Microspheres for sustained release of fenofibrate

Abstract

The present invention relates to biodegradable microspheres for the sustained release of fenofibrate, injectable formulations comprising said microspheres, and their use for the treatment of joint-related disorders, such as osteoarthritis. The present invention further relates to a method for preparing said microspheres.

Description

[Technical field]

[0001] The present invention relates to biodegradable microspheres for the sustained release of fenofibrate, injectable formulations comprising said microspheres, and their use for the treatment of joint-related disorders, such as osteoarthritis. The present invention further relates to a method for preparing said microspheres. [Background technology]

[0002] Osteoarthritis is the most common form of arthritis. It is characterized by joint damage and joint dysfunction. This is because the disease process causes cartilage damage and new bone growth in affected joints, causing stiffness and pain. Osteoarthritis in large joints, such as the knees and hips, can reduce people's mobility and make it difficult or impossible to climb stairs or walk. Osteoarthritis in small joints, such as the hands and fingers, makes normal tasks difficult and painful. Aging is the main risk for patients with cartilage degeneration and osteoarthritis.

[0003] Fenofibrate has been used clinically for the treatment of lipid metabolism dysfunction and is widely known as a therapeutic option for high cholesterol levels. In a recent study, fenofibrate was selected as a suitable candidate for potential treatment against cartilage aging and osteoarthritis (Nogueira-Recalde et al. 2019). The study showed that osteoarthritis patients treated with fenofibrate showed improved physical function and therefore better mobility. This study provided new evidence that activation of peroxisome proliferator-activated receptor alpha (PPARα) by fenofibrate, a PPARα agonist, regulates key mechanisms such as aging and autophagy in chondrocytes and cartilage, thereby preventing cartilage degradation. The data established preclinical evidence for the development of novel disease-modifying therapies targeting lipid metabolism to prevent and treat osteoarthritis.

[0004] However, joints pose a great challenge for drug development. The synovial membrane represents a barrier to the penetration of drugs from the plasma into the joint space, and articular cartilage is avascular, limiting the access of systemically administered drugs. On the other hand, fenofibrate is highly hydrophobic, insoluble in water, and classified as a class II compound in the Biopharmaceutics Classification System due to its low solubility and limited gastrointestinal absorption rate. These characteristics represent a challenge for fenofibrate administration, along with its low synovial bioavailability in the joint. Therefore, there is a need for new formulations suitable for intra-articular injection that can overcome the problems of low drug bioavailability in target tissues and side effects caused by systemic administration.

[0005] Biodegradable microspheres made from polylactic acid-co-glycolic acid copolymer (PLGA) are known in the field of drug delivery. PLGA is a biodegradable polymer made from polylactic acid (PLA) and polyglycolic acid (PGA) and approved by the FDA. It has been widely studied in many medical and pharmaceutical fields due to its good biodegradability and biocompatibility. Microspheres containing PLGA have shown sustained release characteristics due to the mechanism of degradation and diffusion. For example, Klose et al (2009) have shown that localized controlled drug delivery of fenofibrate to the brain is possible by fenofibrate-loaded PLGA microparticles. Grabacka et al (2015) have disclosed in vitro biodegradable PLGA polymer wafers containing fenofibrate, which can ultimately be inserted into the brain cavity after resection of brain tumors.

[0006] The drug release profile of PLGA microsphere preparations depends on certain factors such as the specific characteristics of the drug, the ratio of PLA to PGA, the molecular weight / intrinsic viscosity of the polymer, the loading ratio of drug to polymer, and the size of the microspheres. These constraint factors are related to the physicochemical properties of the formulation and dictate the drug release rate and its pharmacokinetic profile. US2020 / 0297651A1 compares the release profiles of fenofibrate-loaded PLGA microparticles, where PLGA with different molecular weights is used for each particle. Particles containing PLGA with a molecular weight of 34 kDa were selected and tested for toxicity in the retina. Similarly, Qui et al. (2019) explored the sustained therapeutic effect of fenofibrate-loaded biodegradable nanoparticles against diabetic retinopathy and neovascular age-related macular degeneration using optimized fenofibrate PLGA nanoparticles made from PLGA with a molecular weight of 34 kDa, which showed adequate drug loading and sustained drug release for up to 60 days in vitro. The specific preparation method of fenofibrate microgranules using PLGA is disclosed, for example, in CN1302766C and CN100400032C.

[0007] Although various preparations and uses of fenofibrate-loaded microparticles have been described, the use of a mixture of high and low molecular weight PLGA to prepare fenofibrate-loaded microspheres with suitable particle size and optimized loading and controlled release properties has not been suggested to date.Furthermore, the use of such microparticles for the treatment of musculoskeletal disorders, nor their intra-articular administration, has not been described.

[0008] Zilretta®, a PLGA-triamcinolone-acetonide microsphere formulation for knee injection, was approved by the FDA in 2017. Zilretta® has been shown to reduce pain in patients with osteoarthritis of the knee for up to 12 weeks.

[0009] Patients suffering from joint disorders, specifically osteoarthritis, require regular treatment to relieve their discomfort. Daily intake of drugs is cumbersome and good patient compliance is necessary for positive treatment results. Furthermore, systemic delivery of drugs can cause undesirable side effects. To date, no reliable long-term treatment option with fenofibrate that overcomes these drawbacks is available on the market. Therefore, there is a need for a better method of treating joint disorders, such as osteoarthritis, with fenofibrate while minimizing the side effects of the drug observed with systemic delivery. Summary of the Invention

[0010] Accordingly, the present invention provides a biodegradable microsphere, the microsphere comprising: (i) a polylactic-co-glycolic acid copolymer (PLGA) matrix, the matrix comprising at least two PLGAs having different molecular weights (Mw); (ii) having an average diameter of about 40 μm to about 225 μm; (iii) Biodegradable microspheres containing fenofibrate.

[0011] In one embodiment, the microspheres release fenofibrate over a period of at least one month when present in the target tissue.

[0012] In one embodiment, the release of the fenofibrate is controlled release.

[0013] In one embodiment, each of the at least two PLGAs has a viscosity of about 0.16 dl / g to about 1.70 dl / g, preferably, one of the at least two PLGAs has a viscosity of about 0.50 dl / g to about 0.70 dl / g and a second of the at least two PLGAs has a viscosity of about 0.16 dl / g to about 0.24 dl / g.

[0014] In a further embodiment, the at least two PLGAs have a Mw of about 6 kDa to about 90 kDa, preferably one of the at least two PLGAs has a Mw of about 6 kDa to about 18 kDa and a second of the at least two PLGAs has a Mw of about 50 kDa to about 90 kDa.

[0015] In one embodiment, one of the at least two PLGAs has an average molar ratio of lactic acid to glycolic acid of 100:0 to 50:50, preferably one of the at least two PLGAs has an average molar ratio of lactic acid to glycolic acid of 75:25 (PLGA 75:25) and a second of the at least two PLGAs has an average molar ratio of lactic acid to glycolic acid of 50:50 (PLGA 50:50).

[0016] In a further embodiment, the ratio between PLGA 50:50 and PLGA 75:25 is selected from about 90:10, about 80:20 or about 75:25, preferably about 90:10.

[0017] In further embodiments, the average diameter of the microspheres is from about 40 μm to about 175 μm, from about 45 μm to about 155 μm, from about 50 μm to about 125 μm, from about 55 μm to about 90 μm, from about 55 μm to about 70 μm, and most preferably from about 55 μm to about 65 μm.

[0018] In a preferred embodiment, the average diameter of the microspheres is about 60 μm.

[0019] In one preferred embodiment, the microspheres contain from about 0.5 μg to about 20 μg, preferably from about 0.8 μg to about 15 μg, and most preferably from about 1 μg to about 10 μg of fenofibrate per milligram of microsphere.

[0020] In a preferred embodiment, the microspheres comprise (i) a polylactic-co-glycolic acid copolymer (PLGA) matrix, the matrix comprising PLGA 75:25 having a viscosity of about 0.60 dl / g and / or a Mw of about 68 kDa, and PLGA 50:50 having a viscosity of about 0.21 dl / g and / or a Mw of about 13.5 kDa; (ii) has an average diameter of about 55 to 65 μm, preferably about 60 μm; (iii) 1 μg to 10 μg of fenofibrate per milligram of microspheres, preferably 10 μg of fenofibrate per milligram of microspheres.

[0021] In one embodiment, the microspheres, when present in the target tissue, release fenofibrate for at least two months, more preferably at least three months.

[0022] In an even more preferred embodiment, the release is controlled release.

[0023] In a further aspect, the present invention relates to a plurality of biodegradable microspheres as defined in any one of the preceding claims, the microspheres comprising: (i) a d90 particle size value of about 50 μm to about 100 μm, and / or (ii) a d10 particle size value of about 16 μm to about 20 μm, and / or (iii) A plurality of biodegradable microspheres having a d50 particle size value of about 30 μm to about 60 μm.

[0024] In a preferred embodiment of the plurality of biodegradable microspheres, the microspheres comprise: (i) having a d90 particle size value of about 50 μm to about 100 μm; (ii) a polylactic-co-glycolic acid copolymer (PLGA) matrix, the matrix comprising at least two PLGAs having different molecular weights (Mw); (iii) having a therapeutically effective amount of fenofibrate; (iv) preferably releases fenofibrate over a period of at least one month when present in the target tissue.

[0025] In a further preferred embodiment, the at least two PLGAs each have a viscosity of about 0.16 dl / g to about 1.70 dl / g, preferably, one of the at least two PLGAs has a viscosity of about 0.50 dl / g to about 0.70 dl / g and a second of the at least two PLGAs has a viscosity of about 0.16 dl / g to about 0.24 dl / g.

[0026] In yet another embodiment of the multiple biodegradable microspheres, at least two of the PLGAs have a Mw of about 6 kDa to about 90 kDa, preferably, one of the at least two PLGAs has a Mw of about 6 kDa to about 18 kDa and a second of the at least two PLGAs has a Mw of about 50 kDa to about 90 kDa.

[0027] In a further embodiment of the plurality of biodegradable microspheres, one of the at least two PLGAs has an average molar ratio of lactic acid to glycolic acid of 100:0 to 50:50, preferably one of the at least two PLGAs has an average molar ratio of lactic acid to glycolic acid of 75:25 (PLGA 75:25) and a second of the at least two PLGAs has an average molar ratio of lactic acid to glycolic acid of 50:50 (PLGA 50:50).

[0028] In a preferred embodiment, the ratio between PLGA 50:50 and PLGA 75:25 is selected from about 90:10, about 80:20 or about 75:25, preferably about 90:10.

[0029] In further embodiments, the average diameter of the microspheres is from about 40 μm to about 175 μm, from about 45 μm to about 155 μm, from about 50 μm to about 125 μm, from about 55 μm to about 90 μm, from about 55 μm to about 70 μm, and most preferably from about 55 μm to about 65 μm.

[0030] In a preferred embodiment, the average diameter of the microspheres is about 60 μm.

[0031] In one embodiment, the microspheres contain from about 0.5 μg to about 20 μg, preferably from about 0.8 μg to about 15 μg, and most preferably from about 1 μg to about 10 μg of fenofibrate per milligram of microsphere.

[0032] The present invention further relates to an injectable formulation comprising a pharma- ceutically acceptable carrier and a microsphere or a plurality of biodegradable microspheres described herein.

[0033] In a preferred embodiment of the formulation, the microspheres, when present in the target tissue, release fenofibrate for at least 1 month, preferably at least 2 months, more preferably at least 3 months.

[0034] The present invention further relates to the biodegradable microsphere, the plurality of biodegradable microspheres, or the injectable formulation as described herein above for use in the treatment or prevention of joint-related disorders, preferably arthritis, more preferably osteoarthritis.

[0035] In one embodiment, the biodegradable microsphere, a plurality of biodegradable microspheres, or an injectable formulation described herein is administered intra-articularly, preferably, the microsphere, a plurality of microspheres, or an injectable formulation is administered as a single dose.

[0036] The present invention further relates to a kit comprising in separate compartments: (a) a diluent; (b) A kit comprising a biodegradable microsphere or a plurality of biodegradable microspheres according to any one of the preceding claims.

[0037] In a preferred embodiment, the biodegradable microsphere or biodegradable microspheres are present in powder form.

[0038] The present invention further relates to a method for preparing the microspheres described herein, the method comprising a single emulsion-solvent evaporation. In a preferred embodiment of said method, the microspheres are prepared using PLGA at a polymer concentration of about 10-30% (w / w), preferably 15-25% (w / w), more preferably 15-20% (w / w).

[0039] In one embodiment of the method, at least 90% of the fenofibrate is encapsulated within the microspheres.

[0040] The present invention further relates to a method for treating a joint-related disorder in a subject, comprising introducing a biodegradable microsphere, a plurality of biodegradable microspheres, or an injectable formulation described herein into target tissue within or around one or more of the subject's joints, preferably the hip, shoulder, and / or knee joints.

[0041] In one embodiment, the subject is a human or an animal, such as a cat, dog, or horse.

[0042] In a further embodiment, the joint-related disorder is arthritis, preferably osteoarthritis.

[0043] In one embodiment, the method comprises intra-articularly injecting a biodegradable, a plurality of biodegradable microspheres, or an injectable formulation into one or both of the small joints of the subject's knee, shoulder, hip, neck, lower back, hand, or foot. [Brief description of the drawings]

[0044] [Figure 1] Fenofibrate loading for different formulations obtained with a single PLGA (Example 1) The theoretical drug loading was 1% fenofibrate in the PLGA matrix (10 μg fenofibrate / mg PLGA). [Diagram 2]Fenofibrate release profile of different microsphere formulations obtained with a single PLGA prepared in Example 1 over a total period of 90 days. [Diagram 3] Particle size distribution of the microspheres prepared in Example 1. [Figure 4] Fenofibrate loading of formulations with specific MW and optimized polymer ratios prepared in Example 2. The encapsulation efficiency is achieved independently of the PLGA formulation selected. The theoretical drug loading was 1% fenofibrate (10 μg fenofibrate / mg PLGA) in the PLGA matrix. [Diagram 5] Fenofibrate controlled release profiles of different microsphere formulations prepared by PLGA mixtures: PLGA (50:50) 13.5 kDa (0.21 dl / g) and PLGA (75:25) 68 kDa (0.60 dl / g) (Example 2). [Figure 6] Particle size distribution of the microspheres prepared in Example 2. [Figure 7] Intra-articularly administered fenofibrate reduces joint damage in a preclinical model of osteoarthritis in mice. Three-month-old C57Bl / 6J mice underwent osteoarthritis surgery (MMTL+MCL) on the right knee. The experiment included 24 mice (8 mice / each group). (A) Knee joints were analyzed by staining with Safranin O. (B) Histological scores. Values ​​are mean ± SEM. **p<0.01 vs. PLGA 90:10 condition. Magnification: 10x. [Figure 8] Intra-articularly administered fenofibrate reduces joint damage in a preclinical model of osteoarthritis in mice. Three-month-old C57Bl / 6J mice underwent osteoarthritis surgery (LMTL+LCL) on the right knee. The experiment included 24 mice (8 mice / each group). (A) Knee joints were analyzed by staining with Safranin O. (B) Histological scores. Values ​​are mean ± SEM. *p<0.05 vs. PLGA 90:10 condition. Magnification: 10x. [Figure 9]Oral administration of fenofibrate reduces joint damage in a preclinical model of osteoarthritis in mice. Three-month-old C57Bl / 6J mice underwent OA surgery on the right knee. The experiment included 16 mice (8 mice / each group). (A) Knee joints were analyzed by staining with Safranin O. (B) Histological scores. Values ​​are mean ± SEM. *p<0.05 vs. vehicle condition. [Figure 10] Fenofibrate release profile of different microsphere formulations in simulated synovial fluid supplemented with 1% SDS: PLGA(50:50):PLGA(75:25) 90:10 approx. 60 μm, Resomer RG 502 approx. 300 μm, Resomer RG 502 approx. 30 μm over a total period of 39 days. [Figure 11] Regression analysis of the theoretical drug concentrations and the resulting areas under the curves established by the fenofibrate trace. [Figure 12] Fenofibrate release profile of PLGA(50:50):PLGA(75:25) 90:10 microspheres with a mean diameter of about 60 μm in vitro in PBS supplemented with 1% SDS and human synovial fluid from osteoarthritis patients over a total period of 49 days. SSF refers to simulated synovial fluid. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0045] The present invention will be understood more readily by reference to the following detailed description of the preferred embodiments of the invention and the examples included therein.

[0046] As described in more detail above, there is an unmet need for improved long-term options for treating joint disorders, such as osteoarthritis, using fenofibrate while minimizing the side effects of the drug observed with systemic delivery.

[0047] Therefore, the present inventors have developed biodegradable microspheres containing fenofibrate that overcome these drawbacks by providing a sustained release profile in target tissues without reducing therapeutic efficacy compared to routinely administered orally administered fenofibrate. The microspheres of the present invention and their applications are presented in further detail herein below.

[0048] Microspheres of the Invention Polylactic-co-glycolic acid copolymer PLGA is an FDA approved biodegradable copolymer made of polylactic acid (PLA) and polyglycolic acid (PGA). It has been widely observed in many medical and pharmaceutical fields due to its good biodegradability and biocompatibility. Microspheres containing PLGA can exhibit sustained release characteristics due to the mechanism of degradation and diffusion. However, the drug release profile of PLGA microsphere preparations is highly dependent on certain factors such as the specific properties of the drug itself, the ratio of PLA to PGA, the molecular weight / intrinsic viscosity of the polymer, the loading ratio of drug to polymer, and the size of the microsphere. In general, it is known that PLGA with low molecular weight and correspondingly low intrinsic viscosity allows more complete and more rapid release of pharmaceutical agents incorporated in the microparticles than its higher molecular weight and higher intrinsic viscosity counterparts.

[0049] In order to find an ideal composition for the formation of microspheres that can not only encapsulate a significant amount of fenofibrate but also exhibit the desired sustained release profile of the drug over a specific time period, we tested different compositions including single PLGA and a mixture of different PLGAs with different L / G ratios and molecular weights. The data obtained when using single PLGA (Example 1) showed sufficient encapsulation efficiency for the different L / G ratios and particle sizes used (Figure 1), and the controlled release profile showed that almost 20% release of the drug was reached after 3 months (Figure 2). The size and distribution of the microspheres were directly dependent on the molecular weight of the polymer resulting in a higher average diameter for microspheres prepared with medium molecular weight PLGA (Figure 3). The decrease in particle size with decreasing polymer Mw is a result of the decrease in intrinsic viscosity of the polymer since the polymer concentration is constant in each formulation. Thus, the microspheres containing low Mw PLGA met the requirements of high encapsulation efficiency and morphology. However, the release rate was found to be inefficient, since the amount of drug released was not sufficient to provide the desired therapeutic efficacy. Therefore, in order to adjust the release rate while simultaneously ensuring the morphological properties, microspheres were prepared using a mixture of high and low molecular weight PLGA (Example 2). All of these microspheres were characterized by high encapsulation efficiency (Figure 4) and a controlled release profile (Figure 5) that showed a typical triphasic release profile similar to the mixtures tested. The increase in burst release for blended PLGA microspheres can be explained by the polymer miscibility resulting in microspheres with lower density and increased drug diffusion. These profiles provide a higher release rate, with 80% of the drug released after 3 months, allowing to reach the therapeutic concentration in the target tissue required to provide therapeutic efficacy. Meanwhile, the microparticle average size and distribution were clearly dependent on the amount of high Mw PLGA used (Figure 6).Microspheres with a very narrow size distribution are obtained when the ratio of low Mw (PLGA 50:50 13.5 kDa):high MW (PLGA 75:25 68 kDa) used is 90:10. The preferred microspheres are those with a ratio of 90:10, although the other two ratios tested also provided reasonably narrow size distributions. The inventors were further able to show that microparticles containing a combination of different PLGAs disclosed herein provide controlled sustained release over a period of three months, whereas microparticles containing only one PLGA form particles that, instead of providing release over three months as desired, exhibit either a too low release rate, with almost no release occurring during the first month, or a too high release rate, with almost all fenofibrate already released after 39 days (Example 6 and Figure 10). Thus, the diameter and combination of two different PLGAs with different molecular weights are important parameters to ensure the required release profile.

[0050] Accordingly, the present invention provides a biodegradable microsphere, the microsphere comprising: (i) a polylactic-co-glycolic acid copolymer (PLGA) matrix, the matrix comprising at least two PLGAs having different molecular weights (Mw); (ii) having an average diameter of about 40 μm to about 225 μm; (iii) Biodegradable microspheres containing fenofibrate.

[0051] In one embodiment, the microspheres release fenofibrate over a period of at least one month when present in the target tissue.

[0052] In one embodiment, each of the at least two PLGAs has a viscosity of about 0.16 dl / g to about 1.70 dl / g. In a preferred embodiment, one of the at least two PLGAs has a viscosity of about 0.50 dl / g to about 0.70 dl / g, about 0.52 dl / g to about 0.68 dl / g, about 0.54 dl / g to about 0.66 dl / g, about 0.56 dl / g to about 0.66 dl / g, about 0.58 dl / g to about 0.64, or about 0.60 dl / g to about 0.62 dl / g, and a second of the at least two PLGAs has a viscosity of about 0.16 dl / g to about 0.24 dl / g, about 0.18 dl / g to about 0.22 dl / g, or about 0.20 dl / g to about 0.22 dl / g.

[0053] In further embodiments, the at least two PLGAs have a Mw of about 6 kDa to about 90 kDa, about 10 kDa to 80 kDa, or about 12 kDa to 70 kDa.

[0054] In a further preferred embodiment, one of the at least two PLGAs has a Mw of about 6 kDa to about 18 kDa, about 7 kDa to about 17 kDa, about 8 kDa to about 16 kDa, about 9 kDa to about 15 kDa, or about 10 kDa to about 14 kDa, and a second of the at least two PLGAs has a Mw of about 50 kDa to about 90 kDa, about 55 kDa to about 80 kDa, or about 60 kDa to about 70 kDa.

[0055] In one embodiment, the second of the at least two PLGAs has a Mw of about 190-240 kDa, preferably a Mw of about 215 kDa, or a Mw of 76 kDa-115 kDa, preferably 95.5 kDa.

[0056] In one embodiment, one of the at least two PLGAs has an average molar ratio of lactic acid to glycolic acid of about 100:0 to about 40:60, about 90:10 to about 40:60, about 80:20 to about 50:50, preferably one of the at least two PLGAs has an average molar ratio of lactic acid to glycolic acid of about 75:25 (PLGA 75:25), and a second of the at least two PLGAs has an average molar ratio of lactic acid to glycolic acid of about 60:40 to about 40:60, preferably about 50:50 (PLGA 50:50).

[0057] To evaluate the preferred ratio of microspheres used for PLGA50:50 and PLGA75:25, three different ratios of microspheres were prepared as described in Example 2. Thus, in a further embodiment, the ratio between PLGA50:50 and PLGA75:25 is selected from about 90:10, about 80:20 or 75:25, preferably about 90:10, because this ratio shows sufficient encapsulation efficiency and drug release profile while ensuring the required microsphere size diameter and narrow size distribution (as shown in Figure 6).

[0058] In further embodiments, the average diameter of the microspheres is from about 40 μm to about 175 μm, from about 45 μm to about 155 μm, from about 50 μm to about 125 μm, from about 55 μm to about 90 μm, from about 55 μm to about 70 μm, and most preferably from about 55 μm to about 65 μm.

[0059] In a preferred embodiment, the average diameter of the microspheres is about 60 μm.

[0060] In further embodiments, the average diameter of the microspheres is from about 25 μm to about 110 μm, from about 30 μm to about 100 μm, from about 40 μm to about 90 μm, from about 45 μm to about 80 μm, from about 50 μm to about 70 μm, and most preferably from about 55 μm to about 65 μm.

[0061] These ranges are understood to refer to the average diameter of all microparticles in a given population. The diameter of any given individual microparticle will be within a standard deviation above or below the average diameter.

[0062] The drug loading capacity of the microspheres of the present invention was evaluated as described in Example 1. In one preferred embodiment, the microspheres contain about 0.5 μg to about 15 μg, preferably about 1 μg to about 10 μg, of fenofibrate per milligram of microsphere.

[0063] In one embodiment, the fenofibrate contained in the microspheres is 0.1-1% (w / w) of the microparticles, for example, 0.2-0.9% (w / w), 0.3-0.8% (w / w), 0.4-0.7% (w / w), 0.4-0.6% (w / w).

[0064] In a preferred embodiment, the microspheres comprise (i) a polylactic-co-glycolic acid copolymer (PLGA) matrix, the matrix comprising PLGA 75:25 having a viscosity of about 0.60 dl / g and / or a Mw of about 68 kDa, and PLGA 50:50 having a viscosity of about 0.21 dl / g and / or a Mw of about 13.5 kDa; (ii) has an average diameter of about 60 μm; (iii) 1 μg to 10 μg of fenofibrate per milligram of microspheres, preferably 10 μg of fenofibrate per milligram of microspheres; (iv) preferably, when present in a target tissue, it releases fenofibrate over a period of at least 2 months, more preferably, over a period of at least 3 months.

[0065] In a further aspect, the present invention relates to a plurality of biodegradable microspheres as defined in any one of the preceding claims, the microspheres comprising: (i) a d90 particle size value of about 50 μm to about 100 μm, and / or (ii) a d10 particle size value of about 16 μm to about 20 μm, and / or (iii) A plurality of biodegradable microspheres having a d50 particle size value of about 30 μm to about 60 μm.

[0066] In a preferred embodiment of the plurality of biodegradable microspheres, the microspheres comprise: (i) having a d90 value of about 50 μm to about 100 μm; (ii) a polylactic-co-glycolic acid copolymer (PLGA) matrix, the matrix comprising at least two PLGAs having different molecular weights (Mw); (iii) having a therapeutically effective amount of fenofibrate; (iv) preferably releases fenofibrate over a period of at least one month when present in the target tissue.

[0067] As used herein, "d90 particle size" means a particle size distribution such that 90% of the particles have a particle size diameter less than a specified value.

[0068] As used herein, "d50 particle size" means a particle size distribution such that 50% of the particles have a particle size diameter less than a specified value.

[0069] As used herein, "d10 particle size" means a particle size distribution such that 10% of the particles have a particle size diameter less than the specified value.

[0070] The d10, d50 and d90 values ​​may be determined by well-known methods in the art, such as sieve analysis, laser diffraction, optical analysis or optical metrology. The particle size distribution was determined by optical diffraction using a Mastersizer 2000, as described in Example 1. The particle size distribution of microspheres containing ratios of PLGA 50:50 to PLGA 75:25 of about 90:10, about 80:20 or about 75:25 was evaluated as described in the Examples. As can be seen from Figure 6, all three microparticles show a sufficiently sharp peak, but the microspheres containing 90:10 provide the most advantageous particle size distribution. As summarized from the uniformity values ​​ranging from 0 (when all microspheres show the exact same size) to 1 (when all microspheres are different sizes), the 90:10 formulation showed the most satisfactory distribution with a uniformity value of 0.342. The 90:10 formulation was considered to be the preferred one for preparation of fenofibrate microspheres, although microspheres containing ratios of 80:20 and 75:25 were also suitable.

[0071] The d90 particle size range of about 50 μm to about 100 μm includes values ​​of about 45 μm, 50 μm, 55 μm, 60 μm, 70 μm, 75 μm, 80 μm, 85 μm, 90 μm, 95 μm, 100 μm and 105 μm.

[0072] d50 particle size values ​​in the range of about 10 μm to about 30 μm include values ​​of about 8 μm, 9 μm, 10 μm, 11 μm, 12 μm, 13 μm, 14 μm, 15 μm, 16 μm, 18 μm, 19 μm, 20 μm, 21 μm, 22 μm, 23 μm, 24 μm, 25 μm, 26 μm, 27 μm, 28 μm, 29 μm, 30 μm, 31 μm, 32 μm.

[0073] d10 particle size values ​​of 16 μm to about 20 μm include d10 particle size values ​​of about 14 μm, 15 μm 16 μm, 17 μm, 18 μm, 18 μm, 19 μm, 20.0 μm, 21 μm, 22 μm.

[0074] In a further preferred embodiment, each of the at least two PLGAs has a viscosity of approximately 0.16 dl / g to approximately 1.70 dl / g. In a preferred embodiment, one of the at least two PLGAs has a viscosity of approximately 0.50 dl / g to approximately 0.70 dl / g, approximately 0.52 dl / g to approximately 0.68 dl / g, approximately 0.54 dl / g to approximately 0.66 dl / g, approximately 0.56 dl / g to approximately 0.66 dl / g, approximately 0.58 dl / g to approximately 0.64, or approximately 0.60 dl / g to approximately 0.62 dl / g, and a second of the at least two PLGAs has a viscosity of approximately 0.16 dl / g to approximately 0.24 dl / g, approximately 0.18 dl / g to approximately 0.22 dl / g, or approximately 0.20 dl / g to approximately 0.22 dl / g.

[0075] In yet another embodiment of the plurality of biodegradable microspheres, at least two of the PLGA have a Mw of about 6 kDa to about 90 kDa, about 10 kDa to 80 kDa, or about 12 kDa to 70 kDa.

[0076] In a further preferred embodiment, one of the at least two PLGAs has a Mw of about 6 kDa to about 18 kDa, about 7 kDa to about 17 kDa, about 8 kDa to about 16 kDa, about 9 kDa to about 15 kDa, or about 10 kDa to about 14 kDa, and a second of the at least two PLGAs has a Mw of about 50 kDa to about 90 kDa, about 55 kDa to about 80 kDa, or about 60 kDa to about 70 kDa.

[0077] In a further embodiment, one of the at least two PLGAs has a Mw of about 6 kDa to about 18 kDa, about 7 kDa to about 17 kDa, about 8 kDa to about 16 kDa, about 9 kDa to about 15 kDa, about 10 kDa to about 14 kDa, and a second of the at least two PLGAs has a Mw of about 25 kDa to about 40 kDa, about 30 kDa to about 35 kDa.

[0078] In further embodiments of the plurality of biodegradable microspheres, one of the at least two PLGAs has an average molar ratio of lactic acid to glycolic acid of about 100:0 to about 40:60, about 90:10 to about 40:60, about 80:20 to about 50:50, preferably one of the at least two PLGAs has an average molar ratio of lactic acid to glycolic acid of 75:25 (PLGA 75:25) and a second of the at least two PLGAs has an average molar ratio of lactic acid to glycolic acid of 50:50 (PLGA 50:50).

[0079] In a preferred embodiment, the ratio between PLGA 50:50 and PLGA 75:25 is selected from about 90:10, about 80:20 or 75:25, preferably about 90:10.

[0080] In one embodiment, the average diameter of the microspheres is about 30 μm to about 240 μm, about 30 μm to about 235 μm, about 30 μm to about 230 μm, about 35 μm to about 225 μm, about 40 μm to about 225 μm, about 35 μm to about 220 μm, about 35 μm to about 215 μm, about 35 μm to about 210 μm, about 40 μm to about 205 μm, about 40 μm to about 200μm, about 40μm to about 195μm, about 40μm to about 190μm, about 40μm to about 185μm, about 40μm to about 180μm, about 40μm to about 175μm, about 40 μm ~ approx. 170 μm, approx. 40 μm ~ approx. 175 μm, approx. 45 μm ~ approx. 170 μm, approx. 45 μm ~ approx. 165 μm, approx. 45 μm ~ approx. 160 μm, approx. 45 μm ~ approx. 155 μm , about 50 μm to about 150 μm, about 50 μm to about 145 μm, about 50 μm to about 140 μm, about 50 μm to about 135 μm, about 50 μm to about 130 μm, about 50 μm to about 1 25μm, about 50μm to about 120μm, about 50μm to about 115μm, about 50μm to about 110μm, about 50μm to about 105μm, about 50μm to about 100μm, about 50μm Preferably, the thickness is about 55 μm to about 95 μm, about 55 μm to about 90 μm, about 55 μm to about 85 μm, about 55 μm to about 80 μm, 55 μm to about 75 μm, 55 μm to about 70 μm, about 25 μm to about 110 μm, 30 μm to about 100 μm, about 40 μm to about 90 μm, about 45 μm to about 80 μm, about 50 μm to about 70 μm, and most preferably about 55 μm to about 65 μm.

[0081] In one embodiment, the average diameter of the microspheres is about 30 μm, about 35 μm, about 40 μm, about 45 μm, about 50 μm, about 55 μm, about 60 μm, about 65 μm, about 70 μm, about 75 μm, about 80 μm, about 85 μm, about 90 μm, about 100 μm, about 105 μm, about 110 μm, about 115 μm, about 120 μm, about 125 μm, about 130 μm, about 135 μm, about 136 μm, about 137 μm, about 138 μm, about 139 μm, about 200 μm, about 201 μm, about 202 μm, about 203 μm, about 204 μm, about 205 μm, about 206 μm, about 207 μm, about 208 μm, about 209 μm, about 210 μm, about 211 μm, about 212 μm, about 213 μm, about 214 μm, about 215 μm, about 216 μm, about 217 μm, about 218 μm, about 220 μm, about 225 μm, about 226 μm, about 227 μm, about 228 μm, about 230 μm, about 231 μm, about 232 μm, about 233 μm, about 234 μm, about 235 μm, about 236 μm, about 237 μm, about 238 μm, about 240 μm, about 241 μm, about 242 μm, about 243 μm, about 244 μm, about 245 μm 5 μm, about 140 μm, about 145 μm, about 150 μm, about 155 μm, about 160 μm, about 165 μm, about 170 μm, about 175 μm, about 180 μm, about 185 μm, about 190 μm, about 195 μm, about 200 μm, about 205 μm, about 210 μm, about 215 μm, about 220 μm, about 225 μm, about 230 μm, about 235 μm, and about 240 μm.

[0082] In one preferred embodiment, the average diameter of the microspheres is about 60 μm.

[0083] In one embodiment, the microspheres contain from about 0.5 μg to about 20 μg, preferably from about 0.5 μg to about 15 μg, and most preferably about 1 μg to about 10 μg of fenofibrate per milligram of microsphere.

[0084] Pharmaceutical Compositions The present invention also relates to an injectable formulation comprising a pharma- ceutically acceptable carrier and a microsphere or a plurality of biodegradable microspheres described herein.

[0085] The injectable formulations of the present invention and their efficacy in sustained release of fenofibrate in microspheres were evaluated as described in Example 2. Microspheres prepared with a single PLGA showed sustained controlled sustained release of drug over a period of 3 months (see FIG. 2). However, the percentage of drug release was relatively low for all formulations with approximately 20% of the loaded drug released at this time point. Therefore, it was decided to change the structure of the polymer matrix by combining a low molecular weight 50:50 PLGA with a higher molecular weight 75:25 PLGA to prepare microspheres that exhibit a higher total release of drug. As shown in FIG. 5, the resulting microspheres had the highest drug burst release followed by drug diffusion-PLGA erosion controlled drug release that reached approximately 90% release of drug after 3 months. This initial burst release is useful to reach the required therapeutic concentration of the drug while the subsequent controlled release profile allows the drug concentration to remain within the therapeutic window.

[0086] In a preferred embodiment of the formulation, the microspheres exhibit a sustained release profile, when present in the target tissue, that releases fenofibrate over a period of at least 1 month, preferably at least 2 months, and more preferably at least 3 months.

[0087] In a more preferred embodiment, the total release of drug from the microspheres is at least 60%, at least 70%, and preferably at least 80% after 50 days, preferably after 40 days. In a preferred embodiment, the total release of drug from the microspheres is at least 80%, at least 85%, or at least 90% after 60 days, preferably after 70 days, more preferably after 80 days, and most preferably after 90 days.

[0088] Uses and Treatment The present invention further relates to the biodegradable microsphere, the plurality of biodegradable microspheres, or the injectable formulation as described herein above for use in the treatment or prevention of joint-related disorders, preferably arthritis, more preferably osteoarthritis.

[0089] The efficacy of the injectable formulation of the present invention and the sustained release of fenofibrate in microspheres by intra-articular injection was evaluated in two independent preclinical models of osteoarthritis induced by surgery in mice, as further detailed in Example 3 and Example 4. As can be seen from Figure 7A), the mouse knee joints in the vehicle group (PLGA 90:10) showed significant joint damage, including proteoglycan deficiency, loss of the surface layer, and cartilage fibrillation. However, treatment with PLGA 90:10 FN 1ug and PLGA 90:10 FN 10ug significantly reduced the severity of these OA-like changes. Analysis of osteoarthritis lesions by a semi-quantitative scoring system showed a significant reduction in the severity of OA-like changes after sustained release of fenofibrate treatment compared to the vehicle group (P<0.01) (Figure 7B). As can be seen from Figure 8A), the knee joints of mice in the vehicle group (PLGA 90:10) showed significant joint damage. However, treatment with PLGA 90:10 FN 1ug and PLGA 90:10 FN 10ug significantly reduced the severity of experimental osteoarthritis. Analysis of osteoarthritis lesions by semi-quantitative scoring system showed a significant reduction in disease severity after sustained release of fenofibrate treatment compared to the vehicle group (P<0.05) (Figure 8B).

[0090] The inventors could further show that PLGA (50:50):PLGA (75:25) 90:10 microspheres, characterized by high encapsulation efficiency, exhibit sustained release in human synovial fluid from osteoarthritis patients over a total period of 7 weeks (Example 7 and Figure 12). This data is consistent with the controlled release profile of the drug and demonstrates the presence of the drug in the synovial fluid for at least 49 days. Furthermore, the release profile in human synovial fluid was similar to that obtained with 1% SDS supplementation (Figure 12). Thus, the data obtained in mice could be corroborated in human synovial fluid from osteoarthritis patients, indicating that the microspheres of the present invention are suitable for providing therapy in human subjects.

[0091] As discussed above, patients suffering from joint disorders, specifically osteoarthritis, require regular treatment to relieve their discomfort. One way to relieve pain over a long period of time is the long-term daily intake of a drug, which can be cumbersome and requires good patient compliance for positive treatment results. Another drawback of administering a daily dose of a drug is the fact that systemically, as this is usually done by oral dosage forms, the systemic delivery of the drug can cause undesirable side effects. Therefore, local administration at the joint of interest is much more preferred. In the case of fenofibrate, the drug can be administered by injection into the joint, to avoid systemic administration. However, in this case, it is necessary to be able to perform a single injection, which results in a longer treatment period, since it is not feasible for patients to inject into the joint every day. Therefore, the present inventors have developed the microspheres described herein, and injectable formulations containing said microspheres, which use a controlled and sustained release of the active ingredient fenofibrate into the joint, thus providing the possibility of a single injection, which overcomes the drawbacks. To date, no reliable long-term treatment options using fenofibrate are available on the market, and the inventors herein provide a solution to this unmet need by providing a superior method for treating joint disorders, such as osteoarthritis, by using fenofibrate while minimizing the side effects of the drug observed with systemic delivery. Impressively, the inventors were able to show that sustained release was achieved with the microspheres of the present invention for up to three months after administering only a single dose.

[0092] Thus, the present invention also relates to a biodegradable microsphere, a plurality of biodegradable microspheres, or an injectable formulation as described herein, administered intra-articularly, preferably, said microsphere, a plurality of microspheres, or an injectable formulation is administered as a single dose.

[0093] The present invention further relates to a method for treating a joint-related disorder in a subject, comprising introducing a biodegradable microsphere, a plurality of biodegradable microspheres, or an injectable formulation described herein into target tissue within or around one or more of the subject's joints, preferably the hip, shoulder, and / or knee joints.

[0094] In one embodiment, the subject is a human or an animal, such as a cat, dog, or horse.

[0095] In a further embodiment, the joint-related disorder is arthritis, preferably osteoarthritis.

[0096] In one embodiment, the method comprises intra-articularly injecting a biodegradable, a plurality of biodegradable microspheres, or an injectable formulation described herein into one or both of the small joints of the subject's knee, shoulder, hip, neck, lower back, or hand.

[0097] It should be understood that the joints referred to in the description of the present invention relate to any joint that may be affected by a joint-related disorder, such as arthritis. In particular, the joint may be a knee, hip, shoulder, neck, lumbar joint, small joints of the hand or foot.

[0098] kit In one aspect, the present invention provides a kit comprising, in separate compartments: (a) a diluent; (b) a biodegradable microsphere or a plurality of biodegradable microspheres according to any one of the preceding claims.

[0099] The injectable formulation disclosed herein can be provided as a single-dose kit that contains one vial of the microspheres that contain fenofibrate disclosed herein.The microspheres can be provided in powder form that increases the storage stability of the active ingredient and the microspheres that contain it.The second vial provided in the kit can contain a diluent and can be added to the first vial that contains the microspheres immediately before use / administration of the injectable formulation.

[0100] Methods for preparing microparticles In one aspect, the present invention relates to a method for preparing the microspheres described herein, the method comprising a single emulsion-solvent evaporation. The single emulsion-solvent evaporation provides microspheres with the desired properties described herein above, and at the same time is a very simple, economical, reliable and easy to scale-up method. Moreover, the method does not require extreme conditions and avoids the risk of fenofibrate degradation, while ensuring sufficient drug encapsulation. Although the single emulsion-solvent evaporation is well known to those skilled in the art and can be carried out by methods known in the prior art, examples 1 and 2 of the present specification provide one way of applying said method to prepare the microspheres of the present invention.

[0101] In a preferred embodiment of the method, the microspheres are prepared using PLGA at a polymer concentration of about 10-30% (w / w), preferably 15-25% (w / w), more preferably 15-20% (w / w).

[0102] In one embodiment of the method of the present invention, the method allows for encapsulation of at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% of the fenofibrate within the microspheres of the present invention. The combination of a polymer matrix used in conjunction with the use of a single emulsion-solvent evaporation method is preferred and allows for high encapsulation rates as shown herein. EXAMPLES

[0103] Example 1: Development of an extended release formulation containing microspheres and fenofibrate 1.Material PLGA of various lactic acid:glycolic acid ratios (L:G ratios) and molecular weights with the characteristics shown in Table 1 were purchased from Evonik, Ltd. (Essen, Germany). Polyvinyl alcohol (PVA) (87-90% hydrolyzed, average weight molecular weight 30,000-70,000 Da) was obtained from Sigma-Aldrich (St. Louis, USA). High performance liquid chromatography grade dichloromethane (DCM) was purchased from Merck (Germany). All experiments were performed using Milli-Q® water. [Table 1]

[0104] 2. Microsphere Synthesis Fenofibrate-loaded microspheres were obtained by a single emulsion-solvent evaporation method (o / w). For this purpose, a single PLGA polymer (Resomer® RG 502H or Resomer® RG 503H or Resomer® RG 752H or Resomer® RG 753H) was used. To obtain fenofibrate-loaded microspheres, 2 mg of fenofibrate was dissolved in 200 μL of DCM. This solution was added to 0.8 mL of the respective PLGA solution in DCM at 20% w / v and homogenized by vortexing to obtain an oil phase. Then, 4 mL of an aqueous solution prepared with 1% PVA was added to the oil phase and homogenized using a magnetic stirrer at 1000 rpm for 5 minutes to form an emulsion. Finally, the solvent was evaporated by pouring the obtained microspheres into 6 mL of PVA at 0.2% for 12 h under continuous stirring to reach complete dichloromethane evaporation. Finally, the microspheres were washed three times with double distilled water, filtered through a 0.45 μm pore size filter (Pall Corporation, Sigma-Aldrich), lyophilized, and stored at 4° C. until use.

[0105] 3. Particulate characterization The microparticles were characterized in terms of size and size distribution by light diffraction using a Mastersizer (Mastersizer 2000, Malvern Instruments, Malvern, UK). To determine the encapsulation efficiency (EE) of the microspheres, 3 mg of microparticles were dissolved in 3 mL of dichloromethane and centrifuged at 12,000 rpm at 4° C. for 30 minutes. The drug concentration in the supernatant was determined using a validated spectroscopic method. EE was calculated using the following formula:

number

[0106] 4. Fenofibrate Drug Release Profile Drug release studies were performed in simulated synovial fluid supplemented with 1% SDS to ensure immersion conditions. The simulated synovial fluid was prepared according to the procedure described by Marques et al. and [1]. A known weight of microspheres (13 mg) was placed in 4 mL of release medium and incubated in a shaking bath at 37°C and 45 rpm for 3 months. At pre-determined time points, the samples were centrifuged at 1,200 rpm for 4 min and 2 ml of supernatant was collected and replaced with fresh medium. The samples were then homogenized again by vortexing and placed back in the shaking bath. The amount of released fenofibrate was quantified in the collected supernatant using a validated spectroscopic method at 291 nm.

[0107] Example 2: Preparation of fenofibrate-loaded PLGA microspheres using different Mw of PLGA 1.Material PLGA of various lactic acid:glycolic acid ratios (L:G ratios) and molecular weights with the characteristics shown in Table 2 were purchased from Evonik, Ltd. (Essen, Germany). Polyvinyl alcohol (PVA) (87-90% hydrolyzed, average weight molecular weight 30,000-70,000 Da) was obtained from Sigma-Aldrich (St. Louis, USA). High performance liquid chromatography grade dichloromethane (DCM) was purchased from Merck (Germany). All experiments were performed using Milli-Q® water. [Table 2]

[0108] 2. Microsphere Synthesis Similarly as described in Example 1, fenofibrate-loaded microspheres were obtained by a single emulsion-solvent evaporation method (o / w). For this purpose, PLGA of various molecular weights and L:G ratios were combined in different proportions to obtain the desired microspheres with improved properties such as small size, narrow size distribution, high loading efficiency and sufficient fenofibrate release profile. More specifically, Resomer® RG 502H was combined with Resomer® RG 755S in weight:weight ratios of 75:25, 80:20 and 90:10. To obtain fenofibrate-loaded microspheres, 0.2 mg or 2 mg of fenofibrate was dissolved in 200 μL of DCM. This solution was added to 0.8 mL of PLGA solution in DCM at 20% w / v and homogenized by vortex to obtain an oil phase. Then, 4 mL of an aqueous solution prepared with 1% PVA was added to the oil phase and homogenized by vortexing at maximum speed for 1 min. Finally, the solvent was evaporated by pouring the resulting microspheres into 96 mL of PVA at 0.16% for 2 h under continuous stirring. After solvent removal, the microspheres were washed three times with double distilled water, filtered through a 0.45 μm pore size filter (Pall Corporation, Sigma-Aldrich), lyophilized, and stored at 4°C until use.

[0109] 3. Particulate characterization The microparticles were characterized in terms of size and size distribution by light diffraction using a Mastersizer (Mastersizer 2000, Malvern Instruments, Malvern, UK) as described in Example 1.

[0110] 4. Fenofibrate Drug Release Profile Drug release studies were performed in simulated synovial fluid as described in Example 1.

[0111] Example 3: Efficacy of sustained release of fenofibrate in microspheres by intra-articular injection in a preclinical model of osteoarthritis in mice The efficacy of a novel formulation of microspheres for the controlled release of fenofibrate was evaluated on articular tissues by using a surgically induced osteoarthritis model in mice, a predictive preclinical model of the disease that resembles human pathological joint changes occurring in cartilage and synovium.

[0112] 1. Surgery-induced osteoarthritis All animal experiments were performed according to protocols approved by the Institutional Animal Care and Use Committee at Instituto de Investigacion Biomedica de A Coruna (INIBIC). Experimental osteoarthritis was induced in 3-month-old male C57Bl / 6J mice by transverse transection of the medial medial ligament and median collateral ligament (MMTL+MCL) in the right knee as previously described [2]. The left knee was not operated on, only the joint capsule was opened, and was used as a sham control. Body weight was assessed every 2 weeks. Ten weeks after surgery, the mice were euthanized.

[0113] 2. Sustained release of fenofibrate in microspheres via intra-articular administration FN was administered by a single intra-articular injection in two concentrations (1 μg and 10 μg) of drug-loaded microspheres. A sustained release formulation was prepared and gradually released into the joint over at least 3 months. Three test groups were used to evaluate efficacy: Group 1: mice treated with empty microspheres as placebo (control group), Group 2: mice treated with FN-1 μg, and Group 3: mice treated with FN 10 μg. In all groups, treatment was started one week after surgery recovery, meaning that the joint capsule was healed and closed before intra-articular injection.

[0114] 3. Histological Analysis of Mouse Knee Joints Knee joints of mice were fixed in 10% zinc-buffered formalin for 24 hours, decalcified in TBD for 6 hours, and then embedded in paraffin. Serial sections (4 μm) were cut, stained with Safranin O fast green, and examined for histological changes using a semiquantitative scoring system for preclinical evaluation of total joint changes according to the OARSI guidelines [3]. In this system, the score is defined as follows: 0=normal cartilage, 0.5=proteoglycan loss with intact surface, 1=superficial fibrillation without cartilage loss, 2=vertical clefts and superficial layer loss (any % or articular surface area), 3=vertical clefts / erosion to the demineralized layer lesions 1-25% of the quadrant width, 4=lesion reaches demineralized cartilage for 25-50% of the quadrant width, 5=lesion reaches demineralized cartilage for 50-75% of the quadrant width, 6=lesion reaches demineralized cartilage for more than 75% of the quadrant width.

[0115] 4.Statistical analysis Differences between two groups are determined by Student's t-test, while differences between multiple groups are determined by ANOVA with Tukey's multiple comparisons. Data analysis is performed by using Prism 9.0 software. Results are reported as mean ± SEM. A p-value <0.05 is considered significant.

[0116] Example 4: Efficacy of sustained release of fenofibrate in microspheres by intra-articular injection in a preclinical model of osteoarthritis in mice The efficacy of a novel formulation of microspheres for the controlled release of fenofibrate was evaluated on articular tissues by using a surgically induced osteoarthritis model in mice, a predictive preclinical model of the disease that resembles human pathological joint changes occurring in cartilage and synovium.

[0117] 1. Surgery-induced osteoarthritis All animal experiments were performed according to protocols approved by the Institutional Animal Care and Use Committee at Instituto de Investigacion Biomédica de A Coruna (INIBIC). Experimental osteoarthritis was induced in 3-month-old male C57Bl / 6J mice by transverse transection of the transverse medial ligament and transverse collateral ligament (LMTL+LCL) in the right knee as previously described [2]. The left knee was not operated on, only the joint capsule was opened, and was used as a sham control. Body weight was assessed every 2 weeks. Ten weeks after surgery, the mice were euthanized.

[0118] 2. Sustained release of fenofibrate in microspheres via intra-articular administration FN was administered by a single intra-articular injection in two concentrations (1 μg and 10 μg) of drug-loaded microspheres. A sustained release formulation was prepared and gradually released into the joints over at least 3 months. Three test groups were used to evaluate efficacy: Group 1: mice treated with empty microspheres as placebo (control group), Group 2: mice treated with FN-1 μg, and Group 3: mice treated with FN 10 μg. In all groups, treatment began 1 week after surgery recovery.

[0119] 3. Histological Analysis of Mouse Knee Joints Histological joint changes were assessed as described in Example 3.

[0120] 4.Statistical analysis Statistical analyses were determined as described in Example 3.

[0121] Example 5: Efficacy of oral fenofibrate in a preclinical model of surgery-induced osteoarthritis 1. Surgery-induced osteoarthritis All animal experiments were performed according to protocols approved by the Institutional Animal Care and Use Committee at Instituto de Investigacion Biomedica de A Coruna (INIBIC). Experimental osteoarthritis in mouse knee joints was performed as described in Example 3.

[0122] 2. Oral fenofibrate treatment FN was administered daily in drinking water at 100 mg / kg body weight / day. To evaluate efficacy, two test groups were performed: Group 1: mice treated with vehicle (DMSO), Group 2: mice treated with FN 100 mg / kg body weight / day. Treatment began in all groups 3 days after surgery recovery.

[0123] 3. Histological Analysis of Mouse Knee Joints Histological joint changes were assessed as described in Example 3.

[0124] 4.Statistical analysis Statistical analyses were determined as described in Example 3.

[0125] Example 6: Evaluation of Higuchi drug release profiles The aim is to achieve a delayed release that can maintain a constant concentration with the formulation of the present invention over a period of 3 months while ensuring that the initial released drug achieves a therapeutic concentration. Therefore, the inventors evaluated the Higuchi drug release profile at the initial stage of drug release. This constant amount gives information about the initial stage of release, which is a drug diffusion controlled process by the polymer matrix according to the Higuchi equation [4]. The formulations evaluated are shown in Table 3 below, and the formulation selected as optimal according to the previous results is highlighted in bold. [Table 3]

[0126] Figure 10 shows the release rate of three different microspheres prepared as detailed above. One type of microsphere has a diameter of about 30 μm and was prepared according to the formula shown in the last row of Table 3 (Resomer RG 502 about 30 μm). Another type has a diameter of about 300 μm and was prepared according to the conditions shown in the second row starting from the last row (Resomer RG 502 about 300 μm). The third is the preferred microsphere described herein above, comprising PLGA (50:50):PLGA (75:25) 90:10, with an average diameter of about 60 μm.

[0127] As can be seen in Figure 10, (Resomer RG 502 approx. 300 μm) shows that due to too low a release rate, very little or no therapeutic effect is observed during the first month, whereas when the release rate is too high, as in the case of Resomer RG 502 approx. 30 μm microspheres, all of the fenofibrate is already released after 39 days, although it would be released over a 3 month period if desired.

[0128] Example 7: Sustained release of fenofibrate from PLGA microspheres in synovial fluid from patients with OA To demonstrate that the microspheres of the invention are also suitable for therapy in human subjects, the release behavior of the microspheres was tested in synovial fluid from patients suffering from osteoarthritis.

[0129] 1. Human synovial fluid samples from the knee joints of patients diagnosed with osteoarthritis were collected by withdrawing fluid using a needle inserted between the joint gap. Samples from six patients were collected and stored at -80°C until analysis. Samples were then pooled until the experiments were performed.

[0130] Microspheres of PLGA (50:50):PLGA (75:25) 90:10, characterized by an average diameter size of 2.60 μm, were weighed (average release medium volume / mass of microspheres used was 70 μL / mg). The assay was performed in a 1.5 mL tube, containing a total volume of 100 μL of synovial fluid and a total of 1.43 mg of microspheres.

[0131] 3. Microspheres were incubated with the indicated volumes of human synovial fluid at 37° C. in an incubator with medium shaking (200 rpm).

[0132] 4. For LC-MS / MS analysis, synovial fluid was centrifuged at 3000 rpm for 5 min to pellet the microspheres and the supernatant (83% of the initial volume) was collected and stored for drug quantification. Samples were collected weekly (0, 1, 2, 3, 4, 5, 6, 7) for 7 consecutive weeks over a total of 49 days.

[0133] 5. The supernatant was replaced with an equal volume of fresh synovial fluid, homogenized by vortexing for 1 minute, and placed back into the 37° C. incubator with medium shaking (200 rpm).

[0134] 6. The concentration of fenofibrate released from the microspheres was determined by using the LC-MS / MS protocol detailed below. Prior to analysis, samples were diluted with two volumes of acetonitrile (80 μL sample + 160 μL acetonitrile (ACN)). Rolipram was added to each sample as an internal standard. The injection volume used was 4 μL.

[0135] Chromatography conditions The chromatography equipment used was an ACQUITY UPLC H-Class and a Xevo TQD MS System. A BEH C18 1.7 μm 2.1×50 mm (Waters) column was used at a flow rate of 0.6 mL / min.

[0136] Water + 0.1% formic acid was used as solvent A and acetonitrile + 0.1% formic acid was used as solvent B. The gradient program used was as follows: 0-0.1 min 20% B, 0.1-1.0 min 100% B, 1.0-2.0 min 100% B, 2.0-2.1 min 20% B, and 2.1-2.5 min 20% B.

[0137] Electrospray ionization (ESI) was performed in positive mode with a source temperature of 150° C. and a desolvation temperature of 600° C. The capillary voltage was set at 3 kV and the cone voltage at 30 V. The desolvation gas flow was 1100 L / h and the cone gas flow was set at 150 L / h.

[0138] The compound of interest was monitored in multiple reaction monitoring (MRM) mode. The quantification trace used for fenofibrate determination was 361.034>233.01 with a retention time of 1.69 min.

[0139] result A regression analysis was established showing the relationship between the theoretical concentration of the drug and the resulting area under the curve (AUC) for the traces. A range of 12 concentrations (100, 50, 25, 12.5, 6.25, 3.12, 1.56, 0.78, 0.39, 0.19, 0.09, 0.049) was used. A strong relationship between the concentration and AUC data is observed, fitting a linear regression model. R 2 =0.99 (Figure 11).

[0140] PLGA(50:50):PLGA(75:25) 90:10 microspheres with an average diameter of 60 μm and high encapsulation efficiency show controlled drug release in human synovial fluid from osteoarthritis patients over a total period of 7 weeks. As previously observed in simulated synovial fluid supplemented with SDS, an initial burst effect occurs at the first time point, in which 22% of the loaded drug is released in the first week. This initial release leads to a fenofibrate concentration in synovial fluid of 90.8±8.5 μM after one week. Notably, after the first time point, the release rate follows zero-order kinetics with an average release constant of 8 ng / h (R 2 =0.98). The data are consistent with a controlled release profile of the drug and indicate its presence in the synovial fluid over at least 49 days. Furthermore, the release profile in human synovial fluid was similar to that obtained from simulated synovial fluid supplemented with 1% SDS, although a more rapid release could be observed after the second week of the experiment, most likely related to a more rapid degradation of the polymer network associated with the presence of degradative molecules in the diseased synovial fluid (Figure 12).

[0141] List of references: [1] Marques, MRC, Loebenberg, R., & Almukainzi, M. (2011). Simulated Biological Fluids with Possible Application in Dissolution Testing. Dissolution Technologies, 18(3), 15-28. https: / / doi.org / 10.14227 / DT180311P15 [2]Carames B, Hasegawa A, Taniguchi N, et al.Autophagy activation by rapamycin reduces severity of experimental osteoarthritis.Ann Rheum Dis.2012;71:575-81.doi:10.1136 / annrheumdis-2011-200557 [3]Glasson SS,Chambers MG,Van Den Berg WB,et al.The OARSI histopathology initiative - recommendations for histological assessments of osteoarthritis in the mouse.Osteoarthritis Cartilage.2010;18:S17-23.doi:10.1016 / j.joca.2010.05.025 [4]Marcos Luciano Bruschi,Chapter 5 - Mathematical models of drug release,Strategies to Modify the Drug Release from Pharmaceutical Systems,Woodhead Publishing,2015,63-86,ISBN 9780081000922 [5]Klose D.et al:”Fenofibrate-loaded PLGA microparticles:Effects on ischemic stroke”,EUROPEAN JOURNAL OF PHARMACEUTICAL SCIENCES,ELSEVIER AMSTERDAM,NL,vol.37,no.1,11 April 2009(2009-04-11),43-52ページ,XP025992155,ISSN:0928-0987,doi:10.1016 / J.EJPS.2008.12.01 [6]Qiu Fangfang et al:”Fenofibrate-Loaded Biodegradable Nanoparticles for the Treatment of Experimental Diabetic Retinopathy and Neovascular Age-Related Macular Degeneration”,MOLECULAR PHARMACEUTICS,[オンライン]vol.16,no.5,26 March 2019(2019-03-26),1958-1970ページ,XP055932182,US [7]Grabacka M.et al:”Fenofibrate subcellular distribution as a rationale for the intracranial delivery through biodegradable carrier”,Journal of physiology and pharmacology:an official journal of the Polish Physiological Society,1 April 2015(2015-04-01),ページ233-247,XP055938185,Poland [8]Nogueira-Recalde Uxia et al:”Fibrates as drugs with senolytic and autophagic activity for osteoarthritis therapy”,EBIOMEDICINE,[オンライン]vol.45,5 July 2019(2019-07-05),588-605ページ,XP055938558,NL,ISSN:2352-3964.doi:10.1016 / j.ebiom.2019.06.049

Claims

1. Biodegradable microspheres, comprising: (i) a polylactic-co-glycolic acid copolymer (PLGA) matrix, the matrix comprising at least two PLGAs having different molecular weights (Mw); (ii) have an average diameter of about 40 μm to about 225 μm; (iii) Biodegradable microspheres containing fenofibrate.

2. 2. The biodegradable microspheres of claim 1, wherein the at least two PLGAs each have a viscosity of about 0.16 dl / g to about 1.70 dl / g, preferably one of the at least two PLGAs has a viscosity of about 0.50 dl / g to about 0.70 dl / g, and a second of the at least two PLGAs has a viscosity of about 0.16 dl / g to about 0.24 dl / g.

3. 2. The biodegradable microsphere of claim 1, wherein the at least two PLGAs have a Mw of about 6 kDa to 90 kDa, preferably one of the at least two PLGAs has a Mw of about 6 kDa to 18 kDa and a second of the at least two PLGAs has a Mw of about 50 kDa to 90 kDa.

4. 2. The biodegradable microspheres of claim 1, wherein one of the at least two PLGAs has an average molar ratio of lactic acid to glycolic acid of about 100:0 to about 50:50, preferably one of the at least two PLGAs has an average molar ratio of lactic acid to glycolic acid of about 75:25 (PLGA 75:25) and a second of the at least two PLGAs has an average molar ratio of lactic acid to glycolic acid of about 50:50 (PLGA 50:50).

5. 5. The biodegradable microspheres according to claim 4, wherein the ratio between the PLGA 50:50 and the PLGA 75:25 is selected from about 90:10, about 80:20 or about 75:25, preferably about 90:

10.

6. 2. The biodegradable microsphere of claim 1, wherein the microsphere has a diameter of about 40 μm to about 175 μm, about 45 μm to about 155 μm, about 50 μm to about 125 μm, about 55 μm to about 90 μm, about 55 μm to about 70 μm, or about 55 μm to about 65 μm.

7. 2. The biodegradable microspheres of claim 1, wherein the microspheres contain about 0.5 μg to about 20 μg, preferably about 0.8 μg to about 15 μg, and most preferably about 1 μg to about 10 μg of fenofibrate per milligram of microsphere.

8. The microspheres are (i) a polylactic-co-glycolic acid copolymer (PLGA) matrix, the matrix comprising PLGA 75:25 having a viscosity of about 0.60 dl / g and / or a Mw of about 68 kDa and PLGA 50:50 having a viscosity of about 0.21 dl / g and / or a Mw of about 13.5 kDa; (ii) have an average diameter of about 55-65 μm, preferably about 60 μm; (iii) The biodegradable microspheres of claim 1, comprising about 0.5 μg to about 15 μg of fenofibrate per milligram of microsphere, preferably about 1 μg to about 10 μg of fenofibrate per milligram of microsphere.

9. The microspheres are (i) a d90 particle size value of about 50 μm to about 100 μm, and / or (ii) a d10 particle size value of about 16 μm to about 20 μm, and / or (iii) A plurality of biodegradable microspheres according to any one of claims 1 to 8, having a d50 particle size value of from about 30 μm to about 60 μm.

10. An injectable formulation comprising a pharmaceutically acceptable carrier and the microspheres of any one of claims 1 to 8.

11. An injectable formulation comprising a pharmaceutically acceptable carrier and a plurality of biodegradable microspheres according to claim 9.

12. 11. The formulation of claim 10, wherein the microspheres, when present in the target tissue, release fenofibrate for at least 1 month, preferably at least 2 months, more preferably at least 3 months.

13. The formulation of claim 11, wherein the microspheres, when present in the target tissue, release fenofibrate for at least one month, preferably at least two months, and more preferably at least three months.

14. Biodegradable microspheres according to any one of claims 1 to 8 for the treatment or prevention of joint-related disorders, preferably arthritis, more preferably osteoarthritis.

15. A plurality of biodegradable microspheres according to claim 9 for the treatment or prevention of joint-related disorders, preferably arthritis, more preferably osteoarthritis.

16. An injectable formulation according to claim 10 for the treatment or prevention of joint-related disorders, preferably arthritis, more preferably osteoarthritis.

17. An injectable formulation according to claim 11 for the treatment or prevention of joint-related disorders, preferably arthritis, more preferably osteoarthritis.

18. A biodegradable microsphere described in any one of claims 1 to 8, wherein the biodegradable microsphere is administered intra-articularly, preferably the microsphere is administered as a single dose.

19. The biodegradable microspheres of claim 9, wherein the plurality of biodegradable microspheres are administered intra-articularly, preferably the plurality of microspheres are administered as a single dose.

20. The injectable formulation of claim 10, wherein the injectable formulation is administered intra-articularly, preferably wherein the injectable formulation is administered as a single dose.

21. The injectable formulation of claim 11, wherein the injectable formulation is administered intra-articularly, preferably wherein the injectable formulation is administered as a single dose.

22. A kit comprising, in separate compartments: (a) a diluent; (b) the biodegradable microspheres according to any one of claims 1 to 8; Includes a kit.

23. A kit comprising, in separate compartments: (a) a diluent; (b) a plurality of biodegradable microspheres according to claim 9; Includes a kit.

24. 9. A method for preparing microspheres according to any one of claims 1 to 8, said method comprising a single emulsion-solvent evaporation, preferably wherein said microspheres are prepared with PLGA at a polymer concentration of about 10-30% (w / w), preferably 15-25% (w / w), more preferably 15-20% (w / w).

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