Pharmaceutical composition containing STING agonist and application thereof

Abstract

The invention discloses a pharmaceutical composition containing an STING endogenous agonist, and an application of the pharmaceutical composition containing the STING endogenous agonist in preparation of a medicine for treating and / or repairing cerebral ischemic stroke brain injury. The invention also discloses an application of the STING exogenous agonist, or a composition of the STING exogenous agonist and magnesium salt, or a composition of the STING exogenous agonist and a medicine for treating cerebral apoplexy in preparation of a medicine for treating and / or repairing cerebral ischemic stroke brain injury. The drug effect of the pharmaceutical composition containing the STING agonist in the aspects of treating cerebral arterial thrombosis and / or repairing brain injury is remarkably improved, the difficulty of narrow emergency time window is remarkably relieved, the effects of improving cerebral blood circulation and protecting nerves are achieved, and the significant brain function repairing effect is achieved in the follow-up rehabilitation process.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a STING agonist and a composition thereof, a combination drug or a pharmaceutical composition and an application thereof. Background Art

[0002] Cerebral stroke, also known as cerebral infarction, is a disease caused by sudden rupture and bleeding of brain blood vessels or vascular blockage, which causes cerebral ischemia and hypoxia. Stroke is divided into two categories: ischemic stroke and hemorrhagic stroke. Ischemic stroke is caused by occlusive lesions in the external or internal arteries that supply blood to the brain, which fail to obtain timely and sufficient collateral circulation, resulting in insufficient blood supply for the metabolism of local brain tissue.

[0003] At present, the treatment options for acute ischemic stroke include general treatment and specific treatment. General treatment mainly refers to the maintenance and control of physiological indicators. Specific treatment is the key to the treatment of acute ischemic stroke, mainly including improving cerebral blood circulation and neuroprotection. The treatment strategy of neuroprotective drugs is mainly neuroprotective rather than restorative, and is limited by a narrow time window.

[0004] The problems and shortcomings of existing ischemic stroke drugs are that the current first-line clinical drugs, edaravone and its concentrated solution for injection with dexamethasone, are the most effective, but this drug only has the effect of protecting the nerves in the treatment of acute cerebral ischemic injury, and has no obvious effect on the repair of brain nerve function after emergency treatment. The market is in urgent need of drugs that can promote the repair of brain function after treatment, especially drugs that can treat acute cerebral ischemic injury and promote the repair of brain function after treatment.

[0005] The innate immune interferon stimulation pathway (STING pathway) has been widely studied. Immune agonists targeting STING stimulate the upregulation of type I interferon INF-β gene transcription, thereby regulating changes in the expression of multiple cytokines. Cyclic cGMP-AMP dinucleotide synthetase (cGAS) endogenously catalyzes the synthesis of cyclic dinucleotide cGAMP under activation conditions after binding to DNA. Endogenous STING agonist cGAMP stimulates the induction of interferon INF-I through STING as a second messenger, mediates the activation of TBK1 and IRF-3, and then initiates the transcription of type I interferon INF-β gene. The use of STING agonists in immune anti-tumor, anti-Alzheimer's disease and other drugs has been reported. The use of innate immune agonist cGAMP in the preparation of drugs for the prevention and treatment of ischemic cerebrovascular diseases (CN201510322608.0) was also reported by the research group of the inventor of this application in previous years. In our previous studies, using a mouse ischemia model, cGAMP was administered through the tail vein 2 hours after ischemia and 8 hours after reperfusion. It can significantly reduce the percentage of brain ischemia caused by ischemia in mice and improve the behavior of mice after ischemia. Therefore, it was found at that time that cGAMP has the effect of emergency treatment of ischemic brain injury. However, there is no systematic study on whether cGAMP has the effect of repairing brain function after acute treatment of ischemic stroke, nor whether the combination of cGAMP and stroke drugs has a better therapeutic effect or whether the combination has the function of repairing brain function damage caused by ischemic stroke, although clinical first-line stroke drugs such as edaravone have no obvious neurorepair effect. In addition, because the endogenous agonist cyclic dinucleotide cGAMP of STING is a secondary signaling molecule, its short metabolic cycle in the body and easy degradation limit its efficacy and drugability. Based on the crystal structure of the STING molecule, molecular docking simulation calculation technology was used to screen out exogenous agonist compounds with good affinity for STING. Studying the effects of the screened STING exogenous agonists on the treatment of ischemic stroke and repair of brain function is also one of the purposes of this research and invention.

[0006] The treatment plan for acute ischemic stroke mainly includes improving cerebral blood circulation and neuroprotection, and repairing brain nerve function so that it can gradually recover. Another technical difficulty that needs to be overcome is that neuroprotective drugs are mainly protective rather than restorative, and they all have a narrow time limit for the treatment window. The time window for acute thrombolysis in cerebral infarction is very short, 3-4.5 hours, and the optimal window period is often missed. Summary of the invention

[0007] Based on the above-mentioned defects in the prior art, the present invention provides a STING agonist and a composition and application thereof.

[0008] The first aspect of the present invention provides a pharmaceutical composition comprising:

[0009] STING endogenous agonists and magnesium salts;

[0010] The STING endogenous agonist is selected from at least one of 2',3'-cGAMP, c-di-AMP, c-di-GMP, c-di-IMP, c-GMP-IMP and derivatives thereof;

[0011] Preferably, the STING endogenous agonist is 2',3'-cGAMP.

[0012] The magnesium salt is selected from at least one of magnesium sulfate, magnesium chloride, magnesium oxalate, magnesium carbonate, magnesium nitrate, and magnesium ammonium phosphate;

[0013] The molar ratio of the STING endogenous agonist to the magnesium salt is 1:1.5-5.

[0014] Preferably, the pharmaceutical composition further comprises:

[0015] Endogenous STING agonists and drugs for the treatment of stroke;

[0016] The drug for treating stroke is selected from at least one of edaravone, butylphthalide, cebiracem, oxiracetam, piracetam, citicoline, cinepazide, nimodipine, flunarizine, and ganglioside;

[0017] The mass ratio of the drug for treating stroke to the STING endogenous agonist is 2-12:1.

[0018] A second aspect of the present invention provides a pharmaceutical composition comprising:

[0019] STING endogenous agonists, and drugs for the treatment of stroke;

[0020] The STING endogenous agonist is selected from at least one of 2',3'-cGAMP, c-di-AMP, c-di-GMP, c-di-IMP, c-GMP-IMP and derivatives thereof;

[0021] The drug for treating stroke is selected from at least one of edaravone, butylphthalide, cebiracem, oxiracetam, piracetam, citicoline, cinepazide, nimodipine, flunarizine, and ganglioside;

[0022] The mass ratio of the drug for treating stroke to the STING endogenous agonist is 2-8:1.

[0023] The third aspect of the present invention provides use of the above-mentioned pharmaceutical composition in the preparation of a drug for treating and / or repairing ischemic stroke brain damage.

[0024] The fourth aspect of the present invention provides the use of a STING exogenous agonist in the preparation of a drug for preventing, treating or repairing ischemic stroke, wherein the STING exogenous agonist is selected from at least one of the following compounds:

[0025]

[0026] Preferably, the STING exogenous agonist is selected from at least one of the following compounds:

[0027]

[0028] Among them, the molecular formula and systematic nomenclature of the above substances are shown in the following table:

[0029]

[0030]

[0031] A fifth aspect of the present invention provides a pharmaceutical composition comprising:

[0032] STING exogenous agonists and magnesium salts;

[0033] The STING exogenous agonist is selected from at least one of the following compounds:

[0034]

[0035] The magnesium salt is selected from at least one of magnesium sulfate, magnesium chloride, magnesium oxalate, magnesium carbonate, magnesium nitrate, and magnesium ammonium phosphate;

[0036] The molar ratio of the STING exogenous agonist to the magnesium salt is 1:1.5-5.

[0037] Preferably, the STING exogenous agonist is selected from at least one of the following compounds:

[0038]

[0039] The magnesium salt is magnesium sulfate;

[0040] The molar ratio of the STING exogenous agonist to the magnesium salt is 1:1.5-4.

[0041] Preferably, it also includes drugs for treating stroke;

[0042] The drug for treating stroke is selected from at least one of edaravone, butylphthalide, cebiracem, oxiracetam, piracetam, citicoline, cinepazide, nimodipine, flunarizine, and ganglioside;

[0043] The mass ratio of the drug for treating stroke to the STING exogenous agonist is 1-10:1.

[0044] A sixth aspect of the present invention provides a pharmaceutical composition comprising:

[0045] STING exogenous agonists and drugs for the treatment of stroke;

[0046] The STING exogenous agonist is selected from at least one of the following compounds:

[0047]

[0048] The drug for treating stroke is selected from at least one of edaravone, butylphthalide, cebiracem, oxiracetam, piracetam, citicoline, cinepazide, nimodipine, flunarizine, and ganglioside;

[0049] The mass ratio of the drug for treating stroke to the STING exogenous agonist is 1-10:1.

[0050] Preferably, the STING exogenous agonist is selected from at least one of the following compounds:

[0051]

[0052]

[0053] The seventh aspect of the present invention provides the use of the above-mentioned pharmaceutical composition in the preparation of a drug for treating and / or repairing ischemic stroke brain damage.

[0054] The beneficial effects of the present invention include at least:

[0055] The present invention discloses a pharmaceutical composition comprising an endogenous STING agonist and a magnesium salt, and uses of a combination of an endogenous STING agonist and a drug for treating stroke in preparing a drug for treating and / or repairing ischemic stroke brain damage; further disclosed is the use of an exogenous STING agonist, or a combination thereof with a magnesium salt, or a combination thereof with a drug for treating stroke in preparing a drug for treating and / or repairing ischemic stroke brain damage.

[0056] The pharmaceutical composition comprising a STING agonist of the present invention has significantly improved efficacy in treating ischemic stroke and / or repairing ischemic brain damage, not only prolonging the treatment time window of acute ischemic stroke, significantly alleviating the difficulty of a narrow emergency time window, but also having the efficacy of improving cerebral blood circulation and neuroprotection. The inventors of the present invention also unexpectedly found that the pharmaceutical composition of the present invention has brain function repair efficacy in the subsequent rehabilitation process, can gradually repair brain function damage caused by ischemia, significantly improve the treatment effect of stroke, and greatly improve the survival rate and cure rate. Therefore, the pharmaceutical composition comprising a STING agonist of the present invention has broad application prospects.

[0057] The features and advantages of the present invention will be described in detail in the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 This is the ESI-MS image of the cyclic dinucleotide 2',3'-cGAMP in Example 1. DETAILED DESCRIPTION

[0059] In order to make the purpose, technical scheme and beneficial effects of the present invention clearer, the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. Examples of the embodiments are shown in the accompanying drawings. It should be understood that the specific embodiments described in the following embodiments of the present invention are only exemplary descriptions of the specific embodiments of the present invention, and are intended to be used to explain the present invention, but do not constitute a limitation of the present invention.

[0060] The endpoints and any values ​​of the ranges disclosed herein are not limited to the exact ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be regarded as specifically disclosed in this article. It should be noted that in this article, specific numerical values ​​can vary within ±2% as the numerical protection range of this application.

[0061] In the description of the present application, unless otherwise specified, "plurality" and similar words mean two or more than two. In addition, the terms "include", "comprises" and any variations thereof are intended to cover non-exclusive inclusions.

[0062]

Terminology

[0063] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0064] As used herein, definitions of standard chemical terms (eg, groups) can be found in the reference literature in the art.

[0065] Unless otherwise indicated, conventional methods within the technical scope of the art are adopted, such as mass spectrometry, NMR, IR and UV / VIS spectroscopy and pharmacological methods. Unless specifically defined, the terms used in the relevant descriptions of analytical chemistry, organic synthetic chemistry, and drugs and medicinal chemistry herein are known in the art. Standard techniques can be used in chemical synthesis, chemical analysis, drug preparation, preparation and delivery, and in the treatment of patients. For example, the instructions for use of the kit can be utilized by the manufacturer, or the reaction and purification can be implemented according to a manner well known in the art or the description of the present invention. Conventionally, the above-mentioned techniques and methods can be implemented according to conventional methods well known in the art, according to the description in a plurality of summaries and more specific documents cited and discussed in this specification. In this specification, groups and substituents thereof can be selected by those skilled in the art to provide stable structural parts and compounds.

[0066] When substituents are described by conventional chemical formulas written from left to right, the substituents also include chemically equivalent substituents that would result when the formula is written from right to left.

[0067] The section headings used herein are only for the purpose of organizing the article and should not be interpreted as limitations on the subject matter described. All documents or portions of documents cited in this application, including but not limited to patents, patent applications, articles, books, operating manuals and papers, are incorporated herein by reference in their entirety.

[0068] Certain chemical groups defined herein are preceded by a shorthand notation to indicate the total number of carbon atoms present in the group. 1-6 Alkyl refers to an alkyl group as defined below having a total of 1 to 6 carbon atoms. The total number of carbon atoms in the shorthand notation does not include carbons that may be present in substituents of the group being described.

[0069] In addition to the foregoing, when used in the specification and claims of the present application, the following terms have the meanings indicated below unless otherwise specifically stated.

[0070] As used herein, the term "compound of the present invention" or "active ingredient of the present invention" is used interchangeably and includes stereoisomers, enantiomers, or pharmaceutically acceptable salts thereof of the general formula compound. The term also includes racemates, optical isomers, isotopic compounds (such as deuterated compounds) or prodrugs.

[0071] "Stereoisomers" refer to compounds composed of the same atoms, bonded by the same bonds, but having different three-dimensional structures. The present invention is intended to encompass various stereoisomers and mixtures thereof.

[0072] When the compounds of the present invention contain olefinic double bonds, unless specified otherwise, it is intended that the compounds of the present invention include both E- and Z- geometric isomers.

[0073] "Tautomer" refers to an isomer formed when a proton is shifted from one atom of a molecule to another atom of the same molecule. All tautomeric forms of the compounds of the present invention are intended to be encompassed within the scope of the present invention.

[0074] The compounds of the present invention may contain one or more chiral carbon atoms and may therefore give rise to enantiomers, diastereomers and other stereoisomeric forms. Each chiral carbon atom may be defined as (R)- or (S)- based on stereochemistry. The present invention is intended to include all possible isomers, as well as racemates and optically pure forms thereof. The compounds of the present invention may be prepared using racemates, diastereomers or enantiomers as starting materials or intermediates. Optically active isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques, such as crystallization and chiral chromatography.

[0075] Conventional techniques for the preparation / isolation of individual isomers include chiral synthesis from appropriate optically pure precursors, or resolution of the racemate (or the racemate of a salt or derivative) using, for example, chiral high performance liquid chromatography.

[0076] In the present invention, compounds can be replaced with any number of substituents or functional groups to expand their scope. Generally, the term "substituted" appears before or after the term "optional", and the general formula including substituents in the formulation of the present invention refers to replacing hydrogen radicals with designated structural substituents. When multiple positions in a specific structure are substituted by multiple specific substituents, the substituents can be the same or different at each position. The term "substituted" used herein includes all allowed organic compound substitutions. In a broad sense, allowed substituents include non-cyclic, cyclic, branched non-branched, carbocyclic and heterocyclic, aromatic and non-aromatic organic compounds. In the present invention, heteroatom nitrogen can have hydrogen substituents or any allowed organic compounds described above to supplement its valence. In addition, the present invention is not intended to limit the allowed substitution of organic compounds in any way. The present invention believes that the combination of substituents and variable groups is very good in the treatment of diseases in the form of stable compounds. The term "stable" here refers to a stable compound that is tested for a long enough time to maintain the integrity of the compound structure, preferably for a long enough time, and is used for the above purpose herein.

[0077] Pharmaceutical compositions and methods of administration

[0078] The pharmaceutical composition of the present invention is used to prevent and / or treat cancer, immune diseases, metabolic diseases, etc. In the present application, "pharmaceutical composition" refers to a preparation of a compound of the present invention and a medium generally accepted in the art for delivering biologically active compounds to mammals (e.g., humans). The medium includes a pharmaceutically acceptable carrier. The purpose of the pharmaceutical composition is to promote administration of an organism, facilitate the absorption of the active ingredient, and then exert biological activity. The term "pharmaceutical" as used herein refers to a substance (such as a carrier or diluent) that does not affect the biological activity or properties of the compound of the present invention, and is relatively non-toxic, that is, the substance can be applied to an individual without causing adverse biological reactions or interacting with any component contained in the composition in an adverse manner.

[0079] In the present invention, "pharmaceutical excipients" include but are not limited to any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye / colorant, flavoring agent, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent or emulsifier approved by the relevant government regulatory authorities as acceptable for human or livestock use.

[0080] In the present invention, "treatment" should be understood in the broadest sense and may include the meaning of "prevention".

[0081] In the present invention, the term "prevent" includes reducing the likelihood of a disease or condition occurring or becoming worse in a patient.

[0082] In the present invention, the term "treatment" and other similar synonyms include the following meanings:

[0083] (i) preventing a disease or condition from occurring in a mammal, particularly where such mammal is susceptible to the disease or condition but has not yet been diagnosed as having the disease or condition;

[0084] (ii) inhibiting a disease or condition, i.e. arresting its development;

[0085] (iii) alleviate the disease or condition, that is, cause regression of the disease or condition; or

[0086] (iv) alleviating the symptoms caused by the disease or condition.

[0087] In the present invention, the term "effective amount", or "pharmaceutically effective amount" refers to the amount of at least one agent or compound that is sufficient to relieve one or more symptoms of the disease or condition being treated to some extent after administration. The result can be the reduction and / or alleviation of signs, symptoms or causes, or any other desired changes in biological systems. For example, an "effective amount" for treatment is the amount of a composition comprising a compound disclosed herein that is required to provide a clinically significant symptom alleviation effect. Techniques such as dose escalation trials can be used to determine the effective amount suitable for any individual case.

[0088] When using a STING agonist, or a pharmaceutical composition thereof, or a combination thereof, a safe and effective amount of the compound of the present invention is applied to a mammal (including a human) in need of treatment, wherein the dosage during administration is a pharmaceutically effective dosage for preventing and / or treating and / or repairing ischemic stroke brain damage. For a person weighing 60 kg, in some embodiments, if it is for prevention or treatment, when a STING exogenous agonist is used alone, the daily dosage is usually 30 to 60 mg. In some embodiments, if it is for repair, when a STING exogenous agonist is used alone, the daily dosage is usually 50 to 100 mg.

[0089] Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health status, which are all within the skills of a skilled physician.

[0090] In the present invention, the terms "administer", "administer", "drug", etc. refer to methods that can deliver a compound or composition to the desired site for biological action. These methods include, but are not limited to, oral or intravenous injection, intravenous drip, intramuscular injection, subcutaneous injection, and intraventricular direct administration. The STING agonist and its composition, combined drug or pharmaceutical composition of the present invention are in the form of one or more of tablets, capsules, enteric microparticles, granules, suspensions, emulsions, solutions, syrups, and injections.

[0091] In the present invention, "drug combination", "drug combination", "combination therapy", "administration of other treatments", "administration of other therapeutic agents" and the like refer to drug treatments obtained by mixing or combining more than one active ingredient, including fixed combinations and non-fixed combinations of active ingredients. "Fixed combination" refers to the simultaneous administration of at least one compound described herein and at least one synergistic agent to a patient in the form of a single entity or a single dosage form. "Non-fixed combination" refers to the simultaneous administration of at least one compound described herein and at least one synergistic agent to a patient in the form of separate entities, in combination, or sequentially at variable intervals.

[0092] The present invention also provides a treatment method, comprising the steps of administering the STING agonist or a composition thereof, or a combination thereof, described in the present invention to a subject in need of treatment.

[0093] Embodiment 1:

[0094] 1.1. Preparation and characterization of STING endogenous agonist cyclic dinucleotide

[0095] The cyclic dinucleotide 2',3'-cGAMP (hereinafter referred to as cGAMP) was synthesized by cyclic cGMP-AMP dinucleotide synthetase (cGAS) under activation conditions after binding to DNA according to the literature method. The purity was above 98%. (Li P. W, et al., Immunity, 2013, 39 (6), 1019-1031.). ESI-MS results showed that the peak of the target product was 673.0906, which was the cGAMP anion peak.

[0096] 1.2. Preparation and characterization of the combination of STING endogenous agonist cyclic dinucleotide and magnesium salt

[0097] Weigh cyclic dinucleotide cGAMP (1.0 mmol) and magnesium sulfate (1.0 mmol or 2.0 mmol, respectively) and mix them thoroughly in a micro mixer (drive power: 400 W, speed range: 1-360 rpm, screw design: conical, co-rotating / counter-rotating, maximum pressure: 200 bar, feeding method: manual). Mix and extrude the composition.

[0098] Example 2: Study on the effect of the combination of STING endogenous agonist cyclic dinucleotide and magnesium salt on the function of resisting ischemic brain damage

[0099] 2.1 Experimental animals and their groups

[0100] Experimental animals: Male rats weighing 250-280 g were purchased from Southern Model Organisms Technology Co., Ltd. Male rats were randomly divided into 13 groups, with 10 rats in each group. The 13 groups of rats are shown in Table 1:

[0101] In the following Tables 1-9, the substances are separated by " / " when used in combination, and by "+" when used in combination.

[0102] Table 1. Rats grouping and tested drugs

[0103]

[0104] 2.2. MACO model establishment

[0105] Male SD rats weighing 250g-280g were selected for the experiment. They were fasted but not watered for 12 hours before the operation, weighed and numbered, and anesthetized with an intraperitoneal injection of 10% chloral hydrate at a dose of 0.3ml / 100g. After the rats had no pain reaction, the neck skin was prepared and the rats were fixed on a flat board in a supine position. The skin was cut along the middle of the neck to expose the subcutaneous tissue. The fat and neck muscle groups were peeled off with blunt tools such as curved tweezers. The common carotid artery and the adhered vagus nerve were seen, the vagus nerve was peeled off, the internal carotid artery and external carotid artery were separated along the common carotid artery, the proximal end of the external carotid artery and the common carotid artery were ligated, the common carotid artery was cut open, the embolus was first inserted into the internal carotid artery along the common carotid artery, and continued to be pushed into the skull until the black mark exceeded the branches of the internal and external carotid arteries. The internal carotid artery was ligated with a thin wire to prevent the embolus from retreating. The extra thin thread was cut off and the wound was sutured. The rat was placed in a warm place to maintain body temperature until awakening. The timing started from the time when the thread plug blocked the cerebral blood flow. After 2 hours, the thread plug was gently pulled out and the black mark was seen, which means reperfusion was achieved, and the establishment of the rat MACO model was completed. The drug was injected through the rat tail vein at 0 hours, 24 hours, and 48 hours after the rat brain blood was reperfused.

[0106] 2.3. Test drugs and administration methods

[0107] The tested drugs and their dosages are listed in Table 1. The drug solution was normal saline, and the solution was prepared with normal saline solution to the required concentration before use. The drugs were injected through the rat tail vein at 0 hours, 24 hours, 48 ​​hours, and 72 hours after the rat brain blood was reperfused.

[0108] 2.4 TTC brain tissue staining

[0109] After 72 hours of cerebral ischemia and reperfusion, the behavior of the rats was observed and scored. After the rats were anesthetized, blood was collected from the abdominal aorta to reduce the effect of blood in the brain on staining. After the brain was taken, it was placed in a -20℃ refrigerator for 35 minutes and then taken out for coronal sectioning. Each section was about 2 mm, and was stained in 2% TTC solution at 37℃ in the dark for 30 minutes. It was then fixed with 4% paraformaldehyde for 24 hours. The scanner took pictures and the ischemic area was calculated by IMAGE PRO plus. The percentage of cerebral ischemic area = (the sum of the white ischemic area of ​​each section) / (the sum of the brain area of ​​each section) × 100%. According to the behavioral score of the rats within 72 hours of drug administration, the score was scored in a single-blind method, and the score was based on the Zea Longa 4-point scoring standard. The scoring method is as follows: 0 points, the rat is normal, with no symptoms of neurological injury; 1 point, the contralateral forepaw cannot be fully extended; 2 points, turning in circles to the outside; 3 points, leaning relative to the side; 4 points; unable to walk spontaneously, and consciousness is lost.

[0110] 2.5 Experimental Results

[0111] By statistically analyzing the behavioral and brain tissue TTC staining results of rats in each group, the overall results of the model group, positive drug (control) group and combination drug group are shown in Table 2 below.

[0112] Table 2. Results of the efficacy of the tested drugs (as shown in Table 1) on ischemic brain injury

[0113]

[0114]

[0115]

[0116] The results in Table 2 show that the effect of cGAMP in treating ischemic reperfusion brain injury is significantly different under different doses, and the more the dose, the better (the optimal dose is 1 mg / kg). In addition, although magnesium salts (such as MgSO4) themselves have no obvious anti-ischemic brain injury efficacy, the effect of the combination of STING endogenous agonist cGAMP and magnesium salts (such as MgSO4) in treating brain ischemic injury is significantly improved compared with cGAMP alone. Moreover, the molar ratio of the components of the composition is significantly different. Too little MgSO4 has no significant synergistic effect, and too much can cause negative effects such as redness and swelling of the eyes (inflammation) and slow movement. The optimal molar ratio of the composition of STING endogenous agonist cGAMP and magnesium salts (cGAMP:MgSO4) is 1:2.

[0117] Example 3: Study on the effect of the combination of STING endogenous agonist cyclic dinucleotide cGAMP and edaravone and other drugs for treating stroke on the function of resisting ischemic brain damage

[0118] 3.1 Experimental animals and their groups

[0119] Experimental animals: Male rats weighing 250-280 g were purchased from Southern Model Organisms Technology Co., Ltd. Male rats were randomly divided into 14 groups, with 10 rats in each group. The 14 groups of rats were as follows:

[0120] Table 3. Grouping of rats and tested drug combinations

[0121]

[0122]

[0123] 3.2MACO model establishment

[0124] The experimental steps are the same as in Example 2.

[0125] 3.3 Test drugs and administration methods

[0126] The tested drugs and their dosages are listed in Table 3. The drug solution was normal saline, and the solution was prepared with normal saline solution to the required concentration before use. The drugs were injected through the rat tail vein at 0 hours, 24 hours, 48 ​​hours, and 72 hours after the rat brain blood was reperfused.

[0127] 3.4 TTC brain tissue staining

[0128] After 72 hours of cerebral ischemia and reperfusion, the behavior of the rats was observed and scored. After the rats were anesthetized, blood was collected from the abdominal aorta to reduce the effect of blood in the brain on staining. The brain was placed in a -20°C refrigerator for 35 minutes and then taken out for coronal sectioning. Each section was about 2 mm. It was stained in 2% TTC solution at 37°C in the dark for 30 minutes and then fixed with 4% paraformaldehyde for 24 hours. The scanner took pictures and the ischemic area was calculated by IMAGE PRO plus. The percentage of cerebral ischemic area = (the sum of the white ischemic area of ​​each section) / (the sum of the brain area of ​​each section) × 100%.

[0129] 3.5 Experimental Results

[0130] By analyzing the behavioral and brain tissue TTC staining results of rats in each group, the overall results of the model group, positive drug (control) group and combination drug group are shown in Table 4 below.

[0131] Table 4. The efficacy of cGAMP combined with stroke drugs on ischemic brain injury

[0132]

[0133]

[0134] The following conclusions can be drawn from the results in Table 4:

[0135] (1) The STING endogenous agonist cyclic dinucleotide cGAMP has very different performances when used in combination with different anti-stroke drugs. It is not the case that the combination of two effective anti-stroke drugs can enhance the efficacy of anti-brain injury.

[0136] (2) When edaravone is used in combination with cGAMP, the effects vary greatly under different ratios of the two. The best ratio is cGAMP (1 mg) and edaravone (6 mg), which significantly improves the efficacy. Therefore, cGAMP and edaravone have a synergistic effect in the best ratio, but produce toxic side effects when the ratio is not right.

[0137] (3) When cGAMP and butylphthalide, or cGAMP and citicoline were used in combination, the efficacy was significantly improved compared with the single administration of butylphthalide or citicoline under the experimental ratio (1mg:6mg), but the efficacy result was not 1+1 greater than 2, which means that the effects of the two drugs in combination are not simply superimposed, and the pharmacological mechanism is very complex.

[0138] (4) The combination of edaravone and dexcanol showed better pharmacodynamics than edaravone, but when cGAMP was used in combination with dexcanol, the effect was worse, leading to the serious consequence of death of rats. This further shows that when two effective drugs are used in combination, not all of them will have better results as expected.

[0139] (5) After arduous research, the optimal combination of cGAMP and edaravone was discovered, which significantly improved the ideal drug efficacy and laid a foundation for innovative ideas and innovative drugs for subsequent clinical research.

[0140] There are obvious differences in the effects of treating ischemic reperfusion brain injury under different doses. The more the dose, the better. The optimal cGAMP dose is 1 mg / kg. In addition, although magnesium salts (such as MgSO4) themselves have no obvious anti-ischemic brain injury efficacy, the effect of the combination of STING endogenous agonist cGAMP and magnesium salts (such as MgSO4) in treating brain ischemic injury is significantly improved compared with cGAMP alone. Moreover, the molar ratio of the components of the composition is significantly different. Too little MgSO4 has no significant effect, and too much can cause negative effects such as redness and swelling of the eyes. The optimal molar ratio of the composition components (cGAMP:MgSO4) is 1:2. The optimal ratio of cGAMP and edaravone for combined use is 1 mg:6 mg.

[0141] Example 4: Study on the effect of STING endogenous agonist cyclic dinucleotide cGAMP and its combination with stroke drugs on the repair of neurological function in ischemic brain injury

[0142] (1) Experimental animals:

[0143] Healthy male rats, weighing 250-280 g, were purchased from Shanghai South Model Organisms Co., Ltd. and kept in a clean animal room.

[0144] (2) Establishment of rat cerebral ischemia model and experimental methods: Same as Example 2.

[0145] (3) TTC staining

[0146] After achieving 2h reperfusion injury of rat brain ischemia, the drug was administered after the beginning of perfusion, once every 24 hours, and continuously administered for 7 days. Then, 10% chloral hydrate was injected intraperitoneally at a dose of 0.3mL / 100g for anesthesia, and the abdominal aorta was bled to reduce the effect of blood in the brain on staining. The whole brain was immediately removed by decapitation, and then quickly placed in a -20℃ refrigerator for freezing for 20min after being rinsed with physiological saline. The frozen brain was taken out and coronal sliced ​​with a blade, each slice was about 2mm. It was stained in a 2% TTC solution in the dark for 20min, fixed in a 4% paraformaldehyde solution for 6h, and then photographed, and then the area was circled and calculated using Photoshop software, and the percentage of brain ischemia area = (the sum of the white ischemic area of ​​each slice) / (the sum of the brain area of ​​each slice) × 100%.

[0147] (4) Drugs and experimental groups

[0148] Male rats were randomly divided into 8 groups, with 10 rats in each group. The grouping and test drugs of the 8 groups are shown in Table 5:

[0149] Table 5. Rat groups and tested drugs and their dosages

[0150]

[0151]

[0152] Administration: Tail vein injection, once a day, for 7 consecutive days.

[0153] Table 6. Results of the study on the effect of cGAMP and its combination with edaravone on the repair of brain function after ischemic brain injury

[0154]

[0155]

[0156] Among them, the “+” at the beginning of the rightmost row represents weight gain, which reflects the physical condition of the mouse. The more weight gain, the better the physical condition.

[0157] (5) Experimental results

[0158] Through the statistics of TTC staining of brain tissue, body weight and behavioral results of rats in each group, the research results of the model group and the drug-treated group are listed in Table 6, respectively.

[0159] After 7 days of administration, there was no significant improvement in behavioral characteristics and brain ischemic area ratio in the positive control group, edaravone, compared with 72 hours of administration. In other words, edaravone has no obvious brain function repair effect in the rehabilitation stage after stroke treatment. In contrast, cGAMP or a combination of cGAMP and MgSO4 has a significant brain function repair effect after cerebral ischemic injury. When used in combination, the positive control drug, edaravone / decanol, did not show brain function repair effect in the rehabilitation period, although it had a protective therapeutic effect within 72 hours of emergency treatment. The combination of cGAMP / MgSO4 / edaravone has a significant therapeutic effect on brain function repair.

[0160] Example 5: Screening of exogenous STING agonists and the effect of the screened exogenous STING agonists in inhibiting ischemic brain injury

[0161] (1) Screening of exogenous STING agonists

[0162] Virtual screening of STING exogenous agonist strategies and technical methods: First, LibiDock molecular docking is used to preliminarily screen STING agonist compounds, and then CDOCKER molecular docking is used to produce high-precision docking results. Finally, the optimal compound screened is verified by isothermal titration calorimetry (ITC) to obtain STING exogenously screened agonist compounds. The STING agonists screened in this example and their molecular docking and ITC parameters are summarized in Table 7. The molecular structure is shown below.

[0163] Table 7. Screened STING exogenous agonists and their molecular docking and Kd parameters

[0164]

[0165]

[0166] (2) Preparation and characterization of STING agonist compounds

[0167] It should be noted that the various materials and reagents used in this example are commonly used in the art and can be obtained commercially.

[0168] ST45, 2-[(5-methoxy-(imidazo[4,5-B]pyridine)-2-thio)methyl]-7-carboxylic acid methyl ester-quinazolin-4(3H)-one

[0169] 5-Methoxy-2-mercaptoimidazo[4,5-b]pyridine (91 mg, 0.5 mmol), 2-(chloromethyl)-7-carboxylic acid methyl ester quinazolin-4(3H)-one (126 mg, 0.5 mmol) and sodium hydroxide (100 mg, 2.5 mmol) were dissolved in methanol (7 mL) and stirred at room temperature overnight. After post-treatment, the organic solvent methanol was removed under reduced pressure to obtain a crude product, which was purified using a 300-400 mesh silica gel column to obtain the final product 45 (72 mg, 36%) as a white solid. 1 H NMR (400MHz, DMSO) δ13.45(s,1H),12.82(s,1H),8.20(d,J=8.2Hz,1H),8.03(s,1H),7.96(d,J= 8.2Hz,1H),7.79(d,J=8.4Hz,1H),6.62(d,J=8.6Hz,1H),4.53(s,2H),3.90(s,3H),3.85(s,3H). 13 CNMR(101MHz,DMSO)δ166.55,165.62,162.67,161.10,154.71,147.13,144.48,136.80 ,135.33,127.32,127.07,126.03,124.57,121.75,109.74,54.53,53.19,35.81.ESI-MS m / zcalcd forC 18 H 15 N5O4S + 398.0918, found 398.0921[M+H] + .

[0170] ST46, 2-[(5-methoxy-(imidazo[4,5-B]pyridine)-2-thio)methyl]-5-fluoro-quinazolin-4(3H)-one

[0171] 5-Methoxy-2-mercaptoimidazo[4,5-b]pyridine (91 mg, 0.5 mmol), 2-(chloromethyl)-5-fluoroquinazolin-4(3H)-one (107 mg, 0.5 mmol) and sodium hydroxide (100 mg, 2.5 mmol) were dissolved in methanol (7 mL) and stirred at room temperature overnight. After post-treatment, the organic solvent methanol was removed under reduced pressure to obtain a crude product, which was purified using a 300-400 mesh silica gel column to obtain the final product 46 (122 mg, 68%) as a white solid.

[0172] 1H NMR (400MHz, DMSO) δ13.20 (s, 1H), 12.62 (s, 1H), 7.76 (dd, J = 13.9, 7.8Hz, 2H), 7.39 (d ,J=8.1Hz,1H),7.34–7.15(m,1H),6.63(d,J=8.5Hz,1H),4.48(s,2H),3.86(s,3H).13C NMR(101MHz,DMSO)δ162.61,162.18,159.56,158.77,154.74,148.81,147.10,144.62,136.10, 136.00,126.03,122.11,121.84,114.23,114.03,110.91,110.84,109.63,54.54,35.44.ESI-MS m / z calcd for C 16 H 12 FN5O2S + 358.0768, found 358.0754[M+H] + .

[0173] ST55, 2-[(5-chloro-(imidazo[4,5-B]pyridine)-2-thio)methyl]-7-carboxylic acid methyl ester-quinazolin-4(3H)-one

[0174] 5-Chloro-2-mercaptobenzimidazole (92 mg, 0.5 mmol), 2-(chloromethyl)-7-methyl quinazoline-4(3H)-one (126 mg, 0.5 mmol) and sodium hydroxide (100 mg, 2.5 mmol) were dissolved in methanol (7 mL) and stirred at room temperature overnight. After post-treatment, the organic solvent methanol was removed under reduced pressure to obtain a crude product, which was purified by a 300-400 mesh silica gel column to obtain the final product, a white solid ST55 (132 mg, 66%).

[0175] 1H NMR (400MHz, DMSO) δ12.88(s,2H),8.19(d,J=8.2Hz,1H),8.04(d,J=1.4Hz,1H),7.96(dd,J=8.2,1.6H z,1H),7.51(s,1H),7.45(d,J=5.5Hz,1H),7.15(dd,J=8.5,2.0Hz,1H),4.56(s,2H),3.89(s,3H).13C NMR(101MHz,DMSO)δ165.72,161.31,154.06,151.10,147.89,135.92,135.27,133.84, 129.13,127.85,127.20,126.83,124.99,124.59,115.30,113.77,53.14,35.46.ESI-MS m / z calcd for C 18 H 13 ClN4O3S + 401.0470,found401.0467[M+H] + .

[0176] ST64, 2-[(4-methyl-(thiazolo[4,5-B]pyridine)-2-thio)methyl]-7-fluoro-quinazolin-4(3H)-one

[0177] 4-Methyl-2-mercaptobenzothiazole (91 mg, 0.5 mmol), 2-(chloromethyl)-7-fluoroquinazolin-4(3H)-one (106 mg, 0.5 mmol) and sodium hydroxide (100 mg, 2.5 mmol) were dissolved in methanol (7 mL) and stirred at room temperature overnight. After post-treatment, the organic solvent methanol was removed under reduced pressure to obtain a crude product, which was purified by a 300-400 mesh silica gel column to obtain the final product 64 (100 mg, 56%) as a white solid.

[0178] 1H NMR (400MHz, DMSO) δ12.66(s,1H),8.17(dd,J=8.7,6.4Hz,1H),7.82(dd,J=6.0,3.2Hz,1 H),7.38(ddd,J=11.2,9.5,2.5Hz,2H),7.30–7.21(m,2H),4.62(s,2H),2.58(s,3H).13C NMR (101MHz, DMSO) δ167.49,164.99,164.22,161.02,156.09,151.93,149.73,149.60,135.16,131.33, 129.72,129.61,127.36,125.28,119.64,118.14,116.19,115.96,111.87,111.65,35.95,18.23.ESI-MS m / z calcd for C 17 H 12 FN3OS2 + 358.0479,found

[0179] 358.0488[M+H] + .

[0180] Several other exogenous STING agonists, including MSA-2, SR-717, C-178, diABZI, and ADU-S100, were purchased from Sigma.

[0181] Example 6: Study on the effect of STING exogenous agonists in the treatment of ischemic brain damage

[0182] (1) Experimental animals: Healthy male rats, weighing 250-280 g, were purchased from Shanghai South Model Organisms Co., Ltd. and kept in a clean animal room.

[0183] (2) Establishment of rat cerebral ischemia model and experimental methods: Same as Example 2.

[0184] (3) TTC staining

[0185] After achieving 2h reperfusion injury of rat brain ischemia, the drug was administered after the beginning of perfusion, once every 24 hours, and continuously administered for 7 days. Then, 10% chloral hydrate was injected intraperitoneally at a dose of 0.3mL / 100g for anesthesia, and the abdominal aorta was bled to reduce the effect of blood in the brain on staining. The whole brain was immediately cut off and removed, and then quickly placed in a -20℃ refrigerator for 20min after being rinsed with physiological saline. The frozen brain was taken out and coronal sliced ​​with a blade, each slice was about 2mm. It was stained in 2% TTC solution in the dark for 20min, fixed in 4% paraformaldehyde solution for 24h, and then photographed by scanner, and the ischemic area was calculated by IMAGE PRO plus, and the percentage of brain ischemic area = (the sum of the white ischemic area of ​​each slice) / (the sum of the brain area of ​​each slice) × 100%.

[0186] (4) Drugs and experimental groups

[0187] Male rats were randomly divided into 14 groups, with 10 rats in each group. The grouping and test drugs of the 14 groups of rats are shown in Table 8:

[0188] Table 8. Rat groups and tested STING exogenous agonist compounds and their dosages

[0189]

[0190]

[0191] Administration: intraperitoneal injection, once a day, for 7 consecutive days.

[0192] Table 9. Study results of the therapeutic effects of STING exogenous agonist compounds on ischemic brain injury

[0193]

[0194]

[0195]

[0196] (5) Experimental results

[0197] Through the statistics of TTC staining of brain tissue, body weight and behavioral results of rats in each group, the research results of the model group and the drug-treated group are listed in Table 9, respectively.

[0198] The screened STING exogenous agonists are all effective in treating ischemic brain damage, especially ST64, which has a significantly better therapeutic effect on ischemic brain damage than the positive drug edaravone. This shows that the screened STING exogenous agonists have the application prospect of becoming drugs for the treatment and repair of ischemic brain damage.

[0199] Example 7. Acute toxicity study of exogenous STING agonists and their combination with magnesium salts

[0200] Experimental materials: 20 ICR mice (purchased from Shanghai Slake Experimental Animal Co., Ltd., half male and half female, weighing 20-22 g, fed with pelleted feed, free access to food and water.

[0201] Experimental method: ICR mice were intraperitoneally injected with 1g / kg of the screened STING agonist compounds (ST45, ST46, ST55, ST64, MSA-2, diABZI, SR-717, C-178, ADU-S100) (prepared with saline for injection), cGAMP+MgSO4 (1mg:0.36mg) combination, and cGAMP / edaravone (1mg:6mg) according to body weight, and the toxic reactions and deaths of mice within 14 days after administration were observed. The results showed that after intraperitoneal injection of mice, the mice were active normally. No mice died within 14 days after administration. On the 15th day, all mice were killed, dissected, and all organs were examined with the naked eye, and no obvious lesions were found.

[0202] Experimental Results

[0203] The results of the above-mentioned acute toxicity experiments show that the maximum tolerated dose (MTD) of intraperitoneal injection is not less than 1g / Kg, indicating that the screened STING agonist compounds (ST45, ST46, ST55, ST64, MSA-2, diABZI, SR-717, C-178, ADU-S100), cGAMP+MgSO4 (1mg:0.36mg) combination, and cGAMP / edaravone (1mg:6mg) combination have low acute toxicity.

[0204] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention and do not constitute a limitation of the present invention. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A pharmaceutical composition, characterized in that include: STING endogenous agonists and magnesium salts; The STING endogenous agonist is selected from at least one of 2',3'-cGAMP, c-di-AMP, c-di-GMP, c-di-IMP, c-GMP-IMP and derivatives thereof; The magnesium salt is selected from at least one of magnesium sulfate, magnesium chloride, magnesium oxalate, magnesium carbonate, magnesium nitrate, and magnesium ammonium phosphate; The molar ratio of the STING endogenous agonist to the magnesium salt is 1:1.5-5.

2. The pharmaceutical composition according to claim 1, characterized in that Also includes: Endogenous STING agonists and drugs for the treatment of stroke; The drug for treating stroke is selected from at least one of edaravone, butylphthalide, cebixin, oxiracetam, piracetam, citicoline, cinepazide, nimodipine, flunarizine, and ganglioside; The mass ratio of the drug for treating stroke to the STING endogenous agonist is 2-12:

1.

3. A pharmaceutical composition, characterized in that include: Endogenous STING agonists and drugs for the treatment of stroke; The STING endogenous agonist is selected from at least one of 2',3'-cGAMP, c-di-AMP, c-di-GMP, c-di-IMP, c-GMP-IMP and derivatives thereof; The drug for treating stroke is selected from at least one of edaravone, butylphthalide, cebiracem, oxiracetam, piracetam, citicoline, cinepazide, nimodipine, flunarizine, and ganglioside; The mass ratio of the drug for treating stroke to the STING endogenous agonist is 2-8:

1.

4. Use of the pharmaceutical composition according to any one of claims 1 to 3 in the preparation of a medicament for treating and / or repairing ischemic stroke brain damage.

5. Use of an exogenous STING agonist in the preparation of a drug for preventing, treating or repairing ischemic stroke, characterized in that: The STING exogenous agonist is selected from at least one of the following compounds:

6. The use according to claim 5, characterized in that: The STING exogenous agonist is selected from at least one of the following compounds:

7. A pharmaceutical composition, characterized in that include: STING exogenous agonists and magnesium salts; The STING exogenous agonist is selected from at least one of the following compounds: The magnesium salt is selected from at least one of magnesium sulfate, magnesium chloride, magnesium oxalate, magnesium carbonate, magnesium nitrate, and magnesium ammonium phosphate; The molar ratio of the STING exogenous agonist to the magnesium salt is 1:1.5-5.

8. The pharmaceutical composition according to claim 7, characterized in that The STING exogenous agonist is selected from at least one of the following compounds: The magnesium salt is magnesium sulfate; The molar ratio of the STING exogenous agonist to the magnesium salt is 1:1.5-4.

9. The pharmaceutical composition according to claim 7, characterized in that It also includes drugs for treating stroke; The drug for treating stroke is selected from at least one of edaravone, butylphthalide, cebiracem, oxiracetam, piracetam, citicoline, cinepazide, nimodipine, flunarizine, and ganglioside; The mass ratio of the drug for treating stroke to the STING exogenous agonist is 1-10:

1.

10. A pharmaceutical composition, characterized in that include: STING exogenous agonists, and drugs for the treatment of stroke; The STING exogenous agonist is selected from at least one of the following compounds: The drug for treating stroke is selected from at least one of edaravone, butylphthalide, cebiracem, oxiracetam, piracetam, citicoline, cinepazide, nimodipine, flunarizine, and ganglioside; The mass ratio of the drug for treating stroke to the STING exogenous agonist is 1-10:

1.

11. The pharmaceutical composition according to claim 10, characterized in that The STING exogenous agonist is selected from at least one of the following compounds:

12. Use of the pharmaceutical composition according to any one of claims 7 to 11 in the preparation of a medicament for treating and / or repairing ischemic stroke brain damage.

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