Application of cepharanthine in preparation of medicine for treating mycoplasma pneumoniae infection

Abstract

The invention provides application of cepharanthine and pharmaceutically acceptable salts or prodrugs thereof in preparation of drugs for relieving and / or treating mycoplasma pneumoniae infection. Cell experiments and animal experiments show that cepharanthine has an inhibiting effect on mycoplasma pneumoniae, and cepharanthine has certain biological safety. Therefore, the compound can be used for preparing medicines for relieving and / or treating mycoplasma pneumoniae infection.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and particularly relates to the use of cepharanthine in preparing a medicine for alleviating and treating Mycoplasma pneumoniae infection and respiratory inflammation caused by the infection. Background Art

[0002] Mycoplasma pneumoniae (MP) is one of the main pathogens of community-acquired pneumonia (CAP), causing acute upper and lower respiratory tract inflammation and extrapulmonary complications. Its genome is 816,394 base pairs long, comprising 687 genes. It is the smallest prokaryotic organism capable of independent survival in vitro. Due to its limited metabolic capacity, MP requires complex culture components for in vitro propagation. First discovered in patients with atypical pneumonia, MP possesses only a cell membrane, lacking a supporting cell wall. The outermost cell membrane is composed of protein, lipid, and protein. Because its structure is completely different from that of traditional eubacteria, it cannot be observed using ordinary light microscopy and requires electron microscopy. Under electron microscopy, MP cells primarily appear as short, filamentous or dumbbell-shaped cells, with the cell membrane extending outward at one end to form a distinctive structure resembling a wine bottle. Individual MP cells are 0.1-0.2 μm in size and can pass through a 0.45 μm filter. The pneumonia caused by it is called Mycoplasma pneumoniae pneumonia (MPP), and its clinical manifestations are usually mild and self-limited. However, life-threatening pneumonia and even acute respiratory distress syndrome requiring extracorporeal membrane oxygenation have also been reported. In addition, some extrapulmonary symptoms, such as mucositis, hepatitis, encephalitis, hemolysis, or erythema multiforme, are related to infection with Mycoplasma pneumoniae and the formation of autoimmunity or immune complexes, and are closely related to refractory asthma. As a prokaryotic organism, MP has no cell wall, making antibiotics that act on the cell wall ineffective in treating MPP. However, MP is highly sensitive to antibiotics that act on bacterial DNA and protein synthesis, such as macrolides, quinolones, and tetracyclines, which can be used clinically to treat MPP. Clinically, administering tetracycline to children can directly stimulate gastrointestinal function, causing significant intestinal reactions, and is hepatotoxic and can affect dental enamel hypoplasia. Quinolone antibiotics can affect cartilage and bone development in children. Macrolide antibiotics, in addition to lacking these side effects, have low MICs against bacteria, low toxicity, no contraindications in young children, high oral availability, and a once-daily formulation. Therefore, they are considered first-line treatments for pediatric MP infections. However, the incidence of macrolide-resistant MP has been increasing worldwide, particularly in Asia, primarily due to the overuse of macrolides. Reports indicate that 69%-95% of Mycoplasma pneumoniae isolates in China are resistant. MP resistance has become a growing problem, with macrolide-resistant MP strains emerging globally. These strains can lead to inflammation and refractory pneumonia, as well as numerous extrapulmonary complications such as erythema nodosum, liver damage, and central nervous system impairment. Therefore, understanding the mechanisms of resistance and developing targeted therapeutics to combat resistant MP are urgently needed.

[0003] Currently, research on anti-Mycoplasma pneumoniae drugs mainly focuses on the improvement of existing antibiotics, so there is an urgent need to develop more alternative drugs. Summary of the Invention

[0004] The present invention provides cephalanthrin and use of a pharmaceutically acceptable salt or prodrug thereof in preparing a medicament for alleviating and / or treating Mycoplasma pneumoniae infection.

[0005] According to an embodiment of the present invention, the pharmaceutically acceptable salt of Cephalanthus truncatula is selected from the acid addition salts having a nitrogen atom with sufficient basicity in its structure, and the acid addition salts include Cephalanthus truncatula and inorganic acids such as hydrochloric acid, hydrofluoric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, pyrosulfuric acid, phosphoric acid or nitric acid; organic acids such as formic acid, acetic acid, acetoacetic acid, pyruvic acid, trifluoroacetic acid, propionic acid, butyric acid, hexanoic acid, heptanoic acid, undecanoic acid, lauric acid, benzoic acid, salicylic acid, 2-(4-hydroxybenzoyl)benzoic acid, camphoric acid, cinnamic acid, cyclopentanepropionic acid, digluconic acid, 3-hydroxy-2-naphthoic acid , nicotinic acid, pamoic acid, pectinic acid, persulfuric acid, 3-phenylpropionic acid, picric acid, pivalic acid, 2-hydroxyethanesulfonic acid, itaconic acid, sulfamic acid, trifluoromethanesulfonic acid, dodecylsulfuric acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, 2-naphthalenesulfonic acid, naphthalenedisulfonic acid, camphorsulfonic acid, citric acid, tartaric acid, stearic acid, lactic acid, oxalic acid, malonic acid, succinic acid, malic acid, adipic acid, alginic acid, maleic acid, fumaric acid, D-gluconic acid, mandelic acid, ascorbic acid, glucoheptanoic acid, glycerophosphoric acid, aspartic acid, sulfosalicylic acid, hemisulfuric acid or thiocyanic acid.

[0006] According to an embodiment of the present invention, the Mycoplasma pneumoniae infection may lead to respiratory diseases and extrapulmonary complications.

[0007] According to an embodiment of the present invention, the respiratory system diseases include: Mycoplasma pneumoniae pneumonia (primary atypical pneumonia) and upper respiratory tract infection.

[0008] According to an embodiment of the present invention, the upper respiratory tract infection includes pharyngitis, tonsillitis, sinusitis, and otitis media.

[0009] According to an embodiment of the present invention, the extrapulmonary complications include: myocarditis, pericarditis, pericardial effusion, aseptic meningitis, aseptic encephalitis, Guillain-Barré syndrome (GBS), hemolytic anemia, thrombocytopenic purpura, hepatitis, gastroenteritis, erythema multiforme, Stevens-Johnson syndrome (SJS), arthritis or arthralgia.

[0010] According to an embodiment of the present invention, the MIC value of cepharanthin against Mycoplasma pneumoniae is 39.0625 μg / ml.

[0011] According to an embodiment of the present invention, the IC value of Stephania tetrandra against Mycoplasma pneumoniae 50 The value is 21 μM.

[0012] According to an embodiment of the present invention, cepharanthin, its pharmaceutically acceptable salt or prodrug can directly inhibit the P1 protein of Mycoplasma pneumoniae in vitro.

[0013] According to an embodiment of the present invention, cepharanthin, a pharmaceutically acceptable salt or a prodrug thereof can inhibit the inflammatory cytokine storm in lung tissue, for example, reduce the expression of TNF-α, IL-6 and IL-1β.

[0014] The present invention also provides a method for alleviating and / or treating Mycoplasma pneumoniae infection, comprising: administering at least one of cepharanthin, a pharmaceutically acceptable salt or a prodrug thereof to an individual (eg, a human) in need thereof.

[0015] The inventors conducted in vitro anti-pneumoniae experimental tests and found that the MIC value of cephalanthrin against mycoplasma pneumoniae was 39.0625 μg / ml, and the IC 50 The value is 21 μM, and cephalothin can directly inhibit the P1 protein of Mycoplasma pneumoniae in vitro. In addition, the inventors also established an animal model of Mycoplasma pneumoniae nasal infection to evaluate the effect of cephalothin against Mycoplasma pneumoniae. The experimental results showed that high doses (20 mg / kg), medium doses (15 mg / kg) and low doses (10 mg / kg) of cephalothin can inhibit the inflammatory factor storm (TNF-α, IL-6 and IL-1β and other indicators decreased). Therefore, cephalothin, its pharmaceutically acceptable salts or prodrugs can be used to prepare drugs for alleviating and / or treating Mycoplasma pneumoniae infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a test chart of the half-maximal inhibitory concentration of different concentrations of cephalothin to inhibit Mycoplasma pneumoniae.

[0017] Figure 2 Figure 3 shows that cephalothin inhibited the expression of Mycoplasma pneumoniae P1 protein at the protein level.

[0018] Figure 3 This is a test graph showing that cephalothin inhibits the expression of cytokines TNF-α, IL-6, and IL-1β in the lung tissues of mice infected with Mycoplasma pneumoniae (*** indicates P<0.001; **** indicates P<0.0001). DETAILED DESCRIPTION

[0019] The technical solutions of the present invention will be described in further detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are encompassed within the scope of protection that the present invention is intended to protect.

[0020] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0021] Example 1 Test of the Anti-Mycoplasma Pneumoniae Activity of Cephalaenopsisin

[0022] 1. Mycoplasma pneumoniae culture

[0023] The international standard strain of Mycoplasma pneumoniae (ATCC29342 / M129) was kindly provided by Beijing Children's Hospital. PPLO-Medium (Pleuropneumonia-Like Organism Medium) is used, supplemented with serum (typically 20% to 30% horse serum or bovine serum) to provide cholesterol and lipid components to maintain membrane stability. The medium also contains yeast extract, glucose (or other carbon source), phenol red (pH indicator), and necessary antibiotics (such as penicillin) to inhibit contamination by other bacteria. Cultures are then maintained in an incubator at 37°C and 5% CO2.

[0024] 2. Determination of the Minimum Inhibitory Concentration (MIC) of Cephalaenopsis in vitro against MP

[0025] (1) Mycoplasma pneumoniae CCU count

[0026] The Mycoplasma pneumoniae strain was diluted 10-fold. First, prepare a 1.5 mL sterile EP tube, add 900 μL of culture medium, and then add 100 μL of original Mycoplasma pneumoniae, and dilute to 10 in sequence. 10 , use 1 mL of pure culture medium as the negative control and culture in a 37°C incubator for 2 weeks. When the color does not change, the last EP tube that changes from red to yellow is the order of magnitude of Mycoplasma pneumoniae [liquid culture can confirm the bacterial count by the color change of the culture medium (phenol red changes from red to yellow)].

[0027] (2) Preparation of MP working bacterial solution

[0028] The frozen Mycoplasma pneumoniae strains were serially subcultured using the liquid culture method. The third generation was taken and the MP concentration was determined using the color change unit (CCU) method. The strains were quantitatively divided into small tubes and frozen for storage as seed solution. In each subsequent test, one strain was taken and diluted to 10% with MP liquid culture medium.5 CCU / ml, as the working bacterial solution.

[0029] (3) Drug gradient setting

[0030] Stephaniocin was dissolved in DMSO to prepare working solutions of different concentrations (1.25, 0.625, 0.3125, 0.15625, 0.078125, 0.0390625, 0.01953125, 0.00976562, 0.00488281 mg / ml), ensuring that the final DMSO concentration was ≤0.1% (volume fraction).

[0031] (4) Minimum Inhibitory Concentration (MIC) determination

[0032] MIC determination The MIC value of the experimental drug against the clinical isolate of MP was determined by microdilution method. 5 After incubation at a concentration of CCU / ml, the cells were incubated in a 37°C, 5% CO2 incubator for 2 hours. After incubation, 175 μL of the bacterial solution was added to a 96-well plate. Drugs of varying concentrations were then added. Each plate was set up with a positive control, a negative control, and a drug control. Two replicate wells were set up for each sample and sealed with paraffin oil. The plates were cultured in a 37°C, 5% CO2 incubator, and the results were observed and recorded daily. The results were observed after 48 to 96 hours of incubation. The growth status of MP was determined by observing that the growth metabolites of MPs can change the pH of the culture medium, thereby causing the indicator to change color. The minimum inhibitory concentration (MIC) was the minimum inhibitory concentration (MIC) at which the positive control wells changed color (i.e., the phenol red indicator in the culture medium changed from red to orange-yellow) without turbidity and the drug-addition wells no longer changed color.

[0033] MIC test results: The MIC value of cepharanthin against MP standard strain M129 was 39.0625 μg / ml.

[0034] 3. Cephalaenopsis inhibits Mycoplasma pneumoniae IC in vitro 50 Determination

[0035] (1) Preparation of MP working bacterial solution

[0036] The frozen Mycoplasma pneumoniae strains were serially subcultured using the liquid culture method. The third generation was taken and the MP concentration was determined using the color change unit (CCU) method. The strains were quantitatively divided into small tubes and frozen for storage as seed solution. In each subsequent test, one strain was taken and diluted to 10% with MP liquid culture medium. 5 CCU / ml is used as the working bacterial solution.

[0037] (2) Drug gradient setting

[0038] Stephaniocin was dissolved in DMSO to prepare working solutions of different concentrations (0.19, 0.39, 0.78125, 1.5625, 3.125, 6.25, 12.5, 25, 50, and 100 μM), ensuring that the final DMSO concentration was ≤ 0.1% (volume fraction). A negative control group (drug-free) was set up.

[0039] (3) Infection and treatment

[0040] The Mycoplasma pneumoniae strain working solution (diluted to 10 with MP liquid medium) 5 CCU / ml) were inoculated into 1.5 mL sterile EP tubes, co-cultured with various concentrations of cepharanthin, and incubated for 72 hours.

[0041] (4) Detection indicators

[0042] qPCR method: Quantitatively detect the expression level of specific genes (such as P1 adhesin) to reflect the number of mycoplasmas.

[0043] (5) Data Analysis

[0044] After normalization of the relative growth inhibition rate at each drug concentration, nonlinear fitting (four-parameter logistic curve) was performed using GraphPad Prism software to calculate the IC 50 value (drug concentration that makes the growth inhibition rate reach 50%).

[0045] The test results are as follows Figure 1 shown. Figure 1 The experimental results of half-inhibitory concentration showed that different concentrations of cephalothin could inhibit Mycoplasma pneumoniae, and the half-inhibitory concentration IC 50 The value was 21 μM. The test results showed that cephalanthrin has the effect of resisting Mycoplasma pneumoniae infection.

[0046] IC 50 It is the half-maximal inhibitory concentration, which indicates the drug concentration required to inhibit the growth of pathogens by 50%. The smaller the concentration, the better the drug's inhibitory effect on the virus.

[0047] 4. In vitro inhibition of Mycoplasma pneumoniae P1 protein expression by cepharanthin

[0048] The treatment method of cephalanthrin and Mycoplasma pneumoniae is as described in Section 3 above. Cephalanthrin inhibits Mycoplasma pneumoniae IC in vitro. 50 As described in the assay, the cells were incubated at 37°C for 72 hours and the cells were collected by centrifugation for protein extraction.

[0049] Lyse bacteria: Add RIPA lysis buffer (pre-mixed with protease and phosphatase inhibitors at a 1:100 ratio): Lyse on ice for 10 min (1960 μL RIPA + 20 μL PMSF (phenylmethylsulfonyl fluoride) + 20 μL protease inhibitors). Add 200 μL / well, shake on ice for 2 min, collect the sample, and centrifuge at 4°C at 12,000 rpm for 10 min. Collect the supernatant (total protein) and transfer it to a 1.5 ml EP tube. Perform the entire process on ice.

[0050] BCA quantification: Use a BCA kit to determine the protein concentration, adjust to 2 μg / μL, add 5× SDS-PAGE loading buffer, and denature at 100°C for 10 min.

[0051] SDS-PAGE electrophoresis: prepare 8% separation gel (for P1 protein, ∼190 kDa) and 5% stacking gel.

[0052] Load 20 μg of protein and 5 μL of marker into each well. Run at a constant voltage of 80 V (stacking gel) → 120 V (separating gel) until bromophenol blue reaches the bottom of the gel.

[0053] Transfer (wet transfer method): PVDF membrane was pretreated and activated with methanol for 1 min, then immersed in transfer buffer. The transfer conditions were a constant current of 200 mA and 4°C for 90 min (to ensure complete transfer of P1 macromolecules).

[0054] Blocking and antibody incubation: 5% skim milk (prepared in TBST), block on a shaker at room temperature for 1 h.

[0055] Primary antibody incubation: anti-P1 monoclonal antibody (1:1000, diluted in 5% BSA / TBST), incubation at 4°C overnight.

[0056] Secondary antibody incubation: HRP-labeled secondary antibody (1:10,000), shaken at room temperature for 1 h.

[0057] ECL development and data analysis: ECL luminescence was performed by mixing ECL A / B solution in a certain proportion, adding the mixture evenly onto the membrane, and imaging was performed using a chemiluminescence analyzer.

[0058] The test results are as follows Figure 2 shown. Figure 2 The test results showed that cephalanthrin effectively inhibited the expression of P1 protein of Mycoplasma pneumoniae. This indicates that cephalanthrin can inhibit the infection of Mycoplasma pneumoniae. The above results strongly prove the anti-Mycoplasma pneumoniae activity of cephalanthrin.

[0059] 5. Test of Cephalaenopsis in Treating Mice Infected with Mycoplasma Pneumoniae

[0060] SPF BALB / c mice were randomly divided into five groups: normal group, model group (MP), low-dose cepharanthin group (10 mg / kg), medium-dose cepharanthin group (15 mg / kg), and high-dose cepharanthin group (20 mg / kg), with five mice in each group. Except for the normal group, mice in the other groups were inoculated with 50 μL of 1×10 7 CCU / ml Mycoplasma pneumoniae strain model was established, and nasal drops were administered once a day for three consecutive days. After the model was successfully established, the low-dose group (10 mg / kg) of celastrol, the medium-dose group (15 mg / kg) of celastrol, and the high-dose group (20 mg / kg) of celastrol were given 10 mg / kg, 15 mg / kg, and 20 mg / kg, respectively, with 100 μL per mouse by gavage. The normal group and the model group were given 100 μL of 0.9% sodium chloride injection by gavage, and all groups of mice were gavage for 3 days. The pathological changes of lung tissue were observed, and the expression of inflammatory factors TNF-α, IL-6, and IL-1β in the lung tissue of mice was detected.

[0061] The experimental results are as follows Figure 3 . Figure 3 The results showed that MP nasal infection could induce the massive production of inflammatory factors TNF-α, IL-6 and IL-1β, and the expression levels in the lung tissue of mice were significantly higher than those in the normal control group (P<0.001), indicating that the model was successful; and oral administration of the three dose groups of cepharanthin for 3 days could significantly inhibit the production of TNF-α, IL-6 and IL-1β in the lung tissue of model mice (P<0.001).

[0062] In summary, both cell and animal experiments indicate that cephalanthrin has an inhibitory effect on Mycoplasma pneumoniae and has a certain degree of biosafety. Therefore, it can be used to prepare drugs for alleviating and / or treating Mycoplasma pneumoniae infection.

[0063] The above describes exemplary embodiments of the present invention. However, the scope of protection of the present invention is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc. made by those skilled in the art within the spirit and principles of the present invention shall be included in the scope of protection of the present invention.

Claims

1. Use of cephalanthrin, or a pharmaceutically acceptable salt or prodrug thereof, in the preparation of a medicament for alleviating and / or treating Mycoplasma pneumoniae infection.

2. The use according to claim 1, wherein The pharmaceutically acceptable salt of Cephalanthus truncatula is selected from the acid addition salts of a nitrogen atom having a sufficient basicity in its structure, and the acid addition salts include Cephalanthus truncatula and an inorganic acid such as hydrochloric acid, hydrofluoric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, pyrosulfuric acid, phosphoric acid or nitric acid; an organic acid such as formic acid, acetic acid, acetoacetic acid, pyruvic acid, trifluoroacetic acid, propionic acid, butyric acid, hexanoic acid, heptanoic acid, undecanoic acid, lauric acid, benzoic acid, salicylic acid, 2-(4-hydroxybenzoyl)benzoic acid, camphoric acid, cinnamic acid, cyclopentanepropionic acid, digluconic acid, 3-hydroxy-2-naphthoic acid, nicotinic ... pyruvic acid, pyruvic acid, pyruvic acid, pyruvic acid, pyruvic salts formed from 1, 2, 3-dimethylaminopropionic acid, 1, 4-dimethylaminopropionic acid, 1, 4-dimethylaminopropionic acid, 1, 4-dimethylaminopropionic acid, 1, 4-dimethylaminopropionic acid, 1, 4-dimethylaminopropionic acid, 1, 4-dimethylaminopropionic acid, 1, 4-dimethylaminopropionic acid, 1, 4-dimethylaminopropionic acid, 1, 4-dimethylaminopropionic acid, 1, 4-dimethylaminopropionic acid, 1, 4-dimethylaminopropionic acid, 1, 4-dimethylaminopropionic acid, 1, 4-dimethylaminopropionic acid, 1, 4-dimethylaminopropionic acid, 1, 4-dimethylaminopropionic acid, 1, 4-dimethylaminopropionic acid, 1, 4-dimethylaminopropionic acid, 1, 4-dimethylaminopropionic acid, 1, 4-dimethylaminopropionic acid, 1, 4-dimethylaminopropionic acid, 1, 4-dimethylaminopropionic acid, 1, 4-dimethylaminopropionic acid, 1, 4-dimethylaminopropionic acid, 3. The use according to claim 1 or 2, wherein: The Mycoplasma pneumoniae infection can lead to respiratory diseases and extrapulmonary complications.

4. The use according to claim 3, wherein: The respiratory system diseases include: Mycoplasma pneumonia and upper respiratory tract infection.

5. The use according to claim 4, wherein: The upper respiratory tract infection includes pharyngitis, tonsillitis, sinusitis, and otitis media.

6. The use according to claim 3, wherein: The extrapulmonary complications include myocarditis, pericarditis, pericardial effusion, aseptic meningitis, aseptic encephalitis, Guillain-Barré syndrome (GBS), hemolytic anemia, thrombocytopenic purpura, hepatitis, gastroenteritis, erythema multiforme, Stevens-Johnson syndrome (SJS), arthritis or arthralgia.

7. The use according to any one of claims 1 to 6, wherein: The MIC value of cephalanthrin against Mycoplasma pneumoniae was 39.0625 μg / ml.

8. The use according to any one of claims 1 to 7, wherein: IC of cephalanthrin against Mycoplasma pneumoniae 50 The value is 21 μM.

9. The use according to any one of claims 1 to 8, wherein Stephania cephalanthin, its pharmaceutically acceptable salt or prodrug directly inhibits the P1 protein of Mycoplasma pneumoniae in vitro.

10. The use according to any one of claims 1 to 9, wherein: Cephalaenopsis, a pharmaceutically acceptable salt or prodrug thereof, inhibits the inflammatory cytokine storm in lung tissue; Preferably, cepharanthin, a pharmaceutically acceptable salt or prodrug thereof reduces the expression of TNF-α, IL-6 and IL-1β.

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