EA046700B1 - INJECTION ANTIBIOTIC FORMULATION WITH SLOW RELEASE - Google Patents

Description

Field of technology to which the invention relates

The present invention relates to a sustained release formulation, and more particularly to a sustained release formulation that is suitable for poorly soluble antibiotics for veterinary use.

Oral administration of drugs, which is considered the preferred route of administration in medicine, is, for obvious reasons, often not possible in veterinary medicine, especially when it comes to large domestic animals. For similar reasons, administering medications that require multiple dosing often proves difficult or even impractical.

Sustained release of drug following parenteral administration is generally preferred over oral administration in veterinary medicine and allows the treatment of large domestic animals (such as cattle), as well as pets and other animals. Reducing dosing frequency is known to improve patient safety, reduce injection site complications, and improve compliance with drug administration protocols. Sustained release formulations mitigate the bolus effect during injection and thus have a beneficial effect on drug side effects. For some prophylactic applications and therapies, single or infrequent administration has become standard procedure. For example, once-a-month administration exists for most heartworm preventatives, such as Heartguard®, Sentinel®, and Interceptor. Controlled-release parenteral formulations may be in the form of liquids, in situ solids, and solids [Medlicott et al., Advanced Drug Delivery Reviews 2004, 56:1345-1365]. The most popular controlled-release parenteral products include milk enhancer Posilac® (liquid suspension), antibiotic Micotil® (liquid solution), antibiotic Nuflor® (liquid solution) and growth enhancer Revalor® (solid implant).

In recent years, studies involving the use of poloxamers in sustained release formulations have been reported. Poloxamers are nonionic triblock copolymers that consist of relatively hydrophilic polyethylene oxide (PEO) and relatively hydrophobic polypropylene oxide (PPO) blocks arranged in an A-B-A triblock structure: PEO-PPO-PEO. Aqueous poloxamer gels are described, for example, in US Pat. No. 3,740,421. Poloxamers are used as emulsifying agents for intravenous fat emulsions, as solubilizing agents to maintain clarity in elixirs and syrups, and as wetting agents for antibacterial agents. They can also be used in ointment or suppository bases and as binders or coatings for tablets [Sweetman (Ed.), Martindale: The Complete Drug Reference, London: Pharmaceutical Press]. The hydrophobic-lipophilic balance (HLB) of a poloxamer can be characterized by the number of ethylene oxide and propylene oxide units in the copolymer. Due to their amphiphilic nature, poloxamer copolymers exhibit surfactant properties, including the ability to interact with hydrophobic surfaces and biological membranes. In aqueous solutions at concentrations above the critical micelle concentration (CMC), these copolymers self-assemble into micelles. The diameter of poloxamer micelles typically varies from approximately 10 nm to 100 nm. The micelle core consists of hydrophobic PPO blocks, which are separated from the aqueous external environment by a hydrated shell of PEO blocks. The core is capable of containing various therapeutic or diagnostic reagents [Bartrakova & Kabanov, Journal of Controlled Release 2008, 130:98-106]. Typically, poloxamers are designated by the letter P (for poloxamer) followed by three numbers. The first two digits, multiplied by 100, give the approximate molecular weight of the PPO core, and the last digit, multiplied by 10, gives the percentage of PEO. For example, P407 is a poloxamer with a PPO molecular weight of 4000 Da and a PEO content of 70%. According to an additional designation system (used, for example, in connection with the trade names Pluronic® and Lutrol®), the copolymer is designated by a letter that determines its physical form at room temperature: L for liquid, P for paste, F for flakes (solid), for followed by two or three numbers. The first digit (or first two digits in a three-digit number) multiplied by 300 gives the approximate molecular weight of the hydrophobic block, and the last digit multiplied by 10 gives the percentage of polyethylene oxide (PEO). For example, L61 is a liquid poloxamer with a PPO molecular weight of 1800 Da and a PEO content of 10%, which would be designated P181 according to the naming system described above.

US patent application 20090214685 describes a thermoplastic pharmaceutical composition containing botulinum toxin and a biocompatible poloxamer. The pharmaceutical composition can be administered in the form of liquids and gels after introduction into a sustained release drug delivery system from which the botulinum toxin is released over a multi-day period. US Pat. No. 7,008,628 describes a pharmaceutical composition that contains a linear block copolymer, such as a poloxamer, end-modified with a bioadhesive polymer, such

- 1 046700 kim as polyacrylic acid. The polymer is able to aggregate in response to increased temperature. US Pat. No. 7,250,177 discloses gelling poloxamers modified with a cross-linking group, such as acrylate, which can be cross-linked to form a thermosensitive and lipophilic gel useful for drug delivery or tissue coating. Additional prior art includes US Patent No. 5,035,891 and US 2004/0247672. International patent application WO 2012131678, in the name of some of the authors of the invention, relates to sustained release formulations comprising poloxamers in the form of a suspension or other form of undissolved active agent, whereby the disclosed formulations allow the use of higher amounts of active agent in a single administration, when thereby maintaining acceptable dose volumes.

Florfenicol is a broad-spectrum antibiotic used, among other things, for the treatment of swine respiratory disease (SRD). Approved veterinary preparations of florfenicol include injectable formulations typically containing 300 mg/ml. One such approved product for a specified injectable formulation for veterinary use is dissolved in the organic solvent N-methylpyrrolidone (NMP). Several sustained release formulations of florfenicol have been disclosed previously, including Chinese patent application CN 103202802 directed to sustained release formulations that include poloxamers and polysaccharides. The disclosure relates to several different polysaccharides and different loads of florfenicol active agent in these formulations. A pharmacokinetic study of an in-situ forming gel for controlled delivery of florfenicol in pigs is disclosed in Geng et. al. [J. vet. Pharmacol. Therap. 38, 596-600], and demonstrated an increase in the half-life of florfenicol in animal plasma after administration of gels with a 20% loading based on poloxamers and cellulose-based polysaccharide.

There is a need in the art to provide injectable antibiotic formulations that can release drugs in a controlled manner over extended periods of time. There is also a need in the art to provide formulations that will successfully maintain minimum inhibitory concentration levels for various veterinary pathogens. There is another need in the art to provide antibiotic formulations with high drug loads, eg, above 25% and up to about 50%, that can still be administered through conventional syringes.

Brief description of the invention

The stability of the sustained release formulation and the effect that said stability has on the release profile of the active agent in the target organism over time is a critical factor, which in many cases has proven to be an unstable balance between the various components in the formulation. Surprisingly, it was found that the use of a combination of a poloxamer, an organic solvent and optionally a cellulose derivative that is at least partially soluble in organic solvents in a sustained release antimicrobial agent resulted in a stable injectable dispersion formulation having a consistent and reproducible release profile as in vitro and in vivo. Thus, in one aspect, the present invention provides a composition comprising a slightly soluble antimicrobial agent, at least one poloxamer, an organic solvent and a cellulose derivative that is at least partially soluble in organic solvents, and an aqueous medium, said composition being injectable. In addition, it has surprisingly been discovered that at very high active ingredient loadings, eg above 35 wt% or 40 wt%, a combination of poloxamer and organic solvent in water may be sufficient to provide an injectable formulation having a consistent and reproducible release profile. Thus, in another aspect, the present invention provides a composition comprising an antimicrobial agent, at least one poloxamer, an organic solvent and an aqueous medium, wherein the concentration of said antimicrobial agent is greater than 35 wt.% and up to more than 40 wt.%, and wherein said the composition is injectable.

Thus, herein provided is a pharmaceutical composition containing a biologically active agent, a poloxamer, an aqueous carrier and an organic co-solvent, said composition being an injectable composition at room temperature, provided that when the concentration of said active agent is less than 35 wt. %, the composition further contains a cellulose-based compound that is at least partially soluble in organic solvents. In one embodiment, when the drug concentration is greater than 35 wt%, for example, from 35 wt% to 50 wt% or 55 wt%, a cellulose-based material is included. In other embodiments, when the drug concentration is more than 35 wt.%, for example, from 35 wt.% and up to 50 wt.% or 55 wt.%, the composition does not contain cellulose-based material, for example, is from 40 wt. % to 50 wt.%, or from 42.5 wt.% to 50 wt.%, or from 45 wt.% to 50 wt.%. Also provided herein is a pharmaceutical composition containing a biologically active agent, a poloxamer, an aqueous carrier, an organic

- 2 046700 ical co-solvent and cellulose-based compound which is at least partially soluble in organic solvents, wherein said composition is an injectable composition at room temperature, and wherein the concentration of said biologically active agent is greater than 10% by weight and up to 35% by weight .%. The biologically active agent may be selected from florfenicol, lincomycin, tylosin, metronidazole, tilmicosin, spiramycin, erythromycin, tulathromycin, tiamulin, ampicillin, amoxicillin, clavulanic acid, penicillin, streptomycin, trimethoprim, sulfonamide, sulfamethoxazole, pleuromutilin, and vilosin, tilialosin, doxycycline and oxytetracycline. Preferably the biologically active agent is florfenicol. Moreover, preferably, florfenicol may be present in the composition at a loading of from about 25 wt.% to about 50 wt.%. The organic cosolvent may be present in an amount from about 5 wt.% to about 15 wt.%. The cellulose-based material, which is at least partially soluble in organic solvents, may be hydroxypropylcellulose. The organic solvent may be selected from the group consisting of N-methylpyrrolidone (NMP), dimethyl sulfoxide (DMSO), PEG 400, propylene glycol and ethanol. Preferably the organic solvent is N-methylpyrrolidone. In some preferred embodiments, the pharmaceutical composition contains an organic solvent that is N-methylpyrrolidone, and a cellulose-based material that is at least partially soluble in organic solvents that is hydroxypropylcellulose, and the biologically active agent is florfenicol at a concentration of from 25 wt. .% to 50 wt.%. In some other preferred embodiments, the pharmaceutical composition contains an organic solvent that is N-methylpyrrolidone, and florfenicol at a concentration of from 35 wt.% to 50 wt.%. Also provided herein is a pharmaceutical composition, as defined herein, for use in the treatment of a veterinary infection in a non-human animal by administering to said animal a pharmacologically effective dose of an antibiotic in said composition. Preferably, the composition is administered once to said non-human animal during the course of treatment. Moreover, preferably, administration includes intramuscular injection or subcutaneous injection. In some embodiments, the infection may be caused by a swine pathogen.

Brief description of the figures

In fig. 1 schematically shows the release profiles of florfenicol from selected compositions.

In fig. 2 schematically shows the release profiles of florfenicol as a result of the addition of an organic solvent.

In fig. 3 shows the concentrations of florfenicol in blood plasma after a single administration of a composition in accordance with the invention compared to two administrations of a commercial product.

In fig. 4 shows the plasma concentrations of florfenicol after a single administration of additional compositions in accordance with the invention compared to two administrations of a commercial product.

Detailed Description of the Invention

As described above, the sustained release composition of the present invention contains an active biological agent. In some embodiments, said biological agent is preferably an antimicrobial agent that exhibits poor solubility in an aqueous environment. Poor solubility may be understood as defined, for example, in the current US Pharmacopoeia, but may be better understood in the context of composition, as explained in more detail below. In a suitable embodiment, the antimicrobial agent used in the sustained release composition of the present invention is selected from the group consisting of florfenicol, lincomycin, tylosin, metronidazole, tilmicosin, spiramycin, erythromycin, tulathromycin, tiamulin, ampicillin, amoxicillin, clavulanic acid, penicillin, streptomycin, trimethoprim, sulfonamide, sulfamethoxazole, pleuromutilin, avilosin, tylvalosin, doxycycline and oxytetracycline. In some currently preferred embodiments, the antimicrobial agent is florfenicol.

According to the principles of the present invention, the load (ie, the amount of biologically active agent or antimicrobial agent that is introduced into the injectable dosage form) is high, providing a sustained and controlled release over several days. The high loading of the injectable composition according to the invention is facilitated, among other factors, by the presence of a composition containing a biologically active agent, which may be in an insoluble form, thereby forming a dispersion in an aqueous medium. According to the principles of the invention, the antibacterial agent dispersed in the composition is, to some extent, in solid form. Preferably, more than 90% of the drug is in insoluble form, but up to 99.999% of the drug may be in insoluble form. The insoluble form of a drug usually includes basic compounds or salts, especially those having low solubility in water, even though a more soluble salt may be known.

- 3 046700

The load may vary depending on the solid state properties of the active agent. When a drug readily reacts with an aqueous environment or with poloxamer or other surfactants, it may form a paste, that is, a composition that is not easily syringable (not syringable) and/or is not injectable at high loads. In these cases, such drugs can be used at fairly low loadings, for example in the range of 12-20% by weight, but generally a high drug loading is preferred. Thus, in some embodiments, the load is at least about 20% by weight of the injectable composition.

In some other embodiments, the load is from about 25 wt.% to about 30 wt.% of the injectable composition. In some other embodiments, the load is at least about 30% by weight of the injectable composition. In some additional embodiments, the load is from about 30 to about 45 weight percent of the injectable composition. In some further embodiments, the load is from about 35 to about 50 weight percent of the injectable composition. In some embodiments, the load is from about 30 to about 35 weight percent of the injectable composition. In some specific embodiments, when the biologically active agent is florfenicol, the load of florfenicol used for particular applications may be 25-50 wt.%, such as 28-32 wt.%, or 36-42 wt.%, or 44- 48 wt.%.

The biologically active agent forms a dispersion in an aqueous medium with the help of a co-solvent. It should be understood that the biologically active agent must be in the form of a solid, such as a powder. The powder may be in the form of aggregates, granules or coated powder, but preferably the powder is a pure drug substance powder with a defined particle size distribution. In some preferred embodiments, the powder has a particle size of less than about 90 microns, more preferably less than about 50 microns. Sometimes it may be advantageous to also use smaller particle sizes or even micronized powder. Without wishing to be bound by theory, it is believed that a powder with a smaller particle size may increase the peak plasma concentration obtained from a formulation in vivo compared to a conventional drug powder, even if the difference is small or negligible in vitro. Micronized powder or powder with reduced particle size can be obtained directly from the active substance powder, as is generally known in the art, for example, by high impact or high shear grinding, pressure sieving and other methods.

In some preferred embodiments, the biologically active material or antimicrobial agent is released from the in-situ gel of the composition of the present invention over at least 3 days. In some other embodiments, the material is released within 2-3 days. In some additional embodiments, the material is released within 4-5 days. In some embodiments, the material is released over more than 5 consecutive days from a single injectable composition of the present invention. Thus, release can be described in terms of the duration of release rather than any specific rate. Duration of release in vivo can be defined in plasma as concentrations of drug that remain at significant levels over time. In another embodiment, the duration of in vivo release can be defined in the target organ or tissue as drug concentrations that maintain significant levels over time. In particular, since the active agent is an antibiotic, the duration of release can be determined in blood plasma, and the resulting concentrations can be compared with the minimum inhibitory concentrations of antibiotics for specific pathogens. In vitro, by maintaining conditions of sufficient dilution, the duration of drug release can range from about 12 hours to about 3 days, for example, under the conditions described in the Examples section below.

In accordance with some principles of the present invention, the advantageous combination of an organic cosolvent, a poloxamer in an aqueous medium, and a cellulose derivative that is at least partially soluble in organic solvents produces a synergistic effect, providing a stable and controlled release of the biologically active agent over several days. The drug load in formulations containing such a cellulose derivative may be as low as about 5 wt.% or about 10 wt.%. However, depending on the properties of the antibiotic in the solid state, the drug loading can reach 35 wt% or 40 wt%, or 45 wt%, or 47.5 wt%, or even 50 wt%. Moreover, when the active agent is present at a concentration above 35 wt.%, it has surprisingly been found that relatively stable and reproducible drug release kinetics can be achieved using compositions containing a poloxamer, water and an organic co-solvent as defined herein. Although the presence of a cellulose derivative that is at least partially soluble in organic solvents was beneficial even at high drug loadings, the release profiles without the excipient were unexpectedly quite

- 4 046700 stable to meet current USP requirements for variability in drug release from controlled release dosage forms. However, when the drug load is less than 35% by weight, it is preferable that the composition contains a cellulose derivative as described below.

In accordance with some embodiments, the poloxamer described above is selected from the group consisting of poloxamer 407, poloxamer 188, poloxamer 237 and poloxamer 338, and combinations thereof. In some currently preferred embodiments, the poloxamer as described above is poloxamer 407.

The presence of the poloxamer allows the composition to gel at physiological temperature and, therefore, the said poloxamer must be present in a suitable concentration in the injectable composition to ensure the formation of a stable gel, especially in the presence of a large amount of undissolved active substance powder. Accordingly, the concentration of poloxamer, as described above, is more than 8 wt.% of the total weight of the composition. Depending on the nature of the drug, eg particle size, solubility of the drug, its affinity for the poloxamer and the drug loading, the amount of poloxamer can be as low as 7-9 wt% and as high as 16-20 wt%.

The synergistic effect of some embodiments of the present invention is achieved by combining said poloxamer with a unique combination of an organic co-solvent and a cellulose derivative that is at least partially soluble in organic solvents. The chemical compatibility between the cellulose derivative and the organic solvent and the ratio between these two components determine, together with the poloxamer concentration, the release profile of the biologically active agent. Without being limited by any mechanism or theory, it is postulated that while an organic solvent may increase the solubility of a biologically active agent, it also slows down the rate of release of said active agent from the gel composition under physiological conditions due to its effect on the gel itself. It is further postulated that, at least for some drugs, the addition of a cellulose derivative as described above may be responsible for increasing the rate of release of the biologically active agent, and that the organic solvent helps reduce the variability in the overall release profile over time. Although the molecular weight of the cellulose derivative to be used can be selected according to the desired rheological properties and the intended release profile, in accordance with some embodiments of the present invention, the concentration ratio between said cellulose derivative and said organic solvent is typically from about 1:6 to about 1: 20. When the drug is present at a particularly high load, for example, from more than 35 wt.% to more than 40 wt.%, the concentration ratio between said cellulose derivative and said organic solvent can be from about 1:10 to about 1:100.

A cellulose derivative that is at least partially soluble in organic solvents is typically one that is soluble to some appreciable extent in conventional pharmaceutical organic solvents, for example ethanol. Preferably, a suitable derivative forms a clear solution when, for example, 1 gram of the derivative is dissolved in 100 ml of 96% ethanol at room temperature. One suitable cellulose derivative that is at least partially soluble in organic solvents is hydroxypropylcellulose.

Hydroxypropyl cellulose has another useful property in that it is also highly soluble in aqueous solutions at room temperature and becomes less soluble as the temperature increases. Without being limited to any theory or mechanism of action, it is believed that when a composition of the invention is injected into an animal, the solubility of hydroxypropyl cellulose is reduced, which in turn promotes the stability of the resulting gel, resulting in better control of the release of the biologically active agent.

In some related embodiments, the concentration of the cellulose derivative, as described above, is from about 0.5 wt.% to about 1.5 wt.% of the total weight of the injectable composition. In some other embodiments, the concentration of cellulose derivative is from about 0.5 to about 1 weight percent. When the drug is present at a very high load, for example, above 40%, the concentration of cellulose derivative may be from about 0.05 wt.% to about 0.7 wt.%.

In some embodiments, the organic solvent described above is selected from the group consisting of N-methylpyrrolidone (NMP), dimethyl sulfoxide (DMSO), PEG 400, propylene glycol, and ethanol. In some currently preferred embodiments, the organic solvent is NMP.

In some related embodiments, the concentration of the organic solvent, as described above, is from about 1.5 wt.% to about 20 wt.% of the total weight of the injectable composition. In some other embodiments, the organic solvent concentration is from about 3 to about 15 weight percent. In still some other embodiments, the concentration of organic solvent is from about 8 wt.% to about 12 wt.%.

In further suitable embodiments, at least one poloxamer, an organic solvent and a cellulose derivative are dissolved in an aqueous medium. The aqueous medium is typically water, optionally containing additional dissolved additives such as salts and/or buffers. The amount of aqueous medium in the formulation is generally the remainder of 100% of the composition after subtracting the appropriate percentage of the biologically active agent, at least one poloxamer, cellulose derivative, cosolvent and other excipients, if used. Salts may include sodium chloride, calcium chloride or magnesium chloride, and buffers may include mono-, di- or tribasic alkali metal salts and phosphates.

Although the synergistic effect that may be present between the cosolvent, a cellulose derivative that is at least partially soluble in organic solvents, and the poloxamer in an aqueous environment is clearly advantageous when the drug is present at a very high load, e.g., greater than 35 wt.% and up to more than 40 wt.%, the effect of the cellulose derivative on stabilizing the system may become less necessary to obtain a pharmaceutically acceptable composition, for example, exhibiting a release profile with a relative standard deviation of concentration values at each time point below 10%. As shown in the examples below, for example, exclusion of hydroxypropyl cellulose from the florfenicol formulation at 47.5 wt% loading resulted in a moderate burst effect with an increase in relative standard deviation (RSD) at early time points, but also an acceptable release profile.

In accordance with the principles of the invention, the resulting composition is a room temperature (eg, range 15-25°C) or cold (eg, range 2-8°C) injectable formulation that, once injected into the body of an animal (eg, having a temperature above 35°C) is transformed into a gel form, characterized by having a reproducible and well-controlled release profile of the biologically active agent included in it.

In another aspect, the present invention provides a method for preparing sustained release injectable formulations containing an antimicrobial agent, at least one poloxamer, an organic solvent, and a cellulose derivative that is at least partially soluble in organic solvents in an aqueous medium, comprising the following steps: 1) mixing water and an organic solvent (known as a cosolvent) and preferably cooling the resulting mixture, 2) adding sequentially or simultaneously at least one poloxamer and the specified cellulose derivative to the [cold] mixture obtained in step 1, followed by stirring until dissolved; and 3) adding an antimicrobial agent to the resulting mixture.

In some embodiments, the organic solvent described above is selected from the group consisting of N-methylpyrrolidone (NMP), DMSO, PEG (PEG 400), propylene glycol, and ethanol. In some currently preferred embodiments, the organic solvent is NMP.

In some embodiments, the poloxamer described above is selected from the group consisting of poloxamer 407, poloxamer 188, poloxamer 237, poloxamer 338, and combinations thereof. In some currently preferred embodiments, the poloxamer as described above is poloxamer 407.

In some embodiments, the cellulose derivative is hydroxypropylcellulose.

In some embodiments, the antimicrobial agent used in step 3 is selected from the group consisting of florfenicol, lincomycin, tylosin, metronidazole, tilmicosin, spiramycin, erythromycin, tulathromycin, tiamulin, ampicillin, amoxicillin, clavulanic acid, penicillin, streptomycin, trimethoprim, sulfonamide , sulamethoxazole, pleuromutilin, avilosin, tylvalosin, doxycycline, oxytetracycline. In some currently preferred embodiments, the antimicrobial agent is florfenicol.

The term biologically active agent as used herein and in the claims is interchangeable with the term antibacterial agent, drug, or antibiotics.

As used herein and in the claims, the term co-solvent refers to an organic solvent that is mixed with an aqueous carrier or water in the composition of the invention. In some embodiments, the organic solvent described above is selected from the group consisting of N-methylpyrrolidone (NMP), DMSO, PEG 400, propylene glycol, and ethanol.

In a further aspect, there is provided a method of treating veterinary infections or using the compositions in the treatment of veterinary infections by administering to a patient in need thereof at least one injection of sustained release injectable compositions, as generally described herein, containing in an aqueous medium an antimicrobial agent, according to at least

- 6 046700 one poloxamer, an organic solvent and optionally a cellulose derivative that is at least partially soluble in organic solvents. Preferably, the method involves a single administration of the composition, but more than one injection may be used depending on the need and duration of treatment. When working with a veterinary patient, it is preferable to minimize handling of the patient, thereby reducing stress on the animal and the effort required to locate, capture and handle the sick animal. Thus, single administration is preferred. Alternatively, the method includes multiple administrations of the composition, in the event that the number of administrations is lower than what is currently required for the particular biologically active agent.

Administration may involve a single injection or multiple injections at multiple sites if a large volume of injection is required. Due to the advantages of the compositions of the present invention, multiple injection sites may not be required since the poorly soluble drug is present in sufficient quantities in relatively small injection volumes.

Administration is usually an intramuscular injection. However, administration may also be subcutaneous administration, intraperitoneal administration, intradermal or site-specific administration, such as intravulval administration for cows and sheep, intracaudal or ear administration for cattle, intramammary administration, and the like.

Veterinary infections that can be treated in accordance with the invention include infections caused by pathogens in swine, infections in cattle, infections in poultry, infections in domestic animals, or infections in zoo and wild animals.

In some embodiments, the organic solvent described above is selected from the group consisting of N-methylpyrrolidone (NMP), DMSO, PEG 400, propylene glycol, and ethanol. In some currently preferred embodiments, the organic solvent is NMP. In some embodiments, the poloxamer described above is selected from the group consisting of poloxamer 407, poloxamer 188, poloxamer 237, poloxamer 338, and combinations thereof. In some currently preferred embodiments, the poloxamer as described above is poloxamer 407. In some embodiments, the cellulose derivative is hydroxypropylcellulose. In some embodiments, the antimicrobial agent is selected from the group consisting of florfenicol, lincomycin, tylosin, metronidazole, tilmicosin, spiramycin, erythromycin, tulathromycin, tiamulin, ampicillin, amoxicillin, clavulanic acid, penicillin, streptomycin, trimethoprim, sulfonamide sulfamethoxazole, , pleuromutilin, avilosin , tylvalosin, doxycycline, oxytetracycline. In some currently preferred embodiments, the antimicrobial agent is florfenicol.

Examples

Materials and methods.

Florfenicol and N-methylpyrrolidone (NMP) were purchased from Sigma-Aldrich, Israel. Poloxamers 407, 188, 338, and 237 were obtained from your local BASF representative. Amoxicillin, tylosin, Klucel® (hydroxypropylcellulose) polymers, PEG400, and propylene glycol were received as gifts from pharmaceutical companies. Before use, the water was purified on a column and distilled. Sodium chloride was purchased from Merck, Israel.

Unless otherwise stated, florfenicol injectable formulations were prepared as follows.

Measured amounts of water and cosolvent were mixed at room temperature, salts or buffers were added, if present, and stirred until dissolution was achieved. Measured amounts of poloxamer and cellulose derivative were cooled to 4°C in a cold room; Mixtures of water and cosolvent were also cooled separately. The polymers were then added to a mixture of water and cosolvent under the same conditions and stirred vigorously using a magnetic stirrer until a clear solution was obtained. Florfenicol powder (flakes) was then added to the resulting solution and mixed for 24 hours in a cold room to ensure good distribution in the preparation. Alternatively, particularly for high-load formulations, a measured amount of florfenicol was placed in a mortar and triturated geometrically, that is, mixed in the mortar with comparable aliquots of the solution until the entire measured aliquot of the prepared solution was consumed.

Measuring the gelation temperature.

Gelation was measured by inverting a glass tube containing 0.5-1 ml of the composition at increasing temperature. The temperature at which the composition stopped flowing when turned over was considered the initial gelation temperature. Alternatively, for pre-screening, the temperature was raised to 40°C and the time it took for the formulation to gel was recorded.

The gelation temperature was also measured rheometrically using a Physica MCR 101 rheometer from Anton Paar, parallel plate spindles separated by a gap of 200 μm, varying

- 7 046700 temperature at a shear rate of 100 s ^-1 . The second derivative of the viscosity curve gave the most abrupt change in viscosity, which was considered the true gelation point.

Determination of florfenicol.

Florfenicol was determined by HPLC using an HP1090 instrument with a UV detector measuring absorbance at 224 nm. A C-18 250 x 4.6.5 µm column was used, eluting at 1.2 mL/min, with a mobile phase of 25:75 acetonitrile (ACN): deuterium depleted water (DDW). Florfenicol eluted under these conditions after 4-4.5 minutes.

Dissolution testing.

To test the kinetics of florfenicol dissolution from the formulations, 5 ml syringe barrels were cut into 2 ml segments, which served as tube-shaped holders. One side was covered with a sheet of Parafilm® and approximately 2 ml aliquots of the formulation at room temperature were accurately measured into the indicated prepared holder tubes through a 19G needle using a suitable syringe, thereby assessing the injectability of the formulation. The top side was then covered with another sheet of Parafilm® and placed in a preheated 40°C oven for at least 15 minutes to allow gelation. The Parafilm® sheets were then carefully removed, the tube holder was placed in the Sinker Basket dissolution tester sinker and immediately transferred to the Caleva 6ST dissolution tester (USP Apparatus 2) set at 20 rpm at 40°C. The temperature was chosen to match and simulate the body temperature of the target animal (pig). The dissolution medium was phosphate buffer USP, pH 6.8, and a volume of 900 ml was used for each holder tube. Samples were taken from the dissolution medium at predetermined time points and the volume was adjusted with fresh dissolution medium. At the end of testing, the holder tubes were rinsed in the dissolution vessels and mixed vigorously to obtain a recoverable amount of material that could serve as a 100% standard. The percentile of maximum florfenicol concentration at each time point with standard deviation was recorded.

In addition, dissolution of some florfenicol compositions was performed using a 5 USP (paddle over disc) apparatus as follows.

Example 1. Comparative example.

A. To evaluate the effectiveness of the compositions disclosed in Chinese patent application CN 103202802, Example 7 (30% florfenicol) of the said publication was reproduced and tested under the described conditions. Since the publication does not contain enough recommendations regarding the brand of hypromellose used, two brands having an apparent viscosity below 20 cP at the low concentrations tested (HPMC K4M and HPMC K15M) were tested separately. Briefly, poloxamers were accurately measured, cooled, and dissolved in a large portion of cold water at 4°C, followed by the addition of hydroxypropyl methylcellulose (HPMC). The remaining excipients were provided from the original solutions, and the remaining water content was added and mixed thoroughly. Formulation samples having a total amount of 25 grams were prepared, samples prepared using HPMC K4M are referred to as Formulation Sample 1.1, and samples prepared using HPMC K15M are referred to as Formulation Sample 1.2.

To test the beneficial effect of the organic solvent in accordance with the present invention, the same formulations as described above were prepared, this time using approximately 20 wt.% N-methylpyrrolidone as a co-solvent based on the total weight of the solvent (replacing 20% water with an organic solvent), thereby thereby obtaining a sample of drug 1.3 and a sample of drug 1.4, corresponding to HPMC K4M and HPMC K15M, respectively.

It was found that under experimental conditions, that is, at room temperature, samples prepared in accordance with 1.1 and samples prepared in accordance with 1.2 could not be drawn into a syringe even without a needle. This indicates that the specified composition, manufactured in accordance with the disclosure of CN 103202802 (example 7), was not injectable under the described conditions. In order to obtain the release profile and results for the indicated non-injectable formulations, samples were added using a spatula. It should also be noted that the addition of NMP as a co-solvent increased the viscosity beyond practical levels (solid gel even at 4°C), however, samples prepared in accordance with 1.3 and 1.4 were tested for drug release, although they also did not could have been injected.

B. To obtain injectable compositions, formulations with a 20% loading were prepared following the procedure of example 7 and example 6 of the prior art publication CN 103202802. Briefly, florfenicol load was reduced by water. Formulation sample 1.5 included HPMC K15M and pure water, and formulation sample 1.6 included HPMC K15M and 20 wt.% NMP as co-solvent. The resulting formulations, corresponding to Formulations 1.5 and 1.6, containing 20 wt.% florfenicol, were easily administered through the test needle and turned into a gel under the conditions of preparation of the dissolution test sample.

To test the release profile of florfenicol from the formulations described above, despite

- 8 046700 for the lack of injection properties of compositions manufactured in accordance with 1.1-1.4, these compositions were added to test tubes using a spatula according to the usual method of filling semi-solid round molds. The results are presented in table. 1 below.

From the table 1 it can be seen that in general the addition of NMP to a composition containing HPMC accelerates the rate of release of florfenicol from the preparations, and sometimes reduces variability, for example, when comparing preparations 1.1 with 1.3, 1.2 with 1.4, 1.5 with 1.6.

Also, it can be seen that the injectable formulation according to the prior art publication may only have a 20% florfenicol load, which is also confirmed by another publication by the same authors, Z. X. Geng, H. M. Li, J. Tian, T. F. Liu, Z. G. Yu, J Vet Pharmacol Ther, Vol 38, Iss 6, Dec 2015, 596-600). Higher loading cannot be achieved in injectable compositions that use a formulation in accordance with the prior art.

Table 1

A drug 1.1 Preparation 1.2 Preparation 1.3 Preparation 1.4 Preparation 1.5 Preparation 1.6 Time (h) Wednesday SD Wednesday SD Wednesday SD Wednesday SD Wednesday SD Wednesday SD 0.25 2.52 0.51 2.61 0.44 2.61 0.44 2.98 0.38 2.47 2.15 3.94 0.61 0.5 4.25 1.16 5.80 0.79 5.80 0.79 7.39 0.47 5.52 4.35 9.34 0.49 1 6.54 1.49 11.02 2.12 11.02 2.12 15.50 1.06 10.41 7.06 15.25 1D5 1.5 8.34 1.45 15.44 2.92 15.44 2.92 22.30 1.78 15.76 7.09 20.28 1.48 2 9.43 1.60 18.70 3.28 18.70 3.28 27.90 2.65 19.59 6.84 24.13 1.81 3 12.56 3.02 23.94 3.23 23.94 3.23 36.28 3.94 27.20 6.40 30.80 2.96 4 16.40 3.22 28.94 2.33 28.94 2.33 43.82 3.11 32.61 5.57 37.23 4.07 5 20.63 5.31 35.19 4.10 35.19 4.10 49.79 2.01 36.75 4.77 42.60 4.02 6 23.73 5.72 38.59 1.59 38.59 1.59 55.42 2.43 41.78 6.86 48.17 3.10 7 27.12 6.67 42.20 1.93 42.20 1.93 57.69 1.87 43.69 4.76 53.70 2.96 8 30.22 7.38 45.46 1.71 45.46 1.71 63.62 5.77 46.27 4.46 59.21 5.93 24 48.68 9.42 72.25 7.96 72.25 7.96 87.11 7.07 75.23 8.86 85.66 7.62 Max viscosity 44.9 Pass 38.4 Pass 18.3 Pa s 22.9 Pass Min viscosity 0.59 Pass 0.49 Pass Thick gel at 4°C 0.18 Pass 0.27 Pass Geleobr. 1°C 12.9°C 12.8°C 18.4°C 17,GS Range of geleobraz. 12.3- 14.4 12.3 14.8 17.5 -19.9 16.2-19.9

Example 2.

To evaluate the benefits of a florfenicol sustained release formulation in accordance with the principles of the present invention compared to another known sustained release gel formulation disclosed in international patent application WO 2012131678, gels containing 30% by weight florfenicol were prepared. The effect of the cosolvent NMP, the cellulose-based material hydroxypropylcellulose, and their synergistic combination was isolated and studied. All formulations demonstrated gelation at temperatures between 25 and 35°C (individual data below) and release profiles were assessed according to the method described above. The formulations are summarized in the tables below along with their respective release profile data.

Formulation 2.1 corresponds to an embodiment of the present invention and contains a cellulose-based material, hydroxypropylcellulose; Preparation 2.2 demonstrates the effect of the absence of a cosolvent; Preparation 2.3 demonstrates the effect of the absence of hydroxypropylcellulose (Klucel® EF) and the co-solvent NMP; and formulation 2.4 is a reference formulation according to WO2012131678, free of co-solvent and cellulose additive. Formulation 2.5 (an embodiment of the invention) exhibits a lower loading (20 wt.% florfenicol), and preparation 2.6 contains 20% florfenicol and, for comparison with preparation 2.6, a preparation without NMP.

When comparing the results obtained for formulations 2.1 and 2.3, it can be easily seen that the addition of NMP to the formulation according to WO 2012131678 causes a significant reduction in drug release with a significant reduction in the variability between results. In addition, the addition of hydroxypropylcellulose to the formulation according to WO 2012131678 results in a significant reduction in the release rate and a relatively high variability in the release profile. According to the results, only the addition of both components (NMP and HPC) provides a synergistic effect, resulting in lower variability (standard deviation of the mean relative to the mean is lower) and increases drug release compared to pure aqueous formulations 2.2 and 2.3. In addition, it can be seen that formulations having a 20% loading according to the invention produce comparable, but slightly more attenuated, florfenicol release with even less variability than a hypothetical 20% formulation of CN'802 with hypromellose instead of hydroxypropylcellulose (Formulation 1.5).

The results are summarized in table. 2 below and also in FIG. 1. In FIG. 1 shows release profiles

- 9 046700 nia with error bars showing the relative standard deviation (RSD) at each time point. Diamonds (♦) represent formulation 2.1, filled squares () represent formulation 2.2, filled triangles (▲) represent formulation 2.3, and X's (x) represent formulation 2.4, where % FFC indicates cumulative percentile release of florfenicol, and t (h) indicates time , elapsed since the beginning of the experiment, in hours.

table 2

Prepa rat 2.1 Prepa rat 2.2 Great! rat 2.3 Great! rat 2.4 Great! rat 2.5 Prepa] rat 2.6 Mass. (G) % weight/ weight Mass. (G) % weight/ weight Mass. (G) % weight/ weight Mass. (G) % weight/ weight Mass. (G) % weight/ weight Mass. (G) % weight/ weight Florfenicol 7.50 30.00 7.50 30.00 7.50 30.00 7.50 30.00 5.00 20.00 5.00 20.00 Poloxamer 407 3.00 12.00 3.00 12.00 3.13 12.50 3.13 12.00 3.43 13.72 3.43 13.72 Klucel® EF 0.19 0.75 0.19 0.75 ... ... ... — 0.22 0.88 0.22 0.88 DDW 11.81 47.25 14.31 57.25 11.81 47.50 14.38 57.50 13.50 54.00 16.36 65.44 NMP 2.50 10.00 — ... 2.50 10.00 ... — 2.86 11.44 ... ... Time (h) Wednesday standard deviation Wednesday standard deviation Wednesday standard deviation Wednesday standard deviation Wednesday standard deviation Wednesday standard deviation 0.25 1.37 0.50 1.33 0.56 1.05 0.52 9.29 1.05 1.75 0.87 4.40 1.55 0.5 2.80 0.98 2.75 1.24 1.72 0.65 14.97 1.44 3.46 0.31 5.66 2.81 1 5.70 2.02 5.21 2.30 2.89 1,N 21.85 4.01 7.34 0.62 9.69 4.11 1.5 8.76 2.76 7.64 3.57 3.96 1.43 26.20 4.98 11.06 0.74 12.18 4.95 2 11.67 3.86 9.42 4.03 5.48 1.23 28.28 5.34 15.90 1.11 13.85 5.75 3 16.53 4.56 12.39 4.45 8.71 2.23 32.91 5.76 20.70 1.08 16.29 7.76 4 20.39 4.78 15.01 4.77 11.42 3.22 36.29 6.21 26.25 1.17 18.84 8.52 5 24.00 4.61 17.34 4.72 13.53 3.75 39.61 6.67 30.54 1.56 21.09 9.93 6 27.59 4.94 19.64 4.45 15.30 3.88 42.83 7.66 33.59 1.10 23.24 8.98 7 30.34 4.63 21.80 4.20 16.93 3.97 45.93 8.81 36.57 0.64 27.32 8.40 8 33.67 4.68 23.61 4.24 18.49 4.00 48.57 9.02 39.74 0.71 30.22 8.09 24 64.18 4.63 57.38 6.57 40.47 4.47 78.06 11.68 66.69 5.16 59.45 12.58 Rheology Max viscosity 9.69 Pa-s 12.3 Pa s 11.4 Pa s 13 Pa s 8.11 Pass 8.3 Pa s Min viscosity 0.38 Pa-s 0.262 Pa-s 0.181 Pa-s 0.346 Pa-s 0.125 Pa-s 0.0819 Pa-s Gel. t°C 27.1 °C 24°C 27.6°C 23.9°C 27.9°C 25.3°C Gel range 27.0-31.6 23.6-27.1 27-31.2 23.5-27.1 27.5-31.2 24.4-27.5

Example 3.

To evaluate the effect on the composition of the selected cosolvent, NMP, gels were prepared according to Preparation 2.1, and the NMP content was varied from 5 to 20 wt%, yielding Preparations 3.1 (5 wt%) and 3.2 (20 wt%). .

Release data are presented in table. 3 below and the profiles are shown in FIG. 2 with error bars indicating the RSD at each time point. Diamonds (♦) represent preparation 3.1 (indicated as 5 wt%), filled squares () represent preparation 2.1 (indicated as 10 wt%), and filled triangles (▲) represent preparation 3.2 (indicated as 20 wt%), where % FFC indicates the cumulative percentile of florfenicol release, and t (h) indicates the time elapsed since the start of the experiment, in hours.

It can be seen that at 5 wt.% NMP the variability increases, while the release profile remains almost unchanged, while at 20% the release accelerates slightly.

Table 3

Preparation 3.1 Preparation 3.2 Time (h) Wednesday standard deviation Wednesday standard deviation 0.25 2.79 1.06 5.92 0.48 0.5 5.05 1.73 9.14 1.31 1 8.88 3.45 13.81 2.57 1.5 11.49 4.53 17.69 4.17 2 13.67 5.70 21.63 4.57 3 17.21 7.16 28.77 6.72 4 20.37 8.25 33.16 7.55 5 22.74 9.32 37.94 7.03 6 24.86 10.01 43.80 6.41 7 28.79 12.15 46.57 5.34 8 31.25 12.73 51.41 5.88 24 43.06 15.81 76.69 3.56 Rheology Max viscosity 6.16 Pa s 10.9 Pa s Min viscosity 0.23 Pa s 0.39 Pa s Gelation, t°C 26.4°C 21.6°C Gel range 26.2 -29.3 21.1-24.7

- 10 046700

Example 4.

To evaluate the effect on composition of additional cosolvents, gels were prepared according to Preparation 2.1, and NMP was replaced with DMSO ( Preparation 4.1), propylene glycol ( Preparation 4.2), PEG 400 ( Preparation 4.3), or ethanol ( Preparation 4.4).

The release profiles are summarized in Table. 4 below.

It can be easily seen that both DMSO and PEG 400 provide a release profile comparable to NMP, but more significantly reduce the gelation temperature of the solution.

Table 4

Preparation 4.1 Preparation 4.2 Preparation 4.3 Preparation 4.4 Time(h) Wednesday std. deviation Wednesday std. deviation Wednesday std. deviation Wednesday std. deviation 0.25 2.27 0.36 1.38 0.37 2.44 0.69 1.53 0.80 0.5 4.44 0.70 2.36 0.90 4.86 1.35 2.57 1.39 1 8.61 1.18 3.65 1.36 9.52 2.57 3.94 1.96 1.5 11.14 1.00 4.44 1.59 12.93 3.94 4.92 2.27 2 13.09 0.92 5.34 1.63 15.28 4.50 5.86 2.45 3 15.98 1.59 6.89 1.72 19.62 6.25 7.66 2.82 4 18.70 2.62 8.14 1.91 23.53 7.55 9.17 3.37 5 21.61 4.36 9.61 2.08 27.58 8.78 10.84 3.61 6 24.26 5.55 10.82 2.05 30.58 9.32 12.20 3.72 7 27.28 6.75 12.45 2.21 33.89 9.78 13.97 4.29 8 30.14 7.61 13.70 2.42 36.84 10.12 15.49 5.06 24 68.9 7.22 32.8 4.48 69.36 4.96 36.31 11.66 Rheology Max viscosity 10.5 Pa s 10.5 Pa s 8.16 Pa s 4.25 Pa s Min viscosity 0.29 Pa s 0.37 Pa s 0.35 Pa s 0.21 Pa s Geleobr t°C 15.1°C 22.0°C 20.2°C 33.8°C Temp range, gelation 14.7-17.0 21.6-24.7 19.4-22.9 Wide

Example 5.

To demonstrate the effect of the invention in vivo, a pharmacokinetic study was conducted to demonstrate prolonged and effective plasma levels after a single administration of florfenicol in pigs. The study was approved by the Ethics Committee for Animal Research studies of the Hebrew University of Jerusalem. A total of six animals were used, with two female pigs aged 3–4 months. A 20-gauge central venous catheter was inserted into the jugular vein of each pig to facilitate blood collection. All animals received 40 mg/kg of drug 2.1 as a single dose in the first study group, and either 20 mg/kg Nuflor® (Merck Animal Health - 30% florfenicol solution in NMP) administered twice 48 hours apart, or another experimental treatment in the second. group after a washout period of two weeks.

Blood samples were collected before each drug administration (time 0) and 1, 2, 4, 6, 8, 10, 24, 30, 52, 72, 96, 144 and 196 hours after the first administration. Samples were collected in heparinized tubes, and plasma was immediately separated and stored at −20°C until analysis. On the day of analysis, samples were spiked with internal standard (chloramphenicol) and extracted with acetonitrile. The standards were prepared on the same day. Determination of the parent drug, florfenicol, and the major metabolite, florfenicol-amine, was performed using UHPLC-MS/MS (TSQ Quantum Access Max mass spectrometer in positive ion mode using electric spray ionization (ESI) and data acquisition in Multiple reaction monitoring (MRM) results were obtained in duplicate for florfenicol (the parent compound) and florfenicol-amine (the main metabolite).

Data analysis was performed using Microsoft Excel software. Area under the curve (AUC) values were obtained using the trapezoidal rule. Terminal slopes were identified using a semilogarithmic transformation, and the slope was calculated by fitting the curves to the exponential decay data. All further calculations were performed with fitted functions. Deconvolution was not performed due to the complexity of the model, especially for the dual injection groups. For these Nuflor® groups, the final slope data were also used to extrapolate the 48-hour points. Data were calculated using the average curve; ranges of individual values are presented where possible.

Results for a plot of plasma concentrations versus time for the parent florfenicol compound are presented in FIG. 3 for relevant comparisons. The dotted line in each graph indicates the likely maximum MIC value of 90 for typical pathogen targets of porcine respiratory disease. Error bars indicate standard error of the mean. Arrows indicate the time of administration. Diamonds (♦) represent Nuflor, labeled Treatment: Nuflor 20 mg/kg x2, n = 2), and filled squares () represent drug 2.1 (labeled Treatment

- 11 046700

P2.1, 40 mg/kg x1, n = 5), where Concentration (µg/ml) indicates the concentration of florfenicol in the blood plasma, and Time (hours) indicates the time elapsed since the start of the experiment, in hours.

The pharmacokinetic parameters that were obtained for these data are summarized in Table. 5 below.

Table 5

Parameter P2.1 40 mg/kg Nuflor 20 mg/kg x2 Final ti/2 (h) 43.5 (36.7-53.2) 53.1 11(20.3-78.2) AUCinf (µg x h x ^ml'1 ) 224.9 176.0 AUC according to MIC (AUIC) (mcg x h x ml ¹ ) 178.7 84.4 MIC percentile (%) 79.4 47.9 Stach (mcg/ml) 2.24 (1.62-2.79) 2.77 (2.28-3.25) Ttah (h) 10.8 (6-24) 8 (6-10)

The terminal half-life of Nuflor® was calculated after the second injection; data obtained after the first injection show a significantly shorter half-life, indicating rapid clearance in the early stages. The maximum concentration reported for the Nuflor group represents the maximum concentration after the first injection.

It can be readily seen that the formulation of the invention provides higher relevant florfenicol exposure as demonstrated by AUIC and time MIC percentile after a single injection compared to the commercial product.

Example 6.

To evaluate the system's ability to handle ultra-high drug loads, the following florfenicol formulations were also prepared similar to those described herein. Formulation 6.1 contained about 33 wt.% florfenicol, 6.2 about 36 wt.% and 6.3 about 39 wt.%.

The compositions were drawn into a syringe through a 16G needle, then injected, and exhibited reverse thermal behavior, such as gelling when heated and becoming liquid again when cooled. The release profiles and rheological data are summarized in Table. 6 below.

Table 6

Preparation 6.1 Prepa eat 6.2 Preparation 6.3 Weight (g) O/ /0 mass/ weight Weight (g) O/ /o mass/ weight Weight (g) % w/w Florfenicol 8.0 32.6 8.5 36.2 9.25 39.3 Poloxamer 407 h,o 12.2 h,o 12.8 2.5 10.6 Klucel® EF 0.2 0.8 0.2 0.8 0.2 0.8 DDW I,4 46.2 10.1 43.3 10.0 42.6 NMP 2 8.2 1.6 6.9 1.6 6.9 Time (h) Wednesday, SD Wednesday, SD avg. SD 0.25 1.41 0.31 1.59 0.40 2.26 0.47 0.5 2.41 0.35 2.85 0.63 4.28 0.93 1 4.38 0.87 5.41 1.09 7.69 1.17 1.5 5.94 1.32 7.42 1.57 11.33 1.54 2 7.19 1.72 9.36 2.07 14.64 1.32 3 9.33 2.36 13.14 3.10 20.54 1.27 4 11.09 2.71 16.40 4.21 25.56 1.42 5 12.57 3.22 19.10 5.10 29.44 1.77 6 14.27 3.55 21.04 5.31 33.14 1.31 7 15.64 3.55 23.39 6.01 36.91 1.64 8 17.12 4.00 25.23 6.10 40.65 0.84 24 36.45 7.16 59.74 7.54 77.95 5.15 Rheology Max viscosity 13.1 Pass 34.2 Pa s 18.9 Pa s Min viscosity 0.28 Pa s 2.3 Pa s 1.01 Pass Gelationt°C 24.4°C 18.2°C 26.2°C Temp range, gelation 23.9-28.4 18.0-20.3 25.8-28.1

It is easy to see that the formulations formed gels that responded to increased temperature, releasing drug in a controlled manner with low variability, as evidenced by the low relative standard deviation at each point.

Additional compositions were prepared at loadings of 45 wt.% and higher. Florfenicol was sieved through a 50 micron sieve to obtain a smaller particle size fraction. The compositions and results are summarized in table. 7 below.

- 12 046700

Table 7

Preparation 6.4 Preparation 6.5 Preparation 6.6 Preparation 6.7 Preparation 6.8 Weight (g) % w/w Weight (g) % w/w Weight (g) % w/w Weight (g) % w/w Weight (g) % w/w Florfenicol 45.0 45.0 — — — 47.5 47.5 Sifted florfenicol - - 45.0 45.0 45.0 45.0 47.5 47.5 - - Poloxamer 407 12.0 12.0 12.0 12.0 10.0 10.0 9.0 9.0 9.0 9.0 Klucel® EF 0.5 0.5 0.5 0.5 0.5 0.5 0.4 0.4 0.4 0.4 DDW 37.5 37.5 37.5 37.5 39.5 39.5 38.1 38.1 38.1 38.1 NMP 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 Time (h) Wednesday, std. deviation Wednesday, std. deviation Wednesday, std. deviation avg. std. deviation avg. std. deviation 0.5 21.76 3.91 17.64 4.24 10.51 2.38 6.96 1.72 10.03 2.29 1 26.14 5.38 22.02 5.75 13.35 2.50 9.40 1.66 12.35 2.29 2 30.91 5.40 27.45 6.10 18.28 2.67 13.72 1.52 16.42 2.23 4 39.71 5.53 35.80 6.28 27.11 2.88 21.74 1.27 24.02 2.07 6 47.61 5.43 43.25 6.05 35.31 3.04 29.12 1.23 31.11 2.11 24 86.47 5.00 77.75 3.87 76.36 3.09 70.49 1.89 68.40 3.21 48 99.9 1.38 90.8 1.07 95.68 2.37 91.77 2.11 84.41 1.38 Rheology Min viscosity N.P. N.P. 0.95 Pay 0.75 Pa s 0.57 Pa s Max viscosity N.P. N.P. 16.8 Pay 12.9 Pay N.P. Gelationt°C 16.7°C 17,GS 21.5°C 24.2 N.P.

Dissolution testing was performed using the Paddle over Disk method. An amount of approximately 1 g was tested in 900 ml of phosphate buffer USP, pH 6.8, supplemented with 1% CTAB (CTAB). Rheometry was performed after 500 reverse seconds with a gap of 500 μm.

Based on the results obtained, it can be seen that the smaller particle size does not have a negative effect on the release profiles, at very high loadings it may slightly accelerate drug release, and that an ultra-high loading of florfenicol can be obtained in the injectable formulation.

Example 7

Additional compositions were made with a load of 47.5 wt.%. Sifted florfenicol was used as in example 6. The compositions and results are summarized in table. 8 below.

_______________________________________________________________Table 8

Prepa rat 7.1 Preparation 7.2 Preparation 7.3 Preparation 7.4 Weight (g) % weight/ weight Weight (g) % weight/ weight Weight (g) % weight/ weight Weight (g) % weight/ weight Florfenicol* 57 47.5 57 47.5 57 47.5 57 47.5 Poloxamer 407 10.2 8.5 10.2 8.5 10.2 8.5 10.8 9 Klucel® EF 0.12 OD — — 0.12 0.1 0.12 0.1 DDW 46.68 38.9 46.8 39 40.68 33.9 46.08 38.4 NMP 6 5 6 5 12 10 6 5 Time (h) avg. std. deviation avg. std. deviation avg. std. deviation avg. std. deviation 0.5 8.97 3.77 24.31 8.04 7.16 2.42 24.56 2.94 1 11.15 3.98 26.80 7.80 9.72 2.50 27.15 3.53 2 15.40 4.49 30.98 7.35 14.14 2.38 31.38 4.14 4 23.36 5.31 38.21 6.81 22.32 2.18 38.19 4.16 6 30.48 6.02 44.52 6.60 29.72 1.91 44.27 4.26 24 69.82 7.55 77.28 4.67 71.41 0.99 77.95 4.42 48 90.04 4.27 94.71 3.18 92.76 2.09 96.84 1.65 Rheology Min viscosity 0.34 Pa s 0.34 Pa s 0.71 Pa s 0.38 Pa s Max viscosity 10.05 Pa s 9.95 Pa s 7.9 Pa s 12.7 Pa s Geleobr. t°C 27.23°С 29.86°С 28.09°С 27.23°С

* sifted florfenicol.

Based on the results obtained, it can be easily seen that compositions containing 47.5 wt.% florfenicol can be made injectable, for example, with good viscosity at ambient temperature and a suitable gelation temperature.

In addition, it can be seen that even with low amounts of hydroxypropylcellulose (see, for example, formulation 7.1 versus 6.7), the release profile remains stable with a relatively low RSD value (although the variability is actually slightly higher for 7.1).

Quite surprisingly, variability without hydroxypropylcellulose (formulation 7.2) was still within the pharmaceutically acceptable range, although even as little as 0.1% cellulose supplementation significantly reduced variability without negatively affecting the release profile. Moreover, adding more cosolvent (formulation 7.3 compared to 7.1) further improves variability, and even more so when compared to preparation 7.2 without the addition of cellulose.

- 13 046700

Example 8.

To further demonstrate the effect of the invention in vivo, another pharmacokinetic study was conducted to demonstrate prolonged and effective plasma levels after a single administration of florfenicol in pigs.

A total of 20 pigs received in parallel either 40 mg/kg single dose of 6.6-6.8 or 30 mg/kg Nuflor® (Merck Animal Health - 30% solution of florfenicol in NMP), administered according to the manufacturer's recommendations. In addition, a formulation (referred to herein as 8.1) containing 40 wt% florfenicol, 12 wt% poloxamer 407, 0.5 wt% Klucel EF, 5 wt% NMP and 42.5 wt% water, with a gelation temperature of 21.7°C was administered at a dose of 40 mg/kg. The release profile of drug 8.1 under the same conditions as in example 7 is shown in table. 9 below.

Table 9

Time (h) 0 0.5 1 2 4 6 24 48 Average 0 18.85 23.01 26.70 32.97 38.48 64.37 72.78 RSD 0 5.98 6.74 6.51 6.20 5.78 3.05 1.82

Blood samples were collected at time points 0, 0.5, 1, 2, 4, 6, 8, 10, 12, 24, 36, 48, 50, 72, 84, 96, 120, 144, and 168 hours.

In fig. Figure 4 shows a graph of florfenicol concentrations in blood plasma versus time. In fig. Figure 4 shows florfenicol plasma concentrations at each sampling point. Diamonds (♦) indicate Nuflor, filled squares () indicate drug 8.1, filled triangles (▲) indicate drug 6.6 and X-marks (x) indicate drug 6.7, and asterisks (*) indicate drug 6.8, with C (ng/ml) indicates the plasma concentration of florfenicol, and t (h) indicates the time elapsed since the start of the experiment, in hours.

Based on the results obtained, it can be easily seen that the commercially available product is rapidly cleared from the blood of pigs, while all drugs according to the invention maintain plasma levels above 1000 ng/ml for an average of 72 to 84 hours. It is noteworthy that the adjusted per dose, the AUC for therapies was comparable between groups, indicating that bioavailability was not reduced in controlled-release formulations. Peak plasma concentrations were apparently highest for the commercial immediate-release product; however, formulation 6.7 showed a significantly higher peak concentration than formulation 6.8, which differed only in drug particle size.

The time exceeding the minimum inhibitory concentration of Streptococcus suis, a virulent swine pathogen (currently considered 2 μg/ml), in the tested preparations is presented in Table. 10 below.

Table 10

Above MIC Nuflor P8.1 R6.6 R6.7 R6.8 Time (h) 7.44 18.5 27.8 34.2 14.8

It is evident from the results that the tested formulations according to the invention showed excellent results with significant clinical potential for the control of S. suis.

Example 9.

To demonstrate the ability of the compositions according to the invention to release other antibiotics, formulations containing 30% by weight amoxicillin were prepared. Formulation 9.1 contained both a cosolvent and a cellulose derivative at least partially soluble in organic solvents (hydroxypropylcellulose), preparation 9.2 contained only hydroxypropylcellulose and 9.3 none of the additional excipients. The compositions were prepared according to the methods described for florfenicol.

The compositions were drawn into a syringe through a 16G needle, then injected, and exhibited reverse thermal behavior, such as gelling when heated and liquefiing when cooled. Release profile data are summarized in Table. 11 below.

Table 11

Preparation 9.1 Preparation 9.2 Preparation 9.3 Weight (g) % w/w Weight (g) % w/w Weight (g) % w/w Amoxicillin 6.0 thirty 6.0 thirty 6.0 thirty Poloxamer 407 2.4 12 2.4 12 2.4 12 Klucel® EF 0.15 0.75 0.15 0.75 — —

- 14 046700

DDW 9.45 47.25 11.45 57.25 11.6 58 NMP 2.0 10 — — — — std. deviation Wednesday, std. deviation Wednesday, std. deviation Wednesday, std. deviation 0.5 2.99 0.94 2.52 0.45 3.14 0.50 1 6.34 1.40 5.10 0.58 5.61 0.47 1.5 9.26 1.65 7.69 0.55 7.83 0.47 2 11.76 1.81 10.04 0.75 9.68 0.66 3 16.68 2.76 14.12 1.17 12.63 1.18 4 20.90 3.43 19.03 1.63 16.06 1.88 5 25.39 3.16 22.94 2.27 21.59 6.45 6 30.12 5.44 26.56 3.10 21.40 3.31 7 34.14 6.85 29.14 2.93 23.40 2.60 8 36.52 4.86 33.34 5.34 25.65 5.81 24 68.70 7.66 84.69 22.44 77.00 21.35

It is easy to see that the formulations formed gels, releasing drug in a controlled manner with low variability, as evidenced by the low RSD value at each point, but without NMP or Klucel, drug release at a later stage became more erratic, which may indicate the formation of a less stable gel in the absence of both excipients.

Example 10.

To further demonstrate the ability of the compositions in accordance with the invention to release other antibiotics, formulations were prepared containing 15 wt.% tylosin. Formulation 10.1 contained both a cosolvent and a cellulose derivative at least partially soluble in organic solvents (hydroxypropylcellulose), preparation 10.2 contained only hydroxypropylcellulose and 10.3 none of the additional excipients. The compositions were prepared according to the methods described for florfenicol.

The compositions were drawn into a syringe through a 16G needle, then injected, and exhibited reverse thermal behavior, such as gelling when heated and liquefiing when cooled. Release profile data are summarized in Table. 12 below.

Table 12

Preparation 9.1 Preparation 9.2 Preparation 9.3 Weight (g) O/ /O mass/mass Weight (g) O/ /O mass/mass Weight (g) O/ /O mass/mass Tylosin h,o 15 h,o 15 h,o 15 Poloxamer 407 2.92 14.6 2.4 12 2.4 12 Klucel® EF 0.18 0.9 0.15 0.76 — — DDW 11.47 57.3 14.45 72.24 14.6 73 NMP 2.43 12.2 — — — — Time (h) Wednesday SD Wednesday SD Wednesday SD 0.5 11.40 1.80 5.48 0.09 4.83 1.65 1 20.95 1.16 9.33 0.99 8.24 2.96 1.5 35.71 1.47 12.51 1.56 11.26 4.24 2 37.27 1.54 15.63 1.75 14.35 5.54 3 46.75 1.72 21.51 2.09 20.05 7.60 4 52.50 1.28 25.64 2.04 25.18 9.34 5 57.97 1.95 31.04 1.60 30.63 11.46 6 61.14 1.07 36.23 2.67 36.67 13.35 7 70.25 3.08 40.72 4.02 41.42 14.09 8 72.71 3.49 45.10 6.39 46.19 15.39 24 94.05 3.02 97.94 1.60 96.85 2.08

It can be easily seen that the formulations formed gels and released the drug in a controlled manner. Formulation 10.1 had a slightly higher poloxamer content to compensate for NMP's increased drug solubility. The release profile of 10.1 showed low variability, confirmed by the low RSD value at each point, especially at intermediate time points. Formulation 10.2 showed slightly higher variability, but without NMP and Klucel, drug release became more variable.

Claims (11)

1. A pharmaceutical composition containing florfenicol in a concentration of from 25 wt.% to 50 wt.%, a poloxamer, an aqueous carrier and an organic co-solvent selected from the group consisting of N-methylpyrrolidone, dimethyl sulfoxide and polyethylene glycol 400, wherein said composition is an injectable composition at room temperature, provided that when the concentration of said florfenicol is less than 35% by weight, the composition further contains hydroxypropylcellulose. 2. The pharmaceutical composition according to claim 1, in which the concentration of said florfenicol is more than 35 wt.%, and wherein said composition does not contain hydroxypropylcellulose. 3. The pharmaceutical composition according to claim 1, in which the concentration of said florfenicol is more than 35 wt.%, and wherein said composition additionally contains hydroxypropylcellulose. 4. The pharmaceutical composition according to any one of the preceding claims, wherein said organic cosolvent is present in an amount of from about 5 wt.% to about 15 wt.%. 5. The pharmaceutical composition according to any of the preceding claims, wherein said organic co-solvent is N-methylpyrrolidone. 6. The pharmaceutical composition according to any one of the preceding claims, wherein said organic co-solvent is N-methylpyrrolidone and the composition contains hydroxypropyl cellulose and said florfenicol is present in a concentration of from 25 wt.% to 50 wt.%. 7. The pharmaceutical composition according to any of the preceding claims, wherein said organic co-solvent is N-methylpyrrolidone, and wherein the concentration of said florfenicol is from 35% by weight to 50% by weight. 8. Use of a pharmaceutical composition according to any of the preceding claims for the treatment of a veterinary infection in a non-human animal by administering to said animal a pharmacologically effective dose of florfenicol in said composition. 9. Use according to claim 8, wherein said composition is administered once to said non-human animal per course of treatment. 10. Use according to claim 8 or 9, wherein said administration includes intramuscular injection or subcutaneous injection. 11. Use according to any one of claims 8 to 10, wherein said infection is caused by a swine pathogen.