Use of bremelanotide in preparation of drug for treating ischemic stroke

Abstract

Provided in the present invention is use of bremelanotide in the preparation of a drug for preventing and / or treating ischemic stroke. Bremelanotide can ameliorate neurological deficits, reduce the cerebral infarction area, and improve the cerebral blood flow in ischemic stroke.

Description

Application of Bremerlongan in the preparation of drugs for treating ischemic stroke Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of Bremerlongdan in the preparation of drugs for treating ischemic stroke. Background Technology

[0002] Stroke, also known as apoplexy or cerebrovascular accident, is a group of acute cerebrovascular diseases characterized by sudden onset and focal neurological deficits, caused by localized cerebral circulatory disturbances. Due to its high incidence, mortality, disability rate, and recurrence rate, it is one of the three leading causes of death worldwide. Ischemic stroke, also known as cerebral infarction, refers to brain tissue necrosis caused by insufficient blood supply to the brain due to narrowing or occlusion of arteries supplying blood to the brain. Ischemic stroke accounts for 70-80% of all strokes. The pathogenesis of ischemic stroke is complex, involving multiple pathophysiological processes such as thrombosis, embolism, and hypoperfusion. Thrombosis is the most common cause, mainly due to atherosclerosis; embolism is caused by emboli of cardiac or arterial origin blocking arteries supplying blood to the brain; hypoperfusion is caused by low blood pressure or severe vascular stenosis, leading to reduced cerebral blood flow.

[0003] Ischemic stroke occurs when a region of the brain loses its blood supply, triggering an ischemic cascade. The ischemic cascade is a series of biochemical reactions that initiate in the brain and other oxygenated tissues within seconds to minutes of ischemia. Most ischemic neuronal death is due to the activation of chemicals produced during and after the ischemic period. The ischemic cascade typically lasts two to three hours, but can persist for several days even after blood flow returns to normal.

[0004] The ischemic cascade typically features the following characteristics: insufficient blood supply leads to tissue hypoxia, which in turn prevents neurons from producing ATP for energy. The ATP ion transport pumps fail, causing cellular depolarization, which in turn leads to the loss of calcium (Ca) and other ion-carrying capillaries. 2+ Ions, including calcium ions, flow into the cell. Ion pumps are no longer able to transport calcium ions out of the cell, leading to excessively high intracellular calcium levels. The presence of calcium triggers the release of the excitatory amino acid neurotransmitter glutamate. Glutamate stimulates AMPA receptors and calcium... 2+Permeable NMDA receptors open, allowing more calcium to enter cells. Excessive calcium ions entering cells can cause over-excitation, producing harmful chemicals such as free radicals, reactive oxygen species, and calcium-dependent enzymes—a process known as excitotoxicity. Calcium can also lead to the release of more glutamate. If cells die, they release glutamate and toxic chemicals into the surrounding environment. Toxins can poison nearby neurons, while glutamate can cause these neurons to become over-excited. If the brain is reperfused, many factors can contribute to reperfusion injury. The inflammatory response intensifies, and phagocytes engulf damaged but still viable tissue. Harmful chemicals can damage the blood-brain barrier. Large molecules in blood vessels (such as albumin) cross the damaged blood-brain barrier, using osmosis to draw water into brain tissue, causing cerebral edema, leading to brain tissue compression and further damage.

[0005] Currently, the main strategies for treating ischemic stroke include two aspects: first, restoring cerebral reperfusion and improving cerebral blood supply; and second, blocking the injury cascade to prevent neuronal damage. Given the complexity of the pathological mechanisms of ischemic stroke, there are currently no specific drugs for treatment in clinical practice; only symptomatic and supportive medications are used, targeting neuroprotection and improving cerebral blood circulation. Apart from ultra-early thrombolysis, which has a definite therapeutic effect, there is currently no neuroprotective agent with proven efficacy based on evidence-based medicine.

[0006] Bremerlanotide, also known as PT141, is a potent agonist of the MC1R and MC4R melanocortin receptors. MC4R is a 332-amino acid membrane protein. Extensive localization studies in the rodent brain have shown that MC4R mRNA is expressed in multiple brain regions, including the cortex, brainstem, thalamus, hypothalamus, hippocampus, and spinal cord. MC4R is also known as the neural MCR. However, there are no reports of using Bremerlanotide in the treatment of ischemic stroke. Summary of the Invention

[0007] This invention addresses the problems existing in the prior art by providing the application of Bremerlongdan in the preparation of drugs for treating ischemic stroke.

[0008] The use of Bremerlongdan in the preparation of drugs for the prevention and / or treatment of ischemic stroke.

[0009] Preferably, the use of Bremerlongdan in the preparation of drugs to improve neurological deficits.

[0010] Preferably, the use of Bremerlongdan in the preparation of drugs that reduce the area of ​​cerebral infarction.

[0011] Preferably, the use of Bremerlongdan in the preparation of drugs to improve cerebral blood flow in ischemic stroke.

[0012] Preferably, Bremerlongdan is the sole active ingredient in the drug.

[0013] Preferably, the drug further includes pharmaceutically acceptable excipients.

[0014] Preferably, the excipients include one or more of surfactants, buffers, disintegrants, binders, fillers, lubricants, solubilizers, flavorings, and colorings.

[0015] Preferably, the dosage form of the drug includes oral dosage forms and non-gastrointestinal dosage forms.

[0016] Preferably, the oral dosage form is a tablet, capsule, granule, powder, pill, oral liquid preparation or lyophilized powder injection.

[0017] Preferably, the non-gastrointestinal dosage form is an injection dosage form, a respiratory dosage form, a skin dosage form, a mucosal dosage form, or a cavity dosage form.

[0018] This invention provides a novel use of Bremerlongan in the preparation of drugs for treating ischemic stroke. Its mechanism of action is as follows:

[0019] (1) Anti-inflammatory: Bremerlongdan significantly reduces the production of inflammatory factors by activating MC1R and MC4R. In addition to being expressed in keratinocytes and melanocytes, MC1R is also expressed in macrophages, monocytes, lymphocytes, neutrophils and dendritic cells, which can participate in regulating the inflammatory process in the brain and has an anti-inflammatory effect.

[0020] (2) Antioxidant: MC4R agonists can promote antioxidant defense and reduce mitochondrial damage, and prevent ethanol-induced increase of reactive oxygen species (ROS), cytoplasmic calcium homeostasis disorder and mitochondrial potential loss.

[0021] (3) Increase cerebral blood flow: Bremerlongdan increases the density and size of blood vessels in the cerebral cortex of stroke rats, enhances collateral blood flow, restores vascular function and improves cerebral blood flow.

[0022] (4) Protecting neurons: Bremerlongdan significantly increases the number of neurons around the infarct foci, providing neuroprotection to living tissue and playing a neuroprotective role.

[0023] (5) Improve cognitive function: Activation of melanocortin receptors can significantly improve cognitive function.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] This invention provides a novel pharmaceutical use for Bremerlongan: its application in the preparation of drugs for treating ischemic stroke. Studies have found that Bremerlongan can effectively improve behavioral dysfunction, significantly reduce the infarct area, and increase the infarct improvement rate, exhibiting a dose-related effect. This demonstrates the neuroprotective effect of Bremerlongan on ischemic stroke and confirms its potential use as a drug for treating ischemic stroke. Attached Figure Description

[0026] Figure 1 shows the Doppler cerebral blood flow observation of rats in each group 10 minutes after surgery.

[0027] Figure 2 shows the Doppler cerebral blood flow observation of rats in each group 24 hours after surgery.

[0028] Figure 3 shows the Doppler cerebral blood flow observation of rats in each group 48 hours after surgery.

[0029] Figure 4 shows the TTC staining of brain slices from rats in the sham-operated group.

[0030] Figure 5 shows the TTC staining of brain slices from rats in the model group.

[0031] Figure 6 shows the TTC staining of brain sections from rats in the high-dose Bremerlongdan group.

[0032] In Figures 1-3, from left to right, the groups are the sham surgery group, the model group, the high-dose Bremerlongan group, and the low-dose Bremerlongan group. Detailed Implementation

[0033] It is worth noting that the raw materials used in this invention are all commercially available products.

[0034] Example: The therapeutic effect of Bremerlongdan on stroke rats

[0035] 1. Experimental Materials

[0036] 1.1 Drugs: Bremerlongdan, polypeptide content 89%; isoflurane, batch number 2023110302, purchased from Shenzhen Ruiwode Life Technology Co., Ltd.; physiological saline, batch number 123101601, purchased from Zhejiang Tianrui Pharmaceutical Co., Ltd.; 2,35-triphenyltetrazole chloride (TTC), batch number BMX311, purity 98%, purchased from Bid Pharmaceutical.

[0037] 1.2 Drug preparation: Weigh 13.5 mg of Bremerlongdan, add 8.01 mL of physiological saline and mix well until the final volume is 8.01 mL, the drug concentration is 1.5 mg / mL, and record it as solution 1; take 2 mL of solution 1, add 4 mL of physiological saline and mix well until the final volume is 6 mL, the final drug concentration is 0.5 mg / mL.

[0038] 1.3 Experimental animals: 40 male SD rats at the SPF level, weighing about 200 - 220 g, were purchased from Shanghai Slack Experimental Animal Co., Ltd. with the production license number SCXK(Shanghai)2022 - 0004. They were acclimated to the environment for 3 - 5 days, with free access to food and water. The feed was purchased from Jiangsu Xietong Pharmaceutical Biotechnology Co., Ltd., and the water used was high-pressure sterilized filtered water. The environment of the animal room was maintained at a temperature of 20 - 25°C, a humidity of 40 - 70%, and a 12-hour light-dark cycle. The animals were housed five per cage, and the bedding (corn cob bedding, purchased from Suzhou Daichuan Trading Co., Ltd.) was changed twice a week.

[0039] 2. Experimental methods:

[0040] 2.1 Grouping: The SD rats were randomly divided into 4 groups of 10 each, namely the sham operation group, the model group, the low-dose Bremelanotide group, and the high-dose Bremelanotide group.

[0041] 2.2 Animal model establishment: Except for the sham operation group, the experimental animals were anesthetized with isoflurane during the model establishment process, with spontaneous respiration maintained. They were fixed in the supine position on a rat board, the hair on the midline of the neck was removed, and disinfected with 75% alcohol. An incision was made in the midline of the ventral neck of the rat, and the muscles and fascia were separated along the inner edge of the sternocleidomastoid muscle. The right common carotid artery, external carotid artery, and internal carotid artery were separated, and the vagus nerve was also separated from them. The external carotid artery was ligated and transected, and a small beveled incision was made with a vascular scissors at the ligation site of the external carotid artery. The proximal end of the external carotid artery was pulled until it was in a straight line with the internal carotid artery. A nylon thread embolism (model L3200, purchased from Guangzhou Jialing Biotechnology Co., Ltd.) was slowly advanced through the incision of the main trunk of the right external carotid artery towards the direction of the internal carotid artery entering the skull. Marked by the bifurcation of the common carotid artery, when a slight resistance was felt during the advancement, the middle cerebral artery was blocked. About 10 minutes after the obstruction, a Doppler blood flow meter was used to observe the cerebral blood flow in the rats to determine the successful establishment of the model and inclusion in the group. The nylon thread embolism was removed 1.5 hours after the obstruction to complete the ischemic stroke model.

[0042] 2.3 Drug administration: The subcutaneous injection dose of the low-dose Bremelanotide group was 1 mg / kg, and the subcutaneous injection dose of the high-dose Bremelanotide group was 3 mg / kg. The model group was given an equal amount of normal saline. Except for the sham operation group, each group was given the corresponding drug at 15 minutes, 24 hours, and 48 hours after model establishment.

[0043] 2.4 Detection of experimental indicators:

[0044] (1) Zea-Longa neurological function score: Zea-Longa scoring criteria: 0 points: no neurological deficit symptoms; 1 point: inability to fully extend the contralateral forelimb; 2 points: circling towards the hemiplegic side when walking; 3 points: falling towards the hemiplegic side when walking; 4 points: inability to walk spontaneously, consciousness disorder.

[0045] (2) Balance beam test: Rats underwent balance beam training for two consecutive days prior to modeling. The walking beam device consisted of a 1-meter-long horizontal beam with a flat surface, 50 cm above the ground. Rats were encouraged to cross the beam by placing them at the other end of the beam, and training was conducted three times daily. Each animal underwent a balance beam test at 24 h and 48 h after modeling, with a timing of 60 seconds. The time taken for the rat to cross the beam was recorded, and the final result was the average of the three test results.

[0046] (3) Grip strength test: The grip strength of the rat was assessed by holding its tail and placing its forelimbs on a special pull rod assembly (grip dynamometer). The peak force exerted by the animal was displayed and recorded on a digital display. Each animal was tested three times at 24h and 48h after the modeling surgery, and the final result was the average of the three test results.

[0047] (4) Rotary bar test: Before modeling, rats were trained to adapt to the rotating bar by constant speeds of 18 rpm and 25 rpm. Rats that could stay on the rotating axis for 300 s were selected for further study. Each animal underwent a rotation test (30 rpm, 300 s) at 24 h and 48 h after modeling surgery, and the fall time of each rat was recorded.

[0048] (5) Changes in cerebral blood flow: The changes in cerebral blood flow in each animal were measured by Doppler flowmeter at 10 min, 24 h and 48 h after the modeling surgery.

[0049] (6) Brain volume and cerebral infarction area test: At the end of the experiment (52 hours after the modeling surgery), all animals were euthanized by excessive CO2 inhalation, and the whole brain tissue was removed and the brain volume was measured by the water displacement method. The animals in each group were subjected to continuous coronal section TTC staining and photographed. The infarction area was measured using Image-J software and the infarction improvement rate was calculated.

[0050] Experimental data are expressed as mean ± SD. IBM SPSS Statistics 21 software was used for analysis. p < 0.05 was considered statistically significant, and p < 0.01 was considered highly statistically significant. GraphPad Prism 6 was used for plotting.

[0051] 3. Experimental Results

[0052] 3.1 Zea-Longa Neurological Function Scores: The results are shown in Table 1. 24 hours post-surgery, the neurological function score of the sham-operated group was 0.00±0.00; the neurological function score of the model group was 2.80±0.42. Compared with the sham-operated group, there was a statistically significant difference (p<0.01). The neurological function score of the high-dose Bremerlongan group was 2.89±0.33; the neurological function score of the low-dose Bremerlongan group was 2.67±0.50.

[0053] Forty-eight hours post-surgery, the neurological function score of rats in the sham-operated group was 0.00±0.00, while that in the model group was 2.80±0.42. The difference was statistically significant compared to the sham-operated group (p<0.01). The neurological function score in the high-dose Bremerlongan group was 2.78±0.44, and the neurological function score in the low-dose Bremerlongan group was also 2.78±0.44.

[0054] Table 1. Effects of Bremerlongtan on neurological function scores in stroke-affected rats (n=10) Note: ## This indicates that compared with the sham surgery group, p<0.01.

[0055] 3.2 Balance Beam Test Results: The results are shown in Table 2. 24 hours post-surgery, the balance beam time for rats in the sham-operated group was 7.89±0.93 s; the balance beam time for rats in the model group was 60.00±0.00 s. Compared with the sham-operated group, there was a statistically significant difference (p<0.01). The balance beam time for rats in the high-dose Bremerlongdan group was 39.05±11.71 s; the balance beam time for rats in the low-dose Bremerlongdan group was 44.60±11.51 s. Compared with the model group, both the high-dose and low-dose Bremerlongdan groups showed statistically significant differences (p<0.01).

[0056] Forty-eight hours post-surgery, the balance beam time for rats in the sham-operated group was 9.27±1.64 s, while that in the model group was 60.00±0.00 s. This showed a statistically significant difference compared to the sham-operated group (p<0.01). The balance beam time for rats in the high-dose Bremerlongdan group was 43.96±14.02 s, and for those in the low-dose group it was 43.45±12.32 s. Both the high-dose and low-dose Bremerlongdan groups showed statistically significant differences compared to the model group (p<0.01).

[0057] Table 2. Effects of Bremerlongdan on the balance beam in rats with stroke (n=10) Note: ## This indicates that compared with the sham surgery group, p<0.01; ** This indicates that compared with the model group, p < 0.01.

[0058] 3.3 Grasping strength test results: The results are shown in Table 3. 24 hours after surgery, the grasping strength of rats in the sham-operated group was 677.42±45.03g; the grasping strength of rats in the model group was 414.02±26.56g. Compared with the sham-operated group, there was a statistically significant difference (p<0.01). The grasping strength of rats in the high-dose Bremerlongdan group was 512.90±30.78g; the grasping strength of rats in the low-dose Bremerlongdan group was 489.38±44.85g. Compared with the model group, both the high-dose and low-dose Bremerlongdan groups showed statistically significant differences (p<0.01).

[0059] Forty-eight hours post-surgery, the grip strength of rats in the sham-operated group was 692.84±51.82 g, while that in the model group was 412.33±21.25 g. The difference between the sham-operated group and the model group was statistically significant (p<0.01). The grip strength of rats in the high-dose Bremerlongan group was 508.71±37.39 g, and that in the low-dose group was 474.60±50.26 g. Both the high-dose and low-dose Bremerlongan groups showed statistically significant differences compared to the model group (p<0.01).

[0060] Table 3. Effects of Bremerlongan on grip strength in stroke-affected rats (n=10) Note: ## This indicates that compared with the sham surgery group, p<0.01; ** This indicates that compared with the model group, p < 0.01.

[0061] 3.4 Rotator Test Results: The results are shown in Table 4. 24 hours post-operation, the rotator retention time in the sham-operated group was 298.80±3.79 s, while in the model group it was 52.90±16.62 s. These results showed a statistically significant difference compared to the sham-operated group (p<0.01). The rotator retention time in the high-dose Bremerlongan group was 79.56±23.70 s, and in the low-dose group it was 82.33±48.84 s. Compared to the model group, the high-dose Bremerlongan group showed a statistically significant difference (p<0.05).

[0062] Forty-eight hours post-surgery, the rotator retention time in the sham-operated group was 293.50 ± 20.55 s, while that in the model group was 63.30 ± 25.49 s. The difference was statistically significant compared to the sham-operated group (p < 0.01). The rotator retention time in the high-dose Bremerlongan group was 100.22 ± 57.33 s, and in the low-dose Bremerlongan group, it was 96.00 ± 48.48 s.

[0063] Table 4. Effects of Bremerlongan on rotarods in rats with stroke (n=10) Note: ## This indicates that compared with the sham surgery group, p<0.01; * This indicates that compared with the model group, p < 0.05.

[0064] 3.5 Results of Cerebral Blood Flow Changes: Figure 1 shows the Doppler cerebral blood flow observations of rats in the sham-operated group, model group, high-dose Bremerlongan group, and low-dose Bremerlongan group 10 minutes post-operation. Table 5 shows the results of cerebral blood flow changes. As shown in Table 5, 10 minutes post-operation (without drug administration), the difference in cerebral blood flow between the left and right hemispheres in the sham-operated group was 0.48±0.27%; the difference in cerebral blood flow in the model group was 57.12±2.00%. These differences were statistically significant compared to the sham-operated group (p<0.01). The difference in cerebral blood flow in the high-dose Bremerlongan group was 55.76±2.17%; and the difference in cerebral blood flow in the low-dose Bremerlongan group was 56.02±2.05%.

[0065] Figure 2 shows the Doppler cerebral blood flow observations of rats in the sham-operated group, model group, high-dose Bremerlongan group, and low-dose Bremerlongan group 24 hours post-operation. At 24 hours post-operation, the cerebral blood flow difference in the sham-operated group was 0.44±0.20%; the cerebral blood flow difference in the model group was 27.98±14.97%. These differences were statistically significant compared to the sham-operated group (p<0.01). The cerebral blood flow difference in the high-dose Bremerlongan group was 19.06±8.81%; and the cerebral blood flow difference in the low-dose Bremerlongan group was 19.42±18.14%.

[0066] Figure 3 shows the Doppler cerebral blood flow observations of rats in the sham-operated group, model group, high-dose Bremerlongan group, and low-dose Bremerlongan group 48 hours post-operation. At 48 hours post-operation, the cerebral blood flow difference in the sham-operated group was 0.43±0.26%; the cerebral blood flow difference in the model group was 29.49±11.74%. These differences were statistically significant compared to the sham-operated group (p<0.01). The cerebral blood flow difference in the high-dose Bremerlongan group was 23.70±10.94%; and the cerebral blood flow difference in the low-dose Bremerlongan group was 26.77±12.12%.

[0067] Table 5. Effects of Bremerlongan on cerebral blood flow in stroke-affected rats (n=10) Note: ## This indicates that compared with the sham surgery group, p<0.01.

[0068] 3.6 Brain Volume and Infarct Area Test Results: The brain volume results are shown in Table 6. The brain volume of rats in the sham-operated group was 1.467±0.04mL; the brain volume of rats in the model group was 1.667±0.09mL. Compared with the sham-operated group, there was a statistically significant difference (p<0.01). The brain volume of rats in the high-dose Bremerlongan group was 1.650±0.13mL; the brain volume of rats in the low-dose Bremerlongan group was 1.676±0.08mL.

[0069] Table 6. Effects of Bremerlongan on brain volume in rats with stroke (n=10) Note: ## This indicates that compared with the sham surgery group, p < 0.01.

[0070] The results of the cerebral infarction area test are shown in Figures 4-6. Figure 4 shows the TTC staining of brain slices from rats in the sham-operated group, Figure 5 shows the TTC staining of brain slices from rats in the model group, and Figure 6 shows the TTC staining of brain slices from rats in the high-dose Bremerontan group. It can be seen that the cerebral infarction was significantly improved, with an infarction area improvement rate of over 30%.

[0071] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. The use of Bremerlongdan in the preparation of drugs for the prevention and / or treatment of ischemic stroke.

2. The application according to claim 1, characterized in that, Application of Bremerlongdan in the preparation of drugs to improve neurological deficits.

3. The application according to claim 1, characterized in that, Application of Bremerlongdan in the preparation of drugs to reduce the area of ​​cerebral infarction.

4. The application according to claim 1, characterized in that, Application of Bremerlongdan in the preparation of drugs to improve cerebral blood flow in ischemic stroke.

5. The application according to any one of claims 1-4, characterized in that, Bremerlongdan is the only active ingredient in the drug.

6. The application according to claim 1, characterized in that, The drug also includes pharmaceutically acceptable excipients.

7. The application according to claim 6, characterized in that, The excipients include one or more of surfactants, buffers, disintegrants, binders, fillers, lubricants, solubilizers, flavorings, and colorings.

8. The application according to claim 1, characterized in that, The dosage forms of the drug include oral dosage forms and non-gastrointestinal dosage forms.

9. The application according to claim 8, characterized in that, The oral dosage forms are tablets, capsules, granules, powders, pills, oral liquid preparations, gel preparations, or lyophilized powder injections.

10. The application according to claim 8, characterized in that, The non-gastrointestinal dosage forms are injection dosage forms, respiratory dosage forms, skin dosage forms, mucosal dosage forms, or cavity dosage forms.

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