WO2021219112A1 - Isovaleryl spiramycin compound or application of isovaleryl spiramycin composition in preparation of drug for treating sepsis disease - Google Patents

Abstract

An isovaleryl spiramycin compound or an application of an isovaleryl spiramycin composition in the preparation of a drug for treating a sepsis disease. The isovaleryl spiramycin compound is selected from isovaleryl spiramycin I or a derivative thereof, isovaleryl spiramycin II or a derivative thereof, and isovaleryl spiramycin III or a derivative thereof. The isovaleryl spiramycin composition is selected from a combination of at least two of isovaleryl spiramycin I or a derivative thereof, isovaleryl spiramycin II or a derivative thereof, and isovaleryl spiramycin III or a derivative thereof, or carrimycin. The isovaleryl spiramycin compound or the isovaleryl spiramycin composition has a good treatment effect in treating sepsis, and has important social benefits and economic benefits.

Description

Application of isovalerylspiramycin compound or its composition in preparing medicine for treating sepsis disease Technical field

The invention belongs to the field of medicinal chemistry. Specifically, it relates to the application of isovalerylspiramycin compounds or their compositions in the preparation of drugs for treating sepsis diseases.

Background technique

Sepsis and the multiple organ dysfunction syndrome caused by it is currently one of the most common causes of death in clinically critically ill patients. The fatality rate of patients with sepsis is more than 20%, and after the disease progresses to the septic shock stage, the patients die The rate can rise to 40% to 70%. Therefore, the treatment of sepsis has always been a severe challenge facing the intensive care unit. As one of the serious complications of clinical emergency and critical patients, sepsis can induce septic shock and MODS, which are often induced by infection, severe trauma, burns, major surgery and other factors. Sepsis is a pathological process in which inflammation activation and immunosuppression coexist. Its development stage is systemic inflammatory response syndrome-sepsis-septic shock-multiple organ failure. The patient has a long hospital stay, poor prognosis, and high mortality. .

Patients with sepsis often have abnormal blood coagulation function, and the degree of abnormal blood coagulation function is related to the severity of the disease. The disorder of blood coagulation function directly affects the ischemic changes of organs, and is also an important reason for the occurrence of MODS in patients. In patients with sepsis, inflammation and blood coagulation promote each other. Plasma fibrinogen (Fg) is activated and converted into fibrin, and Fg is mostly reduced. The change of Fg in common infectious diseases is often slightly increased or unchanged [ 1]. Studies have shown that platelet count (PLT), prothrombin time (PT), thromboplastin time (APTT), and fibrinogen (Fg) after treatment with blood purification technology, all indicators of blood coagulation and renal function are better than those of each group. Significantly lower before.

Severe sepsis is an acute and critical illness in which various infections cause systemic inflammatory reactions. It is often complicated by acute coagulation dysfunction and acute renal failure, which greatly increases the difficulty of treatment and faces life-threatening patients. Therefore, severe sepsis has a very high death rate. Rate. The pathophysiological cause of sepsis is that the pathogen enters the blood, causing the inflammatory cells in the blood vessel to proliferate and activate, secrete inflammatory mediators, and produce a large number of immunologically active factors. It can also destroy the vascular endothelial cells, initiate the endogenous blood coagulation pathway, and cause diffuseness. Intravascular coagulation, coagulation-anticoagulation system balance is disturbed, but coagulation-related factors can further promote inflammatory cells to further secrete inflammatory factors, leading to such a cycle and rapid deterioration of the disease.

Carrimycin (Carrimycin), also known as Bitespiramycin and Shengjimycin, was developed by the Institute of Biotechnology of the Chinese Academy of Medical Sciences in cooperation with the applicant. The 4″-O-isovaleryltransferase gene (4″-O-isovaleryltransferase gene) was cloned into Streptomyces spiramyceticus, and the spiramycin 4″-OH was acylated and the isovaleryl group was added at the 4″ position. A new type of antibiotic with 4" isovalerylspiramycin as the main component formed by the side chain.

The structural schematic diagram of the main component of climycin is shown in formula (1), which does not represent conformation:

Among them, when R=H, R′=COCH ₂ CH(CH ₃ ) ₂ is isovaleryl spiramycin I; when R=COCH ₃ , R′=COCH ₂ CH(CH ₃ ) ₂ is isovaleryl Spiramycin II; when R=COCH ₂ CH ₃ , R′=COCH ₂ CH(CH ₃ ) ₂ is isovalerylspiramycin III.

The total content of the main active ingredient isovalerylspiramycin (Ⅰ+Ⅱ+Ⅲ) in climycin is not less than 60%, and the total content of acylated spiramycin is not less than 80%. It is a kind of pharmacy. Accepted pharmaceutical composition. The central structure is a 16-membered lactone ring, which is connected with one molecule of folofamine, one molecule of mycosaminoglycan and one molecule of mycaminophen. The difference in the structure of the mycine is that the group attached to the 4" position of mycylmycose is an isovaleryl group instead of a hydroxyl group. The chemical structure, as shown in formula (1), contains more than ten components. At present, the finished product of climycin The composition standard is that the proportion of isovalerylspiramycin Ⅲ ≥ 30%, the total proportion of isovaleryl spiramycin I, II, and III ≥ 60%, the proportion of total acylated spiramycin ≥ 80%, and other unknown components The sum of ≤5%.

Preliminary in vivo and in vitro pharmacodynamic tests have shown that the drug not only has good antibacterial activity ^{against most G + bacteria, but also has a certain effect on some G-} bacteria. The technical indicators are significantly better than those of azithromycin, erythromycin, acetylspiramycin, Medicin, especially the strongest antibacterial activity against Mycoplasma pneumoniae, against erythromycin-resistant bacteria, Neisseria gonorrhoeae, pneumococcus, Staphylococcus aureus, Pseudomonas aeruginosa, Influenza bacillus, Haemophilus influenzae, Bacteroides fragilis , Legionella, Myriobacterium and Clostridium perfringens also have certain antibacterial activity, and there is little cross-resistance to clinical erythromycin-resistant Staphylococcus aureus. Celimycin will be mainly used for the treatment of gram-positive bacterial infections, especially upper respiratory tract infections, and may be used for urinary system infections. So far, there has been no record or report on the treatment of septic diseases by climycin.

In view of this, the present invention is proposed.

Summary of the invention

The technical problem to be solved by the present invention is to overcome the shortcomings of the prior art and provide an application of isovalerylspiramycin compound or its composition in preparing medicines for treating sepsis.

In order to solve the above technical problems, the basic idea of the technical solution adopted by the present invention is:

The first object of the present invention is to provide the use of isovalerylspiramycin compounds or their compositions in the preparation of drugs for the treatment of sepsis.

In a further scheme, septic diseases include systemic inflammatory response, sepsis, severe sepsis, septic shock, organ dysfunction or organ failure caused by infection emergency damage.

In a further scheme, the sepsis disease is induced by coronavirus disease.

In a further scheme, the sepsis disease is induced by the SARS-COV-2 virus, which means that it includes viral sepsis, especially COVID-19-related sepsis.

In a further embodiment, the isovalerylspiramycin compound is selected from isovalerylspiramycin I or its derivatives, isovalerylspiramycin II or its derivatives, isovalerylspiramycin III or its derivatives Things.

In a further embodiment, the isovalerylspiramycin composition is selected from isovalerylspiramycin I or its derivatives, isovalerylspiramycin II or its derivatives, isovalerylspiramycin III or its derivatives A combination of at least two of the derivatives, or colimycin;

In a further aspect, the isovalerylspiramycin-based composition further includes a medically acceptable carrier.

In a further solution, the dosage of the drug is 10-1500 mg/kg, preferably 50-1000 mg/kg, more preferably 100-500 mg/kg.

At present, the main recognized pathogenesis of sepsis includes:

1) Imbalance of inflammatory response

Among them, early pro-inflammatory cytokines are closely related to the occurrence and development of inflammation. These include tumor necrosis factor (TNF)-a, interleukin (IL)-1, IL-6, interferon (IFN)-γ and so on. Among them, TNF-a is the most important pro-inflammatory cytokine in the early stage of inflammation. It plays an important role in the immune defense response and is also a key mediator of endotoxin damage. The role of IL-1 in sepsis is similar to that of TNF-c. It is expressed through IL-1β and initiates an inflammatory response together with TNF-a. IL-6 is an inflammatory mediator produced under the action of IL-1, which can promote the proliferation of T lymphocytes together with TNF-a. The level of IL-6 in plasma can be used as a predictor of the severity of sepsis. A large number of studies have confirmed that the synthesis of pro-inflammatory cytokines in sepsis is closely related to the mitogen-activated protein kinase (MAPK) pathway, which plays a role by activating a variety of downstream transcription factors and protein molecules.

The advanced cytokine high mobility group box-1 protein B1 (HMGB1), as one of the important late inflammatory mediators, can interact with transcription factors, nucleosomes and histones, and participate in transcription regulation, DNA replication, Cell life activities such as cell differentiation. HMGB1 can bind to TLR4, activate nuclear factor (NF)-KB and MAPK and other signal transduction pathways, further promote the production of TNF-a, IL-1, IL-6 and other mediators by cells, and aggravate tissue inflammation and damage.

2) Immune dysfunction

The body secretes a large number of inflammatory mediators in the early stage of sepsis, and then undergoes an immunosuppressive stage during the course of the disease, which is mainly manifested by the anergy of T lymphocyte clones and the negative regulation of immunosuppressive cells (such as Treg), etc. .

3) Blood coagulation dysfunction

The interaction between coagulation dysfunction and inflammation has become a key link in the development and prognosis of sepsis. The process includes the activation of the coagulation system, the inhibition of physiological anticoagulation mechanisms, and the inhibition of the fibrinolytic pathway.

Isovalerylspiramycin I or its derivatives, isovalerylspiramycin II or its derivatives, isovalerylspiramycin III or its derivatives, or colimycin can inhibit PI3K/AKT/m- The TOR signal pathway reduces the level of m-TORC1, thereby inhibiting the protein synthesis of the inflammatory factor NF-KB in its nucleus. At the same time, the inflammatory factors such as IL-4, IL-6, TNF-a are also significantly reduced to achieve anti-inflammatory effects. . As the course of the disease prolongs, patients with sepsis will experience immunosuppressive reactions, including inactivation of macrophages, reduced antigen presentation, and suppression of lymphocyte proliferation activity, resulting in the release of large amounts of anti-inflammatory cytokines. The test of the effect of cleritromycin on immune cells showed that cleritromycin can significantly promote the increase of total T cells (CD3 positive cells) in mice, including both CD4 and CD8 positive cells. This indicates that the isovalerylspiramycin compounds and compositions, especially the anti-infection, anti-inflammatory, and immune-regulating effects of climycin have been further clinically verified, which can bring benefits to patients with sepsis.

The second object of the present invention is a combination product for the treatment of septic diseases, including isovalerylspiramycin I or its derivatives, isovalerylspiramycin II or its derivatives, and isovalerylspiramycin III Or at least one of its derivatives, or colimycin as the first active ingredient of the drug, further comprising a second active ingredient of the drug, and the second active ingredient of the drug is selected from related drugs for the treatment of sepsis;

Preferably, the first active medicament ingredient and the second active medicament ingredient are separate preparations, or they are compounded into one preparation.

Among them, the second active ingredient of the drug includes, but is not limited to, antibiotics, statins, lipid A antagonists, recombinant human bactericidal protein, recombinant human lactoferrin, superantigen antagonists, corticosteroids, recombinant human activated protein C and the like.

After adopting the above technical solution, the present invention has the following beneficial effects compared with the prior art:

The isovalerylspiramycin compound or its composition has a good therapeutic effect in the treatment of sepsis, and has important social and economic benefits.

The specific embodiments of the present invention will be described in further detail below in conjunction with the accompanying drawings.

Description of the drawings

The accompanying drawings are used as a part of the present invention to provide a further understanding of the present invention. The exemplary embodiments of the present invention and the description thereof are used to explain the present invention, but do not constitute an improper limitation of the present invention. Obviously, the drawings in the following description are only some embodiments, and for those of ordinary skill in the art, other drawings can be obtained from these drawings without creative work. In the attached picture:

Figure 1 is the effect of ISP I and LPS on the viability of BV2 cells; A is the effect of ISP I on the viability of BV2 cells, and B is the effect of LPS on the viability of BV2 cells;

Figure 2 shows the effect of ISP I and LPS on NO production in BV2 cells.

Figure 3 shows the effect of ISP I and LPS on IL-6 in BV2 cells.

Fig. 4 is the evaluation result of climycin on the ability of macrophages to phagocytize chicken red blood cells, A is the control group, B is the climycin group, and C is the itraconazole group;

Figure 5 shows the first batch of C57BL/6 mice by intragastric administration for three consecutive days, 50mg/kg, to construct an abdominal inflammation model, and the detection results of abdominal neutrophils (Gr-1 and CD11b double positive cells);

Figure 6 shows the first batch of C57BL/6 mice by intragastric administration for seven days, 50mg/kg, to construct an abdominal cavity inflammation model, and the detection results of abdominal neutrophils (Gr-1 and CD11b double positive cells);

Figure 7 shows the second batch of C57BL/6 mice by intragastric administration for three consecutive days, 50mg/kg, to construct an abdominal cavity inflammation model, and the detection results of abdominal neutrophils (Gr-1 and CD11b double positive cells);

Figure 8 shows the second batch of C57BL/6 mice by intragastric administration for three consecutive days, 50mg/kg, to construct an abdominal inflammation model, and the detection results of the ratio of CD4+ and CD8+ cells in the peripheral blood;

Figure 9 is a histogram of the ratio of CD4+/CD3+ and CD8+/CD3+ cells in peripheral blood in Figure 8;

Figure 10 shows the second batch of C57BL/6 mice by intragastric administration at 50 mg/kg for three consecutive days to construct a model of abdominal inflammation, and the detection results of the proportion of CD3+ cells in the peripheral blood;

Figure 11 is a bar graph of the proportion of CD3+ cells in peripheral blood in Figure 10;

In Figure 12, A is that RAW cells are treated with drugs for 1 hour, and then they are induced to differentiate into M1 type, and the level of TNF-α is detected; B is that RAW cells are treated with drugs for 1 hour, and then they are induced to differentiate into M1 type to detect iNOS. Level; C means that RAW cells are treated with drugs for 1 hour, and then they are induced to differentiate into M2 type, and the level of Arg-1 is detected;

In Figure 13, A is the first addition of cytokines to induce RAW cells to differentiate into M1 macrophages, and corresponding drugs are added to detect the expression of TNF-α; B is the first addition of cytokines to induce RAW cells to differentiate into M1 macrophages , And then add the corresponding drugs to detect the expression of iNOS; C is to add cytokines to induce the differentiation of RAW cells into M2 macrophages, and then add the corresponding drugs to detect the expression of Arg-1;

In Figure 14, A is the first addition of cytokines to induce RAW cells to differentiate into M2 macrophages, and the corresponding drugs are added to detect the expression of TNF-α; B is the first addition of cytokines to induce RAW cells to differentiate into M2 macrophages , And then add the corresponding drugs to detect the expression of iNOS; C is to add cytokines to induce the differentiation of RAW cells into M2 macrophages, and then add the corresponding drugs to detect the expression of Arg-1;

Figure 15 is the diachronic change curve of inflammatory cytokine IL-β (FAS) (mean line graph ± SE thorn);

Figure 16 is the diachronic change curve of inflammatory cytokine IL-4 (FAS) (mean line graph ± SE thorn);

Figure 17 is the diachronic change curve of inflammatory cytokine IL-β (PPS) (mean line graph ± SE thorn);

Figure 18 is the diachronic change curve (PPS) of the inflammatory cytokine IL-4 (mean line graph ± SE spike).

It should be noted that these drawings and text descriptions are not intended to limit the scope of the present invention in any way, but to explain the concept of the present invention for those skilled in the art by referring to specific embodiments.

Detailed ways

In order to make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments will be described clearly and completely in conjunction with the accompanying drawings in the embodiments of the present invention. The following embodiments are used to illustrate the present invention. , But not used to limit the scope of the present invention.

Example 1. Isovalerylspiramycin I or Isovalerylspiramycin II or Isovalerylspiramycin III or Climycin Tablets

Specification: 200mg/350mg

Preparation Process:

Preparation of the tablet core: the main drug and auxiliary materials are respectively passed through a 100 mesh sieve, and the prescription amount isovalerylspiramycin I or isovalerylspiramycin II or isovalerylspiramycin III, microcrystalline cellulose and 1/2 The prescription amount of sodium starch glycolate is mixed uniformly, then 5% povidone K ₃₀ aqueous solution is added to make soft material, granulated with 18 mesh sieve, wet granules are dried at 60℃ under ventilation conditions for 2 hours; after drying, the granules are granulated with 18 mesh sieve , Then add 1/2 prescription amount of sodium starch glycolate and magnesium stearate and mix them uniformly, then press tablets with a dimple die with a diameter of 11mm to obtain a tablet-containing core with a tablet weight of 350mg and a hardness of 6.5kg.

Preparation of coating solution: Weigh the required Opadry II (white), add the required amount of water in the mixing container, add it in portions, after all is added, reduce the stirring speed to make the vortex disappear, and continue to stir 30min, ready.

Preparation of film-coated tablets: put the tablet core in the coating pan and determine the coating conditions. The host speed is 20r/min, the inlet air temperature is 40°C, the outlet air temperature is 30°C, the spray pressure is 0.02Mpa, and the spray flow rate is 1ml. /min for coating, after constant spraying for 1.5h, until the surface of the tablets is smooth and uniform in color, which meets the film coating inspection standard as qualified. The weight gain of the coating is about 5%.

Example 2: Isovalerylspiramycin I or Isovalerylspiramycin II or Isovalerylspiramycin III Tablets (calculated as 10,000 tablets)

prescription:

Original powder of isovalerylspiramycin I or isovalerylspiramycin II or isovalerylspiramycin III 1000g

Low-substituted hydroxypropyl cellulose (5%) 92.5g

Sodium Carboxymethyl Starch (3%) 55.5g

Magnesium stearate (1%) 18.5g

Total starch weight-weight of other raw materials

Total weight 1850g

Preparation process: Weigh an appropriate amount of starch, dilute it to 15% concentration, heat it to a paste, and make an adhesive; main ingredient isovalerylspiramycin I or isovalerylspiramycin II or isovalerylspiramycin III , Auxiliary materials starch, low-substituted hydroxypropyl cellulose, sodium carboxymethyl starch, magnesium stearate, respectively, through a 100 mesh sieve, according to the prescription amount, weigh the required main materials and auxiliary materials; isovalerylspiramycin I, starch 、After mixing the low-substituted hydroxypropyl cellulose thoroughly, use starch paste with 15% starch concentration to make soft material, granulate with 14-mesh sieve, dry at 50-60℃, control moisture within 3-5%, 14-mesh sieve Whole granules, add sodium carboxymethyl starch and magnesium stearate to mix, determine the particle content; calculate the tablet weight according to the particle content, press the tablet (Φ9mm shallow concave punch), check the difference in tablet weight; package after passing the inspection.

Example 3. Capsules of isovalerylspiramycin I or isovalerylspiramycin II or isovalerylspiramycin III (calculated as 10,000 capsules)

prescription:

Original powder of isovalerylspiramycin I or isovalerylspiramycin II or isovalerylspiramycin III 1000g

Weight of starch 1080-Isovalerylspiramycin I original powder

Medicinal No. 3 Capsules 1000 Capsules

Liquid paraffin 50ml

Preparation process: weigh the main material isovalerylspiramycin I or isovalerylspiramycin II or isovalerylspiramycin III, and the auxiliary material medicinal starch according to the amount of the process formula, then put it into the mixer and mix thoroughly 1.5-2 hours; the data obtained by sampling and testing the content should be basically consistent with the theoretical data (the weight of each capsule is about 0.105g), and the qualified medical No. 3 capsules and the mixed raw materials to be filled shall be fully automatic capsules Machine operation requirements, respectively fill in the loader for filling, the filled capsules are tested for difference (within ±10%, <0.3g), the dissolution rate meets the requirements, and the capsules that meet the requirements after the test are put into the polishing machine Add liquid paraffin and light it for 15-20 minutes, and then take it out for inspection of the finished product packaging box.

Example 4, isovalerylspiramycin I or isovalerylspiramycin II or isovalerylspiramycin III dry syrup (calculated by 10,000 bags)

prescription:

Isovalerylspiramycin I or Isovalerylspiramycin II or Isovalerylspiramycin III Original Powder 1250g

Citric acid (0.5%) 15g

Total weight of sucrose-other raw materials

The total weight is about 5000g

Pigment (curcumin) about 1g

Preparation process: isovalerylspiramycin I or isovalerylspiramycin II or isovalerylspiramycin III original powder, citric acid and sucrose are respectively crushed into particles by a high-speed jet mill, 85% passing through 300 mesh, 15% Pass through 180 meshes, then weigh the pulverized fine powder according to the prescription amount and mix it thoroughly for 1-1.5 hours, measure its content, calculate the filling quantity (theoretical filling quantity is 500mg per bag), and then put the mixture into the bagging machine , Pack the aluminum foil paper, divide it according to the operation requirements of the packing machine, the difference of the packing quantity is within ±5%, after packing, carry out the inspection and pass the outer packaging.

Example 5, isovalerylspiramycin I or isovalerylspiramycin II or isovalerylspiramycin III granules (calculated by 10,000 bags)

prescription:

Isovalerylspiramycin I or Isovalerylspiramycin II or Isovalerylspiramycin III Original Powder 1250g

Icing sugar 20000g

Dextrin 9000g

5% PVP-K ₃₀ appropriate amount

Preparation process: isovalerylspiramycin I or isovalerylspiramycin II or isovalerylspiramycin III original powder, powdered sugar, and dextrin through a 120-mesh sieve, and weigh the isovalerylspiramycin according to the prescription amount I. Mix the powdered sugar and dextrin evenly. Use 5% PVP-K ₃₀ mortar to make the above-mentioned materials into soft material. The rocking granule is granulated and dried at 70°C.

Example 6, Isovalerylspiramycin I or Isovalerylspiramycin II or Isovalerylspiramycin III Lyophilized Powder Injection

Weigh 500mg of isovalerylspiramycin I or isovalerylspiramycin II or isovalerylspiramycin III powder and mix it with equimolar adipic acid and dissolve it in 5ml water to obtain a light yellow clear solution, pH Between 4.6-5.6. Then 40mg of mannitol was added as a freeze-dried proppant, and after rapid freezing at low temperature for 9 hours, it was freeze-dried to obtain a light yellow loose mass, which was dissolved in 10ml of sterile water before use.

Example 7. Lyophilized powder injection of climycin

Weigh 500 mg of climycin, mix it with equimolar adipic acid and dissolve it in 5 ml of water to obtain a light yellow clear solution with a pH between 4.6-5.6. Then 40mg of mannitol was added as a freeze-dried proppant, and after rapid freezing at low temperature for 9 hours, it was freeze-dried to obtain a light yellow loose mass, which was dissolved in 10ml of sterile water before use.

Test example one

1. Anti-inflammatory effect of climycin

1. ELISA to detect the effect of climycin on inflammatory factors in various tissues and organs of mice

Experimental Kunming mice were purchased from Experimental Animal Center of Jiangsu University, mouse IL-1β ELISA kit (Invitrogen (88-7013-88)), mouse IL-4 ELISA kit (Invitrogen (88-7044-88) )), other experimental instruments and reagents are the existing conventional instruments and reagents.

Grouping of mice and administration

Calinomycin: dissolving method, add 0.48ml polyethylene glycol 400 to clalimycin several times, then add 2.4μl Tween 80, shake and mix well, then add 1.92ml distilled water (add 200μl each time and shake and mix well. ), respectively formulated to concentrations of 1.44mg/ml, 2.88mg/ml, and 5.76mg/ml.

Azithromycin: first dissolve it with a small amount of absolute ethanol, then add water to make the content of absolute ethanol 10%, and prepare it to a concentration of 1.82mg/ml.

Kunming mice, male, 18-20g in size, 144 mice, after adaptive feeding in the laboratory, the mice weighed about 24g, and they were randomly divided into 6 groups: normal group, model group, and low group (30mg/kg), Kezhong group (60mg/kg), Kegao group (120mg/kg), azithromycin (37.9mg/kg) group. Each component has 8 time points: 0h, 0.5h, 2.5h, 4.5h, 12h, 24h, 48h and 72h. 3 mice at each time point. The mice in the normal group were given no administration or bacteria, and the model group was given no administration of bacteria. Climycin and azithromycin were both administered intragastrically (500μl). The same volume of solvent. After the administration, the mice were killed in batches at different time points.

Model establishment: The concentration of Staphylococcus aureus is determined in the in vitro experiment report. After the concentration is measured, it is resuspended in physiological saline to make the concentration 3×108 CFU/ml, and injected into the tail vein at 24g/100μl injection. The administration started one hour after the injection.

Sample preparation

The mouse eyeballs were taken blood and sacrificed. The tissues and organs were taken and weighed with an electronic balance. Weigh 50mg of each tissue and add it to a 1.5ml EP tube, then add 1ml of pre-cooled PBS, add magnetic beads and homogenize with a homogenizer. Pulp (300Hz, 30s). After standing on ice for 30 minutes, centrifuge at 4°C (10000g, 10min) with a centrifuge, and take the supernatant as the test sample.

Experimental methods The experimental methods are carried out strictly in accordance with the instructions of the ELISA kit, and are briefly described as follows:

Reagent preparation: pH7.35 PBS, Tween 20, prepare a PBS solution containing 0.05% Tween 20 as a washing solution. (If crystals form in the buffer concentrate, heat it gently until completely dissolved). 1. Coating buffer (1X): Dilute PBS (10 times) in deionized water 1:10. 2. Capture antibody: Dilute the capture antibody (250x) 1:250 in coating buffer (1x). 3.5 xELISA/ELISPOT diluent: dilute the concentrated diluent (5x) 1:5 in deionized water. 4. Standard: Recombinant mouse il-1β standard, dissolved in distilled water, and the volume of distilled water added is on the label of the standard vial Specify. The standard solution is prepared 10-30 minutes in advance and mixed thoroughly to ensure complete uniform dissolution (concentration of the reconstituted standard = 1000pg/ml). Standards are prepared fresh and used immediately without storage. 5. Detection antibody: Dilute detection antibody (250x) 1:250 in ELISA/ELISPOT diluent (1x). 6. Enzyme: Dilute HRP concentrate (100x) 1:100 in ELISA/ELISPOT diluent (1x).

^{Experimental steps: 1. Coat the Corning TM} Costar ^TM 9018 ELISA plate with 100 μL of capture antibody per well in the coating buffer (dilute as described in point 1 of the reagent preparation). Seal the ELISA plate and incubate overnight at 4°C. 2. Remove the solution in the well, and rinse with more than 250 microliters of buffer solution 3 times. Allow soaking time (1 minute) in each rinse step to improve the rinse effect. Wipe dry with absorbent paper to remove residual liquid. 3. Add 200 microliters of ELISA/ELISPOT diluent (1X) to each well, and incubate for 1 hour at room temperature. 4. Prepare standard products 30 minutes in advance. 5. Suction and wash at least once with the washing liquid. 6. Add 100ul standards, samples, and blank wells to ELISA/ELISPOT diluent (1X). 7. Seal the plate and incubate at room temperature for 2 hours. 8. Prepare the detection antibody. 9. Follow step 2 to inhale and clean, repeat washing 3-5 times. 10. Add 100 μl/well of diluted detection antibody to all wells. 11. Seal the plate and incubate at room temperature for 1 hour. 12. Prepare HRP. 13. Follow step 2 to inhale and clean, repeat washing 3-5 times. 14. Add 100ul of diluted HRP to each hole. 15. Seal the plate and incubate at room temperature for 30 minutes. 16. Follow step 2 for suction and cleaning, make sure to allow 1 to 2 minutes of soaking time before suction, and repeat the cleaning 5-7 times. 17. Add 100ul of TMB solution to each well. 18. Incubate for 15 minutes at room temperature. 19. Add 50μL stop solution to each well. 20. Read the plate at 450nm. 21. Data collection and processing.

Test results: The effects of climycin on IL-4 factor and IL-1β in various tissues and organs of mice are shown in Table 1 and Table 2, respectively.

Table 1

Table 2

Note: *, P＜0.05; **, P＜0.01; ***, P＜0.001; ****, P＜0.0001.

Experimental conclusion: Climycin has anti-inflammatory effects. Climycin has a significant effect on reducing IL4 factor in lung, kidney, liver and spleen, and its role is more significant in liver and spleen; Climycin is in small intestine , Lung, spleen, liver and kidney all have a significant effect on reducing IL-1β factor, especially in the small intestine and lung.

2. Investigate the effect of isovalerylspiramycin I (ISP I), the main active ingredient of climycin, on the production of IL-6.

Test materials and reagents:

Cell line: mouse microglia BV2 cells were purchased from the National Laboratory Cell Resource Sharing Platform (Beijing)

Isovalerylspiramycin I (Shenyang Tonglian Group Co., Ltd.), lipopolysaccharide (LPS055: B5L6529), trypsin, penicillin, streptomycin, dimethyl sulfoxide (DMSO), methyl thiazole blue (MTT) are all Purchased from Sigma Chemical (St. Louis, MO, USA), DMEM medium was purchased from Gibco Chemical (Grand Island, NY, USA), special grade fetal bovine serum was purchased from South America Lonsera, NO detection kit (Biyuntian Biotechnology Company), ELISA detection Kit (Shanghai Aibixin Biotechnology Company).

The instruments used in this test example can all be conventional instruments in the prior art.

experiment method

Cell culture

BV2 cells were cultured in DMEM medium containing 10% FBS and placed in an incubator at _{37°C and 5% CO 2.} When the cells are cultured to a density of about 90%, they can be passaged and follow-up experiments.

Cell growth inhibition rate determination

The MTT method was used to detect the effect of ISP I on the activity of BV2 cells. MTT (Tetramethylazolium Salt) is a yellow dye that can accept hydrogen ions. The principle of MTT method for detecting cell activity is: succinate dehydrogenase and cytochrome c are present in the mitochondria of living cells. Under the catalysis of these two enzymes, the tetrazolium ring of MTT splits to produce blue-purple formazan crystals, DMSO Or the triple solution can dissolve the crystal, and detect the absorbance at the wavelength of 492nm/630nm, which can be used to detect the activity of the cells.

BV2 cells were seeded in a 96-well plate with a density of 1.6×10 ⁵ cell/ml, 100 μl/well, with six replicate wells in each group, and normal culture for 24 hours before adding medicine. In addition to the negative control group, different concentrations of ISP I were added, and the culture was continued until the specified time. Aspirate and discard the culture solution, add sterile PBS to wash once, aspirate and discard PBS, add 100 μl of the prepared MTT to each well, and continue to incubate for 4 hours. Add 100μl triple solution to it and continue to incubate for 12h, use a micro shaker to oscillate for 3-5min, use a microplate reader to measure the star value (A) at 630nm, and calculate the inhibition rate of ISP I on BV2 cells according to the following formula.

Inhibitoryratio(%)=(A ₆₃₀ , control-A _{630, control} )/(A ₆₃₀ , control-A _{630, blank} )×100

Griess method to detect NO content

BV2 cells were seeded in a 24-well plate at a rate of 1×10 ⁵ /well, and cultured in DMEM medium containing 10% FBS. The culture was continued for 24 hours, and the cells were replaced with serum-free medium and cultured for 6 hours. First, add 250μl of ISP I with final concentrations of 20μM, 10μM, and 5μM to the corresponding wells for pretreatment. One hour later, add LPS with a final concentration of 10μg/ml to the corresponding wells for induction treatment. After placing 5% CO ₂ in a 37°C incubator for 24 hours, the supernatant was taken and stored at -20°C for NO detection.

The determination of NO was carried out in accordance with the instructions, the absorbance was measured at 540nm, and the corresponding NO content was calculated using the standard curve.

ELISA kit to detect cell inflammatory factors

1) Prepare all required reagents and standards; 2) Take out the microplate from the sealed bag that has returned to room temperature, put the unused strips back into the aluminum foil bag, and re-seal; 3) Separate the standards of different concentrations, Add experimental samples or quality control materials to the corresponding wells, 100μL per well. Seal the reaction wells with sealing tape and incubate at room temperature for 2h. 4) Suck off the liquid in the plate and wash the plate with a washing bottle. Add 400μL of washing solution to each well, and then aspirate the washing solution from the plate. Repeat the operation 3 times. Try to absorb the remaining liquid every time the plate is washed will help to get good experimental results. After the last wash, please blot all the liquid in the plate or turn the plate upside down, and pat dry all remaining liquid on absorbent paper; 5) Add 100μL of detection antibody to each microwell. Seal the reaction wells with sealing tape and incubate at room temperature for 2 hours; 6) Repeat step 4 to wash the plate; 7) Add 100 μL of diluted streptavidin-HRP to each microwell, and incubate at room temperature for 20 minutes . Be careful to avoid light; 8) Repeat step 4 to wash the plate; 9) Add 100 μL of chromogenic substrate to each microwell, and incubate at room temperature for 20 minutes. Be careful to avoid light; 10) Add 50μL stop solution to each microwell, the color of the solution in the well will change from blue to yellow. If the color of the solution turns green or the color changes are inconsistent, please tap the microplate to make the solution evenly mixed; 11) Within 30 minutes after adding the stop solution, use a microplate reader to measure the absorbance at 450nm and set 540nm as the calibration wavelength. 12) Calculation result: Take the average value of the corrected absorbance value (OD450-OD540) and the multiple well readings of each standard and sample, and then subtract the average zero standard OD value. The curve can be generated by plotting the logarithm of the standard concentration and the logarithm of the corresponding OD value, and determining the best fit line through regression analysis.

Statistical processing

Statistical software SPSS26.0 was used for data analysis, Excel2016 was used for data summarization and GraphPad to draw charts. Measurement data were expressed in the form of mean±standard deviation (mean±SD). Data comparison between groups was performed by one-way analysis of variance, and P＜0.05 was the difference. There is statistical significance.

test results

The effect of ISP I and LPS on the viability of BV2 cells

After 24 hours of treatment of BV2 cells with different concentrations of ISP I, the MTT test results showed that compared with the untreated group, there was no significant difference in cell viability in the 2.5 μM, 5 μM and 10 μM groups; after 24 hours of treatment of BV2 cells with different concentrations of LPS, the MTT test results showed that: Compared with the untreated group, there was no significant difference in cell viability in the 0.01μg/ml, 0.1μg/ml, 1μg/ml and 10μg/ml groups. As shown in Figure 1 (A is the effect of ISP I on the activity of BV2 cells, B is the effect of LPS on the activity of BV2 cells,)

ISP I inhibits the production of NO induced by LPS

Detecting the level of NO in the cell supernatant when different concentrations of LPS act on cells, the results show that 0.01-10μg/ml LPS can induce NO production. The effect of ISP I on the production of NO in the cell supernatant was tested, and the results showed that ISP I can inhibit the production of NO induced by LPS in a concentration-dependent manner. As shown in Figure 2, in A, when the LPS concentration is 0.01 μg/ml, 0.1 μg/ml, 1 μg/ml, 10 μg/ml, compared with the control group, the NO release amount increases with the increase in concentration. *p<0.05, ***p<0.001vs0 group. In B, when the concentration of ISP I is 2.5 μM, 5 μM and 10 μM, the NO concentration generated in ISP I reduces the concentration-dependent manner, indicating that ISP I can reduce the amount of NO induced by LPS. #p＜0.05, ##p＜0.01vs LPS group, ***p＜0.001vs blank group.

ISP I inhibit LPS-induced IL-6 production

The detection of IL-6 levels in the cell supernatant when different concentrations of LPS acted on the cells showed that 0.01-10 μg/ml LPS can induce IL-6 production. The ELISA method detected the effect of ISP I on the production of IL-6 in the cell supernatant, and the results showed that 5 μM and 10 μM ISP I can significantly inhibit the production of IL-6 induced by LPS. As shown in Figure 3, in A, the concentration of LPS was 0.01 μg/ml, 0.1 μg/ml, 1 μg/ml and 10 μg/ml, and the amount of IL-6 produced by the cells increased. *p<0.05, **p<0.01 vs. 0μg/ml group. In B, when the concentration of ISP I was 2.5 μM, 5 μM, and 10 μM, the concentration of IL-6 decreased in a concentration-dependent manner of ISP I, indicating that ISP I can reduce LPS-induced IL-6. *LPS group*p<0.05, ***p<0.001.

Conclusion: Isovalerylspiramycin I (ISP I), the main active ingredient of climycin, can inhibit the production of inflammatory cytokines IL-6 and NO induced by LSP.

2. The immunomodulatory effect of climycin

Calinomycin enhances the ability of macrophages to engulf chicken red blood cells to a certain extent. The test of the influence of clerisomycin on immune cells showed that: clerisomycin can significantly promote the increase of total T cells (CD3 positive cells) in mice, including both CD4 and CD8 positive cells. The transdifferentiation study of cleritromycin on differentiated macrophages showed that cleritromycin can significantly increase the expression levels of TNF-a and iNos in M2 macrophages, and it is significantly stronger than TNF- induced by LPS+INF- The expression levels of a and iNos are better than itraconazole, and can significantly inhibit the expression level of Arg-1 in M2 macrophages.

1. Evaluation of Calinomycin on the ability of macrophages to engulf chicken red blood cells

OBJECTIVE: To detect whether clinomycin can enhance the function of macrophages in normal mice. The main detection index is the phagocytosis of macrophages. In view of the fact that there is no standard reagent in immunological research, itraconazole has the effect of promoting the polarization of macrophages and enhancing the phagocytosis of macrophages. Therefore, itraconazole was selected as a positive control in the experiment.

Reagents: physiological saline, 6% chicken red blood cells, methanol, acetone, Giemsa staining solution, etc.; consumables: 1ml syringe, ordinary glass slide, gauze, petri dish, etc.

Experimental mice: strain: Balb/c, age: 8-12, source: Chengdu Dashuo Experimental Animal Co., Ltd., quantity: 2 mice in each group.

Experimental procedures: (1) Grouping administration: a) Saline group: same volume as the experimental group, po; b) Climycin group: 50mg/kg, po; c) Itraconazole group: 50mg/kg, po; continuous administration for five days; (2) each mouse was injected with 1ml chicken red blood cells, and the mice were sacrificed after waiting for 30 minutes. (3) Inject 1ml of normal saline into the abdominal cavity, massage to make it evenly distributed, and make the mice prone for 5 minutes. (4) Cut open the abdominal cavity of the mouse, remove the needle with a 1ml syringe, aspirate the abdominal cavity cleaning solution, and drop it on the glass slide, drop two drops on each glass slide, try to ensure the same volume. (5) Put it in a petri dish pad with wet gauze, move to 37° incubator and incubate for 30 min. (6) After incubation, rinse in saline to remove non-adherent cells (preheat the saline in advance), and dry. (7) Fix with 1:1 acetone formaldehyde solution (pre-cooled at -20° in advance). (8) For Giemsa dyeing, first dye A with liquid for 45 seconds, then add liquid B for 4 minutes, gently blow and dye into ripples, mix the two, and then rinse with distilled water to dry. (9) Randomly take the field of view of the microscope, take photos and count, and calculate the phagocytic percentage (10) The phagocytic percentage calculation formula is the number of phagocytic macrophages/total number of macrophages × 100%

results and analysis:

After the mouse ascites cells are stimulated by itraconazole or climycin, the phagocytic macrophages can phagocytize chicken red blood cells (with megakaryocytes) or the chicken red blood cells that accumulate more around. As shown in Fig. 4, A is the control group, B is the climycin group, and C is the itraconazole group.

Compared with the placebo group (NC), the colimycin group (Keli) and itraconazole group (Yiqu) have a certain degree of enhancement in the ability of macrophages to phagocytize chicken red blood cells; however, the colimycin group and the itraconazole group There was no statistically significant difference between itraconazole groups.

Overall, the differences between the components are not obvious. Due to the inevitable subjective intervention of the experiment operator, quantitative judgments are not suitable for small differences; therefore, the macrophage phagocytosis test of fluorescent microspheres is used instead, and the flow method is used for detection to reduce the participation of subjective factors. .

2. Detection of the effect of climycin on the function of neutrophils

The purpose of the experiment: to detect whether climycin can enhance the inflammatory chemotaxis and migration ability of mouse neutrophils, mainly by constructing a mouse abdominal inflammation model, flow cytometric detection of the proportion of abdominal neutrophils, and the detection indicators are CD11b and Gr-1. In this experiment, itraconazole was used as a positive control.

Reagents: sterile PBS, fMLP, sterile HBSS, Gr-1-APC flow cytometry antibody, CD11b-FITC flow cytometry antibody, etc.; consumables: straws, rubber bands, scissors, tweezers, 1ml syringes, 15ml centrifuge tubes, flow tubes, etc. .

Experimental mice: Strain: C57BL/6 mice, age: 8-12, source: Chengdu Dashuo Experimental Animal Co., Ltd., quantity: the first batch of administration for three days, each group of 3 mice, administration for 7 days each Each group has 4 mice. The second batch of repeated experiments was administered with 7 mice in each group for three days.

Experimental steps:

(1) Group administration: d) Normal saline group: the same volume as the experimental group, po; e) Climycin group: 50 mg/kg, po; f) Itraconazole group: 50 mg/kg, po; continuous Administration for three days and seven days; (2) Preparation of fMLP: prepare 100nM fMLP, dilute with PBS, prepare for immediate use, and place on ice after preparation. (3) Intraperitoneal injection: Each mouse was intraperitoneally injected with 100uL100nM pre-chilled fMLP. (4) Collection of ascites cells: 4 hours after injection, the mice were sacrificed, the limbs were fixed, the abdominal skin was cut, a small opening was made in the peritoneum, the peritoneum was fixed with a rubber band and a paper clip, and the peritoneal cavity was repeatedly washed with a straw to suck pre-cooled HBSS. With gentle movements, collect about 8-10ml of ascites and place it on ice. (5) Centrifuge at 1000 rpm for 5 minutes, and discard the supernatant carefully. (6) If there are red blood cells, resuspend in 1ml red blood cell lysis buffer and lyse on ice for 3-5 minutes. (7) Centrifuge at 1000 rpm for 5 min, discard the supernatant, and add 3 ml PBS to wash once. (8) Add 500uLPBS to resuspend. (9) Take 10^6 cells and incubate flow cytometry antibodies: CD11b-FITC and Gr-1-APC in the dark at room temperature. (10) Wash the cells once with PBS, resuspend, place on ice, protect from light, and test on the machine.

Experimental results: The evaluation results of the neutrophil migration ability of colinomycin on the mouse abdominal cavity inflammation model are shown in Figure 5-7. Figure 5-7 shows the peritoneal inflammation model of mice with different administration time and different batches, and the ratio of neutrophils (Gr-1 and CD11b double positive cells) was detected by flow cytometry. Figure 5 shows the first batch of C57BL/6 mice by intragastric administration for three consecutive days, 50mg/kg, to construct an abdominal cavity inflammation model, and the detection results of peritoneal neutrophils (Gr-1 and CD11b double positive cells); Figure 6 shows the first batch of C57BL/ 6 mice were gavaged continuously for seven days, 50mg/kg, the abdominal cavity inflammation model was constructed, and the test results of peritoneal neutrophils (Gr-1 and CD11b double positive cells); Figure 7 shows the second batch of C57BL/6 mice by gavage for three consecutive days, 50mg /kg, construct the abdominal cavity inflammation model, and the test results of abdominal neutrophils (Gr-1 and CD11b double positive cells).

Figure 8-11 shows the detection of T lymphocytes in peripheral blood after the establishment of a mouse abdominal inflammation model three days after administration. Flow cytometry was used to detect the proportion of CD3+, CD4+ and CD8+ cells.

Calinomycin and itraconazole can significantly promote the migration of neutrophils to inflammation sites in mice, and the results are especially obvious in individual mice. Compared with the results of three days and seven days, there is no discovery that continuous medication for seven days can To further strengthen the effect. Climycin and itraconazole can significantly promote the increase of total T cells (CD3 positive cells) in mice, in which both CD4 and CD8 positive cells increase, but itraconazole performs better.

3. The effect of climycin on the differentiation of macrophages

Experimental background and purpose: Macrophages can be divided into two categories: Classically activated macrophages (M1), which are characterized by increased expression of the major histocompatibility complex MHC class II, and nitric oxide (NO) Increased, reactive oxygen species and pro-inflammatory cytokines, such as tumor necrosis factor (Tumornecrosisfactor, TNF), interleukin-1 (IL-1) and interleukin-6 (IL-6), etc. increased. The other type is alternatively activated macrophages (Alternatively activated macrophage, M2), also known as selectively activated macrophages, is a type of macrophages with immunosuppressive activity. Under various stimuli, interleukin-4 (IL-4) levels increase, the expression of interleukin-10 (IL-10) and arginase (Arginase, Arg) increases, leading to increased cell proliferation and collagen production. In the event of infection and cancer, the polarization of M1 cells has a protective effect on human health. Therefore, the purpose of this experiment is to detect whether clalimycin affects the differentiation of macrophages, and then to explore the potential immunomodulatory effects of clalimycin.

Experimental reagents and consumables: Reagents: RAW246.7 cell line, 1640 medium, FBS, RNA extraction kit, reverse transcription kit, SYBR fluorescence quantitative kit. Cytokines: IL-4, INF-γ, LPS; Consumables: Consumables related to cell culture.

Experimental steps:

Option 1: Explore whether the drug will promote or inhibit the polarization process of RAW246.7 cells.

(1) Climycin and itraconazole are prepared as a 10mM mother solution with DMSO for storage. (2) Culture the RAW246.7 cell line in vitro, collect the cells grown in the logarithmic phase and spread a 6-well plate at a density of 1×10^6 cells per well. Three treatments of Traconazole 20uM. (3) After 1h of treatment, each treatment was divided into 2 groups, one group was added with LPS (200ng/ml) and IFN-γ (20ng/ml), and the other group was added with IL-4 (20ng/ml) and cultured for 12h After harvesting cells. (4) Extract total cellular RNA, reverse transcription into cDNA, and detect the RNA level of the corresponding index.

Option 2: Explore whether the drug has an effect on polarized cells.

(1) Climycin and itraconazole are prepared as a 10mM mother solution with DMSO for storage. (2) Culture the RAW246.7 cell line in vitro, use the same method to plate, and add the corresponding cytokines (LPS+IFN-γ or IL-4) to the cells for 12h. (3) After 12 hours, add climycin (20uM) or itraconazole (20uM) to the cells that have been polarized into M1 or M2 type, and act again for 12 hours. (4) Harvest the cells, extract the total RNA of the cells, reverse transcription into cDNA, and detect the RNA level of the corresponding indicators.

Experimental results:

Option 1: Explore whether cleritromycin can promote or inhibit the differentiation of RAW246.7 cells

Figure 12A shows that RAW cells are treated with drugs for 1 hour, and then they are induced to differentiate into M1 type, and the level of TNF-α is detected. Legend: NC (RAW cells, do not do any treatment); PC1 (RAW cells plus LPS+INF-γ, induce RAW cells to differentiate into M1 macrophages); Keli (RAW cells first add colimycin, then add LPS+INF-γ); Yiqu (RAW cells are added with itraconazole first, then LPS+INF-γ); and statistically tested, *P<0.05, **P<0.01, ***P<0.001 .

Figure 12B shows that RAW cells are treated with drugs for 1 hour, and then they are induced to differentiate into M1 type, and the level of iNOS is detected;

Figure 12C shows that RAW cells are treated with drugs for 1 hour, and then they are induced to differentiate into M2 type, and the level of Arg-1 is detected. Note: NC (RAW cells, without any treatment); PC2 (RAW cells with IL-4, induce Differentiation of RAW cells into M2 type macrophages); Keli (RAW cells first add colimycin, then IL-4); Yiqu (RAW cells first add itraconazole, then IL-4); and perform statistics Scientific test, *P<0.05, **P<0.01, ***P<0.001.

Analysis of the results of Scheme 1:

Climycin can increase the expression of TNF-α and iNos, and inhibit the expression of Arg-1. It is suggested that climycin may promote the differentiation and function of M1 macrophages.

Scheme two: A. Explore whether climycin has a transdifferentiation effect on differentiated macrophages

Figure 13A First add cytokines to induce RAW cells to differentiate into M1 macrophages, then add corresponding drugs to detect the expression of TNF-α

Figure 13B First add cytokines to induce RAW cells to differentiate into M1 type macrophages, then add corresponding drugs to detect the expression of iNOS

Figure 13A First add cytokines to induce RAW cells to differentiate into M2 macrophages, then add corresponding drugs to detect the expression of Arg-1

In Figure 13A-13C, NC (RAW cells, without any treatment); PC1 (RAW cells plus LPS+INF-γ, induce RAW cells to differentiate into M1 macrophages); PC2 (RAW cells plus IL-4, induce RAW cells differentiate into M2 type macrophages); Keli ((RAW cells first add LPS+INF-γ to induce RAW cells to differentiate into M1 type macrophages, then add colimycin); Yiqu (RAW cells first add LPS +INF-γ, induce RAW cells to differentiate into M1 type macrophages, plus itraconazole); and statistically tested, *P<0.05, **P<0.01, ***P<0.001.

Experimental results: Consistent with the results obtained on the RAW246.7 cell line, Climycin does not further enhance the expression of TNF-α and iNOS induced by LPS+INF-γ, but can inhibit Arg-1 in M1 macrophages The expression level of colimycin has the potential to enhance the function of M1 macrophages.

Option 2: B to explore whether climycin has a transdifferentiation effect on differentiated macrophages

Figure 14A First add cytokines to induce RAW cells to differentiate into M2 type macrophages, then add corresponding drugs to detect the expression of TNF-α

Figure 14B First add cytokines to induce RAW cells to differentiate into M2 type macrophages, then add corresponding drugs to detect the expression of iNOS

Figure 14C First add cytokines to induce RAW cells to differentiate into M2 type macrophages, then add corresponding drugs to detect the expression of Arg-1

Figure 14A-14C: NC (RAW cells, without any treatment); PC1 (RAW cells plus IL-4, induce RAW cells to differentiate into M2 macrophages); PC2 (RAW cells plus IL-4, induce RAW cells Differentiate into M2 type macrophages); Keli ((RAW cells first add IL-4, induce RAW cells to differentiate into M2 type macrophages, then add colimycin); Yiqu (RAW cells first add IL-4, induce RAW cells were differentiated into M2 type macrophages, plus itraconazole); and statistical tests were performed, *P<0.05, **P<0.01, ***P<0.001.

Experimental results: Climycin can significantly increase the expression levels of TNF-α and iNos in M2 macrophages, and it is significantly stronger than the expression levels of TNF-α and iNos induced by LPS+INF-γ, which is better than that of Itrax Conazole can significantly inhibit the expression level of Arg-1 in M2 macrophages.

Third, the anti-infection effect of climycin

1. The effect of colimycin on carbapenem-resistant Acinetobacter baumannii and carbapenem-resistant Klebsiella pneumoniae

Strains: 1 carbapenem-resistant Acinetobacter baumannii and 1 carbapenem-resistant Klebsiella pneumoniae;

Medium: Staphylococcus: MH medium plus 2% Nacl, incubate at 35-37°C for 24h.

Other strains: conventional MH medium, incubate at 35-37°C for 16-18 and observe the results.

Formula: peptone 1%, beef powder 0.3%, Nacl 0.5%, agar powder 1.2%.

After the preparation of the above-mentioned culture medium is completed, all are placed in an Erlenmeyer flask for autoclave sterilization at 121°C for 15 minutes, and then placed in a refrigerator at 4°C for use after cooling.

Experimental animals:

Kunming mice, half male and half male, weighing 18-22 g, were purchased from Chengdu Dashuo Experimental Animal Co., Ltd. (SPF grade). Feeding conditions: 25°C, humidity 60%. The breeding conditions are SPF-level, and the research process follows the guidelines for the breeding, management and use of laboratory animals.

Drug preparation:

1) Carbapenem-resistant Klebsiella pneumoniae group: Weigh 0.24g of the drug, add 2.4ml Tween 80 and 2.4ml absolute ethanol to dissolve the drug, add distilled water to a total volume of 20ml, at this time the concentration is 12mg/ml . Take 14mL of the medicinal solution and add distilled water to a total volume of 20ml. At this time, the concentration is 8.4mg/ml, which is 70% of the original concentration. By analogy, take 14mL of high-concentration drug solution, add distilled water to a total volume of 20ml, dose interval 1:0.7, a total of 5 groups.

2) Carbapenem-resistant Acinetobacter baumannii group: Weigh 0.45g of the drug, add 4.5ml Tween 80 and 4.5ml absolute ethanol to dissolve the drug, add distilled water to a total volume of 30ml, at this time the concentration is 15mg/ml . Take 24mL of the medicinal solution and add distilled water to a total volume of 30ml. At this time, the concentration is 12mg/ml, which is 80% of the original concentration. By analogy, take 24mL of high-concentration medicinal solution, add distilled water to a total volume of 30ml, dose interval 1:0.8, a total of 6 groups.

Preliminary experiment (determination of drug dose range)

①Bacterial inoculation: Add 3ml LB medium into the centrifuge tube, take out the plate from 4℃, pick 2-3 colonies with an inoculating loop to inoculate the LB medium, and cultivate at 37℃.

② MLD determination: Take healthy Kunming mice, weighing 18-22g, and randomly divide them into several groups, each with 5 mice, both male and female. The bacterial solution was diluted with 5% high-active dry yeast to different concentrations, injected intraperitoneally, 0.5 mL each, observed 7 days after infection, recorded the number of deaths of the mice, and the lowest bacterial count that caused 100% death of the mice was used as MLD. The amount is used as the amount of infectious bacteria in the in vivo protection test.

③Determination of drug dose range: Take healthy Kunming mice, weighing 18-22g, and randomly divide them into several groups, each with 5 mice, both male and female.

Prepare 1MLD with 5% high-active dry yeast, and intraperitoneally inject 0.5mL each. After 1 hour of infecting the mice, gavage the mice with different concentrations of drugs. The administration volume is 0.2mL/10g. Observe for 24 hours and record the death of the mouse. Based on the results, design the dosage of in vivo treatment and protection experiments.

Formal experimental method:

1) Carbapenem-resistant Klebsiella pneumoniae group: healthy Kunming mice, weighing 18-22 g, were randomly divided into 6 groups, each with 10 animals, both male and female. Among the 6 groups, 5 groups are the administration group, and the 1 group is the solvent control group.

2) Carbapenem-resistant Acinetobacter baumannii group: healthy Kunming mice weighing 18-22 g were randomly divided into 7 groups, each with 10 animals, both male and female. Among the 7 groups, 6 groups were the administration group, and the 1 group was the solvent control group.

Prepare 1MLD with 5% high-active dry yeast. All mice were injected intraperitoneally with 0.5mL. One hour after the mice were infected, they were gavage with different concentrations of drugs or solvents. The administration volume was 0.2mL/10g, and the observation was continued for 14 days. The number of deaths of the mice was recorded.

Experimental results:

(1) MLD results (the maximum lethal bacteria count is determined):

table 3

Bacteria name	MLD value
Klebsiella pneumoniae	1×10 8 CFU/mL
Carbapenem-resistant Acinetobacter baumannii	1×10 6 CFU/mL

(2) Determination of drug dosage range:

Table 4

Bacteria name	Drug dosage range
Klebsiella pneumoniae	58-240mg/kg
Carbapenem-resistant Acinetobacter baumannii	98-300mg/kg

(3) The 7-day mortality rate of mice infected with carbapenem-resistant Klebsiella pneumoniae or carbapenem-resistant Acinetobacter baumannii with clinomycin protection is shown in Table 5:

table 5

(4) ED50 results of resistant bacteria (calculated according to Bills method)

Table 6

Bacteria name	ED50
Klebsiella pneumoniae	153.2mg/kg
Carbapenem-resistant Acinetobacter baumannii	112.2mg/kg

Conclusion: Climycin has a good antibacterial effect on carbapenem-resistant Acinetobacter baumannii and carbapenem-resistant Klebsiella pneumoniae, and can significantly improve the survival rate of infected animals.

Four, cytokine related data in the clinical trial of clinomycin against the new crown

Changes in immune-related indicators of patients with new crown after medication (Calinomycin)

The change of immune-related indicators (lymphocyte count, lymphocyte percentage, CD4, CD8 count and percentage, and inflammatory cytokines) from the baseline in the first 1, 3, 5, 7-10 days after the FAS set test group and the control group was used. Comparison of sexual changes, the lymphocyte count was statistically significant from the 7th to 10th day of the baseline change (P=0.003).

Table 7-1

Corresponding to Table 7-1, the diachronic change curve of inflammatory cytokine IL-β (FAS) (mean line graph ± SE spike) is shown in Figure 15; the diachronic change curve of inflammatory cytokine IL-4 (FAS) ) (Mean line graph ± SE burr) as shown in Figure 16.

Table 7-2

Table 8-1

Corresponding to Table 8-1, the diachronic change curve of inflammatory cytokine IL-β (PPS) (mean line graph ± SE spike) is shown in Figure 17; the diachronic change curve of inflammatory cytokine IL-4 (PPS) ) (Mean line graph ± SE burr) as shown in Figure 18.

Table 8-2

The above are only the preferred embodiments of the present invention and do not limit the present invention in any form. Although the present invention has been disclosed as above in preferred embodiments, it is not intended to limit the present invention. Any technology familiar with this patent Without departing from the scope of the technical solution of the present invention, the personnel can use the technical content suggested above to make slight changes or modification into equivalent embodiments with equivalent changes. However, any content that does not deviate from the technical solution of the present invention is based on the technology of the present invention. Essentially, any simple modifications, equivalent changes and modifications made to the above embodiments still fall within the scope of the solution of the present invention.

Claims (10)

1. Application of isovalerylspiramycin compound or its composition in preparing medicine for treating sepsis disease.
2. The application according to claim 1, wherein the septic disease includes systemic inflammatory response, sepsis, severe sepsis, septic shock, organ dysfunction or organ failure caused by infection emergency damage.
3. According to the application of claim 1 or 2, the sepsis disease is induced by coronavirus disease.
4. According to the application of claim 3, the sepsis disease is induced by SARS-COV-2 virus.
5. The use according to any one of claims 1 to 4, wherein the isovalerylspiramycin compound is selected from isovalerylspiramycin I or its derivatives, isovalerylspiramycin II or Its derivatives, isovalerylspiramycin III or its derivatives.
6. The use according to any one of claims 1 to 5, wherein the isovalerylspiramycin composition is selected from isovalerylspiramycin I or its derivatives, isovalerylspiramycin II Or a combination of at least two of its derivatives, isovalerylspiramycin III or its derivatives, or climycin.
7. The use according to claim 6, wherein the isovalerylspiramycin composition further comprises a medically acceptable carrier.
8. The application according to any one of claims 1-7, wherein the dosage of the drug is 10-1500 mg/kg, preferably 50-1000 mg/kg, more preferably 100-500 mg/kg.
9. A combined product for the treatment of septic diseases, characterized in that it comprises isovalerylspiramycin I or its derivatives, isovalerylspiramycin II or its derivatives, isovalerylspiramycin III or its derivatives At least one of the first medicament, or climycin, as the first active ingredient of the medicament; further comprising a second active ingredient of the medicament, and the second active ingredient of the medicament is selected from related medicines used for the treatment of sepsis;
10. The combination product according to claim 1, wherein the first active medicament ingredient and the second active medicament ingredient are separate preparations, or they are compounded into one preparation.

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