Use of sarilmax in the preparation of a drug for preventing platelet aggregation

Abstract

The present application provides a use of sarafloxacin and / or a pharmaceutically acceptable salt of sarafloxacin in the preparation of a drug for inhibiting the collagen receptor glycoprotein VI signal pathway of platelets. Compared with the prior art, according to the results of non-clinical cell tests and animal efficacy tests, sarafloxacin not only plays an anti-thrombosis role in inhibiting collagen-mediated platelet aggregation, but also does not increase the risk of pathological bleeding, which is beneficial to the development of a safe and effective anti-platelet aggregation drug.

Description

Technical Field

[0001] This invention belongs to the pharmaceutical field and relates to a new use of sarrencycline, more specifically to the application of sarrencycline in the preparation of antiplatelet aggregation drugs. Background Technology

[0002] Inhibiting platelet aggregation to prevent and treat thrombosis is a crucial approach in the prevention and treatment of thrombotic diseases. During physiological hemostasis and arterial thrombosis, platelets adhere to the exposed subendothelial matrix of blood vessels, leading to platelet activation, which is the initiation stage of thrombosis. Subsequently, platelet aggregation and release reactions occur, ultimately resulting in thrombosis. Post-adhesion signal transduction and platelet activation depend on the collagen-platelet glycoprotein receptor VI (GPVI) axis. Inhibiting this pathway can prevent platelet activation at an earlier stage, thereby inhibiting thrombosis. In vitro and in vivo experiments have shown that inhibitors or antibodies targeting GPVI can inhibit thrombosis and its inflammatory response without interfering with normal hemostasis, demonstrating both safety and efficacy. This helps address the shortcomings of current clinical medications, namely, the increased risk of bleeding associated with increased antiplatelet aggregation activity. This has spurred the development of various agents that regulate or inhibit GPVI-mediated platelet activation and thrombosis. Currently, these drugs are mainly divided into four categories: competitive inhibitors of GPVI, such as GPVI-Fc; anti-GPVI antibodies, such as antibodies that induce GPVI endocytosis or enzymatic cleavage (consumable antibodies) including JAQ1, mF1201, mF1232, and cF1232; GPVI blockers, which are antibodies that inhibit the function of GPVI, ranging from monoclonal antibodies (mAbs) to their antigen-binding fragments (Fabs) such as 9O12.3, 204-11, OM2, OM4, m-Fab-F, and 1G5; and the last category mainly targets the GPVI signaling pathway, such as curcumin and losartan. Among them, the GPVI competitive inhibitor soluble GPVI-Fc dimer protein (Revacept) has completed phase 1 clinical trials, and the results show that it can safely and effectively inhibit collagen-induced platelet aggregation without prolonging bleeding time.

[0003] Sareccycline is a novel, narrow-spectrum oral tetracycline drug, originally developed by Paratek and Allergen. Its tablet form was approved in the United States in October 2018, primarily for the treatment of non-nodular moderate-to-severe acne vulgaris in patients aged 9 years and older. The exact mechanism of action is not fully understood. Sareccycline is a tetracycline ribosomal protein inhibitor that inhibits protein synthesis through interaction with 70S bacterial ribosomes. However, unlike other tetracyclines, sareccycline's unique C7 extension into the messenger RNA (mRNA) channel directly interacts with the A-codon, thereby interfering with mRNA movement through this channel and / or disrupting the A-codon / anticodon interaction. Its pharmacological effects include in vitro efficacy against *Propionibacterium acnes* and other Gram-positive bacteria, and it exhibits in vitro anti-inflammatory activity. In clinical trials, two phase III, double-blind, randomized controlled trials evaluated the efficacy of sareccycline in treating moderate-to-severe acne vulgaris, showing that the drug is safe, effective, and well-tolerated.

[0004] The structural formula of sarrenzin is as follows:

[0005]

[0006] Through experimental research, the inventors have discovered for the first time that sarrenzin can act as a CPVI inhibitor to exert an antiplatelet aggregation effect and treat related diseases. Summary of the Invention

[0007] Sarecycline, a novel narrow-spectrum oral tetracycline, is used to treat non-nodular moderate to severe acne vulgaris in patients aged 9 years and older. The exact mechanism of action of this treatment is still unclear.

[0008] During our research, we were surprised to find that the drug inhibits the collagen-platelet glycoprotein receptor VI (GPVI) axis pathway, thereby exerting its effect on antithrombotic and cerebrovascular diseases.

[0009] This invention provides the use of sarrencycline and / or pharmaceutically acceptable salts of sarrencycline in the preparation of a drug that inhibits the platelet collagen receptor glycoprotein VI signaling pathway.

[0010] Wherein, the pharmaceutically acceptable salt of sarrencycline is any pharmaceutically acceptable salt known to those skilled in the art. It can be a salt formed by sarrencycline and an inorganic acid or a salt formed by sarrencycline and an organic acid, without any particular limitation. In this invention, one or more of the following are preferred acid addition salts formed by sarrencycline with hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, acetic acid, citric acid, maleic acid, oxalic acid, formic acid, benzoic acid, fumaric acid, succinic acid, tartaric acid, lactic acid, pyruvic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, and trifluoroacetic acid.

[0011] This invention also provides the use of sarrencycline and / or pharmaceutically acceptable salts of sarrencycline in the preparation of antiplatelet aggregation drugs.

[0012] Preferably, the sarrencycline and / or pharmaceutically acceptable salts of sarrencycline inhibit platelet aggregation by suppressing the platelet collagen receptor glycoprotein VI signaling pathway.

[0013] The above-mentioned drugs target GPVI to inhibit platelet activation and exert an anti-platelet aggregation effect.

[0014] Specifically, the platelet collagen receptor is the collagen COL receptor.

[0015] Preferably, the concentration of salpercycline and / or its pharmaceutically acceptable salts in the antiplatelet aggregation process is 1–100 μM, more preferably 3–100 μM, and even more preferably 10–100 μM.

[0016] The sarrencycline and / or pharmaceutically acceptable salts of sarrencycline provided by this invention can be used to prepare drugs for thrombotic diseases and cerebrovascular diseases caused by platelet aggregation.

[0017] The present invention also provides the use of sarrencycline and / or pharmaceutically acceptable salts of sarrencycline in the preparation of medicaments for the treatment and / or prevention of thrombotic diseases.

[0018] Preferably, the thrombotic disease is one or more of arterial thrombotic disease, venous thromboembolism, and thrombotic microangiopathy.

[0019] In this invention, the inhibitory effect of sarrencycline and / or pharmaceutically acceptable salts of sarrencycline on FeCl3-induced arterial thrombosis in rats demonstrates that sarrencycline and / or pharmaceutically acceptable salts of sarrencycline can be used to prepare drugs for the treatment and / or prevention of arterial thrombotic diseases.

[0020] The present invention also provides the use of sarrencycline and / or pharmaceutically acceptable salts of sarrencycline in the preparation of medicaments for the treatment and / or prevention of cerebrovascular diseases.

[0021] The present invention also provides the use of sarrencycline and / or pharmaceutically acceptable salts of sarrencycline in the preparation of medicaments for the treatment and / or prevention of stroke.

[0022] The present invention also provides the use of sarrencycline and / or pharmaceutically acceptable salts of sarrencycline in the preparation of medicaments for the treatment and / or prevention of cerebral small vessel disease.

[0023] The present invention also provides the use of sarrencycline and / or pharmaceutically acceptable salts of sarrencycline in the preparation of medicaments for the treatment and / or prevention of cerebral thrombosis.

[0024] This invention also provides the use of sarrencycline and / or pharmaceutically acceptable salts of sarrencycline in the preparation of medicaments for cerebral ischemia-reperfusion injury.

[0025] In this invention, salpercycline and / or pharmaceutically acceptable salts of salpercycline can improve neurological deficit symptoms and reduce the extent of cerebral infarction in a rat model of focal cerebral ischemia-reperfusion.

[0026] The present invention also provides the use of sarrencycline and / or pharmaceutically acceptable salts of sarrencycline in the preparation of medicaments for arterial stenosis; preferably, the arterial stenosis is carotid artery stenosis, and more preferably, bilateral carotid artery stenosis.

[0027] Specifically, the present invention provides that salpercycline and / or pharmaceutically acceptable salts of salpercycline can improve cerebral blood flow, improve learning and memory abilities, improve brain protein damage, and reduce microglia proliferation in the cortex and hippocampus in mice with bilateral common carotid artery stenosis.

[0028] The present invention also provides a pharmaceutical composition comprising sarrencycline and / or a pharmaceutically acceptable salt of sarrencycline.

[0029] Preferably, pharmaceutically acceptable excipients are also included.

[0030] The pharmaceutical composition can be used to treat thrombotic diseases and cerebrovascular diseases caused by platelet aggregation.

[0031] Preferably, when the pharmaceutical composition is used to treat thrombotic diseases and cerebrovascular diseases caused by platelet aggregation, the amount of the active ingredient sarrencycline and / or pharmaceutically acceptable salts of sarrencycline is 2 to 100 mg / kg, more preferably 3 to 80 mg / kg, and even more preferably 3 to 30 mg / kg.

[0032] Preferably, when the pharmaceutical composition is used to treat arterial stenosis, the amount of the active ingredient sarrencycline and / or a pharmaceutically acceptable salt of sarrencycline is 2 to 100 mg / kg, more preferably 20 to 80 mg / kg, and even more preferably 40 to 80 mg / kg.

[0033] This invention provides the use of sarrencycline and / or pharmaceutically acceptable salts of sarrencycline in the preparation of drugs that inhibit the platelet collagen receptor glycoprotein VI signaling pathway. Compared with the prior art, based on non-clinical cell experiments and animal efficacy studies, sarrencycline's inhibition of collagen-mediated platelet aggregation not only exerts an antithrombotic effect but also does not increase the risk of pathological bleeding, which is beneficial for the development of safe and effective antiplatelet aggregation drugs. Detailed Implementation

[0034] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0035] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, illustrates the application of sarrenzin provided by the present invention in the preparation of antiplatelet aggregation drugs.

[0036] All reagents used in the following examples are commercially available.

[0037] Example 1: Study on the in vitro antiplatelet aggregation activity of sarrencycline

[0038] 1. Materials and Methods

[0039] 1.1 Animals

[0040] Sprague-Dawley (SD) rats, Shanghai Slack Laboratory Animal Co., Ltd.

[0041] 1.2 Reagents and Consumables

[0042]

[0043] 1.3 Preparation and grouping of drug formulations

[0044] One day before the experiment, 4.875 mg of salbutamol powder was accurately weighed and added to 100 μL of DMF. The solution was then pipetted and vortexed until fully dissolved and clear, yielding a 100 mM yellowish-brown stock solution. This stock solution was diluted with DMF to prepare gradient solutions of 40 mM, 12 mM, 4 mM, 1.2 mM, and 0.4 mM. 12 μL of the 100 mM cilostazol stock solution was added to 88 μL of DMF and mixed thoroughly to obtain a 12 mM dilution. Six groups were set up for the experiment: a blank control group, a salbutamol group (1 μM), a salbutamol group (3 μM), a salbutamol group (10 μM), a salbutamol group (30 μM), and a salbutamol group (100 μM). An equal volume of solvent was added to the blank control group. Each group was tested at least six times. In the ADP-induced experiment, a cilostazol final concentration of 30 μM group was added as a positive control.

[0045] 1.4 Preparation of platelet-rich plasma

[0046] Rats were intraperitoneally injected with 7% chloroacetaldehyde hydrate (6 mL / kg). The depth of anesthesia was such that the animals would have a slight response to severe stimulation. The abdominal aorta was separated and the residual fluid on its surface was wiped dry. Blood was collected using a venous lancet and a sodium citrate anticoagulant tube (1:9). The blood was carefully and gently shaken to mix it evenly. The mixture was centrifuged at 180g for 15 min at room temperature. The middle part of the upper liquid was carefully transferred to an EP tube by pipette, avoiding the aspiration of red blood cells and white flocculent material at the interface. The resulting turbid upper liquid was platelet-rich plasma (PRP). Physiological saline was used for zeroing and diluting the PRP.

[0047] 1.5 Determination of platelet aggregation rate

[0048] Connect the semi-automatic platelet aggregator to the power supply and preheat for approximately 30 minutes. Once the temperature reaches approximately 37°C and stabilizes, proceed with the experiment. Dilute physiological saline and PRP solution 1:1 (total volume 290 μL), add a stir bar, and place the saline-containing test cup in the thermostat port for 5 minutes to preheat. Add the test compound to the drug group 2 minutes beforehand. Set the instrument to TEST mode, place the saline test cup in the test channel, and press the ENT key or the corresponding channel key. The instrument will automatically detect the zero point, and the window will display a value, such as P40. After this value stabilizes, press the ENT key or the corresponding channel key again. Remove the saline test cup and place the PRP test cup in it. The window will display another value, such as R30. Press the ENT key or the corresponding channel key again, and the window will display the inducing agent. Use a pipette to add 10 μL of COL or ADP to the bottom of the cup. Press the ENT key or the corresponding channel key, and the instrument will enter the platelet aggregation test state. The window will display the timer. Press the channel key again and the window will display the platelet aggregation rate at that time. The test ends after 5 minutes, and the window will display the maximum platelet aggregation rate.

[0049] The final concentration of COL collagen was 3 μg / mL, and the final concentration of ADP was 5 μmol / L.

[0050] 1.6 Calculation of Platelet Aggregation Inhibition Rate

[0051] Based on the measured maximum aggregation rate (MAR) of the platelet aggregation samples, the platelet aggregation inhibition rate of sarrencycline at each concentration compared to the blank control group was calculated. Calculation formula:

[0052] Platelet aggregation inhibition rate (%) = (MAR) 空白对照组 -MAR 测试药物组 ) / MAR 空白对照组 ×100%.

[0053] 1.7 Data Statistics

[0054] Experimental data are expressed as mean ± standard deviation (Mean ± SD). One-way ANOVA and Dunnett's method were used to test differences between groups. P < 0.05 was considered statistically significant.

[0055] 2 Experimental Results

[0056] 2.1 Effect of sarrocycline on collagen COL-induced platelet aggregation

[0057] As shown in Table 1, compared with the blank control group, salpercycline inhibited platelet aggregation in a concentration-dependent manner within the range of 1–100 μM. One-way ANOVA results showed that salpercycline had a significant anti-platelet aggregation effect starting at 1 μM, with inhibition rates of 9.62%, 22.18%, 33.40%, 65.82%, and 95.19% at concentrations of 1, 3, 10, 30, and 100 μM, respectively. The half-maximal inhibitory concentration (IC50) of salpercycline for inhibiting collagen-induced platelet aggregation was 19.95 μM.

[0058] Table 1. Effects of sarrocycline on COL-induced platelet aggregation

[0059]

[0060] Data are expressed as Mean ± SD. Compared with the model group, *p < 0.05, **p < 0.01, ***p < 0.001.

[0061] 2.2 Effect of sarrocycline on ADP-induced platelet aggregation

[0062] As shown in Table 2, compared with the blank control group, there was no significant difference in platelet aggregation rate at different concentrations of salpercycline, meaning it did not affect ADP-induced platelet aggregation. Cilostazol 30 μM, as a positive control, significantly inhibited platelet aggregation (inhibition rate 25-30%).

[0063] Table 2. Effects of sarrocycline on ADP-induced platelet aggregation

[0064]

[0065] Data are expressed as Mean ± SD. Compared with the model group, *p < 0.05, **p < 0.01, ***p < 0.001.

[0066] Example 2: Effect of sarrenzine on collagen-platelet binding

[0067] 1. Materials and Methods

[0068] 1.1 Laboratory Animals

[0069] Sprague-Dawley (SD) rats, Shanghai Slack Laboratory Animal Co., Ltd.

[0070] 1.2 Reagents and Consumables

[0071]

[0072] 1.3 Platelet Extraction and Processing

[0073] Rats were intraperitoneally injected with 7% chloroacetaldehyde hydrate (6 mL / kg). The depth of anesthesia was such that the animals would have a slight response to severe stimulation. The abdominal aorta was separated and the residual fluid on its surface was wiped dry. Blood was collected using a venous lancet and a sodium citrate anticoagulant tube (1:9). The blood was carefully and gently shaken to mix it evenly. The mixture was centrifuged at 180g for 15 min at room temperature. The middle part of the upper liquid was carefully transferred to an EP tube by pipette, avoiding the aspiration of red blood cells and white flocculent material at the interface. The resulting turbid upper liquid was platelet-rich plasma (PRP). Physiological saline was used for zeroing and diluting the PRP.

[0074] 1.4 Flow cytometry detection

[0075] Platelets (2×10) 8 .mL -1 Incubate with 30 μM salazine or 5 μg / mL Fab 9O12 at 37°C for 10 minutes, then add 10 μg / mL -1 FITC-I collagen was activated at room temperature (RT) for 20 min and then fixed with 2% paraformaldehyde solution (PFA). Samples were analyzed by flow cytometry using a Becton Dickinson FACS101 cytometer.

[0076] 1.5 Data Statistics

[0077] Same as Example 1.

[0078] 2 Experimental Results

[0079] As shown in Table 3, the effect of sarrencycline was further analyzed by inducing platelet aggregation using FITC-conjugated type I collagen. Flow cytometry analysis of the binding of FITC-collagen to platelets showed that 10 μg / mL of FITC collagen could induce platelet aggregation after washing, while sarrencycline significantly inhibited the binding of FITC-collagen to platelets. GPVI antibody Fab 9O12 (50 μg / mL) was used as a positive control.

[0080] Table 3. Effects of sarrocycline on collagen-platelet binding

[0081]

[0082] Data are expressed as Mean ± SD. Compared with the model group, *p < 0.05, ***p < 0.001.

[0083] Example 3: Study on the inhibitory effect of sarrencycline on platelet aggregation in rats (in vivo verification)

[0084] 1. Materials and Methods

[0085] 1.1 Laboratory Animals

[0086] Sprague-Dawley (SD) rats, male, SPF grade, weighing 250-280g.

[0087] 1.2 Test Drug

[0088] Salrecycline and cilostazol are the same as in Example 1.

[0089] 1.3 Experimental Methods

[0090] 1.3.1 Animal grouping and administration

[0091] The experimental animals were divided into six groups: salpercycline group (1 mg / kg), salpercycline group (2 mg / kg), salpercycline group (4 mg / kg), salpercycline group (8 mg / kg), cilostazol group (8 mg / kg), and a normal control group. Salpercycline and cilostazol were administered by gavage in 0.5% CMC-Na solution. Animals in the salpercycline and cilostazol groups were administered the drugs by gavage once 3 hours before blood collection. Animals in the normal control group were given an equal volume of the solvent. Blood was collected from the abdominal aorta.

[0092] 1.3.2 Blood collection and PRP preparation

[0093] Experimental animals were weighed and randomly and equally assigned to each group in a single-blind manner. Rats were administered salbutamol and cilostazol by gavage according to their body weight. Three hours later, rats were anesthetized by intraperitoneal injection of 7% trichloroacetaldehyde hydrate (6 mL / kg). The abdominal aorta was dissected, and any residual fluid and mucosa on its surface were wiped dry. Blood was collected using a venous lancet and sodium citrate anticoagulant tube (1:9), with a volume of 6 mL. The blood was carefully and gently shaken to mix thoroughly and centrifuged at 180g for 15 min at room temperature. The middle portion of the supernatant was carefully transferred to an EP tube, avoiding aspiration of red blood cells. The resulting turbid supernatant was platelet-rich plasma (PRP), which was labeled with the group. Physiological saline was used for zeroing and diluting the PRP.

[0094] 1.3.3 Determination of platelet aggregation rate

[0095] Connect the semi-automatic platelet aggregator to the power supply and preheat for approximately 30 minutes. Once the temperature reaches approximately 37°C and stabilizes, proceed with the experiment. Dilute physiological saline and PRP solution 1:1 (total volume 290 μL), add a stir bar, and place the saline and PRP test cups separately in the thermostat port for 5 minutes to preheat. Add the test compound to the drug group 2 minutes beforehand. Set the instrument to TEST mode, place the saline test cup in the test channel, and press the ENT key or the corresponding channel key. The instrument will automatically detect the zero point, and the window will display a value, such as P40. After this value stabilizes, press the ENT key or the corresponding channel key again. Remove the saline test cup and place the PRP test cup in it. The window will display another value, such as R30. Press the ENT key or the corresponding channel key again, and the window will display the inducing agent. Use a pipette to add 10 μL of COL to the bottom of the cup. Press the ENT key or the corresponding channel key, and the instrument will enter the platelet aggregation test state. The window will display the timer. Press the channel key again and the window will display the platelet aggregation rate at that time. After the 5-minute test, the window will display the maximum platelet aggregation rate.

[0096] The final concentration of COL collagen is 3 μg / mL.

[0097] 1.4 Data Statistics

[0098] Same as Example 1.

[0099] 2 Experimental Results

[0100] Table 4 shows the inhibition rates of collagen-induced platelet aggregation in each group of animals. Compared with the normal control group, sarrencycline at 2 mg / kg significantly inhibited collagen-induced platelet aggregation in rats, and the inhibitory effect was dose-dependent. Compared with the cilostazol group (8 mg / kg), the platelet aggregation inhibition rate of sarrencycline at 4 mg / kg was not significantly different. This indicates that sarrencycline has a superior antiplatelet aggregation effect compared to cilostazol.

[0101] Table 4. Effects of sarrencycline on platelet aggregation in rats (in vivo verification)

[0102]

[0103] Data are expressed as Mean ± SD. Compared with the model group, *p < 0.05, **p < 0.01, ***p < 0.001.

[0104] Example 4: Study on the inhibitory effect of sarrencycline on FeCl3-induced arterial thrombosis in rats

[0105] 1. Materials and Methods

[0106] 1.1 Laboratory Animals

[0107] Sprague-Dawley (SD) rats, male, SPF grade, weighing 250-280g.

[0108] 1.2 Test Drug

[0109] Salariclocycline is the same as in Example 1.

[0110] 1.3 Experimental Methods

[0111] 1.3.1 Animal grouping and administration

[0112] In the thrombosis experiment, animals were randomly and equally divided into 5 groups according to body weight: sham-operated group, model group, salpercycline group (2 mg / kg), salpercycline group (4 mg / kg), and salpercycline group (8 mg / kg). The salpercycline group received the drug via gavage, suspended in 0.5% CMC-Na solution. The sham-operated group and model group received the drug via gavage. The drugs were administered once in the morning and once in the afternoon for 7 consecutive days. The experiment was conducted 30 minutes after the last administration.

[0113] 1.3.2 FeCl3-induced arterial thrombosis in rats

[0114] After anesthetizing rats with 7% chloroacetaldehyde hydrate (6 mL / kg) via intraperitoneal injection, the right common carotid artery was bluntly dissected along the midline of the neck, and a 1 cm long section was inserted. A 0.6 cm wide sealing strip was then placed inside, and a 1.0 cm × 0.5 cm filter paper strip soaked in 20% FeCl3 solution was wrapped around the dissected carotid artery segment and sealed with the sealing strip. The filter paper strip was removed after 15 minutes. After 40 minutes, the blood vessels at both ends of the filter paper strip were ligated, and the segment wrapped in the filter paper strip was precisely cut off. Residual blood was absorbed from the blood vessel with clean filter paper, and the wet weight of the blood vessel containing the thrombus was accurately measured. The blood vessel after removing the thrombus was weighed again, and the difference between the two weights was the mass of the thrombus in the 0.5 cm long segment. In the sham surgery group, physiological saline was used instead of the FeCl3-soaked filter paper strip.

[0115] 1.4 Data Statistics

[0116] Same as Example 1.

[0117] 2 Experimental Results

[0118] Compared with the sham-operated group, the carotid artery model site in the model group rats was filled with thrombi, indicating that FeCl3 can significantly induce carotid artery thrombosis. Compared with the model group, the sarrencycline groups (4 mg / kg and 8 mg / kg) significantly reduced thrombus weight (p < 0.05).

[0119] Table 5. Inhibitory effect of sarrocycline on FeCl3-induced arterial thrombosis in rats

[0120]

[0121] Data are expressed as Mean ± SD. Compared with the model group, *p < 0.05, **p < 0.01.

[0122] Example 5: Pharmacodynamic study of sarrencycline in a rat model of focal cerebral ischemia-reperfusion.

[0123] 1. Materials and Methods

[0124] 1.1 Laboratory Animals

[0125] Sprague-Dawley (SD) rats, male, SPF grade, weighing 280-300g.

[0126] 1.2 Test Drug

[0127] Salariclocycline is the same as in Example 1.

[0128] 1.3 Experimental Methods

[0129] 1.3.1 Preparation of a focal cerebral ischemia-reperfusion model

[0130] Rats were placed in an anesthesia induction chamber (4% isoflurane concentration). The depth of anesthesia was chosen so that the animal would only respond slightly to severe stimulation. The anesthetized rats were secured with elastic bands around their limbs (hind limbs fixed above the knee joint, forelimbs fixed above the wrist joint) and head. The animals were placed in a supine position on the operating table, with an anesthesia hood covering their heads. The isoflurane concentration was maintained at 2.5% to prevent the animal from waking up during the experiment. The fur was shaved from the head towards the chest using an animal shaver, and the skin was disinfected with alcohol. A midline incision was made in the neck, and the subcutaneous tissue was bluntly dissected. The thin fascia on the surface of the anterior cervical triangle was separated, and the lower edge of the clavicle-hyoid muscle was lifted to reveal a longitudinally pulsating artery parallel to this muscle. The carotid shell was opened to expose the bifurcation of the right carotid artery. The right common carotid artery, external carotid artery, and internal carotid artery were separated. The vagus nerve was gently dissected, and the external carotid artery was ligated and cut. The proximal end of the common carotid artery was clamped. An incision was made distal to the ligation suture of the external carotid artery, and a suture was inserted, passing through the bifurcation of the common carotid artery into the internal carotid artery. The suture was then slowly inserted until slight resistance was felt (approximately 20 mm from the bifurcation), thus blocking all blood supply to the middle cerebral artery. The suture was slightly secured below the incision in the external carotid artery with silk suture. The silk suture at the proximal end of the common carotid artery was released, and the animal was simply sutured and placed in a feeding box. After 90 minutes of right-sided cerebral ischemia, the suture was gently removed from the ischemic animal, and reperfusion was performed to restore blood supply. The external carotid artery was ligated with the silk suture used to secure the suture, the skin was sutured, and the area was disinfected. The rat was placed in clean feed, and its general condition and respiration were observed until it recovered from anesthesia. It was then given food and water and fed as usual.

[0131] 1.3.2 Animal grouping and administration

[0132] Experimental animals were divided into five groups: a model group, a salpercycline group (3 mg / kg), a salpercycline group (10 mg / kg), a salpercycline group (30 mg / kg), and an edaravone group (6 mg / kg). After establishing the cerebral ischemia model, animals were randomly assigned to each group in a single-blind manner with equal probability. Animals were administered the drugs once immediately after reperfusion: salpercycline was administered by gavage, and edaravone was administered intravenously. Animals in the model group were given an equal volume of 0.5% CMC-Na by gavage. Neurological deficits were evaluated 24 hours after cerebral ischemia. Animals were then sacrificed, and their brains were harvested, stained, and photographed to determine the infarct area.

[0133] 1.3.3 Neurological deficit symptom score and determination of cerebral infarction area

[0134] A modified Bederson 5-point scale was used to evaluate neurological deficit symptoms. A single-blind method was used to evaluate neurological deficit symptoms in rats after cerebral ischemia. That is, the experiment designer labeled the animals into groups, and the experimenters who scored the neurological deficit symptoms did not know the group assignments of the animals. After the scoring was completed, the scorers submitted the scores of various labels to the designer, who then unblinded the test and obtained the scores of each animal in each experimental group.

[0135] Appendix: Bederson 5-point scale for scoring neurological deficit symptoms

[0136]

[0137] The degree of cerebral infarction was determined using the TTC staining method. After the evaluation of the neurological deficit symptoms of the animals, they were euthanized with CO2, decapitated, and the brain was removed. The olfactory bulb, cerebellum, and lower brainstem were removed. The surface of the brain was rinsed with physiological saline to remove bloodstains, and any residual water was aspirated. The brain was then placed at -20°C for 20 minutes. Immediately after removal, a coronal section was made perpendicularly downward at the plane of visual intersection, and slices were cut every 2 mm posteriorly. The brain slices were incubated in 1% TTC staining solution (37°C for 30 minutes). Normal brain tissue stained deep red, while ischemic brain tissue appeared pale white. After rinsing with physiological saline, the brain slices were quickly arranged in a row from front to back, and any residual water was aspirated before photographing.

[0138] Calculation of cerebral infarction area: The images were processed using ImageJ software, and the corresponding area of ​​the left brain and the area of ​​the non-infarct lesion in the right brain were calculated according to the formula to determine the percentage of infarction range.

[0139] Infarct volume calculation method:

[0140] V = t(A1 + A2 + A3 + ... + An)

[0141] t is the slice thickness, and A is the infarct area.

[0142] %I = 100% × (V) C -VL ) / V C

[0143] %I represents the percentage of infarct volume, V C V represents the brain volume of the control side (left hemisphere). L The volume of the non-infarcted region on the infarcted side (right hemisphere).

[0144] 1.4 Data Statistics

[0145] Experimental data are expressed as mean ± standard deviation (Mean ± SD). One-way ANOVA was performed using GraphPadPrism (7.04) software for each efficacy index, and Dunnett's test was used to test for differences between groups. P < 0.05 was considered statistically significant.

[0146] 2. Experimental Results

[0147] 2.1 Impact on neurological deficit symptoms

[0148] The severity of neurological deficit symptoms in each group of animals is shown in Table 6. One-way ANOVA showed statistical differences among the groups. Compared with the model group, sarrocycline at doses of 3 mg / kg, 10 mg / kg, and 30 mg / kg significantly improved the neurological deficit symptoms in rats, and the effect was dose-dependent (p = 0.036, 0.011, 0.000).

[0149] Table 6. Effects of sarrocycline on neurological deficit symptoms

[0150]

[0151] Data are expressed as Mean ± SD. Compared with the model group, *p < 0.05, **p < 0.01, ***p < 0.001.

[0152] 2.2 Effect on the area of ​​cerebral infarction

[0153] The effects on the area of ​​cerebral infarction are shown in Table 7. One-way ANOVA showed statistically significant differences among the groups. Compared with the model group, the sarrocycline groups at 3 mg / kg, 10 mg / kg, and 30 mg / kg significantly reduced the cerebral infarction area in the model rats (p = 0.047, 0.028, 0.000).

[0154] Table 7. Effect of sarcomatin administration on the area of ​​cerebral infarction.

[0155]

[0156]

[0157] Data are expressed as Mean ± SD. Compared with the model group, **p < 0.01, **p < 0.01, ***p < 0.001

[0158] Example 6: Pharmacodynamic study of sarrencycline in a mouse model of bilateral common carotid artery stenosis.

[0159] 1. Materials and Methods

[0160] 1.1 Laboratory Animals

[0161] C57BL / 6 mouse, male, SPF grade, weight 27-30g.

[0162] 1.2 Test Drug

[0163] Salariclocycline is the same as in Example 1.

[0164] 1.3 Experimental Methods

[0165] 1.3.1 Establishment of a mouse model of bilateral common carotid artery stenosis (BCAS)

[0166] Two to three days prior to the procedure, the neck hair of the mice was shaved. After weighing and recording the experimental animals, the mice were placed in an anesthesia induction chamber (isoflurane concentration 3.5%). The depth of anesthesia was such that severe stimulation elicited a slight response from the animal. The anesthetized mice were secured to their limbs and head with tape, placed in a supine position within the operating area of ​​the stereomicroscope, and an anesthesia hood was placed over their heads. The isoflurane concentration was maintained at 1.5% to prevent the animals from waking up during the experiment.

[0167] After disinfecting the skin with alcohol, a midline incision was made in the neck, and the subcutaneous tissue was bluntly dissected. The thin fascia on the surface of the anterior cervical triangle was separated, and the lower edge of the clavicle-hyoid muscle was lifted to reveal a longitudinally pulsating artery parallel to this muscle. The arterial shell was opened, exposing and carefully separating both common carotid arteries. A silk thread was placed above and below each artery for easy manipulation. The vagus nerve was gently dissected, taking special care not to induce a vagal response. The silk thread was gently pulled to carefully wrap a coil around the common carotid artery, avoiding any hair, fat, or tissue adhesions during the coiling process. The silk thread clamping the common carotid artery was released, and the animal was sutured and placed on a warm blanket. General condition and respiration were observed until the animal recovered from anesthesia. It was then placed in a feeding box, given food and water, and kept in a standard feeding environment. The sham surgery group underwent the same surgical procedure as the model group, except for the use of the coil. During the surgery, any animals exhibiting abnormalities due to anesthesia, surgery, or other reasons were removed and their condition recorded.

[0168] 1.3.2 Animal grouping and administration

[0169] After establishing the BCAS model in experimental animals sequentially, the animals were randomly and equally assigned to each group in a single-blind manner. The experimental animals were divided into five groups: sham-operated group, model group, salpercycline group (40 mg / kg), salpercycline group (80 mg / kg), and cilostazol group (100 mg / kg). Drug administration began on day 3 after model establishment, once daily. All surviving animals were weighed twice weekly for the first two weeks after model establishment, and once weekly thereafter. Salpercycline and cilostazol were both administered by gavage in the form of 0.5% CMC-Na suspension; animals in the model group were given an equal volume of 0.5% CMC-Na by gavage.

[0170] 1.3.3 Cerebral blood flow, behavioral tests, LFB myelin staining pathological score, and IBA-1 immunohistochemical assay.

[0171] 1.3.3.1 Detection of cerebral blood flow (CBF)

[0172] Regional cerebral blood flow (CBF) was measured using a high-resolution laser speckle flow imaging (LSCI) system. Measurements were performed preoperatively and on days 1, 3, 7, and 14 postoperatively. Mice were anesthetized (induced with 3.5% isoflurane and maintained at 1.5%) and placed on the experimental table. Hair was shaved from the head, and the mice were fixed in a prone position. An incision was made along the midline of the brain to expose the skull. The fascia on the skull surface was cleaned. The imaging height and angle of the LSCI system were adjusted, and the imaging position was fixed in real-time display mode. Blood flow throughout the entire brain region was continuously recorded for 5 seconds in imaging mode. The average blood flow within the stable region of interest (ROI) was derived using offline analysis mode. The mouse incisions were sutured, and iodine was applied to prevent infection. Data analysis is expressed as a percentage of baseline values.

[0173] 1.3.3.2 Morris Water Maze

[0174] The Morris water maze test was used to assess the spatial learning and memory abilities of experimental mice. Four weeks post-surgery, mice underwent the Morris water maze test. The maze pool was 120 cm in diameter, 50 cm high, and 29 cm deep. A platform, 6 cm in diameter and 28 cm high, was fixed 1 cm below the water surface in the SE quadrant (target quadrant). A camera was positioned 2 m above the center of the pool, above the bottom, to simultaneously record the mice's movement. The water temperature was maintained at 22 ± 1℃. The experiment lasted six days, including a platform hiding test and a spatial exploration test. The first five days were for the platform hiding test, and the last day was for the spatial exploration test.

[0175] Hidden platform trial

[0176] This experiment lasted for 5 days. The platform was fixed in the target quadrant (SE quadrant). Animals were placed into the water from four different entry points in each quadrant. During training, the animals were gently lowered into the water facing the marked pool wall, and the time it took for the animal to find the platform (escape latency) was recorded. The animal was then allowed to remain on the platform for 10 seconds. If the animal could not find the platform within 60 seconds, the latency was recorded as 60 seconds, and the animal was guided to remain on the platform for 10 seconds. After training, the mice were returned to their cages and kept warm. Training was conducted once daily at each of the four entry points, and the average of the four latency times was used as the daily score for statistical analysis. The entry order for each animal remained consistent each day, but the entry order varied among different animals within the same cage to exclude information exchange between animals in the same group.

[0177] Space exploration experiment (probe trial)

[0178] The platform was removed 24 hours after the concealment experiment. The mice were then entered into the water from the opposite side of the platform, and the time spent in the target quadrant and the number of times the original platform was crossed were recorded within 60 seconds. The animals' spatial orientation ability and its changes during spatial exploration were observed.

[0179] 1.3.3.3Y Maze Experiment

[0180] The Y-maze free-alternation experiment was used to assess the learning and memory abilities of experimental animals. Testing was conducted 6 weeks post-surgery. Mice were brought from the animal facility to the behavioral laboratory beforehand to acclimatize to the experimental environment. The laboratory environment was kept quiet, and the lighting was adjusted to avoid direct, strong light. At the start of the experiment, mice were placed in the center of the Y-maze, facing the same direction, and allowed to freely move between the three arms. The order in which the mice entered and exited each arm was recorded and analyzed using TopscanLite animal behavior analysis software over 5-8 minutes (or manually recorded). Successful entry into three different arms was considered a correct choice. The number of correct alternations (n) and the total number of arm entries (N) for each mouse were recorded, and the correct alternation rate for each mouse was calculated. After each mouse's experiment, excrement was cleaned, and the inside of the Y-maze was wiped with 75% medical alcohol until the alcohol completely evaporated before proceeding to the next mouse to prevent odor from affecting the subsequent mice's movement behavior.

[0181] Correct alternation rate = Number of correct alternations n / (Total number of arm advances N-2)

[0182] 1.3.3.4 LFB myelin staining experiment

[0183] Eight weeks post-surgery, the brains of the animals were perfused with physiological saline and 4% paraformaldehyde and stored at room temperature in 4% paraformaldehyde. The samples were then embedded in paraffin and sectioned to obtain coronal sections (4 μm). The sections were positioned at the beginning of the hippocampus, with the corpus callosum, internal capsule, caudate putamen, and optic tract in the same visual plane.

[0184] Sections were dewaxed and rehydrated, then incubated in a staining jar containing LFB stain (Solarbio, Beijing, China) at 56°C for 2 hours. Excess stain was washed away with 95% ethanol, followed by washing with distilled water. The sections were then separated by Fast Blue differentiation solution for 10 seconds, followed by 70% ethanol for 20 seconds, washed with water, and observed under a microscope until the gray and white matter outlines were clearly defined. Counterstaining was performed with tar violet stain for 30-40 seconds, followed by washing with water. The sections were air-dried in a fume hood, cleared in xylene, and mounted with neutral resin.

[0185] Images were taken using a Leica biological microscope at 200 and 400 magnification. In the sliced ​​field of view, blue represents myelin sheath, and pink-purple represents neurons. The severity of white matter lesions (WML) was assessed by the density of stained axonal fibers.

[0186] WMLs are assessed in five brain regions: optic tract (OPT), internal capsule (IC), caudate nucleus (CPU), and the middle and lateral parts of the corpus callosum. The severity of WMLs is classified as normal (grade 0), disordered nerve fiber arrangement (grade 1), significant vacuolation (grade 2), and loss of myelinated fibers (grade 3).

[0187] 1.3.3.5 IBA-1 Immunohistochemical Detection

[0188] IBA-1 protein is generally considered a marker of microglia in the central nervous system, reflecting neuroinflammation. IBA-1 was selected as an indicator for evaluating neuroinflammation.

[0189] Dewaxing paraffin sections to water: Place sections sequentially in environmentally friendly dewaxing solution I for 10 min, then environmentally friendly dewaxing solution II for 10 min, then environmentally friendly dewaxing solution III for 10 min, followed by anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, anhydrous ethanol III for 5 min, and finally wash with distilled water. Antigen retrieval: See the table above for retrieval procedures. During this process, prevent excessive evaporation of the buffer solution and avoid drying the slides. After natural cooling, place the slides in PBS (pH 7.4) and wash three times on a decolorizing shaker, 5 min each time. (The retrieval solution and conditions are determined based on the tissue). Blocking endogenous peroxidase: Place sections in 3% hydrogen peroxide solution and incubate at room temperature in the dark for 25 min. Then place the slides in PBS (pH 7.4) and wash three times on a decolorizing shaker, 5 min each time. Serum blocking: Add 3% BSA to the histochemistry zone to evenly cover the tissue and block at room temperature for 30 min. (For primary antibodies derived from goat, use rabbit serum for blocking; for other sources, use BSA for blocking). Add primary antibody: Gently shake off the blocking solution, add the primary antibody prepared in PBS at a specific ratio to the slide, and incubate the slide flat in a humidified chamber at 4°C overnight. Add secondary antibody: Place the slide in PBS (pH 7.4) and wash three times on a destaining shaker, 5 min each time. After slightly drying the slide, add the secondary antibody (HRP-labeled) of the corresponding species to the primary antibody in the circle to cover the tissue, and incubate at room temperature for 50 min. DAB staining: Place the slide in PBS (pH 7.4) and wash three times on a destaining shaker, 5 min each time. After slightly drying the slide, add freshly prepared DAB staining solution in the circle, and control the staining time under a microscope. A positive result is brownish-yellow. Rinse the slide with tap water to stop the staining. Counterstain cell nuclei: Counterstain with hematoxylin for about 3 min, wash with tap water, differentiate with hematoxylin differentiation solution for a few seconds, rinse with tap water, use hematoxylin blue solution to return to blue, and rinse with running water. Dehydration and mounting: The sections were sequentially immersed in 75% ethanol for 5 min, 85% ethanol for 5 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, n-butanol for 5 min, and xylene I for 5 min to dehydrate and clear the sections. After removing the sections from the xylene, they were allowed to dry slightly and then mounted with mounting adhesive. Microscopic examination: The results were interpreted under a white light microscope. Hematoxylin staining of cell nuclei appeared blue, and DAB-positive expression appeared brownish-yellow.

[0190] Using a Leica biological microscope, images were taken of the CA1 region of the cortex and hippocampus at magnifications of 10×20 and 10×40. For each slide, five high-power fields (×200) were selected in the cortex and hippocampus for manual counting of IBA-1 positive cells. The average count was taken as the number of positive cells in the sample.

[0191] 1.4 Data Statistics

[0192] Experimental data are expressed as mean ± standard deviation (Mean ± SD). One-way or two-way ANOVA was performed using GraphPadPrism (9.00) software for each efficacy index, and the LSD test was used to examine differences between groups. P < 0.05 was considered statistically significant.

[0193] 2 Experimental Results

[0194] 2.1 Effects on cerebral blood flow in BCAS mice

[0195] Cerebral blood flow (CBF) was measured on days 0, 1, 3, 7, and 14 after BCAS (n = 6–8 / group). The rate of change of CBF over time in each group is shown in Table 8. Two-way ANOVA revealed statistical differences among the groups. The results showed that, compared with the sham-operated group, the CBF in the model group was significantly reduced on days 1, 3, 7, and 14 after BCAS (p < 0.0001). Compared with the model group, both sarrencycline and cilostazol improved the reduction in CBF in mice. The effects of sarrencycline 80 mg / kg (D7, D10, p = 0.002, 0.017) and cilostazol 100 mg / kg (D3, D10, p = 0.032, 0.047) were statistically significant.

[0196] Table 8. Effects of sarrocycline on cerebral blood flow after cerebral ischemia.

[0197]

[0198] Data are expressed as Mean ± SD. Compared with the model group, *p < 0.05, **p < 0.01, ***p < 0.001.

[0199] 2.2 Effects on learning and memory abilities of BCAS mice

[0200] The effects on learning and memory are shown in Tables 9, 10, and 11. Two-way ANOVA revealed statistically significant differences in escape latency among the groups in the water maze experiment. The escape latency of the model group and the sham-operated group differed significantly from that of the sham-operated group starting from day 3 of the hidden platform experiment. Compared to the model group, the escape latency of animals in the cilostazol group was significantly shorter on day 3 of the hidden platform experiment (P = 0.016), and the escape latency of animals in the salpercycline group (80 mg / kg) was significantly shorter on day 5 of the hidden platform experiment (P = 0.008).

[0201] One-way ANOVA of the water maze spatial exploration experiment showed statistically significant differences among the groups. Compared with the sham-operated group, the model group had a significantly shorter time spent in the target quadrant (P = 0.031); compared with the model group, the salazine group (80 mg / kg) had a significantly longer time spent in the target quadrant (P = 0.040). There was no statistically significant difference in the number of times the target quadrant platform was traversed among the groups.

[0202] One-way ANOVA of the Y-maze test showed statistically significant differences among the groups. Compared with the sham-operated group, the model group had a significantly lower rate of correct trajectory alternation in the Y-maze (P = 0.0008). Compared with the model group, the salazine group (80 mg / kg) and the cilostazol group (100 mg / kg) had significantly higher rates of correct trajectory alternation in the Y-maze (P = 0.034, P = 0.044).

[0203] Table 9. Effects of salpercycline on escape latency in mice in a water maze

[0204]

[0205] Data are expressed as Mean ± SD. Compared with the model group, *p < 0.05, **p < 0.01, ***p < 0.001.

[0206] Table 10. Effects of salbutamol on spatial exploration in mouse water maze

[0207]

[0208]

[0209] Data are expressed as Mean ± SD. Compared with the model group, *p < 0.05, **p < 0.01.

[0210] Table 11. Effects of salpercycline on the correct alternation rate in the Y maze of mice

[0211]

[0212] Data are expressed as Mean ± SD. Compared with the model group, *p < 0.05, ***p < 0.001

[0213] 2.3 Effects on brain white matter injury

[0214] White matter lesions in the corpus callosum, caudate putamen, internal capsule, and optic tract regions were observed in mice stained with Luxol firm blue brain sections during the BCAS model. White matter scores (WMLs) for each brain region are shown in Table 12. T-test analysis revealed significantly greater damage to the corpus callosum, internal capsule, and optic tract regions in the BCAS model group compared to the sham-operated group at the experimental endpoint. In mice in the 80 mg / kg salazine group, WMLs scores in the internal capsule and optic tract regions were significantly lower than in the model group (P = 0.042, P = 0.029), indicating improved neuropathological changes. In mice in the 100 mg / kg cilostazol group, WMLs scores in the optic tract region were significantly lower than in the model group (P = 0.021).

[0215] Table 12. Effects of sarrenzine on white matter injury in the brain

[0216]

[0217]

[0218] Data are expressed as Mean ± SD. Compared with the model group, *p < 0.05, **p < 0.01.

[0219] 2.2 Effects on neuroinflammation

[0220] Immunohistochemical staining was performed on microglia in the cerebral cortex and hippocampus of mice in each group. One-way ANOVA showed statistically significant differences in the number of IBA-1 positive cells among the groups (Table 13). Compared with the sham-operated group, the BCAS group showed significantly increased microglia in the cortex and hippocampus (p = 0.017, 0.003). Compared with the model group, sarrocycline (80 mg / kg) significantly reduced microglia in the hippocampus of model mice (p = 0.028). Cilostazol (100 mg / kg) significantly reduced microglia in the cortex and hippocampus of model mice (p = 0.35, 0.018).

[0221] Table 13. Effects of sarrocycline on neuroinflammation

[0222]

[0223] Data are expressed as Mean ± SD. Compared with the model group, *p < 0.05, **p < 0.01.

Claims

1. Use of salpercycline and / or pharmaceutically acceptable salts of salpercycline in the preparation of medicaments for the treatment and / or prevention of thrombotic diseases.

2. The application according to claim 1, characterized in that, The thrombotic disease is one or more of the following: arterial thrombotic disease, venous thromboembolism, and thrombotic microangiopathy.

3. Use of salpercycline and / or pharmaceutically acceptable salts of salpercycline in the preparation of medicaments for the treatment and / or prevention of cerebrovascular diseases caused by platelet aggregation.

4. Use of salpercycline and / or pharmaceutically acceptable salts of salpercycline in the preparation of medicines for the treatment and / or prevention of stroke caused by platelet aggregation.

5. Use of sarrencycline and / or pharmaceutically acceptable salts of sarrencycline in the preparation of drugs for the treatment and / or prevention of cerebral small vessel disease caused by platelet aggregation.

6. Use of sarrencycline and / or pharmaceutically acceptable salts of sarrencycline in the preparation of drugs for the treatment and / or prevention of cerebral thrombosis.

7. Use of salpercycline and / or pharmaceutically acceptable salts of salpercycline in the preparation of drugs for cerebral ischemia-reperfusion injury.

8. Use of salpercycline and / or pharmaceutically acceptable salts of salpercycline in the preparation of drugs for arterial stenosis.

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