Application of elcaindopristat in preparation of anti-sepsis product

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of elcaindopristat in preparation of an anti-sepsis product. According to the application disclosed by the invention, a sepsis mouse model is constructed through cecal ligation-puncture (CLP), and the medicine is administered by taking the elcaindopristat as an active component, so that the fatality rate of the sepsis mouse can be remarkably reduced, the survival rate of the sepsis mouse can be increased, the content of inflammatory factors in serum of the mouse can be reduced, the abnormality of tryptophan-kynurenine metabolites can be improved, and the curative effect of the sepsis mouse can be improved. And morphological abnormalities of lung and intestinal tissues. The icaindopristat is suitable for comprehensive prevention and treatment of sepsis, and has clinical popularization and application potential.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and particularly relates to the application of icardostat in preparing anti-sepsis products. Background Art

[0002] Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection. The pathogenesis is very complex and there is no specific drug for treatment. Sepsis is characterized by high morbidity and mortality, and is accompanied by abnormal immune responses, leading to life-threatening organ dysfunction. Currently, sepsis has become an important public health issue.

[0003] Cecal ligation-perforation (CLP) is the most clinical sepsis model. It uses a non-obstructive way to ligate and puncture the cecum, allowing fecal contents to leak into the abdominal cavity, establishing a mixed bacterial infection with inflammatory sources of necrotic intestinal tissue.

[0004] Epacadostat is an immune checkpoint inhibitor, and its main application area is cancer immunotherapy. It inhibits the differentiation of T cells into regulatory T cells (Treg) by inhibiting the activity of indoleamine 2,3-dioxygenase (IDO), and reduces the exhaustion of cytotoxic T cells (CTLs). In the process of tumor immunotherapy, it is regarded as an immune checkpoint inhibitor to enhance the body's anti-tumor immune response. At present, there is no relevant research on the effect of ecadostat on sepsis. Summary of the invention

[0005] The purpose of the present invention is to provide an application of icardostat in the preparation of an anti-sepsis product, enrich the medical uses of icardostat, fight against sepsis, and be used for the comprehensive prevention and treatment of sepsis.

[0006] The present invention provides the use of icardostat in preparing an anti-sepsis product.

[0007] Preferably, the anti-sepsis product includes a product that reduces sepsis mortality and / or improves sepsis survival rate.

[0008] Preferably, the anti-sepsis product includes a product that reduces sepsis inflammation.

[0009] Preferably, the product for reducing inflammation caused by sepsis includes a product for inhibiting the expression of pro-inflammatory factors caused by sepsis;

[0010] The pro-inflammatory factors include one or more of IL-1β, IL-6 and TNF-α.

[0011] Preferably, the anti-sepsis product includes a product that improves abnormal tryptophan-kynurenine metabolites caused by sepsis;

[0012] The tryptophan-kynurenine metabolites include one or more of tryptophan, kynurenine, quinolinic acid, picolinic acid and xanthuric acid.

[0013] Preferably, the anti-sepsis product includes a product that improves morphological abnormalities of lung and / or intestinal tissues caused by sepsis.

[0014] Preferably, the product includes medicine, reagent or feed.

[0015] The present invention also provides an anti-sepsis drug, wherein the active ingredient of the drug comprises icardostat; the icardostat is prepared as a single-use dosage form at 25 to 100 mg / kg / dose.

[0016] Preferably, the icardostat is formulated as a single-use dosage form at 50 mg / kg / dose.

[0017] Preferably, the drug further comprises a pharmaceutically acceptable excipient.

[0018] Beneficial effects:

[0019] The present invention provides the use of icadostat in the preparation of an anti-sepsis product. The present invention uses icadostat as an active ingredient, can reduce the mortality rate of sepsis, improve the survival rate of sepsis, significantly inhibit the expression of proinflammatory factors (IL-1β, IL-6 and TNF-α) caused by sepsis, reduce inflammation, significantly improve the abnormality of tryptophan-kynurenine metabolites (tryptophan, kynurenine, quinolinic acid, picolinic acid, xanthurenic acid) caused by sepsis, and significantly improve the morphological abnormalities of lung and intestinal tissues. Icardostat is suitable for the comprehensive prevention and treatment of sepsis and is suitable for clinical promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0021] Figure 1 is the survival rate of mice in different treatment groups after cecal ligation and puncture;

[0022] Figure 2 The levels of inflammatory factors in the serum of mice in different treatment groups after cecal ligation and puncture; * indicates p < 0.05; ** indicates p < 0.01; *** indicates p < 0.001;

[0023] Figure 3The results of H&E staining of the lungs of mice in different treatment groups after cecal ligation and puncture;

[0024] Figure 4 H&E staining results of intestinal tissues after cecal ligation and puncture in mice in different treatment groups;

[0025] Figure 5 The contents of tryptophan-kynurenine metabolism-related products in the serum of mice in different treatment groups after cecal ligation and puncture; where * indicates p < 0.05; ** indicates p < 0.01; *** indicates p < 0.001. DETAILED DESCRIPTION

[0026] The present invention provides the use of icardostat in preparing an anti-sepsis product.

[0027] As an embodiment, the product includes a drug, a reagent or a feed. The present invention has no strict requirements on the specific composition of the product. The active ingredient contains icardostat, and other ingredients are conventionally selected according to the type of product, and the product is guaranteed to have an anti-sepsis effect. As an embodiment, the icardostat is the only active ingredient in the product.

[0028] The present invention has no strict requirements on the source of the icardostat, and it can be purchased conventionally. As an embodiment, the CAS number of the icardostat is 1204669-58-8.

[0029] The present invention constructs a sepsis mouse model by cecal ligation-perforation (CLP), and administers icadostat as an active ingredient. The results show that icadostat can reduce the mortality rate of sepsis and / or improve the survival rate of sepsis, reduce sepsis inflammation, improve the abnormal tryptophan-kynurenine metabolites caused by sepsis, and improve the abnormal morphology of lung and / or intestinal tissues caused by sepsis. As an embodiment, the product for reducing sepsis inflammation includes a product that inhibits the expression of proinflammatory factors caused by sepsis. As an embodiment, the proinflammatory factors include one or more of IL-1β, IL-6 and TNF-α; as another embodiment, the proinflammatory factors include IL-1β, IL-6 and TNF-α. As an embodiment, the tryptophan-kynurenine metabolites include one or more of tryptophan, kynurenine, quinolinic acid, picolinic acid and xanthuric acid; as another embodiment, the tryptophan-kynurenine metabolites include kynurenine, quinolinic acid, picolinic acid and xanthuric acid.

[0030] The present invention also provides an anti-sepsis drug, the active ingredient of which includes icardostat; the icardostat is formulated as a single-use dosage form at 25 to 100 mg / kg / dose. As an embodiment, the icardostat is formulated as a single-use dosage form at 50 mg / kg / dose. As an embodiment, the drug also includes a pharmaceutically acceptable excipient. As an embodiment, the dosage form of the drug includes an injection. The present invention has no strict requirements on the type of the pharmaceutically acceptable excipient, and it can be selected according to the conventional dosage form of the drug.

[0031] As an embodiment, the single-use dose of icardostat is calculated according to the unit weight of mice. According to the animal-human drug dose conversion method, as another embodiment, according to the unit weight of humans, the icardostat is formulated as a single-use dosage form at 2.5-10 mg / kg / dose; as another embodiment, according to the unit weight of humans, the icardostat is formulated as a single-use dosage form at 5 mg / kg. As an embodiment, the drug is used by intraperitoneal injection.

[0032] To further illustrate the present invention, the use of icardostat provided by the present invention in preparing anti-sepsis products is described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0033] Example 1

[0034] Effect of Epacadostat (Ep) on the survival rate of septic mice

[0035] 1. Sixty SPF C57BL / 6J male mice aged 6 to 8 weeks were randomly divided into Sham group, EP group, CLP group, CLP+EP25 group, CLP+EP 50 group and CLP+EP 100 group, with 10 mice in each group, and the following treatments were performed:

[0036] Sham group: Normal saline was injected intraperitoneally once 2 hours before modeling and 24 hours after modeling. During modeling, mice were anesthetized with 1% sodium pentobarbital intraperitoneally, and a 1-2 cm longitudinal incision was made in the midline of the abdominal cavity to expose the cecum, which was returned to the abdominal cavity, and the peritoneum and skin were sutured; after surgery, mice were injected with 2 mL of sterile normal saline (0.9%) for fluid resuscitation.

[0037] EP group: The mice were treated with sham surgery in the same manner as the Sham group, except that 50 mg / kg of icadostat was injected intraperitoneally 2 hours before and 24 hours after modeling.

[0038] CLP group: Normal saline was injected intraperitoneally once 2 hours before modeling and 24 hours after modeling. During modeling, mice were anesthetized with 1% sodium pentobarbital intraperitoneally, and a 1-2 cm longitudinal incision was made in the midline of the abdominal cavity to expose the cecum. The cecum was ligated with No. 4 nylon thread at the midpoint of the cecum (accounting for 50% of the cecum), punctured with a No. 21 needle, and then a small amount of feces was squeezed out from the puncture hole. The cecum was returned to the abdominal cavity, and the peritoneum and skin were sutured. After surgery, mice were injected with 2 mL of sterile normal saline (0.9%) for fluid resuscitation.

[0039] CLP+EP 25 group: The sepsis mouse model was established in the same manner as the CLP group, with the only difference being that icardostat was intraperitoneally injected once at a dose of 25 mg / kg mouse 2 hours before and 24 hours after modeling;

[0040] CLP+EP 50 group: The sepsis mouse model was established in the same manner as the CLP group, with the only difference being that icardostat was intraperitoneally injected once at a dose of 50 mg / kg mouse 2 hours before and 24 hours after modeling;

[0041] CLP+EP 100 group: A sepsis mouse model was constructed in the same manner as the CLP group, with the only difference being that icardostat was intraperitoneally injected once at a dose of 100 mg / kg mouse 2 hours before and 24 hours after modeling.

[0042] 2. Statistics Step 1 The survival rate of mice within 7 days after the end of the experiment in each group (i.e., the last injection) was calculated to evaluate the effect of Epacadostat (EP) on the survival rate of septic mice. The results are as follows: Figure 1 And as shown in Table 1.

[0043] Table 1 Survival rate of mice in different treatment groups after cecal ligation and puncture (%)

[0044] Survival rate (%) 0d 1d 2d 3d 4d 5d 6d 7d Sham Group 100 100 100 100 100 100 100 100 EP Group 100 100 100 100 100 100 100 100 CLP Group 100 50 20 0 0 0 0 0 CLP+EP25 100 60 40 30 30 30 30 30 CLP+EP50 100 90 80 70 70 60 60 60 CLP+EP100 100 90 90 70 60 50 50 50

[0045] according to Figure 1 As shown in Table 1, icadolstat can significantly improve the survival rate of mice after cecal ligation and puncture (CLP) Figure 1 ), Icardostat (50mg / kg, 100mg / kg) can reduce the mortality rate of mice with significant sepsis.

[0046] Example 2

[0047] 1. After one week of adaptive feeding, SPF C57BL / 6J male mice aged 6 to 8 weeks were randomly divided into 4 groups, namely Sham group (n=8), CLP group (n=15), EP group (n=8), and CLP+EP 50 mg / kg group (n=15), and the following treatments were performed:

[0048] Sham group: Normal saline was injected intraperitoneally once 2 hours before modeling and 24 hours after modeling. During modeling, mice were anesthetized with 1% sodium pentobarbital intraperitoneally, and a 1-2 cm longitudinal incision was made in the midline of the abdominal cavity to expose the cecum, which was returned to the abdominal cavity, and the peritoneum and skin were sutured; after surgery, mice were injected with 2 mL of sterile normal saline (0.9%) for fluid resuscitation.

[0049] EP group: The mice were treated with sham surgery in the same manner as the Sham group, except that 50 mg / kg of icadostat was injected intraperitoneally 2 hours before and 24 hours after modeling.

[0050] CLP group: Normal saline was injected intraperitoneally once 2 hours before modeling and 24 hours after modeling. During modeling, mice were anesthetized with 1% sodium pentobarbital intraperitoneally, and a 1-2 cm longitudinal incision was made in the midline of the abdominal cavity to expose the cecum. The cecum was ligated with No. 4 nylon thread at the midpoint of the cecum (accounting for 50% of the cecum), punctured with a No. 21 needle, and then a small amount of feces was squeezed out from the puncture hole. The cecum was returned to the abdominal cavity, and the peritoneum and skin were sutured. After surgery, mice were injected with 2 mL of sterile normal saline (0.9%) for fluid resuscitation.

[0051] CLP+EP group: A sepsis mouse model was constructed in the same manner as the CLP group, with the only difference being that icardostat was intraperitoneally injected once at a dose of 25 mg / kg mouse 2 hours before and 24 hours after modeling.

[0052] 2. After the experiment of each group in step 1 was completed, mice were anesthetized with CO2 gas, and venous blood was collected from the mice through the abdominal aorta. After the serum was separated, it was placed in a -80℃ refrigerator for later use.

[0053] The ELISA detection method was used to detect the content of IL-6, TNF-α, and IL-1β in mouse serum. The specific operation steps were carried out according to the instructions of the kit. The specific experimental steps mainly included: (1) Preparation of standard sample; (2) First add 40 μL of sample diluent to the sample well to be tested, and then add 10 μL of sample. Add 50 μL of standard sample to the standard control well, and do not add sample and enzyme-labeled reagent to the blank well; (3) Incubate the sample support plate in a 37°C warm water bath for 30 minutes; (4) Wash the sample wells to be tested 5 times; (5) Discard the washing solution and add 50 μL of enzyme; (6) Incubate the sample support plate in a 37°C warm water bath for 30 minutes; (7) Wash the sample wells to be tested 5 times; (8) Incubate the sample support plate in a 37°C warm water bath for 10 minutes for color development; (9) Add the stop solution and incubate at OD 450 The absorbance value is detected by nm. The specific content test results are as follows Figure 2 And as shown in Table 2.

[0054] Table 2 Serum inflammatory factor levels (pg / mL) in mice after cecal ligation and puncture in different treatment groups

[0055]

[0056]

[0057] according to Figure 2 As shown in Table 2, icadolstat can significantly inhibit the expression of proinflammatory factors (IL-6, TNF-α, IL-1β) caused by sepsis.

[0058] 3. Step 1 After the end of each group of experiments, mice were anesthetized with CO2 gas, the chest cavity was opened, the lungs and colons of the mice were separated, and the residual body fluids were washed with cold PBS. Use formalin for fixation. After 24 hours, xylene-ethanol was used for dehydration and paraffin sample blocks were prepared. The paraffin sample blocks were sliced ​​using a tissue slicer, each slice was 5μm. The samples were rehydrated using xylene-gradient concentration ethanol (ethanol concentration decreased from 100v / v% to 50v / v%). H&E was used for staining, and gradient concentration ethanol (ethanol concentration increased from 50v / v% to 100v / v%)-xylene was used for permeabilization, and the slices were read after sealing. The results showed that compared with the normal group, the lung tissue structure of the model group mice was disordered, the septa were thickened, and there were a large number of inflammatory cells infiltrating; EP intervention did not affect the physiological structure of the lungs of normal mice, but EP could significantly improve the disordered lung tissue structure, thickened septa, and a large number of inflammatory cells infiltrating in mice caused by CLP ( Figure 3 ); at the same time, it can also be found that compared with the normal group, the model group mice can significantly induce changes in colon tissue structure and immune cell infiltration, while EP can significantly improve the changes in colon tissue structure and immune cell infiltration of mice caused by CLP ( Figure 4 ).

[0059] 4. Take 100 μL of the serum prepared in step 2, add 400 μL of extract (methanol / acetonitrile = 1:1, v / v), mix with 10 μL of internal standard (2 μg / mL), take 400 μL of pure methanol to pass through the HLB plate, discard the filtrate; then take 400 μL of extract (water / methanol / acetonitrile = 1:2:2, v / v) to pass through the HLB plate, and discard the filtrate. Centrifuge the treated serum sample at 4°C, 14000 rcf, 15 min, take 400 μL of supernatant to pass through the HLB plate; collect the filtrate, freeze-dry it, and store it at -80°C.

[0060] Agilent 1290 Infinity UHPLC system was used for separation. The standard was placed in an autosampler at 4°C, the column temperature was 40°C, the mobile phase A was 20 mM ammonium formate + 0.2% formic acid aqueous solution, the mobile phase B was 0.2% formic acid methanol solution, the flow rate was 400 μL / min, and the injection volume was 5 μL. The relevant liquid phase gradient was as follows: 0-2 min, phase B was maintained at 10%; 2-9 min, phase B changed linearly from 10% to 100%; 9-11 min, phase B was maintained at 100%; 11-11.5 min, phase B changed linearly from 100% to 10%; 11.5-14 min, phase B was maintained at 10%. Mass spectrometry analysis was performed using a 5500 QTRAP mass spectrometer (SCIEX) in positive / negative ion mode. The 5500QTRAP ESI source positive ion conditions are as follows: source temperature: 450°C; ion Source Gas1 (Gas1): 45; Ion Source Gas2 (Gas2): 45; Curtaingas (CUR): 40; ionSapary VoltageFloating (ISVF): 4500V. The specific analysis results are shown in Figure 5 and Table 3.

[0061] Table 3 Serum tryptophan-kynurenine metabolism-related product contents (ng / mL) in mice after cecal ligation and puncture in different treatment groups

[0062] content Tryptophan Kynurenine Quinolinic acid Picolinic acid Xanthoacic acid Sham Group 7952 142 32 36 9 EP Group 6944 190 65 124 41 CLP Group 8218 110 24 31 10 CLP+EP 7582 117 49 63 26

[0063] according to Figure 5 As can be seen from Table 3, icadolstat can significantly improve the abnormalities of tryptophan-kynurenine metabolites (tryptophan, kynurenine, quinolinic acid, picolinic acid, and xanthuric acid) caused by sepsis.

[0064] Based on the above content, it can be seen that icaridostat can fight sepsis, reduce the mortality rate of sepsis, improve the survival rate, reduce the content of inflammatory factors in serum, and improve the morphology of lung and intestinal tissues and tryptophan-kynurenine metabolism-related products.

[0065] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Use of icardostat in the preparation of anti-sepsis products.

2. The use according to claim 1, characterized in that: The anti-sepsis products include products that reduce sepsis mortality and / or improve sepsis survival rate.

3. The use according to claim 1, characterized in that: The anti-sepsis products include products that reduce sepsis inflammation.

4. The use according to claim 3, characterized in that: The products for reducing inflammation caused by sepsis include products for inhibiting the expression of pro-inflammatory factors caused by sepsis; The pro-inflammatory factors include one or more of IL-1β, IL-6 and TNF-α.

5. The use according to claim 1, characterized in that: The anti-sepsis products include products that improve abnormal tryptophan-kynurenine metabolites caused by sepsis; The tryptophan-kynurenine metabolites include one or more of tryptophan, kynurenine, quinolinic acid, picolinic acid and xanthuric acid.

6. The use according to claim 1, characterized in that: The anti-sepsis products include products that improve morphological abnormalities of lung and / or intestinal tissues caused by sepsis.

7. The use according to any one of claims 1 to 6, characterized in that: The product includes a medicine, a reagent or a feed.

8. An anti-sepsis drug, characterized in that: The active ingredient of the drug includes icandrostat; the icandrostat is formulated into a single-use dosage form at 25 to 100 mg / kg / dose.

9. The drug according to claim 8, characterized in that The icaridostat was formulated as a single-use dosage form at 50 mg / kg / dose.

10. The drug according to claim 8 or 9, characterized in that The drug also includes pharmaceutically acceptable excipients.

Images