WO2019184997A1 - Pharmaceutical composition for preventing or treating sepsis - Google Patents

Abstract

The present invention provides a pharmaceutical composition for preventing or treating sepsis. Disclosed is a use of huperzine A in combination with TIENAM in preparing a drug for preventing or treating sepsis.

Description

Medicinal composition for preventing or treating sepsis Technical field

The invention belongs to the technical field of biomedicine, and in particular relates to a pharmaceutical composition for preventing or treating sepsis.

Background technique

The pathogenesis of sepsis is very complex. It is clinically characterized by inflammation-mediated tissue damage, organ failure, and immunosuppressive state. It is characterized by uncontrolled cytokine production, tissue damage, and intestinal bacterial/toxin migration. The pathogenesis of inflammatory response, immune dysfunction, coagulopathy, multiple organ and systemic changes in the body and their interactions, gene polymorphism and other pathological processes are still unclear. In February 2016, the American Society of Critical Care Medicine (SCCM) and the European Society of Critical Care Medicine (ESICM) jointly released the latest definition and diagnostic criteria for sepsis. It is believed that sepsis is a life-threatening organ dysfunction caused by the body's dysfunctional response to infection. This definition focuses more on the complex pathophysiological responses that occur when the body responds to infections, emphasizing "life-threatening organ dysfunction."

Huperzine A is a highly potent acetylcholinesterase inhibitor (Acetylcholinesterase) Inhibitors, AChEI), clinically Alzheimer's disease (Alzheimer) Disease, AD), enhance learning and memory, improve spatial memory disorder and other aspects have a special effect, the central acetylcholinesterase (Acetylcholinesterase) has high-efficiency, reversible, high selectivity inhibition, can promote the accumulation of acetylcholine (Acetylcholine, Ach). Cholinergic pathway in the relationship between parasympathetic and innate immune systems (Cholinergic In the anti-inflammatory pathway (CAP), the inflammatory stimuli of the afferent vagus afferent brain, through the central nervous system integration, release acetylcholine through the vagal nerve endings, and can specifically bind to cholinergic receptors on immune cells. Inhibition of inflammatory cytokine release, thereby effectively inhibiting local and systemic inflammatory responses; previous studies by the inventors found that HupA has a significant protective effect on the mouse model of sepsis, its protective mechanism and activation of α7nAChR-STAT3 signaling pathway, inhibition of NF- The level of κB phosphorylation is related to the nuclear entry of nuclear factor NF-κB.

TIENAM is a broad-spectrum antibacterial drug and is the drug of choice for clinical treatment of various serious infections. It is very suitable for mixed infection caused by aerobic/anaerobic bacteria or multiple pathogens, and before the pathogen is determined. Early treatment has a good effect; at the same time, it can effectively prevent postoperative infection caused by pollution or potentially contaminated surgery. The main ingredients of TIENAM are imipenem and cilastatin sodium (Cilastatin). Sodium), in which imipenem is the main antibacterial ingredient, as an imipenem antibiotic, imipenem is effective in inhibiting bacterial cell wall synthesis, and is a novel β-lactam antibiotic imipenem. A broader spectrum of bactericidal activity than other antibiotics. Cistatin sodium is a specific enzyme inhibitor whose main function is to increase the concentration of imipenem-producing drugs in the urinary tract and in the body by blocking the metabolism of imipenem in the kidney. Ability to antibacterial.

The present invention has been carefully studied and animal tested, and the results indicate that Huperzine A (HupA) combined with TIENAM has a significant therapeutic effect on sepsis.

technical problem

In the evaluation of the efficacy of drugs for the treatment of sepsis, the mortality rate of the animal model should reach or exceed 50% in order to accurately reflect the therapeutic effect of the drug. Cecal ligation and perforation (Cecal Ligation and Puncture, CLP) The mouse model of sepsis is widely used because of its advantages such as cheap and easy access to animals. CLP can trigger inflammation, immunity, hemodynamics, and biochemical changes very similar to human sepsis. It is the gold standard for current animal models of sepsis. The model of toxemia and bacterial infection can artificially control the dose of infection, stability and reproducibility. However, from the perspective of the clinical pathogenesis of sepsis, there are infectious lesions in the patient's body, and the infected lesion will continuously release the bacteria and endotoxin. Thereby causing a systemic inflammatory response and a change in the state of circulatory metabolism. Transient transient input of endotoxin or flora can cause rapid death in animals and does not mimic clinical features well. The mouse CLP sepsis model destroys the intestinal barrier and causes sustained diffuse release of the intestinal flora, simulating sepsis caused by peritoneal infection caused by perforation of clinical appendicitis or diverticulitis. The advantage is that the condition is not transient, The pathological process of clinically septic patients with systemic inflammatory response syndrome and multiple organ dysfunction syndrome has a better clinical correlation than the induction of LPS or live bacteria and the clinical manifestations of patients with sepsis.

Technical solution

It is an object of the present invention to provide a pharmaceutical composition comprising Huperzine A and Taineng for use in the treatment of sepsis, which is achieved by the following technical solutions:

A pharmaceutical composition for treating sepsis, wherein the dose of Huperzine A is 0.005-5 mg/kg·d, preferably 0.1 mg/kg·d; and the dose of Taineng is 1-500 mg/kg·d, preferably 50 mg/ Kg·d.

The preparation for treating sepsis may be by injection, tablet, lyophilized powder, capsule, film-coated tablet or other suitable dosage form.

Beneficial effect

The combination of Huperzine A and Taineng according to the present invention has obvious therapeutic effects, can inhibit the expression of inflammatory factors in serum to different degrees, and significantly reduce the pathological damage of sepsis to lung, liver and other tissues, and inhibit organ failure. The 7-day survival rate of the cecal ligation and perforation sepsis model mice was increased from 15% to 50% ( P<0.05 ).

DRAWINGS

Figure 1 is a statistical diagram of the effect of Huperzine A HupA combined with antibiotic Taineng TIENAM on the survival rate of CLP sepsis mice;

Figure 2a is a statistical diagram of the effect of HupA combined with antibiotic TIENAM on the expression of inflammatory factor IL-1β in CLP sepsis mice;

Figure 2b is a statistical diagram of the effect of HupA combined with antibiotic TIENAM on the expression of inflammatory factor IL-6 in CLP sepsis mice;

Figure 2c is a statistical diagram of the effect of HupA combined with antibiotic TIENAM on the expression of inflammatory factor TNF-α in CLP septic mice;

Figure 3 is a diagram showing the pathological changes of lung tissue in mice with CLP sepsis; A picture is blank group (Mock group), B picture is saline control group (CLP group), and C picture is sham operation control group (Sham) Group), D picture is HupA treatment group (CLP+A group), F picture is TIENAM group (CLP A+T group) and E picture is HupA+TIENAM combination group (CLP +T group);

Figure 4 is a diagram showing the pathological changes of renal tissue in mice with CLP sepsis; A picture is blank group (Mock group), B picture is saline control group (CLP group), and C picture is sham operation control group (Sham) Group), D picture is HupA treatment group (CLP+A group), F picture is TIENAM group (CLP A+T group) and E picture is HupA+TIENAM combination group (CLP +T group);

Figure 5 shows the expression and distribution of NF-κB (P65) in lung tissue of CLP sepsis mice by immunohistochemistry; A picture is blank control group, B picture is sham operation group, C picture is CLP model group, The D picture is the HupA group, the E picture is the TIMEN group, and the F picture is the HupA+TIMEN group.

Embodiments of the invention

The present invention is further illustrated by the following examples.

Example 1

The test method is as follows:

Huperzine A HupA Antibiotics TIENAM Combined pair CLP The effect of survival rate in septic mice

100 mice were randomly divided into 5 groups, sham control group (Sham group), saline control group (CLP group), HupA treatment group (0.1 mg/kg·d), TIENAM group (50 mg/kg·d). In combination with HupA+TIENAM, 20 mice in each group, all mice were fasted for 12 hours before surgery and were free to drink water. The blank control group did not do any treatment. The mouse model of CLP sepsis was prepared according to the literature. After the sham-operated control group was opened, the cecum was freed, ligated, not perforated, and then the abdominal cavity was closed and the abdomen was sutured. At the end of the operation, 120 mice were subcutaneously injected with 1 ml of 37 ° C pre-warmed physiological saline for fluid resuscitation; intraperitoneal injection, HupA + TIENAM combined group for intraperitoneal injection of TIENAM (50 mg / kg · d) and HupA (0.1 mg/kg·d). The wound was rubbed with lidoca hydrochloride every 12 hours within 3 days after surgery for pain relief; the postoperative behavior of the mice was observed, and the survival time and survival rate of the mice were judged. The observation was performed every 2 hours after 0-24 hours. The survival of the mice was recorded; the survival of the mice was recorded every 4 hours at 24-48 hours; the survival of the mice was recorded every 6 hours at 48-96 hours, and the Kaplan-Meier survival analysis was used for each group of mice. The survival rate was statistically analyzed (log-rank test). In order to avoid the influence of the physiological laws of the mice on the operation, the operation was selected at about 2 o'clock in the afternoon, and the operation time of each mouse should not exceed 10 minutes.

The test results are as follows:

Huperzine A HupA Joint antibiotic TIENAM Correct CLP The effect of survival rate in septic mice

In the CLP sepsis mouse model, HupA was combined with the antibiotic Taineng. The survival rate test results (as shown in Figure 1), combined with HupA or TIENAM alone, CLP sepsis mice for 7 days. The survival rate increased from 15% to 50% ( P < 0.05 ). Experimental observations revealed that after 24 hours of CLP surgery in mice, mice developed typical symptoms of sepsis: standing hair, diarrhea, wilting and squeezing together, and a large number of mice died; CLP surgery in mice for 48 hours After that, the survival rate is stable. The injection of HupA group failed to effectively inhibit the growth of a large number of microorganisms in the peritoneal cavity at 24 hours after surgery, resulting in a large number of deaths in mice, which reached stability 48 hours after surgery. Mice injected with TIENAM had the highest survival rate at 24 hours after surgery, but the survival rate showed a step-down after 48 hours after surgery and reached a steady state at 72 hours after surgery. The use of TIENAM can directly inhibit the growth of various microorganisms leaked from the cecum in the peritoneal cavity of mice, and improve the survival rate of mice within 24 hours after surgery; due to the large expression of inflammatory factors in mice and the accumulation of dead microorganisms, Mice injected with TIENAM group died after 24 hours after surgery, and the survival rate reached stable until 72 hours after surgery. Simultaneous injection of TIENAM and HupA mice showed death at 24 hours after surgery. The inhibition of inflammatory factors by HupA and the inhibition of microbial growth by antibiotics resulted in effective relief of sepsis at 36 hours after surgery.

Example 2

Detection of serum inflammatory factor expression levels

The expression levels of inflammatory factors IL-6, TNF-α, and IL-1β in the serum of peripheral blood were measured according to the procedure of the ELISA kit instructions.

The test results are as follows:

joint TIENAM Effect of cytokine levels in peripheral blood of mice

As shown in Figure 2, the serum levels of TNF-α, IL-1β, IL-6 and other inflammatory factors were lower in the sham-operated mice 48 hours after surgery; the levels of inflammatory factors in the serum of the CLP sepsis mouse model All of them were significantly increased, the difference was significant ( P<0.05 ), and statistically significant. The serum levels of TNF-α, IL-1β and IL-6 in HupA group and HupA+TIENAM group were smaller than those in CLP sepsis. The mouse model was significantly reduced and the difference was statistically significant ( P < 0.01 ).

Example 3

Histopathological examination

At 48 hours after operation, the neck was sacrificed and the lungs and kidneys of the mice were collected. Immediately fixed with 4% paraformaldehyde, numbered, dehydrated, transparent, immersed, embedded, sliced, and dewaxed according to the pathological examination. HE staining was performed to observe the pathological changes of the tissue under an optical microscope.

The test results are as follows:

joint TIENAM Correct CLP Observation of histopathological effects in mice with sepsis

Grouped by experiment, CLP sepsis mice were administered for 48 hours after model administration, and were sacrificed by cervical dislocation. The lungs, liver, spleen and kidney of the main organs of each group were taken for HE staining and their pathological changes were observed under microscope.

(1) As shown in Fig. 3, mice were randomly divided into 6 groups, blank group (Mock group) A map, saline control group (CLP group) B map, sham control group (Sham group) C map, HupA The treatment group (CLP+A group) D map, TIENAM group (CLP A+T group) F map and HupA+TIENAM combined group (CLP + T group) E map were administered by intraperitoneal injection according to the experimental procedure. The rats were sacrificed 48 hours after the operation, and the lung tissues of the mice were collected and stained with HE.

After 48 hours of model administration in CLP septic mice, the lung tissue and alveolar epithelial structure of the blank group and the sham operation group were clear and intact, the alveolar septum was uniform, and no inflammatory cell infiltration was observed; CLP sepsis mice Lung tissue showed alveolar structure destruction, wall parenchyma widened, inflammatory cell infiltration in the alveolar, telangiectasia; lung tissue and alveolar epithelium in HupA group and HupA+TIENAM group were relatively intact, the difference between the two groups was not obvious, and some alveolar septa were not obvious. Thickening, a small amount of inflammatory cell infiltration is still visible.

(2) As shown in Fig. 4, the mice were randomly divided into 6 groups, a blank group (Mock group) A map, a saline control group (CLP group) B map, a sham control group (Sham group) C map, HupA The treatment group (CLP+A group) D map, TIENAM group (CLP A+T group) F map and HupA+TIENAM combined group (CLP + T group) E map were administered by intraperitoneal injection according to the experimental procedure. The rats were sacrificed 48 hours after the operation, and the lung tissues of the mice were collected and stained with HE.

After 24 hours of model administration in CLP sepsis mice, the kidney structure of the kidney tissue of Mock group and TIENAM group was normal, no inflammatory cell infiltration and tubular dilatation were observed, and no abnormal pathological changes were observed; CLP sepsis mice The renal tissue was obviously edematous, and the renal small cystic cavity became small and narrow, the glomerular structure was abnormal, accompanied by a large number of inflammatory cell infiltration; the renal tissue structure of the HupA group and the HupA+TIENAM group was relatively normal, and the glomerular structure Clearer, only mild inflammatory cell infiltration, no renal stenosis and occlusion.

Example 4

joint TIENAM Correct CLP Sepsis in lung tissue of mice NF- κ B ( P65 ) expression

The lung organs were taken out, placed in an oven at 67 ° C, baked, dewaxed to water, washed 3 times with PBS solution for 5 minutes each time; then subjected to antigen retrieval, the antigen recovery solution was boiled in advance, and then the slide was placed Antigen repair solution, take it out after 20 minutes, and let it cool naturally at room temperature; then wash it with PBS three times for 10 minutes each time; after aspirating the PBS solution, add peroxidase blocker to each section and incubate at room temperature. After 10 minutes, wash with PBS solution 3 times for 5 minutes each time; after removing PBS solution, add non-specific stain blocker to each section and block for 30 minutes at room temperature; after removing PBS solution, add the prepared purpose one. The antibody was incubated overnight at 4 ° C; the next day, washed again with PBS for 3 minutes, 5 minutes each time, after removing the PBS solution, each section was further added with biotin-labeled secondary antibody, and incubated at room temperature for 30 minutes; then, Wash again with PBS 3 times for 5 minutes, then incubate with streptomycin avidin-peroxidase solution for 10 minutes at room temperature; wash PBS 3 times for 5 minutes each time; remove PBS solution, each piece The sections were further stained with DAB solution (diaminobenzidine) for 1 minute. The staining was observed under a microscope; after 10 minutes of deionized water washing, hematoxylin was counterstained for 30 seconds, and then rinsed with deionized water for 20 minutes; the sections were dehydrated and dried by gradient alcohol gradients of 95%, 95%, 100%, and 100%. Minutes, xylene was transparent for 5 minutes; air-dried, sealed with a neutral resin, dried, and placed under a microscope.

The test results are as follows:

Immunohistochemical analysis NF- κ B ( P65 )in CLP Expression and distribution of lung tissue in septic mice

As shown in Fig. 5, where A is a blank control group, B is a sham operation group, C is a CLP model group, D is a HupA group, E is a TIMEN group, and F is a HupA+TIMEN group. In the blank control group and the sham operation group, the expression of NF-κB (p65) protein was found in a few lung interstitial cells. In the CLP sepsis model, the expression of p65 protein was significantly increased; after HupA treatment, the expression of P65 protein was lower than that of CLP sepsis model, but there was no change in TIMEN treatment group; combined with HupA+TIMEN In the group, the expression level of p65 protein was significantly decreased, which was comparable to the blank control group and the sham operation group, and the expression of p65 protein was observed only in a few lung interstitial cells.

Claims (3)

1. A pharmaceutical composition for preventing or treating sepsis, characterized in that the pharmaceutical composition comprises Huperzine HupA, Taineng TIENAM, and a pharmaceutically acceptable carrier.
2. The pharmaceutical composition according to claim 1, wherein the oxalipine HupA drug dose is 0.005-5 mg/kg·d; the Taineng TIENAM drug dose is 1-500 mg/kg·d.
3. The pharmaceutical composition according to claim 2, wherein the oxalipine HupA drug dose is 0.1 mg/kg·d; and the Taineng TIENAM drug dose is 50 mg/kg·d.