CN113082013B

CN113082013B - Pharmaceutical application of Napabucasin

Info

Publication number: CN113082013B
Application number: CN202110303156.7A
Authority: CN
Inventors: 毕庶壮; 黄衍强; 杭旭东
Original assignee: Individual
Current assignee: Individual
Priority date: 2021-03-22
Filing date: 2021-03-22
Publication date: 2022-11-25
Anticipated expiration: 2041-03-22
Also published as: CN113082013A

Abstract

The pharmaceutical application of Napabucasin relates to the application of Napabucasin in preparing antibacterial drugs, wherein the bacteria are gram-negative bacteria, gram-positive bacteria or fungi. The Napabucasin has good antibacterial activity on standard sensitive helicobacter pylori, clinical sensitive and drug-resistant helicobacter pylori and the like, the minimum inhibitory concentration range is 0.016 to 0.125 mu g/mL, and the Napabucasin has good killing effect on spherical helicobacter pylori and helicobacter pylori forming a mature biological membrane and has stronger effect than metronidazole. The Napabucasin is not easy to generate drug resistance in the helicobacter pylori resisting process, has a strong killing effect on helicobacter pylori fixedly planted in the stomach of a mouse, and has stronger bactericidal capacity when being used alone or combined with omeprazole compared with a standard triple therapy. In addition, it showed a candida albicans killing effect with the same efficiency as fluconazole in a systemic infection model of candida albicans infected mice.

Description

Pharmaceutical application of Napabucasin

Technical Field

The invention belongs to the field of pharmacy, and provides pharmaceutical application of Napabucasin.

Background

Infectious diseases are serious diseases threatening human life and health, and the problem of microbial drug resistance caused by abuse and overuse of antibiotics has become a public safety crisis facing human beings in common. About 70 million people die from drug-resistant bacteria infection every year worldwide, more than cancer deaths, and therefore there is an urgent need to explore and develop novel anti-infective drugs. In 2017, in 2 months, the WHO releases the 'superbacteria' and '12-strength' list which is most resistant to drugs and most threatening human health in the world for the first time, and the list is used for guiding and promoting the preferential research and development of novel antibiotics.

Helicobacter pylori (Hp) is a microaerophilic gram-negative bacterium that infects about half of the world's population. The Hp infection is the main cause of various stomach diseases such as chronic gastritis and gastric and duodenal ulcers, and is closely related to the occurrence and development of gastric cancer. Many large-scale epidemiological intervention studies have shown that eradication of Hp can prevent the occurrence of gastric cancer. Currently, the treatment regimen recommended by the world health organization for eradicating Hp infection is triple or quadruple therapy, the former being a proton pump inhibitor (omeprazole, etc.) plus two antibiotics (two selected from clarithromycin, amoxicillin, levofloxacin, metronidazole, etc.), the latter being further added with a bismuth agent (bismuth potassium citrate, etc.). However, with the wide application of antibiotics in the treatment of Hp infection in recent years, the problem of Hp resistance is becoming more serious, and new drugs effective to drug-resistant helicobacter pylori are urgently needed.

Candida is one of the normal strains in human body, and can cause candidiasis when systemic or local immunity is reduced, such as after a large amount of antibiotics, glucocorticoids or immunosuppressants and the like. Candida albicans is the main pathogenic bacterium of the disease and can cause skin, mucous membrane and even systemic infection. Studies have indicated that the mortality rate of this severe systemic infection is as high as 40%. In addition to the increasing number of common Candida albicans infections, infections caused by a few rare or rare fungi have also been reported. The development of high-efficiency and low-toxicity antifungal drugs for clinical application is urgent.

Napabucain, developed by Boston biopharmaceutical technology, BBI, usa, is a few STAT3 inhibitors entering phase III clinics. Napabucain can be used for treating cancer stem cells, and has the potential of inhibiting cancer cell metastasis and preventing cancer recurrence in various tumor types.

Disclosure of Invention

The technical problem to be solved is as follows: the invention provides a pharmaceutical application of Napabucasin, which can be used for preparing antibacterial drugs.

The technical scheme is as follows: application of Napabucasin in preparation of antibacterial drugs, wherein the bacteria are gram-negative bacteria, gram-positive bacteria or fungi.

Preferably, the gram-negative bacterium is helicobacter pylori, porphyromonas gingivalis, actinobacillus actinomycetemcomitans, fusobacterium nucleatum, campylobacter jejuni, neisseria gonorrhoeae, haemophilus influenzae, and Moraxella catarrhalis.

Preferably, the gram-positive bacteria are staphylococcus aureus, staphylococcus hemolyticus, staphylococcus epidermidis, bacillus subtilis, bacillus cereus, listeria monocytogenes, streptococcus pneumoniae, clostridium difficile and propionibacterium acnes.

Preferably, the fungus is Candida albicans, candida parapsilosis, candida glabrata, candida krusei, candida viticola, candida tropicalis, cryptococcus neoformans, aspergillus fumigatus, trichophyton rubrum, and Malassezia.

Application of a composition of Napabucasin and a proton pump inhibitor in preparation of a drug resistant or sensitive helicobacter pylori drug.

The proton pump inhibitor is omeprazole.

Application of Napabucasin in preparation of medicine for treating acute and chronic gastritis, gastric ulcer and duodenal ulcer caused by drug-resistant or sensitive helicobacter pylori infection.

A medicine for treating helicobacter pylori infection comprises Napabucasin and omeprazole.

Has the advantages that: napabucasin can be used for preparing medicines for resisting bacterial infection. The Napabucasin shown by the invention has a good killing effect on helicobacter pylori which grows in a floating way and forms a biological membrane, can be used for treating stomach diseases such as acute and chronic gastritis, gastric ulcer and duodenal ulcer caused by drug resistance or sensitive helicobacter pylori infection, has small toxic and side effects, and can effectively relieve the drug resistance problem of the helicobacter pylori.

Drawings

FIG. 1. In vitro induction test of H.pylori G27 strain for resistance to Napabucasin.

FIG. 2 detection of the spheroid deformation of the H.pylori G27 strain under a microscope using amoxicillin induction. A, negative control; b, G27 spherical strain.

FIG. 3. Killing effect of Napabucasin on H.pylori G27 coccoid strain. A, the killing effect of each medicine with the concentration of 1 × MIC on G27 spherical strains respectively; b, killing of G27 coccoid strains after 12 hours treatment with 2 × MIC, 4 × MIC or 8 × MIC concentrations of each drug. Note: NPB, napabucain; MTZ, metronidazole; AMX, amoxicillin; CLR, clarithromycin; LVX, levofloxacin. "-" indicates CFU below detection limit (10 CFU/ml).

FIG. 4. Detection of the anti-H.pylori biofilm activity by Napabucasin. A, determining the activity of Napabucasin for inhibiting biofilm formation by a crystal violet staining method; b, determining the activity of the Napabucasin for destroying the mature biological membrane by a crystal violet staining method; c, detecting the activity of killing helicobacter pylori forming a mature biological membrane by Napabucasin by SYTO9-PI double staining and fluorescence confocal detection; d, detecting the activity of killing helicobacter pylori forming a mature biological membrane by Napabucasin by a plate colony counting method. Note: NPB, napabucain; MTZ, metronidazole. * P <0.05; * P <0.01; * P <0.001.

FIG. 5. Killing effect of Napabucasin on H.pylori NSH57 strain in mice. A, constructing an acute gastritis animal model and a drug treatment flow chart by a helicobacter pylori infected mouse; B. and (3) detecting the colonization amount of helicobacter pylori on gastric mucosa of mice after treatment of different treatment groups. A solvent Control group (Control, 0.5% sodium carboxymethylcellulose +0.2% Tween 80), a triple treatment group (OPZ + AC), and a combined treatment group of omeprazole and Napabucasin (OPZ + NPB) are provided. * P <0.05; * P <0.01.

FIG. 6. Killing effect of Napabucasin against the H.pylori multi-drug resistant strain BHKS159 in mice. A, constructing an acute gastritis animal model and a drug treatment flow chart by a helicobacter pylori infected mouse; B. and (3) detecting the colonization amount of helicobacter pylori on mouse gastric mucosa after treatment of different treatment groups. Control (uninfected mice), solvent Control after BHKS159 infection (Control, 0.5% sodium carboxymethylcellulose +0.2% tween 80), triple treatment (OPZ + AC), omeprazole and napobasin combination treatment (OPZ + NPB), and napobasin treatment (NPB) were assigned. OPZ + NPB and NPB treatment groups were two groups, with NPB administered at 28mg/kg and 7mg/kg, respectively. * P <0.05; * P <0.01; * P <0.001.

FIG. 7. Inhibition of inflammatory factor in mice infected with drug-resistant H.pylori BHKS159 by Napabucasin. The contents of IL-1 beta (A), IL-6 (B) and IL-8 (C) in the serum of each treatment group of mice were measured by ELISA method. Control (uninfected mice), solvent Control (Control, 0.5% sodium carboxymethylcellulose +0.2% tween 80) after BHKS159 infection, triple treatment (OPZ + AC), combined omeprazole and napobasin treatment (OPZ + NPB), and napobasin treatment (NPB) were included. NPB is administered at a dose of 28mg/kg.

FIG. 8. Repair of Napabucasin against gastric mucositis in mice infected with drug-resistant H.pylori BHKS 159. The gastric mucosal tissue was subjected to HE staining and TUNEL staining, respectively, at 100-fold magnification. Control (uninfected mice), solvent Control (Control, 0.5% sodium carboxymethylcellulose +0.2% tween 80) after BHKS159 infection, triple treatment (OPZ + AC), combined omeprazole and napobasin treatment (OPZ + NPB), and napobasin treatment (NPB) were included. NPB is administered at a dose of 28mg/kg.

FIG. 9. Killing effect of Napabucasin on Candida albicans SC5314 strain in mice. Results of measurements of the colonization of the kidney (a) and spleen (B) by candida albicans after treatment in different treatment groups. A solvent Control group (Control, 0.5% sodium carboxymethylcellulose +0.2% tween 80), a fluconazole treatment group (FLC) and a napobucasin treatment group (NPB) were set. * P <0.01.

Detailed Description

The following examples are given to enable a person skilled in the art to fully understand the invention, but do not limit it in any way.

1. Anti-helicobacter pylori effect of Napabucasin

Example 1 Activity assay of Napabucasin against helicobacter pylori in vitro

The anti-helicobacter pylori activity of Napabucasin was determined by measuring the Minimal Inhibitory Concentration (MIC) of Napabucasin against helicobacter pylori.

(1) Material

(1) Chemical products: napabucasin, metronidazole, amoxicillin, clarithromycin, levofloxacin and the like were purchased from MCE.

(2) The strain is as follows: helicobacter pylori standard strains 26695 and G27; other clinical strains were isolated and identified from clinical patient gastric mucosa samples at the affiliated first hospital and affiliated Yifu hospital of Nanjing medical university.

(3) Media and main reagents: brain heart infusion Broth (BHI), colombia medium, selective antibiotics (vancomycin, polymyxin B, trimethoprim), calf serum (FCS), and 100% dimethyl sulfoxide (DMSO).

(4) The main apparatus is as follows: BINDER CB160 three-gas incubator, ultraviolet spectrophotometer, constant temperature shaking table (Thermo), centrifuge, electronic balance, etc.

(5) Consumable material: EP tubes, centrifuge tubes, tip heads, etc.

(2) The method comprises the following steps: minimum inhibitory concentration (MIC, 100. Mu.L system) of Napabucasin against helicobacter pylori was determined by broth dilution method

(1) Stock solutions of 1.6mg/mL Napabucasin and 6.4mg/mL Metronidazole (MTZ) were prepared, respectively, and the solvents were 100% DMSO.

(2) Preparing bacterial liquid: taking the logarithmic growth of helicobacter pylori on the solid plate, making into bacterial suspension with BHI (containing 10% FCS), adjusting the concentration OD ₆₀₀ Is 0.2 (the bacterial concentration is about 1X 10) ⁸ CFU/mL), diluted 10 times, the bacterial load was about 1X 10 ⁷ CFU/mL, spare.

(3) Preparation of 96-well plate: 178. Mu.L of BHI broth (containing 10% FCS) was added to the first well, and 2. Mu.L of Napabucasin stock solution was added thereto, and diluted to the 12 th well by two-fold dilution; the other row of holes is added with metronidazole in the same way.

(4) Inoculating a bacterial liquid: adding 10 μ L of the above-mentioned bacterial suspension to each well (the bacterial concentration per well is about 1.0 × 10) ⁶ CFU/mL), the concentration of Napabucasin is 16. Mu.g/mL, 8. Mu.g/mL, 4. Mu.g/mL, 2. Mu.g/mL, 1. Mu.g/mL, 0.5. Mu.g/mL, 0.25. Mu.g/mL, 0.125. Mu.g/mL, 0.063. Mu.g/mL, 0.031. Mu.g/mL, 0.016. Mu.g/mL and 0.008. Mu.g/mL in this order. Another row was prepared by adding 100. Mu.L of BHI medium (10% FCS) to each well as a sterile control group; in each well, 90. Mu.L of BHI culture solution (containing 10% FCS) and 10. Mu.L of the above-mentioned stock solution were added to each well as a positive control group. Placing the mixture in a three-gas incubator for culture.

(5) And (5) judging a result: the results were read after 48 or 72h incubation with the lowest drug concentration that completely inhibited bacterial growth in the wells as the MIC. The test is only significant when there is significant growth of bacteria in the positive control wells (i.e., no drug) and when the sterile control group is aseptically grown. The experiment was repeated 3 times.

(3) Results

The results are shown in Table 1.

TABLE 1 minimum inhibitory concentration of Napabucasin against H.pylori (. Mu.g/mL)

Note: NPB, napobucasin; MTZ, metronidazole; AMX, amoxicillin; CLR, clarithromycin; LVX, levofloxacin.

As can be seen from the table above, the MIC range of Napabucasin to 15 strains of helicobacter pylori (including 2 standard strains and 13 clinical drug-resistant strains) is 0.016 to 0.125 mu g/mL, which indicates that Napabucasin has strong antibacterial activity to helicobacter pylori in vitro and has good development prospect.

Example 2 determination of Napabucasin induced resistance of H.pylori G27 Strain

(1) Material

The material of example 1.

(2) Method of producing a composite material

The H.pylori G27 strain was plated from fresh solids into 5mL BHI (containing 10% FCS), and the concentration of the strain was adjusted to OD ₆₀₀ And =0.2, adding napobucasin to a final concentration of 0.016 mug/mL (1/2 × MIC), placing the mixture in a three-gas incubator for culturing for 48 hours, observing the growth condition of the bacteria, taking a bacterium solution for passage, continuing the method to induce drug resistance, if the growth is good, multiplying the concentration of the napobucasin, and if the growth is slow or not, maintaining the existing concentration of the napobucasin. A total of 15 drug resistance inductions were performed, with MIC measurements for each generation. The control group is metronidazole, and the method is the same.

(3) Results

The results are shown in FIG. 1. As shown in FIG. 1, during serial passages, the development of resistance to Napabucasin by H.pylori was not observed, whereas H.pylori developed resistance to metronidazole after the first 5 passages, resulting in a 4-fold increase in MIC. These results demonstrate that napobucasin has a very low tendency to induce drug resistance in H.pylori.

Example 3 antibacterial Effect of Napabucasin on helicobacter pylori coccoid in vitro

(1) Material

The material of example 1.

(2) The method comprises the following steps: the antibacterial activity of Napabucasin on helicobacter pylori cocci is detected by a plate colony counting method.

(1) Induction of helicobacter pylori coccoid strains: the H.pylori G27 strain was plated from fresh solids into Brucella broth (containing 2% FCS) and the broth concentration was adjusted to OD ₆₀₀ About =0.4, adding amoxicillin to a final concentration of 0.063 μ G/mL, placing the culture medium in a three-atmosphere incubator for 24 hours, and observing by gram staphyloscopy to determine that all the G27 strains are transformed into coccoid bacteria, while the G27 strains in the control experiment (without adding amoxicillin) are in a screw shape (figure 2).

(2) And (3) drug treatment: centrifuging the spherical strain, discarding the supernatant, adding Brucella broth (2% FCS) and diluting 100-fold to a bacterial concentration of about 1X 10 ⁶ CFU/mL, then adding 1 × MIC, 2 × MIC, 4 × MIC and 8 × MIC of amoxicillin, metronidazole, clarithromycin, levofloxacin and Napabucasin to the mixture respectively, and placing the mixture in a three-gas incubator for culture.

(3) Plate counting: 100 mu L of the bacterial liquid is taken out at the appointed time for gradient dilution, and the bacterial liquid is coated in a Columbia culture blood plate, and then the bacterial liquid is placed in a three-gas incubator for culture for 4 days for colony counting.

(3) Results

The results are shown in FIG. 3. As shown in figure 3, the Napabucasin has extremely strong killing effect on helicobacter pylori cocci in vitro, has higher efficiency than common antibiotics such as amoxicillin, metronidazole, clarithromycin, levofloxacin and the like, and has good development prospect.

Example 4 killing of biofilm-forming helicobacter pylori by Napabucasin

(1) Material

As in the example 1 material.

(2) The method comprises the following steps: the anti-biofilm activity of Napabucasin on helicobacter pylori is detected by crystal violet staining, fluorescence confocal and plate colony counting methods.

(1) Effect of napobucasin on helicobacter pylori biofilm formation: h.pylori G27 strain was cultured overnight in Brucella broth supplemented with 10% FBS, diluted toOD ₆₀₀ 0.15, and various concentrations of napobucasin, metronidazole (as a positive control) or an equal amount of DMSO (as a solvent control) were added thereto, and then plated in a 96-well plate, and after 3 days of culture in a three-atmosphere incubator, the relative amount of the biofilm was measured by a crystal violet staining method.

(2) Disruption of biofilm formation by napobucasin: h.pylori G27 strain was cultured overnight in Brucella broth supplemented with 10% FBS, diluted to OD with the above-mentioned fresh medium ₆₀₀ 0.15, and then plated in a 96-well plate, and after culturing in a three-air incubator for 3 days to form a biofilm, the medium was discarded, and the plate was washed twice with PBS. Different concentrations of napobucasin, MTZ (as a positive control) or equal amounts of DMSO (as a solvent control) were added to fresh brucella broth, incubated in 96-well plates for 24 hours, and the relative amount of biofilm was measured by crystal violet staining.

(3) The effect of Napabucasin on the bacterial activity in the biofilm was examined by fluorescence confocal microscopy. Bacterial viability within biofilms was assessed by using the Live/Dead BacLight bacterial viability kit (Invitrogen, usa) consisting of two fluorescent dyes SYTO9 and Propidium Iodide (PI). The H.pylori G27 strain biofilms were prepared as above and treated with different concentrations of Napabucasin, MTZ (as a positive control) or equal amounts of DMSO (as a solvent control). After 24 hours incubation under the above-mentioned microaerobic conditions, the non-adherent biofilms were washed 3 times with PBS and then stained with two fluorescent dyes in the dark at room temperature for 30 minutes. After rinsing, the images were observed using a confocal laser scanning microscope (LSM 710; carl Zeiss, germany) and randomly examined for more fields of view. SYTO9 and PI distinguish between live cells (green cells) and dead cells (red cells).

(4) The colony counting method detects the influence of Napabucasin on the activity of bacteria in the biological membrane. Biofilms of H.pylori G27 cells were prepared and treated as above. And after 24 hours of treatment under microaerobic conditions, the medium was removed, rinsed 3 times with PBS, and 250 μ l BHI broth was added to each well. The biofilm was peeled off by scraping 15 times with a 200. Mu.l pipette tip on the pore wall. Then repeatedly blowing and sucking to homogenize the scraped biological membrane in the solution. Finally, the homogeneous biofilm bacterial suspension was diluted in duplicate, plated on colombian blood agar plates, and then cultured at 37 ℃ for 4 days under microaerobic conditions. Viable bacterial colonies on the plates were counted and expressed as total number of colonies per ml.

(3) Results

The results are shown in FIG. 4. As shown in FIG. 4A, napabucasin inhibited the formation of H.pylori biofilms, and as shown in FIG. 4B, napabucasin had the ability to disrupt the mature biofilm of H.pylori, and had greater anti-biofilm activity than metronidazole. As shown in FIGS. 4C and 4D, napabucasin killed biofilm-forming H.pylori, which was also more active than metronidazole.

Example 5 test of the killing Effect of Napabucasin against helicobacter pylori NSH57 Strain colonized in the stomach of mice

(1) Material

The strain is a helicobacter pylori mouse domesticated strain NSH57, the mouse is a female C57BL/6 mouse with the SPF grade of 6 weeks old, and other materials are as in example 1.

(2) Method of producing a composite material

(1) In vivo model construction of H.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 obtained from Helicobacter pylori. Antibacterial Agents Chemother [ J]2019;63 And (6) e 00004-19). Taking 10% mouse stomach tissue to detect the colonization of helicobacter pylori, wherein the range of colonization amount is 1 multiplied by 10 ⁵ And (3) the planting is considered to be successful above CFU/g, the helicobacter pylori is planted in the detection results, and the model is successfully constructed.

(2) Grouping: the infection groups successfully molded are averagely divided into 3 groups, namely an omeprazole and Napabucasin (the dosage is 14 mg/kg), an omeprazole and amoxicillin (the dosage is 14 mg/kg) and clarithromycin (the dosage is 7 mg/kg) (standard three-combined group) group and a solvent control group, wherein each group comprises 7 animals; the amount of omeprazole administered in all groups was 138.2mg/kg, and 7 mice not infected with H.pylori were negative control groups.

(3) Administration: the method adopts an intragastric administration method, wherein omeprazole is administered 30 minutes before other medicines, and after the administration, the omeprazole is fasted and the water is forbidden for 4 hours; the weight of the mice is calculated according to the average 20 g/mouse, and the mice are administrated for 3 days for 1 time per day; the solvent control group was given 0.5% sodium carboxymethylcellulose +0.2% tween 80 solution, the volume and the frequency were the same as above.

(4) Mouse treatment: and (3) performing euthanasia 48 hours after the last administration, taking stomach tissues for carrying out isolated culture and identification on helicobacter pylori, and calculating the planting amount.

(3) Results

The results are shown in FIG. 5. As shown in FIG. 5, the omeprazole plus Napabucasin group showed significantly better eradication of H.pylori than the triple combination treatment group (omeprazole plus amoxicillin and clarithromycin) (P < 0.05).

Example 6 test of the killing Effect of Napabucasin against the multidrug-resistant helicobacter pylori BHKS159 Strain colonized in the stomach of mice

(1) Material

The strain was H.pylori mice acclimatized to the strain BHKS159, the mice were female C57BL/6 mice of 6 weeks of age of SPF class, otherwise as in the example 1 material.

(2) Method for producing a composite material

(1) In vivo model construction of H.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 obtained from Helicobacter pylori. Antibacterial Agents Chemother [ J]2019;63 And (6) e 00004-19). Taking 10% mouse stomach tissue to detect the colonization of helicobacter pylori, wherein the range of colonization amount is 1 multiplied by 10 ⁵ And (3) the planting is considered to be successful above CFU/g, the helicobacter pylori is planted in the detection results, and the model is successfully constructed.

(2) Grouping: the experimental group averagely divides the infection group successfully molded into 6 groups, namely two groups of omeprazole and Napabucasin (the dosage is 14mg/kg and 28mg/kg respectively) combination groups with different dosages, two groups of Napabucasin (the dosage is 14mg/kg and 28mg/kg respectively) groups with different dosages, an omeprazole and amoxicillin (the dosage is 28 mg/kg) group and a clarithromycin (the dosage is 14 mg/kg) group and a solvent control group, wherein each group comprises 10 animals; the amount of omeprazole administered in all groups was 138.2mg/kg, and 8 mice not infected with H.pylori were negative control groups.

(3) Administration: by adopting a gastric lavage administration method, omeprazole is administered 30 minutes before other medicines, and after administration, the omeprazole is fasted and the water is forbidden for 4 hours; administering 1 time daily for 3 days; the solvent control group was given 0.5% sodium carboxymethylcellulose +0.2% tween 80 solution, the volume and the frequency were the same as above.

(4) Mouse treatment: performing euthanasia 48 hours after the last administration, taking blood from the orbit, centrifuging to extract serum, and detecting the content of each inflammatory factor by an ELISA method; taking stomach tissue, carrying out isolated culture and identification on helicobacter pylori in half of the tissue, calculating the planting amount, and carrying out pathological section staining on the other half of the tissue. Mice were also weighed daily and the average body weight was calculated.

(3) Results

The combined group of omeprazole and Napabucasin (28 mg/kg) or the single drug group of Napabucasin (28 mg/kg) has obviously better killing effect on helicobacter pylori than the triple combination group of omeprazole, amoxicillin and clarithromycin, as shown in figure 6. The peripheral blood inflammatory factor expression level of mice in the omeprazole and Napabucasin combined group (28 mg/kg) or Napabucasin single drug group (28 mg/kg) is obviously reduced compared with that of the triple treatment group, as shown in figure 7, and the repair capacity of the two groups on mouse gastric mucosa injury is also obviously superior to that of the triple treatment group, as shown in figure 8.

2.Napabucasin against non-helicobacter pylori bacteria

Example 7 Activity assay of Napabucasin against non-helicobacter pylori bacteria in vitro

The antibacterial activity of napobucasin was determined by measuring its Minimal Inhibitory Concentration (MIC) against non-h.pylori bacteria.

(1) Material

(1) Drugs and reagents: napabucasin, metronidazole and vancomycin were purchased from MCE.

(2) The strain is as follows: staphylococcus aureus ATCC 25923 and USA300, staphylococcus hemolyticus ATCC29970, staphylococcus epidermidis ATCC12228, bacillus subtilis 168, bacillus cereus ATCC14579, listeria monocytogenes EGD-e, streptococcus pneumoniae ATCC49619, enterococcus faecalis ATCC29212, enterococcus faecium ATCC19434, clostridium difficile ATCC700057, propionibacterium acnes ATCC11827, porphyromonas gingivalis ATCC BAA-308, actinobacillus actinomycetemcomitans D7S-1, fusobacterium nucleatum ATCC 252511168, campylobacter jejuni NCTC11168, neisseria gonorrhoeae ATCC19424, haemophilus influenzae ATCC 66, moraxella catarrhalis ATCC25238, escherichia coli ATCC25922, pseudomonas aeruginosa PAO1, klebsiella pneumoniae ATCC35657, acinetobacter baumannii ATCC19606, salmonella enterica ATCC14028 are standard or model strains.

(3) Media and main reagents: MH culture solution, MH culture medium, brain-heart infusion culture solution, columbia culture medium and FCS.

(4) The main apparatus is as follows: a constant temperature incubator, an anaerobic incubator, an ultraviolet spectrophotometer, a constant temperature shaking table (Thermo), a centrifuge, an electronic balance, etc.

(2) Method for producing a composite material

The minimal inhibitory concentration of Napabucasin against non-H.pylori bacteria was determined by broth dilution. The detection medium and culture solution are MH medium and culture solution, and the concentration of the culture solution is about 1.0 × 10 ⁵ CFU/mL, the time for interpretation of results was selected to be 24 or 48 hours depending on strain differences, and the other was similar to the method in example 1.

(3) As a result, the

See table 2.

TABLE 2 minimum inhibitory concentrations (μ g/ml) of Napabucasin against non-H.pylori bacterial strains

Note: "-" indicates no detection.

As shown in the above table, napabucasin has growth inhibitory effects on 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, actinobacillus actinomycetemcomitans, fusobacterium nucleatum, campylobacter jejuni, neisseria gonorrhoeae, haemophilus influenzae and Moraxella catarrhalis.

3. Antifungal effect of Napabucasin

Example 8 in vitro antifungal Activity assay of Napabucasin

(1) Material

(1) Chemical product: napabucasin and Amphotericin (AMB) were purchased from MCE.

(2) The strain is as follows: aspergillus fumigatus Af293, trichophyton rubrum ATCC MYA-4438, candida albicans SC5314 and ATCC14053, candida parapsilosis ATCC22019, candida glabrata ATCC36583, candida krusei ATCC6258, candida viticola ATCC200950, candida tropicalis ATCC750, cryptococcus neoformans ATCC MYA-4906 and ATCC 90112, malassezia symptomata CBS7222, malassezia globosa CBS7966, malassezia furfur CBS1878 and Malassezia pachydermans CBS1879 are standard or model strains. Other clinical strains were isolated and identified from clinical specimens at the first hospital affiliated with the university of medical, nanjing, and the second affiliated Yifu hospital.

(3) Media and main reagents: RPMI1640 culture medium, saxabeu medium, FCS, dimethyl sulfoxide (DMSO).

(2) The method comprises the following steps: the minimum inhibitory concentration (MIC, 100. Mu.L system) of Napabucasin against various fungal strains was determined by broth dilution.

(1) Stock solutions of Napabucasin, amphotericin B and fluconazole were prepared at 6.4mg/mL, respectively, and the solvents were 100% DMSO.

(2) Preparing bacterial liquid: preparing Candida albicans growing in logarithmic phase on solid plate into bacterial suspension with RPMI1640 culture solution, and adjusting concentration OD ₅₃₀ Is 0.5 (about 1X 10) ⁶ CFU/mL), diluted 1000-fold, about 1X 10 ³ CFU/mL, spare. Aspergillus fumigatus, trichophyton trichophyton and Malassezia (with specific culture medium added with olive oil) need to be subjected to micro-counting to adjust the concentration of the bacterial liquid; and the neogenesis cryptococcus adjusts bacterial liquid OD ₅₃₀ After 0.5, diluted 10000 times for standby.

(3) Preparation of 96-well plate: adding 178 mu L of RPMI1640 culture solution into the first hole, adding 2 mu L of Napabucasin storage solution, and diluting to the 12 th hole by adopting a double dilution method; in the other two rows of wells, amphotericin B and fluconazole were added in the same manner.

(4) Inoculating bacterial liquid: adding 10 μ L of the above-mentioned bacterial suspension to each well (the bacterial concentration per well is about 1.0 × 10) ² CFU/mL), the drug concentration is 64 μ g/mL, 32 μ g/mL, 16 μ g/mL, 8 μ g/mL, 4 μ g/mL, 2 μ g/mL, 1 μ g/mL, 0.5 μ g/mL, 0.25 μ g/mL, 0.125 μ g/mL, 0.063 μ g/mL, and 0.031 μ g/mL in that order. The other row is added with 100 mu LRPMI1640 culture solution for each well as a sterile control group; in another row, 90. Mu.L of RPMI1640 culture medium and 10. Mu.L of the above-mentioned stock solutions were added to each well, respectively, to prepare a positive control group. Placing the mixture in a constant temperature incubator for cultivation.

(5) And (5) judging a result: the results were interpreted after 24 or 48 hours of incubation, with the lowest drug concentration that completely inhibited fungal growth in the wells being the MIC. The test is only significant when there is significant growth of the fungus in the positive control wells (i.e., no drug) and when the sterile control group is aseptically grown. The experiment was repeated 3 times.

(3) Results

The results are shown in Table 3.

TABLE 3 minimum inhibitory concentration of Napabucasin against fungal strains (. Mu.g/ml)

Note: "-" indicates no detection.

As shown in the above table, napabucasin has the effect of inhibiting the growth of fungi such as Candida albicans, candida parapsilosis, candida glabrata, candida krusei, candida viticola, candida tropicalis, cryptococcus neoformans, aspergillus fumigatus, trichophyton rubrum, malassezia and the like, and the inhibiting activity of the Napabucasin is equivalent to that of amphotericin B.

Example 9 killing of Candida albicans SC5314 Strain in a mouse model of systemic infection by Napabucasin

(1) Material

The strain was Candida albicans SC5314, the mice were SPF grade 8 week old C57BL/6 female mice, and the drug fluconazole was purchased from MCE corporation, otherwise as in the example 8 material.

(2) Method of producing a composite material

(1) Animal(s) productionMolding: culturing Candida albicans SC5314 strain in agar culture medium at 35 deg.C for 24 hr, picking colony to RPMI1640 culture medium, and adjusting bacterial liquid concentration to about 5 × 10 ⁶ CFU/mL, after centrifugation to collect the cells, using an equal volume of PBS heavy suspension. After 125 mul of bacterial liquid is injected into the inner canthus vein plexus of the mouse, candida albicans diffuses in the body of the mouse, causing the whole body infection of the mouse.

(2) Grouping: infected mice successfully molded were divided into 3 groups on average, namely a solvent control group (0.5% sodium carboxymethylcellulose +0.2% tween 80), a fluconazole-treated group and a napobucasin-treated group, each of which contained 10 mice.

(3) Administration: the experimental groups were administered intragastrically, on the day of infection, at the 1 st dose 2 hours after infection, with the 2 nd dose being 10 hours apart. The administration was performed 2 times daily for 3 consecutive days. Mouse body weight was calculated as 20 g/mouse on average. The dose of fluconazole and Napabucasin is 56 mg/kg/day, and the solvent is administered to the control group in the same volume and frequency as above.

(4) Mouse treatment: after 24 hours of the last administration, euthanasia was performed, spleen and kidney tissues were taken, candida albicans isolated culture and identification were performed, and the amount of permanent planting was calculated.

(3) Results

Both Napabucasin and fluconazole can significantly reduce the fungal burden (P < 0.01) in spleen and kidney of mice systemically infected with Candida albicans, and the effect of the fluconazole treated group was comparable to that of the Napabucasin treated group, as shown in FIG. 9.

Claims

The application of Napabucasin in preparing a medicine for resisting helicobacter pylori infection, wherein the Napabucasin compound has the following structure: