Dexamethasone delivery system

Abstract

The present invention relates to a hydrogel formulation which facilitates the controlled release of dexamethasone or a salt thereof over a long period of time. In particular, the hydrogel formulation comprises a cross-linked hyaluronic acid component formulated with a non-cross-linked hyaluronic acid component.

Description

Technical Field

[0001] This invention relates to a hydrogel formulation that facilitates the controlled release of dexamethasone or its salts over a prolonged period. In particular, the hydrogel formulation comprises a cross-linked hyaluronic acid component formulated together with a non-cross-linked hyaluronic acid component. Background Technology

[0002] Osteoarthritis (OA) is a chronic disease characterized by the destruction of articular cartilage, leading to stiffness, pain, swelling, and limited movement. It is the most common joint disease in the United States, currently affecting 12.1% of the adult population. In particular, more than 30 million people in the United States suffer from OA.

[0003] Over the past three decades, corticosteroids have played a crucial role in the multimodal pain management of osteoarthritis. Two different types of injectable steroids—particulate and non-particulate steroids—are primarily used for intra-articular injection in different types of osteoarthritis-related pain. Particulate steroids, such as methylprednisolone acetate, triamcinolone acetonide, and prednisolone acetate, are long-acting but poorly soluble steroids. However, new evidence suggests that particulate steroids are toxic to cartilage and nerve tissue, which will hinder their use. Non-particulate steroids, such as dexamethasone, are less toxic and soluble molecules, making them more suitable for pharmacological application. However, their anti-inflammatory effects are only transient, requiring frequent administration.

[0004] Frequent administration is inconvenient for users and may also be accompanied by discomfort due to repeated injections. To overcome the problem of dexamethasone's short half-life, it is necessary to formulate it appropriately to modify pharmacokinetics and increase efficacy.

[0005] Hyaluronic acid (HA) is an anionic and nonsulfated glycosaminoglycan with several potential advantages as a delivery medium, including its inherent biodegradability in vivo. HA is a naturally occurring long linear polysaccharide consisting of repeating disaccharide units of D-glucuronic acid and N-acetyl-D-glucosamine linked by glycosidic bonds, and is considered a high-value biopolymer with numerous proven and marketed applications in the cosmetics, biomedicine, and pharmaceutical fields.

[0006] Unfortunately, HA typically undergoes rapid degradation after injection into the human body, limiting the potential of biopolymers. Therefore, extensive modification methods, including crosslinking with various crosslinking agents, have been investigated to produce novel and robust HA-based materials with enhanced biostability and improved mechanical properties for a range of applications in medical devices and pharmaceutical products. Thus, the initial basis for crosslinked HA is stability; formulations containing crosslinked HA are currently used in injectable dermal fillers and as adhesive treatments for osteoarthritis. Examples of commercial products for treating OA that provide mechanical lubrication and cushioning of joints include SynVisc, Euflexxa, Hyalgan, and Supartz.

[0007] The proven track record of HA-based materials used in humans makes them attractive candidates for drug delivery systems. However, formulating active substances, such as dexamethasone, in HA-based formulations is more complex than specifically modifying the mechanical properties of medical devices lacking active substances. Specifically, designing formulations that facilitate controlled release of the active substance and thereby ensure that the effective dose remains within the therapeutic window over an extended period is challenging. Rapid and uncontrolled release leads to inefficient delivery of the active substance, requiring higher doses to achieve the desired effect and carrying the risk of potential toxicity and side effects.

[0008] Therefore, it would be advantageous to provide a biocompatible delivery medium that can deliver dexamethasone precisely, effectively, and safely to prolong its anti-inflammatory effect and reduce the frequency of administration. Furthermore, it would be advantageous if such a delivery medium also possessed viscoelasticity similar to synovial fluid in a joint, providing cushioning and ensuring good compatibility with the recipient tissue. Summary of the Invention

[0009] The active pharmaceutical ingredient can be formulated in a carrier medium to improve its pharmacokinetics when administered to subjects. Designing a suitable carrier is a complex task because drug formulations need to meet high standards of safety and efficacy.

[0010] This article presents a hydrogel formulation loaded with dexamethasone, which ensures controlled and prolonged release of dexamethasone to improve patient compliance and prolong anti-inflammatory effects. The hydrogel formulation is based on a mixture of hyaluronic acid (HA)-based hydrogel and non-crosslinked HA. The crosslinked HA in the hydrogel ensures that dexamethasone is retained and released only gradually from the formulation. Adding non-crosslinked HA to the crosslinked HA component allows for control of the formulation's rheology and facilitates fine-tuning of the release profile. The hydrogel formulation is prepared via a multi-step process, including the pulverization and micronization of the HA-based hydrogel component. The resulting hydrogel formulation effectively controls and prolongs the release of dexamethasone.

[0011] Therefore, the object of the present invention relates to a formulation for treating osteoarthritis, which reduces pain and inflammation in the arthritic joints, thereby improving the patient's quality of life and delaying or avoiding the need for joint replacement.

[0012] Specifically, the object of the present invention is to provide a hydrogel formulation that serves both as a viscoelastic supplement for lubricating joints and as a dexamethasone release agent over an extended period of time.

[0013] Therefore, one aspect of the present invention relates to a hydrogel formulation comprising: (i) A first component comprising a hydrogel containing hyaluronic acid (HA) crosslinked with a crosslinking agent. The ratio of HA to crosslinking agent is approximately 10:1% (w / w) to approximately 18:1% (w / w). (ii) a second component, the second component comprising non-crosslinked HA, and (iii) Dexamethasone or its salts, The ratio between the first component and the second component is approximately 60:40% (w / w) to approximately 98:2% (w / w).

[0014] Another aspect of the present invention relates to a hydrogel formulation comprising: (i) A first component comprising a hydrogel containing about 0.5-3% (w / w) of hyaluronic acid (HA) crosslinked with a crosslinking agent. The ratio of HA to crosslinking agent is approximately 10:1% (w / w) to approximately 25:1% (w / w). (ii) A second component comprising approximately 1-6% (w / w) of non-crosslinked HA, and (iii) Dexamethasone or its salts, The ratio between the first component and the second component is approximately 80:20% (w / w) to approximately 95:5% (w / w).

[0015] Another aspect of the present invention relates to a method for preparing the hydrogel formulation described herein, the method comprising the following steps: (i) Provide a solution containing approximately 4-8% (w / w) hyaluronic acid (HA), (ii) The solution is reacted with a crosslinking agent to form a first hydrogel. (iii) Crush the first hydrogel to provide a broken hydrogel. (iv) Add a buffer to the broken hydrogel to provide a neutralized hydrogel. (v) Separating the neutralized hydrogel from the buffer to provide a purified hydrogel. (vi) Micronize the purified hydrogel to provide a first component comprising the hydrogel, the hydrogel containing about 0.5-3% (w / w) of hyaluronic acid (HA) crosslinked with a crosslinking agent. (vii) A second component comprising about 1-6% (w / w) of non-crosslinked HA is added to the first component to provide a hydrogel composition, and (viii) Add dexamethasone or a salt thereof to the hydrogel composition. Thus, the hydrogel formulation is provided.

[0016] Another aspect of the invention relates to the hydrogel formulations described herein that can be obtained by the methods described herein.

[0017] Another aspect of the invention relates to hydrogel formulations as described herein, which can be used as pharmaceuticals.

[0018] Another aspect of the invention relates to hydrogel formulations as described herein for treating, preventing, or inhibiting one or more diseases or conditions, said diseases or conditions being selected from the group consisting of arthritis, tendinitis, synovitis, bursitis, metabolic arthritis, gout, allergic conditions, skin diseases, endocrine disorders, gastrointestinal diseases, blood diseases, tumor diseases, neurological diseases, ophthalmic diseases, kidney diseases, respiratory diseases, dermatological diseases, and pain, preferably arthritis.

[0019] Another aspect of the present invention relates to a reagent kit comprising: (i) Hydrogel formulations as described herein, and (ii) Optional, instructions for use. Attached Figure Description

[0020] Figure 1 The viscoelasticity of the hydrogel formulation was shown compared to the commercially available product Synvisc One.

[0021] Figure 2 The apparatus for measuring the dexamethasone release profile is shown. (A) Schematic diagram of the dexamethasone release system. (B) Actual photograph of the dexamethasone release system. From left to right: Dissolution unit, including water bath and pool module, piston pump, UV spectrophotometer, and media container. (C) Close-up of the pool module containing the hydrogel formulation sample.

[0022] The invention will now be described in more detail. Detailed Implementation

[0023] definition Before outlining the invention in more detail, let us first define a set of terms and conventions: Hyaluronic acid In this article, the term "hyaluronic acid" (HA) refers to a polysaccharide with varying molecular weights composed of D-glucuronic acid and N-acetyl-D-glucosinolate residues. Hyaluronic acid is naturally found on cell surfaces, as a basic extracellular substance in vertebrate connective tissues, in synovial fluid of joints, intraocular fluid, and human umbilical cord tissue.

[0024] Hyaluronic acid is defined herein as a non-sulfated glycosaminoglycan composed of repeating disaccharide units of N-acetylglucosamine (GIcNAc) and glucuronic acid (GIcUA) linked together by alternating β-1,4 and β-1,3 glycosidic bonds. Hyaluronic acid is also referred to as hyaluronic acid or abbreviated as HA. In this document, these terms will also cover conjugate base hyaluronic acid salts, and therefore these terms are used interchangeably herein.

[0025] Cross-linked hyaluronic acid In this document, the term "crosslinked hyaluronic acid" refers to hyaluronic acid crosslinked with a crosslinking agent. Therefore, crosslinked hyaluronic acid is understood to comprise multiple HA polymer chains linked by a crosslinking agent capable of reacting with one or more functional groups in the hyaluronic acid polymer structure. The functional groups in the hyaluronic acid backbone are hydroxyl, carboxylate, and acetamide groups.

[0026] As a non-limiting example, the crosslinking agent divinyl sulfone (DVS) can covalently crosslink hyaluronic acid polymers through the reaction of its vinyl groups with the primary hydroxyl groups of N-acetyl-D-glucosamine.

[0027] The degree of crosslinking can be controlled by adjusting the ratio between hyaluronic acid and the crosslinking agent. A higher amount of crosslinking agent will result in a higher degree of crosslinking, thus leading to a tighter polymer network.

[0028] Hyaluronic acid is preferably cross-linked using a single type of cross-linking agent, but cross-linked hyaluronic acid can also be obtained by reacting it with two or more cross-linking agents.

[0029] Crosslinking agent In this document, the term "crosslinking agent" refers to any compound capable of chemically linking hyaluronic acid polymers together to form a crosslinked polymer network. The reaction between hyaluronic acid and the crosslinking agent is preferably covalent. Therefore, the crosslinking agent preferably contains functional groups capable of forming covalent bonds with the hydroxyl, carboxyl, and / or acetamide functional groups of the hyaluronic acid polymer.

[0030] Crosslinking agents are capable of linking two hyaluronic acid polymers together, and therefore contain at least two functional groups. Some variants of crosslinking agents contain at least two identical functional groups.

[0031] Examples of crosslinking agents include, but are not limited to, divinyl sulfone (DVS), 1,4-butanediol diglycidyl ether (BDDE), polylactic acid, polyethylene glycol, and carboxymethyl cellulose (CMC).

[0032] hydrogel In this paper, the term "hydrogel" refers to a network of macromolecular polymers that swells in aqueous solutions, buffer solutions, or biofluids. The degree of hydration depends on the degree of cross-linking.

[0033] Hydrogels can be processed to modify their properties. In particular, hydrogels can be pulverized and micronized to reduce the particle size of the macromolecular network and to facilitate mixing with non-crosslinked hyaluronic acid, loading of dexamethasone, and viscosity adjustment.

[0034] Controlled release In this article, the term "controlled release" refers to the release of dexamethasone from a hydrogel formulation, where the release occurs over a prolonged period of time.

[0035] The term "controlled release" encompasses different scenarios of prolonged dexamethasone release. That is, dexamethasone is not necessarily released linearly. The term covers situations where a portion of the dexamethasone is released relatively rapidly from the hydrogel formulation, while the remainder is released in a prolonged manner. Prolonged release can be caused by continuous release from the hydrogel formulation or may depend on the gradual degradation of the hydrogel formulation. The term also covers delayed release, where dexamethasone is released only some time after administration. The term also includes sudden release, where a portion of the dexamethasone is released immediately, followed by an optional lag period, and then the remaining dexamethasone is released in a prolonged manner.

[0036] The terms “controlled release” and “extended release” are used interchangeably in this document.

[0037] Average particle size In this paper, the term "average particle size" refers to the D50 value (or median diameter) of the particle size distribution. The D value is a percentile value that can be directly read from the cumulative particle size distribution; therefore, D50 represents the value at which 50% of the particles have a smaller diameter and 50% of the particles have a larger diameter. The D50 value used in this paper refers to the number-average distribution of the particles.

[0038] Particle size distribution can be measured using laser diffraction. Measurements can also be performed using a Malvern Mastersizer 2000 coupled to a Hydro2000 dispersing device. The D50 value can be extracted from these measurements.

[0039] For example, the particle size distribution can be determined by diluting the first component to a concentration of 0.1-2% (w / w) HA using PBS buffer containing 0.5-15% (w / w) NaCl. Measurements were performed with continuous stirring, assuming spherical particles (refractive index 1.343) in the range of 0.02-2000 μm.

[0040] Energy storage modulus In this paper, the term "storage modulus" refers to the storage modulus G', measured in Pascals (Pa), determined by shear experiments; it represents the ability of a viscoelastic material to elastically store energy. At low frequencies, the shear rate is low, thus the medium has a high ability to maintain its original strength. As the frequency increases, the shear rate also increases, leading to an increase in the energy incorporated into the polymer chains. Therefore, the storage modulus increases with frequency.

[0041] The energy storage modulus reported in this article is the value at a frequency of 2.5 Hz.

[0042] Viscous modulus In this paper, the term "viscous modulus" refers to the viscous portion of viscoelastic behavior, which can be considered as the liquid behavior of a sample.

[0043] The slope of the load curve, similar to Young's modulus in a tensile test, is called the storage modulus G' (as described above). The storage modulus is a measure of how much energy must be injected into the sample to deform it. The difference between the "load" and "unload" curves is called the viscous modulus G'. It measures the energy lost during cyclic strain.

[0044] The terms "viscous modulus" and "loss modulus" are used interchangeably.

[0045] about Regardless of whether the term “about” is used in the context of quantities, such as absolute quantities (e.g., numbers, purity, weight, size, etc.), or relative quantities (percentages, equivalents, or proportions), time ranges, and parameters (e.g., temperature, pressure, etc.), it should be understood that these variables are approximate and therefore may differ from the specified actual figures by ±10%, such as ±5%, preferably ±2% (e.g., ±1%). This is true even if these figures are first expressed as percentages (e.g., “about 10%” could mean ±10% of the number 10, i.e., any value between 9% and 11%).

[0046] Dexamethasone delivery system Hyaluronic acid is a natural biopolymer with a linear, unbranched structure, found in many organisms from bacteria to higher animals, including humans. It is biocompatible, non-immunogenic, and readily broken down by the body's natural enzymes, making it a safe compound that has been injected into millions of patients over the past 20 years.

[0047] Hydrogels can be prepared from hyaluronic acid by chemically cross-linking polymers and swelling them in an aqueous medium. HA-based hydrogels have been successfully used as dermal fillers or as a lubricant for treating osteoarthritis. However, using HA-based hydrogels as drug delivery media is more complex because such formulations cannot rely solely on the mechanical properties of hyaluronic acid; interactions with active compounds and the receptor environment must also be considered. Therefore, the commercial success of HA-based hydrogels as drug delivery media is currently limited.

[0048] This article provides a hydrogel formulation loaded with the glucocorticoid dexamethasone, which can be applied to joint sites (e.g., affected joints at the base of the knee, hip, spine, thumb, finger, shoulder, ankle, or big toe) in subjects requiring treatment. The term "dexamethasone" as used herein includes any salt of dexamethasone, unless a specific salt is explicitly mentioned, such as dexamethasone sodium phosphate.

[0049] Hydrogel formulations effectively control the release of dexamethasone and maintain its levels within the therapeutic window—the effective dose between toxic levels (or side effects) and ineffective doses. Therefore, hydrogel formulations can improve patient compliance and safety while optimizing the therapeutic effect obtained from a given dose. This not only potentially reduces treatment costs but also alleviates patient discomfort due to the reduced frequency of administration.

[0050] In particular, hydrogel formulations offer several advantages compared to current treatment options for osteoarthritis, including rapid onset of action, improved pain relief in the early stages of the disease, fewer injections, fewer side effects, and multiple modes of action (analgesia, anti-inflammation, and cartilage protection).

[0051] The hydrogel formulations disclosed herein are based on a mixture of a hydrogel component containing cross-linked HA and a component containing non-cross-linked HA (also known as linear HA). The hydrogel is a three-dimensional network of polymers that can swell in an aqueous medium and retain a significant amount of water while maintaining a well-defined structure. Compared to uncross-linked HA, the hydrogel form exhibits improved resistance to hyaluronidase. The structure of the hydrogel is well-suited for loading dexamethasone, and the release of dexamethasone can be tailored by adjusting the amount of cross-linking agent relative to hyaluronic acid in the hydrogel component. Adding a portion of uncross-linked HA to the hydrogel formulation ensures suitable rheological properties and fine-tuning of the release profile.

[0052] Furthermore, the hydrogel formulation provides lubrication and cushioning, alleviating arthritis-related symptoms. Beyond theoretical considerations, it is further expected that exogenous HA will enhance chondrocyte HA synthesis, prevent cartilage degradation, and promote its regeneration.

[0053] Therefore, one aspect of the present invention relates to a hydrogel formulation comprising: (i) A first component comprising a hydrogel containing hyaluronic acid (HA) crosslinked with a crosslinking agent. The ratio of HA to crosslinking agent is approximately 10:1% (w / w) to approximately 18:1% (w / w). (ii) a second component, the second component comprising non-crosslinked HA, and (iii) Dexamethasone or its salts, The ratio between the first component and the second component is approximately 60:40% (w / w) to approximately 98:2% (w / w).

[0054] It should be understood that the % (w / w) given for cross-linked HA and non-cross-linked HA are relative to the total weight of the first and second components, respectively. During the processing of the first component hydrogel, especially during the swelling process, the HA is diluted, so the final % (w / w) HA of the first component is lower than the % (w / w) HA initially provided during the hydrogel preparation process.

[0055] The ratio between the first and second components can be used to customize the dexamethasone release profile. Hydrogel formulations containing a majority of the first component, which comprises the hydrogel, release dexamethasone more slowly than those containing a majority of the second component, which comprises non-crosslinked HA. The ratio between the first and second components of the hydrogel formulation can also be referred to as the formulation ratio, and it also affects the rheology and injectability of the final hydrogel formulation.

[0056] Therefore, embodiments of the present invention relate to hydrogel formulations as described herein, wherein the ratio between the first component and the second component is about 65:35% (w / w) to about 97:3% (w / w), for example about 70:30% (w / w) to about 96:4% (w / w), for example about 75:25% (w / w) to about 95:5% (w / w), for example about 80:20% (w / w) to about 95:5% (w / w), for example about 85:30% (w / w) to about 95:5% (w / w), preferably about 90:10% (w / w).

[0057] Another embodiment of the invention relates to a hydrogel formulation as described herein, wherein the first component comprises about 0.8-2% (w / w) of HA, for example about 1.0-1.5% (w / w), for example about 1.2-1.4% (w / w).

[0058] Another embodiment of the invention relates to a hydrogel formulation as described herein, wherein the first component comprises about 1.3% (w / w).

[0059] Another embodiment of the invention relates to a hydrogel formulation as described herein, wherein the second component comprises about 1.5-4.5% (w / w) of non-crosslinked HA, for example about 2-4% (w / w), preferably about 3% (w / w).

[0060] Alternatively, the viscosity of the hydrogel formulation can be changed by altering the ratio of HA to crosslinking agent in the first component or by changing the total concentration of HA in the hydrogel formulation. The total concentration of HA can be changed by adjusting the concentrations of crosslinked HA and / or non-crosslinked HA in the hydrogel formulation. If only the total concentration of HA is changed without altering the ratio between crosslinked and non-crosslinked HA, then the amount of HA in both the first and second components should be changed accordingly.

[0061] One embodiment of the present invention relates to a hydrogel formulation as described herein, wherein the concentration of crosslinked HA is from about 0.2% (w / w) to about 2% (w / w), for example from about 0.4% (w / w) to about 1.8% (w / w), for example from about 0.6% (w / w) to about 1.6% (w / w), for example from about 0.8% (w / w) to about 1.4% (w / w), for example from about 1.0% (w / w) to about 1.2% (w / w) based on the total weight of the hydrogel formulation.

[0062] Another embodiment of the invention relates to a hydrogel formulation as described herein, wherein the concentration of crosslinked HA is about 1.1% (w / w) based on the total weight of the hydrogel formulation.

[0063] Another embodiment of the invention relates to a hydrogel formulation as described herein, wherein the concentration of crosslinked HA is from about 5 mg / g to about 20 mg / g, for example from about 8 mg / g to about 15 mg / g, for example from about 10 mg / g to about 12 mg / g, based on the total weight of the hydrogel formulation.

[0064] Another embodiment of the invention relates to a hydrogel formulation as described herein, wherein the concentration of non-crosslinked HA is from about 0.05% (w / w) to about 1% (w / w), for example from about 0.1% (w / w) to about 0.75% (w / w), for example from about 0.2% (w / w) to about 0.5% (w / w), for example from about 0.25% (w / w) to about 0.35% (w / w), based on the total weight of the hydrogel formulation.

[0065] Another embodiment of the invention relates to a hydrogel formulation as described herein, wherein the concentration of non-crosslinked HA is about 0.3% (w / w) based on the total weight of the hydrogel formulation.

[0066] Another embodiment of the invention relates to a hydrogel formulation as described herein, wherein the concentration of non-crosslinked HA is from about 1 mg / g to about 10 mg / g, for example from about 2 mg / g to about 5 mg / g, for example from about 3 mg / g to about 4 mg / g, based on the total weight of the hydrogel formulation.

[0067] Another embodiment of the invention relates to a hydrogel formulation as described herein, wherein the total concentration of HA is from about 10 mg / g to about 25 mg / g, for example from about 12 mg / g to about 20 mg / g.

[0068] Another embodiment of the invention relates to a hydrogel formulation as described herein, wherein the total concentration of HA is from about 15 mg / g to about 17 mg / g.

[0069] The release of dexamethasone from hydrogel formulations can also be modulated by altering the degree of crosslinking (or crosslinking ratio), i.e., the ratio of HA to crosslinking agent in the first component of the hydrogel. By increasing the relative amount of crosslinking agent, the hydrogel will contain a more compact polymer network, thus releasing dexamethasone at a slower rate than a less dense polymer network. Therefore, formulations with a relatively high amount of crosslinking agent are advantageous for applications requiring the longest possible release time. Furthermore, the crosslinking ratio can affect the rheology and injectability of the hydrogel formulation.

[0070] One embodiment of the invention relates to a hydrogel formulation as described herein, wherein the ratio between HA and crosslinking agent is from about 12:1% (w / w) to about 18:1% (w / w), for example from about 14:1% (w / w) to about 18:1% (w / w), for example from about 16:1% (w / w) to about 18:1% (w / w), preferably about 17:1% (w / w).

[0071] Another embodiment of the invention relates to a hydrogel formulation as described herein, wherein the ratio between HA and crosslinking agent is about 10:1% (w / w).

[0072] Crosslinking of hyaluronic acid alters the form of the original HA material and facilitates its transformation into an insoluble hydrogel that can absorb large amounts of water without losing its defined structure. The principle of the HA crosslinking reaction is that polymer chains are covalently linked by reacting with a crosslinking agent, which can be selected from many different compounds, including but not limited to divinyl sulfone (DVS), 1,4-butanediol diglycidyl ether (BDDE), and polyethylene glycol (PEG).

[0073] Therefore, embodiments of the present invention relate to the hydrogel formulations described herein, wherein the crosslinking agent is selected from the group consisting of divinyl sulfone (DVS), 1,4-butanediol diglycidyl ether (BDDE), polylactic acid (PLA), polyethylene glycol (PEG), polyethylene glycol bis(amine), polyethylene glycol diacrylate (PEGDA), polyethylene glycol dimethacrylate (PEGDM), polyethylene glycol diacrylamide (PEGDAA), and polyethylene glycol dimethacrylamide (PEGDMA), carboxymethyl cellulose (CMC), dextran acrylate, dextran methacrylate, dextran glycidylmethacrylate, glycerol dimethacrylate, glycerol 1,3-diglycerolate diacrylate, sorbitol acrylate, and derivatives thereof.

[0074] The preferred crosslinking agent is divinyl sulfone (DVS). With this crosslinking agent, the crosslinking reaction occurs between the primary hydroxyl groups of N-acetyl-D-glucosamine via nucleophilic addition at the vinyl carbon atom of the DVS molecule. DVS contains two vinyl groups and is highly reactive to nucleophilic addition, resulting in a ratio of two moles of HA-disaccharide (N-acetyl-D-glucosamine) to one mole of DVS. Because DVS crosslinking does not involve the bioactive functional groups (carboxylates and acetamides) on the HA molecule, the gel largely retains the natural polyanionic, physicochemical, and biological properties of HA.

[0075] DVS is highly reactive under aqueous alkaline conditions and can therefore react completely instantaneously. The bond formed between HA and DVS is a sulfonyl diethyl ether bond, which is known to be very stable against hydrolysis (degradation) and alkaline treatment.

[0076] A preferred embodiment of the present invention relates to a hydrogel formulation as described herein, wherein the crosslinking agent is divinyl sulfone (DVS).

[0077] The size of HA polymers can vary, affecting the structure of the polymer network in the hydrogel. The size (molecular weight) of HA polymers can be determined by measuring intrinsic viscosity using the Mark Houwink Kuhn Sakurada (MHKS) equation or size exclusion chromatography combined with multi-angle laser scattering (SEC-MALLS).

[0078] This paper has found that certain sizes of HA polymers can be advantageous in providing hydrogels with structures that can effectively load dexamethasone, which is slowly released after application of the hydrogel formulation.

[0079] One embodiment of the present invention relates to a hydrogel formulation as described herein, wherein the molecular weight of the HA in the first and / or second components is from about 500 kDa to about 1500 kDa, for example from about 750 kDa to about 1250 kDa, preferably about 1000 kDa.

[0080] Another embodiment of the invention relates to a hydrogel formulation as described herein, wherein the first component and the second component have the same molecular weight of HA.

[0081] The HA used in hydrogels can, in principle, be any type of HA, including but not limited to HA salts formed with organic or inorganic bases, HA esters formed with alcohols, HA amides, O-sulfated derivatives of HA, deacetylated derivatives of HA, and percarboxylated derivatives of HA. HA esters can form with aliphatic, aryliphatic, alicyclic, aromatic, cyclic, and heterocyclic alcohols. HA amides can be aliphatic, aryliphatic, cycloaliphatic, aromatic, cyclic, and heterocyclic amines.

[0082] Preferably, HA is provided in the form of an inorganic salt. Therefore, one embodiment of the invention relates to a hydrogel formulation as described herein, wherein HA is provided as an inorganic salt selected from the group consisting of sodium hyaluronate, potassium hyaluronate, ammonium hyaluronate, calcium hyaluronate, magnesium hyaluronate, zinc hyaluronate, and cobalt hyaluronate, preferably sodium hyaluronate.

[0083] To avoid precipitation in hydrogel formulations, it is preferable to minimize impurities. In particular, calcium ions can cause undesirable precipitation in hydrogel formulations, such as calcium phosphate precipitation. Therefore, when selecting HA salts and / or aqueous buffers for hydrogel formulations, minimizing the risk of precipitation should be kept in mind.

[0084] Therefore, one embodiment of the present invention relates to a hydrogel formulation as described herein, wherein HA is provided as a calcium-free inorganic salt.

[0085] Another embodiment of the invention relates to a hydrogel formulation as described herein, wherein the hydrogel formulation contains less than 300 ppm, for example less than 250 ppm, for example less than 200 ppm of calcium ions.

[0086] A preferred embodiment of the present invention relates to a hydrogel formulation as described herein, wherein HA is provided in the form of sodium hyaluronate.

[0087] The polymeric properties of the HA hydrogel in the first component result in a very viscous and heterogeneous structure in both the hydrogel and the hydrogel formulation. This texture is well-suited for use in surgical implants or dermal fillers, where large needle sizes may not hinder its application. However, for smaller needle sizes or even spray applications, a very homogeneous hydrogel formulation is required. This can be achieved by micronizing the first component containing the hydrogel before mixing it with the second component, which contains non-crosslinked HA.

[0088] Therefore, embodiments of the present invention relate to hydrogel formulations as described herein, wherein the average particle size of the first component is less than about 1500 μm, for example less than about 1250 μm, for example less than about 1000 μm, for example less than about 750 μm, for example less than about 500 μm.

[0089] Another embodiment of the invention relates to a hydrogel formulation as described herein, wherein the average particle size of the first component is about 50 μm to about 1500 μm, for example about 100 μm to about 1250 μm, for example about 150 μm to about 1000 μm, for example about 200 μm to about 750 μm, for example about 300 μm to about 500 μm.

[0090] The hydrogel formulation comprises a solvent in which a mixture of the first and second components is dispersed. The solvent can be any aqueous solvent that is compatible with the HA hydrogel and biocompatible for administration to a subject. This includes aqueous buffers, including but not limited to PBS buffer. Preferably, the buffer is adjusted to a pH suitable for containing the active compound and within an acceptable range for administration to a subject. Furthermore, the pH is selected to avoid any degradation of hyaluronic acid, for example, within a pH range of 6.5–8.5.

[0091] Therefore, one embodiment of the present invention relates to a hydrogel formulation as described herein, wherein the solvent of the hydrogel formulation is aqueous.

[0092] Another embodiment of the invention relates to a hydrogel formulation as described herein, wherein the pH of the hydrogel formulation is from about pH 6.5 to about pH 8.5, for example from about pH 7 to about pH 8.

[0093] The concentration of dexamethasone or its salts in hydrogel formulations can be adjusted to maintain levels within the therapeutic window for a longer period. Specifically, hydrogel formulations with a higher content of the first component relative to the second component can load higher concentrations of dexamethasone without reaching toxic levels because the release of dexamethasone is slower compared to hydrogel formulations with a relatively lower content of the first component.

[0094] Therefore, hydrogel formulations can be used with a range of different concentrations of dexamethasone. The preferred concentration of dexamethasone is approximately 1 mg / g.

[0095] Therefore, embodiments of the present invention relate to hydrogel formulations as described herein, wherein the concentration of dexamethasone or a salt thereof is from about 0.2 mg / g to about 5 mg / g, for example from about 0.5 mg / g to about 4 mg / g, for example from about 0.75 mg / g to about 2 mg / g, preferably about 1 mg / g, relative to the total weight of the hydrogel formulation.

[0096] Dexamethasone sodium phosphate has been shown to be an effective treatment for osteoarthritis with limited side effects. Chemically, dexamethasone sodium phosphate (DSP) is a water-soluble inorganic ester (9-fluoro-11β,17,21-trihydroxy-16α-methylpregn-1,4-diene-3,20-dione 21-(dihydrogen phosphate) disodium salt).

[0097] Therefore, preferred embodiments of the present invention relate to hydrogel formulations as described herein, wherein the dexamethasone or a salt thereof is dexamethasone sodium phosphate.

[0098] Hydrogel formulations may further contain one or more additional conventional ingredients to improve the physical and / or chemical properties of the formulation. These can improve, for example, the stability, appearance, and / or efficacy of the hydrogel formulation.

[0099] Therefore, embodiments of the present invention relate to hydrogel formulations as described herein, wherein the hydrogel formulation further comprises one or more additives.

[0100] Another embodiment of the invention relates to a hydrogel formulation as described herein, wherein one or more additives are selected from the group consisting of osmotic pressure regulators, pH regulators, thickeners, gelling agents, preservatives, stabilizers, solubilizers, emulsifiers, antioxidants, electrolytes, free radical scavengers, vitamins, fragrances, colorants, pigments, melanin, photoprotective filters, waxes, resins, oils, esters, alcohols, and polyols.

[0101] Another embodiment of the invention relates to a hydrogel formulation as described herein, wherein the hydrogel formulation further comprises a preservative.

[0102] Another embodiment of the present invention relates to a hydrogel formulation as described herein, wherein the preservative is selected from the group consisting of methyl paraben, ethyl paraben, propyl paraben, butyl paraben, benzyl alcohol, chlorobutanol, phenol, m-cresol, chlorocresol, benzoic acid, sorbic acid, thiomersal, phenylmercuric nitrate, bromonitol, propylene glycol, benzalkonium chloride, and benzyl chloride.

[0103] Because hydrogel formulations can be manufactured under sterile conditions, preservatives may be unnecessary for some variants of hydrogel formulations. This can be, for example, but not limited to, single-use hydrogel formulations.

[0104] Therefore, one embodiment of the present invention relates to a hydrogel formulation as described herein, wherein the hydrogel formulation does not contain preservatives.

[0105] Another embodiment of the invention relates to a hydrogel formulation as described herein, wherein the hydrogel formulation further comprises a pharmaceutically acceptable carrier, excipient, or diluent.

[0106] The viscosity of pharmaceutical formulations plays a crucial role in how they are administered to subjects. If a formulation is too viscous, it can significantly limit how it can be administered to a subject, for example, because it can no longer be injected through a needle with a syringe, or it can cause undesirable and unacceptable discomfort to the recipient. The hydrogel formulations described herein contain a portion of non-crosslinked HA to reduce the viscosity of denser hydrogels. If the relative content of crosslinked HA becomes too high, the formulation may become unsuitable for injection, for example. Importantly, increasing the proportion of linear HA promotes the shear-thinning properties of the hydrogel formulation and improves injectability when force is applied to the syringe.

[0107] Storage modulus is a parameter used to describe the viscoelasticity of hydrogel formulations. High cross-linking of the first component will result in a high storage modulus. While a high storage modulus generally leads to a prolonged release profile, too high a storage modulus can cause the hydrogel to behave as a crystalline material, where the loading of the active compound becomes inefficient. Furthermore, this type of inhomogeneous material is unsuitable for injection. Therefore, depending on the application of the hydrogel formulation, it is preferable to obtain a storage modulus suitable for the specific application's desired viscoelasticity, such as providing a hydrogel formulation with the desired cushioning effect.

[0108] For intra-articular application of HA, a storage modulus of approximately 20–200 Pa is generally considered appropriate (see T. C. Laurent (1998), “The Chemistry, Biology and Medical Applications of Hyaluronan and its Derivatives”, pp. 243–253). However, it is also recognized that the storage modulus of knee synovial fluid is approximately 117 Pa, and therefore it is desirable to simulate this value as closely as possible to ensure high biocompatibility with the local tissues receiving the injection.

[0109] It should be understood that for hydrogel formulations that do not contain dexamethasone, the storage modulus referred to in this article was measured at 2.5 Hz.

[0110] Therefore, embodiments of the present invention relate to hydrogel formulations as described herein, wherein the storage modulus of the hydrogel formulation is from about 20 Pa to about 200 Pa, for example from about 50 Pa to about 150 Pa, for example from about 75 Pa to about 125 Pa.

[0111] Preferred embodiments of the present invention relate to hydrogel formulations as described herein, wherein the storage modulus G' of the hydrogel formulation is about 110 Pa to about 130 Pa.

[0112] Another embodiment of the invention relates to a hydrogel formulation as described herein, wherein the storage modulus of the hydrogel formulation is about 100 Pa.

[0113] Similarly, it is desirable to provide a hydrogel formulation with a loss modulus as close as possible to the loss modulus of knee joint synovial fluid (approximately 45 Pa).

[0114] Therefore, embodiments of the present invention relate to hydrogel formulations as described herein, wherein the loss modulus G” of the hydrogel formulation is about 10 Pa to about 80 Pa, for example about 10 Pa to about 50 Pa, for example about 15 Pa to about 70 Pa, for example about 20 Pa to about 60 Pa, preferably about 25 Pa to about 50 Pa.

[0115] Hydrogel formulations are designed for injection into tissues requiring the anti-inflammatory effects of dexamethasone and the cushioning properties of the hydrogel, such as joints. Therefore, a key part of hydrogel formulation design is its injectability (or injectability). The force required to inject the hydrogel formulation into the target tissue should not be too high, as this would cause unacceptable discomfort to the recipient. Generally, injectability can be quantified by the injection force required to dispense the hydrogel formulation through a needle. Acceptable force for intra-articular application is less than 15 N. The injection force also logically depends on the needle size, and therefore on the size of the orifice through which the hydrogel formulation is propelled. Typical needle sizes for osteoarthritis treatment are typically 18 to 22 gauge (Birmingham gauge, G).

[0116] Therefore, one embodiment of the present invention relates to a hydrogel formulation as described herein, wherein the hydrogel formulation is injectable from a 1 mL syringe via a needle of a specification (Birmingham specification) of about 18 G to about 22 G, for example about 20 G to about 22 G, preferably about 22 G.

[0117] Another embodiment of the invention relates to a hydrogel formulation as described herein, wherein the hydrogel formulation can be injected at an injection rate of about 2 mL / min to about 6 mL / min (e.g., about 3 mL / min to about 5 mL / min, preferably about 4 mL / min).

[0118] Another embodiment of the invention relates to a hydrogel formulation as described herein, wherein the hydrogel formulation is injectable with an injection force of less than about 15 N.

[0119] Upon application, dexamethasone is released into the surrounding environment. Over time, all contents of the hydrogel formulation will be released, with the final release time depending on the exact configuration of the hydrogel formulation.

[0120] Therefore, one embodiment of the present invention relates to a hydrogel formulation as described herein, wherein the hydrogel formulation is adapted to release at least 90%, for example at least 95%, for example at least 99% of the dexamethasone or its salt to a subject no earlier than about 8 hours after administration, for example no earlier than about 9 hours, for example no earlier than about 10 hours, for example no earlier than about 11 hours, for example no earlier than about 12 hours.

[0121] Another embodiment of the invention relates to a hydrogel formulation as described herein, wherein the hydrogel formulation is adapted to release at least 99% of dexamethasone to a subject no earlier than about 8 hours after administration.

[0122] The hydrogel formulation described herein is prepared by mixing a first component containing a cross-linked HA hydrogel with a second component containing a non-cross-linked HA, and loading the mixture with dexamethasone. The first component is prepared through a series of steps, including cross-linking the HA with a cross-linking agent, curing and pulverizing the resulting hydrogel, neutralizing and micronizing the broken hydrogel.

[0123] Therefore, one aspect of the present invention relates to a method for preparing a hydrogel formulation as described herein, the method comprising the following steps: (i) Provide a solution containing approximately 4-8% (w / w) hyaluronic acid (HA), (ii) The solution is reacted with a crosslinking agent to form a first hydrogel. (iii) Crush the first hydrogel to provide a broken hydrogel. (iv) Add a buffer to the broken hydrogel to provide a neutralized hydrogel. (v) Separating the neutralized hydrogel from the buffer to provide a purified hydrogel. (vi) Micronize the purified hydrogel to provide a first component comprising the hydrogel, the hydrogel containing about 0.5-3% (w / w) of hyaluronic acid (HA) crosslinked with a crosslinking agent. (vii) A second component comprising about 1-6% (w / w) of non-crosslinked HA is added to the first component to provide a hydrogel composition, and (viii) Add dexamethasone or a salt thereof to the hydrogel composition. Thus, the hydrogel formulation is provided.

[0124] Crosslinking of HA via a crosslinking agent can occur through covalent bonds with the hydroxyl, carboxyl, and / or acetamide functional groups of the hyaluronic acid polymer. For example, under alkaline conditions, crosslinking can be achieved through the primary hydroxyl group of N-acetyl-D-glucosamine in HA. The alkaline agent can be NaOH. HA is dissolved in the alkaline solution by mixing with a turbo mixer. It is preferable to extend the mixing time to 90 minutes at 600 rpm to ensure complete dissolution of HA. However, mixing can be extended to 180 minutes.

[0125] Therefore, one embodiment of the present invention relates to the method as described herein, wherein the solution is alkaline.

[0126] Another embodiment of the invention relates to the method described herein, wherein the pH of the solution is from about pH 9 to about pH 12, for example from about pH 10 to about pH 12.

[0127] Another embodiment of the invention relates to the method as described herein, wherein the pH of the solution is at least 9.

[0128] Another embodiment of the invention relates to the method described herein, wherein the solution comprises NaOH in a concentration of about 0.001 M to about 2.0 M, for example about 0.01 M to about 1.0 M, for example about 0.1 M to about 0.5 M, preferably about 0.2 M.

[0129] The design of the hydrogel formulation was guided by the amount of HA added in the first and second components, respectively. Since the second component was added without any further processing, the amount of HA added in step (vii) directly reflects the amount of uncrosslinked HA in the formulation. In contrast, the HA in the first component underwent numerous processing steps and was therefore "diluted" as part of the processing. Overall, the initial HA content in the solution of step (i) was higher than the HA in the first component of the final hydrogel.

[0130] This article presents the effective range of HA concentrations for producing hydrogel formulations that promote prolonged release. It is important to note that the total HA concentration can be adjusted according to the intended application, such as the injection site. Lowering the HA concentration results in hydrogel formulations with lower viscosity.

[0131] Therefore, embodiments of the present invention relate to the method described herein, wherein the solution comprises about 5-7% (w / w), preferably about 6% (w / w).

[0132] Another embodiment of the invention relates to the method as described herein, wherein the first component comprises about 0.8-2% (w / w), for example about 1.0-1.5% (w / w), for example about 1.2-1.4% (w / w) of HA.

[0133] Another embodiment of the invention relates to the method as described herein, wherein the second component comprises about 1.5-4.5% (w / w), for example about 2-4% (w / w), preferably about 3% (w / w) of non-crosslinked HA.

[0134] To form the first component hydrogel, HA is mixed with a crosslinking agent and vigorously stirred to ensure uniform distribution of the reactants. As part of the processing, it is preferable to allow a period of incubation after mixing to solidify the HA hydrogel, which is then broken down.

[0135] Therefore, one embodiment of the present invention relates to the method described herein, wherein the reaction is carried out in a ratio of about 12:1% (w / w) to about 22:1% (w / w) between HA and crosslinking agent, for example, from about 15:1% (w / w) to about 20:1% (w / w), preferably at about 17:1% (w / w).

[0136] Another embodiment of the invention relates to the method as described herein, wherein the reaction is carried out under stirring.

[0137] Another embodiment of the invention relates to the method described herein, wherein the crosslinking agent is selected from the group consisting of divinyl sulfone (DVS), 1,4-butanediol diglycidyl ether (BDDE), formaldehyde, glutaraldehyde, polyanhydride, polyaldehyde, polyol, carbodiimide, carboxyl chloride, sulfonyl chloride, cellulose, dextran, epichlorohydrin, ethylene glycol, diglycidyl ether, polyglycerol polyglycidyl ether, and diepoxides or polyepoxides.

[0138] Another embodiment of the invention relates to the method as described herein, wherein step (ii) is immediately followed by curing the first hydrogel before pulverization.

[0139] Another embodiment of the invention relates to the method described herein, wherein curing includes heating at about 35°C to about 45°C, for example 40°C, for at least 1 hour, preferably 2 hours.

[0140] The HA hydrogel is broken down into smaller pieces to produce a fragmented hydrogel. Typically, the hydrogel is broken down into 25 × 40 mm pieces. It should be understood that the pieces can be smaller or larger, for example, as low as about 10 mm or as high as about 50 mm. This can be achieved by cutting the hydrogel on a glass, plastic, or metal plate. Alternatively, the hydrogel can be mechanically forced through a cutting screen (also known as a squeeze screen) to break it up. The squeeze screen can have a 25 × 25 mm mesh size.

[0141] Therefore, embodiments of the present invention relate to the method as described herein, wherein the pulverization is performed by cutting.

[0142] After the hydrogel is crushed, the broken hydrogel is rinsed in an excess volume of aqueous solvent. This solvent can be purified water or any suitable type of aqueous buffer with a pH within a specific range. It is used to neutralize the broken hydrogel after initial hydrogel formation in an alkaline solution and to wash away any remaining unreacted crosslinking agents. During neutralization, the HA hydrogel absorbs the aqueous medium and swells to saturation. The time required for saturation of the broken HA hydrogel varies depending on the exact content and degree of crosslinking of the hydrogel, but neutralization and swelling are preferably carried out for 19–25 hours.

[0143] Therefore, one embodiment of the present invention relates to the method as described herein, wherein the broken hydrogel is immediately rinsed after pulverization.

[0144] Another embodiment of the invention relates to the method as described herein, wherein the rinsing is performed by immersing the broken hydrogel in ultrapure water and / or a rinsing buffer, and optionally agitating the immersed broken hydrogel.

[0145] Another embodiment of the invention relates to the method as described herein, wherein the buffer is a phosphate buffer or a saline buffer.

[0146] Another embodiment of the invention relates to the method described herein, wherein the pH of the buffer is from about 5.5 to about 9.

[0147] Another embodiment of the invention relates to the method as described herein, wherein the pH of the buffer is from about 5 to about 7.5, for example from about 6 to about 7, preferably about 6.9.

[0148] Another embodiment of the invention relates to the method as described herein, wherein the volume of the added buffer is at least 3 times the volume of the broken hydrogel, for example, at least 4 times the volume of the broken hydrogel, for example, at least 5 times the volume of the broken hydrogel, for example, at least 6 times the volume of the broken hydrogel, for example, at least 10 times the volume of the broken hydrogel.

[0149] Using a sieve, the still broken neutralized (and swollen) hydrogel is separated from excess and unabsorbed buffer solution. The neutralized hydrogel is placed in the sieve to drain the unabsorbed liquid portion, providing a purified swollen hydrogel from the solution.

[0150] Therefore, one embodiment of the present invention relates to the method as described herein, wherein the separation of the neutralized hydrogel from the buffer solution is achieved by placing the neutralized hydrogel in a sieve and then draining the liquid portion.

[0151] The purified hydrogel is micronized to ensure a homogeneous material that can be applied without difficulty (e.g., clogging of the needle). Therefore, the hydrogel formulations described herein are easily injected using various needle sizes, types, or catheters (22G-30G) without the need for strong force. For a smooth injection profile, a force below 20N is recommended, but preferably below 15N. Micronization can be achieved by extrusion or mixing. The micronized hydrogel is introduced into the hydrogel formulation as a first component.

[0152] Therefore, one embodiment of the present invention relates to the method as described herein, wherein the micronization of the purified hydrogel is carried out by extrusion and / or mixing.

[0153] Another embodiment of the invention relates to the method described herein, wherein the micronization of the purified hydrogel is achieved by extrusion using an extrusion sieve.

[0154] Another embodiment of the invention relates to the method described herein, wherein the extrusion screen has a mesh size of 200-450 μm.

[0155] Another embodiment of the invention relates to the method described herein, wherein micronization is achieved by mixing with a high-shear mixer.

[0156] The micronized hydrogel can optionally be autoclaved before being mixed with a second component containing non-crosslinked HA. Autoclaving will sterilize the hydrogel. However, the optional autoclaving step can also be used to slightly adjust the rheological properties of the hydrogel.

[0157] Therefore, one embodiment of the present invention relates to the method as described herein, wherein the first component and / or the second component is sterilized by autoclaving before the second component is added.

[0158] Another embodiment of the invention relates to the method as described herein, wherein the first component and / or the second component is autoclaved at about 110°C to about 130°C for about 10 minutes to about 20 minutes.

[0159] A first component containing processed HA hydrogel is mixed with a second component containing non-crosslinked HA to form a hydrogel composition. The hydrogel composition is then loaded with dexamethasone to obtain a hydrogel formulation.

[0160] The method described herein will produce a hydrogel formulation that prolongs the release of dexamethasone, thereby making more effective use of the therapeutic window over time.

[0161] Therefore, one aspect of the present invention relates to the hydrogel formulation described herein that can be obtained by the methods described herein.

[0162] The hydrogel formulation described herein is expected to improve the efficiency of existing and future treatments by maximizing the therapeutic effect obtained from a given dose of dexamethasone. The ability to more precisely regulate dexamethasone levels further reduces the risk of side effects and decreases the number of administrations required in the treatment regimen.

[0163] Therefore, one aspect of the present invention relates to hydrogel formulations as described herein, which can be used as pharmaceuticals.

[0164] Hydrogel formulations are delivery systems that facilitate the more effective use of dexamethasone. Therefore, hydrogel formulations are not limited to any specific therapy, but can be used to treat any condition that can be prevented, suppressed, or relieved by the delivery of dexamethasone. These conditions include, but are not limited to, musculoskeletal disorders, acute and chronic inflammatory arthritis, metabolic arthritis, and gout.

[0165] Therefore, another aspect of the present invention relates to hydrogel formulations as described herein for treating, preventing, or inhibiting one or more diseases or conditions, said diseases or conditions being selected from the group consisting of arthritis, tendinitis, synovitis, bursitis, metabolic arthritis, gout, allergic conditions, skin diseases, endocrine disorders, gastrointestinal diseases, blood diseases, tumor diseases, neurological diseases, ophthalmic diseases, kidney diseases, respiratory diseases, dermatological diseases, and pain, preferably arthritis.

[0166] One embodiment of the present invention relates to a hydrogel formulation used as described herein, wherein the arthritis is selected from the group consisting of osteoarthritis, acute or chronic inflammatory arthritis, such as psoriatic arthritis, rheumatoid arthritis, and juvenile arthritis.

[0167] Preferred embodiments of the present invention relate to hydrogel formulations as described herein for the treatment, prevention, or inhibition of osteoarthritis.

[0168] Hydrogel formulations are expected to improve existing treatments associated with inflammatory musculoskeletal conditions, including but not limited to tendinitis, bursitis, fasciitis, neuropathy, and myositis.

[0169] Therefore, one embodiment of the present invention relates to a hydrogel formulation as described herein for treating, preventing or inhibiting one or more inflammatory musculoskeletal conditions.

[0170] Another embodiment of the invention relates to a hydrogel formulation used as described herein, wherein the inflammatory musculoskeletal condition is selected from the group consisting of tendinitis, bursitis, fasciitis, neuropathy, and myositis.

[0171] Another embodiment of the invention relates to a hydrogel formulation used as described herein, wherein the hydrogel formulation is applied to a subject.

[0172] Another embodiment of the invention relates to a hydrogel formulation used as described herein, wherein the subject is a mammal, such as a domestic animal, a livestock animal, or a human, preferably a human.

[0173] Hydrogel formulations are preferably applied to the site of pain or disease, such as painful joints. Therefore, the preferred route of application is intra-articular injection. The injection is performed by directly injecting the substance into the affected joint using a subcutaneous needle.

[0174] Therefore, one embodiment of the present invention relates to a hydrogel formulation as described herein, wherein the route of administration is intra-articular.

[0175] Hydrogel formulations can be administered as a single injection or as a treatment requiring repeated injections. This treatment is suitable for non-bedridden / outpatient care settings.

[0176] Therefore, one embodiment of the present invention relates to a hydrogel formulation as described herein, wherein intra-articular application is performed in a single application or multiple applications.

[0177] Another embodiment of the invention relates to a hydrogel formulation as described herein, wherein intra-articular application is by injection into a joint selected from the group consisting of the hip, knee, shoulder, wrist, ankle, hand, and fingers (preferably the hip or knee).

[0178] Another embodiment of the invention relates to a hydrogel formulation used as described herein, wherein the hydrogel formulation is used as adjunctive therapy for subjects undergoing physical therapy, rehabilitation, and / or undergoing joint or musculoskeletal / orthopedic surgery.

[0179] Another embodiment of the invention relates to a hydrogel formulation used as described herein, wherein at least 90%, for example at least 95%, for example at least 99%, of the dexamethasone or its salt is released from the hydrogel formulation no earlier than about 8 hours after administration, for example no earlier than about 9 hours, for example no earlier than about 10 hours, for example no earlier than about 11 hours, for example no earlier than about 12 hours.

[0180] The hydrogel formulation is conveniently available in kit form, which also includes information on how to administer the formulation. For ease of use, the hydrogel formulation can be pre-filled into one or more syringes.

[0181] Therefore, one aspect of the present invention relates to a kit comprising: (i) Hydrogel formulations as described herein, and (ii) Optional, instructions for use.

[0182] Another embodiment of the invention relates to a kit as described herein, wherein the hydrogel formulation is provided in a syringe.

[0183] Another embodiment of the invention relates to a kit as described herein, wherein the syringe comprises a hypodermic needle.

[0184] Listing or discussing prior art documents in this specification should not be construed as an admission that such documents are part of the prior art or common knowledge.

[0185] Unless the context otherwise requires, preferences, options, and embodiments of a given aspect, feature, or parameter of the invention should be considered as having been disclosed in conjunction with any and all preferences, options, and embodiments of all other aspects, features, and parameters of the invention. This is especially true for the description of hydrogel formulations and all their features, which can readily form part of the final hydrogel formulation obtained by the methods described herein. Embodiments and features of the invention are also outlined in the following items.

[0186] project X1. A hydrogel formulation comprising: (i) A first component comprising a hydrogel containing hyaluronic acid (HA) crosslinked with a crosslinking agent. The ratio of HA to crosslinking agent is approximately 10:1% (w / w) to approximately 18:1% (w / w). (ii) a second component, the second component comprising non-crosslinked HA, and (iii) Dexamethasone or its salts, The ratio between the first component and the second component is approximately 60:40% (w / w) to approximately 98:2% (w / w).

[0187] X2. The hydrogel formulation according to item X1, wherein the ratio between HA and crosslinking agent is about 12:1% (w / w) to about 18:1% (w / w), for example about 14:1% (w / w) to about 18:1% (w / w), for example about 16:1% (w / w) to about 18:1% (w / w).

[0188] X3. The hydrogel formulation according to any one of X1 or X2, wherein the ratio between HA and crosslinking agent is about 17:1% (w / w).

[0189] X4. The hydrogel formulation according to any one of the preceding items, wherein the ratio between the first component and the second component is about 65:35% (w / w) to about 97:3% (w / w), for example about 70:30% (w / w) to about 96:4% (w / w), for example about 75:25% (w / w) to about 95:5% (w / w), for example about 80:20% (w / w) to about 95:5% (w / w), for example about 85:30% (w / w) to about 95:5% (w / w), preferably about 90:10% (w / w).

[0190] X5. The hydrogel formulation according to any one of the preceding items, wherein the ratio between the first component and the second component is about 90:10% (w / w).

[0191] X6. The hydrogel formulation according to any one of the preceding items, wherein the ratio between HA and crosslinking agent is about 17:1% (w / w), and the ratio between the first component and the second component is about 90:10% (w / w).

[0192] X7. The hydrogel formulation according to any one of the preceding items, wherein the first component comprises a hydrogel containing about 0.5-3% (w / w) of hyaluronic acid (HA).

[0193] X8. The hydrogel formulation according to any one of the preceding items, wherein the second component comprises about 1-6% (w / w) of non-crosslinked HA.

[0194] X9. A hydrogel formulation comprising: (i) A first component comprising a hydrogel containing about 0.5-3% (w / w) of hyaluronic acid (HA) crosslinked with a crosslinking agent. The ratio of HA to crosslinking agent is approximately 10:1% (w / w) to approximately 25:1% (w / w). (ii) A second component comprising approximately 1-6% (w / w) of non-crosslinked HA, and (iii) Dexamethasone or its salts, The ratio between the first component and the second component is approximately 80:20% (w / w) to approximately 95:5% (w / w).

[0195] X10. The hydrogel formulation according to item X9, wherein the ratio between the first component and the second component is about 85:15% (w / w) to about 95:5% (w / w), for example about 88:12% (w / w) to about 92:8% (w / w), preferably about 90:10% (w / w).

[0196] X11. The hydrogel formulation according to any one of items X9 or X10, wherein the ratio between HA and crosslinking agent is from about 12:1% (w / w) to about 22:1% (w / w), for example from about 15:1% (w / w) to about 20:1% (w / w), preferably about 17:1% (w / w).

[0197] X12. The hydrogel formulation according to any one of the preceding items, wherein the concentration of crosslinked HA is about 0.2% (w / w) to about 2% (w / w), for example about 0.4% (w / w) to about 1.8% (w / w), for example about 0.6% (w / w) to about 1.6% (w / w), for example about 0.8% (w / w) to about 1.4% (w / w), for example about 1.0% (w / w) to about 1.2% (w / w) based on the total weight of the hydrogel formulation.

[0198] X13. The hydrogel formulation according to any one of the preceding items, wherein the first component comprises about 0.8-2% (w / w), such as about 1.0-1.5% (w / w), such as about 1.2-1.4% (w / w) of HA.

[0199] X14. The hydrogel formulation according to any one of the preceding items, wherein the second component comprises about 1.5-4.5% (w / w), for example about 2-4% (w / w), preferably about 3% (w / w) of non-crosslinked HA.

[0200] X15. The hydrogel formulation according to any one of the preceding items, wherein the crosslinking agent is selected from the group consisting of divinyl sulfone (DVS), 1,4-butanediol diglycidyl ether (BDDE), polylactic acid (PLA), polyethylene glycol (PEG), polyethylene glycol bis(amine), polyethylene glycol diacrylate (PEGDA), polyethylene glycol dimethacrylate (PEGDM), polyethylene glycol diacrylamide (PEGDAA), and polyethylene glycol dimethacrylamide (PEGDMA), carboxymethyl cellulose (CMC), dextran acrylate, dextran methacrylate, dextran glycidyl methacrylate, glyceryl dimethacrylate, glyceryl 1,3-diglyceryl alcohol diacrylate, sorbitan acrylate, and derivatives thereof.

[0201] X16. The hydrogel formulation according to any one of the preceding items, wherein the crosslinking agent is divinyl sulfone (DVS).

[0202] X17. The hydrogel formulation according to any one of the foregoing items, wherein the total concentration of HA is from about 10 mg / g to about 25 mg / g, for example from about 12 mg / g to about 20 mg / g.

[0203] X18. The hydrogel formulation according to any one of the preceding items, wherein the molecular weight of the HA of the first component and / or the second component is about 500 kDa to about 1500 kDa, for example about 750 kDa to about 1250 kDa, preferably about 1000 kDa.

[0204] X19. The hydrogel formulation according to any one of the preceding items, wherein the HA of the first component and the second component have the same molecular weight.

[0205] X20. The hydrogel formulation according to any one of the preceding items, wherein the HA is provided in the form of an inorganic salt selected from the group consisting of sodium hyaluronate, potassium hyaluronate, ammonium hyaluronate, calcium hyaluronate, magnesium hyaluronate, zinc hyaluronate and cobalt hyaluronate, preferably sodium hyaluronate.

[0206] X21. The hydrogel formulation according to any one of the preceding items, wherein the average particle size of the first component is less than about 1500 μm, for example less than about 1250 μm, for example less than about 1000 μm, for example less than about 750 μm, for example less than about 500 μm.

[0207] X22. The hydrogel formulation according to any one of the preceding items, wherein the average particle size of the first component is about 50 μm to about 1500 μm, for example about 100 μm to about 1250 μm, for example about 150 μm to about 1000 μm, for example about 200 μm to about 750 μm, for example about 300 μm to about 500 μm.

[0208] X23. The hydrogel formulation according to any one of the preceding items, wherein the hydrogel formulation contains less than 300 ppm, for example less than 250 ppm, for example less than 200 ppm of calcium ions.

[0209] X24. The hydrogel formulation according to any one of the preceding items, wherein the solvent of the hydrogel formulation is aqueous.

[0210] X25. The hydrogel formulation according to any one of the preceding items, wherein the pH of the hydrogel formulation is from about pH 6.5 to about pH 8.5, for example from about pH 7 to about pH 8.

[0211] X26. The hydrogel formulation according to any one of the preceding items, wherein the concentration of dexamethasone or a salt thereof is from about 0.2 mg / g to about 5 mg / g, for example from about 0.5 mg / g to about 4 mg / g, for example from about 0.75 mg / g to about 2 mg / g, preferably about 1 mg / g, relative to the total weight of the hydrogel formulation.

[0212] X27. The hydrogel formulation according to any one of the preceding items, wherein the dexamethasone or its salt is dexamethasone sodium phosphate.

[0213] X28. The hydrogel formulation according to any one of the preceding items, wherein the hydrogel formulation further comprises a preservative.

[0214] X29. The hydrogel formulation according to item X28, wherein the preservative is selected from the group consisting of methylparaben, ethylparaben, propylparaben, butylparaben, benzyl alcohol, chlorobutanol, phenol, m-cresol, chlorocresol, benzoic acid, sorbic acid, thimerosal, phenylmercuric nitrate, bromonitol, propylene glycol, benzalkonium chloride, and benzyl chloride.

[0215] X30. The hydrogel formulation according to any one of the preceding items, wherein the storage modulus of the hydrogel formulation is about 20 Pa to about 200 Pa, for example about 50 Pa to about 150 Pa, for example about 75 Pa to about 125 Pa.

[0216] X31. The hydrogel formulation according to any one of the preceding items, wherein the storage modulus G' of the hydrogel formulation is from about 50 Pa to about 200 Pa, for example from about 75 Pa to about 175 Pa, for example from about 100 Pa to about 150 Pa, preferably from about 110 Pa to about 130 Pa.

[0217] X32. The hydrogel formulation according to any one of the preceding items, wherein the loss modulus G” of the hydrogel formulation is about 10 Pa to about 80 Pa, for example about 10 Pa to about 50 Pa, for example about 15 Pa to about 70 Pa, for example about 20 Pa to about 60 Pa, preferably about 25 Pa to about 50 Pa.

[0218] X33. The hydrogel formulation according to any one of the foregoing items, wherein the hydrogel formulation is injectable from a 1 mL syringe via a needle, the needle being of a specification (Birmingham specification) of about 22 G to about 30 G, for example about 22 G to about 28 G, for example about 24 G to about 27 G, preferably about 27 G.

[0219] X34. The hydrogel formulation according to any one of the preceding items, wherein the hydrogel formulation is injectable from a 1 mL syringe via a needle, the needle being of a specification (Birmingham specification) of about 18 G to about 22 G, for example about 20 G to about 22 G, preferably about 22 G.

[0220] X35. The hydrogel formulation according to any one of the preceding items, wherein the hydrogel formulation is injectable from a 1 mL syringe via a needle of approximately 22 G (Birmingham specification).

[0221] X36. The hydrogel formulation according to any one of items X33-X35, wherein the hydrogel formulation can be injected at an injection rate of about 2 mL / min to about 6 mL / min, for example about 3 mL / min to about 5 mL / min, preferably about 4 mL / min.

[0222] X37. The hydrogel formulation according to any one of items X33-X36, wherein the hydrogel formulation is injectable with an injection force of less than about 15 N.

[0223] X38. A hydrogel formulation according to any one of items X33-X37, wherein the hydrogel formulation is injectable with an injection force of less than about 15 N, for example less than about 14 N, for example less than about 13 N, for example less than about 12 N, for example less than about 11 N, preferably less than about 10 N.

[0224] X39. The hydrogel formulation according to any one of the preceding items, wherein the hydrogel formulation may be injected from a 1 mL syringe with an injection force of less than about 15 N, for example less than about 14 N, for example less than about 13 N, for example less than about 12 N, for example less than about 11 N, preferably less than about 10 N, through a needle of about 22 G (Birmingham specification).

[0225] X40. The hydrogel formulation according to any one of the preceding items, wherein the hydrogel formulation further comprises a pharmaceutically acceptable carrier, excipient or diluent.

[0226] X41. The hydrogel formulation according to any one of the preceding items, wherein the hydrogel formulation is adapted to release at least 90%, for example at least 95%, for example at least 99% of the dexamethasone or its salt to a subject no earlier than about 8 hours after administration, for example no earlier than about 9 hours, for example no earlier than about 10 hours, for example no earlier than about 11 hours, for example no earlier than about 12 hours.

[0227] Y1. A method for preparing a hydrogel formulation according to any one of the preceding items, the method comprising the following steps: (i) Provide a solution containing approximately 4-8% (w / w) hyaluronic acid (HA), (ii) The solution is reacted with a crosslinking agent to form a first hydrogel. (iii) Crush the first hydrogel to provide a broken hydrogel. (iv) Add a buffer to the broken hydrogel to provide a neutralized hydrogel. (v) Separating the neutralized hydrogel from the buffer to provide a purified hydrogel. (vi) Micronize the purified hydrogel to provide a first component comprising the hydrogel, the hydrogel containing about 0.5-3% (w / w) of hyaluronic acid (HA) crosslinked with a crosslinking agent. (vii) A second component comprising about 1-6% (w / w) of non-crosslinked HA is added to the first component to provide a hydrogel composition, and (viii) Add dexamethasone or a salt thereof to the hydrogel composition. Thus, the hydrogel formulation is provided.

[0228] Y2. The method described in project Y1, wherein the solution is alkaline.

[0229] Y3. The method according to any one of items Y1 or Y2, wherein the pH of the solution is from about pH 9 to about pH 12, for example from about pH 10 to about pH 12.

[0230] Y4. The method according to any one of items Y1-Y3, wherein the pH value of the solution is at least 9.

[0231] Y5. The method according to any one of items Y1-Y4, wherein the solution contains NaOH at a concentration of about 0.001 M to about 2.0 M, for example about 0.01 M to about 1.0 M, for example about 0.1 M to about 0.5 M, preferably about 0.2 M.

[0232] Y6. The method according to any one of items Y1-Y5, wherein the solution contains about 5-7% (w / w), preferably about 6% (w / w).

[0233] Y7. The method according to any one of items Y1-Y6, wherein the first component comprises about 0.8-2% (w / w), for example about 1.0-1.5% (w / w), for example about 1.2-1.4% (w / w) of HA.

[0234] Y8. The method according to any one of items Y1-Y7, wherein the second component comprises about 1.5-4.5% (w / w), for example about 2-4% (w / w), preferably about 3% (w / w) of non-crosslinked HA.

[0235] Y9. The method according to any one of items Y1-Y8, wherein the reaction is carried out at a ratio of about 12:1% (w / w) to about 22:1% (w / w) between HA and crosslinking agent, for example about 15:1% (w / w) to about 20:1% (w / w), preferably about 17:1% (w / w).

[0236] Y10. The hydrogel formulation according to any one of items Y1-Y8, wherein the ratio between HA and crosslinking agent is from about 10:1% (w / w) to about 18:1% (w / w), for example from about 12:1% (w / w) to about 18:1% (w / w), for example from about 14:1% (w / w) to about 18:1% (w / w), for example from about 16:1% (w / w) to about 18:1% (w / w).

[0237] Y11. The method according to any one of items Y1-Y10, wherein the reaction is carried out under stirring.

[0238] Y12. The method according to any one of items Y1-Y11, wherein the crosslinking agent is selected from the group consisting of divinyl sulfone (DVS), 1,4-butanediol diglycidyl ether (BDDE), formaldehyde, glutaraldehyde, polyanhydride, polyaldehyde, polyol, carbodiimide, carboxyl chloride, sulfonyl chloride, cellulose, dextran, epichlorohydrin, ethylene glycol, diglycidyl ether, polyglycerol polyglycidyl ether, and diepoxides or polyepoxides, preferably divinyl sulfone (DVS).

[0239] Y13. The method according to any one of items Y1-Y12, wherein step (ii) is immediately followed by solidification of the first hydrogel before pulverization.

[0240] Y14. The method according to item Y13, wherein curing includes heating at about 35°C to about 45°C, for example 40°C, for at least 1 hour, preferably 2 hours.

[0241] Y15. The method according to any one of items Y1-Y14, wherein the crushing is performed by cutting.

[0242] Y16. The method according to any one of items Y1-Y15, wherein the broken hydrogel is immediately rinsed after pulverization.

[0243] Y17. The method according to item Y16, wherein the rinsing is achieved by immersing the broken hydrogel in ultrapure water and / or rinsing buffer, and optionally agitating the immersed broken hydrogel.

[0244] Y18. The method according to any one of items Y1-Y17, wherein the buffer is a phosphate buffer or a saline buffer.

[0245] Y19. The method according to any one of items Y1-Y18, wherein the pH value of the buffer is from about 5.5 to about 9.

[0246] Y20. The method according to any one of items Y1-Y19, wherein the buffer is added at a volume of at least 3 times, for example at least 4 times, for example at least 5 times, for example at least 6 times, for example at least 10 times the volume of the broken hydrogel.

[0247] Y21. The method according to any one of items Y1-Y20, wherein the neutralized hydrogel is separated from the buffer solution by placing the neutralized hydrogel in a sieve and then draining the liquid portion.

[0248] Y22. The method according to any one of items Y1-Y21, wherein the micronization of the purified hydrogel is carried out by extrusion and / or mixing.

[0249] Y23. The method according to any one of items Y1-Y22, wherein the micronization of the purified hydrogel is achieved by extrusion using an extrusion sieve.

[0250] Y24. The method described in item Y23, wherein the extrusion screen has a mesh size of 200-450 μm.

[0251] Y25. The method according to any one of items Y1-Y22, wherein the micronization is achieved by mixing with a high-shear mixer.

[0252] Y26. The method according to any one of items Y1-Y25, wherein the first component and / or the second component is sterilized by autoclaving before the second component is added.

[0253] Y27. The method according to item Y26, wherein the first component and / or the second component is autoclaved at about 110°C to about 130°C for about 10 minutes to about 20 minutes.

[0254] Z1. The hydrogel formulation according to any one of items X1-X41, which can be obtained by any one of items Y1-Y27.

[0255] W1. A hydrogel formulation according to any one of items X1-X41 or Z1, which is used as a medicine.

[0256] W2. A hydrogel formulation according to any one of items X1-X41 or Z1, used for the treatment, prevention or inhibition of one or more diseases or conditions, said diseases or conditions being selected from the group consisting of arthritis, tendinitis, synovitis, bursitis, metabolic arthritis, gout, allergic conditions, skin diseases, endocrine disorders, gastrointestinal diseases, blood diseases, tumor diseases, neurological diseases, ophthalmic diseases, kidney diseases, respiratory diseases, skin diseases and pain, preferably arthritis.

[0257] W3. The hydrogel formulation according to Project W2, wherein the arthritis is selected from the group consisting of osteoarthritis, acute or chronic inflammatory arthritis, such as psoriatic arthritis, rheumatoid arthritis and juvenile arthritis.

[0258] W4. A hydrogel formulation according to any one of items X1-X41 or Z1, used for the treatment, prevention or inhibition of osteoarthritis.

[0259] W5. The hydrogel formulation used according to any one of items W1-W4, wherein the hydrogel formulation is applied to the subject.

[0260] W6. The hydrogel formulation used according to Project W5, wherein the subject is a mammal, such as a domestic animal, livestock, or human, preferably a human.

[0261] W7. The hydrogel formulation used according to any one of items W1-W6, wherein the route of application is intra-articular application.

[0262] W8. The hydrogel formulation used according to Project W7, wherein the intra-articular application is performed in a single or multiple application manner.

[0263] W9. The hydrogel formulation used according to any one of items W7 or W8, wherein the intra-articular application is an injection into a joint selected from the group consisting of the hip, knee, shoulder, wrist, ankle, hand, and fingers, preferably into the hip or knee joint.

[0264] W10. The hydrogel formulation used according to any one of items W1-W9, wherein the hydrogel formulation is used as adjunctive therapy to subjects receiving physical therapy, rehabilitation and / or undergoing joint or musculoskeletal / orthopedic surgery.

[0265] W11. A hydrogel formulation used according to any one of items W1-W10, wherein at least 90%, for example at least 95%, for example at least 99%, of the dexamethasone or its salt is released from the hydrogel formulation no earlier than about 8 hours after administration, for example no earlier than about 9 hours, for example no earlier than about 10 hours, for example no earlier than about 11 hours, for example no earlier than about 12 hours.

[0266] T1. A reagent kit comprising: (i) The hydrogel formulation according to any one of items X1-X41 or Z1, and (ii) Optional, instructions for use.

[0267] T2. The kit according to item T1, wherein the hydrogel formulation is provided in a syringe.

[0268] T3. The kit according to item T2, wherein the syringe has a needle of approximately 22 G gauge (Birmingham gauge).

[0269] The invention will now be described in more detail in the following non-limiting embodiments.

[0270] Example Example 1: Preparation of hydrogel formulation Hydrogel formulations were prepared using a custom-designed approach to evaluate the potential of the resulting formulations to provide controlled release of dexamethasone.

[0271] method: Sodium hyaluronate (1 MDa) was dissolved in 0.2 M NaOH solution with mechanical stirring via an Ika mixer equipped with a turbine mixer to prepare a 6% (w / w) HA solution. The solution was stirred until no HA clumps were present. Divinyl sulfone (DVS) was added to the solution in an amount to achieve the desired HA:DVS ratio (10:1, 17:1, or 20:1), and then mixed until completely dispersed in the solution.

[0272] The HA:DVS mixture was then transferred to a plastic dish with a uniform layer approximately 25–40 mm thick and cured at 40°C for 2 hours while the dish was covered. After curing, the crosslinked hydrogel was left at ambient temperature for 10 minutes and then cut into small pieces (approximately 25 × 40 mm) using a cutting wheel. The broken hydrogel was transferred to a beaker containing 12 mM PBS (137 mM NaCl, 2.7 mM KCl, 10.2 mM Na₂HPO₄, 2.0 mM KH₂PO₄) at pH 6.9 and swollen for up to 25 hours. Swelling and neutralization were performed to neutralize the pH of the gel and wash away any remaining DVS and possible DVS adducts. The total swelling time for the broken hydrogel was set to 19–25 hours depending on the degree of crosslinking. All gels with a crosslinking degree greater than 10:1 should be neutralized for at least 24 hours, while broken hydrogels with a crosslinking degree less than 10:1 should be neutralized for 19 hours.

[0273] Separate the neutralized hydrogel (pH 6.9–7.3) from the buffer solution through a sieve. Let the hydrogel sit in the sieve for 10 minutes to allow all buffer to seep out. Weigh the remaining hydrogel and, if necessary, correct the mass with buffer to achieve the desired HA concentration in a large-volume purified hydrogel.

[0274] The purified hydrogel was then micronized in large quantities via high-shear mixing or extrusion. Micronization via high-shear mixing was performed using a Silverson mixer connected to an Ultramixer head. Micronization was achieved by extrusion micronization through an extrusion sieve with a mesh size of 200-450 μm. A 1.5 mm support sieve was placed on each side of the extrusion sieve for support. The hydrogel was processed 20 times (10 times in each direction) at a pressure of approximately 6 bar.

[0275] The average particle size of the micronized hydrogel particles was measured using a Malvern Mastersizer 2000 coupled to a Hydro2000 dispersion unit in a buffer solution containing 0.9% NaCl. It was assumed that spherical particles were measured within the range of 0.02–2000 μm.

[0276] Micronized hydrogel particles were autoclaved at 121°C for 15 minutes to provide the first component of the hydrogel formulation. A second component, consisting of non-crosslinked (linear) HA (3% (w / w)) in 12 mM PBS (137 mM NaCl, 2.7 mM KCl, 10.2 mM Na₂HPO₄, 2.0 mM KH₂PO₄) at pH 6.9, was added to this first component to achieve the desired ratio between the first and second components, and the hydrogel composition was autoclaved at 121°C for 15 minutes.

[0277] Hydrogel formulations were prepared for two types of studies. One study aimed to evaluate different degrees of crosslinking (i.e., the ratio of HA to crosslinking agent), while the other aimed to evaluate the ratio of hydrogel components to non-crosslinked HA. The first component of the hydrogel formulation samples was named as follows: PAT (or DX1)-XXX-YZZZ, where "XXX" is the crosslinking agent, Y is the % (w / w) HA added to the first component, and ZZZ is the degree of crosslinking. For example, PAT-DVS-6101 is a sample in which 6% HA is crosslinked with DVS at a ratio of 10:1. It should be understood that 6% is the initial concentration of HA, not the concentration of HA in the first component.

[0278] All hydrogel formulations are loaded with dexamethasone sodium phosphate (DSP).

[0279] The rheological properties of the hydrogel formulation were measured on a Malvern Bohlin CV-100-901 rheometer. Samples were tested under the following conditions: frequency sweep of 0.01–10 Hz, strain of 0.005, 21 points, gap of 1000 μm, operating temperature of 25 °C, and plate-to-plate geometry of 20 mm. Pa readings were taken at 2.5 Hz, as described in the SynVisc information. Storage modulus was measured without DSP loading into the hydrogel formulation to allow for direct comparison with SynVisc products.

[0280] result: According to the data sheet, SynVisc's elastic modulus (storage modulus G') at 2.5 Hz is 111±13 Pa, and its viscous modulus (loss modulus G") is 25±2 Pa (elasticity and viscosity of synovial fluid in the knee joint of people aged 18-27 years, measured at 2.5 Hz using a comparable method: G' = 117±13 Pa; G” = 45±8 Pa).

[0281] The following hydrogel formulation was compared with SynVisc products: PAT-DVS-6171 + 3% (w / w) PAT (90:10 hydrogel: linear HA) in PBS.

[0282] In the apparatus used in this paper, the storage modulus G' of the Synvisc product is 133 Pa, and the viscous modulus G” is 22 Pa. Figure 1 In contrast, the storage modulus G' of the hydrogel formulation is 98 Pa, and the viscous modulus G” is 23 Pa. Figure 1 The figures are similar, indicating that hydrogel formulations are suitable for treating conditions such as osteoarthritis, which is an indication for the use of SynVisc.

[0283] in conclusion: This embodiment demonstrates that hydrogel formulations possess viscoelasticity suitable for treating rheumatic diseases such as osteoarthritis.

[0284] Example 2: Effect of hydrogel formulation on dexamethasone release curve The release of dexamethasone from the hydrogel formulation was measured to evaluate the efficiency of dexamethasone retention and gradual release over time.

[0285] method: The hydrogel formulation was prepared as described in Example 1.

[0286] Dexamethasone sodium phosphate (DSP) was loaded into the hydrogel composition to achieve a final concentration of 1 mg / g. In short, 10 mg of DSP was thoroughly mixed with 10 g of the hydrogel composition and allowed to stand overnight to reach equilibrium.

[0287] A DSP reference sample was prepared at a concentration of 1 mg / g DSP dissolved in 12 mM PBS (137 mM NaCl, 2.7 mM KCl, 10.2 mM Na2HPO4, 2.0 mM KH2PO4) at pH 6.9 in 1.5% (w / w) linear HA.

[0288] The release profile of dexamethasone from the hydrogel formulation was measured using the Sotax USP 4 dissolution system. Figure 2 The system consists of a piston pump (CP7), a dissolution unit (SOTAX CE7), a UV spectrophotometer (Shimadzu UV-1800), and control software (WinSOTAX+).

[0289] Experiments were conducted using the dissolution system settings listed in Table 1.

[0290]

[0291] Table 1: Setup of the Sotax USP 4 dissolution system for release curve measurement.

[0292] result: Dexamethasone sodium phosphate (DSP) measurement data are summarized as endpoint release times in Table 2. These endpoints were obtained from release curves derived from the dissolution system settings.

[0293]

[0294] Table 2: Final release time of dexamethasone sodium phosphate (DSP).

[0295] Compared to formulations containing pure linear HA (Sample 2), the release profile of DSP was delayed when incorporated into hydrogel formulations (Samples 3-8). Release could be further prolonged by increasing the degree of cross-linking (Samples 3-5). Interestingly, visual examination of dialysis cells showed that the hydrogel formulations remained intact even after 100% drug release, suggesting that the hydrogel may also have mechanical / physiological effects in osteoarthritis or similar applications.

[0296] in conclusion: This embodiment demonstrates that the hydrogel formulation described herein can be used for the prolonged and controlled release of dexamethasone sodium phosphate (DSP). Therefore, the hydrogel formulation can serve a dual purpose: providing a buffer to the treatment site and ensuring the controlled release of DSP to the treatment site over a prolonged period.

[0297] Example 3: Rheological properties of hydrogel formulations The viscoelasticity of the hydrogel formulation was tested for different ratios between the first and second components (“formulation ratio”) and between HA and the crosslinking agent (“crosslinking ratio”). For acceptable viscoelastic supplements, the rheological target values ​​should be G'(@2.5 Hz) = 50–200 Pa and G”(@2.5 Hz) = 10–80 Pa. For better and preferred viscoelastic supplements, the rheological target values ​​should be G'(@2.5 Hz) = 75–175 Pa and G”(2.5 Hz) = 10–50 Pa.

[0298] method Two groups of hydrogel formulations were prepared according to Example 1; the first group had a crosslinking ratio kept constant at 17:1 (formulation ratios of 10:90, 50:50, 80:20, 85:15, 90:10, 95:5, 99:1 and 100:0), and the second group had a formulation ratio kept constant at 90:10 (crosslinking ratios of 5:1, 17:1, 20:1 and 25:1).

[0299] Rheological properties of hydrogel formulations in Anton Pao Rheo Compass TM Measurements were performed on a rheometer. Samples were tested under the following conditions: frequency sweep of 0.01–10 Hz, strain of 0.005, 19 points, gap of 1000 μm, operating temperature of 25°C, and plate-to-plate geometry of 25 mm (PP25). Pa readings were taken at 2.5 Hz, as described in the SynVisc information. Storage modulus was measured without loading DSP into the hydrogel formulation to allow for direct comparison with SynVisc products.

[0300] result Table 3 shows the storage modulus and loss modulus at different formulation ratios. The hydrogel formulations FR10, FR50, and FR100 fall outside the acceptable range of G' and G” values. When the crosslinking ratio changes, it is clear that a high content of crosslinking agent (CR5) leads to an increase in storage modulus G' (Table 4), thus the hydrogel formulations do not meet the requirements for acceptable viscoelastic supplements. However, many tested hydrogel formulations (FR80, FR85, FR90, FR95, FR99) exhibit excellent viscoelasticity within the desired range.

[0301]

[0302] Table 3: Storage modulus G' and loss modulus G of hydrogel formulations with different formulation ratios.

[0303]

[0304] Table 4: Storage modulus G' and loss modulus G of hydrogel formulations with different crosslinking ratios.

[0305] in conclusion This embodiment demonstrates that the hydrogel formulation presented herein exhibits excellent viscoelasticity if the formulation ratio and crosslinking ratio are carefully selected. In particular, the second component of non-crosslinked HA is necessary to produce good viscoelasticity.

[0306] Example 4: Injectability of hydrogel formulations The injectability of hydrogel formulations was tested for different ratios between the first and second components (“formulation ratio”) and between HA and the crosslinking agent (“crosslinking ratio”). For hyaluronic acid injections used in the treatment of osteoarthritis, the recommended needle size is typically 18 to 22 gauge (e.g., SynVisc products). This range helps optimize hydrodynamics, resulting in a smoother injection process and greater patient comfort. For patient comfort, the injection force should not exceed 15 N.

[0307] method First, a set of hydrogel formulations were prepared to understand the limitations of the formulations. Formulations were prepared according to Example 1, with the formulation ratio kept constant at 90:10 (crosslinking ratios of 5:1, 17:1, 20:1, and 25:1). This was achieved by dispensing small-sized needles (25G1). 1 / 2 "and G271 1 / 2 They also recorded the injection force and performed stress tests on these formulations.

[0308] Based on observations from stress testing and knowledge from rheological data, a second group of hydrogel formulations was prepared according to Example 1, wherein the crosslinking ratio was kept constant at 17:1 (formulation ratios of 50:50, 85:15, 90:10, 95:5, 99:1, and 100:0). Using small sizes (25g1) 1 / 2 ") and the standard size used in the treatment of osteoarthritis (22G1) 1 / 2 The injection needle was used to dispense these preparations, and the injection force was recorded.

[0309] The injectability of the hydrogel formulation was measured on a texture analyzer from a Micro Stable System. Samples were tested under the following conditions: pre-test speed 10.2 mm / min, test speed 57.0 mm / min, trigger force 1 N. Measurements were taken as force (N), data were collected over 0.7 minutes, and the average force was read over a time range of 0.30–0.40 minutes. The hydrogel formulation was tested using a 2.25 mL HY-PAK BD glass syringe with a 22G needle. 1 / 2 25G 1 1 / 2 "and 27G1 1 / 2 It contains 27G. 1 / 2 "The syringe with the needle was set to an injection force of 2.6 N in this experiment and used as the minimum relevant force."

[0310] result Use small-sized needles (25G1) 1 / 2 "and G271 1 / 2 The injectability stress tests conducted surprisingly revealed a favorable crosslinking ratio (Table 5) at which the injection force was significantly reduced. Not only did adding a large amount of crosslinking agent (CR5) lead to an increase in injection force, but adding small amounts of crosslinking agent (CR20 and CR25) also led to an increase in injection force.

[0311] Unbound by theory, the determined favorable crosslinking ratio is expected to enhance the thixotropic (or shear-thinning) properties of the hydrogel formulation. With high crosslinking agent content, the hydrogel formulation becomes more rigid, thus requiring high injection force. Conversely, with low crosslinking agent content, the hydrogel formulation may become disordered because the internal structure of the hyaluronic acid polymer is less interconnected, to the point that greater force is required to align the polymer material for distribution through the limited volume of the needle.

[0312] Based on this observation, the required injection force was tested in hydrogel formulations with a 17:1 crosslinking ratio and different formulation ratios. This was done using a small needle (25G1). 1 / 2 The first stress test was performed on both. It was clear that hydrogel formulations containing little or no non-crosslinked HA (FR100 and FR99) resulted in high injection force (Table 6) and were not suitable for patients with osteoarthritis (OA).

[0313] Based on stress test injection data (Tables 5 and 6) and rheological data (Example 3, Tables 3 and 4), a needle suitable for patients with osteoarthritis (22G1) was used. 1 / 2 A group of hydrogel formulations were tested. When passed through 22G1 with less than 15N... 1 / 2 "When the needle is dispersed, all hydrogel formulations perform well and produce injection force."

[0314]

[0315] Table 5: Injection force of hydrogel formulations with different crosslinking ratios. Stress test of hydrogel formulations in small-sized needles.

[0316]

[0317] Table 6: Injection force of different formulations compared to hydrogel formulations.

[0318] in conclusion This example demonstrates the existence of an optimal crosslinking ratio range within which the injection force of the hydrogel formulation significantly decreases. Utilizing this knowledge and combining it with specific formulation ratios, hydrogel formulations with excellent rheological properties can be prepared, which can be injected using standard needle sizes.

[0319] References

Claims

1. A hydrogel formulation comprising: (i) A first component comprising a hydrogel containing hyaluronic acid (HA) crosslinked with a crosslinking agent. The ratio of HA to crosslinking agent is approximately 10:1% (w / w) to approximately 18:1% (w / w). (ii) a second component, the second component comprising non-crosslinked HA, and (iii) Dexamethasone or its salts, The ratio between the first component and the second component is approximately 60:40% (w / w) to approximately 98:2% (w / w).

2. The hydrogel formulation according to claim 1, wherein the ratio between HA and crosslinking agent is about 12:1% (w / w) to about 18:1% (w / w), for example about 14:1% (w / w) to about 18:1% (w / w), for example about 16:1% (w / w) to about 18:1% (w / w).

3. The hydrogel formulation according to any one of claims 1 or 2, wherein the ratio of HA to crosslinking agent is about 17:1% (w / w).

4. The hydrogel formulation according to any one of the preceding claims, wherein the ratio between the first component and the second component is about 65:35% (w / w) to about 97:3% (w / w), for example about 70:30% (w / w) to about 96:4% (w / w), for example about 75:25% (w / w) to about 95:5% (w / w), for example about 80:20% (w / w) to about 95:5% (w / w), for example about 85:30% (w / w) to about 95:5% (w / w), preferably about 90:10% (w / w).

5. The hydrogel formulation according to any one of the preceding claims, wherein the ratio between the first component and the second component is about 90:10% (w / w).

6. The hydrogel formulation according to any one of the preceding claims, wherein the ratio between HA and crosslinking agent is about 17:1% (w / w), and the ratio between the first component and the second component is about 90:10% (w / w).

7. The hydrogel formulation according to any one of the preceding claims, wherein the first component comprises a hydrogel containing about 0.5-3% (w / w) hyaluronic acid (HA).

8. The hydrogel formulation according to any one of the preceding claims, wherein the second component comprises about 1-6% (w / w) of non-crosslinked HA.

9. The hydrogel formulation according to any one of the preceding claims, wherein the first component comprises about 0.8-2% (w / w), for example about 1.0-1.5% (w / w), for example about 1.2-1.4% (w / w) of HA.

10. The hydrogel formulation according to any one of the preceding claims, wherein the second component comprises about 1.5-4.5% (w / w), for example about 2-4% (w / w), preferably about 3% (w / w) of non-crosslinked HA.

11. The hydrogel formulation according to any one of the preceding claims, wherein the crosslinking agent is selected from the group consisting of divinyl sulfone (DVS), 1,4-butanediol diglycidyl ether (BDDE), polylactic acid (PLA), polyethylene glycol (PEG), polyethylene glycol bis(amine), polyethylene glycol diacrylate (PEGDA), polyethylene glycol dimethacrylate (PEGDM), polyethylene glycol diacrylamide (PEGDAA), and polyethylene glycol dimethacrylamide (PEGDMA), carboxymethyl cellulose (CMC), dextran acrylate, dextran methacrylate, dextran glycidyl methacrylate, glyceryl dimethacrylate, glyceryl 1,3-diglyceryl alcohol diacrylate, sorbitan acrylate, and derivatives thereof.

12. The hydrogel formulation according to any one of the preceding claims, wherein the crosslinking agent is divinyl sulfone (DVS).

13. The hydrogel formulation according to any one of the preceding claims, wherein the total concentration of HA is from about 10 mg / g to about 25 mg / g, for example from about 12 mg / g to about 20 mg / g.

14. The hydrogel formulation according to any one of the preceding claims, wherein the molecular weight of the HA of the first component and / or the second component is about 500 kDa to about 1500 kDa, for example about 750 kDa to about 1250 kDa, preferably about 1000 kDa.

15. The hydrogel formulation according to any one of the preceding claims, wherein the solvent of the hydrogel formulation is aqueous.

16. The hydrogel formulation according to any one of the preceding claims, wherein the concentration of dexamethasone or a salt thereof is from about 0.2 mg / g to about 5 mg / g, for example from about 0.5 to about 4 mg / g, for example from about 0.75 mg / g to about 2 mg / g, preferably about 1 mg / g, relative to the total weight of the hydrogel formulation.

17. The hydrogel formulation according to any one of the preceding claims, wherein the storage modulus G' of the hydrogel formulation is about 50 Pa to about 200 Pa, for example about 75 Pa to about 175 Pa, for example about 100 Pa to about 150 Pa, preferably about 110 Pa to about 130 Pa.

18. The hydrogel formulation according to any one of the preceding claims, wherein the loss modulus G” of the hydrogel formulation is about 10 Pa to about 80 Pa, for example about 10 Pa to about 50 Pa, for example about 15 Pa to about 70 Pa, for example about 20 Pa to about 60 Pa, preferably about 25 Pa to about 50 Pa.

19. The hydrogel formulation according to any one of the preceding claims, wherein the hydrogel formulation is injectable by a needle in a 1 mL syringe, the needle being of a specification (Birmingham specification) of about 18 G to about 22 G, for example about 20 G to about 22 G, preferably about 22 G.

20. The hydrogel formulation of claim 19, wherein the hydrogel formulation is injectable with an injection force of less than about 15 N, for example less than about 14 N, for example less than about 13 N, for example less than about 12 N, for example less than about 11 N, preferably less than about 10 N.

21. A method for preparing a hydrogel formulation according to any one of the preceding claims, the method comprising the following steps: (i) Provide a solution containing approximately 4-8% (w / w) hyaluronic acid (HA), (ii) The solution is reacted with a crosslinking agent to form a first hydrogel. (iii) Crush the first hydrogel to provide a broken hydrogel. (iv) Add a buffer to the broken hydrogel to provide a neutralized hydrogel. (v) Separating the neutralized hydrogel from the buffer to provide a purified hydrogel. (vi) Micronize the purified hydrogel to provide a first component comprising the hydrogel, the hydrogel containing about 0.5-3% (w / w) of hyaluronic acid (HA) crosslinked with a crosslinking agent. (vii) A second component comprising about 1-6% (w / w) of non-crosslinked HA is added to the first component to provide a hydrogel composition, and (viii) Add dexamethasone or a salt thereof to the hydrogel composition. Thus, the hydrogel formulation is provided.

22. The hydrogel formulation according to any one of claims 1-20, which is obtained by the method according to claim 21.

23. The hydrogel formulation according to any one of claims 1-20 or 22, which is used as a medicine.

24. The hydrogel formulation according to any one of claims 1-20 or 22, for the treatment, prevention or inhibition of osteoarthritis.

25. The hydrogel formulation according to any one of claims 23 or 24, wherein the route of administration is intra-articular.

26. The hydrogel formulation of claim 25, wherein the intra-articular application is an injection into a joint selected from the group consisting of the hip, knee, shoulder, wrist, ankle, hand, and fingers, preferably the hip or knee.

27. A reagent kit comprising: (i) the hydrogel formulation according to any one of claims 1-20 or 22, and (ii) Optional, instructions for use.

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