阅读说明：本技术 异戊酰螺旋霉素类化合物或其组合物在制备治疗脓毒症疾病药物中的应用 (Application of isovaleryl spiramycin compound or composition thereof in preparation of medicines for treating sepsis diseases ) 是由 姜恩鸿 夏明钰 王恒 赵小峰 于 2021-04-30 设计创作，主要内容包括：本发明公开了异戊酰螺旋霉素类化合物或其组合物在制备治疗脓毒症疾病药物中的应用,异戊酰螺旋霉素类化合物选自异戊酰螺旋霉素Ⅰ或其衍生物、异戊酰螺旋霉素Ⅱ或其衍生物、异戊酰螺旋霉素Ⅲ或其衍生物；异戊酰螺旋霉素类组合物选自异戊酰螺旋霉素Ⅰ或其衍生物、异戊酰螺旋霉素Ⅱ或其衍生物、异戊酰螺旋霉素Ⅲ或其衍生物中至少两种的组合,或者可利霉素。异戊酰螺旋霉素类化合物或其组合物在治疗脓毒症方面具有良好的治疗效果,具有重要的社会效益和经济效益。(The invention discloses an application of isovaleryl spiramycin compounds or compositions thereof in preparing medicaments for treating sepsis diseases, wherein the isovaleryl spiramycin compounds are selected from isovaleryl spiramycin I or derivatives thereof, isovaleryl spiramycin II or derivatives thereof, and isovaleryl spiramycin III or derivatives thereof; the isovaleryl spiramycin composition is selected from at least two of isovaleryl spiramycin I or derivatives thereof, isovaleryl spiramycin II or derivatives thereof, isovaleryl spiramycin III or derivatives thereof, or colimycin. The isovaleryl spiramycin compound or the composition thereof has good treatment effect on the aspect of treating sepsis and has important social and economic benefits.)

1. Application of isovaleryl spiramycin compounds or compositions thereof in preparing medicines for treating sepsis diseases.

2. Use according to claim 1, wherein the sepsis disease comprises systemic inflammatory response, sepsis, severe sepsis, septic shock, organ dysfunction or organ failure due to infection-emergent lesions.

3. Use according to claim 1 or 2, wherein the sepsis disease is induced by a coronavirus disease.

4. Use according to claim 3, wherein 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 a derivative thereof, isovalerylspiramycin II or a derivative thereof, isovalerylspiramycin III or a derivative thereof.

6. The use according to any one of claims 1 to 5, wherein the isovalerylspiramycin composition is selected from the group consisting of isovalerylspiramycin I or a derivative thereof, isovalerylspiramycin II or a derivative thereof, a combination of at least two of isovalerylspiramycin III or a derivative thereof, or colimycin.

7. The use of claim 6, wherein said isovalerylspiramycin composition further comprises a pharmaceutically acceptable carrier.

8. The use according to any of claims 1 to 7, wherein the medicament is administered in an amount of 10 to 1500mg/kg, preferably 50 to 1000mg/kg, more preferably 100 to 500 mg/kg.

9. A combination for the treatment of sepsis diseases, comprising at least one of isovalerylspiramycin i or a derivative thereof, isovalerylspiramycin ii or a derivative thereof, isovalerylspiramycin iii or a derivative thereof, or colimycin as a first pharmaceutically active ingredient; also comprises a second medicine active component which is selected from related medicines for treating sepsis.

10. The combination of claim 1, wherein the first pharmaceutically active ingredient and the second pharmaceutically active ingredient are separate formulations or are combined into one formulation.

Technical Field

The invention belongs to the field of medical chemistry, and particularly relates to an application of isovaleryl spiramycin compounds or a composition thereof in preparation of a medicament for treating sepsis diseases.

Background

Sepsis and its induced multiple organ dysfunction syndrome are one of the most common causes of death of patients at present in clinical critical, the mortality rate of patients with sepsis is above 20%, and after the disease progresses to the shock stage of sepsis, the mortality rate of patients can rise to 40% -70%, so the treatment of sepsis is always a serious challenge in intensive care units. Sepsis, one of the serious complications of clinically critical patients, can induce septic shock and MODS, and is often induced by infection, severe trauma, burns, major surgery, and other factors. Sepsis is a pathological process with inflammation activation and immunosuppression, the development stage of sepsis is systemic inflammatory response syndrome-sepsis shock-multiple organ failure, the hospitalization time of a patient is long, the prognosis is poor, and the mortality is high.

Sepsis patients often have abnormal blood coagulation function, the abnormal degree of the blood coagulation function is related to the severity of diseases, and the disorder of the blood coagulation function directly affects the ischemic change of organs and is also an important reason for causing the patients to have MODS. In sepsis patients the inflammatory and clotting reactions are promoted, plasma fibrinogen (Fg) is activated to fibrin, which is often shown to decrease and the common infectious disease Fg changes, often to a small increase or not [1 ]. Research shows that after platelet count (PLT), Prothrombin Time (PT), thromboplastin time (APTT) and fibrinogen (Fg) are treated by a blood purification technology, all indexes of blood coagulation function, renal function and the like are obviously reduced compared with those before treatment of each group.

Severe sepsis is a critical condition in which various infections cause systemic inflammatory reactions, and is often accompanied by acute blood coagulation dysfunction and acute renal failure, so that the treatment difficulty is greatly increased, and patients face life threat, so that severe sepsis has a very high mortality rate. The pathophysiology reason of sepsis is that pathogens enter blood, so that a large amount of inflammatory cells in blood vessels are propagated and activated, inflammatory mediators are secreted, a large amount of immune active factors are generated, blood vessel endothelial cells are damaged, an intrinsic coagulation pathway is started, and diffuse intravascular coagulation and coagulation-anticoagulation system balance disorder is caused.

The colimycin (Carrimycin), also called Bitespiramycin (Bitespiramycin) and Shengmiamycin (Shengjimycin) is a novel antibiotic which is formed by cloning 4 ' isovaleryltransferase gene (4 ' -O-isovaleryltransferase gene) of a carbon-mycin producing strain into a spiramycin producing strain (Streptomyces spiramycin) through a transgenic technology, directionally acylating 4 ' -OH of the spiramycin and adding an isovaleryl side chain to the 4 ' position, wherein the novel antibiotic takes 4 ' isovalerylspiramycin as a main component, and is cooperated with the applicant by the institute of biotechnology of Chinese medical institute.

The structure of the main component of the kelimycin is shown in a formula (1), and does not represent conformation:

wherein, when R ═ H, R ═ COCH₂CH(CH₃)₂Is isovaleryl spiramycin I; when R is COCH₃，R′＝COCH₂CH(CH₃)₂Isovaleryl spiramycin II; when R is COCH₂CH₃，R′＝COCH₂CH(CH₃)₂Is isovaleryl spiramycin III.

The main active components of the spiramycin isovaleryl (I + II + III) in the kelimycin are not less than 60 percent in total content, the spiramycin acylate is not less than 80 percent in total content, and the kelimycin is an acceptable pharmaceutical composition in pharmacy. The central structure is a 16-membered lactone ring which is connected with a molecule of forosamine, a molecule of mycaminose and a molecule of mycaminose, and the main components of the isovaleryl spiramycin I, II and III are structurally different from spiramycin in that a group connected to the 4' position of the mycaminose is isovaleryl instead of hydroxyl. The chemical structure is shown as a formula (1), and the chemical structure comprises more than ten components. The composition standard of the prior finished product of the corrigen is that the content of isovaleryl spiramycin III in the medicine is more than or equal to 30 percent, the sum of the proportions of isovaleryl spiramycin I, II and III is more than or equal to 60 percent, the proportion of total acylated spiramycin is more than or equal to 80 percent, and the sum of other unknown components is less than or equal to 5 percent.

Preliminary in vivo and in vitro pharmacodynamic tests show that the medicine is not only used for most G⁺The bacteria have good antibacterial activity, and can be used for treating part G^-The bacteria also have a certain effect, each technical index is obviously superior to azithromycin, erythromycin, acetylspiramycin and midecamycin, the antibacterial activity to mycoplasma pneumoniae is strongest, the antibacterial activity to erythromycin drug-resistant bacteria, gonococcus, pneumococcus, staphylococcus aureus, pseudomonas aeruginosa, bacillus influenzae, haemophilus influenzae, bacteroides fragilis, legionella, multirow bacillus and clostridium perfringens is certain, and the antibacterial activity to clinical erythromycin drug-resistant staphylococcus aureus only has little cross-resistance. The colimycin is mainly used for treating gram-positive bacteria infectious diseases, particularly upper respiratory tract infection, and possibly urinary system infection and the like. To date, no record or report has been made of the treatment of sepsis with colimycin.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides an application of isovaleryl spiramycin compounds or compositions thereof in preparing medicines for treating sepsis diseases.

In order to solve the technical problems, the invention adopts the technical scheme that:

the invention provides application of isovaleryl spiramycin compounds or compositions thereof in preparing medicines for treating sepsis diseases.

Further aspects, the sepsis disease includes systemic inflammatory response, sepsis, severe sepsis, septic shock, organ dysfunction or organ failure due to infection-emergent lesions.

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

In a further embodiment, the sepsis condition is induced by the SARS-COV-2 virus, i.e. comprises viral sepsis, in particular COVID-19-related sepsis.

In a further embodiment, 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.

In a further embodiment, the isovaleryl spiramycin composition is selected from the group consisting of isovaleryl spiramycin I or a derivative thereof, isovaleryl spiramycin II or a derivative thereof, isovaleryl spiramycin III or a derivative thereof, or a combination of at least two of isovaleryl spiramycin I or a derivative thereof, or colimycin;

in a further embodiment, the isovaleryl spiramycin composition further comprises a pharmaceutically acceptable carrier.

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

Currently recognized major pathogenesis for sepsis includes:

1) imbalance of inflammatory reaction

Among them, early proinflammatory 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) -gamma, and the like. Among them, TNF-a is the most important proinflammatory cytokine in the early stage of inflammation, plays an important role in immune defense reaction, and is also a key mediator of endotoxin damage effect. The role of IL-1 in sepsis bears many similarities to TNF-c, which initiates an inflammatory response through IL-1 β expression and in concert with TNF-a. IL-6 is an inflammatory mediator produced by IL-1 and can promote T lymphocyte proliferation together with TNF-a. The level of IL-6 in plasma can be used as a predictor of the severity of sepsis. Numerous studies have demonstrated that the synthesis of proinflammatory cytokines in sepsis is closely associated with the mitogen-activated protein kinase (MAPK) pathway and acts by activating a variety of downstream transcription factors and protein molecules.

The late-stage cytokine high mobility group box-1protein (HMGB 1) is one of important late-stage inflammation mediators, can interact with transcription factors, nucleosomes and histones, and participates in cell life activities such as transcriptional regulation, DNA replication, cell differentiation and the like. HMGB1 can be combined with TLR4 to activate multiple signal transduction pathways such as Nuclear Factor (NF) -KB and MAPK, and further promote cells to generate mediators such as TNF-a, IL-1 and IL-6, and aggravate tissue inflammatory injury.

2) Immune dysfunction

The body secretes a large amount of inflammatory mediators at the initial stage of sepsis, and then goes through an immunosuppression stage in the course of disease development, which is mainly shown by T lymphocyte clone anergy, negative regulation of immunosuppressive cells (such as Tregs) and the like.

3) Blood coagulation disorders

The mutual influence between blood coagulation dysfunction and inflammation becomes a key link for the occurrence, development and prognosis of sepsis. The processes include activation of the coagulation system, inhibition of physiological anticoagulation mechanisms, and inhibition of the fibrinolytic pathway.

Isovaleryl spiramycin I or a derivative thereof, isovaleryl spiramycin II or a derivative thereof, isovaleryl spiramycin III or a derivative thereof, or colimycin can reduce the level of m-TORC1 by inhibiting a PI3K/AKT/m-TOR signal channel, thereby inhibiting the protein synthesis of an inflammatory factor NF-KB in a cell nucleus, and simultaneously obviously reducing inflammatory factors such as IL-4, IL-6, TNF-a and the like to achieve the anti-inflammatory effect. With prolonged course, sepsis patients develop immunosuppressive reactions, including macrophage inactivation, decreased antigen presentation, and inhibition of lymphocyte proliferation activity, resulting in the release of large amounts of anti-inflammatory cytokines. The effect test of the kelimycin on immune cells shows that the kelimycin can remarkably promote the increase of total T cells (CD3 positive cells) in mice, wherein the CD4 and CD8 positive cells are increased. This shows that isovaleryl spiramycin compounds and compositions, especially the effects of corrigent on infection resistance, inflammation resistance and immunity regulation, have been further clinically verified, and can bring benefits to patients with sepsis.

A second object of the invention is a combination for the treatment of sepsis disease comprising at least one of isovaleryl spiramycin i or a derivative thereof, isovaleryl spiramycin ii or a derivative thereof, isovaleryl spiramycin iii or a derivative thereof, or colimycin as a first pharmaceutically active ingredient and further comprising a second pharmaceutically active ingredient selected from the group consisting of related drugs for the treatment of sepsis;

preferably, the first pharmaceutically active ingredient and the second pharmaceutically active ingredient are separate formulations or are combined into one formulation.

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

After adopting the technical scheme, compared with the prior art, the invention has the following beneficial effects:

the isovaleryl spiramycin compound or the composition thereof has good treatment effect on the aspect of treating sepsis and has important social and economic benefits.

The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention, are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention without limiting the invention to the right. It is obvious that the drawings in the following description are only some embodiments, and that for a person skilled in the art, other drawings can be derived from them without inventive effort. In the drawings:

FIG. 1 shows the effect of ISP I and LPS on the viability of BV2 cells; wherein A is the action result of ISP I on BV2 cell viability, B is the action result of LPS on BV2 cell viability;

FIG. 2 is a graph of the effect of ISP I and LPS on NO production in BV2 cells.

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

FIG. 4 is the results of the evaluation of the ability of the macrophages to phagocytose chicken erythrocytes by the corrigents, A being a control group, B being a corrigent group, and C being an itraconazole group;

FIG. 5 shows the results of peritoneal neutrophilic granulocytes (Gr-1 and CD11b double positive cells) detection in a first batch of C57BL/6 mice which are continuously gazed for three days at 50mg/kg to construct a peritoneal inflammation model;

FIG. 6 shows the results of the peritoneal neutrophilic granulocytes (Gr-1 and CD11b double positive cells) detection in a first batch of C57BL/6 mice which are continuously gazed for seven days at 50mg/kg to construct an abdominal inflammation model;

FIG. 7 shows the results of peritoneal neutrophil (Gr-1 and CD11b double positive cells) detection in a second batch of C57BL/6 mice, which are continuously gavaged for three days at 50mg/kg to construct a peritoneal inflammation model;

FIG. 8 shows the results of the measurement of the ratio of CD4+ and CD8+ cells in peripheral blood, in a second batch of C57BL/6 mice, which were continuously gavaged for three days at 50mg/kg to construct a celiac inflammation model;

FIG. 9 is a bar graph of the proportion of CD4+/CD3+ and CD8+/CD3+ cells in the peripheral blood of FIG. 8;

FIG. 10 shows the results of the measurement of the ratio of CD3+ cells in peripheral blood, in a second batch of C57BL/6 mice, which were subjected to continuous gavage for three days at a dose of 50mg/kg, and a peritoneal inflammation model was constructed;

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

in FIG. 12, A is the level of TNF- α detected by first exposing RAW cells to a drug for 1 hour, then inducing them to differentiate into M1 type; b, adding a medicament into RAW cells for 1 hour, inducing the RAW cells to differentiate towards M1 type, and detecting the level of iNOS; c, adding a medicament into RAW cells for 1 hour, inducing the RAW cells to differentiate towards M2 type, and detecting the level of Arg-1;

in FIG. 13, A is the differentiation of RAW cells into M1-type macrophages induced by adding cytokines, and the expression of TNF- α was detected by adding corresponding drugs; b, adding cell factors to induce RAW cells to differentiate into M1 type macrophages, adding corresponding medicines, and detecting the expression of iNOS; c, adding cell factors to induce RAW cells to differentiate into M2 type macrophages, adding corresponding medicines, and detecting the expression of Arg-1;

in FIG. 14, A is the differentiation of RAW cells into M2-type macrophages induced by adding cytokines, and the expression of TNF- α was detected by adding corresponding drugs; b, adding cell factors to induce RAW cells to differentiate into M2 type macrophages, adding corresponding medicines, and detecting the expression of iNOS; c, adding cell factors to induce RAW cells to differentiate into M2 type macrophages, adding corresponding medicines, and detecting the expression of Arg-1;

FIG. 15 is a time-dependent change curve (FAS) for the inflammatory cytokine IL- β (mean line plot SE prickle);

FIG. 16 is a time-dependent change curve (FAS) of the inflammatory cytokine IL-4 (mean line plot. + -. SE prickle);

FIG. 17 is a plot of the time-dependent change of inflammatory cytokine IL- β (PPS) (mean line plot. + -. SE prickle);

FIG. 18 is a graph of the time-dependent change of inflammatory cytokine IL-4 (PPS) (mean line plot. + -. SE prickle).

It should be noted that the drawings and the description are not intended to limit the scope of the inventive concept in any way, but to illustrate it by a person skilled in the art with reference to specific embodiments.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and the following embodiments are used for illustrating the present invention and are not intended to limit the scope of the present invention.

Example 1 Isovalerylspiramycin I or II or III or a lincomycin tablet

Specification: 200mg/350mg

Tablet core prescription:

the prescription of the coating liquid is as follows:

the preparation process comprises the following steps:

preparation of the tablet core: the main drug and the auxiliary materials are respectively sieved by a 100-mesh sieve, the isovaleryl spiramycin I or isovaleryl spiramycin II or isovaleryl spiramycin III in the prescription amount, the microcrystalline cellulose and the carboxymethyl starch sodium in the prescription amount of 1/2 are mixed evenly, and then 5 percent of polyvidone K is added₃₀Preparing soft material from water solution, granulating with 18 mesh sieve, and drying wet granules at 60 deg.C for 2 hr under ventilation condition; drying, granulating with 18 mesh sieve, adding 1/2 prescription amount of carboxymethyl starch sodium and magnesium stearate, mixing, and tabletting with 11mm diameter shallow concave punch to obtain tablet core containing medicine with weight of 350mg and hardness of 6.5 kg.

Preparing a coating solution: weighing the required opadry II (white), adding the required amount of water into a liquid preparation container, adding the water in several times, reducing the stirring speed after all the water is added, eliminating the vortex, and continuing stirring for 30min to obtain the product.

Preparation of film-coated tablets: and (3) putting the tablet cores into a coating pot, determining coating conditions, coating at the speed of a main engine of 20r/min, the air inlet temperature of 40 ℃, the air outlet temperature of 30 ℃, the spraying pressure of 0.02Mpa and the spraying flow of 1ml/min, and continuously spraying for 1.5h after the constant speed until the surfaces of the tablet cores are smooth and uniform in color and luster, and the tablets are qualified according with the inspection standard of film coating. The weight of the coating is increased by about 5 percent.

Example 2 Isovalerylspiramycin I or II or III (calculated as 10000 tablets)

Prescription:

1000g of raw powder of isovaleryl spiramycin I, isovaleryl spiramycin II or isovaleryl spiramycin III

Low-substituted hydroxypropylcellulose (5%) 92.5g

55.5g sodium carboxymethyl starch (3%)

Magnesium stearate (1%) 18.5g

Total weight of starch-weight of other raw and auxiliary materials

Total weight of 1850g

The preparation process comprises the following steps: weighing a proper amount of starch, diluting to a concentration of 15%, and heating to paste to prepare an adhesive; respectively sieving the main material isovaleryl spiramycin I or isovaleryl spiramycin II or isovaleryl spiramycin III, the auxiliary material starch, the low-substituted hydroxypropyl cellulose, the sodium carboxymethyl starch and the magnesium stearate with a 100-mesh sieve, and weighing the required main material and the auxiliary material according to the prescription amount; mixing isovaleryl spiramycin I, starch and low-substituted hydroxypropyl cellulose uniformly, pasting starch with the concentration of 15% of starch to prepare a soft material, granulating by using a 14-mesh sieve, drying at 50-60 ℃, controlling the water content to be 3-5%, grading by using the 14-mesh sieve, adding sodium carboxymethyl starch and magnesium stearate, mixing, and determining the content of granules; calculating the weight of the tablets according to the content of the particles, tabletting (phi 9mm shallow concave punch), and detecting the difference of the weight of the tablets; and packaging after the inspection is qualified.

Example 3 Isovalerylspiramycin I or II or III capsules (calculated as 10000 capsules)

Prescription:

1000g of raw powder of isovaleryl spiramycin I, isovaleryl spiramycin II or isovaleryl spiramycin III

Starch 1080-Isovalerylspiramycin I raw powder weight

Medicinal No. 3 capsule 1000 granules

Liquid paraffin 50ml

The preparation process comprises the following steps: respectively weighing the main material isovaleryl spiramycin I or isovaleryl spiramycin II or isovaleryl spiramycin III and the auxiliary material medicinal starch according to the process formula amount, and filling the materials into a mixer for fully mixing for 1.5 to 2 hours; the data obtained by sampling and detecting the content is basically consistent with theoretical data (the weight of each capsule is about 0.105g), the qualified pharmaceutical No. 3 capsules and the mixed raw materials to be filled are respectively filled into a filling machine for filling according to the operation requirements of a full-automatic capsule machine, the filled capsules are subjected to difference detection (within +/-10 percent and less than 0.3g), the dissolution rate meets the requirements, the capsules meeting the requirements after detection are put into a polishing machine, liquid paraffin is added for polishing for 15-20 minutes, and then the capsules are taken out for finished product packaging box detection.

Example 4 Isovalerylspiramycin I or II or III dry syrups (calculated as 10000 bags)

Prescription:

1250g of isovaleryl spiramycin I or isovaleryl spiramycin II or isovaleryl spiramycin III raw powder

Citric acid (0.5%) 15g

Sucrose gross weight-other raw and auxiliary materials

A total weight of about 5000g

About 1g of pigment (curcumin)

The preparation process comprises the following steps: the method comprises the steps of crushing isovaleryl spiramycin I, isovaleryl spiramycin II or isovaleryl spiramycin III raw powder, citric acid and cane sugar into particles 85% passing 300 meshes and 15% passing 180 meshes by using a high-speed airflow crusher respectively, weighing the crushed fine powder according to the prescription amount, fully mixing for 1-1.5 hours, measuring the content of the fine powder, calculating the filling amount (the theoretical filling amount is 500mg per bag), filling the mixture into a bagging machine, filling aluminum foil paper, subpackaging according to the operation requirements of the bagging machine, wherein the filling amount difference is within +/-5%, and carrying out inspection and external packaging after the packaging is qualified.

Example 5 Isovalerylspiramycin I or II or III granules (calculated as 10000 bags)

Prescription:

1250g of isovaleryl spiramycin I or isovaleryl spiramycin II or isovaleryl spiramycin III raw powder

20000g powdered sugar

Dextrin 9000g

5％PVP-K₃₀Proper amount of

The preparation process comprises the following steps: isovaleryl spiramycin I or isovaleryl spiramycin II or isovaleryl spiramycin III raw powder, sugar powder and dextrin are sieved by a 120-mesh sieve, the isovaleryl spiramycin I, the sugar powder and the dextrin are weighed according to the prescription amount and are uniformly mixed, and the uniformly mixed materials are mixed by 5 percent of PVP-K₃₀Making the mucilage into soft material, granulating with swing type granule, drying at 70 deg.C, grading, inspecting, and packaging.

Example 6 Isovalerylspiramycin I, isovalerylspiramycin II or isovalerylspiramycin III lyophilized powder for injection

Weighing 500mg of isovaleryl spiramycin I or isovaleryl spiramycin II or isovaleryl spiramycin III raw powder, uniformly mixing the isovaleryl spiramycin I or isovaleryl spiramycin II or isovaleryl spiramycin III raw powder with equimolar adipic acid, dissolving the mixture in 5ml of water to obtain a light yellow clear solution, wherein the pH value of the solution is between 4.6 and 5.6. Adding mannitol 40mg as lyophilized proppant, rapidly freezing at low temperature for 9 hr, freeze drying to obtain yellowish loose block, and dissolving in 10ml sterile water before use.

Example 7 lyophilized powder for injection of Colimycin

Weighing 500mg of the colimycin, uniformly mixing the colimycin with equimolar adipic acid, and dissolving the mixture in 5ml of water to obtain a light yellow clear solution, wherein the pH value is between 4.6 and 5.6. Adding mannitol 40mg as lyophilized proppant, rapidly freezing at low temperature for 9 hr, freeze drying to obtain yellowish loose block, and dissolving in 10ml sterile water before use.

Test example 1

Anti-inflammatory action of colimycin

1. ELISA for detecting effects of kelimycin on inflammatory factors in various tissues and organs of mice

Kunming mice for experiments are purchased from the center of experimental animals of Jiangsu university, and a mouse IL-1 beta ELISA kit (Samerfei (88-7013-88)), a mouse IL-4ELISA kit (Samerfei (88-7044-88)), and other experimental instruments and reagents are all conventional instruments and reagents.

Grouping and administration of mice

And (3) kelimycin: the dissolving method comprises adding 0.48ml polyethylene glycol 400 into some of the kelimycin, adding 2.4 μ l Tween 80, shaking, mixing, adding 1.92ml distilled water (adding 200 μ l each time, shaking, and mixing), and making into 1.44mg/ml, 2.88mg/ml, and 5.76mg/ml concentrations respectively.

Azithromycin: dissolving with small amount of anhydrous ethanol, adding water to make the content of anhydrous ethanol 10%, and making into 1.82 mg/ml.

Kunming mice, male, 18-20g size, 144, mice weighed approximately 24g after laboratory acclimation feeding, and were randomly divided into 6 groups: the normal group, model group, low group (30mg/kg), medium group (60mg/kg), high group (120mg/kg), azithromycin group (37.9 mg/kg). Each component was divided into 8 time points: 0h, 0.5h, 2.5h, 4.5h, 12h, 24h, 48h and 72 h. 3 mice per time point. The normal group of mice is not dosed and is not injected with bacteria, the model group is dosed with bacteria and is not dosed with kelimycin and azithromycin by gavage (500 microliter), the dosage of the first day is doubled, the subsequent normal dosing is carried out every day, and the model group is dosed with solvent with the same volume. Mice were sacrificed in batches at different time points after dosing.

Establishing a model: the concentration of Staphylococcus aureus was determined according to the in vitro test report, and after the concentration was determined, it was resuspended in physiological saline to a concentration of 3X 108CFU/ml, and injected into the tail vein at 24 g/100. mu.l. Administration was started one hour after injection.

Preparation of samples

The mouse eyeballs were bled and sacrificed, tissue and organs were harvested and weighed on an electronic balance, 50mg of each tissue was weighed and added to a 1.5ml EP tube, followed by 1ml of pre-cooled PBS, magnetic beads and homogenate (300Hz, 30s) in a homogenizer. After standing on ice for 30 minutes, it was centrifuged at 4 ℃ by a centrifuge (10000g, 10min), and the supernatant was taken as a test sample.

Experimental methods the experimental procedures were performed exactly according to the ELISA kit instructions and are briefly described as follows:

preparation of reagents: PBS (pH 7.35), Tween 20, PBS solution containing 0.05% Tween 20 was prepared as washing solution. (if crystals form in the buffer concentrate, heat it gently until completely dissolved). 1. Coating buffer (1 ×): PBS (10 fold) was diluted 1:10 in deionized water. 2. Capture antibody: the capture antibody (250x) was diluted 1:250 in coating buffer (1 x). 3.5xELISA/ELISPOT dilutions: the concentrated dilutions (5x) were diluted 1:5 in deionized water. 4. And (3) standard substance: recombinant mouse il-1 β standard, dissolved in distilled water, the volume of distilled water added is noted on the label of the standard vial. Standard solutions were prepared 10-30 minutes in advance and mixed thoroughly to ensure complete uniform dissolution (concentration of recombinant standard 1000 pg/ml). The standards were freshly prepared, used immediately, and not stored. 5. Detecting an antibody: the detection antibody (250X) was diluted 1:250 in ELISA/ELISPOT dilutions (1X). 6. Enzyme: HRP concentrate (100X) was diluted 1:100 in ELISA/ELISPOT dilutions (1X).

The experimental steps are as follows: 1. corning was coated with 100. mu.L of capture antibody per well in coating buffer^TMCostar^TM9018ELISA plates (diluted as described in point 1 of reagent preparation). The ELISA plates were sealed and incubated overnight at 4 ℃. 2. Remove the well and rinse 3 times with greater than 250 microliters of buffer, leave a soak time (1 minute) in each rinse step to improve the rinse effect, dry with absorbent paper, and remove the residual solution. 3. Add 200. mu.l ELISA/ELISPOT dilutions (1X) per well and incubate for 1 hour at room temperature. 4. Standards were prepared 30 minutes in advance. 5. The washing solution is pumped and washed at least once. 6. 100ul of standard, sample, blank wells were added with ELISA/ELISPOT dilutions (1X). 7. Plates were incubated at room temperature for 2 hours. 8. The detection antibody is prepared. 9. And (4) sucking air and cleaning according to the step 2, and repeatedly washing for 3-5 times. 10. To all wells 100 μ l/well diluted detection antibody was added. 11. Plates were incubated for 1 hour at room temperature. 12. HRP was prepared. 13. And (4) sucking air and cleaning according to the step 2, and repeatedly washing for 3-5 times. 14. 100ul of diluted HRP was added to each well. 15. The plates were incubated for 30min at room temperature. 16. Aspiration and rinsing was performed according to step 2, ensuring that 1 to 2 minutes of soaking was left before aspiration, and rinsing was repeated 5-7 times. 17. 100ul of TMB solution was added to each well. Incubate for 15 minutes at room temperature. 19. Add 50. mu.L of stop buffer to each well. 20. Plates were read at 450 nm. 21. And (4) collecting and processing data.

And (3) test results: the results of the effect of kelimycin on IL-4 factor and IL-1. beta. in various tissues and organs of mice are shown in tables 1 and 2, respectively.

TABLE 1

TABLE 2

Note: p < 0.05; p < 0.01; p < 0.001; p < 0.0001.

And (4) experimental conclusion: the kelimycin has an anti-inflammatory effect, has the effect of remarkably reducing IL4 factors in the lung, the kidney, the liver and the spleen, and has more remarkable effect in the liver and the spleen; the kelimycin has obvious IL-1 beta factor reducing effect in small intestine, lung, spleen, liver and kidney, especially in small intestine and lung.

2. The effect of isovalerylspiramycin I (ISP I), the major active ingredient of kelimycin, on IL-6 production was examined.

Test materials and reagents:

cell lines: mouse microglia BV2 cells were purchased from national laboratory cell resources sharing platform (Beijing)

Isovalerylspiramycin I (Shenyang Cogeneration Co., Ltd.), lipopolysaccharide (LPS 055: B5L6529), trypsin, penicillin, streptomycin, dimethyl sulfoxide (DMSO), and methyl thiazole blue (MTT) were all purchased from Sigmachemical (St. Louis, MO, USA), DMEM medium was purchased from Gibco chemical (GrandIsland, NY, USA), extra-grade fetal bovine serum was purchased from Lonsera, N.sub.H., NO detection kit (Biyuntan Biotech), and ELISA detection kit (Shanghai Aibixin Biotech).

The instruments used in the experimental examples were conventional instruments in the prior art.

Test method

Cell culture

BV2 cells were cultured in DMEM medium containing 10% FBS at 37 ℃ in 5% CO₂Cultured in an incubator. When the cell is cultured to the density of about 90%, passage and subsequent experiments can be carried out.

Cell growth inhibition assay

The MTT method was used to examine the effect of ISP I on BV2 cell activity. MTT (tetramethylazoazolium salt) is a yellow dye that can accept hydrogen ions. The MTT method detects the cell activity according to the following principle: succinate dehydrogenase and cytochrome c exist in mitochondria of living cells, tetrazole ring of MTT is cracked under the catalysis of the succinate dehydrogenase and the cytochrome c to generate formazan crystal with blue-purple color, the DMSO or the triple liquid can dissolve the crystal, and the absorbance value is detected at the wavelength of 492nm/630nm, so that the activity of the cells can be detected.

BV2 cells were seeded in 96-well plates at a density of 1.6X 10⁵cell/ml, 100 mul/well, six multiple wells per group, and adding medicine after normal culture for 24 hours. ISP I with different concentrations is added in addition to the negative control group, and the culture is continued for a prescribed time. The culture medium was aspirated off, sterile PBS was added for washing once, PBS was aspirated off, 100. mu.l of the prepared MTT was added to each well, and incubation was continued for 4 h. Adding 100 μ l of triple liquid, culturing for 12h, shaking with a micro-oscillator for 3-5min, measuring the star light value (A) at 630nm with a microplate reader, and calculating the inhibition rate of ISP I on BV2 cells according to the following formula.

Inhibitoryratio(％)＝(A₆₃₀，control-A_{630，control})/(A₆₃₀，control-A_630，blank)×100

Griess method for detecting NO content

BV2 cells at 1X 10⁵And inoculating the cells into a 24-well plate, culturing by adopting a DMEM culture solution containing 10% FBS, continuously culturing for 24 hours, and continuously culturing the cells for 6 hours by replacing the cells with a serum-free culture solution. 250 μ l of ISP I was added to the corresponding wells at final concentrations of 20 μ M, 10 μ M, and 5 μ M for pretreatment, and 1 hour later, LPS at a final concentration of 10 μ g/ml was added to the corresponding wells for induction treatment. Standing for 5% CO₂And after culturing in 37 ℃ culture boxes for 24 hours respectively, taking the supernatant and storing at-20 ℃ for NO detection.

The determination of NO was carried out as specified, the absorbance was determined at 540nm and the corresponding NO content was calculated using a standard curve.

ELISA kit for detecting cell inflammatory factor

1) Preparing all required reagents and standards; 2) taking out the microporous plate from the sealed bag restored to room temperature, putting the unused lath back into the aluminum foil bag, and sealing again; 3) the standard substance, the experimental sample or the quality control substance with different concentrations are respectively added into corresponding holes, and each hole has 100 mu L. The reaction wells were sealed with a sealing plate gummed paper and incubated at room temperature for 2 h. 4) The plate was washed with a wash bottle by aspirating off the liquid in the plate. Wash solution was added at 400. mu.L per well and the plate wash was then blotted. The operation was repeated 3 times. Every time the plate is washed, the residual liquid is sucked away as much as possible, which can help to obtain good experimental results. After the last washing, please suck all the liquid in the plate or invert the plate, and pat all the residual liquid on the absorbent paper; 5) add 100. mu.L of detection antibody to each well. Sealing the reaction hole with sealing plate gummed paper, and incubating at room temperature for 2 hours; 6) repeating the plate washing operation of the step 4; 7) mu.L of diluted streptavidin-HRP was added to each well and incubated for 20 minutes at room temperature. Attention is paid to light protection; 8) repeating the plate washing operation of the step 4; 9) add 100. mu.L of chromogenic substrate to each well and incubate for 20 minutes at room temperature. Attention is paid to light protection; 10) by adding 50. mu.L of stop solution to each well, the color of the solution in the well changes from blue to yellow. If the color of the solution is changed to green or the color change is inconsistent, the microporous plate is tapped to uniformly mix the solution; 11) within 30 minutes after the addition of the stop solution, the absorbance value at 450nm was measured using a microplate reader, and 540nm was set as a calibration wavelength. 12) And (3) calculating the result: the corrected absorbance values (OD450-OD540), the duplicate well readings for each standard and sample were averaged and then the average zero standard OD value was subtracted. Curves can be generated by plotting the logarithm of the concentration of the standard substance and the corresponding OD value, and the best fit line can be determined by regression analysis.

Statistical treatment

Statistical software SPSS26.0 is used for data analysis, Excel2016 is used for data summarization, GraphPad is used for drawing a chart, metering data are expressed in the form of mean plus or minus standard deviation (mean plus or minus SD), data among groups are compared by adopting one-factor variance analysis, and differences with P <0.05 have statistical significance.

Test results

Effect of ISP I and LPS on BV2 cell viability

After BV2 cells were treated for 24h with ISP I at different concentrations, the MTT assay showed: compared with the untreated group, the cell viability of the 2.5 mu M, 5 mu M and 10 mu M groups has no obvious difference; after BV2 cells were treated with LPS at different concentrations for 24h, the MTT assay showed: compared with the untreated group, the cell viability of the groups of 0.01. mu.g/ml, 0.1. mu.g/ml, 1. mu.g/ml and 10. mu.g/ml has no obvious difference. As shown in FIG. 1 (A is the effect of ISP I on the activity of BV2 cell and B is the effect of LPS on the activity of BV2 cell.)

ISP I inhibits LPS-induced NO production

And detecting the level of NO in cell supernatant when LPS with different concentrations acts on cells, wherein the result shows that 0.01-10 mu g/ml of LPS can induce NO generation. The effect of ISP I on the NO production amount in cell supernatant is detected, and the result shows that ISP I can inhibit NO production induced by LPS in a concentration-dependent manner. As shown in FIG. 2, in A, when the LPS concentration was 0.01. mu.g/ml, 0.1. mu.g/ml, 1. mu.g/ml, 10. mu.g/ml, the NO release amount increased with the increase in the concentration as compared with the control group. P <0.05, p <0.001 vs 0. In B, when the concentration of ISP I was 2.5. mu.M, 5. mu.M and 10. mu.M, the concentration of NO produced in ISP I decreased in a concentration-dependent manner, indicating that ISP I can decrease the amount of NO induced by LPS. # p <0.05, # p <0.01 vsLPS group, # p <0.001 vs blank group.

ISP I inhibits LPS-induced IL-6 production

And (3) detecting the level of IL-6 in cell supernatant when LPS with different concentrations acts on cells, wherein the result shows that 0.01-10 mu g/ml of LPS can induce the generation of IL-6. The ELISA method detects the influence of ISP I on the IL-6 production amount in cell supernatant, and the result shows that 5 mu M and 10 mu MISP I can obviously inhibit the IL-6 production induced by LPS. As shown in FIG. 3, in A, the concentration of LPS was 0.01. mu.g/ml, 0.1. mu.g/ml, 1. mu.g/ml and 10. mu.g/ml, respectively, and the amount of IL-6 produced by the cells was increased. P <0.05, p <0.01 vs.0. mu.g/ml group. In B, when ISP I concentration was 2.5. mu.M, 5. mu.M and 10. mu.M, IL-6 concentration was decreased in an ISP I concentration-dependent manner, indicating that ISP I can decrease LPS-induced IL-6. LPS group p <0.05, p < 0.001.

And (4) conclusion: the main active ingredient isovaleryl spiramycin I (ISP I) of the kelimycin can inhibit the generation of inflammatory cytokines IL-6 and NO induced by LSP.

Secondly, the immunoregulation function of the kelimycin

The kelimycin can enhance the ability of macrophages to phagocytose chicken erythrocytes to a certain extent. The detection of the influence of the kelimycin on immune cells shows that: kelimycin significantly promoted an increase in total T cells (CD3 positive cells) in mice, with both CD4 and CD8 positive cells increasing. Transdifferentiation studies of differentiated macrophages with colimycin showed: the kelimycin can obviously improve the expression level of TNF-a and iNos in M2 type macrophages, is obviously stronger than the expression level of TNF-a and iNos induced by LPS + INF-, is better than itraconazole, and can obviously inhibit the expression level of Arg-1 in M2 type macrophages.

1. Assessment of ability of kelimycin to phagocytose chicken erythrocytes by macrophages

The purpose is as follows: and (3) detecting whether the kelimycin can enhance the function of normal mouse macrophage, wherein the main detection index is the phagocytosis of the macrophage. Since there is no standard reagent for the research of immunology by the corrigent, according to the report, itraconazole has the effect of promoting macrophage polarization and enhancing macrophage phagocytosis, and therefore, itraconazole is selected as a positive control in the experiment.

Reagent: normal saline, 6% chicken red blood cells, methanol, acetone, Giemsa dye solution and the like; consumable material: 1ml syringe, common glass slide, gauze, petri dish, etc.

Experimental mice: the strain is Balb/c, the week age is 8-12, the source is: achievement of great achievements, quantity: each group had 2 mice.

The experimental steps are as follows: (1) grouping administration: a) physiological saline group: same volume as experimental group, p.o.; b) group of kelimycin: 50mg/kg, p.o.; c) group of itraconazole: 50mg/kg, p.o.; the administration is continued for five days; (2) each mouse was injected with 1ml of chicken red blood cells and the mice were sacrificed after waiting 30 min. (3) Injecting 1ml of normal saline into abdominal cavity, massaging to make it uniformly distributed, and making the mouse lie prostrate for 5 min. (4) The abdominal cavity of the mouse is cut open, the needle head is removed by a 1ml syringe, the abdominal cavity cleaning solution is sucked out and dripped on the glass slides, two drops are dripped on each glass slide, and the equal volume is ensured as much as possible. (5) Placing into a culture dish with wet gauze, transferring to a 37 ℃ incubator, and incubating for 30 min. (6) After incubation, the cells were rinsed in normal saline to remove nonadherent cells (pre-warmed in normal saline), and air dried. (7) Fixing with 1:1 acetone formaldehyde solution (pre-cooling at-20 deg.C). (8) Dyeing with liquid A for 45s, then adding liquid B for 4min, blowing slightly to dye into ripple shape, mixing the two, washing with distilled water, and air drying. (9) Random field of view by microscope, photographed and counted, and calculated the percent phagocytosis (10) percent phagocytosis calculation formula number of phagocytic cells/total number of macrophages × 100%

Results and analysis:

after the mouse ascites cells are stimulated by itraconazole or the colimycin, macrophages with phagocytosis phagocytose chicken erythrocytes (with megakaryocytes) or chicken erythrocytes with more peripheral aggregation. As shown in fig. 4, a is a control group, B is a clarithromycin group, and C is an itraconazole group.

Both the Keli (Keli) and itraconazole (Yiqu) groups had some degree of enhancement in the ability of macrophages to phagocytose chicken red blood cells compared to the placebo group (NC); however, no statistically significant difference was observed between the group of clarithromycin and the group of itraconazole.

Overall, the individual components are not significantly different. The experiment has inevitable subjective intervention of experiment operators, and quantitative judgment is not suitable for small differences; therefore, the macrophage is used for carrying out phagocytosis test on the fluorescent microsphere, and a flow method is used for detection, so that human subjective factors are reduced.

2. Detection of influence of kelimycin on neutrophil function

Purpose of the experiment: and detecting whether the kelimycin can enhance the inflammatory chemotactic migration capability of the mouse neutrophil, wherein the method is mainly used for detecting the proportion of the mouse neutrophil in the abdominal cavity in a flow mode by constructing a mouse abdominal cavity inflammation model, and the detection indexes are CD11b and Gr-1. Itraconazole was used as a positive control in this experiment.

Reagent: sterile PBS, fMLP, sterile HBSS, Gr-1-APC flow antibody, CD11b-FITC flow antibody, etc.; consumable material: pipette, rubber band, scissors, forceps, 1ml syringe, 15ml centrifuge tube, flow tube, etc.

Experimental mice: strain: c57BL/6 mice, week old: 8-12, source: achievement of great achievements, quantity: the first three days of dosing 3 mice each per group and 7 days of dosing 4 mice each per group. The second replicate experiment was dosed with 7 mice each for three days.

The experimental steps are as follows:

(1) grouping administration: d) physiological saline group: same volume as experimental group, p.o.; e) group of kelimycin: 50mg/kg, p.o.; f) group of itraconazole: 50mg/kg, p.o.; continuous administration for three days and seven days; (2) and (3) preparing fMLP, namely preparing 100 nMLP, diluting with PBS, using the solution as the solution is prepared, and placing the solution on ice after the solution is prepared. (3) And (3) intraperitoneal injection: each mouse was intraperitoneally injected with 100uL100nM of precooled fMLP. (4) Abdomen contracting water cells: after 4 hours of injection, the mice were sacrificed, the four limbs were fixed, the abdominal skin was cut open, a small opening in the peritoneum was cut open, the peritoneum was fixed with a rubber band and a clip, the precooled HBSS were sucked with a straw to repeatedly wash the abdominal cavity, the motion was gentle, approximately 8-10 ml of ascites was collected, and they were placed on ice. (5) Centrifuge at 1000rpm for 5min and carefully discard the supernatant. (6) If red blood cells are present, they are resuspended in 1ml of red blood cell lysate and lysed on ice for 3-5 min. (7) Centrifuge at 1000rpm for 5min, discard the supernatant, and wash once with 3ml PBS. (8) Add 500uLPBS to resuspend. (9) Taking 10^6 cells, incubating the flow antibody at room temperature in the dark: CD11b-FITC and Gr-1-APC. (10) The cells were washed once with PBS, resuspended, placed on ice, protected from light, and tested on the machine.

The experimental results are as follows: the results of evaluating the neutrophil migration ability of the mouse celiac inflammation model by the kelimycin are shown in fig. 5-7. FIGS. 5-7 show the flow measurement of the ratio of neutrophils (Gr-1 and CD11b double positive cells) in the mouse abdominal inflammation model with different administration times and different batches. FIG. 5 shows the results of peritoneal neutrophilic granulocytes (Gr-1 and CD11b double positive cells) detection in a first batch of C57BL/6 mice which are continuously gazed for three days at 50mg/kg to construct a peritoneal inflammation model; FIG. 6 shows the results of the peritoneal neutrophilic granulocytes (Gr-1 and CD11b double positive cells) detection in a first batch of C57BL/6 mice which are continuously gazed for seven days at 50mg/kg to construct an abdominal inflammation model; FIG. 7 shows the results of peritoneal neutrophil (Gr-1 and CD11b double positive cells) detection in a second batch of C57BL/6 mice, which were continuously gavaged for three days at 50mg/kg to construct a peritoneal inflammation model.

FIGS. 8-11 show the detection of T lymphocytes in peripheral blood after three days after the administration of the drug to construct a mouse abdominal inflammation model, and the detection of the proportion of CD3+, CD4+ and CD8+ cells by flow cytometry.

The kelimycin and the itraconazole can remarkably promote the migration of the neutrophils to an inflammation part in a mouse body, and the result in a specific mouse body is particularly obvious; three days compared to seven days, no further enhancement of the effect was found with the continuous administration of seven days. The kelimycin and itraconazole can remarkably promote the increase of total T cells (CD3 positive cells) in mice, wherein the CD4 and CD8 positive cells are increased, but the itraconazole performs better.

3. Effect of Colimycin on macrophage differentiation

Experimental background and purpose: macrophages can be divided into two main categories: classically activated macrophages (M1) are characterized by increased expression of the major histocompatibility complex MHC class ii, increased Nitric Oxide (NO), increased levels of reactive oxygen species and proinflammatory cytokines such as Tumor Necrosis Factor (TNF), interleukin-1 (IL-1) and interleukin-6 (IL-6). Another class is alternatively activated macrophages (M2), also known as selectively activated macrophages, which are a class of macrophages with immunosuppressive activity that increase interleukin-4 (IL-4) levels and increase the expression of interleukin-10 (IL-10) and Arginase (Arg) under multiple stimuli, resulting in increased cell proliferation and collagen production. Polarization of M1 cells has a protective effect on human health when encountered with infection and cancer. Therefore, the purpose of this experiment was to examine whether or not the differentiation of macrophages was affected by colimycin, and further to investigate the potential immunomodulatory effects of colimycin.

Experimental reagent consumables: reagent: RAW246.7 cell line, 1640 culture medium, FBS, RNA extraction kit, reverse transcription kit, SYBR fluorescence quantitative kit. Cytokines: IL-4, INF- γ, LPS; consumable material: cell culture related consumables.

The experimental steps are as follows:

the first scheme is as follows: whether the drug would promote or inhibit the polarization process of RAW246.7 cells was investigated.

(1) The kelimycin and itraconazole were stored in DMSO at 10mM concentration stock. (2) Culturing RAW246.7 cell line in vitro, collecting cells growing in logarithmic phase, spreading in 6-well plate according to density of 1 × 10^6 cells per well, and treating cells with 20uM of colimycin and 20uM of itraconazole without adding drugs. (3) After 1h of action, each treatment was subdivided into 2 groups, one group was supplemented with LPS (200ng/ml) and IFN-. gamma. (20ng/ml), the other group was supplemented with IL-4(20ng/ml), and cells were harvested after 12h of culture. (4) Extracting total RNA of cells, carrying out reverse transcription to obtain cDNA, and detecting the RNA level of a corresponding index.

Scheme II: to investigate whether the drug has an effect on already polarized cells.

(1) The kelimycin and itraconazole were stored in DMSO at 10mM concentration stock. (2) The RAW246.7 cell line was cultured in vitro, plated in the same manner, and the cells were treated with the corresponding cytokines (LPS + IFN-. gamma.or IL-4) for 12 h. (3) After 12h, cells that had been polarized to M1 or M2 were added with either colimycin (20uM) or itraconazole (20uM) and allowed to react for another 12 h. (4) Collecting cells, extracting total RNA of the cells, carrying out reverse transcription to obtain cDNA, and detecting the RNA level of a corresponding index.

The experimental results are as follows:

the first scheme is as follows: whether or not Cochinomycin promotes or inhibits differentiation of RAW246.7 cells was investigated

FIG. 12A shows the TNF-. alpha.levels detected by exposing RAW cells to a drug for 1h and then inducing differentiation to M1. Drawing notes: NC (RAW cells, without any treatment); PC1(RAW cells plus LPS + INF-. gamma., induced differentiation of RAW cells into M1 type macrophages); keli (RAW cells were first supplemented with colimycin, then LPS + INF- γ); yiqu (RAW cells were first added with itraconazole, then LPS + INF- γ); statistical tests were performed, P <0.05, P <0.01, P < 0.001.

FIG. 12B shows the measurement of iNOS levels in RAW cells after 1 hour of drug administration and induced differentiation to M1 type;

FIG. 12C shows that the RAW cells were treated with the drug for 1 hour, and then induced to differentiate into M2 type, and the level of Arg-1 was detected; drawing notes: NC (RAW cells, without any treatment); PC2(RAW cells plus IL-4, induced differentiation of RAW cells into M2 type macrophages); keli (RAW cells plus first colimycin plus then IL-4); yiqu (RAW cells were added itraconazole first, then IL-4); statistical tests were performed, P <0.05, P <0.01, P < 0.001.

Analysis of a result of the protocol:

the kelimycin can increase the expression of TNF-alpha and iNos and inhibit the expression of Arg-1. Suggesting that the kelimycin may promote the differentiation and function of M1 type macrophage.

Scheme II: A. to investigate whether the kelimycin has the transdifferentiation effect on differentiated macrophage

FIG. 13A shows the differentiation of RAW cells into M1-type macrophages induced by cytokine, and the detection of TNF- α expression by the corresponding drugs

FIG. 13B shows the differentiation of RAW cells into M1-type macrophages induced by cytokine, and the addition of drugs to detect iNOS expression FIG. 13A shows the differentiation of RAW cells into M2-type macrophages induced by cytokine, and the addition of drugs to detect Arg-1 expression

In FIGS. 13A-13C, NC (RAW cells, without any treatment); PC1(RAW cells plus LPS + INF-. gamma., induced differentiation of RAW cells into M1 type macrophages); PC2(RAW cells plus IL-4, induced differentiation of RAW cells into M2 type macrophages); keli ((LPS + INF- γ added to RAW cells first, inducing RAW cells to differentiate into M1-type macrophages, then calicheamicin), Yiqu (LPS + INF- γ added to RAW cells first, inducing RAW cells to differentiate into M1-type macrophages, then itraconazole), and statistical tests were performed, P <0.05, > P <0.01, > P < 0.001.

The experimental results are as follows: consistent with the results obtained on the RAW246.7 cell line, the expression of TNF- α and iNOS induced by LPS + INF- γ was not further enhanced by colimycin, but was able to suppress the expression level of Arg-1 in M1-type macrophages, suggesting that the potential role of colimycin in enhancing the function of M1-type macrophages is.

Scheme II: b to investigate whether the kelimycin has transdifferentiation effect on differentiated macrophage

FIG. 14A shows the differentiation of RAW cells into M2-type macrophages induced by cytokine, and the detection of TNF- α expression by the corresponding drugs

FIG. 14B shows that cytokines were added to induce RAW cells to differentiate into M2-type macrophages, and then the corresponding drugs were added to detect iNOS expression

FIG. 14C shows that cytokines are added to induce RAW cells to differentiate into M2-type macrophages, and corresponding drugs are added to detect Arg-1 expression

In FIGS. 14A-14C: NC (RAW cells, without any treatment); PC1(RAW cells plus IL-4, induced differentiation of RAW cells into M2 type macrophages); PC2(RAW cells plus IL-4, induced differentiation of RAW cells into M2 type macrophages); keli ((initial addition of IL-4 to RAW cells, induced differentiation of RAW cells into M2-type macrophages, and then calicheamicin)), Yiqu (initial addition of IL-4 to RAW cells, induced differentiation of RAW cells into M2-type macrophages, and then itraconazole), and statistical tests were performed, P <0.05, P < 0.001.

The experimental results are as follows: the kelimycin can obviously improve the expression level of TNF-alpha and iNos in M2 type macrophages, is obviously stronger than the expression level of TNF-alpha and iNos induced by LPS + INF-gamma, is better than itraconazole, and can obviously inhibit the expression level of Arg-1 in M2 type macrophages.

Anti-infection effect of kelimycin

1. Action of colimycin on carbapenem-resistant acinetobacter baumannii and carbapenem-resistant klebsiella pneumoniae

The strain is as follows: 1 each of carbapenem-resistant acinetobacter baumannii and carbapenem-resistant klebsiella pneumoniae;

culture medium: staphylococcus MH medium was incubated with 2% NaCl at 35-37 ℃ for 24 h.

Other strains are conventional MH culture medium, and are incubated at 35-37 ℃ for 16-18 observation results.

The formula comprises 1% of peptone, 0.3% of beef powder, 0.5% of NaCl and 1.2% of agar powder.

After the preparation of the culture medium, placing the culture medium in a triangular flask, performing autoclaving at 121 ℃ for 15min, cooling, and placing the culture medium in a refrigerator at 4 ℃ for later use.

Experimental animals:

kunming mice, half male and female, weighing 18-22g, were purchased from Wydon laboratory animals Inc. (SPF grade). The breeding conditions are 25 ℃ and 60% of humidity. The feeding conditions were of the SPF grade, and the study procedure followed the guidelines for experimental animal feeding management and use.

Preparing the medicine:

1) the carbapenem-resistant Klebsiella pneumoniae bacterial group is prepared by weighing 0.24g of the drug, adding 2.4ml of Tween 80 and 2.4ml of absolute ethanol to dissolve the drug, and adding distilled water to a total volume of 20ml, wherein the concentration is 12 mg/ml. 14mL of the drug solution was taken out, and distilled water was added thereto to make the total volume 20mL, at which the concentration was 8.4 mg/mL, which was 70% of the original concentration. By analogy, 14mL of high-concentration liquid medicine is taken, distilled water is added until the total volume is 20mL, and the dosage interval is 1:0.7, and 5 groups are formed.

2) The carbapenem-resistant acinetobacter baumannii group is prepared by weighing 0.45g of the drug, adding 4.5ml of tween 80 and 4.5ml of absolute ethanol to dissolve the drug, and adding distilled water until the total volume is 30ml, wherein the concentration is 15 mg/ml. 24mL of the drug solution was taken out, and distilled water was added thereto to a total volume of 30mL, at which the concentration was 12mg/mL, which was 80% of the original concentration. By analogy, 24mL of high-concentration liquid medicine is taken, distilled water is added until the total volume is 30mL, and the dosage interval is 1:0.8, and the total amount is 6 groups.

Preliminary experiments (drug dosage range determination)

Adding 3ml LB culture medium into a centrifugal tube, taking out a plate from 4 ℃, picking 2-3 colonies with an inoculating loop, inoculating into the LB culture medium, and culturing at 37 ℃.

Secondly, MLD measurement, namely taking healthy Kunming mice with the weight of 18-22g, randomly grouping the Kunming mice into a plurality of groups, wherein each group comprises 5 mice and can be used as both male and female. The bacterial solution was diluted with 5% of high activity dry yeast to different concentrations, i.e., 0.5mL each, and observed for 7d after infection, and the number of mouse deaths was recorded so that the minimum bacterial amount causing 100% of mouse deaths was taken as MLD and the bacterial amount was taken as the infectious bacterial amount in the in vivo protection test.

And thirdly, measuring the drug dosage range, namely taking healthy Kunming mice with the weight of 18-22g, randomly grouping the mice into a plurality of groups, wherein each group comprises 5 mice for both male and female.

Preparing 1MLD bacterial dose by using 5% high-activity dry yeast, injecting the bacterial dose into the abdominal cavity, wherein each dose is 0.5mL, after infecting a mouse for 1h, performing intragastric lavage by using medicines with different concentrations, wherein the administration volume is 0.2mL/10g, observing the administration volume for 24h, recording the death number of the mouse, and designing the administration dose of an in-vivo treatment protection experiment according to the result.

The formal experimental method comprises the following steps:

1) carbapenem-resistant Klebsiella pneumoniae group: healthy Kunming mice with the weight of 18-22g are randomly divided into 6 groups, each group contains 10 mice, and the mice can be used for both male and female. Of the 6 groups, 5 groups were administered, and 1 group was a solvent control group.

2) Carbapenem-resistant acinetobacter baumannii group: healthy Kunming mice with the weight of 18-22g are randomly divided into 7 groups, each group contains 10 mice, and the mice can be used for both male and female. 6 of the 7 groups were administered, and 1 group was a solvent control group.

Preparing 1MLD bacterial amount by using 5% high-activity dry yeast, injecting all mice with 0.5mL in an abdominal cavity, infecting the mice for 1h, performing intragastric administration by using medicines or solvents with different concentrations, wherein the administration volume is 0.2mL/10g, continuously observing for 14d, and recording the death number of the mice.

The experimental results are as follows:

(1) MLD results (maximum lethal bacteria quantity determination):

TABLE 3

Name of bacterium	MLD value
		Carbapenem-resistant Klebsiella pneumoniae	1×108CFU/mL
Carbapenem-resistant acinetobacter baumannii	1×106CFU/mL

(2) Determining the dosage range of the medicine:

TABLE 4

Name of bacterium	Dosage range of the drug
		Carbapenem-resistant Klebsiella pneumoniae	58-240mg/kg
Carbapenem-resistant acinetobacter baumannii	98-300mg/kg

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

TABLE 5

(4) ED50 results for drug-resistant bacteria (calculated according to the Bills method)

TABLE 6

Name of bacterium	ED50
		Carbapenem-resistant Klebsiella pneumoniae	153.2mg/kg
Carbapenem-resistant acinetobacter baumannii	112.2mg/kg

And (4) conclusion: the colimycin has good bacteriostatic effect on carbapenem-resistant acinetobacter baumannii and carbapenem-resistant klebsiella pneumoniae in vivo, and can remarkably improve the survival rate of infected animals.

Cytokine-related data in clinical trials of colimycin resistance to neocorolla

Immune-related index change condition of new coronary patient after medication (kelimycin)

Compared with the change of the baseline change value with time by the immune related indexes (lymphocyte count, lymphocyte percentage, CD4, CD8 count and percentage, inflammatory cytokine) at days 1, 3, 5, 7-10 after the administration of the FAS set test group and the control group, wherein the lymphocyte count has statistical significance (P is 0.003) compared with the change of the baseline change value with time at days 7-10.

TABLE 7-1

Corresponding to Table 7-1, the time-dependent change curve (FAS) (mean line plot. + -. SE prickle) of inflammatory cytokine IL- β is shown in FIG. 15; the time-dependent change curve (FAS) (mean line plot. + -. SE prickle) of the inflammatory cytokine IL-4 is shown in FIG. 16.

TABLE 7-2

TABLE 8-1

Corresponding to Table 8-1, the time-dependent change curve (PPS) of the inflammatory cytokine IL- β (mean line plot. + -. SE prickle) is shown in FIG. 17; the time-dependent change curve (PPS) (mean line plot. + -. SE prickle) of the inflammatory cytokine IL-4 is shown in FIG. 18.

TABLE 8-2

Although the present invention has been described with reference to a preferred embodiment, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.