阅读说明：本技术 达托霉素在制备治疗类风湿关节炎药物中的应用 (Application of daptomycin in preparation of medicine for treating rheumatoid arthritis ) 是由 梁淑芳 叶洋 朱璟 于 2019-10-24 设计创作，主要内容包括：本发明涉及达托霉素的用途,具体涉及达托霉素在制备治疗类风湿关节炎药物中的应用,属于生物药物技术领域。本发明解决的技术问题是提供达托霉素的新用途,即达托霉素在制备治疗类风湿关节炎药物中的应用。本发明首次创造性证实了达托霉素对类风湿关节炎的免疫调节活性和疗效,提供了达托霉素作为免疫调节剂治疗类风湿关节炎的新药用用途,为类风湿关节炎的治疗提供了一种全新的选择,应用前景广泛。(The invention relates to an application of daptomycin, in particular to an application of daptomycin in preparing a medicament for treating rheumatoid arthritis, belonging to the technical field of biological medicaments. The invention aims to provide a new application of daptomycin, namely an application of daptomycin in preparing a medicament for treating rheumatoid arthritis. The invention creatively proves the immunoregulation activity and curative effect of daptomycin on rheumatoid arthritis for the first time, provides new medicinal application of daptomycin serving as an immunomodulator to treat rheumatoid arthritis, provides a brand new choice for treating rheumatoid arthritis, and has wide application prospect.)

1. Application of daptomycin in preparing medicine for treating rheumatoid arthritis.

2. The use of daptomycin for the preparation of a medicament for the treatment of rheumatoid arthritis as in claim 1, wherein: the dosage form of the medicine is an injection administration dosage form.

3. A medicine for treating rheumatoid arthritis is characterized in that: the active ingredients comprise daptomycin and methotrexate.

4. The agent for treating rheumatoid arthritis according to claim 3, wherein: the active ingredients are daptomycin and methotrexate.

5. The agent for treating rheumatoid arthritis according to claim 4, wherein: the mass ratio of daptomycin to methotrexate is 10-30: 1.

6. The agent for treating rheumatoid arthritis according to claim 5, wherein: the mass ratio of daptomycin to methotrexate is 20: 1.

Technical Field

The invention relates to an application of daptomycin, in particular to an application of daptomycin in preparing a medicament for treating rheumatoid arthritis, belonging to the technical field of biological medicaments.

Background

Rheumatoid Arthritis (RA) is a chronic, systemic, autoimmune disease characterized primarily by synovial cell proliferation, and abnormal changes in joint synovial structure and function. According to statistics, the prevalence rate of RA in the global range is about 0.5%, wherein the prevalence rate in China is 0.3% -0.5%, and the prevalence rate of females is higher than that of males. RA is a disease caused by multiple factors such as heredity, bacterial infection, endocrine dyscrasia, immune regulation imbalance and the like, and the pathogenesis is complex and can not be cured radically. The current treatment measures include drug treatment, immune purification, functional exercise, surgical treatment and the like. In terms of drug therapy, the drugs used mainly include nonsteroidal drugs, antirheumatic drugs, biological agents, steroidal drugs, etc., which can relieve the clinical symptoms of RA patients and eliminate the inflammatory reaction in the local joints, but also have side effects. Methotrexate (MTX), for example, delays the progression of bone destruction around the joints of RA patients and inhibits synovial inflammatory hyperplasia, but has limited clinical efficacy due to its toxic side effects, especially hepatotoxicity in the case of excessive or prolonged use. Therefore, the development of new therapeutic agents for RA is an important subject to be urgently solved and a great demand for national health.

Daptomycin (Daptomycin, hereinafter referred to as DAP) is a cyclic lipopeptide antibiotic produced by fermentation of streptomyces roseosporus, is usually produced by a biological fermentation method, and is simple and easy to obtain and low in cost compared with biological agents and small molecular compounds. DAP is primarily used for the treatment of gram-positive bacterial infections, approved by the Food and Drug Administration (FDA) in the united states in 2003 for the treatment of complicated skin and skin structure infections, bacteremia, right heart infectious endocarditis, meningitis, diseases associated with urinary tract infections. DAP has a unique antibacterial action mechanism, and can cause sodium and potassium ions to be lost by disturbing the transfer of amino acid by a bacterial cell membrane, so as to block the biosynthesis of bacterial cell wall peptidoglycan, thereby achieving the aim of sterilization. As can be seen, DAP research is mostly focused on the aspects of antibiosis and drug resistance, and no reports on DAP immune action mechanism and RA treatment are found in the existing research.

Disclosure of Invention

The invention aims to provide a new application of daptomycin, namely an application of daptomycin in preparing a medicament for treating rheumatoid arthritis.

Researches show that the daptomycin has immunoregulation activity and curative effect on the rheumatoid arthritis, and can be used for preparing the medicine for treating the rheumatoid arthritis.

Preferably, the medicament is in a form of injection.

The invention also provides a medicament for treating rheumatoid arthritis.

The active ingredients of the medicine for treating rheumatoid arthritis comprise daptomycin and methotrexate.

Preferably, the active ingredients of the medicament for treating rheumatoid arthritis are only daptomycin and methotrexate.

Preferably, the mass ratio of the daptomycin to the methotrexate is 10-30: 1.

Preferably, the mass ratio of daptomycin to methotrexate is 20: 1.

Compared with the prior art, the invention has the following beneficial effects:

the invention creatively proves the immunoregulation activity and curative effect of daptomycin on rheumatoid arthritis for the first time, provides new medicinal application of daptomycin serving as an immunomodulator to treat rheumatoid arthritis, provides a brand new choice for treating rheumatoid arthritis, and has wide application prospect.

Drawings

FIG. 1 shows the effect of different concentrations of DAP on RAW264.7 cells.

FIG. 2 shows the mRNA expression levels of various inflammatory factors in cells, wherein a-c are mouse macrophage models and d-f are human monocyte models.

FIG. 3 shows the amounts of IL-1. beta. and IL-6 inflammatory factors in cell supernatants.

Figure 4 is a graph of DAP alleviating the degree of collagen-induced joint swelling in the RA mouse model. Wherein, figure 4a is the body weight of each group of mice, and figure 4b is the joint score of each group of mice.

FIG. 5 shows the serum content of inflammatory factor MCP-1 in each group of mice.

Detailed Description

Patients with RA are at higher risk of bacterial infection than non-RA patients, and bacterial infection is also one of the mechanisms by which RA occurs and develops. MTX also has a certain immunosuppressive effect as a first-line small-molecule chemical drug for treating RA, and can increase the infection rate of RA patients and the severity of infection diseases (common bacterial infection such as cryptococcosis, herpes zoster and opportunistic infection) after long-term use. Since DAP is metabolized primarily by the kidney and not by human liver microsomes. Thus, DAP is less hepatotoxic to humans than MTX.

According to the invention, through researches of an in vitro cell experiment and an animal experiment, DAP has immunoregulation activity and curative effect on RA, and can be used for preparing a medicament for treating RA.

When the DAP is used for treating rheumatoid arthritis, the DAP can be prepared into various injection administration dosage forms such as intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection, intracavity injection and the like.

In the treatment of RA, DAP may be used, or it may be used in combination with other agents for the treatment of RA, and such combination may be achieved by the simultaneous, sequential or separate administration of the various therapeutic ingredients for the purpose of treating RA.

The invention also provides a medicament for treating rheumatoid arthritis.

The active ingredients of the medicine for treating rheumatoid arthritis comprise DAP and MTX. The active ingredient of such a medicament may be DAP, MTX and other agents for the treatment of RA.

Preferably, the active ingredients of the medicament for treating rheumatoid arthritis are only DAP and MTX.

Through research, the preferable mass ratio of the DAP to the MTX is 10-30: 1.

Preferably, the mass ratio of DAP to MTX is 20: 1.

In order to better understand the essence of the present invention, the pharmacological test and results of DAP will be used to illustrate its novel use in the preparation of a medicament for the treatment of rheumatoid arthritis.

1. Experimental Material

1.1 cell origin

Mouse macrophage RAW 264.7: originated from ATCC, deposited in the national emphasis laboratory of biotherapy, university of sichuan.

Human monocyte THP-1: originated from ATCC, deposited in the national emphasis laboratory of biotherapy, university of sichuan.

1.2 sources of reagents

DMEM, RPM11640, Fetal Bovine Serum (FBS), tryptase (Trypsin) were purchased from Hyclone.

Lipopolysaccharide: purchased from Sigma-Aldrich.

BCA protein quantification reagent: purchased from bi yun tian biotechnology limited.

Reverse transcription Kit (iScript cDNA Synthesis Kit) was purchased from Bio-Rad.

qPCR fluorescent quantitation kit (SsoAdvanced SYBR GreenSupermix): purchased from Bio-Rad.

The mouse TNF-alpha ELISA detection kit, the mouse IL-6ELISA detection kit and the mouse IL-1 beta ELISA detection kit are purchased from Beijing Dake company.

Complete Freund's adjuvant, Incomplete Freund's adjuvant, type II bovine collagen (Immunization graft bone type II collagen) were purchased from Chondrex corporation.

DBA/1J (N000219) mice were purchased from Jiangsu Jiejiaokang Biotech, Inc.

The CBA Flex Set multi-factor detection kit was purchased from BD Biosciences.

Daptomycin (DAP) is available from the company continental.

Methotrexate (MTX) was purchased from Sigma-Aldrich.

2. In vitro cell experiments

(1) MTS assay to detect cytotoxicity of DAP against RAW 264.7:

i, collect RAW264.7 cells in logarithmic phase, adjust cell suspension concentration to make cell density of 96-well plate 5000 per well, add 200. mu.L DMEM medium per well, and fill marginal well with sterile PBS.

II, culturing a 96-well plate in a 5% CO2 incubator at 37 ℃ for 24h, adding DAP with concentration gradient, setting 8 concentrations of 0, 1, 3, 5, 20, 50, 150 and 500ug/ml respectively, and setting 3 multiple wells for each concentration.

III, 96-well plate at 37 ℃ with 5% CO₂Culturing in incubator for 24h, 48h and 72h, and observing cell growth state under inverted microscope.

VI, 10ul of MTS solution is added into each well, the incubation is continued for 2 hours in a cell culture box, and the absorbance of each well is measured at OD490nm of an enzyme linked immunosorbent assay (ELISA), and the result is shown in figure 1.

The results show that 1-5. mu.g/ml DAP does not affect the normal growth of cells in the LPS-induced murine macrophage RAW264.7 inflammation model. Therefore we chose two concentrations of 3. mu.g/ml, 5ug/ml for the subsequent experiments.

(2) Mouse macrophage RAW264.7 and human monocyte THP-1 are used as test cell strains, LPS induction concentration is 1ug/mL, DAP drug concentration of 3ug/mL and 5ug/mL is selected as test concentration, a positive drug control group is MTX, a blank control group is set, after the cells are stimulated by LPS for 5 hours, 3ug/mL, 5ug/mL DAP and 0.45ug/mL of positive drug MTX are respectively given for treatment for 24 hours, and supernatant and cells are respectively collected.

(3) RNA of the tested cells is extracted by using an RNA extraction kit, and mRNA levels corresponding to various inflammatory factors are detected by q-PCR after reverse transcription, and the result is shown in figure 2.

Figure 2 shows that DAP has a significant inhibitory effect on LPS-induced immune cell inflammation. As can be seen from the figure, the mRNA levels of TNF-. alpha.IL-1. beta., and IL-6 in the LPS model group were higher than those in the normal cells (control group), indicating that the cell model was successfully established. Compared with LPS model group, DAP of 3. mu.g/ml and 5ug/ml had significant inhibitory effect on mRNA levels of TNF-. alpha.IL-1. beta., IL-6 in mouse macrophages (FIGS. 2 a-c). In addition, using macrophage derived from human monocyte THP-1 cell induced by LPS as model, 0.45ug/ml MTX had significant inhibitory effect on mRNA levels of TNF-alpha, IL-6, and IL-1 beta, and 3ug/ml and 5ug/ml DAP had significant inhibitory effect on mRNA levels of TNF-alpha, IL-6, and IL-1 beta, and there was no significant difference from the inhibitory effect of positive drug MTX group (FIG. 2 d-f).

(4) The content of inflammatory factors such as TNF-alpha, IL-1 beta, IL-6 and the like in the supernatant was measured by ELISA kit, and the results are shown in FIG. 3.

FIG. 3 shows that DAP reduces the levels of proinflammatory factors IL-1 β, IL-6 in a mouse macrophage inflammation model induced by LPS. ELISA results showed that 5. mu.g/ml DAP decreased IL-1. beta. and IL-6 secretion in cells compared to LPS model group.

The above results indicate that DAP has a better anti-inflammatory effect.

3. Animal experiments

(1) Preparation of the Molding agent

Mixing bovine II type collagen acetic acid solution and Freund's adjuvant in equal volume, and completely emulsifying (emulsifying at 4 deg.C) to obtain modeling agent 1; the bovine type II collagen acetic acid solution and incomplete Freund's adjuvant are mixed in equal volume, completely emulsified and marked as modeling agent 2.

(2) Molding die

The root of the mouse was injected with 100. mu.l of the molding agent 1 at 1-2cm in the skin, and the control group was injected with the same volume of complete Freund's adjuvant. After the day of injection was designated as 0d and 21d, the second molding was performed with the same volume of molding agent 2, and the control group was injected with the same volume of incomplete adjuvant. One more week (i.e., 28d) later, the hind paw of the mouse began to appear inflamed and slowed, indicating successful establishment of the RA animal model. The disease can last for 2 months.

(3) Observation of

After the second molding, observing the mental state, appetite and activity of the mouse; the severity of arthritis was assessed as an arthritis index starting with a second molding (21 d), measured every 3d, with clinical arthritis scoring criteria: no symptom-0, obvious swelling and redness of one toe-1, slight swelling of limbs or swelling of more than two toes-2, obvious swelling and erythema of limbs-3, severe swelling and erythema of limbs-4.

(4) Animal grouping and administration

Mice successfully modeled were randomly and equally divided into 5 groups of 4-5 mice each, feeding environment: 22 +/-1 ℃ and 12h of light/12 h of dark cycle. All operations are performed strictly according to national health and hygiene guidelines. Control group (Normal): only injecting adjuvant, not using medicine intervention, only injecting solvent; model set (CIA): an RA animal model is established, and only a solvent is injected without medicine intervention; ③ MTX group: 0.5mg/kg of positive control drug MTX is injected once every 3 days; fourthly, DAP processing group: according to the cell experiment results and literature, DAP large dose group with 10mg/kg and small dose group with 6mg/kg are set to be injected once every 1 day; sixthly, the combination drug of the DAP and the MTX (MTX 0.5mg/kg + DAP 10mg/kg) is injected once every 3 days, and the DAP is injected once every other day.

(5) Evaluation of degree of joint swelling in RA mouse model

The body weights of the mice at each stage (21 days and 75 days) were measured, and the results are shown in fig. 4a, and it can be seen from fig. 4a that the body weight of the RA mice does not change much after the second immunization, indicating that the body weight of the mice is not significantly affected by the RA model.

Model mouse joint score changes were used to reflect the establishment of RA model mice and the effect of DAP on RA model establishment. FIG. 4b is a graph of joint scores of animal models, and results show that typical RA symptoms such as obvious red swelling, pain and joint movement limitation appear in a CIA model mouse, which indicates that the model is successfully established. Compared with a CIA model group, the DAP (6mg/kg) group has no significant difference, the scores of the DAP (10mg/kg) group, the positive drug MTX (0.5mg/kg) treatment group and the combined drug group are all significantly reduced compared with the CIA model group, meanwhile, the combined drug group has lower score than the MTX single drug group, the red and swollen degree of the toes of the mice is slowly increased, and after continuous treatment, red and swollen relief can be observed on partial toes, which shows that the DAP (10mg/kg) group has significant inhibition effect on arthritis symptoms and has no significant difference with the inhibition effect of the positive drug MTX group. It can be seen that joint swelling in RA mice can be alleviated by continuous high dose DAP treatment or by the combination of MTX and DAP.

(6) Method for detecting content change of inflammatory factors in serum by flow type liquid phase multiple protein quantitative technology

Respectively taking blood from the orbit of an experimental mouse at 5 weeks of administration, collecting blood samples, standing at room temperature for 30min, centrifuging at 3000rpm for 15min, taking supernate, detecting the content of inflammatory factors such as MCP-1 in serum by using a mouse serum multifactor detection kit to analyze and evaluate the treatment effects of a model and an object to be detected, and the method comprises the following steps:

1) and cleaning the instrument, and performing instrument related function detection through the instrument quality control microspheres to ensure that the PE channel, the APC channel and the APC-Cy7 channel have normal functions.

2) And (3) preparing a standard substance, namely pouring all the freeze-dried microspheres of the standard substance into the same test tube, diluting the standard substance by 4ml of diluent, mixing the mixture gently and uniformly, not mixing the mixture violently by vortex, and standing the mixture for 15 minutes after mixing the mixture uniformly.

3) The highest concentration standard is serially diluted in multiple proportions with a diluent.

4) Preparing a capture microsphere: the total amount of diluted capture microspheres required for the experiment was determined, and 50. mu.L of diluted capture microspheres was required for each reaction. The required amount of each capture microsphere was determined and the kit provided 1.0. mu.L of capture microspheres per sample. And determining the volume of the required capture microsphere diluent, uniformly mixing the capture microspheres, transferring the mixed capture microspheres into a new test tube, and marking the mixed capture microspheres as mixed microspheres.

5) And (3) preparing a detection antibody, namely determining the volume of the PE detection reagent to be diluted, and marking the PE detection reagent as the mixed PE detection reagent.

6) Add 50. mu.L of the mixed microspheres to each tube. 50 μ L of the corresponding standard or sample was added to each tube. Mix well and incubate for 1 hour at room temperature in the dark. 50 μ L of the mixed PE detection reagent was added to each tube. Incubate gently and mix at room temperature for 1 hour.

7) 1.0mL of wash solution was added to each tube, 300g was centrifuged for 5 minutes, the supernatant carefully aspirated, and 300ul of wash solution per tube was added to resuspend the microspheres. And (4) performing computer analysis, exporting an FCS data file, and analyzing by using FCAP software. The results are shown in FIG. 5.

A multi-factor detection experiment of mouse serum proves that the content of MCP-1 in a CIA model group is obviously increased in a collagen-induced RA model, which indicates that the establishment of the CIA model is successful. Compared with the CIA model group, the content of MCP-1 in the DAP (6mg/kg) group is not obviously different, but the DAP (10mg/kg) group and the positive drug MTX (0.5mg/kg) treatment group are also obviously different, and the content of MCP-1 in the DAP and MTX combined group is also obviously different from that in the CIA model group, which shows that the DAP (10mg/kg) can reduce the content of MCP-1 in serum of mice and has the equivalent anti-inflammatory effect with the positive drug MTX (0.5mg/kg) group (figure 5). MCP-1 is an important proinflammatory cytokine and plays an important role in the development and progression of RA. Whereas 10mg/kg DAP reduced its secretion, alleviating the inflammatory progression.

Taken together, DAP can reduce the extent of morbidity in RA mice.

Therefore, the invention expands the new pharmacological activity of DAP, finds that DAP can relieve arthritis symptoms to a certain extent, simultaneously prevents and treats serious infection in arthritis treatment, further achieves the aim of relieving RA, and provides a brand new choice for RA treatment.