WO2022057861A1

WO2022057861A1 - Application of diterpene compound or salt thereof in preparation of medicine for preventing and treating acute lung injury

Info

Publication number: WO2022057861A1
Application number: PCT/CN2021/118816
Authority: WO
Inventors: 冯海梅; 李云森; 俞云会; 刘中天; 李玲; 江传亮; 陈子珺; 王梦菲; 张童; 解晴; 王艳萍
Original assignee: 苏州沪云新药研发股份有限公司
Priority date: 2020-09-17
Filing date: 2021-09-16
Publication date: 2022-03-24
Also published as: CN114191423B; CN114191423A

Abstract

A diterpene compound or a pharmaceutically acceptable salt thereof in the preparation of a medicine for preventing and treating acute lung injury. The structure of the diterpene compound is as shown in Formula I. The diterpene compound had favorable preventive and therapeutic effects on a mouse model with a lipopolysaccharide-induced acute lung injury, and improved the state of pathological changes of lung tissues in a mouse with an acute lung injury. The compound is capable of reducing damage from pulmonary edema, reducing damage to the lung blood-air barrier, inhibiting the expression of tumor necrosis factor-α (TNF-α), interleukin 1β (IL-1β), and interleukin 6 (IL-6) in injured lung tissue, and suppressing the infiltration and oxidative stress of neutrophile granulocytes in injured lung tissue.

Description

Application of diterpenoids or their salts in the preparation of medicines for preventing and treating acute lung injury

technical field

The present application relates to the field of biomedicine, in particular to the application of a diterpene compound or a pharmaceutically acceptable salt thereof in the preparation of a medicine for preventing and treating acute lung injury.

Background technique

Acute lung injury (ALI) is a common critical illness with a very high mortality rate, which seriously threatens the lives of critically ill subjects and affects their quality of life. A variety of factors can induce ALI, mainly including ① direct lung injury factors: severe lung infection, inhalation of gastric contents, lung contusion, inhalation of toxic gases, drowning, oxygen poisoning, etc.; ② indirect lung injury factors: severe infection, severe Non-thoracic trauma, acute severe pancreatitis, massive blood transfusion, cardiopulmonary bypass, disseminated intravascular coagulation, etc.

Although the research on acute lung injury has gradually deepened, its pathogenesis is still not fully understood. At present, it is believed that inflammatory response plays a key role in the occurrence and development of acute lung injury. When acute lung injury occurs, the balance of pro-inflammatory and anti-inflammatory responses is disrupted, resulting in the destruction of lung endothelial cells and lung epithelial barriers, and alveolar capillaries. Disruption of membrane integrity, excessive transepithelial migration of neutrophils stimulated by a large number of inflammatory mediators, promoting the release of pro-inflammatory factors and cytotoxic mediators, damage to lung tissue, and severe spillover into the systemic circulation lead to multi-organ failure. High expression of the proinflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) are important events in the early response to acute lung injury, which can disrupt the alveolar-capillary barrier and increase pulmonary edema. High expression of interleukin-6 predisposes acute lung injury subjects to develop multiple organ failure, and circulating IL-6 has been shown to be a good predictor of acute lung injury disease of different etiologies. The local release of cytokines can initiate a series of amplification and regulation effects, resulting in increased neutrophil activation and release of toxic mediators, and the production of a large number of reactive oxygen species, aggravating the severity of acute lung injury and pulmonary edema. Therefore, inhibiting the expression and release of inflammatory factors in the process of acute lung injury, inhibiting neutrophil infiltration and oxidative stress injury is of great significance for the prevention and treatment of acute lung injury.

The strategies for the treatment of acute lung injury in the prior art are still limited, and it is very meaningful to develop more therapeutic approaches for acute lung injury.

SUMMARY OF THE INVENTION

In view of the deficiencies of the prior art, the purpose of the present application is to provide the application of a diterpene compound or a pharmaceutically acceptable salt thereof in the preparation of a medicine for preventing and/or treating acute lung injury.

In order to achieve this purpose, the application adopts the following technical solutions:

The present application finds for the first time that the diterpenoid compound represented by formula I has a significant effect of preventing and treating acute lung injury, it has a good preventive and therapeutic effect on the mouse model of acute lung injury induced by lipopolysaccharide, and can improve the lung tissue of mice with acute lung injury. Pathological changes, reduce pulmonary edema injury, reduce the damage of pulmonary blood gas barrier, inhibit the expression of tumor necrosis factor α, interleukin 1β and interleukin 6 in the lung tissue of lung injury, inhibit the expression of neutrophils in the lung tissue of lung injury. Infiltration and oxidative stress. The above results show that the compounds can be used to prevent and treat acute lung injury, and provide a new idea and drug for the treatment of acute lung injury.

On the one hand, the application provides the application of a diterpene compound or a pharmaceutically acceptable salt thereof in the preparation of a medicine for preventing and/or treating acute lung injury, wherein the diterpene compound has the structure shown in formula I:

The molecular structure of the diterpene compound represented by formula I and the synthesis method thereof have been described in the published texts of patents CN104761460A and WO2016150207A1, which are incorporated herein in their entirety.

In certain embodiments, the pharmaceutically acceptable salts include tartrate, stearate, oxalate, citrate, lactate, sorbate, fumarate, formate, acetic acid Salt, benzoate, benzenesulfonate, ethanesulfonate, resinate, trifluoroacetate, maleate, mesylate, fumarate, amino acid salt, nicotinate, phosphoric acid salt, sulfate, hydrochloride or hydrobromide.

In certain embodiments, the medicament inhibits the expression of TNF-α, IL-1β and IL-6 in lung tissue and bronchoalveolar lavage fluid.

In certain embodiments, the medicament inhibits the activity of myeloperoxidase in lung tissue.

In certain embodiments, the drug inhibits the level of malondialdehyde in lung tissue.

In certain embodiments, the drug promotes superoxide dismutase activity in lung tissue.

In certain embodiments, the drug inhibits an increase in the ratio of wet to dry weight of lung tissue.

In certain embodiments, the drug inhibits the protein content of the bronchoalveolar lavage fluid.

In certain embodiments, the medicament further includes pharmaceutically acceptable excipients.

In certain embodiments, the adjuvants include carriers, diluents, excipients, fillers, binders, wetting agents, disintegrants, emulsifiers, cosolvents, solubilizers, osmotic pressure regulators, surface Any one or a combination of at least two of active agents, coatings, colorants, pH adjusters, antioxidants, bacteriostatic agents, or buffers.

The combination of the at least two kinds, such as the combination of diluent and excipient, the combination of binder and wetting agent, the combination of emulsifier and cosolvent, etc., can be selected in any other combination manner.

In certain embodiments, the dosage form of the medicament is any pharmaceutically acceptable dosage form.

In certain embodiments, the dosage forms include suspensions, granules, capsules, powders, tablets, emulsions, solutions, drop pills, injections, suppositories, enemas, aerosols, sprays, patches or any of the drops.

The present application also provides the use of the diterpene compound represented by formula I or a pharmaceutically acceptable salt thereof in the preparation of a medicament for inhibiting the expression of TNF-α, IL-1β and IL-6 in lung tissue and bronchoalveolar lavage fluid.

The present application also provides the use of the diterpene compound represented by formula I or a pharmaceutically acceptable salt thereof in the preparation of a medicament for inhibiting the activity of myeloperoxidase in lung tissue.

The present application also provides the use of the diterpene compound represented by formula I or a pharmaceutically acceptable salt thereof in the preparation of a medicament for inhibiting the content of malondialdehyde in lung tissue.

The present application also provides the use of the diterpene compound represented by formula I or a pharmaceutically acceptable salt thereof in the preparation of a medicament for promoting the activity of superoxide dismutase in lung tissue.

The present application also provides the use of the diterpene compound represented by formula I or a pharmaceutically acceptable salt thereof in the preparation of a medicament for inhibiting the increase in the ratio of wet to dry weight of lung tissue.

The present application also provides the use of the diterpene compound represented by formula I or a pharmaceutically acceptable salt thereof in the preparation of a medicament for inhibiting the protein content in bronchoalveolar lavage fluid.

In certain embodiments, wherein the lung tissue is acutely injured lung tissue.

In another aspect, the present application provides a method for preventing and/or treating acute lung injury, comprising administering an effective amount of the diterpene compound represented by formula I or a pharmaceutically acceptable salt thereof to a subject in need thereof.

In certain embodiments, wherein said administering comprises a route of administration selected from the group consisting of oral, parenteral, nasal, inhalation, intratracheal, intrapulmonary and intrabronchial, a therapeutically effective amount of the diterpene compound of formula I or its A pharmaceutically acceptable salt is administered to a subject.

In certain embodiments, the dosage of the diterpene compound represented by Formula I or a pharmaceutically acceptable salt thereof is about 0.01 mg to about 5000 mg.

In certain embodiments, the method comprises administering the diterpene compound of formula I or a pharmaceutically acceptable salt thereof to the subject in a dose of 1 to 5 or more times per day .

In certain embodiments, it further comprises at least one additional treatment selected from the group consisting of surgery, steroid therapy, non-steroid therapy, antiviral therapy, antifungal therapy, antimicrobial therapy, immunosuppressive therapy, Anti-infective therapy, anti-hypertensive therapy, nutritional supplement, and any combination thereof.

In certain embodiments, wherein the steroid therapy comprises administration of dexamethasone, beclomethasone propionate, budesonide, and/or fluticasone.

In another aspect, the present application provides a kit comprising the diterpene compound represented by formula I or a pharmaceutically acceptable salt thereof.

In certain embodiments, the kit further comprises a container.

In certain embodiments, the kit further includes instructional material, wherein the instructional material includes treatment or treatment using a route of administration selected from the group consisting of oral, parenteral, nasal, inhalation, intratracheal, intrapulmonary, and intrabronchial. Instructions for preventing acute lung injury in a subject.

Compared with the prior art, the present application has the following beneficial effects:

The present application finds for the first time that the diterpenoid compound represented by formula I has a significant effect of preventing and treating acute lung injury, it has a good preventive and therapeutic effect on the mouse model of acute lung injury induced by lipopolysaccharide, and can improve the lung tissue of mice with acute lung injury. Pathological changes, reduce pulmonary edema injury, reduce the damage of pulmonary blood-gas barrier, inhibit the expression of tumor necrosis factor α, interleukin 1β and interleukin 6 in lung tissue of lung injury, inhibit the expression of neutrophils in lung tissue of lung injury. Infiltration and oxidative stress. The above results show that the compounds can be used to prevent and treat acute lung injury, and provide a new idea and drug for the treatment of acute lung injury.

Other aspects and advantages of the present application can be readily appreciated by those skilled in the art from the following detailed description. Only exemplary embodiments of the present application are shown and described in the following detailed description. As those skilled in the art will recognize, the content of this application enables those skilled in the art to make changes to the specific embodiments disclosed without departing from the spirit and scope of the invention to which this application relates. Accordingly, the drawings and descriptions in the specification of the present application are only exemplary and not restrictive.

Description of drawings

The invention to which this application relates is set forth with particularity characteristic of the appended claims. The features and advantages of the inventions involved in this application can be better understood by reference to the exemplary embodiments described in detail hereinafter and the accompanying drawings. A brief description of the drawings is as follows:

Figure 1 shows the lung histopathological sections of mice in each group in Example 1 of the present application (AF are blank control group, LPS model group, LPS+HY1702 5mg/kg group, LPS+HY1702 10mg/kg group, LPS+HY1702 10mg/kg group in turn +HY1702 30mg/kg group, LPS+dexamethasone (DEX) 5mg/kg group);

Fig. 2 shows the lung wet/dry weight ratio statistics chart of each group of mice in Example 1 of the present application;

Fig. 3 shows the statistic diagram of protein concentration in the BALF of each group of mice in Example 1 of the present application;

Figure 4 shows the statistical graph of the protein content of TNF-α, IL-1β, and IL-6 in the lung tissue of each group of mice in Example 1 of the present application (a, b, and c represent TNF-α, IL-1β, IL-6);

Figure 5 shows a statistical graph of the expression levels of corresponding mRNAs in the BALF of each group of mice in Example 1 of the present application (a, b, and c represent TNF-α, IL-1β, and IL-6 in turn);

Fig. 6 shows the MPO activity statistics diagram in the lung tissue of each group of mice in Example 1 of the present application;

Fig. 7 shows the malondialdehyde (MDA) content statistic diagram in the lung tissue of each group of mice in Example 1 of the present application;

Figure 8 shows a statistical graph of the activity of superoxide dismutase (SOD) in the lung tissue of each group of mice in Example 1 of the present application.

detailed description

The embodiments of the invention of the present application are described below with specific specific examples, and those skilled in the art can easily understand other advantages and effects of the invention of the present application from the contents disclosed in this specification.

Definition of Terms

In this application, the term "pharmaceutically acceptable" generally refers to one or more nontoxic substances that do not interfere with the effectiveness of the biological activity of the active ingredient. Such formulations may generally contain salts, buffers, preservatives, compatible carriers and optionally other therapeutic agents. Such pharmaceutically acceptable formulations may also generally contain compatible solid or liquid fillers, diluents or encapsulating materials suitable for administration to humans. When used in medicine, the salts should be pharmaceutically acceptable salts, but non-pharmaceutically acceptable salts can be conveniently used to prepare pharmaceutically acceptable salts, and they cannot be excluded from the scope of this application. Such pharmacologically and pharmaceutically acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, citric acid, boric acid , formic acid, malonic acid, succinic acid, etc. Pharmaceutically acceptable salts can also be prepared as alkali metal or alkaline earth metal salts, such as sodium, potassium or calcium salts.

In this application, the term "pharmaceutically acceptable excipient" generally refers to a substance that has no persistent adverse effect on the general health of the subject being treated, or does not eliminate the biological activity or properties of the compound, and is relatively non-toxic . Such materials may generally contain salts, buffers, preservatives, compatible carriers and optionally other formulations.

In this application, the term "administration" generally refers to the introduction of the pharmaceutical formulations of the present application into the body of a subject by any route of introduction or delivery. Any method known to those skilled in the art for contacting cells, organs or tissues with the drug may be employed. The administration may include, without limitation, intravenous, intraarterial, intranasal, intraperitoneal, intramuscular, subcutaneous transdermal, or oral. The daily dose may be divided into one, two or more doses in suitable form to be administered at one, two or more times during a certain period of time.

In this application, the term "effective amount" or "effective dose" generally refers to an amount sufficient to achieve, or at least partially achieve, the desired effect. A "therapeutically effective amount" or "therapeutically effective dose" of a drug or therapeutic agent is generally one that, when used alone or in combination with another therapeutic agent, promotes disease regression (this is achieved through a reduction in the severity of disease symptoms, the frequency of asymptomatic periods of the disease) increase in intensity and duration, or as evidenced by the prevention of impairment or disability due to disease). A "prophylactically effective amount" or "prophylactically effective dose" of a drug generally refers to the amount of drug that inhibits the development or recurrence of disease when administered alone or in combination with another therapeutic agent to a subject at risk of disease development or disease recurrence . The ability of a therapeutic or prophylactic agent to promote disease regression or inhibit disease progression or recurrence can be assessed using a variety of methods known to those of skill in the art, such as in human subjects during clinical trials, in animal model systems Efficacy in humans is predicted, or by measuring the activity of an agent in an in vitro assay.

In this application, the term "in need" and similar terms refers to a condition that has been identified as in need of treatment, such as acute lung injury, eg, acute lung injury caused by peritonitis during sepsis, acute lung injury caused by peritonitis during sepsis acute lung injury from venous bacteremia, acute lung injury from soot inhalation, acute lung injury in preterm infants with insufficient surfactant, acute lung injury from oxygen toxicity, or from air pressure from mechanical ventilation Injury-induced acute lung injury in subjects. This determination may be the result of a medical diagnosis. Furthermore, subjects "in need" of the methods of the present application include those subjects who are known or suspected to have been previously diagnosed with acute lung injury.

In this application, the term "subject" generally refers to a human or non-human animal (including mammals) in need of diagnosis, prognosis, amelioration, prevention and/or treatment of a disease, such as a human, non-human primate (simian) , gibbons, gorillas, chimpanzees, orangutans, macaques), livestock (dogs and cats), farm animals (poultry such as chickens and ducks, horses, cows, goats, sheep, pigs) and laboratory animals (mice, rats, rabbits) , guinea pigs). Human subjects include fetal, neonatal, infant, adolescent and adult subjects. Subjects include animal disease models.

In this application, the terms "including", "including", "having", "may", "containing" and variations thereof are generally intended to be open-ended transitional phrases, terms or words that do not preclude additional acts or structures possibility. The term "consisting of" generally means that no other components (or similarly, features, integers, steps, etc.) can be present. Unless the context clearly dictates otherwise, the singular forms such as "a", "an", "the" in English and "a", "an" and "the/the" in Chinese generally include the plural forms of the things they refer to. .

In this application, the term "about" generally means approximately, in the region of, roughly, or around. When the term "about" is used in reference to a range of values, a cut-off or specific value is used to indicate that the stated value can vary by up to 10% from the recited value. Thus, the term "about" can be used to encompass ±10% or less variation, ±5% or less variation, ±1% or less variation, ±0.5% or less variation, or ±0.5% or less variation from a specified value. 0.1% or less variation.

Detailed description of the invention

use

In certain embodiments, the medicament has at least one of the following effects:

1) Can inhibit the expression of TNF-α, IL-1β and IL-6 in lung tissue and bronchoalveolar lavage fluid;

2) Can inhibit the activity of myeloperoxidase in lung tissue;

3) can inhibit the content of malondialdehyde in lung tissue;

4) Can promote the activity of superoxide dismutase in lung tissue;

5) can inhibit the increase of the ratio of wet to dry weight of lung tissue; and

6) Can inhibit the protein content in bronchoalveolar lavage fluid.

For example, the combination of the at least two kinds can be a combination of a diluent and an excipient, a combination of a binder and a wetting agent, a combination of an emulsifier and a cosolvent, etc., and any other combination can be selected.

In certain embodiments, the drug is administered selected from the group consisting of oral, parenteral, nasal, inhalation, intratracheal, intrapulmonary and intrabronchial routes of administration

Oral formulations containing the diterpene compound represented by formula I of the present application or a pharmaceutically acceptable salt thereof may comprise any conventionally used oral forms, including tablets, capsules, buccal forms, lozenges (troches), lozenges (lozenges) and oral liquids, suspensions or solutions. Capsules may contain the active compounds in admixture with inert fillers and/or diluents such as pharmaceutically acceptable starches (eg corn, potato or tapioca), sugar, artificial sweeteners, powdered Cellulose (eg crystalline and microcrystalline cellulose), flour, gelatin, gums, and the like. Useful tablet formulations may be prepared by conventional compaction, wet granulation or dry granulation methods using pharmaceutically acceptable diluents, binders, lubricants, disintegrants, surface modifiers (including surface active agent), suspending or stabilizing agents, including but not limited to magnesium stearate, stearic acid, talc, sodium lauryl sulfate, microcrystalline cellulose, calcium carboxymethyl cellulose, polyvinylpyrrolidone, gelatin , alginic acid, gum arabic, xanthan gum, sodium citrate, complex silicate, calcium carbonate, glycine, dextrin, sucrose, sorbitol, dicalcium phosphate, calcium sulfate, lactose, kaolin, mannitol, chloride Sodium, talc, dry starch and powdered sugar. Surface modifiers include nonionic and anionic surface modifiers. Representative examples of surface modifiers include, but are not limited to, poloxamer 188, benzalkonium chloride, calcium stearate, cetostearyl alcohol, cetomacrogol emulsifying wax, sorbitan esters, gums Silica, Phosphate, Sodium Lauryl Sulfate, Magnesium Aluminum Silicate and Triethanolamine. Oral formulations herein can use standard extended or sustained release formulations to alter the absorption of the active compound. The oral formulation may also include administering the active ingredient in water or fruit juice with suitable solubilizers or emulsifiers as required.

In some cases, it may be desirable to apply the compound directly into the airways in an aerosol form.

The compounds of the present application may also be administered parenterally or intraperitoneally. Solutions or suspensions of these active compounds in free base or pharmaceutically acceptable salt form can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions in glycerol, liquid polyethylene glycols, and mixtures thereof in oils can also be prepared. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. In all cases, the dosage form must be sterile and must be fluid to the extent that ease of syringability exists. It must be stable under the conditions of manufacture and storage and must be protected against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils.

In the present application, transdermal administration is understood to include all administrations on the surface of the body skin and body passage linings, including epithelial and mucosal tissues. Such administration can take the form of lotions, creams, foams, patches, suspensions, solutions and suppositories (rectal and vaginal) using the present compound or a pharmaceutically acceptable salt thereof.

On the other hand, the present application also provides the diterpene compound represented by formula I or a pharmaceutically acceptable salt thereof in the preparation of a drug for inhibiting the expression of TNF-α, IL-1β and IL-6 in lung tissue and bronchoalveolar lavage fluid applications in .

On the other hand, the present application also provides the use of the diterpene compound represented by formula I or a pharmaceutically acceptable salt thereof in the preparation of a medicament for inhibiting the activity of myeloperoxidase in lung tissue.

On the other hand, the present application also provides the use of the diterpene compound represented by formula I or a pharmaceutically acceptable salt thereof in the preparation of a medicament for inhibiting the content of malondialdehyde in lung tissue.

On the other hand, the present application also provides the use of the diterpene compound represented by formula I or a pharmaceutically acceptable salt thereof in the preparation of a medicament for promoting the activity of superoxide dismutase in lung tissue.

On the other hand, the present application also provides the use of the diterpene compound represented by formula I or a pharmaceutically acceptable salt thereof in the preparation of a medicament for inhibiting the increase in the ratio of wet to dry weight of lung tissue.

On the other hand, the present application also provides the use of the diterpene compound represented by formula I or a pharmaceutically acceptable salt thereof in the preparation of a medicament for inhibiting the protein content in bronchoalveolar lavage fluid.

In certain embodiments, the lung tissue is acutely injured lung tissue.

treatment method

On the other hand, the present application also provides a method for preventing and/or treating acute lung injury, comprising administering to a subject in need an effective amount of the diterpene compound shown in formula I or a pharmaceutically acceptable salt thereof .

The regimen of administration may affect what constitutes an effective amount. The therapeutic formulation can be administered to a subject before or after the onset of the disease or condition contemplated herein. Furthermore, several divided and staggered doses may be administered daily or sequentially, or the doses may be infused continuously, or they may be bolus injections. Furthermore, the dosage of the therapeutic agent can be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation.

The medicaments of the present application can be administered to a subject, preferably a mammal, more preferably a human, using known methods, at doses and for periods of time effective to treat the disease or condition contemplated herein. The effective amount of the therapeutic compound necessary to achieve a therapeutic effect may vary depending on factors such as the state of the disease or disorder of the subject; the age, sex, and weight of the subject; disease capacity. Dosage regimens can be adjusted to provide the best therapeutic response. For example, several divided doses may be administered daily, or the dose may be proportionally reduced, as indicated by the exigencies of the therapeutic situation. Non-limiting examples of effective dosage ranges for the treatment of the compounds of the present application are about 0.01 mg/kg body weight/day and 5000 mg/kg body weight/day. One of ordinary skill in the art will be able to study the relevant factors and make a determination of an effective amount of a therapeutic compound without undue experimentation.

The actual dosage level of the active ingredient in the medicaments of the present application can be varied in order to obtain an amount of the active ingredient effective to achieve the desired therapeutic response for a particular subject, drug and mode of administration, while being non-toxic to the subject.

The therapeutically effective amount or dosage of a compound of the present application will depend on the age, sex and weight of the subject, the subject's current medical condition and the progression of the disease or disorder contemplated in the present application.

A physician of ordinary skill in the art, such as a physician or veterinarian, can readily determine and prescribe the effective amount of the drug required. For example, a physician or veterinarian may start a dose of a compound of the present application for use in medicine below the level required to achieve the desired effect and gradually increase the dose until the desired effect is achieved.

A suitable dosage of a diterpene compound of formula I of the present application or a pharmaceutically acceptable salt thereof may be in the range of about 0.01 mg to about 5,000 mg per day, such as about 0.1 mg to about 1,000 mg per day, such as about 1 mg per day to about

500 mg, such as about 5 mg to about 250 mg. The dose may be administered in a single dose or in multiple doses, eg, 1 to 4 or more times per day. When multiple doses are used, the amount of each dose can be the same or different. For example, a dose of 1 mg per day may be administered in two doses of 0.5 mg with an interval of about 12 hours between doses.

The diterpene compound represented by formula I of the present application or a pharmaceutically acceptable salt thereof for administration may be in the range of about 1 μg to about 10,000 mg, about 20 μg to about 9,500 mg, about 40 μg to about 9,000 mg, about 75 μg to about 8,500 mg, about 150 μg to about 7,500 mg, about 200 μg to about 7,000 mg, about 3050 μg to about 6,000 mg, about 500 μg to about 5,000 mg, about 750 μg to about 4,000 mg, about 1 mg to about 3,000 mg, about 10 mg to about 2,500 mg, about 20 mg to about 2,000 mg, about 25 mg to about 1,500 mg, about 30 mg to about 1,000 mg, about 40 mg to about 900 mg, about 50 mg to about 800 mg, about 60 mg to about 750 mg, about 70 mg to about 600 mg, about 80 mg to In the range of about 500 mg, and any and all full or partial increments therebetween.

In some embodiments, the doses of the compounds of the present application are between about 1 mg and about 2,500 mg. In some embodiments, the dose of a compound of the present application for use in the medicaments described herein is less than about 10,000 mg, or less than about 8,000 mg, or less than about 6,000 mg, or less than about 5,000 mg, or less than about 3,000 mg, or less than About 2,000 mg, or less than about 1,000 mg, or less than about 500 mg, or less than about 200 mg, or less than about 50 mg.

In certain embodiments, a medicament of the present application is administered to a subject in a dose of 1 to 5 or more times per day. In another embodiment, a medicament of the present application is administered to a subject in a dose range including, but not limited to, once daily, once every two days, once every three days, once a week, once every two weeks. It will be readily apparent to those skilled in the art that the frequency of administration of the various combinations of the medicaments of the present application will vary from individual to individual, depending on a number of factors including, but not limited to, age, disease or condition being treated, gender, overall health and other factors. Accordingly, this application should not be construed as limited to any particular dosage regimen, and the precise dosage and drug to be administered to any subject is determined by the attending physician taking into account all other factors relevant to the subject.

It will be appreciated that, in non-limiting examples, the amount of the compound to be administered daily may be administered every day, every other day, every 2 days, every 3 days, every 4 days, or every 5 days. For example, with administration every other day, a dose of 5 mg per day may be started on Monday, the first subsequent dose of 5 mg per day may be administered on Wednesday, the second dose of 5 mg per day may be administered on Friday, etc.

In certain embodiments, it further comprises at least one additional treatment selected from the group consisting of surgery, steroid therapy, non-steroid therapy, antiviral therapy, antifungal therapy, antimicrobial therapy, immunosuppressive therapy, Anti-infective therapy, anti-hypertensive therapy, nutritional supplement, and any combination thereof. For example, the steroid therapy may include administration of dexamethasone, beclomethasone propionate, budesonide, and/or fluticasone.

Reagent test kit

In certain embodiments, the kit comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, and the like. The container can be formed from various materials such as glass or plastic. The container holds or contains a drug effective to treat the selected disease or disorder and may have a sterile access port (eg, the container may be an IV bag or vial with a stopper that can be penetrated by a hypodermic needle).

The kit may further comprise a second container with a pharmaceutically acceptable dilution buffer such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles and syringes.

In other embodiments, the kit further comprises at least one additional agent that treats, prevents or alleviates symptoms of acute lung injury, such as dexamethasone, beclomethasone propionate, budesonide, or fluticasone.

In certain embodiments, the kit further includes an applicator and instructional materials for its use. The instructional material may include instructions for preventing or treating acute lung injury, or any other disease or disorder contemplated in this application. The instructional material recites the amount and frequency of the diterpene compound of formula I, or a salt thereof, that should be administered to the subject.

Specifically, the present application provides the following specific embodiments:

1. Use of a small molecule diterpene compound or a salt thereof in the preparation of a medicine for preventing and treating acute lung injury, characterized in that the structure of the small molecule diterpene compound is shown in formula I.

2. The use according to Embodiment 1, wherein the salts include tartrate, stearate, oxalate, citrate, lactate, sorbate, fumarate, and formate , acetate, benzoate, benzenesulfonate, ethanesulfonate, resinate, trifluoroacetate, maleate, mesylate, fumarate, amino acid salt, niacin salt, phosphate, sulfate, hydrochloride or hydrobromide.

3. The use according to Embodiment 1 or 2, wherein the drug inhibits the expression of TNF-α, IL-1β and IL-6 in lung tissue and bronchoalveolar lavage fluid.

4. The use according to any one of Embodiments 1-3, wherein the drug inhibits the activity of myeloperoxidase.

5. The use according to any one of Embodiments 1 to 4, wherein the drug inhibits the content of malondialdehyde in lung tissue.

6. The use according to any one of embodiments 1-5, wherein the drug promotes the activity of superoxide dismutase in lung tissue.

7. The use according to any one of Embodiments 1-6, wherein the drug inhibits the increase of the ratio of wet to dry weight of lung tissue.

8. The use according to any one of embodiments 1-7, wherein the drug inhibits the protein content in bronchoalveolar lavage fluid.

9. The use according to any one of Embodiments 1-8, wherein the dosage form of the medicine is any pharmaceutically acceptable dosage form;

Preferably, the dosage forms include suspensions, granules, capsules, powders, tablets, emulsions, solutions, drop pills, injections, suppositories, enemas, aerosols, sprays, patches or drops any of the .

10. The use according to any one of Embodiments 1-8, wherein the medicine further comprises a pharmaceutically acceptable adjuvant;

Preferably, the adjuvants include carriers, diluents, excipients, fillers, binders, wetting agents, disintegrants, emulsifiers, cosolvents, solubilizers, osmotic pressure regulators, surfactants, Any one or a combination of at least two of coating materials, colorants, pH adjusters, antioxidants, bacteriostatic agents or buffers.

11. Use of the small molecule diterpene compound represented by formula I or its salt in the preparation of a drug for inhibiting the expression of TNF-α, IL-1β and IL-6 in lung tissue and bronchoalveolar lavage fluid.

12. Use of the small-molecule diterpene compound represented by formula I or a salt thereof in the preparation of a medicament for inhibiting the activity of myeloperoxidase.

13. Use of the small-molecule diterpene compound shown in I or its salt in the preparation of a medicament for inhibiting the content of malondialdehyde in lung tissue.

14. Use of the small-molecule diterpene compound represented by formula I or a salt thereof in the preparation of a medicament for promoting the activity of superoxide dismutase in lung tissue.

15. Use of the small-molecule diterpene compound represented by formula I or a salt thereof in the preparation of a medicament for inhibiting the increase in the wet-dry weight ratio of lung tissue.

16. Use of the small-molecule diterpene compound represented by formula I or a salt thereof in the preparation of a medicament for inhibiting protein content in bronchoalveolar lavage fluid.

Without intending to be limited by any theory, the following examples are only to illustrate the use of the diterpene compounds or their salts of the present application, etc., and are not intended to limit the scope of the invention of the present application.

Example

Except for the contents specifically mentioned below, the process, conditions, reagents, test methods, etc. for implementing the present application are all common knowledge and common knowledge in the field, and the present application has no special limited contents. The test methods for which specific conditions are not indicated in each embodiment are usually in accordance with conventional conditions or in accordance with the conditions suggested by the manufacturer.

Unless otherwise specified, all technical and scientific terms used in this specification have the same meanings as commonly understood by those skilled in the art to which this application belongs, but in case of conflict, this specification including definitions shall prevail.

The experimental animals involved in the following examples are SPF grade C57BL/6 mice (8-10 weeks old, male), purchased from Beijing Zhaoyan New Drug Research and Development Technology Co., Ltd., experimental animal license number: SCXK (Su) 2018- 0006).

The reagents involved in the following examples include:

HY1702 solution: provided by Suzhou Huyun New Drug Research and Development Co., Ltd. (molecular weight: 413.98, purity: 98.77%, batch number: 150818), its molecular structure is as follows:

Dexamethasone: purchased from Sangon Bioengineering Co., Ltd. (molecular weight: 392.5, purity: ≥97%, article number: A601187-0005);

MDA, MPO, SOD detection kits: purchased from Nanjing Jiancheng Bioengineering Institute;

ELISA detection kits for TNF-α, IL-1β, and IL-6: purchased from Wuhan Boster Biological Engineering Co., Ltd.;

Micro-sample total RNA extraction kit: purchased from Beijing Tiangen Biochemical Technology Co., Ltd.;

RNA reversal kit and RT-PCR detection kit: purchased from Takara Biotechnology Co., Ltd., Japan.

The data processing methods involved in the following embodiments are:

GraphPad Prism 5.0 software was used to draw and process experimental data. The data were expressed as mean ± standard deviation (Mean ± SEM). The comparison between the two-sample mean and the sample mean and the overall mean was performed by t test. ANOVA was used to test the significance of differences in means of more than one sample; ###P<0.001, ##P<0.01, #P<0.05 compared with the control group; ***P<0.001, **P<0.01, *P<0.05 compared to LPS model group.

Example 1

This example explores the prevention and treatment effect of the small molecule diterpene compounds involved in the application on the acute lung injury mouse model, including the following content:

(1) Establishment, grouping and administration of LPS-induced acute lung injury mouse model

The SPF grade C57BL/6 mice were divided into 6 groups with 6 mice in each group. The blank control group, the LPS model group, the LPS+HY17025mg/kg group, the LPS+HY1702 10mg/kg group, the LPS+HY1702 30mg/kg group, and the LPS+HY1702 30mg/kg group were set up. Dexamethasone (DEX) 5 mg/kg group (gavage administration, administration 1 h before modeling). The mice were anesthetized, and the trachea was surgically exposed, and 5 mg/kg LPS or normal saline was slowly injected along the subchondral space of the mouse epiglottis with an insulin needle, and then the wound was sutured. The mice were sacrificed 6 hours after modeling, and the mouse bronchoalveolar lavage fluid (BALF) and lung tissue were obtained for subsequent index detection.

(2) HE staining

The mouse lung tissue was taken and fixed in 4% formaldehyde solution for 48 hours, then dehydrated, transparent and paraffin-embedded, and sliced into 4 μm thick sections. Then, hematoxylin-eosin (HE) staining was performed, and the mouse lung tissue was observed under a light microscope. Pathological changes (infiltration of inflammatory cells, destruction of alveolar spaces and thickening of alveolar septa, etc.).

(3) ELISA detection

The bronchoalveolar lavage fluid from mice was collected, and the contents of inflammatory cytokines TNF-α, IL-1β and IL-6 in the bronchoalveolar lavage fluid were detected according to the instructions of the kit.

(4) RNA extraction and RT-PCR detection

Lung tissue was collected from mice, RNA was extracted from lung tissue according to the kit instructions, and RT-PCR experiment was performed to detect the mRNA expression of inflammatory cytokines TNF-α, IL-1β and IL-6 in lung tissue.

(5) Determination of protein content in mouse bronchoalveolar lavage fluid

The bronchoalveolar lavage fluid of mice was collected and operated according to the experimental steps of the BCA protein assay kit. The absorbance value at 562 nm was detected by a microplate reader, and the protein concentration in the bronchoalveolar lavage fluid of mice was calculated.

(6) Determination of wet and dry weight ratio of mouse lung tissue

The lung tissue of mice was collected, and the right upper lobe tissue of the mouse was weighed to record the wet weight value, dried in an oven at 70 °C for 72 h, weighed to obtain the dry weight value of the lung tissue, and the wet to dry weight ratio was calculated.

(7) Detection of MPO activity, MDA content and SOD activity

Lung tissue was collected from mice, and the left lung was taken to make tissue homogenate. The MPO activity, MDA content and SOD activity in the lung tissue homogenate were detected according to the kit instructions.

The test results are as follows:

(1) HY1702 alleviates the pathological changes of lung tissue in LPS-induced acute lung injury mice (ALI mice), as shown in Figure 1 (control represents the control group): the lung tissue structure of the control group mice is complete, the alveolar cavity is clear, and the alveoli There was no obvious inflammatory cell infiltration in the septum (Figure 1A); the lung tissue structure of the LPS model group was destroyed, the alveolar septa were thickened, and a large number of inflammatory cells infiltrated (Figure 1B). After the intervention of HY1702, the infiltration of inflammatory cells in the lungs of mice was reduced, and the damage to the lung tissue structure was alleviated to varying degrees (CE in Figure 1). Among them, the HY1702 30 mg/kg group had the greatest improvement; Organization also improved (Fig. 1F). In conclusion, HY1702 can protect the acute lung injury by improving the damage of alveolar wall and reducing the infiltration of inflammatory cells in lung tissue.

(2) HY1702 attenuates LPS-induced pulmonary edema and pulmonary blood-gas barrier damage in ALI mice

Edema is a typical symptom of inflammation, and the lung wet/dry weight ratio can reflect the degree of pulmonary edema. As shown in Figure 2 (control represents the control group), compared with the control group, the ratio of wet to dry weight in the lung tissue of the mice after LPS induction was significantly increased (P<0.001), and the lung tissue of the mice appeared pulmonary edema. Compared with the LPS group, the HY1702 group significantly reduced the wet-dry weight ratio in a dose-dependent manner, and relieved the degree of pulmonary edema in the mice; the degree of pulmonary edema in the DEX group was also significantly relieved. In conclusion, HY1702 can protect ALI mice to a certain extent by alleviating pulmonary edema in acute lung injury.

The protein concentration in BALF can reflect the integrity of the blood-gas barrier in the lung. As shown in Figure 3 (control represents the control group), the protein concentration in the BALF of the LPS group was significantly increased compared with the normal control group. Compared with the LPS group, the HY1702 group inhibited the protein concentration in BALF in a dose-dependent manner, and the protein concentration in the BALF of the mice in the DEX group was also significantly inhibited (P<0.001). It is suggested that HY1702 can alleviate the exudation of protein-rich fluid in the lungs and reduce the damage of the blood-gas barrier in the lungs of ALI mice.

(3) HY1702 inhibits the expression of inflammatory factors in the lung tissue of ALI mice

In the process of inflammation, a large number of pro-inflammatory factors such as TNF-α, IL-1β and IL-6 are released to promote the occurrence of inflammatory factor responses in the body's internal environment. The results of ELISA and RT-PCR showed that compared with the control group, LPS induced the protein content of TNF-α, IL-1β, and IL-6 in the lung tissue of mice (respectively, a, b, c in Figure 4, control represents control group) and the corresponding mRNA in BALF (a, b, c in Figure 5, respectively, control represents the control group), the expression levels were significantly increased; the HY1702 group significantly inhibited TNF-α and IL-1β in a dose-dependent manner compared with the LPS group , IL-6 expression at the protein and mRNA transcription levels. The DEX group also exhibited a significant inhibitory effect. It indicated that HY1702 could prevent and treat acute lung injury by inhibiting the expression of inflammatory cytokines TNF-α, IL-1β and IL-6 in the lung tissue of ALI mice, and further inhibiting the aggravation of inflammatory response.

(4) HY1702 inhibited the activity of myeloperoxidase (MPO) in lung tissue of ALI mice

Neutrophil accumulation is one of the hallmarks of LPS-induced acute lung injury, and MPO activity is a hallmark of neutrophil function and activation. As shown in Figure 6 (control represents the control group): compared with the control group, the MPO activity of the HY1702 group was significantly lower than that of the LPS group, and it was dose-dependently inhibited; the DEX group also showed a significant inhibitory effect on MPO activity. It was shown that HY1702 can alleviate the acute inflammatory response of neutrophil infiltration in lung tissue.

(5) HY1702 inhibits lung oxidative stress in ALI mice

When acute lung injury occurs, a large amount of reactive oxygen species is produced in the body, and the body's defense system is in a state of load, which cannot be cleared in time, resulting in the accumulation of peroxidation products in the body, resulting in a series of oxidation products that damage the body. Detection of oxidation-related products malondialdehyde (MDA) and superoxide dismutase (SOD) in lung tissue can indirectly reflect the body's antioxidant capacity. As shown in Figure 7 and Figure 8 (control represents the control group), compared with the control group, the MDA content in the LPS group was significantly increased, and the SOD activity was significantly decreased; both the HY1702 group and the DEX group could significantly inhibit the MDA content to a certain extent compared with the LPS group. , enhance the activity of SOD. It indicated that HY1702 could regulate the occurrence of inflammatory response by regulating oxidative stress response, thereby protecting against LPS-induced acute lung injury.

The applicant declares that this application illustrates the application of a small molecule diterpene compound or its salt in the preparation of medicines for preventing and treating acute lung injury through the above-mentioned examples, but the application is not limited to the above-mentioned examples, that is, it is not limited to the above-mentioned examples. It means that the present application must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement to the application, the equivalent replacement of each raw material of the product of the application, the addition of auxiliary components, the selection of specific methods, etc., all fall within the scope of protection and disclosure of the application.

The preferred embodiments of the present application have been described in detail above. However, the present application is not limited to the specific details of the above-mentioned embodiments. Within the scope of the technical concept of the present application, various simple modifications can be made to the technical solutions of the present application. These simple modifications All belong to the protection scope of this application.

In addition, it should be noted that the specific technical features described in the above-mentioned specific embodiments can be combined in any suitable manner unless they are inconsistent. The combination method will not be specified otherwise.

Claims

Use of a diterpene compound or a pharmaceutically acceptable salt thereof in the preparation of a medicine for preventing and/or treating acute lung injury, wherein the diterpene compound has the structure shown in formula I:
The use according to claim 1, wherein the pharmaceutically acceptable salts include tartrate, stearate, oxalate, citrate, lactate, sorbate, fumarate, formate, Acetate, benzoate, benzenesulfonate, ethanesulfonate, resinate, trifluoroacetate, maleate, mesylate, fumarate, amino acid salt, nicotinate , phosphate, sulfate, hydrochloride or hydrobromide.
The use according to claim 1 or 2, wherein the drug inhibits the expression of TNF-α, IL-1β and/or IL-6 in lung tissue and bronchoalveolar lavage fluid.
The use according to any one of claims 1-3, wherein the medicament inhibits the activity of myeloperoxidase in lung tissue.
The use according to any one of claims 1-4, wherein the drug inhibits the content of malondialdehyde in lung tissue.
The use according to any one of claims 1-5, wherein the medicament promotes the activity of superoxide dismutase in lung tissue.
The use according to any one of claims 1-6, wherein the drug inhibits an increase in the ratio of wet to dry weight of lung tissue.
The use according to any one of claims 1-7, wherein the drug inhibits protein content in bronchoalveolar lavage fluid.
According to the application according to any one of claims 1-8, the medicine further comprises a pharmaceutically acceptable adjuvant.
The application according to claim 9, wherein the pharmaceutically acceptable auxiliary materials include carriers, diluents, excipients, fillers, binders, wetting agents, disintegrants, emulsifiers, cosolvents, solubilizers Any one or a combination of at least two of solvents, osmotic pressure regulators, surfactants, coating materials, colorants, pH regulators, antioxidants, bacteriostatic agents, or buffers.
According to the application according to any one of claims 1-10, the dosage form of the medicine is any pharmaceutically acceptable dosage form.
The application according to claim 11, the dosage forms include suspensions, granules, capsules, powders, tablets, emulsions, solutions, drop pills, injections, suppositories, enemas, aerosols, sprays, Either patch or drops.
The use of the diterpene compound represented by formula I or a pharmaceutically acceptable salt thereof in the preparation of a medicament for inhibiting the expression of TNF-α, IL-1β and IL-6 in lung tissue and bronchoalveolar lavage fluid.
Use of the diterpene compound represented by formula I or a pharmaceutically acceptable salt thereof in the preparation of a medicament for inhibiting the activity of myeloperoxidase in lung tissue.
Use of the diterpene compound represented by formula I or a pharmaceutically acceptable salt thereof in the preparation of a medicament for inhibiting the content of malondialdehyde in lung tissue.
Use of the diterpene compound represented by formula I or a pharmaceutically acceptable salt thereof in the preparation of a medicament for promoting the activity of superoxide dismutase in lung tissue.
Use of the diterpene compound represented by formula I or a pharmaceutically acceptable salt thereof in the preparation of a medicament for inhibiting the increase in the ratio of wet to dry weight of lung tissue.
Use of the diterpene compound represented by formula I or a pharmaceutically acceptable salt thereof in the preparation of a medicament for inhibiting protein content in bronchoalveolar lavage fluid.
The use according to any one of claims 13-18, wherein the lung tissue is acutely injured lung tissue.
A method for preventing and/or treating acute lung injury, comprising administering an effective amount of a diterpene compound represented by formula I or a pharmaceutically acceptable salt thereof to a subject in need.
The method of claim 20, wherein said administering comprises a route of administration selected from the group consisting of oral, parenteral, nasal, inhalation, intratracheal, intrapulmonary, and intrabronchial to provide a therapeutically effective amount of the diterpene compound of formula I or a pharmaceutically acceptable salt thereof is administered to a subject.
The method of any one of claims 20-21, further comprising at least one additional treatment selected from the group consisting of surgery, steroid therapy, non-steroid therapy, antiviral therapy, antifungal therapy, antifungal therapy Microbial therapy, immunosuppressive therapy, anti-infective therapy, anti-hypertensive therapy, nutritional supplements, and any combination thereof.
The method of claim 22, wherein the steroid therapy comprises administration of dexamethasone, beclomethasone propionate, budesonide and/or fluticasone.
A kit comprising the diterpene compound represented by formula I or a pharmaceutically acceptable salt thereof.