Application of pantethine in treatment of diseases related to excessive activation of dendritic cells

Abstract

The invention discloses an application of pantethine in treatment of diseases related to excessive activation of dendritic cells. The invention discloses an effect of pantethine in regulating activation of dendritic cells and treating psoriasis, provides a new application of pantethine, expands the application range of pantethine, and has important economic benefits and social benefits.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to the application of pantethine in treating diseases related to overactivation of dendritic cells. Background Art

[0002] Dendritic cells (DCs), named for their dendrite-like cell shape, are responsible for initiating adaptive immune responses and therefore act as "sentinels" of the immune system. DCs are bone marrow (BM)-derived leukocytes that are professional and the most efficient antigen-presenting cell type responsible for initiating all antigen-specific immune responses. Therefore, they are master regulators of immune responses by linking the microbial sensing features of the innate immune system with the exquisite specificity of the adaptive response.

[0003] Overactivation of dendritic cell function has been confirmed to be closely related to a variety of infectious diseases, transplant rejection, allergic diseases, and autoimmune diseases, and has increasingly become a target for intervention or treatment of related diseases. Psoriasis, commonly known as psoriasis, is an immune-mediated chronic, recurrent, inflammatory, and systemic disease induced by the combined effects of genetics and the environment. The disease covers all populations, but mainly affects young and middle-aged people, and can greatly affect the patient's physical and mental state. At present, clinical observations of immune abnormalities in psoriasis include enhanced antigen presentation ability of dendritic cells, overactivation of the NF-κB signaling pathway, DCs promoting the expansion of specific TH cell populations (especially TH17 cells, which are the main source of IL-17) and enhanced IL-17 cytokine secretion by producing cytokines such as IL-12 and IL-23, thereby promoting the host's immune response and the infiltration of immune cells. Therefore, overactivation of dendritic cells and T cells plays an important role in the progression of psoriasis.

[0004] So far, great progress has been made in the treatment of diseases related to overactivation of dendritic cells, such as psoriasis, but there are still certain major challenges in the clinic, such as different responses of different patient groups to treatment, side effects of various treatments, and the acceptability of drug prices for different patients. Therefore, finding a safe and effective treatment drug has become an urgent problem to be solved. Summary of the invention

[0005] To make up for the deficiencies of the prior art, the present invention provides the use of pantethine in treating diseases related to overactivation of dendritic cells.

[0006] To achieve the above object, the present invention adopts the following technical solution:

[0007] The first aspect of the present invention provides the use of pantethine or a pharmaceutically acceptable salt thereof in the preparation of a pharmaceutical composition for treating diseases related to excessive activation of dendritic cells.

[0008] Furthermore, the diseases associated with overactivation of dendritic cells include infectious diseases, transplant rejection, allergic diseases, and autoimmune diseases.

[0009] Furthermore, the disease associated with overactivation of dendritic cells is selected from autoimmune diseases.

[0010] Furthermore, the autoimmune disease is selected from psoriasis.

[0011] Furthermore, the treatment of diseases related to overactivation of dendritic cells includes at least one of the following:

[0012] (1) Inhibit the ability of dendritic cells to process and present antigens;

[0013] (2) inhibiting dendritic cell activation;

[0014] (3) Inhibit the ability of dendritic cells to activate T cells.

[0015] Furthermore, the antigen processing and presentation ability of dendritic cells mentioned in (1) is the ability of dendritic cells to present EαEGFP.

[0016] Furthermore, the dendritic cell activation described in (2) is LPS-induced dendritic cell activation.

[0017] Furthermore, the activated co-stimulatory factors include one or more of CD80, CD86, and MHC.

[0018] Furthermore, the MHC includes MHC I and / or MHC II.

[0019] Furthermore, the ability of dendritic cells to activate T cells described in (3) is the proliferation ability of T cells.

[0020] Furthermore, the cytokines related to the proliferation ability of the T cells include IFNγ and / or IL.

[0021] Furthermore, the IL includes IL-17A and / or IL-4.

[0022] A second aspect of the present invention provides a pharmaceutical composition for treating diseases related to excessive activation of dendritic cells, the pharmaceutical composition comprising pantethine or a pharmaceutically acceptable salt thereof.

[0023] Furthermore, the pharmaceutical composition also includes a pharmaceutically acceptable carrier.

[0024] Furthermore, the pharmaceutical composition also includes other active drugs.

[0025] Furthermore, the other active drugs include immunosuppressants, vitamins and their analogs, vitamin A related compounds, corticosteroids, and biological products.

[0026] Furthermore, the vitamins include vitamin A, vitamin B, vitamin C, vitamin D, and vitamin K.

[0027] Furthermore, the dosage form of the pharmaceutical composition includes solid preparations, semisolid preparations or liquid preparations.

[0028] Furthermore, the solid preparation includes tablets, lozenges, capsules, pills or granules.

[0029] Furthermore, the semisolid preparation includes cream, gel, ointment or emulsion.

[0030] Furthermore, the liquid preparation includes a solution or a suspension.

[0031] The third aspect of the present invention provides a method for regulating any of the following substances, the method comprising administering pantethine or a pharmaceutically acceptable salt thereof, wherein the substance comprises any of the following:

[0032] (1) Co-stimulatory factors associated with dendritic cells;

[0033] (2) cytokines associated with T cell proliferation;

[0034] (3) Skin inflammatory factors.

[0035] Furthermore, the co-stimulatory factors described in (1) include one or more of CD80, CD86, and MHC.

[0036] Furthermore, the MHC includes MHC I and / or MHC II.

[0037] Furthermore, the cytokines associated with T cell proliferation described in (2) include IFNγ and / or IL.

[0038] Furthermore, the IL includes IL-17A and / or IL-4.

[0039] Furthermore, the skin inflammatory factors described in (3) include one or more of IL, IFNγ, and TNF-α.

[0040] Furthermore, the IL includes one or more of IL-17, IL-23, and IL-22.

[0041] Furthermore, the IL-17 includes IL-17A and / or IL-17F.

[0042] Furthermore, the method is a non-therapeutic method.

[0043] Advantages and beneficial effects of the present invention:

[0044] The invention discloses the role of pantethine in regulating dendritic cell activation and treating psoriasis, provides a new use of pantethine, expands the application range of pantethine, and has important economic and social benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 1A is a flow cytometry graph, and 1B is a statistical graph of flow cytometry results;

[0046] Figure 2 2A is a graph showing the effect of pantethine on the production of DC maturation and activation costimulatory factors by LPS stimulation in BMDC cells, wherein 2A is a CD80 MFI graph, 2B is a CD86 MFI graph, 2C is a MHC I MFI graph, and 2D is a MHC II MFI graph;

[0047] Figure 3 3A is a flow cytometric graph showing the effect of pantethine on T cell proliferation and cytokine secretion after co-culture of BMDC and OT II T cells, 3B is a CFSE cell statistical graph, 3C is an IFNγ concentration statistical graph, 3D is an IL-4 concentration statistical graph, and 3E is an IL-17A concentration statistical graph;

[0048] Figure 4 4A is a graph showing the effect of pantethine on the IMQ-induced psoriasis mouse model, wherein 4A is a graph showing the mouse model, 4B is a line graph showing the PASI score, 4C is a line graph showing the back skin thickness, 4D is a graph showing the relative expression level of IFNγ, 4E is a graph showing the relative expression level of TNF-α, 4F is a graph showing the relative expression level of IL-23, 4G is a graph showing the relative expression level of IL-17A, 4H is a graph showing the relative expression level of IL-17F, and 4I is a graph showing the relative expression level of IL-22.

[0049] In the figures, each value is mean ± sd.; ns, no significant difference; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001, and the t-test statistical method was used. DETAILED DESCRIPTION

[0050] The definitions of some terms used in this specification are provided below. Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0051] The present invention provides a pharmaceutical composition for treating diseases related to overactivation of dendritic cells, wherein the pharmaceutical composition comprises pantethine or a pharmaceutically acceptable salt thereof.

[0052] In one embodiment of the present invention, the pharmaceutically acceptable salt is a salt of the compound pantethine of the present invention, which is suitable for contact with patient tissue within the scope of reliable medical judgment, and will not produce inappropriate toxicity, irritation, allergic reaction, etc. The main body of pharmaceutically acceptable salts is well known in the art. If a pharmaceutically acceptable salt of pantethine is used in these compositions, those salts are preferably derived from inorganic or organic acids and bases. In such acid salts, the following are included but are not limited to: acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, lucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonic acid Salts, nicotinates, oxalates, pamoates, pectinates, persulfates, 3-phenyl-propionates, picrates, pivalates, propionates, succinates, tartrates, thiocyanates, toluenesulfonates, undecanoates, hydrohalides (e.g., hydrochlorides and hydrobromides), sulfates, phosphates, nitrates, sulfamates, malonates, salicylates, methylene-bis-b-hydroxynaphthoates, gentisates, isethionates, di-p-toluoyl tartrates, ethanesulfonates, cyclohexylsulfamates, quinates, and the like. Pharmaceutically acceptable base addition salts include, but are not limited to, those derived from alkali metal or alkaline earth metal bases or conventional organic bases, such as triethylamine, pyridine, piperidine, morpholine, N-methylmorpholine, ammonium salts, alkali metal salts (such as sodium and potassium salts), alkaline earth metal salts (such as calcium and magnesium salts), salts with organic bases (such as dicyclohexylamine salts, N-methyl-D-glucamine), and salts with amino acids such as arginine, lysine.

[0053] In one embodiment of the present invention, diseases associated with excessive activation of dendritic cells include, but are not limited to, infectious diseases, transplant rejection, allergic diseases, and autoimmune diseases.

[0054] Infectious diseases include infectious diseases caused by non-specific pathogenic microorganisms (such as sepsis, severe pneumonia), bacterial infections, and parasitic infections. Bacterial infections include but are not limited to mycobacterial infections and salmonella infections; parasitic infections include but are not limited to malarial infections and filarial infections.

[0055] Allergic diseases include but are not limited to allergic asthma and Henoch-Schonlein purpura.

[0056] Autoimmune diseases include, but are not limited to, psoriasis, systemic lupus erythematosus, multiple sclerosis, and rheumatoid arthritis.

[0057] In a preferred embodiment of the present invention, the disease associated with excessive activation of dendritic cells is selected from autoimmune diseases.

[0058] In a particular embodiment of the present invention, the autoimmune disease is selected from psoriasis.

[0059] The pharmaceutical composition also includes a pharmaceutically acceptable carrier.

[0060] In one embodiment of the present invention, a pharmaceutically acceptable carrier includes a pharmaceutically acceptable substance, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, which is involved in carrying or transporting the test chemical from one organ or part of the body to another organ or part of the body. Various carriers are "acceptable" in the sense that they are compatible with the other components of the formulation and will not cause harm to the patient. Some examples of substances that can be used as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository wax; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn starch, etc. Rice oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerol, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethanol; (20) phosphate buffer; and (21) other nontoxic compatible substances used in pharmaceutical preparations.

[0061] Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the pharmaceutical compositions.

[0062] Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyphenol (BHA), butylated hydroxyphenol (BHT), lecithin, propyl gallate, α-tocopherol, etc.; and (3) metal chelators, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0063] The pharmaceutical compositions of the present invention include those suitable for oral administration, nasal administration, topical administration (including oral and sublingual administration), rectal administration, vaginal administration, aerosol administration and / or parenteral administration. The composition can be conveniently present in unit dosage form and can be prepared by any method known in the pharmaceutical field. Depending on the subject to be treated, the specific mode of administration, the amount of active ingredient that can be combined with a carrier material to produce a single dosage form can be different. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form is usually the amount of the compound that produces a therapeutic effect. Usually, in addition to 100%, the amount ranges from about 1% to about 99% of the active ingredient, from about 5% to about 70%, or from about 10% to about 30%.

[0064] The dosage form of the pharmaceutical composition includes solid preparation, semisolid preparation or liquid preparation.

[0065] In a solid dosage form (tablet, lozenge, capsule, pill or granule) of the pharmaceutical composition of the present invention for oral administration, the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and / or any of the following: (1) fillers or expanders, such as starch, lactose, sucrose, glucose, mannitol and / or silicic acid; (2) binders, such as carboxymethylcellulose, alginate, gelatin, polyvinyl pyrrolidone, sucrose and / or gum arabic; (3) Humectants such as glycerol; (4) disintegrants such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate; (5) solution retardants such as paraffin; (6) absorption promoters such as quaternary ammonium compounds; (7) wetting agents such as acetyl alcohol and glyceryl monostearate; (8) absorbents such as kaolin and bentonite; (9) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets and pills, the pharmaceutical composition may also contain a buffer. Solid compositions of a similar type may also be used as fillers in soft and hard-filled gelatin capsules using excipients such as lactose or milk sugars and high molecular weight polyethylene glycols.

[0066] Tablets can be prepared by compression or molding with one or more auxiliary ingredients. Compressed tablets can be prepared using a binder (e.g., gelatin or hydroxypropylmethylcellulose), a lubricant, an inert diluent, a preservative, a disintegrant (e.g., sodium starch glycolate or cross-linked sodium carboxymethylcellulose), a surfactant or a dispersant. Molded tablets can be prepared by molding a mixture of powdered active ingredients moistened with an inert liquid diluent in a suitable machine.

[0067] Tablets and other solid dosage forms of the pharmaceutical composition of the present invention, such as lozenges, capsules, pills or granules, can optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings known in the field of pharmacy. Hydroxypropylmethylcellulose, other polymer matrices, liposomes and / or microspheres in different proportions, for example, providing the desired release curve (profile), can also be used to prepare so as to provide a sustained release or controlled release of the active ingredient therein. They can be sterilized, for example, by filtering through a bacteria-retaining filter, or by introducing a sterilizing agent in the form of a sterile solid composition that can be dissolved in sterile water, or by introducing some other sterile injectable medium immediately before use. These compositions can also optionally contain an opacifier, and optionally in a delayed manner, can be a composition that releases the active ingredient only or preferentially in a certain part of the gastrointestinal tract. Examples of implantable compositions that can be used include polymeric substances and waxes. The active ingredient can also be in microcapsule form, if appropriate, in microcapsule form with one or more of the above-mentioned excipients.

[0068] The liquid dosage form (solution or suspension) for oral administration of the pharmaceutical composition of the present invention includes pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage form can also contain inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oil (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofuran methanol, polyethylene glycol and fatty acid esters of sorbitan, and mixtures thereof.

[0069] Besides inert diluents, the oral compositions can include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.

[0070] The pharmaceutical composition also includes other active drugs.

[0071] In one embodiment of the present invention, the other active drugs, such as other drugs for treating psoriasis, include but are not limited to immunosuppressants, vitamin D and its analogs, vitamin A related compounds, corticosteroids, and biological products.

[0072] Among them, immunosuppressants include but are not limited to methotrexate, azathioprine, cyclosporine, fumaric acid, tacrolimus or pimecrolimus and corticosteroids.

[0073] Vitamin D analogs include, but are not limited to, calcitriol, calcipotriol, and tacalcitol.

[0074] Vitamin A related compounds include, but are not limited to, retinoids, tretinoine, isotretinoin, etretinate, acitretine, tazarotene, bexarotene, and adapalene.

[0075] Biological products include but are not limited to alecept, etanercept, and the monoclonal antibodies adalimumab, infliximab, and ustekinumab.

[0076] It is obvious to those skilled in the art that the pharmaceutical composition of the present invention will vary according to the desired effect. Therefore, the optimal content of the pharmaceutical composition of the present invention can be easily determined by those skilled in the art, and can be adjusted according to various factors including the following: the type and severity of the disease, the content of other components contained in the pharmaceutical composition, the type of preparation, the age, weight, general health, sex and diet of the patient, the time of application, the route of administration, the secretion rate of the drug, the duration of treatment and the drug used simultaneously. The amount of the pharmaceutical composition should be determined considering the factors, and it can be applied once a day or in several portions. However, it should be understood that the actual dosage of the active ingredient should be determined considering various relevant factors, including the disease treated, the severity of the disease, the route of application and the weight, age and sex of the patient. Therefore, dosage does not limit the scope of the present invention in any way.

[0077] In one embodiment of the invention, treatment refers to clinical intervention that attempts to change the natural course of the individual being treated, and can be performed for prevention or during clinical pathology. Desirable therapeutic effects include, but are not limited to, preventing disease occurrence or recurrence, alleviating symptoms, attenuating any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and alleviating or improving prognosis.

[0078] In one embodiment of the invention, administration refers to a method of administering a dose / effective amount of a drug or compound or pharmaceutical composition to a subject (e.g., a patient). Administration can be by any suitable means, including parenteral, intrapulmonary and intranasal, and if desired for local treatment, intralesional administration. Parenteral infusion includes, for example, intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Administration can be by any suitable route, for example, by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is short-lived or long-term. Various dosing schedules are contemplated in the present application, including but not limited to single administration or multiple administrations at multiple time points, push administration and pulse infusion.

[0079] The subject refers to any animal, including humans and non-human animals. Non-human animals include all vertebrates, for example, mammals, such as non-human primates (especially higher primates), sheep, dogs, rodents (such as mice or rats), guinea pigs, goats, pigs, cats, rabbits, cattle, and any livestock or pets; and non-mammals, such as chickens, amphibians, reptiles, etc.

[0080] An effective amount means an amount that, when properly administered to a patient, is sufficient to (a) detectably reduce the severity of the condition or disease state being treated; (b) improve or alleviate the symptoms of the patient's disease or condition; or (c) slow or prevent the development of the condition or disease state being treated, or otherwise stabilize or prolong the stable state of the condition or disease state being treated. It should also be understood that the specific dosage and treatment regimen for any particular patient will depend on a variety of factors, including the activity of the specific compound used; the patient's age, weight, general health, sex and diet; administration time; excretion rate; drug combination; the judgment of the treating physician; and the severity of the specific disease being treated.

[0081] The present invention provides a method for regulating any of the following substances, the method comprising administering pantethine or a pharmaceutically acceptable salt thereof, wherein the substance comprises any of the following:

[0082] (1) Co-stimulatory factors associated with dendritic cells;

[0083] (2) cytokines associated with T cell proliferation;

[0084] (3) Skin inflammatory factors.

[0085] In one embodiment of the present invention, regulation or control can be used interchangeably, specifically referring to reducing the level of co-stimulatory factors associated with dendritic cells, reducing the level of cytokines associated with T cell proliferation, and reducing the level of skin inflammatory factors.

[0086] The co-stimulatory factors include but are not limited to one or more of CD80, CD86 and MHC.

[0087] MHC includes, but is not limited to, MHC I and / or MHC II.

[0088] Cytokines associated with T cell proliferation include, but are not limited to, IFNγ and / or IL.

[0089] ILs include, but are not limited to, IL-17A and / or IL-4.

[0090] Skin inflammatory factors include, but are not limited to, one or more of IL (interleukin), IFNγ, and TNF-α.

[0091] IL includes but is not limited to one or more of IL-17, IL-23, and IL-22.

[0092] IL-17 includes but is not limited to IL-17A and / or IL-17F.

[0093] In one embodiment of the invention, the method is a method for non-therapeutic purposes. The method may be to adjust the above-mentioned substances during laboratory research. This application scenario does not involve therapeutic purposes.

[0094] The invention will be further described below in conjunction with specific examples. It should be understood that the specific embodiments described herein are presented by way of example and are not intended to limit the invention. The main features of the invention may be used in a variety of embodiments without departing from the scope of the invention.

[0095] Example

[0096] In the following examples, primary mouse mononuclear dendritic cells (BMDCs) were obtained from the femoral bone marrow of 6-8 week old male C57BL / 6 mice; pantethine (Pan) was purchased from Sigma; GM-CSF and IL-4 were purchased from Peprotech; mouse CD4 T cell separation magnetic bead kit was purchased from Miltenyi Biotec; MACS auto running buffer was purchased from Miltenyi Biotec; LS separation column was purchased from Miltenyi Biotec; magnetic stand was purchased from Miltenyi Biotec; EαEGFP protein was prepared and purified by our laboratory; lipopolysaccharide (LPS) was purchased from Sigma; Imiquimod (IMQ) was purchased from Sichuan Mingxin Pharmaceutical Co., Ltd.; OVA323-339 peptide was purchased from Shenggong Biotechnology Co., Ltd.; OT II transgenic mice (Jackson Laboratory) were purchased from Shanghai South Model Organisms Technology Co., Ltd.; CellTrace TMCFSE cell proliferation kit was purchased from Invitrogen; Brefeldin A solution (1000X) was purchased from Thermo; APC-IFNγ flow cytometry antibody was purchased from Thermo; APC-IL4 flow cytometry antibody was purchased from Biolegend; PE-Cy7-IL-17A flow cytometry antibody was purchased from Biolegend; saponin permeabilization solution was purchased from Thermo; 7-AAD was purchased from BD; PMA / ion stimulator was purchased from Hangzhou Lianke Biotechnology Co., Ltd.; APC-CD11c flow cytometry antibody was purchased from Thermo; FITC-MHC II flow cytometry antibody was purchased from Thermo; PE-CD86 flow cytometry antibody was purchased from Biolegend; PE-CD80 flow cytometry antibody was purchased from Biolegend; FITC-MHC I was purchased from Biolegend; Biotin-Ea52-68peptide bound to I-Ab Monoclonal Antibody (eBioY-Ae (Yae, Y-Ae)), antibody was purchased from eBioscience; APC Streptavidin was purchased from biolegend; PE-CD4 flow cytometry antibody was purchased from biolegend; 4% paraformaldehyde was purchased from promega; DNase I was purchased from Shenggong Biotechnology Co., Ltd.; collagenase IV was purchased from Sigma; dispase II was purchased from Bio-Time; mouse IFNγ ELISA kit, mouse IL-4 ELISA kit, mouse IL-17A ELISA kit were all purchased from biolegend.

[0097] Example 1 Isolation and differentiation of mouse primary dendritic cells (BMDCs)

[0098] 1. Isolation of Mouse Femur

[0099] 1) Place a piece of absorbent paper or a layer of plastic wrap on the foam board.

[0100] 2) The mice were killed by cervical dislocation and placed on absorbent paper or plastic wrap covered with a foam board. The mice were disinfected with alcohol and fixed with the ventral side facing upward.

[0101] 3) Use tweezers to hold the epidermis of the mouse's hind legs, and use scissors to cut upward to the base of the mouse's thigh, and then cut downward to about 1 cm below the mouse's knee and cut it off. Use tweezers to hold the cut lower leg and peel it upward until the mouse's hip joint, exposing a white spherical bone joint. From here, remove the entire femur, remove excess tissue, and soak the femur in PBS.

[0102] 4) Take the other femur using the same method as above.

[0103] 2. Isolation of mononuclear cells from mouse femur

[0104] 1) Preparation of materials: alcohol lamp, 50ml centrifuge tube (for culture medium and PBS), sterile hemostatic forceps, sterile gauze, 1ml syringe, 1640+10% serum+1% double antibody.

[0105] 2) Use sterile hemostatic forceps to clamp a femur from the double antibody, place it on sterile gauze, fold it and wrap it, fix it with both hands, put the two thumbs on the back of the knee joint and break it upwards, the cross section will be plum blossom-shaped, insert the needle tip of the syringe with culture medium into the center of the plum blossom, and push the needle tip down until it can no longer move. The syringe and the femur are placed obliquely against the inner wall of the 50ml centrifuge tube, and the syringe is slowly pushed down to inject the culture medium into the bone marrow cavity, flushing out the cells in the bone marrow, and the color of the bone marrow gradually turns white (take the mononuclear cells in the bone marrow on the other side, the method is the same as above).

[0106] 3) Count the cells taken out and calculate 2×10 6 The cells were inoculated into 6-well plates with a volume of 2 mL per well, and 10 ng / ml GM-CSF and 5 ng / ml IL-4 were added. The cells were differentiated in a 37°C incubator for 6 days. On the third day, each well was supplemented with 2 mL of 1640 medium containing 10 ng / ml GM-CSF and 5 ng / ml IL-4.

[0107] Example 2 Detection of the effect of pantethine on BMDC antigen processing and presentation

[0108] 1. Cell seeding

[0109] 1) Collect the supernatant in the 6-well plate into a 50 mL centrifuge tube and centrifuge (1500 rpm, 5 min); then wash the cells with PBS and centrifuge (1500 rpm, 5 min).

[0110] 2) After centrifugation, discard the supernatant, resuspend the cells in new culture medium, and use a pipette to draw 10 μl for counting. Finally, count 1×10 5 Cells were seeded in 24-well plates.

[0111] 2. Drug pretreatment

[0112] Dissolve pantethine in PBS to prepare a 500 mM stock solution, add the corresponding concentration of pantethine to a 24-well plate, and the final concentration is 2 mM or 5 mM. Keep the volume of PBS added to each well consistent. Incubate at 37°C for 2 hours.

[0113] 3. EαEGFP stimulates BMDCs

[0114] After pretreatment with pantethine, BMDC cells were added with 50 μg / ml EαEGFP and stimulated for 24 h in a 37°C incubator.

[0115] 4. Flow cytometry detection of BMDC antigen processing and presentation ability

[0116] The stimulated BMDCs were collected into a 1.5 ml centrifuge tube, washed with PBS, and the cell pellet was collected by centrifugation at 1500 rpm for 5 min. After washing again with PBS, the pellet was resuspended in 100 μl and the flow cytometry antibody was added according to the proportion in the instruction manual. The cells were incubated on ice or at 4°C for 30 min and tested on a flow cytometer. The antigen presentation ability was evaluated by combining the fluorescence intensity of EGFP and YAe.

[0117] Example 3 Detection of the effect of pantethine on BMDC maturation

[0118] 1. Cell seeding

[0119] 1) Collect the supernatant in the 6-well plate into a 50 mL centrifuge tube and centrifuge (1500 rpm, 5 min); then wash the cells with PBS and centrifuge (1500 rpm, 5 min).

[0120] 2) After centrifugation, discard the supernatant, resuspend the cells in new culture medium, and use a pipette to draw 10 μl for counting. Finally, count 1×10 5 The cells were seeded in a 24-well plate with a volume of 500 μl per well.

[0121] 2. Drug pretreatment

[0122] Dissolve pantethine in PBS to prepare a 500 mM stock solution, add the corresponding concentration of pantethine to a 24-well plate, and the final concentration is 2 mM or 5 mM. Keep the volume of PBS added to each well consistent. Incubate at 37°C for 2 hours.

[0123] 3. LPS stimulation of BMDC

[0124] BMDC cells were pretreated with pantethine and then stimulated with 1 μg / ml LPS in a 37°C incubator for 24 h.

[0125] 4. Flow cytometry detection of BMDC maturity

[0126] The stimulated BMDCs were collected into a 1.5 ml centrifuge tube, washed with PBS, centrifuged at 1500 rpm for 5 min to collect the cell pellet, washed again with PBS, resuspended in 100 μl of the pellet, added with flow cytometry antibodies according to the proportion in the instructions, incubated on ice or at 4°C for 30 min, and the expression of CD80, CD86, MHC I and MHC II was detected by flow cytometry.

[0127] Example 4 Detection of the effect of pantethine on BMDC-activated T cells

[0128] 1. BMDC cell seeding

[0129] 1) Collect the supernatant in the 6-well plate into a 50 mL centrifuge tube and centrifuge (1500 rpm, 5 min); then wash the cells with PBS and centrifuge (1500 rpm, 5 min).

[0130] 2) After centrifugation, discard the supernatant, resuspend the cells in new culture medium, and use a pipette to draw 10 μl for counting. Finally, count 1×10 5 Cells were seeded in 24-well plates.

[0131] 2. Drug pretreatment

[0132] Dissolve pantethine in PBS to prepare a 500 mM stock solution, add the corresponding concentration of pantethine to a 24-well plate, and the final concentration is 2 mM or 5 mM. Keep the volume of PBS added to each well consistent. Incubate at 37°C for 2 hours.

[0133] 3. LPS stimulation of BMDC

[0134] BMDC cells were pretreated with pantethine and then stimulated with 1 μg / ml LPS in a 37°C incubator overnight.

[0135] 4. OVA323-339 peptide stimulates BMDC

[0136] The next day, 10 μg / ml of OVA323-339 peptide was added to the BMDC cells in the 24-well plate and stimulated for 2 h in a 37°C incubator.

[0137] 5. Isolation of OTII T cells

[0138] 1) Isolation of OTII mouse spleen cells: The mouse was killed by cervical dislocation and placed on absorbent paper or plastic wrap covered with a foam board. The mouse was disinfected with alcohol and fixed with the ventral side facing up. The left hand used tweezers to pick up the fur on the abdomen, and the right hand used scissors to cut from bottom to top along the midline. Holding the tweezers in both hands, find the spleen in the lower right corner of the abdomen (about 1 cm, blood red, easy to separate).

[0139] 2) Remove the spleen and place it in a 50 ml centrifuge tube equipped with a 70 μm filter. Add a little MACS auto buffer to soak it. Use the piston end of the syringe to grind on the filter until no red area is visible (add MACS auto buffer to rinse during this period). Collect the filtrate and centrifuge at 1500 rpm for 5 minutes.

[0140] 3) After centrifugation, discard the supernatant and use 1 ml of Gibco ACK lysing buffer to suspend the cell pellet for lysing red blood cells at room temperature for 3 to 5 minutes.

[0141] 4) Add 10 mL of MACS auto buffer to terminate lysis. After mixing, filter with a new 70 μm filter (placed on a new 50 mL centrifuge tube), centrifuge at 1500 rpm for 5 min, discard the supernatant, and resuspend the cell pellet with 1 mL of MACS auto buffer.

[0142] 5) Cell counting: Take 10 μl of the suspension and mix it with 10 μl of trypan blue. Then add 10 μl of the mixture to a cell counting plate and count the cells using a cell counter.

[0143] 6) Antibody for incubation and separation of CD4 T cells: Antibody standard for mouse CD4 T cell separation magnetic bead kit: 2-3×10 7 / ml cells, the total volume of the solution is 100μl, and the corresponding antibody volume is 10μl. Add the antibody volume according to the above ratio, mix well, and incubate at 4℃ with rotation for 5-10min.

[0144] 7) Incubate the magnetic beads for separating CD4 T cells: Take 20 μl / tube and add to the suspension containing antibody and cells (antibody: magnetic beads volume ratio = 1:2), and incubate at 4°C with rotation for 10-15 minutes.

[0145] Take out the magnetic rack and place it on the sterile table, place the LS sorting column on the magnetic rack, use buffer (3 ml / column) to wash the column, and then place a 50 ml centrifuge tube underneath (in preparation for T cell separation).

[0146] 8) After incubating the magnetic beads, add 500 μl of MACS auto buffer to each tube, mix well, and then pass through the column. Then add 3 mL of buffer to wash the column. Wash 3 times, 3 ml each time. Collect the T cell solution in a 50 ml centrifuge tube, centrifuge at 1500 rpm for 3 to 5 min, and discard the supernatant; add 1 ml of buffer to resuspend the precipitate, take 10 μl of the suspension and mix with 10 μl of trypan blue, then take 10 μl and add it to the cell counting plate, and count using a cell counter.

[0147] 6. T cell marker CFSE

[0148] The sorted OTII T cells were washed once with room temperature PBS, centrifuged at 1500 rpm for 3-5 min, the supernatant was discarded, and the serum in the MACS auto buffer was removed; the cells were resuspended with 1 ml PBS, and 5 μM CFSE was added and incubated at 37°C in the dark for 20 min; 5 times the original chromosome volume of the culture medium (containing at least 1% FBS) was added to the cells and incubated for 5 min.

[0149] Pellet the cells by centrifugation and resuspend them in fresh pre-warmed complete medium.

[0150] 1×10 5 cells / well, 100μl / well, i.e. 1×10 6 cells / ml, inoculated into 96 wells.

[0151] 7. Co-culture of DCs and T cells

[0152] The pretreated DC cells were collected into a 1.5 ml centrifuge tube, and the DC cells and CD4+ T cells were co-cultured in a 96-well plate at a ratio of DC:CD4+T cells = 1:2, i.e., 2.5×10 DC cells were used. 4 cell, 100 μl / well: T cells 5×10 4 cell, 100μl / well, 37℃, 72h. About 5h before collecting cells, collect 50μl supernatant from each well and freeze it at -80℃ for subsequent ELISA detection; then add a certain proportion of protein transport inhibitor and PMA / ion mixed stimulator to each well to amplify the cytokine signal that can be detected in the cell. After incubation for 4-5h, collect cells for subsequent flow cytometry analysis.

[0153] 8. Flow cytometry to detect T cell proliferation ability

[0154] CFSE-labeled T cells will spontaneously emit 488 excitation light. As the number of cell proliferation generations increases, the fluorescence intensity will gradually weaken. Analysis of the degree of fluorescence weakening can be used to analyze cell proliferation.

[0155] 9. Flow cytometry to detect T cell cytokine secretion ability

[0156] After co-culture, the cells were collected, washed once with PBS, and labeled with surface CD4 flow cytometry antibodies. After labeling, the cells were fixed with 200 μl / tube of 4% paraformaldehyde at room temperature for 15 to 20 min, then washed with permeabilization solution, and the flow cytometry antibodies required for intracellular staining were prepared with permeabilization solution. After adding antibodies in proportion, the cells were placed at room temperature for intracellular staining for 45 min to 1 h, then washed with permeabilization solution, and the expression of cytokines IFNγ, IL-4 and IL-17A was detected on a flow cytometer.

[0157] 10. ELISA to detect cytokines in co-culture supernatant

[0158] The cell supernatant frozen at -80°C was placed on ice to thaw. After thawing, the contents of cytokines IFNγ, IL-4 and IL-17A were detected using ELISA kits. For the specific process, please refer to the kit instructions.

[0159] Example 5 Effect of pantethine on Imiquimod (IMQ)-induced psoriasis mouse model 1. Establishment of Imiquimod (IMQ)-induced psoriasis mouse model

[0160] The day before the experiment, the mice were shaved on the back using an epilator and depilatory cream, with a back hair removal area of ​​2.5 cm × 2.5 cm. Day 0 was counted from the first administration of the experiment. Starting from day 0, 62.5 mg of imiquimod cream was applied to the shaved area every day. At the same time, the back skin of the mice was observed and scored for disease every day, and the thickness of the back skin was measured. The cream was applied continuously for 6 days, and anesthesia was taken on the 6th day to record the skin lesions. After cervical dislocation, the spleen and draining lymph nodes in the axilla of the mice were separated and placed in PBS. The spleen was used to take pictures to record changes in mass and size, and the lymph nodes were used to analyze changes in the number of T cells and changes in the number and maturity of DCs. The back skin was separated and divided into 2 parts, one part was immersed in 4% paraformaldehyde for immunohistochemical analysis, and the other part was immersed in Trizol for RNA extraction to analyze the level of inflammatory factors.

[0161] 2. Effect of pantethine on imiquimod (IMQ)-induced psoriasis mouse model

[0162] The experiment was divided into three groups: ① control group, intraperitoneal injection of 100 μl PBS per day, application of 62.5 mg vaseline cream per day, ② model group, intraperitoneal injection of 100 μl PBS per day, application of 62.5 mg imiquimod cream per day, ③ drug administration group, intraperitoneal injection of 100 μl pantethine (50 mg / kg) per day, application of 62.5 mg imiquimod cream per day, for 6 consecutive days.

[0163] 3. Extraction of total RNA from skin using Trizol method

[0164] 1) Take fresh animal skin tissue and cut it into pieces and place it in a tissue homogenizer tube (add 2-3 grinding beads at the same time), add 800μl-1ml of pre-cooled Trizol solution to the homogenizer tube, and quickly homogenize it in a tissue grinder. Pause every 30 seconds to check whether the lysis is complete. Repeat about 3 times to fully grind the tissue until it is completely lysed. This step can be frozen at -80℃ until RNA is extracted.

[0165] 2) The lysed tissue mixture was centrifuged at 3000 rpm for 5 min, 500 μl of the supernatant was taken, and 100 μl of chloroform was added, with chloroform:Trizol=1:5.

[0166] 3) Use an oscillator to shake vigorously for 15-30 seconds (purple).

[0167] 4) After standing for 5 minutes, centrifuge at 4 degrees and 15,000 rpm for 10 minutes or 12,000 rpm for 15 minutes (the upper layer is Trziol+RNA, and the lower layer is protein and residue) (Note: the longer the centrifugation time, the more obvious the stratification and the easier it is to separate).

[0168] 5) Pipette the upper transparent liquid into new clean tubes (autoclaved, RNase-free) twice, add 80 μl / 100 μl isopropanol to each tube (isopropanol: supernatant = 1:1), and turn it upside down (to precipitate RNA).

[0169] 6) Let stand for 10 min to precipitate RNA, 4 degrees, 15000 rpm, 10 min.

[0170] 7) Quickly discard the supernatant.

[0171] 8) Add 750 μl DEPC water to each tube and mix with 75% ethanol, invert it (to wash away DNA and protein impurities). Can be frozen at -80 degrees.

[0172] 9) 4 degrees, 15000 rpm, 3 min, repeat steps 8 to 9 once.

[0173] 10) Aspirate and discard the supernatant; aspirate slowly.

[0174] 11) Place the remaining tubes on a cell table covered with plastic wrap to dry.

[0175] 12) Add 65°C preheated DEPC H2O, 16-20 μl / tubes.

[0176] 13) After the RNA is dissolved, measure the RNA concentration and freeze it at -20 degrees.

[0177] 4. Detection of skin cytokine expression at the mRNA level

[0178] Reverse transcription

[0179] system:

[0180] RNA 1μg / 0.5μg (uniform amount), DEPC water 7μl (replenishment), 5×mix 2μl, total volume 10μl.

[0181] program:

[0182] 37 degrees, 15 minutes (reverse transcription); 85 degrees, 5 seconds (enzyme acceptance); 4 degrees, storage; temperature and time depend on the situation.

[0183] qPCR system: 10μl, 3 replicates per sample, add internal reference GAPDH, Tem 0.3μl, primers F+R 0.5μl, H2O 4.2μl, 2×SYBR 5μl, primer / water / mix+SYBR / DNATem mix to seal, mix - centrifuge - program - save - start - name.

[0184] The extracted RNA was reverse transcribed into cDNA as a template to detect the expression of cytokines IL-17A, IL-17F, IL-22, IL-23, IFNγ, and TNF-α in mouse skin.

[0185] Experimental Results

[0186] 1. Effects of pantethine on antigen processing and presentation of BMDCs stimulated by EαEGFP

[0187] Example 2 of the present invention measured the effect of pantethine on the ability of BMDC to process and present antigens after stimulation with EαEGFP protein in primary mouse dendritic cells. Figure 1 shown.

[0188] Depend on Figure 1 It can be seen that BMDC cells can process and present EαEGFP protein after 24h of stimulation, and the ability of DC cells to present antigens is significantly increased compared with the control group. At the same time, 100μM pantethine treatment (Pan+EαEGFP) can significantly inhibit the ability of DC cells to present EαEGFP (p<0.01), indicating that pantethine has a significant inhibitory effect on the antigen processing and presentation ability of BMDC.

[0189] 2. Effect of pantethine on BMDC maturation under LPS stimulation

[0190] To further explore the effect of pantethine on the subsequent activation and maturation of BMDC, Example 3 of the present invention measured the effect of pantethine on the expression of co-stimulatory factors CD80, CD86, MHC I and MHC II in BMDC after lipopolysaccharide (LPS) stimulation in primary dendritic cells of mice. The results are as follows: Figure 2 shown.

[0191] Depend on Figure 2It can be seen that after BMDC was stimulated with LPS, flow cytometry detected that the expression of CD80, CD86, MHC I and MHC II on the cell surface increased significantly compared with the unstimulated group. At the same time, 2mM pantethine (Pan+LPS) could significantly inhibit the expression of these co-stimulatory factors (p<0.01), indicating that pantethine has an inhibitory effect on LPS-induced BMDC activation.

[0192] 3. Effect of pantethine on T cell activation under co-culture conditions of DC and T cells

[0193] Based on the results of the previous DC antigen presentation and maturity test, in order to further clarify the effect of pantethine on DC function, Example 4 of the present invention used a DC and OT II co-culture system to study the effect of pantethine on the subsequent activation of CD4 T cells by DC. Figure 3 shown.

[0194] Depend on Figure 3 It can be seen that compared with untreated BMDC (No DCs), DC treated with OVA peptide (+VehicleDCs) co-cultured with T cells can promote T cell proliferation and related cytokine secretion such as IFNγ, IL-17A, IL-4, and DC treated with LPS and OVA peptide co-cultured with T cells can more significantly promote T cell proliferation and related cytokine secretion. Compared with 2mM pantethine treatment (+Pan DCs), BMDC's ability to activate T cells is significantly reduced, specifically in terms of proliferation ability and cytokine secretion ability.

[0195] 4. Effect of pantethine on IMQ-induced psoriasis in mice

[0196] Example 5 of the present invention uses an imiquimod (IMQ)-induced psoriasis model as an in vivo model to explore the effect of pantethine on autoimmune diseases. Figure 4 shown.

[0197] Depend on Figure 4It can be seen that compared with wild-type (Sham) mice, the back skin of IMQ-induced mice (Vehicle) was significantly thickened, accompanied by scaly dandruff and redness. The expression of inflammatory factors (IL-17A, IL-17F, IL-23, IL-22, IFNγ and TNF-α) in the back skin was significantly increased by qPCR analysis. However, after the mice were given pantethine (Pan), the psoriasis symptoms were significantly alleviated, the thickness was reduced, and the scaly and redness were significantly weakened compared with the IMQ model group mice. The expression of inflammatory factors (IL-17A, IL-17F, IL-23, IL-22, IFNγ and TNF-α) in the back skin was significantly reduced by qPCR analysis. This suggests that pantethine has a significant alleviating effect on IMQ-induced psoriasis.

[0198] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. Use of pantethine or a pharmaceutically acceptable salt thereof in the preparation of a pharmaceutical composition for treating diseases related to overactivation of dendritic cells; Preferably, the diseases associated with overactivation of dendritic cells include infectious diseases, transplant rejection, allergic diseases, and autoimmune diseases; Preferably, the dendritic cell overactivation-related disease is selected from autoimmune diseases; Preferably, the autoimmune disease is selected from psoriasis.

2. The use according to claim 1, characterized in that: The treatment of diseases related to excessive activation of dendritic cells comprises at least one of the following: (1) Inhibit the ability of dendritic cells to process and present antigens; (2) inhibiting dendritic cell activation; (3) Inhibit the ability of dendritic cells to activate T cells.

3. The use according to claim 2, characterized in that: The antigen processing and presentation ability of dendritic cells mentioned in (1) refers to the ability of dendritic cells to present EαEGFP.

4. The use according to claim 2, characterized in that: The dendritic cell activation described in (2) is LPS-induced dendritic cell activation; Preferably, the activated co-stimulatory factors include one or more of CD80, CD86, and MHC; Preferably, the MHC comprises MHC I and / or MHC II.

5. The use according to claim 2, characterized in that: The ability of dendritic cells to activate T cells described in (3) is the proliferation ability of T cells; Preferably, the cytokines related to the proliferation ability of the T cells include IFNγ and / or IL; Preferably, the IL comprises IL-17A and / or IL-4.

6. A pharmaceutical composition for treating diseases related to overactivation of dendritic cells, characterized in that: The pharmaceutical composition comprises pantethine or a pharmaceutically acceptable salt thereof.

7. The pharmaceutical composition according to claim 6, characterized in that The pharmaceutical composition also includes a pharmaceutically acceptable carrier.

8. The pharmaceutical composition according to claim 6, characterized in that The pharmaceutical composition also includes other active drugs; Preferably, the other active drugs include immunosuppressants, vitamins and their analogs, vitamin A related compounds, corticosteroids, and biological products; Preferably, the vitamins include vitamin A, vitamin B, vitamin C, vitamin D, and vitamin K.

9. The pharmaceutical composition according to claim 6, characterized in that The dosage form of the pharmaceutical composition includes solid preparations, semisolid preparations or liquid preparations; Preferably, the solid preparation comprises tablets, lozenges, capsules, pills or granules; Preferably, the semisolid preparation comprises a cream, a gel, an ointment or an emulsion; Preferably, the liquid preparation comprises a solution or a suspension.

10. A method for regulating any of the following substances, characterized in that: The method comprises administering pantethine or a pharmaceutically acceptable salt thereof, wherein the substance comprises any one of the following: (1) Co-stimulatory factors associated with dendritic cells; (2) cytokines associated with T cell proliferation; (3) Skin inflammatory factors; Preferably, the co-stimulatory factors described in (1) include one or more of CD80, CD86, and MHC; Preferably, the MHC comprises MHC I and / or MHC II; Preferably, the cytokines associated with T cell proliferation in (2) include IFNγ and / or IL; Preferably, the IL comprises IL-17A and / or IL-4; Preferably, the skin inflammatory factors described in (3) include one or more of IL, IFNγ, and TNF-α; Preferably, the IL includes one or more of IL-17, IL-23, and IL-22; preferably, the IL-17 includes IL-17A and / or IL-17F.

Images