DESC:TECHNICAL FIELD
The present disclosure relates to the fields of disease management, pharmaceuticals and formulations. Particularly, the present disclosure provides vehicle encapsulated Specialized Pro-resolution Mediators (SPMs). More particularly, provided herein is vehicle encapsulated RvD1. Said vehicle encapsulated SPMs of the present disclosure find application in the prophylaxis and/or treatment of joint related diseases such as osteoarthritis (OA). Further provided in the present disclosure are methods of preparation and application of the vehicle encapsulated SPMs.

BACKGROUND OF THE DISCLOSURE
Osteoarthritis (OA) is the most common joint pathology. According to recent estimates, 303.1 million patients were suffering from Osteoarthritis (OA) in 2020 worldwide. OA is characterized by progressive loss of cartilage, pain, damage to the subchondral bone, and eventual loss of function of the affected joint in humans. Current treatment includes administering glucosamine, glucocorticoids, and other NSAIDS. These treatments often fail to arrest the progressing cartilage deterioration and have several other drawbacks like gastric bleeding and increased propensity to osteoporosis in women. Most OA patients eventually require highly invasive and expensive joint-replacement surgery. Other strategies, like viscosupplementation and oral glucosamine administration, have achieved inconclusive results in humans. Despite the widespread prevalence of the disease, there are no approved disease-modifying OA drugs for human use. Due to the lack of a reliable disease-modifying OA drug, tremendous loss of quality of life and revenue goes unchecked annually.

Factors like old age, diet, obesity, and trauma contribute to inflammation in humans. Cellular changes include decreased chondrocyte viability and increased proliferation, altered matrix synthesis, and increased levels of pro-inflammatory cytokines such as interleukin-1ß (IL-1ß) and tumor necrosis factor (TNF)-a, resulting in elevated production of matrix degrading enzymes and reactive oxygen species. Chronic, low-grade inflammation is a significant driver of OA and is reflected in the surge in the levels of pro-inflammatory cytokines in the synovial fluid and systemic circulation.

Current treatments for the disease rely on symptomatic relief. Targeting chronic low-grade inflammation is one of the viable strategies for treating this disease. Specialized pro-resolution mediators (SPMs) are powerful agents of resolution but are challenging to deliver because of their short half-life. Blockade of inflammation by inhibiting the action of inflammatory cytokines like IL-1ß and TNF-a is considered a viable treatment strategy for OA. However, such approaches have proved sub-therapeutic in human clinical trials. This failure is attributed to the efficient lymphatic drainage that rapidly clears (1-5 h) off the therapeutic molecules from the joint. Some therapeutics like Tanezumab (antibody against nerve growth factor (NGF)) reduce pain in the short term but fail to cease damage to the cartilage.

Since small molecule drugs diffuse rapidly out of the joint, intraarticular delivery of small drugs has not been successful to treat joint-related diseases. The need of the hour is therefore an effective drug or active agent for the treatment of OA.

SUMMARY OF THE DISCLOSURE

Addressing the above identified need in the art for an active agent for facilitating successful intraarticular delivery of small drugs for treatment or management of joint-related diseases, provided herein is a liposome encapsulated specialized pro-resolution mediator (SPM), wherein the liposome has size ranging from about 100nm to about 1µm.

In some embodiments, the liposome is formed by ampiphilic lipids.

In some embodiments, the liposome is formed by lipids selected from a group comprising 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), Egg Phosphatidylcholine (Egg PC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), methoxy-poly(ethyleneglycol)2000-distearoylphosphatidylethanolamine (DSPE-PEG2000), or any combination thereof.

In some embodiments, the lipid(s) forming the liposome further comprises sterol(s). In some embodiments, the sterol is selected from a group comprising cholesterol, beta-Sitosterol, phytosterol and 20-alpha-Hydroxycholesterol or any combination thereof. In an exemplary embodiment, the sterol is cholesterol.

In some embodiments, the liposome is formed by about 40% to about 94% by mole of DPPC; about 1% to about 50% of DSPE-PEG2000; and, optionally about 0% to about 30% of sterol by mole. In some embodiments, the liposome is formed by 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), methoxy-poly(ethyleneglycol)2000-distearoylphosphatidyletholamine (DSPE-PEG2000), and sterol at a ratio of about 85:5:10 by mole.

In some embodiments, the SPM is a resolving family molecule selected from a group comprising Resolvin D1 (RvD1), aspirin-triggered Resolvin D1 and Resolvin E1 or any combination thereof.

In some embodiments, the liposome encapsulated SPM has size ranging from about 300nm to about 500nm.

In some embodiments, the liposome encapsulated SPM comprises SPM at a concentration ranging from about 15 ng/mg of liposomes to about 1200 ng/mg of liposomes.

In some embodiments, the liposome has size ranging from about 150nm to about 1µm.

In an exemplary embodiment, the liposome has size ranging from about 300nm to about 500 nm.

Further provided herein is a method of obtaining SPM loaded liposome comprising –
a) Hydration of thin film(s) of liposome forming lipid(s) with solvent(s) to generate vesicles;
b) Extrusion of the vesicles through a filter to generate liposomes; and
c) Pelleting and re-suspending the liposomes in SPM containing solution
to obtain the SPM loaded liposome.

In some embodiments, the method further comprises washing of the obtained SPM-loaded liposomes to remove excess unloaded SPM from the extra-liposomal environment.

In some embodiments, the liposome forming lipid(s) is selected from a group comprising 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), Egg Phosphatidylcholine (Egg PC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), methoxy-poly(ethyleneglycol)2000-distearoylphosphatidylethanolamine (DSPE-PEG2000), or any combination thereof; and optionally, comprises sterol(s).

In some embodiments, the solvent employed for hydration of the lipid film is selected from solvents such as but not limited to calcium acetate, barium acetate, and magnesium acetate or any combination thereof.

In some embodiments, the extrusion is performed through filters having pore size ranging from about 100 nm to about 5µm.

In a preferred embodiment, the extrusion is performed through filters having pore size ranging from about 150 nm to about 1µm.

In an exemplary embodiment, the extrusion is performed through filters having pore size ranging from about 300 nm to about 500 nm.

In some embodiments, the SPM is a resolving family molecule selected from a group comprising Resolvin D1 (RvD1), aspirin-triggered Resolvin D1 and Resolvin E1 or any combination thereof.

In some embodiments, the SPM containing solution comprises solvent(s) selected from a group comprising sodium sulfate, lithium sulfate, and potassium sulfate acetate or any combination thereof. In some embodiments, the SPM containing solution comprises about 50ng SPM/mL solvent to about 4000ng SPM/mL solvent.

In some embodiments, the solvent employed for hydration has a pH ranging from about 5 to about 7, preferably about 6; and wherein the SPM containing solution has a pH ranging from about 2 to about 4.5, preferably about 4.

In some embodiments, the pelleting and re-suspension of the liposomes in SPM containing solution is at a temperature of about 37°C to about 60°C for about 0.5 hours to about 2 hours.

In some embodiments, the washing is performed with a solvent selected from a group comprising Phosphate Buffered Saline (PBS), Normal Saline (NS), and Hank’s Balanced Salt Solution (HBSS) or any combination thereof.

In some embodiments, the obtained SPM loaded liposome has size ranging from about 150nm to about 1µm.

In some embodiments, the method has encapsulation efficiency of about 10% to about 100%, preferably about 43% to about 99%.

In some embodiments, provided herein is a composition comprising the liposome encapsulated SPM of the present disclosure and one or more pharmaceutically acceptable excipients or additives.
In some embodiments, the composition is a sustained release formulation.

In some embodiments, the composition is formulated into an injectable formulation.

Further provided in the present disclosure is use of the liposome encapsulated SPM of the present disclosure or the composition comprising the liposome encapsulated SPM for the prophylaxis, prevention, management and/or therapeutic treatment of joint related diseases.

In some embodiments, the use of the liposome encapsulated SPM allows sustained release for about 1 day to about 15 days.

In an exemplary embodiment, the use of the liposome encapsulated SPM allows sustained release for about 9 days to about 15 days.

In another embodiment, provided herein is a method of preventing or treating joint related diseases comprising administering the liposome encapsulated SPM of the present disclosure or the composition comprising the liposome encapsulated SPM to a subject in need thereof.

In some embodiments, the administration is by way of injection, preferably an intraarticular injection. In some embodiments, the method reduces joint damage by about 4 fold to about 10 fold as compared to an untreated subject.
In some embodiments, provided herein is a kit comprising one or more of the liposome encapsulated SPM of the present disclosure or the composition comprising the liposome encapsulated SPM, one or more syringes for administration of the liposome encapsulated SPM and optionally, an instruction manual for enabling use of the kit or any combination thereof.

The present disclosure further provides a pre-filled pen or syringe comprising the liposome encapsulated SPM of the present disclosure or the composition comprising the liposome encapsulated SPM.

BRIEF DESCRIPTION OF THE ACCOMPANYING FIGURES
In order that the disclosure may be readily understood and put into practical effect, reference will now be made to exemplary embodiments as illustrated with reference to the accompanying figures. The figures together with detailed description below, are incorporated in and form part of the specification, and serve to further illustrate the embodiments and explain various principles and advantages, where:

Figure 1 depicts characterization and release profile of lipo-RvD1. (A) The size distribution of liposomes as measured using dynamic light scattering. (B) Cryo-TEM micrographs of lipo-RvD1. (C) Size stability of liposomes in phosphate buffered saline (PBS) as measured by DLS; n=3. Data is represented as mean ± SD.

Figure 2 depicts quantification and encapsulation of lipo-RvD1. (A) Plot of the area under the corresponding peak on the chromatogram (area under the curve; AUC) against concentration of RvD1 injected. n= 2 replicates for each concentration. (B) Plot depicting RvD1 loaded as a function of initial gradient provided across the lipid bilayer of the liposomes; n=3-4. ****p<0.0001 versus loading with 50 ng/mg of RvD1 using ANOVA followed by Tukey’s posthoc test. (C) Loading of RvD1 into liposomes containing different cholesterol concentrations; n=3 for each liposome test group. Groups were tested for statistical significance using ANOVA followed by Tukey’s posthoc test. Data is represented as mean ± SD.

Figure 3 depicts stable encapsulation of RvD1. (A) Retention profile of RvD1 in liposomes containing different cholesterol concentrations; n=2-3 replicates per time point in every group. (B) Quantification of in vitro release of RvD1 from lipo-RvD1 when incubated at 37°C at pH 7.4; n=3. Data were represented as mean ± SD.

Figure 4 depicts intraarticular retention studies (A) Fluorescence images of live mice depicting the difference between intraarticular retention of fluorescent liposomes and free dye at day 0 and day 1. (B) Quantification of retention of IA-injected fluorescent liposomes and free dye from the respective joint; n= 4-5 IA injected knee joints per group. **p<0.01 between % fluorescence of free drug and % fluorescence of liposomes using Analysis of Variance (ANOVA) followed by Tukey’s posthoc test. (C) Quantification of IA clearance of different sizes of liposomes as measured by Bruker XTreme II; n=4 injected knee joints per group for every time point. *p <0.05 between respective data from mice joints injected with 350 nm and 900 nm liposomes using ANOVA followed by Tukey’s posthoc test. #p <0.05 between respective data from mice joints injected with 150 nm and 900 nm liposomes using ANOVA followed by Tukey’s posthoc test. Data represented as mean±SEM.

Figure 5 depicts alleviation of cartilage damage upon prophylactic administration of lipo-RvD1. (A) Timeline of the experiment and dosage regime. (B) Plot of weights of animals with respect to time; n=4-6 mice per group for every time point. (C) Characteristic Safranin-O-stained histology sections of different groups of mice (scale bar 200 µm). (D) OARSI scores of Safranin O-stained sections of mice knee joints administered with respective treatment; n=5-7 per group. *p=0.0256 between OARSI scores of sections obtained from untreated and lipo-RvD1 treated knee joints using Kruskal-Wallis test for non-parametric datasets followed by Dunn’s posthoc test. For D, after screening all groups for outliers with the Grubbs test, one point in the lipo-RvD1 treated group proved outlier and was subsequently removed from further analysis. All values are expressed as mean ± SD.

Figure 6 depicts increase in the proportion of M2 macrophages over M1 macrophages in the synovial membrane upon prophylactic administration of lipo-RvD1. (A) IHC images depicting levels of iNOS+ M1 macrophages synovial membrane (scale bar 50 µm). (B) Quantification of iNOS+ M1 macrophages in the synovial membrane; n=4-6 animals per group. (C) IHC images depicting levels of CD206+ M2 macrophages in synovial membrane (scale bar 50 µm). (D) Quantification of CD206+ M2 macrophages in the synovial membrane; n=4-6 per group. *p=0.0153 between the levels of CD206+ cells in synovium obtained from untreated and lipo-RvD1 treated knee joints using ANOVA followed by Tukey’s posthoc test. (E) The ratio of M1/M2 cells in the synovial membrane; n=4-6 animals per group. ****p<0.0001 between the ratios of M1/M2 cells in synovium obtained from untreated and lipo-RvD1 treated knee joints using ANOVA followed by Tukey’s posthoc test. All values are expressed as mean ± SD.

Figure 7 depicts inhibition of activity of catabolic mediators upon prophylactic administration of lipo-RvD1. Representative IHC images of (A) matrix metalloproteinase 13 (MMP13), (B) A disintegrin and metalloproteinase with thrombospondin motifs-5 (ADAMTS5).

Figure 8 depicts protection of cartilage from progressing damage upon therapeutic administration of lipo-RvD1. (A) Timeline for the study and dosage regime. (B) Safranin-O-stained characteristic histological images of different groups of animals (scale bar 200 µm). (C) OARSI scores of Safranin O-stained sections of mice joints administered with respective treatment; n=5-7 per group. **p=0.0058 between OARSI scores of sections obtained from untreated and lipo-RvD1 treated knee joints using Kruskal-Wallis test for non-parametric datasets followed by Dunn’s posthoc test. Values are expressed as mean ± SD.

Figure 9 depicts increase in the proportion of M2 macrophages over M1 macrophages in the synovial membrane upon therapeutic administration of Lipo-RvD1. (A) Characteristic IHC images depicting levels of iNOS+ M1 macrophages in the synovial membrane of respective mice joints (scale bar 50 µm). (B) Quantification of iNOS+ M1 macrophages in the synovial membrane of respective mice joints; n=4-6 animals per group. **p=0.0070 between the levels of iNOS+ cells in synovium obtained from untreated and lipo-RvD1 treated knee joints using ANOVA followed by Tukey’s posthoc test. (C) IHC images depicting levels of CD206+ M2 macrophages in synovial membrane (scale bar 50 µm). (D) Quantification of CD206+ M2 macrophages in the synovial membrane; n=4-6 per group. *p=0.0185 between the levels of cells in synovium obtained from untreated and lipo-RvD1 treated knee joints ANOVA followed by Tukey’s posthoc test. (E) The ratio of M1/M2 cells in the synovial membranes of knee joints administered with respective injections; n=4-6 animals per group. For C, after screening all groups for outliers with the Grubbs test, one point in the DMM group proved outlier and was subsequently removed from further analysis.

Figure 10 depicts inhibition of the activity of catabolic mediators in OA joints by therapeutic administration of lipo-RvD1. Representative IHC images of (A) matrix metalloproteinase 13 (MMP13), (B) A disintegrin and metalloproteinase with thrombospondin motifs-5 (ADAMTS5).

Figure 11 depicts reduction of osteophytes and OA-associated pain by Lipo-RvD1. (A) Characteristic microCT images of mice knee joints administered with respective treatments. Quantification of (B) trabecular spacing, (C) trabecular thickness, and (D) fraction bone volume (bone volume/ total volume) for different treatment groups. For C, **p=0.008 between data from untreated and lipo-RvD1 treated animals using unpaired t-test. For D, *p =0.04 between respective data from untreated and lipo-RvD1 treated animals using ANOVA followed by Tukey’s posthoc test; n=3-4 joints per group. (E) Paw-withdrawal response of different treatment groups as measured by von Frey filaments. **p=0.0023 between respective data from the untreated and lipo-RvD1 treated using Mann-Whitney U-test. Values are expressed as mean ± SD.

Figure 12 depicts release profile of lipo-RvD1 in synovial fluid obtained from OA patients.

Figure 13 depicts (a) weight profiles of mice fed high-fat diet (HFD) and normal diet (ND). Serum levels of (b) cholesterol, (c) LDL, and the serum ratios (d) TC/HDL-c and (e) LDL-c/HDL-c; n=8 lean animals and n=12 obese animals (f) Characteristic safranin-O stained sections of cartilage (scale bar 50 µm). (g) OARSI scores indicating the severity of the joint damage; n=4-8 animals per group. For a, b, c, d, e, and g, *p<0.05, **p<0.01, and ***p<0.001 between the respective groups indicated in the figures using ANOVA followed by Tukey’s posthoc test. Values are expressed as mean ± SD. Scale bar 50 µm.

Figure 14 depicts effect of prophylactic administration of lipo-RvD1 on ObOA mouse model (a) Timeline of the experiment. (b) Characteristic Safranin-O-stained histology sections of different groups of mice (scale bar 200 µm). (c) OARSI scores of Safranin O-stained sections of mice knee joints administered with respective treatment; n=7 joints injected with free RvD1, n=7 DMM joints, n=7 sham joints, and n=7 joints injected with lipo-RvD1. (d) IHC images depicting levels of iNOS+ M1 macrophages synovial membrane (scale bar 50 µm). (e) Quantification of iNOS+ M1 macrophages in the synovial membrane; n=7 DMM joints, n=8 joints treated with free RvD1, n=8 sham joints, and n=8 joints treated with lipo-RvD1. (f) IHC images depicting levels of CD206+ M2 macrophages in synovial membrane (scale bar 50 µm). (g) Quantification of CD206+ M2 macrophages in the synovial membrane; n=7 joints treated with free RvD1, n=7 DMM joints, n=7 sham joints, and n= 8 joints treated with lipo-RvD1. For c and g one point in the lipo-RvD1 group was removed after outlier analysis (Grubbs test). For c, e and g *p<0.05, ***p<0.001, and ****p<0.0001 between the respective groups indicated in the figures using ANOVA followed by Tukey’s posthoc test for parametric datasets or Kruskal-Wallis test followed by Dunn’s posthoc test for nonparametric datasets. Values are expressed as mean ± SD. Scale bar 50 µm.

Figure 15 depicts effect of blank liposomes on joint pathology.

Figure 16 depicts immunohistochemical images of sections stained for expression of the catabolic markers in the ObOA group subjected to prophylactic administration of rvD1, (a)ADAMTS5 and (b)MMP13. Scale bar 50 µm.

Figure 17 depicts effect of therapeutic administration of lipo-RvD1 in ObOA mouse model. (a) Timeline for the study. (b) Safranin-O-stained characteristic histological images of different groups of animals (scale bar 200 µm). (c) OARSI scores of Safranin O-stained sections of mice joints administered with respective treatment; n=6 DMM joints, n=7 joints treated with free RvD1, n=7 sham joints, and n=7 joints treated with lipo-RvD1. (d) Characteristic IHC images depicting levels of iNOS+ M1 macrophages in the synovial membrane of respective mice joints (scale bar 50 µm). (e) Quantification of iNOS+ M1 macrophages in the synovial membrane of respective mice joints; n=6 DMM joints, n=8 joints treated with lipo-RvD1 and sham joint, and n=6 joints treated with free RvD1. (f) IHC images depicting levels of CD206+ M2 macrophages in synovial membrane (scale bar 50 µm). (g) Quantification of CD206+ M2 macrophages in the synovial membrane; n=6 joints treated with free RvD1, n=6 DMM joints, n=8 sham joints, n=7 joints treated with free RvD1, and n=8 joints treated with lipo-RvD1. For c , one point in the lipo-RvD1 group was removed after outlier analysis (Grubbs test). For c, e, and g, *p<0.05, **p<0.01, and ****p<0.0001 between the respective groups indicated in the figures using ANOVA followed by Tukey’s posthoc test for parametric datasets or Kruskal-Wallis test followed by Dunn’s posthoc test for nonparametric datasets. Values are expressed as mean ± SD. Scale bar 50 µm.

Figure 18 depicts Immunohistochemical images of sections stained for expression of the catabolic markers in the ObOA group subjected to therapeutic administration of rvD1, (a)ADAMTS5 and (b)MMP13. Scale bar 50 µm.

Figure 19 depicts Immunohistochemical images of sections stained for expression of ß-catenin when lipo-RvD1 was administered (a)prophylactically and (b)therapeutically. Scale bar 50 µm.

Figure 20 depicts Lipo-RvD1 treated mice show reduced synovitis. (a) Images of stained sections of synovial membranes of joints treated with lipo-RvD1 prophylactically. (b) Thickness of the synovial membrane of joints treated with lipo-RvD1 prophylactically; n=5-8 animals per group. (c) Images of stained sections of synovial membranes of joints treated with lipo-RvD1 therapeutically. (d)Thickness of the synovial membrane of joints treated with lipo-RvD1 therapeutically; n=5-8 animals per group. (e) Magnified images of stained sections of synovial membranes of joints treated with lipo-RvD1 prophylactically. (f) Quantification of cells in the synovial membrane of joints treated with lipo-RvD1 prophylactically. (g) Magnified images of stained sections of synovial membranes of joints treated with lipo-RvD1 therapeutically. (h) Quantification of cells in the synovial membrane of joints treated with lipo-RvD1 therapeutically. For b, d, f, h, *p<0.05, **p<0.01, and ***p<0.0001 between the respective groups indicated in the figures using ANOVA followed by Tukey’s posthoc test. Values are expressed as mean ± SD. Scale bar 50 µm.

Figure 21 depicts analgesic effect of Lipo-RvD1 in (a) prophylactic and (b) therapeutic regimen; n=6-8 mice per group in both studies. For a and b, *p<0.05 and, **p<0.01, ***p<0.001, and ****p<0.0001 between the respective groups indicated in the figures using ANOVA followed by Tukey’s posthoc test. Values are expressed as mean ± SD. Scale bar 50 µm.

DETAILED DESCRIPTION OF THE INVENTION

General definitions
As used herein, the abbreviation ‘RvD1’ has been used in reference to the molecule ‘Resolvin D1’. Reference to RvD1 in the present disclosure encompasses in scope structural and functional analogs of RvD1.

The abbreviation ‘OA’ has been used in reference to Osteoarthritis. The abbreviation ‘IA’ has been used while referring to ‘intraarticular’ injection or retention, to specify local administration or retention of a drug formulation.

‘SPM’ has been used as an abbreviation while referring to ‘Specialized Pro-resolution Mediators’. The terms ‘lipo-SPM’, ‘liposome encapsulated SPM’ and ‘SPM loaded liposome’ have been used interchangeably throughout the present disclosure while referring to SPM encapsulated in liposomes. Similarly, the terms ‘lipo-RvD1’, ‘liposome encapsulated RvD1’ and ‘RvD1 loaded liposome’ have been used interchangeably throughout the present disclosure while referring to RvD1 encapsulated in liposomes.

As used herein, the term ‘active loading’ or obvious variants thereof as used throughout the present disclosure to define the refers to the strategy employed in the present disclosure to load SPMs into liposomes by employing a differential pH gradient across the lipid bilayer to drive the SPM molecule into the intraliposomal space.

As used herein, the term ‘encapsulation efficiency’ refers to the ratio of amount of drug captured into vehicles like liposomes to the total amount drug added during the respective loading.

As used herein, the term ‘subject’ is a vertebrate, such as a mammal, such as a human. Mammals include, but are not limited to, humans, livestock, athletic animals, pets and the like.

As used herein, the term ‘sustained release’ refers to delivery of a specific drug such as SPM in the present disclosure for a prolonged period of time.

As used herein, the term ‘comprising’ when placed before the recitation of steps in a method means that the method encompasses one or more steps that are additional to those expressly recited, and that the additional one or more steps may be performed before, between, and/or after the recited steps. For example, a method comprising steps a, b, and c encompasses a method of steps a, b, x, and c, a method of steps a, b, c, and x, as well as a method of steps x, a, b, and c. Furthermore, the term “comprising” when placed before the recitation of steps in a method does not (although it may) require sequential performance of the listed steps, unless the content clearly dictates otherwise. For example, a method comprising steps a, b, and c encompasses, for example, a method of performing steps in the order of steps a, c, and b, the order of steps c, b, and a, and the order of steps c, a, and b, etc.

With respect to the use of substantially any plural and/or singular terms herein, those having skill in the art can translate from the plural to the singular and/or from the singular to the plural as is appropriate to the context and/or application. The various singular/plural permutations may be expressly set forth herein for sake of clarity. The suffix ‘(s)’ at the end of any term in the present disclosure envisages in scope both the singular and plural forms of said term.

As used in this specification and the appended claims, the singular forms ‘a’, ‘an’ and ‘the’ includes both singular and plural references unless the content clearly dictates otherwise. The use of the expression ‘at least’ or ‘at least one’ suggests the use of one or more elements or ingredients or quantities, as the use may be in the embodiment of the disclosure to achieve one or more of the desired objects or results. As such, the terms ‘a’ (or ‘an’), ‘one or more’, and ‘at least one’ can be used interchangeably herein.

Numerical ranges stated in the form ‘from x to y’ include the values mentioned and those values that lie within the range of the respective measurement accuracy as known to the skilled person. If several preferred numerical ranges are stated in this form, of course, all the ranges formed by a combination of the different end points are also included.

The terms ‘about’ or ‘approximately’ as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, are meant to encompass variations of and from the specified value, such as variations of +/-10% or less, +/-5% or less, +/-1% or less, and +/-0.1% or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier ‘about’ or ‘approximately’ refers is itself also specifically, and preferably, disclosed.

As used herein, the terms ‘include’, ‘have’, ‘comprise’, ‘contain’ etc. or any form said terms such as ‘having’, ‘including’, ‘containing’, ‘comprising’ or ‘comprises’ are inclusive and will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

As regards the embodiments characterized in this specification, it is intended that each embodiment be read independently as well as in combination with another embodiment. For example, in case of an embodiment 1 reciting 3 alternatives A, B and C, an embodiment 2 reciting 3 alternatives D, E and F and an embodiment 3 reciting 3 alternatives G, H and I, it is to be understood that the specification unambiguously discloses embodiments corresponding to combinations A, D, G; A, D, H; A, D, I; A, E, G; A, E, H; A, E, I; A, F, G; A, F, H; A, F, I; B, D, G; B, D, H; B, D, I; B, E, G; B, E, H; B, E, I; B, F, G; B, F, H; B, F, I; C, D, G; C, D, H; C, D, I; C, E, G; C, E, H; C, E, I; C, F, G; C, F, H; C, F, I, unless specifically mentioned otherwise.

Disclosure
Addressing the need in the art for effective means to treat joint-related diseases, the present disclosure provides vehicle encapsulated specialized pro-resolution mediators (SPMs). Said vehicle encapsulated SPMs of the present disclosure are stable, non-toxic and are retained for longer durations in the joints compared to free SPMs, thus generating a high local drug (SPM) concentration.

In some embodiments, the SPM is selected from a group comprising molecules from Resolvin families such as but not limited to Resolvin D1 (RvD1), aspirin-triggered Resolvin D1 and Resolvin E1.

In some embodiments, the encapsulation in carriers is achieved by employment of lipid-based delivery systems such as but not limited to liposomes. Said liposomes, in non-limiting embodiments, are synthetic liposomes.

In some embodiments, provided herein is a liposome encapsulated SPM. In a non-limiting embodiment, the present disclosure provides liposome encapsulated RvD1.

In some embodiments, the encapsulation is nano-encapsulation. In some embodiments, the said nano-encapsulation is facilitated through nano-liposomes. In exemplary embodiments of the present disclosure, the encapsulated SPM is nano-liposome encapsulated RvD1. Thus, the present disclosure, in some embodiments, provides nano-liposome encapsulated RvD1.

Specialized pro-resolution mediators (SPMs) are powerful agents of resolution but are challenging to deliver because of their short half-life. SPMs such as RvD1 polarize macrophages to a pro-resolution M2 phenotype instead of the M1 phenotype. RvD1 is versatile in its activity, and mediates clearance of debris, reduces the influx of phagocytes, and promotes anabolism in chondrocytes. Exogenously administered RvD1 reduces the severity of joint-related diseases such as osteoarthritis, but the short half-life and limited in vivo retention of such molecules limits the molecule’s therapeutic potential.

Said drawback with respect to local retention of the drug is addressed by encapsulation of RvD1 in lipid-based drug delivery systems like liposomes. Liposomes act as efficient drug carriers because of their biodegradability, low toxicity, stability, flexible synthesis methods, and ability to incorporate versatile cargo.

In an exemplary embodiment, the present disclosure provides a liposome encapsulated specialized pro-resolution mediator (SPM), wherein the liposome has size ranging from about 100nm to about 5µm.

One of the objectives of the present disclosure is to facilitate efficient intraarticular delivery of small drugs for the treatment or prophylaxis of OA and ensure their retention at the site of delivery. Without intending to be limited by theory, the liposome characterized by the above defined size allows maximization of intraarticular retention.

In some embodiments, the liposome is formed by ampiphilic lipids.

In some embodiments, the liposome is formed by lipids selected from a group comprising 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), Egg Phosphatidylcholine (Egg PC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), methoxy-poly(ethyleneglycol)2000-distearoylphosphatidylethanolamine (DSPE-PEG2000), or any combination thereof.

In some embodiments, the liposome is formed by lipids selected from a group comprising 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and methoxy-poly(ethyleneglycol)2000-distearoylphosphatidylethanolamine (DSPE-PEG2000) or a combination thereof.

In some embodiments, the lipid(s) forming the liposome further comprises sterol(s).

In some embodiments, the sterol is selected from but not limited to cholesterol, beta-Sitosterol, phytosterol and 20-alpha-Hydroxycholesterol or any combination thereof.

In some embodiments, the sterol is cholesterol. Cholesterol molecules intercalate between the long tails of other lipids and increase the fluidity and permeability of the lipid bilayer that forms the liposomes.

In exemplary embodiments, the liposome is formed by lipids selected from a group comprising dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and methoxy-poly(ethyleneglycol)2000-distearoylphosphatidylethanolamine (DSPE-PEG2000) or a combination thereof, optionally along with one or more sterol(s).

In some embodiments, the liposome is formed by lipids selected from a group comprising dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and methoxy-poly(ethyleneglycol)2000-distearoylphosphatidylethanolamine (DSPE-PEG2000) or a combination thereof, optionally along with cholesterol.

In some embodiments, the liposome is formed by lipids selected from a group consisting of dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and methoxy-poly(ethyleneglycol)2000-distearoylphosphatidylethanolamine (DSPE-PEG2000) or a combination thereof, optionally along with cholesterol.

In some embodiments, the liposome is formed by any one or both lipids selected from a group consisting of dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and methoxy-poly(ethyleneglycol)2000-distearoylphosphatidylethanolamine (DSPE-PEG2000), optionally along with cholesterol.

In some embodiments, the liposome is formed by lipids selected from a group comprising dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and methoxy-poly(ethyleneglycol)2000-distearoylphosphatidylethanolamine (DSPE-PEG2000) or a combination thereof, along with cholesterol.

In some embodiments, the liposome is formed by lipids selected from a group consisting of dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and methoxy-poly(ethyleneglycol)2000-distearoylphosphatidylethanolamine (DSPE-PEG2000) or a combination thereof, along with cholesterol.

In some embodiments, the liposome is formed by any one or both lipids selected from a group consisting of dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and methoxy-poly(ethyleneglycol)2000-distearoylphosphatidylethanolamine (DSPE-PEG2000), along with cholesterol.

In some embodiments, the liposome is formed by dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), methoxy-poly(ethyleneglycol)2000-distearoylphosphatidylethanolamine (DSPE-PEG2000) and cholesterol.

In some embodiments, the liposome comprises cholesterol at a concentration of about 0% to about 30%.

In some embodiments, the liposome comprises cholesterol at a concentration of 10% to about 30%.

In some embodiments, the liposome is formed by 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), methoxy-poly(ethyleneglycol)2000-distearoylphosphatidylethanolamine (DSPE-PEG2000), and cholesterol; wherein the liposome is formed by about 40% to about 94% by mole of DPPC; about 1% to about 50% by mole of DSPE-PEG2000.

In an exemplary embodiment, the liposome is formed by 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), methoxy-poly(ethyleneglycol)2000-distearoylphosphatidylethanolamine (DSPE-PEG2000), and cholesterol at a ratio of about 85:5:10 by mole.

In some embodiments, the liposome has size ranging from about 150nm to about 1µm.

Therefore, in some embodiments, the present disclosure provides a liposome encapsulated specialized pro-resolution mediator (SPM), wherein the liposome has size ranging from about 150nm to about 1µm.

In an exemplary embodiment, the liposome has size ranging from about 300 m to about 500 nm.

Therefore, in some embodiments, the present disclosure provides a liposome encapsulated specialized pro-resolution mediator (SPM), wherein the liposome has size ranging from about 300nm to about 500nm.

Without intending to be limited by theory, while the intended objective of sustained retention or release of SPM at the site of administration is achieved by employing liposomes having size ranging from about 100nm to about 5µm, as shown through data in the working examples, at sizes below about 150nm and above about 1µm, there is a tendency of lower retention of the SPM at the site of administration. In some embodiments, it has been observed through experiments that the optimum retention of the SPM at the site of administration is achieved by employing liposomes having size ranging from about 300 nm to about 500 nm. It will be well understood, therefore, that the size of the liposomes employed for SPM encapsulation may be varied depending on the intended span of retention, by lowering or increasing particle size within the range of about 150nm to about 5µm.

In some embodiments, the SPM loaded liposomes comprise SPM at a concentration ranging from about 15 ng/mg of liposomes to about 1200 ng/mg of liposomes.

In exemplary embodiments, the SPM is RvD1 and the RvD1 loaded liposomes comprise RvD1 at a concentration ranging from about 35.7±16.15 ng/mg of liposomes to about 1065±92 ng/mg of liposomes. Since RvD1 is extremely potent and exerts its function at pico- and nano-molar ranges, lipo-RvD1 with lower loading of about 35.7±16.15 ng/mg of lipid also efficiently achieves the desired results in terms of intraarticular retention of the drug and impedance of joint-related diseases such as but not limited to ortho-arthritis (OA).

The SPM loaded, preferably RvD1 loaded liposomes of the present disclosure allow sustained release of the SPM. In non-limiting embodiments, the SPM loaded liposomes create a depot of the SPM molecules that allows the controlled release of the molecule for up to 11 days in vitro or in vivo.

In some embodiments, the liposomes may further comprise one or more additional agents such as but not limited to imaging agents, co-drugs, excipients. In some embodiments, the liposomes are surface modified liposomes.

The present disclosure further provides a method of obtaining a vehicle encapsulated specialized pro-resolution mediator (SPMs). In some embodiments, the method of obtaining the vehicle encapsulated SPM comprises active loading of the SPM into the vehicle.

In another embodiment, the present disclosure provides a method of obtaining an SPM loaded liposome. In some embodiments, the method of obtaining the SPM loaded liposome comprises active loading of the SPM into the liposome.

In some embodiments, the method of obtaining the SPM loaded liposome comprises establishing a differential pH gradient across the lipid bilayer to drive the SPM molecule into the intraliposomal space.

In some embodiments, the SPM is RvD1 and the method of obtaining the RvD1 loaded liposome follows a remote-loading strategy.

In a non-limiting embodiment, the method of obtaining the SPM loaded liposome comprises –
- Dissolving lipids in defined molar ratios in chloroform
- Depositing lipids in the form of thin films in a round bottom flask by evaporating chloroform
- Hydration of thin films of liposome forming lipids with a solvent to generate vesicle;
- Extrusion of the vesicles through a filter of desired pore size to generate liposome of desired size; and
- Pelleting and re-suspension of the liposomes in SPM containing solution at conditions that facilitate loading of the SPM into the liposomes
- to obtain the SPM loaded liposome.

In some embodiments, the method further comprises washing of the obtained SPM-loaded liposomes to remove the excess unloaded SPM from the extra-liposomal environment.

In some embodiments, SPM is RvD1 and the method of obtaining the RvD1 loaded liposome comprises –
- Hydration of thin films of liposome forming lipids with a solvent to generate vesicle;
- Extrusion of the vesicles through a filter of desired pore size to generate liposome of desired size;
- Pelleting and re-suspension of the liposomes in RvD1 containing solution at conditions that facilitate loading of the RvD1 into the liposomes; and
- Optionally, washing of the obtained RvD1 loaded liposomes.

In some embodiments, the liposome is formed by ampiphilic lipids.

In exemplary embodiments, the liposome is formed by lipids selected from a group comprising dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and methoxy-poly(ethyleneglycol)2000-distearoylphosphatidylethanolamine (DSPE-PEG2000) or a combination thereof, optionally along with one or more sterol(s).

In some embodiments, the liposome is formed by lipids selected from a group comprising dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and methoxy-poly(ethyleneglycol)2000-distearoylphosphatidylethanolamine (DSPE-PEG2000) or a combination thereof, optionally along with cholesterol.

In some embodiments, the liposome is formed by about 40% to about 94% by mole of DPPC; about 1% to about 50% of DSPE-PEG2000; and, optionally about 0% to about 30% of sterol, preferably cholesterol.

In some embodiments, wherein the liposome is formed by 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), methoxy-poly(ethyleneglycol)2000-distearoylphosphatidyletholamine (DSPE-PEG2000), and sterol, preferably cholesterol at a ratio of about 85:5:10 by mole.

In some embodiments, the solvent employed for hydration of the lipid film is selected from solvents such as but not limited to calcium acetate, barium acetate, and magnesium acetate or any combination thereof.

In preferred embodiments, the solvent employed for hydration is calcium acetate having a pH of about 6.

In a non-limiting embodiment, the hydration may be achieved by addition of aqueous solutions, for example through pipettes, to thin films of lipids.

In some embodiments, the extrusion is performed through filters having pore size ranging from about 100 nm to about 5µm.

In a preferred embodiment, the extrusion is performed through filters having pore size ranging from about 150 nm to about 1µm.

In an exemplary embodiment, the extrusion is performed through filters having pore size ranging from about 300 nm to about 500 nm.

In some embodiments, SPM containing solution comprises solvent(s) selected from a group comprising sodium sulfate, lithium sulfate, and potassium sulfate acetate or any combination thereof.

In some embodiments, the SPM containing solution comprises about 50ng SPM/mL solvent to about 4000ng SPM/mL solvent .

In some embodiments, the SPM is RvD1 and the RvD1 containing solution comprises one or more solvents selected from a group comprising sodium sulfate, lithium sulfate, and potassium sulfate or any combination thereof. In some embodiments, the RvD1 containing solution comprises about 50ng RvD1/mL solvent to about 4000ng RvD1/mL solvent. In some embodiments, the SPM containing solution has a pH of about 4.

In exemplary embodiments, the SPM containing solution is an RvD1 containing sodium sulfate solution having a pH of about 4.

In some embodiments, the solvent employed for hydration of thin film(s) of liposome has a pH ranging from about 5 to about 7, preferably about 6; and the SPM containing solution has a pH ranging from about 2 to about 4.5, preferably about 4. In a non-limiting embodiment, said difference in pH creates a differential pH gradient across the lipid bilayer to drive the SPM molecule into the intraliposomal space.

In some embodiments, the pelleting and re-suspension of the liposomes in SPM containing solution performed is at a temperature of about 4°C to about 8°C for about 10 minutes to about 20 minutes. In a non-limiting embodiment, the pelleting is performed by centrifugation at a speed of about 20000g to about 40000g, preferably about 30000g.

In some embodiments, the washing is performed with a solvent selected from a group comprising Phosphate Buffered Saline (PBS), Normal Saline (NS), and Hank’s Balanced Salt Solution (HBSS) or any combination thereof, preferably PBS.

In some embodiments, thin films of lipids are hydrated with about 120 mM calcium acetate (pH about 6) to generate vesicles. These vesicles are extruded through filters of different pore sizes (about 1 µm, about 400 nm, and about 100 nm) to generate liposomes of desired sizes. Finally, the liposomes are pelleted and resuspended in RvD1-containing sodium sulfate solution (pH about 4) and loaded at about 50°C for about 1.5 hours. After loading, the formulation is washed twice in PBS to obtain the rvD1 loaded liposomes.

In some embodiments, the aforesaid method is able to achieve an encapsulation efficiency of about 10% to about 100%.

In exemplary embodiments, the method of the present disclosure provides an encapsulation efficiency of preferably about 43% to about 99%.

The present invention further provides a composition comprising the SPM-loaded vehicles described above. Particularly, provided herein is a composition comprising the RvD1 loaded liposomes of the present disclosure.

In some embodiments, the composition further comprises one or more pharmaceutically acceptable excipients or additives.

In some embodiments, the pharmaceutically acceptable excipients or additives are selected from a group comprising solvents, co-solvents, solubilizing, wetting, suspending, emulsifying or thickening agents, chelating agents, antioxidants, reducing agents, antimicrobial preservatives, buffers, pH adjusting agents, bulking agents, protectants, tonicity adjustors, and other special additives.

In a non-limiting embodiment, the pharmaceutically acceptable excipients or additives are selected from a group comprising saline and phosphate buffered saline (PBS) or a combination thereof.

In some embodiments, the composition is a sustained release formulation.

In some embodiments, the composition is formulated into an injectable formulation. In an embodiment, the composition is formulated as a liquid, ready-to-inject suspension.

In a non-limiting embodiment, the composition is formulated into an injection. In a non-limiting embodiment, the injection may be administered through intraarticular, intramuscular, intravenous or subcutaneous routes.

Said injection, in preferred embodiments, is fit for local administration, at the joint, for treatment or prophylaxis of joint-related diseases. Therefore, in another preferred embodiment, the injection is fit for intraarticular administration.

The SPM-loaded liposomes, preferably the RvD1 loaded liposomes of the present disclosure find application in the prophylaxis and/or treatment of joint related diseases such as but not limited to Osteoarthritis (OA).

Accordingly, the present invention provides a method of preventing or treating joint related diseases such as but not limited to Osteoarthritis (OA) comprising administering the SPM loaded vehicle or the composition comprising the SPM-loaded vehicle as described above to a subject in need thereof.

In some embodiments, the present invention provides a method of preventing or treating joint related diseases such as but not limited to Osteoarthritis (OA) comprising administering the SPM loaded liposome or the composition comprising the SPM-loaded liposome as described above to a subject in need thereof.

In some embodiments, the present invention provides a method of preventing Osteoarthritis (OA) comprising administering the RvD1 loaded liposome or the composition comprising the RvD1-loaded liposome as described above to a subject in need thereof.

In some embodiments, the present invention provides a method of treating Osteoarthritis (OA) comprising administering the RvD1 loaded liposome or the composition comprising the RvD1-loaded liposome as described above to a subject in need thereof.

In a non-limiting embodiment, the method of treating OA comprises administering the SPM loaded liposome or the composition comprising the SPM-loaded liposome as described above to a subject in need thereof at a dosage ranging from about 18 ng/dose to about 36 ng/dose.

In a non-limiting embodiment, the aforesaid methods may be employed for treatment of Osteoarthritis (OA) arising from trauma, injury, age-related wear and tear or obesity.

In some embodiments, the administration is by way of injection. In a non-limiting embodiment, the injection may be administered through intraarticular, intramuscular, intravenous or subcutaneous routes.

In some embodiments, the administration is by way of local injection.

In some embodiments, the injection is an intraarticular injection.

The SPM loaded liposomes show sustained release upon administration. Without intending to be limited by theory, smaller liposomes show longer retention than larger liposomes. This could be due to the saturation of phagocytic clearance by synovial macrophages due to a higher number of smaller particles present in the same weight of lipids compared to larger sized liposomes.

Chronic low-grade inflammation is a major driver of tissue damage in tissue related diseases like OA. Several approaches have attempted to treat OA by arresting the associated inflammation by direct intraarticular injection of the active ingredient (like anti-inflammatory antibodies and ?-3 fatty acids). However, limited in vivo retention of such active ingredients limits their therapeutic potential.

The method of prophylaxis and/or treatment of OA of the present disclosure involving intraarticular delivery of the SPM loaded liposome or the composition comprising the SPM loaded liposome, can help bypass the low oral bioavailability of drugs by generating a high local drug concentration. This allows release of the drug in a sustained fashion, reducing the dosage frequency and total administered dosage.

In an exemplary embodiment, the SPM loaded liposome shows intraarticular retention for at least about 9 days.

In a non-limiting embodiment, the SPM loaded liposome is expected to show intraarticular retention for about 1 days to about 15 days.

In a non-limiting embodiment, the SPM loaded liposome is expected to show intraarticular retention for about 9 days to about 15 days.

In some embodiments, the above method reduces joint damage by about 4 fold to about 10 fold as compared to an untreated subject.

In some embodiments, the above defined methods for prophylaxis/prevention or treatment of OA are employed independently or in combination with other treatment modules for the same or different indication(s).

OA is typically characterized by progressive loss of cartilage, pain, damage to the subchondral bone, and eventual loss of function of the affected joint in humans.

The methods employing the SPM loaded, preferably RvD1 loaded liposomes and/or compositions comprising the said loaded liposomes trigger the preferential polarization of pro-inflammatory M1 cells towards pro-resolution M2 cells, reduce levels of pro-inflammatory catabolic mediators like ADAMTS5 and MMP13, exert analgesic effect for a certain duration post-injection and lead to maintenance of a high percentage of healthy and non-hypertrophic chondrocytes.

In some embodiments, provided herein is a method of prophylaxis of OA, specifically targeted towards OA, comprising administration of the liposome encapsulated SPM or composition comprising the same. Without intending to be limited by theory, the exact source of OA-related allodynia is not known, but the Transient Receptor Potential (TRP) family of mediators is known to play a critical role in response to mechanical stimuli, including those inducing pain. Members of this family, especially TRPV1 and TRPV4, are associated with the severity of pain in OA. SPMs such as RvD1 has been shown to have an anti-nociceptive effect by targeting members of this family, especially TRPV3, TRPV4, and TRPA1. Sustained presence of SPMs such as RvD1 in the affected knee joint may help efficiently alleviate OA-associated pain, especially under a prophylactic regimen. The pain relief could be important translationally as not only would it provide immediate benefit but would also ensure patient compliance.

Taken together, the aforesaid methods reduce the net inflammatory activity in joint related diseases such as OA and allow maintenance of joint integrity.

The present disclosure further provides use of the SPM loaded vehicle or the composition comprising the SPM-loaded vehicle as described above for the prophylaxis or management of joint related diseases such as but not limited to OA.

In preferred embodiments, the present disclosure provides use of the RvD1 loaded liposome or the composition comprising the RvD1 loaded liposome for the prophylaxis or management of joint related diseases such as but not limited to OA.

Further provided herein is the SPM loaded vehicle or the composition comprising the SPM-loaded vehicle for use in the prevention or management of joint related diseases such as but not limited to OA.

In another embodiment, the present disclosure provides the RvD1 loaded liposome or the composition comprising the RvD1 loaded liposome for use in the prevention or management of joint related diseases such as but not limited to OA.

In some embodiments, the present disclosure provides use of the SPM loaded vehicle described above in the manufacture of a medicament for the prophylactic and/or therapeutic treatment of joint related diseases such as but not limited to OA.

In some embodiments, the present disclosure provides use of the RvD1 loaded liposome described above in the manufacture of a medicament for the prophylactic and/or therapeutic treatment of joint related diseases such as but not limited to OA.

In some embodiments, the said use allows sustained release for about 1 day to about 15 days.

In some embodiments, the said use allows sustained release for about 9 days to about 15 days.

In terms of mechanism, without intending to be restricted by this theory, RvD1 polarizes macrophages to a pro-resolution M2 phenotype instead of the M1 phenotype, in both treatment and prophylactic regimens. Ratio of M2/M1 cells increases with the administration of liposome encapsulated RvD1, leading to reduced inflammatory and catabolic markers such as MMP13 and ADAMTS5. The present disclosure further provides a kit having components selected from a group comprising the SPM loaded liposomes as described above, one or more pharmaceutically acceptable carrier(s), the composition comprising the SPM loaded liposomes, means for administration of the SPM loaded liposomes and an instruction manual for enabling use of the kit or any combination thereof.

In some embodiments, the present disclosure provides a kit having components selected from a group comprising the RvD1 loaded liposomes as described above, one or more pharmaceutically acceptable carrier(s), means for administration of the RvD1 loaded liposomes and an instruction manual for enabling use of the kit or any combination thereof.

In some embodiments, the means for administration of the loaded liposome is a syringe.

Accordingly, in some embodiments, the kit of the present disclosure contains components selected from a group comprising the RvD1 loaded liposomes as described above, one or more pharmaceutically acceptable carrier(s), one or more syringes for administration of the RvD1 loaded liposomes and an instruction manual for enabling use of the kit or any combination thereof.

In some embodiments, the kit comprises further components for aftercare of the treated subject such as but not limited to dressing(s), means for temperature regulated compression, topical ointments and additional medication.

The present disclosure further provides pre-filled pens or syringes comprising the SPM loaded vehicle or the composition comprising the SPM-loaded vehicle as described above.

In some embodiments, the present disclosure provides pre-filled pens or syringes comprising the RvD1 loaded liposomes or the composition comprising the RvD1 loaded liposomes as described above.

Said pre-filled pens or syringes allow ease of administration and self-administration of the SPM loaded vehicle, preferably the RvD1 loaded liposomes, for the prophylaxis or management of joint related diseases such as but not limited to OA.

It is to be understood that the foregoing descriptive matter is illustrative of the disclosure and not a limitation. While considerable emphasis has been placed herein on the particular features of this disclosure, it will be appreciated that various modifications can be made, and that many changes can be made in the preferred embodiments without departing from the principles of the disclosure. Those skilled in the art will recognize that the embodiments herein can be practiced with modification within the spirit and scope of the embodiments as described herein. Similarly, additional embodiments and features of the present disclosure will be apparent to one of ordinary skill in art based upon description provided herein.

Descriptions of well-known/conventional methods/steps and techniques are omitted so as to not unnecessarily obscure the embodiments herein. Further, the disclosure herein provides for examples illustrating the above-described embodiments, and in order to illustrate the embodiments of the present disclosure certain aspects have been employed. The examples used herein for such illustration are intended merely to facilitate an understanding of ways in which the embodiments herein may be practiced and to further enable those of skill in the art to practice the embodiments herein. Accordingly, the following examples should not be construed as limiting the scope of the embodiments herein.

EXAMPLES:

EXAMPLE 1: Liposome synthesis
Liposomes were synthesized by the thin-film lipid hydration method. Briefly, the lipids DPPC, DSPE-PEG, and cholesterol were dissolved in chloroform and mixed in their respective molar ratios (85:5:10) in a round bottom flask. The chloroform was evaporated using a rotatory evaporator (DLAB RE100 Pro) for about 45 mins, thus forming thin lipids films. The films generated were hydrated using suitable solutions as per the intended experiments (AF750 in PBS for in vivo retention experiments, calcium acetate for all RvD1 loading experiments) at about 45 °C for about 20 mins. The vesicles were then collected and passed through 1 µm, 400 nm and 100 nm membranes to generate liposomes of a defined size. The sizes and morphology of particles were then measured using dynamic light scattering (DLS) (fig. 1A) and cryo-TEM (fig. 1B). The sizes of liposomes obtained were about 150 nm, about 350 nm, and about 900 nm (fig. 1A). To test the stability of the liposomes, the liposomes were incubated in PBS for up to about 10 days at about 37°C. The sizes of liposomes were measured using Malvern Zetasizer µV. It was observed that the liposomes were stable and maintained their size in PBS for more than about 10 days (fig. 1C).

EXAMPLE 2: Liposome loading
For the purposes of comparison, RvD1 was initially loaded passively by hydrating dry films of DPPC, DSPE:PEG and cholesterol at molar ratio 85:5:10 with about 1 mL of a solution of RvD1 in PBS comprising about 1 µg/mL of RvD1. The concentration of RvD1 entrapped in the liposomes was measured using a standard curve via HPLC (fig. 2A). The encapsulation efficiency when RvD1 was loaded passively was found to be less than about 1%. The low encapsulation efficiency could be because at pH 7.4, majority of the molecules are ionized and have negligible solubility in the lipid bilayer, and total combined intraliposomal volume is about 102–103 fold lower than the total bulk volume of the RvD1 suspension.

To overcome this challenge of low loading, RvD1 was then loaded into the liposomes of Example 1 actively by employing a differential pH gradient across the lipid bilayer to drive the RvD1 molecule into the intraliposomal space.

Thin films of lipids (described in example 1) were hydrated with about 120 mM calcium acetate (pH=6) to generate multilamellar vesicles. These vesicles were extruded through filters of different pore sizes (about 1 µm, about 400 nm, and about 100 nm) to generate liposomes of desired sizes. Finally, the liposomes were pelleted and resuspended in RvD1-containing sodium sulfate solution (pH=4) and loaded at about 50 °C for about 1.5 hours. After loading, the RvD1-loaded liposomes were washed twice in PBS. This strategy provided an encapsulation efficiency of about 71±28% and loading of about 35.7±16.15 ng/mg of lipid.
RvD1 loading in liposomes was tunable and RvD1 were loaded at various different levels – initially starting with about 50ng/mg lipid, about 200ng/mg lipid and about 2000ng/mg lipid to yield liposomes having all the way up to about 1065±92 ng RvD1/mg liposomes) (fig. 2B). Since RvD1 is extremely potent and works at pico- and nano-molar ranges, lipo-RvD1 with lower loading (about 35.7±16.15 ng/mg of lipid) was used for subsequent experiments.

The above experiment was repeated with Resolvin E1 and aspirin-triggered RvD1(AT-RvD1) as candidates for loading into liposomes by the above-described mechanism of active loading. The loading experiments with Resolvin E1 and aspirin-triggered RvD1(AT-RvD1) into liposomes using the active loading strategy revealed high encapsulation efficiencies of about 17.17±0.14% and about 55.5±1.4% respectively.

EXAMPLE 3: Effect of cholesterol concentration on intraliposomal retention
The intraliposomal retention of small-molecule drugs over time directly correlates with their composition, especially cholesterol concentration. In order to understand and ascertain the effect of cholesterol concentration on liposomal loading and drug retention, lipo-RvD1 having about 10%, about 20% and about 30% cholesterol were prepared as per Examples 1 and 2.
While the percentage of cholesterol in the lipid formulations was not found to affect RvD1 loading (Table 1, fig. 2C), 10% cholesterol formulations showed slower release in PBS than other formulations containing higher amounts of cholesterol (Table 2, fig. 3A).

Table 1: Effect of cholesterol on RvD1 loading
10% cholesterol 20% cholesterol 30% cholesterol
Loading
(ng RvD1/mg lipid) 60.06 45.08 51.62
54.93 45.23 50.66
44.44 5.87 30.48

Table 2: Effect of cholesterol on RvD1 retention in liposomes
Time (in days) RvD1 retained (ng/mg lipid)
10% Cholesterol 20% Cholesterol 30% Cholesterol
0 60.06 54.93 44.44 45.08 45.23 5.87 51.62 50.66 30.48
3 8.59 8.55 6.63 5.75 3.27 ND* 4.43 4.37 2.93
6 4.46 5.77 5.875 1.84 2.01 1.42 3.64 3.52 ND
*ND – Not determined
10% cholesterol content was employed for all subsequent experiments.

EXAMPLE 4: In-vitro and in-vivo sustained release studies – effect of liposome on retention
To test temporal release of RvD1 in vitro, the lipo-RvD1 of Example 2 (comprising about 35.7±16.15 ng of RvD1/mg of lipid and 10% cholesterol) were incubated in PBS at 37°C for various time intervals. Drug remaining in the liposomes was quantified using HPLC. It was found that the RvD1 molecules were retained intra-liposomally for at least about 11 days (fig. 3B).

Similarly, to evaluate the sustained release of RvD1 from the lipo-RvD1 of Example 2, in synovium-like conditions, the lipo-RvD1 was incubated in synovial fluid obtained from joints of patients undergoing joint replacement surgery at 37ºC for various time intervals. At intervals of 0, 5 and 10 days, the liposomes were collected, and the drug retained was quantified using HPLC. It was found that the RvD1 molecules were retained intra-liposomally for about 11 days (figure 12).

For testing retention in vivo, the liposomes of Example 1 were synthesized to comprise fluorescent dye AF750. Said dye, loaded into the liposome, allowed for sensitive quantification of fluorescence through live tissues since its emission spectrum has little overlap with tissue autofluorescence. For purposes of comparison, free dye and the encapsulated dye were injected into mice through intraarticular injection. Data showed that intraarticular injected liposomes had significantly higher retention than free dye from day 1 onwards. While more than 90% of the free dye was cleared within 1 day, liposome-encapsulated dye signal was present even after 14 days (fig. 4A, 4B).
The above therefore shows that the liposome encapsulated RvD1 of the present disclosure allows for sustained release of the RvD1 and helps avoid rapid diffusion of the drug out of the joint.

EXAMPLE 5: Effect of particle size
Intraarticular retention of RvD1 loaded liposomes of three different sizes – about 150nm, about 350nm, and about 900 nm prepared as per Examples 1 and 2 was tested by the same method as described in Example 4 – basing reliance on the fluorescent signal from liposomes loaded with fluorescent dyes. Results are depicted in the below table.

Table 3: Effect of liposome size on retention
Time (in days) % Fluorescence retained
150 nm Liposome 350 nm Liposomes 900 nm Liposomes
0 19.7 109 109 162 12.7 291 60.3 35.5 58.7 88.6 69.7 183
1 65.1 50.3 29.9 55.4 14.5 112 122 90.2 14.1 10.9 8.28 32.5
2 49.8 40.9 23.2 41.2 7.04 74.1 60.8 50.5 4.3 4.3 4.44 9.01
3 36.1 35.7 17.3 30.2 3.47 42.1 24.6 29 1.84 3.06 2.64 3.26
6 11.9 9.29 3.4 3.83 1.32 7.84 4.82 8.28 0.628 1.41 0.986 0.526
8 6.87 4.32 1.1 1.43 1.09 6.76 4.17 4.1 0.354 0.89 0.611 0.278
14 0.881 0.52 -0.0146 0.00608 0.00467 1.26 0.439 0.776 -0.166 -0.168 -0.0046 -0.253

Results showed that smaller liposomes (about 100-500 nm, more preferably about 350nm) had longer retention than larger liposomes (about ~900 nm) (Table 3, fig. 4C).

EXAMPLE 6: Prophylactic effect of Lipo-RvD1 on post-traumatic OA
The surgical model of destabilization of the medial meniscus (DMM) is a reliable model for post-traumatic OA (PTOA), which is prevalent in about 12-15% of all osteoarthritis (OA) patients. The medial meniscus is soft fibrocartilage that is located between the articulating surfaces and absorbs mechanical shock. Surgically cutting this tissue results in contact between the two articulating surfaces and visible OA-like changes over about 1-3 months. DMM surgery was performed in mice and a prophylactic dosing regimen was employed by injecting freshly synthesized lipo-RvD1 intraarticularly at weeks 1, 4, and 8 after surgery at a dosage of about 25ng/joint/dose (fig. 5A). Weight monitoring showed no adverse effects as animals in groups continued to gain weight at steady rate (Table 4, fig. 5B).

Table 4: Effect of administration on lipo-RvD1 on weight of mice
Time in weeks Weight of mice (g)
Free RvD1 Untreated Lipo-RvD1 treated
1 24 20 26 22 27 28 27 22 23 25 23 22 25 27 29 29
2 27 21 31 30 29 25 33 27 27 26 23 25 27 29 29 29
3 29 22 33 28 31 33 28 32 27 24 30 25 29 29 30 30
4 28 23 31 32 33 34 29 31 25 32 28 ND ND ND ND ND
5 30 24 31 34 35 35 21 29 31 31 32 ND ND ND ND ND
6 31 24 32 33 35 36 22 23 30 31 31 26 27 30 30 30
7 31 24 ND ND ND ND ND ND ND ND ND 26 26 29 30 31
8 30 24 29 33 33 34 22 30 32 ND ND 26 27 28 30 31
9 ND ND ND ND ND ND ND ND ND ND ND 27 27 27 28 30
10 31 24 27 32 35 ND 31 31 23 ND 33 27 27 28 29 29
11 32 24 26 33 34 ND 24 32 32 ND 33 24 26 27 27 27
12 33 25 25 30 33 ND 25 30 31 ND 36 22 24 26 28 28

Further studies showed that liposomes could impede OA progression by maintaining the overall joint integrity. Specifically, it was observed that the lipo-RvD1 treated mice had a well-maintained matrix in all the cartilage layers compared to free-RvD1 and untreated mice, which in some animals, suffered severe denudation (Table 5, Figure 5D). The parameters of joint damage like cartilage loss, chondrocyte apoptosis, and formation of osteophytes can be quantified on a scale of 0-24 using guidelines laid by Osteoarthritis Research Society International (OARSI). The OARSI scores were calculated post-euthanasia, 3 months post-surgery. These scores are shown in Table 5.

Table 5: OARSI scores
OARSI scores
Sham DMM Free RvD1 Liposomes
0 5 1.33 1.125
2 20 7 4.9
6.8 16 0.57 1.6
0.8 16 5.25 1.6
0.2 10.5 15.33 0.166
ND 14.4 ND 14.300**
ND 7.5 ND ND
ND 6 ND ND
**Outlier (analyzed through Grubbs outlier analysis)

Additionally, the Safranin-O-stained histology sections of different groups of mice also showed a higher percentage of healthy and non-hypertrophic chondrocytes in lipo-RvD1 treated animals (fig. 5C). Untreated DMM mice had higher levels of M1 cells which is indicative of a pro-inflammatory phenotype (Table 6, fig. 6A, 6B).

Table 6: iNOS+ cells in synovium
iNOS+ cells/mm2
DMM Sham Free RvD1 Lipo-RvD1
763.4407 252.3185 585.0752 137.5569
307.027 216.9469 537.2222 239.349
540.5985 122.1505 1006.916 396.1638
680.0812 39.61638 971.5445 269.6114
452.287 ND ND 500.707
469.5274 ND ND 160.9415

Further, Lipo-RvD1 treatment was found to trigger preferential polarization towards M2 cells (Table 7, fig. 6C, 6D).

Table 7: CD206+ cells in synovium
CD206+ cells/mm2
DMM Sham Free RvD1 Lipo-RvD1
429.1775 1000.726 1708.456 1512.025
118.2989 983.3352 1777.235 1109.259
174.5008 38.51592 1353.56 1636.534
236.5978 921.0809 874.8618 792.3276
1136.613 ND ND 2026.213

It was further observed that Lipo-RvD1 successfully decreased the ratio of M1/M2 cells (Table 8, fig. 6E).

Table 8: Ratio of M1/M2 cells
DMM Sham Free RvD1 Lipo-RvD1
1.822301 0.342883 0.335712 0.097204
0.732861 0.294815 0.308254 0.169136
1.290386 0.165994 0.577761 0.279949
1.623325 0.053836 0.557465 0.190521
1.07959 ND ND 0.353824
1.120742 ND ND 0.113729

It was therefore seen that lipo-RvD1 can reduce the net inflammatory activity of the synovium by reducing M1 cells and promoting clearance of debris and other inflammatory factors by increasing pro-resolution M2 cells in the joint more efficiently that free RvD1.

It was also observed that the treated OA joint also had reduced expression of pro-inflammatory catabolic mediators like ADAMTS5 and MMP13 (fig. 7A, 7B).

EXAMPLE 7: Therapeutic effect of Lipo-RvD1 on post-traumatic OA
To test therapeutic efficacy of lipo-RvD1, a treatment regimen was designed wherein the lipo-RvD1 formulation was administered about 4 weeks and about 8 weeks after DMM surgery at a dosage of about 25ng/joint/dose (fig. 8A). This timeline was selected since OA-like changes in cartilage begin to appear within about 2 weeks after the DMM surgery. In this regimen, the number of IA interventions were reduced (two administrations instead of three administrations employed for prophylactic treatments). Results suggest that even though the free RvD1 partially arrested the damage (as seen from Safranin-O-stained sections (fig. 8B)), it was not sufficient to prevent functional deterioration, as observed from OARSI scoring. On the contrary, intraarticular lipo-RvD1 administration was much more effective in treating the damage (Table 9, fig. 8B, 8C).

Table 9: OARSI scores
DMM Sham Free drug Lipo-RvD1
22 2.8 4 1.6
20.4 2 20 7.2
14.00* 3 20 0
22 1.75 17.5 20.000000*
20 4.333333 ND 0.8
ND ND ND 1.857143

Similar to the prophylactic study, lipo-RvD1 treatment decreased the levels of proinflammatory M1 macrophages in the synovial membrane (Table 10, fig. 9A, 9B) while simultaneously increasing the levels of pro-resolution M2 macrophage (Table 11, fig. 9C, 9D).

Table 10: iNOS+ cells in synovium
DMM Sham Free RvD1 Lipo-RvD1
1702.266 54.96773 1737.718 184.1147
2228.421 50.89604 48.25072 41.26706
99.04095 483.7769 74.9156 31.7439
1809.402 218.398 82.53413 27.51138
1542.471 88.72419 ND 24.12535
ND ND ND 62.72594

Table 11: CD206+ cells in synovium
DMM Sham Free RvD1 Lipo-RvD1
11.00455 95.73959 1667.189 2046.846
594.2457 907.8754 1166.482 928.7262
0 797.8298 874.8618 967.7126
528.2184 576.3634 1048.183 779.9475
983.8068 823.9658 ND 1782.737
ND ND ND 1467.457

Besides, results also showed that administration of lipo-RvD1 in a therapeutic regime decreased the ratio of M1/M2 cells in the synovial membrane (Table 12, fig. 9E).

Table 12: Ratio of M1/M2 cells
DMM Sham Free RvD1 Lipo-RvD1
1.778846 0.2521355 0.383792 0.09097525
2.59559 0.2206288 0.5761905 1
3.098392 3.175966 0.592233 0.2420819
2.875087 0.05376344 0.5500668 0.3402778
0.3971015 ND ND 0.2471147
ND ND ND 0.1259364

Catabolic enzymes like MMP13 and ADAMTS5, which are known to be major drivers of damage, were also upregulated in the cartilage of mice that had undergone surgery, as seen in IHC images. It was observed that the lipo-RvD1 treatment reduced the expression of these damaging enzymes (fig. 10A, 10B).

EXAMPLE 8: Effect of Lipo-RvD1 on symptoms of post-traumatic OA
The effect of lipo-RvD1 on two major clinical symptoms associated with OA: osteophytes and pain was analyzed in this experiment. The same dosing regimen as provided in Figure 7 was followed. MicroCT data showed that surgically induced OA resulted in increase of bony growth in the joint which was inhibited by both free and lipo-RvD1 (fig. 11A). Subchondral bone was analyzed for trabecular thickness, spacing and bone vs total volume. The observations are provided in Tables 13-15.

Table 13: Effect of Lipo-RvD1 on symptoms of OA -Trabecular spacing
Trabecular spacing (µm)
DMM Sham Free RvD1 Lipo-RvD1
302.85 452.495 437.091 404.048
360.801 461.621 358.247 403.699
352.798 478.1 363.098 359.565
ND ND ND 468.494

Table 14: Effect of Lipo-RvD1 on symptoms of OA -Trabecular thickness
Trabecular thickness (µm)
DMM Sham Free RvD1 Lipo-RvD1
145.982 136.334 101.659 133.232
159.935 108.712 134.037 139.748
163.562 122.848 127.104 135.638
ND ND ND 129.2

Table 15: Effect of Lipo-RvD1 on symptoms of OA - bone vs total volume
DMM Sham Free RvD1 Lipo-RvD1
0.325248 0.231534 0.188695 0.247975
0.307133 0.190612 0.272276 0.25715
0.31676 0.204423 0.25929 0.273904
ND ND ND 0.216164

It was seen that both free and lipo-RvD1 treatments prevented calcification of ectopic trabecular structures (fig. 11B-D).

Pathological pain (allodynia) is one of the main clinical symptoms of OA. To test if lipo-RvD1 formulations decreased pain in case of post-traumatic OA, the pain threshold of mice was tested using Von Frey filaments. It was observed that administration of lipo-RvD1 was more effective in alleviating the allodynia as compared to free RvD1 and no treatment (Table 16, fig. 11E).

Table 16: Effect of Lipo-RvD1 on symptoms of OA – alleviation of allodynia
Pain Threshold (g)
Sham DMM Lipo-RvD1 Free RvD1
0.4** 1.4 2 1
1.0** 1.4 2 1
8 1.4 4 1
10 0.6 4 2
8 1 4 ND
ND ND 6 ND
ND ND 6 ND

While only DMM operated and free drug administered group had low pain threshold (less than about 2g), lipo-RvD1 injected animals showed close to about 4g which was closer to Sham controls. This analgesic effect of lipo-RvD1 lasted upto about 3 days post injection.

EXAMPLE 8: Setting-up an obesity induced OA (ObOA) mouse model
Obesity is characterized by an increase in body weight due to the storage of excessive fat in adipose tissue. This disorder was modeled in mice by providing ad libitum access to specialized diets which are enriched in fat. The mice were fed with a specialized feed containing 60% fat by calorie content to generate overweight mice. A statistically significant difference was observed between the weights of the normal diet and HFD-fed mice from the 8th week after the commencement of feeding (fig. 13a). Obese patients often show systemic dyslipidemia and upregulated low-density lipoprotein fraction of cholesterol (LDL-c), triglycerides, and cholesterol. Accordingly, the total cholesterol (TC) was also higher in the serum of overweight mice than in their leaner counterparts (figure 13b). LDL-c also increased with dietary fat in mice (fig.13c) and the ratio of both LDL-c (fig. 13d) and TC (fig. 13e) to the high-density lipoprotein fraction of cholesterol (HDL-c) was higher in overweight mice than in their leaner counterparts. Since these parameters were in alignment with those reported in the literature, high fat-fed mice could be successfully classified as obese.

To mimic Obesity related OA (ObOA), DMM surgery (as explained in Example 6) was performed in obese mice and compared to DMM in mice fed with standard diet (11% kCal by fat). More severe damage was found to the cartilage in the obese mice than in their leaner counterparts after DMM surgery (p=0.0311) (fig. 13f, 13g). Furthermore, it was observed that obesity alone was not sufficient to cause OA in mice, as seen from the comparison between the pathologies of lean and obese sham joints, – OA was induced by the DMM surgery and was more severe in obese mice (fig.13f, 13g). The articulating surfaces in both the normal diet DMM and high-fat diet DMMs were severely denuded, with more damage present in the latter; damage in obese mice was about 1.5x more severe compared to their leaner counterparts. This finding was in agreement with earlier studies that showed that obesity-induced inflammatory signaling, and not excessive stress on the joint is the major contributor to the pathology.

Example 9: Lipo-RvD1 as a prophylactic candidate for ObOA treatment
A prophylactic dosing regimen was followed by injecting freshly synthesized lipo-RvD1 intraarticularly (IA) at weeks 1 and 4 after DMM surgery in the mouse model as described in the previous example (figure 14a). Lipo-RvD1 arrested the progressing cartilage damage and maintained the overall joint integrity. Specifically, it was observed that the lipo-RvD1-treated mice had a well-maintained extracellular matrix and showed about 6-to-8-fold reduction in OARSI scores compared to DMM joints (p=0.0001) which had complete loss of articulating cartilage at certain sites of damage (fig.14b, 14c). The stained sections showed a higher percentage of healthy and non-hypertrophic chondrocytes in lipo-RvD1 treated animals compared to DMM and free RvD1 treated mice (fig. 14b, 14c). Administration of free RvD1 did not have any protective effect on the joint and the damage was similar to DMM-only animals.

Several inflammatory diseases have an imbalance between the M1 and M2 macrophages. The ratio of M1/M2 cells is skewed in OA as well and proinflammatory cytokines from M1 macrophages drive cartilage damage. DMM mice had higher levels of M1 cells than sham mice, which indicates the presence of a proinflammatory environment in the synovium (fig. 14d, 14e). Lipo-RvD1 treatment promoted preferential polarization towards M2 cells as compared to DMM mice (p<0.0001) (fig. 14f, 14g).

It was confirmed that the protective effect of lipo-RvD1 was due to the released RvD1 and not the liposome by itself, because blank liposomes did not have improved pathology compared to DMM joints (fig. 15). Overall, it was observed that lipo-RvD1 formulation increased M2 macrophages in the joint which reduced the net inflammatory activity within the synovium and promoted clearance of debris and other inflammatory factors.

Catabolic enzymes like ADAMTS5 and MMP13 released by chondrocytes in OA are considered markers of chondrocyte hypertrophy. Chondrocyte hypertrophy in OA disturbs cartilage homeostasis and is thought to be a factor that is responsible for OA development. Administration of lipo-RvD1 was found to suppress the expression of the catabolic mediators ADAMTS5 and MMP13, thus demonstrating its ability to prevent the formation of hypertrophic chondrocytes in ObOA subjects (fig. 16).

Example 10: Lipo-RvD1 as a therapeutic candidate for ObOA treatment
In clinics, the diagnosis of OA relies on radiographic evidence of joint damage, which is visible only when the damage has progressed significantly. Accordingly, therapeutic formulations are critical for the successful treatment of OA. The therapeutic efficacy of the lipo-RvD1 on ObOA of Example 2 was studied by injecting it at 3 and 6 weeks after DMM surgery. This timeline was chosen as a suitable therapeutic regimen because cartilage damage is known to start within two weeks after DMM surgery (fig. 17a). In this challenging regimen, intraarticular lipo-RvD1 administration was much more effective than free RvD1 (p=0.0006) and DMM-only mice (p=0.0001) in maintaining cartilage health (fig. 17b, 17c).

Slight downregulation of M1 macrophages was observed in the synovial membrane of the DMM treated joints compared to the lipo-RvD1 treated joints (fig. 17d, 17e). The therapeutic regimen of administration also showed increased levels of pro-resolution M2 macrophages (p=0.0010) (figure 17f, 17g) compared to DMM joints. The lipo-RvD1 treatment further reduced the expression of damaging enzymes ADAMTS5 and MMP13 and protected cartilage from degradation more efficiently as compared to free RvD1 (fig. 18).

Wnt signaling is a major player in OA pathology and ß-catenin is a mediator of this signaling and is upregulated in OA. It was observed that lipo-RvD1 administration to OA joints suppressed the expression of ß-catenin in chondrocytes (fig. 19), thus indicating downregulation of Wnt signaling. Said suppression was higher in the lipo-RvD1 joints as compared to the free RvD1 treated joints. This downregulation is further expected to lower the levels of catabolic enzymes like MMP13, ADAMTS4, and ADAMTS5 in chondrocytes. It was therefore concluded that Wnt signaling is one of the pathways affected by RvD1 and that more efficient suppression or downregulation is achievable by lipo-RvD1 as compared to the free drug.

Example 11: Effect of Lipo-RvD1 on Synovitis
Synovitis is a hallmark of OA. Increased leukocyte infiltration to the synovium leads to synovitis and increased production of inflammatory mediators which leads to chondrocyte hypertrophy and blocks anabolism. After DMM surgery, synovitis peaks in early stages before declining but persists at a degree higher than that in sham mice throughout the mid and late stages of PTOA22. It was observed that treating DMM mice with lipo-RvD1 reduced the synovial membrane (SM) thickness (fig. 20 a, b, c, d) and cellularity (fig. 20 e, f, g, h) as compared to DMM-only mice. Synovial membrane from lipo-RvD1-treated joints was similar in thickness to SM in sham joints in both the therapeutic and prophylactic regimens of administration, thus emphasizing the ability of lipo-RvD1 to suppress excessive fibrosis of synovial membrane (fig. 20 a, b, c, d). It was further observed that this result also held true for the total cellularity of the synovial membrane (fig. 20 e, f, g, h).

Taken together, lipo-RvD1 treated joints showed a better ability to prevent cells from infiltrating the inflammatory milieu compared to free RvD1 (fig. 20 g, h).

Example 12: Lipo-RvD1 reduces the incidence of OA-associated allodynia - prophylactic regimen vs. therapeutic regimen
Pathological pain (allodynia) is one of the main clinical symptoms of OA in patients. To test if resolvin formulations decreased pain in both prophylactic regimen and therapeutic regimens, the pain threshold of mice was once again tested using Von Frey filaments. It was observed that administration of lipo-RvD1 was more effective in alleviating the allodynia than free RvD1 injected mice (p=0.0016) in the prophylactic regimen of administration (fig. 21a) as compared to the therapeutic regimen (fig. 21b). While the therapeutic regimen showed that lipo-RvD1 improved the pain threshold of the limb, the difference was not as statistically significant (fig. 21b). The results thus showed that the sustained presence of RvD1 in the affected knee joint helped alleviate OA-associated pain, especially under a prophylactic regimen.

The foregoing description fully reveals the general nature of the embodiments herein that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without departing from the general concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. Therefore, while the embodiments in this disclosure have been described in terms of preferred embodiments, those skilled in the art will recognize that the embodiments herein can be practiced with modification within the spirit and scope of the embodiments as described herein, without departing from the principles of the disclosure.

Any discussion of documents, acts, materials, devices, articles and the like that has been included in this specification is solely for the purpose of providing a context for the disclosure. It is not to be taken as an admission that any or all of these matters form a part of the prior art base or were common general knowledge in the field relevant to the disclosure as it existed anywhere before the priority date of this application.
,CLAIMS:1. A liposome encapsulated specialized pro-resolution mediator (SPM), wherein the liposome has size ranging from about 100nm to about 5µm.
2. The liposome encapsulated SPM as claimed in claim 1, wherein the liposome is formed by lipids selected from a group comprising 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), Egg Phosphatidylcholine (Egg PC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), methoxy-poly(ethyleneglycol)2000-distearoylphosphatidylethanolamine (DSPE-PEG2000), or any combination thereof.
3. The liposome encapsulated SPM as claimed in claim 2, wherein the lipid(s) forming the liposome further comprises sterol(s).
4. The liposome encapsulated SPM as claimed in claim 3, wherein the sterol is selected from a group comprising cholesterol, beta-Sitosterol, phytosterol and 20-alpha-Hydroxycholesterol or any combination thereof cholesterol
5. The liposome encapsulated SPM as claimed in claim 3, wherein the sterol is cholesterol.
6. The liposome encapsulated SPM as claimed in any of claims 1-5, wherein the liposome is formed by about 40% to about 94% by mole of DPPC; about 1% to about 50% of DSPE-PEG2000; and, optionally about 0% to about 30% of sterol by mole.
7. The liposome encapsulated SPM as claimed in any of claims 1-6, wherein the liposome is formed by 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), methoxy-poly(ethyleneglycol)2000-distearoylphosphatidyletholamine (DSPE-PEG2000), and sterol at a ratio of about 85:5:10 by mole.
8. The liposome encapsulated SPM as claimed in any of claims 1-7, wherein the SPM is a resolving family molecule selected from a group comprising Resolvin D1 (RvD1), aspirin-triggered Resolvin D1 and Resolvin E1 or any combination thereof.
9. The liposome encapsulated SPM as claimed in any of claims 1-8, having size ranging from about 150nm to about 1µm.
10. The liposome encapsulated SPM as claimed in any of claims 1-9, having size ranging from about 300nm to about 500nm.
11. The liposome encapsulated SPM as claimed in any of claims 1-10, comprising SPM at a concentration ranging from about 15 ng/mg of liposomes to about 1200 ng/mg of liposomes.

12. A method of obtaining SPM loaded liposome comprising –
a) Hydration of thin film(s) of liposome forming lipid(s) with solvent(s) to generate vesicles;
b) Extrusion of the vesicles through a filter to generate liposomes; and
c) Pelleting and re-suspending the liposomes in SPM containing solution
to obtain the SPM loaded liposome.
13. The method as claimed in claim 12, further comprising washing of the obtained SPM-loaded liposomes to remove excess unloaded SPM from the extra-liposomal environment.
14. The method as claimed in any of claims 12-13, wherein the liposome forming lipid(s) is selected from a group comprising 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), Egg Phosphatidylcholine (Egg PC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), methoxy-poly(ethyleneglycol)2000-distearoylphosphatidylethanolamine (DSPE-PEG2000), or any combination thereof; and optionally, comprises sterol(s).
15. The method as claimed in any of claims 12-14, wherein the solvent employed for hydration of the lipid film is selected from solvents such as but not limited to calcium acetate, barium acetate, and magnesium acetate or any combination thereof.
16. The method as claimed in any of claims 12-15, wherein the extrusion is performed through filters having pore size ranging from about 100 nm to about 5µm.
17. The method as claimed in any of claims 12-16, wherein the SPM is a resolving family molecule selected from a group comprising Resolvin D1 (RvD1), aspirin-triggered Resolvin D1 and Resolvin E1 or any combination thereof.
18. The method as claimed in any of claims 12-17, wherein the SPM containing solution comprises solvent(s) selected from a group comprising sodium sulfate, lithium sulfate, and potassium sulfate acetate or any combination thereof.
19. The method as claimed in any of claims 12-18, wherein the SPM containing solution comprises about 50ng SPM/mL solvent to about 4000ng SPM/mL solvent.
20. The method as claimed in any of claims 12-19, wherein the solvent employed for hydration
has a pH ranging from about 5 to about 7, preferably about 6; and wherein the SPM containing solution has a pH ranging from about 2 to about 4.5, preferably about 4.
21. The method as claimed in any of claims 12-20, wherein the pelleting and re-suspension of the liposomes in SPM containing solution is at a temperature of about 37°C to about 60°C for about 0.5 hours to about 2 hours.
22. The method as claimed in claim 13, wherein the washing is performed with a solvent selected from a group comprising Phosphate Buffered Saline (PBS), Normal Saline (NS), and Hank’s Balanced Salt Solution (HBSS) or any combination thereof.
23. The method as claimed in any of claims 1222, wherein the obtained SPM loaded liposome has size ranging from about 150nm to about 1µm.
24. The method as claimed in any of claims 12-23, having encapsulation efficiency of about 10% to about 100%, preferably about 43% to about 99%.
25. A composition comprising the liposome encapsulated SPM as claimed in claim 1 and one or more pharmaceutically acceptable excipients or additives.
26. The composition as claimed in claim 25, wherein the composition is a sustained release formulation.
27. The composition as claimed in claim 25, wherein the composition is formulated into an injectable formulation.
28. Use of the liposome encapsulated SPM as claimed in claim 1 or the composition as claimed in claim 24 for the prophylaxis, prevention, management and/or therapeutic treatment of joint related diseases.
29. The use as claimed in claim 28, wherein the liposome encapsulated SPM allows sustained release for about 1day to about 15 days.
30. The use as claimed in claim 29, wherein the liposome encapsulated SPM allows sustained release for about 9 days to about 15 days.
31. A method of preventing or treating joint related diseases comprising administering the liposome encapsulated SPM as claimed in claim 1 or the composition as claimed in claim 24 to a subject in need thereof.
32. The method as claimed in claim 31, wherein the administration is by way of injection, preferably an intraarticular injection.
33. The method as claimed in claim 31, wherein the method reduces joint damage by about 4 fold to about 10 fold as compared to an untreated subject.
34. A kit comprising one or more of the liposome encapsulated SPM as claimed in claim 1 or the composition as claimed in claim 26, one or more syringes for administration of the liposome encapsulated SPM and optionally, an instruction manual for enabling use of the kit or any combination thereof.

35. A pre-filled pen or syringe comprising the liposome encapsulated SPM as claimed in claim 1 or the composition as claimed in claim 26.