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Title:
CHITOSAN COMPOSITION
Document Type and Number:
WIPO Patent Application WO/2015/032984
Kind Code:
A1
Abstract:
The application relates to a process for preparing a lypohilized formulation, comprising the steps of: (i) providing (1) a chitosan hydrogel, the chitosan hydrogel being obtainable by providing a chitosan having a degree of deacetylation between 30 and 75%, wherein the chitosan is randomly deacetylated, and a cross-linking agent, wherein the molar ratio of the cross-linking agent to chitosan is 0.2:1 or less based on the number of functional groups in the cross-linking agent and the number of deacetylated amino groups in the chitosan in an aqueous solution, and (2) a liquid dispersed in the hydrogel; cross-linking the composition and isolating the resultant chitosan hydrogel emulsion; and (ii) processing the chitosan hydrogel emulsion to give fragments; and (iii) lypophilizing the fragments of chitosan hydrogel emulsion. Formulations obtainable by the process, and their pharmaceutical uses are also disclosed.

Inventors:
ANDERSSON MATS (SE)
Application Number:
PCT/EP2014/069203
Publication Date:
March 12, 2015
Filing Date:
September 09, 2014
Export Citation:
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Assignee:
VISCOGEL AB (SE)
International Classes:
C08B37/08; A61K8/73; A61K9/51; A61K31/722; A61K47/36; C08J3/075; C08J3/24; C08L5/08
Domestic Patent References:
WO2009056602A12009-05-07
WO2009056602A12009-05-07
WO2011138155A12011-11-10
WO2003011912A12003-02-13
Foreign References:
US5489401A1996-02-06
US5770712A1998-06-23
Other References:
AMBRISH A PANDIT: "Solid Lipid Nanoparticulate Formulation for ifosfamide: Development and Characterization", 4 July 2011 (2011-07-04), pages 1 - 94, XP055152778, Retrieved from the Internet [retrieved on 20141113]
BIOMED RESEARCH INTERNATIONAL 2013, 2013, pages 909045
T. SANNAN ET AL., MAKROMOL. CHEM., vol. 177, 1976, pages 3589 - 3600
X.F. GUO ET AL., JOURNAL OF CARBOHYDRATE CHEMISTRY, vol. 21, 2002, pages 149 - 61
K.M. VARUM ET AL., CARBOHYDRATE POLYMERS, vol. 25, 1994, pages 65 - 70
ANN. PHARM. FR., vol. 58, 2000, pages 47 - 53
IND. ENG. CHEM. RES., vol. 36, 1997, pages 3631 - 3638
MACROMOLECULES, vol. 31, 1998, pages 1695 - 1601
J APPL POLYM SCI, vol. 74, 1999, pages 1093 - 1107
J POLYM SCI A: POLYM CHEM, vol. 38, 2000, pages 2804 - 2814
BIOMATERIALS, vol. 23, 2002, pages 181 - 191
J. POLYM. SCI. PART A: POLYM. CHEM., vol. 38, 2000, pages 474
BULL. MATER. SCI., vol. 29, 2006, pages 233 - 238
MUN ET AL., JOURNAL OF COLLOID AND INTERFACE SCIENCE, vol. 296, 2006, pages 581 - 590
LAPLANTE ET AL., CARBOHYDRATE POLYMERS, vol. 59, 2005, pages 425 - 434
LAPLANTE ET AL., FOOD HYDROCOLLOIDS, vol. 19, 2005, pages 721 - 729
HELGASON ET AL., JOURNAL OF AQUATIC FOOD PRODUCT TECHNOLOGY, vol. 17, no. 3, 2008, pages 216 - 233
Attorney, Agent or Firm:
ELKINGTON AND FIFE LLP (3-4 Holborn Circus, Holborn London EC1N 2HA, GB)
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Claims:
Claims

1. A process for preparing a lypohilized formulation, comprising the steps of:

(i) providing (1 ) a chitosan hydrogel, the chitosan hydrogel being obtainable by providing a chitosan having a degree of deacetylation between 30 and 75%, wherein the chitosan is randomly deacetylated, and a cross-linking agent, wherein the molar ratio of the cross-linking agent to chitosan is 0.2:1 or less based on the number of functional groups in the cross-linking agent and the number of deacetylated amino groups in the chitosan in an aqueous solution, and (2) a liquid dispersed in the hydrogel; cross-linking the composition and isolating the resultant chitosan hydrogel emulsion; and

(ii) processing the chitosan hydrogel emulsion to give fragments; and

(iii) lypophilizing the fragments of chitosan hydrogel emulsion.

2. A process for preparing a lypohilized formulation according to claim 1 , wherein the chitosan has a degree of deacetylation of between 35 and 75 %.

3. A process for preparing a lypohilized formulation according to claim 1 or 2, wherein the chitosan, prior to cross-linking, has a weight average molecular weight of 10-500 kDa.

4. A process for preparing a lypohilized formulation according to any preceding claim, wherein the cross-linking agent is bifunctional.

5. A process for preparing a lypohilized formulation according to any preceding claim, wherein the cross-linking agent is a derivative of squaric acid.

6. A process for preparing a lypohilized formulation according to any preceding claim, wherein the cross-linking is performed at a pH between 6 and 10.

7. A process for preparing a lypohilized formulation according to any preceding claim, wherein the lypohilizing is performed in the presence of a stabilizing agent.

8. A process for preparing a lypohilized formulation according to claim 7, wherein the stabilizing agent is a monosaccharide or an oligosaccharide.

9. A process for preparing a lypohilized formulation according to claim 7, wherein the stabilizing agent is sucrose.

10. A process for preparing a lypohilized formulation according to any one of claims 7 to 9, wherein the stabilizing agent is added before cross-linking the composition.

1 1. A process for preparing a lypohilized formulation according to any one of claims 7 to 9, wherein the stabilizing agent is added after cross-linking the composition and before lypophilizing the fragments of chitosan hydrogel emulsion.

12. A process for preparing a chitosan hydrogel emulsion comprising the process for preparing a lypohilized formulation according to any one of claims 1 to 1 1 , and then addition of an aqueous solution.

13. A lypohilized formulation obtainable by the process of any one of claims 1 to 1 1.

14. An emulsion formulation obtainable by addition of an aqueous solution to a lypohilized formulation according to claim 13.

15. A lypohilized formulation as claimed in claim 13 or an emulsion formulation according to claim 14 for use as a vaccine, in drug delivery, in tissue augmentation, as a cell culture scaffold, for encapsulation of viable cells, in wound healing devices, in orthopaedics, as a biomaterial, for treating urinary incontinence or vesicoureteral reflux, in viscosurgery, in providing living cells to a host organism, as a cosmetic, as a bulking agent, as a thickener, as an additive in the food industry, as a glue, as a lubricants, or as a drilling servicing fluid.

16. A pharmaceutical composition comprising the lypohilized formulation as claimed in claim 13 or an emulsion formulation according to claim 14 and pharmaceutically active ingredients.

17. An immunological agent comprising the lypohilized formulation as claimed in claim 13 or an emulsion formulation according to claim 14 and an antigen, wherein the antigen is optionally covalently bonded to the chitosan.

Description:
Chitosan composition

Field of the invention

This invention relates to advanced formulations containing chitosan. Background of the invention

About 40% of new drug candidates have low aqueous solubility which leads to development of pharmaceutical formulations allowing for solubilization of such drugs. Typical examples of such formulation systems are emulsions and liposomes.

Emulsions are used in many industrial areas e.g. in the pharmaceutical, food, cosmetic, paint, lubricant areas and more. However, emulsions suffer from stability problems due to the inherent thermodynamically unfavorable situation following by exposing large lipophilic surfaces towards large hydrophilic surfaces. Consequently there is a strong drive for phase separation and minimization of contact surfaces between the two immiscible phases.

The poor stability of emulsions is a general industrial problem which leads to high costs due to suboptimal product performance, seen as stability problems. Therefore a lot of techniques are being used to increase the stability of emulsions. Examples of such techniques are use of emulsifiers, minimization of droplet size and stabilization of the aqueous media by addition of viscosity enhancing agents.

An alternative strategy to provide formulations of lipophilic drugs is described in the applicant's earlier patent applications WO2009056602 and WO201 1 138155, the contents of which are incorporated herein in their entirety.

WO2009056602 describes a cross-linkable chitosan composition comprising chitosan having a degree of deacetylation between 30 and 75%, wherein the chitosan is randomly deacetylated, and a cross- linking agent. The hydrogel formed therefrom may be mechanically processed into fragments.

WO201 1 138155 provides a process for providing a cross-linkable chitosan composition comprising chitosan and water; dispersing a liquid in the cross-linkable chitosan composition; and cross-linking the chitosan with a cross-linking agent to form a hydrogel.

Thus, WO201 1 138155 provides chitosan hydrogels which can be used to provide more stable colloidal systems and provides an improvement to existing strategies for formulating lipophilic drugs. The emulsions described in WO201 1 138155 are processable into small, individually separated particles (fragments) having typical sizes from ten to several hundred micrometers. A major difference and advantage of having these large particles, compared to nanoparticles and traditional oil-in-water emulsions is that the oil droplets are surrounded by and embedded in a cross-linked polysaccharide hydrogel matrix. This enables the design of slow drug releasing systems in which different release mechanisms are involved. Lipophilic drugs dissolved in the oil phase will not only have to diffuse out of the oil phase but also need to find their way out through a polysaccharide gel network before they are exposed to the surrounding tissue or media. In addition the oil droplets are protected from enzymatic degradation as long as they are protected by the chitosan surrounding layer. The release of a drug dissolved in the oil phase follows a mixed mechanism in which enzymatic erosion of the particles and diffusion through the polysaccharide network act together. This combination of mechanisms and the possibility of having a large number of particles, each leaking drugs makes it possible to achieve very slow and sustained release of drugs from these emulsions, a most important feature for toxic or rapidly degrading drugs which could cause undesired side effects or result in a too short duration time. Release times up to several weeks have been achieved. This should be compared to e.g. the short release times shown (approximately 80% release within 10 minutes) in BioMed Research International 2013;2013:909045

The hydrogels described in the patent applications above both open for protection and thereby prolong the shelf life of sensitive molecules. Examples of sensitive molecules are vitamins, lipids prone to oxidation, flavonoids, proteins, peptides, gene fragments and molecules sensitive to hydrolysis or enzymatic degradation.

However, with these advantages of having a slow releasing system built up by a vast number of oil droplets localized in particles consisting of a cross-linked polysaccharide hydrogel cause pharmaceutical challenges and often require specific technical solutions. A major difference from nano particles and standard type oil-in-water emulsions is the size of the respective entities. A nano particle has a size between one and 1000 nanometers, and a standard type emulsion, made in homogenizer, typically has an oil droplet size spanning from 100 nm to 500 nm. It is obvious that a nano particle cannot contain oil droplets of this size. Compared to the sizes of nano particles and the size of oil droplets in an oil-in-water emulsion, the particles described in WO201 1 138155 are huge. To further illustrate the difference in size a theoretical mathematical calculation gives that 30 million oil droplets having an average size of 200 nm can be contained in one 100 μιτι particle (containing 25% oil as described in the patent above). Thus it is evident that the surface of these microscopically "gigantic" particles allows for multiple interactions with their surroundings. This could sometimes be problematic and contacting materials must be chosen with care in order to minimize loss due to material interactions. If not treated properly the particles may also interact with each other and form clusters and aggregates. These interactions apply in particular to chitosans having a larger proportion of acetyl groups, compared to standard type commercial chitosans, is their more lipophilic nature. An increased proportion of acetyl groups enable more and stronger hydrophobic interactions. There remains a need in the art for compositions which overcome the above-described disadvantages.

Accordingly, in a first aspect the invention provides a process for preparing a lypohilized formulation, comprising the steps of:

(i) providing (1 ) a chitosan hydrogel, the chitosan hydrogel being obtainable by providing a chitosan having a degree of deacetylation between 30 and 75%, wherein the chitosan is randomly deacetylated, and a cross-linking agent, wherein the molar ratio of the cross-linking agent to chitosan is 0.2:1 or less based on the number of functional groups in the cross-linking agent and the number of deacetylated amino groups in the chitosan in an aqueous solution, and (2) a liquid dispersed in the hydrogel; cross-linking the composition and isolating the resultant chitosan hydrogel emulsion; and

(ii) processing the chitosan hydrogel emulsion to give fragments; and

(iii) lypophilizing the fragments of chitosan hydrogel emulsion.

In a second aspect, the invention provides a lypohilized formulation obtainable by a process comprising the steps of:

(i) providing (1 ) a chitosan hydrogel, the chitosan hydrogel being obtainable by providing a chitosan having a degree of deacetylation between 30 and 75%, wherein the chitosan is randomly deacetylated, and a cross-linking agent, wherein the molar ratio of the cross-linking agent to chitosan is 0.2:1 or less based on the number of functional groups in the cross-linking agent and the number of deacetylated amino groups in the chitosan in an aqueous solution, and (2) a liquid dispersed in the hydrogel; cross-linking the composition and isolating the resultant chitosan hydrogel emulsion; and

(ii) processing the chitosan hydrogel emulsion to give fragments; and

(iii) lypophilizing the fragments of chitosan hydrogel emulsion.

Surprisingly, the applicant has found that lyophilization of chitosan hydrogel emulsions can give further stability to sensitive molecules held therein and thereby opens for new technical combinations in order to make new drug formulations. Moreover, unexpectedly, the applicant has found that these lyophilized hydrogel emulsions can be successfully reconstituted to provide chitosan hydrogel emulsions with the same properties as before lyophilization. In particular, lyophilized chitosan hydrogel emulsions can be reconstituted to provide emulsions with the same desirable slow release profiles.

The present invention will now be described with reference to the accompanying drawings, in which: Fig. 1 shows the dissolution profiles of the compositions according to the invention comprising cyclosporin A; and

Fig. 2 shows a photograph of reconstituted emulsions according to the invention.

The lyophilizated formulations help stabilize molecules that are easily degraded in solution and provide a useful method to make e.g. pharmaceuticals withstand breaks in the "cold chain" distribution, particularly suited for use in developing countries with tropical climate. This technology is particularly applicable to vaccines, because practical handling of vaccines is a big issue, since many vaccines are sensitive to moisture and heat conditions and have to be handled at low temperature in order to maintain a meaningful shelf life. An important feature of such vaccine formulations is that they can be rehydrated within minutes and administered to the patient without delay. This is also true for other drug formulations.

The chitosan hydrogel emulsion comprises a liquid dispersed in the hydrogel. This means that water- immiscible liquid droplets are distributed in a finely divided state throughout the hydrogel. The compositions are therefore analogous to an emulsion where the liquid droplets are dispersed in a chitosan hydrogel continuous phase. By hydrogel is meant a colloidal gel in which water is the dispersion medium.

Chitosan is a linear polysaccharide composed of 1 ,4-beta-linked D-glucosamine and N-acetyl-D- glucosamine residues. Chitosan is produced by alkaline deacetylation of chitin, which is a polymer of a N-acetyl-D-glucosamine found in shells of crustaceans. Chitosan of high molecular weight and/or high degree of N-deacetylation is practically insoluble in water; however its salts with monobasic acids tend to be water-soluble. The average pKa of the glucosamine residues is about 6.8 and the polymer forms water-soluble salts with e.g. HCI, acetic acid, and glycolic acid.

Chitosan is biodegradable, non-toxic and anti-microbial. Furthermore, its cationic and hydrophilic nature makes it attractive in pharmaceutical formulations.

Chitosan is characterised by its molecular weight and degree of deacetylation. Chitosans of different molecular weights and degrees of deacetylation can be produced by varying the conditions of the chitin alkali treatment. Commercially, chitosans are characterised by their viscosity and an average molecular weight is given. Commercially available chitosans typically have molecular weights in the range of 4 to 2,000 kDa and average degrees of deacetylation of 66 to 95%.

Chitosan is polydisperse in its nature, i.e. contains a mixture of different chain lengths. Chitosan used according to the present invention preferably has a viscosity of up to 15,000 mPas prior to cross- linking, preferably from 2 to 10,000 mPas, more preferably from 5 to 2,000 mPas and most preferably from 10 to 1 ,000 mPas when measured as a 1 % w/v solution in 1 % aqueous acetic acid at a temperature of 25°C using a rotating viscometer with a spindle rotating at 20 rpm. The viscosity of the solution is an indication of the average molecular weight of the chitosan, it being understood that chitosan is a polymeric material having a distribution of molecules of varying chain length. The chitosan preferably has a weight average molecular weight of 10 to 500 kDa. Weight average molecular weights can be determined using light scattering techniques. The pattern of the deacetylation of the chitosan is also important for its properties. Commercially available chitosan typically has a block structure, which means that the chitosan includes blocks of N- acetyl-D-glucosamine residues, or blocks of chitin-like polymer. This is because chitin is typically isolated in solid phase processes from crustacean shells. In such processes, in which the shells remain undissolved throughout the process, the shells are treated with strong alkali to give the partially deacetylated chitosan. However, because the chitin is initially in the form of crustacean shell, the hydroxide ions of the alkali tend to act preferentially on the monosaccharide units on the surface of the shell; the monosaccharide units within the centre of the relatively thick shell tend not to see the hydroxide ions and hence retain the N-acetyl substitution pattern.

The solubility of chitosan depends on chitosan chain length, degree of deacetylation, acetyl group distribution within the chains, and external conditions such as ionic strength, pH, temperature, and solvent. Practically, most commercially available, unmodified chitosans have a degree of deacetylation exceeding 80% and are insoluble in aqueous solution when the pH is above approximately 6: above this pH they will precipitate from aqueous solution.

In a hydrogel it is essential that the chitosan and the cross-linked derivative remain in solution and that precipitation thereof is avoided.

The chitosan hydrogels can be made using known methods for cross-linking chitosan. In these methods, the chitosan hydrogels are produced by solubilising chitosan in aqueous solution and cross- linking the chitosan. Thus, commercially available chitosan is cross-linked in aqueous solution at a pH at which the chitosan is soluble, typically in acidic solution, for example pH 4-5. These hydrogels are stable at low pH (pH 5 or less) and are therefore useful in the compositions when a low pH is required for any particular end use. However, when working with triglyceride oils a pH of 4-5 is not optimal for their stability and a higher pH, 6-7, should be used in the surrounding aqueous phase to obtain better long term stability properties when these are used for making emulsions.

Preferably the chitosan hydrogel of the present invention is produced from chitosan that has a degree of deacetylation 75% or less, more preferably 70% or less, more preferably 65% or less, more preferably 60% or less and most preferably 55% or less. Chitin is completely insoluble in water and becomes soluble to some extent when the degree of deacetylation is 30% or more. The chitosan according to the present invention therefore preferably has a degree of deacetylation above 35%, preferred is a degree of deacetylation above 40% and most preferred is a degree of deacetylation above 45%.

Although the chitosan used to produce the hydrogel of the present invention can have a block pattern of deacetylation, preferably the chitosan used to produce the hydrogel of the present invention is randomly deacetylated. That is, the chitosan has a random pattern of acetylated and deacetylated monosaccharide units. One way of determining the nature of the monosaccharides is to determine the nearest-neighbour frequencies using NMR and compare the frequencies obtained with statistical models, see WO 03/01 1912.

Chitosan having a random deacetylation pattern can be produced by treating chitin in solution under carefully controlled conditions, or by fully deacetylating the chitin and then reacetylating in solution to provide the required degree of deacetylation. See T. Sannan et al Makromol. Chem. 177, 3589-3600, 1976; X.F. Guo et al, Journal of Carbohydrate Chemistry 2002, 21 , 149-61 ; and K.M. Varum et al Carbohydrate Polymers 25, 1994, 65-70. The chitosan of the present invention is preferably obtainable by acetylating and/or deacetylating the chitosan in the solution phase to provide a random deacetylation pattern.

Preferably the chitosan used to produce the hydrogel of the present invention has a degree of deacetylation of 75% or less and has a random deacetylation pattern.

Chitosan having a degree of deacetylation below 75% and having a random pattern of deacetylation has higher solubility in water compared to typical commercially available chitosans. The low deacetylated/random chitosans are soluble at higher pH, which means that the cross-linking reaction to produce a hydrogel can take place at higher pH. The advantages of doing this are several. The possibility to use a higher pH is beneficial in terms of substantially increased reactivity of the amino groups on the glucosamine residues. This makes the couplings more efficient and enables the use of much lower concentrations of cross-linking reagents to reach a defined degree of cross-linking, leading to low manufacturing costs. Another benefit is that the side reactions are kept low. Another beneficial and important aspect of using low concentrations of cross-linking agent is that when the formed hydrogels are intended for medicinal use, toxic side effects resulting from interactions of the cross-linker and its biological environment could be minimised.

Although the cross-linking of chitosan having a degree of deacetylation below 75% and having a random pattern of deacetylation can be carried out at acidic pH, for example pH 4 to 5, the cross- linking is preferably performed at pH 6 or above. Even more preferred is to use pH above 6.3. It is also preferred to use a pH that does not to a substantial degree destroy the cross-linking reagent by hydrolysis or via an elimination reaction. Typical conditions for the reaction are alkaline conditions, preferably using a pH below 10, more preferably below 9.5 and most preferably below 9.0. The gels produced are particularly preferred because they have low toxicity and they can be made to degrade rapidly. As mentioned above, the gels do not precipitate when subjected to neutral and alkaline conditions. They also possess a rigidity which allows for further mechanical processing into e.g. injectable so called "crushed gels", useful in a vast number of applications.

Cross-linking agents suitable for use in the present invention comprise at least two reactive sites which are electrophiles designed to react easily with amines. When the cross-linker has two reactive sites it is bifunctional and can thus react with two amino groups e.g. two glucosamine units in different chitosan chains. The distance between the reactive groups may be increased by a spacer moiety. This spacer is often an aliphatic chain or a polyether construct like poly- or oligoethylene glycols. Preferably the cross-linking agent is bi-, tri- or tetrafunctional, although bi- or trifunctional is preferred and bifunctional is most preferred. It is preferred to use bi-functional cross-linkers that easily react at a pH close to or above the pKa (approximately 6.8) of the glucosamines in the polymer chains in high yielding reactions and in which the cross-linking molecule is consumed to a considerable degree. It is also preferred that the cross-linking molecule does not form by-products that have to be removed prior to use. Many cross-linkers are designed to eliminate a leaving group when reacting. In such cases cross-linkers that eliminate non-toxic components are preferred. Typical examples of such cross- linking functionalities are reactive esters, Michael acceptors and epoxides. Suitable cross-linking agents are known and include glycosaminoglycans such as hyaluronic acid and chondroitin sulfate (Ann. Pharm. Fr. 58 47-53, 2000), glutaraldehyde (Ind. Eng. Chem. Res. 36: 3631-3638, 1997), glyoxal (US 5,489,401 ), diethyl squarate (Macromolecules 31 : 1695-1601 , 1998), diepoxides such as diglycidyl ether (US 5,770,712), tripolyphosphate (J Appl Polym Sci 74: 1093-1 107, 1999), genipin (J Polym Sci A: Polym Chem 38, 2804-2814, 2000, Biomaterials. 23, 181-191 , 2002), formaldehyde (J. Polym. Sci. Part A: Polym. Chem. 38, 474, 2000, Bull. Mater. Sci., 29, 233-238, 2006). Preferred cross-linking molecules are ester derivatives of squaric acid, diepoxides and derivatives of acrylamides. Most preferred is diethyl squarate (3,4-diethoxy-3-cyclobutene-1 ,2-dione) and its structurally closely related analogues. Other preferred cross-linkers are 1 ,4-butandiol diglycidylether, derivatives of acrylamide and their structurally closely related analogues.

The structure of the hydrogel is affected by the concentration of chitosan and the amount of cross- linking reagent used. Thus, hydrogels having a higher viscosity can be produced by using a higher concentration of chitosan in the hydrogel, or by increasing the number of cross-links. In general, it is preferred to have higher chitosan concentrations and lower concentrations of cross-linking agent to achieve a gel of the desired nature. It is preferable to minimise the amount of cross-linker used, particularly for pharmaceutical applications, because cross-linkers may cause an immunological response or toxic side reactions if not fully consumed.

The molar ratio of cross-linking agent to chitosan based on the number of functional groups in the cross-linking agent and the number of accessible amino groups in the chitosan is preferably 0.2: 1 or less, more preferably 0.16: 1 or less and most preferably 0.1 :1 or less. The molar ratio is based on the number of groups available for cross-linking on the cross-linker and on the chitosan. For the cross- linker it will depend on the functionality (bi-, tri-, tetrafunctional etc) and on the chitosan to the accessibility of the amino groups (only the deacetylated amino groups will be reactive). Clearly, the number of available amino groups will be determined by the degree of deacetylation of the chitosan.

By way of contrast to the cross-linked hydrogels of the present invention, oil-in-water emulsions based on non-cross-linked chitosan have been proposed (see Mun et al, Journal of Colloid and Interface Science, 2006, 296, 581-590; Laplante et al, Carbohydrate Polymers, 2005, 59, 425-434; Laplante et al, Food Hydrocolloids, 2005, 19, 721-729; and Helgason et al, Journal of Aquatic Food Product Technology, 2008, 17, 3, 216-233). However, these documents disclose a different approach. These documents suggest that in order to provide effective stabilisation, the chitosan should adsorb at the surface of surfactant-stabilised droplets in order to form a multilayer emulsion. However, the large variability in chitosan characteristics such as molecular weight and degree of deacetylation make it difficult to achieve effective stabilisation in this manner. Moreover, it has been found that the compositions of the present invention, which comprise a liquid dispersed in a cross-linked chitosan hydrogel, have improved stability when compared to compositions comprising non-cross-linked chitosan.

The chitosan is preferably present in the composition of the present invention in an amount of 3% by weight or less based on the total weight of chitosan and water in the hydrogel. More preferred is to use an amount of 2% by weight or less. Preferably the amount of chitosan is above 0.3% by weight based on the total weight of chitosan and water in the hydrogel, preferably 0.75% by weight or greater. Water can be present in the hydrogel in amount of up to 99.7% by weight, based on the total weight of the chitosan and water in the hydrogel. However, in many applications a combination of water and one or more other solvents may be used depending on the nature of the intended use of the emulsion systems formed. Examples of such solvents are water-miscible solvents, such as alcohols (e.g. ethanol, glycerol, ethylene glycol or propylene glycol), polyethylene or polypropylene glycols, DMSO, acetone, DMF, glycofuran, methyl pyrrolidone, Transcutol and combinations thereof.

The compositions can optionally include materials that are miscible or soluble in the hydrogel matrix such as preservatives, inorganic salts such as sodium chloride, and buffers.

The compositions comprise a liquid dispersed in the hydrogel. In a preferred embodiment, the composition comprises a water-soluble active agent that is solubilised in the hydrogel. Suitable active agents include water-soluble drugs, vitamins and cosmetic ingredients. The amount of active agent present will vary depending on the type of active ingredient and the end use but the active ingredient may be present in an amount of 0.005 to 15%, preferably 0.1 to 10%, more preferably 1 to 5% by weight, for example, based on the total weight of the composition.

Suitable liquids are immiscible with water and include any liquid that is able to form the dispersed phase in an oil-in-water emulsion. Examples of suitable liquids are well known and include water- immiscible oils, pharmaceutical active agents and excipients, cosmetic ingredients, vitamins, foods, agrochemical active agents and additives, and personal care ingredients.

It has been found that the compositions of the present invention can comprise up to 50% by weight of dispersed liquid, based on the total weight of the composition and still remain stable. The use of cross-linked chitosan significantly increases emulsion stability. This increase in stability allows high proportions of dispersed liquid to be used. The dispersed liquid is preferably present in an amount of 5 to 30% by weight based on the total weight of the composition.

The dispersed liquid may include a mixture of materials provided that the mixture is dispersible in water. For example, the dispersed liquid may comprise a mixture of two or more liquids that are immiscible with water, or a mixture of a water-immiscible liquid and solid particles dispersed in the water-immiscible liquid.

In one preferred embodiment of the present invention, one or more water-insoluble active ingredients are solubilised in the dispersed liquid. According to this embodiment, a water-insoluble drug or vitamin, for example, is solubilised in a water-immiscible liquid that is dispersed in the hydrogel. Many examples of water-insoluble active ingredients are known to the person skilled in the art and include insect repellents; dyes; drugs, for example cytostatic drugs such as paclitaxel, anti-inflammatory agents such as budesonide and immunosuppressant drugs such as cyclosporin; and vitamins such as vitamin D and vitamin A. When the composition includes a drug, for example, the dispersed liquid should be a pharmaceutically acceptable liquid carrier that is immiscible with water. Examples include lipids, e.g. phospholipids, triacyl glycerols, di- and mono alkyl esters of glycerol, and fatty acids, including omega-3 fatty acids such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Typically such oils and lipids are sesame oil, sunflower oil, olive oil, rape seed oil, Miglyol® 812 (caprylic/capric triglyceride), paraffin oil and lanolin.

When the compositions comprise a dispersed liquid, an emulsifier is provided in order to stabilise the liquid droplets. Any emulsifier that is suitable for producing an oil-in-water emulsion can be used. The emulsifier may be anionic, cationic or non-ionic, or a combination thereof. Suitable emulsifiers are well known to the person skilled in the art and include alkyl sulfonates, alkyl sulfosucci nates, phospholipids such as lecithins, proteins, polyethylene glycol-hydrogenated castor oils, copolymers of ethylene oxide and propylene oxide (such as those available under the trade name Pluronic®), polyethylene oxide esters of fatty acids (such as those available under the trade name Myrj®), polyethylene oxide alkyl ethers of fatty alcohols (such as those available under the trade name Brij®), sorbitan fatty acid esters (such as those available under the trade name Span®), alkylphenol ethoxylates (such as those available under the trade name Triton®) and polyethylene oxide sorbitan fatty acid esters (such as those available under the trade name Tween®). In a preferred embodiment, the composition of the present invention further comprises a phospholipid. The phospholipid may advantageously form a liposomal phase which is stabilised by the chitosan hydrogel.

The emulsifier is present in an amount that is suitable for stabilising an oil-in-water emulsion and can be easily determined by the person skilled in the art. It has been surprisingly found that the compositions of the present invention can include relatively low amounts of emulsifier and still give emulsions of higher stability than the corresponding non-cross-linked emulsion. Although not wishing to be bound by theory, it is believed that the use of cross-linked chitosan significantly increases emulsion stability, allowing lower concentrations of emulsifier to be used. The emulsifier can therefore be present in the compositions in an amount of 0.2 to 25% by weight, more preferably 0.2 to 5.0% by weight, based on the weight of the dispersed liquid.

In preferred embodiments it has been found that the amount of emulsifier could be substantially reduced providing compositions of superior stability to the corresponding emulsions comprising non- cross-linked chitosan in which the concentration of the emulsifier was a fivefold higher. In addition, stable compositions of very high lipid content, 50%, may be made, including under conditions normally considered more demanding, such as in a physiological salt concentration.

As discussed hereinabove, the compositions are produced by providing a cross-linkable chitosan composition comprising chitosan and water; dispersing a liquid in the cross-linkable chitosan composition; and cross-linking the chitosan with a cross-linking agent to form a hydrogel. It is believed that the cross-linked chitosan provided in the hydrogel produces a cage-type structure around the dispersed liquid droplets, which helps to prevent aggregation or coalescence of the dispersed material. In the process for producing the hydrogel at least some of the cross-linking should therefore take place after the liquid has been dispersed in the cross-linkable chitosan composition.

The liquid to be dispersed is added to the cross-linkable chitosan composition and the mixture is stirred. High-speed mixers suitable for use in preparing emulsions and colloidal suspensions can be used and these are well known. Homogenisation under high pressure is also commonly used for this purpose.

The cross-linking agent can be added to the cross-linkable chitosan composition before, at the same time or after the liquid to be dispersed. Preferably the cross-linking agent is added to the cross-linkable chitosan composition before the liquid is dispersed in the chitosan composition. This means that the cross-linking reaction commences before the liquid is dispersed in the composition. However, the liquid should be dispersed in the chitosan composition before the cross-linking reaction is complete. According to this preferred embodiment, liquids can easily be dispersed in the cross-linkable chitosan composition simply by stirring with a magnetic stirrer at room temperature: a high speed mixer is not required.

The cross-linkable chitosan must remain solubilised in the aqueous medium while the cross-linking reaction takes place. A discussed hereinabove, the pH can be adjusted to ensure that the chitosan remains soluble. Thus, for many commercially available chitosans, the cross-linking reaction will take place at acidic pH, typically pH 4 to 5. However, the low deacetylated chitosans preferably can be cross-linked at higher pH, typically pH 6 to 10, preferably 6 to 8.

When the composition comprises materials that are miscible or soluble in the hydrogel matrix such as active agents, preservatives, inorganic salts and buffers, these can be conveniently added to the cross-linkable chitosan composition before the liquid is dispersed in the composition and before cross- linking takes place.

When the composition comprises one or more water-insoluble active materials dissolved in the dispersed liquid, the water-insoluble materials are solubilised in the liquid before the liquid is dispersed in the cross-linkable chitosan composition.

The hydrogel is obtained as a block which may be isolated without further treatment. The hydrogel can be processed to provide smaller blocks or fragments using conventional techniques known in the art. This resulting "crushed gel" could be made with various block/fragment sizes depending on the intended use of the crushed gel. When the blocks are made small they become injectable through a fine needle.

In one embodiment, substances can be added to the composition after the composition has been processed into a crushed gel. We prefer the fragments i,e,. particles made from the formulations to have an average particle size ranging from 1 μητι to 1 mm, more preferably 5 μιτι to 500 μιτι, and most preferably 10 μιτι to 250 μιτι.

The viscosity of the gel can be measured with a rheometer such as the Bohlin Gemini VOR instrument, using for measurement cell the cone-plate geometry of 40 mm diameter and a cone angle of 4°, at 25°C.

After the chitosan hydrogel emulsion is processed to give fragments, the fragments are lypophilized.

Freeze drying of the hydrogel emulsions offers a means to increase drug load and at the same time prolong shelf life. In addition, new advanced controlled release formulations are possible, e.g. a freeze dried emulsion in a blister package or as a powder in a gelatin capsule. We also foresee the use of the freeze dried emulsions above in areas outside the traditional pharmaceutical area. Freeze dried food is a billion dollar industry and the possibility to further increase the shelf life of valuable nutrients by use of the invention could be lucrative. Probiotics can be defined as living microbial supplements that can improve the balance of intestinal microorganisms. Good probiotic viability and activity is essential and formulation in an emulsion according to the invention may be a way to maintain the functionality of probiotics. A similar area is vaccination with live viruses and bacteria. The current invention allows for delivery of freeze dried emulsion particles together with such organisms, optionally freeze dried. A very distinct advantage of the stabilized emulsions described in WO201 1 138155 is the high mucoadhesiveness from the chitosan network. This makes the microorganisms stick to the particles and stay in place where administered, e.g. when injected. When given orally the rehydrated particles will not only bind to their microorganism load but also to the lining of the gut and intestines. Most other emulsions (freeze dried and rehydrated) form individually separated oil droplets and can be used for intravenous injections. The emulsion according to the current invention cannot be given systemically but has the advantage of being localized to the area of administration. Like other emulsions it can be injected through very fine needles, e.g. 30G, and be accurately and precisely administered to specific organs.

In one of the examples given in WO2009056602 the applicant described an attempt to freeze dry and re-suspend the particles made from the hydrogels, without a second phase dispersed therein. This attempt failed and it was concluded that a coating of hyaluronic acid was needed in order to make the particles re-suspendable after freeze drying. Nor could the applicant find any guidance in the literature regarding how to freeze dry large particles made from hydrogels to obtain a dry material that could be resuspended in water to give individually separated particles. It was assumed that the particles, with their large surface areas and their multiple points for interactions, hydrophobic, hydrogen bonding and ionic, were too large for being re-suspendable after freeze drying.

Surprisingly, the applicant has found conditions, based on the use of a stabilizing agent (i.e. a cryoprotectant), allow for a rapid rehydration, within minutes, of large hydrogel emulsion particles. Even more surprising is that these rehydrated emulsions maintain their slow release properties and are essentially free from unwanted burst effects.

Preferably the stabilizing agent is a mono or oligosaccharide, for example glucose, lactose, maltose, maltodextrin, mannitol, trehalose, and sucrose, preferably mannitol, trehalose, and sucrose, and most preferably sucrose.

In a first series of experiments the applicant investigated the impact of different stabilizing agents (or cryoprotectants) on the particles described in WO2009056602, containing no oil, and these experiments excluded some of the protectants tested due to either a too slow rehydration or just because of the physical structure obtained after freeze drying, some gave foam like structures.

We also found the amount of cryoprotectant to be important for product performance and rehydration, and that there is an optimum in cryoprotectant concentration with respect to rehydration properties. A too low concentration did not give a homogenous cake, after freeze drying, and the concomitant rehydration became slow. A too high concentration gave a rapid rehydration but caused problems with respect to caught air bubbles in the rehydrated samples.

The stabilizing agent can be added as a solution, for example an aqueous solution.

In one embodiment, the solution has a concentration from 1-25% (w/w), preferably 5-20% (w/w). and most preferably 8-15% (w/w).

The ratio between the emulsion (before freeze drying) and the solution of the stabilizing agent is from 1 : 1 to 1 : 10, preferably 1 :1 to 1 :5. The stabilizing agent e.g. the mono- or oligosaccharides can be added to the aqueous phase prior to cross linking of the emulsion.

Alternatively, the stabilizing agent e.g. the mono- or oligosaccharides can be added to the aqueous phase after cross linking of the emulsion and before lypophilizing the fragments of chitosan hydrogel emulsion

If the stabilizing agent e.g. mono and oligosaccharides is added before the cross linking step, it may then give further stabilization and protection to sensitive molecules harboured in the aqueous phase, e.g. an enzyme, and also give further stabilization of the hydrogel emulsion system during freeze drying.

This means that it is possible to use different stabilizing agents i.e.. cryoprotectants in the particles and between the particles. The inner cryoprotectants (added before cross linking) stabilizes and protects sensitive molecules and or the oil droplets during freeze drying and the function of the outer cryoprotectant (added after cross linking) is essentially to prevent the particles to aggregate and to aid rehydration. The use of different concentrations and different cryoprotectants as inner and an outer cryoprotectants opens for manipulation of product properties. For coating it could be an advantage to keep the outer cryoprotectant concentration low or even excluded.

We found that a process in which the cryoprotectant was added to the dissolved chitosan phase prior to mixing with the oil phase gave a stable gel structure and a mechanically processable material. This material could also be freeze dried and rehydrated. We prefer to use mono- or oligosaccharides as cryoprotectants during freeze drying of chitosan stabilized emulsions in which the chitosan has been further cross linked.

Aliquots of 200 mg of chitosan hydrogel fragments or chitosan hydrogel emulsion fragments were placed in 3 ml silanized vials. Then 800 μΙ of the cryoprotectant solution, e.g. a 12% sucrose solution was added to each sample. The samples were vigorously shaken, using a vortex apparatus, for 30 seconds. Then the samples were frozen in a dry ice/ethanol bath for at least 2 hours. The samples were then transferred to a FreeZone Labconco 2.5 lyophilizer with a collector temperature of -84°C and lyophilized overnight.

The hydrogels of WO2009056602 and WO201 1 138155 offer in themselves a means to stabilize and increase the solubility of sensitive molecules or of molecules of poor solubility. The current invention further improves these properties and open for preparation of dry formulations and powders which can be used as such or in combination with other drugs and excipients. Taken together the freeze drying procedures discussed above can be used for obtaining emulsions in the form of a powder or cake. We foresee the use of such powders in a vast number of products. Areas for their potential use are in pharmaceutical, cosmetic, food, agrochemical or personal care compositions, as a vaccine, a drug delivery agent, a bulking agent, a thickener, a food additive, a probiotic delivery vehicle, a biomaterial component, a coating for medical devices, a paint additive, a paper or pulp additive or a drilling servicing fluid.

Lyophilization offers several advantages since not only stability is improved, it also allows for dry formulations like tablets, capsules, patches, coatings and more. Especially for oral delivery and patient convenience a pill or a capsule is a preferred formulation. Even from an economical point of view such formulations are preferred. There is a huge number of different tablets including sophisticated technology for giving sustained release. Even capsules have different properties and various types are frequently used for oral drug delivery. Commonly used capsules are gelatin capsules. Such capsules are sensitive to moisture and are not always suitable as drug carriers. More advanced capsules have a protecting layer in order to increase their resistance towards moisture. Drugs can also be administered by other dry formulation such as patches and chewing gum, e.g. nicotine gum. For of the above mentioned formulation types water could be problematic and a lyophilized powder would circumvent the problems associated with wet and moisture containing preparations. These lyophilized products may be formulated for e.g. oral, buccal, sublingual, rectal or vaginal delivery or as part of or a coating layer on an orthopedic device or a biomaterial for implantation. Examples of dosage forms are pills, capsules, blisters, powders, and enterocoated capsules. Emulsions may also be lyophilized and reconstituted but two commonly encountered problems with this are a relatively low drug load and a fast release of the drug upon reconstitution (burst effect). In order to avoid coalescence of oil droplets following the freeze drying procedure cryoprotectants are used. Commonly used cryoprotectants are e.g. mono and disaccharides. Desired properties of a freeze dried emulsion are short reconstitution time and maintenance of the characteristics of the original emulsion upon reconstitution e.g. particle size and release properties.

The lypohilized formulation is suitable for use as a vaccine, in drug delivery, in tissue augmentation, as a cell culture scaffold, for encapsulation of viable cells, in wound healing devices, in orthopaedics, as a biomaterial, for treating urinary incontinence or vesicoureteral reflux, in viscosurgery, in providing living cells to a host organism, as a cosmetic, as a bulking agent, as a thickener, as an additive in the food industry, as a glue, as a lubricants, or as a drilling servicing fluid.

The pharmaceutical composition comprising the lypohilized formulation generally includes pharmaceutically active ingredients, such as antiinflammatory agents, e.g., NSAIDs, steroids and opiates, anti-cancer agents, e.g. paclitaxel, doxorubicin and methotrexate, painkillers e.g. lidocain, bupivaccain, peptides, opiates, antibacterial agents e.g. penicillin, and chloramphencol, antipsychotic drugs, e.g. dopamine and its derivatives, and remoxipride, cholesterol lowering drugs e.g. various statins, modern drugs, so called biolocials, e.g. antibodies, antibody fragments and growth factors, nucleic acid DNA and RNA, their derivatives and analogous.

In one embodiment the active ingredient is cyclosporin A, which is is a very potent immune- suppressing drug which is used in transplantation medicine and which has a lot of side effects and is extremely toxic. Individuals receiving cyclosporine A orally require monitoring during administration. A very slow release of cyclosporin A, with no or very small burst effect, will significantly improve clinical treatment protocols. The current invention allows for such administration and some examples are given below. This type of slow release cannot be achieved by use of freeze dried, cryoprotected emulsions made by standard protocols.

As freeze dried powders these formulations can be mixed with other pharmaceutical excipients necessary for making e.g. tablet and capsule formulations. A freeze dried material could also be mixed/blended with other dry agents and compressed to e.g. an implantable biomaterial or device. Another feature of the invention is to enable slow releasing coatings. A material could be soaked or sprayed with the emulsion particles and then freeze dried. A possible application would be coatings leaking antibiotics or cytotoxic agents. Such materials are desired in surgery and drug eluting stents.

The invention also provides an immunological agent comprising the lypohilized formulation according to the invention and an antigen, wherein the antigen is optionally covalently bonded to the chitosan.

The present invention will now be described with reference to the following examples, which are not intended to be limiting. The words particles and fragments are used interchangeably.

Example 1

10.93 g of chitosan (degree of deacetylation 49%) was dissolved in 900 g water by addition of 2M HCI under stirring at room temperature. The pH was then adjusted to neutral with 1 M NaOH and the volume was adjusted to 1000 ml. Then 3,4-diethoxy-3-cyclobutene-1 ,2-dione (2.4 ml 10% solution in ethanol) was added drop wise and the solution was placed in a heating cabinet set to 40°C. After solidification and mechanical processing into particles of average size 10, 100, and 200 μιτι respectively, a smooth crushed hydrogel was obtained.

200 mg of the formed hydrogel fragments were weighed into a vial and 800 μΙ of 5% aqueous glucose was added. The content of the vial was thoroughly mixed and frozen in a dry ice/ethanol bath in a Dewar container. When frozen, the hydrogel suspension was freeze dried at reduced pressure to give a solid residue.

To the freeze dried and solid material 2 ml of Ringer's acetate, pH 5.0, was added and the mixture was vortexed and allowed to swell to give a fully rehydrated and resuspended hydrogel. Example 2

400 mg of the hydrogel fragments (200 μιτι) prepared according to Example 1 were placed in a 3 ml silanized glass vial. Then 600 μΙ of Ringer's acetate (pH 5) was added and the sample was mixed in a Vortex mixer. The vial was then frozen in a Dewar container with a dry ice/ethanol bath and then lyophilized over night to give a white powder in shape of a cake. For rehydration, 1 ml of water was added to the vial, and it was vortexed and left for several hours at room temperature. The vial was then turned sideways, and it was obvious that all the hydrogel had formed one solid block with water around it rather than the original individual particles in suspension.

Example 3

100 mg of the hydrogel fragments (10 μιτι) prepared according to Example 1 were added to each of four 3ml silanized glass vials. Two solutions of glucose (16.7 % and 33 % (w/w) respectively) were prepared. To two of the vials 150 μΙ of the 16.7 % solution was added and to the other to, 150 μΙ of the 33 % solution was added. The vials were mixed in a Vortex mixer and then frozen (as above) and lyophilized. After lyophilization the residue was a hard and foamy material.

Example 4

200 mg of the hydrogel fragments (Ι ΟΟμιτι), prepared according to Example 1 , were added to each of three 3 ml silanized glass vials. Three solutions of 25 % (w/w) of trehalose (A) purified water, (B) Ringer's acetate (pH5) and (C) PBS (pH 7.4) were prepared. To each of the three vials (A, B and C) 800 μΙ of one of the respective solutions was added. The vials were then mixed in a Vortex and then frozen as described above and lyophilized. After lyophilization, all vials had the same appearance: a white solid cake.

For rehydration, 2 ml of purified water was added to each of the vials and they were vortexed for 10 s. After one hour, the samples in A and B had regained their original fragment shape and were suspended in a liquid. In C, the fragments were not yet fully rehydrated (contained air bubbles) and were floating on top of the liquid phase. Staining of the particles with Trypan Blue followed by a mixing in Vortex and another 2.5 h of waiting timed revealed that A gave a homogenous suspension, B showed tendencies to aggregate, but only slightly and C showed a strong tendency of aggregation of the fragments.

Example 5

Three solutions (250 mg/ml) of sucrose (A), glucose (B) and trehalose (C) in Ringer's acetate (pH 5) were prepared. Another solution (100 mg/ml) of mannitol (D) in Ringer's acetate (pH 5) was prepared. Into each of eight 3 ml silanized glass vials, 200 mg of hydrogel particles (Ι ΟΟμιτι), prepared according to Example 1 , were added. To vials A1 and A2, 800 μΙ of sucrose solution was added. To B1 and B2, 800 μΙ of glucose solution was added. To vials C1 and C2, 800 μΙ of trehalose solution was added and to vials D1 and D2, 800 μΙ of mannitol solution was added. All vials were mixed thoroughly with a Vortex mixer and were then frozen as above and lyophilized.

Inspection of the freeze dried samples showed that sucrose, trehalose and mannitol give identical homogenous cakes of dried material. Glucose did not yield a cake, but rather a semi translucent foam. For rehydration, 2 ml of purified water was added to each vial, and they were immediately mixed in a Vortex mixer. The particles were stained with Trypan Blue to be easily distinguished.

The particles in vials A1 and A2 seemed to be rehydrated immediately to form homogenous suspensions. Some air bubbles initially remained inside or on the particles and thus there was a layer of floating particles on top of the suspensions, but after two hours they were completely gone.

Samples B1 and B2 did not rehydrate nearly as fast as the other samples. Large aggregates of material stuck to the inner walls of the vials. After two hours though, the rehydration seemed to be complete and the particles had been dispersed in the suspension. Rehydration of the particles lyophilized in the presence of glucose seems possible but did not work as well as with sucrose. Samples C1 and C2 were rehydrated rapidly, but the process was slower than for samples A1 and A2. The last air bubbles were not gone after 2 hours and the suspension was not homogenous, and the hydrogel particles were still on top of the liquid phase.

Samples D1 and D2 were rehydrated, but the lyophilized cake showed a tendency to break up into fragments of an approximate size of 1 mm rather than to break up into individually separated particles. The rehydration process was markedly slower than for A1 or A2. After two hours, almost all trapped air bubbles were gone, but the particles aggregated, where each aggregate contained approximately 10 particles.

Example 6

Four solutions of sucrose in water (250 [A], 188 [B], 125 [C] and 63 [D] mg/ml respectively) were prepared. To each of eight 3 ml silanized glass vials 200 mg particles (100 μιτι) prepared according to Example 1 were added. To vials A1 and A2, 800 μΙ of solution A was added. To vials B1 and B2, 800 μΙ of solution B was added. To vials C1 and C2, 800 μΙ of solution C was added and to vials D1 and D2, 800 μΙ of solution D was added. All samples were mixed thoroughly with a Vortex mixer to ensure complete homogeneity of the suspensions. The vials were then frozen and lyophilized. After lyophilization, the samples were observed. The A, B and C samples contained homogenous lyophilized cakes. In the D vials the cakes were much less homogenous.

For rehydration, 2ml of water was added to each vial. The vials were also mixed in a Vortex mixer to break up potential aggregates.

The material in all vials was quickly rehydrated but the process was faster in vials C1 and C2 compared to the A and B vials. The amount of trapped air bubbles caught inside or on the particles was significantly lower in the C and D vials than in the corresponding A and B vials.

Example 7

200 mg of particles (100 μιτι) prepared according to Example 1 were placed in a 3 ml silanized glass vial. Then 800 μΙ of a sucrose solution (188 mg/ml) was added, and the vial was mixed in a Vortex mixer. The vial was then frozen and lyophilized as above.

For rehydration of the particles, 2 ml of purified water was added to the vial. Then 200 μΙ of a Trypan Blue solution (0.04 mg/ml) was added to stain the particles. A drop of the suspension was put on a slide and the rehydration was observed over time in a microscope.

Example 8 Preparation of particles containing a protein and a cryoprotectant

Preparation of a chitosan stock solution:

1.0 g of chitosan (DD 49%, Viscosity 365 mPas) was dissolved in 40 ml of water using 1 ml 2M HCI. Once the chitosan was fully dissolved, the pH was adjusted to 6.6 with 1 M NaOH and the total mass of the solution was adjusted to 50g to give a 2% chitosan solution.

Four solutions, A through D, were prepared as described below

(A) 15.0 mg of ovalbumine (OVA) was dissolved in 15.0 ml of purified water. 7.0 g of this solution was then mixed with 7.0 g of the chitosan solution above to give a 1 % chitosan solution containing OVA (1 mg/g).

(B) 1.5 g of sucrose was dissolved in 15.0 g of purified water. 7.0 g of this solution was then mixed with 7.0 g of the chitosan solution above to give a 1 % chitosan solution containing sucrose (50 mg/g).

(C) 3.0 g of sucrose was dissolved in 15.0 g of purified water. 7.0 g of this solution was then mixed with 7.0 g of the chitosan solution above to give a 1 % chitosan solution containing sucrose (100 mg/g).

(D) 7.5 g of sucrose was dissolved in 15.0 g of purified water. 7.0 g of this solution was mixed with 7.0 g of the chitosan solution above to give a 1 % chitosan solution containing sucrose (250 mg/g). Then 3,4-diethoxy-3-cyclobutene-1 ,2-dione (34μΙ 10% solution in ethanol) was added solution was added to each of the solutions A through D above. After mixing, the solutions were sealed in glass vials with snap on lids and left in a heating cabinet (40°C) for two weeks for maturation. The hydrogels formed were then mechanically processed to give 200 μιτι fragments.

To four 3 ml silanized glass vials (A-D) 200 mg hydrogel particles gel of the corresponding type were added together with 800 μΙ of a sucrose solution (12 % (w/w)). The vials were then thoroughly mixed, frozen and lyophilized as earlier described.

For rehydration 800 μΙ of purified water was added into vial A, which gave a quick and homogenous rehydration.

Addition of 2 ml of purified water to vials B through D gave quick and homogenous rehydration in all vials. No difference in rehydration time or appearance was detected between the vials B through D. The ovalbumine containing hydrogel particles were thus rehydrated as efficiently as the hydrogel particles without ovalbumine.

Example 9

A sample (100 mg) including 2.5 g stock solution and 7.5 g activated chitosan was frozen and freeze dried analogously to example 1.

Preparation of chitosan solution 1.25% w/v (100 mL)

Chitosan DD 48% (1.25 g) was added to a beaker equipped with a stir bar. Water (approximately 80 mL) was added and pH adjusted by dropwise addition of hydrochloric acid (2M (aq) ) under constant stirring. When the chitosan had dissolved the pH was adjusted to 6.6 and the volume was adjusted to 100 mL.

Stock solution:

Polysorbate 60 (380 mg) was stirred at room temperature in rape seed oil (25 g) until a transparent solution had formed

After freeze drying a white residue was obtained. Addition of water gave the rehydrated emulsion. Example 10

1.04 g of mannitol was added to 24.91 g of a 1.25% chitosan solution (50% degree of deacetylation). 73 μΙ of a 10% solution of 3,4-diethoxy-3-cyclobutene-1 ,2-dione on ethanol was added and the mixture was stirred for 30 minutes. Budesonide (10.25 mg) was dissolved in 5.13 ml tween 80/miglyol (surfactant to Oil, S/O=0.2 w/w). 4.622 g of the budesonide solution was mixed with 13.86 g of the chitosan solution. The mixture was homogenized in a high pressure homogenizer for 15 minutes at a pressure of 1000 Bars. The sample was placed in a 15 mL Falcon tube and centrifuged for 1 minute at 1000 rpm and then placed in a heating cabinet at 40°C for 17 days.

After mechanical processing into particles of average size 200 μιτι, a smooth crushed viscoelastic emulsion was obtained. 200 mg of this emulsion was weighed into a vial and 800 μΙ of 12% (w/w) aqueous sucrose was added. The content of the vial was thoroughly mixed and frozen. When frozen, the suspended emulsion was lyophilized at reduced pressure to give a solid residue.

To the freeze dried and solid material 1 ml of water for injection was added and the mixture was vortexed and allowed to swell to give a fully rehydrated and resuspended emulsion.

Example 11 A

44.98 mg of cyclosporin A was placed in a 50 mL round bottomed flask and 1.0 mL of ethanol was added. After evaporation to dryness under nitrogen a mixture of tween 80 and miglyol (1 1.251 g S/O=0.2, containing 50 μg Oil Red O/ml oily phase) was added and the mixture was stirred overnight.

41 .06 g chitosan (1 .25% w/w, 50% degree of deacetylation) was treated with 125 μΙ of a 10% solution of 3,4-diethoxy-3-cyclobutene-1 ,2-dione in ethanol for 30 minutes.

3.45 g of sucrose was added to 31 .1 g of the preactivated chitosan solution. Then 10.37 g of the cyclosporin A containing oily phase was mixed with the chitosan solution. The mixture was then homogenized by 5 passages through the high pressure homogenizer at a pressure of 1500 Bars. The resulting emulsion was placed in a 50 ml Falcon tube and was quickly centrifuged at 1000 rpm, and then placed in a heating cabinet at 40°C.

The formed solidified emulsion, Viscomulsion, was then mechanically processed into 200 μιτι fragments. Aliquots of 200 mg of the crushed emulsion were placed in silanized 3 ml vials and 800 μΙ of a 12% (w/w) sucrose solution was added to each vial. The samples were frozen (dry ice/ethanol bath) and lyophilized overnight. The resulting dry emulsion samples were reconstituted to its original volume by adding 874μΙ of water.

The dissolution profiles of the compositions according to examples 1 1A are shown below in Figure 1 (0.2-0.5 g emulsion in 200 ml phosphate buffered saline USP dissolution apparatus 2). It can be seen that the emulsion and the reconstituted emulsion have the same slow release profile. In other words, the lyophilization and reconsitutrion processes have taken place while retaining the beneficial properties of the formulation. Comparative example 11B

24.7 mg of Cyclosporine A was placed in a 50 mL round bottomed flask and 1 .0 mL of ethanol was added. After evaporation to dryness under nitrogen a mixture of miglyol and tween 80 (6.173 g S/O=0.2, containing 50 μg Oil Red O/ml oily phase) was added and the mixture was stirred for 3 hours. 5.55 g of this solution was added to 16.64 g of sucrose solution (10% w/w). The resulting mixture was homogenized in a high pressure homogenizer at a pressure of 1500 Bars for 15 minutes. 1 ml aliquots of the resulting emulsion was placed in a silanized vials and frozen and lyophilized as described above. When the dry emulsion was reconstituted with water a phase separated mixture was obtained, seen as a red oil layer floating on top of an aqueous phase. This sample was of not practical use and an attempt to take a representative sample for release studies indicated a 100% burst effect (expected since Cyclosporine A will be found in the separated oil phase)

The appearance of the reconstituted aqueous emulsion 1 1 B , without chitosan in the aqueous phase, is shown in to the left in Figure 2. A reconstituted emulsion ,1 1A, is shown to the right in the same figure.

Example 12

37.74 g of a 1 .25% (w/w) chitosan solution that had been pre-reacted with 3,4-diethoxy-3-cyclobutene- 1 ,2-dione (134 μΙ of a 10% solution in ethanol) for 20 minutes as described above was mixed with a mixture of miglyol and tween 80 (12.58 g S/O=0.15) containing 50,3 mg of cyclosporin A. The mixture was then homogenized by 4 passages through the high pressure homogenizer at a pressure of 1200 Bars. The resulting emulsion was placed in a 50 ml Falcon tube and was quickly centrifuged at 3000 rpm, and then placed in a heating cabinet at 40°C.

The formed solidified emulsion, Viscomulsion, was then mechanically processed into 200 μιτι fragments. Aliquots of 500 mg of the crushed emulsion were placed in 10 ml vials and 2 ml of a 12% (w/w) sucrose solution was added to each vial. The samples were frozen (dry ice/etanol bath) as described above, and lyophilized overnight. The lyophilized emulsion could easily be reconstituted with purified water to give a homogenous sample. Embodiments of the invention that may be mentioned include:

1. A process for preparing a lypohilized formulation, comprising the steps of:

(i) providing a chitosan hydrogel the chitosan hydrogel being obtainable by providing a chitosan having a degree of deacetylation between 30 and 75%, wherein the chitosan is randomly deacetylated, and a cross-linking agent, wherein the molar ratio of the cross-linking agent to chitosan is 0.2: 1 or less based on the number of functional groups in the cross-linking agent and the number of deacetylated amino groups in the chitosan in an aqueous solution, and cross-linking the composition and isolating the resultant chitosan hydrogel; and

(ii) optionally processing the chitosan hydrogel to give fragments; and

(iii) lypophilizing the chitosan hydrogel or the fragments of chitosan hydrogel.

2. A process for preparing a lypohilized formulation according to 1 , wherein the chitosan has a degree of deacetylation of between 35 and 55%.

3. A process for preparing a lypohilized formulation according to 1 or 2, wherein the chitosan, prior to cross-linking, has a weight average molecular weight of 10-500 kDa.

4. A process for preparing a lypohilized formulation according to any preceding statement, wherein the cross-linking agent is bifunctional.

5. A process for preparing a lypohilized formulation according to any preceding statement, wherein the cross-linking agent has functional groups selected from esters, Michael acceptors, epoxides and combinations thereof.

6. A process for preparing a lypohilized formulation according to any preceding statement, wherein the cross-linking is performed at a pH between 6 and 10.

7. A process for preparing a lypohilized formulation according to any preceding statement, wherein the lypohilizing is performed in the presence of a stabilizing agent.

8. A process for preparing a lypohilized formulation according to 7, wherein the stabilizing agent is a monosaccharide or an oligosaccharide.

9. A lypohilized formulation obtainable by the process of 1 to 8.

10. A lypohilized formulation as described in 8 for use as a vaccine, in drug delivery, in tissue augmentation, as a cell culture scaffold, for encapsulation of viable cells, in wound healing devices, in orthopaedics, as a biomaterial, for treating urinary incontinence or vesicoureteral reflux, in viscosurgery, in providing living cells to a host organism, as a cosmetic, as a bulking agent, as a thickener, as an additive in the food industry, as a glue, as a lubricants, or as a drilling servicing fluid.

1 1. A pharmaceutical composition comprising the lypohilized formulation as described in 9 and pharmaceutically active ingredients.

12. An immunological agent comprising the lypohilized formulation as defined in 9 and an antigen, wherein the antigen is optionally covalently bonded to the chitosan.

13. A process for preparing a lypohilized formulation, comprising the steps of:

(i) providing a composition comprising (1 ) chitosan hydrogel comprising cross-linked chitosan and water; and (2) a liquid dispersed in the hydrogel;

(ii) optionally processing the composition to give fragments; and

(ii) lypohilizing the composition or fragments of the composition.

14. A process for preparing a lypohilized formulation according to 13, wherein the chitosan hydrogel additionally comprises a water-miscible solvent; or a water-soluble preservative, salt, buffer, drug, vitamin, cosmetic, or a mixture thereof.

15. A process for preparing a lypohilized formulation according to 13 or 14, wherein the liquid dispersed in the hydrogel is an oil, a pharmaceutical active agent or excipient, a cosmetic ingredient, a vitamin, a food, an agrochemical active agent or excipient, a personal care ingredient, or a mixture thereof.

16. A process for preparing a lypohilized formulation according to any one of 13 to 15, wherein the dispersed liquid is present in an amount of 5 to 30% by weight based on the total weight of the composition.

17. A process for preparing a lypohilized formulation according to any one of 13 to 16, comprising one or more water-insoluble active ingredients solubilised in the dispersed liquid.

18. A process for preparing a lypohilized formulation according to any one of 13 to 17, wherein the water-insoluble active ingredient is a drug or vitamin.

19. A process for preparing a lypohilized formulation according to any one of 13 to 18, wherein the compisition of step (i) is prepared by a process comprising

providing a cross-linkable chitosan composition comprising chitosan and water; dispersing a liquid in the cross-linkable chitosan composition; and cross-linking the chitosan with a cross-linking agent to form a hydrogel.

20. The process according to 19 wherein the chitosan has a degree of deacetylation of 30 to 75%.

21. The process according to 19 wherein the chitosan has a degree of deacetylation of 40 to 60%.

22. The process according to any one of claims 19 to 21 wherein the chitosan is randomly deacetylated.

23. The process according to any one of 19 to 22 wherein the chitosan, prior to cross-linking, has a molecular weight of 10 to 500 kDa.

24. The process according to any one of 19 to 23 wherein the molar ratio of the cross-linking agent to chitosan is 0.2: 1 or less based on the number of functional groups in the cross-linking agent and the number of deacetylated amino groups in the chitosan.

25. The process according to any one of 19 to 24 wherein the chitosan is present in the cross- linkable chitosan composition in an amount of 3% by weight or less based on the total weight of chitosan and water in the hydrogel.

26. The process according to any one of 19 to 25 wherein the cross-linking is performed at acidic pH.

27. The process according to any one of 19 to 25 wherein the chitosan is randomly deacetylated and has a degree of deacetylation of 30 to 75%, and wherein the cross-linking is performed at a pH from 6 to 10.

28. The process according to any one of 19 to 27 wherein the cross-linking agent is added to the cross-linkable chitosan composition before the liquid is dispersed in the chitosan composition.

29. A lypohilized formulation obtainable by the process of 13 to 28.

30. The lyopohilized formulation according to 29 for use as a vaccine, a drug delivery agent, a cosmetic, a bulking agent, a thickener, a food additive, a paint additive, a paper or pulp additive or a drilling servicing fluid.

31. A pharmaceutical, cosmetic, food, agrochemical or personal care composition comprising the composition as defined in 29.