Use of bicalutamide in the preparation of a medicament for preventing and treating cerebrovascular diseases

Abstract

The present invention discloses the application of bicalutamide in the preparation of drugs for preventing and treating cerebrovascular diseases. The present invention selects a clinically common intracerebral hemorrhage model caused by coagulation system disorder, namely, a thrombin-induced intracerebral hemorrhage model, to explore the improvement effect of bicalutamide on it. Experiments show that bicalutamide inhibits thrombin-induced intracerebral hemorrhage volume, intracerebral hemorrhage area, brain edema, Evans blue leakage and nerve function damage, and has an improvement effect on maintaining the integrity of the blood-brain barrier (BBB). Therefore, bicalutamide can be used to prepare drugs for preventing and treating cerebrovascular diseases.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to the application of bicalutamide in the preparation of drugs for preventing and treating cerebrovascular diseases. Background Art

[0002] Cerebrovascular diseases refer to a general term for a class of diseases caused by cerebrovascular lesions that lead to brain dysfunction, including a series of brain diseases caused by cerebral thrombosis, cerebrovascular rupture, cerebrovascular stenosis or occlusion, intracranial vascular malformations, etc. Cerebrovascular diseases are common intractable diseases that seriously endanger human health and life safety, and are characterized by high incidence, high recurrence rate, high fatality rate, and high disability rate. According to the nature of the disease, they can be divided into ischemic cerebrovascular diseases and hemorrhagic cerebrovascular diseases. Among them, cerebral hemorrhage refers to the hemorrhage caused by the rupture of blood vessels in the non-traumatic brain parenchyma, which is the stroke subtype with the highest clinical mortality rate, and its acute-phase fatality rate is 30% - 40%, seriously threatening human health. Its main causes include hypertension, coagulation system disorders, adverse drug reactions (such as intracranial hemorrhage induced by anticoagulant warfarin and tissue-type plasminogen activator tPA), vascular malformations, etc. In addition, studies have found that cerebral hemorrhage caused by coagulation system disorders is one of the main inducing factors in secondary cerebral hemorrhage, mainly manifested as a large increase in thrombin. Therefore, intervening in thrombin-induced cerebral hemorrhage is very important for preventing and treating cerebral hemorrhage caused by various factors.

[0003] Currently, the treatment of cerebral hemorrhage mainly includes surgical treatment and drug treatment. However, surgical operations have a relatively high risk of side effects, and clinical results show that the recovery effect of patients' neurological function is not good. At the same time, most of the treatment drugs are in the preclinical experiment or clinical trial stage, and the improvement effect is not obvious. Therefore, actively exploring effective drugs for preventing and treating cerebral hemorrhage and providing clinically feasible treatment strategies are the hotspots and difficulties in the research of critically ill cerebrovascular diseases. A large number of studies have shown that the disruption of the BBB (blood-brain barrier) is an important early pathological link in the occurrence and development of cerebrovascular diseases, and maintaining the integrity of the BBB is a potential strategy for treating cerebrovascular diseases.

[0004] Bicalutamide is an anti-androgen drug mainly used for the treatment of advanced prostate cancer, which can relieve symptoms such as dysuria and hematuria caused by prostate cancer. Bicalutamide belongs to non-steroidal androgen receptor inhibitors and can inhibit the action of androgens by binding to androgen receptors in target tissues. Its medications are mainly divided into two categories: one is in combination with luteinizing hormone-releasing hormone (LHRH) analogs or surgical orchiectomy for the treatment of advanced prostate cancer; the other is used to treat patients with locally advanced prostate cancer without distant metastasis who are not suitable for or unwilling to undergo surgical operations or other medical treatments.

[0005]

[0006] Bicalutamide (C18 H 14 F 4 N 2 O 4 S, CAS: 90357-06-5)

[0007] Currently, there is no report on the use of bicalutamide for the treatment of cerebrovascular diseases. Summary of the Invention

[0008] Object of the Invention: The object of the present invention is to provide an application of bicalutamide in the preparation of drugs for preventing and treating cerebrovascular diseases. The present invention has found through research that bicalutamide can improve the maintenance of the integrity of the blood-brain barrier (BBB) by inhibiting thrombin-induced intracerebral hemorrhage volume, intracerebral hemorrhage area, brain edema, Evans blue leakage, and neurological function damage in mice. Therefore, bicalutamide can be used in the preparation of drugs for preventing and treating cerebrovascular diseases.

[0009] The present invention selects an intracerebral hemorrhage model caused by coagulation system disorders that is relatively common clinically, namely a thrombin-induced intracerebral hemorrhage model, to explore the improvement effect of bicalutamide on it, in order to elucidate the possible common mechanisms of cerebrovascular diseases caused by coagulation system disorders and resulting in BBB damage.

[0010] Technical Solution: The object of the present invention is achieved through the following technical solutions:

[0011] The present invention provides an application of bicalutamide in the preparation of drugs for preventing and treating cerebrovascular diseases, and the structural formula of the bicalutamide is as follows:

[0012]

[0013] The bicalutamide can be used as the sole active ingredient to prepare drugs for preventing and treating cerebrovascular diseases.

[0014] The bicalutamide can also be used in combination with other drugs to prepare drugs for preventing and treating cerebrovascular diseases.

[0015] The cerebrovascular diseases are hemorrhagic cerebrovascular diseases caused by coagulation system disorders.

[0016] The present invention also provides an application of bicalutamide in the preparation of drugs for preventing and treating hemorrhagic stroke.

[0017] The bicalutamide improves the maintenance of the integrity of the blood-brain barrier (BBB) by inhibiting thrombin-induced intracerebral hemorrhage volume, intracerebral hemorrhage area, brain edema, Evans blue leakage, and neurological function damage in mice.

[0018] The drugs for preventing and treating cerebrovascular diseases according to the present invention include bicalutamide and pharmaceutically acceptable carriers or excipients.

[0019] The adjuvants include one or more of a wetting agent, an emulsifier, an antioxidant, a preservative, a disintegrant, a diluent, a solubilizer or a binder.

[0020] The wetting agent is selected from at least one of water or ethanol.

[0021] The emulsifier is selected from at least one of Tween series, Span series, glycerol fatty acid esters, pectin, agar, sodium alginate or silica.

[0022] The antioxidant is selected from at least one of ascorbic acid, sulfites, bisulfites, gallic acid and its esters.

[0023] The preservative is selected from at least one of benzoic acid and its salts, sorbic acid and its salts or parabens.

[0024] The disintegrant is selected from at least one of starch, sodium carboxymethyl starch, crospovidone, low-substituted hydroxypropyl cellulose or cross-linked polyvinylpyrrolidone.

[0025] The diluent is selected from at least one of starches, sugars, celluloses or inorganic salts. The solubilizer is selected from one of Tween series, polyoxyethylene fatty alcohol ethers, sulfates or sulfonates.

[0026] The binder is selected from at least one of starch paste, sodium carboxymethyl cellulose, polyvinylpyrrolidone, hydroxypropyl cellulose, methyl cellulose or ethyl cellulose.

[0027] The dosage form of the drug is a granule, a tablet, a capsule, a suspension, an oral liquid, an injection or an infusion. When treating cerebrovascular diseases caused by various reasons, using bicalutamide as the active ingredient and with conventional preparation processes, drugs that can be used clinically are prepared, such as granules, tablets, capsules and oral liquid preparations, etc., which can inhibit the thrombin-induced cerebral hemorrhage volume, cerebral hemorrhage area, cerebral edema, Evans blue leakage and nerve function damage in mice, and improve the maintenance of the integrity of the blood-brain barrier BBB.

[0028] The drugs of the present invention can be administered in various known ways, such as orally, by injection, by inhalation spray. The drugs of the present invention can be administered alone or in combination with other drugs. The oral composition can be any orally acceptable dosage form. The sterile injectable composition can be formulated according to the techniques known in the art, using suitable dispersing agents or wetting agents and suspending agents. Pharmaceutically acceptable carriers and solvents that can be used include water, sodium chloride solution, etc.

[0029] The effective concentration of bicalutamide is 6.5 - 13 mg / kg.

[0030] The actual dosage level of the active ingredient in the medicament of the present invention can be varied to obtain an amount of the active ingredient that is effective in achieving the desired therapeutic response for a particular patient, composition, and mode of administration and that is non-toxic to the patient. The selected dosage level depends on a variety of factors, including the route of administration, the time of administration, the rate of excretion, the duration of treatment, other drugs, compounds, and / or materials used in combination with bicalutamide, the age, sex, weight, general health, and medical history of the patient being treated, and similar factors well known in the medical arts.

[0031] Beneficial effects:

[0032] 1. Bicalutamide as described in the present invention has the effect of maintaining the integrity of the blood-brain barrier and can be used to prepare medicaments for preventing and treating cerebrovascular diseases. The present invention has found through research that after treating with bicalutamide, it can significantly inhibit the amount of cerebral hemorrhage, the area of cerebral hemorrhage, cerebral edema, Evans blue leakage, and neurological function damage induced by thrombin in mice. This indicates that bicalutamide has a significant effect on improving the integrity of the BBB, and it is confirmed that using bicalutamide to prepare medicaments for preventing and treating hemorrhagic stroke has broad application prospects.

[0033] 2. Bicalutamide as described in the present invention belongs to "new use of an old drug", and in the new drug application, it belongs to Class 2 improved drugs, specifically 2.4 preparations with new indications containing known active ingredients. Bicalutamide is a marketed drug, and its safety and quality are guaranteed. Therefore, the present invention has great market development prospects and clinical application prospects. Description of the drawings

[0034] Figure 1 Shows the effects of different doses of bicalutamide on the amount of cerebral hemorrhage. Figure 1 A is a diagram showing the brain tissue, Figure 1 B is a statistical chart of hemoglobin content detection (Bic: bicalutamide tablets, the same below; n = 6, expressed as mean ± SEM, ## P < 0.01 compared with the blank group, *P < 0.05, **P < 0.01 compared with the model group);

[0035] Figure 2 Shows the effects of different doses of bicalutamide on neurological function damage induced by cerebral hemorrhage (n = 6, expressed as mean ± SEM, ## P < 0.01 compared with the blank group, **P < 0.01 compared with the model group);

[0036] Figure 3 Shows the effects of different doses of bicalutamide on the brain water content induced by cerebral hemorrhage (n = 6, expressed as mean ± SEM, ## P < 0.01 compared with the blank group, *P < 0.05, **P < 0.01 compared with the model group);

[0037] Figure 4 Effect of different doses of bicalutamide on Evans blue leakage induced by intracerebral hemorrhage. Figure 4 A is the brain tissue display diagram of the Evans blue experiment. Figure 4 B is the statistical chart of the leakage volume of the Evans blue experiment (n = 6, expressed as mean ± SEM, ## P < 0.01 compared with the blank group, *P < 0.05, **P < 0.01 compared with the model group). Specific implementation mode

[0038] The technical solution of the present invention will be described in detail below through specific embodiments, but the protection scope of the present invention is not limited to the described embodiments.

[0039] For those not specifying specific techniques or conditions in the embodiments, the techniques or conditions described in the literature in this field or according to the product specifications are followed. For reagents or instruments not indicating the manufacturer, they are all conventional products that can be obtained through regular channels.

[0040] For the experimental methods in the following embodiments, unless otherwise specified, they are all conventional methods. For the test materials used in the following embodiments, unless otherwise specified, they are all commercially available products.

[0041] In the embodiments of the present invention:

[0042] Multifunctional anesthesia machine for large and small animals, electric thermostatic blanket, single-arm digital display brain stereotaxic instrument (Beijing Zhongshi Dichuang Technology Development Co., Ltd.); surgical micro scissors, micro forceps, hemostatic forceps (Shanghai Jinzhong Surgical Instrument Factory); micro syringe (Shanghai Guangzheng Medical Instrument Co., Ltd.); sterilized medical suture needle (Chaohu Bixiong Medical Instrument Co., Ltd.); adjustable speed peristaltic pump (Kachuaner Fluid Technology (Shanghai) Co., Ltd.); tissue homogenizer (Jiangsu Jintan Huanyu Science and Technology Instrument Factory); refrigerated centrifuge (Thermo Company, USA); intravenous visible mouse tail injection fixator (Jinan Yiyan Technology Development Co., Ltd.); microplate reader (Bio-Rad Company, USA);

[0043] Bicalutamide is purchased from Shanghai Zhaohui Pharmaceutical Co., Ltd., type VII-S collagenase, Evans blue, TEMED (Sigma Company, USA); isoflurane (Shenzhen Rewod Life Science Co., Ltd.); hemoglobin test kit (Bosch Biotechnology Co., Ltd., USA); formamide (Chenyin Pharmaceutical Co., Ltd.).

[0044] 30 male C57BL / 6J mice, 6 - 8 weeks old, weighing 18 - 22 g, SPF grade, are purchased from Jiangsu Huachuang Xinnuo Pharmaceutical Technology Co., Ltd. (experimental animal production license number SYXK (Su) 2022 - 0013).

[0045] Example 1: Bicalutamide Improves the Hemorrhage Volume in Thrombin-Induced Intracerebral Hemorrhage

[0046] Experimental method:

[0047] 1. Animal grouping, model establishment and administration

[0048] Male C57BL / 6J mice were randomly divided into 5 groups, with 6 mice in each group, namely sham operation group, model group, low, medium and high dose groups of bicalutamide (Bic) (3.25, 6.5, 13 mg / kg). Mice in the sham operation group and the model group were given normal saline 1 h after model establishment, and mice in the low, medium and high dose groups of bicalutamide (Bic) (3.25, 6.5, 13 mg / kg) were given bicalutamide 1 h after model establishment. The volume of normal saline and bicalutamide was given according to the weight of each mouse (0.1 mL / 10 g). After the operation, the mice were allowed to eat and drink freely, and the animals that died due to excessive anesthesia or improper surgical operation during the experiment were excluded. Samples were taken for experiments 24 h after model establishment.

[0049] Establishment of intracerebral hemorrhage model: The C57BL / 6J mice were placed in an anesthesia box and anesthetized with 2% isoflurane. After 2 - 3 min, the mice were taken out and transferred to a stereotaxic apparatus for the brain, fixed in the prone position, and continuously anesthetized with 1% isoflurane. The hair on the head was shaved off, and a 2-cm longitudinal incision was made in the mid-sagittal line of the head. The skin and subcutaneous tissue were incised, and the periosteum was gently wiped with an iodophor cotton ball to expose the skull and the anterior fontanelle. Referring to the mouse brain orientation atlas, the right striatum was located, 2.0 mm to the right of the midline and 1.0 mm in front of the anterior fontanelle. A round hole with a diameter of about 1.0 mm was drilled without damaging the dura mater. A microsyringe was inserted vertically along the drilling direction to a depth of 4 mm, and then 0.5 μL of type VII S collagenase (0.075 U dissolved in 0.5 μL of normal saline) was slowly and evenly injected into the brain. The needle was left in place for 5 min, and then the microsyringe was slowly withdrawn. The skull was sealed with bone wax and the wound was sutured, and the mice were placed back into the cage. The sham operation group was injected with the same volume of normal saline.

[0050] 2. Measuring the intracerebral hemorrhage volume in mice by hemoglobin kit method

[0051] Mice in each group were sacrificed 24 h after ICH (Intracerebral hemorrhage). The mice were perfused from the heart with 1×PBS until the blood was washed out completely. Then the brains were removed and photographed. Excess water was blotted dry and the brains were frozen. After removing the olfactory bulbs, brainstems, and cerebella, the brain tissues were evenly cut into five slices and photographed. The right half of the brain tissue was weighed, and 1 mL of pre-cooled 1×PBS was added to every 0.1 g of the tissue for homogenization. The homogenate was transferred to a 2 mL EP tube and centrifuged (13000 rpm, 30 min, 4 °C). The supernatant (50 μL) was taken and added to a 96-well plate, and 200 μL of hemoglobin detection reagent was added. There were 3 replicate wells in each group. After reacting at room temperature for 5 min, the OD value was measured at a wavelength of 400 nm. The hemoglobin content in the tissue was calculated according to the equation in the kit instructions, and the unit was μg / mg.

[0052] 3. Data processing

[0053] All data were statistically analyzed using Graphpad Prism 8 software, expressed as mean±SEM. For comparisons between two groups, Students’t test was used. When comparing three groups or more, one-way ANOVA was used, and Dunnett’s test was used for the post hoc test. P<0.05 was considered statistically significant.

[0054] Experimental results:

[0055] As Figure 1 shown, compared with the blank group, after stimulation with thrombin, both the hemorrhage area and hemorrhage volume in the model group increased significantly ( ## P<0.01); after administration of different doses of Bic, the results showed that the medium and high dose groups of Bic significantly improved the hemorrhage volume and hemorrhage area induced by thrombin, suggesting that Bic has the effect of improving the hemorrhage volume and hemorrhage area induced by thrombin.

[0056] Example 2 Improvement effect of bicalutamide on neurological function injury in mice with thrombin-induced intracerebral hemorrhage

[0057] Experimental method:

[0058] 1. Animal grouping, model establishment, and drug administration were the same as in Example 1.

[0059] 2. Determination of neurological deficit score

[0060] According to the Clark scoring scale for neurological deficit (see Table 1), mice in each group were evaluated 24 h after ICH. The evaluation indicators included body symmetry, gait, climbing ability, circling behavior, forelimb symmetry, forced circling, and whisker response. The scores of the seven indicators were added together as the final score. The higher the score, the more severe the neurological deficit.

[0061] Table 1 Clark Scoring Scale for Neurological Deficits

[0062]

[0063]

[0064] 3. The data processing is the same as that in Example 1.

[0065] Experimental results:

[0066] The results are as Figure 2 shown. Compared with the blank group, after stimulation with thrombin, the neurological function injury of the mice in the model group increased significantly ( ## P<0.01); after administration of different doses of Bic, the neurological function injury of the mice in the medium and high dose groups was significantly improved ( ** P<0.01), indicating that Bic can improve the neurological function injury induced by thrombin-induced intracerebral hemorrhage.

[0067] Example 3 Effect of Bicalutamide on Improving Brain Water Content in Mice with Thrombin-Induced Intracerebral Hemorrhage

[0068] Experimental method:

[0069] 1. The animal grouping, model establishment and administration are the same as those in Example 1.

[0070] 2. Determination of brain water content

[0071] The mice in each group were sacrificed 24 h after ICH. The whole brain tissue was taken out, rinsed with normal saline, and the excess water was blotted dry with filter paper. The brain tissue was divided into three parts: the injured hemisphere, the contralateral hemisphere and the cerebellum. The weight of each part was recorded as the wet weight. After drying the brain tissue of each part in an oven at 100 °C for 48 h, the weight was measured as the dry weight.

[0072] Brain water content = (wet weight - dry weight) / wet weight × 100%.

[0073] 3. The data processing is the same as that in Example 1.

[0074] Experimental results:

[0075] The results are as Figure 3 shown. Compared with the blank group, after stimulation with thrombin, the brain water content of the mice in the model group increased significantly ( ## P<0.01). After administration of different doses of Bic, the brain water content of the mice in the medium and high dose groups was significantly inhibited ( * P<0.05, ** P<0.01), indicating that Bic can improve the increase in brain water content caused by thrombin-induced intracerebral hemorrhage.

[0076] Example 4: Improvement effect of bicalutamide on Evans blue leakage in thrombin-induced intracerebral hemorrhage mice

[0077] Experimental method:

[0078] 1. Animal grouping, model establishment and drug administration were the same as in Example 1.

[0079] 2. Determination of Evans blue permeability

[0080] At 22 h after ICH, 2% Evans blue solution (0.1 mL / 10 g) was injected into the tail vein of mice in each group. After 2 h, the mice were anesthetized, perfused with 0.9% normal saline from the heart until the blood was washed clean, the brain was taken out, photographed after drying the excess water. The olfactory bulb, brainstem and cerebellum were removed, the right hemisphere of the brain was weighed, 1 mL of precooled formamide was added to every 0.1 g of brain tissue and ground thoroughly, transferred to a 2 mL EP tube, incubated at 60 °C for 18 h and then centrifuged (5000 g, 30 min). 200 μL of the supernatant was taken and added to a 96-well plate, with 3 replicates for each sample. The OD value was measured at a wavelength of 620 nm, and the content was calculated according to the standard curve, with the unit of μg / g.

[0081] Standard curve preparation: Evans blue powder was dissolved in normal saline to prepare solutions with concentrations of 0, 1, 2, 4, 8, 16, 32, 64 μg / ml. The corresponding OD values were measured at a wavelength of 620 nm, and the standard curve was plotted based on the concentration and the corresponding OD values.

[0082] 3. Data processing was the same as in Example 1.

[0083] Experimental results:

[0084] As shown in Figure 4, compared with the blank group, after stimulation with thrombin, the Evans blue leakage level in the model group mice increased significantly ( ## P<0.01). After treatment with different doses of Bic, the medium and high doses of Bic could significantly inhibit the Evans blue leakage level ( * P<0.05, ** P<0.01), indicating that Bic can improve the increase in blood-brain barrier permeability caused by thrombin-induced intracerebral hemorrhage.

[0085] As described above, although the present invention has been shown and described with reference to specific preferred embodiments, it should not be construed as a limitation of the present invention itself. Various changes can be made in its form and details without departing from the spirit and scope of the present invention defined by the appended claims.

Claims

1. Use of bicalutamide in the preparation of a drug for preventing and treating hemorrhagic stroke, characterized in that, the structural formula of the bicalutamide is as follows: 。 2. The use according to claim 1, characterized in that, the bicalutamide is used as the sole active ingredient to prepare a drug for preventing and treating hemorrhagic stroke.

3. The use according to claim 1, characterized in that, the bicalutamide is used in combination with other drugs to prepare a drug for preventing and treating hemorrhagic stroke.

4. The use according to claim 1, characterized in that, the bicalutamide improves the maintenance of the integrity of the blood-brain barrier (BBB) by inhibiting the amount of cerebral hemorrhage, the area of cerebral hemorrhage, cerebral edema, Evans blue leakage and neurological function damage induced by thrombin in mice.

5. The use according to claim 1, characterized in that, the drug comprises bicalutamide and a pharmaceutically acceptable excipient.

6. The use according to claim 5, characterized in that, the excipient comprises one or more of a wetting agent, an emulsifying agent, an antioxidant, a preservative, a disintegrant, a diluent, a solubilizer or a binder.

7. The use according to claim 5, characterized in that, the dosage form of the drug is a granule, a tablet, a capsule, a suspension, an oral liquid or an injection.

8. The use according to claim 1, characterized in that, the effective concentration of the bicalutamide is 6.5 - 13 mg / kg.

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