Use of benzamide derivatives in pharmaceutical preparations for preventing and treating inflammatory injuries

Abstract

The present application relates to the use of benzamide derivatives in pharmaceutical preparations for preventing and treating inflammatory injuries. The applicant has found that benzamide derivatives can be used as pharmaceutical preparations for preventing and / or treating cellular inflammatory damage caused by factors such as radiation or microbial infection. In some of the embodiments, it is demonstrated that benzamide derivatives have the effects of inhibiting cell pyroptosis, promoting the healing of radiation-complex injuries in mice, and reducing tissue inflammatory responses induced by the new coronavirus pseudovirus.

Description

Technical Field

[0001] The present application belongs to the field of medical technology, and specifically relates to the use of benzamide derivatives in pharmaceutical preparations for preventing and treating inflammatory injuries. Background Art

[0002] Pyroptosis is a new type of programmed cell death, which is characterized by dependence on the activation of caspase-1 / 3, hydrolysis of pyroptosis protein or granzyme, and then inducing the release of proinflammatory factors, leading to local or systemic inflammatory response. In the past, the important role of pyroptosis in the occurrence and development of important diseases such as neurodegenerative diseases and tumor immunity has been revealed one after another (Pian yu et al. Signal Transduct Target Ther. 2021; 6(1): 128.), especially since the outbreak of the new crown, the systemic immune inflammatory response caused by viral infection, which in turn causes pyroptosis and tissue damage, has attracted widespread attention (Karki et al. Cell. 2021; 184(1): 149-168.e17.). Due to its complex regulatory mechanism, the initiator and effector factors have significant tissue specificity in different tissues (Kovacs SB, Miao EA. Gasdermins: Effectors of Pyroptosis. Trends Cell Biol. 2017 Sep; 27(9): 673-684.), and the current research and development of pyroptosis inhibitors is still mainly limited to the laboratory research stage.

[0003] Pathogen-associated molecular patterns and damage-associated molecular patterns are the two main molecular mechanisms for initiating the body's innate immune response. Ionizing radiation-induced sterile inflammatory response that initiates immune response is a typical case of damage-associated molecular patterns, while the "inflammatory storm" caused by new coronavirus infection is a vivid manifestation of pathogenic molecular patterns. Both stress factors can cause severe systemic inflammatory responses and have many similarities in pathogenesis and clinical manifestations (Rios CI, et al. Commonalities Between COVID-19 and Radiation Injury. Radiat Res. 2021; 195(1): 1-24.).

[0004] Therefore, there is still a need in the art to develop pharmaceutical preparations for treating or preventing cellular inflammatory damage caused by ionizing radiation and related viral infections. Summary of the invention

[0005] The purpose of the present application is to provide the use of benzamide derivatives in pharmaceutical preparations for preventing and treating inflammatory damage. The examples of the present application found that benzamide derivatives can inhibit cell pyroptosis, thereby preventing and / or treating cell inflammatory damage.

[0006] To achieve the above-mentioned purpose, the present application discloses the use of a benzamide derivative or a pharmaceutically acceptable salt thereof in a pharmaceutical preparation, wherein the pharmaceutical preparation is used to prevent and / or treat inflammatory cell damage, wherein the benzamide derivative has a structural formula shown in Formula I:

[0007]

[0008] In some embodiments of the present application, the cellular inflammatory damage is caused by radiation or microbial infection.

[0009] In some embodiments of the present application, the injury site of the cell inflammatory injury is located in epithelial cells.

[0010] In some embodiments of the present application, the cellular inflammatory damage is caused by ionizing radiation.

[0011] In some embodiments of the present application, the cellular inflammatory damage is caused by viral infection.

[0012] In some embodiments of the present application, the injury site has inflammatory infiltration, pyroptosis, and necrotic cells and / or tissues.

[0013] In some embodiments of the present application, in the pharmaceutical preparation, the content of the benzamide derivative is 5 μM to 50 mM.

[0014] In some embodiments of the present application, the dosage form of the pharmaceutical preparation is selected from any one or more of aqueous infusions, powders, lotions, tinctures, oils, emulsions, ointments, plasters or aerosols.

[0015] In some embodiments of the present application, the pharmaceutical preparation is an oral preparation or an injectable preparation.

[0016] Wherein, oral agent can be made into any oral acceptable preparation form, including but not limited to tablet, capsule, granule, pill, syrup, oral solution, oral suspension and oral emulsion etc.Wherein, the carrier used in tablet usually includes lactose and corn starch, and lubricant such as magnesium stearate can also be added.The diluent used in capsule usually includes lactose and dry corn starch.In oral suspension, active ingredient is usually used by mixing suitable emulsifier and suspending agent.Optionally, some sweeteners, aromatics or coloring agents can be added to the above-mentioned oral preparation form.

[0017] The carriers and solvents that can be used for injections include water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile non-volatile oils such as monoglycerides or diglycerides can also be used as solvents or suspending media.

[0018] In some embodiments of the present application, the pharmaceutically acceptable salt is selected from any one or more of sulfate, phosphate, hydrochloride, hydrobromide, acetate, oxalate, citrate, succinate, gluconate, tartrate, p-toluenesulfonate, benzenesulfonate, methanesulfonate, benzoate, lactate, maleate, lithium salt, sodium salt, potassium salt or calcium salt.

[0019] According to the specific embodiments of the present application: benzamide derivatives or pharmaceutically acceptable salts thereof can be used as pharmaceutical preparations for preventing and / or treating cellular inflammatory damage caused by factors such as radiation or microbial infection. In some of the embodiments, it is demonstrated that they have the effects of inhibiting cell pyroptosis, promoting the healing of radiation-complex injuries in mice, and reducing tissue inflammatory responses induced by the new coronavirus pseudovirus.

[0020] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the following is a detailed description of the preferred embodiments of the present invention in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Photographs showing skin lesions after irradiation of mice treated with benzamide derivatives and DMSO;

[0022] Figure 2 The continuous scoring of skin damage after irradiation of mice treated with benzamide derivatives and DMSO is shown;

[0023] Figure 3 The results of PCR microarray of inflammatory factors in the skin of mice treated with benzamide derivatives and DMSO after irradiation are shown;

[0024] Figure 4 Benzamide derivatives were shown to mitigate cell membrane damage in irradiated skin of mice;

[0025] Figure 5 It shows that benzamide derivatives reduce the fluorescence intensity of pyroporin D hydrolysis products in mouse irradiated skin cells;

[0026] Figure 6 The effects of benzamide derivatives on the expression of pyroptosis markers in irradiated mouse skin cells were shown;

[0027] Figure 7 Benzamide derivatives were shown to enhance the clonogenic capacity of irradiated mouse skin cells;

[0028] Figure 8 It was shown that benzamide derivatives reduce cellular interferon activation caused by COVID-pseudovirus infection;

[0029] Fig. 9 The figure shows the effects of high concentration IRSKIN-1 on important organs of mice after oral gavage or subcutaneous injection. DETAILED DESCRIPTION

[0030] The specific implementation of the present invention is further described in detail below in conjunction with the accompanying drawings and examples. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0032] The terms "prevention and / or treatment" used in this application refer to "preventive treatment and / or therapeutic treatment". As used in the application, the term "treatment" refers to the administration of a compound or pharmaceutical composition for therapeutic purposes. "Treatment of a condition" or for "therapeutic treatment" refers to the administration of treatment to a patient already suffering from a disease to improve the disease or one or more of its symptoms to improve the patient's condition (e.g., by reducing one or more symptoms of inflammation). The term "therapeutic" includes the effect of alleviating the harmful clinical effects of certain inflammatory processes (i.e., the consequences of inflammation, not the symptoms of inflammation). The method of the present application can be used as a primary preventive measure, i.e., to prevent a condition or reduce the risk of developing a condition. Prevention refers to the preventive treatment of patients who may not have fully developed a condition or disorder but are susceptible to the condition or are at risk of the condition. Therefore, in the claims and embodiments, the present application can be used for therapeutic or preventive purposes.

[0033] To further illustrate the technology used to achieve the purpose of the present invention, the following describes detailed methods, techniques, processes and features for determining and identifying the pharmaceutical and therapeutic uses of the compounds in the present invention. The cases provide experimental methods and results for supporting and verifying the animal models used in the present invention. The cases involved all use appropriate control group experiments and statistical analysis methods. The following cases are used to describe but not limit the application of the present invention. The methods and techniques involved in these cases can be used to screen and determine the therapeutic effects of such compound preparations. The same method can be used to evaluate the therapeutic effects of other such compound preparations.

[0034] The present invention is further described in detail below in conjunction with specific examples. The raw materials used in the following examples are all commercially available products unless otherwise specified.

[0035] For the sake of convenience, in the following examples, the benzamide derivative of the present application is named PCD-I1 (pyroptosis / cell death-inhibitor 1), but it is not used to limit the scope of the present invention.

[0036] In the following examples, the chemical formula of PCD-I1 is C 18 H 20 N2O4S, its structural formula is shown in the following formula I:

[0037]

[0038] Example 1 Experimental evidence that subcutaneous injection of PCD-I1 can effectively reduce radiation damage in mouse models In order to evaluate the effect of subcutaneous injection of PCD-I1 on radiation damage to the lower limbs of mice, the following experiment was performed.

[0039] Male C57 BL / 6J mice aged 6-8 weeks purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. were randomly divided into 2 groups (6 mice in each group) after adaptive feeding. The mice were subcutaneously injected with PCD-I1 (50 mM solution dissolved in DMSO, 100 μL / time) and an equal amount of DMSO solution once 1 day and 3 days before irradiation, respectively.

[0040] Among them, after anesthesia by intraperitoneal injection of 1% chloral hydrate at 1% of each mouse's body weight, the mouse's hind limbs were depilated and fixed on a wooden board to avoid accidental movement of the mouse during irradiation.

[0041] The rat buttocks skin was irradiated with 6MeV electron beams generated by the 23EX linear accelerator of Varian Corporation in the United States. The surface was covered with a 1cm thick tissue compensation film and the non-irradiated part was shielded with a lead plate. The irradiated area was about 30mm×40mm, the absorbed dose rate was 750cGy / min, the dose was 45Gy, and the irradiated area was outlined with a marker pen.

[0042] Please refer to Figure 1 , Figure 1 Photographs showing skin lesions in PCD-I1 and DMSO-treated mice after irradiation. (a) and (b) represent DMSO-treated mice, and (c) and (d) represent PCD-I1-treated mice. (a) and (c) are taken 15 days after irradiation, and (b) and (d) are taken 30 days after irradiation.

[0043] according to Figure 1Comparing the irradiated lower limbs of the two groups of mice shown in the figure and observing the skin damage, it can be seen that the morphology and function of the irradiated lower limbs of the mice in the PCD-I1 treatment group recovered significantly faster than those in the control group, indicating that subcutaneous injection of PCD-I1 has the effect of reducing the progression of radiation damage to the lower limbs of mice.

[0044] Please refer to Figure 2 and Figure 3 , Figure 2 It represents the continuous scoring of skin damage in PCD-I1 and DMSO treated mice after irradiation. Figure 3 These represent the results of PCR chip of inflammatory factors in the skin of PCD-I1 and DMSO-treated mice after irradiation. The results showed that the expressions of G-CSF and IL-1b in the skin tissue of mice in the PCD-I1-treated group were increased, and the expressions of PF4, BLC and MIG were decreased.

[0045] According to the experiment of this example, it can be known that compared with the DMSO group, the PCD-I1 subcutaneous injection group can significantly slow down the progression of radiation damage in mice, and its healing and recovery effect is better.

[0046] Example 2 Experimental evidence that PCD-I1 inhibits ionizing radiation-induced cell pyroptosis

[0047] In order to clarify whether PCD-I1 has the effect of inhibiting pyroptosis of mouse skin cells irradiated by electron beams, in this example, mouse cells were treated with 5μM, 10μM, and 20μM PCD-I1 and an equal amount of DMSO for 24 hours before electron beam irradiation, and then a single 20Gy X-ray irradiation was performed. The effect of PCD-I1 on cell membrane damage irradiated by electron beams was evaluated by LDH release method 3 days after irradiation.

[0048] Figure 4 It was shown that PCD-I1 alleviated the damage of irradiated skin cell membranes in mice. Figure 4 As shown, the results showed that pretreatment with PCD-I1 could reduce the cell LDH release rate caused by X-ray irradiation, indicating that PCD-I1 has the effect of reducing cell membrane damage caused by electron beam irradiation.

[0049] To further study whether PCD-I1 has the effect of inhibiting pyroptosis of irradiated skin cells, the cells were treated with 10 μM PCD-I1 and an equal amount of DMSO for 24 hours before irradiation, and then subjected to a single 20 Gy X-ray irradiation. The effect of PCD-I1 on the expression of pyroporin hydrolysis products in irradiated cells was evaluated by immunofluorescence 3 days after irradiation.

[0050] Figure 5 The results show that PCD-I1 reduces the fluorescence intensity of pyroporin D hydrolysis products in skin cells of mice irradiated by electron beams. Figure 5As shown, pretreatment with PCD-I1 can reduce the fluorescence intensity of the hydrolysis product of cell pyroporin D caused by X-ray irradiation. The results show that PCD-I1 has the effect of inhibiting the hydrolysis of pyroporin in irradiated cells.

[0051] The effect of PCD-I1 pretreatment on the expression of proteins related to the pyroptosis signaling pathway in irradiated skin cells was studied by immunoblotting. Figure 6 The results show that PCD-I1 affects the expression of pyroptosis markers in irradiated mouse skin cells, such as Figure 6 As shown in the results, pretreatment with PCD-I1 can reduce the phosphorylation of TKB1, Caspase1, GSDMD and IL1 hydrolysis induced by X-ray irradiation. The results indicate that PCD-I1 has the effect of inhibiting pyroptosis of irradiated cells.

[0052] Example 3 Experimental evidence that PCD-I1 promotes proliferation of irradiated skin cells

[0053] In order to clarify the role of PCD-I1 in promoting the proliferation of irradiated skin cells, in this example, mouse cells were treated with 10 μM PCD-I1 and an equal amount of DMSO for 24 hours before electron irradiation, and then subjected to a single 2Gy, two 2Gy X-ray fractionated irradiations and a single 4Gy Sr 90 irradiation, respectively. The effect of IRSKIN-1 on the proliferation ability of irradiated cells was then evaluated by a clone formation experiment (2500 cells were plated in each well of a six-well plate).

[0054] Figure 7 It was shown that benzamide derivatives enhance the clonogenicity of irradiated mouse skin cells, e.g. Figure 7 As shown, the clone formation area of ​​skin cells irradiated by X-rays and Sr 90 was significantly increased after pretreatment with PCD-I1, and the results showed that PCD-I1 has the effect of promoting the proliferation of irradiated cells.

[0055] Example 4 Experimental evidence that PCD-I1 inhibits interferon response induced by novel coronavirus pseudovirus infection

[0056] In order to clarify the role of PCD-I1 in inhibiting the inflammatory response caused by viral infection, this example establishes a cell model by overexpressing angiotensin-converting enzyme II (ACEII) in human embryonic lung fibroblasts and then infecting them with a new coronavirus pseudovirus, and uses the transcriptional activity of interferon regulatory factor I (IRF1), a key regulatory factor for systemic inflammatory response induced by new coronavirus infection, to respond to pseudovirus infection and cause changes in cellular interferon response. Cells were treated with 10 μM PCD-I1 and an equal amount of DMSO for 24 hours before infection, and then infected with pseudoviruses. The inhibitory effect of PCD-I1 on the interferon response induced by new coronavirus pseudovirus infection was evaluated by dual luciferase experiments at different times after infection.

[0057] Figure 8 It was shown that benzamide derivatives reduce the activation of cellular interferon caused by novel coronavirus pseudovirus infection, such as Figure 8 As shown, the interferon response intensity of HELF cells infected with the new coronavirus pseudovirus was significantly reduced after pretreatment with PCD-I1. The results showed that PCD-I1 has the effect of inhibiting the interferon response induced by the new coronavirus pseudovirus infection.

[0058] Example 5 Experimental evidence that PCD-I1 has no obvious toxicity or side effects on important organs

[0059] To clarify the biosafety of PCD-I1, in this example, C57 mice were gavaged and subcutaneously injected with 100 μL of 10-fold effective dose (10 mM) PCD-I1. The mice were killed one week later, and the heart, liver, spleen, lungs, kidneys and organs of administration (stomach and skin) were removed for HE staining to evaluate the effect of PCD-I1 on damage to important organs.

[0060] Fig. 9 The results show the effects of high concentration IRSKIN-1 on important organs of mice after oral administration or subcutaneous injection. Fig. 9 As shown, oral gavage and subcutaneous injection of high-dose PCD-I1 did not cause significant changes in the HE staining of important organ tissue structures, and the results showed that PCD-I1 has good biosafety.

[0061] Therefore, the above results indicate that the benzamide derivatives of the present application have the effect of alleviating tissue inflammatory damage caused by stress.

[0062] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0063] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. Use of a benzamide derivative or a pharmaceutically acceptable salt thereof in the preparation of a pharmaceutical preparation for preventing and treating inflammatory damage, characterized in that: The pharmaceutical preparation is used for preventing and / or treating cell inflammatory damage, wherein the inflammatory damage is skin damage caused by ionizing radiation, and the benzamide derivative has a structural formula shown in Formula I: 。 2. The use according to claim 1, characterized in that In the pharmaceutical preparation, the content of the benzamide derivative is 5 μM to 50 mM.

3. The use according to claim 1, characterized in that The dosage form of the pharmaceutical preparation is selected from any one or more of aqueous infusion, powder, lotion, tincture, oil, emulsion, ointment, plaster or aerosol.

4. The use according to claim 1, characterized in that The pharmaceutical preparation is a subcutaneous injection.

5. The use according to claim 1, characterized in that The pharmaceutically acceptable salt is selected from any one or more of sulfate, phosphate, hydrochloride, hydrobromide, acetate, oxalate, citrate, succinate, gluconate, tartrate, p-toluenesulfonate, benzenesulfonate, methanesulfonate, benzoate, lactate and maleate.

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