Application of CK2 inhibitor CX4945 in preparation of medicine for preventing and / or treating psoriasis

Abstract

The invention discloses an application of a CK2 inhibitor CX4945 in preparation of medicines for treating skin inflammations such as psoriasis and the like. The CK2 inhibitor CX4945 ointment provided by the invention can be used for remarkably reducing the pathological injury of the skin of a psoriasis mouse and reducing the neutrophil infiltration of the dermis layer of the skin. The medicine can be prepared into external ointment, is locally applied for use, is convenient to use, has no obvious side effect, is high in patient compliance, and can be prepared into a psoriasis treatment medicine.

Description

Technical Field

[0001] This invention belongs to the pharmaceutical field, specifically relating to the application of a CK2 inhibitor CX4945 in the preparation of drugs for the prevention and / or treatment of psoriasis. Background Technology

[0002] Psoriasis is a chronic, relapsing, inflammatory, systemic disease mediated by the immune system and induced by both genetic and environmental factors. Clinically, it manifests as scaly erythematous plaques or patches, localized or widespread. Psoriasis can be complicated by systemic diseases, severely impacting patients' quality of life. Although the exact cause of psoriasis is not fully understood, genetic, immune, and environmental factors play important roles in its development. Genetics is a major risk factor for psoriasis, and the immune pathways associated with interleukin-23 (IL-23) and Th17 helper cells are the core mechanisms of psoriasis pathogenesis. The main goal of clinical treatment for psoriasis is to achieve complete or near-complete clearance of symptoms and skin lesions. This includes controlling and stabilizing the condition, slowing disease progression, inhibiting the aggravation of skin lesions and itching, preventing recurrence and exacerbation, and minimizing short-term and long-term adverse drug reactions. Treatment for mild psoriasis often focuses on topical medications, commonly including vitamin D3 derivatives, retinoids, medium- or high-potency corticosteroids, and calcineurin inhibitors. Moderate to severe psoriasis requires systemic treatment, including a combination of oral or subcutaneous immunosuppressants, biologics, and targeted therapies. Hormonal drugs, immunosuppressants, biologics, and targeted therapies have significant side effects, are expensive, and cannot be used long-term. Conventional topical medications have limited effectiveness. Continued development of drugs with new mechanisms of action is necessary to supplement existing topical medications and alleviate the psychological and physiological burden on patients.

[0003] Protein kinase CK2 is a serine / threonine kinase. The CK2 holoenzyme is a tetramer composed of two catalytic subunits, CK2α and CK2α', and two regulatory subunits. Composition. The two catalytic subunits are encoded by CSNK2A1 (encoding CK2α) and CSNK2A2 (encoding CK2α'), respectively, and the regulatory subunit... Encoded by CSNK2B, the regulatory subunit confers enzyme stability while influencing the substrate specificity of the catalytic subunit. CK2 can regulate various cellular processes, such as transcription and translation. Abnormal CK2 expression and activity are observed in many tumors and are associated with tumor progression and poor prognosis. Although most of our current understanding of CK2 comes from the field of oncology, increasing evidence suggests that CK2 plays a crucial role in immune cell activation, proliferation, and differentiation. CX4945 is the first and only orally administered bioavailable CK2 small molecule inhibitor to enter human clinical trials (structural formula see [link]). Figure 1It has few side effects. In January 2022, the structurally optimized CK2 inhibitor CX-4945 (trade name silverasertib) was designated as an orphan drug by the FDA for the treatment of cholangiocarcinoma. Compared with normal cells, various tumor cells highly express CK2 and become addicted to CK2 in a "non-oncogene addiction" manner, making CK2 a potential anti-tumor target.

[0004] Currently, there are no reports on the use of the CK2 inhibitor CX4945 in the prevention and / or treatment of psoriasis. Summary of the Invention

[0005] The purpose of this invention is to provide a pharmaceutical use for the CK2 inhibitor CX4945.

[0006] The pharmaceutical use of the CK2 inhibitor CX4945 provided by this invention is its application in the preparation of drugs for the prevention and / or treatment of psoriasis.

[0007] Furthermore, the prevention and / or treatment of psoriasis embodies at least one of the following aspects:

[0008] 1) Reduces skin lesions caused by psoriasis;

[0009] 2) Reduce pathological damage to the skin;

[0010] 3) Reduce neutrophil infiltration in the skin;

[0011] 4) Reduce skin thickness;

[0012] 5) Lower the PASI (Psoriasis Area and Severity Index) score.

[0013] The CAS No. of the CX4945 is 1009820-21-6, and its structural formula is as follows: Figure 1 As shown.

[0014] Furthermore, the skin lesions include erythema, papules, patches, plaques, scaling, lichenification, erosion, ulceration, and maceration.

[0015] Furthermore, the drug is a topical medication.

[0016] Furthermore, the topical medication may include creams, lotions, liniments, gels, sprays, etc.

[0017] Taking a cream as an example, the cream may consist of a drug and a cream base.

[0018] According to an embodiment of the present invention, the mass ratio of the drug to the cream base (such as petrolatum) may be 25 mg: 20 g.

[0019] All of the above dosage forms can be prepared using conventional processes in the field of pharmaceutical formulation.

[0020] This invention also provides the use of the CK2 inhibitor CX4945 in the preparation of medicaments for the prevention and / or treatment of diseases related to skin inflammation.

[0021] The skin inflammation-related diseases mentioned include psoriasis, pyoderma gangrenosa, Sweet syndrome, Stevens-Johnson syndrome, toxic epidermal necrolysis, Behçet's disease, hidradenitis suppurativa, neutrophilic urticarial dermatitis, allergic cutaneous vasculitis, pyogenic granuloma, etc.

[0022] The present invention also provides the use of the CK2 inhibitor CX4945 in the preparation of a drug for inhibiting neutrophilic inflammation.

[0023] Furthermore, the inhibition of neutrophilic inflammation is manifested in at least one of the following aspects:

[0024] 1) Reduces the phosphorylation activity of protein kinase CK2 in neutrophils, thereby reducing ROS production levels;

[0025] 2) Reduce the formation of NETs (neutrophil extracellular traps);

[0026] 3) It significantly inhibited the pro-inflammatory effect of NETs on macrophages.

[0027] The present invention also provides a pharmaceutical composition for the prevention and / or treatment of psoriasis.

[0028] The pharmaceutical composition comprises CX4945 as its active ingredient.

[0029] Furthermore, the pharmaceutical composition has at least one of the following effects:

[0030] 1) Reduces skin lesions caused by psoriasis;

[0031] 2) Reduce pathological damage to the skin;

[0032] 3) Reduce neutrophil infiltration in the skin;

[0033] 4) Reduce skin thickness;

[0034] 5) Lower the PASI (Psoriasis Area and Severity Index) score.

[0035] Furthermore, a pharmaceutically acceptable carrier material may be added to the pharmaceutical composition.

[0036] In some embodiments of the present invention, the pharmaceutically acceptable carrier is a cream base.

[0037] The cream base may include white petrolatum, glyceryl monostearate, chitosan, etc.

[0038] The present invention also provides methods for treating and / or preventing diseases or conditions in a subject.

[0039] The method includes administering CX4945 or a pharmaceutical composition containing CX4945 to a subject in need;

[0040] The disease in question is psoriasis;

[0041] The symptoms described are those caused by psoriasis, including skin lesions, neutrophil infiltration, and a significant increase in skin thickness.

[0042] This invention demonstrates the therapeutic effect of CX4945 on psoriatic lesions through the following aspects:

[0043] 1) Imiquimod (IMQ)-induced psoriasis model: An animal model with a significant psoriasis phenotype was established by applying IMQ cream to the dorsal skin and ears of 8- to 10-week-old BALB / c mice. Treatment with CX4945 ointment, applied once daily for 4 days starting on day 4, showed that CX4945 significantly reduced pathological skin lesions. Figure 2 ), neutrophil infiltration was significantly reduced ( Figure 3 ), skin thickness was significantly reduced ( Figure 4 The pathological score PASI score was significantly reduced. Figure 5 ).

[0044] 2) In vitro cell experiments demonstrated that CX4945 can effectively reduce CK2 phosphorylation activity in neutrophils. Figure 6 ), reduce ROS generation level ( Figure 7 Reduce NETs generation ( Figure 8 ), significantly inhibited the pro-inflammatory effect of NETs on macrophages ( Figure 9 ).

[0045] This invention has found that the CK2 inhibitor CX4945 can effectively inhibit neutrophil NET production and significantly reduce the pro-inflammatory effect of NETs on macrophages. Neutrophil infiltration plays an important role in the pathological progression of psoriasis. Therefore, the application of CK2 inhibitors that inhibit neutrophilic inflammation may alleviate skin lesions in psoriasis. Animal experiments of this invention have confirmed this: the applicant found that topical application of CX4945 ointment can significantly alleviate skin lesions in psoriatic mice. Figure 2 ), reducing inflammatory infiltration of neutrophils ( Figure 3 ).

[0046] CX4945 is the only CK2 inhibitor to have entered clinical trials. It boasts a broad safety profile, minimal side effects, and its topical application significantly reduces side effects on other tissues and organs, making it a potential novel treatment for psoriasis. Furthermore, in previous animal studies of CX4945 treatment for tumors, the dosage was typically 25 mg / kg or 75 mg / kg, with good tolerability. The applicant's formulation of CX4945 into a topical ointment for treating psoriasis represents a dosage far lower than that used in cancer patients, resulting in even fewer side effects. Attached Figure Description

[0047] Figure 1 This invention relates to the structural formula of the CK2-specific inhibitor CX4945;

[0048] Figure 2 CX4945 treatment significantly reduced the degree of skin pathological damage in psoriatic mice.

[0049] Figure 3 CX4945 treatment significantly reduced neutrophil infiltration in the skin of psoriatic mice;

[0050] Figure 4 Treatment with CX4945 reduced ear (skin) thickening in a mouse model of imiquimod-induced psoriasis.

[0051] Figure 5 CX4945 treatment significantly reduced the PASI score of the dermatopathology score in an imiquimod-induced psoriasis mouse model.

[0052] Figure 6 CX4945 can significantly reduce the phosphorylation activity of CK2 in neutrophils in vitro;

[0053] Figure 7 CX4945 significantly reduced ROS levels in neutrophils from SLE patients and mouse bone marrow neutrophils in vitro.

[0054] Figure 8 CX4945 significantly reduced NET production in neutrophils of SLE patients in vitro;

[0055] Figure 9 CX4945 significantly reduced the pro-inflammatory effect of NETs generated by neutrophils on macrophages in vitro; A) NETs generated by mouse bone marrow neutrophils pretreated with CX4945 significantly reduced the pro-inflammatory effect on mouse bone marrow-derived macrophages (BMDM); B) NETs generated by neutrophils pretreated with CX4945 significantly reduced the pro-inflammatory effect on peripheral monocyte-derived macrophages (MoDM). Detailed Implementation

[0056] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0057] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0058] Example 1: Efficacy Test of CX4945

[0059] I. Experimental Methods

[0060] 1. Preparation method of CX4945 ointment (single dose calculated as 20g)

[0061] 1) CX4945 was purchased from MedChemExpress (MCE) under the trade name Simitasertib (HY-50855). 50 mg of powder was dissolved in 1 mL of dimethyl sulfoxide (DMSO, Solarbio) solution and thoroughly mixed in a non-contact ultrasonic sample processor to ensure complete dissolution and dispersion of the powder.

[0062] 2) Place 20g of medical petroleum jelly (Haishi Hainuojin Shulike white petroleum jelly) in a sterile round-bottom container, add 500µL of CX4945 solution (50mg / ml), and stir thoroughly with a sterile plastic rod until no liquid is separated.

[0063] 3) The ointment should be stored in a sealed container at room temperature.

[0064] 2. Imiquimod (IMQ)-induced psoriasis model

[0065] 1) Imiquimod (IMQ)-induced psoriasis model: 0.5% imiquimod cream (Idalor) was applied to the epidermis of one ear and a 3*3cm area on the back of 8 to 10-week-old wild-type C57BL / 6N female mice once a day for 5 days.

[0066] 2) On the fourth day of imiquimod application, the mice were divided into two groups (n=5 per group): the CX4945 group and the control group (Vehicle). The CX4945 group was treated with CX4945 ointment applied to the affected area once a day for 5 days. The control group was treated with petroleum jelly. Both groups of mice were euthanized at the end of the 5th day.

[0067] 3) Skin tissue samples: Mice were anesthetized with carbon dioxide and euthanized. The thickness of the skin was measured at the same location on both ears (one ear treated, the other untreated). Skin samples from the ears and back were carefully cut. The skin tissue was frozen in tissue. In an OCT compound, the sections were frozen at -80°C until they were 10 micrometers thick for HE staining.

[0068] 3. In vitro cell experiments

[0069] 1) Peripheral EDTA-anticoagulated blood samples were collected from healthy volunteers. Peripheral neutrophils were purified using Ficoll-pague combined with Dextron sedimentation. Lysis was performed on ice for 30 minutes using RIPA high-efficiency protein lysis buffer (Solarbio, with added protease and phosphatase inhibitors). After centrifugation at 10,000 rpm for 15 minutes, the supernatant was collected as the neutrophil protein sample. Protein quantification was performed using a BCA kit (Thermo). Before protein electrophoresis, 5* protein loading buffer (Solarbio) was added, and the sample was boiled in a 100°C metal bath for 15 minutes.

[0070] 2) Load the sample at a total protein content of 15 micrograms into a 12% precast gel (ACE) and electrophoresis at 150V for 40 minutes; transfer the membrane using the Bio-rad rapid transfer system and a pre-activated PVDF membrane, following the manufacturer's instructions; block the successfully transferred PVDF membrane in 5% skim milk (BD) for 1 hour; incubate overnight at 4°C with primary antibody (antibody sourced from Abcam and Proteintech); wash three times with TBST buffer, then incubate at room temperature with secondary antibody (CST) for 1 hour; wash three times with TBST buffer, then add developing substrate (Millipore) for development.

[0071] 3) ROS generation experiment: Neutrophils from volunteers were collected according to the method in 1). The cells were resuspended with HBSS and pretreated with CX4945 (5μM) for 30 minutes in a cell incubator at 37°C. The probe was labeled according to the instructions of DHR123 (Invitrogen). Fluorescence values ​​were recorded under PMA or LPS conditions every 10 minutes, and the ROS generation curve was plotted.

[0072] 4) NETs generation experiment: Neutrophils were collected from volunteers according to method 1), and cultured in serum-free RPMI 1640 medium at a ratio of 1*10⁻⁶ cells / mL. 6Resuspend cells at a density of / ml; add poly-L-L pretreated cell slides to 24-well cell culture plates (Corning); carefully drop 50 μL of cell suspension into the middle of each cell slide, and incubate at 37°C for 15 minutes to allow cell sedimentation; carefully aspirate and discard the supernatant, add 50 μL of CX4945 (5 μM) or serum-free RPMI 1640 medium, and pretreat at 37°C for 30 minutes; carefully aspirate and discard the supernatant, add 50 μL of PMA (50 nM) and LPS (1 Incubate with 0.00 ng / ml or A23187 (4 μM) at 37°C for 4 hours to stimulate NET generation; add 500 μL of 4% tissue fixative (Solarbio) and fix at room temperature for 20 minutes, then wash three times with PBS buffer, block with 0.2% Gelatin (Solarbio) at room temperature for 1 hour, then stain with primary antibody (anti-NE antibody, Millipore, 1:400) at room temperature for 90 minutes; wash three times with PBS buffer, then stain with secondary antibody (Donkey anti-Rabbit IgG (H+L) Alexa Fluor) TM Stain with Hoechst 33342 (Invitrogen, 1:500) at room temperature for 60 minutes; wash three times with PBS buffer and then stain with Hoechst 33342 (Invitrogen, 1:1000) at room temperature in the dark for 5 minutes; wash three times with PBS buffer and then mount with Prolong (Invitrogen). After the slides are dry, they are photographed and counted under a fluorescence microscope.

[0073] 5) Method for obtaining animal bone marrow neutrophils: When mice were euthanized at the planned experimental time point, femurs were taken. After gently rubbing away the attached muscle and connective tissue on the surface of the femur with gauze, the joint ridges at both ends were cut off with scissors. The bone marrow tissue was gently blown out with a 22G syringe needle and aspirated back and forth to form a single-cell suspension. After filtration, neutrophils were isolated using a mouse neutrophil separation magnetic bead kit (Mitenia) and ROS level generation experiments were performed.

[0074] 6) Culture method of animal bone marrow-derived macrophages (BMDM): Prepare bone marrow single-cell suspension according to the method in 5), resuspend the cells in IMDM medium (supplemented with 10% fetal bovine serum (Gibco), 1% penicillin-streptomycin complex (Gibco) and 50 ng / ml mouse M-CSF (Biolegend)), and culture at 2*10 6 The cells were seeded at a density of 1 / ml onto 12-well cell culture plates (Corning) and incubated at 37°C for 5 days. The culture medium was changed on the third day. On the fifth day, the cell status was observed under a microscope. When the cells reached 90% confluence, stimulation tests could be performed.

[0075] 7) Culture method of human peripheral monocyte-derived macrophages (MoDM): EDTA-anticoagulated peripheral blood was used to separate the upper layer of PBMCs by Ficoll density gradient centrifugation. The cells were resuspended in RPMI medium (supplemented with 10% fetal bovine serum (Gibco), 1% penicillin-streptomycin complex (Gibco), and 50 ng / ml human M-CSF (Biolegend)). Cells were cultured at a rate of 2*10-1. 6 The cells were seeded at a density of 1 / ml onto 12-well cell culture plates (Corning) and incubated at 37°C for 5 days. The culture medium was changed on the third day. On the fifth day, the cell status was observed under a microscope. When the cells reached 90% confluence, stimulation tests could be performed.

[0076] 8) Detection of the pro-inflammatory capacity of NETs: Following the experimental method for generating NETs in 4), 1*10 6 Cells were seeded into 48-well cell culture plates (Corning). Following the method described in section 4), CX4945 pretreatment and NET generation were performed. After 4 hours, the supernatant was carefully discarded, and 100 μL of pre-warmed MNase (Invitrogen, 30 U / ml) was added. The plates were incubated at 37°C for 30 minutes. The supernatant was collected, centrifuged at 5000 rpm for 5 minutes, and the collected supernatant was the NET sample. Protein quantification of CX4945 pretreated and untreated NET samples was performed using a BCA kit (Thermo). Mouse BMDM and human MoDM were stimulated at a protein concentration of 1 μg / ml. After incubation at 37°C for 4 hours, cells were collected, lysed with TRIzol, and RNA was extracted (ZYMO Tissue Cell RNA Extraction Kit). The RNA was reverse transcribed into cDNA (TAKARA Reverse Transcription Kit) and then subjected to qRRCR (PowerTrack SYBR Green). KIT (Invitrogen), using GAPDH (human) / Gapdh (mouse) as internal controls, calculated the relative expression levels of inflammatory cytokines using the ΔΔCT method.

[0077] II. Results

[0078] 1) An imiquimod (IMQ)-induced psoriasis model trial found that CX4945 can significantly reduce pathological skin damage. Figure 2 ), neutrophil infiltration was significantly reduced ( Figure 3 ), skin thickness was significantly reduced ( Figure 4 The pathological score PASI score was significantly reduced. Figure 5 ).

[0079] 2) In vitro cell experiments demonstrated that CX4945 can effectively reduce CK2 phosphorylation activity in neutrophils. Figure 6 ), reduce ROS generation level ( Figure 7 Reduce NETs generation ( Figure 8 ), significantly inhibited the pro-inflammatory effect of NETs on macrophages ( Figure 9 ).

[0080] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. The pharmaceutical use of CX4945 is its application in the preparation of drugs for the prevention and / or treatment of psoriasis; The CAS No. of the CX4945 is 1009820-21-6.

2. The application according to claim 1, characterized in that: The prevention and / or treatment of psoriasis embodies at least one of the following aspects: 1) Reduces skin lesions caused by psoriasis; 2) Reduce pathological damage to the skin; 3) Reduce neutrophil infiltration in the skin; 4) Reduce skin thickness; 5) Lower the PASI score.

3. The application according to claim 1 or 2, characterized in that: The drug is a topical drug; further, the topical drug includes creams, lotions, liniments, gels, and sprays.

4. The use of CX4945 in the preparation of medicaments for the prevention and / or treatment of diseases related to skin inflammation; Furthermore, the skin inflammation-related diseases include psoriasis, pyoderma gangrenosa, Sweet syndrome, Stevens-Johnson syndrome, toxic epidermal necrolysis, Behçet's disease, hidradenitis suppurativa, neutrophilic urticarial dermatitis, allergic cutaneous vasculitis, and pyogenic granuloma.

5. Application of CX4945 in the preparation of drugs that inhibit neutrophilic inflammation.

6. The application according to claim 5, characterized in that: The inhibition of neutrophilic inflammation is manifested in at least one of the following aspects: 1) Reduces the phosphorylation activity of protein kinase CK2 in neutrophils, thereby reducing ROS production levels; 2) Reduce the formation of NETs (neutrophil extracellular traps); 3) It significantly inhibited the pro-inflammatory effect of NETs on macrophages.

7. A pharmaceutical composition for the prevention and / or treatment of psoriasis, wherein the active ingredient comprises CX4945.

8. The composition according to claim 7, characterized in that: The pharmaceutical composition also contains a pharmaceutically acceptable carrier material.

9. A method of treating and / or preventing a disease or condition in a subject, comprising administering CX4945 or a pharmaceutical composition containing CX4945 to a subject in need; The disease in question is psoriasis; The condition described is caused by psoriasis.

10. The method according to claim 9, characterized in that: The symptoms caused by psoriasis include skin lesions, neutrophil infiltration, and a significant increase in skin thickness.

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