WO2015051695A1

WO2015051695A1 - Use of pharmaceutical composition in manufacture of medicaments for treating sepsis and inflammatory lung injury

Info

Publication number: WO2015051695A1
Application number: PCT/CN2014/087043
Authority: WO
Inventors: 张正平; 杨士豹; 华垚; 陈荣; 王鹏; 龚兆龙
Original assignee: 烟台益诺依生物医药科技有限公司
Priority date: 2013-10-12
Filing date: 2014-09-22
Publication date: 2015-04-16
Also published as: CN104546832A

Abstract

The present invention relates to a use of a pharmaceutical composition in the manufacture of medicament for treating related diseases caused by endotoxemia. The pharmaceutical composition comprises 3-methyl-1-phenyl-2-pyrazolin-5-one or pharmaceutically acceptable salt thereof and borneol. The diseases include, but are not limited to, systemic inflammatory response syndrome, sepsis, severe sepsis, septic shock, sepsis related disseminated intravascular coagulation, acute lung injury, acute respiratory distress syndrome, multiple organ failure syndrome, septic brain injury and inflammatory lung injury caused by other reasons, including but not limited to bacterial pneumonia.

Description

Application of a pharmaceutical composition in preparing medicine for treating sepsis and inflammatory lung injury

Technical field

The present invention belongs to the field of pharmaceuticals and relates to the use of 3-methyl-1-phenyl-2-pyrazol-5-one and borneol compositions for the treatment of related diseases caused by endotoxemia caused by various causes.

Background technique

Sepsis is a common complication after severe trauma (burn), shock, and major surgery. It can be further developed into septic shock and multiple organ dysfunction syndrome (MODS). The incidence of sepsis is high, with more than 18 million cases of severe sepsis every year in the world, and 750,000 cases of sepsis in the United States each year, and this number is rising at a rate of 1.5% to 8.0% per year. The condition of sepsis is dangerous and the mortality rate is high. About 14,000 people die every day from the world, and about 215,000 people die each year in the United States. According to foreign epidemiological surveys, the mortality rate of sepsis has exceeded that of myocardial infarction, which has become the main cause of death of non-cardiac people in the intensive care unit. In recent years, despite advances in anti-infective therapy and organ function support technologies, the mortality rate of sepsis is still as high as 30% to 70% (J. Clin. Invest. 2003, 112: 460-467).

The complications of sepsis are clinical manifestations of various stages of sepsis pathophysiology. Common complications include shock, acute lung injury (ALI), acute respiratory distress syndrome (ARDS), and deep vein thrombosis (DVT). Stress ulcer, metabolic acidosis, disseminated intravascular coagulation (DIC) to multiple organ dysfunction syndrome (MODS).

The underlying pathogenesis of sepsis is unclear, involving complex systemic inflammatory network effects, genetic polymorphisms, immune dysfunction, coagulopathy, tissue damage, and host abnormal responses to different infectious pathogenic microorganisms and their toxins. In terms of the system, it is closely related to the multi-system and multi-organ pathophysiological changes. The pathogenesis of sepsis still needs to be further clarified.

Sepsis can be caused by infection in any part, clinically common in pneumonia, peritonitis, cholangitis, urinary tract infection, cellulitis, meningitis, abscess and so on. The most common pathogens of sepsis and septic shock are Gram-negative bacteria (mainly including Escherichia coli, Klebsiella and Pseudomonas aeruginosa). In the past 20 years, Gram-positive bacteria (such as staphylococcus, streptococcus) have been increasing infections and have accounted for about 50% of all cases. Guidelines for the treatment of severe sepsis As always, the basic treatment needs to use antibiotics to control the source of infection.

Oxidative stress during sepsis is reflected by lipid peroxidation and anti-oxidation in plasma. Oxidative damage may be an important cause of cell damage, organ dysfunction, and death. The oxidant causes the cells to produce lipid peroxides, and a large amount of oxygen radicals act on the lipids to raise the lipid peroxides, resulting in tissue damage. Oxygen free radicals cause mitochondrial swelling, mitochondrial dysfunction, lipid peroxidation, inhibition of respiration, and depletion of mitochondrial antioxidants. Long-term exposure to NO in oxidative damage leads to cell damage. This damage occurs in a paracrine or autocrine form, thereby inhibiting cell respiration, resulting in uneven blood flow distribution, increased intestinal permeability, and peroxide. The production of ischemia-reperfusion leads to the production of intestinal superoxide dismutase, which leads to an increase in the level of oxygen free radicals, and a large amount of peroxide is produced from nitric oxide. Increased nitric oxide production means intestinal damage, intestinal dysfunction, and bacterial translocation.

Innate immune cells (including monocytes, macrophages, neutrophils, NK cells, etc.) recognize the Pathogen Associated Molecular Pattern (PAMP) by the indicated Pattern Recognition Receptor (PRR). It includes cell wall components of various bacteria such as lipopolysaccharide, peptidoglycan, muramic acid, flagellin, lipoteichoic acid, etc., which can activate multiple signals in cells and make immune cell intracellular transcription factors (NF-κB, AP1, Fos, c-Jun, etc.) activation, resulting in the release of a large number of cytokines. The lamina propria and mucosa contain a large number of immune cells, including T lymphocytes, B lymphocytes, macrophages, mast cells, and neutrophils. In the case of sepsis, these cells release a large number of cytokines, including IL-1β, IL-4, IL-6, IL-10, IFN-γ, and TNF-α. IFN-γ and TNF-α can increase the permeability of intestinal mucosal epithelial cells under pathological conditions. IFN-γ secreted by intraepithelial lymphocytes is an important factor in the loss of intestinal mucosal function caused by complete parenteral nutrition. IFN-γ leads to an increase in the expression of iNOS, especially when combined with TNF-α and IL-1β. It is currently believed that cytokines increase iNOS resulting in increased permeability of the intestinal mucosa.

A large number of clinical and preclinical evidences indicate that reactive oxygen species (ROS) levels are elevated during the development of sepsis, antioxidants are absent, and oxidative stress markers accumulate. ROS produced by inflammatory cells can kill microorganisms directly, but excessive ROS can cause cell damage (Intensive Care Med. 2000, 26, 474–476). The brain is an immune-active organ, and systemic inflammatory reactions caused by systemic diseases such as sepsis also have a severe impact (Neuro Immuno Modulation 2005, 12, 255–269). In fact, brain tissue is more sensitive to sepsis damage due to its high oxygen consumption rate and low antioxidant capacity (Crit. Care Med. 2008, 36, 1925–1932). Clinically, it has been found in recent years that cognitive function declines in patients with sepsis survival, which in turn affects their quality of life (J Neurol Neurosurg Psychiatry 2013, 84: 62-69).

3-methyl-1-phenyl-2-pyrazoline-5-one (Edaravone, edaravone) as a new powerful free radical scavenger to scavenge hydroxyl radicals (·OH), one Nitric Oxide Free Radical (NO·), Peroxynitrite (ONOO ^- ) (Chem Pharm Bull 2004, 52(2): 186-91; Redox Rep. 2002, 7(4): 219-22.J PharmacolExpTher .2007Jul;322(1):274-81), inhibiting cell peroxidation damage, as an effective neuroprotective agent (free radical scavenger) with wide distribution, half-life, safety, low toxicity, etc. An effective first-line treatment for ischemic stroke (Guidelines for the diagnosis and treatment of acute ischemic stroke in China (2010 edition)). Animal efficacy shows that edaravone has a clear effect on LPS-induced sepsis and CLP-induced sepsis, which can reduce animal mortality and reduce lung and liver damage in animals (J PharmacolExpTher. 2003, 307 ( 1): 74-82; Shock. 2009, 32(6): 586-92; Tohoku J Exp Med. 2011, 223(4): 235-41; Journal of Dalian University of Science, 2012, 34(4): 343-347 ). Patent CN200510009561.1 also shows that edaravone can reduce LPS-induced animal mortality, improve myocardial contractility and reduce oxidative damage. The structure of Edara is as follows:

The borneol is divided into synthetic borneol and natural borneol. Synthetic borneol contains isoborne brain, which has toxic side effects on the human body; natural borneol does not contain isoborneol. Natural borneol belongs to the class of bicyclic monoterpenoids, which can be processed from crystalline crystals of the genus Dryobalanopsaromatica Gaertn.f. resin of the Dipterocarpaceae plant (traditional medicine, 2006, 15(9): 57). Among the volatile oils of plants, Valeriana officinalis, Matricaria chamomilla, Lavandulaofficinalis, and the like (Biochem Pharmacol. 2005, 69(7): 1101-11). Studies have shown that natural borneol has a strong anti-inflammatory effect, possibly by inhibiting the transcription factor NF-κB activation, inhibiting the expression of inflammatory proteins (iNOS and COX-2) and inflammatory cytokines (TNF-α, IL-1β, etc. Release, and thus the role of cytoprotection (Neuroscience. 2011, 176: 408-19; West China Pharmaceutical Journal 2006, 21 (6): 523-526). Natural borneol can penetrate the blood-brain barrier, protect against cerebral infarction area and ischemia-induced inflammatory response, and improve energy metabolism, thereby reducing ischemic brain damage (West China Pharmaceutical Journal 2005, 20 (4): 323-325 Journal of Xinxiang Medical College 2006, 23(1): 23-25). Animal experiments have shown that natural borneol can also improve blood coagulation and antithrombotic activity in rats (Am J Chin Med. 2008; 36(4): 719-27). In addition, natural borneol also enhances the inhibitory amino acid GABA receptor activity (Biochem Pharmacol. 2005, 69(7): 1101-11). The structure of natural borneol is as follows:

The 3-methyl-1-phenyl-2-pyrazolin-5-one and natural borneol mass ratio 4:1 compositions have been tested in clinical trials for the treatment of ischemic stroke. Preclinical animal experiments show that the mass ratio of the two can reduce the area of cerebral infarction in a synergistic ratio of 4:1 to 1:1 (patent CN 101848711 B). According to the pathogenesis of sepsis mentioned above, a combination of 3-methyl-1-phenyl-2-pyrazolin-5-one and natural borneol is selected to exert a mechanism for scavenging free radicals and inhibiting inflammation, and synergistic resistance Excessive inflammatory response during the onset of sepsis, reducing tissue damage, thereby reducing sepsis mortality.

Summary of the invention

An object of the present invention is to provide a pharmaceutical composition for the preparation of a medicament for treating a disease associated with endotoxemia, which comprises 3-methyl-1-phenyl-2-pyrazoline- 5-ketone or a pharmaceutically acceptable salt thereof and borneol, and further, the combination of the drugs can synergistically increase the efficacy of a disease associated with endotoxemia.

The borneol of the present invention comprises natural borneol.

Related diseases of the present invention include systemic inflammatory response syndrome (SIRS), sepsis, severe sepsis, sepsis shock, and sepsis-related diffuse intravascular Coagulation (DIC), acute lung injury (ALI), acute respiratory distress syndrome (ARDS) and multiple organ failure syndrome (MOFS) and septic brain injury, and other causes of inflammatory lung injury, including but not limited to bacteria Pneumonia.

Preferably, the above composition is used for the preparation of a treatment for sepsis, severe sepsis, septic shock, and septic brain injury and inflammatory lung injury.

Use of 3-methyl-1-phenyl-2-pyrazol-5-one Patent Document CN200510009561.1 discloses: tail vein administration of edaravone 1.5, 3, 6 mg/kg can significantly improve LPS-induced pus Symptomatic indicators of toxic rats, including animal survival rate.

The present inventors have found that a combination of 3-methyl-1-phenyl-2-pyrazolin-5-one and natural borneol has a clear therapeutic effect in the treatment of sepsis, than 3-methyl-1-phenyl-2. -Pyrazolin-5-one alone is more effective, especially in reducing Mortality, reduction of inflammatory factors production, improvement of tissue damage, etc. have obvious superiority, can significantly increase survival rate and median survival, reduce excessive inflammatory response in sepsis, thereby reducing organ damage.

Preferably, the weight ratio of 3-methyl-1-phenyl-2-pyrazolin-5-one to natural borneol is from 10:1 to 1:10, and the preferred weight ratio is from 4:1 to 1:4. A further preferred weight ratio is from 4:1 to 2:1, more preferably 10:1, 4:1, 2:1, 1:2, 1:4 or 1:10.

The present invention provides suitable routes of administration and frequency of administration of a combination of 3-methyl-1-phenyl-2-pyrazolin-5-one and natural borneol. Because the patient's inflammatory response is excessive, the condition is critical, and the mortality rate is high, it is best to administer it by intravenous injection, and it is better to administer the drug twice a day for two consecutive days.

DRAWINGS

Figure 1. Effect of composition (4:1) on survival of CLP model mice.

Figure 2-A Effect of different doses of edaravone on the survival rate of CLP model rats.

Figure 2-B Effect of composition (4:1) on survival of CLP model rats.

Figure 3-A Effect of composition (4:1) on plasma TNF-α (A) content in rats with severe sepsis.

Figure 3-B Effect of composition (4:1) on plasma IL-6 (B) levels in rats with severe sepsis.

Figure 4. Effect of different ratio compositions on survival rate in septic rats.

Fig. 5 Pathological changes of lung tissue (400X) at 6h, 12h and 24h after LPS modeling and drug treatment.

Figure 6-A shows the effect of multiple doses of drugs (6 and 24 h after modeling) on the pathology of LPS-induced lung injury.

Figure 6-B shows the effect of multiple doses of drugs (6 and 24 h after modeling) on pathological scores of LPS-induced lung injury.

Figure 7-A shows the effect of drugs on LPS-induced serum inflammatory factor TNF-α

Figure 7-B shows the effect of drugs on LPS-induced serum inflammatory factor IL-6 in mice

Figure 7 shows the effect of drugs on LPS-induced MPO activity in lung tissue of mice.

Figure 8-A shows the effect of drugs on white blood cell counts in lung tissue lavage fluid of LPS-induced model mice.

Figure 8-B shows the effect of drugs on inflammatory factor TNF-α in lung tissue lavage fluid of LPS-induced mouse models.

Figure 8-C shows the effect of drugs on inflammatory factor IL-6 in lung tissue lavage fluid of LPS-induced model mice.

Figure 9-A shows the effect of composition (4:1) on secretion of KC (keratinocyte growth factor) by MLE-12 cells.

Figure 9-B Effect of edaravone on the secretion of KC (keratinocyte growth factor) by MLE-12 cells.

Figure 9-C Effect of natural borneol on secretion of KC (keratinocyte growth factor) by MLE-12 cells.

detailed description

The following examples are illustrative of the invention and are not to be construed as limiting the invention. Mentioned in the examples Edaravone is 3-methyl-1-phenyl-2-pyrazolin-5-one.

Example 1: Replicating mouse CLP sepsis (severe) model

Male BALB/c mice weighing 25-30 g were depilated on the abdomen one day before the experiment. Intraperitoneal injection of pentobarbital sodium 40mg/kg, fixed after anesthesia, disinfected with iodophor; 1.5 incision along the midline of the abdomen to find the cecum, carefully peel the mesentery, ligation of the cecum in the root of the cecum, to avoid ligating the ileum and cecal mesenteric vessels. The cecum was punctured 3 times with a 16-gauge needle and a small amount of intestinal contents was extruded. The cecum was also placed in the abdominal cavity, and the abdominal wall incision was sutured layer by layer. After the operation, the penicillin eye ointment was applied to prevent infection, and the wound was bandaged. The mice recovered their activity capacity about 20 minutes after the operation was completed. Before CLP surgery, the mice were lively and active, the fur was smooth and radiant, the lips and front and rear claws were ruddy, the eating and drinking were active, and the bowel movements were normal. After CLP surgery, the mice showed impotence, vertical hair, lips, and front and rear claws were red. Reduced eating and drinking, loose stools, congestion in the eyes, fever and fever; mice died within 8-30 hours after surgery. At autopsy, multiple organ abnormalities were found and pathological damage occurred. These indicate that the model is successful.

Example 2: Effect of single administration of composition (4:1) on survival rate of CLP model mice

BALB/c mice, male, 64, were randomly divided into sham operation group, sepsis model group, edaravone treatment group (single iv, 6 mg/kg) and composition (4:1) treatment group ( Single iv, 7.5 mg/kg).

The composition (4:1) represents edaravone 3 mg/kg + natural borneol 0.75 mg/kg.

Edaravone injection, specification 5mL: 10mg (including edaravone 10mg), produced by Nanjing Xiansheng Dongyuan Pharmaceutical Co., Ltd.; composition (4:1) injection, the specification is 5mL: 12.5mg (including Edaravone 10 mg and natural borneol 2.5 mg were dissolved in 8% 1,2,-propanediol aqueous solution). Both were diluted with physiological saline to the desired concentration before use. The tail vein was slowly bolused (<5 min) 30 minutes after CLP modeling.

Animal deaths were counted every 4 hours after the above 4 groups of animals. The results showed that edaravone can delay animal death and improve the median survival of the animals. The composition (4:1) can reduce animal mortality and prolong sepsis. The median survival of the mice was superior to the edaravone treatment group (see Figure 1 and Table 1).

Table 1 Median survival of each group of mice (n=16)

Example 3: Replication of rat CLP sepsis model

Male Sprague-Dawley rats, weighing 250-280 g, were anesthetized with intraperitoneal injection of pentobarbital sodium 40 mg/kg, abdomen hair removal, disinfection, and sterile hole towel. Make a 1.5cm incision along the midline of the abdomen, find the cecum, carefully The mesentery is removed and the cecum is ligated at the base of the cecum to avoid ligation of the ileum and cecal mesenteric vessels. The cecum was punctured twice with a 16-gauge needle and a small amount of intestinal contents was extruded. The cecum was also placed in the abdominal cavity, and the abdominal wall incision was sutured layer by layer. Animals were immediately injected subcutaneously with pre-warmed saline 10 mL anti-shock. Before CLP surgery, the rats were active and active, the fur was smooth and smooth, the lips and front and rear claws were ruddy, and the eating and drinking were active, and the bowel movements were normal. After CLP surgery, the rats showed impotence, vertical hair, lips, and front and rear claws were red. Eating and drinking water decreased, and stools were thin; rats died after 12-48 hours. At autopsy, multiple organ abnormalities were found and pathological damage occurred. These indicate that the model is successful.

Example 4: Effect of multiple doses of composition (4:1) on survival rate of CLP model rats

Eighty SD rats were randomly divided into sham operation group, CLP model group, edaravone treatment group (3mg/kg) and composition (4:1) treatment group (3.75mg/kg, including edaravone). 3 mg/kg and natural borneol 0.75 mg/kg). The administration was diluted with physiological saline to a dose of 10 mL/kg. 30 minutes after CLP modeling, the tail vein was injected, followed by tail vein administration once every 12 hours, twice daily (BID, once every 12 hours). Rat deaths were recorded every 4 hours after CLP administration.

Edarabi's new patent document CN200510009561.1 discloses that a single tail vein administration of edaravone at 1.5, 3, and 6 mg/kg can significantly improve the survival rate of LPS-induced sepsis rats. The present study found that edaravone 1.5 and 3 mg/kg (BID) treatment can significantly improve the survival rate of animals, but the high dose of edaravone 6 mg/kg (BID) did not improve the survival rate of the animals, but increased after 24 hours. Animal mortality (see Figure 2-A). The composition (4:1) 3.75mg/kg (BID) treatment effect was significantly better than the edaravone best treatment group (3mg/kg, BID), which not only improved the median survival of the animals, but also improved the severe pus Survival rate of toxic animals (see Figure 2-B and Table 2). This indicates that natural borneol in the composition can increase the efficacy of edaravone in the treatment of sepsis.

Table 2 Median survival of each group of rats (n=20)

Example 5: Effect of composition (4:1) administration on plasma inflammatory factors in septic rats

Twenty-four SD rats were randomly divided into sham operation group, CLP model group, edaravone treatment group (3 mg/kg) and composition (4:1) treatment group (3.75 mg/kg, including edaravone). 3 mg/kg and natural borneol 0.75 mg/kg). 30 minutes after CLP modeling, the tail vein was injected once; after administration, blood was collected from the eyelids at 2h and 6h, respectively, and the plasma was separated and detected by a rat ELISA kit (Excore Co., Ltd.). TNF-α and IL-6 levels. The graph data in Figure 3 is represented as mean±SD; the results of t-test analysis are expressed as: &&&&, p<0.0001, compared with sham surgery; ****, p<0.0001, compared with CLP model group; # #,p<0.01,###, p<0.001, compared with the edaravone 3 mg/kg group.

As shown in Figure 3-A and Figure 3-B, TNF-α (2h) and IL-6 (6h) were significantly elevated in rat plasma after CLP surgery. The levels of TNF-α and IL-6 in the plasma of the edaravone group were significantly decreased, while the levels of TNF-α and IL-6 in the plasma of the composition (4:1) group were lower than those of the edaravone group. This indicates that edaravone can reduce the inflammatory response caused by CLP, and the composition inhibits inflammation significantly better than edaravone, which can better control the excessive inflammatory response in septic rats.

Example 6: Protective effect of composition on cognitive impairment in septic rats

The CLP modeling and resuscitation method is the same as in Example 3. All model rats were injected subcutaneously with antibiotics ceftriaxone 30 mg/kg and clindamycin 25 mg/kg every 6 hours for 3 days as a basic treatment. They were randomly divided into model group and edaravone group (3 mg/ Kg) and composition 4:1 group (3.75 mg/kg, containing edaravone 3 mg/kg, natural borneol 0.75 mg/kg), 15 in each group. A sham operation group (n=15) was also established. The animals in the drug-administered group were given the first tail vein intravenously 30 minutes after CLP, and then administered once a day for 30 days. The model group and the sham operation group were given the corresponding volume of solvent. An inhibitory avoidance test was performed 30 days after modeling.

The inhibitory avoidance test includes learning training and memory testing. The detection device is a 50×25×25 cm box, and the bottom is composed of stainless steel bars of the same diameter (1 mm in diameter), each spaced 1 cm apart, and a platform of 7 cm wide and 2.5 cm high. In the learning training trial, the rats were placed on a platform and the latency of their limbs all falling onto the grid was recorded. Once the animal falls to the grid, it will be subjected to a 2 second 0.4 mA electric shock and then placed back into the squirrel cage. After 24 hours of training and training experiments, a memory test was performed. In addition to no foot shock, the test procedure is consistent with the training time, and the latency (maximum 180 seconds) falling on the grid is used as a detection index for inhibitory avoidance memory.

The effect of edaravone and composition 4:1 on the inhibitory avoidance latency is shown in Table 3. The results of the test were analyzed by Scheffe's one-way analysis of variance. There was no significant difference in the incubation period between the groups (F(3, 56) = 0.116, p = 0.951). In the memory experiment, each group had a significant difference in the gate latency (F(3, 56) = 36.234, p = 0.000). Compared with the model group, edaravone and composition 4:1 were able to significantly extend the latency (F(3,56)=36.234, p=0.000, p=0.000), and the composition was strong in 4:1. In Yida Lafeng. At the same time, the model group also had a very significant difference compared with the sham operation group (F(3, 56) = 36.234, p = 0.000). The above test results suggest that edaravone and composition 4:1 have a significant improvement effect on the inhibitory avoidance memory disorder after CLP in rats, wherein the composition 4:1 is more effective.

Table 3 Effect of Edaravone and Composition 4:1 on Inhibitory Avoidance Latency

Mean ± standard error. ***p<0.001 compared to the model group

Example 7: Effect of different ratios on survival rate in septic rats

Different proportions of compositions, including edaravone: natural borneol mass ratios of 10:1, 4:1, 2:1, 1:2, 1:4 and 1:10, respectively, are shown in Table 4. The solvent of the composition was physiological saline containing 8% 1,2-propanediol. The above composition was diluted with physiological saline to the administration volume before administration.

Table 4 composition configuration table and animal administration volume

* The composition was first dissolved with 1,2-propanediol and then diluted with pre-warmed physiological saline.

108 SD rats were randomly divided into sham operation group, CLP model group, composition (10:1) treatment group (3.3 mg/kg), composition (4:1) treatment group (3.75 mg/kg), combination. (2:1) treatment group (4.5 mg/kg), composition (1:2) treatment group (9 mg/kg), composition (1:4) treatment group (15 mg/kg) and composition (10: 1) Treatment group (33 mg/kg). The doses of each group were edaravone 3 mg/kg and natural borneol were 0.3 mg/kg, 0.75 mg/kg, 1.5 mg/kg, 6 mg/kg, 12 mg/kg and 30 mg/kg, respectively. The mode of administration is tail vein injection, which is administered twice a day (BID, once every 12 hours). CLP was performed in the late sepsis 30 min after the severe sepsis model, followed by tail vein administration once every 12 h (BID); the death of the rats was recorded every 4 h after CLP administration.

As shown in Figure 4 and Table 4, the compositions (4:1 to 1:4) were paired with animals in the different ratio combinations designed. Both survival and median survival rates improved. From the animal survival rate (Fig. 4), it was found that the natural borneol composition in the composition showed two stages: the lower dose of natural borneol increased the edaravone to increase the survival rate of the animal, but the high dose of natural borneol did not use the animal later. Survival, high doses of natural borneol (12, 30 mg / kg, BID) multiple doses may be toxic to severe sepsis animals.

Table 4 Median survival of each group of rats (n=16)

Example 8: Effect of replicating mouse acute lung injury (ALI) model and composition on lung injury

Healthy male C57BL/6 mice, 7-8 weeks, weighing 20-23 g. Eighty-five C57 male mice were randomly assigned to each group of 5-6, and the penis intravenous injection of the composition (4:1) immediately after instillation of LPS 1 mg/kg (12.5 mg/kg, containing edaravone 10 mg) /kg and natural borneol 2.5mg/kg), edaravone (10mg/kg), natural borneol (2.5mg/kg), dexamethasone (5mg/kg) and normal saline, etc., normal control airway Instilled saline. The animals were sacrificed in three batches at 6h, 12h, and 24h after LPS instillation. After ligation of the right lung, the trachea was intubated and the bronchoalveolar lavage fluid (BALF) was performed in 2 mL of normal saline solution. The recovery rate was obtained. Up to 90%, after mixing, the total number of cells was counted, centrifuged at 250 × g for 10 min, and the cell pellet was smeared. After cooling, the cells were stained with Wright's stain for cell sorting. The right lower lobe was fixed with neutral formalin solution (n=3), paraffin sections, and HE staining.

Effect of a single intravenous composition on the pathology of lung tissue in ALI mice:

As shown in Fig. 5, the ALI mice generally observed that the airway instilled into the LPS model mice had a bit of bleeding spots and edema on the surface of the lung tissue, while the physiological control group showed no abnormal changes in the lung appearance. Histopathological sections showed a large amount of neutrophil accumulation in the lumen of the airway and in the lumen of the lumen. A large amount of neutrophil infiltration and erythrocyte exudation were observed in the interstitial space of the lung. There was protein edema in the alveoli, and the edge of the alveolar space was light. A transparent film is formed. LPS induced inflammatory infiltration of lung tissue increased with time, and tissue inflammatory cell infiltration at 24h resulted in structural damage.

When LPS was induced for 6 h, the degree of lung tissue damage in each drug-administered group was lighter than that in the LPS model group. At the same time points after LPS modeling (6, 12 and 24 h, respectively), the overall pathological changes in the composition (4:1) group were lighter than those of edaravone and natural borneol, suggesting that the composition (4:1) was better. Low protection acute lung injury.

Example 9: Effect of multiple intravenous injections on pathology of ALI mice

Male Bal/c mice weighing 20-22 g. Twenty randomly divided into groups, each group of 4-7, after pentobarbital sodium anesthesia, surgical incision tracheal instillation into LPS (10μL / 50μL) suture, respectively, in the tail vein injection of edara 0.5h and 6.5h after modeling Feng (6mg/kg), composition 4:1 (7.5mg/kg, edaravone 6mg/kg, natural borneol 1.5mg/kg) or intraperitoneal injection of dexamethasone DEX (5mg/kg) 3h after modeling The model group was injected with a vehicle control in the tail vein. 24 hours after model establishment, lung tissue was fixed in 10% formalin, and conventional paraffin sections were taken and HE stained. Light microscopy: 1) The alveolar wall has congestion, edema, inflammatory cell infiltration; 2) There is no degeneration or necrosis of the bronchial epithelial cells in the lung, and there is no exudate in the alveolar cavity; 3) Branches and perivascular branches of the bronchus in the lung There is no edema, inflammatory cell infiltration. As shown in Figure 6-A, the results of the microscopic examination are as follows:

(1) Model group (7):

Normal lung tissue consists of alveolar, intrapulmonary bronchial branches, blood vessels and interstitial. The structure is clear. There is no inflammatory exudate in the alveolar cavity and bronchial cavity. There is no inflammatory cell infiltration in the bronchus and blood vessels in the lung. Most mice have alveolar walls. Mild congestion.

Alveolar wall: 7 mice had mild or moderate venous inflammatory cell infiltration in the alveolar wall. The inflammatory cell types were mainly neutrophils and mononuclear macrophages, and a few (3) had mild or slight bleeding locally. In the heavier lesions, the surrounding alveolar cavity is enlarged and is emphysema.

Intrapulmonary bronchus and perivascular tissue: the space around the perivascular tissue of the lung is significantly widened, with irritated red edema fluid (perivascular edema) and infiltration of inflammatory cells of the same type, with varying degrees of severity, mostly mild or mild. 1 moderate.

(2) edaravone group (4 rats): the degree of cell infiltration of lung tissue was significantly reduced compared with the model group

Alveolar wall: 4 mice with mild hyperemia, mild or moderate inflammatory cell infiltration, inflammatory cell type same as before, 3 local minor hemorrhage, 3 mild or mild emphysema.

Intrapulmonary bronchus and perivascular tissue: 2 perivascular tissues have a very small amount of inflammatory cells infiltrated with the same type, and 1 has mild edema.

(3) Composition (4:1) group (4): the degree of cell infiltration of lung tissue inflammation was reduced compared with the model group

Alveolar wall: 4 mice with mild hyperemia, mild or moderate inflammatory cell infiltration, inflammatory cell type same as before, 1 local minor bleeding.

Intrapulmonary bronchus and perivascular tissue: 2 perivascular tissues have a very small amount of inflammatory cell infiltration of the same type, and mild edema.

(4) DEX group (5 rats): The lesions in the positive drug group were significantly relieved compared with the model group. The main lesion was alveolar wall congestion. Infiltration of inflammatory cells

Alveolar wall: 5 mice with mild hyperemia, mild or moderate inflammatory cell infiltration, inflammatory cell type same as before, and 1 mild emphysema.

Intrapulmonary bronchus and perivascular tissue: There were a small or very small amount of inflammatory cells infiltrating into the perivascular tissue of the two lungs, one of which was mildly edematous.

According to the degree of light to heavy, the lesions were recorded as 0.5 (slight), 1 (mild), 2 (moderate), 3 (severe), and no lesions were 0 (negative), and all scores were added. Calculate the average score for each animal in each group. The lung tissue damage scores of the drug groups were shown in Figure 6-B. Both edaravone and the composition (4:1) were able to reduce LPS-induced lung injury.

Example 10: Effect of a single intravenous injection composition on serum inflammatory factors

Male Bal/c mice weighing 20-22 g. Twenty-four randomized groups, 6 in each group, were anesthetized with sodium pentobarbital, and the trachea was instilled into LPS (10 μL/50 μL). The edaravone (6 mg/kg) was injected into the tail vein 0.5 h after modeling. The composition was 4:1 (7.5 mg/kg, wherein edaravone 6 mg/kg, natural borneol 1.5 mg/kg) or intraperitoneal injection of dexamethasone DEX (5 mg/kg), and the model group was injected with a vehicle control in the tail vein.

2 hours after modeling, blood was collected from the eyelids and serum was separated by centrifugation. The serum levels of TNF-α and IL-6 were determined according to the ELISA (Essay) instructions. As shown in Figures 7-A and 7-B, composition 4:1 significantly reduced the levels of inflammatory factors TNF-α and IL-6 in the serum at the early (2 h) induction of LPS, and its effect was due to the edaravone group. The histogram in Figure 7 shows Mean ± SEM (n = 6), One-Way ANOVA analysis results: **, p < 0.01, ***, p < 0.001, compared with the model group; t-test analysis results: & , p<0.05, &&, p<0.01.

Example 11: Effect of multiple intravenous injections on MPO activity in lung tissue

Male Bal/c mice weighing 20-22 g. Thirty randomized groups of 6 rats in each group were anesthetized with sodium pentobarbital, and the trachea was instilled into LPS (10 μL/50 μL). The edaravone was injected into the tail vein at 0.5 h and 6.5 h after modeling. 6mg/kg), composition 4:1 (7.5mg/kg, edaravone 6mg/kg, natural borneol 1.5mg/kg) or intraperitoneal injection of dexamethasone DEX (5mg/kg) 3h after modeling, model A vehicle control was injected into the tail vein.

24 hours after modeling, mice were anesthetized with pentobarbital sodium, the thoracic cavity was opened, the left atrial appendage was cut, and normal saline was injected through the right ventricle to perfuse the blood in the pulmonary circulation until the lungs turned white. The lung tissue was taken out, wrapped in weighing paper, and frozen at -20 °C. The MPO activity of lung tissue of each group was detected according to the myeloperoxidase (MPO) test kit (Nanjing built). As shown in Figure 8, MPO activity in lung tissue of mice was significantly increased after LPS modeling The high, edaravone and composition (4:1) administration groups significantly reduced MPO activity, and the composition (4:1) group had lower MPO activity levels than the edaravone group. The bar graph in Figure 8 shows Mean ± SEM (n = 6), One-Way ANOVA analysis results: *, p < 0.05, **, p < 0.01, ***, p < 0.001, compared to the model group.

Example 12: Effect of multiple intravenous injections on inflammatory cells and inflammatory factors in lung tissue lavage fluid

Male Bal/c mice weighing 20-22 g. Twenty-four randomized groups, 6 rats in each group, were anesthetized with sodium pentobarbital, and the trachea was instilled into LPS (10 μL/50 μL). The edaravone was injected into the tail vein at 0.5 h and 6.5 h after modeling. 6mg/kg), composition 4:1 (7.5mg/kg, edaravone 6mg/kg, natural borneol 1.5mg/kg) or intraperitoneal injection of dexamethasone DEX (5mg/kg) 3h after modeling, model A vehicle control was injected into the tail vein.

24 hours after modeling, the mice were anesthetized with sodium pentobarbital, the trachea was separated, inserted into a silicone tube, and fixed. Pipette 0.6ml PBS (pH=7.2) into the lungs through the catheter, slowly withdraw it, and then slowly inject it back into the lungs, repeat 3 times, and put the collected lavage into 1.5ml EP tube (placed in ice bath in). Repeat the above procedure twice, that is, flush with 1.8 ml of PBS, and finally obtain 1 ml of lavage fluid. The collected lavage fluid was centrifuged at 2000 rpm for 4 minutes at 4 degrees, and the supernatant was stored at -20 ° C for storage. The cell pellet was resuspended in 0.5 ml of PBS, and then added with 0.5 ml of white blood cell staining solution, uniformly stained, fixed for staining, and counted for blood cells. The contents of TNF-α and IL-6 in the supernatant of alveolar lavage fluid were determined according to the ELISA (Essay) instructions.

As shown in Figure 9-A, the number of white blood cells in the alveolar lavage fluid increased after LPS modeling, and the administration group significantly reduced the number of white blood cells, and the composition (4:1) group was significantly better than the edaravone group. . Similarly, for the inflammatory factors TNF-α and IL-6 in the lavage fluid, the composition (4:1) significantly reduced the inflammatory factor content and was significantly better than the edaravone group. In Figures 9-A, B, and C, the histograms represent Mean ± SEM (n = 6), and One-Way ANOVA is as follows: **, p < 0.01, ***, p < 0.001, compared with the model group; -test analysis is as follows: &, p < 0.001.

Example 13: Effect of composition on the function of mouse epithelial cells (MLE-12)

Lung epithelial cells are also important cells of the lung defense barrier, the priming cells of the pulmonary inflammatory response. In this experiment, an LPS-induced mouse lung epithelial cell line (MLE-12) was used to make an inflammatory model. 2×10 ⁵ cell/ml MLE-12 cells were resuspended in complete medium (Gibco RPMI-1640 medium + 4% FBS), and then inoculated into 48-well plates. After 24 hours of culture, the compositions were pre-incubated separately (4: 1), edaravone, natural borneol (final concentration of 1 mM) for 15 min, and then LPS (final concentration 1 μg / mL) induced cells. Supernatants were harvested at 1 h, 3 h, 6 h, 9 h, 12 h, and 24 h after LPS stimulation (n=3). The KC (keratinocyte growth factor) content was determined using R&D's Duo-set kit.

As shown in Figures 10-A, 10-B, and 10-C, the composition (4:1) significantly inhibited the release of KC from LPS-stimulated mouse lung epithelial cells, suggesting an improvement in the initial inflammatory response to LPS-induced acute lung injury. . Edaravone only had a certain effect in the early stage, and the inhibition disappeared after 12h; while the effect of natural borneol on KC began to appear after 9h. This suggests that the composition (4:1) combines the effects of both on KC and exerts a function of inhibiting KC release from 0 to 24 hours after administration.

Claims

Use of a pharmaceutical composition for the preparation of a medicament for treating a disease associated with endotoxemia, the pharmaceutical composition comprising 3-methyl-1-phenyl-2-pyrazolin-5-one or Pharmaceutically acceptable salts and borneol.
Use according to any one of the preceding claims, characterized in that the weight ratio of the 3-methyl-1-phenyl-2-pyrazolin-5-one or a pharmaceutically acceptable salt thereof to borneol It is 10:1 to 1:10.
The use according to claim 6, characterized in that the weight ratio of the 3-methyl-1-phenyl-2-pyrazolin-5-one or a pharmaceutically acceptable salt thereof to the borneol is 4:1. ~1:4.
The use according to Claim 7, characterized in that the weight ratio of the 3-methyl-1-phenyl-2-pyrazolin-5-one or a pharmaceutically acceptable salt thereof to the borneol is 4:1. ~2:1.
The use according to claim 6, characterized in that the weight ratio of the 3-methyl-1-phenyl-2-pyrazolin-5-one or a pharmaceutically acceptable salt thereof to the borneol is 10:1. , 4:1, 2:1, 1:2, 1:4 or 1:10.
The use according to claims 1-5, characterized in that the disease comprises systemic inflammatory response syndrome, sepsis, severe sepsis, septic shock, and sepsis-related diffuse intravascular coagulation, Acute lung injury, acute respiratory distress syndrome or multiple organ failure syndrome and septic brain injury, as well as inflammatory lung injury and bacterial pneumonia caused by other causes.
The use according to claim 6, characterized in that the disease comprises sepsis, severe sepsis, septic shock.
The use according to claim 6, characterized in that the disease comprises septic brain damage and inflammatory lung injury.
The use according to claims 1-5, characterized in that the borneol is a natural borneol.
The use according to claim 6 wherein said borneol is a natural borneol.