Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/007—Pulmonary tract; Aromatherapy
  • A61K9/0073—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
  • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
  • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/02—Inorganic compounds
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
  • A61K47/12—Carboxylic acids; Salts or anhydrides thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/007—Pulmonary tract; Aromatherapy
  • A61K9/0073—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy
  • A61K9/0075—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy for inhalation via a dry powder inhaler [DPI], e.g. comprising micronized drug mixed with lactose carrier particles
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/007—Pulmonary tract; Aromatherapy
  • A61K9/0073—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy
  • A61K9/0078—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy for inhalation via a nebulizer such as a jet nebulizer, ultrasonic nebulizer, e.g. in the form of aqueous drug solutions or dispersions
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/08—Solutions
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/10—Dispersions; Emulsions
  • A61K9/12—Aerosols; Foams
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
  • A61P11/10—Expectorants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B82—NANOTECHNOLOGY
  • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
  • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery

Definitions

• the present invention belongs to the field of medicine, relates to an inhaled preparation of isoglycyrrhizic acid or a salt thereof, and in particular relates to an inhaled preparation of magnesium isoglycyrrhizinate and the use thereof in preparing drugs for treating respiratory system diseases.
• Glycyrrhiza is a commonly used medicinal plant, and main active ingredient thereof is glycyrrhizic acid species, namely 18- ⁇ glycyrrhizic acid and 18- ⁇ glycyrrhizic acid (also known as isoglycyrrhizic acid).
• Chinese patent ZL02111693.8 discloses a new compound magnesium isoglycyrrhizinate (structure shown in Formula I, with molecular weight of 845); proved by a large number of pharmacological and biochemical studies, it can obviously prevent transaminase elevation in animal serum, reduce the liver cell degeneration, necrosis and inflammatory cell infiltration, and promote hepatocyte regeneration in liver-injury animal models caused by different hepatotoxic agents (for example, acute liver injury in rats induced by D-galactosamine, chronic liver injury and liver cirrhosis in rats induced by carbon tetrachloride, and immune liver injury in black mice induced by Gal/FCA), and effect of magnesium isoglycyrrhizinate on resisting liver injury was obviously better than that of natural glycyrrhizic acid.
• hepatotoxic agents for example, acute liver injury in rats induced by D-galactosamine, chronic liver injury and liver cirrhosis in rats
• Glycyrrhiza has extensive pharmacological effects.
• glycyrrhiza and extracts thereof are considered to have many effects such as cardiotonic, eliminating phlegm, relieving cough, relieving asthma, pulmonary protection, broad-spectrum antibacterial and antiviral effect, etc.
• Researches in pharmacodynamics of the active ingredients thereof and new dosage forms have received considerable attention.
• Chinese patent ZL200410041923.8 discloses a magnesium isoglycyrrhizinate gel preparation and the use thereof in preparing drugs for treating psoriasis, chronic eczema dermatitis, contact dermatitis and other allergic skin diseases
• Chinese patent ZL200510106108.X discloses a magnesium isoglycyrrhizinate preparation for intravenous and the use thereof in preparing drugs for treating liver disease
• Chinese patent ZL200510106109.4 discloses an oral preparation of magnesium isoglycyrrhizinate and the use thereof in preparing drugs for treating liver disease, eczema, dermatitis, psoriasis and urticaria
• Chinese patent ZL200510106110.7 discloses an external preparation of magnesium isoglycyrrhizinate and the use thereof in drugs for treating psoriasis, chronic eczema dermatitis, contact dermatitis and other allergic dermatological
• Viral hepatitis is an infectious disease of hepatic pathological changes caused by various hepatitis viruses. Clinically, the main manifestations are loss of appetite, nausea, upper abdominal discomfort, liver pain, and fatigue. Some patients may have jaundice fever and liver enlargement accompanying with liver damage. Chronic viral hepatitis is the case in which the course of viral hepatitis lasts for half a year or more. Magnesium isoglycyrrhizinate is clinically suitable for treating chronic viral hepatitis and improving liver function abnormalities, but patient compliance is poor since it is administered intravenously.
• Sequential therapy also known as “conversion therapy,” is a new treatment proposed by American and European scholars in the 1980s, which refers to that when treating diseases by using drugs, parenteral administration (intravenous injection) is utilized in initial stage for 2 to 3 days, then changed to oral administration after the clinical symptoms is basically stable and improved.
• parenteral administration intravenous injection
• intravenous drip is used for the purpose of timely treatment for patients who cannot be administrated orally, it will inevitably lead to corresponding adverse reactions, such as infusion reaction, vascular stimulation and phlebitis, causing pain to the patients.
• Sequential therapy can shorten the time of intravenous administration and reduce the incidence of adverse reactions related to infusion, which can greatly shorten the hospitalization time of patients, save the expenses of individuals and medical institutions, save limited economic resources, and reduce the related social labor losses caused by hospitalization of patients.
• the clinically recommended sequential therapy for chronic hepatitis is relatively simple, and commonly used method is intravenous infusion of magnesium isoglycyrrhizinate injection plus oral administration of diammonium glycyrrhizinate capsule.
• liver cells are not the only host cells of hepatitis virus, there exists a problem of low bioavailability when using diammonium glycyrrhizinate preparation to treat viral hepatitis, and it has to increase the dosage to achieve the desired therapeutic effect, but the risk of side effects of the drug is also increased at the same time. Therefore, there is an urgent need to find a better pharmaceutical preparation of magnesium isoglycyrrhizinate and to provide a sequential therapy for treating chronic viral hepatitis with high bioavailability.
• Inhaled preparation means a preparation that delivers the drug by a specific device in the form of mist to the respiratory tract and/or lungs to exert a local or systemic effect.
• the properties of the drug itself also have a greater impact on the absorption process.
• Fine particle dose is an important parameter to evaluate the effectiveness of inhaled preparations. Controlling aerodynamic particle size of the powder, changing surface properties, and adjusting the type and size of the carrier can increase fine particle dose and pulmonary deposition ratio, thereby increasing bioavailability.
• the drug particles are highly hygroscopic, aggregation, drug particle enlargement and stratification are very likely to occur during drug preparation and storage, thereby affecting the pulmonary deposition amount of the drug particles, and further affecting the efficacy.
• the drug with proper solubility in the respiratory system (such as respiratory secretion or alveolar fluid) is better absorbed; the absorption of the drug in the lung is passive diffusion, and the lipid-solubility of the drug molecule plays an important role therein.
• lipid-soluble drugs are absorbed by the bilayer lipid membrane of alveolar epithelial cells, so the drug with a larger oil/water partition coefficient could be absorbed more rapidly; the absorption effect of the drug in the lung is also related to the molecular weight, while most of small molecule drugs are absorbed through the pores of the alveolar epithelial cells, so that drugs with a smaller molecular weight are generally absorbed faster.
• the use of magnesium isoglycyrrhizinate prepared as an inhaled preparation for treating diseases has not been reported, and the molecular weight of magnesium isoglycyrrhizinate is moderate, thus preparing it into an inhaled preparation may present a great challenge.
• COPD chronic obstructive pulmonary disease
• the treatment of COPD in a stable phase includes drug treatment and non-drug treatment.
• the main drug treatments are bronchodilators, glucocorticoids, expectorants (mucolytic agents), antioxidants, immunomodulators, vaccines, etc., and long-term regular inhaled glucocorticoids are more suitable for patients with severe COPD (grade III) and very severe COPD (grade IV) (FEV1 ⁇ 50% predicted); this treatment can reduce the frequency of acute exacerbation and improve quality of life.
• Non-drug treatment includes education and management, controlling occupational or environmental pollution, oxygen therapy, rehabilitation therapy and surgical treatment, while the purpose thereof is to relieve symptoms, improve health condition, enhance immunity, prevent disease progression, reduce the number of acute attacks, reduce mortality, improve activity endurance, and improve quality of life.
• Cough, phlegm and asthma are the three common symptoms of the respiratory system, which are present simultaneously and interact with each other, which not only bring pain to the patients, but also is life threatening.
• Expectorant is a drug that can make the sputum thinning, reduce the viscosity and make it easy to cough up. At the same time, it accelerates the mucociliary movement of the respiratory mucosa, improves the sputum transport function, weakens the stimulation to the respiratory mucosa, indirectly relieves cough and asthma, and is beneficial for controlling secondary infections.
• the present invention provides an inhaled preparation of isoglycyrrhizic acid or a salt thereof.
• the salt is magnesium salt, ammonium salt, potassium salt, sodium salt or salt of various amino acids, preferably magnesium salt, ammonium salt, potassium salt or sodium salt. More preferably, it is magnesium salt.
• the inhaled preparation is selected from inhalation aerosol, powder for inhalation, liquid preparation for use in nebulizers, and preparation that can be converted into vapor.
• the inhaled preparation is selected from powder for inhalation or liquid preparation for use in nebulizers.
• the inhaled preparation of the invention is powder for inhalation, comprising: micronized magnesium isoglycyrrhizinate and one or more pharmaceutically acceptable carriers, wherein the partical size of micronized magnesium isoglycyrrhizinate is 0.5-10 ⁇ m.
• the partical size of micronized magnesium isoglycyrrhizinate is 0.5-5 ⁇ m.
• the partical size of magnesium isoglycyrrhizinate is 0.5-10 ⁇ m as used in the present invention means that the particle size of most magnesium isoglycyrrhizinate (Active Pharmaceutical Ingredient, API) is in the range of 0.5-10 ⁇ m, and further, the particle size distribution of magnesium isoglycyrrhizinate is limited to X 10 ⁇ 0.5 ⁇ m, X 80 ⁇ 10 ⁇ m.
• the “X 10 ” in the present invention refers to a particle size with distribution of 10%, that is, the volume content of particles with a particle size smaller than that accounts for 10% of the total particles;
• “X 50 ” refers to a particle size with distribution of 50%, that is, the volume content of particles with a particle size smaller than that accounts for 50% of the total particles;
• “X 80 ” refers to a particle size with distribution of 80%, that is, the volume content of particles with a particle size smaller than that accounts for 80% of the total particles;
• “X 90 ” refers to a particle size with distribution of 90%, that is, the volume content of particles with a particle size smaller than that accounts for 90% of the total particles.
• the “pharmaceutically acceptable carrier” is selected from lactose, mannitol, trehalose or glycine, preferably lactose, further preferably ground lactose, sieved lactose or a mixture of sieved lactose and fine lactose, wherein the particle size distribution of ground lactose ranges from 1 ⁇ m to 350 ⁇ m, preferably with the particle size distribution of X 50 ⁇ 30-110 ⁇ m; the particle size distribution of sieved lactose ranges from 1 ⁇ m to 200 ⁇ m, preferably with the particle size distribution of X 50 ⁇ 35-115 ⁇ m; the particle size distribution of fine lactose ranges from 1 ⁇ m to 60 ⁇ m, preferably with the particle size distribution of X 90 ⁇ 45 ⁇ m.
• ground lactose refers to lactose which is mechanically ground to different degrees of fineness, and the particle size distribution varies depending on different grades of ground lactose; specifically, the particle size distribution ranges from 1 ⁇ m to 350 ⁇ m, with the particle size distribution of X 50 ⁇ 30-110 ⁇ m.
• the term “sieved lactose” as used in the present invention refers to lactose which could have a relatively narrow particle size distribution by sieving; specifically, the particle size distribution thereof ranges from 1 ⁇ m to 200 ⁇ m, with the particle size distribution of X 50 ⁇ 35-115 ⁇ m.
• fine lactose as used in the present invention includes micronized fine lactose and finely ground lactose with a narrow particle size distribution; specifically, the particle size distribution of fine lactose ranges from 1 ⁇ m to 60 ⁇ m, with the particle size distribution of X 90 ⁇ 45 ⁇ m.
• each capsule or bubble-cap comprises 1-50 mg of micronized magnesium isoglycyrrhizinate and 0-50 mg of lactose.
• each capsule or bubble-cap comprises 1-30 mg of micronized magnesium isoglycyrrhizinate and 1-40 mg of lactose.
• the powder for inhalation of the present invention further comprises one or more pharmaceutically acceptable additives.
• the “pharmaceutically acceptable additive” as used in the present invention includes one or more selected from surfactants, lubricants, and flavoring agents.
• the pharmaceutically acceptable additive is surfactant, such as phospholipid, poloxamer.
• the pharmaceutically acceptable additive is lubricant, such as magnesium stearate, micronized silica gel, talcum powder.
• the pharmaceutically acceptable additive is flavoring agent, including natural flavoring agents and synthetic flavors.
• Natural flavoring agent is such as peppermint oil, orange peel oil, cinnamon oil, spearmint oil, mint water, compound orange spirit; synthetic flavor is such as banana flavor, pineapple flavor, and orange flavor.
• the inhaled preparation of the present invention is a liquid preparation for use in nebulizers, comprising magnesium isoglycyrrhizinate, isotonicity adjusting agent, pH adjusting agent and water for injection, with pH of 6.0-8.0; the nebulizer is a continuous nebulizer or a quantitative nebulizer.
• the amount of magnesium isoglycyrrhizinate is from 0.1 mg/ml to 5 mg/ml, preferably from 0.1 mg/ml to 2.5 mg/ml. In some embodiments, the amount of magnesium isoglycyrrhizinate is from 0.1 mg/ml to 0.5 mg/ml. In some embodiments, the amount of magnesium isoglycyrrhizinate is from 0.5 mg/ml to 2.5 mg/ml.
• the isotonicity adjusting agent is one or more selected from glucose, sodium chloride, potassium chloride, mannitol, preferably sodium chloride.
• the pH adjusting agent is one or more selected from sodium hydroxide, ammonium hydroxide, hydrochloric acid, sodium carbonate, sodium bicarbonate, dilute sulfuric acid, citric acid, sodium citrate, acetic acid, tartaric acid, sodium acetate or disodium hydrogen phosphate, preferably ammonium hydroxide or sodium hydroxide.
• the pH is 6.5-7.0.
• the liquid preparation for use in nebulizers is provided in the form of a single dose package, and the single dose package size is 1 ml, 2 ml or 5 ml. It is preferably 2 ml.
• the inhaled preparation is a liquid preparation for use in nebulizers provided in a multi-dose package, and the multi-dose package size is 10 ml, 20 ml or 30 ml.
• the liquid preparation for use in nebulizers comprises flavoring agent, including natural flavoring agents and synthetic flavors.
• Natural flavoring agent is such as peppermint oil, orange peel oil, cinnamon oil, spearmint oil, mint water, compound orange spirit; synthetic flavor is such as banana flavor, pineapple flavor, and orange flavor.
• One object of the present invention is to provide a dosing regimen for an inhaled preparation of isoglycyrrhizic acid or a salt thereof, wherein said regimen comprises that the administration frequency of said inhaled preparation to a subject is selected from: up to three times a day, up to twice a day, up to once a day, and up to once every other day, preferably up to twice a day.
• the present invention provides the use of inhaled preparation of isoglycyrrhizic acid or a salt thereof in preparing drugs for treating chronic viral hepatitis.
• the isoglycyrrhizic acid or a salt thereof is selected from magnesium isoglycyrrhizinate.
• the present invention provides the use of inhaled preparation of magnesium isoglycyrrhizinate in preparing drugs for treating chronic viral hepatitis by sequential therapy, wherein magnesium isoglycyrrhizinate injection and magnesium isoglycyrrhizinate inhaled preparation are used in the sequential therapy.
• the present invention provides a combination comprising magnesium isoglycyrrhizinate inhaled preparation and magnesium isoglycyrrhizinate injection.
• the invention provides a method for treating chronic viral hepatitis, comprising administering a therapeutically effective amount of inhaled preparation of isoglycyrrhizic acid or a salt thereof to a patient with chronic viral hepatitis.
• the inhaled preparation is selected from inhalation aerosol, powder for inhalation, liquid preparation for use in nebulizers, and preparation that can be converted into vapor.
• the inhaled preparation is selected from powder for inhalation or liquid preparation for use in nebulizers.
• the invention provides a kit, comprising an inhaled preparation of isoglycyrrhizic acid or a salt thereof in a package containing one or more single doses, a drug delivery device, an instruction, and a suitable package.
• the inhaled preparation is selected from inhalation aerosol, powder for inhalation, liquid preparation for use in nebulizers, and preparation that can be converted into vapor.
• the inhaled preparation is selected from powder for inhalation or liquid preparation for use in nebulizers.
• the drug delivery device of the liquid preparation for use in nebulizers is a nebulizer, which is a continuous nebulizer or a quantitative nebulizer.
• the instruction relates to a method for treating chronic viral hepatitis, comprising administering a therapeutically effective amount of inhaled preparation of isoglycyrrhizic acid or a salt thereof to a patient with chronic viral hepatitis.
• the present invention provides the use of inhaled preparation of isoglycyrrhizic acid or a salt thereof in preparing drugs for treating chronic obstructive pulmonary disease.
• the isoglycyrrhizic acid or a salt thereof is selected from magnesium isoglycyrrhizinate.
• the inhaled preparation is selected from inhalation aerosol, powder for inhalation, liquid preparation for use in nebulizers, and preparation that can be converted into vapor.
• the inhaled preparation is selected from powder for inhalation or liquid preparation for use in nebulizers; most preferably, the inhaled preparation is selected from liquid preparation for use in nebulizers.
• the inhaled preparation of the present invention is a liquid preparation for use in nebulizers, comprising magnesium isoglycyrrhizinate, isotonicity adjusting agent, pH adjusting agent, and water for injection, with the pH of 6.0-8.0, and the nebulizer is a continuous nebulizer or a quantitative nebulizer.
• the amount of magnesium isoglycyrrhizinate is from 0.1 mg/ml to 5 mg/ml, preferably from 0.1 mg/ml to 2.5 mg/ml. In some embodiments, the amount of magnesium isoglycyrrhizinate is from 0.1 mg/ml to 0.5 mg/ml. In some embodiments, the amount of magnesium isoglycyrrhizinate is from 0.5 mg/ml to 2.5 mg/ml.
• the isotonicity adjusting agent is one or more selected from glucose, sodium chloride, potassium chloride, mannitol, preferably sodium chloride.
• the pH adjusting agent is one or more selected from sodium hydroxide, ammonium hydroxide, hydrochloric acid, sodium carbonate, sodium bicarbonate, dilute sulfuric acid, citric acid, sodium citrate, acetic acid, tartaric acid, sodium acetate or disodium hydrogen phosphate, preferably ammonium hydroxide or sodium hydroxide.
• the pH is 6.5-7.0.
• the liquid preparation for use in nebulizers is provided in the form of a single dose package, and the single dose package size is 1 ml, 2 ml or 5 ml. It is preferably 2 ml.
• the inhaled preparation is provided as a liquid preparation for use in nebulizers in the form of a multi-dose package, and the multi-dose package size is 10 ml, 20 ml or 30 ml.
• the liquid preparation for use in nebulizers comprises flavoring agent, including natural flavoring agent and synthetic flavor.
• Natural flavoring agent is such as peppermint oil, orange peel oil, cinnamon oil, spearmint oil, mint water, compound orange spirit; synthetic flavor is such as banana flavor, pineapple flavor, and orange flavor.
• the administration frequency of said inhaled preparation to a subject is selected from: up to three times a day, up to twice a day, up to once a day, and up to once every other day, preferably up to twice a day.
• the pharmacological effects of the magnesium isoglycyrrhizinate inhaled preparation are evaluated by using trachea administration or aerosolization of the magnesium isoglycyrrhizinate inhaled preparation.
• the pharmacological effects of the magnesium isoglycyrrhizinate inhaled preparation are evaluated by aerosolization of the magnesium isoglycyrrhizinate inhaled preparation once a day and twice a day, with the dosage divided into three doses, namely low dose, medium dose and high dose (respectively 0.5 mg/ml, 1.5 mg/ml and 5 mg/ml).
• the present invention provides a method for treating chronic obstructive pulmonary disease, comprising administering a therapeutically effective amount of inhaled preparation of isoglycyrrhizic acid or a salt thereof to a patient with chronic obstructive pulmonary disease.
• the inhaled preparation is selected from inhalation aerosol, powder for inhalation, liquid preparation for use in nebulizers, and preparation that can be converted into vapor.
• the inhaled preparation is selected from powder for inhalation or liquid preparation for use in nebulizers; most preferably, the inhaled preparation is selected from liquid preparation for use in nebulizers.
• the administration frequency of the inhaled preparation to the subject is selected from: up to three times a day, up to twice a day, up to once a day, and up to once every other day, preferably up to twice a day.
• the invention provides a kit, comprising an inhaled preparation of isoglycyrrhizic acid or a salt thereof in a package containing one or more single doses, a drug delivery device, an instruction, and a suitable package.
• the inhaled preparation is selected from inhalation aerosol, powder for inhalation, liquid preparation for use in nebulizers, and preparation that can be converted into vapor.
• the inhaled preparation is selected from powder for inhalation or liquid preparation for use in nebulizers; and most preferably, the inhaled preparation is selected from liquid preparation for use in nebulizers.
• the drug delivery device of the liquid preparation for use in nebulizers is a nebulizer, which is a continuous nebulizer or a quantitative nebulizer.
• the instruction relates to a method for treating chronic obstructive pulmonary disease, comprising administering a therapeutically effective amount of inhaled preparation of isoglycyrrhizic acid or a salt thereof to a patient with chronic obstructive pulmonary disease.
• the present invention provides the use of inhaled preparation of isoglycyrrhetic acid or a salt thereof in preparing expectorant, wherein the expectorant is a drug that can make the sputum thinning, reduce the viscosity and make it easy to cough up.
• the isoglycyrrhizic acid or a salt thereof is selected from magnesium isoglycyrrhizinate.
• the inhaled preparation is selected from inhalation aerosol, powder for inhalation, liquid preparation for use in nebulizers, and preparation that can be converted into vapor.
• the inhaled preparation is selected from powder for inhalation or liquid preparation for use in nebulizers; most preferably, the inhaled preparation is selected from a liquid preparation for use in nebulizers.
• the inhaled preparation of the present invention is a liquid preparation for use in nebulizers, comprising: magnesium isoglycyrrhizinate, isotonicity adjusting agent, pH adjusting agent, and water for injection, with the pH of 6.0-8.0, and the nebulizer is a continuous nebulizer or a quantitative nebulizer.
• the amount of magnesium isoglycyrrhizinate is from 0.1 mg/ml to 5 mg/ml, preferably from 0.2 mg/ml to 2.5 mg/ml. In some embodiments, the amount of magnesium isoglycyrrhizinate is from 0.2 mg/ml to 0.5 mg/ml. In some embodiments, the amount of magnesium isoglycyrrhizinate is from 0.5 mg/ml to 2.5 mg/ml.
• the isotonicity adjusting agent is onre or more selected from glucose, sodium chloride, potassium chloride, mannitol, preferably sodium chloride.
• the pH adjusting agent is one or more selected from sodium hydroxide, ammonium hydroxide, hydrochloric acid, sodium carbonate, sodium bicarbonate, dilute sulfuric acid, citric acid, sodium citrate, acetic acid, tartaric acid, sodium acetate or disodium hydrogen phosphate, preferably ammonium hydroxide or sodium hydroxide.
• the pH is 6.5-7.0.
• the liquid preparation for use in nebulizers is provided in the form of a single dose package, and the single dose package size is 1 ml, 2 ml or 5 ml. It is preferably 2 ml.
• the inhaled preparation is provided as a liquid preparation for use in nebulizers in the form of a multi-dose package, and the multi-dose package size is 10 ml, 20 ml or 30 ml.
• the liquid preparation for use in nebulizers comprises flavoring agent, including natural flavoring agent and synthetic flavor.
• Natural flavoring agent is such as peppermint oil, orange peel oil, cinnamon oil, spearmint oil, mint water, compound orange spirit; synthetic flavor is such as banana flavor, pineapple flavor, and orange flavor.
• the mouse phenol red excretion assay is used to evaluate the effect of magnesium isoglycyrrhizinate inhaled preparation on tracheal secretion of mice.
• the present invention provides a method for expelling phlegm, comprising administering a therapeutically effective amount of an inhaled preparation of isoglycyrrhizic acid or a salt thereof to a patient having abnormal sputum secretion and/or dysfunction of sputum.
• the inhaled preparation is selected from inhalation aerosol, powder for inhalation, liquid preparation for use in nebulizers, and preparation that can be converted into vapor.
• the inhaled preparation is selected from powder for inhalation or liquid preparation for use in nebulizers; most preferably, the inhaled preparation is selected from a liquid preparation for use in nebulizers.
• the invention provides a kit, comprising an inhaled preparation of isoglycyrrhizic acid or a salt thereof in a package containing one or more single doses, a drug delivery device, an instruction, and a suitable package.
• the inhaled preparation is selected from inhalation aerosol, powder for inhalation, liquid preparation for use in nebulizers, and preparation that can be converted into vapor.
• the inhaled preparation is selected from powder for inhalation or liquid preparation for use in nebulizers; and most preferably, the inhaled preparation is selected from liquid preparation for use in nebulizers.
• the drug delivery device of the liquid preparation for use in nebulizers is a nebulizer, which is a continuous nebulizer or a quantitative nebulizer.
• the instruction relates to a method for expelling phlegm, comprising administering a therapeutically effective amount of inhaled preparation of isoglycyrrhizic acid or a salt thereof to a patient having abnormal sputum secretion, dysfunction of sputum.
• isoglycyrrhizic acid (18 ⁇ ,20 ⁇ )-20-carboxy-11-oxo-30-norolean-12-ene-3 ⁇ -yl-2-O- ⁇ -D-pyranoglucuronyl- ⁇ -D-pyranoglucuronic acid.
• magnesium isoglycyrrhizinate is: magnesium (18 ⁇ ,20 ⁇ )-20-carboxy-11-oxo-30-norolean-12-ene-3 ⁇ -yl-2-O- ⁇ -D-pyranoglucuronyl- ⁇ -D-pyranoglucuronate, the structure thereof shown in Formula I.
• the term further includes hydrates thereof, such as tetrahydrate.
• the inventors unexpectedly discovered that the magnesium isoglycyrrhizinate with medium molecular weight can be prepared into an inhaled preparation, which has a high emptying rate and a high fine particle fraction, and inhalation administration can make magnesium isoglycyrrhizinate rapidly enter the bloodstream in the lung, thereby entering the blood circulation to play a systemic role.
• the bioavailability of magnesium isoglycyrrhizinate inhaled preparation is significantly improved, thereby the dosage and the risk of toxic side effects can be reduced; compared with the injection, the bioavailability of the magnesium isoglycyrrhizinate inhaled preparation is equivalent, but that makes the patient actively or passively receive inhaled preparation, which greatly reduces the pain caused by injection and improves patient compliance. Therefore, the inhaled preparation of the present invention is extremely suitable for sequential use with an injection solution, and the pain and treatment costs brought by injection are reduced while ensuring the efficacy.
• magnesium isoglycyrrhizinate inhaled preparation can alleviate the clinical symptoms of COPD rats, inhibit the infiltration of neutrophils-based inflammatory factors in COPD rats, significantly reduce lung inflammation, improve the role of the bronchial wall, reduce bronchial mucus secretion and improve emphysema, while the experiment proved that it has significant effect on the treatment of COPD.
• magnesium isoglycyrrhizinate different ways of administration of magnesium isoglycyrrhizinate vary the efficacy, and that of the aerosolizing inhalation route (0.4 mg/ml of the magnesium isoglycyrrhizinate liquid preparation for use in nebulizers, 30 min) has the most significantly effect on reducing inflammation.
• each dose group of magnesium isoglycyrrhizinate inhaled preparation significantly lowers the number of white blood cells in the bronchial airway of mice.
• the therapeutic effect of the low dose and medium dose groups are comparable to that of the positive control group, and the therapeutic effect of the high dose group is slightly weaker than that of the low dose and medium dose groups.
• the therapeutic effect of administrating magnesium isoglycyrrhizinate twice a day is better than that of once a day at the same dose.
• mice phenol red excretion method is used to evaluate the effect of magnesium isoglycyrrhizinate inhaled preparation on the tracheal secretion of mice, and it is found that the phenol red excretion of aerosolization administration for 15 min (0.2 mg/ml of the magnesium isoglycyrrhizinate liquid preparation for use in nebulizers) is significantly increased compared with the control group (p ⁇ 0.01).
• Example 1 Magnesium Isoglycyrrhizinate Powder for Inhalation
• the magnesium isoglycyrrhizinate was micronized to obtain samples having the following different particle size ranges.
• Magnesium isoglycyrrhizinate (large particle size) 1 g Lactose A 2 g Amount of preparation 100 capsules
• Magnesium isoglycyrrhizinate (medium particle size) 1 g Lactose A 2 g Amount of preparation 100 capsules
• Magnesium isoglycyrrhizinate (small particle size) 1 g Lactose A 2 g Amount of preparation 100 capsules
• Example 1a Example 1b
• Example 1c Emptying rate 99% 99% 99% Fine particle fraction 13% 20% 32%
• the fine particle dose was an important parameter to evaluate the effectiveness of the inhaled preparation.
• the particle size of the magnesium isoglycyrrhizinate had a crucial influence on the fine particle fraction as the key quality indicator of the powder for inhalation. Therefore, the particle size of the magnesium isoglycyrrhizinate was controlled within the range of 0.5-10 ⁇ m.
• the fine particle fraction was >15%, in line with regulations of the pharmacopoeia.
• Example 2 Magnesium Isoglycyrrhizinate Powder for Inhalation
• Lactose type Description particle size/ ⁇ m Lactose A sieved lactose with a narrow X 10 : 30-60; X 50 : 70-110; X 90 : particle size distribution 110-150 Lactose B X 10 : 7-22; X 50 : 40-70; X 90 : 80-120 Lactose C ground lactose with the average X 10 : 5-15; X 50 : 50-100; X 90 : particle size being strictly 120-160 controlled Lactose D ground lactose with a wide 40%-60% ⁇ 45; 75%-100% ⁇ 100; particle size distribution 90%-100% ⁇ 150; 99.5%-100% ⁇ 315 Lactose E finely ground lactose with a 90%-100% ⁇ 45; 98%-100% ⁇ 63; narrow particle size distribution 100% ⁇ 150 Lactose F micronized fine lactose X 50 ⁇ 5; X 90 ⁇ 10 Note: a typical particle size range of lactose
• Magnesium isoglycyrrhizinate (small particle size) 1 g Lactose B 2 g Amount of preparation 100 capsules
• Magnesium isoglycyrrhizinate (small particle size) 1 g Lactose C 2 g Amount of preparation 100 capsules
• Magnesium isoglycyrrhizinate (small particle size) 1 g Lactose D 2 g Amount of preparation 100 capsules
• Magnesium isoglycyrrhizinate (small particle size) 1 g Lactose E 2 g Amount of preparation 100 capsules
• Magnesium isoglycyrrhizinate (small particle size) 1 g Lactose A 1.5 g Lactose F 0.5 g Amount of preparation 100 capsules
• Magnesium isoglycyrrhizinate (small particle size) 1 g Lactose B 1.5 g Lactose F 0.5 g Amount of preparation 100 capsules
• Example 3 Magnesium Isoglycyrrhizinate Liquid Preparation for Use in Nebulizers
• magnesium isoglycyrrhizinate and sodium chloride were taken and added into 1800 ml of water for injection, stirred until completely dissolved. Then ammonium hydroxide was added to adjust the pH value of the solution to 6.5-7.0, and the obtained solution was added with water for injection to 2000 ml, filtered and sterilized, filled according to 2 ml per package, before a liquid preparation for use in nebulizers containing 10 mg of magnesium isoglycyrrhizinate was obtained.
• magnesium isoglycyrrhizinate and sodium chloride were taken and added into 1800 ml of water for injection, stirred until completely dissolved. Then ammonium hydroxide was added to adjust the pH value of the solution to 6.5-7.0, and the obtained solution was added with water for injection to 2000 ml, filtered and sterilized, filled according to 2 ml per package, before a liquid preparation for use in nebulizer containing 5 mg of magnesium isoglycyrrhizinate was obtained.
• Example 5 Magnesium Isoglycyrrhizinate Liquid Preparation for Use in Nebulizers
• magnesium isoglycyrrhizinate and sodium chloride were taken and added into 1800 ml of water for injection, stirred until completely dissolved. Then ammonium hydroxide was added to adjust the pH value of the solution to 6.5-7.0, and the obtained solution was added with water for injection to 2000 ml, filtered and sterilized, filled according to 2 ml per package, before a liquid preparation for use in nebulizer containing 1 mg of magnesium isoglycyrrhizinate was obtained.
• magnesium isoglycyrrhizinate and sodium chloride were taken and added into 1800 ml of water for injection, stirred until completely dissolved. Then ammonium hydroxide was added to adjust the pH value of the solution to 6.5-7.0, and the obtained solution was added with water for injection to 2000 ml, filtered and sterilized, filled according to 2 ml per package, before a liquid preparation for use in nebulizer containing 0.4 mg of magnesium isoglycyrrhizinate was obtained.
• Example 7 Magnesium Isoglycyrrhizinate Liquid Preparation for Use in Nebulizers
• magnesium isoglycyrrhizinate and sodium chloride were taken and added into 1800 ml of water for injection, stirred until completely dissolved. Then ammonium hydroxide was added to adjust the pH value of the solution to 6.5-7.0, and the obtained solution was added with water for injection to 2000 ml, filtered and sterilized, filled according to 2 ml per package, before a liquid preparation for use in nebulizer containing 0.2 mg of magnesium isoglycyrrhizinate was obtained.
• the rats were randomly divided into two groups, 4 rats in each group, and each rat was inhalation administrated with a single dose of magnesium isoglycyrrhizinate liquid preparation for use in nebulizers (2.5 mg/mL) and intragastric administrated with magnesium isoglycyrrhizinate inhaled preparation (5.0 mg/mL, the amount of isoglycyrrhizic acid is 4.486 mg/mL) respectively.
• Each rat in the inhalation administration group was given 200 ⁇ L of magnesium isoglycyrrhizinate liquid preparation for use in nebulizers (the actual dose given to the rat was 2.24-2.49 mg/kg).
• the dose in the intragastric administration group was 10.0 mg/kg.
• 0.2-0.3 mL of blood was taken from the fundus venous plexus before administration (0 h) and 0.0833 h, 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, 24 h after administration.
• EDTA-K2 was used for anticoagulation, and the plasma was centrifuged. Then 50 ⁇ L was accurately measured, added with 10 ⁇ L internal standard solution to vortex and mixed, then added with 200 ⁇ L methanol, and mixed in high speed vortex mixer for 3 min, centrifuged for 10 min (4° C., 13000 rpm). The supernatant was collected, and 100 ⁇ L thereof was transferred to a 96-well plate. 50 ⁇ L of ultrapure water was added, vortexed, LC-MS/MS was used for detection, and the chromatogram was recorded.
• the relative bioavailability of magnesium isoglycyrrhizinate by inhalation administration for rats was as high as 68052%.
• the bioavailability of magnesium isoglycyrrhizinate by inhalation administration for rats was 87%. Therefore, inhalation administration can significantly improve the bioavailability of magnesium isoglycyrrhizinate by compared with gastrointestinal administration, and the bioavailability of magnesium isoglycyrrhizinate by inhalation administration was basically equal to that by gastrointestinal administration.
• the administration groups continued to be given with smoke stimulation after 30 minutes of administration, and the mental state, breathing, activity, hair luster, weight gain of the rats were recorded every day. 12 hour after the last administration, whole blood was taken from the rat eye for determination of white blood cell count and cell classified comparison. The rats were killed, the trachea and lungs were exposed by thoracotomy, and the morphology of the lungs and trachea was observed with the naked eye. The right lung was ligated at the right main branch, and the left lung was lavaged with normal saline 2 ml ⁇ 3 times. The recovery rate was about 80%.
• the bronchoalveolar lavage fluid (BALF) was prepared for the determination of white blood cell count and classified comparison (white blood cells, neutrophils, lymphocytes and monocytes). Finally, the right lung of rat was fixed with 10% formalin and HE stained, lung injury was observed under microscope, and pathological lesions were scored. All scores were accumulated, and the average score of each animal in each group was calculated (mean ⁇ SD).
• Scoring indicators (1) whether there was mucus and cell blockage in the small airway cavity; (2) whether the small airway epithelium had necrotic erosion; (3) small airway epithelial cells goblet cell metaplasia; (4) small airway epithelial cell squamous metaplasia; (5) small airway wall inflammation cell infiltration; (6) small airway wall fibrous connective tissue hyperplasia; (7) small airway wall smooth muscle hyperplasia; (8) small airway wall pigmentation; and (9) lung emphysema.
• Lesion score according to the degree from light to heavy lesions, it was quantified as slight or very small amount for “0.5 point”, mild or small amount for “1 point”, moderate or more quantity amount for “2 points”, severe or plenty amount for “3 points”, very severe or large amount for “4 points”, and no obvious lesions for 0 point.
• the experimental result data was expressed in the form of mean ⁇ SD.
• Statistical significance was expressed as a P value of less than 0.05.
• the model group was compared with the blank control group, # p ⁇ 0.05, ## p ⁇ 0.01; each administration group of magnesium isoglycyrrhizinate was compared with the model group, *p ⁇ 0.05, **p ⁇ 0.01.
• the experimental results showed that the rats in the blank control group had normal activities, sensitive reactions, body fat, and no symptoms such as cough, sneezing and dyspnea.
• the rats tended to be prone, lack of spirit, stagnation, squint, bunching, unsteady walking, weight gain slowing, and grayish yellow fur, followed by symptoms such as cough, sneezing and dyspnea.
• the experimental results showed that the lung tissue of rats in the blank control group consisted of alveolus, intrapulmonary bronchial branches and interstitial tissues with clear structure, no emphysema, minimal infiltration of inflammatory cells and goblet cell hyperplasia.
• the main lesions in the lung tissue of rats in the model group were interstitial pneumonia, edema around the perivascular tissue, with infiltration of inflammatory cells, increased goblet cells in the bronchial wall of the lung, degeneration and necrosis of bronchial wall cells, and a small amount of exudate in the bronchial lumen, wherein emphysema and inflammatory cell infiltration were particularly evident (p ⁇ 0.01 or p ⁇ 0.05).
• High dose tracheal infusion group, low dose tracheal infusion group and magnesium isoglycyrrhizinate aerosolizing inhalation group can significantly reduce lung inflammation, improve bronchial wall function, reduce bronchial mucus secretion and improve emphysema.
• magnesium isoglycyrrhizinate aerosolizing inhalation group (0.4 mg/ml, 30 min) was the most obvious to reduce inflammation and infiltration.
• mice weighing 18-22 g, were divided into 9 groups, including blank control group, model group, low dose group (0.5 mg/ml, q.d.), medium dose group (1.5 mg/ml, q.d.), high dose group (5.0 mg/ml, q.d.), low dose group (0.5 mg/ml, b.i.d.), medium dose group (1.5 mg/ml, b.i.d.), high dose group (5.0 mg/ml, b.i.d.) of magnesium isoglycyrrhizinate inhaled preparation and positive control (Arformoterol) group.
• the trachea thereof was instilled with 30 ⁇ l the lipopolysaccharide (LPS) to model.
• 10 ml of the drug was administered by nebulizing inhalation device, and the nebulizing time was 30 min. 6 hours after LPS modeling, low dose group (0.5 mg/ml, b.i.d.), medium dose group (1.5 mg/ml, b.i.d.), high dose group (5.0 mg/ml, b.i.d.) and positive control group were continually administrated with 10 ml of drugs by nebulizing inhalation device. 24 hours after modeling, the mice were anesthetized and lung tissues thereof were lavaged. The bronchoalveolar lavage fluid (BALF) was taken to measure the number of inflammatory cells. Some lung tissues were taken for HE staining and the pathological section was used to detect the changes of inflammatory cells.
• BALF bronchoalveolar lavage fluid
• magnesium isoglycyrrhizinate administrated twice a day was better than once a day at the same dose.
• histopathological examination showed that no obvious inflammatory reaction was observed in each dose group of magnesium isoglycyrrhizinate inhaled preparation.
• control group 1 normal saline, NS
• control group 2 normal saline, NS
• positive control group ammonium chloride, 1 g/kg
• drug group 1 0.2 mg/ml of magnesium isoglycyrrhizinate liquid preparation for use in nebulizers, nebulizing for 30 min
• drug group 2 0.2 mg/ml of magnesium isoglycyrrhizinate liquid preparation for use in nebulizers, nebulizing for 15 min.
• mice in control group 1 and the positive control group were intragastrically administered with the corresponding drugs (0.1 ml/10 g), the mice in control group 2 and the drug group 1 were aerosolizing administered with a nebulizer for 30 min, while the mice in drug group 2 were 15 min.
• the administration frequency was once a day for 6 consecutive days. Before the 6th administration, the mice were starved for 16-18 hours, only allowed for drinking water. After 30 minutes of administration, i.p. 1% phenol red physiological in saline solution 0.2 ml/10 g was injected and 30 minutes later, the mice were killed by cervical dislocation.
• the trachea was separated, intubated (6# syringe needle with a smooth tip was inserted into trachea for about 3 mm from laryngeal, fixed by silk thread ligation) and connected with the syringe, and then 0.6 ml of 5% NaHCO 3 was slowly injected into the trachea, then gently sucked out, repeated 3 times, and 3 portions of lavage fluid were combined and centrifuged at 4000 rpm for 5 min, to obtain the supernatant.
• intubated (6# syringe needle with a smooth tip was inserted into trachea for about 3 mm from laryngeal, fixed by silk thread ligation) and connected with the syringe, and then 0.6 ml of 5% NaHCO 3 was slowly injected into the trachea, then gently sucked out, repeated 3 times, and 3 portions of lavage fluid were combined and centrifuged at 4000 rpm for 5 min, to obtain the supernatant.
• phenol red 1.95 mg was weighted and dissolved by adding 5% NaHCO 3 to 3.9 ml, and the obtained solution containing 0.5 mg/ml of phenol red was used as a stock solution.
• 0.1 ml of the stock solution was taken and added with 3.9 ml of 5% NaHCO 3 to achieve the concentration of 12.5 ⁇ g/ml, which was sequentially diluted to 10 ⁇ g/ml, 7.5 ⁇ g/ml, 5 ⁇ g/ml, 2.5 ⁇ g/ml, 1.25 ⁇ g/ml, and 0.625 ⁇ g/ml.
• the phenol red standard curve was produced by colorimetric measuring the optical density (OD) value at the wavelength of 546 nm with an enzyme-labeling instrument.
• the sample OD value at a wavelength of 546 nm was measured by an enzyme-labeling instrument, and the phenol red amount in sample was calculated from the phenol red standard curve.
• the experimental result data was expressed in the form of X ⁇ S, and the statistical significance was expressed with a P value of less than 0.05. Among them, each drug group was compared with the control group 2, ⁇ p ⁇ 0.01. The experimental results were shown in Table 6. The phenol red excretion amount of drug group 2 (0.2 mg/ml of magnesium isoglycyrrhizinate liquid preparation for use in nebulizers, nebulizing for 15 min) was significantly increased compared with control group 2.

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