Methods for treating psoriasis

Abstract

The present invention relates to a method for treating psoriasis. The method comprises administering an effective amount of brillaroxazine, or a pharmaceutically acceptable salt thereof, to a subject in need. Topical administration is the preferred route of administration. Brillaloxazine is effective in reducing one or more signs and / or symptoms of psoriasis.

Description

Technical Field

[0001] The present invention relates to a method for treating psoriasis, comprising administering an effective amount of blisibrolaxazine or a pharmaceutically acceptable salt thereof to a subject that requires them.

Background Art

[0002] Psoriasis is a systemic immune-mediated inflammatory disease with a genetic component, characterized by the recurrent onset of keratinizing, erythematous plaques on the skin (Kamiya 2019, Aleem 2018). The worldwide prevalence is ~125 million people, and this condition appears as phenotypically distinct subtypes, with plaque psoriasis accounting for over 80% (Armstrong 2020, Raharja 2021). It significantly impairs the psychosocial function of patients, reduces the quality of life, and in extreme cases can even cause depression, anxiety, or suicidal thoughts (Marek-josefowicz 2022). A higher prevalence is seen in patients with mental disorders. Pathologically, psoriasis causes an inflammatory skin response through exogenous (e.g., environmental, physical, and lifestyle stress factors) and endogenous (e.g., mental and cardiometabolic stress factors) risk factors (Kamiya, 2019). These stress factors drive the activation of innate immune cells (e.g., dendritic cells) and the differentiation of adaptive immune cells (e.g., from T cells to Thl cells), subsequently releasing inflammatory cytokines (Cantrell, 2018). These cytokines (e.g., tumor necrosis factor [TNF]-α, interferon-γ, and interleukin) cause downstream abnormal proliferation, dysfunctional differentiation, and leukocyte infiltration of damaged keratinocytes (Armstrong, 2020). Systemic cytokine circulation increases the risk of psoriatic arthritis, cardiometabolic diseases, and physiological conditions (Tashiro 2022, Wu 2022, Amin 2020).

[0003] Psoriasis is a persistent, non-infectious autoimmune disease characterized by the development of abnormal skin lesions. These lesions are red, pink, or purple, dry, itchy, and scaly. The severity of psoriasis varies from small, localized patches to complete body coverage.

[0004] The five main types of psoriasis are plaque, guttate, reversal, pustular, and erythrodermic. Plaque psoriasis, also known as psoriasis vulgaris, accounts for about 90% of cases. It typically presents as red patches with white scales on top. The most commonly affected areas of the body are the back of the forearms, the shins, the navel area, and the scalp. Guttate psoriasis has droplet-shaped lesions. Pustular psoriasis presents as small, non-contagious, pus-filled herpes. Reversal psoriasis forms red patches in the folds of skin. Erythrodermic psoriasis occurs when the rash is very widespread and can develop from any of the other types. Most people with psoriasis are affected at some point by their fingernails and toenails. This may include nail depressions or changes in nail color.

[0005] Psoriasis is generally considered a genetic disorder triggered by environmental factors. Symptoms often worsen during winter and with certain medications such as beta-blockers or nonsteroidal anti-inflammatory drugs. Infections and psychological stress may also play a role. The underlying mechanism involves the immune system responding to skin cells. Diagnosis is typically based on signs and symptoms.

[0006] Psoriasis is an autoimmune, chronic, persistent inflammatory skin disease characterized by excessive proliferation of keratinocytes with erythematous plaques, hyperkeratosis, and silvery-covered scales that are symmetrically distributed in areas of the extensor muscles, scalp, and lumbosacral region. The exact cause is unknown, but there are multiple etiologies, including genetics, environmental factors, trauma, infection, medications, and psychological stress. It most commonly causes red, itchy, scaly patches on the knees, elbows, trunk, and scalp.

[0007] The signs and symptoms of psoriasis can vary from person to person. Common signs and symptoms include: • Red spots on the skin covered with thick, silvery scales • Small, scaly spots (commonly seen in children) Dry, cracked skin that may bleed or itch. Itching, burning, or pain • Thickened, perforated, or raised nails • Swollen and stiff joints Includes.

[0008] In people with psoriasis, a very rapid proliferation of keratinocytes occurs, and their migration from the basal layer (basal layer) to the upper layers of the epidermis occurs within four days. Because the skin cells do not peel off immediately, thick, dry patches or plaques form. Very mild psoriasis exists in some people and may not even be suspected as a skin disorder. Very severe psoriasis is seen in others, in some cases the entire body is covered with thick, scaly, red skin. Psoriasis is generally diagnosed in adolescence, despite the fact that it occurs in populations of all age groups, i.e., from childhood to old age. Other causative factors of psoriasis include genetics, sudden changes (mutations) in genes, climate, abnormalities in the immune system, mental or emotional stress, contagion, and trauma.

[0009] Current psoriasis treatments include topical therapy, phototherapy, and systemic therapy, with the latter reserved for severe cases. Topical corticosteroids and vitamin D derivatives remain first-line treatments, both as monotherapy and as a complement to systemic therapy, despite their limited systemic efficacy and long-term side effects (e.g., tachyphylaxis, skin atrophy, adrenal suppression, and skin irritation). Topical treatments can act rapidly and provide local effects with minimal short-term adverse events. Conventional non-biological oral medications (e.g., methotrexate, apremilast, acitretin, or cyclosporine) offer further options for treating a wide range of inflammation, but they are associated with significant toxicity (e.g., hepatotoxicity, nephrotoxicity, hypertension, dyslipidemia, malignancy, and teratogenicity) (Armstrong 2020, Jain 2021).

[0010] Biologics (e.g., TNF and IL inhibitors) target specific immune response components. However, the use of these drugs is limited by the potential for immunogenicity development, the risk of serious infections and malignancies, parenteral administration, and affordability. Treatment inadequacy remains a concern, especially in severe cases and specific populations, given the complexity of managing multi-organ diseases (Raimondo 2017, Feldman 2016). Therefore, there is a need for new, effective therapies with an acceptable safety profile and convenient routes of administration that allow for more individualized treatment approaches (Rendon 2019, Jiang 2023).

[0011] Brillaloxazine (RP5063) is a polymorphic dopamine and 5-HT receptor modulator. Brillaloxazine exhibits high binding affinity to D2-4 and 5-HT1A receptors as a partial agonist, to 5-HT2A as a weak partial agonist or neutral agonist, and to 5-HT2B / 7 as an antagonist, while showing moderate affinity to the serotonin transporter (SERT). Brillaloxazine has established efficacy, safety, and pharmacokinetic profiles from phase 1 and 2 studies in healthy individuals and patients with schizophrenia. Preclinical studies also indicate that this drug inhibits the release of multiple inflammatory cytokines.

[0012] Liposomes are microparticle or colloidal carrier systems, typically with a diameter of 0.025–5.0 μm. Composed of biodegradable, biocompatible components, liposomes offer a unique opportunity for drug delivery within cells or even into individual cellular compartments. Liposomes spontaneously form when lipids are hydrated in an aqueous medium at the transition temperature. Lipids consist of natural and / or synthetic lipids (phospholipids and sphingolipids) and may include other bilayer components such as cholesterol and hydrophilic polymer lipids. Figure 1 shows a typical structure of a liposome.

[0013] The composition of liposomes determines their interaction with blood and tissues. The composition also determines the liposome's net physicochemical properties, namely membrane fluidity, charge density, and steric hindrance permeability. They have been found to be useful carriers for both hydrophilic and hydrophobic drugs. These drug delivery systems are used for the delivery of drugs with varying degrees of lipophilicity, such that water-soluble drugs are encapsulated in the aqueous compartment, and lipid-soluble drugs typically bind to the lipid bilayer or dissolve in the lipid phase. [Brief explanation of the drawing]

[0014] [Figure 1] Figure 1 shows a diagram of the general structure of a liposome. [Figure 2] Figure 2 shows the particle size (Z-mean) and particle distribution index of brillaroxazine liposomes during liposome dispersion, as measured by DLS. [Figure 3] Figure 3 shows the HPLC chromatogram of brillaroxazine liposomes. [Figure 4] Figure 4 shows the HPLC chromatogram of the lipogel sample. The first peak is brillaroxazine, and the second peak is the excipient. [Figure 5] Figure 5 shows a graph of in vitro diffusion studies of lipogel through a membrane. [Figure 6] Figure 6 shows the comparative effects of psoriasis area and severity index (PASI) from day 1 to day 12 in an imiquimod-induced psoriasis mouse model. PASI (p=0.03) between the brillaroxazine lipogel group and the induced psoriasis group from day 3 to day 12. [Figure 7] Figure 7 shows the Baker scores for spurious control, psoriasis, and brillaroxazine lipogel groups in an imiquimod-induced psoriasis mouse model. [Figure 8] Figure 8 shows a 100x magnified image of a skin histological study using H&E staining. [Figure 9] Figure 9 shows a 400x magnified image of a skin histological study using H&E staining. [Figure 10] Figure 10 shows serum TNFα levels in animals from different study groups. [Figure 11] Figure 11 shows the serum KI67 levels in animals of different study groups. [Figure 12] Figure 12 shows the serum TGF-β levels in animals of different study groups.

Mode for Carrying Out the Invention

[0015] The present invention relates to a method for treating psoriasis by administering an effective amount of brexpiprazole to a subject who needs it. Brexpiprazole is effective in reducing one or more signs or symptoms of psoriasis.

[0016] Brexpiprazole is a dopamine-serotonin stabilizer having potent partial agonist activity at dopamine D2, D3 and D4, and serotonin 5-HT1A and 5-HT2A receptors, and antagonist activity at serotonin 5-HT6 and 5-HT7 receptors. By having potent partial agonists at serotonin 5-HT1A and 5-HT2A receptors, brexpiprazole reduces the production of inflammatory mediators such as TNF-α, IFN-γ, IL-1β, IL-6, IL-8, and blocks the activation of nuclear factor-KB, thereby inducing the activation of keratinocytes and causing the deterioration of keratinocytes, worsening the condition.

[0017] Brexpiprazole (free base) is a basic and lipophilic molecule with a molecular weight of 450.36 g / mol. Its chemical structure is shown below.

[0018]

Chemical formula

[0019] Brexpiprazole often forms the HCl salt with a molecular weight of 486.7 g / mol.

[0020] Pharmaceutical composition The present invention provides a pharmaceutical composition comprising one or more pharmaceutically acceptable carriers and brillaloxazine or pharmaceutically acceptable salts thereof. The amount of brillaloxazine or a pharmaceutically acceptable salt thereof in the pharmaceutical composition is generally about 0.01-20%, 0.05-20%, 0.1-20%, 0.1-10%, 0.1-5%, 0.1-2%, 0.2-15%, 0.2-10%, 0.2-5%, 0.2-2%, or 1-5% (w / w) in topical formulations, about 0.1-5% in injectable formulations, 0.1-5% in patch formulations, about 1-90% in tablet formulations, and 1-100% in capsule formulations.

[0021] In one embodiment, brillaroxazine is incorporated into any acceptable carrier, including a cream, gel, lotion, or other type of suspension, which can stabilize the active compound and deliver it to the affected area by topical administration. In another embodiment, the pharmaceutical composition may be in the form of, for example, tablets, capsules, granules, powders, syrups, suppositories, injections, patches, or similar dosage forms. The pharmaceutical composition can be prepared by conventional methods.

[0022] A pharmaceutically acceptable carrier, which is an inert component, can be selected by those skilled in the art using conventional criteria. A pharmaceutically acceptable carrier includes, but is not limited to, non-aqueous-based solutions, suspensions, emulsions, microemulsions, micelle solutions, gels, and ointments. Pharmaceutically acceptable carriers also include physiological saline and aqueous electrolyte solutions; ionic and nonionic osmotic agents such as sodium chloride, potassium chloride, glycerol, and dextrose; pH adjusters and buffers such as hydroxides, phosphoric acid, citric acid, acetic acid, boric acid, and salts of trolamine; antioxidants such as bisulfite, sulfite, metabisulfite, thiosulfite, ascorbic acid, acetylcysteine, cysteine, glutathione, butyrylhydroxyanisole, butyrylhydroxytoluene, tocopherol, and salts of ascorbyl palmitate, acids, and / or bases; surfactants such as lectins and phospholipids, including but not limited to phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol; poloxamer and poloxamine, polysorbate 80, poly The materials include, but are not limited to, polysorbates such as Sorbate 60 and Polysorbate 20, polyethers such as polyethylene glycol and polypropylene glycol; polyvinyls such as polyvinyl alcohol and povidone; cellulose derivatives such as methylcellulose, hydroxypropylcellulose, hydroxyethylcellulose, carboxymethylcellulose and hydroxypropylmethylcellulose and their salts; petroleum derivatives such as mineral oil and white petrolatum; fats such as lanolin, peanut oil, palm oil and soybean oil; mono, di, and triglycerides; acrylic acid polymers such as carboxypolymethylene gel and hydrophobically modified crosslinked acrylic acid copolymers; polysaccharides such as dextran and glycosaminoglycans such as sodium hyaluronate. Other pharmaceutically acceptable carriers include xanthan gum, carrageenan, Avicel RC-591 (in combination with crystalline cellulose), and polyethylene glycol.Alternatively, the active compound may be dissolved or suspended in a pharmaceutically acceptable lipid preparation, such as vegetable oil, coconut oil, or castor oil, as shown by Kalepu et al. (Acta Pharmaceutica Sinica B, 3:361-372, 2013).

[0023] Such pharmaceutically acceptable carriers can be protected from bacterial contamination using well-known preservatives, which include, but are not limited to, benzalkonium chloride, ethylenediaminetetraacetic acid and its salts, benzethonium chloride, chlorhexidine, chlorobutanol, methylparaben, thimerosal, and phenylethyl alcohol, or can be formulated as non-preservative formulations for single or multiple use.

[0024] For example, a tablet or capsule formulation of brillaroxazine may contain other excipients that are not biologically active and do not react with the active compound. Excipients of tablets or capsules may include fillers, binders, lubricants and flow enhancers, disintegrants, wetting agents, and release rate modifiers. Binders are important in tablet formulations as they promote particle adhesion of the formulation. Examples of excipients of tablets or capsules include, but are not limited to, carboxymethylcellulose, cellulose, ethylcellulose, hydroxypropylmethylcellulose, methylcellulose, karaya gum, starch, tragacanth gum, gelatin, magnesium stearate, titanium dioxide, polyacrylic acid, and polyvinylpyrrolidone. For example, a tablet formulation may contain inert components such as colloidal silicon dioxide, crospovidone, hypromellose, magnesium stearate, crystalline cellulose, polyethylene glycol, sodium starch glycolate, and / or titanium dioxide. A capsule formulation may contain inert components such as gelatin, magnesium stearate, and / or titanium dioxide.

[0025] For example, a patch formulation of brillaroxazine may contain several inactive ingredients such as 1,3-butylene glycol, dihydroxyaluminum aminoacetic acid, disodium edetate, D-sorbitol, gelatin, kaolin, methylparaben, polysorbate 80, povidone, propylene glycol, propylparaben, sodium carboxymethylcellulose, sodium polyacrylate, tartaric acid, titanium dioxide, and purified water. The patch formulation may also contain skin permeability enhancers such as lactate esters or diethylene glycol monoethyl ether.

[0026] Topical formulations containing brillaroxazine can take the form of gels, creams, lotions, liquids, emulsions, ointments, sprays, solutions, and suspensions. Inactive components in topical formulations include, but are not limited to, (emollients / penetration enhancers), diethylene glycol monoethyl ether (emollient / penetration enhancer), DMSO (solubility enhancer), silicone elastomer (rheology / texture modifier), caprylic / capric triglyceride (emollient), octicalate (emollient / UV filter), silicone fluid (emollient / diluent), squalene (emollient), sunflower oil (emollient), and silicon dioxide (thickening agent).

[0027] The present invention further provides a gel formulation comprising brillaroxazine liposomes, a gelling agent, and a hydrating agent. In one embodiment, the gel formulation contains brillaroxazine in an amount of 0.005-10%, 0.01-5%, or 0.1-2% by weight. In one embodiment, the formulation has the form of a gel, and the bilayer lipid vesicles are intact and stable in the gel. In one embodiment, the gelling agent is carbomer 940, and the hydrating agent is glycerin.

[0028] Brillaloxazine liposomes In one embodiment, brillaroxazine is incorporated into bilayer lipid vesicles of the liposome composition. In one embodiment, the liposome composition comprises bilayer lipid vesicles encapsulating an aqueous solution, the bilayer lipid vesicles comprising one or more phospholipids, sterols, and brillaroxazine.

[0029] The lipids used in the formation of lipid vesicles typically consist of a lipid mixture mainly composed of phospholipids and sterols. A list of phospholipids commonly used in liposome preparation is provided on page 471 of Szoka et al. (Ann Rev Biophys Bioeng (1980) 9:467). The vesicles are formulated to contain negatively or positively charged lipids such as phosphatidic acid (PA) and phosphatidylglycerol (PG) to provide a desirable surface charge for the reagent vesicles. A small amount (0.1-1 mol%) of antioxidant, such as α-tocopherol, can be added to the lipid mixture to improve stability. A typical lipid mixture used in the formation of brillaloxazine liposomes in this invention includes phosphatidylcholine, cholesterol, and brillaloxazine.

[0030] In one embodiment, the aqueous solution of the liposome composition contains maltodextrin. Maltodextrin consists of D-glucose units linked in a chain of variable length. The glucose units are mainly linked by α(1→4) glycosidic bonds. Maltodextrin typically consists of a mixture of chains of different lengths, ranging from 3 to 17 glucose units. Brillaloxazine liposomes encapsulating maltodextrin may offer a superior drug release profile compared to brillaloxazine liposomes without maltodextrin.

[0031] Brillaloxazine liposomes are first prepared by dissolving vesicle-forming lipids (e.g., brillaloxazine, phosphatidylcholine, cholesterol) in an inert organic solvent or solvent system, such as chloroform and / or ethanol, to form an organic phase solution of lipids. Generally, the inert organic solvent or solvent system is one in which the lipid components can be readily dissolved at a concentration range of approximately 0.5 to 50 mg of lipids / ml. Next, the lipid solution is completely dried to remove the organic solvent, forming a thin lipid film on the surface of the container. After drying, the thin lipid film is then hydrated with an aqueous solution. In preferred embodiments, the aqueous solution contains maltodextrin.

[0032] In one embodiment, the brillaroxazine liposomes contain 10-40% or 20-30% brillaroxazine by weight.

[0033] In one embodiment, the brillaroxazine liposomes contain 20-60% or 30-45% maltodextrin by weight.

[0034] In one embodiment, the average particle size of brillaroxazine liposomes in the formulation is 500-750 nm.

[0035] In one embodiment, the most prominent peak of brillaroxazine liposomes in the formulation has a peak size of 900–1000 nM.

[0036] Methods of treating psoriasis The present invention provides a method for treating psoriasis. The method comprises administering an effective dose of brilaloxazine to a subject in need. As used herein, “effective dose” means an amount effective in treating psoriasis by restoring the condition or reducing the symptoms of psoriasis. The method reduces one or more signs and symptoms selected from the group consisting of red spots of skin covered with thick, silvery scales; small, scaly spots; dry, cracked skin; itchy, burning, or painful skin; thickened, porosity or ridged nails; and enlarged, stiff joints.

[0037] The pharmaceutical composition of the present invention can be administered by topical administration and systemic administration. Topical administration includes local administration. Topical administration is the preferred route of administration.

[0038] In topical administration, brilaloxazine is contained in a topical dosage form and comes into direct contact with psoriatic plaques. Topical delivery involves applying a brilaloxazine preparation to the skin, with the intention of directly treating skin disorders or signs of skin disease, including the pharmacological effects or effects of brilaloxazine on the skin surface. Topical preparations are applied to minimize the flow of brilaloxazine through the skin and maximize its retention on the skin. The therapeutic effect of topical preparations depends on the ability of brilaloxazine to penetrate the skin layers, and subsequently on the physicochemical properties of brilaloxazine, the carrier substrate, and the skin condition. Topical preparations for the treatment of psoriasis can be administered in a wide variety of drug forms: ointments, creams, gels, lotions, sprays, foams, etc. Through topical delivery, brilaloxazine provides control of psoriasis through the serotonin mechanism in cells. As a topical dosage form, a lower dose intensity is sufficient to provide the necessary pharmacodynamic effect at the application site compared to oral dosage forms.

[0039] Conventional semi-fluid dosage forms commonly used have certain limitations in drug delivery due to the barrier properties of the skin. The skin is continuously involved in building an efficient homeostatic barrier. Liposomes are a drug delivery system that can be used for the local delivery of drug molecules. Liposomes are microscopic vesicles containing amphiphilic phospholipids arranged in one or more concentric bilayers that encapsulate a corresponding number of aqueous compartments. In this form, with a spherical shell, liposomes resemble biological membranes. Liposomes contain biodegradable, biocompatible components and offer a unique opportunity to deliver drugs within cells or individual cellular compartments. Therefore, a liposomal-dispersed semi-fluid dosage form of brillaroxazine may represent a significant advance in the delivery of brillaroxazine to deeper layers of the skin in severe psoriatic conditions.

[0040] Systemic administration methods include oral, parenteral (e.g., intravenous, intramuscular, subcutaneous, or rectal), and other systemic routes of administration. In systemic administration methods, the active compound first reaches the plasma and then is distributed to the target tissue.

[0041] In one embodiment, the composition is applied topically to the affected area and rubbed in. The composition is administered topically at least once, twice, or three to four times a day, depending on whether the medical problem and the disease are chronic or acute. Generally, the topical composition contains about 0.01 to 10% (w / w) of the active compound brillaroxazine. For example, the topical composition contains about 0.1 to 2% (w / w) of the active compound. Depending on the size of the affected area, 0.2 to 85 ml, typically 0.2 to 10 ml, of the topical composition is applied individually per dose. The active compound is delivered through the skin to the area of ​​discomfort.

[0042] Those skilled in the art will recognize that a wide variety of delivery mechanisms are also suitable for the present invention.

[0043] This brillaroxazine liposome composition is useful for the treatment of mammals such as humans, horses, and dogs. The present invention is particularly useful for the treatment of humans.

[0044] The following embodiments further illustrate the present invention. These embodiments are intended merely to illustrate the present invention and should not be construed as limiting. [Examples]

[0045] Example 1: Preparation of brillaroxazine liposomes Table 1 shows the formulation composition of brillaroxazine liposomes.

[0046] [Table 1]

[0047] Brillaloxazine liposomes were prepared by lipid hydration. Briefly, phosphatidylcholine and cholesterol were dissolved in a suitable solvent (chloroform and / or ethanol), and brillaloxazine was dissolved in the same solvent. This drug-lipid solution was then dried under reduced pressure at 45-50°C in a rotary evaporator to completely remove the solvent. After all solvent was removed, a thin film formed in a round-bottom flask. The round-bottom flask containing this lipid film was kept under reduced pressure for 12-24 hours to completely remove any trace amounts of solvent present in the thin lipid film. After drying for 12-24 hours, the thin film was hydrated with 66 mL of maltodextrin solution at 60°C (concentration 39.57 mg / mL).

[0048] Example 2: Particle size and zeta potential analysis of brillaroxazine liposomes The liposomes prepared in Example 1 were observed under a light microscope at different magnifications to confirm the hydration process. To confirm the prepared spherical liposomal vesicles, light microscopy observations at different magnifications (10x, 20x, and 40x) were performed throughout the hydration process.

[0049] The particle size of the liposomes was analyzed using the DLS (dynamic light scattering) method for particle size analysis and drug content analysis. The Z-mean (particle size) was measured for the prepared liposomes. Figure 2 shows the particle size (Z-mean) and particle distribution index in the liposome dispersion, as measured by DLS of brillaroxazine liposomes.

[0050] The zeta potential was estimated from the experimentally determined electrophoretic mobility of the particles. The value of the Z potential indicates the stability of the colloidal dispersion.

[0051] Zeta potential (mV) value: • 0-5 - Rapid coagulation or aggregation • 10-30 - Initial unstable state 30-40 - Moderate stability 40-60 - Good stability ·>61—Excellent stability

[0052] Generally, colloidal dispersions with a zeta potential greater than 30mV positive or less than 30mV negative exhibit high stability. The higher the zeta potential (both positive and negative), the greater the stability.

[0053] Table 2 shows the particle size and Z potential analysis of liposomes by DLS method.

[0054] [Table 2]

[0055] Example 3: Measurement of drug content by HPLC The liposome drug content was analyzed by HPLC.

[0056] HPLC system Shimadzu HPLC system (LC-2030C Plus, serial number: L21445711704 AE, Made in Japan), autosampler, UV detector, data acquisition system. An equivalent system can be used as a replacement.

[0057] HPLC column Shimadzu Shim-Pack GIST C18, 5μm, 250x4.6mm column or equivalent.

[0058] Reagent preparation Mobile phase A Dissolve 2.72g of KH2PO4 in 1000mL of ultrapure water (0.02M solution). Adjust the pH to 3.0 with phosphoric acid. Mix the above buffer solution with acetonitrile in a 90:10 ratio. • Filter through a membrane before use.

[0059] Mobile phase B Mix acetonitrile and ultrapure water in a 90:10 ratio. Adjust the pH to 3.0 with phosphoric acid. • Filter through a membrane before use.

[0060] Diluent • To optimize drug content and encapsulation efficiency, prepare a mixture of acetonitrile and phosphate buffer (Section 6.2.1.1) in a ratio of 85:15.

[0061] Sample preparation for drug content and incarnation efficiency Drug content Transfer the required amount of liposome sample (total dispersion) to a volumetric flask and add the required amount of diluent (6.2.3), mixing thoroughly. Maintain this mixture in a sonicator bath at 60°C for 30-45 minutes. Take the required amount of prepared sample and dilute to the required concentration. The assay / drug concentration is 20 μg / ml.

[0062] Import efficiency Place the required amount of liposome sample into a centrifuge tube and centrifuge the liposome dispersion at 10,000 rpm for 30 minutes at 20°C. Remove the supernatant and collect the pellet. Add the required amount of diluent to the liposome pellet and mix well. Hold this mixture in a sonicator tank for 30-45 minutes at 60°C. Take the required amount of the prepared sample and dilute it to a drug concentration of 20 μg / ml.

[0063] Standard solution Prepare a 20 μg / ml standard solution using the above dilution.

[0064] analysis Configure the HPLC using the following parameters: Column: Shimadzu Shim-Pack GIST C18, 5μm, 250x4.6mm column or equivalent. Flow rate: 1mL / min Injection volume: 20μL Detection: UV@215nm Column temperature: 30℃ Execution time: 5-7 minutes HPLC conditions: Mobile phase A 25%, Mobile phase B 75%

[0065] Identification Compare the retention times of the standard / pure drug peak and the sample drug peak.

[0066] result The HPLC chromatogram is shown in Figure 3. The HPLC results indicate a drug content of 96% and a liposome uptake efficiency of 73%.

[0067] The final formulation composition was consistent with brillaroxazine (24.53%), lecithin (34.75%), cholesterol (2.97%), maltodextrin (37.74%), and purified water for lipid membrane hydration.

[0068] Example 4: Preparation of liposome gel formulation Liposome gels were prepared by incorporating a liposome dispersion into a gel formulation. First, a plain gel was prepared, then the liposome dispersion was added and thoroughly mixed to produce a liposome gel or lipogel. Lipogel formulations with various proportions (0.25–1.5% brillaroxazine) were prepared as needed. Table 3 shows the compositions of the lipogel formulations.

[0069] [Table 3]

[0070] The prepared liposome gel formulations were evaluated for their physical appearance and pH. All gel formulations were observed for intact liposomes under a light microscope. The liposome gel formulations had the appearance of a white cream gel, a pH of 5–6, and the presence of liposomes was confirmed by microscopic examination. Furthermore, the liposome particles were intact and stable in all gel formulations.

[0071] Example 5: Lipogel analysis by HPLC The lipogel sample was placed in a volumetric flask, and the diluent was added and mixed thoroughly. The brillaroxazine content was analyzed by HPLC according to the same protocol as in Example 3.

[0072] By comparing the retention times of the pure drug peak and the sample peak, the drug content was calculated to be 95.12%.

[0073] The HPLC chromatogram of the lipogel sample is shown in Figure 4. The first peak is brillaloxazine, and the second peak is the excipient. The brillaloxazine peak is distinct and separated from the excipient peak.

[0074] Example 6: In vitro diffusion / penetration study of lipogel The prepared lipogels were analyzed for drug diffusion / osmosis using a Franz diffusion cell. The Franz diffusion cell was filled with PBS buffer at pH 7.4. A regenerated cellulose dialysis membrane (MW cutoff: 12000-14000), surface-treated and neutralized with PBS at pH 7.4, was placed in the receptor compartment, and the weighed amount of lipogel was placed in the donor compartment. The receptor solution was agitated, and the skin temperature in the diffusion cell was maintained by temperature-controlled water circulation in an outer jacket. At different time intervals, the sample was removed through the sample port and replaced with an equal volume of plain PBS. The removed sample was mixed with an equal volume of HPLC diluent, and the drug content at different time intervals was analyzed. The drug diffusion rate, flux ratio, and osmosis coefficient were calculated for each lipogel formulation. The drug diffusion / release rate over time was plotted using GraphPad Prism version 6.01 software.

[0075] HPLC analysis of drug diffusion samples The HPLC analysis method, system, and column were the same as those described in Example 3, except that a mixture of acetonitrile and phosphate buffer (60:40) was used in the drug diffusion / permeation study.

[0076] Sample preparation for diffusion / permeation sample analysis Mix the diffusion / permeation sample solution recovered from the Franz diffusion apparatus with an equal volume of diluent. Mix thoroughly using a vortex mixer and filter the solution through a syringe filter.

[0077] result The HPLC chromatogram results of the lipogel diffusion sample show a clear and separated brillaroxazine peak.

[0078] Figure 5 shows the graph of the in vitro diffusion study of the lipogel through the membrane. The release profile showed steady and sustained brillaroxazine release from the formulation throughout the entire 8-hour study period.

[0079] Table 4 shows the results of in vitro diffusion studies of lipogel formulations.

[0080] [Table 4]

[0081] Liposome-containing liposomal formulations with maltodextrin exhibit superior drug release profiles and higher flux and osmotic values ​​in in vitro diffusion studies compared to liposome formulations without maltodextrin. This may be due to increased solubility of RP5063 in the liposome gel and an optimal particle size distribution within the formulation.

[0082] Example 7: In vivo preclinical study—psoriasis model Balb / c mice (n=6 / group) were used in this experiment, and the animals were cared for according to protocols approved by the institution's animal ethics committee. Imiquimod cream (5%) was used as the chemical agent to induce psoriatic disease pathology. Psoriasis was induced for 12 days by applying imiquimod to the dorsal side of the shaved skin of the animals in the morning. The test lipogel formulation was applied to the animals at night for 12 days. Imiquimod was applied until the last day of the experiment. All animals were observed for the following parameters: PASI score, Baker score, histological H&E staining, and serum cytokine analysis (TNF-α, KI67, TGF-β). Table 5 shows the animal populations used in the preclinical study.

[0083] [Table 5]

[0084] PASI score Animals were observed daily for signs of imiquimod-induced psoriasis toxicity, and PASI scores were calculated for each group. Figure 6 shows the overall PASI scores from days 1 to 12. The induced group (psoriasis group) showed higher PASI scores than the non-induced control group (spurious control group) (p=0.001). The magnitude difference between these two groups increased from day 3 to day 11. The brilaloxazine lipogel group began to show an increase in PASI scores from day 3, peaking on days 7 and 8, and then decreasing to a plateau level from day 10 to 12. Its score was larger than that of the spurious control group, but did not reach the same level as the psoriasis group. The brilaloxazine lipogel PASI score was consistently lower than that of the induced psoriasis group from day 3 to day 12 (p=0.03). The largest magnitude difference appeared on days 11 and 12.

[0085] Baker score At the end of day 12 of the study period, the animals were euthanized, their skin was collected, and histological examination was performed. Signs of psoriasis toxicity were examined in the skin of each animal, and Baker scores were calculated for each group. Baker scores showed a significant decrease in animals treated with lipogel. Figure 7 shows that topical brilaloxazine significantly treated psoriasis animals (p=0.003).

[0086] Histology (H&E staining) Figure 8 shows a 100x magnified photograph of a dermatologic study. Table 6 shows the observation of the dermatologic sample at 100x magnification.

[0087] [Table 6]

[0088] Figure 9 shows a 400x magnified photograph of a dermatologic study. Table 7 shows the observation of the dermatologic sample at 400x magnification.

[0089] [Table 7]

[0090] In summary, histological assessments included direct observations at 100x and 400x magnification. Tables 6 and 7, and Figures 8 and 9, provide histological observations and H&E staining at both magnifications. Differences were observed between the spurious control group and the induced psoriasis and briraloxazine lipogel groups, and between the latter two groups. Baker score comparisons (Figure 7) reflected significant effects on the spurious control (p=0.001) and briraloxazine lipogel (p=0.003) groups for the psoriasis cohort. Such observations highlight the therapeutic effect of briraloxazine lipogel.

[0091] Serum cytokine levels At the end of the 12th day of the study period, the animals were sacrificed, their blood was collected, and their serum was separated. Serum cytokines TNF-α, KI67, and TGF-β were analyzed by ELISA.

[0092] Figure 10 shows serum TNF-α levels in different study groups of animals. The results show that brillaroxazine reduces serum TNF-α levels in psoriasis-induced animals, bringing TNF-α levels to a level comparable to that of spurious control animals.

[0093] Figure 11 shows serum KI67 levels in different study groups of animals. The results show that brillaroxazine significantly reduced serum KI67 levels in psoriasis-induced animals (p=0.001), and that these levels were comparable to those of the spurious control group.

[0094] Figure 12 shows serum TGF-β levels in different study groups of animals. The results show that the lipogel formulation significantly reduces serum TGF-β levels in psoriasis-induced animals compared to psoriasis control animals (p=0.008).

[0095] The foregoing describes preferred embodiments of the present invention, and it will be understood that modifications are possible without departing from the scope of the invention as described in the claims.

Claims

1. A method for treating psoriasis, comprising administering an effective dose of brilaloxazine, or a pharmaceutically acceptable salt thereof, to a subject in need.

2. The method according to claim 1, wherein brillaroxazine is administered topically to the subject.

3. The method according to claim 2, wherein brillaroxazine is incorporated into the bilayer lipid vesicles of liposomes.

4. The method according to claim 3, wherein the liposome encapsulates an aqueous solution containing one or more phospholipids, sterols, and brillaroxazine or a pharmaceutically acceptable salt thereof, and maltodextrin.

5. The method according to claim 4, wherein the liposome contains 20 to 30% by mass of brillaroxazine.

6. The method according to claim 4, wherein the liposomes are contained in a gel formulation.

7. The method according to claim 4, wherein the gel formulation contains 0.1 to 2% brillaroxazine.

8. The method according to claim 1, wherein brillaroxazine is administered systemically to the subject.

9. The method according to claim 1, wherein the method alleviates one or more signs and symptoms selected from the group consisting of red spots on skin covered with thick, silvery scales; small, scaly spots; dry, cracked skin; itchy, burning, or painful skin; thickened, porosity or ridged nails; and swollen, stiff joints.

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