Methods of treating microbial infections, including mastitis

Abstract

The invention includes methods and compositions for treating mastitis in a subject. The method includes the step of administering, by intramammary delivery, a therapeutically effective amount of a polyether ionophore, or a therapeutically acceptable salt thereof, to the mammary gland of the subject.

Claims

1. A method of treating bacterial mastitis in a subject, the method consisting essentially of administering to a teat canal of the mammary gland of the subject a composition comprising a suspension of a solid dispersion of a therapeutically effective amount of a polyether ionophore selected from the group consisting of: narasin, salinomycin, lasalocid, monensin, semduramicin, maduramicin and laidlomycin, or a therapeutically acceptable salt thereof, as the sole antimicrobial agent, and a water-soluble polymer in an excipient or a gelling substance, wherein the bacterial mastitis is caused by a Gram-positive bacteria of the genus Staphylococcus or Streptococcus, or Mycoplasma bovis, and wherein the therapeutically effective amount is a dose in the range of 100 to 600 mg/teat canal.

2. The method according to claim 1, wherein the bacteria is selected from the group consisting of: Staphylococcus epidermidis, Staphylococcus simulans, Staphylococcus fells, Staphylococcus xylosus, Staphylococcus chromogenes, Staphylococcus warneri, Staphylococcus haemolyticus, Staphylococcus sciuri, Staphylococcus saprophyticus, Staphylococcus hominis, Staphylococcus caprae, Staphylococcus cohnii subsp. cohnii, Staphylococcus cohnii subsp. urealyticus, Staphylococcus capitis subsp. capitis, Staphylococcus capitis subsp. urealyticus, Staphylococcus hyicus, Staphylococcus aureus, Staphylococcus pseudintermedius, Staphylococcus delphini, Staphylococcus schleiferi subsp. coagulans, Staphylococcus aureus subsp. anaerobius, Streptococcus uberis, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus pyogenes, Streptococcus bovis, Streptococcus equi subsp. Zooepidemicus, Streptococcus equinus, and Mycoplasma bovis.

3. The method according to claim 2, wherein the bacteria is antibiotic resistant.

4. The method of claim 1, wherein the composition consists of the polyether ionophore, or the therapeutically acceptable salt thereof, the water-soluble polymer, and the excipient.

5. The method of claim 4, wherein the excipient is intended for rapid release.

6. The method of claim 1, wherein the composition consists of the polyether ionophore, or the therapeutically acceptable salt thereof, the water-soluble polymer, and the gelling substance.

7. The method of claim 1, wherein the gelling substance solidifies the composition or seals the teat canal.

8. A method of treating bacterial mastitis in a subject, the method comprising administering to a teat canal of the mammary gland of the subject a composition consisting of a suspension of a solid dispersion of a therapeutically effective amount of a polyether ionophore as the sole antimicrobial agent, wherein the polyether ionophore is selected from the group consisting of: narasin, salinomycin, lasalocid, monensin, semduramicin, maduramicin and laidlomycin, or a therapeutically acceptable salt thereof, and a water-soluble polymer in an excipient or gelling substance, wherein the bacterial mastitis is caused by a Gram-positive bacteria of the genus Staphylococcus or Streptococcus, or Mycoplasma bovis, and wherein the therapeutically effective amount is a dose in the range of 100 to 600 mg/teat canal.

9. The method of claim 8, wherein the therapeutically effective amount is a dose in the range of 150 to 600 mg/teat canal.

10. The method of claim 8, wherein the bacteria is selected from the group consisting of: Staphylococcus epidermidis, Staphylococcus simulans, Staphylococcus fells, Staphylococcus xylosus, Staphylococcus chromogenes, Staphylococcus warneri, Staphylococcus haemolyticus, Staphylococcus sciuri, Staphylococcus saprophyticus, Staphylococcus hominis, Staphylococcus caprae, Staphylococcus cohnii subsp. cohnii, Staphylococcus cohnii subsp. urealyticus, Staphylococcus capitis subsp. capitis, Staphylococcus capitis subsp. urealyticus, Staphylococcus hyicus, Staphylococcus aureus, Staphylococcus pseudintermedius, Staphylococcus delphini, Staphylococcus schleiferi subsp. coagulans, Staphylococcus aureus subsp. anaerobius, Streptococcus uberis, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus pyogenes, Streptococcus bovis, Streptococcus equi subsp. Zooepidemicus, Streptococcus equinus, and Mycoplasma bovis.

11. The method of claim 8, wherein the bacteria is antibiotic resistant.

12. The method of claim 8, wherein the excipient is intended for rapid release.

13. The method of claim 8, wherein the gelling substance solidifies the composition or seals the teat canal.

14. The method of claim 1, wherein the therapeutically effective amount is a dose in the range of 150 to 600 mg/teat canal.

15. The method of claim 1, wherein the water-soluble polymer is polyvinylpyrrolidone (PVP).

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) Further features of the present invention are more fully described in the following description of several non-limiting embodiments thereof. This description is included solely for the purposes of exemplifying the present invention. It should not be understood as a restriction on the broad summary, disclosure or description of the invention as set out above. The description will be made with reference to the accompanying drawings in which:

(2) FIG. 1 is a table setting out the isolate collection and the vertebrate species source following biochemical identification of the Staphylococcus species including resistance profile according to Example 1;

(3) FIG. 2 is a diagrammatic representation of the 96 well plate layout for the Minimum Inhibitory Concentration Testing according to Example 1;

(4) FIG. 3 is a diagrammatic representation of the Minimum Bactericidal Concentration test with shaded areas representing the placement of 10 μL volumes in duplicate and the direction of the arrow represents the increasing concentration as read clockwise around the plate according to Example 1;

(5) FIG. 4 shows a graph of the Minimum Inhibitory Concentrations for the individual isolates separated into methicillin-sensitive and methicillin-resistant strains; values shown as higher than 128 μg/mL represent strains where no MIC value was obtained within the range of concentrations tested (0.25-128 μg/mL) according to Example 1;

(6) FIG. 5 shows a table illustrating the MIC.sub.50, MIC.sub.90 and MIC ranges for the methicillin-sensitive isolates for ampicillin and the five test compounds according to Example 1;

(7) FIG. 6 shows a table illustrating the MIC.sub.50, MIC.sub.90 and MIC ranges for the methicillin-resistant isolates for ampicillin and the five test compounds according to Example 1;

(8) FIG. 7 shows a graph of the minimum bactericidal concentrations for individual isolates separated into methicillin-sensitive and methicillin-resistant strains; values shown as higher than 128 μg/mL represent strains where no MBC value was obtained within the range of concentrations tested (0.25-128 μg/mL) according to Example 1;

(9) FIG. 8 shows a table showing the MBC.sub.50, MBC.sub.90 and MBC ranges for the methicillin-sensitive isolates for the five test compounds according to Example 1;

(10) FIG. 9 shows a table illustrating the MBC.sub.50, MBC.sub.90 and MBC ranges for the methicillin-resistant isolates for the five test compounds according to Example 1;

(11) FIG. 10 shows a graph illustrating the optical density measurements obtained for the microdilution time-kill assay of ATCC 49775 over 48 hours using various concentrations of ampicillin, LP 1369 and LP 6315 compared to a growth curve according to Example 1;

(12) FIG. 11 shows a graph illustrating the optical density measurements obtained for the microdilution time-kill assay of MSS 1 over 48 hours using various concentrations of ampicillin, LP 1369 and LP 6315 compared to a growth curve according to Example 1;

(13) FIG. 12 shows a graph illustrating the optical density measurements obtained for microdilution time-kill assay of MSS 11 over 48 hours using various concentrations of ampicillin, LP 1369 and LP 6315 compared to a growth curve according to Example 1;

(14) FIG. 13 shows a graph illustrating the optical density measurements obtained for microdilution time-kill assay of MRSA over 48 hours using various concentrations of ampicillin, LP 1369 and LP 6315 compared to a growth curve according to Example 1;

(15) FIG. 14 shows a graph illustrating the number of viable colonies (log10) of ATCC 49775 over 24 hours compared to introduction to one, four and eight times the MIC of LP 1369, one and four times the MIC of ampicillin according to Example 1;

(16) FIG. 15 shows a graph illustrating the number of viable colonies (log10) of MRSA 9 over 24 hours compared to introduction to one, four and eight times the MIC of LP 1369, one and four times the MIC of ampicillin according to Example 1;

(17) FIG. 16 shows a table setting out the change in number of CFU/mL (log10) for ATCC 49775 and MRSA 9 over 24 hours in various concentrations of ampicillin or LP 1369 compared to a growth control according to Example 1;

(18) FIG. 17 shows a graph illustrating the number of viable colonies (log10) of ATCC 49775 over 24 hours compared to introduction to one, four and eight times the MIC of LP 6315, one and four times the MIC of ampicillin according to Example 1;

(19) FIG. 18 shows a graph illustrating the number of viable colonies (log10) of MRSA 9 over 24 hours compared to introduction to one, four and eight times the MIC of LP 6315, one and four times the MIC of ampicillin according to Example 1;

(20) FIG. 19 shows a table illustrating the change in number of CFU/mL (log10) for ATCC 49775 and MRSA 9 over 24 hours in various concentrations of ampicillin or LP 6315 compared to a growth control according to Example 1;

(21) FIG. 20 shows a graph illustrating the optical density readings obtained for the red blood cell toxicity assay for each test compound at various concentrations as well as positive and negative controls and blood only readings according to Example 1; and

(22) FIG. 21 is a table setting out the isolate collection and the dog breed source following biochemical identification of the Staphylococcus pseudintermedius isolates including resistance profile according to Example 2;

(23) FIG. 22 is a table setting out the resistance profile of the Staphylococcus pseudintermedius isolates collected according to Example 2;

(24) FIG. 23 is a table setting out the MIC profile of ampicillin and LP compounds of the Staphylococcus pseudintermedius isolates collected according to Example 2;

(25) FIG. 24 is a diagrammatic representation showing the 96 well microtitre tray layout for Minimum Inhibitory Concentration Testing according to Example 3;

(26) FIG. 25 is a table showing the MIC.sub.50, MIC.sub.90, MIC range and MBC.sub.50, MBC.sub.90, and MBC range of each compound tested according to Example 3;

(27) FIG. 26 is a table showing the MIC.sub.50, MIC.sub.90, MIC range and MBC.sub.50, MBC.sub.90, and MBC range of each compound tested against 14 Staphylococcus aureus isolates according to Example 3;

(28) FIG. 27 is a table showing the MIC.sub.50, MIC.sub.90, MIC range and MBC.sub.50, MBC.sub.90, and MBC range of each compound tested against six coagulase-negative Staphylococcus aureus isolates according to Example 3;

(29) FIG. 28 is a table showing the MIC.sub.50, MIC.sub.90, MIC range and MBC.sub.50, MBC.sub.90, and MBC range of each compound tested against 12 Staphylococcus agalactiae isolates according to Example 3;

(30) FIG. 29 is a table showing the MIC.sub.50, MIC.sub.90, MIC range and MBC.sub.50, MBC.sub.90, and MBC range of each compound tested against six Staphylococcus uberis isolates according to Example 3;

(31) FIG. 30 is a table showing the profiles of the bovine mastitis isolates according to Example 3;

(32) FIG. 31 is a table showing the MICs of individual bovine mastitis isolates according to Example 3;

(33) FIG. 32 is a table showing the MBCs of individual bovine mastitis isolates according to Example 3;

(34) FIG. 33 is a graph showing the weighted UCLs of the concentration of LP1369 in milk at 3 milkings for cows treated at a single occasion with microsized;

(35) FIG. 34 is a graph showing the weighted UCLs of the concentration of LP1369 in milk at 3 milkings for cows treated at a single occasion with nanosized;

(36) FIG. 35 is a graph showing the weighted UCLs of the concentration of LP1369 in milk at 3 milkings for cows treated at a single occasion with PVP;

(37) FIG. 36 is a table presenting the results of the microbial results of milk samples collected at pre-infection, post-infection and post-treatment as discussed in Examples 7 and 8;

(38) FIG. 37 is a graph showing weighted Upper Confidence Limit of the concentration of LP1369 in milk at 4 milkings for quarters treated on 6 occasions (consecutive milkings) with IVP1; and

(39) FIG. 38 is a graph showing Weighted UCLs of the concentration of LP1369 in milk at 4 milkings for non-treated quarters in cows treated in two other quarters on 6 occasions (consecutive milkings) with IVP2

DESCRIPTION OF EMBODIMENTS

(40) General

(41) Before describing the present invention in detail, it is to be understood that the invention is not limited to particular exemplified methods or compositions disclosed herein. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting.

(42) All publications referred to herein, including patents or patent applications, are incorporated by reference in their entirety. However, applications that are mentioned herein are referred to simply for the purpose of describing and disclosing the procedures, protocols, and reagents referred to in the publication which may have been used in connection with the invention. The citation of any publications referred to herein is not to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

(43) In addition, the carrying out of the present invention makes use of, unless otherwise indicated, conventional microbiological techniques within the skill of the art. Such conventional techniques are known to the skilled worker.

(44) As used herein, and in the appended claims, the singular forms “a”, “an”, and “the” include the plural unless the context clearly indicates otherwise.

(45) Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any materials and methods similar to, or equivalent to, those described herein may be used to carry out the present invention, the preferred materials and methods are herein described.

(46) The invention described herein may include one or more ranges of values (e.g. size, concentration, dose etc). A range of values will be understood to include all values within the range, including the values defining the range, and values adjacent to the range that lead to the same or substantially the same outcome as the values immediately adjacent to that value which define the boundary of the range.

(47) The phrase “therapeutically effective amount” as used herein refers to an amount sufficient to inhibit bacterial growth associated with bacterial carriage or mastitis. That is, reference to the administration of the therapeutically effective amount of polyether ionophores according to the methods or compositions of the invention refers to a therapeutic effect in which substantial bacteriocidal or bacteriostatic activity causes a substantial inhibition of mastitis. The term “therapeutically effective amount” as used herein, refers to a nontoxic but sufficient amount of the composition to provide the desired biological, therapeutic, and/or prophylactic result. The desired results include elimination of bacterial carriage or reduction and/or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. An effective amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation. In relation to a pharmaceutical or veterinary composition, effective amounts can be dosages that are recommended in the modulation of a diseased state or signs or symptoms thereof. Effective amounts differ depending on the veterinary composition used and the route of administration employed. Effective amounts are routinely optimized taking into consideration various factors of a particular patient, such as age, weight, gender, etc and the area affected by disease or disease causing microorganisms.

(48) As referred to herein, the terms “microbe” and “microbial” refers to a microscopic organism comprising either a single cell wall clusters of cells and encompasses, but is not limited to, prokaryotes such as bacteria and archaea; and forms of eukaryotes such as protozoan, fungi, algae. Preferably the terms “microbe” and “microbial” refers to prokaryotes and eukaryotes. The prokaryotes may refer to bacteria, such as Staphylococcus spp, Streptocccus spp, Bacillus spp, Enterococcus spp, Listeria spp, Mycoplasma spp, and anaerobic bacteria. The terms may refer to an antibiotic-sensitive strain or an antibiotic-resistant strain. In a preferred embodiment, the terms refer to MRSA. In another preferred embodiment, the terms refer to MRSP.

(49) In one embodiment, the terms “microbe” and “microbial” refer to one or more of coagulase-negative staphylococci (CNS): Staphylococcus epidermidis. Staphylococcus simulans, Staphylococcus felis, Staphylococcus xylosus, Staphylococcus chromogenes, Staphylococcus warneri, Staphylococcus haemolyticus, Staphylococcus sciuri, Staphylococcus saprophyticus, Staphylococcus hominis, Staphylococcus caprae,), Staphylococcus cohnii subsp. Cohnii, Staphylococcus cohnii subsp. urealyticus, Staphylococcus capitis subsp. capitis, Staphylococcus capitis subsp. urealyticus, and Staphylococcus hyicus.

(50) In another embodiment, the terms “microbe” and “microbial” refer to one or more of coagulase-positive staphylococci: Staphylococcus aureus, Staphylococcus pseudintermedius, Staphylococcus delphini, Staphylococcus schleiferi subsp. Coagulans, and Staphylococcus Aureus subsp. anaerobius.

(51) In another embodiment, the bacterial agent is from the Streptococcus genus. For example, the bacterial agent may be selected from the group comprising, but not limited to, Streptococcus uberis, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus pyogenes, Streptococcus bovis, Streptococcus equl subsp. Zooepidemicus, and Streptococcus equinus. The bacteria may be isolated from bovine mastitis.

(52) In another embodiment, the terms “microbe” and “microbial” refer to one or more of bacterial agent of the Baccillus genus: Bacillus melaninogenicus, Bacillus pumilus, Bacillus licheniformis, Bacillus cereus, Bacillus subtilis, and Bacillus anthracis.

(53) In another embodiment, the terms “microbe” and “microbial” refer to one or more of bacterial agent of the Enterococcus genus: Enterococcus faecium, Enterococcus faecalis, and Enterococcus durans.

(54) In another embodiment, the terms “microbe” and “microbial” refer to one or more of bacterial agent of the Listeria genus: such as Listeria monocytogenes.

(55) In another embodiment, the terms “microbe” and “microbial” refer to one or more anaerobic bacteria: Clostridium perfringens, Actinomyces bovis, Propionibacterium acnes, Propionibacterium granulosum, Eubacterium, Peptococcus indolicus, and Peptostreptococcus anaerobius.

(56) In another embodiment, the terms “microbe” and “microbial” refer to one or more species of the Mycoplasma genus: such as Mycoplasma bovis.

(57) In another embodiment, the terms “microbe” and “microbial” refer to one or more fungi of the Malassezia genus.

(58) In another embodiment, the terms “treatment” or “treating” refers to the full or partial removal of the symptoms and signs of the condition. For example, in the treatment of mastitis, the treatment completely or partially removes the signs of mastitis. Preferably in the treatment of mastitis (such as in the treatment of bovines), the treatment reduces the somatic cell count below 280,000 cells/mL (≥a linear score of 5). Preferably the treatment reduces the somatic cell count below 280,000 cells/mL (≥a linear score of 5) by a percentage selected from the group consisting of: by 10%; by 20%; by 50%; by 80%; by 90% and by 95%.

(59) Veterinary and pharmaceutical acceptable salts include salts which retain the biological effectiveness and properties of the compounds of the present disclosure and which are not biologically or otherwise undesirable. In many cases, the compounds disclosed herein are capable of forming acid and/or base salts by virtue of the presence of amino and/or carboxyl groups or groups similar thereto. Veterinary and pharmaceutical acceptable base addition salts can be prepared from, inorganic and organic bases. Salts derived from inorganic bases, include by way of example only, sodium, potassium, lithium, ammonium, calcium and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary and tertiary amines, such as by way of example only, alkyl amines, dialkyl amines, trialkyl amines, substituted alkyl amines, di(subsrituted alkyl) amines, tri(substituted alkyl) amines, alkenyl amines, dialkenyl amines, trialkenyl amines, substituted alkenyl amines, di(substituted alkenyl) amines, tri(substituted alkenyl) amines, cycloalkyl amines, di(cycloalkyl) amines, tri(cycloalkyl) amines, substituted cycloalkyl amines, disubstituted cycloalkyl amines, trisubstituted cycloalkyl amines, cycloalkenyl amines, di(cycloalkenyl) amines, tri(cycloalkenyl) amines, substituted cycloalkenyl amines, disubstituted cycloalkenyl amines, trisubstituted cycloalkenyl amines, aryl amines, diaryl amines, triaryl amines, heteroaryl amines, diheteroaryl amines, triheteroaryl amines, heterocyclic amines, diheterocyclic amines, triheterocyclic amines, mixed di- and tri-amines where at least two of the substituents on the amine are different and are selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, heteroaryl, heterocyclic, and the like. Also included are amines where the two or three substituents, together with the amino nitrogen, form a heterocyclic or heteroaryl group.

(60) Veterinary and pharmaceutical acceptable acid addition salts may be prepared from inorganic and organic acids. The inorganic acids that can be used include, by way of example only, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. The organic acids that can be used include, by way of example only, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like.

(61) The veterinary and pharmaceutical acceptable salts of the compounds useful in the present disclosure can be synthesized from the parent compound, which contains a basic or acidic moiety, by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Lists of suitable salts are found in Remington's Pharmaceutical Sciences. 17th ed., Mack Publishing Company, Easton, Pa. (1985), p. 1418, the disclosure of which is hereby incorporated by reference. Examples of such veterinary acceptable salts are the iodide, acetate, phenyl acetate, trifluoroacetate, acryl ate, ascorbate, benzoate, chlorobenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, methylbenzoate, o-acetoxybenzoate, naphthalene-2-benzoate, bromide, isobutyrate, phenylbutyrate, γ-hydroxybutyrate, β-hydroxybutyrate, butyne-1,4-dioate, hexyne-1,4-dioate, hexyne-1,6-dioate, caproate, caprylate, chloride, cinnamate, citrate, decanoate, formate, fumarate, glycollate, heptanoate, hippurate, lactate, malate, maleate, hydroxymaleate, malonate, mandelate, mesylate, nicotinate, isonicotinate, nitrate, oxalate, phthalate, terephthalate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, propiolate, propionate, phenylpropionate, salicylate, sebacate, succinate, suberate, sulfate, bisulfate, pyrosulfate, sulfite, bisulfite, sulfonate, benzenesulfonate, p-bromophenylsulfonate, chlorobenzenesulfonate, propanesulfonate, ethanesulfonate, 2-hydroxyethanesulfonate, merhanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, p-toluenesulfonate, xylenesulfonate, tartarate, and the like.

(62) It will be understood that the type of microbial infections for which the methods of the invention are intended to be used in treatment include infections in the mammary gland.

(63) Intramammary infections may result in subclinical or clinical mastitis. Subclinical mastitis encompasses an infection without apparent signs of local inflammation or systemic involvement, and may result in transient episodes of abnormal milk or udder inflammation, which are usually asymptomatic. On persistence of infection the mastitis may be termed chronic. Detection may be carried out by examination of milk for somatic cell counts (such as, for example, neutrophils) using standard tests known in the art such as the California Mastitis Test or automated methods provided by dairy herd improvement organizations. Somatic cell counts generally indicate the presence of infection. As an example, cows with a somatic cell count of ≥280,000 cells/mL (≥a linear score of 5) have a >80% chance of being infected. The causative agent of the infection may be identified by bacterial culture of milk according to standard procedures known in the art.

(64) Clinical mastitis encompasses an inflammatory response to infection causing visibly abnormal milk. Indications of inflammation may include changes in the udder (swelling, heat, pain, redness). Mild clinical cases include local signs only. Severe clinical cases include systemic involvement (fever, anorexia, shock) and rapid onset.

(65) The compositions described herein may be formulated for intramammary administration by including such dosage forms in an oil-in-water emulsion, or a water-in-oil emulsion. In such a formulation, the immediate release dosage form is in the continuous phase, and the delayed release dosage form is in a discontinuous phase. The formulation may also be produced in a manner for delivery of three dosage forms as hereinabove described. For example, there may be provided an oil-in-water-in-oil emulsion, with oil being a continuous phase that contains the immediate release component, water dispersed in the oil containing a first delayed release dosage form, and oil dispersed in the water containing a third delayed release dosage form.

(66) The compositions described herein may be in the form of a liquid formulation. The liquid formulation may comprise a solution that includes a therapeutic agent dissolved in a solvent. Generally, any solvent that has the desired effect may be used in which the therapeutic agent dissolves and which can be administered to a subject. Generally, any concentration of therapeutic agent that has the desired effect can be used. The formulation in some variations is a solution which is unsaturated, a saturated or a supersaturated solution. The solvent may be a pure solvent or may be a mixture of liquid solvent components. In some variations the solution formed is an in situ gelling formulation. Solvents and types of solutions that may be used are well known to those versed in such drug delivery technologies.

(67) Also contemplated herein is the intramammary delivery of the compounds according to formulations known in the art. For instance, by intramammary infusion, comprising a polyether ionophore according to the invention, a vegetable oil, an alcohol-soluble fraction of natural lecithin phospholipid material for promoting dispersion of the oil in milk, the phospholipid being selected from the group consisting of phosphatidyl choline and phosphatidyl ethanolamine and mixtures thereof and present in an amount of at least 0.25% in said oil. Such compositions may provide rapid dispersion into milk and short milkout times. Alternatively, a polyether ionophore may be dispersed in an oil consisting of a mixture of triglycerides of palmitic and stearic acid together with polyoxyethylenated cetyl alcohol and stearyl alcohol, and held in an oily medium of mineral, vegetable, synthetic or mixed extraction. Such compositions may speed up release of the antimicrobial agent in the udder, enhancing its biological potential, and reducing milkout time. The intramammary formulation may be in the form of a paste composition comprising a polyether ionophore, fumed silica, a viscosity modifier and a hydrophilic carrier.

(68) In one embodiment, the composition of the invention is formulated for intramammary delivery by formulating the polyether ionophore as a solid dispersion formulated within a intra-mammary formulation. In one further embodiment, the polyether ionophore is dispersed within a water soluble polymer to a form a solid dispersion. The solid dispersion is then formulated into an intramammary formulation by addition to an emollient triester. Viscosity can be adjusted by the addition of a silica-based composition to increase viscosity. In one example, the polyether ionophore is dispersed within polyvinylpyrrolidone K 30 to a form a solid dispsersion. The solid dispersion is then formulated into a intramammary formulation by addition to Crodamol GTCC (which is a fully saturated emollient triester) and the viscosity is adjusted by the addition of Aerosil R972 (Aerosil R972 is a fumed silica aftertreated with dimethyldichlorosilane). The intramammary composition is then dispensed into a syringe for application into the udder of the subject.

(69) In a further aspect, the invention is an intramammary veterinary antimicrobial composition, comprising a therapeutically effective amount of a polyether ionophore formulated in a solid dispersion further formulated into a intramammary formulation. Preferably, the composition comprises a polyether ionophore dispersed within a water soluble polymer to form a solid dispsersion. The solid dispersion is formulated in an intramammary formulation by the addition to an emollient. Viscosity can be adjusted by the addition of a viscosity increasing or decreasing agent. More preferably, the composition comprises polyether ionophore, a water soluble polymer and an emollient. More preferably, the composition comprises polyether ionophore, a water soluble polymer, an emollient and viscosity forming agent. Even more preferably, the composition comprises a polyether ionophore, polyvinylpyrrolidone K 30, Crodamol GTCC and Aerosil R972.

(70) In a preferred embodiment, the composition is a composition selected from the group consisting of:

(71) (1) 600 mg of the LP1369-PVPK30 solid dispersion (150 mg LP1369+450 mg PVPK30)+7% R972+Crodamol GTCC; and

(72) (2) 1200 mg of the LP1369-PVPK30 solid dispersion (300 mg LP1369+900 mg PVPK30)+7% R972+Crodamol GTCC.

(73) In one aspect, the invention is a unit dosage form selected from the group consisting of:

(74) 75 mg Group

(75) Each syringe contains 300 mg of the LP1369-PVPK30 solid dispersion (75 mg LP1369+225 mg PVPK30)+7% R972+Crodamol GTCC; final volume 5 mL.

(76) 150 mg Group

(77) Each syringe contains 600 mg of the LP1369-PVPK30 solid dispersion (150 mg LP1369+450 mg PVPK30)+7% R972+Crodamol GTCC; final volume 5 mL.

(78) 300 mg Group

(79) Each syringe contains 1200 mg of the LP1369-PVPK30 solid dispersion (300 mg LP1369+900 mg PVPK30)+7% R972+Crodamol GTCC; final volume 5 mL.

(80) 600 mg group (300 mg×2)

(81) Each syringe contains 1200 mg of the LP1369-PVPK30 solid dispersion (300 mg LP1369+900 mg PVPK30)+7% R972+Crodamol GTCC; final volume 5 mL.

(82) Intramammary delivery systems for mastitis treatment according to the invention include deliver via the intramammary route primarily for the delivery of compositions of the invention through the teat canal into the udder and close to the site of infection. The composition of the invention may comprise nanomodified active ingredients and be provided in the form of a veterinary acceptable, hydrophilic polymer-based hydrogel intramammary delivery system capable of in-situ gelling. Alternatively, the composition of the invention may be in the form of a mucoadhesive formulation, capable of adhering to the epithelium.

(83) The compositions of the invention may alternatively be formulated using nanotechnology drug delivery techniques such as those known in the art. Nanotechnology-based drug delivery systems have the advantage of improving bioavailability, patient compliance and reducing side effects.

(84) The formulation of the composition of the invention includes the preparation of nanoparticles in the form of nanosuspensions or nanoemulsions, based on compound solubility. Nanosuspensions are dispersions of nanosized drug particles prepared by bottom-up or top-down technology and stabilised with suitable excipients This approach may be applied to the polyether ionophores described herein where the polyether ioinophore has poor aqueous and lipid solubility in order to enhance saturation solubility and improve dissolution characteristics. Saturation solubility will be understood to be a compound-specific constant that depends on temperature, properties of the dissolution medium, and particle size (<1-2 μm).

(85) The composition of the invention may be provided in the form of a nansuspension. For nanosuspensions, the increase in the surface area may lead to an increase in saturation solubility. Nanosuspensions are colloidal drug delivery systems, consisting of particles below 1 μm. Compositions of the invention may be in the form of nanosuspensions including nanocrystalline suspensions, solid lipid nanoparticles (SLNs), polymeric nanoparticles, nanocapsules, polymeric micelles and dendrimers. Nanosuspensions may be prepared using a top-down approach in that larger particles may be reduced to nanometre dimensions by a variety of techniques known in the art including wet-milling and high-pressure homogenisation. Alternatively, nanosuspensions may be prepared using a bottom-up technique in that controlled precipitation of particles may be carried out from solution.

(86) The composition of the invention may be provided in the form of a nanoemulsion. Nanoemulsions are typically clear oil-in-water or water-in-oil biphasic systems, with a droplet size in the range of 100-500 nm, and with compounds of interest present in the hydrophobic phase. The preparation of nanoemulsions may improve the solubility of the polyether ionophores described herein, leading to better bioavailability. Nanosized suspensions may include agents for electrostatic or steric stabilisation such as polymers and surfactants. Compositions in the form of SLNs may comprise biodegradable lipids such as triglycerides, steroids, waxes and emulsifiers such as soybean lecithin, egg lecithin, and poloxamers. The preparation of a SLN preparation may involve dissolving/dispersing drug in melted lipid followed by hot or cold homogenisation. If hot homogenisation is used, the melted lipidic phase may be dispersed in an aqueous phase and an emulsion prepared. This may be solidified by cooling to achieve SLNs. If cold homogenisation is used, the lipidic phase may be solidified in liquid nitrogen and ground to micron size. The resulting powder may be subjected to high-pressure homogenisation in an aqueous surfactant solution.

(87) Compositions of the invention may be in the form of a nanoemulsion. Polyether compounds as described herein may be dissolved in oils/liquid lipid and stabilised into an emulsion formulation. Nanoemulsions may be prepared using high- and low-energy droplet reduction techniques. High-energy methods may include high-pressure homogenisation, ultrasonication and microfluidisation. If the low-energy method is used, solvent diffusion and phase inversion will generate a spontaneous nanoemulsion. Lipids used in nanoemulsions may be selected from the group comprising triglycerides, soybean oil, safflower oil, and sesame oil. Other components such as emulsifiers, antioxidants, pH modifiers and preservatives may also be added.

(88) For mastitis treatment, the intramammary route is primarily used for the delivery of drugs through the teat canal into the udder and close to the site of infection. The composition may be formulated to include nanomodified active ingredients. Accordingly, the composition may be in the form of a hydrophilic polymer-based hydrogel intramammary delivery system, capable of in-situ gelling. Alternatively, the composition may be in the form of a mucoadhesive formulation, capable of adhering to the epithelium. Other routes of administration include topical (such as epicutaneous) and enteral (such as oral).

(89) The composition may be in the form of a controlled-release formulation may include a degradable or non-degradable polymer, hydrogel, organogel, or other physical construct that modifies the release of the polyether ionophore. It is understood that such formulations may include additional inactive ingredients that are added to provide desirable colour, stability, buffering capacity, dispersion, or other known desirable features. Such formulations may further include liposomes, such as emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like. Liposomes for use in the invention may be formed from standard vesicle-forming lipids, generally including neutral and negatively charged phospholipids and a sterol, such as cholesterol.

(90) Throughout this specification, unless the context requires otherwise, the word “comprise” or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

EXAMPLE 1

Antibacterial Activity Against Isolates of Staphylococcus

(91) Specific

(92) As is apparent from the preceding summary of the invention, the invention relates to methods of treatment of mastitis in subjects such as bovines, ovines, caprines, other ruminant species, camelids and equines, and also humans. As is also apparent from the preceding summary of the invention, the invention also relates to compositions used in such methods of treatment of mastitis.

(93) It will be understood that systemic exposure of a subject to be treated according to the methods of treatment of the invention, or the compositions described herein, is to be minimised in order to minimise toxic effects of exposure to therapeutically effective amounts of polyether ionophores. It will be appreciated that the mammary/blood barrier functions as a physical barrier to absorption of therapeutically effective amounts of polyether ionophores, which compounds are understood to remain localised within the tissues and fluids of the mammary gland for localised antimicrobial activity and reduced toxic effects.

Materials and Methods

(94) Bacterial Isolate Collection and Identification

(95) Forty-two isolates of Staphylococcus of varying species and strain type were collected from isolate collections. Biochemical testing including coagulase, latex agglutination testing for protein A, Vogues-Proskauer tests and resistance to polymyxin B were used to identify the species of Staphylococcus. All isolates were also screened for resistance to various antimicrobials commonly used for the treatment of infections. This was performed using the disk diffusion method and resistance standards as outlined by the CLSI. The following antimicrobials were used: amoxicillin-clavulanic acid (30 μg), cephalothin (30 μg), clindamycin (2 μg), enrofloxacin (5 μg), erythromycin (15 μg), gentamicin (10 μg), imipenem (10 μg), oxacillin (1 μg), penicillin G (10 units), tetracycline (30 μg), 1:19 trimethoprim-sulfamethoxazole (25 μg) and vancomycin (30 μg). All strains resistant to oxacillin were found to also be resistant to amoxicillin-clavulanic acid, cephalothin and imipenem and these were determined to be methicillin-resistant strains. All isolate profiles are shown in FIG. 1.

(96) Preparation of Antimicrobials

(97) For each of the five test compounds, a 256 mg/mL stock solution was prepared by dissolving 2.56 grams of compound in 10 mL of dimethyl sulfoxide (DMSO). The resulting solution was then aliquoted into 500 μL volumes and stored at −80° C. until required. A 256 mg/mL stock solution of ampicillin was also prepared by dissolving 0.303 grams of ampicillin (Sigma A-0166) in 10 mL of DMSO. This solution was aliquoted and stored in the same manner as the five test compounds. When these compounds were required, a 256 μg/mL working solution was prepared by diluting 100 μL of stock solution (25.6 mg/mL) in 9.9 mL of cation adjusted Mueller Hinton Broth (CAMHB).

(98) Minimum Inhibitory Concentration Assay

(99) Minimum inhibitory concentration tests were performed according to CLSI Standards (CLSI 2012). 90 μL of one of the test compound solutions, or ampicillin, was added to the end column of a 96 well plate that contained 90 μL of CAMHB in each well. The solutions were then serially diluted across the row, leaving 2 columns for positive and negative controls (FIG. 2). A bacterial suspension was prepared by adding fresh colonies obtained from an overnight culture on Sheep Blood Agar (SBA) to a 9.1 g/L saline solution. This suspension was adjusted to a concentration of between 4×10.sup.8 and 5×10.sup.8 CFU/mL. Concentration of the suspension was determined by measuring optical density (OD) using a spectrophotometer at a wavelength of 600 nm where the correct concentration was determined to have an optical density of between 1.00 and 1.20. 1 mL of this suspension was added to 9 mL of saline before being added to all wells, excluding the negative control wells, in 10 μL volumes giving a final concentration of between 4×10.sup.5 and 5×10.sup.5 CFU/mL in each well. The tests were then incubated for 24 hours at 37° C. and then assessed both visually and using OD readings from a microplate reader at a wavelength of 600 nm. These tests were performed in duplicate but repeated if discrepancies were observed.

(100) The minimum inhibitory concentration (MIC) was determined to be the lowest concentration of antibiotic that prevented growth of bacteria both visually and using OD readings. Direct statistical comparisons between the test compounds and ampicillin could not be performed in light of confidential information restrictions, such as restrictions on disclosure of information relating to the compound structure, such as molecular weight. Instead, MIC values were collated and used to determine the lowest concentration of each compound that was effective against 50% and 90% of isolates, referred to as the MIC.sub.50 and MIC.sub.90 respectively. These values as well as the range of MIC values were then used for direct comparisons between test compounds and for general comparisons with ampicillin.

(101) Minimum Bactericidal Concentration Determination

(102) Following determination of the MIC using the 96 well MIC plate, a variation of the drop plate method was used to determine minimum bactericidal concentration (MBC) for each of the test compounds. These were analysed using samples taken from the MIC plate following incubation. For each compound, a 10 μL drop of each concentration equal to or higher than the MIC was pipetted onto Sheep Blood Agar in a clockwise manner (FIG. 3). Each concentration was pipetted in duplicate with duplicates being pipetted on an inner ring of drops. The plates were incubated at 37° C. overnight and assessed the following day for growth. The MBC was defined as the concentration at which 99.9% of colonies were eradicated, which was visually assessed by a lack of growth on the agar where the drop was placed. Using these data, bactericidal activity could be suggested for some of the compounds. The MBC values for 50% and 90% of the isolates (MBC50 and MBC90) were calculated and assessed along with the MBC range in order to select compounds for further study.

(103) Time-kill Kinetics Assay

(104) Following assessment of MIC and MBC results, two compounds were chosen for analysis using microdilution time-kill assays, LP 1369 and LP 6315 and these were compared to ampicillin. Time-kill assays were performed according to the M26-A guidelines of the CLSI, with modifications. The test compounds and ampicillin were serial diluted in CAMHB across the rows of a 96 well plate and a bacterial suspension was prepared and added in the same manner as the MIC testing. The 96 well plates were then incubated at 37° C. for 48 hours being removed at specific time points and OD for the positive control wells as well as one, four and eight times the MIC of the compound specific to the strain being tested were assessed using a spectrophotometer at a wavelength of 600 nm. The time points assessed were 0, 1, 2, 4, 8, 12, 24 and 48 hours after the addition of the bacterial suspension to the wells. Each test compound was tested in triplicate while ampicillin was tested in duplicate and this test was independently repeated. The bacterial strains used for the microdilution assays were chosen based on bactericidal activity evident during MBC testing. One strain from each of the categories of Staphylococcus in the isolate collection (methicillin-sensitive, methicillin resistant and coagulase negative) were selected as well as the ATCC reference strain for comparison. These strains were strains MSS 1, MSS 11, MRSA 9 and the ATCC 49775 reference strain.

(105) Due to the detection limit of the OD measurements, the time-kill assays were also performed in macrodilution. In 15 mL tubes, 9 mL volumes of the test compounds in CAMHB were prepared at one, four and eight times the MIC concentration and 9 mL volumes of ampicillin were prepared in CAMHB at one and four times the MIC. 1 mL of a 4 to 5×10.sup.6 bacterial suspension (as prepared for MIC testing) was added to each of the tubes as well as a growth control tube containing only 9 mL of CAMHB. These tubes were incubated at 37° C. in an orbital shaker rotating at 100 rpm for 24 hours. At 0, 1, 4, 8, 12 and 24 hours after addition of the bacteria, 100 μL samples were removed from each tube and serial diluted in 9.1 g/L saline solution. The dilutions were then plated in duplicate onto plate count agar and incubated for 24 hours at 37° C. After incubation, viable counts were obtained from the number of colonies visible on the agar and these were used to calculate the number of CFU/mL at each time point. For the macrodilution time-kill analysis, only the ATCC 49775 reference strain and MRSA 9 were assessed in order to further investigate the antibacterial activity against methicillin-resistant strains. These macrodilution time-kill assays were independently repeated and bactericidal activity was defined as a ≥3 log decrease in the number of CFU/mL.

(106) Eukaryotic Cell Toxicity Testing

(107) Erythrocyte haemolysis was used in order to test the toxicity of all five compounds to eukaryotic cells. Blood samples were washed using 9.1 g/L saline solution and centrifuged at 2500 rpm for 10 minutes. This process was repeated until cellular debris and partially lysed cells were removed from solution. 2 mL of the remaining blood cells were suspended in 98 mL of 9.1 g/L saline solution to produce a 2% blood cell solution, which was dispensed in 90 μL volumes into the wells of a 96 well plate. The compound solutions as well as chloramphenicol were prepared from stock solution to a concentration of 256 μg/mL (as described above). Chloramphenicol was used as a negative control for erythrocyte lysis while a ready to use solution of amphotericin B was used as a positive control. 90 μL of the compounds and controls were added to the final well in different rows. These compounds were serially diluted across the row in order to test the different concentrations of antimicrobials. Wells containing only 2% blood solution were also used to assess variation between readings from different columns on the 96 well plates. These tests were incubated at 37° C. for one hour and then assessed for lysis both visually and using a microplate reader to measure optical density at 600 nm wavelengths. Each test was performed in duplicate and then repeated in quadruplicate in order to ensure accuracy.

Results

(108) MIC testing results confirmed antibacterial activity for all test compounds in both methicillin sensitive and methicillin-resistant staphylococci. All test compounds had constantly low MIC values (below or equal to 16 μg/mL) for all strains with the exception of compound LP 9666 as shown in FIG. 4. MIC variation was demonstrated for compound LP 9666 as indicated by the high MIC.sub.90 value and broad MIC range. While the range of MIC values was similar to that of ampicillin, there was no distinction between methicillin-resistant and methicillin-sensitive strains similar to that observed in the MIC values for ampicillin. The MIC.sub.50, MIC.sub.90 and MIC ranges for methicillin-sensitive Staphylococcus strains are shown in FIG. 5. The MIC.sub.50 values for most of the test compounds except for LP 9666 were comparable to ampicillin while the MIC.sub.90 values were considerably lower. This was reflected in the ranges for the test compounds across the strain collection which showed smaller ranges than ampicillin for all the test compounds except LP 9666. A similar trend was observed for methicillin-resistant isolates as shown in FIG. 6. Compounds LP 1088, 1369, 4525 and 6315 demonstrated MIC.sub.50 and MIC.sub.90 values considerably lower than ampicillin as well as narrow MIC ranges. However, while LP 9666 had higher MIC50 and MIC90 values than the other four test compounds, they were considerably lower than ampicillin for the methicillin-resistant strains.

(109) All five test compounds exhibited bactericidal activity in high concentrations but compounds LP 1088, 1369, 4525 and 6315 also showed some bactericidal activity in lower concentrations. Compounds LP 1088 and 1369 exhibited bactericidal activity more consistently for methicillin-sensitive strains while compounds LP 4525 and 6315 exhibited bactericidal activity more consistently for methicillin-sensitive strains, as shown in FIG. 7.

(110) Comparison of the MBC.sub.50, MBC.sub.90 and MBC ranges for the five test compounds for methicillin-sensitive and methicillin-resistant strains are shown in FIGS. 8 and 9, respectively. The MBC.sub.50 and MBC.sub.90 values showed that while some strain-dependent bactericidal activity was evident for the test compounds at low concentrations, most compounds were bacteriostatic for the majority of the strains tested. The greatest bactericidal activity was observed for compounds LP 1369 against methicillin-sensitive isolates and LP 6315 for methicillin-resistant isolates as these demonstrated the lowest MBC.sub.50 values despite their similar MBC ranges.

(111) In the microdilution time-kill assay, both compounds LP 1369 and LP 6315 prevented the growth of the ATCC reference strain over a 48 hour period compared to the growth control (FIG. 10). Some growth was observed after 48 hours for LP 6315 at the MIC but this was still significantly less than that of the growth control. Similar trends were observed for the kill kinetics assays performed for methicillin-sensitive strains MSS 1 and MSS 11 (FIGS. 11 and 12, respectively). Like the ATCC reference strain, all the compounds prevented the growth of bacteria to the levels of the growth control and, for most concentrations, prevented growth above the initial concentration. However, as with the ATCC strain, growth was observed at the MIC of LP 6315 but was observed 24 hours sooner and continued to increase for the final 24 hours. A similar trend was also observed in the MIC for ampicillin for MSS 11 but the increase in the number of-bacteria in the final 24 hours was much steeper than that of the 1×MIC kill kinetics assay for LP 6315. For the time-kill assay for MRSA 9 (FIG. 13), an increase in the number of bacteria after 12 hours was evident for ampicillin and the resulting growth after 48 hours was comparable to the growth control. However, while an increase for 1×MIC of LP 6315 was evident after 24 hours, after 48 hours this growth was no longer observed.

(112) Further time-kill assays in macrodilution showed a relatively constant decrease in the number of viable bacteria over the initial 24 hour period for both test compounds regardless of strain. FIGS. 14 and 15 show that the effect of compound LP 1369 on the number of bacteria was significant compared to the growth control for all the concentrations tested, but that this compound exhibited higher potency at four times the MIC than at eight times the concentration for both MRSA 9 and the ATCC 49775 reference strain. Total reduction in the number of bacteria was assessed, as shown in FIG. 16. As expected, ampicillin was shown to be bactericidal for both strains as the decrease in the number of viable bacteria was >3-log.sub.10 reduction while compound LP 1369 was found to be bacteriostatic for all concentrations as the decrease in the number of viable bacteria was <3-log.sub.10 reduction.

(113) A similar trend to LP 1369 was observed in LP 6315. For the ATCC reference strain (FIG. 17), there was a constant decrease in the number of bacteria over the 24 hour period for all three concentrations of the test compound. For MRSA 9, shown in FIG. 18, all concentrations decreased the number of bacteria for the first 12 hours but an increase was observed between 12 and 24 hours for the MIC. When these reductions were quantified (FIG. 19), ampicillin was shown to be bactericidal as expected while most concentrations of LP 6315 were shown to be bacteriostatic. However, at four times the MIC, LP 6315 was observed to be bactericidal against MRSA as the decrease in colonies was >3-log.sub.10.

(114) The optical density readings from the eukaryotic toxicity assays are shown below in FIG. 20. A decrease in optical density was interpreted as indicative of lysis of the red blood cells. While there was a decrease in optical density for all test compounds in concentrations of 32 μg/mL and higher, for all of the compounds except LP 1369, this decrease was not significant as the decrease was comparable to that of chloramphenicol and as no lysis of red blood cells was visually observed at these concentrations. While the level of lysis was not as high as amphotericin B for compound LP 1369, some lysis of cells was observed within the concentration range 32-128 μg/mL but this was most visually evident at 64 μg/mL.

EXAMPLE 2

Antibacterial Activity Against Skin Lesion Isolates

(115) Isolates of Staphylococcus pseudintermedius were collected from skin lesions of various breeds of dogs. The presence of mec gene and the resistance profile were determined according to the materials and methods described in example 1.

(116) FIG. 21 shows the results obtained for RT-PCR determination of mecA gene presence and resistance profile to various antibiotics. FIG. 22 shows the resistance profile of all the isolates obtained, while FIG. 23 shows the activity (individual results and MIC50, MIC90, MIC mode and MIC range) of ampicillin, LP1369, LP 4525 and LP6315 against the 23 isolates.

EXAMPLE 3

Antibacterial Activity Against Bovine Mastitis Isolates

Summary

(117) Five antimicrobial agents, LP 1088, LP 1369, LP 4525, LP 6315 and LP 9666, were tested against 51 Australian bovine mastitis isolates, primarily pathogenic S. aureus species, S. agalactiaeand S. uberis. LP4525 exhibited the lowest MIC.sub.50 and MIC.sub.90 (0.25 μg/ml and 1 μg/ml respectively). For LP1088, LP1369, LP6315 and LP9666, an MIC.sub.90 of 2 μg/ml, 4 μg/ml, 4 μg/ml and 128 μg/ml was obtained, respectively. All tested antimicrobials elicited MBC values that suggested that these compounds are bacteriostatic against mastitis pathogens. LP4525 appears to be the most promising candidate intramammary antimicrobial agent to treat bovine mastitis cases resulting from infection with Gram-positive bacteria.

Materials and Methods

(118) Bacterial Isolate Collection and Identification

(119) Fifty-one dairy bovine mastitis isolates, encompassing a variety of bacterial species, were isolated from milk samples collected from dairy farms in rural South Australia by the University of Adelaide Ambulatory Clinic. Cellular morphology observed from Gram stains and catalase testing was used to differentiate Staphylococcus, Streptococcus and Corynebacterium species from Gram-negative species. Further biochemical testing including coagulase, Lancefield grouping, esculin hydrolysis and CAMP tests were used to identify the isolates to species level. Where biochemical test results were not definitive in identifying species, amplification and sequencing of the 16S ribosomal RNA gene was used to confirm the identity of the isolates.

(120) Preparation of Antimicrobials

(121) For each of the five test compounds, a 256 ring/ml stock solution was created by dissolving 2.56 grams of compound in 10 ml of dimethyl sulfoxide (DMSO). The resulting solution was then aliquoted into 500 μL volumes and stored at −80° C. A 256 mg/ml stock solution of ampicillin was also created by dissolving 0.303 grams of ampicillin (Sigma A-0166) in 10 ml of DMSO. This solution was aliquoted and stored in the same manner as the five test compounds. When these compounds were required, a 256 μg/ml working solution was created by diluting 100 μl of stock solution (25.6 mg/ml) in 9.9 ml of Cation-Adjusted Mueller Hinton Broth (CAMHB)

(122) Minimum Inhibitory Concentration Assay

(123) Minimum inhibitory concentration tests were performed in the manner outlined by the CLSI (CLSI 2012). Test compounds were dispensed into a 96-well microtitre tray containing 90 μl of CAMHB in 90 μl volumes and serial diluted to obtain a concentration gradient ranging from 128 μg/ml to 0.25 μg/ml (see FIG. 24). For Streptococcus species, CAMHB was replaced with a CAMHB supplement with 4% lysed-sheep blood (4% LSB:CAMHB). 4% LSB:CAMHB was prepared by mixing 5 ml sheep blood to 5 ml milliQ water, and repeated freezing at −20° C. and thawing, followed by centrifuged for 20 min at 7000 rpm. 7 ml of the supernatant was removed and added to 93 mL of CAMB.

(124) Bacterial suspensions were prepared by emulsifying fresh colonies obtained from an overnight culture on Sheep Blood Agar (SBA) in 4 mls of 9.1 g/l physiological saline to an optical density read at 600 nm (OD.sub.600nm) between 1.00 and 1.20. Standardised bacterial suspensions were diluted 1:10 in physiological saline and dispensed into all wells, excluding the negative control wells, in 10 μL volumes giving a final concentration of between 4×10.sup.5 and 5×10.sup.5 CFU/mL in each well. 96-well microtitre trays were incubated for 24 hours at 37° C. in 5% CO.sub.2 and then assessed both visually and using OD readings from a microplate reader at a wavelength of 600 nm. These tests were performed in duplicate and repeated if discrepancies in MIC values were observed between replicates.

(125) The minimum inhibitory concentration (MIC) was determined to be the lowest concentration of antibiotic that prevented growth of bacteria both visually and using OD readings. MIC values were collated and used to determine the lowest concentration of each compound effective against 50% and 90% of isolates, known as the MIC.sub.50 and MIC.sub.90 respectively. These values as well as the range of MIC values were then used for direct comparisons between test compounds and for general comparisons with ampicillin.

(126) Minimum Bactericidal Concentration Determination

(127) Following MIC determination, a variation of the drop plate method was used to determine the minimum bactericidal concentration (MBC) for each of the test compound. MBCs were determined by aliquotting a 10 82 L drop of each concentration from the microtitre plate onto SBA, which were then incubated for 16 hrs at 37° C. The MBC was defined as the concentration at which 99.9% of colonies were eradicated, which was visually assessed by a lack of growth on the agar where the drop was placed. The MBC values for 50% and 90% of the isolates (MBC.sub.50 and MBC.sub.90) were calculated and assessed along with the MBC range in order to select compounds for further study.

Results

(128) Antibacterial activity against the bovine mastitis isolates was observed for all five of the test compounds, LP 1088, LP 1369, LP 4525, LP 6315 and LP 9666. LP4525 demonstrated the lowest MIC90 (1 μg/ml). The values for LP1088, LP1369 and LP6315 were one to two dilutions higher, whereas the value for LP9666 was several dilutions higher (FIG. 25). In contrast to the low MIC.sub.9D values obtained for 4 of the 5 compounds, the across the board high MBC.sub.90 values indicate that all compounds appear for the most part to be bacteriostatic, although bactericidal activity at quite low concentrations above the MIC (2-8 μg/ml) was observed for some isolates. For the MICs and MBCs of individual isolates for all five compounds refer to FIG. 31 and FIG. 32.

(129) When the MIC and MBC data was analysed according to the species of the isolates, it was evident that the MIC.sub.50 and MIC.sub.90 values were lower for Streptococcus species in comparison to the Staphylococcus species (FIGS. 26 to 30). However, the MIC ranges indicate that the differences between the two groups of pathogens are not significant. The MBC data was also highly variable within species, with some staphylococci and streptococci strains effectively killed at concentrations only just above the MIC (eg. for LP4525 and LP6315), but no significant difference was identified between species.

(130) The preliminary results from this study suggest that all five compounds exhibit bacteriostatic activity with some compounds exhibiting strain-dependent bactericidal activity at high concentrations. Although all compounds exhibited some turbidity upon dilution in Cation Adjusted Mueller Hinton Broth, LP1088, LP1369, LP4525 and LP6315 all exhibited low MIC values. LP9666, however, had significantly higher MIC.sub.90 values for each of the groups of mastitis pathogens, and this may be due to the large amount of precipitate formed when diluted in Cation Adjusted Mueller Hinton Broth. LP4525 had the most consistent MIC values for all isolates, and this is evidenced by the small MIC range. LP4525 also has a lower MIC.sub.90 value compared to all compounds. We also found that one Gram-negative bacteria (isolate 2825 in the collection was susceptible to all five compounds.

EXAMPLE 4

Preparation of Intramammary Formulations for Example 5

(131) Four formulations were prepared to formulate LP1369 for the animal studies in Example 5.

(132) Composition of Formulations

(133) Group 1: Vehicles only. Each syringe contains 7% Aerosil R972+Crodamol GTCC; final volume 5 mL

(134) Group 2: ‘Microsized’. Each syringe contains 900 mg micrograde LP1369+7% Aerosil R972+Crodamol GTCC; final volume 5 mL

(135) Group 3: ‘Nanosized’. Each syringe contains 900 mg nanograde LP1369+7% Aerosil R972+Crodamol GTCC; final volume 5 mL

(136) Group 4: ‘PVP’ (which is a solid dispersion). This is a mixture of PVP (polyvinylpyrrolidone K 30 (PVPK30, Sigma, 81420)) and LP1369. Each syringe contains 450 mg LP1369+1350 mg PVP+7% Aerosil R972+Crodamol GTCC; final volume 5 mL. Two syringes (450 mg/syringe×2) were made for each quarter.

(137) Preparation of Intra-mammary Formulations

(138) Group 1: Vehicles only. Ten point five grams of Aerosil R972 was dissolved in 150 ml Crodamol GTCC (which is a fully saturated emollient triester, Chemsupply, CP209).

(139) Group 2: Microsized LP1369 was made by passing LP1369 (>98% purity by HPLC, Bioaustralis fine chemicals, BIA-L1302) through a 75 μm sieve. Twenty seven (27) grams of LP1369 was suspended in Crodamol GTCC and 10.5 grams of R972 was added. The final volume of the suspension was made to 150 ml by Crodamol GTCC. The final concentration of LP1369 is 900 mg/5 mL.

(140) Group 3: Nanosized compound was made by wet milling of LP1369 in Crodamol GTCC (80 mg/ml; 450 mg/5 ml) with <5 mm grinding balls for 30 min at 1000 rpm followed by homogenized for 30 min. Seven percent Aerosil R972 was added to maintain the viscosity. Two syringes (450 mg/syringe×2) were made for each quarter.

(141) Group 4: PVP. Fifty four grams of PVP-LP1369 solid dispersion (13.5 grams of LP1369 and 40.5 grams of PVP) was suspended in Crodamol GTCC and 10.5 grams of Aerosil R972 was added. The final volume of the suspension was made to 150 ml by Crodamol GTCC. The final concentration of LP1369 is 450 mg/5 mL. Two syringes (450 mg/syringe×2) were made for each quarter.

EXAMPLE 5

A Pilot Study to Determine the Residue Depletion Profile of LP1369 in a Developmental Intra-mammary Antibiotic to be Used in Lactating Dairy Cattle

(142) Knowledge of the persistence of drug residues in milk following intramammary treatment is essential in order to determine the milk withholding period or length of time after the last treatment that milk must be discarded to reach a safe concentration. A study in cows treated with three LP1369 formulations was undertaken, monitoring the concentration in milk for 12 milkings (6 days) after administration. Data on concentrations of LP1369 in milk from 4 cows [micronized] (‘Micronised’), 2 cows [nanosized] (‘Nanosized’) and 3 cows (‘PVP’), treated at a single occasions at 1.sup.st, 2.sup.nd, and 12.sup.th milkings after treatment, was provided by the analytical laboratory at the School of Pharmacy, University of South Australia, Adelaide, SA, Australia.

(143) Data Analysis

(144) Undetectable LP1369 concentrations in milk samples were set at 0.0001 mg/L. The milk concentration data was inspected using probability plots, and a log-normal distribution was found to be appropriate for all time-points. Therefore, the concentrations of LP1369 in whole milk were log-transformed before calculating means and standard deviations (weighted by the volume of milk produced by each animal) for each time point, using Microsoft Excel 2010.

Results

(145) Cows Treated with Micronised Formulation

(146) TABLE-US-00001 TABLE 1 Weighted Weighted Total Mean Weighted UCL (upper milk LP1369 Standard confidence volume concentration Deviation limit) Milking information (L) (mg/L) (mg/L) (log scale)* Milking 1 (12 hr) 6.8 11.8533 11.949 1.0738 Milking 2 (24 hr) 14.3 2.6745 1.416 0.4276 Milking 12 (144 hr) 23.0 0.0001 0.000 −4.0000 *weighted by milk yield

(147) The UCLs on the log10 scale appear to decline linearly with time (R.sup.2=0.9980). The fitted line suggests that the UCL will be below −1 (0.010 mg/L on log scale) at 65.3 hours after the last treatment when treated at a single occasion. Refer to FIG. 33.

(148) Cows Treated with Nanosized Formulation

(149) TABLE-US-00002 TABLE 2 Total Weighted milk Mean LP1369 Weighted Weighted volume concentration SD UCL Milking information (L) (mg/L) (mg/L) (log scale)* Milking 1 (12 hr) 7.0 5.2121 3.994 0.7170 Milking 2 (24 hr) 9.4 0.4468 0.321 −0.3499 Milking 12 (144 hr) 25.0 0.0001 0.000 −4.0000 *weighted by milk yield

(150) The UCLs on the log10 scale appear to decline linearly with time (R.sup.2=0.9856). The fitted line suggests that the UCL will be below −1 (0.010 mg/L on log scale) at 61.1 hours after the last treatment when treated at single occasion. Refer to FIG. 34.

(151) Cows Treated with PVP

(152) TABLE-US-00003 TABLE 3 Total Weighted milk Mean LP1369 Weighted Weighted volume concentration SD UCL Milking information (L) (mg/L) (mg/L) (log scale)* Milking 1 (12 hr) 36.1 1.5471 1.059 0.1895 Milking 2 (24 hr) 44.6 0.3748 0.196 −0.4261 Milking 12 (144 hr) 56.7 0.0001 0.000 −4.2283 .sup.#fixed as there was no population *weighted by milk yield

(153) The UCLs on the log10 scale appear to decline linearly with time (R.sup.2=0.9976). The fitted line suggests that the UCL will be below −1 (0.010 mg/L on log scale) at 5.3 hours after the last treatment when treated at single occasion. Refer to FIG. 35.

Conclusion

(154) A significant cost to the dairy farmer is associated with the need to discard milk from treated cows until milk concentrations reach a concentration deemed acceptable by regulatory agencies. The results of this study of LP1369 depletion have determined that a witholding period of 65.3, 61.1 and 5.3 hours, equivalent to 6, 6 and 1 milking(s) for micronised, nanosized and PVP formulations respectively, is expected to be necessary. The solid dispersion (PVP) formulation requires 5 fewer milk discardings.

EXAMPLE 6

Preparation of Intramammary Formulations for Example 7

(155) Three Investigational Veterinary Products (IVP) were prepared to formulate LP1369 for intramammary application for the animal studies in Example 7.

(156) TABLE-US-00004 TABLE 4 ‘IVP1’ Investigational Veterinary Product 1: LP1369 150 mg per syringe in solid dispersion ‘IVP2’ Investigational Veterinary Product 2: LP1369 300 mg per syringe in solid dispersion ‘IVP3’ Investigational Veterinary Product 2: LP1369 600 mg per syringe in solid dispersion

Methods

(157) The preparation of a intramammary formulation of LP1369 is a two step process. The first step is to prepare a solid dispersion of the compound, which is incorporated into an intra-mammary vehicle.

(158) Preparation of Solid Dispersion

(159) Eight grams of LP1369 (>98% purity by HPLC, Bioaustralis fine chemicals, BIA-L1302) and 24 grams of polyvinylpyrrolidone K 30 (PVPK30, Sigma, 81420) were added into a round bottom flask. Methanol (200 ml) was added together with stirring and sonication until LP1369 and PVPK30 were dissolved completely. Methanol was removed using a rotary evaporator at 45° C. under vacuum (˜4-5 h). A solid dispersion formed around the wall of the round bottom flask and was collected, which was crushed out by a spatula and milled by a blender.

(160) Preparation of Intra-mammary Formulations

(161) Thirty grams of PVPK30-LP1369 (equivalent to 7.5 grams LP1369) or 60 grams of PVPK30-LP1369 (equivalent to 15 grams LP1369) solid dispersion was added into a flask. Crodamol GTCC (which is a fully saturated emollient triester, Chemsupply, CP209) was added to make a suspension (close to 250 mL, ˜220 mL). Seventeen point five grams of Aerosil R972 (7% by weight) (Aerosil R972 is a fumed silica aftertreated with dimethyldichlorosilane, and was supplied by Evonik Australia Pty Ltd, catalogue number R972) was then added into the suspension to adjust the viscosity. The suspension was made to 250 mL and resulted in the following concentrations of LP1369; 150 mg/5 mL or 300 mg/5 mL.

(162) For the 600 mg/syringe group, 120 grams of PVPK30-LP1369 (equivalent to 30 grams LP1369) solid dispersion was added into a flask. Crodamol GTCC was added to make a suspension (close to 250 mL, ˜220 mL). Seventeen point five grams of Aerosil R972 (7% by weight) was then added into the suspension to adjust the viscosity. The suspension was made to 250 mL resulting in the concentration of LP1369 600 mg/5 mL. To improve flowability, the solid dispersion was diluted twice into 500 ml with another 17.5 grams of Aerosil R972 added to maintain the viscosity (final R972 7%). Two syringes (300 mg/syringe×2) were made for each quarter. Each syringe (Elm-Plastic, Düsseldorf, Germany: 8 ml Udder Injector Art. No. 808000) was filled up with 5 mL of suspensions and labelled.

(163) Composition of Formulations

(164) 150 mg Group—IVP1

(165) Each syringe contains 600 mg of the LP1369-PVPK30 solid dispersion (150 mg LP1369+450 mg PVPK30)+7% R972+Crodamol GTCC; final volume 5 mL

(166) 300 mg Group—IVP2

(167) Each syringe contains 1200 mg of the LP1369-PVPK30 solid dispersion (300 mg LP1369+900 mg PVPK30)+7% R972+Crodamol GTCC; final volume 5 mL

(168) 600 mg Group (300 mg×2)—IVP3

(169) Each syringe contains 1200 mg of the LP1369-PVPK30 solid dispersion (300 mg LP1369+900 mg PVPK30)+7% R972+Crodamol GTCC; final volume 5 mL

EXAMPLE 7

Efficacy of Investigational Veterinary Products Containing LP1369 in the Treatment of Induced Streptococcus Uberis Clinical Mastitis in Lactating Dairy Cows

(170) The objectives of the study were to estimate preliminary efficacy of two Investigational Veterinary Products (IVPs) containing LP1369 in the treatment of induced Streptococcus uberis clinical mastitis in lactating dairy cows. The specific objectives of this study were to:

(171) (1) Test the preliminary efficacy of each IVP as a treatment of induced clinical mastitis in lactating cattle following experimental challenge via intramammary administration with a known strain of S. uberis.

(172) (2) Compare the preliminary efficacy of the two IVPs in treating induced clinical mastitis in lactating cattle following experimental challenge via intramammary administration with a known strain of S. uberis.

(173) (3) Compare the preliminary efficacy of the two IVPs with a commercial product in treating induced clinical mastitis in lactating cattle following experimental challenge via intramammary administration with a known strain of S. uberis.

(174) The ultimate goal of the study was to provide data on the efficacy of the IVPs.

(175) Clinical mastitis worldwide is the cause of significant losses to the dairy industry. The incidence of clinical mastitis in Australian and New Zealand dairy herds is estimated to be approximately 15% (McDougall, 1999, McDougall et al., 2007a, Petrovski et al., 2009). Streptococcus uberis has been reported as the predominant mastitis-causing organism in Australia (Shum et al., 2009); Petrovski 2013, unpublished) and New Zealand (Laven, 2008, McDougall, 1998, McDougall et al., 2007a, McDougall et al., 2007b). In order to test the efficacy in vivo of antibiotic treatments for mastitis, it is advantageous to treat infected cows. However, this can take a great deal of time in studies that enrol natural infections. Therefore, the use of an established mastitis challenge for lactating cows allows efficacy studies to be completed over a shorter time and with a smaller number of animals than if conducting studies with naturally occurring mastitis. The challenge model has been developed by this group previously and a single strain of S. uberis was selected as preferred challenge strain for further studies.

(176) TABLE-US-00005 TABLE 5 Test sites Test site for animal phase: Dairy Research Centre School of Animal and Veterinary Sciences Roseworthy Campus Roseworthy SA, 2371 Australia Test site for microbiology: Microbiology Laboratory School of Animal and Veterinary Sciences Roseworthy Campus Roseworthy SA, 2371 Australia

Experimental Design

(177) Type and Design of the Study

(178) is the study was a non-randomised study to test the efficacy of the IVPs against clinical, induced mastitis challenge in lactating dairy cows. All cows had two contra-lateral quarters inoculated with a microbial suspension (i.e. Front left and Rear Right or Front Right and Rear Left). Challenged quarters were inoculated using an intramammary administration technique. Each inoculum had a volume of approximately 4 mL and contained approximately 10.sup.6 colony-forming units (cfu) per syringe of the known and well characterised strain of S. uberis.

(179) Cows were milked using portable milking machines twice per day. Clinical examinations of the cow and each quarter were conducted every 12 hours, beginning at the first milking after challenge (hour 12), until hour 180. Prior to the milking before challenge, approximately 2 mL of milk was collected from each of the 4 quarters per cow, using an aseptic collection method for milk sampling testing. The appearance of milk secretions from each quarter was observed, using a dark-coloured plastic container or an old record for changes in consistency, and appearance. The presence of flecks or clots prompted further investigation. As signs of mastitis were detected, milk specimens were collected from the suspected-infected quarter using an aseptic collection method for milk sampling. Milk yield was recorded at each milking for each of the challenged and composite milk yield harvested from both unchallenged (e.g. FR and RL or FL and RR, together) quarters. At strategic time points, milk specimens were collected using a bucket milking system for the composite unchallenged quarters and quarter milking system for each challenged quarter every morning for somatic cell count, protein, fat and lactose percentage testing. Additionally, also at strategic time points, milk specimens were taken for HPLC and microbial inhibition residue testing, e.g. COPAN or Delvotest.

(180) Before and after the completion of each milking, cows were observed for clinical signs associated with acute mastitis: depression, lameness and recumbency. The examination process includes visual observation and clinical examination of the animal, with focus concentrated on the udder. Individual udder quarters were inspected and palpated for clinical signs associated with mastitis, i.e. heat, swelling, redness, tenderness, and all observations were recorded.

(181) Cows/quarters that showed signs consistent with clinical mastitis were immediately treated. As cows/quarters progressively demonstrated signs of mastitis they were treated with IVP1, IVP2, or with reference product (Noroclox LC, Norbrook). Treatment allocation of subsequent individual cows was via ongoing cycling of this pattern. If a second quarter in a single cow showed signs of mastitis at the same or subsequent milking/s, treatment was with the same IVP/Reference product as the first quarter treated for clinical mastitis in that particular cow. Therefore, the number of cows/quarters per group did vary.

(182) TABLE-US-00006 TABLE 6 Schedule of events Study Day Hour relative to inoculation Event 0 −1.0 Aseptic milk sampling all cows 0 −0.5 Milk all cows, observe treated cow, sample all cows 0 0 CHALLENGE ALL COWS 0 0 to 2 Observe all cows 1 12 Clinical observation, milk all cows, collection of specimens and treatment as required 2 24 Clinical observation, milk all cows, collection of specimens and treatment as required 2 36 Clinical observation, milk all cows, collection of specimens and treatment as required 3 48 Clinical observation, milk all cows, collection of specimens and treatment as required 3 60 Clinical observation, milk all cows, collection of specimens and treatment as required 4 72 Clinical observation, milk all cows, collection of specimens and treatment as required 4 84 Clinical observation, milk all cows, collection of specimens and treatment as required 5 96 Clinical observation, milk all cows, collection of specimens and treatment as required 5 108 Clinical observation, milk all cows, collection of specimens and treatment as required 6 120 Clinical observation, milk all cows, collection of specimens and treatment as required 6 132 Clinical observation, milk all cows, collection of specimens and treatment as required 7 144 Clinical observation, milk all cows, collection of specimens and treatment as required 7 156 Clinical observation, milk all cows, collection of specimens and treatment as required 8 168 Clinical observation, milk all cows, collection of specimens and treatment as required 8 180 Clinical observation, milk all cows, collection of specimens and treatment as required

Materials and methods

(183) Animals

(184) Animal Details Species: Bovine Age: 2-10 years old. Breed: Common dairy cattle (Holstein Friesian) Type: Lactating dairy cattle Body weight: Not applicable Number and sex: 14 female Identification: Cows were identified by permanent, uniquely numbered single ear tags at the farm of origin. Additionally, each cow was spray-marked on its hind leg just below the pubic bone with a unique study identification number (e.g. 1, 2, 3 . . . 14) using cans of tailpaint spray of various colours. The same unique study identification number was written on a single, colour-coordinated, plastic leg band that was applied to each cow on a hind pastern. Source: The animals will be obtained from a commercial dairy herd.

(185) Inclusion Criteria

(186) Healthy cows (on clinical observation 3-4 days before purchase)

(187) Four functional quarters (before purchase)

(188) Known somatic cell history (no SCC above 250,000 cells/mL in the last 12 months)

(189) Cows not treated with antimicrobials within 14 days prior to study commencement

(190) Post-inclusion Removal (Withdrawal)

(191) It was not expected that cows would need to be removed from the study due to its short duration. However, cows that were deemed unsuitable for continuation in the study were removed following approval from the Principal Investigator. The reason for any removal was fully documented and justified in the raw data and SR. Any cow that was removed from the study receives appropriate veterinary care. A single cow was removed due to accidental trauma to a rear leg.

(192) Animal Maintenance and Husbandry

(193) Cows were maintained at the test site animal phase for the duration of the study. Cows were acclimatised to the facility for a minimum 7 days before study commencement. Cows were managed under Australian feedlot/housed conditions that are consistent with good farming practice recommendations. Cows were maintained on the total mixed ration and grain-based concentrate used on the farm of origin. Mains water was available for drinking ad libitum from a clean, self-replenishing trough.

(194) Group Allocation and Randomisation

(195) All cows that meet the inclusion criteria were enrolled in the study. The quarter to be challenged was alternated between cows as presented in Table 7 based on the order of cows in the milking shed. The final allocation to challenge was reported in the SR.

(196) TABLE-US-00007 TABLE 7 Treatment regime for each cow Cow order in the milking shed Front Left Front Right Rear Left Rear Right 1 Challenged Unchallenged Unchallenged Challenged 2 Unchallenged Challenged Challenged Unchallenged 3 Challenged Unchallenged Unchallenged Challenged 4 Unchallenged Challenged Challenged Unchallenged 5 Challenged Unchallenged Unchallenged Challenged 6 Unchallenged Challenged Challenged Unchallenged 7 Challenged Unchallenged Unchallenged Challenged 8 Unchallenged Challenged Challenged Unchallenged 9 Challenged Unchallenged Unchallenged Challenged 10 Unchallenged Challenged Challenged Unchallenged 11 Challenged Unchallenged Unchallenged Challenged 12 Unchallenged Challenged Challenged Unchallenged 13 Challenged Unchallenged Unchallenged Challenged 14 Unchallenged Challenged Challenged Unchallenged

(197) Challenge

(198) Challenge Strain

(199) The S. uberis strains used for the challenge was obtained from the library of strains at SAVS Microbiology Laboratory, The University of Adelaide. The selected strain was one that was well characterised and known by prior study to successfully cause clinical mastitis in the period of 36-120 hours after challenge. The strain was phenotypically identified as S. uberis, by means of biochemical tests according to standard microbiological methods.

(200) Challenge Suspension

(201) The preparation of the challenge suspension of the selected S. uberis strains was carried out at the Microbiology Laboratory at SAVS, The University of Adelaide. The procedure for preparation of the challenge suspension followed the laboratory's standard operating procedure.

(202) Experimental Infection

(203) All cows were exposed in two contra-lateral quarters (refer to Table 7) to the challenge suspensions as soon as possible after milking had finished, but not longer than 0.5 hours post-milking. Before inoculation, the teat ends of all four quarters were thoroughly cleaned using dry paper towels and alcohol-soaked cotton swabs or appropriate teat wipes. The challenge inoculum was inoculated via the intramammary route. The entire contents of one syringe was administered to each of the pre-determined, challenged quarters. After administration of the challenge suspension, the quarter was thoroughly massaged to assist dispersion of the suspension in the udder.

(204) Treatment Regime

(205) An accredited veterinarian or appropriately trained personnel undertook all treatments. The Principal Investigator maintained detailed records of the training session of the study personnel, test products used, and dosages administered during the study.

(206) The cows were treated by the intramammary route as per treatment schedule. Treatments were recorded in the “Treatment Record” form.

(207) IVP Products Administration and Frequency

(208) Each individual cow was treated with the recommended dose of one intramammary syringe per quarter on each occasion, on six consecutive instances. For cows/quarters treated with IVP1 and IVP2, each infected quarter was treated immediately following each milking on 6 consecutive occasions, e.g. every 12 hours. The intramammary treatment was administered according to documented standard operating procedures.

(209) Reference Product Administration and Frequency

(210) Each individual cow was treated with the recommended dose of one intramammary syringe per quarter on each occasion, on three instances. For cows/quarters treated with the reference product, each quarter was treated immediately following milking on 3 occasions, 24-hours apart. The intramammary treatment was administered according to documented standard operating procedures.

(211) Concomitant Treatments

(212) The use of any other treatment related to the test product was prohibited during the study. All other treatments should be used only to avoid unnecessary suffering of the animals and need to be justified. In any case, the Principal Investigator must be informed and approve any treatment.

Observations/Measurements/Collection of Specimens

(213) Animal Examination

(214) A Veterinarian examined each cow prior to treatment. The examinations included general observations (specifically body conformation, posture and demeanour), taking rectal temperature and palpation of each quarter of the udders.

(215) Body weights were not be taken during this study.

(216) The cows were observed at least two times daily for any signs of ill health by study personnel throughout the study. Observations were recorded on a Daily Log form. In the case of adverse events (AEs) the Principal Investigator and, where necessary, a veterinarian were notified immediately. All events were recorded.

(217) Each cow was observed for signs consistent with clinical mastitis at each milking starting with Study Day 0 Hour 0 until Study Day 8 Hour 180.

(218) Any cows showing greater than mild signs of discomfort (higher than score 3 as described in documented standard operating procedures) were treated with anti-inflammatory pain relief (ketoprofen) at the recommended dose by the manufacturer.

(219) The results of all observations, whether or not abnormalities are present, were recorded on the observation form.

(220) Clinical Examination Pre-milking and Aseptic Quarter-level Milk Specimens Collection from Cows

(221) Cows were milked and clinical examinations were conducted every 12 hours beginning immediately prior to challenge inoculation (Study Day 0 Hour 0) until Study Day 8 Hour 180. At each milking, prior to quarter milking for challenged and bucket milking for unchallenged quarters, the appearance of the milk secretions was observed, using a dark-coloured plastic container, for changes consistent with mastitis including changes in consistency, appearance and/or presence of flecks or clots. As signs indicative of mastitis occurred, additional milk specimens were collected using an aseptic technique, in accordance with documented standard operating procedures.

(222) All aseptic milk specimens were collected in duplicate.

(223) Observation of the Milk Appearance

(224) Once the initial observations and aseptic specimens had been completed, the appearance of the milk secretions was observed, using a dark-coloured plastic container or an old record, for changes in consistency, appearance and presence of flecks or clots. Milk appearance was assessed based on criteria described in documented standard operating procedures.

(225) Milking

(226) After observations of the milk appearance, cows were milked using portable milking machines. Challenged quarters were milked every 12 hours beginning immediately prior to challenge inoculation (Study Day 0 Hour 0) until Study Day 8 Hour 180 using quarter milkers. Unchallenged quarters of each cow were milked every 12 hours beginning immediately prior to challenge inoculation (Study Day 0 Hour 0) until Study Day 8 Hour 180 using bucket milking.

(227) Milk Yields

(228) Milk yields for challenged and unchallenged quarters were recorded at each milking separately for challenged and composite for unchallenged (e.g. FR and RL or FL and RR, together as a data point for unchallenged quarters) quarters.

(229) Challenged- and Unchallenged- Quarters-level Milk Specimens Collection

(230) Specimens were collected from the pooled milk from the two applicable unchallenged quarters as harvested by bucket milking or at individual quarter for challenged quarters as harvested into the quarter milkers. These specimens were representative of the milk harvested from each cow/quarter/combination of quarters according to documented standard operating procedures and were used as fresh milk analysis specimens. Such collected specimens were used for somatic cell count, protein, fat and lactose percentage testing.

(231) After Milking Examination of the Udders/Cows

(232) After the completion of milking, cows were observed for clinical signs associated with acute mastitis: depression, lameness, recumbency and by checking the rectal temperature. The examination process includee visual observation and clinical examination of the animal concentrated on the udder. However, upon suspicion of an animal being ill, a detailed clinical examination by a registered veterinarian, aiming to diagnose the clinical condition, was undertaken. This may have included a blood sample for appropriate tests (e.g. haematology, biochemistry) if considered necessary.

(233) Udders and individual quarters were inspected and palpated for clinical signs associated with mastitis, i.e. heat, swelling, redness, tenderness. The description of the udder palpation scoring system were according to documented standard operating procedures.

(234) Treatment of Quarters/Cows Diagnosed with Mastitis

(235) As signs of mastitis (changes in milk, hot, swollen and painful udder or palpation score of ≥3 according to documented standard operating procedures) were detected, milk specimens were collected from the suspected-infected quarter using an aseptic collection of milk specimens according to documented standard operating procedures. Quarters diagnosed with mastitis were treated with IVP or reference product. The first cow/quarter showing signs of mastitis was treated with IVP1, thence progressively IVP2 and the reference product. This cycle was repeated as applicable for subsequent infected cows. If a second quarter in a single cow showed signs of mastitis at the same or consecutive milking/s, that cow was treated with the same IVP/reference product in the second quarter as the first. Therefore, the number of cows/quarters per group may vary.

(236) If more than two quarters per cow became infected, an injectable product was used (i.e. Mamazyn) as per label recommendation (e.g. 5 g once a day on three occasions). For cows presenting with signs of acute generalised mastitis, a systemic antibiotic treatment was immediately commenced and, as required, supportive treatment (e.g. non-steroidal anti-inflammatory-ketoprofen) was given. The treatment decision was made by a registered veterinarian in consultation with the Principal Investigator. Quarters continued to be observed at each milking and treated as appropriate.

(237) Assessment of Efficacy

(238) Definition of Efficacy

(239) The product was deemed efficacious if 50% of treated quarters achieved clinical cure within 5 days from initiation of treatment.

(240) Measurement of Efficacy

(241) Clinical Cure

(242) Clinical cure was determined through clinical examination of the cows, udder palpations and observations of the appearance of milk. Clinical cure was defined as:

(243) (1) Palpation score of ≤3 according to documented standard operating procedures, and/or

(244) (2) Milk score of ≤3 according to documented standard operating procedures, and/or

(245) (3) Udder returns to normal temperature, becomes non-swollen, non-painful on touch or redness has disappeared, and/or

(246) (4) The general health of the cow is scored ≤3 according to documented standard operating procedures.

(247) Microbial Cure

(248) The clinical cure was also be determined through microbial culture of aseptic milk samples collected prior to treatment and minimum 7 days after last treatment at quarter level. Microbial cure was defined as:

(249) (1) Isolation of one or two colony types before treatment followed by no growth after treatment.

(250) (2) Isolation of one or two colony types pre-treatment followed by isolation of different colony type/s after treatment.

(251) The efficacy of the IVP/reference product was determined as:

(252) (1) Percentage of quarters with clinical mastitis that achieved clinical cure from the total number of quarters treated with a particular IVP/reference product.

(253) (2) Percentage of quarters with clinical mastitis that achieved microbial cure from the total number of quarters treated with a particular IVP/reference product.

(254) References used for these methods

(255) (1) Laven, R. 2008. Clinical forum: choosing mastitis treatment in the lactating cow: selling or science? UK Vet: Livestock 13(4):29-36.

(256) (2) McDougall, S. 1998. Efficacy of two antibiotic treatments in curing clinical and subclinical mastitis in lactating dairy cows. N. Z. Vet. J. 46(6):226-232.

(257) (3) McDougall, S. 1999. Prevalence of clinical mastitis in 38 Waikato dairy herds in early lactation. N. Z. Vet. J. 47(4):143-149.

(258) (4) McDougall, S., K. E. Agnew, R. Cursons, X. X. Hou, and C. R. Compton. 2007a. Parenteral treatment of clinical mastitis with tylosin base or penethamate hydriodide in dairy cattle. J. Dairy Sci. 90(2):779-789.

(259) (5) McDougall, S., D. G. Arthur, M. A. Bryan, J. J. Vermunt, and A. M. Weir. 2007b. Clinical and bacteriological response to treatment of clinical mastitis with one of three intramammary antibiotics. N. Z. Vet. J. 55(4):161-170.

(260) (6) Petrovski, K., C. Heuer, T. Parkinson, and N. Williamson. 2009. The incidence and aetiology of clinical bovine mastitis on 14 farms in Northland, New Zealand. N. Z. Vet. J. 57(2):109-115.

(261) (7) Shum, L. W. C., C. S. McConnel, A. A. Gunn, and J. K. House. 2009. Environmental mastitis in intensive high-producing dairy herds in New South Wales. Aust. Vet. J. 87(12):469-475.

Results

(262) TABLE-US-00008 TABLE 8 Abbreviations IVP1 Investigational Veterinary Product 1: LP1369 150 mg per syringe in solid dispersion IVP2 Investigational Veterinary Product 2: LP1369 300 mg per syringe in solid dispersion REF Reference product: Norodox containing 200 mg cloxacillin benzathine per syringe UUC Untreated Unchallenged Control: untreated and unchallenged control quarters in each treated cow

(263) Milk appearance before milking

(264) TABLE-US-00009 TABLE 9 Average milk appearance at strip-milk test before milking score and differences per treatment group Average Treatment group appearance score SE Difference to IVP1 0.76 0.09 UUC IVP2 0.76 0.10 UUC REF 0.79 0.07 IVP1, IVP2, UUC UUC −0.13 0.06 IVP1, IVP2, REF

(265) Palpation scores after milking

(266) TABLE-US-00010 TABLE 10 Average palpation scores of quarters immediately after milking and differences per treatment Treatment group Average palpation score SE Difference to IVP1 1.67 0.11 REF, UUC IVP2 1.62 0.11 REF, UUC REF 1 0.08 IVP1, IVP2, UUC UUC 0.51 0.07 IVP1, IVP2, REF

(267) Milk yields

(268) TABLE-US-00011 TABLE 11 Average milk yields and differences per treatment group Average milk yield Treatment group (L/quarter/milking) SE Difference to IVP1 1.69 0.12 IVP2, UUC IVP2 1.38 0.12 IVP1, UUC REF 1.46 0.09 UUC UUC 2.28 0.09 IVP1, IVP2, REF

(269) TABLE-US-00012 TABLE 12 Summary of Clinical Cures and Failures Clinical cure Clinical cure is determined by clinical examinations of each cow, udder palpation and observations of the appearance of the milk. Clinical cure is defined as: 1. Palpation score of ≤3, and/or 2. Milk score of ≤3, and/or 3. Udder returns to normal temperature, becomes non-swollen, non-painful on touch or redness has disappeared, and/or 4. The general health of the cow is scored ≤3 GENERAL PALPATION MILK HEALTH TREATMENT COW QUARTER SCORE SCORE UDDER SCORE STATUS IVP1 1 F ≤3 ≤3 Normal ≤3 Cure IVP1 1 H ≤3 ≤3 Normal ≤3 Cure IVP1 2 F ≤3 ≤3 Normal ≤3 Cure IVP1 2 H ≤3 ≤3 Normal ≤3 Cure IVP1 3 F ≤3 ≤3 Normal ≤3 Cure IVP1 3 H ≤3 ≤3 Normal ≤3 Cure IVP2 4 F ≤3 ≤3 Normal ≤3 Cure IVP2 4 H ≤3 ≤3 Normal ≤3 Cure IVP2 5 F ≤3 ≤3 Normal ≤3 Cure IVP2 5 H ≤3 ≤3 Normal ≤3 Cure IVP2 6 F >3 >3 Abnormal >3 FAIL IVP2 6 H >3 >3 Abnormal >3 FAIL Reference 7 F ≤3 ≤3 Normal ≤3 Cure Reference 7 H ≤3 ≤3 Normal ≤3 Cure Reference 8 F ≤3 ≤3 Normal ≤3 Cure Reference 8 H ≤3 ≤3 Normal ≤3 Cure Reference 9 F >3 >3 Abnormal >3 FAIL Reference 9 H >3 >3 Abnormal >3 FAIL

(270) The Streptococcus uberis challenge in the present study provided sufficient bacterial challenge to overwhelm the commercially available reference product (Noroclox) with only 2 of 3 cows cured. Noroclox is usually expected to be fully effective under conditions of practical field challenge indicating that the challenge in this study was extreme. Despite the significant bacterial challenge, IVP1 (150 mg LP1369) and IVP2 (300 mg LP1369) performed as well as or superior to the reference product, substantiating the high level of activity provided by both formulations against this important and common cause of bovine mastitis. Refer to Table 12.

EXAMPLE 8

Determination of the Microbial Cure Effectiveness of the Treatments Presented in Example 7.

(271) While close inspection of the health of the cow, the appearance of the udder (especially for signs of inflammation—temperature, swelling, pain, redness) and the appearance of the milk can indicate whether or not a clinical cure of mastitis has been achieved, an alternative test is that the treatment demonstrates a microbiological cure. Under appropriate conditions bacteria can invade the udder and cause mastitis. If the mastitis causing organisms are not removed from the infected udder by treatment, then they can remain as a potential sure of reinfection not only for the infected cow but also for other cows in the herd with close contact. Furthermore, the presence of bacteria in milk is not considered desirable from a milk quality and food safety perspective, even though bacteria are likely to be killed during pasteurisation. Therefore, it is highly beneficial to assess the ability of an intramammary treatment to bring about microbiological cure.

(272) Microbiological cure of mastitis is determined through microbial culture of aseptic milk samples collected prior to treatment and a minimum of 7 days after the last treatment at quarter level. Microbial cure is defined as either (i) isolation of one or two colony types before treatment followed by no growth after treatment or (ii) isolation of one or two colony types pre-treatment followed by isolation of different colony type/s after treatment. The efficacy of the IVP/reference product is determined as the percentage of all quarters with clinical mastitis treated with a particular IVP/reference product that achieved microbiological cure.

Materials and Methods

(273) Microbial culture results were generated from milk samples collected at pre-infection, post-infection and post-treatment. Cells containing the digit zero were found to be culture negative. Where a sample was obtained, information on organism identity and number of organisms present (CFU/ml) is presented or the sample is identified as contaminated (see FIG. 36). Remaining cells in the figure indicate the reason a sample was not collected for assessment.

Results

(274) The results are presented in FIG. 36. The reference product (Noroclox) achieved a microbiological cure in 5 of 8 (62.5%) challenged quarters. IVP2 (300 mg LP1369) achieved a microbiological cure in 4 of 6 (66.7%) challenged quarters. IVP1 (150 mg LP1369) achieved a full or partial microbiological cure in 4 of 6 (66.7%) challenged quarters. In this induced Streptococcus uberis-mastitis model, the results show, together with the results presented in Example 7, IVP 1 and IVP2 provided high levels of clinical and microbial cure. These results show effective treatment of mastitis.

EXAMPLE 9

Determining the Residue Depletion Profile of LP1369 in the Treatment of Induced Streptococcus uberis Clinical Mastitis in Lactating Dairy Cows (Example 7)

(275) Data on concentrations of LP1369 in milk from 6 quarters for IVP 1 and IVP 2 treated at 6 occasions (6 consecutive milkings) after treatment was provided by the analytical laboratory at the School of Pharmacy, University of South Australia, Adelaide, SA, Australia. The limit of quantification (LoQ) of the method for LP1369 in milk was 0.088 mg/L. The Maximum Residues level (MRL) for LP1369 is 0.010 mg/kg according to the APVMA, December 2013, Agricultural and Veterinary Chemicals Code Instrument No. 4 (MRL Standard) 2012.

Data Analysis

(276) In milk samples where LP1369 levels were below the LoQ, LP1369 concentration was set to 0.0001 mg/L.

(277) The milk concentration data was inspected using probability plots, and a log-normal distribution was found to be appropriate for all time-points. Therefore, the concentrations of LP1369 in whole milk were log-transformed before calculating means and standard deviations (weighted by the volume of milk produced by each animal) for each time point, using Microsoft Excel 2010.

(278) The determination of the depletion of any substance from milk is affected by the size of the population studied. As the number of sampled cows in this study was small, the g′ factor (a factor that provides a 99% confidence interval so that results are likely to be representative of the population) was applied to the analysis. The possibility of dispersed data was minimised by fixing the g′ factor for a population of 20 animals.

(279) The following assumptions were made. The concentration of the active in milk at milking 1 (time 12 hr) was representative of the average concentrations achieved at milkings after the first, second and the last treatment. Further, the milk volume at milking 1 (time 12 hr) was representative of the average milk volume harvested at milkings after the first, second and the last treatment.

Results

(280) TABLE-US-00013 TABLE 13 Quarters treated with IVP 1 (150 mg active per syringe) Weighted Total Mean milk LP1369 Weighted Weighted volume concentration SD UCL Milking information (L) (mg/L) (mg/L) (log scale)* Milking 1 (12 hr) 6.28 26.1619 11.3811 2.2594 Milking 2 (24 hr) 9.75 1.7359 3.0289 1.3336 Milking 4 (48 hr) 7.00 0.8422 4.3938 1.1010 Milking 6 (72 hr) 9.50 0.1153 0.2720 0.2103 *weighted by milk yield

(281) As shown in FIG. 37, the UCLs on the log10 scale appear to decline linearly with time (R.sup.2=0.9113). The fitted line suggests that the UCL will be below −1 (0.010 mg/L on log scale) at 112.7 hours after the last treatment when treated on 6 consecutive milkings. The extremely high UCL and long residue depletion is most likely due to aberrant result of 1/6 quarters (cow/quarter id 9 FR) being highly positive at 4th milking after the last treatment.

(282) TABLE-US-00014 TABLE 14 Quarters treated (2 per cow) with IVP 2 (300 mg active per syringe) Weighted Total Mean milk LP1369 Weighted Weighted volume concentration SD UCL Milking information (L) (mg/L) (mg/L) (log scale)* Milking 1 (12 hr) 4.94 117.1022 102.3072 2.8663 Milking 2 (24 hr) 5.40 1.5246 4.7322 1.1895 Milking 4 (48 hr) 5.65 0.1376 0.2538 0.0980 Milking 6 (72 hr) 5.75 0.0001 0.0000 −3.3010 *weighted by milk yield

(283) As shown in FIG. 38, the UCLs on the log10 scale appear to decline linearly with time (R.sup.2=0.9558). The fitted line suggests that the UCL will be below −1 (0.010 mg/L on log scale) at 51.7 hours after the last treatment when treated on 6 consecutive milkings.

Conclusion

(284) The results of this study of LP1369 depletion have clearly demonstrated for the first time that LP1369 is cleared rapidly from treated quarters with mastitis. Optimisation of the intramammary formulation can take into consideration the need the maximum efficacy and minimum residence time of LP1369 in milk.