FIELD OF THE INVENTION

The invention relates to new compositions and dosage forms in the field of tissue repair and regeneration, utilizing local drug delivery. Particularly the invention relates to compositions and dosage forms comprising bioactive glass and bisphosphonate, said compositions being suitable for example for tissue repair, improving bone formation and regeneration of tissue. The invention also relates to the method for the manufacture of said compositions and dosage forms, and to the use of said compositions for local delivery of a complex formed of bioactive glass and bisphosphonate, particularly in dental applications, medical devices, combination products, implants and the like.

BACKGROUND

Bioactive glasses are a family of biologically active synthetic materials which, when implanted into living tissue, induce formation of an interracial bond between the material and the surrounding tissue. Bioactive glasses are surface-reactive glass- ceramics designed to induce biological activity that results in the formation of a strong bond between the bioactive glass and living tissue, such as bone. Bioactive glasses have found medical applications in the preparation of synthetic bone graft materials for general orthopedic, craniofacial, maxillofacial and periodontal repair, as well as bone tissue engineering scaffolds. Bioactive glass can interact with living tissue including hard tissue such as bone, and soft connective tissue.

Bioactive glasses are regarded as promising non-toxic materials for tissue regeneration based on for example their distinctive properties of bone bonding, controlled biodegradability and ability to stimulate new bone growth even three times more than hydroxyapatite, when implanted in bone. Bone bonding properties appear to be due to the formation of an apatite layer on the glass surface. Typically the relatively low silicon content and high alkaline content lead to a rapid ion exchange in an aqueous environment. This exchange generally leads to an increase in pH of solution. The initially rapid release of sodium is accompanied by a somewhat slower release of other ion species, mostly calcium and silica. Under certain conditions in solution, these ion species will precipitate onto the glass and onto other nearby surfaces, to form calcium-containing mineral layers. In this case, the outer glass surface itself can transform to apatite layer. The ability to build such a surface is referred to as a measure of the "bioactivity" of the glass. This phenomenon can be described briefly as leaching, dissolution, and precipitation, even though apatite formation is a complex process involving different stages. In the first stage the surface of glass is dealkalized by exchange of cations (Na ⁺ K ⁺ or Ca ⁺ with H ⁺ or H ₃ 0 ⁺ ). This is followed by break-up of the silicate to form silanols; as a result of breaking of Si-O-Si bonds loss of soluble silica in the form of Si(OH) ₄ to solution takes place and formation of silanols happens. At the third stage equilibrium condensation and partial repolymerization of silanols to form a Si0 ₂ -rich gel layer takes place. During next stage an amorphous calcium phosphate layer is formed (rapid migration of Ca+ and P0 ₄ ^3" groups to the surface through Si0 ₂ -rich layer forming CaO-P ₂ 0 ₅ -rich film on the top of the Si0 ₂ -rich layer and further to thicker and denser amorphous layer via the arrival of more ions from bulk glass and absorption of Ca+ and P0 ₄ ³ ions from the surrounding solutions). Finally, in the fifth stage, crystallization of the amorphous calcium phosphate into apatite layer takes place.

The bioactivity of silicate glasses was first observed in soda-calcia-phospho-silica glasses in 1969, resulting in the development of a bioactive glass comprising calcium salts, phosphorous, sodium salts and silicon. These silica glasses comprised Si0 ₂ (40-52%) as the network former, and CaO (10-50%), Na ₂ 0 (10-35%), P ₂ 0 ₅ (2-8%), CaF ₂ (0-25%) and B ₂ 0 ₃ (0-10%). The inclusion of components, such as boron oxide (B ₂ 0 ₃ ) and calcium fluoride (CaF ₂ ) has allowed modification of the properties of the bioactive glass, including the rate of dissolution and the level of bioactivity.

Bisphosphonates are pyrophosphate analogues, which have potent inhibitory effect on bone resorption. They have high affinity for bone minerals and they are selectively uptaken by osteoclasts and strongly inhibit bone resorption by inducing apoptosis in osteoclasts. They are typically used in the treatment of bone disorders, particularly in such as hypercalcemia of cancer, osteoporosis, metastic bone disease, multiple myeloma, fibrous dysplasia and Paget disease.

Each bisphosphonate has its own profile of activity, defined by its unique side chains. The chemical structure of bisphosphonates is presented in the general formula I below. ( I )

R ¹ is typically OH, H or CI. Bisphosphonates can be divided in the following groups: First generation bisphosphonates not presenting nitrogen atoms in R ² side chain; second generation having a nitrogen atom in said alkyl chain; and third generation having a nitrogen atom included within a heterocyclic ring in said side chain. Nitrogen-containing bisphosphonates exhibit more powerful inhibition of bone resorption, and they strongly induce apoptosis of osteoclasts, however they also have increased risk for osteonecrosis. Second generation bisphosphonates, such as pamidronate and alendronate containing a primary nitrogen atom in an alkyl chain have up to hundred times increased anti-resorptive potency when compared with the first generation ones, etidronate and clodronate. Further modifications of the chain, to produce compounds containing tertiary nitrogen groups, such as ibandronate and olpadronate, increase the activity. The most potent bisphosphonates to date are risendronate and zolendronate, containing a nitrogen atom within a heterocyclic group and having activity up to 10 000 times higher than etidronate.

Bisphosphonates have very poor intestinal absorption (1-10%) and low bioavailability (1-3%) through oral or intravenous administration. Inflammation and ulceration of the upper gastrointestinal tract have been reported as adverse effects.

At the pH of the intestine the drug is mostly in acidic form, and if the patient lies down immediately after taking the medicine, there are chances of acid/drug reflux in oesophageal tract causing irritations. Also a vascular osteonecrosis of the jaw has been reported particularly in cancer patients, as a complication correlated with long- term use of systemic bisphosphonates, following dental treatments, including tooth extraction. Particularly zolendronic acid delayed wound healing and inhibited new bone formation, increased bacterial adhesion to hydroxyapatite and proliferation of oral bacteria. As an alternative route for administration of bisphosphonates, local administration by various drug delivery systems have also been suggested. Oliveira A. et al. : Biomimetic Ca-P Coatings Incorporating Bisphosphonates Produced on Starch-Based Degradable Biomaterials; Biomed Mater Res Part B: AppI Biomater (21.8.2009), p. 55-67, describes a method where discs were formed by injection molding of a blend containing 50% by weight starch and ethylene vinyl alcohol. The discs were impregnated for 24 hours with commercial sodium silicate gel, which was then diluted with water, and the discs were removed and dried. In order to achieve apatite formation on the discs they were soaked in a simulated body fluid solution comprising all major inorganic ions present in human blood plasma. Solutions containing 0.064, 0.32, 8 and 16 mg/ml of sodium clodronate were made and 50 μΙ aliquots of said solutions were added to the discs, followed by drying. Accordingly, earlier coating techniques based on plasma-spraying or sol-gel could thus be avoided. It was also found that the concentration of 0.32 mg/ml alendronate was ideal for enhancing cell viability and osteoblastic profile, making it suitable for improving implant-bone integration and in applications where regeneration of the tissue is required.

Capra P, et al. : A Preliminary Study on the Morphological and Release Properties of Hydroxyapatite-Alendronate Composite Materials; J Microencapsulation, 2011; 28(5) : 395-405, relates to composites formed of alendronate solution and hydroxyapatite nanopowder. Said composites could be used for loading into scaffolds for bone regeneration or onto prostheses surface, particularly for the prevention of implant failure.

Local administration of alendronate is suggested in Mondal T. et al. : Poly(L-lactide- co-e—caprolactone) Microspheres Laden with Bioactive Glass-ceramic and

Alendronate Sodium as Bone Regenerative Scaffolds; Mat Sci Engin C 32 (2012) 697-706. Microspheric scaffolds of poly(L-lactide-co-£— caprolactone) were manufactured by mixing the polymer melt with bioactive glass-ceramic granules and alendronate and the mixture was dissolved in dicloromethane. This solution was then added to an aqueous medium containing 5% of polyvinyl chloride and stirred to obtain microspheres, which were washed and dried. Said microspheres are suggested for local delivery of the drug alendronate, for the treatment of osteoporosis like bone defects. Spherical mesoporous microspheres are presented in Zhu M. et al. : An Emulsion- solvent Evaporation Route to Mesoporous Bioactive Glass Microspheres for Bisphosphonate Drug Delivery; J Mater Sci (2012) 47 : 2256-2263. Accordingly, microspheres were manufactured by combining emulsification and evaporation induced self-assembly process, whereby high storage capacity and sustained release of the drug (alendronate sodium) were obtained. In said method bioglass powder was obtained with the emulsion-evaporation procedure. The powder was then immersed in buffered alendronate sodium solution, and the drug loaded particles were dried. The obtained powdery mesoporous miscrospheres were suggested for the use in implants.

In Srisubut S., et al. : Effect of Local Delivery of Alendronate on Bone Formation in Bioactive Glass Grafting in Rats; Oral Surg Oral Med Oral Pathol Oral Radiol Endod (2007); 104(4); el l-el5, the effect of local delivery (single dose) of alendronate in improving bone formation after bioactive glass grafting in rat mandible was studied. Bioactive glass was used as bone graft substitute. The bone graft substitute of bioactive glass (Biogran ^® composed of 45% silicon dioxide, 24.5% calcium oxide, 24.5% sodium oxide and 6% phosphorus pentoxide) was soaked with alendronate (Fosamax ^® ) solution dissolved in saline and it was placed in the bone defect, flaps of the bone defect were repositioned and sutured. Biogran ^® solution in saline without alendronate was used as control. The results suggest that a single dose of topical administration of alendronate with bioactive glass was able to induce more bone regeneration and might be useful for alveolar ridge augmentation and for bone regeneration in periodontal defects.

US 2009/0208428 relates to bioactive glass comprising strontium and silicon dioxide, and to the use of it in the prevention and/or treatment of damage to tissue, optionally in combination with additional materials.

Accordingly, there are compositions known in the art, comprising bioactive glass, typically in the form of dry powders, microspheres, cements, pastes etc and they usually contain other components acting as carriers, such as polymers, or active ingredients. There is still a need for new improved compositions and dosage forms, particularly for local delivery, comprising bioactive glass and bisphosphonate, and to a method for the manufacture of said compositions and dosage forms, where the apatite formation can be accelerated and the adverse effects of bisphosphonates can be avoided. SUM MARY

It is an aim of the present invention to provide new and improved compositions and dosage forms, for the local treatment and/or prevention of bone tissue, cartilage tissue and soft connective tissue damage, and for the prevention of resorption of bone tissue, particularly in the field of dental applications, such as in the treatment and prevention of periodontitis.

Another aim of the present invention is to provide a method for the manufacture of said compositions and dosage forms.

Still another aim of the invention is the use and method of use of said composition and dosage form in the local treatment and/or prevention of bone tissue, cartilage tissue and soft connective tissue damage. The composition and dosage form for the treatment and/or prevention of tissue damage, according to the invention comprises a dry component(s) comprising at least one bioactive glass and an aqueous component(s) comprising at least one aqueous medium, and said dry component comprises at least bisphosphonate or said aqueous component comprises at least one bisphosphonate or both comprise at least one bisphosphonate.

The composition and dosage according to the present invention, comprising a complex formed of at least one bioactive glass and at least one bisphosphonate, and an aqueous medium, is particularly suitable for local delivery, for tissue repair and regeneration, for example for improving bone formation and regeneration of bone tissue, cartilage tissue, soft tissues, including connective tissues and dental tissues. The composition of the present invention provides a surprisingly high rate of apatite deposition and leads to rapid repair and reconstruction of diseased and damaged tissues, including bone tissue, cartilage tissue and soft tissues, as well as wound healing.

In the method for the manufacture of said composition and dosage form dry ingredients are combined with the aqueous medium. The composition and dosage form is particularly useful in the local treatment and/or prevention of bone tissue, cartilage tissue and soft connective tissue damage, and for the prevention of resorption of bone tissue, particularly in the field of dental applications, such as in the treatment and prevention of periodontitis.

Particularly, the ease of manufacture of the composition, the applicability of the composition to various sites of use, suitability for delivering locally the complex formed of at least one bioactive glass and at least one bisphosphonate in the presence of an aqueous medium, and avoiding challenges relating to other administration forms are some examples of the desired benefits achieved by the present invention.

The characteristic features of the invention are presented in the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 depicts changes in pH over the time (hours) for clodronate (clod), bioactive glass (BG) and combination product (BG+clod 200mg).

Figure 2 illustrates a SEM micrograph of some apatite formation on the surface of pure bioactive glass (with saline for 72 hours) in pH 9.45. The bar represents 40 μιη.

Figure 3 illustrates a SEM micrograph of apatite formation on the top of the bioactive glass in combination of clodronate (with saline for 72 hours) in pH 9.45. The bar represents 40 μιη.

Figure 4 illustrates a XRPD diffractogram of the combination product (BG+clod 200mg) and pure clodronate with saline for 15 days. | apatite; * clodronate; + Ca ₂ Si0 ₄ .

Figure 5 illustrates FTIR absorption spectra of clodronate, bioactive glass and combination product with saline for 72 hours (* phosphate groups).

Figure 6 illustrates DSC thermograms of clodronate, combination product and bioactive glass with saline for 72 hours.

Figure 7 presents changes in pH over the time for clodronate, bioactive glass (and combination product with 900 μΙ saline.

Figure 8 presents changes in pH over the time for clodronate, bioactive glass and combination product with 1350 μΙ saline. Figure 9 illustrates a XRPD diffractogram of pure clodronate (Clod), combination product (BGl+clod) containing bioactive glass having particle size < 0.5 mm and 200 mg of clodronate in saline, allowed to stand for 15 days, combination product (BG2+clod) containing bioactive glass having particle size of 0.5-0.8 mm and 200 mg of clodronate in saline, allowed to stand for 15 days, calcium clodronate (CaClod) and hydroxyapatite (Hydapa). The formation of hydroxyapatite and calcium clodronate can be clearly seen in the combination product containing bioactive glass having a bigger particle size, whereas in the combination product containing bioactive glass having particle size below 0.5 mm this was not shown.

DEFINITIONS

Unless otherwise specified, the terms, which are used in the specification and claims, have the meanings commonly used in the field of pharmaceuticals and drug delivery. Specifically, the following terms have the meanings indicated below.

The term "complex" refers here to a combination product formed of bioactive glass and bisphosphonate in an aqueous environment, when bioactive glass is brought into contact with bisphosphonate in an aqueous medium. A rapid change of pH from acidic (even pH around 4) to basic (even pH close to 12) can be observed during formation of said product.

DETAILED DESCRIPTION OF THE INVENTION

There exists a demand for new and improved compositions and dosage forms for local delivery, for the treatment and/or prevention of tissue damage, where for example the problems and adverse events relating to systemic administration of bisphosphonates can be avoided and the apatite formation in connection with bioactive glass can be improved and/or accelerated.

It was surprisingly found that a pharmaceutical composition or dosage form, suitable particularly for local administration, can be obtained from at least one bioactive glass and at least one bisphosphonate in the presence of an aqueous medium.

Said pharmaceutical composition or dosage form, for the treatment and/or prevention of tissue damage, comprises a dry component (s) comprising at least one bioactive glass, and an aqueous component comprising at least one aqueous medium, and said dry component comprises at least bisphosphonate or said aqueous component comprises at least one bisphosphonate or both comprise at least one bisphosphonate.

Said pharmaceutical composition or dosage form can be manufactured with a simple and economic method without the need to use complicated techniques. Surprisingly high and rapid apatite formation was observed with the composition or dosage form, resulting from the complex formed of bisphosphonate and bioactive glass in the presence of an aqueous medium. The bisphosphonate reacts rapidly in an aqueous medium with the bioactive glass to form the active complex. This can be seen as a very clear and rapid change of pH, from acidic to basic. The actual pH values depend on the bisphosphonate and on the bioactive glass. The rate of pH change is also dependent on the particle size of the components, such as the bioactive glass.

The complex (combination product) formed of bisphosphonate and bioactive glass has the ability to enhance and accelerate apatite formation of bioactive glass, and simultaneously bisphosphonate is brought directly to the site where tissue, such as bone repair/regeneration is needed. Accordingly, a highly active complex or combination product is formed of the bioactive glass and bisphosphonate, when brought into contact with the aqueous medium. The surprisingly high enhancing effect on the formation of apatite can particularly be seen already very shortly after the complex is formed, i.e. when the aqueous medium is brought into contact with the bioactive glass and bisphosphonate.

Thus a pharmaceutical composition or dosage form is obtained, comprising a dry component (s) comprising at least one bioactive glass, and an aqueous component comprising at least one aqueous medium, and said dry component comprises at least bisphosphonate or said aqueous component comprises at least one bisphosphonate or both comprise at least one bisphosphonate, optionally in admixture with known excipients, diluents, flavoring agents, aromatizing agents, stabilizers, formulation providing additives, formulation promoting agents commonly used in the pharmaceutical practice.

A dry component (s) comprising at least one bioactive glass, and an aqueous component(s) comprising at least one aqueous medium, and said dry component comprising at least one bisphosphonate or said aqueous component comprising at least one bisphosphonate may be used for the preparation of a pharmaceutical composition or dosage form for local treatment and/or prevention of tissue damage. Suitably the composition or dosage form is administered 0 days - 6 months after combining the dry component(s) and the aqueous component(s). Preferably the composition is administered 50 hours - 6 months after combining the components. In some preferable embodiments the composition is administered after 50 hours, preferably after 80 hours and before 6 months, preferably before 3 months.

Said composition is particularly useful in the treatment of damage to a tissue, for the repair, regeneration, reconstruction of damaged or diseased tissue and/or prevention of damage to the tissue.

The tissue may be bone tissue, cartilage tissue, soft tissues including connective tissues and dental tissues. Said dental tissue may include such as but not limited to calcified dental tissues such as enamel and dentin. The tissues may be animal tissues, preferably mammalian or human tissues.

The composition is suitably provided for humans or animals, such as dogs, cats, horses, sheep, cows, pigs etc. Hydroxyapatite (apatite) is in its most active state when it is formed, i.e. when it is formed or deposited on the surface of bioactive glass. This takes place for example when bioactive glass is brought into contact with body fluids like saliva etc. In the present invention at least one bioactive glass is combined with at least one bisphosphonate to provide a composition, suitable for local delivery. The composition is applied locally at the site where treatment is desired, for example in the dental region. The formed complex in said composition enhances and accelerates the formation of hydroxyapatite. Additionally said composition and bisphosphonate are brought directly to the site where needed, in desired amounts to provide the desired effect.

The pharmaceutical composition or dosage form

Said composition comprises a dry component (s) comprising at least one bioactive glass and an aqueous component comprising at least one aqueous medium, and said dry component(s) comprises at least one bisphosphonate or said aqueous component(s) comprises at least one bisphosphonate or both comprise at least one bisphosphonate, and said aqueous component is brought into contact with the dry component(s) prior to use. Thus, after combining the components (bringing the components into contact with each other) the composition comprises a complex formed of at least one bioactive glass or a combination of bioactive glasses, and at least one bisphosphonate or a combination of bisphosphonates, and at least one aqueous medium.

Suitably the dry component refers here to the bioactive glass(es), as such or combined with the bisphosphonate(s) in dry (solid) form, and the aqueous component refers here to the aqueous medium in liquid form, or alternatively, to the aqueous medium comprising the bisphosphonate(s) dissolved therein. The dry component is suitably reconstituted with the aqueous component prior to use.

Suitably the bioactive glass is selected from any available bioactive glasses, such as but not limited to silica glasses (glasses based on silicon dioxide) where the content of silicon dioxide is no more than 60 wt %. Said silica glasses additionally comprise a source of calcium, such as calcium oxide (CaO), calcium fluoride (CaF ₂ ), calcium carbonate (CaC0 ₃ ), calcium nitrate (Ca(N0 ₃ )2), calcium sulphate (CaS0 ₄ ), calcium silicates, and a source of phosphorus, such as P ₂ 0 ₅ . The glasses may have a variation in the ratio of these components. The silica glasses may optionally comprise one or more other components such as boron oxide (B ₂ 0 ₃ ), sodium oxide (Na ₂ 0), sodium carbonate (Na ₂ C0 ₃ ), sodium nitrate (NaN0 ₃ ), sodium sulphate (Na ₂ S0 ₄ ), sodium silicates, potassium oxide (K ₂ 0), potassium carbonate (K ₂ C0 ₃ ), potassium nitrate (KN0 ₃ ), potassium sulphate (K ₂ S0 ₄ ), potassium silicates, strontium oxide (SrO), strontium carbonate (SrC0 ₃ ), strontium nitrate (SrN0 ₃ ), strontium acetate (Sr(CH ₃ C0 ₂ ) ₂ ), strontium sulphate (SrS0 ₄ ), strontium fluoride (SrF ₂ ), strontium phosphate (Sr ₃ (P0 ₄ ) ₂ ), strontium silicates, magnesium oxide (MgO), magnesium carbonate (MgC0 ₃ ), magnesium nitrate (Mg(N0 ₃ ) ₂ ), magnesium sulphate (MgS0 ₄ ), magnesium silicates, zinc oxide (ZnO), zinc carbonate (ZnC0 ₃ ), zinc nitrate (Zn(N0 ₃ ) ₂ ), zinc sulphate (ZnS0 ₄ ), zinc silicates and any such compounds, including acetates of sodium, potassium, calcium, magnesium or zinc, that decompose to form an oxide.

The bioactive glass may optionally comprise silver, suitably provided as silver oxide in a molar percentage of from 0 up to 1%, 0.75%, 0.5% or 0.25%. The incorporation of silver may provide antibacterial properties to the bioactive glass.

Examples of suitable bioactive glasses are commercially available silica glasses, such as but not limited to S53P4 containing Si0 ₂ 53%, Na ₂ 0 23%, CaO 20% and P ₂ 0 ₄ 4% w/w, and 45S5, containing silicon dioxide Si0 ₂ 45% w/w and a 5 : 1 molar ratio of calcium (source of calcium) to phosphorus (source of phosphorus).

Preferably the bioactive glass is selected from silica glasses comprising from 30 to 60% w/w silicon dioxide, at least one source of calcium, at least one source of phosphorus and optionally at least one source of sodium.

The bioactive glass may be obtained using any techniques known in the art. Typically bioactive glasses are manufactured using methods such as but not limited to melt- derived techniques and sol-gel techniques known in the art. Preferably bioactive glass obtained using melt-derived techniques (referred to as melt-derived bioactive glass) is used in the present invention, whereby impurities originating from the sol- gel technique can be avoided. The bioactive glass may have an average particle size from 0.1 to 2000 μιη.

According to one embodiment suitable for dental application an average particle size from 500 to 800 μιη is suitable. The bioactive glass may comprise fractions having different particle size distributions, whereby for example the rate of bone formation may be controlled and adjusted according to the need.

The composition may comprise 40 to 98% w/w, preferably 45 to 95% w/w of bioactive glass or a combination of bioactive glasses, calculated from the dry matter.

Suitable bisphosphonates are selected from known bisphosphonates, such as but not limited to etidronate, clodronate, tiludronate, pamidronate, alendronate, risedronate, ibandronate, zoledronate, incadronate, olpadronate, neridronate, YH529 (minondronate) and EB-1053. Preferably the bisphosphonate is selected from etidronate and clodronate, particularly preferably it is clodronate. The composition may comprise 2 to 60% w/w of bisphosphonate or a combination of bisphosphonates, preferably 5 to 55% w/w of bisphosphonate, calculated from the dry matter.

The aqueous medium is selected from pharmaceutically acceptable aqueous liquids, such as but not limited to buffered sterile water, saline, water, SBF (=simulated body fluid is a buffered aqueous solution with ion concentrations nearly equal to those of human body plasma), saliva, TRIS-buffer (aqueous solution buffered with tris(hydroxymethyl)aminomethane, pH 7 - 9), and aqueous buffer solutions having pH 4 - 10.

The composition may comprise 5 to 95% w/w of the aqueous medium, preferably 20 to 75% w/w and particularly preferably 30 to 70% w/w, when reconstituted or mixed with the dry component(s).

Additionally the composition may optionally comprise an additive or a combination of additives, excipients, diluents, flavoring agents, aromatizing agents, stabilizers, formulation providing agents and formulation promoting agents, such as but not limited to flavors, sweeteners, aromes, scents, colorants, viscosity increasing agents, surfactants, pH-indicators, antioxidants, preservatives, anti-foaming agents, pH adjusting agents, antimicrobial agents, and antibiotics in amounts well known in the art.

The pharmaceutical dosage form for the treatment and/or prevention of tissue damage, according to the invention comprises the composition, wherein the dry component(s) is provided in a first compartment or vessel and the aqueous component(s) is provided in a second compartment or vessel, the dry component and the aqueous component are brought into contact with each other prior to the administration.

Manufacture of the composition

The bioactive glass or glasses may be mixed or blended, with the bisphosphonate or bisphosphonates using any suitable mixing method. Any method, such as but not limited to spray drying, lyophillization, dry powder mixing, wet granulation, granulation, mixing with turbula, pelleting, extrusion with spheronization may be utilized to obtain the dry component comprising the bioactive glass(es) and bisphosphonate.

In the case the aqueous medium contains the bisphosphonate(s) the bisphosphonate is dissolved in the aqueous medium using mixing and a temperature between 5 to 80°C. Bisphosphonates are typically readily soluble in water. The optional additives and excipients may be incorporated in the dry component or in the aqueous medium, depending on the properties of the additive/excipient. The composition may suitably be formulated as a dosage form or a kit or package, which comprises the dry component in a separate compartment of an application device or in a vessel, and the aqueous component in another separate compartment of the application device or in another vessel. Prior to use the dry component and the aqueous component are brought into contact with each other, whereby they are mixed to form the final composition. The final composition may be used as such or alternatively it may be allowed to stand for 50 to 80 hours after mixing in order to allow the pH change to take place, such as but not limited to from strongly acidic to strongly basic. Particularly when the composition is intended for use in close proximity of tissue sensitive to pH changes or acidic pH, it is preferable to use the composition from 50 to 80 hours after mixing the dry component and the aqueous component. As the pH starts gradually to decrease after 30 days after mixing, indication the decrease of the activity, the composition should preferably be used within 6 months after mixing, preferable within 30 days after mixing and particularly preferably within 15 days after mixing in order to achieve the desired effect without adverse effects.

According to one embodiment, a pH indicator may be incorporated in the composition, mixed in the dry component or in the aqueous component, for providing indication when the pH of the (final) composition after mixing the components has reached a desired range for administration or stabilized to a suitable level. This depends for example of the site of treatment.

The components are suitably packed in containers, such as but not limited to 2-part containers which are as such well known in the art, vessels, syringes or the like, which enable easy and rapid mixing of the components prior to the use. Mixing of the components may result in a suspension or lacquer or gel (mouthwash, varnish etc) or paste (such as toothpaste, filler for cavities, chewing gum etc.) or foam or aerosol, to be applied locally on the desired site in need for the treatment.

Use of the composition

The pharmaceutical composition according to the invention may suitably be used locally in the treatment and/or prevention of damage to a tissue. The composition may be used in medical applications in the field of synthetic bone graft materials for general orthopedic, craniofacial, maxillofacial and periodontal repair, as well as bone tissue engineering scaffolds. The application of the composition provides increased rate of apatite deposition, leading to more rapid repair and reconstitution of diseased and damaged tissue. The composition may also be formulated into toothpaste, dentrifice, chewing gum and mouth wash, as well as filler to treat root cavities and/or to prevent further deterioration of the teeth. The composition may be used of fixation of devices, such as implant, for repairing of bone defects, for remodelling of soft underlining tissue, for promoting new bone formation, for rebuilding of cartilage tissue, reconstruction of cartilage tissue, such as nose and ear repair etc.

The tissue may be animal tissue, suitably mammalian or human tissue, animals being such as but not limited to dogs, cats, horses, sheep, cows and pigs.

The tissue may be bone tissue, cartilage tissue, soft tissues including connective tissues and dental tissues including calcified dental tissues such as but not limited to enamel and dentin. The composition may suitably be used for the treatment of bone fractures, dental cavities, and for the treatment and prevention of periodontal disease, or hypersensitivity in the teeth. Particularly the composition is suitable for use on the dental region, for example as local treatment of periodontitis, for repairing tissue damage, which have not been possible using methods and compositions of the state of the art. When the composition is intended for use in close proximity of tissues sensitive to acidic pH or pH change, it is preferable to use the composition after 50 hours from mixing the aqueous component with the dry components.

The dry component of the composition is brought into contact with the aqueous component prior to the use, such as from 0 to 80 d before the use, suitably 0 - 50 d before the use, for obtaining the final composition. The maximum activity of the formed complex is maintained for at least 6 months, suitably for at least 4 weeks. The final composition may be applied one single time locally or the application may be carried out repeatedly, for example for the treatment and/or prevention of periodontal disease etc.

The composition has several advantages. The bisphosphonate, particularly the clodronate shown in the examples has a clear synergistic effect with the bioactive glass on the formation of apatite, where the bisphosphonate enhances the desired apatite formation on the bioactive glass significantly. Simultaneously the bisphosphonate can be delivered locally to the desired site of action and the adverse effects relating to the oral administration of bisphosphonates can be avoided. Additionally the dosage of bisphosphonate is very low compared to that in oral administration and thus also local adverse effects, such as irritation of mucous membranes can be avoided.

According to preliminary pilot clinical studies comparing the effect of the composition of the invention and bioactive glass the composition of the invention enhances and activates the metabolism in the tissue significantly more than bioactive glass and provides for more effective bone formation.

Also, as can be seen from the examples and figures 7 and 8, the pH increase with the combination product is greater and can be maintained for longer periods of time than that with bioactive glass alone, whereas with the bisphosphonate (clodronate) no pH increase could be noticed at all. Particularly when a combination product comprising the bisphosphonate and bioactive glass having the particle size in the range of 0.5 - 0.8 mm is used the pH increase took place in a controlled manner, which is a clear advantage with tissues sensitive to pH changes, such as dental tissues.

Further, as can be seen in Figure 9, the formation of hydroxyapatite and calcium clodronate can be clearly seen in the combination product containing bioactive glass having a bigger particle size, whereas in the combination product containing bioactive glass having particle size below 0.5 mm this was not shown.

The following examples are illustrative of embodiments of the present invention, as described above, and they are not meant to limit the invention in any way.

EXAMPLES

EXAMPLE 1

Composition comprising bioactive glass and clodronate

The combination product (complex) of bioactive glass (BG) and clodronate was characterized and the effect of clodronate to the bioactivity of BG was studied. The physical and chemical characteristics of the samples were identified by e.g. Scanning electron microscopy (SEM), X-ray powder diffraction (XRPD) and Fourier transform infrared spectroscopy (FTIR) .

Materials

Bioactive glass Bioactive glass (S53P4), BonAlive™ (BonAlive Biomaterials Ltd, former Vivoxid Ltd) contains four oxides (Si0 ₂ 53%, Na ₂ 0 23%, CaO 20% and P ₂ 0 ₅ 4% w/w). It is amorphous, having density of 2.66 g/cm ³ and is odourless white granule in particle size: 0.5-0.8 mm. Bioactive glass was used as dry granules or 1 g bioactive glass moisturized with 900 μΙ of 0.9% saline.

Ciodronate

Ciodronate (PharmaZell GmbH, Ph. Eur.) in the form of disodium salt (CH ₂ CI ₂ 0 ₆ P ₂ Na ₂ 4H ₂ 0 360.9 g/mole) was used.

Mixture of bioactive glass and ciodronate

Combination of 1 g bioactive glass and 200 mg ciodronate was used as dry powder and with 900 μΙ of 0.9% saline added.

Methods

Combination of bioactive glass and ciodronate; preparation of samples

Combination of 1 g bioactive glass and 200 mg ciodronate (BG+clod 200mg) was used as dry powder and with 900 μΙ of 0.9% saline added. For some analysis these wetted samples were dried just before the measurement at RT on the top of a filter paper until no visual sign of moisture was seen on the paper.

The bioactivity process of BG starts when BG is immersed into biological fluids in vivo, simulated body fluids, or other buffered solutions in vitro. Ion leaching and exchange with surrounding solution results bone-like apatite layer formation onto the BG surface. Also the slight alkalinisation is thereby induced. These two phenomena form the basis for the osteoproductive properties of BG. The liquid medium chosen was 0.9% saline, which alone induced only slightly the apatite formation of BG.

Particle Size Measurement

The particle size distribution (PSD) of the granules was determined using a 3D- surface image analysis method (Flashsizer FS3D, Intelligent Pharmaceutics Ltd). Approximately 3 mm ³ of ciodronate powder and bioactive granules were analysed by FS3D. pH

pH measurements (Fieldlab pH, L7137A, Schott Instruments) of the wetted samples (with 900 μΙ of 0.9% saline) were done over the time until time point day 25. Scanning Electron Microscopy (SEM)

Scanning electron micrographs (SEM) were used to document the changes in morphology between the samples. Samples were attached to double-sided carbon tape and coated with 20 nm platinum using an Agar sputter coater B7304 (Agar Scientific Ltd.). Electron micrographs were taken by scanning electron microscopy

(DSM962, Zeiss, and FEI Quanta 250 FEG).

X-ray Powder Diffractometry

The X-ray diffraction patterns of the dry and wetted samples were measured using an X-ray powder diffraction (XRPD) 2theta diffracto meter (Bruker axs D8). The XRPD experiments were performed in symmetrical reflection mode with CuK _a radiation (1.54 A) using Gobel Mirror bent gradient multilayer optics. The scattered intensities were measured with a scintillation counter. The angular range was from 5 to 40° (2Θ) with steps of 0.05° and the measuring time was 6 s/step. All of the samples were measured at room temperature.

Fourier transformed infrared spectroscopy (FTIR)

FTIR spectroscopy was used to analyze dry and wetted samples. The samples were analyzed using Vertex 70 (Bruker Optics Inc.) attenuated total reflectance (ATR) accessory (MIRacle TM ATR, PIKE Technologies, Inc.) by measuring the spectra 20 times with 4 cm ^"1 resolution between 4000-650 cm ^"1 under dry air flow. The spectra were further treated using OPUS 4.0 program (Bruker Optics Inc.). The resulting spectra were baseline corrected to minimize the effect of differing baselines on further data analysis.

Differential Scanning Calorimetry (DSC)

DSC measurements were performed on DSC analyser (model 821 ^e , Mettler Toledo Ag) using STAR software (STAR 5.1, Sun Soft Inc.). The temperature axis of the equipment was calibrated with zinc and indium. The runs were performed under nitrogen gas flow (50 mL/min) in aluminium pans, and the weights of the samples were 5-7 mg. The heating rate was 10°C/min over the temperature range 15-450°C.

Results

Particle Size Measurement

The distribution of the particle size of the BG and clodronate are shown in following

Table 1. The manufacturer of the bioactive glass claims that the particle size varies mostly between 0.5-0.8 mm, whereas in our investigation more than 50% of the used BG had a dimension larger than 750 μιη. This difference in data is understandable due to the different particle size measurement method (sieve analysis by the company).

Table 1. Particle volume size distribution of BG and clodronate pH

The apatite formation on the top of the bioactive glass is related to the ion exchange causing pH increase. In this example the increase of pH was higher in the combination of BG and clodronate than BG alone indicating that the activity of the bioactive glass is stronger in the combination product as can be seen in Fig. 1 representing changes in pH over the time (hours) for clodronate (clod), bioactive glass (BG) and combination product (BG + clod 200mg).

To assure that this phenomenon was not due to the change of the pH caused by clodronate as such, the measurements were repeated in pH adjusted in the combination product to the same level as in pure BG product. The results confirmed that merely pH control does not explain the stronger activity of BG and clondronate. Thus, there is a clear synergy between BG and clodronate, promoting bioactivity. Additionally, the increase of pH sustained much longer in the combination product than in the pure BG, indicating that the reaction and thus bioactivity lasted longer period of time in the combination product of BG and clodronate.

At 72h time point the pH of the combination product and BG was about the same. The pH continued increasing in the combination product, whereas it still gradually decreased in the BG product. At time point of about 15 days (360h) the pH of the combination product started to show decreasing.

SEM

SEM pictures show the difference of the surface of the BG and combination product samples. From SEM images shown in Figs. 2 and 3 it can be seen that there is more Ca-P-rich layer on the top of the BG, which indicates greater apatite formation in the combination product than in the BG. The time point 72 hours was chosen in order to see the changes in morphology at the time the pH in both BG and combination product was about the same (approximately pH 9.45) based on earlier described pH measurements (Fig. 1). Surprisingly, this difference was seen already after 72h. In this example pure BG acted as expected and there is only a small amount of apatite formation seen in SEM micrograph presented in Fig. 2, showing SEM micrographs of some apatite formation on the surface of the pure bioactive glass (with saline for 72 hours) in pH 9.45.

However, in the combination product the apatite formation is also surprisingly extensive already after 3 days, as can be seen in Fig. 3, showing SEM micrograph of apatite formation on the top of the bioactive glass in combination of clodronate (with saline for 72 hours) in pH 9.45. This has not been reported previously.

XRPD

The apatite formation in the combination product was detected also with the XRPD, as can been in Fig. 4, showing a XRPD diffractogram of combination product

(BG+clod 200mg) and pure clodronate with saline for 15 days. | apatite; * clodronate; + Ca ₂ Si0 ₄ . X-ray diffraction analysis clearly showed peaks specific to apatite and clodronate. For example peaks at (2Θ) = 25.9° as well as 31.8°-32.9° may be ascribed to the characteristic peaks of. Additionally, Ca ₂ Si0 ₄ was clearly seen indicating the activity of the bioactive glass. In one stage (stage 4) of the activity of the bioactive glass, it is typical that Ca ²⁺ groups migrate to the surface through the Si0 ₂ -rich layer. In Fig. 4 the diffractogram after 15 days is shown. At the time of 15 days pH for the combination product has achieved its maximum point and is starting to decrease gradually as seen in Fig. 1.

FTIR

The apatite formation was also demonstrated with the FTIR spectra. Fig. 5 illustrates the different IR absorption spectra of bioactive glass, clodronate and combination product of bioactive glass and clodronate with saline for 72 hours. As shown at the time of 72 hours in SEM micrographs (Figs. 2 and 3) there is clear evidence of apatite formation. Clear apatite formation can be seen in Fig. 5, showing FTIR absorption spectra of clodronate, bioactive glass and combination product with saline for 72 hours (* phosphate groups). The FTIR spectrum of the combination product illustrates well-defined phosphate bending vibrational band at 1200-900 cm ^"1 , conforming the formation of a Ca-P-rich layer on the top of the BG, which indicates the apatite formation. IR spectrum shows the characteristic bands of O-H and P-0 reflecting the vibrations of the OH and P0 ₄ groups in the hydroxyapatite. For example, one of the bands for phosphate groups is shown at 1090 cm ^"1 . As expected this phenomena can also be seen in the pure BG spectrum, even not as intense. Additionally, Si-O-Si stretch (1200-1000 cm ^"1 ) can be seen in the combination product and bioactive glass indicating apatite formation.

DSC

Fig. 6 shows the thermal analysis of the studied samples and particularly the DSC thermograms of clodronate, combination product and bioactive glass with saline for 72 hours. The initial endothermic process around 100°C can be attributed to loss of residual water. However the peak seen for clodronate at 136°C seems to disappear in the combination product indicating that there is molecular interaction between BG and clodronate, which has been confirmed already with FTIR spectra (Fig. 5).

Bioactivity of the BG and combination product has been proved with pH change, SEM micrograph, XRPD and FTIR spectra. The SEM micrograph and FTIR and XRPD diffraction clearly showed that there is apatite formation in the combination product. Furthermore, based on pH and SEM data this apatite formation is more extensive in the combination product than BG alone. Thus, it can be concluded that clodronate enhances the activity of the BG. Further, based on for example pH data (Fig. 1) the bioactivity property remains longer in the combination product than BG alone, thus suggesting that combination of clodronate and BG creates a favourable environment for the apatite formation. Thus it can be concluded that clodronate has remarkable ability to enhance the apatite formation of BG and thus promotes the process.

EXAMPLE 2

Comparison of the different clodronate content and particle size of bioactive glass on activity

Materials

Bioactive glass

Bioactive glass (S53P4), BonAlive™ was used in this example. It is amorphous, having density of 2.66 g/cm ³ and is odourless white granule in particle size: 0.5-0.8 mm and <0.5 mm. Bioactive glass was used as dry granules or 1 g bioactive glass moisturized with 900 μΙ or 1350 μΙ of 0.9% saline.

Clodronate

Clodronate (PharmaZell GmbH, Ph. Eur.) CH ₂ CI ₂ 0 ₆ P ₂ Na ₂ 4H ₂ 0 was used . Mixture of bioactive glass and clodronate

Combination of 1 g bioactive glass (particle size: 0.5-0.8 mm and <0.5 mm) and 100 mg, 200 mg or 300 mg clodronate was used as dry powder and with 900 μΙ or 1350 μΙ of 0.9% saline added.

Methods

Combination of bioactive glass and clodronate; preparation of samples

Combination of 1 g bioactive glass and 100 mg, 200 mg or 300 mg clodronate was used as dry powder and with 900 μΙ or 1350 μΙ of 0.9% saline added. For some analysis these wetted samples were dried just before the measurement at RT on the top of a filter paper until no visual sign of moisture was seen on the paper. The aqueous medium was 0.9% saline, which alone induced only slightly the apatite formation of BG. Particle Size Measurement

The particle size distribution (PSD) of the granules was determined using a 3D- surface image analysis method (Flashsizer FS3D, Intelligent Pharmaceutics Ltd.). Approximately 3 mm ³ of both clodronate powder and different bioactive glass granule fractions (0.5-0.8 mm and <0.5 mm) were analysed by FS3D. pH

pH measurements (Fieldlab pH, L7137A, Schott Instruments) of the wetted samples (with 900 μΙ of 0.9% saline) were done over the time until time point day 22 or 24. Scanning Electron Microscopy (SEM)

Scanning electron micrographs (SEM) were used to document the changes in morphology between the samples. Samples were attached to double-sided carbon tape and coated with 20 nm platinum using an Agar sputter coater B7304 (Agar Scientific Ltd.). Electron micrographs were taken by scanning electron microscopy (DSM962, Zeiss, and FEI Quanta 250 FEG).

X-ray Powder Diffractometry

The X-ray diffraction patterns of the dry and wetted samples were measured using an X-ray powder diffraction (XRPD) 2theta diffracto meter (Brukeraxs D8). The XRPD experiments were performed in symmetrical reflection mode with CuK _a radiation

(1.54 A) using Gobel Mirror bent gradient multilayer optics. The scattered intensities were measured with a scintillation counter. The angular range was from 5 to 40° (2Θ) with steps of 0.05° and the measuring time was 6 s/step. All of the samples were measured at room temperature.

Fourier transformed infrared spectroscopy

Fourier transformed infrared (FTIR) spectroscopy was used to analyze dry and wetted samples. The samples were analyzed using Vertex 70 (Bruker Optics Inc.) attenuated total reflectance (ATR) accessory (MIRacle TM ATR, PIKE Technologies, Inc.) by measuring the spectra 20 times with 4 cm ^"1 resolution between 4000-650 cm ^"1 under dry air flow. The spectra were further treated using OPUS 4.0 program (Bruker Optics Inc.). The resulting spectra were baseline corrected to minimize the effect of differing baselines on further data analysis.

Differential Scanning Calorimetry

Differential scanning calorimetric (DSC) measurements were performed on DSC analyser (model 821 ^e , Mettler Toledo Ag) using STAR software (STAR 5.1, Sun Soft Inc). The temperature axis of the equipment was calibrated with zinc and indium. The runs were performed under nitrogen gas flow (50 mL/min) in aluminium pans, and the weights of the samples were 5-7 mg. The heating rate was 10°C/min over the temperature range 15-450°C.

Results

Particle Size Measurement

The distribution of the particle size (volume size) of the different BG fractions and clodronate are shown in Table 2. The particle size distributions of both starting material is basic informative data for the characterization of the used material.

Table 2. Particle size distribution of BG (0.5-0.8 mm and <0.5 mm) and clodronate

pH

In Fig. 7 changes in pH over the time (hours) for clodronate (clod), bioactive glass (BG) and combination product (BG+clod 100 mg or 200mg) with 900 μΙ saline added are presented. In Fig 8 changes in pH over the time (hours) for clodronate (clod), bioactive glass (BG) 0.5-0.8 mm or <0.5 mm and combination product (BG (0.5-0.8 mm or <0.5mm) + clod (100 mg, 200mg or 300mg)) with 1350 μΙ saline added are presented.

While the invention has been described with respect to specific examples including presently preferred modes of carrying out the invention, those skilled in the art will appreciate that there are numerous variations and permutations of the above described embodiments that fall within the spirit and scope of the invention. It should be understood that the invention is not limited in its application to the details of construction and arrangements of the components set forth herein. Variations and modifications of the foregoing are within the scope of the present invention.