New pharmaceutical uses for rilpivirine

Abstract

This application relates to novel pharmaceutical uses of rilpivirine, specifically its application in the preparation of drugs for the prevention and / or treatment of macrophage inflammatory diseases, sepsis, and sepsis-induced acute lung injury. Experiments have demonstrated that rilpivirine can significantly reduce gene expression of inflammatory factors in macrophage inflammation models, exhibits no significant toxicity at effective doses, and has high safety. Furthermore, rilpivirine can significantly alleviate acute lung injury induced by sepsis or lung exposure to bacterial endotoxins by inhibiting the expression of inflammatory factors, thereby improving the effect of acute lung injury in mice and significantly increasing the survival rate of mice with acute lung injury models.

Description

Technical Field

[0001] This application belongs to the field of pharmaceutical technology and relates to new pharmaceutical uses of rilpivirine, specifically the use of rilpivirine in the preparation of medicaments for the prevention and / or treatment of macrophage inflammatory diseases, sepsis, and sepsis-induced acute lung injury. Background Technology

[0002] Sepsis is a critical illness caused by the invasion of pathogenic microorganisms such as bacteria. Clinically, it manifests as a systemic inflammatory response syndrome, accompanied by complications such as septicemia and insufficient organ perfusion. Sepsis can also lead to septic shock, acute lung injury (ALI) / acute respiratory distress syndrome (ARDS), multiple organ failure, and even death. Currently, it is believed that the uncontrolled inflammatory response and immune dysregulation caused by pathogenic microorganism infection are the main causes of sepsis. During septic infection, endotoxins and other substances stimulate immune cells to produce large amounts of inflammatory mediators, prompting the recruitment and activation of inflammatory cells, especially macrophages, in tissues. This further leads to the production of cytokines, chemokines, and oxygen free radicals, forming a cascade reaction that damages or even fails organs such as the lungs, ultimately causing the patient's death.

[0003] Acute lung injury (ALI) is a common and serious clinical condition. ALI often progresses to acute respiratory distress syndrome (ARDS), characterized by pulmonary edema, decreased lung compliance, acute hypoxic respiratory failure, and ultimately death. The pathophysiological mechanism of ALI is believed to be related to dysregulation of lung inflammation, with its main pathological features being increased production of pro-inflammatory factors and increased inflammatory cell infiltration. Therefore, regulating the inflammatory response and improving the pathological state are important directions for the treatment of sepsis-induced acute lung injury and a hot topic in drug development. Summary of the Invention

[0004] Rilpivirine (RPV) is a diarylpyrimidine non-nucleoside reverse transcriptase inhibitor. Clinically, it has been approved for use in combination with other antiretroviral drugs for the treatment of human immunodeficiency virus type 1 (HIV-1). It inhibits HIV-1 replication by non-competitively inhibiting HIV-1 reverse transcriptase. Data suggests that RPV can protect against myocardial ischemia-reperfusion injury in mice by inhibiting abnormal platelet activation.

[0005] This application identified a novel drug, rilpivirine, through cell model screening, which can inhibit macrophage inflammation and can be used to prepare drugs for the prevention and / or treatment of macrophage inflammatory diseases, sepsis, and sepsis-induced acute lung injury.

[0006] One objective of this application is to provide the use of rilpivirine in the preparation of medicaments for the prevention and / or treatment of macrophage inflammatory diseases.

[0007] Another objective of this application is to provide the use of rilpivirine in the preparation of medicaments for the prevention and / or treatment of sepsis and sepsis-induced acute lung injury.

[0008] In some embodiments, the sepsis is induced by bacterial endotoxins.

[0009] Furthermore, in some embodiments, the bacterial endotoxin is a lipopolysaccharide.

[0010] Similarly, in some embodiments, the acute lung injury is induced by bacterial endotoxins. Further, in some embodiments, the bacterial endotoxin is a lipopolysaccharide.

[0011] In some embodiments, the acute lung injury includes at least one of the following: sepsis caused by peripheral inflammation, inflammatory lung injury, bronchial asthma, tracheitis, bronchitis, chronic obstructive pulmonary disease, cor pulmonale, and pulmonary fibrosis.

[0012] In some embodiments, rilpivirine treats acute lung injury by inhibiting the expression of inflammatory factors.

[0013] In some embodiments, the inflammatory factors include TNF-α, IL-1β, and IL-6.

[0014] Another object of this application is to provide the use of a pharmaceutical composition in the preparation of a medicament for the prevention and / or treatment of macrophage inflammatory diseases, said pharmaceutical composition containing a therapeutically effective amount of rilpivirine.

[0015] Another object of this application is to provide the use of a pharmaceutical composition in the preparation of a medicament for the prevention and / or treatment of sepsis and sepsis-induced acute lung injury, said pharmaceutical composition containing a therapeutically effective amount of rilpivirine.

[0016] On the other hand, a pharmaceutical preparation containing a therapeutically effective amount of rilpivirine and a pharmaceutically acceptable carrier, adjuvant, or mediator, the use of said pharmaceutical preparation in the preparation of a medicament for the prevention and / or treatment of macrophage inflammatory diseases, sepsis, and sepsis-induced acute lung injury.

[0017] In this application, the aforementioned compounds and their pharmaceutically acceptable salts, as well as solvates of these compounds (collectively referred to herein as "therapeutic drugs"), may be used alone or preferably in combination with a suitable pharmaceutical carrier or diluent according to standard pharmaceutical methods when administered to mammals. Administration may be via various routes, including oral, non-gastrointestinal, or local administration. Non-gastrointestinal administration as referred to herein includes, but is not limited to, intravenous, intramuscular, intraperitoneal, subcutaneous, and transdermal administration.

[0018] In some embodiments, the pharmaceutical formulation may be an oral formulation, a nebulized inhaler for the lungs or nose, a metered-dose aerosol, or a dry powder inhaler.

[0019] This application demonstrates through experiments that rilpivirine can significantly reduce the gene expression of inflammatory factors in macrophage inflammation models, has no obvious toxicity at effective doses, and is highly safe; furthermore, rilpivirine can significantly alleviate sepsis or acute lung injury induced by exposure of the lungs to bacterial endotoxins, thereby improving the effect of acute lung injury in mice by inhibiting the expression of inflammatory factors, and significantly improving the survival rate of mice in acute lung injury models.

[0020] During the experiment, the dosage of rilpivirine was 1-10 μM per cell.

[0021] Cellular experiments showed that lipopolysaccharide significantly upregulated the mRNA expression of pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 in macrophages, and 10 μM rilpivirine significantly inhibited the expression of inflammation-related genes in macrophage BMDM.

[0022] The advantages of this application are:

[0023] (1) This application is the first to discover that rilpivirine has the effect of inhibiting the expression of inflammatory factors, that is, rilpivirine has good anti-inflammatory activity and no obvious toxicity at effective doses.

[0024] (2) This application is the first to discover that rilpivirine can treat sepsis and acute lung injury caused by sepsis, and has a significant effect on improving pathological damage to the lungs. Attached Figure Description

[0025] Figure 1 This is a schematic diagram illustrating the effect of different concentrations of rilpivirine on the cell viability of BMDM in the embodiments of this application;

[0026] Figure 2 This is a schematic diagram illustrating the effect of rilpivirine at the same concentration on the expression level of inflammatory genes in lipopolysaccharide-stimulated BMDM cells in the embodiments of this application.

[0027] Figure 3This is a microscopic illustration of H&E staining of lung tissue sections from mice with acute lung injury induced by endotracheal instillation of lipopolysaccharide at different concentrations of rilpivirine in the embodiments of this application.

[0028] Figure 4 This is a schematic diagram illustrating the effect of different concentrations of rilpivirine on the pathological damage score of lung tissue in mice with acute lung injury induced by endotracheal instillation of lipopolysaccharide in the embodiments of this application.

[0029] Figure 5 This is a schematic diagram illustrating the effect of different concentrations of rilpivirine on Tnf-α expression in lung tissue of mice with acute lung injury induced by endotracheal instillation with lipopolysaccharide in the embodiments of this application.

[0030] Figure 6 This is a schematic diagram illustrating the effect of rilpivirine on the total protein content in the bronchoalveolar lavage fluid of mice with acute lung injury induced by endotracheal instillation of lipopolysaccharide in the embodiments of this application.

[0031] Figure 7 This is a schematic diagram illustrating the effect of rilpivirine on the ratio of wet to dry weight of the lungs in mice with acute lung injury induced by endotracheal instillation of lipopolysaccharide in the embodiments of this application.

[0032] Figure 8 This is a schematic diagram illustrating the effect of rilpivirine on the survival rate of mice with cecal ligation and puncture simulating sepsis in the embodiments of this application;

[0033] Figure 9 This is a microscopic illustration of H&E staining of lung tissue pathological sections from mice with cecal ligation and puncture simulated by rilpivirine in an embodiment of this application.

[0034] Figure 10 This is a schematic diagram illustrating the effect of rilpivirine on the pathological damage score of lung tissue in mice simulating sepsis by cecal ligation and puncture, as described in this application embodiment. Detailed Implementation

[0035] To further illustrate this application, a series of embodiments are given below. These embodiments are purely illustrative and are only used to specifically describe this application. They should not be construed as limiting this application.

[0036] Drugs and reagents: Rilpivirine used in this experiment was purchased from Taoshu Biotechnology Co., Ltd., lipopolysaccharide (LPS) was purchased from Sigma-Aldrich, and other reagents were commercially available analytical grade reagents.

[0037] Animals: ICR mice and C57BL / 6J mice were purchased from Nanjing Huachuang Biotechnology Co., Ltd.

[0038] Kits: The TNF-α ELISA kit was purchased from Shenzhen Dakowei Biotechnology Co., Ltd., the RNA extraction kit and SYBR reagent were purchased from Novizan Biotechnology Co., Ltd., and the primers were purchased from Genewiz Biotechnology Co., Ltd.

[0039] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0040] Example 1: Cytotoxicity test of rilpivirine;

[0041] 1. Experimental Methods

[0042] (1) Primary macrophage (BMDM) collection: C57BL / 6J mice were euthanized by cervical dislocation and disinfected by whole-body immersion in 75% ethanol. The mice were placed on a board in a supine position with their limbs fixed. The skin of the hind limbs was cut open, and the limbs were cut at the ankle and femoral joints. The muscles and knee joints were removed. The bone marrow was washed with pre-cooled serum-free DMEM using a 1 mL syringe until the leg bones turned white. The washing solution was filtered through a 200-mesh filter and collected in a 15 mL centrifuge tube. The cells were centrifuged at 500×g for 5 min, and the supernatant was discarded. The cell pellet was resuspended in 1 mL of erythrocyte lysis buffer. After erythrocyte lysis for 1 min, 5 mL of DMEM was added to stop the erythrocyte lysis. After centrifugation at 500×g for 5 min, the supernatant was discarded. The lower layer of cells was resuspended in DMEM medium containing 20% ​​fetal bovine serum (FBS) and a final concentration of 25% was added. Macrophage colony-stimulating factor (M-CSF) was added to each well. Cells were spotted at a density of 6×10^5 into 96-well plates and cultured in a cell incubator at 37°C and 5% CO2 for 48 h. Then, M-CSF was added to each well to a final concentration of 25 ng / mL and the cells were cultured for another 48 h. After adhesion and differentiation, the cells became macrophages.

[0043] (2) Cytotoxicity test: After BMDM cells were pretreated with rilpivirine at a certain concentration gradient for 6 hours, cell viability was determined by CCK-8 method and LDH method.

[0044] 2. Experimental Results

[0045] The results are shown in Table 1 and... Figure 1 .

[0046] Table 1. Effects of rilpivirine on BMDM cell viability

[0047]

[0048] 3. Results Analysis

[0049] From Table 1 and Figure 1The results showed that rilpivirine did not exhibit significant toxicity to BMDM cells at different doses.

[0050] Example 2: Effects of rilpivirine on the expression levels of inflammatory genes in primary macrophages;

[0051] Experimental methods

[0052] BMDM cells were added to 24-well plates at a density of 5 × 10^5 cells per well, and M-CSF was added to a final concentration of 25 ng / mL. After the cells adhered and differentiated into macrophages, BMDM cells were pretreated with different concentrations of rilpivirine for 1 hour. After rilpivirine pretreatment, except for the Control group, lipopolysaccharide (LPS) (100 ng / mL) was added and incubated for 3 hours. The supernatant was discarded, and the cells were thoroughly lysed with Trizol (Nanjing Novizan, R401-01). RNA was extracted according to the RNA extraction instructions, reverse transcribed into cDNA, and then amplified using qPCR according to the instructions. Finally, ΔCt was used for statistical calculation.

[0053] Experimental results

[0054] The results are shown in Table 2 and Figure 2 .

[0055] Table 2. Effects of rilpivirine on the expression levels of inflammatory genes in primary macrophages.

[0056]

[0057] Note: *** P <0.001 vs Control; # P <0.05, ## P <0.01, ### P <0.001 vs Model.

[0058] 3. Results Analysis

[0059] From Table 2 and Figure 2 The results showed that lipopolysaccharide could significantly upregulate the expression of inflammation-related genes such as TNF-α and IL-1β in macrophage BMDM, while rilpivirine treatment could significantly alleviate this upregulation trend and inhibit the expression of inflammatory genes in a dose-dependent manner, indicating that rilpivirine has good anti-inflammatory activity.

[0060] Example 3: Evaluation of the efficacy of rilpivirine in improving acute lung injury caused by sepsis;

[0061] 1. Experimental Methods

[0062] Grouping and Modeling of Acute Lung Injury in Mice: Male ICR mice were used as experimental animals and randomly divided into control, model, positive control, and treatment groups according to body weight. The control and model groups were injected with a solvent via tail vein (the solvent was prepared by mixing DMSO, TWEEN-80, and PBS in a ratio of 4:2:94). The positive control group was injected with 2 mg / kg dexamethasone (DXM) via tail vein. The treatment groups were injected with different concentrations of rilpivirine via tail vein. Both dexamethasone and rilpivirine were soluble in the aforementioned solvent and were clear and transparent. Treatment continued for 3 days. One hour after the last administration, except for the control group which received an equal volume of physiological saline via tracheal infusion, the other groups received lipopolysaccharide (LPS) (20 mg / kg) via tracheal infusion. Specifically, mice were anesthetized with 2.5% isoflurane, the neck was disinfected, the neck skin was incised, and the trachea was separated. Except for the control group which received 50 μL of Saline via tracheal infusion, the other groups received 50 μL of LPS (LPS dissolved in Saline). The dosage was 20 mg / kg. mg / kg, and the skin layer was sutured.

[0063] 2. Lung pathology examination

[0064] Six hours after endotracheal infusion of lipopolysaccharide, lungs were fixed with paraformaldehyde. The fixed lungs were then dehydrated, paraffin-embedded, sectioned, and stained with hematoxylin-eosin (H&E). Scoring was performed according to the American Thoracic Society's scoring criteria. Histopathological results and statistical data are shown below. Figure 3 , Figure 4 And Table 3.

[0065] Table 3. Statistical analysis of lung pathological injury scores

[0066]

[0067] 3. Detection of TNF-α content in lung tissue

[0068] Lung tissue was collected 6 hours after endotracheal instillation of lipopolysaccharide. The tissue was homogenized using RIPA lysis buffer, centrifuged, and the protein-containing supernatant was collected. The supernatant was then tested using a commercially available TNF-α ELISA kit. TNF-α content results and statistical data are shown below. Figure 5 And Table 4.

[0069] Table 4. Statistical data on TNF-α inflammatory factors in lung tissue.

[0070]

[0071] Note: * P <0.05, *** P <0.001 vsSham; # P <0.05, ### P <0.001 vs Vehicle.

[0072] 4. Results Analysis

[0073] There was no significant change in body weight of mice after administration of any of the drugs. After 6 hours of intratracheal instillation of lipopolysaccharide, the organ coefficient of the lungs of mice was significantly upregulated, TNF-α in the lung tissue was significantly increased, and the lung tissue was significantly damaged. Compared with the model group, the rilpivirine administration group could significantly downregulate the expression and release of TNF-α in the lungs and had a significant ameliorative effect on lung pathological damage.

[0074] Example 4: The effect of rilpivirine on improving acute lung injury induced by endotracheal instillation of lipopolysaccharide;

[0075] 1. Experimental Methods

[0076] Animal grouping and administration: Male ICR mice were randomly divided into three groups according to body weight: control group, model group, and rilpivirine (5 mg / kg) group. Drug preparation, administration, and modeling methods were the same as in Example 3. After three consecutive days of pre-administration, LPS was administered via tracheal infusion to establish the model 2 hours after the last administration. Six hours after modeling, some mice were sacrificed, and their entire lungs were weighed for wet weight. The entire lungs were then baked at 60℃ for 24 hours, and their dry weight was measured. The dry-wet weight ratio of each group was calculated. Six hours after modeling, the remaining mice underwent bronchoalveolar lavage, and bronchoalveolar lavage fluid (BALF) was collected. The lavage procedure was as follows: After sacrifice, the mice were placed supine with their limbs fixed. The neck skin was incised, the trachea was separated, and a small incision was made at an appropriate location. A 16G needle with a flattened tip was inserted to a depth of 1 cm. The trachea was tied to the needle shaft to form an indwelling needle. 400 mg / kg of the solution was drawn up using a 1 mL syringe. Slowly inject μL of pre-cooled PBS, aspirate twice, and repeat the above operation. Combine the recovered BALF, with a recovery efficiency of 70%–80%. Centrifuge the collected BALF at 500×g, 4℃ for 5 min, collect the supernatant, and determine the total protein content in the supernatant using a BCA kit.

[0077] 2. Experimental Results

[0078] See results Figure 6 , Figure 7 ( *** P <0.001, vs Sham; # P <0.05, vs (Vehicle) and Table 5.

[0079] Table 5. Effects of RPV on acute lung injury induced by endotracheal instillation of lipopolysaccharide

[0080]

[0081] Note: *** P <0.001 vs Control; # P <0.05, ## P <0.01, ### P <0.001 vs Model.

[0082] 3. Results Analysis

[0083] The experimental results showed that after 6 h of intratracheal instillation of lipopolysaccharide, the wet / dry weight ratio of mouse lungs was significantly increased, and the total protein level in bronchoalveolar lavage fluid (BALF) was significantly increased. Compared with the Model group, the rilpivirine administration group significantly reduced the wet / dry weight ratio of mouse lungs and reduced the total protein level in BALF.

[0084] Example 5: Effect of rilpivirine on survival rate of mice with cecal ligation and puncture simulating sepsis;

[0085] 1. Experimental Methods

[0086] Animal grouping and administration: Male ICR mice were randomly divided into three groups according to body weight: a model group, a positive drug dexamethasone (2 mg / kg) group, and a rilpivirine (5 mg / kg) group, with 16 mice in each group. The drug preparation and administration method were the same as in Example 3. One hour after the last administration, cecal ligation and puncture surgery was performed. The specific procedure was as follows: After isoflurane anesthesia and disinfection, a midline incision of about 2 cm was made in the abdomen to open the abdominal cavity. The cecum was located, and the mesentery of its distal end to the large intestine was carefully separated. The distal half of the cecum was tightly ligated with sterile No. 4 silk suture, and a sterile No. 7 needle was used to puncture the ligated cecum at the center of the distal end. The cecum was then pushed back into the abdominal cavity, and the abdominal cavity was closed. The survival status of the mice was checked within 72 hours after modeling, and the survival curve was calculated.

[0087] 2. Experimental Results

[0088] See results Figure 8 (in, *** P <0.001 vs vehicle).

[0089] 3. Results Analysis

[0090] Depend on Figure 8It is evident that 5 mg / kg rilpivirine significantly improves the survival rate of mice simulating sepsis through cecal ligation and puncture.

[0091] Example 6: Effect of rilpivirine on lung injury in mice with cecal ligation and puncture simulating sepsis;

[0092] 1. Experimental Methods

[0093] Animal grouping and administration: Male ICR mice were randomly divided into 3 groups according to body weight: control group, model group and rilpivirine (5 mg / kg) group. The drug preparation, administration method and modeling method were the same as in Example 3.

[0094] 2. Lung pathology examination

[0095] Six hours after the cecal ligation and puncture procedure, the lungs were fixed with paraformaldehyde. The fixed lungs were then dehydrated, embedded in paraffin, sectioned, and stained with hematoxylin-Eosin (H&E).

[0096] 3. Experimental Results

[0097] Histopathological results and statistical data can be found in Figure 9 , Figure 10 .

[0098] 4. Results Analysis

[0099] After cecal ligation surgery, lung tissue was significantly damaged, and the infiltration of immune cells in the lung tissue was significantly increased; the rilpivirine group showed a significant improvement in lung pathological damage compared to the Model group.

[0100] The above description is only a preferred embodiment of this application. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of this application, and these improvements and modifications should also be considered within the scope of protection of this application.

Claims

1. The use of rilpivirine as the sole active ingredient in the preparation of a medicament for the prevention and / or treatment of sepsis and sepsis-induced acute lung injury; wherein rilpivirine treats acute lung injury by inhibiting the expression of inflammatory factors, including TNF-α, IL-1β, and IL-6.

2. The application according to claim 1, characterized in that, The sepsis was caused by bacterial endotoxins.

3. The application according to claim 2, characterized in that, The bacterial endotoxin is a lipopolysaccharide.

4. The application according to claim 1, characterized in that, The acute lung injury was induced by bacterial endotoxins.

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