CN102210712B - A kind of toad skin extract dry powder inhalation agent and its preparation method and application - Google Patents

Abstract

The invention relates to a dry powder inhalant, in particular to a dry powder inhalant prepared from toad skin extract, a preparation method of the dry powder inhalant and application of the dry powder inhalant in preparing a medicament for treating lung tumor. The toad skin extract dry powder inhalant is characterized by consisting of a toad skin extract and medically acceptable auxiliary materials, wherein the mass ratio of the toad skin extract to the auxiliary materials is 1: 0-1: 110. The toad skin extract is prepared into the dry powder inhalant, can directly act on lung diseases, reduces the occurrence of toxic and side effects, improves the bioavailability of the medicine, and can overcome the defects of slow effect taking, liver first-pass effect, systemic toxic and side effects, low bioavailability of the preparation and the like of an oral preparation; meanwhile, the problems of inconvenient use of the injection, poor patient compliance and the like can be solved.

Description

A kind of toad skin extract dry powder inhalation agent and its preparation method and application

Technical field:

The invention relates to a dry powder inhaler, in particular to a dry powder inhaler made of toad skin extract, a preparation method of the dry powder inhaler and an application in the preparation of a drug for treating lung tumors.

Background technique:

Cinobufacin is an extract obtained by extracting and purifying the dried skin of Bufobufo gargarizans Cantor, an animal of the Bufonidae family. It has the effects of detoxification, swelling, and pain relief. etc., the physiologically active substances are mainly bufotoxins and their hydrolysis products bufageins, bufoteinines and so on. At present, cinobufacin injections, oral liquids, and tablets have been used clinically, and have good curative effects on liver cancer, lung cancer, and gastric cancer. In particular, cinobufacin injection, which is a water-soluble preparation processed from the skin of Bufo bufo, has achieved comprehensive effective rates of 44% and 56% in the treatment of primary liver cancer and advanced lung cancer, respectively, and tumor shrinkage rates of 10%. % and 16%, and has a synergistic effect on chemotherapy and radiotherapy. Although there are few clinical adverse reactions of cinobufacin injection, there are still local venous reactions during intravenous infusion, which can cause pain caused by contraction and spasm of infusion veins, and non-specific inflammation of veins caused by long-term stimulation; skin reactions, mainly manifested as urticaria and skin blister-like lesions; secondly, a small number of patients have cardiovascular system reactions such as premature beats, sinus bradycardia, sinus tachycardia, and atrioventricular block, which may be caused by a small amount of bufotoxin in cinobufacin Cardiotoxic reactions produced by ligands. In addition, the injection needs to be used in the hospital, and the patient's compliance is poor. The oral preparation of cinobufacin needs to go through the first-pass effect of the liver, the onset of action is slow, and the bioavailability of the preparation is low.

The lung has a large surface area, and the alveolar wall is composed of a single layer of cells, which makes it easy for drug molecules to enter the blood. Therefore, pulmonary inhalation administration has many advantages such as quick onset of action and small adverse reactions. Dry powder inhalers (drypowder inhalers, DPIs) do not use propellants and solvents in pulmonary inhalation administration, rely on the airflow generated by the patient's spontaneous breathing to atomize the drug powder, and then transfer the drug powder to the lungs, which has the advantages of convenient application, rapid absorption, It has the characteristics of no liver first pass, immediate release and targeted positioning, and is a new type of drug delivery system.

The incidence of lung cancer is rising rapidly all over the world, and it has become the first cause of cancer death. Most of the current anti-tumor drugs are administered by injection. This systemic administration method is not conducive to the accumulation of drugs in the tumor site, resulting in a decrease in the efficacy of the drug and an increase in adverse reactions.

Using localized drug delivery to deliver anti-tumor drugs to tumor patients in the human body can increase the concentration of drugs at the patient site, which has great clinical application value for reducing the toxic and side effects of drugs and improving the therapeutic effect of drugs. The inhalation through the lung administration method can directly deliver the toad skin extract to the focus of lung cancer, which is beneficial to improving the curative effect of the toad skin extract on lung cancer and reducing its toxic and side effects on normal tissues. At present, there is no report on the study of toad skin extract dry powder inhalation with anti-lung tumor effect.

Invention content:

Purpose of the invention: one purpose of the present invention is to provide a lung dry powder inhalation preparation of toad skin extract and a preparation method thereof. Another object of the present invention is to provide the application of the dry powder inhaler of the present invention in the preparation of medicines for treating lung tumors.

For the purpose of the above invention, the present invention provides the following technical solutions:

A kind of toad skin extract dry powder inhalation is characterized in that it is made up of toad skin extract and medically acceptable auxiliary material, the mass ratio of toad skin extract (according to the obtained solid matter amount after drying) and described auxiliary material 1:0-1:110.

The dry powder inhaler of toad skin extract is a powder made by adding auxiliary materials to the toad skin extract, and the powder is agglomerated to form redispersible particles; or the toad skin extract is loaded on the surface of the auxiliary material particles to form particles; the above two The particle size of the seed particles is between 0.5-1000 microns in terms of _D50 .

The toad skin extract is an extract obtained by extracting and purifying the skin of Bufoidae animals, such as Bufo sinensis or Bufo melanocarpi. The preparation method of toad skin extract is:

Take 1 portion of toad skin, add 2-100 times the amount of water, reflux or decoct or extract by ultrasonic or microwave or leaching or percolation for 1-10 times, each time for 0.1-48 hours, combine the water extracts, and concentrate to relative density 1.01-1.50 (60-100°C), add ethanol (65%-100%, W/W) to precipitate, so that the ethanol content reaches more than 50% (W/W), let stand or centrifuge or filter, and take the supernatant The ethanol is recovered from the liquid to obtain the toad skin extract or to obtain the toad skin extract after concentration and drying.

Alternatively, take 1 portion of toad skin, add 2-100 times the amount of water, reflux or decoct or extract by ultrasonic or microwave or leaching or percolation for 1-10 times, each time for 0.1-48 hours, combine the water extracts, and concentrate to The relative density is 1.01-1.50 (60-100°C), add ethanol (65%-100%, W/W) for precipitation, and make the ethanol content reach more than 50% (W/W), let stand or centrifuge or filter, and take Recover ethanol from the supernatant until there is no alcohol smell, add ethanol (65%-100%, W/W) for precipitation, and make the ethanol content reach more than 60% (W/W), let stand or centrifuge or filter, and take the supernatant The ethanol is recovered until there is no alcohol smell, and the toad skin extract is obtained or the toad skin extract is obtained after concentration and drying.

Alternatively, take 1 portion of toad skin, add 2-100 times the amount of water, reflux or decoct or extract by ultrasonic or microwave or leaching or percolation for 1-10 times, each time for 0.1-48 hours, combine the water extracts, and analyze the molecular weight Ultrafiltration with a 500-5000 ultrafiltration membrane to obtain toad skin extract or to obtain toad skin extract after concentration and drying.

Or, take 1 part of toad skin, add 2-100 times the amount of ethanol (1%-100%, W/W) of a certain concentration, reflux or ultrasonic or microwave or leaching or percolation extraction 1-10 times, each time After 0.1-48 hours, combine the extracts, recover ethanol until there is no alcohol smell, add 0-100 times of water, stand still or centrifuge or filter to obtain toad skin extract or to obtain toad skin extract after concentration and drying.

Or, take 1 part of toad skin, add 2-100 times the amount of ethanol (1%-100%, W/W) of a certain concentration, reflux or ultrasonic or microwave or leaching or percolation extraction 1-10 times, each time After 0.1-48 hours, combine the extracts, recover ethanol until there is no alcohol smell, add 0-100 times of water, and ultrafilter through an ultrafiltration membrane with a molecular weight of 500-5000 to obtain toad skin extract or to obtain toad skin extract after concentration and drying .

The auxiliary material is selected from one of sugar, alcohol, amino acid, phospholipid, surface active substance, cyclodextrin, macromolecular substance, magnesium stearate, stearic acid, sodium stearyl fumarate, micropowder silica gel, and talcum powder or several mixtures.

The sugar can be selected from one or more mixtures of galactose, lactose, glucose, fructose, sucrose, trehalose and raffinose; the alcohol can be selected from mannitol, xylitol, maltitol, One or more mixtures of sorbitol; the amino acids can be selected from glycine, aspartic acid, alanine, tryptophan, isoleucine, threonine, glutamic acid, phenylalanine , one or more of leucine, cystine, lysine, proline, arginine; described phospholipids can be selected soybean lecithin, lecithin, phosphatidylcholine, phosphatidylethanolamine, One or more of synthetic phospholipids; the surface active substance can be selected from lung-derived surface active substances such as one or more of dipalmitoylphosphatidylcholine, dilauroylphosphatidylcholine, and cholesterol Mixture; the polymer substance can be selected to meet the safety requirements of the polymer, preferably biodegradable polymer substances such as albumin, starch, polylactic acid, polylactic acid-glycolic acid, polyethylene glycol, poloxamer , hyaluronic acid, sodium hyaluronate, sodium alginate or a mixture of several.

The preferred method is that the auxiliary material is a mixture of lactose particles for inhalation with an average particle size of 45-220 microns and micronized lactose with an average particle size of 1-10 microns, wherein the mass ratio of lactose particles to micronized lactose is 1000:1- 1:1. The most preferred mode is that the mass ratio of lactose granules to micronized lactose is 100:1-7:1.

A method for preparing toad skin extract dry powder inhalation, the preparation steps are as follows:

The toad skin extract is made into powder by crushing or spray drying, and packed into capsules or blisters or dry powder inhalers; the toad skin extract is made into powder by crushing or spray drying, and the powder is uniformly mixed with auxiliary materials to make a powder for inhalation , packed into capsules or blisters or dry powder inhalers; or the toad skin extract and auxiliary materials are evenly mixed, pulverized or spray-dried to make powder for inhalation, packed into capsules or blisters or dry powder inhalers; or toad skin The bark extract and part of the excipients in the prescription are evenly mixed, crushed or spray-dried to make a powder, the powder is evenly mixed with the remaining excipients in the prescription to make a powder for inhalation, and packed into capsules or blisters or dry powder inhalers.

A method for preparing toad skin extract dry powder inhalation, the preparation steps are as follows:

The toad skin extract is made into powder by pulverizing or spray drying, the powder is uniformly mixed with auxiliary materials, agglomerated to form redispersible granules, and packed into capsules or blisters or dry powder inhalers; or the toad skin extract is mixed with auxiliary materials evenly, Make powder by crushing or spray-drying, agglomerate to form redispersible granules, pack into capsules or blisters or dry powder inhalers; or mix toad skin extract and some excipients in the prescription evenly, and make powder by crushing or spray-drying , the powder is evenly mixed with the remaining excipients in the prescription, agglomerated to form redispersible particles, packed into capsules or blisters or dry powder inhalers; or the toad skin extract is made into powder by crushing or spray drying, and the powder is loaded on the surface of the excipient particles Granules are formed and packed into capsules or blisters or dry powder inhalers.

Application of a toad skin extract dry powder inhalation in the preparation of lung tumor medicine.

Beneficial effect:

1. The lung has a large surface area, and the alveolar wall is composed of a single layer of cells, which makes it easy for drug molecules to enter the blood. Therefore, pulmonary inhalation administration has many advantages such as quick onset of action and small adverse reactions. Dry powder inhalers (drypowder inhalers, DPIs) do not use propellants and solvents in pulmonary inhalation administration, rely on the airflow generated by the patient's spontaneous breathing to atomize the drug powder, and then transfer the drug powder to the lungs, which has the advantages of convenient application, rapid absorption, It has the characteristics of no liver first pass, immediate release and targeted positioning, and is a new type of drug delivery system. At present, dry powder inhalers are mostly used to treat respiratory and lung infections, but there is no treatment for lung tumors. At the same time, there is no report on the study of toad skin extract dry powder inhalation with anti-lung tumor effect. At present, toad skin extract injections, oral liquids, and tablets have been used clinically, and have good effects on liver cancer, lung cancer, and gastric cancer. In particular, cinobufacin injection, which is a water-soluble preparation processed from the skin of Bufo bufo, has a comprehensive effective rate of 44% and 56% in the treatment of primary liver cancer and advanced lung cancer, respectively, and the tumor shrinkage rates are respectively 10% and 16%, and have a synergistic effect on chemotherapy and radiotherapy. Although there are few clinical adverse reactions of cinobufacin injection, there are still local venous reactions during intravenous infusion, which can cause pain caused by contraction and spasm of infusion veins, and non-specific inflammation of veins caused by long-term stimulation; skin reactions, mainly manifested as urticaria and skin blister-like lesions; secondly, a small number of patients have cardiovascular system reactions such as premature beats, sinus bradycardia, sinus tachycardia, and atrioventricular block, which may be caused by a small amount of bufotoxin in cinobufacin Cardiotoxic reactions produced by ligands. In addition, the injection needs to be used in the hospital, and the patient's compliance is poor. The oral preparation of toad skin extract needs to go through the first-pass effect of the liver, the onset of action is slow, and the bioavailability of the preparation is low. The dry powder inhalation using toad skin extract is mainly aimed at lung tumors, directly acts on the affected area, and does not have the first-pass effect of the liver.

2. Since the toad skin extract is a mixture, which contains indole bases, cinobufinin, and cinobufaginin, etc., it is found in actual operation that adding a certain ratio of specific excipients can overcome the crushing of the extract to a certain extent. Agglomeration caused by static electricity, cohesive force, etc. generated from time to time reduces hygroscopicity. The applicant has learned through a lot of experiments that especially the mixture of lactose granules and micronized lactose or the powder agglomeration technology can be used as the excipient to make a dry powder inhaler with excellent inhalation performance.

Detailed ways

The present invention will be described in further detail below in conjunction with examples, but the scope of the present invention is not limited by these examples.

Example 1

1. Preparation of toad skin extract: wash dry toad skin, add 10 times the amount of water, decoct twice for 1.5 hours each time, combine the filtrate, concentrate to a relative density of 1.1-1.2 (85°C), and cool to Add ethanol for precipitation at 40°C to make the alcohol content reach 60%, let it stand for 24 hours, take the supernatant to recover the ethanol until it has no alcohol smell, add ethanol to the concentrated solution to make the alcohol content reach 85%, let it stand for 48 hours, filter , recover ethanol to a thick paste, and dry at 60°C to obtain toad skin extract.

2. Preparation of inhalant: the extract of toad skin is pulverized by airflow until the _D50 is below 5 microns, mixed with lactose with a particle size of 45-70 microns in a mass ratio of 1:10, and the drug powder is packed into capsules or blisters. hood or dry powder inhaler for inhalation.

Example 2

1. Preparation of toad skin extract: Take dry toad skin and wash it, add 12 times the amount of water, decoct twice for 2 hours each time, combine the filtrate, concentrate to a relative density of 1.2-1.3 (85°C), and cool to Add ethanol for precipitation at 40°C to make the alcohol content reach 70%, let it stand for 24 hours, take the supernatant solution and recover the ethanol until there is no alcohol smell, add ethanol to the concentrated solution to make the alcohol content reach 85%, let it stand for 48 hours, filter , recovering ethanol until it has no alcohol smell, and spray-drying to obtain toad skin extract powder, the powder D ₅₀ of which is below 5 microns.

2. Preparation of inhalant: mix toad skin extract powder, lactose with a particle size of 45-80 microns, and leucine with a particle size of 20-50 microns in a mass ratio of 10:100:1, pack into Capsules or blisters or dry powder inhalers, get inhalers.

Example 3

1. Preparation of toad skin extract: Take dry toad skin and wash it, add 30 times the amount of water, microwave-assisted extraction 3 times, each time for 1 hour, combine the extracts, and ultrafilter the ultrafiltration membrane with a molecular weight of 1000, and the ultrafiltrate The toad skin extract powder is obtained by spray drying, and the D ₅₀ of the powder is below 5 microns.

2. Preparation of inhalant: pack toad skin extract powder into capsules or blisters or dry powder inhaler to obtain inhaler.

Example 4

1. Preparation of toad skin extract: get the toad skin and wash it, add 12 times the amount of 60% (W/W) ethanol, reflux extraction 2 times, each time for 2 hours, combine the extracts, reclaim the ethanol until there is no alcohol smell, Refrigerate at low temperature for 48 hours, filter, add 0.3% activated carbon (W/V), boil for 30 minutes, filter, and spray dry to obtain toad skin extract powder.

2. Preparation of inhalant: extract powder of toad skin, lactose, mannitol, glycine, dilauroylphosphatidylcholine, lecithin, and magnesium stearate in a mass ratio of 1:5:5:2:0.5:0.5 : mixed in a ratio of 0.1, airflow pulverization until the powder _D50 is below 5 microns, and the drug powder is packed into capsules or blisters or dry powder inhalers to obtain inhalers.

Example 5

1. Preparation of toad skin extract: take toad skin and wash it, add 20 times the amount of absolute ethanol, reflux extraction twice, each time for 1 hour, combine the extracts, recover ethanol, and ultrafilter through an ultrafiltration membrane with a molecular weight of 3000 , the ultrafiltrate was vacuum-concentrated to a specific gravity of 1.20 (45° C.), and the extract was vacuum-dried at 50° C. to obtain toad skin extract.

2. Preparation of inhalation: mix toad skin extract, lactose, and cholesterol in a ratio of 1:8:1, airflow crush until the powder _D50 is below 5 microns, and pack the drug powder into capsules or blisters or dry powder for inhalation In the device, obtain the inhalant.

Example 6

1. Preparation of toad skin extract: get the toad skin and wash it, add 15 times the amount of 60% (W/W) ethanol, reflux extraction 3 times, each time for 1 hour, combine the extracts, reclaim the ethanol until there is no alcohol smell, Add 2 times the amount of water to the concentrated solution, let it stand for 24 hours, centrifuge at high speed, and take the supernatant to obtain toad skin extract.

2. Preparation of inhalant: Add lactose, polyethylene glycol 6000, and albumin to the toad skin extract with 5 times, 1 time, and 2 times of the solid content in the above-mentioned toad skin extract, spray dry, and powder _D50 below 5 microns, the drug powder is packed into capsules or blisters or dry powder inhalers to obtain inhalers.

Example 7

1. Preparation of toad skin extract: take toad skin and wash it, add 10 times the amount of 10% (W/W) ethanol, reflux extraction twice, each time for 1 hour, combine the extracts, reclaim ethanol until there is no alcohol smell, The concentrate is centrifuged at high speed, and the supernatant is taken to obtain toad skin extract, which is spray-dried to obtain the toad skin extract, and the _D50 of the powder is below 5 microns.

2. Preparation of inhalant: mix the drug powder of toad skin extract, mannitol with a particle size of 10-90 microns, and micronized silica gel at a mass ratio of 10:89:1, and pack them into capsules or blisters after mixing evenly. hood or dry powder inhaler for inhalation.

Example 8

1. Preparation of toad skin extract: get the toad skin and wash it, add 10 times the amount of 50% (W/W) ethanol, reflux extraction twice, each time for 1 hour, combine the extracts, reclaim the ethanol until there is no alcohol smell, The concentrate is centrifuged at high speed, and the supernatant is taken to obtain toad skin extract, which is spray-dried to obtain toad skin extract powder.

2. Preparation of inhalant: Mix the toad skin extract drug powder, mannitol, and leucine in a mass ratio of 10:100:3, add an appropriate amount of water after mixing, and spray dry to obtain a powder with a _D50 of 4.8 microns. Mix with a polymer-lined rotary mixer to form loose agglomerates with a _D50 of 390 microns and pack into capsules or blisters or dry powder inhalers to obtain inhalers.

Example 9

1. Preparation of toad skin extract: get the toad skin and wash it, add 10 times the amount of 40% (W/W) ethanol, reflux extraction 3 times, each time for 0.5 hours, combine the extracts, reclaim the ethanol until there is no alcohol smell, The concentrate is centrifuged at high speed, and the supernatant is taken to obtain toad skin extract, which is spray-dried, and the _D50 of the powder is below 5 microns.

2. Preparation of inhalant: mix toad skin extract powder, mannitol, and leucine in a mass ratio of 10:80:2, and mix them with a polymer-lined rotary mixer to make loose agglomerates Thing, the _D50 of agglomerate is 120 microns, is packed in capsule or blister or dry powder inhaler, obtains inhalation.

Example 10

1. Preparation of toad skin extract: take dry toad skin and wash it, add 20 times the amount of water, decoct twice for 2 hours each time, combine the filtrate, concentrate to a relative density of 1.2-1.3 (85°C), and cool to Add ethanol for precipitation at 40°C to make the alcohol content reach 70%, let it stand for 24 hours, take the supernatant to recover the ethanol until it has no alcohol smell, add ethanol to the concentrated solution to make the alcohol content reach 85%, let stand for 48 hours, filter , Recover ethanol until there is no alcohol smell, spray dry, the powder D ₅₀ is below 5 microns.

70乳糖、微粉化乳糖(D₅₀＝5.1微米)按质量比5∶90∶5的比例进行混合，混合均匀后包装入胶囊或泡罩或干粉吸入器内，获得吸入剂。2. Preparation of inhalant: extract powder of toad skin, German Megile

70 Lactose and micronized lactose (D ₅₀ =5.1 micron) are mixed in a mass ratio of 5:90:5, mixed evenly, packed into capsules, blisters or dry powder inhalers to obtain inhalers.

3. Investigation on the lung deposition rate of inhalants

According to the method stipulated in the Pharmacopoeia of the People's Republic of China in 2010, based on the content of bufafoatin in the preparation, the lung deposition rate reached 32.6%. Variety.

Example 11

1. Preparation of toad skin extract: take dry toad skin and wash it, add 20 times the amount of water, decoct twice for 2 hours each time, combine the filtrate, concentrate to a relative density of 1.2-1.3 (85°C), and cool to Add ethanol for precipitation at 40°C to make the alcohol content reach 70%, let it stand for 24 hours, take the supernatant to recover the ethanol until it has no alcohol smell, add ethanol to the concentrated solution to make the alcohol content reach 85%, let stand for 48 hours, filter , Recover ethanol until there is no alcohol smell, spray dry, the powder D ₅₀ is below 5 microns.

70乳糖、微粉化乳糖(D₅₀＝4.9微米)按质量比3∶95∶1的比例进行混合，混合均匀后包装入胶囊或泡罩或干粉吸入器内，获得吸入剂。2. Preparation of inhalant: extract powder of toad skin, German Megile

70 lactose and micronized lactose (D ₅₀ =4.9 microns) are mixed at a mass ratio of 3:95:1, and after being evenly mixed, packed into capsules or blisters or dry powder inhalers to obtain an inhaler.

3. Investigation on the lung deposition rate of inhalants

According to the method stipulated in the Pharmacopoeia of the People's Republic of China in 2010, based on the content of bufafoatin in the preparation, the lung deposition rate reached 30.2%. Variety.

Example 12

1. Preparation of toad skin extract: take dry toad skin and wash it, add 20 times the amount of water, decoct twice for 2 hours each time, combine the filtrate, concentrate to a relative density of 1.2-1.3 (85°C), and cool to Add ethanol for precipitation at 40°C to make the alcohol content reach 70%, let it stand for 24 hours, take the supernatant to recover the ethanol until it has no alcohol smell, add ethanol to the concentrated solution to make the alcohol content reach 85%, let it stand for 48 hours, filter , Recover ethanol until there is no alcohol smell, spray dry, the powder D ₅₀ is below 5 microns.

70乳糖、微粉化乳糖(D₅₀＝5.5微米)按质量比3∶77∶11的比例进行混合，混合均匀后包装入胶囊或泡罩或干粉吸入器内，获得吸入剂。2. Preparation of inhalant: extract powder of toad skin, German Megile

70 lactose and micronized lactose (D ₅₀ =5.5 microns) are mixed at a mass ratio of 3:77:11, and after being evenly mixed, packed into capsules, blisters or dry powder inhalers to obtain an inhaler.

3. Investigation on the lung deposition rate of inhalants

According to the method stipulated in the Pharmacopoeia of the People's Republic of China in 2010, the lung deposition rate of bufafoat in the preparation reached 29.8%. Variety.

Example 13

1. Preparation of toad skin extract: Take dry toad skin and wash it, add 10 times the amount of water, decoct twice for 1 hour each time, combine the filtrate, concentrate to a relative density of 1.3-1.4 (85°C), and cool to Add ethanol for precipitation at 40°C to make the alcohol content reach 60%, let it stand for 48 hours, take the supernatant to recover the ethanol until it has no alcohol smell, add ethanol to the concentrated solution to make the alcohol content reach 80%, let it stand for 48 hours, filter , Recover ethanol until there is no alcohol smell, spray dry, the powder D ₅₀ is below 5 microns.

2. Preparation of inhalant: mix toad skin extract powder and leucine in a ratio of 1:5, the _D50 of the mixed particles is 292 microns, pack into capsules or blisters or dry powder inhalers to obtain inhalation agent.

Example 14

1. Preparation of toad skin extract: wash dry toad skin, add 6 times the amount of water, decoct 5 times, 1 hour each time, combine the filtrate, concentrate to a relative density of 1.05 (70°C), and cool to 40°C , add ethanol for precipitation to make the alcohol content reach 65%, let it stand for 48 hours, take the supernatant to recover the ethanol until it has no alcohol smell, add ethanol to the concentrated solution to make the alcohol content reach 70%, let it stand for 48 hours, filter, and recover Ethanol until there is no alcohol smell, concentrated under reduced pressure, and dried under vacuum to obtain toad skin extract.

2. Preparation of inhalant: mix toad skin extract and leucine in a ratio of 1:1, airflow pulverize, the powder _D50 is below 5 microns, mix the powder with 9 times the amount of mannitol, mix The _D50 of the latter granules is 328 microns and packed into capsules or blisters or dry powder inhalers to obtain inhalers.

Example 15

1. Preparation of toad skin extract: wash dry toad skin, add 30 times the amount of water, decoct for 1 hour, concentrate the extract to a relative density of 1.30 (60°C), cool to 20°C, add ethanol to precipitate, and make The alcohol content reaches 70%, stand still for 24 hours, take the supernatant to recover ethanol until it has no alcohol smell, add ethanol to the concentrated solution to make the alcohol content reach 90%, let it stand for 48 hours, filter, recover the ethanol until it has no alcohol smell, Concentrate under reduced pressure and dry in vacuo to obtain toad skin extract.

2. Preparation of inhalant: mix toad skin extract and poloxamer in a mass ratio of 10:1, airflow pulverize, the powder _D50 is below 5 microns, and then mix the powder with 8 times the amount of lactose granules , 1 times the amount of magnesium stearate is mixed, the _D50 of the mixed granules is 653 microns, packed into capsules or blisters or dry powder inhalers to obtain the inhaler.

Example 16

1. Preparation of toad skin extract: Take toad skin and wash it, add 10 times the amount of water, decoct for 1 hour, concentrate the extract to a relative density of 1.20 (50°C), cool to room temperature, add ethanol for precipitation, and make the alcohol-containing When the concentration reaches 50%, let it stand still for 24 hours, take the supernatant to recover ethanol until it has no alcohol smell, add ethanol to the concentrated solution to make the alcohol content reach 70%, let stand for 48 hours, filter, recover the ethanol until it has no alcohol smell, and depressurize Concentrate and vacuum dry to obtain toad skin extract.

2. Preparation of inhalant: Airflow pulverize the toad skin extract, the powder D ₅₀ is below 5 microns, mix the powder with 20 times the amount of lactose granules, the D ₅₀ of the mixed granules is 286 microns, pack into capsules or Get the inhaler in a blister or dry powder inhaler.

Example 17

The inhalation was obtained according to Examples 1-16, and a pharmacodynamic experiment was carried out, wherein the comparative drugs were cinobufacin injection (commercially available), toad skin injection A and B, and toad skin injection A and B were prepared according to the following method:

Toad skin injection A:

Take dry toad skin and wash it, add 15 times the amount of water, decoct twice for 2 hours each time, combine the filtrate, concentrate to a relative density of 1.2 (85°C), cool to 20°C, add ethanol for precipitation, and make the alcohol content up to 60%, let it stand for 24 hours, take the supernatant to recover ethanol until it has no alcohol smell, add ethanol to the concentrated solution to make the alcohol content reach 85%, let it stand for 48 hours, filter, recover the ethanol until it has no alcohol smell, add water to every One milliliter is equivalent to 5g of the original drug, adjusted to pH 6.5, filtered through a 0.45 micron membrane, potted, and autoclaved to obtain the product.

Toad skin injection B:

Take dry toad skin and wash it, add 20 times the amount of 60% ethanol, reflux extraction twice, each time for 2 hours, combine the extracts, recover the ethanol until it has no alcohol smell, add 3 times the amount of water, refrigerate for 48 hours, filter, Add 0.3% activated carbon (W/V), boil for 30 minutes, filter, add water to equal to 5g of crude drug per milliliter, adjust pH to 7, filter with 0.45 micron membrane, potting, autoclaving to obtain final product.

Drug efficacy experiment:

The rat lung tumor animal model was established by percutaneous intrapulmonary injection of benzopyrene, and 200 model rats were randomly divided into 20 groups, 10 in each group. 1 time a day, medication for 7 days. Administration method: Groups 1-16 are administered by inhalation of the inhalant of Example 1-16, and the dosage is 15 g crude drug/kg; Group 17 is injected intraperitoneally with cinobufacin injection (commercially available), and the dosage is: 15g crude drug/kg; the 18th group intraperitoneally injected toad skin injection A, dosage: 15g crude drug/kg; the 19th group intraperitoneally injected toad skin injection B, dosage: 15g crude drug/kg; In the saline control group, 10 mL/kg was injected intraperitoneally. The rats were sacrificed the next day after drug withdrawal, and the lungs of the rats were dissected to observe the lung lesions.

Experimental results: the incidence of lumps or nodular lesions in the lungs of rats administered with inhalation was the lowest at 10.5%, and the highest was 30.3%; the cinobufacin injection group (commercially available) was 60.5%, and The injection A group was 50.1%, the toad skin injection B group was 60.8%, and the normal saline group was 100%. There was a significant difference in the incidence of lung lesions between toad skin injection group B and normal saline group (P<0.05).

Example 18

The inhalation was obtained according to Examples 1-16, and a pharmacodynamic experiment was carried out, wherein the comparative drugs were cinobufacin injection (commercially available), toad skin injection A and B, and toad skin injection A and B were prepared according to the following method:

Toad skin injection A:

Take dry toad skin and wash it, add 15 times the amount of water, decoct twice for 2 hours each time, combine the filtrate, concentrate to a relative density of 1.2 (85°C), cool to 20°C, add ethanol for precipitation, and make the alcohol content up to 60%, let it stand for 24 hours, take the supernatant to recover ethanol until it has no alcohol smell, add ethanol to the concentrated solution to make the alcohol content reach 85%, let it stand for 48 hours, filter, recover the ethanol until it has no alcohol smell, add water to every One milliliter is equivalent to 5g of the original drug, adjusted to pH 6.5, filtered through a 0.45 micron membrane, potted, and autoclaved to obtain the product.

Toad skin injection B:

Take dry toad skin and wash it, add 20 times the amount of 60% ethanol, reflux extraction twice, each time for 2 hours, combine the extracts, recover the ethanol until it has no alcohol smell, add 3 times the amount of water, refrigerate for 48 hours, filter, Add 0.3% activated carbon (W/V), boil for 30 minutes, filter, add water to equal to 5g of crude drug per milliliter, adjust pH to 7, filter with 0.45 micron membrane, potting, autoclaving to obtain final product.

Drug efficacy experiment:

1. Establishment of VX2 orthotopic lung cancer model in rabbits

1) Production of tumor-bearing rabbits Routinely resuscitated VX2 tumor frozen cell suspension to make 10 ⁶ /mL live cell suspension, and inoculated 1 mL into the lateral muscle of the right hind leg of a rabbit. When a lump with a diameter of about 30 cm was palpable, the tumor-bearing rabbits were made.

2) Preparation of tissue block suspension Under aseptic conditions, the tumor in the lateral muscle of the tumor-bearing rabbit was cut out, and the vigorously growing tumor tissue in the shape of fish was taken and cut into mud. Rinse with RPMI 1640 culture solution and pass through a 30-mesh sieve to remove single cells and too small tissue pieces. The part above the sieve is washed with RPMI 1640 culture solution to pass through a 20-mesh sieve to remove oversized tissue pieces. Take the part under the sieve, centrifuge and precipitate, take 2mL of the precipitate, add RPMI 1640 culture medium to prepare 20mL of tissue block suspension.

3) Intrapulmonary inoculation All rabbits were depilated on the right chest wall. After intramuscular injection of Sumianxin Mixture 0.8mL/kg, fixed after anesthesia, routine disinfection, sterile three-way joints were taken, respectively connected to No. The 1mL syringe of the tumor tissue block and the 1mL syringe of the VX2 tumor tissue block suspension or cell suspension. Under X-ray fluoroscopy, a 12-gauge needle was inserted into the lower lobe of the right lung of the rabbit, and after sucking back no blood, inject 0.4mL of tissue block suspension or cell suspension, and then inject 0.2mL of rabbit's own blood clot, and pull out the needle , the inoculation is completed.

4) Observation of the growth of transplanted tumors From the 3rd day after inoculation, all rabbits underwent CT scanning of the chest with a slice width and thickness of 5 mm every day to observe tumor growth and intrathoracic metastasis.

5) The results of establishing the VX2 orthotopic lung cancer model in rabbits All the rabbits had intrapulmonary tumor growth, and the success rate of inoculation was 100%.

2. Efficacy experiment:

200 successfully modeled rabbits were taken and randomly divided into 20 groups with 10 rabbits in each group. 1 time a day, medication for 12 days. Administration method: Group 1-16 is administered by inhalation of the inhalant of Example 1-16, dosage: 15g crude drug/kg; Group 17 is injected intraperitoneally with cinobufacin injection (commercially available), dosage: 15g crude drug/kg; the 18th group intraperitoneally injected toad skin injection A, dosage: 15g crude drug/kg; the 19th group intraperitoneally injected toad skin injection B, dosage: 15g crude drug/kg; In the saline control group, 10 mL/kg was injected intraperitoneally. Rabbits were killed the next day after drug withdrawal, and the lungs of the rabbits were dissected, and the tumor tissues were stripped and weighed.

Experimental results: the tumor inhibition rate of the rabbits administered by inhalation in each group was 58.7% at the minimum and 64.3% at the highest; 56.3% for the cinobufacin injection group (commercially available), 45.1% for the toad skin injection A group, Toad skin injection group B was 48.9%. Normal saline had almost no inhibitory effect on tumors. , The tumor inhibition rate of normal saline group was significantly different (P<0.05).

Claims (1)

1. A toad skin extract dry powder inhaler is characterized in that it is composed of toad skin extract and medically acceptable auxiliary materials; The preparation of the toad skin extract: wash the dried toad skin, add 20 times the amount of water, decoct twice for 2 hours each time, combine the filtrate, concentrate to a relative density of 1.2-1.3 at 85°C, and cool to 40 ℃, add ethanol for precipitation, so that the alcohol content reaches 70%, let it stand for 24 hours, take the supernatant to recover the ethanol until it has no alcohol smell, add ethanol to the concentrated solution to make the alcohol content reach 85%, let it stand for 48 hours, filter, Recover ethanol until it has no alcohol smell, spray dry, and the D ₅₀ of the powder is below 5 microns; The preparation of the inhalant: mix toad skin extract powder, German MEGGLE InhaLac? 70 lactose, and micronized lactose with a _D50 of 5.1 microns in a mass ratio of 5:90:5, and pack them into Capsules or blisters or dry powder inhalers, get inhalers.