by halogen, NO,, R1t OR1f P02R1t CONHRL NHRi, NRiR2. CONRiR2 or OCOR1f wherein each of R-i and R2is a member independently selected from the group consisting of methyl, ethyl, n-propyl, isopropyl, butyl, t-butyl, isobutyl and phenyl;
and 2 is hydrogen or an acyl group of a hydrocarboncarboxylic acid (preferably a hydrocarbondicarboxyiic acic} having up to 16 carbon atoms or an acyl group cf an amino- hydrocarboncarboxylic acid having up to 12 carbon atoms; and the pharmaceutical^ acceptable salts of said acyl groups.
Included among the halogenated groups contemplated for the moiety R in Formula I are the mono-, di- and tri-fiuoro, the mono-, di- and tri-chloro-, the mono-and di-bromo-, and the iodo-methyl groups as well as the mono- and di-fluoro-, the mono- and di-chloro-, the mono- and di-bromo-, and the iodo-ethyl groups wherein the halogen substituents are preferably on the carbon alpha to the carbonyl function. Also included are mixed dihalogenoalkyl groups in which both halogens are preferably bonded to the carbon alpha to the carbonyl groups, e.g., groups such as fluorochloro-, fiuorobromo-, and chlorobromo-methyl and -ethyl, as well as trihalogen-methyl groups such as dichlorofluoro- and difluorochloromethyL
Also included among the compounds of Formula I are the ester derivatives, e.g. 1-hydrocarboncarboxyiates of Formula I wherein Z is an acyl group of a hydrocarboncarboxylic acid having up to 15 carbon atoms that may be saturated, unsaturated, straight chain or branched chain, aliphatic, cyclic, cyciic-aiiphatic, aromatic, aryl-aliphatic, or alkyl-aromatic and may be substituted by hydroxy, alkoxy

containing irorr, 1 to 5 carbon aioms, carboxyl, NO2, NHRi, NP^RZ: SR1r SOR-,. cr halogen, wherein R1 and R: are as defined aoove.
Other ar.tibacterially active ester derivatives of Formula i are these wherein Z is an acyl croup of an amino acid containing up to 12 carbon atcms that may be saturated, unsaturated, straight chain, branched chain or cyclic, that may contain aromatic grccps and that may be substituted by hydroxy! groups.
Preferred ester derivatives induce those derived from dibasic hydrocarboncarboxylates, e.g. the 1-succinate and 1-palmitate esters, which provide water soluble, pharmaceutical^ acceptable caticnic salts, e.g. the sodium or potassium salts as well as salts with amine, e.g. trimethylamine. Also preferred are ester derivatives of amino acids that provide water soluble, pharmaceutical^ acceptable acic addition salts with minerai or organic acids, e.g. the hydrochloric, or sulfuric acid, or succinic acid addition salts.
As used herein the term "pharmaceutically acceptable salts" thus includes salts wherein the acidic hydrogen in the dibasic hydrocarboncarboxylate esters of this invention is replaced with a cation (e.g. sodium D-(threo)-l-p-nitrophenyl-2-dichloroacetamido-3-fluoro-1 -propyl hemisuccinate) as well as salts wherein the acidic hydrogen forms an acid addition salt with an amine (e.g. D-(threo)-1-p-nitrophenyl-2-dichloroacetamido-3-fluoro-1-propyl hemisuccinate N-trimethylamine salt). Also included are the acid addition salts formed between mineral or organic acids and the amine in the amino acid esters of the compounds of Formula ! (e.g. D-(threo)-1-p-nitrophenyl-2-dichloroacetamido-3-fIuoro-1-propyl giycinate hydrochloride).
Among the pharmaceutically acceptable cationic salts of the dibasic hydrocarboncarboxylate esters included in Formula I are salts of alkaii and alkaline earth metais (e.g., sodium, potassium, calcium, aluminum) and salts with an amine such as triaikyiamines, procaine, dibenzylamine, N-benzyl-beta-phenethylamine,

N,N'-dibenzyletnylenediamine. N-(lower:aikylpiperidines (e.g. N-strypiperidine), sod N-methyi c ucamine.
Preferably R is a halogsnated derivative of methyl or ethyl, Z is a hydrogen, X is phenyl, CCRi or S02Ri, Ri is methyl, and X' is hydrogen. Mcs: preferably R is CHCI2orCHF2.
A preferred antibiotic compound ;s florfenicol (D-(tnreo)-l-p-nethylsulfonyl phenyl-2-dicnlcroacetamido-3-fluoro-1-propanol). Another preferred antibiotic compound is D-(threo)-1-p-methylsulfonyl phenyl-2-difluoroacetanr.idc-3-fluoro-1-propanoi. Processes for the manufacture of these preferred antibiotic compounds, and intermediates useful in such processes, are described in U.S. Patent Nos. 4,311,857; 4,562,918; 4,973,750; 4,876,252; 5,227,494; 4,743,700: 5,567,844; 5,105,009; 5,382,673; 5,352.832; and 5.563,361.
Formulation efforts on the combination product were directed at maintaining the desirable pharmacokinetic profile of florfenicol allowing for one-snot administration to cattle with BRD. A formulation containing 300 mg/mL of florfenicol and 16.5 mg/mL of flunixin, was developed and administered at a dose of 40 mg/kg of florfenicol, and a dose of 2.2 mg/kg of flunixin meglumine. The data presented demonstrated that the bioavailability of florfenicol is unaltered (see Figure 1). Moreover, the flunixin component acted in a clinically equivalent fashion to that of FINADYNE. In a clinical study in which that formulation was compared to the same formulation with twice as much flunixin and NUFLOR alone, the benefit of flunixin therapy was clearly evident. But no incremental benefit of a higher flunixin dose was apparent (see Figure 3).
Clinical studies reconfirmed the weil-established benefits of flunixin in the treatment of bovine respiratory disease in terms of incremental improvement in clinical response, antipyretic response and decreased lung consolidation. Superiority to antibiotic use alone was evident particularly in the critical first 24 hours after diagnosis and treatment (see Figures 4, 5 and 6).

In adc::;on re grearer cenvenience and ease of use, it is believed that a single daily s-': cutaneous administration of a combination product in accordance with the present invention will promote humane animal care by recucing the number of injections needed to treat animais and providing more rapid relief of disease symp:cms. By reducing the number of injections, manpower costs also may be significantly reduced.
in the formulations of the present invention, the concentration of flunixin typically is from about 1 to about 10% by weight, with the preferred level being at from abcut 1.5% to about 3.5%, and an even more preferred level being at least about 1.65% by weight. The concentration of florfenicol or other antibiotic typically is from about 10% to about 50% w/v, with the preferred level between about 20% and about 40% w/v, even more preferred being at least about 30% w/v.
The remaining portion of the formulations of the present invention is a pharmaceutical^ acceptable carrier comprising at least one solvent. The pharmaceutical^ acceptable carrier comprises from about 40% to about 80% of the formulation.
Florfenicoi is generally soluble in aprotic polar solvents such as a pyrrolidone solvent, or NtN-dimethylacetamitie, N.N-dimethylformamide, DMSO, acetone or glycerol formal. Preferred pyrrolidone solvents are N-methyl-2-pyrrolidone and 2-pyrrolidone. Accordingly, such an aprotic polar solvent (or a combination of such solvents) is preferred for use in formulations of the present invention that contain florfenicol or similar antibiotics. Preferably such a solvent is present at about 5% to about 80% by weight of the formuiation. More preferably such a solvent is present at about 10% to about 35% of the formulation.
Other pharmaceutical^ acceptable solvents may be present in the formulations of the present invention. Suitable solvents include water, ethanol, isopropanol, 1,2-propanediol, glycerin, benzyl alcohol, dimethyiisosorbide, triacetin, glycol ethers, propylene glycol and polyethylene glycol (PEG). Particularly

preferred solvents induce PEG having an average molecular weicr.: ce?*veen about 200 and abcu; 400, triacetin. cimethyiiscsorbice, ethanoi, and water, and combinations thereof. These solvents may comprise from 0% :o areut 75% of the formulaticn. Preferably they comprise from about 15% to about 5C%. More preferably they comprise from about 40% to about 55% of the formulation.
The addition of one or more of such additional solvents may be desirable :o reduce the viscosity of the formulation in order to provide a product with workable syringeabiiity. Examples of solvents particularly useful for adjusting the viscosity of the formulations of the present invention include water, ethanoi, isocropanol, propylene glycol, dimethylisosorbide and triacetin, and combinations thereof.
Other inert ingredients can be added to the present composition, as desired. Such ingredients include preservatives, chelating agents, antioxidants and stabilizers. Exemplary preservatives include methyl p-hydroxybenzcate (methylparaben) and propyl p-hydroxybenzoate (propylparaben). Exemplary chelating agents include edetate sodium. Exemplary antioxidants include butylated hydroxyaniscle and sodium monothiogiycerol.
In order to prevent degradation of any of the active ingredients in the formulations of the present invention, the addition of at least one stabilizer has been found to be advantageous. Citric acid is a preferred stabilizer.
In order to prepare the composition of the present invention, the vehicle(s) or a portion of the vehicle(s), are added to the compounding vessel, followed by the remaining excipients and the actives. The mixture is mixed until all solids are dissolved. Additional solvent to bring the composition to final volume may be added if needed. Additives, such as those listed above, may also be included in the vessel and mixed into the formulation (the order of addition is not critical).
The compositions according to the present invention will generally be administered to cattle at from about 1 mg to about 100 mg of the antibacterial per kilogram of body weight per day, and from about 0.5 mg to about 5 mg of flunixin

meglumine oer kilogram of body weigh: per day. Preferably the compositions oi the present invention will be administered to bovines at from abcu: 20 rr,c to aoout 50 mg of the antibacterial per kilogram of body weight. More preferably the dose will be about 40 ma/kg of the antibacterial. Preferably the compositions of the present invention will be administerec at from about 1 mg to about 3 mg flunixin meglumine per kilogram of body weight.
The compositions according to the present invention will generally be administered to swine at a dose of from 15 ma to about 100 mc of the antibacterial per kilogram of body weight per day, and from about 0.5 mg to about 5 mg of flunixin meglumine per kilogram of body weight per day. Preferably the compositions of the present invention will be administered to swine at from about 20 mg to about 50 mg of the antibacterial per kilogram of body weight and about 1 mg to about 2 mg of flunixin meglumine per kg of body weight.
The compositions may be administered once daily or divided into multiple doses. Often only one dose will be sufficient to treat the infection. In some circumstances one dose followed by a second dose 48 hours later will be required to treat the animal. The precise dose will depend on the stage and severity of the infection, the susceptibility of the infecting organism to the composition, and the individual characteristics of the animal species being treated, as will be appreciated by one of ordinary skill in the art.
The compositions according to the present invention are particularly useful for cattle and other bovids, swine, and other large mammals. In.addition to the treatment of bovine respiratory disease, the compositions of this invention are also suitable for the treatment of infectious diseases associated with inflammation such as swine respiratory disease, footrot, acute masliiis, pinkeye (infectious keratoconjunctivitis), acute pneumonia, metritis and enteritis. The dosage regimen for treatment of such diseases would be as described above.

Mastitis is a complex disease thai occurs in lactating females, and is 01 particular economic importance in dairy cows and goats. Several orthogenic agents may be involved, including Staphylococcus aureus, E. coil, and Streptococcus. The acute form of mastitis has a sudden onset, tre udder is enlarged, hot to the touch and tender; usually the affected animal will have a fever. If net treated promptly, the udder may be permanently damaged arc milk production decreased or lost.
Currently, acute mastitis is treated with antibiotics, antiinflammatories and oxytocin. The use of the formulations of the present invention would be an improvement over presently known methods of treating mastitis because it would combine one of the most effective of the standard treatment combinations into a single, conveniently administered formulation.
Pinkeye is an acute infectious disease of cattle, sheep and ether animals that is characterized by inflammation of the tissues of the eye, characterized by inflammation of the tissues of the eye, accompanied by nasal discharge, lacrimation and copious ocular discharge. Affected animals may display extreme discomfort, resulting in a drop in miik production; in extreme cases permanent blindness occurs. The disease, which is caused by Moraxella bovis in cattle, is widespread, especially among range and feedlot cattle, and is of great economic importance to the cattle industry.
Currently, pinkeye is treated by administration of various antibiotics. The use of the formulations of the present invention would be an improvement over presently known methods of treating pinkeye because it would provide good antibiotic therapy along with an NSAID to reduce the ocular inflammation.
Footrot (interdigital phlegmon) is an acute infection of the interdigital space that occurs throughout the world in both zeei and dairy cattle. Fusobacterium necrophorum is the major cause of footrot, although other organisms, including

Bacteraices me'aninogenicus, can be involved. : he maicr symptoms include pain, severe lameness, fever, anorexia, and recuced miik prcductior.
Currently, footrct is treated by antibiotic therapy; recommenced therapy can involve treatment for up to .five days. The use of the formulations c: t.ne present inventicn for the treatment of footrot would be an improvement over presently known treatments because it would provide the proven efficacy of florfenicol (with fewer administrations), along with an NSAID to reduce the inflammation caused by footrot and make the animal feel better.
The compositions of the present invention are also useful for the prevention of these diseases in animals at high risk of developing those diseases. For example, the presently-claimed compositions can be administered to cattle at high risk of developing bovine respiratory disease at the same dosages recommended for treatment of bovine respiratory disease.
The present invention is more particularly described in the following examples which are intended as illustrative only since numerous modifications and variations therein will be apparent to those skilled in the art.

EXAMPLES
EXAMPLE 1 Florenicol/'-lunixin Combination Formulations

The N-methyl-2-pyrrolidone, propylene glycol and approximately 90% of the PEG 300 required were charged to the compounding vessel and mixed well. The florfenicol and flunixin meglumine were added with mixing until all solids were dissolved. The volume was adjusted with the remaining PEG 300,
EXAMPLE 2
Pharmacokinetics of Florfenicol in Combination Product
Formulation 1A of Example 1 (SCH 529752) was assessed in a pilot pharmacokinetic study involving 12 cattle in which 6 received florfenicol (NUFLOR) subcutaneously and 6 received Formulation 1A subcutaneousiy. A dose of 40 mg/kg of florfenicol and a dose of 2.2 mg/kg of flunixin was used. The results are shown in Figure 1. This preliminary study provided good evidence that the pharmacokinetics of florfenicol, the formulation of the present invention, are very similar to that provided by the already-marketed product, NUFLOR.

EXAMPLE 5 Pharmacokinetics of Fiunixin in Combination Product
In a second study, the pharmacokinetic profile of the seccr.c active component in Formulation 1A, flunixin, v.;as examined. The pharmacokinetic profile of flunixin was evaluated following administration to cattle as the approved single entity flunixin meglumine product, FiNADYNE, and as the combination product Formulation 1 A. This study was conducted in four phases using six cattle (3 males, 3 femaies). All animals received FINADYNE intravenously (IV) in the first phase. This was followed, after a washout period, by the administration of FiNADYNE intramuscularly (IM) to 3 cows and FiNADYNE subcutaneously (SQ) to 3 cows. In the next phase the animals were crossed over and received FiNADYNE IM or SQ. In the final phase, all six cattle received the flunixin/florfenicol combination product (Formulation 1A). All animals received fiunixin at a dose of 2.2 mg/kg, regardless of the formulation or route of administration.
Blood samples for determination of fiunixin concentration were collected at 0, 0.17,0.5,0.75, 1,2,3,4,6,3, 12 and 24 hours after dosing. The results are summarized in Table 1 below and in Ficure 2.

At the final phase of the study, the 5 cattle were given Formulation 1A at a dose of 10 mL per 75 kg SQ (equivalent to 40 mg/kg of florfenicol and 2.2 mg/kg of flunixin). The kinetics of flunixin when administered as Formulation 1A are different

from the kinetics of flunixin when administered as FINADYNE. As s^zh, the clinical dcse response of Formulation 1A were £s:eriained in a subsequent study (described beow).
EXA.VPLE4
Clinical Response of Cattle to r!unixin in Combination Product
As the study'of Example 3 demonstrated that the bioavailability of flunixin from the combination formulation (Formication 1A) was lower than the approved single-entity product, FINADYNE, a clinical study was undertaken to compare the clinical response of cattle to flunixin when administered at two different concentrations of flunixin within combination formulations (Formulation 1A and Formulation 13).
Ninety-six (96) head of cattle demonstrating classic symptoms of bovine respiratory disease were randomly allocated to treatment with one of the following three treatments at a dose of 20 mL/150 i Tacle 2

All three treatments were administered subcutaneously once at 40 mg/kg BW of florfenicol (20 mL/150 kg BW). Formulation 1A co-delivered flunixin at a dose of 2.2 mg/kg BW. Formulation 1B co-delivered flunixin at a dose of 4.4 mg/kg BW. Rectal temperature was assessed Et various times throughout the study. Results are shown in Figure 3. The antipyretic response was similar in both treatment groups receiving combination rormulations and superior to NUFLOR at all

timepoints. i: was concluded that :here was no incremental benefit :c increasinc the concentration of flunixin with in the combination formulation.
EXAMPLE 5 Dose Confirmation Study for Flunixin
The optimal dose of flunixin determined in the study of Example 4 was used in a cose confirmation study. Per this study, one-hundred seventy-five (175) beef feeder calves exhibiting clinical signs of acute BRDf were selected. Enrollment was limited to cattle demonstrating the following clinical signs:
• Clinical illness index score of 2 (moderately ill) or 3 (severely iii)
• At least two respiratory characteristics scored as abnormal (polypnea,
dyspnea, coughing, mucopurulent nasal discharge)
• Pyrexia >104.5°F
Upon enrollment the cattle were randomly allocated to one of 3 treatment groups:
Table 3

The objective of this study was to compare the treatment response to a combination formulation of the present invention relative to an approved positive control (FiNADYNE and NUFLOR, each given by their approved route of administration) and negative (NUFLOR aione) control. Efficacy was assessed by scoring of clinical symptoms, rectal temperature and total lung consoiidation.

After enrollment and treatment en Day 0, the calves hac the.: -ectal temperatures taken at approximately 2, z anc 10 hours post-treatment. Calves were then clinically scored crce daily on Days 1, 2, 3 and 4; rectal temperatures were taken once daily after the animal's morning observation.
Treatment success was assessed on Study Day 4. An anirr.ai was classified as a treatment success if the animal had a rectal temperature of £ ir3.5°F, a clinical illness index score Lastly, on Day 4 the calves were humanely euthanized for necropsy and evaluation of pneumonic lesions/lung consolidation. Percent lung consolidation was calculated based en visual and palpation scores of 0, 1, 5, 10, 23, 30, 40, 50, 60, 70, 80, 90 or 100% assigned to each of the eight lung lobes. Tne overall lung consolidation is then calculated using a standard formula (see Shaw, et a/., DISEASES OF SWINE, 8th Ed. 914 (ISU Press, 1999).
Results from this study are summarized in Figures 4, 5 and 5.
The addition of flunixin to treatment resulted in a more rapid decrease in rectal temperature (p The addition of flunixin to treatment resulted in numerically less lung consolidation (p=0.08 [Formulation 1A vs. NUFLOR], p=0.1 [NUFLOR + FINADYNE vs. NUFLOR]). The response is the same whether the flunixin is administered in a separate syringe and given IV (FINADYNE) or co-administered within a subcutaneous injection of Formulation 1A (see Figure 5).

As shc.vn in Figure 6. the addition of flunixin numerically improved the rs:e of success (p=C.1 [Formulation 1A vs. NUFLOR], p=0.3 [NUFLOR - -INADYNE vs. NUFLOR1).
EXAMPLE 6 Florfericol/Hunixin Ccnbinstion Formulations

The NMP ordimethylacetamide, citric acid, propylene glycol, and approximately 90% of the PEG 300 or glycerol formal required for each formulation were charged to the compounding vessel and mixed well. The florfenicol and flunixin meglumine were added with mixing until all solids were dissolved. The volume was adjusted with the remaining PEG 300 or glycerol formal.
EXAMPLE 7
Florfenicol/Flunixin Combination Formulations

The Ni\;p and a portion of the polyethylene glycol 200 are charged to the compounding vessel. Rorfenicol, methyl p-hydroxybenzoate and prtrpyi p-hycroxybenzoate are added to :he vessel and mixed untii dissolved. Water is added, followed by the fiunixin meglumine, and mixed until soiic's zre dissolved. Q.S. to final volume with polyethylene glycol 200 if needed.
Although certain presently preferred embodiments of the invention have been described herein, it will be apparent to those skilled in the art to which the invention pertains that variations and modifications of the described embodiments may be made without departing from the spirit and scope of the invention. Accordingly, ft is intended that the invention be limited only to the extent required by the appended claims and the applicable rules of lav/.

WE CLAIM:
1. A composition for the treatment of microbial infection in an animal
comprising flunixin or one of its pnarmaceutically acceptaoie salts and a compound of Formula I:
FORMULA I
wherein R is a member selected from the group consisting of methyl or ethyl or a halogenated derivative thereof, dihalogenodeuteriomethyl, t-haiogeno-1-deuterioethyi, 1,2-dihalogeno-1-deuterioe:hyl, azidomethyi and methyisulfonylmethyl;
each of X and X' is a member independently selected from the group consisting of N02, SO2R1, SOR1f SRi, SONH2) S02NH2, SONHR1f SO2NHR1, CORi, ORi, Ri, CN, halogen, hydrogen, phenyl, and phenyl substituted by halogen, N02, Rif PO2R1, CONHR1f NHR1( NR^, CONRiR2, OCORi, or OR1f wherein each of Ri and R2 is a member independently selected from the group consisting of methyl, ethyl, n-propyl, isoprcpyl butyl, t-butyl, isobutyl and phenyl;
and Z is hydrogen or an acyl group of a hydrocarboncarboxylic acid having up to 15 carbon atoms or an acyl group of an aminohydrocarboncarboxylic acid having up to 12 carbon atoms; and the pnarmaceutically acceptable salts of said acyl groups.

The composition of Claim 1, wherein R is a halogenatec derivative of methyl or eth'.i.
The composition of Claim 2, wherein R is CHCI2 or CHF:.
The ccmpcsition of Claim 3, wherein Z is hydrogen, X is SC:Ri or phenyl, and X' is hydrogen.
The composition of Claim 4, wherein Ri is methyi.
The composition of Claim 1, further comprising an aproiic polar solvent, wherein the aprotic polar solvent is selected from the group consisting of a pyrrolicone solvent, N,iN-dimethyiacetamide, NtN-dimethylfcrmamide, DMSO, acetone and glycerol formal and combinations thereof.
The composition of Claim 5, wherein the aprotic polar solvent is a pyrrolicone solvent and the pyrrciidone solvent is N-methyl-2-pyrrolidone or 2-pyrrolidone.
The composition of Claim 5 or 7, wherein the aprotic polar solvent is present in an amount of about 5% to about 80%.
The composition of Claim 6 comprising:
a) from about 10% to about 50% w/v florfenicol;
b) from about 1% to about 10% w/v flunixin meglumine;
c) from about 25% to about 60% of the aprotic solvent.
The composition of Claim 6, further comprising a second solvent, wherein the second solvent is selected from the group consisting of water, propylene glycol, polyethylene glycol, triacetin, polyethylene glycol having an average molecular weight between 200 and 400, dimethyl-isosorbide, ethanol,

iscprocanol, glycerin, 1,2-propanediol, glycol ethers, benzyl alcohol, and comcinations thereof.
The composition cf Claim 6. further comprising a stabilizer, wherein the stabilizer is citric acid.
A method of treating a disease selected from the group consisting of bovine respiraiory disease, swine respira:on/ disease, footrot, acute mastitis, pinkeye, metritis and enteritis in an animal comprising the step of subcutaneously administering to an animal in need of such treatment a therapeutically effective amount of the composition of Claim 1 or 6.
A subcutaneous composition for the treatment of microbial infection in an animal comprising;
a) about 300 mg/ml of florfenicol;
b) about 16.5 mg/ml of flunixin or one of its pharmaceutical^ acceptable salts; and
c) from about 5% to about 80% cf an aprotic polar solvent,
wherein said subcutaneous composition exhibits with respect to flunixin a Cmax of about 1500 ng/mL, a Tmax of about 1 hour and an area under the curve of about 6 to about 7 when administered subcutaneously to bovids at a flunixin dose of about 2.2 mg/kg.
The composition of Claim 13, wherein the aprotic polar solvent is selected from the group consisting of a pyrrclidone solvent, N,N-dimethylacetamide, N,N-dimethylformamide, DMSO, acetone and glycerol formal and combinations thereof.

The composition of Claim 14, wherein the aprotic polar solvent is a pyrroiicone solvent and the pyrrolidone solvent is N-methy!-2-pyrolidone or 2-pyrro.idone.
The composition of Claim 13, wherein the sprotic polar solvent is present in an amount of about 5% to about 50%.
The conoosition of Claim 13 comcrisina:
a) from about 10% to about 50% w/v florfenicol;
b) from about 1% to about 10% w/v flunixin meglumine;
c) from about 25% to about 60% of the aprotic solvent.
The composition of Claim 13, further comprising a second solvent, wherein the seoond solvent is selected from the group consisting of water, propylene glycol, polyethylene glycol, triacetin, polyethylene glycol having an average molecular weight between 200 and 400, dimethyl-isosorbide, ethanol, isopropanol, glycerin, 1,2-propaneciol, glycol ethers, benzyl alcohol, and combinations thereof.
The composition of Claim 13, further comprising a stabilizer, wherein the stabilizer is citric acid.
A method of treating a disease selected from the group consisting of bovine respiratory disease, swine respiratory disease, footrot, acute mastitis, pinkeye, metritis and enteritis in an animal comprising the step of subcutaneously administering to an animal in need of such treatment a therapeutically effective amount of the composition of Claim 13.

The method of Claim 12 or 20, wherein treatment results in reduction in a reduction in rectal temperature for the animal.
The method of Claim 21, wherein the reduction in rectal temperature is a reduction of at least 3°F durina the first 24 hours of treatment.
The method of Claim 22, wherein the reduction in rectal temperature is a reduction of at least 3CF during the first 6 hours of treatment.
The method of Claim 12 or 20, wherein treatment results in a reduction in lung consolidation score for the animal, and 42 wherein the reduction in lung consolidation score is to a level of about 14.
A subcutaneous comoosition for the treatment of microbial infection in an animal comprising:
about 300 mg/ml of florfenicol;
about 16.5 mg/mi of flunixin or one of its pharmaceutical^ acceptable salts;
from about 5% to about 80% of an aprotic polar solvent, and
citric acid;
wherein said subcutaneous composition exhibits with respect to flunixin a C max of about 1500 ng/mL, a Tmax of about 1 hour and an area under the curve of about 6 to about 7 when administered subcutaneously to bovids at a flunixin dose of about 2.2 mg/kg.
The composition of Claim 25, wherein the aprotic polar solvent is selected from the group consisting of a pyrroiidone solvent, N,N-dimethylacetamide, N,N-dimethylformamide, DMSO: acetone and glycerol formal and combinations thereof.

The composition of Claim 26, wherein the aprotic polar solvent is a pyrolicone solver.: and the pyrroiicone solvent is N-meihyl-I-pyrrolidcne or 2-pyrrziidone.
The composition of Claim 25 comprising:
d) from about 10% to about 50% v//v florfenicol;
e) from about 1% to about 10% w/v flunixin meglumine;
f) from about 25% to about 60% of the aprotic solvent.
The composition of Claim 25, further comprising a second solvent, wherein the second solvent is selected from the group consisting of water, propylene glycol, polyethylene glycol, triacetin, polyethylene glycol having an average molecular weight between 200 and 400, dimethyl-isosorbide. ethanol, isopropanoi, glycerin, 1,2-propanediol, glycol ethers, benzyl alcohol, and combinations thereof.
A method of treating a disease selected from the group consisting of bovine respiratory disease, swine respiratory disease, footrot, acute mastitis, pinkeye, metritis and enteritis in an animal comprising the step of subcutaneously administering to an animal in need of such treatment a therapeutically effective amount of the composition of Claim 25.

31. A composition for the treatment of microbial infection in an animal substantially as herein described with reference to the accompanying drawings.

Documents:

2604-chenp-2004 abstract-duplicate.pdf

2604-chenp-2004 abstract.pdf

2604-chenp-2004 claims-duplicate.pdf

2604-chenp-2004 description(complete)-duplicate.pdf

2604-chenp-2004 form-26.pdf

2604-chenp-2004 others.pdf

2604-chenp-2004 pct search report.pdf

2604-chenp-2004 petition.pdf

2604-chenp-2004-claims.pdf

2604-chenp-2004-correspondnece-others.pdf

2604-chenp-2004-correspondnece-po.pdf

2604-chenp-2004-description(complete).pdf

2604-chenp-2004-drawings.pdf

2604-chenp-2004-form 1.pdf

2604-chenp-2004-form 18.pdf

2604-chenp-2004-form 3.pdf

2604-chenp-2004-form 5.pdf

2604-chenp-2004-pct.pdf

« Previous Patent

Next Patent »

Patent Number

220040

Indian Patent Application Number

2604/CHENP/2004

PG Journal Number

30/2008

Publication Date

25-Jul-2008

Grant Date

15-May-2008

Date of Filing

19-Nov-2004

Name of Patentee

SCHERING-PLOUGH LTD

Applicant Address

Inventors:

#	Inventor's Name	Inventor's Address
1	VARMA, KANWAL, J
2	FREEHAUF, KEITH, A
3	HUQ, ABU
4	KOHAN, RAUL, E
5	SIMMONS, ROBERT, D

PCT International Classification Number

A61K 31/455

PCT International Application Number

PCT/IB03/02152

PCT International Filing date

2003-05-19

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	60/383.015	2002-05-20	U.S.A.