WO2014096257A1

WO2014096257A1 - Freeze-dried cross-linked hyaluronic acid sponge

Info

Publication number: WO2014096257A1
Application number: PCT/EP2013/077488
Authority: WO
Inventors: Ole Moeller DALL; Morten Jonas Maltesen
Original assignee: Novozymes Biopharma Dk A/S
Priority date: 2012-12-19
Filing date: 2013-12-19
Publication date: 2014-06-26

Abstract

The present invention relates to improved freeze-dried sponges of DVS cross-linked hyaluronic acid hydrogel which has superior swelling properties, an improved method for making such sponges, as well as compositions and uses of such sponges.

Description

Freeze-dried cross-linked hyaluronic acid sponge

FIELD OF THE INVENTION

BACKGROUND OF THE INVENTION

Controlled release (CR) of a drug is a mechanism whereby a drug is released slowly to the environment over time.

The advantages of controlled release medicaments are that they can often be taken less frequently than immediate-release formulations of the same drug, and additionally, they keep steadier levels of the drug in the bloodstream.

Hyaluronic acid (HA) is a natural and linear carbohydrate polymer belonging to the class of non-sulfated glycosaminoglycans. It is composed of beta-1 ,3-/V-acetyl glucosamine and beta-1 ,4-glucuronic acid repeating disaccharide units with a molecular weight (MW) up to approximately 10 MDa. HA is present in hyaline cartilage, synovial joint fluid, and skin tissue, both dermis and epidermis.

WO 2006/056204 describes a method of producing homogeneous hydrogels comprising hyaluronic acid cross-linked with divinyl sulfone (DVS).

US 2007/0036745 also discloses a method of cross-linking hyaluronic acid with divinyl sulfone (DVS).

SUMMARY OF THE INVENTION

The present invention provides improved freeze-dried sponges of DVS cross-linked hyaluronic acid hydrogel which have superior swelling properties, an improved method for making said sponges, as well as compositions and uses of said sponges.

The "swelling capacity" or "degree of swelling" of a freeze-dried sponge is an important parameter in several applications; the two terms are used interchangeably herein. For example, in surgical applications a low swelling capacity provides an advantage when either a swelled sponge material or a dry sponge is administered into human or animal tissue due to its lower bulk volume at its fully swelled stage. This is thought to lead to a lower level of irritation due to the lower physical pressure from the swelled sponge inside the tissue which in turn provides a better comfort for the patient/test animal.

It may also be an advantage to use a material with lower swelling properties then the freeze-dried sponge is used as an in vivo drug delivery matrix or depot loaded with an active pharmaceutical ingredient (API), where a low water content in the depot likely leads to a slower or more linear release of the API, thereby improving its release profile. Accordingly, in a first aspect, the invention provides freeze-dried sponges comprising divinyl sulfone cross-linked hyaluronic acid which has a HA:DVS weight ratio in the range of 8:1 to 100:1 , wherein said sponge has a swelling capacity of no more than 4,200% (w/w) in a 10mM phosphate buffered saline solution at pH 6.9, when measured 50 minutes after immersion of the sponge into the buffer.

A second aspect of the invention relates to powders comprising a crushed, pulverized, micronized, milled or finely ground freeze-dried sponge as defined in the first aspect.

In a third aspect, the invention relates to methods of of producing a freeze-dried sponge comprising DVS cross-linked hyaluronic acid, said method comprising

(a) providing an alkaline solution of hyaluronic acid, or salt thereof;

(b) adding DVS to the solution of step (a) with stirring, wherein the HA:DVS dry weight ratio is in the range of 9:1 to 90:1 , whereby the hyaluronic acid, or salt thereof, is crosslinked with the DVS to form a gel;

(c) treating the gel of step (b) with a buffer, wherein the gel is neutralized, swells and forms a hydrogel comprising hyaluronic acid, or salt thereof, crosslinked with DVS; and

(d) freeze-drying the hydrogel into a freeze-dried sponge,

wherein said sponge has a swelling capacity of no more than 4,200% (w/w) in a 10mM phosphate buffered saline solution at pH 6.9, when measured 50 minutes after immersion of the sponge into the buffer.

Further aspects of the invention relate to compositions and uses of the freeze-dried sponges of the invention.

Earlier aspects of the invention include methods of producing a freeze-dried sponge comprising cross-linked hyaluronic acid and an active pharmaceutical ingredient (API) comprising

(a) providing a gel of cross-linked hyaluronic acid;

(b) treating the gel of step (a) with a buffer to form a hydrogel;

(c) incorporating an active pharmaceutical ingredient (API) into the hydrogel; and

(d) freeze-drying the mixture of step (c) into a freeze-dried sponge. DESCRIPTION OF THE FIGURES

Figure 1 : Shows the re-swelling in PBS buffer over time of freeze-dried sponges prepared from Gel ID 1 in Example 1 and a comparable hydrogel made by a method in the prior art (US 2007/0036745 Example 9).

Figure 2: Shows the re-swelling in PBS buffer over time of freeze-dried sponges prepared from Gel ID 2 in Example 1 and a comparable hydrogel made by a method in the prior art (US 2007/0036745 Example 9). Figure 3: Shows the re-swelling in PBS buffer over time of freeze-dried sponges prepared from Gel ID 3 in Example 1 and a comparable hydrogel made by a method in the prior art (US 2007/0036745 Example 9).

Figure 4: The curve shows the release of a drug (triamcinolone hexacetonide) over 25 days as determined in Example 2.

Figure 5: Shows the viscoelastic properties of Gel ID 1 of Example 1 , measured as outlined in Example 3.

Figure 6: Shows the viscoelastic properties of the prior art gel corresponding to Gel ID

1 of Example 1 , measured as outlined in Example 3.

Figure 7: Shows the viscoelastic properties of Gel ID 2 of Example 1 , measured as outlined in Example 3.

Figure 8: Shows the viscoelastic properties of the prior art gel corresponding to Gel ID

2 of Example 1 , measured as outlined in Example 3.

Figure 9: Shows the viscoelastic properties of Gel ID 3 of Example 1 , measured as outlined in Example 3.

Figure 10: Shows the viscoelastic properties of the prior art gel corresponding to Gel ID

3 of Example 1 , measured as outlined in Example 3.

Figures 1 1A-B: Show scanning electron microscopy pictures of Gel ID 1 and the corresponding prior art gel at x60 and x250 magnifications.

Figures 12A-B: Show scanning electron microscopy pictures of Gel ID 2 and the corresponding prior art gel at x60 and x250 magnifications.

Figures 13A-B: Show scanning electron microscopy pictures of Gel ID 3 and the corresponding prior art gel at x60 and x250 magnifications. DETAILED DESCRIPTION OF THE INVENTION

Herein we provide improved freeze-dried sponges with DVS cross-linked hyaluronic acid which has better swelling properties than already available similar sponges. Accordingly, the first aspect of the invention relates to freeze-dried sponges comprising divinyl sulfone cross-linked hyaluronic acid which has a HA:DVS weight ratio in the range of 8:1 to 100:1 , wherein said sponge has a swelling capacity of no more than 4,200% (w/w) in a 10mM phosphate buffered saline solution at pH 6.9, when measured 50 minutes after immersion of the sponge into the buffer. A second aspect relates to powders comprising a crushed, pulverized, micronized, milled or finely ground freeze-dried sponge as defined in the first aspect.

In a third aspect, the invention also relates to methods of producing the freeze-dried sponges of the first aspect.

The invention further relates to a new type of depot formulation with cross-linked hyaluronic acid and a drug. The formulation is able to deliver slow release of a drug compound over a period of at least 14 days. The formulation is based on cross-linked hyaluronic acid hydrogels mixed with a suspension of a drug. The mixture is then freeze-dried into a form of sponge/fabric/cloth.

The drug may be administrated locally in the body to a specific type of tissue without having a systemic effect of the drug. Alternatively, the drug may be placed in an area in the body with very high fluid circulation to give a systemic effect of the released drug.

Hyaluronic acid

"Hyaluronic acid" is defined herein as an unsulphated glycosaminoglycan composed of repeating disaccharide units of N-acetylglucosamine (GlcNAc) and glucuronic acid (GlcUA) linked together by alternating beta-1 ,4 and beta-1 ,3 glycosidic bonds. Hyaluronic acid is also known as hyaluronan, hyaluronate, or HA. The terms hyaluronan, hyaluronic acid, and HA are used interchangeably herein.

The level of hyaluronic acid may be determined as known in the art, e.g., by using the modified carbazole method (Bitter and Muir, 1962, Anal Biochem. 4: 330-334).

In a preferred embodiment, the hyaluronic acid used according to the present invention is of a very pure quality.

Rooster combs are a commercial source for hyaluronan. Microorganisms are an alternative source. US 4,801 ,539 and EP 0694616 disclose fermentation methods for preparing hyaluronic acid involving a strain of Streptococcus zooepidemicus.

In a preferred embodiment, the hyaluronic acid or salt thereof is of microbial origin, preferably recombinantly produced. In a preferred embodiment the hyaluronic acid or salt thereof is produced by a Gram-positive bacterium such as Bacillus.

In a most preferred embodiment, the hyaluronic acid or salt thereof is produced by Bacillus subtilis. The hyaluronic acid or salt thereof may be produced as disclosed in WO 03/054163.

Hyaluronan synthases have been described from vertebrates, bacterial pathogens, and algal viruses (DeAngelis, P. L, 1999, Cell. Mol. Life Sci. 56: 670-682). WO 99/23227 discloses a Group I hyaluronate synthase from Streptococcus equisimilis. WO 99/51265 and WO 00/27437 describe a Group II hyaluronate synthase from Pasturella multocida. Ferretti et al. disclose the hyaluronan synthase operon of Streptococcus pyogenes, which is composed of three genes, hasA, hasB, and hasC, that encode hyaluronate synthase, UDP glucose dehydrogenase, and UDP-glucose pyrophosphorylase, respectively (Proc. Natl. Acad. Sci. USA. 98, 4658-4663, 2001 ). WO 99/51265 describes a nucleic acid segment having a coding region for a Streptococcus equisimilis hyaluronan synthase.

The host cell may be any Bacillus cell suitable for recombinant production of hyaluronic acid. The Bacillus host cell may be a wild-type Bacillus cell or a mutant thereof. Bacillus cells useful in the practice of the present invention include, but are not limited to, Bacillus agaraderhens, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells. Mutant Bacillus subtilis cells particularly adapted for recombinant expression are described in WO 98/22598. Non- encapsulating Bacillus cells are particularly useful in the present invention.

Since the hyaluronan of a recombinant Bacillus cell is expressed directly to the culture medium, a simple process may be used to isolate the hyaluronan from the culture medium. First, the Bacillus cells and cellular debris are physically removed from the culture medium. The culture medium may be diluted first, if desired, to reduce the viscosity of the medium. Many methods are known to those skilled in the art for removing cells from a culture medium, such as centrifugation or microfiltration. The remaining supernatant may then be filtered, such as by ultrafiltration, in order to concentrate and remove small molecule contaminants from the hyaluronan. Various purification steps may then be performed depending on the required purity of the final product. The hyaluronan may be dried or concentrated by using evaporative techniques known to the person skilled in the art, such as lyophilization or spray drying.

Salts of hyaluronic acid

Any salt of hyaluronic acid may be used according to the present invention. In a preferred embodiment, the salt of hyaluronic acid is an inorganic salt, preferably sodium hyaluronate, potassium hyaluronate, ammonium hyaluronate, calcium hyaluronate, magnesium hyaluronate, zinc hyaluronate, or cobalt hyaluronate.

HA Molecular weight

Any molecular weight may be used according to the invention. Various molecular weight fractions of hyaluronic acid have been described as advantageous for specific purposes.

In a preferred embodiment, the hyaluronic acid or the salt thereof has an average molecular weight in the range of 100-5,000 kDa; more preferably an average molecular weight in the range of 200-4,500 kDa; more preferably an average molecular weight in the range of 300-4,000 kDa; more preferably an average molecular weight in the range of 400-3,500 kDa; more preferably an average molecular weight in the range of 500-3,000 kDa; and even more preferably, an average molecular weight in the range of 600-2,500 kDa. The molecular weight may be determined as known in the art.

Derivatized HA

The hyaluronic acid may be derivatized before it is cross-linked. The Hyaluronic acid may be derivatized as known in the art, e.g., by acrylation (see WO 2007/106738) or by making aryl/alkyl succinic anhydride HA derivatives. Hydrogels

The present invention relates to the use of a freeze-dried sponge based on cross-linked hyaluronic acid hydrogels mixed with a suspension of a drug. The hydrogels are made in the following way:

(a) provide a gel of cross-linked hyaluronic acid; and

(b) treat the gel of step (a) with a buffer to form a hydrogel.

The initial concentration of hyaluronic acid, or a salt thereof, in the method of the present invention, influences the properties of the resulting cross-linked gel, and of the swollen hydrogel.

Therefore, a preferred embodiment of the invention relates to a method, wherein an alkaline solution comprises dissolved hyaluronic acid, or salt thereof, in a concentration of between 0.1 % - 40% (w/v); preferably in a concentration of between 0.1 % - 35% (w/v); preferably in a concentration of between 0.1 % - 30% (w/v); preferably in a concentration of between 0.1 % - 25% (w/v); preferably in a concentration of between 0.1 % - 20% (w/v); preferably in a concentration of between 0.1 % - 15% (w/v); preferably in a concentration of between 0.1 % - 10% (w/v); preferably in a concentration of between 0.2% - 10% (w/v); preferably in a concentration of between 0.3% - 10% (w/v); preferably in a concentration of between 0.4% - 10% (w/v); preferably in a concentration of between 0.5% - 10% (w/v); preferably in a concentration of between 0.6% - 10% (w/v); preferably in a concentration of between 0.7% - 10% (w/v); preferably in a concentration of between 0.8% - 10% (w/v); preferably in a concentration of between 0.9% - 10% (w/v); in particular in a concentration of between 1 .0% - 10% (w/v).

The pH value during the cross-linking reaction also influences the outcome, so in a preferred embodiment, the invention relates to a method, wherein the alkaline solution comprises dissolved sodium hydroxide in a concentration of between 0.001 - 2.0 M, even more preferably, the alkaline solution has a pH of from 9.0 to 12.0.

It is also noteworthy that the concentration of the cross-linking agent has a profound impact on the resulting gels.

Consequently, a preferred embodiment of the invention relates to a freeze-dried sponge which has a HA:DVS weight reatio, or to a method, wherein the DVS cross-linker is added to the solution of hyaluronic acid in a HA:DVS weight ratio, in the range of 9:1 to 90:1 (dry weight); preferably in the range of 10:1 to 80:1 ; more preferably in the range of 1 1 :1 to 80:1 ; even more preferably in the range of 12: 1 to 70:1 ; still more preferably in the range of 13:1 to 60:1 ; yet more preferably in the range of 14:1 to 50:1 ; more preferably in the range of 15:1 to 40:1 ; most preferably in the range of 16:1 to 30:1 (dry weight). An initial period of stirring during and/or immediately after adding the cross-linker to the HA-solution is desirable to achieve satisfactory gelling; preferably the stirring is continued for a period of between 1 - 180 minutes.

A heating step or a curing step may be beneficial after addition of the cross-linker to the solution. In a preferred embodiment we claim:

provide an alkaline solution of hyaluronic acid, or a salt thereof;

add a cross-linker, e.g., DVS to the solution, whereby the hyaluronic acid, or the salt thereof, is cross-linked with the DVS to form a gel;

heat the gel to a temperature in the range of 20°C - 100°C and maintaining the temperature in this range for a period of at least 5 minutes; preferably the solution is heated to a temperature in the range of 25°C - 80°C, more preferably in the range of 30°C - 60°C, and most preferably in the range of 35°C - 55°C; and

treat the gel with a buffer, whereby the gel swells and forms a hydrogel.

Preferably, the heating of the gel is carried out for a period of at least 10 minutes; more preferably for a period of at least 15, 30, 45, 60, 75, 90, 105, 120, 135, 150, 165 or 180 minutes.; preferably without stirring.

Many types of buffers may be suitable for the swelling and neutralizing of the cross- linked gel of the invention. Any physiologically accepted buffer may be used.

In a preferred embodiment, the buffer may be chosen from the group consisting of acetate, sulphate, phosphate, citrate, ammonium, succinate, diethylammonium, imidazole,

Tris, maleate, tetrabutylammonium, barbital, tris(hydroxymethyl)aminomethane, borate, HEPES, glycine, diethanolamine, phthalate, histidine, MES, PBS, and glycylglycine.

In a preferred embodiment, the buffer is PBS (phosphate buffered saline). PBS is a buffer solution commonly used in biological research. It is a water-based salt solution containing sodium chloride, and sodium phosphate. The buffer's phosphate groups help to maintain a constant pH. The osmolarity and ion concentrations of the solution usually match those of the human body (isotonic).

In a preferred embodiment, the buffer comprises a buffer with a pH value in the range of 2.0 - 8.0, preferably in the range of 5.0 - 8.0.

Optimally, a suitable buffer is chosen with a pH value, which results in the swollen hydrogel having a pH value as close to neutral as possible.

In the swelling step, the buffer must have a sufficient volume for it to accommodate the swelling gel until the gel is fully swollen. Accordingly, in a preferred embodiment of the method of the invention, the buffer has a volume of at least 3 times the volume of the gel.

The hydrogel formed may be washed with water, or washed with water and a buffer.

Preferably, the hydrogels are prepared as described in Example 2 in WO 2006/056204.

The hydrogel may be micronized into particles as known in the art. The particle size will typically be in the range of from 0.1 micro-meter to 1 ,000 micro-meter. Active pharmaceutical ingredient (API)

In a preferred embodiment, the freeze-dried sponge of the invention also comprises an active pharmaceutical ingredient and, accordingly, the method comprises comprises a step of incorporating an active pharmaceutical ingredient into the hydrogel prior to freeze-drying.

Preferably the active pharmaceutical ingredient is a peptide, a protein, a hormone, an antibiotic, a fungicide, an analgesic, a vaccine, an anti-inflammatory drug, monoclonal antibodies, or stem cells; even more preferably the active pharmaceutical ingredient is a steroid such as dexamethasone, triamcinolone, triamcinolone acetonide, or triamcinolone hexacetonide, or an analgesic drug, or a nonsteroidal anti-inflammatory drug such as diclofenac, naproxen, or ibuprofen. The active pharmaceutical ingredient (API) of interest may be any compound known in the art.

The API will typically be administered by, e.g., topical administration or by injected administration such as subcutaneous administration, intra muscular administration, intra articular administration, or intra dermal administration, or the API will be inserted/applied during any surgical procedure, or the API will be administered by any other route as known in the art.

In a preferred embodiment, the active pharmaceutical ingredient (API) is a peptide, a protein, a hormone, an antibiotic, a fungicide, an analgesic, a vaccine, an anti-inflammatory drug, monoclonal antibodies, or stem cells.

In a preferred embodiment, the active pharmaceutical ingredient is an anti-inflammatory drug such a steroid such as dexamethasone, triamcinolone, triamcinolone acetonide, or triamcinolone hexacetonide, or an analgesic drug, or a NSAID (nonsteroidal anti-inflammatory drug) such as diclofenac, naproxen, or ibuprofen.

Triamcinolone is a long-acting synthetic corticosteroid. Triamcinolone is used to treat several different medical conditions, such as eczema, psoriasis, arthritis, allergies, ulcerative colitis, lupus, sympathetic ophthalmia, temporal arteritis, uveitis, and ocular inflammation.

Alcohols

The API may, prior to incorporation into the hydrogel, be dissolved in an alcohol or in water.

Any alcohol known in the art may be used, in particular the alcohol may be a mono- alcohol, a di-alcohol, a tri-alcohol, or a poly-alcohol.

The alcohol may be a mono-alcohol such as ethanol, or the alcohol may be a di-alcohol such as propylene glycol (1 ,2-propanediol), or the alcohol may be a tri-alcohol such as propantriol (glycerine or glycerol).

The alcohol may also be a polyethylene glycol (PEG). Polyethylene glycol (PEG) is also classified as a polyether. The structure of PEG is: HO-CH₂-(CH₂-0-CH₂-)_n-CH₂-OH

PEG is also known as polyethylene oxide (PEO) or polyoxyethylene (POE), depending on its molecular weight.

In a preferred embodiment, the alcohol may be a mixture of two or more alcohols, in particular a mixture of two alcohols such as ethanol and propylene glycol.

In one embodiment of the invention, the hydrogel is mixed with a suspension of an API and water, or with a suspension of an API and alcohol(s).

The API of interest may often be a powder. The powder of interest may be suspended into the water or alcohol until the mixture is a homogeneous mixture.

The API of interest may typically be incorporated into the hydrogels by mixing.

Extrusion

The the average particle size of the hydrogels of the invention may be reduced before freeze-drying to provide a more uniform sponge. In a preferrred embodiment the average particle size of the hydrogel is reduced prior to freeze-drying; preferably the average particle size of the hydrogel is reduced to a resulting mean particle size (d0.5) of less than 10,000 μηη; more preferably to a resulting mean particle size (d0.5) of less than 5,000 μηη; and most preferably to a resulting mean particle size (d0.5) of less than 1 ,000 μηη; even more preferably the hydrogel is extruded through a fine mesh prior to freeze-drying; most preferably through a particle size 200 μηη mesh.

Freeze-drying

Products that are to be freeze-dried will typically be present in a pre-frozen form. Freeze-drying takes place under vacuum conditions below the "triple point" (6.2 mbar = 4.6 torr). Here, water will only be present in two phases: ice and vapor. By adding energy to the ice it will sublimate (evaporate) directly into the vapor phase. The vapor is condensed immediately on vapor traps to maintain the vacuum.

Water vapor has easier access through any product cell structure than water, penetrating the product mass to evaporate from the surface of the product as in the case in other drying methods. The gentle escape of water vapor in the freeze-drying process leaves the product close to its original shape and color. The vacuum and temperature used during the freeze-drying depends on the product, its shape, water content, product structure, and composition.

According to the present invention, the sponge may be crushed, pulverized, micronized, milled or finely ground after the freeze-drying. It may also be compressed, flattened or compacted by force.

Swelling capacity or swelling degree The freeze-dried sponges of the invention are characterized by the property of their "swelling capacity", a term used synonymously with the term "swelling degree", or just "swelling (%)" in figures 1 -3. This property is calculated based on the dry weight of a sponge and its swollen weight after immersion in PBS buffer over time, as shown in the Examples. In a preferred embodiment the freeze-dried sponge of the invention has a swelling capacity of no more than 3,500% (w/w) in a 10mM phosphate buffered saline solution at pH 6.9, when measured 23 minutes after immersion of the sponge into the buffer.

Compositions

Various compositions are envisioned as suitable for incorporating the sponges of the invention or powders thereof. These may also comprise a water-soluble excipient; preferably lactose. They may be pharmaceutical compositions comprising an effective amount of a freeze-dried sponge or a powder thereof, together with a pharmaceutically acceptable carrier, excipient or diluent. They may also be cosmetic articles or compositions comprising a freeze-dried sponge or a powder thereof.

Particularly relevant applications are sanitary, medical or surgical articles comprising a freeze-dried sponge or a powder thereof, or a composition as defined in the above; preferably the article is a diaper, a sanitary towel, a surgical sponge, a wound healing sponge, or a part comprised in a band aid or other wound dressing material.

Another application are in the field of medicament capsules or microcapsules, preferably comprising a freeze-dried sponge of the invention or a powder thereof.

Uses

The use of a sponge according to the present invention or a powder thereof may be for the manufacture of a moisturizer or a cosmetic composition or for the manufacture of a medicament for the treatment of angiogenesis, pain, migraine, epilepsy, anxious disorders, inflammatory conditions, infectious diseases, osteoarthritis, for an ophtalmological treatment, for wound treatment, hormonal disorders, cardiovascular diseases, gastro intestinal illnesses or cancer. Relevant illnesses and conditions are:

· Hair loss or baldness

• Pain (acute pains, cancer pains, chronic pains)

• Migraine (acute and preventive treatment)

• Epilepsies (preventive)

• Anxious disorders (long term treatments)

· Inflammatory conditions (acute and preventive treatment with and against steroids, NSAID's, rheumatism, psoriasis, osteoporosis and other autoimmune illnesses)

• Infectious disease (antibiotics and fungicides) • Hormonal disorders (deficiency diseases, e.g., growth hormone)

• Contraceptives

• Cardiovascular diseases (mostly preventive and maintenance dose)

• Chemotherapy (cancer drugs)

· Diabetes (insulin)

• Osteoarthritis

• Heamostasis

• Bone regeneration

EXAMPLES

Example 1

Preparation of DVS-cross-linked HA hydrogels

This example illustrates the preparation of highly homogenous DVS-cross-linked HA hydrogels according to the method of WO06056204 (Gel ID 1 -3, Table 2).

Sodium hyaluronate (1.0 or 2.3 Mda) was dissolved into 0.2 M NaOH with stirring for approx. 2 hours at room temperature (25°C) to give a 5% (w/v) solution. DVS was added to aliquots of the 5% solution under stirring to obtain the desired HA/DVS weight ratios. After stirring at room temperature for 5 min, the gel solutions were kept at room temperature for 10 min, followed by heating at 40°C for 2 hours.

The resulting cross-linked gels were neutralized and swollen in physiological buffered saline (PBS, pH 6.9, 10 mM) with a subsequent buffer shift to 37°C for approx. 30 hours. The weight of the finished swollen gels and their pH values were measured. The visco-elastic properties of the gels were measured on a Physica MCR 301 rheometer (Anton Paar, Osfildern, Germany, see Example 2).

Table 1 : DVS-HA hydrogels of the instant application

Three other DVS cross-linked hydrogels were also prepared using the same starting materials as indicated for Gel ID 1 -3 in table 2, but made by the method outlined in Example 9 of US 2007/0036745, e.g., without a heating step; these hydrogels were termed "prior art hydrogels".

The hydrogels (Gel ID 1 -3) were extruded by pressing each gel 20 times through a fine mesh with a particle size of 200 μηη. This reduced the hydrogel particle size to a mean particle size (d0.5) of less than 1000 μηη.

After hydrogel particle size reduction, the hydrogels were autoclaved in a closed container for 15 minutes at 121 °C with steam pressure of 230 Pa.

30 grammes of each of the autoclaved hydrogels were weighed out and spread in a flat 09 cm round glass or plastic dish in a uniform layer. The dish was closed with a lid and frozen at - 80°C for at least 4 hours. The frozen hydrogel particles were then placed in a freeze-dryer for at least 4 days at maximum (almost perfect) vacuum and lowest possible temperature.

The freeze-dried sponges were either placed directly in an airtight container or ground into a fine powder in a rotary grinding mill set to high speed and then placed in an airtight container.

The freeze-dried sponges were re-swollen, while their swelling capacities were measured over time. A small mesh was placed in a crystallization bowl and 100 mL PBS buffer was added; the wet mesh was weighed (tara). A piece of freeze dried gel was weighed and placed on the mesh and both were immersed in the buffer at time zero (tO). The mesh was withdrawn from the buffer periodically and mesh+gel was weighed to determine the swelling capacity of the freeze-dried sponges over time. The swelling capacity or the degree of swelling was calculated using the formula below and the results are shown in tables 2-4 as well as in figures 1 -3.

Wet weight— Dry weight

Deqree of swellinq = : * 100%

a ' ^a Dry weight

The viscoelastic properties of the hydrogels were also measured, this was done both prior to freeze-drying and after re-swelling. The swelling capacity and viscoelastic properties of the gels of the instant invention were clearly different from those of the prior art. See tables 2-4 as well as figures 1 -3 and 5-10.

We also investigated and compared the freeze-dried sponges visually using scanning electron microscopy at x60 and x250 magnification; the sponges of the invention were clearly different from the sponges made by the prior art method. The sponges of the invention appeared more bubbly and also more uniform; as shown in figures 1 1A-B (Gel ID 1 and the corresponding prior art gel), figures 12A-B (Gel ID 2 and the corresponding prior art gel) and figures 13A-B (Gel ID 3 and the corresponding prior art gel). Table 2. Measurements of the swelling capacity (%; w/w) of Gel ID No. 1 in PBS buffer (see table 1 ) and that of a comparable gel made as disclosed in US 2007/0036745; also shown in

Figure 1 .

Table 3. Measurements of the swelling capacity (%; w/w) of Gel ID No. 2 in PBS buffer (see table 1 ) and that of a comparable gel made as disclosed in US 2007/0036745; also shown in Figure 2.

Time Gel ID 2 Prior art

(min) gel

2 1756,395 231 1 ,365

4 1932,76 2906,076

6 2053,553 3197,57

9 2182,797 3918,728

12 2182,797 4130,665

15 2182,797 4287,062

18 21 16,23 4503,002 21 2573,972 4694,996

26 2573,972 5170,908

33 2482,947 5263,045

39 2486,013 5418,942

53 2544,951 5842,745

1 12 3146,672 6480,057

210 3506,208 6577,198

287 3661 ,78 6214,439

Table 4. Measurements of the swelling capacity (%; w/w) of Gel ID No. 3 in PBS buffer (see table 1 ) and that of a comparable gel made as disclosed in US 2007/0036745; also shown in Figure 3.

Example 2

Freeze-dried cross-linked HA sponge loaded with Triamcinolone hexacetonide

Table 5. Materials

The hydrogels were prepared as described in Example 2 in 7.90

WO 2006/056204. HA:DVS 10:1 , pre-extruded with 16 G1 "

needle.

Method of making the sponge

1 . Weigh out 1.00 gram of both ethanol and propylene glycol in a suitable container and mix well.

2. Weigh out 0.100 gram triamcinolone hexacetonide and mix the powder into the solvents until the mixture is a homogeneous suspension.

3. Weigh out 7.90 gram of the pre-extruded hydrogel in a suitable glass container and add the triamcinolone suspension - mix with a suitable spoon for at least 5 minutes until the gel has a sticky consistency.

4. Spread the gel mixture on to a 05 cm round or square glass dish in a uniform layer.

5. Cover the glass dish and freeze-it at minimum -78°C for 3 hours.

6. Freeze-dry the frozen gel mixture in a freeze-drier for 2-3 days at maximum vacuum and lowest possible temperature.

7. Remove the freeze-dried sponge carefully from the glass dish and compact it between two hard glass plates until the sponge takes a flat and uniform shape.

8. Weigh the sponge and calculate the amount of triamcinolone hexacetonide per amount of sponge. The release of triamcinolone hexacetonide from the HA sponge samples were assessed by a dissolution method using a closed loop system configuration of a USP apparatus 4 from SOTAX.

The system was equipped with 22.6 mm test cells and the entire system was temperature controlled at 37°C. Glass beads were added to fill the bottom of the sample cells, and a sponge sample loaded with 10 mg of triamcinolone hexacetonide was inserted into the cell and covered with glass beads.

PBS buffer at pH 6.8 was used as medium and 1 % SLS and 1 % ethanol was added to the medium to ensure correct sink conditions. The flow rate was set to 4 mL/min and a high stirring speed was used. The released triamcinolone hexacetonide was measured online with UV absorbance at 243 nm.

The analysis proceeded for a period of 26 days and afterwards the absorbance measurements were converted into % drug release and plotted, using the absorbance of 10 mg triamcinolone hexacetonide as 100 % release.

It can be seen from Figure 4 that the drug release was very slow (more than 25 days).

Example 3 Evaluation of visco-elastic properties of DVS-crosslinked HA hydrogels

The rheological measurements were performed on a Physica MCR 301 rheometer (Anton Paar, Ostfildern, Germany) using a plate-plate geometry and at a controlled temperature of 25°C. The visco-elastic behavior of the samples was investigated by dynamic amplitude shear oscillatory tests, in which the material was subjected to a sinusoidal shear strain.

First, strain/amplitude sweep experiments were performed to evaluate the region of deformation in which the linear viscoelasticity is valid. The strain typically ranged from 0.01 to 200% and the frequency was set to 1 Hz. Then, in the linear visco-elastic regions, the shear storage modulus (or elastic modulus G') and the shear loss modulus (or viscous modulus, G") values were recorded from frequency sweep experiments at a constant shear strain (10%) and at a frequency between 0.1 and 10 Hz. The geometry, the NF and the gap were PP 25, 2 and 1 mm, respectively.

G' gives information about the elasticity or the energy stored in the material during deformation, whereas G" describes the viscous character or the energy dissipated as heat. In particular, the elastic modulus gives information about the capability of the sample to sustain load and return in the initial configuration after an imposed stress or deformation. In all experiments, each sample was measured at least three times.

The results (Figures 5-10) showed that for all hydrogels:

· G'>G" and

G' is ALMOST independent of the frequency.

In case of the hydrogels with a higher degree of cross-linking, G' is one order of magnitude higher than G", indicating that this sample behaves as a strong gel material. Briefly, the overall rheological response is due to the contributions of physical and chemical crosslinks, and to topological interactions among the HA macromolecules. The interactions among the chains bring about a reduction of their intrinsic mobility that is not able to release stress, and consequently the material behaves as a three-dimensional network, where the principal mode of accommodation of the applied stress is by network deformation. Moreover, these hydrogels ware more elastic than that with a lower degree of cross-linking (i.e. higher ratio of HA DVS: 48:1 ). Indeed, the higher the number of permanent covalent cross-links, the larger the number of entanglements, and therefore the higher the elastic response of the hydrogel.

Claims

1 . A freeze-dried sponge comprising divinyl sulfone cross-linked hyaluronic acid which has a HA:DVS weight ratio in the range of 8:1 to 100:1 , wherein said sponge has a swelling capacity of no more than 4,200% (w/w) in a 10mM phosphate buffered saline solution at pH 6.9, when measured 50 minutes after immersion of the sponge into the buffer.

2. The freeze-dried sponge of claim 1 which also comprises an active pharmaceutical ingredient; preferably the active pharmaceutical ingredient is a peptide, a protein, a hormone, an antibiotic, a fungicide, an analgesic, a vaccine, an anti-inflammatory drug, monoclonal antibodies, or stem cells; even more preferably the active pharmaceutical ingredient is a steroid such as dexamethasone, triamcinolone, triamcinolone acetonide, or triamcinolone hexacetonide, or an analgesic drug, or a nonsteroidal anti-inflammatory drug such as diclofenac, naproxen, or ibuprofen.

3. The freeze-dried sponge of claim 1 or 2 which has a HA:DVS weight ratio in the range of 9:1 to 90:1 ; preferably in the range of 10:1 to 80:1 ; more preferably in the range of 1 1 :1 to 80:1 ; even more preferably in the range of 12:1 to 70:1 ; still more preferably in the range of 13:1 to 60:1 ; yet more preferably in the range of 14:1 to 50:1 ; more preferably in the range of 15:1 to 40:1 ; most preferably in the range of 16:1 to 30:1 .

4. The freeze-dried sponge of any preceding claim which has a swelling capacity of no more than 3,500% (w/w) in a 10mM phosphate buffered saline solution at pH 6.9, when measured 23 minutes after immersion of the sponge into the buffer.

5. The freeze-dried sponge of any preceding claim, wherein the hyaluronic acid has been recombinantly produced; preferably in a Bacillus host cell.

6. The freeze-dried sponge of any preceding claim which has been compressed, flattened or compacted by force.

7. A powder comprising a crushed, pulverized, micronized, milled or finely ground freeze- dried sponge as defined in any preceding claim.

8. A method of producing a freeze-dried sponge comprising DVS cross-linked hyaluronic acid, said method comprising

(a) providing an alkaline solution of hyaluronic acid, or salt thereof; (b) adding DVS to the solution of step (a) with stirring, wherein the HA:DVS dry weight ratio is in the range of 9:1 to 90:1 , whereby the hyaluronic acid, or salt thereof, is crosslinked with the DVS to form a gel;

(d) freeze-drying the hydrogel into a freeze-dried sponge,

9. The method of claim 8, wherein the hyaluronic acid, or a salt thereof, has an average molecular weight in the range of 100 to 5,000 kDa.

10. The method of claim 8 or 9, wherein the hyaluronic acid has been recombinantly produced; preferably in a Bacillus host cell.

1 1. The method of any of claims 8 - 10, wherein the alkaline solution comprises dissolved sodium hydroxide in a concentration of between 0.001 - 2.0 M.

12. The method of any of claims 8 - 1 1 , wherein the HA:DVS dry weight ratio is in the range of 10:1 to 80:1 ; more preferably in the range of 1 1 :1 to 80:1 ; even more preferably in the range of 12:1 to 70: 1 ; still more preferably in the range of 13: 1 to 60:1 ; yet more preferably in the range of 14:1 to 50:1 ; more preferably in the range of 15:1 to 40:1 ; most preferably in the range of 16:1 to 30:1 .

13. The method of any of claims 8 - 12, wherein the solution is heated in a step following step (b) but before step (c) to a temperature in the range of 20°C - 100°C and maintaining the temperature in this range for a period of at least 5 minutes; preferably the solution is heated to a temperature in the range of 25°C - 80°C, more preferably in the range of 30°C - 60°C, and most preferably in the range of 35°C - 55°C.

14. The method of claim 13, wherein the solution is heated for a period of at least 10 minutes; preferably for a period of at least 15, 30, 45, 60, 75, 90, 105, 120, 135, 150, 165 or 180 minutes.

15. The method according to any of claims 8 - 14, wherein the buffer is selected from the group consisting of acetate, sulphate, phosphate, citrate, ammonium, succinate, diethylammonium, imidazole, Tris, maleate, tetrabutylammonium, barbital, tris(hydroxymethyl)aminomethane, borate, HEPES, glycine, diethanolamine, phthalate, histidine, MES, PBS, and glycylglycine.

16. The method according to any of claims 8 - 15 which also comprises a step of incorporating an active pharmaceutical ingredient into the hydrogel prior to freeze-drying; preferably the active pharmaceutical ingredient is a peptide, a protein, a hormone, an antibiotic, a fungicide, an analgesic, a vaccine, an anti-inflammatory drug, monoclonal antibodies, or stem cells; even more preferably the active pharmaceutical ingredient is a steroid such as dexamethasone, triamcinolone, triamcinolone acetonide, or triamcinolone hexacetonide, or an analgesic drug, or a nonsteroidal anti-inflammatory drug such as diclofenac, naproxen, or ibuprofen.

17. The method according to any of claims 8 - 16, wherein the average particle size of the hydrogel is reduced prior to freeze-drying; preferably the average particle size of the hydrogel is reduced to a resulting mean particle size (d0.5) of less than 1 ,000 μηη.

18. The method of claim 17, wherein the hydrogel is extruded through a fine mesh prior to freeze-drying; preferably through a particle size 200 μηη mesh.

19. The method of any of claims 8 - 18, wherein the freeze-dried sponge is crushed, pulverized, micronized, milled, finely ground, compressed, flattened or compacted by force.

20. A composition comprising a freeze-dried sponge or a powder thereof as defined in any of claims 1 - 7.

21 . The composition according to claim 20, which also comprises a water-soluble excipient; preferably lactose.

22. A pharmaceutical composition comprising an effective amount of a freeze-dried sponge or a powder thereof, as defined in any of claims 1 - 7, together with a pharmaceutically acceptable carrier, excipient or diluent.

23. A cosmetic article or composition comprising a freeze-dried sponge or a powder thereof as defined in any of claims 1 - 7 or a composition as defined in any of claims 20 - 22.

24. A sanitary, medical or surgical article comprising a freeze-dried sponge or a powder thereof as defined in any of claims 1 - 7, or a composition as defined in any of claims 20 - 23; preferably the article is a diaper, a sanitary towel, a surgical sponge, a wound healing sponge, or a part comprised in a band aid or other wound dressing material.

25. A medicament capsule or microcapsule comprising a freeze-dried sponge or a powder thereof as defined in any of claims 1 - 7 or a composition as defined in any of claims 20 - 23.

26. Use of a freeze-dried sponge or a powder thereof as defined in any of claims 1 - 7, or a composition as defined in any of claims 20 - 23, for the manufacture of a medicament for the treatment of osteoarthritis.

27. Use of a freeze-dried sponge or a powder thereof as defined in any of claims 1 - 7, or a composition as defined in any of claims 20 - 23, for the manufacture of a medicament for an ophtalmological treatment.

28. Use of a freeze-dried sponge or a powder thereof as defined in any of claims 1 - 7, or a composition as defined in any of claims 20 - 23, for the manufacture of a medicament for the treatment of cancer.

29. Use of a freeze-dried sponge or a powder thereof as defined in any of claims 1 - 7, or a composition as defined in any of claims 20 - 23, for the manufacture of a medicament for the treatment of a wound.

30. Use of a freeze-dried sponge or a powder thereof as defined in any of claims 1 - 7, or a composition as defined in any of claims 20 - 23, for the manufacture of a medicament for angiogenesis.

31 . Use of a freeze-dried sponge or a powder thereof as defined in any of claims 1 - 7, or a composition as defined in any of claims 20 - 23, for the manufacture of a moisturizer or a cosmetic composition.

32. Use of a freeze-dried sponge or a powder thereof as defined in any of claims 1 - 7, or a composition as defined in any of claims 20 - 23, for the manufacture of a medicament for the treatment of hair loss or baldness.

33. Use of a freeze-dried sponge or a powder thereof as defined in any of claims 1 - 7, or a composition as defined in any of claims 20 - 23, in a cosmetic treatment.