Veterinary compositions for mastitis

Abstract

The present invention relates to veterinary compositions for the treatment and / or prevention of mastitis in cattle, as well as to methods of treating and / or preventing mastitis in cattle.

Description

[0001] VETERINARY COMPOSITIONS FOR MASTITIS

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to veterinary compositions for the treatment and / or prevention of mastitis in cattle, as well as to methods of treating and / or preventing mastitis in cattle.

[0004] BACKGROUND

[0005] Clinical mastitis is an inflammatory condition of the mammary gland typically caused by bacterial infection. In dairy cows it can result in swelling, redness, heat and pain in the udder leading to production of poor-quality milk and reduction of yield and, in severe cases, septicaemia and death. In contrast, subclinical mastitis is mammary disease which does not result in visible inflammation and is largely asymptomatic other than causing reduction in milk yield and elevation of somatic cell count. Subclinical mastitis is detected by the routine measurement of somatic cell counts in milk samples, the accepted upper limit for infection-free glands being 200,000 cells per millilitre.

[0006] Clinical mastitis is usually seen in lactating cows. Nonetheless, despite innate protection of each udder quarter provided by lactoferrin, hydrolytic enzymes and immunoglobulins in mammary secretions, as well as the keratin plug in the streak canal, the majority of infections detected in lactating cows are acquired during the preceding dry period. Indeed, each udder quarter is at greatest risk of infection in the early dry period when the gland is still undergoing involution and a keratin plug has not yet formed. Results from a study by Dingwell et. al (Prev Vet Med. 2004 Apr 30;63(1-2):75-89. doi: 10.1016 / j.prevetmed.2004.01. 012. PMID: 15099718, incorporated herein by reference in its entirety) suggested 47% of teats are without a keratin plug one week after dry off and 23% remain open even after 6 weeks. In these udder quarters, therefore, a physical barrier preventing ingress of bacteria into the gland does not exist. Although secreted proteins largely limit pathogen multiplication and prevent manifestation of clinical mastitis in the dry period, they are unable to prevent the establishment of quiescent infection which can become fulminant during the subsequent period of lactation.

[0007] Historically, blanket dry cow therapy was employed to counteract infection, in which long acting intramammary antibiotic products were routinely employed at dry off to eliminate subclinical infections and to prevent new infections from being acquired during the ensuing dry period. Thus, the antibiotic products were used both for therapy and prophylaxis. However, due to concerns regarding antimicrobial resistance and the requirement for appropriate antibiotic stewardship, the indiscriminate use of broad-spectrum antibiotics has been discontinued. Currently, intra mammary antibiotics are permitted at dry off in those udder quarters known to be infected, identified as a result of evaluation of somatic cell count and by microbiological assessment, but in non-infected udder quarters alternative means of protection are required.

[0008] The realisation that many teats remained open without a functional keratin plug during the early dry period led to the development of teat sealants to provide protection for non-infected quarters in which antibiotic administration is not permitted. There are two types of teat sealants: external teat sealants encompassing materials that adhere to the external surface of the teat and internal teat sealants comprising inert formulations of bismuth salts. External teat sealants have a poor retention and thus need to be replaced regularly, meaning that they are not widely used. In contrast, internal teat sealants have been widely adopted and have become an integral component of dairy cow management in the dry period.

[0009] Cao et al (J. Dairy Sci. 90:3980-3985, 2007) describe the administration of nisin, an antimicrobial peptide, to lactating dairy cows with clinically mastitic quarters. The results were similar to those obtained from gentamicin which was used as a comparator in the trial. Huang et al (J. Dairy Sci. 105:3530-3543) have reported that nisin K exerted an anti-inflammatory effect by inhibiting the ERK1 / 2 and p38 MAPK signalling pathway and promoting the blood-milk barrier on liposaccharide (LPS)-induced mastitis.

[0010] Galvao et al (Applied and Environmental Microbiology 86 18 e1066-20, 2020) compared the administration of microparticles containing chitosan to the uterus of cows to the administration of ceftiofur and reported that “our study indicates with a high degree of confidence that chitosan microparticles, tested as described here, are detrimental to uterine health”.

[0011] EP-3265139-B describes a self-assembled microparticle comprising an acid of formula HOOC- (CH2)n-COOH wherein n is at least 5 and not more than 40 and an organic base in which the molar ratio of acid groups to basic groups is from 0.6 to 1.4:1, the microparticle being obtainable by a process comprising contacting the said acid and base in aqueous solution, wherein the acid is insoluble in the aqueous solution and the organic base is soluble in the aqueous solution wherein the microparticle comprises a multi-lamellar structure. The claimed microparticles may be formed by the process of forming microparticles described in EP-3265139-B (in particular in Example 7 of EP-3265139-B).

[0012] Bismuth subnitrate is a commonly used internal teat sealant. However, it is a recognised risk of the use of bismuth subnitrate that pathogens may be inadvertently introduced into the mammary gland. In addition, bismuth subnitrate is carried over into milk when the cow’s dry period ends. Therefore, there is a need for an internal teat sealant which minimises these issues.

[0013] BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 compares the log reduction in bacterial biofilm cell concentration which is observed upon addition of sterile water (the control formulation), a suspension containing microparticles without surface-bound e-polylysine and a suspension containing microparticles with surfacebound e-polylysine.

[0015] SUMMARY OF INVENTION

[0016] The present invention provides a new approach to treating and preventing mastitis in cattle which comprises sealing the animal’s teats with microparticles. Preferably, the microparticles have antimicrobial activity for providing prevention and treatment of mastitis in cattle. Importantly, the antimicrobial microparticles remain in situ in the lowest part of the teat in the vicinity of streak canal. Thus, the present invention provides a new approach to preventing intra mammary infections in cows during their dry period.

[0017] In a first aspect of the invention, there is provided a sterile veterinary formulation for use in treating mastitis in cattle, wherein the formulation comprises a suspension of a microparticle which comprises a veterinary acceptable saturated or unsaturated di-carboxylic acid that contains between seven and twenty-eight carbon atoms and a first organic polycationic polymer and to which a second organic polycationic polymer is bound, wherein the microparticle is present in an amount of 5% to 60% on a weight to volume basis. Preferably, the microparticle is antimicrobial.

[0018] In a second aspect of the invention, there is provided a method of treating mastitis in cattle, the method comprising administering a sterile veterinary formulation which comprises a suspension of a microparticle which comprises a veterinary acceptable saturated or unsaturated di-carboxylic acid that contains between seven and twenty-eight carbon atoms and a first organic polycationic polymer and to which a second organic polycationic polymer is bound, wherein the microparticle is present in an amount of 5% to 60% on a weight to volume basis. Preferably, the microparticle is antimicrobial.

[0019] In a third aspect of the invention, there is provided a sterile veterinary formulation comprising a suspension of a microparticle which comprises a veterinary acceptable saturated or unsaturated di-carboxylic acid that contains between seven and twenty-eight carbon atoms and a first organic polycationic polymer and to which a second organic polycationic polymer is bound, wherein the microparticle is present in an amount of 5% to 60% on a weight to volume basis. Preferably, the microparticle is antimicrobial.

[0020] In a fourth aspect of the invention, there is provided a mixture of (a) microparticles which comprise a veterinary acceptable saturated or unsaturated di-carboxylic acid that contains between seven and twenty-eight carbon atoms and an organic polycationic polymer and (b) microparticles which comprise a veterinary acceptable saturated or unsaturated di-carboxylic acid that contains between seven and twenty-eight carbon atoms and a first organic polycationic polymer to which a second organic polycationic polymer is bound, wherein the ratio of (b) to (a) with no bound polymer is 10:90 to 90:10 on a weight to weight basis. In some embodiments, there is provided a veterinary formulation comprising this mixture. Preferably, the microparticle defined in (b) is antimicrobial.

[0021] In any of the above aspects of the invention, the second organic polycationic polymer may be an organic antimicrobial polycationic polymer.

[0022] The present invention solves the aforementioned problems because the microparticles remain in situ in the teat of cattle and therefore act as a teat sealant, while also exhibiting antimicrobial activity. Advantageously, the presence of an organic antimicrobial polymer which is bound to the external surface of the microparticle provides particularly effective antimicrobial activity. Thus, the formulations of the invention can be used to provide effective prevention of bacterial infection in cattle with non-infected udder quarters without requiring the use of antibiotics. DEFINITIONS

[0023] The terms “comprising”, “including” and “includes” as used herein means that at least all of the listed elements must be present, but other elements that are not mentioned may also be present.

[0024] The term “microparticle” as used herein refers to a particle which has a size on the order of micrometres, such as from 0.05 to 50pm. In preferred embodiments, the particle has a size of between 0.1 and 5pm. In particularly preferred embodiments, the particle has a size of between 0.1 and 1 pm.

[0025] The term “first microparticle” as used herein refers to microparticles which comprise a veterinary acceptable saturated or unsaturated di-carboxylic acid that contains between seven and twentyeight carbon atoms (preferably between 8 and 20 carbon atoms) and a first organic polycationic polymer and to which a second organic polycationic polymer is bound. The second organic polycationic polymer is preferably bound to the external surface and may be an organic antimicrobial polycationic polymer.

[0026] The term “second microparticle” as used herein refers to microparticles which comprise a veterinary acceptable saturated or unsaturated di-carboxylic acid that contains between seven and twenty-eight carbon atoms (preferably between 8 and 20 carbon atoms) and an organic polycationic polymer. The second microparticles may be obtained by a process comprising contacting the di-carboxylic acid with the polycationic polymer in aqueous solution.

[0027] The term “external surface” as used herein with respect to microparticles refers to the outer surface of the microparticles, i.e. the surface which faces outward.

[0028] The term “polycationic” as used herein means that more than one cationic centre is present. In other words, more than one positive charge is present. Thus, the term “polycationic polymer” refers to a polymer which has more than one positive charge.

[0029] The term “treating” as used herein with reference to treating mastitis encompasses treating and / or preventing mastitis.

[0030] The term “biodegradable” as used herein with respect to microparticles means that they can be broken down by naturally occurring enzymes in the environment.

[0031] DETAILED DESCRIPTION

[0032] The present invention provides a new approach to treating and preventing mastitis in cattle which comprises sealing the animal’s teats with microparticles. Preferably, the microparticles have antimicrobial activity for providing prevention and treatment of mastitis in cattle. Importantly, the antimicrobial microparticles remain in situ in the lowest part of the teat in the vicinity of streak canal. Thus, the present invention provides a new approach to preventing intra mammary infections in cows during their dry period. In a first aspect of the invention, there is provided a sterile veterinary formulation for use in treating mastitis in cattle, wherein the formulation comprises a suspension of first microparticles as herein defined, wherein the suspension comprises first microparticles in an amount of 5% to 60% on a weight to volume basis.

[0033] In a second aspect of the invention, there is provided a method of treating mastitis in cattle, the method comprising administering a sterile veterinary formulation which comprises a suspension of first microparticles as herein defined.

[0034] In a third aspect of the invention, there is provided a sterile veterinary formulation comprising a suspension of first microparticles as herein defined, optionally in the presence of second microparticles as herein defined, wherein the suspension comprises first microparticles or a mixture of first microparticles with second microparticles in an amount of 5 % to 60% on a weight to volume basis.

[0035] In some embodiments, the second organic polycationic polymer is an organic antimicrobial polycationic polymer. In yet further embodiments, the organic polycationic polymer of the second microparticle and the second organic polycationic polymer of the first microparticle are both organic antimicrobial polycationic polymers.

[0036] In a fourth aspect of the invention, there is provided a mixture of first microparticles as herein defined and second microparticles as herein defined in a ratio of 10:90 to 90:10 on a weight to weight basis. Preferably, the first microparticles and second microparticles are in a ratio of 40:60 to 60:40 on a weight to weight basis, preferably about 1:1 on a weight to weight basis. In some embodiments, there is provided a veterinary formulation comprising this mixture.

[0037] The suspension comprises 5 to 60% of first microparticles and optionally second microparticles on a weight to volume basis, preferably 15 to 60%, more preferably 20 to 50%, and more preferably still about 30%. Advantageously, this quantity is sufficient for the first microparticles and second microparticles if they are present to coagulate at their interface with milk in the cow’s udder after administration, trapping microparticle suspension beneath this interface, abutting the streak canal and causing the teat to be sealed.

[0038] In preferred embodiments, the formulation comprises a mixture of first microparticles and second microparticles in an amount of 5% to 60% on a weight to volume basis, wherein the ratio of first microparticles to second microparticles is 90:10 to 10:90 on a weight to weight basis.

[0039] This combination of first microparticles and second microparticles is particularly advantageous because microparticles without bound organic antimicrobial polycationic polymer (i.e. the second microparticles) provide superior interactions with milk compared to first microparticles. In particular, they form a 3-dimensional mousse-like structure at their interface with milk in the cow’s udder after administration, trapping microparticle suspension beneath this interface, abutting the streak canal and causing the teat to be sealed. This provides a particularly effective milk coagulation and thus effective teat sealing. Thus, providing a suspension which comprises a mixture of first microparticles and second microparticles results in a particularly advantageous combination of antimicrobial properties and effective milk coagulation. Similarly, the provision of a mixture of first microparticles and second microparticles provides these advantageous properties.

[0040] In some embodiments, the formulation comprises a first microparticle to which the second organic polycationic polymer is bound in two or more different amounts.

[0041] In some embodiments, the formulation additionally comprises a gelling agent. In some embodiments, the gelling agent is selected from polyethylene oxides, carboxymethyl cellulose, hydroxypropyl methylcellulose, sodium alginate and xanthan gum and combinations thereof. The gelling agent advantageously improves the ability of the formulation to act as a teat sealant. Xanthan gum and sodium methyl cellulose are preferred gelling agents. If a gelling agent is present in the formulation then it is suitably present at an amount of 0.05% to 5.0% on a weight to volume basis.

[0042] In some embodiments, the formulation further comprises a veterinary acceptable carrier. The carrier may be selected from mineral oil, liquid paraffin, white soft paraffin, water and combinations thereof and is preferably water. It will be appreciated that the formulations of the invention may additionally comprise further components which could be readily identified by the skilled person. For example, the formulations may comprise a thickener, such as aluminium stearate, or a formulation aide such as colloidal silica.

[0043] The saturated or unsaturated di-carboxylic acid contains between seven and twenty-eight carbon atoms and in preferred embodiments between eight and twenty carbon atoms. In particularly preferred embodiments, the saturated or unsaturated di-carboxylic acid contains between nine and fifteen carbon atoms, more preferably between nine and thirteen carbon atoms. In preferred embodiments, the di-carboxylic acid is saturated. In preferred embodiments, the saturated or unsaturated di-carboxylic acid is one which is present in the human / mammalian food chain, or which is already used in human or veterinary medicine. The saturated or unsaturated di-carboxylic acid may be selected from pimelic acid, suberic acid, azelaic acid, brassylic acid, sebacic acid and dodecanedioic acid. Preferably, the saturated or unsaturated di- carboxylic acid is selected from brassylic acid, sebacic acid, azelaic acid and combinations thereof. In particularly preferred embodiments, it is sebacic acid.

[0044] Suitable organic polycationic polymers include veterinary acceptable quarternary ammonium compounds, polypeptides and carbohydrates. Veterinary acceptable quarternary ammonium compounds may include didecyldimethylammonium chloride and / or poly(diallyldimethylammonium chloride)). Suitable polypeptides include anti-microbial peptides as described in Lei et al (Am J Transl Res 2019; 11(7); 3919-3931), which is incorporated by reference in its entirety. It is believed that these cationic peptides exert their activity through their positive charges interacting with negatively charged cell membranes through electrostatic interactions. Suitable carbohydrates include chitosan. The most suitable anti-microbial organic polycationic polymers include nisin and e-polylysine, preferably e-polylysine. In some embodiments, the molar ratio of the veterinary acceptable saturated or unsaturated di-carboxylic acid to organic polycationic polymer in the second microparticles is 95:5 to 25:75, preferably 40:60 to 60:40, more preferably around 1 :1.

[0045] Particularly preferred first microparticles and second microparticles are formed from sebacic acid and e-polylysine. In preferred embodiments, the first microparticles comprise sebacic acid and e-polylysine and have further e-polylysine bound to them. Preferably, the further e-polylysine is external to the microparticles, more preferably bound to their external surface. As demonstrated in Examples 1-5 below, microparticles formed from a combination of sebacic acid and e-polylysine provide antimicrobial effects and remain in situ in the teat of cattle. They therefore act as an effective teat sealant which provides protection against infection in cattle.

[0046] In preferred embodiments, the first organic polycationic polymer contained in the first microparticle and / or the organic polycationic polymer in the second microparticle if present is the same as the second organic polycationic polymer in the first microparticle. In preferred embodiments, this second organic polycationic polymer is £-polylysine.

[0047] In preferred embodiments, the second microparticles comprise the same saturated or unsaturated dicarboxylic acid, organic polycationic polymer as the first microparticles. Thus in preferred embodiments, the first microparticles and second microparticles are formed from sebacic acid and £-polylysine.

[0048] In preferred embodiments, the formulation comprises a mixture of first microparticles and second microparticles, wherein the ratio of first microparticles to second microparticles is 90:10 to 10:90 on a weight to weight basis, preferably a ratio of 75:25 to 25:75 on a weight to weight basis. In preferred embodiments, the ratio is 40:60 to 60:40 on a weight to weight basis, preferably 45:55 to 55:45 on a weight to weight basis, e.g. 49:51 to 51 :49 on a weight to weight basis. Most preferably, the ratio is 50:50 on a weight to weight basis.

[0049] In some embodiments, the second microparticles and first microparticles of the invention are biodegradable. This is advantageous because it ensures that they are broken down naturally in the environment and do not cause damage to the cattle’s surroundings when they are administered.

[0050] Microparticles of the present invention may be formed by contacting a long chain fatty dicarboxylic acid with a solution of organic polycationic polymer. Microparticles of the present invention may be formed in suspension by contacting a long chain fatty dicarboxylic acid with a solution of organic polycationic polymer. This process is described in EP3265139B, which is incorporated by reference in its entirety. Specifically, the process is described in Example 7 of EP3265139B. This process may be used with any of the acids listed in Example 4 of EP3265139B (i.e. pimelic acid, suberic acid, azelaic acid, sebacic acid and dodecanedioic acid). Further, the process may make use of any of the quaternary ammonium compounds listed in Example 15 of EP3265139B (i.e. didecyldimethylammonium chloride and poly(diallyldimethylammonium chloride)).

[0051] Microparticles may then be further derivatised by bringing them into contact with a solution of the second organic polycationic polymer in the presence of a cross-linking agent such as a carbodiimide and or hydroxybenzotriazole. Suitable carbodiimide cross-linking agents include 1- (3-dimethylaminopropyl)-3-ethylcarbodiimide.

[0052] The first microparticles and second microparticles may conveniently be prepared as dry powders. The dry powders may be prepared from a suspension prepared as above by spray drying or lyophilisation.

[0053] The suspensions of the present invention are formed by suspending the first microparticles and second microparticles if they are present in a veterinary acceptable carrier, optionally in the presence of a conventional formulation aide. When second microparticles are present, they may be mixed with the first microparticles prior to suspending in the veterinary acceptable carrier or by suspending one or both of the second microparticles or first microparticles in carriers and then mixing.

[0054] The formulations of the invention may be administered to the cattle by any suitable means. In some embodiments, the formulation is administered by intra mammary infusion. In preferred embodiments, the formulation is administered during the cow’s dry period. Preferably, the formulation is administered via the udder streak canal. Administration during the cow’s dry period provides a prophylactic effect whereby future infections associated with milking can be prevented.

[0055] In some embodiments, the inventive formulation may be administered to the cow in a volume of 1 ml to 10 ml, preferably 2ml to 5ml. In preferred embodiments, the formulation comprises a suspension which comprises first microparticles in an amount of about 30% on a weight by volume basis.

[0056] The invention also provides the following numbered embodiments. In these embodiments, the term “Microparticle” refers to microparticles which comprise a veterinary acceptable saturated or unsaturated di-carboxylic acid that contains between seven and twenty-eight carbon atoms and an organic polycationic polymer. The Microparticles may be obtained by a process comprising contacting the di-carboxylic acid with the polycationic polymer in aqueous solution. In these embodiments, the term “Derivatised Microparticle” refers to a Microparticle to which an organic antimicrobial polycationic polymer is bound. The organic antimicrobial polycationic polymer is preferably bound to the external surface of the Microparticle.

[0057] 1. A sterile veterinary formulation for use in treating mastitis in cattle, wherein the formulation comprises a suspension of Derivatised Microparticles as herein defined, wherein the suspension comprises Derivatised Microparticles in an amount of 5% to 60% on a weight to volume basis. 2. A method of treating mastitis in cattle, the method comprising administering a sterile veterinary formulation which comprises a suspension of Derivatised Microparticles as herein defined.

[0058] 3. The veterinary formulation for use according to embodiment 1 or the method of embodiment 2, wherein the formulation is administered during the dry period of the cow.

[0059] 4. The veterinary formulation for use according to embodiment 1 or 3 or the method of embodiment 2, wherein the formulation comprises a mixture of Derivatised Microparticles and Microparticles as herein defined in an amount of 5% to 60% on a weight to volume basis, wherein the ratio of Derivatised Microparticles to Microparticles is 90:10 to 10:90.

[0060] 5. The veterinary formulation for use according to embodiment 1 , 3 or 4 or the method of embodiment 2 or 4, wherein the organic polycationic polymer contained in the Derivatised Microparticle and / or Microparticle if present is the same as the organic antimicrobial polycationic polymer which is bound to the Microparticle.

[0061] 6. The veterinary formulation for use according to any of embodiments 1 , 3, 4 or 5 or the method of embodiments 2, 4 or 5 wherein the Derivatised Microparticles and Microparticles if present are biodegradable.

[0062] 7. The veterinary formulation for use or the method according to any of the preceding embodiments, wherein the molar ratio of the fatty dicarboxylic acid to organic polycationic polymer in the Microparticle is 95:5 to 25:75, preferably 40:60 to 60:40, more preferably around 1 :1.

[0063] 8. The veterinary formulation for use or the method according to any of the preceding embodiments, wherein the suspension comprises 20% to 50% of Derivatised Micropartices or a mixture of Derivatised Microparticles and Microparticles on a weight to volume basis, preferably 30% on a weight to volume basis.

[0064] 9. The veterinary formulation for use or the method according to any of the preceding embodiments, further comprising a veterinary acceptable carrier selected from mineral oil, liquid paraffin, white soft paraffin, water and combinations thereof, preferably wherein the carrier is water.

[0065] 10. The veterinary formulation for use or the method according to any of the preceding embodiments, wherein the saturated or unsaturated carboxylic acid is selected from brassylic acid, sebacic acid, azelaic acid and combinations thereof, preferably wherein the long chain fatty carboxylic acid is sebacic acid.

[0066] 11. The veterinary formulation for use or the method according to any of the preceding embodiments, wherein the organic polycationic polymer is selected from organic polymeric amines, quaternary ammonium compounds, polypeptides, carbohydrates, or combinations thereof. 12. The veterinary formulation for use or the method according to any of the preceding embodiments, wherein the anti-microbial organic polycationic polymer is selected from nisin, e-polylysine and chitosan, preferably wherein the organic polycationic polymer is e-polylysine.

[0067] 13. The veterinary formulation for use or the method according to any of the preceding embodiments, wherein the Derivatised Microparticle and Microparticle if present is formed from sebacic acid and e-polylysine.

[0068] 14. A sterile veterinary formulation comprising a suspension of Derivatised Microparticles as herein defined, optionally in the presence of Microparticles as herein defined, wherein the suspension comprises Derived Microparticles or a mixture of Derivatised Microparticles with Microparticles in an amount of 5% to 60% on a weight to volume basis.

[0069] 15. A mixture of Derivatised Microparticles as herein defined and Microparticles as herein defined in a ratio of 10:90 to 90:10.

[0070] 16. The mixture of embodiment 15, wherein the Derivatised Microparticles and Microparticles are in a ratio of 40:60 to 60:40, preferably about 1:1.

[0071] 17. A veterinary formulation comprising the mixture of embodiment 15 or 16.

[0072] 18. The veterinary formulation of embodiment 14, having the characteristics described in any of embodiments 1-13 above.

[0073] EXAMPLES

[0074] Example 1 : Preparation of microparticles a)(i) Preparation of second microparticles

[0075] Second microparticles were prepared according to the method of Example 7 of EP 3265 139 B1 , which is incorporated by reference in its entirety. In particular, sebacic acid (14.235g,) and 4-methylmorpholine (NMM) (15.66g), were dissolved in water, e-polylysine (21.889g,) was dissolved in water and added to the above solution of Sebacic acid / NMM. The mixture was stirred for two hours and then filtered through a 0.5pm membrane. Dimethylaminopropyl-3- ethylcarbodiimide (3.233g) and hydroxybenzotriazole 40.468g) were then added. The mixture was stirred for less than one minute and left overnight. Multilamellar second microparticles had formed.

[0076] Aa)(ii) Preparation of lyophilised second microparticles

[0077] The suspension from (a)(i) was subjected to semi-membrane filtration and the product lyophilised to give 14g of lyophilised second microparticles. b) Preparation of first microparticles e-polylysine (21.889g), dimethylaminopropyl-3-ethylcarbodiimide (15.75g) and hydroxybenzotriazole (1.25g) were added to the suspension from (a)(i) and the mixture stirred for less than one minute and then left to stand for two hours. The product was filtered through a 53 pm mesh and the resultant suspension subjected to semi-membrane filtration and lyophilisation as before to give 14g of lyophilised first microparticles.

[0078] Example 2: Assessment of bactericidal activity of microparticles

[0079] Second microparticles and first microparticles were prepared according to the method in Example 1. A 10% w / v suspension of each of these microparticles was prepared by addition of sterile water and mixing. The bactericidal properties of the two types of microparticle suspension was compared using an Escherichia coli biofilm assay with sterile water acting as control (the results are provided in Figure 1). Both types of microparticle were found to have antimicrobial properties and displayed greater bactericidal effects than the control. However, first microparticles exhibited greater activity than second microparticles.

[0080] Example 3: Comparison of antimicrobial activity of microparticle mixtures

[0081] The microparticles described in Example 1 were combined in different proportions and suspended to provide two mixtures. In particular, second microparticles were combined with first microparticles in ratios of 50:50 and 25:75 on a weight to weight basis to provide a final microparticle concentration in each case of 50% w / v in sterile deionised water. The antimicrobial properties of the mixtures were evaluated in the context of Streptococcus uberis, Staphylococcus aureus and Escherichia coli using standard assays at a commercial microbiology laboratory. Sterile deionised water was included as a control. The bacterial count pre-treatment and after 24 hours of incubation was evaluated in each case and the results are shown in Table 1 below.

[0082] Table 1: Antibacterial effect of microparticle mixtures with varying ratios of first microparticles and second microparticles

[0083] Both mixtures were found to be antimicrobial with respect to each microorganism. Of the two mixtures, however, the mixture which contained the greater proportion of first microparticles exhibited the greater bactericidal activity. Example 4: In situ persistence within an in vitro udder

[0084] The microparticles of Example 1 were combined with water in equal amounts as well as in 75:25 and 25:75 ratios on a weight to weight basis to provide a three different 30% aqueous pastes. An artificial udder was constructed with four blind ending projections below a central reservoir which was open at the top. A 5ml volume of microparticle paste was placed into each teat-like projection after which whole milk was introduced into the reservoir such that contact was made between the paste and the milk. The reservoir was sealed, suspended and gently agitated periodically over three days. Subsequently, the reservoir was drained and the contents of each teat were examined. In all cases the paste was found to remain in situ in the lowest portion of the teat.

[0085] Example 5: In vivo challenge study to assess efficacy

[0086] 25.95g of each of the first microparticles and the second microparticles of Example 1 were suspended and mixed in sterile water until a final volume of 173ml was achieved to give 30% w / v aqueous paste. The paste was dispensed into 5ml syringes which were sealed with syringe caps pending use.

[0087] Ten cows identified as free of intra mammary pathogens by observation of somatic cell count and by microbiological assessment were dried off. One quarter in each udder was randomly chosen as the control which received no treatment. The other three quarters of each udder were infused via the streak canal with 5ml of the 30% microparticle. After two days, five cows were artificially challenged via the streak canal with a well characterised strain of Streptococcus uberis which reliably causes mild infection. A further 5 cows were challenged in a similar manner seven days after infusion with the antimicrobial paste. Duplicate samples of mammary fluid were obtained by manual expression from each quarter two weeks after dry off and these underwent microbiological assessment. Infection with the challenge microorganism was found to be present in all control quarters that had not received microparticle paste, but both fluid samples were free of Streptococcus uberis in 13 of the treated quarters despite bacterial challenge. The 100% rate of infection with the challenge microorganism in the control quarters revealed the challenge to be robust and the 43% freedom from Streptococcus uberis in the treated quarters revealed the antimicrobial paste to be capable of preventing intra mammary infection. For context, the published results of a similar challenge study (Twomey DP et. al, “Protection against Staphylococcus aureus mastitis in dairy cows using a bismuth-based teat seal containing the bacteriocin, lacticin”, 3147. J Dairy Sci. 2000 Sep;83(9):1981-8. doi: 10.3168 / jds.S0022-0302(00)75075-2. PMID: 11003227, incorporated herein by reference in its entirety) revealed infection to be present in 100% of quarters infused with bismuth subnitrate internal teat sealant mixed with Lacticin 3147 and this indicated failure to provide protection against the bacterial challenge.

[0088] Example 6: Mousse formation

[0089] Syringes of second microparticles and first microparticles were prepared according to Example 5. Paste-like suspensions of first microparticles / second microparticles were created by suspending quantities of the microparticles in water. Due to the different properties of the two materials, the suspension concentration varied depending on the composition. The concentrations are shown in Table 2 below. It can be seen that higher concentrations of first microparticles were needed to make a thick paste compared with second microparticles.

[0090] Table 2: Concentrations of first microparticles and second microparticles in each of the paste-like suspensions

[0091] Crude artificial cow teats were created using 5ml syringes, luer lock caps and O-rings made from plunger rubber. Each was filled with one of the suspensions described above before being placed in the finger of a latex glove. Full fat milk containing blue food colouring was added to provide a milk reservoir above the artificial teat to mimic the gland cistern. The gloves were tied off and suspended from a pole which was gently agitated by being carried over a distance of 2km to create an artificial udder.

[0092] The artificial udders were left undisturbed for 24 hours and the syringes were then removed from the artificial udders and examined. In the cases of the suspensions made entirely from the second microparticles and entirely from the first microparticles, some loss of material from the confines of the syringes was evident. Conversely, for the suspensions made from a combination of the two materials, a stable mousse like interface between the microsphere suspension and the coloured milk was present. The quality and durability of the mousse created with a 50:50 mix of batches was superior to that made with the unbalanced mixture.

[0093] Example 7: Gelling agents

[0094] To improve the stability and viscosity of the formulated microparticle suspension, an initial screen of different viscosity modifiers was performed using high and low excipient concentrations. The microparticle suspensions and viscosity modifiers are provided in Table 3. Included in the list of viscosity modifiers were xantham gum, carboxymethyl cellulose, sodium alginate, and poloxamer P188. Assessment of the resulting formulations was achieved subjectively by consideration of appearance and syringeability and objectively by measurement of viscosity using an Anton Paar MCR 302 Rheometer. These assessments were made following 40°C storage at T=0 and at T=2 weeks.

[0095] Table 3: Formulated microparticle suspensions assessed for appearance, syringeability and viscosity The best viscosity modifiers for inclusion in the microparticle formulation that provided the desired properties under the conditions of assessment were xanthan gum at 0.4% and sodium carboxymethyl cellulose at 1.1%. These formulations demonstrated consistent viscosity, maintained appearance, and were syringeable under the testing conditions, making them the most suitable candidates for further formulation development and stability studies.

[0096] The present disclosure provides exemplary embodiments of the invention and is not intended to be limiting. It will be appreciated that various other modifications and variations of the invention are also possible.

Claims

CLAIMS1. A sterile veterinary formulation for use in treating mastitis in cattle, wherein the formulation comprises a suspension of microparticles which comprise a veterinary acceptable saturated or unsaturated di-carboxylic acid that contains between seven and twenty-eight carbon atoms and a first organic polycationic polymer and to which a second organic polycationic polymer is bound, wherein the microparticles are present in an amount of 5% to 60% on a weight to volume basis.

2. The veterinary formulation for use according to claim 1, wherein the formulation is administered during the dry period of the cow.

3. The veterinary formulation for use according to claim 1 or 2, wherein the formulation comprises a mixture of (a) microparticles which comprise a veterinary acceptable saturated or unsaturated di-carboxylic acid that contains between seven and twenty-eight carbon atoms and an organic polycationic polymer and (b) microparticles which comprise a veterinary acceptable saturated or unsaturated di-carboxylic acid that contains between seven and twenty-eight carbon atoms and a first organic polycationic polymer to which a second organic polycationic polymer is bound, wherein the mixture is present in an amount of 5% to 60% on a weight to volume basis, wherein the ratio of (b) to (a) is 90:10 to 10:90 on a weight to weight basis.

4. The veterinary formulation for use according to any of claims 1-3, wherein the formulation is antimicrobial.

5. The veterinary formulation for use according to claim 3, wherein the organic polycationic polymer in (a) is antimicrobial.

6. The veterinary formulation for use according to any of claims 1-5, wherein the first organic polycationic polymer and / or the second organic polycationic polymer is / are antimicrobial; preferably wherein the second organic polycationic polymer is antimicrobial.

7. The veterinary formulation for use according to any of claims 1 to 6, wherein the microparticles are biodegradable.

8. The veterinary formulation for use according to claim 3 or any of claims 4-7 when dependent upon claim 3, wherein the molar ratio of the veterinary acceptable saturated or unsaturated di-carboxylic acid to organic polycationic polymer in (a) is 95:5 to 25:75, preferably 40:60 to 60:40, more preferably around 1:1.

9. The veterinary formulation for use according to claims 1 or 2 or claims 4 or 6-7 when dependent upon claims 1 or 2, wherein the suspension comprises 15% to 60%, suitably 20% to 50% of microparticles on a weight to volume basis.

10. The veterinary formulation for use according to claim 3 or claims 4-8 when dependent upon claim 3, wherein the suspension comprises 15% to 60%, suitably 20% to 50% of a mixture of microparticles (a) and (b) on a weight to volume basis, and preferably 25% to 50% on a weight to volume basis.

11. The veterinary formulation for use according to any of the preceding claims, further comprising a veterinary acceptable carrier selected from mineral oil, liquid paraffin, white soft paraffin, water and combinations thereof.

12. The veterinary formulation for use according to claim 11, wherein the veterinary acceptable carrier is water.

13. The veterinary formulation for use according to any preceding claim, further comprising a gelling agent.

14. The veterinary formulation for use according to claim 13, wherein the gelling agent is selected from xanthan gum and sodium carboxymethyl cellulose in an amount of 0.05% to 5.0% on a weight to volume basis, preferably 0.3% to 1.2% on a weight to volume basis.

15. The veterinary formulation for use according to any preceding claim, wherein the veterinary acceptable saturated or unsaturated di-carboxylic acid contains between eight and twenty carbon atoms.

16. The veterinary formulation for use according to any previous claim, wherein the veterinary acceptable saturated or unsaturated di-carboxylic acid is selected from brassylic acid, sebacic acid, azelaic acid and combinations thereof; preferably wherein the veterinary acceptable saturated or unsaturated di-carboxylic acid is sebacic acid.

17. The veterinary formulation for use according to any of the preceding claims, wherein: a) the first or second organic polycationic polymer is selected from organic polymeric amines, quaternary ammonium compounds, polypeptides, carbohydrates, or combinations thereof; or b) the first or second organic polycationic polymer is selected from nisin, e-polylysine and chitosan, preferably wherein the first or second organic polycationic polymer is e-polylysine.

18. The veterinary formulation for use according to claim 3 or any of claims 4-8 or 10-17 when dependent upon claim 3, wherein: a) the organic polycationic polymer in (a) is selected from organic polymeric amines, quaternary ammonium compounds, polypeptides, carbohydrates, or combinations thereof; or b) the organic polycationic polymer in (a) is selected from nisin, e-polylysine and chitosan, preferably wherein the organic polycationic polymer in (a) is e-polylysine.

19. A sterile veterinary formulation for use in treating mastitis in cattle, wherein the formulation comprises a suspension of microparticles which comprise a veterinary acceptable saturated or unsaturated di-carboxylic acid that contains between seven and twenty-eight carbon atoms and an organic polycationic polymer and to which an organic antimicrobial polycationic polymer is bound, wherein the microparticles are present in an amount of 5% to 60% on a weight to volume basis.

20. A sterile veterinary formulation comprising a suspension of microparticles which comprise a veterinary acceptable saturated or unsaturated di-carboxylic acid that contains between seven and twenty-eight carbon atoms (preferably between eight and twenty carbon atoms) and a first organic polycationic polymer and to which a second organic polycationic polymer is bound, wherein the microparticles are present in an amount of 5% to 60% on a weight to volume basis.

21. A sterile veterinary formulation according to claim 20, further comprising microparticles which comprise a veterinary acceptable saturated or unsaturated di-carboxylic acid that contains between seven and twenty-eight carbon atoms (preferably between eight and twenty carbon atoms) and an organic polycationic polymer.

22. A mixture of (a) microparticles which comprise a veterinary acceptable saturated or unsaturated di-carboxylic acid that contains between seven and twenty-eight carbon atoms (preferably between eight and twenty carbon atoms) and a first organic polycationic polymer to which a second organic polycationic polymer is bound and (b) microparticles which comprise a veterinary acceptable saturated or unsaturated di-carboxylic acid that contains between seven and twenty-eight carbon atoms (preferably between eight and twenty carbon atoms) and an organic polycationic polymer, wherein the ratio of (a) to (b) is 10:90 to 90:10 on a weight to weight basis.

23. The mixture of claim 22, wherein the ratio of (a) to (b) is about 1 :1 on a weight to weight basis.

24. The sterile veterinary formulation of claims 20 or 21 or the mixture of claims 22 or 23, wherein: a) the veterinary acceptable saturated or unsaturated di-carboxylic acid is selected from brassylic acid, sebacic acid, azelaic acid and combinations thereof; preferably wherein the veterinary acceptable saturated or unsaturated di-carboxylic acid is sebacic acid; and / or b) the first or second organic polycationic polymer is selected from nisin, e-polylysine and chitosan, preferably wherein the first or second organic polycationic polymer is e-polylysine.

25. The sterile veterinary formulation of claim 21 , wherein the organic polycationic polymer is selected from nisin, e-polylysine and chitosan, preferably wherein the organic polycationic polymer is e-polylysine.

26. The mixture of claims 22 or 23, wherein the organic polycationic polymer in (b) is selected from nisin, e-polylysine and chitosan, preferably wherein the organic polycationic polymer in (b) is e-polylysine.

Images