Application of norhalman in the preparation of antibacterial agents

Abstract

This invention discloses the application of norharmane in the preparation of antibacterial agents. In this invention, the quorum sensing inhibitor Norharmane, when used in combination with azithromycin, exhibits a significant synergistic effect. It can inhibit the expression of bacterial-associated virulence factors without inhibiting bacterial growth, thereby weakening bacterial virulence and demonstrating good antibacterial efficacy against drug-resistant Pseudomonas aeruginosa in vivo. In mouse models of Pseudomonas aeruginosa pneumonia, it significantly improves the survival rate of infected mice and reduces the bacterial load in the lungs. Furthermore, it reduces the levels of inflammatory factors in the lungs and improves alveolar structural damage. While achieving the same antibacterial effect, the dosage can be significantly reduced, thereby minimizing drug side effects. The combination of Norharmane and azithromycin supports the development of antibacterial drugs against drug-resistant bacteria or as adjunctive antibiotic therapy.

Description

(I) Technical Field

[0001] This invention relates to the field of pharmaceutical science, specifically to the application of a quorum sensing inhibitor, Norharmane, in combination with azithromycin in the preparation of antibacterial agents. (II) Background Technology

[0002] Bacterial infections have long been a major health challenge for humanity, but this problem has been effectively addressed since the advent of antibiotics. However, with the widespread use and indiscriminate misuse of antibiotics, antibiotic resistance has gradually emerged, with many antibiotics developing resistance within years of their market launch. Simultaneously, the development of new antibiotics has slowed. According to the 2016 Global Review of Antimicrobial Resistance, if the current situation is not improved, 10 million people worldwide will die annually from drug-resistant bacterial infections by 2050. The WHO's 2020 Global Health Estimates report still indicates that the current need for anti-infective drugs remains unmet.

[0003] Therefore, there is an urgent need to find new targets to develop new antibacterial drugs that can resist bacterial resistance. (III) Summary of the Invention

[0004] The purpose of this invention is to provide an application of northalmonium in the preparation of antibacterial agents. The combination of northalmonium, a small molecule compound with quorum sensing inhibitory activity, and azithromycin significantly reduces the virulence factors of *Pseudomonas aeruginosa*, significantly improves the survival rate of mice with pneumonia, reduces the bacterial load in the lungs of mice, lowers the levels of inflammatory factors in the lungs of mice, and improves alveolar structural damage. In clinical trials, it also showed good therapeutic effects in patients with *Pseudomonas aeruginosa*-induced cystic fibrosis. When used in combination, the two compounds exhibit synergistic effects in various bacterial infection models in mice, including a *Pseudomonas aeruginosa* pneumonia model, an *Escherichia coli* thigh infection model, and a *Staphylococcus aureus* peritonitis model; and significantly reduces the dosage while achieving the same antibacterial effect.

[0005] The technical solution adopted in this invention is:

[0006] This invention provides the application of norhalman in the preparation of an antibacterial agent, wherein the antibacterial agent is a mixture of norhalman and azithromycin.

[0007] Preferably, the norhalman acts in the form of hydrochloride, the azithromycin acts in the form of azithromycin hydrochloride, and the norhalman hydrochloride and azithromycin hydrochloride are mixed in a mass ratio of 3:1-4, more preferably 3:2.7.

[0008] The structural formula of Norharmane is:

[0009]

[0010] The structural formula of the azithromycin is:

[0011]

[0012] Preferably, the antibacterial agent is a drug-resistant Pseudomonas aeruginosa pneumonia inhibitor, and the drug-resistant Pseudomonas aeruginosa is drug-resistant Pseudomonas aeruginosa (GDMCC No: 61027).

[0013] Preferably, the antibacterial agent is a drug for treating cystic fibrosis.

[0014] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0015] 1. In this invention, the quorum sensing inhibitor Norharmane, when used in combination with azithromycin, exhibits good antibacterial effects against drug-resistant Pseudomonas aeruginosa in vivo. In mouse models of Pseudomonas aeruginosa pneumonia, it significantly improves the survival rate of infected mice, reduces the bacterial load in the lungs, and decreases the levels of inflammatory factors in the lungs, while also improving alveolar structural damage.

[0016] 2. Both Norharmane and azithromycin can inhibit the expression of bacterial-associated virulence factors without inhibiting bacterial growth, thereby weakening bacterial virulence and having the potential to prevent Pseudomonas aeruginosa from developing drug resistance.

[0017] 3. When Norharmane is used in combination with azithromycin, it has a significant synergistic effect in a variety of bacterial infection models, including Pseudomonas aeruginosa pneumonia, Escherichia coli thigh infection, and Staphylococcus aureus peritonitis. While achieving the same antibacterial effect, the dosage can be significantly reduced, thereby reducing the side effects of the drug.

[0018] 4. Norharmane, in combination with azithromycin, supports the development of antibacterial drugs against antibiotic resistance or as an adjunct therapy for antibiotics. (iv) Description of the attached drawings

[0019] Figure 1 Effects of Norharmane and azithromycin on survival rate in mice with a Pseudomonas aeruginosa pneumonia model.

[0020] Figure 2 Effects of Norharmane and azithromycin on bacterial load in the lungs of mice with a Pseudomonas aeruginosa pneumonia model.

[0021] Figure 3 Scanning electron microscopy of mouse lung tissue.

[0022] Figure 4 Levels of inflammatory factors in mouse lung homogenate. (V) Detailed Implementation

[0023] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

[0024] Example 1: Antibacterial effect of combined application of norhalamic acid and azithromycin in a mouse model of Pseudomonas aeruginosa pneumonia.

[0025] 1. Materials

[0026] 1.1 Medicines and Reagents

[0027] Norharmane, Shanghai Bid Pharmaceutical Co., Ltd.;

[0028] Azithromycin, Ron Reagent Co., Ltd.;

[0029] Physiological saline, Shanghai Yuanye Biotechnology Co., Ltd.;

[0030] Concentrated hydrochloric acid, Sinopharm Chemical Reagent Co., Ltd.;

[0031] Cyclophosphamide, Shanghai Bid Pharmaceutical Co., Ltd.;

[0032] Tryptone, Shanghai Aladdin Reagent Co., Ltd.;

[0033] Yeast extract, Sinopharm Chemical Reagent Co., Ltd.;

[0034] Agar, Sinopharm Chemical Reagent Co., Ltd.;

[0035] Sodium chloride, Sinopharm Chemical Reagent Co., Ltd.;

[0036] Methanol, Shanghai Titan Technology Co., Ltd.;

[0037] Disodium hydrogen phosphate, Shanghai Maclean Biochemical Technology Co., Ltd.

[0038] Sodium hydroxide, Sinopharm Chemical Reagent Co., Ltd.

[0039] Sterile PBS buffer, BioDer Biotech Co., Ltd.

[0040] Norhalmanine hydrochloride (norhalmanine·HCl) injection: Dissolve 100 mg of norhalmanine in 7-8 mL of methanol, add 1-2 mL of excess concentrated hydrochloric acid, and stir magnetically at room temperature for 6 h. After the reaction is complete, evaporate to dryness to remove methanol, excess hydrochloric acid and water from the system to obtain 123 mg of norhalmanine hydrochloride solid. Prepare fresh with physiological saline according to the required concentration and filter through a 0.22 μm filter membrane.

[0041] Azithromycin hydrochloride injection: First, prepare 1 mol / L hydrochloric acid aqueous solution and 1 mol / L disodium hydrogen phosphate aqueous solution for later use. Weigh 100 mg of azithromycin solid, add 4 ml of physiological saline, stir to suspend, and slowly add the hydrochloric acid aqueous solution dropwise to allow for complete reaction. Then adjust the pH to 6.5-8.0 with Na2PO4. Filter through a 0.22 μm filter membrane, and add physiological saline to the filter membrane to a final volume of 10 ml. Dilute with physiological saline according to the required concentration before use.

[0042] Culture medium:

[0043] (1) LB liquid culture medium: NaCl 10g / L, peptone 10g / L, yeast extract 5g / L, solvent is water. After mixing evenly, adjust the pH to 7.2±0.2 with 1mol / L NaOH solution at 25℃. Dispense into 250mL Erlenmeyer flasks and incubate at 121℃, 1×10 5 Sterilize at 20 min under Pa for later use.

[0044] (2) LB agar medium: NaCl 10g / L, peptone 10g / L, yeast extract 5g / L, solvent is water. After mixing evenly, add agar powder with a mass concentration of 1.5%-2.0%. Adjust the pH to 7.2±0.2 with 1mol / L NaOH solution at 25℃. Continue at 121℃ for 1×10⁻⁶ ppm. 5 Sterilize at 20 min under pressure, then pour into plates for later use.

[0045] 1.2 Instruments

[0046] SW-CJ-IFB Clean Workbench, Suzhou Antai Air Technology Co., Ltd.;

[0047] LDZF-75L-I Vertical Pressure Steam Sterilizer, Shanghai Shenan Medical Instrument Factory;

[0048] LRH-250 Biochemical Incubator, Shanghai Yiheng Scientific Instruments Co., Ltd.;

[0049] ZS-AR Thermostatic Incubator Shaker, Zhejiang Huayuan Instrument Co., Ltd.;

[0050] YH-M3001 Electronic Balance, Dongyang Yingheng Intelligent Equipment Co., Ltd.;

[0051] BCE224i-ICCN analytical balance, Sartorius GmbH, Germany;

[0052] DELTA320 benchtop pH meter, Mettler Toledo Instruments (Shanghai) Co., Ltd.;

[0053] IKA C-MAG HS7 Magnetic Stirrer, AKA Instrument Equipment Co., Ltd.;

[0054] IKA RV3eco rotary evaporator, AKA Instrument Equipment Co., Ltd.;

[0055] 1.3 Experimental strains

[0056] Experimental strain: Pseudomonas aeruginosa C218, deposited at Guangdong Provincial Microbial Culture Collection Center, accession number GDMCC No: 61027, deposited on May 13, 2020, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Institute of Microbiology, postcode 510075, and published in patent application CN11235801A.

[0057] 1.4 Laboratory Animals

[0058] Healthy, nonspecific pathogen-free (SPF) ICR mice (20±2g) were obtained from Hangzhou Hangsi Biotechnology Co., Ltd. (Hangzhou, China). The mice were housed at a temperature of 20–26℃ and a relative humidity of 40%–70%, with a light-dark cycle every 12 hours. The mice had free access to food and water.

[0059] 2. Content and Methods

[0060] 2.1 Effect of Norharmane combined with azithromycin on survival rate of mice with Pseudomonas aeruginosa pneumonia model

[0061] (1) Preparation of bacterial culture

[0062] The cryopreservation solution of *Pseudomonas aeruginosa* GDMCC No. 61027, stored at -80℃, was thawed at room temperature and streaked onto LB agar. It was incubated at 37℃ for 24 hours. A single, well-developed colony was then inoculated again and incubated at 37℃ for 24 hours to ensure the strain was in the growth phase. A single colony was then selected and inoculated into LB liquid medium and incubated overnight at 37℃ with shaking at 200 rpm. The bacterial concentration was adjusted to OD500 with sterile physiological saline. 600 It is 0.5-0.8, for backup.

[0063] (2) Inducing immunosuppression

[0064] After one week of acclimatization, all mice were intraperitoneally injected with cyclophosphamide to induce immunosuppression. The dose was 150 mg / kg / day for the first four days and 100 mg / kg / day for the fifth day.

[0065] (3) Establishment of pneumonia model

[0066] The model was established the day after the immunosuppressive induction was completed. Mice were infected by intranasal inoculation: the mice were held upright, and 20 μl of bacterial solution was dripped into one nostril. After 5-10 minutes, another 20 μl was dripped into the other nostril. The general condition of the mice after model induction was observed, such as rapid breathing, hair standing on end, and trembling, to confirm that the model was successfully established.

[0067] (4) Grouping and administration

[0068] Twenty-seven female model mice were randomly divided into a model control group (n=8), a Norharmane·HCl 12mg / kg group (n=8), an azithromycin hydrochloride group (22mg / kg, n=8), and a combination drug group (Norharmane·HCl 3mg / kg + azithromycin hydrochloride 2.7mg / kg, n=3).

[0069] The treatment group received subcutaneous injections of different doses of the drug, while the model control group received an equal volume of physiological saline subcutaneously. Drug administration began 24 hours after inoculation with the bacterial culture and continued every 12 hours for one week. Changes in food intake, activity, fur, and respiration were observed during this period, and mouse mortality was recorded.

[0070] 2.2 Effects of Norharmane combined with azithromycin on alveolar structure, pulmonary bacterial load, and pulmonary inflammatory factor levels in mice with Pseudomonas aeruginosa pneumonia

[0071] (1) Preparation of bacterial culture

[0072] The cryopreservation solution of *Pseudomonas aeruginosa* GDMCC No. 61027, stored at -80℃, was thawed at room temperature and streaked onto LB agar. It was incubated at 37℃ for 24 hours. A single, well-developed colony was then inoculated again and incubated at 37℃ for 24 hours to ensure the strain was in its growth phase. A single colony was then selected and inoculated into LB liquid medium and incubated overnight at 37℃ with shaking at 200 rpm. The bacterial concentration was adjusted to 3.0 × 10⁻⁶ with sterile physiological saline. 9 CFU mL -1 ,spare.

[0073] (2) Inducing immunosuppression

[0074] After one week of acclimatization, all mice were intraperitoneally injected with cyclophosphamide to induce immunosuppression. The dose was 150 mg / kg / day for the first four days and 100 mg / kg / day for the fifth day.

[0075] (3) Establishment of pneumonia model

[0076] The model was established the day after immunosuppression induction was completed. Mice were infected by intranasal inoculation: the mice were upright, and 20 μl of bacterial solution was dripped into one nostril. After 5-10 minutes, another 20 μl was dripped into the other nostril. The general condition of the mice after model induction was observed, such as rapid breathing, hair standing on end, and trembling, to confirm that the model was successfully established.

[0077] (4) Grouping and administration

[0078] Thirty-two model mice were randomly divided into four groups of eight mice each, with half males and half females: a model control group, a Norharmane·HCl 12 mg / kg group, an azithromycin hydrochloride 22 mg / kg group, and a combination therapy group (Norharmane·HCl 3 mg / kg + azithromycin hydrochloride 2.7 mg / kg).

[0079] The treatment group received different doses of the drug via subcutaneous injection, while the model control group received an equal volume of physiological saline subcutaneously. The drugs were administered 2 hours after inoculation with the bacterial culture.

[0080] (5) Scanning electron microscopy

[0081] Mice were sacrificed 24 hours after drug administration, and lung tissue was aseptically collected. A small piece of lung tissue was cut and washed with physiological saline. The lung tissue was fixed overnight in 2.5% glutaraldehyde at 4°C, then the glutaraldehyde was discarded, and the tissue was washed 2-3 times with physiological saline to remove residual glutaraldehyde. After washing, the tissue was dehydrated sequentially at room temperature with 50%, 70%, 90%, and 100% ethanol, with each concentration dehydrating for 15 minutes. After dehydration, the ethanol was discarded, tert-butanol was added, and the tissue was placed in a -20°C freezer for pre-freezing. It was then thoroughly dried using a freeze dryer and observed and photographed using a scanning electron microscope. Additional lung tissue samples were taken to detect bacterial load and inflammatory factor levels in the lungs.

[0082] (6) Colony count

[0083] The remaining lung tissue was weighed and homogenized thoroughly with 5 ml of sterile PBS. After homogenization, the tissue was serially diluted, and 100 μl of the diluted homogenate was evenly spread on LB agar medium and incubated overnight at 37°C. Colony counts were then performed visually.

[0084] 3. Results

[0085] 3.1 Effect of Norharmane combined with azithromycin on survival rate of mice with Pseudomonas aeruginosa pneumonia model

[0086] See results Figure 1Compared with the model control group, Norharmane·HCl at a dose of 12 mg / kg improved the survival rate of mice with Pseudomonas aeruginosa pneumonia, and its therapeutic effect was superior to azithromycin hydrochloride. Furthermore, the combined administration was more effective than Norharmane·HCl or azithromycin hydrochloride alone, and the dosage was significantly lower than that of either drug alone. This indicates that Norharmane can protect mice from lethal Pseudomonas aeruginosa pneumonia and has a synergistic effect with azithromycin.

[0087] 3.2 Effects of Norharmane combined with azithromycin on alveolar structure, pulmonary bacterial load, and inflammatory factor levels in mice with Pseudomonas aeruginosa pneumonia

[0088] Effects on mouse alveolar structure are shown in Figure 2 In the saline group, the alveolar walls of mice were significantly thickened and the alveolar structure was damaged, but these phenomena were improved after Norharmane·HCl and combined drug treatment.

[0089] Effects on bacterial load in mouse lungs are shown in [reference needed]. Figure 3 Treatment with 12 mg / kg Norharmane·HCl significantly reduced the bacterial load in the lungs of mice compared to the saline group, while azithromycin showed no significant reduction effect. The combined use of both drugs had efficacy comparable to 12 mg / kg Norharmane·HCl, but at more than half the required dosage, and the combined treatment group was significantly more effective than azithromycin. This indicates that Norharmane has a good protective effect against Pseudomonas aeruginosa pneumonia in mice and exhibits a synergistic effect with azithromycin, thus reducing the required dosage.

[0090] Effects on the levels of inflammatory factors in mouse lung homogenate are shown in the figure. Figure 4 Treatment with 12 mg / kg CJW1·HCl and the combined administration group significantly reduced the levels of IL-6 and IL-8 in mouse lung homogenate, while 22 mg / kg azithromycin hydrochloride had no significant effect on reducing IL-8 levels; and the reduction effect of the combined administration group was comparable to that of CJW1·HCl or azithromycin hydrochloride alone, but the dose was much lower.

[0091] The results above indicate that Norharmane has a certain antibacterial effect in mice with Pseudomonas aeruginosa pneumonia, and when used in combination with azithromycin, it has a significant synergistic effect on a variety of bacteria, thereby greatly reducing the dosage of the drug.

[0092] While the foregoing has described specific embodiments of the present invention, the scope of protection of the present invention is not limited thereto. Based on the technical solutions of the present invention, various changes, modifications, substitutions, simplifications, and applications that can be made by those skilled in the art without creative effort should be considered equivalent substitution conditions and are all included within the scope of protection of the present invention.

Claims

1. The application of a combination of norhalamic acid and azithromycin in the preparation of an antibacterial agent, characterized in that, The antibacterial agent is drug-resistant Pseudomonas aeruginosa (… Pseudomonas aeruginosa GDMCC No: 61027 Antibacterial agent.

2. The application as described in claim 1, characterized in that, The norhalman acts in the form of hydrochloride, the azithromycin acts in the form of azithromycin hydrochloride, and the norhalman hydrochloride and azithromycin hydrochloride are mixed in a mass ratio of 3:1-4.

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