WO2022178957A1 - Pharmaceutical use of napabucasin - Google Patents
Pharmaceutical use of napabucasin Download PDFInfo
- Publication number
- WO2022178957A1 WO2022178957A1 PCT/CN2021/086431 CN2021086431W WO2022178957A1 WO 2022178957 A1 WO2022178957 A1 WO 2022178957A1 CN 2021086431 W CN2021086431 W CN 2021086431W WO 2022178957 A1 WO2022178957 A1 WO 2022178957A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- napabucasin
- helicobacter pylori
- candida
- drug
- gram
- Prior art date
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- DPHUWDIXHNQOSY-UHFFFAOYSA-N napabucasin Chemical compound O=C1C2=CC=CC=C2C(=O)C2=C1OC(C(=O)C)=C2 DPHUWDIXHNQOSY-UHFFFAOYSA-N 0.000 title claims abstract description 107
- 229950011456 napabucasin Drugs 0.000 title claims abstract description 106
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- SUBDBMMJDZJVOS-UHFFFAOYSA-N 5-methoxy-2-{[(4-methoxy-3,5-dimethylpyridin-2-yl)methyl]sulfinyl}-1H-benzimidazole Chemical compound N=1C2=CC(OC)=CC=C2NC=1S(=O)CC1=NC=C(C)C(OC)=C1C SUBDBMMJDZJVOS-UHFFFAOYSA-N 0.000 claims abstract description 22
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/10—Antimycotics
Definitions
- the invention belongs to the field of pharmacy and provides the pharmaceutical use of Napabucasin.
- Hp Helicobacter pylori
- Hp is a microaerobic gram-negative bacterium that infects about half of the world's population.
- Helicobacter pylori infection is the main cause of a variety of gastric diseases such as chronic gastritis and gastric and duodenal ulcers, and is closely related to the occurrence and development of gastric cancer.
- gastric diseases such as chronic gastritis and gastric and duodenal ulcers
- Several large-scale epidemiological intervention studies have shown that eradication of H. pylori can prevent the occurrence of gastric cancer.
- the treatment regimen recommended by the World Health Organization to eradicate H. pylori infection is triple or quadruple therapy.
- the former is a proton pump inhibitor (omeprazole, etc.) plus two antibiotics (clarithromycin, amoxicillin, levofloxacin, and metronidium). azole, etc. to choose two), the latter then add bismuth (bismuth potassium citrate, etc.).
- Candida is one of the normal strains of the human body. When the systemic or local immunity is weakened, such as the extensive use of antibiotics, glucocorticoids or immunosuppressants, candidiasis can be caused.
- Candida albicans is the main pathogen of the disease, which can cause skin, mucous membranes and even systemic infections. Studies have pointed to the fatality rate of this severe systemic infection as high as 40%. In addition to the increasing number of common Candida albicans infections, infections caused by rare or uncommon fungi have also been reported. It is imperative to develop high-efficiency and low-toxic antifungal drugs for clinical use.
- Napabucasin developed by Boston Biomedical (BBI), is one of the few STAT3 inhibitors that has entered Phase III clinical trials. Napabucasin can be used to treat cancer stem cells and has the potential to inhibit cancer cell metastasis and prevent cancer recurrence in a variety of tumor types.
- the present invention provides a pharmaceutical application of Napabucasin, which can be used for the preparation of antibacterial drugs.
- the above-mentioned Gram-negative bacteria are Helicobacter pylori, Porphyromonas gingivalis, Actinobacter actinomycetemcomitans, Fusobacterium nucleatum, Campylobacter jejuni, Neisseria gonorrhoeae, Haemophilus influenzae and catarrhal Pull bacteria.
- the above-mentioned Gram-positive bacteria are Staphylococcus aureus, Staphylococcus hemolyticus, Staphylococcus epidermidis, Bacillus subtilis, Bacillus cereus, Listeria monocytogenes, Streptococcus pneumoniae, Clostridium difficile and Propionibacterium acnes.
- the above-mentioned fungi are Candida albicans, Candida parapsilosis, Candida glabrata, Candida cruzi, Candida portuguese, Candida tropicalis, Cryptococcus neoformans, Aspergillus fumigatus, Trichophyton rubrum and Malassezia.
- the above-mentioned proton pump inhibitor is omeprazole.
- a drug for the treatment of Helicobacter pylori infection consisting of Napabucasin and omeprazole.
- Napabucasin can be used to prepare medicines for antibacterial infections.
- the Napabucasin shown in the present invention has a good killing effect on planktonic growth and biofilm-forming Helicobacter pylori, and can be used for the treatment of acute and chronic gastritis, gastric, tet It can effectively alleviate the drug resistance problem of Helicobacter pylori, and has little side effects.
- FIG. 3 The killing effect of Napabucasin on Helicobacter pylori G27 spherical strain.
- A The killing effect of each drug at the concentration of 1 ⁇ MIC on G27 globus strains;
- B the killing effect of each drug at the concentration of 2 ⁇ MIC, 4 ⁇ MIC or 8 ⁇ MIC on G27 globus strain after 12 hours of treatment.
- NPB Napabucasin
- MTZ metronidazole
- AMX amoxicillin
- CLR clarithromycin
- LVX levofloxacin.
- “*" indicates CFU below detection limit (10 CFU/ml).
- FIG. 4 Detection of Napabucasin anti-Helicobacter pylori biofilm activity.
- A Crystal violet staining was used to determine the activity of Napabucasin in inhibiting biofilm formation;
- B Crystal violet staining was used to determine the activity of Napabucasin to destroy mature biofilms;
- C SYTO9-PI double staining and fluorescence confocal detection were used to detect Napabucasin killing and forming mature biofilms Activity of Helicobacter pylori;
- D Plate colony counting method to detect the activity of Napabucasin to kill Helicobacter pylori forming mature biofilm.
- NPB Napabucasin
- MTZ Metronidazole. *, P ⁇ 0.05; **, P ⁇ 0.01; ***, P ⁇ 0.001.
- FIG. 5 The killing effect of Napabucasin on Helicobacter pylori NSH57 strain in mice.
- A Helicobacter pylori-infected mice to establish an acute gastritis animal model and drug treatment flow chart;
- B The detection results of Helicobacter pylori colonization in the gastric mucosa of mice after treatment in different treatment groups.
- the solvent control group Control, 0.5% sodium carboxymethyl cellulose+0.2% Tween 80
- OPZ+AC triple therapy group
- FIG. 6 The killing effect of Napabucasin on the multidrug-resistant strain of Helicobacter pylori BHKS159 in mice.
- A Helicobacter pylori-infected mice to establish an acute gastritis animal model and drug treatment flow chart;
- B The detection results of Helicobacter pylori colonization in the gastric mucosa of mice after treatment in different treatment groups.
- Set up control group uninfected mice), solvent control group (Control, 0.5% sodium carboxymethyl cellulose + 0.2% Tween 80) after BHKS159 infection, triple treatment group (OPZ + AC), omeprazole and Napabucasin combination treatment group (OPZ+NPB) and Napabucasin treatment group (NPB).
- FIG. 7 Inhibitory effect of Napabucasin on inflammatory factors in mice infected with drug-resistant Helicobacter pylori BHKS159.
- the serum levels of IL-1 ⁇ (A), IL-6(B) and IL-8(C) were detected by ELISA method.
- Set up control group uninfected mice
- solvent control group Control, 0.5% sodium carboxymethyl cellulose + 0.2% Tween 80
- OPZ + AC triple treatment group
- omeprazole and Napabucasin combination treatment group OPZ+NPB
- Napabucasin treatment group Napabucasin treatment group
- Figure 8 The repairing effect of Napabucasin on gastric mucosal inflammation in mice infected with drug-resistant Helicobacter pylori BHKS159.
- the gastric mucosa tissue was stained with HE and TUNEL, respectively, at a magnification of 100 times.
- Set up control group uninfected mice
- solvent control group Control, 0.5% sodium carboxymethyl cellulose + 0.2% Tween 80
- BHKS159 infection triple treatment group
- omeprazole and Napabucasin combination treatment group OPZ+NPB
- Napabucasin treatment group NPB
- the dose of NPB administered was 28 mg/kg.
- Figure 9 The killing effect of Napabucasin on Candida albicans SC5314 strain in mice. . Detection results of Candida albicans colonization in mouse kidney (A) and spleen (B) after treatment in different treatment groups.
- a solvent control group Control, 0.5% sodium carboxymethyl cellulose + 0.2% Tween 80
- FLC fluconazole treatment group
- NPB Napabucasin treatment group
- the anti-H. pylori activity of Napabucasin was determined by determining the minimum inhibitory concentration (MIC) of Napabucasin against Helicobacter pylori.
- 2Strains Helicobacter pylori standard strains 26695 and G27; other clinical strains were isolated and identified from the gastric mucosa samples of clinical patients in the First affiliated Hospital of Nanjing Medical University and the affiliated Yifu Hospital.
- BHI Brain Heart Infusion
- Columbia medium selective antibiotics (vancomycin, polymyxin B, trimethoprim), calf serum (FCS) and 100 % Dimethyl sulfoxide (DMSO).
- selective antibiotics vancomycin, polymyxin B, trimethoprim
- FCS calf serum
- DMSO Dimethyl sulfoxide
- 2Preparation of bacterial solution take the log phase growth of Helicobacter pylori on the solid plate to make a bacterial suspension with BHI (containing 10% FCS), and adjust the concentration OD 600 to 0.2 (the bacterial concentration is about 1 ⁇ 10 8 CFU/mL ), diluted 10 times, the bacterial volume was about 1 ⁇ 10 7 CFU/mL, and it was used for later use.
- 396-well plate preparation add 178 ⁇ L of BHI culture medium (containing 10% FCS) to the first well, add 2 ⁇ L of Napabucasin stock solution, and dilute to the 12th well by double dilution method; add metronidazole in the same way to the other row of wells azoles.
- 4Inoculation bacterial solution take 10 ⁇ L of the above-mentioned standby bacterial solution and add it into each well (the concentration of bacteria in each well is about 1.0 ⁇ 10 6 CFU/mL), and the concentrations of Napabucasin are 16 ⁇ g/mL, 8 ⁇ g/mL, and 4 ⁇ g/mL. , 2 ⁇ g/mL, 1 ⁇ g/mL, 0.5 ⁇ g/mL, 0.25 ⁇ g/mL, 0.125 ⁇ g/mL, 0.063 ⁇ g/mL, 0.031 ⁇ g/mL, 0.016 ⁇ g/mL, and 0.008 ⁇ g/mL.
- NPB Napabucasin
- MTZ metronidazole
- AMX amoxicillin
- CLR clarithromycin
- LVX levofloxacin
- Example 1 material Such as Example 1 material.
- Example 1 material Such as Example 1 material.
- 2Drug treatment Centrifuge the spherical strain and discard the supernatant, add Brucella broth (containing 2% FCS) to dilute 100 times to a bacterial concentration of about 1 ⁇ 10 6 CFU/mL, and then add 1 ⁇ MIC, 2 ⁇ MIC, 4 ⁇ MIC and 8 ⁇ MIC final concentrations of amoxicillin, metronidazole, clarithromycin, levofloxacin and Napabucasin were cultured in a three-gas incubator.
- Brucella broth containing 2% FCS
- Napabucasin has a strong killing effect against Helicobacter pylori in vitro, and its efficiency is stronger than that of commonly used antibiotics such as amoxicillin, metronidazole, clarithromycin and levofloxacin, and has a good development prospect.
- Example 4 The killing effect of Napabucasin on Helicobacter pylori that has formed biofilm
- Example 1 material Such as Example 1 material.
- Napabucasin The destruction effect of Napabucasin on the formed biofilm: Helicobacter pylori G27 strain was cultured overnight in Brucella broth supplemented with 10% FBS, diluted with the above fresh medium to an OD 600 of 0.15, and then spread on a 96-well plate , after 3 days in a three-gas incubator to form a biofilm, the medium was discarded, and the plate was washed twice with PBS.
- Different concentrations of Napabucasin, MTZ (as a positive control) or an equal amount of DMSO (as a solvent control) were added to fresh Brucella broth, added to a 96-well plate and cultured for 24 hours, and the biofilm was detected by crystal violet staining. relative amount.
- the non-adherent biofilms were washed 3 times with PBS and then stained with two fluorescent dyes for 30 min at room temperature in the dark. After rinsing, images were observed using a confocal laser scanning microscope (LSM710; Carl Zeiss, Germany) and additional fields were randomly examined. SYTO9 and PI distinguish between live cells (green cells) and dead cells (red cells).
- Biofilms of H. pylori G27 cells were prepared and treated as above. After 24 hours of treatment under micro-aerobic conditions, the medium was removed, washed three times with PBS, and then 250 ⁇ l of BHI medium was added to each well. The biofilm was peeled off by scraping the walls of the wells with a 200 ⁇ l pipette tip 15 times. The scraped biofilm was then homogenized in the solution by repeated pipetting. Finally, the homogeneous biofilm bacterial suspension was double-diluted, plated on Columbia blood agar plates, and then incubated at 37°C for 4 days under microaerobic conditions. The viable bacterial colonies on the plates were counted and expressed as total colonies per milliliter.
- Napabucasin can inhibit the formation of Helicobacter pylori biofilm
- Napabucasin has the ability to destroy the mature biofilm of Helicobacter pylori, and its anti-biofilm activity is stronger than that of metronidazole.
- Napabucasin can kill the biofilm-forming Helicobacter pylori, and its activity is also stronger than that of metronidazole.
- the strain is Helicobacter pylori mouse domesticated strain NSH57, the mice are SPF grade 6-week-old female C57BL/6 mice, and other materials are as in Example 1.
- 2Grouping Divide the infected group with successful modeling into 3 groups on average, namely the omeprazole plus Napabucasin (administration dose of 14mg/kg) group, and the omeprazole plus amoxicillin (administration dose of 14mg/kg) group. and clarithromycin (administration dose of 7mg/kg) group (standard triple combination group), solvent control group, 7 in each group; omeprazole dose in all groups was 138.2mg/kg, no infection of Helicobacter pylorus 7 mice were negative control group.
- mice were fasted for 4 hours after administration; the average weight of mice was 20g/mice, and the administration was once a day , for 3 consecutive days; the solvent control group was given 0.5% sodium carboxymethyl cellulose + 0.2% Tween 80 solution, the capacity and frequency were the same as above.
- mice treatment 48 hours after the last administration, euthanasia was performed, and gastric tissue was collected for the isolation, culture and identification of Helicobacter pylori, and the colonization amount was calculated.
- the strain is Helicobacter pylori mouse domesticated strain BHKS159, the mice are SPF grade 6-week-old female C57BL/6 mice, and other materials are as in Example 1.
- omeprazole was administered 30 minutes before other drugs, fasting for 4 hours after administration; once a day for 3 consecutive days; solvent control group was given 0.5 % Sodium carboxymethyl cellulose + 0.2% Tween 80 solution, the capacity and frequency are the same as above.
- mice 48 hours after the last administration, euthanize, collect blood from the orbit and centrifuge to extract serum, and detect the content of various inflammatory factors by ELISA method; take gastric tissue, and half of the tissue is separated, cultured and identified for Helicobacter pylori, and colonization is calculated. The other half of the tissue was stained for pathological sections. At the same time, mice were weighed daily and the average body weight was calculated.
- the expression levels of inflammatory factors in the peripheral blood of mice in the omeprazole plus Napabucasin combination group (28mg/kg) or Napabucasin single-agent group (28mg/kg) were significantly lower than those in the triple therapy group, as shown in Figure 7, and these two groups had no significant effect.
- the repair ability of rat gastric mucosal injury was also significantly better than that of the triple therapy group, as shown in Figure 8.
- Example 7 In vitro activity assay of Napabucasin against non-Helicobacter pylori bacteria
- the antibacterial activity of Napabucasin was determined by determining the minimum inhibitory concentration (MIC) of Napabucasin against non-Helicobacter pylori bacteria.
- Napabucasin, metronidazole and vancomycin were purchased from MCE Company.
- 2Strains Staphylococcus aureus ATCC 25923 and USA300, Staphylococcus hemolytica ATCC29970, Staphylococcus epidermidis ATCC12228, Bacillus subtilis 168, Bacillus cereus ATCC14579, Listeria monocytogenes EGD-e, Streptococcus pneumoniae ATCC49619, Enterococcus faecalis ATCC29212, Enterococcus faecalis ATCC19434, Clostridium difficile ATCC700057, Propionibacterium acnes ATCC11827, Porphyromonas gingivalis ATCC BAA-308, Actinobacter actinomycetemcomitans D7S-1, Fusobacterium nucleatum ATCC25586, Curvature jejunum Bacillus NCTC11168, Neisseria gonorrhoeae ATCC19424, Haemophilus influenzae ATCC49766, Moraxella catarrhalis ATCC2523
- MH medium 3Medium and main reagents: MH medium, MH medium, brain-heart infusion medium, Columbia medium and FCS.
- the minimum inhibitory concentration of Napabucasin against non-Helicobacter pylori bacteria was detected by broth dilution method.
- the medium and medium used for the detection were MH medium and medium, and the bacterial concentration was about 1.0 ⁇ 10 5 CFU/mL.
- the time for interpretation of the results was 24 or 48 hours according to the difference of the strains. Others were similar to the methods in Example 1. .
- Napabucasin is effective against Gram-positive bacteria such as Staphylococcus aureus, Staphylococcus hemolyticus, Staphylococcus epidermidis, Bacillus subtilis, Bacillus cereus, Listeria monocytogenes, Streptococcus pneumoniae, Clostridium difficile and Propionibacterium acnes and Gram-negative bacteria such as Porphyromonas gingivalis, Actinobacter actinomyces, Fusobacterium nucleatum, Campylobacter jejuni, Neisseria gonorrhoeae, Haemophilus influenzae, and catarrhalis Moraxella, etc., all have the effect of inhibiting growth.
- Gram-positive bacteria such as Staphylococcus aureus, Staphylococcus hemolyticus, Staphylococcus epidermidis, Bacillus subtilis, Bacillus cereus, Listeria monocytogenes, Streptococcus pneumoniae, Clostridium difficile and Propion
- Napabucasin and Amphotericin (AMB) were purchased from MCE Company.
- 2Strains Aspergillus fumigatus Af293, Trichophyton rubrum ATCC MYA-4438, Candida albicans SC5314 and ATCC14053, Candida parapsilosis ATCC22019, Candida glabrata ATCC36583, Candida cruzi ATCC6258, Candida portuguese ATCC200950, Candida tropicalis ATCC750 , Cryptococcus neoformans ATCC MYA-4906 and ATCC 90112, Malassezia sphaeroides CBS7222, Malassezia globosa CBS7966, Malassezia furfur CBS1878 and Malassezia pachyderma CBS1879 are standard or model strains. Other clinical strains were isolated and identified from clinical samples at the First affiliated Hospital of Nanjing Medical University and the affiliated Yifu Hospital.
- RPMI1640 medium Shabao weak medium
- FCS dimethyl sulfoxide
- 2Preparation of bacterial solution Take Candida albicans growing in logarithmic phase on the solid plate to make bacterial suspension with RPMI1640 culture solution, adjust the concentration OD 530 to 0.5 (about 1 ⁇ 10 6 CFU/mL), and dilute it 1000 times, about 1 ⁇ 10 3 CFU/mL, for use.
- Aspergillus fumigatus, Trichophyton and Malassezia using a specific medium added with olive oil
- Cryptococcus neoformans adjusts the bacterial solution OD 530 to 0.5, diluted 10,000 times for use.
- 396-well plate preparation firstly add 178 ⁇ L of RPMI1640 culture medium to the first well, then add 2 ⁇ L of Napabucasin stock solution, and dilute to the 12th well by double dilution method; add amphotericin B and fluconin to the other two wells in the same way. azoles.
- 4Inoculation bacterial liquid take 10 ⁇ L of the above-mentioned standby bacterial liquid and add it to each hole (the concentration of bacteria in each hole is about 1.0 ⁇ 10 2 CFU/mL), and the drug concentrations are 64 ⁇ g/mL, 32 ⁇ g/mL, 16 ⁇ g/mL, 8 ⁇ g/mL, 4 ⁇ g/mL, 2 ⁇ g/mL, 1 ⁇ g/mL, 0.5 ⁇ g/mL, 0.25 ⁇ g/mL, 0.125 ⁇ g/mL, 0.063 ⁇ g/mL and 0.031 ⁇ g/mL.
- Napabucasin is effective against Candida albicans, Candida parapsilosis, Candida glabrata, Candida cruzi, Candida portugalii, Candida tropicalis, Cryptococcus neoformans, Aspergillus fumigatus, Trichophyton rubrum and Malassezia Bacteria and other fungi, all have the effect of inhibiting growth, and its inhibitory activity is comparable to that of amphotericin B.
- Example 9 Killing effect of Napabucasin on Candida albicans SC5314 strain in a mouse systemic infection model
- the strain is Candida albicans SC5314, the mice are SPF grade 8-week-old C57BL/6 female mice, the drug fluconazole is purchased from MCE Company, and other materials are as in Example 8.
- mice The infected mice that were successfully modeled were divided into 3 groups on average, namely the solvent control group (0.5% sodium carboxymethyl cellulose + 0.2% Tween 80), the fluconazole treatment group and the Napabucasin treatment group. group of 10 mice.
- mice The experimental group was given intragastric administration. The first administration was 2 hours after the infection on the day of infection, and the second administration was administered at an interval of 10 hours. Administer 2 times a day for 3 consecutive days. The weight of mice was calculated as an average of 20 g/mice. The doses of fluconazole and Napabucasin were both 56 mg/kg/day, and the control group was given a solvent with the same volume and frequency.
- mice 24 hours after the last administration, euthanize, take spleen and kidney tissues, isolate, culture and identify Candida albicans, and calculate the colonization amount.
Abstract
A pharmaceutical use of Napabucasin, relating to an application thereof in the preparation of an antibacterial drug. The bacteria are gram-negative bacteria, gram-positive bacteria or fungi. Napabucasin has a good antibacterial activity against standard sensitive helicobacter pylori, clinical sensitive and drug-resistant helicobacter pylori, etc., has a minimum inhibitory concentration of 0.016-0.125 μg/mL, has a good killing effect on spherical helicobacter pylori and helicobacter pylori which forms a mature biological membrane, and has a stronger effect than metronidazole. Napabucasin is not prone to be drug-resistant in the process of resisting helicobacter pylori, and has a strong killing effect on helicobacter pylori implanted in the stomach of the mouse, and the use of Napabucasin alone or in combination with omeprazole has a strong sterilization capability than the standard triple therapy method. In addition, Napabucasin shows the effect of killing Candida albicans on the same efficiency of fluconazole in a systemic infection model of the Candida albicans infected mouse.
Description
本发明属于制药领域，提供Napabucasin的制药用途。The invention belongs to the field of pharmacy and provides the pharmaceutical use of Napabucasin.
感染性疾病是威胁人类生命和健康的重大疾病，滥用和过度使用抗生素造成的微生物耐药问题已成为人类共同面临的公共安全危机。全球每年约70万人死于耐药菌感染，多于癌症死亡人数，因此迫切需要探索和研发新型的抗感染药物。2017年2月，WHO首次发布世界上最具耐药性、最能威胁人类健康的“超级细菌”“12强”清单，用于指导和促进新型抗生素的优先研发。Infectious diseases are major diseases that threaten human life and health. The problem of microbial resistance caused by the abuse and overuse of antibiotics has become a public security crisis faced by mankind. About 700,000 people die from drug-resistant bacterial infections every year, more than cancer deaths. Therefore, there is an urgent need to explore and develop new anti-infective drugs. In February 2017, WHO released the world's most drug-resistant "superbugs" and "Top 12" list for the first time to guide and promote the priority research and development of new antibiotics.
幽门螺杆菌(Helicobacter pylori,Hp)是一种微氧革兰氏阴性菌，感染全球约一半的人口。Hp感染是多种胃部疾病如慢性胃炎和胃、十二指肠溃疡发生的主要原因，同时与胃癌的发生发展密切相关。多个大规模流行病学干预研究表明，根除Hp可以预防胃癌的发生。目前，世界卫生组织推荐的根除Hp感染的治疗方案是三联或四联疗法，前者即质子泵抑制剂(奥美拉唑等)加两种抗生素(克拉霉素、阿莫西林、左氧氟沙星和甲硝唑等选二种)，后者再添加铋剂(枸橼酸铋钾等)。但近年来随着抗生素在Hp感染治疗中的广泛应用，Hp的耐药问题日益严重，迫切需要对耐药幽门螺杆菌有效的新药。Helicobacter pylori (Hp) is a microaerobic gram-negative bacterium that infects about half of the world's population. Helicobacter pylori infection is the main cause of a variety of gastric diseases such as chronic gastritis and gastric and duodenal ulcers, and is closely related to the occurrence and development of gastric cancer. Several large-scale epidemiological intervention studies have shown that eradication of H. pylori can prevent the occurrence of gastric cancer. At present, the treatment regimen recommended by the World Health Organization to eradicate H. pylori infection is triple or quadruple therapy. The former is a proton pump inhibitor (omeprazole, etc.) plus two antibiotics (clarithromycin, amoxicillin, levofloxacin, and metronidium). azole, etc. to choose two), the latter then add bismuth (bismuth potassium citrate, etc.). However, with the widespread application of antibiotics in the treatment of H. pylori infection in recent years, the problem of H. pylori drug resistance has become increasingly serious, and new drugs that are effective against drug-resistant Helicobacter pylori are urgently needed.
念珠菌属是人体正常菌株之一，当全身或局部免疫力下降，如大量使用抗生素、糖皮质激素或免疫抑制剂等之后可引发念珠菌病。白色念珠菌是此病的主要病原菌，可引起皮肤、粘膜甚至全身感染。有研究指出这种严重的全身感染的致死率高达40％。除了常见的白色念珠菌感染的数量不断增多，一些少见或罕见真菌引起的感染也时有报道。研制高效低毒的抗真菌药提供给临床是当务之急。Candida is one of the normal strains of the human body. When the systemic or local immunity is weakened, such as the extensive use of antibiotics, glucocorticoids or immunosuppressants, candidiasis can be caused. Candida albicans is the main pathogen of the disease, which can cause skin, mucous membranes and even systemic infections. Studies have pointed to the fatality rate of this severe systemic infection as high as 40%. In addition to the increasing number of common Candida albicans infections, infections caused by rare or uncommon fungi have also been reported. It is imperative to develop high-efficiency and low-toxic antifungal drugs for clinical use.
Napabucasin由美国波士顿生物制药技术公司(Boston Biomedical,BBI)开发，是为数不多的进入III期临床的STAT3抑制剂。Napabucasin可用于治疗癌症干细胞，具有在多种肿瘤类型抑制癌细胞转移和防止癌症复发的潜力。Napabucasin, developed by Boston Biomedical (BBI), is one of the few STAT3 inhibitors that has entered Phase III clinical trials. Napabucasin can be used to treat cancer stem cells and has the potential to inhibit cancer cell metastasis and prevent cancer recurrence in a variety of tumor types.
发明内容SUMMARY OF THE INVENTION
解决的技术问题：本发明提供一种Napabucasin的制药用途，可用于抗菌药物的制备。Technical problem to be solved: The present invention provides a pharmaceutical application of Napabucasin, which can be used for the preparation of antibacterial drugs.
技术方案：Napabucasin在制备抗菌药物中的应用，上述菌为革兰氏阴性菌、革兰氏阳性菌或真菌。Technical scheme: the application of Napabucasin in the preparation of antibacterial drugs, the above bacteria are Gram-negative bacteria, Gram-positive bacteria or fungi.
优选的，上述革兰氏阴性菌为幽门螺杆菌、牙龈卟啉单胞菌、伴放线放线杆菌、具核梭杆菌、空肠弯曲杆菌、淋病奈瑟菌、流感嗜血杆菌和卡他莫拉菌。Preferably, the above-mentioned Gram-negative bacteria are Helicobacter pylori, Porphyromonas gingivalis, Actinobacter actinomycetemcomitans, Fusobacterium nucleatum, Campylobacter jejuni, Neisseria gonorrhoeae, Haemophilus influenzae and catarrhal Pull bacteria.
优选的，上述革兰氏阳性菌为金黄色葡萄球菌、溶血性葡萄球菌、表皮葡萄球菌、枯草芽孢杆菌、蜡样芽孢杆菌、单核细胞增生李斯特氏菌、肺炎链球菌、艰难梭菌和痤疮丙酸杆菌。Preferably, the above-mentioned Gram-positive bacteria are Staphylococcus aureus, Staphylococcus hemolyticus, Staphylococcus epidermidis, Bacillus subtilis, Bacillus cereus, Listeria monocytogenes, Streptococcus pneumoniae, Clostridium difficile and Propionibacterium acnes.
优选的，上述真菌为白色念珠菌、近平滑念珠菌、光滑念珠菌、克鲁斯念珠菌、葡萄牙念珠菌、热带念珠菌、新生隐球菌、烟曲霉、红色毛癣菌和马拉色菌。Preferably, the above-mentioned fungi are Candida albicans, Candida parapsilosis, Candida glabrata, Candida cruzi, Candida portuguese, Candida tropicalis, Cryptococcus neoformans, Aspergillus fumigatus, Trichophyton rubrum and Malassezia.
Napabucasin与质子泵抑制剂的组合物在制备抗耐药性或敏感性幽门螺杆菌药物中的应用。Application of the combination of Napabucasin and proton pump inhibitor in the preparation of anti-drug-resistant or sensitive Helicobacter pylori medicine.
上述质子泵抑制剂为奥美拉唑。The above-mentioned proton pump inhibitor is omeprazole.
Napabucasin在制备治疗耐药性或敏感性幽门螺杆菌感染导致的急、慢性胃炎、胃、十二指肠溃疡药物中的应用。Application of Napabucasin in the preparation of medicines for treating acute and chronic gastritis, gastric and duodenal ulcers caused by drug-resistant or sensitive Helicobacter pylori infection.
治疗幽门螺杆菌感染的药物，组分为Napabucasin和奥美拉唑。A drug for the treatment of Helicobacter pylori infection, consisting of Napabucasin and omeprazole.
有益效果：Napabucasin可用于制备抗菌感染的药物。本发明展示的Napabucasin对浮游生长的和形成生物膜的幽门螺杆菌都有很好的杀灭作用，可用于治疗耐药性或敏感性幽门螺杆菌感染导致的急、慢性胃炎、胃、十二指肠溃疡等胃部疾病，而且毒副作用小，能有效缓解幽门螺杆菌耐药性问题。Beneficial effects: Napabucasin can be used to prepare medicines for antibacterial infections. The Napabucasin shown in the present invention has a good killing effect on planktonic growth and biofilm-forming Helicobacter pylori, and can be used for the treatment of acute and chronic gastritis, gastric, tet It can effectively alleviate the drug resistance problem of Helicobacter pylori, and has little side effects.
图1.体外诱导检测幽门螺杆菌G27菌株对Napabucasin的耐药性。Figure 1. In vitro induction and detection of Napabucasin resistance of Helicobacter pylori G27 strains.
图2.利用阿莫西林诱导，显微镜下检测幽门螺杆菌G27菌株的球形变。A,阴性对照；B,G27球形菌株。Figure 2. Spheroidization of Helicobacter pylori G27 strain was detected under a microscope using amoxicillin induction. A, negative control; B, G27 spherical strain.
图3.Napabucasin对幽门螺杆菌G27球形菌株的杀灭作用。A,1×MIC浓度的各药物分别对G27球形菌株的杀灭作用；B,2×MIC、4×MIC或8×MIC浓度的各药物在12小时处理后对G27球形菌株的杀灭作用。注：NPB,Napabucasin；MTZ,甲硝唑；AMX,阿莫西林；CLR，克拉霉素；LVX，左氧氟沙星。“*”表示CFU在检测极限以下(10CFU/ml)。Figure 3. The killing effect of Napabucasin on Helicobacter pylori G27 spherical strain. A, The killing effect of each drug at the concentration of 1×MIC on G27 globus strains; B, the killing effect of each drug at the concentration of 2×MIC, 4×MIC or 8×MIC on G27 globus strain after 12 hours of treatment. Note: NPB, Napabucasin; MTZ, metronidazole; AMX, amoxicillin; CLR, clarithromycin; LVX, levofloxacin. "*" indicates CFU below detection limit (10 CFU/ml).
图4.Napabucasin抗幽门螺杆菌生物膜活性检测。A,结晶紫染色法测定Napabucasin抑制生物膜形成的活性；B,结晶紫染色法测定Napabucasin破坏成熟生物膜的活性；C,SYTO9-PI双染色和荧光共聚焦检测Napabucasin杀灭形成成熟生物膜的幽门螺杆菌的活性；D，平板菌落计数法检测Napabucasin杀灭形成成熟生物膜的幽门螺杆菌的活性。注：NPB,Napabucasin；MTZ,甲硝唑。*，P<0.05；**，P<0.01；***，P<0.001。Figure 4. Detection of Napabucasin anti-Helicobacter pylori biofilm activity. A, Crystal violet staining was used to determine the activity of Napabucasin in inhibiting biofilm formation; B, Crystal violet staining was used to determine the activity of Napabucasin to destroy mature biofilms; C, SYTO9-PI double staining and fluorescence confocal detection were used to detect Napabucasin killing and forming mature biofilms Activity of Helicobacter pylori; D, Plate colony counting method to detect the activity of Napabucasin to kill Helicobacter pylori forming mature biofilm. Note: NPB, Napabucasin; MTZ, Metronidazole. *, P<0.05; **, P<0.01; ***, P<0.001.
图5.Napabucasin在小鼠体内对幽门螺杆菌NSH57菌株的杀灭作用。A,幽门螺杆菌感染小鼠构建急性胃炎动物模型和药物治疗流程图；B.不同治疗组治疗后幽门螺杆菌在小鼠胃粘膜定植量的检测结果。设溶剂对照组(Control，0.5％羧甲基纤维素钠+0.2％吐温80)、三联治疗组(OPZ+AC)、奥美拉唑和Napabucasin联用治疗组(OPZ+NPB)。*，P<0.05；**，P<0.01。Figure 5. The killing effect of Napabucasin on Helicobacter pylori NSH57 strain in mice. A, Helicobacter pylori-infected mice to establish an acute gastritis animal model and drug treatment flow chart; B. The detection results of Helicobacter pylori colonization in the gastric mucosa of mice after treatment in different treatment groups. The solvent control group (Control, 0.5% sodium carboxymethyl cellulose+0.2% Tween 80), the triple therapy group (OPZ+AC), and the omeprazole and Napabucasin combined therapy group (OPZ+NPB) were set. *, P<0.05; **, P<0.01.
图6.Napabucasin在小鼠体内对幽门螺杆菌多重耐药菌株BHKS159的杀灭作用。A,幽门螺杆菌感染小鼠构建急性胃炎动物模型和药物治疗流程图；B.不同治疗组治疗后幽门螺杆菌在小鼠胃粘膜定植量检测结果。设对照组(未感染小鼠)、BHKS159感染后的溶剂对照组(Control,0.5％羧甲基纤维素钠+0.2％吐温80)、三联治疗组(OPZ+AC)、奥美拉唑和Napabucasin联用治疗组(OPZ+NPB)和Napabucasin治疗组(NPB)。OPZ+NPB和NPB治疗组分别有两组，NPB的给药剂量分别为28mg/kg和7mg/kg。*，P<0.05；**，P<0.01；***，P<0.001。Figure 6. The killing effect of Napabucasin on the multidrug-resistant strain of Helicobacter pylori BHKS159 in mice. A, Helicobacter pylori-infected mice to establish an acute gastritis animal model and drug treatment flow chart; B. The detection results of Helicobacter pylori colonization in the gastric mucosa of mice after treatment in different treatment groups. Set up control group (uninfected mice), solvent control group (Control, 0.5% sodium carboxymethyl cellulose + 0.2% Tween 80) after BHKS159 infection, triple treatment group (OPZ + AC), omeprazole and Napabucasin combination treatment group (OPZ+NPB) and Napabucasin treatment group (NPB). There are two groups in the OPZ+NPB and NPB treatment groups, respectively, and the doses of NPB are 28 mg/kg and 7 mg/kg, respectively. *, P<0.05; **, P<0.01; ***, P<0.001.
图7.Napabucasin对耐药幽门螺杆菌BHKS159感染的小鼠炎症因子的抑制作用。用ELISA方法分别检测了各处理组小鼠血清中IL-1β(A)、IL-6(B)和IL-8(C)的含量。设对照组(未感染小鼠)、BHKS159感染后的溶剂对照组(Control,0.5％羧甲基纤维素钠+0.2％吐温80)、三联治疗组(OPZ+AC)、奥美拉唑和Napabucasin联用治疗组(OPZ+NPB)和Napabucasin治疗组(NPB)。NPB的给药剂量为28mg/kg。Figure 7. Inhibitory effect of Napabucasin on inflammatory factors in mice infected with drug-resistant Helicobacter pylori BHKS159. The serum levels of IL-1β(A), IL-6(B) and IL-8(C) were detected by ELISA method. Set up control group (uninfected mice), solvent control group (Control, 0.5% sodium carboxymethyl cellulose + 0.2% Tween 80) after BHKS159 infection, triple treatment group (OPZ + AC), omeprazole and Napabucasin combination treatment group (OPZ+NPB) and Napabucasin treatment group (NPB). The dose of NPB administered was 28 mg/kg.
图8.Napabucasin对耐药幽门螺杆菌BHKS159感染的小鼠胃粘膜炎症的修复作用。胃粘膜组织分别进行HE染色和TUNEL染色，放大100倍。设对照组(未感染小鼠)、BHKS159感染后的溶剂对照组(Control,0.5％羧甲基纤维素钠+0.2％吐温80)、三联治疗组(OPZ+AC)、奥美拉唑和Napabucasin联用治疗组(OPZ+NPB)和Napabucasin治疗组(NPB)。NPB的给药剂量为28mg/kg。Figure 8. The repairing effect of Napabucasin on gastric mucosal inflammation in mice infected with drug-resistant Helicobacter pylori BHKS159. The gastric mucosa tissue was stained with HE and TUNEL, respectively, at a magnification of 100 times. Set up control group (uninfected mice), solvent control group (Control, 0.5% sodium carboxymethyl cellulose + 0.2% Tween 80) after BHKS159 infection, triple treatment group (OPZ + AC), omeprazole and Napabucasin combination treatment group (OPZ+NPB) and Napabucasin treatment group (NPB). The dose of NPB administered was 28 mg/kg.
图9.Napabucasin在小鼠体内对白色念珠菌SC5314菌株的杀灭作用。.不同治疗组治疗后 白色念珠菌在小鼠肾脏(A)和脾脏(B)的定植量检测结果。设溶剂对照组(Control,0.5％羧甲基纤维素钠+0.2％吐温80)、氟康唑治疗组(FLC)和Napabucasin治疗组(NPB)。**，P<0.01。Figure 9. The killing effect of Napabucasin on Candida albicans SC5314 strain in mice. . Detection results of Candida albicans colonization in mouse kidney (A) and spleen (B) after treatment in different treatment groups. A solvent control group (Control, 0.5% sodium carboxymethyl cellulose + 0.2% Tween 80), a fluconazole treatment group (FLC) and a Napabucasin treatment group (NPB) were set. **, P<0.01.
下面的实施例可使本专业技术人员可全面的理解本发明，但不以任何方式限制本发明。The following examples enable those skilled in the art to fully understand the present invention, but do not limit the present invention in any way.
一、Napabucasin的抗幽门螺杆菌作用1. Anti-Helicobacter pylori effect of Napabucasin
实施例1 Napabucasin体外抗幽门螺杆菌的活性测定Example 1 Determination of in vitro anti-Helicobacter pylori activity of Napabucasin
通过测定Napabucasin对幽门螺杆菌的最低抑菌浓度(MIC)来确定其抗幽门螺杆菌的活性。The anti-H. pylori activity of Napabucasin was determined by determining the minimum inhibitory concentration (MIC) of Napabucasin against Helicobacter pylori.
(1)材料(1)Material
①化学品：Napabucasin、甲硝唑、阿莫西林、克拉霉素和左氧氟沙星等购于MCE公司。①Chemicals: Napabucasin, metronidazole, amoxicillin, clarithromycin and levofloxacin were purchased from MCE Company.
②菌株：幽门螺杆菌标准菌株26695和G27；其他临床菌株为南京医科大学附属第一医院和附属逸夫医院从临床患者胃粘膜样本中分离鉴定所得。②Strains: Helicobacter pylori standard strains 26695 and G27; other clinical strains were isolated and identified from the gastric mucosa samples of clinical patients in the First Affiliated Hospital of Nanjing Medical University and the Affiliated Yifu Hospital.
③培养基和主要试剂：脑心浸液培养液(BHI)、哥伦比亚培养基、选择性抗生素(万古霉素、多粘菌素B、甲氧苄氨嘧啶)、小牛血清(FCS)和100％二甲基亚砜(DMSO)。③Medium and main reagents: Brain Heart Infusion (BHI), Columbia medium, selective antibiotics (vancomycin, polymyxin B, trimethoprim), calf serum (FCS) and 100 % Dimethyl sulfoxide (DMSO).
④主要仪器：BINDER CB160三气培养箱、紫外分光光度计、恒温摇床(Thermo)、离心机、电子天平等。④Main instruments: BINDER CB160 three-gas incubator, UV spectrophotometer, constant temperature shaker (Thermo), centrifuge, electronic balance, etc.
⑤耗材：EP管、离心管、Tip头等。⑤Consumables: EP tube, centrifuge tube, Tip, etc.
(2)方法：采用肉汤稀释法检测Napabucasin对幽门螺杆菌的最低抑菌浓度(MIC，100μL体系)(2) Method: The minimum inhibitory concentration (MIC, 100 μL system) of Napabucasin against Helicobacter pylori was detected by broth dilution method.
①分别配备1.6mg/mL的Napabucasin和6.4mg/mL的甲硝唑(MTZ)储存溶液，溶剂均为100％DMSO。① Prepare 1.6 mg/mL Napabucasin and 6.4 mg/mL metronidazole (MTZ) stock solutions, respectively, and the solvents are both 100% DMSO.
②菌液制备：取固体平板上对数期生长的幽门螺杆菌用BHI(含10％FCS)制作成菌悬液，调整浓度OD
600为0.2(菌量浓度约为1×10
8CFU/mL)，稀释10倍，菌量约为1×10
7CFU/mL，备用。
②Preparation of bacterial solution: take the log phase growth of Helicobacter pylori on the solid plate to make a bacterial suspension with BHI (containing 10% FCS), and adjust the concentration OD 600 to 0.2 (the bacterial concentration is about 1×10 8 CFU/mL ), diluted 10 times, the bacterial volume was about 1×10 7 CFU/mL, and it was used for later use.
③96孔板制备：第一孔先加BHI培养液(含10％FCS)178μL，再加2μL的Napabucasin储存溶液，采用二倍稀释法稀释至第12孔；另外一排孔以同样方法加甲硝唑。③96-well plate preparation: add 178 μL of BHI culture medium (containing 10% FCS) to the first well, add 2 μL of Napabucasin stock solution, and dilute to the 12th well by double dilution method; add metronidazole in the same way to the other row of wells azoles.
④接种菌液：取10μL上述备用菌液加入上述每孔中(每孔菌量浓度约为1.0×10
6CFU/mL)内，Napabucasin的浓度依次为16μg/mL、8μg/mL、4μg/mL、2μg/mL、1μg/mL、0.5μg/mL、0.25μg/mL、0.125μg/mL、0.063μg/mL、0.031μg/mL、0.016μg/mL和0.008μg/mL。另外一排 每孔分别加100μL BHI培养液(含10％FCS)，作为无菌对照组；再一排每孔分别加90μL BHI培养液(含10％FCS)和10μL上述备用菌液，作为阳性对照组。放置三气培养箱培养。
④Inoculation bacterial solution: take 10 μL of the above-mentioned standby bacterial solution and add it into each well (the concentration of bacteria in each well is about 1.0×10 6 CFU/mL), and the concentrations of Napabucasin are 16 μg/mL, 8 μg/mL, and 4 μg/mL. , 2 μg/mL, 1 μg/mL, 0.5 μg/mL, 0.25 μg/mL, 0.125 μg/mL, 0.063 μg/mL, 0.031 μg/mL, 0.016 μg/mL, and 0.008 μg/mL. In the other row, add 100 μL of BHI culture solution (containing 10% FCS) to each well, as a sterile control group; in the other row, add 90 μL of BHI culture solution (containing 10% FCS) and 10 μL of the above-mentioned standby bacterial solution to each well, as a positive control group. Place the three-gas incubator for culture.
⑤结果判断：培养48或72h后判读结果，以在小孔内完全抑制细菌生长的最低药物浓度为MIC。当阳性对照孔(即不含药物)内细菌明显生长以及无菌对照组无菌生长时，试验才有意义。重复3次试验。⑤Result judgment: Interpret the results after 48 or 72 hours of culture, and take the lowest drug concentration that completely inhibits bacterial growth in the small well as the MIC. The test is only meaningful when there is significant bacterial growth in the positive control wells (ie, no drug) and when the sterile control group is sterile. The experiment was repeated 3 times.
(3)结果(3) Results
结果见表1。The results are shown in Table 1.
表1 Napabucasin对幽门螺杆菌的最低抑菌浓度(μg/mL)Table 1 The minimum inhibitory concentration of Napabucasin against Helicobacter pylori (μg/mL)
注：NPB,Napabucasin；MTZ,甲硝唑；AMX,阿莫西林；CLR，克拉霉素；LVX，左氧氟沙星。Note: NPB, Napabucasin; MTZ, metronidazole; AMX, amoxicillin; CLR, clarithromycin; LVX, levofloxacin.
从上表可知，Napabucasin对15株幽门螺杆菌(包括2株标准菌株和13株临床耐药菌株)的MIC范围为0.016～0.125μg/mL，表明Napabucasin体外对幽门螺杆菌具有很强的抗菌活性，具有良好的开发前景。It can be seen from the above table that the MIC of Napabucasin against 15 strains of Helicobacter pylori (including 2 standard strains and 13 clinical drug-resistant strains) ranged from 0.016 to 0.125 μg/mL, indicating that Napabucasin has strong antibacterial activity against Helicobacter pylori in vitro , with good development prospects.
实施例2 Napabucasin诱导幽门螺杆菌G27菌株产生耐药性的测定Example 2 Determination of drug resistance of Helicobacter pylori G27 strain induced by Napabucasin
(1)材料(1)Material
如实施例1材料。Such as Example 1 material.
(2)方法(2) Method
将幽门螺杆菌G27菌株从新鲜固体平板接种到5mL BHI(含10％FCS)中，调整菌液浓度至OD
600＝0.2左右，再添加Napabucasin至终浓度0.016μg/mL(1/2×MIC)，放置三气培养箱培养48小时，观察细菌生长情况，并取菌液传代，继续上述方法诱导耐药，如生长良好， 则倍增Napabucasin浓度，如生长缓慢或不生长，则维持现有Napabucasin浓度。共进行15代耐药诱导，每代检测MIC。对照组为甲硝唑，方法相同。
Inoculate the Helicobacter pylori G27 strain from a fresh solid plate into 5 mL of BHI (containing 10% FCS), adjust the concentration of the bacterial solution to OD 600 = about 0.2, and then add Napabucasin to a final concentration of 0.016 μg/mL (1/2×MIC) , place the three-gas incubator for 48 hours, observe the bacterial growth, and take the bacterial liquid for passage, continue the above method to induce drug resistance, if the growth is good, then double the Napabucasin concentration, if the growth is slow or no growth, then maintain the existing Napabucasin concentration . A total of 15 generations of drug resistance were induced, and MIC was detected in each generation. The control group was metronidazole, and the method was the same.
(3)结果(3) Results
结果见图1。由图1所示，在连续传代期间，未观察到幽门螺杆菌对Napabucasin耐药性的形成，而幽门螺杆菌在前5次传代后发展出对甲硝唑的抗性，导致MIC增加了4倍。这些结果证明Napabucasin在幽门螺杆菌中具有极低的诱导耐药性的趋势。The results are shown in Figure 1. As shown in Figure 1, the development of H. pylori resistance to Napabucasin was not observed during serial passages, while H. pylori developed resistance to metronidazole after the first 5 passages, resulting in an increase of 4 in the MIC. times. These results demonstrate that Napabucasin has a very low tendency to induce resistance in H. pylori.
实施例3 Napabucasin体外对球形幽门螺杆菌的抗菌作用Example 3 Antibacterial effect of Napabucasin on Helicobacter pylori in vitro
(1)材料(1)Material
如实施例1材料。Such as Example 1 material.
(2)方法：采用平板菌落计数法检测Napabucasin对球形幽门螺杆菌的抗菌活性。(2) Methods: The antibacterial activity of Napabucasin against Helicobacter pylori was detected by plate colony counting method.
①幽门螺杆菌球形菌株的诱导：将幽门螺杆菌G27菌株从新鲜固体平板接种到布鲁氏肉汤培养液(含2％FCS)，调整菌液浓度至OD
600＝0.4左右，再添加阿莫西林至终浓度0.063μg/mL，放置三气培养箱培养24小时，革兰氏染色镜检观察确定G27菌株全部转变为球形菌，而对照实验(未添加阿莫西林)中G27菌株为螺杆状(图2)。
①Induction of Helicobacter pylori spherical strain: Inoculate Helicobacter pylori G27 strain from fresh solid plate into Brucella broth (containing 2% FCS), adjust the concentration of the bacterial solution to OD 600 = about 0.4, then add Amo The final concentration of cillin was 0.063 μg/mL, and it was placed in a three-gas incubator for 24 hours. Gram staining and microscopic observation confirmed that the G27 strains were all transformed into spherical bacteria, while the G27 strains in the control experiment (without adding amoxicillin) were screw-shaped. (figure 2).
②药物处理：离心球形菌株弃上清，添加布鲁氏肉汤培养液(含2％FCS)稀释100倍至菌量浓度约为1×10
6CFU/mL，然后分别加入1×MIC、2×MIC、4×MIC和8×MIC终浓度的阿莫西林、甲硝唑、克拉霉素、左氧氟沙星和Napabucasin，放置三气培养箱培养。
②Drug treatment: Centrifuge the spherical strain and discard the supernatant, add Brucella broth (containing 2% FCS) to dilute 100 times to a bacterial concentration of about 1×10 6 CFU/mL, and then add 1×MIC, 2 ×MIC, 4×MIC and 8×MIC final concentrations of amoxicillin, metronidazole, clarithromycin, levofloxacin and Napabucasin were cultured in a three-gas incubator.
③平板计数：在指定时间取出100μL菌液进行梯度稀释，并涂布于哥伦比亚培养血平板中，再放置三气培养箱培养4天进行菌落计数。③ Plate count: Take out 100 μL of bacterial solution at the designated time for gradient dilution, spread it on Columbia culture blood plate, and then place it in a three-gas incubator for 4 days for colony counting.
(3)结果(3) Results
结果见图3。由图3所示，Napabucasin体外对抗幽门螺杆菌球形菌有极强的杀灭作用，效率强于常用抗生素如阿莫西林、甲硝唑、克拉霉素和左氧氟沙星等，具有良好的开发前景。The results are shown in Figure 3. As shown in Figure 3, Napabucasin has a strong killing effect against Helicobacter pylori in vitro, and its efficiency is stronger than that of commonly used antibiotics such as amoxicillin, metronidazole, clarithromycin and levofloxacin, and has a good development prospect.
实施例4 Napabucasin对已形成生物膜的幽门螺杆菌的杀灭作用Example 4 The killing effect of Napabucasin on Helicobacter pylori that has formed biofilm
(1)材料(1)Material
如实施例1材料。Such as Example 1 material.
(2)方法：采用结晶紫染色、荧光共聚焦和平板菌落计数法检测Napabucasin对幽门螺杆菌的抗生物膜活性。(2) Methods: The anti-biofilm activity of Napabucasin against Helicobacter pylori was detected by crystal violet staining, confocal fluorescence and plate colony counting.
①Napabucasin对幽门螺杆菌生物膜形成的影响：将幽门螺杆菌G27菌株在添加10％FBS的布鲁氏菌肉汤中过夜培养，用上述新鲜培养基稀释至OD
600为0.15，并向其中加入不 同浓度的Napabucasin、甲硝唑(作为阳性对照)或等量的DMSO(作为溶剂对照)，然后铺在96孔板，置于三气培养箱中培养3天后，用结晶紫染色法检测生物膜的相对量。
①Effect of Napabucasin on Helicobacter pylori biofilm formation: Helicobacter pylori G27 strain was cultured overnight in Brucella broth supplemented with 10% FBS, diluted with the above fresh medium to an OD 600 of 0.15, and added different Concentrations of Napabucasin, metronidazole (as a positive control) or an equivalent amount of DMSO (as a solvent control) were then spread on a 96-well plate, placed in a three-gas incubator for 3 days, and the biofilm was detected by crystal violet staining. relative amount.
②Napabucasin对已形成生物被膜的破坏作用：将幽门螺杆菌G27菌株在添加10％FBS的布鲁氏菌肉汤中过夜培养，用上述新鲜培养基稀释至OD
600为0.15，然后铺在96孔板，置于三气培养箱中培养3天以形成生物膜后，弃去培养基，然后用PBS清洗平板两次。在新鲜布鲁氏菌肉汤中添加不同浓度的Napabucasin、MTZ(作为阳性对照)或等量的DMSO(作为溶剂对照)，加入96孔板中继续培养24小时，用结晶紫染色法检测生物被膜的相对量。
②The destruction effect of Napabucasin on the formed biofilm: Helicobacter pylori G27 strain was cultured overnight in Brucella broth supplemented with 10% FBS, diluted with the above fresh medium to an OD 600 of 0.15, and then spread on a 96-well plate , after 3 days in a three-gas incubator to form a biofilm, the medium was discarded, and the plate was washed twice with PBS. Different concentrations of Napabucasin, MTZ (as a positive control) or an equal amount of DMSO (as a solvent control) were added to fresh Brucella broth, added to a 96-well plate and cultured for 24 hours, and the biofilm was detected by crystal violet staining. relative amount.
③荧光共聚焦显微镜检测Napabucasin对生物膜中细菌活性的影响。生物膜内的细菌活力通过使用Live/Dead BacLight细菌活力试剂盒(Invitrogen，美国)进行评估，该试剂盒由两种荧光染料SYTO9和碘化丙啶(PI)组成。如上制备幽门螺杆菌G27菌株生物膜，用不同浓度的Napabucasin、MTZ(作为阳性对照)或等量的DMSO(作为溶剂对照)处理。在上述微氧条件下孵育24小时后，用PBS洗涤未黏附的生物膜3次，然后用两种荧光染料在室温下黑暗中染色30分钟。漂洗后，使用共聚焦激光扫描显微镜(LSM710；Carl Zeiss，德国)观察图像，并随机检查更多视野。SYTO9和PI区分活细胞(绿色细胞)和死亡细胞(红色细胞)。③ The effect of Napabucasin on bacterial activity in biofilm was detected by fluorescence confocal microscopy. Bacterial viability within biofilms was assessed by using the Live/Dead BacLight Bacterial Viability Kit (Invitrogen, USA), which consists of two fluorescent dyes, SYTO9 and propidium iodide (PI). Helicobacter pylori G27 strain biofilms were prepared as above and treated with various concentrations of Napabucasin, MTZ (as a positive control) or an equivalent amount of DMSO (as a solvent control). After 24 h incubation under the above-mentioned microaerobic conditions, the non-adherent biofilms were washed 3 times with PBS and then stained with two fluorescent dyes for 30 min at room temperature in the dark. After rinsing, images were observed using a confocal laser scanning microscope (LSM710; Carl Zeiss, Germany) and additional fields were randomly examined. SYTO9 and PI distinguish between live cells (green cells) and dead cells (red cells).
④菌落计数法检测Napabucasin对生物膜中细菌活性的影响。如上制备和处理幽门螺杆菌G27细胞的生物膜。并在微氧条件下处理24小时后，去除培养基，用PBS冲洗3次，然后向每孔中加入250μl BHI培养液。用200μl的移液器顶端在孔壁上刮15次，从而将生物膜剥离。然后反复吹吸使刮过的生物膜在溶液中均质。最后，均匀的生物被膜菌悬液进行倍比稀释，铺在哥伦比亚血液琼脂平板上，然后在37℃的微氧条件下培养4天。对平板上的活细菌菌落进行计数，并以每毫升菌落总数表示。④The effect of Napabucasin on bacterial activity in biofilm was detected by colony counting method. Biofilms of H. pylori G27 cells were prepared and treated as above. After 24 hours of treatment under micro-aerobic conditions, the medium was removed, washed three times with PBS, and then 250 μl of BHI medium was added to each well. The biofilm was peeled off by scraping the walls of the wells with a 200 μl pipette tip 15 times. The scraped biofilm was then homogenized in the solution by repeated pipetting. Finally, the homogeneous biofilm bacterial suspension was double-diluted, plated on Columbia blood agar plates, and then incubated at 37°C for 4 days under microaerobic conditions. The viable bacterial colonies on the plates were counted and expressed as total colonies per milliliter.
(3)结果(3) Results
结果如图4。由图4A所示，Napabucasin可抑制幽门螺杆菌生物膜的形成，由图4B所示，Napabucasin具有破坏幽门螺杆菌成熟生物膜的能力，且抗生物膜活性强于甲硝唑。由图4C和4D所示，Napabucasin可杀灭形成生物膜的幽门螺杆菌，其活性也强于甲硝唑。The results are shown in Figure 4. As shown in Figure 4A, Napabucasin can inhibit the formation of Helicobacter pylori biofilm, and as shown in Figure 4B, Napabucasin has the ability to destroy the mature biofilm of Helicobacter pylori, and its anti-biofilm activity is stronger than that of metronidazole. As shown in Figures 4C and 4D, Napabucasin can kill the biofilm-forming Helicobacter pylori, and its activity is also stronger than that of metronidazole.
实施例5 Napabucasin对小鼠胃内定植的幽门螺杆菌NSH57菌株的杀灭效果检测Example 5 Detection of the killing effect of Napabucasin on Helicobacter pylori NSH57 strains colonized in the stomach of mice
(1)材料(1)Material
菌株为幽门螺杆菌小鼠驯化菌株NSH57，小鼠为SPF级别的6周龄雌性C57BL/6小鼠，其他如实施例1材料。The strain is Helicobacter pylori mouse domesticated strain NSH57, the mice are SPF grade 6-week-old female C57BL/6 mice, and other materials are as in Example 1.
(2)方法(2) Method
①幽门螺杆菌NSH57菌株感染小鼠的体内模型构建：参考文章(Huang Y,Hang X,Jiang X,Zeng L,Jia J,Xie Y,Li F,Bi H.In Vitro and In Vivo Activities of Zinc Linolenate,a Selective Antibacterial Agent against Helicobacter pylori.Antimicrob Agents Chemother[J].2019；63(6):e00004-19)。取10％小鼠胃组织检测幽门螺杆菌定植，定植量范围为1×10
5CFU/g以上认为定植成功，检测结果均有幽门螺杆菌定植，模型构建成功。
①Construction of an in vivo model of Helicobacter pylori NSH57 strain-infected mice: Reference article (Huang Y, Hang X, Jiang X, Zeng L, Jia J, Xie Y, Li F, Bi H. In Vitro and In Vivo Activities of Zinc Linolenate , a Selective Antibacterial Agent against Helicobacter pylori. Antimicrob Agents Chemother[J]. 2019;63(6):e00004-19). 10% mouse gastric tissue was taken to detect the colonization of Helicobacter pylori. The colonization amount was more than 1×10 5 CFU/g, and the colonization was considered to be successful. The detection results showed the colonization of Helicobacter pylori.
②分组：将造模成功的感染组平均分成3组，分别为奥美拉唑加Napabucasin(给药量为14mg/kg)组、奥美拉唑加阿莫西林(给药量14mg/kg)和克拉霉素(给药量为7mg/kg)组(标准三联组)、溶剂对照组，每组7只；所有组中奥美拉唑的给药量为138.2mg/kg，未感染幽门螺杆菌的7只小鼠为阴性对照组。②Grouping: Divide the infected group with successful modeling into 3 groups on average, namely the omeprazole plus Napabucasin (administration dose of 14mg/kg) group, and the omeprazole plus amoxicillin (administration dose of 14mg/kg) group. and clarithromycin (administration dose of 7mg/kg) group (standard triple combination group), solvent control group, 7 in each group; omeprazole dose in all groups was 138.2mg/kg, no infection of Helicobacter pylorus 7 mice were negative control group.
③给药：采取灌胃给药法，奥美拉唑先于其他药物前30分钟给药，给药后禁食禁水4小时；小鼠体重按照平均20g/只计算，每天给药1次，连续3天；溶剂对照组给0.5％羧甲基纤维素钠+0.2％吐温80溶液，容量、次数与上相同。③Administration: The oral administration method was adopted, omeprazole was administered 30 minutes before other drugs, and the mice were fasted for 4 hours after administration; the average weight of mice was 20g/mice, and the administration was once a day , for 3 consecutive days; the solvent control group was given 0.5% sodium carboxymethyl cellulose + 0.2% Tween 80 solution, the capacity and frequency were the same as above.
④小鼠处理：最后一次给药48小时后进行安乐死，取胃组织进行幽门螺杆菌的分离培养和鉴定，并计算定植量。④ Mice treatment: 48 hours after the last administration, euthanasia was performed, and gastric tissue was collected for the isolation, culture and identification of Helicobacter pylori, and the colonization amount was calculated.
(3)结果(3) Results
结果如图5。由图5所示，奥美拉唑加Napabucasin组对幽门螺杆菌菌的杀灭明显优于三联治疗组(奥美拉唑加阿莫西林和克拉霉素)(P<0.05)。The results are shown in Figure 5. As shown in Figure 5, the killing of Helicobacter pylori in the omeprazole plus Napabucasin group was significantly better than that in the triple therapy group (omeprazole plus amoxicillin and clarithromycin) (P<0.05).
实施例6 Napabucasin对小鼠胃内定植的多重耐药幽门螺杆菌BHKS159菌株的杀灭效果检测Example 6 Detection of the killing effect of Napabucasin on the multidrug-resistant Helicobacter pylori BHKS159 strain colonized in the stomach of mice
(1)材料(1)Material
菌株为幽门螺杆菌小鼠驯化菌株BHKS159，小鼠为SPF级别的6周龄雌性C57BL/6小鼠，其他如实施例1材料。The strain is Helicobacter pylori mouse domesticated strain BHKS159, the mice are SPF grade 6-week-old female C57BL/6 mice, and other materials are as in Example 1.
(2)方法(2) Method
①幽门螺杆菌BHKS159菌株感染小鼠的体内模型构建：参考文章(Huang Y,Hang X,Jiang X,Zeng L,Jia J,Xie Y,Li F,Bi H.In Vitro and In Vivo Activities of Zinc Linolenate,a Selective Antibacterial Agent against Helicobacter pylori.Antimicrob Agents Chemother[J].2019；63(6):e00004-19)。取10％小鼠胃组织检测幽门螺杆菌定植，定植量范围为1×10
5CFU/g以上认为定植成功，检测结果均有幽门螺杆菌定植，模型构建成功。
①Construction of an in vivo model of Helicobacter pylori BHKS159 strain-infected mice: Reference article (Huang Y, Hang X, Jiang X, Zeng L, Jia J, Xie Y, Li F, Bi H. In Vitro and In Vivo Activities of Zinc Linolenate , a Selective Antibacterial Agent against Helicobacter pylori. Antimicrob Agents Chemother[J]. 2019;63(6):e00004-19). 10% mouse gastric tissue was taken to detect the colonization of Helicobacter pylori. The colonization amount was more than 1×10 5 CFU/g, and the colonization was considered to be successful. The detection results showed the colonization of Helicobacter pylori.
②分组：实验组将造模成功的感染组平均分成6组，分别为两组不同给药剂量的奥美拉唑加Napabucasin(给药量分别为14和28mg/kg)联用组、两组不同给药剂量的Napabucasin(给药量分别为14mg/kg和28mg/kg)组，奥美拉唑加阿莫西林(给药量28mg/kg)和克拉 霉素(给药量为14mg/kg)组和溶剂对照组，每组10只；所有组中奥美拉唑的给药量为138.2mg/kg，未感染幽门螺杆菌的8只小鼠为阴性对照组。② Grouping: In the experimental group, the infected group with successful modeling was divided into 6 groups on average. Different doses of Napabucasin (14mg/kg and 28mg/kg respectively) group, omeprazole plus amoxicillin (28mg/kg) and clarithromycin (14mg/kg) ) group and solvent control group, with 10 mice in each group; the dose of omeprazole in all groups was 138.2 mg/kg, and 8 mice that were not infected with Helicobacter pylori were the negative control group.
③给药：采取灌胃给药法，奥美拉唑先于其他药物前30分钟给药，给药后禁食禁水4小时；每天给药1次，连续3天；溶剂对照组给0.5％羧甲基纤维素钠+0.2％吐温80溶液，容量、次数与上相同。③Administration: by gavage administration, omeprazole was administered 30 minutes before other drugs, fasting for 4 hours after administration; once a day for 3 consecutive days; solvent control group was given 0.5 % Sodium carboxymethyl cellulose + 0.2% Tween 80 solution, the capacity and frequency are the same as above.
④小鼠处理：最后一次给药48小时后进行安乐死，眼眶取血离心提取血清，用ELISA法检测各炎症因子的含量；取胃组织，一半组织进行幽门螺杆菌的分离培养和鉴定，计算定植量，另一半组织进行病理切片染色。同时每天进行小鼠称重并计算平均体重。④ Treatment of mice: 48 hours after the last administration, euthanize, collect blood from the orbit and centrifuge to extract serum, and detect the content of various inflammatory factors by ELISA method; take gastric tissue, and half of the tissue is separated, cultured and identified for Helicobacter pylori, and colonization is calculated. The other half of the tissue was stained for pathological sections. At the same time, mice were weighed daily and the average body weight was calculated.
(3)结果(3) Results
奥美拉唑加Napabucasin联用组(28mg/kg)或Napabucasin单药组(28mg/kg)对幽门螺杆菌的杀灭效果明显优于三联治疗组(奥美拉唑加阿莫西林和克拉霉素)，如图6。奥美拉唑加Napabucasin联用组(28mg/kg)或Napabucasin单药组(28mg/kg)中小鼠外周血的炎症因子表达水平较三联治疗组明显下降，如图7，且这两组对小鼠胃粘膜损伤的修复能力也明显优于三联治疗组，如图8。Omeprazole plus Napabucasin combination group (28mg/kg) or Napabucasin monotherapy group (28mg/kg) had significantly better killing effect on Helicobacter pylori than triple therapy group (omeprazole plus amoxicillin and clarithromycin) element), as shown in Figure 6. The expression levels of inflammatory factors in the peripheral blood of mice in the omeprazole plus Napabucasin combination group (28mg/kg) or Napabucasin single-agent group (28mg/kg) were significantly lower than those in the triple therapy group, as shown in Figure 7, and these two groups had no significant effect. The repair ability of rat gastric mucosal injury was also significantly better than that of the triple therapy group, as shown in Figure 8.
二、Napabucasin抗非幽门螺杆菌细菌的作用2. The effect of Napabucasin against non-Helicobacter pylori bacteria
实施例7 Napabucasin体外抗非幽门螺杆菌细菌的活性测定Example 7 In vitro activity assay of Napabucasin against non-Helicobacter pylori bacteria
通过测定Napabucasin对非幽门螺杆菌细菌的最低抑菌浓度(MIC)来确定其抗菌活性。The antibacterial activity of Napabucasin was determined by determining the minimum inhibitory concentration (MIC) of Napabucasin against non-Helicobacter pylori bacteria.
(1)材料(1)Material
①药品和试剂：Napabucasin、甲硝唑和万古霉素购于MCE公司。①Drugs and reagents: Napabucasin, metronidazole and vancomycin were purchased from MCE Company.
②菌株：金黄色葡萄球菌ATCC 25923和USA300、溶血性葡萄球菌ATCC29970、表皮葡萄球菌ATCC12228、枯草芽孢杆菌168、蜡样芽孢杆菌ATCC14579、单核细胞增生李斯特菌EGD-e、肺炎链球菌ATCC49619、粪肠球菌ATCC29212、屎肠球菌ATCC19434、艰难梭菌ATCC700057、痤疮丙酸杆菌ATCC11827、牙龈卟啉单胞菌ATCC BAA-308、伴放线放线杆菌D7S-1、具核梭杆菌ATCC25586、空肠弯曲杆菌NCTC11168、淋病奈瑟菌ATCC19424、流感嗜血杆菌ATCC49766、卡他莫拉菌ATCC25238、大肠杆菌ATCC25922、铜绿假单胞菌PAO1、肺炎克雷伯氏菌ATCC35657、鲍曼不动杆菌ATCC19606、肠沙门氏菌肠亚种ATCC14028为标准或模式菌株。②Strains: Staphylococcus aureus ATCC 25923 and USA300, Staphylococcus hemolytica ATCC29970, Staphylococcus epidermidis ATCC12228, Bacillus subtilis 168, Bacillus cereus ATCC14579, Listeria monocytogenes EGD-e, Streptococcus pneumoniae ATCC49619, Enterococcus faecalis ATCC29212, Enterococcus faecalis ATCC19434, Clostridium difficile ATCC700057, Propionibacterium acnes ATCC11827, Porphyromonas gingivalis ATCC BAA-308, Actinobacter actinomycetemcomitans D7S-1, Fusobacterium nucleatum ATCC25586, Curvature jejunum Bacillus NCTC11168, Neisseria gonorrhoeae ATCC19424, Haemophilus influenzae ATCC49766, Moraxella catarrhalis ATCC25238, Escherichia coli ATCC25922, Pseudomonas aeruginosa PAO1, Klebsiella pneumoniae ATCC35657, Acinetobacter baumannii ATCC19606, Salmonella enterica Intestinal subsp. ATCC14028 is the standard or type strain.
③培养基和主要试剂：MH培养液、MH培养基、脑心浸液培养液、哥伦比亚培养基和FCS。③Medium and main reagents: MH medium, MH medium, brain-heart infusion medium, Columbia medium and FCS.
④主要仪器：恒温培养箱、厌氧培养箱、紫外分光光度计、恒温摇床(Thermo)、离心 机、电子天平等。④Main instruments: constant temperature incubator, anaerobic incubator, UV spectrophotometer, constant temperature shaker (Thermo), centrifuge, electronic balance, etc.
(2)方法(2) Method
采用肉汤稀释法检测Napabucasin对非幽门螺杆菌细菌的最低抑菌浓度。检测所用培养基、培养液为MH培养基、培养液，菌液浓度约为1.0×10
5CFU/mL，结果判读的时间根据菌株差异选择24或48小时，其他均与实施例1中方法相似。
The minimum inhibitory concentration of Napabucasin against non-Helicobacter pylori bacteria was detected by broth dilution method. The medium and medium used for the detection were MH medium and medium, and the bacterial concentration was about 1.0×10 5 CFU/mL. The time for interpretation of the results was 24 or 48 hours according to the difference of the strains. Others were similar to the methods in Example 1. .
(3)结果(3) Results
见表2。See Table 2.
表2.Napabucasin对非幽门螺杆菌细菌菌株的最低抑菌浓度(μg/ml)Table 2. Minimum inhibitory concentrations (μg/ml) of Napabucasin against non-Helicobacter pylori bacterial strains
注：“—”表明未检测。Note: "—" indicates not detected.
由上表所示，Napabucasin对革兰氏阳性菌如金黄色葡萄球菌、溶血性葡萄球菌、表皮葡萄球菌、枯草芽孢杆菌、蜡样芽孢杆菌、单核细胞增生李斯特氏菌、肺炎链球菌、艰难梭菌和痤疮丙酸杆菌以及革兰氏阴性菌如牙龈卟啉单胞菌、伴放线放线杆菌、具核梭杆菌、空肠弯曲杆菌、淋病奈瑟菌、流感嗜血杆菌和卡他莫拉菌等，都有抑制生长的作用。As shown in the table above, Napabucasin is effective against Gram-positive bacteria such as Staphylococcus aureus, Staphylococcus hemolyticus, Staphylococcus epidermidis, Bacillus subtilis, Bacillus cereus, Listeria monocytogenes, Streptococcus pneumoniae, Clostridium difficile and Propionibacterium acnes and Gram-negative bacteria such as Porphyromonas gingivalis, Actinobacter actinomyces, Fusobacterium nucleatum, Campylobacter jejuni, Neisseria gonorrhoeae, Haemophilus influenzae, and catarrhalis Moraxella, etc., all have the effect of inhibiting growth.
三、Napabucasin抗真菌的作用3. Antifungal effect of Napabucasin
实施例8 Napabucasin体外抗真菌的活性测定Example 8 Determination of in vitro antifungal activity of Napabucasin
(1)材料(1)Material
①化学品：Napabucasin和两性霉素(AMB)购于MCE公司。①Chemicals: Napabucasin and Amphotericin (AMB) were purchased from MCE Company.
②菌株：烟曲霉Af293、红色毛癣菌ATCC MYA-4438、白色念珠菌SC5314和ATCC14053、近平滑念珠菌ATCC22019、光滑念珠菌ATCC36583、克鲁斯念珠菌ATCC6258、葡萄牙念珠菌ATCC200950、热带念珠菌ATCC750、新生隐球菌ATCC MYA-4906和ATCC 90112、合轴马拉色菌CBS7222、球形马拉色菌CBS7966、糠秕马拉色菌CBS1878和厚皮马拉色菌CBS1879为标准或模式菌株。其他临床菌株为南京医科大学附属第一医院和附属逸夫医院从临床样本中分离鉴定所得。②Strains: Aspergillus fumigatus Af293, Trichophyton rubrum ATCC MYA-4438, Candida albicans SC5314 and ATCC14053, Candida parapsilosis ATCC22019, Candida glabrata ATCC36583, Candida cruzi ATCC6258, Candida portuguese ATCC200950, Candida tropicalis ATCC750 , Cryptococcus neoformans ATCC MYA-4906 and ATCC 90112, Malassezia sphaeroides CBS7222, Malassezia globosa CBS7966, Malassezia furfur CBS1878 and Malassezia pachyderma CBS1879 are standard or model strains. Other clinical strains were isolated and identified from clinical samples at the First Affiliated Hospital of Nanjing Medical University and the Affiliated Yifu Hospital.
③培养基和主要试剂：RPMI1640培养液、沙保弱培养基、FCS、二甲基亚砜(DMSO)。③Medium and main reagents: RPMI1640 medium, Shabao weak medium, FCS, dimethyl sulfoxide (DMSO).
(2)方法：借助肉汤稀释法检测Napabucasin对多种真菌菌株的最低抑菌浓度(MIC，100μL体系)。(2) Method: The minimum inhibitory concentration (MIC, 100 μL system) of Napabucasin against various fungal strains was detected by broth dilution method.
①分别配备6.4mg/mL的Napabucasin、两性霉素B和氟康唑储存溶液，溶剂均为100％DMSO。① Prepare 6.4 mg/mL Napabucasin, Amphotericin B, and Fluconazole stock solutions, all in 100% DMSO.
②菌液制备：取固体平板上对数期生长的白色念珠菌用RPMI1640培养液制作成菌悬液，调整浓度OD
530为0.5(约1×10
6CFU/mL)，稀释1000倍，约为1×10
3CFU/mL，备用。烟曲霉、毛癣菌以及马拉色菌(用加入橄榄油的特定培养基)需要进行显微计数调整菌液浓度；而新生隐球菌调整菌液OD
530为0.5后，稀释10000倍，备用。
②Preparation of bacterial solution: Take Candida albicans growing in logarithmic phase on the solid plate to make bacterial suspension with RPMI1640 culture solution, adjust the concentration OD 530 to 0.5 (about 1×10 6 CFU/mL), and dilute it 1000 times, about 1×10 3 CFU/mL, for use. Aspergillus fumigatus, Trichophyton and Malassezia (using a specific medium added with olive oil) need to be microscopically counted to adjust the bacterial concentration; while Cryptococcus neoformans adjusts the bacterial solution OD 530 to 0.5, diluted 10,000 times for use.
③96孔板制备：第一孔先加RPMI1640培养液178μL，再加2μL的Napabucasin储存溶液，采用二倍稀释法稀释至第12孔；另外两排孔以同样方法分别加两性霉素B和氟康唑。③96-well plate preparation: firstly add 178 μL of RPMI1640 culture medium to the first well, then add 2 μL of Napabucasin stock solution, and dilute to the 12th well by double dilution method; add amphotericin B and fluconin to the other two wells in the same way. azoles.
④接种菌液：取10μL上述备用菌液加入上述每孔中(每孔菌量浓度约为1.0×10
2CFU/mL)内，药物浓度依次为64μg/mL、32μg/mL、16μg/mL、8μg/mL、4μg/mL、2μg/mL、1μg/mL、0.5μg/mL、0.25μg/mL、0.125μg/mL、0.063μg/mL和0.031μg/mL。另外一排每孔分别加100μL RPMI1640培养液，作为无菌对照组；再一排每孔分别加90μL RPMI1640培养液和10μL上述备用菌液，作为阳性对照组。放置恒温培养箱培养。
④Inoculation bacterial liquid: take 10 μL of the above-mentioned standby bacterial liquid and add it to each hole (the concentration of bacteria in each hole is about 1.0×10 2 CFU/mL), and the drug concentrations are 64 μg/mL, 32 μg/mL, 16 μg/mL, 8 μg/mL, 4 μg/mL, 2 μg/mL, 1 μg/mL, 0.5 μg/mL, 0.25 μg/mL, 0.125 μg/mL, 0.063 μg/mL and 0.031 μg/mL. In the other row, add 100 μL of RPMI1640 culture solution to each well as a sterile control group; in the other row, add 90 μL of RPMI1640 culture solution and 10 μL of the above-mentioned standby bacterial solution to each well as a positive control group. Place in a constant temperature incubator.
⑤结果判断：培养24或48小时后判读结果，以在小孔内完全抑制真菌生长的最低药物浓度为MIC。当阳性对照孔(即不含药物)内真菌明显生长以及无菌对照组无菌生长时，试验才有意义。重复3次试验。⑤Result judgment: Interpret the results after culturing for 24 or 48 hours, and take the lowest drug concentration that completely inhibits the growth of fungi in the small well as the MIC. The test is only meaningful when there is significant fungal growth in the positive control wells (ie, no drug) and when the sterile control group is sterile. The experiment was repeated 3 times.
(3)结果(3) Results
结果见表3。The results are shown in Table 3.
表3.Napabucasin对真菌菌株的最低抑菌浓度(μg/ml)Table 3. Minimum inhibitory concentrations (μg/ml) of Napabucasin against fungal strains
注：“—”表明未检测。Note: "—" indicates not detected.
由上表所示，Napabucasin对白色念珠菌、近平滑念珠菌、光滑念珠菌、克鲁斯念珠菌、葡萄牙念珠菌、热带念珠菌、新生隐球菌、烟曲霉、红色毛癣菌和马拉色菌等真菌，都有抑制生长的作用，其抑制活性与两性霉素B相当。As shown in the table above, Napabucasin is effective against Candida albicans, Candida parapsilosis, Candida glabrata, Candida cruzi, Candida portugalii, Candida tropicalis, Cryptococcus neoformans, Aspergillus fumigatus, Trichophyton rubrum and Malassezia Bacteria and other fungi, all have the effect of inhibiting growth, and its inhibitory activity is comparable to that of amphotericin B.
实施例9 Napabucasin对小鼠全身感染模型中的白色念珠菌SC5314菌株的杀灭作用Example 9 Killing effect of Napabucasin on Candida albicans SC5314 strain in a mouse systemic infection model
(1)材料(1)Material
菌株为白色念珠菌SC5314，小鼠为SPF级别的8周龄C57BL/6雌性小鼠，药品氟康唑 购于MCE公司，其他如实施例8材料。The strain is Candida albicans SC5314, the mice are SPF grade 8-week-old C57BL/6 female mice, the drug fluconazole is purchased from MCE Company, and other materials are as in Example 8.
(2)方法(2) Method
①动物造模：将白色念珠菌SC5314菌株在沙氏琼脂培养基中35℃培养24小时后，挑取菌落至RPMI1640培养基中，调整菌液浓度约为5×10
6CFU/mL，离心收集菌体后用等体积PBS重悬。经小鼠内眦静脉丛注射125μl菌液，白色念珠菌经血在小鼠体内扩散，造成小鼠全身感染。
①Animal modeling: After culturing the Candida albicans SC5314 strain in Sabouraud agar medium at 35°C for 24 hours, pick colonies into RPMI1640 medium, adjust the concentration of the bacterial solution to about 5×10 6 CFU/mL, and collect by centrifugation The cells were then resuspended with an equal volume of PBS. 125μl of bacterial solution was injected through the venous plexus of the inner canthus of the mice, and the menstrual blood of Candida albicans spread in the mice, causing systemic infection of the mice.
②分组：将造模成功的感染小鼠平均分为3组，分别为溶剂对照组(0.5％羧甲基纤维素钠+0.2％吐温80)、氟康唑治疗组和Napabucasin治疗组，每组10只小鼠。② Grouping: The infected mice that were successfully modeled were divided into 3 groups on average, namely the solvent control group (0.5% sodium carboxymethyl cellulose + 0.2% Tween 80), the fluconazole treatment group and the Napabucasin treatment group. group of 10 mice.
③给药：实验组灌胃给药，感染当天在感染2小时后第1次给药，间隔10小时第2次给药。每天给药2次，连续3天。小鼠体重按照平均20g/只计算。氟康唑和Napabucasin的给药量都为56mg/kg/天，对照组给予溶剂，容量、次数同上。③Administration: The experimental group was given intragastric administration. The first administration was 2 hours after the infection on the day of infection, and the second administration was administered at an interval of 10 hours. Administer 2 times a day for 3 consecutive days. The weight of mice was calculated as an average of 20 g/mice. The doses of fluconazole and Napabucasin were both 56 mg/kg/day, and the control group was given a solvent with the same volume and frequency.
④小鼠处理：最后一次给药24小时后，进行安乐死，取脾和肾组织，进行白色念珠菌分离培养和鉴定，并计算定植量。④ Treatment of mice: 24 hours after the last administration, euthanize, take spleen and kidney tissues, isolate, culture and identify Candida albicans, and calculate the colonization amount.
(3)结果(3) Results
Napabucasin和氟康唑均能显著降低白色念珠菌全身感染小鼠脾脏和肾脏中的真菌载量(P＜0.01)，且氟康唑治疗组和Napabucasin治疗组的效果相当，如图9。Both Napabucasin and fluconazole could significantly reduce the fungal load in the spleen and kidney of C. albicans systemically infected mice (P<0.01), and the effects of fluconazole-treated and Napabucasin-treated groups were comparable, as shown in Figure 9.
Claims (8)
- Napabucasin在制备抗菌药物中的应用，所述菌为革兰氏阴性菌、革兰氏阳性菌或真菌。The application of Napabucasin in the preparation of antibacterial drugs, the bacteria are Gram-negative bacteria, Gram-positive bacteria or fungi.
- 根据权利要求1所述的应用，其特征在于，所述革兰氏阴性菌为幽门螺杆菌、牙龈卟啉单胞菌、伴放线放线杆菌、具核梭杆菌、空肠弯曲杆菌、淋病奈瑟菌、流感嗜血杆菌和卡他莫拉菌。The application according to claim 1, wherein the gram-negative bacteria are Helicobacter pylori, Porphyromonas gingivalis, Actinobacter acini, Fusobacterium nucleatum, Campylobacter jejuni, Neisseria gonorrhoeae Sericella, Haemophilus influenzae and Moraxella catarrhalis.
- 根据权利要求1所述的应用，其特征在于，所述革兰氏阳性菌为金黄色葡萄球菌、溶血性葡萄球菌、表皮葡萄球菌、枯草芽孢杆菌、蜡样芽孢杆菌、单核细胞增生李斯特氏菌、肺炎链球菌、艰难梭菌和痤疮丙酸杆菌。The application according to claim 1, wherein the Gram-positive bacteria are Staphylococcus aureus, Staphylococcus hemolyticus, Staphylococcus epidermidis, Bacillus subtilis, Bacillus cereus, Listeria monocytogenes Streptococcus pneumoniae, Clostridium difficile, and Propionibacterium acnes.
- 根据权利要求1所述的应用，其特征在于，所述真菌为白色念珠菌、近平滑念珠菌、光滑念珠菌、克鲁斯念珠菌、葡萄牙念珠菌、热带念珠菌、新生隐球菌、烟曲霉、红色毛癣菌和马拉色菌。The application according to claim 1, wherein the fungus is Candida albicans, Candida parapsilosis, Candida glabrata, Candida cruzi, Candida portuginosa, Candida tropicalis, Cryptococcus neoformans, Aspergillus fumigatus , Trichophyton rubrum and Malassezia.
- Napabucasin与质子泵抑制剂的组合物在制备抗耐药性或敏感性幽门螺杆菌药物中的应用。Application of the combination of Napabucasin and proton pump inhibitor in the preparation of anti-drug-resistant or sensitive Helicobacter pylori medicine.
- 根据权利要求5所述的应用，其特征在于，所述质子泵抑制剂为奥美拉唑。The use according to claim 5, wherein the proton pump inhibitor is omeprazole.
- Napabucasin在制备治疗耐药性或敏感性幽门螺杆菌感染导致的急、慢性胃炎、胃、十二指肠溃疡药物中的应用。Application of Napabucasin in the preparation of medicines for treating acute and chronic gastritis, gastric and duodenal ulcers caused by drug-resistant or sensitive Helicobacter pylori infection.
- 治疗幽门螺杆菌感染的药物，其特征在于组分为Napabucasin和奥美拉唑。The medicine for treating Helicobacter pylori infection is characterized in that the components are Napabucasin and omeprazole.
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