NZ747155B2 - Injectable composition for delivery of a biologically active agent - Google Patents

Abstract

The invention relates to an injectable composition for rapid and sustained delivery of a biologically active agent and to uses of the injectable composition in the treatment or prevention of a condition in a subject. The injectable composition comprises a water-in-oil emulsion having an aqueous phase dispersed in an oil phase. The aqueous phase comprises a plurality of hydrogel particles and an aqueous liquid and a biologically active agent is contained in the hydrogel particles and in the aqueous liquid.

Description

INJECTABLE COMPOSITION FOR DELIVERY OF A BIOLOGICALLY ACTIVE AGENT cal Field The present invention relates to an injectable composition for delivery of a biologically active agent. In particular, the present invention relates to an injectable composition comprising multiple phases for rapid and sustained delivery of a ically active agent. The invention also s to uses of the injectable composition in the treatment or prevention of a condition.

Background Biologically active agents may be incorporated in a number of different dosage forms for administration by a number of different routes. These dosage forms may be for example, tablets, capsules, sprays, ointments or patches for delivery of the active agent by routes such as oral, transmucosal or transdermal routes. However, for a variety of reasons, many ically active agents may not be effectively delivered using routes such as the oral, transmucosal or transdermal routes. This may be because the biologically active agent is tible to degradation by enzymes or stomach acid, or is insufficiently absorbed into the systemic circulation due to molecular size and/or charge.

As such, a number of biologically active agents are most ly administrated by injection. Administration by injection allows an active agent to rapidly enter the systemic circulation and to by-pass the digestive system and first-pass metabolism by the liver. r, repeated injections of an active agent over a period of time may be necessary in order to achieve or in a desired effect in vivo. For example, immunisation may e multiple vaccinations, boosters and high doses of vaccine generally to be administered, which can result in increased cost to both industry and the end-users.

Sustained release compositions are of interest in biomedical ations where maintenance of a systemic level of an active agent over a period of time is desired. For injected biologically active , sustained release compositions can help to reduce the frequency of injection and increase the duration of action of the active agent or reduce adverse side effects.

D:\DOCUME~1\CTS\docbases\PTO_REP\config\temp_sessions\2008900423662999969\Complete Specifications.docx A number of injectable sustained release compositions have been described. For example, one form of injectable sustained release composition utilises small diameter polymer particles for the delivery of an encapsulated drug. Such polymer particles are often formed from synthetic degradable polymers such as poly(lactic acid), lycolic acid) or poly(lactic-co-glycolic acid), which breakdown in a biological environment, leading to release of the encapsulated drug over an extended period of time. Drug release may therefore be reliant on the rate of breakdown of the polymer, which may not always give a d kinetic profile.

There s a need to develop an injectable composition that can provide a desired release profile for a biologically active agent.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and ising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

The discussion of documents, acts, materials, devices, articles and the like is ed in this specification solely for the purpose of providing a context for the present invention. It is not ted or represented that any or all of these matters formed part of the prior art base or were common general dge in the field relevant to the present invention as it existed before the priority date of each claim of this application.

Summary of the Invention The present invention provides an injectable composition comprising a biologically active agent that is formulated to provide for initial rapid e of the ically active agent, followed by sustained ry of the ical active agent, to a subject in vivo.

In one aspect, the present invention provides an injectable composition for rapid and sustained delivery of a biologically active agent comprising: a water-in-oil emulsion having an aqueous phase sed in an oil phase, the D:\DOCUME~1\CTS\docbases\PTO_REP\config\temp_sessions\2008900423662999969\Complete Specifications.docx aqueous phase comprising a ity of hydrogel particles and an s liquid; and a biologically active agent in the hydrogel particles and in the aqueous liquid of the aqueous phase, wherein when administered, the injectable ition provides rapid and sustained delivery of the biologically active agent in vivo.

In some embodiments, the injectable composition of the invention may further se an adjuvant. The adjuvant may be present in the oil phase or the aqueous phase of the water-in-oil emulsion of the injectable composition. In one ment, the adjuvant is present in the oil phase. The adjuvant may be an adjuvanting oil forming the oil phase of the emulsion.

The el particles of the aqueous phase of the water-in-oil emulsion comprise a biocompatible material. In one embodiment, the hydrogel particles comprise a crosslinked polysaccharide.

Crosslinked polysaccharide in the hydrogel les preferably comprises a polysaccharide and a crosslinking agent. In one embodiment, the crosslinking agent comprises functional groups that participate in non-covalent bonding interactions with the polysaccharide.

In one set of embodiments, the crosslinked polysaccharide in the hydrogel particles may comprise a glycosaminoglycan (GAG).

The polysaccharide present in the hydrogel particles may be ed from the group consisting of an, alginate, hyaluronic acid, cellulose, chondroitin sulphate, an sulphate, keratan sulphate, heparin and derivatives thereof. The hydrogel particles may comprise a mixture of two or more such polysaccharides and/or derivatives thereof.

In one embodiment, the crosslinked polysaccharide in the hydrogel particles comprises a polysaccharide ed from the group consisting of chitosan, te, chondroitin sulphate, and mixtures thereof.

D:\DOCUME~1\CTS\docbases\PTO_REP\config\temp_sessions\2008900423662999969\Complete Specifications.docx Chitosan in the hydrogel particles may be crosslinked with a phosphate compound, preferably a tripolyphosphate (TPP) such as sodium tripolyphosphate. te and oitin te in the hydrogel particles may each be inked with a divalent cation derived from an alkaline earth metal. In some particular ments, alginate and chondroitin sulphate in the el particles are crosslinked with calcium (Ca2+) or magnesium (Mg2+) cations.

The hydrogel particles present in the injectable composition have an average diameter in the range of from about 10 nm to 20 ?m, preferably in the range of from about 50 nm to about 5 ?m.

In one embodiment, the hydrogel particles further comprise an aqueous insoluble alkaline earth metal phosphate. Preferably, the aqueous insoluble alkaline earth metal phosphate is hydroxyapatite.

In some embodiments, the injectable composition comprises one or more coated hydrogel particles.

In some embodiments, the water-in-oil emulsion of the injectable composition comprises a surfactant. The surfactant can help to facilitate formation of a stable emulsion composition. In one preference, the injectable composition comprises a non-ionic surfactant.

The injectable composition is useful for the delivery of a biologically active agent and may comprise a range of biologically active agents. In accordance with the ion, the biologically active agent is present in the aqueous phase of the water-in-oil emulsion.

In particular, the biologically active agent is in the hydrogel les and in the aqueous liquid of the aqueous phase.

In one ment, the biologically active agent is ed from the group consisting of a hormone, an antimicrobial, a therapeutic antibody, a cytokine, a fusion D:\DOCUME~1\CTS\docbases\PTO_REP\config\temp_sessions\2008900423662999969\Complete Specifications.docx protein, a virus, a bacteria or bacteria fragment, a vaccine and an antigen. In a particular embodiment the biologically active agent is one or more selected from vaccine antigens and hormones. The injectable composition comprises at least one, and may comprise more than one, biologically active agent belonging to one or more of these classes.

In one set of embodiments, the biologically active agent is an antigen. In particular embodiments, the antigen may be Bm86 or TSOL18.

In another set of ments, the biologically active agent is a hormone. In particular embodiments, the hormone may be somatotropin or luteinising hormonereleasing hormone .

In one form of the able composition of embodiments described herein the biologically active agent in the hydrogel particles is conjugated to the particles.

The present invention also provides a method of delivering a biologically active agent to a subject, the method comprising the step of administering an injectable composition of any one of the embodiments described herein to the subject by injection.

In particular, the injectable composition of the invention provides rapid delivery of an initial primary dose of the biologically active agent followed by longer term sustained delivery of the active agent to a subject, in vivo.

The present invention r provides a method of treating or preventing a disease or disorder in a subject comprising the step of administering an injectable ition of any one of the ments described herein to the subject by injection.

In one embodiment, the injectable composition may be suitably used for the treatment or tion of a microorganism infection.

In another embodiment, the injectable ition may be suitably used for the treatment or tion of a viral infection.

In another aspect the present invention provides a process for preparing an UME~1\CTS\docbases\PTO_REP\config\temp_sessions\2008900423662999969\Complete Specifications.docx injectable composition of embodiments described herein for rapid and sustained delivery of a biologically active agent, the process comprising the steps of: providing a first aqueous composition comprising a first hydrogel forming component and a second aqueous composition comprising a second hydrogel forming component, at least one of the first aqueous composition and the second aqueous composition comprising a biologically active agent; combining the first aqueous composition with a lipophilic composition comprising an oil to form an emulsified composition; and combining the second aqueous composition with the emulsified ition under conditions allowing the first hydrogel forming component to react with the second hydrogel forming ent to form a plurality of hydrogel particles in situ and y provide an injectable composition comprising a in-oil emulsion comprising an aqueous phase dispersed in an oil phase, the aqueous phase sing a plurality of hydrogel particles and an s liquid, and wherein the biologically active agent is ned in the el particles and in the aqueous liquid of the aqueous phase of the water-in-oil emulsion.

Further aspects appear below in the detailed description.

Brief ption of the s Embodiments of the invention will now be described with reference to the following non-limiting figures in which: Figure 1 is a schematic representation showing an injectable composition comprising an antigen in accordance with one embodiment of the invention.

Figure 2 is a graph showing the antibody titre to Bm86 (ELISA 1:1600 dilution) with a comparative formulation representing a conventional anti-tick composition administered subcutaneously in two injections consisting of primary dose (day 0) and a booster injected after 4 weeks (2×50 µg Bm86/dose), and with an injectable composition of an ment of the ion administered subcutaneously as a single injection.

Figure 3 are graphs showing the amount of Bm86 released over time from a D:\DOCUME~1\CTS\docbases\PTO_REP\config\temp_sessions\2008900423662999969\Complete Specifications.docx model bulk chitosan hydrogel, with release sed as (A) the amount of Bm86 detected in the aqueous phase, including the syneresis liquid, and (B) a percentage of the initial Bm86 loading in the composition.

Figure 4 is a graph showing the release of Bm86 over an 8 week period from samples of model bulk crosslinked chitosan hydrogel with different crosslinking (TTP) densities. Column labels are "S" for the initial syneresis release after 24 hours, with the numbers enting the week number after composition formation, and total amount of Bm86 released over the cumulative 8 week period shown as a percentage.

Figure 5 illustrates graphs showing the syneresis release of cytochrome c, albumin, myoglobin and pST from samples of model bulk crosslinked chitosan hydrogels at various loading levels expressed as concentration in the syneresis liquid (A-D) and percentage of the initial loading (E), and the onship between the syneresis release and the ctric point of the loaded protein (F).

Figure 6 shows images of (A) an emulsion composition prior to the in situ formation of hydrogel les, (B) an injectable composition of an embodiment of the invention with hydrogel particles in situ, and (C) the able composition of (B) after storage at 4 °C for 5 months.

Figure 7 shows micrographs of: (A) and (B) an injectable composition of embodiments of the invention comprising hydrogel particles, (C) an injectable composition of an embodiment of the invention after storage at 4 °C for 5 months indicating the ity of the composition, and (D) the hydrogel particles after removal of the aqueous phase from the ition by evaporation.

Figure 8 is a graph showing the sis release of porcine somatotropin (pST) after a 12 hour period from samples of model bulk alginate – Ca2+ hydrogels with s crosslinking (Ca2+) densities and pST loading levels, expressed as a percentage of initial pST loading.

D:\DOCUME~1\CTS\docbases\PTO_REP\config\temp_sessions\2008900423662999969\Complete Specifications.docx Figure 9 is a graph showing the e of porcine tropin (pST) over a 28 day period from samples of model bulk alginate – Ca2+ hydrogels formed with various amounts of PEG porogen and having various pST loading levels, expressed as (A) quantitative release, and (B) release as percentage of the total pST loading.

Figure 10 is a graph showing changes in viscosity under applied shear stress for samples of injectable itions of ments of the invention comprising crosslinked chitosan hydrogel particles with various crosslinking densities and aqueous phase volume ratio.

Figures 11 shows compositional diagrams illustrating regions of low ity that correspond to the stresses indicated by the black arrows in Figure 10 for (A) an injectable composition of one embodiment prepared with chitosan, TPP and montanide oil, and (B) an injectable composition of another embodiment prepared with chitosan/hydroxyapatite and TPP/chondroitin sulphate in ide oil.

Figure 12 is a graph showing the immune response in sheep over a period of 400 days exhibited by a comparative formulation comprising Bm86 representing a conventional anti-tick composition administered subcutaneously in two injections consisting of primary dose (day 0) and a booster injected after 4 weeks (2×50 µg Bm86/dose), and by an able ition of one embodiment stered subcutaneously as a single injection.

Figure 13 is a graph illustrating syneresis release of TSOL18 from various model uncoated and coated chitosan based hydrogels over 1 week.

Figure 14 is a graph illustrating syneresis release of TSOL18 from various model inked and non-crosslinked chitosan hydrogels coated with a crosslinked alginate or chondroitin sulphate coating over 1 day.

Figure 15 shows a micrograph of an injectable composition containing TSOL18 in accordance with an embodiment of the invention, with a 1:10 dilution of the ition (in inset) showing particles of microhydrogel in the emulsion.

D:\DOCUME~1\CTS\docbases\PTO_REP\config\temp_sessions\2008900423662999969\Complete Specifications.docx Figure 16 shows (A) a tic representation of a porcine somatotropin (pST) loaded alginate hydrogel particle bearing a chitosan coating, (B) a schematic representation of the barrier effect of the coating on the e of (pST), (C) light microscope micrograph of an injectable composition comprising alginate hydrogel coated with chitosan in accordance with one embodiment of the ion, and (D) the sample shown in (C) diluted 1:10 in sesame oil selectively showing partially formed and broken chitosan shells.

Figure 17 is a graph rating the initial fractionation (protein partition) of porcine somatotropin (pST), albumin and Bm86 after hydrogel formation and the completion of sis process, where electrostatic binding of pST, Bm86 and albumin with a model bulk chitosan – HAp hydrogel formed with 0.08 M TPP in 1% ChS crosslinking is measured at equilibrium.

Detailed Description of the ion The present invention is based on the finding that both rapid and sustained delivery of a biologically active agent in vivo is able to be ed through the use of an injectable composition as described herein.

The injectable composition of the invention is able to deliver an initial primary dose of the biologically active agent, followed by more sustained delivery of the active agent over a period of time, which reduces the need for repeated injections.

In one aspect the present invention provides an injectable composition for rapid and sustained delivery of a biologically active agent comprising: a water-in-oil on having an aqueous phase dispersed in an oil phase, the aqueous phase comprising a plurality of hydrogel particles and an aqueous liquid; and a ically active agent in the hydrogel particles and in the aqueous liquid of the aqueous phase, wherein when administered, the injectable composition provides rapid and sustained delivery of the biologically active agent in vivo.

In the context of the present ion, "rapid" delivery relates to delivery of the biologically active agent ately upon stration of the injectable composition to D:\DOCUME~1\CTS\docbases\PTO_REP\config\temp_sessions\2008900423662999969\Complete Specifications.docx a t, or y thereafter. The rapid delivery provides for an initial dose of the biologically active agent immediately or shortly after injection of the composition of the invention to elicit an initial therapeutic effect.

"Sustained" delivery s to delivery of the biologically active agent subsequent to the initial rapid delivery. Sustained delivery thus occurs over a time period that is substantially longer than rapid delivery. The sustained delivery provides for a dose of the biologically active agent over a longer term and therefore prolongs the therapeutic effect provided by the active agent. In some embodiments, sustained delivery may be for a time period of at least 24 hours. In particular embodiments, sustained delivery may be for time period of at least several days or weeks, or even months.

It is believed that the ability of the injectable composition to provide for rapid and sustained delivery of a biologically active agent is assisted by the composition comprising multiple phases. The desired in vivo delivery profile of the ically active agent is thus influenced by the different phases in the composition.

In particular embodiments, the injectable composition of the invention comprises at least three phases. In some embodiments, the injectable composition may comprise more than three phases, such as four, five, six or more phases, in total. The injectable composition of the invention may be regarded as a hasic ition.

In one embodiment, the injectable composition of the invention is triphasic.

The injectable composition of the ion comprises a water-in-oil emulsion. A skilled person would understand that a water-in-oil emulsion comprises an oil phase and an aqueous phase dispersed in the oil phase. The oil phase forms the uous phase of the emulsion while the aqueous phase forms the dispersed phase.

The aqueous phase of the water-in-oil emulsion comprises an aqueous liquid. The aqueous phase also comprises a plurality of el particles, which are sed in the aqueous liquid. Thus the aqueous liquid and the hydrogel les each form part of the aqueous phase of the water-in-oil emulsion.

UME~1\CTS\docbases\PTO_REP\config\temp_sessions\2008900423662999969\Complete Specifications.docx In some embodiments, the injectable ition of the invention comprises at least three phases and these may be a first phase, a second phase and a third phase. The first phase may be provided by the oil phase of the water-in-oil emulsion. At least two r phases may be provided by the s phase of the water-in-oil emulsion. In particular, the aqueous liquid and the hydrogel particles of the aqueous phase of the emulsion may provide a second phase and a third phase of the injectable composition, respectively.

The oil phase of the water-in-oil emulsion comprises at least one logically acceptable oil and may comprise a mixture of such oils. Physiologically acceptable oils are generally hydrophobic and liquid at a temperature between 20° C and 40° C.

A range of le physiologically acceptable oils may be used in the injectable ition of the invention.

In some embodiments, the oil phase may comprise one or more oils selected from the group consisting of fatty acids; fatty acid esters; esters of polyethylene glycols, for example mono- and di-esters; hydrocarbon oils, for example natural hydrocarbon oils; and steroids, for example cholesterol.

In one embodiment, the oil phase may se one or more oils selected from fatty acids; fatty acid esters; esters of polyethylene glycols; and hydrocarbon oils.

Suitable fatty acids and fatty acid esters may be those having an aliphatic saturated or rated chain comprising from 6 to 24 carbon atoms. Unsaturated aliphatic chains may be mono- or poly-unsaturated. Some particular fatty acids may be long chain C12-C24 fatty acids e.g. C15-C22 fatty acids, and medium chain C6-C12 fatty acids.

Among these, include poly-unsaturated fatty acids such as omega-3 oils, for example, eicosapentanoic acid (EPA), docosohexaenoic acid (DHA), alpha-linoleic acid (ALA).

Combinations of such compounds are also contemplated. Fatty acid esters may be triglycerides, as well as esters of glycerol (particularly tri-esters) with a combination of fatty acids and lower molecular weight acids e.g. succinic acid (fatty acid triglycerides are D:\DOCUME~1\CTS\docbases\PTO_REP\config\temp_sessions\2008900423662999969\Complete Specifications.docx a particular example of glycerides). Oils containing triglycerides may also contain monoand /or cerides, e.g. as a minor part of the glyceride content (less than 50 mol %).

In some embodiments, the oil phase may comprise a e of oils, for example fatty acid macrogolglycerides, also known as polyoxylglycerides, which are mixtures of fatty acid monoesters, diesters and triesters of glycerol and fatty acid monoesters and diesters of polyethylene glycol; examples are oleoyl macrogolglycerides and linoeoyl macrogolglycerides.

Suitable hydrocarbon oils may be mineral oils or terpenes.

Particular terpenes may be triterpenes such as, for example, squalene.

Particular mineral oils may comprise a mixture of l hydrocarbon chains of different lengths, including small chains (= C14) and longer chain (> C14) hydrocarbon lengths. Examples of mineral oils of a pharmaceutical grade include light liquid paraffin and light l oil. ative or additional oils, which may be included in the oil phase in combination with or in place of the above oils include plant or vegetable oils, such as peanut oil, safflower oil, sunflower oil, soya bean oil, cottonseed oil, chaulmoogra oil, corn oil, jojoba oil, pesic oil, olive oil, sesame oil, almond oil, castor oil, canola oil, linseed oil, ne and coconut oil; fish oils such as shark oil, orange roughy oil, en oil and cod liver oil; animal oils such as mink oil, lard oil and chicken fat oil; and synthetic oils such as ethyl oleate.

In some ments, the oil phase may comprise a physiologically acceptable oil having adjuvanting properties. The physiologically acceptable oil is therefore an adjuvant and may be referred to as an adjuvanting oil.

In other embodiments, the oil phase may comprise a non-adjuvanting physiologically acceptable oil (i.e. a passive oil). In such embodiments, the oil does not possess adjuvanting properties. The passive oil may be regarded as being chemically inert.

D:\DOCUME~1\CTS\docbases\PTO_REP\config\temp_sessions\2008900423662999969\Complete Specifications.docx However, if desired, an adjuvant may be contained in the passive oil. Such adjuvants are preferably oleophilic or oil soluble and may be dissolved or suspended in the oil.

The oil phase is derived from oils used to e the injectable composition of the invention. The oil phase may be derived from commercially available preparations containing combinations of oils, optionally with other components. For example, cially ble emulsion ations comprising a physiologically acceptable oil may be used to produce the oil phase of the injectable composition. Such commercial emulsion preparations may bly contain one or more emulsifiers, in which case the emulsifier may also be present in the composition of the invention. Examples of commercial preparations of oil adjuvants include the Montanide series of adjuvants from Seppic. A particular example is Montanide ISA 61, which comprises a light mineral oil and an emulsifier sing mannitol and oleic acid. Other commercial formulations such as Fluad® (Novartis) and Pandemrix® (GSK), which contain MF59 and AS03 oil adjuvants, may also be used to form the oil phase.

The water-in-oil emulsion of the injectable composition also comprises an aqueous phase. In accordance with the ements of the invention, the aqueous phase comprises a plurality of hydrogel particles and an aqueous liquid.

A skilled person would understand that the term gel particles" refers to discrete colloidal portions of hydrogel material. Hydrogel materials are polymer es in a gel state that are swollen or hydrated by an aqueous liquid.

The hydrogel particles of the injectable ition described herein will generally be low modulus, soft materials comprising a low solids content and high water content. In some embodiments, the hydrogel particles may have a Young's modulus in the range of from about 5 to 700 kPa. The el particles may further have a water t of at least 60%.

In some embodiments, the hydrogel particles may be microhydrogels. The term "microhydrogels" is used herein as a reference to te portions of hydrogel having at least one dimension in the nanometer (nm) to micrometer (µm) range. Such D:\DOCUME~1\CTS\docbases\PTO_REP\config\temp_sessions\2008900423662999969\Complete Specifications.docx microhydrogels may be nanometer or micrometer sized droplets sing or composed of hydrated polymer gel.

The el particles are dispersed in and may be surrounded by the aqueous liquid.

Generally, the aqueous liquid comprises water.

It is contemplated that other compounds or ents may be present in the aqueous liquid if desired. For example, in some embodiments the aqueous liquid may comprise an adjuvant, which can be dissolved or suspended in the liquid. When used, such adjuvants are generally hydrophilic adjuvants.

The hydrogel particles of the s phase are biocompatible and are formed from biocompatible materials. Biocompatibility is a concept known to those in the art.

Biocompatible nces are those that elicit acceptable immune responses. Accordingly, as used herein the term "biocompatible" refers to a substance or component that is biologically compatible such that it substantially does not elicit an adverse immune, toxic or injurious se in vivo, or adversely integrates with a particular cell type or tissue.

The el particles can be colloidal portions of hydrogel material having a diameter in the range of from about 10 nm to 20 ?m, preferably from about 50 nm to about ?m. Hydrogel particles according to such embodiments may be referred to herein as microhydrogels. Hydrogel particle diameter may be controlled by the choice of the oil phase and the choice of any surfactant or fier used to prepare the able composition. el particle diameter may be ascertained using a range of optical techniques, such as dynamic light scattering, light microscopy and confocal laser scanning microscopy.

The hydrogels particles preferably comprise or are composed of a crosslinked polymer. In addition to being biocompatible, the crosslinked polymer is hilic and amenable to aqueous solvation.

D:\DOCUME~1\CTS\docbases\PTO_REP\config\temp_sessions\2008900423662999969\Complete Specifications.docx A range of crosslinked polymers may be suitable for the el particles.

Polymers in the el may be chemically or physically crosslinked.

Crosslinked polymers suitable for the hydrogel particles may be produced when a first component and a second component react or interact to form a three-dimensional olecular k ure that is held together via intermolecular bonds. The macromolecular network can be regarded as a polymer matrix.

The first component may be a polymer component, while the second component may be a crosslinking component. The crosslinking ent may be provided by a crosslinking agent. The crosslinking agent may be a small molecule or a macromolecule, such as a further polymer.

Crosslinking between the polymer component and the crosslinking component may occur via physical, covalent or non-covalent bonds.

Crosslinked polymers suitable for the hydrogel particles may comprise or be formed from polymers and crosslinking agents of natural or synthetic origin.

In one set of embodiments, the hydrogel particles in emulsion may be formed with a neutral polymer, which is crosslinked via physical, covalent or non-covalent bonds.

A neutral r may be one that has no ionisable functional groups. Consequently, hydrogel particles formed with a neutral polymer may carry no net charge at physiological In one set of embodiments, the el particles may be formed with a charged polymer, which is crosslinked via non-covalent bonds. The charged polymer may be a polyelectrolyte. The charged polymer may be selected from a cationic polymer, an anionic polymer, a zwitterionic polymer, or a combination thereof.

Hydrogel particles formed with a charged polymer may comprise groups that are ble, such that the el particles carry a net charge at physiological pH. d hydrogel particles can influence both the uptake and release of a biologically active agent D:\DOCUME~1\CTS\docbases\PTO_REP\config\temp_sessions\2008900423662999969\Complete Specifications.docx [Link] https://en.wikipedia.org/wiki/Galactose [Link] https://en.wikipedia.org/wiki/Iduronic_acid [Link] https://en.wikipedia.org/wiki/Glucuronic_acid [Link] https://en.wikipedia.org/wiki/Uronic_acid [Link] https://en.wikipedia.org/wiki/N-Acetylgalactosamine [Link] https://en.wikipedia.org/wiki/N-acetylglucosamine [Link] https://en.wikipedia.org/wiki/N-acetylglucosamine [Link] https://en.wikipedia.org/wiki/Amino_sugar [Link] https://en.wikipedia.org/wiki/Disaccharide [Link] https://en.wikipedia.org/wiki/Polysaccharide through electrostatic interactions, as well as the kinetics of release. Hydrogel particles formed with a charged polymer may therefore affect both the initial release of the biologically active agent as well as its longer term ned release.

In one ment, the hydrogel particles se a crosslinked biopolymer.

Biopolymers may be polymeric molecules obtained from, or derived from, natural sources.

In one embodiment, the hydrogel particles se a crosslinked polysaccharide.

The crosslinked polysaccharide is formed when at least one ccharide is combined with at least one crosslinking agent.

In one embodiment, the crosslinked polysaccharide may comprise a glycosaminoglycan (GAG). Glycoaminoglycans are unbranched ccharides based on a repeating disaccharide unit. The disaccharide unit can t of an amino sugar (N- acetylglucosamine or N-acetylgalactosamine) and either a uronic sugar (glucuronic acid or iduronic acid) or galactose. GAGs may also be esterified by sulphur containing groups.

Some examples of glycosaminoglycans are chondroitin sulphate, dermatan sulphate, keratan sulphate, and heparin. In one preference, the hydrogel particles se a crosslinked glycosaminoglycan.

Hydrogel particles comprising a inked polysaccharide may comprise a polysaccharide selected from the group consisting of chitosan, alginate, hyaluronic acid, cellulose, chondroitin te, dermatan sulphate, keratan sulphate, and heparin, as well as salts thereof and derivatives thereof. Polysaccharides such as chitosan, alginate, hyaluronic acid, chondroitin sulphate and cellulose may possess adjuvanting properties, and can assist in modulating the e of the biologically active agent from the injectable composition in vivo.

Crosslinked polysaccharide in the hydrogel particles may be d (i.e. carry a net positive or negative charge) or uncharged.

In one preference, the crosslinked polysaccharide comprises a polysaccharide selected from oitin sulphate, alginate and chitosan, a salt thereof or a tive D:\DOCUME~1\CTS\docbases\PTO_REP\config\temp_sessions\2008900423662999969\Complete Specifications.docx thereof.

Derivatives of chitosan may be chitosan-dextran sulphate, N-trimethyl an, N-carboxymethyl chitosan, and lipo- and glycoconjugated derivatives of oligochitosans.

Polysaccharides are combined with crosslinking agents to form a three- dimensional crosslinked macromolecule. It would be appreciated that the polysaccharide and the crosslinking agent must be e of interacting with one another in order to form the crosslinked polymer. Accordingly, the polysaccharide and the crosslinking agent may each comprise a chemical moiety bearing a functionality that is capable of crosslinking.

Suitable crosslinking agents may comprise or be composed of a chemical moiety that is capable of interacting with a functional group t on the polysaccharide via covalent or non-covalent bonding mechanisms. In some embodiments, the chemical moiety may bear onal groups that are capable of interacting with functional groups present on the polysaccharide via covalent or valent bonding isms.

In one form, suitable crosslinking agents may be capable of ipating in non- covalent bonding interactions with the polysaccharide. The crosslinking agents may comprise functional groups that can participate in such non-covalent g interactions.

The polysaccharide may se an ionisable functional group (such as an amino, carboxylic acid or nate , that is capable of forming a cationic or anionic functional group, and the crosslinking agent may comprise a complementary ionisable functionality that is capable of bonding with one or more functional groups of the polysaccharide through non-covalent interactions. Thus the ing crosslinked polysaccharide may be crosslinked via electrostatic or ionic bonds.

Ionisable functional groups in the polysaccharide may impart a net charge to the crosslinked polysaccharide if some of the ionisable functionalities remain free and not bound with a complementary crosslinking agent. These free ionisable functional groups may be available to interact with an oppositely charged biologically active agent contained in the hydrogel particles via non-covalent interactions such as electrostatic interactions.

D:\DOCUME~1\CTS\docbases\PTO_REP\config\temp_sessions\2008900423662999969\Complete Specifications.docx This could aid in the retention of the active agent in the hydrogel particles and thus influence the delivery profile of the biologically active agent over the time period where sustained release is desired.

In one ment, the hydrogel particles comprise crosslinked chitosan.

Chitosan is a linear polyaminosaccharide composed of randomly distributed ß-(1- 4)-linked D-glucosamine (a deacetylated unit) and N-acetyl-D-glucosamine (an acetylated unit). The degree of deacetylation (% DA) can be determined by NMR spectroscopy, and the % DA in commercial chitosan is in the range %.

Chitosan is biocompatible, enzymatically biodegradable (for example by lysozyme hydrolysis), and non-toxic (its degradation products are vely nonimmunogenic and non-carcinogenic).

The amino group in chitosan has a pKa value of approximately 6.5. Thus, chitosan is positively charged (i.e. the amino groups are protonated) and soluble in acidic to neutral solution with a charge density ent on pH and the % DA-value. In other words, chitosan can act as a positively charged polyelectrolyte under physiological conditions and thus has appropriate onality to be crosslinked with a crosslinking agent.

Chitosan suitable for use in the hydrogel particles may be of a range of molecular weights. In an embodiment, the chitosan is a low molecular weight chitosan having a molecular weight (Mw) of between 40-250 kDa. In some embodiments, the Mw is ably in the range of 50-200 kDa and even more preferably 100-180 kDa.

Chitosan may be crosslinked by a range of crosslinking agents or compounds.

Suitable crosslinking agents or compounds may se c functionalities. The anionic groups are capable of interacting with positively charged amino groups in chitosan to produce a crosslinked an that is held er via non-covalent electrostatic bonds.

In one form, the hydrogel particles may comprise chitosan inked with a D:\DOCUME~1\CTS\docbases\PTO_REP\config\temp_sessions\2008900423662999969\Complete Specifications.docx phosphate compound. The ate compound can be selected from those suitably functionalised as to promote intermolecular crosslinking between chains of chitosan. The phosphate compound may be suitably chosen to facilitate rapid (spontaneous) crosslinking and thus hydrogel formation when combined with the chitosan. Suitable crosslinking phosphate compounds include tripolyphosphate, and salts thereof. Commonly known salts of tripolyphosphate include sodium tripolyphosphate and potassium yphosphate.

Sodium tripolyphosphate (STPP, sometimes STP or sodium triphosphate or TPP), with formula Na5P3O10, is a polyphosphate of sodium. It is the sodium salt of triphosphoric acid. In one preference, the crosslinking agent is TPP.

In some embodiments, an may be inked by compounds comprising electrophilic functional groups. A skilled person would appreciate that the electrophilic group may react with nucleophilic amino groups in the chitosan, resulting in the formation of a covalent carbon-carbon bond between the inosaccharide and the crosslinking agent. Suitable crosslinking agents may comprise electrophilic groups selected from ketone, aldehyde and epoxide functional groups. In one preference, the crosslinking compound may be selected from glutaraldehyde and epichlorohydrin.

Crosslinked an useful for the hydrogel les may comprise chitosan and a inking agent in a suitable molar ratio. In some embodiments, it may be desirable to vary the level of crosslinking by adjusting the molar ratio of chitosan to crosslinking agent.

Variation in the crosslink density may be used to modify the physical ties of the el particles and/or to modulate the release of a bioactive agent from the hydrogel particles.

In some embodiments, the molar ratio of chitosan to inking to chitosan agent is from about 12:1 to about 50:1, preferably from about 25:1 to about , more preferably from about 37:1 to about 1030:1.

In some embodiments, it may be preferable for the crosslinked chitosan to comprise a relatively low crosslinking ratio (i.e. a low molar concentration of crosslinking agent) as this may result in ionised groups in the chitosan remaining charged and thus available to ipate in non-covalent interactions with a biologically active agent D:\DOCUME~1\CTS\docbases\PTO_REP\config\temp_sessions\2008900423662999969\Complete Specifications.docx contained in the hydrogel particles.

In other embodiments, it may be preferable for the crosslinked chitosan to comprise a relatively high crosslinking ratio (i.e. excess crosslinking agent relative to polysaccharide). A high crosslink density may in some ments ageously assist with rapid polymer network and hydrogel formation as well as help to improve the mechanical properties of the hydrogel particles and/or help produce more uniform logy.

In one embodiment, the hydrogel particles comprise crosslinked alginate.

Alginate is naturally occurring polysaccharide that is ed from seaweed and is composed of a block copolymer comprising covalently linked blocks comprising (1 -4)- linked ß-D-mannuronate (M) and a-L-guluronate (G) es. The proportion and distribution of M and G may determine the physical and chemical properties of the alginate.

Alginate has biocompatibility and low toxicity and can undergo crosslinking and gelation under mild conditions. At neutral pH ximately pH 7), alginate may be anionic and carry a net negative charge.

Alginate le for use in the hydrogel particles may be of a range of molecular weights. In some embodiments, alginate may have a molecular weight in the range of from about 40 to 270 kDa. In an embodiment, the alginate may have medium molecular weight, which gives a ity of >2000 cP at a concentration of 2% in water at 25 °C.

Crosslinking of alginate may be induced by combining the alginate with a positively charged molecule or compound, such as a . Alkaline earth metal compounds may provide a source of divalent cations and thus such compounds may be used as crosslinking agents to crosslink the alginate. Alkaline earth metal compounds useful as crosslinking agents may be m or magnesium compounds. In one embodiment the alginate may be crosslinked electrostatically with s such as calcium (Ca2+) or magnesium (Mg2+) cations.

D:\DOCUME~1\CTS\docbases\PTO_REP\config\temp_sessions\2008900423662999969\Complete Specifications.docx In one preference, the hydrogel particles comprise te crosslinked with calcium cations. Calcium s may participate in selective ionic bonding with guluronate residues in alginate chains to induce gel formation and crosslinking via noncovalent bonding interactions.

Calcium cations may be provided by a range of calcium compounds as crosslinking agents. Suitable calcium compounds may be selected from calcium chloride (CaCl2), calcium sulphate (CaSO4) and calcium carbonate (CaCO3). In one preference, the calcium compound is calcium chloride.

Crosslinked alginate useful for the hydrogel particles may comprise alginate and a cation (such as a calcium cation) in a suitable molar ratio. In some embodiments, it may be desirable to vary the level of crosslinking by adjusting the ratio of alginate to cation.

For instance, it has been found that stable injectable itions comprising hydrogel particles in a water-in-oil emulsion can be prepared by combining sesame oil with 2% alginate in water and 5.6% CaCl2 in water at volumetric ratios of 2:1:0.2 to 2:1:1 and 2:0.5:0.2 to 2:0.5:1 (based on oil : alginate solution : CaCl2 solution).

The crosslinking of alginate with a cation also neutralises the negative charge carried by alginate at neutral pH. This neutralisation of negative charge allows hydrogel particles comprising alginate to efficiently contain negatively charged biologically active agents, which might otherwise be difficult to achieve due to the potential for unfavourable electrostatic interactions between the negatively charged biologically active agent and the c alginate polymer.

Variations in cation concentration can be used to alter the crosslink density of the alginate containing hydrogel particles and this can be used to modify the physical properties of the hydrogel particles and/or to te the release of a bioactive agent from the hydrogel les. For example, when the tration of cation is icient to lise all vely charged groups in the alginate, the alginate may therefore have residual anionic . Hydrogel particles comprising the alginate may thus carry a net D:\DOCUME~1\CTS\docbases\PTO_REP\config\temp_sessions\2008900423662999969\Complete Specifications.docx negative charge at logical pH. In some embodiments, this al net negative charge may advantageously help to modulate the uptake and release of a biologically active agent from the hydrogel particle. For example, in the case of a positively charged ically active agent, a net negative charge carried by a el particle may help to inhibit rapid release of the active agent so that more sustained release of the agent can be achieved over a prolonged period of time.

In some embodiments, the el particles may comprise or be composed of a glycosaminoglycan (GAG). Glycosaminoglycans (GAGs) are long unbranched polysaccharides containing a repeating disaccharide unit. The repeating disaccharide unit contains either of two modified sugar (N-acetylgalactosamine or N-acetylglucosamine), and lly a uronic acid (glucuronate or iduronate). GAGs may also be esterified by sulphur ning groups.

In one set of embodiments, the hydrogel particles may comprise or be composed of at least one glycosaminoglycan (GAG) selected from the group ting of chondroitin, hyaluronate, keratan, dermatan, heparin, and derivatives thereof, such as chondroitin sulphate, sodium hyaluronate, keratan sulphate, dermatan te, and heparin sulphate.

In one embodiment, the glycosaminoglycan (GAG) is oitin te.

Chondroitin sulphate is a sulphated glycosaminoglycan composed of an unbranched polysaccharide chain of alternating sugars (N-acetyl-galactosamine and glucuronic acid).

The sulphate is covalently attached to the sugar. If some glucuronic acid residues are ized into L-iduronic acid, the resulting disaccharide is then referred to as dermatan sulphate. Since the molecule has multiple negative charges at physiological pH (approximately pH 7), a cation is present in salts of chondroitin sulphate. Commercial preparations of chondroitin sulphate typically are the sodium salt.

Chondroitin sulphate is a major component of the extracellular matrix, and is important in maintaining the structural integrity of the tissue. It is also an important structural component of cartilage, as part of aggrecan, and provides much of its resistance to compression through the tightly packed and highly charged te groups of D:\DOCUME~1\CTS\docbases\PTO_REP\config\temp_sessions\2008900423662999969\Complete Specifications.docx chondroitin sulphate.

A chondroitin chain can have over 100 individual sugars, each of which can be sulphated in variable positions and quantities. Each monosaccharide may be left hated, sulphated once, or sulphated twice. Most commonly, the hydroxyls of the 4 and 6 positions of the N-acetyl-galactosamine are sulphated, with some chains having the 2 position of glucuronic acid sulphated. tion is mediated by specific sulphotransferases. Sulphation in these different positions confers specific biological activities to chondroitin glycosaminoglycan .

Some old classification terminology exists as follows: Chondroitin te A— tion site is carbon 4 of the N-acetylgalactosamine sugar (also known as chondroitin- 4-sulphate); Chondroitin sulphate B—an old name for dermatan sulphate, which is no longer classified as a form of chondroitin sulphate; Chondroitin sulphate C—sulphation site is carbon 6 of the N-acetyl-galactosamine sugar (also known as chondroitin sulphate); Chondroitin sulphate D—sulphation sites are carbon 2 of the glucuronic acid and 6 of the N-acetylgalactosamine sugar (also known as chondroitin-2,6-sulphate); and Chondroitin sulphate E—sulphation sites are carbons 4 and 6 of the N-acetylgalactosamine sugar (also known as chondroitin-4,6-sulphate). All such derivatives are encompassed herein as roitin sulphate" as contemplated for use in the present invention.

Chondroitin sulphate useful for inclusion in the hydrogel particles may have an average molecular weight of from about 5,000 Da to about 150,000 Da, from about 10,000 Da to about 50,000 Da, or from about 10,000 Da to about 40,000 Da. Other molecular weights can however be used.

When the hydrogel particles comprise or are composed of a glycosaminoglycan (GAG) such as oitin sulphate, a net charge can be carried by the hydrogel particles at logical pH and this charge may ageously assist with modulating the release of a biologically active agent contained in the particles. In particular, chondroitin sulphate may interact with a positively charged biologically active agent to provide higher entrapment and ion of the active agent in the hydrogel particles. Electrostatic interactions between the negatively charged chondroitin te and a positively charged D:\DOCUME~1\CTS\docbases\PTO_REP\config\temp_sessions\2008900423662999969\Complete Specifications.docx biologically active agent can also help to limit ure release of the active agent or improve the erm release profile of the active from the hydrogel particle.

Furthermore, chondroitin te may have immunoregulatory effects, and may also interact with polysaccharides such as chitosan via non-covalent bonding interactions to contribute to the crosslinking of the polysaccharide.

In some embodiments, the hydrogel particles may comprise or be composed of a crosslinked glycosaminoglycan.

Sulphated glycosaminoglycans such as chondroitin sulphate may be crosslinked with a suitable crosslinking agent. In one embodiment, the crosslinking agent may be a cation, such as a divalent cation sourced from alkaline earth metal compounds. In one embodiment, the chondroitin sulphate may be crosslinked with cations such as calcium (Ca2+) or magnesium (Mg2+).

In one embodiment, the hydrogel les comprise chondroitin sulphate crosslinked with calcium cations. Calcium s may be provided by a range of calcium compounds as crosslinking agents. Suitable calcium compounds may be selected from calcium chloride (CaCl2), calcium sulphate (CaSO4) and calcium carbonate (CaCO3). In one preference, the calcium compound is calcium chloride.

Crosslinked chondroitin sulphate useful for the hydrogel particles may se chondroitin sulphate and a cation (such as a calcium cation) in a suitable molar ratio. In some embodiments, the molar ratio of chondroitin sulphate to cation may be from 4:1 to 1:10.

Similar to te discussed above, variations in cation tration can be used to alter the crosslink density and charge of the crosslinked glycosaminoglycan ning hydrogel particles. For example, when the GAG is sulphated GAG (for example chondroitin sulphate), the concentration of cation may be insufficient to neutralise all vely charged groups in the GAG and thus the GAG may carry a net negative charge at physiological pH due to residual anionic groups. This residual net ve charge may D:\DOCUME~1\CTS\docbases\PTO_REP\config\temp_sessions\2008900423662999969\Complete Specifications.docx advantageously help to modulate the uptake, retention and release of a positively charged biologically active agent from the hydrogel particle according to mechanisms discussed above.

In some embodiments, hydrogel particles of the injectable composition of the ion may comprise a mixture of polysaccharides, such as mixture of two different glycosaminoglycans, or a mixture of a glycosaminoglycan and at least one other polysaccharide. At least one of the polysaccharides is crosslinked. In some embodiments, the crosslinked polysaccharide can be crosslinked chitosan, crosslinked alginate or inked chondroitin sulphate, as described .

In one form, the hydrogel particles comprise a mixture of oppositely charged polysaccharides. For example, the hydrogel particles may comprise a mixture of chitosan and chondroitin sulphate. In such ments, negatively charged sulphate groups on the chondroitin sulphate may interact with positive amino groups present in chitosan via electrostatic interactions to thereby form a crosslinked macromolecule. The crosslinked olecule may be considered to be a hybrid crosslinked polysaccharide that is composed of a mixture of chitosan and chondroitin sulphate. The crosslinked macromolecule, which is composed of at least two electrostatically linked polysaccharides, therefore forms the crosslinked polymer matrix of the hydrogel particles. Other es of oppositely charged polysaccharides are contemplated, such as a mixture of chitosan and alginate.

In some embodiments, a d polysaccharide that is e of interacting with r polysaccharide of opposite charge may be regarded herein as a crosslinking agent.

In some other embodiments, when the hydrogel particles comprise a aminoglycan (GAG), the GAG may not form part of the crosslinked network structure of the hydrogel polymer. In such embodiments, the GAG may be present as an additive component in the hydrogel particles. As an additive, the glycosaminoglycan may act to help modify the physical or mechanical properties of the hydrogel particles.

UME~1\CTS\docbases\PTO_REP\config\temp_sessions\2008900423662999969\Complete Specifications.docx In particular, the glycosaminoglycan (GAG) can enable the el particles to become more pliable, with greater c like properties. This may be beneficial when the hydrogel particles also comprise an aqueous ble alkaline earth metal phosphate as described below, as the inclusion of a glycosaminoglycan (GAG) in the particles may enable the mechanical properties of the particles to be adjusted so as to e structural stability and in vivo performance. s in el particle properties may be due to the glycosaminoglycan acting to prevent crystallisation and growth of the alkaline earth metal phosphate, y reducing the rigidity of the particles.

In one set of embodiments, hydrogel les comprising a glycosaminoglycan may se chondroitin sulphate in an amount of up to 2% (w/v). In some embodiments, the hydrogel particles may comprise chondroitin sulphate in an amount of up to 1% (w/v).

When the hydrogel particles comprise a crosslinked macromolecule composed of electrostatically linked chondroitin sulphate and chitosan, the amount of chondroitin te and chitosan may preferably be in a 1:2 weight ratio. An injectable formulation containing such hydrogel particles can be prepared by emulsifying a an aqueous solution containing 2% (w/v) chitosan with an oil, to which a second aqueous solution containing 1% (w/v) chondroitin sulphate is added under continuous shear to give 1:2 chondroitin sulphate : chitosan weight ratio microhydrogel particles in a in-oil emulsion.

If desired, the hydrogel particles may comprise one or more additional ents. The additional components may be used to modify the chemical and/or physical properties of the hydrogel particles present in the injectable composition of the invention.

In one form, the hydrogel particles may further comprise a biocompatible aqueous insoluble alkaline earth metal ate and/or a biocompatible proteoglycan.

Aqueous insoluble alkaline earth metal phosphates may be included in the hydrogel particles to modify the physical or mechanical properties of the hydrogel particles. For ce, the aqueous insoluble alkaline earth metal phosphates may be used D:\DOCUME~1\CTS\docbases\PTO_REP\config\temp_sessions\2008900423662999969\Complete Specifications.docx to se the ty to the particles.

In some embodiments, the hydrogel particles may comprise aqueous insoluble phosphates of calcium and magnesium. Doped calcium phosphates, such as Mg2+, Zn2+, Na+, CO32- and SiO44- doped calcium phosphates, are also contemplated.

In one embodiment the aqueous insoluble alkaline earth metal phosphate is apatite.

Apatite is a group of phosphate minerals and es fluorapatite, Ca5(PO4)F3; patite, Ca5(PO4)3Cl; atite, Ca5(PO4)3Br and hydroxyapatite, Ca5(PO4)3(OH) (which are also often usually written Ca10(PO4)6(OH, F, Cl, Br)2 to denote that the crystal unit cell comprises two molecules). Hydroxyapatite crystallizes in the hexagonal l system. It has a specific y of 3.1-3.2 and has a hardness of 5 on the Mohs hardness scale. Hydroxyapatite can be found in teeth (enamel) and bones. About 70% of bone is comprised of hydroxyapatite.

In a preferred embodiment the hydrogel particles may further comprise hydroxyapatite.

In one set of embodiments, the hydrogel les may comprise hydroxyapatite in an amount of up to 0.1% (1 mg/ml).

In one set of embodiments, the hydrogel particles may se hydroxyapatite and chitosan at a weight ratio of hydroxyapatite to chitosan of 3:20, more preferably a weight ratio of 1:20 yapatite : chitosan. The latter ratio can be achieved using an aqueous 2% chitosan solution containing hydroxyapatite at a concentration of 1 mg / mL in the preparation of the injectable composition of the invention. yapatite may also be capable of modifying crosslinking reactions used to form the crosslinked polymer of the hydrogel particles through charge interactions with one or more components of the crosslinked polymer. For example, excess of PO43- ions from hydroxyapatite at mildly acidic pH may modify the ability of chitosan to interact with D:\DOCUME~1\CTS\docbases\PTO_REP\config\temp_sessions\2008900423662999969\Complete Specifications.docx a crosslinking agent.

Proteoglycans are a specific group of compounds that have at least one aminoglycan chain ed to a protein. Such compounds may comprise multiple negative charges at physiological pH (approximately pH 7). The el particles may comprise at least one proteoglycan. Mixtures of two or more glycans are also contemplated. An example of a proteoglycan is aggrecan.

In one set of embodiments, the hydrogel particles may be porous. In such ments, one or more of the hydrogel particles in the injectable composition comprises at least one pore and may comprise a plurality of pores. In one form, each hydrogel particle comprises a plurality of pores. Pores in the hydrogel particles may help to modify the release of the biologically active agent that is in the particles. In some embodiments, hydrogel particles comprising pores may release biologically active agent present therein at a faster rate.

Hydrogel particles in the able composition of the invention may be ed hydrogel particles or coated hydrogel particles. The able composition may comprise a e of uncoated and coated hydrogel particles.

In one set of embodiments, the injectable composition comprises one or more coated hydrogel particles. Thus hydrogel particles as described herein may be coated. The injectable composition of the invention may therefore comprise at least one coated hydrogel particle in the s phase. In some embodiments, the injectable composition of the invention may se a plurality of coated hydrogel particles in the aqueous phase.

Coated hydrogel particles may comprise an inner hydrogel component forming a core and an outer ent of material that covers at least a portion of the core. The outer component can be regarded as a coating for the inner core.

The coating of a coated hydrogel particle may be a distinct layer of material that covers at least a portion of a hydrogel core. In some embodiments, the coating may be a D:\DOCUME~1\CTS\docbases\PTO_REP\config\temp_sessions\2008900423662999969\Complete Specifications.docx shell that encapsulates and ns the hydrogel core.

When present, the coating generally forms part of the el particle per se and is attached to the inner core of the le. Attachment of the coating to the core may be via physical or chemical means.

The presence of a coating may be determined using various optical, imaging or spectroscopic techniques. For e, the coating may be visually ed microscopically.

It is thought that the coating forms due to phase separation of a component material from the mixture of components used to form the injectable composition of the ion in accordance with methods described herein. The separated component material preferentially locates at the surface to form the coating while the remaining components of the mixture can form the core of the hydrogel particle.

As an example, a coated hydrogel particle with a polysaccharide coating may be formed when a crosslinker diffuses from the core microhydrogel to the surface of the core and reacts with a polysaccharide in solution in the environment to give a crosslinked gel layer around the core.

In another example, a coated hydrogel particle may be formed when two oppositely charged polymers interact electrostatically to form an interpenetrated network (IPN), where one of the rs is d in the core of the hydrogel and the other r is located in solution in the microhydrogel environment.

In one set of embodiments, the coating may be ed to a microhydrogel particle core by chemical means. Exemplary chemical means can be chemical bonds that may be formed between the coating and the hydrogel particle core. In some embodiments, at least a n of the coating is bound to at least a portion of the polymer material component in the hydrogel core via covalent or non-covalent bonds. Non-covalent bonds may be electrostatic bonds or hydrogen bonds.

D:\DOCUME~1\CTS\docbases\PTO_REP\config\temp_sessions\2008900423662999969\Complete Specifications.docx In another set of embodiments, the coating may be attached to a microhydrogel particle core via physical means. In some embodiments, at least a portion of the coating is physically interlaced with at least a portion of the polymer material component in the hydrogel particle core on a molecular scale. In some embodiments, a portion of the coating is physically entangled with a portion of the polymer material of the hydrogel core.

The region of entanglement between the coating and the hydrogel particle core can resemble an interpenetrating polymer network.

The coating can advantageously help to control the passage of the biologically active agent from the hydrogel particle into the aqueous liquid portion of the aqueous phase of the water-in-oil emulsion and hence into the surrounding environment for release in vivo. In some embodiments, the coating can assist to reduce the rate of release of the biologically active agent from the el particle and thus help retard premature e of the active agent, or help promote sustained release of the active agent over a period of time.

Control over the e of the ically active agent may be achieved through a number of mechanisms, including by the coating per se, having adjustable porosity, or by the coating ng a net charge.

The coating of a coated hydrogel particle may further be crosslinked or non- crosslinked. The degree of crosslinking (if any) may provide a further mechanism for controlling the passage of a biologically active agent from the hydrogel particle.

A coating of a coated hydrogel particle may comprise or be ed of a patible material.

In some embodiments, the coating may comprise a biocompatible polymer material. Such r materials can be patible, hydrophilic and amenable to aqueous solvation. A range of patible polymers may be suitable for the coating.

In one embodiment, the coating may comprise a polysaccharide. The polysaccharide may be selected from any one of the polysaccharides described herein. In D:\DOCUME~1\CTS\docbases\PTO_REP\config\temp_sessions\2008900423662999969\Complete Specifications.docx one form, the coating comprises a polysaccharide selected from the group consisting of chitosan, alginate, hyaluronic acid, chondroitin sulphate, cellulose, dermatan sulphate, keratan sulphate and heparin sulphate, as well as salts thereof and derivatives thereof.

Such polysaccharides, salts and derivatives thereof are described .

In some embodiments it can be preferable for the polysaccharide in the coating to be different from the polysaccharide in the core of the hydrogel particle. For example, when a hydrogel particle core comprises a crosslinked chitosan, the coating may comprise te or a glycosaminoglycan such as chondroitin sulphate. Similarly, when the hydrogel particle core comprises a crosslinked alginate or crosslinked chondroitin sulphate, the coating on the hydrogel particle core may comprise chitosan. Other combinations of ccharide are also contemplated.

At physiological pH (approximately pH 7), polysaccharides may carry a net charge, which depending on the composition of the polysaccharide, may be positive or ve. Consequently, a coating comprising such polysaccharides may be charged at physiological pH. The charged coating can influence the rate of release of a biologically active agent from the hydrogel particle, particularly where the active agent is also d.

For example, a coating having a net negative charge may interact with a positively charged biologically active agent ostatically and in this manner can help to te the e of the active agent from the el particle into the s liquid of the aqueous phase (i.e. syneresis).

In some embodiments, the coating may comprise a polysaccharide that is positively charged at physiological pH. The vely charged polysaccharide can impart a positive charge to the coating. An exemplary positively d polysaccharide is chitosan.

When chitosan is present in the coating, the core of the hydrogel particle may and preferably will comprise a different polysaccharide, which is not chitosan. In one preference, the hydrogel particle core comprises negatively charged ccharide such as alginate or a glycosaminoglycan such as chondroitin sulphate. The negatively charged polysaccharide may aid in the attachment of the positively d coating to the core D:\DOCUME~1\CTS\docbases\PTO_REP\config\temp_sessions\2008900423662999969\Complete Specifications.docx component of the hydrogel particle.

In one embodiment, the coating comprises a polysaccharide that is vely d at physiological pH. The negatively charged polysaccharide can impart a negative charge to the coating. Exemplary negatively charged polysaccharides may be selected from te and sulphated glycosaminoglycans (sulphated . Sulphated GAGs may be oitin sulphate, keratan te, dermatan sulphate and heparin sulphate. When the coating comprises a negatively charged polysaccharide, in some embodiments it can be preferable for the core of the hydrogel particle to comprise a positively charged ccharide. In one preference, the hydrogel particle core comprises chitosan. The positively charged polysaccharide may aid in the attachment of the negatively charged coating to the core of the hydrogel particle.

A coating of a coated hydrogel particle may be crosslinked or non-crosslinked. A crosslinked coating may se a crosslinked polysaccharide, while a non-crosslinked coating may comprise a polysaccharide that is not crosslinked.

Non-crosslinked and crosslinked coatings may be ed to a hydrogel particle core via physical or chemical means as described herein.

Crosslinked polysaccharides suitable for use in a crosslinked coating may comprise a polysaccharide as described herein crosslinked with a suitable crosslinking agent. A skilled person would be able to select a suitable crosslinking agent for a selected polysaccharide. Exemplary polysaccharides include chitosan, alginate and sulphated GAGs such as oitin sulphate and suitable crosslinking agents for these polysaccharides are bed herein.

In some ments, when the coating comprises a charged polysaccharide (either positively or negatively charged), the hydrogel particle core may comprise a charged compound that is capable of interacting with the d polysaccharide in the coating in order to facilitate attachment of the coating to the core. The charged compound may be selected to be complementary to the charged polysaccharide in the g. For D:\DOCUME~1\CTS\docbases\PTO_REP\config\temp_sessions\2008900423662999969\Complete Specifications.docx instance, a positively charged nd may be incorporated in the hydrogel particle core when a negatively charged polysaccharide is in the coating, and vice versa.

In one embodiment, the coating comprises chitosan. In such embodiments, the hydrogel particle may comprise a phosphate compound. The phosphate compound can interact with amino groups present in chitosan in order to attach the chitosan to the hydrogel through non-covalent electrostatic bonds. The phosphate compound may be tripolyphosphate, and salts thereof, such as sodium tripolyphosphate (TPP) and potassium tripolyphosphate. In one preference, the phosphate compound is TPP.

In one embodiment, the coating ses alginate or a sulphated glycosaminoglycan such as chondroitin sulphate. In such embodiments, the hydrogel particle core may comprise a cation, preferably a divalent cation. Exemplary cations may be calcium (Ca2+) or magnesium (Mg2+) s. The cation in the el particle core can ct with anionic groups such as ylic acid or sulphate groups present in polysaccharide in order to attach the polysaccharide to the core.

When a charged compound is used to facilitate the attachment of a coating to the hydrogel particle core, it may not be necessary for the r (e.g. the polysaccharide) in the core of the hydrogel particle to be charged. For example, the hydrogel core may comprise or be composed of an uncharged polysaccharide. In these circumstances, the ing of the coating to the hydrogel le core is achieved via the charged compound contained in the core.

Additionally, when a hydrogel particle ses a charged compound (such as a phosphate compound or a divalent cation), the charged compound may diffuse from the core of the hydrogel particle into the coating. Depending on the polysaccharide in the coating, the charged compound may act as a crosslinking agent for the ccharide and in this way help promote the formation of a crosslinked ccharide in the coating. A crosslinked coating may have greater thickness than a non-crosslinked coating and thus might provide a further avenue for modulating the ned release of a biologically active agent from the coated hydrogel particle over a period of time.

Crosslinked polysaccharides useful for a coating in a coated el particle may D:\DOCUME~1\CTS\docbases\PTO_REP\config\temp_sessions\2008900423662999969\Complete Specifications.docx comprise polysaccharide and a crosslinking agent in a suitable molar ratio. In some embodiments, it may be desirable to vary the level of inking by ing the molar ratio of polysaccharide to inking agent. Variation in the crosslink density may be used to modify the physical properties of the coating, such as the ty or the net charge of the coating. In turn, this may help to modulate the release of a bioactive agent from the hydrogel particle to the aqueous liquid portion of the aqueous phase of the able composition.

In some embodiments, it may be preferable for a crosslinked polysaccharide in the coating to comprise a relatively low crosslinking ratio (i.e. a low molar concentration of crosslinking agent) as this may result in ionised groups in the polysaccharide remaining charged at physiological pH and thus available to participate in non-covalent interactions with a ically active agent contained in the hydrogel particles. Non-covalent interactions between the ically active agent and the coating can also help to modulate the release profile of the biologically active agent from the hydrogel particle over a period of time.

In some embodiments, the molar ratio of crosslinking agent to polysaccharide in a coating comprising a crosslinked polysaccharide is from about 1:1 to about 50:1, preferably from about 2:1 to about 30:1, more preferably from about 5:1 to about 20:1.

In some ments, the coating of a coated hydrogel particle may comprise an hilic compound. An amphiphilic compound in the g may help to stabilise the water-in-oil emulsion or may help to modulate the release of the biologically active agent from the injectable composition of the invention. In one embodiment, the amphiphilic compound is a lecithin.

In some embodiments, the coating of a coated hydrogel particle is . Porous coatings may be formed through the incorporation of a porogen during the manufacture of the injectable composition of the invention or by varying crosslinked density in the case of a crosslinked coating.

Porogens of different size may be used to e coatings of different porosity.

D:\DOCUME~1\CTS\docbases\PTO_REP\config\temp_sessions\2008900423662999969\Complete Specifications.docx An exemplary porogen is poly(ethylene glycol) (PEG). PEG compounds of different molecular weight can produce passages (pores) of ent size in the coating. ary poly(ethylene glycol) may have a lar weight in a range of from about 200 to 100,000 Da.

The injectable composition of the invention comprises at least one biologically active agent, which is desired to be administered to a subject.

As used herein, the term "biologically active agent" asses any le of synthetic or natural , which is able to elicit a desired physiological effect in vivo. For example, a biologically active agent may be a drug compound or a vaccine having use in the treatment or prevention of a disease or condition, especially one in which the delivery of an immediate dose is desired ed by prolonged delivery over a period of time to a subject.

In accordance with the invention, the injectable composition comprises a biologically active agent in the aqueous phase of the water-in-oil emulsion. More particularly, the biologically active agent is contained in the aqueous liquid and in the hydrogel particles of the aqueous phase. ingly, it may be considered that two phases of the injectable composition comprise the biologically active agent. It is believed that the presence of the biologically active agent in the aqueous liquid and in the hydrogel particles facilitates the ability of the able ition to provide for rapid and sustained delivery of the active agent, as further described below.

When the injectable composition comprises coated hydrogel particles, the biologically active agent may be situated in the core or in the coating of the hydrogel particles. In some embodiments, the biologically active agent may be situated in both the core and the coating of the coated hydrogel particles.

The injectable ition of the invention may comprise a range of biologically active agents.

In some embodiments, hydrophilic biologically active agents may be preferred.

D:\DOCUME~1\CTS\docbases\PTO_REP\config\temp_sessions\2008900423662999969\Complete Specifications.docx The biologically active agent may be selected from non-limiting s of active agents including hormones, antimicrobials, therapeutic antibodies, cytokines, fusion proteins, antigens, viruses, bacteria, bacteria fragments, vaccines and hormones. The injectable composition of the invention may comprise one or more biologically active agents selected from one or more of these classes.

Biologically active agents may carry a net charge at physiological pH, which can be indicated by the isoelectric point (pI) of the active agent. Alternatively, biologically active agents may have no net charge (i.e. neutral).

When the injectable ition comprises two or more biologically active agents, the biologically active agents may belong to the same class of active agent or to ent classes of active agent. Each biologically active agent may also be ndently selected at each occurrence. es may be peptide es such as insulin and somatotropin, or steroid hormones such as corticosteroids, estrogens, progestogens and androgens.

A "peptide hormone" is a peptide or protein that has an effect on the endocrine system of a subject.

A specific example of a peptide hormone that may be contained in and delivered by the injectable ition of the invention is somatotropin. Somatotropin stimulates the growth, cell reproduction and cell regeneration in humans and non-human animals and is important in growth and development.

Therapeutic antibodies may be infliximab, adalimumab, nituximab, zumab, daclizumab or basiliximab.

Fusion proteins may be etanercept.

An "antigen" is a compound which, when introduced into a human or non-human UME~1\CTS\docbases\PTO_REP\config\temp_sessions\2008900423662999969\Complete Specifications.docx , will result in the formation of antibodies against the antigen and cell-mediated immunity.

Antigens are commercially available or may be prepared using known procedures and techniques. Representative antigens may include, but are not limited to, natural, recombinant or tic ts derived from viruses, bacteria, fungi, parasites and other infectious agents including prions. Examples of antigens also include human antigens which might be desirable to use in prophylactic or therapeutic vaccines e.g. which are involved in or relevant to autoimmune diseases, in ular autoantigens; hormones; tumour antigens; and allergens. The microbial (e.g. viral or bacterial) products can be components which the organism produces or can be induced to produce e.g. by enzymatic cleavage or can be components of the organism that were produced by recombinant DNA techniques that are well known to those of ordinary skill in the art.

Some specific examples of antigens that may be contained in and delivered by the injectable composition of the invention are TSOL18 antigen, Bm86 antigen, H. contortus n, antigens for Old World orm fly omya bezziana) and antigens for bluetongue virus.

A "vaccine" is a preparation that is used to stimulate the immune system to produce antibodies against one or more specific agents. The vaccine may prevent, treat or suppress the progression of a condition, disease or disorder that is caused directly or indirectly or exacerbated by an infectious or infecting agent or any other agent.

One specific vaccine that may be contained in and delivered by the injectable composition of the invention is luteinizing hormone-releasing hormone (LHRH) e.

The vaccine may be used to suppress uction in animals and form the basis for castration or immunocontraception.

In one form, the injectable composition comprises one or more biologically active agents selected from the group consisting of e antigens and hormones.

In some embodiments, the able composition of the ion comprises an D:\DOCUME~1\CTS\docbases\PTO_REP\config\temp_sessions\2008900423662999969\Complete Specifications.docx adjuvant. The term "adjuvant" as used herein refers to a compound or substance that enhances a subject's physiological response to a biologically active agent. For example, in the case of an antigen, an adjuvant may act to enhance a subject's immune response to the n by increasing antibodies and thus the ity of the immune se.

An adjuvant can therefore help to promote a more effective physiological response to a biologically active agent in a subject, compared to the administration of the biologically active agent alone or without the adjuvant.

In some embodiments, an adjuvant may act to modify the release of a biologically active agent in vivo. The modulated release can provide a more durable or higher level of delivery using smaller amounts or fewer doses of the biologically active agent, ed to if the biologically active agent were administered alone or without the adjuvant.

The adjuvant can be present in the water-in-oil emulsion of the injectable composition and may be in the oil phase or in the aqueous phase of the emulsion. In some embodiments of the able composition, the oil phase and aqueous phase of the emulsion may each comprise an nt.

In one embodiment of the injectable composition, the aqueous phase of the water- in-oil emulsion comprises at least one adjuvant. In such embodiments, the adjuvant is ably hydrophilic and may be water soluble.

When present in the aqueous phase, the adjuvant may be in the aqueous liquid and/or in the hydrogel particles of the aqueous phase. For example, the adjuvant may be dissolved in the aqueous liquid of the aqueous phase. Additionally or alternatively, the adjuvant may be contained in the hydrogel particles or be incorporated as part of the chemical composition or structure of the el les of the aqueous phase. For example, chitosan may have adjuvanting ties and thus an adjuvant may be introduced into the injectable composition through the use of hydrogel les comprising crosslinked chitosan in the aqueous phase.

Hydrophilic adjuvants that may be incorporated in the aqueous phase of the D:\DOCUME~1\CTS\docbases\PTO_REP\config\temp_sessions\2008900423662999969\Complete Specifications.docx water-in-oil emulsion may be selected from the group consisting of alum, the water soluble extract of Mycobacterium smegmatis, synthetic N-acetyl-muramyl-l-alanyl-disoglutamine , monoacyl lipopeptides and ligands for Toll-like receptors. Such adjuvants may be orated in the aqueous liquid and/or within hydrogel particles of the aqueous phase.

In other embodiments of the injectable composition, the oil phase of the water-in- oil emulsion comprises at least one adjuvant. In such embodiments, the adjuvant is preferably lipophilic and is at least oil compatible and may be oil soluble.

In some embodiments, the oil per se can be an adjuvant and thus the oil phase comprises an adjuvanting oil. The use of an adjuvanting oil may be desirable as it avoids the need to incorporate a separate adjuvanting compound in the injectable composition of the invention. Examples of adjuvanting oils are described herein.

In alternative embodiments, the oil phase may comprise a lipophilic nt dissolved or suspended in a non-adjuvanting (passive) oil.

Various nts are known to those skilled in the art. Adjuvants useful for the injectable composition of the invention may be inorganic adjuvants or organic adjuvants.

A skilled person would appreciate that the selection of a particular adjuvant might depend on the biologically active agent to be delivered to a subject, the e or er to be treated by the active agent, and the release profile desired for the active agent.

Some examples of specific adjuvants include incomplete s adjuvant (IFA), nt 65 (containing peanut oil, mannide monooleate and aluminium monostearate), oil emulsions, Ribi adjuvant, the ic polyols, polyamines, Avridine, Quil A, saponin, MPL, QS-21, mineral gels, and ium salts such as aluminium ide and aluminium phosphate. Other examples include oil-in-water emulsions such as SAF-1, SAF-0, MF59, Seppic ISA720, and other particulate adjuvants such as ISCOMs and ISCOM .

D:\DOCUME~1\CTS\docbases\PTO_REP\config\temp_sessions\2008900423662999969\Complete Specifications.docx The injectable ition of the invention may optionally comprise one or more additional components. The additional components may be, for example, salts or ions to adjust the pH or ionic th of the aqueous phase, surfactants, emulsifiers, and the like.

When present, the onal components may be contained in the oil phase and/or the aqueous phase of the water-in-oil emulsion described herein, including in the hydrogel particles and/or the aqueous liquid of the aqueous phase. A skilled person would understand that the additional components may be dissolved or suspended in the oil phase or aqueous phase, depending on the lipophilicity or hydrophilicity of a ular component.

In one set of embodiments, the injectable composition of the ion may comprise at least one surfactant. Surfactants may help to reduce or prevent complete phase separation of the aqueous and oil phases and thus aid in the production of a more stable water-in-oil emulsion.

Surfactants may be present in an amount of up to 5% by weight of the injectable composition. In some embodiments, the tant may be present in an amount of up to 1% by weight of the injectable ition.

Surfactants utilised in the able composition of the invention may have a low hydrophilic–lipophilic balance (HLB) value. For instance, the surfactant may have a HLB of 6 or less. Additionally, surfactants with high HLB ratio may be used in combination with low HLB surfactants to give low HLB surfactant mixture to optimise the stability of the water-in-oil emulsion.

Various pharmaceutically acceptable surfactants are known to one skilled in the art. ceutically acceptable surfactants may be anionic, cationic, zwitterionic or ic.

In one set of embodiments, the injectable composition of the invention further comprises a non-ionic tant. A range of non-ionic surfactants may be employed.

Exemplary non-ionic surfactants may be selected from sorbitan esters, polysorbates and poloxamers.

D:\DOCUME~1\CTS\docbases\PTO_REP\config\temp_sessions\2008900423662999969\Complete Specifications.docx Preferred surfactants may be selected from Tween 80 (Polysorbate 80), glycol and glycerol esters, polyoxyethylene/propylene glycols such as PEG-35 castor oil, sorbitan derivatives such as polysorbates-20, 40, 60, 65, 80 and 85, sorbitan monooleate, sorbitan urate, sorbitan monopalmitate, sesquioleate, trioleate, tristearate, saccharide and polysaccharide based surfactants.

The injectable ition of the invention can be prepared using equipment and techniques suitable for g emulsion compositions, in particular, water-in-oil emulsions.

In one aspect, the present invention provides a process for ing an injectable ition for rapid and sustained delivery of a biologically active agent, the process comprising the steps of: providing a first aqueous composition comprising a first hydrogel forming component and a second aqueous composition sing a second hydrogel forming component, at least one of the first aqueous composition and the second aqueous composition comprising a biologically active agent; combining the first aqueous composition with a lipophilic composition comprising an oil to form an emulsified composition; and combining the second s composition with the emulsified composition under conditions allowing the first hydrogel forming component to react with the second hydrogel forming component to form a plurality of hydrogel particles in situ and thereby provide an injectable composition comprising a water-in-oil emulsion comprising an aqueous phase dispersed in an oil phase, the aqueous phase comprising a plurality of hydrogel les and an aqueous liquid, and wherein the ically active agent is contained in the hydrogel particles and in the aqueous liquid of the aqueous phase of the water-in-oil on.

In forming the injectable composition, the first el forming component is one selected from a r and a crosslinking agent while the second hydrogel forming component is the other selected from a polymer and a crosslinking agent. That is, when the first el forming component is selected to be a polymer, then the second hydrogel D:\DOCUME~1\CTS\docbases\PTO_REP\config\temp_sessions\2008900423662999969\Complete Specifications.docx forming component is a crosslinking agent, and vice-versa. Polymers and crosslinking agents suitable for g hydrogel particles have been described herein.

The aqueous compositions and the lipophilic composition may be ed under shear, to form the hydrogel particles and the water-in-oil emulsion.

In one embodiment, the injectable composition of the invention may be prepared by lly combining a first aqueous composition comprising a polysaccharide in an aqueous t with a ilic composition comprising a physiologically acceptable oil and a surfactant, and emulsifying the first aqueous composition in the lipophilic composition under shear. A second aqueous composition comprising a crosslinking agent in an aqueous solvent is then combined with the initial emulsified mixture with continuous agitation or shear. The crosslinking agent reacts with the polysaccharide and spontaneously crosslinks the polysaccharide to result in colloidal portions of crosslinked el being formed in situ. The resulting composition is therefore a water-in-oil emulsion containing a plurality of hydrogel particles. The formed hydrogel particles are dispersed in the aqueous solvent, which forms the aqueous liquid portion of the aqueous phase of the emulsion. Crosslinking of the polysaccharide and the formation of hydrogel les containing the crosslinked polysaccharide occurs without the need for additional curing mechanisms or apparatus (e.g. by UV, IR, heat). Crosslinking and hydrogel particle formation may occur rapidly.

Advantageously, it has been found that the resulting injectable composition is stable, with the in-oil emulsion and the dispersion of hydrogel particles in the emulsion remaining stable over a number of weeks. In l, a stable emulsion would not exhibit phase separation, ation or itation of the composition components over a desired period of time. In one set of embodiments, the injectable composition of the invention may be stable for more than 6 months at ambient room temperature.

The order in which the s compositions containing the crosslinking agent and polysaccharide are mixed with the lipophilic composition may be reversed. That is, the first aqueous composition may comprise a crosslinking agent (e.g. TPP) and the ilic composition comprising the oil may lly be emulsified with the crosslinking D:\DOCUME~1\CTS\docbases\PTO_REP\config\temp_sessions\2008900423662999969\Complete Specifications.docx agent containing aqueous composition. Subsequently, a second aqueous composition comprising a polysaccharide (e.g. chitosan) may then be added to this initial fied composition to e the injectable composition of the invention.

In some embodiments, when the hydrogel particles comprise a mixture of polysaccharides, the injectable composition may be prepared by initially combining a first aqueous composition comprising a first polysaccharide in an aqueous solvent with a lipophilic composition comprising a physiologically able oil, and emulsifying the first aqueous composition in the lipophilic composition under shear. A second aqueous composition comprising a second ccharide in an aqueous solvent is then combined with the initial fied mixture with uous agitation or shear. Preferably, the second polysaccharide is of opposite charge to the first polysaccharide. The first and second ccharides then crosslink via intermolecular electrostatic interactions to result in colloidal ns of crosslinked hydrogel being formed in situ.

In some embodiments, an aqueous composition used in the preparation of the injectable composition may comprise a mixture of hydrogel forming components of similar electrostatic charge. For instance, an aqueous composition may comprise a mixture of negatively charged polysaccharides such as alginate and chondroitin sulphate. If desired, such aqueous compositions can also include an ionic inking agent with the same charge, such as TPP. An l emulsion ning this aqueous composition can be combined with another aqueous composition containing a el forming component of opposite charge, for example, a vely d polysaccharide such as chitosan. In desired, the chitosan constituent may comprise other positively charged polymers and/or crosslinker species. Combining these different aqueous compositions containing components of opposite charge in an emulsion can then allow formation of an injectable composition comprising microhydrogel particles having a combination of different polysaccharides that are crosslinked electrostatically.

During production of the injectable composition, formation of the hydrogel particle by crosslinking a hydrogel forming polymer in the presence of the biologically active agent can involve two processes: formation of the crosslinked hydrogel network and compression of the network driven by increasing ink density, which continues until D:\DOCUME~1\CTS\docbases\PTO_REP\config\temp_sessions\2008900423662999969\Complete Specifications.docx the balance between the c and elastic forces of the polymer chains is reached. The aqueous liquid phase expelled by the reduction in volume of the hydrogel during crosslinking, termed syneresis liquid, contains free ive s not physically bound or encapsulated by the el polymer chains, i.e. those exceeding the binding capacity of the crosslinked chains. Notably, the binding capacity of the crosslinked polymer chains may be lower than that of the chains prior to crosslinking due to ic conformational changes imposed on the component arising from compression during network formation.

The physical equilibrium established between (i) bioactive species bound to the hydrogel polymer chains and (ii) free species in aqueous liquid where the s liquid is both al to the el network in the aqueous phase of the emulsion and within the swollen hydrogel itself, is characterised by the respective g coefficient of the system.

In embodiments where the able composition comprises coated hydrogel particles, the first aqueous composition and the second s composition used in the preparation of the injectable composition may each optionally further comprise a coating forming component. In one embodiment, the second aqueous composition comprises a coating forming component. In a further embodiment, both the first aqueous composition and the second aqueous composition comprise coating forming ents.

When the first aqueous composition is emulsified with the lipophilic composition and the resulting emulsion subsequently combined with the second aqueous composition, the first and second hydrogel forming components react to form hydrogel particles in situ in the composition. Meanwhile, the coating forming component that is also present in the emulsified reaction mixture forms a coating that at least lly covers the surface of one or more of the hydrogel particles. The coating is thus also formed in situ on the surface of the hydrogel particles.

As an example, the injectable composition of the invention may be prepared by initially combining a first aqueous composition comprising a first hydrogel forming component with a lipophilic composition comprising a physiologically acceptable oil and a surfactant, and emulsifying the first aqueous composition and the lipophilic ition under shear. A second aqueous composition comprising a second hydrogel forming ent and a g forming component in an aqueous solvent is then combined with D:\DOCUME~1\CTS\docbases\PTO_REP\config\temp_sessions\2008900423662999969\Complete Specifications.docx the initial fied mixture with uous agitation or shear. The first hydrogel forming component reacts with the second hydrogel forming component to result in colloidal portions of el being formed in situ while the coating g component forms a coating on the colloidal hydrogel. The coating is located at the surface of the hydrogel particle. Aportion of the coating may be physically interlaced with the underlying hydrogel particle core to attach the coating to the el core. Alternatively, the coating may be attached (e.g. chemically attached) to the ying hydrogel core.

The resulting composition is therefore a water-in-oil emulsion containing one or more coated hydrogel particles dispersed in the aqueous liquid portion of the aqueous phase of the emulsion.

As described herein, in some embodiments a crosslinked coating may be formed on the hydrogel particles. In such embodiments, a coating forming component may be incorporated in each of the aqueous compositions used to form the injectable composition of the invention. The coating forming components react together to form a crosslinked As an example, the injectable composition of the invention may be prepared by initially combining a first aqueous composition comprising a first hydrogel g component and a first coating forming component with a lipophilic ition comprising a physiologically acceptable oil and a surfactant. The mixture is then emulsified under shear. A second aqueous composition comprising a second hydrogel forming ent and a second coating forming component in an aqueous solvent is then combined with the initial emulsified mixture with continuous agitation or shear. The first hydrogel forming component reacts with the second hydrogel forming component to form colloidal portions of hydrogel in situ. Meanwhile, the first g forming component reacts with the second g forming component to form a crosslinked g in situ on the colloidal hydrogel.

Where a crosslinked coating is desired to be , it is preferable that the components in each of the first aqueous composition and the second aqueous composition do not react with one another. Rather, it is preferred that reaction n the components in these compositions only takes place after the first and second aqueous compositions are D:\DOCUME~1\CTS\docbases\PTO_REP\config\temp_sessions\2008900423662999969\Complete Specifications.docx combined together and emulsified with the lipophilic composition.

In the above described processes, one or more compositions selected from the group consisting of the first aqueous ition, the second aqueous composition and the lipophilic composition may comprise an adjuvant. Accordingly, an adjuvant may be contained in at least one of the entioned compositions used to prepare the injectable composition of the invention.

In one embodiment, the lipophilic composition comprises an adjuvant. Such ments would result in the injectable ition of the invention comprising an nt in the oil phase of the water-in-oil emulsion.

In one embodiment, the lipophilic composition comprises a physiologically acceptable adjuvanting oil. Examples of adjuvanting oils are described herein.

In one embodiment, the lipophilic composition comprises a physiologically acceptable passive oil and at least one lipophilic nting compound or substance that is dissolved or dispersed in the passive oil.

In some embodiments an adjuvant may be provided by at least one adjuvanting nd or substance being dissolved in or sed in the first aqueous composition and/or the second aqueous composition used to prepare the injectable composition. Such adjuvanting substances are generally hydrophilic. Examples of hydrophilic adjuvanting compounds are described herein.

As an illustration with reference to one of the embodiments described herein, a first aqueous composition sing chitosan may be combined with a lipophilic composition comprising an adjuvanting oil (e.g. Montanide ISA61) under ion or shear. A second aqueous composition comprising sodium tripolyphosphate (TPP) is then added to the resulting emulsified ition in order to crosslink the chitosan. The addition of the TPP to the initial composition occurs dropwise, under continuous shear. A uniform water-in-oil emulsion formulation that comprises colloidal portions of el composed of crosslinked chitosan in the s phase of the emulsion is then formed.

D:\DOCUME~1\CTS\docbases\PTO_REP\config\temp_sessions\2008900423662999969\Complete Specifications.docx As an illustration of a further embodiment bed herein, when coated hydrogel particles are desired, a first aqueous composition comprising alginate and sodium tripolyphosphate (TPP) may be ed with a lipophilic composition comprising an adjuvanting oil (e.g. Montanide ISA61) under agitation or shear. A second s composition comprising chitosan and calcium chloride (CaCl2) is then added to the resulting emulsified composition under continuous shear. Calcium cations interact with the alginate to ink the alginate and form colloidal portions of alginate–Ca2+ hydrogel.

Meanwhile, chitosan is crosslinked with TPP to produce a crosslinked an coating on the colloidal hydrogel. The resulting composition is a water-in-oil emulsion ation comprising colloidal portions of coated hydrogel in the dispersed aqueous phase. The coated hydrogel is composed of crosslinked alginate core and a inked chitosan coating When preparing the injectable composition of the invention, the biologically active agent may be contained in the first aqueous composition and/or in the second aqueous ition described . In one preference, the first aqueous composition comprises the biologically active agent. The inclusion of the biologically active agent in the first aqueous composition may be preferred as this may promote more efficient distribution of the active agent in the emulsion and consequently, in the final injectable composition.

The first aqueous composition, the lipophilic composition and the second aqueous composition may be combined in any suitable tric ratio. In one specific embodiment, the first aqueous composition ses a crosslinking agent and a biologically active agent, the lipophilic composition comprises an adjuvanting oil and the second aqueous solution comprises a polysaccharide and the volumetric ratio between the first s composition : lipophilic composition : second aqueous composition is 0.5:1.2:1.8.

During the preparation of the injectable composition, a portion of the ically active agent s encapsulated in the hydrogel particles that are formed in situ. A further portion of the biologically active agent is not encapsulated in the hydrogel particles D:\DOCUME~1\CTS\docbases\PTO_REP\config\temp_sessions\2008900423662999969\Complete Specifications.docx but remains in the s solvent, which then forms the aqueous liquid of the aqueous phase of the water-in-oil emulsion.

As the hydrogel particles comprising the biologically active agent are prepared ex vivo, product reproducibility may be improved.

If other components are desired to be present in the injectable composition, such components may be incorporated in one or more of the first aqueous composition, the second aqueous composition and/or in the lipophilic composition used to prepare the injectable composition. For example, in one embodiment, an s insoluble alkaline earth metal phosphate such as hydroxyapatite may be incorporated in the aqueous composition comprising the polysaccharide, while a biocompatible proteoglycan may be incorporated in the aqueous composition comprising the crosslinking agent.

If porous hydrogel particles are desired, one or more of the first aqueous composition, the second aqueous composition and/or the lipophilic ition used to prepare the injectable ition may further comprise a porogen.

In some embodiments, at least one selected from the first aqueous composition and the second s composition comprises a porogen.

A n is a substance that can be used to form an opening or pore in a material. Since the porogen is used to form pores in the hydrogel particles, it can be preferable for the porogen to be a hydrophilic substance.

The reaction between a polymer and a crosslinking agent to form hydrogel les may proceed in the presence of a n. The porogen does not ipate in the hydrogel forming reaction and thus the hydrogel material forms around the porogen.

Accordingly, the porogen acts as a template for the pores in the hydrogel les. After the hydrogel particles are formed, the porogen may leach from the particles and be removed, thereby leaving pores in the hydrogel particles.

A range of porogens may be ed to form porous hydrogel particles. An D:\DOCUME~1\CTS\docbases\PTO_REP\config\temp_sessions\2008900423662999969\Complete Specifications.docx example of a suitable porogen is poly(ethylene glycol) (PEG). A skilled person would understand that PEG is a hydrophilic polymer that is compatible with an aqueous solvent.

Poly(ethylene glycol) is useful as a porogen and may have a molecular weight in a range of from about 200 to 100,000 Da. In one ment, the poly(ethylene glycol) has a molecular weight of about 35,000 Da.

Porogens for forming porous hydrogel particles may be used in a suitable .

The quantity of porogen may be dependent on the type of porogen selected and the degree of porosity desired for the hydrogel particles. In one set of ments, a porogen may be present in at least one selected from the first aqueous composition and the second aqueous composition in an amount up to 2 wt %.

Additionally, surfactants can be employed in the process and when used, suitable surfactants may be incorporated in one or more of the first s ition, the second s composition and/or in the lipophilic ition used to prepare the injectable composition. In one embodiment, the lipophilic ition used to form the oil phase of the water-in-oil emulsion of the able composition comprises a surfactant.

The injectable composition of the invention may be prepared in a one-step process, which provides a simple and effective method of production. Conventional manufacturing equipment and apparatus can be used to prepare the composition, which aids in reducing production costs.

The injectable ition of ments described herein, which may be prepared in ance with processes described herein, is capable of being administered to a subject for the delivery of the biologically active agent. It is suitably administered in a single injection. Advantageously, the injectable composition may be used as prepared, without the need for additional isolation, purification or formulation steps to be performed.

For instance, the present invention avoids the need to isolate the hydrogel particles and to re-formulate the isolated hydrogel particles in a suitable pharmaceutically acceptable carrier or vehicle prior to their stration to a subject. However, a skilled person would appreciate that processes such as sterilisation may be carried out in on to the D:\DOCUME~1\CTS\docbases\PTO_REP\config\temp_sessions\2008900423662999969\Complete Specifications.docx able composition to ensure that it complies with relevant safety or regulatory requirements.

The injectable composition of embodiments described herein is of a viscosity that is suitable for administration by ion to a subject. It has been found that the injectable composition can exhibit shear thinning behaviour when shear stress is applied, such as when the ition is injected through the lumen of a needle. Shear thinning that occurs under applied shear stress can reduce the viscosity of the composition.

The release profile of a biologically active agent from the injectable composition includes a short-term (i.e. rapid) and a long-term (i.e. sustained) portion. In some embodiments, the release profile of the biologically active agent can be controlled or modulated by components of the injectable composition influencing the passage of the active agent by various mechanisms as described herein.

The short-term release profile allows a primary dose of the biologically active agent to be rapidly red. It is believed that the primary dose is ed by that portion of the biologically active agent that is not contained in the hydrogel particles but is instead present in the aqueous liquid of the aqueous phase of the water-in-oil emulsion. As the biologically active agent is not bound or encapsulated in the solid hydrogel particles (i.e. it is free of the hydrogel particles), the active agent in the aqueous liquid is able to be rapidly delivered to a subject in vivo.

It is believed that the ry of a primary dose of the biologically active agent can occur when a droplet of the aqueous liquid containing the active agent separates from the dispersed s phase and is orted through the continuous oil phase of the water-in-oil emulsion. The aqueous liquid t may be encapsulated by an oil film during its transport h the oil phase. The oil encapsulated aqueous droplet containing the biologically active agent is then delivered to the surrounding physiological environment, where the active agent contained in the droplet can be released as an initial primary dose.

This rapid delivery of a primary dose of the biologically active agent occurs D:\DOCUME~1\CTS\docbases\PTO_REP\config\temp_sessions\2008900423662999969\Complete Specifications.docx immediately upon administration of the injectable composition to a subject, or shortly fter (i.e. within s of administration).

In some embodiments, release of the biologically active agent may be modulated by interactions that the biologically active agent may have with hydrogel particles in the injectable composition. For example, if the biologically active agent and hydrogel polymer matrix each carry a net charge at physiological pH, electrostatic interactions between the biologically active agent and el polymer matrix may influence the release kinetics of the active agent. Similarly, a charged coating may also interact with a charged biologically active agent electrostatically and thus influence the e of the active agent.

In one form of the injectable composition of embodiments described herein the biologically active agent in the hydrogel particles may be conjugated to the particles.

Conjugation may be via covalent or non-covalent interactions. The biologically active agent may be conjugated to the crosslinked polymer matrix of the hydrogel particle and/or to a coating covering the hydrogel particle. In one embodiment, the biologically active agent is conjugated via non-covalent interactions, such as electrostatic interactions. This may occur when the biologically active agent is charged at physiological pH and the polymer matrix of the el le and/or the coating on the hydrogel particle bears an opposite .

In some embodiments, the short-term release profile of the biologically active agent is linear and may be of zero order.

The delivery of the biologically active agent to the physiological environment can also be modulated by an adjuvant in the injectable composition. In embodiments where the oil phase of the water-in-oil emulsion comprises an adjuvant such as an adjuvanting oil, the oil film nding the aqueous droplet will also comprise the adjuvant. In such embodiments, the adjuvant in the oil film will help to modulate the release of the ically active agent from the s liquid droplet to the subject's logical environment.

D:\DOCUME~1\CTS\docbases\PTO_REP\config\temp_sessions\2008900423662999969\Complete Specifications.docx More prolonged (i.e. sustained) delivery of the biologically active agent may be achieved after delivery of the primary dose via release of that portion of the active agent, which is contained in the hydrogel particles of the able composition. Accordingly, the particles of el may act as a depot for the biologically active contained within.

Release of quantities of the active agent from the particles over time enables the injectable ition of the invention to deliver a biologically active agent to a subject over a sustained period of time, thus providing for longevity of response.

In order to e for ned delivery, the biologically active agent contained within the hydrogel les passes from the hydrogel particles into the aqueous liquid of the aqueous phase. Once in the aqueous , the biologically active agent may then be delivered to a subject's physiological nment via the same oil film encapsulated aqueous droplet mechanism described above for delivery of the y dose of active agent.

Passage of the ically active agent from the hydrogel particles to the aqueous liquid may be assisted by a concentration gradient being established for the active agent or by differences in osmotic pressure between the hydrogel les and the aqueous liquid, which might encourage the nt of the active agent from the hydrogel particles to the aqueous liquid.

In particular, rapid and sustained delivery of the biologically active agent to a physiological environment can be influenced by a dynamic equilibrium being established for the active agent. In that regard, it is believed that the injectable composition of the invention can provide a partition system facilitating the establishment of an equilibrium between active agent bound to or contained in the hydrogel particles and active agent free in the aqueous liquid of the dispersed aqueous phase of the water-in-oil emulsion. The partition, resulting from the binding equilibrium, es an initial rapid release of free syneresis active agent, giving the priming dose, which in the presence of an adjuvant can trigger innate immunity responses (i.e. macrophages, dendritic cells). The bioactive species physically bound to the network chains of the hydrogel provides longer term trickle feed (i.e. sustained release) of the active agent either by re-establishment of further "free" state species re-establishing the binding equilibrium or by uptake of the micro-hydrogels by D:\DOCUME~1\CTS\docbases\PTO_REP\config\temp_sessions\2008900423662999969\Complete Specifications.docx antigen presenting cells (APCs) by such processes as phagocytosis (if hydrogel is greater than ~1 µm), micropinocytosis (if hydrogel is less than 1µm) or endocytosis (if hydrogel is nanoscale ~50-60nm). Microhydrogels containing antigen can further trigger the adaptive immune system inducing the differentiation of the T cells to T helper and T killer cells resulting into production of specific antibodies (i.e., adaptive immunity).

In some embodiments, where electrostatic interactions dominate designed partition, consisting of an brium between the active agent bound to the biopolymer chains of the hydrogel and the active agent free in the dispersed aqueous phase of the water-in-oil emulsion, the composition of the biopolymer can be adjusted to tailor the proportion of ive agent deliverable in the rapid (aqueous) and sustained (bound) In some embodiments, the bioavailability profile of the biologically active agent may involve modulation of the presentation of the active agent to the in vivo environment.

For example, presentation of the active agent can be ted via the internalization of the hydrogel particles in hages and dendritic cells followed by the lysing of the hydrogel to release the active agent into the cytoplasm of the antigen presenting cells (APC).

Effective short term and long term release of the biologically active agent can be achieved without the need to apply an external stimulus to trigger release of the active agent from the injectable composition.

A skilled person would also appreciate that the morphology of the hydrogel particles may change after injection intramuscularly or subcutaneously. Pressure d by the surrounding ical tissue may result in packing of the hydrogel les in the water-in-oil emulsion at the injection site with a small amount of emulsion liquid only nding and filling the spaces n hydrogel particles. This results in the filtrating out of the aqueous and oil liquids which contains the biologically active agent, to be delivered as the primary dose upon injection of the ition to a subject.

Hydrogel particles may also contract over time after injection, resulting in the D:\DOCUME~1\CTS\docbases\PTO_REP\config\temp_sessions\2008900423662999969\Complete Specifications.docx ion of aqueous liquid contained within the particles, hence resulting in the ically active agent being expelled from the particles and into the surrounding aqueous environment. The biologically active agent ing contained in the hydrogel particles may be expelled from the particles at a slower rate and thus contribute to providing sustained delivery of the active agent.

Passage of the biologically active agent from the hydrogel particles to the aqueous liquid may also be modulated by a coating present on one or more of the hydrogel particles of the injectable composition. Depending on its composition and possible ctions with a biologically active agent, a coating may impede the movement the active agent from the hydrogel particles into the aqueous liquid and thus help to promote sustained release of the active agent from the able composition over a prolonged period of time.

In some embodiments the materials used to prepare the hydrogel particles may further assist in the ned delivery of the ically active agent as such materials may have adjuvanting properties and thus act as adjuvants to help modulate the passage of the active agent from the hydrogel particles to the aqueous liquid. For example, chitosan may have adjuvanting properties and hydrogel particles comprising crosslinked chitosan may be capable of influencing the release of the biologically active agent and enhance immune response.

Modulation of the release of the biologically active agent may involve an increase or a decrease in the rate of active agent delivery to the physiological environment.

As used herein, the phrase "sustained release" means that the rate of release of the agent to the subject is slower than would occur if the agent were administered to the subject directly.

In one example, an injectable composition of the present invention ses Bm86 as an active agent and is able to e an immune response to Bm86 over a period of at least 6 months in vivo.

In an embodiment, ic components of the injectable composition may be D:\DOCUME~1\CTS\docbases\PTO_REP\config\temp_sessions\2008900423662999969\Complete Specifications.docx present in the following % wt range: polysaccharide (0.05-3%): crosslinking agent (0.05- 3%): aqueous insoluble ne earth metal ate (0.0-3% subject to particle size distribution): glycosaminoglycans (GAGs) from about (0.0-3%).

The injectable composition of embodiments described herein may have a low tion of solids in the water-in-oil emulsion.

One advantage of the injectable composition of the invention is that it enables fewer injections to be administered to a subject. For example, for conditions that would ly require daily injections under current conventional regimes, the present invention may allow the substantially the same physiological effects and benefits to be achieved with weekly injections.

Additionally, regimes requiring an initial injection to be followed up by one or more subsequent injections or r injections may be simplified, as the extended bioavailability provided by the able composition of the present invention means that an effective physiological benefits may be achieved with a single injection, thus obviating the need for subsequent or booster injections to be administered. For instance, it has been found that the injectable composition of the present invention is able to induce effective protective immunity (i.e. dy levels) in a subject following a single injection t the need for subsequent follow up single or le injections. Furthermore, an effective level of immunity was maintained over a number of weeks. In some embodiments, an effective level of immunity could be maintained over a period of several months, and in one embodiment, immunity may be ined for more than a year.

The ability to reduce the number of injections may therefore afford increased convenience to a subject receiving the injections, as well as cost savings to the manufacturer and the consumer.

In use, the injectable composition may be contained in a e chamber and injected through the lumen of a needle for administration to a subject. For example, the injectable composition may be stered via a gauge 23 needle.

UME~1\CTS\docbases\PTO_REP\config\temp_sessions\2008900423662999969\Complete Specifications.docx A further advantage of the injectable ition of the ion that the ition can be tailored to contain different tions of hydrogel particles and aqueous liquid in the aqueous phase of the composition. In this manner, the invention can control the proportion of biologically active agent contained in the hydrogel particles and in the aqueous liquid respectively. This in turn could influence the amount of active agent that would available for rapid (short term) and sustained (long term) delivery. r control over the release of the biologically active agent can also be achieved through the appropriate selection of material used to form the hydrogel particles, as well as through the formation of a coating on the hydrogel particles. As discussed above, the r in the hydrogel particles and a coating on the hydrogel particles each have the potential to interact with a selected biologically active agent and thus may modulate the release of that biologically active agent from the hydrogel particles into the aqueous liquid component of the aqueous phase of the injectable composition.

In still a further aspect the invention provides a method of delivering a biologically active agent to a subject comprising the step of administering an injectable composition as described herein to the subject by ion.

The injection may be aneous, intramuscular or intraperitoneal. Preferably, administration is via subcutaneous injection.

The injectable composition of the invention has application in the administration of a biologically active agent, such as a pharmaceutical drug or e.

The injectable composition of the invention may be stered to a subject in order to treat or prevent a disease or condition. As used herein the terms "treating" and "preventing" mean any treatment of prevention of a disease or condition in a subject.

"Treatment" and ntion" includes: (a) controlling or inhibiting the disease or condition, i.e., arresting its development or progression; or (b) relieving or ameliorating the ms of the disease or ion, i.e., cause regression of the symptoms of the disease or condition. The effect may be prophylactic or therapeutic in terms of a partial or complete cure of the disease or condition.

D:\DOCUME~1\CTS\docbases\PTO_REP\config\temp_sessions\2008900423662999969\Complete Specifications.docx "Disease" as used herein is a general term used to refer to any departure from health in which a subject suffers and which can be treated or prevented using a microhydrogel-depot, which provides prolonged release of an active agent. A "condition" refers to an abnormal function of part of the body of a subject and which can be treated or prevented using a microhydrogel-depot which provides prolonged e of an active agent.

In use, the injectable composition of the invention provides rapid and sustained release of a biologically active agent to a subject in vivo. Accordingly, upon administration to a subject, the injectable composition rapidly provides an initial primary dose of the ically active agent to the subject followed by more sustained (i.e. trickle) dosing of the active agent over a longer period of time.

In another aspect the present invention provides a method of treating or preventing a disease or disorder in a t comprising the step administering an injectable composition of one or more embodiments as described herein to the subject by injection.

In some embodiments, the disease or disorder is a microorganism infection and the injectable composition of the invention may be used to treat or prevent the microorganism infection. Microorganisms may e bacteria, fungi and viruses.

In some ments, the e or disorder is a viral infection and the injectable composition of the invention may be used to treat or prevent the viral infection.

In some embodiments, the disease or disorder is a parasite infestation and the able composition of the invention may be used to treat, control or prevent the infestation. For e, the injectable composition may be used to control a tick infestation.

In other embodiments, the injectable composition may be used to inhibit a normal condition, (e.g. reproductive condition) that is arrested in ss or pment.

D:\DOCUME~1\CTS\docbases\PTO_REP\config\temp_sessions\2008900423662999969\Complete Specifications.docx The subject in which a disease or ion is to be treated or prevented may be a human or an animal of economical importance and/or social importance to humans, for instance, carnivores other than humans (such as cats and dogs), swine (pigs, hogs, and wild boars), ruminants (such as cattle, oxen, sheep, giraffes, deer, goats, bison, and camels), , and birds including those kinds of birds that are endangered, kept in zoos, and fowl, and more particularly domesticated fowl, e.g., poultry, such as turkeys, chickens, ducks, geese, guinea fowl, and the like, as they are also of economical importance to humans.

The term does not denote a particular age. Thus, both adult and newborn subjects are intended to be covered.

In particular embodiments, the subject is a livestock animal, such as , sheep or pigs. In such ments, the injectable composition may be considered to be a nary composition, and the ically active agent contained in the composition is selected for the ent or prevention of a disease or condition in the livestock .

The present invention also provides use of an injectable composition of one or more embodiments as described herein in the manufacture of a medicament for the treatment or prevention of a disease or disorder in a subject.

In some embodiments of the method or use described herein, the biologically active agent is an n. In particular, the antigen may be Bm86 or TSOL18.

In other embodiments of the method or use described herein, the biologically active agent is a hormone. In particular, the hormone may be somatotropin or luteinizing hormone-release hormone (LHRH).

As used in the subject specification, the singular forms "a", "an" and "the" include plural aspects unless the context clearly dictates otherwise. Thus, for example, reference to a "virus" includes a single viral particle as well as two or more viral les, "a gene" includes a single gene or two or more genes. Reference to "the invention" includes single or multiple aspects of the invention.

UME~1\CTS\docbases\PTO_REP\config\temp_sessions\2008900423662999969\Complete Specifications.docx Unless defined otherwise, all technical and scientific terms used herein have the same gs as commonly understood by one of ordinary skill in the art to which this invention s. Although any als and methods similar or equivalent to those described herein can be used to practice or test the present invention, the preferred materials and methods are now described.

The invention will now be described with reference to the following examples.

However, it is to be understood that the examples are ed by way of illustration of the invention and that they are in no way limiting to the scope of the invention.

Examples Materials and methods Low molecular weight chitosan having Mw of 164 kDa, 75–85% degree of deacetylation and a viscosity of 0.026 Pa.s for a 1 wt % on in 1% acetic acid was obtained from Sigma-Aldrich. Hydroxyapatite (HAp) (Type I, Suspension in 0.001 M phosphate buffer, pH 6.8; . 25% solids) was obtained from Sigma-Aldrich. This suspension was sed in the chitosan solution using a high shear mixer (MICCRA and DS-8/P stator rotor – Stator; spike head8 mm) at 20000 rpm for 5 min to produce 1 mg HAp/mL. Sodium tripolyphosphate pentabasic (TPP) (technical grade, 85%) and chondroitin sulphate A sodium salt (ChS) (from bovine a lyophilized , BioReagent) were obtained from Sigma-Aldrich. The adjuvant Montanide ISA61 VG was from Seppic SA (Paris La Defense, France). Sesame oil, Tween 80, and medium viscosity sodium alginate (brown algae) were from Sigma-Aldrich. Anti-cattle tick Bm86 antigen (Bm86), anti-tapeworm TSOL18 antigen (TSOL18) and porcine somatotropin (pST) were produced and characterised as reported in the following literature: (1) Bm86: Willadsen, P. et al, The Journal of Immunology, 1989. 143(4): p. 1346-1351; (2) : Gauci, C. and M.W. Lightowlers, Molecular and mical Parasitology, 2003. 127(2): p. 193- 198; (3) pST: Ouyang, J. et al, Protein Expression and Purification, 2003. 32(1): p. 28-34.

D:\DOCUME~1\CTS\docbases\PTO_REP\config\temp_sessions\2008900423662999969\Complete Specifications.docx Injectable Vaccine Compositions with Bm86 Antigen Three ent component solutions were prepared, each having a range of concentrations: Part [A]: Bm86 (0 µg, 50 µg, 100 µg or 200 µg) dissolved in an aqueous solution of 2% chitosan or 2% chitosan with 1 mg hydroxyapatite (HAp)/mL Part [B]: tripolyphosphate (TPP) solution (0.04M, 0.08M, 0.16M or 0.32M) or TPP solution (0.04M, 0.08M, 0.16M or 0.32M) with 1% oitin sulphate (ChS) Part [C]: Montanide ISA61 VG To prepare the vaccine composition, a desired quantity of solution [A] was initially emulsified with a quantity of solution [C] using MICCRA high shear emulsifier at ,000 rpm rate for 2-3 min. A desired quantity of solution [B] was then added se under constant shearing at the same rate as before (2-3 min) to form injectable composition. The consistency of the emulsion was examined by withdrawing in a syringe using a 23 gauge needle to assess the injectability of the composition.

Examples 1 to 6 – the effect of inker (TPP) solution volume In these examples, 1 mL of [A] containing HAp was emulsified in 2 mL of [C] then various s of [B] with 0.08 M TPP containing ChS was added dropwise under continuous shearing. The volumes of [B] were 0.5 mL, 1 mL, 1.5 mL, 2 mL, 2.5 mL, and 3 mL. The consistency of the emulsion was tested using the e and a 23 gauge needle after 10-15 min rest. The hydrogel particle formation was med by centrifuging the emulsion at 14000g for 5 min and observation of the hydrogel particles, aqueous phase and oil phase. The results are shown in Table 1. In unstable compositions, the emulsion collapsed, or inverted over time to an oil–in-water emulsion.

UME~1\CTS\docbases\PTO_REP\config\temp_sessions\2008900423662999969\Complete Specifications.docx Table 1 - itions prepared with 0.08M TPP in 1% ChS as solution [B] Example Vol [A] (ml) Vol [B] (ml) Vol [C] (ml) Comments 1 1 0.5 2 Stable on 2 1 1 2 Stable emulsion 3 1 1.5 2 Stable emulsion 4 1 2 2 Stable emulsion 1 2.5 2 Unstable 6 1 3 2 Unstable Examples 7 to 24 – the effect of the crosslinker (TPP) concentration In these examples, 1 mL of [A] containing HAp was emulsified in 2 mL of [C] then various volumes of [B] with 0.04 M, 0.16 M or 0.32 M TPP containing ChS were added dropwise under continuous shearing. Various volumes of [B] (0.5 mL, 1 mL, 1.5 mL, 2 mL, 2.5 mL or 3 mL) were then gradually added and the consistency of the emulsion was tested using the syringe and 23 gauge needle after 10-15 min rest. The hydrogel particle formation was med as above. The results are shown in Tables 2, 3 and 4.

Table 2 – Compositions prepared with 0.04M TPP in 1% ChS as solution [B] Example Vol [A] (ml) Vol [B] (ml) Vol [C] (ml) Comments 7 1 0.5 2 Stable emulsion 8 1 1 2 Stable emulsion 9 1 1.5 2 Stable emulsion 1 2 2 Stable emulsion 11 1 2.5 2 Unstable 12 1 3 2 Unstable D:\DOCUME~1\CTS\docbases\PTO_REP\config\temp_sessions\2008900423662999969\Complete Specifications.docx Table 3 – Compositions prepared with 0.16M TPP in 1% ChS as solution [B] Example Vol [A] (ml) Vol [B] (ml) Vol [C] (ml) ts 13 1 0.5 2 Stable emulsion 14 1 1 2 Stable emulsion 1 1.5 2 Stable emulsion 16 1 2 2 Stable emulsion 17 1 2.5 2 Unstable 18 1 3 2 Unstable Table 4 – Compositions ed with 0.32M TPP in 1% ChS as solution [B] Example Vol [A] (ml) Vol [B] (ml) Vol [C] (ml) Comments 19 1 0.5 2 Stable emulsion 1 1 2 Stable emulsion 21 1 1.5 2 Stable emulsion 22 1 2 2 Stable emulsion 23 1 2.5 2 Unstable 24 1 3 2 Unstable Examples 25 to 32 – the effect of polymer (chitosan) solution volume In these examples, 2 mL of [A] containing HAp was emulsified in 2 mL of [C] then [B] (0.16 M or 0.32 M TPP) containing ChS was added dropwise to the emulsion under uous shearing. Various s of [B] (0.5 mL, 1 mL, 1.5 mL, and 2 mL) were employed and the injectability and consistency of emulsion was tested. The results are shown in Tables 5 and 6.

D:\DOCUME~1\CTS\docbases\PTO_REP\config\temp_sessions\2008900423662999969\Complete Specifications.docx Table 5 – Compositions prepared with 0.16M TPP in 1% ChS as solution [B] Example Vol [A] (ml) Vol [B] (ml) Vol [C] (ml) Comments 2 0.5 2 Stable emulsion 26 2 1 2 Stable emulsion 27 2 1.5 2 Unstable 28 2 2 2 Unstable Table 6 – Compositions prepared with 0.32M TPP in 1% ChS as solution [B] e Vol [A] (ml) Vol [B] (ml) Vol [C] (ml) Comments 29 2 0.5 2 Stable emulsion 2 1 2 Stable emulsion 31 2 1.5 2 Unstable 32 2 2 2 Unstable Examples 33 to 44 – the emulsification order Vaccine compositions containing Bm86 were produced using the following component solutions: Part [A2]: An aqueous solution of 2% chitosan with 1 mg yapatite (HAp)/mL Part [B2]: Bm86 (0 µg, 50 µg, 100 µg or 200 µg) dissolved in an aqueous TPP solution (0.16M or 0.32M) with 1% chondroitin sulphate (ChS) Part [C]: Montanide ISA61 VG A quantity of component solution [B2] was emulsified in oil [C] first, then the chitosan ent solution [A2] was added to produce the final microhydrogel in n-oil emulsion. In these es Bm86 was incorporated in the ChS-TPP component solution [B2].

In these examples, 1 mL of [B2] (with 0.16 or 0.32 M TPP) was emulsified with 2 D:\DOCUME~1\CTS\docbases\PTO_REP\config\temp_sessions\2008900423662999969\Complete Specifications.docx mL of [C] then various volumes of [A2] (0.5 mL, 1 mL, 1.5 mL, 2 mL, 2.5 mL or 3 mL) were added dropwise under continuous shearing. The results are shown in Tables 7 and 8.

Compositions forming a mousse were found to be uninjectable using 23G × 33 mm syringe Table 7 – Compositions ed with 0.16 M TPP in 1% ChS as solution [B2] Example Vol [A2] (ml) Vol [B2] (ml) Vol [C] (ml) Comments 33 0.5 1 2 Stable on 34 1 1 2 Stable emulsion 1.5 1 2 Stable emulsion 36 2 1 2 Stable emulsion 37 2.5 1 2 Unstable - mousse 38 3 1 2 Unstable - mousse Table 8 – itions prepared with 0.32 M TPP in 1% ChS as solution [B2] Example Vol [A2] (ml) Vol [B2] (ml) Vol [C] (ml) Comments 39 0.5 1 2 Stable emulsion 40 1 1 2 Stable emulsion 41 1.5 1 2 Stable emulsion 42 2 1 2 Stable emulsion 43 2.5 1 2 Unstable - mousse 44 3 1 2 Unstable - mousse Example 45 - Injectable vaccine composition with Bm86 antigen An injectable vaccine composition containing Bm86 antigen was prepared from the ing component solutions as follows: Part [C]: Montanide ISA61 VG D:\DOCUME~1\CTS\docbases\PTO_REP\config\temp_sessions\2008900423662999969\Complete Specifications.docx Part [D]: The required dose of Bm86 (for example 400 µg Bm86/mL) dissolved in an aqueous solution of 0.32 M TPP with 1% chondroitin te (ChS) Part [E]: 2% chitosan solution with 1 mg hydroxyapatite (HAp)/mL A quantity (1 mL) of solution [D] was emulsified in 2.4 mL of Montanide ISA61 VG oil [C] using high shear (20,000 rpm, 2 min). To this emulsion, 3.6 mL of solution [E] was added drop wise under the same shear rate produced a stable injectable water-in-oil on ning crosslinked chitosan hydrogel microparticles.

Example 46 - Injectable composition with somatotropin Three different ent solutions were prepared: Part [F]: Alginate on in water (1 or 2% alginate) with porcine somatotropin (pST) (300, 1000 or 3000 µg/mL) as peptide e active agent Part [G]: Calcium chloride on in water (2.8% or 5.6%) as the crosslinker Part [H]: Tween 80 in sesame oil (50 mg in 1 mL) A quantity (1 mL) of solution [F] was emulsified in 2 mL of [H] using high shear 0 rpm, 2 min). To this emulsion, 0.5 mL of solution [G] was added drop wise under the same shear rate. A stable and able water-in-oil emulsion containing crosslinked alginate hydrogel particles was produced with a lipophilic composition having a 50 mg Tween 80/mL sesame oil concentration.

Example 47 - Injectable composition with somatotropin In this example an aqueous sodium alginate composition and a lipophilic sesame oil composition were ed as in [F] and [H] in Example 46 and an aqueous chitosan composition was prepared as the crosslinker for the negatively charged alginate as follows: Part [I]: 2% Chitosan solution in 1% acetic acid in water A quantity (1 mL) of solution [F] was emulsified in 2 mL of [H] using high shear (20,000 rpm, 2 min). To this emulsion, 1 mL of solution [I] was added drop wise under the same shear rate which produced a stable and injectable water-in-oil emulsion D:\DOCUME~1\CTS\docbases\PTO_REP\config\temp_sessions\2008900423662999969\Complete Specifications.docx comprising hydrogel particles formed with alginate crosslinked with chitosan.

Example 48 – Model Bulk Hydrogel composition with somatotropin Three ent component solutions were prepared, each having a range of concentrations: Part [J]: Alginate solution in water (0.5, 1 or 2% te) with porcine somatotropin (pST) (500, 1000 or 3000 µg/mL) as peptide e active agent Part [K]: Calcium chloride solution in water (0.3, 0.7, 1.4 or 2.8%) as the crosslinker A quantity (1 mL) of on [J] was crosslinked using 1 mL of solution [K] to afford pST loaded bulk hydrogel. The initial syneresis (12 hours) release of pST from the model bulk hydrogel is given in Figure 8.

Example 49 – Model Bulk Hydrogel ition with somatotropin Different component solutions were prepared: Part [L]: Alginate solution in water (1% alginate) with porcine somatotropin (pST) (500, 1000 or 3000 µg/mL) as peptide hormone active agent and PEG (35 kDa) porogen (0, 0.5, 1, or 2 wt % PEG) Part [K]: Calcium chloride solution in water (0.3, 0.7, 1.4 or 2.8%) as the crosslinker A quantity (1 mL) of solution [L] was crosslinked using 1 mL of solution [K] to afford pST loaded bulk hydrogel. After the removal of the sis initial liquid long term release of pST (4 weeks) from the model bulk hydrogel was monitored by refreshing the hydrogel surrounding liquid using PBS solution every 48 hours and measuring the pST in PBS. The release profile is given in Figure 9.

Animal Trials Dorper sheep were used as models to evaluate the immunological performance of the Bm86-hydrogel in oil system in a study under approval of The University of land Animal Ethics Committee. Sheep were female, 5 – 7 months old, and 12 – 16 kg weight at the start of the trial. The sheep trial consisted of two groups with four animals per group. The first group was a positive control inoculated subcutaneously with two injections of a comparative formulation representing a conventional anti-tick formulation, D:\DOCUME~1\CTS\docbases\PTO_REP\config\temp_sessions\2008900423662999969\Complete Specifications.docx which was given as an initial primary dose followed by a booster dose after 4 weeks. Each dose contained an emulsion of aqueous 50 µg Bm86 in 1.2 mL Montanide ISA61 VG adjuvanting oil. The second group was inoculated with an able Bm86 vaccine composition ed in accordance with the procedure of Example 45. In brief, one dose of microgel in emulsion formulation prepared by dissolving 200 ug Bm86 and 50 mg ChS in 0.5 mL of 0.32 M TPP and emulsified in 1.2 mL Montanide ISA60 VG, then 1.8 mL of (2% chitosan – 1mg HAp/mL) was emulsified in this initial primary emulsion to give 200 ug Bm86 and 3.5 mL per dose. A single dose of the Bm86 composition was administered by subcutaneous injection. Anti-Bm86 titres were measured as IgG, and determined by ELISA assay every two weeks. The trial results are shown in Figures 2 and 12 (in Figure 12, Ab units relative to negative control group at 1/25600 dilution).

The trial results show that: ? The Bm86 vaccine composition of the invention produces protective immunity (antibody (Ab) levels) in a single vaccine injection not requiring follow up single or le booster injections.

? With the ed vaccine antigen (Bm86) the antibody titre levels at day 150 of the trial were higher than the comparative formulation, which was administered using 2 injections.

? At up to 150 days of the trial, the vaccine composition established a period of antitick immunity not achieved by the comparative formulation. The antibody response ued at tive levels up to one year after a single injection.

? The immune response with the vaccine ition as described in Example 45 is higher than the ative formulation, which es protective levels only after day 28.

? Over a trial duration of more than 400 days, antibody titre levels remained icantly higher than the comparative formulation, demonstrating a longer term immune response following a single injection of the vaccine composition.

D:\DOCUME~1\CTS\docbases\PTO_REP\config\temp_sessions\2008900423662999969\Complete Specifications.docx In vitro Release Results Release of biologically active agent from a model bulk chitosan hydrogel (primer dose) The ability to e an initial release of Bm86 from a an el was tested by monitoring the concentration of Bm86 in the aqueous phase immediately after mixing the polymer and crosslinker solutions and hydrogel formation.

Figure 3 shows results from a study to assess the in vitro release of Bm86 from a model bulk chitosan hydrogel over a period of 144 hours. The bulk hydrogel is composed by mixing of 2% chitosan – HAp (1 mg/mL) solution and 0.08 M TPP – ChS (1%) solution, with ent initial loading concentrations of Bm86. As seen in Figure 3(A), the concentration of Bm86 ed from the hydrogel particles into the surrounding aqueous liquid remains constant for different samples containing Bm86 concentrations of 50, 100, and 200 ?g/mL in solution, giving a relative initial release of ~55 – 60% of the Bm86 into the aqueous liquid phase, which is available as a primary dose (Figure 3(B)).

The effect of crosslinking on long term release rate The g coefficient of Bm86 to hydrogel is mainly dependant on the electrostatic interaction between the protein due to its isoelectric point (pI) and the r chains electrostatic charge in the hydrogel. This results in an equilibrium between Bm86 free in the syneresis liquid and bound to the hydrogel in the initial formation stage. This is a dynamic equilibrium, so when the free Bm86 is removed and the media is refreshed, bound Bm86 tends to be released from the hydrogel to establish a new equilibrium via a transport process. Here we studied the long term release of Bm86 from a chitosan model hydrogel at various crosslinking densities and Bm86 loading concentrations by refreshing the hydrogel aqueous environment with PBS weekly. This is a simulation of the in vivo environment where ellular fluid is continuously refreshed.

The release rate of Bm86 from a bulk chitosan hydrogel prepared with different polymer crosslinking density (0.04 M, 0.08 M or 0.16 M TPP concentrations) was measured over an 8 week period. In these examples, model bulk hydrogels were formed by mixing equal volumes of solutions containing (a) 2% chitosan with 1 mg HAp/mL and various trations of Bm86, and (b) 1% ChS with various trations of the UME~1\CTS\docbases\PTO_REP\config\temp_sessions\2008900423662999969\Complete Specifications.docx crosslinker TPP (0.04M, 0.08M or 0.16M). The amount of Bm86 detected in the initial sample (syneresis release) gave the initial amount of Bm86 that is rapidly released by the composition as a primary dose. Then e media, i.e. PBS, was replaced with fresh solution every week. Subsequent samples collected weekly gave a progressive release profile. The results are shown in Figure 4. As seen in Figure 4, the inking density of the hydrogel can influence both the concentration of Bm86 released in the primary dose as well as the longer term sustained release. Additionally, the total amount of Bm86 released over the cumulative 8 week period (shown as a percentage) indicates that a significant proportion of Bm86 is released over the sustained release phase. At higher crosslinking density, where [TPP] = 0.16 M is used, a significant portion of the Bm86 antigen remains entrapped in the hydrogel at the end of the 8 week period.

The effect protein isoelectric point on g and initial syneresis release The effect of protein isoelectric point on e rate during the initial rapid release (syneresis) phase was investigated using a range of model proteins. In these examples, model bulk hydrogels were formed by mixing equal volumes of solutions containing (a) 2% chitosan with 1 mg HAp/mL and s concentrations of different proteins, and (b) 1% ChS with 0.8 M TPP. Figure 5 gives the release rates for the ns cytochrome C, myoglobin, albumin and the hormone pST from a bulk hydrogel formed with chitosan crosslinked with 0.08 M TPP at various model protein g levels and expressed as tration in the syneresis liquid (A-D) and percentage of the initial loading (E), and the relationship n the syneresis release and the isoelectric point of the loaded n (F).

Additionally, Figure 17 shows the partition of proteins between the bound to the hydrogel state and free in the syneresis liquid state. Bm86, pST, and albumin were used to rate this process at syneresis equilibrium in hydrogel formed by mixing a 2% chitosan – 1 mg HAp/mL constituent with the 0.08 M TPP in 1% chondroitin sulphate crosslinking constituent. Proteins were incorporated in the chitosan containing constituent. This shows: ? This brium is mainly a function of the protein isoelectric point (pI), that is, the binding of the protein to the hydrogel is mainly due to electrostatic interactions.

? The protein partition in the tration ranges studied here gave a constant coefficients value which means that the protein concentrations used here are much D:\DOCUME~1\CTS\docbases\PTO_REP\config\temp_sessions\2008900423662999969\Complete Specifications.docx lower than the binding ty of the hydrogel.

Hydrogel microparticles in a in-oil emulsion An initial water-in-oil emulsion formed with an aqueous solution of tripolyphosphate in adjuvant oil is shown in Figure 6(A). On addition of a chitosan ning solution, crosslinked hydrogel microparticles formed within water droplets in the water-in-oil emulsion (Figure 6(B)). The hydrogel containing water-in-oil emulsion remained stable for 5 months at 4°C, with no phase separation t (Figure 6(C)).

A composition containing hydrogel microparticles formed with chitosan (2%), HAp (1 , TPP (0.32 M) and ChS (1%)) in a water-in-oil emulsion with Montanide ISA61 as the oil phase was analysed by optical microscopy. Results are shown in Figure 7. As seen in Figures 7(A) and 7(B), the hydrogel particles in the water-in-oil emulsion are contained in aqueous droplets. In this , the aqueous droplets were determined to be approximately 1 to 4 µm in diameter. Figure 7(C) shows the emulsion after storage for months at 4 °C. The aqueous phase was then removed from the emulsion by solvent evaporation, to give hydrogel particles of approximately 1 to 2 µm only dispersed in a continuous oil phase (Figure 7(D)).

Release of ically active agent from model bulk te hydrogel (primer dose) The release rate of e somatotropin (pST) from samples of bulk alginate hydrogel prepared in accordance with Example 48 was measured over 28 day period. The different hydrogel samples had different concentrations of alginate (varying from 0.5% to 2%), different crosslinking density (0.3, 0.7, 1.4 or 2.8% CaCl2 concentrations) and different initial loads of pST (varying from 500 to 30000 . The amount of pST detected in the initial sample esis release) during the initial 12 hour period gave the amount of pST that is rapidly released by the composition as a primary dose. The resulting syneresis release given as a tage of initial pST loading is shown in Figure 8. As can be seen in Figure 8 the syneresis release as a percentage of the pST loading increased with increasing loading level and crosslinking degree and decreased with increasing alginate concentration. This provides variations in the syneresis release between 1-28% of the pST loading, enting control of the primary injection dose in the formulation.

D:\DOCUME~1\CTS\docbases\PTO_REP\config\temp_sessions\2008900423662999969\Complete Specifications.docx Longer term release of biologically active agent from model bulk alginate hydrogel Longer term release of pST from alginate hydrogel samples was also observed over 4 weeks. The alginate hydrogel samples were prepared in accordance with e 49. In these trials, after removal of the syneresis liquid the release media, i.e. PBS, was hed every 48 hours. Subsequent samples collected gave a progressive release profile.

The results are shown in Figure 9. Figure 9A shows pST release on a weight basis while Figure 9B shows release as a percentage of the initial pST g. Linear release rates were observed during the initial 2 weeks, which then plateaued over the following 2 weeks.

Porous bulk alginate hydrogels prepared with PEG (35kDa) as an added porogen (at concentrations of 0, 0.5, 1 or 2 wt % ratio) exhibited a modified longer term release rate. As a percentage of the initial loading after 2 weeks a total 29 to 44% of the pST is Viscosity of model able itions To investigate composition viscosity and injectability, model injectable compositions without biologically active agent were prepared. Model injectable compositions were prepared with three different component solutions, with each component solutions: Part [A1]: 2% chitosan or 2% chitosan with 1 mg hydroxyapatite (HAp)/mL Part [B1]: tripolyphosphate (TPP) solution (0.32M) or TPP solution (0.32M) with 1% chondroitin sulphate (ChS) Part [C1]: Montanide ISA61 VG In these es, 2 mL of [A1] (with or without HAp) was emulsified in 2 mL of [C1] then various volumes of [B1] (with or without ChS) were added dropwise under continuous shearing to produce microgel in emulsion formulation having the constituents [A1] : [B1] : [C1] in the following tric ratios: 2:2;0; 2:2:0.5; 2:2:1; 2:2:2:; 2:2:3; 2:2:4 and 2:2:5. The emulsion flow behaviour and stability of the model able compositions was evaluated using stress rheology.

Figure 10 gives the stress sweep measurements of the model able D:\DOCUME~1\CTS\docbases\PTO_REP\config\temp_sessions\2008900423662999969\Complete Specifications.docx compositions where the volumetric ratios of the constituent components were varied between 2:2:0 to 2:2:5. As seen in Figure 10, for injectable itions formed with higher concentrations of the crosslinker TPP, viscosity can decrease with increasing shear stress due to shear thinning behaviour.

Figure 11 (A) gives the variation of flow viscosity for model injectable compositions formed using [A1] contains 2% chitosan and [B1] contains 0.32M TPP, where the volumetric ratios of [A1], [B1] and [C1] were varied to survey the overall stability of the model injectable composition and its flow our, using viscosity ?* (Pa.s) as the control parameter. This variation in viscosity with composition is presented as ternary diagrams at three different shear stresses (0.2 Pa, 2 Pa and 45 Pa).

Flow viscosity measurements were also ted on model injectable compositions formed using [A1] contains 2% chitosan with 1 mg/ml HAp and [B1] contains 0.32M TPP with 1% ChS, where the volumetric ratios of [A1], [B1] and [C1] compositions were varied. The results shown in Figure 11 (B) give corresponding flow behaviour, viscosity ?* (Pa.s), of the different model injectable compositions at shear stresses of 0.2 Pa, 2 Pa and 45 Pa.

TSOL18 antigen in model bulk an-based hydrogel TSOL18 is anti-tapeworm n with positive charge (pI 9.65), contains 112 amino acids, with molecular mass of 12.8 kDa. In Examples 50 to 55, model bulk hydrogel particles containing TSOL18 were prepared. The els comprise TPP- crosslinked chitosan in their core. In some examples, other components, such as te, chondroitin sulphate and hydroxyapatite were also contained in the hydrogel, ing a composite hydrogel material.

Example 50 – Model bulk Chitosan – TPP hydrogel with TSOL18 The following component solutions were ed: Part [M]: 2% chitosan in 1% AcOH Part [N]: Tripolyphosphate (TPP) solution (0.08M) D:\DOCUME~1\CTS\docbases\PTO_REP\config\temp_sessions\2008900423662999969\Complete Specifications.docx In this example, TSOL18 was incorporated in component solution [M] at a concentration of 0.5mg /mL. A quantity of [M] containing TSOL18 was then combined with an equal volume of [N] to form a TSOL18 loaded bulk chitosan hydrogel.

Example 51 - Model bulk Chitosan – TPP hydrogel with TSOL18 The component solutions [M] and [N] of e 50 were used in this example.

However, TSOL18 was incorporated into ent solution [N] instead at a tration of 0.5mg /mL. A quantity of [M] was combined with an equal volume of [N] containing TSOL18, to form a TSOL18 loaded bulk chitosan hydrogel.

Example 52 – Model bulk (Chitosan – HAp – TSOL18) hydrogel and chondroitin te coating The following component solutions were prepared: Part [O]: 2% chitosan in 1% AcOH with 1 mg hydroxyapatite (HAp)/mL Part [P]: yphosphate (TPP) solution (0.08M) with 1% chondroitin sulphate (ChS).

In this example, TSOL18 was incorporated in component solution [O] at a concentration of 0.5mg /mL. A quantity of [O] containing TSOL18 was then combined with an equal volume of [P] to form a TSOL18 loaded bulk chitosan el core with a chondroitin te (ChS) coating. The g was non-crosslinked. .

Example 53 - Model bulk (Chitosan – HAp) hydrogel with chondroitin sulphate – TSOL18 coating The component solutions [O] and [P] of Example 52 were used in this example.

However, TSOL18 was incorporated into component solution [P] at a concentration of 0.5mg/mL instead. A quantity of [O] was combined an equal volume of [P] ning TSOL18, to form a bulk chitosan hydrogel with a non-crosslinked chondroitin sulphate (ChS) coating. In this example, TSOL18 was incorporated in the ChS coating around the chitosan hydrogel core.

Example 54 – Model bulk (Chitosan – HAp - TSOL) hydrogel with alginate coating D:\DOCUME~1\CTS\docbases\PTO_REP\config\temp_sessions\2008900423662999969\Complete Specifications.docx The following component solutions were ed: Part [O]: 2% chitosan in 1% AcOH with 1 mg hydroxyapatite (HAp)/mL Part [Q]: yphosphate (TPP) solution ) with 1% alginate.

In this example, TSOL18 was incorporated in component solution [O] at a concentration of 0.5mg / mL. A ty of [O] containing TSOL18 was then combined with an equal volume of [Q] to form a coated TSOL18 loaded bulk chitosan hydrogel.

Here an alginate g formed around the chitosan hydrogel loaded with TSOL18.

Example 55 - Model bulk (Chitosan – HAp) hydrogel with alginate-TSOL18 g The component solutions [O] and [Q] of Example 54 were used in this example.

However, TSOL18 was incorporated into component solution [Q] at a concentration of 0.5mg/mL instead. A quantity of [O] was combined with an equal volume of [Q] containing TSOL18, to form a bulk an hydrogel with an alginate coating. In this example, the TSOL18 was incorporated in the alginate coating around the chitosan hydrogel core.

Release of TSOL18 from model bulk hydrogel The initial syneresis release of TSOL18 (monitored over 1 week) from the bulk hydrogel s prepared in Examples 50 to 55 is shown in Figure 13. The following results can be seen in Figure 13: ? Example 50: The syneresis liquid ned 54% of the TSOL18 after 4 hours and remained the same after 24 hours. The release of TSOL18 increased to 78% after 1 week.

? Example 51: The syneresis liquid contained 75% of the TSOL18 after 4 hours, which then reduced to 72% at 24 hours. The release of TSOL18 further decreased to 57% after 1 week.

? Example 52: The syneresis liquid contained 44% of the TSOL18 after 4 hours and remained the same after 24 hours. The release of TSOL18 increased to 50% after 1 week, showing the effect of the chondroitin sulphate coating in modulating the release of TSOL18.

D:\DOCUME~1\CTS\docbases\PTO_REP\config\temp_sessions\2008900423662999969\Complete Specifications.docx ? Example 53: The syneresis liquid contained 52% of the TSOL18 after 4 hours, which remained the same at 24 hours. The release of TSOL18 decreased to 45% after 1 week, showing the strong binding between TSOL18 and chondroitin sulphate in the ? Example 54: The l syneresis release of TSOL18 from the bulk hydrogel (monitored over 1 week) is given in Figure 13. As seen in Figure 13, the syneresis liquid ned 34% of the TSOL18 after 4 hours, which increased to 49% after 24 hours. The release of TSOL18 reached an equilibrium at 66% after 1 week.

? Example 55: The sis liquid contained 69% of the TSOL18 as measured after 4 hours, which then reduced to 66% after 24 hours and further reduced to 53% after 1 week. This shows that the initial interaction between TSOL18 and alginate enhances the incorporation of TSOL18 in the g around the chitosan hydrogel.

Injectable compositions containing TSOL18 antigen in hydrogels with an coating Injectable compositions containing chitosan coated hydrogels containing TSOL18 were prepared in accordance with Examples 56 to 59. The coated hydrogels were made by injecting ChS, alginate, ChS - TPP, or alginate - TPP constituent solutions into a chitosan - CaCl2 constituent solution to form TSOL18 containing coated hydrogel particles having a chitosan coating as an outer layer. It is envisaged that the coated hydrogel could e greater entrapment of TSOL18 and lower syneresis (i.e. lower free antigen in aqueous phase) due to the highly positively charged TSOL18 interacting electrostatically with negatively d ChS or alginate in the hydrogel core.

Model bulk hydrogel with TSOL18 and an coating The ing component solutions were prepared and used in the following examples 56 to 59: Part [AC]: 1% CaCl2 in 1% chitosan in 1% AcOH Part [BT] 2% ChS with TSOL18 (0.5 mg /mL) Part [CT] 2% ChS – 0.01 M TPP with TSOL18 (0.5 mg /mL) Part [DT] 2% alginate with TSOL18 (0.5 mg /mL) Part [ET] 2% alginate – 0.01 M TPP with TSOL18 (0.5 mg /mL) D:\DOCUME~1\CTS\docbases\PTO_REP\config\temp_sessions\2008900423662999969\Complete Specifications.docx In each of the Examples, component solutions [BT], [CT], [DT] or [ET] were injected into [AC] to form a hydrogel having a chitosan g as a skin on the formed el.

Calcium chloride was co-solubilized with the chitosan constituent to provide quick gel formation. The negatively charged cross-linker TPP can be included in the negatively charged ChS or alginate to help with formation of the chitosan coating and to increase its thickness if necessary. e 56 – Bulk Chondroitin Sulphate Hydrogel with Chitosan Coating A volume of [BT] was injected into an equal volume of [AC]. This produced a ChS – Ca2+ hydrogel containing TSOL18 and with a chitosan coating. In this e, the chitosan coating is not crosslinked.

Example 57 - Bulk Chondroitin Sulphate Hydrogel with Crosslinked an Coating A volume of [CT] was injected into an equal volume of [AC]. This produced a ChS – Ca2+ hydrogel containing TSOL18 and with a crosslinked an coating, where the chitosan is crosslinked with TPP.

Example 58: - Bulk Alginate - Ca el with Chitosan Coating A volume of [DT] was injected into an equal volume of [AC]. This produced an alginate – Ca2+ hydrogel containing TSOL18 and with a chitosan coating. In this example, the chitosan coating is not inked.

Example 59 - Bulk Alginate - Ca Hydrogel with Crosslinked Chitosan Coating A volume of [ET] was injected into an equal volume of [AC]. This produced an alginate – Ca2+ hydrogel containing TSOL18 and with a crosslinked chitosan coating. In this example, TPP is used to form the crosslinked chitosan coating.

Syneresis e of TSOL18 from Coated Bulk Hydrogel The syneresis release of TSOL18 from the coated bulk hydrogel samples of Examples 56 to 59 into the aqueous liquid was measured after 4 and 24 hours. The results are shown in Figure 14.

D:\DOCUME~1\CTS\docbases\PTO_REP\config\temp_sessions\2008900423662999969\Complete Specifications.docx As seen in Figure 14, the sample of Example 56 ted a constant syneresis release value of 24% of the initial amount of TSOL18 at 4 and 24 hours. Meanwhile, the syneresis release value of Example 57 was constant at 42% of the initial amount of TSOL18. This represents an increase in comparison to Example 56. It is thought that this increase could be due to an se in the porosity of the chitosan – TPP g allowing more of the TSOL18 to diffuse through to the aqueous liquid.

In Example 58, no TSOL18 syneresis into aqueous phase was observed after 4 and 24 hours. In comparison, in Example 59, TSOL18 syneresis release from this sample was increased compared to Example 58. A constant value of TSOL18 syneresis into the aqueous phase was measured after 4 and 24 hours at 6%. This se may be due to an increase in the porosity of the chitosan – TPP coating. These examples also show that the interaction between TSOL18 and alginate is much stronger than the interaction between TSOL18 and chondroitin sulphate.

Example 60 - able composition with chitosan coated alginate el particles containing TSOL18 The following component solutions were prepared and used: Part [A3]: 2.8% CaCl2 in 2% chitosan in 1% AcOH Part [D3] 2% alginate with TSOL18 (300 µg /mL) Part [F1] Sesame oil with 20 mg Span 80/mL A volume of [D3] containing TSOL18 was fied in [F] using high shear emulsifier.

To this primary emulsion a volume of [A3] was added drop wise under constant shear.

The ratio of the ent solutions [F1]:[D3]:[A3] was 2:1:0.5. The resulting composition is shown in Figure 16.

Injectable composition with TSOL18 anti-tapeworm vaccine The following ent solutions were used to prepare various injectable compositions ning TSOL18 loaded hydrogels: Part [M]: 2% chitosan in 1% AcOH Part [N]: Tripolyphosphate (TPP) solution (0.08M) Part [O]: 2% chitosan in 1% AcOH with 1 mg hydroxyapatite (HAp)/mL D:\DOCUME~1\CTS\docbases\PTO_REP\config\temp_sessions\2008900423662999969\Complete Specifications.docx Part [P]: Tripolyphosphate (TPP) solution (0.08M) with 1% chondroitin sulphate (ChS).

Part [Q]: Tripolyphosphate (TPP) solution (0.08M) with 1% alginate.

Example 61 – Injectable composition sing Chitosan – TPP hydrogel with TSOL18 In this example, TSOL18 was incorporated in component solution [M] at a concentration of 0.5mg /mL A volume of [M] containing TSOL18 (0.25 mL) was emulsified in 1 mL of Montanide ISA 61 VG oil using high shear emulsifier. To this y emulsion 0.25 mL of [N] was added drop wise under constant shear. The resulting composition was a stable uniform emulsion with below 1 µm droplet diameter.

Example 62 – Injectable composition sing Chitosan – TPP hydrogel with TSOL18 TSOL18 was incorporated in component solution [N] at a concentration of 0.5mg /mL. A volume of [M] (0.25 mL) was emulsified in 1 mL of ide ISA 61 VG oil using high shear emulsifier. To this primary emulsion 0.25 mL of [N] containing TSOL18 was added drop wise under constant shear. The resulting composition was a stable uniform emulsion with below 1 µm droplet diameter. An example is shown in Figure 15.

Example 63 – Injectable composition comprising (Chitosan – HAp) – (ChS – TPP) hydrogel with TSOL18 TSOL18 was incorporated in ent solution [O] at a concentration of 0.5mg /mL. A volume of [O] containing TSOL18 (0.25 mL) was emulsified in 1 mL of Montanide ISA 61 VG oil using high shear emulsifier. To this y emulsion 0.25 mL of [P] was added drop wise under constant shear. The resulting injectable composition was a stable uniform emulsion with below 1 µm droplet diameter. The hydrogel particles in the composition comprise a inked chitosan-HAp core and a coating comprising chondroitin sulphate.

Example 64 - able composition comprising (Chitosan – HAp) – (ChS – TPP) hydrogel with TSOL18 D:\DOCUME~1\CTS\docbases\PTO_REP\config\temp_sessions\2008900423662999969\Complete Specifications.docx The ent solutions [O] and [P] of Example 62 were used in this example.

However, TSOL18 was incorporated into component solution [P] at a concentration of 0.5mg/mL d. A volume of [O] containing TSOL18 (0.25 mL) was emulsified in 1 mL of Montanide ISA 61 VG oil using high shear emulsifier. To this primary emulsion 0.25 mL of [P] was added drop wise under constant shear. The resulting injectable composition was a stable uniform emulsion with below 1 µm droplet diameter. The el particles in the composition comprise a crosslinked chitosan-HAp core and a coating comprising chondroitin sulphate.

Example 65 – Injectable composition comprising san – HAp) – (Alginate – TPP) hydrogel with TSOL18 TSOL18 was incorporated in ent solution [O] at a concentration of 0.5mg / mL. A volume of [O] containing TSOL18 (0.25 mL) was emulsified in 1 mL of Montanide ISA 61 VG oil using high shear emulsifier. To this primary emulsion 0.25 mL of [Q] was added drop wise under constant shear. The resulting injectable composition was a stable uniform emulsion with below 1 µm t diameter. The hydrogel particles in the composition comprise a crosslinked chitosan-HAp core and coating comprising Example 66 - able composition comprising (Chitosan – HAp) – (Alginate – TPP) hydrogel with TSOL18 The component solutions [O] and [Q] of e 64 were used in this example.

However, TSOL18 was incorporated into component solution [Q] at a concentration of 0.5mg/mL instead. 0.25 mL of [O] was emulsified in 1 mL of Montanide ISA 61 VG oil using high shear emulsifier. To this primary emulsion 0.25 mL of [Q] was added drop wise under constant shear. The resulting injectable composition was a stable uniform emulsion with below 1 µm droplet diameter. The hydrogel particles in the ition comprise a crosslinked chitosan-Hap core and a coating comprising alginate.

Injectable composition containing chitosan coated el particles and TSOL18 anti-tapeworm vaccine The following component solutions were used to prepare various injectable compositions containing TSOL18 loaded coated hydrogels: D:\DOCUME~1\CTS\docbases\PTO_REP\config\temp_sessions\2008900423662999969\Complete Specifications.docx Part [AC]: 1% CaCl2 in 1% chitosan in 1% AcOH Part [BT] 2% ChS with TSOL18 (0.5 mg /mL) Part [CT] 2% ChS – 0.01 M TPP with TSOL18 (0.5 mg /mL) Part [DT] 2% alginate with TSOL18 (0.5 mg /mL) Part [ET] 2% te – 0.01 M TPP with TSOL18 (0.5 mg /mL) Example 67 – Injectable composition comprising chitosan coated chondroitin sulphate hydrogel with TSOL18 A volume of [BT] was emulsified in 1 mL of Montanide ISA 61 VG oil using high shear fier. To this primary emulsion 0.25 mL of [AC] was added drop wise under constant shear. The resulting composition was a stable uniform emulsion with below 1 µm droplet diameter. This produced an injectable composition having ChS – Ca2+ hydrogel particles containing TSOL18 and with a chitosan coating.

Example 68 – Injectable composition comprising crosslinked chitosan coated chondroitin sulphate hydrogel with TSOL18 A volume of [CT] was emulsified in 1 mL of Montanide ISA 61 VG oil using high shear emulsifier. To this primary emulsion 0.25 mL of [AC] was added drop wise under constant shear. The resulting composition was a stable uniform emulsion with below 1 µm t diameter. This produced an able composition having ChS – Ca2+ el particles containing TSOL18 and with a crosslinked chitosan – TPP coating.

Example 69 - Injectable composition comprising chitosan coated alginate hydrogel with TSOL18 A volume of [DT] was emulsified in 1 mL of ide ISA 61 VG oil using high shear emulsifier. To this primary emulsion 0.25 mL of [AC] was added drop wise under constant shear. The resulting composition was a stable uniform emulsion with below 1 µm droplet diameter. This produced an injectable composition having alginate – Ca2+ el particles containing TSOL18 and with a non-crosslinked chitosan coating.

Example 70 – Injectable composition sing crosslinked an coated alginate el with TSOL18 D:\DOCUME~1\CTS\docbases\PTO_REP\config\temp_sessions\2008900423662999969\Complete Specifications.docx A volume of [ET] was fied in 1 mL of Montanide ISA 61 VG oil using high shear emulsifier. To this primary emulsion 0.25 mL of [AC] was added drop wise under constant shear. The resulting composition was a stable uniform emulsion with below 1 µm droplet diameter. This produced an injectable composition having alginate – Ca2+ hydrogel particles containing TSOL18 and with a crosslinked chitosan – TPP coating.

It is to be understood that various other modifications and/or alterations may be made without departing from the spirit of the t invention as outlined herein.

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Claims (21)

Claims

1. An injectable composition for rapid and sustained delivery of a biologically active agent comprising: a water-in-oil emulsion comprising an aqueous phase dispersed in a oil phase, the aqueous phase comprising a plurality of hydrogel particles and an aqueous liquid; a biologically active agent in the hydrogel particles and in the aqueous liquid of the s phase, n when administered, the injectable composition provides rapid and sustained delivery of the biologically active agent in vivo, and wherein the hydrogel particles comprise a crosslinked polysaccharide.

2. A composition according to claim 1, wherein the composition further comprises an adjuvant.

3. A ition according to claim 2, wherein the oil phase of the emulsion comprises the adjuvant.

4. A composition according to any one of claims 1 to 3, wherein the hydrogel particles comprise a polysaccharide crosslinked with a inking agent having functional groups that participate in non-covalent bonding interactions with the polysaccharide.

5. A composition ing to any one of claims 1 to 4, wherein the crosslinked ccharide comprises a polysaccharide selected from the group consisting of chitosan, alginate, hyaluronic acid, cellulose, chondroitin sulphate, dermatan sulphate, keratan sulphate, heparin, and derivatives thereof, and mixtures thereof.

6. A composition according to any one of claims 1 to 5, n the crosslinked polysaccharide comprises an crosslinked with a phosphate nd.

7. A composition ing to any one of claims 1 to 5, wherein the crosslinked polysaccharide comprises alginate or chondroitin sulphate and said alginate or chondroitin te is crosslinked with a divalent cation derived from an alkaline earth metal.

8. A ition according to any one of claims 1 to 7, n the hydrogel particles have an average particle diameter in the range of from about 10 nm to 20 ?m, ably in the range of from about 50 nm to about 5 ?m.

9. A composition according to any one of claims 1 to 8, wherein the hydrogel particles further comprise an aqueous insoluble alkaline earth metal ate.

10. A composition according to any one of claims 1 to 9, wherein one or more of the plurality of hydrogel les comprise a coating.

11. A composition according to any one of claims 1 to 10, wherein the biologically active agent is ed from the group consisting of a hormone, an antimicrobial, a therapeutic antibody, a cytokine, a fusion protein, a virus, a bacteria or bacteria fragment, a vaccine and an antigen.

12. A composition according to any one of claims 1 to 11, wherein the biologically active agent in the hydrogel particles is conjugated to the particles.

13. A method of delivering a biologically active agent to a non-human subject, the method comprising the step of administering the composition of any one of claims 1 to 12 to the t by injection.

14. A method according to claim 13 wherein the biologically active agent is an antigen.

15. A method according to claim 14 wherein the antigen is Bm86 or TSOL18.

16. A method according to claim 13 wherein the biologically active agent is a hormone.

17. A method according to claim 16 wherein the hormone is somatotropin or luteinising hormone-releasing hormone (LHRH).

18. A method of treating or preventing a disease or disorder in a non-human subject comprising the step administering the composition of any one of claims 1 to 16 to the nohuman t by ion.

19. A method according to claim 18, wherein the e or disorder is a microorganism infection.

20. A method according to claim 18, wherein the disease or disorder is a viral infection.

21. A process for preparing an injectable ition for rapid and sustained delivery of a biologically active agent, the process comprising the steps of: providing a first aqueous composition comprising a first hydrogel forming component and a second s composition comprising a second hydrogel forming component, at least one of the first aqueous composition and the second aqueous composition comprising a biologically active agent; combining the first aqueous composition with a lipophilic composition comprising an oil to form an emulsified composition; and combining the second aqueous ition with the emulsified composition under conditions allowing the first hydrogel forming ent to react with the second el forming component to form a plurality of hydrogel les in situ and thereby provide an injectable composition comprising a water-in-oil emulsion comprising an aqueous phase dispersed in an oil phase, the aqueous phase comprising a plurality of hydrogel particles and an aqueous liquid, and wherein the biologically active agent is contained in the hydrogel particles and in the aqueous liquid of the aqueous phase of the in-oil emulsion, and wherein the hydrogel particles comprise a crosslinked polysaccharide. -