Use of aminothiazole derivatives in the preparation of immune inflammation drugs

Abstract

The present invention provides a use of an aminothiazole derivative in the preparation of an immune inflammation drug. The drug of the present invention has obvious effects on surface immune inflammatory dermatitis such as psoriasis, neurodermatitis, atopic dermatitis eczema, allergic dermatitis, urticaria, seborrheic dermatitis and alopecia, abnormal scar formation, pathological pigmentation, etc., and is safe and non-toxic. There are also surface inflammations such as: chronic rhinitis, conjunctivitis, dry eyes, allergic asthma, chronic bronchitis, immune inflammation caused by various mucosal and epidermal injuries, and various mucosal and epidermal chronic inflammations. Immune inflammatory diseases also include other chronic, refractory, immune inflammatory diseases such as autoimmune diseases (such as diabetes, systemic lupus erythematosus), various immune chronic inflammatory diseases (such as chronic colitis, allergies), ischemia-reperfusion injury, chronic inflammation of the nervous system (such as neurodegeneration, Heimer's syndrome, depression, etc.), etc.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the use of an aminothiazole derivative in the preparation of an immune inflammation drug. Background Art

[0002] Medicine has long proven that inhibitory regulation of the body's immune system is the key to treating a variety of diseases, such as organ transplant rejection, autoimmune diseases, inflammatory diseases, ischemia-reperfusion injury, etc.

[0003] Chronic refractory inflammatory diseases can manifest in various organs and form a variety of inflammatory diseases, such as autoimmune diseases, various immune inflammatory diseases, chronic inflammatory diseases, etc. Surface inflammation refers to inflammation occurring on the surface of the skin or mucous membranes, including infectious inflammation and immune inflammation caused by immune disorders. Immune surface inflammation often develops into chronic refractory inflammation. Immune surface inflammation is a large category of refractory immune inflammatory diseases. Due to the characteristics of the surface tissue, it is easy for a large number of inflammatory stimuli and a large number of inflammatory cells to accumulate. In the process of cell pyroptosis, the inflammation continues to expand, and the vicious cycle makes the surface inflammation protracted. Immune surface inflammation includes dermatitis and mucosal inflammation, such as dermatitis, rhinitis, conjunctivitis, mucosal and epidermal damage inflammation, etc. Chronic vicious cycle immune response is considered to be the key step in the occurrence of refractory surface inflammation. In the process of the occurrence and development of refractory surface dermatitis, the activation of various immune cells such as macrophages, neutrophils and mast cells rich in surface tissues will lead to the synthesis and secretion of various inflammatory cytokines, attracting more inflammatory cells to gather in the surface tissues, triggering a series of subsequent vicious cycles. This is the chronic immune response and the main reason for the prolonged and unhealed surface inflammation. Therefore, inhibiting inflammation and interrupting the vicious cycle have become the key to the treatment of immune-inflammatory surface inflammation, but it is not limited to the above-mentioned inflammation. Similar vicious cycles also exist in autoimmune diseases, various immune-inflammatory diseases, and chronic inflammatory diseases.

[0004] Pyroptosis is a new inflammatory mechanism that plays an important role in various inflammations. When cells undergo pyroptosis, the cell membrane is damaged, the cells swell and rupture, and a large number of inflammatory factors are released, leading to a severe local inflammatory response and recruiting a large number of inflammatory cells to gather locally, forming a vicious cycle of inflammation. The classical pyroptosis pathway believes that pathogenic stimuli such as LPS and HMGB1 can bind to RAGE receptors, enter cells through endocytosis, activate Pro-Caspase-11, and lead to pyroptosis (see Appendix). Figure 1 ).

[0005] The prior art provides an aminothiazole derivative, whose structure and function are disclosed in the Chinese invention patent CN201110049687.4 "2-aminothiazole derivatives and preparation methods and applications". This type of small molecule compound inhibits myeloid differentiation protein (MYD-88) and blocks the activation of NF-кB, thereby producing an immunosuppressive effect, and has therapeutic effects in anti-transplant rejection, anti-autoimmune diseases, anti-ischemia reperfusion injury, anti-chronic inflammatory response, and anti-endotoxinemia.

[0006] However, our experiments found that other NF-кB inhibitors do not have a good effect in chronic refractory inflammatory diseases, and there are currently no commercially available or clinically effective inflammatory drugs targeting NF-кB. Therefore, in this study, we further studied the cell pyroptosis process and discovered a new effective conduction pathway for this type of aminothiazole derivatives in inhibiting cell pyroptosis. Summary of the invention

[0007] The purpose of the present invention is to provide the use of the above-mentioned aminothiazole derivatives in the preparation of immune inflammatory drugs. The aminothiazole derivatives of the present invention have a strong inhibitory effect on pyroptosis. By inhibiting the assembly and activation of inflammasomes and the occurrence of pyroptosis of innate immune cells (mainly macrophages), they can not only effectively inhibit inflammation, but also block the vicious cycle of inflammation, and obtain good therapeutic effects in a series of pharmacodynamic tests of immune inflammatory surface inflammation.

[0008] The basic molecular structure of the aminothiazole derivative small molecule compound described in the present invention is as follows:

[0009]

[0010] In the formula, R1 is phenyl or substituted aromatic group; R2 is benzyl, substituted benzyl, phenyl or substituted phenyl; and n=0, 1 or 2.

[0011] Our mechanism study confirmed for the first time another pyroptosis activation pathway, which activates NF-кB from the classic TLR / MyD88 activation pathway, and then activates pyroptosis through the NLRP3–Caspase-1–GSDMD-N pathway. Further experiments found that this type of aminothiazole derivative can inhibit the expression of NLRP3 in immune cells, and through the secondary inhibition of Caspase-1, cell pyroptosis is inhibited. It can also inhibit the release of inflammatory factors IL-1b and IL-18 by reducing the pathways of Pro-IL-1b and Pro-IL-18 in cells. (See Example 2 for related experiments)

[0012] Therefore, this type of aminothiazole derivatives can be used as cell pyroptosis inhibitors, NLRP3 inflammasome inhibitors, caspase-1 / caspase-11 inhibitors, and Gasdermin D (GSDMD-N, the N-terminus of GSDMD) inhibitors.

[0013] The aminothiazole derivatives of the present invention have extremely small molecules, stable structures, can penetrate cell membranes, and can be used both in vivo and in vitro. Other uses of internal preparations of this type of compound also include other chronic, refractory, immune inflammatory diseases such as autoimmune diseases (such as diabetes, systemic lupus erythematosus), various immune chronic inflammatory diseases (such as chronic colitis, allergies), ischemia-reperfusion injury, chronic inflammation of the nervous system (such as neurodegeneration, Heimer's syndrome, depression, etc.), etc.

[0014] Such aminothiazole derivatives include N-(4-phenylthiazol-2-yl)-3-(4-benzylpiperazin-1-yl)propionamide (product number INNA1605 of Yingnei Company), N-(4-(3-methoxyphenyl)thiazol-2-yl)-2-(4-(4-methylphenyl)piperazin-1-yl)acetamide (product number INNA1602), N-(4-phenylthiazol-2-yl)-2-(4-benzylpiperazin-1-yl)acetamide (product number INNA1608); N-(4-(3-methoxyphenyl)thiazol-2-yl)-2-(4-(4-methylphenyl)piperazin-1-yl)acetamide (product number INNA1609); ) thiazol-2-yl)-3-(4-benzylpiperazin-1-yl) propionamide (product number INNA1609); N-(4-(4-methoxyphenyl)thiazol-2-yl)-3-(4-(4-methylphenyl)piperazin-1-yl) propionamide (product number INNA1611); N-(4-phenylthiazol-2-yl)-2-(4-benzylpiperazin-1-yl) acetamide (product number INNA1612) are drugs with the same efficacy, stability and safety verified by the inventor, and the structural formula is as follows:

[0015]

[0016]

[0017] Preliminary studies have shown that small molecule compounds of the aminothiazole derivative class can inhibit the synthesis and secretion of inflammatory factors, and their therapeutic effects are basically the same. This patent mainly uses INNA1605 and non-infectious superficial dermatitis as examples for illustration.

[0018] The present invention also discloses specific examples of the aminothiazole derivatives as external preparations (ointments, liniments, sprays, patches, drops), which are used for surface immune inflammation and other refractory immune diseases as internal preparations (oral and intravenous preparations), such as various immune inflammatory dermatitis (psoriasis, neurodermatitis, atopic dermatitis, eczema, allergic dermatitis, urticaria, and similar dermatitis).

[0019] Beneficial effects of the present invention: In animal experiments and sympathetic treatment of patients, it is fully confirmed that the drug has excellent curative effect in many immune inflammatory diseases, including: 1. It has obvious effects on surface immune inflammatory dermatitis such as psoriasis, neurodermatitis, atopic dermatitis eczema, allergic dermatitis, urticaria, seborrheic dermatitis and alopecia, abnormal scar formation, pathological pigmentation, etc., and is safe and non-toxic. There are also other types of immune surface inflammation as follows but not limited to: chronic rhinitis, conjunctivitis, dry eye, allergic asthma, chronic bronchitis, immune inflammation caused by various mucosal and epidermal injuries, and various mucosal and epidermal chronic inflammations. 2. Immune inflammatory diseases also include other chronic, refractory, immune inflammatory diseases such as autoimmune diseases (such as diabetes, systemic lupus erythematosus), various immune chronic inflammatory diseases (such as chronic colitis, allergy), ischemia-reperfusion injury, chronic inflammation of the nervous system (such as neurodegeneration, Heimer's syndrome, depression, etc.), etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Diagram of the mechanism of macrophage activation of pyroptosis.

[0021] Figure 2 It is a series of aminothiazole-derived small molecule compounds (INNA1602 (TJ-2), INNA1605 (TJ-5), INNA1608 (TJ-8), INNA1609 (TJ-9), INNA1611 (TJ-11), INNA16012 (TJ-12)) that can inhibit LPS-induced macrophages from secreting a large number of inflammatory factors and have the same inflammatory factor inhibitory effect.

[0022] Figure 3 INNA1605 (TJ-5) inhibits pyroptosis of bone marrow-derived macrophages. A. Apoptosis of bone marrow-derived macrophages in each group (PI + C. LDH concentration in the supernatant of bone marrow-derived macrophages in each group. DF. Expression of GSDMD and GSDMD-N proteins in bone marrow-derived macrophages in each group.

[0023] Figure 4INNA1605 (TJ-5) inhibits the classical and non-classical pyroptosis pathways of bone marrow-derived macrophages and reduces the synthesis and secretion of multiple inflammatory factors. AC. Expression of NLRP3 inflammasome and its component proteins and non-classical pyroptosis-related proteins in bone marrow-derived macrophages of each group. D. Synthesis and secretion of multiple inflammatory factors in bone marrow-derived macrophages of each group.

[0024] Figure 5 In the figure, the effect of INNA1605 ointment on the treatment of psoriasis in Balb / c mice. a. Normal control group, b. IMQ model group, c. INNA1605 treatment group.

[0025] Figure 6 Showing skin histopathological changes after INNA1605 treatment of psoriasis.

[0026] Figure 7 It was shown that INNA1605 could inhibit the MAPK signaling pathway in the psoriasis model.

[0027] Figure 8 These are the results of MPO (neutrophil) immunohistochemistry and CD86 (macrophage) immunohistochemistry in skin tissue of psoriasis after INNA1605 treatment.

[0028] Fig. 9 INNA1605 inhibits mast cell infiltration and proliferation in psoriasis models. A. H&E staining of mouse skin tissue under high magnification. B. C-kit (CD117) staining results of mast cell-specific markers in mouse skin tissue.

[0029] Fig.10 INNA1605 significantly reduced the expression of CD80.

[0030] Fig.11 INNA1605 inhibits the expression of psoriasis intradermal lymphokines (IL-1beta, IL-6, IL-17a, TNF-alpha, MyD88 mRNA, Cxcl2 mRNA).

[0031] Fig.12 The gross appearance and weight of spleen in each group after INNA1605 treatment of psoriasis, flow cytometry graphs of spleen-derived T cells and their subsets in each group, and flow cytometry ratio statistics.

[0032] Fig.13 H&E pathological images and keratinized skin thickness measurement statistics to show that INNA1605 treatment reduces the excessive keratinization and proliferation of keratinocytes in the skin tissue of psoriasis mice.

[0033] Fig.14Pathological changes of atopic dermatitis (eczema) in mice treated with INNA1605.

[0034] Fig.15 The expression levels of inflammatory factors in the skin of mice with atopic dermatitis (eczema) after INNA1605 treatment.

[0035] Fig.16 The changes of inflammatory factors in atopic dermatitis (eczema) in mice at different times: the models were tested on days 8, 10, and 12 respectively. IL-4 and IL-13 increased on the 8th day, and reached a higher level on days 10-12 (the expression level was almost the same as the internal reference); the expression levels of TSLP and MyD88 reached the highest peak before the 8th day, and the expression decreased on days 10-12.

[0036] Fig.17 INNA1605 treatment inhibits colon mucosal inflammation and colon mucosal epithelial cell proliferation. A. H&E pathological changes of colon mucosa. B. Neutrophil infiltration of colon mucosa, i.e. MPO immunohistochemical pathological staining. C&D. BrdU staining of colon membrane and Ki-67 immunohistochemical pathological conditions, i.e., the proliferation of colon mucosal epithelial cells in each group.

[0037] Fig.18 INNA1605 ointment for the treatment of allergic dermatitis caused by insect bites (compassionate treatment clinical study).

[0038] Fig.19 A compassionate care clinical study of INNA1605 ointment in the treatment of neurodermatitis.

[0039] Fig. 20 INNA1605 ointment for the treatment of eczema (compassionate treatment clinical study).

[0040] Fig.21 INNA1605 ointment for the treatment of urticaria (sympathetic treatment clinical study).

[0041] Fig. 22 INNA1605 ointment for the treatment of psoriasis (compassionate treatment clinical study).

[0042] Fig.23 INNA1605 ointment for the treatment of proteinosis dermatitis (compassionate treatment clinical study).

[0043] Fig.24 INNA1606 (TJ-M2010-6) can prevent the onset of type 1 diabetes in NOD mice. If the drug is used in the early stage of the disease, it can also treat type 1 diabetes that has already occurred. The mechanism is that MyD88 inhibitors can significantly reduce insulitis and save pancreatic islet cells.

[0044] Fig.25 INNA1605 (TJ-M2010-5) significantly reduced the body weight of high-fat diet obese mice.

[0045] Fig.26 INNA1605 (TJ-M2010-5) significantly inhibits R848-induced B cell (lupus erythematosus-like cell) activation and differentiation into plasma cells.

[0046] Fig. 27 INNA1605 (TJ-M2010-5) significantly inhibits R848-induced B cells (lupus erythematosus-like cells) from secreting autoimmune antibodies and inflammatory factors.

[0047] Fig.28 INNA1605 (TJ-M2010-5) significantly alleviates ischemia-reperfusion injury secondary to acute cerebral infarction, reducing the volume of brain tissue necrosis in acute cerebral infarction by 80%.

[0048] Fig.29 INNA1605 (TJ-M2010-5) improves the cognitive ability of APP / PS1 mice or mice with Heimer's syndrome (water maze test).

[0049] Fig.30 INNA1605 (TJ-M2010-5) significantly reduced Aβ deposition in the brain of APP / PS1 mice with Alzheimer's syndrome. Representative immunohistochemical images of the whole brain (A), cortex (B), and hippocampus (C) of each group of mice at 9 months of age and the percentage of Aβ plaque area (D). (A bar = 1000 μm; BC bar = 200 μm) (###p<0.001vs Control group; *p<0.05vs APP / PS1 group; N = 3control group; N = 5APP / PS1 and APP / PS1+INNA1605 groups).

[0050] Fig.31 INNA1605 (TJ-M2010-5) significantly inhibits the activation of brain microglia in APP / PS1 mice, thereby reducing inflammation of brain neural tissues. Representative immunohistochemical images of the whole brain (A), cortex (B), and hippocampus (C) of each group of mice at 9 months of age and the area percentage of Iba-1-positive microglia (D). (Abar=1000μm; BC bar=200μm) (#p<0.05,###p<0.001vs Control group; *p<0.05,**p<0.01vs APP / PS1 group; N=3control group; N=5APP / PS1 and APP / PS1+INNA1605 groups).

[0051] Fig.32 INNA1605 (TJ-M2010-5) prevents depressive behavior and inflammation in mice INNA1605 (TJ-M2010-5) prevents depressive behavior and inflammation induced by the coronavirus spike protein (CSDS- / SARS-907COV-2Spike RBD).

[0052] Fig.33 Diagram of the mechanism by which INNA1605 (TJ-M2010-5) prevents depressive behavior and inflammation induced by the coronavirus spike protein (CSDS- / SARS-907COV-2Spike RBD).

[0053] Fig.34 INNA1605 (TJ-M2010-5) made skin transplantation that could not be successfully transplanted successful (Balb / c mouse skin transplanted into B6 mice).

[0054] Fig.35 INNA1605 (TJ-M2010-5) was used for a short period of time after surgery to induce permanent immune tolerance in mouse heart transplantation. A. TJ-M2010-5 was used for two weeks after allogeneic heart transplantation (Balb / c mouse heart transplanted to B6 mouse), and 60% of the heart transplants were not rejected. B. Long-term surviving recipient mice received a second transplant of the same donor mouse heart (Balb / c mouse heart transplanted to B6 long-term surviving mouse), and 100% of the transplants were not rejected, confirming the development of immune tolerance.

[0055] Fig.36 INNA1605 (TJ-M2010-5) significantly inhibits the growth of liver cancer (H22 cancer cells subcutaneously implanted). Specific implementation methods

[0056] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments. The materials used in the embodiments can be obtained from commercial channels.

[0057] Example 1: A series of aminothiazole-derived small molecule compounds [INNA1602 (TJ-M2010-2, TJ-2), INNA1605 (TJ-M2010-5, TJ-5), INNA1608 (TJ-M2010-8, TJ-8), INNA1609 (TJ-M2010-9, TJ-9) INNA1611 (TJ-M2010-11, TJ-11), INNA16012 (TJ-M2010-12, TJ-12)] inhibit the secretion of inflammatory factors by macrophages induced by LPS.

[0058] In vitro macrophage grouping:

[0059] Mouse bone marrow-derived macrophages were cultured in vitro.

[0060] The experiment was divided into 8 groups, namely:

[0061] Control group (mouse bone marrow-derived macrophages without any treatment), LPS group (macrophages stimulated with 500ng / ml LPS for 24h), LPS+TJ-2 group (30μmol / L TJ-2 drug stimulation 2h before LPS stimulation), LPS+TJ-5 group (30μmol / L TJ-5 drug stimulation 2h before LPS stimulation), LPS+TJ-8 group (30μmol / L TJ-8 drug stimulation 2h before LPS stimulation), LPS+TJ-9 group (30μmol / L TJ-9 drug stimulation 2h before LPS stimulation), LPS+TJ-11 group (30μmol / L TJ-11 drug stimulation 2h before LPS stimulation) and LPS+TJ-12 group (30μmol / L TJ-12 drug stimulation 2h before LPS stimulation). After 24h of LPS stimulation, the cell culture supernatant was removed and the concentrations of various inflammatory factors in the supernatant were detected by ELISA.

[0062] The experimental results obtained (see attached Figure 2 ), the results showed that all aminothiazole-derived small molecule series compounds [INNA1602 (TJ-2), INNA1605 (TJ-5), INNA1608 (TJ-8), INNA1609 (TJ-9) INNA1611 (TJ-11), INNA16012 (TJ-12)] can inhibit the secretion of multiple inflammatory factors in macrophages including IL-1β, Il-6, TNF-α, IL-18, IL-17, etc., and the small molecule compounds INNA1602 (TJ-2), INNA1605 (TJ-5), INNA1608 (TJ-8), INNA1609 (TJ-9) INNA1611 (TJ-11), INNA16012 (TJ-12) have the same inflammatory factor inhibitory effect.

[0063] Example 2: INNA1605 (TJ-M2010-5, JT-5) inhibits pyroptosis of bone marrow-derived macrophages in vitro.

[0064] Preparation and grouping of bone marrow-derived macrophage pyroptosis model:

[0065] Model preparation: Bone marrow-derived macrophages were stimulated with LPS (500 ng / ml) for 6 hours and with ATP (5 mmol / L) for 2 hours, and the cells and their cell supernatants were collected for detection.

[0066] The experiment was divided into 4 groups:

[0067] Control group (normal culture of cell culture medium),

[0068] Model group (LPS and ATP were added to the culture medium for stimulation),

[0069] TJ-5 (10 μmol / L) group (TJ-5, 10 μmol / L, was added 2 h before LPS stimulation),

[0070] TJ-5 (30 μmol / L) group (TJ-5, 30 μmol / L, was added 2 hours before LPS stimulation).

[0071] At the end of the experiment, cells and their supernatants were collected for detection.

[0072] The experimental results obtained (see attached Figure 3 ) as shown in the results, the PI of bone marrow-derived macrophages after LPS and ATP stimulation + The number of cells increased significantly, and the amount of LDH secreted in the cell culture supernatant also increased significantly. INNA1605 (TJ-M2010-5, JT-5) could inhibit the apoptosis of bone marrow-derived macrophages induced by LPS and ATP, and reduce the secretion of LDH (see Appendix Figure 2 ), suggesting that INNA1605 can inhibit the death of bone marrow-derived macrophages induced by LPS and ATP. INNA1605 significantly inhibited the activation and cleavage of GSDMD in bone marrow-derived macrophages induced by LPS and ATP, suggesting that INNA1605 can inhibit the pyroptosis of bone marrow-derived macrophages (Appendix Figure 3 D, F). In addition, INNA1605 can block the assembly and activation of NLRP3 inflammasome, inhibiting both classical and non-classical cell pyroptosis pathways (see Appendix Figure 4 A), thereby inhibiting the maturation and release of Pro-IL-1b and Pro-IL-18 in bone marrow-derived macrophages, interrupting the inflammatory cascade, and reducing the secretion of multiple inflammatory factors (Appendix Figure 4 BE)

[0073] Example 3: INNA1605 (TJ-M2010-5, JT-5) is used to treat psoriasis in mice (Balb / c).

[0074] Preparation and grouping of mouse psoriasis model:

[0075] Model preparation: The back skin of 6-8 week-old male Balb / c mice was shaved and 5% imiquimod (IMQ) cream was applied once a day for 7 consecutive days.

[0076] The experiment was divided into 3 groups:

[0077] Psoriasis control group (no treatment, psoriasis model group), TJ-M2010-5 systemic medication group (intraperitoneal injection) and local medication group (ointment group). Specific treatment methods are as follows:

[0078] TJ-M2010-5 systemic medication group: Balb / c mouse psoriasis model was taken and TJ-M2010-5 was injected intraperitoneally at 50 mg / kg once a day for 2 consecutive weeks.

[0079] TJ-M2010-5 topical medication group: Balb / c mouse psoriasis model was used, and 1% TJ-M2010-5 ointment was applied through the skin once a day for 2 consecutive weeks.

[0080] The experimental results obtained (see attached Figure 5 ) The results showed that the treatment effect was excellent, whether it was systemic or local administration, with significant improvement in 7 days and a cure in 14 days. Both treatments can significantly reduce inflammatory cells in the skin (see Appendix Figure 6 ), the abnormally thickened stratum corneum of psoriasis can basically restore its normal tissue structure after treatment (see attached Figure 6 ). According to the in vitro study of Example 2, INNA1605 treatment may inhibit the assembly and activation of NLRP3 inflammasomes in psoriasis models, inhibit the maturation and release of Pro-IL-1β and IL-18, and ultimately reduce the pyroptosis of skin tissue cells (see Example 2). The reduction of cell pyroptosis directly interrupts the explosive vicious cycle of skin inflammation. INN1605 also inhibits the MAPK signaling pathway (see Appendix Figure 7 ), the direct result of which is a reduction in the infiltration and activation of immune cells including macrophages, neutrophils and mast cells (see Appendix Figure 8-10 ), further inhibiting the synthesis and secretion of inflammatory factors (see Appendix Fig.11 ); The reduction of innate immune cell infiltration will inevitably lead to the inhibition of antigen presentation to adaptive immune cells. Therefore, INN1605 can improve splenomegaly in psoriasis and correct the disorder of splenic T cells and their subsets (see Appendix Fig.12 ); INN1605 can also inhibit the excessive keratinization and proliferation of skin keratinocytes, and the skin thickness is significantly thinned. (See attached Fig.13 ).

[0081] Example 4: INNA1605 (TJ-M2010-5, TJ-5) is used to treat atopic dermatitis / eczema in mice (C57).

[0082] Preparation and grouping of mouse psoriasis model:

[0083] Model preparation: 2% M7309 (calcipotriol) ointment was applied to the ears of 6-8 week-old male C57 mice once a day for 10 consecutive days.

[0084] The experiment was divided into 3 groups:

[0085] Eczema control group (no treatment, eczema model group), TJ-M2010-5 systemic medication group (intraperitoneal injection) and local medication group (ointment group). Specific treatment methods are as follows:

[0086] TJ-M2010-5 systemic medication group: mice with eczema model were given intraperitoneal injection of TJ-M2010-5, 50 mg / kg, once a day for 2 weeks.

[0087] TJ-M2010-5 topical medication group: mice with eczema model were treated with 1% TJ-M2010-5 ointment applied to the skin once a day for 2 weeks.

[0088] The experimental results obtained (see attached Fig.14 ) The results show that topical medication has excellent therapeutic effects. The abnormally thickened stratum corneum of dermatitis can basically restore its normal tissue structure after treatment, and the inflammation is significantly reduced (see Appendix Fig.13 ). Various inflammatory factors are reduced (see attached Fig.15 ), and over time, various inflammatory factors showed peak and decrease changes (see Appendix Fig.16 ).

[0089] Example 5: INNA1605 (TJ-M2010-5, TJ-5) is used to treat azoxymethane / dextran sulfate sodium (AOM / DSS)-induced inflammatory colon cancer in mice (C57).

[0090] Preparation and grouping of mouse inflammatory colon cancer model:

[0091] Model preparation: Inflammatory colon cancer model was induced by intraperitoneal injection of AOM (10 mg / Kg) / DSS (2.5%) into male C57 mice aged 6-8 weeks.

[0092] The experiment was divided into 2 groups:

[0093] Inflammatory colon cancer model group (no treatment, i.e. AOM / DSS group) and TJ-M2010-5 medication group (intraperitoneal injection). The specific treatment methods are as follows:

[0094] TJ-M2010-5 treatment group: 2 days before C57 mice were intraperitoneally injected with AOM (10 mg / Kg) / DSS (2.5%) to induce inflammatory colon cancer, TJ-M2010-5 was intraperitoneally injected at 50 mg / kg once a day for 10 consecutive weeks.

[0095] The experimental results obtained (see attached Fig.17) showed that the model group had severe ulcerative colitis, and 100% of them became cancerous. The results showed that after the administration of TJ-M2010-5, the therapeutic effect was excellent, and the inflammatory tissue damage of the colon mucosa of the treated mice was significantly reduced (Appendix Fig.17 A), neutrophil infiltration was significantly reduced (attached Fig.17 B), colitis disappeared. Moreover, after TJ-M2010-5 treatment, the proliferation of epithelial cells in the colon mucosa of mice was significantly inhibited, mucosal thickening was improved, and 100% of them were free of cancer.

[0096] Example 6: 1% INNA1605 ointment is used in a clinical study of dermatitis cases under compassionate treatment to treat atopic dermatitis / eczema, neurodermatitis, allergic dermatitis, urticaria, proteinosis dermatitis, etc.

[0097] Safety assessment: Preclinical trial results show that INNA1605 has low toxicity to the body. When directly injected intravenously at a dose of 30 mg / kg or more, liver enzyme toxicity will be increased, but at 10 mg / kg, there will be no toxicity. When the skin ointment is applied to the largest body area, only a trace drug concentration of 2-3 ng / ml can be detected in the blood, indicating that the amount absorbed through the skin is negligible and will not cause toxic side effects to the body.

[0098] Compassionate treatment: For some patients with long-term recurrent attacks, no effective treatment, and extreme pain, compassionate treatment will be provided at their initiative and strong request, after the physician's safety assessment and under close observation.

[0099] Compassionate treatment clinical study: Since these cases are only based on clinical research and observation under safety and compassionate treatment, they cannot be used for NMPA registration and declaration purposes, but only as research data for the purpose of evaluating the efficacy of the ointment.

[0100] 6.1.1% INNA1605 ointment for the treatment of allergic dermatitis caused by mosquito bites (sympathetic treatment clinical study)

[0101] Diagnosis: Allergic dermatitis and ulceration caused by mosquito bites

[0102] Enrollment time: May 12, 2019

[0103] Treatment process: A 34-year-old male had dermatitis caused by mosquito bites, with long-term itching and ulceration. He did not use other drugs. He used 1% INNA1605 cream for half a month and recovered and stopped taking the drug.

[0104] Side effects: None

[0105] Efficacy evaluation: Excellent, cured.

[0106] (See attached Fig.18 ).

[0107] 6.2.1% INNA1605 ointment for the treatment of neurodermatitis (sympathetic treatment clinical study)

[0108] Diagnosis: Neurodermatitis (multiple)

[0109] Enrollment time: October 20, 2019

[0110] Treatment process: A 58-year-old woman suffered from neurodermatitis on the back of the hairline, neck and shoulder blades, with redness, swelling and itching. Other drugs were ineffective. After using 1% INNA1605 cream for 3 weeks, the redness and swelling subsided, the itching disappeared, and the skin returned to normal. She was completely cured and has not relapsed so far.

[0111] Side effects: None.

[0112] Efficacy evaluation: Excellent, cured.

[0113] (See attached Fig.19 ).

[0114] 6.3.1% INNA1605 ointment for the treatment of eczema (sympathetic treatment clinical study)

[0115] Diagnosis: Atopic dermatitis (eczema) (multiple)

[0116] Enrollment time: May 25, 2020

[0117] Treatment process: A 30-year-old woman had multiple dermatitis / eczema on both lower limbs for many years, severe itching, constant scratching, broken skin and scabs. Long-term use of other drugs including hormone plasters were ineffective, and the disease recurred repeatedly. After two weeks of using INNA1605 (1%) cream on the lesions after enrollment, multiple medication sites on both legs no longer scratched, deposited pigment faded, and the skin quality basically returned to normal. After stopping the medication, there has been no recurrence so far.

[0118] The patient said: The anti-itching effect is immediate, the scars are reduced after healing, and it has a whitening effect.

[0119] Side effects: None.

[0120] Efficacy evaluation: Excellent, cured.

[0121] (See attached Fig. 20 ).

[0122] 6.4.1% INNA1605 ointment for the treatment of urticaria (sympathetic treatment clinical study)

[0123] Diagnosis: Urticaria (multiple)

[0124] Enrollment time: September 7, 2020

[0125] Treatment process: A 54-year-old man had urticaria in many parts of his body, which recurred repeatedly, with severe itching, redness and swelling, and he could not help scratching until it broke. Long-term use of various other drugs including hormone plasters were ineffective, and the recurrences did not heal for many years. He had used benvimod cream, which was not only ineffective, but also caused the side effect of "heart pain".

[0126] After using 1% INNA1605 cream for 12 days after enrollment, the application site no longer itched, there was no ulceration, the redness and swelling were significantly reduced, and the skin returned to normal. The patient was completely cured and has not relapsed so far.

[0127] Side effects: None.

[0128] Efficacy evaluation: Excellent, cured.

[0129] (See attached Fig.21 ).

[0130] 6.5.1% INNA1605 ointment for the treatment of psoriasis

[0131] Diagnosis: severe psoriasis.

[0132] Enrollment time: September 17, 2019

[0133] Treatment process: A 69-year-old man suffered from severe leg psoriasis for more than ten years. Long-term treatment was ineffective, including various hormone ointments and other treatments. The disease recurred and could not be cured. After only two weeks of using INNA1605 cream after enrollment, the itching disappeared, the surface scales disappeared, the whole surface became smooth, the brown-red patches became lighter, the redness and swelling subsided, and the patient felt good without any side effects. However, due to the limited ointment prepared at the time, there was no ointment after only two weeks of use, and the medication was forced to be interrupted (the standard course of treatment should be more than 3 months). The patient lost contact with the follow-up and did not continue treatment.

[0134] Side effects: None.

[0135] Efficacy evaluation: excellent.

[0136] (See attached Fig. 22 ).

[0137] 6.6.1% INNA1605 ointment for the treatment of proteinosis dermatitis

[0138] Diagnosis: proteinosis dermatitis.

[0139] Enrollment time: March 20, 2021

[0140] Treatment history: A 60-year-old man had proteinosis dermatitis for decades, which caused itching, hyperplasia, and extreme disfigurement, and he could not receive any medication.

[0141] After enrollment, the use of INNA1605 (1%) cream quickly relieved the itching and reduced the dermatitis. After one month of treatment, the symptoms were significantly alleviated. The patient is currently continuing the treatment and is required to receive treatment for more than three months.

[0142] Side effects: None.

[0143] Efficacy evaluation: excellent.

[0144] (See attached Fig.23 ).

[0145] The above experimental results directly show that the aminothiazole small molecule compound INNA1605 ointment has obvious efficacy in treating psoriasis, atopic dermatitis (eczema), allergic dermatitis, neurodermatitis, urticaria and other immune inflammatory dermatitis, and can achieve a complete cure. It will be a broad-spectrum, special-effect immune inflammatory dermatitis ointment, which can almost fill the market gap for many refractory skin diseases.

[0146] Example 7: Preparation of 2-aminothiazole derivative cream formulation

[0147] Take 1% INNA1605 cream as an example 1.1 Matrix preparation:

[0148] Preparation of oil phase: Add 10 ml (8.7 g) of liquid paraffin, 7.0 g of stearic acid, 4.0 g of white vaseline and 3.0 g of octadecyl alcohol into a beaker, heat to about 70°C with stirring until the oil phase is dissolved, and keep warm for later use.

[0149] Preparation of aqueous phase: Add 0.1 g of ethyl hydroxybenzoate, 0.2 g of sodium dodecyl sulfate, 0.40 ml (0.4 g) of triethanolamine, 10.0 ml of water, and 1.0 ml (1.0 g) of VE into a beaker, heat to 70°C, stir until the aqueous phase is dissolved, and keep warm for later use.

[0150] Preparation of flavor phase: 12 ml (15.2 g) of glycerol, 0.2 ml of flavor, and 0.5 ml (0.4 g) of azone are mixed and stirred evenly to obtain the flavor phase.

[0151] Matrix preparation process: add the aqueous phase to 49.8 ml of water at about 50°C while stirring, slowly add the oil phase to the above aqueous solution while stirring, continue stirring and emulsifying for 3 hours, add the flavor phase after cooling to room temperature, continue stirring for 30 minutes to 1 hour, until a milky white semi-solid solid is formed, and the matrix is ​​solidified for use.

[0152] 1.2 Preparation of 1% INNA1605 cream (grinding method)

[0153] 1.0 g of the finely ground (passed through a 100 mesh sieve) INNA1605 compound was placed in a mortar and added to the prepared matrix in equal amounts in portions until evenly ground. The preparation methods of other INNA1605 cream preparations with different contents were the same as above, except that the drug content was changed (e.g., for a 2% INNA1605 cream preparation, 2 g of INNA1605 compound was added to the corresponding matrix according to the above method).

[0154] Example 8: Preparation of 2-aminothiazole derivative ointment preparation.

[0155] Take the preparation of 1% INNA1602 ointment as an example

[0156] Preparation method: Grind the drug and pass it through a 100-mesh sieve for later use; heat and melt lanolin (9.0%), vaseline (85.0%), and stearic acid (4.5%), cool to 50°C, add azone (transdermal absorption enhancer, 1.5%), stir evenly, and cool to room temperature for later use. Then add the drug paste in equal amounts in batches, grinding while adding until it cools to room temperature.

[0157] The preparation methods of ointment preparations of other 2-aminothiazole compounds such as INNA1605, INNA1608, INNA1609, INNA1611, INNA1612, etc. are the same as above.

[0158] Example 9: INNA1606 (TJ-M2010-6, TJ-6) is used to treat type I diabetes in mice.

[0159] Preparation and grouping of mouse type 1 diabetes model:

[0160] Model preparation: NOD mice were purchased from Beijing Weitonglihua Biotechnology Co., Ltd. NOD mice will spontaneously develop autoimmune diabetes (type I diabetes).

[0161] The experiment was divided into 2 groups:

[0162] NOD control group (no treatment, model group);

[0163] INNA1606 (TJ-M2010-6, TJ-6) preventive medication group (oral gavage): NOD mice were gavaged with TJ-M2010-6 (50 mg / kg) daily from 8 to 30 weeks of age, or until the onset of diabetes. The control group mice were simultaneously given the same dose of drug solvent;

[0164] INNA1606 (TJ-M2010-6, TJ-6) immediate medication group (oral gavage): NOD mice were given twice immediately on the first day they were diagnosed with diabetes, and then TJ-M2010-6 was administered twice a day (50 mg / kg) for the next four weeks of observation; the NOD control group was also given an equal amount of drug solvent;

[0165] INNA1606 (TJ-M2010-6, TJ-6) delayed medication group (oral administration by gavage): The treatment dose and duration were the same as those of the immediate treatment group, while the NOD mice were given TJ-M2010-6 treatment one week after the onset of diabetes. The NOD control group was also given immediate treatment and delayed treatment groups. All NOD mice were observed 4 weeks after the onset of diabetes.

[0166] The experimental results obtained (see attached Fig.24 ), the results showed that INNA1606 (TJ-M2010-6, TJ-6) treatment significantly reduced the proportion of mice still diagnosed with type 1 diabetes at the end of the experiment (Appendix Fig.24 A), and both immediate and delayed treatment were effective in treating type 1 diabetes in mice (Appendix Fig.24 C), while INNA1606 plays a therapeutic role in type 1 diabetes by inhibiting pancreatic islet inflammation (Appendix Fig.24 D, E). Meanwhile, INNA1606 can significantly reduce the body weight of obese mice fed a high-fat diet (see Appendix Fig.25 ), also suggesting its potential in treating diabetes.

[0167] Example 10: INNA1605 (TJ-M2010-5, TJ-5) can correct the immune disorder of lupus-like B cells induced by R848.

[0168] In vitro lupus-like B cell immune disorder model preparation and grouping:

[0169] Model preparation: Mouse spleen-derived B cells were purified using a magnetic bead sorting kit and then cultured in vitro. The stimulatory factor CD40L was added to maintain the long-term survival of B cells in vitro. B cells were stimulated with R848 (500 ng / ml) for 48 hours to induce lupus-like B cell immune disorder.

[0170] The experiment was divided into 4 groups:

[0171] Control group (no treatment, normal cultured B cells);

[0172] R848 group (model group): B cells were stimulated with R848 (500 ng / ml) for 48 h;

[0173] TJ-5 (10 μM) group: B cells were pre-stimulated with TJ-5 (10 μmol / L) for 3 h, and then stimulated with R848 (500 ng / ml) for 48 h;

[0174] TJ-5 (20 μM) group: B cells were pre-stimulated with TJ-5 (20 μmol / L) for 3 hours, and then stimulated with R848 (500 ng / ml) for 48 hours.

[0175] After R848 stimulation, cells and cell culture supernatant were collected for detection.

[0176] The experimental results obtained (see attached Fig.26 / 27), the results showed that R848 stimulated B cells and caused B cells to activate and differentiate into plasma cells, produce a large number of autoimmune antibodies, and synthesize and secrete a large number of inflammatory factors; INNA1605 (TJ-M2010-5, TJ-5) could inhibit the abnormal activation and differentiation of B cells (see Appendix Fig.26 ) and inhibit the secretion of B cell autoantibodies and inflammatory factors (see Appendix Fig. 27 ), INNA1605 can correct a series of immune disorders of lupus-like B cells induced by R848 and has the potential to treat systemic lupus erythematosus.

[0177] Example 11: INNA1605 (TJ-M2010-5, TJ-5) has a significant therapeutic effect on reperfusion injury after acute cerebral infarction.

[0178] Preparation and grouping of mouse model of acute cerebral infarction and reperfusion injury:

[0179] Model preparation: Adult male C57BL / 6 mice (8-10 weeks old, weighing 22-25g) were fasted for 8 hours before surgery and had free access to water. Anesthesia was performed by intraperitoneal injection of 1% sodium pentobarbital solution. Middle cerebral artery occlusion (MCAO) model: The mouse was fixed in the supine position, and the head of the mouse was kept tilted to its left side. Disinfectant alcohol was sprayed on the middle area of ​​the neck, and the hair on the neck was shaved with a blade. The middle right side of the neck was cut vertically, and the skin and thyroid gland were bluntly separated. Be careful not to damage the vagus nerve and parathyroid glands. The common carotid artery (CCA) was carefully separated and exposed, and the right external carotid artery (ECA) and internal carotid artery (ICA) were gradually separated upward to expose the CCA and ICA. The distal part of the ECA was ligated with silk thread. At the heart end, the ECA proximal end is lined, and a small incision is made in the ECA with precision ophthalmic scissors. The silicone-coated thread plug is slowly inserted into the ICA, the silk thread is ligated and fixed, the artery clamp is loosened, and the thread plug is slowly inserted. The insertion is stopped after a certain resistance. At the same time, the Doppler blood flow meter monitors the significant decrease in cerebral blood flow in the middle cerebral artery (MCA) area on the ischemic side. After 1 hour of ischemia, the embolism line is slowly withdrawn, the ECA is ligated, the neck skin is sutured, and the mouse is placed in a 32°C incubator. After the mouse wakes up, 1 ml of normal saline is injected intraperitoneally for fluid replacement. The surgical steps of the sham group (Sham group) are basically the same as those of the model group, but the MCA is not blocked. After the animal wakes up, it is returned to the cage and fed freely.

[0180] The experiment was divided into 4 groups:

[0181] Sham group: The surgical procedures and postoperative treatment were the same as those in the model group, but the MCA was not blocked;

[0182] Model group (I / R group): MCAO model was established, and the operation was as above without special treatment;

[0183] Vehicle control group: After the suture was removed, an equal amount of normal saline was immediately injected intravenously

[0184] Medication group (I / R+INNA1605 15 mg / kg group): After the suture was removed, 150 ul of 2.5 mg / ml INNA1605 solution (15 mg / kg) was immediately injected intravenously.

[0185] After 1h of ischemia and 24h of reperfusion, the brain tissues of the mice were removed for pathological detection and subsequent molecular biological analysis.

[0186] The experimental results obtained (see attached Fig.28), the results showed that INNA1605 (TJ-M2010-5, TJ-5) significantly reduced the infarct volume of acute cerebral infarction in mice, and the reduction degree could reach an astonishing 80%, which can be said to have basically reached the level of curing reperfusion injury after acute cerebral infarction. It can be seen that INNA1605 (TJ-M2010-5, TJ-5) has great potential for the treatment of acute cerebral infarction.

[0187] Example 12: The therapeutic effect of INNA1605 (TJ-M2010-5, TJ-5) on Alzheimer's disease.

[0188] Alzheimer's disease mouse model preparation and grouping:

[0189] Model preparation: APP / PS1 transgenic mice were purchased from Shanghai Slake Laboratory Animal Co., Ltd. The transgenic mice spontaneously developed Alzheimer's disease when they were bred to 8 to 9 months of age and no external treatment was required.

[0190] The experiment was divided into 3 groups:

[0191] Drug group (APP / PS1+INNA1605 group): APP / PS1 transgenic mice were given 50 mg / Kg / day of INNA1605 (twice a day, 25 mg / Kg each time, once by intraperitoneal injection and once by gavage);

[0192] Model group (APP / PS1 group): APP / PS1 transgenic mice were given an equal dose of solvent (ddH2O);

[0193] Control group: C57BL / 6 mice were given an equal dose of solvent (ddH2O).

[0194] When the mice were 9 months old, Morris water maze test was performed to evaluate the spatial learning and memory ability of APP / PS1 mice. After the behavioral test, after deep anesthesia by intraperitoneal injection of sodium pentobarbital (100 mg / kg), some mice were fully anesthetized by thoracotomy to fully expose the heart and aorta. A 20 ml syringe with an infusion needle was inserted from the apex of the heart into the aorta, the needle was fixed, the right atrium was cut, and 30 ml of 4 ° C saline was perfused through the aorta until most of the blood flowed out. The hippocampus and cortex tissues were carefully and quickly separated and removed, and stored at -80 ° C for subsequent biochemical analysis. The remaining mice were first perfused with 30 ml of 4 ° C saline, and then perfused with 30 ml of pre-cooled 4% paraformaldehyde, and then the whole brain was removed and fixed with 4% paraformaldehyde for 48 hours. After dehydration, transparency, paraffin embedding, and sectioning, immunohistochemical analysis was performed.

[0195] The experimental results obtained (see attached Fig.29 / 30 / 31), the results showed that the memory of APP / PS1 transgenic mice treated with INNA1605 (TJ-M2010-5, TJ-5) was better than that of APP / PS1 transgenic mice given only ddH2Od, although it did not reach the memory level of normal mice (i.e., the control group) (see Appendix Fig.29 ); We also found through immunohistochemistry that APP / PS1 transgenic mice treated with INNA1605 had Fig.30 A) Cortex (see attached Fig.30 B) Hippocampus (see attached Fig.30 C) The Aβ deposition in the brain of the mice was significantly less than that in the model group. Moreover, the Aβ deposition in the whole brain of the mice treated with NNA1605 was significantly reduced (see Appendix Fig.31 A) Cortex (see attached Fig.31 B) Hippocampus (see attached Fig.31 C) The area of ​​Iba-1-positive microglia in the region was also significantly reduced compared with the model group, suggesting that INNA1605 can be used as a new drug for the treatment of Alzheimer's disease both in terms of efficacy and mechanism.

[0196] The above experimental results directly show that the aminothiazole small molecule compound INNA1605 ointment has obvious efficacy in treating psoriasis, atopic dermatitis (eczema), allergic dermatitis, neurodermatitis, urticaria and other immune inflammatory dermatitis, and can achieve a complete cure. It will be a broad-spectrum, special-effect immune inflammatory dermatitis ointment, which can almost fill the market gap for many refractory skin diseases.

[0197] Example 13: The aminothiazole derivative INNA1605 is used for preventing and treating depression.

[0198] Model mice were male C57BL / 6J (6-7 weeks old, 18-20g in weight) and male CD-1 (6-8 years old) mice. The model of CSDS was established: C57BL / 6J mice were attacked by different CD-1 rats for 10 minutes every day for 10 consecutive days. At night, the two types of mice were kept in the same cage separated by a perforated transparent glass to maintain the stress of being attacked. The experiment confirmed that INNA1605 (TJ-M2010-5) significantly improved the induced depressive behavior and brain neuroinflammation (see Fig.32 , 33 ).

[0199] Example 14: The aminothiazole derivative INNA1605 is used to prevent and treat transplant rejection.

[0200] The transplant paired mice were: donor mice were balb / c mice; recipient mice were B6 mice. The donor's skin or heart was transplanted to the recipient. After the skin transplant, INNA1605 (TJ-M2010-5) was continuously used for 2 weeks, and then the drug was stopped. About 38% of the transplanted skin was permanently not rejected. After TJ-M2010-5 was used for two weeks after the allogeneic heart transplant, 60% of the heart transplants were permanently not rejected. In these surviving recipient mice that did not reject for a long time, the same donor mouse heart was transplanted for the second time. No treatment was required after the transplant. As a result, 100% of the hearts were not rejected, confirming the generation of immune tolerance against allogeneic antigens (see Fig.34 , 35 ).

[0201] Example 15: The aminothiazole derivative INNA1605 is used to prevent and treat liver cancer.

[0202] H22 hepatocellular carcinoma cells were implanted subcutaneously in balb / c mice and allowed to grow freely. The animals were divided into two groups, a group treated with INNA1605 (TJ-M2010-5) and a control group without drug. The tumor size was measured. The results showed that the tumor in the group treated with INNA1605 (TJ-M2010-5) was significantly smaller than that in the control group (see Fig.36 ).

Claims

1. Use of an aminothiazole derivative in the preparation of a drug for treating ischemia-reperfusion injury secondary to cerebral infarction, wherein the specific structure of the aminothiazole derivative is N-(4-phenylthiazol-2-yl)-3-(4-benzylpiperazine-1-yl)propionamide.

2. Use of an aminothiazole derivative in the preparation of a drug for treating depression, wherein the specific structure of the aminothiazole derivative is N-(4-phenylthiazol-2-yl)-3-(4-benzylpiperazin-1-yl)propionamide.

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