阅读说明：本技术 1-乙基-3，7-二甲基黄嘌呤在制备治疗肺炎药物中的应用 (Application of 1-ethyl-3, 7-dimethyl xanthine in preparation of medicine for treating pneumonia ) 是由 黄文� 张伯礼 李幼平 张俊华 于 2020-04-26 设计创作，主要内容包括：本发明涉及1-乙基-3,7-二甲基黄嘌呤在制备治疗肺炎药物中的应用,属于医药技术领域。本发明提供了1-乙基-3,7-二甲基黄嘌呤或其盐在制备治疗肺炎的药物中的用途。药理实验结果显示,1-乙基-3,7-二甲基黄嘌呤的各种剂型均能明显抑制病毒、细菌、支原体感染引发的肺炎,降低感染小鼠的肺指数,降低TNF-α、IL-8、IL-10、IL-17等炎性因子水平,提高IFN-γ水平,改善外周血的白细胞和中性粒细胞计数,表明1-乙基-3,7-二甲基黄嘌呤具有强效抗肺炎的作用。(The invention relates to application of 1-ethyl-3, 7-dimethyl xanthine in preparing a medicine for treating pneumonia, and belongs to the technical field of medicines. The invention provides application of 1-ethyl-3, 7-dimethylxanthine or salt thereof in preparing a medicament for treating pneumonia. Pharmacological experiment results show that 1-ethyl-3, 7-dimethylxanthine in various dosage forms can obviously inhibit pneumonia caused by virus, bacteria and mycoplasma infection, reduce pulmonary index of infected mice, reduce the levels of inflammatory factors such as TNF-alpha, IL-8, IL-10, IL-17 and the like, improve IFN-gamma level, and improve the count of leukocytes and neutrophils in peripheral blood, and the 1-ethyl-3, 7-dimethylxanthine has a strong anti-pneumonia effect.)

1. Use of a compound of formula i or a salt thereof in the manufacture of a medicament for the treatment of pneumonia:

2. use according to claim 1, characterized in that: the pneumonia is viral pneumonia, bacterial pneumonia and mycoplasma pneumonia.

3. Use according to claim 2, characterized in that: the viral pneumonia is pneumonia caused by influenza virus and coronavirus infection; preferably, the influenza virus is an influenza a virus; preferably, the coronavirus is SARS-CoV or SARS-CoV-2.

4. Use according to claim 2, characterized in that: the bacterial pneumonia is pneumonia caused by Klebsiella pneumoniae infection.

5. Use according to claim 2, characterized in that: the mycoplasma pneumonia is pneumonia caused by mycoplasma pneumoniae infection.

6. Use according to claim 1, characterized in that: at least one of the following is satisfied:

the drug reduces the pulmonary index;

the medicine can relieve lung lesion;

the medicine reduces the pneumonia death rate;

the drug increases IFN-gamma levels in lung tissue;

the agent reduces TNF-alpha levels in lung tissue;

the medicament reduces the level of peripheral blood leukocytes and/or neutrophils;

the medicine can reduce the level of IL-8, IL-10 and IL-17 in serum.

7. Use according to any one of claims 1 to 6, characterized in that: the medicine is a preparation prepared by taking a compound shown in a formula I or a salt thereof as an active ingredient and adding pharmaceutically acceptable auxiliary materials or auxiliary ingredients.

8. Use according to claim 7, characterized in that: the preparation is an oral preparation, a nasal mucosa administration preparation, an oral mucosa administration preparation or an injection preparation.

9. Use according to claim 7, characterized in that: the preparation is common tablets, sustained-release agents, controlled-release agents, effervescent tablets, granules, capsules, oral liquid, nasal spray, sublingual tablets or injection.

Technical Field

The invention relates to application of 1-ethyl-3, 7-dimethyl xanthine in preparing a medicine for treating pneumonia, and belongs to the technical field of medicines.

Background

Pneumonia is an important disease threatening human health, and has high morbidity and mortality. Viral, bacterial, mycoplasma infections are the major cause of pneumonia. For example, mycoplasma pneumoniae is a pathogen between bacteria and viruses, and mycoplasma pneumoniae is a common acquired pneumonia, i.e., inflammation of the lungs caused by mycoplasma pneumoniae. Immune damage is an important pathogenesis of mycoplasma infection. Pneumonia caused by mycoplasma pneumoniae can cause various extrapulmonary complications such as encephalitis, myocarditis, hepatitis, blood system diseases and the like, and the pneumonia causes great harm to human bodies.

Research shows that the microbial toxin can induce the combination of cell factor and inflammation medium, start intracellular signal transduction path and promote the synthesis of proinflammatory cell factors, such as TNF, IL-1, IL-6, etc. Cytokines play an important role in the pathogenesis of severe pneumonia. Thus, simple antimicrobial therapy does not significantly and rapidly ameliorate the inflammatory response, and inhibition of pro-inflammatory cytokine production can alter the clinical symptoms of pneumonia and reduce mortality. The effective regulation and control of the inflammatory reaction of the organism have important significance for improving the treatment effect of the pneumonia.

Disclosure of Invention

The invention aims to provide application of 1-ethyl-3, 7-dimethyl xanthine in preparing a medicine for treating pneumonia.

The invention provides an application of a compound shown as a formula I or a salt thereof in preparing a medicament for treating pneumonia:

further, the pneumonia is viral pneumonia, bacterial pneumonia and mycoplasma pneumonia.

Further, the viral pneumonia is pneumonia caused by influenza virus and coronavirus infection.

Preferably, the influenza virus is an influenza a virus.

Preferably, the coronavirus is SARS-CoV or SARS-CoV-2.

Further, the bacterial pneumonia is pneumonia caused by Klebsiella pneumoniae infection.

Further, the mycoplasma pneumonia is pneumonia caused by mycoplasma pneumoniae infection.

Further, the drug decreases the pulmonary index.

Further, the medicament alleviates lung lesions.

Further, the drug reduces pneumonia mortality.

Further, the drug increases IFN- γ levels in lung tissue.

Further, the drug reduces TNF- α levels in lung tissue.

Further, the medicament reduces peripheral blood leukocyte and/or neutrophil levels.

Further, the medicine reduces the level of IL-8, IL-10 and IL-17 in serum.

Furthermore, the medicine is a preparation prepared by taking the compound shown in the formula I or the salt thereof as an active ingredient and adding pharmaceutically acceptable auxiliary materials or auxiliary ingredients.

Further, the preparation is an oral preparation, a nasal mucosa administration preparation, an oral mucosa administration preparation or an injection preparation.

Furthermore, the preparation is common tablets, sustained release agents, controlled release agents, effervescent tablets, granules, capsules, oral liquid, nasal spray, sublingual tablets or injection.

In a preferred embodiment of the invention, the medicament comprises a therapeutically effective amount of the 1-ethyl-3, 7-dimethylxanthine as the active ingredient and one or more inert, non-toxic, pharmacologically suitable excipients. Preferably, the excipient is selected from at least one of a carrier, a solvent, an emulsifier, a dispersant, a wetting agent, a binder, a stabilizer, a colorant, a perfume, and a flavoring agent.

The effective dose of the 1-ethyl-3, 7-dimethyl xanthine can vary according to the administration mode, the age and the weight of a patient, the severity of the disease and other relevant factors, and the recommended dose is 100-1000 mg/time for oral administration and 1-3 times per day; the recommended dosage of the injection is 15-45 mg/time, and 1 time per day; the recommended dose for the aerosol inhalation administration is 500-1000 mg/time and 1-3 times per day.

The invention provides application of 1-ethyl-3, 7-dimethyl xanthine in preparing a medicine for treating pneumonia. Pharmacological experiment results show that the 1-ethyl-3, 7-dimethyl xanthine can obviously inhibit pneumonia caused by virus, bacteria and mycoplasma infection, reduce pulmonary index of infected mice, reduce the levels of inflammatory factors such as TNF-alpha, IL-8, IL-10, IL-17 and the like, improve IFN-gamma level, improve the count of leukocytes and neutrophils in peripheral blood, and show that the 1-ethyl-3, 7-dimethyl xanthine has a strong anti-pneumonia effect. The application of the invention can provide a new medicine source for the treatment of pneumonia, and has potential significant economic effect and social benefit. The preparation prepared from the 1-ethyl-3, 7-dimethyl xanthine has the prospect of being applied as a medicament for treating pneumonia, is expected to become an innovative traditional Chinese medicine for treating pneumonia with high efficiency and low toxicity, and has wide industrialization prospect.

Detailed Description

The invention provides an application of a compound shown as a formula I or a salt thereof in preparing a medicament for treating pneumonia:

the inventor of the invention uses coffee as a lead compound to modify and optimize the structure of the compound, so as to obtain a medicament for treating pneumonia, which has better activity and lower toxicity. Through the investigation of structure-effect relationship, alkyl substituents on nitrogen atoms at positions 1, 3 and 7 are key groups for ensuring the anti-pneumonia effect of xanthine. The inventors have further screened a variety of different alkyl groups, which ultimately provided 1-ethyl-3, 7-dimethylxanthines of formula I, which exhibited the best anti-pneumonic effects. In addition, the treatment effect of the 1-ethyl-3, 7-dimethyl xanthine on a lung inflammation model is obviously better than that on pancreatitis, hepatitis and rheumatoid arthritis models, and the 1-ethyl-3, 7-dimethyl xanthine has great potential for being developed into anti-pneumonia medicines.

The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

The compound CF is 1-ethyl-3, 7-dimethyl xanthine, and the chemical structure is shown as the formula I:

caffeine (caffeine) and compounds a, b, c, d for pharmacodynamic comparisons were each as follows:

EXAMPLE 1 preparation of a conventional tablet of the invention

Mixing CF crystal with starch of about 1/3, adding starch slurry, mixing to obtain soft material, granulating with 16 mesh sieve, drying at 60 deg.C, sieving with 14 mesh sieve, grading, adding the rest starch (dried at 105 deg.C at 100 deg.C) and microcrystalline cellulose, sieving with 14 mesh sieve, and tabletting.

EXAMPLE 2 preparation of an injection of the invention

CF powder is added into pure water to prepare a solution with the concentration of 5mg/mL, filtered and sterilized by a disposable filter membrane with the diameter of 0.22 mu m, and packaged into a sterilized ampere bottle for later use.

EXAMPLE 3 preparation of sprays of the invention

Dissolving CF powder in pure water, and adding xylitol and ethyl maltol as flavoring agent.

EXAMPLE 4 preparation of sustained Release agent of the present invention

The weight range of CF in the sustained-release tablet (based on 1 tablet) prepared by the present invention is 60-120 mg.

A. 60-120mg of CF powder, 20-75mg of diluent microcrystalline cellulose, 50-150mg of filler starch and 10-50mg of sustained-release material carboxymethyl cellulose are sieved and mixed uniformly;

B. mixing the mixed medicinal powder with 5-75% ethanol solution or 0.5-5% hypromellose as binder for 12-18 min to obtain soft material;

C. granulating the soft material with 16 mesh nylon sieve, drying at 60 deg.C, taking out, air drying, grading with 14 mesh iron wire sieve, adding 3-4mg magnesium stearate as lubricant for glidant, and mixing;

D. sampling and measuring the content, calculating the weight of the tablet, tabletting and coating the outer layer of the tablet core with a film coat according to a conventional method.

EXAMPLE 5 preparation of controlled Release agent of the present invention

60-120mg of CF compound powder, 200mg of microcrystalline cellulose, 70mg of K4M type hydroxypropyl methylcellulose, 65mg of K15M type hydroxypropyl methylcellulose and 70mg of K100M type hydroxypropyl methylcellulose are added into a mixer, mixed for 10min at 10r/min, added with 5mg of magnesium stearate and 2.5mg of silicon dioxide, mixed for 3min at 10r/min, and tabletted by a rotary tabletting machine, wherein the hardness control range of the tablet is 50-80N. The dosage form provides an in vitro dissolution according to USP Apparatus Type Il (paddle method) of about 5% to about 50% CF released after 1 hour when tested at 100rpm using 1000ml of distilled water containing 2.0% sodium lauryl sulfate maintained at a temperature of about 37 ± 0.5 ℃ as dissolution medium; about 40% to about 85% CF is released after 6 hours; and no less than about 70% CF is released after 12 hours.

EXAMPLE 6 preparation of effervescent tablets of the invention

(1) Taking CF compound powder, adding part of filler, and drying for later use;

(2) respectively drying boric acid and effervescent agent for later use;

(3) calcining Alumen;

(4) pulverizing the above materials, adding disintegrating agent, mixing, adding binder, mixing, granulating, grading, adding anhydrous alcohol solution of Borneolum Syntheticum, Camphora, and benzalkonium bromide, mixing, and making into tablet.

EXAMPLE 7 preparation of sublingual tablets of the invention

Firstly, preparing blank granules from lactose and powdered sugar by using 17% starch slurry, then mixing a 10% CF ethanol solution (fed in 120%) into the fine powder of the blank granules (less than 30 days), sieving the mixture twice by using a 10-mesh sieve, drying the mixture for 50 to 60 minutes at a temperature of below 40 ℃, then uniformly mixing the dried mixture with the blank granules and magnesium stearate prepared in advance, and tabletting the mixture to obtain the finished product.

EXAMPLE 8 preparation of granules of the invention

Mixing CF powder, sugar powder and dextrin, granulating with water or aqueous ethanol, drying, grading, packaging, inspecting, and storing.

EXAMPLE 9 preparation of oral liquid of the invention

Adding CF powder into purified water, heating for dissolving, adding Mel and active carbon, boiling, filtering while hot, adding citric acid into the filtrate, adding purified water to adjust to the preparation amount, packaging, and sterilizing.

EXAMPLE 10 preparation of capsules of the invention

Taking CF powder, adding dextrin by an equivalent incremental method, uniformly mixing, adding 70% ethanol to prepare a soft material which is kneaded into a mass by hands and then is dispersed by pressing, extruding and granulating through a sieve with 14-22 meshes, drying at 60-80 ℃, grading, and filling into No. 2 capsules.

The beneficial effects of the invention are proved by the pharmacodynamic experiments below.

Test example 1 therapeutic Effect of the Compound of the present invention on viral pneumonia

(A) Material

C57 mouse, ribavirin, CF tablet, CF injection, CF spray, CF slow release agent, CF controlled release agent, CF effervescent tablet, CF sublingual tablet, CF granule, CF oral liquid, caffeine, compound a, b, C, d, influenza A virus mouse lung adaptation strain FM1, TNF-alpha ELISA kit, animal interferon INF-gamma ELISA kit, formaldehyde and ethanol.

(II) grouping and molding

C57 mice, randomly divided into 57 groups of 12 mice each, 684 total mice, Normal group (Normal), Model group (Model), Ribavirin group (Ribavirin); CF tablet group, caffeine tablet group, compound a tablet group, compound b tablet group, compound c tablet group, compound d tablet group; CF injection group, caffeine injection group, compound a injection group, compound b injection group, compound c injection group and compound d injection group; CF spray group, caffeine spray group, compound a spray group, compound b spray group, compound c spray group, compound d spray group; a CF sustained-release agent, a caffeine sustained-release group, a compound a sustained-release group, a compound b sustained-release group, a compound c sustained-release group and a compound d sustained-release group; a CF controlled release agent group, a caffeine controlled release group, a compound a controlled release group, a compound b controlled release group, a compound c controlled release group and a compound d controlled release group; CF effervescent tablet group, caffeine effervescent tablet group, compound a effervescent tablet group, compound b effervescent tablet group, compound c effervescent tablet group and compound d effervescent tablet group; the compound c is selected from the group consisting of a CF sublingual tablet group, a caffeine sublingual tablet group, a compound a sublingual tablet group, a compound b sublingual tablet group, a compound c sublingual tablet group and a compound d sublingual tablet group; CF particle group, caffeine particle group, compound a particle group, compound b particle group, compound c particle group, compound d particle group; CF oral liquid group, caffeine oral liquid group, compound a oral liquid group, compound b oral liquid group, compound c oral liquid group and compound d oral liquid group. After adaptive feeding for 2 days, virus inoculation is started, influenza virus is inoculated on the 1 st day, normal mice are inoculated with normal saline by nasal drip, the other mice are inoculated with 50 mu L of influenza A H1N1 strain FM1 (the hemagglutination titer is 1:320) by nasal drip, after 24 hours, the sustained-release group is administered by intragastric administration, the injection group is administered by tail vein injection, and the spray group is administered by nasal drip. And (3) drug intervention treatment on days 2 to 8, wherein the normal group and the model group of mice are administrated by pouring, spraying or tail vein injection of normal saline with the volume of 10mL/kg, and the mice of the ribavirin group are administrated by pouring, spraying or tail vein injection of ribavirin with the same volume, and the administration dose of each group is 58.5mg/kg, which is equivalent to the clinical equivalent dose of human. The administration was continued until day 8. The systemic responses including hair, activity, defecation, feeding, respiration, etc. were observed daily for each group of mice after infection with virus, and the daily body mass and death of the mice were recorded. On the 9 th day after the infection of the mouse virus, the mouse is sacrificed, the body mass is weighed, the whole lung is taken out and washed by physiological saline, the water is absorbed by clean filter paper, the lung mass is weighed, and the lung index, the lung index inhibition rate, the lung tissue inflammatory cytokine TNF-alpha, the animal interferon INF-gamma and the death rate are calculated.

Lung index ═ lung mass/body mass × 100%

Lung index inhibition rate ═ [ model group average lung index-administration group average lung index ]/[ model group average lung index-blank control group average lung index ] × 100%

(III) the instrument

A dryer, a centrifuge, an optical microscope, a blood gas analyzer, a microplate reader, an electronic balance, a turbidimetric tube, and a 0.5mL/1.5mL Eppendorf tube.

(IV) data statistics

Data were processed using SPSS 18.0 and the data were expressed as x + -s or x, with statistical significance given a difference of P < 0.05.

(V) results of the respective groups

1. General observations

The normal group mice had good mental status and had acute response to the external environment. After 2 days of virus infection, the mice in the model group gradually appear the conditions of diet reduction and urine volume reduction, hair is easy to fall off, breath is short, sleepiness, conglobation is curled, aversion to cold and trembling, activity is dull, and spirit is listened.

2. Pulmonary index inhibition rate, mortality rate

TABLE 1

3. Inflammatory factor TNF-alpha of lung tissue and interferon INF-gamma of animals

TABLE 2

Test example 2 therapeutic Effect of the Compound of the present invention on bacterial pneumonia

(A) Material

The kit comprises a Klebsiella pneumoniae standard strain, cefoperazone/sulbactam for injection, heparin sodium, ketamine hydrochloride, 0.5% dexamethasone sodium phosphate, rat IL-8, an IL-10ELISA kit and sterile physiological saline.

(II) Instrument

A dryer, a centrifuge, an optical microscope, a blood gas analyzer, a microplate reader, an electronic balance, a turbidimetric tube, and a 0.5mL/1.5mL Eppendorf tube.

(III) grouping and Molding

Healthy Standard (SD) rats (180-220 g in body mass) aged 6 weeks are randomly grouped, 12 rats in each group account for 696 rats and are divided into a Normal group (Normal), a Model group (Model), dexamethasone sodium phosphate, cefoperazone, sulbactam control group and cefoperazone, sulbactam control group; CF tablet group, caffeine tablet group, compound a tablet group, compound b tablet group, compound c tablet group, compound d tablet group; CF injection group, caffeine injection group, compound a injection group, compound b injection group, compound c injection group and compound d injection group; CF spray group, caffeine spray group, compound a spray group, compound b spray group, compound c spray group, compound d spray group; a CF sustained-release agent, a caffeine sustained-release group, a compound a sustained-release group, a compound b sustained-release group, a compound c sustained-release group and a compound d sustained-release group; a CF controlled release agent group, a caffeine controlled release group, a compound a controlled release group, a compound b controlled release group, a compound c controlled release group and a compound d controlled release group; CF effervescent tablet group, caffeine effervescent tablet group, compound a effervescent tablet group, compound b effervescent tablet group, compound c effervescent tablet group and compound d effervescent tablet group; the compound c is selected from the group consisting of a CF sublingual tablet group, a caffeine sublingual tablet group, a compound a sublingual tablet group, a compound b sublingual tablet group, a compound c sublingual tablet group and a compound d sublingual tablet group; CF particle group, caffeine particle group, compound a particle group, compound b particle group, compound c particle group, compound d particle group; the CF oral liquid group, the caffeine oral liquid group, the compound a oral liquid group, the compound b oral liquid group, the compound c oral liquid group and the compound d oral liquid group are 58 groups in total, and 12 animals are bred adaptively for 3d, and then inoculation is started. Weighing rats in the model group, the positive drug control group and each administration group, performing ketamine anesthesia, injecting 0.3ml of Klebsiella pneumoniae into a trachea through annular subchondral puncture, enabling the bacteria to directly enter the lung, putting 6 groups of rats back to a mouse cage after inoculation is completed, and after 24 hours, performing intragastric administration on the granule group, performing tail vein injection administration on the injection group, and performing nasal drip on the spray group; and 6 days to 10 days of drug intervention treatment, wherein rats in a normal group and a model group are irrigated, sprayed or injected with normal saline as a solvent in a volume of 10mL/kg, mice in an administration group are irrigated, sprayed through a nasal cavity or injected with CF, caffeine or compound a, b, c and d preparations in the same volume through tail veins, and the administration dose of each group is 58.5mg/kg, which is equivalent to the clinical equivalent dose of human. Respectively inoculating with the bacterial liquid, collecting 1.5mL of carotid blood on days 6, 8 and 10, standing whole blood, centrifuging at 4 ℃ for 10min at the speed of 3000r/min, collecting serum, storing at-80 ℃ for testing, and testing the levels of leukocyte, neutrophil granulocyte, IL-8 and IL-10.

(IV) data statistics

Using SPSS 18.0 to process data for data measurementIs represented by P<A difference of 0.05 is statistically significant.

(V) results of the respective groups

1. General observations

The normal rats had good mental status and had acute response to the external environment. After the rats in the model group are infected with Klebsiella pneumoniae 2d, the mental retardation, the activity and the diet are gradually reduced, the urine volume is reduced, the respiration is short, the sleepiness is realized, and the body shape is gradually thinned.

2. Pulmonary index suppression and mortality

TABLE 3

3. Leukocyte and neutrophil of peripheral blood table 4

4. Peripheral blood inflammatory factors IL-8 and IL-10

TABLE 5

Test example 3 therapeutic Effect of the Compound of the present invention on Mycoplasma pneumonia

(A) Material

0.9% physiological saline, diethyl ether, absolute ethyl alcohol, PBS buffer solution, alcohol cotton ball, neutral gum, arabic gum solution, polylysine, IL-10, IL-17Elisa kit; glucose 50% injection, penicillin, and PPLO culture medium.

(II) Instrument

Biological safety cabinet, desiccator, centrifuge, optical microscope, blood gas analyzer, ELIASA, electronic balance, turbidimeter, 0.5mL/1.5mL Eppendorf pipe.

(III) grouping and Molding

BALB/C mice (20 +/-2 g) are randomly grouped into 12 mice per group, and 684 mice are counted and divided into a Normal group (Normal), a Model group (Model) and an azithromycin control group; a CF capsule group, a caffeine capsule group, a compound a capsule group, a compound b capsule group, a compound c capsule group and a compound d capsule group; a CF slow release group, a caffeine slow release group, a compound a slow release group, a compound b slow release group, a compound c slow release group and a compound d slow release group; a CF controlled release group, a caffeine controlled release group, a compound a controlled release group, a compound b controlled release group, a compound c controlled release group and a compound d controlled release group; CF effervescent tablet group, caffeine effervescent tablet group, compound a effervescent tablet group, compound b effervescent tablet group, compound c effervescent tablet group and compound d effervescent tablet group; the compound c is selected from the group consisting of a CF sublingual tablet group, a caffeine sublingual tablet group, a compound a sublingual tablet group, a compound b sublingual tablet group, a compound c sublingual tablet group and a compound d sublingual tablet group; CF particle group, caffeine particle group, compound a particle group, compound b particle group, compound c particle group, compound d particle group; CF oral liquid group, caffeine oral liquid group, compound a oral liquid group, compound b oral liquid group, compound c oral liquid group, compound d oral liquid group; CF injection group, caffeine injection group, compound a injection group, compound b injection group, compound c injection group and compound d injection group; CF spray group, caffeine spray group, compound a spray group, compound b spray group, compound c spray group, compound d spray group; for a total of 57 groups. Inoculation was started 5d after acclimatization. The model group, the azithromycin control group and the administration group are infected with 20ul of 1 × 106CFU/ml mycoplasma pneumoniae by a nasal drip method, the drug intervention treatment is carried out after 24 hours, the content of the capsule group is dissolved in pure water to form suspension, the gastric perfusion administration is carried out, the injection group is carried out by tail vein injection administration, and the spray group is used for nasal drip. Normal group and model group mice were drenched, sprayed or injected with tail vein physiological saline in a volume of 10mL/kg, mice of azithromycin group were drenched, nasal spray or injected with tail vein with the same volume of azithromycin, caffeine, compound a, b, c, d group mice were drenched, nasal spray or injected with the same volume of the corresponding compound. The dose administered in each group was 58.5mg/kg, corresponding to a clinically equivalent dose in humans. Detecting IL-10 and IL-17 levels in mouse serum at different time points by an ELISA method, weighing and killing each group of mice after fasting for 9 hours on the 7 th day after model building, taking lung tissues of the mice, washing the lung tissues by normal saline, absorbing water by clean filter paper, weighing the lung mass, and calculating the lung index and the lung index inhibition rate.

Lung index ═ lung mass/body mass × 100%

Lung index inhibition rate ═ [ model group average lung index-administration group average lung index ]/[ model group average lung index-blank control group average lung index ] × 100%

(IV) data statistics

Using SPSS 18.0 to process data for data measurementIs represented by P<A difference of 0.05 is statistically significant.

(V) results of the respective groups

1. General observations

The normal group of mice had good spirit, glossy coat, normal activity, eating and respiration, increasing physical quality, white and glossy lung tissue and no inflammatory cell infiltration during the observation period. The model group mice have reduced activity after model building, general mental state, lusterless fur, reduced diet and water intake, accelerated respiratory rate, slowed growth speed of physique, deep red lung tissue color, congestion and edema, damaged lung tissue epithelial cells, obviously thickened and edema of alveolar walls, fractured and obviously widened alveolar spaces, interstitial inflammatory edema of lung and inflammatory cell infiltration.

2. Pulmonary index and pulmonary index inhibition rate

As can be seen from Table 6, the body mass of the model group mice is obviously reduced, the lung mass is obviously increased, and the lung index is obviously increased compared with the normal group mice. The lung index of the model group is 0.95 percent, and is obviously increased (P is less than 0.05) compared with the lung index (0.67 percent) of the normal group, which indicates that the mouse infection virus model in the experiment is reliable. The lung index of the azithromycin group is 1.59 percent and is obviously lower than that of the model group (P <0.05), which indicates that the positive drug treatment model is reliable. The lung index of the CF capsule group is 0.68%, the CF slow-release group is 0.71%, the CF controlled-release group is 0.66%, the CF effervescent tablet group is 0.69%, the CF sublingual buccal tablet group is 0.69%, the CF granule group is 0.67%, the CF oral liquid group is 0.67%, the CF injection group is 0.73%, and the CF spray group is 0.71%, which are all obviously lower than those of the model group. The effective concentration range of the CF oral preparation, injection and spray can effectively improve the pulmonary index and the inhibition rate of the pulmonary index, the inhibition effect of the CF oral preparation, the injection and the spray is obviously stronger than that of caffeine and compounds a, b, c and d, and the CF oral preparation, the injection and the spray can be used together with anti-mycoplasma medicaments to improve the treatment effect.

3. IL-10 and IL-17 levels in mouse serum

As can be seen from Table 6, the serum IL-10 and IL-17 contents of the mice of the positive drug control group and each administration group are obviously lower than those of the model group (P < 0.05). Therefore, the CF can effectively inhibit the pulmonary inflammatory reaction caused by mycoplasma infection by various dosage forms, and has better effect than caffeine and the compounds a, b, c and d.

Test example 4 therapeutic Effect of the Compounds of the present invention on various inflammatory diseases

Inflammation is a fundamental biological response of the body to the invasion of pathogenic microorganisms, promoting the repair of damaged cells and tissues and preventing their further damage. However, excessive inflammatory responses can also lead to damage and necrosis of tissue organs, and even organs throughout the body. At present, inflammation is a common disease and frequently encountered disease threatening human health, and for the treatment of inflammation, glucocorticoid steroid anti-inflammatory drugs and traditional non-steroid anti-inflammatory drugs are commonly used. Even though the existing anti-inflammatory drugs can effectively control infectious inflammation and non-infectious inflammation and effectively eliminate functional damage disorder caused by inflammation, the long-term use of the existing anti-inflammatory drugs can cause the functional decline of adrenal cortex and other complications. The clinical application of the conventional anti-inflammatory drugs is greatly limited due to the disadvantages of poor selectivity and significant side effects. In recent years, with the continuous and deep research on inflammation mechanism and the wide application to molecular biology technical analysis, a new anti-inflammatory drug with good curative effect and small side effect is urgently needed to be developed in clinic.

Pneumonia is one of the major respiratory diseases, with high morbidity and mortality. Severe pneumonia often causes respiratory failure disease leading to death, and inflammatory cytokines play an important role in the pathogenesis, so that effective control of the levels thereof is one of the important means for treating pneumonia. Viral, bacterial, mycoplasma infections are the major cause of pneumonia. Microbial infection induces the binding of cytokines and inflammatory mediators, activates the immune system of the body, causes the massive activation of inflammatory cells, and releases a large amount of cytokines. Simple antimicrobial therapy does not provide rapid relief from inflammation and disease, while inhibition of cytokine production and release can significantly improve the clinical symptoms of pneumonia and reduce mortality.

Pancreatitis is a common gastrointestinal disease, acute pancreatitis is a common digestive system disease with high morbidity and mortality, and acute pancreatitis of children is an increasingly common problem. Although the current diagnostic and therapeutic levels are constantly increasing, the incidence, recurrence and mortality of acute pancreatitis remains high. For the treatment of acute pancreatitis, no specific treatment scheme is available at present, and according to Chinese diagnosis and treatment guidelines for acute pancreatitis (2014) and American gastroenterology society' acute pancreatitis treatment guidelines (2013), the main treatment schemes comprise nutrition support, antibiotic treatment, surgical treatment and Chinese and Western medicine combined treatment.

The liver, as an important organ of the body, bears the main physiological functions of the body, such as metabolism, detoxification, secretion, etc., and is a lymph organ with unique immunological characteristics, which participates in natural immunity and adaptive immune response, and if liver dysfunction occurs, various immune diseases can be produced. Autoimmune hepatitis is a chronic liver disease with indefinite etiology, obvious autoimmune phenomenon and inflammatory necrosis as the main pathological change, and the disease has wide epidemiology, seriously harms human health and brings great economic loss. Autoimmune hepatitis can lead to elevated serum transaminases, circulating autoantibodies, hyper-gammaglobulinemia, changes in hepatic histology and chronic inflammatory liver disease characterized by immunosuppressive responses, which can lead to cirrhosis and liver failure. Therefore, it is necessary to research a drug having a significant anti-immune hepatitis effect.

Rheumatoid arthritis is a systemic autoimmune disease mainly characterized by chronic destructive joint diseases, mainly damages articular cartilage, bones and joint capsules, and can cause consequences such as joint deformity and function loss in severe cases. Rheumatoid arthritis is characterized by synovitis and its resultant destruction of articular cartilage and bone, which ultimately can lead to joint deformity. The treatment method mainly comprises general treatment, drug treatment, surgical operation, other treatment and the like; however, at present, it is not clear which antirheumatic should be selected for treating rheumatoid arthritis to improve the disease condition. If rheumatoid arthritis is not treated timely and effectively, the disability rate is high, so that the treatment of rheumatoid arthritis mainly aims at controlling the disease condition and improving the joint function and prognosis, and the principles of early treatment, combined medication and individualized treatment are emphasized.

The experimental example proves that the treatment effect of the 1-ethyl-3, 7-dimethyl xanthine on the pulmonary inflammation is obviously superior to the treatment effect on pancreatitis, hepatitis and rheumatoid arthritis models.

(A) Material

Influenza A virus mouse lung adapted strain FM1, TNF-alpha ELISA kit, animal interferon INF-gamma ELISA kit, formaldehyde and ethanol.

(II) grouping and molding

72C 57 mice were randomly divided into 6 groups of 12 mice each, namely a Normal group (Normal), a Model group (Model), a pneumonia group, a pancreatitis group, a hepatitis group and a rheumatoid arthritis group, and inoculated molding was started after adaptive feeding for 2 days.

And (3) pneumonia model: ketamine is used for anesthesia, 0.1ml of influenza A mouse lung adaptive strain FM1 is injected into a trachea through the puncture of the cricoid subchondral, and the influenza A mouse lung adaptive strain FM1 directly enters the lung and is put back to a mouse cage after the inoculation is finished.

Pancreatitis model: and (4) carrying out NaT molding, fasting for 12h before experiment, and carrying out operation in a sterile environment without water prohibition. Intraperitoneal injection of 3.5% sodium pentobarbital, anesthesia, sterilizing the operation site with alcohol cotton ball, placing sterile gauze on the operation site, performing laparotomy at the width of two fingers under the xiphoid process, starting to cut down after seeing the liver, extending a cotton swab soaked in normal saline into the abdominal cavity, finding duodenum at the back of the liver, turning out, placing on sterile gauze, finding pancreaticobiliary duct and duodenal papilla, penetrating a hole on the duodenum with a 1ml syringe needle in the direction aligned with the papilla, inserting a 24G indwelling needle hose into the pancreaticobiliary duct along the papilla, closing the common bile duct under the liver with an arterial vascular clamp, injecting sodium taurocholate (concentration of 3.8%) at a dose of 0.1ml/100G and a speed of 0.1ml/1min, dripping normal saline onto the exposed pancreas and duodenum during the process, keeping moist, slowly taking out the hose after the injection is finished, closing the common bile duct continuously, taking off the vascular artery clamp after 3min, taking down the duodenum, and suturing the abdominal cavity in a double-layer mode.

Hepatitis model: ConA solution (15mg/kg) was injected into the tail vein, and an equal volume of physiological saline was injected into the control group.

Arthritis model: 10% chloral hydrate solution (3.5mL/kg) was injected intraperitoneally and fixed in the supine position after anesthesia was complete. 0.1mL of 1g/L sodium iodoacetate solution is injected into the knee joint cavity. The animals were driven for 30min each day from day 5, and were allowed to move freely in their cages the rest of the time.

The experiments in this group were administered by intravenous injection with injection solution, continuously until day 8. The systemic responses including hair, activity, defecation, feeding, respiration, etc. were observed daily for each group of mice after infection with virus, and the daily body mass and death of the mice were recorded. On day 9 post-mouse viral infection, mice were sacrificed, weighed and evaluated for inflammatory cytokines TNF-. alpha.IL-6 and mortality.

(III) the instrument

A dryer, a centrifuge, an optical microscope, a blood gas analyzer, a microplate reader, an electronic balance, a turbidimetric tube, and a 0.5mL/1.5mL Eppendorf tube.

(IV) data statistics

Data were processed using SPSS 18.0 and the data were expressed as x + -s, with differences of <0.05 being statistically significant.

(V) results of the respective groups

1. General observations

The normal group mice had good mental status and had acute response to the external environment. After 2 days of virus infection, the mice in the model group gradually appear the conditions of diet reduction and urine volume reduction, hair is easy to fall off, breath is short, sleepiness, conglobation is curled, aversion to cold and trembling, activity is dull, and spirit is listened.

2. Mortality rate

The normal group has no death, the model group has higher death condition, and the death rate reaches 41.67 percent; the mortality rate of the pneumonia group is 13.33 percent, the mortality rate of the pancreatitis group is 23.45 percent, the mortality rate of the hepatitis group is 21.56 percent, and the mortality rate of the rheumatoid arthritis group is 22.15 percent, which indicates that the concentration is in an effective concentration range. CF can reduce the mortality of a model group, wherein CF (1-ethyl-3, 7-dimethyl xanthine) intervenes in the mortality of a pneumonia group, a pancreatitis group is 6.66%, a hepatitis group is 10.7% and a rheumatoid arthritis group is 11.07%.

3. Serum inflammatory factors TNF-alpha and IL-6

Table 7 therapeutic effect of CF on different inflammatory diseases (n-12,)

test example 5 therapeutic Effect of the Compound of the present invention on novel coronavirus pneumonia COVID-19

(A) Material

0.9% physiological saline, diethyl ether, absolute ethyl alcohol, PBS buffer solution, alcohol cotton ball, neutral gum, arabic gum solution, polylysine, IL-10, IL-17Elisa kit; glucose 50% injection, penicillin, and PPLO culture medium.

(II) Instrument

Biological safety cabinet, desiccator, centrifuge, optical microscope, blood gas analyzer, ELIASA, electronic balance, turbidimeter, 0.5mL/1.5mL Eppendorf pipe.

(III) grouping and Molding

BALB/C mice (20 +/-2 g) are randomly divided into 12 mice in each group, 708 mice in total are divided into a Normal group (Normal), a Model group (Model) and a ribavirin control group; ribavirin + hydrocortisone; hydrocortisone group; a CF capsule group, a caffeine capsule group, a compound a capsule group, a compound b capsule group, a compound c capsule group and a compound d capsule group; a CF slow release group, a caffeine slow release group, a compound a slow release group, a compound b slow release group, a compound c slow release group and a compound d slow release group; a CF controlled release group, a caffeine controlled release group, a compound a controlled release group, a compound b controlled release group, a compound c controlled release group and a compound d controlled release group; CF effervescent tablet group, caffeine effervescent tablet group, compound a effervescent tablet group, compound b effervescent tablet group, compound c effervescent tablet group and compound d effervescent tablet group; the compound c is selected from the group consisting of a CF sublingual tablet group, a caffeine sublingual tablet group, a compound a sublingual tablet group, a compound b sublingual tablet group, a compound c sublingual tablet group and a compound d sublingual tablet group; CF particle group, caffeine particle group, compound a particle group, compound b particle group, compound c particle group, compound d particle group; CF oral liquid group, caffeine oral liquid group, compound a oral liquid group, compound b oral liquid group, compound c oral liquid group, compound d oral liquid group; CF injection group, caffeine injection group, compound a injection group, compound b injection group, compound c injection group and compound d injection group; CF spray group, caffeine spray group, compound a spray group, compound b spray group, compound c spray group, compound d spray group; for a total of 59 groups. Ribavirin, 58.5 mg/kg; hydrocortisone, 25 mg/kg; the compound CF, the caffeine and the compounds a, b, c and d are all 58.5 mg/kg; inoculation was started 2d after acclimatization. Except for the mice in the blank control group, the mice in each group were infected with COVID-19 virus (30 μ L) by nasal instillation. After 24h, performing drug intervention treatment, dissolving the contents of the tablet group in pure water to form suspension, and performing intragastric administration; tail vein injection administration of injection group; nasal drip of spray group; the slow release agent group is administered by intragastric administration; the controlled release agent group is administered by intragastric administration; the effervescent tablet is administered by intragastric administration. Normal group mice were drenched, sprayed or injected tail vein with normal saline at a volume of 10mL/kg, and the administration was continued up to day 8. On day 9 after the infection with mouse virus, the mice were sacrificed, the body mass was weighed, the whole lung was taken out and washed with physiological saline, the water was removed with a clean filter paper, the lung mass was weighed, and the lung index, the lung index inhibition rate, the lung tissue inflammatory cytokine NF- κ B, TNF- α and the mortality were calculated.

Lung index ═ lung mass/body mass × 100%

Lung index inhibition rate ═ [ model group average lung index-administration group average lung index ]/[ model group average lung index-blank control group average lung index ] × 100%

(IV) data statistics

Using SPSS 18.0 to process data for data measurementIs represented by P<A difference of 0.05 is statistically significant.

(V) results of the respective groups

1. General observations

The normal group of mice had good spirit, glossy coat, normal activity, eating and respiration, increasing physical quality, white and glossy lung tissue color and no inflammatory cell infiltration during observation period. The model group mice have reduced activity after model building, general mental state, lusterless fur, reduced diet and water intake, accelerated respiratory rate, slowed growth speed of physique, deep red lung tissue color, congestion and edema, damaged lung tissue epithelial cells, obviously thickened and edema of alveolar walls, fractured and obviously widened alveolar spaces, interstitial inflammatory edema of lung and inflammatory cell infiltration.

2. Degree of lung disease

As shown in Table 8, in terms of lung disease reduction rate of mice, different formulations of the compound CF can reduce lung disease to a certain extent, and the curative effect is higher than that of the ribavirin + hydrocortisone group, the ribavirin group and the hydrocortisone group.

3. Pulmonary index suppression and mortality

As can be seen from Table 8, the mouse infection virus model established by the experiment is reliable, wherein the lung index of the compound CF injection group is remarkably reduced (P is less than 0.05) compared with the model group, which indicates that different formulations of the compound CF can alleviate lung lesion caused by influenza virus and the medicament has certain curative effect. The normal group has no death, the model group has higher death condition, and the death rate reaches 46.67 percent. Compared with the model group, the compound CF has the tendency of reducing the mortality rate of virus-infected mice in different dosage form groups. In addition, the compound CF injection has better effect of reducing the death rate.

TABLE 8

4. Lung tissue inflammatory factor NF-kappa B, TNF-alpha

TABLE 9

Test example 6 therapeutic Effect of the Compound of the present invention on pneumonia caused by SARS-CoV infection

(A) Material

0.9% physiological saline, diethyl ether, absolute ethyl alcohol, PBS buffer solution, alcohol cotton balls, neutral gum, arabic gum, polylysine, NF-kB Elisa kit and TNF-alpha Elisa kit; glucose 50% injection, penicillin, and PPLO culture medium.

(II) Instrument

Biological safety cabinet, desiccator, centrifuge, optical microscope, blood gas analyzer, ELIASA, electronic balance, turbidimeter, 0.5mL/1.5mL Eppendorf pipe.

(III) grouping and Molding

BALB/C mice (20 +/-2 g) are randomly divided into 12 mice in each group, 708 mice in total are divided into a Normal group (Normal), a Model group (Model) and a ribavirin control group; ribavirin + hydrocortisone; hydrocortisone group; a CF capsule group, a caffeine capsule group, a compound a capsule group, a compound b capsule group, a compound c capsule group and a compound d capsule group; a CF slow release group, a caffeine slow release group, a compound a slow release group, a compound b slow release group, a compound c slow release group and a compound d slow release group; a CF controlled release group, a caffeine controlled release group, a compound a controlled release group, a compound b controlled release group, a compound c controlled release group and a compound d controlled release group; CF effervescent tablet group, caffeine effervescent tablet group, compound a effervescent tablet group, compound b effervescent tablet group, compound c effervescent tablet group and compound d effervescent tablet group; the compound c is selected from the group consisting of a CF sublingual tablet group, a caffeine sublingual tablet group, a compound a sublingual tablet group, a compound b sublingual tablet group, a compound c sublingual tablet group and a compound d sublingual tablet group; CF particle group, caffeine particle group, compound a particle group, compound b particle group, compound c particle group, compound d particle group; CF oral liquid group, caffeine oral liquid group, compound a oral liquid group, compound b oral liquid group, compound c oral liquid group, compound d oral liquid group; CF injection group, caffeine injection group, compound a injection group, compound b injection group, compound c injection group and compound d injection group; CF spray group, caffeine spray group, compound a spray group, compound b spray group, compound c spray group, compound d spray group; for a total of 59 groups. Ribavirin, 58.5 mg/kg; hydrocortisone, 25 mg/kg; the compound CF, the caffeine and the compounds a, b, c and d are all 58.5 mg/kg; inoculation was started 2d after acclimatization. Except for the mice in the blank control group, which were infected with SARS-CoV (30. mu.L) by nasal drip. After 24h, performing drug intervention treatment, dissolving the contents of the tablet group in pure water to form suspension, and performing intragastric administration; tail vein injection administration of injection group; nasal drip of spray group; the slow release agent group is administered by intragastric administration; the controlled release agent group is administered by intragastric administration; the effervescent tablet is administered by intragastric administration. Normal group mice were drenched, sprayed or injected tail vein with normal saline at a volume of 10mL/kg, and the administration was continued up to day 8. On day 9 after the infection with mouse virus, the mice were sacrificed, the body mass was weighed, the whole lung was taken out and washed with physiological saline, the water was removed with a clean filter paper, the lung mass was weighed, and the lung index, the lung index inhibition rate, the lung tissue inflammatory cytokine NF- κ B, TNF- α and the mortality were calculated.

Lung index ═ lung mass/body mass × 100%

Lung index inhibition rate ═ [ model group average lung index-administration group average lung index ]/[ model group average lung index-blank control group average lung index ] × 100%

(IV) data statistics

Using SPSS 18.0 to process data for data measurementIs represented by P<A difference of 0.05 is statistically significant.

(V) results of the respective groups

1. General observations

The normal group of mice had good spirit, glossy coat, normal activity, eating and respiration, increasing physical quality, white and glossy lung tissue color and no inflammatory cell infiltration during observation period. The model group mice have reduced activity after model building, general mental state, lusterless fur, reduced diet and water intake, accelerated respiratory rate, slowed growth speed of physique, deep red lung tissue color, congestion and edema, damaged lung tissue epithelial cells, obviously thickened and edema of alveolar walls, fractured and obviously widened alveolar spaces, interstitial inflammatory edema of lung and inflammatory cell infiltration.

2. Degree of lung disease

As shown in Table 10, in terms of lung disease reduction rate of mice, different formulations of the compound CF can reduce lung disease to a certain extent, and the curative effect is higher than that of the ribavirin + hydrocortisone group, the ribavirin group and the hydrocortisone group.

3. Pulmonary index suppression and mortality

As can be seen from Table 10, the experimentally established mouse model for viral infection is reliable, wherein the lung index of the CF injection composition is significantly reduced (P <0.05) compared with that of the model composition, which indicates that different formulations of CF compound can alleviate lung diseases caused by influenza virus and that the drug has a certain curative effect. The normal group has no death, the model group has higher death condition, and the death rate reaches 49.72 percent. Compared with the model group, the compound CF has the tendency of reducing the mortality rate of virus-infected mice in different dosage form groups. In addition, the compound CF injection has better effect of reducing the death rate.

Watch 10

4. Lung tissue inflammatory factor NF-kappa B, TNF-alpha

TABLE 11

The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited by the foregoing examples, which are provided to illustrate the principles of the invention, and that various changes and modifications may be made without departing from the spirit and scope of the invention, which is also intended to be covered by the appended claims.