WO2021078246A1 - Pharmaceutical composition for preventing or treating sepsis, kit, use thereof and treatment method thereof - Google Patents

Abstract

In order to overcome the problems of poor therapeutic effects and large side effects of existing treatment methods and therapeutic medicines for sepsis, provided is a pharmaceutical composition for preventing or treating sepsis, comprising mitochondria having physiological activity as an active ingredient. Also provided is a kit comprising the described pharmaceutical composition. Furthermore, provided are use of mitochondria in the preparation of a medicine, a pharmaceutical composition or a kit for preventing or treating sepsis, and a method for preventing or treating sepsis. Different administration modes (such as intraperitoneal injection IP, intravenous injection IV and subcutaneous injection SC) and administration dosages are set, active mitochondria and inactivated mitochondria are transplanted to treat a mouse having sepsis. It has been discovered that transplanting autologous or allogeneic active mitochondria can improve the survival rate of the mice by 30-50% during the construction process of a mouse sepsis model induced by Escherichia coli, having significant therapeutic effects.

Description

Pharmaceutical composition, kit for preventing or treating sepsis, application and treatment method thereof

This application is based on the U.S. invention patent with application number US 62/925,253 filed on October 24, 2019, and claims priority.

Technical field

The invention belongs to the field of biopharmaceuticals, and specifically relates to a pharmaceutical composition, a kit for preventing or treating sepsis, and an application and treatment method thereof.

Background technique

Sepsis is an uncontrolled response of the host to infection and causes life-threatening organ dysfunction. There are approximately 48.9 million sepsis cases worldwide each year, of which 11 million deaths from sepsis, with a mortality rate of 22.5%. There is currently no specific medicine for sepsis, and conventional treatments have limited effects, including: fluid resuscitation, the use of broad-spectrum or narrow-spectrum antibiotics, surgery to remove the source of infection, and "symptomatic treatment."

Fluid resuscitation is to give patients liquids such as crystalloid fluid through Early, goal-directed therapy (EGDT), so that the patient's central venous pressure and other physiological indicators meet the corresponding standards, so as to reduce the mortality of patients with sepsis. The disadvantage of this method is that the dosage is difficult to determine, and the dosage window is narrow. Many clinical studies have shown that EGDT cannot reduce the mortality of patients with sepsis, and excessive fluid can easily cause damage to the kidneys, heart and other organs. Antibiotics are used in the treatment of sepsis in two ways. One is the direct use of broad-spectrum antibiotics in the early stage of sepsis. The disadvantage of this method is that it is prone to produce drug-resistant bacteria and has greater side effects on the human body. The second is to obtain biological evidence through bacterial culture, and then target antibiotic treatment. The shortcomings of this method are obvious. Sepsis needs to be administered as soon as possible, but the microorganisms are cultured for a long time, and the culture results are not necessarily positive, and there are many multi-drug resistant bacteria, and the antibiotic treatment effect is very poor. In addition, the common shortcoming of the two methods is that antibiotics can easily further aggravate the disorder of the immune system and cannot prevent sepsis. If sepsis is caused by surgical infection, the lesion can be removed or drained by surgery, but the disadvantage of this method is that it is difficult to determine the source of the infection. "Symptomatic treatment" of sepsis refers to, for example, the use of glucocorticoids to control blood pressure, insulin to control blood sugar, and oxygen delivery to control blood oxygen content. The main basis of this method is that the patient's physiological indicators are lower or higher than a certain alert value and given corresponding drugs for regulation. The treatment effect of sepsis is not good, and it cannot prevent sepsis. It can only be used as an auxiliary treatment method. . In addition, the mitochondria of patients with sepsis are damaged and the utilization of oxygen is low. The excess oxygen easily combines with electrons to form reactive oxygen species (ROS), which increases oxygen stress and exacerbates the condition and prognosis of patients with sepsis.

Although the true cause and mechanism of sepsis are still unclear so far, studies have shown that the metabolism of sepsis is strong in the early stage, and the large amount of ROS produced by high-intensity respiration is one of the important reasons for mitochondrial damage. As the main organelle for aerobic respiration of eukaryotic cells, mitochondria are the main place to provide energy. At present, most researches on the cutting-edge treatment of diseases caused by mitochondrial damage are to use mitochondria as therapeutic targets to enhance their functions. There is no research on directly applying mitochondria to the prevention or treatment of sepsis.

Summary of the invention

In view of the problems of poor therapeutic effects and large side effects of existing sepsis treatment methods and therapeutic drugs, the present invention provides a pharmaceutical composition, kit, and application and treatment thereof for preventing or treating sepsis. method.

The technical solutions adopted by the present invention to solve the above technical problems are as follows:

In one aspect, the present invention provides a pharmaceutical composition for preventing or treating sepsis, which includes physiologically active mitochondria as an active ingredient.

On the other hand, the present invention also provides a kit for preventing or treating sepsis, which includes the above-mentioned pharmaceutical composition.

On the other hand, the present invention also provides the application of mitochondria in the preparation of medicines, pharmaceutical compositions or kits for preventing or treating sepsis.

Optionally, the source of the mitochondria is autologous, allogeneic or heterologous, and combinations thereof.

Optionally, the source of the mitochondria is allogeneic.

Optionally, the mitochondria are separated and extracted from cells or tissues, and the cells include somatic cells, germ cells, stem cells, and combinations thereof, and the tissues include heart, liver, spleen, kidney, brain, and combinations thereof.

Optionally, the mitochondria are extracted and isolated from the tissues of the heart, liver, spleen, kidney and combinations thereof.

Optionally, the pharmaceutical composition further includes a solvent.

Optionally, the concentration of the mitochondria is 0.1 μg/ml to 900 mg/ml.

Optionally, the solvent in the pharmaceutical composition includes physiological saline, phosphate buffer, culture fluid, tissue fluid, phospholipid or amino acid solution with drug properties, and combinations thereof.

Optionally, the pharmaceutical composition further includes one or more of insulin, antibiotics, antiviral drugs, antifungal drugs, glucocorticoids, and cardiotonic drugs.

On the other hand, the present invention also provides a method for preventing or treating sepsis, including the following steps:

A pharmaceutical composition including mitochondria having physiological activity as an active ingredient is administered to the patient.

Optionally, the administration modes of the pharmaceutical composition include intravenous injection, arterial injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, intradermal injection, oral administration, sublingual administration, external application, inhalation, and oral, eye, and urinary Reproductive system mucosal administration and combinations thereof.

Optionally, the method for preventing or treating sepsis further includes symptomatic and supportive therapy.

Optionally, the method for preventing or treating sepsis further includes other symptomatic and supportive therapies, and the other symptomatic and supportive therapies include rehydration, cardiotonia, boosting, oxygen inhalation, assisted ventilation, enteral nutrition support, and parenteral nutrition. Support, ECG monitoring and their combination.

Mitochondria originated from archaea 1.5 billion years ago, so they have an "infection ability" similar to that of intracellular bacteria. When co-cultured with cells in vitro, mitochondria can enter cells efficiently. Inspired by the characteristics of mitochondria and the profound understanding of the importance of mitochondria to life, the inventor of the present invention realized that mitochondrial administration can be used as an effective means to treat sepsis, which can directly restore the energy supply level of the patient's body, thereby prolonging the patient's energy supply level. Survival, to achieve the purpose of treatment. In order to verify this hypothesis, the inventors used E. coli or mouse fecal diluent to construct a mouse sepsis model, isolated and extracted mitochondria from other mice, and performed a variety of administration methods on the mouse sepsis model. Mitochondria were applied, and the results showed that the mortality of mice was effectively reduced.

Compared with existing treatment methods, the use of mitochondria as the active ingredient in the treatment of sepsis in the present invention has the following advantages:

1. Stop the increase of oxygen stress and directly solve the problem of mitochondrial energy supply in patients with sepsis;

2. Mitochondria are natural medicines with low side effects and can be extracted from allogeneic cells, which will not cause or have only a weak immune rejection reaction when entering the patient's body;

3. Due to the characteristics of mitochondria, it is easy to enter the cell and play a role;

4. Easy to obtain mitochondria;

5. The earlier the medication is used, the better the effect, and it can even prevent the occurrence of sepsis.

Description of the drawings

Fig. 1 is a fluorescence image of active mitochondria provided in Example 1 of the present invention and mitochondria inactivated by two methods after being stained with MitoTracker Red under a confocal microscope.

Figure 2 is a graph showing the survival rate of septic mice constructed by intraperitoneal injection of E. coli by IP administration of mitochondria provided in Example 2 of the present invention.

Fig. 3 is a graph showing the survival rate of septic mice constructed by subcutaneous injection of Escherichia coli by IP administration of mitochondria provided in Example 3 of the present invention.

Fig. 4 shows the survival rate of the septic mice (male) provided in Example 4 of the present invention after inactivated or activated mitochondria were injected through the tail vein.

Fig. 5 is the result of the survival rate of the septic mice (female) provided in Example 4 of the present invention after inactivated or activated mitochondria were injected through the tail vein.

Fig. 6 is the result of the survival rate of the septic mice provided in Example 5 of the present invention after inactivated or activated mitochondria after intraperitoneal injection.

Fig. 7 is the result of the survival rate of septic mice after intramuscular injection of inactivated or activated mitochondria provided in Example 6 of the present invention.

Figure 8 is the result of the survival rate of the septic mice provided in Example 7 of the present invention after inactivated or activated mitochondria after intraperitoneal injection.

Fig. 9 is the result of the survival rate of the septic mice provided in Example 8 of the present invention after inactivated or activated mitochondria after intraperitoneal injection.

Fig. 10 is the result of the survival rate of the septic mice provided in Example 9 of the present invention after inactivated or activated mitochondria derived from 293T cells were injected through the tail vein.

Fig. 11 shows the survival rate of the septic mice provided in Example 10 of the present invention after a single injection of inactivated or activated mitochondria through the tail vein.

Figure 12 is the result of the survival rate of septic mice constructed by intraperitoneal injection of mouse fecal solution provided in Example 11 of the present invention after a single intraperitoneal injection of inactivated or activated mitochondria.

Figure 13 is the result of the survival rate of the septic mice provided in Example 12 of the present invention after inactivated or activated mitochondria after intraperitoneal injection.

Fig. 14 is the result of the survival rate of mice in the case of prior tail vein injection of inactivated or activated mitochondria and then induced sepsis in Example 13 of the present invention.

Detailed ways

In order to make the technical problems, technical solutions and beneficial effects solved by the present invention clearer, the following further describes the present invention in detail with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described here are only used to explain the present invention, but not to limit the present invention.

In the description of the present invention, the term "isolated and extracted" refers to mitochondria or a composition containing mitochondria (for example, cytoplasm), which has been physically separated or removed from its natural biological environment. The isolated mitochondria or the composition containing mitochondria may be purified during the isolation process, or need not be purified.

The term "autologous" refers to a biological composition obtained from the same organism.

The term "allo" refers to organisms of different genotypes in the same genus.

The term "heterologous" refers to organisms of different genotypes in different species.

The term "sepsis" refers to the life-threatening organ function damage caused by the imbalance of the host's response to infection, including infections (bacteria, fungi, mycoplasma, parasites, viruses and other infections), host reactions (inflammatory reactions and involving multiple systems Non-immune reactions) and organ dysfunction.

The term "mitochondria with physiological activity" refers to mitochondria that can perform basic physiological activities, such as energy conversion, and also includes mitochondria that dormant under certain conditions but whose physiological activity is not destroyed. As an example, the physiological activity of mitochondria can be defined by the in vitro respiration rate of mitochondria. At the same time, various in vitro methods for assessing the physiological functions of mitochondria can also be used, including spectrophotometric enzyme determination, bioluminescence measurement of ATP production, MitoTracker staining intensity, JC- 1 Dyeing and so on. For example, various specific antibodies against mitochondrial proteins can be used in immunocytochemistry and Western blot analysis. These antibodies include ATP synthase subunits, cytochrome c and cytochrome c oxidase, PGC-1 and mtTFA. Two-dimensional polyacrylamide gel electrophoresis can be used, followed by Western blotting to analyze the content of mitochondrial protein.

The present invention provides a pharmaceutical composition for treating sepsis, which includes physiologically active mitochondria as an active ingredient.

On the other hand, the pharmaceutical composition can be administered in advance to achieve the effect of preventing sepsis. Therefore, the present invention also provides a pharmaceutical composition for preventing sepsis, which includes physiologically active mitochondria as an active ingredient.

In some embodiments, the source of the mitochondria is autologous, allogeneic or xenogeneic, and combinations thereof.

In some specific embodiments, the source of the mitochondria is allogeneic.

In some other specific embodiments, the source of the mitochondria is autologous or heterologous, and combinations thereof.

In some embodiments, the mitochondria are isolated and extracted from cells or tissues.

Mitochondria can be mitochondria obtained from non-human mammals (such as mice, rabbits, pigs, sheep, goats, cattle, and higher primates), or mitochondria obtained from humans. Specifically, mitochondria may be mitochondria isolated from cells or tissues. In a specific embodiment, the cells or tissues are all cells or tissues in vitro.

The cell may be any one selected from the group consisting of somatic cells, germ cells, stem cells, and combinations thereof. For example, mitochondria may be mitochondria obtained from somatic cells, germ cells, or stem cells. In addition, the mitochondria may be normal mitochondria obtained from cells with normal mitochondrial biological activity. In addition, the mitochondria may be mitochondria cultured in vitro.

The tissues include heart, liver, spleen, kidney, brain, and combinations thereof.

In some specific embodiments, the mitochondria are extracted and isolated from tissues of heart, liver, spleen, kidney, and combinations thereof.

In some embodiments, the pharmaceutical composition further includes a vehicle.

In some embodiments, the concentration of the mitochondria is 0.1 μg/ml to 900 mg/ml.

In a more specific embodiment, the concentration of the mitochondria may be 0.1 μg/ml, 0.15 μg/ml, 0.6 μg/ml, 1 μg/ml, 2 μg/ml, 5 μg/ml, 10 μg/ml, 20 μg/ml, 50μg/ml, 100μg/ml, 200μg/ml, 500μg/ml, 1mg/ml, 5mg/ml, 10mg/ml, 20mg/ml, 50mg/ml, 100mg/ml, 300mg/ml, 500mg/ml, 700mg/ ml or 900mg/ml.

Those skilled in the art should understand that the selection of vehicles, including physiologically acceptable compounds, according to needs, and the selection of suitable carriers and their formulations according to the route of administration of the composition are well known to those skilled in the art.

In some embodiments, the solvent in the pharmaceutical composition includes physiological saline, phosphate buffer, culture fluid, tissue fluid, phospholipid or amino acid solution with drug properties, and combinations thereof.

In some embodiments, the pharmaceutical composition further includes a stabilizer.

The stabilizer includes glucose, sucrose, fetal bovine serum, ADP, ATP, amino acids, glycerol, propylene glycol, sodium glycocholate, cholesterol, mannitol, albumin or sodium citrate and combinations thereof.

In some embodiments, the pharmaceutical composition further includes other active agents with disease prevention or treatment effects.

The active agent may be an agent for synergistic prevention or treatment of sepsis, or an agent for prevention or treatment of other diseases.

In some embodiments, the pharmaceutical composition further includes one or more of antibiotics, antiviral drugs, antifungal drugs, glucocorticoids, insulin, and cardiotonic drugs. Another embodiment of the present invention provides a kit for preventing or treating sepsis, including the pharmaceutical composition as described above.

Another embodiment of the present invention provides the use of mitochondria in the preparation of drugs, pharmaceutical compositions or kits for preventing or treating sepsis.

The characteristic requirements of the mitochondria as a medicine, pharmaceutical composition or kit for the prevention or treatment of sepsis are consistent with the description in the above-mentioned pharmaceutical composition and kit, and will not be repeated.

Another embodiment of the present invention provides a method for preventing or treating sepsis, including the following steps:

A pharmaceutical composition including mitochondria having physiological activity as an active ingredient is administered to the patient.

In some embodiments, the administration mode of the pharmaceutical composition includes intravenous injection, arterial injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, intradermal injection, oral administration, sublingual administration, topical application, inhalation, and oral and eye , Urogenital system mucosal administration and combinations thereof.

In some embodiments, the method for preventing or treating sepsis further includes other symptomatic and supportive therapies. The other symptomatic and supportive therapies include fluid rehydration, cardiotonic, boosting, oxygen inhalation, assisted ventilation, enteral nutrition support, and intestinal support. External nutritional support, ECG monitoring and their combination.

The following is the content of the priority text US 62/925,253 required by the present invention, which is incorporated into this application as a reference.

Escherichia coli (DH 5α) was cultured in TB medium and harvested by centrifugation. Prepare a 20% E. coli solution (20% E. coli, 30% glycerol, 50% TB medium, calculated by volume). The same batch of E. coli is frozen and stored at -80°C in the refrigerator. Kunming mice (KM mice) were used to test the toxicity of different batches of E. coli, including male and female mice.

Mitochondria were isolated and extracted from the heart, liver, kidney and spleen of Kunming mice. In short, these organs were homogenized in 1x PBS using an electronic homogenizer, and centrifuged at 1015 g for 10 min in a 50 mL centrifuge tube. Subsequently, the supernatant was transferred to a new 50ml centrifuge tube and centrifuged at 14610g for 10min. The supernatant was discarded, the pellets were combined and resuspended in DMEM.1xPS.10% FBS (DMEM is high glucose, containing 1x Pen-Strep and 10% FBS) cell culture medium. In order to use inactive mitochondria as a negative control, each gram of mitochondrial pellet was resuspended in 5ml PBS and 2.5ml 75% ethanol. Let it stand at room temperature for 5 min, and then centrifuge at 14610 g for 10 min to obtain inactivated mitochondria (IAM). The inactivated mitochondria were then resuspended in DMEM.1xPS.10% FBS cell culture medium at the same concentration as the active mitochondria.

In order to induce the establishment of a mouse sepsis model by intraperitoneal injection (IP), E. coli was diluted with 1xPBS and injected into Kunming mice through the intraperitoneal cavity. The dose of E. coli that killed about 50% of Kunming mice within 6 hours Construct a murine sepsis model. Subsequently, using the same number of male and female mice weighing 20-26g, by intraperitoneal injection (IP), with 0.1ml/10g 50% mt.DMEM (50% active mitochondria in DMEM.1xPS.10% FBS culture Resuspension in the basal medium) or 50% IAM.DMEM (50% inactivated mitochondria are resuspended in DMEM.1xPS.10% FBS medium) dose administration, record the death of mice every 6 hours, and continue to observe 24 hours.

In order to induce the establishment of a sepsis model in mice by subcutaneous injection (SC), the amount of Escherichia coli that killed more than 50% of Kunming mice within 5 days was diluted into TB, and Kunming Xiao was administered by subcutaneous injection (SC). mouse. Use equal amounts of female and male mice weighing about 20-26g, with 0.1ml/10g of 50% mt.DMEM (50% active mitochondria body weight suspended in DMEM.1xPS.10% FBS solution) or 50% IAM. The dose of DMEM (50% inactivated mitochondria resuspended in DMEM.1xPS.10% FBS solution) was administered by IP, once a day for 5 consecutive days. For 7 days, the death of mice was recorded every day.

The test results are shown in Figure 2 and Figure 3. Figure 2 shows the construction of a Kunming mouse sepsis model induced by intraperitoneal injection (IP) of Escherichia coli, and it was administered in high glucose DMEM.1xPS.10% FBS by IP or IV methods. 50% active mitochondria diluted in solution (Treatment) or inactivated mitochondria (Control). Figure 3 shows the construction of a Kunming mouse sepsis model induced by subcutaneous injection (SC) of Escherichia coli, and 50% active mitochondria diluted in high glucose DMEM.1xPS.10% FBS solution by intraperitoneal injection (IP) ( Treatment or inactivation of mitochondria (Control).

The results show that the administration of mitochondria through IP or IV can effectively reduce the mortality of sepsis in mice.

The present invention will be further illustrated by specific examples below.

Extraction of active mitochondria

Visceral extraction of mitochondria: After the mice were sacrificed by cervical dislocation, the heart, liver, spleen, and kidney were taken in a 50mL centrifuge tube, and 1×PBS (phosphate buffered saline solution) was added at 20mL/piece, and then added to the final concentration of 5mM EDTA according to the volume. (Ethylenediaminetetraacetic acid) and a final concentration of 0.2mM PMSF, homogenize in a homogenizer at 6000rpm for 2min. After homogenization, centrifuge at 1015g, 4℃ for 10min. Take the supernatant and centrifuge at 14600g for 10 min at 4°C. The supernatant is discarded, the precipitate is filtered and dried, and then weighed. The mitochondrial solution is prepared with the required solution directly or after inactivation.

Newborn mice extract mitochondria: take newborn mice within 1-3 days, sterilize with alcohol, cut into 4-5 cuts and place in a beaker, add 1×PBS (phosphate buffered saline solution) at 10mL/piece, and then add according to the volume The final concentration is 5mM EDTA (ethylenediaminetetraacetic acid) and the final concentration is 0.2mM PMSF, homogenized in a homogenizer at 6000rpm for 2min. After homogenization, dispensed into 50mL centrifuge tubes, centrifuged at 1015g, 4℃ for 10min. Take the supernatant and centrifuge at 14600g for 10 min at 4°C. The supernatant was discarded, the precipitate was filtered and dried and weighed, and the mitochondrial solution was prepared with the required solution directly or after inactivation.

Mitochondrial inactivation treatment

Ethanol inactivation: In the above mitochondrial extraction step, 14,600g, centrifuged at 4°C for 10 minutes, discard the supernatant, filter the precipitate and weigh it, add 5 times 1×PBS according to the weight, and add 75% at the volume of 50%. Mix it with ethanol, leave it at room temperature for 5 minutes, then centrifuge at 14600g for 10 minutes, discard the supernatant, filter the precipitate, and weigh it. According to the weight, use DMEM.1xPS.10% FBS cell culture medium to configure 500mg/mL inactivated mitochondria (Inactivated Mitochondria). , IAM) solution.

Freeze-thaw inactivation: place the active mitochondrial solution extracted in the above mitochondrial extraction step in a refrigerator at -80°C or room temperature, and freeze and thaw three times repeatedly to obtain an inactivated mitochondrial solution.

Example 1

Mitochondria were extracted from Kunming mouse heart, liver, spleen, kidney and other organs. Active mitochondria were prepared using DMEM.1xPS.10% FBS to prepare 50mg/mL mt.DMEM. Inactivated mitochondria were inactivated by ethanol and three freeze-thaw methods. For inactivation, use DMEM.1xPS.10% FBS to prepare 50mg/mL IAM.DMEM. Using MitoTracker Red staining (dilution ratio 1:5000), MitoTracker Red enters through the mitochondrial membrane potential and remains in the mitochondrial matrix. A strong signal indicates good mitochondrial activity, and no signal indicates loss of mitochondrial membrane potential, which is a manifestation of mitochondrial inactivation. The results are shown in Figure 1, where A is the active mitochondria, B is the mitochondria inactivated by freezing and thawing three times, and C is the mitochondria inactivated by alcohol. It can be seen that the fluorescent bright spots of active mitochondria are brighter and more than those inactivated by the two methods, and there are almost no bright spots in B and C, indicating that the mitochondria can be almost completely inactivated by freezing and thawing three times and alcohol.

Monitoring experiment on the therapeutic effect of mitochondrial administration on sepsis in mice

Example 2

Example 2 is a detailed description of the example in the priority document. The mouse sepsis model is induced by the dose of intraperitoneal injection (IP) of Escherichia coli in Kunming mice with a body weight of 20-26 g that kills about 50% within 6 hours. , E. coli was diluted with 1×PBS and injected by IP into 24 male Kunming mice of equal weight (12 in the control group and 12 in the treatment group). Mitochondria are extracted from mouse internal organs. 500mg/mL mt.DMEM (50% active mitochondria resuspended in DMEM.1xPS.10% FBS) or 500mg/mL IAM.DMEM (50% inactivated mitochondria resuspended in DMEM) at a dose of 0.1ml/10g , Administered by IP. Death records are recorded every 6 hours for 24 hours.

The results are shown in Figure 2. Intraperitoneal injection of 50% mitochondria can increase the survival rate of mice by about 17%.

Example 3

Example 3 is a detailed description of the example in the priority document. The mouse sepsis model is induced by subcutaneous injection (SC) of E. coli in Kunming mice by killing about 50% of the weight of 20-26g in 5 days. , E. coli was diluted with 1×PBS and injected by SC into 24 male Kunming mice of equal weight (12 in the control group and 12 in the treatment group). Mitochondria are extracted from mouse internal organs. 500mg/mL mt.DMEM (50% active mitochondria resuspended in DMEM.1xPS.10% FBS) or 500mg/mL IAM.DMEM (50% inactivated mitochondria resuspended in DMEM) at a dose of 0.1ml/10g , Administered by IP, once a day for 5 consecutive days. The deaths were recorded every day for 7 days. The results are shown in Figure 3. Intraperitoneal injection of 50% mitochondria can increase the survival rate of mice by about 40%.

Example 4

The Kunming mice were randomly divided into two groups (male: 24 in the control group, 24 in the treatment group, female: 8 in the treatment group, 8 in the control group), the weight range of 22-36g, the treatment group and the control group were paired in pairs. The difference is within 1g. The E. coli solution with a concentration of 2mg/0.1mL was injected intraperitoneally with 0.1mL/10g into the mice, and the mice developed sepsis symptoms 2 hours later. The inactivated or active mitochondria extracted from newborn mice were diluted with PBS into a 25mg/mL mitochondrial suspension, and the mice with sepsis symptoms were injected with 0.05mL/10g tail vein, and the control group was injected with IAM.PBS for treatment The group was injected with mt.PBS, and the survival of the mice was continuously observed until the 7th day. The results are shown in Figures 4 and 5. A single injection of 25 mg/mL mitochondria in the tail vein can increase the survival rate of mice by about 17%-25%.

Example 5

34 male Kunming mice were randomly divided into two groups (the treatment group and the control group each had 17), the weight range was 22-36g (the treatment group and the control group were paired, the weight difference was within 1g), and the concentration was 2mg/0.1mL The Escherichia coli strain was injected intraperitoneally with 0.1mL/10g into the mice, and the mice developed sepsis symptoms 2 hours later. The inactivated or active mitochondria extracted from newborn mice were diluted with DMEM into a 250mg/mL mitochondrial suspension, and injected intraperitoneally with 0.1mL/10g to mice with sepsis symptoms. The control group was injected with IAM.DMEM, and the treatment group was injected with IAM.DMEM. Mt.DMEM was injected and repeated intraperitoneal injections with the same dose on the second and third days, and the survival of the mice was continuously observed until the 7th day. The results are shown in Figure 6. Intraperitoneal injection of mitochondria for 3 consecutive days can increase the survival rate of mice by about 30%.

Example 6

34 male Kunming mice were randomly divided into two groups (the treatment group and the control group each had 17), the weight range was 22-36g (the treatment group and the control group were paired, the weight difference was within 1g), and the concentration was 2mg/0.1mL The Escherichia coli bacteria solution was injected into the left abdominal cavity of 0.1mL/10g into the mice, and the mice developed sepsis symptoms 2 hours later. Inactivated or active mitochondria extracted from newborn mice were diluted with DMEM into a 250mg/mL suspension, and then injected into the right abdominal cavity of the mice at 0.1mL/10g. The control group was injected with IAM.DMEM, and the treatment group was injected with IAM.DMEM. mt.DMEM, repeated intraperitoneal injection of the same dose on the second and third days, and continued observation of the survival of the mice until the seventh day. The results are shown in Figure 7. Intraperitoneal injection of mitochondria for 3 consecutive days can increase the survival rate of mice by about 30%.

Example 7

Twenty-six male Kunming mice were randomly divided into two groups (13 in the treatment group and 13 in the control group), with a weight range of 20-26g (the treatment group and the control group were paired with a weight difference of less than 1g), and the concentration was 2mg/0.1mL The Escherichia coli bacteria solution was injected intraperitoneally with 0.1 mL/10 g into the mice, and the mice developed sepsis symptoms 2 hours later. The inactivated or active mitochondria extracted from newborn mice were diluted with DMEM into a 500mg/mL mitochondrial suspension, and then injected into the sepsis-symptomatic mice with 0.05mL/10g right thigh muscle, and the control group was injected with IAM. DMEM, the treatment group was injected with mt.DMEM, and the survival of the mice was continuously observed until day 7. The results are shown in Figure 8. A single intramuscular injection of mitochondria can increase the survival rate of mice by about 30%.

Example 8

76 female Kunming mice were randomly divided into two groups (38 in the treatment group and 38 in the control group), with a weight range of 20-27g (the treatment group and the control group were paired with a weight difference of less than 1g), and the concentration was 10mg/0.1mL The Escherichia coli bacteria solution was injected subcutaneously into the neck of 0.1mL/10g mice, and the mice developed sepsis symptoms 4 hours later. The inactivated or active mitochondria extracted from newborn mice were diluted with DMEM into a 250mg/mL mitochondrial suspension, and then injected intraperitoneally with 0.1mL/10g to mice with sepsis symptoms. The control group was injected with IAM.DMEM for treatment The group was injected with mt.DMEM, and repeated intraperitoneal injections on the second and third days. The survival of the mice was continuously observed until the 7th day. The results are shown in Figure 9. Intraperitoneal injection of mitochondria for 3 consecutive days can increase the survival rate of mice by about 31.5%.

Example 9

Thirty male Kunming mice were randomly divided into two groups (15 in the treatment group and 15 in the control group), with a weight range of 22-30g (the treatment group and the control group were paired with a weight difference of less than 1g), and the concentration was 2mg/0.1mL The Escherichia coli strain was injected intraperitoneally with 0.1mL/10g into the mice, and the mice developed sepsis symptoms 2 hours later. The inactivated or activated mitochondria extracted from 293T cells were diluted with DMEM into a 50mg/mL mitochondrial suspension, and then injected into the sepsis-symptomatic mice with 0.05mL/10g tail vein, and the control group was injected with IAM.DMEM for treatment The group was injected with mt.DMEM, and the survival of the mice was continuously observed until the 7th day. The results are shown in Figure 10. A single tail vein injection of mitochondria can increase the survival rate of mice by about 17.5%.

Example 10

Forty male Kunming mice were randomly divided into two groups (20 in the treatment group and 20 in the control group), with a weight range of 22-30g (the treatment group and the control group were paired with a weight difference of less than 1g), and the concentration was 2mg/0.1mL The Escherichia coli bacteria solution of 0.1mL/10g was injected subcutaneously into the neck of the mice, and the mice developed sepsis symptoms 2 hours later. The mitochondria extracted from newborn mice, after inactivation or alcohol inactivation, are diluted with DMEM.10% FBS solution containing 20% sucrose at a concentration of 200 mg/mL, and placed in the program cooling box for one week at -80 degrees. the above. After thawing, the mice with sepsis symptoms were injected with 0.05mL/10g tail vein. The control group was injected with IAM.DMEM, and the treatment group was injected with mt.DMEM. The survival of the mice was continuously observed until the 7th day. The results are shown in Figure 11. A single tail vein injection of mitochondria can increase the survival rate of mice by about 19%.

Example 11

Fifty male Kunming mice were randomly divided into two groups (25 in the treatment group and 25 in the control group), with a weight range of 22-30g (the treatment group and the control group were paired with a weight difference of less than 1g), and the concentration was 5% mice The stool solution was injected intraperitoneally with 0.12mL/10g into the mice, and the mice developed sepsis symptoms 4 hours later. The inactivated or active mitochondria extracted from newborn mice were diluted with DMEM into a 250mg/mL mitochondrial suspension, and then injected intraperitoneally with 0.1mL/10g to mice with sepsis symptoms. The control group was injected with IAM.DMEM for treatment The group was injected with mt.DMEM, and the survival of the mice was continuously observed until the 7th day. The results are shown in Figure 12. A single intraperitoneal injection of mitochondria can increase the survival rate of mice by about 22%.

Example 12

29 male Kunming mice were randomly divided into two groups (15 in the treatment group and 14 in the control group), with a weight range of 22-30g (the treatment group and the control group were paired with a weight difference of less than 1g), and the concentration was 10mg/0.1mL The Escherichia coli bacteria solution was injected subcutaneously in the neck of 0.1mL/10g into the mice, and the mice developed sepsis symptoms 4 hours later. The inactivated or active mitochondria extracted from newborn mice were diluted with DMEM into a 250mg/mL mitochondrial suspension, and then injected intraperitoneally with 0.1mL/10g to mice with sepsis symptoms. The control group was injected with IAM.DMEM for treatment The group was injected with mt.DMEM and repeated intraperitoneal injections on the 2nd to 5th days. The survival of the mice was continuously observed until the 7th day. The results are shown in Figure 13. Intraperitoneal injection of mitochondria for 5 consecutive days can increase the survival rate of mice by about 53%.

Monitoring experiment on the preventive effect of mitochondrial administration on sepsis in mice

Example 13

Forty-four male Kunming mice were randomly divided into two groups (22 in the treatment group and 22 in the control group), with a weight range of 22-31g (the treatment group and the control group were paired in pairs, and the weight difference was within 1g). The inactivated or active mitochondria extracted from newborn mice were diluted with 1×PBS into a 50mg/mL mitochondrial suspension, and the mice were injected into the tail vein at 0.05mL/10g body weight respectively. The control group was injected with IAM.DMEM for treatment The group was injected with mt.DMEM, 4 hours after the administration, the E. coli solution with a concentration of 2mg/0.1mL was injected intraperitoneally with 0.1mL/10g into the mice, and the survival of the mice was continuously observed until the 7th day. The results are shown in Figure 14. The tail vein injection of active mitochondria can reduce the death rate of sepsis by 30% 24 hours after the induction of sepsis, and reduce the death rate by 11% after the seventh day.

The above descriptions are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modification, equivalent replacement and improvement made within the spirit and principle of the present invention shall be included in the protection of the present invention. Within range.

Claims (14)

1. A pharmaceutical composition for preventing or treating sepsis, which is characterized by comprising physiologically active mitochondria as an active ingredient.
2. A kit for preventing or treating sepsis, which is characterized by comprising the pharmaceutical composition according to claim 1.
3. Application of mitochondria in preparing medicines, pharmaceutical compositions or kits for preventing or treating sepsis.
4. The application according to claim 3, characterized in that the source of the mitochondria is autologous, allogeneic or xenogeneic and combinations thereof.
5. The application according to claim 3, wherein the source of the mitochondria is allogeneic.
6. The application according to claim 3, wherein the mitochondria are separated and extracted from cells or tissues, and the cells include somatic cells, germ cells, stem cells and combinations thereof, and the tissues include heart, liver, spleen, kidney , Brain and its combination.
7. The application according to claim 3, wherein the mitochondria are extracted and isolated from tissues of heart, liver, spleen, kidney and combinations thereof.
8. The use according to claim 3, wherein the pharmaceutical composition further comprises a solvent.
9. The application according to claim 8, wherein the concentration of the mitochondria is 0.1 μg/ml to 900 mg/ml.
10. The application according to claim 8, wherein the solvent in the pharmaceutical composition includes physiological saline, phosphate buffer, culture fluid, tissue fluid, phospholipid or amino acid solution with drug properties, and combinations thereof.
11. The application according to claim 3, wherein the pharmaceutical composition further comprises one or more of antibiotics, antiviral drugs, antifungal drugs, insulin, glucocorticoids, blood pressure drugs, and cardiotonic drugs .
12. A method for preventing or treating sepsis, which is characterized in that it comprises the following steps:

    A pharmaceutical composition including mitochondria having physiological activity as an active ingredient is administered to the patient.
13. The method according to claim 12, characterized in that the administration mode of the pharmaceutical composition comprises intravenous injection, arterial injection, intraperitoneal injection, intramuscular injection, intradermal injection, subcutaneous injection, oral administration, sublingual administration, topical application , Inhalation and administration through the mucous membranes of the oral cavity, eyes, genitourinary system and combinations thereof.
14. The method according to claim 12, wherein the method for preventing or treating sepsis further comprises other symptomatic and supportive therapies, and the other symptomatic and supportive therapies include rehydration, cardiotonia, boosting, oxygen inhalation, and assisted ventilation , Enteral nutrition support, parenteral nutrition support, ECG monitoring and their combination.

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