CN111840302A - Application of two compounds in preparing anti-rheumatoid arthritis medicine - Google Patents

Abstract

The invention discloses application of two compounds in preparing a medicine for resisting rheumatoid arthritis. The invention discovers that the compounds 1 and 2 shown in the figure 1 have the activity for treating the rheumatoid arthritis similar to that of the positive drug methotrexate, so that the compounds 1 and 2 shown in the figure 1 have the prospect of being developed into the drugs for treating the rheumatoid arthritis.

Description

Application of two compounds in preparing anti-rheumatoid arthritis medicine

Technical Field

The invention belongs to the field of medicines, and particularly relates to application of two compounds in preparation of an anti-rheumatoid arthritis medicine.

Background

Rheumatoid Arthritis (RA) is a chronic systemic connective tissue disease, belonging to the category of "maladies" in traditional chinese medicine, and is pathologically characterized by synovial hyperplasia leading to degradation of articular cartilage and bone, resulting in functional disability. RA patients are distributed in the world, the morbidity of the RA patients is different from one another by certain ethnicity, the prevalence rate of the RA patients in different populations is 0.18-1.07%, the prevalence rate of the RA patients in China is 0.32-0.36%, the disability rate of the RA patients is more than that of the RA patients in males, the RA patients seriously affect the life quality of the RA patients, and the RA patients are listed as one of five diseases which have the greatest influence on human health and the highest medical consumption in the future in recent years.

RA pathogenesis is complex, multiple action targets and links exist, the complete elucidation is not achieved at present, the common opinion is related to individual genetic susceptibility, environmental factors and dysregulated immune response, and how to effectively prevent and treat RA is a hot point of attention of researchers.

Disclosure of Invention

The invention aims to provide application of two compounds in preparing a medicine for resisting rheumatoid arthritis.

The above purpose of the invention is realized by the following technical scheme:

use of a compound of the following chemical structure for the preparation of a medicament against rheumatoid arthritis:

a pharmaceutical preparation for resisting rheumatoid arthritis is prepared from the above compounds as active ingredients and pharmaceutically acceptable adjuvants as carriers by making into pharmaceutically acceptable dosage forms.

Further, the auxiliary material is solid, liquid or semisolid.

Further, the dosage form is injection, tablet, capsule, pill, powder or oral liquid.

Has the advantages that:

the invention discovers that the compounds 1 and 2 shown in the figure 1 have the activity for treating the rheumatoid arthritis similar to that of the positive drug methotrexate, so that the compounds 1 and 2 shown in the figure 1 have the prospect of being developed into the drugs for treating the rheumatoid arthritis.

Drawings

FIG. 1 is the chemical structural formulas of Compound 1 and Compound 2;

figure 2 is the mean arthritis index for each group of mice, where: comparing the administration group with the model group (. + -.) p <0.05, (. + -.) p <0.01, (. + -.) p < 0.001;

FIG. 3 is the mean body weight change of the mice in each group;

FIG. 4 is a photograph of the left hind limb of representative groups of mice taken before sacrifice on day 42;

fig. 5 is the left hind limb footpad thickness for each group of mice, where: comparing the administration group with the model group (. + -.) p <0.05, (. + -.) p <0.01, (. + -.) p < 0.001;

FIG. 6 is a H & E diagram of representative joints of mice in each group.

Figure 7 is a H & E score for each group of mouse joints, where: comparing (. about.). sup.p <0.01 in the administration group with the model group;

Detailed Description

The following detailed description of the present invention is provided in connection with the accompanying drawings and examples, but not intended to limit the scope of the invention.

First, test materials

1. Test drugs and reagents

Methotrexate tablets: 2.5 mg/tablet of Shanghai medicine Xinyi pharmaceutical factory Co., Ltd; the chemical structures of compound 1 and compound 2 are shown in FIG. 1, the former is from Tokyo Kasei Kogyo Co., Ltd, and the latter is from Steraloids company of USA; bovine type II collagen: chondrex, USA; complete Freund's Adjuvant (CFA): chondrex, USA; incomplete Freund's Adjuvant (IFA): chondrex corporation, USA.

2. Animal for test

Male DBA/1 mice: SPF grade, 18-22g weight, supplied by shanghai slyke test animals ltd, certification number: 20170005025510, test animal production license number: SCXK (Shanghai) 2017-.

3. Feed for test

Complete nutrition pellet feed: provided by cooperative medical biotechnology limited of Jiangsu province, lot number: 20200409.

4. test conditions

Room temperature 20 + -2 deg.C, humidity 55-65%, proper illumination, and good ventilation. Animal laboratory use license number: SCXK (Su) 2017-0069.

5. Pharmaceutical and reagent formulations

Grinding methotrexate 1 tablet (2.5mg), adding 16.67mL of 0.5% CMC-Na solution, suspending to obtain suspension containing 0.15mg of methotrexate per mL, and performing intragastric administration according to 0.1mL/10 g; precisely weighing 1100mg of the compound, adding 1mL of DMSO for dissolving, subpackaging and storing per 100 μ l of DMSO (-20 ℃), adding 2.4mL of 0.5% CMC-Na solution into 100 μ l of mother liquor before administration until each mL contains 14mg of the compound, and performing intragastric administration according to 0.1mL/10 g; precisely weighing 5mg of compound 2, adding 1mL of DMSO for dissolving, subpackaging and storing at 120 μ l (-20 ℃), adding 2.88mL of 0.5% CMC-Na solution into 120 μ l of mother liquor before administration until each mL contains 20.2mg of compound, and performing intragastric administration at 0.1mL/10 g; bovine type II collagen immune emulsifier: sucking 6mL of 2mg/mL bovine II type collagen, adding the bovine II type collagen into a sterile test tube, then adding 6mL of Freund's complete adjuvant according to the proportion of 1:1, pushing and pulling back and forth by using an injector to ensure that the bovine II type collagen and the adjuvant are fully emulsified and uniformly mixed until the liquid is milky white, and dropping the mixture into clear water until the emulsion drops keep the original state and are not dispersed. The process is carried out on ice to prevent collagen deformation; bovine type II collagen immunopotentiating emulsifier: sucking 6mL of 2mg/mL bovine II type collagen, adding the bovine II type collagen into a sterile test tube, then adding 6mL of Freund's incomplete adjuvant according to the proportion of 1:1, pushing and pulling back and forth by using an injector to ensure that the bovine II type collagen and the adjuvant are fully emulsified and uniformly mixed until the liquid is milky white, and dropping the mixture into clear water until the emulsion drops keep the original state and are not dispersed. The process was performed on ice to prevent collagen deformation.

Second, test method

1. Model building method for rheumatoid arthritis model

For the first immunization, 10mL of bovine type II collagen (2mg/mL) was precisely aspirated, and 10mL of Freund's complete adjuvant was added as 1: preparing immune reagent according to the ratio, emulsifying and mixing evenly (the whole process is carried out on ice). Injecting subcutaneous injection at 1-2cm distance from the root of mouse tail at multiple points, administering 200 μ L physiological saline solution when molding each mouse in normal group, and administering 200 μ L immunoreagent to each mouse in molding group. A second boost was performed 21 days after the first immunization. 10mL of bovine type II collagen (2mg/mL) was precisely aspirated, and 10mL of Freund's incomplete adjuvant was added at a volume ratio of 1: preparing immune enhancing agent in the ratio, emulsifying and mixing (the whole process is carried out on ice). Injecting subcutaneously at multiple points 1-2cm from the root of mouse tail, administering 200 μ L physiological saline to each mouse in normal group, and administering 200 μ L immunopotentiating agent to each mouse in model group.

2. Group administration and detection

The molded mice were randomly divided into four groups: the model group, methotrexate group (i.e., positive drug group), compound 1 group, and compound 2 group were simultaneously set as controls, and normal group was given physiological saline only in the above-mentioned manner without molding, for a total of five groups of 8 mice each. The administration method for each group is as follows:

normal group: equivalent 0.5% CMC-Na solution;

model group: equivalent 0.5% CMC-Na solution;

methotrexate group: 1.5mg/kg, 0.1mL/10 g;

compound group 1: 40mg/kg, 0.1mL/10 g;

compound group 2: 2mg/kg, 0.1mL/10 g.

Methotrexate groups were administered once every three days, and the remaining groups were administered daily at the dose for a total of 3 weeks.

The weight of each group of mice was measured before the second immunization and every 3 days after the second immunization, the thickness of the left hind limb and foot pad of the mice was measured with a vernier caliper, and the plantar arthritis index of the mice was scored. The degree of lesion of each joint was rated according to 5, 0: no red swelling; 1: red swelling is localized to one toe; 2: red and swollen with more than one toe; 3: red swelling involves all toes and instep; 4: the whole paw and ankle joint are severely inflamed. The Mean Arthritis Index (MAI) is the sum of the arthritis scores of all mice in each dose group divided by the number of mice in that group. The difference in body weight of the mice was the body weight on the other days (24, 27, 30, 33, 36, 39, 42) minus the body weight on day 21 divided by the body weight on day 21. And taking the joint of the mouse to make an H & E pathological section. The examination indexes under the optical microscope are as follows: (1) whether articular surface cartilage is damaged; (2) whether the synovium is hyperplastic; (3) whether there is inflammatory cell infiltration in the synovium; (4) whether there is exudate in the joint cavity; (5) whether pannus is significantly increased; (6) whether articular cartilage and bone marrow are affected. According to the degree of the pathological changes, the semi-quantitative score is 0-4, the mild score is 0.5, the mild score is 1, the moderate score is 2, the severe score is 3, the severe score is 4, the non-pathological tissue is marked as 0, all the scores are accumulated, and the average score of each group is calculated.

3. Statistical method

Studeng' stest, () p <0.05, () p <0.01, () p <0.001 was used.

Third, test results

1. Mean arthritis index in each group of mice

The average arthritis indexes of the mice in each group are shown in table 1 and figure 2, the mice in a normal group have no joint lesion symptoms, the joint lesion degree of the mice in a model group is the most serious, and the average arthritis index gradually increases along with the test progress; compared with the model group, the average arthritis index of the mice in the methotrexate group is obviously reduced, which shows that the positive methotrexate inhibits the joint lesion degree of the model-making mice; compared with the model group, the average arthritis indexes of the mice in the compound 1 group and the compound 2 group are also obviously reduced, which indicates that the compound 1 and the compound 2 play a role in inhibiting the joint lesion of the rheumatoid arthritis model mice similar to that of the positive drugs.

TABLE 1 mean arthritis index in each group of mice

2. Weight change of mice in each group

As shown in table 2 and fig. 3, the weight of mice in the model group was reduced and the weight change of mice in the methotrexate group, compound 1 group, and compound 2 group was improved as compared with the normal group.

TABLE 2 mean body weight differences for the groups of mice

3. Swelling change in the feet of mice in each group

The thickness of the left hind limb foot pad of each group of mice is shown in table 3 and fig. 5, the mice in the normal group have no foot swelling, the mice in the model group have obvious foot swelling, and the swelling degree gradually increases along with the test progress; compared with the model group, the foot swelling degree of the mice in the methotrexate group is obviously relieved, which shows that the positive methotrexate inhibits the foot swelling of the model-making mice; compared with the model group, the foot swelling degree of the mice in the compound 1 group and the compound 2 group is also obviously relieved, which shows that the compound 1 and the compound 2 play the similar role of inhibiting the foot swelling of the rheumatoid arthritis model mice as the positive drugs. Figure 4 is a photograph of the left hind limb of representative groups of mice taken 42 days before sacrifice.

TABLE 3 average left hind limb and foot pad thickness (mm) for each group of mice

4. Pathological detection of joints of mice in each group

FIG. 6 is a graph of representative joints H & E of mice in each group, the H & E score of the joints of the mice in each group is shown in FIG. 7, and FIGS. 6 and 7 illustrate that: compared with the normal ratio, the articular lesion of the mice in the model group is severe, and the H-score is highest; compared with the model group, H-score is remarkably reduced in the methotrexate group, the compound 1 group and the compound 2 group. The compound 1 and the compound 2 are proved to play the similar effect of inhibiting the joint lesion of the rheumatoid arthritis model mouse as the positive medicine.

The above test results show that the compounds 1 and 2 shown in fig. 1 have activity for treating rheumatoid arthritis similar to that of the positive drug methotrexate, and thus the compounds 1 and 2 shown in fig. 1 have a promising prospect for the development of drugs for treating rheumatoid arthritis.

The above-described embodiments are intended to be illustrative of the nature of the invention, but those skilled in the art will recognize that the scope of the invention is not limited to the specific embodiments.

Claims (4)

1. Use of a compound of the following chemical structure for the preparation of a medicament against rheumatoid arthritis:

2. a pharmaceutical preparation for resisting rheumatoid arthritis is characterized in that: the compound as claimed in claim 1 is used as an active ingredient, and pharmaceutically acceptable excipients are used as carriers to prepare pharmaceutically acceptable dosage forms.

3. The pharmaceutical formulation of claim 2, wherein: the auxiliary material is solid, liquid or semisolid.

4. The pharmaceutical formulation of claim 2, wherein: the preparation formulation is injection, tablet, capsule, pill, powder or oral liquid.

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