Adenosine-coated liposome

Abstract

The invention provides a liposome wrapping adenosine. The liposomes may be formed from sphingomyelin or a combination of sphingomyelin and 1, 2-dimyristoyl-sn-glycerol-3-phosphocholine (DMPC) or a combination of sphingomyelin and 1, 2-dimyristoyl-sn-glycerol-3-phosphoglycerol (DMPG) or a combination of sphingomyelin, DMPG and DMPC. The liposome wrapping adenosine can be used for inducing cartilage regeneration, treating osteoarthritis, alleviating arthralgia, and / or slowing, preventing and / or reversing progressive structural tissue damage associated with osteoarthritis, or treating osteoarthritis, rheumatoid arthritis, acute gouty arthritis and / or synovitis. The liposome can release adenosine for two weeks at most.

Description

[0001] Cross - reference to related applications

[0002] This application claims the benefit of priority of U.S. Provisional Patent Application No. 62 / 828,916, filed on April 3, 2019, the disclosure of which is incorporated herein by reference. Background of the Invention

[0003] Osteoarthritis (OA) is a disease characterized by cartilage loss and is the most common type of arthritis, affecting 151 million people worldwide, including nearly 10% of the population in the United States and other industrialized countries. Age, prior trauma, obesity, and genetics are some of the risk factors for this degenerative joint disease. The incidence of OA increases with age, and the resulting pain, loss of joint function and mobility, social isolation, and significant reduction in quality of life make OA a disease with high medical and social impact. OA can affect any joint, but most commonly affects the knee, hip, and hand. The incidence of OA is highest in the knee joint, both in women (47%) and men (40%). Current treatment options are unsatisfactory and do not address the root cause. Most treatments are palliative and include the use of non - steroidal anti - inflammatory drugs (such as ibuprofen), narcotic analgesics, exercise, and acupuncture. The FDA has also approved some specific treatments for OA, including corticosteroids (anti - inflammatory agents) and hyaluronic acid (lubricating, pain - relieving), all of which are administered by intra - articular (IA) injection. While these injectable agents can relieve symptoms, none are restorative.

[0004] The purinergic system plays a key role in maintaining cartilage homeostasis. Adenosine acts on its A2A receptor (A2AR) and is an important autocrine homeostatic factor for maintaining chondrocytes and cartilage homeostasis. Adenosine is an endogenous physiological regulator, and its intracellular and extracellular concentrations are strictly controlled by oxygen consumption, cellular stress, and mitochondrial function. Extracellular adenosine mainly comes from the hydrolysis of ATP (mainly but not exclusively through the extracellular enzymes CD39 and CD73), and mediates its effects by activating G protein-coupled receptors (A1R, A2AR, A2BR, and A3R). These adenosine receptors are highly conserved evolutionarily, and their expression and functions also tend to be conserved. Adenosine has long been thought to regulate inflammatory and immune responses, and previous studies have demonstrated the importance of adenosine and its receptors in osteoblasts, osteoclasts, and bone marrow homeostasis. Previous studies have shown that adenosine receptors can also regulate the physiology and pathology of chondrocytes in response to inflammatory stimuli in rodent, equine, bovine, and human chondrocytes, but the specific receptors involved have not been determined. Although purine metabolism in horses is different from that in other species, the removal of endogenous adenosine (by adding adenosine deaminase) or the blockade of A2AR leads to cartilage degradation in equine cartilage explants because adenosine deaminase, which is present in lymphocytes, plasma, and extracellular fluid in most species, is absent in equine lymphocytes or serum. It has been reported that in a chemically induced OA model, the stimulation of A3R can reduce the progression of OA, mainly because of the anti-inflammatory effect of A3R agonists. However, the half-life of adenosine is only a few seconds. Summary of the Invention

[0005] The present disclosure provides an injectable formulation. Methods of making and using the injectable formulation are also disclosed. The injectable formulation comprises liposomes and physiological saline, wherein the liposomes are metastable and encapsulate adenosine. Brief Description of the Drawings

[0007] To more fully understand the nature and objects of the present disclosure, reference is made to the following detailed description taken in conjunction with the accompanying drawings.

[0008] Figure 1 Shown is the adenosine retention of liposomes formed by RgnA09.

[0009] Figure 2 Shown is a microscopic image of a liposome suspension formed by RgnA09.

[0010] Figure 3 Shown is a histogram of the approximate diameter of liposomes formed by RgnA09.

[0011] Figure 4 Shown is the adenosine retention of liposomes formed by RgnA10.

[0012] Figure 5The microscopic image of the liposome suspension formed by RgnA10 is shown.

[0013] Figure 6 The histogram of the approximate diameter of the liposomes formed by RgnA10 is shown.

[0014] Figure 7 The proliposome lyophilizate is shown.

[0015] Figure 8 The microscopic image of the liposome suspension of the prior art is shown. The liposome suspension contains obvious signs of crystallized adenosine, and the spheres are believed to be oil.

[0016] Figure 9 The pain data recorded using the incapacitance test is shown.

[0017] Figure 10 The HPLC chromatogram of the material isolated in Example 1 is shown.

[0018] Figure 11 Shown is Figure 10 The UV spectrum of the material isolated in

[0019] Figure 12 The initial burst release of RgnA09 and RgnA10 is shown.

[0020] Figure 13 The adenosine release kinetics of (left) RgnA09-MLV and (right) RgnA10-MLV within 24 hours are shown.

[0021] Figure 14 The rotarod pain test using (left) RgnA09 and (right) RgnA10 at 60 days (after six injections) is shown.

[0022] Figure 15 The effects of six injections of (A) RgnA09 and (B) RgnA10 at different concentrations on joint inflammation are shown. The administration method is ipsilateral - contralateral. Statistics: one-way (Brown-Forsythe and Welch) ANOVA. *P < 0.05 v / s vehicle, P < 0.05 v / s saline, +P < 0.05 v / s 0.3 mg and 1 mg.

[0023] Figure 16Shown are representative safranin-O stained sections of the affected rat tibias after treatment with vehicle or 3 doses of liposomal adenosine. In vehicle-treated animals, there was a marked reduction in chondroitin proteoglycan and surface irregularities of the cartilage. In rats treated with RgnA09, there was a dose-dependent improvement in chondroitin proteoglycan and disappearance of cartilage wear, accompanied by an increase in surface cartilage. In rats treated with RgnA10, the effect was strongest in those treated with the highest dose (3 mg / ml), although protection of cartilage was also observed at lower doses. Detailed Description

[0024] Although the claimed subject matter will be described in terms of particular embodiments, other embodiments including those that do not provide all of the advantages and features set forth herein are also within the scope of this disclosure. Various structural, logical, and process steps may be changed without departing from the scope of this disclosure.

[0025] Unless otherwise specified, all ranges provided herein include all values falling within the range, down to the tenth decimal place.

[0026] This disclosure provides an injectable formulation. Methods of making and using the injectable formulation are also disclosed herein.

[0027] In one aspect, this disclosure provides an injectable formulation comprising liposomes and saline, wherein the liposomes encapsulate adenosine.

[0028] Liposomes can contain i) sphingomyelin or ii) a combination of sphingomyelin and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) or iii) a combination of sphingomyelin and 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) or iv) a combination of sphingomyelin, DMPG, and DMPC. In various examples, the liposomes contain 70-100% by mass of sphingomyelin. Liposomes containing less than 100% by mass of sphingomyelin can further contain up to 30% by mass (such as the remainder) of DMPC or DMPG or a combination of DMPC and DMPG. In one embodiment, the liposomes can contain 70-99.9% by mass of sphingomyelin and 0.1-30% by mass (such as the remainder) of DMPC or 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) or a combination of DMPC and DMPG. In one embodiment, the liposomes contain 75-100% by mass of sphingomyelin. Liposomes containing less than 100% by mass of sphingomyelin can further contain up to 25% by mass (such as the remainder) of DMPC or DMPG or a combination of DMPC and DMPG. In one embodiment, the liposomes contain 75-99.9% by mass of sphingomyelin and 0.1-25% by mass (such as the remainder) of DMPC or DMPG or a combination of DMPC and DMPG. For example, the liposomes can contain 75, 80, 85, 90, 95, 96, 97, 98, 99, and 99.9% of sphingomyelin, with the remainder being DMPC, DMPG, or a combination thereof. The mass percentages refer to the total mass of the phospholipids.

[0029] The diameter and / or average diameter of the liposomes can be from 50 nm to 150 μm, including all values of 0.1 nm and the ranges therebetween (such as 50 nm - 1 μm, 50 nm - 750 μm, 50 - 500 nm, 50 - 250 nm, 50 - 100 nm, 100 nm - 1 μm, 100 - 750 nm, 100 - 500 nm, 100 - 250 nm, 1 - 150 μm, 1 - 100 μm, 1 - 50 μm, 1 - 40 μm, 1 - 30 μm, 1 - 25 μm, 1 - 20 μm, 1 - 10 μm, 1 - 5 μm). For example, the diameter and / or average diameter of the liposomes can be 50 nm, 75 nm, 100 nm, 250 nm, 500 nm, 1 μm, 10 μm, 25 μm, 30 μm, 40 μm, 50 μm, 75 μm, or 100 μm. In one embodiment, at least 60, at least 70, at least 80, at least 90, at least 95, at least 96, at least 97, at least 98, at least 99, at least 99.9, or 100% of the liposomes have a diameter of 50 nm - 1 μm, 50 nm - 750 μm, 50 - 500 nm, 50 - 250 nm, 50 - 100 nm, 100 nm - 1 μm, 100 - 750 nm, 100 - 500 nm, 100 - 250 nm, 1 - 150 μm, 1 - 100 μm, 1 - 50 μm, 1 - 40 μm, 1 - 30 μm, 1 - 25 μm, 1 - 20 μm, 1 - 10 μm, 1 - 5 μm. In one embodiment, there are no liposomes with a diameter greater than 150 μm. In one embodiment, less than 1% of the liposomes have a diameter greater than 150 μm. In some embodiments, the liposomes can be produced by the ethanol injection method, and the resulting liposomes can be smaller than those formed by other methods.

[0030] Prior to the release of adenosine, the liposomes of the present disclosure can be in a metastable state. Due to greater stability at the delivery site, metastable liposomes can provide enhanced release. Metastable liposomes a) have a relative diameter not equal to 1 (such as the metastable liposomes are not perfect circles or spheres); b) are large enough such that the swelling stress associated with membrane bending is not sufficient to overcome the conformational equilibrium tendency of the liposomes; and c) have a longest linear dimension (such as diameter) of 100 nm - 150 μm, including each value of 0.1 nm and the ranges therebetween. Such liposomes collapse (such as shrink or contract) into a smaller stable form when subjected to a temperature of 35 - 45 °C (including all values of 0.1 °C and the ranges therebetween) (such as contact with a reservoir having that temperature) (such as 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 °C) (such as about (or slightly above) 40 °C). In one embodiment, the longest linear dimension (such as diameter) of the smaller stable liposomes is 50 nm - 110 μm, including each value of 0.1 nm and the ranges therebetween. Additionally, the ratio of the volume enclosed by the liposomes at 25 °C to the volume enclosed by the liposomes after heating to a temperature above the gel-fluid phase transition of one or more of the lipids forming the liposomes is greater than 10. Metastable liposomes containing a hydrophile can collapse at 35 - 45 °C (including all values of 0.1 °C and the ranges therebetween) (such as 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45 °C) (such as about 40 °C) so as to release (such as gradually release) its payload (such as adenosine) upon such collapse (such as shrinkage or contraction). Metastable liposomes are described in U.S. Patent Publication No. 2016 / 0263031 (the relevant portions of which are incorporated herein by reference for convenience). Metastable liposomes may be referred to simply as liposomes.

[0031] The liposomes can be formulated together with one or more excipients. The formulation can be in the form of a liquid or a gel, preferably a liquid, for injection applications.

[0032] The liposomes are formed from one or more lipids, which can be neutral, anionic, or cationic at physiological pH. Examples of types of lipids include, but are not limited to, sterols and lipids such as cholesterol, phospholipids, lysolipids, lysophospholipids, sphingolipids, or PEGylated lipids. In one embodiment, the carbon chain length of the phospholipid is C 10 -C 22 In one embodiment, the carbon chain length of the phospholipid is C 14 -C 20. Suitable lipids include, but are not limited to, phosphatidylcholine (PC) (such as egg PC, soy PC) and phosphatidylglycerol. Examples of PC include, for example, 1,2-dioleoylphosphatidylcholine (DOPC), 1,2-distearoylphosphatidylcholine (DSPC), 1,2-dipalmitoylphosphatidylcholine (DPPC), and 1,2-dimyristoylphosphatidylcholine (DMPC). A variety of phosphatidylglycerols can be used. Non-limiting examples of phosphatidylglycerol include 1,2-dioleoylphosphatidylglycerol (DOPG), 1,2-distearoylphosphatidylglycerol (DSPG), 1,2-dipalmitoylphosphatidylglycerol (DPPG), and 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG).

[0033] In one embodiment, the phospholipid is sphingomyelin, or a combination of sphingomyelin with DMPC or DMPG. The total lipid concentration can be 7 - 12 mg / mL, including all values of 0.01 mg / mL and the ranges therebetween. In one embodiment, the total lipid concentration is 8 - 10 mg / mL. In one embodiment, the total lipid concentration is 8 mg / mL or 10 mg / mL. In one embodiment, the liposome contains sphingomyelin, DMPC, and DMPG, and the ratio of DMPC to DMPG is from 6:4 to 8:2. In one embodiment, the ratio of DMPC to DMPG is 7:3.

[0034] The liposome has an aqueous compartment. The aqueous compartment can contain water and adenosine. The adenosine concentration can be 0.1 - 7 mg / mL, including all values of 0.01 mg / mL and the ranges therebetween. In one embodiment, the adenosine concentration can be 0.1 - 4 mg / mL. In one embodiment, the adenosine concentration is 3 mg / mL.

[0035] Methods for manufacturing metastable liposomes are described herein. In one embodiment, dehydrated metastable liposomes are prepared from a homogeneous dispersion of phospholipids, preferably sphingomyelin, in a water / tert-butanol (TBA) co-solvent system, with the ratio of mg of phospholipid to mL of water / TBA being 2:1. Various ratios of water to TBA can be employed (such as 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 9:2, 7:2, 5:2, 3:2, 10:3, 8:3, 7:3, 5:3 (water:TBA)). The isotropic single-phase liposome solution is lyophilized to produce a dehydrated liposome powder in a sterile vial. After removing water and TBA from the vial, the lyophilization step produces empty lipid vesicles or dehydrated liposomes. Upon addition of a pharmaceutically acceptable carrier (such as water, saline, or PBS), the lyophilized product spontaneously forms a large metastable liposome dispersion. The ratio of lipid to TBA is an important factor affecting the size and polydispersity of the resulting liposome formulation.

[0036] In one embodiment, dehydrated metastable liposomes (e.g., RgnA09) are prepared from a solution comprising a dispersion of a plurality of phospholipids in a TBA / water cosolvent system with a volume ratio of water to TBA of 1:1. For example, for a solution containing 100 mg of phospholipids, 1 - 50 mL (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 25, 30, 40, or 50 mL) of a cosolvent system with a TBA:water volume ratio of 1:1 is used. The plurality of phospholipids can be a mixture of 75% sphingomyelin (by mass) and 25% PC / PG mixture (by mass), where the PC / PG mixture comprises 70% (by mass) DMPC and 30% (by mass) DMPG (e.g., in the total plurality of phospholipids comprising sphingomyelin and PC / PG mixture, 70% (by mass) of the plurality of phospholipids is sphingomyelin, 17.5% (by mass) of the plurality of phospholipids is DMPC, and 7.5% (by mass) is DMPG). The resulting solution containing the plurality of phospholipids is lyophilized to produce dehydrated liposome powder in a sterile vial. Thereafter, the lyophilized product (such as the dehydrated liposome powder) can be rehydrated with a solution containing adenosine (e.g., for 100 mg of phospholipids, 10 mL of an aqueous solution containing adenosine (such as physiological saline containing adenosine, where the concentration of adenosine is 0.1 - 7 mg / mL (such as 3 mg / mL)) to rehydrate the lyophilized product).

[0037] In one embodiment, dehydrated metastable liposomes (e.g., RgnA10) are prepared from a solution comprising a dispersion of phospholipids in a TBA / water cosolvent system with a volume ratio of water to TBA of 3:2. For example, for a solution containing 100 mg of phospholipids, 1 - 50 mL (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 25, 30, 40, or 50 mL) of a cosolvent system with a water:TBA volume ratio of 3:2 is used. The phospholipid can be sphingomyelin. The resulting solution containing the phospholipids is lyophilized to produce dehydrated liposome powder in a sterile vial. Thereafter, the lyophilized product (such as the dehydrated liposome powder) can be rehydrated with a solution containing adenosine (e.g., for 100 mg of phospholipids, 10 mL of an aqueous solution containing adenosine (such as physiological saline containing adenosine, where the concentration of adenosine is 0.1 - 7 mg / mL (such as 3 mg / mL)) to rehydrate the lyophilized product).

[0038] The liposomes of the present disclosure can be manufactured by various methods. For example, the manufacturing methods include but are not limited to the emulsion method, reverse evaporation method, detergent depletion method, and ethanol injection method. Various other methods known in the art are also included within the scope of the present disclosure.

[0039] Liposome-adenosine suspensions can be prepared by the methods of the present disclosure. The dehydrated liposome powder is hydrated by adding an adenosine solution and then mixed. For example, 10 mL of an adenosine solution (such as a 3 mg / mL adenosine solution in physiological saline (such as 0.9 mass% sodium chloride (9 mg NaCl per mL of water))) is added to a vial containing 100 mg of dehydrated liposome powder. The resulting liposomes containing adenosine can be multilamellar. The longest linear dimension (such as diameter) of the liposomes containing adenosine can be 50 nm - 150 μm, including all values of 0.1 nm and the ranges therebetween (such as 50 nm - 1 μm, 50 nm - 750 μm, 50 - 500 nm, 50 - 250 nm, 50 - 100 nm, 100 nm - 1 μm, 100 - 750 nm, 100 - 500 nm, 100 - 250 nm, 1 - 150 μm, 1 - 100 μm, 1 - 50 μm, 1 - 40 μm, 1 - 30 μm, 1 - 25 μm, 1 - 20 μm, 1 - 10 μm, 1 - 5 μm).

[0040] The selected dose depends on the desired therapeutic effect, the route of administration, and the desired treatment duration. The general dose level administered to a mammal (such as an individual) is 0.001 - 10 mg / kg body weight per day. Generally, the dose for intravenous injection or infusion can be lower.

[0041] The composition can also be administered by routes such as oral, parenteral (intramuscular, intraperitoneal, intravenous (IV) or subcutaneous injection), transdermal (passive or using iontophoresis or electroporation), or transmucosal (nasal, vaginal, rectal or sublingual), and can be formulated into dosage forms suitable for each route of administration. In one embodiment, the formulation is directly injected into the joint of an individual.

[0042] The metastable liposomes containing adenosine of the present disclosure have several advantages. For example, the metastable liposomes can slowly release adenosine, thereby prolonging the biological activity of the delivered adenosine and / or reducing the required dose.

[0043] Dosage units of metastable liposome preparations of different sizes can be used. The dosage units of the dry powder containing dehydrated metastable proliposome lyophilizate or an aqueous solution of adenosine or other hydrophilic active agents can be reconstituted in a container with a pharmaceutically acceptable carrier. Preferably, the pharmaceutically acceptable carrier is an aqueous carrier. Suitable dosage units include but are not limited to 0.1 - 1 mg, 1 - 3 mg, 3 - 10 mg, 10 - 20 mg, and 20 - 50 mg. Suitable dosage unit concentrations include but are not limited to 0.05 mg / mL - 10 mg / mL, preferably 0.05 mg / mL - 5 mg / mL, more preferably 0.05 mg / mL - 3.5 mg / mL.

[0044] The injectable formulation of the present disclosure can be used to induce cartilage regeneration, treat osteoarthritis, relieve joint pain, and / or slow down, arrest, and / or reverse progressive structural tissue damage associated with osteoarthritis in an individual in need of treatment. In one example, the individual may have or be suspected of having osteoarthritis, rheumatoid arthritis, acute gouty arthritis, and / or synovitis. The method of inducing cartilage regeneration, treating osteoarthritis, alleviating joint pain, and / or slowing down, arresting, and / or reversing progressive structural tissue damage associated with osteoarthritis in an individual in need of treatment includes administering the injectable formulation of the present disclosure to the individual in need of treatment.

[0045] In various embodiments, the individual is a human or non-human mammal. Examples of non-human mammals include, but are not limited to, agricultural animals (such as farm animals) (e.g., cows, pigs, sheep, etc.) and pets, service, or sport animals (e.g., horses, dogs, cats, etc.). Other non-limiting examples of individuals include rabbits, rats, and mice.

[0046] After administration to a human in need of treatment, adenosine is released from the liposome for up to 2 weeks. In one embodiment, after administration of the injectable formulation, adenosine is released from the liposome within 1 second to 1 hour (such as 1 minute to 1 hour) after administration to the individual. In one embodiment, at least a portion of adenosine (such as 1-20% of adenosine) is released from the liposome within 1 minute to 1 hour after administration to the individual. In one embodiment, at least a portion of adenosine (such as 1-20% of adenosine) is released within 1 second, 5 seconds, 10 seconds, 20 seconds, 30 seconds, 40 seconds, 50 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, or 10 minutes after administration to the individual.

[0047] The injectable formulation can be administered by intra-articular injection into the joint of an individual. The injectable formulation can be administered in one or multiple injections. The formulation can be administered multiple times (such as up to ten times), for example, once every 10 days or longer.

[0048] The method steps described in the various embodiments and examples disclosed herein are sufficient to carry out the method of the present invention. Thus, in one embodiment, the method consists essentially of the combination of the method steps disclosed herein. In an embodiment, the method consists of such steps.

[0049] The following statements describe various non-limiting examples of the present disclosure.

[0050] Statement 1. A preparation (such as an injectable preparation) comprising physiological saline and one or more liposomes, wherein the one or more liposomes comprise one or more lamellae (such as one or more multilamellar liposomes), wherein the liposomal lamellae comprise 70 - 100% by mass of sphingomyelin, and when the sphingomyelin is less than 100% by mass, the remainder is (such as up to 30% by mass) 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) or 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) or a combination of DMPC and DMPG, wherein the liposomes (a) have a diameter of 50 nm - 150 μm, including all values of 0.1 nm and the ranges therebetween (such as 50 nm - 1 μm, 50 nm - 750 μm, 50 - 500 nm, 50 - 250 nm, 50 - 100 nm, 100 nm - 1 μm, 100 - 750 nm, 100 - 500 nm, 100 - 250 nm, 1 - 150 μm, 1 - 100 μm, 1 - 50 μm, 1 - 40 μm, 1 - 30 μm, 1 - 25 μm, 1 - 20 μm, 1 - 10 μm, 1 - 5 μm); and (b) encapsulate adenosine in the aqueous compartment of the liposome. The one or more liposomes have a diameter of 50 nm - 100 μm. The one or more liposomes have a diameter of 100 nm - 150 μm.

[0051] Statement 2. The preparation (such as an injectable preparation) according to Statement 1, wherein the liposomes are in a metastable state.

[0052] Statement 3. The preparation (such as an injectable preparation) according to Statement 1, wherein adenosine or a portion thereof is released for up to two weeks, or after administration to a joint of an individual, adenosine or a portion thereof is released for up to two weeks.

[0053] Statement 4. The preparation (such as an injectable preparation) according to any one of the foregoing statements, further comprising an excipient.

[0054] Statement 5. The preparation (such as an injectable preparation) according to any one of the foregoing statements, wherein the adenosine concentration is 0.1 - 7 mg / mL.

[0055] Statement 6. The preparation (such as an injectable preparation) according to Statement 5, wherein the adenosine concentration is 0.1 - 4 mg / mL.

[0056] Statement 7. The preparation (such as an injectable preparation) according to any one of the foregoing statements, wherein the ratio of DMPC to DMPG is 6:4 to 8:2.

[0057] Statement 8. The preparation (such as an injectable preparation) according to Statement 7, wherein the ratio of DMPC to DMPG is 7:3.

[0058] Statement 9. A preparation (such as an injectable preparation) as described in any of the foregoing statements, wherein the total lipid concentration is 7-12 mg / mL.

[0059] Statement 10. A preparation (such as an injectable preparation) as described in any of the foregoing statements, wherein the liposome collapses (such as shrinks or reduces in size) at a temperature of 35-45 °C, the temperature including all values of 0.1 °C and the ranges therebetween (such as 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45 °C) (such as about 40 °C) to release its payload.

[0060] Statement 11. A preparation (such as an injectable preparation) as described in any of the foregoing statements, wherein adenosine is released into the joint of an individual within 1 second to 1 hour (such as within 1 minute to 1 hour) after administration.

[0061] Statement 12. A preparation (such as an injectable preparation) as described in Statement 11, wherein at least a portion of adenosine is released into the joint of an individual within 1 second to 1 hour (such as within 1 minute to 1 hour) after administration.

[0062] Statement 13. A preparation (such as an injectable preparation) as described in Statement 11 or Statement 12, wherein at least 1-20% of adenosine is released into the joint of an individual within 1 second to 1 hour (such as within 1 minute to 1 hour) after administration.

[0063] Statement 14. A preparation (such as an injectable preparation) as described in any of Statements 11-13, wherein at least a portion of adenosine or at least 1-20% of adenosine is released within 1 second, 5 seconds, 10 seconds, 20 seconds, 30 seconds, 40 seconds, 50 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes or 10 minutes after administration to the joint of an individual.

[0064] Statement 15. A method for inducing cartilage regeneration and / or treating osteoarthritis in an individual in need thereof, comprising administering to the individual a preparation (such as an injectable preparation) as described in any of the foregoing statements.

[0065] Statement 16. A method for alleviating joint pain in an individual in need thereof, comprising administering to the individual a preparation (such as an injectable preparation) as described in any of Statements 1-14.

[0066] Statement 17. A method for slowing, halting and / or reversing progressive structural tissue damage associated with osteoarthritis in an individual in need thereof, comprising administering to the individual a preparation (such as an injectable preparation) as described in any of Statements 1-14.

[0067] Statement 18. A method as described in any of Statements 15-17, wherein the preparation (such as an injectable preparation) is administered to the joint of the individual by intra-articular injection.

[0068] Statement 19. The method according to any one of statements 15 - 18, wherein the injectable preparation is administered by one or more injections.

[0069] Statement 20. The method according to any one of statements 15 - 19, wherein the injectable preparation is administered multiple times (such as up to 10 times) every 10 days.

[0070] Statement 21. The method according to any one of statements 15 - 20, wherein the individual has osteoarthritis, rheumatoid arthritis, acute gouty arthritis, and / or synovitis.

[0071] Statement 22. The method according to any one of statements 15 - 21, wherein the individual is a human or non - human mammal. Examples of non - human mammals include, but are not limited to, agricultural animals (such as farm animals) (e.g., cows, pigs, sheep, etc.) and pet, service, or sport animals (e.g., horses, dogs, cats, etc.). Other non - limiting examples of individuals include rabbits, rats, and mice.

[0072] The following examples are intended to illustrate the invention. They are not intended to be limiting in any way.

[0073] Example 1

[0074] This example describes the liposomes of the present disclosure.

[0075] Since the half - life of adenosine is only a few seconds, the liposome - adenosine used in the above study was freshly prepared daily. A series of storage - stable formulation protocols were developed and evaluated based on lipid components, dissolution efficiency, and retention characteristics. It was determined whether cholesterol / stabilizer should be included and these variables were optimized. The percentage of adenosine bound to liposomes in the total preparation was measured and could be increased or decreased by reducing or increasing the amount of adenosine solution used to hydrate a fixed amount of pre - liposome lyophilizate. In the resulting liposome particles formed by hydrating the pre - liposome lyophilizate with adenosine solution, the ratio of adenosine to (adenosine + lipid) depends on the adenosine concentration in the solution rather than the volume used.

[0076] All formulations were hydrated with water containing 7 mg / mL adenosine.

[0077] Conditions

[0078]

[0079] Data analysis

[0080]

[0081] Two formulations, RgnA09 (75% sphingomyelin, 17.5% DMPC, 7.5% DMPG) and RgnA10 (100% sphingomyelin), were tested to evaluate their ability to bind and release adenosine over time. Liposomes were formed in sterile glass vials containing 100 mg of phospholipid powder. The liposomes were mixed with 10 mL of sterile adenosine solution (3 mg / mL in physiological saline) provided by a pre-filled plastic syringe. Samples (100 μL) of the lipid-adenosine suspension were incubated at 37 °C in phosphate-buffered saline for 0, 1 hour, 2 hours, 1 day, and 2, 5, 7, 10 days. At the end of each incubation time, the samples were centrifuged at 23,000 g for 15 minutes at 4 °C. The supernatant was removed, and the liposome particles were resuspended in a physiological saline solution containing 0.5% Triton-X100. The adenosine concentration in the remaining intact liposomes was quantified by high-performance liquid chromatography (HPLC).

[0082] Figure 1 and Figure 4 The percentage of adenosine retention in the two liposomal formulations is shown. No significant difference was found between RgnA09 and RgnA10. A slightly higher retention rate of RgnA09 was observed at time point zero, and the retention rate increased over time points. The freshly prepared liposome suspension (time 0) showed adenosine retention rates of 21% and 19% for RgnA09 and RgnA10, respectively. After 1 hour of incubation, the retention rates of both formulations decreased to 4%, and decreased slowly over time, reaching 1.4% and 2% (corresponding to RgnA09 and RgnA10, respectively) on day 10, equivalent to 159 μM and 227 μM of adenosine.

[0083] These results suggest that both liposomal formulations are good reservoirs for encapsulating and slowly releasing adenosine at concentrations sufficient to activate A2A adenosine receptors in vivo.

[0084] Pre-liposome lyophilizates can be rehydrated in concentrated solutions of hydrophilic pharmacological agents such as adenosine. During the rehydration process, multilamellar liposome particles are produced that contain the hydrophilic agent in the aqueous compartments of the liposomes. The particles are "large", approximately 30 microns, and are metastable, so they will collapse into a dense form in a high-heat environment (about 40 °C). On the other hand, if these particles are confined within a locally enclosed compartment, such as the bursa of the human knee joint synovium, the particles can achieve sustained release of the pharmacological agent. Therefore, the contained hydrophilic agent is not affected by degrading enzymes in the physiological environment.

[0085] Large metastable multilamellar lipid particles can be generated by preparing a proliposome lyophilizate by dissolving lipids (including at least 50% sphingomyelin) and up to 50% non-sphingomyelin phosphatidylcholine in a water-tert-butanol (60:40 v / v) mixture prior to lyophilization. 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) are used as non-sphingomyelin in a ratio of 70%:30%.

[0086] Example 2

[0087] This example describes the liposomes of the present disclosure.

[0088] The provided liposome-adenosine laboratory formulation was analyzed using cross-polarized microscopy. The results are shown in Figure 8 .

[0089] When observed under the microscope, the liposome suspension exhibited signs of significantly crystallized adenosine. It is believed that the liposome suspension may not contain a large amount of liposome content and is mainly an emulsion. The spherical objects in the image may be oil. The composition of the formulation contains a significant proportion (60%) of soybean oil, which would not be a component of the liposome formulation but would be a component of the emulsion.

[0090] Considering the solubility of adenosine in water, which is 7 mg / mL, the laboratory formulation requires adding 300 mg of adenosine to 10 mL of physiological saline. It is speculated that only 70 mg of adenosine will dissolve, leaving 230 mg of adenosine in crystalline form. It is thought that when the emulsion is centrifuged, most of the particles may be crystalline adenosine. The particles of the provided formulation do not pass through HPLC because their size decreases with each washing until nothing remains. We have looked at publications using this formulation and found that only the supernatant was analyzed by HPLC. The publication states that 73% of the adenosine was retained in the liposomes. However, the amount in the particles was inferred by measuring the adenosine concentration in the supernatant. However, this HPLC measurement of the supernatant is consistent with the case where 70 mg of adenosine is dissolved in 10 mL of physiological saline at its maximum solubility in water, and the remaining 230 mg of adenosine is crystallized in the particles. It is speculated that some adenosine-containing liposomes were formed, but it is uncertain whether they are present in this formulation.

[0091] An analysis of the technique for preparing the proliposome lyophilizate of liposome adenosine was conducted to study the capture amount of adenosine. The advantage of this technique is that it is a stable and sterile process, which is ideal for drug preparation. This technique involves reconstructing a sterile proliposome lyophilizate in the presence of an active agent. Figure 7 A photograph of the proliposome lyophilizate is shown.

[0092] The pre-liposome lyophilized product appears as a white, fluffy powder in a sterile vacuum vial. The rehydration process involves injecting a concentrated adenosine solution into the vial. Subsequently, the powder dissolves within 30 seconds, sometimes instantaneously, and may require gentle swirling. Thereafter, the resulting liposome suspension can be withdrawn from the vial using a syringe. It should be noted that due to the vacuum inside the vial, the adenosine solution will be rapidly absorbed by the vial upon insertion of the syringe. The ideal concentration of the adenosine solution for rehydration is its maximum solubility in water, i.e., 7 mg / mL. The diluent used is Sterile Water for Injection (SWFI), but normal saline and buffer solutions can also be used. The microscopic image of the resulting liposome suspension is shown in Figure 5 .

[0093] The average diameter of each particle is approximately 50 microns. In this example, we rehydrated 80 mg of the powder in 11 mL of a 7 mg / mL adenosine solution (in SWFI). It should be noted that the above microscopic image is of the diluent to enable observation of individual liposome particles.

[0094] The HPLC method for measuring adenosine and lipid content was prototyped. The liposome preparation was centrifuged to separate the particles from the supernatant. The particles were completely dissolved in methanol. The lipid portion of the liposome is relatively insoluble in acetonitrile, while adenosine is soluble in acetonitrile. Therefore, the HPLC method includes an initial mobile phase of water / acetonitrile, which first elutes adenosine from the HPLC column (C18 column), and then elutes the lipid with a methanol mobile phase. A standard was run for each reagent: adenosine and lipid. The dissolved particles were injected and the results were compared with those standards. The particle chromatogram is shown in Figure 10 .

[0095] The retention time of adenosine in this method is 1.42 minutes, while that of the lipid is 9.75 minutes. The wavelengths at which these two analytes are detected also differ: 260 nm and 203 nm for adenosine and lipid, respectively. Figure 11 These spectra are shown.

[0096] The lipid [SM] standard is 4 mg / mL and the adenosine [ADO] standard is 1 mg / mL. The peak areas generated from the standard runs are as follows.

[0097]

[0098] To replace the calibration curve, consider the following.

[0099] [SM] = SM / 22,465 / 4 = SM / 5,616

[0100] [ADO] = ADO / 25,338

[0101] Subsequently, the particles were assayed in triplicate, and the results of the two peaks obtained were as follows.

[0102]

[0103] The functional relationship between the proportion of adenosine in the particles (defined as [ADO] / ([ADO]+[SM])) and the proportion of chromatographic peak areas (ADO / SM) can be obtained as follows.

[0104] [ADO]=ADO / 25,338

[0105] [SM]=SM / 5616

[0106]

[0107] When applied to the chromatogram results, the following values were obtained.

[0108]

[0109] Example 3

[0110] This example describes the method of use of the injectable formulation of the present disclosure.

[0111] Rats with established OA received an intra-articular injection of normal saline (100 μL), while the other 8 groups of animals received Ade in 2 different liposomal formulations at doses of 3, 1, 0.3, and 0 mg / mL. The first injection was performed 4 weeks after ACL rupture. The animals received an injection every 10 days for a total of 6 times. The knee joint swelling was measured before each injection as a measure of joint inflammation. Pain tests were performed on the rats at baseline (before the first injection), 5 days after the 3rd injection, and finally at 57 days, i.e., just before sacrifice (7 days after the last injection). The joints after sacrifice were analyzed by histology and uCT.

[0112] 10 treatment groups

[0113] 2 formulations × 4 doses = 8 treatment groups

[0114] 1 positive control (Rgn01)

[0115] 1 negative control (normal saline)

[0116] RgnA01The preparation was carried out according to the method described by Corciulo et al. in "Endogenous adenosine maintains cartilage homeostasis and exogenous adenosine inhibits osteoarthritis progression", Nat Commun., May 11, 2017; 8:15019.

[0117] Liposomes were freshly prepared one day before injection. Ethanol was added to soybean oil containing adenosine or adenosine plus an adenosine receptor antagonist. The lipid phase containing phosphatidylcholine and cholesterol (molar ratio 1:0.5) was added to the aforementioned solution and emulsified at 15,000 r.p.m. for 10 minutes. Thereafter, physiological saline and glycerol were added together to the lipid phase and homogenized at 15,000 r.p.m. for 20 minutes, followed by sonication for 1 minute at 100% duty cycle.

[0118] PTOA rats were randomly assigned to the experimental group. Pain tests were performed before the start of the experiment (4 weeks after ACL rupture) and 5 days after the 3rd injection. Pain behavior was measured by the weight-bearing asymmetry between the ipsilateral and contralateral hindlimbs using a bipedal balance meter. After the hyperalgesia test, the animals were placed in a rodent restraint device and allowed to stand on their hind paws. The hindlimbs rested on two weight-averaging platform pads. When the animals removed their weight from each pad, the device made 3 - 4 consecutive measurements within 12 seconds to record the average weight (grams). The average value for each animal was used for statistical analysis.

[0119] Example 4

[0120] This example describes the method for preparing the liposomes of the present disclosure and the release kinetics of the liposomes of the present disclosure.

[0121] Preparation of pre-liposome lyophilizate:

[0122] Conditions

[0123]

[0124] The total content of all vials was 100 mg, the filling volume was 5 mL, and the filling concentration was 20 mg / ml of solvent.

[0125] For RgnA09, 75 mg of SM, 17.5 mg of DMPC and 7.5 mg of DMPG were dissolved in 5 mL of a 1:1 (v / v) mixture of water and tert-butanol (TBA). The solution was lyophilized according to the following parameters (first frozen at -40 °C for 30 minutes, then subjected to primary drying at 10 °C under a vacuum of 200 μ for 20 hours, and then secondary drying at 20 °C for 4.5 hours), and kept in a vacuum-sealed vial. Subsequently, the lyophilizate was rehydrated with 40 mg of pure water at room temperature (25 °C).

[0126] For RgnA10, 100 mg of pure sphingomyelin (SM) was dissolved in 5 mL of a 3:2 (v / v) mixture of water and tert-butanol (TBA). The solution was lyophilized according to the following parameters (first frozen at -40 ° for 30 minutes, then subjected to primary drying at 10 ° under a vacuum of 200 μ for 20 hours, and then secondary drying at 20 ° for 4.5 hours), and kept in a vacuum-sealed vial. Subsequently, the lyophilizate was rehydrated with 40 mg of pure water at room temperature (25 °).

[0127] Preparation of adenosine stock solution: The adenosine stock solution was prepared by dissolving pure adenosine powder into physiological saline (0.9% saline). 50 mL of 0.9% saline was transferred to a sterile centrifuge tube, and then 150 mg of adenosine was weighed and transferred into the saline to form a stock solution of 3 mg / ml. The solution was mixed by intermittent vigorous vortexing over a period of at least 30 minutes. Subsequently, the solution was filtered into a new 50 mL centrifuge tube, and undissolved adenosine particles were removed using a 0.2 μm sterile syringe filter to obtain a solution containing dissolved monomeric adenosine. The solution was prepared at room temperature and stored refrigerated (2 - 8 °C) after use.

[0128] Preparation of liposome-adenosine suspension: The liposome solution was first prepared as follows. A glass vial containing 100 mg of freeze-dried lipid powder with the appropriate composition was used. Subsequently, each vial of lipid was hydrated by injecting 10 mL of adenosine stock solution (3 mg / ml in saline) and vortexing vigorously. 100 mg of lipid was dissolved in 10 mL of buffer, and a solution with a total lipid of 10 mg / mL was expected. The liposomes formed were expected to be multilamellar, with sizes between 1 - 10 μm, and there might be some larger and smaller liposomes.

[0129] 24-hour in vitro release by dialysis: Preparation of dialysis cassette: The dialysis cassette is used according to the manufacturer's recommended protocol. Briefly, the dialysis cassette is filled with 5 mL of 20% EtOH (a mixture of 200-proof ethanol and DI water in a 1:4 ratio) and allowed to float in a glass beaker containing 500 mL of 20% EtOH for 10 minutes. No stir bar or agitation is used in this step. After that, the dialysis cassette is emptied with a pipette and filled with 5 L of DI water, 500 mL of the volume is discarded and replaced with 500 mL of DI water, and the dialysis cassette is allowed to float in DI water for another 20 minutes (without agitation). After removing 5 mL of DI water, the dialysis cassette is considered suitable for use.

[0130] Preparation of dialysis chamber: A 1 L glass beaker is filled with 500 mL of 0.9% saline as the external buffer. A magnetic stir bar is added to each glass beaker.

[0131] The dialysis cassette is filled with 3 mL of a suitable test solution, either 1) a pure adenosine stock solution or 2) a multilamellar liposome + Ade solution.

[0132] After that, the filled dialysis cassette is placed in a foam floating ring and allowed to float in 500 mL of 0.9% saline, with one dialysis cassette in each 500 mL container. The stirring plate is adjusted to maintain uniform stirring, without splashing and without forming leaks / vortices that may affect the dialysis cassette, and the stirring speed is 250 - 300 rpm. The zero time point is defined as the time when the filled cassette containing the sample is first placed in the beaker and stirring is started.

[0133] Samples of the reflux solution (the solution inside the dialysis cassette) are collected at multiple time points, including first gently aspirating with a 1 mL pipette to mix the solution inside the dialysis cassette, and then removing 50 μL and transferring it to a pre-labeled 1.5 mL Eppendorf tube.

[0134] Analysis of adenosine concentration is carried out by measuring the UV / Vis absorbance at 260 nm using a NanoDrop OneC spectrophotometer (baseline correction at 750 nm is on, automated path length is off). 0.9% saline is used as the blank during the measurement. The UV / Vis measurement is carried out by dropping 2 μL of the sample onto the instrument pedestal, and a total of n = 3 measurements are made for each sample condition time point (3 × 2 μL volume, and the pedestal is wiped with lint-free cloth between each measurement).

[0135] 10-day in vitro release kinetics: The liposome lyophilized product in a sterile glass vial was mixed with a sterile adenosine solution (3 mg / ml in physiological saline) and provided in a pre-filled plastic syringe (customized from Mycoscience Inc.). Samples (100 μl) of the lipid-adenosine suspension were incubated at 37 °C in phosphate-buffered physiological saline for 0, 1, 2 hours, 1, 2, 5, 7, 10 days. At the end of each incubation time, the samples were centrifuged at 23,000×g for 15 minutes at 4 °C. The supernatant was removed and the liposome particles were resuspended in a physiological saline solution containing 0.5% Triton-X100. The adenosine concentration in the remaining intact liposomes was quantified by high-performance liquid chromatography (HPLC).

[0136] Animal research : Rats with established OA received an intra-articular injection of physiological saline (100 μl), while the other 8 groups of animals would receive Ade in 2 different liposomal formulations at doses of 3, 1, 0.33, and 0 mg / ml. The first injection was performed 4 weeks after ACL rupture. The animals received an injection every 10 days for a total of 6 times. Knee joint swelling was measured before each injection as a measure of joint inflammation. Pain tests were performed on the rats at baseline (before the first injection), 30 days after the 3rd injection, and finally at 57 days, i.e., just before sacrifice (7 days after the last injection). Pain tests and motor tests were performed, namely the bipedal balance test (measuring the weight-bearing asymmetry between the ipsilateral and contralateral hindlimbs by a bipedal balance meter) and the rotarod test (the time the rats could continuously run on the rotarod before falling off). The joints after sacrifice were analyzed by histology and uCT.

[0137] 24-hour in vitro release by dialysis: Figure 13 The release of non-liposomal adenosine over time is shown. However, RgnA09 achieved a higher overall dose of 21.86% to 37.90% because the higher retention rate of the drug in the liposomes enabled slow release of liposome-adenosine, as well as some burst releases around 2 hours and 16 hours, achieving higher doses of 25.76% and 37.90%. On the other hand, RgnA10 achieved a higher overall dose of 26.61% to 49.27% because the higher retention rate of the drug in the liposomes enabled slow release of liposome-adenosine, as well as some burst releases around 1 hour, 2 hours, and 3 hours, achieving higher doses of 48.89%, 39.21%, and 29.65%.

[0138] 10-day in vitro release kinetics: Figure 12Shows the percentage of adenosine retention in two liposomal formulations. Adenosine had an initial burst release (RgnA09 was 1096 μM; RgnA10 was 2014 μM). No significant differences were found between the RgnA09 and RgnA10 formulations. Freshly prepared liposome suspensions (time 0) showed an adenosine retention rate of 21% for RgnA09 and 19% for RgnA10. Figure 1 and Figure 4 Showed that after 1 hour of incubation, the retention rate of both formulations decreased to 4%, and decreased slowly over time, reaching 1.4% and 2% (corresponding to RgnA09 and RgnA10 respectively) on day 10, equivalent to 159 μM and 227 μM of adenosine. These results indicate that both liposomal formulations are good reservoirs for encapsulating and slowly releasing adenosine at concentrations sufficient to activate A2A receptors in vivo.

[0139] Animal studies: We further tested the efficacy of the newly developed formulations in a rat model of post-traumatic OA (PTOA). As described above, rats develop OA after ACL rupture. PTOA rats were randomly assigned to the experimental groups. Before starting the experiment, a bipedal stance and pain test were performed. The animals were divided into 10 groups and received 0 (empty liposomes / carrier), 0.3, 1, or 3 mg / ml of adenosine, saline, or the formulations of RgnA09 or RgnA10 as previously described by Corciulo et al. The animals received injections once every 10 days for a total of 6 times. Knee joint swelling was measured before each injection as a measure of joint inflammation. Pain tests were performed on the rats at baseline (before the first injection), at 30 days (midpoint of the treatment protocol), and immediately before sacrifice (7 days after the last injection). The joints after sacrifice were analyzed by histology and μCT. Pain behavior was measured by the weight-bearing asymmetry between the ipsilateral and contralateral hindlimbs using a bipedal balance meter and a rotarod test.

[0140] The animals were placed in a rodent restrainer and stood on their hind paws with their hindlimbs resting on two equally weighted platform pads. When the animals removed their weight from each pad, the device made 3 - 4 consecutive measurements within 12 seconds to record the average weight (grams). The average value for each individual animal was used for analysis. Pain was also measured by the rotarod test, which provides an assessment of motor function in the presence of knee joint pressure and stress. The rats were placed on an accelerating rotarod, and the failure to stay on top of the rod was determined and used for further analysis.

[0141] Based on bipedal balance testing, treatment with intra-articular vehicle and 1 mg / mL and RgnA09, between 0.3 mg and 3 mg of RgnA10, resulted in a significant reduction in pain behavior in the treated animals. Additionally, at 30 days (after 3 injections), we observed a stable dose-response trend for both formulations in reducing joint pain, with all doses of Rgn09 being significantly different from the vehicle, and 3 mg of Rgn10 being different from Rgn01 and the vehicle( Figure 9 ). The rotarod test also showed a dose-response trend and a difference for the highest dose of Rgn10 (3 mg / ml) at 60 days( Figure 14 ). Additionally, we observed significant changes in joint inflammation for both formulations, with some doses showing significant differences from the vehicle and saline after 6 injections. For both formulations at 3 mg / ml, joint inflammation decreased steadily over time( Figure 15 ). The highest dose used for both Rgn09 and Rgn10 was 3 mg / ml.

[0142] Figure 16 Representative safranin-O stained sections of the affected rat tibiae after treatment with vehicle or 3 doses of liposomal adenosine are shown. In vehicle-treated animals, there was a significant reduction in chondral proteoglycans and chondral surface irregularities. In rats treated with RgnA09, there was a dose-dependent improvement in chondral proteoglycans and disappearance of chondral wear, accompanied by an increase in surface cartilage. In rats treated with RgnA10, the effect was strongest in those treated with the highest dose (3 mg / ml), although chondral protection was also observed at lower doses.

[0143] Conclusion: The new formulations of liposomal adenosine are equally effective, and even more effective, in relieving pain and swelling in OA knees and, at the same time, protecting and enhancing cartilage. Both formulations are effective, and the in vitro release of adenosine from multilamellar vesicles is superior to non-liposomal or free adenosine.

[0144] Example 5

[0145] This example describes a method of using the liposomes of the present disclosure.

[0146] Storage-stable formulations RgnA09 and RgnA10: A series of storage-stable formulation options were developed and evaluated based on lipid components, dissolution efficiency, and retention characteristics. RgnA09 and RgnA10 can be rehydrated in a concentrated solution of hydrophilic adenosine. During the rehydration process, multilamellar liposome particles are generated that contain adenosine in the aqueous compartment of the liposome. The liposome size is ∼10 - 100 microns and is metastable, so it will collapse into a dense form at high temperatures (∼40 °C). When the liposomes are confined within a locally enclosed compartment, such as the bursa of the human knee joint synovium, sustained release of adenosine can be achieved. RgnA09 and RgnA10 were tested to evaluate their ability to bind and release adenosine over time. The liposome freeze-dried product in a sterile glass vial was mixed with a sterile adenosine solution (3 mg / mL, in physiological saline) and provided in a pre-filled plastic syringe (customized from Mycoscience Inc.). Samples (100 μL) of the lipid-adenosine suspension were incubated at 37 °C in phosphate-buffered saline for 0, 1, 2 hours, 1, 2, 5, 7, 10 days. At the end of each incubation time, the samples were centrifuged at 23,000 × g for 15 minutes at 4 °C. The supernatant was removed, and the liposome particles were resuspended in a physiological saline solution containing 0.5% Triton-X100. The adenosine concentration in the remaining intact liposomes was quantified by high-performance liquid chromatography (HPLC). Figure 1 , 4 and 12 show the percentage of adenosine retention in the two liposomal formulations. There is an initial burst release of adenosine (1096 μM for RgnA09; 2014 μM for RgnA10). No significant difference was found between the RgnA09 and RgnA10 formulations. The freshly prepared liposome suspension (time 0) showed an adenosine retention rate of 21% for RgnA09 and 19% for RgnA10. After 1 hour of incubation, the retention rate of both formulations decreased to 4% and slowly decreased over time, reaching 1.4% and 2% on day 10 (corresponding to RgnA09 and RgnA10, respectively), equivalent to 159 μM and 227 μM of adenosine. These results indicate that both liposomal formulations are good reservoirs for encapsulating and slowly releasing adenosine at concentrations sufficient to activate A2A receptors in vivo.

[0147] The efficacy of the newly developed formulation was further tested in a post-traumatic OA (PTOA) rat model. As described above, rats develop OA after ACL rupture. PTOA rats were randomly assigned to the experimental groups. Before starting the experiment, a bipedal balance and pain test was performed. The animals were divided into 10 groups and received 0 (empty liposome / carrier), 0.3, 1, or 3 mg / ml of adenosine, saline, or the formulation, such as previously described by Corciulo et al., of RgnA09 or RgnA10. The animals received injections every 10 days for a total of 6 times. Knee joint swelling was measured before each injection as a measure of joint inflammation. Pain tests were performed on the rats at baseline (before the first injection), at 30 days (midpoint of the treatment protocol), and immediately before euthanasia (7 days after the last injection). The joints after euthanasia were analyzed by histology and uCT. Pain behavior was measured by the weight-bearing asymmetry between the ipsilateral and contralateral hindlimbs using a bipedal balance meter ( Figure 9 ).

[0148] After the hyperalgesia test, the animals were placed in a rodent restraint device and stood on their hind paws with their hindlimbs resting on two weight-averaging platform pads. When the animals removed their weight from each pad, the device made 3 - 4 consecutive measurements within 12 seconds to record the average weight (grams). The average value for each animal was used for analysis. Motor ability was also measured by a rotarod test, which provided an assessment of motor function in the presence of knee joint pressure and stress. The rats were placed on an accelerating rotarod, and the failure to stay on top of the rod was determined and used for further analysis (data not shown). Based on the bipedal balance test, there was a strong dose interaction between the carrier and 1 mg / mL for RgnA09, and between 0.3 mg and 3 mg for RgnA10. Additionally, at 30 days (after 3 injections), we observed a stable dose-response trend for both formulations in reducing joint pain. All doses of Rgn09 were significantly different from the carrier, while 3 mg of Rgn10 was different from Rgn01 and the carrier. The rotarod test also showed a dose-response trend and a difference for the highest dose of Rgn10 (3 mg / ml) at 60 days. Additionally, we observed significant changes in joint inflammation for both formulations, with some doses showing significant differences from the carrier and saline after 6 injections. For both formulations at 3 mg / ml, there was a stable decrease in joint inflammation over time ( Figure 15 ). Rgn09 and Rgn10 can be used at a maximum dose of 3 mg / ml.

[0149] Although the invention has been described with respect to one or more particular embodiments, it should be understood that other embodiments of the invention may be practiced without departing from the scope of the invention.

Claims

1. An injectable preparation comprising physiological saline and liposomes, said liposomes comprising one or more lamellae, wherein, the liposome lamellae comprise 70 - 100% by mass of sphingomyelin, and when the sphingomyelin is less than 100% by mass, the remainder is 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG), wherein the ratio of DMPC to DMPG is from about 6:4 to about 8:2, wherein the liposomes (a) have a diameter of 50 nm - 150 μm; and (b) encapsulate adenosine in the aqueous compartment of the liposome.

2. The injectable preparation according to claim 1, wherein, the liposomes are in a metastable state.

3. The injectable preparation according to claim 1, wherein, the release of adenosine or a portion thereof lasts up to two weeks.

4. The injectable preparation according to claim 1, further comprising an excipient.

5. The injectable preparation according to claim 1, wherein, the preparation is suitable for intra-articular injection.

6. The injectable preparation according to claim 1, wherein, the adenosine concentration is 0.1 - 7 mg / mL.

7. The injectable preparation according to claim 5, wherein, the adenosine concentration is 0.1 - 4 mg / mL.

8. The injectable preparation according to claim 1, wherein, the total lipid concentration is 7 - 12 mg / mL.

9. The injectable preparation according to claim 1, wherein, the diameter of one or more liposomes is 50 nm - 100 μm.

10. The injectable preparation according to claim 6, wherein, the diameter of one or more liposomes is 100 nm - 150 μm.

Images