Application of abacavir in preparation of medicine for treating rheumatoid arthritis

Abstract

The invention relates to application of abacavir in preparation of a medicine for treating rheumatoid arthritis, and belongs to the technical field of biological medicine. The abacavir is found to be capable of relieving related symptoms of rheumatoid arthritis for the first time, and particularly, abacavir is proved to be capable of inhibiting joint swelling and bone erosion of mice and relieving pathological phenotypes in a collagen-induced arthritis mouse model; meanwhile, in a peripheral blood mononuclear cell (PBMCs) sample of a patient with rheumatoid arthritis, abacavir is found to be capable of reducing expression of interferon stimulated gene (ISGs) family related genes, so that abacavir can be used as a novel therapeutic drug for rheumatoid arthritis. In conclusion, abacavir can be used as a medicine for treating rheumatoid arthritis and has very high clinical application value.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to the application of abacavir in preparing a drug for treating rheumatoid arthritis. Background Art

[0002] Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by inflammatory infiltration of the synovial membranes. Initial symptoms include morning stiffness, swelling, and pain, followed by gradual destruction of articular cartilage, ultimately leading to joint deformity and loss of function, which in turn affects the patient's normal joint function and limits their mobility. RA is incurable and has a high disability rate. Untreated RA patients may experience joint erosion and destruction within two years of onset. Therefore, early diagnosis and treatment can effectively control disease progression and alleviate symptoms such as joint damage.

[0003] The goal of rheumatoid arthritis treatment is to alleviate the disease or reduce disease activity. Timely and effective intervention can slow down the progression of the disease and improve the long-term prognosis of patients. Currently, the drug treatment of rheumatoid arthritis mainly includes nonsteroidal anti-inflammatory drugs, anti-rheumatic drugs (csDMARDs), glucocorticoids and biological agents. However, the above drugs have the following problems: (1) Current treatment drugs mainly target acquired immune responses, such as biological agents TNF-α inhibitors. Methotrexate (MTX), as the first-line drug, is often combined with other drugs and biological agents to treat RA, but this treatment method has high economic costs and large differences in efficacy between individuals. (2) About 69.7% of RA patients use hormones for a long time, and 11.3% of RA patients rely on hormone monotherapy. This treatment method can cause serious side effects such as osteoporosis and infection, and has limited effect on repairing existing bone erosion. (3) Currently, there is limited understanding of the etiology of RA. Although the above drugs are widely used in clinical treatment of RA, the individual differences in drug efficacy are large. Some patients still do not respond to the drugs and need to frequently change treatment plans. (4) csDMARDs have a slow onset of action, requiring weeks or even months to be effective, which prolongs the treatment cycle for RA patients. In addition, some drugs mainly inhibit inflammation but have limited effects on repairing existing bone erosion. Therefore, there is an urgent need to explore more selective drugs to improve the prognosis of RA patients, reduce disability rates, and enhance the quality of life of patients. Summary of the Invention

[0004] Therefore, the technical problem to be solved by the present invention is to overcome the problem in the prior art of lacking a drug that is effective in treating rheumatoid arthritis.

[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 a pharmaceutically acceptable excipient.

[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 associated with the interferon-stimulated gene (ISG) family. ISG expression levels are significantly elevated in the synovial fluid of patients with rheumatoid arthritis, and therefore ISGs are often used as targets for RA diagnosis and treatment. This study is the first to discover that abacavir can inhibit the expression of genes associated with the interferon-stimulated gene (ISG) family, thereby alleviating symptoms associated with rheumatoid arthritis.

[0015] Furthermore, ISGs family-related genes include the gene encoding 2',5'-oligoadenylate synthetase 1 ( OAS1 ), interferon-induced genes ( IFI44L ), the gene encoding myxovirus resistance protein 1 ( MX1 2',5'-oligoadenylate synthetase 1 is an antiviral protein induced by interferon. It activates the RNase L (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-inducible genes are antiviral genes whose expression is induced by interferon. Myxovirus resistance protein 1 is an antiviral protein induced by type I and type III interferons.

[0016] The third object of the present invention is to provide a use of abacavir in preparing a product for treating arthritis.

[0017] Furthermore, the arthritis includes rheumatoid arthritis.

[0018] The fourth object of the present invention is to provide a use of abacavir in preparing a product for preventing arthritis.

[0019] Beneficial effects of the present invention:

[0020] This study is the first to demonstrate that abacavir can treat rheumatoid arthritis. Specifically, in a collagen-induced arthritis (CIA) mouse model, the study demonstrated that abacavir can inhibit joint swelling and bone erosion, alleviating pathological phenotypes. Furthermore, in PBMC samples from RA patients, abacavir was found to reduce the expression of ISG genes. In summary, this study demonstrates for the first time that abacavir can be used in the treatment and prevention of rheumatoid arthritis, demonstrating its high clinical therapeutic value. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0023] Figure 3 The average arthritis index of the control group, control + abacavir group, arthritis mice group and arthritis mice + abacavir group is shown in the figure, where *** indicates P <0.001, the same below;

[0024] Figure 4 is the lesion score graph of the control group, control + abacavir group, arthritis mouse group, and arthritis mouse + abacavir group;

[0025] Figure 5 Figure 1 is the results of hematoxylin & eosin (H&E) and safranin O staining of joint tissues of mice in the control group, control + abacavir group, arthritis mice group, and arthritis mice + abacavir group. The black arrows indicate synovial hyperplasia.

[0026] Figure 6 This is the experimental flow chart of 72-hour abacavir treatment after isolating PBMCs from RA patients, where “-” indicates no abacavir treatment and “+” indicates abacavir treatment, the same below;

[0027] Figure 7 Is it before and after 72 hours of abacavir treatment OAS1 、 IFI44L and MX1 Relative expression level changes. DETAILED DESCRIPTION

[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 the present invention and implement it. However, the embodiments are not intended to limit the present invention.

[0029] Example 1

[0030] 1. Experimental Materials

[0031] 8-week-old DBA / 1J mice (DBA / 1J mice immunized with type II collagen can induce severe autoimmune polyarthritis, thus generating collagen-induced arthritis (CIA) mice. CIA mice share immunological and pathological characteristics with rheumatoid arthritis patients and are a commonly used mouse model of rheumatoid arthritis), bovine type II collagen, complete Freund's adjuvant (CFA), incomplete Freund's adjuvant (IFA), phosphate-buffered saline (PBS), syringes, small animal computed tomography system (Micro-CT scanning system), 4% paraformaldehyde, xylene, ethanol, hematoxylin & eosin staining (H&E staining) kit, safranin O staining kit, microscope, etc.

[0032] 2. Experimental steps

[0033] Eight-week-old healthy DBA / 1J mice were selected and divided into four groups, with 8-9 mice in each group, namely the control group, the control + abacavir group, the arthritis mice group, and the arthritis mice + abacavir group.

[0034] Arthritis mice and arthritis mice plus abacavir groups underwent immunization induction. The following induction procedures were performed: Bovine type II collagen (Chondrex, 2 mg / mL) and complete Freund's adjuvant (CFA) were mixed in equal volumes and thoroughly emulsified on ice using a homogenizer. 100 μL of the emulsion was injected into the tail base of mice in the arthritis and arthritis mice plus abacavir groups for primary immunization. Twenty-one days later, complete Freund's adjuvant (CFA) was replaced with incomplete Freund's adjuvant (IFA), and booster immunizations were performed using the same protocol.

[0035] Mice in the arthritis mice + abacavir group were treated with abacavir after the immune induction. Abacavir was injected at a dose of 2 mg / mL, with 50 μL injected into each mouse (25 μL injected into each of the two hind limbs), once every 3 days for 10 consecutive injections ( Figure 1 The mice in the control + abacavir group were not immunized and injected with PBS in the same manner.

[0036] After immune induction, the arthritis mice were injected with PBS at a dose of 2 mg / mL, with 50 μL injected into each mouse (25 μL into each hind limb), every three days for 10 consecutive injections. The control group of mice did not undergo immune induction and received PBS injections using the same method.

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

[0038] Morphological analysis: The morphology of the mouse paws was observed every 3 days, and the arthritis index was calculated based on the degree of swelling of the mouse limbs. The scoring criteria for the arthritis index of a single paw were: no swelling (0 points), mild swelling (1 point), swelling of the toe joints and mild swelling of the paws (2 points), severe swelling and redness of the ankle joints and paws (3 points), and severe swelling and redness of all paws including the ankle joints (4 points). Figure 3 This is the total score for all four paws.

[0039] Imaging analysis: Mice were dissected and their ankle and toe joints were scanned with a Micro-CT scanning system to observe bone destruction in the ankle and toe joints. Joint lesions were scored based on the degree of erosion and destruction of the joints. The scoring criteria for ankle and toe joint bone lesions on a single paw are: no erosion (0 points), mild erosion (1 point), moderate erosion (2 points), and severe erosion (3 points). Figure 4 This is the total score for all four paws.

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

[0041] 3. Experimental Conclusion

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

[0043] Example 2

[0044] 1. Experimental Materials

[0045] Ethylenediaminetetraacetic acid (EDTA) anticoagulant blood collection tubes, density gradient centrifugation tubes (SepMate™ tubes), lymphocyte separation medium (Lymphoprep), RNA extraction kit, Dulbecco's modified Eagle's medium (DMEM high glucose medium), phosphatidylserine (PS), phosphate buffered saline (PBS), fetal bovine serum (FBS), triton X-100, RNase-free pipette tips, centrifuge, micro-biological detector, real-time fluorescence quantitative PCR instrument, cell culture incubator, clean bench, etc.

[0046] 2. Experimental steps

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

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

[0049] RNA was extracted using an RNA extraction kit (column method), and the absorbance of RNA at 260 nm, 280 nm, and 230 nm was measured using a micro-spectrophotometer (Nanodrop) to calculate and evaluate the purity. Agarose gel electrophoresis was used to detect the purity and integrity of the RNA. The sample mRNA was reverse transcribed into cDNA. The reaction system contained total RNA, polythymidine (Oligo dT), random primers, reverse transcriptase, and RNase inhibitors. PCR amplification was performed using specific primers. The green fluorescent nucleic acid dye (SYBE Green) bound to double-stranded DNA. As the number of cycles increased, the amplified target gene increased exponentially, and the fluorescence signal continued to increase. Fluorescence was detected and analyzed using a fluorescence quantitative PCR instrument, and the housekeeping gene was detected. Gapdh ( Gapdh is the gene encoding glyceraldehyde-3-phosphate dehydrogenase) as an internal reference, and 2 -△△Ct The expression of genes related to the interferon-stimulated gene (ISGs) family was quantitatively analyzed by the method. The genes related to the ISGs family detected (including the gene encoding 2',5'-oligoadenylate synthetase 1 ( OAS1 ), interferon-induced genes ( IFI44L ), the gene encoding myxovirus resistance protein 1 ( MX1 The primer sequences are shown in Table 1.

[0050] Table 1 Primer sequences for detecting ISGs

[0051] 3. Experimental Conclusion

[0052] PBMCs were isolated from RA patients and then treated with abacavir (n = 12) ( Figure 6 The results showed that abacavir treatment resulted in decreased expression of ISGs in PBMCs in more than half of the patients ( Figure 7 ). Therefore, abacavir can be used as a treatment for rheumatoid arthritis.

[0053] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will appreciate that other variations or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. Use of abacavir in the preparation of a drug for preventing or treating rheumatoid arthritis, characterized in that: The drug uses abacavir as an active ingredient and is prepared into a pharmaceutically acceptable dosage form using pharmaceutically acceptable excipients.

2. A drug for treating rheumatoid arthritis, characterized in that: The drug is abacavir and a pharmaceutically acceptable carrier or excipient.

3. The drug according to claim 2, characterized in that The pharmaceutically acceptable carrier is selected from any one of a solvent, an emulsifier, a suspending agent, a disintegrant, a binder, an excipient, a stabilizer, a diluent, a gelling agent, a preservative, a lubricant and a surfactant.

4. The drug according to claim 2, characterized in that The excipient is selected from at least one of hydroxypropyl methylcellulose, hydroxypropyl cellulose, povidone, polyethylene glycol, ethyl cellulose, liposome, methacrylic acid copolymer, polyvinyl acetate, carboxymethyl ethyl cellulose, carboxymethyl cellulose phthalate, hydroxypropyl methyl cellulose phthalate, hydroxypropyl methyl cellulose acetate succinate, polyacrylic acid resin, polycarboxyvinyl, alginate, carrageenan, carboxylic acid lactone, gum, polyvinyl alcohol, pregelatinized starch, cross-linked starch, sodium carboxymethyl starch, dextrin, polyethylene oxide, chitosan, chitosan, ion exchange resin and collagen.

5. The drug according to claim 2, characterized in that The drug is in a pharmaceutically acceptable dosage form, and the dosage form is selected from any one of tablets, capsules, injections, granules, suspensions and solutions.

6. The use of abacavir in preparing a product for treating arthritis, characterized in that: The arthritis treatment product is abacavir.

7. The use according to claim 6, characterized in that The arthritis includes rheumatoid arthritis.

8. Use of abacavir in preparing a product for preventing arthritis, characterized in that: The arthritis prevention product is abacavir.

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