RU2822218C1 - Gold clusters, compositions and methods of treating ischemic cerebral stroke - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to treatment of cerebral diseases. Disclosed is the use of a ligand-bound gold cluster for the treatment of ischemic cerebral stroke in a subject, where the gold cluster bound to the ligand contains a gold nucleus and a ligand bound to the gold nucleus, wherein the gold nucleus has a diameter in range of 0.5–3 nm; ligand is one selected from a group consisting of L-cysteine and derivatives thereof, D-cysteine and derivatives thereof, cysteine-containing oligopeptides and derivatives thereof and other thiol-containing compounds; L-cysteine and its derivatives are selected from the group consisting of L-cysteine, N-isobutyryl-L-cysteine (L-NIBC) and N-acetyl-L-cysteine (L-NAC), and where D-cysteine and its derivatives are selected from the group consisting of D-cysteine, N-isobutyryl-D-cysteine (D-NIBC) and N-acetyl-D-cysteine (D-NAC); cysteine-containing oligopeptides and derivatives thereof are cysteine-containing dipeptides, cysteine-containing tripeptides, cysteine-containing tetrapeptides or cysteine-containing pentapeptides; and wherein the other thiol-containing compounds are selected from the group consisting of 1-[(2S)-2-methyl-3-thiol-1-oxopropyl]-L(D)-proline, thioglycolic acid, mercaptoethanol, thiophenol, D-3-trolovol, N-(2-mercaptopropionyl)-glycine, dodecyl mercaptan, 2-aminoethane thiol (CSH), 3-mercaptopropionic acid (MPA) and 4-mercaptobenic acid (p-MBA).

EFFECT: invention provides the use of the gold cluster in the treatment of ischemic cerebral stroke.

6 cl, 17 dwg, 1 tbl, 4 ex

Description

FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

[1] The present invention relates to the field of treatment of diseases of the brain, in particular to gold clusters (AuCs) associated with ligands, to a composition comprising gold clusters associated with ligands, to the use of gold clusters associated with ligands for the preparation of drugs for treatment of ischemic cerebral stroke, as well as methods of using gold clusters associated with ligands and compositions for the treatment of ischemic cerebral stroke.

BACKGROUND OF THE ART

[2] A stroke occurs when a blood vessel is either blocked by a blood clot or ruptured. There are three types of stroke: hemorrhagic cerebral stroke, ischemic cerebral stroke, and transient ischemic attack (TIA).

[3] Hemorrhagic cerebral stroke is caused by a ruptured blood vessel that prevents blood from flowing to the brain. Common symptoms include sudden weakness, paralysis of any part of the body, difficulty speaking, vomiting, difficulty walking, coma, loss of consciousness, stiff neck and dizziness. There is no specialized treatment.

[4] Ischemic cerebral stroke, also known as cerebral ischemia and cerebral ischemia, is one of the most common pathologies in humans and is one of the leading causes of death and disability. Ischemic stroke accounts for about 87 percent of all stroke cases. Ischemic cerebral stroke is the result of a blockage, such as a blood clot or plaque, in the artery supplying blood to the brain. The blockage occurs in the neck or skull and reduces the flow of blood and oxygen to the brain, leading to damage or death of brain cells. If circulation is not restored quickly, brain damage may be permanent.

[5] The characteristic symptoms of ischemic cerebral stroke depend on which area of the brain is affected. Common symptoms of most cases of ischemic cerebral stroke are vision problems, weakness or paralysis of the limbs, dizziness and vertigo, confusion, loss of coordination and

drooping of the face on one side. Once symptoms appear, it is important to begin treatment as quickly as possible to reduce the chance that the damage will become permanent.

[6] The main treatment for ischemic cerebral stroke is intravenous administration of tissue plasminogen activator (t-PA), which breaks down blood clots. To be effective, t-PA must be administered within four and a half hours of the stroke onset. However, t-PA causes bleeding, so patients should not receive t-PA if they have a history of hemorrhagic stroke, cerebral hemorrhage, or recent major surgery or head trauma. Long-term treatment includes aspirin or an anticoagulant to prevent further blood clots.

[7] Amani et al. said that OX26@GNPs formed by conjugating OX26-PEG to the surface of 25 nm colloidal gold nanoparticles significantly increased the infarcted brain tissue, while free GNPs and PEGylated GNPs had no effect on the infarct intensity; the study results showed that OX26@GNPs are not suitable for the treatment of ischemic stroke.

[8] Zheng et al. said that in their OGD/R injury rat model, 20 nm Au-NPs increased cell viability, reduced neuronal apoptosis and oxidative stress, and improved mitochondrial respiration. However, in the study by Zheng et al. also demonstrated that 5 nm Au-NPs showed opposite effects, not suitable for the treatment of ischemic stroke.

[9] TIA occurs due to a temporary blood clot. Common symptoms include weakness, numbness or paralysis on one side of the body, slurred or confused speech, blindness and dizziness. There is no specialized treatment.

[10] Currently, there remains a need for effective methods and drugs for the treatment of ischemic cerebral stroke.

DISCLOSURE OF THE INVENTION

[11] The present invention discloses the use of ligand-linked gold clusters for the treatment of ischemic cerebral stroke in a subject.

[12] In certain embodiments of the present invention, a gold cluster linked

with a ligand, wherein the ligand-bound gold cluster includes a gold core and a ligand bound to the gold core.

[13] In certain therapeutic applications, the gold core has a diameter in the range of 0.5-3 nm. In certain embodiments, the gold core has a diameter in the range of 0.5-2.6 nm.

[14] In certain therapeutic applications, the ligand is selected from the group consisting of L-cysteine and its derivatives, D-cysteine and its derivatives, cysteine-containing oligopeptides and derivatives thereof, and other thiol-containing compounds.

[15] In certain therapeutic applications, L-cysteine and its derivatives are selected from the group consisting of L-cysteine, N-isobutyryl-L-cysteine (L-NIBC) and N-acetyl-L-cysteine (L-NAC), and D-cysteine and its derivatives are selected from the group consisting of D-cysteine, N-isobutyryl-D-cysteine (D-NIBC) and N-acetyl-D-cysteine (D-NAC).

[16] In certain therapeutic applications, 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).

[17] In certain therapeutic applications, 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).

[18] In certain therapeutic applications, 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 tetrapeptide (GSCR) and glycine-L(D)-cysteine-L(D)-serine-L(D)-arginine tetrapeptide (GCSR).

[19] In certain therapeutic applications, the cysteine-containing oligopeptides and derivatives thereof are a cysteine-containing pentapeptide, wherein the cysteine-containing pentapeptides are selected from the group consisting of cysteinyl-

asparaginyl-glutaminyl-valinyl-asparagine (CDEVD) and asparginyl-glutaminyl-valinyl-asparaginyl-cysteine (DEVDC).

[20] In certain therapeutic applications, other thiol-containing compounds are selected from the group consisting of 1-[(2S)-2-methyl-3-thiol-1-oxopropyl]-L(D)-proline, thioglycolic acid, mercaptoethanol, thiophenol , D-3-trolovol, N-(2-mercaptopropionyl)-glycine, dodecylmercaptan, 2-aminoethanethiol (CSH), 3-mercaptopropionic acid (MPA) and 4-mercaptobenic acid (p-MBA).

[21] The objectives and advantages of the application of the claimed invention will be disclosed from the following detailed description of preferred embodiments thereof in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[22] Preferred embodiments of the present invention will be described below with reference to the drawings, in which like numerals indicate like elements.

[23] In FIG. Figure 1 shows ultraviolet-visible (UV) spectra, transmission electron microscope (TEM) images, and particle size distribution diagrams of gold nanoparticle-modified L-NIBC ligand (L-NIBC-AuNPs) with different particle sizes.

[24] In FIG. Figure 2 shows ultraviolet-visible (UV) spectra, TEM images, and particle size distribution diagrams of gold clusters bound to L-NIBC ligand (L-NIBC-AuCs) with different particle sizes.

[25] In FIG. Figure 3 shows the infrared spectra of L-NIBC-AuCs with different particle sizes.

[26] In FIG. Figure 4 shows UV, IR, TEM and particle size distribution diagrams of CR ligand-bound gold clusters (CR-AuCs).

[27] In FIG. Figure 5 shows UV, IR, TEM, and particle size distribution diagrams of RC ligand-bound gold clusters (RC-AuCs).

[28] In FIG. Figure 6 shows UV, IR, TEM and particle size distribution diagrams of the ligand 1-[(2S)-2-methyl-3-thiol-1-oxopropyl]-L-proline (i.e. Cap) bound to gold clusters ( Cap-AuCs).

[29] In FIG. Figure 7 shows UV, IR, TEM and particle size distribution diagrams of gold clusters bound to the GSH ligand (GSH-AuCs).

[30] In FIG. Figure 8 shows UV, IR, TEM and particle size distribution diagrams of gold clusters bound to the D-NIBC ligand (D-NIBC-AuCs).

[31] In FIG. Figure 9 shows UV, IR, TEM and particle size distribution diagrams of gold clusters bound to the ligand L-cysteine (L-Cys-AuCs).

[32] In FIG. Figure 10 shows UV, IR, TEM and particle size distribution diagrams of gold clusters bound to the ligand 2-aminoethaneethiol (CSH-AuCs).

[33] In FIG. Figure 11 shows UV, IR, TEM and particle size distribution diagrams of gold clusters bound to 3-mercaptopropionic acid ligand (MPA-AuCs).

[34] In FIG. Figure 12 shows UV, IR, TEM and particle size distribution diagrams of gold clusters bound to the ligand of 4-mercaptobenic acid (p-MBA-AuCs).

[35] In FIG. 13 shows UV, TEM and particle size distribution diagrams of gold clusters bound to 4-cysteinyl-asparaginyl-glutaminyl-valinyl-asparagine (CDEVD) ligand (CDEVD-AuCs).

[36] In FIG. 14 shows UV, TEM and particle size distribution diagrams of gold clusters bound to 4-asparginyl-glutaminyl-valinyl-asparaginylcysteine (DEVDC) ligand (DEVDC-AuCs).

[37] In FIG. Figure 15 shows the evaluation of the results of the reflex behavior of rats in each group (in the histogram at each time point from left to right: placebo group (blank), model control group, group A1 with low dosage, group A1 with high dosage, group A2 with low dosage, group A2 high dosage, low dosage group A3, high dosage group A3, low dosage group A4, high dosage group A4, low dosage group B1, high dosage group B1, low dosage group B2 and high dosage group B2 dosage).

[38] In FIG. Figure 16 shows the percentage of rat brain damage in each group (in the histogram, from left to right: placebo group (blank), model control group, low-dose group A1, high-dose group A1, low-dose group A2, high-dose group A2, low dosage group A3, high dosage group A3, low dosage group A4, high dosage group A4, low dosage group B1, high dosage group B1, low dosage group B2 and high dosage group B2).

[39] In FIG. 17 shows exemplary images of TTC staining of rat brain tissue after administration of gold cluster drugs and gold nanoparticles, where (1) placebo group;

(2) model control group; (3) low-dose group A1; (4) high-dose group A1; (5) low dose group B1; (6) group B1 with high dosage.

DETAILED DESCRIPTION OF IMPLEMENTATION OPTIONS

[40] The present invention will be better understood with reference to the following detailed description of certain embodiments of the invention.

[41] In this application, where references are made to various publications, the descriptions of these publications are incorporated herein in their entirety to more fully describe the prior art to which the present invention relates.

[42] As used herein, “administration” means oral (“po”) administration, suppository administration, topical administration, intravenous (“iv”), intraperitoneal (“ip”), intramuscular noe ("im"), intralesional, intrahypocampal, intracerebroventricular, intranasal or subcutaneous (“sc”) administration, or implantation of a sustained release device, such as a mini-osmotic pump or a corrosive implant, into the subject. Administration is by any route, including parenteral and transmucosal (eg, oral, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, but is not limited to, intravenous, intramuscular, intraarterial, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial. Other routes of administration include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, etc.

[43] By “systemic administration” and “systematic administration” is meant a method of administering a compound or composition to a mammal such that the compound or composition is delivered to sites of the body, including the target site of pharmaceutical action, through the circulatory system. Systemic administration includes, but is not limited to, oral, intranasal, rectal, and parenteral (i.e., intramuscular, intravenous, intraarterial, transdermal, and subcutaneous administration other than the digestive tract), with the caveat that systemic administration herein does not include direct administration to a site in the brain other than through the circulatory system, such as intrathecal administration and intracranial administration.

[44] “Treatment” and “therapy” as used herein mean slowing the onset, arresting or reversing the progression, alleviating or preventing the disease or condition to which the term applies, or one or more symptoms of such disease or condition.

[45] “Patient,” “subject,” or “individual” is used interchangeably to mean a mammal, such as a human or other mammal, including primates (e.g., macaques, common chimpanzees, pongos), domesticated mammals (e.g., felids, canines), agricultural mammal (eg, bovine, ovine, porcine, equine) and laboratory mammals or rodents (eg, rat, mouse, lagomorph, hamster, guinea pig).

[46] Gold clusters (AuCs) are a special form of gold that exists between gold atoms and gold nanoparticles. AuCs are less than 3 nm in size and consist of only a few hundred gold atoms, which leads to the destruction of the face-centered cubic structure of gold nanoparticles. As a result, AuCs exhibit molecule-like discrete electronic properties with a distinct HOMO-LUMO gap, in contrast to the continuous or quasi-continuous energy levels of gold nanoparticles. This leads to the disappearance of the surface plasmon resonance effect and the corresponding plasmon resonance absorption band (520 ± 20 nm) in the UV-Vis spectrum, which is possessed by conventional gold nanoparticles.

[47] The present invention discloses AuC bound to ligands.

[48] In certain embodiments of the invention, the ligand-bound AuC includes a ligand and a gold core, wherein the ligand is bound to the gold core. The binding of ligands to the gold core means that the ligands form solution-stable complexes with the gold core through covalent bonding, hydrogen bonding, electrostatic force, hydrophobic force, van der Waals force, etc. In some embodiments, the diameter of the gold core is between 0.5 and 3 nm. In some embodiments, the diameter of the gold core is 0.5 - 2.6 nm.

[49] In some embodiments, the AuC bound to the ligands is a thiol-containing compound or oligopeptide. In some embodiments, the ligand binds to the gold core to form AuC bound to the ligand via an Au-S bond.

[50] In some embodiments, the ligand is, but is not limited to, L-cysteine, D-cysteine, or a cysteine derivative. In some variants

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).

[51] In certain embodiments, the ligand is, but is not limited to, a cysteine-containing oligopeptide and derivatives thereof. In certain embodiments, the cysteine-containing oligopeptide is a cysteine-containing dipeptide. In certain 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-histidine dipeptide (CH). In certain embodiments, the cysteine-containing oligopeptide is a cysteine-containing tripeptide. In certain 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 certain embodiments, the cysteine-containing oligopeptide is a cysteine-containing tetrapeptide. In certain 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 certain embodiments, the cysteine-containing oligopeptide is a cysteine-containing pentapeptide. In certain embodiments, the cysteine-containing pentapeptide is cysteinyl-asparaginyl-glutaminyl-valinyl-asparagine (CDEVD) or asparginyl-glutaminyl-valinyl-asparaginyl-cysteine (DEVDC).

[52] In certain embodiments of the invention, the ligand is a thiol-containing compound. In certain embodiments, the thiol-containing compound is 1-[(2S)-2-methyl-3-thiol-1-oxopropyl]-L(D)-proline, thioglycolic acid, mercaptoethanol, thiophenol, D-3-trolovol, dodecyl mercaptan, 2-Aminoethanethiol (CSH), 3-mercaptopropionic acid (MPA) or 4-mercaptobenic acid (p-MBA).

[53] The present invention discloses a pharmaceutical composition for the treatment of ischemic cerebral stroke. In certain embodiments, the subject is a human. In certain embodiments, the subject is a pet, such as a dog.

[54] In certain embodiments, the pharmaceutical composition includes AuC bound to ligands as described above and a pharmaceutically acceptable excipient. In certain embodiments, the excipient is a phosphate buffered solution or physiological saline solution.

[55] The present invention discloses the use of the above-described ligand-linked AuCs for the preparation of a drug for the treatment of ischemic cerebral stroke.

[56] The present invention discloses the use of the above-described ligand-linked AuCs for treating a subject with ischemic cerebral stroke or a method of treating a subject with ischemic cerebral stroke using the above-described ligand-linked AuCs. In certain embodiments, the method of treatment includes administering to a subject a pharmaceutically effective amount of AuCs bound to ligands. The pharmaceutically effective amount can be determined by routine in vivo studies. In certain embodiments, the pharmaceutically effective amount of AuCs bound to the ligands is at least 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, 0.5 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, or 100 mg/kg/day .

[57] The following examples are provided solely for the purpose of demonstrating the basic principles of the present invention; they are in no way intended to limit the scope of the present invention.

[58] Embodiments

[59] Embodiment 1: Preparation of Ligand-Bound AuCs

[60] 1.1 Dissolve HAuCl4 in methanol, water, ethanol, n-propanol or ethyl acetate to obtain solution A, in which the concentration of HAuCl4 is 0.01~0.03M;

[61] 1.2 Dissolve the ligand in the solvent to obtain solution B, in which the ligand concentration is 0.01~0.18 M; the ligand includes, but is 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 their derivatives, including, but not limited to, dipeptides, tripeptide, tetrapeptide, pentapeptide and other peptides,

containing cysteine, 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 tripeptide ( PCR), L(D)-glutathione (GSH), glycine-L(D)-serine-L(D)-cysteine-L(D)-arginine tetrapeptide (GSCR), glycine-L(D)-cysteine-L (D)-serine-L(D)-arginine tetrapeptide (GCSR), cysteinyl-asparaginyl-glutaminyl-valinyl-asparagine (CDEVD) and asparginyl-glutaminyl-valinyl-asparaginyl-cysteine (DEVDC), 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-trolovol, dodecylmercaptan, 2-aminoethanethiol ( CSH), 3-mercaptopropionic acid (MPA) and 4-mercaptobenic acid (p-MBA); 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, pentanol, butyl acetate, tributyl methyl ether, isopropyl acetate, dimethyl sulfoxide, ethyl formate, isobutyl acetate, methyl acetate, 2-methyl-1-propanol and propyl acetate;

[62] 1.3 Mix solution A and solution B so that the molar ratio between HAuCl4 and ligand is 1:(0.01~100), stir in an ice bath for 0.1~48h, add 0.025~0.8M solution of NaBH4 in water, ethanol or methanol, continue stirring in an ice water bath and carry out the reaction for 0.1~12 hours. The molar ratio between NaBH4 and the ligand is 1:(0.01~100).

[63] 1.4 Using MWCO 3K~30K ultrafiltration tubes, centrifuge the reaction solution at 8000~17500 rpm by gradient for 10~100 min after the end of the reaction to precipitate ligand-bound AuCs with different media P a r t s i m e r . The aperture of filtration membranes for ultrafiltration tubes with different MWCO directly determines the size of AuCs bound to ligands that can pass through the membranes . If desired, this step can be skipped.

[64] 1.5 Dissolve the precipitate of ligand-bound AuCs in various average particle sizes obtained in step (1.4) in water, place it in a dialysis bag, and dialyze in water at room temperature for 1~7 days.

[65] 1.6 Freeze-dry the ligand-bound AuCs within 12~24 hours after dialysis to obtain a powder or flocculating substance, i.e. AuCs bound to ligands.

[66] The authors unexpectedly found that the particle size of the powder or flocculant substance obtained by the above method was less than 3 nm (generally distributed in the range of 0.5-2.6 nm). There is no obvious absorption peak at 520 nm. The resulting powder or floc was found to be AuCs bound to ligands.

[67] Embodiment 2: Preparation and characterization of AuCs bound to various ligands

[68] 2.1 Preparation of AuCs bound to L-NIBC, i.e. L-NIBC-AuCs

[69] Using the L-NIBC ligand as an example, the preparation and characterization of AuCs bound to the L-NIBC ligand are described in detail.

[70] 2.1.1 Weigh 1.00 g of HAuCl4 and dissolve in 100 ml of methanol to obtain 0.03 M solution A;

[71] 2.1.2 Weigh 0.57 g of L-NIBC and dissolve it in 100 ml of glacial acetic acid (acetic acid) to obtain 0.03 M solution B;

[72] 2.1.3 Measure 1 ml of solution A, mix it with 0.5 ml, 1 ml, 2 ml, 3 ml, 4 ml or 5 ml of solution B, respectively (i.e. molar ratio between HAuCl4 and L-NIBC is 1:0.5, 1:1, 1:2, 1:3, 1:4, 1:5, respectively), carry out the reaction in an ice bath with stirring for 2 hours, quickly add 1 ml of freshly prepared 0.03 M (prepared by weighing 11.3 mg of NaBH4 and dissolving it in 10 ml of ethanol) of ethanol NaBH4 solution, when the solution turns from bright yellow to colorless, continue the reaction for 30 min after the solution turns dark brown and add 10 ml acetone to complete the reaction.

[73] 2.1.4 After the reaction, the reaction solution is subjected to gradient centrifugation to obtain L-NIBC-AuCs powder with different particle sizes. Specific method: After completion of the reaction, the reaction solution is transferred into a 50 ml MWCO 30K ultrafiltration tube, centrifuged at 10,000 rpm for 20 min, and the retentate in the inner tube is dissolved in ultrapure water to obtain a powder with a particle size of about 2.6 nm. The mixed solution in the outer tube is then transferred to a 50 mL ultrafiltration tube with MWCO 10K and centrifuged at 13,000 rpm for 30 min. The retentate in the inner tube is dissolved in ultrapure water to obtain a powder with a particle size of about 1.8 nm. The mixed solution in the outer tube is then transferred to a 50 ml ultrafiltration tube with MWCO 3K and

centrifuge at 17,500 rpm for 40 minutes. The retentate in the inner tube is dissolved in ultrapure water to obtain a powder with a particle size of about 1.1 nm.

[74] 2.1.5 Precipitate the powder of three different particle sizes obtained by gradient centrifugation, remove the solvent, dry the crude product N2, dissolve it in 5 ml of ultrapure water, place it in a dialysis bag (MWCO is 3KDa), place the dialysis bag in 2 L of ultrapure water, the water is changed every two days, dialysis is carried out for 7 days, freeze-dried and stored for future use.

[75] 2.2 Characterization of L-NIBC-AuCs

[76] A characterization experiment was conducted on the powder obtained above (L-NIBC-AuCs). Meanwhile, L-NIBC ligand-modified gold nanoparticles (L-NIBC-AuNPs) are used as a control. The method for preparing gold nanoparticles with the L-NIBC ligand is given in the source (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).

[77] 2.2.1 Morphological examination using transmission electron microscope (TEM)

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

[79] Four TEM images of L-NIBC-AuNPs are shown in panels B, E, H, and K of Fig. 1; three TEM images of L-NIBC-AuCs are shown in panels B, E and H of Fig. 2.

[80] Images in FIGS. 2 show that each of the L-NIBC-AuCs samples has uniform particle size and good dispersibility, and the average diameter of L-NIBC-AuCs (related to the diameter of the gold core) is 1.1 nm, 1.8 nm and 2.6 nm respectively, which is consistent with the results in panels C, F, and I of Fig. 2. For comparison, the L-NIBC-AuNPs samples have a larger particle size. Their average diameter (relative to the diameter of the gold core) is 3.6 nm, 6.0 nm, 10.1 nm, and 18.2 nm, respectively, which is completely consistent with the results in panels C, F, I, and L of Fig. 1.

[81] 2.2.2 Ultraviolet (UV)–visible (vis) absorption spectra

[82] Test powders (L-NIBC-AuCs sample and L-NIBC-AuNPs sample) were dissolved in ultrapure water to a concentration of 10 mg L-1, and then UV-vis absorption spectra were measured at room temperature. The measurement range was 190-1100 nm, the sample cuvette was a standard quartz cell with an optical path of 1 cm, and the control cuvette was filled with ultrapure water.

[83] UV-vis absorption spectra of four samples of L-NIBC-AuNPs of different sizes are shown in panels A, D, G and J of Fig. 1, and the statistical distribution of particle sizes is shown in panels C, F, I and L of FIG. 1; UV-vis absorption spectra of three L-NIBC-AuCs samples of different sizes are shown in panels A, D and G of FIG. 2, and the statistical distribution of particle sizes is shown in panels C, F and I of FIG. 2.

[84] In FIG. Figure 1 shows that due to the surface plasmon effect of L-NIBC-AuNPs, an absorption peak was observed at a wavelength of about 520 nm. The position of the absorption peak depends on the particle size. With a particle size of 3.6 nm, the UV absorption peak appears at 516 nm; when the particle size is 6.0 nm, the UV absorption peak appears at 517 nm; when the particle size is 10.1 nm, the UV absorption peak appears at 520 nm, and when the particle size is 18.2 nm, the absorption peak appears at 523 nm. None of the four samples has an absorption peak above 560 nm.

[85] In FIG. Figure 2 shows that in the UV absorption spectra of three L-NIBC-AuCs samples with different particle sizes, the surface plasmon effect absorption peak at 520 nm disappeared, and two obvious absorption peaks appeared above 560 nm, and the positions of the absorption peaks varied slightly depending on the sizes AuCs particles. This is explained by the fact that AuCs exhibit molecular-like properties due to the decomposition of the face-centered cubic structure, which leads to a discontinuity in the density of AuCs compounds, energy level splitting, disappearance of the plasmon resonance effect, and the appearance of a new absorption peak in the long-wavelength direction. It can be concluded that all three powder samples with different particle sizes obtained above are AuCs bound to ligands.

[86] 2.2.3 Fourier transform infrared spectroscopy

[87] Infrared spectra were measured on a Bruker VERTEX80V Fourier transform infrared spectrometer in total reflection mode in solid powder under high vacuum. The scanning range is 4000-400 cm-1, the number of scans is 64. For example, L-NIBC-AuCs samples, test samples are taken

were dry L-NIBC-AuCs powder with three different particle sizes, and the control sample was pure L-NIBC powder. The results are shown in Fig. 3.

[88] In FIG. Figure 3 shows the infrared spectrum of L-NIBC-AuCs with different particle sizes. Compared with pure L-NIBC (bottom curve), the S-H stretching vibration of L-NIBC-AuCs with different particle sizes completely disappeared at 2500-2600 cm-1, while other characteristic peaks of L-NIBC were still observed, which confirms that L-NIBC molecules were successfully coupled to the surface of AuCs through the Au-S bond. The figure also shows that the infrared spectrum of AuCs bound to ligands is independent of their size.

[89] AuCs bound to other ligands were prepared in a similar manner to that described above, except that the solvent for solution B, the feed ratio between HAuCl4 and the ligand, the reaction time, and the amount of NaBH4 added were 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 CR dipeptide, RC dipeptide or 1-[(2S)-2-methyl-3-thiol-1-oxopropyl]-L-proline is used as the ligand, water is selected as the solvent, etc.; other stages are similar, so further details are not given here.

[90] In the context of the present invention, a series of ligand-bound AuCs were prepared by the method described above. Ligands and characteristics of the preparation method are given in Table 1.

[91] Table 1. Production characteristics of AuCs bound to various ligands according to the present invention

Parameter Time reactions Time V Ligand Solvent used for solution B Relationship between HAuCl4 and ligand reactions in an ice bath with stirring until
add Molar ratio between HAuCl4 and NaBH4 ice bath while stirring
and after I'm NaBH4 added nia NaBH4 1 L-cysteine Vinegar
acid 1:3 2h 1:2 0.5h

2 D-cysteine Vinegar
acid 1:3 2h 1:2 0.5h 3 N-acetyl-L-cysteine Ethanol 1:4 1h 1:1 0.5h 4 N-acetyl-D-cysteine Ethanol 1:4 1h 1:1 0.5h 5 L-NIBC Water 1:4 0.5h 1:2 0.5h 6 D-NIBC Water 1:4 0.5h 1:2 0.5h 7 Other derivatives
cysteine Solvent 1:(0.1~100) 0.5h~24h 1:
(0.1~100) 0.1~24h 8 CR Water 1:4 22h 2:1 0.5h 9 R.C. Water 1:4 20h 2:1 0.5h 10 H.C. Water 1:3 12h 1:2 2h eleven CH Ethanol 1:4 16h 1:3 3h 12 GSH Water 1:2 12h 1:1 3h 13 KCP Water 1:3 15h 1:2 1h 14 PCR Water 1:4 16h 1:3 2h 15 GSCR Water 1:4 16h 1:3 1.5h 16 GCSR Water 1:3 12h 1:2 2h 17 CDEVD Water 1:7 1h 1:0.1 0.5h 18 DEVDC Water 1:7 1h 1:0.1 0.5h 19 Other
cysteine-containing oligopeptides Solvent 1:(0.1~100) 0.5h~24h 1: (0.1~100) 0.1~24h 20 1-[(2S)-2-methyl-3-
diol-1-oxopropyl]-L-proline Water 1:8 2h 1:7 1h 21 Mercaptoethanol Ethanol 1:2 2h 1:1 1h 22 Thioglycolic
acid Vinegar
acid 1:2 2h 1:1 1h 23 Thiophenol Ethanol 1:5 5h 1:1 1h 24 D-3-trovolol Water 1:2 2h 1:1 1h 25 N-(2-
mercaptopropionyl) - glycine Water 1:2 2h 1:1 1h 26 Dodecyl mercaptan Methanol 1:5 5h 1:1 1h 27 2-aminoethanethiol
(CSH) Water 1:5 2h 8:1 0.5h 28 3-
Mercaptopropionic acid (MPA) Water 1:2 1h 5:1 0.5h 29 4-mercaptobenzoic
acid (p-MBA) Water 1:6 0.5h 3:1 2h thirty Other
thiol-containing compounds Solvent 1:(0.01~100) 0.5h~24h 1: (0.1~100) 0.1~24h

[92] The samples listed in Table 1 were prepared by the above methods. The characteristics of eleven (11) different ligand-bound AuCs are shown in FIG. 4 (CR-AuCs), in FIG. 5 (RC-AuCs), in Fig. 6 (Cap-AuCs) (Cap stands for 1-[(2S)-2-methyl-3-thiol-1-oxopropyl]-L-proline), FIG. 7 (GSH-AuCs), in FIG. 8 (D-NIBC-AuCs), FIG. 9 (L-Cys-AuCs), in FIG. 10 (CSH-AuCs), in FIG. 11 (MPA-AuCs), in FIG. 12 (p-MBA-AuCs), in FIG. 13 (CDEVD-AuCs) and FIG. 13 (DEVDC-AuCs). In fig. Figures 4-12 show UV spectra (panel A), infrared spectra (panel B), TEM images (panel C), and particle size distribution (panel D). In fig. Figures 13 and 14 show UV spectra (panel A), TEM images (panel B), and particle size distribution (panel C).

[93] The results show that the diameters of AuCs bound to various ligands obtained in Table 1 are less than 3 nm. The UV spectra also show the disappearance of the peak at 520 ± 20 nm and the appearance of an absorption peak at other positions. The position of the absorption peak can vary depending on the ligands and particle sizes, as well as the structure. In certain cases, there is no absorption peak, mainly due to the formation of mixtures of AuCs with different particle sizes and structures or some special AuCs that move the absorption peak position outside the UV-vis spectrum. Meanwhile, the Fourier transform infrared spectra also show the disappearance of the infrared absorption peak of the thiol ligand (between the dashed lines in panel B of Figure 4-8), while other infrared characteristic peaks persist, presumably because all ligand molecules were successfully bound with gold atoms to form AuCs bound to ligands, and in the present invention, AuCs bound to the ligands listed in Table 1 were successfully prepared.

[94] Embodiment 3: Conducting an ischemic cerebral stroke study in an animal model

[95] 3.1 Test samples

[96] Gold Clusters:

[97] A1: L-NIBC ligand-bound gold clusters (L-NIBC-AuCs), size distribution in the range of 0.5-3.0 nm;

[98] A2: gold clusters bound to the ligand L-cysteine (L-Cys-AuCs), size distribution in the range of 0.5-3.0 nm;

[99] A3: gold clusters bound to the ligand N-acetyl-L-cysteine (L-NAC-AuCs), size distribution in the range of 0.5-3.0 nm; And

[100] A4: DEVDC ligand bound gold clusters (DEVDC-AuCs), size distribution in the range of 0.5-3.0 nm.

[101] Gold nanoparticles:

[102] B1: L-NIBC-bound gold nanoparticles (L-NIBC-AuNPs), size distribution range 6.1 ± 1.5 nm; And

[103] B2: L-NAC bound gold nanoparticles (L-NAC-AuNPs), size distribution range 9.0±2.4 nm.

[104] All test samples were prepared in accordance with the method described above with minor modifications, and their quality was assessed using the methods described above.

[105] 3.2 Research protocols

[106] 3.2.1 Creation of a model of middle cerebral artery occlusion (MCAO) in rats and administration of test substances

[107] Male Sprague Dawley (SD) SPF rats (220–260 g) were purchased from Shanghai Shrek Experimental Animal Co., Ltd. All rats underwent a 7-day period of adaptation to environmental conditions before the start of the study. The rats were randomly divided into 14 groups (n=10), including a placebo group, a model control group, a low (2 mg/kg rat body weight) and a high dose (10 mg/kg rat body weight) group of A1 gold cluster drugs, A2, A3 and A4, as well as a group of low (2 mg/kg rat body weight) and high dose (10 mg/kg rat body weight) gold nanoparticles B1 and B2. On the day of the study, rats were administered 10% chloral hydrate (350 mg/kg body weight) as anesthesia. The right common carotid artery, internal carotid artery, and external carotid artery were exposed through a midline incision. An 18 mm ± 0.5 mm suture was inserted into the internal carotid artery (ICA) through the external carotid artery (ECA) until the regional blood supply to the MCA was blocked, resulting in cerebral infarction. After 1.5 hours, the suture was removed to the entrance to the ICA for reperfusion. Preoperative and post-embolization cerebral blood flow (CBF) values were measured using a flowmeter. Animals whose CBF was consistently decreased (rCBF ≥ 70%) were considered to be a successful model of middle cerebral artery occlusion (MCAO). After reperfusion, rats were intraperitoneally administered drugs or vehicles (saline) for 0 h, 24 h, 48 h, and 72 h, respectively. Neurological behavioral indicators were assessed at 0, 24, 48, 72 and 96 hours. The study was stopped 96 hours after surgery. Brain sampling and TTC staining were performed after euthanasia. Brain slice images were taken and the percentage of cerebral infarct area was calculated.

[108] 3.2.2 Neurological assessment

[109] 0 points: no difference from ordinary rats; 1 point: extension of the right front paw is not straight, the head is thrown back to the opposite side; 2 points: walking in intermittent circles in open space; 3 points: walking in continuous circles in an open space; 4 points: loss of consciousness when walking, falling to one side; 5 points: death.

[110] 3.2.3 Area of brain lesion (TTC staining)

[111] Rats were euthanized by carbon dioxide inhalation. The brain was removed and placed in a coronal slice bath (2 mm). Staining was carried out with 2% TTC in the dark at room temperature. After photographing, the area of the brain lesion was measured using ImageJ software. Percentage of brain lesion area (%) = (contralateral hemisphere area - (ipsilateral hemisphere area - brain lesion area)) / contralateral hemisphere area × 100%.

[112] 3.2.4 Statistical analysis

[113] Statistical analysis was performed using Graph Pad Prism Software 7.0 (California, USA). Data were expressed as mean ± statistical error, and statistical analysis was performed using Dunnett's test. P < 0.05 indicates statistical significance.

[114] 3.3 Results

[115] 3.3.1 Cerebral circulation in the area of ischemic brain damage

[116] A decrease in cerebral blood flow in rats of more than 70% (reduced cerebral blood flow, rCBF≥70%) indicates the successful creation of the OSMA model. In all other groups, with the exception of the placebo group, the rCBF rate was more than 70%, with an average of about 80%, indicating the successful creation of the OSMA model.

[117] 3.3.2 Effect of study drugs on neurological parameters in rats

[118] In FIG. Figure 15 shows the assessment of neurological parameters in rats in each group (in the histogram at each time point, from left to right: placebo group (blank), model control group, low-dose group A1, high-dose group A1, low-dose group A2, group A2 high dosage, low dosage group A3, high dosage group A3, low dosage group A4, high dosage group A4, low dosage group B1, high dosage group B1, low dosage group B2 and high dosage group B2 dosage). Rats in the placebo group had normal neurological scores and an outcome score of 0;

rats in the model control group showed severe behavioral functional defects at 0 h, 24 h, 48 h, 72 h, and 96 h after surgery (compared with the placebo group, P < 0.001, ###). Compared with the control group, the neurological scores in the low-dose groups A1, A2, A3, A4 and high-dose groups were not significantly improved 24 hours after surgery. At 48 hours after surgery, the neurological scores in the low-dose groups A1, A2, A3, and A4 and the high-dose groups began to decline, but there was no statistical difference (compared with the control group, P > 0.05). At 72 hours post-surgery, the neurological scores of animals treated with the four drugs were further reduced, with significant differences observed between low-dose group A1, high-dose group A1 and high-dose group A2 (compared with the control group, P < 0.05, *). At 96 hours postoperatively, significant differences were observed for all low-dose groups and high-dose groups of the four drugs (compared with control group, P < 0.05, *). These results suggest that all four gold cluster drugs can significantly improve neurological outcomes after brain injury caused by ischemic stroke, and this effect is dose-dependent to a certain extent.

[119] Compared with the control model group, the low and high dosage groups of gold nanoparticles B1 and B2 did not show significant improvement in neurological parameters in MACO rat model at 24 hours, 48 hours, 72 hours and 96 hours after surgery, indicating that that gold nanoparticles cannot significantly restore behavioral functions caused by ischemic cerebral stroke.

[120] 3.3.3.3 Effect of each drug on the area of brain damage in the MACO rat model

[121] In FIG. Figure 16 shows the percentage of brain lesion area of rats in each group (in the histogram from left to right: placebo group (blank), model control group, low-dose group A1, high-dose group A1, low-dose group A2, high-dose group A2 , Low Dosage Group A3, High Dosage Group A3, Low Dosage Group A4, High Dosage Group A4, Low Dosage Group B1, High Dosage Group B1, Low Dosage Group B2 and High Dosage Group B2). In the placebo group, brain tissue was normal and no infarction was observed; the affected area was 0%.

The area of brain damage in the model control group was 44.7% ± 4.5% (P < 0.001, ###). Compared with the control group, the percentage of brain lesion area in the low-dose and high-dose groups A1, A2, A3, A4 obviously decreased, but the low-dose groups did not show a significant difference, while the high-dose groups showed a significant difference. difference (compared to control group, P < 0.05, *). Taking group A1 as an example, the lesion area in the low-dose group decreased from 44.7 ± 4.5% to 36.0 ± 4.0% (compared with the control group, P > 0.05), and in the high-dose group decreased to 27.8 ± 3.4% (compared to the control group, P < 0.05, *).

[122] In FIG. Figure 17 shows exemplary images of TTC staining of brain tissue of rats with OSMA after administration of gold cluster preparations, represented by group A1, and gold nanoparticles, represented by group B1. In fig. 17, (1) placebo group; (2) model control group; (3) low-dose group A1; (4) high-dose group A1; (5) low dose group B1; (6) group B1 with high dosage. As can be seen in FIG. 17, rats in the placebo group did not develop cerebral infarction, while those in the model control group experienced extensive cerebral infarction (white part of the figure on the right). The area of brain damage after the administration of low dosage of the drug to group A1 decreased (the white part on the right decreased), while the area of brain damage decreased significantly with the introduction of the high dosage of drug A1 (the white part on the right decreased significantly), while the administration of low and high dosage of drug B1 had no effect on the area of brain damage (the white part on the right did not decrease). Groups A2, A3 and A4 showed a similar effect as A1 in reducing the lesion area, while the result in group B2 was similar to group B1: the lesion area did not decrease.

[123] Other ligand-bound AuCs have similar effects in the treatment of ischemic cerebral stroke, but their effects vary within certain limits. They will not be described in detail in this section.

[124] Embodiment 4: Animal model studies of hemorrhagic cerebral stroke

[125] 4.1 Regent

[126] Gold clusters bound to the L-cysteine ligand (L-Cys-AuCs), particle size distribution in the range of 0.5-3.0 nm; gold nanoparticles associated with L-NIBC (L-NIBC-AuNPs), particle size distribution in the range of 6.1±1.5 nm.

[127] 4.2 Study protocol and results

[128] Rats are anesthetized and placed in a stereotaxic frame. On day 0, type VII collagenase was stereotactically injected into the right striatum (coordinates: 0.0 mm rostral and 3.0 mm lateral to bregma, 5.5 mm inferior to the skull) at a rate of 0.4 μl/min for 5 min. The study drugs are administered intravenously from days 0 to 4 at a dosage of 10 mg/kg of rat weight. Locomotion was assessed on the third day. On the fourth day, the rats were sacrificed and analyzed and histochemical staining was performed. The study drug AuCs and gold nanoparticles showed similar results in hemorrhagic cerebral stroke, indicating the absence of a clear therapeutic effect.

[129] Although the present invention has been disclosed in terms of specific embodiments, the embodiments are intended to be illustrative and not to limit the scope of the invention. Alternative embodiments of the present invention will be apparent to those of ordinary skill in the field to which the present invention relates. Such alternative embodiments are considered to be included within the scope of the present invention. Accordingly, the scope of the present invention is defined by the accompanying claims and is confirmed by the above description.

Links

Amani H., Mostafavi E., Mahmoud Reza Alebouyeh MR, Arzaghi H., Akbarzadeh A., Pazoki-Toroudi H., Webster TJ Would Colloidal Gold Nanocarriers Present An Effective Diagnosis Or Treatment For Ischemic Stroke? Int 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):1549-1566.

Claims (13)

1. Use of a ligand-linked gold cluster for the treatment of ischemic cerebral stroke in a subject, wherein the ligand-linked gold cluster contains: golden core; And ligand bound to the gold core; the gold core has a diameter in the range of 0.5-3 nm; where the ligand is one selected from the group consisting of L-cysteine and its derivatives, D-cysteine and its derivatives, cysteine-containing oligopeptides and derivatives thereof, and other thiol-containing compounds; L-cysteine and its derivatives are selected from the group consisting of L-cysteine, N-isobutyryl-L-cysteine (L-NIBC) and N-acetyl-L-cysteine (L-NAC), and wherein D-cysteine and its derivatives selected from the group consisting of D-cysteine, N-isobutyryl-D-cysteine (D-NIBC) and N-acetyl-D-cysteine (D-NAC); cysteine-containing oligopeptides and derivatives thereof are cysteine-containing dipeptides, cysteine-containing tripeptides, cysteine-containing tetrapeptides or cysteine-containing pentapeptides; and wherein the other thiol-containing compounds are selected from the group consisting of 1-[(2S)-2-methyl-3-thiol-1-oxopropyl]-L(D)-proline, thioglycolic acid, mercaptoethanol, thiophenol, D-3-trolovol , N-(2-mercaptopropionyl)-glycine, dodecylmercaptan, 2-aminoethanethiol (CSH), 3-mercaptopropionic acid (MPA) and 4-mercaptobenic acid (p-MBA). 2. Application according to claim 1, where the gold core has a diameter in the range of 0.5-2.6 nm. 3. Use according to claim 1, where 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). 4. Use according to claim 1, where 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). 5. Use according to claim 1, wherein the cysteine-containing tetrapeptides are selected from the group consisting of glycine-L(D)-serine-L(D)-cysteine-L(D)-arginine tetrapeptide (GSCR) and glycine-L(D) -cysteine-L(D)-serine-L(D)-arginine tetrapeptide (GCSR). 6. Use according to claim 1, wherein the cysteine-containing pentapeptides are selected from the group consisting of cysteinyl-asparaginyl-glutaminyl-valinyl-asparagine (CDEVD) and asparginyl-glutaminyl-valinyl-asparaginyl-cysteine (DEVDC).