Pharmaceutical composition comprising an amino acid composition in purified hyaluronic acid

Abstract

The current invention relates to a pharmaceutical composition for local or parenteral administration, more specifically an injectable solution comprising of an amino acid composition dissolved in purified hyaluronic acid or a salt or ester thereof, wherein said hyaluronic acid has a molecular weight between 500 kDa and 4000 kDa and wherein said injectable solution comprises a pH between 6.4 and 7.8. In a further aspect, the present invention also relates to use of aforementioned pharmaceutical composition in the treatment of a musculoskeletal disease, such as osteoarthritis, in a subject.

Description

[0001] PHARMACEUTICAL COMPOSITION COMPRISING AN AMINO ACID COMPOSITION IN PURIFIED HYALURONIC ACID

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to a pharmaceutical composition for local or parenteral administration, more specifically an injectable solution comprising an amino acid composition dissolved in purified hyaluronic acid or a salt or ester thereof. In a further aspect, the present invention also relates to use of aforementioned pharmaceutical composition in the treatment of a musculoskeletal disease, such as osteoarthritis.

[0004] BACKGROUND

[0005] Osteoarthritis (OA) is one of the most frequently occurring joint disorders in veterinary practice. Osteoarthritis is characterized by the progressive loss of articular cartilage that leads to chronic pain and functional restrictions in affected joints. The most abundant macromolecules of the extracellular matrix of cartilage are type II collagen, aggrecan and hyaluronan (HA), and chondrocytes are the only cellular components.

[0006] Under normal physiologic conditions, chondrocytes maintain an equilibrium between anabolic and catabolic activities and express various proteolytic enzymes such as aggrecanases and matrix metalloprotainases (MMPs), which mediate a very low matrix turnover responsible for cartilage remodeling. In pathologic conditions such as OA, however, production of these enzymes increases considerably, resulting in aberrant cartilage destruction.

[0007] Weight management, tailored exercise and medical treatments for OA such as nonsteroidal anti-inflammatory drugs (NSAIDs) and corticosteroids are mainly focused on pain relief and treatment of the inflammatory reaction, but are not able to slow down the disease progression or reverse the pathological condition. Persistent high doses of certain older generation NSAIDs are associated with gastro-intestinal, renal and hepatic abnormalities. Corticosteroids provide a rather short pain relief and could possibly induce further cartilage damage. Therefore, there is a very high demand for effective and long-term treatment options in OA. Intra-articular (IA) hyaluronic acid injections are widely used for over three decades in the treatment of OA. Compositions comprising hyaluronic acid and amino acids are known (for instance described in W02024080954, W02023042120, US2017326089 and US2012237610). However controversies exist regarding its safety and efficacy, the number of injections and courses, type of preparation, duration of its effects, and combining it with other drugs or molecules.

[0008] Recent research for OA treatment has focused on finding agents aimed at slowing or reversing OA progression.

[0009] The present invention aims to resolve at least some of the problems and disadvantages mentioned above.

[0010] SUMMARY OF THE INVENTION

[0011] The present invention and embodiments thereof serve to provide a solution to one or more of above-mentioned disadvantages. To this end, the present invention relates to a pharmaceutical composition for local or parenteral administration according to claim 1. More particularly, the invention relates to a pharmaceutical composition for local or parenteral administration, said pharmaceutical composition being an injectable solution comprising an amino acid composition dissolved in purified hyaluronic acid or a salt or ester thereof, wherein said hyaluronic acid has a molecular weight between 500 kDa and 4000 kDa and wherein said injectable solution comprises a pH between 6.4 and 7.8.

[0012] By combining hyaluronic acid or a salt or ester thereof and an amino acid composition into one injectable solution, said hyaluronic acid having a molecular weight between 500 kDa and 4000 kDa and said injectable solution having a pH between 6.4 and 7.8, a pharmaceutical composition with optimal viscoelastic and bio-active properties can be obtained for use in the treatment of a musculoskeletal disease, such as osteoarthritis.

[0013] Preferred embodiments of the pharmaceutical composition are shown in any of the claims 2 to 11.

[0014] In a further aspect, the present invention relates to a use according to claim 12. More particular, the present invention relates to use of aforementioned pharmaceutical composition in the treatment of a musculoskeletal disease, preferably a bone disease or a joint disease in a subject.

[0015] DETAILED DESCRIPTION OF THE INVENTION

[0016] The present invention concerns a pharmaceutical composition for local or parenteral administration, said pharmaceutical composition being an injectable solution comprising an amino acid composition dissolved in purified hyaluronic acid or a salt or ester thereof, wherein said hyaluronic acid has a molecular weight between 500 kDa and 4000 kDa and wherein said injectable solution comprises a pH between 6.4 and 7.8. Such a pharmaceutical composition has optimal viscoelastic and bio-active properties for use in the treatment of a musculoskeletal disease, such as osteoarthritis.

[0017] Unless otherwise defined, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, term definitions are included to better appreciate the teaching of the present invention.

[0018] As used herein, the following terms have the following meanings:

[0019] "A", "an", and "the" as used herein refers to both singular and plural referents unless the context clearly dictates otherwise. By way of example, "a compartment" refers to one or more than one compartment.

[0020] "About" as used herein referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, is meant to encompass variations of + / -20% or less, preferably + / -10% or less, more preferably + / -5% or less, even more preferably + / -1% or less, and still more preferably + / -0.1% or less of and from the specified value, in so far such variations are appropriate to perform in the disclosed invention. However, it is to be understood that the value to which the modifier "about" refers is itself also specifically disclosed.

[0021] "Comprise", "comprising", and "comprises" and "comprised of" as used herein are synonymous with "include", "including", "includes" or "contain", "containing", "contains" and are inclusive or open-ended terms that specifies the presence of what follows e.g. component and do not exclude or preclude the presence of additional, non-recited components, features, element, members, steps, known in the art or disclosed therein.

[0022] Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order, unless specified. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.

[0023] The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within that range, as well as the recited endpoints.

[0024] Whereas the terms "one or more" or "at least one", such as one or more or at least one member(s) of a group of members, is clear per se, by means of further exemplification, the term encompasses inter alia a reference to any one of said members, or to any two or more of said members, such as, e.g., any >3, >4, >5, >6 or >7 etc. of said members, and up to all said members.

[0025] Unless otherwise defined, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, definitions for the terms used in the description are included to better appreciate the teaching of the present invention. The terms or definitions used herein are provided solely to aid in the understanding of the invention.

[0026] Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment, but may. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention, and form different embodiments, as would be understood by those in the art. For example, in the following claims, any of the claimed embodiments can be used in any combination.

[0027] "Osteoarthritis" (OA), also referred to as "Degenerative Joint Disease" (DJD), is a progressively worsening inflammation of the joint caused by the deterioration of cartilage. In a healthy joint, cartilage acts as a cushion to allow the joint to move smoothly through its full range of motion. In cases of osteoarthritis, this cartilage cushion begins to breakdown because of factors such as age, injury, repetitive stress, or disease. The loss of this protective cushion results in pain, inflammation, decreased range of motion, and the development of bone spurs. While any joint in the body can develop osteoarthritis, the condition most commonly affects the limbs and lower spine. For animals such as dogs and horses, typical visual signs of OA may include stiffness, lameness, or difficulty getting up; lethargy; reluctance to run, jump, or play; weight gain; irritability or changes in behavior; pain when petted or touched; difficulty posturing to urinate or defecate, or having accidents in the house; and / or loss of muscle mass over the limbs and spine.

[0028] "Molecular weight" as used herein refers to the sum of the atomic masses of all atoms in a molecule, based on a scale in which the atomic masses of hydrogen, carbon, nitrogen, and oxygen are 1, 12, 14, and 16, respectively.

[0029] In the context of hyaluronic acid (HA), the molecular weight can vary widely, depending on its source and method of production. HA can have molecular weights ranging from 10 kDa (kilodaltons) to over 1000 kDa, which can affect its physical and biological properties, such as viscosity, tissue penetration, and interaction with cellular receptors.

[0030] As used herein, "molecular weight of HA" refers to a weighted average of a HA sample. As used herein, "molecular weight of a collagen tripeptide (Ctp)" refers to a weighted average of said Ctp sample.

[0031] "Hyaluronic acid (HA) component" as used herein refers to either HA or a salt or ester of HA, as used in the pharmaceutical composition of the current invention. "Amino acid component" as used herein refers to either a composition comprising single amino acids or to a composition comprising collagen tripeptides, as used in the pharmaceutical composition of the current invention.

[0032] The terms "patient" or "subject", are used interchangeably and refer to a mammalian subject to be treated. Preferably, the mammal is a canine or an equine, such as a dog or a horse.

[0033] "Canine" or "canines" in the present invention refers to dog-like carnivorans of the Canidae family. A member of this family is called a canid. There are three subfamilies found within the canid family, which are the extinct Borophaginae and Hesperocyoninae, and the extant Caninae. The Caninae are known as canines, and include domestic dogs, wolves, foxes, coyotes, jackals and other extant and extinct species.

[0034] Said equine may be any horse-like animal of the Equidae family, preferably of the genus Equus, such as from the species E. caballus (including the myriad domestic strains), E. zebra, E. burchelli, and E. grevyi (zebras) or E. asinus and E. hemionus (wild asses). Said equine is preferably a horse of E. caballus.

[0035] The term "effective amount" as used herein refers to the minimum amount or concentration of a compound or composition that is effective to prevent or reduce the symptoms or to ameliorate the condition of a disease. "Effective amount" as used herein can thus be used in the context of prophylactic, metaphylactic and / or therapeutic treatment.

[0036] Detailed description

[0037] Osteoarthritis (OA) is characterized by the progressive loss of articular cartilage that leads to chronic pain and functional restrictions in affected joints. The most abundant macromolecules of the extracellular matrix of cartilage are type II collagen, aggrecan and hyaluronan (HA), and chondrocytes are the only cellular components.

[0038] Weight management, tailored exercise and medical treatments for OA such as nonsteroidal anti-inflammatory drugs (NSAIDs) and corticosteroids are mainly focused on pain relief and treatment of the inflammatory reaction, but are not able to slow down the disease progression or reverse the pathological condition. Persistent high doses of certain older generation NSAIDs are associated with gastro-intestinal, renal and hepatic abnormalities. Corticosteroids provide a rather short pain relief and could possibly induce further cartilage damage. Therefore, there is a very high demand for effective and long-term treatment options in OA.

[0039] Recent research for OA treatment has focused on finding agents aimed at slowing or reversing OA progression.

[0040] HA is one of the principal components of cartilage matrix, and intra-articular injection of HA is widely used in the treatment of OA. Exogenous HA has been shown to delay degradation of cartilage by inhibiting glycosaminoglycan release from cartilage tissue and has anti-inflammatory effects by suppressing expression of MMPs and IL-ip. MMPs are involved in progressive cartilage destruction in arthritis, and MMP-13 activity has been implicated as playing a central role in cartilage degeneration in OA. Intra-articular (IA) hyaluronic acid injections are widely used for over three decades in the treatment of OA. However controversies exist regarding its safety and efficacy, the number of injections and courses, type of preparation, duration of its effects, and combining it with other drugs or molecules.

[0041] Type II collagen, one of the extracellular matrix components of cartilage, plays a crucial role in its tensile property, which allows formation of a fibrillar meshwork. This fibril meshwork also binds and traps proteoglycans and noncollagenous glycoproteins. All collagens consist of three polypeptide chains, termed a chains, characterized by repeating glycine-X-Y sequences. Position X is often occupied by proline or lysine and position Y by hydroxyproline or hydroxylysine. Glycine is required at every third position to allow the close packing of a chains within the triple helix. Availability of a pool of these amino acids may thus improve production of type II collagen by chondrocytes and facilitate collagen reorganization, subsequently decreasing destruction of cartilage in OA.

[0042] In a first aspect, the invention provides a pharmaceutical composition for local or parenteral administration, said pharmaceutical composition being an injectable solution comprising an amino acid composition dissolved in purified hyaluronic acid or a salt or ester thereof, wherein said hyaluronic acid has a molecular weight between 500 kDa and 4000 kDa and wherein said injectable solution comprises a pH between 6.4 and 7.8. The inventors have unexpectedly observed that by combining hyaluronic acid or a salt or ester thereof and an amino acid composition into one injectable solution, said hyaluronic acid having a molecular weight between 500 kDa and 4000 kDa and said injectable solution having a pH between 6.4 and 7.8, a pharmaceutical composition with optimal viscoelastic and bio-active properties can be obtained.

[0043] The existence of polar and apolar segments in the polymer structure affords hyaluronic acid the capability to chemically interact with various agents, such as amino acids. As such, besides the bio-active properties of hyaluronic acid (delaying degradation of cartilage, as discussed above), it further functions as a carrier to deliver the amino acid composition to the site of injury.

[0044] Bacterial technology makes it possible to obtain high-purity hyaluronic acid with low protein and endotoxin levels. In a preferred embodiment, the produced HA has a low degree of impurity and is at least 90%, more preferably at least 95% pure. The purified HA component is injectable grade and ultrapure.

[0045] The amino acid composition is dissolved in said purified hyaluronic acid or a salt or ester thereof.

[0046] In an embodiment, the HA component is first dissolved in a solution to obtain a HA solution. In an embodiment, the HA component is a white or almost white, very hygroscopic powder. In a preferred embodiment, the resulting HA solution is clear, has an absorbance at 600 nm of less than 0.01 (0.33% [w / v] solution, dried). In a preferred embodiment, the HA component is dissolved in phosphate buffered saline (for instance by reconstitution and sterilization by filtration with 0.2-pm syringe filters). In an embodiment, the pH of the HA component (0.5% in water, on dried product) is between 5.0 and 8.5. In an embodiment, the intrinsic viscosity of the HA solution is between 1.5 and 2.3 m3 / kg. In an embodiment, the absorbance of the HA component at 260 nm (0.33% solution, on dried product) comprises less than 0.5 (presence of nucleic acids). In an embodiment, the HA component comprises less than 0.1% protein, less than 0.5% chlorides and / or less than 80 ppm iron. In an embodiment, loss on drying of the HA component is less than 20.0%. In an embodiment, residual solvent (ethanol) in the HA component is less than 0.5%. In an embodiment, total aerobic microbial count (TAMC) in the HA component is less than 102CFU / g. In an embodiment, bacterial endotoxins (gel clot) in the HA component is less than 0.05 lU / mg. The amino acid composition is dissolved in the obtained HA solution. As described above, by dissolving the amino acid composition in the hyaluronic acid solution, the resulting injectable solution has better viscoelastic properties, allowing to better retain the amino acids at the site of injection (for instance where the defective chondrocytes are present). This enhances the uptake of the amino acid composition by the chondrocytes allowing them to increase the collagen II production.

[0047] The pharmaceutical composition of the current invention has optimal viscoelastic properties for injection and maintaining the pharmaceutical composition at the desired location for sustained release of the amino acid composition.

[0048] There is a relationship between the viscosity and both molecular weight and concentration of hyaluronic acid. For instance, a two-fold increase / decrease of hyaluronic acid concentration or molecular weight leads to a ten-fold respective change of the zero shear viscosity of the hyaluronic acid solution.

[0049] As described above, the hyaluronic acid component in the pharmaceutical composition of the current invention has a molecular weight between 500 kDa and 4000 kDa. In a preferred embodiment, said pharmaceutical composition comprises between 5-20 mg / ml of said hyaluronic acid or a salt or ester thereof.

[0050] In an embodiment, the pharmaceutical composition of the current invention has an intrinsic viscosity between 0.2-10 m3 / kg, more preferably between 0.2-5 m3 / kg, more preferably between 0.5-2.3 m3 / kg as measured by capillary viscometry at 25°C (according to the European Pharmacopoeia chapter 2.2.9). In a preferrred embodiment, the pharmaceutical composition of the current invention has an intrinsic viscosity between 1.6-1.8 m3 / kg as measured by capillary viscometry at 25°C (according to the European Pharmacopoeia chapter 2.2.9).

[0051] In a preferred embodiment, the pharmaceutical composition of the current invention has an intrinsic viscosity between 2.8 to 3.2 m3 / kg, preferably between 2.8 and 3.1 m3 / kg, more preferably between 2.85 and 3.00 m3 / kg, such as 2.9 m3 / kg as measured by capillary viscometry at 25°C (according to the European Pharmacopoeia chapter 2.2.9).

[0052] The increase in the molecular weight and concentration of hyaluronic acid in polymer solutions leads to the reinforcement of the three-dimensional network of the polymer. Consequently, it results in an increase in the solution viscosity and viscoelasticity. These parameters are important, especially for artificial synovial fluids, because such viscoelastic matter is able to successfully absorb mechanical energy and preserve cartilage from damage or fretting.

[0053] Hyaluronic acid with high molecular weight possesses a longer relaxation time (passage from a mainly viscous behavior to an elastic behavior). With decrease in the molecular weight of hyaluronic acid, it takes less time for a three-dimensional polymer network to untwine and demonstrate principally viscous behavior. The behavior difference between lower and higher molecular weight hyaluronic acid could also be explained by variation in the entwinement of the polymer chains.

[0054] Note that the structure of hyaluronic acid in solution is very sensitive to pH; the addition of acids or alkalis leads to the displacement of the balance between repelling and attractive forces of polymer chains. When the pH is more than 11.0 and less than 4.0, hyaluronic acid depolymerizes.

[0055] Besides the molecular weight and the concentration of the hyaluronic acid, also the pH affects the viscoelasticity of the composition. Increasing pH causes the solution to change from a dominant viscous to elastic behaviour. A pharmaceutical composition comprising an amino acid composition and a hyaluronic acid or a salt or ester thereof with a molecular weight between 500 kDa and 4000 kDa, which has a pH between 6.4 and 7.8 has the optimal viscoelastic properties to allow the pharmaceutical composition to be used in injectable biomaterials. A higher pH will increase the elastic behaviour of the pharmaceutical composition (making it for instance more suitable for use as a hydrogel for wound dressings). On the other hand, a pharmaceutical composition that is too viscous (for instance because of a too low pH), will also be suboptimal for injection.

[0056] Furthermore, the inventors found that the pharmaceutical composition of the current invention is optimal for intracellular uptake of the amino acid composition in cells responsible for collagen production. Without wishing to be bound to theory, it seems that a pH between 6.4 and 7.8 facilitates endocytosis of the amino acid composition (clathrin-mediated endocytosis and / or endocytosis by means of caveolae and cytoskeleton components). A higher intracellular uptake of the amino acid composition, will allow a higher production of type II collagen (chondrocytes) or type I collagen (tenocytes or meniscocytes). In an embodiment, the pharmaceutical composition has a pH between 6.5 and 7.5, more preferably between 6.7 and 7.2, such as between 6.8 and 7.0, for instance 6.9. In an embodiment, the pharmaceutical composition has a pH between 6.5-6.7, for instance between 6.5-6.6 or between 6.6-6.7. In an embodiment, the pharmaceutical composition has a pH between 6.7-7.2, for instance between 6.8-6.9, between 6.9-7.0, between 7.0-7.1, between 7.1-7.2. In an embodiment, the pharmaceutical composition has a pH between 7.2-7.5, for instance between 7.2-7.3, between 7.3-7.4 or between 7.4-7.5. In an embodiment, the pharmaceutical composition has a pH of 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4 or 7.5. In a preferred embodiment, the pharmaceutical composition has a pH of 6.9.

[0057] As described above, all collagens consist of three polypeptide chains, termed a chains, characterized by repeating glycine-X-Y sequences. Position X is often occupied by proline (Pro) or lysine (Lys) and position Y by hydroxyproline (Hyp) or hydroxylysine (Hyl). Availability of a pool of these amino acids in the pharmaceutical composition of the current invention improves the production of collagen and facilitates collagen reorganization, for instance the production of type II collagen by chondrocytes, subsequently decreasing destruction of cartilage in OA.

[0058] The amino acid composition in the current invention provides a pool of these amino acids.

[0059] In an embodiment, said amino acid composition comprises a pool of single amino acids. In a preferred embodiment, said amino acid composition of the current invention comprises glycine, lysine, hydroxy lysine, proline and / or hydroxyproline. In a preferred embodiment, said amino acid composition comprises glycine, proline and hydroxyproline at a combined concentration of at least 10%.

[0060] In a preferred embodiment, said amino acid composition of the current invention comprises glycine, proline and hydroxyproline ata combined concentration of at least 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%.

[0061] In an embodiment, said amino acid composition of the current invention comprises glycine at a concentration of at least 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29% or at least 30 % of the total amino acid content. In an embodiment, said amino acid composition of the current invention comprises proline at a concentration of at least 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29% or at least 30 % of the total amino acid content. In an embodiment, said amino acid composition of the current invention comprises hydroxyproline at a concentration of at least 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29% or at least 30 % of the total amino acid content.

[0062] In an embodiment, the amino acid composition of the current invention comprises glycine, proline and hydroxyproline at an equimolar concentration. In an alternative embodiment, the amino acid composition of the current invention comprises glycine, proline and hydroxyproline at a non-equimolar concentration, wherein at least one of glycine, proline or hydroxyproline is present in a different concentration than the other(s).

[0063] In a preferred embodiment, said amino acid composition comprises a collagen tripeptide (Ctp).

[0064] Collagen tripeptide (Ctp) is a collagen hydrolysate comprising a highly purified, nonantigenic and low-allergenic tripeptide fraction containing Gly-Xaa-Yaa sequences. As described above, availability of a pool of amino acids in the pharmaceutical composition of the current invention improves the production of collagen and facilitates collagen reorganization.

[0065] It has been shown that Ctp exerts beneficial effects on bone fracture healing by increasing type I collagen gene expression of osteoblastic cells. Furthermore, Ctp stimulates chondrocyte type II collagen gene expression in a previously performed in vitro study.

[0066] In addition, Ctp modulates the inflammatory process. Without wishing to be bound to theory, Ctp for instance binds the IL1 receptor and TGF-pi in joint fluid and downregulates PgE2, MMP3 and NO. Furthermore, Ctp inhibits macrophage activity, resulting in less biological degradation of HA. As such, Ctp will result in less chondrocyte apoptosis because of the reduced inflammatory response and because of the regenerative effect of Ctp. The purity of Ctp can be analyzed by any suited method known from the art, such as using HPLC with a Superdex peptide gel filtration column (GE Healthcare UK Ltd., England) and quantitation by integrating the absorbance at 214 nm of the major peak. The composition of tripeptide components is expressed as the area ratio (%).

[0067] The tripeptide content in a collagen hydrolysate may be determined by various analytical methods. In one approach, UHPLC-MS / MS in multiple reaction monitoring mode is used to detect individual Gly-Xaa-Yaa tripeptides with reference standards, and the content is expressed as a weight percentage of the total hydrolysate. Alternatively, size-exclusion chromatography with ultraviolet or evaporative light scattering detection can be employed, whereby the tripeptide fraction (250-450 Da) is integrated and expressed as an area percentage of the chromatographic signal. Tripeptides may also be quantified following derivatization with reagents such as OPA or FMOC, with separation by HPLC or capillary electrophoresis and expression as a relative peak area percentage. High-resolution mass spectrometry can further be used to profile Gly-Xaa-Yaa tripeptides, with the content expressed as relative ion abundance or converted to weight percentage using calibration standards. In addition, ultrafiltration combined with colorimetric assays such as TNBS or OPA provides a global estimate of the low-molecular-weight fraction, with the tripeptide content expressed as a weight percentage based on calibration with tripeptide standards.

[0068] In an embodiment, the tripeptide content is determined by size-exclusion chromatography with UV detection at 214 nm, reporting tripeptide content as area % of the chromatogram. The (Superdex) Peptide gel filtration column separates peptides based on molecular size. Detection at 214 nm monitors the peptide bond absorbance. Quantitation by integration of the absorbance peak area gives the relative abundance of the tripeptide fraction. Expressing the composition as an area ratio (%) corresponds to reporting the relative chromatographic peak area percentage of tripeptides compared to all detected peptide fractions.

[0069] In an embodiment, the Ctp does not heat coagulate. In an embodiment, the Ctp has a pH between 4.6 and 7.0. In an embodiment, the Ctp has less than 20 ppm heavy metals. In an embodiment, the Ctp has less than 1 ppm arsenic. In an embodiment, the Ctp has less than 10.0% loss on drying. In an embodiment, the Ctp has less than 2.0% residue on ignition. In an embodiment, the Ctp has between 12.0 and 22.0% nitrogen content. In an embodiment, the Ctp is a powder, for instance an odorless powder. In a preferred embodiment, the Ctp is soluble in cold water or other solutions.

[0070] In a preferred embodiment, the collagen hydrolysate comprises at least 10 % of tripeptide content, said tripeptides having a Glycine(Gly)-Xaa-Yaa sequence. In a preferred embodiment, said Ctp has at least 25% tripeptide content.

[0071] In an embodiment, the collagen hydrolysate comprises at least 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% of tripeptide content.

[0072] In a preferred embodiment, Ctp major components include peptides such as Glycine-Proline-Hydroxyproline (Gly-Pro-Hyp), Glycine-Proline-Alanine (Gly-Pro-Ala), and Glycine-Alanine-Hydroxyproline (Gly-Ala-Hyp). In a preferred embodiment, the tripeptide Glycine-Proline-Hydroxyproline is comprised in said Ctp at a concentration of at least 10% of the total tripeptide content.

[0073] In an embodiment, the tripeptide Glycine-Proline-Hydroxyproline (Gly-Pro-Hyp) is comprised in said CTP at a concentration of at least 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49% or at least 50% of the total tripeptide content.

[0074] Ctp can include other tripeptides such as Glycine-Proline-Serine (Gly-Pro-Ser), Glycine-Proline-Lysine (Gly-Pro-Lys), Glycine-Proline-Proline (Gly-Pro-Pro), Glycine-Proline-Arginine (Gly-Pro-Arg), Glycine-Proline-Hydroxylysine (Gly-Pro-Hyl), Glycine-Proline-Glutamine (Gly-Pro-GIn), Glycine-Alanine-Alanine (Gly-Ala-Ala), Glycine-Alanine-Arginine (Gly-Ala-Arg), Glycine-Alanine-Aspartic acid (Gly-Ala-Asp), Glycine-Alanine-Lysine (Gly-Ala-Lys), Glycine-Alanine-Serine (Gly-Ala-Ser), Glycine-Serine-Hydroxyproline (Gly-Ser-Hyp), Glycine-Serine-Alanine (Gly-Ser-Ala), Glycine-Lysine-Aspartic acid (Gly-Lys-Asp), and Glycine-Glutamic acid-Glutamine (Gly-Glu-GIn). In an embodiment, CTP can also contain (at low percentages) dipeptides such as Glycine-Proline (Gly-Pro), Proline-Hydroxyproline (Pro-Hyp), Alanine-Hydroxyproline (Ala-Hyp), and Hydroxyproline-Glycine (Hyp-Gly), and peptides composed of four or more amino acids.

[0075] Ctp is absorbed intracellularly and stimulates the ribosomes to produce more collagen depending on the local cell type. Chondrocytes for instance produce Col II, III, IV and VII; whereas meniscocytes produce Col I and Col III.

[0076] In an embodiment, said collagen tripeptide has a molecular weight below 500 Da, more preferably below 450 Da, more preferably below 400 Da, more preferably below 350 da, more preferably below 300 Da. In an embodiment, said collagen tripeptide has a molecular weight between 20 and 50 Da, between 50 and 100 Da, between 100 and 150 Da, between 150 and 200 Da, between 200 and 250 Da or between 250 Da and 300 Da.

[0077] By limiting the molecular weight of the Ctp, the intracellular uptake in the target cells (for instance chondrocytes, tenocytes, or meniscocytes) is enhanced. These cells are responsible for collagen I (tenocytes, meniscocytes) and collagen II (chondrocytes) production. As described above, availability of a pool of amino acids in the pharmaceutical composition of the current invention improves the production of collagen and facilitates collagen reorganization. For instance, it has been shown that Ctp can enhance type I collagen production of human osteoblastic cells and hyaluronic acid production in human dermal fibroblasts and that supplementation of culture medium with tripeptide induced type II collagen synthesis predominantly by chondrocytes.

[0078] In an embodiment, Ctp can increase the number of type II collagen-positive chondrocytes and / or Ctp can enhance type II collagen production of chondrocytes. Furthermore, the deposition of newly synthesized collagen necessary for cartilage reorganization may be enhanced by availability of tripeptides. The current invention thus enables sufficient supply of tripeptide to reach the target cells. For instance, tripeptides can be supplied to the chondrocytes, presumably by diffusion through the cartilage matrix via the synovial fluid and various transporter systems. Deposition of newly synthesized type II collagen necessary for cartilage anabolism may therefore be enhanced by both availability of tripeptides and activation of chondrocytes' synthetic ability, subsequently reducing cartilage degradation. The promotion of type II collagen synthesis by Ctp may contribute predominantly to the prevention of cartilage destruction.

[0079] Ctp can be derived from different sources (e.g. fish, cow, pig, chicken), primarily through the hydrolysis of collagen.

[0080] Marine sources, specifically the skin and scales of fish such as tilapia, cod, and salmon, are a rich source of collagen tripeptides. Fish-derived CTP (CTP-F) is considered the most effective due to its smaller molecular size, high bioavailability, and faster absorption. Collagen can also be extracted from the skin and bones of cows (bovine) and pigs (Porcine). These sources provide larger collagen molecules that, when hydrolyzed, yield collagen peptides, though their absorption rate is generally slower than marine sources. Chicken sternum and cartilage are additional sources of collagen. These are rich in type II collagen and can be used for joint health supplements.

[0081] As such, in a preferred embodiment, said collagen tripeptide is prepared from fish skin (CTP-F).

[0082] Collagen derived from fish has a lower molecular weight compared to bovine or porcine sources. As described above, said collagen tripeptide preferably has a molecular weight less than 300 Da. Fish skin is a byproduct of the fishing industry, making it a more sustainable and environmentally friendly source of collagen. Utilizing fish skin reduces waste and creates a valuable product from an otherwise discarded resource. Fish collagen is generally considered safer for people with allergies than bovine or porcine collagen. Since marine collagen is free from the risks associated with animal-borne diseases (e.g., BSE or mad cow disease), it is preferred by people seeking cleaner, safer, and more hypoallergenic options.

[0083] In veterinary medicine, collagen-derived therapeutics (based on bovine or porcine collagen) are known. Animals that have previously received such bovine- or porcine-derived products may become sensitized to mammalian collagen epitopes, increasing the risk of an allergic or hypersensitivity reaction upon re-exposure to collagen fragments of similar origin. By contrast, fish-derived collagen hydrolysates comprising collagen tripeptides (CTP) are of non-mammalian origin and lack mammalian epitopes. Accordingly, the use of fish-derived CTP in horses, dogs or other animals provides a therapeutic composition with a reduced risk of eliciting allergic responses, particularly in subjects that have previously been treated with bovine- or porcine-derived collagen therapies.

[0084] As described above, HA is also one of the principal components of cartilage matrix. The endogenous HA in the joint depolymerizes from a high molecular weight (6,500-10,900 kDa) to a lower molecular weight (2,700-4,500 kDa) as OA progresses, reducing the mechanical and viscoelastic properties of the synovial fluid in the affected joint. Exogenous HA injections have therefore been employed clinically to attenuate the macerated activities of OA patients' depolymerized endogenous HA. Although the exogenous HA may not fully repair and replace the characteristics and activities of the synovial fluid's depolymerized endogenous HA, it may provide enough pain relief through a variety of methods. Synthesis of proteoglycan and / or glycosaminoglycan, anti-inflammatory action, and viscoelasticity maintenance are among these processes.

[0085] Exogenously injected HA for instance stimulates the production of HA (3000-5000 kDa) by B synoviocytes in the joint for at least 4 weeks, leading to an increased HA production. As such, the exogenously injected HA results in a sustained HA increase in the injected joint. Besides the physical presence of the exogenously injected HA (3 weeks), endogenic HA production by B synoviocytes can cause a sustained increase in HA levels for up to 6 months. Furthermore, the biological clearance of HA by the cellular immune response (macrophages) is inhibited by Ctp (the amino acid composition of the current injectable solution), which aids in maintaining the elevated levels of HA even more.

[0086] Long-term HA lubrication results in smoother joint flexion / movement and less longterm cartilage damage (less chondrocyte apoptosis). In horses, one injection of the pharmaceutical composition according to the current invention can for instance have 2-6 months effect depending on the training level of the horse.

[0087] Traditionally, hyaluronic acid is extracted from animal sources such as rooster combs and cocks' combs. However, the difficulty in controlling animal tissue, high costs, and ethical concerns associated with animal-derived HA have led to the development of microbial production methods. Apart from the animal of origin, hyaluronic acid can be separated based on bacteria, for example, from Streptococcus genus (uberis, equisimilis, zooepidermicus, pyogenes, equi), Pasteurella multocida, and Corynebacterium glutamicum; from the green algae Chlorella purposely infected by the Chlorovirus; Saccharomycetes (Cryptococcus neoformans); and from molluscan shellfish, such as the bivalve mollusc Mytilus galloprovincialis.

[0088] Microorganisms such as bacteria and yeast have been used to produce HA through fermentation. Bacterial technology makes it possible to obtain high-purity hyaluronic acid with low protein and endotoxin levels. In a preferred embodiment, the produced HA has a low degree of impurity and is at least 90%, more preferably at least 95% pure.

[0089] The selection of the appropriate microorganism to produce hyaluronic acid is an important factor in the microbial production process. Each microorganism has its own unique advantages and disadvantages in terms of production performance and profitability. When choosing a microorganism to produce hyaluronic acid, it is essential to consider the specific requirements of the final application of this molecule. The molecular weight of hyaluronic acid depends on the source. Consequently, hyaluronic acid from animal materials has a very high molecular weight (up to 20,000 kDa). For example, rooster combs contain hyaluronic acid with 1200 kDa, the navel cords— 3400 kDa, bovine vitreous humors— 770-1700 kDa. By contrast, bacterial hyaluronic acid has a molecular weight between 1000 and 4000 kDa; however, the enzymatic technique makes it possible to obtain polysaccharides with a range of molecular weight between 550 kDa and 2500 kDa.

[0090] The microorganism must be able to produce high yields of hyaluronic acid, be easy to cultivate, and have a low cost of production. When considering the profitability and possible applications of the hyaluronic acid produced by each microorganism, it is essential to consider factors such as production costs, purity of the final product, and market demand. For example, the higher-molecular-weight hyaluronic acid produced by Streptococcus has significant potential in the medical field due to its excellent biocompatibility, making it a high-value product. Meanwhile, the lower-molecular-weight hyaluronic acid produced by Bacillus has excellent potential in the cosmetic industry, with demand for hyaluronic acid-based cosmetics continuing to grow. Group A and C streptococci, namely Streptococcus zooepidemicus, have been the most explored strain in the production process of microbial hyaluronic acid and have obtained the best results. Streptococcus zooepidemicus, a Gram-positive bacteria, is one of the most widely used organisms for the production of HA due to its high hyaluronic acid production rate and ease of cultivation.

[0091] Other metabolically engineered microorganisms can also be used, such as Bacillus subtilis, Corynebacterium glutamicum, Escherichia Coli, Lactococcus lactis, Pichia pastoris, and Kluyveromyces lactis. It has been implied that the source of HA may have an impact on the therapy's efficacy.

[0092] In an embodiment, said hyaluronic acid is produced by bacterial fermentation using Streptococcus zooepidemicus.

[0093] Streptococcus zooepidemicus produces a HA having the desired molecular weight and with an excellent biocompatibility, making it suitable for medical applications such as joint lubrication. In addition, the production process does not require the use of toxic chemicals or solvents, resulting in a pure and safe end product

[0094] As described above, the biological effects of hyaluronic acid depend heavily on molecular weight. Hyaluronic acid with molecular weights from 0.4 to 4.0 kDa acts as an inducer of heat shock proteins, and has a non-apoptotic property. Polysaccharides with a molecular weight equal to 6-20 kDa possess immunostimulatory, angiogenic, and phlogotic activities. Hyaluronic acid with a molecular weight of 20-200 kDa takes part in biological processes such as embryonic development, wound healing and ovulation. By contrast, high molecular weight hyaluronic acid (>500 kDa) has anti-angiogenic activity, and can function as a space filler and a natural immunologic depressant. The hyaluronic acid component comprised in the pharmaceutical composition of the current invention has a molecular weight between 500 kDa and 4000 kDa.

[0095] In an embodiment, the hyaluronic acid component comprised in the pharmaceutical composition of the current invention has a molecular weight between 750 kDa and 4000 kDa, between 750 kDa and 3500 kDa, between 750 kDa and 3000 kDa, between 750 kDa and 2500 kDa, between 750 kDa and 2000 kDa, between 750 kDa and 1500 kDa or between 750 kDa and 1200 kDa. In an embodiment, the hyaluronic acid component comprised in the pharmaceutical composition of the current invention has a molecular weight between 1000 kDa and 4000 kDa, between 1250 kDa and 4000 kDa, between 1500 kDa and 4000 kDa, between 1750 kDa and 4000 kDa or between 2000 kDa and 4000 kDa.

[0096] In a preferred embodiment, said hyaluronic acid has a molecular weight between 800 kDa and 1200 kDa. Such a molecular weight of HA provides an optimal pharmaceutical composition for injection at the site of injury, especially in canines and equines.

[0097] In an embodiment, said pharmaceutical composition comprises a hyaluronic acid salt, preferably sodium hyaluronate. Sodium hyaluronate is the sodium salt of hyaluronic acid. Since the molecule typically exists in vivo in its polyanionic form, it is commonly referred to as hyaluronan. It is a natural complex sugar of the glycosaminoglycan family and is a polymer of disaccharides, themselves composed of D-glucuronic acid and D-N-acetylglucosamine, linked via alternating 13-1,4 and 13-1,3 glycosidic bonds. Hyaluronan can be 25,000 disaccharide repeats in length. Polymers of hyaluronan can range in size from 5,000 to 20,000,000 Da in vivo. It is unique among glycosaminoglycans in that it is non-sulfated. Sodium hyaluronate is the sodium salt of hyaluronic acid, making it more stable and water-soluble than the acid form. This allows it to dissolve easily in water-based products like gels and injections. Hyaluronic acid, in its pure form, is less stable and more prone to degradation. It does not dissolve as readily in water, limiting its effectiveness in many formulations.

[0098] The pharmaceutical composition as defined herein may furthermore comprise further additives or additional compounds. Further additives which may be included in the pharmaceutical composition are emulsifiers, such as, for example, Tween®; wetting agents, such as, for example, sodium lauryl sulfate; colouring agents; pharmaceutical carriers; stabilizers; antioxidants; preservatives; serological substances, such as platelet rich plasma (PRP) or alpha 2 macroglobulins (A2MG) etc. Sterile injectable forms of the pharmaceutical compositions may be an aqueous or oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents.

[0099] In an embodiment, said pharmaceutical composition further comprises a pharmaceutically and pharmacologically acceptable liquid to disperse the hyaluronic acid component and amino acid component of the pharmaceutical composition of the current invention. As such, a pharmaceutical composition for parenteral administration, such as parenteral injection, can be obtained. In an embodiment, said pharmaceutical composition comprises a sterile liquid. Said sterile liquid should be safe for parenteral administration to a mammalian patient.

[0100] In an embodiment, the HA component is first dissolved in a solution to obtain a HA solution. In a preferred embodiment, the HA component is dissolved in phosphate buffered saline (for instance by reconstitution and sterilization by filtration with 0.2-pm syringe filters).

[0101] In an embodiment, the pharmaceutical composition comprises between 0.05-0.5 mg / ml, preferably between 0.1-0.3 mg / ml, such as between 0.15-0.25 mg / ml phosphate buffer.

[0102] In an embodiment, the pharmaceutical composition comprises between 1.0-30.0 mg / ml, preferably between 5.0-20 mg / ml, such as between 7.5-12.5 mg / ml, for instance 9.0 mg / ml sodium chloride.

[0103] In a further embodiment, the amino acid composition is dissolved in the obtained HA solution. As described above, by dissolving the amino acid composition in the hyaluronic acid solution, the resulting injectable solution has better viscoelastic properties, allowing to better retain the amino acids at the site of injection (for instance where the defective chondrocytes are present). This enhances the uptake of the amino acid composition by the chondrocytes allowing them to increase the collagen II production.

[0104] As described above, the injectable solution comprises a pH between 6.4 and 7.8. A lower pH will have a negative effect on the quality of the amino acids and on the cells present at the injection site (for instance the chondrocytes) which are responsible for the collagen production. At this pH range intracellular uptake of the amino acid composition is enhanced and the cellular production of collagen is enhanced.

[0105] The pharmaceutical composition of the current invention typically comprises a "safe and effective amount or dose" of the components of the pharmaceutical composition, particularly of the HA component and the amino acid component as defined herein. As used herein, a "safe and effective amount or dose" means an amount of the components as defined herein as such that is sufficient to significantly induce a positive modification of a disease or disorder or to prevent a disease, for instance osteoarthritis. At the same time, however, a "safe and effective amount or dose" is small enough to avoid serious side-effects and to permit a sensible relationship between advantage and risk. The determination of these limits typically lies within the scope of sensible medical judgment.

[0106] In an embodiment, a safe and effective amount or dose of said hyaluronic acid component in said pharmaceutical composition is between 0.1 and 200 mg, preferably between 0.1 and 100 mg, more preferably between 10 and 100 mg, such as between 10 and 100 mg, between 10 and 90 mg, between 10 and 80 mg, between 10 and 70 mg, between 10 and 60 mg, between 10 and 50 mg, between 10 and 40 mg, between 10 and 30 mg, between 10 and 20 mg, between 20 and 100 mg, between 20 and 90 mg, between 20 and 80 mg, between 20 and 70 mg, between 20 and 60 mg, between 20 and 50 mg, between 20 and 40 mg, between 20 and 30 mg, between 30 and 100 mg, between 30 and 90 mg, between 30 and 80 mg, between 30 and 70 mg, between 30 and 60 mg, between 30 and 50 mg, between 30 and 40 mg, between 40 and 100 mg, between 40 and 90 mg, between 40 and 80 mg, between 40 and 70 mg, between 40 and 60 mg, between 40 and 50 mg, between 50 and 100 mg, between 50 and 90 mg, between 50 and 80 mg, between 50 and 70 mg, between 50 and 60 mg, between 60 and 100 mg, between 60 and 90 mg, between 60 and 80 mg, between 60 and 70 mg, between 70 and 100 mg, between 70 and 90 mg, between 70 and 80 mg, between 80 and 100 mg, between 80 and 90 mg or between 90 and 100 mg.

[0107] In an embodiment, a safe and effective amount or dose of said amino acid component in said pharmaceutical composition is between 0.1 and 200 mg, preferably between 0.1 and 100 mg, more preferably between 10 and 100 mg, such as between 10 and 100 mg, between 10 and 90 mg, between 10 and 80 mg, between 10 and 70 mg, between 10 and 60 mg, between 10 and 50 mg, between 10 and 40 mg, between 10 and 30 mg, between 10 and 20 mg, between 20 and 100 mg, between 20 and 90 mg, between 20 and 80 mg, between 20 and 70 mg, between 20 and 60 mg, between 20 and 50 mg, between 20 and 40 mg, between 20 and 30 mg, between 30 and 100 mg, between 30 and 90 mg, between 30 and 80 mg, between 30 and 70 mg, between 30 and 60 mg, between 30 and 50 mg, between 30 and 40 mg, between 40 and 100 mg, between 40 and 90 mg, between 40 and 80 mg, between 40 and 70 mg, between 40 and 60 mg, between 40 and 50 mg, between 50 and 100 mg, between 50 and 90 mg, between 50 and 80 mg, between 50 and 70 mg, between 50 and 60 mg, between 60 and 100 mg, between 60 and 90 mg, between 60 and 80 mg, between 60 and 70 mg, between 70 and 100 mg, between 70 and 90 mg, between 70 and 80 mg, between 80 and 100 mg, between 80 and 90 mg or between 90 and 100 mg.

[0108] In an embodiment, one dosage of said pharmaceutical composition has a volume of about 0.5-5 ml. In an embodiment, one dosage of said pharmaceutical composition has a volume of about 0.5 ml, 1.0 ml, 1.5 ml, 2.0 ml, 2.5 ml, 3.0 ml, 3.5 ml, 4.0 ml, 4.5 ml or 5.0 ml.

[0109] In an embodiment, said pharmaceutical composition comprises between 1-100 mg / ml, more preferably between 2-50 mg / ml, more preferably between 2-40 mg / ml, more preferably between 2-30 mg / ml, more preferably between 2-20 mg / ml of said hyaluronic acid or a salt or ester thereof.

[0110] In a preferred embodiment, said pharmaceutical composition comprises between 5-20 mg / ml of said hyaluronic acid or a salt or ester thereof. In an embodiment, said pharmaceutical composition comprises between 5-6 mg / ml, between 6-7 mg / ml, between 7-8 mg / ml, between 8-9 mg / ml, between 9-10 mg / ml, between 10-11 mg / ml, between 11-12 mg / ml, between 12-13 mg / ml, between 13-14 mg / ml, between 14-15 mg / ml, between 15-16 mg / ml, between 16-17 mg / ml, between 17-18 mg / ml, between 18-19 mg / ml or between 19-20 mg / ml of said hyaluronic acid or a salt or ester thereof.

[0111] In an embodiment, said pharmaceutical composition comprises between 5-5.5 mg / ml, between 5.5-6 mg / ml, between 6-6.5 mg / ml, between 6.5-7 mg / ml, between 7-7.5 mg / ml, between 7.5-8 mg / ml, between 8-8.5 mg / ml, between 8.5-9 mg / ml, between 9-9.5 mg / ml, between 9.5-10 mg / ml, between 10-10.5 mg / ml, between 10.5-11 mg / ml, between 11-11.5 mg / ml, between 11.5-12 mg / ml, between 12-12.5 mg / ml, between 12.5-13 mg / ml, between 13-13.5 mg / ml, between 13.5-14 mg / ml, between 14-14.5 mg / ml, between 14.5-15 mg / ml, between 15-15.5 mg / ml, between 15.5-16 mg / ml, between 16-16.5 mg / ml, between 16.5-17 mg / ml, between 17-17.5 mg / ml, between 17.5-18 mg / ml, between 18-18.5 mg / ml, between 18.5-19 mg / ml, between 19-19.5 mg / ml or between 19.5-20 mg / ml of said hyaluronic acid or a salt or ester thereof.

[0112] In an embodiment, said pharmaceutical composition comprises at least 5 mg / ml, at least 6 mg / ml, at least 7 mg / ml, at least 8 mg / ml, at least 9 mg / ml, at least 10 mg / ml, at least 11 mg / ml, at least 12 mg / ml, at least 13 mg / ml, at least 14 mg / ml, at least 15 mg / ml, at least 16 mg / ml, at least 17 mg / ml, at least 18 mg / ml or at least 19 mg / ml of said hyaluronic acid or a salt or ester thereof.

[0113] In an embodiment, said pharmaceutical composition comprises at most 20 mg / ml, at most 19 mg / ml, at most 18 mg / ml, at most 17 mg / ml, at most 16 mg / ml, at most 15 mg / ml, at most 14 mg / ml, at most 13 mg / ml, at most 12 mg / ml, at most 11 mg / ml, at most 10 mg / ml, at most 9 mg / ml, at most 8 mg / ml, at most 7 mg / ml, at most 6 mg / ml, at most 5 mg / ml, at most 4 mg / ml or at most 3 mg / ml of said hyaluronic acid or a salt or ester thereof.

[0114] In a preferred embodiment, said pharmaceutical composition comprises between 15-25 mg / ml of said hyaluronic acid or a salt or ester thereof. In an embodiment, said pharmaceutical composition comprises between 15-20 mg / ml, such as 15-16 mg / ml, between 16-17 mg / ml, between 17-18 mg / ml, between 18-19 mg / ml, between 19-20 mg / ml. In an embodiment, said pharmaceutical composition comprises between 20-25 mg / ml, such as between 20-21 mg / ml, between 21-22 mg / ml, between 22-23 mg / ml, between 23-24 mg / ml or between 24-25 mg / ml of said hyaluronic acid or a salt or ester thereof.

[0115] In an embodiment, said pharmaceutical composition comprises approximately 15 mg / ml, 16 mg / ml, 17 mg / ml, 18 mg / ml, 19 mg / ml, 20 mg / ml, 21 mg / ml, 22 mg / ml, 23 mg / ml, 24 mg / ml or 25 mg / ml of said hyaluronic acid or a salt or ester thereof.

[0116] In an embodiment, said pharmaceutical composition comprises between 2-50 mg / ml, more preferably between 2-40 mg / ml, more preferably between 2-30 mg / ml, more preferably between 2-20 mg / ml of said amino acid composition.

[0117] In a preferred embodiment, said pharmaceutical composition comprises between 5-20 mg / ml of said hyaluronic acid or a salt or ester thereof.

[0118] In a preferred embodiment, said pharmaceutical composition comprises between 5-20 mg / ml of said amino acid composition.

[0119] In an embodiment, said pharmaceutical composition comprises between 5-6 mg / ml, between 6-7 mg / ml, between 7-8 mg / ml, between 8-9 mg / ml, between 9-10 mg / ml, between 10-11 mg / ml, between 11-12 mg / ml, between 12-13 mg / ml, between 13-14 mg / ml, between 14-15 mg / ml, between 15-16 mg / ml, between 16-17 mg / ml, between 17-18 mg / ml, between 18-19 mg / ml or between 19-20 mg / ml of said amino acid composition.

[0120] In an embodiment, said pharmaceutical composition comprises between 5-5.5 mg / ml, between 5.5-6 mg / ml, between 6-6.5 mg / ml, between 6.5-7 mg / ml, between 7-7.5 mg / ml, between 7.5-8 mg / ml, between 8-8.5 mg / ml, between 8.5-9 mg / ml, between 9-9.5 mg / ml, between 9.5-10 mg / ml, between 10-10.5 mg / ml, between 10.5-11 mg / ml, between 11-11.5 mg / ml, between 11.5-12 mg / ml, between 12-12.5 mg / ml, between 12.5-13 mg / ml, between 13-13.5 mg / ml, between 13.5-14 mg / ml, between 14-14.5 mg / ml, between 14.5-15 mg / ml, between 15-15.5 mg / ml, between 15.5-16 mg / ml, between 16-16.5 mg / ml, between 16.5-17 mg / ml, between 17-17.5 mg / ml, between 17.5-18 mg / ml, between 18-18.5 mg / ml, between 18.5-19 mg / ml, between 19-19.5 mg / ml or between 19.5-20 mg / ml of said amino acid composition.

[0121] In an embodiment, said pharmaceutical composition comprises at least 5 mg / ml, at least 6 mg / ml, at least 7 mg / ml, at least 8 mg / ml, at least 9 mg / ml, at least 10 mg / ml, at least 11 mg / ml, at least 12 mg / ml, at least 13 mg / ml, at least 14 mg / ml, at least 15 mg / ml, at least 16 mg / ml, at least 17 mg / ml, at least 18 mg / ml or at least 19 mg / ml of said amino acid composition.

[0122] In an embodiment, said pharmaceutical composition comprises at most 20 mg / ml, at most 19 mg / ml, at most 18 mg / ml, at most 17 mg / ml, at most 16 mg / ml, at most 15 mg / ml, at most 14 mg / ml, at most 13 mg / ml, at most 12 mg / ml, at most 11 mg / ml, at most 10 mg / ml, at most 9 mg / ml, at most 8 mg / ml, at most 7 mg / ml, at most 6 mg / ml, at most 5 mg / ml, at most 4 mg / ml or at most 3 mg / ml of said amino acid composition.

[0123] In a preferred embodiment, said pharmaceutical composition comprises between 15-35 mg / ml of said amino acid composition.

[0124] In an embodiment, said pharmaceutical composition comprises between 15-20 mg / ml, such as between 15-16 mg / ml, between 16-17 mg / ml, between 17-18 mg / ml, between 18-19 mg / ml, between 19-20 mg / ml. In an embodiment, said pharmaceutical composition comprises between 20-25 mg / ml, such as between 20-21 mg / ml, between 21-22 mg / ml, between 22-23 mg / ml, between 23-24 mg / ml, between 24-25 mg / ml. In an embodiment, said pharmaceutical composition comprises between 25-30 mg / ml, such as between 25-26 mg / ml, between 26-27 mg / ml, between 27-28 mg / ml, between 28-29 mg / ml, between 29-30 mg / ml. In an embodiment, said pharmaceutical composition comprises between 30-35 mg / ml, such as between 30-31 mg / ml, between 31-32 mg / ml, between 32-33 mg / ml, between 33-34 mg / ml, between 34-35 mg / ml of said amino acid composition. In an embodiment, said pharmaceutical composition comprises approximately 15 mg / ml, 16 mg / ml, 17 mg / ml, 18 mg / ml, 19 mg / ml, 20 mg / ml, 21 mg / ml, 22 mg / ml, 23 mg / ml, 24 mg / ml, 25 mg / ml, 26 mg / ml, 27 mg / ml, 28 mg / ml, 29 mg / ml, 30 mg / ml, 31 mg / ml, 32 mg / ml, 33 mg / ml, 34 mg / ml, 35 mg / ml of said amino acid composition.

[0125] In a preferred embodiment, the pharmaceutical composition comprises 10 mg / ml of said hyaluronic acid or a salt or ester thereof and 10 mg / ml of said amino acid composition.

[0126] In a preferred embodiment, the pharmaceutical composition comprises 20 mg / ml of said hyaluronic acid or a salt or ester thereof and 20 mg / ml of said amino acid composition.

[0127] In a preferred embodiment, the particle size of the pharmaceutical composition is less than 0.45 pm, preferably less than 0.30 pm, and more preferably less than or equal to 0.22 pm.

[0128] In a preferred embodiment, the density of the pharmaceutical composition at 20-25 °C is between 900 and 1200 g / cm3, more preferably between 950 and 1150 g / cm3, more preferably between 1000 and 1050 g / cm3, more preferably between 1.010 and 1.030 g / cm3, more preferably between 1.016 and 1.022 g / cm3, and more preferably between 1.018 and 1.020 g / cm3, with a most preferred value of about 1.0199 g / cm3.

[0129] In a preferred embodiment, the turbidity of the pharmaceutical composition is less than 3.0 NTU, preferably less than 2.0 NTU, and more preferably less than 1.0 NTU.

[0130] In a preferred embodiment, the total heavy metal content of the pharmaceutical composition is less than 50 pg / g, preferably less than 25 pg / g, and more preferably less than or equal to 10 pg / g.

[0131] In a preferred embodiment, the osmolality of the pharmaceutical composition is between 200 and 400 mOsmol / kg, preferably between 230 and 320 mOsmol / kg, and more preferably between 240 and 270 mOsmol / kg, with a most preferred value of about 250 mOsmol / kg.

[0132] The pharmaceutical composition must comply with sterility testing and shows no growth in the sterility test. Furthermore, the bacterial endotoxin content, measured by the (Limulus Amebocyte Lysate test) LAL test in accordance with the European Pharmacopoeia, is less than 1.0 EU / mg.

[0133] In an embodiment, said pharmaceutical composition is formulated in a vial or in a pre-filled syringe, said vial or syringe comprising one or more dosages for administration to said subject.

[0134] In an embodiment, said pharmaceutical composition comprises additional bio-active substances such as platelet-rich plasma (PRP), alpha 2 macroglobulins (A2MG), Interleukin 1 receptor antagonist (IRAP), stem cells, triamcinolone (TA), betamethasone (CS), poly deoxyribonucleotide (PDRN) or dexamethasone (DX). These are known to have additional beneficial functions during downstream applications of the pharmaceutical composition according to the current invention. Mixing of the pharmaceutical composition comprising HA and an amino acid component with such substances may in some cases be desirable to increase the effectiveness of the pharmaceutical composition or create a synergistic effect.

[0135] In a specific embodiment, said pharmaceutical composition comprises 10 mg / ml sodium hyaluronate and 10 mg / ml CTP-F present in a sterile liquid, wherein said hyaluronic acid has a molecular weight between 500 kDa and 4000 kDa and wherein said pharmaceutical composition comprises a pH between 6.4 and 7.8.

[0136] In a preferred embodiment, the pharmaceutical composition of the invention comprises 20 mg / ml purified hyaluronic acid, 20 mg / ml amino acid composition (preferably a collagen hydrolysate comprising at least 10% tripeptide content), 9 mg / ml sodium chloride and 0.15-0.25 mg / ml phosphate buffer. In a preferred embodiment, said pharmaceutical composition has a pH between 6.5-7.5 (preferably 6.9), an intrinsic viscosity between 2.8-3.2 m3 / kg (preferably 2.9 m3 / kg), a particle size below 0.22 pm, a density of 1.0199 g / cm3, a turbidity below 1.0 NTU and a osmolality between 200-400 mOsmol / kg. In a preferred embodiment, the pharmaceutical composition of the invention comprises 20 mg / ml purified hyaluronic acid, 30 mg / ml amino acid composition (preferably a collagen hydrolysate comprising at least 10% tripeptide content), 9 mg / ml sodium chloride and 0.15-0.25 mg / ml phosphate buffer. In a preferred embodiment, said pharmaceutical composition has a pH between 6.5-7.5 (preferably 6.9), an intrinsic viscosity between 2.8-3.2 m3 / kg (preferably 2.9 m3 / kg), a particle size below 0.22 pm, a density of 1.0199 g / cm3, a turbidity below 1.0 NTU and a osmolality between 200-400 mOsmol / kg.

[0137] In a further aspect, the invention relates to aforementioned pharmaceutical composition for use in the treatment of a musculoskeletal disease, preferably a bone disease or a joint disease in a subject.

[0138] As used herein, the term "subject" refers to any animal (e.g., a mammal), including, but not limited to, humans, non-human primates, rodents, and the like, to which the compositions as disclosed herein are administered. The subject is preferably a mammal and the pharmaceutical composition is preferably used in the veterinary field. In a preferred embodiment, said subject is a canine. In another preferred embodiment, said subject is an equine.

[0139] Such subjects may include, without limitation, those that have been diagnosed with said condition, those prone to develop said condition and / or those in whom said condition is to be prevented.

[0140] In an embodiment, said subject to be treated is unresponsive to a previous treatment, for instance a corticosteroid treatment or a treatment based on hyaluronic acid (injection) alone, hence not combined with an amino acid composition.

[0141] A disorder of the musculoskeletal system may include a disorder of the joint, cartilage, ligament, tendon, tendon sheath or a combination of different connective tissues.

[0142] Pathologies in the musculoskeletal system in present invention are, but not limited to (osteo)arthritis, tendinitis, fibromyalgia, bone fractures. These disorders or injuries can often be multifactorial; aging, trauma, mechanical forces, conformation, hormonal and genetic factors contributing to varying degrees. Preferably, the pharmaceutical composition of current invention will be used in a subject in need of treatment of said musculoskeletal disease.

[0143] In addition, the pharmaceutical composition of current invention may alleviate the symptoms of a subject diagnosed with musculoskeletal disease.

[0144] In an embodiment, the current invention relates to a pharmaceutical composition for use in the treatment of osteoarthritis. Osteoarthritis is a progressively worsening inflammation of the joint caused by the deterioration of cartilage. In a healthy joint, cartilage acts as a cushion to allow the joint to move smoothly through its full range of motion. In cases of osteoarthritis, this cartilage cushion begins to break down because of factors such as age, injury, repetitive stress, disease or genetic predisposition. The loss of this protective cushion results in pain, inflammation, decreased range of motion, and the development of bone spurs. Diagnosis is typically based on signs and symptoms, with medical imaging and other tests used to support or rule out other problems.

[0145] Many subjects diagnosed with or suffering from osteoarthritis show (visual) signs of lameness and / or joint pain. Therefore, the invention also relates to the pharmaceutical composition as described in any of the previous embodiments, which are used in the in the treatment of lameness and / or joint pain in subjects diagnosed with or suffering from osteoarthritis, or as a method for treating lameness and / or joint pain in subjects diagnosed with or suffering from osteoarthritis, or for use in the preparation of a medicament for the treatment of lameness and / or joint pain in subjects diagnosed with or suffering from osteoarthritis. Said pharmaceutical composition is preferably intra-articularly administered.

[0146] Treatment of lameness and / or joint pain comprises the prevention, the reduction, the mitigation, the amelioration and / or the reversion of said lameness and / or joint pain in the subject diagnosed with or suffering from osteoarthritis.

[0147] In an embodiment, the current invention relates to a pharmaceutical composition for use in the prevention of cranial cruciate ligament (CCL) rupture. The cranial cruciate ligament is one of the most important stabilizers inside the knee joint, the middle joint in the back leg. Rupture of the cranial cruciate ligament is one of the most common reasons for hind limb lameness, pain, and subsequent knee arthritis. While the clinical signs associated with CCL rupture vary, the condition invariably causes rear limb dysfunction and pain. Trauma accounts for a minority of CCL ruptures, whereas progressive degeneration of the ligament has been attributed to a variety of factors that may be broadly classified as genetic, conformational, environmental, immune-mediated, and inflammatory. Diagnosing complete tears of the CCL is easily accomplished using a combination of gait observations, physical examination findings, and radiography (X-rays). By contrast, partial CCL tears may be more challenging to diagnose. Use of the current pharmaceutical composition for treating and / or preventing partial rupture of the cranial cruciate ligament in a subject such as an animal, significantly reduces signs of ligament degeneration and helps prevent complete cranial cruciate ligament rupture and reduces the incidence of contralateral disease in subjects with unilateral CCL rupture.

[0148] In an embodiment, the current invention relates to a pharmaceutical composition for use in the treatment of tendinopathies, such as digital flexor tendinopathy and suspensory ligament tendon rupture. Tendinopathy is a type of tendon disorder that results in pain, swelling, and impaired function. Digital flexor tendinopathy is a term used to describe tears, calcifying tendinopathy, tendinosis and / or injuries in and around the tendon of the flexor digitorum muscles, and is a cause of forelimb lameness. The digital flexor tendons are important passive stabilizers of the equine lower limb, and are responsible for limb extension and advancing the limb. Injury to the digital flexor tendon causes inflammation. Tearing of the tendon fibers and the resulting inflammation can lead to mineralization and calcification of the tendon, which are a source of pain and lameness. Depending on the trauma, the severity of the lesion may vary considerably, leading to stretching, small or partial lacerations or a complete rupture.

[0149] In a preferred embodiment, said musculoskeletal disease is osteoarthritis.

[0150] As described above, the pharmaceutical composition of current invention is dispersed in a pharmaceutically and pharmacologically acceptable liquid to obtain a pharmaceutical composition for local or parenteral administration, such as local or parenteral injection.

[0151] In an embodiment, said pharmaceutical composition is administered to said subject by means of parenteral administration, such as intravenous administration (for instance intravenous injection).

[0152] Preferably the pharmaceutical composition is configured for local administration, such as intraosseous, periosseous, intra-articular, periarticular administration, or for intratendon, peritendon, intraligament, or periligament administration. These modes of administration will strongly depend on the site of injury, being for example a joint, ligament, tendon or tendon sheet. The site of delivery of the pharmaceutical composition is typically at or near the site of tissue damage. The site of tissue damage is determined by well-established methods, including imaging studies, known by a skilled person.

[0153] In a particular embodiment the pharmaceutical composition is configured for intraosseous or periosseous administration. Intraosseous administration or delivery generally refers to a method whereby a treatment is delivered, directly or indirectly, into the bone (trabecular or cortical). Peri-osseous administration or delivery generally refers to a method whereby a treatment is delivered in the surroundings of a bone (especially around the fracture / damage site).

[0154] In another particular embodiment, the pharmaceutical composition is configured for intra-articular or periarticular administration. Intra-articular administration or delivery generally refers to a method whereby a treatment is delivered, directly or indirectly, into the synovial capsule of an articulating joint.

[0155] Peri-articular administration or delivery generally refers to a method whereby a treatment is delivered in the surroundings of the synovial capsule of an articulating joint and / or the subchondral bone.

[0156] In an embodiment, the pharmaceutical composition is configured for intratendon or peritendon administration.

[0157] In an embodiment, the pharmaceutical pharmaceutical composition is configured for intraligament or periligament administration. A person skilled in the art is familiar with the indications for and complications associated with these injection techniques. As described above, the pharmaceutical composition provides optimal viscoelastic and bio-active properties, allowing to reduce the frequency of administration. Without limitation, a typical dose of the pharmaceutical composition to be administered may range from about 0.1 to 5.0 ml per injection, preferably between 1.0 and 5.0 ml per injection, such as 2 ml per injection. This amount is suitable for parenteral injection, such as intra-articular or periarticular injection.

[0158] In an embodiment, one dosage of said pharmaceutical composition has a volume of about 1.0 to 5.0 ml. In another or further embodiment, one dosage of said pharmaceutical composition has a volume of maximally about 5.0 ml, maximally about 4.0 ml, maximally about 3.0 ml, maximally about 2.0 ml or maximally about 1.0 ml. In an embodiment, the volume of the pharmaceutical composition which is administered per injection to a patient is adapted in accordance with the patient's body weight.

[0159] In a specific embodiment, when the subject to which the pharmaceutical composition is to be administered is an equine, one dosage of said pharmaceutical composition has a volume between 2.0 and 5.0 ml, such as 2.0, 2.5, 3.0, 3.5, 4.0, 4.5 or 5.0 ml.

[0160] In a specific embodiment, when the subject to which the pharmaceutical composition is to be administered is a canine, one dosage of said pharmaceutical composition has a volume between 1.0 and 3.0 ml, such as 1.0, 1.5, 2.0, 2.5 or 3.0 ml.

[0161] In an embodiment, said pharmaceutical composition of the current invention is administered once.

[0162] In an embodiment, said pharmaceutical composition of the current invention is administered at least twice, at least three times, at least four times, at least five times, preferably with intervals.

[0163] In an embodiment, the treatment comprises: multiple administrations of the pharmaceutical composition of the current invention, for example multiple intraarticular administrations per canine or equine patient, wherein said multiple doses are administered at various time points, including but not limited to one or more of the following time points 1 day apart, 2 days apart, 3 days apart, 4 days apart, 5 days apart, 6 days apart, 7 days (1 week) apart, 2 weeks apart, 3 weeks apart, 4 weeks apart, 5 weeks apart, 6 weeks apart, 7 weeks apart, 8 weeks apart, 3 months apart, 4 months apart, 5 months apart, 6 months apart, 7 months apart, 8 months apart, 9 months apart, 10 months apart, 11 months apart, 1 year apart or more than 1 year apart.

[0164] In an embodiment, the pharmaceutical composition of the current invention is administered multiple times over a period of 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks or more than 24 weeks, such as 7 months, 8 months, 9 months, 10 months, 11 months or 12 months.

[0165] Preferably each dose is administered 2-3 months apart, for a period of 6 months. In an embodiment, the invention relates to a pharmaceutical composition for use in the treatment of osteoarthritis in equines, wherein: the treatment comprises a step of administering, preferably intra-articularly, a first amount of said pharmaceutical composition comprising a total dose of 20-100 mg of the hyaluronic acid component and 20-100 mg of the amino acid component per patient, one dosage of said pharmaceutical composition having a volume between 2-5 ml,

[0166] the treatment further comprises a step of administering, preferably intraarticularly, a second amount of said pharmaceutical composition comprising a total dose of 20-100 mg of the hyaluronic acid component and 20-100 mg of the amino acid component per patient, one dosage of said pharmaceutical composition having a volume between 2-5 ml,

[0167] - the treatment further comprises a step of administering, preferably intra-articularly, one or more further amounts of said pharmaceutical composition comprising a total dose of 20-100 mg of the hyaluronic acid component and 20-100 mg of the amino acid component per patient, one dosage of said pharmaceutical composition having a volume between 2-5 ml,

[0168] - the treatment further comprises a step of administering, preferably intra-articularly, a last amount of said pharmaceutical composition comprising a total dose of 20-100 mg of the hyaluronic acid component and 20-100 mg of the amino acid component per patient, one dosage of said pharmaceutical composition having a volume between 2-5 ml,

[0169] wherein said second amount is administered 2-3 months after the first amount and each further and last amount is administered 2-3 months after the previous amount and wherein said last amount is administered 6 months after the first amount.

[0170] As such, in an embodiment, said second amount is administered 8 weeks, 9 weeks, 10 weeks, 11 weeks or 12 weeks after the first amount and each further and last amount is administered 8 weeks, 9 weeks, 10 weeks, 11 weeks or 12 weeks after the previous amount.

[0171] In an embodiment, the invention relates to a pharmaceutical composition for use in the treatment of osteoarthritis in canines, wherein:

[0172] the treatment comprises a step of administering, preferably intra-articularly, a first amount of said pharmaceutical composition comprising a total dose of 10-60 mg of the hyaluronic acid component and 10-60 mg of the amino acid component per patient, one dosage of said pharmaceutical composition having a volume between 1-3 ml, the treatment further comprises a step of administering, preferably intraarticularly, a second amount of said pharmaceutical composition comprising a total dose of 10-60 mg of the hyaluronic acid component and 10-60 mg of the amino acid component per patient, one dosage of said pharmaceutical composition having a volume between 1-3 ml,

[0173] - the treatment further comprises a step of administering, preferably intra-articularly, one or more further amounts of said pharmaceutical composition comprising a total dose of 10-60 mg of the hyaluronic acid component and 10-60 mg of the amino acid component per patient, one dosage of said pharmaceutical composition having a volume between 1-3 ml,

[0174] - the treatment further comprises a step of administering, preferably intra-articularly, a last amount of said pharmaceutical composition comprising a total dose of 10-60 mg of the hyaluronic acid component and 10-60 mg of the amino acid component per patient, one dosage of said pharmaceutical composition having a volume between 1-3 ml,

[0175] wherein said second amount is administered 2-3 months after the first amount and each further and last amount is administered 2-3 months after the previous amount and wherein said last amount is administered 6 months after the first amount.

[0176] As such, in an embodiment, said second amount is administered 8 weeks, 9 weeks, 10 weeks, 11 weeks or 12 weeks after the first amount and each further and last amount is administered 8 weeks, 9 weeks, 10 weeks, 11 weeks or 12 weeks after the previous amount.

[0177] In an embodiment, said second dose, further doses and / or last dose are identical to the first dose. In another embodiment, said second dose, further doses and / or last dose are lower than the first dose. In yet another embodiment, said second dose, further doses and / or last dose are higher than the first dose.

[0178] A qualified physician will be able to determine the dose and amount of injections taking into account the size of the patient. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. The pharmaceutical composition for use according to the current invention, possibly together with further components as described above, will by preference be stored at room temperature (freezing creates crystal formation resulting in disturbed 3D polymerization and composition). These conditions allow a save storage of the composition, and enable the components to keep their biological and physical characteristics during storage. In a more preferred embodiment, the pharmaceutical composition for use of the invention can be stored for at least 12 months at room temperature.

[0179] In an embodiment, administration of the pharmaceutical composition of the current invention is combined with other therapies, such as low-level laser therapy (LLLT).

[0180] EXAMPLES

[0181] The present invention will now be further exemplified with reference to the following examples. The present invention is in no way limited to the given examples.

[0182] EXAMPLE 1: the pharmaceutical composition according to an embodiment of the invention for use in the treatment of osteoarthritis in dogs

[0183] The objective of this study is to evaluate the potential of intra -articular injections of the pharmaceutical composition as treatment for dogs suffering from naturally occurring articular pain, unresponsive to the current standard therapies. The clinical effect and safety of the treatment are evaluated 1 and 3 weeks and 3 and 6 months after the first injection.

[0184] A total of 5 joints are treated with the investigational product (IVP). Patient inclusion is restricted by the following inclusion criteria: joint pain in one or multiple joints for several days / weeks, non-responsiveness to conservative therapies, confirmed lameness (see Table 2), confirmed pain by anamnesis, joint pain associated signs confirmed by radiography (RX) or other imaging modalities and completion of Canine Brief Pain Inventory (CBPI) guestionnaires including pain severity score (PSS)>3 and pain interference score (PIS) >3 (see Table 1).

[0185] Patients with following conditions and treatments are excluded from the study: sprains, pregnancy, other diseases that could influence the clinical study, PSS<3 and PIS<3, changes in dog's regular medical treatment, corticosteroid administrations within the washout period, or an ongoing corticosteroid treatment. All dogs are observed for uncommon behavior, posture and the occurrence of potential adverse events, such as worsening of lameness, joint distention or skin allergy at the injection site, at five evaluation points during the study (Day 0, 1 week, 3 weeks, 3 months and 6 months after the first injection). Evaluations are performed by a veterinarian with at least 5 years of practical experience in the field of canine orthopedics. Owners, who are well informed, are tasked to report the occurrence of potential adverse events in between evaluation points. All regular medical treatments are continued during the study.

[0186] The pharmaceutical composition used (the IVP) comprises CTF-P (10 mg / ml) dissolved in sodium hyaluronate (lOmg / ml) in 2ml volume with a pH between 6.4 and 7.8. The hyaluronic acid component in the pharmaceutical composition has a molecular weight between 800 kDa and 1000 kDa, the CTP-F has a molecular weight below 300 Da.

[0187] The pharmaceutical composition was injected once at timepoint 0.

[0188] At the evaluation points, the effect of the treatment is investigated by means of a CBPI score (Table 1) and lameness evaluation (Table 2). The CBPI consists of eleven questions with a scoring system ranging from 0 to 10. Hereby the first four questions are used for scoring severity of the pain, the next six questions are used to determine pain interference and the last question gauges the overall quality of life of the dog.

[0189] Table 1: The canine brief pain inventory (CBPI) scoring

[0190] # Description Question Scoring

[0191] 1 Score the pain at its worst in the last 7 days 0-10

[0192] 2 Pain Score the pain at its least in the last 7 days 0-10 severity Score the pain at its average in the last 7

[0193] 3 0-10

[0194] score (PSS) days

[0195] 4 Score the pain as it is right now 0-10

[0196] Score pain interference with general

[0197] 5 0-10

[0198] activity

[0199] Score pain interference with enjoyment of

[0200] 6 0-10

[0201] life

[0202] Pain

[0203] Score pain interference with ability to rise

[0204] 7 interference 0-10

[0205] to standing from lying down

[0206] score (PIS)

[0207] 8 Score pain interference with ability to walk 0-10

[0208] 9 Score pain interference with ability to run 0-10

[0209] Score pain interference with ability to climb

[0210] 10 0-10

[0211] stairs, curbs, doorsteps, etc.

[0212] Overall Poor Score the dog's overall quality of life over

[0213] 11 quality of Fair the last 7 days

[0214] life Good

[0215]

[0216] Very good Excellent

[0217]

[0218] Table 2: Lameness scoring

[0219] Parameter Score Definition

[0220] 1 Stands, walks and trots normally

[0221] Stands normally, slight painful gait when 2

[0222] trotting

[0223] Stands normally, slight painful gait when 3

[0224] Lameness walking

[0225] Stands normally, evident painful gait when 4

[0226] walking

[0227] Stands abnormally, evident painful gait when 5

[0228] walking

[0229]

[0230] 5 joints were injected in total. No clinical adverse effects was detected in any of the dogs treated with the IVP. A clinical improvement was observed in 3 out of 5 joints at all time points, showing improvement of CBPI score of at least 2 scores and improvement of lameness of at least 1 score.

[0231] EXAMPLE 2: the pharmaceutical composition according to an embodiment of the invention for use in the treatment of osteoarthritis in horses

[0232] The objective of this study is to evaluate the potential of intra-articular injections of the pharmaceutical composition as treatment for horses suffering from naturally occurring articular pain, unresponsive to the current standard therapies. The clinical effect and safety of the treatment are evaluated 1 week, 4 weeks, 3 months and 6 months after the first injection.

[0233] A total of 20 joints are treated with the investigational product (IVP). Patient inclusion is restricted by the following inclusion criteria: joint pain in one or multiple joints for several days / weeks, non-responsiveness to conservative therapies, confirmed lameness, confirmed pain by anamnesis, joint pain associated signs confirmed by radiography (RX) or other imaging modalities. As a control group, standard of care was administered in the contralateral limb of 5 horses. Patients with following conditions and treatments are excluded from the study: sprains, pregnancy, other diseases that could influence the clinical study, changes in horses' regular medical treatment, corticosteroid administrations within the washout period, or an ongoing corticosteroid treatment. All horses are observed for uncommon behavior, posture and the occurrence of potential adverse events, such as worsening of lameness, joint distention or skin allergy at the injection site, at four evaluation points during the study ( 1 week, 4 weeks, 3 months and 6 months after the first injection). Evaluations are performed by a veterinarian with at least 5 years of practical experience in the field of equine orthopedics. Owners, who are well informed, are tasked to report the occurrence of potential adverse events in between evaluation points. All regular medical treatments are continued during the study.

[0234] The pharmaceutical composition used (the IVP) comprises CTF-P (10 mg / ml) dissolved in sodium hyaluronate (lOmg / ml) in 2ml volume with a pH between 6.4 and 7.8. The hyaluronic acid component in the pharmaceutical composition has a molecular weight between 800 kDa and 1000 kDa, the CTP-F has a molecular weight below 300 Da.

[0235] The pharmaceutical composition was injected once at timepoint 0.

[0236] 20 joints were injected in total. No clinical adverse effects was detected in any of the equines treated with the IVP. At the evaluation points, the effect of the treatment is investigated and scored by an orthopedic examination and lameness evaluation. In the treatment group joint effusion was on average decreased from 3 / 4 to 0 / 4 or 1 / 4 (see Table 3 below) and lameness was on average decreased from 2 / 5 or 3 / 5 to 0 / 5 or 1 / 5 (see Table 2 above) at all time points after injection. No or mild improvement was seen in the control group.

[0237] Table 3: joint effusion scoring

[0238] Parameter Score Definition

[0239] 0 None

[0240] 1 Mild signs (only at site of injection)

[0241] Joint effusion 2 Moderate signs (mild swelling entire joint)

[0242] 3 Severe sings (severe swelling entire joint)

[0243] 4 Extreme (peri-articular swelling)

[0244]

[0245] The present invention is in no way limited to the embodiments described in the examples. On the contrary, methods according to the present invention may be realized in many different ways without departing from the scope of the invention.

Claims

CLAIMS1. A pharmaceutical composition for local or parenteral administration, said pharmaceutical composition being an injectable solution comprising an amino acid composition dissolved in purified hyaluronic acid ora salt or ester thereof, wherein said hyaluronic acid has a molecular weight between 500 kDa and 4000 kDa and wherein said injectable solution comprises a pH between 6.4 and 7.8.

2. The pharmaceutical composition according to claim 1, wherein said amino acid composition comprises a collagen tripeptide (CTP), said collagen tripeptide comprising a collagen hydrolysate comprising at least 10% of tripeptide content, said tripeptides having a Glycine-Xaa-Yaa sequence.

3. The pharmaceutical composition according to claim 2, wherein the tripeptide Glycine-Proline-Hydroxyproline is comprised in said CTP at a concentration of at least 10% of the total tripeptide content.

4. The pharmaceutical composition according to claim 1, wherein said amino acid composition comprises a pool of single amino acids.

5. The pharmaceutical composition according to claim 4, wherein said amino acid pool comprises glycine, (hydroxy)lysine and / or (hydroxy)proline.

6. The pharmaceutical composition according to any of the previous claims, wherein said pharmaceutical composition comprises a hyaluronic acid salt, preferably sodium hyaluronate.

7. The pharmaceutical composition according to any of the previous claims, wherein said pharmaceutical composition comprises between 5-20 mg / ml of said hyaluronic acid or a salt or ester thereof.

8. The pharmaceutical composition according to any of the previous claims, wherein said pharmaceutical composition comprises between 5-20 mg / ml of said amino acid composition.

9. The pharmaceutical composition according to any of the previous claims, wherein said collagen tripeptide has a molecular weight less than 300 Da.

10. The pharmaceutical composition according to any of the previous claims 2-3 and 6-9, wherein said collagen tripeptide is prepared from fish skin (CTP-F).

11. The pharmaceutical composition according to any of the previous claims, wherein said pharmaceutical composition has an intrinsic viscosity between 0.5-2.3 m3 / kg as measured by capillary viscometry at 25°C.

12. The pharmaceutical composition according to any one of the preceding claims 1 to 11 for use in the treatment of a musculoskeletal disease, such as osteoarthritis, wherein said pharmaceutical composition is administered to a subject in need of treatment.

13. The pharmaceutical composition for use according to claim 12, wherein said use is in the veterinary field, and wherein said subject is preferably a canine or an equine.

14. The pharmaceutical composition for use according to any of the previous claims 12-13, wherein said pharmaceutical composition is administered to said subject by means of local administration, such as intra-articular or periarticular or intratendinous or peritendinous injection, or parenteral administration, such as intravenous injection.

15. The pharmaceutical composition for use according to any of the previous claims 12-14, wherein said pharmaceutical composition is formulated in a vial or in a pre-filled syringe, said vial or syringe comprising one or more dosages for administration to said subject.

16. The pharmaceutical composition for use according to any of the previous claims 12-15, wherein said composition is repeatedly administered to said subject, preferably with a time interval of 2-3 months between each administration and this for a period of at least 6 months.

17. The pharmaceutical composition for use according to any of the previous claims 12-16, wherein said subject to be treated is unresponsive to previouscorticosteroid treatment or a treatment based on hyaluronic acid alone, hence not combined with an amino acid composition.

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