Compositions and methods for the treatment of multiple sclerosis

Abstract

Disclosed is the pharmaceutical use of a gold cluster for treating multiple sclerosis in a subject, the gold cluster comprising a gold core and a ligand bound to the gold core.

Description

[Technical field]

[0001] The present invention relates to the field of multiple sclerosis, and in particular to compositions and methods for the treatment of multiple sclerosis. [Background technology]

[0002] Multiple sclerosis (MS), meaning "scar tissue in multiple areas," is an autoimmune disease in which the immune system attacks the myelin sheath that surrounds and protects nerve fibers in the central nervous system (CNS), causing inflammation. When the myelin sheath or nerve fibers are damaged or destroyed by MS, the damage to areas of the CNS can cause a variety of neurological symptoms that vary in type and severity in MS patients. There are four types of MS: clinically isolated syndrome (CIS), relapse-remitting MS (RRMS), primary progressive MS (PPMS), and secondary progressive MS (SPMS). Common symptoms include muscle weakness, numbness and tingling, Lhermitte's sign, bladder problems, bowel problems, fatigue, dizziness and vertigo, sexual dysfunction, spasticity and muscle spasms, tremors, vision problems, changes in gait and mobility, emotional changes and depression, learning and memory problems, as well as pain.

[0003] The cause of multiple sclerosis is still unknown, but it is thought that a combination of genetic susceptibility, immune system abnormalities, and environmental factors leads to the disease.

[0004] Although conventional medicines are effective in slowing progression by altering how the immune system works or relieving symptoms during flares when symptoms worsen, new drugs and methods for the treatment of MS remain needed. Summary of the Invention [Problem to be solved by the invention]

[0005] It is an object of the present invention to provide a pharmaceutical composition for the treatment of multiple sclerosis in a subject.

[0006] A particular embodiment of the present invention is a medicament for treating multiple sclerosis. A composition, the pharmaceutical composition comprising: Gold Cluster (AuC) Includes wherein the AuC comprises a gold core and a ligand bound to the gold core; Pharmaceutical Compositions It is.

[0007] The above Pharmaceutical Compositions In certain embodiments, the gold core has a diameter of less than 3 nm, while in certain embodiments, the gold core has a diameter in the range of 0.5 to 2.6 nm.

[0008] The above Pharmaceutical Compositions In certain embodiments, the ligand is a member 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.

[0009] The above Pharmaceutical Compositions In certain embodiments, the 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 the 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).

[0010] The above Pharmaceutical Compositions In certain embodiments, said cysteine-containing oligopeptides and their derivatives are cysteine-containing dipeptides, cysteine-containing tripeptides or cysteine-containing tetrapeptides.

[0011] The above Pharmaceutical CompositionsIn certain embodiments, the cysteine-containing dipeptide is selected from the group consisting of L-cysteine-L-arginine dipeptide (CR), L-arginine-L-cysteine ​​dipeptide (RC), L-histidine-L-cysteine ​​dipeptide (HC), and L-cysteine-L-histidine dipeptide (CH).

[0012] The above Pharmaceutical Compositions In certain embodiments, the cysteine-containing tripeptide is selected from the group consisting of glycine-L-cysteine-L-arginine tripeptide (GCR), L-proline-L-cysteine-L-arginine tripeptide (PCR), L-lysine-L-cysteine-L-proline tripeptide (KCP), and L-glutathione (GSH).

[0013] The above Pharmaceutical Compositions In a particular embodiment, said cysteine-containing tetrapeptide is selected from the group consisting of glycine-L-serine-L-cysteine-L-arginine tetrapeptide (GSCR) and glycine-L-cysteine-L-serine-L-arginine tetrapeptide (GCSR).

[0014] The above Pharmaceutical Compositions In certain embodiments, the other thiol-containing compound is selected from the group consisting of 1-[(2S)-2-methyl-3-thiol-1-oxopropyl]-L-proline, thioglycolic acid, mercaptoethanol, thiophenol, D-penicillamine (D-3-trolovol), N-(2-mercaptopropionyl)-glycine, and dodecyl mercaptan.

[0015] The objects and advantages of this invention will become apparent from the following detailed description of the preferred embodiment taken in conjunction with the accompanying drawings. [Brief description of the drawings]

[0016] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Preferred embodiments according to the present invention will now be described with reference to the drawings, in which like reference numbers indicate like elements.

[0017] [Figure 1] Figure 1 shows ultraviolet-visible (UV) spectra, transmission electron microscope (TEM) images, and particle size distribution diagrams of gold nanoparticles modified with the ligand L-NIBC (L-NIBC-AuNPs) with various particle sizes.

[0018] [Diagram 2] The ultraviolet-visible (UV) spectra, TEM images, and particle size distribution diagrams of ligand L-NIBC-bound gold clusters (L-NIBC-AuCs) with various particle sizes are shown.

[0019] [Diagram 3] 1 shows infrared spectra of L-NIBC-AuCs with various particle sizes.

[0020] [Figure 4] The UV spectrum, infrared spectrum, TEM image, and particle size distribution map of the gold clusters bound to the ligand CR (CR-AuCs) are shown.

[0021] [Diagram 5] The UV spectrum, infrared spectrum, TEM image, and particle size distribution map of the gold clusters bound with the ligand RC (RC-AuCs) are shown.

[0022] [Figure 6] The UV spectrum, infrared spectrum, TEM image, and particle size distribution diagram of the ligand 1-[(2S)-2-methyl-3-thiol-1-oxopropyl]-L-proline (i.e., Cap)-bound gold clusters (Cap-AuCs) are shown.

[0023] [Figure 7] The UV spectrum, infrared spectrum, TEM image, and particle size distribution diagram of gold clusters bound to the ligand GSH (GSH-AuCs) are shown.

[0024] [Figure 8]The UV spectrum, infrared spectrum, TEM image, and particle size distribution diagram of gold clusters bound to the ligand D-NIBC (D-NIBC-AuCs) are shown.

[0025] [Figure 9] The UV spectrum, infrared spectrum, TEM image, and particle size distribution diagram of gold clusters bound to the ligand L-cysteine ​​(L-Cys-AuCs) are shown.

[0026] [Figure 10] Graph showing clinical scores of EAE.

[0027] [Figure 11] FIG. 1 shows the levels of (a) TNF-α, (b) IL-17 and (c) IFN-γ in the spinal cord, where 1 is the naive group, 2 is the saline treatment group, 3 is the prednisone treatment group, 4 is the 50 mg / kg L-Cys-AuCs treatment group, 5 is the 20 mg / kg L-Cys-AuCs treatment group and 6 is the 5 mg / kg L-Cys-AuCs treatment group.

[0028] [Figure 12] (a) is a photograph of HE staining of the spinal cord showing infiltration of immune cells into the spinal cord, and (b) is a bar graph showing the histological scale of inflammation, where 1 is the naive group, 2 is the saline treatment group, 3 is the prednisone treatment group, 4 is the 50 mg / kg L-Cys-AuCs treatment group, 5 is the 20 mg / kg L-Cys-AuCs treatment group, and 6 is the 5 mg / kg L-Cys-AuCs treatment group. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] The present invention may be understood more readily by reference to the following detailed description of specific embodiments of the invention.

[0030] Throughout this application, publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.

[0031] Gold clusters (AuCs) are a special form of gold that lies between gold atoms and gold nanoparticles. AuCs are less than 3 nm in size and consist of a few to hundreds of gold atoms, which disrupts the face-centered cubic stacking structure of gold nanoparticles. As a result, AuCs exhibit a molecular-like discretized electronic structure with a different HOMO-LUMO gap, unlike the continuous or quasi-continuous energy levels of gold nanoparticles. This causes the disappearance of the surface plasmon resonance effect of conventional gold nanoparticles and the corresponding plasmon resonance absorption band (520 ± 20 nm) in the uv-vis spectrum.

[0032] The present invention provides ligand-conjugated AuC.

[0033] In certain embodiments, the ligand-bound AuC comprises a ligand and a gold core, the ligand being bound to the gold core. In certain embodiments, 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.

[0034] In certain embodiments, the ligand of the ligand-conjugated AuC is a thiol-containing compound or an oligopeptide, hi certain embodiments, the ligand is bound to a gold core via an Au-S bond to form the ligand-conjugated AuC.

[0035] 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).

[0036] In certain embodiments, the ligand is, but is not limited to, a cysteine-containing oligopeptide and its derivatives. In certain embodiments, the cysteine-containing oligopeptide is a cysteine-containing dipeptide. In certain embodiments, the cysteine-containing dipeptide is L-cysteine-L-arginine dipeptide (CR), L-arginine-L-cysteine ​​dipeptide (RC), or L-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-cysteine-L-arginine tripeptide (GCR), L-proline-L-cysteine-L-arginine tripeptide (PCR), or L-glutathione (GSH). In certain embodiments, the cysteine-containing oligopeptide is a cysteine-containing tetrapeptide. In a particular embodiment, said cysteine-containing tetrapeptide is glycine-L-serine-L-cysteine-L-arginine tetrapeptide (GSCR) or glycine-L-cysteine-L-serine-L-arginine tetrapeptide (GCSR).

[0037] In certain embodiments, the ligand is a thiol-containing compound, hi certain embodiments, the thiol-containing compound is 1-[(2S)-2-methyl-3-thiol-1-oxopropyl]-L-proline, thioglycolic acid, mercaptoethanol, thiophenol, D-penicillamine, or dodecyl mercaptan.

[0038] The present invention provides a pharmaceutical composition for the treatment of multiple sclerosis in a subject. In certain embodiments, the subject is a human. In certain embodiments, the subject is a pet animal, such as a dog.

[0039] In certain embodiments, the pharmaceutical composition comprises the ligand-bound AuC disclosed hereinabove and a pharmacologically acceptable excipient. In certain embodiments, the excipient is a phosphate buffer or saline.

[0040] The present invention provides the use of the ligand-conjugated AuCs disclosed hereinabove in the manufacture of a medicament for the treatment of multiple sclerosis in a subject.

[0041] The present invention provides the use of the ligand-conjugated AuCs disclosed above for treating multiple sclerosis in a subject, or a method of treating multiple sclerosis in a subject using the ligand-conjugated AuCs disclosed above.In certain embodiments, the treatment method comprises administering a pharmacologic effective amount of the ligand-conjugated AuCs to the subject.The pharmacologic effective amount can be confirmed by routine in vivo studies.

[0042] The following examples are provided merely for the purpose of illustrating the principles of the present invention, and are not intended to limit the scope of the invention in any way.

[0043] Working Example

[0044] 1. Preparation of ligand-bound AuCs

[0045] 1.1 HAuCl4 was dissolved in methanol, water, ethanol, n-propanol, or ethyl acetate to obtain solution A with a HAuCl4 concentration of 0.01 to 0.03 M.

[0046] 1.2 The ligand was dissolved in the solvent to obtain solution B having a ligand concentration of 0.01 to 0.18 M. The ligands include 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 (L-cysteine-L-arginine dipeptide (CR), L-arginine-L-cysteine ​​dipeptide (RC), L-cysteine-L-histidine (CH), glycine-L-cysteine-L-arginine tripeptide (GCR), L-proline ... cysteine-containing peptides, such as, but not limited to, dipeptides, tripeptides, tetrapeptides and other cysteine-containing peptides, such as, but not limited to, L-glutathione (GSH), glycine-L-serine-L-cysteine-L-arginine tetrapeptide (GSCR) and glycine-L-cysteine-L-serine-L-arginine tetrapeptide (GCSR); and other thiol-containing compounds, such as, but not limited to, one or more of 1-[(2S)-2-methyl-3-thiol-1-oxopropyl]-L-proline, thioglycolic acid, mercaptoethanol, thiophenol, D-penicillamine and dodecyl mercaptan. The solvent was 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, t-butyl methyl ether, isopropyl acetate, dimethyl sulfoxide, ethyl formate, isobutyl acetate, methyl acetate, 2-methyl-1-propanol, and propyl acetate.

[0047] 1.3 Solution A and solution B were mixed so that the molar ratio of HAuCl4 to the ligand was 1: (0.01-100), and the mixture was stirred in an ice bath for 0.1-48 hours, and then a 0.025-0.8M NaBH4 solution in water, ethanol, or methanol was added, and the mixture was stirred in an ice bath for 0.1-12 hours. The molar ratio of NaBH4 to the ligand was 1: (0.01-100).

[0048] 1.4 After the reaction was completed, the reaction solution was centrifuged for 10-100 minutes at a gradient of 8000-17500 r / min using an ultrafiltration tube with a MWCO of 3K-30K to obtain ligand-bound AuCs precipitates with different average particle sizes. The pores of the filtration membranes of the ultrafiltration tubes with different MWCOs directly determined the sizes of the ligand-bound AuCs that could pass through the membranes. This step may be omitted if desired.

[0049] 1.5 The ligand-bound AuCs precipitates with different average particle sizes obtained in step (1.4) were dissolved in water, loaded into a dialysis bag, and dialyzed in water at room temperature for 1-7 days.

[0050] 1.6 After dialysis, the ligand-bound AuCs was freeze-dried for 12 to 24 hours to obtain the ligand-bound AuCs as a powder or aggregated substance.

[0051] As can be seen from the measurements, the particle size of the powder or aggregated material obtained by the above method was less than 3 nm (overall, distributed over a range of 0.5-2.6 nm). There was no obvious absorption peak at 520 nm. It was found that the obtained powder or aggregate was ligand-bound AuCs.

[0052] 2. Preparation and identification of AuCs bound with different ligands

[0053] 2.1 Preparation of L-NIBC-bound AuCs, i.e., L-NIBC-AuCs

[0054] Taking the ligand L-NIBC as an example, the preparation and confirmation of L-NIBC-bound AuCs are described in detail below.

[0055] 2.1.1 1.00 g of HAuCl4 was weighed out and dissolved in 100 mL of methanol to obtain a 0.03 M solution A.

[0056] 2.1.2 0.57 g of L-NIBC was weighed out and dissolved in 100 mL of glacial acetic acid (acetic acid) to obtain a 0.03 M solution B.

[0057] 2.1.3 1 mL of solution A was weighed out and mixed with 0.5 mL, 1 mL, 2 mL, 3 mL, 4 mL, or 5 mL of solution B, respectively (i.e., the molar ratios of HAuCl4 and L-NIBC were 1:0.5, 1:1, 1:2, 1:3, 1:4, and 1:5, respectively), and reacted in an ice bath with stirring for 2 hours. When the solution turned from light yellow to colorless, 1 mL of freshly prepared 0.03 M (prepared by weighing 11.3 mg of NaBH4 and dissolving it in 10 mL of ethanol) NaBH4 ethanol solution was promptly added. After the solution turned dark brown, the reaction was continued for 30 minutes, and 10 mL of acetone was added to stop the reaction.

[0058] 2.1.4 After the reaction, the reaction solution was subjected to gradient centrifugation to obtain L-NIBC-AuCs powders with different particle sizes. The specific method is as follows. That is, after the reaction is completed, the reaction solution was transferred to an ultrafiltration tube with a MWCO of 30K and a volume of 50mL, centrifuged at 10,000r / min for 20 minutes, and the retentate in the inner tube was dissolved in ultrapure water to obtain a powder with a particle size of about 2.6nm. Then, the mixture in the outer tube was transferred to an ultrafiltration tube with a volume of 50mL and a MWCO of 10K, and centrifuged at 13,000r / min for 30 minutes. The retentate in the inner tube was dissolved in ultrapure water to obtain a powder with a particle size of about 1.8nm. Then, the mixture in the outer tube was transferred to an ultrafiltration tube with a volume of 50mL and a MWCO of 3K, and centrifuged at 17,500r / min for 40 minutes. The retentate in the inner tube was dissolved in ultrapure water to obtain a powder with a particle size of approximately 1.1 nm.

[0059] 2.1.5 The powders with three different particle sizes obtained by gradient centrifugation were precipitated, the solvent was removed respectively, the crude products were blown dry with N2, dissolved in 5mL of ultrapure water, placed in a dialysis bag (MWCO 3KDa), the dialysis bag was placed in 2L of ultrapure water, the water was changed every other day, dialyzed for 7 days, freeze-dried, and kept for future use.

[0060] 2.2 Identification of L-NIBC-AuCs

[0061] Identification experiments were carried out on the powder (L-NIBC-AuC) obtained above. At the same time, gold nanoparticles modified with the ligand L-NIBC (L-NIBC-AuNPs) were used as a control. The preparation method of gold nanoparticles with the ligand L-NIBC is referred to references (W. Yan, L. Xu, C. Xu, W. Ma, H. Kuang, L. Wang and NAKotov, Journal of American Chemical Society 2012, 134, 15114; X. Yuan, B. Zhang, Z. Luo, Q. Yao, DT Leong, N. Yan and J. Xie, Angewandte Chemie International Edition 2014, 53, 4623).

[0062] 2.2.1 Morphology observation using a transmission electron microscope (TEM)

[0063] The test powders (L-NIBC-AuCs sample and L-NIBC-AuNPs sample) were dissolved in ultrapure water to 2 mg / L to prepare the test samples, and the hanging drop method was used to prepare the test samples. More specifically, 5 μL of the sample was dropped onto an ultrathin carbon film, and the film was allowed to evaporate naturally until there were no water droplets, and the morphology of the sample was observed using a JEM-2100F STEM / EDS field emission high-resolution TEM.

[0064] Four TEM images of L-NIBC-AuNPs are shown in Figure 1 B, E, H, and K, and three TEM images of L-NIBC-AuCs are shown in Figure 2 B, E, and H.

[0065] As shown in the images in Figure 2, each L-NIBC-AuCs sample had uniform particle size and good dispersibility, and the average diameters of L-NIBC-AuCs (referred to as the diameter of the gold core) were 1.1 nm, 1.8 nm, and 2.6 nm, respectively, which were in good agreement with the results in Figure 2C, F, and I. In contrast, the L-NIBC-AuNPs sample had larger particle sizes. Their average diameters (referred to as the diameter of the gold core) were 3.6 nm, 6.0 nm, 10.1 nm, and 18.2 nm, respectively, which were in good agreement with the results in Figure 1C, F, I, and L.

[0066] 2.2.2 Ultraviolet (UV)-visible (vis) absorption spectrum

[0067] 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 The UV-vis absorption spectrum was measured at room temperature. The scanning range was 190-1100 nm, the sample cell was a standard quartz cuvette with a path length of 1 cm, and the reference cell was filled with ultrapure water.

[0068] The UV-vis absorption spectra of four L-NIBC-AuNPs samples with different sizes are shown in Figure 1A, D, G, and J, and the statistical distribution of particle sizes is shown in Figure 1C, F, I, and L. The UV-vis absorption spectra of three L-NIBC-AuCs samples with different sizes are shown in Figure 2A, D, and G, and the statistical distribution of particle sizes is shown in Figure 2C, F, and I.

[0069] As shown in FIG. 1, due to the surface plasmon effect, L-NIBC-AuNPs had an absorption peak at about 520 nm. The position of the absorption peak was related to the particle size. When the particle size was 3.6 nm, the UV absorption peak appeared at 516 nm, when the particle size was 6.0 nm, the UV absorption peak appeared at 517 nm, when the particle size was 10.1 nm, the UV absorption peak appeared at 520 nm, and when the particle size was 18.2 nm, the absorption peak appeared at 523 nm. None of the four samples had an absorption peak above 560 nm.

[0070] As shown in Figure 2, the UV absorption spectra of L-NIBC-AuCs samples with different particle sizes showed the disappearance of the absorption peak due to the surface plasmon effect at 520 nm, and two obvious absorption peaks appeared above 560 nm, with slight differences in the positions of the absorption peaks depending on the particle size of AuCs. This is because AuCs exhibits molecular-like properties due to the collapse of the face-centered cubic structure, which results in discontinuity in the density of states of AuCs, splitting of the energy levels, disappearance of the plasmon resonance effect, and the appearance of a new absorption peak in the long-wave direction. It can be concluded that all three powder samples with different particle sizes obtained above were ligand-bound AuCs.

[0071] 2.2.3 Fourier transform infrared spectroscopy

[0072] Infrared spectra were measured using a Bruker VERTEX80V Fourier transform infrared spectrometer in solid powder high vacuum total reflection mode. The scan range was 4000–400 cm. -1 The number of scans was 64. Taking the L-NIBC-AuCs sample as an example, the test samples were L-NIBC-AuCs dry powders with three different particle sizes, and the control sample was pure L-NIBC powder. The results are shown in Figure 3.

[0073] Figure 3 shows the infrared spectra of L-NIBC-AuCs with different particle sizes. Compared with pure L-NIBC (lower curve), the SH stretching vibrations of L-NIBC-AuCs with different particle sizes are all located in the range of 2500 to 2600 cm -1 The infrared spectrum of the ligand-bound AuCs was completely disappeared at 37°C, whereas the other characteristic peaks of L-NIBC still remained, proving that the L-NIBC molecules were successfully bound to the surface of AuCs via Au-S bonds. This figure also showed that the infrared spectrum of the ligand-bound AuCs was independent of their size.

[0074] AuCs bound with other ligands were prepared in the same manner as above, except that the solvent of solution B, the ratio of HAuCl4 to ligand, reaction time, and the amount of NaBH4 added were slightly adjusted. Other steps were also performed in the same manner, for example, when L-cysteine, D-cysteine, N-isobutyryl-L-cysteine ​​(L-NIBC) or N-isobutyryl-D-cysteine ​​(D-NIBC) was used as the ligand, acetic acid was selected as the solvent, and when dipeptide CR, dipeptide RC or 1-[(2S)-2-methyl-3-thiol-1-oxopropyl]-L-proline was used as the ligand, water was selected as the solvent, and further details are omitted here.

[0075] The present invention prepared a series of ligand-conjugated AuCs by the above-mentioned method. The ligands and preparation process parameters are shown in Table 1.

[0076] Table 1. Preparation parameters of AuCs with different ligands in the present invention. JPEG0007620632000001.jpg226164JPEG0007620632000002.jpg32164

[0077] The samples listed in Table 1 were confirmed by the above method. The properties of five different ligand-conjugated AuCs are shown in Figure 4 (CR-AuCs), Figure 5 (RC-AuCs), Figure 6 (Cap-AuCs) (Cap is 1-[(2S)-2-methyl-3-thiol-1-oxopropyl]-L-proline), Figure 7 (GSH-AuCs), and Figure 8 (D-NIBC-AuCs). Figures 4-8 show UV spectra (A), infrared spectra (B), TEM images (C), and particle size distribution (D).

[0078] As a result, it was found that the diameters of the AuCs bound to different ligands obtained from Table 1 were all less than 3 nm. The ultraviolet spectrum also showed the disappearance of a peak at 520±20 nm and the appearance of an absorption peak at other positions. The position of this absorption peak differed depending on the ligand, particle size, and structure. In certain situations, a special absorption peak was not formed, mainly due to the formation of a mixture of AuCs with different particle sizes and structures, or the position of the absorption peak was moved outside the range of the ultraviolet-visible spectrum by a certain special AuCs. On the other hand, in the Fourier transform infrared spectrum, the infrared absorption peak of the thiol of the ligand (between the dotted lines in Figures 4 to 8 B) disappeared, but all other infrared characteristic peaks were retained, indicating that all ligand molecules were successfully fixed on the surface of AuCs, and the present invention was successful in obtaining AuCs bound to the ligands listed in Table 1.

[0079] 3. Animal testing

[0080] 3.1 Materials and animals

[0081] 3.1.1 Test sample

[0082] L-Cys-AuCs with gold core diameters in the range of 0.5-1.5 nm (1.0 ± 0.5 nm) were used as test samples, and the preparation method followed the above method with slight modifications. Figure 9 shows the UV spectrum, infrared spectrum, TEM image, and particle size distribution diagram of the gold clusters (L-Cys-AuCs) bound with the ligand L-cysteine.

[0083] 3.1.2 Positive control sample

[0084] Prednisone (Shanghai Yuanye Biotechnology Co., Ltd.).

[0085] 3.1.3 Dosage Formulation

[0086] The required amount of prednisone was weighed out, the appropriate amount of saline was added, and the formulation was gently stirred to ensure proper mixing. L-Cys-AuCs was weighed out, the appropriate amount of saline was added, and the suspension was thoroughly mixed by stirring. All compounds were made fresh each day.

[0087] 3.1.4 Experimental animals

[0088] C57BL / 6N female mice aged 7 to 9 weeks were used. Animals were housed and maintained according to regulations.

[0089] JPEG0007620632000003.jpg67161

[0090] 3.1.6 Flow

[0091] On day 0, mice were randomly divided into groups according to body weight, and then injected subcutaneously into the left and right flanks with 200 μg myelin oligodendrocyte glycoprotein (MOG) peptide in complete Freund's adjuvant (CFA), 100 μl per site. Pertussis toxin (PTX) was injected by intraperitoneal administration at 0 and 48 hours after MOG immunization. Compounds were given from day 1 to day 28 according to Table 2. Animals (n=10) were scored daily for clinical signs of EAE. At the end of the experiment, spinal cords were stained with HE (n=5) and analyzed for TNF-α, IL-17, and IFN-γ by ELISA kits.

[0092] 3.2 Results

[0093] Animals were scored daily for clinical signs of EAE according to the EAE clinical scoring criteria (Table 3). As shown in Figure 10, L-Cys-AuCs at 50 mg / kg (50 mpK), 20 mg / kg (20 mpK) and 5 mg / kg (5 mpK) prevented paralysis in EAE mice starting on day 17. Prednisone was the positive control and significantly reduced clinical signs of EAE starting on day 13.

[0094] JPEG0007620632000004.jpg33140

[0095] TNF-α, IL-17 and IFN-γ analysis of spinal cord by ELISA kit. TNF-α, IL-17 and IFN-γ are indicators of inflammation. L-Cys-AuCs at 50 mg / kg, 20 mg / kg and 5 mg / kg significantly reduced the production of these inflammatory factors in EAE mice (Figure 11).

[0096] HE staining of the spinal cord was used to study the infiltration of immune cells into the spinal cord. During the induction of EAE, mononuclear inflammatory cells infiltrated the spinal cord and contributed to the paralysis of EAE mice, HE staining showed a significant increase in inflammatory cells in EAE mice, and L-Cys-AuCs at 50 mg / kg, 20 mg / kg, and 5 mg / kg significantly reduced immune cell infiltration (Figure 12(a)) and histological scale of inflammation (Figure 12(b)).

[0097] Other sizes of L-Cys-AuCs and other ligand-conjugated AuCs of different sizes are also effective in suppressing EAE, with some differences in their effects, which will not be described in detail here.

[0098] [Industrial Applicability]

[0099] The ligand-bound AuCs can be used to treat multiple sclerosis. They are suitable for industrial applications.

Claims

1. A pharmaceutical composition for treating multiple sclerosis, the pharmaceutical composition comprising gold clusters (AuC), the AuC comprising: Gold core and a ligand bound to said gold core; Including, The ligands include L-cysteine, N-isobutyryl-L-cysteine ​​(L-NIBC), N-acetyl-L-cysteine ​​(L-NAC), D-cysteine, N-isobutyryl-D-cysteine ​​(D-NIBC), N-acetyl-D-cysteine ​​(D-NAC), L-cysteine-L-arginine dipeptide (CR), L-arginine-L-cysteine ​​dipeptide (RC), L-histidine-L-cysteine ​​dipeptide (HC), L-cysteine-L-histidine dipeptide (CH), glycine-L-cysteine ​​dipeptide (Gc), and L-cysteine-L-histidine dipeptide (Hc).

1. A pharmaceutical composition comprising a compound selected from the group consisting of glycine-L-arginine tripeptide (GCR), L-proline-L-cysteine-L-arginine tripeptide (PCR), L-lysine-L-cysteine-L-proline tripeptide (KCP), L-glutathione (GSH), glycine-L-serine-L-cysteine-L-arginine tetrapeptide (GSCR), glycine-L-cysteine-L-serine-L-arginine tetrapeptide (GCSR), thioglycolic acid, mercaptoethanol, and D-penicillamine.

2. 2. The pharmaceutical composition of claim 1, wherein the gold core has a diameter of less than 3 nm.

3. 2. The pharmaceutical composition of claim 1, wherein the gold core has a diameter in the range of 0.5 to 2.6 nm.

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