CN109701000B - Novel use of gold cluster molecules - Google Patents

Abstract

The invention relates to a new application of gold cluster molecules, in particular to an application of gold cluster molecules in preparing a medicament for preventing, relieving and/or treating rheumatoid arthritis. The invention proves that the gold cluster molecules have positive anti-inflammatory and antirheumatic effects in animal models, which shows the great potential of the gold cluster molecules as a new category of rheumatoid arthritis therapeutic agents. More importantly, the gold cluster molecule has no significant toxic properties in vivo, and this safety profile is highly beneficial for rheumatoid arthritis patients who recover their symptoms once treatment is discontinued, as it produces no or little side effects.

Description

Novel use of gold cluster molecules

Technical Field

The invention relates to a new application of gold cluster molecules, belonging to the field of treatment of rheumatoid arthritis.

Background

Rheumatoid arthritis is characterized by persistent inflammation, pain and joint swelling caused by synovial cell proliferation. The skilled person has made great efforts to find a solution for treating this disease. Although inflammation is involved in causing rheumatoid arthritis, many classical anti-inflammatory drugs are ineffective in preventing or treating rheumatoid arthritis, patients with rheumatoid arthritis fail to respond to many classical anti-inflammatory drugs, and the disease at different stages responds differently to treatment. For detailed test results, see K.Phadke et al, Immunopharmacology, titled "Evaluation of the Effect of various Anti-Arthritic Drugs in Type II Collagen-Induced Mouse arthritis model" (Evaluation of the Effects of varied Anti-inflammatory Drugs on Type II Collagen-Induced Mouse arthritis model,1985, Vol.10, p.51-60), the entire contents of which are incorporated by reference.

Although the potential and sometimes significant toxicity problems have not been solved to a great extent, gold therapy involving oral gold drugs, Auranofin (Auranofin), in injectable and oral forms, remains an effective treatment for rheumatoid arthritis.

The orally administered auranofin form is a white, tasteless crystalline solid that is insoluble in water. The powder is unstable and should be protected from light and heat. The chemical complex alloy (I) is the active ingredient in auranofin with the chemical name 2,3,4,6-tetra-o-acetyl-1-thio- β -D-glucopyranoso-S- (triethyl-phosphine) -gold (2,3,4,6-tetra-o-acetyl-1-thio- β -D-glucopyranosato-S- (trietyl-phosphine) -gold), where the triethylphosphine group stabilizes the gold (I) thiol complex (fig. 2A, 2B and 2C). Auranofin has many side effects. Diarrhea is the most common report because the drug alters the absorption of salts and water through the colon and during intestinal transit. Other side effects include severe rashes, proteinuria, eye problems, aplastic anemia, and hematologic toxicity that patients have reported to experience.

Rheumatoid arthritis remains the most common disease associated with progressive disability, systemic complications, and early death. Current disease-modifying therapies produce only limited or partial responses. There is a lack of reliable predictive therapeutic response and toxicity. Since synovial inflammation tends to persist, sustained relief is almost impossible to achieve for rheumatoid arthritis, and continuous drug therapy is required, and therefore, there is an urgent need in the art to develop safe therapeutic agents for the treatment of rheumatoid arthritis.

Disclosure of Invention

In view of the above-mentioned real need, it is an object of the present invention to provide a novel method for treating rheumatoid arthritis.

The experiment of the invention proves that the gold cluster molecules (also called metal gold cluster molecules, gold cluster compound molecules, metal gold cluster compound molecules, gold cluster complexes and metal gold cluster complexes) have positive anti-inflammatory and antirheumatic effects in animal models, which shows the great potential of the gold cluster molecules as a new category of rheumatoid arthritis therapeutic agents. More importantly, the gold cluster molecule has no significant toxic properties in vivo, and this safety profile is highly beneficial for rheumatoid arthritis patients who recover their symptoms once treatment is discontinued, as it produces no or little side effects.

Thus, the present invention provides the use of a gold cluster molecule for the preparation of a medicament for the prevention, alleviation and/or treatment of rheumatoid arthritis and/or anti-inflammatory; wherein the gold cluster molecule has gold (0)_m(stabilizers)_nThe structural formula is shown in the specification, wherein m and n respectively represent 0-valence gold and the number of stabilizers, and the numbers of the stabilizers are integers which are not zero, and the stabilizers are used for stabilizing the 0-valence gold.

In some embodiments, the stabilizer comprises a group capable of stabilizing a 0-valent gold atom, the group including, for example, one or more of a thiol group, a guanidino group, an amide group, an imidazole group, a selenol group, a phosphine group, an amino group, an amine group, a carboxyl group, and a hydroxyl group.

In some embodiments, the stabilizing agent comprises one or more of PEG comprising the group, PLGA comprising the group, a protein, a sugar, a nucleic acid, a peptide, e.g., comprising one or more of polylysine, polyarginine, polyasparagine, polyaspartic acid, polyaspartate, polyglutamate, and glutathione; the polyaspartate is, for example, polyaspartate sodium salt.

In some embodiments, the gold cluster molecule has the structural formula Au_m- (peptide)_nOr (peptide)_n-Au_mWherein m and n respectively represent the number of gold atoms and the number of peptides in the gold cluster molecule, and are integers which are not zero.

In some embodiments, the peptide is one or more of SEQ ID NO 1, SEQ ID NO 2 (glutathione), SEQ ID NO 3, SEQ ID NO 4, or a serum protein;

SEQ ID NO：1：Cys-Cys-Tyr-Gly-Gly-Pro-Lys-Lys-Lys-Arg-Lys-Pro-Gly；

SEQ ID NO：2：Glu-Cys-Gly；

SEQ ID NO：3：Cys-Cys-Tyr；

SEQ ID NO：4：Cys-Tyr。

preferably, the serum protein is a human serum protein.

In some embodiments, the Au is gold_m- (peptide)_nOr (peptide)_n-Au_mIs Au_1～2000(SEQ ID NO：1)_1～2500、Au_1～2000(SEQ ID NO：2)_1～2500、Au_1～2000(SEQ ID NO：3)_1～2500、Au_1～2000(SEQ ID NO： 4)_1～2500Or HSA-Au_1～10000Said HSA represents human serum albumin, preferably Au₂₅(SEQ ID NO：1)₉、 Au₂₅(SEQ ID NO：2)₁₈、Au₅(SEQ ID NO：3)₃、Au₂₅(SEQ ID NO：4)₁₈Or HSA-Au₄₀₀₀。

In some embodiments, the dosage form of the medicament comprises an oral dosage form, a liquid suspension, or an injection. In some embodiments, the injectable formulation comprises an intraluminal injection, an intravenous injection, or an intramuscular injection.

In some embodiments, the intraluminal injection is an intraperitoneal injection, preferably, the intraperitoneal injection is an intraperitoneal injection liquid suspension.

The medicament of the invention can be prepared into a suitable dosage form for a subject according to the actual needs by the prior art, and can be orally administered when the medicament is an oral preparation, and can be intraperitoneally, intramuscularly or intravenously administered when the medicament is an injection preparation. The experiments herein performed in animal models in accordance with the present invention show that intraperitoneally administered gold cluster molecules exhibit significant anti-inflammatory and antirheumatic effects. Therefore, the gold cluster molecules are preferably prepared into injections, and are more preferably prepared into intraperitoneal injections.

In some embodiments, the gold cluster molecule has the structural formula Au_m- (peptide)_nOr (peptide)_n-Au_mWherein m and n respectively represent the number of gold atoms and the number of peptides in the gold cluster molecule and are integers which are not zero;

the peptide is SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4;

SEQ ID NO：1：Cys-Cys-Tyr-Gly-Gly-Pro-Lys-Lys-Lys-Arg-Lys-Pro-Gly；

SEQ ID NO：2：Glu-Cys-Gly；

SEQ ID NO：3：Cys-Cys-Tyr；

SEQ ID NO：4：Cys-Tyr；

preferably, the Au is_m- (peptide)_nOr (peptide)_n-Au_mIs Au₂₅(SEQ ID NO：1)₉、Au₂₅(SEQ ID NO： 2)₁₈、Au₅(SEQ ID NO：3)₃；Au₂₅(SEQ ID NO：4)₁₈；

The dosage form of the medicine is an intraperitoneal injection or a skin smearing preparation.

That is, the present invention discloses methods of administering gold cluster molecules in the prevention, alleviation and/or treatment of rheumatoid arthritis.

In one embodiment, stable gold cluster molecules are first obtained and a liquid suspension of a therapeutically effective amount of gold cluster molecules is administered to the abdominal cavity of an animal with rheumatoid arthritis to alleviate the development and progression of the disease.

In one embodiment, a liquid suspension of gold cluster molecules is administered orally to an animal with rheumatoid arthritis in a therapeutically effective amount to ameliorate the development and progression of the disease.

The terms "first," "second," "third," and "fourth," etc., may be used herein to distinguish one element from another, but are not necessarily used to describe a particular sequential or chronological order. It is to be understood that the terms so used are interchangeable. Furthermore, the terms "comprises," "comprising," "includes," "including," "has," "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, apparatus, or composition that comprises a list of features is not necessarily limited to those features but may include other features not expressly listed or inherent to such process, method, article, apparatus, or composition.

The term "gold cluster" refers to a gold atom cluster molecule in which the gold atoms form a geometric crystal structure. Gold geometric crystal structures can often be stabilized by forming non-covalent metallic bonds with thiol, guanidino, amide, imidazole, selenol, phosphine, amino, amine, carboxyl, hydroxyl groups contained in the stabilizer molecule. Gold cluster molecules typically fluoresce under UV excitation. The gold cluster molecules can be prepared according to conventional techniques, typically in the presence of stabilizer molecules in the reaction solution. For example, reacting gold (I) or gold (III) salt with a stabilizer comprising thiol, guanidino, amide, imidazole, selenol, phosphine, amino, amine, carboxyl, hydroxyl groups to form gold cluster molecules, the stabilizer in the reaction solution forming a non-covalent metallic bond with gold (0) for stabilization, the stabilizer molecules including, but not limited to, one or more of PEG containing said groups, PLGA containing said groups, proteins, sugars, nucleic acids, peptides, for example, including one or more of polylysine, polyarginine, polyasparagine, polyaspartic acid, polyaspartate, polyglutamate, and glutathione; the polyaspartate is, for example, polyaspartate sodium salt; the sugar is, for example, a polysaccharide; the peptide is, for example, a protein.

The term "Au_m- (peptide)_nOr (peptide)_n-Au_m(Au_m-peptide_nOr peptide_n-gold_m) "or" gold-peptide cluster complex "or" gold-peptide cluster "or" gold (0)_m-peptides_n(gold(0)_m-peptide_n) "or" peptide_n-gold_m(peptide_n-gold_m) "molecules are mutually useful, these are gold cluster molecules which are stable by forming with a peptide or polypeptide containing a thiol group, a guanidino group, an amide group, an imidazole group, a selenol group, a phosphine group, an amino group, an amine group, a carboxyl group, a hydroxyl group, wherein m and n each represent the number of gold atoms in the complex and the number of encapsulated molecules.

The term "stabilizer" may also be referred to as a "gold cluster capping molecule," which refers to a thiol-, guanidino-, amide-, imidazole-, selenol-, phosphine-, amino-, carboxyl-, hydroxyl-containing peptide or polymer molecule that can form a non-covalent metallic bond with a gold atom, thereby stabilizing the gold cluster geometry. These molecules include lipids, glutathione, polylysine, polyarginine, polyaspartamide, polyaspartic acid sodium salt, polyglutamate, PEG (polyethylene glycol) containing such groups, PLGA (polylactic-co-glycolic acid) containing such groups, proteins, polysaccharides, nucleic acids and their degradation products, biopolymer molecules from digestive extracts of various biological sources including plants, animals, bacteria and fungi, in standard form readily available from commercial sources.

The gold cluster molecules of the present invention are structurally different from conventional colloidal gold particles (gold colloidal particles).

The term "colloidal gold" refers to high density gold particles formed from densely packed gold atoms. Which are spherical gold particles of various sizes (fig. 1). The number of gold atoms of the colloidal gold particles is not accurately predictable. In typical colloidal gold formulations, the size of the gold particles produced is diverse over a wide range; such gold particles do not have single photon excited fluorescence. The most common method of colloidal gold preparation is to react trisodium citrate as a reducing agent with a tetrachloroaurate in an aqueous solution and dehydrate it by heating to temperatures elevated near boiling or reflux temperatures, see fress, g, "controlled nucleation for adjusting particle size in monodisperse gold suspensions" (controlled nucleation for particle size in monodispersed gold suspensions, Nature physics, sci.1973,241:20-22), the entire contents of which are incorporated by reference.

Unlike colloidal gold (FIG. 1), which has densely packed solid gold particles under an electron microscope and has various sizes, gold cluster molecules, or Au_m- (peptide)_nGold clusters that are stable and have well-defined molecular formula, size and geometry (fig. 3A, 3B and 3C). Their physical appearance and properties are different because they have different UV light absorption spectra and mass spectra. The difference in their structures is evidenced by the presence of absorption spectra and fluorescence shown in fig. 3A (fig. 3B). The mass spectrum shows the exact molecular formula. For example, in FIG. 3C, 25The Au atom forms a cluster that binds 9 peptides.

Gold cluster molecules are also different from gold salts. In another comparison, the gold chemical 2,3,4,6-tetra-o-acetyl-1-thio- β -D-glucopyranosate-S- (triethyl-phosphine) -gold in auranofin is shown in fig. 2A, 2B and 2C, the gold atom is in an oxidized state and is unable to form a metallic bond.

Gold cluster molecules have a metal surface that can form a non-covalent but stable metal bond with a chemical group such as a thiol. A typical preparative reaction is shown in figure 3. See also U.S. patent No. 8383919 to Gao, X, which is incorporated by reference in its entirety. The gold cluster complex is stabilized by polymer molecules containing thiol, guanidino, amide, imidazole, selenol, phosphine, amino, amine, carboxyl, hydroxyl groups, collectively referred to as stabilizers (gold cluster encapsulating molecules), which may be peptides containing thiol, guanidino, amide, imidazole, selenol, phosphine, amino, amine, carboxyl, hydroxyl groups. As shown by example in fig. 3C, where the gold-peptide cluster preparation is composed of gold clusters with a fixed number of gold atoms, where each gold cluster molecule is non-covalently bound to a limited number of thiol-containing peptides. However, these non-covalent metallic bonds formed between the peptide and the gold crystalline atom will allow other cysteine-or selenocysteine-rich proteins or peptides to competitively bind in vivo, making the gold cluster complex a good non-toxic non-invasive therapeutic candidate for modulating the protein levels of the body's content of cysteine or selenocysteine.

The term "rheumatoid arthritis" refers to a disease that is generally characterized by synovial inflammation and proliferation ("swelling"), autoantibody production, cartilage and bone destruction ("deformity"). Rheumatoid arthritis in patients is the result of a complex interaction between genotype and environmental triggers. It usually has a high level of cellular and humoral immune response to type II collagen. Multiple arthritis can be induced in rats and mice by intradermal injection of type II collagen emulsified in freund's adjuvant. Various animal models have been established based on type II collagen-induced inflammation. Some inbred mice (e.g., MRL/lpr) are known to develop autoimmune disease with age (similar to human systemic lupus erythematosus) and these mice have also been reported to spontaneously develop arthritis and have humoral responses to type I and type II collagen. It has been reported that the sensitivity of mice to arthritis and the immune response to collagen are controlled by Major Histocompatibility Complex (MHC) genes, and that certain specific epitopes of human leukocyte antigen genes confer specific susceptibility. This may be because of the predisposing T cell lineage selection, the alteration of antigen presentation or peptide affinity plays a role in promoting an autoreactive adaptive immune response.

In summary, the present invention provides the use of gold cluster molecules in the manufacture of a medicament for the prevention, alleviation and/or treatment of rheumatoid arthritis, i.e. the present invention relates to metallic Au (0) -cluster-complexing molecules, in particular to the use of Au (0) -peptide cluster molecules as therapeutic agents for the prevention, alleviation, treatment, cure and/or inhibition of the symptoms and development of rheumatoid arthritis in animals, which indicates that gold cluster molecules have positive anti-inflammatory and antirheumatic effects in animal models, a great potential as a new category of therapeutic agents for rheumatoid arthritis. More importantly, the gold cluster molecule has no significant toxic properties in vivo, and this safety profile is highly beneficial for rheumatoid arthritis patients who recover their symptoms once treatment is discontinued, as it produces no or little side effects.

Drawings

The disclosed application will be described with reference to the accompanying drawings, which show important exemplary embodiments of the invention and which are incorporated in the specification hereof by reference, wherein:

figure 1 is a bar graph illustration of the size distribution showing the size diversity of the prior art colloidal gold formulations.

Fig. 2A is the chemical structure of the gold compound (active ingredient of auranofin).

Fig. 2B is an absorption spectrum of a gold compound (active ingredient of auranofin).

Fig. 2C is a mass spectrum of gold compound (active ingredient of auranofin).

FIG. 3 visually illustrates an exemplary reaction process for generating gold (0) -peptide cluster molecules.

FIG. 3A is a schematic representation of the present inventionThe peptide using the peptide of SEQ ID NO. 1₉-gold₂₅Absorption spectrum of the composite molecule.

FIG. 3B is the peptide of FIG. 3A of the present invention₉-gold₂₅Fluorescence spectrum of the complexed molecules.

FIG. 3C is the peptide of FIG. 3A of the present invention₉-gold₂₅Mass spectrum of the complexed molecules.

FIG. 4A is a peptide of the present invention using the peptide of SEQ ID NO. 2₁₈-gold₂₅Absorption spectrum of the composite molecule.

FIG. 4B is the peptide of FIG. 4A of the present invention₁₈-gold₂₅Fluorescence spectrum of the complexed molecules.

FIG. 4C is the peptide of FIG. 4A of the present invention₁₈-gold₂₅Mass spectrum of the complexed molecules.

FIG. 5 shows HAS-gold of the present invention₄₀₀₀Absorption spectrum of the composite molecule.

FIG. 6 shows photographs of hind limbs of mice of different experimental groups of the present invention.

FIG. 7 shows radiographs of hind limbs of mice of different experimental groups of the present invention.

FIG. 8 shows histological staining of hind limb joint sections of mice of different experimental groups of the invention.

Figure 9 graphically shows the amount of gold in the limb joint area for the different experimental groups of figure 6.

FIG. 10 graphically shows the amount of the inflammatory cytokine IL-6 factor in the limb joint area of the different experimental groups of FIG. 6.

FIG. 11 graphically shows the amount of the inflammatory cytokine IL-1 β factor in the limb joint area of the different experimental groups of FIG. 6.

FIG. 12 graphically shows the amount of inflammatory cytokine TNF-. alpha.factor in the limb joint area of the different experimental groups of FIG. 6.

FIG. 13 is a peptide of the present invention using the peptide of SEQ ID NO. 3₃-gold₅Mass spectrum of the complexed molecules.

FIG. 14 is a peptide of the peptide of SEQ ID NO 3 prepared by the present invention₃-gold₅The compound molecule inhibits inflammatory reaction of macrophage.

FIG. 15 is a peptide of the peptide of SEQ ID NO 3 prepared by the present invention₃-gold₅The compound molecule inhibits inflammatory reaction of macrophage, and secretes cell factor and chemical factor for inducing or aggravating rheumatoid arthritis.

Detailed Description

The numerous innovative teachings of the present application will be described with particular reference to presently preferred embodiments (by way of example and not of limitation). The present invention describes several embodiments, and all statements below should not be considered as limiting the general claims.

For simplicity and clarity of illustration, the drawing figures show the general manner of construction, and descriptions and details of well-known features and techniques may be omitted to avoid unnecessarily obscuring the invention. Furthermore, the features in the drawings are not necessarily to scale, emphasis instead being placed upon illustrating embodiments of the invention.

Preparation of gold (0) -peptide cluster molecules

In preparing stable gold cluster molecules using peptides containing tyrosine or cysteine residues, the peptides also act as stabilizers for the metallic gold cluster structure. Other conjugated polymer molecules may also be used as stabilizing agents, including polylysine, polyarginine, polyaspartamide, polyaspartic acid sodium salt, polyglutamate, PEG containing such groups, PLGA containing such groups, proteins, glutathione, polysaccharides, nucleic acids and their degradation products, biopolymer molecules from digestive extracts of various biological sources, including plants, animals, bacteria and fungi, in standard form readily available from commercial sources.

Example 1

The examples of the present invention were performed using the disclosed peptide sequence (SEQ ID NO:1) Cys-Cys-Tyr-Gly-Gly-Pro-Lys-Lys-Lys-Arg-Lys-Pro-Gly. See Liu, R. et al, "Induce tumor cell Apoptosis via a specific target Thioredoxin Reductase 1(TrxR1) and inhibit Its active gold cluster (The Au Cluster industry cell Apoptosis via specific Targeting of Thioredoxin Reductase 1(TrxRl) and supporting Its Activity, chem. The chemical reaction is as follows:

peptide-TyrOH + OH^-+HAuCl₄→ peptide-Tyr ═ O + Au₂₅(peptide)₉(I)

All chemicals were purchased from Sigma-Aldrich unless otherwise noted. Ultrapure water was used throughout the experiment. Peptides were synthesized with a purity of 95% by solid phase method (China Peptides co. ltd.). All glassware was washed with aqua regia, then rinsed with ultra pure water and ethanol, and HAuCl was added under vigorous stirring at room temperature₄Aqueous solution (25mM, 80. mu.L) was added slowly to a 5mL vial of peptide solution (1.06mM, 1880. mu.L), followed by the slow addition of NaOH (0.5M, 40. mu.L) over 30 seconds to give a final pH of about 10. The samples were sealed without any interference and stored in the dark for 15 hours to yield the product. The resulting product was dialyzed (dialysis tube MWCO ═ 500) for 12 hours to remove free HAuCl₄And NaOH, and the sample was further concentrated by an ultrafiltration tube (Millipore, MWCO:3000) to remove free peptide. The gold (0) -peptide cluster molecules obtained were suspended in water and kept in the dark at 4 ℃ for further testing. The structure was tested by UV and fluorescence spectroscopy and mass spectrometry.

FIGS. 3A and 3B show that the absorbance and fluorescence emission peaks are at 281nm and 650nm, respectively. The mass spectrometry (FIG. 3C) results indicated that the resulting gold complex was a 25 gold cluster with 9 binding peptide molecules, i.e., Au₂₅(peptide)₉。

Example 2

Using chemical reaction (II), a gold (0) -metal peptide cluster molecule was prepared using the peptide (SEQ ID NO:2) Glu-Cys-Gly (GSH) according to a procedure similar to example 1. See Luo, Z, et al, "Emission From Induced polymerization of Au (I) -Thiolate Complexes to Ultra-bright Au (0) @ Au (I) -Thiolate Core-shell clusters" (From Aggregation-Induced Emission of Au (I) -Thiolate Complexes to Ultra bright Au (0) @ Au (I) -Thiolate Core-shells, J am. chem. Soc.,2012,134, 16662-16670).

GSH+OH^-+HAuCl₄→Au₂₅(SG)₁₈+GS-SG (II)

FIG. 4C shows the resulting molecular formula Au₂₅(SG)₁₈Mass spectrum of the gold-peptide cluster molecules of (1). FIGS. 4A and 4B show Au₂₅(SG)₁₈Absorbance and fluorescence emission spectra of cluster molecules. The absorbance and maximum fluorescence emission peaks were at 294nm and 607nm, respectively.

Example 3

A metallic gold-peptide cluster complex sample was prepared using Human Serum Albumin (HSA) by the following chemical reaction.

HSA-TyrOH+OH^-+HAuCl₄→HSA-Tyr＝O+HSA-Au₄₀₀₀(III)

All chemicals were purchased from Sigma-Aldrich unless otherwise noted. Ultrapure water was used throughout the experiment. All glassware was washed with aqua regia, then rinsed with ultra pure water and ethanol, and HAuCl was added under vigorous stirring at room temperature₄Aqueous solution (5mL,10mM,37 ℃) was added to HSA solution (5mL,5mg/mL,37 ℃). After 2 minutes, NaOH solution (0.5mL,1M) was introduced and the reaction allowed to proceed for 12 hours with vigorous stirring at 37 ℃. After the reaction, the sample was concentrated by a dialysis tube (MWCO:100kDa) to remove unreacted free HSA, NaOH and HAuCl₄. The obtained HSA-bound Au (0) was suspended in water and kept in the dark at 4 ℃. The UV-VIS spectrum (fig. 5) and HSA-bound gold cluster molecules showed a characteristic absorption peak at about 520nm due to characteristic local Surface Plasmon Resonance (SPR).

Example 4

The examples in the present invention use the disclosed peptide sequence (SEQ ID NO:3) Cys-Cys-Tyr. The chemical reaction is as follows:

peptide-TyrOH + OH^-+HAuCl₄→ peptide-Tyr ═ O + Au₅(peptide)₃

All chemicals were purchased from Sigma-Aldrich unless otherwise noted. Ultrapure water was used throughout the experiment. Peptides were synthesized with a purity of 95% by solid phase method (China Peptides co. ltd.). All glassware was washed with aqua regia, then rinsed with ultra pure water and ethanol, and HAuCl was added under vigorous stirring at room temperature₄Aqueous solution (25mM, 80. mu.L) was added slowly to a 5mL vial of peptide solution (1.06mM, 1880. mu.L), followed by the slow addition of NaOH (0.5M, 40. mu.L) over 30 seconds to give a final pH of about 11. The samples were sealed without any interference and stored in the dark for 15 hours to yield the product. Dialyzing the resultant product12 hours (dialysis tube MWCO 500) to remove free HAuCl₄And NaOH, and the sample was further concentrated by an ultrafiltration tube (Millipore, MWCO:3000) to remove free peptide. The gold (0) -peptide cluster molecules obtained were suspended in water and kept in the dark at 4 ℃ for further testing. By testing the structure by mass spectrometry, FIG. 13 shows that the resulting gold complex is a 5-gold cluster with 3 binding peptide molecules, i.e., Au₅(peptide)₃。

Animal experiment 1

Collagen-induced arthritis in mice is a standard animal model for the study of rheumatoid arthritis and other polyarthritis. DBA/1 male mice weighing 20-22 grams were purchased from Beijing Huafukang Biotech GmbH, China. Type II collagen and complete freund adjuvant were purchased from Chondrex inc, Redmond, WA, USA. Auranofin was purchased from Sigma, USA, dexamethasone was purchased from tianjin jinyao amino acids ltd, china. Gold (Au)₂₅(peptide)₉Cluster molecules were prepared according to example 1.

Type II collagen was dissolved in 0.1mM acetic acid solution and emulsified with an equal volume of complete Freund's adjuvant to make 1.0mg/ml type II collagen emulsion. One week after rest and acclimation, DBA/l male mice were grouped, each group consisting of 10 mice. Each animal was injected intradermally with 100. mu.g of type II collagen emulsion at the 2-3cm base of the tail. On day 21, a second booster dose of 100 μ g type II collagen solution was injected. A negative control group of mice was injected with an equal amount of 0.9% sodium chloride solution.

On day 22, each group of mice was given a different drug once a day for 28 days (4 weeks) until day 49 (end of week 7). Groups of mice were examined for inflammation and status. Group 1 (normal group) and negative control group (non-drug treated control group) mice received 0.9% sodium chloride solution in the stomach; group 2 mice received 0.5mg/kg body weight dexamethasone (Dex) by oral administration; group 3 mice received 1mg/kg body weight of auranofin orally; group 4 mice received 50mg/kg body weight gold orally (i.g.)₂₅(peptide)₉Cluster solution (gold synthesized in example 1)₂₅(peptide)₉Prepared as a solution with a gold concentration of 5mg/ml, 0.2 ml per mouse orally); group 5 mice were placed in the abdominal cavity(i.p.) received 5mg/kg body weight of gold₂₅(peptide)₉Cluster solution (gold synthesized in example 1)₂₅(peptide)₉Prepared as a solution with a gold concentration of 1 mg/ml, 0.1 ml per mouse).

TABLE 1 Effect of treatment on mouse body weight (g) (mean. + -. standard deviation)

^##:p<0.01 for the normal group; p<0.05；**:p<0.01 for the drug untreated group.

Intradermal injection of collagen type II immunization induced weight loss in all groups 1 week after the second enhanced collagen injection (week four) compared to untreated mouse groups. The anti-inflammatory dexamethasone did not reverse weight loss, while all gold agent treated groups of mice had slightly recovered weight.

Figure 6 shows a picture of hindlimb swelling in each animal group at the end of the 7 week experiment. Swelling began to become visible around 4 weeks, which after the second collagen injection, typically peaked 2 to 3 weeks after the second collagen injection, i.e. at weeks 5-6. In fig. 6, a shows the paw of a normal mouse without swelling; b shows drug-free treated immunized mice in which type II collagen induces swelling and deformity around toes, claws, and ankles; c shows Dex treated mice with no significant swelling; d shows auranofin treated mice with swelling and no significant improvement; e shows gold for oral administration₂₅(peptide)₉The cluster molecule mouse still has obvious swelling, but the condition is slightly good; f shows i.p. application of gold₂₅(peptide)₉Cluster molecular mice, whose swelling was significantly reduced and whose deformation was improved.

Inflammation was assessed by visual scoring method, where individual paw mice were graded by 0-4 as follows:

0 minute: no red swelling and swelling;

1 minute: mild erythema of the toes;

and 2, dividing: toe joint and paw swelling;

and 3, dividing: swelling below the ankle;

and 4, dividing: all ankles and paws were swollen.

The limbs of each mouse were examined and scored and the scores of all limbs of each mouse were added together.

The evaluation results of all groups are shown in Table 2.

TABLE 2 anti-inflammatory drug treatment Effect

^##:p<0.01 for the normal group; p<0.05；**:p<0.01 for the drug untreated group.

Type II collagen induced significant inflammation and severe deformities in the paw and limbs of untreated mice throughout the entire experimental period from 5 weeks to 7 weeks. In the second week of treatment, the swelling and inflammation in the group of mice receiving oral administration of 0.5mg/kg body weight dexamethasone was immediately inhibited; mice receiving oral administration of 1mg/kg body weight auranofin had a slightly decreased swelling score; receiving oral administration of 50mg/kg body weight gold₂₅(peptide)₉The mice in the cluster solution showed a score superior to auranofin, but developed considerable inflammation after 4 weeks of treatment; at 3 weeks of treatment, 5mg/kg body weight gold intraperitoneally was received₂₅(peptide)₉The swelling and joint deformity of the mice in the cluster solution improved significantly, and the improvement was significantly better with increasing treatment time.

Referring to fig. 7, the treatment effect showing the change in bone density of the paw joint at the end of 7 weeks of drug treatment is illustrated by a picture of a 3D CT scan image of each group of mice. Panel a shows the paw of a normal mouse without osteolysis; b shows drug untreated immunized mice, wherein IICollagen-induced arthritis causes significant bone deformity and osteolysis in the toe joint; c shows that Dex treatment prevented bone deformation and no significant osteolysis at the end of week 7; d shows auranofin-treated mice and e shows oral administration of gold₂₅(peptide)₉Cluster molecule mice, both groups of mice still undergoing bone deformation and osteolysis at the end of week 7; f shows i.p. application of gold₂₅(peptide)₉The clustered mice had significantly improved bone density, no significant osteolysis, and only slight bone deformation, compared to untreated arthritic mice. Thus, intraperitoneal administration of gold-peptide-cluster molecules showed significant efficacy for anti-inflammatory as well as anti-rheumatic effects. Especially when administered for long periods of time.

To assess soft tissue damage around the joint area, the joint tissues of the hind limbs of each mouse group were then fixed, embedded, sectioned, and stained with hematoxylin and eosin to examine histological and histopathological changes at the joints. Fig. 8 shows the staining results of each mouse group. A is a picture of the normal joint area without synovial macrophage invasion; b is a picture of type II collagen-induced arthritic joints without any treatment, where there is significant bone erosion and soft tissue damage, extensive synovial macrophage infiltration; c is a picture of the joints of Dex treated mice, with no significant bone erosion and no significant synovial macrophage invasion; d shows the joint picture of the mice treated by auranofin, obvious soft tissue injury in the joint area and a large amount of synovial membrane macrophage infiltration; e and F are gold by oral or i.p. administration, respectively₂₅(peptide)₉Pictures of cluster molecule treated joint regions, with significantly reduced bone erosion in F and no significant number of synovial macrophage infiltrates; oral administration of the gold-peptide-cluster in E showed reduced soft tissue damage and less macrophage infiltration. Thus, oral and intraperitoneal administration of gold-peptide-cluster molecules both exhibited significant anti-inflammatory and antirheumatic effects, however, the intraperitoneal use of gold₂₅(peptide)₉Clustering molecules is a significantly more efficient process.

Referring to fig. 9, the amount of gold was measured at the arthritic joints of various groups of treated mice. This shows that intraperitoneal (group 5) administration of the gold agent enables higher amounts of gold to be delivered to the arthritic joints compared to oral administration (group 4). Less but comparable amounts of gold were also observed for auranofin treatment (group 3).

Referring to FIGS. 10 and 11, the amounts of proinflammatory cytokines IL-6 and IL-1 β were measured at arthritic joints in various treated mouse groups, gold was administered orally (group 4) and intraperitoneally (group 5)₂₅(peptide)₉Both clusters reduced both cytokines to levels similar to that of Dex (group 2), while both cytokines remained high in auranofin-treated mice (group 3).

Referring to fig. 10, the amount of the proinflammatory cytokine TNF was measured at arthritic joints of various treated groups of mice. It was shown that intra-abdominal administration of gold compared to other treatment methods₂₅(peptide)₉Clusters significantly reduced TNF production (group 5), although Dex (group 2) was most effective, gold was administered orally₂₅(peptide)₉Clusters also reduced the amount of TNF (group 4), indicating gold₂₅(peptide)₉The clusters can exert anti-inflammatory and antirheumatic effects.

Cell experiment 2

The macrophage plays an important role in rheumatoid arthritis, the macrophage shows inflammatory reaction, the cell secretes inflammatory factors including NO chemical inflammatory factor, TNF- α cell inflammatory factor, IL-1 β cell inflammatory factor and IL-6 cell inflammatory factor, the inflammatory factors secreted by the macrophage can induce and aggravate rheumatoid arthritis, therefore, the macrophage shows inflammatory reaction is one of the biological mechanisms of rheumatoid arthritis and other autoimmune diseases, the invention utilizes Au synthesized in the example 4₅-peptides₃Inhibiting the inflammatory response of macrophages, see figure 14. As can be seen from FIG. 14, the macrophage inflammatory response was observed with the addition of different concentrations of gold cluster molecules (gold concentrations: 5,10,20, 50. mu. mol/L, respectively) to the macrophage culture medium in which the inflammatory response occurredThrough molecular biology experiments, as shown in figure 15, the ability of macrophages to induce or aggravate rheumatoid arthritis or other autoimmune diseases is reduced by adding different concentrations of gold cluster molecules (gold concentrations are 5,10,20 and 50 micromole per liter, respectively), inflammatory factors (including NO chemical inflammatory factors, TNF- α cellular inflammatory factors, IL-1 β cellular inflammatory factors and IL-6 cellular inflammatory factors) secreted by macrophages is gradually reduced, and the experiments show that the gold cluster molecules play a molecular mechanism for inhibiting the rheumatoid arthritis diseases from the level of cells and molecules.

Safety evaluation 3

Kunming mice with the weight of 20 g/mouse are selected. The mice are divided into 12 groups, each group comprises 10 mice, each male and female half, and each group is injected with gold cluster molecules in the abdominal cavity, the dosage of the gold cluster molecules from the mice in the 12 groups in the example 2 is as follows according to the content of gold: (1000, 700, 490, 343, 240, 150, 105, 73.5, 51.5, 36, 25, 17.5) mg/kg. Mice were observed for a toxic response time of 2 weeks following dosing, with results: after administration, no death, acceptable mental status of the mice in each group, and no significant weight loss occurred. Therefore, the safety of the gold clusters is high.

As will be recognized by those skilled in the art, the innovative concepts described in the present application can be modified and varied over a very wide range of applications, and the scope of patented subject matter is therefore not limited by any of the specific exemplary teachings given. It is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

Claims (8)

1. Use of gold cluster molecules in the manufacture of a medicament for the prevention, alleviation and/or treatment of rheumatoid arthritis; wherein the gold cluster molecule has a structural formula of Au_m- (peptide)_nOr (peptide)_n-Au_mWherein m and n respectively represent the number of gold atoms and the number of peptides in the gold cluster molecule and are integers which are not zero, wherein m is selected from 1-2000, and n is selected from 1-2500;

the peptide is one or more of SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3 and SEQ ID NO 4;

SEQ ID NO：1：Cys-Cys-Tyr-Gly-Gly-Pro-Lys-Lys-Lys-Arg-Lys-Pro-Gly；

SEQ ID NO：2：Glu-Cys-Gly；

SEQ ID NO：3：Cys-Cys-Tyr；

SEQ ID NO：4：Cys-Tyr。

2. use according to claim 1, wherein the Au is_m- (peptide)_nOr (peptide)_n-Au_mComprises the following steps:

Au₂₅(peptide)₉And the peptide is SEQ ID NO: 1;

Au₂₅(peptide)₁₈And the peptide is SEQ ID NO: 2;

Au₅(peptide)₃And the peptide is SEQ ID NO: 3; or

Au₂₅(peptide)₁₈And the peptide is SEQ ID NO: 4.

3. the use of claim 1 or 2, wherein the medicament is in a dosage form comprising an oral dosage form, a liquid suspension, or an injection.

4. Use according to claim 3, wherein the injection comprises an intracavitary, intravenous or intramuscular injection or a dermal or intramuscular injection.

5. Use according to claim 4, wherein the intraluminal injection is an intraperitoneal injection.

6. The use of claim 5, wherein the intraperitoneal injection is an intraperitoneal injection of a liquid suspension.

7. The use of claim 1, wherein the gold cluster molecule has the formula Au_m- (peptide)_nOr (peptide)_n-Au_mWherein m and n respectively represent the gold cluster moleculesThe number of gold atoms and the number of peptides of (a) are integers different from zero, wherein m is selected from 1 to 2000, and n is selected from 1 to 2500;

the peptide is SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4;

SEQ ID NO：1：Cys-Cys-Tyr-Gly-Gly-Pro-Lys-Lys-Lys-Arg-Lys-Pro-Gly；

SEQ ID NO：2：Glu-Cys-Gly；

SEQ ID NO：3：Cys-Cys-Tyr；

SEQ ID NO：4：Cys-Tyr；

the dosage form of the medicine is an intraperitoneal injection or a skin smearing preparation.

8. Use according to claim 7, wherein the Au is_m- (peptide)_nOr (peptide)_n-Au_mComprises the following steps:

Au₂₅(peptide)₉And the peptide is SEQ ID NO: 1;

Au₂₅(peptide)₁₈And the peptide is SEQ ID NO: 2;

Au₅(peptide)₃And the peptide is SEQ ID NO: 3; or

Au₂₅(peptide)₁₈And the peptide is SEQ ID NO: 4.

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