Use of GGC in the preparation of a medicament for preventing and / or treating rheumatoid arthritis

Abstract

The present invention relates to a new use of γ-glutamylcysteine (GGC), specifically to the application of GGC in the preparation of drugs for treating rheumatoid arthritis (RA) and a pharmaceutical composition for treating rheumatoid arthritis. The pharmaceutical composition for treating rheumatoid arthritis can be made into tablets, capsules, granules, drops, granules, ointments or injections by adding general pharmaceutical adjuvants to GGC with a purity of more than 95% (mass percentage). The results of pharmacological experiments show that GGC can significantly improve the joint swelling of rheumatoid arthritis, reduce the arthritis index, reverse the pathological changes of arthritis, and has no serious disadvantages of commonly used clinical drugs, indicating that GGC has a strong effect against rheumatoid arthritis. Therefore, GGC can be used to prepare drugs for treating rheumatoid arthritis. The present invention provides a new drug source for the treatment of rheumatoid arthritis.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and mainly relates to the use of GGC in the preparation of drugs for preventing and / or treating rheumatoid arthritis. Background Art

[0002] Rheumatoid arthritis (RA) is a multi-systemic inflammatory autoimmune disease mainly involving peripheral joints, and its etiology is unknown. Rheumatoid arthritis is the most common inflammatory joint disease clinically, seriously affecting the functional status, quality of life and life expectancy of patients. Due to its chronic characteristics, related complications and long-term dysfunction, it has a huge impact on society and economy. At present, the treatment of rheumatoid arthritis mainly relies on drug treatment (common drugs are non-steroidal anti-inflammatory drugs, newly marketed biological agents such as TNF-α inhibitors), but the toxic and side effects are obvious. Therefore, there is an urgent need to develop new anti-rheumatoid arthritis drugs.

[0003] γ-glutamylcysteine (GGC) is the direct precursor and limiting substrate of glutathione (GSH) biosynthesis, and is the reduced form of de-glycine-glutathione. Since GGC can be directly absorbed by a variety of cells, it can effectively relieve the substrate inhibition of γ-glutamylcysteine synthetase (GSHⅠ) in the process of GSH biosynthesis, and increase the substrate concentration of the catalytic reaction of glutathione synthetase (GSHⅡ). Therefore, compared with directly supplying glutathione and cysteine, it can more effectively increase the intracellular glutathione concentration. In recent years, the production methods of GGC mainly include 3 types: yeast fermentation method, chemical synthesis method and CCT catalytic synthesis method. At present, GGC has important application prospects in industries such as medicine, food and feed additives, and cosmetics. The pure product of GGC is yellow / white powder, which is extremely hygroscopic, and its solubility in water can reach 70% W / V. 1 g of powder is dissolved in 10 mL of water to obtain a solution with a pH of 3, and its properties are stable in a solution with a pH of 3-8. Its molecular weight is 250.27, and its chemical structural formula is shown in Formula Ⅰ:

[0004]

[0005] As a known monomer compound, although GGC has an exact molecular formula and structural formula, its research activity is not high. At present, there are very few reports on the pharmacological effects of GGC. The pharmacological research reports are mainly on aspects such as antioxidant, increasing GSH level, treating colitis and improving Alzheimer's disease. So far, the application of GGC as a monomer in the preparation of drugs for treating rheumatoid arthritis has not been reported, and the present invention comes from this. Summary of the Invention

[0006] The object of the present invention is to invent a new compound for preparing drugs for treating rheumatoid arthritis.

[0007] Specifically, the technical solution of the present invention is as follows:

[0008] In a first aspect of the present invention, the use of GGC or its geometric isomers, pharmaceutically acceptable salts, esters, solvates, hydrates in the preparation of drugs for preventing and / or treating RA is disclosed, and the structural formula of the GGC is shown in Formula I:

[0009]

[0010] In a second aspect of the present invention, a pharmaceutical composition is disclosed, and the pharmaceutical composition comprises GGC or its geometric isomers, pharmaceutically acceptable salts, esters, solvates, hydrates.

[0011] Preferably, the active ingredients of the pharmaceutical composition comprise GGC or its geometric isomers, pharmaceutically acceptable salts, esters, solvates, hydrates.

[0012] More preferably, the only active ingredient in the pharmaceutical composition is GGC or its geometric isomers, pharmaceutically acceptable salts, esters, solvates, hydrates.

[0013] Preferably, the pharmaceutical composition further comprises one or more pharmaceutically acceptable inert and non-toxic excipients.

[0014] More preferably, the excipient is a carrier, solvent, emulsifier, dispersant, wetting agent, binder, stabilizer, colorant or flavoring.

[0015] Preferably, the pharmaceutical composition is a tablet, capsule, powder, drop, freeze-dried product, granule, ointment or injection.

[0016] Preferably, the preparation method of the pharmaceutical composition comprises: adding pharmaceutical adjuvants to GGC to prepare tablets, capsules, powders, drops, freeze-dried products, granules, ointments or injections.

[0017] More preferably, the mass fraction of GGC in the pharmaceutical composition is at least 95%; the pharmaceutical adjuvants are excipients.

[0018] In a third aspect of the present invention, the use of the above-mentioned pharmaceutical composition in the preparation of drugs for preventing and / or treating RA is disclosed.

[0019] In some preferred embodiments of the present invention, the pharmaceutical composition for treating rheumatoid arthritis can be prepared into tablets, capsules, powders, drops, freeze-dried products, granules, ointments or injections by adding general pharmaceutical adjuvants to GGC with a purity of more than 95% (mass percentage).

[0020] The pharmaceutical composition according to the invention can act systemically and / or locally, and for this purpose, it can be administered in a suitable manner, for example, by oral, parenteral, pulmonary or nasal routes, and the composition according to the invention can be administered in dosage forms suitable for these administration routes.

[0021] Suitable for oral administration are dosage forms that act according to the state of the art and rapidly and / or in an improved manner release the pharmaceutical composition of the invention, and that contain the pharmaceutical composition of the invention in crystalline and / or amorphous and / or dissolved form, such as tablets (uncoated or coated tablets, which for example have a coating that resists gastric juice or delays dissolution or does not dissolve, and release the composition according to the invention), tablets that rapidly disintegrate in the mouth, or films, film / lyophilizates, capsules (such as hard or soft capsules), dragees, granules, pellets, powders, emulsions, suspensions, aerosols or solutions.

[0022] Parenteral administration can be carried out avoiding the absorption step (such as intravenous, intra-arterial, intracardiac, intraspinal or intralumbar or intra-articular) or including absorption simultaneously (such as intramuscular, subcutaneous, intradermal, transdermal or intraperitoneal). Suitable dosage forms for parenteral administration methods are in particular preparations for injection and infusion in the form of solutions, suspensions, emulsions, lyophilizates or sterile powders.

[0023] Suitable for another administration route are, for example, pharmaceutical forms for inhalation, such as powder inhalers or nebulizers, or pharmaceutical forms that can be administered nasally, such as drops, solutions or sprays.

[0024] The pharmaceutical composition according to the invention can be converted into the said dosage forms. This can be carried out in a manner known per se by mixing with inert, non-toxic, pharmacologically suitable excipients. These excipients particularly include carriers (such as microcrystalline cellulose, lactose, mannitol, starch), solvents (such as liquid polyethylene glycol), emulsifiers and dispersants or wetting agents (such as sodium lauryl sulfate, polyoxy sorbitan oleate, propylene glycol), binders (such as polyvinylpyrrolidone), synthetic and natural polymers (such as albumin), stabilizers (such as antioxidants, such as ascorbic acid), colorants (such as inorganic pigments, such as iron oxides) and masking flavors and odors.

[0025] The effective dose of GGC can vary according to the mode of administration, the age and weight of the patient, the severity of the disease and other relevant factors. The recommended dose for oral administration is 50 - 200 mg / time, 2 - 3 times a day; the recommended dose for parenteral administration is 20 - 120 mg / time, once a day.

[0026] The technical solution of the present invention is: to establish a Collagen-induced arthritis (CIA) mouse model through a collagen induction method, and for the first time, GGC is selected as a therapeutic drug to study its effect on the joint pathological changes in CIA mice, and to observe the prevention and treatment effects of GGC on collagen-induced arthritis, so as to provide an experimental basis for the prevention and treatment of rheumatoid arthritis by GGC.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] (1) It provides the use of GGC in highly efficient anti-rheumatoid arthritis, which can significantly reduce the severity of arthritis, such as joint swelling and arthritis index, and reverse joint pathological damage, with no obvious toxic and side effects, and has great clinical application value.

[0029] (2) The present invention provides the use of GGC in highly efficient anti-rheumatoid arthritis, and this use has potential significant economic effects. The preparation prepared from GGC or its pharmaceutically acceptable carrier has the application prospect of a drug for treating rheumatoid arthritis. Developed and prepared in accordance with the national innovative drug approval method, it is expected to become a new type 1 highly efficient and low-toxic drug for treating rheumatoid arthritis with independent intellectual property rights in China, and the industrialization prospect is clear. Description of the Drawings

[0030] Figure 1 It is a representative picture of a mouse on the 51st day after the first immunization of a CIA mouse; [[ID= / / 16]] [[ID= / / 17]]

[0031] [[ID= / / 18]] Figure 2 It is the effect of GGC on the joint inflammation score and foot thickness of CIA mice;

[0032] Figure 3 It is a representative Micro-CT reconstruction image of the hind paws of mice in each group;

[0033] Figure 4 It is the effect of GGC on the related bone mass score of CIA mice;

[0034] Figure 5 It is a picture of the pathological tissue changes in the joint parts of mice in each group;

[0035] Figure 6 It is the immunohistochemical map of the joint tissues of mice in each group and the quantitative map of (caspase-1 and IL-1β) in immunohistochemistry;

[0036] Among them, the Healthy group is the healthy control group, the CIA group is the model group, and the GGC group is the γ-glutamylcysteine administration group, and the administration dose is 100 mg / kg / day. Detailed Implementation Modes

[0037] The technical solutions of the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but the present invention is not limited to the scope of the described embodiments.

[0038] For the experimental methods without specific conditions indicated in the following embodiments, they are carried out according to conventional methods and conditions, or selected according to the product instructions. The reagents and raw materials used in the present invention are all commercially available.

[0039] I. Materials

[0040] 1. Experimental animals

[0041] Eighteen male DBA / 1 mice, 8 weeks old, weighing 18 - 25 g, of SPF grade, were provided by Beijing Vital River Laboratory Animal Technology Co., Ltd. During the experiment, the temperature in the breeding room was 20 ± 3.2 °C, and the relative humidity was 65 - 75%. They had free access to food and water. All animal experiments were carried out with the permission of the Laboratory Animal Welfare and Ethics Committee of Soochow University.

[0042] 2. Drugs and reagents:

[0043] 2.1 Main drugs and reagents

[0044] GGC, white powder, with a content of ≥95%, was provided by Shanghai yuanye Bio-Technology Co., Ltd. The drug was stored in a -20 °C refrigerator. The solvent was pure water when used, and it was prepared freshly before use.

[0045] Type II bovine collagen, complete Freund's adjuvant, and incomplete Freund's adjuvant (Chondrex, USA); paraformaldehyde, EDTA, DAB chromogenic agent (Solarbio, China); HE, safranin - O fast green staining kit (Sigma); Caspase - 1 antibody (sc - 56036, Santa Cruz Biotechnology), IL - 1β antibody (sc - 52012, Santa Cruz Biotechnology).

[0046] 2.2 Instruments

[0047] Electronic analytical balance, model EL104, produced by Shanghai Yueping Scientific Instruments Co., Ltd.; vernier caliper; small animal Micro - CT (SkyScan 1176, Aartselaar, Belgium), paraffin embedding machine (Leica, Germany), paraffin slicing machine (Leica, Germany), LSM 800 laser confocal microscope (Zeiss, Germany), a set of surgical instruments, etc.

[0048] II. Experimental methods

[0049] 1. Model establishment and drug administration

[0050] Eighteen male DBA / 1 mice were weighed, numbered, and randomly divided into three groups: the Healthy group, the CIA model group, and the GGC administration group, with 6 mice in each group. Collagen-induced arthritis mouse models were established in both the CIA group and the GGC group through secondary immunization. In the first immunization, a mixed emulsion of bovine type II collagen solution (2 mg / mL) and complete Freund's adjuvant (4 mg / mL) in equal volumes was subcutaneously injected into the tails of the mice. Twenty-one days after the first immunization, the mice were boosted with bovine type II collagen solution emulsified with incomplete Freund's adjuvant. The GGC administration group was orally administered GGC at a dose of 100 mg / kg (body weight) daily, and the other two groups were given the same amount of blank solvent, pure water.

[0051] 2. Measurement of joint score and hind paw swelling degree

[0052] The mice were scored every three days after the second immunization. Each hind paw was given a score of 0 - 4: 0, normal; 1, toe swelling; 2, ankle joint and / or tarsal bone swelling; 3, moderate swelling of the ankle joint and / or tarsal bone or mild swelling of both; 4, severe swelling of the entire paw. Before the primary immunization (day 0) and on day 51 after the primary immunization (day 51), the thickness of the mice's paws was measured using a vernier caliper. The increase in paw thickness was defined as (thickness 51 - thickness 0) / thickness 0 × 100 (%).

[0053] 3. Specimen collection

[0054] Treatment of joints: At the end of the experiment, the hind legs of the mice (including the hind paws and knee joint parts) were taken and fixed in 10% paraformaldehyde for more than 24 hours. One hind leg was used for histopathological observation, and the other was used for imaging observation (Micro-CT).

[0055] 4. Histopathological observation

[0056] The hind legs of the mice (hind paws and knee joint parts) were fixed with 10% paraformaldehyde, then decalcified by soaking in 10% EDTA decalcifying solution for four weeks, embedded in paraffin, sectioned at 6 μm, and then stained with HE, safranin-O fast green. Evaluation was performed based on microscopic changes in the tissue such as inflammatory cell infiltration, pannus hyperplasia, cartilage degradation and destruction, and loss of proteoglycans.

[0057] 5. Evaluation of Micro-CT

[0058] The mouse hind paws fixed with paraformaldehyde were placed on the Micro-CT scanning bed for scanning. The parameters of the X-ray were set as a current of 500 μA, a voltage of 50 kV, and a scanning amount of 9 μm per layer. The CTAn analysis software was used to further process the images.

[0059] 6. Immunohistochemical study

[0060] 6-μm sections of the knee joint were selected. After antigen repair with trypsin, the sections were incubated overnight at 4 °C with Caspase-1 and IL-1β antibodies. After incubation with the primary antibody overnight at 4 °C, the sections were incubated with the secondary antibody at room temperature for 1 hour and stained in 100 μL of DAB solution for 2-6 min, and photographed and observed under a microscope.

[0061] 7. Statistical methods

[0062] Sigmaplot V12.5, Prism V8.2.1 and R program V4.1.1 were used for statistical analysis.

[0063] III. Implementation results

[0064] Example 1 Effect of GGC on the volume of mouse joints and feet

[0065] Experimental mice were treated orally with GGC, and their joint inflammation scores and hind paw thickness were statistically analyzed. The results showed that the hind paws of the mice in the model group had obvious redness, swelling and joint stiffness, while the lesion degree of the mice in the GGC treatment group was significantly reduced, as shown in Figure 1 . Compared with the model group, the joint inflammation score and the severity of hind paw swelling in the mice in the GGC (100 mg / kg) treatment group were significantly reduced, as shown in Figure 2 .

[0066] Example 2 Effect of GGC treatment on joint bone destruction

[0067] The hind paws of the mice were examined by Micro-CT. The hind paws of the mice were scanned by Micro-CT, and it was also found that the mice in the model group had obvious joint destruction. The degree of joint and bone destruction in the mice in the GGC (100 mg / kg) treatment group was significantly reduced, as shown in Figure 3 . Correspondingly, the quantitative analysis of BV / TV and total porosity, the histological synovitis score (HSS) of the knee joint, and the knee joint OARSI score results showed that GGC could effectively inhibit bone erosion activity, cartilage damage and the formation of pannus, as shown in Figure 4 .

[0068] Example 3 Effect of GGC on joint synovial tissue

[0069] At the end of the experiment, the hind paws and knee joints of the mice were taken, paraffin sections were made and stained with HE and safranin-O fast green. The results observed under a confocal microscope showed that there were obvious symptoms such as inflammatory cell infiltration, pannus formation, cartilage destruction and bone erosion in the synovium of the mice in the CIA model group. The degree of inflammatory cell infiltration in the mice in the GGC (100 mg / kg) treatment group was reduced, the number of pannus was significantly decreased, and the degrees of cartilage destruction and bone erosion were also significantly reduced, as shown inFigure 5 .

[0070] Example 4 Effect of GGC on the Expression Levels of Caspase-1 and IL-1β in Joints

[0071] Immunohistochemistry was used to detect the expression levels of caspase-1 and IL-1β in the joint specimens of each group of mice. Microscopic observation results showed that the expression levels of caspase-1 and IL-1β in the mice treated with GGC were significantly reduced by more than 80%. Representative immunohistochemical staining of caspase-1 and IL-1β is shown in Figure 6 .

[0072] Summarize:

[0073] 1. After continuous oral administration of GGC for 51 days, GGC (100 mg / kg) can significantly reduce the severity of hind paw swelling in CIA mice.

[0074] 2. Micro-CT examination showed that after 51 days of continuous administration, GGC (100 mg / kg) could significantly improve the degree of joint destruction.

[0075] 3. Pathological histological observation results showed that after GGC treatment, symptoms such as inflammatory cell infiltration, pannus formation, cartilage destruction and bone erosion in the mouse joints were significantly alleviated.

[0076] 4. Immunohistochemical observation results showed that the expression levels of caspase-1 and IL-1β in the joints of mice were significantly reduced after GGC treatment.

[0077] In summary, GGC has a certain preventive and therapeutic effect on inflammatory cell infiltration, bone destruction, and articular cartilage erosion in arthritis of CIA mice.

[0078] The present invention can be further illustrated by the following experimental examples.

[0079] Example 1 of preparing medicament:

[0080] Take 20g of GGC monomer compound, add 280g of medicinal starch, mix the two thoroughly, and prepare 1000 capsules, each capsule weighing 0.3g and containing 20mg of GGC.

[0081] Example 2 of preparing medicament:

[0082] Take 100 g of GGC monomer compound, add 200 g of medicinal starch, mix the two thoroughly, and make 1000 tablets, each weighing 0.3 g and containing 100 mg of GGC.

[0083] Example 3 of preparing medicament:

[0084] Take 20 g of the GGC monomer compound, add 100 mL of 1,2-propanediol, fully dissolve it, dilute it to 1000 mL with sterile injection water, mix well and then dispense it into 1000 ampoules, 1 mL per ampoule, containing 20 mg of GGC.

[0085] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. Use of GGC or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating rheumatoid arthritis, wherein the structural formula of GGC is shown as Formula I: Ⅰ 。

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