New use of exemestane

Abstract

The present patent application discloses a new use of exemestane, and specifically discloses the use of exemestane in treating diseases with reduced nerve function, including cerebral ischemia-reperfusion injury, cerebral infarction, Parkinson's disease, etc. In long-term research, it is found that exemestane has a neuroprotective effect, can prevent or delay the death of nerve cells, and has a therapeutic effect on diseases with reduced nerve function, such as cerebral ischemia-reperfusion injury and cerebral infarction.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a novel pharmaceutical use of exemestane. Background Technology

[0002] Cerebral ischemia-reperfusion injury (CIS) is a common disease that seriously threatens human health, characterized by high morbidity, high mortality, and high disability rates. The pathogenesis of CIS-reperfusion injury is highly complex, and exploring this mechanism has always been a hot topic and focus of research in neuroscience. Currently, the most effective treatments for ischemic stroke are thrombolysis and anticoagulation, but these treatments have strict time windows and contraindications, meaning that most patients often do not have the opportunity to undergo thrombolytic therapy. Therefore, finding neuroprotective drugs that prevent or delay neuronal death has broader prospects and greater significance. Summary of the Invention

[0003] In order to overcome the shortcomings of the prior art, the purpose of this invention is to provide a new pharmaceutical use for exemestane. This invention discovers that exemestane has a neuroprotective effect, which can prevent or delay nerve cell death and has a therapeutic effect on diseases with weakened neurological function such as cerebral ischemia-reperfusion injury and cerebral infarction.

[0004] The technical solution adopted in this invention is as follows:

[0005] Exemestane is used in products for the treatment of neurological disorders.

[0006] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0007] Exemestane is an antitumor drug with the chemical name 6-methyleneandrost-1,4-diene-3,17-dione and the chemical formula C60. 20 H 24 O2, its structural formula is:

[0008] Through long-term research, this invention has discovered that exemestane has a neuroprotective effect, which can prevent or delay nerve cell death and has a therapeutic effect on diseases with weakened neurological function such as cerebral ischemia-reperfusion injury, cerebral infarction, and Parkinson's disease.

[0009] In a preferred embodiment of the present invention, exosomes are used to load exemestane. This method utilizes exosomes for exemestane loading and delivery, which offers high biocompatibility, protects exemestane from rapid degradation, and allows for slow release, resulting in better therapeutic efficacy.

[0010] In a preferred embodiment of the present invention, exogenous loading is used to load exemestane. Exogenous loading has advantages such as relatively simple experimental operation, short experimental cycle, and more stable and controllable batch-to-batch results, making it particularly suitable for scale-up production.

[0011] In a preferred embodiment of the present invention, the exosomes are extracellular vesicles. Extracellular vesicles (EVs) are characterized by low toxicity, high biocompatibility, and low immunogenicity. Their phospholipid bilayer structure can protect their contents from rapid degradation. Furthermore, EVs typically carry specific receptors on parental cells, exhibiting a certain degree of targeting, which facilitates targeted drug delivery. EVs also have the advantage of being easily modified artificially, allowing them to be endowed with specific functions. Using EVs as delivery carriers for exemestane can achieve better therapeutic effects.

[0012] In a preferred embodiment of the present invention, the diseases causing weakened neurological function include cerebral ischemia-reperfusion injury and cerebral infarction.

[0013] In a preferred embodiment of the present invention, the product is a pharmaceutical and / or health product.

[0014] In a preferred embodiment of the present invention, the product is one of an injection, suspension, powder, tablet, or granule. When necessary, one or more pharmaceutically acceptable carriers may be added to the above product, including diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption enhancers, surfactants, adsorbents, lubricants, etc., commonly used in the pharmaceutical field. All of the above dosage forms can be prepared according to conventional methods in the pharmaceutical field. Attached Figure Description

[0015] Figure 1 The images are taken by a digital camera after TTC staining according to an embodiment of the present invention;

[0016] Figure 2 This is a detection image of the present invention after HE staining under a high-power optical microscope (×400);

[0017] Figure 3 This is a detection image of the embodiment of the present invention after Nissl staining under a high-power optical microscope (×400). Detailed Implementation

[0018] Typical embodiments embodying the features and advantages of the present invention will be specifically described in the following description. It should be understood that the present invention can have various variations in different embodiments without departing from the scope of the present invention, and the descriptions and illustrations herein are for illustrative purposes only and not intended to limit the present invention.

[0019] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0020] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0021] The exemestane used in the following examples is a DMSO solution of exemestane. The manufacturer of exemestane is Selleck, catalog number S1196.

[0022] The exosomes used in the following examples were provided by Beijing Siluosai Company and are extracellular vesicle exosomes.

[0023] This embodiment discloses the use of exemestane in products for treating neurological disorders.

[0024] The drug-loaded exosome construction method used in the following examples:

[0025] Using existing exogenous loading methods, the required exosomes and exemestane were mixed at a volume ratio of 300:1. The mixture was incubated at room temperature in the dark for 30 minutes. After incubation, the exosomes were purified by centrifugation to remove free exemestane. The encapsulation efficiency and loading amount were determined by nanoflow cytometry and HPLC.

[0026] The groups were as follows: sham surgery (SHAM) group plus exosomes alone, sham surgery (SHAM) group loaded with exemestane exosomes, MCAO group plus exosomes alone, and MCAO group loaded with exemestane exosomes.

[0027] Sham surgery (SHAM) group: Arterial embolization was performed without inserting a suture embolism, and the rest of the procedures were the same as those in the MCAO group.

[0028] The rat MCAO group model construction method used in the following examples:

[0029] (1) After weighing the rats, they were anesthetized by intraperitoneal injection of 3.5% chloral hydrate (1ml / 100g).

[0030] (2) After anesthetizing the rat, fix it in a supine position on the operating table, shave and disinfect the neck, and make an incision about 2 cm long in the middle.

[0031] (3) Separate the blood vessels and vagus nerve to expose the left common carotid artery, external carotid artery and internal carotid artery (be careful not to damage the vagus nerve).

[0032] (4) Ligate the external carotid artery and the proximal end of the common carotid artery, and tie a single knot suture at the distal end of the common carotid artery. Clamp the internal carotid artery with an arterial clamp.

[0033] (5) Make a small notch in the center above the ligation point of the common carotid artery, and slowly push the prepared nylon suture into the internal carotid artery through the incision while releasing the arterial clamp on the internal carotid artery.

[0034] (6) When the suture thrombus is advanced to a length of 18mm-20mm (from the bifurcation of the common carotid artery), resistance can be clearly felt. At this time, the opening of the middle cerebral artery is blocked, and the suture thrombus is fixed.

[0035] (7) After 1 hour of blockage, loosen the single knot, slowly pull out the nylon suture, ligate the distal end of the common carotid artery, restore blood circulation, and achieve reperfusion.

[0036] (8) Suture the wound and clean and disinfect it. The rat's body temperature was maintained at around 37°C during and after the operation. The rats after modeling were placed in a cage with clean bedding, given sufficient food and water, and the room temperature was maintained at around 25°C.

[0037] MCAO group model success criteria:

[0038] ① Pain in the contralateral forelimb, slow or absent retraction;

[0039] ② When hanging upside down with the tail raised, the opposite forelimb bends towards the chest;

[0040] ③ When walking, lean your body to the opposite side or turn in a circle to the opposite side;

[0041] ④ Horner's syndrome occurs on the contralateral side.

[0042] The occurrence of any of the above conditions indicates that the MCAO model has been successfully established.

[0043] The administration method used in the following examples:

[0044] After the online thrombus is removed, the wound is sutured, and 100ug / animal of simple exosomes or drug-loaded exosomes is injected via the tail vein.

[0045] For rats treated with the above drugs, the infarct volume was measured by TTC staining, and the morphological changes of the brain tissue 24 h after ischemia-reperfusion were assessed by HE staining and Nissl staining.

[0046] TTC staining to determine the volume of cerebral infarction in rats

[0047] The following method was used for TTC staining assay:

[0048] (1) After 24 hours of reperfusion, the brain was quickly anesthetized, decapitated, and the cranial cavity was opened to remove the complete brain.

[0049] (2) Place the brain in a -20°C freezer for 20 minutes.

[0050] (3) Place the frozen brain in the groove of the meninges, remove the front and back ends, and cut a piece every 2 mm, for a total of 5 pieces.

[0051] (4) Place 5 brain slices in 2% TTC solution, stain in the dark for 15 minutes, then turn the brain slices over and continue staining in the dark for another 15 minutes.

[0052] (5) The stained brain slices were transferred to 4% paraformaldehyde solution for fixation for 24 hours.

[0053] (6) Place brain slices on filter paper, then capture images using a digital camera, and use ImageJ to calculate and analyze the cerebral infarction area. The formula for calculating the cerebral infarction area after edema is: {[total infarction volume - (volume of ipsilateral hemisphere - volume of contralateral hemisphere)] / volume of contralateral hemisphere} × 100%.

[0054] The results of TTC staining to determine the infarct volume in rats are shown in the figure. Figure 1 As shown in the figure. Compared with injecting exosomes alone after MCAO modeling, injecting exosomes loaded with exemestane after MCAO modeling significantly reduced the volume of cerebral infarction in rats, indicating that exemestane can effectively prevent or delay neuronal death.

[0055] HE staining to determine morphological changes in brain tissue

[0056] HE staining assay was performed using the following method:

[0057] (1) After reperfusion for 24 hours, brain tissue was obtained by perfusion with 4% paraformaldehyde. The brain tissue was then fixed by immersion in 4% paraformaldehyde solution for 24 hours, followed by paraffin embedding and preparation of 4µm sections.

[0058] (2) Dewaxing with xylene, followed by hydration with ethanol at various levels: The tissue sections were soaked in xylene I and xylene II for 10 min respectively. Then the sections were hydrated by soaking in 100%, 95%, 80%, and 70% ethanol for 2 min respectively, and then rinsed with tap water for 1 min.

[0059] (3) Hematoxylin staining: Immerse the sections in hematoxylin for 8-10 min, rinse with tap water for 1 min, and differentiate with 1% hydrochloric acid alcohol for 30 s. Then rinse with tap water for 1 min, and place the sections in saturated lithium carbonate for 2 min for blueing. Finally, soak in tap water for 15 min.

[0060] (4) Eosin staining: Place the slide in eosin for 2 min, rinse with tap water for 1 min, and then quickly place it in a gradient of alcohols for dehydration.

[0061] (5) Seal the film. After clearing with xylene, seal the film with neutral resin, being careful not to create air bubbles.

[0062] The results of morphological changes in brain tissue determined by HE staining are shown below. Figure 2 As shown. Figure 2 HE staining results showed that, compared with the two SHAM groups, the MCAO group had brain tissue edema, disordered cell arrangement, loose cytoplasm, and nuclear pyknosis. The MCAO + exosome-only group showed more severe brain tissue edema and diffuse vacuolation in the interstitial area, which was improved by injection of drug-loaded exosomes.

[0063] Nissl staining counts of intact neurons

[0064] Nissl staining and counting were performed using the following method:

[0065] (1) After reperfusion for 24 hours, brain tissue was obtained by perfusion with 4% paraformaldehyde. The brain tissue was then fixed by immersion in 4% paraformaldehyde solution for 24 hours, followed by paraffin embedding and preparation of 4µm sections.

[0066] (2) Dewaxing with xylene, followed by hydration with ethanol at various levels: The tissue sections were soaked in xylene I and xylene II for 10 min respectively. Then the sections were hydrated by soaking in 100%, 95%, 80%, and 70% ethanol for 2 min respectively, and then rinsed with tap water for 1 min.

[0067] (3) Staining: Immerse the slices in tar purple for 15 minutes and rinse with tap water for 1 minute.

[0068] (4) Color separation: After placing the slices in 95% ethanol for 30 seconds, they are then quickly placed in a gradient of alcohols for dehydration.

[0069] (5) Seal the film. After clearing with xylene, seal the film with neutral resin, being careful not to create air bubbles.

[0070] (6) Images were acquired under a high-power microscope (×400) using a standard optical microscope. The number of intact neurons was counted in a unit area (0.25×0.25mm). The results were expressed as number / mm. 2 express.

[0071] The results of counting intact neurons using Nissl staining are shown in the figure. Figure 3 As shown. Figure 3 The results showed that the neuronal structure of the brain tissue in both SHAM groups was intact and clear, with a regular distribution of Nissl bodies. Compared with the SHAM group, the brain tissue structure of the two MCAO groups was loose, the cell arrangement was disordered, and the Nissl body staining was unclear. After MCAO modeling, injection of exosomes alone would make the tissue structure even looser, the number of intact neurons would be significantly reduced, neurons would atrophy, nuclei would condense and fragment, and a large number of Nissl bodies would be lost, while injection of drug-loaded exosomes would improve this phenomenon.

[0072] The above embodiments are merely preferred embodiments of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-substantial changes and substitutions made by those skilled in the art based on the present invention shall fall within the scope of protection claimed by the present invention.

Claims

1. The use of exemestane in the preparation of drugs for the treatment of neurological disorders; Exemestane was loaded using exosomes, which were extracellular vesicles. The diseases that cause weakened neurological function are cerebral ischemia-reperfusion injury and cerebral infarction.

2. The application according to claim 1, characterized in that: The drug is one of the following: injection, suspension, powder, tablet, or granule.

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