CN111298097A - Application of cortistatin14 in preparation of drugs for treating autoimmune inflammatory diseases - Google Patents

Abstract

The invention discloses an application of a tumor necrosis factor antagonist cortistatin14 in preparing autoimmune inflammatory disease treatment drugs, wherein the cortistatin14 can be directly combined with a tumor necrosis factor TNF- α cell surface receptor to antagonize the function of TNF- α and play a protective role in autoimmune inflammatory diseases such as psoriasis and the like, and an intermolecular interaction experimental result shows that the cortistatin14 can be directly combined with a cell surface receptor TNFR of a key molecule TNF- α of various autoimmune diseases including psoriasis.

Description

Application of cortistatin14 in preparation of drugs for treating autoimmune inflammatory diseases

Technical Field

The invention relates to the field of medicine, in particular to application of cortistatin14 in preparation of a medicament for treating autoimmune inflammatory diseases.

Background

At present, autoimmune inflammatory diseases such as psoriasis and the like are common in clinic, patients generally need long-term drug therapy, wherein TNF- α antagonist drugs are the most common drugs with definite therapeutic effect [1] and are widely applied worldwide, clinical TNF- α antagonist drugs are prepared from bioengineering drugs, the related drugs have few types, the patient selection margin is small, the synthesis cost of the related drugs is high, and economic burden is caused to the patients needing long-term application.

1.Dervieux,T.,Kremer,J.M.&Weinblatt,M.E.(2019)Differing contributionof methotrexate polyglutamates to adalimumab blood levels as compared withetanercept,Annals of the rheumatic diseases.78,1285-1286.

2.Murray,K.M.&Dahl,S.L.(1997)Recombinant human tumor necrosis factorreceptor(p75)Fc fusion protein(TNFR:Fc)in rheumatoid arthritis,The Annals ofpharmacotherapy.31,1335-8.

3.Moulis,G.,Pugnet,G.,Costedoat-Chalumeau,N.,Mathian,A.,Leroux,G.,Boutemy,J.,Espitia,O.,Bouillet,L.,Berthier,S.,Gaultier,J.B.,Jeandel,P.Y.,Konate,A.,Mekinian,A.,Solau-Gervais,E.,Terrier,B.,Wendling,D.,Andry,F.,Garnier,C.,Cathebras,P.,Arnaud,L.,Palmaro,A.,Cacoub,P.,Amoura,Z.,Piette,J.C.,Arlet,P.,Lapeyre-Mestre,M.&Sailler,L.(2018)Efficacy and safety of biologicsin relapsing polychondritis:a French national multicentre study,Annals of therheumatic diseases.77,1172-1178.

Disclosure of Invention

The invention aims to provide an application of a tumor necrosis factor antagonist cortistatin14 in preparing a medicament for treating autoimmune inflammatory diseases, wherein the cortistatin14 serving as a TNF- α antagonist medicament has the effects of relieving inflammatory reactions and improving the conditions of inflammatory diseases such as psoriasis and the like, in-vitro experiments show that the cortistatin14 has no risk of increasing tumors and the like, and the medicament is safe for long-term use.

The technical scheme of the invention is as follows:

cortistatin14 (Cortistatin14, abbreviated as CST-14), CAS: 186901-48-4. Its molecular weight is 21 kD.

The amino acid sequence of CST-14 is:

Pro-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Ser-Ser-Cys-Lys

(Disulfide bridge: Cys2-Cys13), which is also an advantage over other TNF antagonists. Meanwhile, the synthesis purity of the compound can reach over 99.5 percent at present, and the purity meets the requirement of medicine preparation.

The invention provides an application of a tumor necrosis factor antagonist cortistatin14 in preparing autoimmune inflammatory disease treatment drugs, in particular, the application of the cortistatin14 serving as the tumor necrosis factor antagonist to antagonize TNF- α.

In particular to the application in preparing the psoriasis treatment medicine.

Also includes the application in preparing the medicine for treating rheumatoid arthritis and osteoarthritis.

And predictable use in the treatment of lupus erythematosus.

The medicine also comprises a pharmaceutically acceptable carrier, an auxiliary agent or a diluent.

The form of the medicament is selected from one of the following forms: sprays, aerosols, solutions, lotions, gels, ointments, pastes, emulsions and suspensions.

Preferably, the form of the medicament is selected from one of the following: cream and ointment.

For an ointment, the ointment comprises an agent selected from the group consisting of: stearic acid, stearyl alcohol, cetyl alcohol, glycerol, water, and mixtures thereof, and the medicament has a physiologically acceptable pH.

For a cream, the cream comprises an agent selected from the group consisting of: stearic acid, stearyl alcohol, cetyl alcohol, glycerol, water, and mixtures thereof, and the medicament has a physiologically acceptable pH.

The medicament is preferably in an injection form, and the administration mode is preferably nanoparticle-mediated local administration.

In addition, cortistatin-17 belongs to another existing form of cortistatin in animals and humans, and studies show that cortistatin-14 and cortistatin-17 have similarities in function, so that cortistatin-17 has the possibility of developing and applying in autoimmune diseases in the future.

The results of intermolecular interaction experiments in vitro and in vivo animal model experiments show that cortistatin14 (CST-14) can directly bind to the cell surface receptor of TNF- α, antagonize the function of TNF- α, and exert a protective effect in autoimmune inflammatory diseases such as psoriasis, etc. CST-14 belongs to a novel TNF- α antagonist drug, can be directly synthesized by the prior art, thereby reducing the cost.

Drawings

FIG. 1 is a graph of the visual therapeutic effect of cortistatin14 in mouse dermatitis

FIG. 2 is a psoriasis score for the effect of cortistatin14 on treatment of mouse dermatitis

FIG. 3 is the CO-IP measured ability of cortistatin14 to bind to TNF

FIG. 4 is a solid phase binding assay for determining the binding ability of cortistatin14 to the tumor necrosis factor receptors TNFR1 and TNFR2

FIG. 5 is a graph of the effect of cortistatin14 on macrophage polarization process in animal models

Figure 6 is a histological study examining the effect of cortistatin14 in reducing epidermal proliferation and inflammatory cell infiltration in a mouse model of dermatitis.

FIG. 7 is a visual comparison of MRL/lpr knockout mouse models in systemic lupus erythematosus animal experiments

FIG. 8 comparison of spleen volumes in MRL/lpr knockout mouse model for systemic lupus erythematosus animal experiments

FIG. 9 comparison of body weights of MRL/lpr knockout mice model in systemic lupus erythematosus animal experiments

FIG. 10 osteosarcoma HOS cell scratch test

FIG. 11 statistical analysis of osteosarcoma HOS cell scratch experiment

FIG. 12EdU experiment

Detailed Description

Sources of experimental animals, reagents, media and buffers referred to in the following examples:

cortisan 14 (purity 98.4%, CAS:186901-48-4, amino acid sequence Pro-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Ser-Ser-Cys-Lys (Disulfide bridge: Cys2-Cys13)) (Gill Biochemical Co., Ltd.)

BL6/C57 male mouse (Shandong university animal center)

PBS buffer (Biyuntian biological reagent company)

F4/80 antibody, CD86 antibody, CD206 antibody (Thermo Fisher, Pierce)

RIPA cell protein extraction lysate (Thermo Fisher, Pierce)

Protease inhibitor (Beijing Solaibao Tech Co., Ltd.)

BCA protein quantification kit (Shanghai Yanxi Biotech Co., Ltd.)

Complete EDTA-Free (Roche biomedicine)

Xylene (national drug group chemical reagent Co., Ltd.)

Neutral gum (Shanghai Tantake skill Co., Ltd.)

Concentrated hydrochloric acid (national drug group chemical reagent Co., Ltd.)

Eosin (Shanghai Tantake skill Co., Ltd.)

Hematoxylin (Shanghai Tantake skill Co., Ltd.)

Methanol (national medicine group chemical reagent Co., Ltd.)

Citrate buffer (0.01M, pH 6.0) (Biotechnology engineering, Shanghai Co., Ltd.)

10% NGS (biological engineering Shanghai share Co., Ltd.)

Hydrogen peroxide (H2O2) (national drug group chemical Co., Ltd.)

BSA (Biotechnology Shanghai GmbH)

Absolute ethyl alcohol (national medicine group chemical reagent Co., Ltd.)

Cell counter was purchased from Thermo Fisher, USA

Microscope from Shanghai Caikang optical instruments Ltd

Centrifuge from Jinan Olaibo medical instruments Ltd

Electronic balance from medical instruments ltd, denna olabo

Enzyme-linked immunosorbent assay (ELISA) instrument purchased from Beijing Meihua apparatus science and technology Limited

Flow cytometry was purchased from BD Co

Ice machine from Jinan Ou Laibo medical instruments Ltd

Ultra pure water system is commercially available from Jinan Oolabo medical instruments Ltd

Vortex mixer from Jinan Olaibo medical devices, Inc

Biocare 3000 available from GE health care

1. Construction of mouse model for skin inflammation

A mouse model of imiquimod (imiquimod) induced skin inflammation was established in 10 week old wild type BL6/C57 male mice (total of 42). First, all mice were shaved of their dorsal skin with a razor and depilatory cream. Wild type mice (14) from the blank group were daily coated with petrolatum cream on shaved back skin and injected intraperitoneally with sterile PBS buffer. Wild type mice (14) of the experimental treatment group were then coated daily with 62.5mg of 5% imiquimod cream on shaved back skin and injected intraperitoneally with exogenous cortistatin14 (1 μ g/200 μ l) for 1 week. Wild type mice (14) in the scytotis group were daily coated with 62.5mg of 5% imiquimod cream on shaved back skin and injected intraperitoneally with equal amounts of sterile PBS buffer. Based on the psoriasis-like skin area severity index, an assessment differentiating the severity of skin inflammation was made using clinical scores as previously reported. In short, erythema, scaling and thickening were scored 0 to 4 (0: none; 1: mild; 2: moderate; 3: significant; 4: severe). Cumulative scores (ratio: 0-12) of these three parameters are shown to assess the severity of skin inflammation. Two experimenters do not influence each other by adopting a double-blind method and independently score. After 1 week, all groups of mice were euthanized with an excess of 10% chloral hydrate (qiu hospital, shandong university, china) and dorsal skin specimens were collected for subsequent testing.

Fig. 1 and 2 are graphs of the results of this experiment, showing that psoriasis dermatitis in mice has significant relief from treatment with cortistatin14 by visual photographs and psoriasis scores.

2. Flow cytometry analysis of macrophages

To determine macrophage subpopulations, mouse skin samples from an imiquimod-induced mouse model of skin inflammation were ground to a single cell suspension, filtered through a 200 mesh screen and stained for flow cytometry. Briefly, fluorescently conjugated antibodies (anti-F4/80, anti-CD 86, anti-CD 206) were added to a single cell suspension (100. mu.l). After gentle mixing, the samples were incubated at 4 ℃ for 20 minutes and then analyzed using a flow cytometer. Macrophage subpopulation analysis was determined by analyzing the percentage of positive cells and the intensity of the positive signal. Flow cytometer parameter setting: FITC is detected through an FITC channel (FL-1), the excitation wavelength is 488nm, and the emission wavelength is 530 nm; PI is detected through FL-2 channel, excitation wavelength 488nm, emission wavelength 630 nm.

Fig. 5 shows that, by flow cytometry, cortistatin14 has a good therapeutic effect in dermatitis by regulating macrophage polarization to M2 subtype with anti-inflammatory function.

3. Tissue slice preparation

The skin of mice from all groups was fixed in 10% formalin for at least 72 hours at room temperature. Sequentially dehydrating the tissue with 50% ethanol (60 min), 70% ethanol (60 min), 85% ethanol (60 min), 95% ethanol (60 min), 100% ethanol (30 min); sequentially treating the mixture with ethanol, xylene (60 minutes) and xylene (60 minutes); then clear with xylene and paraffin (60 min), paraffin (80 min); the tissue was placed in a cassette, filled with paraffin, and then placed on the cold plate of a paraffin embedding machine. Placing the embedded tissue paraffin block on a microtome and sectioning the tissue to a thickness of about 4 μm; the organized paraffin pieces were lightly smeared in water at 42 ℃. After the glass is completely flattened, a clean glass sheet is used for gently pulling up the slices; the sections were placed on glass slides, numbered, and baked in an oven at 68 ℃ for at least 6 hours.

4. Hematoxylin/eosin staining

The slices were dewaxed with a conventional fat-soluble solvent to water (xylene twice, 15 min/each time; 100% alcohol 5 min; 95% alcohol 5 min; 75% alcohol 5 min; 50% alcohol 5 min), then stained with hematoxylin staining solution for 5min, rinsed with clear water, then stained with eosin staining solution for 5min, rinsed with clear water, dehydrated (50% alcohol 5 min; 75% alcohol 5 min; 95% alcohol 5 min; 100% alcohol 5 min; xylene twice, 15 min/each time), after which the slices were air-dried, they were sealed with neutral gum, and observed and analyzed under an optical microscope.

5. Immunohistochemical staining

Tissue sections were deparaffinized and hydrated. I.e., 8 minutes of xylene, 8 minutes of absolute ethanol, 8 minutes of 95% ethanol, 8 minutes of 80% ethanol, and 8 minutes of 75% ethanol in succession. After 8 minutes in 70% ethanol, the sections were rinsed 4 times for 5 minutes each; next, the deparaffinized and hydrated sections were placed in a 3% hydrogen peroxide solution and reacted at 37 ℃ for 20 minutes to block endogenous peroxidase. The antigen was repaired by washing 4 times with double distilled water each time for 5 minutes. The citrate buffer was placed in a metal heater and boiled. Boil for 15 minutes, turn off power and hold for 15 minutes. Naturally cooling to room temperature; PBS was then washed 5 times each for 5 minutes, the surrounding tissue was wiped, 5% goat serum was added, non-specific antigen was blocked, reaction was performed at room temperature for 1 hour, then CD68 antibody was added drop-wise to the tissue slices and incubated overnight in a wet box at 4 ℃; the next day, sections were removed, incubated in an incubator at 37 ℃ for 1 hour, rinsed 5 times with PBS every 15 minutes, and an enhanced horseradish peroxidase-conjugated secondary antibody was added and incubated at room temperature for 2 hours. Excess secondary antibody was washed with PBS (5 times for 5 minutes each); dropwise adding a freshly prepared DAB coloring solution, observing under an optical microscope, and taking on a brown yellow color, and washing with PBS to stop color development; next, hematoxylin counterstaining was performed. The stained sections were placed in modified hematoxylin stain and stained for 5 minutes. The staining was observed under an optical microscope. The sections were then separated with 0.2% hydrochloric acid and washed with running water. Finally, the slices were sequentially rinsed with 70% alcohol for 10 minutes, 75% alcohol for 8 minutes, 80% alcohol for 8 minutes, 90% alcohol for 8 minutes, absolute ethanol for 8 minutes, xylene for 8 minutes, and xylene for 8 minutes. After dehydrating for 2 minutes, the tissue surrounding the tissue was wiped and a neutral gel was dropped, and the cover glass was placed under an optical microscope for observation.

Fig. 6 is a graph showing the results of the above experiments 4 and 5, and a graph showing HE staining results showing that cortistatin14 treatment has an excellent alleviating effect on the infiltration of inflammatory cells such as macrophages into the skin.

Panel B is an immunohistochemical staining using CD68 immunohistochemistry as a marker molecule for macrophages, confirming the function of cortistatin-14 to improve inflammatory cell infiltration in the psoriasis model.

CO-IP and Western blot detection

180. mu.l of RIPA lysate was removed, and 200ul of tissue lysate was prepared by adding 10ul of protease inhibitor and 10ul of phosphatase inhibitor. Then placing the frozen chondrocyte sample taken out from a refrigerator at the temperature of-80 ℃ in an EP tube, adding the prepared tissue lysate, and placing the EP tube in a homogenizer pre-cooled by liquid nitrogen for homogenization; the homogenization time and number are set. After complete homogenization of the cells, the EP tubes were removed, centrifuged at 12000rpm, centrifuged at 15 ℃ for 15 minutes, and the supernatant was transferred to a new 1.5ml EP tube. About 20ul of cell lysis supernatant was left and boiled with 2Xloading buffer for 5min to obtain input group. Agarose beads (S beads) were aliquoted into new EP tubes in advance, using tips cut off to aspirate beads, and ensuring consistent amounts of beads in each tube, carefully aspirating the supernatant, adding protein A antibodies and cell lysed supernatant. 1mg of protein lysate was added to 25ul of suspension containing 1: 1S beads and 2ug of protein A antibody. Shaking table incubation at 4 deg.C for 2-4h, Binding, 1400rx1min, and centrifuging at 4 deg.C. Sucking the supernatant by a vacuum pump or a pipette, paying attention to no suction of the precipitate, adding 800ul of NETN, inverting and mixing up and down to ensure that the precipitate at the bottom is suspended, centrifuging, washing beads for three times, discarding the supernatant for the last time, sucking up residual liquid by a spotting gun head, adding 15ul of 2xloading buffer for boiling for 5min to be used as Co-IP group spotting, and adding 10ul of 2xloading buffer to the residual precipitate for boiling once again to be used as IP group spotting.

The electrophoresis tank is placed on an electrophoresis device, and the power supply is turned on, and the positive electrode and the negative electrode are installed. The voltage was adjusted to about 150V to maintain a constant voltage. When the bromophenol blue label moved to the bottom of the gel, the power was turned off and the running buffer was poured back into the vial. Preparing 1000mL of membrane transfer buffer solution and precooling at 4 ℃; soaking 2 pieces of thick filter paper (about 8 × 10cm in size) and nitrocellulose membrane 30 min before electrophoresis, and soaking sponge clips in the transfer buffer; immersing the glass plate in the transfer solution, carefully taking out the glass plate with a plastic spatula, taking the gel off the glass, and removing all the concentrated gel and unnecessary separating gel; assembling a film transfer interlayer according to the sequence of sponge-filter paper-gel-nitrocellulose film-filter paper-sponge; placing the transfer clip into a transfer tank to ensure that the gel side of the transfer clip faces the cathode and the side of the membrane faces the anode, adding an appropriate amount of buffer solution to the transfer tank to ensure that the transfer clip is completely immersed in water, and placing the transfer clip into an ice box; inserting the black electrode lead into a cathode socket of the film transfer device, inserting the red electrode lead into an anode socket, and connecting the anode lead and the cathode lead to corresponding power supply outputs; placing the film rotating device on ice, turning on a power supply, setting the transfer condition of the instrument to be constant current 240mA, and setting the film transfer time to be 90 min; after the transfer, the film was taken out from the transfer apparatus and transferred to a cassette, the press plate was carefully opened to transfer the PVDF film layer by tweezers, the nitrocellulose PVDF film was taken out, and a corner was cut at the upper right near the film as a mark. Blocking and antibody incubation: the transferred nitrocellulose membrane was taken out, washed with TBST for 1 minute, and the transfer solution remaining on the membrane was washed off and then placed face up in a 5% nonfat dry milk blocking solution. The membrane was cut to size and ready for primary antibody incubation at room temperature on a shaker for 1 hour. Primary antibody incubation: and (3) diluting the primary antibody, soaking the membrane in a proper amount of diluted primary antibody, and putting the membrane into a refrigerator at 4 ℃ overnight (8-12 h). Washing a primary antibody: the membrane was removed and washed with TBST on a shaker for 5min 3 times. And (3) secondary antibody incubation: the concentration of the goat anti-rabbit secondary antibody and the goat anti-mouse secondary antibody is 1: 10000 secondary antibody dilution. The membrane was immersed in an appropriate amount of diluted secondary antibody and incubated at room temperature for 1 hour. Washing a secondary antibody: after removal of the membrane, it was washed 5min × 3 times with TBST on a shaker. And (3) developing: the same amount of the hypersensitive ECL chemiluminescent kit was taken and mixed well with liquid a and liquid B, shielded from light, and then the nitrocellulose membrane was placed on a black film, right side up, and an appropriate amount of the chromogenic mixture was applied to the film with a pipette at room temperature for development and imaging in a gel imaging system.

FIG. 3 shows that, in chondrocytes, cortistatin-14 can form a complex with cell surface tumor necrosis factor receptors TNFR1 and TNFR2, and thus, cortistatin-14 binds to TNFR1 and TNFR 2.

Fig. 3 and 4 are graphs showing the results of direct binding of cortistatin14 to tumor necrosis factor receptors TNFR1 and TNFR 2. FIG. 3 shows that cortistatin14 is bound to TNF receptor by antibody detection as measured by CO-IP, and FIG. 4 shows that A-B is measured by solid phase binding assay, and that cortistatin14 has direct binding ability to TNF receptor.

Through the experiments, the CST-14 can be directly combined with the TNFR of the cell surface receptor of TNF- α, has exact curative effect on an imiquimod induced psoriasis animal model, and effectively promotes the macrophage to convert to an M2 anti-inflammatory subtype, meanwhile, the cortistatin-14 is a polypeptide molecule, the molecular weight of which is 21kD, is obviously smaller than that of a previous similar preparation (etanercept, infliximab, adalimumab and the like), and the single dosage can be reduced in comparison.

7. Animal experiments

Animal experiments show that the molecule is used for a long time (the mouse experiment lasts for more than 3 months) and no obvious organ damage and mouse behavior change are observed.

In addition, mouse experiments were also performed for lupus erythematosus, another common autoimmune inflammatory disease. FIG. 7, 8 and 9 show that the CST treatment has a relieving effect on lupus occurrence of mice, and the body weight of the mice has no obvious change after the cortistatin-14 treatment for 3 months, as shown by an animal experiment MRL/lpr gene knockout mouse model of systemic lupus erythematosus.

In FIG. 7, it is shown that SLE facial plaque alopecia and skin lesions are significantly reduced, in FIG. 8 splenomegaly is shown in the non-treated group and cortistatin-14 is shown in the treated group. Figure 9 shows no significant difference in body weight between the two groups.

Meanwhile, the early in vitro cell data show that cortistatin-14 stimulates tumor cells to produce no obvious sign of migration and proliferation. In FIG. 10, osteosarcoma HOS cell scratch assay; FIG. 11, statistical analysis of scratches, cortistatin-14 did not significantly promote migration; figure 12EdU experiment, cortistatin-14 stimulation did not significantly stimulate HOS cell proliferation compared to control.

Both tests are commonly known in the art and are not described herein.

All data are expressed as mean standard deviations of at least three independent experiments. Statistical analysis for two groups of data statistical analysis using paired t-tests more than two groups of data statistical analysis using one-way analysis of variance.

More importantly, the CST-14 is simple to prepare, the preparation method is pure and mature, the preparation method can be directly synthesized, and the early-stage similar drugs are all biological agents, so that the cost of the CST-14 can be obviously reduced compared with other similar TNF- α inhibitors, and the economic burden of patients is reduced.

While one embodiment of the present invention has been described in detail, the description is only a preferred embodiment of the present invention and should not be taken as limiting the scope of the invention. All equivalent changes and modifications made within the scope of the present invention shall fall within the scope of the present invention.

Claims (9)

1. The application of cortistatin14 in preparing the medicine for treating autoimmune inflammatory diseases.

2. The application of the cortistatin14 in preparing the medicine for treating the autoimmune inflammatory diseases is characterized in that the cortistatin14 is used as a tumor necrosis factor antagonist and is realized by antagonizing the function of TNF- α.

3. Use of cortistatin14 in the manufacture of a medicament for the treatment of psoriasis.

4. Application of cortistatin14 in preparing medicaments for treating rheumatoid arthritis and osteoarthritis.

5. Application of cortistatin14 in preparing medicine for treating lupus erythematosus.

6. The use of any one of claims 1 to 5, wherein the medicament further comprises a pharmaceutically acceptable carrier, adjuvant or diluent.

7. The use according to any one of claims 1 to 5, wherein the medicament is in a form selected from one of: sprays, aerosols, solutions, lotions, gels, ointments, creams, salves, pastes, emulsions, suspensions.

8. The use according to any one of claims 1 to 5, wherein the medicament is preferably in an injectable dosage form.

9. Use according to any one of claims 1 to 5, wherein the mode of administration is preferably nanoparticle-mediated topical administration.

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