Application of fullerene and fullerene derivative in preparation of medicine

Abstract

The invention relates to the field of biological medicines, in particular to application of fullerene and fullerene derivatives in preparation of medicines, and the medicines are used for preventing, relieving, assisting in treating or treating intracranial hemorrhagic diseases. The invention finds that the fullerene and the fullerene derivative have excellent blood brain barrier crossing capability, can remarkably reduce neurotoxicity caused by intracranial hemorrhagic diseases such as cerebral hemorrhage and accelerate hematoma absorption, can be used for preparing medicines for preventing, relieving, assisting in treating or treating the intracranial hemorrhagic diseases, and has a wide application prospect. A new strategy and a solution scheme are provided for treatment of related diseases such as intracranial hemorrhagic diseases, and the application prospect is wide.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to the use of fullerenes and fullerene derivatives in the preparation of drugs. Background Technology

[0002] Fullerene is a type of carbon atom composed entirely of carbon atoms linked by sp... 2 Molecules formed by hybridization have unique cage-like structures, commonly including C1 and C2. 60 C 70 Fullerene derivatives are products of chemical modification of fullerene molecules. There are many kinds of fullerene derivatives, the most common of which are metal fullerenes. At present, fullerenes and fullerene derivatives have been widely used in many fields such as optics, electricity, magnetism, and biomedicine due to their unique cage-like structure and physicochemical properties.

[0003] Intracranial hemorrhage disorders are a group of critical illnesses caused by the rupture of blood vessels in the brain, resulting in bleeding into the brain parenchyma, ventricles, or subarachnoid space, accompanied by disruption of the blood-brain barrier (BBB). Intracerebral hemorrhage (ICH) is the core subtype of ICH, accounting for 10-15% of all stroke cases, characterized by high rates of disability and mortality. Currently, the clinical manifestations of ICH are diverse, mainly depending on the location of the hemorrhage and the size of the hematoma. Common symptoms include sudden focal neurological deficits, such as contralateral limb weakness, sensory loss, and speech impairment. If the hematoma extends into the ventricular system, it can also lead to hydrocephalus and increased intracranial pressure. For the treatment of ICH and similar brain diseases, the effectiveness of drug therapy largely depends on the safe and effective delivery of drugs to the brain parenchyma. However, due to the strong blocking function of the blood-brain barrier, most drugs cannot directly enter the brain parenchyma via intravenous injection to achieve the desired therapeutic effect.

[0004] Therefore, developing or exploring a drug that can cross the blood-brain barrier is of great significance for the treatment of intracranial hemorrhage diseases such as cerebral hemorrhage. Summary of the Invention

[0005] This invention aims to at least partially address one of the technical problems existing in the prior art. To this end, this invention provides the use of fullerenes and fullerene derivatives in the preparation of pharmaceuticals. This invention discovers that fullerenes and fullerene derivatives possess excellent blood-brain barrier crossing ability, while significantly reducing neurotoxicity caused by intracranial hemorrhage and other intracranial hemorrhage diseases, and accelerating hematoma absorption. They can be used to prepare drugs for the prevention, relief, adjuvant treatment, or treatment of intracranial hemorrhage diseases, providing new strategies and solutions for the treatment of intracranial hemorrhage and related diseases, with broad application prospects.

[0006] This invention proposes the use of fullerenes and fullerene derivatives in the preparation of pharmaceuticals. According to embodiments of the invention, the pharmaceuticals are used for the prevention, relief, adjunctive treatment, or treatment of intracranial hemorrhage. Based on the uses described in the embodiments of the invention, fullerenes and fullerene derivatives possess excellent blood-brain barrier crossing capabilities, while also significantly reducing neurotoxicity caused by intracranial hemorrhage and other intracranial hemorrhage, and accelerating hematoma absorption. Therefore, by utilizing fullerenes and fullerene derivatives to prepare pharmaceuticals for the prevention, relief, adjunctive treatment, or treatment of intracranial hemorrhage, a new strategy and solution are provided for the treatment of intracranial hemorrhage and related diseases, with broad application prospects.

[0007] According to embodiments of the present invention, the above-described uses may also have the following additional technical features:

[0008] According to an embodiment of the present invention, the fullerene is a hollow fullerene.

[0009] According to an embodiment of the present invention, the hollow fullerene has the general structural formula C0. 2m Where 30≤m≤60.

[0010] According to a preferred embodiment of the present invention, the hollow fullerene has the general structural formula C0. 2m Where 30≤m≤45.

[0011] According to embodiments of the present invention, the fullerene derivative includes one or more of carboxyl-modified fullerene, alanine-modified fullerene, amino-modified fullerene, alkyl-modified fullerene, sulfonic acid-modified fullerene, and metallofullerene.

[0012] According to an embodiment of the present invention, the fullerene derivative is a carboxyl-modified fullerene, wherein the carboxyl-modified fullerene has an addition number of 1 to 4 and a carboxyl number of 1 to 8.

[0013] According to an embodiment of the present invention, the fullerene derivative is a metallofullerene, and the general structural formula of the metallofullerene is M@C 2n M2@C 2n MA@C 2n M3N@C 2nM2C2@C 2n M2S@C 2n M2O@C 2n and M x A 3-x N@C 2n One or more of the following; wherein @ indicates that an atom is embedded inside the carbon cage of the fullerene, M and A are metal elements, and M and A are selected from any one of scandium, yttrium and lanthanide metal elements, 30≤n≤60, 0≤x≤3.

[0014] According to embodiments of the present invention, the intracranial hemorrhage disease includes one or more of primary cerebral hemorrhage, traumatic cerebral hemorrhage, neoplastic cerebral hemorrhage, hemorrhagic stroke, and amyloid angiopathy.

[0015] According to embodiments of the present invention, the drug also has at least one of the following uses: crossing the blood-brain barrier; promoting microglia processing or digestion of red blood cells; promoting the absorption of intracerebral hematomas; and regulating the phagosome signaling pathway of phagocytes to promote hematoma absorption.

[0016] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0017] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0018] Figure 1 This is a graph showing the characterization and determination results of the fullerene water-soluble derivative TMF sample in Example 1 of the present invention, where A represents the fullerene water-soluble derivative TMF (C 70 The synthetic flow chart of (-COOH), B is the tetraaddition esterification product C. 70 The infrared spectrum characterization results of the [C(COOCH2CH3)2]4 sample, where C represents the fullerene water-soluble derivative TMF (C 70 Infrared spectral characterization results of the COOH sample;

[0019] Figure 2 This is a graph showing the characterization and determination results of the alanine-modified fullerene derivative sample in Example 1 of the present invention;

[0020] Figure 3 This is a graph showing the characterization and measurement results of the TMF-Cy5 sample in Example 1 of this invention;

[0021] Figure 4 Cy5-C in Embodiment 1 of the present invention 70 -Graph showing the characterization and measurement results of Ala samples;

[0022] Figure 5 The images show the IVIS imaging results of the mouse brains in each group in Example 2 of this invention. * indicates P<0.05, ** indicates P<0.01. A shows the IVIS imaging results of the mouse brains in each group. B shows the statistical results of the brain fluorescence intensity of the IVIS imaging of the mouse brains in each group. C shows the IVIS imaging results of the mouse brains in each group after cardiac perfusion and non-cardiac perfusion dissection.

[0023] Figure 6 The images show the in vivo imaging and brain tissue fluorescence imaging results of mice in each group in Example 3 of this invention. In the images, ns indicates no significant difference between groups, * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001. A shows the IVIS imaging results of the brain of mice in each group at different time points; B shows the statistical results of the fluorescence intensity of IVIS imaging of the brain of mice in each group at different time points; C shows the IVIS imaging results of the brain of mice in each group after cardiac perfusion 3 h or 24 h after drug administration; D shows the statistical results of the fluorescence intensity of IVIS imaging of the brain after cardiac perfusion 3 h after drug administration; and E shows the statistical results of the fluorescence intensity of IVIS imaging of the brain after cardiac perfusion 24 h after drug administration.

[0024] Figure 7 The figures shown are the brain fluorescence intensity monitoring results of mice in each group after 7 days of continuous administration of TMF-Cy5 in Example 4 of this invention. A represents the brain IVIS imaging results of mice in each group at different time points after 7 days of continuous administration; B represents the statistical results of brain IVIS imaging fluorescence intensity at different time points after 7 days of continuous administration; C represents frozen section images of mice after 7 days of continuous administration of TMF-Cy5 for different durations; and D represents the results of 7 days of continuous administration of Cy5-Cy5. 70 - Frozen sections of Ala at different days, with blue markings for cell nuclei and yellow markings for TMF-Cy5;

[0025] Figure 8 In the intravenous injection of C in Example 5 of the present invention 70 -COOH and oral C 60 The figure shows a comparison of the treatment effects of cerebral hemorrhage. ** indicates P < 0.01, *** indicates P < 0.001, and **** indicates P < 0.0001. A shows MRI slices of mice in each group at 7 and 14 days after cerebral hemorrhage. The numbers in the lower right corner of the figure represent the hematoma volume in mm. 3 B is a statistical chart showing the proportion of hematoma absorption in each group of mice during the period of 7 to 14 days; C is a statistical chart showing the drop time of each group of mice in the rotarod fatigue test after 14 days.

[0026] Figure 9The images show the KEGG analysis results of the hemorrhage side brain tissue of mice in each group 14 days after cerebral hemorrhage in Example 6 of this invention. Among them, A is the KEGG enrichment result of the ICH group relative to the control group, B is the KEGG enrichment result of the TMF treatment group relative to the ICH group, C is the heatmap of the changes in the phagosome-related protein set of the TMF treatment group relative to the ICH group, D is the GSEA analysis result of the phagosome pathway of the protein with significant changes in expression in the TMF treatment group relative to the ICH group, and E is the KEGG Mapper map of the phagosome pathway of the protein with significant changes in expression in the TMF treatment group relative to the ICH group. Red indicates that the protein expression is upregulated, and green indicates that the protein expression is downregulated.

[0027] Figure 10 The graph shows the fluorescence intensity measurement results of BV-2 cells in each group in Example 7 of the present invention. In this graph, A is a schematic diagram of the experimental procedure of Example 7, B is a graph showing the fluorescence intensity measurement results of BV-2 cells in each group, and the percentage on the right indicates the proportion of BV2 lineage cells containing erythrocytes in that group. Detailed Implementation

[0028] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0029] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0030] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0031] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this invention, but do not exclude other aspects.

[0032] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0033] Terms and Definitions

[0034] In this article, the term "fullerene" refers to a spherical molecule composed of carbon atoms with a unique cage-like structure, resembling a soccer ball, hence also known as a fullerene. Common fullerenes include C464-C ... 60 C 70 It possesses good electrical conductivity, optical properties, and chemical stability.

[0035] In this article, the term "hollow fullerene" specifically refers to a classical fullerene (such as C1) that is not filled with any atoms or molecules. 60 C 70 The carbon cage is a vacuum or contains only trace amounts of gas (such as He).

[0036] In this paper, the term "fullerene derivative" refers to a series of compounds formed by chemically modifying fullerene molecules. Fullerene derivatives are diverse. For example, introducing a carboxyl group (-COOH) onto a fullerene molecule yields carboxyl-modified fullerene; covalently linking alanine to a fullerene molecule yields alanine-modified fullerene; introducing an amino group (-NH2) onto a fullerene molecule yields amino-modified fullerene; attaching an alkyl chain (such as methyl, ethyl, etc.) to a fullerene molecule yields alkyl-modified fullerene; introducing a sulfonic acid group (-SO3H) onto a fullerene molecule yields sulfonic acid-modified fullerene; and embedding metal atoms or metal ions into the carbon cage of a fullerene molecule or attaching them to its surface yields metallofullerene.

[0037] In this document, the term "ultra-dry tetrahydrofuran" refers to tetrahydrofuran that has undergone special treatment and has extremely low moisture content, and is used as an organic solvent and chemical reaction medium in the embodiments of this invention.

[0038] In this document, the term "ultra-dry o-xylene" refers to o-xylene that has undergone special treatment and has extremely low moisture content, and is used as an organic solvent and chemical reaction medium in the embodiments of this invention.

[0039] In this document, the term "sulfo-Cyanine5 amine" refers to a specific fluorescent dye derivative, specifically the amine form of the Sulfo-Cyanine5 dye, used for labeling and detecting biomolecules. In embodiments of this invention, it is used to label fullerenes and fullerene derivatives to track their distribution and metabolism in vivo using fluorescence imaging techniques, thereby assessing their ability to cross the blood-brain barrier and their residence and clearance characteristics in brain tissue.

[0040] In this document, the term "Ex / Em:640 / 680" refers to the excitation wavelength of 640 nm and the emission wavelength of 680 nm in the specific detection of IVIS imaging in the embodiments of the present invention.

[0041] In this document, the term "anterior fontanelle" is located anterior to the intersection of the midline of the head and the coronal suture of the skull. In embodiments of the invention, it serves as a reference marker to determine the location and direction of surgery for precise operation.

[0042] In this article, the term "crossing the blood-brain barrier" refers to the process by which drugs and other substances pass through multiple barriers formed by the endothelial cells and their tight junctions, the basement membrane, and the foot processes of astrocytes from the blood circulation to enter the brain parenchyma. This process overcomes the high selectivity, low permeability, and efflux pump mechanism of the blood-brain barrier and is a key link in the treatment of central nervous system diseases.

[0043] Uses in drug preparation

[0044] This invention proposes the use of fullerenes and fullerene derivatives in the preparation of pharmaceuticals. According to embodiments of the invention, the pharmaceuticals are used for the prevention, relief, adjunctive treatment, or treatment of intracranial hemorrhage. Based on the uses described in the embodiments of the invention, fullerenes and fullerene derivatives possess excellent blood-brain barrier crossing capabilities, while also significantly reducing neurotoxicity caused by intracranial hemorrhage and other intracranial hemorrhage, and accelerating hematoma absorption. Therefore, by utilizing fullerenes and fullerene derivatives to prepare pharmaceuticals for the prevention, relief, adjunctive treatment, or treatment of intracranial hemorrhage, a new strategy and solution are provided for the treatment of intracranial hemorrhage and related diseases, with broad application prospects.

[0045] According to an embodiment of the present invention, the fullerene is a hollow fullerene. This further clarifies the structural characteristics of the fullerene applicable to the uses of the present invention. Hollow fullerenes possess a unique cage-like structure, exhibiting excellent ability to cross the blood-brain barrier, while also being able to encapsulate or carry other therapeutic molecules or drugs across the blood-brain barrier, thereby achieving prevention, relief, adjunctive treatment, or treatment of intracranial hemorrhage.

[0046] According to an embodiment of the present invention, the hollow fullerene has the general structural formula C0. 2m Where 30≤m≤60. Thus, hollow fullerene structures within this structural range possess suitable size and stability, ensuring their ability to cross the blood-brain barrier while providing sufficient space for encapsulating and carrying drug molecules, ensuring effective drug delivery and release in brain tissue, thereby achieving the prevention, relief, adjunctive treatment, or treatment of intracranial hemorrhage.

[0047] According to a preferred embodiment of the present invention, the hollow fullerene has the general structural formula C0.2m Where 30≤m≤45. Therefore, further optimization of the structure of hollow fullerenes can improve their performance, resulting in better effects in crossing the blood-brain barrier, drug loading, and release, thus enabling more efficient prevention, relief, adjunctive treatment, or treatment of intracranial hemorrhage.

[0048] According to embodiments of the present invention, the fullerene derivative includes one or more of carboxyl-modified fullerenes, alanine-modified fullerenes, amino-modified fullerenes, alkyl-modified fullerenes, sulfonic acid-modified fullerenes, and metallofullerenes. Therefore, the specific modification type of the fullerene derivative can be adjusted according to different application requirements and drug administration methods, further enhancing the drug's stability and biocompatibility in vivo, and improving therapeutic efficacy and safety. For example, carboxyl-modified fullerenes, administered intravenously, can more effectively cross the blood-brain barrier, promote microglia processing or digestion of erythrocytes, induce M1-type microglia polarization towards M2 type, and regulate the phagosome signaling pathway within phagocytes to promote hematoma absorption, thereby more efficiently achieving the prevention, relief, adjuvant treatment, or treatment of intracranial hemorrhage.

[0049] It should be noted that when fullerenes and their derivatives, and drugs containing fullerenes and their derivatives, are used to prevent, alleviate, assist in the treatment or treatment of intracranial hemorrhage, the administration methods include, but are not limited to, intravenous injection, nasal administration, oral administration, transdermal administration, intramuscular injection, subcutaneous injection, etc. Any administration method that can deliver fullerenes and their derivatives, and drugs containing fullerenes and their derivatives, to the lesion site is within the scope of protection of this invention.

[0050] It should be noted that, although the embodiments of the present invention only target typical hollow fullerene C 60 C 70 The functions of carboxyl-modified fullerenes and alanine-modified fullerenes have been verified. However, the scope of protection of this invention is not limited to the specific types of fullerenes and fullerene derivatives verified. In addition to the fullerenes and their derivatives mentioned above, other fullerenes and their derivatives with similar chemical structures and physiological functions, such as amino-modified fullerenes, alkyl-modified fullerenes, sulfonic acid-modified fullerenes, and metallofullerenes, are also applicable to the prevention, relief, adjunctive treatment, or treatment of intracranial hemorrhage diseases. All fullerenes and their derivatives should fall within the scope of protection of this invention.

[0051] According to embodiments of the present invention, the fullerene derivative is a carboxyl-modified fullerene, wherein the carboxyl-modified fullerene has an addition number of 1 to 4 and a carboxyl group number of 1 to 8. Thus, carboxyl modification endows the fullerene molecule with good water solubility and biocompatibility, enabling it to cross the blood-brain barrier more efficiently and distribute in brain tissue, thereby more effectively promoting hematoma absorption and reducing neurotoxicity. Exemplarily, the carboxyl-modified fullerene has an addition number of 1, 2, 3, or 4, preferably 3 to 4; and the carboxyl group number is 1, 2, 3, 4, 5, 6, 7, or 8, preferably 6 to 8.

[0052] According to an embodiment of the present invention, the fullerene derivative is a metallofullerene, and the general structural formula of the metallofullerene is M@C 2n M2@C 2n MA@C 2n M3N@C 2n M2C2@C 2n M2S@C 2n M2O@C 2n and M x A 3-x N@C 2n One or more of the following: where @ indicates that an atom is embedded within the carbon cage of the fullerene; M and A are metallic elements, and both M and A are selected from scandium, yttrium, and lanthanides, where 30 ≤ n ≤ 60, and 0 ≤ x ≤ 3. Thus, metallofullerenes combine the unique properties of metallic elements with the ability of fullerene molecules to cross the blood-brain barrier, thereby playing a special role in the prevention, relief, adjuvant treatment, or treatment of intracranial hemorrhage, such as enhancing antioxidant capacity and regulating immune responses, thus providing more options and possibilities for the treatment of intracranial hemorrhage.

[0053] According to embodiments of the present invention, the intracranial hemorrhage includes one or more of primary cerebral hemorrhage, traumatic cerebral hemorrhage, neoplastic cerebral hemorrhage, hemorrhagic stroke, and amyloid angiopathy. Therefore, drugs containing fullerenes and fullerene derivatives have good therapeutic effects on various types of intracranial hemorrhage, and can effectively intervene in intracranial hemorrhage caused by different etiologies and pathological mechanisms, showing broad application prospects.

[0054] According to embodiments of the present invention, the drug also has at least one of the following uses: crossing the blood-brain barrier; promoting microglia processing or digestion of red blood cells; promoting the absorption of intracerebral hematomas; and regulating the phagosome signaling pathway of phagocytes to promote hematoma absorption. Therefore, drugs containing fullerenes and fullerene derivatives can effectively cross the blood-brain barrier for drug delivery, allowing the drugs to directly act on the lesions in the brain, increasing the drug concentration in brain tissue, and further enhancing the therapeutic effect. Furthermore, drugs containing fullerenes and fullerene derivatives accelerate hematoma absorption by promoting the processing and digestion of erythrocytes by microglia, reducing the pressure and damage of the hematoma on brain tissue, and alleviating neurotoxicity. In addition, drugs containing fullerenes and fullerene derivatives also reduce inflammation-mediated secondary brain injury by inducing microglia to polarize from the pro-inflammatory M1 type to the anti-inflammatory M2 type, thus promoting brain tissue repair and functional recovery. Furthermore, drugs containing fullerenes and fullerene derivatives further enhance the phagocytic and digestive capacity of phagocytes on hematoma components by regulating the phagosome signaling pathway, promoting hematoma absorption more efficiently, reducing the pressure and damage of the hematoma on brain tissue, and alleviating neurotoxicity. The phagocytes mentioned here include macrophages, neutrophils, and microglia.

[0055] It should be noted that although the embodiments of the present invention only verified the relevant functions of fullerenes and fullerene derivatives for cerebral hemorrhage, the use of fullerenes and fullerene derivatives in the preparation of drugs is not limited to this. Based on their unique advantages such as being able to cross the blood-brain barrier, promote microglia to process or digest red blood cells, promote the absorption of cerebral hematoma, and regulate phagosome-related signaling pathways in phagocytes, they can also be used to prevent, alleviate, assist in the treatment or treatment of other diseases involving brain inflammation and tissue repair. Any disease situation that requires drug delivery to the brain and exertion through / across the blood-brain barrier should fall within the protection scope of the present invention.

[0056] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0057] Example 1: Synthesis and Characterization of Fullerene Derivatives

[0058] 1. Synthesis and characterization of carboxylated fullerene derivatives

[0059] Using the Binger reaction principle, under nitrogen (N2) protection, 4g of sodium hydride (CAS: 7646-69-7) and 40mL of ultra-dry tetrahydrofuran (CAS: 109-99-9) were stirred for 10min, and then 8mL of diethyl bromomalonate (CAS: 685-87-0) was slowly added dropwise, releasing heat and gas. Stirring was continued until the reaction was complete and the solution turned yellow, thus obtaining reaction solution I.

[0060] 0.5g C 70 Fullerene solid powder (CAS: 115383-22-7) was dissolved in 100 mL of ultra-dry o-xylene (CAS: 95-47-6) and added at a constant rate to the yellow reaction solution I. The mixture was stirred for 2 h, and the reaction was monitored by TLC thin-layer chromatography. Once the reaction was complete, reaction mixture I was obtained.

[0061] The reaction mixture I was filtered to remove the solids, and the filtrate was collected. Hexane (CAS: 110-54-3) was added to the filtrate to induce precipitation. The precipitate was collected by centrifugation, dissolved in toluene (CAS: 108-88-3), and separated using a silica gel column chromatography. The tetraaddition esterification product C was collected. 70 [C(COOCH2CH3)2]4 sample; and the tetraaddition esterification product C obtained was detected by MALDI-TOF mass spectrometry and 1H NMR spectroscopy. 70 After determining the molecular structure of the [C(COOCH2CH3)2]4 sample, subsequent operations were performed.

[0062] The tetraaddition esterification product C, whose molecular structure has been confirmed, 70 The [C(COOCH2CH3)2]4 sample was evaporated to dryness using a rotary evaporator, then dissolved in toluene. Under nitrogen (N2) protection, 6 g of sodium hydride was added and stirred at 60°C for 6 h to ensure complete reaction. After the reaction was complete, excess sodium hydride was quenched with excess anhydrous ethanol to precipitate fullerene disalt. The precipitate was collected by centrifugation and washed three times with anhydrous ethanol. The precipitate was then dissolved in ultrapure water and dialyzed using a dialysis bag with a molecular weight cutoff of 3500. After lyophilization, the water-soluble fullerene derivative TMF(C) was obtained. 70 [C(COOH)2]4, C 70 The samples were analyzed using the COOH group; and the water-soluble fullerene derivative TMF samples were characterized by particle size, 1H NMR and infrared spectroscopy.

[0063] Characterization and determination results of fullerene water-soluble derivative TMF samples are shown in the figure. Figure 1 .

[0064] 2. Synthesis and characterization of alanine-modified fullerene derivatives

[0065] Choose C 7050 mg of fullerene solid powder and 3.6 g of β-alanine (CAS: 107-95-9) were added to a round-bottom flask, and 50 mL of 14% sodium hydroxide (CAS: 1310-73-2) solution was slowly added. After ultrasonic dispersion, the mixture was stirred vigorously at 80 °C for 4–8 h. After the reaction was complete, the reactants were precipitated with a large amount of anhydrous ethanol. The precipitate was obtained by centrifugation and washed 2–3 times with anhydrous ethanol. After washing, the product was dissolved in ultrapure water. The dissolved product was then dialyzed to remove salts using a dialysis bag with a molecular weight cutoff of 1000. After lyophilization, alanine-modified fullerene derivative (C) was obtained. 70 -Ala) samples; and alanine-modified fullerene derivative samples were characterized and analyzed using infrared spectroscopy and other methods.

[0066] The characterization and determination results of alanine-modified fullerene derivative samples are shown in the figure. Figure 2 .

[0067] 3. Fluorescent labeling synthesis of two fullerene derivative samples

[0068] (1) Synthesis of fullerene water-soluble derivative TMF samples with fluorescent labeling

[0069] Weigh 7 mg of the fullerene water-soluble derivative TMF sample obtained in step 1 and dissolve it in morpholine ethanesulfonic acid buffer (0.1 M, pH = 6.0). Add 11 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (CAS: 7084-11-9) and stir for 30 min. Then add 6.5 mg of N-hydroxysuccinimide (CAS: 6066-82-6) and continue stirring for 2 h. Afterward, adjust the pH of the reaction solution to between 7 and 8 using 0.1 M sodium hydroxide solution, and then add sulfo-Cyanine 5-amine dissolved in DMSO. 5 mg (CAS: 2230212-27-6) was added and stirred at room temperature in the dark for 48 hours. Then, the product was dialyzed using a dialysis bag with a molecular weight cutoff of 3500 to remove excess salt and other substances. The product was then separated using a G-25 dextran gel column. The first green solution obtained was the Cy5-labeled fullerene water-soluble derivative TMF (TMF-Cy5, Cy5-C). 70 The TMF-Cy5 sample was characterized and analyzed by UV absorption spectroscopy.

[0070] The characterization results of the TMF-Cy5 sample are shown in the figure. Figure 3 .

[0071] (2) Fluorescent labeling synthesis of alanine-modified fullerene derivative samples

[0072] 7 mg of the alanine-labeled fullerene derivative sample obtained in step 2 was dissolved in morpholine ethanesulfonic acid buffer (0.1 M, pH = 6.0), and 11 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (CAS: 7084-11-9) was added and stirred for 30 min. Then, 6.5 mg of N-hydroxysuccinimide (CAS: 6066-82-6) was added and stirring continued for 2 h. The pH of the reaction solution was then adjusted to between 7 and 8 using 0.1 M sodium hydroxide solution. Finally, 5 mg of sulfo-Cyanine5amine dissolved in DMSO was added, and the mixture was stirred at room temperature for 48 h. Afterward, the product was dialyzed using a dialysis bag with a molecular weight cutoff of 3500 to remove excess salt and other substances. The product was then separated using a G-25 dextran gel column. The first green solution obtained was the Cy5-labeled alanine-labeled fullerene derivative (Cy5-C). 70 -Ala) sample, and analyzed Cy5-C by UV absorption spectroscopy. 70 - Ala samples were characterized and analyzed.

[0073] Cy5-C 70 The characterization results of the Ala sample are shown below. Figure 4 .

[0074] Example 2: Evaluation of blood-brain barrier crossing after intravenous and nasal administration

[0075] Intravenous administration (IV) and intranasal administration (IN) are common routes of drug delivery to the brain. This study investigated the brain distribution of these two administration methods by administering drugs with the same fluorescence intensity. The specific procedures are as follows:

[0076] The TMF-Cy5 sample prepared in step 3-(1) of Example 1 was dissolved in PBS to obtain TMF-Cy5 solutions with concentrations of 0.3 mg / mL and 1.5 mg / mL, respectively. Then, 12 healthy male mice (C57BL / 6, approximately 24g each) were randomly divided into 3 groups: control group, IV administration group, and IN administration group. The control group mice were treated by intravenous injection of 200 μL PBS. The IV administration group mice were administered 200 μL of 0.3 mg / mL TMF-Cy5 solution via intravenous injection. The IN administration group mice were administered 40 μL of 1.5 mg / mL TMF-Cy5 solution via nasal drip in four divided doses, with 10 μL administered alternately to the left and right nostrils each time. After 1.5 h of administration, IVIS imaging (Ex / Em: 640 / 680) was performed on the brains of the mice in each group.

[0077] The brains of mice that had undergone in vivo IVIS imaging were dissected. Two mice that had not undergone cardiac perfusion were euthanized and their brains removed. The other two mice underwent cardiac perfusion: the anesthetized mice were fixed to the operating table with their abdomens facing upwards. An abdominal incision was made, extending along the lateral abdominal wall towards the head to expose the heart in the thoracic cavity. The right atrial appendage was then cut open, and a 15mL syringe needle was inserted into the left atrium, leaving the needle in the left ventricle. A syringe containing 15mL of physiological saline was gently injected to replace the mouse's blood throughout its body until the fluid flowing from the right atrial appendage was clear and transparent physiological saline, thus completing the perfusion.

[0078] IVIS imaging results of the brains of mice in each group are shown in the figure. Figure 5 .

[0079] The results showed that intravenous (IV) administration resulted in a much higher drug distribution in the brain than intranasal (IN) administration, and the brain still showed a high drug distribution even after cardiac perfusion with saline.

[0080] Example 3: TMF-Cy5 can reside in the brain for a long time.

[0081] Thirty healthy male C57BL / 6 mice, weighing approximately 24g, had their head hair removed beforehand with depilatory cream. They were then randomly divided into five groups: control, TMF-Cy5-IN, TMF-Cy5-IV, sulfo-Cyanine5amine-IN, and sulfo-Cyanine5amine-IN. The amine-IV (Cy5-IV) administration groups were divided into several groups. The control group received 200 μL of PBS intravenously. The TMF-Cy5-IN group received 40 μL of TMF-Cy5 solution (1.5 mg / mL) via nasal instillation in four divided doses. The TMF-Cy5-IV group received 200 μL of TMF-Cy5 solution (0.3 mg / mL) intravenously. The Cy5-IN group received 40 μL of sulfo-Cyanine5 solution with the same Cy5 fluorescence intensity concentration as the 1.5 mg / mL TMF-Cy5 solution via nasal instillation. The Cy5-IV group mice were administered 200 μL of sulfo-Cyanine5amine solution with the same Cy5 fluorescence intensity concentration as 0.3 mg / mL TMF-Cy5 solution via intravenous injection. Subsequently, IVIS imaging was performed at 1, 3, 6, and 24 h after simultaneous administration to observe the fluorescence distribution of Cy5 in the head. Brain tissue was dissected after cardiac perfusion at 3 h and 24 h for IVIS imaging.

[0082] The results of in vivo imaging and brain tissue fluorescence imaging of each group of mice are shown in the figure. Figure 6 .

[0083] The results showed that both TMF-Cy5 and Cy5 could enter the bloodstream and were distributed in the brain after both intravenous (IV) and intranasal (IN) administration. However, Cy5 had a significantly shorter residence time in the blood than TMF-Cy5. Excluding errors caused by the breaking of chemical bonds in Cy5, the fluorescence of Cy5 in the brain was TMF-Cy5. Among the mice in each group, TMF-Cy5 had the largest brain residence amount after intravenous (IV) administration and was slowly metabolized over time.

[0084] The above results demonstrate that intravenous (IV) administration of TMF-Cy5 is a method of administration that can reach the brain and is effective for a long period of time.

[0085] Example 4: Carboxyl-modified fullerenes have higher brain penetration efficiency than alanine-modified fullerenes.

[0086] Thirty healthy male C57BL / 6 mice, weighing approximately 24g, had their head hair removed with depilatory cream beforehand. They were then randomly divided into five groups: a control group, a TMF-Cy5 treatment group, and a Cy5-C... 70 -COOH) and Cy5-C 70 -Ala treatment group (Cy5-C 70 -Ala), where the control group mice were treated by intravenous injection of 200 μL PBS; Cy5-C 70 Mice in the -COOH group were administered 200 μL of 1.5 mg / mL TMF-Cy5 solution intravenously; Cy5-C 70 The Ala group mice were administered 200 μL of Cy5-C with the same fluorescence intensity as the TMF-Cy5 solution via intravenous injection. 70 -Ala solution; administered once daily for 7 consecutive days (7d), during which the fluorescence intensity in the brains of mice in each group was continuously monitored, and the fluorescence intensity in the brains was observed by confocal imaging using frozen sections.

[0087] The results of intracranial fluorescence intensity monitoring in mice of each group after 7 days of continuous administration are shown in the figure. Figure 7 .

[0088] The results show that: with Cy5-C 70 -COOH, Cy5-C 70 With continuous administration of Ala, the accumulation of fullerene drugs in the mouse brain increased, including carboxyl-modified fullerene drugs (Cy5-C). 70 -COOH) is more effective than alanine-modified fullerene drugs (Cy5-C) with the same fluorescence intensity. 70-Ala) has a higher amount of drug residing in the brain.

[0089] Example 5: Carboxyl-modified fullerenes are more effective in treating cerebral hemorrhage than unmodified oral fullerenes.

[0090] 1. Construction of a mouse model of cerebral hemorrhage

[0091] To establish a stable mouse model of intracerebral hemorrhage (ICH), C57BL / 6 mice (male, approximately 24g) were anesthetized via autologous blood injection. Their heads were then fixed using a stereotaxic apparatus. After shaving the hair on the top of their heads and disinfecting the area, a 1cm incision was made along the midline of the scalp. The anterior fontanelle was identified as the origin, and a mark was made 2.3mm and 0.5mm anterior to the origin. An injection hole with a diameter of approximately 1mm was drilled, and 50μL of fresh autologous blood was collected via the femoral vein. Immediately after injection, a 36G needle (purchased from WPI) was used to slowly inject the blood into the mouse brain tissue at a depth of 3.7mm. The needle was withdrawn slowly upwards after a 20-minute pause to prevent backflow of blood. Thus, the mouse model of intracerebral hemorrhage (ICH) was successfully established, and ICH model mice were obtained.

[0092] After successfully establishing a mouse model of brain hemorrhage, magnetic resonance imaging (MRI) was chosen to characterize the hematoma absorption. First, an appropriate characterization method was selected for testing. The test results showed that SWI (Susceptibility-Weighted Imaging) could more clearly display the hematoma site in the mouse ICH model compared to T2W (T2-weighted imaging). Therefore, a combination of T2W and SWI was used to characterize the hematoma volume in the ICH model mice.

[0093] 2. Intravenous injection of C 70 -COOH and oral C 60 Comparison of treatment effects for cerebral hemorrhage

[0094] To verify the results of step 1 of Example 1, the water-soluble fullerene derivative TMF(C) was prepared. 70 The therapeutic effect of the (-COOH) sample in ICH model mice was investigated by administering it intravenously to ICH model mice and comparing it with oral C 60 The effects were compared, and the hematoma volume was calculated using MRI-SWI sequences and Slicer software after model construction in step 1. The ICH model mice were then divided into the ICH group (ICH), the TMF treatment group (ICH+TMF), and the oral C group based on the hematoma volume. 60 Treatment group (ICH+C)60 Mice in the ICH group were treated with intravenous injection of 200 μL of PBS; mice in the TMF treatment group were treated with intravenous injection of 40 mg / kg / day of TMF; and mice were given oral C 60 The treatment group mice were administered medication daily by feeding them a solution containing 0.4% C. 60 The mice were fed a specific diet; in addition, a control group and a sham group were set up. The control group mice were treated with intravenous injection of 200 μL PBS; the sham group mice were treated in the same way as the ICH group mice, except that they did not receive an injection of 50 μL of fresh autologous blood (i.e., the skin was cut, marked, and drilled during the model mouse construction process, but no blood was injected); all the mice in the above groups were given intravenous injection / oral administration 24 hours after modeling, and the hematoma volume was characterized by MRI-SWI sequence at 7 and 14 days of treatment. The fall time of the rotarod fatigue test was measured for each group of mice on day 14.

[0095] Intravenous injection of C 70 -COOH and oral C 60 A comparison of the treatment effects of cerebral hemorrhage can be found in [the table]. Figure 8 .

[0096] The results showed that the fullerene water-soluble derivative TMF(C) obtained using step 1 of Example 1 was effective. 70 During a 7-14 day treatment cycle of intravenous injection of COOH samples, it significantly promoted hematoma absorption and increased the hematoma absorption rate, and its therapeutic effect was comparable to that of oral C. 60 (ICH+C 60 The group showed a significant improvement; furthermore, compared with the ICH group and the sham group, the use of C 70 -COOH, C 60 Both treatments significantly increased the duration of the mice's time on the roller in the roller drop test, indicating that intravenous injection of C... 70 -COOH or oral C 60 Both can promote the recovery of balance and endurance in mice and treat nerve damage. Meanwhile, intravenous injection of C... 70 -COOH has a significantly better therapeutic effect than oral C 60 .

[0097] Example 6: Carboxyl-modified fullerenes may promote hematoma absorption by regulating lysosomal phagocytosis-related functions.

[0098] Brain tissue from the hemorrhage side of mice 14 days after cerebral hemorrhage in Example 5 (tissue sources included ICH group mice, control group mice and TMF treatment group mice) was subjected to proteomics detection, and proteins with significant expression differences (|log2FC|>1.2, P<0.05, considered to be significantly different) were subjected to KEGG analysis.

[0099] The results of KEGG analysis of brain tissue from the hemorrhage side in mice 14 days after cerebral hemorrhage are shown below. Figure 9 .

[0100] The results showed that, compared with the control group mice, the expression of common ICH pathways such as apoptosis and necrocytosis was upregulated in the ICH group mice, further demonstrating the realism and reliability of the model in simulating ICH; and after intravenous injection of C 70 Following COOH treatment, the "Phagosome" signaling pathway showed significant changes in upregulated genes among mice in the TMF treatment group. This pathway is a major regulatory pathway for the digestion of phagocytic contents by phagocytes. Among these pathways, microglia / macrophages play the most important role in hematoma absorption and are also the cell type with the most phagosomes. Therefore, it is inferred that C... 70 -COOH may promote hematoma absorption by regulating phagosome-related signaling pathways within phagocytes.

[0101] Example 7: Carboxyl-modified fullerenes promote the digestive function of phagocytes

[0102] 1. Preparation and labeling of red blood cells

[0103] Fresh red blood cells were extracted from healthy mice in the control group of Example 5 and incubated at 56°C for 7 min to destroy the enzyme activity on the surface of the red blood cells. Then, the red blood cells were co-incubated with DIO cell membrane dye (green fluorescence) at 37°C for 30 min to label the red blood cell membrane. The red blood cells were then washed with PBS 2-3 times to remove unbound dye and obtain green fluorescent labeled red blood cells.

[0104] 2. Co-incubation of green fluorescently labeled red blood cells with BV-2 cells

[0105] Mouse microglia BV-2 cells (purchased from Fenghui Biotechnology) were used at a rate of 2 × 10⁻⁶. 5Cells were seeded in 6-well plates, and then the green fluorescently labeled red blood cells obtained in step 1 were added to the 6-well plates in a ratio of 100:1 (green fluorescently labeled red blood cells: BV-2 cells) to ensure that the red blood cells were evenly distributed around the BV-2 cells. The plates were then incubated at 37°C in a 5% CO2 incubator for 3 hours to allow the BV-2 cells to fully engulf the green fluorescently labeled red blood cells, thus obtaining a cell mixture.

[0106] 3. C 70 -COOH treatment of BV-2 cells

[0107] Remove the supernatant from the cell mixture in step 2, and wash the cells 2-3 times with PBS to remove unphagocytosed red blood cells; then add PBS at concentrations of 0 μg / mL (control), 12.5 μg / mL, 25 μg / mL, 50 μg / mL, and 100 μg / mL, respectively. This is in contrast to the fullerene water-soluble derivative TMF (C) obtained in step 1 of Example 1. 70 The cells were incubated with a (-COOH) solution at 37°C in a 5% CO2 incubator for 5 hours. After incubation, BV-2 cells in each group were digested with trypsin and washed 2-3 times with PBS. The cell suspension was collected, and the culture medium and C were removed by centrifugation. 70 -COOH, and finally the fluorescence intensity in BV-2 cells of each group was detected by flow cytometry. Since red blood cells are labeled with DIO dye as green fluorescence, the decrease in fluorescence intensity indicates that the red blood cells have been digested by BV-2 cells.

[0108] The fluorescence intensity measurements of BV-2 cells in each group are shown below. Figure 10 .

[0109] The results show that: with C 70 As the concentration of COOH increased, the fluorescence intensity in BV-2 cells gradually decreased after incubation.

[0110] The above results indicate that: C 70 -COOH can promote the digestion of phagocytosed red blood cells.

[0111] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0112] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. Use of fullerenes and fullerenes derivatives for the preparation of a medicament, characterized in that, The drug is used for preventing, alleviating, assisting treatment or treating intracranial hemorrhagic diseases.

2. Use according to claim 1, characterized in that, The fullerene is a hollow fullerene. Optionally, the structure general formula of the hollow fullerene is C 2m wherein 30≤m≤60; Preferably, the structure general formula of the hollow fullerene is C 2m wherein 30≤m≤45.

3. Use according to claim 1, characterized in that, The fullerene derivative includes one or more of carboxyl-modified fullerene, alanine-modified fullerene, amino-modified fullerene, alkyl-modified fullerene, sulfonic acid-modified fullerene and metal fullerene.

4. Use according to claim 3, characterized in that, The fullerene derivative is carboxyl-modified fullerene, the number of addition of the carboxyl-modified fullerene is 1-4, and the number of carboxyl is 1-8.

5. Use according to claim 3, characterized in that, The fullerene derivative is a metallofullerene, the metallofullerene having a general structure of one or more of M@C 2n , M2@C 2n , MA@C 2n , M3N@C 2n , M2C2@C 2n , M2S@C 2n , M2O@C 2n , and M x A 3-x N@C 2n . Wherein, @ indicates that an atom is embedded inside the carbon cage of the fullerene, M and A are metal elements, and the M and A are each selected from any one of scandium, yttrium and lanthanide metal elements, 30≤n≤60, and 0≤x≤3.

6. Use according to claim 1, characterized in that, The intracranial hemorrhagic disease includes one or more of primary cerebral hemorrhage, traumatic cerebral hemorrhage, tumor cerebral hemorrhage, hemorrhagic cerebral stroke and amyloid cerebral angiopathy.

7. Use according to claim 1, characterized in that, The drug also has at least one of the following purposes: Crossing the blood-brain barrier; Promoting microglial cells to process or digest red blood cells; Promoting the absorption of hematoma in the brain; Regulating the Phagosome signal pathway of phagocytes to promote hematoma absorption.

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