AU2015268626A1 - Injectable compositions for mastitis comprising an NSAID and an antibiotic in a non-aqueous solvent - Google Patents

Abstract

James & Wells Reference: 131431 AU/76 This invention relates to an injectable composition when used for the treatment of a microbial infection in a mammary gland of an animal, wherein the composition includes a) a non-steroidal anti-inflammatory drug (NSAID); b) an antibiotic selected from the group consisting of a beta lactam antibiotic and macrolide antibiotic; characterised in that the composition includes a non-aqueous solvent, and the NSAID and antibiotic in the composition are dissolved in the non aqueous solvent.

Description

James & Wells Ref: 131431AU/76 METHOD OF TREATMENT OF INFECTION TECHNICAL FIELD The invention relates to a composition for the treatment of infection, and most preferably, although not specifically for the treatment of mastitis. 5 BACKGROUND ART The present invention will be discussed in relation to a means by which infection within an animal can be treated. While reference throughout the specification shall be made to the treatment of farm animals such as dairy cattle, it should be appreciated that principles behind the 10 present invention can be applied to other animals including humans. Throughout this specification, specific examples are provided in relation to the treatment of mastitis. It should also be appreciated that the scope of the invention is not limited to mastitis, and can relate to substantially any form of bacterial infection in an animal. 15 Reference throughout the specification should be now made to the infection as being bacterial in nature, and in particular relating to the mastitis resulting from this bacterial infection. Typically, antibiotics are introduced to animals to combat bacterial infections. This may be via a number of administration routes, including oral and topical. A 20 preferred application is through injection near the infected site (such as the udder). The requirement to administer antibiotics is an expensive one. Firstly, the medication itself is expensive. 1 James & Wells Ref: 131431AU/76 Secondly, with cases such as mastitis which adversely affects milk production, there is a milk withholding time in which the animal's milk production is wasted through discard of contaminated milk. Across the herd, this can lead to the loss of thousands of dollars a year. Similarity, there are regulations governing the 5 introduction of animal products into the food chain (whether eggs or meat) from animals having been recently treated with antibiotics. Therefore, it would be desirable if an antibiotic treatment could be given that is effective with regard to bacterial infections with minimal downtime in terms of discarded milk. 10 Mastitis is a costly disease which affects production level of animals, both immediate (milk) and long term (shortened productive life with early culling risk), as well as compromising animal welfare and increasing stress for animal handlers (milkers) - especially during peak lactation. There is a need to find new treatments which are more convenient to administer, 15 but are still efficacious. This may result in better compliance and better treatment outcomes (cure rates). Injectable antibiotics have the advantage of treating all four quarters of the udder (vs intramammary which treat only one quarter) provided that high enough concentrations of active are achieved to cure the causative bacteria. 20 Minimum Inhibitory Concentrations (MIC) of antibiotic required to inhibit specific bacteria signify the target concentration for mastitis treatment products. Products which fail to achieve the desired MIC are likely to fail to cure mastitis infection. For example, Staphylococcus aureus can results in hard-to-cure mastitis and for which antibiotics such as Tylosin containing products are routinely prescribed. 25 However, little work has been done internationally to establish the MIC for Tylosin 2 James & Wells Ref: 131431AU/76 vs S. aureus. Most MICs are established using Erythromycin, which is less commonly used for treatment of mastitis. The MIC for erythromycin vs S. aureus has been reported to be 0.5pg/ml, whereas the MIC for Tylosin vs S. aureus is 2 pg/ml. Essentially, this means the 5 concentration of Tylosin required to effectively treat S. aureus is approximately 4 fold that which is required of erythromycin. Indeed, pharmacokinetic studies have shown that a 20% Tylosin injectable preparation only achieves peak concentrations of cf 1.4 pg/ml during treatment thus failing to achieve the required therapeutic concentrations (MIC). As such 10 either a larger dose and/or a higher concentration of active (>20%) needs to be administered to achieve milk concentrations above MIC. Therefore there is a need to develop improved antibiotic treatments (not only for Tylosin, but also other antibiotics) to help avoid the need for larger dosages or higher concentrations of active (>20%) to achieve the necessary MIC not only for 15 S. aureus, but also for other causes of bacterial infection. Also, there is a need to develop compositions wherein excipients or additional actives are included to help the antibiotic act more effectively at treating the bacterial infection. In the past, co-administration of an NSAID with antibiotic treatment has been found 20 to be effective in improving antibiotic response. However, difficulties with combining these actives in a stable composition have led to the two actives typically to be delivered separately. This is a general inconvenience, and it would be preferred to provide a single composition with both NSAIDs and an antibiotic. It can also be important consider animal welfare. Infections such as mastitis are 25 painful. Therefore, it has become common practice to treat not only the infection, 3 James & Wells Ref: 131431AU/76 but also the pain associated with it using active agents such as an NSAID. This combination treatment is relatively new in the industry. US 2005/0277634 discloses a combination of NSAID (meloxicam) and an antibiotic (penethamate hydroiodide) in a suspension formulation for injection to treat 5 mastitis. They discuss a synergistic reaction between the NSAID and antibiotic, to the effect that lower levels of antibiotic are needed to achieve a level above the MIC. Preferably, the higher the concentration of NSAID in the composition, the better the antibiotic appears to function. However, there are considerable disadvantages to a suspension formulation. 10 These include potential caking, problems with dispersability before use, difficulty in delivery, site reaction/pain after injection and poor absorption of the active(s). Thus, it would be preferable to provide an NSAID and antibiotic combination as a stable composition in solution. Furthermore, providing a solution composition where the NSAID is loaded to improve the effectiveness of the antibiotic would be 15 beneficial. Yet, this has not been achieved, due to significant problems such as those discussed below. In general, a significant problem associated with many injectable compositions can be the development of a site reaction and/or pain caused to the animal upon either intramuscular or subcutaneous delivery. 20 This site reaction and pain on injection can often be due to the active agent, such as the antibiotic Tylosin. However, such problems can be exacerbated by other excipients in the compositions, typically solvents which act as carriers or solubilisers for the active agent(s). This problem is well documented in the review article Strickley et al., Pharmaceutical Research, Vol., 21 No.2 February 2004 titled 25 "Solubilizing Excipients in Oral and Injectable Formulations". 4 James & Wells Ref: 131431AU/76 For this reason, veterinary chemists often aim to develop compositions as solutions with as low a concentration and/or volume of solvent(s) in an injectable composition as possible. As a consequence, this can lower the ability to achieve higher concentrations of the active agent(s), lower the stability of the composition 5 as a whole, and ultimately lower the animal's likely success from treatment. Therefore, veterinary chemists have preferred to use aqueous-based compositions to avoid site reactions and pain upon injection. Whilst this can be effective in reducing side effects such as site reaction/pain on delivery, aqueous based compositions can again lead to instability of the active 10 agent(s), resulting in a shorter shelf-life of the composition (and ultimately bio availability). It can also limit the ability to retain higher concentrations of active agent(s). It can then be necessary to then revert back to suspensions as discussed in US 2005/0277634. WO 02/41899 (WO'899) discloses pour-on or injectable compositions including an 15 antibiotic and an analgesic both dissolved in solvent. However, the exemplified types antibiotics (florfenicol, gentamicin and oxytetracycline) are distinguishable from those which are the focus of the compositions of the present invention, namely beta lactam and macrolide antibiotics. Indeed, it is known that beta lactam and macrolide antibiotics are particularly 20 unstable and are likely to react with NSAIDs such as flunixin. Despite difficulties in combining beta lactam and macrolide antibiotics with NSAIDs, such antibiotics are very useful in treating microbial infections in such as that seen in mastitis. No stability data is provided in WO'899 suggesting that many of the compositions, particularly those with a higher active agent concentration (such as that in Example 5 James & Wells Ref: 131431AU/76 4), would not be storage stable. Indeed, studies conducted by the inventors have shown similar compositions were unstable. It is an object of the present invention to address the foregoing problems or at least to provide the public with a useful choice. 5 All references, including any patents or patent applications cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a 10 number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents form part of the common general knowledge in the art, in New Zealand or in any other country. Throughout this specification, the word "comprise", or variations thereof such as "comprises" or "comprising", will be understood to imply the inclusion of a stated 15 element, integer or step, or group of elements integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. Further aspects and advantages of the present invention will become apparent from the ensuing description which is given by way of example only. 20 DISCLOSURE OF INVENTION According to a first aspect of the present invention there is provided an injectable composition when used for the treatment of a microbial infection in a mammary gland of an animal, wherein the composition includes 6 James & Wells Ref: 131431AU/76 a. a non-steroidal anti-inflammatory drug (NSAID); b. an antibiotic selected from the group consisting of a beta lactam antibiotic and macrolide antibiotic; characterised in that: 5 the composition includes a non-aqueous solvent, and the NSAID and antibiotic in the composition are dissolved in the non-aqueous solvent. According to a further aspect of the present invention there is provided a method of treating an animal for an internal microbial infection 10 characterised by the step of administering a composition substantially as described above by injection. According to a further aspect of the present invention there is provided a use of a composition substantially as described above in the manufacture of a medicament for the treatment of a microbial infection. 15 A method of preparing a composition substantially as described above characterised by the step of adding at least one non-aqueous based solvent to the composition. 7 James & Wells Ref: 131431AU/76 Discussion of the advantages provided by the invention It has been recognized by the inventors that substantial inflammation in and around an infection site may hinder the effectiveness of an antibiotic as this reduces the ability to reach and act on the bacteria. 5 In the past, NSAIDs have been administered more-so to treat inflammation and/or pain. The inventors have identified an important synergistic effect by administering an NSAID and an antibiotic together in a single injectable composition. This synergistic effect may allow the antibiotic to treat the microbial infection more effectively than if antibiotic was administered alone. Similarly, the inventors have 10 found that the combination of the NSAID and antibiotic in a single formulation has a heightened synergistic effect which is not present if the animal is treated with NSAID and antibiotic in two separate formulations. This may be partially due to the ability to provide the two actives together in a stable composition. The inventors consider that, without being limited to the proposed mode of action, 15 the synergistic interaction may result from the NSAID decreasing the inflammation in and around the site of the infection (at the udder in the case of mastitis) such that the NSAID allows effective distribution of the antibiotic within the otherwise inflamed site. Additionally the synergistic effect may be partially attributed to the NSAID binding 20 endo-toxins (endotoxaemia), which the inventors otherwise found hindered antibiotic effectiveness. For instance, G-negative bacteria, which produce endo-toxins, are associated with mastitis. Such bacteria can cause systemic illness (fever and endotoxaemia). Although this is currently relatively uncommon in New Zealand (less that 1% 25 incidence), the increased use of feed-pads and herd homes may result in higher 8 James & Wells Ref: 131431AU/76 incidence rates of mastitis caused by G-negative pathogens. Thus the present formulation is envisioned to become an important component of infection treatment regimes in the future, for example in mastitis. Additionally, the synergistic effect may be further enhanced as the NSAID may act 5 to reduce fever (pyrexia) to allow better effectiveness of the antibiotic as well as safety/comfort of the animal. During experimentation with certain NSAIDs and antibiotics, the inventors recorded a synergistic effect as discussed above. After conducting the above experiments and identifying the advantageous result, a skilled person would appreciate that the 10 synergistic result would be expected for common types of antibiotics, not just those specific examples tested. For example, it would be expected that all beta lactam and macrolide antibiotics would display the same synergistic effect when in combination with an NSAID. It is known that both types of antibiotics have a similar mode of action (cellular protein synthesis inhibition). 15 A further significant advantage of the combination identified by the inventors may be the ability of the NSAID to improve animal welfare due to the reduction of pain and inflammation which can result from internal infections such as mastitis. Also, the ability to provide two different actives in one combination formulation may help to overcome the need for delivery of multiple compositions, storage of 20 compositions, costs, packaging and so forth. The inventors were able to identify a particular solvent type that aided the combination of the two actives, which previously were found to be very difficult to combine in a stable solution. In the past, suspensions were used in order to combine such actives together in a single composition. 25 The inventors identified a further, unexpected, synergistic effect between the two actives as the concentration of the NSAID was increased, as discussed below. 9 James & Wells Ref: 131431AU/76 The aim of increasing the concentration of NSAID was to improve the synergistic effect initially identified by the inventors to help improve antibiotic effectiveness at the site of infection. Active agents have recommended dosages based on what has been established 5 as effective. Thus when two agents are combined, the combination should provide the appropriate doses of each active. For example, for the recommended dosage of a particular antibiotic (e.g. Tylosin, 10 mg/kg) a concentration of is 20% Tylosin is often used in the composition. This high concentration of Tylosin can often be the limiting factor in such a composition as it can cause significant irritation on 10 injection. In order to provide concomitant dose of an NSAID (e.g. Ketoprofen), a concentration of 6% w/v is preferred. Indeed, compatibility of dosage regimes between the multiple actives used can be an important aspect of the successfulness of the active composition to provide the correct amounts of active when administered. 15 However, when the inventors attempted to provide a composition with 6% Ketoprofen, the composition was not stable with an aqueous based solvent. With other NSAIDs tested, similarly poor stability results were seen in an aqueous based composition if NSAIDs were included at required concentrations to reflect recommended dosages. 20 Without wanting to revert to suspensions, the inventors had to consider other avenues. To compensate for an increased concentration of NSAID, the inventors utilised a non-aqueous solvent system in attempt to provide a stable composition in solution at various conditions (e.g. temperatures above 40C). However, it was expected, as 25 generally understood in the art, that reverting to non-aqueous solvents would lead 10 James & Wells Ref: 131431AU/76 to increased site reaction and/or pain on delivery compared to aqueous-based compositions. Surprisingly, however, studies from the inventors identified that such site reaction/pain was not increased beyond that seen in commercially available 5 compositions (e.g. Tylan and Ketofen) which are aqueous-based compositions. Without wanting to being limited to a particular mode of action, it is thought that the increased NSAID synergistically helps to alleviate the inflammation/pain locally at the site of injection. Therefore, it is possible that the additional amounts of NSAID are counter-acting against the harsher solvents which would normally increase site 10 reaction and pain on injection. Therefore, unexpectantly, the inventors identified that an antibiotic and an NSAID can be included together in a stable composition in solution with an increased concentration of NSAID greater than normally achievable than when combined in an aqueous based system. Importantly, this was achievable without resulting in an 15 increased site reaction or pain on delivery compared to industry standards (Tylan and Ketofen). A downstream effect of this discovery was that the concentration (and ultimately dosage) of the NSAID and even the antibiotic may be increased without causing additional site reaction/pain on delivery. This synergistic effect at the sight of 20 infection may then also be enhanced, improving the overall treatment outcome. Preferred embodiments of the composition The composition of the present invention is provided as an injectable liquid formulation. Throughout the specification the term liquid formulation should be taken as meaning any therapeutic formulation that is of a "syringable" consistency. 11 James & Wells Ref: 131431AU/76 This is the mode of administration preferred by many farmers and veterinarians, particularly as localised application of a NSAID can be of greater effect when treating an infection site such as mammary glands (mastitis). Preferably, the liquid formulation has a syringable viscosity at lower temperatures. 5 The liquid formulation is a solution. This should be taken as meaning a composition wherein all the excipients and active agents are substantially solubilised in the solution, and not in suspension. As previously discussed, a solution provides many advantages over suspensions. For example, there are no issues with caking, re-suspension, or difficulties with injection. 10 The solution may have one or more phases. Non-steroidal anti-inflammatory drug Throughout the specification the term non-steroidal anti-inflammatory drug (NSAID) should be taken as meaning any drugs or active compound with anti inflammatory effects without using steroids. 15 There are many different types of NSAIDs known in the art. It should be considered within the scope of the invention to utilise any one or combination of known NSAIDs or those of which are identified in the future. Preferably, the NSAID is selected from the group consisting of Carprofen, Naproxen, Ibuprofen, Ketoprofen, Piroxicam, Diclofenac, Etodolac, Flunixin, 20 Deracoxib, Meloxicam, Celecoxib, Rofecoxib, and combinations thereof. Most preferably, the NSAID is selected from the group consisting of Flunixin, Carprofen, Ketoprofen and combinations thereof. These NSAID were found by the inventors to show the greatest synergistic effect when in combination with the antibiotics as claimed. Additionally, these NSAIDs 12 James & Wells Ref: 131431AU/76 have all been shown to be effective anti-inflammatory agents in bovine medicine, both from an efficacy and safety perspective. Preferably, the NSAID is present in the liquid formulation at a concentration of 1 15% w/v. As discussed previously, it may be preferable to increase the 5 concentration of NSAID to improve the synergistic effect between the two actives. Although the benefits of doing so would be well appreciated, providing such a combination in a stable composition in solution has not been achieved, let alone one where the composition shows no greater site reaction/pain on delivery compared to aqueous based compositions. 10 Preferably, the NSAID flunixin meglumine is present in the composition at a concentration of approximately 7.3% w/v. This may provide approximately 4.4 % Flunixin in the composition. This is as per dose rate in combination with Tylosin. The preferred approximate concentration of each NSAID may also be dependent on it's anti-inflammatory efficacy and safety profiles, as well as the concentration of 15 antibiotic used in the composition. Preferably, the NSAID Meloxicam is present in the composition at a concentration of approximately 1 % w/v. Preferably, the NSAID Carprofen is present in the composition at a concentration of approximately 3% w/v. 20 Preferably, the NSAID Ketoprofen is present in the composition at a concentration of approximately 4% to 10 % w/v. For example, when the concentration of the antibiotic Tylosin is at 20% w/v in the composition, the corresponding Ketoprofen concentration is preferably 6% w/v. If the Tylosin concentration is increased to 30% w/v, a preferred concentration of Ketoprofen is 9% w/v. 13 James & Wells Ref: 131431AU/76 Alternatively, the NSAID is selected from natural forms of anti-inflammatory agents. For example, these may include green-lipped mussel extract, omega 3, and the like. Antibiotic 5 Throughout the specification the term antibiotic should be taken as meaning a substance or compound, either natural, synthetic or semi-synthetic, that kills bacteria (bactericidal) and/or inhibits bacterial growth (bacteriostatic). The antibiotic in the composition is selected from a group consisting of macrolide and beta-lactam antibiotics. 10 Throughout this specification, the term macrolide antibiotic should be taken as meaning any antibiotic which activity stems from the presence of a macrolide ring to which one or more deoxy sugars, usually cladinose and desosamine, may be attached. The lactone rings are usually 14-, 15-, or 16-membered. Macrolide-class antibiotics are typically used in veterinary medicine and are 15 considered to share a common mode of action. It is considered that macrolide antibiotics' mode of action involves interference with protein synthesis, for instance by blocking translation at the ribosome. Preferably, the macrolide-class antibiotic is selected from Azithromycin, Clarithromycin, Dirithromycin, Erythromycin, Roxithromycin, Telithromycin, 20 Carbomycin A, Josamycin, Kitasamycin, Midecamicine/midecamicine acetate, Oleandomycin, Spiramycin, Troleandomycin, and Tylosin/tylocine. Most preferably, the macrolide antibiotic is Tylosin. 14 James & Wells Ref: 131431AU/76 Tylosin is a macrolide-class antibiotic used in veterinary medicine to treat bacterial infections in a wide range of species and has a high margin of safety. A further advantage of Tylosin is that it is licensed for mastitis therapy in New Zealand. Tylosin has a wide spectrum of activity against gram positive bacteria including 5 Staphylococci, Streptococci, Corynebacteria, and Erysipelothrix. It also has activity towards gram negative Campylobacter coli, and some spirochaetes. It is also active against Mycoplasma species. Injectable Tylosin formulations can cause pain, inflammation, and itchiness around the injection site. Therefore, it is important to be able to reduce the above side 10 effects when Tylosin is injected. The provision of an antibiotic such as Tylosin together with a NSAID has an additional advantage in that the NSAID may help to avoid the side effects the Tylosin. This reduction in side effects may be aided by the synergistic effect between the NSAID and the antibiotic when administered in one formulation. Throughout this specification, the term beta lactam antibiotic 15 should be taken as meaning any antibiotic including a beta lactam ring in its structure. For example, this may include penicillin derivatives (penams), cephalosporins (cephems), monobactams, and carbapenems. It is considered that the mode of action for beta lactam antibiotics involves attacking bacterial cell walls. The beta-lactam antibiotics may be provided with 20 beta-lactamase inhibitors such as clavulanic acid. Preferably, the beta lactam antibiotic is penethamate. In a recent study, it was found that penethamate has a similar efficacy to Tylosin (a particularly preferred type of antibiotic used for the present invention). An advantage common to both beta lactam and macrolide antibiotics over other 25 antibiotic types is that the former types are weak bases and are lipophilic. This 15 James & Wells Ref: 131431AU/76 may result in higher concentrations in bodily fluids such as milk than in plasma. As such, they are more likely than less lipophilic compounds to achieve concentrations greater than the minimum inhibitory concentration (MIC). Unexpectantly, these types of antibiotics appear to show a synergistic effect with 5 the NSAID. However this synergistic effect was not observed with other antibiotic types. Preferably, the antibiotic is present in the liquid formulation at a concentration between 10-35% w/v, and most preferably approximately 20.0 - 30.0 % w/v. Typically, a higher antibiotic concentration will lead to a lesser volume of antibiotic 10 needed to administer per dose, which may be more convenient for farmers and more comfortable for smaller animals. By increasing the NSAID levels in the composition, it may also be possible to increase the antibiotic concentration in the composition without increased site reaction on delivery (similar to the effect seen when the inventors reverted to a non-aqueous based solvent, also expected to 15 cause increased site reaction/pain). For example, the inventors acknowledge that if the treatment is for Staph. aureus, the amount of antibiotic (e.g. Tylosin) may be increased up to 60% w/v to account for the minimum inhibitory concentration (MIC). Preferably, the ratio of NSAID to antibiotic (w/v) in the liquid formulation is 20 approximately 1:3, respectively. This may be a particularly applicable ratio if the NSAID is flunixin. However if the NSAID is a different type of NSAID, such as meloxicam or carprofen, the ratio may vary. Solvent Throughout this specification the term solvent should be taken as meaning any 25 solvent or combination of different solvents which are present in the composition. 16 James & Wells Ref: 131431AU/76 Preferably, the solvent has one or more of the following features: - non-aqueous; - a dielectric point above 30 at 25 C; - a boiling point above 1OOC; and/or 5 - classified as a dipolar aprotic solvent. Most preferably, the composition is entirely non-aqueous. The inventors tested a range of different aqueous-based compositions which were found to be quite unstable under high temperature stress and often even at room temperature. For example, Tylosin was found to be particularly unstable when combined with an 10 NSAID in an aqueous based formulation. However if the NSAID and antibiotic combination were combined in a non-aqueous solvent based system, the composition remained stable, especially at lower temperatures (e.g. 2-150C). Moving from an aqueous based composition to one which included only non aqueous based solvents was found to be most effective in providing stable 15 compositions in solution. More-so, an entirely non-aqueous based solvent composition allowed the inventors to increase the concentration of the NSAID beyond what would normally be possible in an aqueous based system. Despite the unexpected increase in site reaction, this did not occur in studies performed by the inventors. 20 A non-aqueous solvent having a dielectric point above 30 at 250C also appeared to be beneficial for the composition's stability. Non-aqueous solvents such ethanol (having dielectric point of 25) was a poor solvent for the present invention and did not provide the same level or solubility/stability as those with a dielectric point above 30. Similarly classed solvents would be expected to have similar beneficial 25 effects. 17 James & Wells Ref: 131431AU/76 Most preferably, the non-aqueous solvent has a dielectric point between 30 and 50. propylene glycol (32.1), NMP (32.2), DMSO (46.7) and DMA (38) were all found to improve stability of the active. Surprisingly such solvents were not seen to increase the site reaction/pain on delivery compared to aqueous based 5 compositions (possibly due to secondary synergistic effect discussed above). Similarly classed solvents would be expected to have similar beneficial effects. Similarly, a non-aqueous solvent having a boiling point above 1OOC was also found to provide improved stability whilst also not increasing site reaction on delivery. Again, ethanol and water were both poor solvents. Instead, solvents 10 such as propylene glycol (BP = 1880C), glycerine formal (BP = 190-1950C), NMP (BP = 202-2040C), DMSO (BP = 1890C), DMA (BP =164-1660C) all were seen to have the beneficial advantages discussed above. Similarly classed solvents would be expected to have similar beneficial effects. Furthermore, inclusion of at least one non-aqueous solvent broadly classified as a 15 dipolar aprotic solvent was found to be particularly advantageous. For example, NMP was seen as a very useful solvent for improving stability yet not increasing site reaction, despite it being considered an irritating solvent and one which causes site reaction on injection. Preferably, the non aqueous solvent is selected from N Methyl-2-Pyrrolidone (NMP), glycerine formal, propylene glycol, and benzyl alcohol. 20 Solvents such as NMP and propylene glycol, which were found to be particularly effective in stabilising the composition and allowing higher concentrations of NSAID, are also known to be high irritants when injected. Therefore, it was particularly surprising to find these solvents did not lead to increased site reaction when the composition was injected. 25 Preferably, the liquid formulation includes at least two different non-aqueous based solvents. 18 James & Wells Ref: 131431AU/76 More preferably, the liquid formulation includes a first and second non-aqueous based solvent, wherein the first solvent acts as a solubilising agent, and wherein the second solvent acts as a carrier solvent. For example, the inventors have identified that a combination of NMP and 5 propylene glycol provide particularly stable actives in the formulation. The inclusion of NMP, glycerine formal and/or benzyl alcohol may be used as solubilising agent for the actives, whereas a solvent such as propylene glycol may be used as a carrier in the composition to provide improved stability. Preferably, only a minimal amount of solubilising agent, such as NMP, is used. 10 The amount of solubilising agent may vary depending on the type and solubility of the active agents in the composition. Preferably, the amount of solubilising agent in the composition is less than 40% w/v. More preferably, the amount of solubilising agent in the composition is 15 approximately 30% w/v. In the present invention, NMP preferably is instead only used at minimal levels and merely as a solubilising agent(s). This is in contrast to Examples 1-4 of WO 02/41899 (WO'899), wherein the main solvent is NMP, and the composition is made to volume with the NMP (in most cases equating to approximately 60% w/v 20 or more in the final composition). The simple use of NMP as a main solvent (disclosed on page 4 of WO'899 as being up to 95% w/v of the final composition) reflects the antibiotics used, i.e. not beta lactam or macrolide type antibiotics. Preferably, the amount of carrier solvent in the composition is at least 30% w/v. More preferably, the amount of carrier solvent in the formulation is at least 40% 25 w/v. 19 James & Wells Ref: 131431AU/76 Preferably, the ratio of the first solvent to the second solvent is between 1:10 and 1:1. More preferably, the ratio of the first solvent to the second solvent is approximately between 1:2 and 1:1. Carrier solvents such as propylene glycol are much harsher as a solvent and are 5 understood to cause more site reaction on injection than NMP. Based on what was generally understood in the art, this is also possibly a further reason why disclosures such as WO'899 may not have directed the reader to using a carrier solvent such as propylene glycol, and instead relied primarily on large amounts of NMP. 10 It is thought that this carrier solvent in the present invention is what helps to provide good stability of the beta-lactam/macrolide antibiotic and NSAID in the composition. WO'899 does not teach towards use of a carrier solvent feature in any way, let alone towards the types of antibiotics used in the present invention. Furthermore, the solvent combinations disclosed in WO'899 would not provide the 15 same stability results as those of the present invention, even if macrolide/beta lactam antibiotics were used instead of the more stable antibiotic/NSAID combinations exemplified in WO'899. As known by the inventors, beta lactam and macrolide antibiotics are notoriously unstable with flunixin, the NSAID focused on in WO'899. In studies conducted by 20 the inventors, similar compositions to those disclosed in WO'899 showed poor stability. Instead, the compositions of the present invention, which are limited to macrolide/beta lactam antibiotics, are shown to have good stability in a range of conditions (seen in Example 6 of the Best Modes section). 20 James & Wells Ref: 131431AU/76 Preferably, the non-aqueous based solvent accounts for over 10% w/v of the composition. More preferably, the non-aqueous based solvent accounts for between 30% w/v to 80% of the composition. As discussed previously, such high levels of non-aqueous 5 solvent (and particularly a carrier solvent in comparison to the solubilising agent) were beneficial in providing more stable compositions and increased concentrations of NSAID/antibiotic, yet it was expected to lead to significantly increased site reaction and pain compared to aqueous based compositions. Yet, this did not transpire. 10 Antioxidant Preferably, the liquid formulation includes at least one antioxidant. Preferably, the antioxidant is selected from sodium formaldehyde sulphoxylate or butylated hydroxyl toluene (BHT). However, a skilled person would appreciate that substantially any antioxidant may be used in the present invention and should not 15 be considered beyond the scope of the invention. Preferably, the antioxidant is present in the liquid formulation at a concentration between 0.01 - 0.3 % w/v, and most preferably approximately 0.05% w/v. Particularly preferred examples of formulations are detailed in the Best Modes section of the specification. 20 Preferred embodiments of the method of treatment Throughout the specification the term administration should be taken as meaning the delivery of actives in the composition to the site of infection, either as a result of local or systemic application (i.e. substantially throughout the blood stream and body of the animal). 21 James & Wells Ref: 131431AU/76 Preferably, the composition is for treating an internal infection. Throughout this specification, the term internal infection should be taken as meaning a site inside the body, which includes bodily cavities which are accessible from outside the body. For instance, this includes cavities such as the mouth, teat canal, udder, 5 anus, vagina and so forth. Preferably, the composition is administered systemically. Examples of systemic administration may include delivery via injection, bolus, or drench, wherein the actives may then be distributed through the animal's bloodstream to the site of infection and/or inflammation. 10 Most preferably, the composition is administered via injection. It should be noted that the injection may occur locally near or at the site of infection whilst still resulting in good systemic delivery of the active. The composition may be injected subcutaneously or intramuscularly. Preferably, the composition is used to treat clinical mastitis. However in some 15 embodiments the composition may be used for the prevention of early stage mastitis. The use of NSAID in a composition to treat mastitis is a new concept, with considerable advantages as discussed throughout this specification. However the inventors acknowledge the composition may be used to treat substantially any type of internal microbial infection and/or associated 20 inflammation/pain. Preferably, the composition is used to treat non-human animals such as cows, sheep, goats or other animals commonly used in the dairy industry. Preferred embodiments of Dosage Regimes 22 James & Wells Ref: 131431AU/76 Preferably, the composition is administered with a dosage of 5-20 mg/kg/day (by weight drug/animal) antibiotic and 0.2 mg - 4 mg/kg/day NSAID. In preliminary trials, this dosage appears to provide the MIC needed for Tylosin (2 pg/ml), such that a therapeutic level of antibiotic is provided. However, due to the 5 synergistic effect identified between the antibiotic and NSAID, the antibiotic may be more effective than normal such that lower levels of antibiotic are required whilst still effectively treating the bacterial infection. The inventors found the amount of NSAID will vary depending on the type. For instance, the preferred approximate dosage of each NSAID may be dependent on 10 it's anti-inflammatory efficacy and safety profiles. Preferably, the dosage of Flunixin is between 1-3 mg/kg/day. Most preferably, the dosage of Flunixin is approximately 2.2 mg/kg/day. Preferably, the dosage of Meloxicam is between 0.1-1 mg/kg/day. Most preferably, the dosage of Meloxicam is approximately 0.5 mg/kg/day. 15 Preferably, the dosage of Ketoprofen is between 2-4 mg/kg/day. Most preferably, the dosage of Ketoprofen is approximately 3 mg/kg/day. Preferably, the dosage of Carprofen is between 0.5-2.5 mg/kg/day. Most preferably, the dosage of Carprofen is approximately 1.4 mg/kg/day. The treatment method may utilise a flexible dosage approach to allow adjustments 20 in treating the disease based on its type and severity. For example, the inventors note that a flexipack may be particularly useful for applying this flexible dosage approach. A flexipack is typically a container made from a mixture of high and low density polyethylene. Such packaging is readily available from many manufacturers. 23 James & Wells Ref: 131431AU/76 Preferred Embodiments of the Method of Preparation As already discussed in this specification, a major advantage of using non aqueous solvents is that the concentration of actives in the composition can be increased. This has numerous benefits, including being able to administer a 5 greater quantity of active to the animal in a lower volume. This may help to decrease administration time, and discomfort to the animal during injection. Furthermore, a lower volume dosage is beneficial for storage as it takes up less space. Preferably, the NSAID in the composition is increased to a concentration (% w/v) 10 which would not be storage stable at room temperature in a corresponding aqueous based composition. Preferably, the NSAID is Ketoprofen and wherein the concentration of Ketoprofen is above 4% w/v. Preferred aspects of the manufacturing process are now discussed below. 15 Preferably, a first and second non-aqueous solvent is added to a mixing container prior to the addition of either the NSAID or antibiotic. The first and second non-aqueous solvents chosen may depend on the type, number or concentration of NSAID and antibiotic used in the formulations. Examples of first and second non aqueous solvents are provided in the Best 20 Modes section. However, these examples are not intended to be limiting. In some embodiments, additional non aqueous solvents may be added. Alternatively, it may be appropriate to only include one non-aqueous solvent. 24 James & Wells Ref: 131431AU/76 Preferably, the non-aqueous solvent(s) are heated to approximately 55-800C prior to the addition of either the NSAID or antibiotic. The temperature used may depend on the specific actives used in the composition. Preferably, the antibiotic is added to the non aqueous solvent(s) and dissolved 5 prior to the addition of the NSAID. The NSAID may then be added and dissolved into the mixture of dissolved antibiotic and non aqueous solvent(s). It was found by the inventors that this step wise addition of solvents, antibiotic and finally NSAID provided improved solubility and stability of the composition. Preferably, the temperature is maintained at 55-80C throughout the step-wise 10 addition of the NSAID and antibiotic to the formulation. The composition containing the NSAID and antibiotic is then cooled to approximately room temperature (e.g. between 20-30C) before making up to a final volume (qs) with a non aqueous solvent and being mixed for approximately 10-20 minutes. 15 The composition may then be stored at approximately 40C prior to administration. Some advantages of the present invention include: - Allows synergistic relationship between NSAID and antibiotic (NSAID improves effectiveness of antibiotic). - Use of a non aqueous solvent helps to overcome issues of incompatibility 20 between actives, and in particular instability of many antibiotics. - Use of a non aqueous solvent allows the composition to be provided as a stable composition in solution (not a suspension) over a range of storage conditions. - A solution avoids caking, the need for re-suspension of the composition 25 James & Wells Ref: 131431AU/76 prior to delivery, and problems with injection (clogging of the needle, and potential pain to the animal from the particulate matter). - Use of non aqueous solvents allows the concentration of the NSAID to be increased beyond levels obtainable in an aqueous based composition. 5 Despite the expected increase in site reaction and pain on delivery due to the known traits of non-aqueous solvents, an increase in these side effects were not seen. - The increase in the NSAID obtained may also help to lower the site reaction caused, not only by the solvents, but potentially also the antibiotic (tylosin is 10 a known irritant). Potentially, the concentration of tylosin in the composition may be increased due to the lower site reaction on injection. - By allowing increased concentration of NSAID in the composition, the synergy seen between the two actives at the site of infection is thought to be increased. By increasing the effectiveness of the antibiotic, lower 15 concentrations of antibiotic could potentially be used to provide the same result. - Avoidance of water in the composition prevents the need to adjust the composition to a desired pH, removing additional steps in the manufacturing process. 20 - Reduces pain and inflammation which may be associated with internal infections. - Improves efficiency of administration (only one composition opposed to two). For example, the combination of antibiotic (e.g. tylosin) and NSAID in a single product negates the need for multiple injections and will be easier 25 to administer with better compliance and improved treatment outcomes. 26 James & Wells Ref: 131431AU/76 - Flexible delivery options. - Preferred administration (injection) allows improved drug distribution - NSAID improves animal welfare while antibiotic is acting - In addition to reducing pain and fever, NSAIDs may bind with the 5 endotoxins (G-negative bacteria) improving treatment outcomes, and as such will become an important component of mastitis treatment regimens in the future. - Formulation likely to be particularly effective in treating Gram-negative mastitis which is expected to rise in prevalence in New Zealand over the 10 coming years. BEST MODES FOR CARRYING OUT THE INVENTION Example 1 Table 1 below illustrates a number of initial formulations trialed by the inventors in accordance with the present invention. 15 Below are comments on the suitability of the formulations trialed. 27 James & Wells Ref: 131431AU/76 Table 1: Tylosin/Flunixin combinations F1 F2 F3 F4 F5 F6 F7 F8 # Ingredients %w/v %w/v %w/v %w/v %w/v %w/v %w/v %w/v 1. Tylosin base ** 20.4 -- 20.4 20.4 20.4 20.4 20.4 20.4 Flunixin meglumine 2. (7.3 % provides 4.4% Flunixin) -- 8.3 7.3 7.3 7.3 7.3 7.3 7.3 3. Sodium formaldehyde sulphoxylate -- 0.05 0.05 0.05 --- -- -- - 4. Butylated hydroxyl toluene (BHT) -- -- -- -- 0.05 0.05 0.05 0.05 5. Di sodium edetate -- 0.1 0.1 0.1 -- 0.1 0.1 - 6. N-Methyl -2-Pyrrolidone (NMP) -- -- -- -- -- -- -- 20 7. Glycerine formal stabilised -- -- -- -- 40 -- 4.18 - 18%v 8. Propylene glycol 52 /v 52 52 qs 52 52 qs 9. Phenol Liquified -- 0.25 -- -- -- -- -- - 10. Benzyl alcohol 4.18 0 4.18 4.18 -- 4.18 1 1 Monoethanolamine 11. (for pH adjustment) -- qs qs -- -- -- -- - Sodium hydroxide 12. (for pH adjustment) -- -- -- qs -- qs qs - Hydrochloric acid 13. for pH adjustment) -- qs qs qs -- qs qs - 14. Water for Injection qs qs qs qs -- qs qs - ** 2% overages added *** For PH adjustment between (8- 9.5) F1 An existing formulation of Tylosin base injection which is registered with 5 ACVM. F2 An existing formulation of Flunixin injection (Brand Name: Fluxamine injection) which is registered with ACVM. 28 James & Wells Ref: 131431AU/76 F3 Product color turned to dark brown within 48hrs at room temperature. F4 pH adjustment was achieved with 20% sodium hydroxide. Product color remained stable at 4 and 400C. F5 To avoid a pH issue, the product was formulated using glycerine formal + 5 propylene glycol. Product color remained stable at 4 and 400C. F6 Antioxidant was changed to BHT instead of sodium formaldehyde sulfoxylate. Product color remained stable at 4 and 400C. F7 Glycerine formal stabilised was incorporated to stabilize the formulation. Product color remained stable at 4 and 400C. 10 F8 Product color remained stable at 4 and 400C. Summary Formulation F5 appeared the most stable of the Tylosin/Flunixin formulations. One method of preparing the F5 formulation is provided below. Manufacturing Procedure for formulation F5 In a clean and dry manufacturing vessel: 1. Load required quantity of glycerine formal stabilised. 2. Add and mix 25% of propylene glycol. 3. Heat the mixture to 70 -80C. 4. Add and dissolve Tylosin Base and mix well (maintain the temp at 70 800C). 5. Add and dissolve flunixin meglumine and mix well (maintain the 29 James & Wells Ref: 131431AU/76 temperature at 70-800C). 6. Cool the bulk to below 300C. 7. Make up the final volume with propylene glycol and mix well for 10 minutes. Example 2 Two other formulations (F9 and F10) were trialed, containing Tylosin with either Carprofen or Meloxicam. These formulations and manufacturing procedures are provided below. 5 Table 2: Tylosin/Carprofen and Tylosin/Meloxicam Combinations F9 F10 SI No Ingredients %w/v %w/v 1 Tylosin base** 21 21 2 Carprofen** 3 0 4 Meloxicam** 0 1.05 5 Butylated hydroxytoulene (BHT) 0.05 0.05 6 NMP 20 20 7 Benzyl alcohol 1 1 8 Propylene glycol qs Qs ** 5% averages added 30 James & Wells Ref: 131431AU/76 Manufacturing procedure for F9 and F10 1. In a clean and dry manufacturing vessel. 2. Load required quantity of NMP. 3. Add and mix benzyl alcohol. 4. Add and mix (35%) of propylene glycol. 5. Heat the mixture to 70 -80C. 6. Add and dissolve Tylosin Base and mix well (maintain the temperature at 70-80C). 7. Add and dissolve meloxicam/carprofen and mix well (maintain the temperature at 70 80C). 8. Cool the bulk to below 300C. 9. Make up the final volume with propylene glycol and mix well for 10 minutes. 31 James & Wells Ref: 131431AU/76 Example 3 The inventors then trialed a combination containing Tylosin/Ketoprofen. SI no (Fw/ ) Ingredients w 1 Tylosin base ** 21 Ketoprofen BP 2 4.08 3 Propylene glycol 52 4 Benzyl alcohol 4.18 Sodium hydroxide 10%w/v solution (for pH 5 adjustment) qs Hydrochloric acid solution 5%w/v (if 6 required) qs water for 7 Injection qs ** 5% averages added (label claim 20%) *** 2% averages added (label claim 4%) In this aqueous based composition, the Tylosin/Ketoprofen was stable. However, when the inventors increased the concentration of Ketoprofen above 4% w/v, the composition lost its stability in an aqueous based composition. 32 James & Wells Ref: 131431AU/76 Manufacturing procedure for F11 1. In a clean manufacturing vessel, load the required quantity of propylene glycol, benzyl alcohol and mix well. 2. Heat the mixture to 60-700C. 3. Add and dissolve Tylosin Base and mix well (maintain the temperature at 60-700C). Check the clarity of the solution. 4. Add and dissolve Ketoprofen and mix well. Cool the bulk to room temperature. 5. Check pH. It should be between 8.8 to 9.5. If required, adjust the pH between 8.8-9.5 using 10% w/v sodium hydroxide solution or 5% w/v hydrochloric acid. 6. Make up the final volume with water for injection and mix well for 10-15 minutes. 7. Check pH. It should be between 8.8-9.5. 33 James & Wells Ref: 131431AU/76 Example 4 The inventors aimed to provide a composition containing higher levels of NSAID Ketoprofen than seen in Example 3, together with Tylosin. The rationale was that an increased amount of NSAID would synergistically aid the antibiotic's 5 effectiveness at the site of infection, whilst also providing increased pain relief to the animal. Without wanting to revert to a suspension, the inventors had to trial stronger (non aqueous) based solvents in order to stabilize the NSAID at higher loading. Achieving a stable composition in solution with successful. However, using non 10 aqueous solvents was expected to result in the composition causing a stronger site reaction and pain upon injection. SI no (F12) Ingredients % w/v 1 Tylosin base ** 21 2 Ketoprofen BP 6 3 Butylated Hydroxytoluene (BHT) 0.05 4 Benzyl Alcohol 1 5 N-Methyl-2-Pyrrolidone (NMP) 30 6 Propylene glycol To 100 ml ** 5% averages added (label claim 20%) 34 James & Wells Ref: 131431AU/76 Manufacturing procedure for F12 1. In a clean and dry manufacturing vessel, load required quantity of N-Methyl 2-Pyrrolidone (NMP). 2. Load the required quantity of benzyl alcohol to the vessel and mix well. 5 3. Load Propylene Glycol (35% of the batch size) to the vessel and mix well. 4. Heat the bulk to 55-650C. 5. Add and dissolve Butylated Hydroxy toluene (BHT) with mixing. 6. Add and dissolve Tylosin base with mixing (maintaining the temperature at 55-650C). 10 7. Add and dissolve Ketoprofen with mixing. 8. Check the clarity of the solution. 9. Make up the volume to 98% of the batch size with Propylene Glycol and mix well. 10. Cool the bulk to 25-30 0 C with mixing. 15 11. Make up the final volume with Propylene Glycol and mix well. 35 James & Wells Ref: 131431AU/76 Example 5: Preliminary Animal studies to test site reaction on injection A study was conducted to compare therapeutic equivalence and side effects (site reaction and pain on delivery) between a test composition containing 20% Tylosin and 6% Ketoprofen (as per Example 4) and industry standards. 5 The industry standards were Tylan (Elanco Animal Health, Auckland, New Zealand) and Ketofen (Merial New Zealand, Auckland, New Zealand) when co administered into the muscle of lactating dairy cattle. Tylan has 20% w/v Tylosin; with a prescribed dosage of 10 mg/kg body weight. Ketofen has 10% w/v ketoprofen with a prescribed dosage of 3 mg/kg. Both Tylan and Ketofen are 10 aqueous based compositions. Twenty cattle at various stages of lactation, ranging in age from 4 to 6 years old were chosen from a herd of approximately 1200 cattle. Animals were blocked in pairs and randomly allocated to Group 1 or Group 2. The study was a two-way cross-over in which each group was treated with each of two 15 treatments over the duration of the animal phase of the study, thus giving 20 animals treated for each treatment period. Dosage was kept constant between two Groups, namely 10 mg/kg for Tylosin, 3 mg/kg for Ketoprofen. Following milking on Day 0, cattle were treated based on individual live weight. Blood samples were collected at 11 time points and recorded in real time over a 24 20 hour period. As indicated below, the studies revealed bioequivalence between the test composition and the industry standards. 36 James & Wells Ref: 131431AU/76 03 I *2 U 5 10' is 2) 25 30 Ha' pest4 eatment Solid line represents Tylosin serum levels from using the test composition from Example 4. Dotted line represents Tylosin serum levels from using Tylan. 5 4 Q ,2 0 1 152 25 20 Hours post-treatment Solid line represents Ketoprofen serum levels from using the test composition from Example 4. Dotted line represents Ketoprofen serum levels from using Ketofen. 37 James & Wells Ref: 131431AU/76 In brief, the study results showed similar levels of bioequivalence between two compositions when dosages were kept constant. This illustrates that the combination composition has good bioavailability compared to industry standards which are administered separately. This in itself is a major advantage. 5 A second major advantage identified from this study, contrary to expectations, was that the test composition did not show any added adverse reactions (site reaction or pain to the animal) compared to the industry standards (Tylan and Ketofen). This was unexpected because the non-aqueous solvent used in the test composition in Example 4 was thought to cause adverse side effects upon 10 injection. Considering that the dosage remained the same in the study in Group 1 and Group 2, it may be expected that an increased dosage of NSAID (e.g. Ketoprofen) beyond 3 mg/kg could be achieved whilst avoiding increased (or even decreasing) site reaction/pain compared to currently available compositions. Increased dosage through increased concentration of NSAID may be achieved 15 because a non-aqueous solvent is used to stabilise the active in the composition. 38 James & Wells Ref: 131431AU/76 Example 6: Stability trials A stability study was conducted on three 4.5 L batches of compositions exemplified in Example 4. The batches were packed in 100 ml glass amber vials and stored at the designated 5 storage conditions of 25 C/60& RH, 30 0 C/65%RH and 40 0 C/75%RH. The physical and chemical characteristics of the product were recorded at regular intervals as recommended by the ACVM. As shown in the tables below, the compositions were stable for at least six months at 25 0 C/60%RH and 30 0 C/65%RH. 10 Storage Conditions: 25 0 C/60%RH Test Specification Initial 3 Months 6 Months Description Clear golden yellow, slightly Complies Complies Complies viscous solution Relative 1.050 - 1.100 1.079 1.079 1.078 density Tylosin base 180 - 230 mg/ml 206 207 203 (Total) Ketoprofen 54 - 66 mg/ml 59.1 59.6 59.3 Syringeability (only for - Complies Complies Complies stability) No growth in Fluid Thioglycollate Medium after 14 days incubation at 30 - 35 0 C Sterility (BP) No growth in Soyabean-Casein Complies - Complies Digest Medium after 14 days incubation at 20 - 25 0 C 39 James & Wells Ref: 131431AU/76 Storage Conditions: 30 0 C/65%RH Test Specification Initial 3 Months 6 Months Description Clear golden yellow, slightly Complies Complies Complies viscous solution Relative 1.050 - 1.100 1.079 1.079 1.079 density Tylosin base 180 - 230 mg/ml 206 205 200 (Total) Ketoprofen 54 - 66 mg/ml 59.1 59.6 59.8 Syringeability (only for - Complies Complies Complies stability) No growth in Fluid Thioglycollate Medium after 14 days incubation at 30 - 350C Sterility (BP) No growth in Soyabean-Casein Complies - Complies Digest Medium after 14 days incubation at 20 - 25 0 C Storage Conditions: 40 0 C/75%RH Test Specification Initial 1 Month 3 Months 6 Months Description slghty viscous sluon Complies Complies Complies Complies Relative 1.050 - 1.100 1.079 1.085 1.079 1.078 density Tylosin base 180 - 230 mg/ml 206 207 201 184 (Total) Ketoprofen 54 - 66 mg/ml 59.1 59.7 59.3 58.9 Syringeability (only for - Complies Complies Complies Complies stability) No growth in Fluid Thioglycollate Medium after 14 days incubation at 30 - 35 0 C Sterility (BP) No growth in Soyabean- Complies - - Complies Casein Digest Medium after 14 days incubation at 20 - 25 0 C 40 James & Wells Ref: 131431AU/76 Storage Conditions: 25 0 C/60%RH Test Specification Initial 3 Months 6 Months Description Clear golden yellow, slightly Complies Complies Complies viscous solution Relative 1.050 - 1.100 1.075 1.079 1.078 density Tylosin base 180 - 230 mg/ml 210 210 205 (Total) Ketoprofen 54 - 66 mg/ml 60.2 59.9 60.3 Syringeability (only for - Complies Complies Complies stability) No growth in Fluid Thioglycollate Medium after 14 days incubation at 30 - 350C Sterility (BP) No growth in Soyabean-Casein Complies - Complies Digest Medium after 14 days incubation at 20 - 25 0 C Storage Conditions: 30 0 C/65%RH Test Specification Initial 3 Months 6 Months Description Clear golden yellow, slightly Complies Complies Complies viscous solution Relative 1.050 - 1.100 1.075 1.079 1.078 density Tylosin base 180 - 230 mg/ml 210 208 200 (Total) Ketoprofen 54 - 66 mg/ml 60.2 59.8 59.9 Syringeability (only for - Complies Complies Complies stability) No growth in Fluid Thioglycollate Medium after 14 days incubation at 30 - 35 0 C Sterility (BP) No growth in Soyabean-Casein Complies - Complies Digest Medium after 14 days incubation at 20 - 25 0 C 5 41 James & Wells Ref: 131431AU/76 Storage Conditions: 40 0 C/75%RH Test Specification Initial 1 Month 3 Months 6 Months Description sl viscous soluto n Complies Complies Complies Complies Relative 1.050 - 1.100 1.075 1.084 1.079 1.078 density Tylosin base 180 - 230 mg/ml 210 209 198 183 (Total) Ketoprofen 54 - 66 mg/ml 60.2 59.7 60.2 58.9 Syringeability (only for - Complies Complies Complies Complies stability) No growth in Fluid Thioglycollate Medium after 14 days incubation at 30 - 35 0 C Sterility (BP) No growth in Soyabean- Complies - - Complies Casein Digest Medium after 14 days incubation at 20 - 25 0 C Aspects of the present invention have been described by way of example only and 5 it should be appreciated that modifications and additions may be made thereto without departing from the scope thereof as defined in the appended claims. 42

Claims (35)

1. An injectable composition when used for the treatment of a microbial infection in a mammary gland of an animal, wherein the composition includes a. a non-steroidal anti-inflammatory drug (NSAID); b. an antibiotic selected from the group consisting of a beta lactam antibiotic and macrolide antibiotic; c. a carrier characterised in that the composition includes at least two non-aqueous solvents, wherein at least one non-aqueous solvent is a solubilising agent; and at least one non-aqueous solvent is a carrier, wherein the amount of carrier in the composition is more than the amount of solubilising agent in the composition; wherein the solubilising agent has the property of dissolving the NSAID and/or antibiotic more readily than a carrier; and wherein the NSAID and antibiotic are dissolved in the at least two non aqueous solvents.

2. The composition as claimed in claim 1 wherein the composition is non aqueous. 43 James & Wells Reference: 131431 AU/76

3. The composition as claimed in claim 1 or claim 2 wherein the non aqueous solvent has one or more of the following features: a) has a dielectric point above 30 at 250C b) has a boiling point above 100 C and/or c) is a dipolar aprotic solvent.

4. The composition as claimed in any one of the above claims wherein the at least two non aqueous solvents are selected from the group consisting of N-Methyl-2-Pyrrolidone (NMP), dimethyl sulfoxide (DMSO), dimethylacetamide (DMA) glycerine formal, propylene glycol, benzyl alcohol, and combinations thereof.

5. The composition as claimed in claim 4 wherein the first non aqueous solvent is either glycerine formal or NMP and wherein the second non aqueous solvent is propylene glycol.

6. The composition as claimed in any one of the above claims wherein the NSAID is selected from Carprofen, Naproxen, Ibuprofen, Ketoprofen, Piroxicam, Diclofenac, Etodolac, Flunixin, Feracoxib, Meloxicam, Celecoxib, Rofecoxib, and combinations thereof.

7. The composition as claimed in any one of the above claims wherein the NSAID is selected from Flunixin, Meloxicam, Carprofen, Ketoprofen and combinations thereof.

8. The composition as claimed in any one of the above claims wherein the NSAID is present at a concentration of 1 - 15% w/v.

9. The composition as claimed in claim 7 wherein the concentration of 44 James & Wells Reference: 131431 AU/76 Flunixin is approximately 7.3 % w/v, the concentration of Meloxicam is approximately 1.0 % w/v, the concentration of Carprofen is approximately 3.0 % w/v or the concentration of Ketoprofen is approximately 4 - 10 % w/v.

10. The composition as claimed in any one of the above claims wherein the macrolide class antibiotic is selected from Azithromycin, Clarithromycin, Dirithromycin, Erythromycin, Roxithromycin, Telithromycin, Carbomycin A, Josamycin, Kitasamycin, Midecamicine/midecamicine acetate, Oleandomycin, Spiramycin, Troleandomycin, and Tylosin/tylocine, and combinations thereof.

11. The composition as claimed in any one of the above claims wherein the macrolide antibiotic is Tylosin.

12. The composition as claimed in any one of the above claims wherein the beta lactam antibiotic is penethamate.

13. The composition as claimed in any one of the above claims wherein the antibiotic is present at a concentration between 10 - 35% w/v.

14. The composition as claimed in any one of the above claims wherein the antibiotic is present at a concentration of approximately 20.0% w/v.

15. The composition as claimed in any one of claims 1 to 14 wherein the ratio of NSAID to antibiotic in the composition is approximately 1:3 w/v.

16. The composition as claimed in any one of the above claims wherein the composition includes at least one anti-oxidant.

17. The composition as claimed in claim 16 wherein the anti-oxidant is 45 James & Wells Reference: 131431 AU/76 selected from sodium formaldehyde sulphoxylate or butylated hydroxyl toluene (BHT), and combinations thereof.

18. The composition as claimed in either claim 16 or 17 wherein the anti oxidant is present in the composition at a concentration of between 0.01 - 0.3 % w/v.

19. A method of treating a non-human animal for an internal microbial infection wherein there is provided a method of treating a non-human animal for an internal microbial infection characterised by the step of administering a composition as claimed in any one of claims 1 to 20 by injection to the animal.

20. The method as claimed in claim 19 wherein the composition is injected subcutaneously.

21. The method as claimed in claim 19 or 20 wherein the non-human animal is treated with a dosage 5 - 10 milligrams antibiotic per kg of non-human animal per day (mg/kg/day) and 0.2 -4 mg/kg/day of NSAID.

22. The method as claimed in any one of claims 19 to 21 wherein the non human animal is treated with 5-10 mg/kg/day antibiotic and any one of the following approximate dosages for the NSAID: a. 2.2 mg/kg/day Flunixin b. 0.5 mg/kg/day Meloxicam 46 James & Wells Reference: 131431 AU/76 c. 3 mg/kg/day Ketoprofen d. 1.4 mg/kg/day Carprofen.

23. The method as claimed in any one of claims 19 to 22 wherein the composition is used to treat clinical mastitis and / or early stage mastitis.

24. A use of a composition substantially as claimed in any of claims 1 to 18 in the manufacture of a medicament for the treatment of a microbial infection.

25. A method of preparing a composition when used for the treatment of a microbial injection in the mammary gland containing an NSAID and a antibiotic selected from the group consisting of a macrolide and beta lactam antibiotic characterised by the steps of a) adding at least two non aqueous solvents to the composition, wherein at least one non-aqueous solvent is a solubilising agent and at least one non-aqueous solvent is a carrier, wherein the amount of carrier in the composition is more than the amount of solubilising agent in the composition; b) dissolving the NSAID and antibiotic in the at least two non aqueous solvents.

26. The method as claimed in claim 25 wherein the NSAID in the composition is increased to a concentration (% w/v) which would not be storage stable at room temperature in a corresponding aqueous based composition.

27. The method as claimed in claim 25 or claim 26 wherein the NSAID is 47 James & Wells Reference: 131431 AU/76 Ketoprofen and wherein the concentration of Ketoprofen is above 4% w/v.

28. The method as claimed in any one of claims 25 to 27 wherein the at least two non aqueous solvents are added to a mixing container prior to the addition of either the NSAID or antibiotic.

29. A method as claimed in any one of claims 25 to 28 wherein the carrier is added to the solubilising agent prior to the addition of either the NSAID or antibiotic.

30. A method as claimed in any one of claims 25 to 29 wherein the composition is maintained between 60-80C until after the NSAID and antibiotic have been substantially dissolved in the at least two non aqueous solvents.

31. A method as claimed in any one of claims 25 to 30 wherein the antibiotic is added to the at least two non aqueous solvents and dissolved prior to the addition of the NSAID.

32. A method as claimed in any one of claims 25 to 31 wherein the NSAID is added and dissolved into the mixture of dissolved antibiotic and at least two non aqueous solvents.

33. A method as claimed in any one of claims 25 to 31 wherein the composition containing the dissolved NSAID and antibiotic is cooled to approximately 300C before making up to a final volume (qs) with a non aqueous solvent and mixed for approximately 10 minutes.

34. A composition as substantially herein described and with reference to the formulations F3-F1O shown in Examples 1-3 in the Best Modes section. 48 James & Wells Reference: 131431 AU/76

35. A method of preparing a composition including an NSAID and an antibiotic substantially as herein described and with reference to the manufacturing procedure outlined in Examples 1 -3 the Best Modes section. 49