CN116421739A

CN116421739A - Gold clusters, compositions and methods for treating cerebral stroke

Info

Publication number: CN116421739A
Application number: CN202110938143.7A
Authority: CN
Inventors: 孙涛垒
Original assignee: Shenzhen Profound View Pharma Tech Co Ltd
Current assignee: Shenzhen Profound View Pharma Tech Co Ltd
Priority date: 2020-11-27
Filing date: 2021-08-16
Publication date: 2023-07-14

Abstract

Ligand-bound gold clusters and compositions containing ligand-bound gold clusters are useful in the treatment of stroke and in the preparation of medicaments for the treatment of stroke. A method for treating cerebral apoplexy.

Description

Gold clusters, compositions and methods for treating cerebral stroke

Technical Field

The present invention relates to the field of brain disease treatment, and in particular to ligand-bound gold clusters (AuCs), compositions comprising ligand-bound gold clusters, and methods of using the ligand-bound gold clusters and compositions to prepare a medicament for treating stroke and using the ligand-bound gold clusters and compositions to treat stroke.

Background

Cerebral stroke occurs when a blood vessel is blocked by a blood clot or suffers from rupture. Cerebral stroke is of three types, namely cerebral hemorrhagic stroke, cerebral ischemic stroke and Transient Ischemic Attacks (TIA).

Cerebral hemorrhagic stroke is caused by rupture of blood vessels and prevention of blood flow to the brain. Common symptoms include sudden weakness, paralysis of any part of the body, inability to speak, vomiting, difficulty walking, coma, loss of consciousness, stiffness of the neck, and dizziness. No specific medicine exists at present.

Cerebral ischemic stroke is one of the most common diseases in humans and is the leading cause of death and disability. Cerebral ischemic stroke accounts for about 87% of all strokes. Cerebral ischemic stroke is caused by blockage of blood clots, plaques and the like in arteries supplying blood to the brain, and the blockage occurs in the neck or the skull, so that the blood flow and oxygen of the brain are reduced, and brain cells are damaged or dead. Brain damage may be permanent if blood circulation is not quickly restored.

The specific symptoms of cerebral ischemic stroke depend on which region of the brain is affected. Common symptoms of most ischemic strokes include vision problems, weakness of limbs or paralysis, dizziness and dizziness, confusion, loss of coordination ability, and one-sided sagging. Once symptoms appear, it is critical that the treatment be received as soon as possible, so that permanent damage is unlikely to occur.

Cerebral ischemic stroke has limited therapeutic means. The primary therapeutic agents currently in clinical use, such as tissue plasminogen activator (tPA), act by breaking down thrombus; however, tPA must be administered intravenously within 4 half hours after the onset of stroke to be effective. However, tPA can lead to bleeding, and thus cannot be treated with tPA if the patient has a history of hemorrhagic stroke, cerebral hemorrhage, recent major surgery, or head injury. Long-term treatment includes aspirin or anticoagulants to prevent further thrombosis.

Amani et al disclose that nanoparticles ox26@gnps formed by combining OX26-PEG with the surface of 25nm colloidal gold nanoparticles can significantly increase infarcted brain tissue, and that naked GNPs and pegylated GNPs have no effect on infarcted volume; the results show that: ox26@gnps are not suitable for the treatment of cerebral ischemic stroke.

Zheng et al disclose that 20nmAu-NPs increase cell viability, alleviate neuronal apoptosis and oxidative stress, and improve mitochondrial respiration in its OGD/R injured rat model. Zheng et al, however, also demonstrated that 5nm gold nanoparticles have opposite effects and cannot be used for the treatment of ischemic stroke.

TIA is caused by temporary thrombosis. Common symptoms include weakness, numbness or paralysis of one side of the body, blurred or confused speech, blindness and dizziness, and no specific drugs are currently available.

Treatment of stroke must be timely to avoid or alleviate neurological dysfunction or death in patients caused by injury or death of brain nerve cells due to cerebral ischemia or cerebral hemorrhage. Cerebral hemorrhagic stroke and TIA have no specific therapeutic drugs; the main drugs for treating cerebral ischemic stroke, such as tPA, must be decided to use the drugs for treating cerebral ischemic stroke after a cerebral ischemic stroke is diagnosed in a cerebral apoplexy patient instead of hemorrhagic stroke because of bleeding; this diagnostic procedure takes up valuable time for timely treatment of cerebral ischemic stroke.

There remains a need for better strategies and medicaments for treating stroke, particularly medicaments that have therapeutic effects on both cerebral hemorrhagic and cerebral ischemic strokes. However, since the causes of cerebral hemorrhagic and cerebral ischemic stroke are essentially different, the development of all drugs in the literature is either directed to cerebral hemorrhagic or cerebral ischemic stroke, and therefore, the development of a drug that can treat both cerebral hemorrhagic and cerebral ischemic stroke is an unforeseen challenge or can be said to be a forbidden zone in the industry.

Disclosure of Invention

The invention provides the use of ligand-bound gold clusters in the treatment of stroke in a patient, a method of treating stroke in a patient using ligand-bound gold clusters, and the use of ligand-bound gold clusters in the manufacture of a medicament for treating stroke in a patient.

Some embodiments of the invention utilize ligand-bound gold clusters to treat stroke in a patient; wherein the ligand-bound gold cluster comprises a gold core, and a ligand bound to the gold core; wherein cerebral stroke includes cerebral ischemic stroke and Transient Ischemic Attacks (TIA).

In some embodiments of this therapeutic use, the gold core has a diameter of 0.5-3nm. In some embodiments, the gold core has a diameter of 0.5-2.6nm.

In some embodiments of this therapeutic use, the ligand is one selected from the group consisting of L-cysteine and derivatives thereof, D-cysteine and derivatives thereof, cysteine-containing oligopeptides and derivatives thereof, and other thiol-containing compounds.

In some embodiments of this therapeutic use, the L-cysteine and derivatives thereof are selected from the group consisting of L-cysteine, N-isobutyryl-L-cysteine (L-NIBC) and N-acetyl-L-cysteine (L-NAC), and the D-cysteine and derivatives thereof are selected from the group consisting of D-cysteine, N-isobutyryl-D-cysteine (D-NIBC) and N-acetyl-D-cysteine (D-NAC).

In some embodiments of this therapeutic use, the cysteine-containing oligopeptides and derivatives thereof are cysteine-containing dipeptides, wherein the cysteine-containing dipeptides are selected from the group consisting of L (D) -cysteine-L (D) -arginine dipeptide (CR), L (D) -arginine-L (D) -cysteine dipeptide (RC), L (D) -histidine-L (D) -cysteine dipeptide (HC), and L (D) -cysteine-L (D) -histidine dipeptide (CH).

In some embodiments of this therapeutic use, the cysteine-containing oligopeptides and derivatives thereof are cysteine-containing tripeptides, wherein the cysteine-containing tripeptides are selected from the group consisting of glycine-L (D) -cysteine-L (D) -arginine tripeptide (GCR), L (D) -proline-L (D) -cysteine-L (D) -arginine tripeptide (PCR), L (D) -lysine-L (D) -cysteine-L (D) -proline tripeptide (KCP) and L (D) -Glutathione (GSH).

In some embodiments of this therapeutic use, the cysteine-containing oligopeptides and derivatives thereof are cysteine-containing tetrapeptides, wherein the cysteine-containing tetrapeptides are selected from the group consisting of glycine-L (D) -serine-L (D) -cysteine-L (D) -arginine tetrapeptides (GSCR) and glycine-L (D) -cysteine-L (D) -serine-L (D) -arginine tetrapeptides (GCSR).

In some embodiments of this therapeutic use, the cysteine-containing oligopeptide and derivatives thereof are cysteine-containing pentapeptides, wherein the cysteine-containing pentapeptides are selected from the group consisting of cysteine-aspartic acid-glutamic acid-valine-aspartic acid (CDEVD) and aspartic acid-glutamic acid-valine-aspartic acid-cysteine (devcd).

In some embodiments of this therapeutic use, the other thiol-containing compound is selected from the group consisting of 1- [ (2S) -2-methyl-3-thiol-1-oxopropyl ] -L (D) -proline, thioglycolic acid, mercaptoethanol, thiophenol, D-3-mercaptovaline, N- (2-mercaptopropionyl) -glycine, dodecyl mercaptan, 2-aminoethanethiol, 3-mercaptopropionic acid, and 4-mercaptobenzoic acid.

Some embodiments of the invention use a ligand-bound gold cluster to prepare a medicament for treating stroke in a subject, wherein the ligand-bound gold cluster comprises a gold core and a ligand bound to the gold core; wherein cerebral stroke includes cerebral ischemic stroke and Transient Ischemic Attacks (TIA).

In some embodiments of this preparation use, the gold core has a diameter of 0.5-3nm. In some embodiments, the gold core has a diameter of 0.5-2.6nm.

In some embodiments of this preparation use, the ligand is one selected from the group consisting of L-cysteine and derivatives thereof, D-cysteine and derivatives thereof, cysteine-containing oligopeptides and derivatives thereof, and other thiol-containing compounds.

In some embodiments of this preparation use, the L-cysteine and derivatives thereof are selected from the group consisting of L-cysteine, N-isobutyryl-L-cysteine (L-NIBC) and N-acetyl-L-cysteine (L-NAC), and the D-cysteine and derivatives thereof are selected from the group consisting of D-cysteine, N-isobutyryl-D-cysteine (D-NIBC) and N-acetyl-D-cysteine (D-NAC).

In some embodiments of this preparation use, the cysteine-containing oligopeptides and derivatives thereof are cysteine-containing dipeptides, wherein the cysteine-containing dipeptides are selected from the group consisting of L (D) -cysteine-L (D) -arginine dipeptide (CR), L (D) -arginine-L (D) -cysteine dipeptide (RC), L (D) -histidine-L (D) -cysteine dipeptide (HC), and L (D) -cysteine-L (D) -histidine dipeptide (CH).

In some embodiments of this preparation use, the cysteine-containing oligopeptides and derivatives thereof are cysteine-containing tripeptides, wherein the cysteine-containing tripeptides are selected from the group consisting of glycine-L (D) -cysteine-L (D) -arginine tripeptide (GCR), L (D) -proline-L (D) -cysteine-L (D) -arginine tripeptide (PCR), L (D) -lysine-L (D) -cysteine-L (D) -proline tripeptide (KCP) and L (D) -Glutathione (GSH).

In some embodiments of this preparation use, the cysteine-containing oligopeptides and derivatives thereof are cysteine-containing tetrapeptides, wherein the cysteine-containing tetrapeptides are selected from the group consisting of glycine-L (D) -serine-L (D) -cysteine-L (D) -arginine tetrapeptides (GSCR) and glycine-L (D) -cysteine-L (D) -serine-L (D) -arginine tetrapeptides (GCSR).

In some embodiments of this preparation use, the cysteine-containing oligopeptide and derivatives thereof are cysteine-containing pentapeptides, wherein the cysteine-containing pentapeptides are selected from the group consisting of cysteine-aspartic acid-glutamic acid-valine-aspartic acid (CDEVD) and aspartic acid-glutamic acid-valine-aspartic acid-cysteine (devc).

In some embodiments of this preparation use, the other thiol-containing compound is selected from the group consisting of 1- [ (2S) -2-methyl-3-thiol-1-oxopropyl ] -L (D) -proline, thioglycolic acid, mercaptoethanol, thiophenol, D-3-mercaptovaline, N- (2-mercaptopropionyl) -glycine, dodecyl mercaptan, 2-aminoethanethiol, 3-mercaptopropionic acid, and 4-mercaptobenzoic acid.

The objects and advantages of the present invention will become apparent from the following detailed description of the preferred embodiments, taken in conjunction with the accompanying drawings.

Drawings

Preferred embodiments according to the present invention will now be described with reference to the accompanying drawings, wherein like reference numerals refer to like elements.

FIG. 1 shows ultraviolet-visible (UV) spectra, transmission Electron Microscope (TEM) images, and particle size distribution diagrams (); (a) shows UV spectra of 3.6 nm L-NIBC-AuNPs; (B) displaying TEM images of 3.6 nm L-NIBC-AuNPs; (C) shows a particle size distribution profile of 3.6 nm L-NIBC-AuNPs; (D) shows UV spectra of 6.0 nm L-NIBC-AuNPs; (E) shows TEM images of 6.0 nm L-NIBC-AuNPs; (F) shows a particle size distribution profile of 6.0 nm L-NIBC-AuNPs; (G) shows UV spectra of 10.1 nm L-NIBC-AuNPs; (H) shows TEM images of 10.1 nm L-NIBC-AuNPs; (I) shows a particle size distribution profile of 10.1 nm L-NIBC-AuNPs; (J) UV spectra showing 18.2 nm L-NIBC-AuNPs; (K) displaying TEM images of 18.2 nm L-NIBC-AuNPs; (L) shows a particle size distribution plot of 18.2 nm L-NIBC-AuNPs.

FIG. 2 shows Ultraviolet Visible (UV) spectra, TEM images, and particle size distribution diagrams of ligand L-NIBC-bound gold clusters (L-NIBC-AuCs) having different particle sizes; (a) shows UV spectra of 1.1 nm L-NIBC-AuCs; (B) displaying TEM images of 1.1 nm L-NIBC-AuCs; (C) shows a particle size distribution profile of 1.1 nm L-NIBC-AuCs; (D) shows UV spectra of 1.8 nm L-NIBC-AuCs; (E) shows TEM images of 1.8 nm L-NIBC-AuCs; (F) shows a particle size distribution profile of 1.8 nm L-NIBC-AuCs; (G) shows UV spectra of 2.6 nm L-NIBC-AuCs; (H) a TEM image showing 2.6 nm L-NIBC-AuCs; (I) shows a particle size distribution profile of 2.6 nm L-NIBC-AuCs.

FIG. 3 shows the IR spectra of L-NIBC-AuCs at 1.1 nm, 1.8 nm, 2.6 nm.

FIG. 4 shows UV, IR, TEM and particle size distribution diagrams of ligand CR-binding gold clusters (CR-AuCs); (a) shows the UV spectrum of CR-AuCs; (B) shows the infrared spectrum of CR-AuCs; (C) displaying TEM images of CR-AuCs; (D) shows the particle size distribution of CR-AuCs.

FIG. 5 shows UV, IR, TEM and particle size distribution diagrams of ligand RC-bonded gold clusters (RC-AuCs); (a) shows the UV spectrum of RC-AuCs; (B) shows the infrared spectrum of RC-AuCs; (C) displaying TEM images of RC-AuCs; (D) shows the particle size distribution of RC-AuCs.

FIG. 6 shows UV, IR, TEM and particle size distribution diagrams of ligand 1- [ (2S) -2-methyl-3-thiol-1-oxopropyl ] -L-proline (i.e., cap) bound gold clusters (Cap-AuCs); (a) shows UV spectra of Cap-AuCs; (B) shows the infrared spectrum of Cap-AuCs; (C) displaying TEM images of Cap-AuCs; (D) shows the particle size distribution profile of Cap-AuCs.

FIG. 7 shows UV, IR, TEM and particle size distribution diagrams of ligand GSH-bound gold clusters (GSH-AuCs); (a) shows UV spectra of GSH-AuCs; (B) shows the infrared spectrum of GSH-AuCs; (C) displaying TEM images of GSH-AuCs; (D) shows particle size distribution of GSH-AuCs.

FIG. 8 shows UV, infrared, TEM and particle size distribution diagrams of ligand D-NIBC-bound gold clusters (D-NIBC-AuCs); (a) shows UV spectra of D-NIBC-AuCs; (B) shows the infrared spectrum of D-NIBC-AuCs; (C) displaying a TEM image of D-NIBC-AuCs; (D) shows the particle size distribution of D-NIBC-AuCs.

FIG. 9 shows UV, IR, TEM and particle size distribution diagrams of ligand L-cysteine-bound gold clusters (L-Cys-AuCs); (a) shows UV spectra of L-Cys-AuCs; (B) shows the infrared spectrum of L-Cys-AuCs; (C) displaying a TEM image of L-Cys-AuCs; (D) shows the particle size distribution of L-Cys-AuCs.

FIG. 10 shows UV, IR, TEM and particle size distribution diagrams of ligand 2-aminoethanethiol-bound gold clusters (CSH-AuCs); (a) shows the UV spectrum of CSH-AuCs; (B) shows the infrared spectrum of CSH-AuCs; (C) displaying TEM images of CSH-AuCs; (D) shows the particle size distribution of CSH-AuCs.

FIG. 11 shows UV, IR, TEM and particle size distribution diagrams of ligand 3-mercaptopropionic acid-bound gold clusters (MPA-AuCs); (a) shows UV spectra of MPA-AuCs; (B) shows the infrared spectrum of MPA-AuCs; (C) displaying TEM images of MPA-AuCs; (D) shows the particle size distribution of MPA-AuCs.

FIG. 12 shows UV, IR, TEM and particle size distribution diagrams of ligand 4-mercaptobenzoic acid-bound gold clusters (p-MBA-AuCs); (a) shows UV spectra of p-MBA-AuCs; (B) shows the infrared spectrum of p-MBA-AuCs; (C) displaying TEM images of p-MBA-AuCs; (D) shows the particle size distribution of p-MBA-AuCs.

FIG. 13 shows UV, TEM and particle size distribution diagrams of ligand 4-cysteine-aspartic acid-glutamic acid-valine-aspartic acid (CDEVD) -bound gold clusters (CDEVD-AuCs); (a) shows the UV spectrum of CDEVD-AuCs; (B) displaying TEM images of CDEVD-AuCs; (C) shows the particle size distribution of CDEVD-AuCs.

FIG. 14 shows UV, TEM and particle size distribution diagrams of ligand 4-aspartic acid-glutamic acid-valine-aspartic acid-cysteine (DEVDC) -bound gold clusters (DEVDC-AuCs); (a) shows UV spectra of devc-AuCs; (B) displaying TEM images of the devc-AuCs; (C) shows the particle size distribution profile of DEVDC-AuCs.

Fig. 15 shows the neurobehavioral scores of the rats in each group (in the histogram of each time point, the sham operation group (blank), the model control group, the A1 low dose group, the A1 high dose group, the A2 low dose group, the A2 high dose group, the A3 low dose group, the A3 high dose group, the A4 low dose group, the A4 high dose group, the B1 low dose group, the B1 high dose group, the B2 low dose group, and the B2 high dose group, respectively, from left to right).

Fig. 16 shows the percentage of cerebral infarct size in each group (in bar graph, from left to right, sham operation group (blank), model control group, A1 low dose group, A1 high dose group, A2 low dose group, A2 high dose group, A3 low dose group, A3 high dose group, A4 low dose group, A4 high dose group, B1 low dose group, B1 high dose group, B2 low dose group, B2 high dose group), respectively.

FIG. 17 is a representative graph of TTC staining of brain tissue of MCAO rats after administration of a cash-cluster drug and administration of gold nanoparticles, wherein (1) the sham surgery group; (2) model control group; (3) A1 low dose group; (4) A1 high dose group; (5) B1 low dose group; (6) B1 high dose group.

FIG. 18 presents results of mNSS neurological function scoring experiments at various times after molding is completed, wherein a normal control group (NC); SHAM surgery group (SHAM); model control group (IVH); drug a low dose administration group (AL); drug a high dose administration group (AH); drug B low dose administration group (BL); drug B high dose administration group (BH); (a) mNSS neurological function score after 12 hours of modeling; (B) mNSS neurological function score after 1 day of modeling; (C) mNSS neurological function score after 2 days of modeling; (D) mNSS neurological function score after 4 days of modeling; (E) mNSS neurological function score after 7 days of modeling; * **: p <0.001 relative to the IVH group.

FIG. 19 shows Evansan's levels in supernatant of rat brain homogenates of each group in an Evansan staining test for Blood Brain Barrier (BBB) permeability; wherein, normal control group (NC); SHAM surgery group (SHAM); model control group (IVH); drug a low dose administration group (AL); drug a high dose administration group (AH). * : p <0.05 relative to IVH group rats; * *: p <0.01 relative to IVH group rats; * **: p <0.001 relative to IVH group rats.

FIG. 20 presents the water content of rat brain tissue measured by dry and wet weight method; wherein, normal control group (NC); SHAM surgery group (SHAM); model control group (IVH); drug a low dose administration group (AL); drug a high dose administration group (AH). * : p <0.05 relative to IVH group rats; * *: p <0.01 relative to IVH group rats; * **: p <0.001 relative to IVH group rats.

Fig. 21 presents representative H & E stained pictures of brain tissue of different groups of rats. (a) NC group; (B) a SHAM group; (C) group IVH; (D) group AL; (E) AH group.

Fig. 22 presents representative pictures of iNOS immunofluorescent staining of brain tissue of different groups of rats. (a) NC group; (B) a SHAM group; (C) group IVH; (D) group AL; (E) AH group.

FIG. 23 shows MMP9 protein expression in brain tissue of rats of each group in WB experiments, wherein the normal control group (NC); SHAM surgery group (SHAM); model control group (IVH); drug a low dose administration group (AL); drug a high dose administration group (AH). (a) MMP9 protein strips from 6 parallel samples; (B) Relative protein expression of MMP9 protein (GAPDH as reference). * : p <0.05 relative to IVH group rats.

FIG. 24 presents the MDA levels of rat brain tissue for each group, wherein the normal control group (NC); SHAM surgery group (SHAM); model control group (IVH); drug a low dose administration group (AL); drug a high dose administration group (AH). * **: p <0.001 relative to the IVH group; * *: p <0.01 relative to the IVH group.

FIG. 25 presents the SOD levels of brain tissue of rats in each group, wherein the normal control group (NC); SHAM surgery group (SHAM); model control group (IVH); drug a low dose administration group (AL); drug a high dose administration group (AH). * **: p <0.001 relative to the IVH group.

FIG. 26 presents representative photographs of ultra-thin sections of brain tissue from each group; (a) NC group; (B) a SHAM group; (C) group IVH; (D) group AL; (E) AH group.

Detailed Description

The invention may be understood more readily by reference to the following detailed description of some embodiments of the invention.

Where publications are cited, the disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.

As used herein, "administration" refers to oral ("po") administration, suppository administration, topical contact administration, intravenous administration ("iv"), intraperitoneal administration ("ip"), intramuscular administration ("im"), intralesional administration, intrahippocampal administration, lateral ventricular administration, nasal administration or subcutaneous administration ("sc") or implantation of a sustained release device such as a micro osmotic pump or erodible implant into a subject. Administration may be by any route including parenteral and transmucosal (e.g., oral, nasal, vaginal, rectal or transdermal). Parenteral administration includes, for example, intravenous, intramuscular, arteriole, intradermal, subcutaneous, intraperitoneal, ventricular and intracranial. Other modes of delivery include, but are not limited to, use of liposomal formulations, intravenous infusion, transdermal patches, and the like.

The term "systemic administration" refers to a method of administering a compound or composition to a mammal such that the compound or composition is delivered to a site in the body, including the target site of drug action, through the circulatory system. Systemic administration includes, but is not limited to, oral, intranasal, rectal, and parenteral administration (i.e., administration by other routes than the digestive tract, such as intramuscular, intravenous, arterial, transdermal, and subcutaneous), provided that, as used herein, systemic administration does not include administration directly to the brain region by other means than the circulatory system, such as intrathecal injection and intracranial administration.

As used herein, the term "treating" refers to delaying the occurrence or slowing/reversing the progression of, or alleviating/preventing a disease or condition for which the term is applicable.

The terms "patient," "subject" or "individual" interchangeably refer to mammals, such as humans or non-human mammals, including primates (e.g., macaques, apes, gibbons), domestic mammals (e.g., felines, canines), agricultural mammals (e.g., cows, sheep, pigs, horses), and laboratory mammals or rodents (e.g., rats, mice, lagomorphs, hamsters, guinea pigs).

Ligand-bound gold clusters (AuCs) are a special form of gold that exists between Jin Yuanzi and gold nanoparticles. The gold core size of the ligand-bound gold clusters is less than 3nm, consisting of only a few to hundreds of gold atoms, resulting in the collapse of the face-centered cubic stacked structure of gold nanoparticles. Thus, unlike gold nanoparticles, which have continuous or quasi-continuous energy levels, gold clusters exhibit a molecularly discrete electronic structure with different HOMO-LUMO gaps. This results in the surface plasmon resonance effect possessed by conventional gold nanoparticles and the corresponding disappearance of the plasmon resonance absorption band (520±20 nm) in the uv-visible spectrum.

The present invention provides ligand-bound gold clusters.

In some embodiments, the ligand-bound gold cluster comprises a ligand and a gold core, wherein the ligand is bound to the gold core. Binding of the ligand to the gold core means that the ligand forms a complex with the gold core that is stable in solution by covalent bonds, hydrogen bonds, electrostatic forces, hydrophobic forces, van der waals forces, and the like. In some embodiments, the gold core has a diameter of 0.5-3nm. In some embodiments, the diameter of the gold core is in the range of 0.5-2.6 nm.

In some embodiments, the ligand of the ligand-bound gold cluster is a thiol-containing compound or oligopeptide. In some embodiments, the ligand is bonded to the gold core through an Au-S bond to form a ligand-bonded gold cluster.

In some embodiments, the ligand is, but is not limited to, L-cysteine, D-cysteine, or a cysteine derivative. In some embodiments, the cysteine derivative is N-isobutyryl-L-cysteine (L-NIBC), N-isobutyryl-D-cysteine (D-NIBC), N-acetyl-L-cysteine (L-NAC), or N-acetyl-D-cysteine (D-NAC).

In some embodiments, the ligand is, but is not limited to, cysteine-containing oligopeptides and derivatives thereof.

In some embodiments, the cysteine-containing oligopeptide is a cysteine-containing dipeptide. In some embodiments, the cysteine-containing dipeptide is L (D) -cysteine-L (D) -arginine dipeptide (CR), L (D) -arginine-L (D) -cysteine dipeptide (RC), or L (D) -cysteine-L (D) -histidine dipeptide (CH).

In some embodiments, the cysteine-containing oligopeptide is a cysteine-containing tripeptide. In some embodiments, the cysteine-containing tripeptide is glycine-L (D) -cysteine-L (D) -arginine tripeptide (GCR), L (D) -proline-L (D) -cysteine-L (D) -arginine tripeptide (PCR), or L (D) -Glutathione (GSH). In some embodiments, the cysteine-containing oligopeptide is a cysteine-containing tetrapeptide. In some embodiments, the cysteine-containing tetrapeptide is glycine-L (D) -serine-L (D) -cysteine-L (D) -arginine tetrapeptide (GSCR) or glycine-L (D) -cysteine-L (D) -serine-L (D) -arginine tetrapeptide (GCSR). In some embodiments, the cysteine-containing oligopeptide is a cysteine-containing pentapeptide. In some embodiments, the cysteine-containing pentapeptide is cysteine-aspartic acid-glutamic acid-valine-aspartic acid (CDEVD), or aspartic acid-glutamic acid-valine-aspartic acid-cysteine (devcd).

In some embodiments, the ligand is a thiol-containing compound. In some embodiments, the thiol-containing compound is 1- [ (2S) -2-methyl-3-thiol-1-oxopropyl ] -L (D) -proline, thioglycolic acid, mercaptoethanol, thiophenol, D-3-mercaptovaline, dodecyl thiol, 2-aminoethanethiol, 3-mercaptopropionic acid, or 4-mercaptobenzoic acid.

The present invention provides a pharmaceutical composition for treating cerebral ischemic stroke in a subject, wherein the cerebral stroke includes cerebral ischemic stroke, and Transient Ischemic Attack (TIA). In some embodiments, the subject is a human. In some embodiments, the subject is a pet animal, such as a dog.

In some embodiments, the pharmaceutical composition comprises a ligand-bound gold cluster as disclosed above and a pharmaceutically acceptable excipient. In some embodiments, the excipient is phosphate buffered solution or physiological saline.

The present invention provides the use of the ligand-bound gold clusters disclosed above for the manufacture of a medicament for treating stroke in a subject, wherein stroke comprises cerebral ischemic stroke, and Transient Ischemic Attacks (TIA).

The present invention provides methods for treating stroke in a subject using the ligand-bound gold clusters disclosed above, or using the ligand-bound gold clusters disclosed above, wherein stroke comprises cerebral ischemic stroke, and Transient Ischemic Attacks (TIA). In some embodiments, the method of treatment comprises administering to the subject a pharmaceutically effective amount of the ligand-bound gold cluster. Pharmaceutically effective amounts can be determined by routine in vivo studies. In some embodiments, the pharmaceutically effective amount of ligand-bound gold cluster is 0.001 mg/kg/day, 0.005 mg/kg/day, 0.01 mg/kg/day, 0.05 mg/kg/day, 0.1 mg/kg/day, 0.05 mg/kg/day, 1 mg/kg/day, 2 mg/kg/day, 3 mg/kg/day, 4 mg/kg/day, 5 mg/kg/day, 6 mg/kg/day, 7 mg/kg/day, 8 mg/kg/day, 9 mg/kg/day, 10 mg/kg/day, 15 mg/kg/day, 20 mg/kg/day, 30 mg/kg/day, 40 mg/kg/day, 50 mg/kg/day, 60 mg/kg/day, 70 mg/kg/day, 80 mg/kg/day, 90 mg/kg/day, or 100 mg/kg/day.

The following examples are provided only to illustrate the principles of the present invention; they are in no way intended to limit the scope of the invention.

Examples

Example 1 preparation of ligand-bound gold clusters

1.1 HAuCl ₄ Dissolving in methanol, water, ethanol, n-propanol or ethyl acetate to obtain solution A, wherein HAuCl ₄ The concentration of (2) is 0.01-0.03M;

1.2, dissolving the ligand in a solvent to obtain a solution B, wherein the concentration of the ligand is 0.01-0.18M; ligands include, but are not limited to, L-cysteine, D-cysteine and other cysteine derivatives such as N-isobutyryl-L-cysteine (L-NIBC), N-isobutyryl-D-cysteine (D-NIBC), N-acetyl-L-cysteine (L-NAC) and N-acetyl-D-cysteine (D-NAC), cysteine-containing oligopeptides and derivatives thereof including, but not limited to, dipeptides, tripeptides, tetrapeptides, pentapeptides and other cysteine-containing peptides such as L (D) -cysteine-L (D) -arginine dipeptide (CR), L (D) -arginine-L (D) -cysteine dipeptide (RC), L (D) -cysteine L (D) -histidine (CH), glycine-L (D) -cysteine-L (D) -arginine tripeptide (GCR), L (D) -proline-L (D) -cysteine-L (D) -arginine-L (GSL) -arginine-L (D) -arginine dipeptide (GSL (D) -arginine-L (GSL) -arginine (D) -arginine (GSL-L) L (GSL), glycine-L (D) -cysteine-L (D) -serine-L (D) -arginine tetrapeptide (GCSR), cysteine-aspartic acid-glutamic acid-valine-aspartic acid pentapeptide (CDEVD) and aspartic acid-glutamic acid-valine-aspartic acid-cysteine pentapeptide (devcd c), and other thiol-containing compounds such as one or more of 1- [ (2S) -2-methyl-3-thiol-1-oxopropyl ] -L (D) -proline, thioglycolic acid, mercaptoethanol, thiophenol, D-3-mercaptovaline, dodecyl mercaptan, 2-aminoethanethiol, 3-mercaptopropionic acid, and 4-mercaptobenzoic acid; the solvent is one or more of methanol, ethyl acetate, water, ethanol, n-propanol, pentane, formic acid, acetic acid, diethyl ether, acetone, anisole, 1-propanol, 2-propanol, 1-butanol, 2-butanol, amyl alcohol, butyl acetate, tributylmethyl ether, isopropyl acetate, dimethyl sulfoxide, ethyl formate, isobutyl acetate, methyl acetate, 2-methyl-1-propanol and propyl acetate;

1.3 mixing solution A and solution B to HAuCl ₄ The molar ratio to the ligand is 1: (0.01-100), stirring in ice bath for 0.1-48h, adding 0.025-0.8M NaBH ₄ And (3) continuously stirring water, ethanol or methanol solution in ice water bath for reaction for 0.1-12h. NaBH ₄ The molar ratio to the ligand is 1: (0.01-100);

1.4 after the reaction, centrifuging the reaction solution for 10-100min at 8000-17500r/min by using an MWCO 3K-30K ultrafiltration tube to obtain ligand-bonded gold cluster precipitates with different average particle diameters. The pores of the filtration membranes of the ultrafiltration tubes of different MWCO directly determine the size of the gold clusters that can be bound by the ligands of the membrane.

This step may optionally be omitted;

1.5 dissolving the ligand-bound gold cluster precipitates with different average particle diameters obtained in the step (1.4) in water, placing in a dialysis bag, and dialyzing in water at room temperature for 1-7 days;

1.6 lyophilization of ligand-bound gold clusters after dialysis for 12-24h to give powdered or flocculant material, i.e., ligand-bound gold clusters.

As detected, the particle size of the powdered or flocculant substance obtained by the aforementioned method is less than 3nm (typically distributed between 0.5 and 2.6 nm). There is no distinct absorption peak at 520 nm. The obtained powder or floe was determined to be ligand-bound gold clusters.

Example 2 preparation and identification of gold clusters bound with different ligands

2.1 preparation of L-NIBC-conjugated gold clusters, i.e., L-NIBC-AuCs

The preparation and identification of ligand L-NIBC-bound gold clusters is described in detail using ligand L-NIBC as an example.

2.1.1 weighing 1.00g of HAuCl ₄ This was dissolved in 100mL of methanol to give 0.03M solution A;

2.1.2 weighing 0.57g L-NIBC, dissolving it in 100mL glacial acetic acid (acetic acid) to obtain 0.03M solution B;

2.1.3 1mL of solution A is taken and mixed with 0.5mL, 1mL, 2mL, 3mL, 4mL, or 5mL of solution B (i.e., HAuCl) ₄ The molar ratio with L-NIBC is 1:0.5, 1: 1. 1: 2. 1: 3. 1: 4. 1: 5) The reaction was stirred in an ice bath for 2h, and when the solution changed from bright yellow to colorless, 1.1 mL freshly prepared 0.03M (11.3 mg NaBH was weighed ₄ And dissolved in 10mL ethanol) NaBH ₄ The reaction was continued for 30 minutes after the solution turned dark brown with ethanol solution and quenched by the addition of 10mL of acetone.

2.1.4, carrying out gradient centrifugation on the reaction solution to obtain L-NIBC-AuCs powder with different particle sizes. The specific method comprises the following steps: after the completion of the reaction, the reaction solution was transferred to a ultrafiltration tube having a volume of 50mL and an MWCO of 30K, centrifuged at 10000r/min for 20min, and the retentate in the inner tube was dissolved in ultrapure water. A powder having a particle size of about 2.6nm was obtained. The mixed solution in the outer tube was then transferred to a ultrafiltration tube having a volume of 50mL and an MWCO of 10K and centrifuged at 13,000r/min for 30 minutes. The retentate in the inner tube was dissolved in ultrapure water to obtain a powder having a particle size of about 1.8 nm. The mixed solution in the outer tube was then transferred to a ultrafiltration tube of volume 50mL and MWCO 3K and centrifuged at 17,500r/min for 40 minutes. The retentate in the inner tube was dissolved in ultrapure water to obtain a powder having a particle size of about 1.1 nm.

2.1.5 precipitation of three powders with different particle diameters obtained by gradient centrifugation, respectively removing the solvent, drying the crude product with N2, dissolving in 5mL of ultrapure water, placing into a dialysis bag (MWCO is 3 kDa), placing into 2L of ultrapure water, changing water every other day, dialyzing for 7 days, and freeze-drying for later use.

2.2 identification of L-NIBC-AuCs

The powder (L-NIBC-AuCs) obtained above was subjected to an identification experiment. Meanwhile, ligand L-NIBC modified gold nanoparticles (L-NIBC-AuNP) were used as controls. Preparation of gold nanoparticles with L-NIBC ligand reference (W.Yan, L.Xu, C.Xu, W.Ma, H.Kuang, L.Wang and N.A.Kotov, journal of the American Chemical Society 2012,134,15114; X.Yuan, B.Zhang, Z.Luo, Q.Yao, D.T.Leong, N.Yan and J.Xie, angewandte Chemie International Edition 2014,53,4623).

2.2.1 morphology by Transmission Electron Microscopy (TEM)

Test powders (L-NIBC-AuCs sample and L-NIBC-AuNPs sample) were dissolved in ultrapure water to 2mg/L as a sample, and then test samples were prepared by the hanging drop method. More specifically, 5. Mu.L of the sample was dropped on an ultrathin carbon film, naturally volatilized until the water drop disappeared, and then the morphology of the sample was observed by JEM-2100F STEM/EDS field emission high-resolution TEM.

Four TEM images of L-NIBC-AuNP are shown in panels B, E, H and K of FIG. 1; three TEM images of L-NIBC-AuCs are shown in FIG. 2, panel B, panel E and panel H.

The image in fig. 2 shows that each L-NIBC-AuCs sample has a uniform particle size and good dispersibility, and that the average diameters (refer to the diameter of the gold core) of the L-NIBC-AuCs are 1.1nm, 1.8nm and 2.6nm, respectively, in complete agreement with the results in panels C, F and I of fig. 2. In contrast, the L-NIBC-AuNPs sample had a larger particle size. Their average diameters (referred to as the diameter of the gold nuclei) were 3.6nm, 6.0nm, 10.1nm and 18.2nm, respectively, which are in good agreement with the results in panels C, F, I and L of FIG. 1.

2.2.2 Ultraviolet (UV) -visible (vis) absorption Spectrum

The test powders (L-NIBC-AuCs sample and L-NIBC-AuNPs sample) were dissolved in ultrapure water to a concentration of 10mg.multidot.L-1, and the UV-vis absorption spectrum was measured at room temperature. The scanning range is 190-1100nm, the sample cell is a standard quartz cuvette, the optical path is 1cm, and the reference cell is filled with ultrapure water.

UV-vis absorption spectra of four L-NIBC-AuNP samples with different sizes are shown in panels a, D, G and J of fig. 1, and statistical distributions of particle sizes are shown in panels C, F, I and L of fig. 1; UV-vis absorption spectra of three L-NIBC-AuCs samples with different sizes are shown in panels a, D and G of fig. 2, and statistical distributions of particle sizes are shown in panels C, F and I of fig. 2.

FIG. 1 shows that L-NIBC-AuNP has an absorption peak at about 520nm due to the surface plasmon effect. The position of the absorption peak is related to the particle size. When the particle size is 3.6nm, a UV absorption peak appears at 516 nm; when the particle diameter is 6.0nm, a UV absorption peak appears at 517 nm; the UV absorption peak appears at 520nm when the particle size is 10.1nm, and at 523nm when the particle size is 18.2 nm. None of the four samples had any absorption peak above 560 nm.

FIG. 2 shows that in the ultraviolet absorption spectrum of the L-NIBC-bonded gold cluster samples with three different particle sizes, the surface plasmon absorption peak at 520nm disappears, two distinct absorption peaks appear above 560nm, and the positions of the absorption peaks are slightly different from the particle sizes of the gold clusters. This is because gold clusters exhibit molecular-like properties due to collapse of the face-centered cubic structure, which results in discontinuity of state density of gold clusters, energy level splitting, disappearance of plasmon resonance effect, and appearance of new absorption peaks in the long-wave direction. It can be concluded that all three of the powder samples of different particle sizes obtained above were ligand-bound gold clusters.

2.2.3 Fourier transform Infrared Spectroscopy

The infrared spectrum is measured by using solid powder high vacuum total reflection mode on a VERTEX80V type Fourier transform infrared spectrometer manufactured by Bruker, and the scanning range is 4000-400cm ^-1 Scanned 64 times. Taking an L-NIBC-bonded gold cluster sample as an example, the test sample is three L-NIBC-bonded gold cluster dry powders with different particle sizes, and the control sample is pure L-NIBC powder. The results are shown in FIG. 3.

FIG. 3 is an infrared spectrum of L-NIBC-bonded gold clusters with different particle sizes. S-H stretching vibrations between 2500-2600cm-1 were completely eliminated for L-NIBC-bound gold clusters of different particle sizes compared to pure L-NIBC (bottom curve), while other characteristic peaks for L-NIBC were still observed. The successful binding of the L-NIBC molecule to the gold cluster surface through gold-sulfur bonds was demonstrated. The figure also shows that the infrared spectrum of ligand-bound gold clusters is independent of its size.

Gold clusters bound by other ligands were prepared in a similar manner as described above except that the solvent of solution B, HAuCl ₄ Feed ratio to ligand, reaction time and NaBH added ₄ Slightly adjusted, for example: when L-cysteine, D-cysteine, N-isobutyryl-L-cysteine (L-NIBC) or N-isobutyryl-D-cysteine (D-NIBC) is used as the ligand, acetic acid is selected as the solvent; when using dipeptide CR, dipeptide RC or 1- [ (2S) -2-methyl-3-mercapto-1-oxopropyl ]-when L-proline is the ligand, water is chosen as solvent, and so on; other steps are similar and therefore no further details are provided herein.

The present invention prepares and obtains a series of ligand-bound gold clusters by the above-described method. The parameters of the ligand and the preparation method are shown in table 1.

TABLE 1 preparation parameters of gold clusters bound by different ligands according to the invention

The samples listed in table 1 were confirmed by the foregoing method. The characteristics of 11 different ligand-bound gold clusters are shown in FIG. 4 (CR-AuCs), FIG. 5 (RC-AuCs), FIG. 6 (Cap-AuCs) (Cap represents 1- [ (2S) -2-methyl-3-mercapto-1-oxopropyl ] -L-proline), FIG. 7 (GSH-AuCs), FIG. 8 (D-NIBC-AuCs), FIG. 9 (L-Cys-AuCs), FIG. 10 (CSH-AuCs), FIG. 11 (MPA-AuCs), FIG. 12 (p-MBA-Au Cs), FIG. 13 (CDEVD-AuCs), FIG. 14 (DEVDC-AuCs). Fig. 4-12 show UV spectra (a), infrared spectra (B), TEM images (C) and particle size distribution (D). Fig. 13-14 show UV spectra (a), TEM images (B) and particle size distribution (C).

The results show that the diameter of the gold clusters bound by the different ligands obtained in Table 1 is less than 3nm. The uv spectrum also shows the disappearance of peaks at 520±20nm and the appearance of absorption peaks at other positions, which vary with ligand and particle size and structure, and there are cases where no special absorption peaks occur, mainly because a mixture of a plurality of gold clusters different in size and structure or some special gold clusters make the positions of absorption peaks out of the conventional uv-visible absorption spectrum measurement range. At the same time, the fourier transform infrared spectrum also shows the disappearance of the thiol infrared absorption peaks of the ligand (between the dashed lines of panels B in fig. 4-8), while the other infrared characteristic peaks remain, indicating that the ligand molecules have successfully bound to the gold atoms to form ligand-bound gold clusters, indicating that the present invention successfully achieved ligand-bound gold clusters as set forth in table 1.

Example 3 cerebral ischemic stroke animal model test

3.1 test sample

Gold clusters:

a1: the size of the L-NIBC ligand-combined gold cluster is 0.5-3.0nm;

a2: gold clusters bound by L-Cys ligand with a size of 0.5-3.0nm;

a3: gold clusters bound by L-NAC ligand, with a size of 0.5-3.0nm;

a4: the size of the DEVDC ligand-bound gold clusters is 0.5-3.0nm.

Gold nanoparticles:

b1: L-NIBC modified gold nanoparticles with a size of 6.1+ -1.5 nm;

b2: L-NAC modified gold nanoparticles with a size of 9.0.+ -. 2.4nm.

The preparation methods of all test samples were slightly modified with reference to the previous methods; their quality was identified by the method described above.

3.2 test methods

3.2.1 establishment of rat cerebral middle artery infarction model and administration of test substance

Male SPF-grade Sprague Dawley (SD) rats (220-260 g) were purchased from Shanghai Laike laboratory animals Co. The environment was adapted for 7 days prior to the experiment. All rats were randomly divided into 14 groups (10 per group) of sham surgery, model control, low (2 mg/Kg rat weight) and high dose groups (10 mg/Kg rat weight) of gold cluster drugs A1, A2, A3 and A4, and low (2 mg/Kg rat weight) and high dose groups (10 mg/Kg rat weight) of gold nanoparticles B1 and B2, respectively. On the day of the experiment, rats were anesthetized with 10% chloral hydrate (350 mg/kg body weight), right common carotid artery, internal carotid artery and external carotid artery were exposed through a midline incision, a embolic wire was inserted through the External Carotid Artery (ECA), 18mm±0.5mm into the Internal Carotid Artery (ICA) until the blood supply in the MCA area was blocked, cerebral infarction occurred, and after 1.5h the embolic wire was withdrawn from the inlet end of ECA for reperfusion. Preoperative basal Cerebral Blood Flow (CBF) and post-embolic cerebral blood flow were measured separately using a blood flow meter. Animals with a sustained decrease in CBF (rCBF. Gtoreq.70%) were considered successful in modeling the rat middle cerebral artery infarct model (Middle cerebral artery occlusion, MCAO). The rats were re-perfused, and the study was then scored by intraperitoneal injection at 0h, 24h, 48h, 72h, neuro-behavioural at 0h, 24h, 48h, 72h, 96h, and the experiment was terminated at 96h post-operation, brain collection and TTC staining were performed after euthanasia, brain photographs were taken, and the percentage of cerebral infarct area was calculated.

3.2.2 neurobehavioral scoring

0 point: is not different from normal mice; 1, the method comprises the following steps: the right front claw is not stretched straight, and the head rotates to the opposite side; 2, the method comprises the following steps: walking in open field for discontinuous rotation; 3, the method comprises the following steps: walking in open field to make continuous turn; 4, unconscious walking and paralysis to one side; 5, the method comprises the following steps: death.

3.2.3 infarct size (TTC staining)

The rat was euthanized by carbon dioxide inhalation, the brain was taken and placed in a brain tank for coronal section (2 mm), 2% TTC was stained at normal temperature in the dark, and infarct size was analyzed with imageJ after photographing. Percentage of infarct area (%) = (contralateral hemisphere area- (ipsilateral hemisphere area-infarct area))/contralateral hemisphere area x 100%.

3.2.4 statistical analysis

Statistical analysis was performed using Graph Pad Prism software 7.0 (CA, US). Data expressed as mean ± standard error statistical analysis the Dunnett test was used for one-way analysis of variance. P <0.05 is statistically significant for the differences.

3.3 experimental results

3.3.1 cerebral ischemia area cerebral blood flow

The cerebral blood flow of the rat cerebral ischemia area is reduced by more than 70 percent (reduction cerebral blood flow, rCBF is more than or equal to 70 percent), and the MACO model is considered to be successful, and the rCBF of the rest groups except the sham operation group is more than 70 percent, and the average rCBF is about 80 percent, which indicates that the model is successful in all the groups.

3.3.2 effects of each test drug on rat neuro-behaviours

Fig. 15 shows the neurobehavioral scores of the rats in each group (in the histogram of each time point, the sham operation group (blank), the model control group, the A1 low dose group, the A1 high dose group, the A2 low dose group, the A2 high dose group, the A3 low dose group, the A3 high dose group, the A4 low dose group, the A4 high dose group, the B1 low dose group, the B1 high dose group, the B2 low dose group, and the B2 high dose group, respectively, from left to right). The nerve behaviors of rats in the sham operation group are normal, and the behavioristic score is 0; model control rats showed severe behavioural functional deficits at 0h, 24h, 48, 72h and 96h post-surgery (# relative to sham P < 0.001). The neurobehavioral scores of the A1, A2, A3, A4 low dose and high dose groups were not significantly improved 24 hours post-surgery compared to the model control group. At 48h post-surgery, the neurobehavioral scores of the A1, A2, A3 and A4 low and high dose groups all began to drop, but were not statistically different (relative to model control group P > 0.05). At 72h post-surgery, the neurobehavioral scores of the four drugs were further reduced, with A1 low dose group, A1 high dose group and A2 high dose group exhibiting significant differences (P <0.05 relative to model control group). At 96h post-surgery, significant differences occurred in both the low and high dose groups for the four drugs (P <0.05 relative to the model control group). The results show that the four gold cluster medicaments can obviously improve the neurobehavioral functional defects caused by ischemic cerebral apoplexy, and the effect shows a certain dose dependence.

Compared with the model group, the administration of the gold nanoparticles B1, B2 in low and high dose groups did not significantly improve the neurobehavioral scores of MACO model rats 24h, 48 h, 72h and 96h after the operation, showing that the gold nanoparticles could not significantly improve the behavioral disorder caused by cerebral ischemic stroke.

3.3.3 Effect of each subject on MACO model rat cerebral infarction area

Fig. 16 shows the percentage of cerebral infarct size in each group (in bar graph, from left to right, sham operation group (blank), model control group, A1 low dose group, A1 high dose group, A2 low dose group, A2 high dose group, A3 low dose group, A3 high dose group, A4 low dose group, A4 high dose group, B1 low dose group, B1 high dose group, B2 low dose group, B2 high dose group), respectively. The brain tissue of the rats in the sham operation group is normal, no infarction occurs, and the infarct area is 0%. The model control group had a clear cerebral infarction with an infarct size of 44.7% ± 4.5% (relative to sham P <0.001, # # #). There was a significant decrease in the percentage of cerebral infarctions in the low and high dose groups of A1, A2, A3, A4 compared to the model control group, but none of the low dose groups showed significant differences, whereas the high dose group showed significant differences (P <0.05 relative to the model control group). Taking A1 as an example, the infarct size was reduced from 44.7±4.5% to 36.0±4.0% (relative to model control group P > 0.05) in the low dose group, and to 27.8±3.4% (relative to model control group P < 0.05).

Fig. 17 shows a representative graph of TTC staining of brain tissue of MCAO rats after administration of gold cluster drug and administration of gold nanoparticles, taking A1 and B1 drugs as examples, wherein the white area is a cerebral infarction area. In fig. 17, (1) a sham surgery set; (2) model control group; (3) A1 low dose group; (4) A1 high dose group; (5) B1 low dose group; (6) B1 high dose group. As can be seen from fig. 17, in the sham operated rats, no cerebral infarction occurred, while in the model control rats, no significant cerebral infarction occurred (white right portion), the cerebral infarction area was reduced (white right portion was reduced) by the administration of the A1 drug at a low dose, the cerebral infarction area was significantly reduced (white right portion was significantly reduced) by the administration of the A1 drug at a high dose, and the cerebral infarction area was hardly affected by the administration of the B1 drug at a low dose and the administration of the high dose (white right portion was not reduced at all). A2, A3 and A4 show a remarkable effect of reducing infarct size similar to that of A1, while B2 is similar to that of B1, and has no effect on reduction of infarct size.

Other ligand-bound gold clusters also have the same effect of treating cerebral ischemic stroke, with their effects differing. They will not be described in detail here.

Example 4 cerebral hemorrhagic stroke animal model test

4.1 materials and methods

4.1.1 test drugs

The medicine A is an L-NIBC modified gold cluster (L-NIBC-AuNCs), and the diameter of a gold core is in the range of 0.5-3.0 nm;

drug B: the diameter of the gold core is in the range of 0.5-3.0 nm.

4.1.2 animals and groupings

140 SD female rats, 8 weeks old, weight 190-220g; rats were housed adaptively for 7 days in an SPF environment and then randomized into 7 groups:

(1) Normal Control (NC) group 20, average body weight 214.0 ±5.1g;

(2) The SHAM (SHAM) group had an average body weight of 213.5+ -6.5 g;

(3) Model control (IVH) group 20, average body weight 214.7 + -6.1 g;

(4) Drug a low dose (4 mg/kg rat body weight) Administration (AL) group 20, average body weight 212.5±7.3g;

(5) Drug a high dose (10 mg/kg rat body weight) Administration (AH) group 20, average body weight 212.1±6.8g;

(6) Drug B low dose (4 mg/kg rat body weight) administration (BL) group 20, average body weight 213.2±6.3g;

(7) Drug B high dose (10 mg/kg rat body weight) administration (BH) group 20, average body weight 211.2.+ -. 7.1g.

There were no significant statistical differences in body weight between groups.

4.1.3, molding, dosing and testing methods:

rats were modeled using an intracerebroventricular hemorrhage model (intraventricular hemorrhage, IVH):

For IVH group and rats of each administration group, 2% of the non-barbital abdominal cavity is injected into anesthetized rats (50 mg/kg), the head skin is prepared after anesthesia, the prone position is fixed on a stereotactic instrument, the stereotactic instrument is regulated, the incisors of the rats are fixed on the incisors of the incisors, so that front and back fontanels are positioned on the same horizontal line, the earstems are regulated, the heads of the rats cannot move, the skin of the head skin is cut in the middle after disinfection, 3% of hydrogen peroxide is used for ablating periosteum, the front and back fontanels and coronary slits are exposed, 0.2mm is used for the front fontanel and the back fontanel, 3mm is used for the side of the midline, a small hole with the diameter of about 1mm is drilled by a dental drill, the brain tissue and the dura mater is not damaged, the tail is cleaned by warm water heating at 40 ℃, after congestion, 50 mu l of non-anticoagulated arterial blood is taken by a 1ml injector, the syringe is fixed on the stereotactic instrument, the microinjectors are needle is inserted to 6mm through holes, a two-time autologous arterial blood injection method is adopted, 10 mu l of the arterial blood is injected first, then stopped from the blood is injected into the skin, the skin is cut in the middle, the bone membrane is degraded, the bone membrane is exposed, the front fonned and back fonned, the front fonned, the fond is and coronary slit is left for 0mm, the wound is slowly, the wound is stopped for 4 mm, and the needle wound is completely and stopped after the needle is completely and is closed by 4 mm.

After the rats are awake, performing behavioral observation, and observing the modeling condition of the rats by adopting mNSS neural function scoring. The rats in the AL group and the AH group are administered 1 time a day, 2 days, 3 days, 4 days, 5 days, 6 days and 7 days by intraperitoneal injection, wherein the doses of the rats in the AL group and the AH group are respectively 4 mg/kg of body weight. NC group and SHAM group rats were injected with equal volumes of saline by intraperitoneal injection at the corresponding time points. All rats were evaluated for neurological function by scoring for neurological deficit mNSS at 12h,24h,3d,5d,7d time points after intraperitoneal injection, respectively.

4.1.4 detection of blood brain Barrier permeability by Evans blue dye method

The treated rats were given Evans Blue Stain (0.5%) by intravenous injection, and the eyes and skin of the rats appeared Blue. After 0.5-1 hour, the rats are sacrificed and brain tissues are harvested. The brain tissue was placed in a 1.5mL centrifuge tube, 1mL PBS was added, and the brain tissue was homogenized rapidly with a tissue homogenizer, and centrifuged. The supernatant was taken, and an equal amount of trichloroacetic acid was added thereto for incubation at 4 ℃. Centrifuging for 15min. The solution was taken and the absorbance at 620nm (OD value) was measured by a spectrophotometer. The OD values of standard evans blue of known different gradients were measured simultaneously and a standard curve was drawn. And calculating the Evansan content of the sample to be detected according to the standard curve.

4.1.5 brain tissue Water content test

Dry and wet weight method is adopted. The rat after ischemia reperfusion for 24 hours is subjected to excessive anesthesia, the brain is taken after head breakage, the olfactory bulb, cerebellum and low-level brainstem are removed, the left and right hemispheres of the brain are separated, the wet weight is immediately weighed, and then the rat is put into an electric oven at 110 ℃ for 24 hours to be baked, and then the dry weight of brain tissue is rapidly weighed. Brain water content (%) = (wet weight-dry weight)/wet weight×100% was calculated.

4.1.6, H & E staining

Rat brain tissue was removed and placed in an embedding cassette. Sequentially carrying out the steps of dehydration, transparency, wax dipping, embedding, slicing, baking and the like, and then carrying out H & E dyeing: taking out the slices from the oven, treating the slices twice with xylene for 15 minutes each time, then sequentially treating the slices with 100%, 95%, 80% and 70% ethanol for 5 minutes respectively, and then treating the slices with ultrapure water for 5 minutes each time to complete the dewaxing step; dyeing with hematoxylin dye solution for 5 minutes, and flushing with ultrapure water for 3 times; after washing for 10 minutes with tap water, the nuclei are turned blue, and are dyed with 0.5% of reddish dye liquor for 5 minutes, after washing for 5 times with tap water, the slices are dried in a 60 ℃ oven, and finally the slices are sealed with neutral resin. The observation and image acquisition were performed with an optical microscope at 200 times the field of view.

4.1.7 Immunofluorescence (IF) detection

Taking out rat brain tissue, placing into an embedding box, sequentially dehydrating, transparentizing, immersing in wax, embedding, and slicing paraffin, baking and the like. The slices were removed from the oven and subjected to dewaxing. Then, the antigen is repaired, cleaned, penetrated, treated by the primary antibody of iNOS, treated by the secondary antibody of the coat anti-Rabbit, stained by DAPI and the like. Finally, the steps of baking and sealing are carried out. Observations and image acquisitions were made with a fluorescence microscope at 200 x field of view.

4.1.8 Western-Blot (WB) assay

Pretreatment of protein samples: taking out frozen tissue from the refrigerator, weighing a proper amount of tissue, adding a lysis solution containing a protease inhibitor according to a ratio of 1:9, vibrating at 4 ℃ until the tissue blocks are all broken, putting the tissue blocks on ice for 20min, centrifuging at 12000rpm for 20min at 4 ℃, and taking the supernatant. Protein concentration was determined using BCA protein concentration assay kit. And (3) adjusting the protein concentration according to the concentration measurement result to ensure the consistency of the protein concentration among different groups, boiling at 95 ℃ for 5min, loading samples, and preserving the rest samples at-80 ℃.

The WB experimental operation was then performed as follows:

preparing an electrophoresis gel: after the glass plate is cleaned and wiped, the glass plate is fixed on a glue making device, and then the separation glue is prepared, the separation glue is poured into the gap of the glass plate to a proper height, and absolute ethyl alcohol is used for covering the separation glue until the glue is completely polymerized. The absolute ethanol was decanted, gently rinsed with double distilled water and blotted dry with filter paper. Then adding concentrated gel to proper height, and inserting comb teeth. And taking out the comb teeth after the concentrated glue is completely polymerized.

Loading electrophoresis: the treated samples were loaded in a total amount of 40 μg per well. Concentrating the gel with 80V of each gel, separating the gel with 120V, and performing electrophoresis with constant voltage power supply; judging the position of the target protein according to the relative positions of the molecular weight of the pre-dyed Marker and the molecular weight of the target protein, and stopping electrophoresis separation when the target protein is positioned at the optimal separation position of the lower 1/3 of the separation gel surface.

Transferring: and (3) putting the sheared PVDF film into methanol for soaking for 10 seconds, and then putting the PVDF film into a new film transfer liquid for later use. The gel was taken out, the target band was cut off according to Marker, washed with distilled water, and PVDF membrane and filter paper of the same size as the PAGE gel were cut and immersed in electrotransport buffer together. Sequentially placing the black plate, the fiber pad, the filter paper, the gel, the PVDF film, the filter paper, the fiber pad and the white plate, clamping the plates, and then placing the plates into a film transfer instrument, wherein one side of the black plate is compared with a black negative electrode. And filling electrotransfer liquid into the transfer film tank to start transferring film. The transfer was carried out at 4℃in an ice bath under conditions of 200mA, 90min (0.45 μm film) or 200mA, 60min (0.2 μm film).

Closing: the membrane was washed 3 times with PBST, and then the PVDF membrane was blocked with TBST blocking solution containing 5% milk, shaking at room temperature for 2h.

An antibody: the corresponding primary antibody was diluted with TBST containing 3% bsa, and PVDF membranes were immersed in the primary antibody incubation solution and incubated overnight at 4 ℃. Wherein MMP9 is as defined in 1:1000 dilution, iNOS at 1:2000 dilution. After the completion, TBST was sufficiently washed for 3 times, 10 min/time to wash out the excess primary antibody.

And (2) secondary antibody: the secondary antibody is marked by HRP diluted (1:5000) by a sealing solution, so that the PVDF film is soaked in the secondary antibody incubation solution, and the shaking table is incubated for 1h at room temperature. After the completion, the PVDF membrane was thoroughly washed with TBST 3 times for 10 min/time to wash out the excess secondary antibody.

Color development exposure: and uniformly mixing the enhancement solution in the ECL kit with the stable peroxidase solution according to the ratio of 1:1 to prepare working solution, dripping a proper amount of working solution on the PVDF film, and exposing by adopting a full-automatic chemiluminescence image analysis system.

Membrane elution and regeneration: after the end of exposure, the PVDF film was washed thoroughly with TBST 3 times for 5 min/time. Adding a proper amount of membrane regeneration liquid, soaking the PVDF membrane in the membrane regeneration liquid, and carrying out elution by a shaking table at room temperature for 20 min. After the completion, the PVDF membrane was thoroughly washed with TBST 3 times for 5 min/time to wash off the excess membrane regenerated liquid.

And (3) sealing again, incubating the internal reference, combining the secondary antibody, and exposing, wherein the specific flow is the same as that of the experimental process. Wherein both beta-tubulin and GAPDH are respectively as follows: diluting with 5000 a.

The exposure results were analyzed for gray values using Image J software.

4.1.9 detection of lipid peroxidation

Sample processing: adding PBS (mass volume ratio of 1:9) with corresponding volume into the tissue, fully homogenizing, incubating on ice for 10min, centrifuging at 4000rpm for 10min, and taking the supernatant to be tested.

The test was performed using Malondialdehyde (MDA) kit. Blank, standard, assay and control tubes were prepared for the instruction procedure. Absorbance (OD) values at 532nm were used. The MDA content in the supernatant to be tested in the tissue was calculated according to the following formula: MDA content (nmol/mL) = (measured OD value-control OD value)/(standard OD value-blank OD value) ×standard concentration (10 nmol/mL).

4.1.10 superoxide dismutase (SOD) index detection

The SOD kit is adopted for testing according to instructions. Calculated according to the following formula: SOD inhibition (%) = ((control well OD value-control blank well OD value) - (assay well OD value-assay blank well OD value))/(control well OD value-control blank well OD value) ×100%; SOD activity (U/mgprot) =sod inhibition rate ++50% x reaction dilution ++test sample protein concentration (mgprot/mL).

4.1.11 electron microscope detection

Tissue pieces were taken at about 1 cubic millimeter. After fixation, dehydration, embedding and solidification, slicing by an ultra-thin slicer with the thickness of 70nm. Then double-stained with 2% uranium acetate-lead citrate. The images were observed and photographed by transmission electron microscopy.

4.2 test results

FIG. 18 presents results of mNSS neurological function scoring experiments at various times after molding is completed, wherein a normal control group (NC); SHAM surgery group (SHAM); model control group (IVH); drug a low dose administration group (AL); drug a high dose administration group (AH); drug B low dose administration group (BL); drug B high dose administration group (BH).

As shown in panel a of fig. 18, the results showed that the average mNSS neurological function score was: normal Control (NC) group (0.0±0.0), SHAM operation (SHAM) group (0.0±0.0), model control (IVH) group (14.7±1.2), drug a low dose (AL) group (14.7±1.0), drug a high dose (AH) group (14.4±1.1), drug B low dose (BL) group (14.7±1.3), drug B high dose (BH) group (14.7±1.1), NC group and SHAM group have significant statistical differences (P <0.001, ×) compared to the IVH group, and the remaining groups have no significant statistical differences compared to the IVH group.

As shown in panel B of FIG. 18, the results showed that the average score of mNSS nerve function was found to be NC group (0.0.+ -. 0.0), SHAM group (0.0.+ -. 0.0), IVH group (14.3.+ -. 1.3), AL group (14.6.+ -. 1.1), AH group (14.3.+ -. 1.0), BL group (14.9.+ -. 0.9), BH group (14.3.+ -. 1.0), NC group and SHAM group were significantly statistically different from IVH group (P < 0.001), and the other groups were not significantly statistically different from IVH.

As shown in panel C of fig. 18, the results showed that the average score of mNSS neurological function was: NC (0.0±0.0), SHAM (0.0±0.0), IVH (11.0±0.9), AL (8.6±1.5), AH (7.7±1.7), BL (9.9±1.8), BH (10.2±1.9), NC, SHAM, AL and AH all have significant statistical differences (P <0.001,) compared to IVH. While BL and BH groups have slightly smaller average values than IVH groups, no statistical differences occur.

As shown in panel D of fig. 18, the results showed that the average score of mNSS neurological function was: NC group (0.0±0.0), SHAM group (0.0±0.0), IVH group (8.0±1.5), AL group (6.0±1.8), AH group (6.1±1.7), BL group (7.2±1.8), BH group (7.0±1.8), BL and BH groups were not significantly statistically different from IVH group, and the other groups were significantly statistically different from model group (P < 0.001).

As shown in panel E of fig. 18, the results showed that the average mNSS neurological function score was: NC group (0.0±0.0), SHAM group (0.0±0.0), IVH group (5.4±1.7), AL group (2.9±0.9), AH group (2.8±1.0), BL group (4.5±1.8), BH group (4.1±1.6), BL and BH groups have no significant statistical difference from the IVH group, and the other groups have significant statistical difference from the model group (P < 0.001). As shown in fig. 1E.

The results show that the mNSS nerve function score of IVH-model rats can be remarkably improved from the 2 nd day after model making, and the results show that the drug A is used for treating IVH-model rats and has excellent effects. However, drug B showed no significant difference (P > 0.05) in mNSS scores from day 2, although there was no significant difference relative to group IVH, indicating that drug B was not effective in treatment of IVH-model rats.

To further investigate the therapeutic effect of drug a on IVH-model rats, we developed the study from multiple angles.

FIG. 19 is a test result of Evanskian staining for Blood Brain Barrier (BBB) permeability. It can be seen that after staining with evans, the brain tissue of rats in group IVH had significantly higher levels of evans (P <0.001,/x) than in NC and SHAM groups. The brain tissue of the mice in both the low dose (AL) and high dose (AH) groups had significantly lower levels of evans than in the IVH group (P < 0.05; P < 0.01). This result demonstrates that IVH modeling results in a substantial increase in blood brain barrier permeability in rats, whereas both low and high doses of drug A significantly improve the increase in blood brain barrier permeability in rats resulting from IVH modeling.

Cerebral edema caused by IVH modeling is an important cause of increased intracranial pressure, cerebral herniation and death of animals. The rat brain edema condition can be evaluated by detecting the water content of the rat brain tissue by a dry-wet weight method. FIG. 20 is a graph showing the results of a dry and wet weight measurement of the water content of brain tissue of rats. By analysis, the brain tissue water content of the NC group rats and SHAM group rats is obviously lower than that of IVH model rats (P <0.001, X), and the brain tissue water content of the drug A low dose Administration (AL) group and the brain tissue water content of the high dose Administration (AH) group rats are also obviously lower than that of IVH group rats (P <0.05, X; P <0.01, X). These results demonstrate that both low and high doses of drug A significantly improve the condition of animal brain edema caused by IVH modeling, thereby improving the prognosis of the animal.

Fig. 21 is a representative picture of an H & E staining test. The results showed that NC group showed some gaps around blood vessels and cells (panel A of FIG. 21), SHAM group showed slight injury (panel B of FIG. 21), IVH group showed significant increase of gaps around blood vessels and cells of brain tissue, cell degeneration occurred, and cavitation, indicating severe brain injury (panel C of FIG. 21), and both low dose Administration (AL) and high dose Administration (AH) of drug A (panel D of FIG. 21) significantly reduced brain injury in IVH-molded rats.

injury-Inducible Nitric Oxide Synthase (iNOS) is expressed after brain injury. FIG. 22 is a typical picture of iNOS immunofluorescent staining of rat brain tissue. It can be seen that the IVH group (panel C of FIG. 22) had higher iNOS positive expression, and the remaining groups, including NC group (panel A of FIG. 22), SHAM group (panel B of FIG. 22), AL group (panel D of FIG. 22) and AH group (panel E of FIG. 22) were each reduced to a different extent than the IVH group, indicating that IVH modeling increased iNOS expression in rat brain tissue, while both low and high concentrations of drug A reduced iNOS expression and had a protective effect on brain injury.

Gelatinase B (MMP 9) is not or is underexpressed in normal brain tissue, but after IVH modeling causes brain injury, MMP9 plays an important role in angiogenic cerebral edema and other brain injury-related processes by disrupting the blood brain barrier. FIG. 23 shows MMP9 protein expression in brain tissue of each group of rats, wherein panel A of FIG. 23 shows MMP9 protein bands from 6 parallel samples obtained by WB assay, and panel B of FIG. 23 shows relative protein expression levels of MMP9 (with GAPDH as reference). It can be seen that the relative expression of MMP9 in brain tissue was significantly lower in NC, SHAM, AL and AH rats than in IVH rats, and that the differences were significant (P <0.05 relative to IVH). These results demonstrate that both low and high doses of drug A significantly reduced brain damage in model rats resulting in increased MMP9 protein expression, demonstrating the protective effect of drug A on brain damage.

Malondialdehyde (MDA) is a final product of peroxidation reaction of free radicals and cell membrane unsaturated fatty acid, is an important biomarker for lipid oxidative damage, can indirectly reflect the degree of tissue peroxidation damage, has the level closely related to the severity of clinical symptoms in cerebral stroke incidence, and has important clinical significance in cerebral stroke diagnosis, treatment and prognosis. FIG. 24 shows the expression level of MDA in brain tissue of each group of rats. It can be seen that MDA expression was significantly lower in both NC, SHAM, AL and AH groups than in IVH group, indicating that brain damage caused by IVH modeling resulted in a significant increase in brain MDA expression (both NC and SHAM groups were less than 0.001, P relative to IVH group), whereas both low and high concentration drug a administration significantly reduced MDA expression associated with brain damage (P was less than 0.01, P and 0.001, respectively, relative to IVH group).

Superoxide dismutase (SOD) is the most effective radical scavenging enzyme in the body, and SOD produced in the body can reflect the condition of radicals in the body. Studies have shown that SOD expression and activity in hemorrhagic stroke brain tissue are significantly lower than normal brain tissue. FIG. 25 shows the expression levels of SOD in brain tissue of rats in each group. The results showed that both NC, SHAM, AL and AH showed significantly higher SOD expression than the IVH group, indicating that brain damage due to IVH modeling caused a significant decrease in SOD expression in the brain (both NC and SHAM groups were less than 0.001, P relative to the IVH group), whereas both low and high doses of drug a significantly increased SOD expression in the brain (both P were less than 0.001, P relative to the IVH group).

FIG. 26 shows the results of transmission electron microscopy experiments on ultra-thin sections of brain tissue.

In NC group, glial cell morphology is normal or slightly irregular, and the nucleus (N) is irregularly oval; the endoplasmic reticulum (M) is in the form of a short circle or a short rod, is structurally intact, has slightly blurry internal ridges, and has slightly distended the Endoplasmic Reticulum (ER) (panel a of fig. 26).

In SHAM group, glial cell morphology is slightly irregular, and nucleus (N) is oval; the endoplasmic reticulum (M) is dumbbell-shaped, with blurred internal ridges, and the Endoplasmic Reticulum (ER) is slightly dilated (panel B of fig. 26).

In the IVH group, glial cells are irregularly shaped, and nuclei (N) are irregularly shaped; the cytoplasmic mitochondria (M) are rounded, markedly swollen, with a markedly distended Endoplasmic Reticulum (ER), seen with Autophagosomes (AP) (panel C of fig. 26). Indicating that IVH modeling results in severe damage to cells of brain tissue.

In group AL, glial cells were slightly irregular in morphology and nuclei (N) were oval; the endoplasmic mitochondria (M) were rounded or oval, slightly swollen, with partial internal ridge rupture, slightly distended Endoplasmic Reticulum (ER), and a small amount of lipofuscin (L) was seen (panel D of fig. 26). It is demonstrated that the administration of drug A at low doses can well improve or repair the cell damage of brain tissue caused by IVH modeling.

In the AH group, glial cell morphology is basically regular or slightly irregular, and the nucleus (N) is oval; the endoplasmic mitochondria (M) are oval or long rod-shaped, clear in internal ridges, intact in structure, slightly dilated in the Endoplasmic Reticulum (ER), and a small amount of lipofuscin (L) is partially visible (E panel of fig. 26). Demonstrating that high doses of drug A can improve or repair cell damage to brain tissue caused by IVH modeling, and brain tissue cells are indistinguishable from NC groups.

The results show that the gold cluster using L-NIBC as the ligand has unexpected treatment effect on hemorrhagic stroke, and can be used for developing medicaments for treating cerebral stroke with blood flow.

Industrial applicability

Ligand-bound gold clusters are useful in the treatment of stroke. They are suitable for industrial applications.

Reference to the literature

Amani H,Mostafavi E,Mahmoud Reza Alebouyeh MR,Arzaghi H,Akbarzadeh A, Pazoki-ToroudiH,Webster TJ.Would Colloidal Gold Nanocarriers Present An Effective Diagnosis Or Treatment For Ischemic StrokeInt J Nanomedicine.2019 Oct 7；14:8013-8031.

Zheng Y,Wu Y,Liu Y,Guo Z,Bai T,Zhou P,Wu J,Yang Q,Liu Z,Lu X.Intrinsic Effects of Gold Nanoparticles on Oxygen-Glucose Deprivation/Reperfusion Injury in Rat Cortical Neurons.Neurochem Res.2019 Jul；44(7):1549-156。

Claims

1. Use of a ligand-bound gold cluster in the treatment of stroke in a patient, wherein the ligand-bound gold cluster comprises:

a gold core; and

a ligand that binds to the gold core;

wherein the cerebral stroke includes cerebral ischemic stroke and transient ischemic attack TIA.

2. The therapeutic use according to claim 1, wherein the gold core has a diameter of 0.5-3nm.

3. The therapeutic use according to claim 1, wherein the gold core has a diameter of 0.5-2.6nm.

4. The therapeutic use according to claim 1, wherein the ligand is one selected from the group consisting of L-cysteine and derivatives thereof, D-cysteine and derivatives thereof, cysteine-containing oligopeptides and derivatives thereof and other thiol-containing compounds.

5. Therapeutic use according to claim 4, characterized in that said L-cysteine and derivatives thereof are selected from the group consisting of L-cysteine, N-isobutyryl-L-cysteine (L-NIBC) and N-acetyl-L-cysteine (L-NAC), said D-cysteine and derivatives thereof being selected from the group consisting of D-cysteine, N-isobutyryl-D-cysteine (D-NIBC) and N-acetyl-D-cysteine (D-NAC).

6. The therapeutic use according to claim 4, wherein the cysteine-containing oligopeptide and derivatives thereof are cysteine-containing dipeptides selected from the group consisting of L (D) -cysteine-L (D) -arginine dipeptide (CR), L (D) -arginine-L (D) -cysteine dipeptide (RC), L (D) -histidine-L (D) -cysteine dipeptide (HC) and L (D) -cysteine-L (D) -histidine dipeptide (CH).

7. The therapeutic use according to claim 4, wherein the cysteine-containing oligopeptide and derivatives thereof are cysteine-containing tripeptides selected from the group consisting of glycine-L (D) -cysteine-L (D) -arginine tripeptide (GCR), L (D) -proline-L (D) -cysteine-L (D) -arginine tripeptide (PCR), L (D) -lysine-L (D) -cysteine-L (D) -proline tripeptide (KCP) and L (D) -Glutathione (GSH).

8. The therapeutic use according to claim 4, wherein the cysteine-containing oligopeptide and derivatives thereof are cysteine-containing tetrapeptides selected from the group consisting of glycine-L (D) -serine-L (D) -cysteine-L (D) -arginine tetrapeptides (GSCR) and glycine-L (D) -cysteine-L (D) -serine-L (D) -arginine tetrapeptides (GCSR).

9. The therapeutic use according to claim 4, wherein the cysteine-containing oligopeptide and derivatives thereof are cysteine-containing pentapeptides selected from the group consisting of cysteine-aspartic acid-glutamic acid-valine-aspartic acid (CDEVD) and aspartic acid-glutamic acid-valine-aspartic acid-cysteine (devc).

10. The therapeutic use according to claim 4, wherein the other thiol-containing compound is selected from the group consisting of 1- [ (2S) -2-methyl-3-thiol-1-oxopropyl ] -L (D) -proline, thioglycolic acid, mercaptoethanol, thiophenol, D-3-mercaptovaline, N- (2-mercaptopropionyl) -glycine, dodecyl mercaptan, 2-aminoethanethiol, 3-mercaptopropionic acid and 4-mercaptobenzoic acid.

11. Use of a ligand-bound gold cluster in the manufacture of a medicament for treating stroke in a patient, wherein the ligand-bound gold cluster comprises:

A gold core; and

a ligand that binds to the gold core;

12. The use according to claim 11, characterized in that the diameter of the gold core is 0.5-3nm.

13. The use according to claim 11, characterized in that the diameter of the gold core is 0.5-2.6nm.

14. The use according to claim 11, wherein the ligand is one selected from the group consisting of L-cysteine and derivatives thereof, D-cysteine and derivatives thereof, cysteine-containing oligopeptides and derivatives thereof and other thiol-containing compounds.

15. The use according to claim 14, characterized in that said L-cysteine and derivatives thereof are selected from the group consisting of L-cysteine, N-isobutyryl-L-cysteine (L-NIBC) and N-acetyl-L-cysteine (L-NAC), said D-cysteine and derivatives thereof being selected from the group consisting of D-cysteine, N-isobutyryl-D-cysteine (D-NIBC) and N-acetyl-D-cysteine (D-NAC).

16. The use according to claim 14, wherein the cysteine-containing oligopeptide and derivatives thereof are cysteine-containing dipeptides selected from the group consisting of L (D) -cysteine-L (D) -arginine dipeptide (CR), L (D) -arginine-L (D) -cysteine dipeptide (RC), L (D) -histidine-L (D) -cysteine dipeptide (HC) and L (D) -cysteine-L (D) -histidine dipeptide (CH).

17. The use according to claim 14, wherein the cysteine-containing oligopeptide and derivatives thereof are cysteine-containing tripeptides selected from the group consisting of glycine-L (D) -cysteine-L (D) -arginine tripeptide (GCR), L (D) -proline-L (D) -cysteine-L (D) -arginine tripeptide (PCR), L (D) -lysine-L (D) -cysteine-L (D) -proline tripeptide (KCP) and L (D) -Glutathione (GSH).

18. The use according to claim 14, wherein the cysteine-containing oligopeptide and derivatives thereof are cysteine-containing tetrapeptides selected from the group consisting of glycine-L (D) -serine-L (D) -cysteine-L (D) -arginine tetrapeptides (GSCR) and glycine-L (D) -cysteine-L (D) -serine-L (D) -arginine tetrapeptides (GCSR).

19. The use according to claim 14, wherein the cysteine-containing oligopeptide and derivatives thereof are cysteine-containing pentapeptides selected from the group consisting of cysteine-aspartic acid-glutamic acid-valine-aspartic acid (CDEVD) and aspartic acid-glutamic acid-valine-aspartic acid-cysteine (devc).

20. The use according to claim 14, wherein the other thiol-containing compound is selected from the group consisting of 1- [ (2S) -2-methyl-3-thiol-1-oxopropyl ] -L (D) -proline, thioglycolic acid, mercaptoethanol, thiophenol, D-3-mercaptovaline, N- (2-mercaptopropionyl) -glycine, dodecyl mercaptan, 2-aminoethanethiol, 3-mercaptopropionic acid and 4-mercaptobenzoic acid.