Dry powder formulations and methods for treating pulmonary diseases

Related application This application is in the interest of U.S. Patent Application No. 61 / 431,242 filed January 10, 2011, the benefit of U.S. Patent Application No. 61 / 387,925 filed September 29, 2010, and August 30, 2010. Claims the benefit of US Patent Application No. 61 / 378,146 filed in, and the entire teachings of the above application are incorporated herein by reference.

Pulmonary delivery of therapeutic agents may have some advantages over other delivery methods. Such advantages include rapid onset of action, convenience of self-administration by the patient, possibility of reducing side effects of the drug, easy delivery by inhalation, elimination of needles and the like. Inhalation therapy can provide an easy-to-use drug delivery system in the clinic of inpatients or outpatients, resulting in a very rapid onset of drug action and minimal side effects. Become.

A metered dose inhaler (MDI) is used to deliver the therapeutic agent to the airways. MDIs are generally suitable for administration of therapeutic agents that can be formulated in volatile liquids under pressure as solid dry particles for inhalation. The opening of the valve releases the suspension at a relatively high rate. The liquid then volatilizes, leaving behind an aerosol of fast-moving dry particles containing a therapeutic agent. MDI reliably delivers the drug to the upper and middle airways, but is limited because it usually delivers only low doses in a single actuation. However, the sites where lung diseases such as asthma and infections develop are often the bronchioles and alveoli.

Delivery of liquid aerosols is one of the oldest forms of drug delivery to the lungs. Liquid aerosols are usually produced by an air jet atomizer, which expels compressed air from a small orifice at high speed, and the Bernoulli effect creates a low pressure at the outlet. See, for example, US Pat. No. 5,511,726. This low pressure is used to draw the aerosolizing liquid out of the second tube. This liquid is accelerated in the air stream into small droplets. The disadvantage of this standard aerosol design is that the size of the primary droplets is relatively large, causing the primary droplets to collide with the baffle to produce secondary droplets of inhalation size. Often it is necessary that the size of the liquid aerosol droplets is not uniform, that a significant amount of chemical is recirculated, and that the density of the small inhalation liquid aerosol droplets in the inhalation air is high. It can be mentioned that it is low.

The ultrasonic atomizer uses a flat or concave piezoelectric disk submerged in the stored liquid, and the piezoelectric disk resonates with the surface of the stored liquid to form a liquid cone, and aerosol particles are scattered from the surface. (US Patent Application Publication No. 2006/0249144 and US Patent No. 5,551,416). High-concentration aerosols can be obtained because no air flow is required in the process of aerosolization, but the fabrication of piezoelectric parts is expensive and the efficiency of aerosolization of suspensions is low, so the active drug is placed in water or saline. Must be dissolved in low concentration. The relatively new liquid aerosol technology allows the liquid to be aerosolized to pass through micron-sized pores to produce smaller, more uniform, dry liquid particles for inhalation. See, for example, US Pat. No. 6,131,570; US Pat. No. 5,724,957; and US Pat. No. 6,098,620. Disadvantages of this technique include the high cost of piezoelectric and fine mesh parts, as well as the fouling of pores with residual salts and solid suspensions.

So far, dry powder inhalation has relied on mixing lactose to allow the administration of inhalable-sized particles, but it is not dispersible on its own. This process is inefficient and is known to be useless with some drugs. Some groups are trying to remedy these shortcomings by developing dry powder inhaler (DPI) formulations that are breathable, dispersible and do not require lactose mixing. Dry powder formulations for inhalation therapy are US Pat. No. 5,993,805 to Sutton et al.; US Pat. No. 6,9216527 to Platz et al.; International Publication No. 0000176 to Robinson et al.; International Publication No. 9916419 to Tarara et al.; Bot et al. It is described in International Publication No. 0000215; US Pat. No. 5,855,913 to Hanes et al. And US Pat. Nos. 6,136,295 and 5,874,064 to Edwards et al.

The clinical application of powder inhalation delivery is to produce dry powders with suitable particle size, particle density and dispersibility, to store dry powders in a dry state, and to efficiently inhale dry particles in the air. It has been limited due to the difficulty of developing convenient portable devices that can be distributed to. Another limiting factor for long-term storage of dry powders was the difficulty in maintaining stable physicochemical properties over time. Furthermore, the particle size of the dry powder for inhalation delivery is inherently limited by the fact that smaller dry particles for inhalation are less likely to disperse in the air. Dry powder formulations have advantages over cumbersome liquid dosage forms and propellant formulations, but tend to aggregate and reduce fluidity, which reduces dispersibility and efficiency of powder inhalation therapy. For example, Van der Waals interaction and capillary condensing action between particles are known to contribute to the aggregation of dry particles. Hickey, A et al., Factors Influencing the Dispersion of Dry Powders as Aerosols , Pharmaceutical Technology, August, 1994.

To overcome the adhesion between particles, Batycky et al. In US Pat. No. 7,182,961 measured geometric volume median with a laser diffractometer such as HELOS (manufactured by Sympatec, Princeton, NJ). It teaches the production of so-called "aerodynamically light inhalation particles" with a diameter (VMGD) greater than 5 microns (μm). See Batycky et al., Column 7, lines 42-65. Another approach to improve particle dispersibility for inhalation with an average particle size of less than 10 μm is to add a water-soluble polypeptide or suitable complement (including amino acid complements such as leucine) to the total composition weight. Add in an amount of 50% to 99.9%. Eljamal et al., US Pat. No. 6,582,729, 4th, 12th-19th and 5th, 55th-6th, 31st. However, this approach reduces the amount of active agent that can be delivered with a given amount of powder. Therefore, it may be necessary to increase the amount of dry powder to achieve the desired therapeutic result, for example the need for multiple inhalations and / or frequent administration. Yet another approach uses a device that applies a mechanical force to the particles, such as the pressure of a compressed gas, to break the adhesion between the particles during or just before administration. See, for example, U.S. Pat. No. 7,601,336 to Lewis et al., No. 6,737,044 to Dickinson et al., No. 6,546,928 to Ashurst et al., Or U.S. Patent Application Publication No. 20090208582 to Johnston et al.

A further limitation common to each of the above methods is that the aerosol agent produced usually contains significant amounts of inert carriers, solvents, emulsifiers, propellants and other non-pharmaceutical substances. In general, large amounts of non-pharmaceutical material are required to efficiently form dry inhalation particles of a size that can be delivered to the alveoli (eg, less than 5 microns, preferably less than 3 microns). However, large amounts of non-pharmaceutical material reduce the purity and amount of active drug substance that can be delivered. Therefore, it is practically impossible to accurately administer a large amount of active drug to a patient for systemic delivery by the above method.

Therefore, the formation of highly dispersible small particle size aerosols is still required. In addition, in order to maximize the quality of the drug in a given delivery container, it is necessary to produce powders with high mass density and drug density. In addition, there is a need for methods of producing aerosols containing higher amounts of drugs and less non-pharmaceutical substances. Finally, there is a need for a method that allows patients to rapidly administer a unit dose with one or two small breaths.

The present invention describes calcium (Ca) as an active or inactive ingredient.²⁺) Shall be an inhalation dry powder consisting of dry particles containing one or more divalent metal cations, and a dry powder containing inhalation particles. Preferably, the dry particles for inhalation are small, dense and highly dispersible, as described in detail herein.

In one aspect, the dry inhalation powder is a dry inhalation particle comprising a salt of a divalent metal cation, a salt of a monovalent metal cation, one or more additional therapeutic agents, and optionally a co-formant. The ratio of divalent metal cations to monovalent metal cations is about 8: 1 (molar: mol) to about 2: 1 (molar: mol), about 4: 1 (molar: mol) to about 2. 1 (molar: mol) or 3.9: 1 (molar: mol) to about 2: 1 (molar: mol). As shown herein, dry particles for inhalation containing calcium and sodium ions in the above ranges provide excellent effects. Therefore, such types of formulations may provide the therapeutic effect of divalent metal cations and additional therapeutic agents. Preferably, the salt of the divalent metal cation is a calcium salt such as calcium lactate, calcium sulfate, calcium carbonate, calcium citrate and combinations thereof. Preferably, the salt of the monovalent metal cation is a lithium salt, a potassium salt or a sodium salt. In some embodiments, the salt of the monovalent metal cation is a sodium salt selected from the group consisting of sodium chloride, sodium citrate, sodium lactate, sodium sulphate and combinations thereof. If an excipient is present, the excipient may be present at about 1% (w / w) to about 40% (w / w). Suitable excipients are selected from the group consisting of sugars, polysaccharides, sugar alcohols, amino acids and any combination thereof. In certain embodiments, the excipient is selected from leucine, maltodextrin, mannitol and any combination thereof. Additional therapeutic agents contain from about 0.01% (w / w) to about 90% (w / w) dry particles for inhalation. Suitable additional therapeutic agents are described herein and suitable agents are LABA, short-acting β-agonists, corticosteroids, LAMA, antibiotics, DNase, sodium channel blockers and these. It is independently selected from the group consisting of combinations of. Dry particles for inhalation have a geometric volume median diameter (VMGD) of about 10 microns or less as measured by laser diffraction (RODOS / HELOS system);

In another aspect, the dry inhalation powder comprises dry inhalation particles containing a calcium salt and a sodium salt, Ca.²⁺And Na⁺The ratio is about 8: 1 (mol: mol) to about 2: 1 (mol: mol), 4: 1 (mol: mol) to about 2: 1 (mol: mol) or 3.9: 1 (mol: mol). ) ~ About 2: 1 (molar: mol). As shown herein, dry inhaled particles containing calcium and sodium ions in the above ranges provide excellent effects in a particular disease model. The calcium salt can be selected from the group consisting of calcium lactate, calcium sulfate, calcium carbonate, calcium citrate and combinations thereof. The sodium salt can be selected from the group consisting of sodium chloride, sodium citrate, sodium lactate, sodium sulfate and combinations thereof. If necessary, the dry powder for inhalation of this embodiment may further contain 1% (w / w) to 40% (w / w) of the excipient of the dry powder. Suitable excipients are selected from the group consisting of sugars, polysaccharides, sugar alcohols, amino acids and any combination thereof. In some embodiments, the excipient is selected from leucine, maltodextrin, mannitol and any combination thereof. The dry powder of this embodiment may further comprise additional therapeutic agents such as LABA, short-acting β-agonists, corticosteroids, LAMA, antibiotics, DNase, sodium channel blockers and combinations thereof. Dry particles for inhalation are measured by laser diffraction (RODOS / HELOS system) and have a geometric volume median diameter (VMGD) of about 10 microns or less; a dispersibility ratio (1 / 4bar) of 2.0 or less; less than 5.6 microns. Fine particle fraction (FPF) of at least 45%, fine particle fraction (FPF) of less than 3.4 microns at least 30%, aerodynamic mass median diameter (MMAD) of about 7 microns or less, tap density 0.45 g / cc Greater than, and / or heat of solution is between about -10 kcal / mol ~ 10 kcal / mol. Preferably, calcium cations are present at least about 5% by weight of the dry powder for inhalation.

In another aspect, the dry inhalation powder comprises dry inhalation particles comprising a salt of a divalent metal cation, one or more therapeutic agents, and optionally a complementary agent, and the dry inhalation particles are a laser. Geometric volume median diameter (VMGD) of 10 mm or less, dispersibility ratio of 2.0 or less (1 / 4 bar), and about 0.4 g / cc to about 1.2 g / cc as measured by diffraction (RODOS / HELOS system). Has a tap density. In some embodiments, the dry particles for inhalation have a tap density of about 0.5 g / cc to about 1.2 g / cc. In some embodiments, the salt of the divalent metal cation does not have antibacterial activity, antiviral activity, anti-inflammatory activity and biological activity selected from the group consisting of combinations thereof. Suitable divalent metal cation salts for the dry powder of this embodiment are magnesium salts such as magnesium lactate and magnesium sulfate. In certain embodiments, the dry particles for inhalation are a) about 20% (w / w) to about 90% (w / w) magnesium salts and about 0.01% (w / w) to about 20% (w / w). w) Therapeutic agents; b) Approximately 20% (w / w) to approximately 80% (w / w) magnesium salts and approximately 20% (w / w) to approximately 60% (w / w) therapeutic agents; Or c) Contains about 5% (w / w) to about 40% (w / w) magnesium salt and about 60% (w / w) to about 95% (w / w) therapeutic agent, dry particles for inhalation The total of all the ingredients of is 100% by weight. Preferably, the dry particles for inhalation contain 3% (w / w) or more of magnesium ions. Dry powder for inhalation is about 0. It may contain from 01% (w / w) to about 80% (w / w) of excipients. Suitable excipients are selected from the group consisting of sugars, polysaccharides, sugar alcohols, amino acids and any combination thereof. In some embodiments, the excipient is selected from leucine, maltodextrin, mannitol and any combination thereof. The dry powder of this embodiment may further comprise additional therapeutic agents such as LABA, short-acting β-agonists, corticosteroids, LAMA, antibiotics, DNase, sodium channel blockers and combinations thereof. Dry particles for inhalation are measured by laser diffraction (RODOS / HELOS system) and have a geometric volume median diameter (VMGD) of about 10 microns or less; a dispersibility ratio (1 / 4bar) of 2.0 or less; less than 5.6 microns. Fine particle fraction (FPF) of at least 45%, fine particle fraction (FPF) of less than 3.4 microns at least 30%, aerodynamic mass median diameter (MMAD) of about 7 microns or less, tap density 0.45 g / cc Greater than, and / or heat of solution is between about -10 kcal / mol ~ 10 kcal / mol. Preferably, divalent metal cations are present at least about 5% by weight of the dry inhalation powder.

The invention also relates to dry inhaled powders or particles for use in treatment (eg, treatment, prevention or diagnosis) as described herein. The present invention also uses the dry particles or powders for inhalation described herein for the treatment, prevention or mitigation of contact infections described herein, as well as respiratory diseases and / as described herein. Or it may be used in the manufacture of drugs for the treatment, prevention or diagnosis of infectious diseases.

The present invention also relates to methods of reducing inflammation, including administering an effective amount of a dry powder for inhalation into the respiratory tract of a patient in need thereof, as described herein. Inflammation can be associated with asthma, chronic obstructive pulmonary disorder (COPD) or cystic fibrosis (CF).

The present invention also relates to a method of treating a respiratory disease, comprising administering an effective amount of a dry powder for inhalation to the respiratory tract of a patient in need thereof, as described herein. ..

The present invention also comprises administering an effective amount of dry particles or powders for inhalation to the airways of the subject in need thereof, including respiratory diseases such as asthma, airway hyperresponsiveness, seasonal allergic allergies, bronchi. It also relates to methods for treating dilatation, chronic bronchitis, emphysema, chronic obstructive pulmonary disease, cystic fibrosis, etc. The present invention also comprises administering an effective amount of dry particles or powders for inhalation into the airways of the subject in need thereof, including chronic lung diseases such as asthma, airway hyperresponsiveness, seasonal allergic allergies, bronchi. It also relates to methods for treating or preventing acute exacerbations such as dilatation, chronic bronchitis, emphysema, chronic obstructive pulmonary disease, and cystic fibrosis.

<figref num="1A-1">It is a table which shows the characteristic of the dry powder prepared from the feedstock preparations I, II, III and IV described in Examples 1 to 3 and 14. FIG. 1A shows the spray drying parameters used for spray drying the powder.</figref><figref num="1A-2">It is a table which shows the characteristic of the dry powder prepared from the feedstock preparations I, II, III and IV described in Examples 1 to 3 and 14. FIG. 1A shows the spray drying parameters used for spray drying the powder.</figref><figref num="1B-1">FIG. 1B shows the results of HPLC representing the percent content of calcium ions in the powder, the results of the density including tap density and bulk density, and the results of the Karl Fischer analysis representing the percentage content of water in the powder.</figref><figref num="1B-2">FIG. 1B shows the results of HPLC representing the percent content of calcium ions in the powder, the results of the density including tap density and bulk density, and the results of the Karl Fischer analysis representing the percentage content of water in the powder.</figref><figref num="1C">Figure 1C shows the fine particle fraction (FPF) data of the powder collected using the two-stage (ACI-2) Andersen cascade impactor and the percentage mass of the powder.</figref><figref num="1D">Figure 1D shows the fine particle fraction (FPF) data and percent mass of the powder collected using the 8-stage (ACI-8) Andersen cascade impactor.</figref><figref num="1E">Figure 1E shows data for aerodynamic median mass diameter (MMAD) and FPF (based on total and recovered doses).</figref><figref num="1F-1">Figure 1F shows the geometric volume median diameter (DV50) measured by the Spraytec instrument, the geometric standard deviation (GSD) and the FPF less than 5.0 microns (FPF &lt;5.0 μm), and HELOS with RODOS attachments. Shows data on the geometric or volumetric particle size distribution (VMGD, also called x50 / dg or x50), GSD, and 1 / 4bar and 0.5 / 4bar information.</figref><figref num="1F-2">Figure 1F shows the geometric volume median diameter (DV50) measured by the Spraytec instrument, the geometric standard deviation (GSD) and the FPF less than 5.0 microns (FPF &lt;5.0 μm), and HELOS with RODOS attachments. Shows data on the geometric or volumetric particle size distribution (VMGD, also called x50 / dg or x50), GSD, and 1 / 4bar and 0.5 / 4bar information.</figref><figref num="2">It is a graph which shows the comparison of the average of tap density and bulk density in the particles prepared from feedstock preparations I, II and III and placebo.</figref><figref num="3">Geometric volume median diameter (x50) particles at different dispersion (regulator) pressures measured using a laser diffractometer (HELOS with RODOS) (prepared from feedstock formulations I-III and placebo) It is a graph which shows the comparison between.</figref><figref num="4">Between particles prepared from feedstock formulations I (identified as PUR111 (citrate)), II (identified as PUR112 (sulfate)) and III (identified as PUR113 (lactate)) and placebo It is a graph which shows the comparison of the average FPF obtained by ACI-2 and ACI-8 in.</figref><figref num="5-1">It is an electron microscope image of formulation I (Fig. 5A); formulation III (Fig. 5B).</figref><figref num="5-2">FIG. 3 is an electron micrograph of Formulation II (Fig. 5C); and Formulation IV (Fig. 5D).</figref><figref num="6A">It is a table which shows the characteristic of the dry powder prepared by the feed raw material preparations 1-9. Formulation 1 in FIG. 6 corresponds to Formulation III-B of Example 2. Formulation 4 in FIG. 6 corresponds to Formulation IB of Example 1. Formulation 7 in FIG. 6 corresponds to Formulation II-B of Example 3. Abbreviations for table headings are found elsewhere herein. All powders in FIG. 6 were produced using a Buchi spray dryer.</figref><figref num="6B">It is a table which shows the characteristic of the dry powder prepared by the feed raw material preparations 1-9. Formulation 1 in FIG. 6 corresponds to Formulation III-B of Example 2. Formulation 4 in FIG. 6 corresponds to Formulation IB of Example 1. Formulation 7 in FIG. 6 corresponds to Formulation II-B of Example 3. Abbreviations for table headings are found elsewhere herein. All powders in FIG. 6 were produced using a Buchi spray dryer.</figref><figref num="7">It is a schematic diagram of a passage model .</figref><figref num="8">It is a graph which shows the result of the bacterial passage model exposed to a dry powder. Calcium sulfate-containing powder (4.5 μg Ca / cm²The delivery dose) reduced the migration of bacteria via the sodium alginate mimic. FIG. 8B is a graph showing the results of a bacterial passage model exposed to dry powder. The calcium salt dry powder prepared and tested from the feedstock formulations A to E contained 0 μg, 4.3 μg, 6.4 μg or 10 μg of calcium. Calcium sulfate-containing powder (4.3 μg Ca / cm²Delivery dose), calcium acetate-containing powder (10 μg Ca / cm)²Delivery dose) and calcium lactate-containing powder (6.4 μg Ca / cm)²The delivery dose) reduced the migration of bacteria via the sodium alginate mimic.</figref><figref num="9">It is a graph which shows the dose-dependent effect on the influenza A / WSN / 33 (H1N1) infection of the dry powder for inhalation prepared from the feedstock preparations 10-1 to 10-4 in Example 10A.</figref><figref num="10">It is a graph which shows the dose-dependent effect of the dry powder for inhalation prepared in Example 10B on influenza A / Panama / 99/2007 (H3N2) infection.</figref><figref num="11">FIG. 5 is a graph showing that a dry powder formulation consisting of a calcium salt and sodium chloride reduces the severity of influenza in ferrets. FIG. 11A shows a comparison of changes in body temperature of ferrets treated with calcium citrate powder compared to control individuals. FIG. 11B shows a comparison of changes in body temperature of ferrets treated with calcium sulfate powder compared to control individuals. FIG. 11C shows a comparison of changes in body temperature of ferrets treated with calcium lactate powder compared to control individuals. FIG. 11D shows the change in body temperature from baseline for each individual using the area under the curve during the test period (d0 to d10). The data represent mean ± SEM for each group (Student's t-test shows p = 0.09 for leucine control and lactate groups).</figref><figref num="12">FIG. 5 is a graph showing that a dry powder formulation consisting of different excipients (mannitol, maltodextrin), calcium lactate and sodium chloride reduced influenza titers at higher concentrations than formulation III powder alone.</figref><figref num="13">It is a graph which shows that the effect of the calcium dry powder preparation is different with respect to various viral pathogens. Calu-3 cells not exposed to the formulation were used as controls and compared to Calu-3 cells exposed to Formulation I, Formulation II and Formulation III. The concentration of virus released by cells exposed to each aerosol formulation was quantified. The symbols represent the mean and standard deviation of the two wells for each test.</figref><figref num="14">It is a graph which shows the release dose of the formulation III powder of three different capsule filling weights (25 mg, 60 mg, 75 mg) at various inhalation energies.</figref><figref num="15">FIG. 5 is a graph showing particle size distribution of calcium lactate (formulation III) powder released from different inhalers, characterized by volume median diameter (Dv50) and plotted against added inhalation energy. The addition of energy does not cause further deagglomeration of the released powder, so a consistent Dv50 value as the energy value decreases indicates good dispersion of the powder.</figref><figref num="16">It is a figure which shows the high resolution XRPD pattern of the formulation I powder. This pattern indicates that Formulation I powder is composed of a combination of crystalline sodium chloride, low crystalline or amorphous calcium citrate, and a potentially calcium chloride-rich layer.</figref><figref num="17">It is a figure which shows the comparison of the XRPD pattern with the crystal reflection by the formulation I powder and NaCl.</figref><figref num="18">It is a figure which shows the superimposition of the temperature cycle type DSC thermogram of preparation I. By periodic DSC, a glass transition temperature of about 167 ° C was observed in the calcium-rich amorphous phase.</figref><figref num="19">It is a figure which shows the high-resolution XRPD pattern of the formulation II powder. This pattern indicates that Formulation III powder is composed of a combination of crystalline sodium chloride, low crystalline or amorphous calcium lactate, and a potentially calcium chloride-rich phase.</figref><figref num="20">It is a figure which shows the comparison of the XRPD pattern with the crystal reflection by the formulation II powder and NaCl.</figref><figref num="21">It is a figure which shows the superimposition of the temperature cycle type DSC thermogram of preparation II. By periodic DSC, a glass transition temperature of about 144 ° C was observed in the calcium-rich amorphous phase.</figref><figref num="22">It is a figure which shows the high-resolution XRPD pattern of the formulation IV powder.</figref><figref num="23">It is a figure which shows the comparison of the XRPD pattern with the crystal reflection by the formulation IV powder and NaCl.</figref><figref num="24">It is a figure which shows the superimposition of the temperature cycle type DSC thermogram of preparation IV. Periodic DSC observed a glass transition temperature of about 134 ° C in the calcium-rich amorphous phase.</figref><figref num="25A">FIG. 25A is a diagram showing a high resolution XRPD pattern of Formulation II powder. This pattern indicates that the formulation II powder contains some crystalline calcium salt-containing material (calcium sulfate) in addition to crystalline sodium chloride. FIG. 25B is a diagram showing a comparison of XRPD patterns between Formulation II powder and crystal reflection by NaCl.</figref><figref num="25B">FIG. 25A is a diagram showing a high resolution XRPD pattern of Formulation II powder. This pattern indicates that the formulation II powder contains some crystalline calcium salt-containing material (calcium sulfate) in addition to crystalline sodium chloride. FIG. 25B is a diagram showing a comparison of XRPD patterns between Formulation II powder and crystal reflection by NaCl.</figref><figref num="26">It is a figure which shows the superimposition of the temperature cycle type DSC thermogram of preparation II. By periodic DSC, a glass transition temperature of about 159 ° C was observed in the calcium-rich amorphous phase.</figref><figref num="27A">It is a figure of Raman spectrum. FIG. 27A shows the Raman spectra of the six particles of the formulation I sample superimposed.</figref><figref num="27B">FIG. 27B shows the spectrum 389575-6 with the background removed and superimposed on the Raman spectra of calcium citrate tetrahydrate, sodium citrate and leucine.</figref><figref num="27C">Figure 27C shows the Raman spectra of the eight particles of the Formula II sample superimposed.</figref><figref num="27D">FIG. 27D shows the spectrum 388369-4 stripped of background and superimposed on the Raman spectra of calcium sulphate, calcium sulphate dihydrate, anhydrous sodium sulphate and leucine.</figref><figref num="27E">FIG. 27E shows the Raman spectra of the 12 particles of the Formulation III sample superimposed.</figref><figref num="27F">FIG. 27F shows the spectra 389576-7 and 389576-12 with the background removed and superimposed on the Raman spectra of calcium lactate pentahydrate and leucine.</figref><figref num="27G">FIG. 27G shows an superimposed Raman spectrum of 12 particles of the Formula IV sample.</figref><figref num="27H">Figure 27H shows the Raman spectrum of calcium lactate pentahydrate with the background removed from spectrum 389577-9.</figref><figref num="28">It is a graph which shows the result of the dry powder volume particle size of the dry powder | preparation II (calcium sulfate) prepared while increasing the solid concentration from the liquid feed material which performed premixing and the liquid feed material which performed static mixing. In the premixed feedstock, the particle size distribution became wider (GSD increased) and the volume median diameter increased significantly (× 50) as the solid concentration increased. In the statically mixed feedstock, the particle size distribution is constant even when the solid concentration increases, but the volume median diameter increases slightly as expected from the increase in the solid concentration.</figref><figref num="29">It is a graph which shows the result of the volume particle size of the preparation II (calcium sulfate) spray-dried powder prepared while increasing the solid concentration from the liquid feed material which performed premixing and the liquid feed material which performed static mixing. The particle size distribution of the premixed feedstock widens as the solid concentration increases, but the particle size distribution of the statically mixed feedstock remains narrow as the solid concentration increases. is there. Triangles are 5g / L, static mixing; squares are 5g / L, premixing; diamonds are 30g / L, static mixing; circles are 30g / L, premixing.</figref><figref num="30">It is a graph which shows the result of the aerosol characterization of the formulation II (calcium sulfate) spray-dried powder prepared while increasing the solid concentration from the premixed liquid feedstock and the statically mixed liquid feedstock.</figref><figref num="31-1">It is a graph which shows the change of the fine particle fraction (FPF) of the preparations of preparation I (calcium citrate), preparation II (calcium sulfate) and preparation III (calcium lactate) during the stability test at the time of use under extreme conditions. The graph compares changes in FPF (total dose) &lt;5.6 microns (%) over time in the chamber under extreme temperature and humidity conditions (30 ° C, 75% RH). The numbers in the legend show the true value at time zero. The plot shows the variation in response to the change compared to time zero. FIG. 31B is a graph showing changes in the volumetric particle size of the preparations of preparation I (calcium citrate), preparation II (calcium sulfate) and preparation III (calcium lactate) during the in-use stability test under extreme conditions. .. The graph compares the volume median diameter with respect to the elapsed time in the chamber under extreme temperature and humidity conditions (30 ° C, 75% RH). The numbers in the legend show the true value at time zero. The plot shows the variation in response to the change compared to time zero.</figref><figref num="31-2">Figures 31C and 31D consist of control calcium chloride: sodium chloride: leucine powder and calcium lactate: sodium chloride powder containing (i) lactose, (ii) mannitol or (iii) maltodextrin as excipients. It shows similar data for the second set of spray-dried formulations. Figure 31C compares the change in FPF (total dose) &lt;5.6 microns (%) with respect to the elapsed time in the chamber for extreme temperature and humidity conditions (30 ° C, 75% RH) of the powder in the second set of powders. It was done. The numbers in the legend show the true value at time zero. The plot shows the variation in response to the change compared to time zero. FIG. 31D is a graph showing the change in volume particle size of the second set of powders during the in-use stability test under extreme conditions. The graph compares the volume median diameter with respect to the elapsed time in the chamber under extreme temperature and humidity conditions (30 ° C, 75% RH). The numbers in the legend show the true value at time zero. The plot shows the variation in response to the change compared to time zero.</figref><figref num="32">It is a graph which shows the powder stability of various powders measured by the volume particle size when exposed to the condition of about 40% RH for a maximum of one week.</figref><figref num="33">It is a graph which shows the volume particle diameter of various powders when exposed to the condition of about 40% RH for a maximum of one week. This figure is the same as Figure 32, except that the chloride has been removed for better detail.</figref><figref num="34">It is a graph which shows the typical TGA thermogram of the formulation I.</figref><figref num="35">It is a graph which shows the heat of dissolution obtained at the time of dissolution of preparations I to III. Formulations I-III showed a significant reduction in heat of solution compared to raw calcium chloride dihydrate and calcium chloride: sodium chloride: leucine control powder.</figref><figref num="36">It is a graph which shows the result of the in vivo pneumonia test. Individuals treated with Formula II (calcium sulphate) showed 5-fold lower bacterial titers, and individuals treated with Formula I (calcium citrate) showed 10.4-fold lower bacterial titers, treated with Formula III (calcium lactate). The individual showed a 5.9-fold lower bacterial titer.</figref><figref num="37">It is a table which shows the composition of a typical dry powder preparation.</figref><figref num="38-1">It is a graph which shows the result of an in vivo influenza test. The graph shows the effect of three different doses (0.1 mg, 0.3 mg and 0.9 mg) of Formulation III on body temperature (Fig. 38A and 38B) and body weight (Fig. 38C) 10 days after infection. The data show that Formulation III is dose-dependently effective in treating ferret influenza.</figref><figref num="38-2">It is a graph which shows the result of an in vivo influenza test. The graph shows the effect of three different doses (0.1 mg, 0.3 mg and 0.9 mg) of Formulation III on body temperature (Fig. 38A and 38B) and body weight (Fig. 38C) 10 days after infection. The data show that Formulation III is dose-dependently effective in treating ferret influenza.</figref><figref num="39">It is a graph which shows the effect of the preparation III in the OVA mouse model of allergic asthma. The data show that Formulation III reduces asthma-related inflammatory cells (eosinophils).</figref><figref num="40-1">It is a graph which shows the effect of the preparation III on the inflammation induced in the tobacco smoke (TS) model of chronic obstructive pulmonary disease (COPD). The data show that Formulation III significantly reduces COPD-related inflammatory cells.</figref><figref num="40-2">It is a graph which shows the effect of the preparation III on the inflammation induced in the tobacco smoke (TS) model of chronic obstructive pulmonary disease (COPD). The data show that Formulation III significantly reduces COPD-related inflammatory cells.</figref><figref num="41">It is a graph which shows the dispersibility of preparations III, IV and V. The release doses of Formulations III, IV and V (Fig. 41A) and the geometric median volume (Fig. 41B) are expressed as a function of inhalation energy. The data show that Formulation IV and Formulation V behave similarly and disperse slightly better than Formulation III.</figref><figref num="42">It is a graph which shows the result of the test of the bacterial pneumonia in the mouse model using the preparations III, IV and V. The data show that Formulation III suppresses bacterial pneumonia more effectively than Formulation IV and Formula V.</figref><figref num="43">It is a graph which shows the effect of the preparation III, the preparation IV and the preparation V in the mouse OVA model of allergic asthma. The results show that Formulation III reduces total cell count (Fig. 43A) and eosinophil count (Fig. 43B) more effectively than Formulation IV and Formula V.</figref><figref num="44">It is a graph which shows the dispersibility of preparations III, VI, VII and VIII. The release dose of the formulation is expressed as a function of inhaled energy. The data show that all powders disperse well and formulations III, VI and VIII show higher dispersibility than formulation VII.</figref><figref num="45">It is a graph which shows the solid state characteristic of formulation VII and formulation VIII. Figure 45A shows the high resolution XRPD patterns of both Formulation VII and Formulation VIII, and the two XRPD patterns show that these powders consist of crystalline leucine, amorphous calcium lactate and sodium chloride. ing. FIG. 45B shows the mDSC graphs of both formulations, and the two graphs show the glass transition temperatures of formulation VII (91 ° C) and formulation VIII (107 ° C).</figref><figref num="46">FIG. 5 is a graph showing the results of a test for bacterial pneumonia in a mouse model using dry powders with fixed calcium doses and various calcium to sodium molar ion ratios. The data show that all types of dry powder are effective in controlling bacterial pneumonia.</figref><figref num="47">It is a graph which shows the result of the in vivo influenza test in a ferret. The graph shows the effect of Formulation III and Formulation VI on body temperature (Fig. 47A) and body weight (Fig. 47B) 10 days after infection. The data show that both Formulation III and Formulation VI are effective in treating ferret influenza.</figref><figref num="48">It is a graph which shows the effect in the allergic asthma OVA mouse model of the dry powder which has a fixed calcium dose and various calcium and sodium molar ion ratios. The data show that all formulations reduce total cell count (Fig. 48A) and eosinophil cell count (Fig. 48B), and that dry powders with higher molar ratios of calcium to sodium are more effective.</figref><figref num="49-1">It is a graph which shows the effect of the preparation III and VII on the inflammation induced in the TS model of COPD. The data show that both Formulation III and Formulation VII significantly reduced COPD-related inflammatory cells, and that once-daily administration (QD) of Formulation III was as effective as twice-daily administration (BID). It shows that.</figref><figref num="49-2">It is a graph which shows the effect of the preparation III and VII on the inflammation induced in the TS model of COPD. The data show that both Formulation III and Formulation VII significantly reduced COPD-related inflammatory cells, and that once-daily administration (QD) of Formulation III was as effective as twice-daily administration (BID). It shows that.</figref><figref num="50">Treatment of TS mice with Formulations III and VII once daily showed a significant reduction in two important neutrophil chemokines, KC and MIP2.</figref><figref num="51">Treatment of TS mice with Formulation VIII twice daily showed a significant reduction in neutrophil inflammation, represented by cell number, at the lowest dose tested. When OVA-sensitized mice were treated with Formulation VIII and then infected with rhinovirus, a significant reduction in neutrophil inflammation, represented by cell number, was observed.</figref><figref num="52">When mice were treated with Formulation VIII and then stimulated with metacholine chloride (MCh), no significant increase in airway resistance was observed compared to the simulated treatment group stimulated with MCh.</figref><figref num="53">It is shown that when mice were treated with preparations XI and 48-A and then stimulated with metacholine chloride (MCh), airway resistance was reduced compared to when the pseudo (placebo B) treated group was stimulated with MCh. It is a graph.</figref><figref num="54">Graph showing that when mice were treated with XIV and 48-B and then stimulated with metacholine chloride (MCh), airway resistance was reduced compared to when the pseudo (placebo B) treated group was stimulated with MCh. Is.</figref>

The present invention partially comprises a dry inhalation powder that delivers one or more divalent metal cations such as calcium as an active ingredient, and divalent metal cations contained in the powder (eg,). Regarding dry particles for inhalation (containing calcium). The present invention also relates to one or more monovalent cations (Na).⁺It also relates to dry particles for inhalation containing (etc.) and dry powders containing particles for inhalation.

In one aspect, the inhalation dry powder and dry particles of the present invention can be small, dispersible inhalation particles rich in divalent metal cations (eg, calcium). For example, dry particles can contain a high percentage of salts of divalent metal cations (ie, the salts of divalent metal cations are concentrated) and / or dissociate to more than 2 mol per mol of salt. It may contain salts of divalent metal cations that release divalent metal cations.

Dry powders and particles for inhalation dissociate to release 1 mol of divalent metal cations per mol of salt, or because they contain high molecular weight anions, they dissociate and have a relatively small mass of divalent. It may contain a high percentage of divalent metal cation salts (eg, calcium salts) that give rise to cations. Thus, in some embodiments, the dry powders and particles for inhalation of the present invention may be rich in salts of divalent metal cations (eg, calcium salts) and may be small and dispersible.

In another aspect, the dry powder and dry particles for inhalation have a high mass density (eg, tap density or envelope density of about 0.4 g / cc or at least about 0.45 g / cc, 0.5 g / cc, 0.6 g / cc). , 0.7g / cc or 0.8g / cc), small size and dispersibility.

The dry particles for inhalation may be large or small, for example, dry powders have a geometric particle size (VMGD) between 0.5 and 30 microns. Optionally, the dry powder MMAD can be between 0.5 and 10 microns, more preferably between 1 and 5 microns. If the dry particles are small, the particles have a tap density of optionally between 0.4 g / cc to 1.2 g / cc or 0.55 g / cc to 1.0 g / cc. If the dry particles are large, the particles have a geometric particle size (VMGD) between 5 and 30 microns (more preferably between 10 and 30 microns) and optionally a tap density of 0.01 g / g. It can be between cc and 0.4 g / cc or between 0.05 g / cc and 0.25 g / cc.

A dry powder for inhalation that contains small particles, is dispersed in the air, and is preferably concentrated (eg, rich in active ingredient) is a leap from conventional wisdom. It is well known that as the particle size decreases, the tendency for particles to aggregate or aggregate increases. See, for example, Hickey et al., Factors Influencing the Dispersion of Dry Powders as Aerosols, Pharmaceutical Technology, August, 1994.

As described herein, the present invention provides a dry inhalation powder containing small inhalation particles that disperse in the air in the absence of additional energy sources other than the inhalation of interest. Therefore, the dry inhalation powder and the dry inhalation particles may be subjected to mechanical force during or just before administration, without the particles or powder containing large amounts of inactive ingredients (eg, excipients). It can be used in therapy by using a device that disrupts aggregated or agglomerated particles. For example, a device such as a passive dry powder inhaler may be used to deliver the dry powder or dry particles.

Inhalation dry powders and inhalation particles of the present invention are also generally rich in active ingredient (s), ie divalent metal cations (eg, calcium-containing salts (s)). For example, as described herein, when the excipient is contained within the dry powder or particles for inhalation, the excipient is a small component (eg, about 50% by weight or less, preferably about 20). Weight% or less, about 12% by weight or less, about 10% by weight or less, about 8% by weight or less, or less). However, in some embodiments, more excipients may be included. Thus, in one aspect, the inhalation particles are small and highly dispersible, as well as large amounts of divalent metal cations, such as calcium (Ca).²⁺) Can be contained. Therefore, a smaller amount of powder may be administered to deliver the desired dose of divalent metal cations (eg, calcium). For example, the desired dose of calcium can be administered with one or two inhalants from a capsule or blister inhaler.

Inhalation dry powders and particles that are small, dispersive and rich (eg, rich in divalent cations, rich in divalent cation salts and / or high mass density) provide therapeutic use advantages. Bring. For example, the subject can inhale a small amount of dry powder to deliver the desired therapeutically effective amount of divalent metal cations (eg, calcium).

Definition As used herein, the term "dry powder" refers to a composition containing finely dispersed dry particles for inhalation that can be dispersed in an inhaler and then inhaled by a subject. Such dry powders or particles may contain less than about 25%, less than about 20% or less than about 15% solvent such as water, be substantially free of solvents such as water, or may be anhydrous. ..

As used herein, the term "dry particles" contains about 25% or less, about 20% or less, or about 15% or less of a solvent such as water, or is it substantially free of solvents such as water. Refers to particles for inhalation, which can be anhydrous.

As used herein, the term "for inhalation" refers to dry particles or powders suitable for delivery to the subject's airways by inhalation (eg, lung delivery). The dry powder or particles for inhalation have an aerodynamic mass median diameter (MMAD) of less than about 10 microns, preferably less than about 5 microns.

The term "small" as used herein to describe dry particles for inhalation refers to particles with a geometric volume median diameter (VMGD) of about 10 microns or less, preferably about 5 microns or less. Point to.

As used herein, the term "administration" or "administrating" of dry inhalation particles refers to the introduction of dry inhalation particles into the airways of interest.

As used herein, the term "airway" refers to the upper respiratory tract (eg, nasal passage, nasal cavity, throat, pharynx), respiratory airway (eg, larynx, trachea, bronchi, bronchial bronchi) and lung (eg, respiratory bronchi). Includes bronchi, alveolar canal, alveolar sac, alveolar).

The term "dispersible" is a term used to describe the properties of dry powders or particles that are dispersed in an aerosol for inhalation. As used herein, the dispersibility of dry powder or particles is measured by HELOS / RODOS, with a geometric volume median diameter (VMGD) measured at a dispersion pressure of 1 bar (ie, regulator pressure). It is expressed as the quotient divided by VMGD measured at a dispersion pressure of 4 bar (ie, the pressure of the controller), or the quotient of VMGD at 0.5 bar divided by VMGD at 4 bar. In the present specification, these quotients are referred to as "1 / 4bar" and "0.5 / 4bar", respectively, and the diversification correlates with the smallness of the quotient. For example, 1 / 4bar HELOS / VMGD of dry inhalation particles or powder emitted at about 1 bar from the orifice of a RODOS dry powder disperser (or equivalent technology) measured by a laser diffraction system such as HELOS. Refers to the same dry inhalation particles or powder measured by RODOS at 4 bar divided by VMGD. Therefore, the ratio of 1/4 bar or 0.5 / 4 bar of highly dispersible dry powder or dry particles is close to 1.0. Highly dispersible powders have a weak tendency to agglomerate, aggregate, or clump, and / or even if agglomerated, agglomerated, or agglomerated, are easily released from the inhaler and inhaled into the subject. Is fragmented or deagglomerated. Further, the dispersibility can be evaluated by measuring the magnitude discharged from the inhaler as a function of the flow velocity. As the flow velocity from the inhaler decreases, the amount of energy available in the airflow that is transmitted to the powder and acts as a force to disperse it decreases. For highly dispersible powders, their particle size distribution is aerodynamically characterized by aerodynamic mass median diameter (MMAD) or geometrically by its VMGD, substantially like about 15-60 LPM. Does not increase over the range of typical flow rates of human inhalation.

As used herein, the terms "FPF (&lt;5.6)", "FPF (&lt;5.6 microns)" and "fine particle fraction less than 5.6 microns" are dry particles with aerodynamic particle size less than 5.6 microns. Refers to the fraction of the sample. For example, FPF (&lt;5.6) is a two-stage collapsing Andersen Cascade Impactor (ACI) for inhalation in which the mass of dry inhalation particles deposited on stage 1 and collection filters is weighed into a capsule delivered to the instrument. It can be determined by dividing by the mass of dry particles. This parameter can also be specified as "FPF_TD (&lt;5.6)" where TD means total dose. Similar measurements can be made using an 8-stage ACI. FPF_TD (&lt;5.6) can be estimated from the entire 8-stage dataset, although the 8-stage ACI cutoff at a standard 60 L / min flow rate is different. The FPF can also be determined by calculating the results of the 8-stage ACI by the USP method using doses collected in the ACI rather than in the capsule.

As used herein, the terms "FPF (&lt;3.4)", "FPF (&lt;3.4 microns)" and "fine particle fraction less than 3.4 microns" refer to a group of aerodynamic particle sizes less than 3.4 microns. Refers to a fraction of dry particles for inhalation. For example, FPF (&lt;3.4) divides the mass of dry inhalation particles deposited on the two-stage collapsing ACI collection filter by the total mass of dry inhalation particles weighed into the capsule delivered to the instrument. Can be determined by This parameter can also be specified as "FPF_TD (&lt;3.4)" where TD means total dose. Similar measurements can be made using an 8-stage ACI. The FPF can also be determined by calculating the results of an 8-stage ACI by the USP method using doses collected in ACI rather than in capsules.

As used herein, the terms "FPF (&lt;5.0)", "FPF (&lt;5.0 microns)" and "fine particle fraction less than 5.0 microns" refer to a group of aerodynamic particle sizes less than 5.0 microns. Refers to a fraction of dry particles for inhalation. For example, FPF (&lt;5.0) can be determined by estimating from the entire 8-stage dataset using an 8-stage ACI at a standard 60 L / min flow rate. This parameter can also be specified as "FPF_TD (&lt;5.0)" where TD means total dose. When used with a geometric particle size distribution as obtained by a Malvern Spraytec, Malvern Mastersizer or Sympatec Helos particle size analyzer, "FPF (&lt;5.0)" is a group of geometric particle size less than 5.0 micrometers. Refers to a fraction of dry particles for inhalation.

As used herein, the terms "FPD (&lt;4.4)", "FPD &lt;4.4 μm", "FPD (&lt;4.4 microns)" and "fine particle dose less than 4.4 microns" have an aerodynamic particle size of 4.4. Refers to a group of dry powder particles for inhalation that are less than a micrometer. For example, FPD &lt;4.4 μm is a single dose deposited on filters and stages 6, 5, 4, 3 and 2 using an 8-stage ACI at a standard 60 L / min flow rate, placed in the ACI and activated. Can be determined by summing the masses of the powders of.

As used herein, the term "release dose" or "ED" refers to an indicator of formulation delivery after an injection or dispersion event from a suitable inhaler. More specifically, in a dry powder formulation, the ED is a measure of the proportion of powder out of the unit dose packaging to the powder present in the mouthpiece of the inhaler. ED is defined as the ratio of the dose delivered by the inhaler to the nominal dose (ie, the mass of powder per unit dose placed in a suitable inhaler prior to injection). ED is an experimentally measured parameter, USP Section 601 Aerosols, Metered-Dose Inhalers and Dry Powder Inhalers, Delivered-Dose Uniformity, Sampling the Delivered Dose from Dry Powder Inhalers, United States Pharmacopia convention, Rockville, MD, It can be determined using the method of Rev. 13, 222-225, 2007. This method mimics administration to a patient in It uses a set of in vitro devices.

As used herein, the term "capsule release powder mass" or "CEPM" refers to the amount of dry powder formulation released from a capsule or dose unit container during an inhalation operation. CEPM is usually measured by a gravimetric method by weighing the capsule before and after the inhalation operation and determining the mass of the missing powder formulation. CEPM can be expressed in milligrams of mass of lost powder or as a percentage of the mass of the first powder filled into the capsule prior to the inhalation operation.

As used herein, the term "effective amount" increases the amount of agent required to achieve the desired effect, eg, the surface and / or volume viscoelasticity of airway mucus (eg, airway lining fluid). Increases gelation of airway mucus (eg, in surface and / or volume gelling), increases surface tension of airway mucus, increases elasticity of airway mucus (eg, surface elasticity and / or volume elasticity), Increase the surface viscosity of airway mucus (eg, surface viscosity and / or volumetric viscosity), reduce the amount of inhaled particles, reduce the uptake of pathogens (eg, bacteria, viruses) or the vital capacity, symptoms (eg, fever) , Coughing, squeezing, nasal leakage, diarrhea, etc.), controlling the development of infections, suppressing viral replication, or improving or preventing respiratory function (eg, 1 second forced vital capacity (FEV1) and / Or forced vital capacity for 1 second as part of forced vital capacity (FEV1) to improve FEV1 / FVC), or enough to stimulate the natural immunity of the airway epithelium. The actual effective amount for a particular application may depend on the particular dry powder or particles, the method of administration, and the age, weight, general health of the subject, and the severity of the symptoms or condition being treated. The appropriate amount and schedule of dry powders and particles to administer to a particular patient can be determined by a clinician with conventional skills based on these and other considerations.

As used herein, the term "pharmaceutically acceptable excipient" means that an excipient can be taken up by the lungs without significant toxic effects on the lungs. Such excipients are generally recognized as safe (GRAS) by the US Food and Drug Administration.

All salts referred to herein include anhydrous and all hydrated forms of salt.

Dry powder and dry particles The present invention relates to one or more divalent metal cations, such as beryllium (Be).²⁺), Magnesium, (Mg²⁺), Calcium (Ca²⁺), Strontium (Sr²⁺), Barium (Ba²⁺), Radium (Ra²⁺) Or iron (ferrous iron ion, Fe²⁺) Etc. as an active ingredient, and the present invention relates to dry powder for inhalation and dry particles. Active divalent metal cations (eg, calcium) are generally present in dry powders and dry particles in the form of salts, which can be crystalline or non-crystalline. The dry powder and dry particles may optionally contain additional salts (eg, monovalent salts such as sodium, potassium and lithium salts), therapeutically active substances or pharmaceutically acceptable complements.

In some embodiments, the dry powder and dry particles for inhalation are salts of one or more Group IIA elements (ie, one or more beryllium salts, magnesium salts, calcium salts, barium salts, radium salts or any of these. Combination) is contained. In a more specific embodiment, the dry powder and dry particles for inhalation contain one or more calcium salts, magnesium salts or any combination thereof. In certain embodiments, the dry powder for inhalation and the dry particles contain one or more calcium salts. In other specific embodiments, the dry powder and dry particles for inhalation contain one or more magnesium salts.

Suitable beryllium salts include, for example, beryllium phosphate, beryllium acetate, beryllium tartrate, beryllium citrate, beryllium gluconate, beryllium maleate, beryllium succinate, sodium beryllium malate, beryllium α-bromocamfer sulfonate, beryllium acetyl. Acetonate, beryllium formate or any combination thereof can be mentioned.

Suitable magnesium salts include, for example, magnesium fluoride, magnesium chloride, magnesium bromide, magnesium iodide, magnesium phosphate, magnesium sulfate, magnesium sulfite, magnesium carbonate, magnesium oxide, magnesium nitrate, magnesium borate, magnesium acetate, etc. Magnesium Citrate, Magnesium Gluconate, Magnesium Maleate, Magnesium Succinate, Magnesium Marite, Magnesium Taurate, Magnesium Orotate, Magnesium Glycinate, Magnesium Naphthenate, Magnesium Acetylacetonate, Magnesium Tirate, Magnesium Hydroxide, Steeric Acid Magnesium, magnesium hexafluorosilicate, magnesium salicylate or any combination thereof can be mentioned.

Suitable calcium salts include, for example, calcium chloride, calcium sulfate, calcium lactate, calcium citrate, calcium carbonate, calcium acetate, calcium phosphate, calcium alginate, calcium stearate, calcium sorbate, calcium gluconate and the like.

Suitable strontium salts include, for example, strontium chloride, strontium phosphate, strontium sulfate, strontium carbonate, strontium oxide, strontium nitrate, strontium acetate, strontium tartrate, strontium citrate, strontium gluconate, strontium maleate, strontium succinate. Strontium malate, L-type and / or D-type strontium aspartate, strontium fumarate, L-type and / or D-type strontium glutamate, strontium glutamate, strontium lactate, strontium L-treonate, strontium malonate Examples thereof include strontium (organic metal chelate), strontium ascorbate, strontium butyrate, strontium clodronate, strontium ibandronate, strontium salicylate, strontium acetylsalicylate or any combination thereof.

Suitable barium salts include, for example, barium hydroxide, barium fluoride, barium chloride, barium bromide, barium iodide, barium sulfate, (S) barium sulfide, barium carbonate, barium peroxide, barium oxide, barium nitrate, etc. Barium acetate, barium tartrate, barium citrate, barium gluconate, barium maleate, barium succinate, barium malate, barium glutamate, barium oxalate, barium malate, barium naphthenate, barium acetylacetonate, barium formate, benzoate Barium acid, pt-butyl barium benzoate, barium adipate, barium pimerate, barium sverate, barium azelaine, barium sebacate, barium phthalate, barium isophthalate, barium terephthalate, barium anthranilate, barium mandelate, Examples include barium salicylate, barium titanate or any combination thereof.

Suitable radium salts include, for example, radium fluoride, radium chloride, radium bromide, radium iodide, radium oxide, radium nitride or any combination thereof.

Suitable iron (ferrous) salts include, for example, ferrous sulfate, ferrous oxide, ferrous acetate, ferrous citrate, ammonium ferrous citrate, ferrous gluconate. , Ferrous oxalate, ferrous fumarate, ferrous maleate, ferrous malate, ferrous lactate, ferrous ascorbate, ferrous erythrobate, ferrous glycerate Includes iron, ferrous pyruvate or any combination thereof.

In one aspect, the dry particles of the present invention are small, preferably rich in divalent metal cations (eg, calcium) and dispersible. In another aspect of the invention, the dry particles are small and the salt of the divalent metal cation is concentrated (eg, at least about 30% or at least about 40% (w / w) of the salt of the divalent metal cation). Including) and dispersibility. In a further aspect of the invention, the dry particles are small, have a high mass density (eg, tap density, envelope density), and are dispersible. In this last aspect, the particles may be rich in salts of divalent metal cations (eg, calcium, magnesium) or may be low in charge of metal cation salts in the formulation.

Generally, the dry particles of the present invention have a VMGD of about 10 μm or less (for example, about 0.1 μm to about 10 μm) as measured by HELOS / RODOS at 1.0 bar. Preferably, the dry particles of the invention have a VMGD of about 9 μm or less (eg, about 0.1 μm to about 9 μm), about 8 μm or less (eg, about 0.1 μm to about 8 μm), as measured by HELOS / RODOS at 1.0 bar. About 7 μm or less (for example, about 0.1 μm to about 7 μm), about 6 μm or less (for example, about 0.1 μm to about 6 μm), about 5 μm or less (for example, less than 5 μm, about 0.1 μm to about 5 μm), about 4 μm or less (for example) , 0.1 μm to about 4 μm), about 3 μm or less (for example, 0.1 μm to about 3 μm), about 2 μm or less (for example, 0.1 μm to about 2 μm), about 1 μm or less (for example, 0.1 μm to about 1 μm), about 1 μm ~ It is about 6 μm, about 1 μm to about 5 μm, about 1 μm to about 4 μm, about 1 μm to about 3 μm, or about 1 μm to about 2 μm.

In another aspect, the dry particles of the present invention are large, preferably calcium-rich and dispersible. In another aspect of the invention, the inhalation particles are large, dispersible, and have a relatively low charge of divalent cations and divalent cation salts, eg, 50% divalent cation salt. The following (w / w). Generally, the dry particles of the present invention have a VMGD of about 30 μm or less (for example, about 5 μm to about 30 μm) as measured by HELOS / RODOS at 1.0 bar. Preferably, the dry particles of the present invention have a VMGD of about 25 μm or less (eg, about 5 μm to about 25 μm), about 20 μm or less (eg, about 5 μm to about 20 μm), about 15 μm as measured by HELOS / RODOS at 1.0 bar. The following (eg, about 5 μm to about 15 μm), about 12 μm or less (eg, about 5 μm to about 12 μm), about 10 μm or less (eg, about 5 μm to about 10 μm), or about 8 μm or less (eg, 6 μm to about 8 μm). ..

Further, the dry particles of the present invention are dispersible regardless of whether the particles are small or large, and 1/4 bar and / or 0.5 / 4 bar are about 2.2 or less (for example, about 1.0 to about 2.2). Or about 2.0 or less (for example, about 1.0 to about 2.0). Preferably, the dry particles of the present invention have 1 / 4bar and / or 0.5 / 4bar of about 1.9 or less (eg, about 1.0 to about 1.9), about 1.8 or less (eg, about 1.0 to about 1.8), about 1.7 or less. (For example, about 1.0 to about 1.7), about 1.6 or less (for example, about 1.0 to about 1.6), about 1.5 or less (for example, about 1.0 to about 1.5), about 1.4 or less (for example, about 1.0 to about 1.4), about 1.3 or less (eg, less than 1.3, about 1.0 to about 1.3), about 1.2 or less (eg, 1.0 to about 1.2), about 1.1 or less (eg, 1.0 to about 1.1 μm), or the dry particles of the invention , 1 / 4bar is about 1.0.

Alternatively or additionally, the dry particles for inhalation of the present invention can have an MMAD of about 10 microns or less, such as an MMAD of about 0.5 to about 10 microns. Preferably, the dry particles of the invention have an MMAD of about 5 microns or less (eg, about 0.5 micron to about 5 microns, preferably about 1 micron to about 5 microns) and about 4 microns or less (eg, about 1 micron to about 1 micron). 4 microns), about 3.8 microns or less (eg, about 1 micron to about 3.8 microns), about 3.5 microns or less (eg, about 1 micron to about 3.5 microns), about 3.2 microns or less (eg, about 1 micron to about 3.2 microns) ), About 3 microns or less (for example, about 1 micron to about 3.0 microns), about 2.8 microns or less (for example, about 1 micron to about 2.8 microns), about 2.2 microns or less (for example, about 1 micron to about 2.2 microns), It is about 2.0 microns or less (eg, about 1 micron to about 2.0 microns) or about 1.8 microns or less (eg, about 1 micron to about 1.8 microns).

Alternatively or in addition, the dry powders and particles for inhalation of the present invention have an FPF (FPF &lt;5.6 μm) of less than about 5.6 microns, at least about 20%, at least about 30%, at least about 40%, preferably at least about. It can be 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65% or at least about 70%.

Alternatively or additionally, the dry powders and particles of the invention have an FPF of less than 5.0 microns (FPF_TD &lt;5.0 μm) of at least about 20%, at least about 30%, at least about 45%, preferably at least about 40%. At least about 45%, at least about 50%, at least about 60%, at least about 65% or at least about 70%. Alternatively or in addition, the dry powders and particles of the invention have an FPF (FPF_ED &lt;5.0 μm) release dose of less than 5.0 microns, at least about 45%, preferably at least about 50%, at least about 60%, at least about. 65%, at least about 70%, at least about 75%, at least about 80% or at least about 85%. Alternatively or in addition, the dry powders and particles of the invention have an FPF of less than about 3.4 microns (FPF &lt;3.4 μm) of at least about 20%, preferably at least about 25%, at least about 30%, at least about 35%. , At least about 40%, at least about 45%, at least about 50% or at least about 55%.

Alternatively or additionally, the inhalation dry powder and dry particles of the present invention have a tap density of about 0.1 g / cm.³~ About 1.0g / cm³Is. For example, small, dispersible dry particles are about 0.1 g / cm.³~ About 0.9g / cm³, About 0.2g / cm³~ About 0.9g / cm³, About 0.2g / cm³~ About 0.9g / cm³, About 0.3g / cm³~ About 0.9g / cm³, About 0.4g / cm³~ About 0.9g / cm³, About 0.5g / cm³~ About 0.9g / cm³Or about 0.5g / cm³~ About 0.8g / cm³, Greater than about 0.4g / cc, greater than about 0.5g / cc, greater than about 0.6g / cc, greater than about 0.7g / cc, about 0.1g / cm³~ About 0.8g / cm³, About 0.1g / cm³~ About 0.7g / cm³, About 0.1g / cm³~ About 0.6g / cm³, About 0.1g / cm³~ About 0.5g / cm³, About 0.1g / cm³~ About 0.4g / cm³, About 0.1g / cm³~ About 0.3g / cm³, 0.3g / cm³Has a tap density of less than. In a preferred embodiment, the tap density is greater than about 0.4 g / cc. In another preferred embodiment, the tap density is greater than about 0.5 g / cc. Alternatively, the tap density is less than about 0.4 g / cc.

Alternatively or in addition, the dry inhalation powders and dry particles of the present invention may have a water or solvent content of less than about 25% by weight, less than about 20% by weight, less than about 15% by weight of the dry inhalation particles. For example, the dry inhalation particles of the present invention have a water or solvent content of less than about 25% by weight, less than about 20% by weight, less than about 15% by weight, less than about 13% by weight, less than about 11.5% by weight, about 10%. Less than% by weight, less than about 9% by weight, less than about 8% by weight, less than about 7% by weight, less than about 6% by weight, less than about 5% by weight, less than about 4% by weight, less than about 3% by weight, about 2% by weight It can be less than, about 1% by weight, or anhydrous. The dry particles for inhalation of the present invention have a water or solvent content greater than about 1% and less than about 6%, greater than about 1.5% and less than about 5.5%, greater than about 2% and less than about 5%, about. It can be 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5% or about 5%.

As described herein, some dry inhalable particles of the invention are one or more divalent metal cations, generally present in the form of salts (eg, crystalline and / or non-crystalline). (For example, calcium (Ca²⁺)) Is contained as an active ingredient. Suitable calcium salts that may be present in the dry inhalable particles of the present invention include, for example, calcium chloride, calcium sulfate, calcium lactate, calcium citrate, calcium carbonate, calcium acetate, calcium phosphate, calcium alginate, calcium stearate, sorbic acid. Calcium, calcium gluconate and the like can be mentioned. In certain preferred embodiments, the dry powder or particles of the invention do not contain calcium phosphate, calcium carbonate, calcium alginate, calcium stearate or calcium gluconate. In another preferred embodiment, the dry powder or dry particles of the present invention include calcium citrate, calcium lactate, calcium chloride, calcium sulfate or any combination thereof. In another preferred embodiment, the dry powder or dry particles comprises any combination of calcium citrate, calcium lactate or salts thereof. In another preferred embodiment, the dry powder or dry particles contain calcium carbonate. In a further aspect, the dry powder or particles include any combination of calcium citrate, calcium lactate, calcium sulfate, calcium carbonate or salts thereof. A suitable calcium salt is calcium lactate. In certain preferred embodiments, the dry powders or particles of the invention do not contain calcium chloride. If desired, the dry inhalation particles of the invention contain a salt of a divalent metal cation (eg, a calcium salt) and one or more additional salts, such as sodium, potassium, magnesium, calcium, It further contains one or more non-toxic salts of elements such as aluminum, silicon, scandium, titanium, vanadium, chromium, cobalt, nickel, copper, manganese, zinc, tin and silver elements. Preferably, the dry particles contain at least one calcium salt and at least one monovalent cation salt (eg, sodium salt).

Suitable sodium salts that may be present in the dry particles for inhalation of the present invention include, for example, sodium chloride, sodium citrate, sodium sulfate, sodium lactate, sodium acetate, sodium hydrogencarbonate, sodium carbonate, sodium stearate, ascorbic acid. Examples thereof include sodium, sodium benzoate, sodium dihydrogen phosphate, sodium phosphate, sodium hydrogen sulfite, sodium borate, sodium gluconate, sodium metasilicate and the like. In a preferred embodiment, the dry powder and dry particles include sodium chloride, sodium citrate, sodium lactate, sodium sulphate or any combination thereof.

Suitable lithium salts include, for example, lithium chloride, lithium bromide, lithium carbonate, lithium nitrate, lithium sulfate, lithium acetate, lithium lactate, lithium citrate, lithium aspartate, lithium gluconate, lithium malate, lithium ascorbate. , Lithium orotate, lithium succinate and / or combinations thereof.

Suitable potassium salts include, for example, potassium chloride, potassium bromide, potassium iodide, potassium hydrogencarbonate, potassium nitrite, potassium persulfate, potassium sulfite, potassium hydrogen sulfite, potassium phosphate, potassium acetate, potassium citrate, glutamate. Included are potassium, dipotassium guanylate, potassium gluconate, potassium malate, potassium ascorbate, potassium sorbate, potassium succinate, sodium tartrate and any combination thereof.

In another aspect of the invention, the dry powder for inhalation or the dry particles for inhalation are suitable for use as carrier particles for delivering a therapeutic agent. In these embodiments, the dry powder for inhalation 1) has no pharmacological effect on its own (eg magnesium (Mg)).²⁺)) Or 2) Non-therapeutic amount (eg, less than therapeutic amount, eg low% divalent metal cation salt (eg less than about 20% (w / w), 15% (w) Inhalation containing one or more divalent metal cations present in less than / w), less than 10% (w / w), less than 5% (w / w) or less than 3% (w / w))) Contains dry particles for use. Preferably, the pharmacological effect is an antibacterial activity, an antiviral activity, an anti-inflammatory activity and a biological activity selected from combinations thereof. Whether or not the divalent metal cation itself has such a pharmacological effect is disclosed and exemplified herein in. It can be easily evaluated using a vivo model. For example, if the divalent metal cations used herein result in a reduction of less than 50% of colony forming units recovered from the lung in the mouse model of bacterial pneumonia disclosed in Example 26. , It has no antibacterial activity. If the divalent metal cations used herein result in a reduction of less than 50% in nasal lavage virus titers in the ferret model of influenza infection disclosed in Example 11, it is antiviral activity. Does not have. If the divalent metal cations used herein result in less than 15% reduction in lung-recovered neutrophils in the COPD cigarette smoke mouse model disclosed in Example 30, it is Has no anti-inflammatory activity. Except for replacing the divalent metal cations to be tested with formulations in the examples, the models and tests are performed substantially as described herein. These models can also be used to evaluate the therapeutic effect of divalent metal cations such as calcium cations. For example, if the amount of calcium filling in the dry powder is low, such dry powder cannot be adequately administered to the subject by inhalation in the amount required to deliver an effective amount of calcium ions. There is no possibility of a therapeutic effect. Therefore, such powders contain an amount of calcium ions that does not provide a therapeutic effect.

Suitable magnesium salts that may be present in this type of dry inhalable particles of the invention include, for example, magnesium fluoride, magnesium chloride, magnesium bromide, magnesium iodide, magnesium phosphate, magnesium sulfate, magnesium sulfite, carbon dioxide. Magnesium, Magnesium Oxide, Magnesium Nitrate, Magnesium Borate, Magnesium Acetate, Magnesium Citrate, Magnesium Gluconate, Magnesium Maleate, Magnesium Succinate, Magnesium Appleate, Magnesium Taurate, Magnesium Orotate, Magnesium Glycinate, Magnesium Naphthenate , Magnesium acetylacetonate, magnesium formate, magnesium hydroxide, magnesium stearate, magnesium hexafluorosilicate, magnesium salicylate or any combination thereof. In a preferred embodiment, the dry powder or particles include magnesium sulfate, magnesium lactate, magnesium chloride, magnesium citrate and magnesium carbonate. Suitable magnesium salts are magnesium sulfate and magnesium lactate.

Suitable divalent metal salts (eg, calcium salts) have one or more, preferably two or more of the following characteristics: (i) can be processed into dry particles for inhalation, (ii) to high humidity. It has sufficient physicochemical stability in the dry powder form to facilitate the production of dispersible and physically stable powders under various conditions including exposure, (iii) dissolves immediately upon deposition in the lungs. For example, half the mass of divalent metal cations can dissolve in less than 30 minutes, less than 15 minutes, less than 5 minutes, less than 2 minutes, less than 1 minute or less than 30 seconds, (iv) high dissolution endothermic or dissolution It does not have properties that can lead to poor tolerability or adverse events, such as endothermic (ΔH), eg, ΔH below about -10 kcal / mol or above about 10 kcal / mol. Conversely, suitable ΔH is between about -9kcal / mol and about 9kcal / mol, between about -8kcal / mol and about 8kcal / mol, between about -7kcal / mol and about 7kcal / mol, and about -6kcal / mol. Between mol ~ about 6kcal / mol, about -5kcal / mol ~ about 5kcal / mol, about -4kcal / mol ~ about 4kcal / mol, about -3kcal / mol ~ about 3kcal / mol, about- Between 2kcal / mol and about 2kcal / mol, it is about -1kcal / mol to about 1kcal / mol or about 0kcal / mol.

Suitable divalent metal cation salts (eg, calcium salts) may have the desired solubility properties. In general, very or moderately soluble divalent metal cation salts (eg, calcium salts) are preferred. For example, suitable divalent metal cation salts (eg, calcium salts) contained in dry particles for inhalation and dry powders have at least a solubility in distilled water at room temperature (20-30 ° C), 1 bar. About 0.4 g / L, at least about 0.85 g / L, at least about 0.90 g / L, at least about 0.95 g / L, at least about 1.0 g / L, at least about 2.0 g / L, at least about 5.0 g / L, at least about 6.0g / L, at least about 10.0g / L, at least about 20g / L, at least about 50g / L, at least about 90g / L, at least about 120g / L, at least about 500g / L, at least about 700g / L or at least about It can be 1000g / L. Preferably, the salt of the divalent metal cation has a solubility greater than about 0.90 g / L, greater than about 2.0 g / L, or greater than about 90 g / L. Suitable divalent metal cation salts include calcium and magnesium salts.

If necessary, dry particles and dry powders of the present invention containing salts of divalent metal cations that are poorly soluble in water (eg, calcium salts) may be prepared. As described herein, prior to or at the same time as spray drying, a salt of the desired divalent metal cation (eg, a salt of the desired divalent metal cation) is subjected to anion exchange using a different, more soluble salt feedstock. Calcium salts) may be produced to prepare such dry particles and dry powders. Alternatively, the suspension may be sent to a spray dryer to produce dry inhalation powder and dry inhalation particles.

The dry powders and particles of the present invention may contain a high percentage of active ingredient (eg, divalent metal cations (eg, calcium)) in the composition and may be rich in divalent metal cations. Dry particles are 3% or more, 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 50% or more, 60% or more, 70%. It may contain 75% or more, 80% or more, 85% or more, 90% or more or 95% or more of active ingredients.

A salt of a divalent metal cation (eg, a calcium salt) dissociates and more than 2 mol of a divalent metal cation (eg, Ca) per mole of salt²⁺) Occurs, which is advantageous. Such salts can be used to produce dry powders and particles for inhalation that are rich in divalent metal cations (eg, calcium). For example, from 1 mole of calcium citrate, 3 moles of Ca upon dissolution²⁺Produces. It is also generally preferred that the salt of the divalent metal cation (eg, the calcium salt) is a low molecular weight salt and / or contains a low molecular weight anion. Salts of low molecular weight divalent metal cations, such as calcium salts containing calcium ions and low molecular weight anions, are divalent cations compared to high molecular weight salts and salts containing high molecular weight anions. Ions (eg Ca²⁺) Is rich. In general, the molecular weight of a divalent metal cation salt (eg, calcium salt) is less than about 1000 g / mol, less than about 950 g / mol, less than about 900 g / mol, less than about 850 g / mol, less than about 800 g / mol, about. Less than 750g / mol, less than about 700g / mol, less than about 650g / mol, less than about 600g / mol, less than about 550g / mol, less than about 510g / mol, less than about 500g / mol, less than about 450g / mol, about 400g / mol It is preferably less than mol, less than about 350 g / mol, less than about 300 g / mol, less than about 250 g / mol, less than about 200 g / mol, less than about 150 g / mol, about 125 g / mol or less than about 100 g / mol. In addition, or in general, it is preferred that the divalent metal cations (eg, calcium ions) make up the majority of the weight of the total salt of the divalent metal cations. In general, divalent metal cations (eg, calcium ions) are at least 10% of the total weight of the salt, at least 16%, at least 20%, at least of the total weight of the divalent metal cation salt (eg, calcium salts). It preferably accounts for 24.5%, at least 26%, at least 31%, at least 35% or at least 38%.

Alternatively or in addition, the dry particles for inhalation of the present invention are divalent metal cations (Ca).²⁺) May include a suitable divalent metal cation salt (eg, calcium salt), wherein the weight ratio of the divalent metal cation (eg, calcium ion) to the total weight of the salt is about 0.1 ~. It is between about 0.5. For example, the weight ratio of divalent metal cations (eg, calcium ions) to the total weight of the salt is between about 0.15 and about 0.5, between about 0.18 and about 0.5, between about 0.2 and about 5, and about 0.25. Between about 0.5, between about 0.27 and about 0.5, between about 0.3 and about 5, between about 0.35 and about 0.5, between about 0.37 and about 0.5, or between about 0.4 and about 0.5.

Alternatively or in addition, the dry inhalation particles of the present invention are divalent cations (eg, Ca) in an amount of at least about 5% by weight of the dry inhalation particles.²⁺) Can contain salts of divalent metal cations (eg, calcium salts). For example, the dry inhalation particles of the present invention are at least about 7% by weight, at least about 10% by weight, at least about 11% by weight, at least about 12% by weight, at least about 13% by weight, and at least about 14% by weight. %, At least about 15%, at least about 17%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, At least about 50% by weight, at least about 55% by weight, at least about 60% by weight, at least about 65% by weight, or at least about 70% by weight of divalent cations (eg, Ca).²⁺) May include salts of divalent metal cations (eg, calcium salts).

Alternatively or in addition, the dry inhalation particles of the present invention are dry particles for inhalation in an amount of at least about 5% by weight of divalent metal cations (eg, Ca).²⁺, Be²⁺, Mg²⁺, Sr²⁺, Ba²⁺, Fe²⁺) Containing salts of divalent metal cations and in an amount of at least about 3% by weight of dry particles for inhalation (eg, Na)⁺, Li⁺, K⁺) Can also be contained (eg, sodium salt, lithium salt, potassium salt). For example, the dry inhalation particles of the present invention are at least about 7% by weight, at least about 10% by weight, at least about 11% by weight, at least about 12% by weight, at least about 13% by weight, and at least about 14% by weight. %, At least about 15%, at least about 17%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, At least about 50% by weight, at least about 55% by weight, at least about 60% by weight, at least about 65% by weight, or at least about 70% by weight of divalent cations (eg, Ca).²⁺) Containing salts of divalent metal cations (eg, calcium salts) that produce at least about 3% by weight, at least about 4% by weight, at least about 5% by weight, at least about 6% by weight, at least about about dry particles for inhalation. 7% by weight, at least about 8% by weight, at least about 9% by weight, at least about 10% by weight, at least about 11% by weight, at least about 12% by weight, at least about 14% by weight, at least about 16% by weight, at least about 18% by weight %, At least about 20% by weight, at least about 22% by weight, at least about 25% by weight o, at least about 27% by weight, at least about 29% by weight, at least about 32% by weight, at least about 35% by weight, at least about 40% by weight , A sodium salt of a monovalent salt (Na) that produces at least about 45% by weight, at least about 50% by weight, or at least about 55% by weight of a monovalent anion.⁺) Can be further contained.

Alternatively or additionally, the dry particles for inhalation of the present invention contain a salt of a divalent metal cation and a monovalent cation, wherein the divalent cation is a component of one or more salts of the dried particles. It is present in an amount of at least 5% by weight and has a weight ratio of divalent cations to monovalent cations of about 50: 1 (ie, about 50: about 1) to about 0.1: 1 (ie, about 0.1: about 1). Is. The weight ratio of the divalent metal cation to the monovalent cation is the divalent metal cation salt contained in the dried particles and the divalent metal cation and the monovalent cation contained in the monovalent salt, respectively. It is based on the amount of. As a specific example, the weight ratio of divalent metal cations to monovalent cations is about 0.2: 1, about 0.3: 1, about 0.4: 1, about 0.5: 1, about 0.6: 1, about 0.7: 1, about. 0.8: 1, about 0.86: 1, about 0.92: 1, about 1: 1, about 1.3: 1, about 2: 1, about 5: 1, about 10: 1, about 15: 1, about 20: 1, about 25: 1, about 30: 1, about 35: 1, about 40: 1, about 45: 1 or about 50: 1, about 20: 1 to about 0.1: 1, about 15: 1 to about 0.1: 1, about It is 10: 1 to about 0.1: 1 or about 5: 1 to about 0.1: 1.

Alternatively or additionally, the dry inhalation particles of the present invention may contain a salt of a divalent metal cation and a salt of a monovalent cation, where the salt of the divalent metal cation and the monovalent cation. The salt contains chloride ion, citrate ion or sulfate ion as counter ions and is a divalent metal cation (for example, Ca).²⁺, Be²⁺, Mg²⁺, Sr²⁺, Ba²⁺, Fe²⁺) And monovalent cations (eg Na⁺, Li⁺, K⁺) Mole: mol ratio is about 50: 1 (ie, about 50 to about 1) to about 0.1: 1 (ie, about 0.1 to about 1). The molar ratio of divalent metal cations to monovalent cations is the divalent metal cations and monovalent metal cations contained in the divalent metal cation salt and the monovalent cation salt contained in the dried particles, respectively. It is based on the amount of valence cations. Preferably, the divalent metal cation is present as a component of the salt of one or more divalent metal cations in an amount of at least 5% by weight of the dry particles for inhalation. As a specific example, divalent metal cations and monovalent cations are contained in dry particles for inhalation at about 8.0: 1, about 7.5: 1, about 7.0: 1, about 6.5: 1, about 6.0: 1, about 5.5 :. 1, about 5.0: 1, about 4.5: 1, about 4.0: 1, about 3.5: 1, about 3.0: 1, about 2.5: 1, about 2.0: 1, about 1.5: 1, about 1.0: 1, about 0.77: 1, about 0.65: 1, about 0.55: 1, about 0.45: 1, about 0.35: 1, about 0.25: 1 or about 0.2: 1, about 8.0: 1 to about 0.55: 1, about 7.0: 1 to about 0.55: 1, about 6.0: 1 to about 0.55: 1, about 5.0: 1 to about 0.55: 1, about 4.0: 1 to about 0.55: 1, about 3.0: 1 to about 0.55: 1, about 2.0: 1 to about 0.55: It exists in a molar ratio of 1 or about 1.0: 1 to about 0.55: 1.

Preferably, a divalent metal cation (eg, Ca)²⁺, Be²⁺, Mg²⁺, Sr²⁺, Ba²⁺, Fe²⁺) And monovalent cations (eg Na⁺, Li⁺, K⁺) Mole: molar ratio is about 16.0: 1.0 ~ about 1.0: 1.0, about 16.0: 1.0 ~ about 2.0: 1.0, about 8.0: 1.0 ~ about 1.0: 1.0, about 4.0: 1.0 ~ about 1.0: 1.0, about It is from 4: 0: 1.0 to about 2.0: 1.0. More preferably, divalent metal cations and monovalent cations are present in the dry particles for inhalation in a molar ratio of about 8.0: 1.0 to about 2.0: 1.0 or about 4.0: 1.0 to about 2.0: 1.0. Most preferably, the divalent metal cation is Ca²⁺And the monovalent cation is Na⁺Is.

Suitable dry particles for inhalation contain at least one calcium salt selected from the group consisting of calcium lactate, calcium citrate, calcium sulfate and calcium chloride, and also contain sodium chloride.

Calcium citrate, calcium sulphate and calcium lactate allow processing by spray drying on dry inhalation powders and are hygroscopic despite having sufficient water solubility to promote dissolution upon deposition in the lungs. Allows the production of dry powders with high calcium salt fillings, which are low and physically relatively stable when exposed to normal and high humidity. Calcium citrate, calcium sulphate and calcium lactate also have a much lower heat of dissolution than calcium chloride, which is useful for administration to the airway, and citrate, sulphate and lactate are safe and pharmaceutical. There is no problem even if it is contained in the composition.

Thus, in addition to any combination of features and properties described herein, the dry inhalation particles of the present invention may contain at least about 51% by weight of the dry inhalation particles in total at least about 51% by weight of one or more salts. Here, one or more salts are independently selected from the group consisting of calcium and sodium, and lactate (C).₃H₅O₃^-), Chloride ion (Cl^-), Citric acid ion (C₆H₅O₇^3-) And sulfate ion (SO)₄^2-) Consists of anions selected from the group, but at least one salt is a calcium salt. For example, the dry inhalation particles of the present invention in total are at least about 55% by weight, at least about 60% by weight, at least about 65% by weight, at least about 70% by weight, at least about 75% by weight, at least about about. It may contain one or more salts of 80% by weight, at least about 85% by weight, at least about 90% by weight, at least about 91% by weight, at least about 92% by weight, or at least about 95% by weight.

Alternatively or in addition, the dry inhalation particles of the present invention may contain a calcium salt and a sodium salt, wherein the calcium cation is a component of one or more calcium salts, at least 5% by weight of the dry particles. The weight ratio of calcium and sodium ions is about 50: 1 (ie, about 50: about 1) to about 0.1: 1 (ie, about 0.1: about 1). The weight ratio of calcium ions to sodium ions is based on the amount of calcium ions and sodium ions contained in the calcium salt and the sodium salt contained in the dry particles, respectively. As a specific example, the weight ratio of calcium ion to sodium ion is about 0.2: 1, about 0.3: 1, about 0.4: 1, about 0.5: 1, about 0.6: 1, about 0.7: 1, about 0.8: 1, about. 0.86: 1, about 0.92: 1, about 1: 1, about 1.3: 1, about 2: 1, about 5: 1, about 10: 1, about 15: 1, about 20: 1, about 25: 1, about 30: 1, about 35: 1, about 40: 1, about 45: 1 or about 50: 1, about 20: 1 to about 0.1: 1, about 15: 1 to about 0.1: 1, about 10: 1 to about It is 0.1: 1 or about 5: 1 to about 0.1: 1.

Alternatively or in addition, the dry inhalation particles of the present invention may contain a calcium salt and a sodium salt, wherein the calcium salt and the sodium salt are counterions such as chloride ion, lactate ion, citrate ion or It contains sulfate ions and has a molar: molar ratio of calcium to sodium of about 50: 1 (ie, about 50: about 1) to about 0.1: 1 (ie, about 0.1: about 1). The molar ratio of calcium to sodium is based on the amount of calcium and sodium contained in the calcium salt and sodium salt contained in the dry particles, respectively. Preferably, calcium is present as a component of one or more calcium salts in an amount of at least 5% by weight of dry inhalation particles. As a specific example, calcium and sodium are contained in dry particles for inhalation at about 8.0: 1, about 7.5: 1, about 7.0: 1, about 6.5: 1, about 6.0: 1, about 5.5: 1, about 5.0: 1, About 4.5: 1, about 4.0: 1, about 3.5: 1, about 3.0: 1, about 2.5: 1, about 2.0: 1, about 1.5: 1, about 1.0: 1, about 0.77: 1, about 0.65: 1, About 0.55: 1, about 0.45: 1, about 0.35: 1, about 0.25: 1 or about 0.2: 1, about 8.0: 1 ~ about 0.55: 1, about 7.0: 1 ~ about 0.55: 1, about 6.0: 1 ~ About 0.55: 1, about 5.0: 1 ~ about 0.55: 1, about 4.0: 1 ~ about 0.55: 1, about 3.0: 1 ~ about 0.55: 1, about 2.0: 1 ~ about 0.55: 1 or about 1.0: 1 ~ It is present in a ratio of about 0.55: 1 molar.

If desired, the dry inhalation particles described herein may contain a physiologically or pharmaceutically acceptable carrier or excipient. For example, a pharmaceutically acceptable excipient may be any of the standard carbohydrate, sugar alcohol and amino acid carriers known in the art to be useful excipients for inhalation therapy, alone or. Included in any desired combination. These excipients are generally relatively free-flowing particles that do not condense or polymerize when in contact with water, are toxicologically non-toxic when inhaled as a dispersed powder, and are associated with active agents. There are few interactions between them that adversely affect the desired physiological effects of the salts of the invention. Carbohydrate excipients useful in this regard include monosaccharides and polysaccharides. Typical monosaccharides include carbohydrate modifiers such as dextrose (anhydrous and monohydrate; also called glucose and glucose monohydrate), galactose, mannitol, D-mannose, sorbose and the like. Typical disaccharides include lactose, maltose, sucrose, trehalose and the like. Raffinose and the like can be mentioned as a typical trisaccharide. Other carbohydrate excipients include maltodextrins and cyclodextrins such as 2-hydroxypropyl-β-cyclodextrin, which can be used as needed. Typical sugar alcohols include mannitol and sorbitol.

Suitable amino acid complements form powders under standard formulation processing techniques and are any natural amino acids, including non-polar (hydrophobic) amino acids and polar (uncharged, positively charged and charged) amino acids. Such amino acids are pharmaceutical grade and are generally recognized as safe by the US Food and Drug Administration (GRAS). Representative examples of non-polar amino acids include alanine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan and valine. Representative examples of uncharged polar amino acids include cystine, glycine, glutamine, serine, threonine and tyrosine. Typical examples of positively charged polar amino acids include arginine, histidine and lysine. Aspartic acid and glutamic acid are typical examples of loaded amino acids. These amino acids are generally available from commercial sources such as Aldrich Chemical Company, Inc., Milwaukee, Wis. Or Sigma Chemical Company, St. Louis, Mo., Which offer pharmaceutical grade products.

Suitable amino acid complements, such as the hydrophobic amino acid leucine, may be present in the dry particles of the present invention in an amount of about 74% by weight or less of the dry particles for inhalation. For example, the dry particles for inhalation of the present invention are about 5% by weight to about 30% by weight, about 10% by weight to about 20% by weight, about 5% by weight to about 20% by weight, about 50% by weight or less, about 45% by weight. % Or less, about 40% by weight or less, about 35% by weight or less, about 30% by weight or less, about 25% by weight or less, about 20% by weight or less, about 18% by weight or less, about 16% by weight or less, about 15% by weight or less , About 14% by weight or less, about 13% by weight or less, about 12% by weight or less, about 11% by weight or less, about 10% by weight or less, about 9% by weight or less, about 8% by weight or less, about 7% by weight or less, about It may contain up to 6% by weight, about 5% by weight or less, about 4% by weight or less, about 3% by weight or less, about 2% by weight or less, or about 1% by weight or less.

Suitable carbohydrate excipients such as maltodextrin and mannitol may be present in the dry particles of the present invention in an amount of about 74% by weight or less of the dry particles for inhalation. For example, the dry particles for inhalation of the present invention are about 50% by weight or less, about 45% by weight or less, about 40% by weight or less, about 35% by weight or less, about 30% by weight or less, about 25% by weight or less, about 20% by weight. % Or less, about 18% by weight or less, about 16% by weight or less, about 15% by weight or less, about 14% by weight or less, about 13% by weight or less, about 12% by weight or less, about 11% by weight or less, about 10% by weight or less , About 9% by weight or less, about 8% by weight or less, about 7% by weight or less, about 6% by weight or less, about 5% by weight or less, about 4% by weight or less, about 3% by weight or less, about 2% by weight or less It may contain up to 1% by weight of maltodextrin. In some preferred embodiments, the dry particles contain leucine, maltodextrin, mannitol and an excipient selected from any combination thereof. In certain embodiments, the excipient is leucine, maltodextrin or mannitol.

In certain embodiments, the dry inhalable particles of the invention are (a) calcium lactate in an amount of at least about 30% by weight, at least about 40% by weight, at least about 45% by weight, or at least about 50% by weight of the dry particles. , Calcium salt selected from calcium citrate or calcium sulphate, and (b) sodium salts such as sodium chloride in an amount of at least about 25% by weight or at least about 30% by weight of the dried particles, and the book. It may have any of the properties or characteristics described in the specification. If desired, excipients such as leucine, maltodextrin, mannitol or any combination thereof are present in an amount of about 74% by weight or less or about 50% by weight or less or about 20% by weight or less of the dry particles. May be. For example, the dry inhalation particles of the present invention are (a) about 30% to about 65% by weight, about 40% to about 65% by weight, or about 45% to about 65% by weight of calcium. Salt and (b) sodium salts such as sodium chloride in an amount of about 25% to about 60% or about 30% to about 60% by weight of dry particles, and (c) about 20% by weight of dry particles. Hereinafter, more preferably, it comprises an amount of about 10% by weight or less of the dry particles, such as leucine, maltodextrin, mannitol or any combination thereof, and (d) any of those described herein. Can have characteristics or characteristics such as 1 / 4bar, 0.5 / 4bar, VMGD, MMAD, FPF and the like.

In other embodiments, the dry inhalable particles of the invention are (a) calcium lactate in an amount of at least about 30% by weight, at least about 40% by weight, at least about 45% by weight, or at least about 50% by weight of the dry particles. , Calcium salt selected from calcium citrate or calcium sulphate, and (b) sodium chloride in an amount between about 2% to about 20% by weight or between about 3.5% by weight and about 10% by weight of dry particles. And may contain any of the properties or characteristics described herein. If desired, excipients such as leucine, maltodextrin, mannitol or any combination thereof are present in an amount of about 74% by weight or less or about 50% by weight or less or about 20% by weight or less of the dry particles. May be. For example, the dry particles for inhalation of the present invention are (a) calcium in an amount of about 30% to about 65% by weight, about 40% to about 65% by weight, or about 45% to about 65% by weight of the dry particles. Salts and (b) sodium salts such as sodium chloride in an amount between about 2% to about 20% by weight or between about 3.5% by weight and about 10% by weight of dry particles, and (c) dry particles. Includes up to about 20% by weight, more preferably no more than about 10% by weight of dry particles of excipients such as leucine, maltodextrin, mannitol or any combination thereof, and (d) herein. It may have any of the properties or characteristics described in, such as 1 / 4bar, 0.5 / 4bar, VMGD, MMAD, FPF and the like.

In some embodiments, the dry particles for inhalation contain salts of divalent metal ions and monovalent salts and are characterized by crystalline and non-crystalline inclusions of the particles. For example, dry particles for inhalation are a mixture of a non-crystalline and crystalline content, such as a non-crystalline salt-rich phase of divalent metal ions and a crystalline monovalent salt or complementary phase. May include. This type of dry particles for inhalation offers several advantages. For example, as described herein, the crystalline phase (eg, crystalline sodium chloride and / or crystalline leucine) can contribute to the stability and dispersibility of the dried particles in the dry state. Amorphous phases (eg, amorphous calcium salts) can facilitate the rapid uptake and dissolution of particles in water upon deposition in the airway. This is because relatively high water solubility salts (such as sodium chloride) present in the dry particles are crystalline, and relatively low water solubility salts (such as calcium citrate) are in the non-crystalline state in the dry particles. It is especially advantageous if it is present in.

The amorphous phase is also characterized by a high glass transition temperature (Tg), which is at least 90 ° C, at least 100 ° C, at least 110 ° C, at least 120 ° C, at least 125 ° C, at least 130 ° C, At least 135 ° C, at least 140 ° C, between 120 ° C and 200 ° C, between 125 ° C and 200 ° C, between 130 ° C and 200 ° C, between 120 ° C and 190 ° C, Between 125 ° C and 190 ° C, between 130 ° C and 190 ° C, between 120 ° C and 180 ° C, between 125 ° C and 180 ° C, or between 130 ° C and 180 ° C. is there.

In some embodiments, the dry particles for inhalation contain an amorphous phase rich in salts of divalent metal cations and a crystalline phase of the monovalent salt, and the ratio of the amorphous phase to the crystalline phase (w :). w) is about 5:95 ~ about 95: 5, about 5:95 ~ about 10:90, about 10:90 ~ about 20:80, about 20:80 ~ about 30:70, about 30:70 ~ about 40:60, about 40:60 ~ about 50:50, about 50:50 ~ about 60:40, about 60:40 ~ about 70:30, about 70:30 ~ about 80:20 or about 90:10 ~ about 95: 5. In another embodiment, the dry particles for inhalation contain an amorphous phase rich in salts of divalent metal cations and a crystalline phase of monovalent salts, and the weight ratio of the amorphous phase to the particles (w: w). ) Is about 5:95 ~ about 95: 5, about 5:95 ~ about 10:90, about 10:90 ~ about 20:80, about 20:80 ~ about 30:70, about 30:70 ~ about 40 : 60, about 40:60 ~ about 50:50, about 50:50 ~ about 60:40, about 60:40 ~ about 70:30, about 70:30 ~ about 80:20 or about 90:10 ~ about 95 : 5. In another embodiment, the dry particles for inhalation contain an amorphous phase rich in salts of divalent metal cations and a crystalline phase of monovalent salts, and the weight ratio of the crystalline phase to the particles (w: w). Is about 5:95 ~ about 95: 5, about 5:95 ~ about 10:90, about 10:90 ~ about 20:80, about 20:80 ~ about 30:70, about 30:70 ~ about 40: 60, about 40:60 ~ about 50:50; about 50:50 ~ about 60:40, about 60:40 ~ about 70:30, about 70:30 ~ about 80:20 or about 90:10 ~ about 95: It is 5.

In some embodiments, the dry particles for inhalation are calcium salts such as calcium citrate, calcium sulphate, calcium lactate, calcium chloride or any combination thereof, and sodium chloride, sodium citrate, sodium sulphate, sodium lactate. Or contains a sodium salt such as any combination thereof, wherein the dry particles for inhalation contain a non-crystalline phase rich in calcium salt and a phase of crystalline sodium salt. In certain embodiments, the calcium salt-rich amorphous phase contains calcium citrate and at least some calcium chloride, calcium lactate and at least some calcium chloride, or calcium sulfate and at least some calcium chloride. Including. In some embodiments, the dry particles for inhalation contain an amorphous phase rich in calcium salt and a crystalline phase of sodium salt, and the ratio of amorphous phase to crystalline phase (w: w) is about 5. : 95 ~ about 95: 5, about 5:95 ~ about 10:90, about 10:90 ~ about 20:80, about 20:80 ~ about 30:70, about 30:70 ~ about 40:60, about 40 : 60 ~ about 50:50, about 50:50 ~ about 60:40, about 60:40 ~ about 70:30, about 70:30 ~ about 80:20 or about 90:10 ~ about 95: 5. In another embodiment, the dry particles for inhalation contain an amorphous phase rich in calcium salt and a crystalline phase of sodium salt, and the weight ratio (w: w) of the amorphous phase to the particles is about 5 :. 95 ~ about 95: 5, about 5:95 ~ about 10:90, about 10:90 ~ about 20:80, about 20:80 ~ about 30:70, about 30:70 ~ about 40:60, about 40: It is about 60 to about 50:50, about 50:50 to about 60:40, about 60:40 to about 70:30, about 70:30 to about 80:20, or about 90:10 to about 95: 5. In another embodiment, the dry particles for inhalation contain an amorphous phase rich in calcium salt and a crystalline phase of sodium salt, and the weight ratio (w: w) of the crystalline phase to the particles is about 5:95. ~ About 95: 5, about 5:95 ~ about 10:90, about 10:90 ~ about 20:80, about 20:80 ~ about 30:70, about 30:70 ~ about 40:

Preferably, the dry particles for inhalation are 1/4 bar or 0.5 / 4 bar 2 or less, as described herein. For example, 1 / 4bar or 0.5 / 4bar is 1.9 or less, 1.8 or less, 1.7 or less, 1.6 or less, 1.5 or less, 1.4 or less, 1.3 or less, 1.2 or less, 1.1 or less or about 1.0. Alternatively or in addition, dry particles for inhalation have an MMAD of about 5 microns or less. Alternatively or in addition, the dry particles for inhalation can have a VMGD of between about 0.5 and about 5 microns, or a VMGD of between about 5 and about 20 microns. Alternatively or in addition, the dry particles for inhalation may have a heat of solution not exceeding about -l0 kcal / mol (eg, between -l0 kcal / mol and l0 kcal / mol).

As described herein, dry particles for inhalation may further comprise excipients such as leucine, maltodextrin or mannitol. The excipient may be crystalline or amorphous, or may be present in a combination of these forms. In some embodiments, the excipient is amorphous or mostly amorphous. In some embodiments, the dry particles for inhalation are substantially crystalline.

As described herein, the surface Raman mapping spectrum of dry inhalation powders containing excipients (ie, leucine, maltodextrin) showed that the excipients were not concentrated on the particle surface. And indicates that the excipient is evenly distributed throughout the particle or is not exposed on the particle surface. In particular, leucine excipients have been reported to improve dispersibility when concentrated on the particle surface. See, for example, US2003 / 0186894. Therefore, it seems that leucine does not act as a dispersion promoter in this way. Thus, in the dry inhalation particles of the invention containing a excipient (eg, leucine), the excipient may be distributed within the particle rather than on the surface of the particle, or may be distributed throughout the particle (eg, uniform). Can be distributed in). For example, in some specific embodiments, the dry particles for inhalation of the present invention do not produce the characteristic peaks indicating the presence of excipients (eg, leucine) on Raman spectroscopy. In a more specific embodiment, the dry inhalation powder containing leucine has a peak characteristic of leucine on Raman spectroscopy (eg, 1340 cm).^-1Does not occur.

As described herein, some powders of the present invention have poor fluidity. But surprisingly, these powders are highly dispersible. This is surprising because both fluidity and dispersibility are known to be negatively affected by agglomeration or agglomeration of particles. Therefore, it was unexpected that particles with poor fluidity would be highly dispersible.

In addition to any of the features and properties described herein in any combination, dry inhalation particles may have excellent dispersibility despite their poor fluidity. For example, dry particles for inhalation have Hausner ratios greater than 1.35 (eg, 1.4 or higher, 1.5 or higher, 1.6 or higher, 1.7 or higher, 1.8 or higher, 1.9 or higher, 2.0 or higher) and 2 or lower, 1.9 or lower, It can also have 1/4 bar or 0.5 / 4 bar of 1.8 or less, 1.7 or less, 1.6 or less, 1.5 or less, 1.4 or less, 1.3 or less, 1.2 or less, 1.1 or less or about 1.0.

In addition to any of the features and properties described herein in any combination, dry inhalation particles may have a heat of solution that is not highly exothermic. Preferably, simulated lung epithelial lining fluid with metal-containing particulate matter.Health Phys. 36,447-448; or Sun, G. 2001. Oxidative interactions of synthetic lung epithelial lining fluid with metal-containing particulate matter. Am J Physiol Lung Cell Mol Physiol.281, described in L807-L815) is used in an isothermometer at pH 7.4, 37 ° C to determine heat of solution. For example, dry particles for inhalation may have a heat of solution that is less exothermic than the heat of solution of calcium chloride dihydrate, eg, above about -10 kcal / mol, above about -9 kcal / mol, about-. More than 8kcal / mol, more than about -7kcal / mol, more than about -6kcal / mol, more than about -5kcal / mol, more than about -4kcal / mol, more than about -3kcal / mol, about -2kcal / It can have a heat of solution greater than mol, greater than about -1 kcal / mol, or from about -10 kcal / mol to about 10 kcal / mol. Dry particles for inhalation may have a heat of fusion of about -8 kcal / mol to about 8 kcal / mol, about -6 kcal / mol to about 6 kcal / mol or about -4 kcal / mol to about 4 kcal / mol.

If desired, salt preparations may include one or more additional agents, such as expectorants or mucolytics, surfactants, antibiotics, antivirals, antihistamines, antitussives, bronchial dilators, anti-inflammatory agents, steroids, etc. It may contain vaccines, adjuvants, expectorants, polymers, or therapeutic agents useful for the chronic maintenance of cystic fibrosis (CF). If desired, the additional agent may be mixed with the dry powder of the salt formulation or spray dried with it.

In some embodiments, the salt formulation may contain an agent that disrupts and / or disperses the biofilm. Examples of suitable agents that promote biofilm disruption and / or dispersion include certain amino acid stereoisomers such as D-leucine, D-methionine, D-tyrosine, D-tryptophan (Kolodkin-). Gal, I., D. Romero et al., D-amino acids trigger biofilm disassembly. Science 328 (5978): 627-629). For example, all or part of leucine in the dry powders described herein containing leucine can be D-leucine.

Examples of suitable expectorants or mucolytics are MUC5AC and MUC5B mucins, DNAase, N-acetylcysteine (NAC), cysteine, nacysteline, dornase α, gelsolin, heparin, heparin sulfate, P2Y2 stimulants (eg UTP). , INS365), nedocromyl sodium, hypertonic saline and mannitol.

Suitable surfactants include L-α-dipalmitoylphosphatidylcholine (DPPC), diphosphatidylglycerol (DPPG), 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (DPPS), 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) , Fat alcohol, polyoxyethylene-9-lauryl ether, surfactant, sorbitan trioleate (Span 85), glycocholic acid, surfactin, poloxomers, sorbitan fatty acid ester, tyroxapol, phospholipids and alkylated sugars Can be mentioned.

If desired, the salt formulation may contain antibiotics. Antibiotics may be suitable for the treatment of any desired bacterial infection, and salt preparations containing the antibiotic can be used to reduce the spread of the infection within or between patients. For example, salt preparations for treating bacterial pneumonia or VAT include antibiotics such as macrolides (eg azithromycin, clarithromycin and erythromycin), tetracyclines (eg doxicyclins, tigecyclins), fluoroquinoloxacins (eg gemifloxacin). , Levofloxacin, ciprofloxacin and mocifloxacin (), cephalosporin (eg, ceftriaxime, defotaxime, ceftadidim, cepepim), optionally β-lactamase inhibitors (eg, sulbactam, tazobactam and club) Aminoglycosides (eg, amicacin, albecacin, gentamycin) such as penicillin (eg, amoxycillin, amoxycillin / clarithromycin, ampicillin, piperacillin and ticarcillin) with added (lanoic acid), eg ampicillin-sulbactam, piperacillin-tazobactam and ticarcillin / clarithromycin. , Canamycin, neomycin, netylmycin, paromomycin, rhodostreptomycin, streptomycin, tobramycin and apramycin), penem or carbapenem (eg, dripenem, eltapenem, imipenem and meropenem), monobactam (eg, azuthreonum), oxazolidinone (eg, oxazolidinone) , Glycopeptide antibiotics (eg, teravancin), tuberculosis-mycobacterium antibiotics, etc. may be further included.

If desired, the salt preparation may contain a drug for treating an infection caused by Mycobacterium, such as Mycobacterium tuberculosis. Suitable agents for treating infections caused by Mycobacterium tuberculosis (eg, M. tuberculosis) include aminoglycosides (eg, capreomycin, kanamycin, streptomycin), fluoroquinolones (eg, ciprofloxacin, levofloxacin, moxifloxacin). Syn), isozianid and isozianid analogs (eg, ethionamide), aminosalicylic acid, cycloserine, diallylquinolin, ethambutol, pyrazinamide, prothionamide, rifampin and the like.

If desired, salt preparations include suitable antiviral agents such as oseltamivir, zanamavir, amantidin, rimantadine, ribavirin, ganciclovir, valganciclovir, foscabir, cytogam® (cytomegalovirus immunoglobulin), pleconaryl. , Lupintrivir, paribizmab, motabizumab, citarabin, docosanol, denotibir, cidofovir and acyclovir and the like. The salt preparation may contain suitable anti-influenza agents such as zanamivir, oseltamivir, amantadine or rimantadine.

Suitable antihistamines include clemastine, asalastine, loratadine, fexofenadine and the like.

Suitable antitussives include benzonatate, benproperine, clobutinal, diphenhydramine, dextromethorphan, divenate, fedrilate, glaucine, oxalamine, piperidion, opiods such as codeine.

As a suitable bronchodilator, short-acting β₂Stimulant, long-acting β₂Stimulants (LABA), long-acting muscariner antagonists (LAMA), LABA and LAMA combinations, methylxanthines, short-acting anticholinergic agents (sometimes called short-acting antimuscariners), long-acting antimuscariners Acting-type bronchodilators and the like can be mentioned.

Appropriate short-acting β₂Stimulants include albuterol, epinephrine, pirbuterol, levalvterol, metaproteronol, maxair and the like.

Examples of albuterol sulfate preparations (also called salbutamol) are Inspiryl (AstraZeneca Pic), Salbutamol SANDOZ (SanofI-Aventis), Asmasal clickhaler (Vectura Group Pic), Ventolin® (Glaxo SmitfiKline Pic), Salbutamol GLAND (Glaxo). SmitfiKline Pic), Airomir® (Teva Pharmaceutical Industries Ltd.), ProAir HFA (Teva Pharmaceutical Industries Ltd.), Salamol (Teva Pharmaceutical Industries Ltd.), Ipramol (Teva Pharmaceutical Industries Ltd), Albuterol sulfate TEVA (Teva Pharmaceutical) Industries Ltd) and the like. Examples of epinephrine include Epinephine Mist KING (King Pharmaceuticals, Inc.). Examples of pirbuterol as pirbuterol acetate include Maxair® (Teva Pharmaceutical Industries Ltd.). Examples of levosalbutarol include Xopenex® (Sepracor). Examples of metaproteronol preparations as metaproteronol sulfate include Alupent® (Boehringer Ingelheim GmbH).

Suitable LABAs include salmeterol, formoterol and isomers (eg alformoterol), clembuterol, tulobuterol, vilanterol (Revolair ), indacaterol, carmoterol, isoproterenol, procaterol, bambuterol, milbetarol, olodaterol and the like. Can be mentioned.

Examples of salmeterol preparations include salmeterol xinafoate as Serevent® (Glaxo SmithKline Pic), Inaspir (Laboratorios Almirall, SA), Advair® HFA (Glaxo SmithKline PLC), Advair Diskus® (registered trademark). Glaxo SmithKline PLC, Therapy Inc), Plusvent (Laboratorios Almirall, SA), Salmeterol as VR315 (Novartis, Vectura Group PLC), etc. Examples of formoterol and isomers (eg, alformoterol) include Foster (Chiesi Farmaceutici SpA), Atimos (Chiesi Farmaceutici SpA, Nycomed Intemaional Management), Flutiform® (Abbott Laboratories, Skye Pharma). PLC), MFF258 (Novartis AG), Formoterol clickhaler (Vectura Group PLC), Formoterol HFA (Skye Pharma PLC), Oxis® (Astrazeneca PLC), Oxis pMDI (Astrazeneca), Foradil® Aerolizer (Novartis, Schering) -Plough Corp, Merck), Foradil® Certihaler (Novartis, SkyePharma PLC), Symbicort® (Astra Zeneca), VR632 (Novartis AG, Sandoz International GmbH), MFF258 (Merck &amp; Co Inc, Novartis AG), Alvesco Combo (Nycomed International Management GmbH, SanofI-Aventis, Sepracor Inc), Mometazone Floate (Schering-Plough) Corp) and so on. Examples of clenbuterol include Ventipulmin® (Boehringer Ingelheim). Examples of tulobuterol include Hokunalin Tape (Abbott Japan Co., Ltd., Maruho Co., Ltd.). Examples of vilanterol include Revolair (Glaxo SmithKline PLC) and GSK64244 (GlaxoSmithKline PLC). Examples of indacaterol include QAB149 (Novartis AG, SkyePharma PLC), QMF149 (Merck &amp; Co Inc) and the like. Examples of carmoterol are CHF4226 (Chiese Farmaceutici SpA, Mitsubishi Tanabe Pharma Corporation), CHF5188 (Chiesi Farmaceutici) SpA) and so on. Examples of isoproterenol sulfate include Aludrin (Boehringer Ingelheim GmbH). Examples of Procaterol include Meptin clickhaler (Vectura Group PLC). An example of Bambuterol is Bambec (AstraZeneca PLC). Examples of milveterol include GSK159797C (GlaxoSmithKline PLC) and TD3327 (Theravance Inc). Examples of Olodaterol include BI1744CL (Boehringer Ingelheim GmbH).

Examples of LAMA include tiotropium (Spiriva), tropium chloride, glycopyrrolate, aclidinium, ipratropium and the like.

Examples of tiotropium preparations include Spiriva® (Boehringer-Ingleheim, Pfizer). Examples of glycopyrrolate include Robinul® (Wyeth-Ayerst), Robinul® Forte (Wyeth-Ayerst), NVA237 (Novartis) and the like. Examples of acridinium include Eklira® (Forest Labaoratories, Almirall).

Examples of combinations of LABA and LAMA include indacaterol and glycopyrrolate, formoterol and glycopyrrolate, indacaterol and tiotropium, olodaterol and tiotropium, vilanterol and LAMA, and the like.

Examples of the combination of indacaterol and glycopyrrolate include QVA149A (Novartis). Examples of the combination of formoterol and glycopyrrolate include PT003 (Pearl Therapeutics). Examples of combinations of olodaterol and tiotropium include BI1744 and Spirva (Boehringer Ingelheim). Examples of combinations of vilanterol and LAMA include GSK573719 and GSK642444 (Glaxo SmithKline PLC).

Examples of methylxanthine include aminophylline, ephedrine, theophylline, oxtriphyllin and the like.

Examples of aminophylline preparations include Aminophylline BOEHRINGER (Boehringer Ingelheim GmbH). Examples of ephedrine include Bronkaid® (Bayer AG), Broncholate (SanofI-Aventis), Primatene® (Wyeth), Tedral SA®, Marax (Pfizer Inc) and the like. Examples of theophylline include Euphyllin (Nycomed International Management GmbH) and Theo-dur (Pfizer Inc, Teva Pharmacetuical Industries Ltd). Examples of octrephyllin include Choledyl SA (Pfizer Inc).

Examples of short-acting anticholinergic agents include ipratropium bromide and oxitropium bromide.

Examples of ipratropium bromide preparations include Atrovent® / Apovent / Inpratropio (Boehringer Ingelheim GmbH), Ipramol (Teva Pharmaceutical Industries Ltd) and the like. Examples of oxitropium bromide include Oxivent (Boehringer Ingelheim GmbH).

Suitable anti-inflammatory agents include leukotriene inhibitors, phosphodiesterase 4 (PDE4) inhibitors, and other anti-inflammatory agents.

Suitable leukotriene inhibitors include montelukast (cystinyl leukotriene inhibitor), masilcast, zafirlukast (leukotriene D4 and E4 receptor inhibitor), pranlukast, diroyton (5-lipoxygenase inhibitor) and the like.

Examples of montelukast preparations (cystinyl leukotriene inhibitors) include Singulair® (Merck &amp; Co Inc), loratadine, montelukast sodium SCHERING (Schering-Plough Corp), MK0476C (Merck &amp; Co Inc) and the like. .. An example of Masilcast is MCC847 (AstraZeneca PLC). Examples of zafirlukast (leukotriene D4 and E4 receptor inhibitors) include Accorate (AstraZeneca PLC). An example of pranlukast is Azlaire (Schering-Plough Corp). Examples of Zileuton (5-LO) include Zyflo® (Abbott Laboratories), Zyflo CR® (Abbott Laboratories, SkyePharma PLC), Zileuton ABBOTT LABS (Abbott). Laboratories) and so on. Suitable PDE4 inhibitors include cilomilast, roflumilast, ogremirast, tofimilast and the like.

Examples of cilomilast preparations include Ariflo (GlaxoSmithKline PLC). Examples of roflumilast include Daxas® (Nycomed International Management GmbH, Pfizer Inc) and APTA2217 (Mitsubishi Tanabe Pharma Corporation). An example of Ogre Mirast is GRC3886 (Forest Laboratories Inc). Examples of Tofimilast include Tofimilast PFIZER INC (Pfizer Inc).

Examples of other anti-inflammatory agents include omalizumab (anti-IgE immunoglobulin, Daiichi Sankyo Company, Limited), Zolair (anti-IgE immunoglobulin, Genentech Inc, Novartis AG, Roche Holding Ltd), Solfa (LTD4 antagonist and phosphodiesterase inhibition). Drug, Takeda Pharmaceutical Company Limited), IL-13 and IL-13 receptor inhibitors (eg AMG-317, MILR1444A, CAT-354, QAX576, IMA-638, Anrukinzumab, IMA-026, MK-6105, DOM-0910, etc.), IL- 4 and IL-4 receptor inhibitors (eg Pitrakinla, AER-003, AIR-645, APG-201, DOM-0919, etc.), IL-1 inhibitors such as canaquinumab, AZD1981 (CRTh2 receptor antagonist, Astra Zeneca) ), CRTh2 receptor antagonists, neutrophil elastase inhibitors such as AZD9668 (AstraZeneca neutrophil elastase inhibitor), GW856553X Losmapimod (P38 kinase inhibitor, GlaxoSmithKline PLC), Arofylline LAB ALMIRALL (PDE) -4 Inhibitors, Laboratorios Almirall, SA), ABT761 (5-LO Inhibitors, Abbott Laboratories), Zyflo® (5-LO Inhibitors, Abbott Laboratories), BT061 (anti-CD4 mAb, Boehringer Ingelheim GmbH), Corus (inhaled lidocaine that reduces eosinophils, Gilead Sciences Inc), Prograf® (IL-2 mediated T cell activation inhibitor, Astellas Pharma), Bimosiamose PFIZER INC (Selectin Inhibitor, Pfizer Inc), R411 (α4β1 / α4β7 Integrin Antagonist, Roche Holdings Ltd), Tilade® (Inflammatory Mediator Inhibitor, SanofI-Aventis), Orenica (T) Cell co-stimulation inhibitors, Bristol-Myers Squibb Company, Soliris® (Anti-C5, Alexion Pharmaceuticals Inc), Entorken® (Farmacija doo), Excellair® (Syk kinase siRNA, ZaBeCor Pharmaceuticals, Baxter International Inc), KB003 (anti-GMCSF mAb, KaloBios Pharmaceuticals), cromolyn sodium (inhibits the release of mast cell mediators): Cromolyn sodium BOEHRINGER (Boehringer Ingelheim GmbH), Cromolyn sodium TEVA (Teva Pharmaceutical Industries Ltd), Intal (SanofI) -Aventis), BI1744CL (orodaterol (β2-adrenaline receptor antagonist) and thiotropium, Boehringer Ingelheim GmbH), NFK-B inhibitor, CXR2 antagonist, HLE inhibitor, HMG-CoA reductase inhibitor, etc.

Also, as anti-inflammatory agents, inflammatory molecules such as cytokines (eg IL-1, IL-4, IL-5, IL-6, IL-9, IL-13, IL-18, IL-25, IFN-α , IFN-β, etc.), CC chemokines CL1 to CCL28 (some of these are known as MCP-1, CCL2, RANTES, for example), CXC chemokines CXCL1 to CXCL17 (some of these) Depends on, for example, IL-8, MIP-2), growth factors (eg, GM-CSF, NGF, SCF, TGF-β, EGF, VEGF, etc.) and / or their respective receptors. Also included are compounds that inhibit / reduce cellular signaling.

Some examples of the above anti-inflammatory antagonists / inhibitors include ABN912 (MCP-1 / CCL2, Novartis AG), AMG761 (CCR4, Amgen Inc), Enbrel® (TNF, Amgen Inc, Wyeth), huMAb OX40L GENENTECH (TNF Super Family, Genentech Inc, AstraZeneca PLC), R4930 (TNF Super Family, Roche Holding Ltd), SB683699 / Firategrast (VLA4, Glaxo SmithKline PLC), CNT0148 (TNFα, Centocor, Inc, Johnson &amp; Johnson, Schering -Plough Corp); Canaquinumab (IL-1β, Novartis); Israpafant MITSUBISHI (PAF / IL-5, Mitsubishi Tanabe Pharma Corporation); IL-4 and IL-4 receptor antagonists / inhibitors: AMG317 (Amgen Inc), BAY169996 (Bayer AG), AER-003 (Aerovance), APG-201 (Apogenix); IL-5 and IL-5 receptor antagonists / inhibitors: MEDI563 (AstraZeneca PLC, Medlmmune, Inc), Bosatria® (GlaxoSmithKline PLC) ), Cinquil® (Ception Therapeutic), TMC120B (Mitsubishi Tanabe Pharma Corporation), Bosatria (GlaxoSmithKline PLC), Reslizumab SCHERING (Schering-Plough Corp); MEDI528 (IL-9, AstraZeneca, Medlmmune, Inc); IL- 13 and IL-13 receptor antagonists / inhibitors: TNX650 GENENTECH (Genentech), CAT-354 (AstraZeneca PLC, Medlmmune), AMG-317 (Takeda Pharmaceutical Company Limited), MK6105 (Merck &amp; Co) Inc), IMA-026 (Wyeth), IMA-638, Anrukinsumab (Wyeth), MILR1444A / Revrikizumab (Genentech), QAX576 (Novartis), CNTO-607 (Centocor), MK-6105 (Merck, CSL); IL-4 And IL-13 dual inhibitors: AIR645 / ISIS369645 (ISIS Altair), DOM-0910 (Glaxo SmithKline, Domantis), Pitrakinla / AER00l / Aerovant (Aerovance Inc), AMG-317 (Amgen), etc. Be done.

Suitable steroids include adrenocortical steroids, a combination of adrenocortical steroids and LABA, a combination of adrenocortical steroids and LAMA, a combination of adrenocortical steroids and LABA and LAMA, and the like.

Suitable corticosteroids include budesonide, fluticasone, flunisolide, triamcinolone, beclomethasone, mometasone, ciclesonide, dexamethasone and the like.

Examples of budesonide preparations include Captisol-Enabled for spraying (registered trademark budesonide solution (AstraZeneca PLC), Pulmicort (registered trademark) (AstraZeneca PLC), Pulmicort (registered trademark) Flexhaler (AstraZeneca Pic), Pulmicort (registered trademark) HFA- MDI (AstraZeneca PLC), Pulmicort Respules® (AstraZeneca PLC), Inflammide (Boehringer Ingelheim GmbH), Pulmicort® HFA-MDI (SkyePharma PLC), Unit Dose Budesonide ASTRAZENECA (AstraZeneca PLC), Budesonide Farmaceutici SpA), CHF5188 (Chiesi Farmaceutici SpA), Budesonide ABBOTT LABS (Abbott) Laboratories), Budesonide clickhaler (Vestura Group PLC), Miflonide (Novartis AG), Xavin (Teva Pharmaceutical Industries Ltd.), Budesonide TEVA (Teva Pharmaceutical Industries Ltd.), Symbicort® (AstraZeneca KK, AstraZeneca PLC), VR632 (Novartis AG, Sandoz International GmbH) and others.

Examples of fluticazone propionate preparations include Flixotide Evohaler (GlaxoSmithKline PLC), Flixotide Nebules (Glaxo SmithKline Pic), Flovent® (GlaxoSmithKline Pic), Flovent® Diskus (GlaxoSmithKline PLC), Flovent® HFA (GlaxoSmithKline PLC), Flovent® Rotadisk (Glaxo SmithKline PLC), Advair® HFA (GlaxoSmithKline PLC, Theravance Inc), Advance Diskus® (GlaxoSmithKline PLC, Therapy Inc.), VR315 (Novartis) AG, Vectura Group PLC, Sandoz International GmbH), etc. Other fluticasone preparations include Flusonal (Laboratorios) Fluticasone as Almirall, SA), GW685698 (GlaxoSmithKline PLC, Thervance Inc.), Plusvent (Laboratorios Almirall, SA), Fluticasone as Flutiform® (Abbott Laboratories, Skye Pharma PLC), etc.

Examples of flunisolide preparations include Aerobid® (Forest Laboratories Inc), Aerospan® (Forest Laboratories Inc) and the like. Examples of triamcinolone include Triamcinolone ABBOTT LABS (Abbott Laboratories), Azmacort® (Abbott Laboratories, Sanof I-Aventis), and the like. Examples of beclomethasone dipropionate include Beclovent (GlaxoSmithKline PLC), QVAR® (Johnson &amp; Johnson, Schering-Plough Corp, Teva Pharmacetucial Industries Ltd), Asmabec clickhaler (Vectura Group PLC), Beclomethasone TEVA (Teva Pharmaceutical Industries). Ltd), Vanceril (Schering-Plough Corp), BDP Modulite (Chiesi Farmaceutici SpA), Clenil (Chiesi Farmaceutici SpA), Beclomethasone dipropionate TEVA (Teva Pharmaceutical Industries Ltd), etc. Examples of mometasone are QAB149 Mometasone furoate (Schering-Plough Corp), QMF149 (Novartis AG), Fomoterol fumarate / mometoasone furoate (Schering-Plough Corp), MFF258 (Novartis AG, Merck &amp; Co Inc), Asmanex®. Examples include Twisthaler (Schering-Plough Corp). An example of ciclesonide is Alvesco® (Nycomed International). Management GmbH, Sepracor, SanofI-Aventis, Tejin Pharma Limited), Alvesco (registered trademark) Combo (Nycomed International Management GmbH, Sanofi-Aventis), Alvesco (registered trademark) HFA (Nycomed Intenational Management GmbH, Sepracor Inc), etc. .. Examples of dexamethasone include DexPak® (Merck), Decadron® (Merck), Adrenocot, CPC-Cort-D, Decaject-10, Solurex and the like. Other corticosteroids include Etiprednol dicloacetate TEVA (Teva Pharmaceutical Industries Ltd).

Examples of combinations of corticosteroids and LABA include salmeterol and fluticazone, formoterol and budesonide, formoterol and fluticazone, formoterol and mometasone, and indacaterol and mometasone.

Examples of salmeterol and fluticasone include Plusvent (Laboratorios Almirall, SA), Advair® HFA (Glaxo SmithKline PLC), Advair® Diskus (GlaxoSmithKline PLV, Theravance Inc), VR315 (Novartis AG, Vectura Group PLC). , Sandoz International GmbH), etc. Examples of vilanterol and fluticasone include GSK642444 and fluticasone. Examples of formoterol and budesonide include Symbicort® (AstraZeneca PLC), VR632 (Novartis AG, Vectura Group PLC) and the like. Examples of formoterol and fluticasone are Flutiform® (Abbott Laboratories, Skye Pharma). PLC) and the like. Examples of formoterol and mometasone include Dulera® / MFF258 (Novartis AG, Merck &amp; Co Inc). Examples of indacaterol and mometasone include QAB149 Mometasone furoate (Schering-Plough Corp) and QMF149 (Novartis AG). Examples of the combination of corticosteroids and LAMA include fluticazone and tiotropium, budesonide and tiotropium, mometasone and tiotropium, salmeterol and tiotropium, formoterol and tiotropium, indacatorol and tiotropium, and viranterol and tiotropium. Combinations of corticosteroids with LAMA and LABA include, for example, fluticasone, salmeterol and tiotropium.

Other anti-asthma molecules include: ARD111421 (VIP stimulant, AstraZeneca PLC), AVE0547 (anti-inflammatory agent, SanofI-Aventis), AVE0675 (TLR stimulant, Pfizer, SanofI-Aventis), AVE0950 (Syk inhibitor, SanofI-Aventis), AVE5883 (NK1 / NK2) Antagonists, SanofI-Aventis), AVE8923 (tryptase β inhibitor, Sanofi-Aventis), CGS21680 (adenosine A2A receptor stimulant, Novartis AG), ATL844 (A2B receptor antagonist, Novartis AG), BAY443428 (tryptase inhibitor) , Bayer AG), CHF5407 (M3 receptor inhibitor, Chiesi Farmaceutici SpA), CPLA2 inhibitor WYETH (CPLA2 inhibitor, Wyeth), IMA-638 (IL-13 antagonist, Wyeth), LAS100977 (LABA, Laboratorios Almirall, SA), MABA (M3 and β2 receptor antagonist, Chiesi Farmaceutici SpA), R1671 (mAb, Roche Holding Ltd), CS003 (Neurokinin Receptor Antagonist, Daiichi Sankyo Company, Limited), DPC168 (CCR Antagonist, Bristol-Myers Squibb), E26 (Anti-IgE, Genentech Inc), HAE1 (Genentech), IgE Inhibitor AMGEN (Amgen Inc), AMG853 (CRTH2 and D2 Receptor Antagonists, Amgen), IPL576092 (LSAID, SanofI-Aventis), EPI2010 (Antisense Adenosine 1, Chiesi Farmaceutici SpA), CHF5480 (PDE) -4 Inhibitor, Chiesi Farmaceutici SpA), KI04204 (Corticosteroid, Abbott Laboratories), SVT47060 (Laboratorios Salvat, SA), VML530 (Leukotriene Synthesis Inhibitor, Abbott) Laboratories), LAS35201 (M3 receptor antagonist, Laboratorios Almirall, SA), MCC847 (D4 receptor antagonist, Mitsubishi Tanabe Pharma Corporation), MEM1414 (PDE-4 inhibitor, Roche), TA270 (5-LO inhibitor, Chugai Pharmaceutical Co Ltd), TAK661 (eosiophilic inhibitor, Takeda Company Limited), TBC4746 (VLA-4 antagonist, Schering-Plough Corp), VR694 (Vectura Group PLC), PLD177 (steroid, Vectura Group) PLC), KI03219 (corticosteroid + LABA, Abbott Laboratories), AMG009 (Amgen Inc), AMG853 (D2 receptor antagonist, Amgen Inc);

AstraZeneca PLC: AZD1744 (CCR3 / histamine-1 receptor antagonist, AZD1419 (TLR9 antagonist), Must Cell inhibitor ASTRAZENECA, AZD3778 (CCR antagonist), DSP3025 (TLR7 antagonist), AZD1981 (CRTh2 receptor antagonist), AZD5985 (CRTh2 antagonist), AZD8075 (CRTh2 antagonist), AZD1678, AZD2098, AZD2392, AZD3825 AZD8848, AZD9215, ZD2138 (5-LO inhibitor), AZD3199 (LABA);

GlaxoSmithKline PLC: GW328267 (adenosine A2 receptor stimulant), GW559090 (α4 integrin antagonist), GSK679586 (mAb), GSK597901 (adrenalinergic β2 stimulant), AM103 (5-LO inhibitor), GSK256006 (PDE4 inhibitor) ), GW842470 (PDE-4 inhibitor), GSK870086 (glucocorticoid stimulant), GSK159802 (LABA), GSK256066 (PDE-4 inhibitor), GSK642444 (LABA, adrenalinergic β2 agonist), GSK64244 / Revolair ( Fluticasone / Vilanterol), GSK799943 (corticosteroid), GSK573719 (mAchR antagonist) and GSK573719.

Pfizer Inc: PF3526299, PF3893787, PF4191834 (FLAP antagonist), PF610355 (adrenalinergic β2 stimulant), CP664511 (α4p1 / VCAM-1 interaction inhibitor), CP609643 (α4p1 / VCAM-1 interaction inhibitor) , CP690550 (JAK3 inhibitor), SAR21609 (TLR9 stimulant), AVE7279 (Th1 switch), TBC4746 (VLA-4 antagonist); R343 (IgE receptor signaling inhibitor), SEP42960 (adenosine A3 antagonist);

SanofI-Aventis: MLN6095 (CrTH2 inhibitor), SAR137272 (A3 antagonist), SAR21609 (TLR9 antagonist), SAR389644 (DPI receptor antagonist), SAR398171 (CRTH2 antagonist), SSR161421 (adenosine A3 receptor antagonist) ;

Merck &amp; Co Inc: MK0633, MK0633, MK0591 (5-LO inhibitor), MK886 (leukotriene inhibitor), BI01211 (VLA-4 antagonist); Novartis AG: QAE397 (long-acting corticosteroid), QAK423 , QAN747, QAP642 (CCR3 antagonist), QAX935 (TLR9 stimulant), NVA237 (LAMA).

Suitable expectorants include guaifenesin, guaiacolculfonate, ammonium chloride, potassium iodide, tyroxapol, antimony sulfide and the like.

Suitable vaccines include nasal inhaled influenza vaccines.

Suitable macromolecules include proteins and large peptides with therapeutic, prophylactic or diagnostic activity, polysaccharides and oligosaccharides, and DNA and RNA nucleic acid molecules, and analogs thereof. Examples of the protein include antibodies such as monoclonal antibodies. Nucleic acid molecules include genes, antisense molecules such as siRNA that bind to complementary DNA, RNAi, shRNA, microRNAs, RNAs or ribosomes that inhibit transcription or translation. Suitable macromolecules have a molecular weight of at least 800 Da, at least 3000 Da or at least 5000 Da.

High molecular weight drugs selected for systemic application: Ventavis® (Iloprost), calcitonin, erythropoetin (EPO), factor IX, granulocyte colony stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor ( GM-CSF), growth hormone, insulin, interferon-α, interferon-β, interferon-gamma, luteinizing hormone-releasing hormone (LHRH), follicular stimulating hormone (FSH), hairy neurotrophic factor, growth hormone-releasing factor ( GRF), insulin-like growth factor, insulinotropin, interleukin-1 receptor antagonist, interleukin-3, interleukin-4, interleukin-6, macrophage colony stimulating factor (M-CSF), thymosin α1, IIb / IIIa inhibitor, α-1 antitrypsin, anti-RSV antibody, paribizumab, motabizumab, and ALN-RSV, cystic fibrosis transmembrane regulatory factor (CFTR) gene, deoxyribonuclease (DNase), heparin, bactericidal / permeabilizing Sex-enhancing protein (BPI), anti-cytomegalovirus (CMV) antibody, interferon-1 receptor antagonist, etc. GLP-1 analogs (liraglutide, exenatide, etc.), domain antibodies (dAbs), pramlintide acetate (Symlin), leptin analogs, Synagis (palivizumab, MedImmune) and cisplatin.

Useful therapeutic agents of choice for chronic maintenance of CF include antibiotics / macrolide antibiotics, bronchodilators, inhaled LABA, and agents that promote airway excretion. Examples of suitable antibiotics / macrolide antibiotics include tobramycin, azithromycin, ciprofloxacin, colistin, aztreonam and the like. Another example of an antibiotic / macrolide is levofloxacin. An example of a suitable bronchodilator is an inhaled short-acting β such as salbutamol.₂Examples include stimulants. Examples of suitable inhaled LABAs include salmeterol, formoterol and the like. Drugs that Promote Airway Secretion Excretion Suitable examples include Pulmozyme (Dornase alfa, Genentech), hypertonic saline, DNase, heparin and the like. Useful therapeutic agents of choice for the prevention and / or treatment of CF include VX-770 (Vertex Pharmaceuticals) and amiloride.

Useful therapeutic agents of choice for the treatment of idiopathic pulmonary fibrosis include meterimumab (CAT-192) (TGF-β1 mAb inhibitor, Genzyme), Aerovant (AER001, Pitrakinla) (IL-13). / IL-4 protein double antagonist, Aerovance), Aeroderm (pegged Aerovant, Aerovance), microRNA, RNAi and the like.

In a preferred embodiment, the dry powder for inhalation or the dry particles for inhalation include antibiotics such as teravancin, tuberculosis-mycobacterial antibiotic, tobramycin, azithromycin, ciprofloxacin, colistin and the like. In a further preferred embodiment, the inhalation dry powder or inhalation dry particles comprises levofloxacin. In a further preferred embodiment, the inhalation dry powder or inhalation dry particles comprises aztreonam or a pharmaceutically acceptable salt thereof (ie, Cayston®). In a more preferred embodiment, the dry inhalation powder or dry inhalation particles are free of tobramycin. In a more preferred embodiment, the dry inhalation powder or dry inhalation particles do not contain levofloxacin. In a more preferred embodiment, the dry inhalation powder or dry inhalation particles do not contain Cayston®.

In a preferred embodiment, the dry inhalation powder or dry inhalation particles are LABA, such as salmeterol, formoterol and isomers (eg, alformoterol), clenbuterol, tulobuterol, vilanterol (Revolair ), indacaterol, carmoterol. , Isoproterenol, Procaterol, Bambuterol, Milveterol and the like. In a more preferred embodiment, the dry inhalation powder or dry inhalation particles comprises formoterol. In a more preferred embodiment, the dry inhalation powder or dry inhalation particles comprises salmeterol. If the dry powder is intended for the treatment of CF, suitable additional therapeutic agents are short-acting β-agonists (eg, albuterol), antibiotics (eg, levofloxacin), recombinant human deoxyribonuclease I. (For example, Dornase α, also known as DNase), sodium channel blockers (eg, amiloride) and combinations thereof.

In a preferred embodiment, the inhalation dry powder or inhalation dry particles include LAMA, such as tiotropium, glycopyrrolate, aclidinium, ipratropium and the like. In a further preferred embodiment, the dry inhalation powder or dry inhalation particles comprises tiotropium.

In a preferred embodiment, the dry powder for inhalation or the dry particles for inhalation include corticosteroids such as budesonide, fluticasone, flunisolide, triamcinolone, bechrometasone, mometasone, ciclesonide, dexamethasone and the like. In a further preferred embodiment, the dry inhalation powder or dry inhalation particles comprises fluticasone.

In a preferred embodiment, the dry powder for inhalation or the dry particles for inhalation comprises a combination of two or more of the following; LABA, LAMA and corticosteroids. In a more preferred embodiment, the inhalation dry powder or inhalation dry particles comprises fluticasone and salmeterol. In a more preferred embodiment, the dry powder for inhalation or the dry particles for inhalation comprises fluticasone, salmeterol and tiotropium.

When an additional therapeutic agent is administered to a patient with the dry powder or dry particles disclosed herein, the agent and the dry powder or dry particles are administered and added so that the pharmacological activity is substantially duplicated. Therapeutic agents can be administered to the patient prior to, substantially simultaneously with, or thereafter the dry powder or dry particles described herein. For example, a LABA such as formoterol or a short-acting β-agonist such as albuterol may be administered to the patient prior to administration of the dry powder or dry particles described herein.

In a preferred embodiment, the dry inhalation powder or dry inhalation particles are surfactants such as L-α-dipalmitoylphosphatidylcholine (DPPC), diphosphatidylglycerol (DPPG), 1,2-dipalmitoyl-sn. -Glycero-3-phospho-L-serine (DPPS), 1,2-dipalmitoyl-sn-Glycero-3-phosphocholine (DSPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) ), 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), fatty alcohol, polyoxyethylene-9-lauryl ether, surfactant, sorbitan trioleate (Span85), glycolic acid, surfactin, poloxomers, sorbitan Free of fatty acid esters, tyroxapols, phospholipids or alkylated sugars.

In general, dry particles and powders for inhalation preferably do not contain salts, excipients or other active ingredients having a molecular weight greater than about 1 kilodalton (1000 Dalton, Da). For example, the inhalable particles of the present invention preferably have a molecular weight greater than 1 kDa, greater than about 900 Da, greater than about 800 Da, greater than about 700 Da or greater than about 600 Da, proteins, polypeptides, oligos. Contains no peptides, nucleic acids or oligonucleotides.

The dry inhalation powder or dry inhalation particles described herein may be hygroscopic because they contain salts. Therefore, it is desirable to store or maintain the dry inhalation powder and the dry inhalation particles under conditions that prevent the powder from hydrating. For example, if it is desirable to prevent hydration, the relative humidity of the storage environment is less than 75%, less than 60%, less than 50%, less than 40%, less than 30%, less than 25%, less than 20%, less than 15%, Humidity should be less than 10% or less than 5%. Dry inhalation powder and dry inhalation particles can be packaged under these conditions (eg, in sealed capsules, blisters, visals).

The present invention is also produced by preparing a solution, emulsion or suspension of raw materials according to the method described in the present specification and spray-drying the raw materials, and is produced by inhalation dry powder or inhalation drying. Also related to particles. The raw materials are (a) at least about 25% by weight (eg, of the total solute used to prepare the raw material), calcium salts such as calcium lactate or calcium chloride, and (b) at least about 1% by weight (eg,). It can be prepared with sodium salts such as sodium citrate, sodium chloride or sodium sulphate, in an amount of (of the total solute used in the preparation of the feedstock). If desired, one or more excipients such as leucine may be added to the raw material in an amount of about 74% by weight or less (eg, of the total solute used in the preparation of the raw material). For example, the calcium salt used in the preparation of the raw material is at least about 30% by weight, at least about 35% by weight, at least about 40% by weight, at least about 50% by weight, and at least about 60% by weight of the total solute used in the preparation of the raw material. Or it can be at least about 70% by weight. The sodium salt used in the preparation of the raw material is, for example, at least about 2% by weight, at least about 3% by weight, at least about 4% by weight, at least about 5% by weight, at least about 6% by weight of the total solute used in the preparation of the raw material. , At least about 7% by weight, at least about 8% by weight, at least about 9% by weight, at least about 10% by weight, at least about 20% by weight, at least about 25% by weight, at least about 30% by weight, at least about 40% by weight, at least It can be in an amount of about 50% by weight, at least about 55% by weight or at least about 65% by weight. The shape-forming agent added to the raw material is, for example, about 50% by weight or less, about 30% by weight or less, about 20% by weight or less, about 10% by weight or less, about 9% by weight or less, about 9% by weight or less of the total solute used for preparing the raw material. 8% by weight or less, about 7% by weight or less, about 6% by weight or less, about 5% by weight or less, about 4% by weight or less, about 3% by weight or less, about 2% by weight or less, or about 1% by weight or less. obtain. Alternatively, the excipient is about 10% to about 90% by weight, about 10% to about 50% by weight, about 2% of the total solute used in the preparation of the raw material.

In certain embodiments, the inhalation dry powder or inhalation dry particles of the invention are (1) about 10.0% leucine by weight percent of the total dry solute, about 35.1% calcium chloride, and about 54.9% citric acid. Obtained by preparing a raw material containing a dry solute containing sodium (a) and one or more solvents (b) suitable for dissolving the solute and forming the raw material, and (2) spray-drying the raw material. be able to. In another embodiment, the inhalation dry powder or inhalation dry particles of the present invention are (1) about 10.0% leucine by weight percent of the total dry solute, about 58.6% calcium lactate, and about 31.4% chloride. Obtained by preparing a raw material containing a dry solute containing sodium (a) and one or more solvents (b) suitable for dissolving the solute and forming the raw material, and (2) spray-drying the raw material. be able to. In another embodiment, the dry inhalation powder or dry inhalation particles of the present invention are (1) about 10.0% leucine by weight percent of the total dry solute, about 39.6% calcium chloride, and about 50.44% sulphate. Obtained by preparing a raw material containing a dry solute containing sodium (a) and one or more solvents (b) suitable for dissolving the solute and forming the raw material, and (2) spray-drying the raw material. be able to. In another embodiment, the dry inhalation powder or dry inhalation particles of the present invention are (1) about 10.0% maltodextrin by weight percent of the total dry solute, about 58.6% calcium lactate, and about 31.4%. By preparing a raw material containing a dry solute containing sodium chloride (a) and one or more solvents (b) suitable for dissolving the solute and forming the raw material, and (2) spray-drying the raw material. Obtainable. In another embodiment, the inhalation dry powder or inhalation dry particles of the present invention are (1) with about 20.0% leucine by weight percent of the total dry solute, about 75.0% calcium lactate, and about 5. A raw material containing a dry solute containing 0% sodium chloride (a) and one or more solvents (b) suitable for dissolving the solute and forming the raw material was prepared, and (2) the raw material was spray-dried. It can be obtained by letting it. In another embodiment, the inhalation dry powder or inhalation dry particles of the present invention are (1) about 37.5% leucine by weight percent of the total dry solute, about 58.6% calcium lactate and about 3.9% chloride. Obtained by preparing a raw material containing a dry solute containing sodium (a) and one or more solvents (b) suitable for dissolving the solute and forming the raw material, and (2) spray-drying the raw material. be able to. In another embodiment, Ca on a molar basis²⁺And Na⁺The ratio of is about 8: 1 to about 2: 1. As described herein, various methods known in the art (eg, static mixing, bulk mixing) can be used to mix solutes and solvents to prepare raw materials. If necessary, other suitable mixing methods may be used. For example, the ingredients may contain additional ingredients that cause or promote mixing. For example, carbon dioxide causes foaming and can act to promote the physical mixing of solutes and solvents. Since various salts of carbonic acid or hydrogen carbonate can promote the same action as that produced by carbon dioxide, they may be used in the preparation of the raw materials of the present invention. If desired, the resulting suspension may be spray dried if the solid components (solutes) of the formulation are not sufficiently soluble in the solvent or if they begin to precipitate from the solution prior to spraying.

In a preferred embodiment, the dry inhalation powder or dry inhalation particles of the present invention have an aerosol property that allows the dry inhalation particles to be efficiently delivered to the respiratory system without the use of propellants. Has.

In certain embodiments, the dry inhalation powder or dry inhalation particles of the present invention can be produced by an ion exchange reaction. In certain embodiments of the invention, two saturated or subsaturated solutions are placed in a static mixer to obtain a saturated or supersaturated statically mixed solution. Preferably, the mixed solution is supersaturated. The two solutions may be aqueous or organic, but are preferably substantially aqueous. The statically mixed solution is then placed in the spray unit of the spray dryer. In a preferred embodiment, the statically mixed solution is immediately placed in the nebulizer unit. Some examples of atomizer units include bifluid nozzles, rotary atomizers or pressure nozzles. Preferably, the atomizer unit is a two-fluid nozzle. In one embodiment, the bifluid nozzle is an internal mixing nozzle, i.e., one in which the gas collides with the liquid material before it exits the outermost orifice. In another embodiment, the bifluid nozzle is an external mixing nozzle, i.e., where the gas collides with the liquid material after it exits the outermost orifice.

Salts of divalent metal cations (eg, calcium, magnesium) can be co-prepared with excipients and optionally salts of monovalent metal cations and / or additional therapeutic agents to form dry particles for inhalation. Suitable complements include, for example, sugars (eg, lactose, trehalose, maltodextrin), polysaccharides (eg, dextrin, maltdextrin, dextran, raffinose), sugar alcohols (eg, mannitol, xylitol, sorbitol) and amino acids (eg, mannitol, xylitol, sorbitol). For example, glycine, alanine, leucine, isoleucine). Other suitable complements include, for example, dipalmitoylphosphatidylcholine (DPPC), diphosphatidylglycerol (DPPG), 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (DPPS), 1, 2-Dipalmitoyl-sn-glycero-3-phosphatidylcholine (DSPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), fat Alcohol, polyoxyethylene-9-lauryl ether, surfactant, sorbitan trioleate (Span85), glycolic acid, surfactin, poloxomers, sorbitan fatty acid ester, tyroxapole, phospholipids, alkylated sugars, sodium phosphate, Maltodextrin, human serum albumin (eg, recombinant human serum albumin), biodegradable polymer (eg PLGA), dextran, dextrin, citric acid, sodium citrate and the like.

Preferably, the complement is selected from one or more of the following; sugars (eg lactose, trehalose), polysaccharides (eg dextrin, maltdextran, dextran, raffinose), sugar alcohols (eg mannitol, etc.) Xylitol, sorbitol) and amino acids (eg, glycine, alanine, leucine, isoleucine). More preferably, the excipient is selected from one or more of the following: leucine, mannitol and maltodextrin. In one aspect of the invention, the coformant is a phospholipid such as dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylglycerol (DPPG), 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (DPPS). ), 1,2-Dipalmitoyl-sn-glycero-3-phosphatidylcholine (DSPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1-palmitoyl-2-oleoylphosphatidylcholine (DSPE) Not POPC). In another aspect of the invention, the excipient is not a carboxylic acid or a salt form thereof, such as citric acid, sodium citrate.

The dried particles of the present invention can be mixed with other therapeutic agents or co-prepared with other therapeutic agents in order to maintain the characteristic high dispersibility of the dry particles and dry powders of the present invention. Such mixed or co-prepared formulations deliver therapeutic divalent metal cations (eg, calcium ions) and one or more additional therapeutic agents, or one or more that are not divalent metal cations. It can provide dry particles, which are carrier particles that deliver the therapeutic agent, and can be produced by various methods. For example, the dry particles for inhalation of the present invention may be mixed with additional therapeutic agents, or the components of the dry particles and dry powders described herein may be added to additional therapeutic agents, eg, any of those disclosed herein. Dry powders can be produced by co-spray drying with one or a combination of additional therapeutic agents and the like. The mixed dry powder contains the dry powder and dry particles described herein and particles containing additional therapeutic agents. Suitable additional therapeutic agents are LABA (eg, formoterol, salmeterol), short-acting β-agonists (eg, albuterol), corticosteroids (eg, fluticasone), LAMA (eg, tiotropium), antibiotics (eg, thiotropium). For example, levofloxacin, tobramycin) and combinations thereof. If the dry powder is intended for the treatment of CF, suitable additional therapeutic agents are short-acting β-agonists (eg, albuterol), antibiotics (eg, levofloxacin), recombinant human deoxyribonuclease I. (For example, Dornase α, also known as DNase), sodium channel blockers (eg, amiloride) and combinations thereof.

As described herein as an example, dry powders containing certain divalent metal cations (eg, calcium ions) have antiviral, antibacterial and anti-inflammatory activity. These activities also contain salts of monovalent metal cations (eg, sodium chloride), and the ratio of divalent metal cations to monovalent metal cations (molar: mol) is within a specific range. Is enhanced in. For example, it contains a calcium salt (eg, calcium lactate) and a sodium salt (eg, sodium chloride), and the ratio of calcium ions to sodium ions (mol: mol) is about 1: 1 to about 16: 1, about 1. 1 to about 8: 1, about 1: 1 to about 4: 1, about 1: 1 to about 3.9: 1, about 1: 1 to about 3.5: 1, about 2: 1 to about 8: 1, about 2 Dry powders of 1 to about 4: 1, about 2: 1 to about 3.9: 1, about 2: 1 to about 3: 5 or about 4: 1 are better than those of other proportions of calcium and sodium salts. Can also have excellent activity.

Thus, co-prepared dry powders that can be administered to a subject to benefit from divalent metal cations (eg, calcium) and other therapeutic agents are salts of divalent metal cations (eg, calcium salts). And may include dry particles for inhalation comprising a salt of a monovalent metal cation (eg, a sodium salt), one or more additional therapeutic agents, and optionally a co-formant. Preferably, the ratio of calcium ions to sodium ions (molar: mol) in such dry inhalation particles is within one or more of the above ranges (eg, the ratio is about 4: 1). obtain). This can be done in several ways, for example, with additional therapeutic agents, divalent salts and monovalents of the dry powders and dry particles described herein (eg, any of the specific formulations described herein). This can be achieved by co-spray drying with the salt component and, if present, optionally with all or part of the excipient component. For example, in some embodiments, the dry particles may contain 0% to about 1% of excipient.

It contains a salt of a divalent metal cation (eg, a calcium salt), a salt of a monovalent metal cation (eg, a sodium salt), one or more additional therapeutic agents, and optionally a complement. The dry particles for inhalation, in which the ratio of the divalent metal cation to the monovalent metal cation is within one or more of those described herein, may contain any desired amount of therapeutic agent. In general, it is desirable to maintain a high filling of divalent metal cation salts (eg, calcium salts) in dry inhalation particles (eg, at least about 50% (w / w) of calcium salts), but in addition. If a high filling of the therapeutic agent is desired, the dry particles for inhalation should contain a relatively small amount of salt of the divalent metal cation (eg, about 10% to about 50%) and the desired divalent metal. It may contain a sufficient amount of monovalent cation salt to obtain the ratio of cations to monovalent metal cations.

In some embodiments, the dry particles for inhalation contain a calcium salt, a sodium salt, and an additional therapeutic agent, wherein the additional therapeutic agent is from about 0.01% (w / w) to about 10% (w / w). w / w), about 0.01% (w / w) ~ about 20% (w / w), about 0.01% ~ about 90%, about 20% (w / w) ~ about 90% (w / w), about 20% (w / w) ~ 80% (w / w), 20% (w / w) ~ 60% (w / w), 20% (w / w) ~ 50% (w / w / w), about 50% (w / w) ~ about 90% (w / w), about 50% (w / w) ~ about 80% (w / w), about 60% (w / w) ~ about 90 It exists at a concentration of% (w / w) or about 60% (w / w) to about 80% (w / w), and the ratio of calcium ion to sodium ion (mol: mol) is about 1: 1 to about 16. 1, about 1: 1 to about 8: 1, about 1: 1 to about 4: 1, about 1: 1 to about 3.9: 1, about 1: 1 to about 3.5: 1, about 2: 1 to about 8 1, about 2: 1 to about 4: 1, about 2: 1 to about 3.9: 1, about 2: 1 to about 3: 5 or about 4: 1. The dry particles for inhalation are preferably small (eg, VMGD at 1.0 bar less than 10 μm, preferably less than 5 μm) and dispersible (ie, as described herein, 1 /. The ratio of 4 bar and / or 0.5 / 4 bar is 2.2 or less). Preferably, the MMAD of the dry particles for inhalation is from about 0.5 μm to about 10 μm, more preferably from about 1 μm to about 5 μm. Preferably, the dry particles for inhalation are also rich in calcium and / or have a tap density of about 0.4 g / cc to about 1.2 g / cc, preferably about 0.55 g / cc to about 1. It is between 0g / cc. The therapeutic agents of these embodiments are preferably LABA (eg, formoterol, salmeterol), short-acting β-agonists (eg, albuterol), corticosteroids (eg, fluticasone), LAMA (eg, tiotropium). , Antibiotics (eg, levofloxacin) and one or more agents independently selected from the group consisting of combinations thereof. If the dry powder is intended for the treatment of CF, suitable additional therapeutic agents are short-acting β-agonists (eg, albuterol), antibiotics (eg, levofloxacin), recombinant human deoxyribonuclease I. (For example, Dornase α, also known as DNase), sodium channel blockers (eg, amiloride) and combinations thereof.

In a more specific embodiment, the dry particles for inhalation contain a calcium salt (eg, calcium lactate), a sodium salt (eg, sodium chloride), and an additional therapeutic agent, wherein the additional therapeutic agent. , About 20% (w / w) ~ about 90% (w / w), about 20% (w / w) ~ about 80% (w / w), about 20% (w / w) ~ about 60% ( w / w), about 20% (w / w) ~ about 50% (w / w), about 50% (w / w) ~ about 90% (w / w), about 50% (w / w) ~ Antibiotics present at concentrations of about 80% (w / w), about 60% (w / w) to about 90% (w / w) or about 60% (w / w) to about 80% (w / w) It is a substance (eg, levofloxacin), and the ratio of calcium ion to sodium ion (molar: mol) is about 1: 1 to about 16: 1, about 1: 1 to about 8: 1, about 1: 1 to about 4: 1, about 1: 1 to about 3.9: 1, about 1: 1 to about 3.5: 1, about 2: 1 to about 4: 1, about 2: 1 to about 3.9: 1, about 2: 1 to about 3: 5 or about 4: 1.

If it is desired to retain the relative proportions of divalent salts, monovalent salts and modifiers of any particular dry powder and dry particle formulation described herein, add to the solution of the components of the dry powder. A therapeutic agent may be added and the resulting solution may be spray dried to produce dry particles. For such particles, with the addition of additional therapeutic agents, the amount of divalent salt, monovalent salt and excipient in the dry particles is greater than the amount in the dry powder or dry particles described herein, respectively. Less. In one example, the formulation can contain up to about 20% (w / w) of additional therapeutic agent, with proportionally reduced amounts of each of the divalent salt, monovalent salt and excipient. However, the ratio of divalent salt: monovalent salt: amount of excipient (wt%) is the same as for the dry powder or dry particles described herein. In another example, the formulation may contain up to about 6% (w / w) of additional therapeutic agent. In a further example, the formulation may contain up to about 1% (w / w) of additional therapeutic agent.

In a typical embodiment, the dry particles are based on formulation VIII, with one or more additional therapeutic agents of about 6% (w / w) or less, and about 70% to about 75% (w / w). Calcium lactate, about 3% to about 5% (w / w) sodium chloride, and about 17% to about 20% (w / w) leucine. In other typical embodiments, the dry particles are based on Formulation VII with one or more additional therapeutic agents of about 6% (w / w) or less and about 45.0% to about 58.6% (w / w). It contains w) calcium lactate, about 1.9% to about 3.9% (w / w) sodium chloride, and about 27.5% to about 37.5% (w / w) leucine. In a more typical embodiment, the dry particles are based on Formulation VIII, with one or more additional therapeutic agents of about 20% (w / w) or less and about 60% to about 75% (w / w). ), Approximately 2% to approximately 5% (w / w) sodium chloride, and approximately 15% to approximately 20% (w / w) leucine. In other typical embodiments, the dry particles are based on Formulation VII with one or more additional therapeutic agents of about 20% (w / w) or less and about 54.6% to about 58.6% (w / w). It contains w) calcium lactate, about 1.9% to about 3.9% (w / w) sodium chloride, and about 34.5% to about 37.5% (w / w) leucine. If the additional treatment is potent, 0. Small amounts, such as 01% to about 1% (w / w), may be used and the composition of the dry particles is substantially the same as formulation VIII or VII. The additional therapeutic agent can be any of the additional therapeutic agents described herein. Suitable additional therapeutic agents are LABA (eg, formoterol, salmeterol), short-acting β-agonists (eg, albuterol), corticosteroids (eg, fluticasone), LAMA (eg, tiotropium), antibiotics (eg, thiotropium). For example, levofloxacin, tobramycin) and combinations thereof. If the dry powder is intended for the treatment of CF, suitable additional therapeutic agents are short-acting β-agonists (eg, albuterol), antibiotics (eg, levofloxacin), recombinant human deoxyribonuclease I. (For example, Dornase α, also known as DNase), sodium channel blockers (eg, amiloride) and combinations thereof.

For dry powders containing additional therapeutic agents, all or part of the excipient component in the dry powders or dry particles described herein may be replaced with one or more additional therapeutic agents. This approach is particularly advantageous for additional therapeutic agents that require relatively high effective doses, for example, less potent, with the beneficial effects of calcium cations in the airways and additional therapeutic agents (s). Produces dry particles that have the beneficial effect of. In a typical example, the dry particles are based on Formulation VIII, with one or more additional therapeutic agents of about 0.01% to about 20% (w / w) and about 75% (w / w). It contains calcium lactate, about 5% (w / w) sodium chloride, and about 20% (w / w) or less leucine. In other typical embodiments, the dry particles are based on formulation VII, with one or more additional therapeutic agents of about 0.01% to about 37.5% (w / w) and about 58.6% (w / w). It contains w) calcium lactate, about 3.9% (w / w) sodium chloride, and about 37.5% (w / w) or less leucine. The additional therapeutic agent can be any of the additional therapeutic agents described herein. Suitable additional therapeutic agents are LABA (eg, formoterol, salmeterol), short-acting β-agonists (eg, albuterol), corticosteroids (eg, fluticasone), LAMA (eg, tiotropium), antibiotics (eg, thiotropium). For example, levofloxacin, tobramycin) and combinations thereof. Specific examples of this type of dry powder are disclosed herein as formulations X to XX.

In one aspect, salts of divalent cations (eg, calcium, magnesium) can be co-prepared with active substances other than calcium to produce small, highly dispersible powders or large, porous particles. Optionally, these particles may also contain a monovalent cationic salt (eg, sodium, potassium) and optionally a excipient (eg, leucine, maltodextrin, mannitol, lactose). The components can be mixed (eg, mixed as one solution, statically mixed as two solutions) so that they form a single particle after spray drying.

Some of the dry powders for inhalation of the present invention have no pharmacological effect or have no therapeutic effect by themselves (for example, an amount less than the therapeutic amount, for example, a salt of a low% divalent metal cation). (For example, less than about 20% (w / w), less than 15% (w / w), less than 10% (w / w), less than 5% (w / w) or less than 3% (w / w)), etc. ) Contains dry particles for inhalation containing divalent metal cations or salts thereof. For example, dry particles for inhalation may contain magnesium ions or magnesium salts such as magnesium lactate, magnesium sulfate, magnesium citrate, magnesium carbonate, magnesium chloride, magnesium phosphate or any combination thereof. Magnesium lactate and magnesium sulfate are preferred. Dry particles for inhalation of this type may be large and dispersible, but are preferably small and dispersible and have a high mass density as described herein (eg, tap density or envelope density). high). The use of such particles as carrier particles for delivering other therapeutic agents, for example by mixing with a therapeutic agent or by incorporating the therapeutic agent into the particles (eg, by co-spray drying). Can be done. Suitable therapeutic agents that can be delivered using these types of particles are LABA (eg, formoterol, salmeterol), short-acting β-agonists (eg, formoterol, salmeterol), especially when co-spray-dried with other particle components. For example, albuterol), corticosteroids (eg fluticasone), LAMA (eg tiotropium), antibiotics (eg levofloxacin, tobramycin) and combinations thereof. If the dry powder is intended for the treatment of CF, suitable additional therapeutic agents are short-acting β-agonists (eg, albuterol), antibiotics (eg, levofloxacin), recombinant human deoxyribonuclease I. (For example, Dornase α, also known as DNase), sodium channel blockers (eg, amiloride) and combinations thereof. In addition, dry grains for inhalation

The relative ratio of the divalent metal cation or salt thereof, the therapeutic agent and any excipient, for example, of the dry powder contained in one or two capsules or blister (eg, 50 mg capsules, 40 mg capsules). By inhalation, a sufficient amount of therapeutic agent is selected to be contained in the dry powder so that an effective amount of therapeutic agent is conveniently administered to the subject. Therefore, the amount of therapeutic agent is from about 0.01% (w / w) with a strong therapeutic agent (or low molecular weight therapeutic agent) such as tiotropium to low potency like many antibiotics (eg levofloxacin). It can vary up to about 90% (w / w) with (or high molecular weight) therapeutic agents. For example, LABA (eg, formoterol, salmeterol), corticosteroids (eg, fluticasone) and LAMA (eg, tiotropium) are generally highly effective, with dry particles for inhalation of about 0.01% (w / w) to about 20. % (W / w), preferably about 0.01% (w / w) to about 10% (w / w) or about 0.01% (w / w) to about 5% (w / w) of these therapeutic agents May contain (ie, alone or in any combination). Antibiotics are generally relatively ineffective and require relatively high doses to be therapeutically effective. Therefore, dry particles for inhalation may contain from about 10% (w / w) to about 99% (w / w) of antibiotics. Preferably, the dry particles for inhalation containing the antibiotic are about 10% (w / w) to about 80% (w / w), about 25% (w / w) to about 80% (w / w) or Contains about 25% (w / w) to about 75% (w / w) antibiotics.

In such dry particles for inhalation, salts of one or more divalent metal cations and compensators in an amount sufficient to obtain the desired particle properties (eg, size, dispersibility, tap density). There are (eg, monovalent salts, sugars, polysaccharides, sugar alcohols, amino acids and any combination thereof) (in% (w / w)). In general, the amount of salt of divalent metal cations in dry inhalation particles is sufficient to obtain at least about 5% (w / w) of dry metal divalent cations, eg, dry inhalation particles. Can contain from about 20% to about 90% (w / w) salts of divalent metal cations. Dry particles contain about 5% to about 95% divalent metal cations, about 5% to about 90%, about 5% to about 85%, about 5% to about 80%, about 5% to about 75%, About 5% to about 70%, about 5% to about 65%, about 5% to about 60%, about 5% to about 55%, about 5% to about 50%, about 5% to about 45%, about 5 % ~ About 40%, about 5% ~ about 35%, about 5% ~ about 30%, about 5% ~ about 25%, about 5% ~ about 20%, about 5% ~ about 15%, about 5% ~ It may contain about 10% or about 5% to about 8%. In a preferred embodiment, the dried particles contain from about 5% to about 20% divalent cations, and in a more preferred embodiment, the dried particles contain from about 5% to about 15% divalent cations. Excipients are generally present in dry particles for inhalation in an amount of 0% to about 50%, preferably about 10% to about 50%.

Thus, in some embodiments, the present invention comprises magnesium salts, therapeutic agents, and optionally excipients (eg, monovalent metal salts, sugars, polysaccharides, sugar alcohols, amino acids and any combination thereof). It is a dry powder for inhalation containing the dry particles for inhalation contained therein. The dry particles for inhalation are preferably small (eg, VMGD at 1.0 bar less than 10 μm, preferably less than 5 μm) and dispersible (1 / 4bar and / or 0.5 / 4bar as described herein). Is 2.2 or less). Preferably, the MMAD of the dry particles for inhalation is from about 0.5 μm to about 10 μm, more preferably from about 1 μm to about 5 μm. Preferably, the dry particles for inhalation are also dense, with a tap density of between about 0.4 g / cc and about 1.2 g / cc, preferably between about 0.55 g / cc and about 1.0 g / cc. The magnesium salt can be magnesium lactate, magnesium sulfate, magnesium citrate, magnesium carbonate, magnesium chloride, magnesium phosphate or any combination thereof. In a preferred embodiment, the magnesium salt is magnesium lactate or magnesium sulfate. The therapeutic agents of these embodiments are preferably LABA (eg, formoterol, salmeterol), short-acting β-agonists (eg, albuterol), corticosteroids (eg, fluticasone), LAMA (eg, tiotropium). , Antibiotics (eg, levofloxacin) and one or more agents independently selected from the group consisting of combinations thereof. If the dry powder is intended for the treatment of CF, suitable additional therapeutic agents are short-acting β-agonists (eg, albuterol), antibiotics (eg, levofloxacin), recombinant human deoxyribonuclease I. (For example, Dornase α, also known as DNase), sodium channel blockers (eg, amiloride) and combinations thereof.

In a more specific embodiment, the dry powder for inhalation contains at least about 5% (w / w) magnesium ions, 1) about 5% to about 45% therapeutic agent, and about 20% to about 90 magnesium salts. % And the therapeutic agent about 0.01% to about 20%; 2) The complementary agent about 0.01% to about 30%, the magnesium salt about 20% to about 80%, and the therapeutic agent about 20% to about 60%; or 3 ) Contains dry particles for inhalation containing about 0.01% to about 20% of the complementary agent, about 20% to about 60% of the magnesium salt, and about 60% to about 99% of the therapeutic agent. The dry particles for inhalation are preferably small (eg, VMGD at 1.0 bar less than 10 μm, preferably less than 5 μm) and dispersible (1/4 bar and / or 0.5 / as described herein). 4bar is 2.2 or less). Preferably, the MMAD of the dry particles for inhalation is from about 0.5 μm to about 10 μm, more preferably from about 1 μm to about 5 μm. Preferably, the dry particles for inhalation are also dense, with a tap density of about 0.4 g / cc to about 1.2 g / cc, preferably about 0.55 g / cc to about 1. It is between 0g / cc. The magnesium salt can be magnesium lactate, magnesium sulfate, magnesium citrate, magnesium carbonate, magnesium chloride, magnesium phosphate or any combination thereof. In a preferred embodiment, the magnesium salt is magnesium lactate or magnesium chloride. The therapeutic agents of these embodiments are preferably LABA (eg, formoterol, salmeterol), short-acting β-agonists (eg, albuterol), corticosteroids (eg, fluticasone), LAMA (eg, tiotropium). , Antibiotics (eg, levofloxacin) and one or more agents independently selected from the group consisting of combinations thereof. If the dry powder is intended for the treatment of CF, suitable additional therapeutic agents are short-acting β-agonists (eg, albuterol), antibiotics (eg, levofloxacin), recombinant human deoxyribonuclease I. (For example, Dornase α, also known as DNase), sodium channel blockers (eg, amiloride) and combinations thereof.

Alternatively, the particles may be large, for example, dry powders have a geometric particle size (VMGD) between 5 and 30 microns. Optionally, the particles are large and the tap density can be between 0.01 g / cc and 0.4 g / cc or between 0.05 g / cc and 0.3 g / cc. For particles with small or large VMGD, the MMAD of the dry powder is between 0.5 and 10 microns, more preferably between 1 and 5 microns.

In another aspect, the dry particles of the present invention are large, porous and dispersible. The size of the dry particles can be expressed in various ways. The particles can have a VMGD of between 5 μm and 30 μm or between 5 μm and 20 μm and a tap density of less than 0.5 g / cc, preferably less than 0.4 g / cc.

How to prepare dry powder and dry particles Dry particles and powders for inhalation can be prepared using any suitable method. Suitable methods for preparing dry powders and particles for inhalation have traditionally existed in the art and such methods include single and double emulsion solvent evaporation, spray drying, grinding (eg, jet grinding). , Mixing, solvent extraction, solvent evaporation, phase separation, simple and composite coaselation, interfacial polymerization, supercritical carbon dioxide (CO)₂), Appropriate methods, including ultrasonic crystallization, nanoparticle agglomeration formation, other suitable methods and any combination thereof. Dry particles for inhalation may be produced using a method for producing microspheres or microcapsules known in the art. These methods can be used under conditions that result in the formation of dry inhalation particles with the desired aerodynamic properties (eg, aerodynamic and geometric particle sizes). If desired, dry inhalation particles with properties such as desired size and density may be sorted using a suitable method such as sieving.

The dry particles for inhalation are preferably spray-dried. Suitable spray drying techniques are described, for example, in the "Spray Drying Handbook" by K. Masters, John Wiley &amp; Sons, New York (1984). Generally, in spray drying, the heat of heated air or a hot gas such as nitrogen is used to evaporate the solvent from the droplets formed by the spraying of a continuously supplied liquid. When hot air is used, at least some of the moisture in the air is removed before use. When nitrogen is used, the nitrogen gas can flow in a "dry" state, i.e., without adding excess water vapor to the gas. Nitrogen or air moisture levels can be set as needed before starting spray drying at a fixed value above "dry" nitrogen. If necessary, equipment such as spray dryers used to prepare dry particles, such as jet grinders, are in-line geometric particle size analyzers, which determine the geometric particle size of dry particles for inhalation during manufacture. And / or may include an in-line aerodynamic particle size analyzer that determines the aerodynamic particle size of dry inhalation particles during manufacture.

In spray drying, a solution, emulsion or suspension containing the components of the dried particles to be produced in a suitable solvent (eg, aqueous solvent, organic solvent, aqueous-organic mixture or emulsion) is sprayed into a drying tank by a spraying device. To do. For example, a nozzle or rotary atomizer can be used to spray the solution or suspension into the drying tank. The nozzle may be a two-fluid nozzle with an internal mixing setting and an external mixing setting. For example, a rotary atomizer with 4 or 24 bladed wheels can be used. Examples of suitable spray dryers that may be equipped with a rotary sprayer or nozzle are the Mobile Minor Spray Dryer or Model, both manufactured by Niro (Denmark). PSD-1 can be mentioned. The actual spray drying conditions will depend in part on the composition of the spray dried solution or suspension and the flow rate of the raw material. One of ordinary skill in the art will be able to determine suitable conditions based on the composition of the solution, emulsion or suspension to be spray dried, desired particle properties and other factors. Generally, the inlet temperature to the spray dryer is about 90 ° C to about 300 ° C, preferably about 220 ° C to about 285 ° C. Another suitable range is between 130 ° C and about 200 ° C. The outlet temperature of the spray dryer depends on factors such as the supply temperature and the characteristics of the raw material to be dried. Generally, the outlet temperature is from about 50 ° C to about 150 ° C, preferably from about 90 ° C to about 120 ° C or from about 98 ° C to about 108 ° C. Another preferred range is between 65 ° C and about 110 ° C, more preferably from about 75 ° C to about 100 ° C. If desired, the produced dry inhalation particles may be fractionated by volume, eg, using a sieve, or by aerodynamic magnitude, eg, using a cyclone. And / or may be separated by density using techniques known to those skilled in the art.

In order to prepare dry particles for inhalation of the present invention, generally, a solution, emulsion or suspension (that is, raw material) containing a desired component of a dry powder is prepared and spray-dried under appropriate conditions. .. Preferably, the concentration of solids dissolved or suspended in the feedstock is at least about lg / L, at least about 2 g / L, at least about 5 g / L, at least about 10 g / L, at least about 15 g / L, at least about about. 20g / L, at least about 30g / L, at least about 40g / L, at least about 50g / L, at least about 60g / L, at least about 70g / L, at least about 80g / L, at least about 90g / L or at least about 100g / L It is L. Feeding materials can be obtained by dissolving or suspending suitable ingredients (eg, salts, excipients, other active ingredients) in suitable solvents to prepare a single solution or suspension. Solvents, emulsions or suspensions can be prepared using any suitable method, such as drying and / or bulk mixing of liquid components or static mixing of liquid components to form a formulation. For example, a static mixer is used to mix a hydrophilic component (eg, an aqueous solution) and a hydrophobic component (eg, an organic solution) to form a formulation. The formulation is then sprayed into droplets, which are dried to form dry particles for inhalation. Preferably, the spraying step is performed immediately after mixing the components in a static mixer. Alternatively, the spraying step is performed with a bulk mixed solution.

In one example, dry inhalation particles containing calcium citrate, sodium chloride and leucine are prepared by spray drying. Prepare the first phase containing an aqueous solution of sodium citrate and leucine. Prepare a second phase containing calcium chloride in a suitable solvent. One or both solutions may be heated separately as needed to ensure that the ingredients dissolve. The first and second phases are then mixed in a static mixer to form a mixture. The mixture is spray dried to form dry particles for inhalation.

Any suitable solvent, such as an organic solvent, an aqueous solvent or a mixture thereof, can be used to prepare the feedstock or ingredients of the feedstock. Suitable organic solvents that can be used include, but are not limited to, alcohols such as ethanol, methanol, propanol, isopropanol, butanol and the like. Other organic solvents include, but are not limited to, perfluorocarbo, dichloromethane, chloroform, ether, ethyl acetate, methyl tert-butyl ether and the like. Examples of the co-solvent that can be used include an aqueous solvent and an organic solvent such as the above-mentioned organic solvent without particular limitation. Aqueous solvents include water and buffer solutions.

The feedstock or ingredients of the feedstock can have any desired properties such as pH, viscosity. If necessary, a pH buffer may be added to the solvent or co-solvent or the mixture formed. Generally, the pH of the mixture is in the range of about 3 to about 8.

Dry particles and powders for inhalation can be prepared and then separated by, for example, filtration or centrifugation using a cyclone to obtain a particle sample with a preselected particle size distribution. For example, more than about 30%, more than about 40%, more than about 50%, more than about 60%, more than about 70%, more than about 80%, or more than about 90% dry particles in the sample. Has a particle size within the selected range. The selected range containing a particular percentage of dry inhalation particles can be in any size range, such as VMGD of about 0.1 to about 3 microns, as described herein.

The particle size of dry inhalation particles, such as VMGD, can be determined by an electrical sensing band device such as Multisizer IIe (Coulter Electronic, Luton, Beds, England), or a HELOS system (Sympatec, Princeton, NJ) or Mastersizer system (Malvern, Worcestershire). , UK) can be measured using a laser diffractometer. Other instruments for measuring geometric particle size are known in the art. The particle size of the dry inhalation particles in the sample varies depending on factors such as particle composition and synthetic method. The particle size distribution of dry inhalation particles in the sample can be selected to allow optimal deposition within the target site within the respiratory system.

Time-of-flight (TOF) measurements can be used to experimentally determine aerodynamic particle size. For example, an instrument such as the Aerosol Particle Sizer (APS) Spectrometer (TSI Inc., Shoreview, MN) can be used to measure aerodynamic particle size. APS measures the time it takes for an individual dry inhalation particle to pass between two fixed laser beams.

The aerodynamic particle size may also be determined directly experimentally using conventional gravity settling methods, which measure the time required for a sample of dry inhalation particles to settle a particular distance. To do. Indirect methods for measuring the aerodynamic mass medial diameter include the Andersen Cascade Impactor and the Multistage Liquid Impinger (MSLI) method. Methods and instruments for measuring aerodynamic particle size are known in the art.

Tap density is accepted as an approximate measure of envelope mass density that characterizes particles. The envelope mass density of a statistically isotropic particle is defined as the mass of the particle divided by the envelope volume of the smallest sphere that can contain the particle inside. Features that can contribute to low tap density include irregular surface texture, high particle cohesiveness and porous structure. Tap density is similar to Dual Platform Microprocessor Controlled Tap Density Tester (Vankel, NC), GeoPyc equipment (Micrometrics Instrument Corp., Norcross, GA) or SOTAX Tap Density Tester model TD2 (SOTAX Corp., Horsham, PA). It can be measured using a device known to those skilled in the art. USP Bukf Density and Tapped Density, United States Pharmacopia The tap density may be determined using the method convention, Rockville, MD, 10th Edition Addendum, 4950-4951, 1999.

A fine particle fraction can be used as one method for characterizing the aerosol performance of the dispersed powder. The fine particle fraction represents the particle size distribution of dry particles for inhalation in the air. Gravimetric analysis with a cascade impactor is a method of measuring the particle size distribution or fine particle fraction of dry inhaled particles in the air. The Andersen Cascade Impactor (ACI) is an 8-stage impactor capable of separating aerosols into nine different fractions based on aerodynamic magnitude. The size cutoff at each stage depends on the flow velocity at which the ACI is operated. ACI consists of multiple stages consisting of a series of nozzles (ie, jet plates) and collision surfaces (ie, collision discs). At each stage, the aerosol stream passes through the nozzle and collides with the surface. Of the dry suction particles in the aerosol stream, particles with sufficient inertia collide with the plate. Small dry inhalation particles that do not have enough inertia to collide with the plate remain in the aerosol stream and are transported to the next stage. The aerosol velocity in the nozzle increases with each successive stage of ACI, gradually allowing small inhalation dry particles to be collected at each successive stage.

If necessary, the fine particle fraction may be measured using a two-stage collapsing ACI. The two-stage collapsing ACI consists of only the upper two stages of the eight-stage ACI, stage 0 and stage 2, and the final collection filter, and is capable of collecting two different powder fractions. Specifically, the two-stage disintegrating ACI is calibrated so that the powder fraction collected in stage 2 consists of dry inhalation particles with an aerodynamic particle size greater than 3.4 microns and less than 5.6 microns. To do. Therefore, the powder fraction that passes through stage 2 and deposits on the final collection filter consists of dry inhalation particles with an aerodynamic particle size of less than 3.4 microns. The airflow in such calibration is about 60 L / min. FPF (&lt;5.6) has been shown to correlate with a fraction of powder that can reach the patient's lungs, while FPF (&lt;3.4) can reach deep into the patient's lungs. It has been shown to correlate with various powders. Such a correlation provides a quantitative index that can be used for particle optimization.

It is possible to estimate the release dose using ACI, which is referred to herein as the weight-measured recovery dose and the analytical recovery dose. "Weighed recovery dose" is defined as the ratio of powder weight to nominal dose as measured by ACI's all-stage filters. "Analytical recovery dose" is defined as the ratio of powder recovered from all stages of ACI, all stages of filters and inspiratory port washes to the nominal dose. FPF_TD (&lt;5.0) is the ratio of the interpolation amount of powder deposited below 5.0 μm in ACI to the nominal dose. FPF_RD (&lt;5.0) is the ratio of the interpolation amount of powder deposited below 5.0 μm by ACI to the recovery dose by weight measurement or analysis.

Another way to estimate the release dose is to determine the amount of powder that comes out of the container, eg, capture or blister, when the dry powder inhaler (DPI) is activated. This takes into account the percentage of powder that comes out of the capsule, but does not take into account any powder that is deposited in the DPI. The mass of powder released is the difference between the weight of the capsule with the dose before inhaler activation and the weight of the capsule after inhaler activation. This measurement is sometimes referred to as capsule release powder mass (CEPM) or sometimes "shot weight".

The multistage liquid impinger (MSLI) is another device that can be used to measure particulate fractions. The multi-stage liquid impinger operates on the same principle as ACI, but instead of 8 stages, MSLI has 5 stages. In addition, each stage of MSLI consists of a glass frit moistened with ethanol instead of a solid plate. The moistened stage is used to prevent possible particle bounce and re-scattering when using ACI.

The geometric particle size distribution of the dry powder for inhalation after being released from the dry powder inhaler (DPI) can be measured using a laser diffracting instrument such as Malvern Spraytec. The inhaler adapter is placed in a closed bench arrangement, the DPI is airtightly sealed, and the injected aerosol is vertically crossed through the laser beam as an internal flow. In this way, the vacuum pressure can generate a known flow velocity from the DPI to empty the DPI. For samples obtained, usually at 1000 Hz during the inhalation time, the geometric particle size distribution of the resulting aerosol was measured with a photodetector and DV50, GSD, FPF &lt;5.0 μm were measured over the entire inhalation time. Get the average.

The present invention also relates to a method for producing a dry powder for inhalation containing dry particles for inhalation containing calcium citrate or calcium sulfate. This method involves preparing a) a first liquid feedstock containing an aqueous solution of calcium chloride and a second liquid feedstock containing an aqueous solution of sodium sulfate or sodium citrate, and b) with a first liquid feedstock. Mixing with a second liquid feedstock, the resulting anion exchange reaction produces a mixture of calcium sulfate and sodium chloride, or a saturated or supersaturated solution containing calcium citrate and sodium chloride, and c). b) Includes spray-drying the saturated or supersaturated solution produced in step 2 to produce dry particles for inhalation. The first liquid feedstock and the second liquid feedstock can be batch mixed or preferably statically mixed. In some embodiments, the resulting mixture is mixed, preferably within 60 minutes, within 30 minutes, within 15 minutes, within 10 minutes, within 5 minutes, within 4 minutes, within 3 minutes, 2 from static mixing. Spray dry within minutes, within 1 minute, within 45 seconds, within 30 seconds, within 15 seconds, within 5 seconds.

The present invention also relates to dry inhalation powders or dry inhalation particles produced using any of the methods described herein.

The dry inhalation particles of the present invention may also be characterized by the chemical stability of the salt or modifier contained in the dry inhalation particles. The chemical stability of the component salts can affect the important characteristics of the inhalation particles, including shelf life, suitable storage conditions, acceptable dosing environment, biocompatibility and salt efficacy. .. Chemical stability can be characterized using techniques known in the art. An example of a technique that can be used to assess chemical stability is reverse phase high performance liquid chromatography (RP-HPLC). The dry particles for inhalation of the present invention generally contain a long-term stable salt.

If desired, the dry inhalation particles and dry powders described herein may be further treated to increase stability. An important feature of medicinal dry powders is whether they are stable under different temperature and humidity conditions. Since the unstable powder absorbs the surrounding moisture and aggregates, the particle size distribution of the powder changes.

Excipients such as maltodextrin may be used to make more stable particles and powders. Maltodextrin can act as a stabilizer for the amorphous phase and prevent the components from changing from amorphous to crystalline. Alternatively, if aggregates are formed during the crystallization process, in a controlled manner to assist the particle crystallization process, for example by passing the particles through a cyclone to separate the aggregates (eg,). The post-treatment stage (in a high humidity bug house) can be used for the resulting powder that may be further treated to restore its dispersibility. Another possible approach is to optimize the surrounding process conditions that result in the production of more crystalline and thus more stable particles. Another approach is to attempt to produce more stable salt forms with different excipients or at varying levels of excipients.

The dry particles and powders for inhalation described herein are suitable for inhalation therapy. Dry particles for inhalation can be produced with raw materials, surface roughness, diameter and tap density suitable for local delivery to selected areas such as the deep lungs of the respiratory system or the upper and middle respiratory tract.

The energy required to perform the inhalation operation can be calculated to correlate the dispersion of powder from inhalers at different inhalation rates, volumes, and resistances. The intake energy is the equation E = R²Q²It can be calculated from V. In the formula, E is the intake energy expressed in joules and R is kPa.^1/2The resistance of the inhaler expressed in / LPM, Q is the steady flow velocity expressed in L / min, and V is the volume of intake air expressed in L.

The dry powders and particles for inhalation described herein add total inhalation energy of less than about 2 joules, less than about 1 joule, less than about 0.8 joules, less than about 0.5 joules or less than about 0.3 joules to the dry powder inhaler. The dry powder inhaler is characterized by a high release dose (eg, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% CEPM). For example, if the total inhalation energy of less than about 1 joule (eg, less than about 0.8 joules, less than about 0.5 joules, less than about 0.3 joules) is added to the dry powder inhaler, about 50 mg or about 40 mg in the dry powder inhaler. A release dose of CEPM of at least 75%, at least 80%, at least 85%, at least 90%, at least 95% of Formula I or Formula II contained in a unit dose container containing the appropriate formulation of. When a total inhalation energy of about 0.28 joules is applied to the dry powder inhaler, at least the inhalation dry powder contained in the dry powder inhaler in a unit dose container containing about 50 mg or about 40 mg of inhalation dry powder. A release dose of about 70% CEPM can be achieved. The unit dose container may be filled with dry powder and the unit dose container may be filled with at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90%. Unit dose containers are capsules (eg, sizes 000, 00, 0E, 0, 1, 2, 3 and 4, with volumes of 1.37 ml, 950 μl, 770 μl, 680 μl, 480 μl, 360 μl, 270 μl and 200 μl, respectively). It may be. Alternatively, the unit dose container may be a blister. The blister may be packaged as a single blister or as part of a set of blister, such as 7 blister, 14 blister, 28 blister or 30 blister.

A healthy adult population is 0.02 kPa^1/2/ LPM and 0.055kPa^1/2The maximum inspiratory flow velocity (PIFR) measured by Clarke et al. (Journal of Aerosol Med, 6 (2), p.99-110, 1993) for the flow velocity Q obtained from the two types of inhaler resistance of / LPM. A study by Tiddens et al. (Journal of Aerosol Med, 19 (4), p.456-) found that the average inhaled volume of adults by the FDA guidelines for dry powder inhalers and various DPIs was 2.2 L. When used with an inhalation volume of 2 L based on both 465,2006), it is expected that inhalation energy ranging from 2.9 joules for easy inhalation to 22 joules for maximum inspiration can be achieved.

It is expected that mild, moderate and severe adult COPD patients can achieve maximum inhalation energies of 5.1-21 joules, 5.2-19 joules and 2.3-18 joules, respectively. This is also due to the use of PIFR measurements for the flow velocity Q in the inspiratory energy equation. The achievable PIFR in each group is a function of inhaler resistance at which inhalation takes place. Using a study by Broeders et al. (Eur Respir J, 18, p.780-783, 2001), each resistance was 0.021 kPa.^1/2/ LPM and 0.032kPa^1/2We predicted the maximum and minimum PIFR achievable with two types of dry powder inhalers that are / LPM.

Similarly, adult asthma patients are expected to be able to achieve a maximum inhalation energy of 7.4-21 joules based on the same assumptions as the PIFR data from the COPD population and Broeders et al.

For example, healthy adults and children, COPD patients, asthma patients over 5 years of age, and CF patients can add sufficient inhalation energy to empty and disperse the dry powder formulation of the invention. For example, a 50 mg dose of Formula I or Formula II was found to require only 0.28 joules to empty 70% or more of the filling weight with a single inhalation. It was calculated that the entire adult patient population listed above could achieve more than 2 joules, more than 7 times the inhaled energy required. For example, in a fully deagglomerated 25 mg dose of Formula II, to empty 80% of the filling weight with a single inhalation, as indicated by its Dv50 within 1 micrometer at much higher inhalation energy. It turns out that only 0.16 joules are needed. The entire adult patient population listed above was calculated to be able to achieve more than 2 joules, which is more than an order of magnitude higher than the required inhalation energy.

The advantage of the present invention is the production of a powder that disperses well at various flow rates and is less dependent on the flow rate. The dry particles and powders of the present invention allow easy and passive use of DPI for a wide patient population.

Method The inhalation dry powder of the present invention and the inhalation dry particles of the present invention are for administration to the respiratory tract. The dry powders and dry particles of the invention are administered to subjects in need thereof for respiratory (eg, lung) disorders such as asthma, airway hyperresponsiveness, seasonal allergic allergies, bronchial dilatation, chronic bronchitis. , Pulmonary emphysema, chronic obstructive pulmonary disease, cystic fibrosis, pulmonary parenchymal inflammatory condition, etc., and acute exacerbations of the above chronic diseases, such as viral infections (eg, influenza virus, parainfluenza virus, respiratory polynuclear virus) , Rhinovirus, adenovirus, metapneumovirus, coxsackie virus, echovirus, coronavirus, herpesvirus, cytomegalovirus, etc., bacterial infections (eg, Streptococcus pneumoniae, commonly referred to as Streptococcus pneumoniae), Staphylococcus -Aureus (Staphylococcus aureus), Burkholderis species, Streptococcus agaractia (Streptococcus) agalactiae), Haemophilus influenzae, Haemophilus parainfluenzae, Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, Moraxella catarrhala, Moraxella catarrhala, Moraxella catarrhala, Moraxella catarrhala Chlamydophila pneumoniae, Mycoplasma pneumoniae, Legionella pneumophila, Serratia marcescens, Mycobacterium tuberosis, Mycobacterium tuberculosis, Mycoplasma pneumoniae, Serratia marcescens, Mycobacterium tuberosis Pertussis, etc.), fungal infections (eg, Histoplasma capsulatum, Cryptococcus neoformans, Pneumocystis jiroveci, Coccidioidomycosis, etc. Due to infectious diseases (eg, Toxoplasma gondii, Strongyloides stercoralis, etc.), or environmental allergens and irritants (eg, aerial allergens, including pollen and cat dimples, aerial particulates, etc.) The exacerbations caused can be treated and / or prevented.

The dry powders and dry particles of the present invention are administered to subjects in need thereof for airway infections such as pneumonia (nosocomial pneumonia, nosocomial pneumonia (nosocomial infectious pneumonia, HAP; medical related pneumonia, HCAP)). , Artificial Respirator-Related Pneumonia (VAP)), Artificial Respiratory-Related Bronchial Bronchitis (VAT), Bronchitis, Group (eg Post-Intubation Group and Infectious Group), Tuberculosis, Influenza, Storm and Virus Infections (eg) , Influenza virus, parainfluenza virus, respiratory polynuclear virus, rhinovirus, adenovirus, metapneumovirus, coxsackie virus, echovirus, coronavirus, herpesvirus, cytomegalovirus, etc., bacterial infections (eg, generally pneumococcus) Streptococcus pneumoniae, Staphylococcus aureus, Streptococcus agalactiae, Haemophilus influenzae), Haemophilus parainfluenzae, Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, Moraxella catarrhalis, Moraxella catarrhalis, Claraxella catarrhalis Mycoplasma pneumoniae, Legionella pneumophila, Serratia marcescens, Mycobacterium tuberculosis, Bordetella pertussis, etc. (For example, Histoplasma capsulatum), Cryptococcus neoformans, Pneumocystis jiroveci, Cocccidioides immitis, etc.) or parasitic infections (eg, Toxoplasma gondii) Strongyloides stercoralis)), or environmental allergens and irritants (eg, air allergens, air microparticles, etc.), etc. can be treated and / or prevented, and / or their contact transmission can be reduced. Similarly, for inhalation to prevent or treat chronic infections such as bacterial colonization and biomembrane formation that are common in persons with chronic respiratory diseases such as cystic fibrosis and chronic obstructive pulmonary disease. Dry particles or dry powders can be administered to subjects in need of it. Although not bound by any particular theory, the dry inhalation particles or dry powders described herein are ion-controlled ion channels, such as TRP channels (eg, TRPV, TRPC, etc.). Bacteria by activating ion channels such as TRPM, TRPA channels) to mediate antibacterial defense, eg, final induction such as secretion of antibacterial peptides (eg, α-, β-, θ-defensins). It is believed that infections can be prevented and / or treated.

Dry particles and powders for inhalation can be administered to alter the biophysical and / or biological properties of the mucosal lining of the airways (eg, airway lining fluid) and underlying tissues (eg, airway epithelium). .. Such properties include, for example, gelation of the mucous surface, surface tension of the mucosal inner layer, surface elasticity and / or viscosity of the mucosal inner layer, volumetric elasticity and / or viscosity of the mucosal inner layer. Although not bound by any particular theory, the benefits provided by the inhaled dry particles or dry powders and methods described herein (eg, therapeutic and preventive benefits) are inhalation dry. Ca provided by calcium cations (dry particles for inhalation or calcium salts in dry powder) in the airways (eg, lung mucus or airway lining fluid) after administration of particles or dry powder²⁺) Is considered to be due to the increase.

Dry powder and dry particles for inhalation can be administered to increase the rate of mucous pili clearance. Clearance of microorganisms and inhaled particles is an important function of the respiratory tract to prevent exposure to respiratory infections and potentially harmful substances or their systemic absorption. This is done as an integrated function by epithelial cells, mucus secretory cells and immune response cells present on the surface of the airways. Central to this function are the pili in the epithelial cells on the surface of the airways, which, by synchronizing and waving, transport the underlying liquid mucus coating proximally (towards the mouth). That is. The transported mucus coating exits the respiratory tract and is swallowed or exhaled as sputum.

Dry powder and dry particles for inhalation can be administered to assist all of the above functions. The dry powder for inhalation and the dry particles increase the viscoelasticity of the surface, so that the microorganisms and particles are retained on the surface of the airway mucus coating, from which they are not systemically exposed to the host. Dry powder and particles induce water / liquid transport from airway epithelial cells, reducing the viscosity of the liquid layer around the pili, causing the pili to undulate and move the overlying mucus coating. Increases the efficiency of removal. In addition, dry particles and dry powders containing calcium salts as pharmacologically active substances increase both the number of undulations of the pili and the force or momentum of the pili to contract, and the clearance rate of the mucilage flow overlying the pili. To increase.

Mucofimbria clearance is a safe inhalation radioisotope (eg, technitium (eg, technitium).^99mIt is measured by a well-established technique that quantitatively measures the function and speed of clearance using the solution preparation of Tc)). Radioisotopes are quantitatively measured by scintigraphy from outside the body. Continuous measurements over minutes to hours allow evaluation of clearance rate and drug vs. baseline / control value results.

In some embodiments, the present invention treats lung diseases such as asthma, airway hyperresponsiveness, seasonal allergic allergies, bronchiectasis, chronic bronchitis, emphysema, chronic obstructive pulmonary disease, cystic fibrosis and the like. A method, the method comprising administering an effective amount of the dry particles or powders for inhalation described herein into the respiratory tract of the subject in need thereof.

In another aspect, the present invention relates to pulmonary parenchymal inflammatory / fibrotic diseases such as idiopathic pulmonary fibrosis, inflammatory conditions of the lung interstitial (eg sarcoidosis, allergic interstitial pneumonia (eg farmer's lung)). Fibrogenic dust interstitial diseases (eg, asbestos pneumonia, siliceous pneumonia, berylium pneumonia), eosinophil granulomatosis / histiocytosis X, collagen vascular diseases (eg, rheumatoid arthritis, sclerosis, wolf ), A method of treating and / or reducing the severity of Wegener's granulomatosis, etc., which requires an effective amount of dry particles or powders for inhalation as described herein. Including administration to the airways of.

In other aspects, the invention presents an acute exacerbation of chronic lung diseases such as asthma, airway hyperresponsiveness, seasonal allergic allergies, bronchiectasis, chronic bronchitis, emphysema, chronic obstructive pulmonary disease, cystic fibrosis. A method of treating or preventing, which comprises administering an effective amount of the dry particles or powders for inhalation described herein into the respiratory tract of the subject in need thereof.

In another aspect, the invention is a method of treating, preventing and / or reducing contact transmission thereof for respiratory tract infections, the method of which is an effective amount of dry inhaled particles or dried as described herein. It involves administering the powder to the airways of the subject in need of it.

In yet another aspect, the invention is a method of reducing inflammation, which administers an effective amount of the dry particles or powders for inhalation described herein into the respiratory tract of a subject in need thereof. Including to do. Therefore, with dry particles and powders for inhalation, acute and / or chronic inflammation and especially asthma, airway hypersensitivity, seasonal allergic allergies, bronchial dilatation, chronic bronchitis, emphysema, chronic obstructive pulmonary disease ( Extensive prevention or treatment of inflammation that is characteristic of numerous lung diseases and conditions, including COPD), cystic fibrosis (CF), pulmonary parenchymal inflammatory diseases / conditions, etc. Administration of dry particles and dry powder, both of which play a central role in the etiology of the disease, the inflammation specific to diseases such as asthma, COPD and CF, and the increase in inflammation caused by the acute exacerbation of these diseases. Both can be prevented or treated.

In certain embodiments of the methods described herein, the dry powder or particles for inhalation described herein are administered to a patient who has been pretreated with a bronchodilator, or at the same time as the bronchodilator. Administer. If the patient is pretreated with a bronchodilator, after the bronchodilator, when the onset of bronchodilator effect becomes apparent, or more preferably maximized, dry powder or particles for inhalation It is preferable to administer. Short-acting β such as salbutamol₂The stimulant may be administered from about 10 minutes to about 30 minutes, preferably about 15 minutes before administration of the dry powder or particles for inhalation. Short-acting β like salbutamol₂Pretreatment with stimulants is particularly preferred for CF patients. Some patients may already be taking bronchodilators such as LABA (eg, formoterol). Patients with COPD often take LABA to manage their illness. Patients taking LABA have already experienced some bronchial relaxation due to the action of LABA, so further bronchodilation (eg, short-acting β)₂May not or may not require (with stimulants). For these types of patients, dry powder or particles for inhalation may be administered, eg, in a single formulation, at substantially the same time as or at the same time as LABA.

Inhalation dry particles and dry powder into the airways of the subject in need of it, in any suitable method, such as infusion techniques and / or dry powder inhalers (DPI) or metered dose inhalers (MDI). It can be administered using an inhaler or the like. For example, inhalers, Spinhaler® (Fisons, Loughborough, UK), Rotahers®, Diskhaler® and Diskus® disclosed in U.S. Pat. Nos. 4,995,385 and 4,069,819. (GlaxoSmithKline, Research Triangle Technology Park, North Carolina), FlowCapss® (Hovione, Loures, Portal), Inhalators® (Boehringer-Ingelheim, Germany), Aerolizer® (Novartis, Switzerland), and Many DPIs are available, including other DPIs known to those skilled in the art.

Inhalers (eg, DPIs) are generally capable of delivering the maximum amount of dry powder or dry particles in a single inhalation, which is a blister containing dry particles or dry powder in the inhaler. , Capacities of means such as capsules (eg, sizes 000, 00, 0E, 0, 1, 2, 3 and 4, with volumes of 1.37 ml, 950 μl, 770 μl, 680 μl, 480 μl, 360 μl, 270 μl and 200 μl, respectively). Related to. Therefore, two or more inhalations may be required for delivery of the desired dose or effective amount. Preferably, each dose administered to the subject in need contains an effective amount of dry particles or powder for inhalation and is administered using about 4 or less inhalations. For example, each dose of dry particles or powder for inhalation can be administered in one inhalation or two, three or four inhalations. Dry particles and powders for inhalation are preferably administered in a single respiratory actuation step with a respiratory actuated DPI. When using this type of device, the subject's inhalation energy disperses the inhalation dry particles and at the same time draws them into the airways.

The dry particles or powder for inhalation can be delivered to the desired site in the respiratory tract by inhalation, if desired. It is well known that particles with an aerodynamic particle size of about 1 micron to about 3 microns can be delivered deep into the lungs. Particles with a larger aerodynamic particle size, eg, about 3 microns to about 5 microns, can be delivered to the middle and upper respiratory tract.

In certain embodiments, a dry powder formulation is administered to the small airways. In such embodiments, the dry powder is preferably VMDG and / or MMAD suitable for delivery to the airways, eg, about 0.5 μm to about 3 μm, about 0.75 μm to about 2 μm or about 1 μm to about 1.5 μm. Contains inhalation particles, such as VMGD and / or MMAD.

When certain dry powders containing divalent metal salts as active ingredients are administered, it is believed that at least some dry powders for inhalation may deposit in the oral cavity, creating an unpleasant "salty" sensation. Be done. Because of this sensation, it is expected that the patient will not follow the treatment instructions or discontinue treatment. The advantage of the dry powder for inhalation of the present invention is that due to its small size and high dispersibility, there is less deposition in the oral cavity and the unpleasant salty sensation is reduced or suppressed.

In dry powder inhalers, oral deposition is determined by inertial collisions and is therefore characterized by the number of Stokes in the aerosol (De Haan et al., Journal of Aerosol Science, 35 (3), 309-331, 2003). If the shape of the inhaler, the respiratory pattern and the shape of the oral cavity, and the number of Stokes are the same, the deposition in the oral cavity is mainly influenced by the aerodynamic size of the inhaled powder. Therefore, factors that cause the deposition of powder in the oral cavity include the particle size distribution of individual particles and the dispersibility of the powder. If the MMAD of individual particles is too large, for example above 5 μm, the percentage of powder deposited in the oral cavity will increase. Similarly, a low dispersibility of the powder indicates that the particles exit the dry powder inhaler and then enter the oral cavity as aggregates. The agglomerated powder aerodynamically behaves like individual particles of the same size as the agglomerates, so even if the individual particles are small (eg, MMAD is 5 microns or less), the particle size distribution of the inhaled powder. Can result in MMAD greater than 5 μm and enhanced oral deposition.

Therefore, the particles are small (eg, MMAD less than 5 microns, eg between 1 and 5 microns) and highly dispersible (eg 1 / 4bar or 0.5 / 4bar is 2.0, preferably less than 1.5). It is desirable to have the powder of. More preferably, the dry inhalation powder is from dry inhalation particles with an MMAD of between 1 and 4 microns or between 1 and 3 microns and a 1/4 bar of less than 1.4 or less than 1.3, preferably less than 1.2. Become.

The absolute geometric particle size of the particles measured at 1 bar using the HELOS system is not important as long as the envelope density of the particles is sufficient to accommodate the MMAD in one of the ranges listed above. However, MMAD is VMGD multiplied by the square root of the envelope density (MMAD = VMGD x sqrt (envelope density)). If it is desired to deliver high doses of salt using a fixed volume dosing vessel, particles with higher envelope density are desirable. The high envelope density allows a large amount of powder to be contained in a fixed volume dosing vessel. Preferred envelope densities are greater than 0.1 g / cc, 0.25 g / cc, 0.4 g / cc, 0.5 g / cc and 0.6 g / cc.

The dry inhalation powders and particles of the present invention can be used as compositions suitable for drug delivery via the respiratory system. For example, such compositions contain dry particles for inhalation of the invention and one or more other dry particles or powders, such as another active agent, or one or more pharmaceutically acceptable supplements. It may contain a mixture such as dry particles or powders that substantially consist of the excipient.

Dry powders and particles for inhalation suitable for use in the methods of the invention pass through the upper respiratory tract (ie, the mesopharynx and laryngeal tract), the lower respiratory tract, which later includes the trachea that branches into the bronchi and alveoli, and later into the respiratory bronchi. It is possible to travel through the dividing terminal bronchi until it reaches the final respiratory area, alveoli or deep lungs. In one embodiment of the invention, most of the dry powder or particles for inhalation are deposited deep in the lungs. In another embodiment of the invention, delivery is primarily to the middle respiratory tract. In another embodiment delivery is to the upper respiratory tract.

The dry particles or powders for inhalation of the present invention can be delivered by inhalation at various parts of the respiratory cycle (eg, laminar flow during intermediate respiration). The advantage of the highly dispersible dry powders and dry particles of the present invention is that they can target deposition in the respiratory tract. For example, respiratory controlled propellant delivery has recently been developed in liquid aerosol delivery (Dalby et al., In Inhalation Aerosols, Hickey, 2007, p. 437). In this case, the sprayed droplets are emitted only during certain parts of the respiratory cycle. Droplets are released towards the beginning of the inhalation cycle for delivery to the deep lungs, whereas they are released towards the end of inhalation for deposition in the middle respiratory tract.

The highly dispersible powders of the present invention provide the advantage of also targeting the timing of drug delivery within the respiratory cycle and its location in the human lung. Since the dry powder for inhalation of the present invention can be rapidly dispersed, such as during a short period of a typical inhalation operation, the timing of powder dispersion is controlled to deliver the aerosol at a specific time in the inhalation. Is possible.

With a highly dispersible powder, the entire dose of aerosol can be dispersed at the onset of inhalation. When the patient's inhalation flow rate increases to the maximum inspiratory flow rate, the highly dispersible powder already begins to disperse at the onset of the increase, and all doses can be dispersed in the first part of inhalation. Since the air inhaled at the start of inhalation is ventilated deep into the lungs, it is preferable to disperse most of the aerosol in the first part of the inhalation in order to deposit it deep in the lungs. Similarly, for deposition in the middle respiratory tract, it may be achieved to disperse a high concentration of aerosol in the air ventilated to the middle respiratory tract by rapidly dispersing the dose from mid to late inhalation. .. This can be done by a number of mechanical and other means, such as a switch that is operated by time, pressure or flow velocity and turns the patient's inhaled air into a powder to be dispersed only when the switch conditions are met. it can.

For specific therapeutic applications, for example, Gonda, I., "Aerosols for delivery of therapeutic and diagnostic agents to the respiratory tract," Critical Reviews in Therapeutic Drug Carrier Systems, 6: 273-313 (1990); and Moren, Aerosol doses, formulations and delivery systems can be selected as described in "Aerosol Dosage Forms and Formulations", Aerosols in Medicine, Principles, Diagnosis and Therapy, Moren et al., Esevier, Amsterdam (1985).

As described herein, the therapeutic and prophylactic effect of inhaled dry particles and dry powder is the result of an increase in the amount of calcium in the airways (eg, lungs) after administration of the inhaled dry particles and dry powder. Is considered to be. Therefore, the amount of calcium supplied will vary depending on the particular salt selected and the dose may be based on the desired amount of calcium delivered to the lungs. For example, 1 mole of calcium chloride (CaCl)₂) Dissociates and 1 mol of Ca²⁺1 mol of calcium citrate produces 3 mol of Ca²⁺Can occur.

Generally, the effective amount of pharmaceutical product is about 0.001 mg Ca.²⁺/ kg Weight / Dose ~ Approximately 2mg Ca²⁺/ kg body weight / dose, approx. 0.002 mg Ca²⁺/ kg Weight / Dose ~ Approximately 2mg Ca²⁺/ kg body weight / dose, approx. 0.005 mg Ca²⁺/ kg Weight / Dose ~ Approximately 2mg Ca²⁺/ kg body weight / dose, approx. 0.01 mg Ca²⁺/ kg Weight / Dose ~ Approximately 2mg Ca²⁺/ kg body weight / dose, approx. 0.01 mg Ca²⁺/ kg Weight / Dose ~ Approximately 60mg Ca²⁺/ kg body weight / dose, approx. 0.01 mg Ca²⁺/ kg Weight / Dose ~ Approximately 50mg Ca²⁺/ kg body weight / dose, approx. 0.01 mg Ca²⁺/ kg Weight / Dose ~ About 40mg Ca²⁺/ kg body weight / dose, approx. 0.01 mg Ca²⁺/ kg Weight / Dose ~ About 30mg Ca²⁺/ kg body weight / dose, approx. 0.01 mg Ca²⁺/ kg Weight / Dose ~ Approximately 20mg Ca²⁺/ kg body weight / dose, approx. 0.01 mg Ca²⁺/ kg Weight / Dose ~ Approximately 10mg Ca²⁺/ kg body weight / dose, approx. 0.01 mg Ca²⁺/ kg Weight / Dose ~ Approximately 5mg Ca²⁺/ kg body weight / dose, approx. 0.01 mg Ca²⁺/ kg Weight / Dose ~ Approximately 2mg Ca²⁺/ kg body weight / dose, approx. 0.02 mg Ca²⁺/ kg Weight / Dose ~ Approximately 2mg Ca²⁺/ kg body weight / dose, approx. 0.03 mg Ca²⁺/ kg Weight / Dose ~ Approximately 2mg Ca²⁺/ kg body weight / dose, approx. 0.04 mg Ca²⁺/ kg Weight / Dose ~ Approximately 2mg Ca²⁺/ kg body weight / dose, approx. 0.05 mg Ca²⁺/ kg Weight / Dose ~ Approximately 2mg Ca²⁺/ kg body weight / dose, about 0.1 mg Ca²⁺/ kg Weight / Dose ~ Approximately 2mg Ca²⁺/ kg body weight / dose, about 0.1 mg Ca²⁺/ kg Weight / Dose ~ Approximately 1mg Ca²⁺/ kg body weight / dose, about 0.1 mg Ca²⁺/ kg Weight / Dose ~ 0.5mg Ca²⁺/ kg body weight / dose, approx. 0.2 mg Ca²⁺/ kg Weight / Dose ~ 0.5mg Ca²⁺/ kg body weight / dose, about 0.18mg Ca²⁺/ kg body weight / dose, approx. 0.001 mg Ca²⁺/ kg body weight / dose, approx. 0.005 mg Ca²⁺/ kg body weight / dose, approx. 0.01 mg Ca²⁺/ kg body weight / dose, approx. 0.02 mg Ca²⁺/ kg body weight / dose or about 0.5 mg Ca²⁺Deliver a dose of / kg body weight / dose.

In some embodiments, the amount of calcium delivered to the airways (eg, lungs, respiratory airways) is about 0.001 mg Ca.²⁺/ kg Weight / Dose ~ Approximately 2mg Ca²⁺/ kg body weight / dose, approx. 0.002 mg Ca²⁺/ kg Weight / Dose ~ Approximately 2mg Ca²⁺/ kg body weight / dose, approx. 0.005 mg Ca²⁺/ kg Weight / Dose ~ Approximately 2mg Ca²⁺/ kg body weight / dose, approx. 0.01 mg Ca²⁺/ kg Weight / Dose ~ Approximately 2mg Ca²⁺/ kg body weight / dose, approx. 0.01 mg Ca²⁺/ kg Weight / Dose ~ Approximately 60mg Ca²⁺/ kg body weight / dose, approx. 0.01 mg Ca²⁺/ kg Weight / Dose ~ Approximately 50mg Ca²⁺/ kg body weight / dose, approx. 0.01 mg Ca²⁺/ kg Weight / Dose ~ About 40mg Ca²⁺/ kg body weight / dose, approx. 0.01 mg Ca²⁺/ kg Weight / Dose ~ About 30mg Ca²⁺/ kg body weight / dose, approx. 0.01 mg Ca²⁺/ kg Weight / Dose ~ Approximately 20mg Ca²⁺/ kg body weight / dose, approx. 0.01 mg Ca²⁺/ kg Weight / Dose ~ Approximately 10mg Ca²⁺/ kg body weight / dose, approx. 0.01 mg Ca²⁺/ kg Weight / Dose ~ Approximately 5mg Ca²⁺/ kg body weight / dose, approx. 0.01 mg Ca²⁺/ kg Weight / Dose ~ Approximately 2mg Ca²⁺/ kg body weight / dose, approx. 0.02 mg Ca²⁺/ kg Weight / Dose ~ Approximately 2mg Ca²⁺/ kg body weight / dose, approx. 0.03 mg Ca²⁺/ kg Weight / Dose ~ Approximately 2mg Ca²⁺/ kg body weight / dose, approx. 0.04 mg Ca²⁺/ kg Weight / Dose ~ Approximately 2mg Ca²⁺/ kg body weight / dose, approx. 0.05 mg Ca²⁺/ kg Weight / Dose ~ Approximately 2mg Ca²⁺/ kg body weight / dose, about 0.1 mg Ca²⁺/ kg Weight / Dose ~ Approximately 2mg Ca²⁺/ kg body weight / dose, about 0.1 mg Ca²⁺/ kg Weight / Dose ~ Approximately 1mg Ca²⁺/ kg body weight / dose, about 0.1 mg Ca²⁺/ kg Weight / Dose ~ 0.5mg Ca²⁺/ kg body weight / dose, approx. 0.2 mg Ca²⁺/ kg Weight / Dose ~ 0.5mg Ca²⁺/ kg body weight / dose, about 0.18mg Ca²⁺/ kg body weight / dose, approx. 0.001 mg Ca²⁺/ kg body weight / dose, approx. 0.005 mg Ca²⁺/ kg body weight / dose, approx. 0.01 mg Ca²⁺/ kg body weight / dose, approx. 0.02 mg Ca²⁺/ kg body weight / dose or about 0.5 mg Ca²⁺/ kg Body weight / dose.

In other embodiments, the amount of calcium delivered to the upper respiratory tract (eg, nasal cavity) is about 0.001 mg Ca.²⁺/ kg Weight / Dose ~ Approximately 2mg Ca²⁺/ kg body weight / dose, approx. 0.002 mg Ca²⁺/ kg Weight / Dose ~ Approximately 2mg Ca²⁺/ kg body weight / dose, approx. 0.005 mg Ca²⁺/ kg Weight / Dose ~ Approximately 2mg Ca²⁺/ kg body weight / dose, approx. 0.01 mg Ca²⁺/ kg Weight / Dose ~ Approximately 2mg Ca²⁺/ kg body weight / dose, approx. 0.01 mg Ca²⁺/ kg Weight / Dose ~ Approximately 60mg Ca²⁺/ kg body weight / dose, approx. 0.01 mg Ca²⁺/ kg Weight / Dose ~ Approximately 50mg Ca²⁺/ kg body weight / dose, approx. 0.01 mg Ca²⁺/ kg Weight / Dose ~ About 40mg Ca²⁺/ kg body weight / dose, approx. 0.01 mg Ca²⁺/ kg Weight / Dose ~ About 30mg Ca²⁺/ kg body weight / dose, approx. 0.01 mg Ca²⁺/ kg Weight / Dose ~ Approximately 20mg Ca²⁺/ kg body weight / dose, approx. 0.01 mg Ca²⁺/ kg Weight / Dose ~ Approximately 10mg Ca²⁺/ kg body weight / dose, approx. 0.01 mg Ca²⁺/ kg Weight / Dose ~ Approximately 5mg Ca²⁺/ kg body weight / dose, approx. 0.01 mg Ca²⁺/ kg Weight / Dose ~ Approximately 2mg Ca²⁺/ kg body weight / dose, approx. 0.02 mg Ca²⁺/ kg Weight / Dose ~ Approximately 2mg Ca²⁺/ kg body weight / dose, approx. 0.03 mg Ca²⁺/ kg Weight / Dose ~ Approximately 2mg Ca²⁺/ kg body weight / dose, approx. 0.04 mg Ca²⁺/ kg Weight / Dose ~ Approximately 2mg Ca²⁺/ kg body weight / dose, approx. 0.05 mg Ca²⁺/ kg Weight / Dose ~ Approximately 2mg Ca²⁺/ kg body weight / dose, about 0.1 mg Ca²⁺/ kg Weight / Dose ~ Approximately 2mg Ca²⁺/ kg body weight / dose, about 0.1 mg Ca²⁺/ kg Weight / Dose ~ Approximately 1mg Ca²⁺/ kg body weight / dose, about 0.1 mg Ca²⁺/ kg Weight / Dose ~ 0.5mg Ca²⁺/ kg body weight / dose, approx. 0.2 mg Ca²⁺/ kg Weight / Dose ~ 0.5mg Ca²⁺/ kg body weight / dose, about 0.18mg Ca²⁺/ kg body weight / dose, approx. 0.001 mg Ca²⁺/ kg body weight.

In addition, if the dry particles and powder for inhalation contain sodium salts, the dry particles and dry powder for inhalation should be about 0.001 mg Na.⁺/ kg Weight / Dose ~ Approximately 10mg Na⁺/ kg body weight / dose, approx. 0.01 mg Na⁺/ kg Weight / Dose ~ Approximately 10mg Na⁺/ kg body weight / dose, approx. 0.1 mg Na⁺/ kg Weight / Dose ~ Approximately 10mg Na⁺/ kg body weight / dose, approx. 1.0 mg Na⁺/ kg Weight / Dose ~ Approximately 10mg Na⁺/ kg body weight / dose, approx. 0.001 mg Na⁺/ kg Weight / Dose ~ Approximately 1mg Na⁺/ kg body weight / dose, approx. 0.01 mg Na⁺/ kg Weight / Dose ~ Approximately 1mg Na⁺/ kg body weight / dose, approx. 0.1 mg Na⁺/ kg Weight / Dose ~ Approximately 1mg Na⁺/ kg Body weight / dose, approx. 0.2-approx. 0.8 mg Na⁺/ kg body weight / dose, about 0.3-about 0.7 mg Na⁺/ kg body weight / dose or about 0.4-about 0.6 mg Na⁺It can be administered in an amount sufficient to deliver a dose of / kg body weight / dose.

In some embodiments, the amount of sodium delivered to the airway (eg, lung, respiratory airway) is from about 0.001 mg / kg body weight / dose to about 10 mg / kg body weight / dose, about 0.01 mg / kg body weight / dose. ~ About 10 mg / kg body weight / dose, about 0.1 mg / kg body weight / dose ~ About 10 mg / kg body weight / dose, about 1 mg / kg body weight / dose ~ about 10 mg / kg body weight / dose, about 0.001 mg / kg body weight / dose ~ About 1 mg / kg body weight / dose, about 0.01 mg / kg body weight / dose ~ about 1 mg / kg body weight / dose, about 0.1 mg / kg body weight / dose ~ about 1 mg / kg body weight / dose, about 0.2 ~ about 0.8 mg / kg body weight / dose, about 0.3 to about 0.7 mg / kg body weight / dose or about 0.4 to about 0.6 mg / kg body weight / dose.

In other embodiments, the amount of sodium delivered to the upper airway (eg, nasal cavity) is from about 0.001 mg / kg body weight / dose to about 10 mg / kg body weight / dose, from about 0.01 mg / kg body weight / dose to about 10 mg. / kg body weight / dose, about 0.1 mg / kg body weight / dose ~ about 10 mg / kg body weight / dose, about 1 mg / kg body weight / dose ~ about 10 mg / kg body weight / dose, about 0.001 mg / kg body weight / dose ~ about 1 mg / kg body weight / dose, about 0.01 mg / kg body weight / dose ~ about 1 mg / kg body weight / dose, about 0.1 mg / kg body weight / dose ~ about 1 mg / kg body weight / dose, about 0.2 ~ about 0.8 mg / kg body weight / The dose is about 0.3 to about 0.7 mg / kg body weight / dose or about 0.4 to about 0.6 mg / kg body weight / dose.

Appropriate dosing intervals to achieve the desired therapeutic effect depend on considerations such as the severity of the condition (eg, infection), the subject's general health, and the subject's resistance to dry particles and powders for inhalation. Can be determined on the basis. Based on the above and other considerations, the clinician may determine the appropriate dosing interval. In general, dry particles and powders for inhalation are administered once, twice or three times daily as needed.

If necessary or indicated, the dry particles and powders for inhalation described herein may be administered with one or more other therapeutic agents. Other therapeutic agents are suitable routes, such as oral, parenteral (eg, intravenous, intraarterial, intramuscular or subcutaneous injection), topical, inhalation (eg, intrabronchial, intranasal or oral inhalation, intranasal drop). Agent), intrarectally, transvaginally, etc. may be administered. The dry particles and powder for inhalation may be administered prior to administration of the other therapeutic agent, substantially simultaneously with the administration of the other therapeutic agent, or after administration of the other therapeutic agent. Preferably, the dry particles and powders for inhalation and the other therapeutic agent are administered so that their pharmacological activities substantially overlap.

Another advantage provided by the dry inhalation powders and dry inhalation particles described herein is that the efficiency of administration can be increased as a result of the hygroscopic growth of the particles in the lung due to the hygroscopic growth of the particles. .. The tendency of the partially amorphous, salty compositions of the present invention to absorb water at high humidity may also be advantageous for their in vivo deposition profile. By rapidly absorbing moisture at high humidity, such formulations can absorb moisture from the humid air in the respiratory tract and undergo hygroscopic growth when migrating to the lungs. This allows for an effective increase in aerodynamic particle size during the transition to the lungs, further promoting deposition in the airways. Example

Concrete example The materials used in later examples and their sources are listed below. Calcium chloride dihydrate, calcium lactate pentahydrate, sodium chloride, L-leucine, maltodextrin, mannitol, lactose and trehalose from Sigma-Aldrich (St. Louis, MO) or Spectrum Chemicals (Gardena, CA) Obtained from; Sodium Sulfate from EMD Chemicals (Gibbstown, NJ), Sigma-Aldrich (St. Louis, MO) or Spectrum Chemicals (Gardena, CA); Sodium Citrate Dihydrate from JTBaker Obtained from (Phillipsburg, NJ), Mallinckrodt Baker (Phillipsburg, NJ) or Spectrum Chemicals (Gardena, CA). Ultrapure water was obtained from a purification system (Millipore Corp., Billerica, MA).

Method: Geometric particle diameter and volume diameter Laser diffraction techniques were used to determine the median volume (× 50 or Dv50), also known as the median volume (VMGD). The instrument consisted of a HELOS diffractometer and a RODOS dry powder disperser (Sympatec, Inc., Princeton, NJ). The RODOS disperser applies a shearing force to the powder sample, which is controlled by the regulatory pressure of the incoming compressed dry air (usually set at 1.0 bar). The pressure setting can be varied to vary the amount of energy used to disperse the powder. For example, the regulation pressure can be varied from 0.2 bar to 4.0 bar and the orifice ring pressure can be varied from 5.00 mbar to 115.00 mbar. Disperse the powder sample from the microspatula into the RODOS funnel. The dispersed particles travel through the laser beam, and the resulting diffracted light pattern is collected by a series of detectors, usually using an R1 lens. The Fraunhofer diffraction model is then used to transform the set of diffractions into a volume-based particle size distribution, based on the fact that smaller particles scatter light at larger angles. The geometric standard deviation (GSD) of the geometric volume mean diameter was also determined using this method.

The median volume can also be measured using a method of discharging powder from a dry powder inhaler device. The device consisted of "Spraytec", a Spraytec laser diffractive particle size measurement system (Malvern, Worcestershire, UK). The powder formulation was manually filled into size 3 HPMC capsules (Capsugel V-Caps) and the filling weight was measured by a weighing method using a chemical scale (Mettler Tolerdo XS 205). Specific resistance 0.036kPa^1/2LPM^-1A capsule-type passive dry powder inhaler (RS-01 Model 7, High resistance Plastiape SpA) was used. A timer-controlled solenoid valve (TPK2000, Copley Scientific) with a flow control valve was used to set the flow velocity and suction volume. The capsule was placed in a dry powder inhaler, punctured, and the inhaler was hermetically connected to the inlet of a laser diffraction particle size analyzer. Steady air flow rates in the system were initiated using the TPK2000 and the particle size distribution was measured by Spraytec at 1 kHz for at least 2 seconds to the total inhalation time. The calculated particle size distribution parameters included volume median diameter (Dv50), geometric standard deviation (GSD), and particle fraction (FPF) of particles less than 5 micrometers in diameter. At the end of the inhalation time, the dry powder inhaler was opened to remove the capsule and reweighed to calculate the mass of powder released from the capsule within the inhalation time (capsule release powder mass, ie CEPM).

The above statement regarding the use of Spraytec was for a use called "closed bench placement". Instead, Spraytec can be used in an "open bench arrangement". In the open bench arrangement, the capsule was placed inside the dry powder inhaler, punctured, and the inhaler was sealed inside the cylinder. The cylinder was connected to a positive pressure air source, the steady air flow in the system was measured again with a mass flow meter, and its duration was controlled by a timer-controlled solenoid valve. In an open bench arrangement, the outlet of the dry powder inhaler was exposed to room pressure and the resulting aerosol jet passed through the laser of a diffractive particle size analyzer (Spraytec) before being collected by the aspirator. As in the closed bench arrangement, a solenoid valve was used to initiate a steady air flow velocity in the system and Spraytec measured the particle size distribution at 1 kHz for a minimum of 2 seconds per inhalation operation. The data measured by Spraytec and reported in the Examples are in a closed bench arrangement, unless otherwise noted.

Geometric particle size or volume diameter after release Medium Volume Volume (Dv50) (Geometric Medium Volume Diameter (VMGD)) of Powder After Ejection from Dry Powder Inhaler Using Laser Diffraction Techniques by Spraytec Diffractometer (Malvern, Inc., Worcestershire, UK) ) Was also determined. Capsule-type dry powder inhaler (RS01 Model 7 High) filled with powder into size 3 capsules (V-Caps, Capsugel) Resistance, Plastiape, Italy) or DPI was installed and the DPI was connected to the Spraytec inhaler adapter using an airtight seal. Steady air velocity was generated within the DPI for a period of time, usually 2 seconds, at 60 L / min, controlled by a timer-controlled solenoid (TPK2000, Copley, Scientific, UK). Alternatively, the airflow velocity generated in the DPI was sometimes set to 15 L / min, 20 L / min or 30 L / min. The injected aerosol then passed vertically through the laser beam as an internal stream. The geometric particle size distribution of the resulting aerosol was calculated by software based on the scattering patterns measured by photodetectors for samples obtained, typically at 1000 Hz, during the inhalation time. The average of Dv50, GSD, FPF &lt;5.0 μm measured over the entire inhalation time was then calculated.

Fine particle fraction The aerodynamic properties of the powder diffused from the inhaler, Mk-II 1 ACFM Andersen Cascade Impactor (Copley Scientific) Limited, Nottingham, UK). The device was operated under controlled environmental conditions of 22 ± 2 ° C and 30 ± 5% relative humidity (RH). The instrument consisted of eight stages that separated the aerosol particles based on inertial collisions. At each stage, an aerosol stream passes through a series of nozzles and collides with the corresponding collision plate. Particles with sufficiently small inertial force proceed to the next stage with the aerosol flow, and the remaining particles collide with the plate. The aerosol passes through the nozzle at an increasing velocity in each successive stage, and aerodynamically small particles are collected on the plate. After the aerosol has passed the final stage, the smallest remaining particles are collected by a filter called the "last collection filter". Gravimetric and / or chemical analysis can then be performed to determine the particle size distribution. It is also possible to evaluate two aerodynamic particle size cut points in a short working time by using a cascade impactor with a small number of stages, which is also called a collapse type cascade impactor. In this collapsing cascade impactor, stages other than those required to form a fraction of fine particles and coarse particles are removed.

By using collision technology, it was possible to collect two or eight separated powder fractions. Capsules (HPMC, size 3; Shionogi Qualicaps, Madrid, Spain or Capsugel Vcaps, Peapack, NJ) are filled to about half the powder and are a handheld breath-actuated dry powder inhaler (DPI) device with high resistance RS. -01 It was installed at DPI (Plastiape, Osnago, Italy). The capsule was perforated and operated at a flow rate of 60.0 L / min for 2.0 seconds to draw the powder into the cascade impactor. At this flow rate, the calibration cutoff diameters for the eight stages are 8.6, 6.5, 4.4, 3.3, 2.0, 1.1, 0.5 and 0.3 microns, and for the two stages of the low number of cascade impactors, the cutoff diameter is 5.6 microns. And 3.4 microns. A filter was placed in the device to collect the fractions and the amount of powder colliding with the filter was determined by weight measurement or chemical measurement by HPLC as shown in the table. The powder mass recovered from the desired stage of the impactor was divided by the total particle mass in the capsule to calculate the fine particle fraction below the effective cutoff aerodynamic particle size of the total dose of powder (FPF_TD). .. Results are reported as a fine particle fraction &lt;5.6 microns (FPF &lt;5.6 microns) and a fine particle fraction &lt;3.4 microns (FPF &lt;3.4 microns). Alternatively, the particulate fraction for the powder recovery or release dose may be calculated by dividing the powder mass recovered from the desired stage of the impactor by the total powder mass recovered.

Aerodynamic particle size The information obtained by the Andersen Cascade Impactor was used to determine the aerodynamic mass median diameter (MMAD). For each stage, the cumulative mass below the stage cutoff diameter is calculated and normalized by the powder recovery dose. The powder MMAD is then calculated by linear interpolation of the stage cutoff diameter that sets the 50th percentile limit.

Fine particle dose The information obtained by ACI was used to determine the particulate dose. A single dose powder applied to ACI and deposited on the final collection filter and stages 6, 5, 4, 3 and 2 with a cumulative mass of less than 4.4 microns (FPD &lt;4.4 μm). be equivalent to.

Capsule release powder mass Information obtained from the Andersen Cascade Impactor test was used to determine a measure of powder release characteristics. The weight of the filled capsule was recorded at the start of operation, and the final capsule weight was recorded after the end of operation. The difference in weight represented the amount of powder released from the capsule (CEPM, or capsule-released powder mass). Capsule shell The release dose was calculated by dividing the amount of powder released by the mass of particles in the first capsule. The reference CEPM was measured at 60 L / min, but it was also measured at 15 L / min, 20 L / min, or 30 L / min.

Tap density Tap densities were measured using two methods. (1) First, according to USP &lt;616&gt;, but a 1.5cc micropipette (Eppendorf AG, Hamburg, Germany), or a polyethylene cap (Kimble Chase, Vineland,) for attaching to both ends to hold the powder. A modified method was used that required a small amount of powder, replacing a 0.3 cc portion (Grenier Bio-One, Monroe, NC) of a disposable serological polystyrene micropipette with NJ). (2) USP &lt;616&gt; using a 100cc graduated cylinder was used. Equipment known to those skilled in the art for measuring tap density is not particularly limited, but is a Dual Platform Microprocessor Controlled Tap Density Tester (Vankel, Cary, NC) or GeoPyc equipment (Micrometrics Instrument). Corp., Norcross, GA). Tap density is a standard approximate measure of envelope mass density. The envelope mass density of an isotropic particle is defined as the mass of the particle divided by the envelope volume of the smallest sphere that can contain the particle inside.

Bulk density Prior to the tap density measurement, the bulk density was estimated by dividing the weight of the powder by the volume of the powder estimated using a volumetric device.

Hausner ratio The Hausner ratio is a dimensionless number and was calculated by dividing the tap density by the bulk density. The Hausner ratio is a numerical value that correlates with the fluidity of the powder.

Scanning electron microscopy (SEM) SEM was performed using a FEI Quanta 200 scanning electron microscope (Hillsboro, Oregon) equipped with an Everhart Thornley (ET) detector. Image collection and analysis were performed using xTm (v.2.01) and XT Docu (v.3.2) software, respectively. The magnification was confirmed using the NIS traceable standard. Each sample was prepared for analysis by placing a small amount on a carbon attachment tab supported on an aluminum table. Each sample was then sputter coated with Au / Pd at approximately 20 mA and 0.13 mbar (Ar) for 75 seconds using a Cressington 108 automatic Sputter Coater. Data collection parameters are displayed in the information bar at the bottom of each image. The magnification reported for each image was calculated during the initial data collection. The scale bar reported below each image is accurate when resizing and should be used when sizing.

Preparation of liquid feedstock for spray drying In order to spray dry the uniform particles, it is necessary to solubilize the components of interest into a solution or suspend them into a uniform and stable suspension. Certain calcium salts, such as calcium chloride, calcium acetate and calcium lactate, have sufficient water solubility to prepare a suitable spray dry solution. However, other calcium salts such as calcium sulphate, calcium citrate and calcium carbonate have low solubility in water. Table 1 lists the solubility of typical calcium salts in water. Due to such low solubility, it was necessary to develop a feedstock for preparation in order to prepare a solution or suspension that can be spray-dried. These solutions or suspensions contained a combination of salts in a suitable solvent, namely normal water, as well as a mixture of ethanol and water or other solvents already described herein.

<tables num="1"><img id="000002" he="104" wi="128" file="JP5877201B2_D0001.tif" img-format="tif" img-content="drawing" /></tables>

As already mentioned, calcium chloride is highly water-soluble. Sodium salts such as sodium sulphate, sodium citrate and sodium carbonate are also very soluble in water. As detailed in the examples below, calcium chloride and a sodium salt (starting material) are mixed in a solution or suspension to give a stable calcium salt in the final dry powder form. When calcium chloride and a sodium salt are mixed in solution, a precipitation reaction occurs between the calcium and the anions resulting from the sodium salt, which can produce the desired calcium salt (ie, CaCl).₂+ 2NaXX CaXX + 2NaCl). In this case, the maximum solid concentration at which a clear solution or stable suspension was maintained was used for spray drying. Certain calcium salts were sufficiently soluble in water and were spray dried alone. The same idea can be applied, for example, to magnesium salts with magnesium chloride, potassium salts with potassium chloride, and sodium salts.

A "starting material" may be provided in a molar concentration at which all precipitation reactions are completed (referred to as "complete reactions"). Table 2 further lists the weight percent of calcium ions in a typical calcium salt.

<tables num="2"><img id="000003" he="100" wi="128" file="JP5877201B2_D0001.tif" img-format="tif" img-content="drawing" /></tables>

Alternatively, excess calcium chloride may be added to the incomplete or "incomplete reaction" so that a given amount of calcium chloride is present in the final powder form. Calcium chloride is hygroscopic, but its high water solubility allows it to be included in small amounts in the final product to increase the solubility of the final product, allow adjustment of the dissolution profile, and sodium ions present in the formulation, etc. It can be useful for increasing the relative ratio of calcium ions to cations. To facilitate formulation development, the required calcium chloride to sodium salt molar ratio was converted to the calcium chloride to sodium salt mass ratio. In the example of calcium citrate (ie calcium chloride + sodium citrate), the precipitation reaction proceeds as follows: 3CaCl₂+ 2Na₃C₆H₅O₇ Ca₃(C₆H₅O₇)₂+ 6 NaCl.

This reaction yields Ca and Na ions with a molar ratio of 1: 2. To complete the reaction, 3 moles of calcium chloride and 2 moles of sodium citrate are required. Multiply the number of moles of salt by the molecular weight of the salt in grams per mole to convert to mass and weight ratio in grams: With calcium chloride: 3 mol of CaCl₂× 111g / mol = 333g CaCl₂ With sodium citrate: 2 mol of Na₃C₆H₅O₇× 258g / mol = 516g of Na₃C₆H₅O₇。

Therefore, for a complete reaction, CaCl with a weight ratio of 1: 1.55 or 39:61₂: Na₃C₆H₅O₇is required. Solubilized in these ratios and spray dried to produce "pure salt" formulations. In addition, dry powders were made with additional excipients such as leucine or lactose. The ratio of calcium salt to sodium salt was maintained to result in a "complete reaction". For example, in a 50% (w / w) leucine formulation, the rest is a salt like calcium citrate (ie, CaCl).₂: Na₃C₆H₅O₇), Where the CaCl is 39:61₂: Na3C₆H₅O₇Weight ratio is maintained. Therefore, in this reaction, 50% (w / w) leucine and 19.5% (w / w) CaCl₂And 30.5% (w / w) Na₃C₆H₅O₇Will be added. In the spray drying step, the salt and other excipients are dissolved or suspended in a solvent (ie, water). The solid concentration (w / v) can be selected according to the solubility of various components. In the preparation of citrate, the concentration limit of calcium citrate was 0.95 mg / mL, so a concentration of 5 mg / mL was appropriate. Therefore, 5 g of solid (ie 2.5 g of leucine, 0.975 g of calcium chloride and 1.525 g of sodium citrate) was dissolved in 1 L of ultrapure water.

In addition, the weight of the hydrated starting material water must be taken into account when preparing the spray-dried solution. The ratio used in the formulation was based on the molecular weight of the anhydrous salt. For certain salts, the hydrated form is more readily available than the anhydrous form. In this case, the initially calculated ratio needs to be adjusted using a multiplier to correlate the molecular weight of the anhydrous salt with the molecular weight of the hydrate. An example of this calculation is given below.

In the above example, calcium chloride has an anhydrous molecular weight of 110.98 g / mol and dihydrate has a molecular weight of 147.01 g / mol. Sodium citrate has an anhydrous molecular weight of 258.07 g / mol and dihydrate has a molecular weight of 294.10 g / mol.

The multiplier is similar to the ratio of molecular weights of dihydrate to anhydrous, for example, calcium chloride is 1.32 and sodium citrate is 1.14. Therefore, when adjusted to the dihydrate form, leucine is 2.5 g, calcium chloride dihydrate is 1.287 g (ie 0.975 g x 1.32), and sodium citrate dihydrate is 1.738 g (ie 1.525 g x). It became 1.14), and these were melted and spray-dried.

Spray drying using Niro spray dryer Dry powder was produced by spray drying with a Niro Mobile Minor spray dryer (GEA Process Engineering Inc., Columbia, MD) and the powder was collected from cyclones, product filters, or both. Using a parallel flow difluid nozzle from Niro (GEA Process Engineering Inc., Columbia, MD) or a bifluid nozzle from Spraying Systems (Carol Stream, IL) with a gas cap 67147 and a liquid cap 2850SS. The liquid feedstock was sprayed, but other two-fluid nozzle devices can also be used. For example, the bifluid nozzle may be in an internal mixer or an external mixer. Further spraying techniques include rotary spray nozzles or pressure nozzles. A static mixer (Charles Ross) using a gear pump (Cole-Parmer Instrument Company, Vernon Hills, IL) to introduce the liquid feedstock directly into or just before the two-fluid nozzle. &amp; Son Company, Hauppauge, NY). Further liquid supply technology includes pressurized containers. Nitrogen or air can be used as a drying gas as long as at least some of the moisture in the air is removed before use. (Ii) Pressurized nitrogen or pressurized air may be used as the spraying gas supplied to the fluid nozzle. The inlet temperature of the process gas can range from 100 ° C to 300 ° C, the outlet temperature can range from 50 ° C to 120 ° C, and the rate of the liquid feedstock can range from 20 mL / min to 100 mL / min. The gas supplied to the bifluid atomizer depends on the nozzle selection, with the Niro parallel flow bifluid nozzle in the range of 8 kg / hour to 15 kg / hour, the pressure set in the range of 0.5 bar to 2.0 bar, and the gas. Spraying with cap 67147 for liquid and cap 2850SS for liquid For Systems bifluid nozzles, it can range from 40 / min to 100g / min. For example, with the Niro bifluid nozzle described above, it can be in the range of 5 kg / hour to 50 kg / hour. The gas spray rate may be set to obtain a particular gas / liquid mass ratio, which directly affects the size of the droplets produced. The pressure in the drying drum can be in the range of +3 "WC ~ -6" WC. The spray-dried powder can be recovered in the container at the outlet of the cyclone, on a cartridge or baghouse filter, or from both the cyclone and the cartridge or baghouse filter.

Spray drying using Buchi spray dryer Dry powders were prepared by spray drying with a B-290 Mini Spray Dryer (BUCHI Labortechnik AG, Flawil, Switzerland) and the powders were recovered from standard or high performance cyclones. In this system, a Buchi B-296 dehumidifier was used to stabilize the temperature and humidity of the air used for spray drying. In addition, when the relative humidity in the room exceeded 30% RH, an external LG dehumidifier (Model 49007903, LG Electronics, Englewood Cliffs, NJ) was always in operation. A Buchi bifluid nozzle with a diameter of 1.5 mm was used for spraying the liquid feed material. The inlet temperature of the process gas can range from 100 ° C to 220 ° C, the outlet temperature can range from 80 ° C to 120 ° C, and the flow rate of the liquid feedstock can range from 3 mL / min to 10 mL / min. Bifluid spray gas is 25mm ~ 45mm (300LPH ~ 530LPH), suction speed is 70% ~ 100% (28m)³/ Hour ~ 38m³/ Hour) can be in the range.

Table 3 lists the feedstock formulations used in the preparation of some of the dry powders described herein.

<tables num="3"><img id="000004" he="122" wi="129" file="JP5877201B2_D0001.tif" img-format="tif" img-content="drawing" /></tables>

The expected final dry powder composition is shown in Table 4. These compositions are based on the expectation that the above ion exchange reactions will proceed to completion in formulations I and III. Although not bound by any particular theory, the evaporation of droplets that occurs during spray drying results in the first precipitation of the most insoluble salts, namely calcium citrate and calcium sulphate, respectively, in formulations I and II, respectively. It is expected to promote this.

<tables num="4"><img id="000005" he="69" wi="129" file="JP5877201B2_D0001.tif" img-format="tif" img-content="drawing" /></tables>

Description of placebo: A placebo formulation containing 100 weight percent leucine or 98 weight percent leucine and 2 weight percent sodium chloride was prepared by spray drying. In the batch step, an aqueous phase was prepared by dissolving leucine in ultrapure water with continuous stirring until the raw material was completely dissolved in water at room temperature. In the static mixing step, the ultrapure water was divided in half and half of the required total leucine was dissolved in each amount of water. The solution was then spray dried using a Niro or Buchi spray dryer. For placebo formulations, two batches (A and B) of feedstock were prepared and spray dried. The total solid concentration was 15 g / L for batch A and 5 g / L for batch B. Niro Mobile The process conditions used for spray drying of batch A (placebo A) with a Minor spray dryer were the same as those used for spray drying of formulation IA of Example 1. The process conditions used for spray drying of batch B (placebo B) are the same as those used for spray drying of the formulation IC of Example 1 except that the outlet temperature of formulation placebo B is about 82 ° C. there were. Further information on the process conditions and properties of the powders and / or particles of the formulations placebo A and placebo B prepared in this example is given in the table or in the graphs shown in Figures 1A-IF and 2-4.

Example 1 This example describes the preparation of a dry powder using Formulation I: 10.0 weight percent leucine, 35.1 weight percent calcium chloride and 54.9 weight percent sodium citrate as feedstock.

In the batch step, an aqueous phase was prepared by dissolving leucine, then sodium citrate dihydrate, and finally calcium chloride dihydrate in ultrapure water. The solution or suspension was continuously stirred throughout the process until the raw material was completely dissolved in water at room temperature. In the static mixing step, the sodium salt and the calcium salt were kept in separate solutions. The ultrapure water was divided in half and half of the required total leucine was dissolved in each amount of water. Sodium citrate dihydrate was dissolved in one aqueous phase and calcium chloride dihydrate was dissolved in the second aqueous phase. The solution or suspension was continuously stirred throughout the process until the ingredients were completely dissolved in water at room temperature. The solution or suspension was then spray dried using a Niro or Buchi spray dryer. For each formulation, feedstocks for three batches (A, B and C) were prepared and spray dried. Details regarding the preparation of liquid feedstock for each of the three batches are shown in Table 5, where the total solid concentration is reported as the sum of the weights of the dissolved anhydrous materials. Particles of batches A and D were prepared on a Niro spray dryer using the feedstock of batches A and D, respectively. Particles of batches B and C were prepared on a Buchi spray dryer using the corresponding feedstock.

<tables num="5"><img id="000006" he="69" wi="128" file="JP5877201B2_D0001.tif" img-format="tif" img-content="drawing" /></tables>

Dry powder for batch A (IA) was produced by spray drying with a Niro Mobile Minor spray dryer (GEA Process Engineering Inc., Columbia, MD) and the powder was collected from the product cartridge filter. A parallel flow type two-fluid nozzle with a 1.0 mm insertion part of Niro (GEA Process Engineering Inc., Columbia, MD) was used for spraying the liquid feed material. A static mixer (Charles Ross &amp; Son) just before introducing the liquid feedstock into the two-fluid nozzle using a gear pump (Cole-Parmer Instrument Company, Vernon Hills, IL). Supplyed to Company, Hauppauge, NY). Nitrogen was used as the drying gas. The inlet temperature of the process gas was set to 282 ° C and the measured outlet temperature was about 98 ° C. The gas supplied to the two-fluid atomizer was set at a flow rate of 14.5 kg / hour and a pressure of 2 psi, the flow rate of the process gas was set at 85 kg / hour and a pressure of 25 psi, and the pressure in the drying drum was -2 "WC. The total flow rate of the feedstock was 70 mL / min and each stream was fed at 35 mL / min. Spray-dried powder was collected from a product collection cartridge filter.

Prepare dry powders of batch B (IB) and batch C (IC) by spray drying with a Buchi B-290 Mini spray dryer (BTJCHI Labortechnik AG, Flawil, Switzerland) equipped with a Buchi bifluid nozzle with a diameter of 1.5 mm. , Collected powder from high performance cyclone. In this system, a Buchi B-296 dehumidifier was used to stabilize the temperature and humidity of the air used for spray drying. The inlet temperature of the process gas was set to 220 ° C, and the flow rate of the liquid feedstock was set to 6.7 mL / min for formulation IB and 7 mL / min for formulation IC. The outlet temperature was about 108 ° C for formulation IB and about 95 ° C for formulation IC. The second-rate spray gas was 40 mm and the aspirator speed was 90%.

Batch D (ID) dry powder was produced by spray drying with a Niro Mobile Minor spray dryer (GEA Process Engineering Inc., Columbia, MD) and the powder was collected from the product filter membrane. A two-fluid nozzle from Spraying Systems (Carol Stream, IL) equipped with a gas cap 67147 and a liquid cap 2850SS was used to spray the liquid feedstock. A static mixer (Charles Ross &amp; Son) just before introducing the liquid feedstock into the two-fluid nozzle using a gear pump (Cole-Parmer Instrument Company, Vernon Hills, IL). Supplyed to Company, Hauppauge, NY). Nitrogen was used as the drying gas. The inlet temperature of the process gas was set to about 265 ° C and the measured outlet temperature was about 99 ° C. The gas supplied to the two-fluid atomizer was set to a flow rate of 80 g / min, the flow rate of the process gas was set to 80 kg / hour, and the pressure in the drying drum was -2 "WC. The total flow rate of the liquid feed material was 66 mL / min. Minutes, each stream was fed at 33 mL / min. Spray-dried powder was collected from the product collection filter membrane.

Table 6 summarizes some of the physical properties of the particles obtained in the four different batches (formulations IA, IB, IC and ID). In addition to the data listed in Table 5, detailed data on dry powders prepared from feedstock IA is summarized below. The average particle fraction (FPF) measured by the Andersen cascade impactor in all eight stages by gravimetric analysis was 56.2% for FPFs &lt;5.6 microns and 41.7% for FPFs &lt;3.4 microns. Aerodynamic particle size was also measured by gravimetric analysis by ACI at all stages. The mean aerodynamic mass median diameter (MMAD) was 2.72 microns. The particle size was determined by laser diffraction with a HELOS / RODOS particle size measuring device, and the average value of the median volume (× 50) at a pressure of 1 bar was 2.57 microns. Furthermore, this powder behaved relatively independently of the flow velocity, as evidenced by the ratio of x50 measured at 0.5 bar to x50 measured at 4.0 bar, which was 1.19. The value of 1/4 bar of these powders was 1.17.

Further characteristics of the dry powder prepared from the feedstock formulation ID are summarized below. The average particle fraction (FPF) measured by the Andersen cascade impactor in all eight stages by gravimetric analysis was 58.8% for FPFs &lt;5.6 microns and 46.7% for FPFs &lt;3.4 microns. Aerodynamic particle size was also measured by gravimetric analysis by ACI at all stages. The average aerodynamic mass median diameter (MMAD) was 2.38 microns. The particle size was determined by laser diffraction with a HELOS / RODOS particle size measuring device, and the average value of the median volume (× 50) at a pressure of 1 bar was 2.45 microns. Furthermore, this powder behaved relatively independently of the flow velocity, as evidenced by the ratio of x50 measured at 0.5 bar to x50 measured at 4.0 bar, which was 1.12. The value of 1 / 4bar of these powders was 1.09.

<tables num="6"><img id="000007" he="29" wi="129" file="JP5877201B2_D0001.tif" img-format="tif" img-content="drawing" /></tables>

Further information on the powder and / or particle properties of Formulation IA prepared in this example is given in the table or in the graphs shown in Figures 1A-IF and 2-4. In Figure ID, GSD represents the geometric standard deviation. In Figure IF, Dv50 represents the geometric median volume (VMGD) measured by the Spraytec instrument and V represents the volume. SEM was performed as described above (Fig. 5A).

Example 2 This example describes the preparation of a dry powder using Formulation III: 10.0 weight percent leucine, 58.6 weight percent calcium lactate and 31.4 weight percent sodium chloride as feedstock.

In the batch step, an aqueous phase was prepared by dissolving leucine, then sodium chloride, and finally calcium lactate pentahydrate in ultrapure water. The solution or suspension was continuously stirred throughout the process until the raw material was completely dissolved in water at room temperature. For calcium lactate preparations, four batches (A, B, C and D) of feedstock were prepared and spray dried. Details regarding the preparation of liquid feedstock for each of the four batches are shown in Table 7, where the total solid concentration is reported as the sum of the weights of the dissolved anhydrous materials. Particles of batches A and D were prepared on a Niro spray dryer using the feedstock of batches A and D, respectively. The process conditions used for spray drying of batch A (III-A) are the same as those used for spray drying of formulation IA of Example 1, and the process conditions used for spray drying of batch D (III-D). Was the same as the conditions used for spray drying of the formulation ID of Example 1. Particles of batches B and C, Buchi using the corresponding feed material It was prepared with a Mini spray dryer, but the process conditions were the same as those used for spray drying of Formulations IB and IC of Example 1 except for the following steps. The flow rate of the liquid feed material was set to 5.2 mL / min for Formula III-B and 6 mL / min for Formula III-C. The outlet temperature was about 91 ° C to 109 ° C for Formulation III-B and about 100 ° C for Formulation III-C.

<tables num="7"><img id="000008" he="69" wi="129" file="JP5877201B2_D0001.tif" img-format="tif" img-content="drawing" /></tables>

Table 8 summarizes some of the physical properties of the particles obtained in the four different batches (formulations III-A, III-B, III-C and III-D). In addition to the data listed in Table 8, detailed data on dried particles prepared from feedstock preparation III-A can be summarized as follows. The average particle fraction (FPF) measured by the Andersen cascade impactor in all eight stages by gravimetric analysis was 55.3% for FPFs &lt;5.6 microns and 39.7% for FPFs &lt;3.4 microns. Aerodynamic particle size was also measured by gravimetric analysis by ACI at all stages. The mean aerodynamic mass median diameter (MMAD) was 2.89 microns. The particle size was determined by laser diffraction with a HELOS / RODOS particle size measuring device, and the average value of the median volume (× 50) at a pressure of 1 bar was 1.51 microns. Furthermore, this powder behaved relatively independently of the flow velocity, as evidenced by the ratio of x50 measured at 0.5 bar to x50 measured at 4.0 bar, which was 1.12. The value of 1 / 4bar of these powders was 1.08.

Further characteristics of the dry powder prepared from the feedstock preparation III-D are summarized below. The average particulate fraction (FPF) measured by the Andersen cascade impactor in all eight stages by gravimetric analysis was 62.2% FPF &lt;5.6 microns and 45.3% FPF &lt;3.4 microns. Aerodynamic particle size was also measured by gravimetric analysis by ACI at all stages. The mean aerodynamic mass median diameter (MMAD) was 2.72 microns. The particle size was determined by laser diffraction with a HELOS / RODOS particle size measuring device, and the average value of the median volume (× 50) at a pressure of 1 bar was 1.47 microns. Furthermore, this powder behaved relatively independently of the flow velocity, as evidenced by the ratio of x50 measured at 0.5 bar to x50 measured at 4.0 bar, which was 1.08. The value of 1 / 4bar of these powders was 1.03.

<tables num="8"><img id="000009" he="29" wi="129" file="JP5877201B2_D0001.tif" img-format="tif" img-content="drawing" /></tables>

Further information on the properties of the powder and / or particles of Formulation III prepared in this example is given in the table or in the graphs shown in Figures 1A-IF and 2-4. SEM was performed as described above (Fig. 5B).

Example 3 This example describes the preparation of a dry powder using Formulation II: 10 weight percent leucine, 39.6 weight percent calcium chloride and 50.4 weight percent sodium sulfate as feedstock.

In the batch step, an aqueous phase was prepared by dissolving leucine, then sodium sulfate, and finally calcium chloride dihydrate in ultrapure water. The solution or suspension was continuously stirred throughout the process until the raw material was completely dissolved in water at room temperature. In the static mixing step, the sodium salt and the calcium salt were kept in separate solutions. The ultrapure water was divided in half and half of the required total leucine was dissolved in each amount of water. Sodium sulphate was dissolved in one aqueous phase and calcium chloride dihydrate was dissolved in the second aqueous phase. The solution or suspension was continuously stirred throughout the process until the ingredients were completely dissolved in water at room temperature. The solution or suspension was then spray dried using a Niro or Buchi spray dryer. For each formulation, four batches (A, B, C and D) of feedstock were prepared and spray dried. Details regarding the preparation of liquid feedstock for each of the four batches are shown in Table 9, where the total solid concentration is reported as the sum of the weights of the dissolved anhydrous materials. Particles of batches A and D were prepared on a Niro spray dryer using the feedstock of batches A and D, respectively. Particles of batches B and C were prepared on a Buchi spray dryer using the corresponding feedstock. The process conditions used for spray drying of batch A (II-A) are the same as those used for spray drying of formulation IA of Example 1, and the process conditions used for spray drying of batch D (II-D). Was the same as the conditions used for spray drying of the formulation ID of Example 1. Particles of batches B and C were prepared in a Buchi Mini spray dryer using the corresponding feedstock, but the process conditions were used in the spray drying of formulations IB and IC of Example 1 except for the following steps: It was the same as the condition. The flow rate of the liquid feed material was set to 8.3 mL / min for Formula II-B and 7 mL / min for Formula II-C. The outlet temperature was about 83 ° C for Formulation II-B and about 92 ° C for Formulation II-C. For Formulation II-B, the aspirator was set to 80%.

<tables num="9"><img id="000010" he="69" wi="129" file="JP5877201B2_D0001.tif" img-format="tif" img-content="drawing" /></tables>

Table 10 summarizes the physical properties of the particles obtained in the four different batches (formulations II-A, II-B, II-C and II-D). In addition to the data listed in Table 10, detailed data on dry powders prepared from feedstock formulation II-A are summarized below. The average particulate fraction (FPF) measured by the Andersen cascade impactor in all eight stages of gravimetric analysis was 68.7% of FPFs &lt;5.6 microns and 51.5% of FPFs &lt;3.4 microns. Aerodynamic particle size was also measured by gravimetric analysis by ACI at all stages. The average aerodynamic mass median diameter (MMAD) was 2.59 microns. The particle size was determined by laser diffraction with a HELOS / RODOS particle size measuring device, and the average value of the median volume (× 50) at a pressure of 1 bar was 2.50 microns. Furthermore, this powder behaved relatively independently of the flow velocity, as evidenced by the ratio of x50 measured at 0.5 bar to x50 measured at 4.0 bar, which was 1.47. The value of 1 / 4bar of these powders was 1.42.

Further characteristics of the dry powder prepared from the feedstock preparation II-D are summarized below. The average particulate fraction (FPF) measured by the Andersen cascade impactor in all eight stages by gravimetric analysis was 77.9% FPF &lt;5.6 microns and 68.3% FPF &lt;3.4 microns. Aerodynamic particle size was also measured by gravimetric analysis by ACI at all stages. The mean aerodynamic mass median diameter (MMAD) was 2.17 microns. The particle size was determined by laser diffraction with a HELOS / RODOS particle size measuring device, and the average value of the median volume (× 50) at a pressure of 1 bar was 1.90 microns. Furthermore, this powder behaved relatively independently of the flow velocity, as evidenced by the ratio of x50 measured at 0.5 bar to x50 measured at 4.0 bar, which was 1.17. The value of 1 / 4bar of these powders was 1.63.

<tables num="10"><img id="000011" he="29" wi="128" file="JP5877201B2_D0001.tif" img-format="tif" img-content="drawing" /></tables>

Further information on the properties of the powder and / or particles of Formulation II prepared in this example is given in the table or in the graphs shown in Figures 1A-IF and 2-4. SEM was performed as described above (Fig. 5C).

Example 4 This example describes the dose release of formulation batch IB, II-B and III-B powders from a dry powder inhaler under indoor and high conditions.

Method: Spray-dried powders of 3 different formulations (IB, II-B and III-B) up to about half of size 2 HPMC capsules (Quali-V, Qualicaps, Whitsett, NC) (13-30 mg depending on powder) ) Was filled. The capsule was loaded into one of the four capsule-type DPIs after the capsule was punctured so that the capsule was properly punctured. The capsule was loaded horizontally into the inhaler and then connected to a custom chamber. A pressure converter was connected to each dry powder inhaler to monitor the flow velocity through the inhaler during the test. At the start of the test, the operation of drawing an air flow of 45 L / min into each inhaler at once in 0.3 seconds was performed three times at 1-minute intervals. Each time it is pulled in at once, the air drawn through the inhaler rotates the capsule, the powder is released from the capsule into the air, and the four subs whose bottom is formed by three tissue culture wells arranged in a row. Entered one of the chambers. During a total of three times, the aerosol cloud was able to stay for one minute before the next withdrawal, and the total volume of air drawn into the aspirator was 0.68 L. Flow regulator (TPK-2000, MSP) for duration and total air velocity Adjusted with Corporation, Shoreview, MN) and recorded with an air mass flow meter (model number 3063, TSI Inc., Shoreview, MN). Pre-calibrated pressure sensors (model numbers ASCX01DN, Honeywell International Inc., Morristown, NJ) were used to monitor the airflow velocities of individual inhalers, and the signals were converted to flow velocities by a custom Lab-view code. In one case, the custom chambers were placed on the laboratory table in room conditions, and in the other two cases, the stability test chambers (Darwin Chambers) were set at 37 ° C and 90% RH. Placed in Company, St.Louis, MO). Stability test In the first case in the chamber, under indoor conditions, the capsule is pierced and loaded into the inhaler, the chamber door is opened, and about 30 seconds after the capsule is placed in the chamber, the inhaler is installed. It was attached and the flow velocity was activated. In the second case, the capsule is first placed in the stability test chamber without puncturing for 3 minutes, then removed from the chamber, punctured and loaded under room conditions, and entered into the chamber a second time. It was installed and operated in the chamber within 30 seconds. After each test, the capsules were removed from the inhaler and weighed and used to calculate the percentage of powder released from the capsules. For each of the three sets of powder formulations tested, two 12-well tissue culture plates (each plate required four capsules with four inhalers delivering the powder to each of the three wells. ) Was exposed to the powder, and a total of 8 capsules were released for each powder at each temperature and humidity setting.

As shown in Table 11 below, for all three powder batches (IB, II-B and III-B), the average amount of powder released from the capsule based on the weight change of the capsule was 99%. It exceeds.

<tables num="11"><img id="000012" he="39" wi="128" file="JP5877201B2_D0001.tif" img-format="tif" img-content="drawing" /></tables>

Example 5 In this example, the dispersion and density characteristics of the leucine formulations of formulations IA, II-A, III-A and placebo summarized in Table 12 are described. All the data in Table 12 are also shown in Figures 1A-1E. As is clear from the results shown in Table 12, all formulations are highly dispersible, which means that their measured particle size is relatively independent of pressure in HELOS / RODOS. As shown in Table 12, the ratio of the volume median diameter obtained at a low dispersion pressure (0.5 bar or 1.0 bar) to the volume median diameter obtained at a high dispersion pressure (4.0 bar) is used as an index of dispersibility. Can be used. Such a value is called a 0.5bar / 4.0bar ratio or a 1.0bar / 4.0bar ratio.

The tap density was determined by the modified USP &lt;616&gt; method using a 1.5 cc microcentrifuge tube, and the mean tap densities at 1,000 taps were 0.29, 0.69, 0.34 and 0.04 g / cc, respectively. .. The MMADs measured by the Andersen Cascade Impactor (ACI) for all stages (8 stages) were 2.72, 2.89, 2.59 and 4.29 μm, respectively. FPFs measured by ACI at all stages were 41.7%, 39.7%, 51.5% and 17.4% below 3.4 μm, respectively, and 56.2%, 55.3%, 68.7% and 32.5% below 5.6 μm, respectively. The particle size was determined by laser diffraction, and the mean volume median diameter (x50) at a pressure of 1 bar was 2.57 microns, 1.51 microns, 2.50 microns and 6.47 microns, respectively. The values at pressure values of 0.5 bar, 2.0 bar and 4.0 bar are listed in Table 12. Furthermore, as can be seen from the ratio of × 50 measured at 0.5 bar and × 50 measured at 4.0 bar shown in Table 12, the powder behaved relatively independently of the flow velocity. The values were 1.19, 1.12, 1.47 and 1.62, respectively. Also, since the value of the ratio of 1.0 bar to 4.0 bar is another measure of the dependence on the flow velocity, this is also shown in the table for comparison with other techniques.

<tables num="12"><img id="000013" he="98" wi="128" file="JP5877201B2_D0001.tif" img-format="tif" img-content="drawing" /></tables>

Example 6 In this example, the preparation of dry powder using the feedstock preparations 6.1 to 6.9 listed in Table 13 below will be described.

<tables num="13"><img id="000014" he="50" wi="128" file="JP5877201B2_D0001.tif" img-format="tif" img-content="drawing" /></tables>

The general mode of preparation of dry powders in this example is above, except that all dry powders of this example were spray dried using a Buchi B-290 spray dryer equipped with a high performance cyclone. It is the same as that described with respect to the powder of the Example. Formulations 6.1, 6.4 and 6.7 of this example correspond to Formulations III-B, IB and II-B of the above examples, respectively.

The physical properties of the powder and / or particles obtained in this example are summarized in the tables of Figures 6A and 6B. Formulations 6.1-6.9 in Table 13 correspond to Formulations 6.1-6.9 in Figures 6A and 6B, respectively. In Figure 6A, x50 and Dv50 represent the volume median or geometric volume median (VMGD), and GSD represents the geometric standard deviation. In FIG. 6B,% yield represents a percentage of the weight of the product recovered in a collection bottle mounted on a high performance cyclone divided by the weight of the solute in the feedstock. All other abbreviations are found elsewhere herein.

Example 7 This example describes the dose release of the powder prepared by feedstock formulations 6.1-6.9 from a dry powder inhaler under indoor and high conditions. Some of this data is also described in Example 4.

Method: Nine feedstock formulations 6.1-6.9 spray-dried powders were separately filled to about half (13-30 mg depending on the powder) of size 2 HPMC capsules (Quali-V, Qualicaps, Whitsett, NC). The capsule was loaded into one of the four capsule-type DPIs after the capsule was punctured so that the capsule was properly punctured. The capsule was loaded horizontally into the inhaler and then connected to a custom chamber. A pressure converter was connected to each dry powder inhaler to monitor the flow velocity through the inhaler during the test. At the start of the test, the operation of drawing an air flow of 45 L / min into each inhaler at once in 0.3 seconds was performed three times at 1-minute intervals. Each time it is pulled in at once, the air drawn through the inhaler rotates the capsule, the powder is released from the capsule into the air, and the four subs whose bottom is formed by three tissue culture wells arranged in a row. Entered one of the chambers. During a total of three times, the aerosol cloud was able to settle for 1 minute before the next withdrawal, and the total volume of air drawn into the aspirator was 0.68 L. Flow regulator (TPK-2000, MSP) for duration and total air velocity Adjusted with Corporation, Shoreview, MN) and recorded with an air mass flow meter (model number 3063, TSI Inc., Shoreview, MN). Pre-calibrated pressure sensors (model numbers ASCX01DN, Honeywell International Inc., Morristown, NJ) were used to monitor the airflow velocities of individual inhalers, and the signals were converted to flow velocities by a custom Lab-view code. In one case, the custom chambers were placed on the laboratory table in room conditions, and in the other two cases, the stability test chambers (Darwin Chambers) were set at 37 ° C and 90% RH. Placed in Company, St.Louis, MO). Stability test In the first case in the chamber, under indoor conditions, the capsule is pierced and loaded into the inhaler, the chamber door is opened, and about 30 seconds after the capsule is placed in the chamber, the inhaler is installed. It was attached and the flow velocity was activated. In the second case, the capsule is first placed in the stability test chamber without puncturing for 3 minutes, then removed from the chamber, punctured and loaded under room conditions, and entered into the chamber a second time. It was installed and operated in the chamber within 30 seconds. After each test, the capsules were removed from the inhaler and weighed and used to calculate the percentage of powder released from the capsules. For each of the three sets of powder formulations tested, two 12-well tissue culture plates (each plate required four capsules with four inhalers delivering the powder to each of the three wells. ) Was exposed to the powder, and a total of 8 capsules were released for each powder at each temperature and humidity setting.

As shown in Table 14 below, in all nine powder batches (obtained using feedstock formulations 6.1-6.9), the average amount of powder released from the capsules based on capsule weight variation was 98. It exceeds%.

<tables num="14"><img id="000015" he="61" wi="109" file="JP5877201B2_D0001.tif" img-format="tif" img-content="drawing" /></tables>

Example 8 In this example, the results of a short-term stability test conducted on the dry powder prepared according to the feedstock preparations 6.1, 6.4 and 6.7 are described.

An important property of pharmaceutical dry powder is its stability under various temperature and humidity conditions. One property that can destabilize the powder is that the tendency of the powder to absorb the surrounding moisture makes it easier for the particles to form agglomerates, and the apparent particle size of the powder changes under the same dispersion conditions. The spray-dried powder was stored under a certain range of conditions for a period of 1 week to 3 months or more, and the particle size distribution was tested regularly. Storage conditions include sealed capsules in vials at 25 ° C, 60% RH, sealed capsules in vials at 40 ° C, 75% RH, room temperature, sealed capsules at 40% RH, 30 ° C, 65. Included were capsules opened at% RH and capsules opened at 30 ° C, 75% RH. Size 3 HPMC capsules (Quali-V, Qualicaps, Whitsett, NC) were half filled with each dry powder. Spraytec (Malvern Instruments) capable of dispersing dry powder from an inhalation cell device Inc., Westborough, MA) One sample was immediately tested with a laser diffractive spray particle size measurement system. Each powder prepared using feedstock solutions 6.1, 6.4 and 6.7 was packed into approximately 16 capsules. Capsules were stored in laboratories with controlled humidity and temperature conditions (approximately 23-28% RH) and outside the laboratory with varying temperatures and relative humidity (approximately 40-75% RH). Stability tests set to these conditions for capsules stored under storage conditions of 60% RH at 25 ° C, 75% RH at 40 ° C, 65% RH at 30 ° C and 75% RH at 30 ° C. Stored in chambers (Darwin Chambers Company, St. Louis, MO). At a specific point in time (range 30 minutes to 3 months), for 1 to 3 capsules under each condition, the geometric particle size distribution characteristics are measured by Spraytec, and the aerodynamic particle size characteristics are determined by ACI-. Tested in 2.

The powder in sealed capsules generally placed in vials was stable for long periods of more than 3 months. Powder in open capsules not placed in vials showed agglomeration when exposed to high humidity conditions. Stability data are summarized in Table 15 below.

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Example 9 This example describes a bacterial passage assay performed using dry powders prepared using feedstock formulations A through E shown in Table 16.

Method: A transit model was used to test the effect of the aerosolized dry powder formulation on bacterial migration within the mucus. In this model, 200 μL of 4% sodium alginate (Sigma-Aldrich, St.Louis, MO) was added to the apical surface of a 12 mm Costar Transwell membrane (Corning, Lowell, MA; pore size 3.0 μm) before making a dry powder formulation. Exposed. The dry powder was aerosolized into the chamber using a dry powder blower (Penn-Century, Inc., Philadelphia, PA) and settled by gravity for 5 minutes. After this exposure, 10 μL of Klebsiella pneumoniae (about 10 in saline)⁷CFU / mL) was added to the apical surface of the mimic. At various time points after the addition of bacteria, aliquots of basal buffer were taken, serially diluted and seeded on a blood agar plate to determine the number of bacteria in each aliquot. A schematic diagram of this method is shown in FIG. The concentration of salt delivered to each Transwell was quantified by HPLC. For this purpose, the empty wells of a 12-well cell culture plate next to each Transwell and exposed to the same dose of formulation were washed with sterile water, diluted 1: 1 with acetic acid and in each powder. The calcium salt was solubilized.

The effect of calcium-containing powders on the transfer of K. pneumoniae within a sodium alginate mucus mimic was tested. Dry powder formulations containing calcium salts with different solubility profiles along with leucine and sodium chloride were screened for activity. Table 16 (bottom) lists the feedstock formulations of the powders tested. Due to dosing and detection limits in the transit model, the formulations described in the above examples were filled with 10.0% (w / w) leucine, whereas this composition was filled with 50.0% (w / w) leucine. Needed to be filled. Calcium to sodium molar ratios for each formulation were selected with the goal of a 1: 1 molar ratio, but did not need to be very low relative to the relative weight of any particular salt. Therefore, the lactate, citrate and acetate formulations used to maintain weights of sodium chloride and calcium chloride in excess of about 10% by weight, respectively, in these formulations are not in a 1: 1 molar ratio. It was.

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The results of this test are shown in Figures 8A and 8B. These two different figures represent two different sets of experiments performed under the same conditions. From the leucine control and sulfate data, it is possible to compare the two sets of experiments relatively. Powders containing sulfuric acid, lactic acid and acetic acid as anions, i.e., dry powders prepared from feedstock formulations A, D and E, respectively, reduced bacterial migration within the mimic, whereas anions. No effect was seen with powders containing carbonic acid and citric acid, i.e. dry powders prepared from feedstock formulations B and C. These findings correlate with the previously known solubility of calcium salts in water, and it is alginic acid that carbonates and citrates failed to inhibit the migration of K. pneumoniae. It has been shown that it may be related to the solubility of these powders on the surface of sodium mimetics. This conclusion is also concluded that the exchange reaction already described is completed during spray drying and the forms of calcium salts in formulations A-E are calcium sulfate, calcium carbonate, calcium citrate, calcium lactate and calcium acetate, respectively. It is also based on a reasonable assumption. The solubility of these salts is in the order of calcium carbonate &lt;calcium citrate &lt;calcium sulfate &lt;calcium lactate &lt;calcium acetate, from small to large. (See Table 1 above.)

Example 10 In this example, the performance of the dry powder in reducing virus replication using a virus replication model will be described.

This example describes a series of dose-response tests for different dry powders prepared from feedstock formulations composed of different calcium salts. Dry powders were made using leucine, calcium salts (milk salts or hydrochlorides) and sodium salts (salts, sulfates, citrates or carbonates). The listed feedstock formulations 10-1, 10-2 and 10-3 were spray dried on a Buchi B-290 mini spray dryer. In this system, a Buchi B-296 dehumidifier was used to stabilize the temperature and humidity of the air used for spray drying. The feedstock formulation 10-4 was spray-dried with a Niro Mobile Minor spray dryer using nitrogen in an open cycle manner.

Four liquid feedstocks were prepared with the following ingredients and ratios (weight percentages) listed in Table 17.

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Due to the limitations of administration and detection in the virus replication model, the formulations described in the above examples were loaded with 10.0% (w / w) leucine, whereas this composition was loaded with 50.0% (w / w) leucine. Needed to be filled. Calcium to sodium molar ratios for each formulation were selected with the goal of a 1: 1 molar ratio, but did not need to be very low relative to the relative weight of any particular salt. Therefore, the lactate and citrate formulations used to maintain weights of sodium chloride and calcium chloride in excess of about 10% by weight, respectively, in these formulations were not in a 1: 1 molar ratio.

Formulations 10-1, 10-2 and 10-3 were spray dried at a solid feedstock concentration of 5 g / L, but the exact amount of salt and excipient dissolved in ultrapure water and its specific volume It was various. Using process settings such as: Inlet temperature at 220 ° C, liquid flow rate at about 10 mL / min, room conditions at 19-21% RH at 23.2-24.6 ° C, and 30% RH at 3-5 ° C. Dehumidifier air. Outlet temperatures, cyclone and aspirator velocities varied. Formulation 10-1 was spray dried with a high performance cyclone at 80% aspirator and 93 ° C outlet temperature. Dry powder formulations 10-2 and 10-3 were prepared using a conventional cyclone at 100% aspirator and outlet temperature 111-115 ° C. Formulation 10-4 was spray dried with a solid concentration of 2.7 g / L and a process setting such as: 140 ° C inlet temperature, 75 ° C outlet temperature, 30 mL / min liquid feed feed rate, 100 kg. Process gas flow rate at / hour, atomizer gas flow rate of 20 g / min, and spray drying drum chamber pressure of -2 "WC.

The effects of preparations 1 to 4 were tested using a cell culture model of influenza infection. Calu-3 cells (American Type Culture Collection, Manasas, VA) until confluent (membrane completely covered with cells) on permeable membrane (12 mm Transwells; pore diameter 0.4 μm, Corning Lowell, MA) Incubate, remove apical medium, 37 ° C / 5% CO₂By culturing in, gas-phase liquid-phase interface (ALI) culture was established. By each experiment, cells were cultured in ALI for more than 2 weeks. Prior to each experiment, the apical surface of each Transwell was washed 3 times with PBS (Hyclone, Logan, UT). Calu-3 cells were exposed to dry powder using a proprietary dry powder deposition chamber. Capsules were filled with various amounts of each powder to expose the cells to equal doses of calcium. The amount of calcium delivered by each powder was matched to calculate high, medium and low filling weights (4.23 mg, 1.06 mg and 0.35 mg). Under the conditions of each dry powder tested, the empty, fully filled and post-exposure weights of the two capsules were weighed to determine the powder release dose. The pre- and post-exposure capsule filling weights determined by HPLC measurements and the calcium concentrations delivered to the cells are shown in Table 18 (bottom). Immediately after exposure, the basal surface medium (Transwell bottom medium) was replaced with new medium. In each test, triple wells were exposed to dry powder from each feedstock formulation. A second cell culture plate was exposed to the same dry powder from the feedstock formulation to quantify the total amount of salt or calcium delivered to the cells. 1 hour after exposure, cells were infected with 10 μL influenza A / WSN / 33/1 (H1N1) or influenza A / Panama / 2007/99 (H3N2) with a multiplicity of infection of 0.1-0.01 (0.1-0.01 per cell). Infected with virions). After 4 hours of aerosol treatment, the apical surface was washed to remove excess dry powder and unbound virus and cells were cooled at 37 ° C, 5% CO.₂Incubated for another 20 hours. Twenty-four hours after aerosol treatment, the virus released on the apical surface of infected cells was collected in medium or PBS, and the virus concentration in the apical lavage fluid was measured by TCID.₅₀(50% tissue culture infection amount) Quantified by assay. TCID₅₀The assay is a standard endpoint dilution assay used to quantify the amount of virus present in a sample.

Dry powder formulations were tested in cell culture models and their effect on influenza A / WSN / 33/1 infection was evaluated (Table 18). Equal amount of calcium ions (Ca²⁺) Was delivered, the desired filling weight was calculated for each dry powder formulation. The empty, fully filled and post-exposure Qualicap capsules were weighed to determine the release dose. Three sets of wells were exposed to each capsule, after which the wells were washed. HPLC analysis of these samples was performed to deliver Ca to the cells.²⁺The amount of was determined.^*Indicates that two capsules were used to achieve the desired filling weight.^aIs n = 3,^bIndicates that n = 1.

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Example 10A Dry powders prepared from feedstock formulations 10-1 to 10-4 reduce influenza A / WSN / 33/1 (H1N1) infection in a dose-dependent manner.

To test the effect of the dry powder formulation on influenza infection, cell culture model Calu-3 cells were exposed to four different dry powder formulations, each consisting of 50% leucine, calcium salt and sodium chloride. Virus infection was assessed by quantifying the amount of virus replication over a 24-hour period. The specific powders tested were listed in Table 18 (above) and contained carbonates, lactates, sulfates and citrates. Capsules were filled to the appropriate filling weight prior to administration to expose cells to equal amounts of calcium in each of the four calcium-containing powders. Cells that were not exposed to the formulation (Air) were used as control cells.

As shown in FIG. 9, each powder showed a dose-dependent reduction in influenza infection, but the degree of effect varied among the four test powders. At low calcium concentrations, calcium lactate was the most effective, indicating that calcium lactate was the most potent of the test powders. At high concentrations of calcium, calcium lactate and calcium citrate powders showed comparable efficacy. Further testing of calcium citrate powder at higher concentrations may show that calcium citrate powder is the most effective powder. Calcium sulphate powder showed an intermediate effect and was comparable to calcium citrate at some concentrations. Calcium carbonate had the least effect on viral replication at the highest concentrations (less than 10-fold). It should be noted that calcium carbonate has the lowest solubility of the test powders.

As shown in FIG. 9, the dry powder prepared for this example reduces influenza infection in a dose-dependent manner. Calu-3 cells not exposed to the formulation were used as controls and compared to Calu-3 cells exposed to dry powder formulations at various packing weights. The concentration of virus released by cells exposed to each aerosol formulation was quantified. The bars represent the mean and standard deviation of the triple wells under each condition. After the test, the data were statistically analyzed by one-way ANOVA and Tukey multiple comparison.

Example 10B Dry powders prepared from feedstock formulations 10-1 to 10-4 in Table 19 reduce influenza A / Panama / 2007/99 (H3N2) infection in a dose-dependent manner.

To further extend these tests, the same powder was tested on a second influenza strain [Influenza A / Panama / 2007/99 (H3N2)]. Similar to Example 10A, Calu-3 cells were exposed to four different dry powder formulations, each consisting of 50% leucine, a calcium salt and sodium chloride. Virus infection was assessed by quantifying the amount of virus replication over a 24-hour period. The specific powders tested were listed in Table 19 (bottom) and contained carbonates, lactates, sulfates and citrates. Capsules were filled to the appropriate filling weight prior to administration to expose cells to equal amounts of calcium in each of the four calcium-containing powders. Cells that were not exposed to the formulation (Air) were used as control cells.

As shown in FIG. 10, similar efficacy is seen with this strain, calcium lactate is the most effective, calcium citrate and calcium sulfate have an intermediate effect, and calcium carbonate has the least effect. There was only. These data support the widespread activity of dry Ca: Na powders against multiple influenza strains.

Dry powders prepared from feedstock preparations 10-1 to 10-4 were tested in a cell culture model and their effect on influenza A / Panama / 99/2007 (H3N2) infection was evaluated (Table 19). Equal amount of Ca²⁺The desired filling weight was calculated for each dry powder formulation to deliver. The empty, fully filled and post-exposure Qualicap capsules were weighed to determine the release dose. Three sets of wells were exposed to each capsule and then the wells were washed. HPLC analysis of these samples was performed to deliver Ca to the cells.²⁺The amount of was determined.

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As shown in FIG. 10, the dry powder prepared for this example reduces influenza A / Panama / 99/2007 (H3N2) infection in a dose-dependent manner. Calu-3 cells not exposed to the formulation (0 μg Ca)²⁺/cm²) Was used as a control and compared to Calu-3 cells exposed to dry powder formulations at different packing weights and thus at different calcium concentrations. HPLC measurements of calcium in the wash solution of empty plates exposed to each condition were used to determine the concentration of calcium delivered to cells in each experiment for each packing weight. TCID the concentration of virus released by cells exposed to each aerosol formulation 24 hours after administration.₅₀Quantified by assay. Each data point represents the mean and standard deviation of the triple wells under each condition.

Example 11. In vivo influenza model In this example, a dry powder formulation consisting of a calcium salt and sodium chloride is shown to reduce the severity of influenza infection in ferrets. The formulations tested are shown in Table 20. Control ferrets were exposed to a powder consisting of 100% leucine under the same exposure conditions. In in vitro preliminary studies, this control powder had no effect on virus replication. Calcium powder and controls (Formula I, Formulation II, Formulation III and leucine controls) were aerosolized with a Palas Rotating Brush Generator 1000 solid particle disperser (RBG, Palas GmbH, Karlsruhe, Germany). Approximately 0.2 mg of ferret (n = 8 per group) Exposure to Ca / kg, the severity of infection was assessed over time. Each formulation was dispersed by nasal exposure method 1 hour before infection, 4 hours after infection, and then by BID for 4 days (d1-4). The test was completed on the 10th day. Body temperature was measured twice daily, starting on day 0 of the study. Ferrets infected with influenza usually show elevated body temperature within 2 days of infection, lose weight during the study period, and show clinical signs of infections such as somnolence and sneezing. These changes are consistent with increased titers of influenza virus flowing from the nasal passages and increased rhinitis.

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Four days before the test, a microchip was placed subcutaneously on the right splenic abdomen of the ferret and the rest under the shoulder. A BMDS electronic proximity reader (WRS-6007; Biomedic Data Systems) during the test period with a transponder chip (IPTT-300 Implantable Programmable Temperature and Identification Transponder; Bio Medic Data Systems, Inc, Seaford, Delaware 19973). Inc, Seaford, Delaware) can be used to identify ferrets and provide subcutaneous body temperature data. Subcutaneous body temperature measured 3 to 1 day ago was used as the baseline temperature to calculate changes from baseline for each individual during the test period. Treatment with a dry powder formulation of leucine (excipient), calcium lactate (formulation III) and NaCl had a significant effect on elevated body temperature (Fig. 11C). Changes in mean body temperature in this group were maintained below baseline measurements during the study, and area under the curve (AUC) measurements were approximately 5-fold lower than controls (Figure 11D). The other two powders tested showed less pronounced effect, limited to that the effect was different from the control on a particular day of the test. Especially in the Ca citrate and Ca sulfate treated groups, the body temperature on the third day of the test was lower than that of the control individual (FIGS. 11A and 11B, respectively), and in the Ca sulfate group, the body temperature was lower during the last three days of the test.

Example 12 In this example, it is shown that a dry powder formulation consisting of various excipients reduces influenza infection at higher doses than a formulation consisting of leucine.

To assess the effect of excipients on in vitro efficacy, we used an influenza replication model to test two dry powder formulations with different excipients (Table 21) and their efficacy. Was compared with Formulation III (containing leucine). These formulations contained the same concentrations of calcium lactate and sodium chloride and the same weight percentage of excipient (10%).

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Calu-3 cells not exposed to the formulation were used as controls and compared to Calu-3 cells exposed to a dry powder consisting of calcium lactate and a different excipient than sodium chloride. 10 μg Ca with 3 different filling weights of mannitol and maltodextrin powder²⁺/cm²~ 30μg Ca²⁺/cm²Covered the dose range between. The concentration of virus released by cells exposed to each aerosol formulation was quantified (Fig. 12). Each data point represents the mean and standard deviation of the dual wells at each concentration. After the test, the data were statistically analyzed by one-way ANOVA and Tukey multiple comparison. The low dose data for each powder is representative of two independent experiments.

Both mannitol-containing and maltodextrin-containing preparations reduced influenza infection in a dose-dependent manner, but were significantly less potent than leucine-containing powders. 14.8 μg Ca²⁺/cm²Dosage of leucine-containing powder 2.9 ± 0.2 log influenza infection₁₀TCID₅₀A similar dose (12.2 μg Ca) compared to a reduction of / mL²⁺/cm²) Mannitol powder 0.85 ± 0.0 log infection₁₀TCID₅₀Reduced by / mL, maltodextrin powder (11.9 μg Ca)²⁺/cm²) Had no effect on replication (Fig. 12). Higher dose (&gt; 27 μg Ca²⁺/cm²) But the maximum reduction with mannitol (1.9 ± 0.50 log)₁₀TCID₅₀Maximum reduction (2.2 ± 0.14 log) with / mL) and maltodextrin₁₀TCID₅₀/ mL) did not reach that of leucine powder. It should be noted that previous studies with 100% leucine powder did not show the effect of the excipient alone on virus replication. These data indicate that the nature of the excipient affects the effectiveness of calcium-containing preparations.

Example 13 In this example, the effect of a dry powder formulation containing calcium salt, calcium lactate, calcium sulfate or calcium citrate powder on the treatment of influenza, parainfluenza or rhinovirus is shown.

Powders of Formulation I, Formulation II and Formulation III were produced by spray drying using a Mobile Minor spray dryer (Niro, GEA Process Engineering Inc., Columbia, MD). All solutions had a solid concentration of 10 g / L and were prepared using the ingredients listed in Table 22. Leucine and calcium salt were dissolved in DI water, and leucine and sodium salt were separately dissolved in DI water, and the two solutions were stored in separate containers. The liquid feedstock was sprayed using a parallel flow two-fluid nozzle (Niro, GEA Process Engineering Inc., Columbia, MD). A static mixer (Charles Ross &amp; Son) just before introducing the liquid feedstock into the two-fluid nozzle using a gear pump (Cole-Parmer Instrument Company, Vernon Hills, IL). Supplyed to Company, Hauppauge, NY). Nitrogen was used as the drying gas, and dry compressed air was supplied to the two-fluid nozzle as the spraying gas. The inlet temperature of the process gas was 282 ° C, the outlet temperature was 98 ° C, and the rate of the liquid feedstock was 70 mL / min. The gas supplied to the two-fluid atomizer was about 14.5 kg / hour. The pressure in the drying chamber was -2 "WC. Spray dried products were collected from the filter device into the container.

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The effects of dry powder preparations were evaluated using a cell culture model of influenza A / Panama / 2007/99, human parainfluenza type 3 (hPIV3) or rhinovirus (Rv16) infection. This model has already been described in detail (see Example 10) and uses Calu-3 cells grown at the gas-phase-liquid phase interface as a model for influenza infection of airway epithelial cells. Calu-3 cells were exposed to dry powder using a dry powder deposition chamber. Calcium ions (Ca) delivered to each well²⁺) Was determined by HPLC using dry powder recovered from the empty wells of the cell culture plate. Table 23 shows the concentration of calcium deposited in each test.

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One hour after exposure, cells were infected with 10 μL of influenza A / Panama / 99/2007 with a multiplicity of infection of 0.1-0.01 (0.1-0.01 virions per cell) and 0.1 to human parainfluenza type 3 (hPIV3). Infected with ~ 0.01 multiplicity of infection (0.1-0.01 virions per cell) or 10 μL of rhinovirus (Rvl6) with 0.1-0.01 multiplicity of infection (0.1-0.01 virions per cell) I let you. After 4 hours of dry powder treatment, the apical surface was washed to remove excess formulation and unbound virus and cells were isolated at 37 ° C, 5% CO.₂Incubated for another 20 hours. The next day (24 hours after infection), the virus released on the apical surface of infected cells was collected in a medium, and the virus concentration in the apical lavage fluid was measured by TCID.₅₀(50% tissue culture infection amount) Quantified by assay. TCID₅₀The assay is a standard endpoint dilution assay used to quantify the amount of virus present in a sample. For each of the three powders, three different Ca²⁺Calu-3 cells were exposed to the dose and replication of each virus was evaluated.

influenza In influenza, all three powders significantly reduce viral titers to comparable levels at the highest doses tested, i.e. Formula I, Formula II and Formula III, 3.25, 3.80 and 3.95 log, respectively.₁₀TCID₅₀The virus titer was reduced to / mL (Fig. 13A). At the highest doses tested, these powders showed comparable activity against influenza, but at lower doses, the most effective powders are Formula III (leucine, calcium lactate and chloride). It is important to note that it consisted of sodium). Formulation III at 3.70 and 3.75 logs at low and medium doses₁₀TCID₅₀Low doses of Formulation I and Formulation II reduced virus titers by / mL by 2.50 and 2.95 log, respectively.₁₀TCID₅₀Only / mL, medium doses Formula I and Formula II have viral titers of 2.65 and 3.30 log, respectively.₁₀TCID₅₀Reduced by / mL.

Parainfluenza Formulation I, Formulation II and Formulation III were tested against parainfluenza in similar dose ranges. Parainfluenza titers in cell cultures treated with Formulation II were comparable to control cells at doses of calcium similar to those used in influenza experiments (Figure 13B), which is calcium sulfate based. It has been shown that the formulation can only be active against specific pathogens. In contrast, treatment with Formulation I and Formulation III showed a dose-dependent reduction in parainfluenza infection. At high doses, Formulation I and Formulation III were 2.70 and 4.10 log, respectively, compared to control cells.₁₀TCID₅₀Reduced infection by / mL. Similarly, Formulation III was more effective than Formulation I at the medium dose tested, but neither formulation reduced infection at the lowest dose tested (Figure 13B; Table 25). Overall, these data indicate that calcium-based dry powder formulations effectively reduce parainfluenza infectivity. These effects are specific to a particular calcium salt, and the effective dose range is very different from that seen in influenza.

Rhinovirus Influenza and parainfluenza are enveloped viruses. The same powder was tested against rhinovirus to test the broad activity of the dry calcium powder formulation and extend its findings to non-enveloped viruses. All three formulations reduced rhinovirus to some extent, with Formula III powder showing the highest activity (Fig. 13C). Treatment with Formulation III was significant 2.80 log at the highest dose tested₁₀TCID₅₀There was a decrease in virus in / mL. This low-dose and medium-dose powder titers 1.15 and 2.10 logs, respectively, compared to control cells.₁₀TCID₅₀Reduced by / mL. Treatment with Formulation I and Formulation II also reduced rhinovirus infection, to a lesser extent than with Formulation III. At the highest dose tested, formulation I was 1.70 log₁₀TCID₅₀Reduced infection by / mL, formulation II 1.60 log₁₀TCID₅₀Reduced infection by / mL. Overall, these results indicate that calcium-based dry powder formulations can be widely applied to a variety of viral infections.

The above data suggest that by increasing the delivery dose of the dry calcium powder formulation, the formulation exhibits higher activity than was already seen at low doses. All three powders tested reduced influenza infection, but the calcium lactate-based formulation (formulation III) was more potent than the calcium sulphate formulation (formulation II) and the calcium citrate formulation (formulation I). In addition, the virus titers were most reduced when treated with Formulation III across all three viral strains. High-dose formulation I reduced the viral titers of all three viral strains, but the effect was much more pronounced in influenza and parainfluenza, a mechanism that may be associated with viral strain specificity. Suggests the difference. Treatment with Formulation II was active against parainfluenza, but was more active against both influenza and rhinovirus, which means that certain calcium counterions may have some role in the optimal activity of the formulation. It suggests that.

Example 14. Calcium lactate, sodium chloride, maltodextrin dry powder This example describes the preparation of a dry powder with feedstock formulation IV: 10.0 weight percent maltodextrin, 58.6 weight percent calcium lactate and 31.4 weight percent sodium chloride.

In the batch process, an aqueous phase was prepared by dissolving maltodextrin, then calcium lactate pentahydrate, and finally sodium chloride in ultrapure water. The solution was continuously stirred throughout the process until the ingredients were completely dissolved in water at room temperature. For the maltodextrin and calcium lactate formulations, three batches (A, B and C) of feedstock were prepared and spray dried. Details regarding the preparation of each of the three batch liquid feedstocks are shown in Table 24, where the total solid concentration is reported as the sum of the weights of the dissolved anhydrous materials. The solution or suspension was then spray dried using a Buchi spray dryer. For each formulation, feedstocks for three batches (A, B and C) were prepared and spray dried. Particles of batches A, B and C, Buchi using the corresponding feed material It was prepared with a Mini spray dryer, but the process conditions were the same as those used for spray drying of Formulations IB and IC of Example 1 except for the following steps. The flow rate of the liquid feed material was set to 5.2 mL / min for Formulation IV-A and Formulation IV-B and to 5.6 mL / min for Formulation IV-C. The outlet temperature was about 90 ° C to 98 ° C for Formulation IV-A, about 100 ° C for Formulation IV-B, and about 100 ° C to 106 ° C for Formulation IV-C.

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Table 25 summarizes some of the physical properties of the particles obtained in the three different batches (formulations IV-A, IV-B and IV-C). In addition to the data listed in Table 25, detailed data on dry powders prepared from feedstock formulation IV-A are summarized below. The average particulate fraction (FPF) measured by gravimetric analysis with a collapsing two-stage Andersen cascade impactor was 71.3% FPF less than 5.6 microns and 47.5% FPF less than 3.4 microns. The particle size was determined by laser diffraction with a HELOS / RODOS particle size measuring device, and the average value of the median volume (× 50) at a pressure of 1 bar was 1.40 microns. Furthermore, this powder behaved independently of the flow velocity, as evidenced by the ratio of x50 measured at 0.5 bar to x50 measured at 4.0 bar, which was 1.04. The 1 / 4bar value of these powders was 1.00, indicating that the particles were highly dispersible.

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Further information on the properties of the powder and / or particles of Formulation IV prepared in this example is given in the table or in the graphs shown in Figures 1A-IF.

Example 15: Dispersibility In this example, a dry containing a powder of calcium lactate, calcium sulphate or calcium citrate when delivered from different dry powder inhalers by various inhalation operations and compared to a similarly dispersed conventional micronized formulation. Shows the dispersibility of powder preparations.

The dispersibility of various powder formulations was investigated by measuring the geometric particle size and percentage of the powder released from the capsule when inhaled by a dry powder inhaler at a typical flow rate used by the patient. For multiple powder formulations, particle size distribution and changes in packed capsule weight were measured as a function of flow velocity, inhalation volume and packed weight in the two passive dry powder inhalers.

The powder formulation was manually filled into size 3 HPMC capsules (Capsugel V-Caps) and the filling weight was measured by a weighing method using a chemical scale (Mettler Tolerdo XS 205). Formulation I (lot number 26-190-F) 25 mg and 35 mg, Formula III (lot number 69-191-1) 25 mg, 60 mg and 75 mg, Formula II (lot number 65-009-F) 25 mg and 40 mg, Spray-dried leucine powder (lot number 65-017-F) was filled with 10 mg and atomized albuterol sulfate (Cirrus lot number 073-001-02-039A) with a filling weight of 25 mg. Specific resistance over the typical range of dry powder inhaler resistance 0.020 kPa^1/2/ LPM and 0.036kPa^1/2Two types of capsule-type passive dry powder inhalers with / LPM (RS-01 Model 7, Low resistance Plastiape SpA and RS-01 Model 7, High resistance Plastiape SpA) were used. Timer control type solenoid valve with flow control valve (TPK2000, Copley) Scientific) was used to set the flow velocity and inhalation volume. Capsules were placed in a suitable dry powder inhaler and punctured, and the inhaler was hermetically connected to the inlet of a laser diffractive particle size analyzer (Spraytec, Malvern). Steady air flow rates in the system were initiated using the TPK2000 and the particle size distribution was measured by Spraytec at 1 kHz for at least 2 seconds to the total inhalation time. The calculated particle size distribution parameters included volume median diameter (Dv50), geometric standard deviation (GSD), and particle fraction (FPF) of particles less than 5 micrometers in diameter. At the end of the inhalation time, the dry powder inhaler was opened, the capsule was removed and reweighed to calculate the mass of powder released from the capsule within the inhalation time. In each test condition, quintuplets of capsules were measured and averaged for Dv50, FPF and capsule release powder mass (CEPM) results.

Calculate the energy required to perform the inhalation operation to correlate powder dispersions from inhalers at different inhalation rates, volumes, and resistances, and provide particle size and dose release data for inhalation energy. Plotted. Inhalation energy E = R²Q²Calculated as V, but in the formula, E is the intake energy expressed in joules and R is kPa.^1/2The resistance of the inhaler expressed in / LPM, Q is the steady flow velocity expressed in L / min, and V is the volume of intake air expressed in L.

FIG. 14 shows the doses released from capsules of Formula III powder with three different capsule filling weights using both high resistance and low resistance RS-01 dry powder inhalers. For each filling weight, the stable inhalation range is a high resistance inhaler with a total volume of 2L (R = 0.036kPa).^1/2From the maximum energy condition of 9.2 joules, which corresponds to a flow rate of 60 L / min by / LPM), the volume is reduced to 1 L, the flow rate is reduced to 15 L / min, and the inhaler resistance is R = 0.020 kPa.^1/2It was up to low energy reduced to / LPM. As can be seen from FIG. 14, under the highest energy conditions tested, Formula III out of the capsule with a single inhalation at all three filling weights of 25, 60 and 75 mg, the total mass of the powder filled in the capsule. .. With a filling weight of 25 mg, an average of more than 80% of the filling weight is produced under all inhalation conditions up to 0.16 joules. At 60 mg, the capsule release dose is less than 80% of the filling weight at 0.36 joules. At a capsule filling weight of 75 mg, the capsule release dose is less than 80% of the filling weight at 1.2 joules.

Figure 14 shows two different packing weights, 25 mg and 40 mg, ground in a jet mill to an average particle size of 1.8 micrometers, manually filled in size 3 capsules and dispersed in a high resistance RS-01 inhaler. Granulated albuterol sulfate formulations have also been shown. At an inhalation energy of 9.2 joules (2 L with a stable inhalation of 60 L / min), the average CEPM is above 80% of the capsule fill weight (at a 25 mg fill weight), as seen in both 25 mg and 40 mg fill weights. 93%, 84% with a 40 mg filling weight). However, at all measured lower energies, CEPM was below 10 mg (less than 30% of the capsule fill weight) at both fill weights, unilaterally decreasing with reduced inhalation energy.

FIG. 15 shows the particle size distribution of Formula III powder released from the inhaler, characterized by volume median diameter (Dv50) and plotted against the applied inhalation energy. The fact that the value of Dv50 does not change even if the energy value decreases means that the powder is well dispersed because the released powder does not further disaggregate even if energy is added. Shown. At all high energy values, the Dv50 values of the three fill weights of 75, 60 and 25 mg did not change, and even when dropped to 0.51 joules, the Dv50 was below 2 micrometers for all three fill weights (figure). 15). Given that inhalation in the range of 0.5-1.2 joules did not adequately release the powder from the capsule at 60 mg and 75 mg packing weights (Figure 14), the released powder was still well dispersed by DPI. It is clear that this was the case (Fig. 15). In this range, the size of Dv50 has not increased much, which can be expected if the released powder contains a large amount of aggregates and the dispersion is not good.

Figure 15 shows 25 mg (x) and 40 mg (+) crushed to an average particle size of 1.8 micrometers with a jet mill, manually filled into size 3 capsules and dispersed with a high resistance RS-01 inhaler. Filled weight atomized albuterol sulfate formulations are also shown. At an inhalation energy of 9.2 joules (2 L with a stable inhalation of 60 L / min), the average Dv50 is below 2 micrometers for both fill weights (1.8 μm each), as seen with both 25 mg and 40 mg fill weights. And 1.6 μm), which indicates good dispersion and relatively low agglomerates. However, at all measured lower energies, Dv50 increased to more than 2 micrometers (3.9 μm and 3.1 μm, respectively) and decreased monotonically with decreasing inspiratory energy, which is primary. It shows agglomeration of particles and poor dispersibility.

Additional powders were tested under all test conditions to determine mean CEPM and Dv50 (Table 26). These results indicate that the powder formulation can be completely released and deagglomerated with inhalation energy up to about 0.5 joules.

<tables num="26"><img id="000027" he="165" wi="129" file="JP5877201B2_D0001.tif" img-format="tif" img-content="drawing" /></tables><img id="000028" he="166" wi="128" file="JP5877201B2_D0001.tif" img-format="tif" img-content="drawing" /><img id="000029" he="146" wi="128" file="JP5877201B2_D0001.tif" img-format="tif" img-content="drawing" />

Example 16: Analysis of particles in solid state AX ray powder diffraction High-resolution X-ray powder diffraction (XRPD) and differential scanning calorimetry (DSC) were used to analyze the amorphous / crystalline content and polymorphism of formulations I, II, III and IV. In XRPD, phase identification was performed to identify any crystalline phase observed in each XRPD pattern. XRPD patterns were collected using PANalytical's X'Pert Pro diffractometer (Almelo, The Netherlands). Samples were analyzed using Cu radiation generated with an Optix long fine focus source. Source Cu using a multi-layer mirror with elliptical steps KαX rays were applied to the sample, passed through, and collected in a detector. The sample was sandwiched between 3 micron thick membranes, transmitted geometrically analyzed and rotated for optimum orientation statistics. Beam stops were optionally used with helium purge to minimize the background generated by air scattering. Axle divergence was minimized by using solar slits for the incident and diffracted beams. Diffraction patterns were collected using a highly sensitive X'Celerator at the scan position, 240 mm from the sample. The data collection parameters of each diffraction pattern are displayed above the image of each pattern in Attached Table C. Prior to the analysis, a silicon sample (NIST standard reference material 640c) was analyzed to confirm the position of the peak of silicon 1 1 1. Calculate patterns of possible crystalline components (including anhydrous and hydrated forms) at the Cambridge Structural Database or the International Center for Diffraciton It was obtained from the Data (ICDD) Database and compared with the pattern obtained in the experiment. The crystalline component was qualitatively determined. In order to evaluate the tendency of the powder to recrystallize due to short-term exposure to high humidity, XRPD was also performed on the powder that had been left under the condition of 75% RH for 3 to 4 hours in the Dynamic Vapor Sorption system. ..

Differential scanning calorimetry (DSC) was performed using a differential scanning calorimetry Q2000 (New Castle, DE) from TA Instruments. The sample was placed in an aluminum DSC pan and the weight was accurately recorded. Data collection and processing parameters are displayed on each thermogram. Indium metal was used as the calibration standard material. Glass transition temperature (T_g) Is reported from the inflection point of the transition or half the height of the transition. Standard mode DSC experiments were first performed on the powder of interest to assess the overall thermal behavior of the powder. Periodic mode DSC experiments were also performed to identify the occurrence of glass transitions in these powders in the temperature range of interest identified by standard DSC thermograms.

Surprisingly, calcium and sodium salts with a mixture of amorphous and crystalline contents with optimized properties for dispersibility and stability in the dry state and dissolution and water absorption properties in the hydrated state. A powder with a high content was produced. As shown in FIGS. 16 and 17, Formulation I powder is composed of a combination of crystalline sodium chloride, low crystalline or amorphous calcium citrate, and a potentially calcium chloride-rich phase. This was observed by XRPD (as evidenced by the absence of any peaks characteristic of calcium salt morphology in this powder and the absence of any peaks characteristic of leucine). As shown in FIG. 18, a glass transition temperature of about 167 ° C was observed in the calcium-rich amorphous phase in the periodic DSC, which is the condition under which this amorphous phase is standard. It has been shown to be relatively stable to conversion to crystalline at (25 ° C, 30% RH). Crystalline sodium chloride present in the powder in the dry state can increase the dispersibility and stability of the powder. Low crystalline or amorphous calcium salts present in Formula I powder can also promote rapid water absorption and dissolution of Formula I formulation during lung deposition (ie, even though crystalline sodium chloride is readily soluble). In contrast, calcium citrate is sparingly soluble). Soluble particles or powders dissolve rapidly. Poorly soluble particles or powders are slow to dissolve.

Similar results were seen with powder formulation III and formulation IV. As shown in FIGS. 19 and 20, Formulation III powder is composed of a combination of crystalline sodium chloride, low crystalline or amorphous calcium lactate, and a potentially calcium chloride-rich phase. Was observed by XRPD (as evidenced by the absence of any peaks characteristic of calcium salt morphology in this powder and the absence of any peaks characteristic of leucine). As shown in FIG. 21, a glass transition temperature of about 144 ° C was observed in the calcium-rich amorphous phase in the periodic DSC, which is the condition under which this amorphous phase is standard. It has been shown to be relatively stable to conversion to crystalline at (25 ° C, 30% RH). For Formula IV powder containing 10% maltodextrin or 10% leucine (see Figures 22 and 23), similar results are seen in XRPD data and in Figure 24 showing the glass transition temperature at about 134 ° C. Was done.

In contrast, Preparation II preparation showed the presence of some crystalline calcium salt-containing material (calcium sulfate) in addition to crystalline sodium chloride (see Figures 25A and 25B). However, the powder still had a fairly high content of the calcium-rich amorphous phase, as evidenced by the presence of a glass transition temperature of about 159 ° C by DSC (see Figure 26).

B. Surface Raman mapping For samples of formulations I-IV, surface mapping Raman experiments were performed to determine the nature of the chemical composition on the surface of the particles containing these formulations. Raman map spectra were obtained with a Renishaw in Via Ramascope (Gloucestershire, UK) Leica equipped with a DM LM microscope (Wetzlar, Germany). The equipment was calibrated using a standard silicon wafer. Samples of aluminum-coated microscope slides were prepared for analysis. The excitation wavelength using a high-power near-infrared diode laser source was 785 nm. Data collection for Formulation I, Formulation III and Formulation IV was a static scan with an exposure time of 30 seconds and an integration count of 10 times. The data collection of Formulation II was a long scan with an exposure time of 60 seconds and a cumulative number of times. Images with a 50x objective were obtained using a Philips ToUcam Pro II camera (model PCVC 840K) (Amsterdam, the Netherlands). Renishaw WiRE for data collection and processing 3.1 (Service Pack 9) Software (Gloucestershire, UK) was used.

Raman spectra of six particles were obtained from the Formula I sample, which are superimposed and shown in FIG. 27A. Spectral files 389575-1 and 389575-6 are approximately 1450, 965 and 850 cm^-1It is characterized by the presence of a weak peak in. These peaks are only identified as very weak features in the spectral file 389575-6 and are not detected in the remaining spectral data files. FIG. 27B removes background from spectrum 389575-6 and overlays it with Raman spectra of calcium citrate tetrahydrate, sodium citrate and leucine. Sample spectra are approximately 1450 and 850 cm common to leucine and citrate.^-1It shows the peak at. The sample spectrum is about 965 cm further^-1It also shows a peak in, which is consistent with a relatively strong peak in the spectrum of citrate (ie, calcium citrate tetrahydrate and sodium citrate). 1340 cm characteristic of leucine^-1No peak was observed in the sample spectrum.

Raman spectra of eight particles were obtained from the Formula II sample, which are superimposed and shown in Figure 27C. The spectrum of whole particles is about 1060 cm^-1It is characterized by the presence of peaks in. In spectrum file 388369-4, an additional about 670 cm^-1A peak is also observed in. 670 cm^-1Peaks are also observed in spectral data files 388369-1, 3 and 8 after background removal (not shown). Figure 27D is the Raman spectrum of calcium sulfate, calcium sulfate dihydrate, anhydrous sodium sulfate and leucine with the background removed from spectrum 388369-4. 520 cm in sample spectrum with background removed^-1What is considered to be the third peak is shown in the vicinity. At a position similar to the peak characteristic of the sulfate ion shown, 1060 cm^-1And 670 cm^-1There are peaks, but they do not exactly overlap. 1060 cm of sample spectrum^-1And 670 cm^-1The frequency of peaks in is consistent with the stretch and bending modes of the sulfate ion functional groups, respectively. No peaks due to leucine are observed in the spectrum of the particles.

Raman spectra of 12 particles were obtained from the Formula III sample, which are superimposed and shown in FIG. 27E. The spectrum of whole particles is about 1045 cm^-1And 860 cm^-1It is characterized by the presence of peaks in. In addition, various spectra of approximately 1450, 1435, 1125, 1095, 930 and 775 cm^-1Peaks can also be observed in, but these are generally 1045 cm.^-1It correlates relatively strongly with the strong peak in. Figure 27F shows the Raman spectra of calcium lactate pentahydrate and leucine with background removed from spectra 389576-7 and 389576-12. There is a good correspondence between the sample spectrum and the calcium lactate pentahydrate spectrum. However, the sample spectra are about 1345, 1170, 960, 830 and 760 cm.^-1Further weak peaks are shown in the calcium lactate pentahydrate spectrum. Similar peaks are found in the leucine reference spectrum, but with slightly different relative intensities and frequencies.

Raman spectra of 12 particles were obtained from the Formula IV sample, which are superimposed and shown in Figure 27G. The spectrum of whole particles is about 1045 cm^-1It is characterized by the presence of peaks in. The spectrum of all particles except file 389577-2 is about 860 cm.^-1Also shows a peak. In addition, various spectra of approximately 1450, 1435, 1125, 1095, 930 and 775 cm^-1Peaks can also be observed in, but these are generally 1045 cm.^-1It correlates relatively strongly with the strong peak in. Figure 27H shows the Raman spectrum of calcium lactate pentahydrate with the background removed from spectrum 389577-9. There is a good correspondence between the sample and the spectrum of calcium lactate pentahydrate. Peaks due to maltodextrin (not shown) are not observed in the sample spectrum.

Therefore, in the Raman surface mapping analysis, the surface composition of each preparation I to IV is occupied by the presence of various calcium salts (calcium citrate in preparation I, calcium sulfate in preparation II, calcium lactate in preparations III and IV). It is shown that. This is different from reports of using leucine as a dispersion accelerator to increase the dispersibility of the powder, which is concentrated on the surface of the particles constituting the powder and is aerosolized, in the case of formulations I to III. ing. In the formulations disclosed herein, it appears that leucine does not act as a dispersion enhancer in this way, which is the case with formulation III (leucine-containing calcium lactate) with respect to surface inclusions and dispersibility. It is clear from the fact that similar results were observed with IV (calcium lactate containing maltodextrin).

Example 17: Ion exchange reaction in spray drying of saturated calcium citrate and calcium sulfate A saturated or supersaturated stock of calcium sulfate or calcium citrate aqueous solution was prepared for spray drying using calcium chloride and sodium sulfate or calcium chloride or sodium citrate as starting materials. The following two methods: (i) premix both salts with water, and (ii) store the calcium and sodium salts in separate aqueous solutions and perform static mixing in-line just before spray drying. Thereby, a total solid concentration in the range of 5 g / L to 30 g / L was prepared. All prepared liquid feedstocks are saturated or supersaturated amounts of calcium sulphate (here, the dissolution limit of calcium sulphate in water is 2.98 g / L) and saturated or supersaturated amounts of calcium citrate (here, quen The dissolution limit of calcium sulfate in water is 0.96 g / L). Precipitation reaction of calcium chloride and sodium sulfate proceeds to the end (CaCl₂+ Na₂SO₄ CaSO₄Considering + 2 NaCl), the corresponding final concentrations of calcium sulfate are listed in Table 24. In addition, the precipitation reaction of calcium chloride and sodium citrate (3CaCl)₂+ 2Na₃C₆H₅O₇ Ca₃(C₆H₅O₇)₂Similar results for +6 NaCl) are shown in Table 27.

<tables num="27"><img id="000030" he="63" wi="128" file="JP5877201B2_D0001.tif" img-format="tif" img-content="drawing" /></tables>

Spray drying with a Mobile Minor spray dryer (Niro, GEA Process Engineering Inc., Columbia, MD) produced 44 weight percent calcium chloride and 56 weight percent sodium sulfate formulations. Liquid feedstocks with solid concentrations in the range of 5 g / L to 30 g / L were prepared. In the feedstock for premixing, a sodium salt and then a calcium salt were dissolved in DI water with constant stirring on a magnetic stirring plate. For the feedstock for static mixing, the calcium salt was dissolved in DI water, and the sodium salt was separately dissolved in DI water, and the two solutions were stored in separate containers with constant stirring. The liquid feedstock was sprayed using a parallel flow two-fluid nozzle (Niro, GEA Process Engineering Inc., Columbia, MD). Gear pump (Cole-Parmer Instrument Company, Vernon Using Hills, IL), the liquid feedstock is fed directly to the bifluid nozzle for premixed feedstocks, or the static mixer (for static mix feedstocks) just before introduction into the bifluid nozzle. Supplied to Charles Ross &amp; Son Company, Hauppauge, NY). Nitrogen was used as the drying gas, and dry compressed air was supplied to the two-fluid nozzle as the spraying gas. The inlet temperature of the process gas was 240-250 ° C, the outlet temperature was 94-98 ° C, and the rate of the liquid feedstock was 50-70 mL / min. The gas supplied to the two-fluid atomizer was approximately 11 kg / hour. The pressure in the drying chamber was -2 "WC. The spray-dried product was collected from the cyclone and laser diffracted with RODOS-equipped HELOS to obtain volumetric particle size and a collapsing two-stage ACL. The aerosol characteristics were analyzed.

Solution stability and clarity were evaluated for the premixed feedstock. At a total solid concentration of 5 g / L, where the final calcium sulphate concentration was just above the dissolution limit of calcium sulphate, the solution was clear for 30 minutes of mixing and spray drying. As the total solid concentration increased and the final calcium sulfate concentration far exceeded the dissolution limit, the feedstock became cloudy and precipitated. At 10 g / L, the liquid becomes slightly turbid, at 20 g / L, the liquid becomes clarified for about 5 to 10 minutes and then gradually becomes turbid in 10 minutes, and at 30 g / L, the liquid becomes clarified for about 2 minutes after mixing. The condition was maintained, and after about 5 minutes, precipitation began to appear.

The premixed liquid feedstock and the statically mixed liquid feedstock were spray dried and the resulting dry powder was collected from the cyclone. The results obtained from HELOS with RODOS are shown in Fig. 28, and the typical particle size distribution is shown in Fig. 29. An increase in particle size is expected as the solid concentration of the feedstock increases (as seen in statically mixed feedstocks), but a significant increase in particle size and particle size in the premixed feedstock. Spreading the distribution is not desirable.

The results of aerosol characterization of dry powders using disintegrating ACI are shown in FIG.

Unstable solutions with persistent precipitation can adversely affect reproducible particle formation during spray drying and can result in a wide particle size distribution. The presence of a supersaturated clear solution at high solid concentrations for 2-10 minutes indicates that static mixing of the solution can achieve a high spray drying capacity while obtaining a reproducible narrow particle size distribution. Shown.

Results similar to those shown in Formulation 1 containing 10.0 weight percent leucine, 35.1 weight percent calcium chloride, and 54.9 weight percent sodium citrate (Formulation IA) were demonstrated with calcium citrate. Was done. The precipitation reaction will result in a formulation containing 10.0 weight percent leucine, 52.8 weight percent calcium citrate, and 37.2 weight percent sodium chloride. At a total solid concentration of 10 g / L, the final calcium citrate concentration can be 5.3 g / L, which exceeds the dissolution limit of 0.96 g / L in calcium citrate water. As can be seen from the characteristics of the spray-dried powder (Figs. 1A-1E and 2-4), inhalation particles with a narrow particle size distribution were obtained from this supersaturated solution.

Example 18 Small, dispersible particles were produced from leucine-containing and leucine-free calcium-containing and magnesium-containing and sodium-only formulations.

The powders listed below were spray dried with Buchi B-290 using a high performance cyclone equipped with air with an air supply rate of 30 mm, a suction device with a speed of 90% and a small glass collection container. The inlet temperature was 220 ° C and the outlet temperature was between 96 ° C and 102 ° C. The solid concentration was 5 g / L, and the following components were added after completely dissolving one component at a time, all in the order listed below, and mixed in DI water. 18-1) Lactose 10.0%, magnesium chloride 30.6%, sodium citrate 59.4%, Ca: Na ratio = 1: 2 18-2) Magnesium lactate 63.4%, sodium chloride 36.6%, Ca: Na ratio = 1: 2 18-3) Leucine 10.0%, magnesium lactate 58.4%, sodium chloride 31.6%, Ca: Na ratio = 1: 2 18-4) Leucine 50.0%, calcium lactate 50% 18-5) Leucine 10%, sodium chloride 90% 18-6) Leucine 60%, sodium chloride 40% 18-7) Salbutamol 10.0%, calcium lactate 58.6%, sodium chloride 31.4% 18-8) Salbutamol 90.0%, calcium lactate 5.9%, sodium chloride 3.1%

The results of characterization of these powders are shown in Table 28 below. All eight powders showed good dispersibility with respect to the 0.5 / 4 and 1/4 ratios of x50. FPF &lt;5.6 microns was at least 18.7% -75.6%.

<tables num="28"><img id="000031" he="136" wi="129" file="JP5877201B2_D0001.tif" img-format="tif" img-content="drawing" /></tables>

Following the same steps as above, various spray dryer systems (Buchi, LabPlant and Niro systems) were used to further produce some typical calcium-free formulations. The results of characterization of the resulting powder are selected and shown in Table 29 (cells not filled with numbers indicate that no numbers were measured for that powder).

<tables num="29"><img id="000032" he="126" wi="128" file="JP5877201B2_D0001.tif" img-format="tif" img-content="drawing" /></tables>

Further examples of compositions containing no excipients or no leucine excipients using various spray dryer systems (Buchi, Labplant and Niro based systems) according to the same steps as above. I made some. The results of the characterization of the resulting powder are selected and shown in Table 30 (cells without numbers indicate that no numbers were measured for that powder).

<tables num="30"><img id="000033" he="85" wi="128" file="JP5877201B2_D0001.tif" img-format="tif" img-content="drawing" /></tables><img id="000034" he="200" wi="128" file="JP5877201B2_D0001.tif" img-format="tif" img-content="drawing" /><img id="000035" he="21" wi="127" file="JP5877201B2_D0001.tif" img-format="tif" img-content="drawing" />

Data for characterization of small, dispersible leucine / calcium-containing powder compositions further produced using the Buchi or Niro spray drying system according to the same steps as above are shown in Table 31 (numerical values are entered). No cell indicates that the powder was not measured).

<tables num="31"><img id="000036" he="178" wi="128" file="JP5877201B2_D0001.tif" img-format="tif" img-content="drawing" /></tables><img id="000037" he="148" wi="129" file="JP5877201B2_D0001.tif" img-format="tif" img-content="drawing" />

Example 19 Pure calcium chloride was spray dried at an inlet temperature of 180 ° C using the LabPlant spray drying system. The liquid feedstock was composed of calcium chloride dihydrate with a solid concentration of 20 g / L in DI water. Water was condensed in the collection container because calcium chloride was deliquescent and the powder could not be collected. Since pure calcium chloride has very high hygroscopicity, it was considered impossible to spray dry from an aqueous solution if the water content in the discharged drying gas was high. Next, the liquid feedstock was changed to 70% ethanol to reduce the humidity of the exhaust gas, keeping the solid concentration at 20 g / L, the inlet temperature at 200 ° C, and the outlet temperature at 69 ° C. Even so, the water condensed in the collection container and the powder was apparently moist. It was concluded that calcium chloride is so hygroscopic that it cannot be spray dried unless it is mixed with other salts or excipients to reduce the calcium chloride content of the final powder.

Pure magnesium chloride was spray dried on the Labplan system at an inlet temperature of 195 ° C and an outlet temperature of 68 ° C. The liquid feedstock was composed of magnesium chloride hexahydrate with a solid concentration of 20 g / L in DI water. The dry powder in the collection vessel was apparently moist, with a median diameter of 21 microns as measured by the HELOS / RODOS system. Next, the liquid feedstock was changed to 70% ethanol to reduce the humidity of the exhaust gas, keeping the solid concentration at 50 g / L, the inlet temperature at 200 ° C, and the outlet temperature at 74 ° C. This magnesium chloride powder was apparently not moist and had a volume median diameter of 4 microns, but the powder had a granular appearance with a fine particle fraction of less than 5.6 microns, which was 19% for inhalation. It was shown to be poorly suited.

Example 20: Large, porous particles<tables num="32"><img id="000038" he="4" wi="82" file="JP5877201B2_D0001.tif" img-format="tif" img-content="drawing" /></tables><img id="000039" he="177" wi="128" file="JP5877201B2_D0001.tif" img-format="tif" img-content="drawing" />

Example 21: Stability Dry powders were tested for stability in use under extreme temperature and humidity conditions defined at 30 ° C, 75% RH (ICH, Climatic Zone XIV). Capsules were filled with approximately 25 mg of Formulation I, Formulation II and Formulation III. The capsules were left open and then placed in the stability test chamber for 15 and 30 minutes under defined conditions. The capsules were taken out and sealed at an appropriate time, and the aerodynamic particle size distribution (aPSD) was examined using a collapsing two-stage ACI, and the geometric particle size distribution (gPSD) was examined using Malvern Spraytec. Both tests were performed at 60 LPM for 2 seconds. Each time point was repeated with n = 2. The results were compared with powder aPSD / gPSD data at room temperature, 25-30% RH.

Fine particle fraction less than 5.6 microns for the entire dose (FPFTD) of all formulations (Formula I, Formulation II and Formulation III) after 30 minutes of exposure to extreme temperature and humidity conditions (30 ° C, 75% RH). Showed a change of less than +/- 5% from the same fine particle fraction under standard conditions (22 ° C, 25-30% RH). With respect to gPSD, Formulation I showed an increase of about 30% after 30 minutes, while Formulation II remained nearly stable and Dv50 of Formulation III decreased by about 15% after 30 minutes.

After 30 minutes of exposure at 30 ° C and 75% RH, there was a slight change in the aerosol properties of the three formulations, but the change in geometric particle size was more pronounced (Fig. 31A and). 31B). The particle size of Formulation I (calcium citrate) increased by about 30%, while the particle size of Formulation III (calcium lactate) increased by about 15%. The particle size of Formulation II (calcium sulfate) was slightly reduced.

The additional formulations tested were calcium chloride powder (leucine 38.4%, calcium chloride 0% to about 1%, sodium chloride 31.6%), and formulation III formulation (excipient 10.0%, calcium lactate 58.6%, sodium chloride 31.4%). ), Three types of calcium lactate powders using different excipients (lactouse, mannitol, maltodextrin).

After 30 minutes of exposure to extreme temperature and humidity conditions (30 ° C, 75% RH), microparticle fractions &lt;5.6 microns relative to the total dose of maltodextrin (formulation IV) and mannitol formulations are generally standard. A change of less than +/- 10% was shown from the same fine particle fraction under the conditions (22 ° C, 25 to 30% RH). Particle fractions smaller than 5.6 microns relative to the total dose of calcium chloride powder and lactose preparation were affected by a decrease of more than 50% and an increase of about 20%, respectively (Figure 31C). The result of gPSD is the opposite, Dv of calcium chloride powder and lactose preparation after 30 minutes.₅₀The change in mannitol was less than +/- 10%, whereas the mannitol preparation Dv₅₀Increased by 30% to 60% during the study (Fig. 31D). Maltodextrin Dv₅₀Was not tested.

Example 22. Short-term stability at 30% and 40% RH at room temperature The spray-dried powder was stored at room temperature at about 30% and 40% RH for 1 week, and the particle size distribution was examined regularly. Each dry powder was half-filled in size 3 HPMC capsules (Quali-V, Qualicaps, Whitsett, NC). One sample was immediately examined with a Spraytec (Malvern Instruments Inc., Westborough, MA) laser diffracted spray particle size measurement system capable of dispersing dry powder from the inhaler using an inhaler cell device. Each powder was packed into about 16 capsules. Half of the capsules are stored in the laboratory (about 23-28% RH) under controlled humidity and temperature conditions, and the other half are stored outside the laboratory (about 38-40% RH) where the temperature and relative humidity change. did. At a specific time point (t = 1 hour, 2 hours, 4 hours, 24 hours, 48 hours, 1 week), the volume particle size distribution of one capsule in the room and one capsule outside the laboratory was adjusted by Spraye. Examined.

The results of formulation selection containing the combination of 50% leucine and the indicated calcium chloride and sodium salts are shown in Figures 32 and 33. Formulations containing calcium chloride and sodium chloride showed significant aggregation after exposure to high humidity conditions. With acetate preparations, the results at the beginning varied. Sulfate, citrate and carbonate formulations showed good relative stability throughout the test period.

When stored at room temperature and 40% RH, the dry powder formulation containing calcium chloride and sodium chloride became unstable 1 hour after exposure, and the results of particle size also varied with acetate formulation. The particle size of the sulfate and lactate powders increased slightly, while the particle size of the carbonate and citrate powders decreased slightly. The stability of chloride-only and acetate-containing formulations was considered unsuitable for further experimentation.

Example 23. Fluidity of powder The fluidity of the powders of formulations I, II, III and IV was also evaluated using conventional methods in the art for characterizing the fluidity of the powder. Flodex Powder Fluidity Testing Equipment (Hanson Research) The liquidity index of each powder was determined using Corp., Model 21-101-000). In each test, all samples were added using a stainless steel funnel towards the center of the cylinder trap door. Care was taken not to disturb the powder column in the cylinder. After waiting for the flocure to form for about 30 seconds, the trap door was opened to prevent the device from vibrating as much as possible. When the powder falls through the trap door, a hole can be seen through the cylinder from above, and if the remaining powder in the cylinder falls while forming an inverted cone, it is considered that the test has passed and the hole is open. The test was considered unsuccessful if it was not visible or if the powder fell straight without remaining in a cone. The flow disc was tested until the smallest hole size was found through which the powder passed and gave a positive test result. Two more tests were performed with the smallest size flow disk, with positive results being obtained on three out of a total of three. The liquidity index (FI) is reported for this smallest pore size.

Bulk density and tap density were determined using SOTAX's Tap Density Tester model TD2. In each test, all samples were placed in a 100 mL graduated cylinder weighed using a stainless steel funnel. Mass and initial volume of powder (V) according to USP I method₀) Was recorded, and the cylinder was attached to the anvil and operated. The first time, tap the cylinder using Tap Count 1 (500 taps), and the obtained volume V_aWas recorded. The second time, using Tap Count 2 (750 taps), a new volume V_b1Got V_b1Is V_aIf it exceeds 98% of, the test is completed, and if it does not exceed, V_bnIs V_bn-1Tap Count 3 (1250 taps) was used repeatedly until it exceeded 98% of. Powder bulk density (d_B), Tap density (d_T), Hausner ratio (H) and compressibility index (C) were calculated. The latter two are standard measures of powder fluidity. H is the tap density divided by the bulk density, and C is 100 × (1- (bulk density divided by the tap density)). Skeleton density was measured by Micromeritics' analytical service with Accupyc II 1340, which determines the volume of the powder using helium gas substitution technology. With this instrument, the volume of each sample was measured excluding the gaps in the bulk powder and the open porosity of the individual particles that the gas came into contact with. This volume still contained internal (closed) porosity. The density was calculated using this measured deposition and the sample weight determined using a scale. Volume was measured 10 times for each sample and skeletal density (d)_S) Was reported with the standard deviation as the average of 10 calculated densities.

The results of these density and fluidity tests are shown in Tables 34 and 35. All four powders tested have Hausner ratios and compressibility indices described in the art as being characteristic of powders with extremely poor fluidity (see, eg, USP &lt;1174&gt;). ). Therefore, it is surprising that these powders are highly dispersible and have good aerosol properties as described herein.

<tables num="33"><img id="000040" he="29" wi="128" file="JP5877201B2_D0001.tif" img-format="tif" img-content="drawing" /></tables>

<tables num="34"><img id="000041" he="26" wi="128" file="JP5877201B2_D0001.tif" img-format="tif" img-content="drawing" /></tables>

The USP &lt;1174&gt; mentioned above states that dry powders with Hausner ratios greater than 1.35 have poor fluidity. Both fluidity and dispersibility are negatively affected by particle agglutination or aggregation. Therefore, it is unexpected that powders with a Hausner ratio of 1.75 to 2.31 are highly dispersible.

Example 24. Water content and hygroscopicity The water content of the powders of Formulations I, II, III and IV was determined by both thermogravimetric analysis (TGA) and Karl Fischer analysis. Thermogravimetric analysis (TGA) was performed using a TA Instruments Q5000 IR thermogravimetric analyzer (New Castle, DE). The sample was placed in an aluminum sample pan and inserted into a TG furnace. Data collection and processing parameters are displayed on each thermogram. Nickel and Alumel were used as calibration reference materials. In TGA, the water content was determined from the weight loss of the sample when heated to a temperature of 150 ° C (in TGA, the spray-drying solvent used was 100% water, so the volatile components in these powders. Assuming that only water exists). A typical TGA thermogram of powder formulation I is shown in Figure 34. Water coulometric Karl Fischer (KF) analysis was determined using a Mettler Toledo DL39 KF titrator (Greifensee, Switzerland). Hydranal-Coulomat The sample was placed in a titration vessel containing AD and mixed for 10 seconds until dissolved. Then electrochemical oxidation: 2I^- I₂The sample was titrated by a generating electrode where iodine is produced by + 2e. In general, one range measurement and two iterations were performed to ensure reproducibility. Table 35 shows a summary of the powdered water content data using these methods.

<tables num="35"><img id="000042" he="34" wi="128" file="JP5877201B2_D0001.tif" img-format="tif" img-content="drawing" /></tables>

Dynamic steam adsorption (DVS) step mode experiments were performed to determine the hygroscopicity and water absorption capacity of the powders of Formulations I, II, III and IV, raw calcium chloride dihydrate, and 38.4% leucine and CaCl.₂Calcium chloride: sodium chloride 1: 2 control powder produced by spray drying of a formulation containing 30.0% and NaCl 31.6% (good in spray dried powder without deliquescent in collection vessel immediately after spray drying The maximum filling level of calcium chloride that can be incorporated was determined to be 30% by weight). For DVS operating conditions, the powder was first equilibrated at 0% RH, then exposed to 30% RH for 1 hour, and then exposed to 75% RH for 4 hours. Table 36 shows the mass% of water absorption of each powder. As can be seen from Table 36, both the raw calcium chloride dihydrate and the control powder are extremely hygroscopic, absorbing about 14-15% water and 75% RH when exposed to 30% RH for 1 hour. After 4 hours of exposure to, it absorbed well more than 100% of its own mass. In contrast, powders of Formulations I, II, III and IV absorb less than 2.5% water when exposed to 30% RH for 1 hour and 14% to 33% when exposed to 75% RH for 4 hours. did.

<tables num="36"><img id="000043" he="45" wi="128" file="JP5877201B2_D0001.tif" img-format="tif" img-content="drawing" /></tables>

Example 25: Heat of dissolution The samples of preparations I to III were dissolved in HBSS buffer to obtain heat of solution, and (i) a control powder consisting of 30% calcium chloride, 31.6% sodium chloride and 38.4% leucine, and (ii) unprocessed calcium chloride di Compared with hydrate and (iii) raw leucine. The same method was also used to obtain heat of fusion for formulations VII and VIII.

As shown in Table 37, formulations I, II and III containing equal molar numbers of calcium ions were tested on calcium-containing samples. The results are also shown in Fig. 35. As can be seen from the data shown in FIG. 35, formulations I-III showed a significant reduction in heat of solution compared to raw calcium chloride dihydrate and control calcium powder. Calcium chloride dihydrate has a large dissolution calorific value and is known to release a large amount of heat when it comes into contact with water. Under certain circumstances, such as when a large amount of caloric salt, such as calcium chloride dihydrate, dissolves rapidly, a large amount of heat can be released and cause burns. Therefore, there is concern about the safety of contact between the mucosal surface and calcium chloride dihydrate. Such safety concerns can be mitigated by producing powders such as Formulations I-III, which do not have a high dissolution calorific value and are less likely to cause unwanted exothermic effects.

In Table 37, the ΔH (kcal / mol) of formulations I, II, III, VII and VIII were -6.9, -8.3, -4.3, -3.6 and -4.4. Anhydrous calcium chloride was used as a control in two experiments, the first experiment testing formulations I, II and III, and the second experiment testing formulations VII and VIII. Anhydrous calcium chloride had a ΔH (kcal / mol) of -12.1 in the first experiment and a ΔH (kcal / mol) of -8.4 in the second experiment.

The heats of fusion of preparations I, II and III were relatively lower than those of anhydrous calcium chloride, and similarly, the heats of fusion of preparations VII and VIII were also relatively lower than those of anhydrous calcium chloride. Therefore, dry powder formulations VII and VIII, which do not generate a large amount of heat of dissolution, are unlikely to cause an undesired heat-generating effect.

<tables num="37"><img id="000044" he="120" wi="128" file="JP5877201B2_D0001.tif" img-format="tif" img-content="drawing" /></tables>

Example 26. In vivo pneumonia model Cultures on trypsin soy agar (TSA) blood plate, 37 ° C, 5% C0₂Bacteria were prepared by growing overnight in. OD single colony in sterile PBS₆₀₀After resuspending to about 0.3, it was diluted 1: 4 with sterile PBS (about 2 x l0).⁷Colony forming unit (CFU) / mL). 50 μL of bacterial suspension (approximately 1 x l0) by intratracheal infusion of mice under anesthesia⁶Infected with CFU).

C57BL6 mice were exposed to an aerosolized liquid formulation by systemic exposure using a high power atomizer or Pari LC Sprint atomizer connected to a pie chamber cage, each containing up to 11 individuals. Mice were treated with a dry powder formulation (Table 38) for 2 hours before infection with S. pneumoniae. As a control, mice were exposed to the same amount of 100% leucine powder. Twenty-four hours after infection, mice were euthanized by pentobarbital injection, lungs were harvested and homogenized in sterile PBS. Lung homogenate samples were serially diluted with sterile PBS and seeded on TSA blood agar plates. The next day, I counted the CFUs.

Calcium dry powder treated individuals showed a decrease in bacterial titers 24 hours after infection compared to control individuals. Specifically, individuals treated with a preparation consisting of calcium sulfate and sodium chloride (formulation II) showed a 5-fold lower bacterial titer, and individuals treated with a preparation consisting of calcium citrate and sodium chloride (formulation I). Showed 10.4 times lower bacterial titer, and individuals treated with a formulation consisting of calcium lactate and sodium chloride (Formula III) showed 5.9 times lower bacterial titer (Fig. 36).

<tables num="38"><img id="000045" he="33" wi="129" file="JP5877201B2_D0001.tif" img-format="tif" img-content="drawing" /></tables>

The data presented here indicate that highly dispersible dry powders containing salts of divalent metal cations can be produced and used in the treatment of bacterial and viral infections.

Example 27.3 Stability test under refrigerated, standard and accelerated conditions for 7 months Fill formulation I-III into size 3 HPMC capsules (Shionogi Qualicaps, Madrid, Spain), (i) refrigerate at 2-8 ° C, (ii) capsule with desiccant at 25 ° C / 60% RH. Physical stability tests were performed for 3 months using representative samples placed under the conditions of storage and (iii) storage of capsules with desiccant at 40 ° C / 75% RH. FPF &lt;5.6, FPF &lt;3.4, Dv50 (Spraytec) and water content (Karl Fischer) were monitored up to the third month. As shown in Table 39, each of the formulations I to III showed good stability with respect to the physical properties evaluated under each of the above conditions.

<tables num="39"><img id="000046" he="176" wi="129" file="JP5877201B2_D0001.tif" img-format="tif" img-content="drawing" /></tables>

For formulations I and III, the stability of the bulk powder stored with the desiccant in the vial was also tested. Dry powder was produced by spray drying with a Niro Mobile Minor spray dryer (GEA Process Engineering Inc., Columbia, MD) and the powder was collected from a product filter. The liquid feedstock was sprayed using a two-fluid nozzle from Spraying Systems (Carol Stream, IL) equipped with a gas cap 67147 and a liquid cap 2850SS. A static mixer (Charles Ross &amp; Son) just before introducing the liquid feedstock into the two-fluid nozzle using a gear pump (Cole-Parmer Instrument Company, Vernon Hills, IL). Directly supplied to Company, Hauppauge, NY). (Ii) Pressurized nitrogen or pressurized air may be used as the spraying gas supplied to the fluid nozzle. The inlet temperature of the process gas was 282 ° C, the outlet temperature was about 98 ° C, and the rate of the liquid feedstock was 70 mL / min. The speed of the process gas was set to 80 kg / hour and the speed of the spray gas was set to 80 g / min. The gas spray rate may be set to obtain a particular gas / liquid mass ratio, which directly affects the size of the droplets produced. The pressure in the drying drum was -2 "WC. The spray-dried powder was collected with a powder collection filter. The liquid feedstock was prepared by dissolving in ultrapure water at a solid concentration of 15 g / L. there were.

Formulations I, II and III are then placed in bulk in 20 mL scintillation vials (Kimble, Vineland, NJ) and (i) in a Dri-Shield 3000 foil pouch (3M, Sanford, NC) desiccant (MoistureBarrierBags.com; Refrigerate at 2-8 ° C with Concord, NC), (ii) at 25 ° C / 60% RH with desiccant (Fischer Scientific, Pittsburgh, PA) in Desi-Vac container (Control Company, Friendswood, TX) Storage, (iii) A 6-month physical stability test of the powder was performed using a representative sample placed under the conditions of storage in a Desi-Vac container with a desiccant at 40 ° C / 75% RH. ..

FPF_TD (%) &lt;5.6 μm, FPF_TD (%) &lt;3.4 μm, Dv50 (Spraytec), calcium and sodium content (HPLC), and water content (Karl Fischer) under the conditions (i) and (ii). Monitoring was performed up to the second month and up to the sixth month under the condition of (iii). As shown in Tables 41, 42 and 43, each of Formulations I and III showed good stability with respect to the physical properties evaluated under each of the above conditions.

<tables num="40"><img id="000047" he="91" wi="128" file="JP5877201B2_D0001.tif" img-format="tif" img-content="drawing" /></tables><tables num="41"><img id="000048" he="93" wi="129" file="JP5877201B2_D0001.tif" img-format="tif" img-content="drawing" /></tables>

The liquid feedstock was prepared in batches by dissolving leucine, then calcium lactate, and finally sodium chloride in ultrapure water. All chemicals were obtained from Spectrum Chemicals (Gardena, CA). The solution was continuously stirred throughout the process until the ingredients were completely dissolved in water at room temperature. Dry powder was produced by spray drying with a Niro Mobile Minor spray dryer (GEA Process Engineering Inc., Columbia, MD) and the powder was collected from a product filter. The liquid feedstock was sprayed using a two-fluid nozzle from Spraying Systems (Carol Stream, IL) equipped with a gas cap 67147 and a liquid cap 2850SS. A static mixer (Charles Ross &amp; Son) just before introducing the liquid feedstock into the two-fluid nozzle using a gear pump (Cole-Parmer Instrument Company, Vernon Hills, IL). Directly supplied to Company, Hauppauge, NY). Nitrogen was used as the drying gas. The inlet temperature of the process gas was 263 ° C to 267 ° C, the outlet temperature was 98 ° C to 100 ° C, and the rate of the liquid feed material was 66 mL / min. The speed of the process gas was set to 80 kg / hour and the speed of the spray gas was set to 80 g / min. The gas spray rate may be set to obtain a particular gas / liquid mass ratio, which directly affects the size of the droplets produced. The pressure in the drying drum was -2 "WC. The spray-dried powder was collected with a powder collection filter. The liquid feedstock was prepared by dissolving in ultrapure water at a solid concentration of 15 g / L. there were.

Representative of Formula II being manually filled in size 3 HPMC capsules (Capsugel, Greenwood, NC) and bulk in a 20 mL HDPE bottle (Nolato, Trollhattan, Sweden) with a desiccant (2.4 g silica gel) in the cap. A 3-month physical stability test was performed using a standard sample. Package the bottle in a heat-sealed Dri-Shield 3000 foil pouch (3M, Sanford, NC), (i) refrigerate at 2-8 ° C, (ii) 25 ° C / 60% RH, (iii) 40 ° Stored under C / 75% RH conditions.

FPF_TD (%) &lt;5.6 μm, FPF_TD (%) &lt;3.4 μm, Dv50 (Spraytec), calcium and sodium content (HPLC), and water content (Karl Fischer) up to 3 months under all conditions I monitored it. As shown in Table 42, Formulation II is sensitive to the reduction in water content caused by the desiccant in the cap, but nevertheless showed good stability with respect to the physical properties evaluated under each of the above conditions.

<tables num="42"><img id="000049" he="59" wi="128" file="JP5877201B2_D0001.tif" img-format="tif" img-content="drawing" /></tables>

In addition, the long-term stability of formulation III, which was placed in a vial with a desiccant and covered, was further tested. Formula III is manually filled in size 3 HPMC capsules (Capsugel, Greenwood, NC) and placed in 20 mL scintillation vials (Kimble, Vineland, NJ) and (i) Detergent sponge (Fischer Scientific, Pittsburgh, PA). In a PE bag (Fischer Scientific, Pittsburgh, PA), refrigerated as a bulk powder at 2-8 ° C without encapsulation, (ii) in a Desi-Vac container (Control Company, Friendswood, TX) with a desiccant ( Typical samples placed under the conditions of storage at 25 ° C / 60% RH with Fischer Scientific, Pittsburgh, PA) and (iii) storage at 40 ° C / 75% RH with desiccant in Desi-Vac containers. It was used for a 6-month physical stability test.

FPF_TD (%) &lt;5.6 μm, FPF_TD (%) &lt;3.4 μm, Dv50 (Spraytec) and water content (Karl Fischer) were monitored up to 6 months under all conditions. As shown in Table 43, Formulation III showed good stability with respect to the physical properties evaluated under each of the above conditions.

<tables num="43"><img id="000050" he="75" wi="128" file="JP5877201B2_D0001.tif" img-format="tif" img-content="drawing" /></tables>

Example 28. Effect of Dry Powder Dose on Effect of Dry Powder on Ferret Influenza It has already been shown in an in vivo model of ferret influenza that a dry powder containing calcium and sodium reduces the severity of influenza (see Example 11). This ferret influenza model was used to test the effect of dose increase in Formulation III (leucine 10.0%, calcium lactate 58.6%, sodium chloride 31.4%; calcium ion 10.8%). Control ferrets were exposed to a powder consisting of 100% leucine under the same exposure conditions, but in vitro this control powder had no effect on viral replication. Palas Rotating Brush Generator 1000 Solid Particle Disperser (RBG, Palas) with several doses of Formulation III and leucine control powder Aerosolized with GmbH, Karlsruhe, Germany) and ferret (n = 8 per group) 1 hour before infection, 4 hours after infection, then twice daily (BID) for 4 days (Days 1-4) Day), exposed by the nose exposure method. The test was completed on the 10th day. Nasal lavage fluid samples were taken on days 1, 2 and 4 of the test, and subcutaneous body temperature and body weight were measured twice daily starting from day 0 of the test. Body temperatures measured 3, 2 and 1 day ago were used as baseline temperatures to calculate changes from baseline for each individual during the test period. The number of inflammatory cells and viral titers of the nasal wash sample were determined. Ferrets infected with influenza usually show an increase in body temperature within 2 days of infection, decrease in weight loss during the study period, and show clinical signs of infections such as somnolence and sneezing. These changes are consistent with increased titers of influenza virus flowing from the nasal passages and increased rhinitis.

The mean ± SEM of changes in body temperature in the control group and individuals receiving various doses of Formulation III is shown (Fig. 38A). Individuals treated with Formulation III showed less elevated body temperature on the two peak fever days (Days 2 and 5) compared to control-treated individuals. As shown in FIG. 38B, on days 2 and 5 after infection, formulation III treated individuals showed dose-dependently low body temperature. On both days, the control individual showed the largest increase in body temperature (Leu in Figure 38B). In addition, the mean ± SEM of body temperature changes in individuals treated with Formulation III was lower than in control individuals. Ferrets treated with the highest dose of Formulation III lost less severe weight (kinetics and maximum weight loss) and recovered faster than controls (Figure 38C). Similar to body temperature results, control individual body weight changes on day 2 post-infection were much greater than ferret treated with Formulation III, and increased calcium doses were associated with decreased changes in body temperature (Fig. 38C). ..

Overall, the data showed that Formulation III could reduce the severity of influenza infections in ferrets in a dose-dependent manner.

Example 29. Effect of dry powder on a mouse model of asthma Asthma is a disorder characterized by recurrent seizures of dyspnea, wheezing, coughing and chest tightness, with individual differences in the severity and frequency of the seizures, which can be fatal or fatal. There is also. Asthma is caused by inflammation of the airways of the lungs and is a seizure symptom caused by chronic respiratory distress (chronic asthma) or a number of provoking events such as environmental irritation, allergen exposure, cold air, exercise or emotional stress. It can be any of the intermittent diseases characterized by (intermittent asthma).

To assess the effects of calcium / sodium preparations on inflammation, specifically, to assess whether these preparations can further exacerbate inflammation or, conversely, be therapeutically safe to administer, respiratory disorders. A model-based calcium / sodium formulation was tested. To investigate the role of calcium / sodium preparations, a mouse model of allergic asthma with ovalbumin (OVA) as the allergen was used. In this model, mice are sensitized with OVA for 2 weeks and then stimulated with aerosol. The next stimulus by the OVA induces airway inflammation, resulting in changes in lung function. The basic change in inflammation is an increase in lung eosinophil count. Similar changes in lung inflammation and lung function are also observed in humans with asthma. Mice were sensitized and stimulated with OVA, as shown below.

<tables num="44"><img id="000051" he="33" wi="127" file="JP5877201B2_D0001.tif" img-format="tif" img-content="drawing" /></tables>

Sensitization was performed by intraperitoneal injection of OVA and albumin. Stimulation was achieved by systemic exposure to the sprayed 1% OVA solution for 20 minutes. Two different doses (low dose = 0.24 mg Ca) from day 27 to day 29²⁺/ kg and high dose = 0.48 mg Ca²⁺Treatment with dry powder of formulation III (leucine 10%; calcium lactate 58.6%, sodium chloride 31.4%; calcium ion 10.8%) or 100% leucine 1 hour before or 4 hours after OVA stimulation, and 30 Treatment was performed twice on the day. The dose was varied by varying the number of capsules used for each exposure. Treatment was performed using a capsule dry powder inhaler system in a systemic exposure chamber. Mice were euthanized on the final day of the study (Day 31) and bronchoalveolar lavage (BAL) was performed. The total number of cells per BAL was determined. In addition, the percentage and total number of macrophages, polymorphonuclear cells (neutrophils), lymphocytes and eosinophils were determined by banding. The data represent mean ± SD of 4-5 mice per group and represent at least two different trials.

Surprisingly, 0.48mg Ca²⁺Treatment of mice at / kg reduced total BAL cell count and BAL eosinophil count to statistically significant levels compared to control individuals (one-way ANOVA; Tukey's multiple trial) (Figure 39A and 39B). Similarly, low dose formulation III (0.24 mg Ca)²⁺The number of eosinophils was significantly reduced by (/ kg), but the high dose (0.48 mg Ca)²⁺It did not reach the level of / kg). Therefore, treatment of mice with a dry powder formulation consisting of calcium lactate and sodium chloride reduced airway inflammation in a dose-response manner.

Therefore, it has been discovered that the dry powder formulation of the present invention reduces airway inflammation and reduces the degree of eosinophilia, rather than exacerbating inflammation. This result cannot be explained solely by the biophysical mechanism of action, as it was previously observed that the transfer of soluble factors such as Derp1 or OVA in mucosal substances was not inhibited after calcium exposure. Therefore, this finding indicates that the salt preparations of the present invention have unexpected anti-inflammatory properties and may serve as monotherapy or in combination with other asthma agents in the treatment of asthma or asthma-related symptoms. It is a thing.

Example 30. Effect of dry powder on a mouse model of COPD Chronic obstructive pulmonary disease (COPD) is a progressive disease with pulmonary dysfunction, primarily resulting from smoking. Patients with COPD are also prone to exacerbations often associated with infectious pathogens and acute inflammation. These exacerbations further reduce lung function, which increases the frequency and severity of the next exacerbation.

Animal models of COPD have been developed to study both disease and therapeutic potential. Animal models of tobacco smoke (TS) exposure have been established to facilitate testing of new therapeutic agents and assess acute airway inflammation after TS exposure (Churg, A. et al., Am J Physiol Lung Cell Mol Physiol 294). (4): L612-631, 2008; Churg, A. and JL Wright, Proc Am Thorac Soc 6 (6): 550-552, 2009; Fox, JC and Fitzgerald MR, Curr Opin Pharmacol 9 (3): 231-242 , 2009).

Therefore, a test was conducted to evaluate the effect of a dry powder preparation consisting of a calcium salt and a sodium salt on pneumonia induced by TS exposure. The 4-day TS exposure model below was used.

<tables num="45"><img id="000052" he="45" wi="92" file="JP5877201B2_D0001.tif" img-format="tif" img-content="drawing" /></tables>

Mice (C57BL6 / J) were exposed to TS for up to 45 minutes per day for 4 consecutive days with systemic exposure. On the day of TS exposure, mice were treated with Formulation III (leucine 10.0%, calcium lactate 58.6%, sodium chloride 31.4%; calcium ion 10.8%) 1 hour before and 6 hours after exposure. Formulation III was administered using a systemic exposure method and a capsule delivery system. A dry powder of 100% leucine was used as a control powder. The outline of the study schedule and estimated delivery doses are shown below and in Table 44. The p38MAP kinase inhibitor ADS110836 was used as a reference (Example 11 of WO 2009/098612) and was administered by the intranasal route.

<tables num="46"><img id="000053" he="37" wi="128" file="JP5877201B2_D0001.tif" img-format="tif" img-content="drawing" /></tables>

Various doses of calcium were delivered by increasing the number of capsules used. Dose was calculated by collecting samples from the pie cage system on a fiberglass filter at 1 LPM. The aerosol collected on the filter was collected and the calcium concentration was determined by HPLC. Using this data, the aerosol concentration of calcium ions (E)_c) Was calculated and then used to determine the estimated dose level. Estimated dose level (D_L) Is the equation: D_L= E_c-Derived from RMV / T / BW, in the formula, RMV is the individual's minute ventilation (0.21LPM), T is the exposure time, and BW is the individual's body weight (kg). The estimated dose obtained is then adjusted for the inhalable aerosol fraction determined based on the particulate fraction (FPF; less than 5.6 μm mass%).

Mice were euthanized by intraperitoneal overdose of barbituric acid anesthetic 24 hours after the final exposure to air (pseudo) or TS on day 5. Bronchoalveolar lavage (BAL) was performed using 0.4 mL of phosphate buffered saline (PBS). Using slides prepared with cytospin, cells recovered from BAL were counted and cell discrimination was performed. The number of inflammatory cells in the BAL fluid of individuals exposed to TS for 4 days was determined. Individuals exposed to TS were then exposed to Formula III or 100% leucine control dry powder. Leucine-treated individuals exposed to TS showed a 9-fold increase in total cell count compared to air-treated individuals who also received control powder (Fig. 40A). The magnitude of this increase indicated the degree of inflammation observed 4 days after TS exposure. Additional populations were exposed to calcium-containing dry powder at increasing doses. The dose was increased by increasing the number of capsules used at each exposure.

As shown in Figure 40A, formulation III treatment reduced the total number of cells in the BAL solution in a dose-response manner compared to the control group (14% reduction at low dose, 32% reduction at medium dose, high dose). 45% decrease). At the highest dose tested, the reduction was comparable to the positive control p38MAPK inhibitor treatment (51% reduction). In addition, Formulation III significantly reduced the number of macrophages (Fig. 40B), neutrophils (Fig. 40C) and lymphocytes (Fig. 40D) in BAL samples, with the largest reduction percentage in neutrophils and lymphocytes. It was observed. Even low doses of calcium (1 capsule) reduce neutrophils and lymphocytes to statistically significant levels, and at high doses these cell types are comparable to positive control compounds (p38 inhibitors). It should be noted that it has decreased to the level of calcium.

In addition, the data showed that aerosol delivery of dry powder formulations of calcium and sodium salts could limit inflammation and had a general anti-inflammatory effect. The magnitude of the effect is comparable to other drugs known to be effective in this model. The data show that a dry powder formulation consisting of calcium and sodium salts can be used to treat COPD, as well as other drugs used to treat COPD (eg, ICS, bronchodilators (LABA / LAMA), p38MAPK inhibition). It was suggested that the combined use with agents, PDE4 inhibitors, antibody therapeutic agents, NF-κB inhibitors, etc.) could increase the efficacy. In order to determine the specific cell type reduced by this treatment, cell discrimination counts were performed on the same BAL sample. It should be noted that the inflammation characteristic of this model is characterized by an increase in macrophages and neutrophils, with a slight increase in lymphocytes and epithelial cells.

Example 31. Stability of dry powder formulations containing various excipients The stability of a dry powder consisting of calcium lactate and sodium chloride and further containing other excipients (eg, maltodextrin and mannitol) was tested as previously described (Example 37). The composition of these formulations is shown in Table 45.

<tables num="47"><img id="000054" he="61" wi="128" file="JP5877201B2_D0001.tif" img-format="tif" img-content="drawing" /></tables>

A dry powder was produced from a liquid feedstock prepared in batches by dissolving the excipient (mannitol or maltodextrin) in ultrapure water, then calcium lactate, and finally sodium chloride. All chemicals were obtained from Spectrum Chemicals (Gardena, CA). The solution was continuously stirred throughout the process until the ingredients were completely dissolved in water at room temperature. The solid concentration was 5 g / L in ultrapure water.

Dry powders of Formulations IV and V were produced by spray drying on a Buchi B-290 Mini spray dryer (BUCHI Labortechnik AG, Flawil, Switzerland) and the powders were collected from high performance cyclones in glass containers with a plastic cover. .. A Buchi B-296 dehumidifier was used in this system. A Buchi bifluid nozzle with a diameter of 1.5 mm was used to spray the liquid feed material. The second-rate spray gas was set to 40 mm and the speed of the aspirator was set to 90%. Indoor air was used as the drying gas. The inlet temperature of the process gas was 220 ° C, the outlet temperature was 99 ° C to 104 ° C, and the flow rate of the liquid feed material was 5 mL / min to 6 mL / min.

Formulations IV and V are then manually filled into size 3 HPMC capsules (Capsugel, Greenwood, NC) or placed in bulk in 20 mL scintillation vials (Kimble, Vineland, NJ) and (i) dried. Store refrigerated at 2-8 ° C in a PE bag (Fischer Scientific, Pittsburgh, PA) with agent sponge (Fischer Scientific, Pittsburgh, PA), (ii) Drying agent (Fischer Scientific, at 25 ° C / 60% RH) Capsules were stored in Desi-Vac containers (Control Company, Friendswood, TX) with Pittsburgh, PA) and capsules were stored in Desi-Vac containers with desiccant at (iii) 40 ° C / 75% RH. A 6-month physical stability test was performed using a representative sample.

FPF_TD (%) &lt;5.6 μm, FPF_TD (%) &lt;3.4 μm, Dv50 (Spraytec) and water content (Karl Fischer) were monitored up to 6 months under all conditions. As shown in Table 46, formulations IV and V both showed an increase of FPF_TD (%) &lt;5.6 μm and FPF_TD (%) &lt;3.4 μm, but the change was less than 20% from the time-zero value. .. Formulation IV showed good stability with respect to Dv50, while Formulation V showed a 20% increase in Dv50 under conditions (ii) and (iii). Formulation V showed good stability in water content under conditions (ii) and (iii) and showed a reduction in water content of more than 20% under condition (i). Formulation IV also showed a reduction in water content of more than 20% in (i) and (ii) and a stable water content in condition (iii). These results indicated that Formulations IV and V were sensitive to reduced water content when stored with desiccants. The decrease in water content can be the root cause of the decrease in particle size seen as a result of cascade collisions.

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Example 32. Dispersibility of a dry powder formulation containing leucine, mannitol or maltodextrin The dispersibility of dry powder formulations III, IV and V is encapsulated by inhalation with a dry powder inhaler using a typical flow rate used by the patient, as already described (see Example 15). It was evaluated by measuring the geometric particle size and percentage of the powder released from. Changes in particle size distribution and packed capsule weight were measured as a function of flow velocity, suction volume and packed weight in a passive dry powder inhaler. At each flow rate, the mass of powder released from the quintuplets was measured and the average of Dv (50) and CEPM results was calculated.

Figure 41A shows a passive high resistance dry powder inhaler (RS-01 Model 7, High Resistance, Plasticape SpA, resistance 0.036 kPa).^1/2LPM^-1) Indicates the dose released from the capsule containing Formulation III, IV or V at a filling weight of 50 mg. As can be seen from FIG. 41A, all three powders came out of the capsule well. CEPM as a function of inhalation energy of Formulations IV and V was comparable, with more than 90% of powder released at 15-20 LPM, while Formulation III was 25 LPM with CEPM of approximately 90%. FIG. 41B shows the particle size distribution of Formulations III, IV and V as released from the inhaler, characterized by a volume median diameter plotted against added inhalation energy, Dv (50). Consistent with CEPM, Formulations IV and V have similar Dv (50) characteristics, but Formulation III has a larger particle size at low flow rates (Fig. 41B). Overall, the Dv (50) value of the dry powder formulation showed that the powder was well dispersed without agglomeration.

Example 33. Effect of salt preparations containing leucine, mannitol or maltodextrin in a mouse model of bacterial pneumonia The anti-infective effect of calcium / sodium-containing dry powder was tested in an in vivo mouse model of bacterial pneumonia (see Example 26). A dry powder containing calcium lactate, sodium chloride and leucine (formulation III), maltodextrin (formulation IV) or mannitol (formulation V), gel capsules (Capsugel Vcaps , size) to a suitable weight. 00CS) Filled into 2 pieces. The filled capsules were stored in a laboratory desiccator until treatment.

Cultures on trypsin soy agar (TSA) blood plate, 37 ° C, 5% C0₂Serotype 3 Streptococcus pneumoniae was prepared by growing overnight in. Absorbance of a single colony at 600 nm (OD₆₀₀) Was resuspended in sterile PBS until about 0.3, then diluted 1: 2 with sterile PBS (about 4 x l0).⁷Colony forming unit (CFU) / mL).

C57BL6 mice were treated with leucine dry powder or the above dry powder (Ca dose 0.24 mg / kg) preparation for 2.5 minutes / capsule by systemic exposure. A dry powder aerosol was generated using a capsule delivery system connected to a top-loaded pie chamber cage, each containing up to 11 individuals. Total dry powder treatment was performed at 10 psi, 7 scfh (approximately 2.8 L / min). After 2 hours of dry powder treatment, mice were anesthetized by intratracheal infusion 50 μL (approximately 2 × 10) of S. pneumoniae suspension.⁶Infected with CFU). Twenty-four hours after infection, mice were euthanized by pentobarbital injection, lungs were harvested and homogenized in sterile PBS. Lung homogenate samples were serially diluted with sterile PBS and seeded on TSA blood agar plates. Agar plates were incubated overnight at 37 ° C and the next day CFUs were counted to quantify pulmonary bacterial loading.

The lung bacterial load of each individual is shown in FIG. Each circle represents the data of one individual, and the bar represents the geometric mean of the group. Data were normalized to leucine controls for each experiment. The data are pooled from two independent experiments. The treatment group was compared with the leucine control group by bilateral Student's t-test. Compared to control individuals, all three mice treated with the dry powder formulation showed reduced bacterial titers 24 hours after infection. Individuals treated with a formulation consisting of calcium lactate, sodium chloride and leucine (formulation III) showed 5.9-fold lower bacterial titers, and individuals treated with mannitol-containing powder and maltodextran-containing powder had approximately 2-fold lower bacterial loadings. Indicated. These data showed that while dry calcium-sodium powders containing leucine, maltodextran and mannitol were all effective, leucine-containing powders (formulation III) were most effective in treating bacterial infections.

Example 34. Effect of dry powder containing leucine, mannitol or maltodextrin in a mouse OVA model of allergic asthma Calcium / sodium preparations, also including leucine, mannitol or maltodextrin, were further evaluated for their ability to suppress the inflammatory cell response associated with allergic asthma. The powder was tested on the OVA mouse model already described (see Example 29). Briefly, mice were sensitized with OVA and then stimulated with OVA to induce airway inflammation similar to that found in humans with asthma. Mice were treated with leucine alone, Formula III, Formula IV or Formula V by systemic exposure 1 hour before or 4 hours after OVA stimulation on days 27-29, and this treatment was performed twice on day 30. .. BAL was performed on day 31, and the total number of BAL cells and eosinophils was determined by banding. The data represent the standard deviation of 4-5 mice per group and represent at least two different trials. The leucine control had slightly lower cell counts, but the total cell count (Figure 43A) and eosinophil count (Figure 43B) of mice treated with Formula III were much lower than those of mice treated with Formula IV or Formula V. It is clear that this is a leucine-containing dry calcium / sodium powder that was most effective in controlling asthma-related inflammation.

Example 35. Characteristics of powders containing various amounts of leucine and various molar ratios of calcium and sodium ions. The ingredients of the dry powder salt formulation can affect both the stability and efficacy of the formulation. A dry powder formulation was prepared to confirm the effect of increased leucine levels and increased molar ratios of calcium and sodium ions on the dry powder.

A liquid feedstock for powder was prepared in batches by dissolving leucine, then calcium lactate, and finally sodium chloride in ultrapure water. All chemicals were obtained from Spectrum Chemicals (Gardena, CA). The solution was continuously stirred throughout the process until the ingredients were completely dissolved in water at room temperature.

Formulation III was prepared from the same lot as previously described (see Example 27). All other powders were spray dried on a Buchi B-290 Mini spray dryer (BUCHI Labortechnik AG, Flawil, Switzerland) and collected from a high performance cyclone in a 60 mL glass container. A Buchi B-296 dehumidifier was used in this system. In addition, when the relative humidity in the room exceeded 30% RH, an external LG dehumidifier (Model 49007903, LG Electronics, Englewood Cliffs, NJ) was always in operation. A Buchi bifluid nozzle with a diameter of 1.5 mm was used to spray the liquid feed material. The second-rate spray gas was set to 40 mm and the speed of the aspirator was set to 90%. Indoor air was used as the drying gas. The inlet temperature of the process gas was 220 ° C, the outlet temperature was 94 ° C to 102 ° C, and the flow rate of the liquid feedstock was 4.9 mL / min to 5.3 mL / min. The solid concentration was 10 g / L when dissolved in ultrapure water.

The powder produced is characterized as already described (eg, with respect to particle size, water content) and the results of that characterization are shown in Table 47.

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Example 36. Stability of dry powder containing calcium lactate, sodium chloride and leucine in various ratios The stability of some of the powders produced was then evaluated under the conditions already described. Physical stability for 2 months using representative samples of Formulation III, VI, and three additional formulations containing various leucine fills and Ca: Na with molar ion ratios of 2: 1 and 8: 1. A sex test was performed. Hand-fill the dry powder into size 3 HPMC capsules (Capsugel, Greenwood, NC), place in 20 mL scintillation vials (Kimble, Vineland, NJ), and place in Dri-Shield 3000 foil pouches (3M, Sanford, NC). It was heat-sealed and stored under the conditions of (i) 2-8 ° C, (ii) 25 ° C / 60% RH and (iii) 40 ° C / 75% RH.

Monitor FPF_TD (%) &lt;5.6 μm, FPF_TD (%) &lt;3.4 μm, Dv50 (Spraytec), calcium / sodium content (HPLC) and water content (Karl Fischer) up to 2 months under all conditions did. As shown in Table 48, all formulations showed good stability under each of the above conditions with respect to the evaluated physical properties.

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Example 37. Aerosol properties of dry powder containing various amounts of leucine and various molar ratios of calcium and sodium ions Furthermore, the dispersibility of the dry powder was evaluated. The dry powders tested are shown in Table 49.

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The dispersibility of the dry powder formulation when delivered from a dry powder inhaler at various inhalation flow rates and volumes was evaluated. This dispersibility was investigated by measuring the geometric particle size and percentage of the powder released from the capsule when inhaled with a dry powder inhaler using a typical flow velocity used by the patient. Changes in particle size distribution and packed capsule weight for multiple powder formulations were measured as a function of flow velocity, inhaled volume and packed weight in a passive dry powder inhaler.

The powder formulation was manually filled into size 3 HPMC capsules (Capsugel V-Caps) and the filling weight was measured by a weighing method using a chemical scale (Mettler Tolerdo XS 205). Formulations III, VI, VII and VIII were packed with a filling weight of 50 mg. Specific resistance 0.036kPa^1/2LPM^-1A capsule-type passive dry powder inhaler (RS-01 Model 7, High Resistance, Plastiape SpA) was used. Timer-controlled solenoid valve (TPK2000, Copley) with flow control valve with in-line mass flow meter (TSI model 3063) Scientific) was used to set the flow velocity and inhalation volume. Capsules were placed in a dry powder inhaler, punctured, the inhaler sealed in a cylinder, and the DPI outlet pointed to a laser diffractive particle size analyzer (Spraytec, Malvern) in an open bench arrangement. A solenoid valve was used to initiate a steady air flow rate in the system and the particle size distribution was measured at 1 kHz by Spraytec during one inhalation operation for a minimum of 2 seconds. The calculated particle size distribution parameters included volume median diameter (Dv50), geometric standard deviation (GSD), and particle fraction (FPF) of particles less than 5 micrometers in diameter. At the end of the inhalation time, the dry powder inhaler was opened, the capsule was removed and reweighed to calculate the mass of powder released from the capsule within the inhalation time. In each test condition, quintuplets of capsules were measured and averaged for Dv50, FPF and capsule release powder mass (CEPM) results.

Calculate the energy required to perform the inhalation operation to correlate powder dispersions from inhalers at different inhalation rates, volumes, and resistances, and provide particle size and dose release data for inhalation energy. Plotted. Inhalation energy E = R²Q²Calculated as V, but in the formula, E is the intake energy expressed in joules and R is kPa.^1/2The resistance of the inhaler expressed in / LPM, Q is the steady flow velocity expressed in L / min, and V is the volume of intake air expressed in L.

FIG. 44 shows the doses of Formulations III, VI, VII and VIII released from the capsule using a high resistance RS-01 dry powder inhaler with a capsule filling weight of 50 mg. For each powder, 2 L of inhalation was used at a high flow rate condition of 60 LPM, which corresponds to a maximum energy condition of 9.2 joules. For the other three flow rates 30, 20, and 15 LPM, 1 L inhalation volume was used. As can be seen from FIG. 44, for all four formulations, under the highest energy conditions tested, the total mass of the powder filled in the capsule came out of the capsule in a single inhalation. Formulation III produced more than 80% of the filling weight under all inhalation conditions tested. For formulations VI and VIII, the capsule release dose was less than 80% of the packed weight at 0.29 joules. For Formulation VII, the capsule release dose was less than 80% of the packed weight at 0.51 joules.

Table 50 lists the particle size distributions of the released formulations III, VI, VII and VIII powders, characterized by Dv50 and GSD as a function of added flow velocity and inhalation energy. The fact that the value of Dv50 does not change even if the energy value decreases means that the powder is well dispersed because the released powder does not further disaggregate even if energy is added. Shown. The Dv50 values of all four formulations remained unchanged, with an average increase in Dv50 of less than 2 micrometers from the highest inhalation energy condition (and thus the highest dispersion) to an inhalation energy of 0.29 joules. In Formulation VIII, mean Dv50 did not increase by 2 micrometers from baseline over the entire range tested, with the largest increase being 1.4 micrometers (from 2.1 micrometers) when inhalation energy decreased from 9.2 joules to 0.29 joules. It was 3.5 micrometers). In this range, the size of Dv50 has not increased much, which can be expected if the released powder contains a large amount of aggregates and the dispersion is not good.

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We also evaluated the aerodynamic particle size distribution of the dry powder formulation when delivered from the dry powder inhaler to the extent suitable for deposition in the respiratory tract. The aerodynamic particle size distribution of the four powder formulations was measured by characterizing the powder with an 8-stage Anderson Cascade Impactor (ACI). The dry powder formulation was manually filled into size 3 HPMC capsules (Capsugel V-Caps) and the filling weight was measured by a weighing method using a chemical scale (Mettler Tolerdo XS 205). Formulations III, VI and VII were packed with a packing weight of 50 mg and Formulation VIII was packed with a filling weight of 40 mg. The powder was dispersed within the cascade impactor using a reloadable capsule passive dry powder inhaler (RS-01 Model 7, High Resistance, Plastiape, Osnago, Italy). Two capsules were used for each measurement, and in each capsule the dry powder inhaler (DPI) was operated twice at 60 LPM for 2 L of air. Timer control type solenoid valve with flow control valve (TPK2000, Copley) Scientific) was used to set the flow velocity and inhalation volume. ACI measurements were repeated 3 times for Formulations VII and VIII, 5 times for Formulation VI, and 8 times for Formulation III. The impactor stage, intake port (IP), inlet cone (EC) and post-filter (F) were washed with volumetricized water and the calcium ion concentration of the wash was assayed by HPLC. In Formulation III, the inlet cone was not washed. The particle size distribution of the released powder, MMAD, GSD and particle doses &lt;4.4 micrometers (FPD &lt;4.4 μm) were averaged between iterations and summarized in Table 51. Formulations III, VI and VII are for two capsules with a powder filling weight of 50 mg, which is the Ca filled in the capsule.²⁺It was equivalent to 10.8 mg. In formulation VIII, Ca of this formulation²⁺Due to its high content, the same 10.8 mg Ca²⁺Was contained in two capsules of 40 mg of packed powder.

The low standard deviation of all values in the table indicates that all four formulations have a reproducible particle size distribution (Table 51). Ca filled in 2 capsules in all iterations of all 4 formulations²⁺Of these, more than 85% were recovered within the cascade impactor. Both of these indicate that administration of the formulation from DPI was less variable and that powder residues in capsules and DPI were consistently low, and that the measured particle size distribution was not a single sample of dose. , Suggesting that it was characteristic of all delivered doses. In this study, particle doses less than 4.4 micrometers accounted for a significant proportion of the filling dose, with particle doses ranging from 2.0 mg to 5.4 mg of the 10.8 mg filled calcium, such inhalable doses. All four formulations have. The maximum GSD of the four formulations was 2.1, and the degree of polydispersity of the particle size distribution was relatively low compared to the typical dry powder formulation for inhalation.

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Example 38. Solid state properties of powder formulations VII and VIII High resolution X-ray powder diffraction (XRPD) was also used to analyze the amorphous / crystalline content and polymorphism of formulations VII and VIII. In XRPD, phase identification was performed to identify any crystalline phase observed in each XRPD pattern. XRPD patterns were collected using PANalytical's X'Pert Pro diffractometer (Almelo, The Netherlands). Samples were analyzed using Cu radiation generated with an Optix long fine focus source. Source Cu using a multi-layer mirror with elliptical steps KαX rays were applied to the sample, passed through, and collected in a detector. The sample was sandwiched between 3 micron thick membranes, transmitted geometrically analyzed and rotated for optimum orientation statistics. A beam stop was used to minimize the background generated by air scattering. Axle divergence was minimized by using solar slits for the incident and diffracted beams. Diffraction patterns were collected using a highly sensitive X'Celerator at the scan position, 240 mm from the sample. Scans over 3-60 ° were obtained with a step size of 0.017 ° and a step time of 70 seconds. As shown in Figure 45A, peaks at about 6 °, 19 °, 24 °, 31 ° and 33 ° characteristic of leucine (leucine scan not shown) are seen in the Diffractogram of Formulation VII and crystallize. It was shown that leucine was present in this powder (the peak at about 44 ° in each scan was due to the sample holder). The crystalline peaks characteristic of calcium lactate pentahydrate or sodium chloride were not observed in the differential grams of formulations VIII and VII, indicating that these components may be present in the two powders in amorphous form. I was there.

Modulated differential scanning calorimetry (mDSC) experiments were performed using the TA Instruments DSCQ200 system. About 10 mg of sample was placed in a closed pan. The mDSC conditions were up to 250 ° C with equilibrium at 0 ° C, heating rate 2 ° C / min, amplitude 0.32 ° C and modulation at a period of 60 seconds. The glass transition temperature was determined by the inflection point of the stepwise change vs. temperature curve of the reversible heat flow rate. Using this method, the glass transition temperature (Tg) of formulation VIII was determined to be approximately 107 ° C and the glass transition temperature (Tg) of formulation VII was determined to be approximately 91 ° C (Fig. 45B).

Example 39. Effect of molar ratio of calcium and sodium ions on the effect of dry powder in a mouse model of bacterial pneumonia Dry powders of various calcium to sodium molar ratios were also tested for their ability to reduce bacterial infections in mouse models of pneumonia. By systemic exposure (see Example 33), the leucine powder or calcium dose was fixed at 0.24 mg / kg and the molar ratio of calcium to sodium was 1: 0, 16: 1, 8: 1, 4: C57BL6 mice were treated with different dry powders of 1, 2: 1, 1: 1 and 1: 2. Two hours after treatment, mice were serotyped 3 Streptococcus Infected with pneumonia) and euthanized 24 hours after infection, the bacterial load in the lungs was evaluated as previously described (see Examples 26 and 34). Pulmonary bacterial loading of mice in each group is determined and presented as a percentage of control mouse bacterial loading. As shown in Figure 46A, any calcium / sodium molar ratio (1: 1 to 16: 1) calcium / sodium-containing dry powder reduced bacterial loading in S. pneumonia-infected mice. ..

In addition, the ability of the dry powders of the invention to treat mice already infected with bacteria was evaluated. Therefore, leucine, or a dry powder with a molar ratio of calcium to sodium of 4: 1 (0.31 Ca mg / kg) (formulation VIII), was applied 2 hours before S. pneumonia infection (prevention, Figure 46B). Alternatively, it was administered to mice 4 hours later (treatment, FIG. 46B). Compared to leucine-treated mice, Formulation VIII was able to reduce the bacterial load on mice not only when administered prior to bacterial infection, but also when administered after the mice had already been infected with bacteria. Therefore, the data showed that dry calcium / sodium powder could be used to treat not only infections afflicted after salt preparation treatment, but also existing and / or established bacterial and viral infections.

Example 40. Effects of leucine filling and calcium: sodium molar ion ratio on ferret influenza treatment Dry powders with a calcium to sodium molar ion ratio of 1: 2 (formulation III) and 8: 1 (formulation VI) are also effective in reducing the severity of influenza in the ferret influenza model (see Example 28). Tested. By nasal exposure method, ferret (n = 8) was added to 100% leucine control powder, 0.1 mg / kg, 0.3 mg / kg or 0.9 mg / kg formulation III (leucine 10.0%, calcium lactate 58.6%, sodium chloride 31.4). %; Calcium ion 10.8%), or 0.3 mg / kg formulation VI (leucine 39.4%, calcium lactate 58.6%, sodium chloride 2.0%; calcium ion 10.8%). Ferrets were exposed to powder 1 hour before infection, 4 hours after infection, and then twice daily (BID). The subcutaneous body temperature and body weight of the individual were measured twice a day starting from the 0th day of the test, and the body temperature measured 1 to 3 days before the test was used as a baseline for calculating the change in body temperature.

Ferrets treated with control leucine powder showed a typical increase in body temperature on days 2 and 5 after influenza infection. On the other hand, both Formulation III and Formulation VI suppressed this increase in body temperature compared to the control individual (Fig. 47A). In addition, Formulation VI reduced the severe weight loss commonly seen in influenza-infected ferrets, and Formulation III dose-response reduced weight loss. Therefore, both powders were able to reduce the severity of ferret influenza and could be used to treat viral infections.

Example 41. Effects of salt preparations with various calcium: sodium molar ion ratios on mouse allergic asthma Dry powder formulations with various calcium and sodium ion molar ratios and fixed calcium doses (0.24 mg / kg) were tested in an OVA mouse model of allergic asthma (Example 29). After sensitizing mice with ovoalbumin, on days 27-29, 1 hour and 4 hours after stimulation of sensitized mice with OVA, and twice on day 30, leucine powder or Ca: Mice were treated by systemic exposure with dry powders with Na molar ion ratios of 8: 1, 4: 1, 2: 1, 1: 1 or 1: 2. Bronchoalveolar lavage was performed on day 31, and the total number of cells and eosinophils was determined by banding. The data represent the standard deviation of 4-5 mice per group and represent at least two different trials. Dry powders with a high ratio of calcium ions to sodium ions, i.e. Ca: Na molar ratios of 8: 1, 4: 1 and 2: 1, total cell count (Figure 48A) and eosinophil count (Figure 48B). The effect of reducing both was the greatest. These data suggested that the molar ratio of calcium and sodium ions may be involved in the broad anti-inflammatory effects of dry powder formulations.

Example 42. Effect of dry powders with different molar ratios of calcium and sodium ions on TS mouse-related inflammation To determine the effect of other calcium-sodium powders and once-daily dosing regimens (QDs), the previously described 4-day tobacco smoke (TS) mouse model (see Example 30) was used. The same test used was performed. Formulation III (leucine 10.0%, calcium lactate 58.6%, sodium chloride 31.4%; calcium ion 10.8%; Ca: Na molar ratio 1: 2) and Formulation VII (leucine 37.6%, calcium lactate 58.6%, sodium chloride 4%; calcium Ion 10.8%; Ca: Na molar ratio 4: 1) was tested with a COPD model. Formulation VII was used to deliver two different doses of calcium by increasing the number of capsules used. The dose was calculated as previously described (see Example 30). Six groups of mice were exposed to TS daily for 4 days. Each group received one of the following treatments: twice daily (BID), 1 hour before and 6 hours after TS exposure by systemic powder inhalation, with Formula III, Formula VII or leucine control excipients. Administered; Formulation III was administered in a once-daily regimen (QD) 1 hour prior to TS exposure and a leucine-only control powder was administered 6 hours after TS exposure; 1 hour prior to TS exposure, p38. The inhibitor ADS110836 was administered by the intranasal route (in). An additional group (simulated) was exposed to air instead of TS for the same period and leucine control powder was administered by BID 1 hour before and 6 hours after air exposure. Twenty-four hours after the final exposure to air (pseudo) or TS on day 5, the barbituric acid anesthetic was overdosed intraperitoneally to euthanize the mice and phosphate buffered saline (PBS). Bronchoalveolar lavage (BAL) was performed using 0.4 mL. Using slides prepared with cytospin, cells recovered from BAL were counted and cell discrimination was performed.

Leucine-treated individuals exposed to TS showed a 10-fold increase in total cell number compared to air-treated individuals that were also treated with the control powder. In contrast, the positive control reference compound, p38MAPK, suppressed inflammation (Fig. 49A). Similar to the previous time, treatment with preparation III at about 1.68 mg Ca ions / kg twice daily (BID) significantly reduced the total cell count to 45% of the control individuals. A single treatment (QD) with the same dose of calcium alone 1 hour prior to TS exposure resulted in a similar reduction in total cell count (51%) (Fig. 49A). In preparation VII, the total number of cells in the BAL solution decreased in a dose-response manner (45% decrease at the dose of 0.68 mg Ca / kg, 1.41 mg) as compared with the control group. 58% reduction at Ca / kg dose). In addition, both Formulation III and Formulation VII significantly reduced the number of inflammatory cells, including macrophages (Fig. 49B), neutrophils (Fig. 49C) and lymphocytes (Fig. 49D), resulting in macrophages and neutrophils. On the other hand, the maximum effect was seen. In fact, Formulation VII reduced neutrophil and macrophage cell numbers more than the positive control reference compound p38MAPK inhibitor ADS110836. Surprisingly, the lower dose of Formulation VII reduced the number of inflammatory cells to the same level as the higher dose of Formulation III, despite about three times less delivery of calcium ions. Similarly, of all treatments, high dose formulation VII showed the largest reduction in neutrophils.

Overall, the data show that dry calcium-sodium powder has a significant impact on reducing airway inflammation and is a particularly suitable treatment for the treatment / prevention of inflammation associated with respiratory diseases such as asthma, COPD and CF. Was showing. Furthermore, the fact that once-daily and twice-daily treatments had comparable effects indicated that the once-daily treatment regimen could be used for treatment.

Example 43. Dry powder reduces expression of inflammatory chemokines / cytokines In diseases such as allergic asthma and COPD, inflammatory cells such as eosinophils, macrophages and neutrophils flow into the airway lumen in response to external stimuli, causing the cells to receive cytokines and / or chemokines. It is caused by the release. This cytokine / chemokine signaling induces chemotaxis of inflammatory cells into the airway lumen. Using the previously described COPD tobacco smoke (TS) mouse model, a study was conducted to determine whether calcium-containing dry powder provides both reduction of inflammation and regulation of inflammatory cytokine / chemokine expression. Mice were exposed to TS for 4 consecutive days and treated with Formulation III or Formulation VII once daily, 1 hour prior to TS exposure. Control individuals were exposed to a 100% leucine dry powder formulation and the second control group was treated with leucine but not TS. Mice were euthanized, bronchoalveolar lavage (BAL) was performed, and BAL samples were assayed for a panel of 13 different cytokines and chemokines involved in inflammation. Protein levels were evaluated in a multiplex assay using Luminex technology and the concentration of each protein was determined from a standard curve. Data are analyzed by one-way ANOVA for excipients^*The p-values for which p &lt;0.05 are shown below each group. KC and MIP2 are two important neutrophil chemokines that perform functions similar to human IL-8. Exposure to TS upregulated the expression of KC and MIP2 (see Figures 50A and 50B, Leu (air) and Leu bars). Treatment with Formulation III or VII reduced BAL levels of KC (Fig. 50A) and MIP2 (Fig. 50B) compared to leucine-treated individuals. The data are similar to the effect of these same formulations on lung chemotaxis of neutrophils in the same individual, and one mechanism by which dry powder formulations reduce neutrophil inflammation is neutrophils. It was shown to be through a decrease in chemokine levels that mobilize the lungs. In addition, these data showed that treatment with calcium-containing preparations regulated the biochemical and biological responses of airway epithelium and airway macrophages.

Example 44. Acute exacerbations of mouse allergic asthma pathogens are treated with dry powder Acute exacerbations in asthma and COPD patients are major causes of pulmonary dysfunction, morbidity and death. Rhinovirus infection is associated with a large number of acute exacerbations in patient populations with both diseases. The calcium-containing dry powder formulation reduced rhinovirus infection in cultured epithelial cells (see Example 13 and Figure 13C). The non-infection of mouse cells because the major lineage of rhinovirus does not bind to mouse ICAM-1 has hampered the mouse preclinical model of rhinovirus. Recently, a mouse model of rhinovirus infection using a small line (RV1B) has been described (Bartlett NW et al., Nat Med. 2008 Feb; 14 (2): 199-204). Bartlett et al. Have described both rhinovirus infection in naive mice and rhinovirus infection in ovalbumin-stimulated mice as models of acute exacerbations. These two models were used to evaluate the effect of dry calcium-sodium powder on rhinovirus infection and inflammation. The rhinovirus exacerbation model is shown below.

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BALB / c mice (n = 5) were treated with different doses of Formulation VIII for 3 days with BID prior to intranasal infection with RV1B. On the day of infection, mice were treated 1 hour before and 4 hours after infection. Twenty-four hours after infection, lung inflammation was assessed by total cell count and discriminant cell count in bronchoalveolar lavage samples. Formulation VIII significantly reduced total inflammatory cell count and neutrophil count compared to leucine-controlled individuals at the lowest dose tested (Fig. 51A). To extend this finding to exacerbation-like models, mice were sensitized with OVA using standard protocols (see Example 29) and dosed with BID on a daily basis of OVA stimulation. Mice were infected with RV1B 1 hour after the last OVA stimulation. Twenty-four hours after infection, lung inflammation was assessed by total cell count and discriminant cell count in bronchoalveolar lavage samples. Rhinovirus infection was associated with increased neutrophil inflammation compared to uninfected controls (Fig. 51B). Formulation VIII reduced its neutrophil inflammation compared to leucine-controlled individuals (one-way ANOVA; Tukey's multiple trial) (Fig. 51B). Overall, these data show that inhaled dry calcium powder can reduce the frequency and severity of acute exacerbations in patients with respiratory disease by partially reducing inflammation associated with infection. Was there.

Example 45. Calcium-containing dry powder does not cause airway hyperresponsiveness In respiratory diseases and conditions, inhalation of foreign particles often has adverse effects on the small airways of the lungs. This causes airway constriction, increases airway resistance, respiratory work and, in extreme cases, can pose a significant risk to the patient's health. Therefore, it is important that inhalation therapy does not produce any unintended consequences such as bronchoconstriction, especially if the airways are inflamed or responsive. Therefore, a test was conducted to determine whether a calcium-sodium preparation (formulation VIII) had an adverse effect on airway bronchoconstriction. Airway resistance was evaluated using two-chamber plethysmography. Briefly, the mouse is fixed to a conical retainer and then consists of two closed chambers, a chamber surrounding the head and a chamber surrounding the torso, with an airtight seal between the two chambers. I put it in. The airflow in each chamber was measured with a respiratory airflow meter, and the specific airway resistance (sRaw), which is a direct measure of the airway inner diameter, was calculated as a function of the delay time between flow signals. To accurately determine the effect of Formulation VIII on sRaw, obtain baseline sRaw measurements for 5 minutes and then place mice in high dose Formulation VIII (0.90 mg Ca).²⁺/ kg) was exposed. Mice exposure to dry powder was performed using a whole body exposure chamber using a capsule dry powder inhaler system. After treatment, sRaw measurements were obtained after 5 minutes of treatment. Mice were then exposed to and inhaled metacholine chloride in increasing doses by spraying metacholine chloride (MCh) in 0.9% sodium chloride into the head chamber for 10 seconds. The experimental procedure is shown below.

<tables num="55"><img id="000063" he="59" wi="128" file="JP5877201B2_D0001.tif" img-format="tif" img-content="drawing" /></tables>

The head chamber was cleaned after each dose of MCh (0, 6.25, 12.5, 25 and 50 mg / ml) and sRaw was obtained for an additional 5 minutes. The average of sRaw for each 5 minutes was calculated for each individual and normalized to the baseline sRaw. The same procedure was performed on two additional groups of mice, where the first group was treated with 100% leucine dry powder instead of formulation VIII and the second group was simulated with dry air only. Treatment was given.

Surprisingly, treatment with Formulation VIII (and leucine) made sRaw almost indistinguishable from sham treatment, and even statistically indistinguishable from sham treatment (Fig. 52). In fact, when mice were exposed to spray saline and inhaled (MCh 0 mg / ml), the degree of increase in sRaw was greater than that seen during dry powder treatment. In each group, sRaw increased with increasing MCh dose, but there was no significant difference in sRaw between treatment groups.

Overall, the data showed that calcium dry powder treatment had little effect on sRaw in healthy unstimulated airways, and that calcium dry powder had no adverse effect on airway response during bronchoconstriction. Unexpectedly, the increase in sRaw was greater than in Formulation VIII when inhaled with a 0.9% sodium chloride solution, which is widely used as a diluent in inhaled drug therapy. These results show that calcium-containing dry powder is unlikely to unintentionally constrict the airways as some currently approved therapies (eg, mannitol inhalation therapy for cystic fibrosis). It clearly shows that it can be used as a safe and effective treatment for conditions such as COPD, asthma and CF.

Example 46. In vivo sheep mucus pili clearance test using formulation VIII Liquid and dry powder formulations were evaluated using the established Sheep Mucus Fimbria Clearance (MCC) model. MCC was evaluated by measuring the clearance of lung Tc99m-labeled sulfur colloidal aerosols delivered by inhalation in 4 healthy sheep. Treatment Immediately after exposure to the aerosol, a radiolabeled sulfur colloidal aerosol was delivered to each sheep by the same aerosol delivery system and continuous images were collected to determine the MCC.

Using a Pari LC jet nebulizer that operates in a manner that exposes only one sheep, Formulation 46-A (8 times more tonic than isotonic, in water, CaCl₂9.4% (w / v), NaCl 0.62% (w / v)) was delivered. The atomizer was connected to a dosimeter system consisting of a solenoid valve and a compressed air source (20 psi). Connect the output port of the atomizer to a T-piece with a ventilator (Harvard vSphere Inc., Holliston, MA) attached to one end. The system was activated for 1 second at the beginning of the ventilator inhalation cycle with an inspiratory / expiratory ratio of 1: 1 and a velocity of 20 breaths / minute. The spray formulation was delivered with a tidal volume of 300 ml. The atomizer was filled with 4 mL of Formulation 46-A and dried. The dry powder formulation VIII was delivered by the same exposure method except that a rotary brush generator (RBG1000, Palas) was used instead of the atomizer to generate the dry powder aerosol. The aerosol was continuously generated by RBG and the dry powder formulation VIII was administered for 15 minutes.

Immediately after treatment, aerosolized technetium-labeled sulfur colloid (99mTC-SC) was delivered using the same aerosol exposure method as for liquid treatment. The sheep were supported by a movable retainer and a cuffed endotracheal tube was intubated to keep them awake and kept awake during the test.

Immediately after spraying 99mTC-SC, extubate the sheep and place the sheep in a natural standing position under a gamma-ray camera (Dyna Cam, Picker Corp., Nothford, CT) so that the field of view of the image is perpendicular to the spinal cord of the sheep. I stood in a posture. After obtaining the baseline images, continuous images were obtained at 5-minute intervals during the first hour. All the obtained images were saved on a computer for analysis. The area of interest of the image corresponding to the individual's right lung was tracked and counts were recorded. The left lung was excluded from the analysis because the corresponding image overlapped the stomach and swallowed radiolabeled mucus could affect the count. The counts were corrected for decay and the clearance was expressed as a percentage of the existing radioactivity reduced from the baseline image.

Delivery doses of both formulations were measured in vitro by drawing in expiratory airflow with a respiratory simulator system and collecting filter samples at the distal end of the tracheal tube. For the dry powder of Formulation VIII, calcium deposited on 10 filter samples for 1.5 minutes each was assayed by HPLC to determine the average calcium deposition rate. This 15-minute delivery dose for 50 kg sheep is 0.5 mg Ca²⁺It was calculated as / kg. For liquid formulation 46-A, the calcium content of the filter sample for 1.5 minutes was reasserted by HPLC and the delivery dose when 4 mL of the solution was dried was 0.5 mg Ca per 50 kg sheep.²⁺It was calculated as / kg. These doses measured correspond to the doses delivered from the distal end of the tracheal tube to the sheep during the procedure.

Each formulation was tested in 4 different sheep. The sheep mucous pilus clearance model is a well-established model, and its excipient clearance is usually measured to be about 5-10% 60 minutes after delivery of the radioactive aerosol (eg, Coote et al., 2009, See JEPT 329: 769-774). It is known in the art that an average clearance measurement above about 10% 60 minutes after baseline indicates increased clearance for the model. Both dry powder formulation VIII and liquid formulation 46-A showed increased mucous pili clearance in the sheep model, with mean ± standard error of clearance 60 minutes after baseline being 16.7% ± 2.7 of baseline radioactivity, respectively. % And 18.9% ± 1.2%.

Mucofimbria clearance was found to increase over 60 minutes after administration. For example, the 10-minute clearances for Formulations VIII and 46-A are 2.9 ± 2.3% baseline and 4.5 ± 1.4% baseline, respectively, and the 20-minute clearance is 4.6 ± 2.8% baseline and baseline, respectively. 9.4 ± 1.8% of line, clearance after 30 minutes is 7.7 ± 4.0% of baseline and 10.6 ± 1.7% of baseline, respectively, clearance after 40 minutes is 12.1 ± 2.5% of baseline and baseline, respectively. Clearance after 13.6 ± 0.1% and 50 minutes is 13.1 ± 2.6% at baseline and 14.5 ± 1.2% at baseline, respectively, and clearance after 60 minutes is 16.7 ± 2.7% at baseline and 18.9 ± at baseline, respectively. It is 1.2%.

The data presented here indicate that mucous pili clearance can be increased with calcium salt-based dry powders and hypertonic formulations.

Example 47. In vivo canine mucus pili clearance test The purpose of this study was to evaluate liquid and dry powder formulations in a canine mucous fimbria clearance (MCC) model. MCC was evaluated in a crossover study in 6 healthy male beagle dogs by removal of lung Tc99m-labeled sulfur colloidal aerosol delivered by inhalation by continuous imaging immediately after treatment or control aerosol exposure.

Formulation 47-A (tonicity isotonic) using a Pari LC jet atomizer that operates in a manner that exposes only one dog, using a two-way valve and a two-phase ventilator (Holliston, MA). CaCl in water at a concentration eight times that of₂9.4% (w / v), NaCl 0.62% (w / v)) was delivered. A dry powdered calcium-based formulation (Formulation III) was delivered in a similar exposure manner, except that a rotary brush generator (RBG1000, Palas) was used, or a dry powder blower was used at the lowest dose. In this model, untreated and isotonic sodium chloride (0.9% w / v) was used as the negative control and hypertonic saline (7% w / v) was used as the positive control. All doses were delivered with a duration of 15 minutes, with formulation 47-A delivered with a duration of 7.5 minutes and the lowest dose of formulation III delivered from a dry powder blower with 4 bolus deliveries. Dogs were anesthetized with propofol during exposure and imaging, and mechanical ventilation was provided during exposure. Immediately after exposure to the radiolabel, continuous planar images were collected every 2 minutes until 11 minutes later and then every 5 minutes until about 33 minutes later. The target region of the lung was analyzed, the amount of remaining activity was determined as a function of time, and the linear regression parameters of the matching radioactive clearance rate were calculated.

Harvard pump was operated prior to intubation in the dog to recover the treated aerosol from the end of the intubation tube, aerosol concentration (weighing method or chemical method by HPLC) and particle size distribution (APS, TSI, model 3321). It was determined. Minute ventilation (RMV) was calculated inversely to calculate the inhaled and deposited dose (Bide et al., 2000, J. Appl. Toxicol. 20: 273-290). Next, the estimated dose was calculated using the following formula: Dose = (C × RMV × T × DF) / BW. In the formula, C is the concentration of the test sample in the exposed air, T is the exposure time, and BW. Is body weight and deposition rate (DF = 30%) (Guyton AC, 1974, American Journal of Physiology 150: 70-77). The aerosol concentration, calculated delivery dose and MCC rate obtained are shown in Table 52.

<tables num="56"><img id="000064" he="60" wi="129" file="JP5877201B2_D0001.tif" img-format="tif" img-content="drawing" /></tables>

High-dose formulation III was found to have a significantly different (p &lt;0.05) -0.326% / min gradient from untreated baseline over a 32-minute image interval. Medium dose formulation III (gradient -0.291% / min), formulation 47-A (gradient = -0.285% / min) treatment and 7% hypertonic saline (gradient = -0.281% / min) treatment are all significant levels It was found that p &lt;0.10 was significantly different from the untreated baseline, indicating that the mucilage hair clearance rate was increased in dogs compared to the untreated one. Medium and high doses of Formulation III increased clearance to at least comparable to 7% hypertonic saline. The 7.5 minute administration of Formulation 47-A increased mucous fimbria clearance to the same extent as the 15 minute administration of 7% hypertonic saline, even with similar tonicity and half duration of administration.

Example 48. Calcium-containing dry powder combined with other active substances A. Powder preparation A feedstock solution was prepared and used to produce a dry powder consisting of raw dry particles containing calcium lactate, sodium chloride and optionally leucine and other active agents. Table 53 lists the ingredients of the feedstock formulation used to prepare the dry powder consisting of dry particles. The weight percentage on an anhydrous basis is described.

<tables num="57"><img id="000065" he="117" wi="129" file="JP5877201B2_D0001.tif" img-format="tif" img-content="drawing" /></tables>

Feeding material solutions were prepared according to the parameters in Table 54.

<tables num="58"><img id="000066" he="191" wi="129" file="JP5877201B2_D0001.tif" img-format="tif" img-content="drawing" /></tables>

The dry powders of Formulations X to XX were prepared by spray drying with a Buchi B-290 Mini spray dryer (BUCHI Labortechnik AG, Flawil, Switzerland) and collected from a high performance cyclone in a 60 mL glass container. The system used a Buchi B-296 dehumidifier with an external LG dehumidifier (Model 49007903, LG Electronics, Englewood Cliffs, NJ) running at all times. A Buchi bifluid nozzle with a diameter of 1.5 mm was used to spray the liquid feed material. The second-rate spray gas was set to 40 mm and the speed of the aspirator was set to 90%. Indoor air was used as the drying gas and the spraying gas. Details regarding spray drying conditions are given in Table 55 below.

<tables num="59"><img id="000067" he="201" wi="128" file="JP5877201B2_D0001.tif" img-format="tif" img-content="drawing" /></tables>

B. Powder characterization The physical and aerosol properties of the powder are summarized in Tables 57, 58, 59 and 60 below. Numerical values with ± represent the standard deviation of the reported numerical values. Table 56 shows FPF for all formulations_TDIt shows that &lt;3.4 μm was above 18%. Formulations X, XI, XIV, XV, XVI, XVII, XVIII and XIX are FPF respectively_TD&lt;3.4 μm was above 25%. Formulations X, XI, XV and XVI are FPF respectively_TD&lt;3.4 μm was above 30%. FPF for all formulations_TD&lt;5.6 μm was above 40%. Formulations X, XI, XIV, XV, XVI, XVII, XVIII and XIX are FPF respectively_TD&lt;5.6 μm was above 50%. Formulation XV is FPF_TD&lt;5.6 μm was above 60%. The tap density of all preparations exceeded 0.45 g / cc. Formulations X, XII, XIII, XIV, XV, XVII, XVIII, XIX and XX each had tap densities above 0.5 g / cc. Formulations X, XIII, XIV, XVII, XVIII, XIX and XX each had a tap density above 0.65 g / cc. The Hausner ratio of all formulations was above 1.8. Formulations XII, XIV, XV, XVI, XVIII and XIX each had a Hausner ratio above 2.0. Formulations XV, XVI and XIX each had a Hausner ratio of 2.4 or higher.

<tables num="60"><img id="000068" he="78" wi="129" file="JP5877201B2_D0001.tif" img-format="tif" img-content="drawing" /></tables>

Table 57 shows that the geometric particle size (Dv50) of all formulations was less than 3.5 μm at a dry powder inhaler flow rate of 60 LPM. Formulations X, XIII, XIV, XV, XVI, XVII, XVIII, XIX and XX had a Dv50 of less than 2.5 μm at 60 LPM. The Dv50 of all formulations was less than 6.0 μm at 15 LPM. Formulations X, XIII, XIV, XV, XVII, XVIII, XIX and XX had a Dv50 of less than 4.6 μm at 15 LPM. Formulations XIV, XV, XVII, XVIII, XIX and XX had a Dv50 of less than 4.0 μm at 15 LPM.

<tables num="61"><img id="000069" he="76" wi="128" file="JP5877201B2_D0001.tif" img-format="tif" img-content="drawing" /></tables>

Table 58 shows that the capsule release particle mass (CEPM) of all formulations was above 94% at 60 LPM. Formulations X, XI, XII, XIV, XV, XVI, XVII, XVIII, XIX and XX each had an CEPM of above 97% at 60 LPM. CEPM for all formulations except XI was above 80% at 15 LPM. Formulations XII, XIV, XV, XVI, XVIII, XIX and XX each had an CEPM of above 90% at 15 LPM.

<tables num="62"><img id="000070" he="80" wi="124" file="JP5877201B2_D0001.tif" img-format="tif" img-content="drawing" /></tables>

Table 59 shows that the Dv50 of all formulations measured using RODOS at the 1.0 bar setting was less than 2.5 μm. Formulations X, XIII, XIV, XV, XVI, XVII and XVIII each had a Dv50 of less than 2.2 μm. Formulations X, XIII, XV, XVI and XVII each had a Dv50 of less than 2.0 μm. The RODOS ratio of 0.5 / 4 bar of all the measured preparations was less than 1.2. The RODOS ratio of 1/4 bar of all the measured preparations was less than 1.1.

<tables num="63"><img id="000071" he="93" wi="128" file="JP5877201B2_D0001.tif" img-format="tif" img-content="drawing" /></tables>

C. Anti-inflammatory effect of co-formulation (formulation XI) of calcium salt with fluticasone propionate and salmeterol xinafoate in an OVA mouse model of allergic asthma Formulation XI was evaluated in a mouse model of allergic asthma using ovalbumin (OVA) as the allergen. This model has already been described and illustrated in Example 29.

In this model, mice are sensitized to OVA for 2 weeks and then stimulated with OVA with liquid aerosol (Example 29). This stimulation induces pneumonia and increases airway hyperresponsiveness in response to airway stimulation. The basic change in inflammation is an increase in lung eosinophil count. Similar changes in lung inflammation and lung function have been observed in humans with asthma.

Balb / c mice were sensitized and stimulated with OVA by the sensitization protocol described in Example 29. Mice placebo B dry powder (leucine 98%, NaCl 2%, w / w on an anhydrous basis), formulation 48-A (leucine 30%, NaCl 65.4%, fluticasone propionate 4.0% and salmeterol xinafoate 0.13%, anhydrous Treatment with base w / w) and formulation XI (calcium lactate 75.0%, leucine 15.31%, NaCl 5.0%, fluticasone propionate 4.0% and salmeterol xinafoate 0.58%, anhydrous base w / w). Treatment was performed in a systemic exposure chamber using a capsule dry powder inhaler system. On the final day of the study (Day 31), mice were euthanized and bronchoalveolar lavage (BAL) was performed. The total number of cells per BAL was determined. In addition, the percentages and total number of eosinophils, neutrophils, macrophages and lymphocytes were determined by banding.

The effect of preparation XI on inflammation was evaluated. (Ohta, S et al. (2010), Effect of tiotropium bromide on airway inflammation and remodeling in a mouse model of asthma, Clinical and Experimental Allergy 40: 1266-1275) and (Riesenfeld, EP (2010), Inhaled salmeterol and Based on literature such as / or fluticasone alters structure / function in a murine model of allergic airways disease , Respiratory Research, 11:22), fluticasone propionate (FP) determined eosinophil and total cell counts in mouse OVA models. It is known to reduce.

The effect of co-formulation of FP and calcium salt preparations was not yet known in the art. Therefore, formulation XI was tested. The results in Table 60 show that at similar doses (mg FP / kg mouse body weight), in the reduction of eosinophils and total cell count, Formulation XI formulated FP without calcium salts (Preparation 48- It shows that it was as effective as A).

<tables num="64"><img id="000072" he="33" wi="129" file="JP5877201B2_D0001.tif" img-format="tif" img-content="drawing" /></tables>

D. Effect of co-formulation of calcium salt and salmeterol xinafoate and co-formulation of calcium salt and tiotropium bromide (formulas XI and XVII, respectively) on specific airway resistance in mouse OVA model As described in Example 29, mouse sensitization with OVA followed by OVA stimulation of mice. Mice sensitized and stimulated with OVA show an increase in airway hyperresponsiveness that can be measured as a change in airway resistance after bronchial induction, in addition to changes in inflammation. A lung function test was performed 1 hour after the treatment on the 30th day. In this test, the specific airway resistance (sRaw) of mice was measured. Baseline sRaw was measured for 5 minutes. MCh is then delivered by spraying within the head chamber, increasing the concentration of MCh with doses of 0 mg / ml, 50 mg / ml or 100 mg / ml of metacholine (MCh) to assess lung function. MCh stimulation was applied to mice.

Mice were stimulated and their lung function tested according to the method described in Example 45. References such as (Schutz, N. (2004), "Prevention of bronchoconstriction in sensitized guinea pigs: efficacy of common prophylactic drugs", Respir Physiol Neurobiol 141 (2): 167-178) and (Ohta, S. et al. (2010) ), "Effect of tiotropium bromide on airway inflammation and remodeling in a mouse model of asthma", Clinical and Experimental Allergy From 40: 1266-1275), both salmeterol xinafoate (SX) and tiotropium bromide (TioB) increase lung function in animals and humans stimulated with inhaled metacholine chloride (MCh) dissolved in 0.9% sodium chloride. It was known to reduce the sRaw value.

The effects of SX and TioB on sRaw were known from the literature, but the effect of co-formulation of SX and TioB preparations with calcium salts was unclear. Formulation XI (calcium lactate 75.0%, leucine 15.31%, NaCl 5.0%, fluticasone propionate 4.0% and salmeterol xinafoate 0.58%, w / w on an anhydrous basis), XIV (calcium lactate 75.0%, leucine 19.89%, NaCl5 .0% and tiotropium bromide 0.113%, w / w on an anhydrous basis), 48-A (leucine 30%, NaCl 65.4%, fluticasone propionate 4.0% and salmeterol xinafoate 0.13%, w / w on an anhydrous basis w) and 48-B (leucine 34.47%, NaCl 65.42% and tiotropium bromide 0.113%, w / w on an anhydrous basis) were tested. Non-calcium-containing formulations 48-A and 48-B were tested to contrast the effects of calcium-containing formulations XI and XIV, respectively. The results obtained in the lung function test for formulations XI and XIV are shown in FIGS. 53 and 54, respectively. These data indicate that calcium-containing formulation XIV is consistent with positive control formulation 48-B and completely eliminates airway hyperresponsiveness in response to metacholine stimulation in the OVA model of allergic asthma. Treatment with Formulation XI was inconsistent with the reduction in sRaw achieved with Formulation 48-A, but variability within the group treated with Formulation XI overlapped with variability in Formulation 48-A, with an average reduction of placebo. It was lower than that seen in B.

E. Effect of co-formulation (formulation I) of calcium salt with fluticasone propionate and salmeterol xinafoate in LPS mouse model of acute lung injury In this study, a mouse model of acute lung injury was used to test the effect of calcium / sodium preparations in combination with other therapeutic agents on pneumonia. Lipopolysaccharide (LPS) isolated from Pseudomonas aeruginosa was aerosolized and exposed to mice. This stimulus caused pneumonia and altered lung function. The basic change in inflammation was an increase in lung eosinophil count. Similar changes in lung inflammation and lung function were also observed in humans suffering from acute lung injury.

Mice were exposed to spray LPS 1.12 mg / ml for 30 minutes with systemic exposure. One hour after LPS exposure, a dry powder formulation XI (calcium lactate 75.0%, leucine 15.31%, NaCl 5.0%, fluticasone propionate 4.0% and xinaho) was used in a systemic exposure chamber using a capsule dry powder inhaler system. Treatment with salmeterol acid 0.58%, w / w) on an anhydrous basis was performed. About 0.32mg Ca²⁺Mice were treated with two 90 mg capsules corresponding to / kg lung delivery. To compare the effects of calcium salt-containing and calcium-free preparations, additional populations were added to Formulation 48-A (leucine 30%, NaCl 65.4%, fluticasone propionate 4.0% and salmeterol xinafoate). An additional powder consisting of 0.13%) was exposed in the same amount (ie, fluticasone mg / kg body weight). Another group of individuals was treated with two 30 mg capsules of placebo B control powder (leucine 98%, NaCl 2%). Three hours after the dry powder treatment, all mice were euthanized, whole lung lavage was performed, and total cell count and cell discrimination count were performed.

As shown in Table 61, treatment of mice with formulation XI significantly reduced total cell and neutrophil counts in BAL fluid compared to individuals exposed to placebo B, resulting in inflammatory cells. It decreased more than the calcium-free preparation 48-A. Therefore, in the LPS model of acute lung injury, treatment of mice with formulation XI significantly reduced lung inflammation.

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Antibacterial effect of co-formulation of calcium salt and levofloxacin in F. Pseudomonas aeruginosa mouse model The effect of formulation XVII was evaluated in vivo using a mouse model of bacterial infection. Injection of cyclophosphamide (100 mg / Kg) induced neutropenia on days -4 and -1. 2 ml of bacteria (Pseudomonas aeruginosa) Luria It was grown overnight at 37 ° C in Bertani broth and about 5000 CFU per mouse was delivered in 50 μl PBS by intranasal administration. Four hours after infection, mice were subjected to placebo B powder (leucine 98%, NaCl 2%), formulation 48-C (leucine 27%, NaCl 52% and levofloxacin) using a systemic exposure chamber using a capsule dry powder inhaler system. 20%) and formulation XVII (calcium lactate 75.0%, NaCl 5.0%, levofloxacin 20%). The next day, the individual was euthanized and the lungs and spleen were removed and homogenized to determine lung and systemic bacterial loading, respectively. The homogenate was serially diluted on trypsin soybean agar plates and incubated overnight at 37 ° C. The next day, colony forming units were counted and CFU / ml for each lung and pancreas was calculated.

The results are shown in Table 62. Formulations XVII and 48-C 5log in lung compared to placebo B treated individuals₁₀It was found to significantly reduce bacterial loading, more than CFU and almost 100-fold in the spleen. Therefore, treatment of mice with Formulation XVII significantly reduces lung and systemic bacterial loading during Pseudomonas aeruginosa infection. From these data, it was found that the presence of calcium in the levofloxacin dry powder formulation did not adversely affect the effect of levofloxacin. This is a surprising result, given the literature that states that magnesium / calcium-based antacids adversely affect the bioavailability of levofloxacin absorbed from the gastrointestinal tract (Flor, S. et al.). (1990), "Effects of Magnesium-Aluminum Hydroxide and Calcium Carbonate Antacids on Bioavailability of Ofloxacin", Antimicrobial Agents and Chemotherapy 34 (12): 2436-2438) and (Pai, MP. et al. (2006), Altered steady state pharmacokinteics of levofloxacin in adult cystic fibrosis patients receiving calcium carbonate, J. Cyst. Fibros., Aug; 5 (3) : 153-7). (Ofloxacin is a racemic mixture of 50% levofloxacin, which is known to be biologically active, and 50% of its enantiomers.)

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G. Calcium salt and protein co-formulation (formulation XVIII) results in both pulmonary topical and systemic delivery of protein In this study, whether a protein can be used for lung delivery using a calcium-containing dry powder formulation, and whether this dry powder can be used for systemic protein delivery, is determined by formulation XVIII (calcium lactate 75.0%, Judgment was made using leucine 17.5%, sodium chloride 5.0%, bovine immunoglobulin G (IgG) 2.5%, w / w) on an anhydrous basis.

In this study, mice were treated with Formulation XVIII using a systemic exposure chamber using a capsule dry powder inhaler system. Mice were then treated with 2 capsules, 4 or 6 capsules of Formulation XVIII, and another population was treated with 6 capsules of placebo B control powder (leucine 98%, NaCl 2%). Placebo controls were performed to confirm non-cross-reactivity with native mouse proteins in bovine IgG assay and serum or bronchoalveolar lavage (BAL). Immediately after DP treatment, the individual was euthanized, BAL was performed and serum was collected. Wash solutions and sera were then assayed for bovine IgG using a commercially available ELISA kit. The results are shown in Table 63. Placebo B (n = 3 individuals, data not reported in the table) is below the detectable range of the assay, which means that there is cross-reactivity between bovine IgG and native mouse protein in serum or BAL. It showed that there was no. It can be seen that IgG delivered to the lungs gradually increased as the number of capsules delivered to the mice increased. In addition, treatment with 2 or 4 capsules of Formulation XVIII slightly increased the serum IgG content to the extent that it was within the detection limit of the ELISA kit, whereas treatment with 6 capsules resulted in approximately 100 ng / ml IgG. Increased to IgG. Assuming a serum volume of approximately 2 ml, treatment with 6 capsules of Formulation XVII showed that an average of 200 ng of IgG was delivered systemically. This indicated that the calcium-containing dry powder could be used for systemic protein delivery.

<tables num="67"><img id="000075" he="44" wi="128" file="JP5877201B2_D0001.tif" img-format="tif" img-content="drawing" /></tables>

Example 49. Magnesium-containing dry powder combined with an active drug A. Powder preparation A feedstock solution was prepared to produce a dry powder consisting of raw dry particles containing a magnesium salt, optionally a non-salt excipient, and at least one active agent. Table 64 lists the ingredients of the feedstock formulation used to prepare the dry powder consisting of dry particles. The weight percentage on an anhydrous basis is described.

<tables num="68"><img id="000076" he="46" wi="129" file="JP5877201B2_D0001.tif" img-format="tif" img-content="drawing" /></tables>

Feeding material solutions were prepared according to the conditions in Table 65.

<tables num="69"><img id="000077" he="69" wi="128" file="JP5877201B2_D0001.tif" img-format="tif" img-content="drawing" /></tables>

Dry powders of formulations XXI to XXIV were prepared by spray drying in a Buchi B-290 Mini spray dryer (BUCHI Labortechnik AG, Flawil, Switzerland) and collected from a high performance cyclone in a 60 mL glass container. The system used a Buchi B-296 dehumidifier with an external LG dehumidifier (Model 49007903, LG Electronics, Englewood Cliffs, NJ) running at all times. A Buchi bifluid nozzle with a diameter of 1.5 mm was used to spray the liquid feed material. The second-rate spray gas was set to 40 mm and the speed of the aspirator was set to 90%. Air was used as the drying gas and the spraying gas. Details regarding spray drying conditions are given in Table 66 below.

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B. Powder characterization The physical and aerosol properties of the powder are summarized in Tables 68, 69 and 70 below. Numerical values with ± represent the standard deviation of the reported numerical values. Table 67 shows FPF for all formulations._TDIt shows that &lt;3.4 μm was above 25%. Formulations XXI, XXII and XXIII are FPF respectively_TD&lt;3.4 μm was above 35%. Formulations XXII and XXIII are FPFs, respectively._TD&lt;3.4 μm was above 39%. FPF for all formulations_TD&lt;5.6 μm was above 50%. Formulations XXI, XXII and XXIII are FPF_TD&lt;5.6 μm was above 60%. Formulation XXIII is FPF_TD&lt;5.6 μm was above 68%. The tap density of all preparations exceeded 0.70 g / cc. The tap densities of formulations XXII and XXIII were above 0.90 g / cc, respectively. The Hausner ratio of all formulations was above 1.7. Formulations XXII and XXIII had Hausner ratios above 2.0.

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Table 68 shows that the geometric particle size (Dv50) of all formulations was less than 2.2 μm at a dry powder inhaler flow rate of 60 LPM. Formulations XXI, XXII and XXIII had a Dv50 of less than 2.0 μm at 60 LPM. Formulations XXI, XXII and XXIII had a Dv50 of less than 2.5 μm at 15 LPM.

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Table 69 shows that the capsule release particle mass (CEPM) of all formulations was above 97% at 60 LPM. The CEPM of all preparations was 15 LPM, and the CEPM was higher than 80%. Formulations XXI, XXII and XXIII each had a CEPM of above 92% at 15 LPM. Formulations XXII and XXIII each had a CEPM of above 97% at 15 LPM.

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Table 70 shows that the Dv50 of all formulations measured using RODOS at the 1.0 bar setting was less than 2.2 μm. Formulations XXI, XXIII and XXIV each had a Dv50 of less than 1.9 μm. Formulation XXIV had a Dv50 of 1.57 μm. The RODOS ratio of 0.5 / 4 bar of all the measured preparations was less than 1.2. The RODOS ratio of 1/4 bar of all the measured preparations was less than 1.1.

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Example 50. Effect of magnesium-containing preparations on eosinophilic airway inflammation and lung function in an allergic inflammation asthma model A. Inflammation A dry powder formulation XXV containing leucine (37.5%), magnesium lactate (58.3%) and sodium chloride (4.2%) was evaluated in a mouse model of allergic asthma using ovalbumin (OVA) as the allergen. Example 29 describes this model and illustrates the dosing protocol.

In this model, Balb / c mice were sensitized to OVA for 2 weeks and then stimulated with OVA liquid aerosol (Figure 29). This stimulation induces pneumonia and increases airway hyperresponsiveness in response to airway stimulation. The basic change in inflammation was an increase in lung eosinophil count. Similar changes in lung inflammation and lung function have been observed in humans with asthma.

Mice were treated with placebo B dry powder (leucine 98%, NaCl 2%, w / w on an anhydrous basis) or formulation XXV. Treatment was performed in a whole body exposure chamber using a capsule dry powder inhaler system. On the final day of the study (Day 31), mice were euthanized and bronchoalveolar lavage (BAL) was performed. The total number of cells per BAL was determined. In addition, the percentages and total number of eosinophils, neutrophils, macrophages and lymphocytes were determined by banding.

The effect of the preparation XXV on inflammation was evaluated. Based on the literature, magnesium was not known to reduce eosinophil and total cell numbers in the mouse OVA model. This finding was confirmed in this experiment. As reported in Table 71, there were no significant differences in eosinophil and total cell numbers between formulation XXV and placebo B.

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B. Lung function The sensitization of mice by OVA and the subsequent stimulation of mice by OVA were as described above. In addition to increased eosinophilic inflammation, these OVA-sensitized mice also had increased airway hyperresponsiveness, which was measurable as a change in airway resistance after bronchial induction. Literature (Okayama, H. et al. (1987), "Bronchodilating effect of intravenous magnesium sulfate in bronchial asthma", JAMA, Feb Based on 27; 257 (8): 1076-8), intravenous administration of magnesium sulfate has been shown to reduce bronchoconstriction. In a preclinical model, it was unclear whether inhaled delivery of magnesium salts to the airways had a similar effect on bronchoconstriction. To test the effect of formulated XXV on reducing mouse susceptibility to airway hyperresponsiveness, a lung function test was performed 1 hour after treatment on day 30. In this test, the specific airway resistance (sRaw) of mice was measured. Baseline sRaw was measured for 5 minutes. MCh is then delivered by spraying within the head chamber while increasing the concentration of MCh with doses of 0 mg / ml, 25 mg / ml or 50 mg / ml of metacholine (MCh) to assess lung function. MCh stimulation was applied to mice.

Mice were stimulated and their lung function tested according to the method described in Example 45. From a literature review, formulation XXV may be effective in reducing air hypersensitivity in animals and humans stimulated with inhaled metacholine chloride (MCh) in 0.9% sodium chloride, resulting in reduced sRaw values. Was considered.

Table 72 shows the results of lung function tests for formulation XXV and placebo B. These data indicate that magnesium-containing formulation XXV was consistent with the negative control placebo and that formulation XXV was not similar to the literature results that intravenous administration of magnesium sulphate reduced bronchoconstriction. ..

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Formulation XXV was tested in a mouse model of allergic inflammation, an OVA model. Formulation XXV was found not to significantly reduce eosinophil count or total inflammatory cell count compared to placebo B. Similarly, formulation XXV was tested to elucidate its role in lung function. Formulation XXV was found not to significantly reduce susceptibility to MCh stimulation compared to placebo B.

Example 51. Effect of dry powder containing magnesium on inflammation associated with TS mice To determine the effect of magnesium preparations on a COPD-like model of pneumonia, a 4-day tobacco smoke (TS) mouse model was used. This model has already been described in Example 30. Formulation XXVI (leucine 19.6%, magnesium lactate 75.0%, sodium chloride 5.4%) and Formulation VIII (leucine 20.0%, calcium lactate 75.0%, sodium chloride 5.0%) were tested in a COPD-like model. In this experiment, the doses of calcium and magnesium administered to mice were matched on a μmol salt / kg basis, and the doses were achieved by delivering 6 capsules each of formulations XXVI and VIII. The dose was calculated as previously described (see Example 30). Six groups of mice were exposed to TS daily for 4 days. Each group received one of the following treatments: Formulation XXVI, Formulation VIII or leucine control excipient once daily (QD), 1 hour prior to TS exposure by systemic powder inhalation; One hour prior to TS exposure, the p38 inhibitor ADS110836 was administered by the intranasal route (in). An additional group (simulated) was exposed to air instead of TS for the same period and leucine control powder was administered by BID 1 hour prior to air exposure. Twenty-four hours after the final exposure to air (pseudo) or TS on day 5, the barbituric acid anesthetic was overdosed intraperitoneally to euthanize the mice and phosphate buffered saline (PBS). Bronchoalveolar lavage (BAL) was performed using 0.4 mL. Using slides prepared with cytospin, cells recovered from BAL were counted and cell discrimination was performed.

Leucine-treated individuals exposed to TS showed an 8.4-fold increase in total cell count compared to air-treated individuals that were also treated with control powder. Similar to the previous time, QD treatment with preparation VIII at about 1.68 mg Ca ion / kg significantly reduced the total cell count to 53% of the control individuals. Treatment with the same dose of Mg ions / kg did not show a statistically significant reduction in total cell number (Table 73). Similar results were seen with inflammatory cell numbers in macrophages (Table 73), neutrophils (Table 73) and lymphocytes (Table 73), i.e., in Formulation VIII, the cell numbers of each type were statistically significant. It decreased by the amount. In contrast, formulation XXVI did not reduce total cell count, neutrophil count or lymphocyte count to statistically significant levels, with 65% of the 65% seen after treatment with formulation VIII with similar salt molar numbers. There was only a slight decrease in macrophage numbers (21%) at levels well below the decrease. As expected, the p38MAPK inhibitor ADS110836 reduced the number of cells in each cell type by a statistically significant amount (Table 73).

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Overall, the data show that dry calcium-based powder has a significant effect on reducing airway inflammation and is particularly suitable for the treatment / prevention of neutrophil inflammation associated with respiratory diseases such as asthma, COPD and CF. It shows that it is a cure. The data further indicate that magnesium-based dry powder has no significant effect on reducing airway inflammation and is particularly suitable for the treatment / prevention of neutrophil inflammation associated with respiratory diseases such as asthma, COPD and CF. It shows that it is not a law.

Example 52. Comparison of Calcium-Containing Dry Powder and Magnesium-Containing Dry Powder for Treatment of Acute Lipopolysaccharide Inflammation In this study, a mouse model of acute lung injury was used to test the effects of calcium dry powder and magnesium dry powder on lung inflammation. Lipopolysaccharide (LPS) isolated from Pseudomonas aeruginosa was aerosolized and exposed to mice. This stimulus caused pneumonia and altered lung function. A fundamental change in inflammation was an increase in lung neutrophil count, and similar changes in lung inflammation and lung function were also observed in humans suffering from acute lung injury.

The purpose of this study was to evaluate the effects of calcium lactate dry powder and magnesium lactate dry powder on pneumonia. In the course of this study, it was found that both calcium powder and magnesium lactate powder significantly reduced pneumonia.

Mice were exposed to spray LPS 1.12 mg / ml for 30 minutes with systemic exposure. 1 hour after LPS exposure, using a whole body exposure chamber and a capsule dry powder inhaler system, placebo B (leucine 98%, NaCl 2%) dry powder, formulation VIII (leucine 20%, calcium lactate 75%, NaCl 5%) Alternatively, mice were treated with formulation XXVII (leucine 20%, magnesium lactate 75%, NaCl 5%). Approximately 0.32 mg Ca from dry powder containing calcium lactate²⁺Mice were treated with two 90 mg capsules corresponding to / kg lung delivery. Three hours after the dry powder treatment, all mice were euthanized, whole lung lavage was performed, and total cell count and cell discrimination count were performed.

As shown in Table 74, treatment of mice with either Formulation VIII or XXVI significantly reduced total cell and neutrophil counts in BAL fluid compared to individuals exposed to placebo powder. This indicates that both calcium lactate dry powder and magnesium lactate dry powder can be effective treatments for pneumonia.

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The patents, patent applications, patent gazettes and published articles cited herein are all incorporated herein by reference in their entirety.