Application of combination of chlorogenic acid and sivelestat sodium in prevention and treatment of infectious diseases

Abstract

The invention discloses application of chlorogenic acid combined with sivelestat sodium in prevention and treatment of infectious diseases. Through animal in-vivo experiments, the protective effect of chlorogenic acid (CGA) and sivelestat sodium (SIV) on mouse lethality caused by klebsiella pneumoniae (Kpn) infection is verified in a constructed model of sepsis caused by klebsiella pneumoniae infection, the survival rate of infected mice can be increased by combining chlorogenic acid (CGA) and sivelestat sodium (SIV), the weight loss of the mice caused by infection can be resisted, and the curative effect of the sivelestat sodium (SIV) on the mice caused by klebsiella pneumoniae (Kpn) can be improved. Experiments prove that continuous intraperitoneal injection of chlorogenic acid does not cause significant change of hemogram-related indexes, indicating that chlorogenic acid has good safety. According to the scheme of combining the chlorogenic acid with the sivelestat sodium, through the advantages of precise targeted anti-inflammation, two-way immune regulation, anti-infection and immune synergy and the like, the defects of an existing immune regulation technology for bacterial sepsis are comprehensively overcome, and a new optimization direction is provided for clinical treatment.

Description

Technical Field

[0001] This application relates to the application of chlorogenic acid in combination with cevelexostat sodium in the prevention and treatment of infectious diseases, and belongs to the field of biomedical technology. Background Technology

[0002] Infectious diseases, in severe cases, can lead to sepsis. Sepsis, an infection-induced immune-mediated organ dysfunction, has seen progress in early anti-infection and fluid resuscitation bundled therapies, but the lack of targeted immunomodulatory measures results in a poor prognosis, particularly in long-term survival. Its core pathological basis is a biphasic dynamic imbalance of "cytokine storm-immune paralysis": early pathogen stimulation triggers a cascade release of pro-inflammatory factors, leading to multi-organ damage; later, immune cell apoptosis and upregulation of immune checkpoint molecules lead to immunosuppression, making secondary infections more likely. Current immunomodulatory therapies mainly focus on three areas: anti-inflammation, immune activation, and cell regulation, but these techniques have significant limitations and are ill-suited to the complex immune imbalances caused by bacterial infections. Anti-inflammatory modulatory techniques primarily utilize glucocorticoids and TNF-α / IL-6 antagonists. The former, by inhibiting the NF-κB pathway and blocking the release of inflammatory factors induced by bacterial toxins, suffers from target nonspecificity, potentially suppressing the body's antibacterial immune response and easily leading to infection spread. Furthermore, the lack of rapid detection technology for bacterial toxins and inflammatory stages makes it difficult to personalize the timing and dosage of medication. The latter, limited by technology, can only block a single inflammatory factor pathway and cannot cover the multifactorial synergistic inflammatory network triggered by bacterial infection, resulting in limited therapeutic efficacy. Immune activation technologies target bacterial-induced immune paralysis, including PD-1 / PD-L1 inhibitors and IL-7 infusion. The former lacks precise technology for assessing the immune status after bacterial infection, making it difficult to select suitable patients, and indiscriminate activation may trigger a rebound of the cytokine storm. The latter is limited by immature dosage control technology, resulting in a narrow therapeutic window; excessive dosage can exacerbate immune damage, while insufficient dosage fails to rebuild antibacterial immune function. Moreover, exogenous cytokines have short half-lives and lack technologies for long-term effects. Cell therapy technologies such as mesenchymal stem cells suffer from deficiencies in preparation and quality control techniques, insufficient targeted homing capabilities, and difficulty in directing their migration to sites of inflammation caused by bacterial infections. Furthermore, the lack of techniques to assess the synergistic effect of cells and antibacterial immunity leads to unstable treatment outcomes. A common technological shortcoming lies in the lack of real-time immunophenotyping technology and multi-target synergistic regulation technology for bacterial infections, making it impossible to achieve precise synergy between "anti-infection and immune regulation." Summary of the Invention

[0003] The purpose of this invention is to address the shortcomings of the prior art by providing the application of chlorogenic acid combined with cevelexta sodium in the prevention and treatment of infectious diseases.

[0004] To achieve the above objectives, this application adopts the following technical solution:

[0005] This application provides chlorogenic acid (chemical formula C). 16 H 18 O9 (CAS No. 327-97-9) combined with cevelexta sodium (CAS No. 150374-95-1, molecular formula C) 20 H 21 The application of N2NaO7S (molecular weight 456.44) in the preparation of drugs for the prevention and treatment of sepsis and related diseases.

[0006] In some embodiments, the infection and related disease include bacterial infection and bacterial sepsis caused by bacterial infection.

[0007] In some embodiments, the drug comprises an active ingredient and pharmaceutically acceptable excipients, wherein the active ingredient is chlorogenic acid combined with cevelexatol sodium.

[0008] In some embodiments, the pharmaceutically acceptable excipient is one or more of the following: diluent, binder, wetting agent, lubricant, disintegrant, solvent, emulsifier, cosolvent, preservative, pH adjuster, osmotic pressure adjuster, surfactant, coating material, antioxidant, and buffer.

[0009] In some embodiments, the dosage form of the drug is any one of solution, powder, tablet, capsule, suspension, and emulsion.

[0010] Compared with the prior art, this application has the following beneficial effects: 1) The combination drug regimen provided in this application can achieve precise targeted anti-inflammatory treatment while taking into account immune protection: Cevelexat sodium can specifically inhibit neutrophil elastase activated by bacterial toxins, blocking the vascular endothelial damage and cascade release of inflammatory factors mediated by it, without affecting the phagocytic and bactericidal function of neutrophils; Chlorogenic acid selectively downregulates the expression of pro-inflammatory factors (such as TNF-α and IL-6) and upregulates the level of anti-inflammatory factors (such as IL-10) by regulating the NF-κB and MAPK signaling pathways, thus avoiding the broad-spectrum suppression of immune function by traditional anti-inflammatory drugs and reducing the risk of superinfection; 2) The combination of the two has bidirectional immunomodulatory properties, without relying on complex typing techniques: During the cytokine storm, chlorogenic acid and cetuximab sodium synergistically inhibit excessive inflammatory response; after entering the immune paralysis phase, chlorogenic acid can promote lymphocyte proliferation, enhance the ability of macrophages to phagocytose bacteria, and improve immune cell apoptosis and functional exhaustion caused by bacterial infection; cetuximab sodium, on the other hand, creates conditions for immune function repair by reducing damage to the inflammatory microenvironment; this bidirectional "anti-inflammatory-immune-promoting" effect can be adapted to patients with bacterial sepsis at different immune stages, avoiding the drawbacks of traditional techniques with "single regulatory direction"; 3) The combination of the two can achieve a synergistic effect of anti-infection and immune regulation: Chlorogenic acid (CGA) itself has the activity of directly inhibiting common septic pathogens such as Staphylococcus aureus, Klebsiella pneumoniae and Escherichia coli, which can help clear bacterial load and reduce toxin release; Chlorogenic acid (CGA) targets and blocks the immune damage caused by bacterial toxins. The two form a closed loop of "antibacterial and toxin reduction - immune regulation", which solves the core problem of "separation of immune regulation and anti-infection" in traditional technology. 4) Chlorogenic acid combined with cevelexat sodium as a small molecule drug combination regimen does not require complicated cell culture and quality control processes, thus avoiding batch differences and tumorigenic risks in cell therapy; at the same time, the two can directly act on the systemic inflammatory microenvironment and immune cells, exerting their regulatory effects without relying on targeted modification technology, making clinical application more convenient and safer.

[0011] In summary, chlorogenic acid combined with cetylexadestatin sodium overcomes the shortcomings of existing immunomodulatory technologies for bacterial sepsis through its advantages of precise targeted anti-inflammatory effects, bidirectional immune regulation, and synergistic effects between anti-infection and immunity, providing a new direction for clinical treatment optimization. Attached Figure Description

[0012] Figure 1 Establishment of a sepsis model induced by Klebsiella pneumoniae: survival of mice after modeling with different concentrations of KP002 bacterial suspension of the same strain.

[0013] Figure 2 Validation of the optimal infection dose in the Klebsiella pneumoniae sepsis model: survival of mice after modeling with different concentrations of Klebsiella pneumoniae bacterial suspension of the same strain.

[0014] Figure 3 Detection results of Klebsiella pneumoniae in the lungs during sepsis caused by intraperitoneal infection with Klebsiella pneumoniae.

[0015] Figure 4 Survival curves of mice in the combination group of chlorogenic acid (CGA) and cevelexostat sodium (SIV) and each control group; p < 0.05 p < 0.01, p < 0.001, p < 0.0001, NS: no significant difference; PBS control group (CON), infection model group (KP), chlorogenic acid group (C), cevelexostat sodium group (S), and chlorogenic acid and cevelexostat sodium combination group (C+S).

[0016] Figure 5 The curves showing the changes in body weight of mice in the chlorogenic acid (CGA) combined with cevelexatol sodium (SIV) treatment group and each control group; where KP is the infection model group, CON is the PBS control group, C is the chlorogenic acid intervention group, S is the cevelexatol sodium intervention group, and C+S is the chlorogenic acid combined with cevelexatol sodium intervention group.

[0017] Figure 6 Survival curves of mice in the CGA combined with cevelexat sodium group and the control group; where KP is the infection model group and C+S is the chlorogenic acid combined with cevelexat sodium intervention group.

[0018] Figure 7 To verify the changes in body weight in mice treated with chlorogenic acid (CGA) combined with cevelexostat sodium (SIV) and in the infection group; where KP is the infection model group and C+S is the chlorogenic acid combined with cevelexostat sodium intervention group.

[0019] Figure 8 Blood routine test results of mice in the chlorogenic acid combined with cevelexostatin intervention group and the infection model group; where KP is the infection model group, C+S is the chlorogenic acid combined with cevelexostatin intervention group; NEU represents neutrophils, PLT represents platelets, WBC represents white blood cells, and RBC represents red blood cells.

[0020] Figure 9 The effect of chlorogenic acid (CGA) combined with cevelexostat sodium (SIV) intervention at different time points on the survival rate of mice with sepsis induced by Klebsiella pneumoniae infection; KP is the infection model group, C+S is the chlorogenic acid combined with cevelexostat sodium intervention group, the parentheses indicate different intervention time points, d represents days, and h represents hours. Detailed Implementation

[0021] To make the technical solution of this application clearer and easier to understand, preferred embodiments are described in detail below with reference to the accompanying drawings.

[0022] Unless otherwise specified, the test methods or experimental methods described in the following examples are conventional methods; unless otherwise specified, the reagents and materials are obtained from conventional commercial sources or prepared by conventional methods.

[0023] Example 1 A sepsis model was established in mice by intraperitoneal injection (ip) of Klebsiella pneumoniae solution. 1) Klebsiella pneumoniae ( Klebsiella pneumoniae,KPN strain Kpn002 was frozen at -80°C. In a biosafety cabinet, a small amount of frozen bacterial solution (surface frost is sufficient, no need to thaw completely) was scraped off with a sterile inoculation loop from the KPN strain stored at -80°C. The solution was then streaked in four zones on Columbia blood agar plates and MacConkey agar plates and incubated overnight at 37°C in a 5% CO2 incubator for 18–24 h before isolating single colonies.

[0024] 2) Pick a single colony from the plate and dissolve it in sterile PBS. After thorough mixing, take 200 μL of the bacterial solution and use an enzyme-linked immunosorbent assay (ELISA) analyzer to detect the OD of the bacterial solution. 600nm The OD value is approximately 0.5. The ELISA analyzer uses sterile PBS for OD measurement. 600nm Perform a blank control.

[0025] 3) OD 600nm The original bacterial culture at approximately 0.5 μL was serially diluted with PBS solution; eight 1.5 mL EP tubes were placed, and 900 μL of sterile PBS was added to each EP tube using a pipette. 100 μL of the original bacterial culture was added to the first tube and mixed thoroughly (10...). -1 Then, draw 100 μL of bacterial culture from the first tube into the second tube and mix well (10 μL). -2 ), and so on, until the last EP tube (10) is diluted. -8 ).

[0026] 4) Select a dilution to 10 -6 10 -7 10 -8 For bacterial suspensions of different concentrations, plate counting was performed. 100 μL of bacterial suspension was pipetted onto LB solid medium and spread using a disposable spreader. Two plates were spread for each dilution. The plates were then inverted and incubated at 37°C for 18–24 hours. Colony morphology was observed.

[0027] 5) Select plates with colony counts between 30 and 300, and record the average colony count for each dilution; calculate the colony count (CFU / mL / OD) as equal to the average colony count × dilution factor × 10.

[0028] 6) According to the experimental requirements, concentrate the prepared bacterial suspension with sterile PBS to the appropriate multiple for modeling.

[0029] A lethal infection model was established in mice by intraperitoneal injection of 1×10^7~5×10^8 CFU of Kpn002, with more than half of the mice dying within 24 hours. Figures 1-2 As shown. Experimental results: By preparing bacterial solutions of different concentrations, a stable and reproducible mouse sepsis model induced by intraperitoneal infection with Klebsiella pneumoniae leading to damage to organs such as the lungs and liver was successfully established. The optimal concentration of bacterial solution for intraperitoneal injection was determined to be 1 × 10^7 CFU / mouse. Figure 2As shown; Klebsiella pneumoniae can be detected in the lungs after infection, such as... Figure 3 As shown.

[0030] Example 2 Protective effect of chlorogenic acid (CGA) combined with cevelexostat sodium (SIV) against lethality in mice infected with Klebsiella pneumoniae (Kpn). Male C57BL / 6J SPF mice aged 6–8 weeks were purchased from the Experimental Animal Center of Putuo Hospital affiliated with Shanghai University of Traditional Chinese Medicine. The mice were randomly divided into 5 groups of 10 mice each. The intervention groups were as follows: control group (CON), model group (KP), chlorogenic acid group (C), cetelestatin sodium group (S), and a combination of chlorogenic acid and cetelestatin sodium group (C+S). Survival rate and clinical scores included: recording 7-day survival rate and monitoring daily indicators such as body weight, mental status, coat color, and activity level.

[0031] Objective: To compare the effects of chlorogenic acid, cevelexostat sodium, and their combined intervention on the survival rate of mice infected with Klebsiella pneumoniae. Methods: SPF-grade male C57BL / 6 mice were randomly divided into five groups: control group, KP model group, CGA intervention group, SIV intervention group, and CGA+SIV combined group. Intervention dosage: Chlorogenic acid was 50 mg / kg, and cevelexostat sodium was 50 mg / kg. Intervention strategy: CGA was administered daily for 6 consecutive days. On day 7, Klebsiella pneumoniae was induced through intraperitoneal infection, and daily administration continued after modeling. All SIV administrations were given 1 hour before intraperitoneal injection for Klebsiella pneumoniae modeling, and daily administration continued after modeling. Observation indicators: Activity status, survival, and weight changes of mice in each intervention group after infection. Details are as follows: ① Control group (abbreviated CON): 200 μL of normal saline was injected intraperitoneally.

[0032] ② Sepsis model group (KP): Intraperitoneal injection of Kpn bacterial solution (Kpn bacterial count: 1×10⁻⁶) 7 CFU / mouse, 200 uL).

[0033] ③ Chlorogenic acid (CGA) intervention in sepsis group (CGA+KP group): intraperitoneal injection of CGA and KPN bacterial solution (CGA dosage: 50mg / kg, 100 μL injection; KPN bacterial quantity: 1×10^7 CFU / mouse, 200 μL).

[0034] ④ Severexana sodium (SIV) intervention group (SIV+KP group): Intraperitoneal injection of SIV + KP bacterial solution (SIV concentration: 50mg / kg, 100 μL; Kpn bacterial count: 1×10^7 CFU / mouse, 200 μL).

[0035] ⑤CGA+SIV intervention group (CGA+SIV+KP group): Intraperitoneal injection of SIV + intraperitoneal injection of KP bacterial solution (CGA concentration: 50 mg / kg, SIV concentration: 50 mg / kg, injection volume: 100 μL; Kpn bacterial count: 1×10^7 CFU / mouse, injection volume: 200 μL).

[0036] The chlorogenic acid group received pre-treatment (7 days before infection) and combination therapy (7 days before CGA infection, 1 hour before SIV infection). Subsequent administration: every 12-24 hours for 7 consecutive days.

[0037] The results showed that chlorogenic acid combined with cetylexadecitar sodium significantly improved the survival rate of KP-infected mice and reduced clinical scores, such as... Figure 4 As shown.

[0038] Example 3 To verify the protective efficacy of chlorogenic acid (CGA) combined with cefulexostat sodium (SIV) against lethal Klebsiella pneumoniae infection. Objective: To evaluate the effect of long-term pretreatment with chlorogenic acid (CGA) combined with cevelexostat sodium (SIV) on the survival status of mice infected with Klebsiella pneumoniae in a lethal manner. Methods: SPF-grade male C57BL / 6 mice were randomly divided into a KP model group and a CGA and SIV combined intervention group. Intervention doses: CGA 50 mg / kg, SIV 50 mg / kg. Intervention measures: Klebsiella pneumoniae infection was induced on day 7 after CGA administration for 6 days. SIV was administered 1 hour before Klebsiella pneumoniae infection. Post-infection observation: Survival rate and body weight were monitored for 7 consecutive days. Results: Chlorogenic acid combined with cevelexostat sodium significantly improved the mortality rate and body weight loss in mice induced by intraperitoneal injection of Klebsiella pneumoniae. Figures 5-7 As shown.

[0039] Example 4 Evaluation of blood routine parameters in mice infected with Klebsiella pneumoniae by long-term chlorogenic acid (CGA) pretreatment SPF-grade C57BL / 6 male mice were divided into a sepsis model group and a chlorogenic acid (CGA) combined with cevelexostat sodium (SIV) intervention group (n=5 in each group). Intervention doses: CGA 50 mg / kg, SIV 50 mg / kg; Intervention measures: CGA administration for 6 days, followed by Klebsiella pneumoniae infection on day 7; SIV administration for 1 hour prior to Klebsiella pneumoniae infection; Specimen collection: Blood was collected in EDTA·K2 anticoagulant tubes on day 7 before Klebsiella pneumoniae infection and analyzed using a Sismerco hematology analyzer. Results showed that continuous intraperitoneal injection of chlorogenic acid did not cause significant changes in blood cell-related indicators, such as... Figure 8 As shown.

[0040] Example 5 Determine the effect of chlorogenic acid (CGA) combined with cevelexostat sodium (SIV) intervention at different time points on the survival rate of mice with sepsis induced by Klebsiella pneumoniae infection. Experimental Methods: SPF-grade C57BL / 6 male mice were divided into four groups. CGA (50 mg / kg) and SIV (50 mg / kg) were administered simultaneously on days 7, 3, and 1 before infection with Klebsiella pneumoniae, and 1 hour after model establishment. Survival rate and body weight were observed and recorded for 7 consecutive days. Analysis of the results concluded that the combination of chlorogenic acid and cetylexadecistat sodium, administered on the first 3 days after infection, achieved the best protective effect. Figure 9 As shown.

[0041] The above description is merely a preferred embodiment of this application and is not intended to limit this application in any form or substance. It should be noted that those skilled in the art can make several improvements and additions without departing from this application, and these improvements and additions should also be considered within the scope of protection of this application.

Claims

1. Application of chlorogenic acid combined with cevelexat sodium in the preparation of a solution for the prevention and treatment of sepsis and related infections.

2. The application according to claim 1, characterized in that, The infections and related diseases include bacterial infections and bacterial sepsis caused by bacterial infections.

3. The application according to claim 1, characterized in that, The drug comprises an active ingredient and pharmaceutically acceptable excipients, wherein the active ingredient is chlorogenic acid combined with cevelexatol sodium.

4. The application according to claim 3, characterized in that, The pharmaceutically acceptable excipients are one or more of the following: diluents, binders, wetting agents, lubricants, disintegrants, solvents, emulsifiers, cosolvents, preservatives, pH adjusters, osmotic pressure adjusters, surfactants, coating materials, antioxidants, and buffers.

5. The application according to any one of claims 1 to 4, characterized in that, The dosage form of the drug is any one of solution, powder, tablet, capsule, suspension and emulsion.

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