CN111991394A

CN111991394A - Application of isoquinoline derivative in preparation of medicine for treating sepsis

Info

Publication number: CN111991394A
Application number: CN202011007170.4A
Authority: CN
Inventors: 吕奔; 唐怡庭
Original assignee: Individual
Current assignee: Individual
Priority date: 2020-09-23
Filing date: 2020-09-23
Publication date: 2020-11-27
Also published as: CN112656793A

Abstract

The invention discloses an application of an isoquinoline derivative in preparing a medicament for treating sepsis, wherein the structure of the isoquinoline derivative is as follows:

the isoquinoline derivative has a certain treatment effect on sepsis and endotoxemia, can effectively improve the survival rate of mice with sepsis and endotoxemia, and provides a new treatment method and a new medicine for clinically treating sepsis.

Description

Application of isoquinoline derivative in preparation of medicine for treating sepsis

Technical Field

The invention relates to the field of biological medicines, and in particular relates to application of an isoquinoline derivative in preparation of a medicine for treating sepsis.

Background

Sepsis (sepsis, also known as sepsis) is a critical condition characterized by multiple organ failure induced by infection, characterized by an acute onset and a severe condition with a mortality rate of up to 40%. Common clinical symptoms include fever, accelerated respiratory rate and heartbeat, and unconsciousness, and severe sepsis can cause insufficient blood flow to supply tissues and even cause organ failure and septic shock, which seriously threatens the life safety of patients. At present, early fluid resuscitation, early use of antibiotics and organ function support, etc. treatment, reduce the mortality rate of sepsis to some extent, but sepsis remains the leading cause of death in Intensive Care Unit (ICU) patients.

The pathogenesis of sepsis is quite complex and in recent years, new sepsis pathogenesis has been revealed in several studies: caspase-11 mediated activation of inflammatory bodies to induce apoptosis (Pyrtopsis) plays an important role in sepsis^[1-7]. These studies found that Caspase-11 is an intracellular receptor for bacterial endotoxins (LPS)^[1-4]After LPS enters into cytoplasm, the LPS can be directly combined with Caspase-11 and can activate the Caspase-11; the activated Caspase-11 shears downstream Gasderm D (GSDMD) protein into peptide segments with membrane breaking function, and the peptide segments are gathered on cell membranes to form cell membrane pore channels, so that cell swelling and cell lysis are caused; this process is accompanied by the release of a number of proinflammatory cytokines such as interleukin-1 alpha (IL-1 alpha) and interleukin-1 beta (IL-1 beta), and the like^[5-7]. The IL-1 alpha or IL-1 beta gene is knocked out, so that the survival rate of an endotoxemia model mouse is not influenced; however, the knockout of Caspase-11 or GSDMD gene can obviously improve the survival rate of mice in sepsis and endotoxemia^[1-7]. The above studies show that: caspase-11 mediated cell apoptosis plays an extremely important role in the lethal link of sepsis and endotoxemia. The research of relevant mechanisms finds that: caspase-11 is mainly expressed in mononuclear macrophages and vascular endothelial cells. Activation of mononuclear macrophage Caspase-11 in sepsis induces cell apoptosis to generate a large amount of arachidonic acid, resulting in increase of systemic vascular permeability and massive extravasation of intravascular fluid^[1-7](ii) a Microcirculation disturbance caused by cell apoptosis induced by Caspase-11 activation of vascular endothelial cells^[8](ii) a Finally, the body is subjected to low volume shock, and multiple organ function failure and even death are caused. The isoquinoline compounds have an isoquinoline parent nucleus structure and have various pharmacological activities. Isoquinoline and derivatives have various biological activities and are widely applied to the fields of medicines, chemical engineering, dyes and the like.

At present, medicines for treating sepsis mainly take nonspecific treatment such as glucocorticoid to prevent and treat organ failure, shock and other symptomatic treatment. While treatment methods to control infection can alleviate the symptoms of sepsis and prolong the life of the patient, they are both temporary and permanent. Therefore, the development of new sepsis therapeutic drugs for application in clinical therapy is of great significance. There is no report on the use of isoquinoline derivatives for the treatment of sepsis.

Reference documents:

1.Kayagaki N,Warming S,Lamkanfi M,Vande Walle L,Louie S,Dong J,Newton K,Qu Y,Liu J,Heldens S,Zhang J,Lee WP,Roose-Girma M,Dixit VM.Non-canonical inflammasome activation targets caspase-11.Nature.2011,479(7371):117-121.

2.Hagar JA,Powell DA,Aachoui Y,Ernst RK,Miao EA.Cytoplasmic LPS activates caspase-11:implications in TLR4-independent endotoxic shock.Science.2013,341(6151):1250-1253.

3.Kayagaki N,Wong MT,Stowe IB,Ramani SR,Gonzalez LC,Akashi-Takamura S,Miyake K,Zhang J,Lee WP,Muszynski A,Forsberg LS,Carlson RW,Dixit VM.Noncanonical inflammasome activation by intracellular LPS independent of TLR4.Science.2013,341(6151):1246-1249.

4.Shi J,Zhao Y,Wang Y,Gao W,Ding J,Li P,Hu L,Shao F.Inflammatory caspases are innate immune receptors for intracellular LPS.Nature.2014,514(7521):187-192.

5.Kayagaki N,Stowe IB,Lee BL,O'Rourke K,Anderson K,Warming S,Cuellar T,Haley B,Roose-Girma M,Phung QT,Liu PS,Lill JR,Li H,Wu J,Kummerfeld S,Zhang J,Lee WP,Snipas SJ,Salvesen GS,Morris LX,Fitzgerald L,Zhang Y,Bertram EM,Goodnow CC,Dixit VM.Caspase-11cleaves gasdermin D for non-canonical inflammasome signalling.Nature.2015,526(7575):666-671.

6.Shi J,Zhao Y,Wang K,Shi X,Wang Y,Huang H,Zhuang Y,Cai T,Wang F,Shao F.Cleavage of GSDMD by inflammatory caspases determines pyroptotic cell death.Nature.2015,526(7575):660-665.

7.Liu X,Zhang Z,Ruan J,Pan Y,Magupalli VG,Wu H,Lieberman J.Inflammasome-activated gasdermin D causes pyroptosis by forming membrane pores.Nature.2016Jul 7；535(7610):153-8.

8.Cheng KT,Xiong S,Ye Z,Hong Z,Di A,Tsang KM,Gao X,An S,Mittal M,Vogel SM,Miao EA,Rehman J,Malik AB.Caspase-11-mediated endothelial pyroptosis underlies endotoxemia-induced lung injury.J Clin Invest.2017Nov 1；127(11):4124-4135.

disclosure of Invention

The present invention is directed to solving at least one of the problems of the prior art. Therefore, the invention provides the application of the isoquinoline derivative in the preparation of the medicament for treating the sepsis, and the medicament has a good treatment effect on the sepsis.

The invention also provides application of the isoquinoline derivative in preparation of an inhibitor in a sepsis related pharmacological pathway.

Use of an isoquinoline derivative according to an embodiment of the first aspect of the invention in the preparation of a medicament for the treatment of sepsis, the isoquinoline derivative having the formula:

the application of the isoquinoline derivative in the preparation of the medicament for treating sepsis according to the embodiment of the invention has at least the following beneficial effects: the isoquinoline derivative is used for preparing a medicament for treating sepsis, and can improve the survival rate of a human body or an animal after sepsis or endotoxemia infection.

Sepsis also includes sepsis-associated diseases in the present invention, such as endotoxemia (endoxemia), severe sepsis (severe septis), and septic shock (septic shock). All belong to symptoms such as systemic infection caused by gram-negative bacteria and further systemic multi-organ dysfunction caused by gram-negative bacteria, and the pathogenic causes and the treatment methods of the symptoms are similar.

According to some embodiments of the invention, the sepsis therapeutic agent is an agent capable of increasing the survival rate of an infected sepsis animal. According to some embodiments of the invention, the sepsis therapeutic agent is an agent capable of inhibiting apoptosis of cells in an infected sepsis animal.

Apoptosis (Pyroptosis), also known as inflammatory necrosis, is a programmed cell death that is characterized by a constant swelling of cells until the cell membrane is ruptured, resulting in the release of cellular contents that in turn activate a strong inflammatory response. Cell apoptosis is an important natural immune response in the body and plays an important role in combating infection. Cell apoptosis is characterized by dependence on inflammatory caspases (primarily Caspase-1, 4, 5, 11) with release of a number of proinflammatory cytokines.

Further, inhibition of cellular apoptosis is inhibition of Caspase11 dependent cellular apoptosis.

Further, the inhibition of cell apoptosis is the inhibition of cell apoptosis induced by Caspase11 activated by LPS and HMGB.

Further, the cell is a macrophage.

According to some embodiments of the invention, the sepsis therapeutic agent is an agent capable of inhibiting the secretion of proinflammatory cytokines in an infected sepsis animal.

Further, the proinflammatory cytokines include IL-1 α and IL-1 β.

According to some embodiments of the invention, the medicament further comprises a pharmaceutically acceptable carrier or excipient.

According to some embodiments of the invention, the medicament is formulated into any one of the pharmaceutically acceptable formulations as required, for example, into an oral administration formulation: tablets, capsules, pills, granules, dripping pills, oral preparations and the like; preparing a rectal administration preparation: suppositories, enemas; preparing an injection preparation: intramuscular injection preparations, intravenous injection preparations, and the like.

The use of an isoquinoline derivative according to a second aspect of the invention for the preparation of an inhibitor of a sepsis-associated pharmacological pathway, the isoquinoline derivative having the formula:

also provides the application of the isoquinoline derivative in the preparation of a cell apoptosis inhibitor; or

The application of the isoquinoline derivative in preparing proinflammatory cytokine inhibitors.

According to some embodiments of the invention, the proinflammatory cytokines comprise IL-1 α and IL-1 β.

Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.

Drawings

FIG. 1 shows the results of an LDH assay for the compound of example 1 of the present invention;

FIG. 2 shows the results of ELISA assay for detecting cytokine of the compound of example 1 of the present invention, wherein (a) is an IL-1. alpha. content test pattern, and (b) is an IL-1. beta. content test pattern;

FIG. 3 is a graph showing the statistical results of the effect of the compound of example 2 on the survival rate of endotoxemia-affected mice;

FIG. 4 is a graph of the statistics of the effect of the compounds of example 1 on the survival of septic mice.

Detailed Description

In order to explain technical contents, achieved objects, and effects of the present invention in detail, the following description is made with reference to the accompanying drawings in combination with the embodiments.

Unless otherwise specified, the reagents and materials used in the following examples are commercially available. The compounds used in the following examples are isoquinoline derivatives, purchased from selelck, having the formula:

example 1: primary macrophage apoptosis induced by LH (LPS + HMGB1) activation of Caspase11 by compound

High mobility group Protein B1((high-mobility group box 1Protein, HMGB1) is a highly conserved and widely expressed Protein in mammals, is mainly present in nuclei under normal conditions, is released to The outside of cells under stress conditions such as infection and plays an important pathogenic role in diseases or tissue damage (Regulation of post-translational modifications of HMGB1 reduced immunity. Antioxed Redox Signal 201620; 24 (620-34.). The inventors found that The HMGB1 released by hepatocytes in sepsis can transport LPS in circulation to The cytosol of endothelial cells and macrophages and initiate The LPS-11 mediated apoptosis (The HMGB differentiation Protein 1-11-Protein, HMGB 753. The expression of LPS in liver cells in The course of Sepsis-11-8 Protein kinase 19. Sep # 35. The inventors identified LPS receptor in The course of liver 19. 23, release large amounts of HMGB1 into the blood; these released HMGB1 in turn bind LPS in the blood circulation, and the HMGB1-LPS complex enters the lysosomes of vascular endothelial cells or macrophages through RAGE receptor-mediated endocytosis; under the acidic environment of lysosomes, HMGB1 can directly act on lysosome membranes to cause the rupture of lysosomes, so that LPS in the lysosomes leaks to cytoplasm, and Caspase-11 is finally activated and cell apoptosis is started. The discovery not only reveals a novel mechanism causing the sepsis, but also suggests that the HMGB1-Caspase-11 pathway is a potential drug intervention target spot in the sepsis, so that the inventor constructs an in vitro screening platform for primary macrophage apoptosis induced by LPS + HMGB1 activated Caspase11, and the inhibition of the HMGB1-Caspase11 pathway cell apoptosis by the compound is researched to prove the inhibitory activity of the compound on the sepsis.

1. The experimental steps are as follows: wild Type (WT) mice and Casp11KO mice were extracted as primary peritoneal macrophages, RPMI-1640 complete medium was resuspended at a cell density of 1 × 10^ 6/ml in 500. mu.l/well seed 24 well plates, cells were washed 2 times with DPBS after overnight adherence and replaced with 1640 serum-free medium. The compound of example 1 is added into cells at final concentrations of 5 mu M and 10 mu M respectively for preincubation for 1h, then a mixture of 1 mu g/ml of Lipopolysaccharide (LPS) and 1400ng/ml of high mobility group protein (HMGB) (preincubation for 20min at room temperature in advance) is added for stimulating the cells overnight, and supernatant is collected for 16-18 h to detect the content of Lactate Dehydrogenase (LDH) (judging the cell death rate) and ELISA experiments (detecting the content of proinflammatory cytokines IL-1 alpha and IL-1 beta).

The specific experimental operations were as follows:

1) extracting primary abdominal cavity macrophages of the mice: 3% thioglycollate medium (3 ml/mouse) is injected into the abdominal cavity, after 3-4 days, 10ml of RPMI1640 medium is injected into the abdominal cavity of the mouse, the abdominal cavity lavage fluid is pumped back by a syringe after the abdomen of the mouse is slightly kneaded, the operation is repeated for 1 time, the mouse is collected in a centrifuge tube, and the mouse is centrifuged at 800rpm for 5 minutes, and the supernatant is discarded. Resuspending cells in 5-10 ml of RPMI1640 complete medium, adjusting the cell concentration to 1x 10^ 6/ml after counting cells under a microscope, seeding in a 96-well plate by 100 mul/well, and adhering the wall overnight.

2) Adding medicine for stimulation: washing the cells for 2 times by using DPBS to remove non-adherent suspension cells, replacing the cells with 1640 serum-free culture medium, adding LPS 1 mu g/ml and HMGB 1400ng/ml (pre-incubation for 20min at room temperature) to stimulate the cells overnight after pre-treating the compound for 1H at the final concentration of 10 mu M/hole (comprising a control group: Crtl-without LPS and HMGB; L-with LPS 1 mu g/ml; H-with HMGB 1400 ng/ml);

3) collecting cell supernatant to detect the content of Lactate Dehydrogenase (LDH), cell factors IL-1 alpha and IL-1 beta.

a. Cell death rate assay (lactate dehydrogenase LDH assay):

the experimental steps are as follows: 100 μ l of LDH release reagent was added to the control group, and RPMI-1640 medium was added to make up the volume of the control group to the original volume and mixed well, followed by incubation at room temperature for 1 hour. The supernatant was collected into 1.5ml EP tubes, placed in a 4-degree centrifuge, centrifuged at 500rpm for 5min, and then transferred to new EP tubes. And (3) preparing an LDH detection working solution: lactic acid solution, iodonitrotetrazolium chloride (INT) solution (1X), and enzyme solution are mixed according to the ratio of 1:1: 1. Taking a 96-well plate, firstly adding 80 mu l of RPMI-1640 culture medium into each well, then adding 60 mu l of each sample to be detected, and then respectively adding 60 mu l of LDH detection working solution into each well. Mix well and incubate in the dark at room temperature (about 25 ℃) for 30 min. The absorbance was then measured at 490 nm. The two-wavelength measurement is performed using either 600nm or a wavelength greater than 600nm as a reference wavelength.

Data processing: calculated according to the following formula (the absorbance of each group should be subtracted by the absorbance of the background blank control well):

cell death (%) × (treated sample absorbance-sample control well absorbance)/(absorbance for maximum enzyme activity of cells-sample control well absorbance) × 100.

Elisa assay (cytokine assay): and respectively detecting the expression of IL-1 alpha and IL-1 beta in the supernatant sample by an ELISA detection kit. The ELISA plates were coated with IL-1 α and IL-1 β, respectively, overnight at 4 ℃. 0.05% PBST was washed 3 times for 1 min/time. 1 × Assay buffer was blocked for 1h at RT. Washing 0.05% PBST for 3 times, 1 min/time, adding corresponding sample to be tested and cytokine standard, and incubating for 2h at room temperature on a shaking table. 0.05% PBST was washed 3 times for 1 min/time. Adding corresponding detection antibody and incubating for 1h in a shaking table at room temperature. 0.05% PBST was washed 3 times for 1 min/time. Finally adding an HRP shaker to incubate for 30min at room temperature. TMB developed after 5 washes with 0.05% PBST, and the development was stopped with 2M sulfuric acid. Absorbance at 450nm was measured.

2. The experimental results are as follows: the experimental results of the cytotoxicity experiments of this example are shown in fig. 1 (in the figure, L is LPS only, H is HMGB only, LH is LPS and HMGB added, LH +5 μ M is the group to which 5 μ M compound is added, and LH +10 μ M is the group to which 10 μ M compound is added), and as can be seen from fig. 1, the LDH content in the cell supernatant of the experimental group (LH group) is significantly increased and depends on caspase11 protein, as compared with the control group or LPS alone and HMGB1 alone; however, the LDH content in the cell supernatant is greatly reduced with the addition of the compound, and the compound has good effect when the concentration of the compound is 5 mu M, which indicates that the addition of the compound can reduce the cell death rate, namely inhibit macrophage apoptosis induced by Caspase 11. The ELISA assay of this example is shown in FIG. 2, (a) is an IL-1 alpha content test chart, and (b) is an IL-1 beta content test chart, which shows that the cell supernatant of the experimental group (LH group) has significantly increased contents of cytokines IL-1 alpha and IL-1 beta, and is dependent on caspase11 protein, compared with the control group or the LPS alone and HMGB1 alone; when the compound is added, the content of proinflammatory cytokines IL-1 alpha and IL-1 beta is obviously reduced, which indicates that the compound inhibits the release of the proinflammatory cytokines induced by Caspase 11.

Example 2: protective effect of compound on endotoxemia mice

1. The experimental steps are as follows: selecting mice with the weight of 25-30 g, and dividing the mice into an LPS + DMSO group, an LPS + compound group and a DMSO complete control group. The compounds were dissolved in DMSO at a mother liquor concentration of 40 mg/ml. The compound is injected with LPS 10mg/kg and 100 mul/piece after intraperitoneal injection for 1h in advance at a dose of 4mg/kg and 100 mul/piece; the LPS + DMSO group is DMSO 100 μ l/one, and LPS 10mg/kg, 100 μ l/one is injected before 1h intraperitoneal injection; the DMSO complete control group was injected intraperitoneally with DMSO 100. mu.l. The 5-day survival rate of each group of mice was observed.

2. The experimental results are as follows: the experimental result of this example is shown in fig. 3, and it can be seen from fig. 3 that the survival rate of endotoxemia-treated mice is improved after the compound is used, which proves that the compound has a certain therapeutic effect on endotoxemia.

Example 1: protective effect of compound on sepsis mice

Cecal Ligation and Perforation (CLP) model experiment:

1. the experimental steps are as follows: selecting a mouse with the weight of 25-30 g, anesthetizing, making a lower abdominal incision to find the cecum, ligating the cecum from the distal end of the cecum to 50% of the ileocaecal region (CLP moderate model), performing opposite-penetrating ligation with a sterile needle to ligate the cecum, extruding a proper amount of intestinal contents from each hole, and then gently placing the pulled cecum back into the abdominal cavity to close the abdomen. The mice were post-operatively subcutaneously injected with pre-warmed physiological saline (1 ml/mouse) at 37 ℃ on both sides of the back and re-warmed to revival. The compound is injected into the abdominal cavity for 1h after operation at 4mg/kg, namely a CLP + compound group, and the CLP group is injected with DMSO with the same volume after operation. The 5-day survival rate of each group of mice was observed.

2. The experimental results are as follows: the experimental result of this example is shown in fig. 4, and it can be seen from fig. 4 that the survival rate of sepsis mice is improved after the compound is used, which proves that the compound has a certain therapeutic effect on sepsis.

In conclusion, the isoquinoline derivative provided by the invention has a certain treatment effect on sepsis and endotoxemia, and can effectively improve the survival rate of mice with sepsis and endotoxemia; meanwhile, the pharmacological action of the isoquinoline derivative on treating sepsis can be found by inhibiting macrophage apoptosis induced by Caspase11 and inhibiting the release of proinflammatory cytokines IL-1 alpha and IL-1 beta through the experiment. Provides a new treatment method and a new treatment medicine for clinical treatment of sepsis.

The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all equivalent changes made by using the contents of the present specification and the drawings, or applied directly or indirectly to the related technical fields, are included in the scope of the present invention.

Claims

1. The application of an isoquinoline derivative in the preparation of a medicament for treating sepsis, wherein the structure of the isoquinoline derivative is shown as follows:

2. the use according to claim 1, wherein the sepsis therapeutic agent is an agent that increases the survival of an infected sepsis animal.

3. The use according to claim 1, wherein the sepsis therapeutic agent is an agent capable of inhibiting apoptosis of cells in an animal infected with sepsis.

4. The use according to claim 1, wherein the sepsis therapeutic agent is an agent capable of inhibiting the secretion of pro-inflammatory cytokines in an animal infected with sepsis.

5. The use according to claim 4, wherein the proinflammatory cytokines comprise IL-1 α and IL-1 β.

6. The use according to any one of claims 1 to 5, wherein the medicament for the treatment of sepsis further comprises a pharmaceutically acceptable carrier or adjuvant.

7. The use according to any one of claims 1 to 5, wherein the medicament for the treatment of sepsis is formulated as required to be pharmaceutically acceptable.

8. The application of an isoquinoline derivative in the preparation of a cell apoptosis inhibitor is characterized in that the structure of the isoquinoline derivative is as follows:

9. the application of an isoquinoline derivative in the preparation of an inhibitor of proinflammatory cytokines, wherein the structure of the isoquinoline derivative is as follows:

10. the use according to claim 9, wherein the proinflammatory cytokines comprise IL-1 α and IL-1 β.