CN111228243B

CN111228243B - Tobramycin liposome for aerosol inhalation and preparation method thereof

Info

Publication number: CN111228243B
Application number: CN201911347292.5A
Authority: CN
Inventors: 张树荣; 严贤龙; 薛琦; 田霞; 牛璐; 王振恒
Original assignee: Zhuhai Essex Bio Pharmaceutical Co ltd
Current assignee: Zhuhai Essex Bio Pharmaceutical Co ltd
Priority date: 2019-12-24
Filing date: 2019-12-24
Publication date: 2022-02-18
Anticipated expiration: 2039-12-24
Also published as: CN111228243A

Abstract

The invention discloses a tobramycin liposome for aerosol inhalation, which consists of the following components: 0.1-15.0 percent of tobramycin, 0.5-36.0 percent of phospholipid, 0.05-20.0 percent of stabilizing agent, 0.01-10.0 percent of charge modifier, 0.01-5.0 percent of antioxidant, 5.0-50.0 percent of organic phase medium and the balance of aqueous phase medium; compared with oral administration, the tobramycin liposome for aerosol inhalation can directly transfer the medicament to the respiratory tract, has quick absorption and quick action, and improves the medicament concentration of the respiratory tract; the bioavailability and stability are high, and the safety is good; the preparation process is simple, easy to prepare and reasonable, has stable performance, and creates conditions for realizing industrial productization.

Description

Tobramycin liposome for aerosol inhalation and preparation method thereof

Technical Field

The invention relates to the technical field of medicinal preparations, in particular to tobramycin liposome for aerosol inhalation and a preparation method thereof.

Background

Tobramycin is an aminoglycoside antibiotic naturally produced by streptomyces and is bactericidal at concentrations equal to or slightly greater than its inhibitory concentration, mainly by inhibiting protein synthesis leading to changes in the permeability of the cell membrane, progressive destruction of the cell envelope and ultimately cell death. The antibiotic is aminoglycoside antibiotic with less clinical drug resistance, has good antibacterial activity on most enterobacteriaceae bacteria and staphylococcus, has the most prominent characteristic of high efficiency on gram-negative bacteria, particularly pseudomonas aeruginosa, is suitable for the infection of adult and children over 6 years old (including 6 years old) with bronchiectasis, respiratory machine-related pneumonia (VAP) and pulmonary pseudomonas aeruginosa, haemophilus, staphylococcus aureus, klebsiella pneumoniae and escherichia coli of cystic fibrosis patients, and is used as a first-line treatment drug for the early infection of the pulmonary pseudomonas aeruginosa; the antibacterial effect on pseudomonas aeruginosa is 3-5 times stronger than that of gentamicin, and the defect is that the antibacterial activity is concentration-dependent, when lung is infected, a large dose of intravenous injection or oral administration is needed to enable the effective concentration of the pseudomonas aeruginosa to be reached at the infected part, but the high dose can cause adverse reactions such as renal poisoning, otoxicity and the like.

Only a small amount of drugs can enter lung tissues after the tobramycin is intravenously administered, the effect of clinically treating nosocomial infectious pneumonia is poor, but when the method of atomizing and inhaling the tobramycin is used, the drugs are directly conveyed into the lung tissues through throats, trachea and bronchus to reach infected focuses, the height in the lung tissues is increased to reach a peak value and lasts for a certain time, then the drugs are gradually diffused into blood, the tobramycin is uniformly distributed in alveoli, and the tobramycin has good antibacterial effect.

Tobramycin inhalation solution (trade name TOBI) was first approved by the Food and Drug Administration (FDA) for recurrent lung infections in patients with cystic fibrosis. Epidemiological statistical analysis shows that the pulmonary cystic fibrosis is a gene-defective disease, and the patients are infected repeatedly due to the colonization of lung pseudomonas aeruginosa, the gene defect is rare in Asian population, while the non-cystic fibrosis bronchiectasis has higher incidence in China and other east Asian countries. Clinical application researches find that the therapeutic effect of acute infection treatment of bronchiectasis can be improved by inhalation of tobramycin in an atomizing mode, the reason is that bronchiectasis is chronic airway inflammation, repeated cough and purulent sputum are taken as clinical manifestations, infection is taken as a common cause of aggravation of the disease, bacteria which are separated and cultured in sputum of a bronchiectasis patient most frequently are haemophilus influenzae, streptococcus pneumoniae, staphylococcus aureus and pseudomonas aeruginosa, and the pseudomonas aeruginosa is also the most intractable infectious pathogen most frequently.

The inhalation administration is an administration method which skillfully combines the aerosol technology with the anatomical, physiological and histological characteristics of the respiratory system and plays a local or systemic effect, and has the characteristics of quick response, less adverse reaction and the like. In recent years, with the increasing prevalence of respiratory diseases, such as asthma, bronchiectasis, ventilator-associated pneumonia, Chronic Obstructive Pulmonary Disease (COPD), cystic fibrosis, and the like, inhalation therapy for these diseases has become a global consensus.

The aerosol inhalant uses compressed air or oxygen, ultrasonic waves and electric oscillation to provide energy to form and release aerosol, and enters the respiratory tract and the lung for deposition by an inhalation method, thereby achieving the effect of quickly and effectively treating the local respiratory tract and simultaneously having a certain effect of humidifying and diluting airway secretions. The advantages of aerosol inhalation administration can be summarized roughly in the following points:

(1) can increase the water content of mucus in the air passage, and has the functions of humidifying the air passage and diluting sputum, thereby being beneficial to sputum drainage.

(2) In vitro and in vivo deposition experiments prove that the medicine can be directly delivered to the diseased part of the respiratory tract after being atomized by an inhalation device, even can reach the deep part of the lower respiratory tract, and has rapid and direct action.

(3) The aerosol inhalation has small dosage, obviously reduces the toxic and side effects of the medicine, and is particularly suitable for children and old people. For example, various hormone-related complications are often generated when hormones are used in the whole body, so that the quality of life is reduced, the influence of the hormones on the whole body is mainly related to the accumulation amount of the hormones, the whole body accumulation amount of a patient can be obviously reduced by inhaling the hormones, and the occurrence of the hormone-related complications is greatly reduced.

(4) When the aerosol is inhaled, a user can breathe quietly, the inhalation technique or the cooperation is not the key influencing the curative effect any more, and the aerosol is suitable for children to take medicine or severe patients.

(5) The aerosol inhalation can deliver a larger dose of drug without propellant initiation.

Tobramycin can be formulated for administration by inhalation by nebulisation, providing an attractive alternative to parenteral administration, delivering high concentrations of tobramycin directly to the infected area within the bronchi while minimising systemic bioavailability, reducing systemic absorption and systemic toxicity of the drug, for use in the treatment of pseudomonas aeruginosa and/or other susceptible bacterial infections associated with pulmonary diseases such as cystic fibrosis.

Disclosure of Invention

The invention aims to exert the advantages of tobramycin in treatment of patients with ventilator-associated pneumonia and cystic fibrosis and overcome the problems of low bioavailability and the like of the traditional injection and oral administration, and the invention has the advantages that sputum antagonism is generated after inhalation administration, namely mucin in sputum is combined with drugs to influence the antibacterial activity of the drugs, and in addition, the tobramycin has high water solubility, is difficult to pass through cell membranes, is unstable in heat and the like.

In order to achieve the purpose, the invention adopts the technical scheme that:

a tobramycin liposome for aerosol inhalation is composed of the following components: 0.1-15.0 percent of tobramycin, 0.5-36.0 percent of phospholipid, 0.05-20.0 percent of stabilizing agent, 0.01-10.0 percent of charge modifier, 0.01-5.0 percent of antioxidant, 5.0-50.0 percent of organic phase medium and the balance of aqueous phase medium;

wherein the mass ratio of the phospholipid to the cholesterol is 1: 1-10: 1;

the mass ratio of tobramycin to phospholipid is 1: 0.5-1: 6;

the volume ratio of the aqueous phase medium to the organic phase medium is 0.5: 1-8: 1;

tobramycin includes salts of tobramycin in terms of the content of tobramycin, i.e., tobramycin salts are converted to tobramycin.

The phospholipid is one or more of natural phospholipid, hydrogenated phospholipid, synthetic phospholipid and polyethylene glycol modified derivatives thereof.

The stabilizer is one or more of cholesterol, oleic acid, lauric acid, palmitic acid, stearic acid, arachidic acid, capric acid, caproic acid, adipic acid, povidone, ethyl lactate, benzyl benzoate, citric acid, cholic acid and salts thereof, deoxycholic acid and salts thereof;

the charge modifier is one or more of hyaluronic acid, chitosan, modified chitosan, sodium carboxymethylcellulose, carbomer, sodium alginate, amphotericin B, polyamine, stearylamine and arginine;

the antioxidant is one or more of disodium ethylene diamine tetraacetate, vitamin E, vitamin C, L-cysteine, sodium sulfite, hydrogen sulfite, sodium metabisulfite, sodium thiosulfate, citric acid, tea polyphenol, thimerosal, propylene glycol, glycerol and boric acid;

the organic phase medium is one or more of ethanol, methanol, ether, chain, cyclic or aromatic organic alcohol containing 4-7 carbon atoms, chloroform and dichloromethane;

the water phase medium is water for injection, pure water, phosphate water solution, glucose sodium chloride water solution, and lactobionic acid, fumaric acid, maleic acid, citric acid, malic acid, aspartic acid, boric acid, acetic acid, hydrochloric acid or sulfuric acid, or other organic acids, and buffer pairs with certain buffer capacity.

Further, the tobramycin liposome for aerosol inhalation is composed of the following components: 0.1-15.0 percent of tobramycin, 0.5-36.0 percent of phospholipid, 0.05-20.0 percent of stabilizing agent, 0.01-10.0 percent of charge modifier, 0.01-5.0 percent of antioxidant, 5.0-50.0 percent of organic phase medium and the balance of aqueous phase medium;

wherein the mass ratio of the phospholipid to the cholesterol is 6: 1;

the mass ratio of tobramycin to phospholipid is 1: 2;

the volume ratio of the aqueous phase medium to the organic phase medium was 3: 1.

Further, the modified chitosan is water-soluble chitosan obtained by carboxymethylation, sulfonation or hydroxypropylation and glycolation, and the water-soluble chitosan is further subjected to hydrophobic modification with acyl chloride, alkyl halide and glycidyl ether to obtain the amphiphilic chitosan derivative.

Further, the aqueous phase medium is a buffer solution with the pH value of 4.0-8.0.

Further, the tobramycin liposome for aerosol inhalation is composed of the following components: tobramycin 0.1-15.0%, egg yolk lecithin 0.5-36.0%, cholesterol 0.05-20.0%, carboxylated chitosan 0.01-10.0%, vitamin E0.01-5.0%, ethanol 5.0-50.0%, and phosphate buffer solution with pH of 7.0 for the rest;

wherein the mass ratio of the phospholipid to the cholesterol is 6: 1;

the mass ratio of tobramycin to phospholipid is 1: 2;

The invention discloses a preparation method of tobramycin liposome for aerosol inhalation, which comprises the following steps:

1) precisely weighing phospholipid, a stabilizer and an antioxidant in the prescribed amount, dissolving the phospholipid, the stabilizer and the antioxidant in an organic phase medium, wherein the raw materials account for 10-40% of the total amount, and dissolving the raw materials in water bath at 25-80 ℃ to form an organic phase solution;

2) accurately weighing tobramycin and a charge modifier in the formula amount, dissolving the tobramycin and the charge modifier in an aqueous phase medium, wherein the raw materials account for 60-90% of the total amount and are used as an aqueous phase;

3) slowly adding the organic phase into the water phase stirred at the constant temperature of 25-80 ℃, and stirring for 20-60 min;

4) and (3) evaporating under reduced pressure to remove the organic solvent, and then filtering with a 0.45-micrometer microporous filter membrane to obtain tobramycin liposome solution.

Further, the preparation method of the tobramycin liposome for atomization and inhalation comprises the following steps:

1) accurately weighing egg yolk lecithin, cholesterol and vitamin E in a prescription amount, dissolving the egg yolk lecithin, cholesterol and vitamin E in an organic phase medium, wherein the total amount of the raw materials accounts for 10-40%, and dissolving the raw materials in a water bath at 25-80 ℃ to form an organic phase solution;

2) precisely weighing tobramycin and chitosan with the prescription amount, dissolving the tobramycin and the chitosan in phosphate buffer solution with the pH value of 5.0-7.0, wherein the raw materials account for 60-90% of the total amount and are used as a water phase;

The application of the invention also discloses a preparation method of the tobramycin liposome for aerosol inhalation, which is characterized by comprising the following steps:

1) and (3) precisely weighing the phospholipid, the stabilizer and the antioxidant according to the prescription amount, dissolving the phospholipid, the stabilizer and the antioxidant in an organic phase medium, and completely dissolving the phospholipid, the stabilizer and the antioxidant in an ultrasonic mode to obtain an organic phase.

2) And precisely weighing a prescription amount of tobramycin dissolved aqueous phase medium as an aqueous phase.

3) And injecting the water phase into the organic phase, and performing water bath ultrasonic treatment for 2-15 min to obtain the stable emulsion.

Then transferring the mixture into a specific evaporation container, carrying out water bath at 25-80 ℃ for 100r min < -1 >, and carrying out reduced pressure evaporation to remove the organic solvent to obtain a viscous colloidal solution; and then adding a proper amount of aqueous medium for elution, and after complete elution, filtering the solution by using a 0.45-micron microporous filter membrane to obtain the tobramycin liposome solution.

Further, the preparation method of the tobramycin liposome for atomization and inhalation is characterized by comprising the following steps:

1) egg yolk lecithin, cholesterol and vitamin E in the prescribed amount are precisely weighed, dissolved in a proper amount of ethanol and completely dissolved by ultrasonic to serve as an organic phase.

2) Tobramycin with the prescription dose is precisely weighed and dissolved in phosphate buffer solution with the pH value of 5.0-7.0 to be used as a water phase.

The technical scheme of the invention takes phospholipid and cholesterol as a liposome system, optimizes a screening prescription process, prepares tobramycin liposome, generates fog drops by aerosol through an atomizing device, and achieves the administration effect of atomizing and inhaling the tobramycin liposome into the lung, and is mainly characterized in that:

1. selection of phospholipid species

Phospholipids are the basic materials of liposomes and determine the physicochemical properties of liposomes. The type and amount of phospholipids is critical to the preparation of liposomes. The phospholipids are divided into natural phospholipids and synthetic phospholipids according to the source, the natural phospholipids are mainly lecithin (phosphatidylcholine, PC), the common lecithin is soybean lecithin and yolk lecithin, and the natural PC has different lengths and saturation of two fatty chains. The unsaturated fatty chains of the plant phospholipid are many, and the plant phospholipid is in a liquid state at normal temperature and is easy to oxidize; the animal phospholipid has many saturated fatty chains, is solid at normal temperature, and is not easy to oxidize. The synthesized phospholipid is obtained by chemical synthesis reaction, mainly comprises DPPP (dipalmitoylphosphatidylcholine), DPPE (dipalmitoylphosphatidylethanolamine), DSPC (distearoylphosphatidylcholine) and the like, is generally a saturated fatty chain, and has the characteristics of stable property, high purity, strong oxidation resistance, stable finished product and the like.

The two natural phospholipids are respectively the soybean lecithin and the egg yolk lecithin, the two natural phospholipids have equivalent influence on the encapsulation efficiency, the encapsulation efficiency is improved when the phospholipids are increased, the encapsulation efficiency cannot be improved when the phospholipids sufficiently wrap the medicine, the particle size is possibly influenced by the excessive phospholipids, and the encapsulation efficiency is highest when the concentration of the phospholipids is about 120 mg/ml.

2. Phospholipid/cholesterol mass ratio

The cholesterol is a common lipid for preparing the liposome and can increase the rigidity of a lipid membrane and the stability of the liposome, and the cholesterol is an amphiphilic molecule and is composed of a hydroxyl polar head and 4 steroid rings in planar conformation connected with a short hydrocarbon chain at the other end. The liposomal bilayer is generally composed of spaced, oriented arrays of phospholipid and cholesterol molecules. When the cholesterol content is increased to stabilize the liposome membrane, but the cholesterol is below the phospholipid phase transition temperature, it acts as a fluidization effect and instead increases fluidity. As a result of the investigation, it was found that the encapsulation efficiency was increased as the phospholipid/cholesterol mass ratio was increased, and the phospholipid/cholesterol ratio had a significant influence on the encapsulation efficiency, but when the phospholipid was too high and the cholesterol was too low, the liposome leaked. The optimal phospholipid/cholesterol mass ratio is 6: 1.

3. Mass ratio of drug to phospholipid

The mass ratio of the medicine to the phospholipid has great influence on the encapsulation efficiency and the stability of the liposome, and if the dosage is too large, the encapsulation capacity of the phospholipid is exceeded. As can be seen from the research, when the tobramycin liposome is prepared, the entrapment rate of the liposome is increased along with the reduction of the drug/phospholipid ratio, but when the entrapment rate is reduced to 1:2, the entrapment rate tends to be flat and constant, and the increase of the phospholipid to the entrapment rate has no obvious change, so that the optimal drug/phospholipid mass ratio is 1: 2.

4. Chitosan as charge modifier

The charges affect the stability of the liposome, the aggregation of the liposome is related to the Zeta potential, the higher the Zeta potential is, the larger the electrostatic repulsion among liposome particles is, the less aggregation is easy to occur, and the better the stability is. Because the cell membrane is negatively charged, the positively charged liposome can be well combined with cells and open the tight connection between the cells, so that the liposome is easy to adhere and enter the cells.

Stearyl amine is a commonly used cationic modifier, but generally, ionic surfactants are limited in their utility due to their strong hemolytic activity.

The other natural only positively charged basic polysaccharide is chitosan, which is degradable by lysozyme in vivo, has no toxicity, good biocompatibility and low immunological rejection, is a heteropolymer with glucosamine and acetylglucosamine as units, can be obtained by deacetylation of chitin to different degrees, and is the only naturally occurring cationic polysaccharide. Due to deacetylation, the amino groups in the chitosan structure make it positively charged in acidic solution and interact with the negatively charged amino acid residues of mucin via hydrogen bonds or ions, thus having mucoadhesive properties that increase the bioavailability of the drug and reduce the frequency of administration. The invention wraps carboxylated chitosan on the surface of the liposome through electrostatic adsorption, so that the liposome is charged positively, the permeability and targeting property of the liposome to lung cell membranes are improved, and the curative effect of the medicament is further improved.

At present, the chitosan is subjected to carboxymethylation, sulfonation or hydroxypropylation and glycolysis to obtain water-soluble chitosan, and the water-soluble chitosan is further subjected to hydrophobic modification with acyl chloride, alkyl halide and glycidyl ether to obtain an amphiphilic chitosan derivative, wherein carboxylated chitosan is easily soluble in water.

The encapsulation efficiency is higher along with the addition of the chitosan, which shows that the addition of the carboxylated chitosan has a larger influence on the encapsulation efficiency, and the encapsulation effect of the liposome can be obviously improved, probably because the existence of phosphate groups and/or amino groups of phospholipid causes the phospholipid to mostly carry negative charges, the carboxylated chitosan is a natural water-soluble polysaccharide with positive charges, is dissolved in a water phase, tobramycin is also water-soluble, the tobramycin is adhered to an oil-water interface membrane due to the potential difference, so that the encapsulation efficiency is improved, when the amount of the carboxylated chitosan is increased along with the increase of the amount of the carboxylated chitosan, the steric hindrance space adhered to the surface of the phospholipid is saturated, so that a platform area gradually appears, and researches show that when 0.7 percent of carboxylated chitosan has the highest encapsulation efficiency, but the charge reaches +39mV, and the higher potential has a certain influence on the stability of the liposome, when 0.5% of carboxylated chitosan is added, the encapsulation efficiency is higher, the charge reaches +27.9mV within a reasonable range of 20-30 mV, and the properties of the lipid agent such as appearance, particle size, encapsulation efficiency and the like are not changed. Therefore, the final tobramycin liposome solution is optimally formulated to contain 0.5% of carboxylated chitosan.

5. Antioxidant agent

The liposome is composed of phospholipid as main carrier, which is easy to oxidize, and antioxidant should be added to ensure its chemical stability and avoid oxidative degradation, wherein common fat-soluble antioxidant is vitamin E, water-soluble antioxidant is vitamin C and L-cysteine, and vitamin C is easy to photodegrade and has slightly poor stability. In the preparation process, vitamin E is mixed with phospholipid, L-cysteine is dissolved with water phase, and the tobramycin liposome containing vitamin E or L-cysteine is respectively prepared.

6. Selection of the type of organic solvent

The organic solvent is mainly used for dissolving phospholipid and cholesterol, and different organic solvents have different dissolving effects on lipid, so that the encapsulation efficiency of the liposome is influenced. In the research, ethanol and diethyl ether are selected as organic solvent investigation objects, and liposomes prepared from the ethanol and the diethyl ether are respectively compared. Finally, the encapsulation efficiency of the ethanol used as the organic solvent is obviously higher than that of the ether, so the ethanol is selected as the organic solvent for the liposome injection method.

The invention has the following beneficial effects:

(1) the product is liposome, the main component of alveolus is lipid, wherein phospholipid accounts for 80% of lipid component, and the liposome is composed of phospholipid, and has good compatibility with the two, and remarkably improved bioavailability.

(2) By adopting liposome technology, the medicament can effectively permeate stubborn pseudomonas biomembranes, has strong bactericidal activity, avoids local irritation, enhances curative effect, reduces toxic and side effects, can reach higher concentration of lung tissues and has good safety.

(3) Compared with oral administration, the aerosol inhalation technology can directly deliver the medicament to the respiratory tract, has quick absorption and quick action, improves the concentration of the medicament in the respiratory tract, increases the curative effect and simultaneously minimizes the exposure of the whole body, thereby reducing the toxic and side effects of the whole body, avoiding the first pass effect of the liver, having high bioavailability and reducing the dosage; compared with injection administration, inhalation administration can improve the compliance of patients and reduce or avoid part of adverse drug reactions; compared with a pressure quantitative inhalant and a dry powder inhalant, the inhalant has the characteristics of simple prescription, good dose flexibility, small influence of inhalation mode on inhalation effect and the like.

(4) Tobramycin with poor fat solubility can be wrapped in the liposome to avoid being combined with mucin in the sputum, so that the bioavailability and the stability of the tobramycin are improved; meanwhile, the sustained delivery of tobramycin to lung tissues is realized based on a liposome encapsulation technology, a long-acting slow release effect is achieved, 1 time daily inhalation administration can be realized, the current marketed medicine TOBI is replaced for 2 times daily, the administration frequency is reduced, and the compliance of patients is enhanced.

(5) The liposome technology of the research realizes the room temperature storage of the product, does not need the refrigeration storage at 2-8 ℃, improves the stability in the room temperature storage, and reduces the transportation and storage cost.

(6) The preparation process of the research is simple, easy and reasonable to prepare and stable in performance, and conditions are created for realizing industrial productization.

(7) The research simulates that the tobramycin liposome for atomization inhalation is atomized and then subjected to in-vitro evaluation in the lung, and shows that the mass median particle diameter (MMAD) of the tobramycin liposome for atomization inhalation is smaller than 3 mu m in the storage and use processes of the tobramycin liposome, the optimal range of the particle diameter value of the inhaled fogdrop in the lung is reached, the in-vitro effective deposition rate (FPF) reaches more than 70 percent, and the particle diameter, the Zeta potential and the entrapment rate are not obviously changed before and after atomization, which shows that the tobramycin liposome has good lung absorbability, so that most of tobramycin liposome is deposited in the lung and plays a pharmacodynamic treatment effect.

Drawings

FIG. 1 is a graph comparing the encapsulation efficiency of a tobramycin liposome preparation process;

FIG. 2 is a graph of the amount of drug deposited in each part of the NGI for three batches prepared in example 1;

FIG. 3 is a comparison of the amount of tobramycin liposome deposited in various parts of the NGI in comparison to conventional inhalation solutions;

FIG. 4 is a in vitro release profile study of three batches of samples prepared in example 1 with the original research (TOBI).

Detailed Description

In order to make the technical means, the creation characteristics, the achievement purposes and the effects of the invention easy to understand, the invention is further described with the specific embodiments.

Example 1

The tobramycin liposome for aerosol inhalation comprises the following components in percentage by mass:

the preparation method comprises the following steps:

1) precisely weighing the protein lecithin, cholesterol and vitamin E according to the prescription amount, dissolving the protein lecithin, cholesterol and vitamin E in absolute ethyl alcohol (accounting for 25 percent of the total amount), and dissolving the mixture in water bath at 55 ℃ to form an organic phase solution;

2) accurately weighing tobramycin and chitosan in the prescribed amount, dissolving in phosphate buffer solution (accounting for 75% of the total amount) with pH of 7.0, and using as water phase;

3) slowly adding the organic phase into the water phase stirred by magnetic force at the constant temperature of 55 ℃ by using an injector, and stirring for 30 min;

4) evaporating under reduced pressure to remove organic solvent, and filtering with 0.45 μm microporous membrane to obtain tobramycin liposome. The pattern is shown in figure 1.

Example 2

the preparation method comprises the following steps:

1) egg yolk lecithin, cholesterol and vitamin E in the prescribed amount are precisely weighed, dissolved in a proper amount of ethanol (25 percent) and completely dissolved by ultrasonic to be used as an organic phase.

2) The prescribed amount of tobramycin was precisely weighed and dissolved in phosphate buffer at pH7.0 as the aqueous phase.

3) Injecting the water phase into the organic phase, and performing ultrasonic treatment in water bath for 4min to obtain stable emulsion.

4) Transferring to a round-bottom flask, and carrying out water bath at 50 ℃ for 100r min^-1Evaporating under reduced pressure to remove organic solventTo a viscous, colloidal solution. And then adding a proper amount of aqueous medium for elution, and after complete elution, filtering the solution by using a 0.45-micron microporous filter membrane to obtain the tobramycin liposome solution.

Example 3

the preparation method comprises the following steps:

1) egg yolk lecithin, cholesterol and vitamin E in the prescribed amount are precisely weighed and placed in a round bottom flask, and a proper amount of ethanol (25%) is added for ultrasonic dissolution.

2) Placing on a rotary evaporator, heating in 50 deg.C water bath for 100 r.min-1, and performing rotary evaporation under reduced pressure to remove organic solvent to make lipid form a film on the bottle wall.

3) Weighing tobramycin according to the prescription amount, dissolving the tobramycin into phosphate buffer solution with the pH value of 7.0, adding the tobramycin into a round-bottom flask, continuously rotating and hydrating completely to obtain milky suspension, carrying out ultrasonic treatment on the milky suspension for 5min (working for 3s and intermittent for 3s) by using a 100W probe, and filtering the milky suspension by using a 0.45-micrometer microporous filter membrane to obtain the tobramycin liposome.

Example 4

the preparation method comprises the following steps:

2) Then placing on a rotary evaporator, carrying out water bath at 50 ℃ for 100 r.min < -1 >, and carrying out rotary evaporation under reduced pressure to remove all the organic solvent, so that the lipid forms a layer of film on the bottle wall.

3) Adding citric acid solution to hydrate phospholipid membrane, and filtering with 0.45 μm microporous membrane to obtain blank liposome. And then, regulating the pH value to be neutral by using a 150mmol/L sodium carbonate solution, adding a prescription amount of tobramycin aqueous solution, incubating in a water bath at 50 ℃ for 20min, and cooling to room temperature to obtain the tobramycin liposome.

Example 5

Compared with other common methods, the method disclosed by the invention takes the example 1 and the example 2 as examples, and has higher encapsulation efficiency.

The encapsulation efficiency of the four liposome preparation methods (i.e., examples 1-4) was calculated and compared according to the encapsulation efficiency determination method, and the results are shown in the following table and fig. 1.

TABLE several encapsulation efficiency comparisons of Tobramycin liposomes preparation methods (n ═ 3)

As can be seen from the above table, the encapsulation efficiency of the film dispersion method is the lowest, and during the preparation process, the film dispersion method also has obvious precipitation phenomenon, which indicates that the film dispersion method is not suitable for water-soluble drugs, so the method is omitted; the liposome prepared by the injection method and the reverse evaporation method has higher entrapment rate, the method has certain advantages in the aspect of wrapping water-soluble drugs, the method is most simple and convenient to operate, the process steps are fewer, the requirement on equipment is not high, and the tobramycin liposome with the most ideal entrapment rate can be easily prepared under the limited conditions of a laboratory, so that the injection method and the reverse evaporation method can be determined to be used as the preparation method for the optimized research of the tobramycin liposome process and the prescription.

Example 6

the preparation method comprises the following steps:

the preparation method is the same as that of the embodiment 1, and the finished product of the microemulsion eye drops of the embodiment 6 is obtained.

Example 7

the preparation method comprises the following steps:

the preparation method is the same as that of example 1, and the tobramycin liposome solution of example 7 is obtained.

Example 8

the preparation method comprises the following steps:

the preparation method is the same as that of example 1, and the tobramycin liposome solution of example 8 is obtained.

Example 9

the preparation method comprises the following steps:

the preparation method is the same as that of example 1, and the tobramycin liposome solution of example 9 is obtained.

Example 10

the preparation method comprises the following steps:

the preparation method is the same as that of example 1, and the tobramycin liposome solution of example 10 is obtained.

Example 11

the preparation method comprises the following steps:

the preparation method is the same as that of example 1, and the tobramycin liposome solution of example 11 is obtained.

Example 12

the preparation method comprises the following steps:

the preparation method is the same as that of example 1, and the tobramycin liposome solution of example 12 is obtained.

Example 13

the preparation method comprises the following steps:

the preparation method is the same as that of example 1, and the tobramycin liposome solution of example 13 is obtained.

Example 14

the preparation method comprises the following steps:

2) accurately weighing tobramycin and chitosan in the prescribed amount, dissolving in phosphate buffer solution (accounting for 75% of the total amount) with pH5.0, and using as water phase;

4) evaporating under reduced pressure to remove organic solvent, and filtering with 0.45 μm microporous membrane to obtain tobramycin liposome.

Example 15

the preparation method comprises the following steps:

1) precisely weighing the protein lecithin, cholesterol and vitamin E according to the prescription amount, dissolving the protein lecithin, cholesterol and vitamin E in absolute ethyl alcohol (accounting for 25 percent of the total amount), and dissolving the mixture in water bath at 25 ℃ to form an organic phase solution;

3) slowly adding the organic phase into the water phase stirred by magnetic force at a constant temperature of 25 ℃ by using an injector, and stirring for 30 min;

Example 16

the preparation method comprises the following steps:

1) precisely weighing the protein lecithin, cholesterol and vitamin E according to the prescription amount, dissolving the protein lecithin, cholesterol and vitamin E in absolute ethyl alcohol (accounting for 25 percent of the total amount), and dissolving the mixture in water bath at 80 ℃ to form an organic phase solution;

3) slowly adding the organic phase into the water phase stirred by magnetic force at constant temperature of 80 ℃ by using an injector, and stirring for 30 min;

Example 17

the preparation method comprises the following steps:

5) precisely weighing the protein lecithin, cholesterol and vitamin E according to the prescription amount, dissolving the protein lecithin, cholesterol and vitamin E in absolute ethyl alcohol (accounting for 25 percent of the total amount), and dissolving the mixture in water bath at 55 ℃ to form an organic phase solution;

6) accurately weighing tobramycin and chitosan in the prescribed amount, dissolving in phosphate buffer solution (accounting for 75% of the total amount) with pH of 7.0, and using as water phase;

7) slowly adding the organic phase into the water phase stirred by magnetic force at the constant temperature of 55 ℃ by using an injector, and stirring for 60 min;

8) evaporating under reduced pressure to remove organic solvent, and filtering with 0.45 μm microporous membrane to obtain tobramycin liposome.

Example 18

the preparation method comprises the following steps:

3) slowly adding the organic phase into the water phase stirred by magnetic force at the constant temperature of 55 ℃ by using an injector, and stirring for 10 min;

Example 19: performance detection

The finished products obtained in example 1 are respectively subjected to various tests, the results of the physicochemical property measurement are shown in the following tables 1-2, and the in vitro release curve is shown in the attached figure 2. After long-term stability test at 25 ℃ and accelerated stability test at 40 ℃ for 6 months, the appearance of the product is uniform liquid, and the particle size, zeta potential, content, encapsulation efficiency, leakage rate, oxidation index and in-vitro release degree are not significantly changed and are all in the qualified range. The content of the marketed TOBI is significantly degraded at the accelerated temperature of 40 ℃, the release rate is obviously faster than that of the product, the content is slightly reduced at the long-term temperature of 25 ℃, and the release rate is obviously faster than that of the product at the 2 h. The tobramycin liposome is considered to have better thermal stability and certain slow release effect than the marketed TOBI. The product can be stored at normal temperature and has no influence on quality.

TABLE 1-1 accelerated stability test investigation results of this product

TABLE 1-2 TOBI accelerated stability test investigation results for marketed drugs

TABLE 2-1 Long-term stability test investigation results of this product

TABLE 2-2 Long-term stability test investigation results of marketed drug TOBI

The above embodiments are preferred embodiments of the present invention, and all processes similar to the present invention and equivalent variations should be considered to fall within the scope of the present invention.

Example 20: lung in vitro evaluation of tobramycin liposome solution for aerosol inhalation

1 delivery Rate and Total delivery determination

Examine the in vitro assessment of tobramycin liposome solutions for nebulization in the lungs. Taking 20170801 batches, 20170802 batches and 20170803 batches of the product and stability test samples thereof, adopting a PARI BOY SX suit atomizer to respectively test the effective delivery dose of an adult mode and a child mode, wherein the adult mode (the tidal volume is 500ml, the breathing ratio is 1:1, the breathing frequency is 15 times/min), and the child mode (the tidal volume is 155ml, the breathing ratio is 1:2, and the breathing frequency is 15 times/min); the detection results are shown in table 20-1, and the effective delivery dose (delivery rate and total delivery amount) of the product in the adult mode and the child mode has no obvious difference between the accelerated long-term stability in batches, and has good and basically consistent batch-to-batch reproducibility.

TABLE 20-1 stability comparison of delivery Rate and Total amount delivered

2 Fine particle dose and particle size distribution test

Taking a stability sample of the product, adopting NGI equipment, PARI (BOY SX + LC SPRINT), setting the pump flow rate to be 15L/min, measuring the fine particle dose and the particle size distribution, and counting the test result of each batch (n is 3), wherein the drug content in MMAD, GSD, FPF, NGI parts, artificial throat and the like of the atomized solution is mainly obtained. The in vitro atomization characteristic evaluation of the product and the related stability conditions is carried out according to the above determination indexes, and the detection results are shown in Table 20-2 and FIG. 2. Meanwhile, a small sample of ordinary tobramycin aerosol inhalation solution is prepared by taking tobramycin as a raw material and sodium chloride and sulfuric acid as auxiliary materials, the particle size distribution of the small sample is inspected by NGI, and the result is shown in figure 3 when the small sample is compared with the product.

TABLE 20-2 NGI stage drug deposition amounts for three samples (day 0)

From the statistical analysis of the data chart of the drug deposition amount of each part stage of the NGI in the attached fig. 2-3, the three batches of tobramycin liposome for atomization inhalation have no significant difference (P <0.05) in each stage, and show that the particle size and distribution of the droplets are substantially consistent. In addition, the comparison of the particle size distribution of the tobramycin liposome inhalation solution at each part measured by NGI after being atomized by using a PARIBOYSX atomizer and the result measured after the tobramycin common inhalation solution is atomized shows that the particle size distribution proportion is basically consistent, which indicates that the particle size and the distribution of droplets are not obviously influenced when the tobramycin liposome is prepared into liposome, and the possible reason is that the particle size of the tobramycin liposome is in the nanometer level range, and the particle size of the atomized aerodynamic particles is in the micrometer level, so the particle size and the distribution of the atomized particles are not influenced.

The drug contents of each stage of the product obtained by the three repeated tests of 20170801 batches, 20170802 batches and 20170803 batches were introduced into CITDAs software of Copley corporation to obtain three parameter results of FPF, MMAD and GSD, and the results are shown in Table 20-3.

TABLE 20-3 Fine particle dose and particle size distribution results (n ═ 3, X. + -. S)

Table 20-3 shows that the stability of the product in batches and in batches is not significantly different between FPF and MMAD for 6 months, which indicates that the prescribed process of the product is relatively stable and uniform in external characterization of nebulization, and that the MMAD and FPF mainly react to aerodynamic particle size, which depends on external equipment factors such as nebulization cup, nebulizer flow rate, NGI measurement conditions, etc., and they are different depending on respiratory airflow, temperature, humidity, performance of nebulizer, etc. The data show that the MMAD of the product is less than 3 μm, and the MMAD reaches the optimal lung inhalation droplet size value; the percentage of the FPF with the effective deposition amount is more than 70 percent, which shows that the medicine fog drops which can be deposited in the bronchioles and alveoli account for more than 70 percent, and most of the medicine fog drops enter the lungs to achieve the effective treatment effect; GSD is geometric standard deviation, is an index of variation of the particle diameter of the droplets, is the square root of the ratio of the corresponding aerodynamic particle diameters when the cumulative drug distribution rate is 84.1 percent and 15.9 percent respectively, indicates that the inhalant is monodisperse and has narrower distribution when the GSD is closer to 1, indicates that the inhalant is polydisperse when the GSD is more than 1.2, and the GSD of the product is more than 1.2, so the product is a polydisperse system, and is consistent with the fact that research reports that the particle diameter of the droplets atomized by using a spray atomizer of Pari is 1.2-6.9 mu m and the droplets are heterogeneous.

3. Change of physicochemical properties of tobramycin liposome before and after atomization

The key of liposome pulmonary administration is to deliver drug-containing liquid particles to the lung, and the research on the aerodynamic particle size after atomization proves that the liposome pulmonary administration drug has good absorbability and the feasibility of preparing atomized inhalation liquid for clinical application, but different from other atomized solutions, the liposome pulmonary administration drug mainly aims to produce a slow release effect, and whether the stability (particle size, Zeta potential and entrapment rate) of the liposome pulmonary administration drug changes in the atomization process is particularly important, and the problem that the drug administration of the aerosolized lung of the tobramycin liposome needs to be considered is also solved.

Putting 5mL of the liposome of the product into an atomizing cup, connecting the atomizing cup with an entrance joint of NGI, atomizing by using a jet atomizer, then collecting liquid obtained from each part of the NGI, mixing, diluting by about 2 times, and then carrying out liposome nanometer particle size and Zeta potential measurement on a laser particle size analyzer (PSS-Z3000). Each sample is subjected to parallel operation and measurement for 3 times, then the changes of the particle size and the Zeta potential of the tobramycin liposome before and after atomization are compared, the entrapment rate of the collected mixed solution is measured by adopting an ultrafiltration centrifugation method, and the comparison data results before and after atomization are shown in a table 20-4.

TABLE 20-4 Tobramycin liposomes particle size, Zeta potential and encapsulation efficiency before and after atomization (n ═ 3, X. + -. S)

As can be seen from the above table, the increase of the average particle size of the tobramycin liposome before and after atomization is about 10-20nm, the reduction amplitude of the Zeta potential value is between 2 and 4mV, the reduction amplitude of the encapsulation efficiency is within 10 percent, the reason is probably that a shearing force is generated in the atomization process of the jet atomizer, so that the tobramycin liposome in atomization is likely to generate the phenomena of aggregation, partial capsulorhexis and leakage, the reduction of the Zeta potential also reflects that the physical and chemical stability of the tobramycin liposome in the atomization process is slightly influenced, however, the average particle size, Zeta potential and encapsulation efficiency before and after atomization do not change greatly from the analysis of the general trend, therefore, no significant influence is generated, and further shows that the atomized inhalation solution prepared into the liposome nano-disperse system by using the tobramycin as a model medicament can be used for a pulmonary inhalation administration route.

In vitro release degree determination experiment of tobramycin liposome

The in vitro release degree of the composition is measured by a dialysis bag method, which comprises the following specific steps:

weighing 2ml tobramycin liposome, adding into dialysis bag, placing into rotary basket, placing into dissolution cup according to dissolution determination requirement of Chinese pharmacopoeia 2015 edition, pouring 500ml phosphate buffer solution with pH7.0 preheated to 37 deg.C as release medium into the cup, and adjusting rotation speed to 50 r.min^-1The temperature was 37 ℃. 2ml of the sample (2 ml of the release medium is supplemented) are respectively sampled at 0.5 h, 1 h, 2h, 4 h, 6 h, 8 h, 10 h and 12h, the sample is centrifuged and analyzed by an HPLC method, the cumulative release amount of the drug at each time point is determined by an external standard method, 2ml of the original ground product TOBI (tobramycin inhalation solution) is measured and added into a dialysis bag, the measurement is carried out by the same method, and a drug release curve is drawn and shown in figure 4.

Claims

1. The tobramycin liposome for aerosol inhalation is characterized by comprising the following components: 0.1-15.0 percent of tobramycin, 0.5-36.0 percent of phospholipid, 0.05-20.0 percent of stabilizing agent, 0.01-10.0 percent of charge modifier, 0.01-5.0 percent of antioxidant, 5.0-50.0 percent of organic phase medium and the balance of aqueous phase medium;

wherein the mass ratio of the phospholipid to the cholesterol is 1: 1-10: 1;

the mass ratio of tobramycin to phospholipid is 1: 0.5-1: 6;

the tobramycin comprises tobramycin salt, and the tobramycin salt is calculated according to the content of the tobramycin, namely, the tobramycin salt is converted into the tobramycin;

the preparation method of the tobramycin liposome for atomization and inhalation comprises the following steps:

1) precisely weighing phospholipid, a stabilizer and an antioxidant in a prescription amount, dissolving the phospholipid, the stabilizer and the antioxidant in an organic phase medium, wherein the raw materials account for 10-40% of the total amount, and dissolving the raw materials in water bath at 25-80 ℃ to form an organic phase solution;

4) evaporating under reduced pressure to remove organic solvent, and filtering with 0.45 μm microporous membrane to obtain tobramycin liposome solution;

the phospholipid is one or more of natural phospholipid, hydrogenated phospholipid, synthetic phospholipid and polyethylene glycol modified derivatives thereof;

the stabilizer is one or more of cholesterol, oleic acid, lauric acid, palmitic acid, stearic acid, arachidic acid, capric acid, caproic acid, adipic acid, polyvidone, ethyl lactate, benzyl benzoate, citric acid, cholic acid and its salts, and deoxycholic acid and its salts;

the antioxidant is one or more of disodium ethylene diamine tetraacetate, vitamin E, vitamin C, L-cysteine, sodium sulfite, sodium bisulfite, sodium metabisulfite, sodium thiosulfate, citric acid, tea polyphenol, thimerosal, propylene glycol, glycerol and boric acid;

the organic phase medium is one or more of ethanol, methanol, diethyl ether, chain, cyclic or aromatic organic alcohol containing 4-7 carbon atoms, chloroform and dichloromethane;

the water phase medium is water for injection, pure water, phosphate water solution, glucose sodium chloride water solution, and lactose acid, fumaric acid, maleic acid, citric acid, malic acid, aspartic acid, boric acid, acetic acid, hydrochloric acid or sulfuric acid, or other organic acids, and buffer pairs thereof with certain buffer capacity.

2. The tobramycin liposome for aerosol inhalation according to claim 1, wherein the tobramycin liposome for aerosol inhalation is composed of: 0.1-15.0 percent of tobramycin, 0.5-36.0 percent of phospholipid, 0.05-20.0 percent of stabilizing agent, 0.01-10.0 percent of charge modifier, 0.01-5.0 percent of antioxidant, 5.0-50.0 percent of organic phase medium and the balance of aqueous phase medium;

wherein the mass ratio of the phospholipid to the cholesterol is 6: 1;

the mass ratio of tobramycin to phospholipid is 1: 2;

3. The tobramycin liposome for aerosol inhalation according to claim 1, wherein the modified chitosan is carboxymethylated, sulfonated or hydroxypropylated, and glycolated to obtain water-soluble chitosan, and the water-soluble chitosan is further hydrophobically modified with acyl chloride, alkyl halide and glycidyl ether to obtain an amphiphilic chitosan derivative.

4. The tobramycin liposome for aerosol inhalation of claim 1, wherein the aqueous medium is a buffer having a pH of 4.0 to 8.0.

5. The tobramycin liposome for aerosol inhalation according to claim 1, wherein the tobramycin liposome for aerosol inhalation is composed of: tobramycin 0.1-15.0%, egg yolk lecithin 0.5-36.0%, cholesterol 0.05-20.0%, carboxylated chitosan 0.01-10.0%, vitamin E0.01-5.0%, ethanol 5.0-50.0%, and phosphate buffer solution with pH of 7.0 for the rest;

wherein the mass ratio of the phospholipid to the cholesterol is 6: 1;

the mass ratio of tobramycin to phospholipid is 1: 2;

6. The process for preparing tobramycin liposomes for nebulisation inhalation according to claim 1, comprising the steps of:

1) accurately weighing egg yolk lecithin, cholesterol and vitamin E in the prescribed amount, dissolving the egg yolk lecithin, cholesterol and vitamin E in an organic phase medium, wherein the total amount of the raw materials accounts for 10-40%, and dissolving the raw materials in a water bath at 25-80 ℃ to form an organic phase solution;

4) and (3) removing the organic solvent by reduced pressure evaporation, and then filtering the organic solvent by using a 0.45-micron microporous filter membrane to obtain the tobramycin liposome solution.

7. A preparation method of tobramycin liposome for aerosol inhalation is characterized by comprising the following steps:

1) accurately weighing phospholipid, a stabilizer and an antioxidant in a prescription amount, dissolving the phospholipid, the stabilizer and the antioxidant in an organic phase medium, and performing ultrasonic complete dissolution to obtain an organic phase;

2) accurately weighing a prescription amount of tobramycin soluble aqueous phase medium as an aqueous phase;

3) injecting the water phase into the organic phase, and carrying out water bath ultrasonic treatment for 2-15 min to obtain a stable emulsion;

4) transferring the mixture into a specific evaporation container, carrying out water bath at the temperature of between 25 and 80 ℃ for 100r min < -1 >, and carrying out reduced pressure evaporation to remove the organic solvent to obtain a viscous colloidal solution; and then adding a proper amount of aqueous medium for elution, and after complete elution, filtering the solution by using a 0.45-micron microporous filter membrane to obtain the tobramycin liposome solution.

8. The process for preparing tobramycin liposomes for nebulisation and inhalation according to claim 7, comprising the steps of:

1) accurately weighing egg yolk lecithin, cholesterol and vitamin E in a prescription amount, dissolving in a proper amount of ethanol, and performing ultrasonic complete dissolution to obtain an organic phase;

2) precisely weighing tobramycin with the prescription amount, dissolving the tobramycin in phosphate buffer solution with the pH value of 5.0-7.0, and taking the tobramycin as a water phase;