Use of abacavir in the preparation of a medicament for the treatment of rheumatoid arthritis

Abstract

The application relates to application of abacavir in preparation of a drug for treating rheumatoid arthritis, and belongs to the technical field of biological medicine. It is found for the first time that abacavir can relieve the related symptoms of rheumatoid arthritis, and specifically, it is proved in a collagen-induced arthritis mouse model that abacavir can inhibit joint swelling and bone erosion of the mouse, and relieve the pathological phenotype; meanwhile, it is found in peripheral blood mononuclear cell (PBMCs) samples of rheumatoid arthritis patients that the use of abacavir can reduce the expression of interferon stimulated gene (ISG) family related genes, which indicates that the abacavir can be used as a new type of drug for treating rheumatoid arthritis. In summary, the abacavir can be used as a drug for treating rheumatoid arthritis, and has high clinical application value.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to the application of abacavir in the preparation of a drug for treating rheumatoid arthritis. BACKGROUND

[0002] Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by inflammatory infiltration of the synovial membrane of the joint. The early symptoms are morning stiffness, swelling and pain in the joints, and gradually the destruction of the articular cartilage occurs, eventually leading to joint deformity and loss of function, and further affecting the normal joint function of the patient, resulting in limited activity of the patient. RA cannot be cured and has a high rate of disability. Patients with RA who have not been treated according to the standard may have joint erosion and destruction within 2 years of onset. Therefore, early diagnosis and treatment can effectively control the progression of the disease and reduce symptoms such as joint damage.

[0003] The goal of the treatment of rheumatoid arthritis is to reduce the activity of the disease or to achieve remission. Timely and effective intervention can slow down the progression of the disease and improve the long-term prognosis of the patient. At present, the drug treatment of rheumatoid arthritis mainly includes non-steroidal anti-inflammatory drugs, anti-rheumatic drugs (csDMARDs), glucocorticoids and biological agents, etc. However, the above-mentioned drugs have the following problems: (1) The current treatment drugs mainly target acquired immune responses, such as biological agents TNF-α inhibitors. Methotrexate (MTX), as the first choice drug, is often combined with other drugs and biological agents for the treatment of RA, but this treatment method has high economic cost and large individual differences in efficacy. (2) About 69.7% of RA patients use hormones for a long time, and 11.3% of RA patients rely on hormone monotherapy, which can cause serious side effects such as osteoporosis and infection, and has limited effect on repairing the bone erosion that has occurred. (3) The etiology and pathogenesis of RA is not fully understood, and although the above-mentioned drugs are widely used in the treatment of RA, there are still large individual differences in drug efficacy, and some patients do not respond to the drugs, and frequent changes in treatment regimens are required. (4) csDMARDs take effect slowly and need several weeks or even months to see results, which prolongs the treatment cycle of RA patients, and some drugs mainly inhibit inflammation, but have limited effect on repairing the bone erosion that has occurred. Therefore, it is urgent to explore more selective drugs to improve the prognosis of RA patients, reduce the rate of disability and improve the quality of life of patients. SUMMARY

[0004] Therefore, the technical problem to be solved by the present application is to overcome the lack of an effective drug for treating rheumatoid arthritis in the prior art.

[0005] To solve the above technical problems, the application provides application of abacavir in preparation of a drug for treating rheumatoid arthritis.

[0006] A first object of the application is to provide application of abacavir in preparation of a drug for preventing or treating rheumatoid arthritis.

[0007] Further, the drug takes abacavir as an active ingredient, and is prepared into a pharmaceutically acceptable dosage form by using pharmaceutically acceptable excipients.

[0008] A second object of the application is to provide a drug for treating rheumatoid arthritis, which comprises abacavir.

[0009] Further, the drug further comprises a pharmaceutically acceptable carrier or excipient.

[0010] Further, the pharmaceutically acceptable carrier is selected from any one of a solvent, an emulsifier, a suspending agent, a disintegrating agent, a binding agent, an excipient, a stabilizing agent, a diluent, a gelling agent, a preservative, a lubricant, and a surfactant.

[0011] Further, the excipient is at least one of hydroxypropyl methylcellulose, hydroxypropyl cellulose, povidone, polyethylene glycol, ethyl cellulose, a liposome, a methacrylic acid copolymer, polyvinyl acetate, carboxymethyl ethyl cellulose, carboxymethyl cellulose phthalate, hydroxypropyl methyl cellulose phthalate, hydroxypropyl methyl cellulose acetate succinate, a polyacrylic acid resin, a carboxyvinyl polymer, an alginate, a carrageenan, a carboxylic acid lactone, a gum, a polyvinyl alcohol, a pregelatinized starch, a crosslinked starch, sodium carboxymethyl starch, dextrin, a polyethylene oxide, chitosan, chitosan, an ion exchange resin, and collagen.

[0012] Further, the administration route of the drug comprises oral administration, intraperitoneal injection, subcutaneous injection, intravenous injection, or intramuscular injection.

[0013] Further, the drug is in a pharmaceutically acceptable dosage form, and the dosage form is selected from any one of a tablet, a capsule, an injection, a granule, a suspension, and a solution.

[0014] Furthermore, abacavir can inhibit the expression of genes related to the interferon-stimulated gene (ISG) family. The expression level of ISGs is significantly elevated in the synovial fluid of rheumatoid arthritis (RA) patients, thus ISGs are often used as targets for RA diagnosis and treatment. This invention is the first to discover that abacavir can inhibit the expression of genes related to the interferon-stimulated gene (ISG) family, thereby alleviating related symptoms of rheumatoid arthritis.

[0015] Furthermore, ISGs family-related genes include the gene encoding 2',5'-oligoadenylate synthase 1 (… OAS1 ), interferon-induced gene ( IFI44L ), the gene encoding myxoviral drug resistance protein 1 ( MX1 Among them, 2',5'-oligoadenylate synthase 1 is an antiviral protein induced by interferon. It activates the RNase L pathway by catalyzing the synthesis of 2',5'-oligoadenylate from adenosine triphosphate (ATP), thereby inhibiting viral replication by degrading viral and host RNA. Interferon-induced genes are antiviral genes expressed in response to interferon. Myxovirus resistance protein 1 is an antiviral protein induced by type I and type III interferon.

[0016] A third objective of this invention is to provide an application of abacavir in the preparation of arthritis treatment products.

[0017] Furthermore, the arthritis mentioned includes rheumatoid arthritis.

[0018] The fourth objective of this invention is to provide an application of abacavir in the preparation of arthritis prevention products.

[0019] The beneficial effects of this invention are:

[0020] This invention is the first to discover that abacavir can treat rheumatoid arthritis. Specifically, this invention demonstrated in a collagen-induced arthritis (CIA) mouse model that abacavir can inhibit joint swelling and bone erosion, alleviating the pathological phenotype. Furthermore, it found that abacavir can reduce the expression of ISG genes in PBMCs samples from RA patients. In summary, this invention is the first to discover that abacavir can be used for the treatment and prevention of rheumatoid arthritis, and has high clinical therapeutic value. Attached Figure Description

[0021] Figure 1 This is a schematic diagram illustrating the construction process of arthritis mice + abacavir group mice;

[0022] Figure 2 These are morphological and scanned images of the hind paws of mice in the control group, control + abacavir group, arthritis mouse group, and arthritis mouse + abacavir group.

[0023] Figure 3 This is a graph showing the mean arthritis index of mice in the control group, control + abacavir group, arthritis mouse group, and arthritis mouse + abacavir group. *** indicates... P <0.001, the same applies below;

[0024] Figure 4 These are lesion score charts for the control group, control + abacavir group, arthritis mouse group, and arthritis mouse + abacavir group.

[0025] Figure 5 The images show the results of hematoxylin and eosin (H&E) staining and safranin O staining of joint tissues from the control group, control + abacavir group, arthritis mouse group, and arthritis mouse + abacavir group. The black arrows indicate synovial hyperplasia.

[0026] Figure 6 This is a flowchart of an experiment in which PBMCs were isolated from RA patients and treated with abacavir for 72 hours. In the flowchart, "-" indicates that no abacavir treatment was given and "+" indicates that abacavir treatment was given. The same applies below.

[0027] Figure 7 It is around 72 hours after abacavir treatment. OAS1 , IFI44L and MX1 Graph showing the change in relative expression levels. Detailed Implementation

[0028] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0029] Example 1

[0030] 1. Experimental materials

[0031] Eight-week-old DBA / 1J mice (DBA / 1J mice can be immunized with type II collagen to induce severe autoimmune polyarthritis, thus obtaining collagen-induced arthritis (CIA) mice. CIA mice share common immunological and pathological characteristics with rheumatoid arthritis patients and are a commonly used mouse model of rheumatoid arthritis), bovine type II collagen, Freund's complete adjuvant (CFA), Freund's incomplete adjuvant (IFA), phosphate-buffered saline (PBS), syringes, small animal computed tomography (Micro-CT) scanning system, 4% paraformaldehyde, xylene, ethanol, hematoxylin and eosin (H&E) staining kit, safranin O staining kit, microscope, etc.

[0032] 2. Experimental Procedure

[0033] Select 8-week-old healthy DBA / 1J mice, divide the DBA / 1J mice into four groups, each group of 8-9, respectively, control group, control + abacavir group, arthritis mouse group and arthritis mouse + abacavir group.

[0034] The arthritis mouse group and the arthritis mouse + abacavir group are immunized and induced, and the immunization and induction process is as follows: after mixing bovine type II collagen (Chondrex, 2 mg / mL) with Freund's complete adjuvant (CFA) in equal volume, emulsify it thoroughly on ice with a homogenizer, inject 100 uL of the emulsion into the base of the mouse tail of the arthritis mouse group and the arthritis mouse + abacavir group for primary immunization. 21 days later, replace Freund's complete adjuvant (CFA) with Freund's incomplete adjuvant (IFA), and use the same method for booster immunization.

[0035] The mice in the arthritis mouse + abacavir group are treated with abacavir after the end of immunization induction, and the abacavir is injected at a dose of 2 mg / mL, 50 uL per mouse (25 uL for each hind limb), every 3 days, for a total of 10 injections. Figure 1 The mice in the control + abacavir group are not immunized and are injected with PBS in the same manner.

[0036] The mice in the arthritis mouse group are injected with PBS after the end of immunization induction, and the PBS is injected at a dose of 2 mg / mL, 50 uL per mouse (25 uL for each hind limb), every 3 days, for a total of 10 injections. The mice in the control group are not immunized and are injected with PBS in the same manner.

[0037] The effects of abacavir on the pathological phenotype of CIA mice are systematically studied by morphological, imaging and immunohistochemical methods.

[0038] Morphological analysis: observe the morphology of the mouse paw every 3 days, and obtain the arthritis index according to the swelling degree of the mouse limbs. The arthritis index scoring standard for a single paw is: no swelling (0 points), mild swelling (1 point), toe joint swelling and mild paw swelling (2 points), severe swelling of the ankle joint and paw (3 points), and severe swelling of all paws including the ankle joint (4 points). Figure 3 The total score for four paws.

[0039] Imaging analysis: dissect the mouse and take the mouse ankle joint and toe joint for Micro-CT scanning system scanning, observe the bone destruction of the ankle joint and toe joint, and score the joint lesion according to the erosion and destruction degree. The lesion scoring standard for the ankle joint and toe joint bone on a single paw is: no erosion (0 points), mild erosion (1 point), moderate erosion (2 points), and severe erosion (3 points). Figure 4 The total score for four paws.

[0040] Histopathological analysis: Mouse ankle joints were taken, fixed with 4% paraformaldehyde, decalcified, embedded in paraffin, and stained with hematoxylin and eosin (H&E staining) and safranin O staining. The lesions were evaluated, and the evaluation indicators included inflammatory cell infiltration, synovial hyperplasia, pannus formation and destruction of cartilage or bone.

[0041] 3. Experimental Conclusions

[0042] This study investigated the effects of abacavir on collagen-induced arthritis (CIA) mice by administering intra-articular injections of abacavir every three days. Figure 1 The treatment effect was monitored by measuring clinical, imaging, and histological changes. Results showed that, compared with the arthritis mouse group, the arthritis mouse + abacavir group exhibited significantly reduced redness and swelling in the hind paws, and significant relief of joint damage. Figure 2 Consistent with the hind paw phenotype of mice, the arthritis index scores of the arthritis mice + abacavir group were also lower. Figure 3 The lesion scores of the arthritis mice + abacavir group were also significantly lower than those of the arthritis mice group. Figure 4 Furthermore, histopathological analysis of the ankle joints of arthritis mice in the abacavir group showed a significant reduction in the formation of synovial granulomas, which are typically associated with cartilage and bone erosion. Figure 5 The results for the control group and the control + abacavir group were similar, indicating that abacavir has no side effects. In conclusion, abacavir can alleviate joint swelling and bone erosion in CIA mice, thereby relieving arthritis.

[0043] Example 2

[0044] 1. Experimental materials

[0045] Ethylenediaminetetraacetic acid (EDTA) anticoagulant blood collection tubes, density gradient centrifuge tubes (SepMate™ tubes), lymphocyte separation medium (Lymphoprep), RNA extraction kits, Duchenne modified Eagle medium (DMEM high glucose medium), phosphatidylserine (PS), phosphate buffered saline (PBS), fetal bovine serum (FBS), polyethylene glycol octylphenyl ether (Triton X-100), RNase-free pipette tips, centrifuges, micro-volume bioassay analyzers, real-time quantitative PCR instruments, cell culture incubators, clean benches, etc.

[0046] 2. Experimental Procedure

[0047] After obtaining informed consent from the patients, samples were obtained from 12 patients with rheumatoid arthritis from the First Affiliated Hospital of Soochow University. After blood samples were collected, they were placed in citrate anticoagulant tubes and peripheral blood mononuclear cells (PBMCs) were obtained by density gradient centrifugation (Ficoll).

[0048] PBMCs were cultured in a 37℃, 5% CO2 incubator. Each group was divided into two parts: one part was treated with abacavir and the other part was not treated with abacavir as a control. After 2 days of treatment, PBMCs were collected and RNA was extracted for real-time quantitative PCR detection.

[0049] RNA was extracted using an RNA extraction kit (column method). The absorbance of RNA at 260 nm, 280 nm, and 230 nm was measured using a Nanodrop spectrophotometer to calculate and assess purity. Agarose gel electrophoresis was used to detect RNA purity and integrity. Sample mRNA was reverse transcribed into cDNA. The reaction system contained total RNA, polythymidine (Oligo dT), random primers, reverse transcriptase, and an RNase inhibitor. PCR amplification was performed using specific primers. The green fluorescent nucleic acid dye (SYBE Green) bound to the double-stranded DNA. With increasing cycle number, the amplified target gene increased exponentially, and the fluorescence signal continuously strengthened. Fluorescence was detected and analyzed using a quantitative real-time PCR instrument, focusing on housekeeping genes. Gapdh ( Gapdh (The gene encoding glyceraldehyde-3-phosphate dehydrogenase) was used as an internal control, employing 2... -△△Ct The expression of genes related to the interferon-stimulated gene (ISG) family was quantitatively analyzed using this method. The genes related to the ISG family detected included those encoding 2',5'-oligoadenylate synthase 1. OAS1 ), interferon-induced gene ( IFI44L ), the gene encoding myxoviral drug resistance protein 1 ( MX1 The primer sequences for ) are shown in Table 1.

[0050] Table 1 Primer sequences for detecting ISGs

[0051]

[0052] 3. Experimental Conclusions

[0053] PBMCs were isolated from RA patients and then treated with abacavir (n=12) Figure 6 The results showed that abacavir treatment led to a decrease in ISG expression in PBMCs in more than half of the patients. Figure 7 Therefore, abacavir can be used as a treatment for rheumatoid arthritis.

[0054] Obviously, the above embodiments are merely example for clearly illustrating, and are not limitation to the embodiments. For ordinary skilled in the art, other different forms of changes or variations can be made on the basis of the above description. Here, all the embodiments need not and can not be exhausted. The obvious changes or variations derived therefrom are still within the scope of the present invention.

Claims

1. Use of abacavir for the preparation of a medicament for the prophylaxis or treatment of rheumatoid arthritis, characterized in that, The drug is prepared in a pharmaceutically acceptable dosage form using abacavir as an active ingredient and pharmaceutically acceptable excipients.

2. Use according to claim 1, characterized in that, The excipient is at least one selected from the group consisting of hydroxypropyl methylcellulose, hydroxypropyl cellulose, povidone, polyethylene glycol, ethyl cellulose, methacrylic acid copolymer, polyvinyl acetate, carboxymethyl ethyl cellulose, carboxymethyl cellulose phthalate, hydroxypropyl methyl cellulose phthalate, hydroxypropyl methyl cellulose acetate succinate, polyacrylic acid resin, carboxyvinyl polymer, alginate, carrageenan, carboxylic acid lactone, gum, polyvinyl alcohol, pregelatinized starch, crosslinked starch, sodium carboxymethyl starch, dextrin, polyethylene oxide, chitosan, chitinous chitosan, ion exchange resin, and collagen.

3. Use according to claim 1, characterized in that, The dosage form is at least one selected from the group consisting of tablets, capsules, injections, granules, suspensions, and solutions.

Images