Pharmaceutical application of P2Y11 specific inhibitor and composition containing P2Y11 specific inhibitor

Abstract

The invention discloses pharmaceutical application of a P2Y11 specific inhibitor and a composition containing the P2Y11 specific inhibitor, and belongs to the technical field of biological medicine. The invention discloses application of NF157 in preparation of drugs related to treatment of HD5-promoted pathogenic infection for the first time. In the aspect of pathogen infection treatment, NF157 can specifically inhibit combination of P2Y11 and HD5, so that the effect that HD5 regulates cytoskeleton rearrangement to promote filamentous pseudopod generation is eliminated, and the NF157 becomes a key link for cutting off pathogen infection. By preventing the cells from generating filamentous pseudopod, the NF157 effectively blocks the capturing path of bacteria, and has a remarkable intervention effect on part of pathogens which are lack of adhesion devices and flagellum and are invaded by relying on cell pseudopod capturing.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to pharmaceutical applications of P2Y11 specific inhibitors and compositions containing the same. Background Art

[0002] Human α-defensin 5 (HD5) belongs to the category of intestinal defensins and has multi-level immune activity. It is not only a physical barrier of the intestinal epithelium that separates the intestinal flora from the immune system cells, but also a chemical barrier that plays a key role in maintaining intestinal homeostasis and microbial flora. However, under certain physiological conditions, HD5 can enhance the ability of pathogens to infect and invade the host. The human purinergic receptor P2Y11 has been identified as the binding receptor of HD5. This receptor family is known for its natural ligands such as ADP and ATP, and is widely expressed in various tissues of the human body, especially in human intestinal epithelial tissues. After HD5 binds to the cell surface P2Y11 receptor, it can regulate the rearrangement of the cytoskeleton, promote the production of a large number of filopodia, and then capture bacteria, achieving highly specific and efficient infection of the human intestine.

[0003] Current treatments for pathogens include antibiotics and antiviral drugs, but these methods lack the specificity to target the specific mechanism by which HD5 promotes pathogen infection. In addition, traditional anti-infective drugs usually have multiple targets and lack specificity, making it difficult to achieve optimal results in the process of HD5 regulating skeleton rearrangement to capture pathogens.

[0004] The P2Y11 specific inhibitor NF157 is a highly selective P2Y11 antagonist with a pKi value of 7.35. As a specific inhibitor of the P2Y11 receptor, NF157 has a significant inhibitory effect on P2Y11, while its inhibitory effect on P2Y1 and P2Y2 is weaker. Currently, there are no reports on the relationship between NF157 and HD5. Summary of the invention

[0005] In order to overcome the above-mentioned disadvantages of the prior art, the object of the present invention is to provide pharmaceutical applications of a P2Y11 specific inhibitor and a composition containing the same.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions: The present invention discloses the use of a P2Y11 specific inhibitor in the preparation of a drug for treating, inhibiting or alleviating pathogen infection in an individual suffering from pathogen infection or at risk of developing / acquiring pathogen infection.

[0007] The "pathogenic infection" described in the present invention refers to an infectious disease caused by an organism being infected with certain pathogenic microorganisms, which mainly include bacteria, fungi and viruses.

[0008] Among them, common bacteria include hemolytic streptococci, Staphylococcus aureus, Streptococcus pneumoniae, Klebsiella pneumoniae, Mycobacterium tuberculosis, Salmonella typhi, Bacillus anthracis, Shigella, etc. Bacterial infection can lead to a series of diseases such as colds, otitis media, tracheitis, bronchitis, pneumonia, tuberculosis, typhoid fever, and bacillary dysentery.

[0009] The "cell surface P2Y11 receptor" described in the present invention is a purinergic receptor, a member of the P2Y family. The P2Y11 receptor is widely expressed in a variety of tissues and cells, including the spleen, lymph nodes and 25 other tissues. The P2Y11 receptor plays an important role in cell signal transduction and is involved in regulating a variety of physiological and pathological processes.

[0010] Preferably, the P2Y11 specific inhibitor is NF157.

[0011] Further preferably, NF157 can block the binding of HD5 to the P2Y11 receptor and reduce the infection ability of pathogenic microorganisms under the action of HD5.

[0012] Preferably, the pathogen is a pathogenic microorganism that relies on pseudopodia for capture and invasion.

[0013] It should be noted that the pseudopodia described in the present invention refers to "HD5-promoted pseudopodia", which can capture pathogenic microorganisms that lack adhesion devices or flagella. In other words, this type of pathogenic microorganisms relies on pseudopodia extended by cells to capture them and invade cells.

[0014] Further preferably, the pathogenic microorganism is Shigella.

[0015] The present invention also discloses the use of NF157 as a P2Y11 specific inhibitor in the preparation of medicines for treating diseases related to pathogenic infection.

[0016] Preferably, NF157 can block the binding of HD5 to the P2Y11 receptor and reduce the infection ability of pathogens under the action of HD5.

[0017] Preferably, the diseases include conjunctivitis, gastroenteritis and bacillary dysentery.

[0018] The present invention also discloses a pharmaceutical composition for treating diseases related to pathogenic bacteria infection, comprising NF157 and a pharmaceutically acceptable carrier or excipient, wherein the diseases include conjunctivitis, gastroenteritis and bacillary dysentery.

[0019] Preferably, NF157 is capable of blocking the binding of HD5 to the P2Y11 receptor.

[0020] Compared with the prior art, the present invention has the following beneficial effects: The present invention discloses for the first time the new use of the P2Y11 receptor inhibitor NF157. Using NF157 in the preparation of drugs related to the treatment of HD5-promoted pathogenic infections is a new use. In terms of responding to pathogenic infections, the use of NF157 is an innovative solution revealed for the first time by the present invention. It can specifically inhibit the binding of P2Y11 and HD5, thereby eliminating the role of HD5 in regulating cytoskeleton rearrangement and promoting the generation of filopodia, becoming a key link in cutting off pathogenic infections. By preventing cells from generating filopodia, NF157 effectively blocks the bacterial capture pathway, and has a significant intervention effect on some pathogens that lack adhesion devices and flagella and rely on cell pseudopodia capture for invasion.

[0021] The present invention is verified by in vitro cell experiments, intestinal microfluidic chips and guinea pig conjunctival infection experiments. In the presence of HD5, NF157 can significantly inhibit its effect on the generation of cell filopodia, thereby successfully inhibiting the invasion of Shigella to host cells. Compared with the prior art, NF157 has the following significant advantages: First, high targeting. NF157 can specifically target the interaction between HD5 and the P2Y11 receptor, can accurately intervene in the specific mechanism of pathogen infection, and greatly reduce the interference with other normal physiological functions. Second, specific inhibition. As a specific inhibitor of P2Y11, NF157 targets a specific infection mechanism, thus providing a new application path for NF157, that is, a new pharmaceutical use for specific pathogenic infections, which can provide new ideas and methods for conquering pathogenic infection research. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The effect of NF157 on the efficiency of Shigella invasion into HeLa cells; Figure 2 For fluorescence microscopy analysis of the effect of NF157 on Sf301 TSAR activation; Figure 3 For the application of NF157 in the intestinal microfluidic chip infection model; Figure 4 Application of NF157 in an in vivo guinea pig conjunctivitis infection model. DETAILED DESCRIPTION

[0023] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0024] It should be noted that the terms "first", "second", etc. in the specification and claims of the present invention and the above-mentioned drawings are used to distinguish similar objects, and are not necessarily used to describe a specific order or sequence. It should be understood that the data used in this way can be interchanged where appropriate, so that the embodiments of the present invention described herein can be implemented in an order other than those illustrated or described herein. In addition, the terms "including" and "having" and any variations thereof are intended to cover non-exclusive inclusions, for example, a process, method, system, product or device that includes a series of steps or units is not necessarily limited to those steps or units that are clearly listed, but may include other steps or units that are not clearly listed or inherent to these processes, methods, products or devices.

[0025] The present invention is further described in detail below in conjunction with the accompanying drawings: The present invention relates to a new use of the drug NF157, which aims to provide a new strategy to combat pathogenic infections promoted by human intestinal alpha defensin 5 (HD5). NF157 is a specific P2Y11 receptor inhibitor with high selectivity. HD5 triggers the reorganization of the cytoskeleton by interacting with the P2Y11 receptor, thereby prompting the cell to form pseudopodia to capture bacteria, thereby promoting the invasion of pathogens.

[0026] The present invention studies the efficacy of NF157 under different conditions, and confirms in multiple experimental models: in vitro cell culture infection model, intestinal microfluidic chip infection model, guinea pig conjunctivitis infection model, that NF157 can block the binding of HD5 to P2Y11 receptor, and effectively reduce the infection ability of pathogens under the action of HD5. Therefore, NF157 provides a new drug use for the preparation of drugs for treating HD5-mediated pathogenic infections, that is, it opens up a new direction for drug research and development in related fields. Through this strategy, the invasion of pathogens can be reduced, and more effective therapeutic drugs can be provided to patients.

[0027] The "intestinal microfluidic chip technology" described in the following experiment of the present invention is a new experimental platform that combines microfluidic technology and intestinal physiology research. Based on the design principle of microfluidic chips, it controls the flow of fluids and the exchange of substances through microscale channels to simulate the complex environment in the intestine. This technology uses micromachining technology to make chips with tiny channels. The channel size is usually between tens and hundreds of microns, which can accommodate intestinal cells, microorganisms and related biomolecules, providing new methods and ideas for intestinal disease research, drug screening and nutrition research.

[0028] 1. Anti-bacterial infection effect of NF157 in in vitro cell culture model 1) Cell culture and treatment HeLa cells (adherent cells) were cultured in DMEM low-glucose medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin double antibiotics at 37°C and 5% carbon dioxide. 5 Cells / well were seeded in 24-well plates, and the culture conditions were the same as above. When cells were starved, DMEM low-glucose medium without serum and double antibody was used and cultured in a 37°C and 5% carbon dioxide incubator.

[0029] Three replicates were set for each experimental group. Cells were first pretreated with 20 μM NF157 (purchased from Tocris Bioscience Cat#: 2450) for 30 minutes, and then treated with HD5 for 15 minutes. At the same time, Sf301 bacteria (1×10 6 CFU). The HD5 and bacterial co-culture plates were centrifuged at 500 g for 10 minutes.

[0030] The invasion assay was performed as follows: After incubation of bacteria and cells for 40 minutes, the cells were treated with a medium containing 100 μg / mL gentamicin for 20 minutes and then washed twice. For immunofluorescence samples, the cells were fixed with 4% paraformaldehyde for 20 minutes, perforated with 0.1% Triton X-100 for 5 minutes, and washed three times with PBS. Next, the cells were stained with phalloidin TRITC (10 μg / mL) in the dark for 30 minutes, then sealed with a DAPI-containing sealing medium, and baked in a 37°C oven for 30 minutes and stored at 4°C. For colony counting, the cells were lysed with 1 mL of sterile water for 20 minutes, and repeatedly blown during the lysis process. After diluting the lysate with PBS, 10 μL was aspirated for spotting, placed in a 37°C bacterial incubator for about 9 hours, and colony counting was performed. For adhesion experiments, samples were prepared directly after bacterial cell co-culture.

[0031] 2) Data analysis: Bacterial adhesion was defined as the total number of bacteria adhering to the cells, expressed as a percentage of the input bacterial load; bacterial invasion was defined as the total number of bacteria within the cells, also expressed as a percentage of the input bacterial load. Bacterial invasion efficiency was calculated as the number of bacterial invasions divided by the number of bacterial adhesions (results are shown in Table 2). Figure 1 as shown).

[0032] Through cell immunofluorescence images, the number of bacteria invading cells in a field of view (Sf301 invades into the cell, the type III secretion system is activated, and the bacteria appear green) and the total number of bacteria (Sf301 appears red) are calculated, and the ability of bacterial infection and invasion is judged by the ratio of green to red. The colony counting results showed that in the presence of HD5, NF157 significantly reduced the invasion / adhesion ratio of Shigella that had already adhered. The activation of T3SS (Type ⅢSecretion System) was observed using a laser confocal microscope, and it was found that NF157 significantly reduced the green / red ratio of TSAR-containing bacteria adhered to HeLa cells. (The results are shown in Figure 2 shown) 2. Anti-bacterial infection effect of NF157 in intestinal microfluidic chip model In order to simulate the in vivo intestinal environment in vitro, the present invention uses intestinal microfluidic chip technology, which can accurately reproduce the microvilli structure and barrier function of the intestine. First, a three-dimensional model of the intestine was constructed using a microfluidic chip, which can accurately simulate the microstructure of intestinal epithelial cells. Subsequently, infection experiments were carried out using Shigella in the logarithmic growth phase to simulate the process of Shigella infection in vivo, thereby providing a highly simulated in vitro platform for studying the pathogenic mechanism of Shigella and evaluating the effect of drugs.

[0033] The microfluidic chip device is made of PDMS (polydimethylsiloxane) and is divided into two upper and lower chambers separated by a porous polyester membrane. The pore size of the porous polyester membrane is 0.4 μm and the pore density is 4×10 6 Hole / cm 2, the membrane thickness is 10 μm. The volume of the top chamber is about 1 μL (dimensions are 10 mm×1 mm×100 μm), and the volume of the bottom chamber is about 2.5 μL (dimensions are 10 mm×1 mm×250 μm). First, the device was cleaned and sterilized with 70% ethanol, then rinsed with sterile water, injected with air to remove residual liquid, and exposed to ultraviolet light for 30 minutes for sterilization. Next, the chip was coated with 10 μg / mL poly-L-lysine, incubated at 37 °C for 30 minutes, and washed three times with PBS. After that, the chip was coated with 50 μg / mL bovine plasma fibronectin (Fibronectin) and incubated at 37 °C for 30 minutes. Finally, a mixture of 50 μg / mL Matrigel and 100 μg / mL type I collagen was coated at 37 °C for 30 minutes.

[0034] CACO-2 cells were collected at 8×10 6 The cells were inoculated in the upper chamber at a concentration of 10 cells / mL and left to stand for 4 hours to promote adhesion. After adhesion, the culture medium was perfused at a rate of 20 µL / h for 3 days. In the Shigella infection cell experiment, the cells were first pretreated with 20 μM NF157 for 30 minutes, and then replaced with a culture medium containing 4 μM HD5 for 15 minutes. Next, Shigella pretreated with HD5 was added to the intestinal chip at an MOI=1 and infected at a flow rate of 30 μL / h for 2 hours. Finally, 100 μg / L gentamicin was used at a flow rate of 30 μL / h for 30 minutes to terminate the infection. After the infection, the cells were fixed with 4% paraformaldehyde for 20 minutes, perforated with 0.1% Triton X-100 for 5 minutes, and washed 3 times with PBS. After that, the cells were stained with phalloidin TRITC (10 μg / mL) in the dark for 30 minutes, and then sealed with a sealing medium containing DAPI.

[0035] Photograph samples of intestinal microfluidic chip infection. The laser confocal microscope uses the Z-axis layer scanning function to obtain the 3D structure of the intestine formed in the intestinal microfluidic chip and observe images at different depths. The Z-axis selects 1 μm for layer scanning. The height of the intestinal structure formed in the chip is about 30 μm. The immunofluorescence image is processed in ImageJ. In ImgaeJ, merge the three channels of the image (green is the bacterial channel, red is the cytoskeleton channel, and blue is the nucleus channel), set the Voxel depth to 1 μm in Image>Properties, and then select the desired area in Image>Stacks>orthogonalviews. The result is as follows: Figure 3As shown, the results showed that most Shigella gathered and invaded the intestinal crypt area. In the presence of NF157, the invasion of bacteria by HD5 was effectively reduced, and the bacterial clusters and the number of bacteria gathered in the intestinal crypts were significantly reduced.

[0036] 3. Effect of NF157 on antibacterial infection in guinea pig conjunctivitis model in vivo The guinea pig conjunctivitis model is a method for detecting bacterial invasion and has been widely used to distinguish invasive Escherichia coli from non-invasive Escherichia coli. In the present invention, SPF-grade female Dunkin Hartley guinea pigs aged 6 to 8 weeks and weighing about 250 g were selected. The guinea pigs were randomly divided into four groups, each with 10, namely the control group, HD5 group, NF157 group, and NF157+HD5 group.

[0037] First, 10 μL of DMEM culture medium containing 20 μM NF157 was dripped into the eyes of guinea pigs in the NF157 group and the NF157+HD5 group for 30 minutes. Then, 4 μM HD5 was added to the eyes of Shigella spp. (1×10 6 CFU / each eye) and the mixture was dripped into the eyes of guinea pigs in the HD5 group and the NF157+HD5 group. The control group and the NF157 group were dripped with the same amount of DMEM medium containing only Shigella. For three consecutive days, the eyes of the two groups of guinea pigs treated with NF157 were dripped with DMEM culture medium containing 20 μM NF157.

[0038] Over the next three days, the degree of inflammation of the guinea pig conjunctiva was observed and recorded, and graded according to the following criteria: Grade 0 means no disease or only mild irritation; Grade 1 means mild conjunctivitis; Grade 2 means moderate non-purulent conjunctivitis; Grade 3 means severe purulent conjunctivitis. The results showed that NF157 significantly reduced the symptoms of conjunctivitis caused by HD5-enhanced infection at all observation time points. Through this model, the therapeutic effect of NF157 on HD5-enhanced Shigella infection can be evaluated, providing valuable data for further drug development and research.

[0039] In summary, the present invention discloses a new use of the drug NF157, which is used as a specific P2Y11 receptor inhibitor for treating pathogenic infections promoted by human intestinal alpha defensin 5 (HD5). Through a series of in vitro and in vivo experimental models, the present invention confirms that NF157 can block the binding of HD5 to the P2Y11 receptor, thereby effectively reducing the infectious ability of pathogens under the action of HD5.

[0040] In an in vitro cell culture model, NF157 showed significant anti-bacterial infection effects and was able to reduce the invasion of Shigella on HeLa cells. In addition, through the intestinal microfluidic chip model, the present invention simulated the in vivo intestinal environment and proved that NF157 had an inhibitory effect on Shigella invasion in the simulated intestinal microenvironment, reducing the aggregation and invasion of bacteria in the intestinal crypt area.

[0041] In the in vivo guinea pig conjunctivitis model, NF157 also showed therapeutic effects against Shigella infection, significantly alleviating the conjunctivitis symptoms caused by HD5-enhanced infection. These results suggest that NF157 not only provides a new therapeutic strategy for treating HD5-mediated pathogenic infections, but also opens up a new direction for drug development in related fields.

[0042] The above contents are only for explaining the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.

Claims

1. Use of a P2Y11-specific inhibitor in the preparation of a medicament for treating, inhibiting or alleviating a pathogen infection in an individual suffering from a pathogen infection or at risk of developing / acquiring a pathogen infection.

2. The use according to claim 1, characterized in that The P2Y11 specific inhibitor is NF157.

3. The use according to claim 2, characterized in that NF157 can block the binding of HD5 to P2Y11 receptor and reduce the infection ability of pathogenic microorganisms under the action of HD5.

4. The use according to any one of claims 1 to 3, characterized in that: The pathogen is a pathogenic microorganism that relies on pseudopodia to capture and invade.

5. The use according to claim 4, characterized in that The pathogenic microorganism is Shigella.

6. Use of NF157 as a P2Y11 specific inhibitor in the preparation of drugs for the treatment of diseases related to pathogenic infections.

7. The use according to claim 5, characterized in that NF157 can block the binding of HD5 to P2Y11 receptor and reduce the infection ability of pathogens under the action of HD5.

8. The use according to claim 5, characterized in that Such diseases include conjunctivitis, gastroenteritis and bacillary dysentery.

9. A pharmaceutical composition for treating diseases related to pathogenic bacteria infection, characterized in that: The invention comprises NF157 and a pharmaceutically acceptable carrier or excipient, wherein the diseases include conjunctivitis, gastroenteritis and bacillary dysentery.

10. The pharmaceutical composition for treating diseases related to pathogenic bacteria infection according to claim 9, characterized in that: NF157 can block the binding of HD5 to the P2Y11 receptor.

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