WO2019041405A1 - 伊班膦酸钠的用途及粉雾剂和制备方法 - Google Patents

伊班膦酸钠的用途及粉雾剂和制备方法 Download PDF

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WO2019041405A1
WO2019041405A1 PCT/CN2017/102755 CN2017102755W WO2019041405A1 WO 2019041405 A1 WO2019041405 A1 WO 2019041405A1 CN 2017102755 W CN2017102755 W CN 2017102755W WO 2019041405 A1 WO2019041405 A1 WO 2019041405A1
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ibandronate
powder
particle size
pulverization
chronic obstructive
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PCT/CN2017/102755
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English (en)
French (fr)
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陈燃
陆泉
栗波
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杭州旦承医药科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A61K31/662—Phosphorus acids or esters thereof having P—C bonds, e.g. foscarnet, trichlorfon
    • A61K31/663—Compounds having two or more phosphorus acid groups or esters thereof, e.g. clodronic acid, pamidronic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/007—Pulmonary tract; Aromatherapy
    • A61K9/0073—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy

Definitions

  • the present invention is in the field of pharmacy and formulation, and relates to ibandronate for inhalation administration, a process for its preparation and use.
  • Ibandronate sodium (IBA), whose chemical name is 1-hydroxy-3-(methylpentylamine)-propane-1,1-bisphosphonate, has the molecular formula C9H22NO7P2 ⁇ Na, and has a molecular weight of 341.21. Commonly used in a molecule of water, the molecular formula is C9H22NNaO7P2 ⁇ H2O, molecular weight of 359.23, is a third-generation bisphosphonate drug bone resorption inhibitor.
  • the US FDA approved the prevention and treatment of postmenopausal osteoporosis in women.
  • the US market name is BONIVA
  • the Chinese name is Bonoli
  • the clinical dose is about 150 mg/tablet/month for oral administration and 3 mg/3 months for intravenous injection.
  • the average absolute oral bioavailability of ibandronate tablets is approximately 0.6%, which inhibits osteoclast activity, bone resorption and degradation by binding to skeletal hydroxyapatite.
  • Ibandronate has many side effects, including upper gastrointestinal disorders, dysphagia, esophagitis, esophageal or gastric ulcers, diarrhea, abdominal pain, nausea, constipation, etc. Others such as hypocalcemia, Mineral metabolism disorders, influenza-like syndrome, headache, dizziness, rash, joint pain, mandibular necrosis, etc.
  • Chronic obstructive pulmonary disease (“slow obstructive pulmonary disease” or “COPD”) is a chronic lung disease characterized by incomplete reversible airflow limitation. It is often clinically characterized by recurrent cough, cough, and difficulty breathing. Symptoms, usually characterized by progressive progression, include most chronic bronchitis and emphysema. As the disease progresses, the airway remodels, eventually developing into irreversible airflow obstruction, or coexisting with asthma. Asthma (full name: bronchial asthma) is a chronic respiratory disease, usually caused by airway inflammation, divided into acute bronchoconstriction, leading to repeated attacks of wheezing, chest tightness, difficulty breathing, and cough.
  • the overall prevalence of COPD in China is 8.2%, with a male prevalence rate of 12.4% and a female prevalence rate of 5.1%.
  • chronic obstructive pulmonary disease will be one of the main causes of human incapacity.
  • Chronic obstructive lung is the second leading cause of death in the world, second only to heart disease, cerebrovascular disease and acute lung infection. 4. And the mortality rate is increasing year by year, and the number of deaths due to chronic obstructive pulmonary disease reached 3 million in 2004.
  • Bronchodilators are the drug of choice for the treatment of chronic obstructive pulmonary disease and asthma.
  • the current Chinese market is estimated to be 10 billion yuan. It is expected to maintain an annual growth rate of approximately 20%.
  • Anticholinergic agents including Tiotropium Bromide, etc.
  • ⁇ 2-receptor agonists including salbutamol, salmeterol, salmeterol, etc.
  • bronchodilators for all age-related chronic obstructive pulmonary disease (2017 version of chronic obstruction) Global Strategy for the Diagnosis, Treatment and Prevention of Pulmonary Diseases, GOLD2017).
  • Inhaled corticosteroids are the most commonly used anti-inflammatory drugs for asthma and chronic obstructive pulmonary disease. Asthma is more responsive to bronchodilators (bronchodilators) and inhaled corticosteroids than chronic obstructive pulmonary disease.
  • bronchodilators bronchodilators
  • inhaled corticosteroids chronic obstructive pulmonary disease.
  • eosinophils are the main airway inflammatory cells of asthma, while in chronic obstructive lungs, the main airway inflammatory cells are neutrophils and macrophages.
  • glucocorticoids are significant for asthma and not for chronic obstructive pulmonary disease.
  • GOLD2017 recommends long-acting bronchodilators and inhaled corticosteroids in patients with severe and very severe chronic obstructive pulmonary disease, but does not advocate inhaled corticosteroids in patients with mild to moderate chronic obstructive pulmonary disease, nor does it advocate any degree of chronic obstructive pulmonary disease in patients.
  • Inhaled glucocorticoid treatment alone because not only the improvement of symptoms is not obvious, but also the risk of inducing pneumonia. Therefore, there is currently no anti-inflammatory drug in the field of chronic obstructive pulmonary therapy that can effectively improve pathological conditions.
  • the root cause of COPD is inflammation.
  • the severity of symptoms in patients is mainly affected by inflammatory pathological factors. It is not always related to the degree of airflow limitation. Inhalation of bronchodilator can relieve airflow obstruction, but it cannot replace antibiotics. The role of inflammatory drugs. At the same time, patients need long-term medication, and the low side effects of drugs are also very important to improve the quality of life of patients. The development of anti-inflammatory drugs with good curative effect and low side effects has fundamentally improved the pathological condition of the lungs of patients, which is an important development direction of slow obstructive pulmonary therapy and has great room for development.
  • ibandronate has no research report for the treatment of respiratory diseases that are not associated with bone. Ibandronate has no research reports on inhaled dosage forms such as powders or atomized droplets.
  • the present invention provides a new therapeutic use and a new dosage form of ibandronate, and directly transports ibandronate to the respiratory tract by means of a powder spray or atomized droplets. Play bronchodilator and anti-inflammatory anti-emphysema effect.
  • the present invention provides a use of ibandronate in the treatment of chronic obstructive pulmonary disease and asthma.
  • the ibandronate is administered by inhalation.
  • the powder mist production process is simple, the drug loading amount is large, and the use is convenient--portable and self-use, and is currently the mainstream inhalation type.
  • Atomized droplet inhalation administration may be a better choice for patients who have difficulty using a powder/dry powder inhaler. This part of the patients includes frail elderly, low inspiratory flow rate, serious illness, or infants.
  • the preparation method of the ibandronate inhalation administration and the realization of the new therapeutic use provided by the present invention include two methods, such as a powder spray and an atomized droplet.
  • the preparation method of the inhalation administration of the ibandronate may be prepared by a pulverization method or a spray drying method.
  • the pulverization processing or the spray drying process only the Ibandronate raw material drug having the purity meets the requirements may be used, or the auxiliary materials such as lactose, leucine, ammonium hydrogencarbonate or the like may be simultaneously added.
  • the particle size distribution, solubility, bulk density, angle of repose, atomization performance and moisture absorption of the powder must be considered in order to achieve curative effect.
  • Characteristics of granules in the respiratory tract 5 ⁇ m ⁇ 20 ⁇ m mainly deposited in the trachea, bronchi and bronchioles, ⁇ 5 ⁇ m can enter the bronchioles and deposit in the alveoli.
  • Conventional use of powder is intended to enter the alveoli and then enter the blood, and the powder from the mouth to the lungs through the trachea, bronchial and other long-term moist respiratory tract, and particles with a particle size of 2.5 ⁇ m or more are easily deposited.
  • the particle size (d90) In the respiratory tract, it can not enter the alveoli, so the particle size (d90) generally needs to be controlled below 5 ⁇ m, and it is often necessary to use excipients with good water solubility, low irritation and good biocompatibility to facilitate drug diffusion into the alveoli, reducing and avoiding Deposition in the respiratory tract. These excipients, such as surfactants, may cause damage to the lungs when used for a long time.
  • excipients such as surfactants
  • the particle size of the powder and atomized droplets is suitable for control in the range of 2.5 ⁇ m ⁇ 10 ⁇ m.
  • a preferred embodiment is d90 ⁇ 7 ⁇ m. More preferably, the particle size is controlled at d90 ⁇ 5 ⁇ m, and the particle size is uniform, and the distribution peak on the particle size distribution map displayed by the particle size analyzer is sharp.
  • the ibandronate powder micropowder micro powder processing can be carried out by a pulverization method, such as using a jet mill, and the ibandronate powder is processed into a fine powder having a particle size of d90 ⁇ 7 ⁇ m by pulverization once or more.
  • the pulverization loading amount is 50 g to 1000 g
  • the pulverization pressure is 0.7 MPa
  • the pulverization time is 2.5 h to 3.5 h
  • the pulverization times are 2 times.
  • the ibandronate powder micropowder micropowder processing can also be carried out by a spray drying method, and the ethanol aqueous solution in which ibandronate is dissolved is processed into a fine powder having a particle size of d90 ⁇ 7 ⁇ m by a spray dryer.
  • the spray drying parameter is such that the concentration of ibandronate is 2.0 g/L, the concentration of ammonium hydrogencarbonate is 5.0 g/L, the concentration of ethanol is 40%, the inlet temperature is 160 ° C, 100% is aspirated, and the feed rate is 15 ml. /min, spray rate 500 L / h, outlet temperature of about 70 ° C.
  • the ibandronate sodium powder is mixed into the capsules alone or in combination with an appropriate amount of other auxiliary materials such as lactose or leucine, and is administered once a day.
  • the preparation method of the ibandronate sodium inhalation administration may also be that the ibandronate sodium is dissolved in a phosphate buffer solution (PBS) or other aqueous solution having a pH of 5.0 to pH 7.0, and the atomized droplets are passed through. The way to proceed.
  • PBS phosphate buffer solution
  • the ibandronate sodium solution for nebulization only the ibandronate raw material having the purity meets the requirements may be used, or the auxiliary materials such as propylene glycol and disodium edetate may be added at the same time.
  • the atomizing device can be a conventional device, such as the Ning YZB1093-2009 drug atomizer produced by Ningbo Haishu Medical Products Factory.
  • sodium ibandronate is dissolved in PBS to obtain an aqueous solution for nebulization of concentration ⁇ 1 mg/ml, and the solution is added to the injection site of the drug atomizer for atomization, and the atomized droplet size is ⁇ 7 ⁇ m.
  • the gas flow rate was ⁇ 0.1 ml/min and the administration time was 1 min.
  • the present invention has the following advantages:
  • the inhaled administration of ibandronate of the present invention has an airway anti-inflammatory and a rapid and efficient diastolic airway function of an existing bronchodilator such as an anticholinergic drug or a ⁇ 2 receptor agonist.
  • Therapeutic effect of pathological improvement such as emphysema.
  • the inhaled administration of ibandronate of the present invention has an anti-inflammatory function of inhaled glucocorticoids, and also has a therapeutic effect against pathological improvement such as emphysema, and is particularly suitable for use in patients with chronic obstructive pulmonary disease.
  • ibandronate of the present invention is mainly deposited in the respiratory tract of the lung to directly exert the effects of promoting bronchiectasis and airway anti-inflammatory and pathological improvement.
  • Intravenous metabolism therefore, low systemic side effects, good safety, suitable for long-term use, and because it is a treatment and prevention drug for osteoporosis, it is expected to become the first choice for life-long use of COPD and asthma patients Good medicine.
  • Figure 1 is a laser particle size analyzer measurement of an IBA-3 sample.
  • Figure 2 is a SEM electron micrograph of an IBA-3 sample.
  • Figure 3 is a measure of airway resistance after treatment with inhaled ibandronate in mice with chronic obstructive lung disease.
  • Figure 4 shows lung dynamic compliance after treatment with inhaled ibandronate in mice with chronic obstructive lung disease
  • Figure 5A is a result of H&E staining of lung tissue in untreated chronic obstructive lung mice.
  • Figure 5B is a graph showing H&E staining of lung tissue in mice with chronic obstructive lung disease treated with ibandronate.
  • Figure 6 shows the changes in protein signal of airway smooth muscle cells treated with ibandronate.
  • Embodiments of the present invention provide sodium ibandronate for administration by inhalation, which may be a powder containing a fine powder of ibandronate or an atomization solution in which an appropriate amount of ibandronate is dissolved. Aqueous solution.
  • auxiliary means any other ingredient which can be added for the purpose of promoting the preparation process or atomization performance in the preparation of the inhaled administration of ibandronate of the present invention, and for the preparation of ibandronate bulk drug
  • the impurities contained in the time are not considered as excipients.
  • the purity of ibandronate in the ibandronate bulk drug used in the embodiment of the present invention is ⁇ 98%.
  • the fine powder or droplet size distribution of the inhaled administration of ibandronate satisfies the following condition: d90 ⁇ 7 ⁇ m.
  • d90 is: the particle size corresponding to the cumulative particle size distribution of a sample reaching 90%, and its physical meaning is that the particle having a particle size smaller than the value accounts for 90%.
  • the inhaled administration of sodium ibandronate is prepared by pulverizing an ibandronate drug substance.
  • the inhaled administration of sodium ibandronate is prepared by spray drying an ibandronate drug substance.
  • the inhaled administration of ibandronate is obtained by dissolving the ibandronate bulk drug in an isotonic aqueous solution of pH 5.0 to pH 7.0 to obtain a mist having a concentration of ⁇ 1 mg/ml.
  • the aqueous solution for inhalation is obtained by atomizing the atomizer to form an atomized droplet.
  • Embodiments of the present invention also provide the use of the inhaled administration of ibandronate of the present invention for the treatment of chronic obstructive pulmonary disease and asthma.
  • the dosage is from 0.5 mg to 5 mg per day, administered in the form of a powder inhalation or inhalation of nebulized droplets.
  • the Ibandronate raw material drug in accordance with the national standard is used as the raw material, and the preparation method is prepared by the pulverization method.
  • a single Ibandronate raw material drug may be used, or an appropriate amount of lactose or other may be incorporated.
  • the pulverization method is one of the common methods for preparing a powder spray, but has been gradually replaced by a spray drying method. The main reason is that compared with the spray drying method, the fine powder prepared by the pulverization method has a large particle size, 2 to 3 ⁇ m is the pulverization limit of the method, and the shape of the fine powder is irregular.
  • the pulverization can also generate static electricity.
  • the drug After the micronized powder, the drug has a high surface free energy, and the powder particles are easily aggregated, resulting in enhanced viscosity of the drug micropowder and poor fluidity, which affects the redispersion of the drug after being released from the micropowder inhaler.
  • the violent comminution of the comminution method may also cause adverse physicochemical properties of the drug.
  • amorphous crystals may be formed on the surface of the fractured crystal.
  • the pulverization method is advantageous in that the fine powder has a large particle size and an irregular shape, and may also be advantageous for reducing the amount of blood absorbed into the alveoli and thereby reducing the side effects on the body;
  • the process is simple, the production cycle is short, and the chance of microbial contamination is low.
  • an auxiliary material such as a solvent, as long as the pulverization pressure and time are properly controlled, the main drug is not easily deteriorated.
  • this powder is designed for local administration of the respiratory tract, while minimizing the absorption into the alveoli, does not require strong fluidity, and ibandronate is a simple molecular drug with a simple physical and chemical properties. It can withstand the processing of the pulverization method.
  • the pulverization conditions were mainly based on the operating conditions of the tiotropium bromide inhalation powder, and the QS100 type jet mill was used for the test.
  • the pulverization pressure is controlled at 0.7 MPa, and the single continuous pulverization time does not exceed 3.5 hours. Under the pulverization conditions, ibandronate did not deteriorate.
  • the fine powder particle size was measured using a Mastersizer 2000 laser particle size analyzer, and the results are shown in Table 1 below.
  • the particle size distribution is related to the number of pulverizations under the pulverization conditions in which the pulverization pressure is controlled at 0.7 MPa and the single continuous pulverization time is not more than 3.5 hours.
  • the particle size distribution fluctuated greatly, and d90>7 ⁇ m failed to achieve the desired powder particle size.
  • the particle size d90 can be controlled to ⁇ 5 ⁇ m, and the results of the two batch processes are very similar, indicating that the pulverization effect remains stable under the condition that the pulverization pressure is controlled at 0.7 MPa, and the pulverization is performed twice, and the particle size d90 can be stably maintained.
  • Figure 1 is a particle size distribution of the IBA-3 sample measured by the MASTERSIZER-2000 laser particle size analyzer. It can be seen that the main peak of the distribution is sharp, but the portion with a particle size of less than 1 ⁇ m has a small secondary peak distribution.
  • Figure 2 is a SEM electron microscopy analysis of the IBA-3 sample.
  • Solubility 10 mg of the IBA-3 sample was weighed and placed in a stoppered test tube containing 2 ml of water, and shaken for 1 minute to observe the dissolution. The powder was completely dissolved and the solution was clear, indicating that the solubility was acceptable.
  • the bulk density is the density obtained by dividing the mass of the powder by the volume V of the container occupied by the powder.
  • the density measured after a certain regular vibration or tapping is called the tap density ⁇ tap .
  • Porosity refers to the ratio of the volume of voids formed in the matrix to the total volume. After the micronization treatment of the drug, the bulk density and porosity of the drug change greatly, which may cause the density difference between the drug and the auxiliary material, which causes difficulty in mixing uniformity. Therefore, micronized drugs should be tested for bulk density and porosity.
  • the volume measured by filling the powder into the container includes the true volume of the powder, the voids in the particles, the voids between the particles, and the like.
  • the shape and size of the measuring container, the filling speed of the material, and the filling method affect the volume of the powder.
  • the density measured is the loosest bulk density without applying any external force
  • the density measured by applying an external force to make the powder in the tightest filling state is called the tightest packing density.
  • the tap density changes with the number of tapping times, and the tap density measured when the final oscillation volume is constant is the tightest packing density.
  • Angle of repose The angle of repose is measured by taking a small funnel with a diameter of about 6 cm and a diameter of about 0.4 cm. It is fixed on the iron frame, and a white paper is placed under the funnel. The lower end of the funnel and the height of the paper are 4-5cm, slowly pour the powder from the top of the funnel into the funnel. When the powder to be leaked is close to the funnel outlet, measure the height of the cone and the diameter of the lower end of the powder, calculate the tangent of the angle of repose, and further calculate the angle of repose. .
  • the critical relative humidity (CRH) determination method is divided into two steps:
  • the ibandronate sodium powder of the present invention can be applied in the form of a capsule.
  • the size of the capsule used can be any type, and the preferred model number is less than 0, usually 4th.
  • Ibandronate capsules are not only easy to industrialize, but also easy to carry.
  • the amount of ibandronate powder in the capsule is constant, ensuring accurate inhalation dose during use, which can effectively prevent patients from taking more or less suction.
  • the micropowder is filled into a capsule in a single dose of 0.5 mg to 5.0 mg, and sealed and stored. Use in a single capsule when using. Good anti-hygroscopic effect. Satisfactory emptying and atomization performance can still be achieved in an environment where the relative humidity reaches 90%.
  • the ibandronate micropowder can be mixed with several times the mass of the carrier lactose (30 ⁇ m ⁇ 100 ⁇ m) and then filled.
  • the atomization effect and the residual amount after use produce a good synergistic effect, in particular, the hygroscopicity of the ibandronate powder aerosol can be reduced.
  • Capsule stability mainly concerned with the effect of water absorption of capsule shell on the performance of the preparation
  • the piloted ibandronate inhalation powder is accurately weighed (W1), placed in a special inhaler (manufactured by Shanghai Tianping Pharmaceutical Factory), the capsule is perforated, and then inhaled.
  • the unit is connected to a 5000ml glass bottle with a switch knob that is in the off position.
  • the vacuum was applied at a flow rate of 60 l/min, and the above knob was turned on, and the powder in the capsule was ejected from the inhaler three times in succession.
  • the powder forms a uniform smoke, no large particles are present after deposition, indicating that the atomization performance is excellent; if most of the powder is atomized, there is only a small amount of particles at the bottom of the bottle, and the atomization performance is moderate. If most of the powder is not atomized, it is deposited in the form of a block at the bottom of the bottle, indicating poor atomization performance.
  • the used capsule shell was taken out from the inhalation device, accurately weighed (W2), and the residual powder on the inner wall of the capsule was wiped off with a small brush, and the weight of the capsule (W3) was weighed.
  • the calculation method of the residual amount of the capsule contents is: [(W2-W3)/(W1-W3)] ⁇ 100%
  • Humidity effect and capsule wall adhesion test Take Ibandronate sodium inhalation powder, accurately weighed, set the relative humidity of 75% at room temperature at 25 °C, take it out after 24 hours, and weigh the capsule again. The powder was then poured out, the change in powder properties was observed, and the residual amount of the powder in the capsule was measured by the above method to understand the adhesion of the capsule wall.
  • the content of the capsule provided by the invention consists of a mixture of ibandronate micropowder or ibandronate micropowder having a particle size of d90 ⁇ 7 ⁇ m and an appropriate amount of adjuvant lactose.
  • the preferred ibandronate micropowder has a particle size d90 ⁇ 5 ⁇ m, and the most preferred fine powder has a particle size controlled at d90 ⁇ 5 ⁇ m, and the distribution peak is sharp.
  • the solubility, bulk density, angle of repose and emptying rate of the micropowder are good, the atomization performance is good, and it is not easy to absorb moisture during processing, storage and use.
  • the powder for treating chronic obstructive pulmonary disease and asthma is inhaled to the center of the airway to produce a predetermined therapeutic effect.
  • a commercially available inhalation powder device for treating chronic obstructive pulmonary disease and asthma can be utilized.
  • the device actually functions to crush the capsule.
  • the device for squeezing the capsule can be used repeatedly.
  • the contents of one capsule can be discarded after the light is absorbed.
  • the ibandronate capsule of the present invention can also be opened by hand, and the fine powder can function by entering the respiratory tract by the action of airflow during breathing. This facilitates self-administration by patients in outdoor or emergency situations.
  • mice Perform airway intubation on the mice, place them in the closed compartment of the instrument, and connect the relevant equipment lines and pipelines.
  • Figure 3 shows the results of airway resistance (sRaw) measurement. It can be seen that with the inhalation of ibandronate, the airway resistance of mice with chronic obstructive lung decreased, when the concentration of ibandronate solution was ⁇ 1.25mg/ml (1.25mg/ml, 2.5mg/ml, 5mg/ml), slow. Airway resistance in lung-blocking mice decreased to 60% when not administered, reflecting that inhaled administration of ibandronate promoted airway relaxation in mice with chronic obstructive pulmonary disease.
  • sRaw airway resistance
  • FIG. 4 Simultaneous lung dynamic compliance measurements ( Figure 4), when the concentration of Ibandronate solution ⁇ 2.5 mg / ml (2.5 mg / ml, 5 mg / ml), lung dynamic compliance in mice with chronic obstructive pulmonary disease (Cdyn Increased by about 90%, reflecting that inhaled administration of ibandronate not only improved airway resistance, but also significantly improved lung tissue elasticity in mice with chronic obstructive lung disease, which significantly improved emphysema.
  • Figure 5A is a result of H&E staining of lung tissue in untreated chronic obstructive lung mice.
  • Figure 5B is a graph showing H&E staining of lung tissue in mice with chronic obstructive lung disease treated with ibandronate.
  • COPD untreated chronic obstructive lung mice
  • Ibandronate chronic obstructive lung mice treated with ibandronate. It can be seen from the staining results that the alveolar wall was significantly thickened after treatment with ibandronate, indicating that the improvement of alveolar elastic function, that is, the emphysema condition, was improved.
  • ibandronate The following is the treatment of airway smooth muscle cells with ibandronate, followed by Western Blot (WB) to determine the changes in airway smooth muscle contraction/diastolic regulation of upstream signaling protein molecules.
  • WB Western Blot
  • ibandronate when the concentration of ibandronate (IBAedronate) was increased to 12.5 ⁇ M or higher, MLC phosphorylation was significantly inhibited, and the amount of protein kinase MLCK and the upstream ROCK upstream thereof were significantly decreased. It has been shown that ibandronate promotes relaxation of airway smooth muscle by inhibiting the ROCK-MLCK-MLC phosphorylation signaling pathway of airway smooth muscle contraction.
  • ibandronate raw material drug 1000 g was taken and loaded into a QS100 type pulverizer.
  • the pulverization conditions were a pulverization pressure of 0.7 MPa, and the mixture was pulverized once for 2 hours to obtain a fine powder of ibandronate.
  • the micro-powder has no adhesion, no clumps, and good dispersion.
  • the fine powder sample was taken, and the fine powder powder was subjected to particle size measurement using a Malvern Instruments Ltd. laser particle size analyzer in the United Kingdom. As a result, the sample had a particle size d90 of 5.55 ⁇ m, a d50 of 2.73 ⁇ m, and a d10 of 0.90 ⁇ m.
  • the raw material of ibandronate before pulverization and the powder after pulverization were determined by molybdenum blue colorimetry.
  • the method is to take 8 mg of the raw material medicine or powder, and prepare a solution having a concentration of ibandronate equivalent to 0.08 mg/ml, and measure the absorbance of the corresponding wavelength according to the analysis method.
  • the powder absorbance after pulverization (0.258 ⁇ 0.035) was comparable to that of the raw material before pulverization (0.266 ⁇ 0.042), and the content of ibandronate was not significantly changed.
  • ibandronate raw material drug 1000 g was taken and loaded into a QS100 type pulverizer.
  • the pulverization conditions were a crushing pressure of 0.7 MPa, and the mixture was pulverized twice.
  • the first pulverization time was 1.5 hours, and the second pulverization time was 2 hours.
  • a fine powder of ibandronate was obtained.
  • the micro-powder has no adhesion, no clumps, and good dispersion.
  • the fine powder sample was taken, and the fine powder powder was subjected to particle size measurement using a Malvern Instruments Ltd. laser particle size analyzer in the United Kingdom. As a result, the sample had a particle size d90 of 2.73 ⁇ m, a d50 of 1.61 ⁇ m, and a d10 of 0.58 ⁇ m.
  • the raw material of ibandronate before pulverization and the powder after pulverization were determined by molybdenum blue colorimetry.
  • the method is to take 8 mg of the raw material medicine or powder, and prepare a solution having a concentration of ibandronate equivalent to 0.08 mg/ml, and measure the absorbance of the corresponding wavelength according to the analysis method.
  • the powder absorbance after pulverization (0.249 ⁇ 0.026) was comparable to the bulk drug before pulverization (0.266 ⁇ 0.042), and the content of ibandronate was not significantly changed.
  • ibandronate 0.5 g was dissolved in 200 ml of water, 2.5 g of ammonium hydrogencarbonate was dissolved in 100 ml of water, two solutions were mixed, and 200 ml of ethanol was added to form a 40% aqueous solution of ethanol for spraying.
  • the ammonium bicarbonate is completely degraded and volatilized during the spray drying process, so that small pores are formed in the finally formed solid powder of ibandronate.
  • the B-290 spray dryer is equipped with a 0.7 mm 2-liquid nozzle.
  • the conditions were: inlet temperature of 160 ° C, 100% aspiration, feed rate of 15 ml / min, spray rate of 500 L / h.
  • the dry powder sample is stored in the desiccator at room temperature.
  • the outlet temperature is about 70 °C.
  • ibandronate micropowder 5 mg was weighed into a balance and filled into a No. 4 capsule. Load 100 capsules continuously and record the actual charge of the capsule, which is accurate to one decimal place.
  • the liquids in the first-stage distribution bottle and the second-distribution bottle were respectively taken up to 1 ml, and the OD710 was also determined by molybdenum blue colorimetric method.
  • the amount of powder deposited in the distribution bottle was also obtained according to the standard curve and the liquid volume (10 ml) in the distribution bottle. Divided by the number of capsules (30) and the average charge (5 mg), the first-stage distribution bottle deposition rate was 62.4%, and the secondary distribution bottle deposition rate was 20.2%.
  • the liquids in the first-stage distribution bottle and the second-distribution bottle were respectively taken up to 1 ml, and the OD710 was also determined by molybdenum blue colorimetric method.
  • the amount of powder deposited in the distribution bottle was also obtained according to the standard curve and the liquid volume (10 ml) in the distribution bottle. Divided by the number of capsules (30) and the average charge (5 mg), the first-stage distribution bottle deposition rate was 42.6%, and the secondary distribution bottle deposition rate was 40.8%.

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Abstract

伊班膦酸钠治疗慢性阻塞性肺疾病和/或哮喘的用途以及包含伊班膦酸钠的粉雾剂或雾化液滴；所述粉雾剂中伊班膦酸钠微粉的粒度为2.5μm-10μm，粒度分布d90≤7μm；所述雾化液滴为伊班膦酸钠溶于PBS溶液中，雾化液滴颗粒大小≤7μm。

Description

伊班膦酸钠的用途及粉雾剂和制备方法
本发明属于药物学和制剂学领域，涉及用于吸入给药的伊班膦酸钠、其制备方法和用途。
伊班膦酸钠（Ibandronate sodium，IBA）, 其化学名称为1-羟基-3-(甲基戊基胺)-丙烷-1,1-双膦酸钠，分子式为C9H22NO7P2·Na，分子量为341.21；常用含1个分子水的，分子式为C9H22NNaO7P2·H2O，分子量为359.23，是一种第三代的双膦酸盐药物骨吸收抑制剂。2003年美国FDA批准用于妇女停经后骨质疏松症的防治，在美国市场的商品名为BONIVA，中文名是邦罗力，有口服片剂和静脉注射液2种剂型。临床剂量是口服约150mg/片/月，静脉注射是3mg/3个月。伊班膦酸钠片的平均绝对口服生物利用度大约为0.6%，通过与骨骼羟基磷灰石结合而抑制破骨活性、骨质的再吸收及降解。伊班膦酸钠的副作用较多，包括可引起上消化道紊乱，表现为吞咽困难、食道炎、食道或胃溃疡，还可以引起腹泻、腹痛、恶心、便秘等，其他如低钙血症、矿物质代谢障碍、流感样综合征、头痛、头晕、皮疹、关节痛、下颌骨坏死等。
慢性阻塞性肺疾病（简称“慢阻肺”或“COPD”），是以不完全可逆的气流受限为特征的慢性肺部疾病，临床上常表现为反复发作的咳嗽、咳痰、呼吸困难等症状，通常呈现出进行性进展的特点，包括了绝大部分慢性支气管炎和肺气肿。随着疾病发展，导致气道重构，最终发展为不可逆性气流阻塞，或与哮喘重叠共存发生。哮喘（全称：支气管哮喘)是一种慢性呼吸道疾患,一般是由气道炎症引起的气流受限分为急性支气管狭窄，导致喘鸣、胸闷、呼吸困难、咳嗽的反复发作。慢阻肺和哮喘在全球范围内严重威胁着公众的健康。目前全球已有2.1亿慢阻肺患者和约3亿哮喘患者。慢阻肺和哮喘患者在中国分别达到4000万人和3000万人。由钟南山院士主持的一项流行病学调查结果显示，目前中国慢阻肺的总体患病率为8.2%，其中男性患病率为12.4%，女性患病率为5.1%。据世界卫生组织估计，在社会环境和治疗延误的影响下，慢阻肺将是人类丧失工作能力的主要病因之一。慢阻肺在全球疾病死亡原因中，仅次于心脏病、脑血管病和急性肺部感染，与艾滋病一起并列第 4 位。且死亡率正在逐年增加，因慢阻肺死亡的人数在2004年就达到300万。
慢阻肺和哮喘由于需要终生每天用药和定期备药，素有医药费用高的特点。支气管舒张剂是慢阻肺和哮喘治疗的首选药物。目前中国市场估计是100亿元。预计保持约20%的年增长率。抗胆碱能制剂（包括噻托溴铵Tiotropium Bromide等）和β2-受体激动剂（包括沙丁胺醇Salbutamol、沙美特罗Salmeterol等）是目前所有年龄慢阻肺的首选支气管舒张剂（2017年版慢性阻塞性肺疾病诊断、治疗和预防全球策略，GOLD2017）。与增加某一种支气管舒张剂的剂量相比，联合使用不同的支气管舒张剂可以提高药效和减少相应的副作用。吸入糖皮质激素是哮喘和慢阻肺最常用的抗炎药物。哮喘比慢阻肺对支气管舒张剂（支气管扩张剂）和吸入糖皮质激素的反应更为明显。哮喘和慢阻肺的炎症机理上存在本质差异：嗜酸性粒细胞是哮喘主要的气道炎症细胞，而在慢阻肺则主要的气道炎症细胞是中性粒细胞和巨噬细胞，这是糖皮质激素的抗炎药效对于哮喘明显而对于慢阻肺则不明显的原因。GOLD2017推荐重度和极重度慢阻肺患者联合使用长效支气管舒张剂和吸入糖皮质激素，但不主张轻度和中度慢阻肺患者吸入糖皮质激素，也不主张任何程度的慢阻肺患者单独吸入糖皮质激素治疗，原因是不仅对症状改善不明显，反而有诱发肺炎的风险。因此，慢阻肺治疗领域目前缺乏能有效改善病理状况的抗炎药物。慢阻肺的根源是炎症，患者症状的严重程度主要还是受到炎症病理因素的影响，并不一定总是和气流受限的程度相关，吸入支气管舒张剂能缓解气流阻塞症状，但并不能取代抗炎药物的作用。同时，患者需要长期用药，药物的低副作用对于改善患者生活质量也非常重要。发展疗效好、副作用低的抗炎药物，从根本上改善患者肺部的病理状况，是慢阻肺治疗的重要发展方向，具有很大的发展空间。
目前伊班膦酸钠没有用于与骨质不相关的呼吸系统疾病治疗应用的研究报道，伊班膦酸钠也没有吸入剂型如粉雾剂或者雾化液滴的研究报道。
本发明为了克服现有技术的至少一个不足，提供伊班膦酸钠新的治疗用途及新的剂型，通过粉雾剂或雾化液滴的方式，将伊班膦酸钠直接输送沉积在呼吸道发挥支气管舒张和抗炎抗肺气肿功效。
为了实现上述目的，本发明的发明人做了大量研究和实践工作，具体的本发明采用了以下技术方案：
本发明提供了一种伊班膦酸钠在慢性阻塞性肺疾病和哮喘治疗药物中的用途。
进一步的，所述伊班膦酸钠采用吸入给药。
肺部吸入给药的剂型中，粉雾剂的生产工艺简单，载药量大，使用方便--可便携自行使用，是目前主流的吸入剂型。雾化液滴吸入给药对于使用粉雾剂/干粉吸入器存在困难的患者则可能是更佳选择。这一部分患者包括年老体弱、吸气流速较低，疾病病情较重，或婴幼儿等。为此，本发明提供的伊班膦酸钠吸入给药的制备方法和新的治疗用途的实现途径包括粉雾剂和雾化液滴等2种方式。
所述的伊班膦酸钠吸入给药的制备方法，可以是通过粉碎法也可以是通过喷雾干燥法制备成粉雾剂。粉碎加工或喷雾干燥加工过程中，可以只使用纯度符合要求的伊班膦酸钠原料药，也可以同时加入辅料，如乳糖、亮氨酸、碳酸氢铵等。根据粉雾剂的设计用途，药粉的粒度分布、溶解性、堆密度、休止角、雾化性能、吸湿变化是达到疗效所必须考虑的。颗粒在呼吸道沉积特点：5μm~20μm主要沉积在气管、支气管和细小支气管，≤5μm可以进入细小支气管及在肺泡沉积。常规使用的粉雾剂的给药目标是进入到肺泡然后进入血液，而药粉从口腔到肺部要经过气管、支气管等相当长一段潮湿的呼吸道，而粒度在2.5μm以上的颗粒很容易沉积在呼吸道中而不能进入肺泡，因此粒度（d90）一般需控制在5μm以下，并且经常需要选用水溶性好、刺激性小、生物相容性好的辅料，以利于药物扩散进入肺泡，减少和避免在呼吸道中的沉积。这些辅料如表面活性剂等，长期使用，可能会对肺部产生损害。我们考虑到慢阻肺和哮喘的主要症状发生在支气管、细小支气管等小气道及肺泡区域，粒度适中且覆盖一定粒度范围（2.5μm ~10μm）的药粉可以沉积在支气管、细小支气管及肺泡直接发挥功效，因此，粉雾剂和雾化液滴的粒度比较适合控制在2.5μm ~10μm这个范围。优选方案是d90≤7μm。更优选的方案，粒度控制在d90≤5μm，且粒径大小均匀，粒度仪显示的粒度分布图上分布峰尖锐。
所述的伊班膦酸钠粉雾剂微粉加工可以采用粉碎法，如采用气流粉碎机，通过1次或1次以上的粉碎，将伊班膦酸钠药粉加工成粒度d90≤7μm的微粉。优选的，粉碎上样量为50g~1000g，粉碎压力为0.7Mpa，粉碎时间为2.5h~3.5h，粉碎次数为2次。
所述的伊班膦酸钠粉雾剂微粉加工也可以采用喷雾干燥法，将溶解有伊班膦酸钠的乙醇水溶液通过喷雾干燥机加工成粒度d90≤7μm的微粉。优选的，喷雾干燥参数为将伊班膦酸钠浓度为2.0g/L，碳酸氢铵浓度为5.0g/L，乙醇浓度为40%，进口温度为160℃，100%吸出，进料速率15ml/min，喷雾速率500L/h，出口温度约70℃。
进一步，将伊班膦酸钠微粉以0.5mg~5mg的装药量单独或与适量的乳糖或亮氨酸等其它辅料混合装入胶囊，每天用药一次。
所述的伊班膦酸钠吸入给药的制备方法，也可以是将伊班膦酸钠溶解于磷酸盐缓冲液（PBS）或者其它pH5.0~pH7.0的水溶液，通过雾化液滴的方式进行。用于雾化吸入的伊班膦酸钠溶液制备过程中，可以只使用纯度符合要求的伊班膦酸钠原料药，也可以同时加入辅料，如丙二醇、依地酸二钠等。雾化装置可以采用常规装置，如宁波海曙医疗用品厂生产的宁因YZB1093-2009药物雾化器。优选的，将伊班膦酸钠溶于PBS，得到浓度≥1mg/ml的雾化吸入用水溶液，将该溶液加入药物雾化器进样处，进行雾化，雾化液滴颗粒大小≤7μm，气体流速~0.1ml/min，给药时间1min。
与现有技术相比，本发明具有以下优势：
本发明的吸入给药的伊班膦酸钠，在具备抗胆碱能类药物、β2受体激动剂等现有支气管舒张剂的快速高效舒张气道功能的同时，还具有气道抗炎和抗肺气肿等病理改善的治疗作用。
本发明的吸入给药的伊班膦酸钠，在具备吸入糖皮质激素抗炎功能的同时，还具有抗肺气肿等病理改善的治疗作用，尤其适合慢阻肺患者使用。
本发明的吸入给药的伊班膦酸钠，主要沉积在肺呼吸道中直接发挥促支气管扩张和气道抗炎及病理改善功效。入血代谢迅速，因此，系统性副作用低，安全性好，适合长期使用，而且因为本身是一种骨质疏松治疗和预防药物，所以有望发展成为慢阻肺和哮喘患者终生使用的首选必备良药。
图1是IBA-3样品的激光粒度仪测定图。
图2是IBA-3样品的SEM电镜图。
图3是慢阻肺小鼠采用吸入伊班膦酸钠治疗后气道阻力测定。
图4是慢阻肺小鼠采用吸入伊班膦酸钠治疗后肺动态顺应性测定
图5A是未治疗的慢阻肺小鼠的肺组织H&E染色结果。
图5B是慢阻肺小鼠经伊班膦酸钠治疗后肺组织H&E染色结果。
图6是气道平滑肌细胞经伊班膦酸钠处理后蛋白质信号变化情况。
具体实施方式
为让本发明的上述描述和其它目的、特征和优点能更明显易懂，下文特举较佳实施例，并配合附图，作详细说明如下。
本发明实施方案提供了用于吸入给药的伊班膦酸钠，其剂型可以是成份为伊班膦酸钠微粉的粉雾剂，也可以是溶解有适量伊班膦酸钠的雾化用的水溶液。
应理解，上述吸入给药的伊班膦酸钠可以包含有辅料。辅料的含义是指在制备本发明的吸入给药的伊班膦酸钠时，可以添加有利于促进制备过程或雾化性能等目的的任何其它的成分，而对于制备伊班膦酸钠原料药时含有的杂质不被认为是辅料。
优选地，本发明实施方案中使用的伊班膦酸钠原料药中伊班膦酸钠的纯度≥98%。
根据本发明优选实施方案，该吸入给药的伊班膦酸钠的微粉或液滴粒度分布满足以下条件：d90≤7μm。本说明书中d90的含义为：一个样品的累计粒度分布数达到90%时所对应的粒度，它的物理意义是粒度小于该数值的颗粒占90%。
根据本发明的一个实施方案，该吸入给药的伊班膦酸钠，通过将伊班膦酸钠原料药粉碎制备得到。根据另一实施方案，该吸入给药的伊班膦酸钠，通过将伊班膦酸钠原料药喷雾干燥制备得到。根据第三个实施方案，该吸入给药的伊班膦酸钠，通过将伊班膦酸钠原料药溶解于pH5.0-pH7.0的等渗水溶液中，得到浓度≥1mg/ml的雾化吸入用水溶液，经雾化器雾化操作形成雾化液滴而得到。
本发明实施方案还提供了本发明的吸入给药的伊班膦酸钠用于治疗慢性阻塞性肺疾病和哮喘的用途。
根据优选实施方案，用于治疗慢性阻塞性肺疾病和哮喘时，用药量为0.5mg~5mg每天，以粉雾剂吸入的形式或雾化液滴吸入的形式给药。
本发明的吸入给药的伊班膦酸钠的粉雾剂的制备方法之一如下：
采用符合国家标准的伊班膦酸钠原料药为原料，用粉碎法进行生产制备，在微粉的生产制备过程中可以采用单一的伊班膦酸钠原料药，也可以掺入适量的乳糖或其它辅料。粉碎法是制备粉雾剂的常用方法之一，但已经渐渐被喷雾干燥法所替代。主要原因是与喷雾干燥法相比，粉碎法制备得到的微粉粒度较大，2～3μm是该法的粉碎极限，而且微粉外形不规则。粉碎作用还可产生静电，药物经微粉化后，具有较高的表面自由能，粉粒易聚集成团，导致药物微粉粘性增强，流动性差，影响药物从微粉吸入器中释放后的再分散。粉碎法剧烈的粉碎作用还可能会使药物发生不利的物理化学性质的改变。特别是在断裂的晶体表面可能产生不定型化。然而，发明人研究认为，对于呼吸道局部用药，粉碎法的有利之处在于：微粉粒度较大和外形不规则，也可能有利于减少药物进入肺泡吸收入血量从而有利于减少对于机体的副作用；制备过程简单，生产周期较短，微生物污染的机会低；另外，因为不需要使用溶剂等辅料，只要粉碎压力和时间控制得当，则主药不容易发生变质等。我们考虑到本粉雾剂设计目的是进行呼吸道局部用药，同时尽量减少进入肺泡吸收，不需要很强的流动性，而且伊班膦酸钠是结构简单的小分子药物，物理化学性质比较稳定，可以耐受粉碎法的加工过程。粉碎条件主要参考噻托溴铵吸入粉雾剂的操作条件，采用了QS100型气流粉碎机进行试验。粉碎压力控制在0.7MPa，单次连续粉碎时间不超过3.5小时。在该粉碎条件下，伊班膦酸钠不发生变质。采用Mastersizer2000激光粒度测定仪进行微粉粒度测定，结果如下表1。
样品编号 粉碎设备 上样量（g） 粉碎时间（h） 粉碎压力(Mpa) 粉碎次数 D90(μm) D50(μm) D10(μm)
IBA-1 QS100 1000 1.5 0.7 1 5.55 2.73 0.90
IBA-2 QS100 1000 2.0 0.7 1 4.74 2.42 0.84
IBA-3 QS100 1000 3.5 0.7 2 2.73 1.61 0.58
IBA-4 QS100 1000 3.5 0.7 2 2.92 1.63 0.73
由上表1可见，在粉碎压力控制在0.7Mpa，单次连续粉碎时间不超过3.5小时的粉碎条件下，粒度分布与粉碎次数相关。粉碎1次，粒度分布波动较大，d90＞7μm，没能达到理想的粉雾剂粒度。粉碎2次，则可以把粒度d90控制在＜5μm，且2批加工的结果非常相似，表明在粉碎压力控制在0.7MPa 的条件下，粉碎效果保持稳定，粉碎2次，能够稳定地保持粒度d90控制在＜5μm，且分布峰尖锐规则，分布情况比较理想。图1是IBA-3样品的MASTERSIZER-2000激光粒度仪测定的粒度分布图，可见分布主峰尖锐，但粒度小于1μm的部分有一个小的次级峰分布。图2是IBA-3样品的SEM电镜分析结果。
我们对IBA-3样品进行了溶解度、茚三酮法鉴定、钼蓝比色法测定、微生物污染情况及各种物理化学性质测定。
2.1 溶解性：称取IBA-3样品10mg，放入装有2ml水的带塞试管中，振荡1分钟，观察溶解情况。粉末能够完全溶解且溶液澄清，显示溶解性为合格。
性质鉴定：分别取原料药和IBA-3样品各10mg，加水2ml溶解后，再加0.2%茚三酮试液1ml，混合，加热煮沸10分钟，均显紫红色。
2.2 钼蓝比色法测定：
称取IBA-3样品10mg，加水适量溶解，加水分次移入100ml量瓶中，置40℃水浴中加热20分钟，并不断振摇，放冷至室温，加水稀释至刻度，作为供试品溶液；另外称取伊班膦酸钠原料药10mg，置100ml量瓶中，加水适量溶解，并用水稀释至刻度，摇匀，作为对照品溶液。精密量取供试品溶液与对照品溶液各5ml，分别置25ml量瓶中，各加过硫酸铵溶液（1%）8ml，置水浴中加热20分钟，放冷至室温，加钼酸铵溶液（取钼酸铵7.5g，加水100ml溶解，加5mol/L硫酸溶液100ml，混匀）2.0ml，摇匀，放置15分钟后，加对甲氨基酚硫酸盐溶液（取对甲氨基酚硫酸盐0.5g，加15％亚硫酸氢钠溶液195ml，加20％亚硫酸钠溶液5ml，摇匀）2ml，摇匀，放置15分钟后，加34％醋酸钠溶液5ml，加水至刻度，摇匀。在710nm的波长处分别测定吸光度，结果2个供试品溶液与对照品溶液吸光度相当，显示IBA-3样品不存在主药降解。
2.4 微生物检验：
称取IBA-3样品3mg，溶于10ml纯化水中。按《中华人民共和国药典》 2010年第二部附录XI J 微生物限度检验法进行检验。检验结果细菌菌落数≤2个/ml；霉菌、酵母菌菌落数=0个/ml。检测结果为合格。
2.5 堆密度、休止角、孔隙率、临界相对湿度：
堆密度、孔隙率：堆密度是指粉体质量除以该粉体所占容器的体积V求得的密度。填充粉体时，经一定规律振动或轻敲后测得的密度称振实密度（tap density）ρtap。孔隙率是指基体中形成的空隙体积占总体积的比率。药物进行微粉化处理后，其堆密度、孔隙率均发生较大的变化，可能造成药物与辅料的密度差，造成混合均匀性上的困难。所以微粉化的药物应该进行堆密度和孔隙率测定。将粉体装入容器中所测得的体积包括粉体真体积、粒子内空隙、粒子间空隙等，因此测量容器的形状、大小、物料的装填速度及装填方式等影响粉体体积。将粉体装填于测量容器时不施加任何外力所测得密度为最松堆密度，施加外力而使粉体处于最紧充填状态下所测得密度叫最紧堆密度。振实密度随振荡（tapping）次数而发生变化，最终振荡体积不变时测得的振实密度即为最紧堆密度。
将一定量的粉体样品填装于改制的5ml量筒，读取初始体积V初,轻敲并观察量筒中粉体的体积变化，直到体积无明显变化为止（一般要敲击2500次），记录下终体积V终。加样前后的质量差值即为所加入粉体的质量w。
堆密度pb=w/V初，振实密度ptap=w/V终，孔隙率=1-堆密度/振实密度
休止角：休止角的测量方法为，取口径约6cm，管径(内径)约0.4cm的小漏斗一个，固定于铁架台上，漏斗下方铺一张白纸，漏斗的下端与纸的高度为4-5cm，将药粉从漏斗上方缓慢倒入漏斗，待漏下的药粉接近漏斗出口时，测量该药粉圆锥体的高度和下端直径，据此计算出休止角的正切，并进一步计算出休止角。
临界相对湿度的测定。药物在进行微粉化处理后，由于比表面积的增大，吸湿性可能明显发生变化，而水分又是粉雾剂严格控制的检查项目，所以应该测定微粉化药物的临界相对湿度（Critical Relative Humidity，CRH）。
临界相对湿度(CRH)测定方法分两步：
第一、制作吸湿曲线：
称取药物2mg，平铺于干燥至恒重的扁形称量瓶中（厚约2mm），干燥至恒重，精密称定，打开瓶盖，置盛有浓硫酸溶液或其他盐溶液（相对湿度75%）的玻璃干燥器中，于25℃的恒温箱中保存，分别于2、4、8、12、24……或其他时间段取出称量瓶，精密称定，计算吸湿百分率，以吸湿率对时间作图即得到吸湿曲线，找到吸湿平衡时对应的时间T天（或小时）。
第二、CRH的测定：
称取药物2mg 6份，同上操作，置盛有不同浓度的浓硫酸溶液或其他盐溶液的玻璃干燥器中，于25摄氏度的恒温箱中保存T天，取出称量瓶，精密称定，计算吸湿百分率，以吸湿量对相对湿度作图的吸湿平衡曲线，在拐点作两切线，切线焦点对应的相对湿度，即为CRH。
IBA-3样品实验测定结果总结于表2。
本发明所述伊班膦酸钠粉雾剂可以胶囊的形式应用。所用的胶囊的大小可以是任何型号的，优选的型号为小于0号，通常选4号。伊班膦酸钠胶囊不仅能方便地进行工业化生产，而且便于携带。胶囊中伊班膦酸钠粉末的量是一定的，确保使用时吸入剂量的准确，可有效地防止患者多吸或少吸。将微粉按0.5mg~5.0mg的单剂量装入胶囊中，密封保存。使用时按单胶囊取用。抗吸湿效果好。在相对湿度达到90%的环境中仍然能达到满意的排空率和雾化性能。为了便于工业大量生产自动灌装的需要，可以将伊班膦酸钠微粉与若干倍质量的载体乳糖（30μm~100μm）混匀后进行灌装。雾化效果及使用后的残留量产生良好的协同作用，尤其是可以减少伊班膦酸钠粉雾剂的吸湿性。
3.1 胶囊的含量均匀性测定。方法：抽样30个胶囊，天平称量，并采用钼蓝比色法确定胶囊中伊班膦酸钠的含量。
3.2 胶囊的排空率测定。方法：按2010版中国药典（二部）附录IL方法进行。排空率要求≥90%
胶囊稳定性，主要关注胶囊囊壳吸水对制剂性能的影响
方法：采取加速试验。取胶囊3批（30个/批），置于恒温恒湿箱（40±2℃，75±5%相对湿度）中保存，分别于0、1、2、3月末取样，考察外观、排空率、沉积率、含量。
3.3 粉末残留量和雾化性能：将试制的伊班膦酸钠吸入粉雾剂精密称重(W1)，放入专用吸入器（上海天平制药厂生产）中，将胶囊打孔，再将吸入器与5000ml玻璃瓶相连，连接处配备开关旋钮，此旋钮始处于关闭位置。以60l/min气流量抽真空，打开上述旋钮，胶囊中的药粉即从吸入器中喷出，连续三次。若粉末形成均匀烟雾，沉积后无大的颗粒存在，说明雾化性能优良；若大部分粉末得到雾化，瓶底仅有少量颗粒，雾化性能中等。若大部分粉末未得到雾化，而成块状在瓶底沉积，说明雾化性能差。将用过的胶囊壳从吸入装置中取出，精密称重(W2)，以小毛刷将胶囊内壁的残余粉末拭净，再称空胶囊重量(W3)。胶囊内容物粉末残留量的计算方法为：[(W2-W3)/(W1-W3)]×100％
湿度影响和胶囊壁沾附试验：取伊班膦酸钠吸入粉雾剂，精密称定，25℃室温下置相对湿度75％环境中，24小时后取出，再次称量胶囊重量。然后将粉末倒出，观察粉末性状的变化，并按上法测定胶囊中粉末的残留量以了解胶囊壁沾附情况。
下表是筛选伊班膦酸钠粉雾剂配方的实验情况：
处方1 处方2 处方3 处方4 处方5
伊班膦酸钠微粉 5mg 5mg 2mg 1mg 0.5mg
乳糖 - 15mg 18mg 19mg 19.5mg
溶解性 合格 合格 合格 合格 合格
堆密度 0.50±0.03 1.25±0.15 1.29±0.20 1.13±0.10 1.23±0.12
休止角 44.5±0.7 42.8±0.8 43.7±0.5 43.7±0.9 43.5±0.9
排空率 90.7% 94.5% 94.7% 94.2% 94.8%
囊壳粉末残留量 9.3% 5.5% 5.3% 5.8% 5.2%
胶囊剂在75％相对湿度下的变化情况 +3.5% +3.4% +3.5% +3.8% +3.7%
雾化性能 良好 良好 良好 良好 良好
一级分布瓶沉积率 62.4% 42.6% 42.8% 41.6% 41.5%
二级分布瓶沉积率 20.2% 40.8% 40.2% 41.5% 41.9%
以上由IBA-3混合组成的胶囊剂的性质差别不大。采用乳糖用作辅料能够增进二级分布瓶沉积率，说明乳糖能够促进伊班膦酸钠微粉流动性。
本发明所提供的胶囊剂的内容物由粒度d90≤7μm的伊班膦酸钠微粉或伊班膦酸钠微粉与适量的辅料乳糖混合组成。优选的伊班膦酸钠微粉的粒度d90≤5μm，最优选的微粉的粒度控制在d90≤5μm，且分布峰尖锐。微粉的溶解性、堆密度、休止角、排空率都较好，雾化性能良好，在加工、贮存、使用过程中不易吸湿。一般治疗慢阻肺和哮喘的药粉吸入到气道较中心处即可产生预定疗效。因为按本发明制备的伊班膦酸钠粉雾剂的雾化效果良好，所以可以利用市售的治疗慢阻肺和哮喘的吸入粉末装置。例如使用上海天平制药厂的吸入器。该装置实际上起的是将胶囊挤破的作用。所述挤破胶囊的装置可以反复使用。一颗胶囊内容物吸光后即可将空壳丢弃，下一次使用时，向该装置内再填入一颗胶囊。本发明的伊班膦酸钠胶囊剂也可以用手打开，其中的微粉可借助呼吸时的气流作用进入呼吸道而发挥作用。这有利于室外或紧急情况下患者自行给药。
下面是采用吸入给药的伊班膦酸钠对慢阻肺小鼠进行治疗的肺功能测定结果。
慢阻肺小鼠建模采用烟熏结合弹性蛋白酶灌注构建。建模大约 7天后经肺功能和病理测定，确认已建立稳定的慢阻肺症状。使用FinePointe RC 气道阻力与肺顺应性检测系统（Buxco公司）进行治疗反应测定。步骤如下：
1.检查仪器密封性，调整基线。
2.对小鼠实行气道插管，置入仪器封闭舱中，连接相关设备线路和管道。
3.测定肺功能参数，根据以下时间段，使用PBS和不同浓度的药物（浓度由低到高），进行相关数据采集：
A:适应阶段5min
B:取20μlPBS或药物加入仪器放样处，进行雾化1min
C: 对PBS或药物雾化反应的数据测定5min
D:恢复阶段3min后，进行下一组药物浓度测定反应
4.导出数据为excel表格，分析数据。
图3为气道阻力（sRaw）测定结果。可见随着吸入伊班膦酸钠，慢阻肺小鼠气道阻力下降，当伊班膦酸钠溶液浓度≥1.25mg/ml（1.25mg/ml、2.5mg/ml、5mg/ml），慢阻肺小鼠气道阻力下降到未用药时的60%，反映伊班膦酸钠的吸入给药促进了慢阻肺小鼠气道舒张。同时进行的肺动态顺应性测定显示（图4），当当伊班膦酸钠溶液浓度≥2.5mg/ml（2.5mg/ml、5mg/ml），慢阻肺小鼠的肺动态顺应性（Cdyn）上升了约90%，反映伊班膦酸钠的吸入给药不仅改善了气道阻力状况，同时显著改善了慢阻肺小鼠肺组织弹性，也即显著改善了其肺气肿状况。图5A是未治疗的慢阻肺小鼠的肺组织H&E染色结果。图5B是慢阻肺小鼠经伊班膦酸钠治疗后肺组织H&E染色结果。其中COPD：未经治疗的慢阻肺小鼠，Ibandronate：经伊班膦酸钠治疗后的慢阻肺小鼠。从染色结果可以看出经伊班膦酸钠治疗后，肺泡壁明显增厚，显示肺泡弹性功能的改善也即肺气肿状况得到了改善。
下面是采用伊班膦酸钠处理气道平滑肌细胞，然后采用Western Blot (WB)测定气道平滑肌收缩/舒张调控上游信号蛋白质分子的变化情况。结果如图6显示，当伊班膦酸钠（IBAedronate）浓度提高到12.5μM或更高，MLC磷酸化受到显著抑制，同时，其上游的蛋白激酶MLCK和更上游的ROCK的量都显著下降，显示伊班膦酸钠通过抑制气道平滑肌收缩的ROCK-MLCK-MLC磷酸化信号通路而促进气道平滑肌的舒张。
实施例
实施例1
一步粉碎法
取伊班膦酸钠原料药1000g，上样到QS100型粉碎机中。粉碎条件为，0.7MPa粉碎压力，2小时，粉碎1次，得到伊班膦酸钠微粉。初步观察，微粉无粘连，无团块，分散良好。
取微粉样品约1g，使用英国Malvern Instruments Ltd.激光粒度测定仪对微粉粉末进行了粒度测定，结果上述样品粒度d90为5.55μm，d50为2.73μm，d10为0.90μm。
为了考察制备过程是否引起伊班膦酸钠的破坏降解，以钼蓝比色法对粉碎前伊班膦酸钠原料药与粉碎后的粉末进行了测定。方法为取原料药或粉末各8mg，制成伊班膦酸钠浓度相当于0.08mg/ml的溶液，按分析方法进行相应波长的吸光度测定。结果，粉碎后的粉末吸光度（0.258±0.035）与粉碎前的原料药（0.266±0.042）相当，可见伊班膦酸钠的含量无明显的改变。
实施例2
二步粉碎法
取伊班膦酸钠原料药1000g，上样到QS100型粉碎机中。粉碎条件为0.7MPa粉碎压力，粉碎2次。第一次粉碎时间1.5小时，第二次粉碎时间2小时。得到伊班膦酸钠微粉。初步观察，微粉无粘连，无团块，分散良好。
取微粉样品约1g，使用英国Malvern Instruments Ltd.激光粒度测定仪对微粉粉末进行了粒度测定，结果上述样品粒度d90为2.73μm，d50为1.61μm，d10为0.58μm。
为了考察制备过程是否引起伊班膦酸钠的破坏降解，以钼蓝比色法对粉碎前伊班膦酸钠原料药与粉碎后的粉末进行了测定。方法为取原料药或粉末各8mg，制成伊班膦酸钠浓度相当于0.08mg/ml的溶液，按分析方法进行相应波长的吸光度测定。结果，粉碎后的粉末吸光度（0.249±0.026）与粉碎前的原料药（0.266±0.042）相当，可见伊班膦酸钠的含量无明显的改变。
实施例3
喷雾干燥法
将0.5g的伊班膦酸钠溶解在200ml水中，2.5g碳酸氢铵溶解在100ml水中，混合2个溶液，再加入200ml乙醇混合形成40%乙醇水溶液用于喷雾。碳酸氢铵在喷雾干燥的过程中完全降解挥发，使得最后形成的伊班膦酸钠固体微粉中产生小孔。
B-290型喷雾干燥机，装备有0.7mm的2-液喷头。条件是：进口温度为160℃，100%吸出，进料速率15ml/min，喷雾速率500L/h。干粉样品室温下保存在干燥器里。出口温度约70℃。
实施例4
雾化液滴
将0.5g的伊班膦酸钠溶解于100ml pH7.0的磷酸盐缓冲液（PBS）中，将该溶液加入药物雾化器进样处，进行雾化，雾化液滴颗粒大小≤7μm，气体流速~0.1ml/min。对慢阻肺小鼠的给药时间1min。5min后，以FinePointe RC 气道阻力与肺顺应性检测系统（Buxco公司）进行肺功能测定。结果显示，气道阻力（sRaw）下降约40%，动态顺应性（Cdyn）增强约90%。
实施例5
单一的伊班膦酸钠粉雾剂胶囊
以天平称取5mg伊班膦酸钠微粉，装入4号胶囊中。连续装入100个胶囊，记录胶囊实际装药量，精确到小数点后一位。
抽取30颗胶囊，采用中国药典（2010二部）方法（附录XH/附录89）进行雾粒分布测定，并将胶囊壳统一回收浸入10ml纯化水中，搅拌溶解残留药粉后，取出1ml液体，以钼蓝比色法测定OD710，再根据标准曲线换算得到伊班膦酸钠浓度，乘以总体积（10ml），除以胶囊用量数（30），再除以胶囊平均装药量（5mg），得到胶囊内伊班膦酸钠微粉平均残留量为9.3%，因此，平均胶囊排空率为90.7%。分别吸取一级分布瓶和二级分布瓶中液体各1ml，同样进行钼蓝比色法测定OD710，同样根据标准曲线和分布瓶中液体体积（10ml）换算得到分布瓶中沉积的药粉量，再除以胶囊用量数（30）和平均装药量（5mg），得到一级分布瓶沉积率为62.4%，二级分布瓶沉积率为20.2%。
实施例6
混合有乳糖辅料的伊班膦酸钠粉雾剂胶囊
以天平分别称取700mg伊班膦酸钠微粉、2.1g乳糖，置入50ml离心管中，盖紧管盖后剧烈震荡5min，然后反复颠倒混匀2500次，然后，以天平称量，按20mg/颗，其中含有伊班膦酸钠微粉5mg，装入4号胶囊中。连续装入100个胶囊，记录胶囊实际装药量，精确到小数点后一位。
抽取30粒胶囊，采用中国药典（2010二部）方法（附录XH/附录89）进行雾粒分布测定，并将胶囊壳统一回收浸入10ml纯化水中，搅拌溶解残留药粉后，取出1ml液体，以钼蓝比色法测定OD710，再根据标准曲线换算得到伊班膦酸钠浓度，乘以总体积（10ml），除以胶囊用量数（30），再除以胶囊平均装药量（5mg），得到胶囊内伊班膦酸钠微粉平均残留量为5.5%，因此，平均胶囊排空率为94.5%。分别吸取一级分布瓶和二级分布瓶中液体各1ml，同样进行钼蓝比色法测定OD710，同样根据标准曲线和分布瓶中液体体积（10ml）换算得到分布瓶中沉积的药粉量，再除以胶囊用量数（30）和平均装药量（5mg），得到一级分布瓶沉积率为42.6%，二级分布瓶沉积率为40.8%。

Claims (10)

  1. 伊班膦酸钠在治疗慢性阻塞性肺疾病和/或哮喘的用途。
  2. 如权利要求1所述的用途，其特征在于，所述伊班膦酸钠吸入给药。
  3. 如权利要求1所述的用途，其特征在于，伊班膦酸钠为粉雾剂或雾化液滴。
  4. 如权利要求2所述的用途，其特征在于，伊班膦酸钠粉雾剂还包含药学上可接受的辅料。
  5. 具有权利要求1所述用途的伊班膦酸钠粉雾剂，其特征在于，伊班膦酸钠粉雾剂中伊班膦酸钠微粉的粒度为2.5μm ~10μm。
  6. 具有权利要求5所述用途的伊班膦酸钠粉雾剂，其特征在于，伊班膦酸钠粉雾剂中伊班膦酸钠微粉的粒度分布为d90≤7μm。
  7. 具有权利要求5所述用途的伊班膦酸钠粉雾剂，其特征在于，伊班膦酸钠粉雾剂中伊班膦酸钠微粉的粒度控制在d90≤5μm，且粒径大小均匀。
  8. 如权利要求5所述伊班膦酸钠粉雾剂，其特征在于，所述粉雾剂采用粉碎法或者喷雾干燥法制备。
  9. 具有权利要求1所述用途的伊班膦酸钠粉雾剂的制备方法，其特征在于，所述粉雾剂采用粉碎法，粉碎压力为0.7MPa，单次连续粉碎时间不超过3.5小时，粉碎两次。
  10. 具有权利要求1所述用途的伊班膦酸钠雾化液滴，其特征在于，所述伊班膦酸钠雾化液滴为伊班膦酸钠溶于PBS溶液中，雾化液滴颗粒大小≤7μm。
PCT/CN2017/102755 2017-09-04 2017-09-21 伊班膦酸钠的用途及粉雾剂和制备方法 WO2019041405A1 (zh)

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CN1852719A (zh) * 2003-09-19 2006-10-25 辉瑞产品公司 包括2－亚烷基－19－去甲－维生素d衍生物和二膦酸盐的组合的药物组合物和方法
CN101083979A (zh) * 2004-11-30 2007-12-05 维克特拉有限公司 药物制剂
CN101522032A (zh) * 2006-11-21 2009-09-02 帝国制药美国公司 二膦酸吸入制剂及其使用方法
US20160039852A1 (en) * 2013-04-02 2016-02-11 The University Of Sheffield Therapeutic uses of bisphosphonates

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CN101083979A (zh) * 2004-11-30 2007-12-05 维克特拉有限公司 药物制剂
CN101522032A (zh) * 2006-11-21 2009-09-02 帝国制药美国公司 二膦酸吸入制剂及其使用方法
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