Adenosine-encapsulated liposomes

Abstract

A liposome encapsulating adenosine is provided herein. Liposomes can be formed by a combination of sphingomyelin or 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. Liposomes encapsulating adenosine can be used to induce cartilage regeneration, treat osteoarthritis, relieve joint pain, and / or slow down, prevent and / or reverse the progressive structural tissue damage associated with osteoarthritis, or treat osteoarthritis, rheumatoid arthritis, acute gouty arthritis and / or synovitis. Liposomes can release adenosine for up to two weeks.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

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

[0003] Osteoarthritis (OA), a disease characterized by cartilage loss, 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, previous trauma, obesity, and genetics are among 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 a 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 knees, hips, and hands. OA has the highest incidence in the knee, both in women (47%) and men (40%). Current treatment options are unsatisfactory and do not address the underlying problem. Treatment is mostly palliative and includes the use of nonsteroidal anti-inflammatory drugs (such as ibuprofen), narcotic analgesics, exercise, and acupuncture. The FDA has also approved a number of specific treatments for OA, including corticosteroids (anti-inflammatory agents) and hyaluronic acid (lubricant, analgesic), all of which are administered via intra-articular (IA) injections. Although these injectables can relieve symptoms, none are restorative.

[0004] The purinergic system plays a key role in maintaining cartilage homeostasis. Adenosine, acting at its A2A receptor (A2AR), is an important autocrine homeostatic factor in maintaining chondrocyte and cartilage homeostasis. Adenosine is an endogenous physiological regulator whose intracellular and extracellular concentrations are tightly controlled by oxygen consumption, cellular stress, and mitochondrial function. Extracellular adenosine is derived primarily from the hydrolysis of ATP (primarily but not exclusively through the exoenzymes CD39 and CD73), and mediates its effects through the activation of G protein-coupled receptors (A1R, A2AR, A2BR, and A3R). These adenosine receptors are highly conserved evolutionarily, and their expression and function also tend to be conserved. Adenosine has long been known to modulate 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 also regulate chondrocyte physiology and pathology in response to inflammatory stimuli in rodent, horse, bovine, and human chondrocytes, but the specific receptors involved have not been identified. Although purine metabolism in horses differs from that in other species, removal of endogenous adenosine (by addition of adenosine deaminase) or blockade of A2ARs results in cartilage degradation in horse cartilage explants because adenosine deaminase, which is present in lymphocytes, plasma, and extracellular fluids of most species, is not present in lymphocytes or serum of horses. In a chemically induced OA model, stimulation of A3Rs has been reported to reduce the progression of OA, primarily due to the anti-inflammatory effects 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 preparation. The present disclosure also discloses methods for making and using the injectable preparation. The injectable preparation comprises liposomes and physiological saline, wherein the liposomes are metastable and encapsulate adenosine.

[0006] BRIEF DESCRIPTION OF THE DRAWINGS

[0007] For a fuller understanding of 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 adenosine retention by liposomes formed with RgnA09.

[0009] Figure 2 Shown are microscopic images of liposome suspensions formed by RgnA09.

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

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

[0012] Figure 5Shown are microscopic images of liposome suspensions formed by RgnA10.

[0013] Figure 6 Shown is a histogram of the approximate diameters of liposomes formed by RgnA10.

[0014] Figure 7 Shown is a pre-liposomal lyophilisate.

[0015] Figure 8 Shown is a microscope image of a prior art liposome suspension. The liposome suspension contains clear evidence of crystallized adenosine and the spherical objects are believed to be oil.

[0016] Fig. 9 Shown are pain data recorded using the incapacitance test.

[0017] Fig.10 Shown is an HPLC chromatogram of the material isolated in Example 1.

[0018] Fig.11 Shown Fig.10 UV spectra of the separated materials.

[0019] Fig.12 Shown is the initial burst release of RgnA09 and RgnA10.

[0020] Fig.13 Shown are the adenosine release kinetics of (left) RgnA09-MLV and (right) RgnA10-MLV over 24 hours.

[0021] Fig.14 Shown are rotarod pain tests using (left) RgnA09 and (right) RgnA10 at 60 days (after six injections).

[0022] Fig.15 Shown are the effects of 6 injections of (A) RgnA09 and (B) RgnA10 at different concentrations on joint inflammation. Administration was ipsilateral-contralateral. Statistics: One-way (Brown-Forsythe and Welch) ANOVA. *P<0.05 v / s vehicle, P<0.05v / s normal saline, +P<0.05v / s 0.3mg and 1mg.

[0023] Fig.16Shown are representative Safranin-O stained sections of affected rat tibiae after treatment with vehicle or 3 doses of liposomal adenosine. In vehicle treated animals, cartilage proteoglycans and cartilage surface irregularities were significantly reduced. In RgnA09 treated rats, there was a dose-dependent improvement in cartilage proteoglycans and a disappearance of cartilage wear, with an increase in surface cartilage. In RgnA10 treated rats, the effect was greatest 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 substance will be described in terms of specific embodiments, other embodiments, including embodiments that do not provide all of the advantages and features set forth herein, are also within the scope of the present disclosure. Various structural, logical, and process step changes may be made without departing from the scope of the present disclosure.

[0025] Unless otherwise indicated, all ranges provided herein include all values ​​within that range, to the tenth decimal place.

[0026] The present disclosure provides an injectable formulation. Also disclosed herein are methods of making and using the injectable formulation.

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

[0028] Liposomes may include i) sphingomyelin or ii) 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 multiple examples, liposomes include 70-100% by mass of sphingomyelin. Liposomes including less than 100% by mass of sphingomyelin may further include up to 30% by mass (such as the rest) of DMPC or DMPG or a combination of DMPC and DMPG. In one embodiment, liposomes may include 70-99.9% by mass of sphingomyelin and 0.1-30% by mass (such as the rest) of DMPC or 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) or a combination of DMPC and DMPG. In one embodiment, liposomes include 75-100% by mass of sphingomyelin. The liposome comprising the sphingomyelin less than 100 mass % can further comprise the DMPC or DMPG or the combination of DMPC and DMPG up to 25 mass % (such as the remainder). In one embodiment, liposome comprises the sphingomyelin of 75-99.9 mass % and the DMPC or DMPG or the combination of DMPC and DMPG of 0.1-25 mass % (such as the remainder). For example, liposome can comprise 75,80,85,90,95,96,97,98,99 and 99.9% sphingomyelin, and the remainder is DMPC, DMPG or its combination.Mass percent refers to the gross mass of phospholipid.

[0029] The diameter and / or average diameter of the liposomes can be 50 nm-150 μm, including all values ​​of 0.1 nm and ranges therebetween (e.g., 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 50nm-1 μm, 50nm-750 μm, 50-500nm, 50-250nm, 50-100nm, 100nm-1 μm, 100-750nm, 100-500nm, 100-250nm, 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, no liposomes have a diameter greater than 150 μm. In one embodiment, less than 1% of the liposomes have a diameter greater than 150 μm. In various embodiments, liposomes can be produced by ethanol injection, and the resulting liposomes can be smaller than liposomes formed by other methods.

[0030] Before releasing adenosine, the liposomes of the present disclosure may be in a metastable state. Metastable liposomes can provide enhanced release due to greater stability at the delivery site. Metastable liposomes a) have a relative diameter not equal to 1 (e.g., metastable liposomes are not perfectly round or spherical); b) are large enough so that the expansion stress associated with membrane bending is insufficient to overcome the conformational equilibrium tendency of the liposomes; and c) the longest linear dimension (e.g., diameter) is 100nm-150μm, including each 0.1nm value and range therebetween. Such liposomes collapse (e.g., shrink or contract) into a smaller stable form when subjected to a temperature of 35-45°C (including all 0.1°C values ​​and ranges therebetween) (e.g., 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45°C) (e.g., about (or slightly above) 40°C). In one embodiment, the longest linear dimension (such as diameter) of the smaller stable liposome is 50nm-110μm, including each 0.1nm value and range therebetween. In addition, the volume enclosed by the liposome at 25°C is greater than 10 relative to the volume enclosed by the liposome after heating to a temperature exceeding the gel-fluid phase transition of one or more lipids forming the liposome. Metastable liposomes containing hydrophilic agents can collapse at 35-45°C (including all 0.1°C values ​​and 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 cargo (such as adenosine) during such collapse (such as contraction or shrinkage). Metastable liposomes are described in U.S. Patent Publication No. 2016 / 0263031 (for ease of reference, the relevant parts thereof are incorporated herein). Metastable liposomes can be referred to as liposomes.

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

[0032] Liposomes are formed from one or more lipids, which can be neutral, anionic or cationic at physiological pH. Examples of lipid types include, but are not limited to, sterols and lipids such as cholesterol, phospholipids, lysolipids, lysophospholipids, sphingosine lipids 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) (e.g., egg PC, soybean 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 phosphatidylglycerols 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 sphingomyelin and DMPC or DMPG, or a combination thereof. The total lipid concentration can be 7-12 mg / mL, including all 0.01 mg / mL values ​​and 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 comprises sphingomyelin, DMPC and DMPG, and the ratio of DMPC to DMPG is 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 may contain water and adenosine. The adenosine concentration may be 0.1-7 mg / mL, including all values ​​of 0.01 mg / mL and ranges therebetween. In one embodiment, the adenosine concentration may be 0.1-4 mg / mL. In one embodiment, the adenosine concentration is 3 mg / mL.

[0035] Methods for making metastable liposomes are described herein. In one embodiment, dehydrated metastable liposomes are prepared from homogeneous dispersions of phospholipids, preferably sphingomyelin, in a water / tert-butyl alcohol (TBA) cosolvent system, with mg phospholipids relative to mL water / tert-butyl alcohol in a ratio of 2:1. Various ratios of water to TBA (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)) can be used. The isotropic single-phase liposome solution is freeze-dried to produce dehydrated liposome powder in a sterile vial. After removing water and TBA from the vial, the freeze-drying step produces empty lipid vesicles or dehydrated liposomes. Add a pharmaceutically acceptable carrier (e.g., water, saline or PBS), and the freeze-dried product will spontaneously form a large metastable liposome dispersion. The ratio of lipid to TBA is an important factor affecting the size and polydispersity of the resulting liposome preparations.

[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 co-solvent system, wherein the volume ratio of water to TBA is 1: 1. For example, for a solution comprising 100 mg of phospholipids, 1-50 mL (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 25, 30, 40, or 50 mL) of a co-solvent system having a volume ratio of TBA: water of 1: 1 is used. The multiple phospholipids can be a mixture of 75% sphingomyelin (by mass) and 25% PC / PG mixture (by mass), wherein the PC / PG mixture includes 70% (by mass) DMPC and 30% (by mass) DMPG (e.g., in the total multiple phospholipids containing sphingomyelin and PC / PG mixture, 70% (by mass) of the multiple phospholipids are sphingomyelin, 17.5% (by mass) of the multiple phospholipids are DMPC, and 7.5% (by mass) are DMPG). The obtained solution containing multiple phospholipids is freeze-dried to produce a dehydrated liposome powder in a sterile vial. Thereafter, the lyophilizate (e.g., 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 (e.g., a physiological saline containing adenosine, wherein the concentration of adenosine is 0.1-7 mg / mL (e.g., 3 mg / mL)) is used to rehydrate the lyophilizate).

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

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

[0039] Liposome-adenosine suspensions can be prepared by the methods disclosed herein. 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 a physiological saline solution (such as 0.9 mass % sodium chloride (9 mg NaCl per mL 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 (e.g., diameter) of the adenosine-containing liposomes can be 50 nm-150 μm, including all values ​​of 0.1 nm and ranges therebetween (e.g., 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 dosage depends on the desired therapeutic effect, the route of administration and the desired treatment time. The general dosage level for mammals (such as individuals) is 0.001-10 mg / kg body weight per day. Generally speaking, the dosage for intravenous injection or infusion can be lower.

[0041] The composition can also be administered orally, parenterally (intramuscularly, intraperitoneally, intravenously (IV) or subcutaneously), transdermally (passive or using iontophoresis or electroporation) or transmucosally (nasal, vaginal, rectal or sublingual), and can be formulated into dosage forms suitable for each route of administration. In one embodiment, the formulation is injected directly into a joint of the individual.

[0042] The metastable liposomes containing adenosine disclosed herein have several advantages. For example, the metastable liposomes can slowly release adenosine, thereby extending the biological activity of the delivered adenosine and / or reducing the required dosage.

[0043] Dosage units of metastable liposome preparations of different sizes can be used. Dosage units containing a dry powder of a dehydrated metastable pre-liposome lyophilizate or an aqueous solution of adenosine or other hydrophilic active agent 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, and more preferably 0.05 mg / mL-3.5 mg / mL.

[0044] The injectable formulations disclosed herein can be used to induce cartilage regeneration, treat osteoarthritis, relieve joint pain, and / or slow down, prevent and / or reverse the progressive structural tissue damage associated with osteoarthritis in an individual in need of treatment. In one example, the individual may suffer from or be suspected of suffering from osteoarthritis, rheumatoid arthritis, acute gouty arthritis and / or synovitis. The method of inducing cartilage regeneration, treating osteoarthritis, alleviating joint pain and / or slowing down, preventing and / or reversing the progressive structural tissue damage associated with osteoarthritis in an individual in need of treatment includes administering the injectable formulations disclosed herein to an individual in need of treatment.

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

[0046] After administration to a person in need of treatment, adenosine is released from the liposomes for up to 2 weeks. In one embodiment, after administration of the injectable formulation, adenosine is released from the liposomes within 1 second to 1 hour (e.g., 1 minute to 1 hour) after administration to the individual. In one embodiment, at least a portion of the adenosine (e.g., 1-20% of the adenosine) is released from the liposomes within 1 minute to 1 hour after administration to the individual. In one embodiment, at least a portion of the adenosine (e.g., 1-20% of the adenosine) is released from the liposomes 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 to a joint of an individual by intra-articular injection. The injectable formulation can be administered in one or more injections. The formulation can be administered multiple times (e.g., 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 perform the method of the present invention. Therefore, in one embodiment, the method is substantially composed 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 formulation (e.g., an injectable formulation) comprising saline and one or more liposomes, wherein the one or more liposomes comprise one or more lamellae (e.g., one or more multilamellar liposomes), wherein the liposome lamellae comprise 70-100% by weight of sphingomyelin, and when the sphingomyelin is less than 100% by weight, the remainder is (e.g., up to 30% by weight) 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) The diameter of the liposome is 50nm-150μm, including all 0.1nm values ​​and ranges therebetween (such as 50nm-1μm, 50nm-750μm, 50-500nm, 50-250nm, 50-100nm, 100nm-1μm, 100-750nm, 100-500nm, 100-250nm, 1-150μm, 1-100μm, 1-50μm, 1-40μm, 1-30μm, 1-25μm, 1-20μm, 1-10μm, 1-5μm); and (b) encapsulating adenosine in the aqueous compartment of the liposome. The diameter of one or more liposomes is 50nm-100μm. The diameter of one or more liposomes is 100nm-150μm.

[0051] Statement 2. A formulation (eg, an injectable formulation) as described in Statement 1, wherein the liposomes are in a metastable state.

[0052] Statement 3. The formulation (eg, an injectable formulation) of Statement 1, wherein adenosine or a portion thereof is released for up to two weeks, or after administration to a joint of a subject, adenosine or a portion thereof is released for up to two weeks.

[0053] Statement 4. A formulation (eg, an injectable formulation) as described in any of the preceding statements, further comprising an excipient.

[0054] Statement 5. The formulation (eg, an injectable formulation) of any of the preceding statements, wherein the adenosine concentration is 0.1-7 mg / mL.

[0055] Statement 6. The formulation (eg, an injectable formulation) of Statement 5, wherein the adenosine concentration is 0.1-4 mg / mL.

[0056] Statement 7. A formulation (eg, an injectable formulation) as described in any of the preceding statements, wherein the ratio of DMPC to DMPG is from 6:4 to 8:2.

[0057] Statement 8. A formulation (such as an injectable formulation) as described in Statement 7, wherein the ratio of DMPC to DMPG is 7:3.

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

[0059] Statement 10. A formulation (e.g., an injectable formulation) as described in any of the preceding statements, wherein the liposomes collapse (e.g., shrink or shrink) at a temperature of 35-45°C, including all values ​​of 0.1°C and ranges therebetween (e.g., 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45°C) (e.g., about 40°C, to release their cargo).

[0060] Statement 11. The formulation (eg, an injectable formulation) of any of the preceding statements, wherein adenosine is released to the subject's joints within 1 second to 1 hour (eg, 1 minute to 1 hour) after administration.

[0061] Statement 12. The formulation (eg, an injectable formulation) of Statement 11, wherein at least a portion of the adenosine is released to the joints of the subject within 1 second to 1 hour (eg, 1 minute to 1 hour) after administration.

[0062] Statement 13. The formulation (eg, an injectable formulation) of Statement 11 or Statement 12, wherein at least 1-20% of the adenosine is released to the subject's joints within 1 second to 1 hour (eg, 1 minute to 1 hour) after administration.

[0063] Statement 14. A formulation (e.g., an injectable formulation) as described in any of Statements 11-13, wherein at least a portion of the adenosine or at least 1-20% of the 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 a joint of an individual.

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

[0065] Statement 16. A method of reducing joint pain in an individual in need of treatment, comprising administering to the individual a formulation (eg, an injectable formulation) as described in any one of Statements 1-14.

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

[0067] Statement 18. The method of any one of Statements 15-17, wherein the formulation (eg, injectable formulation) is administered to a joint of the individual by intra-articular injection.

[0068] Statement 19. The method of any of Statements 15-18, wherein the injectable formulation is administered as one or more injections.

[0069] Statement 20. The method of any of Statements 15-19, wherein the injectable formulation is administered multiple times (eg, up to 10 times) every 10 days.

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

[0071] Statement 22. The method of any of Statements 15-21, wherein the subject is a human or a non-human mammal. Examples of non-human mammals include, but are not limited to, agricultural animals (e.g., farm animals) (e.g., cows, pigs, sheep, etc.) and pets, service or sports animals (e.g., horses, dogs, cats, etc.). Other non-limiting examples of subjects include rabbits, rats, and mice.

[0072] The following examples are intended to illustrate the present 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] Because the half-life of adenosine is only a few seconds, the liposome-adenosine used in the above-mentioned studies was freshly prepared every day. A series of storage-stable formulation schemes based on lipid components, dissolution efficiency and retention characteristics were developed and evaluated. It was determined whether cholesterol / stabilizers should be included and these variables were optimized. The percentage of liposome-bound adenosine in the total formulation was measured, which can 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 relative 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] condition

[0078]

[0079]

[0080] Data analysis

[0081]

[0082] 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 saline) provided by a prefilled plastic syringe. Samples (100 μL) of the lipid-adenosine suspension were incubated in phosphate-buffered saline at 37°C for 0, 1 hour, 2 hours, 1 day, and 2, 5, 7, and 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 saline solution containing 0.5% Triton-X100. The adenosine concentration in the remaining intact liposomes was quantified by high performance liquid chromatography (HPLC).

[0083] Figure 1 and Figure 4 The percentage retention of adenosine in the two liposome formulations is shown. No significant differences were found between RgnA09 and RgnA10. A slightly higher retention was observed for RgnA09 at time point zero, and the retention increased over time points. Freshly prepared liposome suspensions (time 0) showed 21% and 19% adenosine retention for RgnA09 and RgnA10, respectively. After 1 hour of incubation, the retention for both formulations dropped to 4% and slowly decreased over time, reaching 1.4% and 2% (for RgnA09 and RgnA10, respectively) on day 10, equivalent to 159 μM and 227 μM adenosine.

[0084] These results indicate that both liposomal formulations are good depots for encapsulating and slowly releasing adenosine at concentrations sufficient to activate the A2A adenosine receptor in vivo.

[0085] Pre-liposome lyophilisates can be rehydrated in concentrated solutions of hydrophilic pharmacological agents, such as adenosine. During the rehydration process, multilamellar liposome particles are produced, which contain the hydrophilic agent in the aqueous compartment of the liposomes. The particles are "large", about 30 microns, and metastable, so they collapse into a dense form under high heat conditions (about 40° C.). On the other hand, if these particles are confined to a locally closed compartment, such as the bursa of the synovial membrane of the human knee joint, they can achieve a sustained release of the pharmacological agent. Therefore, the contained hydrophilic agent is not affected by degrading enzymes in a physiological environment.

[0086] The pre-liposome lyophilisate producing these large metastable multilamellar lipid particles can be manufactured by dissolving lipids (including at least 50% sphingomyelin) and up to 50% of the non-sphingomyelin phospholipid choline in a mixture of water-tert-butyl alcohol (60:40 v / v) before lyophilization. 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) are used as non-sphingomyelins in a ratio of 70%:30%.

[0087] Example 2

[0088] This example describes the liposomes of the present disclosure.

[0089] The laboratory preparation of liposome-adenosine provided was analyzed using crossed polarization microscopy. The results are shown in Figure 8 .

[0090] When viewed under a microscope, the liposomal suspension showed evidence of clear crystallized adenosine. It is believed that the liposomal suspension may not contain a significant amount of liposomal content and is primarily an emulsion. The spherical objects in the image are likely oil. The composition of the formulation contains a significant proportion (60%) of soybean oil, which would not be a component of the liposomal formulation, but would be a component of the emulsion.

[0091] Consider the solubility of adenosine in water, which is 7 mg / mL. The laboratory preparation calls for adding 300 mg of adenosine to 10 mL of saline. It is assumed 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 preparation are not passed through HPLC because each time the particles are washed, their size decreases until nothing remains. We have reviewed a publication using this preparation and found that only the supernatant was analyzed by HPLC. The publication states that 73% of the adenosine is 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 70 mg of adenosine being dissolved in 10 mL of saline at its maximum solubility in water, with the remaining 230 mg of adenosine crystallized in the particles. It is assumed that some liposomes containing adenosine are formed, but it is uncertain whether they are present in this preparation.

[0092] An analysis of the pre-liposomal lyophilisate technique for preparing liposomal adenosine was performed to study the entrapment of adenosine. The advantage of this technique is that it is a stable and sterile process, ideal for drug preparation. The technique involves reconstituting a sterile pre-liposomal lyophilisate in the presence of an active agent. Figure 7 A photograph of a pre-liposomal lyophilisate is shown.

[0093] The pre-liposomal lyophilisate appears as a white fluffy powder in a sterile vacuum vial. The rehydration process involves injecting the concentrated adenosine solution into the vial. The powder then dissolves within 30 seconds, sometimes instantly, and gentle swirling may be required. The resulting liposomal suspension can then be removed from the vial via a syringe. It should be noted that since the vial is in a vacuum state, the adenosine solution will be quickly absorbed by the vial after the syringe is inserted. The ideal concentration of adenosine solution for rehydration is its maximum solubility in water, which is 7 mg / mL. Sterile water for injection (SWFI) is used as the diluent, but normal saline and buffered solutions can also be used. A micrograph of the resulting liposomal suspension is shown in Figure 5 .

[0094] The average diameter of each particle was about 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 microscope images above are of dilutions so that individual liposome particles can be observed.

[0095] An HPLC method for measuring adenosine and lipid content was prototyped. The liposome preparation was centrifuged and the particles were separated from the supernatant. The particles were completely dissolved with methanol. The lipid portion of the liposomes is relatively insoluble in acetonitrile, while adenosine is soluble in acetonitrile. Therefore, the HPLC method included an initial mobile water / acetonitrile phase to first wash the adenosine out of the HPLC column (C18 column), followed by a methanol mobile phase to wash out the lipids. One standard was run for each reagent: adenosine and lipid. The dissolved particles were put into the run and the results were compared to those standards. The particle chromatogram is shown in Fig.10 .

[0096] The retention time of adenosine in this method is 1.42 minutes, while the retention time of lipids is 9.75 minutes. The wavelengths at which the two analytes are detected are also different: 260 nm for adenosine and 203 nm for lipids. Fig.11 These spectra are shown.

[0097] The lipid [SM] standard was 4 mg / mL and the adenosine [ADO] standard was 1 mg / mL. The peak areas generated by the standard run are as follows.

[0098]

[0099] In place of a calibration curve, consider the following.

[0100] [SM] = SM / 22,465 / 4 = SM / 5,616

[0101] [ADO] = ADO / 25,338

[0102] Afterwards, the particles were tested in triplicate and the results of the two peaks were as follows.

[0103]

[0104] The functional relationship between the ratio of adenosine in the particles (defined as [ADO] / ([ADO]+[SM])) and the ratio of the chromatographic peak areas (ADO / SM) can be obtained as follows.

[0105] [ADO] = ADO / 25,338

[0106] [SM]=SM / 5616

[0107]

[0108]

[0109]

[0110] When applied to the chromatogram results, the following values ​​were obtained.

[0111]

[0112] Example 3

[0113] This example describes the method of using the injectable formulation of the present disclosure.

[0114] Rats with established OA received intra-articular injections of saline (100 μL), while the other 8 groups of animals received 2 different liposomal formulations of Ade at doses of 3, 1, 0.3, and 0 mg / mL. The first injection was performed 4 weeks after ACL rupture. Animals received injections every 10 days for a total of 6 times. Knee swelling was measured before each injection as a measure of joint inflammation. Rats were tested for pain at baseline (before the first injection), 5 days after the third injection, and finally at 57 days, just before sacrifice (7 days after the last injection). Post-sacrifice joints were analyzed by histology and uCT.

[0115] 10 treatment groups

[0116] 2 preparations x 4 doses = 8 treatment groups

[0117] 1 positive control (Rgn01)

[0118] 1 negative control (normal saline)

[0119] 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", Nature Communications (Nat Commun.), 2017 May 11;8:15019.

[0120] Liposomes were freshly prepared one day before injection. Ethanol was added to soybean oil containing adenosine or adenosine plus adenosine receptor antagonists. A lipid phase containing phosphatidylcholine and cholesterol (molar ratio of 1:0.5) was added to the previous solution and emulsified at 15,000 rpm for 10 minutes. Afterwards, physiological saline and glycerol were added to the lipid phase together and homogenized at 15,000 rpm for 20 minutes, followed by sonication at 100% duty cycle for 1 minute.

[0121] PTOA rats were randomly assigned to experimental groups. 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 performed by measuring the weight-bearing asymmetry between the ipsilateral and contralateral hindlimbs with a bipedal balance meter. After the hyperalgesia test, the animals were placed in a rodent restrainer and allowed to stand with their hind paws. The hind limbs rested on two weight-averaged platform pads. When the animals removed their weight from each pad, the device performed 3-4 continuous measurements within 12 seconds to record the average weight (grams). The mean value of each animal was used for statistical analysis.

[0122] Example 4

[0123] This example describes the method of preparing the liposomes of the present disclosure and the release kinetics of the liposomes of the present disclosure.

[0124] Preparation of pre-liposomal lyophilisate:

[0125] condition

[0126]

[0127]

[0128] The total content of all vials is 100 mg, the fill volume is 5 mL, and the fill concentration is 20 mg / ml solvent.

[0129] For RgnA09, 75 mg SM, 17.5 mg DMPC and 7.5 mg DMPG were dissolved in 5 mL of a 1:1 (volume ratio) mixture of water and tert-butyl alcohol (TBA). The solution was lyophilized according to the following parameters (first frozen at -40°C for 30 minutes, followed by primary drying at 10°C for 20 hours under 200 micro vacuum, followed by secondary drying at 20°C for 4.5 hours) and kept in a vacuum-sealed vial. Afterwards, the lyophilized material was rehydrated with 40 mg of purified water at room temperature (25°C).

[0130] For RgnA10, 100 mg of pure sphingomyelin (SM) was dissolved in 5 mL of a 3:2 (volume ratio) mixture of water and tert-butyl alcohol (TBA). The solution was lyophilized according to the following parameters (first freezing at -40° for 30 minutes, followed by primary drying at 10° for 20 hours under 200 micro vacuum, followed by secondary drying at 20° for 4.5 hours) and kept in vacuum-sealed vials. Afterwards, the lyophilizate was rehydrated with 40 mg of pure water at room temperature (25°).

[0131] Preparation of adenosine stock solution: Adenosine stock solution was prepared by dissolving pure adenosine powder into saline (0.9% saline). 50 mL of 0.9% saline was transferred to a sterile centrifuge tube, after which 150 mg of adenosine was weighed and transferred into the saline to form a 3 mg / ml stock solution. The solution was mixed by intermittent vigorous vortexing over a period of at least 30 minutes. Afterwards, 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.

[0132] Preparation of liposome-adenosine suspension: Liposome solutions were first prepared as follows, using glass vials containing 100 mg of lyophilized lipid powder of appropriate composition. Each vial of lipid was then hydrated by injecting 10 mL of adenosine stock solution (3 mg / ml in saline) into the vial and vortexing vigorously. Dissolving 100 mg of lipid in 10 mL of buffer was expected to produce a solution with 10 mg / mL total lipid. The liposomes formed were expected to be multilamellar and between 1-10 μm in size, with some larger and smaller liposomes possible.

[0133] 24-hour in vitro release by dialysis: Preparation of dialysis cassettes: The dialysis cassettes were used according to the manufacturer's recommended protocol. Briefly, the dialysis cassettes were filled with 5 mL of 20% EtOH (200-proof ethanol mixed with DI water in a ratio of 1:4) and allowed to float in a glass beaker containing 500 mL of 20% EtOH for 10 minutes. No stirring bar or stirring was used in this step. Afterwards, the dialysis cassette was emptied with a pipette and filled with 5 L of DI water, 500 mL of the volume was discarded and replaced with 500 mL of DI water, and the dialysis cassette was allowed to float in DI water for another 20 minutes (without stirring). Afterwards, the dialysis cassette was considered suitable for use after removing 5 mL of DI water.

[0134] Prepare the dialysis chambers: Fill 1 L glass beakers with 500 mL of 0.9% saline as the external buffer. Add a magnetic stir bar to each glass beaker.

[0135] Dialysis cassettes were filled with 3 mL of the appropriate test solution, either 1) pure adenosine stock solution, or 2) multilamellar liposomes + Ade solution.

[0136] Afterwards, place the packed cassettes in a foam float and allow them to float in 500mL of 0.9% saline, with one cassette in each 500mL container. Adjust the stirring plate to maintain uniform stirring, without splashing, and without forming leaks / vortices that may affect the cassettes, and the stirring speed is 250-300rpm. The zero time point is determined as the time when the packed cassette with sample is first placed in the beaker and stirring begins.

[0137] The retentate (the solution in the cassette) was sampled at various time points by first mixing the solution in the cassette by gentle pipetting with a 1 mL pipette, followed by removal of 50 μL and transfer to a pre-labeled 1.5 mL Eppendorf tube.

[0138] Adenosine concentrations were analyzed using a NanoDrop OneC spectrophotometer measuring UV / Vis absorbance at 260 nm (baseline correction at 750 nm on, automated path length off). 0.9% saline was used as a blank. UV / Vis measurements were performed with 2 μL of sample dripped onto the instrument base, with a total of n=3 measurements per sample condition time point (3×2 μL volumes, base wiped with lint-free cloth between each measurement).

[0139] 10-day in vitro release kinetics: Liposome lyophilisates in sterile glass vials were mixed with sterile adenosine solution (3 mg / ml in saline) and provided in pre-filled plastic syringes (custom-made from Mycoscience Inc.). Samples (100 μl) of the lipid-adenosine suspension were incubated in phosphate-buffered saline at 37°C for 0, 1, 2 hours, 1, 2, 5, 7, and 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 saline solution containing 0.5% Triton-X100. The adenosine concentration in the remaining intact liposomes was quantified by high performance liquid chromatography (HPLC).

[0140] Animal studies : Rats with established OA receive intra-articular injections of saline (100 μl), while the other 8 groups of animals will receive 2 different liposomal formulations of Ade at doses of 3, 1, 0.33, and 0 mg / ml. The first injection was performed 4 weeks after ACL rupture. 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. Rats were tested for pain at baseline (before the first injection), 30 days after the third injection, and finally at 57 days, just before execution (7 days after the last injection). Pain tests and motor tests were performed, namely the bipedal balance test (weight-bearing asymmetry between the ipsilateral and contralateral hindlimbs was measured by a bipedal equilibrator) and the rotarod test (how long the rat can continue to run on the rotating rod before falling off). The joints after execution will be analyzed by histology and uCT.

[0141] 24-hour in vitro release by dialysis: Fig.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 of the drug in the liposomes allowed the liposome-adenosine to be released slowly, and 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 of the drug in the liposomes allowed the liposome-adenosine to be released slowly, and some burst releases around 1 hour, 2 hours, and 3 hours, achieving higher doses of 48.89%, 39.21%, and 29.65%.

[0142] 10-day in vitro release kinetics: Fig.12The percent retention of adenosine in the two liposome formulations is shown. There was an initial burst of adenosine (1096 μM for RgnA09; 2014 μM for RgnA10). No significant differences were found between the RgnA09 and RgnA10 formulations. The freshly prepared liposome suspension (time 0) showed 21% adenosine retention for RgnA09 and 19% adenosine retention for RgnA10. Figure 1 and Figure 4 The results showed that after 1 hour of incubation, the retention rate of both preparations decreased to 4%, and then slowly decreased over time, reaching 1.4% and 2% on day 10 (corresponding to RgnA09 and RgnA10, respectively), which corresponded to 159 μM and 227 μM adenosine. These results indicate that both liposomal preparations are good reservoirs for encapsulating and slowly releasing adenosine at concentrations sufficient to activate A2A receptors in vivo.

[0143] Animal studies: We further tested the efficacy of the newly developed formulation in a rat model of post-traumatic OA (PTOA). Rats developed OA after ACL rupture as described above. PTOA rats were randomly assigned to experimental groups. Bipedal balance and pain testing was performed before the start of the experiment. Animals were divided into 10 groups and received 0 (empty liposomes / vehicle), 0.3, 1, or 3 mg / ml of adenosine, saline, or formulations of RgnA09 or RgnA10 as previously described by Corciulo et al. 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. Rats were tested for pain at baseline (before the first injection), at 30 days (mid-term of the treatment regimen), and just before sacrifice (7 days after the last injection). Joints after sacrifice were analyzed by histology and uCT. Pain behavior was performed by measuring the weight-bearing asymmetry between the ipsilateral and contralateral hindlimbs using a bipedal balance meter and rotarod test.

[0144] Animals are placed in rodent restrainers, standing with hind paws, with hind limbs resting on two weight average platform pads. When animals remove their weight from each pad, the device performs 3-4 continuous measurements in 12 seconds to record the average weight (gram). The mean value of each animal is used for analysis. Pain is also measured by rotating rod test, which provides an assessment of motor function with knee joint pressure and stress. Rats are placed on an accelerating rotating rod, and the situation of failing to stay on the top of the rod is determined, and used for further analysis.

[0145] Based on the bipedal balance test, pain behavior was significantly reduced in animals treated with intra-articular vehicle and 1 mg / mL RgnA09, and between 0.3 mg and 3 mg of RgnA10. In addition, at 30 days (after 3 injections), we observed a stable dose-response trend in reducing joint pain for both formulations, with all doses of Rgn09 being significantly different from vehicle, and 3 mg of Rgn10 being different from Rgn01 and vehicle ( Fig. 9 The rotarod test also showed a dose-response trend and differences at the highest dose of Rgn10 (3 mg / ml) at 60 days ( Fig.14 In addition, we observed significant changes in joint inflammation for both formulations, with some doses showing significant differences from vehicle and saline after 6 injections. Both formulations at 3 mg / ml showed a steady decrease in joint inflammation over time ( Fig.15 ). Rgn09 and Rgn10 were both used at the highest dose of 3 mg / ml.

[0146] Fig.16 Shown are representative Safranin-O stained sections of affected rat tibiae after treatment with vehicle or 3 doses of liposomal adenosine. In vehicle treated animals, cartilage proteoglycans and cartilage surface irregularities were significantly reduced. In RgnA09 treated rats, there was a dose-dependent improvement in cartilage proteoglycans and cartilage wear also disappeared, with an increase in surface cartilage. In RgnA10 treated rats, the effect was greatest in those treated with the highest dose (3 mg / ml), although protection of cartilage was also observed at lower doses.

[0147] Conclusion: The new formulation of liposomal adenosine is equally effective or even more effective in relieving pain and swelling in OA knee joints, while protecting and strengthening cartilage. Both formulations are effective, and the in vitro release of adenosine from multilamellar vesicles is superior to that of non-liposomal or free adenosine.

[0148] Example 5

[0149] This example describes methods of using the liposomes of the present disclosure.

[0150] Storage-stable formulations RgnA09 and RgnA10: A series of storage-stable formulation schemes based on lipid composition, dissolution efficiency and retention properties were developed and evaluated. RgnA09 and RgnA10 can be rehydrated in a concentrated solution of hydrophilic adenosine. During the rehydration process, multilamellar liposome particles are produced, which contain adenosine in the aqueous compartment of the liposome. The liposome size is ~10-100 microns and is metastable, so it collapses into a dense form under high heat conditions (~40°C). When the liposomes are confined to a locally closed compartment, such as the bursa of the human knee synovium, sustained release of adenosine can be achieved. RgnA09 and RgnA10 were tested to evaluate their ability to bind and release adenosine over a period of time. The liposome lyophilisate in a sterile glass vial was mixed with a sterile adenosine solution (3 mg / mL in saline) and provided in a pre-filled plastic syringe (customized from Mycoscience Inc.). Samples (100 μL) of the lipid-adenosine suspension were incubated in phosphate-buffered saline at 37°C for 0, 1, 2 hours, 1, 2, 5, 7, and 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 pellet was resuspended in 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 Figures 1 and 12 show the percentage retention of adenosine in the two liposomal formulations. There was an initial burst of adenosine (1096 μM for RgnA09; 2014 μM for RgnA10). No significant differences were found between the RgnA09 and RgnA10 formulations. Freshly prepared liposome suspensions (time 0) showed 21% adenosine retention for RgnA09 and 19% adenosine retention for RgnA10. After 1 hour of incubation, retention for both formulations dropped to 4% and slowly decreased over time, reaching 1.4% and 2% on day 10 (for RgnA09 and RgnA10, respectively), corresponding to 159 μM and 227 μM 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.

[0151] The newly developed formulation was further tested for efficacy in a rat model of post-traumatic OA (PTOA). As described above, rats developed OA after ACL rupture. PTOA rats were randomly assigned to experimental groups. Before starting the experiment, bipedal balance and pain testing were performed. Animals were divided into 10 groups and received 0 (empty liposomes / vehicle), 0.3, 1, or 3 mg / ml of adenosine, saline, or formulations of RgnA09 or RgnA10 as previously described by Corciulo et al. 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. Rats were tested for pain at baseline (before the first injection), at 30 days (mid-term of the treatment regimen), and just before execution (7 days after the last injection). Joints after execution were analyzed by histology and uCT. Pain behavior was performed by measuring the asymmetry of weight bearing between the ipsilateral and contralateral hindlimbs using a bipedal balance meter ( Fig. 9 ).

[0152] After the hyperalgesia test, animals were placed in rodent restrainers, stood with hind paws, and hind limbs rested on two weight average platform pads. When animals removed their weight from each pad, the device carried out 3-4 continuous measurements in 12 seconds to record average weight (gram). The mean value of each animal was used for analysis. Motor ability is also measured by rotating rod test, which provides the assessment of motor function with knee joint pressure and stress. Rats are placed on the accelerating rotating rod, and the situation of failing to stay on the top of the rod is determined, and is used for further analysis (data not shown). Based on the bipedal balance test, for RgnA09, between vehicle and 1mg / mL, for RgnA10, between 0.3mg and 3mg, there is a strong dose interaction. In addition, at 30 days (after injection 3 times), we observed that two preparations have a stable dose-response trend in reducing joint pain, and all doses of Rgn09 are significantly different from vehicle, while 3mg of Rgn10 is different from Rgn01 and vehicle. The rotarod test also showed a dose-response trend and differences at the highest dose of Rgn10 (3 mg / ml) at 60 days. In addition, we observed significant changes in joint inflammation for both formulations, with some doses showing significant differences from vehicle and saline after 6 injections. Both formulations at 3 mg / ml showed a steady decrease in joint inflammation over time ( Fig.15 ). Rgn09 and Rgn10 can be used at a maximum dose of 3 mg / ml.

[0153] Although the invention has been described with respect to one or more specific embodiments, it should be understood that other embodiments of the invention may be implemented without departing from the scope of the invention.

Claims

1. An injectable formulation comprising physiological saline and liposomes, wherein the liposomes comprise one or more lamellar bodies, wherein: The liposome layer contains 70-100% by mass of sphingomyelin, and when the sphingomyelin is less than 100% by mass, the rest 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 7:3, wherein the liposome (a) 50 nm to 150 μm in diameter; and (b) Adenosine is encapsulated in the aqueous compartment of liposomes.

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

3. The injectable preparation according to claim 1, wherein Adenosine or a portion thereof is released for up to two weeks.

4. The injectable formulation of 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 was 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 was 7-12 mg / mL.

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

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

11. The injectable preparation according to claim 1, wherein The temperature at which liposomes collapse is 35-45°C.

12. The injectable preparation according to claim 1, wherein At least a portion of the adenosine is released within 1 second to 1 hour after administration to a joint of a subject.

13. The injectable preparation according to claim 12, wherein At least a portion of the adenosine is released within 1 minute to 1 hour after administration to a joint of a subject.

14. The injectable preparation according to claim 13, wherein At least 1-20% of the adenosine is released within 1 minute to 1 hour after administration to the joint of the individual.

15. The injectable preparation according to claim 12, wherein At least a portion of the adenosine or at least 1-20% of the adenosine is released within 1 second to 10 minutes after administration to a joint of a subject.

16. Use of the injectable formulation of claim 1 in the preparation of a pharmaceutical composition for i) inducing cartilage regeneration and / or ii) relieving joint pain and / or inflammation and / or iii) slowing down and / or preventing and / or reversing progressive structural tissue damage, wherein administration of the pharmaceutical composition comprises administering the injectable formulation of claim 1 to an individual, wherein, i) cartilage regeneration is induced and / or ii) joint pain and / or inflammation is relieved or partially relieved and / or iii) progressive structural tissue damage is slowed or partially slowed and / or prevented or partially prevented and / or reversed or partially reversed.

17. The use according to claim 16, wherein Injectable formulations are administered to a joint of a subject by intra-articular injection.

18. The use according to claim 16, wherein Injectable formulations are administered in one or more injections.

19. The use as claimed in claim 16, wherein: The injectable formulation is administered multiple times, with each administration occurring every 10 days.

20. The use according to claim 16, wherein The subject suffers from osteoarthritis, rheumatoid arthritis, acute gouty arthritis, and / or synovitis.

21. The use according to claim 17, wherein An individual is a human or a non-human mammal.

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