阅读说明：本技术 氯喹那多的新应用 (Novel application of chloroquinate ) 是由 胡文辉 孙平 杨忠金 陈燕红 陈秀会 梁淑丽 熊兮 于 2021-07-22 设计创作，主要内容包括：本发明涉及一种氯喹那多的新应用。本发明中通过氯喹那多对NLRP3炎症小体抑制作用的体外研究表明,氯喹那多能有效抑制NLRP3炎症小体的活化,能抑制炎症细胞因子IL-1β和Caspase-1的成熟和分泌。另外,动物试验证明,氯喹那多对于NLRP3相关疾病具有良好的防治作用,尤其是针对银屑病,具有显著的防治效果。(The invention relates to a new application of cloquindol. In-vitro research on the inhibition effect of the cloquindol on the NLRP3 inflammasome shows that the cloquindol can effectively inhibit the activation of the NLRP3 inflammasome and can inhibit the maturation and secretion of inflammatory cytokines IL-1 beta and Caspase-1. In addition, animal experiments prove that the chloroquinate has good prevention and treatment effects on NLRP 3-related diseases, and particularly has remarkable prevention and treatment effects on psoriasis.)

1. The application of the chloroquinate in preparing the medicine for preventing and/or treating NLRP3 inflammasome related diseases.

2. The use according to claim 1, wherein the NLRP3 inflammasome-related disease is psoriasis.

3. The use according to claim 1, wherein the NLRP3 inflammasome-related disease is atopic dermatitis, uv-induced sunburn of the skin, obesity, type 2 diabetes, atherosclerosis, gout, multiple sclerosis, alzheimer's disease, parkinson's disease, hepatitis, silicosis, rheumatoid arthritis, contact hypersensitivity, depression, non-alcoholic fatty liver disease, alcoholic liver disease or kidney disease.

4. The use according to claim 1, wherein the NLRP3 inflammasome-related disease is familial cold autoinitis syndrome, M μ ckle-Wells syndrome, chronic infant neurocutaneous and joint syndrome, neonatal onset multiple system inflammatory disease, amyotrophic lateral sclerosis or asthma or acute respiratory distress syndrome.

5. The use of claim 1, wherein the chloroquinar is capable of inhibiting NLRP3 inflammasome activation.

6. The use of claim 1, wherein the chloroquinar pluripotency inhibits Caspase-1 maturation or secretion.

7. The use of claim 1, wherein said chloroquinar is capable of inhibiting IL-1 β maturation or secretion.

8. The use according to any one of claims 1 to 7, wherein the medicament is in the form of a capsule, granule, injection, pill, syrup, powder, paste, emulsion, solution, suspension or tincture.

9. The medicine for preventing and/or treating NLRP3 inflammasome-related diseases is characterized by comprising an active substance and pharmaceutically acceptable auxiliary materials, wherein the active substance comprises cloquindol.

10. The pharmaceutical according to claim 9, wherein the excipient is at least one of an excipient, a filler, a compatibilizer, a binder, a humectant, a disintegrant, a slow-dissolving agent, an absorption accelerator, an adsorbent, a diluent, a solubilizer, an emulsifier, a lubricant, a wetting agent, a suspending agent, a flavoring agent, and a perfume.

Technical Field

The invention relates to the field of medicines, in particular to a new application of chloroquinate.

Background

The NLRP3 inflammasome is a Pattern Recognition Receptor (PRR), which is an inflammatory activation platform for Caspase-1. The NLRP3 inflammasome is a multi-protein complex assembled by receptor protein NLRP3, adaptor protein ASC (apoptosis-associated plaque binding protein) and Caspase1 as key proteins. After the immunogen is stimulated to be activated, Caspase1 can be activated, so that the cleavage and maturation of cell factor precursors such as Pro-IL-1 beta and the like are promoted; meanwhile, activated Caspase-1 can also trigger cell apoptosis. The NLRP3 inflammasome can recognize exogenous pathogen-associated molecular patterns (PAMPs) such as lipopolysaccharide LPS, viral RNA, etc., or endogenous damage-associated molecular patterns (DAMPs) such as DNA, endotoxin, uric acid, ATP, a β, and cell debris, etc. The abnormal appearance of the substances is at a certain position outside or inside cells, so that the homeostasis of tissues is stimulated to activate NLRP3 inflammasome to resist damage of exogenous or endogenous factors to the body, but the over-activated NLRP3 inflammasome can cause the development of various diseases, such as psoriasis, gout, hereditary Cryopyrin-related periodic fever syndrome, multiple sclerosis, Alzheimer disease, Parkinson disease, sepsis and the like. Although many inhibitors have some relief in basic studies on NLRP3 inflammasome-related diseases, treatment of related diseases by targeting NLRP3 inflammasome has been demonstrated in several disease models.

Some small molecule compounds and metabolites have been reported to be effective in inhibiting NLRP3 inflammasome, such as MCC950(Coll, et al.,2016, Nat Med), ω -3 fatty acids (Yan, et al.,2013, Immunity), sulforaphane (Yang, et al.,2016, Sci Rep), isoliquiritigenin (Honda, et al.,2014, J Leukoc Biol), 3, 4-methyldioxy- β -nitrostyrene (3, 4-methyldioxy-nitrostyrene) (He, et al.,2014, J Biol Chem), β -hydroxybutyric acid (Youm,2015, Nat Med), and the like. Although the compounds are expected to be drugs for treating diseases related to NLRP3 inflammasome, long-term drug efficacy, drug safety evaluation and clinical tests are needed, and no drug which is effective against NLRP3 inflammasome is available so far.

In view of the above, there is a need in the art for clinical drugs that inhibit the activation of NLRP3 inflammasome. At present, no report is found on whether the cloquindol can prevent and treat the NLRP3 inflammasome-related diseases.

The structural formula of the chloroquinadol is shown asCloquindol is a topical antibacterial agent used for treating skin infection, has been clinically used as an ointment for treating local skin infection for a long time alone or in combination with corticoids, and research and practice on the cloquindol prove the safety of the cloquindol. Chloroquinate has been demonstrated to have significant antibacterial, antitubercular, antiviral, antiparasitic activity, and current research on chloroquinate focuses mainly on its antibacterial effect. The pharmacological mechanism of the cloquindol in preventing and treating NLRP3 inflammasome-related diseases is not reported.

Disclosure of Invention

Based on the above, the invention aims to provide an application of cloquindol in preparing a medicament for preventing and/or treating NLRP3 inflammasome-related diseases.

The specific technical scheme is as follows:

the application of the chloroquinate in preparing the medicine for preventing and/or treating NLRP3 inflammasome related diseases.

In some of these embodiments, the NLRP3 inflammasome-related disease is psoriasis.

In some of these embodiments, the NLRP3 inflammasome-related disease is atopic dermatitis, uv-induced sunburn of the skin, obesity, type 2 diabetes, atherosclerosis, gout, multiple sclerosis, alzheimer's disease, parkinson's disease, hepatitis, silicosis, rheumatoid arthritis, contact hypersensitivity, depression, non-alcoholic fatty liver disease, alcoholic liver disease, or kidney disease.

In some of these embodiments, the NLRP3 inflammatory-corpuscle-associated disease is familial cold autoinitis syndrome, M μ ckle-Wells syndrome, chronic infant neurocutaneous and joint syndrome, neonatal onset multiple system inflammatory disease, amyotrophic lateral sclerosis or asthma, or acute respiratory distress syndrome.

In some of these embodiments, wherein the chloroquinalder inhibits NLRP3 inflammasome activation.

In some of these embodiments, the cloquindol inhibits Caspase-1 maturation or secretion.

In some of these embodiments, wherein the chloroquinar is capable of inhibiting IL-1 β maturation or secretion.

In some of these embodiments, the medicament is in the form of a capsule, granule, injection, pill, syrup, powder, paste, emulsion, solution, suspension, or tincture.

The invention also aims to provide a medicament for preventing and/or treating NLRP3 inflammasome-related diseases, which comprises an active substance and pharmaceutically acceptable auxiliary materials, wherein the active substance comprises cloquindol.

In some of these embodiments, the excipient is at least one of an excipient, a filler, a compatibilizer, a binder, a humectant, a disintegrant, a slow-dissolving agent, an absorption accelerator, an adsorbent, a diluent, a solubilizer, an emulsifier, a lubricant, a wetting agent, a suspending agent, a flavoring agent, and a fragrance.

Compared with the prior art, the invention has the following beneficial effects:

the invention provides a new application of cloquindol in a medicament for preventing and/or treating NLRP3 inflammation body related diseases. In-vitro research on the inhibition effect of the cloquindol on the NLRP3 inflammasome shows that the cloquindol can effectively inhibit the activation of the NLRP3 inflammasome and can inhibit the maturation and secretion of inflammatory cytokines IL-1 beta and Caspase-1. In addition, animal experiments prove that the chloroquinate has good prevention and treatment effects on NLRP 3-related diseases, particularly has obvious prevention and treatment effects on psoriasis.

Drawings

FIG. 1: (A) the structural formula of the chloroquinate is shown in the specification, and CQD is an abbreviation of the chloroquinate; (B, C, D) Cloquinadol (CQD) can inhibit Nigericin (Nigericin), ATP and urate crystal (MSU) in concentration-dependent manner to induce the secretion of IL-1 beta.

FIG. 2: the CCK8 experiment detects that the Chloroquinate (CAQ) has small toxicity to cells in vitro.

FIG. 3: detection of cellular total lactate dehydrogenase activity showed that Chloroquinate (CQD) was able to inhibit LDH release.

FIG. 4: immunoblotting (WB) performed to detect that Chloroquinate (CQD) can inhibit activation of Caspase-1 and secretion of IL-1 beta induced by Nigericin in a concentration-dependent manner.

FIG. 5: cloquindol (CQD) was effective in reducing the back skin erythema score, the scaling score, the skin thickness score, and the total score in an imiquimod-induced psoriasis mouse model.

FIG. 6: hematoxylin-eosin (HE) staining showed that Cloquindol (CQD) was able to reduce the thickness of the dorsal epidermis in an imiquimod-induced psoriasis mouse model.

FIG. 7: cloquinate (CQD) reduces the release of IL-1 β, IL-17 in the dorsal skin of an imiquimod-induced psoriasis mouse model. Wherein A is the expression change of IL-1 beta and B is the expression change of IL-17.

Detailed Description

Experimental procedures according to the invention, in which no particular conditions are specified in the following examples, are generally carried out under conventional conditions, or under conditions recommended by the manufacturer. The various chemicals used in the examples are commercially available.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

The terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusions. For example, a process, method, apparatus, article, or device that comprises a list of steps is not limited to only those steps or modules listed, but may alternatively include other steps not listed or inherent to such process, method, article, or device.

The "plurality" referred to in the present invention means two or more. "and/or" describes the association relationship of the associated objects, meaning that there may be three relationships, e.g., a and/or B, which may mean: a exists alone, A and B exist simultaneously, and B exists alone. The character "/" generally indicates that the former and latter associated objects are in an "or" relationship.

The present invention will be described in further detail with reference to specific examples.

Test materials used in the following examples: unless otherwise specified, all were purchased from conventional biochemical stores. Cloquinado (abbreviation: CQD) was purchased from TargetMol (cat # T0908). LPS and ATP were purchased from Sigma. Nigericin (Nigericin) was purchased from Invitrogen. The LDH detection kit is purchased from Biyuntian. Anti-murine IL-1 β antibody (AF-401-NA) was purchased from R & D. anti-NLRP 3 antibody (AG-20B-0014), anti-ASC antibody (AG-25B-0006) and anti-Caspase-1 antibody (AG-20B-0042) were purchased from Adipogen. Anti-beta-actin antibody (P30002) was purchased from Abmart corporation. ELISA kits (IL-1. beta., IL-17) were purchased from Invitrogen. J774A.1 cells were purchased from jennio-bio. Imiquimod cream was purchased from Sichuan mingxin lidi. Petrolatum cream was purchased from Shanghai Shang dynasty imperial Weikang. C57BL/6J female mice were purchased from the center of laboratory animals, Guangdong province.

Example 1

Cloquinadol (CQD) inhibits secretion of IL-1 beta

1. The first day, well-grown J774A.1 cells were seeded into 96-well plates at 5X 10 cells per well³(ii) individual cells;

2. observing cells on the next day, discarding the supernatant, adding 100 μ L of bacterial Lipopolysaccharide (LPS) (1 μ g/ml) prepared by DMEM medium containing 10% serum into each well, acting for 5h, adding Chloroquinalder (CQD) (chloroquinalder is dissolved by dimethyl sulfoxide in advance to prepare 10mM mother solution, and diluting to the desired concentration by adopting the culture medium, wherein the final concentration of the chloroquinalder is 2 μ M, 4 μ M and 6 μ M in sequence for 1h, and then adding Nigericin (Nigericin, the final concentration of 10 μ M) and ATP (final concentration of 5mM) for 1h or MSU (final concentration of 300 μ g/ml) for 12 h;

3. after the treatment in step 2, the cell culture supernatant was aspirated and the IL-1. beta. content was determined according to the ELISA kit instructions, the results are shown in FIG. 1.

The results in FIG. 1 show that under the combined action of LPS pretreatment and a second signal Nigericin, ATP or MSU, inflammasome is activated and secretes mature IL-1 beta into the supernatant, with the addition of different concentrations of Chloroquinaldo (CQD), the maturation and secretion of IL-1 beta can be effectively inhibited, and the inhibition effect is dose-dependent within a certain concentration range.

Example 2

CCK8 cytotoxicity assay

1. J774A.1 cells were plated at 5X 10 per well³The individual cells were seeded in 96-well plates and placed in 5% CO₂And cultured overnight in a cell culture box at 37 ℃.

2. The next day, the growth state of the cells was observed to be good, and the medium was discarded. The blank control group was added with 100. mu.L of complete medium (98% DMEM + 2% FBS), and the administration group was added with Cloquindol (CQD) at different concentrations (final concentrations of cloquindol were 2.5. mu.M, 5. mu.M, and 10. mu.M), respectively, for 24 hours.

3. After the treatment in step 2, the cells were washed with sterile PBS 1 time, and 10% CCK-8 solution (100. mu.L/well) was added to all groups, and incubated in a cell incubator for 1 hour.

4. Before detection, bubbles in the discharge hole are prevented from influencing an experimental result, and the absorbance value of a detection sample with the wavelength of 450nm is set on an enzyme-labeling instrument. The results are shown in FIG. 2.

The results in FIG. 2 show that the cells treated with different concentrations of Chloroquinate (CQD) were less toxic to the cells for 24 h.

Example 3

Cloquinadol (CQD) inhibits the release of Lactate Dehydrogenase (LDH)

1. J774A.1 cells were plated at 5X 10 per well³One was inoculated into a 96-well plate and cultured overnight in a cell incubator.

2. mu.L of LPS (1. mu.g/ml) prepared in DMEM medium containing 10% serum was added to each well, and after 5 hours of action, 150. mu.L of opti-MEM medium containing the concentration of Chloroquinaldi (CQD) (2. mu.M, 4. mu.M, 6. mu.M, respectively) was added to each well of the administration group, the same volume of opti-MEM medium was added to each well of the administration group for 1 hour of action, and Nigericin (final concentration of 10. mu.M) was added for 1 hour of stimulation.

3. According to the instructions of the LDH kit, 120 mu L of supernatant of each well is respectively sucked and placed in a new 96-well plate, 60 mu L of LDH working solution is added into each well, and then the well is wrapped by tinfoil paper and placed on a shaking table for reaction for 30 min.

4. Setting the detection wavelength of the microplate reader to be 490nm and the reference wavelength to be 600nm, and detecting the absorbance value of the microplate reader. The results are shown in FIG. 3.

The results in FIG. 3 show that Chloroquinate (CQD) significantly inhibits the release of lactate dehydrogenase. Activation of NLRP3 inflammasome leads to immune cell apoptosis, and thus the extent of cellular apoptosis can be assessed by measuring the amount of lactate dehydrogenase in the supernatant.

Example 4

Cloquinadol (CQD) inhibits activation of NLRP3 inflammasome

1. The first day, J774A.1 cells were seeded onto 6-well plates, 2X 10 per well⁶(ii) individual cells;

2. plating overnight, discarding the supernatant, adding 1mL of DMEM medium containing 10% serum and bacterial Lipopolysaccharide (LPS) (1. mu.g/mL) into each well for stimulation for 5h, then discarding the supernatant, adding opti-MEM medium containing CQD (the concentrations are 2. mu.M, 4. mu.M and 6. mu.M respectively) with different concentrations for treatment for 1h, and adding Nigericin (the final concentration is 10. mu.M) for treatment for 1 h;

3. after the treatment in step 2, respectively extracting supernatant protein and cell total protein, and performing western blot analysis by using anti-IL-1 beta antibody, anti-Caspase-1 antibody, anti-NLRP 3 antibody and anti-ASC antibody, wherein the result is shown in figure 4.

The results in FIG. 4 show that under the combined action of LPS pretreatment and Nigericin as a second signal, inflammasome is activated, caspase-1 and IL-1 β mature and are secreted into the supernatant, and with the addition of different concentrations of Chloroquinaldo (CQD), caspase-1 and IL-1 β maturation and secretion are effectively inhibited, and the inhibition effect is dose-dependent over a certain concentration range.

Example 5 Chloroquindol (CQD) inhibits symptoms in psoriasis model mice

1. Selecting 6-8 week-old C57BL/6 male mice, weighing and randomly dividing into 6 groups, respectively: a negative control group, a positive control group (methotrexate group), a model group, and an administration group (5mg/kg, 15 mg/kg).

2. And (5) molding. All mice were housed in the animal center for one week to acclimate to the experiment. After one week, all mice were shaved on their backs and had an area of 2cm by 4 cm. The negative control group was administered with 62.5mg of vaseline per mouse, and the remaining groups were administered with 62.5mg of IMQ per mouse once a day for 6 days, and the mice were administered with the drugs by intraperitoneal injection 1h after administration of vaseline and IMQ (imiquimod).

3. And (4) administration. The weight of each mouse was weighed and recorded before the injection of the drug, and the administered group was intraperitoneally injected with cloquinalder, the positive control group was injected with methotrexate (1mg/kg), and the negative control group and the model group were injected with the same solvent, which was 2% dimethyl sulfoxide + 20% polyethylene glycol + 78% physiological saline, and the administration was continued for 6 days.

Score was recorded 4. Weighing the weight of the mouse before smearing vaseline and IMQ every day, photographing and recording the skin of the mouse so as to observe the change conditions of scales, red spots and skin thickness on the exposed skin surface of the mouse, and timely recording and scoring.

5. The materials are obtained. After 6 days of administration, mice were subjected to cervical dislocation for death, and a part of the skin was put into a 1.5ml centrifuge tube and then frozen in liquid nitrogen. And fixing another part of skin in paraformaldehyde, and performing immunohistochemistry and HE staining to observe pathological changes.

6. And (6) detecting. After step 5, the skin tissue frozen in about 40mg of liquid nitrogen was taken and tested for IL-1. beta. and IL-17 expression by Elisa.

The results in figure 5 show that cloquindol significantly improved the erythema, scaling and wrinkling of the skin of imiquimod-induced psoriatic mice compared to the model group.

FIG. 6 shows the results of a model group of cutaneous parakeratosis with the epidermal skin extending downward in the shape of spikes; the dermis is seen with telangiectasia hyperemia, inflammatory cell infiltration. This condition can be significantly improved after treatment with cloquindol.

The results are shown in FIG. 7. The results in FIG. 7 show that chloroquinate effectively inhibited the expression levels of IL-1 β and IL-17 in the skin.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.