RU2832089C2 - Gold clusters, compositions and methods of treating depression - Google Patents

Abstract

FIELD: medicine; pharmaceutics.

SUBSTANCE: invention relates to the field of medicine and pharmacology, namely the use of a ligand-bound gold cluster for the treatment of depression, where said gold cluster includes a gold nucleus; and a ligand bound to a gold nucleus; where the gold nucleus has a diameter in range of 0.5-3 nm; ligand is one of selected from a group consisting of L-cysteine and its derivatives, D-cysteine and its derivatives, cysteine-containing oligopeptides and their derivatives 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); 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 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).

EFFECT: disclosed is the use of a ligand-bound gold cluster for treating depression in a subject.

6 cl, 19 dwg, 1 tbl, 1 ex

Description

FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

[0001] The present invention relates to the field of psychotherapy, in particular to gold clusters (AuCs) bound to ligands, compositions comprising gold clusters, the use of gold clusters bound to ligands for the production of drugs for the treatment of depression, as well as to methods in which gold clusters bound to ligands and compositions for the treatment of depression are used.

LEVEL OF TECHNOLOGY

[0002] Depression is a mental disorder with a high prevalence among people, reaching 21% of the world's population, and causes severe symptoms including sadness, anger, frustration, hopelessness, anxiety, irritability, lack of motivation and guilt.

[0003] Although a number of factors are thought to be involved, including biological differences, brain chemistry, hormones, heredity, and chronic inflammation, the exact factor that causes depression, or the exact mechanism by which any factor causes depression, is not known, posing significant challenges to research and development of treatments for depression.

[0004] Currently, antidepressants are used to treat depression, which directly affect brain chemistry and are thought to achieve their therapeutic effect by correcting the chemical dysregulation that causes depression. These include tricyclic antidepressants, selective serotonin reuptake inhibitors (SSRIs, such as fluoxetine, paroxetine, sertraline, fluvoxamine, citalopram, and escitalopram), and serotonin-norepinephrine reuptake inhibitors (SNRIs, such as venlafaxine and duloxetine).

[0005] Studies have attempted to investigate the effects of nanoparticles on depression. Zinc oxide (NanoZnO) with a size of 20–80 nm reduced the immobility time in the forced swim test (FST) in a mouse model of lipopolysaccharide (LPS)-induced depression (Xie 2012). Iron nanoparticles (NPs) with a size of 20 nm alleviated depressive symptoms in a rat model of LPS-induced depression (Saeidienik 2018). However, nanosilver (nanoAg) with a size of 10 nm, when orally administered at a dose of 0.2 mg/kg body weight, caused morphological abnormalities in the myelin sheaths, exhibiting toxicity to the central nervous system (CNS) in rats (Dabrowska-Bouta 2016). _Al2O3 nanoparticles ( _NPs ) administered via the respiratory route induced depressive-like behavior in female mice (Zhang 2015). It is clear that previous studies do not provide a unified opinion and recommendations regarding the effect of nanoparticles on the course of depression.

[0006] There remains a need to develop an effective method and drugs for the treatment of depression.

DISCLOSURE OF THE ESSENCE OF THE INVENTION

[0007] The present invention relates to gold clusters linked to ligands for use in the treatment of depression in a subject.

[0008] In certain embodiments of the present invention, a gold cluster linked to a ligand is used to treat depression in a subject, wherein the gold cluster linked to the ligand comprises a gold core and a ligand linked to the gold core.

[0009] 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.

[0010] In certain therapeutic applications, the ligand is a ligand 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.

[0011] In certain therapeutic uses, 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).

[0012] In certain therapeutic embodiments, 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).

[0013] In certain therapeutic embodiments, 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).

[0014] In certain therapeutic embodiments, 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).

[0015] In certain therapeutic embodiments, 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 asparaginyl-glutaminyl-valinyl-asparaginyl-cysteine (DEVDC).

[0016] In certain therapeutic embodiments, 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-trolovole, N-(2-mercaptopropionyl)-glycine, dodecyl mercaptan, 2-aminoethanethiol (CSH), 3-mercaptopropionic acid (MPA), and 4-mercaptobenic acid (p-MBA).

[0017] The objects and advantages of the invention will be revealed from the following detailed description of preferred embodiments thereof taken in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF DRAWINGS

[0018] Preferred embodiments of the present invention will be described hereinafter with reference to the Figures, in which like numerals designate like elements.

[0019] Fig. 1 shows ultraviolet-visible (UV) spectra, transmission electron microscope (TEM) images, and particle size distribution diagrams of L-NIBC ligand modified with gold nanoparticles (L-NIBC-AuNPs) with different particle sizes.

[0020] Fig. 2 shows ultraviolet-visible (UV) spectra, TEM images, and particle size distribution diagrams of L-NIBC ligand-linked gold clusters (L-NIBC-AuCs) with different particle sizes.

[0021] Fig. 3 shows the infrared spectra of L-NIBC-AuCs with different particle sizes.

[0022] Figure 4 shows UV, IR, TEM and particle size distribution diagrams of gold clusters bound to the CR ligand (CR-AuCs).

[0023] Figure 5 shows UV, IR, TEM and particle size distribution diagrams of gold clusters bound to the RC ligand (RC-AuCs).

[0024] Figure 6 shows UV, IR, TEM, and particle size distribution diagrams of gold clusters bound to the ligand 1-[(2S)-2-methyl-3-thiol-1-oxopropyl]-L-proline (i.e., Cap).

[0025] Figure 7 shows UV, IR, TEM and particle size distribution diagrams of gold clusters bound to the GSH ligand (GSH-AuCs).

[0026] Figure 8 shows UV, IR, TEM, and particle size distribution diagrams of gold clusters bound to the D-NIBC ligand (D-NIBC-AuCs).

[0027] Figure 9 shows UV, IR, TEM, and particle size distribution diagrams of gold clusters bound to the ligand L-cysteine (L-Cys-AuCs).

[0028] Figure 10 shows UV, IR, TEM and particle size distribution diagrams of gold clusters bound to the 2-aminoethanediol ligand (CSH-AuCs).

[0029] Figure 11 shows UV, IR, TEM and particle size distribution diagrams of gold clusters bound to the 3-mercaptopropionic acid ligand (MPA-AuCs).

[0030] Figure 12 shows UV, IR, TEM and particle size distribution diagrams of gold clusters bound to the 4-mercaptobenic acid ligand (p-MBA-AuCs).

[0031] Figure 13 shows UV, TEM, and particle size distribution diagrams of gold clusters linked by the 4-cysteinyl-asparaginyl-glutaminyl-valinyl-asparagine (CDEVD) ligand.

[0032] Figure 14 shows UV, TEM, and particle size distribution diagrams of gold clusters linked by the 4-aspartic acid-glutamic acid-valine-aspartic acid-cysteine (DEVDC) ligand (DEVDC-AuCs).

[0033] Fig. 15 shows a histogram showing the results of forced swimming tests.

[0034] Fig. 16 shows a histogram showing the results of the tail suspension tests.

[0035] Fig. 17 shows the results of the social behavior test of mice with chronic social stress: A - the time of social interaction T1 in mice with a normal stress level and model mice in the social interaction zone in the first stage of the test; B - the time of social interaction T2 in mice with a normal stress level and model mice in the social interaction zone in the second stage of the test; C - the ratio of social interaction T2/T1 in mice with a normal stress level and model mice in the social behavior test. Data are presented as Mean ± Std. Error, ** P < 0.01 and *** P < 0.001 compared to normal mice in the normal control group.

[0036] Fig. 18 shows the results of the social behavior test of mice in the A1 drug administration group: A - at the first stage of the test - the time of social interaction T1 of mice of the normal control group, the model control group, and the A1 drug administration group in the social interaction zone; B - at the second stage - the time of social interaction T2 of mice of the normal control group, the model control group, and the A1 drug administration group in the social interaction zone; C - the coefficient of social interaction T2/T1 of mice of the normal control group, the model control group, and the A1 drug administration group in the social behavior test. The data are presented in the format Mean ± Std. Error, # P<0.05 compared with the normal control group, * P<0.05 compared with the model control group.

[0037] Fig. 19 shows the results of the elevated plus maze test on mice in the drug A4 administration group: A is the walking time of the normal control group, the model control group, and the drug A4 administration group in the open arm; B is the walking distance of the normal control group, the model control group, and the drug A4 administration group in the open arm; C is the walking time of the normal control group, the model control group, and the drug A4 administration group in the open arm; D is the percentage of the walking time in the open arm to the walking time in the open arm + closed arm (TO%) of the normal control group, the model control group, and the drug A4 administration group; E is the percentage of the walking distance in the open arm to the total walking distance (DO%) for the normal control group, the model control group, and the drug A4 administration group; F - percentage of time spent moving in the open arm and total time spent moving in the closed arm (EO%) for the normal control group, the model control group and the mice in the A4 drug administration group. The data in the figure are presented as Mean ± Standard Error, # P<0.05 compared with the normal control group, * P<0.05 compared with the model control group.

DETAILED DESCRIPTION OF IMPLEMENTATION OPTIONS

[0038] The following detailed description of some embodiments of the invention will provide a better understanding of the present invention.

[0039] In this application, where references are made to various publications, the disclosures of these publications are incorporated herein in their entirety to more fully describe the state of the art to which the present invention pertains.

[0040] As used herein, "administration" means oral ("ro") administration, suppository administration, topical administration, intravenous ("iv"), intraperitoneal ("ip"), intramuscular ("im"), intralesional, intrahippocampal, intracerebroventricular, intranasal, or subcutaneous ("sc") administration, or implantation of a sustained release device such as a mini-osmotic pump or an 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, 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.

[0041] By "systemic administration" and "systemic administration" is meant a method of administering a compound or composition to a mammal such that the compound or composition is delivered to areas of the body, including a target site of pharmaceutical action, via the circulatory system. Systemic administration includes, but is not limited to, oral, intranasal, rectal, and parenteral (i.e., administration not through the digestive tract, but intramuscular, intravenous, intraarterial, transdermal, and subcutaneous) administration, with the proviso that, as used herein, systemic administration does not include direct administration to an area of the brain other than via the circulatory system, such as intrathecal administration and intracranial administration.

[0042] By "treatment" and "therapy" as used herein is meant slowing the onset, arresting or reversing the progression of, alleviating or preventing the disease or condition to which such term applies, or one or more symptoms of such disease or condition.

[0043] By "patient," "subject," or "individual" is meant interchangeably a mammal, such as a human or other mammal, including primates (e.g., macaques, common chimpanzees, pongos), domesticated mammals (e.g., felines, dogs), farm mammals (e.g., bovines, ovines, pigs, horses), and laboratory mammals or rodents (e.g., rats, mice, lagomorphs, hamsters, guinea pigs).

[0044] Gold clusters (AuCs) are a special form of gold that exists between gold atoms and gold nanoparticles. AuCs are smaller than 3 nm in size and consist of only a few to 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 clearly defined 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 exhibited by conventional gold nanoparticles.

[0045] The present invention relates to AuCs bound to ligands.

[0046] In certain embodiments, AuC bound to ligands comprises a ligand and a gold core, wherein the ligand is bound to the gold core. Binding of ligands to the gold core means that the ligands form solution-stable complexes with the gold core via covalent bonding, hydrogen bonding, electrostatic force, hydrophobic force, van der Waals force, etc. In certain embodiments, the diameter of the gold core is between 0.5 and 3 nm. In certain embodiments, the diameter of the gold core is between 0.5 and 2.6 nm.

[0047] In certain embodiments, the ligand-bound AuC is a thiol-containing compound or an oligopeptide. In certain embodiments, the ligand binds to the gold core to form ligand-bound AuC via an Au-S bond.

[0048] In certain embodiments, the ligand is, but is not limited to, L-cysteine, D-cysteine, or a cysteine derivative. In certain 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).

[0049] 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 asparaginyl-glutaminyl-valinyl-asparaginyl-cysteine (DEVDC).

[0050] In certain embodiments, 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-trolovole, dodecyl mercaptan, 2-aminoethanethiol (CSH), 3-mercaptopropionic acid (MPA), or 4-mercaptobenic acid (p-MBA).

[0051] The present invention relates to a pharmaceutical composition for treating depression. In certain embodiments, the subject is a human. In certain embodiments, the subject is a domestic animal, such as a dog.

[0052] In certain embodiments, the pharmaceutical composition comprises AuC linked to ligands as described above and a pharmaceutically acceptable excipient. In certain embodiments, the excipient is a phosphate buffered saline solution or a physiological saline solution.

[0053] The present invention discloses the use of the above-described AuCs linked to ligands for the preparation of a medicament for the treatment of depression.

[0054] The present invention discloses the use of the above-described AuCs linked to ligands for treating a subject with depression or a method of treating a subject with depression using the above-described AuCs linked to ligands. In certain embodiments, the method of treatment comprises administering to the subject a pharmaceutically effective amount of the AuCs linked to ligands. A pharmaceutically effective amount can be determined using routine in vivo assays. In certain embodiments, the pharmaceutically effective amount of AuCs bound to 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, 100 mg/kg/day, 200 mg/kg/day, 300 mg/kg/day, 400 mg/kg/day, 500 mg/kg/day, 600 mg/kg/day, 700 mg/kg/day, 800 mg/kg/day, 900 mg/kg/day, or 1000 mg/kg/day. [0055] The following examples are provided solely for the purpose of demonstrating the basic teachings of the present invention; they are in no way intended to limit the scope of the present invention.

[0056] Embodiments

[0057] Embodiment 1: Obtaining AuCs bound to ligands

[0058] 1.1 Dissolve HAuCl ₄ in methanol, water, ethanol, n-propanol or ethyl acetate to obtain solution A, in which the concentration of HAuCl ₄ is 0.01~0.03M;

[0059] 1.2 Dissolve the ligand in the solvent to obtain solution B, in which the concentration of the ligand 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 derivatives thereof, 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), cysteine-asparaginyl-glutaminyl-valinyl-asparagine (CDEVD) and asparaginyl-glutaminyl-valine-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, dodecyl mercaptan, 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;

[0060] 1.3 Mix solution A and solution B so that the molar ratio between HAuCl ₄ and the ligand is 1:(0.01~100), stir in an ice bath for 0.1~48 h, add 0.025~0.8 M NaBH ₄ solution in water, ethanol or methanol, continue stirring in an ice water bath and carry out the reaction for 0.1~12 h. The molar ratio between NaBH ₄ and the ligand is 1:(0.01~100);

[0061] 1.4 Using 3K~30K MWCO ultrafiltration tubes, centrifuge the reaction solution at 8000~17500 rpm under gradient for 10~100 min after completion of the reaction to obtain a precipitate of ligand-bound AuCs with different average particle sizes. The aperture of the filtration membranes for the ultrafiltration tubes with different MWCOs directly determines the size of the ligand-bound AuCs that can pass through the membranes. This step can be omitted if desired;

[0062] 1.5 Dissolve the precipitate of ligand-bound AuCs in different 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;

[0063] 1.6 Freeze-drying of the ligand-bound AuCs is carried out for 12~24 h after dialysis to obtain a powder or flocculating substance, i.e., the ligand-bound AuCs.

[0064] The authors unexpectedly found that the particle size of the powder or flocculant substance obtained by the above-described method is less than 3 nm (generally distributed within the range of 0.5-2.6 nm). There is no obvious absorption peak at 520 nm. It was found that the obtained powder or floc is AuCs bound to ligands.

[0065] Embodiment 2: Preparation and Characterization of AuCs Linked to Different Ligands

[0066] 2.1 Preparation of AuCs linked to L-NIBC, i.e. L-NIBC-AuCs

[0067] Using the example of the L-NIBC ligand, the preparation and characterization of AuCs bound to the L-NIBC ligand is described in detail.

[0068] 2.1.1 Weigh 1.00 g of HAuCl ₄ and dissolve in 100 ml of methanol to obtain 0.03 M solution A;

[0069] 2.1.2 Weigh 0.57 g L-NIBC and dissolve it in 100 ml glacial acetic acid (acetic acid) to obtain 0.03 M solution B;

[0070] 2.1.3 Measure out 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., the molar ratio between HAuCl ₄ 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 h, when the solution turns from bright yellow to colorless, quickly add 1 ml of freshly prepared 0.03 M (prepared by weighing 11.3 mg NaBH ₄ and dissolving it in 10 ml of ethanol) ethanol solution of NaBH ₄ , continue the reaction for 30 min after the solution turns dark brown, and add 10 ml of acetone to complete the reaction.

[0071] 2.1.4 After the reaction, the reaction solution is subjected to gradient centrifugation to obtain L-NIBC-AuCs powder having different particle sizes. Specific method: after completion of the reaction, the reaction solution is transferred to a 50 mL ultrafiltration tube with MWCO 30K, centrifuged at 10000 rpm for 20 min, and the retentate in the inner tube is dissolved in ultrapure water to obtain a powder having a particle size of about 2.6 nm. Then, the mixed solution in the outer tube is transferred to a 50 mL ultrafiltration tube with MWCO 10K, and centrifuged at 13000 rpm for 30 min. The retentate in the inner tube is dissolved in ultrapure water to obtain a powder having a particle size of about 1.8 nm. Then, the mixed solution in the outer tube was transferred to a 50 ml ultrafiltration tube with MWCO 3K and centrifuged at 17,500 rpm for 40 min. The retentate in the inner tube was dissolved in ultrapure water to obtain a powder with a particle size of about 1.1 nm.

[0072] 2.1.5 Precipitate the powder of three different particle sizes obtained by gradient centrifugation, remove the solvent, dry the crude product with N ₂ , 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, change the water every two days, dialyze for 7 days, freeze-dry and store for further use.

[0073] 2.2 Characterization of L-NIBC-AuCs

[0074] The characterization experiment was conducted for the above-obtained powder (L-NIBC-AuCs). Meanwhile, gold nanoparticles modified with L-NIBC ligand (L-NIBC-AuNPs) were used as a control. The method for producing gold nanoparticles with L-NIBC ligand was given in the 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).

[0075] 2.2.1 Morphological examination using transmission electron microscopy (TEM)

[0076] The test powders (L-NIBC-AuCs sample and L-NIBC-AuNPs sample) were dissolved in ultrapure water to 2 mg/L as samples, and then the test samples were prepared by the hanging drop method. More specifically, 5 μL of the samples were dropped onto the ultrathin carbon film, naturally evaporated until the water droplet disappeared, and then the morphology of the samples was observed using a JEM-2100F STEM/EDS high-resolution field emission TEM.

[0077] 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.

[0078] The images in Fig. 2 show that each of the L-NIBC-AuCs samples has a uniform particle size and good dispersity, and the average diameter of the L-NIBC-AuCs (related to the diameter of the gold core) is 1.1 nm, 1.8 nm and 2.6 nm, respectively, which are consistent with the results in panels C, F and I of Fig. 2. In comparison, the L-NIBC-AuNPs samples have a larger particle size. Their average diameter (related to the diameter of the gold core) is 3.6 nm, 6.0 nm, 10.1 nm and 18.2 nm, respectively, which are completely consistent with the results in panels C, F, I and L of Fig. 1.

[0079] 2.2.2 Ultraviolet (UV) - visible (vis) absorption spectra

[0080] The 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 the 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.

[0081] The UV-vis absorption spectra of four L-NIBC-AuNPs samples 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; The 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.

[0082] Fig. 1 shows that due to the surface plasmon effect, the absorption peak of L-NIBC-AuNPs was observed at a wavelength of about 520 nm. The position of the absorption peak depends on the particle size. When the particle size is 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.

[0083] Fig. 2 shows that in the UV absorption spectra of three L-NIBC-AuCs samples with different particle sizes, the absorption peak of the surface plasmon effect at 520 nm disappeared, and two obvious absorption peaks appeared above 560 nm, and the positions of the absorption peaks slightly changed depending on the particle sizes of AuCs. This is explained by the fact that AuCs exhibits molecule-like properties due to the decomposition of the face-centered cubic structure, which leads to a gap in the compound density of AuCs, splitting of the energy level, 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 ligand-bound AuCs.

[0084] 2.2.3 Fourier Transform Infrared Spectroscopy

[0085] The infrared spectra were measured on a Bruker VERTEX80V Fourier transform infrared spectrometer in the total reflection mode in solid powder under high vacuum. The scanning range was 4000-400 cm ^-1 , the number of scans was 64. L-NIBC-AuCs samples were taken as examples, the test samples 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.

[0086] Fig. 3 shows the infrared spectrum of L-NIBC-AuCs with different particle sizes. Compared with pure L-NIBC (bottom curve), the SH 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, confirming that the L-NIBC molecules were successfully bound to the surface of AuCs through Au-S bonding. It can also be seen from the figure that the infrared spectrum of AuCs bound with ligands is independent of their size.

[0087] AuCs bound with other ligands were prepared in a manner similar to that described above, except that the solvent for solution B, the ratio between HAuCl ₄ and ligand, the reaction time, and the amount of NaBH ₄ 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 the dipeptide CR, dipeptide RC, or 1-[(2S)-2-methyl-3-thiol-1-oxopropyl]-L-proline is used as the ligand, water is selected as the solvent, etc.; other steps are similar, so further details are not given here.

[0088] In the context of the present invention, a series of ligand-bound AuCs were prepared by the above-described method. The ligands and characteristics of the preparation method are given in Table 1.

[0090] 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), Fig. 5 (RC-AuCs), Fig. 6 (Cap-AuCs) (Cap stands for 1-[(2S)-2-methyl-3-thiol-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-AuCs), Fig. 13 (CDEVD-AuCs) and Fig. 14 (DEVDC-AuCs). Fig. 4-12 show the UV spectra (panel A), infrared spectra (panel B), TEM images (panel C), and particle size distribution (panel D). Fig. 13 and 14 show the UV spectra (panel A), TEM images (panel B), and particle size distribution (panel C).

[0091] The results show that the diameters of AuCs bound to the different 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, the absorption peak is absent, 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 ligand thiol (between the dotted lines in panel B of Fig. 4-8), while other infrared characteristic peaks remain, presumably because all the ligand molecules were successfully bound to gold atoms to form ligand-bound AuCs, and in the present invention, the ligand-bound AuCs listed in Table 1 were successfully obtained.

[0092] Embodiment 3. Test samples for animal studies

[0093] A1: gold clusters bound to the L-NIBC ligand (L-NIBC-AuCs), gold core diameter in the range of 0.5-3.0 nm.

[0094] A2: gold clusters linked to the ligand N-acetyl-E-cysteine (L-NAC-AuCs), gold core diameter in the range of 0.5-3.0 nm.

[0095] A3: CR ligand-linked gold clusters (CR-AuCs), gold core diameter in the range of 0.5-3.0 nm.

[0096] A4: gold clusters linked to the L-cysteine ligand (L-C-AuCs), gold core diameter in the range of 0.5-3.0 nm.

[0097] A5: gold clusters bound to the DEVDC ligand (DEVDC-AuCs), gold core diameter in the range of 0.5-3.0 nm.

[0098] B1: L-NIBC-linked gold nanoparticles (L-NIBC-AuNPs), size distribution in the range of 6.2±1.2 nm.

[0099] All test samples were prepared according to the method described above with minor modifications, and their quality was assessed using the methods described above.

[0100] Embodiment 4. Forced Float Test (FST)

[0101] In the FST test, mice were placed in a cylindrical barrel with a water depth of 18 cm and a water temperature of 26°C. The mice swam in the barrel for 6 minutes. During this time, the device records the swimming time of the mice, and the swimming time for the last 4 minutes is used for data analysis. The immobility ratio (i.e., swimming time) (%) is calculated as 100 x immobility time / testing time. The higher the immobility ratio, the higher the degree of depression in the mice.

[0102] All data were analyzed using prism 6.01 (one-way ANOVA plus Dunnett's multiple comparison test).

[0103] 84 ICR mice were randomly divided into the following 7 groups (n-12): C, normal saline control group; A1, drug (L-NIBC-AuCs) administration group; A2, drug (L-NAC-AuCs) administration group; A3, drug (CR-AuCs) administration group; A4, drug (L-C-AuCs) administration group; A5, drug (DEVDC-AuCs) administration group; B1, drug (L-NIBC-AuNPs) administration group. All drugs were dissolved in normal saline. Mice in groups A1, A2, A3, A4, A5, and B1 were intraperitoneally injected with the drug once a day at a dose of 20 mg/kg body weight, the injection volume was 50 μL, and mice in the control group were intraperitoneally injected with the same volume of normal saline. On the 7th day (day 7), the drug was administered after the mice had adapted to laboratory conditions for 60 minutes, and the forced swim test (FST) was performed 30 minutes after administration.

[0104] Fig. 15 shows the results of the forced swimming test in mice in the control group and each of the drug administration groups. In the control group of mice, the immobility ratio was 67.8%±3.9%; in the gold cluster A1, A2, A3, A4, and A5 administration groups, the immobility ratio was 52.0%±5.7%, 48.7%±4.0%, 43.9%±4.9%, 47.9%±5.2%, or 50.2%±5.0%, respectively, which were significantly lower than those in the control group (*: P<0.05; **: P<0.01; ***: P<0.001). In contrast, no significant change was observed in the group of mice administered with gold nanoparticles B1 compared with the control group.

[0105] Embodiment 5: Tail Suspension Test

[0106] In the tail suspension test (TST), the tail tip of mice, 2 cm long, is attached to a horizontal wooden stick to cause the animals to hang. The distance between the mice and the environment is 5 cm. The immobility time of the mice (in seconds) is observed and recorded for 6 minutes. The longer the immobility time, the higher the degree of depression in the mice. All data are analyzed as described above.

[0107] Eighty-four ICR mice participated in the tail suspension test. The treatment schedule for groups was the same as the forced swim test described above.

[0108] Fig. 16 shows the results of the tail suspension test in the mice in the control group and each of the drug administration groups. In the control group, the immobility time of the mice was 140.3±12.4 seconds, while in the gold cluster administration groups A1, A2, A3, A4 and A5, the immobility time of the mice was 90.2±11.1 seconds, 70?4±11.4 seconds, 88.0±10.6 seconds, 68.8±12.2 seconds and 75.5±9.7 seconds, respectively, significantly decreased compared with the control group (*: P<0.05; **: P<0.01; ***: P<0.001). In contrast, no significant difference was observed in the gold nanoparticle administration group B1 compared with the control group.

[0109] The results show that five gold cluster drugs have significant therapeutic effects on depression, but gold nanoparticles are ineffective.

[0110] Embodiment 6: Social Stress Test in an Animal Model

[0111] 6.1 Modeling and drug administration

[0112] The animal model of social stress simulates the situation in which humans experience frustration from isolation and helplessness in everyday interactions by allowing aggressive CD-1 mice to attack C57BL/6J (abbreviated C57) mice for a short period of time, and allowing C57 mice to be threatened and fearful of aggressive CD-1 mice for an extended period of time. When the mice are repeatedly exposed to social failure pressure, this causes overt depressive-like manifestations characterized by lack of interest, anxiety, and socially avoidant behavior. This model is an animal model that is closer to the etiology of human depression.

[0113] First, normal C57 mice were tested against aggressive CD-1 mice.

[0114] (1) Aggressive CD-1 mice were placed on one side of a mouse cage separated by a perforated transparent partition and kept for 24 hours;

[0115] (2) Normal C57 mice were placed in the same cage with aggressive CD-1 mice according to their numbering and stimulated for 5 minutes (avoiding injury); then the C57 mice were removed and placed in the other side of the same cage, which was divided by a perforated transparent partition (the two sides were separated by a transparent partition), so that the C57 mice could see the aggressive CD-1 mice and smell the CD-1 mice; the C57 mice and CD-1 mice were kept together overnight for 24 hours;

[0116] (3) During the experiment, each C57 mouse was continuously exposed to stress for 20 days. For 20 days, C57 mice from different model groups were placed in cages with aggressive CD-1 mice according to their numbering for daily stimulation. During the 20 days, no more than one contact was made between any two mice (i.e., there were no repeated contacts to avoid habituation);

[0117] (4) In the normal control group, C57 mice separated by transparent partitions were used instead of aggressive CD-1 mice, and the C57 mice in the normal control group were changed daily according to the numbering;

[0118] (5) At the end of the last day of the simulation, the C57 model mice and normal control C57 mice were kept in single cages separately for 24 hours and then behavioral tests were performed.

[0119] On the 15th day of social stress, the C57 model mice were administered drugs A1, A2, A3, A4, A5, and B1 by intraperitoneal injection once a day at a dose of 20 mg/kg for 9 consecutive days, resulting in an A1 administration group, an A2 administration group, an A3 administration group, an A4 administration group, an A5 administration group, and a B1 administration group. Starting from the 7th day of drug administration (the 21st day from the start of the model), the behavioral tests were performed 1 hour after drug administration. The normal control group and the model control group received the same volume of saline, and the behavioral tests were performed on the corresponding days. There were 15 mice in each group.

[0120] 6.2. Behavioural tests

[0121] The first behavioral test was a social behavior test, which was used to detect social avoidance behavior (a typical sign of depression) in mice. This test was conducted on the 14th and 21st day from the start of the modeling, respectively. The first test on the 14th day was conducted to assess the success of the modeling, and the second on the 21st day to assess the effect of the drugs on the social behavior of the mice.

[0122] The social behavior test consisted of two stages, each lasting 2.5 min with an interval of 30 s. In the first stage (no-target stage), a gas-permeable cylinder with a radius of 4 cm is placed on the side of the open area, and the area within 8 cm from the center of the cylinder is defined as the interaction zone (IZ). The time spent in the interaction zone by successfully modeled C57 mice was recorded and designated as T1. In the second stage (target), a CD-1 mouse, which had no contact with the mouse during the modeling stage, is placed in the cylinder, providing visual and olfactory interaction between them (but not allowing physical contact), the time of visual and olfactory interaction between them at this stage in the interaction zone is recorded and designated as T2. The ratio of T2 to T1 (T2/T1) is called the social interaction coefficient (SIC). The lower this value, the more obvious the social avoidance behavior. And vice versa, the better the antidepressant ability of the drug.

[0123] The second behavioral test was the elevated plus maze test, which was used to detect anxiety-like behavior (another characteristic feature of depression) in mice. The elevated plus maze test was started on day 23 of the simulation. Model C57 mice were placed on the elevated plus maze platform, and the moving time, moving distance, and moving time of the mice in each arm were observed and recorded for 5 minutes. The following data were calculated: (1) the percentage of time spent by mice in the open arm to the time spent by mice in both the open and closed arms: TO% = 100% * time spent in open arm / (time spent in open arm + time spent in closed arm); (2) the percentage ratio of the distance of movement in the open arm to the total distance of movement: DO%=100%*distance of movement in the open arm/(distance of movement in the open arm + distance of movement in the closed arm); (3) the percentage ratio of the number of shuttle movements in the open arm to the total number of shuttle movements: EO%=100%*number of shuttle movements in the open arm/(number of shuttle movements in the open arm + number of shuttle movements in the closed arm). The higher the specified values, the higher the anti-anxiety effect of the drug.

[0124] All data were analyzed using prism 6.01 (one-way ANOVA plus Dunnett's multiple comparison test).

[0125] 6.3. Research results

[0126] 6.3.1 Successful Model Creation

[0127] The social behavior ability of the mice was tested on the 14th day (1 day before the drug administration). The results are shown in Fig. 17. Figs. 17A to 17C show the results of T1, T2, and T2/T1 of the mice in the social behavior test. The results showed that the T2/T1 value of the model mice was significantly lower than that of the normal mice, and the difference was highly significant (P<0.001, ***), indicating that the social ability of the model mice was significantly lower than that of the normal mice, and the model was successfully established.

[0128] 6.3.2. Effect of drugs on social behavior of model mice

[0129] Fig. 18 shows the effect of gold cluster drugs on the social behavior test of mice with chronic social stress, using drug A1 as an example. The results showed that the five gold cluster drugs A1-A5 could significantly improve the T2/T1 ratio in the model mice, and the difference was significant compared with the control group (P<0.05, *), indicating that the five gold cluster drugs could effectively improve social behavioral ability and have significant antidepressant effect. However, compared with the control group, the T2/T1 value in the B1 group did not improve, indicating that there was no antidepressant effect.

[0130] Fig. 19 shows the results of the effect of gold cluster preparations on mice with chronic social stress in the elevated plus maze test using preparation A4 as an example. The results showed that on the 23rd day of the model, five gold cluster preparations A1-A5 could significantly increase the time of movement in the open arm (FIG. 19A), the distance of movement in the open arm (FIG. 19B), the time of movement in the open arm (FIG. 19C), the percentage (TO%) of time in the open arm to the time in the open arm + closed arm (FIG. 19D), the percentage (DO%) of the distance of movement in the open arm to the total distance of movement (FIG. 19E) and the percentage (EO%) of the time of movement in the open arm to the total time of movement (FIG. 19F). Using the example of drug A4, it can be noted that the values of TO% and EO% in the drug administration group had significant differences compared to the model control group (P<0.05, *). These results indicate that gold cluster drugs have a pronounced anti-anxiety effect. However, drug B1 did not improve the test results, which indicates that gold nanoparticles larger than 3.0 nm do not affect the anxiety-like behavior of model mice.

[0131] The obtained results indicate that gold cluster-based drugs can significantly improve social behavior and anxiety-like behavior in model mice and can be used as antidepressants. However, gold nanoparticles larger than 3.0 nm do not have such an effect and cannot be used to develop antidepressant drugs.

[0132] Similar effects are also exhibited by other ligand-bound AuCs of various sizes, although their effects vary to some extent. They are not described in detail here.

[0133] Although the present invention has been disclosed with reference to specific embodiments, it is intended that the embodiments be illustrative and not limiting of the scope of the invention. Alternative embodiments of the present invention will be apparent to those of ordinary skill in the art to which the present invention pertains. 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 appended claims and is confirmed by the description given above.

Links

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Claims (13)

1. Use of a ligand-bound gold cluster for treating depression in a subject, wherein the ligand-bound gold cluster comprises: golden core; and gold core-linked ligand ; where the gold core has a diameter in the range of 0.5-3 nm; the ligand is one selected from the group consisting of L-cysteine and its derivatives, D-cysteine and its derivatives, cysteine-containing oligopeptides and their derivatives, 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); 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 their derivatives are cysteine-containing dipeptides, cysteine-containing tripeptides, cysteine-containing tetrapeptides or cysteine-containing pentapeptides; and 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-aminoethanethiol (CSH), 3-mercaptopropionic acid (MPA) and 4-mercaptobenic acid (p-MBA). 2. The use according to claim 1, wherein the gold core has a diameter in the range of 0.5-2.6 nm. 3. The use according to claim 1, 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). 4. The use according to claim 1, 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). 5. The 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. The use according to claim 1, wherein the cysteine-containing pentapeptides are selected from the group consisting of cysteinyl-asparaginyl-glutaminyl-valinyl-asparagine (CDEVD) and asparaginyl-glutaminyl-valinyl-asparaginyl-cysteine (DEVDC).