NZ769844A - Method of treatment of infection - Google Patents

Abstract

The disclosure relates to an injectable composition 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) N-Methyl-2-pyrrolidone (NMP) as a solubilising agent; and (d) Propylene glycol as 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 non-aqueous solvents of N-Methyl-2-pyrrolidone and propylene glycol.

Description

James & Wells Reference: DIV6/110 METHOD OF TREATMENT OF INFECTION TECHNICAL FIELD The invention relates to a composition for the treatment of ion, and most preferably, although not specifically for the treatment of is.

BACKGROUND ART The present ion 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 present invention can be applied to other animals including humans. Throughout this ication, specific examples are provided in relation to the ent of mastitis. It should also be iated that the scope of the invention is not limited to mastitis, and can relate to substantially any form of bacterial infection in an animal.

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 l. A preferred ation 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.

Secondly, with cases such as mastitis which adversely affects milk production, there is a James & Wells Reference: 131431DIV6/110 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 tions governing the introduction of animal products into the food chain (whether eggs or meat) from animals having been recently d 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.

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) - ally during peak lactation.

There is a need to find new ents which are more convenient to administer, but are still efficacious. This may result in better ance and better treatment es (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 ive bacteria.

Minimum Inhibitory Concentrations (MIC) of antibiotic required to inhibit specific ia 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 otics such as Tylosin containing products are routinely prescribed. However, little work has been done internationally to establish the MIC for Tylosin vs S. aureus. Most MICs are established using Erythromycin, which is less commonly used for treatment of James & Wells Reference: DIV6/110 mastitis.

The MIC for erythromycin vs S. aureus has been reported to be 0.5μg/ml, whereas the MIC for Tylosin vs S. aureus is 2 μg/ml. ially, this means the concentration of n required to effectively treat S. aureus is approximately 4-fold that which is required of omycin.

Indeed, cokinetic studies have shown that a 20% Tylosin injectable preparation only es peak concentrations of cf 1.4 μg/ml during treatment thus failing to achieve the required therapeutic concentrations (MIC). A s such 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 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 otic treatment has been found 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 otic.

It can also be important consider animal welfare. Infections such as mastitis are painful.

Therefore, it has become common practice to treat not only the infection, but also the pain associated with it using active agents such as an NSAID. This combination James & Wells Reference: DIV6/110 treatment is relatively new in the industry.

US 2005/0277634 discloses a ation of NSAID (meloxicam) and an antibiotic hamate hydroiodide) in a suspension ation for injection to treat mastitis.

They s 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 otic appears to function.

However, there are considerable disadvantages to a suspension formulation. These include potential caking, ms with dispersability before use, difficulty in delivery, site reaction/pain after injection and poor absorption of the (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 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.

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 ley et al., Pharmaceutical Research, Vol., 21 No.2 February 2004 titled “Solubilizing Excipients in Oral and Injectable Formulations”.

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 James & Wells Reference: 131431DIV6/110 possible. As a consequence, this can lower the ability to achieve higher concentrations of the active agent(s), lower the stability of the composition 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 ry, aqueous based compositions can again lead to instability of the active 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 9) discloses n or injectable itions including an antibiotic and an analgesic both dissolved in solvent. r, 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 unstable and are likely to react with NSAIDs such as flunixin. Despite difficulties in combining beta lactam and macrolide otics 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 ting that many of the compositions, ularly those with a higher active agent concentration (such as that in Example 4), would not be e stable. Indeed, studies ted 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 James & Wells Reference: 131431DIV6/110 provide the public with a useful choice.

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 nces 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 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 dge in the art, in New Zealand or in any other country.

Throughout this specification, the word “comprise”, or variations thereof such as “comprises” or ising”, will be understood to imply the inclusion of a stated element, integer or step, or group of elements rs 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.

DISCLOSURE OF INVENTION According to a first aspect of the present invention there is provided an injectable ition 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 ting of a beta lactam antibiotic and ide antibiotic; James & Wells Reference: 131431DIV6/110 characterised in that: the composition includes a ueous solvent, and the NSAID and antibiotic in the composition are dissolved in the non-aqueous solvent.

According to a r aspect of the present invention there is provided a method of treating an animal for an internal microbial infection characterised by the step of administering a composition substantially as described above by injection. ing 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 ment for the treatment of a ial infection.

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.

BRIEF PTION OF DRAWINGS Further aspects of the present invention will become apparent from the ensuing description which is given by way of example only and with reference to the accompanying gs in which: Figure 1 describes the Tylosin serum levels from using the test composition from Example 4 (solid line) versus from using Tylan (dotted line); and Figure 2 describes the Ketoprofen serum levels from using the test composition from Example 4 (solid line) versus from using Ketofen (dotted line).

Discussion of the advantages provided by the invention James & Wells Reference: 131431DIV6/110 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.

In the past, NSAIDs have been administered more-so to treat inflammation and/or pain.

The inventors have identified an ant 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 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 ations. This may be partially due to the ability to provide the two actives together in a stable composition.

The ors consider that, without being limited to the proposed mode of action, 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 istic effect may be partially attributed to the NSAID binding xins (endotoxaemia), which the inventors otherwise found hindered antibiotic effectiveness.

For instance, G-negative ia, which produce oxins, are associated with mastitis. Such bacteria can cause systemic illness (fever and xaemia). Although this is currently relatively uncommon in New Zealand (less that 1% incidence), the increased use of feed-pads and herd homes may result in higher nce 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 James & Wells Reference: 131431DIV6/110 example in mastitis.

Additionally, the synergistic effect may be further enhanced as the NSAID may act 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 ments and identifying the advantageous result, a skilled person would appreciate that the synergistic result would be expected for common types of otics, 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).

A further significant advantage of the combination fied 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 le compositions, storage of compositions, costs, ing and so forth. The inventors were able to identify a particular solvent type that aided the combination of the two actives, which usly 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.

The ors identified a further, unexpected, synergistic effect between the two actives as the tration of the NSAID was increased, as discussed below. The aim of increasing the concentration of NSAID was to improve the synergistic effect initially identified by the ors to help improve antibiotic effectiveness at the site of infection.

James & Wells Reference: 131431DIV6/110 Active agents have recommended dosages based on what has been established 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 ition. This high concentration of Tylosin can often be the limiting factor in such a composition as it can cause significant tion on 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 ant aspect of the successfulness of the active composition to provide the t amounts of active when administered.

However, when the inventors ted to provide a composition with 6% Ketoprofen, the composition was not stable with an s based t. With other NSAIDs tested, similarly poor stability s were seen in an aqueous based composition if NSAIDs were included at required concentrations to reflect recommended dosages.

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 nonaqueous solvent system in attempt to provide a stable composition in solution at various conditions (e.g. temperatures above 4°C). However, it was ed, as generally understood in the art, that reverting to non-aqueous solvents would lead 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 itions (e.g. Tylan and Ketofen) which are aqueous-based itions.

Without wanting to being limited to a particular mode of action, it is thought that the sed NSAID synergistically helps to alleviate the inflammation/pain locally at the James & Wells nce: 131431DIV6/110 site of injection. Therefore, it is possible that the additional amounts of NSAID are counter-acting against the harsher ts which would normally increase site reaction and pain on injection.

Therefore, ctantly, 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 . Importantly, this was achievable without resulting in an 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 infection may then also be enhanced, improving the overall treatment outcome.

Preferred ments 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 eutic ation that is of a “syringable” consistency.

This is the mode of administration preferred by many s 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 ity at lower temperatures.

The liquid formulation is a solution. This should be taken as meaning a composition wherein all the ents and active agents are substantially solubilised in the solution, and not in suspension. As previously discussed, a solution provides many advantages over suspensions. For e, there are no issues with caking, re-suspension, or [Link] http://en.wikipedia.org/wiki/Anti-inflammatory [Link] http://en.wikipedia.org/wiki/Glucocorticoid James & Wells Reference: 131431DIV6/110 difficulties with injection.

The on 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.

There are many different types of NSAIDs known in the art. It should be considered within the scope of the ion 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 ting of Carprofen, Naproxen, Ibuprofen, Ketoprofen, Piroxicam, Diclofenac, Etodolac, Flunixin, 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 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 sed previously, it may be able to increase the concentration of NSAID to improve the synergistic effect n the two actives. Although the ts of doing so would be well iated, providing such a combination in a stable composition in solution has not been achieved, let alone one where the composition James & Wells Reference: DIV6/110 shows no greater site reaction/pain on delivery compared to aqueous based compositions.

Preferably, the NSAID flunixin ine is present in the composition at a tration 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 ent on it’s anti-inflammatory efficacy and safety profiles, as well as the concentration of antibiotic used in the composition.

Preferably, the NSAID Meloxicam is t 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.

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 ofen is 9% w/v.

Alternatively, the NSAID is ed from natural forms of anti-inflammatory agents. For example, these may include green-lipped mussel extract, omega 3, and the like.

[Link] file:///C:/wiki/Deoxy_sugar [Link] //C:/wiki/Cladinose [Link] file:///C:/wiki/Desosamine [Link] http://en.wikipedia.org/wiki/Azithromycin [Link] http://en.wikipedia.org/wiki/Clarithromycin [Link] http://en.wikipedia.org/wiki/Dirithromycin [Link] http://en.wikipedia.org/wiki/Erythromycin [Link] http://en.wikipedia.org/wiki/Roxithromycin [Link] http://en.wikipedia.org/wiki/Telithromycin [Link] http://en.wikipedia.org/w/index.php?title=Carbomycin_A&action=edit&redlink=1 [Link] /en.wikipedia.org/wiki/Josamycin [Link] http://en.wikipedia.org/wiki/Kitasamycin [Link] http://en.wikipedia.org/w/index.php?title=Midecamicine&action=edit&redlink=1 [Link] http://en.wikipedia.org/w/index.php?title=Midecamicine_acetate&action=edit&redlink=1 [Link] http://en.wikipedia.org/wiki/Oleandomycin [Link] http://en.wikipedia.org/wiki/Spiramycin [Link] http://en.wikipedia.org/wiki/Troleandomycin [Link] http://en.wikipedia.org/wiki/Tylosin [Link] http://en.wikipedia.org/w/index.php?title=Tylocine&action=edit&redlink=1 [Link] http://en.wikipedia.org/wiki/Macrolide [Link] http://en.wikipedia.org/wiki/Antibiotic James & Wells Reference: 131431DIV6/110 Throughout the specification the term antibiotic should be taken as meaning a substance or nd, either natural, synthetic or semi-synthetic, that kills ia (bactericidal) and/or inhibits bacterial growth (bacteriostatic).

The antibiotic in the composition is selected from a group ting of macrolide and beta-lactam antibiotics.

Throughout this specification, the term macrolide otic 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 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 otic is selected from Azithromycin, Clarithromycin, Dirithromycin, Erythromycin, Roxithromycin, Telithromycin, Carbomycin A, Josamycin, Kitasamycin, Midecamicine/midecamicine acetate, Oleandomycin, Spiramycin, Troleandomycin, and Tylosin/tylocine.

Most preferably, the macrolide antibiotic is Tylosin.

Tylosin is a macrolide-class antibiotic used in nary 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.

[Link] http://en.wikipedia.org/wiki/Staphylococci [Link] http://en.wikipedia.org/wiki/Streptococci [Link] http://en.wikipedia.org/wiki/Corynebacteria [Link] http://en.wikipedia.org/wiki/Erysipelothrix [Link] http://en.wikipedia.org/w/index.php?title=Campylobacter_coli&action=edit&redlink=1 [Link] http://en.wikipedia.org/wiki/Spirochaete James & Wells Reference: 131431DIV6/110 n has a wide spectrum of activity against gram positive bacteria including Staphylococci, Streptococci, Corynebacteria, and Erysipelothrix. It also has ty towards gram ve Campylobacter coli, and some spirochaetes. It is also active against Mycoplasma species. able Tylosin formulations can cause pain, inflammation, and itchiness around the injection site. Therefore, it is important to be able to reduce the above side s when Tylosin is injected. The provision of an antibiotic such as n 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 otic when administered in one formulation. Throughout this specification, the term beta lactam antibiotic should be taken as meaning any antibiotic including a beta lactam ring in its structure. For e, this may include llin 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 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 antibiotic types is that the former types are weak bases and are lipophilic. This 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 tory concentration (MIC).

James & Wells Reference: 131431DIV6/110 Unexpectantly, these types of antibiotics appear to show a synergistic effect with the NSAID. r this synergistic effect was not observed with other antibiotic types.

Preferably, the antibiotic is present in the liquid formulation at a concentration between -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 needed to administer per dose, which may be more ient 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 ed to 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 otic (w/v) in the liquid formulation is approximately 1:3, respectively. This may be a particularly able ratio if the NSAID is in.

However if the NSAID is a different type of NSAID, such as meloxicam or carprofen, the ratio may vary.

Throughout this specification the term solvent should be taken as meaning any solvent or combination of different solvents which are present in the ition.

Preferably, the solvent has one or more of the following features: - non-aqueous; James & Wells Reference: 131431DIV6/110 - a dielectric point above 30 at 25°C; - a boiling point above 100°C; and/or - classified as a dipolar c 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, n was found to be particularly unstable when combined with an NSAID in an aqueous based formulation. However if the NSAID and otic combination were combined in a nonaqueous solvent based , the composition remained stable, ally at lower temperatures (e.g. 2-15°C).

Moving from an aqueous based composition to one which ed only non-aqueous based ts was found to be most effective in providing stable 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.

A non-aqueous solvent having a dielectric point above 30 at 25°C also appeared to be beneficial for the composition’s ity. Non-aqueous solvents such ethanol (having dielectric point of 25) was a poor solvent for the t 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 effects.

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 James & Wells nce: 131431DIV6/110 improve stability of the active. Surprisingly such solvents were not seen to increase the site reaction/pain on delivery ed to aqueous based itions (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 100°C was also found to provide improved stability whilst also not increasing site reaction on delivery. Again, l and water were both poor solvents. Instead, solvents such as propylene glycol (BP = 188°C), glycerine formal (BP = 190-195°C), NMP (BP = 202-204°C), DMSO (BP = 189°C), DMA (BP =164-166°C) all were seen to have the beneficial ages discussed above. Similarly classed solvents would be expected to have similar beneficial effects.

Furthermore, ion of at least one non-aqueous solvent broadly classified as a dipolar c 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-MethylPyrrolidone (NMP), glycerine , propylene glycol, and benzyl alcohol.

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 ularly surprising to find these solvents did not lead to increased site reaction when the composition was injected.

Preferably, the liquid formulation includes at least two ent non-aqueous based solvents.

James & Wells nce: 131431DIV6/110 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 ors have identified that a combination of NMP and propylene glycol provide ularly stable actives in the formulation. The inclusion of NMP, ine formal and/or benzyl alcohol may be used as solubilising agent for the actives, whereas a t 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. The amount of solubilising agent may vary depending on the type and solubility of the active agents in the composition.

Preferably, the amount of lising agent in the composition is less than 40% w/v.

More preferably, the amount of solubilising agent in the composition is approximately % w/v.

In the present invention, NMP ably 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 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 ition) 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% w/v.

James & Wells Reference: 131431DIV6/110 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 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 , and instead relied primarily on large amounts of NMP.

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 s use of a carrier t feature in any way, let alone towards the types of antibiotics used in the present ion.

Furthermore, the t combinations disclosed in WO’899 would not provide the 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 otics are notoriously unstable with flunixin, the NSAID focused on in . In s conducted by the ors, 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).

Preferably, the non-aqueous based solvent accounts for over 10% w/v of the James & Wells Reference: 131431DIV6/110 ition.

More preferably, the ueous based solvent accounts for between 30% w/v to 80% of the composition. As discussed previously, such high levels of ueous solvent (and particularly a carrier solvent in comparison to the solubilising agent) were beneficial in ing 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.

Antioxidant Preferably, the liquid formulation includes at least one antioxidant.

Preferably, the idant 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 be considered beyond the scope of the ion.

Preferably, the antioxidant is t in the liquid formulation at a concentration between 0.01 – 0.3 % w/v, and most preferably imately 0.05% w/v.

Particularly preferred examples of formulations are detailed in the Best Modes section of the specification.

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).

James & Wells Reference: 131431DIV6/110 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 es which are accessible from outside the body. For instance, this es cavities such as the mouth, teat canal, udder, anus, vagina and so forth.

Preferably, the composition is administered systemically. es of systemic administration may include delivery via ion, bolus, or drench, n the actives may then be distributed through the animal’s bloodstream to the site of ion and/or inflammation.

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 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 hout this specification.

However the inventors acknowledge the composition may be used to treat substantially any type of internal ial infection and/or associated 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 s Preferably, the composition is administered with a dosage of 5-20 mg/kg/day (by weight James & Wells Reference: 131431DIV6/110 nimal) antibiotic and 0.2 mg - 4 mg/kg/day NSAID.

In inary trials, this dosage appears to provide the MIC needed for n (2 µg/ml), such that a therapeutic level of antibiotic is provided. However, due to the 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 it’s antiinflammatory 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.

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 day. Most preferably, the dosage of fen is approximately 1.4 mg/kg/day.

The treatment method may utilise a flexible dosage approach to allow adjustments in ng the disease based on its type and severity. For example, the ors note that a flexipack may be particularly useful for applying this flexible dosage ch. A flexipack is typically a ner made from a mixture of high and low density polyethylene. Such packaging is readily available from many manufacturers.

Preferred Embodiments of the Method of Preparation James & Wells Reference: 131431DIV6/110 As y discussed in this specification, a major advantage of using non-aqueous ts is that the concentration of actives in the composition can be increased. This has numerous benefits, including being able to administer a 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 ition is increased to a concentration (% w/v) 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 ofen is above 4% w/v.

Preferred aspects of the manufacturing process are now discussed below.

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. es of first and second non aqueous solvents are provided in the Best 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.

Preferably, the non-aqueous solvent(s) are heated to imately C prior to the addition of either the NSAID or antibiotic. The ature used may depend on the specific actives used in the composition.

James & Wells nce: 131431DIV6/110 Preferably, the antibiotic is added to the non aqueous solvent(s) and dissolved 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 on of ts, antibiotic and finally NSAID provided improved solubility and stability of the composition.

Preferably, the ature is maintained at 55-80°C throughout the step-wise addition of the NSAID and antibiotic to the formulation.

The ition containing the NSAID and antibiotic is then cooled to approximately room temperature (e.g. between 20-30°C) before making up to a final volume (qs) with a non aqueous solvent and being mixed for approximately 10-20 minutes.

The ition may then be stored at approximately 4°C 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 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 , the need for re-suspension of the composition 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 ts allows the concentration of the NSAID to be increased beyond levels obtainable in an aqueous based composition. Despite James & Wells Reference: 131431DIV6/110 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 ed may also help to lower the site reaction caused, not only by the solvents, but potentially also the antibiotic (tylosin is a known irritant). Potentially, the concentration of n in the composition may be increased due to the lower site reaction on injection.

- By allowing sed concentration of NSAID in the composition, the synergy seen between the two actives at the site of ion is thought to be increased.

By increasing the effectiveness of the antibiotic, lower concentrations of otic could potentially be used to provide the same result.

- Avoidance of water in the ition prevents the need to adjust the composition to a desired pH, removing additional steps in the manufacturing process.

- Reduces pain and inflammation which may be associated with internal infections.

- Improves ency 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 le injections and will be easier to administer with better compliance and improved treatment outcomes.

- 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 endotoxins (G- negative bacteria) improving treatment outcomes, and as such will become an James & Wells Reference: 131431DIV6/110 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 ence in New d over the 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 ion.

Below are comments on the suitability of the formulations trialed.

James & Wells Reference: 131431DIV6/110 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 % es 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 yl toluene (BHT) -- -- -- -- 0.05 0.05 0.05 0.05 . Di sodium edetate -- 0.1 0.1 0.1 -- 0.1 0.1 -- 6. N-Methyl Pyrrolidone (NMP) -- -- -- -- -- -- -- 20 7. Glycerine formal ised -- -- -- -- 4.18 -- 8. Propylene glycol 52 /v 52 52 qs 52 52 qs 9. Phenol Liquified -- 0.25 -- -- -- -- -- -- . Benzyl alcohol 4.18 0 4.18 4.18 -- 4.18 1 1 hanolamine *** 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 ACVM.

F2 An existing formulation of Flunixin injection (Brand Name: Fluxamine injection) James & Wells Reference: 131431DIV6/110 which is registered with ACVM.

F3 Product color turned to dark brown within 48hrs at room ature.

F4 pH adjustment was ed with 20% sodium hydroxide. Product color remained stable at 4 and 40°C.

F5 To avoid a pH issue, the product was formulated using ine formal + propylene glycol. Product color remained stable at 4 and 40°C.

F6 Antioxidant was changed to BHT instead of sodium formaldehyde sulfoxylate.

Product color remained stable at 4 and 40°C.

F7 Glycerine formal stabilised was orated to stabilize the formulation. Product color remained stable at 4 and 40°C.

F8 Product color remained stable at 4 and 40°C.

Summary Formulation F5 appeared the most stable of the n/Flunixin formulations. On e 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 -80°C.

James & Wells Reference: 131431DIV6/110 4. Add and ve Tylosin Base and mix well (maintain the temp at 70- 80°C).

. Add and dissolve flunixin meglumine and mix well (maintain the temperature at 70-80°C). 6. Cool the bulk to below 30°C. 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.

Table 2: Tylosin/Carprofen and n/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 Butylated hydroxytoulene (BHT) 0.05 0.05 6 NMP 20 20 James & Wells Reference: 131431DIV6/110 7 Benzyl l 1 1 8 Propylene glycol qs Qs ** 5% overages added 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 l. 4. Add and mix (35%) of propylene glycol.

. Heat the mixture to 70 -80°C. 6. Add and dissolve Tylosin Base and mix well (maintain the temperature at 70-80°C). 7. Add and dissolve meloxicam/carprofen and mix well (maintain the temperature at 70- 80°C). 8. Cool the bulk to below 30°C.

James & Wells Reference: 131431DIV6/110 9. Make up the final volume with propylene glycol and mix well for 10 minutes.

Example 3 The inventors then trialed a combination containing n/Ketoprofen.

Sl no (F11) Ingredients %w/w 1 Tylosin base ** 21 Ketoprofen BP 2 *** 4.08 3 Propylene glycol 52 4 Benzyl alcohol 4.18 Sodium hydroxide %w/v solution (for pH ment) qs Hydrochloric acid solution %w/v (if 6 required) qs water for 7 ion qs ** 5% overages added (label claim 20%) *** 2% overages 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.

James & Wells Reference: 131431DIV6/110 Manufacturing ure 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 60-70°C. 3. Add and dissolve Tylosin Base and mix well (maintain the temperature at 60- 70°C).

Check the clarity of the solution. 4. Add and ve Ketoprofen and mix well. Cool the bulk to room temperature.

. Check pH. It should be between 8.8 to 9.5. If ed, adjust the pH between 8.8-9.5 using 10% w/v sodium hydroxide solution or 5% w/v hloric 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.

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 otic’s 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-aqueous solvents was expected to result in the composition causing a stronger site on and pain upon injection.

James & Wells Reference: 131431DIV6/110 Sl no (F12) Ingredients % w/v 1 Tylosin base ** 21 2 Ketoprofen BP *** 6 3 Butylated Hydroxytoluene (BHT) 0.05 4 Benzyl Alcohol 1 N-MethylPyrrolidone (NMP) 30 6 Propylene glycol To 100 ml ** 5% overages added (label claim 20%) cturing procedure for F12 1. In a clean and dry manufacturing vessel, load ed quantity of N-Methyl Pyrrolidone (NMP). 2. Load the required quantity of benzyl l to the vessel and mix well. 3. Load Propylene Glycol (35% of the batch size) to the vessel and mix well. 4. Heat the bulk to 55-65°C.

. Add and dissolve Butylated Hydroxy toluene (BHT) with mixing. 6. Add and dissolve Tylosin base with mixing (maintaining the temperature at 55- 65°C). 7. Add and dissolve Ketoprofen with mixing. 8. Check the clarity of the solution.

James & Wells Reference: 131431DIV6/110 9. Make up the volume to 98% of the batch size with Propylene Glycol and mix well.

. Cool the bulk to 25-30°C with mixing. 11. Make up the final volume with Propylene Glycol and mix well.

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% n and 6% Ketoprofen (as per Example 4) and industry rds.

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 mg/kg body weight. Ketofen has 10% w/v ofen with a prescribed dosage of 3 mg/kg. Both Tylan and Ketofen are aqueous based compositions.

Twenty cattle at various stages of lactation, g in age from 4 to 6 years old were chosen from a herd of imately 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 d with each of two 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 n, 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 hour As indicated in Figures 1-2, the studies revealed bioequivalence between the test James & Wells Reference: 131431DIV6/110 ition and the industry rds.

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 ilability ed to industry standards which are administered separately. This in itself is a major advantage.

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 ) compared to the ry standards (Tylan and Ketofen).

This was unexpected because the non-aqueous t used in the test composition in Example 4 was thought to cause adverse side effects upon 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 h increased concentration of NSAID may be achieved because a non-aqueous solvent is used to stabilise the active in the composition.

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 storage conditions of 25°C/60& RH, 30°C/65%RH and 40°C/75%RH.

The physical and chemical characteristics of the product were recorded at regular intervals as recommended by the ACVM.

James & Wells Reference: 131431DIV6/110 As shown in the tables below, the compositions were stable for at least six months at °C/60%RH and 5%RH.

Storage Conditions: 25°C/60%RH Test Specification Initial 3 Months 6 Months Clear golden yellow, slightly Description 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°C Sterility (BP) No growth in Soyabean-Casein Complies - Complies Digest Medium after 14 days tion at 20 - 25°C e Conditions: 30°C/65%RH Test Specification Initial 3 Months 6 Months Clear golden , slightly Description Complies Complies Complies viscous solution Relative 1.050 – 1.100 1.079 1.079 1.079 density n 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 - 35°C Sterility (BP) No growth in Soyabean-Casein Complies - Complies Digest Medium after 14 days incubation at 20 - 25°C James & Wells Reference: DIV6/110 Storage Conditions: 40°C/75%RH Test Specification Initial 1 Month 3 Months 6 Months Clear golden yellow, Description Complies Complies Complies Complies slightly viscous solution Relative 1.050 – 1.100 1.079 1.085 1.079 1.078 density n base 180 – 230 mg/ml 206 207 201 184 (Total) Ketoprofen 54 – 66 mg/ml 59.1 59.7 59.3 58.9 Syringeabilit y (only for - Complies Complies Complies Complies stability) No growth in Fluid Thioglycollate Medium after 14 days incubation at 30 - 35°C Sterility (BP) Complies - - Complies No growth in Soyabean- Casein Digest Medium after 14 days incubation at 20 - 25°C Storage Conditions: 25°C/60%RH Test Specification Initial 3 Months 6 Months Clear golden yellow, slightly ption es 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 tion at 30 - 35°C Sterility (BP) No growth in Soyabean-Casein es - Complies Digest Medium after 14 days incubation at 20 - 25°C James & Wells Reference: 131431DIV6/110 Storage Conditions: 30°C/65%RH Test Specification Initial 3 Months 6 Months Clear golden yellow, ly Description 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) ofen 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°C Sterility (BP) No growth in Soyabean-Casein Complies - Complies Digest Medium after 14 days incubation at 20 - 25°C Storage Conditions: 40°C/75%RH Test Specification l 1 Month 3 Months 6 Months Clear golden yellow, Description Complies Complies Complies Complies slightly viscous solution 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 Syringeabilit y (only for - es Complies Complies Complies No growth in Fluid Thioglycollate Medium after 14 days incubation at 30 - 35°C Sterility (BP) Complies - - Complies No growth in Soyabean- Casein Digest Medium after 14 days incubation at 20 - 25°C James & Wells Reference: 131431DIV6/110 Aspects of the present invention have been described by way of example only and it should be appreciated that modifications and additions may be made thereto without departing from the scope thereof as defined in the ed claims.

James & Wells Reference: 131431DIV6/110

Claims (6)

1. An able composition when used for the treatment of a microbial infection in a mammary gland of a non-human animal, wherein the composition includes a. a non-steroidal anti-inflammatory drug (NSAID); b. an otic ed from the group consisting of a beta lactam antibiotic and macrolide antibiotic; c. N-MethylPyrrolidone (NMP); and d. Propylene glycol; n the amount of propylene glycol in the composition is more than the amount of NMP in the composition; and wherein the NSAID and antibiotic are dissolved in the NMP and propylene glycol.

2. The composition as d in claim 1 wherein the composition is non-aqueous.

3. The composition as claimed in any one of the above claims wherein the NSAID is Ketoprofen.

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

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

6. The composition as claimed in any one of the above claims wherein the antibiotic James & Wells Reference: 131431DIV