BR112018071007B1 - INJECTABLE COMPOSITION, PROCESS FOR PREPARING AN INJECTABLE COMPOSITION, AND, USE OF AN INJECTABLE COMPOSITION - Google Patents

Abstract

The invention relates to an injectable composition for rapid and prolonged delivery of a biologically active agent and to uses of the injectable composition in treating or preventing a condition in an individual. The injectable composition comprises a water-in-oil emulsion having an aqueous phase dispersed in an oily 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 the aqueous liquid.

Description

TECHNICAL FIELD

[01] The present invention relates to an injectable composition for delivering a biologically active agent. In particular, the present invention relates to an injectable composition comprising multiple phases for rapid and prolonged delivery of a biologically active agent. The invention also relates to the uses of the injectable composition in the treatment or prevention of a condition.

FUNDAMENTALS

[02] Biologically active agents can be incorporated into numerous different dosage forms for administration by numerous different routes. These dosage forms may be, for example, tablets, capsules, sprays, ointments or patches for distributing the active agent by routes such as oral, transmucosal or transdermal routes. However, for a variety of reasons, many biologically active agents may not be effectively distributed using routes such as oral, transmucosal, or transdermal routes. This may be because the biologically active agent is susceptible to degradation by enzymes or stomach acid, or is insufficiently absorbed into the systemic circulation due to molecular charge and/or size.

[03] As such, numerous biologically active agents are predominantly administered by injection. Administration by injection allows an active agent to rapidly enter the systemic circulation and bypass the digestive system and first-pass metabolism through the liver. However, repeated injections of an active agent over a period of time may be necessary in order to achieve or maintain a desired effect in vivo. For example, immunization may require, in general, multiple vaccinations, multiple boosters and multiple high doses of vaccine to be administered, which can result in high costs for both the industry and end users.

[04] Sustained-release compositions are of interest in biomedical applications in which maintenance of a systemic level of an active agent over a period of time is desired. For injected biologically active agents, extended-release compositions can help reduce injection frequency and increase the duration of action of the active agent or reduce adverse side effects.

[05] Numerous injectable extended-release compositions have been described. For example, one form of injectable sustained-release composition uses small diameter polymeric particles to deliver an encapsulated drug. Such polymeric particles are often formed from decomposable synthetic polymers such as poly(lactic acid), poly(glycolic acid) or poly(lactic acid-co-glycolic acid), which decompose in a biological environment, resulting in the release of the encapsulated drug over an extended period of time. Drug release may therefore be dependent on the rate of polymer decomposition, which may not always provide a desired kinetic profile.

[06] There remains a need to develop an injectable composition that can provide a desired release profile for a biologically active agent.

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

[08] Discussion of documents, actions, materials, devices, articles and the like is included in this specification solely for the purpose of providing a context for the present invention. It is not suggested or asserted that any or all of these matters formed part of the basis of the prior art or were common general knowledge in the field relevant to the present invention as if they existed prior to the priority date of each claim of this application.

SUMMARY OF THE INVENTION

[09] The present invention provides an injectable composition comprising a biologically active agent that is formulated to provide rapid initial release of the biologically active agent, followed by prolonged distribution of the biological agent, to an individual in vivo.

[10] In one aspect, the present invention provides an injectable composition for rapid and prolonged delivery of a biologically active agent comprising: a water-in-oil emulsion having an aqueous phase dispersed in an oily phase, the aqueous phase comprising a plurality of hydrogel particles and an aqueous liquid; and a biologically active agent in the hydrogel particles and in the aqueous liquid of the aqueous phase, and when administered, the injectable composition provides rapid and prolonged distribution of the biologically active agent in vivo.

[11] In some embodiments, the injectable composition of the invention may additionally comprise an adjuvant. The adjuvant may be present in the oil phase or in the aqueous phase of the water-in-oil emulsion of the injectable composition. In one embodiment, the adjuvant is present in the oil phase. The adjuvant may be an adjuvant oil forming the oily phase of the emulsion.

[12] The hydrogel particles of the aqueous phase of the water-in-oil emulsion comprise a biocompatible material. In one embodiment, the hydrogel particles comprise a cross-linked polysaccharide.

[13] The cross-linked polysaccharide in the hydrogel particles preferably comprises a polysaccharide and a cross-linking agent. In one embodiment, the crosslinking agent comprises functional groups that participate in non-covalent bonding interactions with the polysaccharide.

[14] In one set of embodiments, the cross-linked polysaccharide in the hydrogel particles may comprise a glycosaminoglycan (GAG).

[15] The polysaccharide present in the hydrogel particles can be selected from the group consisting of chitosan, alginate, hyaluronic acid, cellulose, chondroitin sulfate, dermatan sulfate, keratan sulfate, heparin and derivatives thereof. The hydrogel particles may comprise a mixture of two or more polysaccharides and/or derivatives thereof.

[16] In one embodiment, the cross-linked polysaccharide in the hydrogel particles comprises a polysaccharide selected from the group consisting of chitosan, alginate, chondroitin sulfate and mixtures thereof.

[17] Chitosan in hydrogel particles can be cross-linked with a phosphate compound, preferably a tripolyphosphate (TPP) such as sodium tripolyphosphate.

[18] Alginate and chondroitin sulfate in the hydrogel particles can each be cross-linked with a divalent cation derived from an alkaline earth metal. In some specific embodiments, alginate and chondroitin sulfate in the hydrogel particles are cross-linked with calcium (Ca2+) or magnesium (Mg2+) cations.

[19] The hydrogel particles present in the injectable composition have an average diameter in the range of about 10 nm to 20 μm, preferably in the range of about 50 nm to about 5 μm.

[20] In one embodiment, the hydrogel particles additionally comprise an aqueous-insoluble alkaline earth metal phosphate. Preferably, the aqueous-insoluble alkaline earth metal phosphate is hydroxyapatite.

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

[22] In some embodiments, the water-in-oil emulsion of the injectable composition comprises a surfactant. The surfactant can help facilitate the formation of a stable emulsion composition. In one preference, the injectable composition comprises a nonionic surfactant.

[23] The injectable composition is useful for delivering a biologically active agent and can comprise a variety of biologically active agents. According to the invention, 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 particles and the aqueous liquid of the aqueous phase.

[24] In one embodiment, the biologically active agent is selected from the group consisting of a hormone, an antimicrobial, a therapeutic antibody, a cytokine, a fusion protein, a virus, a bacterium or a bacterium fragment, a vaccine and a antigen. In a specific 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.

[25] In one set of embodiments, the biologically active agent is an antigen. In specific embodiments, the antigen may be Bm86 or TSOL18.

[26] In another set of embodiments, the biologically active agent is a hormone. In specific embodiments, the hormone may be somatotropin or luteinizing hormone-releasing hormone (LHRH).

[27] In one form of the injectable composition of the embodiments described herein, the biologically active agent in the hydrogel particles is conjugated to the particles.

[28] The present invention also provides a method of delivering a biologically active agent to an individual, the method comprising the step of administering to the individual, by injection, an injectable composition of any of the embodiments described herein. In particular, the injectable composition of the invention provides rapid delivery of an initial primary dose of the biologically active agent followed by longer-lasting sustained delivery of the active agent to an individual, in vivo.

[29] The present invention further provides a method of treating or preventing a disease or disorder in an individual comprising the step of administering to the individual, by injection, an injectable composition of any of the embodiments described herein.

[30] In one embodiment, the injectable composition can be suitably used for the treatment or prevention of a microorganism infection.

[31] In another embodiment, the injectable composition can be suitably used for the treatment or prevention of a viral infection.

[32] In another aspect the present invention provides a process for preparing an injectable composition of the embodiments described herein for rapid and prolonged delivery of a biologically active agent, the process comprising the steps of: providing a first aqueous composition comprising a first forming component of hydrogel 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 composition under conditions that allow the first hydrogel-forming component to react with the second hydrogel-forming component to form a plurality of hydrogel particles in situ and thereby produce an injectable composition comprising an emulsion of water-in-oil 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 the aqueous liquid of the phase water-in-oil emulsion.

[33] Other aspects appear below in the detailed description.

BRIEF DESCRIPTION OF FIGURES

[34] Embodiments of the invention will now be described with reference to the following non-limiting figures in which:

[35] Figure 1 is a schematic representation showing an injectable composition comprising an antigen according to an embodiment of the invention.

[36] Figure 2 is a graph showing the antibody titer to Bm86 (ELISA, 1:1,600 dilution) with a comparative formulation representing a conventional anti-tick composition administered subcutaneously in two injections consisting of a primary dose (day 0) and a booster injected after 4 weeks (2x50 μg of Bm86/dose) and with an injectable composition of an embodiment of the invention administered subcutaneously as a single injection.

[37] Figure 3 are graphs showing the amount of Bm86 released over time from a bulk model chitosan hydrogel, with release expressed as (A) the amount of Bm86 detected in the aqueous phase, including the syneresis liquid and (B) A percentage of the initial loading of Bm86 in the composition.

[38] Figure 4 is a graph showing release of Bm86 over a period of 8 weeks from cross-linked chitosan bulk model hydrogel samples with different cross-link densities (TTP). Column labels are "S" for initial syneresis release after 24 hours, with the numbers representing the week number after composition formation and total amount of Bm86 released during the 8-week cumulative period shown as a percentage.

[39] Figure 5 illustrates graphs showing the syneresis release of cytochrome C, albumin, myoglobin and pST ("porcine SomatoTropin", porcine somatotropin) from cross-linked chitosan bulk model hydrogel samples at various loading levels expressed as concentration in the net of syneresis (A-D), the percentage of initial loading (E), and the relationship between the release of syneresis and the isoelectric point of the charged protein (F).

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

[41] 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 stability of the composition and (D) the hydrogel particles after removing the aqueous phase of the composition by evaporation.

[42] Figure 8 is a graph showing the release of porcine somatotropin syneresis (pST) after a period of 12 hours from alginate - Ca2+ bulk model hydrogel samples with various crosslinking densities (Ca2+) and various loading levels of pST, expressed as a percentage of the initial pST loading.

[43] Figure 9 is a graph showing the release of porcine somatotropin (pST) over a period of 28 days from alginate - Ca2+ bulk model hydrogel samples formed with various amounts of PEG porogen ("Poly(Ethylene Glycol)" , poly(ethylene glycol)) and having various levels of pST loading, expressed as (A) quantitative release and (B) release as a percentage of total pST loading.

[44] Figure 10 is a graph showing changes in viscosity under applied shear stress for samples of injectable compositions of embodiments of the invention comprising cross-linked chitosan hydrogel particles with various cross-link densities and aqueous phase volume ratios.

[45] Figures 11 show compositional diagrams illustrating the regions of low viscosity that correspond to the stresses indicated by the black arrows in Figure 10 for (A) an injectable composition of a modality prepared with chitosan, TPP and Montanide oil (trademark) and ( B) an injectable composition of another modality prepared with chitosan/hydroxyapatite and TPP/chondroitin sulfate in Montanide oil.

[46] Figure 12 is a graph showing the immune response in sheep over a period of 400 days presented by a comparative formulation comprising Bm86 representing a conventional anti-tick composition administered subcutaneously in two injections consisting of a primary dose (day 0) and a booster injected after 4 weeks (2x50 μg of Bm86/dose) and by an injectable composition of one modality administered subcutaneously as a single injection.

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

[48] Figure 14 is a graph illustrating the syneresis release of TSOL18 from various cross-linked and non-cross-linked chitosan model hydrogels coated with cross-linked alginate or chondroitin sulfate coating for 1 day.

[49] Figure 15 shows a micrograph of an injectable composition containing TSOL18 according to an embodiment of the invention, with a 1:10 dilution of the composition (in insect) showing microhydrogel particles in the emulsion.

[50] Figure 16 shows (A) a schematic representation of a porcine somatotropin (pST)-loaded alginate hydrogel particle having a chitosan coating, (B) a schematic representation of the barrier effect of the coating on the release of ( pST), (C) light microscope micrograph of an injectable composition comprising alginate hydrogel coated with chitosan in accordance with an embodiment of the invention and (D) the sample shown in (C) diluted 1:10 in sesame oil selectively showing partially formed and ruptured chitosan films.

[51] Figure 17 is a graph illustrating the initial fractionation (protein partitioning) of porcine somatotropin (pST), albumin and Bm86 after hydrogel formation and the completeness of the syneresis process, where the electrostatic binding of pST, Bm86 and albumin with a chitosan bulk model hydrogel - HAp formed with 0.08 M TPP cross-linking agent in 1% ChS is measured at equilibrium.

DETAILED DESCRIPTION OF THE INVENTION

[52] The present invention is based on the discovery that both rapid and prolonged distribution of a biologically active agent in vivo is capable of being achieved through the use of an injectable composition as described herein.

[53] The injectable composition of the invention is capable of delivering an initial primary dose of the biologically active agent, followed by more prolonged distribution of the active agent over a period of time, which reduces the need for repeated injections.

[54] In one aspect the present invention provides an injectable composition for rapid and prolonged delivery of a biologically active agent comprising: a water-in-oil emulsion having an aqueous phase dispersed in an oily phase, the aqueous phase comprising a plurality of hydrogel particles and an aqueous liquid; and a biologically active agent in the hydrogel particles and in the aqueous liquid of the aqueous phase, and when administered, the injectable composition provides rapid and prolonged distribution of the biologically active agent in vivo.

[55] In the context of the present invention, "rapid" distribution refers to the distribution of the biologically active agent immediately after administration of the injectable composition to an individual, or after a short time. Rapid release provides an initial dose of the biologically active agent immediately or a short time after injection of the composition or invention to produce an initial therapeutic effect.

[56] "Prolonged" distribution refers to the distribution of the biologically active agent subsequent to the initial rapid distribution. Prolonged distribution, therefore, occurs over a period of time that is substantially longer than that of rapid distribution. Prolonged delivery delivers a dose of the biologically active agent over a longer time and therefore prolongs the therapeutic effect produced by the active agent. In some embodiments, the extended distribution may be over a period of time of at least 24 hours. In specific embodiments, the extended distribution may be over a period of time of at least several days or several weeks, or even several months.

[57] It is believed that the ability of the injectable composition to provide rapid and prolonged distribution of a biologically active agent is aided by the composition comprising multiple phases. The desired in vivo distribution profile of the biologically active agent is therefore influenced by the different phases in the composition.

[58] In specific 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 can be considered as a multiphase composition.

[59] In one embodiment, the injectable composition of the invention is three-phase.

[60] The injectable composition of the invention comprises a water-in-oil emulsion. A person skilled in the art 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 continuous phase of the emulsion while the water phase forms the dispersed phase.

[61] The aqueous phase of the water-in-oil emulsion comprises an aqueous liquid. The aqueous phase also comprises a plurality of hydrogel particles, which are dispersed in the aqueous liquid. In this way, the aqueous liquid and the hydrogel particles each form part of the aqueous phase of the water-in-oil emulsion.

[62] In some embodiments, the injectable composition 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 can be provided by the oil phase of the water-in-oil emulsion. At least two other phases may be provided by the aqueous phase of the water-in-oil emulsion. In particular, the aqueous liquid and hydrogel particles of the aqueous phase of the emulsion can provide a second phase and a third phase of the injectable composition, respectively.

[63] The oily phase of the water-in-oil emulsion comprises at least one physiologically acceptable oil and may comprise a mixture of such oils. Physiologically acceptable oils are, in general, hydrophobic and liquid at a temperature between 20°C and 40°C.

[64] A variety of suitable physiologically acceptable oils can be used in the injectable composition of the invention.

[65] In some embodiments, the oily phase may comprise one or more oils selected from the group consisting of fatty acids; fatty acid esters; esters of poly(ethylene glycols), for example monoesters and diesters; hydrocarbon oils, for example natural hydrocarbon oils; and steroids, for example cholesterol.

[66] In one embodiment, the oily phase may comprise one or more oils selected from fatty acids; fatty acid esters; poly(ethylene glycols) esters; and hydrocarbon oils.

[67] Suitable fatty acids and fatty acid esters may be those having a saturated or unsaturated aliphatic chain comprising 6 to 24 carbon atoms. Unsaturated aliphatic chains can be monounsaturated or polyunsaturated. Some specific fatty acids may be C12-C24 long chain fatty acids e.g. C15-C22 fatty acids and C6-C12 medium chain fatty acids. These include polyunsaturated fatty acids such as omega-3 oils, for example, eicosapentanoic acid (EPA, "EicosaPentanoic Acid"), docosahexaenoic acid (DHA, "DocosaHexaenoic Acid"), alpha-linoleic acid (ALA, " AlphaLinoleic Acid"). Combinations of such compounds are also considered. Fatty acid esters can be triglycerides, and also glycerol esters (particularly triesters) with a combination of fatty acids and lower molecular weight acids e.g. succinic acid (triglycerides of fatty acids are a specific example of glycerides). . Oils containing glycerides may also contain monoglycerides and/or diglycerides, for example, as a minor part of the glyceride content (less than 50 mol%).

[68] In some embodiments, the oily phase may comprise a mixture of oils, for example macrogolglycerides of fatty acids, also known as polyoxylglycerides, which are mixtures of monoesters, diesters and triesters of glycerol fatty acids and monoesters and diesters of fatty acids. poly(ethylene glycol); examples are oleoyl-macrogolglycerides and linoeoyl-macrogolglycerides.

[69] Suitable hydrocarbon oils may be mineral oils or terpenes.

[70] Specific terpenes can be triterpenes such as squalene.

[71] Specific mineral oils may comprise a mixture of several hydrocarbon chains of different lengths, including short chain hydrocarbon lengths (< C14) and longer chain hydrocarbon lengths (> C14). Examples of mineral oils of a pharmaceutical grade include light liquid paraffin and light mineral oil.

[72] Alternative or additional oils that may be included in the oily phase in combination with or in place of the above oils include vegetable or plant oils, such as peanut oil, safflower oil, sunflower oil, soybean oil, cottonseed, chaulmoogra oil, corn oil, jojoba oil, pesic oil, olive oil, sesame oil, almond oil, castor oil, canola oil, linseed oil, squalene and coconut oil ; fish oils such as shark oil, orange roughy fish oil (Hoplostethus atlanticus), yellowtail oil and cod liver oil; animal oils such as mink oil, lard oil and chicken fat oil; and synthetic oils such as ethyl oleate.

[73] In some embodiments, the oily phase may comprise a physiologically acceptable oil having adjuvant properties. The physiologically acceptable oil is therefore an adjuvant and can be called an adjuvant oil.

[74] In other embodiments, the oily phase may comprise a non-adjuvant physiologically acceptable oil (i.e., a passive oil). In such embodiments, the oil does not have adjuvant properties. Passive oil can be considered as chemically inert. 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.

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

[76] The water-in-oil emulsion of the injectable composition also comprises an aqueous phase. According to the requirements of the invention, the aqueous phase comprises a plurality of hydrogel particles and an aqueous liquid.

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

[78] The hydrogel particles of the injectable composition described herein are generally made of soft, low-modulus materials comprising a low solids content and a high water content. In some embodiments, the hydrogel particles may have a Young's modulus in the range of about 5 kPa to 700 kPa. The hydrogel particles may additionally have a water content of at least 60%.

[79] In some embodiments, the hydrogel particles may be microhydrogels. The term "microhydrogels" is used herein as a reference to separate hydrogel portions having at least one dimension in the range of nanometer (nm) to micrometer (μm). Such microhydrogels may be nanometer or micrometer sized droplets comprising or composed of hydrated polymeric gel.

[80] The hydrogel particles are dispersed in and may be surrounded by the aqueous liquid.

[81] In general, aqueous liquid comprises water.

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

[83] Aqueous phase hydrogel particles are biocompatible and are formed from biocompatible materials. Biocompatibility is a concept known to those in the art. Biocompatible substances are those that provoke 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, toxic or harmful immune response in vivo, or adversely integrates with a specific tissue or cell type.

[84] Hydrogel particles can be colloidal portions of hydrogel material having a diameter in the range of about 10 nm to 20 μm, preferably from about 50 nm to about 5 μm. Hydrogel particles according to such embodiments may be referred to herein as microhydrogels. The hydrogel particle diameter can be controlled by the choice of the oil phase and the choice of any surfactant or emulsifier used to prepare the injectable composition. Hydrogel particle diameter can be assessed using optical techniques such as dynamic light scattering, light microscopy, and confocal laser scanning microscopy.

[85] The hydrogel particles preferably comprise or are composed of a cross-linked polymer. In addition to being biocompatible, the cross-linked polymer is hydrophilic and susceptible to aqueous solvation.

[86] A variety of cross-linked polymers can be suitable for hydrogel particles. The polymers in the hydrogel can be chemically or physically cross-linked.

[87] Cross-linked polymers suitable for hydrogel particles can be produced when a first component and a second component react or interact to form a three-dimensional macromolecular network structure that is held together via intermolecular bonds. The macromolecular network can be considered as a polymeric matrix.

[88] The first component may be a polymeric component, while the second component may be a crosslinking component. The crosslinking component may be provided by a crosslinking agent. The crosslinking agent can be a small molecule or a macromolecule, such as an additional polymer.

[89] Cross-linking between the polymeric component and the cross-linking component can occur via physical bonds, covalent or non-covalent.

[90] Cross-linked polymers suitable for hydrogel particles can comprise or be formed from polymers and cross-linking agents of natural or synthetic origin.

[91] In one set of embodiments, emulsion hydrogel particles can be formed with a neutral polymer, which is cross-linked via physical bonds, covalent or non-covalent. A neutral polymer can be one that has no ionizable functional groups. Consequently, hydrogel particles formed with a neutral polymer may not carry net charge at physiological pH.

[92] In one embodiment, hydrogel particles can be formed with a charged polymer, which is cross-linked via non-covalent bonds. The charged polymer may be a polyelectrolyte. The charged polymer can be selected from a cationic polymer, an anionic polymer, a zwitterionic polymer, or a combination thereof.

[93] Hydrogel particles formed with a charged polymer can comprise groups that are ionizable, so that the hydrogel particles carry a net charge at physiological pH. Charged hydrogel particles can influence both the absorption and release of a biologically active agent through electrostatic interactions and also the release kinetics. Hydrogel particles formed with a charged polymer can therefore affect both the initial release of the biologically active agent and also its long-lasting sustained release.

[94] In one embodiment, the hydrogel particles comprise a cross-linked biopolymer. Biopolymers can be polymeric molecules obtained from, or derived from, natural sources.

[95] In one embodiment, the hydrogel particles comprise a cross-linked polysaccharide. Cross-linked polysaccharide is formed when at least one polysaccharide is combined with at least one cross-linking agent.

[96] In one embodiment, the cross-linked polysaccharide may comprise a glycosaminoglycan (GAG). Glycosaminoglycans are unbranched polysaccharides based on a disaccharide repeating unit. The disaccharide unit may consist of an aminosugar (N-acetylglucosamine or N-acetylgalactosamine) and either a uronic sugar (glucuronic acid or iduronic acid) or galactose. GAGs can also be esterified by sulfur-containing groups. Some examples of glycosaminoglycans are chondroitin sulfate, dermatan sulfate, keratan sulfate and heparin. In a preference, the hydrogel particles comprise a cross-linked glycosaminoglycan.

[97] Hydrogel particles comprising a cross-linked polysaccharide may comprise a polysaccharide selected from the group consisting of chitosan, alginate, hyaluronic acid, cellulose, chondroitin sulfate, dermatan sulfate, keratan sulfate and heparin, and also salts thereof. and derivatives thereof. Polysaccharides such as chitosan, alginate, hyaluronic acid, chondroitin sulfate and cellulose may have adjuvant properties and may help modulate the release of the biologically active agent from the injectable composition in vivo.

[98] The cross-linked polysaccharide in the hydrogel particles may be charged (i.e., carry a net positive or negative charge) or may be uncharged.

[99] In a preference, the cross-linked polysaccharide comprises a polysaccharide selected from chondroitin sulfate, alginate and chitosan, a salt thereof or a derivative thereof.

[100] Chitosan derivatives can be chitosan-dextran sulfate, N-trimethylchitosan, N-carboxymethylchitosan and lipo- and glycoconjugated oligochitosan derivatives.

[101] Polysaccharides are combined with cross-linking agents to form a three-dimensional cross-linked macromolecule. It would be recognized that the polysaccharide and the crosslinking agent need to be able to interact with each other to form the crosslinked polymer. Accordingly, the polysaccharide and the crosslinking agent may each comprise a chemical moiety having a functionality that is capable of crosslinking.

[102] Suitable crosslinking agents may comprise or be composed of a chemical moiety that is capable of interacting with a functional group present in the polysaccharide via covalent or non-covalent bonding mechanisms. In some embodiments, the chemical moiety may have functional groups that are capable of interacting with functional groups present in the polysaccharide via covalent or non-covalent bonding mechanisms.

[103] In one form, suitable crosslinking agents may be capable of participating in non-covalent bonding interactions with the polysaccharide. Crosslinking agents may comprise functional groups that may participate in such non-covalent bonding interactions.

[104] The polysaccharide may comprise an ionizable functional group (with an amino, carboxylic acid or sulfonate group), which is capable of forming a cationic or anionic functional group, and the crosslinking agent may comprise a complementary ionizable functionality that is capable of binding. one or more functional groups of the polysaccharide through non-covalent interactions. In this way, the resulting cross-linked polysaccharide can be cross-linked via electrostatic or ionic bonds.

[105] Ionizable functional groups on the polysaccharide can impart a net charge to the cross-linked polysaccharide if some of the ionizable functionalities remain free and unbound to a complementary cross-linking agent. These free ionizable functional groups may be available to interact with an oppositely charged biologically active agent contained in the hydrogel particles via noncovalent interactions such as electrostatic interactions. This could aid retention of the active agent in the hydrogel particles and thus influence the distribution profile of the biologically active agent during the time period in which sustained release is desired.

[106] In one embodiment, the hydrogel particles comprise cross-linked chitosan.

[107] Chitosan is a linear polyaminosaccharide composed of randomly distributed β-(1-4)-linked D-glucosamine (a deacetylated unit) and randomly distributed N-acetyl-D-glucosamine (a acetylated unit). The degree of deacetylation (%DA) can be determined by NMR spectroscopy and the %DA in commercial chitosan is in the range of 60-100%.

[108] Chitosan is biocompatible, enzymatically biodegradable (for example by hydrolysis by lysozyme) and non-toxic (its degradation products are relatively non-immunogenic and non-carcinogenic).

[109] The amino group in chitosan has a pKa value of approximately 6.5. Therefore, chitosan is positively charged (i.e., the amino groups are protonated) and is soluble in acidic to neutral solutions with a charge density dependent on pH and % DA value. In other words, chitosan can act as a positively charged polyelectrolyte under physiological conditions and thus has appropriate functionality to be cross-linked with a cross-linking agent.

[110] Chitosan suitable for use in hydrogel particles can have a variety of molecular weights. In one embodiment, the chitosan is a low molecular weight chitosan having a molecular weight (MW) of between 40 kDa and 250 kDa. In some embodiments, the MW is preferably in the range of 50-200 kDa and even more preferably 100-180 kDa.

[111] Chitosan can be cross-linked by a variety of compounds or cross-linking agents. Suitable compounds or crosslinking agents may comprise anionic functionalities. The anionic groups are able to interact with the positively charged amino groups in chitosan to produce a cross-linked chitosan that is held together via non-covalent electrostatic bonds.

[112] In one form, the hydrogel particles may comprise chitosan cross-linked with a phosphate compound. The phosphate compound can be selected from those suitably functionalized to promote intermolecular cross-linking between the chitosan chains. The phosphate compound can be suitably chosen to facilitate rapid (spontaneous) cross-linking and thus hydrogel formation when combined with chitosan. Suitable cross-linking phosphate compounds include tripolyphosphate and salts thereof. Commonly known salts of tripolyphosphate include sodium tripolyphosphate and potassium tripolyphosphate. Sodium tripolyphosphate (STPP, "Sodium TriPolyPhosphate", sometimes STP or sodium triphosphate or TPP), with the formula Na5P3O10, is a sodium polyphosphate. It is the sodium salt of triphosphoric acid. In a preference, the crosslinking agent is TPP.

[113] In some embodiments, chitosan can be cross-linked by compounds comprising electrophilic functional groups. A person skilled in the art would recognize that the electrophilic group can react with electrophilic amino groups in chitosan, resulting in the formation of a carbon-carbon covalent bond between the polyaminosaccharide and the crosslinking agent. Suitable crosslinking agents may comprise electrophilic groups selected from ketone, aldehyde and epoxide functional groups. In a preference, the crosslinking compound can be selected from glutaraldehyde and epichlorohydrin.

[114] Cross-linked chitosan useful for hydrogel particles can comprise chitosan and a cross-linking agent in a suitable molar ratio. In some embodiments, it may be desirable to vary the level of crosslinking by adjusting the ratio of moles of chitosan to moles of crosslinking agent. Variation in crosslinking density can be used to modify the physical properties of the hydrogel particles and/or to modulate the release of a bioactive agent from the hydrogel particles.

[115] In some embodiments, the ratio of moles of chitosan to moles of crosslinking agent is from about 12:1 to about 50:1, preferably from about 25:1 to about 2,040:1, more preferably from about from 37:1 to about 1,030:1.

[116] In some embodiments, it may be preferable for the cross-linked chitosan to comprise a relatively low cross-linking ratio (i.e., a low molar concentration of cross-linking agent) because this may result in ionized groups on the chitosan remaining charged and thus available. to participate in non-covalent interactions with a biologically active agent contained in the hydrogel particles.

[117] In other embodiments, it may be preferable for the cross-linked chitosan to comprise a relatively high cross-linking ratio (i.e., excess cross-linking agent relative to the polysaccharide). A high crosslink density can in some embodiments advantageously aid rapid formation of the polymer network and hydrogel and also help improve hydrogel mechanical properties and/or help produce more uniform morphology.

[118] In one embodiment, the hydrogel particles comprise cross-linked alginate.

[119] Alginate is a naturally occurring polysaccharide that is isolated from seaweed and is composed of a block copolymer comprising covalently linked blocks comprising β-D-mannuronate (M) and α-L-guluronate (G) residues ( 1-4)-connected. The proportion and distribution of M and G can determine the physical and chemical properties of the alginate.

[120] Alginate has biocompatibility and low toxicity and can experience crosslinking and gelation under mild conditions. At neutral pH (approximately pH 7), alginate can be anionic and carry a net negative charge.

[121] Alginate suitable for use in hydrogel particles can be of a variety of molecular weights. In some embodiments, the alginate may have a molecular weight in the range of about 40 kDa to 270 kDa. In one embodiment, the alginate may have medium molecular weight, which gives a viscosity of >2,000 cP (mPa.s) at a concentration of 2% in water at 25°C.

[122] Alginate crosslinking can be induced by combining alginate with a positively charged compound or with a positively charged molecule, such as a cation. Alkaline earth metal compounds can provide a source of divalent cations, and thus such compounds can be used as crosslinking agents to crosslink alginate. Alkaline earth metal compounds useful as crosslinking agents may be calcium or magnesium compounds. In one embodiment, the alginate can be electrostatically cross-linked with cations such as calcium (Ca2+) or magnesium (Mg2+) cations.

[123] In a preference, the hydrogel particles comprise alginate cross-linked with calcium cations. Calcium cations can participate in selective ionic bonding with guluronate residues in alginate chains to induce gel formation and cross-linking via noncovalent bonding interactions.

[124] Calcium cations can be provided by a variety of calcium compounds as crosslinking agents. Suitable calcium compounds can be selected from calcium chloride (CaCl2), calcium sulfate (CaSO4) and calcium carbonate (CaCO3). In one preference, the calcium compound is calcium chloride.

[125] The cross-linked alginate useful for hydrogel particles can 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 alginate to cation ratio.

[126] For example, it has been discovered that stable injectable compositions 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 in volumetric ratios of 2:1:0.2 to 2:1:1 and 2:0.5:0.2 to 2:0.5:1 (oil-based : alginate solution : CaCl2 solution).

[127] Crosslinking alginate with a cation can also neutralize the negative charge brought by alginate at neutral pH. This negative charge neutralization allows hydrogel particles comprising alginate to efficiently contain negatively charged biologically active agents, which might, under other conditions, be difficult to obtain due to the potential for unfavorable electrostatic interactions between the negatively charged biologically active agent and the polymer. anionic alginate.

[128] Variations in cation concentration can be used to alter the crosslinking density of alginate-containing hydrogel particles and this can be used to modify the physical properties of the hydrogel particles and/or to modulate the release of a bioactive agent from the particles. of hydrogel. For example, when the cation concentration is insufficient to neutralize all of the negatively charged groups in the alginate, the alginate may therefore have residual anionic groups. Hydrogel particles comprising alginate can thus carry a net negative charge at physiological pH. In some embodiments, this residual net negative charge may advantageously help modulate the absorption and release of a biologically active agent from the hydrogel particle. For example, in the case of a positively charged biologically active agent, a net negative charge carried by a hydrogel particle can help inhibit rapid release over an extended period of time.

[129] In some embodiments, the hydrogel particles may comprise or be composed of a glycosaminoglycan (GAG). Glycosaminoglycans (GAGs) are long unbranched polysaccharides containing a disaccharide repeating unit. The disaccharide repeating unit contains either two modified sugars (N-acetylgalactosamine or N-acetylglucosamine) and generally a uronic acid (glucuronate or iduronate). GAGs can also be esterified by sulfur-containing groups.

[130] In one embodiment, the hydrogel particles may comprise or be composed of at least one glycosaminoglycan (GAG) selected from the group consisting of chondroitin, hyaluronate, keratan, dermatan, heparin and derivatives thereof, such as chondroitin sulfate, hyaluronate sodium, keratan sulfate, dermatan sulfate and heparin sulfate.

[131] In one embodiment, the glycosaminoglycan (GAG) is chondroitin sulfate. Chondroitin sulfate is a sulfated glycosaminoglycan composed of an unbranched polysaccharide chain of alternating sugars (N-acetyl-galactosamine and glucuronic acid). Sulfate is covalently linked to sugar. Some glucuronic acid residues are epimerized into L-iduronic acid, the resulting disaccharide is then called dermatan sulfate. Since the molecule has multiple negative charges at physiological pH (approximately pH 7), a cation is present in chondroitin sulfate salts. Commercial preparations of chondroitin sulfate typically are the sodium salt.

[132] Chondroitin sulfate is a main component of the extracellular matrix and is important in maintaining the structural integrity of the tissue. It is also an important component of cartilage, as part of aggrecan, and provides much of its compressive strength to the highly charged and tightly packed sulfate groups of chondroitin sulfate.

[133] A chondroitin chain can have more than 100 individual sugars, each of which can be sulfated in varying positions and amounts. Each monosaccharide can be left unsulfated, sulfated once, or sulfated twice. Most commonly, the 4- and 6-position hydroxyls of N-acetyl-galactosamine are sulfated, with some chains having the 2-sulfated position of glucuronic acid. Sulfation is mediated by specific sulfotransferases. Sulfation at these different positions imparts specific biological activities to chondroitin glycosaminoglycan chains.

[134] There is some old classification terminology as follows: the sulfation site of chondroitin A sulfate is the 4th carbon of the sugar N-acetylgalactosamine (also known as chondroitin-4-sulfate); chondroitin sulfate B — an old name for dermatan sulfate, which is no longer classified as a form of chondroitin sulfate; the sulfation site of chondroitin sulfate C is carbon 6 of the sugar N-acetyl-galactosamine (also known as chondroitin-6-sulfate); the sulfation sites of chondroitin sulfate D are carbon 2 of glucuronic acid and carbon 6 of the sugar N-acetylgalactosamine (also known as chondroitin-2,6-sulfate); and the sulfation sites of chondroitin sulfate E are carbons 4 and 6 of the sugar N-acetylgalactosamine (also known as chondroitin-4,6-sulfate). All such derivatives are included herein as "chondroitin sulfate" as considered for use in the present invention.

[135] The chondroitin sulfate useful for inclusion in the hydrogel particles can have an average molecular weight of 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 of 40,000 Da. Other molecular weights can, however, be used.

[136] When the hydrogel particles comprise or are comprised of a glycosaminoglycan (GAG) such as chondroitin sulfate, a net charge can be brought by the hydrogel particles at physiological pH and this charge can advantageously help to modulate the release of a biologically active agent. active ingredient contained in the particles. In particular, chondroitin sulfate can interact with a positively charged biologically active agent to provide higher capture and retention of the active agent in the hydrogel particles. Electrostatic interactions between the negatively charged chondroitin sulfate and a positively charged biologically active agent may also help limit premature release of the active agent or improve the long-term release profile of the active agent from the hydrogel particle.

[137] Furthermore, chondroitin sulfate may have immunoregulatory effects and may also interact with polysaccharides such as chitosan via non-covalent bonding interactions to contribute to polysaccharide cross-linking.

[138] In some embodiments, the hydrogel particles may comprise or be composed of a cross-linked glycosaminoglycan.

[139] Sulfated glycosaminoglycans such as chondroitin sulfate can be cross-linked with a suitable cross-linking agent. In one embodiment, the crosslinking agent may be a cation, such as a divalent cation from alkaline earth metal compounds. In one embodiment, chondroitin sulfate can be cross-linked with cations such as calcium (Ca2+) or magnesium (Mg2+).

[140] In one embodiment, the hydrogel particles comprise chondroitin sulfate cross-linked with calcium cations. Calcium cations can be provided by a variety of calcium compounds as crosslinking agents. Suitable calcium compounds can be selected from calcium chloride (CaCl2), calcium sulfate (CaSO4) and calcium carbonate (CaCO3). In one preference, the calcium compound is calcium chloride.

[141] The cross-linked chondroitin sulfate useful for hydrogel particles can comprise chondroitin sulfate and a cation (such as a calcium cation) in a suitable molar ratio. In some embodiments, the ratio of moles of chondroitin sulfate to moles of calcium can be from 4:1 to 1:10.

[142] Similar to alginate discussed above, variations in cation concentration can be used to alter the crosslinking density and charge of hydrogel particles containing crosslinked glycosaminoglycan. For example, when GAG is sulfated GAG (e.g. chondroitin sulfate), the cation concentration may be insufficient to neutralize all of the negatively charged groups on GAG and thus GAG may carry a net negative charge at physiological pH due to to residual anionic groups. This residual net negative charge can advantageously help modulate the absorption, retention of a positively charged biologically active agent from the hydrogel particle according to the mechanisms discussed above.

[143] In some embodiments, the hydrogel particles of the injectable composition of the invention may comprise a mixture of polysaccharides, such as a mixture of two different glycosaminoglycans, or a mixture of a glycosaminoglycan and at least one other polysaccharide. At least one of the polysaccharides is cross-linked. In some embodiments, the cross-linked polysaccharide may be cross-linked chitosan, cross-linked alginate, or cross-linked chondroitin sulfate, as described herein.

[144] 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 sulfate. In such embodiments, negatively charged sulfate groups in chondroitin sulfate can interact with the positive amino groups present in chitosan via electrostatic interactions to thus form a cross-linked macromolecule. The cross-linked macromolecule can be considered a hybrid of the cross-linked polysaccharide that is composed of a mixture of chitosan and chondroitin sulfate. The cross-linked macromolecule, which is composed of at least two electrostatically linked polysaccharides, therefore forms the cross-linked polymeric matrix of the hydrogel particles. Other mixtures of oppositely charged polysaccharides are considered, such as a mixture of chitosan and alginate.

[145] In some embodiments, a charged polysaccharide that is capable of interacting with another oppositely charged polysaccharide can be considered here as a crosslinking agent.

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

[147] In particular, glycosaminoglycan (GAG) can allow hydrogel particles to become flexible, with excellent properties similar to plastic properties. This can be beneficial when the hydrogel particles also comprise an aqueous-insoluble alkaline earth metal phosphate as described below, because the inclusion of a glycosaminoglycan (GAG) in the particles can allow the mechanical properties of the particles to be adjusted so as to improve the structural stability and in vivo performance. Changes in hydrogel particle properties may be due to glycosaminoglycan acting to prevent crystallization and growth of the alkaline earth metal phosphate, thereby reducing particle rigidity.

[148] In one embodiment, hydrogel particles comprising a glycosaminoglycan can comprise chondroitin sulfate in an amount of up to 2% (w/v). In some embodiments, the hydrogel particles may comprise chondroitin sulfate in an amount of up to 1% (w/v).

[149] When the hydrogel particles comprise a cross-linked macromolecule composed of electrostatically linked chondroitin sulfate and chitosan, the amount of chondroitin sulfate and chitosan can preferably be in a weight ratio of 1:2. An injectable formulation containing such hydrogel particles can be prepared by emulsifying an aqueous solution containing 2% (w/v) chitosan with an oil, to which a second aqueous solution containing 1% (w/v) chondroitin sulfate is added. added under continuous shear to obtain hydrogel microparticles in a chondroitin sulfate:chitosan weight ratio of 1:2 in a water-in-oil emulsion.

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

[151] In one form, the hydrogel particles may additionally comprise a biocompatible aqueous-insoluble alkaline earth metal phosphate and/or a biocompatible proteoglycan.

[152] Aqueous-insoluble alkaline earth metal phosphates can be included in hydrogel particles to modify the physical or mechanical properties of the hydrogel particles. For example, alkaline earth metal phosphate insoluble in aqueous media can be used to increase particle rigidity.

[153] In some embodiments, the hydrogel particles may comprise calcium and magnesium phosphates insoluble in aqueous media. Doped calcium phosphates, such as calcium phosphates doped with Mg2+, Zn2+, Na+, CO32- and SiO44-, are also considered.

[154] In one embodiment, the alkaline earth metal phosphate insoluble in aqueous media is apatite.

[155] Apatite is a group of phosphate-based minerals and includes fluoroapatite, Ca5(PO4)F3; chloroapatite, Ca5(PO4)3Cl; bromoapatite, Ca5(PO4)3Br and hydroxyapatite, Ca5(PO4)3(OH) (which are also commonly written as Ca10(PO4)6(OH, F, Cl, Br)2 to denote that the crystalline unit cell comprises two molecules). Hydroxyapatite crystallizes in the hexagonal crystal system. It has a specific gravity 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.

[156] In a preferred embodiment the hydrogel particles may additionally comprise hydroxyapatite.

[157] In one embodiment, the hydrogel particles may comprise hydroxyapatite in an amount of up to 0.1% (1 mg/mL).

[158] In one embodiment, the hydrogel particles may comprise hydroxyapatite and chitosan in a ratio between the weight of hydroxyapatite and the weight of chitosan of 3:20, more preferably in a ratio between the weight of hydroxyapatite and the weight of chitosan. of 1:20. The latter ratio can be achieved by using a 2% aqueous solution of chitosan containing hydroxyapatite at a concentration of 1 mg/mL in preparing the injectable composition of the invention.

[159] Hydroxyapatite may also be capable of modifying the crosslinking reactions used to form the crosslinked polymer of hydrogel particles through charge interactions with one or more components of the crosslinked polymer. For example, excess PO43- ions from hydroxyapatite at slightly acidic pH can modify the ability of chitosan to interact with a crosslinking agent.

[160] Proteoglycans are a specific group of compounds that have at least one glycosaminoglycan chain attached to a protein. Such compounds may comprise multiple negative charges at physiological pH (approximately pH 7). The hydrogel particles may comprise at least one proteoglycan. Mixtures of two or more proteoglycans are also considered. An example of a proteoglycan is aggrecan.

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

[162] The hydrogel particles in the injectable composition of the invention may be uncoated hydrogel particles or coated hydrogel particles. The Injectable Composition May Comprise A Mixture Of Coated And Uncoated Hydrogel Particles.

[163] In one embodiment, the injectable composition comprises one or more coated hydrogel particles. In this way, the hydrogel particles as described herein can be coated. The injectable composition of the invention may therefore comprise at least one hydrogel particle coated in the aqueous phase. In some embodiments, the injectable composition of the invention may comprise a plurality of hydrogel particles coated in the aqueous phase.

[164] The coated hydrogel particles may comprise an internal core-forming hydrogel component and an external material component that coats at least a portion of the core. The outer component can be considered as a coating for the inner core.

[165] The coating of a coated hydrogel particle may be a layer other than the material that coats at least a portion of a hydrogel core. In some embodiments, the coating may be a film that encapsulates and contains the hydrogel core.

[166] When present, the coating generally forms part of the particle per se and is attached to the inner core of the particle. The attachment of the coating to the core can be via physical or chemical means.

[167] The presence of a coating can be determined using various optical, imaging or spectroscopic techniques. For example, the coating can be visually observed microscopically.

[168] It is considered 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 invention in accordance with methods described herein. The separated component material preferentially localizes to the surface to form the coating while the remaining components of the mixture can form the core of the hydrogel particle.

[169] As an example, a hydrogel particle coated with a polysaccharide coating can be formed when a cross-linking agent diffuses from the core microhydrogel to the core surface and reacts with a polysaccharide in solution in the environment to obtain a layer of cross-linked gel around the core.

[170] In another example, a coated hydrogel particle can be formed when two oppositely charged polymers interact electrostatically to form an interpenetrated network (IPN), where one or more of the polymers are located in the core of the hydrogel and the another polymer is located in solution in the microhydrogel environment.

[171] In one set of embodiments, the coating can be attached to a microhydrogel particle core by chemical means. Exemplary chemical means can be chemical bonds formed between the coating and the core of the hydrogel particle. In some embodiments, at least a portion of the coating is linked to at least a portion of the polymeric material component via covalent or non-covalent bonds. Non-covalent bonds can be hydrogen bonds or electrostatic bonds.

[172] In another set of embodiments, the coating can be attached to the core of the microhydrogel particle via physical means. In some embodiments, at least a portion of the coating is physically intertwined with at least a portion of the polymeric material component in the core of the hydrogel particle on a molecular scale. In some embodiments, a portion of the coating is physically interwoven with at least a portion of the hydrogel core polymeric material. The interlocking region between the shell and the core of the hydrogel particle may resemble an interpenetrating polymer network.

[173] The coating can advantageously help 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, consequently, into the surrounding environment for internal release. alive. In some embodiments, the coating may help to reduce the rate of release of the biologically active agent from the hydrogel particle and thereby help to delay premature release of the active agent, or help to promote prolonged release of the active agent over a period of time. of time.

[174] Control over the passage of the biologically active agent can be accomplished through numerous mechanisms, including by the coating per se, having adjustable porosity, or by the coating carrying a liquid charge.

[175] The coating of a coated hydrogel particle can be additionally cross-linked or non-cross-linked. The degree of cross-linking (if any) may provide another mechanism for controlling the passage of a biologically active agent from the hydrogel particle.

[176] A coating of a coated hydrogel particle may comprise or be composed of a biocompatible material.

[177] In some embodiments, the coating may comprise a biocompatible polymeric material. Such polymeric materials can be biocompatible, hydrophilic and susceptible to aqueous solvation. A variety of biocompatible polymers may be suitable for coating.

[178] In one embodiment, the coating may comprise a polysaccharide. The polysaccharide can be selected from any of the polysaccharides described herein. In one form, the coating comprises a polysaccharide selected from the group consisting of chitosan, alginate, hyaluronic acid, chondroitin sulfate, cellulose, dermatan sulfate, keratan sulfate and heparin sulfate and also salts thereof and derivatives thereof. Such polysaccharides, salts and derivatives thereof are described herein.

[179] In some embodiments it may 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 core of the hydrogel particle comprises a cross-linked chitosan, the coating may comprise alginate or a glycosaminoglycan such as chondroitin sulfate. Similarly, when the hydrogel particle core comprises a cross-linked alginate or a cross-linked chondroitin sulfate, the coating on the hydrogel particle core may comprise chitosan. Other polysaccharide combinations are also considered.

[180] At physiological pH (approximately pH 7), polysaccharides can carry a net charge, which depending on the composition of the polysaccharide, can be positive or negative. Consequently, a coating comprising such polysaccharides can be charged at physiological pH. The charged coating can influence the rate of release of a biologically active agent from the hydrogel particle, particularly if the active agent is also charged. For example, a coating having a net negative charge may interact electrostatically with a positively charged biologically active agent and, in this manner, may help to modulate the passage of the active agent from the hydrogel particle into the aqueous liquid of the aqueous phase (i.e., syneresis).

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

[182] When chitosan is present in the coating, the core of the hydrogel particle can and preferably will comprise a different polysaccharide, which is not chitosan. In a preference, the core of the hydrogel particle comprises a negatively charged polysaccharide such as alginate or a glycosaminoglycan such as chondroitin sulfate. The negatively charged polysaccharide can help in attaching the positively charged coating to the core component of the hydrogel particle.

[183] In one embodiment, the coating comprises a polysaccharide that is negatively charged at physiological pH. The negatively charged polysaccharide can impart a negative charge to the coating. Exemplary negatively charged polysaccharides can be selected from alginate and sulfated glycosaminoglycans (sulfated GAGs). Sulfated GAGs can be chondroitin sulfate, keratan sulfate, dermatan sulfate and heparin sulfate. When the coating comprises a negatively charged polysaccharide, in some embodiments it may be preferable for the core of the hydrogel particle to comprise a positively charged polysaccharide. In one preference, the core of the hydrogel particle comprises chitosan. The positively charged polysaccharide can help in attaching the negatively charged coating to the core of the hydrogel particle.

[184] A coating of a coated hydrogel particle can be cross-linked or non-cross-linked. A cross-linked coating may comprise a cross-linked polysaccharide, while a non-cross-linked coating may comprise a polysaccharide that is uncross-linked.

[185] Cross-linked and non-cross-linked coatings can be attached to a hydrogel particle core via physical or chemical means as described herein.

[186] Cross-linked polysaccharides suitable for use in a cross-linked coating may comprise a polysaccharide as described herein cross-linked with a suitable cross-linking agent. A skilled person would be able to select a suitable crosslinking agent for a selected polysaccharide. Exemplary polysaccharides include chitosan, alginate and sulfated GAGs such as chondroitin sulfate and suitable crosslinking agents for these polysaccharides are described herein.

[187] In some embodiments, when the coating comprises a charged polysaccharide (either positively or negatively charged), the core of the hydrogel particle may comprise a charged compound that is capable of interacting with the charged polysaccharide in the coating for the purpose of facilitating fixing the cladding to the core. The loaded compound can be selected to be complementary to the polysaccharide loaded in the coating. For example, a positively charged compound may be incorporated into the core of the hydrogel particle when a negatively charged polysaccharide is in the shell and vice versa.

[188] 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 fix the chitosan to the hydrogel through non-covalent electrostatic bonds. The phosphate compound can be tripolyphosphate and salts thereof, such as sodium tripolyphosphate (TPP) and potassium tripolyphosphate. In one preference, the phosphate compound is TPP.

[189] In one embodiment, the coating comprises alginate or a sulfated glycosaminoglycan such as chondroitin sulfate. In such embodiments, the core of the hydrogel particle may comprise a cation, preferably a divalent cation. Exemplary cations can be calcium (Ca2+) or magnesium (Mg2+) cations. The cation in the core of the hydrogel particle can interact with anionic groups such as carboxylic acid or sulfate groups present in the polysaccharide for the purpose of attaching the polysaccharide to the core.

[190] When a charged compound is used to facilitate attachment of a coating to the core of the hydrogel particle, it may not be necessary for the polymer (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. Under these circumstances, the anchoring of the coating to the core of the hydrogel particle is accomplished via the charged compound contained in the core.

[191] Additionally, when the hydrogel particle comprises a charged compound (such as a phosphate compound or a divalent cation), the charged compound can 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 cross-linking agent for the polysaccharide and, in this way, may help to promote the formation of a cross-linked polysaccharide in the coating. A cross-linked coating may be thicker than a non-cross-linked coating and thus could provide an additional possibility to modulate the sustained release of a biologically active agent from the coated hydrogel particle over a period of time.

[192] Cross-linked polysaccharides useful for a coating on a coated hydrogel particle can comprise polysaccharide and a cross-linking agent in a suitable molar ratio. In some embodiments, it may be desirable to vary the level of crosslinking by adjusting the ratio of moles of polysaccharide to moles of crosslinking agent. Variation in crosslink density can be used to modify the physical properties of the coating, such as the porosity or net charge of the coating. As a result, this can help modulate the release of a bioactive agent from the hydrogel particle into the aqueous liquid portion of the aqueous phase of the injectable composition.

[193] In some embodiments, it may be preferable for a cross-linked polysaccharide in the coating to comprise a relatively low cross-linking ratio (i.e., a low molar concentration of cross-linking agent) because this may result in ionized groups on the remaining charged polysaccharide at physiological pH and , thus available to participate in non-covalent interactions with a biologically active agent contained in the hydrogel particles. Noncovalent interactions between the biologically active agent and the coating can also help modulate the release profile of the biologically active agent from the hydrogel particle over a period of time.

[194] In some embodiments, the ratio of moles of cross-linking agent to moles of polysaccharide in a coating comprising a cross-linked 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.

[195] In some embodiments, the coating of a coated hydrogel particle may comprise an amphiphilic compound. An amphiphilic compound in the coating can help stabilize the water-in-oil emulsion or can help modulate the release of the biologically active agent from the injectable composition of the invention. In one embodiment, the amphiphilic compound is lecithin.

[196] In some embodiments, the coating of a coated hydrogel particle is porous. Porous coatings can be formed by incorporating a porogen during the manufacture of the injectable composition of the invention or by varying the crosslink density in the case of a crosslinked coating.

[197] Porogens of different sizes can be used to produce coatings of different porosity. An exemplary porogen is poly(ethylene glycol) (PEG). PEG compounds of different molecular weights can produce different sized passages (pores) in the coating. Exemplary poly(ethylene glycol) may have a molecular weight in a range of about 200 Da to 100,000 Da.

[198] The injectable composition of the invention comprises at least one biologically active agent, which is desired to be administered to an individual.

[199] As used herein, the term "biologically active agent" encompasses any molecule of synthetic or natural origin, which is capable of producing a desired physiological effect in vivo. For example, a biologically active agent may be a pharmaceutical compound or a vaccine having use in treating or preventing a disease or condition, especially one in which immediate dose delivery is desired followed by prolonged delivery over a period of time. an individual.

[200] According to 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 hydrogel particles of the aqueous phase. Consequently, it can be considered that the 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 hydrogel particles facilitates the ability of the injectable composition to provide rapid and prolonged distribution of the active agent, as further described below.

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

[202] The injectable composition of the invention can comprise a variety of biologically active agents.

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

[204] The biologically active agent can be selected from non-limiting classes of active agents including hormones, antimicrobials, therapeutic antibodies, cytokines, fusion proteins, antigens, viruses, bacteria, bacterial 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.

[205] Biologically active agents can bring 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., be neutral).

[206] When the injectable composition comprises two or more biologically active agents, the biologically active agents may belong to the same class of active agent or to different classes of active agent. Each biologically active agent can also be independently selected at each occurrence.

[207] Hormones can be peptide hormones such as insulin and somatotropin, or steroid hormones such as corticosteroids, estrogens, progestogens and androgens.

[208] A "peptide hormone" is a peptide or protein that has an effect on an individual's endocrine system.

[209] A specific example of a peptide hormone that can be contained in and released by the injectable composition of the invention is somatotropin. Somatotropin stimulates growth, cell reproduction, and cell regeneration in humans and nonhuman animals and is important in growth and development.

[210] Therapeutic antibodies can be infliximab, adalimumab, nituximab, alemtuzumab, daclizumab, or basiliximab.

[211] Fusion proteins can be etanercept.

[212] An "antigen" is a compound that, when introduced into a human or non-human animal, will result in the formation of antibodies against the antigen and cell-mediated immunity.

[213] Antigens are commercially available or can be prepared using known procedures and known techniques. Representative antigens may include, but are not limited to, natural, recombinant or synthetic products derived from viruses, bacteria, fungi, parasites and other infectious agents including prions. Examples of antigens also include human antigens that could be desirable for use in prophylactic or therapeutic vaccines, for example, that are involved in or relevant to autoimmune diseases, in particular autoantigens; hormones; tumor antigens; and allergens. Microbial products (e.g., viral or bacterial) may be components that the organism produces or can be induced to produce, for example, by enzymatic cleavage, or they may be components of the organism that have been produced by recombinant DNA techniques that are well known to those persons commonly skilled in the art.

[214] Some specific examples of antigens that may be contained in and released by the injectable composition of the invention are TSOL18 antigen, Bm86 antigen, H. contortus antigen, Old World screwworm fly (Chrysomya bezziana) antigens and H. blue tongue.

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

[216] A specific vaccine that can be contained in or be contained in anxiety disorder by the injectable composition of the invention is the luteinizing hormone-releasing hormone (LHRH) vaccine. The vaccine can be used to suppress reproduction in animals and form the basis for immunocastration and immunocontraception.

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

[218] In some embodiments, the injectable composition of the invention comprises an adjuvant. The term "adjuvant", as used herein, refers to a compound or substance that enhances an individual's physiological response to a biologically active agent. For example, in the case of an antigen, an adjuvant may act to intensify an individual's immune response to the antigen by increasing antibodies and, thus, to increase the longevity of the immune response.

[219] An adjuvant may therefore help to promote a more effective physiological response to a biologically active agent in an individual, compared to administration of the biologically active agent alone or without the adjuvant.

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

[221] The adjuvant may 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 injectable composition, the oily phase and aqueous phase of the emulsion may each comprise an adjuvant.

[222] 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 preferably hydrophilic and may be water soluble.

[223] 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 chemical structure of the aqueous phase hydrogel particles. For example, chitosan may have adjuvant properties and, therefore, an adjuvant may be introduced into the injectable composition through the use of hydrogel particles comprising cross-linked chitosan in the aqueous phase.

[224] Hydrophilic adjuvants that can be incorporated into the aqueous phase of the water-in-oil emulsion can be selected from the group consisting of alum, the water-soluble extract of Mycobacterium smegmatis, N-acetyl-muramyl-1-alanyl-d- synthetic isoglutamine, monoacyl lipopeptides and ligands for Toll-like receptors. Such adjuvants may be incorporated into the aqueous liquid and/or within the hydrogel particles of the aqueous phase.

[225] In other embodiments of the injectable composition, the oily 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.

[226] In some embodiments, the oil per se may be an adjuvant and, thus, the oily phase comprises an adjuvant oil. The use of an adjuvant oil may be desirable because it avoids the need to incorporate a separate adjuvant compound into the injectable composition of the invention. Examples of adjuvant oils are described herein.

[227] In alternative embodiments, the oil phase may comprise a lipophilic adjuvant dissolved or suspended in a non-adjuvant (passive) oil.

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

[229] A skilled person would recognize that the selection of a specific adjuvant could depend on the biologically active agent to be delivered, the disease or disorder to be treated by the active agent and the desired release profile for the active agent.

[230] Some examples of specific adjuvants include Incomplete Freund's Adjuvant (IFA), Adjuvant 65 (containing peanut oil, mannid monooleate and aluminum monostearate), oily emulsions, Ribi's adjuvant, the Pluronic polyols, polyamines, Avridine, Quil A, saponin, MPL, QS-21, mineral gels and aluminum salts such as aluminum hydroxide and aluminum 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 matrix.

[231] The injectable composition of the invention may optionally comprise one or more additional components. Additional components may be, for example, salts or ions for adjusting the pH or ionic strength of the aqueous phase, surfactants, emulsifiers and the like. When present, additional 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 in the aqueous liquid of the aqueous phase. A skilled person would understand that additional components may be dissolved or suspended in the oil phase or the aqueous phase, depending on the lipophilicity or hydrophilicity of a specific component.

[232] In one embodiment, the injectable composition of the invention may comprise at least one surfactant. Surfactants can help reduce or prevent complete phase separation of the water and oil phases and thus help produce a more stable water-in-oil emulsion.

[233] Surfactants may be present in an amount of up to 5% by weight of the injectable composition. In some embodiments, the surfactant may be present in an amount of up to 1% by weight of the injectable composition.

[234] The surfactants used in the injectable composition of the invention may have a low hydrophilic-lipophilic balance (HLB) value. For example, the surfactant may have an HLB of 6 or less. Additionally, high HLB ratio surfactants can be used in combination with low HLB surfactants to produce low HLB surfactant mixture to optimize the stability of the water-in-oil emulsion.

[235] Various pharmaceutically acceptable surfactants are known to a person skilled in the art. Pharmaceutically acceptable surfactants can be anionic, cationic, zwitterionic or nonionic.

[236] In one set of embodiments, the injectable composition of the invention additionally comprises a nonionic surfactant. A variety of nonionic surfactants can be used. Exemplary non-ionic surfactants can be selected from sorbitan esters, polysorbates and poloxamers.

[237] Preferred surfactants can 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 monolaurate, sorbitan monopalmitate, sorbitan sesquioleate, sorbitan trioleate, sorbitan triestearate and surfactants based on saccharide and polysaccharide.

[238] The injectable composition of the invention can be prepared using equipment and techniques suitable for forming emulsion compositions, in particular, water-in-oil emulsions.

[239] In one aspect, the present invention provides a process for preparing an injectable composition for rapid and prolonged 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 composition under conditions that allow the first hydrogel-forming component to react with the second hydrogel-forming component to form a plurality of hydrogel particles in situ and thereby produce an injectable composition comprising an emulsion of water-in-oil 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 the aqueous liquid of the phase water-in-oil emulsion.

[240] In forming the injectable composition, the first hydrogel-forming component is one selected from a polymer and a cross-linking agent while the second hydrogel-forming component is the other selected from a polymer and a cross-linking agent. That is, when the first selected hydrogel-forming component is a polymer, then the second hydrogel-forming component is a crosslinking agent and vice versa. Suitable polymers and crosslinking agents for forming hydrogel particles have been described herein.

[241] The aqueous compositions and the lipophilic composition can be combined under shear to form the hydrogel particles and the water-in-oil emulsion.

[242] In one embodiment, the injectable composition of the invention can be prepared initially by combining a first aqueous composition comprising a polysaccharide in an aqueous solvent with a lipophilic composition comprising a physiologically acceptable oil and a surfactant and emulsifying the first aqueous composition into the composition lipophilic under shear. A second aqueous composition comprising a crosslinking agent in an aqueous solvent is then combined with the initial emulsified mixture under continuous stirring or continuous shear. The cross-linking agent reacts with the polysaccharide and spontaneously cross-links the polysaccharide to result in colloidal cross-linked hydrogel moieties 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. Cross-linking of the polysaccharide and the formation of hydrogel particles containing the cross-linked polysaccharide occur without the need for additional curing apparatus or mechanisms (e.g., by UV (ultraviolet radiation), IR (infrared radiation), heat). Cross-linking and hydrogel particle formation can occur rapidly.

[243] Advantageously, it has been found that the resulting injectable composition is stable, with the water-in-oil emulsion and the dispersion of hydrogel particles in the emulsion remaining stable for numerous weeks. In general, a stable emulsion would not exhibit phase separation, aggregation or precipitation of the components of the composition over a desired period of time. In a number of embodiments, the injectable composition of the invention may be stable for more than 6 months at room temperature.

[244] The order in which the aqueous compositions containing the crosslinking agent and the polysaccharide are mixed with the lipophilic composition can be reversed. That is, the first aqueous composition may comprise a crosslinking agent (e.g., TPP) and the lipophilic composition comprising the oil may initially be emulsified with the aqueous composition containing the crosslinking agent. Subsequently, a second aqueous composition comprising a polysaccharide (e.g., chitosan) may then be added to this initial emulsified composition to produce the injectable composition of the invention.

[245] In some embodiments, when the hydrogel particles comprise a mixture of polysaccharides, the injectable composition can be prepared initially by combining a first aqueous composition comprising a first polysaccharide in an aqueous solvent with a lipophilic composition comprising a physiologically acceptable oil and emulsification of the first aqueous composition into the lipophilic composition under shear. A second aqueous composition comprising a second polysaccharide in an aqueous solvent is then combined with the initial emulsified mixture under continuous stirring or continuous shear. Preferably, the second polysaccharide is of opposite charge to the charge of the first polysaccharide. The first and second polysaccharides then cross-link via intermolecular electrostatic interactions to result in colloidal cross-linked hydrogel moieties being formed in situ.

[246] In some embodiments, an aqueous composition used in preparing the injectable composition may comprise a mixture of hydrogel-forming components of similar electrostatic charge. For example, an aqueous composition may comprise a mixture of negatively charged polysaccharides such as alginate and chondroitin sulfate. If desired, such aqueous compositions may also include an ionic crosslinking agent with the same charge, such as TPP. An initial emulsion containing this aqueous composition can be combined with another aqueous composition containing an oppositely charged hydrogel-forming component, for example, a positively charged polysaccharide such as chitosan. If desired, the chitosan constituent may comprise other positively charged polymers and/or other positively charged crosslinking species. The combination of these different aqueous compositions containing oppositely charged components in an emulsion can then allow the formation of an injectable composition comprising hydrogel microparticles having a combination of different polysaccharides that are electrostatically cross-linked.

[247] During production of the injectable composition, the formation of the hydrogel particle by cross-linking a hydrogel-forming polymer in the presence of the biologically active agent may involve two processes: formation of the cross-linked hydrogel network and compression of the network driven by increased cross-linking density, which continues until equilibrium is reached between the osmotic and elastic forces of the polymer chains. The aqueous liquid phase expelled by the reduction in hydrogel volume during cross-linking, called syneresis liquid, contains free bioactive species not physically bound or encapsulated by the hydrogel polymer chains, that is, those that exceed the binding capacity of the cross-linked chains. Notably, the binding capacity of cross-linked polymer chains may be lower than that of chains before cross-linking due to entropic conformational changes imposed on the component arising from compression during network formation. The physical balance established between (i) the bioactive species bound to the hydrogel polymer chains and (ii) the free species in the aqueous liquid is both external to the hydrogel network in the aqueous phase of the emulsion and internal to the swollen hydrogel itself, is characterized by the respective system binding coefficient.

[248] In embodiments in which the injectable composition comprises coated hydrogel particles, the first aqueous composition and the second aqueous composition used in preparing the injectable composition may optionally each additionally comprise a coating-forming component. In one embodiment, the second aqueous composition comprises a coating-forming component. In another embodiment, both the first aqueous composition and the second aqueous composition comprise coating-forming components.

[249] When the first aqueous composition is emulsified with the lipophilic composition and the resulting emulsion is 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 partially coats 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.

[250] By way of example, the injectable composition of the invention can be prepared initially by 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 composition under shear. A second aqueous composition comprising a second hydrogel-forming component and a coating-forming component in an aqueous solvent is then combined with the initial emulsified mixture under continuous stirring or continuous shear. The first hydrogel-forming component reacts with the second hydrogel-forming component to result in colloidal hydrogel portions being formed in situ while the coating-forming component forms a coating on the colloidal hydrogel. The coating is located on the surface of the hydrogel particle. A portion of the coating may physically interlock with the core of the underlying hydrogel particle to secure the coating to the hydrogel core. Alternatively, the coating may be attached (e.g., chemically fixed) to the underlying 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.

[251] As described herein, in some embodiments a cross-linked coating can be formed on the hydrogel particles. In such embodiments, a coating-forming component may be incorporated into each of the aqueous compositions used to form the injectable composition of the invention. The coating-forming components react with each other to form a cross-linked coating.

[252] As an example, the injectable composition of the invention can be prepared initially by combining a first aqueous composition comprising a first hydrogel-forming component and a first coating-forming component with a lipophilic composition comprising a physiologically acceptable oil and a surfactant. The mixture is then emulsified under shear. A second aqueous composition comprising a second hydrogel-forming component and a second coating-forming component in an aqueous solvent is then combined with the initial emulsified mixture under continuous stirring or continuous shear. The first hydrogel-forming component reacts with the second hydrogel-forming component to form colloidal hydrogel moieties in situ. Meanwhile, the first coating-forming component reacts with the second coating-forming component to form a cross-linked coating in situ on the colloidal hydrogel.

[253] If it is desired that a cross-linked coating be formed, it is preferable that the components in each of the first aqueous composition and the second aqueous composition do not react with each other. More precisely, it is preferred that the reaction between the components in these compositions occurs only after the first aqueous composition and the second aqueous composition are combined and emulsified with the lipophilic composition.

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

[255] In one embodiment, the lipophilic composition comprises an adjuvant. Such embodiments would result in the injectable composition of the invention comprising an adjuvant in the oily phase of the water-in-oil emulsion.

[256] In one embodiment, the lipophilic composition comprises a physiologically acceptable adjuvant oil. Examples of adjuvant oils are described herein.

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

[258] In some embodiments an adjuvant may be provided by at least one adjuvant compound or an adjuvant substance being dissolved or dispersed in the first aqueous composition and/or the second aqueous composition used to prepare the injectable composition. Such adjuvant substances are, in general, hydrophilic. Examples of hydrophilic adjuvant compounds are described herein.

[259] As an illustration with reference to one or more of the embodiments described herein, a first aqueous composition comprising chitosan can be combined with a lipophilic composition comprising an adjuvant oil (e.g., Montanide ISA61) under stirring or shearing. A second aqueous composition comprising sodium tripolyphosphate (TPP) is then added to the resulting emulsified composition for the purpose of cross-linking the chitosan. The addition of TPP to the initial composition occurs by dripping, under continuous shear. A uniform water-in-oil emulsion formulation is then formed which comprises colloidal portions of hydrogel composed of chitosan cross-linked in the aqueous phase of the emulsion.

[260] As an illustration of another embodiment described herein, when coated hydrogel particles are desired, a first aqueous composition comprising alginate and sodium tripolyphosphate (TPP) can be combined with a lipophilic composition comprising an oil adjuvant (e.g., Montanide ISA61) under agitation or shear. A second aqueous 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 cross-link the alginate and form colloidal moieties of alginate-Ca2+ hydrogel. Meanwhile, chitosan is cross-linked with TPP to produce a cross-linked chitosan coating on colloidal hydrogel. The resulting composition is a water-in-oil emulsion formulation comprising colloidal hydrogel portions coated in the dispersed aqueous phase. The coated hydrogel is composed of a cross-linked alginate core and a cross-linked chitosan shell.

[261] When preparing the injectable composition of the invention, the biologically active agent can be combined with the first aqueous composition and/or the second aqueous composition described here. In a 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 because this may favor more efficient distribution of the active agent in the emulsion and consequently, in the final injectable composition.

[262] The first aqueous composition, the lipophilic composition and the second aqueous composition can be combined in any suitable volumetric ratio. In a specific embodiment, the first aqueous composition comprises a cross-linking agent and a biologically active agent, the lipophilic composition comprises an adjuvant oil, and the second aqueous solution comprises a polysaccharide and the volumetric ratio of the first aqueous composition: lipophilic composition: second aqueous composition is 0.5:1.2:1.8.

[263] During preparation of the injectable composition, a portion of the biologically active agent becomes encapsulated in the hydrogel particles that are formed in situ. Another portion of the biologically active agent is not encapsulated in the hydrogel particles but remains in the aqueous solvent, which then forms the aqueous liquid of the aqueous phase of the water-in-oil emulsion.

[264] Since the hydrogel particles comprising the biologically active agent are prepared ex vivo, product reproducibility can be improved.

[265] If it is desired for other components to be present in the injectable composition, such components can be incorporated into one or more of the first aqueous composition, the second aqueous composition and/or the lipophilic composition used to prepare the injectable composition. For example, in one embodiment, an aqueous-insoluble alkaline earth metal phosphate such as hydroxyapatite can be incorporated into the aqueous composition comprising the polysaccharide, while a biocompatible proteoglycan can be incorporated into the aqueous composition comprising the crosslinking agent.

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

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

[268] A porogen 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 may be preferable for the porogen to be a hydrophilic substance.

[269] The reaction between a polymer and a crosslinking agent to form hydrogel particles can be carried out in the presence of a porogen. The porogen does not participate in the hydrogel formation reaction and, therefore, hydrogel material forms around the porogen. Consequently, the porogen acts as a template for the pores in the hydrogel particles. After the hydrogel particles are formed, the porogen can leach from the particles and be removed, thus leaving pores in the hydrogel particles.

[270] A variety of porogens can be used to form porous hydrogel particles. An example of a suitable porogen is poly(ethylene glycol) (PEG). A person skilled in the art would understand that PEG is a hydrophilic polymer that is compatible with an aqueous solvent.

[271] Poly(ethylene glycol) is useful as a porogen and can have a molecular weight in a range of about 200 Da to 100,000 Da. In one embodiment, the poly(ethylene glycol) has a molecular weight of about 35,000 Da .

[272] Porogens for forming porous hydrogel particles can be used in a suitable solvent. The amount of porogen may be dependent on the type of porogen and the degree of porosity desired for the hydrogel particles. In one set of embodiments, a porogen may be present in at least one of the first aqueous composition and the second aqueous composition in an amount of up to 2% by weight.

[273] Furthermore, surfactants can be used in the process and when used, suitable surfactants can be incorporated into one or more of the first aqueous composition, the second aqueous composition and/or the lipophilic composition used to prepare the injectable composition. In one embodiment, the lipophilic composition used to form the oily phase of the water-in-oil emulsion of the injectable composition comprises a surfactant.

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

[275] The injectable composition of the embodiments described herein, which can be prepared according to the processes described herein, is capable of being administered to an individual for delivery of the biologically active agent. It is adequately administered in a single injection. Advantageously, the injectable composition can be used as prepared, without the need for additional isolation, purification or formulation steps to be carried out. For example, the present invention avoids the need to isolate hydrogel particles and to reformulate the isolated hydrogel particles in a pharmaceutically acceptable vehicle or carrier prior to administering them to a subject. However, a skilled person would recognize that processes such as sterilization may be carried out in relation to the injectable composition to ensure that it complies with relevant safety or regulatory requirements.

[276] The injectable composition of the embodiments described herein is of a viscosity that is suitable for administration by injection to an individual. It has been discovered that the injectable composition can exhibit shear fluidizing behavior when shear is applied, such as when the composition is injected through the lumen of a needle. Shear fluidization that occurs under applied shear stress can reduce the viscosity of the composition.

[277] The release profile of a biologically active agent from the injectable composition includes a short-acting (i.e., rapid) portion and a long-acting (i.e., prolonged) portion. In some embodiments, the release profile of the biologically active agent can be controlled or modulated by components of the injectable composition that influence the passage of the active agent through various mechanisms as described herein.

[278] The short-term release profile allows a primary dose of the biologically active agent to rapidly relieve anxiety disorder. It is believed that the primary dose is provided by that portion of the biologically active agent that is not contained in the hydrogel particles but, rather, is present in the aqueous liquid of the aqueous phase of the water-in-oil emulsion. Since the biologically active agent is not bound to or encapsulated in the solid hydrogel particles (i.e., it is free from the hydrogel particles), the active agent in the aqueous liquid is capable of being rapidly distributed to an individual in vivo.

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

[280] This rapid distribution of a primary dose of the biologically active agent occurs immediately after administration of the injectable composition to an individual, or shortly thereafter (i.e., within minutes after administration).

[281] In some embodiments, the release of the biologically active agent can be modulated by interactions that the biologically active agent can have with hydrogel particles in the injectable composition. For example, if the biologically active agent and the polymeric hydrogel matrix each carry a net charge at physiological pH, electrostatic interactions between the biologically active agent and the polymeric hydrogel matrix can influence the release kinetics of the active agent. Similarly, a charged coating can also interact with an electrostatically charged biologically active agent and thereby influence the release of the active agent.

[282] In one form of the injectable composition of the embodiments described herein, the biologically active agent in the hydrogel particles can be conjugated to the particles. Conjugation can be via covalent or non-covalent interactions. The biologically active agent can be conjugated to the cross-linked polymeric matrix of the hydrogel particle and/or to a coating that coats the hydrogel particle. In one embodiment, the biologically active agent is conjugated via non-covalent interactions, such as electrostatic interactions. This can occur when the biologically active agent is charged at physiological pH and the polymeric matrix of the hydrogel particle and/or the coating on the hydrogel particle has an opposite charge.

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

[284] The distribution of the biologically active agent to the physiological environment can also be modulated by an adjuvant in the injectable composition. In embodiments in which the oily phase of the water-in-oil emulsion comprises an adjuvant such as an adjuvant oil, the oily film surrounding the oily droplet will also comprise the adjuvant. In such embodiments, the adjuvant in the oily film will help modulate the release of the biologically active agent from the aqueous liquid droplet into the individual's physiological environment.

[285] More prolonged (i.e., longer) delivery of the biologically active agent can 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 injectable composition. Consequently, the hydrogel particles can act as a "depot" for the biologically active agent contained therein. Releasing quantities of the active agent from the particles over time allows the injectable composition of the invention to deliver a biologically active agent to an individual over an extended period of time, thereby providing longevity of response.

[286] For the purpose of providing prolonged destruction, the biologically active agent contained within the hydrogel particles passes from the hydrogel particles into the aqueous liquid of the aqueous phase. When within the aqueous liquid, the biologically active agent can then be delivered to the physiological environment of an individual via the same oily film-encapsulated aqueous droplet mechanism described above for delivering the primary dose of active agent.

[287] The passage of the biologically active agent from the hydrogel particles to the aqueous liquid can be aided by a concentration gradient that is established for the active agent or by differences in osmotic pressure between the hydrogel particles and the aqueous liquid, which could favor the movement of the active agent from the hydrogel particles to the aqueous liquid.

[288] In particular, rapid and prolonged distribution of the biologically active agent to a physiological environment can be influenced by a dynamic equilibrium that is established for the active agent. In this regard, it is believed that the injectable composition of the invention can provide a partition system that facilitates the establishment of an equilibrium between the active agent bound to or contained in the hydrogel particles and the free active agent in the aqueous liquid of the dispersed aqueous phase. of the water-in-oil emulsion. Partition, resulting from binding equilibrium, provides a rapid initial release of free syneresis active agent, giving the primo-dose (primary dose), which in the presence of an adjuvant can elicit innate immune responses (i.e., macrophages, dendritic cells ). Bioactive species physically bound to the chains of the hydrogel network provide longer lasting trickle feed (i.e. sustained release) of the active agent either by re-establishing other species in the "free" state establishing binding equilibrium or by absorbing the micro- hydrogels by antigen-presenting cells (APCs) by such processes as phagocytosis (if the hydrogel is larger than ~1 μm), micropinocytosis (if the hydrogel is smaller than 1 μm), or endocytosis (if the hydrogel is is in the nanoscale of ~50-60nm). Antigen-containing microhydrogels can additionally trigger the adaptive immune system by inducing the differentiation of T-cells into helper T-cells and killer T-cells resulting in the production of specific antibodies (i.e., adaptive immunity).

[289] In some embodiments, in which electrostatic interactions dominate the planned partition, consisting of an equilibrium between the active agent bound to the biopolymeric chains of the hydrogel and the free active agent in the dispersed aqueous phase of the water-in-oil emulsion, the composition of the biopolymer can be adjusted to adapt to the proportion of bioactive agent deliverable in the rapid (aqueous) and prolonged (bound) delivery stages.

[290] In some embodiments, the bioavailability profile of the biologically active agent may involve modulating the presentation of the active agent to the in vivo environment. For example, active agent presentation can be modulated via internalization of hydrogel particles into macrophages and dendritic cells followed by lysis of the hydrogel to release the active agent into the cytoplasm of antigen-presenting cells (APC).

[291] Effective short-term and long-term release of the biologically active agent can be accomplished without the need to apply an external stimulus to cause the release of the active agent from the injectable composition.

[292] A skilled person would also recognize that the morphology of the hydrogel particles may change after intramuscular or subcutaneous injection. The pressure exerted by the surrounding biological tissue can result in packing of the hydrogel particles into the water-in-oil emulsion at the injection site with a small amount of emulsion liquid just surrounding and filling the spaces between the hydrogel particles. This results in the separation by filtration of the oily liquid and aqueous liquid containing the biologically active agent, to be released as the primary dose after injecting the composition into an individual.

[293] Hydrogel particles may also contract over time after injection, resulting in the expulsion of aqueous liquid contained within the particles, consequently resulting in the expulsion of the biologically active agent from the particles and into the surrounding aqueous environment. . The remaining biologically active agent contained in the hydrogel particles can be expelled from the particles at a slower rate and thus contributes to providing prolonged distribution of the active agent.

[294] The passage of the biologically active agent from the hydrogel particles to the aqueous liquid can 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 interactions with a biologically active agent, a coating may prevent the movement of the active agent from the hydrogel particles into the aqueous liquid and thus help to promote prolonged release of the active agent from the injectable composition at the time of over an extended period of time.

[295] In some embodiments, the materials used to prepare the hydrogel particles may further aid prolonged delivery of the biologically active agent because such materials may have adjuvant properties and, therefore, act as adjuvants to help modulate the passage of the active agent from the hydrogel particles into the aqueous liquid. For example, chitosan may have adjuvant properties and hydrogel particles comprising cross-linked chitosan may be able to influence the release of the biologically active agent and enhance the immune response.

[296] Modulation of the release of the biologically active agent may involve an increase or decrease in the rate of distribution of the active agent to the physiological environment.

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

[298] In one example, an injectable composition of the present invention comprises Bm86 as an active agent and is capable of providing an immune response to Bm86 over a period of at least 6 months in vivo.

[299] In one embodiment, specific components of the injectable composition may be present in the following weight % ranges: polysaccharide (0.05-3%): crosslinking agent (0.05-3%): insoluble alkaline earth metal phosphate aqueous medium (0.0-3% subject to particle size distribution): glycosaminoglycans (GAGs) of about (0.0-3%).

[300] The injectable composition of the embodiments described herein may have a low proportion of solids in the water-in-oil emulsion.

[301] An advantage of the injectable composition of the invention is that it allows fewer injections to be administered to an individual. For example, for conditions that would normally require daily injections under current conventional regimens, the present invention may allow substantially the same physiological effects and benefits to be achieved with weekly injections.

[302] Additionally, regimens requiring an initial injection to be followed by one or more subsequent injections or booster injections can be simplified, because the prolonged bioavailability provided by the injectable composition of the present invention means that effective physiological benefits can be achieved with a single injection. , therefore avoiding subsequent or booster injections from being administered. For example, it has been discovered that the injectable composition of the present invention is capable of inducing effective protective immunity (i.e., antibody levels) in an individual after a single injection without the need for subsequent follow-up with a single injection or with multiple injections. Even more, an effective level of immunity was maintained for numerous weeks. In some embodiments, an effective level of immunity could be maintained over a period of several months and in one embodiment, immunity could be maintained for more than a year.

[303] The ability to reduce the number of injections can therefore provide increased convenience for an individual receiving injections, and also cost savings for the manufacturer and consumer.

[304] In use, the injectable composition may be contained in a syringe barrel and injected through the lumen of a needle for administration to an individual. For example, the injectable composition can be administered via a 23 gauge needle.

[305] Another advantage of the injectable composition of the invention is that the composition can be adapted to contain different proportions of hydrogel particles and aqueous liquid in the aqueous phase of the composition. In this way, the invention can control the proportion of biologically active agent contained in the hydrogel particles and the aqueous liquid respectively. This, in turn, could influence the amount of active agent that would be available for rapid (short-term) and prolonged (long-term) delivery.

[306] Additional control over the release of the biologically active agent can also be carried out through the appropriate selection of material used to form the hydrogel particles, and also through the formation of a coating on the hydrogel particles. As discussed above, the polymer 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 can 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.

[307] In yet another aspect, the invention provides a method of delivering a biologically active agent to an individual comprising the step of administering an injectable composition, as described herein, to the individual by injection.

[308] The injection can be subcutaneous, intramuscular or intraperitoneal. Preferably, administration is via subcutaneous injection.

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

[310] The injectable composition of the invention can be administered to an individual for the purpose of treating or preventing a disease or condition. As used herein the terms "treat" and "prevent" mean any treatment or prevention of a disease or condition in an individual. "Treatment" and "prevention" include: (a) controlling or inhibiting the disease or condition, that is, stopping its development or progression; or (b) alleviate or improve the symptoms of the disease or condition, that is, 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.

[311] "Disease", as used herein, is a general term used to refer to any deviation in health from which an individual suffers and which can be treated or prevented by the use of a microhydrogel "depot", which provides prolonged release of an active agent. A "condition" refers to an abnormal function of part of an individual's body that can be treated or prevented using a microhydrogel "depot" that provides prolonged release of an active agent.

[312] In use, the injectable composition of the invention provides rapid and prolonged release of a biologically active agent to an individual in vivo. Accordingly, upon administration to an individual, the injectable composition rapidly delivers an initial primary dose of the biologically active agent to the individual followed by more prolonged (i.e., drip) dosing of the active agent over a longer period of time.

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

[314] In some embodiments, the disease or disorder is an infection by a microorganism and the injectable composition of the invention can be used to treat or prevent infection by a microorganism. Microorganisms can include bacteria, fungi and viruses.

[315] In some embodiments, the disease or disorder is a viral infection and the injectable composition of the invention can be used to treat or prevent the viral infection.

[316] In some embodiments, the disease or disorder is a parasitic infestation and the injectable composition of the invention can be used to treat, control or prevent the infestation. For example, the injectable composition can be used to control a tick infestation.

[317] In other embodiments, the injectable composition can be used to inhibit a normal condition, (e.g., reproductive condition) whose progress or development is interrupted.

[318] The individual in whom a disease or condition is to be treated or prevented may be a human or an animal of economic importance and/or social importance to humans, for example, carnivores other than humans (such as cats and dogs), swine (pigs, fattening pigs and wild boars), ruminants (such as cattle, cattle, sheep/rams, giraffes, deer, goats, bison and camels), horses and birds/poultry including those types of birds/birds that are at risk endangered, kept in zoos and wild or domestic chicken birds and more particularly domestic chicken birds, for example, meat birds, such as turkeys, chicks/chickens/hens, ducks, geese, guinea fowl and the like, because they are, also, of economic importance for humans. The term does not denote a specific age. In this way, it is intended that newborns and adults are covered.

[319] In specific embodiments, the individual is a farm animal, such as cattle, sheep/rams, or pigs. In such embodiments, the injectable composition may be considered a veterinary composition and the biologically active agent contained in the composition is selected for the treatment or prevention of a disease or condition in the farm animal.

[320] The present invention also provides the 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 an individual.

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

[322] 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-releasing hormone (LHRH).

[323] As used in this specification, the singular forms "a", "an", "the" and "a" include the respective plural forms unless the context clearly dictates otherwise. Therefore, for example, reference to "a virus" includes a single viral particle and also two or more viral particles, "a gene" includes a single gene or two or more genes. Reference to "the invention" includes an aspect or multiple aspects of the invention.

[324] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by a person commonly skilled in the art to which this invention belongs. Although any materials and methods similar or equivalent to those described herein may be used to practice or test the present invention, the preferred materials and methods are now described.

[325] The invention will now be described with reference to the following examples. However, it should be understood that the examples are provided by way of illustration of the invention and that they are not, in any way, limiting the scope of the invention.

EXAMPLES Materials and methods

[326] Low molecular weight chitosan having a MW of 164 kDa, a degree of deacetylation of 75-85% and a viscosity of 0.026 Pa.s for a 1% by weight solution in 1% acetic acid was obtained from Sigma-Aldrich . (HAp) (Type I, Suspension in 0.001 M phosphate buffer solution, pH 6.8; approximately 25% solids) was obtained from Sigma-Aldrich. This suspension was dispersed in the chitosan solution using a high shear mixer (MICCRA with DS-8/P dispersion tool with 8 mm outer diameter stator and cutter knife type rotor) at 20,000 rpm for 5 min to yield 1 mg of HAp/mL. Sodium tripolyphosphate pentabasic (TPP) (technical grade, 85%) and chondroitin A sulfate (ChS) sodium salt (from lyophilized bovine trachea powder, BioReagent) were obtained from Sigma-Aldrich. Montanide ISA61 VG adjuvant oil was obtained from Seppic SA (Paris La Defense, France). Sesame oil, Tween 80, and medium viscosity sodium alginate (from brown algae) were obtained from Sigma-Aldrich. Bovine tick anti-Bm86 antigen (Bm86), tapeworm anti-TSOL18 antigen (TSOL18), and porcine somatotropin (pST) were produced and characterized as reported in the following literature: (1) Bm86: Willadsen, P. et al., The Journal of Immunology, 1989. 143(4): p. 1346-1351; (2) TSOL18: Gauci, C. and M.W. Lightowlers, Molecular and Biochemical Parasitology, 2003. 127(2): p. 193-198; (3) pST: Ouyang, J. et al., Protein Expression and Purification, 2003. 32(1): p. 28-34.

Injectable Vaccine Compositions with Bm86 Antigen

[327] Three solutions with different components 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% chondroitin sulfate (ChS), Part [C]: Montanide ISA61 VG Oil.

[328] To prepare the vaccine composition, a desired amount of solution [A] was initially emulsified with an amount of solution [C] using MICCA high shear emulsifier at the speed of 20,000 rpm for 2-3 min. A desired amount of solution [B] was then added dropwise under constant shear at the same rate as before (2-3 min) to form the injectable composition. The consistency of the emulsion was examined by aspiration with a syringe using a 23 gauge needle to assess the injectability of the composition. Examples 1 to 6: The Effect of Crosslinking Solution Volume (TPP)

[329] In these examples, 1 mL of [A] containing HAp was emulsified in 2 mL of [C] then several volumes of [B] with 0.08 M TPP containing ChS were added dropwise under continuous shear. 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 syringe and a 23 gauge needle after standing for 10-15 min. Hydrogel particle formation was confirmed by centrifuging the emulsion at 14,000 g for 5 min and observing 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. Table 1 - Compositions prepared with 0.08 M TPP in 1% ChS as solution [B]

Examples 7 to 24: The Effect of Cross-linker Concentration (TPP)

[330] 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 dripping under continuous shear. 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 needle. 23 gauge after resting for 10-15 min. Hydrogel particle formation was confirmed 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] Table 3 - Compositions prepared with 0.16M TPP in 1% ChS as solution [B] Table 4 - Compositions prepared with 0.32M TPP in 1% ChS as solution [B]

Examples 25 to 32: The Effect of Volume of Polymer Solution (Chitosan)

[331] 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 continuous shear . Various volumes of [B] (0.5 mL, 1 mL, 1.5 mL and 2 mL) were used and the injectability and consistency of the emulsion were tested. The results are shown in Tables 5 and 6. Table 5 - Compositions prepared with 0.16M TPP in 1% ChS as solution [B] Table 6 - Compositions prepared with 0.32M TPP in 1% ChS as solution [B]

Examples 33 to 44: The Emulsification Order

[332] Vaccine compositions containing Bm86 were produced using the following component solutions: Part [A2]: An aqueous solution of 2% chitosan with 1 mg hydroxyapatite (HAp)/mL, Part [B2]: Bm86 (0 μg, 50 μg, 100 μg or 200 μg) dissolved in an aqueous solution of TPP (0.16M or 0.32M) with 1% chondroitin sulfate (ChS), Part [C]: Montanide ISA61 VG Oil.

[333] An amount of component solution [B2] was emulsified in oil [C] first, then chitosan component solution [A2] was added to produce the final water-in-oil emulsion microhydrogel. In these examples Bm86 was incorporated into the ChS-TPP component solution [B2].

[334] In these examples, 1 mL of [B2] (with 0.16 M or 0.32 M TPP) was emulsified with 2 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 shear. The results are shown in Tables 7 and 8. It was found that the mousse-forming compositions are not injectable using a 23G x 33 syringe needle. Table 7 - Compositions prepared with 0.16 M TPP in 1% ChS as solution [B2 ] Table 8 - Compositions prepared with 0.32 M TPP in 1% ChS as solution [B2]

Example 45: Injectable Vaccine Composition with Bm86 Antigen

[335] An injectable vaccine composition containing the Bm86 antigen was prepared from the following component solutions, as follows: Part [C]: Montanide ISA61 VG Oil, Part [D]: The required dose of Bm86 (e.g. 400 μg of Bm86/mL) dissolved in an aqueous solution of 0.32 M TPP with 1% chondroitin sulfate (ChS), Part [E]: 2% chitosan solution with 1 mg of hydroxyapatite (HAp)/mL.

[336] An amount (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 dropwise under the same shear rate to produce a stable and injectable water-in-oil emulsion containing hydrogel microparticles with cross-linked chitosan.

Example 46: Injectable Composition with Somatotropin

[337] Three different component solutions were prepared: Part [F]: Alginate solution in water (1% or 2% alginate) with porcine somatotropin (pST) (300 μg/mL, 1,000 μg/mL, or 3,000 μg/mL ) as the active agent peptide hormone, Part [G]: Calcium chloride solution in water (2.8% or 5.6%) as the crosslinker, Part [H]: Tween 80 in sesame oil (50 mg in 1 mL).

[338] An amount (1 mL) of solution [F] was emulsified in 2 mL of [H] using high shear (20,000 rpm, 2 min). To this emulsion, 0.5 mL of solution [G] was added dropwise under the same shear rate. A stable, injectable water-in-oil emulsion containing cross-linked alginate hydrogel particles was produced with a lipophilic composition having a concentration of 50 mg Tween 80/mL sesame oil.

Example 47: Injectable Composition with Somatotropin

[339] In this example an aqueous sodium alginate composition and a lipophilic sesame oil composition were prepared 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 (AcOH) in water.

[340] An amount (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 dropwise under the same shear rate, which produced a stable and injectable water-in-oil emulsion comprising hydrogel particles formed with alginate cross-linked with chitosan.

Example 48: Bulk Model Hydrogel Composition with Somatotropin

[341] Three different component solutions were prepared, each having a range of concentrations: Part [J]: Alginate solution in water (0.5%, 1% or 2% alginate) with porcine somatotropin (pST) ( 500 μg/mL, 1,000 μg/mL, or 3,000 μg/mL) as active agent peptide hormone, Part [K]: Calcium chloride solution in water (0.3, 0.7, 1.4, or 2.8% ) as the crosslinker.

[342] An amount (1 mL) of solution [J] was cross-linked using 1 mL of solution [K] to produce the bulk hydrogel loaded with pST. The initial syneresis release (12 hours) of pST from the bulk model hydrogel is shown in Figure 8.

Example 49: Bulk Model Hydrogel Composition with Somatotropin

[343] Different component solutions were prepared: Part [L]: Alginate solution in water (1% alginate) with porcine somatotropin (pST) (500 μg/mL, 1,000 μg/mL or 3,000 μg/mL) as active agent peptide hormone and PEG porogen (35 kDa) (0 wt% PEG, 0.5 wt%, 1 wt%, or 2 wt%), Part [K]: Calcium chloride solution in water (0. 3%, 0.7%, 1.4% or 2.8%) as the crosslinker.

[344] An amount (1 mL) of solution [L] was cross-linked using 1 mL of solution [K] to produce the bulk hydrogel loaded with pST. After removal of the initial syneresis liquid, long-term (4 weeks) release of pST from the bulk model hydrogel was monitored by renewing the liquid surrounding the hydrogel using phosphate-buffered saline (SSTF) every 48 hours and measuring the pST in SSTF. The release profile is shown in Figure 9.

Animal Testing

[345] Dorper sheep were used as models to evaluate the immunological performance of the hydrogel-Bm86 in oil system in a study under the approval of the Animal Ethics Committee of the University of Queensland (Australia). The sheep were female, 5-7 months old and weighing 12-16 kg at the start of the test. The sheep test 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, which was administered as an initial primary dose followed by a booster dose after 4 weeks. Each dose contained an emulsion of 50 μg aqueous solution of Bm86 in 1.2 mL of Montanide ISA61 VG adjuvant oil. The second group was inoculated with an injectable Bm86 vaccine composition prepared according to the procedure of Example 45. Briefly, a dose of microgel in emulsion formulation prepared by dissolving 200 μg of Bm86 and 50 mg of ChS in 0.5 mL of 0.32 M TPP and emulsified in 1.2 mL of Montanide ISA60 VG, then 1.8 mL of (2% chitosan - 1 mg HAp/mL) was emulsified into this initial primary emulsion to produce 200 μg of Bm86 and 3.5 mL per dose. A single dose of the Bm86 composition was administered by subcutaneous injection. Anti-Bm86 antibody titers were measured as IgG and determined by ELISA assay every two weeks. The test results are shown in Figures 2 and 12 (in Figure 12, Ab units (antibody) relative to the negative control group at 1/25,600 dilution).

[346] The test results show that: • The Bm86 vaccine composition of the invention produces protective immunity (antibody (Ab) levels) in a single vaccine injection not requiring a single booster injection or multiple follow-up booster injections. • With the selected vaccine antigen (Bm86) antibody titer levels on day 150 of the test were higher than those of the comparative formulation, which was administered using 2 injections. • Within 150 days of the test, the vaccine composition established a period of anti-tick immunity not achieved by the comparative formulation. The antibody response continued at protective levels for up to a year after a single injection. • The immune response with the vaccine composition as described in Example 45 is higher than that of the comparative formulation, which produces protective levels only after day 28. • During a test duration of more than 400 days, the Antibody titer levels remained significantly higher than those of the comparative formulation, demonstrating a longer-lasting immune response after a single injection of the vaccine composition.

IN VITRO Release Results Release of Biologically Active Agent from a Chitosan Bulk Model Hydrogel (Primo-Dose)

[347] The ability to achieve an initial release of Bm86 from a chitosan hydrogel was tested by monitoring the concentration of Bm86 in the aqueous phase immediately after mixing the polymer and crosslinker solutions and hydrogel formation.

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

The Effect of Crosslinking on Long-Lasting Release Velocity

[349] The binding coefficient of Bm86 to the hydrogel is predominantly dependent on the electrostatic interaction between the protein due to its isoelectric point (pI) and the electrostatic charge of the polymer chains in the hydrogel. This results in an equilibrium between free Bm86 in the syneresis liquid and bound to the hydrogel at the initial formation stage. This is a dynamic equilibrium, so that when free Bm86 is removed and the medium is renewed, 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 model chitosan hydrogel at various cross-linking densities and various Bm86 loading concentrations by weekly refreshing the aqueous environment of the hydrogel with SSTF. This is a simulation of the renewal of the in vivo environment where intercellular fluid is continually renewed.

[350] The release rate of Bm86 from a bulk chitosan hydrogel prepared with different cross-linking densities (TPP concentrations of 0.04 M, 0.08 M or 0.16 M) was measured over a period of 8 weeks . In these examples, bulk model hydrogels were formed by mixing equal volumes of solutions containing (a) 2% chitosan with 1 mg HAp/mL and various concentrations of Bm86 and (b) 1% ChS with various concentrations of the crosslinker TPP (0.04M, 0.08M or 0.16M). The amount of Bm86 detected in the initial sample (syneresis release) gave an initial amount of Bm86 that is rapidly released by the composition as a primary dose. Then the release medium, i.e. SSTF, was replaced with a new 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 cross-linking density of the hydrogel can influence both the concentration of Bm86 released in the primary dose and the longer duration sustained release. Furthermore, the total amount of Bm86 released during the 8-week cumulative period (shown as a percentage) indicates that a significant proportion of Bm86 is released throughout the extended release phase. At higher cross-linking density, in which [TPP] = 0.16 M is used, a significant portion of the Bm86 antigen remains captured within the hydrogel at the end of the 8-week period.

The Effect of Protein Isoelectric Point on Loading and Release of Initial Syneresis

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

[352] Furthermore, Figure 17 shows the partition of proteins between the state bound to the hydrogel and the free state in the syneresis liquid. Bm86, pST and albumin were used to illustrate this process in equilibrium syneresis in a hydrogel formed by mixing a 2% chitosan constituent - 1 mg HAp/mL with the crosslinking constituent 0.08 M TPP in 1% chondroitin sulfate. The proteins were incorporated into the chitosan-containing constituent. This shows: • This balance is predominantly a function of the isoelectric point (pI) of the protein, i.e. the binding of the protein to the hydrogel is mainly due to electrostatic interactions. • Protein partitioning into the concentration ranges studied here gave a constant coefficient value which means that the protein concentrations used here are much lower than the binding capacity of the hydrogel.

Hydrogel Microparticles in a Water-in-Oil Emulsion

[353] An initial water-in-oil emulsion formed with an aqueous solution of tripolyphosphate in oil adjuvant is shown in Figure 6(A). Upon addition of a chitosan-containing solution, cross-linked hydrogel microparticles formed within water droplets in the water-in-oil emulsion (Figure 6(B)). The water-in-oil emulsion containing hydrogel remained stable for 5 months at 4°C, with no obvious phase separation (Figure 6(C)).

[354] A composition containing hydrogel microparticles formed with chitosan (2%), HAp (1 mg/mL), TPP (0.32 M) and ChS (1%)) in a water-in-oil emulsion with Montanide ISA61 as the oil phase was analyzed by optical microscopy. The 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 sample, the aqueous droplets were determined to be approximately 1 μm to 4 μm in diameter. Figure 7(C) shows the emulsion after storage for 5 months at 4°C. The aqueous phase was then removed from the emulsion by evaporation of the solvent, to produce hydrogel particles of approximately 1 μm to 2 μm just dispersed in a continuous oil phase (Figure 7(D)).

Release of Biologically Active Agent from Alginate Bulk Model Hydrogel (Primo-Dose)

[355] The release rate of porcine somatotropin (pST) from alginate bulk hydrogel samples prepared according to Example 48 was measured over a period of 28 days. The different hydrogel samples had different alginate concentrations (ranging from 0.5% to 2%), different cross-linking densities (CaCl2 concentrations of 0.3%, 0.7%, 1.4% or 2.8% ) and different initial pST loadings (ranging from 500 μg/mL to 30,000 μg/mL). The amount of pST detected in the initial sample (syneresis release) during the initial 12 h period gave an amount of pST that is rapidly released from the composition as a primary dose. The resulting syneresis release given as a percentage of initial pST loading is shown in Figure 8. As can be seen in Figure 8 the syneresis release as a percentage of pST loading increased with increasing loading level and degree of cross-linking. increasing and decreasing with increasing alginate concentration. This provides variations in syneresis release between 1% and 28% of the pST loading, representing control of the primary injection dose in the formulation.

Longer-Lasting Release of Biologically Active Agent from Alginate Bulk Model Hydrogel

[356] Longer-lasting release of pST from alginate hydrogel samples was also observed over 4 weeks. Alginate hydrogel samples were prepared according to Example 49. In these tests, after removing the syneresis liquid, the release medium, i.e., SSTCF, was renewed every 48 hours. Subsequent samples collected gave a progressive release profile. The results are shown in Figure 9. Figure 9A shows the release of pST on a weight basis while Figure 9B shows the release as a percentage of the initial pST loading. Linear release rates were observed during the initial 2 weeks, which then remained constant during the following 2 weeks.

[357] Bulk porous alginate hydrogels prepared with PEG (35kDa) as an added porogen (in concentrations of 0%, 0.5%, 1% or 2% by weight) showed a modified longer duration release rate. As a percentage of the initial loading after 2 weeks, a total of 29% to 44% of the pST is released.

Viscosity of Model Injectable Compositions

[358] To investigate the viscosity and injectability of the composition, model injectable compositions without biologically active agent were prepared. Model injectable compositions were prepared with three different component solutions, with solutions of each component: Part [A1]: 2% chitosan solution or 2% chitosan solution with 1 mg hydroxyapatite (HAp)/mL. Part [B1]: Tripolyphosphate (TPP) solution (0.32M) or TPP solution (0.32M) with 1% chondroitin sulfate (ChS), Part [C1]: Montanide ISA61 VG.

[359] In these examples, 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 shear to produce the microgel in emulsion formulation having the constituents [A1] : [B1] : [C1] in the following volumetric 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 stability and emulsion flow behavior of the model injectable compositions were evaluated using stress rheology.

[360] Figure 10 provides voltage sweep measurements of model injectable compositions in which the volumetric ratios of the constituent components were varied between 2:2:0 and 2:2:5. As seen in Figure 10, for injectable compositions formed with higher concentrations of the TPP crosslinker, viscosity may decrease with increasing shear stress due to shear fluidization behavior.

[361] Figure 11 (A) shows the variation in flow viscosity for the model injectable compositions formed using [A1] contains 2% chitosan and [B1] contains 0.32M TPP, with the volumetric ratios of [A1] , [B1] and [C1] were varied to examine the overall stability of the model injectable composition and its flow behavior, using viscosity n* (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).

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

TSOL18 Antigen in Chitosan-Based Bulk Model Hydrogel

[363] TSOL18 is a positively charged anti-tapeworm antigen (pI 9.65), contains 112 amino acids, with a molecular mass of 12.8 kDa. In Examples 50 to 55, bulk model hydrogel particles containing TSOL18 were prepared. The hydrogels comprise chitosan cross-linked with TPP in their core. In some examples, other components such as alginate, chondroitin sulfate and hydroxyapatite were also contained in the hydrogel, producing a composite hydrogel material.

Example 50: Chitosan Bulk Model Hydrogel - TPP with TSOL18

[364] The following component solutions were prepared: Part [M]: 2% chitosan solution in 1% AcOH, Part [N]: Tripolyphosphate (TPP) solution (0.08M).

[365] In this example, TSOL18 was incorporated into the solution of component [M] at a concentration of 0.5 mg / mL. An amount of [M] containing TSOL18 was then combined with an equal volume of [N] to form a bulk model chitosan hydrogel loaded with TSOL18.

Example 51: Chitosan Bulk Model Hydrogel - TPP with TSOL18

[366] Component solutions [M] and [N] from Example 50 were used in this example. However, TSOL18 was instead incorporated into the [N] component solution at a concentration of 0.5 mg/mL. An amount of [M] was combined with an equal volume of [N] containing TSOL18, to form a chitosan bulk model hydrogel loaded with TSOL18.

Example 52: Bulk Model Hydrogel (Chitosan - HAp - TSOL18) and with Chondroitin Sulfate Coating

[367] The following component solutions were prepared: Part [O]: 2% chitosan solution in 1% AcOH with 1 mg hydroxyapatite (HAp)/mL, Part [P]: Tripolyphosphate (TPP) solution (0. 08M) with 1% chondroitin sulfate (ChS).

[368] In this example, TSOL18 was incorporated into the [O] component solution at a concentration of 0.5mg/mL. An amount of [O] containing TSOL18 was then combined with an equal volume of [P] to form a TSOL18 core-loaded chitosan bulk model hydrogel with a chondroitin sulfate (ChS) coating. The coating was not crosslinked.

Example 53: Bulk Model Hydrogel (Chitosan - HAp) with Chondroitin Sulfate Coating - TSOL18

[369] The [O] and [P] component solutions from Example 52 were used in this example. However, TSOL18 was instead incorporated into the [P] component solution at a concentration of 0.5 mg/mL. An amount of [O] was combined with an equal volume of [P] containing TSOL18, to form a bulk chitosan hydrogel with a non-cross-linked chondroitin sulfate (ChS) coating. In this example, TSOL18 was incorporated into the ChS coating around the chitosan hydrogel core.

Example 54: Bulk Model Hydrogel (Chitosan - HAp - TSOL) with Alginate Coating

[370] The following component solutions were prepared: Part [O]: 2% chitosan solution in 1% AcOH with 1 mg hydroxyapatite (HAp)/mL, Part [Q]: Tripolyphosphate (TPP) solution (0. 08M) with 1% alginate.

[371] In this example, TSOL18 was incorporated into the [O] component solution at a concentration of 0.5mg/mL. An amount of [O] containing TSOL18 was then combined with an equal volume of [Q] to form a coated TSOL18-loaded chitosan hydrogel. Here an alginate coating formed around the TSOL18-loaded chitosan hydrogel.

Example 55: Bulk Model Hydrogel (Chitosan - HAp) with Alginate-TSOL18 Coating

[372] The [O] and [Q] component solutions from Example 54 were used in this example. However, TSOL18 was instead incorporated into the [Q] component solution at a concentration of 0.5 mg/mL. An amount of [O] was combined with an equal volume of [Q] containing TSOL18, to form a bulk chitosan hydrogel with an alginate coating. In this example, TSOL18 was incorporated into the alginate coating surrounding the chitosan hydrogel core.

TSOL18 Release from Bulk Model Hydrogel

[373] The initial syneresis release of TSOL18 (monitored over 1 week) from the bulk hydrogel samples prepared in Examples 50 to 55 is shown in Figure 13. The following results can be seen in Figure 13: • Example 50: The liquid of syneresis contained 54% of TSOL18 after 4 hours and remained the same after 24 hours. TSOL18 release increased to 78% after 1 week. • Example 51: The syneresis liquid contained 75% TSOL18 after 4 hours, which then reduced to 72% within 24 hours. TSOL18 release further decreased to 57% after 1 week. • Example 52: Syneresis liquid contained 44% of TSOL18 after 4 hours and remained the same after 24 hours. TSOL18 release increased to 50% after 1 week, showing the effect of chondroitin sulfate coating in modulating TSOL18 release. • Example 53: The syneresis liquid contained 52% of 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 sulfate in the coating. • Example 54: The initial syneresis release of TSOL18 from the bulk hydrogel (monitored over 1 week) is shown in Figure 13. As seen in Figure 13, the syneresis liquid contained 34% of the TSOL18 after 4 hours, which increased to 49 % after 24 hours. TSOL18 release reached equilibrium at 66% after 1 week. • Example 55: Syneresis liquid contained 69% TSOL18 when 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 into the coating around the chitosan hydrogel.

Injectable Compositions Containing TSOL18 Antigen in Chitosan-Coated Hydrogels

[374] Injectable compositions containing chitosan-coated hydrogels containing TSOL18 were prepared according to Examples 56 to 59. The coated hydrogels were prepared by injecting solutions of constituent ChS, alginate, ChS - TPP, or alginate - TPP into a solution of chitosan constituent - CaCl2 to form coated hydrogel particles containing TSOL18 having a chitosan coating as an outer layer. It is predicted that the coated hydrogel could provide greater TSOL18 capture and lower syneresis (i.e., less free antigen in the aqueous phase) due to the electrostatic interaction of highly positively charged TSOL18 with alginate or negatively charged ChS in the hydrogel core.

Bulk Model Hydrogel with TSOL18 and Chitosan Coating

[375] The following component solutions were prepared and used in the following Examples 56 to 59: Part [AC]: 1% CaCl2 solution in 1% chitosan in 1% AcOH, Part [BT] 2% ChS solution with TSOL18 ( 0.5mg/mL), Part [CT] 2% ChS Solution -0.01 M TPP with TSOL18 (0.5mg/mL) Part [DT] 2% Alginate Solution with TSOL18 (0.5mg/mL) Part [ET] 2% alginate solution - 0.01 M TPP with TSOL18 (0.5mg/mL). In each of the Examples, solutions of component [BT], [CT], [DT] or [ET] were injected into [AC] to form a hydrogel having a chitosan coating as a film over the formed hydrogel. Calcium chloride was co-solubilized with the constituent chitosan to promote rapid gel formation. The negatively charged TPP crosslinker can be included in the negatively charged ChS or alginate to aid in the formation of the chitosan coating and to increase its thickness if necessary.

Example 56: Chondroitin Sulfate Bulk Hydrogel with Chitosan Coating

[376] 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 example, the chitosan coating is non-crosslinked.

Example 57: Chondroitin Sulfate Bulk Hydrogel with Cross-Linked Chitosan Coating

[377] One volume of [CT] was injected into an equal volume of [AC]. This produced a ChS - Ca2+ hydrogel containing TSOL18 and with a cross-linked chitosan coating, in which the chitosan is cross-linked with TPP.

Example 58: Bulk Alginate Hydrogel - Ca2+ with Chitosan Coating

[378] One 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 non-crosslinked.

Example 59: Bulk Alginate Hydrogel - Ca2+ with Cross-Linked Chitosan Coating

[379] One volume of [ET] was injected into an equal volume of [AC]. This produced an alginate - Ca2+ hydrogel containing TSOL18 and with a cross-linked chitosan coating. In this example, TPP is used to form the cross-linked chitosan coating.

Syneresis Release of TSOL18 from Coated Bulk Hydrogel

[380] The release of TSOL18 syneresis from the coated bulk hydrogel samples of Examples 56 to 59 into the aqueous liquid was measured after 4 hours and 24 hours. The results are measured in Figure 14.

[381] As seen in Figure 14, the sample of Example 56 showed a constant syneresis release value of 24% of the initial amount of TSOL18 at 4 hours 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 compared to Example 56. It is considered that this increase could be due to an increase in the porosity of the chitosan - TPP coating which allows more TSOL18 to diffuse through the coating into the aqueous liquid.

[382] In Example 58, no syneresis of TSOL18 into the phase was observed after 4 hours and 24 hours. In comparison, in Example 59, the release of TSOL18 syneresis from this sample was increased compared to Example 58. A constant value of TSOL18 syneresis into the aqueous phase was increased after 4 hours and 24 hours at 6%. This increase 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 sulfate.

Example 60: Injectable Composition with Chitosan-Coated Alginate Hydrogel Particles Containing TSOL18

[383] The following component solutions were prepared and used: Part [A3]: 2.8% CaCl2 solution in 2% chitosan in 1% AcOH, Part [D3] 2% alginate solution with TSOL18 (300 μg/mL ), Part [F1] Sesame oil with 20 mg of Span 80/mL. A volume of [D3] containing TSOL18 was emulsified in [F] using high shear emulsifier. A volume of [A3] was added dropwise to this primary emulsion under constant shear. The ratio of component solutions [F1]:[D3]:[A3] was 2:1:0.5. The resulting composition is shown in Figure 16.

Injectable Composition with TSOL18 Anti-Tenia Vaccine

[384] The following component solutions were used to prepare various injectable compositions containing TSOL18-loaded hydrogels: Part [M]: 2% chitosan solution in 1% AcOH, Part [N]: Tripolyphosphate (TPP) solution (0. 08M), Part [O]: 2% chitosan solution in 1% AcOH with 1 mg hydroxyapatite (HAp)/mL, Part [P]: Tripolyphosphate (TPP) solution (0.08M) with 1% sulfate chondroitin (ChS). Part [Q]: Tripolyphosphate (TPP) solution (0.08M) with 1% alginate.

Example 61: Injectable Composition Comprising Chitosan Hydrogel - TPP with TSOL18

[385] In this example, TSOL18 was incorporated into the solution of component [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 primary emulsion 0.25 mL of [N] was added dropwise under constant shear. The resulting composition was a stable uniform emulsion with a droplet diameter of less than 1 μm.

Example 62: Injectable Composition Comprising Chitosan Hydrogel - TPP with TSOL18

[386] TSOL18 was incorporated into the [N] component solution at a concentration of 0.5 mg / mL. A volume of [M] (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 [N] containing TSOL18 was added dropwise under constant shear. The resulting composition was a stable uniform emulsion with a droplet diameter of less than 1 μm. An example is shown in Figure 15.

Example 63: Injectable Composition Comprising Hydrogel (Chitosan - HAp) - (ChS - TPP) with TSOL18

[387] TSOL18 was incorporated into the [O] component solution 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 [P] was added dropwise under constant shear. The resulting injectable composition was a stable uniform emulsion with a droplet diameter of less than 1 μm. The hydrogel particles in the composition comprise a cross-linked chitosan core - HAp and a coating comprising chondroitin sulfate.

Example 64: Injectable Composition Comprising Hydrogel (Chitosan - HAp) - (ChS - TPP) with TSOL18

[388] The [O] and [P] component solutions from Example 62 were used in this example. However, TSOL18 was instead incorporated into the [P] component solution at a concentration of 0.5 mg/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 [P] was added dropwise under constant shear. The resulting injectable composition was a stable uniform emulsion with a droplet diameter of less than 1 μm. The hydrogel particles in the composition comprise a cross-linked chitosan core - HAp and a coating comprising chondroitin sulfate.

Example 65: Injectable Composition Comprising Hydrogel (Chitosan - HAp) - (Alginate - TPP) with TSOL18

[389] TSOL18 was incorporated into the [O] component solution 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 dropwise under constant shear. The resulting injectable composition was a stable uniform emulsion with a droplet diameter of less than 1 μm. The hydrogel particles in the composition comprise a cross-linked chitosan core - HAp and a coating comprising alginate.

Example 66: Injectable Composition Comprising Hydrogel (Chitosan - HAp) - (Alginate - TPP) with TSOL18

[390] The [O] and [Q] component solutions from Example 64 were used in this example. However, TSOL18 was instead incorporated into the [Q] component solution at a concentration of 0.5 mg/mL. 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 dropwise under constant shear. The resulting injectable composition was a stable uniform emulsion with a droplet diameter of less than 1 μm. The hydrogel particles in the composition comprise a core of cross-linked chitosan - HAp and a coating comprising alginate.

Injectable Composition Containing Chitosan-Coated Hydrogel Particles and TSOL18 Anti-Tenia Vaccine

[391] The following component solutions were used to prepare various injectable compositions containing TSOL18-loaded hydrogels: Part [AC]: 1% CaCl2 solution in 1% chitosan in 1% AcOH, Part [BT] 2% ChS solution with TSOL18 (0.5 mg/mL), Part [CT] 2% ChS solution - 0.01 M TPP with TSOL18 (0.5mg/mL), Part [DT] 2% alginate solution with TSOL18 (0.5mg /mL) Part [ET] 2% alginate solution - 0.01 M TPP with TSOL18 (0.5mg/mL).

Example 67: Injectable Composition Comprising Chitosan Coated Chondroitin Sulfate Hydrogel with TSOL18

[392] A volume of [BT] 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 dropwise under constant shear. The resulting composition was a stable uniform emulsion with a droplet diameter of less than 1 μm. This produced an injectable composition having ChS - Ca2+ hydrogel particles containing TSOL18 and with a chitosan coating.

Example 68: Injectable Composition Comprising Cross-Linked Chitosan Coated Chondroitin Sulfate Hydrogel Containing TSOL18

[393] 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 dropwise under constant shear. The resulting composition was a stable uniform emulsion with a droplet diameter of less than 1 μm. This produced an injectable composition having ChS - Ca2+ hydrogel particles containing TSOL18 and with a cross-linked chitosan - TPP coating.

Example 69: Injectable Composition Comprising Chitosan Coated Alginate Hydrogel with TSOL18

[394] A volume of [DT] 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 dropwise under constant shear. The resulting composition was a stable uniform emulsion with a droplet diameter of less than 1 μm. This produced an injectable composition having alginate - Ca2+ hydrogel particles containing TSOL18 and with a non-crosslinked chitosan coating.

Example 70: Injectable Composition Comprising Cross-Linked Chitosan Coated Alginate Hydrogel Containing TSOL18

[395] A volume of [ET] 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 dropwise under constant shear. The resulting composition was a stable uniform emulsion with a droplet diameter of less than 1 μm. This produced an injectable composition having alginate hydrogel particles - Ca2+ containing TSOL18 and with a cross-linked chitosan - TPP coating.

[396] It should be understood that various other modifications and/or changes can be made without deviating from the spirit of the present invention as described herein.

Claims (21)

1. Injectable composition for rapid and prolonged delivery of a biologically active agent, characterized in that it comprises: a water-in-oil emulsion comprising an aqueous phase dispersed in an oily phase, the aqueous phase comprising a plurality of hydrogel particles and an aqueous liquid; and a biologically active agent in the hydrogel particles and in the aqueous liquid of the aqueous phase, wherein, when administered, the injectable composition provides rapid and prolonged distribution of the biologically active agent in vivo, wherein the hydrogel particles comprise a cross-linked polysaccharide selected from from chitosan cross-linked with a phosphate compound, and alginate cross-linked with a divalent cation derived from an alkaline earth metal; wherein the hydrogel particles have a mean particle diameter in the range of 10 nm to 20 μm, and wherein the biologically active agent is selected from the group consisting of a hormone, an antimicrobial, a therapeutic antibody, a cytokine, a protein of fusion, a virus, a bacterium or a fragment of bacteria, a vaccine and an antigen. 2. Composition according to claim 1, characterized by the fact that said injectable composition exhibits shear fluidization when injected through the lumen of a needle. 3. Composition according to claim 1 or 2, characterized in that the composition additionally comprises an adjuvant. 4. Composition according to claim 3, characterized in that the oily phase of the emulsion comprises an adjuvant. 5. Composition according to claim 3 or 4, characterized in that the oily phase of the emulsion comprises an adjuvant oil. 6. Composition according to any one of claims 1 to 5, characterized in that the cross-linked polysaccharide comprises chitosan cross-linked with a tripolyphosphate. 7. Composition according to any one of claims 1 to 5, characterized in that the cross-linked polysaccharide comprises alginate cross-linked with a divalent cation derived from an alkaline earth metal. 8. Composition according to any one of claims 1 to 7, characterized in that the hydrogel particles have an average particle diameter in the range of 50 nm to 5 μm. 9. Composition according to any one of claims 1 to 8, characterized in that the hydrogel particles additionally comprise an alkaline earth metal phosphate insoluble in aqueous media. 10. Composition according to claim 9, characterized by the fact that the aqueous-insoluble alkaline earth metal phosphate is hydroxyapatite. 11. Composition according to any one of claims 1 to 10, characterized in that one or more of the plurality of hydrogel particles comprises a coating. 12. Composition according to any one of claims 1 to 11, characterized in that the water-in-oil emulsion comprises a surfactant. 13. Composition according to any one of claims 1 to 12, characterized in that the biologically active agent is one or more selected from the group consisting of vaccine antigens and hormones. 14. Composition according to any one of claims 1 to 13, characterized in that the biologically active agent in the hydrogel particles is conjugated with the particles. 15. Composition according to any one of claims 1 to 14, characterized in that the biologically active agent is an antigen. 16. Composition according to claim 15, characterized by the fact that the antigen is Bm86 or TSOL18. 17. Composition according to any one of claims 1 to 14, characterized in that the biologically active agent is a hormone. 18. Composition according to claim 17, characterized by the fact that the hormone is somatotropin or luteinizing hormone releasing hormone (LHRH). 19. Process for preparing an injectable composition for rapid and prolonged delivery of a biologically active agent as defined in any one of claims 1 to 18, the process characterized by the fact that it comprises 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 composition under conditions that allow the first hydrogel-forming component to react with the second hydrogel-forming component to form a plurality of hydrogel particles in situ and thereby produce an injectable composition comprising an emulsion of water-in-oil 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 the aqueous liquid of the phase aqueous water-in-oil emulsion, and wherein the hydrogel particles comprise a cross-linked polysaccharide selected from chitosan cross-linked with a phosphate compound, and alginate cross-linked with a divalent cation derived from an alkaline earth metal; wherein the hydrogel particles have a mean particle diameter in the range of 10 nm to 20 μm, and wherein the biologically active agent is selected from the group consisting of a hormone, an antimicrobial, a therapeutic antibody, a cytokine, a protein of fusion, a virus, a bacterium or a fragment of bacteria, a vaccine and an antigen. 20. Process according to claim 19, characterized by the fact that said injectable composition exhibits shear fluidization when injected through the lumen of a needle. 21. Use of an injectable composition as defined in any one of claims 1 to 18, characterized by the fact that it is in the manufacture of a medicament for the treatment or prevention of a disease or disorder in an individual, wherein the disease or A disorder is an infection caused by a microorganism, preferably an infection caused by a virus.