Methods for treating psoriasis

Abstract

The present invention relates to a method for treating psoriasis. The method comprises administering to a subject in need thereof an effective amount of bulaxin or a pharmaceutically acceptable salt thereof. The topical application is a preferred route of application. Bralaxin is effective in reducing one or more signs and / or symptoms of psoriasis.

Description

Technical Field

[0002] This invention relates to a method for treating psoriasis, the method comprising administering to a subject in need an effective amount of bromerazine or a pharmaceutically acceptable salt thereof. Background Technology

[0004] Psoriasis is a systemic, immune-mediated inflammatory disease characterized by recurrent episodes of hyperkeratotic, erythematous plaques on the skin (Kamiya 2019, Aleem 2018). The global prevalence of this condition is approximately 125 million, manifesting in different phenotypic subtypes, with plaque psoriasis accounting for over 80% (Armstrong 2020, Raharja 2021). The disease severely impairs patients' psychosocial functioning, reduces their quality of life, and in extreme cases can lead to depression, anxiety, or even suicidal thoughts (Marek-Josefowicz 2022). A higher prevalence appears to exist among patients with mental illness.

[0005] Pathologically, psoriasis triggers an inflammatory skin response through both external (e.g., environmental, physical, and lifestyle stressors) and internal (e.g., psychological and cardiometabolic stressors) risk factors (Kamiya 2019). These stressors drive the activation of innate immune cells (e.g., dendritic cells) and the differentiation of adaptive immune cells (e.g., T cells differentiating into Th1 cells), which subsequently release pro-inflammatory cytokines (Cantrell 2018). These cytokines (e.g., tumor necrosis factor [TNF]-α, interferon-γ, and interleukins) lead to abnormal proliferation, dysfunctional differentiation, and leukocyte infiltration downstream of diseased keratinocytes (Armstrong 2020). Systemic cytokine circulation increases the risk of psoriatic arthritis, cardiovascular metabolic diseases, and physiological conditions (Tashiro 2022, Wu 2022, Amin 2020).

[0006] Psoriasis is a long-term, non-contagious autoimmune disease characterized by raised areas of abnormal skin. These areas are red, pink, or purple, dry, itchy, and scaly. The severity of psoriasis varies from small localized patches to widespread coverage.

[0007] The five main types of psoriasis are plaque, guttate, inverted, pustular, and erythrodermic. Plaque psoriasis (also known as common psoriasis) 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, lower legs, navel area, and scalp. Guttate psoriasis has teardrop-shaped lesions. Pustular psoriasis presents as small, non-infectious, pus-filled blisters. Inverted psoriasis forms red patches in skin folds. Erythrodermic psoriasis occurs when the rash becomes very widespread and can develop from any other type. Most people with psoriasis will experience involvement of their fingernails and toenails at some point. This may include pitting or changes in nail color.

[0008] Psoriasis is generally considered a genetic disorder triggered by environmental factors. Symptoms typically worsen in winter and with the use of certain medications (such as beta-blockers or NSAIDs). Infections and psychological stress may also play a role. The underlying mechanism involves the immune system's response to skin cells. Diagnosis is usually based on physical signs and symptoms.

[0009] Psoriasis is an autoimmune, chronic, residual inflammatory skin disease characterized by excessive proliferation of keratinocytes, accompanied by erythematous plaques, hyperkeratosis, and silvery scales—symmetrically distributed in common areas such as the extensor muscles, scalp, and lumbosacral region. The exact cause is unclear, but several contributing factors exist, including genetics, environmental factors, trauma, infection, medications, and psychological stress. It causes red, itchy, scaly patches, most commonly found on the knees, elbows, trunk, and scalp.

[0010] The signs and symptoms of psoriasis can vary from person to person. Common signs and symptoms include: ● Red patches on the skin covered with thick, silvery scales. ● Small scaly spots (common in children). ● Dry, cracked skin that may bleed or itch. ● Itching, burning, or pain, ● Thickened, concave, or raised nails ● Swelling and stiff joints.

[0011] Keratinocytes proliferate very rapidly in people with psoriasis, and they migrate from the basal layer of the skin (the basal layer) to the upper epidermis within four days. Because the skin cannot shed these cells quickly enough, thick, dry patches or plaques form. Some people have very mild psoriasis, which may not even be suspected of being a skin condition. Very severe psoriasis can also be seen in others, sometimes involving a full-body coverage of scaly, thick, red skin. Although psoriasis can occur in all age groups (from pediatrics to geriatrics), it is usually diagnosed during adolescence. Other contributing factors to psoriasis include genetics, sudden gene alterations (mutations), climate, immune system abnormalities, mental or emotional stress, infections, and wounds.

[0012] Current treatments for psoriasis include topical therapy, phototherapy, and systemic therapy, with the latter reserved for severe cases. Despite limitations in systemic efficacy and long-term side effects (e.g., rapid resistance, skin atrophy, adrenal suppression, and skin irritation), topical corticosteroids and vitamin D derivatives remain first-line complements to monotherapy and systemic therapy. Topical treatments act rapidly and exert local effects with minimal short-term adverse events. Conventional non-biological oral agents (e.g., methotrexate, apremilast, atracin, or cyclosporine) offer more options for treating widespread inflammation; however, they are associated with significant toxicities (e.g., hepatotoxicity, nephrotoxicity, hypertension, dyslipidemia, malignancy, and teratogenicity). (Armstrong 2020, Jain 2021)

[0013] Biologics (e.g., TNF and IL inhibitors) target specific components of the immune response. However, their use is limited by potential immunogenicity, risks of severe infection and malignancy, parenteral administration, and affordability. Given the complexity of managing multisystem diseases, undertreatment remains a problem, particularly in severe cases and specific populations (Raimondo 2017, Feldman 2016). Therefore, there is a need for new, effective treatments with acceptable safety profiles and convenient routes of administration, allowing for more personalized approaches (Rendon 2019, Jiang 2023).

[0014] Bripraxacillin (RP5063) is a multimodal dopamine and 5-HT receptor modulator. Bripraxacillin exhibits high binding affinity for D2-4 and 5-HT1A receptors as partial agonists, 5-HT2A as a weak partial agonist or neutral antagonist, and 5-HT2B / 7 as an antagonist, and moderate affinity for the serotonin transporter (SERT). In Phase 1 and Phase 2 studies in healthy volunteers and patients with schizophrenia, bripraxacillin demonstrated established efficacy, safety, and pharmacokinetic profiles. Furthermore, preclinical work indicates that this agent inhibits the release of multiple pro-inflammatory cytokines.

[0015] Liposomes are microparticle or colloidal carrier systems, typically ranging from 0.025 μm to 5.0 μm in diameter. Composed of biodegradable, biocompatible components, liposomes offer a unique opportunity to deliver drugs into cells or even single-cell compartments. Liposomes spontaneously form when lipids are hydrated in an aqueous medium at a transition temperature. Lipids consist of natural and / or synthetic lipids (phospholipids and sphingolipids) and may contain other bilayer components such as cholesterol and hydrophilic polymeric lipids. Figure 1 A representation of the general structure of liposomes is shown.

[0016] The composition of liposomes determines their interaction with blood and tissues. The composition determines the net physicochemical properties of liposomes, 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, used to deliver drugs with different lipophilicities (such as water-soluble drugs), are encapsulated in aqueous compartments; lipophilic drugs are typically bound to a lipid bilayer or dissolved in the lipid phase. Attached Figure Description

[0018] Figure 1 A diagram showing the general structure of liposomes is provided.

[0019] Figure 2 Particle size (Z-mean) and particle partition index in liposome dispersions measured by DLS of Brirazacin liposomes are shown.

[0020] Figure 3 The HPLC chromatogram of Brisarazacin liposomes is shown.

[0021] Figure 4 The HPLC chromatogram of the lipid gel sample is shown. The first peak is bromelain, while the second peak is the excipient.

[0022] Figure 5 The graph shows the in vitro diffusion curve of the lipid gel across the membrane.

[0023] Figure 6The comparative effects of psoriasis area and severity index (PASI) on day 1 through day 12 in an imiquimod-induced psoriasis mouse model are shown. PASI, between the bromelain lipogel and the induced psoriasis group from day 3 to day 12 (p=0.03).

[0024] Figure 7 Baker scores are shown for the sham control group, psoriasis group, and brevicornu lipid gel group in an imiquimod-induced psoriasis mouse model.

[0025] Figure 8 This image shows a 100x magnified view of a skin histological study obtained through H&E staining.

[0026] Figure 9 The image shown is a 400x magnified image of a skin histological study stained with H&E.

[0027] Figure 10 Serum TNF-α levels in animals from different study groups are shown.

[0028] Figure 11 Serum KI67 levels in animals from different study groups are shown.

[0029] Figure 12 Serum TGF-β levels in animals from different study groups are shown. Detailed Implementation

[0031] This invention relates to a method for treating psoriasis by administering an effective amount of bromelain to a subject in need. Brirelain effectively reduces one or more signs or symptoms of psoriasis.

[0032] Brisarazazon is a dopamine-serotonin system stabilizer with potent partial agonist activity against dopamine D2, D3, and D4 receptors, as well as serotonin 5-HT1A and 5-HT2A receptors, and antagonist activity against serotonin 5-HT6 and 5-HT7 receptors. Through its potent partial agonist activity against serotonin 5-HT1A and 5-HT2A receptors, brisarazazon reduces the production of inflammatory mediators such as TNF-α, IFN-γ, IL-1β, IL-6, and IL-8, and prevents activation of nuclear factor-κB, thereby inducing keratinocyte activation, triggering keratinocyte degeneration, and exacerbating psoriasis symptoms.

[0033] Brirazazan (free base) is a basic and lipophilic molecule with a molecular weight of 450.36 g / mol. Its chemical structure is shown below.

[0034]

[0035] Brisaracin is usually in the form of an HCl salt with a molecular weight of 486.7 g / mol.

[0036] Pharmaceutical Composition

[0037] This invention provides pharmaceutical compositions comprising one or more pharmaceutically acceptable carriers and bromerazine or a pharmaceutically acceptable salt thereof. The amount of bromerazine or a pharmaceutically acceptable salt thereof in the pharmaceutical composition is typically: for topical formulations, about 0.01% to 20%, or 0.05% to 20%, or 0.1% to 20%, or 0.1% to 10%, or 0.1% to 5%, or 0.1% to 2%, or 0.2% to 15%, or 0.2% to 10%, or 0.2% to 5%, or 0.2% to 2%, or 1% to 5% (w / w); for injectable formulations, about 0.1% to 5%; for patch formulations, 0.1% to 5%; for tablet formulations, about 1% to 90%; and for capsule formulations, 1% to 100%.

[0038] In one embodiment, bromelain is incorporated into any acceptable carrier, including creams, gels, lotions, or other types of suspensions, which stabilize the active compound and deliver it to the affected area via topical application. In another embodiment, the pharmaceutical composition may be in dosage forms such as tablets, capsules, granules, fine granules, powders, syrups, suppositories, injectable solutions, or patches. The above pharmaceutical compositions can be prepared using conventional methods.

[0039] Pharmaceutically acceptable carriers are inactive ingredients that can be selected by those skilled in the art using conventional standards. Pharmaceutically acceptable carriers include, but are not limited to, non-aqueous solutions, suspensions, emulsions, microemulsions, micelle solutions, gels, and ointments. Pharmaceutically acceptable carriers may also contain a variety of ingredients, including, but not limited to, saline and aqueous electrolyte solutions; ionic and nonionic permeabilizers, such as sodium chloride, potassium chloride, glycerol, and dextran; pH adjusters and buffers, such as salts of hydroxides, phosphates, citrates, acetates, and borates; and triethanolamine; antioxidants, such as bisulfites, sulfites, metabisulfites, thiosulfites, ascorbic acid, acetylcysteine, cysteine, glutathione, butylated hydroxyanisole, butylated hydroxytoluene, tocopherol, and salts, acids, and / or bases of palmitic acid ascorbate; and surfactants, such as lecithin and phospholipids, including but not limited to phosphatidylcholine, phosphatidylethanolamine, and phosphatidylcholine. Inositol; poloxamer and poloxamine, polysorbates (such as polysorbate 80, polysorbate 60 and polysorbate 20), polyethers (such as polyethylene glycol and polypropylene glycol); polyethylene, 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, soybean oil; monoglycerides, diglycerides and triglycerides; acrylic polymers, such as carboxymethyl polymethylene gel and hydrophobically modified crosslinked acrylic copolymers; polysaccharides (such as dextran) and glycosaminoglycans (such as sodium hyaluronate). Other pharmaceutically acceptable carriers include xanthan gum, carrageenan, Avicel RC-591 (a combination of microcrystalline cellulose and polysaccharides), and polyethylene glycol. Alternatively, the active compound may be soluble or suspended in pharmaceutically acceptable lipid formulations, such as those described by Kalepu et al. (Acta Pharmaceutica Sinica B, 3:361-372, 2013), for example, vegetable oils, coconut oil, castor oil, etc.

[0040] Such pharmaceutically acceptable carriers can be preserved with well-known preservatives to prevent bacterial contamination, including but not limited to benzalkonium chloride, ethylenediaminetetraacetic acid and its salts, benzyl chloride, chlorhexidine, chlorobutanol, methylparaben, thimerosal, and phenylethanol, or can be formulated as non-preservative preparations for single or multiple use.

[0041] For example, tablet or capsule formulations of brevicornu may contain other excipients that are non-biologically active and do not react with the active compound. Excipients for tablets or capsules may include fillers, binders, lubricants and flow aids, disintegrants, wetting agents, and release rate modifiers. Binders promote the adhesion of formulation particles and are important for tablet formulations. Examples of excipients for tablets or capsules include, but are not limited to, carboxymethyl cellulose, cellulose, ethyl cellulose, hydroxypropyl methylcellulose, methyl cellulose, guilarin, starch, tragacanth gum, gelatin, magnesium stearate, titanium dioxide, poly(acrylic acid), and polyvinylpyrrolidone. For example, tablet formulations may contain inactive ingredients such as colloidal silica, crocidolone, hydroxypropyl methylcellulose, magnesium stearate, microcrystalline cellulose, polyethylene glycol, sodium starch-hydroxyacetate, and / or titanium dioxide. Capsule formulations may contain inactive ingredients such as gelatin, magnesium stearate, and / or titanium dioxide.

[0042] For example, brevicornu transdermal patches may contain inactive ingredients such as 1,3-butanediol, aluminum dihydroxyaminoacetate, disodium EDTA, D-sorbitol, gelatin, kaolin, methylparaben, polysorbate 80, povidone, propylene glycol, propylparaben, sodium carboxymethyl cellulose, sodium polyacrylate, tartaric acid, titanium dioxide, and purified water. Transdermal patches may also contain skin permeability enhancers such as lactates or diethylene glycol monoethyl ether.

[0043] Topical formulations containing brevicornu may be in the form of gels, creams, lotions, liquids, emulsions, ointments, sprays, solutions, and suspensions. Inactive ingredients in topical formulations may include, but are not limited to, emollients / penetration enhancers, diethylene glycol monoethyl ether (emollients / penetration enhancers), DMSO (solubility enhancers), silicone elastomers (rheology / texture modifiers), caprylic / capric triglycerides (emollients), octyl salicylate (emollients / UV filters), silicone fluids (emollients / diluents), squalene (emollients), sunflower oil (emollients), and silica (thickeners).

[0044] This application also provides a gel formulation comprising brevicornu liposomes, a gelling agent, and a humectant. In one embodiment, the gel formulation comprises 0.005% to 10% by weight, 0.01% to 5% by weight, or 0.1% to 2% by weight of brevicornu. In one embodiment, the formulation has a gel appearance, and the bilayer lipid vesicles are intact and stable within the gel. In one embodiment, the gelling agent is carbomer 940, and the humectant is glycerin.

[0045] Brirasaqin Liposome

[0046] In one embodiment, bromelain is incorporated into a bilayer lipid vesicle of the liposome composition. In another embodiment, the liposome composition comprises a bilayer lipid vesicle encapsulating an aqueous solution, wherein the bilayer lipid vesicle comprises one or more phospholipids, sterols, and bromelain.

[0047] Lipids used to form lipid vesicles typically comprise a mixture of lipids primarily composed of phospholipids and sterols. A list of commonly used phospholipids in liposome preparation can be found on page 471 of Szoka et al. (Ann Rev Biophys Bioeng (1980) 9:467). Vesicles can be formulated to contain negatively or positively charged lipids, such as phosphatidic acid (PA) and phosphatidylglycerol (PG), to provide the desired surface charge on the reagent vesicles. Small amounts of antioxidants, such as α-tocopherol (0.1 mol% to 1 mol%), can be added to the lipid mixture to increase stability. A typical lipid mixture used to form the brevicornuate liposomes of the present invention includes phosphatidylcholine, cholesterol, and brevicornuate.

[0048] In one embodiment, the aqueous solution of the liposome composition comprises maltodextrin. Maltodextrin consists of D-glucose units linked in chains of variable length. The glucose units are primarily linked by α(1→4) glycosidic bonds. Maltodextrin typically consists of a mixture of chains with lengths of three to 17 glucose units. Brirazazanol liposomes encapsulating maltodextrin provide a better drug release profile than brirazazanol liposomes without maltodextrin.

[0049] Brirazacin liposomes are prepared by first dissolving vesicle-forming lipids (e.g., brirazacin, phosphatidylcholine, cholesterol) in an inert organic solvent or solvent system (e.g., 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 component is readily soluble, with a concentration ranging from about 0.5 mg lipid / mL to 50 mg lipid / mL. The lipid solution is then completely dried to remove the organic solvent, forming a thin lipid film on the container surface. After drying, the thin lipid film is then hydrated with an aqueous solution. In a preferred embodiment, the aqueous solution contains maltodextrin.

[0050] In one embodiment, the briraxacin liposome contains 10% to 40% or 20% to 30% briraxacin.

[0051] In one embodiment, the Brirazazin liposome comprises 20% to 60% or 30% to 45% maltodextrin.

[0052] In one embodiment, the average particle size of the brenrazazine liposomes in the formulation is between 500 nm and 750 nm.

[0053] In one embodiment, the strongest peak of the brenrazacin liposome in the formulation has a peak size of 900 nM to 1000 nM.

[0054] Methods for treating psoriasis

[0055] This application provides a method for treating psoriasis. The method comprises administering an effective amount of bromelain to a subject in need. As used herein, an "effective amount" is an amount that effectively treats psoriasis by improving the pathological condition or reducing the symptoms of psoriasis. The method alleviates one or more signs and symptoms selected from the group consisting of: erythematous patches of skin covered with thick, silvery scales; small scaly spots; dry and cracked skin; itching, burning, or pain of the skin; thickened, pitted, or raised nails; and swollen and stiff joints.

[0056] The pharmaceutical compositions of the present invention can be administered by partial or systemic application. Partial application includes local application. Local application is the preferred route of administration.

[0057] In topical application, bromelain can be contained in a topical formulation and applied directly to psoriatic plaques. Topical delivery is the application of a bromelain formulation to the skin to treat the dermal manifestations of a skin condition or disease directly, with the aim of encapsulating the pharmacological or therapeutic effects of bromelain on the skin surface. Topical formulations are applied to minimize the flux of bromelain through the skin and maximize its retention on the skin. The therapeutic effect of a topical formulation depends on the ability of bromelain to penetrate the skin layers, which in turn depends on the physicochemical properties of bromelain, the carrier matrix, and the skin condition. Topical formulations for treating psoriasis can be administered in various pharmaceutical forms: ointments, creams, gels, lotions, sprays, foams, etc. Through topical delivery, bromelain provides its serotonin mechanism within cells to control psoriasis. As a topical formulation, a lower dose intensity than an oral formulation is sufficient to provide the desired pharmacodynamic effect at the application site.

[0058] Conventional semi-solid dosage forms have certain limitations in drug delivery due to the barrier properties of the skin. The skin is continuously involved in the construction of an efficient homeostatic barrier. Liposomes are drug delivery systems 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 surrounding an equal number of aqueous compartments. In this form, liposomes resemble biological membranes as a spherical shell. Liposomes contain biodegradable, biocompatible components and offer a unique opportunity to deliver drugs into cells or even individual cellular compartments. Therefore, the semi-solid dosage form of brirasacrazine liposome dispersion offers a significant advancement in the delivery of brirasacrazine to deeper layers of the skin in severe psoriasis conditions.

[0059] Systemic administration includes oral, parenteral (such as intravenous, intramuscular, subcutaneous, or rectal) routes and other systemic routes. In systemic administration, the active compound first reaches the plasma and then distributes to the target tissues.

[0060] In one embodiment, the composition is applied topically to the affected area and rubbed in. Depending on whether the medical problem and disease pathology are chronic or acute, the composition is applied topically at least once or twice daily, or three to four times daily. Generally, the topical composition contains about 0.01% to 10% (w / w) of the active compound, brevicornuate. 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, each dose consists of 0.2 mL to 85 mL, typically 0.2 mL to 10 mL, of the topical composition applied to the individual. The active compound penetrates the skin and is delivered to the site of discomfort.

[0061] Those skilled in the art will recognize that various delivery mechanisms are also applicable to this invention.

[0062] The brevicornu liposomes of the present invention can be used to treat mammalian subjects, such as humans, horses, and dogs. The present invention is particularly useful for treating humans.

[0063] The following examples further illustrate the present invention. These examples are intended to illustrate the invention only and should not be construed as limiting.

[0064] Example

[0065] Example 1. Preparation of Brisaracin liposomes

[0066] Table 1 shows the formulation composition of Brirazacin liposomes.

[0067] Table 1.

[0068]

[0069] Brisaracin liposomes were prepared via a fat hydration method. In short, phosphatidylcholine and cholesterol were dissolved in a suitable solution (chloroform and / or ethanol), and brisaracin was dissolved in the same solvent. The drug-lipid solution was then dried under vacuum at 45°C to 50°C in a rotary evaporator to completely remove the solvent. Once all solvent was removed, a thin film was formed in a round-bottom flask. The round-bottom flask containing the lipid film was held under vacuum for 12 to 24 hours to completely remove any trace solvent present in the thin lipid film. After drying for 12 to 24 hours, the film was hydrated with 66 mL of a 60°C maltodextrin solution (concentration 39.57 mg / mL).

[0070] Example 2. Particle size and zeta potential analysis of brevicornuate liposomes

[0071] The liposomes prepared in Example 1 were observed under an optical microscope at different magnifications to confirm their formation during the hydration process. Throughout the hydration process, optical microscopy was performed at different magnifications (10x, 20x, and 40x) to confirm the formation of spherical liposome vesicles.

[0072] The particle size of liposomes was analyzed using DLS (dynamic light scattering) method, which is used for particle size analysis and drug content determination. The Z-mean (particle size) of the prepared liposomes was measured. Figure 2 Particle size (Z-mean) and particle partition index in liposome dispersions measured by DLS of Brirazacin liposomes are shown.

[0073] The zeta potential is estimated from the experimentally measured electrophoretic mobility of particles. The zeta potential value indicates the stability of the colloidal dispersion.

[0074] ζ potential (mV) value: ● 0 to 5 - Rapid coagulation or flocculation ● 10 to 30 - Initial Instability ● 30 to 40 - Moderate stability ● 40 to 60 - Good stability ● >61-Excellent stability

[0075] Generally speaking, colloidal dispersions with a zeta potential greater than +30mV or less than -30mV exhibit high stability. The higher the zeta potential (both positive and negative), the better the stability.

[0076] Table 2 shows the particle size and zeta potential analysis of liposomes using the DLS method.

[0077] Table 2.

[0078] Example 3. Measurement of drug content by HPLC

[0079] The liposomal drug content was analyzed by HPLC.

[0080] HPLC system

[0081] The system includes a Shimadzu HPLC system (LC-2030C Plus, serial number: L21445711704 AE, made in Japan), an autosampler, a UV detector, and a data acquisition system. Equivalent systems can be used as alternatives.

[0082] HPLC column

[0083] Shimadzu Shim-Pack GIST C18, 5µm, 250×4.6mm column or equivalent.

[0084] Reagent preparation

[0085] Mobile phase A

[0086] ● Dissolve 2.72g KH2PO4 in 1000mL of ultrapure water (0.02M solution).

[0087] ● Adjust the pH to 3.0 using phosphoric acid.

[0088] Mix 90 parts of the above buffer solution with 10 parts of acetonitrile.

[0089] ● Filter through a membrane before use.

[0090] Mobile phase B

[0091] ● Mix 90 parts acetonitrile with 10 parts ultrapure water. Adjust the pH to 3.0 using phosphoric acid.

[0092] Filter through a membrane before use.

[0093] diluent

[0094] ● Prepare a mixture of acetonitrile and phosphate buffer (Section 6.2.1.1). The drug concentration / encapsulation efficiency is (85:15).

[0095] Sample preparation targeting drug content and conversion efficiency

[0096] Drug content

[0097] Place the required amount of liposome sample (complete dispersion) in a volumetric flask, add the required volume of diluent (6.2.3), and mix thoroughly. Incubate the mixture in a water bath sonicator at 60°C for 30 to 45 minutes. Take the required volume of the prepared sample and dilute it to the desired concentration. The assay / drug concentration is 20 μg / mL.

[0098] Incorporation efficiency

[0099] Place the required amount of liposome sample in a centrifuge tube and centrifuge the liposome dispersion at 10,000 rpm for 30 minutes at 20°C. Remove the supernatant and collect the precipitate. Add the required volume of diluent to the liposome precipitate and mix thoroughly. Incubate the mixture in a water bath sonicator at 60°C for 30 to 45 minutes. Take the required volume of the prepared sample and dilute it to a drug concentration of 20 μg / mL.

[0100] standard solutions

[0101] Prepare a 20 μg / mL standard solution using the above diluent.

[0102] analyze

[0103] Use the following parameters to set up the HPLC:

[0104] Column: Shimadzu GIST C18, 5µm, 250×4.6mm column or equivalent

[0105] Flow rate: 1 mL / min

[0106] Injection volume: 20 μL

[0107] Detection: 215nm ultraviolet light

[0108] Column temperature: 30℃

[0109] Running time: 5 to 7 minutes

[0110] HPLC conditions: Mobile phase A, 25%; Mobile phase B, 75%.

[0111] Identification

[0112] Compare the retention times of the standard / pure drug peak and the sample drug peak.

[0113] result

[0114] HPLC chromatogram as shown Figure 3 As shown in the figure. HPLC results indicate that the drug content is 96%, and the efficiency of drug incorporation into liposomes is 73%.

[0115] The final formulation composition was consistent with that of bromerazine (24.53%), lecithin (34.75%), cholesterol (2.97%), maltodextrin (37.74%), and purified water for lipid membrane hydration.

[0116] Example 4. Preparation of liposome gel formulation

[0117] Liposome gels are prepared by incorporating liposome dispersions into gel formulations. First, a plain gel is prepared, then the liposome dispersion is added and thoroughly mixed to obtain a liposome gel or lipid gel. Liposome gel formulations are prepared in varying percentages (0.25% to 1.5% brevicornuate) as needed. Table 3 shows the composition of the lipid gel formulations.

[0118] Table 3.

[0119] The physical appearance and pH of the prepared liposome gel formulations were evaluated. All gel formulations were observed under an optical microscope to obtain intact liposomes within the gel formulations. The liposome gel formulations had a white, creamy gel appearance, with a pH ranging from 5 to 6, and microscopic examination revealed the presence of liposomes. Furthermore, the liposome particles were intact and stable in all gel formulations.

[0120] Example 5. Analysis of lipid gels by HPLC

[0121] The lipid gel sample was placed in a volumetric flask, diluted, and mixed thoroughly. The content of bromelain was analyzed by HPLC according to the same protocol as in Example 3.

[0122] By comparing the retention times of the pure drug peak and the sample peak, the drug content was calculated to be 95.12%.

[0123] The HPLC chromatogram of the lipid gel sample is shown in Figure 4 The first peak is brevicornuate, and the second peak is excipient. The brevicornuate peak is clear and separated from the excipient peak.

[0124] Example 6. In vitro diffusion / permeation study of lipid gels

[0125] Drug diffusion / permeation of the prepared lipogels was analyzed using a Franz diffusion cell. The Franz diffusion cell was filled with pH 7.4 PBS buffer. A surface-treated and pH 7.4 PBS-neutralized regenerated cellulose dialysis membrane (molecular weight cutoff: 12,000 to 14,000) was placed on the recipient compartment, and a weighed amount of lipogel was placed in the donor compartment. The recipient solution was stirred using a magnetic stirrer, and the skin temperature in the diffusion cell was maintained by circulating temperature-controlled water in the outer jacket. Samples were removed through the sample port at different time intervals and replaced with the same volume of plain PBS. The removed samples were mixed with an equal volume of HPLC diluent, and the drug content was analyzed at different time intervals. The percentage of drug diffusion, flux, and permeation coefficient for each lipogel formulation were calculated. Graph Pad Prism version 6.01 software was used to plot the relationship between time and percentage of drug diffusion / release.

[0126] HPLC analysis of drug diffusion samples

[0127] The HPLC analysis method, system, and column were the same as described in Example 3, except that a mixture of acetonitrile and phosphate buffer (60:40) was used for drug diffusion / permeation studies.

[0128] Sample preparation for diffusion / permeation sample analysis

[0129] An equal volume of diluent was mixed with the diffusion / permeation sample fluid taken from the Franz diffusion apparatus. The mixture was thoroughly mixed in a apex mixer and then filtered through a syringe filter.

[0130] result

[0131] The HPLC chromatogram of the lipid gel diffusion sample showed clear and separated Brira saqin peaks.

[0132] Figure 5 The in vitro diffusion study curves of the lipid gel across the membrane are shown. The release curves demonstrate stable and sustained release of bromerazine from the formulation throughout the 8-hour study period.

[0133] Table 4 shows the results of in vitro diffusion studies of the lipid gel formulation.

[0134] Table 4.

[0135] In in vitro diffusion studies, liposome gel formulations containing maltodextrin showed better drug release profiles and higher flux and permeability values ​​than liposomes without maltodextrin. This is likely due to the increased solubility of RP5063 in the liposome gel and the optimal particle size distribution in the formulation.

[0136] Example 7. In vivo preclinical study - psoriasis model

[0137] This experiment used BALB / c mice (n=6 / group) and the animals were housed according to protocols approved by the institution's animal ethics committee. Imiquimod cream (5%) was used as an inducing chemical for the pathology of psoriasis. Psoriasis was induced by applying imiquimod to the back of shaved skin in the morning for 12 consecutive days. The test lipid gel formulation was applied to the animals at night for 12 consecutive days. Imiquimod was applied until the last day of the experiment. The following parameters were observed in all animals: PASI score, Baker score, histological H&E staining, and serum cytokine analysis (TNF-α, KI67, TGF-β). Table 5 shows the animal groups used for the preclinical study.

[0138] Table 5.

[0139] PASI score

[0140] Observe the animals daily for signs of imiquimod-induced psoriasis virus and calculate the PASI score for each group. Figure 6 The composite PASI scores from day 1 to day 12 are illustrated. The induced group (psoriasis group) showed a higher PASI score than the non-induced control group (sham control group) (p=0.001). From day 3 to day 11, the amplitude difference between the two groups became larger. The brevicornu lipogel group increased the PASI score from day 3, peaked on days 7 and 8, and decreased to a plateau level from days 10 to 12. The score amplitude was higher than the sham control group, but not to the same level as the psoriasis group. From day 3 to day 12, the brevicornu lipogel PASI score was consistently lower than the PASI score in the induced psoriasis group (p=0.03). The largest amplitude difference appeared on days 11 and 12.

[0141] Baker rating

[0142] At the end of day 12 of the study period, animals were euthanized, skin was collected, and histological analysis was performed. Signs of psoriatic virulence were examined on the skin of each animal, and a Baker score was calculated for each group. Baker scores showed a significant reduction in the lipid gel treatment group. Figure 7 The study demonstrated that topical brenrazan preparations significantly (P=0.003) treated psoriasis in animals.

[0143] Histology (H&E staining)

[0144] Figure 8Images of skin histology studies at 100x magnification are shown. Table 6 describes the observations of skin histology samples at 100x magnification.

[0145] Table 6.

[0146] Figure 9 Images of skin histology studies at 400x magnification are shown. Table 7 presents the observations of skin histology samples at 400x magnification.

[0147] Table 7.

[0148] In summary, histological evaluation included direct observation at magnifications of 100x and 400x. (Tables 6 and 7, and...) Figure 8 and Figure 9 Histological observation and H&E staining at two magnifications were provided. Differences were observed between the sham control group with induced psoriasis and the Brilazazin lipid gel group, as well as between the latter two groups. Baker score comparison ( Figure 7 This reflects the significant difference between the sham control (p=0.001) and the Brisarazazin lipogel (p=0.003) groups compared to the psoriasis cohort. This observation highlights the therapeutic effect of Brisarazazin lipogel.

[0149] Serum cytokine levels

[0150] At the end of the 12-day study period, animals were euthanized, blood was collected, and serum was separated. Serum cytokines TNF-α, KI-67, and TGF-β were analyzed by ELISA.

[0151] Figure 10 Serum TNF-α levels in animals from different study groups are shown. The results indicate that bromelain reduces serum TNF-α levels in psoriasis-induced animals, and these TNF-α levels are comparable to those in the sham control group.

[0152] Figure 11 Serum KI67 levels in animals from different study groups are shown. The results indicate that bromelain significantly reduced (P=0.001) serum KI67 levels in psoriasis-induced animals, and the serum KI67 levels were comparable to those in the sham control group.

[0153] Figure 12 Serum TGF-β levels in animals from different study groups are shown. The results indicate that the lipid gel formulation significantly reduced (P=0.008) serum TGF-β levels in psoriasis-induced animals compared to the psoriasis control group.

[0154] It should be understood that the foregoing describes preferred embodiments of the invention and that modifications may be made thereto without departing from the scope of the invention as set forth in the claims.

Claims

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

2. The method of claim 1, wherein brenrazan is locally administered to the subject.

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

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

5. The method of claim 4, wherein the liposomes comprise 20% to 30% by weight of brevicornuate.

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

7. The method of claim 4, wherein the gel formulation comprises 0.1% to 2% brevicornuate.

8. The method of claim 1, wherein brenrazan is administered systemically to the subject.

9. The method of claim 1, wherein the method alleviates one or more signs and symptoms selected from the group consisting of: erythema of skin covered with thick, silvery scales; small scaly spots; dry and cracked skin; itching, burning, or pain of said skin; thickened, pitted, or raised nails; and swollen and stiff joints.

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