CN117643588A - Application of nitrofurantoin in preparation of anti-rheumatoid arthritis drugs - Google Patents

Abstract

The invention provides the application of nitrofurantoin in preparing anti-rheumatoid arthritis drugs, and belongs to the field of medicinal chemistry. Rheumatoid arthritis is typically characterized by synovial lining cell proliferation, which was significantly reduced by nitrofurantoin. In addition, nitrofurantoin can also significantly inhibit osteoporosis and bone loss caused by rheumatoid arthritis, significantly reduce the expression of inflammatory factors (IL-1β, IL-6, IL-17, IL-23), and promote anti-inflammatory factors. Expression of inflammatory cytokines (IL‑10). The present invention applies nitrofurantoin, which is originally an antibacterial drug, to the treatment of rheumatoid arthritis, enriches the types of medications for rheumatoid arthritis, and is expected to provide a new path for the treatment of rheumatoid arthritis.

Description

Application of nitrofurantoin in the preparation of anti-rheumatoid arthritis drugs

Technical field

The invention belongs to the field of medicinal chemistry, and particularly relates to the application of nitrofurantoin in the preparation of anti-rheumatoid arthritis drugs.

Background technique

Inflammation is a defensive response process of living tissues with vascular systems to damaging factors, and it is closely related to many diseases. Studying inflammation and related phenomena (including the establishment of inflammation models, the discovery of inflammatory response factors, the mechanism of action of response factors and drugs, and the relationship between inflammation and other diseases, etc.) is important for drug development, early diagnosis of cancer, research on many physiological processes, and cancer treatment etc. have important guiding significance.

Rheumatoid arthritis is a relatively common inflammatory disease and is a type of autoimmune disease. The incidence of this disease in the population is about 1%, affecting at least more than 30 million people worldwide. It mainly affects women and increases the risk of heart disease, lymphoma, diabetes, stroke and lung disease, and may reduce the life expectancy of patients. shortened by 10 to 15 years. At present, the pathogenesis of rheumatoid arthritis has not yet been clarified, but many breakthrough discoveries about new cells, molecules, and pathways in related fields have been reported in top international academic journals, and the market size of rheumatoid arthritis also showed significant growth. Currently, drugs used for rheumatoid arthritis are mainly divided into non-steroidal anti-inflammatory drugs, disease-improving anti-rheumatic drugs, including targeted drugs, biological agents, etc. For example, related drugs targeting tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and non-receptor tyrosine kinase (JAK) have shown effective clinical effects. However, these drugs usually have low response effects and can easily cause toxic side effects such as viral infections and malignant tumors, and their use is limited in long-term clinical use. Therefore, the development of new therapeutic drugs for rheumatoid arthritis is of great scientific and practical significance.

Nitrofurantoin's English name is Nitrofurantoin, its chemical formula is C ₈ H ₆ N ₄ O ₅ , and its CAS number is 67-20-9. It is an antibacterial drug with broad-spectrum antibacterial activity and can be used clinically for urinary tract infections caused by sensitive bacteria. Systemic infections, etc., its clinical effectiveness and safety have been clinically confirmed. At present, there are no reports on the use of nitrofurantoin in the treatment of rheumatoid arthritis.

Contents of the invention

The invention provides the use of nitrofurantoin in preparing anti-rheumatoid arthritis drugs. Applying nitrofurantoin, originally an antibacterial drug, to the treatment of rheumatoid arthritis has enriched the types of drugs for the treatment of rheumatoid arthritis and is expected to provide a new path for the treatment of rheumatoid arthritis.

The present invention is realized through the following technical solutions:

The invention provides the use of nitrofurantoin in preparing anti-rheumatoid arthritis drugs.

Based on the same inventive concept, the present invention provides a medicine for treating rheumatoid arthritis, and the active ingredient of the medicine includes nitrofurantoin.

Based on the same inventive concept, the present invention provides the application of nitrofurantoin in the preparation of drugs for inhibiting synovial lining cell proliferation.

Based on the same inventive concept, the present invention also provides the use of nitrofurantoin in preparing drugs for inhibiting osteoporosis.

Based on the same inventive concept, the present invention also provides the application of nitrofurantoin in preparing drugs for inhibiting bone loss.

One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

1. The application of nitrofurantoin of the present invention in the preparation of anti-rheumatoid arthritis drugs. This application applies nitrofurantoin, which is originally an antibacterial drug, to the treatment of rheumatoid arthritis, enriching the types of drugs for treating rheumatoid arthritis, and is expected to be A new approach to the treatment of rheumatoid arthritis.

2. The application of nitrofurantoin in the preparation of anti-rheumatoid arthritis drugs according to the present invention. Nitrofurantoin can significantly improve the symptoms of rheumatoid arthritis in mice, significantly reduce the proliferation of synovial lining cells, and can also significantly inhibit rheumatoid joints. Osteoporosis and bone loss caused by inflammation, and significantly reduce the expression of inflammatory factors (IL-1β, IL-6, IL-17, IL-23), while promoting the expression of anti-inflammatory cytokines (I L-10), therefore, nitrofurantoin can be used in the treatment of rheumatoid arthritis.

Description of drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained based on these drawings without exerting creative efforts.

Figure 1 shows the therapeutic effect of nitrofurantoin on rheumatoid arthritis.

Figure 2 shows the effect of nitrofurantoin on different inflammatory factors.

Detailed ways

The present invention will be described in detail below with reference to specific implementation modes and examples, from which the advantages and various effects of the present invention will be more clearly presented. Those skilled in the art should understand that these specific implementation modes and examples are used to illustrate the present invention, but not to limit the present invention.

Throughout this specification, unless otherwise specifically stated, the terms used herein are to be understood as having the meaning commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is any conflict, this manual takes precedence.

Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased in the market or prepared by existing methods.

The overall idea of the present invention is as follows:

At present, there is a lack of therapeutic drugs for rheumatoid arthritis. Therefore, the development of new target drugs for rheumatoid arthritis has very important scientific and practical significance.

Nitrofurantoin is an antibacterial drug with broad-spectrum antibacterial activity. It can be used clinically for urinary tract infections caused by sensitive bacteria. Its clinical effectiveness and safety have been clinically confirmed. At present, there are no reports on the use of nitrofurantoin in the treatment of rheumatoid arthritis.

The inventor found through extensive experiments that nitrofurantoin can significantly improve the symptoms of rheumatoid arthritis in mice, including: significantly reducing the proliferation of synovial lining cells, significantly inhibiting osteoporosis and bone loss caused by rheumatoid arthritis, and significantly Reduce the expression of inflammatory factors (IL-1β, IL-6, IL-17, IL-23) and promote the expression of anti-inflammatory cytokines (IL-10). Based on this, the present invention proposes to apply nitrofurantoin, which is originally an antibacterial drug, to the treatment of rheumatoid arthritis. It enriches the types of drugs for the treatment of rheumatoid arthritis and is expected to provide a new path for the treatment of rheumatoid arthritis.

The application of nitrofurantoin in the preparation of anti-rheumatoid arthritis drugs according to the present application will be described in detail below with reference to examples and experimental data.

Example 1

This embodiment conducts an animal model test of nitrofurantoin for the treatment of rheumatoid arthritis, including the following steps:

(1) Animal information: Animals were obtained from Hunan Slack Jingda Experimental Animal Co., Ltd., 20 C57 mice, female, 4-6 weeks old, raised under specific pathogen free (SPF) conditions. The breeding environment is a temperature of 22 to 26°C, a relative humidity of 50% to 60%, and artificial light for 12 hours each. Adaptation feeding for 7 days.

(2) After adaptive feeding, the mice were randomly divided into blank control group (n=5) and rheumatoid arthritis model group (n=15). Among them, the blank control group: normal feeding without any operation; the rheumatoid arthritis model group (n=15): the common modeling method of rheumatoid arthritis is adopted, that is, type II collagen and Freund's complete adjuvant are fully Mix well to make a 2mg/mL emulsion. Inject 50uL/animal into the left and right foot pads respectively. Boost immunity after 1 week.

(3) Group intervention. After the modeling was completed, the rheumatoid arthritis model group (n=15) was randomly divided into model group (n=5), nitrofurantoin group (n=5), and baricitinib group (n=5); among which Model group: Normal feeding without any operation. Nitrofurantoin group: Nitrofurantoin was injected into the tail vein, the drug dose was 2 mg/kg, the drug concentration was 0.4 mg/mL, the injection volume was 0.1 mL/animal, once every 2 days, and the intervention was continuous for 40 days. Baricitinib group: Baricitinib was injected into the tail vein, the drug dose was 2 mg/kg, the drug concentration was 0.4 mg/mL, the injection volume was 0.1 mL/animal, once every 2 days, and the intervention was continuous for 40 days. During the intervention, daily weight weighing and arthritis scoring tests were performed.

(4) Obtain materials. After the intervention, the mice were anesthetized with 1% sodium pentobarbital, and blood and hind limb paws were collected. If it is found that the animal is not dead, use 100mg/kg sodium pentobarbital to anesthetize until there is no heartbeat and breathing and then confirm the death of the animal.

(5)Micro-CT test: Instrument model: Skyscan 1276Burker

Fixation and preservation of specimens: First, put the prepared specimens into 4% paraformaldehyde solution for 24-48 hours; then, remove the specimens and rinse them three times with phosphate buffer; finally, put the specimens into 75% alcohol solution. Save it for later use and send it for Micro-CT scan.

(6)H&E dyeing:

I. When cutting tissue, sharp knives and scissors should be used. When cutting tissue blocks, start from the root of the knife and pull backwards to cut through the tissue. Place the fixed mouse left paw tissue with the plane to be cut facing down at the bottom of the plastic embedding box, and follow the usual steps for dehydration and wax embedding.

II. Slicing: Before slicing, freeze the embedded tissue sample at -20°C. Once the tissue reaches appropriate hardness, it can be sliced. The thickness of the slice is 3 μm. If it is too thick, the patch will not be firm and it is not conducive to observation under the microscope. Embedding creates certain edges on the upper and lower sides of the tissue, which facilitates continuous slicing and avoids tissue shrinkage when unfolding the slices.

III. Expand and bake slices: Expand slices in water bath, fish out the slices, and carefully attach the slices to the glass slide. When attaching the slices, your hands should be steady and your hands should extend downward. Control the slices, then put the slices into the display device to unfold the slices. Pay attention to make the slices free of bubbles, flat and tight, and bake the slices in time.

IV.H&E staining: 1) Routinely dewax the tissue sections to water; 2) Wash slightly with water for 1-2 minutes; 3) Stain with hematoxylin semen for 3-6 minutes; 4) Wash away the hematoxylin semen with running water for 1-2 minutes; 5) 1% Hydrochloric acid alcohol 1-3s; 6) Slightly washed with water for 1-2s; 7) Blue-promoting liquid to return to blue for 5-10s; 8) Rinse with running water for 15-30s; 9) Dye with 0.5% eosin solution for 2-3 minutes; 10) Slightly washed with distilled water 1-2s; 11) 80% ethanol 15-30s; 12) 95% ethanol 15-30s; 13) absolute ethanol 1-2s; 14) xylene (I) 2-3s; 15) xylene (II) 2 -3s; 16) Neutral gum sealing.

V. Image collection: Take pictures through the microscope, and the Leica Application Suite image system collects the relevant parts of the sample.

(7) Serum inflammatory factor test:

a. Dilution of standard substance: Prepare 6 small test tubes and number them in sequence. First add 110uL of standard diluent to each small test tube, then take 110uL of the original concentration standard and add it to a numbered test tube. Mix well; then add 110uL from this test tube to the second test tube and mix thoroughly; then take 110uL from this test tube and add it to the third test tube and mix thoroughly; then take 110uL from this test tube and add the fourth test tube. into the test tube and mix thoroughly; then take 110uL from the test tube and add it to the fifth test tube and mix thoroughly; then take 110uL into the test tube and discard it. The sixth test tube was used as the No. 0 standard.

b. Add sample: Set standard wells, blank wells, and sample wells on the enzyme-labeled coated plate. Add 50uL of standards of different concentrations to the standard wells in sequence; first add 40uL of sample to the sample well, and then add 10uL of biotin-labeled antibody.

c. Add enzyme and incubate: Except for the blank wells, add 50uL of enzyme-labeled reagent to each well, seal the plate with a sealing film and incubate it in a 37°C incubator for 30 minutes.

d. Washing: Dilute the concentrated washing solution 30 times with distilled water, carefully remove the sealing film, discard the liquid in the wells, spin dry, fill each well with washing solution, let it stand for 30 seconds and then discard, repeat this 5 times. Pat dry.

e. Color development: First add 50 uL of chromogen A to each well, then add 50 uL chromogen B, shake gently to mix, and develop color for 10 minutes at 37°C in the dark.

f. Termination and measurement: Add 50uL of stop solution to each well to terminate the reaction; zero the blank wells and measure the absorbance (OD value) of each well at 450nm wavelength. The measurement should be performed within 15 minutes after adding the stop solution.

The test results are shown in Figure 1.

After the experiment, three groups of data were randomly selected for statistical analysis. The results are as follows: Figure 1A shows the establishment of a mouse model of rheumatoid arthritis and the schematic diagram of drug intervention, as well as the scores and results of rheumatoid arthritis in different groups of mice. thickness. As can be seen from the figure, nitrofurantoin can significantly improve the symptoms of rheumatoid arthritis, and its efficacy is equivalent to that of the commercial drug baricitinib. Figure 1B shows Micro-CT images of the paws of mice in different groups and related bone analysis parameter results. It can be seen from the figure that the rheumatoid arthritis group has the typical characteristics of bone loss, and the number of trabeculae (Tb, N) and bone mineral density (BMD) are both reduced, indicating that the mice with rheumatoid arthritis have produced more bone loss. Severe osteoporosis and bone loss; while the CT images, Tb, N and BMD values of the drug intervention group (i.e. nitrofurantoin group and baricitinib group) were significantly improved or increased compared with the rheumatoid arthritis group. High, indicating that nitrofurantoin has better efficacy. Figure 1C shows H&E staining pictures of joints of mice in different groups. It can be seen from the picture that the rheumatoid arthritis group (i.e., RA model group) produced obvious synovial lining cell proliferation (Synovial lining cells). hyperplasia); while the proliferation of the drug intervention group was significantly inhibited. Figure 2 shows the concentrations of typical inflammatory factors or anti-inflammatory cytokines in the serum of mice in different groups. The results show that the drug group significantly reduced the expression of inflammatory factors (IL-1β, IL-6, IL-17, IL-23 ), and promotes the expression of anti-inflammatory cytokines (IL-10).

The above results all show that nitrofurantoin can well relieve the symptoms of rheumatoid arthritis, inhibit the expression of various inflammatory factors, and has good curative effect on rheumatoid arthritis.

Finally, it should also be noted that the terms "comprises," "comprises," or any other variation thereof are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that includes a list of elements includes not only those elements, but also It also includes other elements not expressly listed or that are inherent to the process, method, article or equipment.

Although the preferred embodiments of the present invention have been described, those skilled in the art will be able to make additional changes and modifications to these embodiments once the basic inventive concepts are apparent. Therefore, it is intended that the appended claims be construed to include the preferred embodiments and all changes and modifications that fall within the scope of the invention.

Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the invention. In this way, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and equivalent technologies, the present invention is also intended to include these modifications and variations.

Claims (5)

1. Application of nitrofurantoin in the preparation of anti-rheumatoid arthritis drugs. 2. A medicine for treating rheumatoid arthritis, characterized in that the active ingredient of the medicine includes nitrofurantoin. 3. Application of nitrofurantoin in the preparation of drugs that inhibit the proliferation of synovial lining cells. 4. Application of nitrofurantoin in the preparation of drugs for inhibiting osteoporosis. 5. Application of nitrofurantoin in the preparation of drugs for inhibiting bone loss.