Pharmaceutical applications of P2Y11-specific inhibitors and compositions containing them

Abstract

This invention discloses the pharmaceutical applications of a P2Y11-specific inhibitor and compositions containing it, belonging to the field of biomedical technology. This invention discloses for the first time the use of NF157 in the preparation of drugs related to the treatment of HD5-induced pathogen infection. In combating pathogen infection, NF157 can specifically inhibit the binding of P2Y11 to HD5, thereby eliminating the role of HD5 in regulating cytoskeleton rearrangement and promoting filopodia formation, thus becoming a key link in interrupting pathogen infection. By preventing the formation of filopodia, NF157 effectively blocks the bacterial capture pathway, showing significant intervention effects on some pathogens that lack adhesion devices and flagella and rely on cellular pseudopodia capture for invasion.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the pharmaceutical application of P2Y11 specific inhibitors and compositions containing them. Background Technology

[0002] Human enteric defensin 5 (HD5) belongs to the intestinal defensins family and possesses multi-layered immune activity. It serves as both a physical barrier of the intestinal epithelium separating the gut microbiota from immune system cells and a crucial chemical barrier for maintaining intestinal homeostasis and the microbial community. However, under specific 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 for HD5. This receptor family is known for its natural ligands, such as ADP and ATP, and is widely expressed in various human tissues, especially abundant in human intestinal epithelial tissue. After HD5 binds to the cell surface P2Y11 receptor, it regulates cytoskeleton rearrangement, promoting the production of numerous filopodia, thereby capturing bacteria and achieving highly specific and efficient infection of the human gut.

[0003] Current treatments targeting pathogens include antibiotics and antiviral drugs, but these methods lack specificity regarding the particular mechanism by which HD5 promotes pathogen infection. Furthermore, traditional anti-infective drugs typically have multiple targets and lack specificity, making it difficult to achieve optimal results in addressing HD5's regulatory framework rearrangement to capture pathogens.

[0004] NF157, a highly selective P2Y11 antagonist, has a pKi value of 7.35. As a specific inhibitor of the P2Y11 receptor, NF157 exhibits significant inhibitory effects on P2Y11, while its inhibitory effects on P2Y1 and P2Y2 are weaker. Currently, there are no reported studies on the interaction between NF157 and HD5. Summary of the Invention

[0005] In order to overcome the shortcomings of the prior art, the present invention aims to provide pharmaceutical applications of P2Y11 specific inhibitors and compositions comprising them.

[0006] To achieve the above objectives, the present invention employs the following technical solution:

[0007] This invention discloses the use of P2Y11 specific inhibitors in the preparation of medicaments for treating, inhibiting or reducing pathogenic infections in individuals suffering from or at risk of developing / acquiring pathogenic infections.

[0008] The "pathogenic infection" described in this invention refers to an infectious disease caused by the body being infected with a certain pathogenic microorganism, which mainly includes bacteria, fungi and viruses.

[0009] Common bacteria include hemolytic streptococci, Staphylococcus aureus, Streptococcus pneumoniae, Klebsiella pneumoniae, Mycobacterium tuberculosis, Salmonella typhi, Bacillus anthracis, and Shigella. Bacterial infections can lead to a range of diseases such as colds, otitis media, tracheitis, bronchitis, pneumonia, tuberculosis, typhoid fever, and bacterial dysentery.

[0010] The "cell surface P2Y11 receptor" described in this invention is a purinergic receptor belonging to the P2Y family. The P2Y11 receptor is widely expressed in various tissues and cells, including the spleen, lymph nodes, and 25 other tissues. The P2Y11 receptor plays a crucial role in cell signaling, participating in the regulation of various physiological and pathological processes.

[0011] Preferably, the P2Y11 specific inhibitor is NF157.

[0012] More preferably, NF157 can block the binding of HD5 to the P2Y11 receptor and reduce the infectivity of pathogenic microorganisms under the action of HD5.

[0013] Preferably, the pathogen is a pathogenic microorganism that relies on pseudopodia to capture and invade.

[0014] It should be noted that the pseudopodia mentioned in this invention refer to "HD5 cell-extending pseudopodia," which are capable of grasping pathogenic microorganisms that lack their own adhesion mechanisms or flagella. In other words, these types of pathogenic microorganisms rely on cell-extending pseudopodia to capture them and thus invade the cell.

[0015] More preferably, the pathogenic microorganism is Shigella.

[0016] This invention also discloses the application of NF157 as a specific inhibitor of P2Y11 in the preparation of drugs for treating pathogen-related diseases.

[0017] Preferably, NF157 can block the binding of HD5 to the P2Y11 receptor and reduce the infectivity of pathogens under the action of HD5.

[0018] Preferably, the diseases include conjunctivitis, gastroenteritis, and bacterial dysentery.

[0019] The present invention also discloses a pharmaceutical composition for treating diseases related to pathogenic bacterial infections, comprising NF157 and a pharmaceutically acceptable carrier or excipient, said diseases including conjunctivitis, gastroenteritis and bacterial dysentery.

[0020] Preferably, NF157 can block the binding of HD5 to the P2Y11 receptor.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] This invention discloses for the first time a novel application of the P2Y11 receptor inhibitor NF157: its use in the preparation of drugs related to the treatment of HD5-induced pathogen infection, representing a completely new application. In the response to pathogen infection, the use of NF157 is an innovative approach revealed for the first time in this invention. It specifically inhibits the binding of P2Y11 to HD5, thereby eliminating the role of HD5 in regulating cytoskeleton rearrangement and promoting filopodia formation, thus becoming a key link in interrupting pathogen infection. By preventing the formation of filopodia, NF157 effectively blocks the bacterial capture pathway, showing significant intervention effects on some pathogens that lack adhesion devices and flagella and rely on cellular pseudopodia capture for invasion.

[0023] This invention was validated through in vitro cell experiments, intestinal microfluidic chip experiments, and guinea pig conjunctival infection experiments. In the presence of HD5, NF157 significantly inhibited its effect on the formation of filamentous pseudopodia, thereby successfully inhibiting Shigella invasion of host cells. Compared with existing technologies, NF157 has the following significant advantages: First, high targeting. NF157 specifically targets the interaction between HD5 and the P2Y11 receptor, enabling precise intervention in specific mechanisms of pathogen infection and greatly reducing interference with other normal physiological functions. Second, specific inhibition. As a specific inhibitor of P2Y11, NF157 targets a specific infection mechanism, thus providing a novel application for NF157—a new pharmaceutical use targeting specific pathogen infections—offering new ideas and methods for tackling pathogen infection research. Attached Figure Description

[0024] Figure 1 The effect of NF157 on the efficiency of Shigella invasion of HeLa cells;

[0025] Figure 2 To analyze the effect of NF157 on Sf301 TSAR activation using fluorescence microscopy;

[0026] Figure 3 Application of NF157 in an intestinal microfluidic chip infection model;

[0027] Figure 4 Application in the in vivo guinea pig conjunctivitis infection model of NF157. Detailed Implementation

[0028] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0029] It should be noted that the terms "first," "second," etc., in the specification, claims, and accompanying drawings of this invention are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that such data can be interchanged where appropriate so that the embodiments of the invention described herein can be implemented in orders other than those illustrated or described herein. Furthermore, the terms "comprising" and "having," and any variations thereof, are intended to cover a non-exclusive inclusion; for example, a process, method, system, product, or apparatus that comprises a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to such processes, methods, products, or apparatus.

[0030] The present invention will now be described in further detail with reference to the accompanying drawings:

[0031] This invention relates to novel uses of the drug NF157, aiming to provide a new strategy against pathogenic infections promoted by human intestinal alpha-defensin 5 (HD5). NF157 is a specific, highly selective P2Y11 receptor inhibitor. HD5, through its interaction with the P2Y11 receptor, triggers cytoskeleton remodeling, which in turn promotes the formation of pseudostools to capture bacteria, thereby facilitating pathogen invasion.

[0032] This invention, through studying the efficacy of NF157 under different conditions, demonstrated in various experimental models—including an in vitro cell culture infection model, an intestinal microfluidic chip infection model, and a guinea pig conjunctivitis infection model—that NF157 can block the binding of HD5 to the P2Y11 receptor, effectively reducing the infectivity of pathogens under the influence of HD5. Therefore, NF157 provides a new pharmaceutical use for the preparation of drugs to treat HD5-mediated pathogenic infections, opening a new direction for drug development in related fields. This strategy can reduce pathogen invasion and provide patients with more effective treatments.

[0033] The "intestinal microfluidic chip technology" described in the following experiments of this invention is a novel experimental platform combining microfluidic technology and intestinal physiology research. Based on the design principles of microfluidic chips, it controls fluid flow and substance exchange through microscale channels to simulate the complex environment within the intestine. This technology utilizes microfabrication techniques to create chips with microchannels, typically ranging in size from tens to hundreds of micrometers, capable of accommodating intestinal cells, microorganisms, and related biomolecules. This provides new methods and approaches for intestinal disease research, drug screening, and nutritional research.

[0034] 1. Antibacterial infection effect of NF157 in in vitro cell culture models

[0035] 1) Cell culture and processing

[0036] HeLa cells (adherent cells) were cultured in DMEM low-glucose medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin dual antibiotics at 37 °C and 5% CO2. For cell seeding, 1 × 10⁶ cells were cultured... 5 Cells were seeded per well in 24-well plates under the same conditions as described above. For cell starvation treatment, DMEM low-glucose medium without serum and antibiotics was used, and the cells were cultured at 37°C in a 5% CO2 incubator.

[0037] Each experimental group was set up in triplicate. Cells were first pretreated with 20 μM NF157 (purchased from Tocris Bioscience Cat#:2450) for 30 minutes, followed by treatment with HD5 for 15 minutes. Simultaneously, Sf301 bacteria in logarithmic growth phase (1×10⁻⁶ cells / year) were added. 6 CFU). Centrifuge the well plates containing the HD5 and bacterial co-culture system at 500 g for 10 minutes.

[0038] The invasion assay was performed as follows: After co-incubating bacteria and cells for 40 minutes, the cells were treated with a culture medium containing 100 μg / mL gentamicin for 20 minutes, followed by two washes. For immunofluorescence sample preparation, the cells were fixed with 4% paraformaldehyde for 20 minutes, punched with 0.1% Triton X-100 for 5 minutes, and washed three times with PBS. Next, the cells were stained with TRITC (10 μg / mL) in the dark for 30 minutes, mounted with DAPI-containing mounting medium, and baked at 37 ℃ for 30 minutes, then stored at 4 ℃. For colony counting, the cells were lysed with 1 mL of sterile water for 20 minutes, with repeated pipetting during the lysis process. After diluting the lysis buffer with PBS, 10 μL was spotted onto a plate and incubated at 37 ℃ for approximately 9 hours before colony counting. For adhesion assays, samples were prepared directly after bacterial cell co-culture.

[0039] 2) Data Analysis:

[0040] Bacterial adhesion is defined as the total number of bacteria adhering to a cell, expressed as a percentage of the input bacterial count; bacterial invasion is defined as the total number of bacteria within the cell, also expressed as a percentage of the input bacterial count. Bacterial invasion efficiency is calculated by dividing the number of invading bacteria by the number of adhering bacteria (results are shown in the image). Figure 1 (As shown).

[0041] Using immunofluorescence imaging, the number of bacteria invading cells in a single field of view (Sf301 invades the cell, Type III Secretion System is activated, bacteria appear green) and the total number of bacteria (Sf301 appears red) were calculated. The ratio of green to red was used to determine the bacterial infection and invasion capabilities. Colony counting results showed that in the presence of HD5, NF157 significantly reduced the invasion / adhesion ratio of already adhered Shigella. Laser confocal microscopy was used to observe the activation of the T3SS (Type III Secretion System), revealing that NF157 significantly reduced the green / red ratio of TSAR-containing bacteria adhering to HeLa cells. (Results are shown below) Figure 2 (As shown)

[0042] 2. Antibacterial infection effect of NF157 in intestinal microfluidic chip model

[0043] To simulate the in vivo intestinal environment in vitro, this invention employs 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, precisely simulating the microscopic structure of intestinal epithelial cells. Subsequently, infection experiments were conducted using Shigella bacteria in their logarithmic growth phase to simulate the in vivo infection process, thus providing a highly simulated in vitro platform for studying the pathogenic mechanisms of Shigella and evaluating drug efficacy.

[0044] The microfluidic chip device is made of PDMS (polydimethylsiloxane) and consists of upper and lower chambers separated by a porous polyester membrane. The porous polyester membrane has a pore size of 0.4 μm and a pore density of 4 × 10⁻⁶. 6 Hole / cm 2The membrane thickness was 10 μm. The top cavity had a volume of approximately 1 μL (dimensions: 10 mm × 1 mm × 100 μm), and the bottom cavity had a volume of approximately 2.5 μL (dimensions: 10 mm × 1 mm × 250 μm). First, the device was cleaned and sterilized with 70% ethanol, then rinsed with sterile water, air was injected to remove residual liquid, and the device was exposed to UV sterilization for 30 minutes. Next, the chip was coated with 10 μg / mL poly-L-lysine and incubated at 37 °C for 30 minutes, followed by washing three times with PBS. Then, the chip was coated with 50 μg / mL bovine plasma fibronectin and incubated at 37 °C for 30 minutes. Finally, the chip was coated with a mixture of 50 μg / mL Matrigel and 100 μg / mL type I collagen at 37 °C for 30 minutes.

[0045] CACO-2 cells were used at a rate of 8 × 10 6 Cells were seeded at a concentration of [number] cells / mL in the intestinal microarray and allowed to stand for 4 hours to promote adhesion. After adhesion, culture medium was perfused at a rate of 20 µL / h for 3 days. In the Shigella infection assay, cells were first pretreated with 20 μM NF157 for 30 minutes, then treated with medium containing 4 μM HD5 for 15 minutes. Next, the HD5-pretreated Shigella was added to the intestinal microarray at an MOI of 1 and infected at a flow rate of 30 μL / h for 2 hours. Finally, infection was terminated by treatment with 100 μg / L gentamicin at a flow rate of 30 μL / h for 30 minutes. After infection, cells were fixed with 4% paraformaldehyde for 20 minutes, punched in 0.1% Triton X-100 for 5 minutes, and washed three times with PBS. Afterward, cells were stained with TRITC (10 μg / mL) in the dark for 30 minutes and then mounted with DAPI-containing mounting medium.

[0046] Intestinal microfluidic chip-infected samples were imaged. Laser confocal microscopy, using Z-axis tomography, acquired the 3D intestinal structure formed within the microfluidic chip, observing images at different depths. A 1 μm Z-axis was selected for tomography, revealing an intestinal structure approximately 30 μm in height within the chip. Immunofluorescence images were then processed in ImageJ. In ImageJ, the three channels of the image were merged (green for bacteria, red for the cytoskeleton, and blue for the nucleus). In Image>Properties, the Voxel depth was set to 1 μm, and then the desired region was selected in Image>Stacks>orthogonalviews. The results are as follows: Figure 3As shown, the results indicated that most Shigella bacteria aggregated and invaded the intestinal crypts. The presence of NF157 effectively reduced the invasive effect of HD5 on bacteria, significantly decreasing both the number of bacterial clusters and bacteria in the intestinal crypts.

[0047] 3. The antibacterial effect of NF157 in an in vivo guinea pig conjunctivitis model.

[0048] The guinea pig conjunctivitis model is a method for detecting bacterial invasion and has been widely used to differentiate between invasive and non-invasive Escherichia coli. In this invention, SPF-grade female Dunkin' Hartley guinea pigs aged 6-8 weeks and weighing approximately 250 g were selected. The guinea pigs were randomly divided into four groups of 10 each: a control group, an HD5 group, an NF157 group, and an NF157+HD5 group.

[0049] First, 10 μL of DMEM culture medium containing 20 μM NF157 was instilled into the eyes of guinea pigs in the NF157 group and the NF157+HD5 group for 30 minutes per eye. Then, 4 μM HD5 was added to Shigella bacteria in logarithmic growth phase (1×10⁻⁶). 6 A mixture of CFU / eye was added to the eyes of guinea pigs in the HD5 and NF157+HD5 groups. The control and NF157 groups received the same volume of DMEM medium containing only Shigella. For three consecutive days, the eyes of guinea pigs treated with NF157 were instilled with DMEM medium containing 20 μM NF157.

[0050] Over the next three days, the degree of conjunctival inflammation in guinea pigs was observed and recorded, and graded according to the following criteria: grade 0 indicated no disease or only mild irritation; grade 1 indicated mild conjunctivitis; grade 2 indicated moderate non-purulent conjunctivitis; and grade 3 indicated severe purulent conjunctivitis. The results showed that NF157 significantly reduced conjunctivitis symptoms caused by HD5-enhanced Shigella infection at all observation time points. This model allows for the evaluation of the therapeutic effect of NF157 on HD5-enhanced Shigella infection, providing valuable data for further drug development and research.

[0051] This invention reveals a novel use of NF157 as a specific P2Y11 receptor inhibitor for the treatment of pathogenic infections promoted by human intestinal alpha-defensin 5 (HD5). Through a series of in vitro and in vivo experimental models, this invention demonstrates that NF157 can block the binding of HD5 to the P2Y11 receptor, thereby effectively reducing the infectivity of pathogens under the influence of HD5.

[0052] In an in vitro cell culture model, NF157 exhibited significant antibacterial infection effects, reducing Shigella invasion of HeLa cells. Furthermore, using an intestinal microfluidic chip model, this invention simulated the in vivo intestinal environment, demonstrating that NF157 inhibits Shigella invasion within the simulated intestinal microenvironment, reducing bacterial aggregation and invasion in intestinal crypt regions.

[0053] In an in vivo guinea pig conjunctivitis model, NF157 also demonstrated a therapeutic effect against Shigella infection, significantly alleviating conjunctivitis symptoms caused by HD5-enhanced infection. These results indicate that NF157 not only provides a new therapeutic strategy for treating HD5-mediated pathogenic infections but also opens up new directions for drug development in related fields.

[0054] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. The use of the P2Y11 specific inhibitor NF157 in the preparation of medicaments for treating, inhibiting, or reducing pathogenic infections in individuals suffering from or at risk of developing / acquiring such infections, characterized in that, The pathogen is Shigella, a pathogenic microorganism that relies on pseudopodia to capture and invade.

2. The application as described in claim 1, characterized in that, NF157 can block the binding of HD5 to the P2Y11 receptor and reduce the infectivity of pathogens under the action of HD5.

3. The application of NF157 as a specific inhibitor of P2Y11 in the preparation of drugs for treating pathogen-related infections, characterized in that... The pathogen is Shigella, a pathogenic microorganism that relies on pseudopodia to capture and invade.

4. The application as described in claim 3, characterized in that, NF157 can block the binding of HD5 to the P2Y11 receptor and reduce the infectivity of pathogens under the action of HD5.

5. The application as described in claim 3, characterized in that, The diseases mentioned include conjunctivitis, gastroenteritis, and bacterial dysentery.

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