TWI782181B - Liposomal formulation for joint lubrication - Google Patents

Abstract

The present invention provides a pharmaceutical composition for the lubrication of joints, the pharmaceutical composition comprising a non-ionic tonicity agent comprising a polyol, and liposomes comprising at least one membrane comprising at least one phospholipid (PL) selected from a glycerophospholipid (GPL), said GPL having two C₁₂ -C₁₈ hydrocarbon chains, being the same or different, and sphingomyelin (SM) having a C₁₂ -C₁₈ hydrocarbon chain, the pharmaceutical composition being essentially free of an additional pharmaceutically active agent, wherein the at least one membrane has a phase transition temperature in the range of about 20ºC to about 39ºC and the joint has a joint temperature which is above the phase transition temperature.

Description

Liposome formulations for joint lubrication

The present invention relates to liposomal pharmaceutical compositions in which phospholipids themselves are the only active agents, and their therapeutic use for joint lubrication.

Joint dysfunction affects a very large proportion of the population. Adequate biological lubrication is a prerequisite for proper joint mobility, and adequate biological lubrication is important for preventing and improving degenerative changes in joints.

A common joint dysfunction is osteoarthritis (OA), which affects more than 20 million people in the United States alone. Current treatment focuses on reducing joint overload, physical therapy, and pain and inflammation relief, usually by systemic or intra-articular administration of drugs.

Articular cartilage forms the smooth, strong, elastic, and flexible surface that helps bones move. The synovial space is filled with highly viscous synovial fluid (SF), which contains hyaluronic acid (HA) and glycoprotein lubricin. HA, a polymer of D-glucuronic acid and DN-acetylglucosamine, is extremely unstable and degrades under the inflammatory conditions of OA (Nitzan, DW, Kreiner, B. and Zeltser, R. TMJ lubrication system: its effect on the joint function, dysfunction, and treatment approach. Compend. Contin. Educ. Dent. 25 , 437-444 (2004); Yui, N., Okano, T. and Sakurai, Y. Inflammation responsive degradation of crosslinked hyaluronic acid gels. J. Control. Release 22 , 105-116 (1992)). Lubricant is composed of about 44% protein, about 45% carbohydrate and about 11% phospholipid (PL), of which about 41% is phosphatidylcholine (PC), about 27% is phosphatidylethanolamine (PE) and About 32% is sphingomyelin. These PLs are called "surfactant phospholipids" (SAPLs).

Boundary lubrication, in which layers of lubricant molecules separate opposing surfaces, occurs under load in the joint. Several different substances have been proposed as native boundary lubricants in articular cartilage, including HA and lubricin. Pickard et al. and Schwartz and Hills demonstrated that phospholipids, defined as surface-active phospholipids of lubricins, promote joint lubrication in articular cartilage (Pickard, JE, Fisher, J., Ingham, E. and Egan, J. Investigation into the effects of proteins and lipids on the frictional properties of articular cartilage. Biomaterials 19 , 1807-1812 (1998); Schwarz, IM and Hills, BA Surface-active phospholipid as the lubricating component of lubricin. Br. J. Rheumatol. 37 , 21-26 ( 1998)). Hills and colleagues demonstrated that OA joints are deficient in SAPL and that injection of the surfactant phospholipid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) into the joints of OA patients resulted in improved mobility that lasted for a long time. Up to 14 weeks without serious side effects (Vecchio, P., Thomas, R. and Hills, BA Surfactant treatment for osteoarthritis. Rheumatology (Oxford) 38 , 1020-1021 (1999); Gudimelta, OA, Crawford, R. and Hills, BA Consolidation responses of delipidized cartilage. Clin. Biomech. 19 , 534-542 (2004)). In another study, using a unique cryogenic cartilage preservation technique, Watanabe et al. observed lipid spherical vesicles on the surface of healthy cartilage, hypothesizing that they play a major role in lubrication (Watanabe, M. et al. Ultrastructural study of upper surface layer in rat articular cartilage by “in vivo cryotechnique” combined with various treatments. Med. Elect. Microsc. 33 , 16-24 (2000)). Kawano et al. and Forsey et al. used animal models to demonstrate that the combination of high molecular weight HA (about 2000 kDa) and DPPC can improve the lubricating ability of the latter (Kawano, T. et al. Mechanical effects of the intraarticular administration of high molecular weight hyaluronic acid plus Phospholipid on synovial joint lubrication and prevention of articular cartilage degeneration in experimental osteoarthritis. Arthritis Rheum. 48 , 1923-1929 (2003); Forsey, RW et al. The effect of hyaluronic acid and phospholipid based lubricants on friction with friction Biomaterials 27 , 4581-4590 (2006)).

US Patent No. 6,800,298 discloses polydextrose-based hydrogel compositions containing lipids, specifically phospholipids, for lubricating mammalian joints.

US Patent Application 2005/0123593 relates to a composition for intra-articular administration for the treatment of osteoarthritis comprising glycosaminoglycan encapsulated in a liposomal delivery system.

US Patent No. 8,895,054 relates to a method of joint lubrication and/or prevention of cartilage wear using liposomes consisting essentially of a phospholipid membrane having a phase transition temperature in the range of about 20°C to about 39°C.

Commercially available pharmaceutical compositions for the prevention and treatment of osteoarthritis based on hyaluronic acid as active ingredient include, among others, Antalvisc®, Kartilage® and Kartilage® Cross. In addition to HA, the pharmaceutical compositions also include mannitol. Mannitol was found to have the ability to reduce HA degradation under oxidative stress and thus can be used to significantly prolong the intra-articular residence time of injected HA and improve the viscous replenishment effectiveness of HA-based intra-articular injections (M. Rinaudo, B. . Lardy, L. Grange and T. Conrozier, Polymers 2014 , 6, 1948-1957). A clinical study comparing the safety and efficacy of intra-articular viscosupplements versus high MW HA alone (Bio-HA) in patients with knee osteoarthritis demonstrated pain relief and improved function over a six-month period with Manna The viscosupplement of sugar alcohol was no less effective than its comparator Bio-HA and did not induce more side effects into (Conrozier, Thierry et al. The Knee , 2016 , 23 (5), 842-848). Eymard et al (Eymard F, Bossert M, Lecurieux R, Maillet B, Chevalier X et al, (2016) Addition of Mannitol to Hyaluronic Acid may Shorten Viscosupplementation Onset of Action in Patients with Knee Osteoarthritis: Post-Hoc Analysis of A Double- blind, Controlled Trial. J Clin Exp Orthop 2: 21) and Conrozier, T. et al. (Role of high concentrations of mannitol on the stability of hyaluronan in an oxidative stress model induced by xanthine/xanthine oxidase Osteoarthritis and Cartilage, Vol. 22 , S478) also studied the effect of mannitol on the efficacy of HA-based viscous replenisher compositions. Ferraccioli et al. demonstrated that UV irradiation of synovial fluid can be a helpful method for screening drugs for free radical scavenging, since it induces a decrease in viscosity caused by free radical production. The viscosity protection of human synovial fluid is mediated by superoxide dismutase, mannitol and catalytic enzymes (GF Ferraccioli, U. Ambanelli, P. Fietta, N. Giudicelli, C. Giori, Decrease of osteoarthritic synovial fluid viscosity by means of uv illumination: A method to evaluate the free radical scavenging action of drugs, Biochemical Pharmacology, 30, (13) 1981, 1805-1808).

International patent application WO2012/001679 relates to injectable pharmaceutical formulations for alleviating or reducing joint irritation or reducing the exacerbation of existing joint inflammation, formulated for intra-articular injection, comprising active polyol ingredients, the The polyol active ingredient is xylitol. The efficacy of xylitol was compared with that of mannitol and glycerol for prevention of joint irritation, and it was found that solutions of mannitol or glycerol did not prevent irritation when injected into the knee of rabbits by intra-articular injection.

U.S. Patent Application 2014/0038917 relates to a sterile and injectable aqueous formulation, in the form of a gel, for intra-articular space administration to an intra-articular joint of an individual, comprising: hyaluronic acid or a salt thereof and a polyol, preferably Sorbitol (concentration equal to or greater than 7 mg/ml).

International Patent Application WO2003/000191 relates to compositions and methods for the treatment of arthritis comprising a combination of one or more glycosaminoglycans and one or more hyaluronidase inhibitors, wherein the hyaluronidase inhibitors may be selected from sulfuric acid Heparin, polydextrose sulfate and xylose sulfate, and hyaluronic acid and hyaluronidase inhibitors can be co-encapsulated in liposomes.

Injectable HA compositions are known to have various side effects such as difficulty in movement, muscle pain or stiffness, joint pain, and joint swelling or redness. Some side effects of Antalvisc® include transient pain and swelling of the injected joint after injection.

Thus, there remains an unmet need for effective pharmaceutical compositions for joint lubrication that will provide long-acting action while reducing the incidence of side effects associated with intra-articular administration.

The present invention provides a liposomal formulation for introduction into synovial joints to provide lubrication, thereby reducing pain and irritation and improving or restoring joint mobility. Liposome formulations are specifically adapted for intra-articular delivery. The pharmaceutical composition of the present invention comprises, as an active ingredient, a liposome comprising a phospholipid membrane having a phase transition temperature slightly lower than a physiological temperature. Thus, when administered to a synovial joint, the liposomes are in a liquid disordered (LD) phase. The pharmaceutical composition further comprises a tonicity agent, which is a polyol. Tonicity agents are used to reduce local irritation by preventing osmotic shock at the site of application.

The present invention is based in part on the unexpected discovery that nonionic tonicity agents added to liposome compositions provide enhanced lubrication compared to ionic tonicity agents. Specifically, the addition of mannitol to liposome formulations improved the lubricating efficacy of the composition, whereas the use of sodium chloride resulted in a decrease in the lubricating efficiency of the liposomes. The effect of mannitol is even more surprising considering the fact that the pharmaceutical composition does not contain any other pharmaceutically active agent than the phospholipid itself and in particular does not contain hyaluronic acid, the activity of which is known to be enhanced by the addition of polyols . Addition of different non-ionic polyols, including glycerol in particular, also resulted in an enhanced lubricity of the liposome formulations compared to the use of sodium chloride, albeit to a lesser extent.

Therefore, according to a first aspect, the present invention provides a pharmaceutical composition comprising a tonicity agent comprising a polyol; and a liposome comprising at least one membrane comprising at least one selected from the group consisting of glycerophospholipids ( GPL) and a phospholipid (PL) of sphingomyelin (SM), the GPL has two identical or different C ₁₂ -C ₁₈ hydrocarbon chains, and the sphingomyelin has a C ₁₂ -C ₁₈ hydrocarbon chain, wherein the at least one membrane has A phase transition temperature in the range of about 20°C to about 39°C; wherein the pharmaceutical composition is substantially free of other pharmaceutically active agents. The pharmaceutical composition is suitable for lubrication of mammalian joints having a temperature above the phase transition temperature. According to some embodiments, the tonicity agent is non-ionic. According to other embodiments, the polyol is selected from mannitol and glycerol. According to a particular embodiment, the tonicity agent comprises mannitol.

In another aspect, there is provided a method for lubricating a joint in a mammal, the method comprising: administering to a cavity of the joint a pharmaceutical composition comprising a tonicity agent comprising a polyol; and at least one Liposomes of membranes, the at least one membrane comprising at least one phospholipid (PL) selected from glycerophospholipids (GPL) and sphingomyelin (SM), the GPL having two identical or different C ₁₂ -C ₁₈ hydrocarbon chains and the sphingomyelin Phospholipids have C ₁₂ -C ₁₈ hydrocarbon chains, wherein the at least one membrane has a phase transition temperature in the range of about 20°C to about 39°C; wherein the pharmaceutical composition is substantially free of other pharmaceutically active agents, wherein the joint has a high Joint temperature at phase transition temperature. According to some embodiments, the tonicity agent is non-ionic. According to other embodiments, the polyol is selected from mannitol and glycerol. According to a particular embodiment, the tonicity agent comprises mannitol.

In another aspect, there is provided a method for treating joint pain or irritation in a subject having a joint disorder, the method comprising lubricating the subject's joint by administering a pharmaceutical composition into a cavity of the joint, the subject The pharmaceutical composition comprises a tonicity agent comprising a polyol; and a liposome comprising at least one membrane comprising at least one phospholipid (PL) selected from glycerophospholipids (GPL) and sphingomyelin (SM), the GPL has two identical or different C ₁₂ -C ₁₈ hydrocarbon chains and the sphingomyelin has C ₁₂ -C ₁₈ hydrocarbon chains, wherein the at least one membrane has a phase transition temperature ranging from about 20°C to about 39°C; wherein The pharmaceutical composition is substantially free of other pharmaceutically active agents, and wherein the joint has a joint temperature higher than the phase transition temperature. According to some embodiments, the tonicity agent is non-ionic. According to other embodiments, the polyol is selected from mannitol and glycerol. According to a particular embodiment, the tonicity agent comprises mannitol.

In another aspect, the present invention provides a use of a tonicity agent comprising a polyol; and a liposome consisting essentially of at least one membrane comprising at least one selected from the group consisting of glycerophospholipids (GPL) and Phospholipids (PL) of sphingomyelin (SM), the GPL has two identical or different C ₁₂ -C ₁₈ hydrocarbon chains and the sphingomyelin has C ₁₂ -C ₁₈ hydrocarbon chains, wherein the at least one membrane has To a phase transition temperature in the range of about 39°C, the tonicity agent is used in the manufacture of a pharmaceutical composition for lubricating mammalian joints having a temperature above the phase transition temperature, wherein the pharmaceutical composition is substantially free of other pharmaceuticals active agent. According to some embodiments, the tonicity agent is non-ionic. According to other embodiments, the polyol is selected from mannitol and glycerol. According to a particular embodiment, the tonicity agent comprises mannitol.

In some embodiments, the polyol does not include xylitol.

In some embodiments, the polyol is present in the pharmaceutical composition in a weight percentage ranging from about 5% (by weight) to about 50% (by weight) of the dry weight of the pharmaceutical composition. In some embodiments, mannitol is present in the pharmaceutical composition in a weight percentage ranging from about 20% (by weight) to about 40% (by weight) of the dry weight of the pharmaceutical composition. In some embodiments, glycerin is present in the pharmaceutical composition in a weight percent in the range of about 5% (by weight) to about 25% (by weight) of the dry weight of the pharmaceutical composition. In some embodiments, the phospholipid is present in the pharmaceutical composition in a weight percentage ranging from about 50% (by weight) to about 95% (by weight) of the dry weight of the pharmaceutical composition.

In some embodiments, the pharmaceutical composition further comprises a fluid medium, wherein the liposomes are dispersed or suspended in the fluid medium. In other embodiments, the polyol is dispersed or dissolved in the fluid medium. In other embodiments, mannitol is dissolved in the fluid medium. The fluid medium can be selected from buffers and water. In certain embodiments, the buffer comprises histidine buffer or phosphate buffered saline. The various possibilities represent separate embodiments of the invention. In certain embodiments, the buffer comprises a histidine buffer.

In some embodiments, pharmaceutical compositions are in the form of pharmaceutically acceptable suspensions comprising liposomes suspended in a fluid medium.

According to some embodiments, the concentration of polyol inside the liposome is substantially the same as the concentration of polyol in the medium outside the liposome.

In some embodiments, the polyol is present in the pharmaceutical composition in a weight percentage ranging from about 0.05% (by weight) to about 10% (by weight) of the total weight of the pharmaceutical composition. In other embodiments, the weight percent of polyol is in the range of about 0.1% (by weight) to about 7% (by weight). In other embodiments, the weight percent of polyol is in the range of about 1% (by weight) to about 5% (by weight).

In some embodiments, mannitol is present in the pharmaceutical composition at a weight percentage ranging from about 0.1% (by weight) to about 7% (by weight) of the total weight of the pharmaceutical composition. In other embodiments, the weight percent of mannitol ranges from about 1% (by weight) to about 7% (by weight).

In some embodiments, glycerin is present in the pharmaceutical composition in a weight percentage ranging from about 0.05% (by weight) to about 5% (by weight) of the total weight of the pharmaceutical composition. In other embodiments, the weight percentage of glycerol is in the range of about 0.5% (weight ratio) to about 5% (weight ratio).

In some embodiments, the pharmaceutical composition has an osmolality in the range of about 200 to about 600 mOsm. In certain embodiments, the pharmaceutical composition has an osmolality of about 300 mOsm. In certain such embodiments, the pharmaceutical composition is isotonic.

In some embodiments, the pH of the pharmaceutical composition is about 5-8.

In some embodiments, the weight ratio between liposomes and polyol is in the range of about 15:1 to about 1:1. In other embodiments, the weight ratio between liposomes and mannitol ranges from about 10:1 to about 1:1. In other embodiments, the weight ratio between liposomes and glycerol is in the range of about 15:1 to about 2:1.

According to some embodiments, the liposome has more than one membrane. In certain such embodiments, the liposomes are multilamellar vesicles (MLVs).

In some embodiments, GPL comprises two acyl chains. In other embodiments, the chains are selected from the group consisting of C ₁₄ , C ₁₅ , C ₁₆ and C ₁₈ acyl chains. In certain embodiments, at least one of the hydrocarbon chains is a saturated hydrocarbon chain. In other embodiments, both hydrocarbon chains are saturated.

In some embodiments, PL is phosphatidylcholine (PC). In other embodiments, at least one membrane comprises 1,2-dimyristyl- sn -glycero-3-phosphocholine (DMPC).

In some embodiments, at least one membrane further comprises PC selected from the group consisting of: 1,2-disalmityl- sn -glycero-3-phosphocholine (DPPC), 1,2-dipentadecanoyl Acyl- sn -glycero-3-phosphocholine (C15), 1,2-distearoyl- sn -glycero-3-phosphocholine (DSPC) and N-palmitoyl-D-erythro Sphingosine phosphorylcholine (D-erythro C16). In some embodiments, the molar percentage of DMPC in at least one film ranges from about 10% to about 75%.

In some embodiments, at least one film comprises DMPC and DPPC. In other embodiments, the molar percentage ratio of DMPC to DPPC ranges from about 25:75 to about 70:30. In certain embodiments, the molar percentage ratio of DMPC to DPPC is about 45:55.

In some embodiments, at least one membrane comprises DMPC and C15. In other embodiments, the molar percentage ratio of DMPC to C15 is in the range of about 25:75 to about 45:55.

In some embodiments, at least one film comprises DMPC and DSPC. In other embodiments, the molar percentage ratio of DMPC to DSPC is about 75:25.

In some embodiments, at least one membrane comprises DMPC and D-erythro C16. In other embodiments, the molar percentage ratio of DMPC to D-erythro C16 ranges from about 10:90 to about 25:75.

In some embodiments, at least one membrane comprises C15.

The total concentration of phospholipids in the pharmaceutical composition according to some embodiments of the invention is in the range of about 50 to about 300 mM. In certain embodiments, the total concentration of phospholipids ranges from about 100 mM to about 200 mM.

In some embodiments, the phospholipid is present in the pharmaceutical composition in a weight percentage ranging from about 0.5% (by weight) to about 30% (by weight) of the total weight of the pharmaceutical composition. In other embodiments, the weight percent of phospholipids ranges from about 3% (by weight) to about 30% (by weight).

In some embodiments, liposomes have an average diameter between about 0.5 µm and about 10 µm.

In certain embodiments, at least one film has a phase transition temperature of about 30°C to about 35°C.

In some embodiments, the temperature of the joint is in the range of about 1-15°C above the phase transition temperature.

In some presently preferred embodiments, the pharmaceutical composition is substantially free of hyaluronic acid.

In certain embodiments, the liposome consists essentially of at least one membrane comprising at least one phospholipid (PL), as described in detail above.

In some embodiments, the pharmaceutical composition comprises MLV liposomes; mannitol; and histidine buffer, the membrane of the liposomes consisting essentially of DMPC and DPPC. In other embodiments, DMPC is present in the pharmaceutical composition at a weight percentage ranging from about 1% (by weight) to about 10% (by weight) of the total weight of the pharmaceutical composition. In other embodiments, DPPC is present in the pharmaceutical composition at a weight percentage ranging from about 2% (by weight) to about 12% (by weight) of the total weight of the pharmaceutical composition. In other embodiments, mannitol is present in the pharmaceutical composition in a weight percentage ranging from about 1% (by weight) to about 7% (by weight) of the total weight of the pharmaceutical composition.

In some embodiments, joint lubrication is used to treat joint disorders or symptoms resulting therefrom. In other embodiments, the joint disorder is selected from the group consisting of: arthritis, osteoarthritis, osteoarthritis in patients with rheumatoid arthritis, traumatic joint injury, joint lock, sports injury, arthrocentesis Posterior state, arthroscopic surgery, open joint surgery and joint replacement. The various possibilities represent separate embodiments of the invention. In certain embodiments, the pharmaceutical composition is used to reduce knee joint pain in osteoarthritis patients.

In some embodiments, lubrication is used to prevent joint wear.

According to a particular embodiment, the pharmaceutical composition is a parenteral pharmaceutical composition comprising a suspension of liposomes. The pharmaceutical composition can be in a form suitable for administration by intra-articular injection, arthroscopic administration, or by surgery. The various possibilities represent separate embodiments of the invention.

Pharmaceutical compositions according to various embodiments of the invention may be administered in a dose of about 0.5 ml to about 10 ml. In other embodiments, the pharmaceutical composition is administered in a dose of about 1 ml to about 6 ml. In certain embodiments, the pharmaceutical composition is administered in a dose of about 3 ml.

In some embodiments, one dosage unit of the pharmaceutical composition comprises from about 20 mg to about 350 mg of mannitol. In a certain embodiment, one dosage unit of the pharmaceutical composition comprises about 120 mg mannitol.

In some embodiments, one dosage unit of the pharmaceutical composition comprises from about 50 mg to about 1000 mg of phospholipid. In some embodiments, one dosage unit of the pharmaceutical composition comprises from about 50 mg to about 500 mg DPPC. In some embodiments, one dosage unit of the pharmaceutical composition comprises about 40 mg to about 300 mg DMPC.

Other embodiments and full scope of applicability of the invention will become apparent from the detailed description provided hereinafter. It should be understood, however, that the detailed description and specific examples, while indicating the preferred embodiment of the invention, are provided for illustration only, since the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. Variations and alterations within.

The present invention provides liposomal formulations for lubricating mammalian joints that provide reduced pain and irritation and are capable of improving or restoring joint mobility and reducing joint wear. The pharmaceutical compositions can also be used to treat, manage or prevent joint disorders or conditions. The pharmaceutical composition according to the principles of the present invention is based on a liposome composition with a defined phase transition temperature of the liposome membrane, which is lower than that of the joint.

As used herein, in some embodiments, the term "phase transition temperature" refers to the temperature at which a liposome undergoes an ordered solid (SO) to disordered liquid (LD) phase transition. The phase transition temperature of liposomes can be assessed by differential scanning calorimetry (DSC). Various parameters of the DSC thermogram that can be examined to assess the phase transition temperature include T _on , which represents the temperature at which the SO-LD phase transition begins, and T _off , which represents the temperature at which the SO-LD phase transition ends during the heating scan , and T _p and T _m , which denote the temperatures at which the maximum change in heat capacity occurs during the pre-transition (T _p ) and main transition (T _m ), respectively.

It has previously been demonstrated that multilamellar vesicular liposomes (with two C ₁₂ -C ₁₆ hydrocarbon chains) composed of various PCs are activated at slightly above (eg, about 1°C, 2°C, 3°C, 5°C, 8°C, 11°C and Sometimes as high as about 15°C) SO to LD phase transition temperature is an effective cartilage lubricant and wear reducing agent (as described in detail in US Pat. No. 8,895,054, the contents of which are incorporated herein by reference in their entirety). The pharmaceutical composition of the present invention further comprises a non-ionic tonicity agent, which increases the osmolality of the composition.

It was unexpectedly found that the lubrication efficiency of pharmaceutical compositions comprising non-ionic polyols is significantly higher compared to pharmaceutical compositions comprising ionic tonicity agents, and in particular sodium chloride salts. A positive effect of polyols compared to sodium chloride is not foreseen at all, since the pharmaceutical composition of the invention is not based on hyaluronic acid, the activity of which is known to be enhanced by the addition of polyols.

The inventors also confirmed that the addition of mannitol does not change the phase transition temperature of liposome membranes. Without wishing to be bound by theory or mechanism of action, it can be hypothesized that the unexpected effect of polyol addition is not directly related to the phase transition temperature of liposomes, which has been previously reported as an essential feature related to the lubricating ability of liposomes.

Therefore, according to an aspect of the present invention, there is provided a pharmaceutical composition comprising a tonicity agent comprising a polyol; and a liposome comprising at least one selected from glycerophospholipids (GPL) or sphingolipids (SPL) A phospholipid (PL) having a phase transition temperature in the range of about 20°C to about 39°C, wherein the pharmaceutical composition is substantially free of other pharmaceutically active agents. In some embodiments, the pharmaceutical composition is used to lubricate the joints of a mammal.

In another aspect, there is provided a method for lubricating a mammalian joint, the method comprising: administering to a cavity of the joint a pharmaceutical composition comprising: a tonicity agent comprising a polyol; and liposomes , which comprises at least one phospholipid (PL) selected from glycerophospholipids (GPL) or sphingolipids (SPL) and has a phase transition temperature in the range of about 20°C to about 39°C, wherein the pharmaceutical composition is substantially free of other pharmaceutical active agent.

In another aspect, there is provided a method for treating pain or irritation in a joint of a subject having a joint disorder, the method comprising lubricating the subject's joint by administering a pharmaceutical composition into a cavity of the joint , the pharmaceutical composition comprising: a tonicity agent comprising a polyhydric alcohol; and a liposome comprising at least one phospholipid (PL) selected from glycerophospholipids (GPL) or sphingolipids (SPL) and having a phospholipid (PL) at about 20 A phase transition temperature in the range of C to about 39 C, wherein the pharmaceutical composition is substantially free of other pharmaceutically active agents.

In another aspect, the present invention provides a use of a tonicity agent comprising a polyol and a liposome comprising at least one phospholipid (PL) selected from glycerophospholipids (GPL) or sphingolipids (SPL) And having a phase transition temperature in the range of about 20°C to about 39°C, the tonicity agent is used in the preparation of a pharmaceutical composition for lubricating mammalian joints, wherein the pharmaceutical composition is substantially free of other pharmaceutically active agents.

As used herein, in some embodiments, the term "tonicity agent" refers to a tonicity agent suitable for use in pharmaceutical compositions for intra-articular injection.

In some embodiments, the tonicity agent is non-ionic. In some embodiments, the polyols are linear polyols. In some embodiments, the polyol is a cyclic polyol. Non-limiting examples of nonionic polyols suitable for use in the pharmaceutical compositions of the present invention include mannitol, glycerol, dextrose, lactose, and trehalose.

In some presently preferred embodiments, the polyol is mannitol. Mannitol is a well-known and low-cost excipient commonly used by formulators in various types of pharmaceutical compositions. As mentioned above, mannitol has been used in combination with hyaluronic acid in pharmaceutical compositions for joint lubrication. It has also been reported that mannitol is suitable for cryopreservation of liposomes (Talsma H, van Steenbergen MJ, Salemink PJ, Crommelin DJ, Pharm Res. 1991 , 8 (8): 1021-6).

In some embodiments, the tonicity agent comprises glycerin.

In some embodiments, the pharmaceutical composition comprises a combination of polyols, ie, a combination of mannitol and glycerol. Pharmaceutical compositions may further include polyols in combination with other tonicity agents.

In some embodiments, the polyol does not include xylitol.

It should be emphasized that according to some presently preferred embodiments, the tonicity agent is not encapsulated within liposomes. As used herein, in some embodiments, the term "encapsulation" refers to the concentration of the tonicity agent within the liposome being significantly higher than the concentration in the medium external to the liposome. The term "inside the liposome" is understood to encompass at least one internal aqueous phase of the liposome. The term "concentration" may include osmolarity. As used herein, in some embodiments, the term "substantially higher than" refers to a concentration difference of at least about 90%. In some embodiments, the polyol is not encapsulated within liposomes. In other embodiments, the mannitol is not encapsulated within liposomes. In other embodiments, the glycerol is not encapsulated within liposomes.

According to other embodiments, the concentration of the tonicity agent inside the liposome is substantially the same as the concentration of the tonicity agent in the medium outside the liposome. As used herein, in some embodiments, the term "substantially the same" refers to concentrations that differ by less than about 15%. In other embodiments, the term "substantially the same" refers to concentrations that differ by less than about 10%, less than about 5%, less than about 2.5%, or less than about 1%. The various possibilities represent separate embodiments of the invention.

According to other embodiments, the concentration of polyol inside the liposome is substantially the same as the concentration of polyol in the medium outside the liposome. According to other embodiments, the concentration of mannitol inside the liposome is substantially the same as the concentration of mannitol in the medium outside the liposome. According to other embodiments, the concentration of glycerol inside the liposome is substantially the same as the concentration of glycerol in the medium outside the liposome.

In some embodiments, the liposomes are not lyophilized. In other embodiments, the liposomes are not lyophilized and/or thawed prior to administration to the joint.

In some embodiments, the pharmaceutical composition further comprises a fluid medium. In some embodiments, liposomes are dispersed or suspended in the fluid medium. In other embodiments, the tonicity agent is dissolved or dispersed in the fluid medium. In other embodiments, mannitol or glycerol is dissolved in the fluid medium. In some embodiments, pharmaceutical compositions are in the form of pharmaceutically acceptable suspensions comprising liposomes suspended in a fluid medium.

Also provided is a pharmaceutically acceptable suspension comprising a pharmaceutical composition according to the various embodiments above and further comprising a fluid medium.

In some embodiments, the polyol is present in an amount ranging from about 5% (by weight) to about 50% (by weight) or about 10% (by weight) to about 40% (by weight) of the dry weight of the pharmaceutical composition. Percentages are present in pharmaceutical compositions. In certain embodiments, the polyol is present in the pharmaceutical composition in a weight percent of about 30% (weight ratio) based on the dry weight of the pharmaceutical composition. In other embodiments, the polyol is present in the pharmaceutical composition at a weight percent of about 15% (by weight) based on the dry weight of the pharmaceutical composition.

In some embodiments, mannitol is present in the range of about 10% (by weight) to about 50% (by weight) or about 20% (by weight) to about 50% (by weight) of the dry weight of the pharmaceutical composition % by weight exists in the pharmaceutical composition. In other embodiments, mannitol is present in the pharmaceutical composition in a weight percent of about 30% (weight ratio) of the dry weight of the pharmaceutical composition.

In some embodiments, glycerol is present in an amount ranging from about 5% (by weight) to about 35% (by weight) or about 5% (by weight) to about 25% (by weight) of the dry weight of the pharmaceutical composition. Percentages are present in pharmaceutical compositions. In other embodiments, glycerin is present in the pharmaceutical composition at a weight percent of about 15% (weight ratio) based on the dry weight of the pharmaceutical composition.

In some embodiments, the liposome-forming phospholipids are present in an amount of about 50% (by weight) to about 95% (by weight) or about 60% (by weight) to about 85% (by weight) of the dry weight of the pharmaceutical composition The weight percent within the range is present in the pharmaceutical composition. In other embodiments, the phospholipid is present in the pharmaceutical composition in a weight percent of about 70% (weight ratio) of the dry weight of the pharmaceutical composition. In other embodiments, the phospholipid is present in the pharmaceutical composition at a weight percent of about 85% (weight ratio) based on the dry weight of the pharmaceutical composition.

As used herein, in some embodiments, the term "dry weight" refers to the weight of a pharmaceutical composition excluding fluid media. In other embodiments, the term "dry weight" refers to the weight of the pharmaceutical composition excluding water.

In some embodiments, compositions of the invention are pharmaceutically acceptable. As used herein, in some embodiments, the term "pharmaceutically acceptable" refers to any formulation that is safe and provides the desired route of administration for the active ingredients in effective amounts used in the present invention. deliver. The term also refers to the use of buffered formulations in which the pH is maintained at a specific desired value in the range of pH 4.0 to pH 9.0, depending on the stability of the compound and the route of administration.

Thus, the fluid medium may be selected from buffers, water and saline solutions. In some embodiments, the fluid medium comprises a buffer. In certain embodiments, the buffer comprises histidine buffer or phosphate buffered saline. The various possibilities represent separate embodiments of the invention. The concentration of histidine may range from about 0.5 mg/ml to about 10 mg/ml. In certain embodiments, the concentration of histidine is about 2 mg/ml. In some embodiments, the concentration of histidine ranges from about 1 mM to about 50 mM. In certain embodiments, the concentration of histidine is about 10 mM. Histidine may be present in the composition as a dissolved hydrochloric acid or acetate salt. In certain embodiments, the pharmaceutical composition further comprises minor amounts of mineral acids, such as hydrochloric acid.

The pH of the pharmaceutical composition may range between about 5 and about 8. In some embodiments, the pH ranges between about 6 and about 7. In certain embodiments, the pH of the pharmaceutical composition is about 6.5.

In some embodiments, the concentration of polyol in the pharmaceutical composition ranges from about 0.5 to about 100 mg/ml. In other embodiments, the concentration of polyol ranges from about 1 to about 70 mg/ml. In other embodiments, the concentration of polyol is in the range of about 2.5 to about 60 mg/ml. In other embodiments, the concentration of polyol is in the range of about 5 to about 50 mg/ml. In other embodiments, the concentration of polyol is in the range of about 30 to about 50 mg/ml. In certain embodiments, the concentration of polyol ranges from about 5 to about 30 mg/ml.

In some embodiments, the concentration of mannitol in the pharmaceutical composition ranges from about 1 mg/ml to about 70 mg/ml. In other embodiments, the concentration of mannitol ranges from about 10 mg/ml to about 70 mg/ml. In other embodiments, the concentration of mannitol ranges from about 10 mg/ml to about 50 mg/ml. In certain embodiments, the concentration of mannitol is about 40 mg/ml. In other embodiments, the concentration of mannitol is about 20 mg/ml.

In some embodiments, the concentration of glycerol in the pharmaceutical composition ranges from about 0.5 mg/ml to about 50 mg/ml. In other embodiments, the concentration of glycerol ranges from about 1 mg/ml to about 40 mg/ml. In other embodiments, the concentration of glycerol is in the range of about 5 mg/ml to about 30 mg/ml. In certain embodiments, the concentration of glycerol is about 20 mg/ml. In other embodiments, the concentration of glycerol is about 10 mg/ml.

In some embodiments, the concentration of polyol in the pharmaceutical composition ranges from about 50 to about 500 mM. In other embodiments, the concentration of polyol is in the range of about 100 to about 400 mM. In other embodiments, the concentration of polyol is in the range of about 200 to about 300 mM. The polyol may be selected from mannitol and glycerol.

In some embodiments, the polyol is present in an amount of about 0.05% (weight ratio) to about 10% (weight ratio), about 0.1% (weight ratio) to about 7% (weight ratio), about 0.5% (weight ratio) of the total weight of the pharmaceutical composition. % (by weight) to about 10% (by weight) or about 1% (by weight) to about 5% (by weight) is present in the pharmaceutical composition. In certain embodiments, the weight percentage of polyol is about 4% (weight ratio). In other embodiments, the weight percentage of polyol is about 2% (weight ratio).

In some embodiments, mannitol is present in an amount of about 0.1% (weight ratio) to about 7% (weight ratio), about 0.5% (weight ratio) to about 10% (weight ratio) or about 0.1% (weight ratio) of the total weight of the pharmaceutical composition. A weight percentage in the range of 1% (by weight) to about 7% (by weight) is present in the pharmaceutical composition. In certain embodiments, the weight percentage of mannitol is about 4% (weight ratio).

In some embodiments, glycerin is present in an amount ranging from about 0.05% (by weight) to about 5% (by weight) or from about 0.5% (by weight) to about 5% (by weight) of the total weight of the pharmaceutical composition. Percentages are present in pharmaceutical compositions. In certain embodiments, the weight percentage of glycerin is about 2% (weight ratio).

As used herein, in some embodiments, the term "total weight" refers to the weight of a pharmaceutical composition comprising a fluid medium. In other embodiments, the term "total weight" refers to the weight of a pharmaceutically acceptable suspension.

In some embodiments, the pharmaceutical composition has an osmolality in the range of about 200 to about 600 mOsm. In other embodiments, the pharmaceutical composition has an osmolality in the range of about 250 to about 500 mOsm. In other embodiments, the pharmaceutical composition has an osmolality in the range of about 250 to about 400 mOsm. In certain embodiments, the pharmaceutical composition has an osmolality of about 300 mOsm. In certain such embodiments, the pharmaceutical composition is isotonic.

In some embodiments, the weight ratio between liposomes and polyols ranges from about 30:1 to about 1:2. In other embodiments, the weight ratio between liposomes and polyol is in the range of about 15:1 to about 2:1. In other embodiments, the weight ratio between liposomes and polyol is in the range of about 10:1 to about 2:1. In other embodiments, the weight ratio between liposomes and polyol is in the range of about 6:1 to about 2:1. In other embodiments, the weight ratio between liposomes and polyol is in the range of about 10:1 to about 6:1.

In some embodiments, the weight ratio between liposomes and mannitol ranges from about 10:1 to about 1:1. In other embodiments, the weight ratio between liposomes and mannitol ranges from about 6:1 to about 2:1. In certain embodiments, the weight ratio between liposomes and mannitol is about 4:1.

In some embodiments, the weight ratio between liposomes and glycerol ranges from about 15:1 to about 2:1. In other embodiments, the weight ratio between liposomes and glycerol is in the range of about 12:1 to about 2:1. In other embodiments, the weight ratio between liposomes and glycerol is in the range of about 10:1 to about 6:1.

In some embodiments, mineral acids or bases can be used to adjust the pH of the pharmaceutical composition. Non-limiting examples of suitable inorganic bases include sodium hydroxide and potassium hydroxide. The various possibilities represent separate embodiments of the invention.

According to some embodiments of the invention, GPL comprises a phosphorylcholine headgroup (phosphatidylcholine, PC-based lipid) or a phosphoglycerol headgroup (phosphatidylglycerol, PG-based lipid), and SPL is a ceramide (with N-acylsphingosine of the phosphorylcholine head group, also known as N-acylsphingosine-phosphocholine (SM-based lipid)).

PC and SM are zwitterionic phospholipids having a cationic choline and an anionic diester phosphate moiety (making up the phosphorylcholine head group). The hydrophobic portion of PC and PG includes 2 hydrocarbon (eg, acyl and alkyl) chains. SM also has two hydrophobic hydrocarbon chains, one of which is the chain of the sphingosine base itself and the other is the N-acyl chain. The shapes of PC, SM and PG in which the hydrocarbon chain exceeds 12 carbon atoms are all cylindrical because their packing parameters are in the range of 0.74-1.0. It forms lipid bilayers that become highly hydrated and vesicled above the SO to LD phase transition temperature to form lipid vesicles (liposomes). PC and PG liposome bilayers can be in the form of an ordered solid (SO) phase or a disordered liquid (LD). The transition between SO to LD phases involves an endothermic, first-order phase transition, known as the primary phase transition. _Tm is the temperature at which the largest change in heat capacity change during SO to LD phase transition occurs. The _Tm and temperature range of the SO to LD phase transition of PL depend inter alia on the PL hydrocarbon chain composition. In the LD phase (but not the SO phase), the charged phosphocholine and phosphoglycerol headgroups are highly hydrated.

In some embodiments, the term "phase transition temperature" refers to _Tm . In other embodiments, the term "phase transition temperature" refers to the temperature range of phase transition from SO to LD.

Furthermore, it should be noted that PG and SM have _Tm similar to that of corresponding PC (substituted hydrocarbon _chains of the same length). For example, the T _m of 1,2-dimyristyl- sn -glycero-3-phosphoglycerol (DMPG) is the same as the T _m of DMPC, i.e. 23°C, and 1,2-dipalmitin The T _m of phosphonyl- sn -glycero-3-phosphoglycerol (DPPG) or N-palmitoyl SM is consistent with that of _DPPC , ie 41°C. Thus, although the examples below mainly use PC-based lipids, the PL according to the invention can also be PG- or SM-based lipids.

In accordance with the principles of the present invention, a mixture of two or more PLs (e.g. two different PCs, PC and PG, two different PGs, two SMs, PC or PG and SM, etc.) The mixture formed in the healthy joint area or the dysfunctional joint) should be in the LD state.

In some specific embodiments, the liposomes comprise PC. In other specific embodiments, the liposomes comprise a combination of two different PCs. In other specific embodiments, the liposomes comprise a combination of PC and SM.

In some embodiments, the liposome is characterized in that it comprises at least one membrane comprising at least one phospholipid (PL) selected from the group consisting of glycerophospholipids (GPL) and sphingolipids (SPL), the glycerophospholipids having two identical or a different C ₁₂ -C ₁₈ hydrocarbon chain and the sphingolipid has a C ₁₂ -C ₁₈ hydrocarbon chain. The phase transition temperature at which the ordered solid (SO) to disordered liquid (LD) phase transition occurs is in the temperature range of about 20°C to about 39°C. Liposomes are used to lubricate joints with joint temperatures above the phase transition temperature. Therefore, liposomes exhibit the LD phase in the joint.

It should be noted that the above conditions are cumulative, that is, the PL contained in the liposomes (single PL or a combination of PLs with other PLs) is selected such that the liposomes will have a SO- LD phase transition temperature.

GPL or SPL may have an alkyl, alkenyl or acyl _C12 to _C18 hydrocarbon chain. In the case of the GPL, the two chains can be the same or different. In some embodiments, the GLP has a C ₁₂ -C ₁₆ hydrocarbon chain. In other embodiments, the SPL has a C ₁₂ -C ₁₆ hydrocarbon chain.

A specific embodiment relates to pharmaceutical compositions comprising liposomes containing GPL or SPL having at least one C ₁₄ acyl chain. Another specific embodiment relates to pharmaceutical compositions comprising liposomes containing GPL or SPL having at least one C ₁₅ acyl chain. Another specific embodiment relates to pharmaceutical compositions comprising GPLs having _at least one of _C14 , _C15 , C16 and _C18 acyl chains. Another specific embodiment relates to pharmaceutical compositions comprising liposomes containing SPL with C ₁₆ acyl chains. Other embodiments relate to pharmaceutical compositions comprising a combination of any of the above liposomes.

In some embodiments, at least one C ₁₂ -C ₁₈ or C ₁₂ -C ₁₆ hydrophobic chain is saturated. In other embodiments, both the C ₁₂ -C ₁₈ and C ₁₂ -C ₁₆ hydrophobic chains are saturated.

In one embodiment, the C ₁₂ -C ₁₈ or C ₁₂ -C ₁₆ hydrophobic chains are unsaturated.

Non-limiting examples of phospholipids that may be present in liposomes according to the principles of the invention include 1,2-dimyristoyl- sn -glycero-3-phosphocholine (DMPC, _Tm about 24°C); 1,2-Dipalmitoyl- sn -glycero-3-phosphocholine (DPPC, T m is 41.4°C); 1,2-Dipentadecyl- sn -glycero-3-phosphocholine (C15 , T _m is 33.0 ℃); 1,2-distearoyl- sn -glycero-3-phosphocholine (DSPC, T _m is 55 ℃); and N-palmitoyl-D-erythrosheath Aminophosphorylcholine (D-erythro C16, _Tm 41.0°C). _Tm values for various PC-based lipids can be found in "Thermotropic Phase Transitions of Pure Lipids in Model Membranes and Their Modifications by Membrane Proteins", John R. Silvius, Lipid-Protein Interactions, John Wiley & Sons, Inc., New York, 1982 and Lipid Thermotropic Phase Transition Data Base - LIPIDAT and Marsh (1990).

According to some embodiments, when a mixture of two or more PLs is used, the molar ratio of the two or more PLs is designed such that when the pharmaceutical composition is administered to a joint, the _Tm of the combination provides Liposomes in LD phase form. In other embodiments, the molar ratio is selected to provide liposomes with a phase transition temperature in the range of about 20°C to about 39°C.

In some embodiments, the liposomes comprise DMPC. In other embodiments, the liposomes consist essentially of DMPC. In other embodiments, at least one membrane of the liposome consists essentially of DMPC. In other embodiments, liposomes consisting essentially of DMPC include a tonicity agent at substantially the same concentration inside the liposome as in the medium outside the liposome. In other embodiments, liposomes consisting essentially of DMPC include polyols at substantially the same concentration inside the liposomes as in the medium outside the liposomes. In other embodiments, liposomes consisting essentially of DMPC include mannitol at substantially the same concentration inside the liposome as in the medium outside the liposome.

In some embodiments, the pharmaceutical composition comprises liposomes comprising DMPC in combination with another PC. In some embodiments, the pharmaceutical composition comprises liposomes comprising DMPC in combination with SPM.

In some embodiments, the molar percentage of DMPC in the liposome membrane ranges from about 5% to about 100%. In other embodiments, the molar percentage of DMPC in the liposome membrane ranges from about 5% to about 80%. In other embodiments, the molar percentage of DMPC in the liposome membrane is between about 10% to about 75%, about 15% to about 70%, about 20% to about 65%, about 25% to about 60%, about 30% to about 55%, about 35% to about 50%, about 5% to about 15%, about 20% to about 30%, about 5% to about 30%, about 40% to about 50%, or about 70% to about 80%. The various possibilities represent separate embodiments of the invention. In some exemplary embodiments, the molar percentage of DMPC in the liposome membrane is about 10%. In other exemplary embodiments, the molar percentage of DMPC in the liposome membrane is about 25%. In other exemplary embodiments, the molar percentage of DMPC in the liposome membrane is about 45%. In other exemplary embodiments, the molar percentage of DMPC in the liposome membrane is about 75%.

In some embodiments, the liposomes comprise a combination of DMPC and DPPC. In other embodiments, the liposomes consist essentially of DMPC and DPPC. In other embodiments, at least one membrane of the liposome consists essentially of DMPC and DPPC. In other embodiments, liposomes consisting essentially of DMPC and DPPC include a tonicity agent at substantially the same concentration inside the liposome as in the medium outside the liposome. In other embodiments, liposomes consisting essentially of DMPC and DPPC include polyols at substantially the same concentration inside the liposomes as in the medium outside the liposomes. In other embodiments, liposomes consisting essentially of DMPC and DPPC include mannitol at substantially the same concentration inside the liposome as in the medium outside the liposome.

In some embodiments, the molar percentage ratio of DMPC to DPPC ranges from about 25:75 to about 70:30. In other embodiments, the molar percentage ratio of DMPC to DPPC ranges from about 30:70 to about 65:25, from about 35:65 to about 60:30, or from about 40:60 to about 55:45. The various possibilities represent separate embodiments of the invention. In certain embodiments, the molar percentage ratio of DMPC to DPPC is about 45:55. In other embodiments, the molar percentage ratio of DMPC to DPPC is about 25:75.

In some embodiments, the phase transition temperature of liposomes comprising a combination of DMPC and DPPC ranges between about 33°C and about 37°C.

In some embodiments, the liposomes comprise DMPC and C15 in combination. In other embodiments, the liposomes consist essentially of DMPC and C15. In other embodiments, at least one membrane of the liposome consists essentially of DMPC and C15. In other embodiments, liposomes consisting essentially of DMPC and C15 include a tonicity agent at substantially the same concentration inside the liposome as in the medium outside the liposome. In other embodiments, liposomes consisting essentially of DMPC and C15 include polyols at substantially the same concentration inside the liposomes as in the medium outside the liposomes. In other embodiments, the liposomes consisting essentially of DMPC and C15 include mannitol at substantially the same concentration inside the liposome as the mannitol in the medium outside the liposome.

In some embodiments, the molar percentage ratio of DMPC to C15 ranges from about 15:85 to about 55:45. In other embodiments, the molar percentage ratio of DMPC to C15 is in the range of about 25:75 to about 45:55. In certain embodiments, the molar percentage ratio of DMPC to C15 is about 45:55. In other embodiments, the molar percentage ratio of DMPC to C15 is about 25:75.

In some embodiments, the phase transition temperature of the liposomes comprising the combination of DMPC and C15 is in the range of between about 29°C and about 31°C.

In some embodiments, at least one film comprises DMPC and DSPC. In other embodiments, the liposomes consist essentially of DMPC and DSPC. In other embodiments, at least one membrane of the liposome consists essentially of DMPC and DSPC. In other embodiments, liposomes consisting essentially of DMPC and DSPC include a tonicity agent at substantially the same concentration inside the liposome as in the medium outside the liposome. In other embodiments, liposomes consisting essentially of DMPC and DSPC include polyols at substantially the same concentration inside the liposomes as in the medium outside the liposomes. In other embodiments, liposomes consisting essentially of DMPC and DSPC include mannitol at substantially the same concentration inside the liposome as the concentration of mannitol in the medium outside the liposome.

In some embodiments, the molar percentage ratio of DMPC to DSPC is about 75:25.

In some embodiments, the phase transition temperature of liposomes comprising a combination of DMPC and DSPC is about 27°C.

In some embodiments, the liposomes comprise DMPC in combination with D-erythro C16. In other embodiments, the liposomes consist essentially of DMPC and D-erythro C16. In other embodiments, at least one membrane of the liposome consists essentially of DMPC and D-erythro C16. In other embodiments, liposomes consisting essentially of DMPC and D-erythro C16 include a tonicity agent at substantially the same concentration inside the liposome as in the medium outside the liposome. In other embodiments, liposomes consisting essentially of DMPC and D-erythro C16 include polyols at substantially the same concentration inside the liposomes as in the medium outside the liposomes. In other embodiments, liposomes consisting essentially of DMPC and D-erythro C16 include mannitol at substantially the same concentration inside the liposome as in the medium outside the liposome.

In some embodiments, the molar percentage ratio of DMPC to D-erythro C16 ranges from about 5:95 to about 50:50. In other embodiments, the molar percentage ratio of DMPC to D-erythro C16 is about 10:90 to about 45:55, about 10:90 to about 40:60, about 10:90 to about 35:65, about 10:90 to about 30:70 or about 10:90 to about 25:75. The various possibilities represent separate embodiments of the invention. In some embodiments, the molar percentage ratio of DMPC to D-erythro C16 ranges from about 5:95 to about 50:50. In some exemplary embodiments, the molar percentage ratio of DMPC to D-erythro C16 is about 10:90. In other exemplary embodiments, the molar percentage ratio of DMPC to D-erythro C16 is about 25:75.

In some embodiments, the phase transition temperature of the liposomes comprising the combination of DMPC and D-erythro C16 is in the range between about 27°C and 32°C.

In some embodiments, the liposomes comprise C15. In other embodiments, the liposomes consist essentially of C15. In other embodiments, at least one membrane of the liposome consists essentially of C15. In other embodiments, liposomes consisting essentially of C15 include a tonicity agent at substantially the same concentration inside the liposome as in the medium outside the liposome. In other embodiments, liposomes consisting essentially of C15 include polyols at substantially the same concentration inside the liposomes as in the medium outside the liposomes. In other embodiments, the liposomes consisting essentially of C15 include mannitol at substantially the same concentration inside the liposome as the concentration of mannitol in the medium outside the liposome.

The total PL concentration in the pharmaceutical composition according to some embodiments of the invention is in the range of about 20 mM to about 500 mM. In other embodiments, the concentration ranges from about 50 mM to about 300 mM. In other embodiments, the concentration ranges from about 100 mM to about 200 mM. In other embodiments, the concentration ranges from about 130 mM to about 170 mM. In certain embodiments, the total PL concentration is about 150 mM.

In some embodiments, the total PL concentration ranges from about 10 mg/ml to about 500 mg/ml. In other embodiments, the concentration ranges from about 30 mg/ml to about 300 mg/ml. In other embodiments, the concentration ranges from about 50 mg/ml to about 200 mg/ml. In certain embodiments, the total PL concentration is about 100 mg/ml.

In some embodiments, the phospholipids forming liposomes are present in an amount of about 0.1% (by weight) to about 40% (by weight), about 0.5% (by weight) to about 30% (by weight) of the total weight of the pharmaceutical composition , about 3% (by weight) to about 30% (by weight) or about 1% (by weight) to about 20% (by weight) by weight in the pharmaceutical composition. In certain embodiments, the liposome-forming phospholipids are present in the pharmaceutical composition at a weight percent of about 10% (by weight).

According to some embodiments, liposomes suitable for use in the pharmaceutical compositions of the invention do not include membrane active sterols, such as cholesterol, in their bilayers. It should be noted that the pharmaceutical composition of the present invention preferably does not contain propylene glycol. Furthermore, it should be noted that the pharmaceutical composition of the present invention preferably does not contain polydextrose.

Furthermore, it should be emphasized that the lipid system used in the pharmaceutical composition of the present invention serves as the active ingredient itself and not as a carrier for a certain pharmaceutically active agent. Thus, and as mentioned above, pharmaceutical compositions according to the principles of the present invention are substantially free of other pharmaceutically active agents. As used herein, in some embodiments, the term "substantially free of other pharmaceutically active agents" means that the pharmaceutical composition includes less than a therapeutically effective amount of a pharmaceutically active agent known for use in joint lubrication, Treating joint dysfunction, reducing joint pain, irritation and/or wear, or any combination thereof. As used herein, in some embodiments, the term "known for" means that the pharmaceutically active agent is approved for the indicated use in the present invention. In other embodiments, the term "known for" means that the pharmaceutically active agent will be approved for the indicated use in the future. In other embodiments, the term "known for use" means that the pharmaceutically active agent is mentioned in the scientific literature and/or patents as being suitable for the indicated use.

In some embodiments, the pharmaceutical compositions of the present invention do not include pharmaceutically active agents as lubricants, such as glycosaminoglycans, or pharmaceutically acceptable salts, esters or derivatives thereof, among others. In certain embodiments, the glycosaminoglycan is hyaluronic acid or a salt or ester containing hyaluronic acid. In certain embodiments, the hyaluronic acid is not encapsulated within liposomes. Alternatively or additionally, hyaluronic acid should not be dispersed in the fluid medium. In some presently preferred embodiments, the pharmaceutical composition is substantially free of hyaluronic acid or a pharmaceutically acceptable salt or ester thereof. In some embodiments, the term "substantially free" as used in conjunction with hyaluronic acid means that the pharmaceutical composition includes less than a therapeutically effective amount of hyaluronic acid or a salt or ester thereof. In other embodiments, the term "substantially free" means that the pharmaceutical composition includes less than a detectable amount of hyaluronic acid or a salt or ester thereof.

In some embodiments, the pharmaceutical composition of the present invention does not include a pharmaceutically active agent as a lubricant selected from superficial zone protein (SZP), lubricin, proteoglycan 4, and analogs and derivatives thereof .

In some embodiments, the pharmaceutical compositions of the present invention do not include pharmaceutically active agents as anti-inflammatory agents, such as xylitol, betamethasone, prednisolone, piroxicam, aspirin ( aspirin), flurbiprofen, disalicylic acid (+)-N-{4-[3-(4-fluorophenoxy)phenoxy]-2-cyclopenten-l-yl} -N-hydroxyurea, diflunisal, ibuprofen, fenoprofen, fenamate, ketoprofen, nabumetone ( nabumetone, naproxen, diclofenac, indomethacin, sulindac, tolmetin, etodolac, ketorolac , oxaprozin, celecoxib, meclofenamate, mefenamic acid, oxyphenbutazone, phenylbutazone , salicylates (salicylates) or phytosphingosine type reagents.

In some embodiments, the pharmaceutical compositions of the invention do not include pharmaceutically active agents that are antiviral agents, such as acyclovir, nelfinavir, or virazole.

In some embodiments, the pharmaceutical composition of the present invention does not include a pharmaceutically active agent that is an antibiotic, including antibiotics belonging to the following families: penicillines, cephalosporins, aminoglycosidics , macrolides, carbapenem and penem, β-lactam monocycle, β-lactamase inhibitors and tetracyclines, polypeptide antibiotics, chloramphenicol ) and derivatives, poly-etheric ionophores and quinolones. Non-limiting examples of such antibiotics include ampicillin, dapsone, chloramphenicol, neomycin, cefaclor, cefadroxil, cephalexin cephalexin, cephradine, erythromycin, clindamycin, lincomycin, amoxicillin, ampicillin, pammicillin (bacampicillin), carbenicillin, dicloxacillin, cyclacillin, picloxacillin, hetacillin, methicillin, naphthalene Nafcillin, oxacillin, penicillin G, penicillin V, ticarcillin, rifampin, tetracycline, fusidic acid, lincomicyn , novobiocine and spectinomycin.

In some embodiments, the pharmaceutical compositions of the present invention do not include pharmaceutically active agents such as benzalkonium chloride or chlorhexidine as anti-infective agents.

In some embodiments, the pharmaceutical compositions of the invention exclude pharmaceutically active agents that are steroids. As used herein, the term "steroid" refers to naturally occurring steroids and their derivatives, as well as synthetic or semi-synthetic steroid analogs that possess steroid-like activity. The steroid can be a glucocorticoid or a corticosteroid. Examples of specific natural and synthetic steroids include, but are not limited to: aldosterone, beclomethasone, betamethasone, budesonide, cprednisol , cortisone, cortivazol, deoxycorticosterone, desonide, desoximetasone, dexamethasone, difluorocortolone ), fluclorolone, flumethasone, flunisolide, fluocinolone, fluocinonide, fluocortin butyl, fluocoride fluorocortisone, fluorocortolone, fluorometholone, flurandrenolone, fluticasone, halcinonide, hydrocortisone, ectomethasone (icomethasone), meprednisone, methylprednisolone, paramethasone, prednisolone, prednisone, tixocortol, or triamcinolone Triamcinolone and its respective pharmaceutically acceptable salts or derivatives.

According to some embodiments, phospholipids are used as the sole active ingredient in the pharmaceutical compositions of the invention.

According to some embodiments, the pharmaceutical composition consists essentially of a nonionic tonicity agent comprising a polyol and liposomes, as described herein. In some embodiments, the term "consisting essentially of refers to a composition whose active ingredient is only the indicated active ingredient (i.e., liposomes), however, other compounds may be included, such Other compounds are used to stabilize, maintain or control the osmolarity, viscosity and/or pH of the formulations, but are not directly involved in the therapeutic effects of liposomes and/or phospholipids. In some embodiments, the term "composition" refers to a composition comprising liposomes, a tonicity agent, and a pharmaceutically acceptable vehicle or excipient.

Pharmaceutical compositions according to various embodiments of the invention may be sterilized and optionally mixed with adjuvants such as preservatives, stabilizers, wetting agents, synthetic emulsifiers, other useful Salts, dyes and/or aromatic substances and the like that affect osmotic pressure.

GPL, SPL, or combinations thereof form liposomes, preferably liposomes having an average diameter of greater than about 0.3 μm, greater than about 0.5 μm, greater than about 0.8 μm, or greater than about 1 μm. Liposomes can have an average diameter of less than about 10 μm, 8 μm, 7 μm, 6 μm or 5 μm. The various possibilities represent separate embodiments of the invention. According to some embodiments, the liposomes have an average diameter in the range between about 0.3 μm and 10 μm. According to other embodiments, the liposomes have an average diameter in the range between about 0.5 μm and 9 μm. According to other embodiments, the liposomes have an average diameter in the range between about 1 μm and 8 μm. According to other embodiments, the liposomes have an average diameter in the range between about 3 μm and 5 μm.

The terms "average diameter" and "average particle size" are used interchangeably herein and, in some embodiments, refer to the average diameter of liposomes derived from a particle size distribution based on a number distribution model. In some embodiments, these terms refer to the mean diameter of liposomes derived from a particle size distribution based on a volume distribution model. In other embodiments, these terms refer to the mean diameter of liposomes derived from a particle size distribution based on a surface area distribution model. The particle size distribution can be determined inter alia by laser light diffraction and/or the Coulter Counter method.

Liposomes can be single membrane liposomes or, according to some embodiments, can be multilamellar vesicular (MLV) liposomes. According to other embodiments, the liposomes may also be large multivesicular (LMVV) or dehydrated rehydrated vesicular (DRV) liposomes.

In some presently preferred embodiments, the liposomes are multilamellar vesicles (MLVs). In certain such embodiments, the liposomes have more than one membrane.

According to one embodiment, the MLV is defined by an average diameter in the range between 0.3 μm and 10 μm. According to another embodiment, the MLV is defined by an average diameter in the range between 0.5 μm and 9 μm. According to other embodiments, the MLV is defined by an average diameter in the range between about 1 μm and 8 μm. According to other embodiments, the MLV is defined by an average diameter in the range between about 3 μm and 5 μm.

In certain embodiments, the pharmaceutical composition comprises a polyol and MLV liposomes, the membranes of which consist essentially of DMPC and DPPC. The polyol may be selected from mannitol and glycerol. In another embodiment, a pharmaceutical composition comprises mannitol and MLV liposomes, the membranes of which consist essentially of DMPC and DPPC. In another embodiment, the concentration of mannitol ranges from about 1 to about 70 mg/ml. In another embodiment, the pharmaceutical composition has an osmolality in the range of about 200 to about 600 mOsm. In another embodiment, the weight ratio between liposomes and mannitol is in the range of about 6:1 to about 2:1.

In some embodiments, the pharmaceutical composition comprises mannitol and MLV liposomes, the membranes of which consist essentially of DMPC and DPPC. In some embodiments, DMPC is present in the pharmaceutical composition at a weight percentage ranging from about 20% (by weight) to about 40% (by weight) of the total weight of the pharmaceutical composition. In a certain embodiment, DMPC is present in the pharmaceutical composition at a weight percentage of about 30% (weight ratio). In some embodiments, DPPC is present in the pharmaceutical composition at a weight percentage ranging from about 30% (by weight) to about 60% (by weight) of the dry weight of the pharmaceutical composition. In a certain embodiment, DPPC is present in the pharmaceutical composition at a weight percentage of about 40% (weight ratio). In some embodiments, mannitol is present in the pharmaceutical composition in a weight percentage ranging from about 20% (by weight) to about 40% (by weight) of the dry weight of the pharmaceutical composition. In a certain embodiment, mannitol is present in the pharmaceutical composition at a weight percentage of about 30% (weight ratio).

In some embodiments, the pharmaceutical composition comprising mannitol and liposomes whose membranes consist essentially of DMPC and DPPC further comprises a histidine buffer as a fluid medium. In other embodiments, DMPC is present in the pharmaceutical composition at a weight percentage ranging from about 1% (by weight) to about 10% (by weight) of the total weight of the pharmaceutical composition. In a certain embodiment, DMPC is present in the pharmaceutical composition at a weight percentage of about 4% (weight ratio). In some embodiments, DPPC is present in the pharmaceutical composition at a weight percentage ranging from about 2% (by weight) to about 12% (by weight) of the total weight of the pharmaceutical composition. In a certain embodiment, DPPC is present in the pharmaceutical composition at a weight percentage of about 5% (weight ratio). In some embodiments, mannitol is present in the pharmaceutical composition at a weight percentage ranging from about 1% (by weight) to about 7% (by weight) of the total weight of the pharmaceutical composition. In a certain embodiment, mannitol is present in the pharmaceutical composition at a weight percentage of about 4% (weight ratio).

Pharmaceutical compositions according to various embodiments of the present invention can be used for the preparation of replacements for naturally occurring cartilage PL, ie as joint lubricants and/or wear reducing agents.

It should be noted that joint temperature in patients with reduced joint lubrication or joint wear, such as osteoarthritis, changes as the disease progresses [Hollander, JL; Moore, R., Studies in osteoarthritis using Intra-Articular Temperature Response to Injection of Hydrocortisone. Ann. Rheum. Dis. 1956 , 15, (4), 320-326]. In fact, this temperature change was used as a clinical tool for the assessment of osteoarthritis [Thomas, D.; Ansell, BM; Smith, DS; Isaacs, RJ, Knee Joint Temperature Measurement using a Differential Thermistor Thermometer. Rheumatology 1980 , 19, (1), 8-13]. In the hand joints of osteoarthritis patients, temperature variations in the range of about 28 to about 33°C were demonstrated [Varju, G.; Pieper, CF; Renner, JB; Kraus, VB, Assessment of hand osteoarthritis: correlation between thermographic and radiographic methods. Rheumatology 2004 , 43, 915-919], while the temperature of a healthy temporomandibular joint (TMJ) varies in the range of about 35 to 37°C [Akerman, S.; Kopp, S., Intra-articular and skin surface temperature of human temporomandibular joint. Scand. J. Dent. Res. 1987 , 95, (6), 493-498].

Therefore, according to the principle of the present invention, it is necessary and indeed presupposed that the PL or its mixture should be in LD phase in situ at the joint area lubricated therewith. In some embodiments, the excursion temperature (upper limit) of the SO to LD phase transition of the liposome is no more than 15°C higher than the in situ (ie, in the joint) temperature, which is in the range of about 20°C to about 39°C. According to the principles of the present invention, liposomes are formed from GPL, SPL, or combinations thereof, and thus the SO to LD phase transition temperatures described above relate to liposomes formed from GPL, SPL, and combinations thereof, thereby providing wherein PL or The mixture was liposomes in LD phase.

In certain embodiments, nonionic tonicity agents comprising polyols do not affect the phase transition temperature of liposomes. In other embodiments, the phase transition temperature of the liposomes in combination with the nonionic tonicity agent comprising a polyol differs by no more than about 10% from the phase transition temperature of the liposomes alone. In other embodiments, the phase transition temperature of the liposomes in combination with the nonionic tonicity agent comprising a polyol differs by no more than about 5% from the phase transition temperature of the liposomes alone.

The pharmaceutical composition of the present invention can be used to treat, alleviate, arrest, prevent, manage or cure any joint disorder associated with joint dysfunction or the symptoms caused thereby. As used herein, the term "joint disorder" shall always mean any ailment (congenital, autoimmune or otherwise), injury or disease of the joint area which causes degeneration, pain, decreased mobility, inflammation of the joint , irritation or physiological disruption and dysfunction. The condition can be associated with decreased joint secretions and lubrication and can result from complications of knee and hip replacements.

The joint according to the principles of the present invention may be any of the knee, hip, ankle, shoulder, elbow, tarsal, carpal, interphalangeal, and intervertebral. The various possibilities represent separate embodiments of the invention. In some embodiments, the joint is a knee joint.

Specific joint conditions include, but are not limited to, joint discharge and/or insufficient lubrication resulting from arthritis, including rheumatoid arthritis, osteoarthritis, osteoarthritis in patients with rheumatoid arthritis, traumatic joint injury (including sports injuries), joint locking (such as in the temporomandibular joint (TMJ)), post-arthrocentesis states, arthroscopic surgery, open joint surgery, joints in mammals (preferably humans) (such as the knee or hip replacement). A preferred condition to be treated or prevented by using the pharmaceutical composition of the present invention is osteoarthritis.

In certain embodiments, the pharmaceutical composition is used to reduce knee joint pain in patients with osteoarthritis.

The pharmaceutical composition of the present invention can be used as a prophylactic measure to prevent future damage or degeneration. For example, pharmaceutical compositions can be administered intra-articularly to an athlete intermittently throughout his life to minimize the risk of pressure-related injury or cartilage degeneration.

The pharmaceutical compositions of the present invention may be administered with or as an adjuvant to anti-inflammatory agents, analgesics, muscle relaxants, antidepressants, or joint-promoting agents commonly used in the treatment of conditions associated with joint stiffness, such as arthritis. Potion of lubrication. Combination treatment approaches are advantageous in reducing side effects associated with agents commonly used to prevent, manage or treat conditions associated with decreased joint lubrication, such as osteoarthritis, such as nonsteroidal, anti-inflammatory drugs (NSAIDs) . In addition to enhancing safety, combination therapy approaches may also be beneficial in improving therapeutic efficacy.

In some embodiments, the pharmaceutical compositions are in a form suitable for parenteral administration. Parenteral administration of the pharmaceutical composition of the present invention into the joint cavity of a patient can be performed by a method selected from the group consisting of intra-articular injection, arthroscopic administration or surgical administration. Accordingly, in some embodiments, pharmaceutical compositions are formulated in a form suitable for administration by a route selected from intra-articular injection, arthroscopic administration, or surgical administration. An advantageous feature of the disclosed pharmaceutical compositions is the presence of a tonicity agent, which adjusts the osmolarity of the liposomal composition to physiological values, thereby reducing side effects associated with intra-articular administration.

Pharmaceutical compositions according to various embodiments of the invention may be administered in a dose of about 0.5 ml to about 10 ml. In other embodiments, the pharmaceutical composition is administered in a dose of about 1 ml to about 6 ml. In certain embodiments, the pharmaceutical composition is administered in a dose of about 3 ml.

In some embodiments, one dosage unit of the pharmaceutical composition comprises from about 20 mg to about 350 mg of mannitol. In some embodiments, one dosage unit of the pharmaceutical composition comprises from about 40 mg to about 250 mg of mannitol. In a certain embodiment, one dosage unit of the pharmaceutical composition comprises about 120 mg mannitol. In another embodiment, one dosage unit of the pharmaceutical composition comprises about 40 mg mannitol. In another embodiment, one dosage unit of the pharmaceutical composition comprises about 250 mg mannitol.

In some embodiments, one dosage unit of the pharmaceutical composition comprises from about 50 mg to about 1000 mg of phospholipid. In some embodiments, one dosage unit of the pharmaceutical composition comprises from about 100 mg to about 800 mg of phospholipid. In a certain embodiment, one dosage unit of the pharmaceutical composition comprises about 300 mg phospholipid. In another embodiment, one dosage unit of the pharmaceutical composition comprises about 100 mg of phospholipid. In another embodiment, one dosage unit of the pharmaceutical composition comprises about 600 mg phospholipids.

In some embodiments, one dosage unit of the pharmaceutical composition comprises from about 30 mg to about 550 mg DPPC. In some embodiments, one dosage unit of the pharmaceutical composition comprises from about 50 mg to about 500 mg DPPC. In a certain embodiment, one dosage unit of the pharmaceutical composition comprises about 180 mg DPPC. In another embodiment, one dosage unit of the pharmaceutical composition comprises about 60 mg DPPC. In another embodiment, one dosage unit of the pharmaceutical composition comprises about 365 mg DPPC.

In some embodiments, one dosage unit of the pharmaceutical composition comprises from about 20 mg to about 450 mg DMPC. In some embodiments, one dosage unit of the pharmaceutical composition comprises about 40 mg to about 300 mg DMPC. In a certain embodiment, one dosage unit of the pharmaceutical composition comprises about 140 mg DPPC. In another embodiment, one dosage unit of the pharmaceutical composition comprises about 45 mg DPPC. In a certain embodiment, one dosage unit of the pharmaceutical composition comprises about 275 mg DPPC.

The pharmaceutical compositions may be dispensed in vials or single injections, or in any other manner convenient for practical use.

Individuals expected to receive administration of the pharmaceutical compositions of the invention include mammals such as, but not limited to, humans and other primates.

Throughout the description and claims of this specification, the singular forms "a" and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to "PL" is a reference to one or more PLs and "liposome" refers to one or more liposomes. Throughout the descriptions and claims of this specification, unless the context clearly dictates otherwise, plural forms of words also include a single reference. It should be noted that the term "and" or the term "or" is generally used in its sense including "and/or" unless the content clearly dictates otherwise.

In addition, throughout the description of this specification and the scope of the patent application, the words "comprise" and "containing" and the variant forms of these words, such as "comprising (comprising/comprises)", mean "including (but not limited to)" and is not intended (and does not) to exclude other parts, additives, components, integers or steps.

As used herein, when referring to measurable values such as amounts, time intervals, and the like, the term "about" is meant to encompass ±10%, more preferably ±5%, and even more preferably ±1% of the specified value. % and more preferably ±0.1%, since such deviations are suitable for carrying out the disclosed method.

The following examples are presented to more fully illustrate some embodiments of the invention. However, it should not be construed as limiting the broad scope of the invention in any way. Numerous variations and modifications of the principles disclosed herein can readily be conceived by those skilled in the art without departing from the scope of the invention.

EXAMPLES Materials and Methods Materials 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC 14:0, Catalog Number: 556200), 1,2-Dipalmitoyl-sn-glycerol- 3-Phosphocholine (DPP C 16:0, catalog number: 556300) was obtained from Lipoid (Ludwigshafen, Germany). 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC 18:0, catalog number: 850365P), 1,2-Dipentadecyl-sn-glycero-3-phosphocholine (C15 (also abbreviated herein as PC(15:0)), catalog number: 850350P) was obtained from Avanti Polar Lipids (Alabaster, AL, USA). N-palmitoyl-D-erythrosphingosine phosphorylcholine (palmitoyl sphingomyelin, D-erythro C16 (also abbreviated herein as C16 SPM), catalog number: 16608050) was obtained from Bio -Acquired from Lab ltd. (Jerusalem, Israel).

High purity water (18.2 megaOhm electrical resistance) was obtained using a WaterPro PS HPLC/Ultrafilter Hybrid System (Labconco, Kansas City, MO). HPLC grade ethanol was obtained from BioLab Ltd, Jerusalem, Israel. L-histidine monohydrochloride monohydrate, sodium hydroxide (NaOH) and d-mannitol were all obtained from Merck (Darmstadt, Germany). Glycerol was obtained from Merck, catalog number 1.04093. Sodium chloride was obtained from J. T. Baker, catalog number 4058-02.

Methods A hypotonic composition containing MLV liposomes was prepared by dissolving the desired mixture of phospholipids in 2.5 ml ethanol to a concentration of 180 mM. The solution was stirred by vortexing and placed in a water bath (60°C) for about 20 minutes. Repeat the stirring several times until the phospholipids are completely dissolved. The ethanol solution was transferred to 10 ml of warm 10 mM or 7.5 mM histidine buffer (pH 6.5), mixed vigorously by vortexing for 2 minutes to hydrate the lipids and form a dispersion of the desired MLV.

5 cycles of centrifugation and cold buffer exchange at 4°C to remove ethanol. For the liposome system composed of PC, centrifugation was performed at 3000 rpm for 40 minutes in the 1st cycle and 30 minutes at 4000 rpm in subsequent cycles. For the liposome system containing SPL, centrifugation was performed at 4000 rpm, wherein in the first cycle, two 50-minute centrifugations were performed with overnight rest between centrifugations, and in the second and third cycles , and centrifuged twice for 40 minutes at 4000 rpm. Cycles 4 and 5 were performed at 4000 rpm for 60 minutes. Monitoring for ethanol removal was performed by molar osmolality measurements. After each cold buffer exchange, pellets were resuspended using a sterile dropper to loosen sticky pellets, then tubes were tightly closed and vortexed for 2 minutes. The centrifugation process and solution replacement were repeated until the osmolality in the mixture was less than 50 mOsm. The osmolality of HB (10 mM, 7.5 mM) was measured and found to be 26 and 19 mOsm, respectively. MLVs were stored at 2-8°C until analysis.

Preparation of Isotonic Compositions Comprising MLV Liposomes and Tonicity Agents Liposomes were prepared as described above. The ethanolic solution of liposomes was transferred to 10 ml of warmed 13.5 mM histidine buffer (pH 6.5) containing a tonicity agent selected from mannitol, glycerol and sodium chloride and mixed vigorously by vortexing for 2 minutes to hydrate the lipids and form the desired dispersion of MLV.

The concentrations of tonicity agents in the histidine buffer were as follows: Glycerol - 235 mM; Mannitol - 234 mM; and NaCl - 131 mM.

The centrifugation process and solution replacement were repeated until the osmolality in the mixture was about 300 mOsm. Compositions were stored at 2-8°C until analysis.

MLV liposomes were characterized using a modified Bartlett method to measure phospholipid concentrations [Barenholz, Y. and S. Amselem, Quality control assays in the development and clinical use of liposome-based formulations . Liposome technology, 1993. 1 : pp. 527-616]. Liposome size distribution was determined by a laser diffraction particle size analyzer (LS13320 Beckman Coulter) capable of measuring particle sizes in the range of 40 nm to 2 mm. Size distribution determinations were also performed using the Coulter counting method, which is based on measuring the change in conductivity as particles suspended in a conductive fluid pass through a small pore.

Cartilage -on-cartilage ex vivo models for evaluation of lubrication efficiency were obtained from donors (ages: male 70 years old and female 68, 72, 81, 87 , 98 years old) of normal articular cartilage. Tissues were frozen at -20°C until analysis. Synovial fluid was collected from 8 donors at Hadassah Medical Center, Jerusalem.

Reagents used to prepare cartilage samples included Superglue (cyanoacrylate adhesive, 3 g), NaCl (Bio Lab LTD, Israel, 19030291 catalog number: 19030291, lot number: 57747) and ethanol (Frutarom, Israel, catalog number: 5551640, Batch number: 26141007).

Samples were prepared at the laboratory of Cartilage and Joints Diseases, Department of Biomedical Engineering Technion, IIT, Haifa, Israel. Tested at Shamban & Microsystems Tribology Laboratories of Department of Mechanical Engineering Technion, IIT, Haifa, Israel. The internal setup for friction measurements was equipped with a dynamometer with a strain gauge measurement system (HBM Z8, Germany) and LabView software (National Instruments, USA).

The device settings are as follows. Preparation and fixation of cartilage plugs. From each cartilage, 10-20 cartilage plugs with a diameter of 4 mm or 8 mm were prepared. These cartilage plugs were randomly assigned to various test formulations. 8 mm cartilage plugs were mounted in the device on the fixed holder and immersed in a solution containing 2 ml of synovial fluid (SF) and test solution in a 1:1 ratio (v/v). A 4 mm cartilage plug was secured to the upper piston.

Measurements: The friction test was repeated several times in the presence of different test samples. For each measurement, the upper cartilage plug was placed over the bottom cartilage plug and after a dwell interval of several seconds, the coefficient of friction was measured. For any given pair of cartilage plugs, no fewer than 10 independent measurements were performed, with the cartilage plugs being rotated prior to each subsequent test in all replicates to provide similar conditions.

Static coefficient of friction was measured under specific friction conditions of applied load, sliding velocity, residence time and temperature, as shown in Table 1: Table 1 : Cartilage model experimental conditions on cartilage: normal load 30N sliding speed 1 mm s ^-1 Dwell time (load duration before sliding begins) 5 seconds sliding distance 5 mm Lubricant temperature 32 to 34°C

Differential Scanning Calorimetry (DSC) Measurements To determine the _Tm of the different liposome systems, samples were scanned using a MicroCal ^™ VP-DSC GE Healthcare Life Sciences (Uppsala, Sweden, now owned by Malvern UK). Samples of MLV-containing HB and a pharmaceutical composition comprising liposomes and mannitol (at a concentration of about 20 mM phospholipids, With HB or with HB and mannitol in reference cells). Each sample under study was scanned three times at the same rate - increasing the temperature from 10°C to 75°C (scan 1), decreasing the temperature from 75°C to 10°C (scan 2) and ramping the temperature again from 10°C Ramp to 75°C (3rd scan). The calorimetric data were processed by Origin® 7.0 software. T _on and T _off of the main phase transitions were determined by extrapolating straight lines to define the temperature range of the main phase transitions. For system F1, due to the broad "shoulders" in peak 2, an additional analysis was performed by fitting the model: MN2State.

Example 1 - Effect of Tonicity Agents on the Lubrication Properties of Liposomal Pharmaceutical Compositions Cartilage lubrication by pharmaceutical compositions comprising tonicity agents and liposomes was evaluated using a suprachondral cartilage ex vivo model. The suprachondral cartilage ex vivo model provides an experimental system to test the relative effects of biolubricant formulations on the static coefficient of friction. This type of measurement can indicate the ability of different lubricants to reduce the coefficient of friction of cartilage. The suprachondral cartilage ex vivo model utilizes a setup in which two fixed plugs of human cartilage are loaded one on top of the other while submerged in different lubricant solutions. The device realizes the measurement of static friction between two cartilage samples [Merkher Y et al. "A rational human joint friction test using a human cartilage-on-cartilage." Tribology Letters (2006): 29-36]. This model has been used in the past to compare the friction coefficients of different liposome compositions [Sivan S et al. "Liposomes act as effective biolubricants for friction reduction in human synovial joints." Langmuir (2010): 1107-16].

The experiments of the present invention were designed to determine the relative lubricity properties of lipid formulations containing tonicity agents, as reflected by static coefficient of friction measurements, and compare them to hypotonic liposome formulations. Table 2 presents the pharmaceutical compositions tested. The liposome combination chosen was DMPC/DPPC with a molar percentage ratio of 45:55.

Table 2: Hypotonic and Isotonic Liposome Compositions Formulation number Phospholipids (molar percentage ratio) Tonicity agent fluid medium 1 DMPC/DPPC (45:55) - Histidine buffer 2 DMPC/DPPC (45:55) glycerin Histidine buffer 3 DMPC/DPPC (45:55) Mannitol Histidine buffer 4 DMPC/DPPC (45:55) NaCl Histidine buffer

A liposome composition comprising 183 mg DPPC and 136 mg DMPC was dispersed in 3 ml 10 mM histidine buffer (HB), pH 6.5. The liposome composition comprising 10 mM histidine buffer had an osmolarity of about 50 mOsm (Table 3). Therefore, to increase the osmolarity to an isotonic level of about 300 mOsm, the concentration of the tonicity agent should be adjusted to provide about 250 mM of solute.

Mannitol was added in an amount of 120 mg (4% by weight or 40 mg/ml) to form an isotonic composition. Glycerol was added in an amount of 61 mg (2% by weight or 20 mg/ml). Sodium chloride was added in an amount of 21 mg.

Table 3 summarizes the physicochemical properties of the different liposome compositions. Liposome compositions were prepared with three different tonicity agents, each contributing equally to the total osmolality of the formulation. The isotonicity of these formulations is about 300 mOsm. A comparison between ionic (NaCl) and non-ionic (mannitol and glycerol) tonicity agents was performed. Hypotonic liposome compositions (less than 50 mOsm) without tonicity agents were also tested. The effect of tonicity agents on the lubricity of liposome compositions was assessed using a suprachondral cartilage model setup, as described in Materials and Methods .

Table 3: Physicochemical Properties of Liposome Compositions Isotonic formulation buffer Mannitol glycerin NaCl pH 6.6 6.4 6.4 6.4 Molar Osmolality (mOsm) 49 307 299 289 Particle size by volume (µm) 5.8 4.4 3.8 4.5 Particle Size by Surface Area (µm) 3.4 2.8 2.9 3.2 Particle size in number (µm) 1.3 1.5 1.7 1.8 Analysis method DMPC (mM) 66.4 67.5 70.2 69.6 Analysis method DPPC (mM) 81.7 82.1 84.7 83.3 Phospholipid concentration (mM) 148 150 155 153 Molar percentage ratio DMPC/DPPC 55.2/44.8 45.1/54.9 45.3/54.7 45.5/54.5 Hemolysomyristylphosphatidylcholine (%) NMT ^* 0.5 ND ^** ND ND Lysopalmitoylphosphatidylcholine (%) NMT0.5 ND ND ND Mysterious Acid (%) 0.27 ND ND ND Palmitic acid(%) 0.27 0.06 0.04 ND total impurities LT ^*** 0.2 0.06 0.04 ND ^* NMT - not more than ^** ND - not detected ^*** LT - less than

Table 4 presents the preliminary results of the average static coefficient of friction obtained in the experiments. The precision of repeated measures was similar in all formulations tested, as reflected by the relative SD.

Table 4: Lubrication Properties of Liposome Compositions formulation buffer glycerin Mannitol NaCl repeat times 53 55 56 50 Average Static Friction Coefficient 0.093 0.075 0.064 0.098 SD 0.025 0.020 0.022 0.026

A comparison of the formulations showed that the formulation containing mannitol exhibited the lowest static coefficient of friction compared to the other isotonic formulations. The lowest static coefficient of friction indicates higher lubricating properties of the mannitol formulation. It was unexpectedly found that the static coefficient of friction obtained when using the mannitol formulation was about 30% lower than that obtained when using the hypotonic liposome composition without the tonicity agent. Glycerin formulations also provided better lubrication (average static coefficient of friction about 20% higher compared to hypotonic formulations). In contrast, increasing the ionic tension of liposomal composition reagents did not enhance their lubricity.

Example 2- Effect of tonicity agent on the thermotropism of liposome pharmaceutical composition The experiments of the present invention are designed to determine whether the addition of tonicity agent will affect the thermotropism and thermodynamic parameters of the liposome composition, including the SO-LD phase transition (T _on → Range of T _off ), T _p , T _m , T _1/2 and ΔH. T _on and T _off represent the temperature at the beginning and end of SO-LD phase transformation during the heating scan, T _p and T _m represent the maximum heat capacity occurring before the transformation (T _p ) and during the main transformation (T _m ) Change, T _1/2 represents the temperature (width) range at half-height of the endothermic peak representing the enthalpy change during the SO-LD phase transition, and ΔH is the area under the curve representing the total enthalpy change during the SO-LD phase transition .

Two types of liposome compositions were chosen and each composition was tested in the presence and absence of mannitol (as tonicity agent). The liposome compositions tested are presented in Table 5.

Table 5: Hypotonic and Isotonic Liposome Compositions Phospholipids (molar percentage ratio (when applicable)) Tonicity agent fluid medium DMPC/DPPC (45:55) - Histidine buffer DMPC/DPPC (45:55) Mannitol Histidine buffer 1,2-Dipentadecyl- sn -glycero-3-phosphocholine (C15) - Histidine buffer 1,2-Dipentadecyl- sn -glycero-3-phosphocholine (C15) Mannitol Histidine buffer

A DMPC/DPPC liposome composition containing 183 mg DPPC and 136 mg DMPC was dispersed in 3 ml 10 mM histidine buffer (HB), pH 6.5. The C15 liposome composition contained 212 mg (70.6 mg/ml) phospholipids. Mannitol was added in an amount of 120 mg (4% by weight) to form an isotonic composition.

Table 6 summarizes the physicochemical properties of the different liposome compositions.

Table 6: Physicochemical properties of liposome compositions. Phospholipids Tonicity agent Total PC (mM) MLV Molar Osmolality (mOsm) Size distribution by volume Average mean (µm) Median value (µm) SD DMPC/DPPC - 105 34 2.7 2.18 1.73 DMPC/DPPC Mannitol 91 288 4.24 2.70 4.61 C15 - 102.7 28 3.97 2.59 3.96 C15 Mannitol 99.3 272 3.1 2.61 1.96

For determination of thermotropism and thermodynamic parameters (T _on →T _off , T _m , T _1/2 , ΔH) of different systems, samples were scanned using MicroCal™ VP-DSC (GE Healthcare Life Sciences, Uppsala, Sweden). The calorimetric data were processed by Origin® 7.0 software. The way of determining the T _on → T _off range is described in Materials and methods .

Table 7 presents the thermotropism characterization of the liposome compositions tested as assessed by DSC scans.

Table 7: Thermotropism characterization of liposome compositions. Phospholipids Tonicity agent Thermotropism Characterization Peak 1 (before transition) Peak 2 (phase transition) T _on (°C) T _off (°C) T _p (℃) T _1/2 (°C) ΔH (Cal/mole) T _on (°C) T _off (°C) T _m (°C) T _1/2 (°C) ΔH (Cal/mole) DMPC/DPPC - 17.8 24.7 21.0 2.2 1030.5 30.44 35.19 33.6 3.5 9782.1 DMPC/DPPC Mannitol 17.6 24.5 21.0 2.5 962.7 30.49 35.42 33.7 3.3 10707.7 C15 - 21.7 27.3 24.6 2.2 1316.9 33.03 35.16 34.3 1.3 6716.9 C15 Mannitol 20.5 27.0 24.5 2.5 1009.1 32.66 35.29 34.4 1.5 6081.3

The results are summarized in Table 7 and Figures 1 and 2 indicate the effect of mannitol deficiency on the thermotropism of DMPC/DPPC 45/55 molar ratio MLV and 100 molar % C15 MLV.

Example 3 - Phase Transition Temperatures of Liposome Compositions The present study was configured to evaluate the thermotropism and thermodynamic parameters of various liposome compositions and in particular to look for those with phase transition temperatures in the range of 20°C to 39°C. Liposome combination. Because the addition of mannitol does not affect the thermotropism of liposomes (as demonstrated in Example 2), the phase transition temperatures of the various liposome compositions tested in the present study should be similar to those of the corresponding isotonic compositions .

The liposome compositions tested are presented in Table 8.

Table 8: Liposome Composition system Phospholipids (mole%) buffer Expected T _m DMPC DPPC DSPC C15 D-red C16 A1 0 100 - - - HB 41°C B1 10 90 - - - HB ＜41℃ C1 25 75 - - - HB ＜＜41℃ D1 45 55 - - - HB ~34℃ F1 75 - 25 - - HB ＜＜55℃ G1 45 - - 55 - HB ＜＜35℃ H1 25 - - 75 - HB ＜35℃ A2 - - - - 100 HB ~41℃ B2 10 - - - 90 HB ＜41℃ C2 25 - - - 75 HB ＜41℃ D2 75 - - - 25 HB ＜＜41℃ E2 90 - - - 10 HB ＜＜41℃ F2 100 - - - - HB ~24℃ G2 - - - 100 - HB ~34℃

The different MLV systems were characterized for size distribution, molar osmolality, and total PC concentration. The results are summarized in Tables 9-12.

Table 9: Physicochemical properties of MLV of different DPPC:DMPC mixtures System PC Ratio (Mole%) Total PC (mM) MLV Molar Osmolality (mOsm) Size distribution by volume Average mean (µm) Median value (µm) SD A1 DPPC 100 93 31 3.12 2.4 2.38 B1 DMPC/DPPC 10/90 111 29 3.10 2.34 2.31 C1 DMPC/ 25/75 107 27 3.92 2.64 4.03 D1 DMPC/DPPC 45/55 105 34 2.7 2.18 1.73 F2 DMPC 100 136.5 32 2.93 1.87 3.02

Based on the osmolality results described in this table, the ethanol content was less than 0.1% for all MLV systems. Based on the liposome/water partition coefficient, it is mostly in the aqueous phase.

Table 10: Physicochemical properties of MLV of DSPC:DMPC mixtures System PC Ratio (Mole%) Total PC (mM) MLV Molar Osmolality (mOsm) Size distribution by volume Average mean (µm) Median value (µm) SD F1 DMPC/DSPC 75/25 69 50 3.72 3 2.77

Based on the osmolality results described in this table, the ethanol content was less than 0.2%. Based on the liposome/water partition coefficient, it is mostly in the aqueous phase. This MLV represents the major lipid loss that occurs during ethanol removal.

Table 11: Physicochemical properties of MLV of different C15:DMPC mixtures System PC Ratio (Mole%) Total PC (mM) MLV Molar Osmolality (mOsm) Size distribution by volume Average mean (µm) Median value (µm) SD G2 PC (15:0) 100 102.7 28 3.97 2.59 3.96 G1 DMPC/PC15 45/55 103 40 4.57 3.44 3.84 H1 DMPC/PC15 25/75 97 34 3.06 2.16 2.38

Based on the osmolality results described in this table, the ethanol content was less than 0.1% for all MLV systems. Based on the liposome/water partition coefficient, it is mostly in the aqueous phase.

Table 12: Physicochemical properties of MLV of different DMPC/D-erythro C16 mixtures System PC Ratio (Mole%) Total PC (mM) MLV Molar Osmolality (mOsm) Size distribution by volume Average mean (µm) Median value (µm) SD A2 D-Red C16 100 53.9 20 4.04 2.66 4.38 B2 DMPC/D-Red C16 10/90 76.6 20 3.31 2.27 3.25 C2 DMPC/D-Red C16 25/75 85.7 twenty one 3.37 2.31 3.18 D2 DMPC/C16 D-Red C16 75/25 90.4 twenty four 2.67 2.05 1.83 E2 DMPC/D-Red C16 10/90 56.6 twenty two 3.7 3.03 2.5 F2 DMPC 100 136.5 32 2.93 1.87 3.02

Based on the osmolality results described in this table, the ethanol content was less than 0.1% for all MLV systems. Based on the liposome/water partition coefficient, it is mostly in the aqueous phase.

Thermotropism and thermodynamic parameters of liposome combinations were assessed as described in Example 2 and Materials and Methods . Table 13 summarizes the thermotropism characterization results for the liposome combinations tested and Figure 3 shows the SO-LD phase transition temperature range of MLV for different phospholipid mixtures evaluated by DSC scans.

Table 13: Characterization of thermotropism of MLV of different mixtures evaluated by DSC scans PC Ratio (Mole%) Thermotropism Characterization Peak 1 (before transition) Peak 2 (phase transition) T _on (°C) T _off (°C) T _p (℃) T _1/2 (°C) ΔH (Cal/mole) T _on (°C) T _off (°C) T _m (°C) T _1/2 (°C) ΔH (Cal/mole) DPPC:DMPC mixture DPPC (100) 32.3 37.3 34.9 2.2 1983.5 40.44 42.45 41.8 1.3 7993.7 DMPC/DPPC (10/90) 28.3 33.8 30.8 2.9 1666.2 37.77 40.91 40.0 1.9 9982.4 DMPC/DPPC (25/75) 22.6 29.8 25.8 3.2 1429.3 34.24 38.23 37.4 2.8 10363.0 DMPC/DPPC (45/55) 17.8 24.7 21.0 2.2 1030.5 30.44 35.19 33.6 3.5 9782.1 DMPC (100) 11.4 16.4 14.0 1.0 683.7 23.24 25.24 24.4 1.1 5394.7 DSPC:DMPC mixture DMPC/DSPC (75/25) 11.4 17.4 14.2 4.0 369.5 24.67 34.6 27.1 2.7 10199.9 C15:DMPC mixture C15 (100) 21.7 27.3 24.6 2.2 1316.9 33.03 35.16 34.3 1.3 6716.9 DMPC/C15 (45/55) 15.9 21.0 18.1 1.7 1059.7 28.03 30.28 29.4 1.3 7767.7 DMPC/C15 (25/75) 17.9 22.9 20.4 1.2 851.1 30.35 32.12 31.3 1.2 7107.5 D-erythro C16/DMPC mixture SPM (100) 30.2 38.8 33.9 0.8 941.5 41.14 43.24 41.8 1.0 7967.0 D-Red C16/DMPC (90/10) 32.5 36.0 33.8 0.7 200.2 38.36 41.91 39.7 2.2 7503.7 D-Red C16/DMPC (75/25) 22.3 25.8 23.8 0.5 97.5 34.7 39.77 36.5 3.1 6758.1 D-Red C16/DMPC (25/75) 13.9 19.1 16.6 2.9 126.9 30.91 36.19 32.1 3.2 7304.5 D-Red C16/DMPC (10/90) 11.4 14.8 13.0 1.3 233.9 26.68 29.72 27.5 1.7 7592.0 DMPC (100) 11.4 16.4 14.0 1.0 683.7 23.24 25.24 24.4 1.1 5394.7

Various liposome combinations can be found, including for example DMPC/DPPC (25/75), DMPC/DPPC (45/55), DMPC/DSPC (75/25), DMPC/C15 (45/55), DMPC/C15 ( 25/75), D-erythro C16/DMPC (75/25), D-erythro C16//DMPC (25/75) and D-erythro C16/DMPC (10/90), with the required 20 The phase transition temperature of the liposome membrane in the temperature range of ℃ to 39 ℃.

Example 4 - Evaluation of liposome compositions by pin -on-disc cartilage wear test Knee friction conditions were simulated in vitro by the pin-on-disc test using porcine cartilage pins sliding against CoCrMo discs. Pin-on-disk testing was performed with an OrthoPOD machine from Advanced Mechanical Technology Inc. (AMTI), Watertown, MA 02472-4800, USA. Heat the machine with a thermostat to ensure that the temperature inside the liquid is 37±3°C. Fill the cylindrical container with 20 mL of test liquid. The force exerted by each individual fixed arm was verified at 3, 10, 30, 50, and 100 N by a pin centered on the plate using a Mecmesin force gauge.

Harvest cartilage pins from pork shoulders. Use a spatula to open the joint capsule to expose the cartilage surface. Collect at least ten cylindrical pins using a hollow punch with a 5.0 mm inner diameter. Six pins (with appropriate length and minimum surface inclination) from the same animal were selected for each pin-on-disc wear test.

The lubricating ability of the pharmaceutical composition of the present invention was evaluated by measuring subchondral bone loss and height loss 12 hours after the wear test. Six cartilage pins were used per test, with three pins removed after 6 hours and another three pins retested for 6 hours. In addition to determining pin weight and mass loss, the cartilage surface was analyzed by optical mapper before and after the test. The devices used were S neox interferometry and confocal microscopy (Sensofar, Spain). For the determination of the roughness parameters, more than 12 spectral line profiles were extracted from the surface morphology after demolding. For clarification of surface inclination, such as positioning of pins, profiles were extracted from 5X confocal measurements along the north-south direction, with the highest layer facing south. For enhanced clarity, the profiles were smoothed and offset using Kaleidagraph 4.0.

A liposome-based composition comprising mannitol (formulation no. 3 from Table 2) was tested in the wear test. This composition was compared with a protein-containing liquid (containing 30 g/l of calf serum protein, EDTA and NaN ₃ ), as used in the hip mimic test (ISO 14242-1).

Results Abrasion Test A pin-on-disk test in a protein-containing fluid showed wear of the cartilage. The average mass loss of pins increased from 22 mg 6 hours after testing to 26 mg 12 hours after testing. The increase in the mean height loss of the pins was more pronounced, 0.6 mm after 6 hours of wear and 1.1 mm after 12 hours of wear. The extracted profile (Fig. 4) demonstrates the flattening of the cartilage surface after 6 hours of abrasion. Some of the cartilage material moves sideways and forms an outer bulge that is only loosely connected to the original pin. Therefore, the actual mass loss is underestimated since the measured weight includes the protruding part of the connection. After 12 hours of wear, the remaining three pins showed areas where the cartilage had worn through and subchondral bone had emerged (Figure 4 and Figures 5A, 5B).

After removal, the cartilage pins in the liposome composition (Formulation No. 3) showed signs of wear. After 6 hours of wear, the average mass loss of the pin was 14 mg and remained at this level after another 6 hours of wear. At both time points, the height loss was about 0.3 to 0.4 mm. After 6 hours of abrasion, the cartilage surface was flattened (Figure 6 and Figures 7A, 7B) and the cartilage material formed a bulge around the center. After 12 hours of wear, the cartilaginous surface of the remaining pins remained intact and did not exhibit areas where subchondral bone emerged (Figs. 8A, 8B).

Unlike the pins in the liposome composition, where the cartilage remained intact throughout the test, the cartilage pins in the protein liquid exhibited the emergence of subchondral bone after the 12 hour abrasion test. Attrition appeared to slow down in the liposome composition, as no mass loss or height loss was observed between 6 and 12 hours.

The wear results are further illustrated in Figures 9A (mass loss) and 9B (height loss). A comparison of the two liquids confirmed that the liposome composition caused less loss of mass and height of the cartilage pins.

Roughness Measurements Roughness parameters were determined from a series of extracted profiles based on interferometry with a DI 10X objective. Measurements are taken at the center of the pin that is well within the contact surface. Figures 10A and 10B show an exemplary overview after demolding of the underlying plane.

The selected roughness parameters Ra, Rk, Rpk and Rvk are shown in FIG. 11 . For the wear test in protein-based liquids, a very significant increase of 2 (p<0.01 ) in these parameters due to wear can be observed. As an example, for worn pins, the average roughness Ra increases from 0.5±0.2 µm at t=0 to 1.6±0.4 µm, and the core roughness Rk increases from 1.4±0.5 µm to 4.5±1.1 µm. For the abrasion test in liposome compositions, a small but significant increase in roughness parameters was observed except for Rpk (p>0.2). The average roughness Ra increased from 0.4±0.2 µm to 0.8±0.3 µm and the core roughness Rk increased from 0.9±0.4 µm to 2.4±0.8 µm.

Those skilled in the art should understand that the present invention is not limited to what has been specifically shown and described above. Rather, the scope of the invention includes combinations and subcombinations of the various features described above as well as variations and modifications. Accordingly, the present invention is not considered to be limited to the particular described embodiments, and the scope and concept of the present invention will be better understood by reference to the following claims.

Figure 1 shows untreated differential scanning calorimetry (DSC) thermograms of isotonic and hypotonic liposome compositions comprising DMPC/DPPC. Figure 2 shows untreated differential scanning calorimetry (DSC) thermograms of isotonic and hypotonic liposome compositions comprising C15. Figure 3 is a bar graph showing the SO to LD phase transition temperature range of liposomes comprising different phospholipid mixtures as assessed by DSC scans. The gray area indicates the temperature range of 20°C-39°C. Figure 4 shows the profiles of cartilage pins before and after abrasion tests in protein-based fluids. The location where subchondral bone emerges is marked by an arrow. Scales do not show effective altitude as profile is offset for better visibility. Figures 5A -5B show optical images of cartilage pin No. 9 in a protein-based fluid before ( Figure 5A ) and 12 hours after ( Figure 5B ) the abrasion test. In addition to the outer cartilage ring, subchondral bone (indicated by arrows) emerges where the cartilage wears away. Figure 6 shows the profile of cartilage pins before and after abrasion testing in liposome compositions. Scales do not show effective altitude as profile is offset for better visibility. Figures 7A - 7B show optical images of No. 14 cartilage pins in liposome composition before ( Figure 7A ) and 6 hours after abrasion testing ( Figure 7B ). Figures 8A-8B show optical images of cartilage pin No. 17 in liposome composition before ( Figure 8A ) and 12 hours after abrasion testing ( Figure 8B ). 9A-9B show a graphical comparison of mass loss ( FIG . 9A ) and height loss ( FIG. 9B ) for protein-based and liposome compositions. Error bars are a rough estimate of the accuracy of the measurements. Figures 10A-10B show profiles of cartilage surfaces before and after abrasion testing in protein-based liquid ( Figure 10A ) and liposome compositions ( Figure 10B ). Figure 11 shows the roughness parameters Ra (●), Rk (♦), Rpk before (t=0) and after (6 hours and 12 hours) the abrasion test for pins No. 13 to 18 tested in the liposome composition (▲) and Rvk (▼), where Ra is the arithmetic mean deviation of the roughness profile and Rk is the core roughness depth (roughness depth excluding values of the highest peak (Rpk) and lower valley (Rvk)).

Claims (20)

A pharmaceutical composition formulated to provide lubrication to mammalian joints in dosage units consisting essentially of liposomes consisting essentially of 1,2-dimyristyl-sn - composed of glycerol-3-phosphocholine (DMPC) and 1,2-dipalmityl-sn-glycero-3-phosphocholine (DPPC), wherein the liposomes are multilamellar vesicles (MLV), the MLV has at least one membrane and a phase transition temperature in the range of about 20°C to about 39°C; mannitol in an amount sufficient to reduce local irritation by preventing osmotic shock at the site of administration of the composition; and a fluid medium, which is selected from a buffer and water, wherein the liposomes are suspended in the fluid medium; wherein the pharmaceutical composition is substantially free of hyaluronic acid and any other pharmaceutically active agent; wherein the pharmaceutical composition provides a Lubrication of mammalian joints at the temperature of the phase transition temperature, wherein the presence of mannitol also improves the lubricating effect of the composition; wherein the mannitol ranges from about 1% (by weight) to about 7% (by weight) The weight percent within is present in the total weight of the pharmaceutical composition, wherein the weight ratio between the liposomes and mannitol is in the range of about 6:1 to about 2:1; and wherein the dosage of the pharmaceutical composition The unit has about 30 mg to about 550 mg DPPC, about 20 mg to about 450 mg DMPC, and about 20 mg to about 350 mg mannitol. The composition of claim 1, wherein DMPC is present in the pharmaceutical composition at a weight percentage in the range of about 1% (weight ratio) to about 10% (weight ratio), and DPPC is present in the pharmaceutical composition at about 2% (weight ratio) Ratio) to about 12% (weight ratio) in the range of weight percentages. The composition according to claim 1, wherein the buffer is a histidine buffer. The composition of claim 1, which has an osmolality in the range of about 200 to about 600 mOsm. The composition according to claim 1, which has a pH value of about 5-8. The composition of claim 1, wherein the molar percentage ratio of DMPC to DPPC is in the range of about 25:75 to about 70:30. The composition according to claim 6, wherein the molar percentage ratio of DMPC and DPPC is about 45:55. The composition according to claim 1, wherein the liposomes have an average diameter between about 0.5 μm and about 10 μm. The composition of claim 1, wherein the at least one film has a phase transition temperature of about 30°C to about 35°C. The composition according to claim 1, wherein the mammalian joint temperature is about 1-15°C higher than the phase transition temperature. The composition according to claim 1, wherein the liposome is in the form of a suspension. The composition of claim 1, wherein the composition is in a form suitable for administration selected from the group consisting of intra-articular injection, arthroscopic administration, and surgical administration. The composition according to claim 1, wherein the lubrication is used for the treatment, management or prevention of a joint disorder or condition or a symptom caused by a joint disorder or condition. A pharmaceutical composition formulated to provide lubrication to mammalian joints in dosage units consisting essentially of liposomes consisting essentially of 1,2-dimyristyl-sn - composed of glycerol-3-phosphocholine (DMPC) and 1,2-dipalmityl-sn-glycero-3-phosphocholine (DPPC), wherein the liposomes are multilamellar vesicles (MLV), The MLV has at least one membrane and a phase transition temperature in the range of about 20°C to about 39°C; mannitol in an amount sufficient to obtain an osmolality in the range of about 200 to about 600 mOsm; and A fluid medium of an amino acid buffer, wherein the liposomes are suspended in the fluid medium; wherein the composition has a pH of about 5-8; wherein the liposomes have an average diameter between about 0.5 μm and about 10 μm ; wherein the pharmaceutical composition is substantially free of hyaluronic acid and any other pharmaceutically active agent; wherein the pharmaceutical composition provides lubrication to mammalian joints having a temperature higher than the phase transition temperature, wherein the presence of mannitol also improves the combination The lubricating effect of objects; Wherein DMPC is present in the pharmaceutical composition at a weight percentage ranging from about 1% (weight ratio) to about 10% (weight ratio), and DPPC is present at about 2% (weight ratio) to about 12% (weight ratio) The weight percent in the range and mannitol are present in the total weight of the pharmaceutical composition at a weight percent in the range of about 1% (weight ratio) to about 7% (weight ratio); wherein the liposomes and mannose The weight ratio between alcohols is in the range of about 6:1 to about 2:1; and wherein the dosage unit of the pharmaceutical composition has about 20mg to about 350mg mannitol, about 30mg to about 550mg DPPC, and about 20mg to about 450mg DMPC. A use of the pharmaceutical composition according to any one of claims 1 to 13, which is used for the preparation of a medicament for treating pain in the joints of mammals and/or reducing local irritation of the joints of mammals, wherein the treatment of mammals Pain in joints and/or reducing local irritation of joints in mammals comprises: administering a pharmaceutical composition according to any one of claims 1 to 13 to the cavity of the joint to provide joint lubrication, and due to the presence of Mannitol prevents osmotic shock at the site of administration. The use according to claim 15, wherein the pharmaceutical composition is administered by a route selected from the group consisting of intra-articular injection, arthroscopic administration and surgical administration. The use according to claim 15, wherein the pharmaceutical composition is in the form of a parenteral pharmaceutical composition having a suspension of liposomes and administered at a dose of about 0.5ml to about 10ml. A use of the pharmaceutical composition as claimed in claim 14, which is used to prepare a medicament for treating the pain of the joints of mammals and/or reducing the local irritation of the joints of mammals, wherein the Treating pain in the joints of mammals and/or reducing local irritation in the joints of mammals comprises: administering the pharmaceutical composition according to claim 14 to the cavity of the joint to provide joint lubrication, and due to the presence of mannose in the composition alcohol to prevent osmotic shock at the site of administration. The use according to claim 18, wherein the pharmaceutical composition is administered by a route selected from the group consisting of intra-articular injection, arthroscopic administration and surgical administration. The use according to claim 18, wherein the pharmaceutical composition is in the form of a parenteral pharmaceutical composition having a suspension of liposomes and administered at a dose of about 0.5ml to about 10ml.

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