Pharmaceutical compositions comprising sting agonists and uses thereof

Abstract

This invention discloses pharmaceutical compositions containing endogenous STING agonists, and the use of these compositions in the preparation of medicaments for treating and / or repairing ischemic stroke brain injury; it also discloses the use of exogenous STING agonists, or compositions thereof with magnesium salts, or compositions thereof with stroke-treating pharmaceutical compositions in the preparation of medicaments for treating and / or repairing ischemic stroke brain injury. The pharmaceutical compositions containing STING agonists of this invention significantly improve efficacy in treating ischemic stroke and / or repairing brain injury, not only significantly alleviating the difficulty of a narrow emergency window, but also improving cerebral blood circulation and neuroprotection, and exhibiting significant brain function repair effects during subsequent rehabilitation.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to STING agonists and their compositions, combination therapies or pharmaceutical compositions, and their applications. Background Technology

[0002] Stroke, also known as cerebrovascular accident, is a disease caused by the sudden rupture and bleeding of blood vessels in the brain, or by blockage of blood vessels leading to ischemia and hypoxia in the brain. Stroke is divided into two main categories: ischemic stroke and hemorrhagic stroke. Ischemic stroke occurs when there is an occlusion in the external or internal arteries supplying blood to the brain, preventing timely and sufficient collateral circulation and causing a shortage of blood supply to the local brain tissue for metabolic needs.

[0003] Current treatment options for acute ischemic stroke include general management and specific therapies. General management primarily refers to the maintenance and control of physiological parameters. Specific therapies are key to treating acute ischemic stroke and mainly include improving cerebral blood circulation and neuroprotection. Neuroprotective drug treatment strategies are primarily neuroprotective rather than reparative, and are limited by a narrow time window.

[0004] The existing problems and limitations of ischemic stroke medications are as follows: Edaravone and its combination with dextromethorphan for injection are the most effective first-line clinical drugs. However, these drugs only have neuroprotective effects in treating acute ischemic stroke and their effect on restoring brain function after emergency treatment is not significant. There is an urgent need in the market for drugs that can promote the repair of brain function after treatment, especially drugs that can both treat acute ischemic stroke and promote the repair of brain function after treatment.

[0005] The innate immune interferon stimulation pathway (STING pathway) has been extensively studied. STING-targeted immune agonists upregulate the transcription of the type I interferon INF-β gene, thereby regulating changes in the expression of various cytokines. Cyclic cGMP-AMP dinucleotide synthase (cGAS), under activation conditions after binding to DNA, endogenously catalyzes the synthesis of cyclic dinucleotide cGAMP. The endogenous STING agonist cGAMP acts as a second messenger, mediating the activation of TBK1 and IRF-3 through STING stimulation of interferon INF-I, thereby initiating the transcription of the type I interferon INF-β gene. The application of STING agonists in immunotherapy, anti-tumor drugs, and anti-Alzheimer's drugs has been reported. The application of the 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 inventors of this application a few years ago. In our previous study, using a mouse model of local ischemia, cGAMP was administered via the tail vein 2 hours after ischemia and 8 hours after reperfusion. This significantly reduced the percentage of ischemic brain area and improved the behavioral patterns of mice after ischemia. Therefore, we initially concluded that cGAMP had an efficacy in the emergency treatment of ischemic brain injury. However, we did not systematically study whether cGAMP had a role in repairing brain function after acute treatment of ischemic stroke, nor did we investigate whether combining cGAMP with stroke drugs would have better therapeutic effects or whether the combined use had a repair function for brain function damage caused by ischemic stroke, even though first-line clinical stroke drugs such as edaravone did not have significant neuroreparative effects. Furthermore, because the endogenous agonist of STING, cyclic dinucleotide cGAMP, is a secondary signaling molecule with a short metabolic cycle and is easily degraded in vivo, its efficacy and drug-likeness are limited. Based on the crystal structure of the STING molecule, molecular docking simulation technology was used to screen out exogenous agonist compounds with good affinity for STING. One of the purposes of this study is to investigate the effects of the screened exogenous STING agonists on the treatment of ischemic stroke and the repair of brain function.

[0006] Treatment for acute ischemic stroke primarily involves improving cerebral blood circulation and neuroprotection, while also aiming to repair brain nerve function and promote gradual recovery. A key technical challenge is that neuroprotective drugs are primarily protective rather than restorative, and they all have a narrow therapeutic window. The window for acute thrombolysis in stroke is extremely short, only 3-4.5 hours, often causing patients to miss the optimal treatment window. Summary of the Invention

[0007] In view of the deficiencies of the prior art, the present invention provides STING agonists, compositions thereof, and applications.

[0008] A 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 their derivatives;

[0011] Preferably, the endogenous agonist of STING 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] STING endogenous agonists and drugs for treating stroke;

[0016] The medication for treating stroke is selected from at least one of edaravone, butylphthalide, septoprolol, piracetam, cytidine diphosphate choline, cinnarizine, nimodipine, flunarizine, and gangliosides.

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

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

[0019] STING endogenous agonists, and drugs for treating 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 their derivatives;

[0021] The medication for treating stroke is selected from at least one of edaravone, butylphthalide, septoprolol, piracetam, cytidine diphosphate choline, cinnarizine, nimodipine, flunarizine, and gangliosides.

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

[0023] A third aspect of the invention provides the use of the pharmaceutical composition described above in the preparation of a medicament for treating and / or repairing brain injury from ischemic stroke.

[0024] A fourth aspect of the present invention provides the use of an exogenous STING agonist in the preparation of a medicament for the prevention, treatment, or repair of ischemic stroke, wherein the exogenous STING agonist is selected from at least one of the following compounds:

[0025]

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

[0027]

[0028] The molecular formulas and systematic names of the above substances are shown in the table below:

[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 exogenous STING agonist to the magnesium salt is 1:1.5-5.

[0037] Preferably, the exogenous STING 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 exogenous STING agonist to the magnesium salt is 1:1.5-4.

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

[0042] The medication for treating stroke is selected from at least one of edaravone, butylphthalide, septoprolol, piracetam, cytidine diphosphate choline, cinnarizine, nimodipine, flunarizine, and gangliosides.

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

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

[0045] STING exogenous agonists and drugs for treating stroke;

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

[0047]

[0048] The medication for treating stroke is selected from at least one of edaravone, butylphthalide, septoprolol, piracetam, cytidine diphosphate choline, cinnarizine, nimodipine, flunarizine, and gangliosides.

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

[0050] Preferably, the exogenous STING 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-described pharmaceutical composition in the preparation of a medicament for treating and / or repairing brain injury from ischemic stroke.

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

[0055] This invention discloses a pharmaceutical composition comprising an endogenous STING agonist and a magnesium salt, and the use of the endogenous STING agonist in combination with a drug for treating stroke / the composition in the preparation of a drug for treating and / or repairing brain injury from ischemic stroke; it also discloses the use of an exogenous STING agonist, or a combination thereof with a magnesium salt, or a composition thereof with a drug for treating stroke, in the preparation of a drug for treating and / or repairing brain injury from ischemic stroke.

[0056] The pharmaceutical composition containing the STING agonist of this invention significantly improves the efficacy in treating ischemic stroke and / or repairing ischemic brain injury. It not only prolongs the treatment window for acute ischemic stroke, significantly alleviating the difficulty of a narrow emergency treatment window, but also improves cerebral blood circulation and provides neuroprotection. The inventors also unexpectedly discovered that the pharmaceutical composition of this invention has brain function repair effects during subsequent rehabilitation, gradually repairing brain function damage caused by ischemia, significantly improving the treatment effect of stroke, and greatly increasing survival and cure rates. Therefore, the pharmaceutical composition containing the STING agonist of this invention has broad application prospects.

[0057] The features and advantages of the present invention will be described in detail in the following detailed description section. Attached Figure Description

[0058] Figure 1 The image shows the ESI-MS image of the cyclic dinucleotide 2',3'-cGAMP in Example 1. Detailed Implementation

[0059] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the invention will be further described in detail below with reference to 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 invention are merely illustrative examples of specific implementations of the invention and are intended to explain the invention, but do not constitute a limitation thereof.

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

[0061] In the description of this application, unless otherwise stated, the terms "multiple / areas" and similar terms mean two / a kind or more. Furthermore, the terms "comprising," "including," and any variations thereof are intended to cover non-exclusive inclusion.

[0062]

Terminology Explanation

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

[0064] As used herein, definitions of standard chemical terms (such as groups) can be found in the references in this field.

[0065] Unless otherwise stated, conventional methods within the scope of the art, such as mass spectrometry, NMR, IR, and UV / VIS spectroscopy, and pharmacological methods, are employed. Unless specifically defined, the terminology used herein in the relevant descriptions of analytical chemistry, organic synthetic chemistry, and pharmaceutical and medicinal chemistry is known in the art. Standard techniques can be used in chemical synthesis, chemical analysis, drug preparation, formulation and delivery, and in the treatment of patients. For example, reactions and purifications can be carried out using the manufacturer's instructions for use of kits, or in accordance with methods known in the art or the description of this invention. The techniques and methods described above can generally be carried out according to conventional methods well known in the art, based on the descriptions in several summary and more specific documents cited and discussed in this specification. In this specification, groups and their substituents can be selected by those skilled in the art to provide stable structural moieties and compounds.

[0066] When a substituent is described by a conventional chemical formula written from left to right, the substituent also includes chemically equivalent substituents obtained when the structural formula is written from right to left.

[0067] The chapter headings used in this document are for organizational purposes only and should not be construed as limiting the subject matter. All references or portions thereof cited in this application, including but not limited to patents, patent applications, articles, books, manuals, and papers, are incorporated herein by reference in their entirety.

[0068] In this paper, certain chemical groups are preceded by simplified symbols to indicate the total number of carbon atoms present in the group. For example, C 1-6 Alkyl refers to an alkyl group having a total of 1 to 6 carbon atoms as defined below. The total number of carbon atoms in the simplified symbol does not include carbons that may be present in substituents of the group.

[0069] Except as otherwise specified, when used in the specification and claims of this application, the following terms shall have the following meanings.

[0070] As used herein, the terms "compound of the invention" or "active ingredient of the invention" are used interchangeably and include stereoisomers, enantiomers, or pharmaceutical salts thereof of the general formula compound. The term also includes racemic mixtures, optical isomers, isotopic compounds (such as deuterated compounds), or prodrugs.

[0071] "Stereoisomers" are compounds composed of identical atoms bonded by the same bonds, but with different three-dimensional structures. This invention will cover various stereoisomers and mixtures thereof.

[0072] When the compounds of the present invention contain alkene double bonds, unless otherwise stated, the compounds of the present invention are intended to contain both E- and Z- geometric isomers.

[0073] "Tautomer" refers to an isomer formed when a proton is transferred from one atom of a molecule to another atom of the same molecule. All tautomer forms of the compounds of this invention are also included within the scope of this invention.

[0074] The compounds of this invention may contain one or more chiral carbon atoms, and thus can produce enantiomers, diastereomers, and other stereoisomers. Each chiral carbon atom may be defined as (R)- or (S)- based on stereochemistry. This invention aims to include all possible isomers, as well as their racemic and optically pure forms. The compounds of this invention can be prepared using racemic, diastereomer, or enantiomers as starting materials or intermediates. Optically active isomers can be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques, such as crystallization and chiral chromatography.

[0075] Conventional techniques for preparing / separating individual isomers include chiral synthesis from suitable optically pure precursors, or resolution of racemates (or racemates of salts or derivatives) using, for example, chiral high-performance liquid chromatography.

[0076] In this invention, compounds can be expanded to include any number of substituents or functional groups. Generally, the term "substitution," whether preceding or following the term "optional," in the formulations of this invention, refers to the substitution of a hydrogen radical with a substituent of a specified structure. When multiple positions in a particular structure are replaced by multiple specific substituents, each position of the substituent can be the same or different. The term "substitution" as used herein includes all permissible substitutions in organic compounds. In a broad sense, permissible substituents include acyclic, cyclic, branched-unbranched, carbocyclic, and heterocyclic, aromatic and non-aromatic organic compounds. In this invention, heteroatomic nitrogen may be supplemented with hydrogen substituents or any permissible organic compound described above to complete its valence state. Furthermore, this invention is not intended to limit permissible substituted organic compounds in any way. This invention considers the combination of substituents and variable groups to be beneficial in the treatment of diseases in the form of stable compounds. The term "stable" here means having a stable compound that, when tested for a sufficiently long time, maintains sufficient structural integrity, preferably remaining effective for a sufficiently long time, and is used herein for the aforementioned purposes.

[0077] Pharmaceutical Compositions and Administration

[0078] The pharmaceutical compositions described in this invention are intended for the prevention and / or treatment of cancer, immune diseases, metabolic diseases, etc. In this application, "pharmaceutical composition" refers to a formulation of the compounds of this invention with a medium generally accepted in the art for delivering bioactive compounds to mammals (e.g., humans). This medium includes pharmaceutically acceptable carriers. The purpose of the pharmaceutical composition is to facilitate administration to the organism, thereby promoting the absorption of the active ingredient and the exertion of its bioactivity. The term "pharmaceutical" as used herein refers to a substance (such as a carrier or diluent) that does not affect the bioactivity or properties of the compounds of this invention and is relatively non-toxic, i.e., the substance can be administered to an individual without causing an adverse biological response or interacting adversely with any component contained in the composition.

[0079] In this invention, "pharmaceutical excipients" include, but are not limited to, any adjuvant, carrier, excipient, flow aid, sweetener, diluent, preservative, dye / coloring agent, flavoring agent, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent or emulsifier that is permitted by the relevant government regulatory authority to be acceptable for human or livestock use.

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

[0081] In this invention, the term "prevention" includes reducing the likelihood of a patient developing or worsening a disease or symptom.

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

[0083] (i) To prevent the occurrence of disease or condition in mammals, especially when such mammals are susceptible to the disease or condition but have not yet been diagnosed with it;

[0084] (ii) To suppress a disease or symptom, that is, to curb its development;

[0085] (iii) To alleviate a disease or symptom, that is, to cause the condition of the disease or symptom to subside; or

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

[0087] In this invention, the term "effective amount" or "pharmaceutical effective amount" refers to an amount of at least one drug or compound that, when taken, is sufficient to alleviate, to some extent, one or more symptoms of the disease or condition being treated. The result may be a reduction and / or relief of signs, symptoms, or causes, or any other desired change in the biological system. For example, an "effective amount" for treatment is the amount of a composition comprising the compounds disclosed herein that is clinically necessary to provide significant symptom relief. Effective amounts suitable for any individual case can be determined using techniques such as dose escalation testing.

[0088] When using STING agonists, or pharmaceutical compositions thereof, or in combination, it is to provide a safe and effective amount of the compounds of the invention to mammals (including humans) requiring treatment, wherein the dose administered is a pharmaceutically considered effective dose for the prevention and / or treatment and / or repair of ischemic stroke brain injury. For a person weighing 60 kg, in some embodiments, the daily dose is typically 30 to 60 mg when using an exogenous STING agonist alone for prevention or treatment. In some embodiments, the daily dose is typically 50 to 100 mg when using an exogenous STING agonist alone for repair.

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

[0090] In this invention, the terms "application," "administration," "medication," etc., refer to methods capable of delivering a compound or composition to the desired site for biological action. These methods include, but are not limited to, treatment via one or more of the following routes of administration: oral or intravenous injection, intravenous infusion, intramuscular injection, subcutaneous injection, or direct intraventricular injection. The STING agonists and compositions of this invention, when used in combination or as pharmaceutical compositions, are available in one or more of the following dosage forms: tablets, capsules, enteric-coated microparticles, granules, suspensions, emulsions, solutions, syrups, and injections.

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

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

[0093] Example 1:

[0094] 1.1 Preparation and Characterization of STING Endogenous Agonist Cyclic Dinucleotides

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

[0096] 1.2 Preparation and Characterization of Combinations of STING Endogenous Agonist Cyclic Dinucleotides and Magnesium Salts

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

[0098] Example 2: Study on the effect of a combination of STING endogenous agonist cyclic dinucleotide and magnesium salt on anti-ischemic brain injury function.

[0099] 2.1 Laboratory animals and their grouping

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

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

[0102] Table 1. Grouping of rats and test drugs

[0103]

[0104] 2.2 Establishment of the MACO Model

[0105] Male SD rats weighing 250g-280g were selected for the experiment. They were fasted for 12 hours before the procedure but allowed free access to water. They were weighed and numbered, and anesthetized with an intraperitoneal injection of 10% chloral hydrate at a dose of 0.3ml / 100g. After the rats showed no pain response, their necks were prepared, and they were fixed supine on a flat plate. The skin was cut along the midline of the neck to expose the subcutaneous tissue. Using blunt tools such as curved forceps, the fat and neck muscles were dissected, revealing the common carotid artery and the adhered vagus nerve. The vagus nerve was dissected, and the internal and external carotid arteries were separated along the common carotid artery. The external and proximal ends of the common carotid arteries were ligated. The common carotid artery was cut open, and the embolus was inserted first, advancing along the common carotid artery into the internal carotid artery. The embolus continued to advance into the cranium until the black markers passed beyond the branches of the internal and external carotid arteries. A thin suture was used to ligate the internal carotid artery to prevent embolism. Excess sutures were cut and the wound sutured. The rat was placed in a warm environment to maintain body temperature until awakening. Timing began after the suture plug blocked cerebral blood flow. Two hours later, the suture plug was gently pulled out, and a black mark was observed, indicating reperfusion, thus establishing the rat MACO model. The drug was administered via tail vein injection at 0, 24, and 48 hours after cerebral blood reperfusion.

[0106] 2.3. Test Drug and Administration Method

[0107] The test drugs and their dosages are listed in Table 1. The drug lysin was physiological saline, which was prepared into a solution of the required concentration using physiological saline solution before use. The drugs were administered via tail vein injection at 0, 24, 48, and 72 hours after cerebral reperfusion in rats.

[0108] 2.4. TTC brain tissue staining

[0109] 72 hours after cerebral ischemia-reperfusion, the behavior of rats was observed and scored. After anesthetizing the rats, blood was collected from the abdominal aorta to reduce the influence of blood in the brain on staining. The brain tissue was then frozen at -20°C for 35 minutes and sectioned coronally, approximately 2 mm in size. Staining was performed in 2% TTC solution at 37°C in the dark for 30 minutes, followed by fixation with 4% paraformaldehyde for 24 hours. Images were taken using a scanner, and the ischemic area was calculated using Image Pro Plus. The percentage of ischemic area was calculated as: (sum of white ischemic areas in each section) / (sum of brain slice areas in each section) × 100%. Scoring was based on the average behavior of the rats within 72 hours of drug administration, using a single-blind method and referencing the Zea Longa 4-point scoring system. The scoring method was as follows: 0 points: normal rat, no neurological damage symptoms; 1 point: unable to fully extend the contralateral forepaw; 2 points: circling outwards; 3 points: tilting to the side; 4 points: unable to walk spontaneously, loss of consciousness.

[0110] 2.5 Experimental Results

[0111] Based on the statistical analysis of the behavioral and TTC staining results of the 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. Efficacy of the test drugs (as shown in Table 1) on ischemic brain injury

[0113]

[0114]

[0115]

[0116] Table 2 shows that the efficacy of cGAMP in treating ischemia-reperfusion brain injury varies significantly at different dosages, and higher doses are not necessarily better (the optimal dose is 1 mg / kg). Furthermore, although magnesium salts (such as MgSO4) themselves do not have significant anti-ischemic brain injury effects, the combination of the STING endogenous agonist cGAMP and a magnesium salt (such as MgSO4) significantly improves the efficacy of treating ischemic brain injury compared to cGAMP alone. Moreover, the molar ratio of the components in this combination varies significantly; too little MgSO4 has no significant synergistic effect, while too much can cause adverse effects such as eye redness (inflammation) and bradykinesia. The optimal molar ratio of the STING endogenous agonist cGAMP to a magnesium salt (cGAMP:MgSO4) is 1:2.

[0117] Example 3: Study on the effect of combined use of STING endogenous agonist cyclic dinucleotide cGAMP and edaravone and other stroke treatment drugs on anti-ischemic brain injury function.

[0118] 3.1 Laboratory animals and their grouping

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

[0120] Table 3. Grouping of rats and combination of tested drugs

[0121]

[0122]

[0123] 3.2 Establishment of the MACO Model

[0124] The experimental procedure is the same as in Example 2.

[0125] 3.3 Test drug and administration method

[0126] The test drugs and their dosages are listed in Table 3. The drug lysin was prepared as physiological saline and was dissolved in physiological saline solution to the required concentration before use. The drugs were administered via tail vein injection at 0, 24, 48, and 72 hours after cerebral reperfusion in rats.

[0127] 3.4 TTC brain tissue staining

[0128] 72 hours after cerebral ischemia-reperfusion, the behavior of rats was observed and scored. After anesthetizing the rats, blood was collected from the abdominal aorta to reduce the influence of blood in the brain on staining. After the brain was collected, it was frozen in a -20℃ freezer for 35 minutes and then sectioned into coronal sections, each about 2 mm. The sections were stained in 2% TTC solution at 37℃ in the dark for 30 minutes, and then fixed with 4% paraformaldehyde for 24 hours. The images were taken with a scanner and the ischemic area was calculated using IMAGE PRO plus. The percentage of cerebral ischemic area = (sum of white ischemic area area of ​​each section) / (sum of brain slice area of ​​each section) × 100%.

[0129] 3.5 Experimental Results

[0130] Based on the statistical analysis of the behavioral and TTC staining results of rats in each group, the overall results of the model group, positive drug (control) group, and combined drug group are shown in Table 4 below.

[0131] Table 4. Efficacy of cGAMP combined with stroke drugs in ischemic brain injury

[0132]

[0133]

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

[0135] (1) The endogenous agonist cyclic dinucleotide cGAMP of STING showed significant differences when used in combination with different anti-stroke drugs, and it is not true that the combination of two effective anti-stroke drugs can enhance the anti-brain injury efficacy.

[0136] (2) When edaravone is used in combination with cGAMP, the effects vary greatly depending on the ratio of the two. The optimal ratio is cGAMP (1 mg) to edaravone (6 mg), which significantly improves the efficacy. Therefore, cGAMP and edaravone have a synergistic effect when used in the optimal ratio, but toxic side effects occur when the ratio is incorrect.

[0137] (3) When cGAMP is used in combination with butylphthalide or cGAMP with cidylcholine, under the experimental ratio (1mg:6mg), the efficacy is significantly higher than that of butylphthalide or cidylcholine alone. However, the efficacy result is not 1+1 greater than 2, indicating that the effects of the two drugs are not simply superimposed when used in combination. The pharmacological mechanism is very complex.

[0138] (4) Edaravone combined with dextran showed better efficacy than edaravone. However, when cGAMP was combined with dextran, the effect was worse, leading to the death of rats. This further illustrates that the combined use of two effective drugs does not always yield the expected better results.

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

[0140] The efficacy of cGAMP in treating ischemia-reperfusion brain injury varies significantly depending on the dosage; more is not always better. The optimal cGAMP dose is 1 mg / kg. Furthermore, although magnesium salts (such as MgSO4) themselves do not have significant anti-ischemic brain injury effects, the combination of the endogenous agonist cGAMP and magnesium salts (such as MgSO4) significantly improves the efficacy of treating ischemic brain injury compared to cGAMP alone. Moreover, the molar ratio of the components in this combination varies significantly; too little MgSO4 has no significant effect, while too much can cause side effects such as eye redness and swelling. The optimal molar ratio of the components (cGAMP:MgSO4) is 1:2. The optimal ratio for combined cGAMP and edaravone is 1 mg:6 mg.

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

[0142] (1) Laboratory animals:

[0143] Healthy male rats, weighing 250-280 grams, were purchased from Shanghai Southern Model Biotechnology Co., Ltd. and housed in the cleaning agent animal room.

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

[0145] (3) TTC staining

[0146] After inducing 2-hour cerebral ischemia-reperfusion injury in rats, the drug was administered every 24 hours for 7 consecutive days, starting from the beginning of perfusion. Then, rats were anesthetized by intraperitoneal injection of 10% chloral hydrate at a dose of 0.3 mL / 100 g. Bloodletting was performed from the abdominal aorta to reduce the influence of blood in the brain on staining. The rats were immediately decapitated and the intact brain was removed, rinsed with physiological saline, and then rapidly frozen at -20°C for 20 min. The frozen brain was then sectioned coronally using a scalpel, with each section approximately 2 mm in size. The sections were stained in 2% TTC solution in the dark for 20 min, fixed in 4% paraformaldehyde solution for 6 h, and photographed. The area was then delineated and calculated using Photoshop software. The percentage of ischemic area was calculated as: (sum of white ischemic areas in each section) / (sum of brain slice areas in each section) × 100%.

[0147] (4) Drugs and experimental grouping

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

[0149] Table 5. Rat grouping, test drugs and their dosages

[0150]

[0151]

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

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

[0154]

[0155]

[0156] In this context, the "+" at the beginning of the rightmost row represents weight gain, which reflects the mouse's physical condition; the greater the weight gain, the better the mouse's health.

[0157] (5) Experimental Results

[0158] Based on the statistical analysis of TTC staining of rat brain tissue, body weight, and behavioral results in each group, the research results of the model group and the drug-treated group are listed in Table 6.

[0159] In the positive control group, edaravone showed no significant improvement in behavioral characteristics or cerebral ischemia-to-area ratio 7 days after administration, compared to 72 hours after administration. This indicates that edaravone does not have a significant effect on brain function repair during the rehabilitation phase after stroke treatment. In contrast, cGAMP or a combination of cGAMP and MgSO4 has a significant effect on brain function repair after ischemic brain injury. When used in combination, the positive control drug, edaravone / dexcanol, did not show any effect on brain function repair during the rehabilitation period, although it had a protective effect within 72 hours of emergency treatment. The combination of cGAMP / MgSO4 / edaravone showed significant efficacy in brain function repair.

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

[0161] (1) Screening of exogenous STING agonists

[0162] Virtual screening strategy and techniques for exogenous STING agonists: First, LibiDock molecular docking was used for initial screening of STING agonist compounds. Then, CDOCKER molecular docking was employed to produce high-precision docking results. Finally, the optimal selected compounds were verified by isothermal titration calorimetry (ITC) to obtain the exogenous STING agonist compounds. The STING agonists screened in this embodiment, along with their molecular docking and ITC parameters, are summarized in Table 7. The molecular structures are shown below.

[0163] Table 7. Screened exogenous STING 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 embodiment are all commonly used materials and reagents in the art and can be obtained commercially.

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

[0169] 5-Methoxy-2-mercaptoimidazo[4,5-b]pyridine (91 mg, 0.5 mmol), methyl 2-(chloromethyl)-7-carboxylate 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 overnight at room temperature. The reaction was then post-treated by removing the organic solvent methanol under reduced pressure to obtain a crude product. The crude product was purified using a 300-400 mesh silica gel column to give a final product, a white solid 45 (72 mg, 36%). 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-fluoroquinazoline-4(3H)-one (107 mg, 0.5 mmol), and sodium hydroxide (100 mg, 2.5 mmol) were dissolved in methanol (7 mL) and stirred overnight at room temperature. The reaction was then post-treated by removing the organic solvent methanol under reduced pressure to obtain a crude product. The crude product was purified using a 300-400 mesh silica gel column to give a final product, a white solid 46 (122 mg, 68%).

[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, methyl 2-[(5-chloro-(imidazo[4,5-B]pyridine)-2-thio)methyl]-7-carboxylate-quinazolin-4(3H)-one

[0174] 5-Chloro-2-mercaptobenzimidazole (92 mg, 0.5 mmol), methyl 2-(chloromethyl)-7-carboxylate 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 overnight at room temperature. The reaction was then post-treated by removing the organic solvent methanol under reduced pressure to obtain the crude product. The crude product was purified using a 300-400 mesh silica gel column to give 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, found 401.0467 [M+H] + .

[0176] ST64, 2-[(4-methyl-(thiazo[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-fluoroquinazoline-4(3H)-one (106 mg, 0.5 mmol), and sodium hydroxide (100 mg, 2.5 mmol) were dissolved in methanol (7 mL) and stirred overnight at room temperature. The reaction was then post-processed, and the organic solvent methanol was removed under reduced pressure to obtain the crude product. The crude product was purified using a 300-400 mesh silica gel column to give the final product, a white solid 64 (100 mg, 56%).

[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] 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 role of exogenous STING agonists in the treatment of ischemic brain injury

[0182] (1) Experimental animals: Healthy male rats, weighing 250-280 grams, were purchased from Shanghai Southern Model Biotechnology Co., Ltd. and housed in the cleaning agent animal room.

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

[0184] (3) TTC staining

[0185] After inducing 2 hours of cerebral ischemia-reperfusion injury in rats, the drug was administered every 24 hours for 7 consecutive days, starting from the beginning of perfusion. Then, rats were anesthetized by intraperitoneal injection of 10% chloral hydrate at a dose of 0.3 mL / 100 g. Bloodletting was performed from the abdominal aorta to reduce the influence of blood in the brain on staining. The rats were immediately decapitated and the intact brain was removed, rinsed with physiological saline, and then rapidly frozen at -20°C for 20 minutes. The frozen brain was then sectioned coronally using a scalpel, with each section approximately 2 mm in size. The sections were stained in 2% TTC solution in the dark for 20 minutes, fixed in 4% paraformaldehyde solution for 24 hours, and then photographed. The ischemic area was calculated using Image Pro Plus: percentage of ischemic area = (sum of white ischemic areas in each section) / (sum of brain slice areas in each section) × 100%.

[0186] (4) Drugs and experimental grouping

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

[0188] Table 8. Grouping of rats and test STING exogenous agonist compounds and their dosages

[0189]

[0190]

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

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

[0193]

[0194]

[0195]

[0196] (5) Experimental Results

[0197] The results of TTC staining of rat brain tissue, body weight, and behavioral findings in each group are statistically analyzed. The research results of the model group and the drug-treated group are listed in Table 9.

[0198] The screened exogenous STING agonists were all effective against ischemic brain injury, especially ST64, which showed significantly better therapeutic effects than the positive control drug edaravone. This indicates that the screened exogenous STING agonists have the potential to become drugs for the treatment and repair of ischemic brain injury.

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

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

[0201] Experimental Methods: ICR mice were intraperitoneally injected with 1 g / kg of selected STING agonist compounds (ST45, ST46, ST55, ST64, MSA-2, diABZI, SR-717, C-178, ADU-S100) (prepared with physiological saline for injection), a cGAMP+MgSO4 (1 mg:0.36 mg) combination, or a cGAMP / edaravone (1 mg:6 mg) combination. Toxicity and mortality were observed within 14 days after administration. Results: Mice showed normal activity after intraperitoneal injection. No mice died within 14 days. On day 15, all mice were sacrificed, and autopsies revealed no obvious lesions in any organs.

[0202] Experimental results

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

[0204] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and do not constitute a limitation on the present invention. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, including combining various technical features in any other suitable manner. These simple modifications and combinations should be regarded as the content disclosed in the present invention and all fall within the protection scope of the present invention.

Claims

1. A pharmaceutical composition, characterized in that, include: STING endogenous agonists and magnesium salts; The endogenous agonist of STING is 2',3'-cGAMP; The magnesium salt is magnesium sulfate; The molar ratio of the STING endogenous agonist to the magnesium salt is 1:1.5-3.

5.

2. The pharmaceutical composition according to claim 1, characterized in that, Also includes: Medications for treating stroke; The drug used to treat stroke is edaravone; The mass ratio of the drug for treating stroke to the endogenous agonist STING is 6:

1.

3. The use of the pharmaceutical composition according to claim 1 or 2 in the preparation of a medicament for treating and / or repairing brain injury from ischemic stroke.

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