Pharmaceutical Composition for the Treatment of Osteoarthritis

Abstract

The present disclosure provides methods and compositions for treating osteoarthritis, comprising hyaluronic acid (HA) hydrogel in combination with saturated fatty acids. In some embodiments, aspects of the present disclosure relate to pharmaceutical compositions comprising HA hydrogel in combination with steroids. In some embodiments, the pharmaceutical compositions can be administered locally, topically, and by injection. In some embodiments, the pharmaceutical compositions can be administered by intra-articular injection.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This patent application claims priority to U.S. Provisional Patent Application No. 63 / 441,415, filed January 26, 2023, and U.S. Provisional Patent Application No. 63 / 509,085, filed June 20, 2023, each of which is incorporated by reference in its entirety. [Background technology]

[0002] Inflammation creates a significant global disease burden. For example, osteoarthritis affects 25 to 35 million people in the United States alone. Inflammation within articular cartilage and bone is thought to cause osteoarthritis, which results in chronic pain and functional disability that gradually worsens with age. Symptomatic knee osteoarthritis occurs in more than 10% of people over the age of 60, and knee osteoarthritis reduces mobility more than any other medical condition in older adults.

[0003] Current pharmaceutical interventions for osteoarthritis are limited to analgesics, nonsteroidal anti-inflammatory drugs (NSAIDs), and intra-articular steroid injections, each of which exhibits limiting or adverse side effects. Given these pharmaceutical limitations, chronic knee osteoarthritis often leads to progressive functional disability that ultimately requires total joint replacement. The increasing prevalence of knee osteoarthritis in aging and obese populations suggests a growing clinical need for safe and effective pharmaceutical interventions to delay and potentially eliminate the need for orthopedic surgery.

[0004] Chronic inflammation and / or osteoarthritis can be treated with, for example, corticosteroids, NSAIDs, and tumor necrosis factor (TNF) inhibitors. Corticosteroids are generally passively transported into the cytoplasm, bind to intracellular steroid receptors, and then transport to the nucleus, where they exert their effects through gene expression. NSAIDs generally inhibit the activity of cyclooxygenase enzymes, while TNF inhibitors typically bind to TNF, preventing ligand-receptor binding and suppressing two important inflammatory signaling cascades. However, targeting specific pathological signaling pathways alone may not reverse the harm caused by chronic or hyperactive inflammation alone. Identifying novel strategies to treat inflammation overall or upstream modalities is desirable; strategies that prevent or reverse underlying pathologies could facilitate a paradigm shift in how physicians manage inflammation.

[0005] An attractive target for treating chronic inflammatory diseases such as osteoarthritis is the cytoplasmic multiprotein complex known as the inflammasome. Inflammasomes assemble during inflammation and activate caspase-1-mediated release of the proinflammatory cytokines interleukin-1β (IL-1β) and interleukin-18 (IL-18). Furthermore, inflammasome activation generally initiates other inflammatory signaling cascades. Therefore, inflammasome inhibitors could potentially quell several different inflammatory pathways and even prevent inflammation from progressing. Inflammasome-targeting drugs are in clinical development (see, for example, Marchetti et al., "OLT1177, a β-sulfonyl nitrile compound, safe in humans, inhibits the NLRP3 inflammasome and reverses the metabolic cost of inflammation," PROC. NAT'L ACAD. SCI. USA 2018; 115(7)). Nevertheless, identifying inflammasome agonists that are effective in inhibiting multiple inflammatory signaling pathways remains challenging. Such agonists are likely to offer therapeutic benefit in the treatment of chronic inflammatory diseases, including osteoarthritis.

[0006] Pain associated with inflammation can be directly treated with analgesics, such as opiates, which generally target opioid receptors in the brain. Local analgesics include NSAIDs, capsaicin, and lidocaine. Capsaicin targets vanilloid receptors, while lidocaine targets voltage-gated sodium channels. Additionally, N-methyl-D-aspartate (NMDA) receptor agonists are known to exhibit analgesic effects, such as ketamine and nitrous oxide. NMDA receptor agonists are generally not prescribed for long-term pain management or for the treatment of pain associated with inflammation.

[0007] The NMDA receptor is a glutamate receptor and calcium ion channel. When two glutamates bind to the NMDA receptor, the calcium ion channel is activated, increasing calcium permeability. Agonists such as ketamine and nitrous oxide block the calcium channel. NMDA receptor activation may contribute to the development and maintenance of chronic pain states by inducing sensitization of pain-sensing neurons, and thus NMDA receptors play a role in synaptic plasticity and pain.

[0008] Glutamate, which functions as a neurotransmitter when bound to NMDA receptors, is also an amino acid building block of proteins and a precursor and metabolite in numerous other biochemical pathways. Glutamate is notably the transamination product of α-ketoglutarate (AKG), an intermediate in the citric acid cycle, and many different enzymes interconvert glutamate and AKG.

[0009] The citric acid cycle (also known as the Krebs cycle) is a series of enzymatic reactions that occur in mitochondria and generate energy in the form of adenosine triphosphate (ATP). Fatty acid beta-oxidation breaks down fatty acids to produce acetyl-CoA, which then enters the citric acid cycle in mitochondria to generate ATP. AKG is not formed directly from fatty acids. Fatty acids first undergo beta-oxidation to produce acetyl-CoA, which can then be converted to AKG. The enzyme glutamate dehydrogenase can catalyze the conversion of glutamate to AKG and NADPH, which is involved in cellular processes such as fatty acid synthesis and antioxidant defense. It remains unclear whether pharmacological manipulation of the citric acid cycle or its intermediates, for example by modulating glutamate levels, can affect NMDA receptor activation and produce therapeutic effects. Summary of the Invention

[0010] Various aspects of the present disclosure relate to pharmaceutical compositions comprising hyaluronic acid (HA) hydrogel in combination with saturated fatty acids (e.g., decanoic acid (capric acid), octanoic acid (caprylic acid), and other medium-chain fatty acids). In some embodiments, aspects of the present disclosure relate to pharmaceutical compositions comprising HA hydrogel in combination with a steroid (e.g., dexamethasone or its esters). In some embodiments, the pharmaceutical composition may be administrable locally, topically, or by injection. In some specific embodiments, the pharmaceutical composition may be administrable by intra-articular injection. [Brief explanation of the drawings]

[0011] [Figure 1] Figure 1 is a bar graph showing a dose-dependent increase in aggrecan (ACAN) immunofluorescence staining (FU) of human osteoarthritic chondrocyte cultures in response to increasing millimolar (mM) concentrations of decanoic acid (DA) compared to vehicle controls. ACAN is a proteoglycan unique to articular cartilage, which is important for the proper function and load-bearing capacity of cartilage. Data are presented as fluorescence from photographic images captured with an epifluorescence microscope, with the ACAN signal (FU) normalized to the signal of 4',6-diamidino-2-phenylindole (DAPI) nuclear stain, referred to as FU / DAPI.

[0012] [Figure 2] Figure 2 is a bar graph showing a dose-dependent increase in the relative expression of type II collagen alpha 1 (Col2a1) in human osteoarthritic chondrocyte cultures in response to increasing millimolar (mM) concentrations of decanoic acid (DA). Col2a1 is the major component of hyaline cartilage. Data are shown as ΔΔ relative expression calculations obtained from quantitative real-time reverse transcription polymerase chain reaction (real-time RT-PCR) amplification calls for Col2a1 mRNA concentrations detected in treatment groups relative to the vehicle control group and normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH), actin, and 18S ribosomal RNA transcripts.

[0013] [Figure 3] Figures 3A and 3B show microscopic images of human chondrocyte spheroids after 4 days of incubation with or without decanoic acid. One of the main modes of action observed after treating primary human chondrocytes cultured on hyaluronic acid hydrogels with decanoic acid is increased condensation and spheroid formation. Figure 3A shows human chondrocytes cultured on hyaluronic acid hydrogels incubated for 4 days without decanoic acid; small spheroids settled through the gel to the bottom of the culture well. Figure 3B shows human chondrocyte spheroids incubated for 4 days with decanoic acid; under these conditions, the spheroids formed an interconnected network, suspending the spheroids in the hyaluronic acid hydrogel on the bottom of the culture well.

[0014] [Figure 4] Figures 4A and 4B are microscopic images of human chondrocyte spheroids after 20 days of incubation with or without decanoic acid. Figure 4A shows a human chondrocyte culture incubated for 20 days without decanoic acid, with free cells and spheroids settling to the bottom of the culture well to form a confluent monolayer. In contrast, Figure 4B shows that a human chondrocyte culture incubated with 1 mM decanoic acid for 20 days resulted in increased condensation and the development of large spheroids, which may represent increased differentiation.

[0015] [Figure 5] Figure 5 is a bar graph showing the concentration of IL-1β released from cultures of THP-1 monocytic cells differentiated into macrophage-like cells with phorbol 12-myristate 13-acetate (PMA), then activated with lipopolysaccharide (LPS), and subsequently further incubated with ATP in the presence of vehicle, dexamethasone (Dex) positive control, or decanoic acid at concentrations ranging from 0.19 mM to 6 mM. The graph shows a dose-dependent inhibition of IL-1β concentrations in cultures treated with decanoic acid.

[0016] [Figure 6] Figures 6A and 6B are bar graphs showing the viability of differentiated THP-1 cells differentiated into macrophage-like cells with PMA and then exposed to vehicle, dexamethasone positive control, or decanoic acid at concentrations ranging from 0.19 mM to 6 mM. Figure 6A shows the viability of resting cells, and Figure 6B shows the viability of activated cell cultures shown in Figure 5. Cells treated with 6 mM decanoic acid showed a significant decrease in viability, while lower concentrations of decanoic acid showed no significant change in cell viability compared to the vehicle control.

[0017] [Figure 7] FIG. 7 is a graph showing the reduction in IL-1β concentrations in macrophage-like THP-1 cells treated with decanoic acid relative to vehicle control after stimulation with the "first hit" CL075 alone or the "second hit" CL075 followed by both ATP to induce IL-1β release.

[0018] [Figure 8] Figure 8 is a graph showing the synergistic decrease in IL-1β concentrations for macrophage-like THP-1 cells treated with hyaluronic acid and decanoic acid versus hyaluronic acid alone, after stimulation with CL075 to induce IL-1β release, or after incubation with both CL075 for 24 hours followed by ATP for 1 hour to induce IL-1β release. CL075 is a Toll-like receptor 7 / 8 agonist that increases IL-1β release via an inflammasome-mediated pathway.

[0019] [Figure 9] FIG. 1 is a graph showing cell viability of macrophage-like THP-1 cells treated with hyaluronic acid and decanoic acid versus hyaluronic acid alone following a "first hit model" in which cells were stimulated with CL075 alone for 24 hours, or the "second hit model" described above in which cells were stimulated with CL075 followed by incubation with 1 mM ATP for 1 hour to induce IL-1β release. DETAILED DESCRIPTION OF THE INVENTION

[0020] Various aspects of the present disclosure relate to methods of enhancing the effectiveness of hyaluronic acid (HA) hydrogels, such as Synvisc, by incorporating compounds that can 1) reduce inflammation and inflammasome activity, and 2) promote chondrocyte condensation, redifferentiation, and cartilage formation.

[0021] Inflammasomes are multiprotein complexes that assemble within the cytosol of cells in response to various innate immune stimuli (e.g., pathogen-associated molecular pattern molecules (PAMPs) and damage-associated molecular pattern molecules (DAMPs)). These pinwheel-like structures function as scaffolds for the dimerization and activation of inflammatory proteases known as caspases. Inflammasomes promote the maturation of IL-1β and GasDermin D, along with other cellular insults (e.g., ATP release and potassium efflux). Thus, IL-1β concentrations directly correlate with inflammasome activity, and compounds that decrease IL1-β typically decrease inflammasome activity.

[0022] Inflammation contributes to the pathology of osteoarthritis (OA). IL-1β, for example, promotes synovitis, cartilage loss, osteophyte formation, and chondrocyte dedifferentiation. Macrophages are the main source of IL-1β. Some embodiments of the present disclosure relate to compositions that inhibit the release of IL-1β by macrophages and increase collagen production (e.g., as measured by Col2a1 expression). Such compositions can advantageously treat OA by reducing inflammation and disease progression, promoting cartilage development by redifferentiating osteoarthritic chondrocytes into healthy chondrocytes, and restoring lost collagen.

[0023] Various embodiments of the present disclosure relate to pharmaceutical compositions comprising an HA hydrogel and a saturated fatty acid (e.g., a medium-chain fatty acid).

[0024] Compositions containing the fatty acid decanoic acid and hyaluronic acid can advantageously reduce inflammasome-mediated inflammation. Without limiting the present disclosure or any claims arising from it, fatty acids can bind to and inhibit intracellular inflammasomes, which can reduce IL-1β signaling and corresponding inflammation. Without limiting the present disclosure or any claims arising from it, fatty acids bind to the lipopolysaccharide-binding site on the inflammasome caspase activation and recruitment domain (CARD), which inhibits inflammasomes. Without limiting the present disclosure or any claims arising from it, fatty acids can also function as a carbon source for the citric acid cycle, which can increase the concentration of the citric acid cycle intermediate AKG, which improves nitrogen transport and exhibits antioxidant properties. Without limiting the present disclosure or any claims arising from it, fatty acids can also bind to a specific locus on the NACHT domain of the NLRP3 inflammasome, which has ATPase activity, to inhibit NLRP3 inflammasome activation. For example, inhibition of the NACHT domain locus by the small molecule MCC950 is known to inhibit the activation of the NLRP3 inflammasome. Without limiting this disclosure or any claims arising from this disclosure, saturated fatty acids can also inhibit the NLRP3 inflammasome by binding to the NACHT domain.

[0025] Regardless of the exact mechanism of action, the examples presented below suggest that (1) the fatty acid decanoic acid modulates human monocytes and / or macrophages, reducing IL-1β signaling, and (2) decanoic acid independently modulates human osteoarthritic chondrocytes, promoting their redifferentiation. Each of these effects has an independent positive impact on osteoarthritis. Similar medium-chain saturated free fatty acids are expected to show similar effects as their monoglyceride counterparts.

[0026] In some embodiments, the pharmaceutical composition is effective in reducing inflammation, reducing the progression of osteoarthritis, and / or redifferentiating osteoarthritic chondrocytes.In some particular embodiments, hyaluronic acid and saturated fatty acid are synergistically effective in reducing inflammasome-mediated inflammation.In some particular embodiments, hyaluronic acid and saturated fatty acid are synergistically effective in reducing IL-1β.

[0027] In some embodiments, the HA is a high molecular weight HA having a molecular weight of at least 500 kDa (kilodaltons). In some specific embodiments, the HA is a high molecular weight HA having a molecular weight of at least 1000 kDa. In some very specific embodiments, the HA is a high molecular weight HA having a molecular weight of at least 5000 kDa. SYNVISC® (Sanofi-Aventis, United States), for example, contains HA having an average molecular weight of about 6000 kDa.

[0028] In some embodiments, the saturated fatty acid is selected from hexanoic acid (caproic acid), octanoic acid (caprylic acid), decanoic acid (capric acid), dodecanoic acid (lauric acid), tetradecanoic acid (myristic acid) and hexadecanoic acid (palmitic acid).In some particular embodiments, the saturated fatty acid is octanoic acid or decanoic acid.In some very particular embodiments, the saturated fatty acid is decanoic acid.

[0029] In some embodiments, the pharmaceutical composition comprises saturated fatty acids at a concentration of at least 10 μM (micromolar) and at most 5 mM (millimolar). In some specific embodiments, the pharmaceutical composition comprises saturated fatty acids at a concentration of at least 10 μM and at most 1 mM. In some very specific embodiments, the pharmaceutical composition comprises saturated fatty acids at a concentration of at least 25 μM and at most 500 μM. Concentrations of either decanoic acid or octanoic acid greater than 5 mM are toxic, as shown, for example, in Figures 6A and 6B.

[0030] In some embodiments, the saturated fatty acid has a conjugate base that is a carboxylate. In some embodiments, the composition comprises a carboxylate, and the carboxylate is the conjugate base of the fatty acid. In some embodiments, the fatty acid comprises a conjugate base that is a carboxylate, and the composition comprises a carboxylate. Decanoic acid, for example, has a pKa (negative logarithm of the acid dissociation constant) of about 4.9, meaning that an aqueous phase containing decanoic acid will generally also contain its conjugate base, decanoate, at least at neutral pH. Various compositions of the present disclosure contain water, and a portion of the decanoic acid dissolved in water is deprotonated to form dissolved decanoate. The solubility of decanoic acid in water is about 150 parts per million by mass (ppm), and compositions of the present disclosure are generally characterized by a concentration of decanoic acid that exceeds its solubility in water. Therefore, the concentration of decanoate in various compositions of the present disclosure is lower than that which could be determined based on pKa alone.

[0031] In some embodiments, the composition comprises a total concentration of fatty acid and carboxylate of at least 300 ppm by mass and at most 3% by mass.In some specific embodiments, the composition comprises a total concentration of fatty acid and carboxylate of at least 1,000 ppm by mass and at most 1.5% by mass.In some even more specific embodiments, the composition comprises a total concentration of fatty acid and carboxylate of at least 1,133 ppm by mass and at most 1.02% by mass.In some very specific embodiments, the composition comprises a total concentration of fatty acid and carboxylate of at least 1,700 ppm by mass and at most 6,800 ppm by mass.A low total concentration of fatty acid and carboxylate shows lower efficacy, and a high total concentration carries the risk of toxicity, and this concentration-dependent effect is evident in the following example section, for example, Figures 5, 6A and 6B.Therefore, the therapeutic window of fatty acid concentration is somewhat narrow.

[0032] In some embodiments, the composition comprises one or more of sodium cations, potassium cations, and chloride anions. In some particular embodiments, the composition comprises each of sodium cations, potassium cations, and chloride anions.

[0033] Various aspects of the present disclosure relate to pharmaceutical compositions comprising HA hydrogels and monoglyceride esters of saturated fatty acids (monoglycerides). At least a portion of the free fatty acids are converted to monoglycerides in vivo. Without limiting this disclosure or any claims arising therefrom, the monoglycerides exhibit activity against osteoarthritis inflammation. Free fatty acids are first converted to monoglycerides in vivo by acetyl-CoA synthetase, which converts free fatty acids, coenzyme A (CoA), and ATP into adenosine monophosphate (AMP), pyrophosphate, and a thioester of the free fatty acid and CoA. Monoglyceride acyltransferase then converts the thioester and glycerol back into monoglycerides and CoA.

[0034] In some embodiments, the monoglyceride is selected from monohexanoin (monocaproin), monooctanoin (monocaprylin), monodecanoin (monocaprin), monododecanoin (monolaurin), tetradecanoin (monomyristin), and hexadecanoin (monopalmitin). In some particular embodiments, the monoglyceride is selected from monooctanoin and monodecanoin.

[0035] In some embodiments, the pharmaceutical composition comprises monoglyceride at a concentration of at least 10 μM and at most 5 mM. In some particular embodiments, the pharmaceutical composition comprises monoglyceride at a concentration of at least 10 μM and at most 1 mM. In some very particular embodiments, the pharmaceutical composition comprises monoglyceride at a concentration of at least 25 μM and at most 500 μM. Without limiting the present disclosure or any claims arising from this disclosure, concentrations of either monocaprin or monocaprylin above 5 mM are considered toxic based on the toxicity of decanoic acid, for example, as shown in Figures 6A and 6B.

[0036] Various aspects of the present disclosure relate to pharmaceutical compositions comprising a hyaluronic acid hydrogel and a corticosteroid.

[0037] In some embodiments, the pharmaceutical composition is effective for reducing inflammation, reducing the progression of osteoarthritis, and / or redifferentiating osteoarthritic chondrocytes.In some specific embodiments, hyaluronic acid and corticosteroids are synergistically effective for reducing inflammasome-mediated inflammation.In some specific embodiments, hyaluronic acid and corticosteroids are synergistically effective for reducing IL-1β.In some specific embodiments, hyaluronic acid and corticosteroids are synergistically effective for increasing Col2a1.

[0038] In some embodiments, corticosteroid is selected from dexamethasone, dexamethasone phosphate, dexamethasone acetate, dexamethasone palmitate, other fatty acid esters of dexamethasone, cortisol, cortisone, triamcinolone, prednisone, prednisolone, methylprednisolone and betamethasone.In some particular embodiments, corticosteroid is dexamethasone, dexamethasone phosphate or dexamethasone acetate.In some particular embodiments, corticosteroid is dexamethasone fatty acid ester, for example dexamethasone palmitate.In some very particular embodiments, corticosteroid is dexamethasone phosphate.

[0039] In some embodiments, the pharmaceutical composition comprises at least 1 mg (milligram) and at most 10 mg of corticosteroid.In some specific embodiments, the pharmaceutical composition comprises at least 1 mg and at most 5 mg of corticosteroid.In some very specific embodiments, the pharmaceutical composition comprises at least 2 mg and at most 5 mg of corticosteroid.For example, a dose of about 3 or 4 mg of dexamethasone phosphate is generally effective for treating osteoarthritis of the knee when administered as an intra-articular injection into the knee.

[0040] In some embodiments, the pharmaceutical composition comprises a corticosteroid at a concentration of at least 400 pM (picomolar) and at most 20 mM.

[0041] In some embodiments, the pharmaceutical composition comprises HA at a concentration of at least 0.01% and at most 1%.

[0042] Various aspects of the present disclosure relate to syringes containing the pharmaceutical compositions described elsewhere in this disclosure.

[0043] Various aspects of the present disclosure relate to methods of treating osteoarthritis in a human patient, comprising administering to the human patient a therapeutically effective amount of a pharmaceutical composition described elsewhere in this disclosure.

[0044] In some embodiments, administration is local administration near the joint affected by osteoarthritis.In some specific embodiments, administration is selected from local administration and injection.In some very specific embodiments, administration is intra-articular injection.

[0045] In some embodiments, the method comprises injecting the pharmaceutical composition into a joint of a patient. In some particular embodiments, the method comprises injecting the pharmaceutical composition into a joint of a patient, the joint being affected by osteoarthritis.

[0046] In some embodiments, the joint is selected from the knee, hip, and shoulder. In some specific embodiments, the joint is the knee.

[0047] In some embodiments, the method comprises re-injecting the patient's joint with a second therapeutically effective amount of the pharmaceutical composition 5 to 25 days after the initial injection. In some particular embodiments, the method comprises re-injecting the patient's joint with a second therapeutically effective amount of the pharmaceutical composition 5 to 15 days after the initial injection.

[0048] In some embodiments, the pharmaceutical composition is effective for reducing prostaglandin E2 (PGE2) concentration in patients.In some specific embodiments, the pharmaceutical composition is effective for reducing PGE2 concentration in patients' osteoarthritis-affected joints, for example, after injecting the pharmaceutical composition into or adjacent to the joint.In some very specific embodiments, hyaluronic acid and saturated fatty acid are synergistically effective for reducing PGE2 concentration in patients.In some very specific embodiments, hyaluronic acid and monoglyceride are synergistically effective for reducing PGE2 concentration in patients.In some very specific embodiments, hyaluronic acid and corticosteroid are synergistically effective for reducing PGE2 concentration in patients.

[0049] In some embodiments, the pharmaceutical composition is effective in reducing inflammasome-mediated inflammation in patients.In some particular embodiments, the pharmaceutical composition is effective in reducing inflammasome-mediated inflammation in patients' osteoarthritis-affected joints, for example, after injecting the pharmaceutical composition into or adjacent to a joint.In some very particular embodiments, hyaluronic acid and saturated fatty acid are synergistically effective in reducing inflammasome-mediated inflammation in patients.In some very particular embodiments, hyaluronic acid and monoglycerides are synergistically effective in reducing inflammasome-mediated inflammation in patients.In some very particular embodiments, hyaluronic acid and corticosteroids are synergistically effective in reducing inflammasome-mediated inflammation in patients.

[0050] In some embodiments, the pharmaceutical composition is effective for increasing IL-1β concentration in patients.In some specific embodiments, the pharmaceutical composition is effective for increasing IL-1β concentration in the joints of patients affected by osteoarthritis, for example, after injecting the pharmaceutical composition into or adjacent to the joint.In some very specific embodiments, hyaluronic acid and saturated fatty acid are synergistically effective for reducing IL-1β concentration in patients.In some very specific embodiments, hyaluronic acid and monoglyceride are synergistically effective for reducing IL-1β concentration in patients.In some very specific embodiments, hyaluronic acid and corticosteroid are synergistically effective for reducing IL-1β concentration in patients.

[0051] In some embodiments, the pharmaceutical composition is effective in increasing Col2a1 in a patient. In some specific embodiments, the pharmaceutical composition is effective in increasing Col2a1 in a patient's osteoarthritis-affected joint, for example, after injecting the pharmaceutical composition into or adjacent to a joint. In some very specific embodiments, hyaluronic acid and saturated fatty acids are synergistically effective in increasing Col2a1 in a patient. In some very specific embodiments, hyaluronic acid and monoglycerides are synergistically effective in increasing Col2a1 in a patient. In some very specific embodiments, hyaluronic acid and corticosteroids are synergistically effective in increasing Col2a1 in a patient.

[0052] In some embodiments, the pharmaceutical composition is effective in inhibiting cyclooxygenase in patients, for example, COX-1 or COX-2.In some particular embodiments, the pharmaceutical composition is effective in inhibiting cyclooxygenase in patients' osteoarthritis-affected joints, for example, after injecting the pharmaceutical composition into or adjacent to the joint.In some very particular embodiments, hyaluronic acid and saturated fatty acid are synergistically effective in inhibiting cyclooxygenase in patients.In some very particular embodiments, hyaluronic acid and monoglyceride are synergistically effective in inhibiting cyclooxygenase in patients.In some very particular embodiments, hyaluronic acid and corticosteroid are synergistically effective in inhibiting cyclooxygenase in patients.

[0053] In some embodiments, the pharmaceutical composition is effective for redifferentiating osteoarthritic chondrocytes in patients.In some particular embodiments, the pharmaceutical composition is effective for redifferentiating osteoarthritic chondrocytes in the joints of patients affected by osteoarthritis, for example, after injecting the pharmaceutical composition into or adjacent to the joint.In some very particular embodiments, hyaluronic acid and saturated fatty acid are synergistically effective for redifferentiating osteoarthritic chondrocytes in patients.In some very particular embodiments, hyaluronic acid and monoglyceride are synergistically effective for redifferentiating osteoarthritic chondrocytes in patients.In some very particular embodiments, hyaluronic acid and corticosteroid are synergistically effective for redifferentiating osteoarthritic chondrocytes in patients.

[0054] The following examples provide a framework for practicing various aspects of the present disclosure, and the examples do not limit any aspect of the disclosure or any claims issuing from this patent document. [Example]

[0055] Example 1 The combination of HA and decanoic acid increases the expression of SOX9, ACAN, and Col2a1 in human osteoarthritic chondrocytes. Chondrocytes are a cell type unique to joints that maintain the structural integrity of cartilage by producing extracellular matrix proteins. One of the hallmarks of osteoarthritis is an imbalance between the anabolic and catabolic functions of chondrocytes. Repeated mechanical injury and inflammation alter chondrocyte differentiation, resulting in irregular collagen types and proteolytic enzyme production.

[0056] The process by which chondroprogenitor cells mature into chondrocytes is called chondrogenesis. Growth factors (e.g., TGF-β) and transcription factors (e.g., SOX9) promote changes in transcriptional programming that induce cartilage production. One of the hallmarks of this process is the condensation of these cells into tightly packed nodules or spheroids.

[0057] Human osteoarthritic chondrocytes were derived from osteoarthritic tissue and cultured in the presence of decanoic acid for 24 days with weekly medium changes. Chondrocytes were layered on HA hydrogel (SYNVISC®, Sanofi-Aventis, United States). After 24 days, cells were fixed with paraformaldehyde and immunostained for SOX9, Col2a1, and ACAN.

[0058] Exposure of dense monolayers of osteoarthritic chondrocytes to decanoic acid increased nuclear staining of SOX9 in a dose-dependent manner compared with cultures containing vehicle, 0.1 mM decanoic acid, 0.5 mM decanoic acid, and 1 mM decanoic acid. Cell cultures containing 1 mM decanoic acid showed increased spheroid development or condensation, and the resulting spheroids also stained strongly for SOX9. The correlation between decanoic acid concentration and SOX9 expression suggests that decanoic acid promotes chondrocyte redifferentiation.

[0059] Exposure of high-density monolayer osteoarthritic chondrocytes to decanoic acid resulted in a dose-dependent increase in aggrecan (ACAN) staining relative to cultures containing vehicle, 0.1 mM decanoic acid, 0.5 mM decanoic acid, and 1 mM decanoic acid. Exposure of low-density monolayer osteoarthritic chondrocytes to decanoic acid also resulted in a dose-dependent increase in intracellular staining of ACAN, with the most intense staining observed at 1 mM decanoic acid (Figure 1). Furthermore, chondrocyte spheroids in HA hydrogels showed strong ACAN immunostaining when exposed to 1 mM decanoic acid.

[0060] Exposure of high-density monolayer osteoarthritic chondrocytes to decanoic acid resulted in dose-dependent punctate staining of Col2a1 for cultures containing vehicle, 0.1 mM decanoic acid, 0.5 mM decanoic acid, and 1 mM decanoic acid, with peak staining observed at 0.5 mM decanoic acid. Exposure of low-density monolayer osteoarthritic chondrocytes to decanoic acid similarly resulted in strong punctate staining of Col2a1 for chondrocytes cultured with 1 mM decanoic acid.

[0061] These results suggest that the combination of high molecular weight hyaluronan and decanoic acid is effective in redifferentiating osteoarthritic chondrocytes.

[0062] Example 2 The combination of HA and decanoic acid increases Col2a1 expression and promotes distinct spheroid morphology in human osteoarthritic chondrocytes. Human osteoarthritic chondrocytes were derived from osteoarthritic tissue and cultured in the presence of decanoic acid for 20 days, with medium changes every 2–3 days. Chondrocytes were layered on HA hydrogel (SYNVISC®, Sanofi-Aventis, United States). After 20 days, hyaluronidase was added to release the cells from the hydrogel, and the cells were harvested and total RNA was isolated.

[0063] Real-time RT-PCR was performed to compare gene expression relative to GAPDH, actin, and 18S ribosomal RNA transcripts using ΔΔCT analysis. At 0.5 mM decanoic acid, Col2a1 mRNA increased 1-fold, and at 1 mM decanoic acid, Col2a1 mRNA increased 7-fold (Figure 2).

[0064] Microscopic examination of these chondrocyte models after 4 days of culture revealed distinct morphological differences in the presence and absence of decanoic acid, which promoted an interconnected network of spheroids. The interconnected network allowed the chondrocyte spheroids to exist in suspension within the HA hydrogel; in contrast, in the absence of decanoic acid, the spheroids drifted to the surface below the hydrogel (Figures 3A and 3B).

[0065] Examination of the chondrocyte spheroids after 20 days of culture revealed further morphological differences in the presence and absence of decanoic acid. Control chondrocytes formed a monolayer on the bottom of the culture well (Figure 4A). Addition of decanoic acid resulted in the development or condensation of large spheroids, indicating increased differentiation (Figure 4B).

[0066] These findings suggest that the combination of high molecular weight hyaluronan and decanoic acid is effective in redifferentiating osteoarthritic chondrocytes.

[0067] Example 3 Decanoic acid inhibits IL-1β release from human macrophage-like cells. The human monocytic cell line THP-1 was differentiated into macrophage-like cells with PMA for 72 hours. After differentiation, cells were exposed to decanoic acid at concentrations ranging from 0.2 mM to 6 mM at 25,000 cells / well in 96-well tissue culture plates. Cells were then activated with 100 ng / mL lipopolysaccharide (LPS) for 24 hours, followed by 10 mM adenosine triphosphate (ATP) for an additional hour. LPS is a toll-like receptor 4 (TLR4) agonist, and ATP activates sodium channels to promote inflammasome activity. The resulting IL-1β release was quantified by enzyme-linked immunosorbent assay (ELISA).

[0068] Cells cultured with decanoic acid showed dose-dependent inhibition of inflammasome-IL-1β release. Decanoic acid at 1.5 and 3 mM concentrations showed superior inhibition compared to the steroid dexamethasone, which was statistically significant (Figure 5). Resting cells did not release detectable IL-1β. Cell density was then assessed in both resting and activated cultures using a colorimetric MTS assay at optical density at 490 nm (Figures 6A and 6B). 6 mM decanoic acid showed an unfavorable effect on cell density, suggesting that decanoic acid has a narrow therapeutic window. Furthermore, viability results indicate that IL-1β inhibition at concentrations of decanoic acid below 3 mM does not correlate with cell density.

[0069] Similar results were observed when TLR7 / 8 agonist CL075 was used instead of LPS. TLR7 / 8 agonists increase inflammasome activity and IL-1β release through a pathway distinct from that of TLR4 agonists. To assess this response, THP-1 monocytic cells were differentiated to a macrophage-like state using PMA for 72 hours. Decanoic acid (final concentration 1 mM) or diluent control was added, and cells were stimulated with the TLR7 / 8 agonist CL075. The cells were then incubated at 37°C for 24 hours. To further activate the inflammasome, ATP was then added to wells designated as "second hits," and the cultures were incubated for an additional hour. Hyaluronidase was added to selected wells for 15 minutes, and all media was collected for IL-1β ELISA quantification. The results are shown in Figure 7. The addition of decanoic acid reduced IL-1β levels by 77% compared to the absence of ATP, and by 91% with the addition of ATP. Taken together, these results suggest that decanoic acid can inhibit monocyte- and macrophage-mediated inflammation through different signaling pathways.

[0070] Example 4 The combination of HA and decanoic acid inhibits IL-1β release from human macrophage-like cells. To evaluate macrophage responses in vitro, THP-1 monocytic cells were differentiated into a macrophage-like state using PMA for 72 hours and then placed on a thin layer of HA hydrogel (SYNVISC®, Sanofi-Aventis, United States), which was then immediately covered with fresh medium. Decanoic acid (final concentration 1 mM) or diluent control was then added, and the cells were stimulated with the TLR7 / 8 agonist CL075. The cells were incubated at 37°C for 24 hours, and the cultures were then incubated with ATP for an additional hour.

[0071] To demonstrate the synergistic effect between HA and decanoic acid, experiments were performed with and without HA hydrogel, and the results are shown in Figure 8. Hyaluronic acid alone reduced IL-1β by 29% after CL075 and ATP, while the combination of hyaluronic acid and decanoic acid reduced IL-1β by 92%.

[0072] A colorimetric assay confirmed that the inhibition of IL-1β release was not caused solely by a decrease in cell number. Tetrazolium colorimetric dye was added to the cultures at the end of the experiment, and cell number was measured by optical density at 490 nm. Optical density decreased by 18% when decanoic acid was added to the wells in the absence of ATP and by 25% in the presence of ATP; these differences were much smaller than the observed decrease in IL-1β concentration (Figure 9).

[0073] Example 5 The combination of hyaluronic acid and dexamethasone synergistically reduces MMP13 expression in osteoarthritic fibroblast-like chondrocytes. Osteoarthritic fibroblast-like chondrocytes were cultured for 2 to 28 days in the presence of either 0.1% hyaluronic acid, 0.1 μM dexamethasone, or both 0.1% hyaluronic acid and 0.1 μM dexamethasone. The medium was changed every 2 to 3 days. Chondrocytes cultured for 2, 14, or 28 days were analyzed by real-time RT-PCR to assess matrix metallopeptidase 13 (MMP13) expression.

[0074] MMP13, also known as collagenase-3, is an endopeptidase that plays a key role in the degradation and remodeling of extracellular matrix. Under normal physiological conditions, MMP13 is tightly regulated and is involved in collagen turnover. However, in osteoarthritis, overexpression and dysregulated activity of MMP13 leads to excessive collagen degradation and cartilage destruction.

[0075] The change in MMP13 expression, calculated by ΔΔCT relative to GAPDH expression, demonstrated an unexpected synergistic effect between 0.1% hyaluronic acid and 0.1 μM dexamethasone, as shown in Table 1 below. The synergistic effect observed on day 14 was greater than additive, and the synergistic effect observed on day 28 was more than an order of magnitude greater than additive. These results suggest that the combination of hyaluronic acid and dexamethasone can synergistically reverse collagen degradation in osteoarthritis. [Table 1]

Claims

1. A hyaluronic acid (HA) hydrogel comprising HA having a molecular weight of at least 500 kDa and water; and Medium-chain fatty acids selected from hexanoic acid (caproic acid), octanoic acid (caprylic acid), decanoic acid (capric acid), and dodecanoic acid (lauric acid).

1. A pharmaceutical composition for use in a method for treating osteoarthritis, comprising: A pharmaceutical composition, wherein the composition is effective in reducing inflammation, reducing the progression of osteoarthritis, and redifferentiating osteoarthritic chondrocytes.

2. the medium chain fatty acid is octanoic acid or decanoic acid; and HA and medium-chain fatty acids are synergistically effective in reducing inflammasome-mediated inflammation.

2. The pharmaceutical composition of claim 1.

3. the medium chain fatty acid is octanoic acid or decanoic acid; and HA and medium-chain fatty acids are synergistically effective in reducing interleukin-1β.

2. The pharmaceutical composition of claim 1.

4. 10. The pharmaceutical composition of claim 1, comprising a medium-chain fatty acid at a concentration of at least 10 μM and at most 1 mM.

5. 10. The pharmaceutical composition of claim 1, comprising a medium-chain fatty acid at a concentration of at least 100 μM and at most 1 mM.

6. 10. A method of treating osteoarthritis in a human patient, comprising administering to the patient a therapeutically effective amount of the pharmaceutical composition of claim 1.

7. 7. The method of claim 6, comprising injecting the pharmaceutical composition into a joint of the patient.

8. 8. The method of claim 7, comprising re-injecting the patient's joint with a second therapeutically effective amount of the pharmaceutical composition 5 to 25 days after the injection.

9. 7. The method of claim 6, wherein the HA and medium-chain fatty acids are synergistically effective in reducing inflammasome-mediated inflammation.

10. 7. The method of claim 6, wherein the HA and the medium chain fatty acid are synergistically effective in reducing interleukin-1β levels in the patient.

11. 7. The method of claim 6, wherein the HA and medium chain fatty acids are synergistically effective in increasing type II collagen alpha 1 (Col2a1) in the patient.

12. A hyaluronic acid (HA) hydrogel comprising HA having a molecular weight of at least 500 kDa and water; and Corticosteroids 10. A pharmaceutical composition for use in a method for treating osteoarthritis, comprising:

13. 13. The pharmaceutical composition of claim 12, wherein the corticosteroid is selected from dexamethasone or an ester thereof, the ester being selected from dexamethasone acetate, dexamethasone phosphate, dexamethasone palmitate, and other fatty acid esters of dexamethasone.

14. 14. The pharmaceutical composition of claim 13, wherein the corticosteroid is dexamethasone phosphate.

15. 15. The pharmaceutical composition of claim 14, comprising at least 1 mg and at most 5 mg of dexamethasone phosphate.

16. 13. The pharmaceutical composition of claim 12, wherein the corticosteroid is selected from cortisol, cortisone, triamcinolone, prednisone, prednisolone, methylprednisolone, and betamethasone.

17. 13. A method of treating osteoarthritis in a human patient, comprising administering to the patient a therapeutically effective amount of the pharmaceutical composition of claim 12.

18. 18. The method of claim 17, comprising injecting the pharmaceutical composition into a joint of the patient.

19. 18. The method of claim 17, wherein the HA and the corticosteroid are synergistically effective in reducing inflammasome-mediated inflammation in the patient.

20. 18. The method of claim 17, wherein the HA and the corticosteroid are synergistically effective in reducing interleukin-1β levels in the patient.

21. 18. The method of claim 17, wherein the HA and the corticosteroid are synergistically effective in increasing type II collagen alpha 1 (Col2a1) in the patient.

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