Hydrogel method of production and use

Abstract

The current invention relates to a method of production of a biocompatible, injectable crosslinked hydrogel wherein a first and a second water-soluble polymer are mixed with water or a buffer resulting in an aqueous solution, wherein the aqueous solution is further transferred into a container and subjected to ionizing radiations, wherein the water-soluble polymers are poly(N-vinylpyrrolidone) (PVP) and poly(acrylic acid) (PAA).

Description

[0001] HYDROGEL METHOD OF PRODUCTION AND USE

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to the method of production of a sterile, crosslinked, injectable hydrogel. The hydrogel is particularly suited to be used as a synovial fluid substitute in subjects affected by osteoarthritis. The synovial fluid substitute described here addresses the limitations of previous hydrogels. It shows improved resistance to degradation from enzymes like hyaluronidase or reactive oxygen species (ROS).

[0004] BACKGROUND

[0005] Osteoarthritis (OA) is a painful, debilitating joint disease with no known cure. It is characterized by pain, swelling, crepitus (a crackling, crinkly, or grating feeling or sound under the skin), and a decreased range of motion in affected joints. In humans, it affects the hands, knees, hips, spine, and other joints.

[0006] OA is not limited to humans but also affects a wide range of animal species. Similar to humans, animals with OA experience joint pain, stiffness, and reduced mobility of pets such as cats, dogs, and horses.

[0007] The common symptoms for all types of OA include varied levels of pain. Pain symptoms can be treated with anti-inflammatory drugs such as NSAIDs. Another possibility is an injection of steroids directly into the affected joint. Specific treatment depends on the underlying cause of the OA and its level of progression. Common to the various types of existing treatment is that they all have their disadvantages, e.g. short-term treatment benefits, toxicity, and side-effects like gastrointestinal, cardiovascular, or chondral risks.

[0008] Visco-supplementation is the process of injecting a gel-like substance into the joint. The substance is thought of as an additive to the joint fluid, thus lubricating the cartilage and improving joint flexibility. Substances used in intra-articular visco- supplementation include hyaluronians (Hyalagan®, Monovisc®, Synvisc®, Synvisc- One®, Durolan®, Euflexxa®, or Supartz®), poly-sulfated glycosaminoglycans (PSGAGS) such as Adequan® or polyacrylamide, such as Arthrosamid®. Visco- supplementation based on hyaluronic acid and glycosaminoglycans, requires ongoing injections, as benefits are only temporary because the currently used substances are degradable within weeks to a few months. Moreover, some of these substances are synthesized by complex processes, are toxic or can induce other side effects. In particular, polyacrylamide leads to concerns about long-term safety and possible complications, considering that it is a known allergen.

[0009] There remains a need for visco-supplements that are:

[0010] - resistant to degradation,

[0011] - biocompatible,

[0012] - adequately simulating the rheological behavior of the synovial fluid,

[0013] - easy and cost-effective in the manufacturing process,

[0014] - side effects free,

[0015] - characterized by mucin-like properties w.r.t. friction,

[0016] - show no interference with synovial fluid nutrients, so that they have sufficient persistence to remove the need for frequent reapplication.

[0017] The invention thereto aims to provide a solution to majority of the problems mentioned above.

[0018] SUMMARY OF THE INVENTION

[0019] The present invention and embodiments thereof serve to provide a solution to one or more of the above-mentioned disadvantages. To this end, the present invention relates to a method of producing a sterile, injectable crosslinked hydrogel, according to claim 1. The method as disclosed herein allows for a direct crosslinking of poly(N- vinylpyrrolidone) (PVP) and poly(acrylic acid) (PAA) in water or PBS into a two- component hydrogel and the sterilization of the obtained composition directly in its final storage container. As such, the method is straightforward and eliminates steps necessary to follow with other methods, in particular, purification steps to remove cross-linkers, monomers, or other additives.

[0020] Preferred embodiments of the method are shown in any of the claims 2 to 7.

[0021] In a second aspect, the present invention relates to a pharmaceutical composition comprising an injectable crosslinked hydrogel, according to claim 8. The hydrogel has improved viscosity properties compared to known products and is particularly effective to be used as visco-supplement. Preferred embodiments of the pharmaceutical composition are shown in any of the claims 9 to 12. In a third aspect, the present invention relates to the use of the pharmaceutical composition in the treatment of OA, according to claim 13, and preferred embodiments are shown in claims 14 to 17.

[0022] The invention relates in a final aspect to a kit, according to claim 18.

[0023] FIGURES

[0024] Figure 1 shows the apparent viscosity of DAH1-10, DAH1-12, and DAH2-12, hydrogels formulated according to an embodiment of the invention, in comparison to the apparent viscosity of Synvisc-One® over a range of stress values.

[0025] Figure 2 depicts the apparent viscosity of DAH1-10, DAH1-12, and DAH2-12, hydrogels formulated according to embodiments of the invention, in comparison to Synvisc-One®, plotted against varying shear rates.

[0026] DETAILED DESCRIPTION OF THE INVENTION

[0027] The present invention concerns the method of production of a crosslinked, injectable, and biocompatible hydrogel. The hydrogel is particularly suited to be used as a synovial fluid substitute in subjects affected by osteoarthritis. The synovial fluid substitute as disclosed herein, addresses the shortcomings of prior art hydrogels, such as resistance to degradation due to reduced depolymerization by enzymes like hyaluronidase or reactive oxygen species (ROS), high biocompatibility, and accurately replicating the rheological properties of synovial fluid.

[0028] 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.

[0029] As used herein, the following terms have the following meanings:

[0030] “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. “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.

[0031] “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 specify 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.

[0032] 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.

[0033] The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within that range, as well as the recited endpoints.

[0034] The expression “% by weight", “weight percent", “%wt" or “wt%", here and throughout the description unless otherwise defined, refers to the relative weight of the respective component based on the overall weight of the formulation.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] The invention relates to the method of production of an injectable crosslinked and biocompatible hydrogel wherein a first and a second water-soluble polymer are mixed with water or a buffer, resulting in an aqueous solution, wherein the aqueous solution is subjected to ionizing irradiation. It is preferred that the water-soluble polymers are poly(N-vinylpyrrolidone) (PVP) and poly(acrylic acid) (PAA).

[0039] PVP and PAA are water-soluble synthetic polymers that offer unique characteristics for crosslinking into hydrogels by ionizing radiation. PVP and PAA have both a flexible main polymer chains built by -CH2- units with pendant groups allowing the formation of strong hydrogen bonds between both polymers as well as with water molecules. PAA possesses carboxyl functional groups along its molecular chain, which contribute to its pH responsiveness. PVP-PAA bipolymeric system is also known to show mucoadhesive properties. Both PVP and PAA are biocompatible and have good resistance to degradation. Nonetheless, they can be engineered to be biodegradable, enabling the gradual degradation of the products over time. All these characteristics recommend PAA and PVP as suitable constituents for injectable hydrogels to be used as visco-supplement in the treatment of osteoarthritis. In an embodiment of the method, as disclosed herein, the PVP and PAA are independently mixed with water or a buffer resulting in a first and a second aqueous solution and said first and second aqueous solutions are further combined into a container and subjected to ionizing radiations.

[0040] In an alternative embodiment, PVP is dissolved in water or a buffer forming a first aqueous solution and ulterior, PAA is dissolved into the first aqueous solution. In yet another alternative embodiment, PAA is dissolved in water or a buffer, forming a first aqueous solution and ulterior, PVP is dissolved into the first aqueous solution

[0041] In an embodiment of the method, as disclosed herein, the pH of the first and the second aqueous solutions is between 6.8 and 7.6, preferably between 7.0 and 7.4, and most preferably around 7.2. The inventors observed that adjusting the pH of the PVP and PAA comprising solutions allows the fine-tuning of the rheological properties of the hydrogel. In addition, a pH above pKa of poly(acrylic acid) prevents the formation of the slurry during the production process. In some embodiments, the pH is between 6.9 and 7.6, between 7.0 and 7.6, between 7.1 and 7.6, between 7.2 and 7.6, between 7.3 and 7.6, between 7.4 and 7.6, or between 7.5 and 7.6. Alternatively, the pH is between 6.8 and 7.5, between 6.8 and 7.4, between 6.8 and 7.3, between 6.8 and 7.2, between 6.8 and 7.1, between 6.8 and 7.0, or between 6.8 and 6.9.

[0042] In an alternative embodiment, the PVP and PAA are mixed together with water or a buffer.

[0043] In a further embodiment of the method, as disclosed herein, the total concentration of both polymers in the solution is between 0.5 wt.% and 25 wt.%, more preferably between 1 wt.% and 20 wt.%., more preferably between 5 wt.% and 15 wt.%. Alternatively, the concentration of PVP or PAA is at least 0.5 wt.%, 1 wt.%, 1.5 wt.%, 2 wt.%, 2.5 wt.%, 3 wt.%, 3.5 wt.%, 4 wt.%, 4.5 wt.%, 5 wt.%, 5.5 wt.%, 6 wt.%, 6.5 wt.%, 7 wt.%, 7.5 wt.%, 8 wt.%, 8.5 wt.%, 9 wt.%, 9.5 wt.%, 10 wt.%, 10.5 wt.%, 11 wt.%, 11.5 wt.%, 12 wt.%, 12.5 wt.%, 13 wt.%, 13.5 wt.%, 14 wt.%, 14.5 wt.%, 15 wt.%, 15.5 wt.%, 16 wt.%, 16.5 wt.%, 17 wt.%, 17.5 wt.%, 18 wt.%, 18.5 wt.%, 19 wt.%, 19.5 wt.%, 20 wt.%, 20,5 wt.%, 21 wt.%, 21.5 wt.%, 22 wt.%, 22.5 wt.%, 23 wt.%, 23.5 wt.%, 24 wt.%, 24.5 wt.% or 25 wt.%.

[0044] In an embodiment, the total concentration of the water-soluble polymers in said aqueous solutions is between 0.5 wt.% and 25 wt.%, between 0.5 wt.% and 20 wt.%, between 0.5 wt.% and 15 wt.%, between 0.5 wt.% and 12 wt.%, between

[0045] 0.5 wt.% and 10 wt.%, or between 0.5 wt.% and 5 wt.%.

[0046] In an alternative embodiment, the total concentration of the water-soluble polymers in said aqueous solutions is between 0.5 wt.% and 20 wt.%, between 0.5 wt.% and 15 wt.%, between 0.5 wt.% and 10 wt.%, or between 0.5 wt.% and 5 wt.%.

[0047] In an embodiment of the method, as disclosed herein, the PAA to PVP weight ratio in the hydrogel is between 0.01 and 4. Alternatively, the ratio is at least 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0 preferably the ratio is between 0.01 and 0.2.

[0048] In some embodiments of the method as disclosed herein, the ratio between PAA and PVP in the hydrogel is 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19 or 0.2.

[0049] Surprisingly, the inventors observed that a ratio between PAA and PVP in the hydrogel of about 1 : 10, is crucial for ensuring the synovial fluid-like properties of the hydrogel such as viscosity and high uniformity.

[0050] Without wishing to be bound to theory, the adjustment of the ratios between PAA and PVP results in different rheological properties of the obtained hydrogel. The ratios between PAA and PVP may thus be changed in the current method to result in a hydrogel with lower or higher viscosity than the human synovial fluid viscosity and, thus a product that may be applied in veterinary medicine.

[0051] In an embodiment of the method, as disclosed herein, ionizing radiation is selected from gamma rays or UV rays, preferably gamma rays. Ionizing radiation is used for crosslinking PAA and PVP to form hydrogels. This crosslinking process involves subjecting the aqueous solution comprising the polymers to high-energy ionizing radiation. The ionizing radiation causes radiolysis of water, resulting in the formation of very reactive hydroxyl radicals that further interact with polymer chain via hydrogen atom abstraction and formation of macroradicals on polymer chains of PVP and PAA. The radicals on the polymer chains of PVP and PAA then recombine with each other to form new chemical bonds, resulting in a three-dimensional network structure within the hydrogel. In a further embodiment, the total absorbed dose of gamma irradiation is between

[0052] 20 and 30 kGy. In some embodiments, the dose of gamma irradiation is between

[0053] 21 and 30 kGy, between 22 and 30 kGy, between 23 and 30 kGy, between 24 and 30 kGy, between 25 and 30 kGy, between 26 and 30 kGy, between 27 and 30 kGy, between 28 and 30 kGy, or between 29 and 30 kGy. Alternatively, the dose of gamma irradiation is between 20 and 29 kGy, between 20 and 28 kGy, between 20 and 27 kGy, between 20 and 26 kGy, between 20 and 25 kGy, between 20 and 24 kGy, between 20 and 23 kGy, between 20 and 22 kGy, or between 29 and 30 kGy. In a preferred embodiment, the dose of gamma irradiation is 25 kGy. The ionizing radiation sterilizes the hydrogel during the crosslinking process, reducing the risk of microbial contamination and ensuring the hydrogel's suitability for biomedical applications. The method as disclosed herein is particularly advantageous, as the crosslinking and sterilization happen in the same step and in the container where the final product, namely the injectable hydrogel, is to be stored and from which is to be delivered to the pat. The method is, thus, particularly time and cost-effective.

[0054] In a preferred embodiment of the method, as disclosed herein, prior to the irradiation, the aqueous solution is transferred into an irradiation container. The container, where the aqueous solution comprising the polymers is subjected to irradiation is a syringe or a cannula. However, it would be obvious to the skilled person that any other containers that may be closed by means of a system impervious to water and to gas, may be used with the method disclosed herein. These containers may be glass, silicone, or medical-grade polymers, like polypropylene.

[0055] In an embodiment of the method as disclosed herein, the polymers, PAA and PVP, are dissolved into water, such as deionized water, or a buffer, such as PBS buffer. The polymers may be dissolved independently, and then the resulting polymers containing aqueous solutions may be mixed, or they may be successively dissolved in the same aqueous solution. The total weight of polymers in the aqueous solution is between 0.5 wt.% and 25 wt.%, and the PAA to PVP weight ratio in the hydrogel is between 0.01 and 4. In a further embodiment, the pH of the aqueous solutions is adjusted to between 6.8 and 7.8. In yet a further embodiment, the aqueous solution comprising dissolved PAA and PVP is transferred to a container, such as a syringe or a cannula and closed tightly. The container is then subjected to gamma irradiation at 25 kGy absorbed dose. The method allows a one-step synthesis and sterilization of the hydrogel with desired rheological properties, being thus simple to apply and time and cost-effective.

[0056] In a second aspect, the current disclosure relates to a pharmaceutical composition comprising an injectable crosslinked hydrogel wherein said hydrogel comprises PVP and PAA and wherein the PAA to PVP weight ratio in said hydrogel is between 0.01 and 4, preferably 0.1.

[0057] In some embodiments, the PAA to PVP weight ratio in the hydrogel is 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19 or 0.2.

[0058] PAA and PVP are biocompatible and have good resistance to degradation. Nonetheless, they can be engineered to be biodegradable. The inventors observed that crosslinked PAA and PVP are stable substitutes for synovial fluid, and do not present the toxicity associated with the use of monomers. The ratio between PAA and PVP is crucial for the visco-supplementation properties, such as apparent and complex viscosity and elastic and viscous moduli, of the hydrogel. Moreover, the crosslinked PAA and PVP-based hydrogel of the invention is more resistant to degradation during the inflammatory phase than currently available products based on hyaluronic acid. The glycosidic bond of hyaluronic acid is build by oxygen linkage and thus is weak when attacked by reactive oxygen forms or hyaluronidase and easily depolymerizes. PVP and PAA have carbon-based backbone and thus it requires much longer exposure to reactive oxygen species to be degraded.

[0059] The viscosity of a composition is a measure of its resistance to gradual deformation under stress. For liquids or gels, it corresponds to the informal notion of "thickening".

[0060] The shear or apparent viscosity is commonly measured using viscometers or rheometers, which apply controlled shear stress to the fluid and measure its resulting shear rate.

[0061] In an embodiment of the pharmaceutical composition as disclosed herein, the shear viscosity of the injectable hydrogel, measured at a shear rate of 0 / s and 37 °C temperature, is between 1 and 1000 Pa.s, preferably between 20 and 200 Pa.s. Moreover, the hydrogel as disclosed herein, shows smooth shear thinning with an increase in shear rate. In an embodiment, the shear viscosity measured at a shear rate of 0 / s and of 37 °C temperature is between 1 and 750 Pa.s, 1 and 500 Pa.s, 1 and 250 Pa.s, 1 and 200 Pa.s, 1 and 100 Pa.s, 1 and 50 Pa.s, 1 and 20 Pa.s, 1 and

[0062] 15 Pa.s, 1 and 10 Pa.s, or 1 and 900 Pa.s.

[0063] Alternatively, the shear viscosity measured at a shear rate of 0 / s and 37 °C temperatures between 5 and 1000 Pa.s, 10 and 1000 Pa.s, 15 and 1000 Pa.s, 20 and 1000 Pa.s, 50 and 1000 Pa.s, 100 and 1000 Pa.s, 250 and 1000 Pa.s, 500 and 1000 Pa.s, or 750 and 1000 Pa.s.

[0064] In yet another alternative embodiment, the shear viscosity of the injectable hydrogel, measured at a shear rate of 0 / s and 37 °C temperature, is between 1 and 5000 Pa.s, 5 and 5000 Pa.s, 10 and 5000 Pa.s, 20 and 5000 Pa.s, 50 and 5000 Pa.s, 100 and 5000 Pa.s, 250 and 5000 Pa.s, 500 and 5000 Pa.s, 750 and 5000 Pa.s, 1000 and 5000 Pa.s, 1500 and 5000 Pa.s, 2000 and 5000 Pa.s, 2500 and 5000 Pa.s, 3000 and 5000 Pa.s, 3500 and 5000 Pa.s, 4000 and 5000 Pa.s, or between 4500 and 5000 Pa.s,

[0065] In yet another alternative embodiment, the shear viscosity of the injectable hydrogel, measured at a shear rate of 0 / s and 37 °C temperature, is between 1 and 4500 Pa.s, 1 and 4000 Pa.s, 1 and 3500 Pa.s, 1 and 3000 Pa.s, 1 and 2500 Pa.s, 1 and 2000 Pa.s, 1 and 1500 Pa.s, 1 and 1000 Pa.s, 1000 and 5000 Pa.s, 1500 and 5000 Pa.s, 2000 and 5000 Pa.s, 2500 and 5000 Pa.s, 3000 and 5000 Pa.s, 3500 and 5000 Pa.s, 4000 and 5000 Pa.s, or between 4500 and 5000 Pa.s.

[0066] In yet another alternative embodiment, the shear viscosity of the injectable hydrogel, measured at a shear rate of 0 / s and 37 °C temperature, is between 1 and 10000 Pa.s, 10 and 10000 Pa.s, 100 and 10000 Pa.s, 500 and 10000 Pa.s, 1000 and 10000 Pa.s, 1500 and 10000 Pa.s, 2000 and 10000 Pa.s, 2500 and 10000 Pa.s, 3000 and 10000 Pa.s, 3500 and 10000 Pa.s, 4000 and 10000 Pa.s, 4500 and 10000 Pa.s, 5000 and 10000 Pa.s, 6000 and 10000 Pa.s, 7000 and 10000 Pa.s, 8000 and 10000 Pa.s, or 9000 and 10000 Pa.s.

[0067] In yet another alternative embodiment, the shear viscosity of the injectable hydrogel, measured at a 1 and 9500 Pa.s, 1 and 9000 Pa.s, 1 and 8500 Pa.s, 1 and 8000 Pa.s, 1 and 7500 Pa.s, 1 and 7000 Pa.s, 1 and 6500 Pa.s, 1000 and 9000 Pa.s, 1500 and 8500 Pa.s, 2000 and 8000 Pa.s, 2500 and 7500 Pa.s, 3000 and 7000 Pa.s, 3500 and 6500 Pa.s, 4000 and 6000 Pa.s, or 4500 and 5500 Pa.s. Complex viscosity is a rheological parameter used to describe the behavior of viscoelastic materials, which exhibit both viscous (fluid-like) and elastic (solid-like) properties. Complex viscosity measures how the material's viscosity changes while the rheometer applies an oscillatory shear deformation to the material at a constant frequency while varying the angular velocity. The rheometer measures the response of the material to the oscillatory shear deformation. It records the applied stress or strain and the corresponding phase shift between the stress and strain. From this data, the complex viscosity of the material can be calculated at each angular velocity.

[0068] The complex viscosity, q*, is represented as q* = q' + iq", where q' is the real component (resistance to flow) and q" is the imaginary component (elasticity). The values of q' and q" obtained from the measurements at different angular velocities provide insights into the material's viscoelastic behavior and its ability to store and dissipate energy during deformation.

[0069] In an embodiment of the pharmaceutical composition, the complex viscosity of the crosslinked hydrogel, measured at 0.1 Hz frequency and 37 °C temperature, is between 1 and 200 Pa.s. In some embodiments of the pharmaceutical composition, the complex viscosity of the crosslinked hydrogel, measured at 0.1 Hz frequency and 37 °C temperature, is between 20 and 100 Pa.s. The inventors observed that the hydrogel as disclosed herein show high complex viscosity, similar to that observed for linear and cross-linked hyaluronic acids. High complex viscosity indicates good viscoelastic properties of the hydrogel for application as a synovial fluid substitute. For example, low values of complex viscosity in microgels obtained from monomers could potentially not give the right response of the fluid to walking or running and the formulation collapses under pressure. This is in contrast with the hydrogel of the current disclosure, wherein the polymers are combined into a continuous network of covalently crosslinked chains.

[0070] In a further embodiment of the pharmaceutical composition as disclosed herein, said injectable crosslinked hydrogel is produced in, stored in and delivered from a syringe or a cannula. However, it would be obvious to the skilled person that any other containers that may be closed by means of a system impervious to water and to gas, and that withstands the irradiation conditions, may be used with the method disclosed herein. These containers may be glass, silicone, or medical-grade polypropylene. The hydrogel is preferably crosslinked by gamma irradiation, as disclosed in any of the previous embodiments, and the hydrogel is sterile in a preferred embodiment. The hydrogel is thus synthetized and sterilized in a single step, in the container that is used for further storage and application.

[0071] In an embodiment, the pharmaceutical composition further comprises at least one additional compound. The hydrogel can be mixed with bioactive pharmaceutical agents, like drugs, vitamins, excipients, analgesics, anti-inflammatory agents, antioxidants. It is preferred that the pharmaceutical composition further comprises chondroprotective agents such as but not limited to glucosamine, chondroitin sulfate, curcumin, or growth factors.

[0072] In a third aspect, the disclosure relates to a pharmaceutical composition comprising an injectable crosslinked hydrogel wherein said hydrogel comprises PVP and PAA and wherein the PAA to PVP weight ratio in said hydrogel is between 0.01 and 4 for use as a medicament.

[0073] In an embodiment, the pharmaceutical composition is used for the prevention and / or the treatment of osteoarthritis in a subject in need thereof. The pharmaceutical composition for use has the properties of the composition disclosed in any of the previous embodiments.

[0074] The inventors surprisingly observed that the pharmaceutical composition comprising a crosslinked hydrogel wherein PVP and PAA are within the ratios of the disclosure, possesses exceptional rheological properties that make it particularly effective as a synovial fluid substitute in the treatment of OA. Advantageously, the hydrogel is resistant to degradation, requiring a less frequent administration. In addition, the hydrogel as used herein is potentially less toxic due to the use of PAA and PVP, compared with hydrogels based on monomers.

[0075] In an embodiment, the pharmaceutical composition is used for treating a human or an animal.

[0076] Osteoarthritis is classified in five levels of severity, based on the Kellgren-Lawrence (KL) grading system which is a radiographic classification system used to assess the severity of osteoarthritis (OA). The KL grading system consists of five grades, ranging from 0 to 4, each representing a different level of osteoarthritic changes in the knee joint: KL grade 0 (no osteoarthritis), KL grade 1 (doubtful osteoarthritis), KL grade 2 (mild osteoarthritis), KL grade 3: (moderate osteoarthritis) and KL grade 4 (severe osteoarthritis). The pharmaceutical composition as disclosed herein may be used for treatment of humans diagnosed with KL grades 1, 2, 3 or 4. Further, the pharmaceutical composition is suitable for treatment of male or female human subjects, under the age of 70 or over the age of 70 and having a BMI categorized as underweight, normal, overweight or obese.

[0077] Osteoarthritis can equality other animal species, particularly those with weightbearing joints and long lifespans. Veterinary indications for the use of the pharmaceutical composition as disclosed herein includes dogs, cats, horses, camels, cattle, large wild species such as elephants, bears, primates.

[0078] Osteoarthritis is relatively common in dogs, especially in older and large breed dogs. It can affect weight-bearing joints like hips, knees (stifles), and elbows, causing pain and reduced mobility.

[0079] Cats, especially older or obese cats, can also suffer from osteoarthritis. It often affects the hips, elbows, and spine, leading to stiffness and discomfort.

[0080] Osteoarthritis in horses is often referred to as degenerative joint disease (DJD). It affects weight-bearing joints like the knees (carpi), hocks, and fetlocks, leading to lameness and decreased performance.

[0081] Osteoarthritis can occur in cattle, particularly in dairy cows, due to the heavy workload and stress on their joints. Elephants, especially those living in captivity, may develop osteoarthritis due to the significant weight they bear on their large joints. Captive bears or older wild bears can also develop osteoarthritis, especially in the hips and shoulders. Certain primates, such as chimpanzees, gorillas, and orangutans, may experience osteoarthritis as they age. Some reptiles, like turtles and tortoises, can develop osteoarthritis in their joints over time. Avian species, especially those with a long lifespan, may develop osteoarthritis in their legs and feet. Various zoo animals, including large cats, apes, and pachyderms, may benefit from osteoarthritis treatment to improve their quality of life.

[0082] In an embodiment of the use, the injectable crosslinked hydrogel is administered by injection into the intraarticular cavity in replacement or in addition to deficient synovial fluid. The hydrogel is preferably sterile.

[0083] The injection of the hydrogel may be performed under local anaesthesia, but local anaesthesia is not necessarily required. However, the procedure is preferably performed under sterile conditions. Any hair covering the injection area is cropped and the skin thoroughly rinsed, e.g. with chlorhexidine and ethanol (e.g. 3 times interchangeably). Then, the cannula is inserted into the joint cavity and it is checked by aspiration that it is placed properly intraarticularly. Aspiration is used to remove potential swelling after the medical procedure. Generally, the joint is emptied for at least the amount of liquid which it has been decided to inject and the desired amount of the hydrogel is then injected.

[0084] In an embodiment of the pharmaceutical composition for use, between 1 ml and 6 ml of injectable crosslinked hydrogel is administered by injection into the intraarticular cavity. In some embodiments, between 1 ml and 5.5 ml, between 1 ml and 5 ml, between 1 ml and 4.5 ml, between 1 ml and 4 ml, between 1 ml and 3.5 ml, between 1 ml and 3 ml, between 1 ml and 2.5 ml, between 1 ml and 2 ml, or between 1 ml and 1.5 ml, preferably between 1 ml and 3 ml injectable crosslinked hydrogel is administered by injection into the intraarticular cavity.

[0085] Alternatively, between 1.5 ml and 6 ml, between 2 ml and 6 ml, between 2.5 ml and 6 ml, between 3 ml and 6 ml, between 3.5 ml and 6 ml, between 4 ml and 6 ml, between 4.5 ml and 6 ml, between 5 ml and 6 ml, or between 5.5 ml and 6 ml injectable crosslinked hydrogel is administered by injection into the intraarticular cavity.

[0086] The current invention also covers a kit comprising a pharmaceutical composition comprising an injectable crosslinked hydrogel wherein said hydrogel comprises PVP and PAA and wherein the PAA to PVP weight ration in said hydrogel is between 0.01 and 4, packaged in a syringe or a cannula. The pharmaceutical composition of said has the properties of the composition disclosed in any of the previous embodiments

[0087] However, it is obvious that the invention is not limited to this application. The method according to the invention can be applied for the treatment of any other medical indication, such as reconstruction, plastic or cosmetic surgery or treatments, where a hydrogel with the rheological properties of the current composition would be beneficial.

[0088] The invention is further described by the following non-limiting examples which further illustrate the invention, and are not intended to, nor should they be interpreted to, limit the scope of the invention. EXAMPLES

[0089] Example 1. Hydrogel preparation

[0090] The hydrogel according to the invention (HG1) is prepared by dissolving pharmagrade poly(N-vinylpyrrolidone) polymer and poly(acrylic acid), both of low or moderate molar mass, in deionized water or PBS buffer, so that the final concentration of PVP and PAA in the aqueous solution is between 1 wt.% and 25 wt.%. The pH of the aqueous solutions is adjusted to 7.0-7.4. Finally, a syringe is filled with the mixture and irradiated with gamma radiation for sterilization and simultaneous crosslinking. The total gamma radiation absorbed dose tested are 20, 25 and 30 kGy and the process is done in a closed package, making it a one-step synthesis and sterilization. Hydrogels comprising PAA to PVP weight ratio of 0.01, 0.1 and 4 are produced.

[0091] The hydrogel according to the prior art (Pielka et al., 2004) (HG2) is prepared from monomers of N-vinylpyrrolidone and acrylic acid. Said monomers are distilled, and the resulting mixture is saturated with inert gas. The mixture is afterwards subjected to radiation-induced cross-linking polymerization using gamma radiation and a total absorbed dose of 25 kGy. The solid, hard blocks obtained from this process is warmed up at 120°C for at least 2 hours before being crushed and milled to obtain a microgel fraction of the desired diameter. Finally, the product is sterilized with 25 kGy, and the microgels are dissolved to prepare a solution with the desired rheological properties, similar to Synvisc One®.

[0092] Example 2. Rheological properties of the hydrogel: apparent viscosity

[0093] All rheological measurements are taken at a temperature of 37°C. Solvent trap covers were employed in all rheological testing to minimize the drying of the samples at the exposed geometry edge. A rotational rheometer from TA Instruments (DAHR2) with a 40mm / 2° cone-plate system with a measuring gap of 53 pm between cone and plate is used for testing all HG1 samples. A rotational rheometer from Thermo Haake with a 035 / 2° titanium cone-plate system with a measuring gap of 0.109 mm between cone and plate is used for testing HG2 samples. Samples are equilibrated for at least 200 seconds but not longer than 500 seconds at 37°C before analysis. The apparent viscosity analysis is conducted in a controlled stress or rate mode. In a controlled stress viscosity profiling measurement, the samples are subjected to a shear sweep from 0.1 Pa to 100 Pa, logarithmically spaced, with 8 points per decade of shear stress. In controlled rate analysis, following a 60-second equilibration time at 37°C and 30-second pre-shear at 0.1 s-1, the samples are exposed to a shear rate sweep ranging from 0.1 s1to 8000 s ~1, logarithmically scaled, with 5 points per decade of shear rate. Three types of hydrogel samples are tested; HG1 and HG2 are prepared according to Example 1 and HG3 is a commercial synovial liquid substitute (Synvisc-One®).

[0094] Results: It is expected that, HG2 which is obtained through a multi-step process involving monomers characterized by toxicity, has a complex apparent viscosity profile, with steep changes in the viscosity curve, including shear thinning followed by shear thickening behavior. In contrast, HG1, according to the invention, is expected to show smooth shear thinning with an increase in shear rate, similar to the commercial HG3.

[0095] Example 3. Rheological properties of the hydrogel: complex viscosity, elastic and viscous moduli

[0096] A rotational rheometer from TA Instruments (DAHR2) with a 40mm / 2° cone-plate system with a measuring gap of 53 pm between cone and plate is used for testing the complex viscosity, elastic and viscous moduli of HG1 samples. The complex viscosity, elastic and viscous moduli of HG2 samples are measured using a rotational rheometer from Thermo Haake with a C35 / 2° titanium cone-plate system with a measuring gap of 0.109 mm between cone and plate. The samples are equilibrated for at least 120 seconds but not longer than 500 seconds at 37°C before analysis.

[0097] The complex viscosity is measured by exposing samples to oscillatory frequency sweeps, 0.1% strain, 100 rad / s to 0.1 rad / s, logarithmically scaled, with 4 points per decade of frequency. Three types of hydrogel samples were tested; HG1 and HG2 were prepared according to Example 1 and HG3, a commercial synovial liquid substitute (Synvisc-One®).

[0098] Results: It is expected that HG2 lacks viscoelastic properties as expressed by complex viscosity, which is expected to be lower than the complex viscosity of HG1 or HG3. Example 4. Tribological properties of the hydrogel: friction coefficient.

[0099] HG1 and HG3 samples are applied to a silicone sheet and spread to a thickness of 1000 pm using a spreader bar. A defined axial load of IN is imposed and the angular velocity is swept from 0.05 rad / s to 20 rad / s. The coefficient of friction is calculated as the ratio of sliding force to load force and plotted as a function of sliding speed to generate a Stribeck curve. The lower substrate is held at 37°C throughout the testing performed in duplicate.

[0100] Results: HG1 is expected to show the same good tribological properties as HG3, as expressed by the friction coefficient.

[0101] Example 5. Rheological properties of the hydrogel exposed to hyaluronidase

[0102] The apparent viscosity is determined according to Example 2 in hydrogel samples comprising hyaluronidase. Hyaluronidase is an enzyme responsible for breaking down hyaluronic acid. It is responsible for the metabolism of hyaluronic acid in the human body, including joints and is used herein as a model for degradation of synovial fluid substitutes. Other mechanism of HA degradation is induced by reactive oxygen species (ROS).

[0103] Four types of hydrogel samples are tested; HG1 prepared according to Example 1, HG1 treated with hyaluronidase, HG3, a commercial synovial liguid substitute (Synvisc-One®) and HG3 treated with hyaluronidase.

[0104] Results: Both versions of HG1, simple or treated with hyaluronate are expected to show the same level of apparent viscosity and smooth shear thinning with an increase in shear rate, similar to the commercial HG3, demonstrating that it is more resistant to degradation than currently available products based on hyaluronic acid such as HG3.

[0105] Example 6. Cytotoxicity of the hydrogel

[0106] Cytotoxicity of HG1 product is performed according to ISO-10993 on human fibroblasts with XTT test kit from Biological Industries and on mouse fibroblast cell line with MTT test kit. Cytotoxicity tests are performed in three-time intervals: 24, 48 and 72 hours for human fibroblasts and after 72 hours for mouse fibroblasts. Results: The viability of human fibroblast is, on average, above 90% and a composition is considered non-toxic if cell viability is above 70%. It is expected that all the HG1 hydrogels tested are non-toxic. Additionally, it is expected that fibroblasts will not show changed morphology in the optical picture.

[0107] Example 7. Clinical investigation

[0108] A clinical investigation of injection of PAA to PVP crosslinked hydrogel obtained as described in Example 1, in subjects with knee osteoarthritis, is performed. The subjects are injected with either one injection of 1 to 3mL PAA and PVP crosslinked hydrogel (HG1), or one injection of 1 to 3 mL Synvisc-One® (HG3).

[0109] The participating subjects are followed from baseline to week 104 answering a questionnaire relating to pain and stiffness, prior to the injection as well as on the following weeks: 4, 12 / 13, 26, 52, 104.

[0110] It is expected that both pain and stiffness is reduced in the subjects treated with HG1 compared to the subjects treated with HG2.

[0111] Example 8. Comparison testing rheological properties of various hydrogels

[0112] Three hydrogels were prepared according to the embodiments of the present invention, following the procedure described in Example 1 for HG1.

[0113] More specifically, DAH2-12 was prepared by mixing 2.5 wt% 30 kDa PVP with 50 kDa PAA and deionized water with pH = 7.2, and wherein the PAA to PVP ratio is 1 : 10 (0.1).

[0114] DAH1-12 was prepared by mixing 2.5 wt% 30 kDa PVP with 50 kDa PAA and PBS, and wherein the PAA to PVP ratio is 1 : 10 (0.1).

[0115] DAH1-10 was prepared by mixing 2.5 wt% 30 kDa PVP with 50 kDa PAA and PBS, and wherein the PAA to PVP ratio is 1 :20 (0.05).

[0116] The rheological properties of DAH2-12, DAH1-12 and DAH1-10 as well as of commercial hydrogels Synvisc-One®, Cingal® and Monovisc® were determined or provided by literature. The apparent viscosity was determined using the method described in Example 2; the zero-shear viscosity was evaluated from controlled stress measurements using Carreau-Yasuda model fit. The complex viscosity was determined using the method described in Example 3.

[0117] The storage modulus and loss modulus were determined from oscilatory frequency sweep measurements, using the method described in Example 3

[0118] The friction coefficient was determined using tribological analysis as described in Example 4.

[0119] The results of the rheological properties measurements are displayed in Table 1 and Figures 1 and 2.

[0120] Results

[0121] The DAH1-12 hydrogel exhibited much higher apparent viscosity, and the DAH2-12 hydrogel showed comparable zero-shear viscosity, when compared to the commercial product Synvisc-One® (Table 1 and FIG. 1). This suggests that the DAH1-12 and DAH2-12 hydrogels have stronger resistance to flow at low shear rates, resulting in better joint cushioning properties. As shown in FIG. 1, the rheological behavior of DAH hydrogels can be modified by altering the composition of the hydrogels or the dissolving medium (water vs. PBS). It is worth noting that this flexibility in the preparation of DAH hydrogels with different properties allows the manufacturing of different products for tailored applications. Furthermore, DAH1-10, DAH1-12, and DAH2-12 exhibit lower complex viscosity compared to Synvisc-One® and Cingal®, lower storage modulus compared to Synvisc-One® but comparable to Cingal®, and higher than Monovisc®. This indicates that they have satisfactory elastic properties for load-bearing applications, such as visco supplementation.

[0122] In addition, referencing Figure 2, it is evident that DAH hydrogels exhibit shear thinning behavior. While the viscosity values at low shear rates are lower than those of Synvisc One, at moderate and very high shear rates, the DAH flow curves approach that of Synvisc One.

[0123] This equilibrium between high viscosity at rest and appropriate flow under shear emphasizes DAH as a versatile hydrogel suitable for both effective cushioning and ease of injection. Table 1. Results of rheological properties measurements for various hydrogels

[0124] The storage modulus and loss modulus values in Table 1 for all three DAH hydrogels (DAH2-12, DAH1-12, and DAH1-10) show that they exhibit a strong elastic response to the load. The G" value is lower than that of Synvisc-One® and Cingal®, but higher than that of Monovisc®, indicating that DAH hydrogels still offer sufficient elasticity to support joint function. The loss modulus value, which represents the viscous or 'liquid-like' behavior, is lower than that of Synvisc-One® but higher than that of Monovisc®, demonstrating a good balance between flowability and resistance to deformation. DAH hydrogels are generally more elastic than liquidlike.

[0125] The friction coefficient of all three DAH hydrogels (Table 1) is lower (0.09-0.1) than that of Synvisc-One® (0.153) but comparable to the Cingal formulation. This suggests that DAH is suitable for reducing friction within the joint, similar to hyaluronic acid, a natural synovial fluid constituent. The lower friction coefficient of DAH hydrogels can be beneficial in osteoarthritic joints, as they can facilitate smoother joint movement, potentially reducing wear and tear on the joint surfaces and, consequently, decreasing inflammation.

Claims

CLAIMS1. A method of production of an injectable crosslinked hydrogel wherein a first and a second water-soluble polymer are mixed with water or an aqueous buffer resulting in an aqueous solution, wherein the aqueous solution is subjected to ionizing irradiation, characterized in that the water-soluble polymers are poly(N-vinylpyrrolidone) (PVP) and poly(acrylic acid) (PAA).

2. Method according to claim 1, wherein the total concentration of the water soluble polymers in said aqueous solutions is between 0.5 wt.% and 25 wt.%.

3. Method according to any of the claims 1 or 2, wherein the pH of the aqueous solution is pH 6.8-7.6.

4. Method according to any of the claims 1 to 3, wherein the PAA to PVP weight ratio in the hydrogel is between 0.01 and 4, preferably 0.1.

5. Method according to claims 1 to 4, wherein the ionizing radiation is selected from gamma rays or UV rays, preferably gamma.

6. Method according to any of the previous claims, wherein the total absorbed dose of gamma irradiation is between 20 and 30 kGy.

7. Method according to any of the previous claims, wherein prior to the irradiation, the aqueous solution is transferred into an irradiation container such as a syringe or a cannula.

8. A pharmaceutical composition comprising an injectable crosslinked hydrogel obtainable according to any of the claims 1 to 7, wherein said hydrogel comprises PVP and PAA and wherein the PAA to PVP weight ratio in said hydrogel is between 0.01 and 4.

9. The pharmaceutical composition according to claim 8, wherein the apparent viscosity of said hydrogel, measured at a shear rate of 0 / s and 37 °C temperature, is between 10 and 5000 Pa.s.

10. The pharmaceutical composition according to any of the claims 8 or 9, wherein the complex viscosity of said injectable crosslinked hydrogel, measured at 1 Hz frequency and 37 °C temperature, is between 1 and 200 Pa.s.

11. The pharmaceutical composition according to any of the claims 8 to 10, wherein said injectable crosslinked hydrogel is produced in, stored in and delivered from a syringe or a cannula.

12. The pharmaceutical composition according to any of the claims 8 to 11, wherein said composition further comprises at least one additional compound.

13. The pharmaceutical composition according to claims 8 to 12, for use as a medicament.

14. The pharmaceutical composition for use according to claim 13 in the prevention and / or the treatment of osteoarthritis in a subject in need thereof.

15. The pharmaceutical composition for use according to claim 14, wherein the subject is a human or an animal.

16. The pharmaceutical composition for use according to any of the claims 13 to15, wherein the injectable crosslinked hydrogel is administered by injection into the intraarticular cavity in replacement or in addition to deficient synovial fluid.

17. The pharmaceutical composition for use according to any of the claims 13 to16, wherein between 1 and 6 ml of injectable crosslinked hydrogel is administered by injection into the intraarticular cavity.

18. A kit comprising a pharmaceutical composition according to any of the claims8 to 12, packaged in a syringe or a cannula.

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