Pharmaceutical composition containing ibudilast nanoparticles for the treatment of osteoarthritis

Abstract

A pharmaceutical composition comprising bovine serum albumin (BSA) nanoparticles loaded with ibudilast for the treatment of osteoarthritis, the composition comprising: a) Ibudilast in an amount of 10 to 50 mg, b) bovine serum albumin in an amount of 50 to 150 mg / ml, c) Glutaraldehyde as crosslinking agent in an amount of 8%, d) sucrose in an amount of 5% as a cryoprotectant, e) water and ethanol as solvents, f) wherein the nanoparticles have a particle size of about 241 nm, a zeta potential of -38.1 ± 1.2 mV, a polydispersity index (pdi) of 0.09 ± 0.007, and an entrapment efficiency of about 69%, g) wherein the composition is effective in reducing inflammatory markers, increasing anti-inflammatory cytokines and improving antioxidant defense in osteoarthritic conditions.

Description

The present invention relates to a nanoparticle pharmaceutical composition of ibudilast for the treatment of osteoarthritis. It is specifically focused on modulating proinflammatory cytokines and increasing serum antioxidant activity. The invention aims to improve therapeutic efficacy and stability for better treatment of osteoarthritis.Osteoarthritis (OA) is a progressive degenerative joint disease characterized by chronic inflammation, cartilage degradation and oxidative stress. Conventional treatment possibilities such as nonsteroidal antirheumatic agents (NSAIDs) and corticosteroids alleviate the symptoms but frequently lead to undesirable effects on prolonged use. Moreover, these treatments do not address the underlying inflammatory and oxidative stress mechanisms that contribute to the progression of the disease, thus requiring the development of a more effective and targeted therapeutic approach.Ibudilast, a phosphodiesterase (PDE) inhibitor, has demonstrated anti-inflammatory and neuroprotective properties, making it a promising candidate for the treatment of OA. However, its poor solubility and bioavailability limit its therapeutic potential. Systemic administration of ibudilast results in suboptimal drug concentrations in the affected joint, reducing its effectiveness. Moreover, the instability of ibudilast in biological fluids complicates its clinical application, which illustrates the need for an optimized formulation for improving drug delivery and therapeutic effect.To overcome these challenges, the present invention proposes a nanoparticle-based formulation of ibudilast that improves solubility, stability, and targeted delivery. The nanoformulation facilitates controlled release of the drug, enhances cellular uptake, and effectively modulates proinflammatory cytokines while increasing serum antioxidant activity. This novel approach aims to provide a superior therapeutic alternative for osteoarthritis which allows prolonged relief of symptoms with minimal side effects and thus improves results for patients.Another object of the present disclosure is that the formulation provides a controlled and prolonged release of ibudilast for a prolonged therapeutic effect.Another object of the present disclosure is to achieve better inclusion, reduce drug loss, and increase efficacy.Another object of the present disclosure is to improve drug absorption and penetration at the target site by the administration of nanoparticles.Another object of the present disclosure is to effectively reduce pro-inflammatory cytokines (TNF-α, IL-1β, and IL-10).Another object of the present disclosure is to restore antioxidant markers (CAT, SOD, GSH) and reduce lipid peroxidation (MDA).Another object of the present disclosure is to reduce paw edemaAnother object of the present disclosure is the local administration of drugs that minimizes systemic side effects as compared to oral administration.Another object of the present disclosure is non-invasive transdermal administration that improves patient compliance and comfort.Another object of the present disclosure is to improve the haematological parameters in the treatment of osteoarthritis.Another object of the present disclosure is the use of BSA as a natural polymer which provides minimal toxicity and better compatibility with biological systems.The present invention relates generally to ibudilast-loaded bovine serum albumin (BSA) nanoparticles for targeted and sustained drug delivery in the treatment of osteoarthritis.In one embodiment of the present invention, the formulation comprises ibudilast (10-50 mg), BSA (50-150 mg), glutaraldehyde (0.5-2% v / v), ethanol and sucrose (1-5% w / v) as a stabilizer.In another embodiment of the invention, the optimized nanoparticles have a particle size of about 241 nm, a zeta potential of -38.1 mV, and a polydispersity index (PDI) of 0.09, which ensures uniform dispersion and stability.A further embodiment of the invention consists in the nanoparticles reaching a high active ingredient inclusion rate of 69%, which improves the active ingredient loading and reduces the waste.Another embodiment of the invention is that the formulation provides a controlled release of ibudilast over a period of 24 hours, thereby maintaining therapeutic levels for prolonged anti-inflammatory activity.Another embodiment of the invention consists in the fact that the nanoparticles significantly lower the levels of TNF-α, IL-1β and IL-10 and thus improve the symptoms of osteoarthritis.Another embodiment of the invention is that the formulation restores the antioxidant markers (CAT, SOD, GSH) and reduces lipid peroxidation (MDA), thereby reducing oxidative stress in arthritis.Another embodiment of the invention is designed for transdermal administration. The nanoparticles improve skin penetration and provide localized and effective treatment of osteoarthritis.The present invention relates to the composition of ibudilast-loaded bovine serum albumin (BSA) nanoparticles designed for improved antiarthritic activity by controlled drug delivery. The nanoparticles are formulated by the desolvation process, in which ibudilast is encapsulated in a BSA matrix and subsequently cross-linked with glutaraldehyde to ensure structural stability. This formulation allows sustained release of drug and improves therapeutic results as the level of drug is maintained over a longer period of time. The use of BSA as a biocompatible carrier increases the stability of the medicament, reduces systemic toxicity and ensures targeted delivery to inflammatory articular tissue. The nanoparticles are designed for topical application and provide localized drug delivery to the affected areas, thereby minimizing systemic side effects and improving patient compliance.The invention further comprises an optimized nanoparticle formulation having improved physicochemical properties such as uniform particle size, high efficiency of the active ingredient inclusion and improved stability. The negatively charged nanoparticles prevent aggregation and ensure better dispersion in the physiological environment. Moreover, the formulation contributes to the reduction of inflammatory responses by modulating important cytokines such as TNF-α, IL-1β and IL-10 and simultaneously restoring antioxidant defense mechanisms. The invention provides a novel approach for the treatment of osteoarthritis by the combination of nanotechnology with an effective anti-inflammatory drug which provides for prolonged therapeutic effect and improved patient results.EXAMPLE 1: CompositionA pharmaceutical composition comprising ibudilast-loaded bovine serum albumin (BSA) nanoparticles, the composition comprising: a) ibudilast in an amount of 10-50 mg, b) bovine serum albumin in an amount of 50-150 mg / ml, c) glutaraldehyde as a cross-linking agent in an amount of 8%, d) sucrose in an amount of 5% as a cryoprotective agent, e) water and ethanol as a solvent, f) wherein the nanoparticles have a particle size of about 241 nm, a zeta potential of -38. 1 ± 1.2 mv, a polydispersity index (pdi) of 0.09 ± 0.007 and an inclusion efficiency of about 69%, g) wherein the composition is effective in reducing inflammatory markers, increasing anti-inflammatory cytokines and improving antioxidant rejection in osteoarthritic conditions.EXAMPLE 2: Preparation of Ibudilast loaded BSA NanoparticlesThe desolvation procedure was used with minimal changes to produce ibudilast-containing BSA nanoparticles which were then cross-linked with glutaraldehyde as previously reported. After dissolving BSA in water, 0.1 M NaOH was used to adjust the pH to 9.0. Ibudilast was dissolved in ethanol and gradually added to the aqueous BSA solution with stirring by a magnetic stirrer until the mixture became turbid. The ethanol was added stepwise at 800 μL / min. To crosslink the particles, 8% glutaraldehyde was added to the cofactor and cured for 24 hours. After ultracentrifugation to filter the nanoparticles, the mixture was washed twice with ultra pure water. The precipitate was resuspended in 5% sucrose and freeze dried. Process supernatants were collected for further analysis.EXAMPLE 3: Particle Size, Zeta Potential, and Polydispersity MeasurementsThe particle size of the formulated ibudilast-loaded nanoparticles was determined using a Malvern Zeta Size based on dynamic light scattering. For uniform dispersion, the nanoparticles were suspended in deionized water and sonicated for 10 minutes prior to measurement. Each analysis was performed in triplicate to ensure accuracy.The optimized ibudilast loaded bovine serum albumin nanoparticles were analyzed and their particle size was determined to be 241 nm.The stability of nanoparticles in dispersion is influenced by the electrostatic repulsion between the particles, which is reflected in the zeta potential. Higher zeta potential values enhance rejection between the nanoparticles and reduce the likelihood of aggregation. The optimized ibudilast nanoparticles had a zeta potential of -38.1±1.2 mV, which indicates good stability. The negative charge is due to albumin being produced at a pH above its isoelectric point, resulting in ionization of the carboxyl terminus and a negative charge. In addition, the polydispersity index (PDI) was 0.09±0.007, which indicates a uniform size distribution within the nanoparticles.EXAMPLE 4: Determination of the Efficacy of the Active Ingredient InclusionTo evaluate the efficiency of the active ingredient inclusion (EA), the nanoparticle formulations were centrifuged and the supernatants were analyzed for the ibudilast content using a Shimadzu UV spectrophotometer. The EA was calculated from the following formula:The results showed that the optimized ibudilast loaded bovine serum albumin nanoparticles had an inclusion efficiency of 69%.EXAMPLE 5: In vitro Drug ReleaseApproximately 20 mg of ibudilast-loaded nanoparticles were placed in a dialysis membrane bag (8 kDa molecular cut-off) and dispersed in phosphate buffered saline (PBS, pH 7.4). The bag was immersed in 100 ml of PBS and maintained at 37±0.5°C with constant stirring at 50 U / min. At predefined intervals, 0.5 ml of the releasing medium was removed and replaced with an equal volume of fresh PBS. The concentration of the liberated ibudilast was quantified by means of UV spectroscopy and all measurements were carried out in triplicate.The albumin nanoparticles released ibudilast in two phases: First, a burst effect was observed, then the drug was released continuously for 12 hours. About 40% of the nanoparticles were released in a rapid burst within 4 hours and then gradually over 12 hours. Both the agent on the surface of the nanoparticles and the unbound, unencapsulated agent could be the cause of the initial burst effect. After 12 hours, the nanoparticle dispersion had an active ingredient release of 63%. In contrast, in the single Ibudilast dispersion, an immediate release push of 42.24% was observed in the first hour. The release rate was more than 5.5 times higher than when dispersed with the nanoparticles. The release of ibudilast from the simple dispersion was 84.32% after 2 hours and 87.92% after 4 hours when the release rate was found to be uniform. The sustained release pattern may be caused by the active ingredient present in the nanoparticle matrix.EXAMPLE 6: In Vivo StudyAdult male Wistar albino rats (250-300 g) were obtained from India for the evaluation of antiarthrosis activity. The animals were housed in well aerated cages with sterile rice hull litter at controlled temperature (25±1°C) and humidity (45-60%) and a 10-15 hour light-dark cycle. They had free access to commercial food and tap water.A total of 24 male Wistar rats (250-300 g) were used for the MIA-induced osteoarthritis model. The animals were acclimated under standard laboratory conditions for 10 days prior to testing. The study followed the CPCSEA guidelines (Registration Number RIPS / IAEC / 2023-24 / 08 / 08). Osteoarthritis (OA) was induced in 18 rats by a one-time intra-articular injection of 3 mg MIA into the left knee joint in a volume of 50 μl under anaesthesia (5% Na thiopentone), while six normal control rats received 50 μl of saline. Animals were divided into four groups (n=6): Group A (normal control, received saline intra-articular), Group B (drug control, MIA-induced OA without treatment), Group C (single ibudilast dispersion treatment), and Group D (optimized ibudilast charged nanoparticle treatment). Treatment took 21 days, with groups C and D each applying 10 mg of the drug twice daily to the left knee after shaving the area 24 hours prior to OA induction.Blood collection and determination of the haematological parameters:At the end of the study, the overnight fasting animals were anesthetized with halothane and blood samples were taken from the retro-orbital sinus. Each sample was divided into two parts: the first part was used to determine haematological parameters such as red blood cells (RBC), white blood cells (WBC) and hemoglobin, while the second part was tested for arthritis related mediators such as tumor necrosis factor (TNF-α), interleukin-6 (IL-6), IL-10 and oxidative stress markers such as catalase (CAT), superoxide dismutase (SOD), glutathione (GSH) and malondialdehyde (MDA). The serum was separated for further analysis by centrifugation at 10,000 U / min for 10 minutes.In the rats treated with both the clean dispersion and the nanoparticle formulation, the white blood cell count decreased and hemoglobin (Hb) and red blood cell count increased compared to the arthritis control group. After topical application, hematology changes including an increase in Hb and a decrease in leukocyte count were observed. The reduction in Hb during arthritis is likely due to reduced erythropoietin levels, reduced bone marrow response to erythropoietin, and premature erythrocyte destruction. Moreover, arthritis is associated with a moderate increase in leukocyte count due to IL-1β-mediated stimulation of colony stimulating factors. The present study demonstrates that topical treatments with nanoparticles contribute to normalization of leukocyte count, indicating their potential for alleviating inflammatory responses in osteoarthritis.The MIA-induced osteoarthritis group exhibited a significant increase in serum levels of TNF-α, IL-1β and IL-10 as compared with the normal control group. In contrast, treatment with both pure ibudilast dispersion and ibudilast loaded nanoparticles resulted in a marked decrease in TNF-α, IL-1β and IL-10 levels. This suggests that nanoparticle formulations effectively modulate pro-inflammatory and anti-inflammatory cytokines and contribute to an improved profile of inflammation in osteoarthritic conditions.The content of antioxidants in serum including catalase (CAT), superoxide dismutase (SOD) and glutathione (GSH) was significantly lower in the MIA control group compared to the normal control group, indicating oxidative stress. Chronic treatment with a pure Ibudilast dispersion and with Ibudilast-loaded nanoparticles led to a significant recovery of these antioxidant values. In addition, malonic dialdehyde (MDA), a marker of lipid peroxidation and oxidative stress, was markedly increased in the MIA control group. However, treatment with ibudilast formulations led to a marked reduction in MDA values, which emphasizes their potential to counteract oxidative damage in osteoarthritis.Examples1. A pharmaceutical composition comprising ibudilast-loaded bovine serum albumin (BSA) nanoparticles for the treatment of osteoarthritis, the composition comprising: a) ibudilast in an amount of 10 to 50 mg, b) bovine serum albumin in an amount of 50 to 150 mg / ml, c) glutaraldehyde as a cross-linking agent in an amount of 8%, d) sucrose in an amount of 5% as a cryoprotective agent, e) water and ethanol as a solvent, f) wherein the nanoparticles have a particle size of about 241 nm, a zeta potential of -38. 1 ± 1.2 mv, a polydispersity index (pdi) of 0.09 ± 0.007 and an inclusion efficiency of about 69%, g) wherein the composition is effective in reducing inflammatory markers, increasing anti-inflammatory cytokines and improving antioxidant rejection in osteoarthritic conditions.

Claims

A pharmaceutical composition comprising ibudilast-loaded bovine serum albumin (BSA) nanoparticles for the treatment of osteoarthritis, the composition comprising: a) ibudilast in an amount of 10 to 50 mg, b) bovine serum albumin in an amount of 50 to 150 mg / ml, c) glutaraldehyde as a cross-linking agent in an amount of 8%, d) sucrose in an amount of 5% as a cryoprotective agent, e) water and ethanol as a solvent, f) wherein the nanoparticles have a particle size of about 241 nm, a zeta potential of -38. 1 ± 1.2 mv, a polydispersity index (pdi) of 0.09 ± 0.007 and an inclusion efficiency of about 69%, g) wherein the composition is effective in reducing inflammatory markers, increasing anti-inflammatory cytokines and improving antioxidant rejection in osteoarthritic conditions.The pharmaceutical composition of claim 1, wherein the nanoparticles are configured for sustained release and achieve controlled ibudilast release over a period of 24 hours in a phosphate buffered saline (PBS) medium at pH 7.4.The pharmaceutical composition of claim 1, wherein the nanoparticles have enhanced anti-inflammatory activity by significantly reducing TNF-α, IL-1β and IL-10 levels in an osteoarthritis model.The pharmaceutical composition of claim 1, wherein the formulation enhances antioxidant rejection by restoring catalase (CAT), superoxide dismutase (SOD), and glutathione (GSH) levels while reducing malonic dialdehyde (MDA) levels in the treatment of osteoarthritis.The pharmaceutical composition of claim 1, wherein the formulation reduces paw edemaThe pharmaceutical composition of claim 1, wherein the formulation enhances haematological parameters in osteoarthritis treatmentThe pharmaceutical composition of claim 1, wherein the ibudilast loaded BSA nanoparticles are formulated for topical application in the treatment of osteoarthritis and provide improved permeation through the skin for localized anti-inflammatory activity.